Université de Montréal
A Mutagenesis Study on Photoreaction Centen of Two Purple
Photosynthetic Bacteria
Par
Shiming Zhang
Départment de Biochimie
Faculté de Médecine
These pksentée Ia Faculté d a étuàcs sup6rieures
En vue de Pobtention du grade de
Philosophiae Doctor (PhD.)
En Biochimie
Septembre 1999
Sbimiig Zhang
7
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Université de Montréal
Faculté dcs Chides supérieum
Cette thése institutée
A Mutagenesis Study on Photoreaction Centers of Two Purple
Photosynthetic Bacteria
Préscatde par
Shiming Zbang
A été evalué par un jury composé des personnes suivantes:
Président-rapporteur
Lang, Bernd Fmnz
Directeur de recherche
Gingras, C a brie1
Membre du jury
Hallenkit, Patrick
Examinateur externe
Beatty, J. Thomas
Représentant du doyen de la FES
Chakmbarti, Saroj
Thbeacceptee k: 22
décembre 1999
The photosynthetic ceaction center (RC) of purple bacteria is a membram-bound
pigment-pmtein complex serving as the seat of the primary charge sejwation required for the
subsequent elecûon transfer aaoss the photosynthetic membranes by which light mergy is
converted to chemical energy. RCs of purple bacteria consist of thm protein subunits: L, M
and H. The L and M subunits bind four bacteriochiorophylls (Bchl), two bacteriopheophcopbytins
(Bph), two quinones (Q), and a femus nonheme imn atom (Fe). Some RCs contain an extra
four-heme cytochrome as a fourth subunit. The crystal structure of the RC has been obtained
in two purple bacterial species at high resolution. Our current understanding of the
mechanism of light induced electron transfer in RC has greatly benefited h m that work.
Molecular biological and genetic studies, mainly in nonsulfur purple bacteria, have
produced plentifil Monnation about the sequence, the organization and the expression of the
photosynthesis genes. The genes encociing the subunits L and M @ujL and puFi) of RC are
organized as an operon @un.The H subunit coding-gene is located 45 kb away from the pu/
operon aa detennined in Rb. cupsuIatus. in contrast to the rich information obtained h m
nonsulfur purple bacteria, the study of sulfut purple photosynthetic bacteria did not receive
the attention they desme. As a consequence, liale genetic idonnation is available about the
photosynthetic genes h m these organisrns.
This thesis pfesents wodc wîth sulfur and nonsulfur purple photosynthetic bacteria
Chapter [I and III fncus on a sulfur purple photosynthetic bacteriun (ATCC3 1ISI), which
was essignecl to the genus Ectothiorhodospira ((Et.),but of unidentifid species. Chapter IV
and V describe the expeiiments with Rhodospirillum mbmm (k.
rubnun) S1 strain.
To gain insight into the gemtic information of the photosynthesis genes in s u k
purple bacteria, we decided to shdy the pfoperon in Ectothiorhdospira sp. A 16s rDNA
sequence analysis was used to determine the phylogenetic celationship of ATCC31751 with
other species of Ectothiorhodospira. nie entire 16s rRNA cding gene was obtained by two
runs of Polymerase Chain Reaction (PCR), including one run of inverse PCR Sequence
alignment showed that 16s rDNA of ATCC 31751 siund 99.3% identity without any gap
with Ect. shposhnibvii ~ ~ ~ ~Thmfore
2 3 4 ATCC
.
31751 was identifieci as a strain of k t .
shaposhnikovil. Fmm this strain, the pufoperon genes were cloned and sequenced. Sequence
aaalysis mvealed the p m c e of five puf gents ('pz@, A, L, A4 and C)in the same order as
those in non-sulf'ur purple bacteria An extension of amund 20 amino acid residues at the Ct e i n u s of the M subunit was detected in this species as found in other species whose RCs
have four-heme cytochrome C as a fourtb subunit.
The residue of glutamine was found at
LI04 in this strain, instead of glutamic acid residue in other purple bactaal RCs, which
sfsould be responsible for the blue sbiA of the Qx absorption band of
@A
(monomeric
bacteriopheophytin at A pathway) h m 546 nm to 540 m.An open reading fiame (ORFQ')
was located before pu@.
The insertion of a Lanemycin mistance gene in this ORF caused a
80% decrease in both the amount of B880 complex and the photochernical activity of RC.
This decrease could not be complemented in hUItS by wild type ORFQ', suggesting that the
ORFQ' region might contain a cis-acting element for pu/ operm expression. A phylogenetic
study baseci on the amino acid sequence of the L and
M subunits iadicateù that this sulfur
purple bacteriun of the y subgroup is closest to another species in the same subgroup. But
some species in the a subpup are closer to this y subgroup species than to the species within
the a subgmup, indicating the occurrence of the lateral gene transfer h e e n the ancesfors of
these bacteria.
Because the cofactors in the two lines and the L and M subunits are m g e d in an
approximate C2 symrnetry, discrimination of the electron üansfer between the two lines
appears largely due to the diffennt redox potentiai of the cofactors in the two ihes, cspcially
the two mmomeric bacteriochîomphylls (BA and Be). It is our interest to see how the
properties of this site, especiaiiy the d o x potential, influence the RC fùnction in Eci.
shaposhnihvii ATCC3 1751. The selected target residue Ml 80 histidine serves as the sixth
iigand midue for the Mg of Be. The change of His to Lcu is expected to bring about the
pigment replacement of Bchl by Bph, and then d u c e the redox potential at this site. Even
though it grows vigorously in the light, this bacterium hardly grows in the dark. This makes it
difficult to carry out mutagenesis studies by commonly used in tram complementation
strategy. Therefore, in this bacterium, a new strategy was developod to introduce the
Ml 80H(CAT)+M18OL(CTT)
mutation on the chromosomal DNA and the mutation was
selected with the help of a second selectable d e r , kanamycin raistance gene (Kaf). While
the insertion of Km' had only a marginal affect on ce11 p w t h , the Nll8oHLmutation causeci
70% reâuction in the growth rate. The absorption spectnun end pigment analysis on the
purified ~ 1 8 RC
0 sample
~
indicateâ that the replacement of Bchl by Bph at the Be site in
the ~ 1 8 RC
0 was
~ ~successful. A new absorption component at 790 nm appearrd in the
mutant RC. As a result of its photo-bleaching pmperty and its resemblance to a similar
absorption component in a native 3Bph RC of C h i o r o f l ~
awantianu, thîs new component
was assigmd as the blueshifted high energy band that is located at 800 am in the wild type
RC.
Chapter IV descriks the efforts to elucidate the fùnction of the very conservecl Ntexminai region of the L subunit in Rhodospiriilum mbmm, Sequence anaiysis indicated that
the most conserved region in the RC subunits is located at the N-temiinal c e g h of the L
subunit, rather than the pigment binding pockets (Ejélanger and Gingras 1988). They M e r
suggested that this region might serve as a membrane targeting scqwnce for the L subunit. To
investigate the mles of these conserved nsidues by mutagenesis, apuf deletioa mutaat of Rr.
mbmm was tirst constructeci. The successful restoration of photosynthesis in this mutant by
wild type puf operon genes dlowed us to test the effects on the photosynthesis of the
mutations of the consecved midues in the N-tenainal region of L subunit. in total, 15
mutations including a 17 residue deletion mutation were introduced into this region. The
results indicated that this region is indispensable for photosynthesis. Many conserved residues
are important for the membrane integration of the L subunit, ventjing the suggestion
pmposed by Btlanger and Gingras (1988). Accorâhg to their tolerance to substiMon, these
residws could be classified into three categories. The resiôues in the f h t category are very
swsitive to substitution, including L6E, L23D,L25Y,L28P and L30Y. The residues in the
second category are tolerant, to various extents, to substitution, including L7R, LlOR and
L12R The residues in the third category include L18G.LI9G,L9Y and LI 1V. The mutation
of these residues did not affect the growth rate, the levels of photochernicd activity of RC or
the amount of L subunit. Therefore, the reason for theù conservation h u g h the long course
of evolution is not clear for the moment. The tesults h m two double mutants and one
quadruple mutant suggested an interaction between the nsidues 23D,2SW and 28P, 30Y in a
U-tum structure, which might be an indication that the secondary structure is involved in
a
recognition p m e s s for membrane targeting.
nie previous stuâies with Rî. rirbrum demonstr~ttedthat puh and its upstrpem gene
GU5 are transcribed hto two diff'int mRNA molecules, even though they are sepatated by
only 25 bases. To address the h c t i o n of these two genes, a deletion of the puh was designed
to avoid interference with the transcription of 0 1 15. The d t i n g pl, deletion mutant was
photosynthetically incompetent. The mutant containeci a similar amount of 8880 complex as
the wild type, but only 11% of the photachemically active RC in the wild type. The âeficiency
in the mutant couid k complemented in trans by the wild type p h gene alone. nie 0 1 15
deletion mutant was photosynthetically comptent, but with a reduced gmwth rate (67% of
the wild type). The mutation in O115 affecteci the 8880 complex content (33% of the
wildtype) but not the RC content. A pseudo-cevertant of the puh deletion mutant was isolated,
which was photosynthetically competent but with a much slowei growîh rate than the wildtype strain. Sequence searching did not detect mutation in the L a d M st~cturalgenes.
Therefore, the second mutation must be located in another locus. Since the pseudo-revertant
contained a similar level of photochernical activity of
RC as the parent mutant, the second
mutation seems to suppnss the deficient fùnction nlated to elecmn transfer in the parent
mutant.
Le centre photochimique (CP) des bactdries pourpres (rhodadcs) est un complexe
prothe-pigmeat 1%
la mmbane qui se trouve B €üe le siège de La s6pacatioa de charge
primaire et du traasfert d'dlectn,n suôdquent i travers les membranes photosynthétiques
par lequel l'tiiagie lumineuse est convertie en Cnergie chimique. Les CP des ihodacCes
contiennent trois sous-unités pmtdiques: L,M et H,qui lient quatre bactétiochlorophylles
(Bchl), deux bactériopbéophytirm @ph), deux quim,nes a un atome de â r krreux nonhème (Fe). Queiques CP contiennent une quatriém sous-unité, soit un cytochme
t6trahémique suppltmentaire. La structure crystalline des CP a et6 oôtenue chez deux
espèces de rbodaaks, a une haute résolutioa Notre compréhension actuelle du dcaaisme
de transfett tlectronique d u i t par la lurnière doit beaucoup B ces travaux Les Ctudes
mutagtnétiques ont montré que certains résidus d'acides amids jouaient un rôle dans la
détermination des propriétés des cofacteurs. Cepeidant, il faut mter que ces résidus
pounaient agir i un autre niveau, tel que la locaiisation membranriite des sous-unit&, ou la
régulation des interactions entre sous-unitds.
Les Chdes de gbnétique et de biobgie mkulaire,
rQüsCes surtout chez les
aîhioihodafées, ont dom! beaucoup d'informafion quant B la séquencesl ' o r g ~ t i o n et
,
ia régulation de I'expteSSiOn des g h e s de la pbotosyntbe. Les gènes codanî pour les
sous-unités L et M
@u/Z a puFI) du CP sont ocgaaists en opCron M.Il a été montré
que, cbcz Rb. cupstllurr~r~
le gène codant pour la sous-dt
H est sauC A 4 kb de l'opéron
puJ Contrairement aux athiorhodacées, les thiorhodacces photosynthétiques ont été peu
étudiées. Par conséquent, mus davons que trb peu d'information quand ii l'ocganilcatian
des génes photosyashctisues de ces organhms.
Cette
tbtse présente des travaux réalkés avec des thiorhodacées et des
athiorhodacces photosyiithetiques. Les chapitres II et III traitent diuw thiorbodacee
photosynthétique (ATCC 31751), qui appartient au g e m Ectothiorhodospira ( k t . ) ,
mais dont î'espèce n'a pas Ctd identifiée. Les chapitres IV et V décrivent, quant 1eux, les
expériences tCalisCesavec la souche SI de Rhodospirillum r u b m (RF. rubruni).
Afin d'élucider l'information génétique des gènes de la photosynthèse chez les
thiorfrodacées, nous avons dkidC d'étudier l'opéron puf chez Ectothiorhodospira q..
Des
Ctudes de compataison phylogédtique entre ATCC 31751 et d'autres espèces de
Ectothiorhodaspira ont dté d a M e s B l'aide de l'analyse de la séquence du DNA
nisomipw 16s. Le g&necomplet du RNA nisocnique 16s a été obtenu en effectuant
deux cycles de réaction de polym&ation en chaîne (PCR), dont un en PCR inversé.
L'analyse des séquences a permis de determiner que le DNA nisomique 16s de ATCC
3 1751 est identique B 99.3%avec Ect. shuposhnibvii D S M ~sans
~ ~aucun
,
déadage dans
l'alignement. ATCC 31751 a donc étC identifik comme Ctant une souche de Ecf.
shpshnihvii. Par la suite, les g & mde l'o'optmnpufontet6 cionCs et séquencés partir
de cette souche. L'analyse de dquellce a montré La présence de cinq génes daas l'opéron
pS@ufB,A, L. Met C), situés dans le dane or& que chez les atbioibodadcs. Une
extension d'environ 20 acides aminés du &té C-terniinal de la sms-unif6 M a 6té obsmée
cha cette espèce, tout comm chez les autres espèces qui possèdent k cytochrome C
tCtrahérnique comme quatrihe sous-unité du CP. La préseme, chez cette souche, d'un
résidu de glutamine B la position 104 de la sous-unité L, au ku d'un W u d ' d e
g b a m k p tel qu'on ie rencontre dam d'autres CP de rhodacees. semble responsable du
décaiage hypsochrom, de 546 & 540
am. de la
bande d'absorption Qx de
@A
(mombactdriophéophytiœ dans la branck A). L'anaîyse de séqueace a pexmis de
reconnaître un caâre de iecture ouvert (ORFQ')saut en amont de
gène de cé&tamx
m.L'iiisertion d'un
la kammycine dans cet ORF rtsulie en me diminution de 80% iila
fois daas La quantitd du complexe 8880 et daDs Pactivitb photochimique du CP. Cette
diminution ne pouvait Ctre compkmatée en tram par la présence du gène ORFQ' sauvage
sur un pbmîde, ce qui suggere que La région ORFQ'pourrait contenir un tIément agissant
en cis pour contrôler l'expressionde l'opéron pu/. Une analyse phylogdnétique baste sur la
séquence des peptides L et M montre que l'espèce La plus proche de cette bactérie pourpre
du sous-groupe y est une autre espèce du même sous-groupe. Cepenàant, certaines
espèces du sous-groupe a sont plus proches du cette espèce du groupe y que de d'autres
e q h s du sous-groupe a,ce qui indique qu'un transfert gdnétique latdral a eu lieu entre
les ancêtres de ces bact6rîes.
Par rapport A la voie active du tmdèrt C h n i q u e (branche A), pour laquelle Les
interactions pmtémeS-groupemnts pmsthétjques ont faa l'objet d'études poussées, la voie
dencieuse (branche B) a C t t rrletiwmnt négligée. La bsctCriochlomphyik momdrique
(BB)est adjacente A la paire Spaciaie du CP.Nous avons chercbl B d6tenniner comment les
pmpriCt& de a pigment idlueilcc~ltla photosynthèse chez Ect. sluposhnihvii ATCC
3 1751. Bien qu'elle se muliiplie rapidement A la lumiàe, cette bacténe pousse B peine dans
l'ohscuritd. Cette partiCulant6 rcnd plus difficüe La réalisation d'dhdes de mutagénèse, qui
font couiamment appel B la straîégie de wmpIémeutation en tram Cest pourquoi, chez
cette btdrie, une nouvek stratdgie a Ctt devebppée dans le but de wristniire un mutant
résultant du remplacement du gkie. Le résidu-cibie, ~ i 8 0 * , sert de sixième ligand pour
le Mg de Bs. Le rempiacemeat de l'histidiae par un tésidu non-polein tel que la leucine
devrait Muire k nrnpiacexnent du pigment Bchl par Bph. La mutation de CAT ( ~ 1 8 0 9
B CCT (~18drJ).introduite d a m le DNA chromosomique, a Ctd dlectiorilde avec succès
en utilisant comme marqueur de sélection un géne de I.esistance A la lcsnamycine (Km?,
inséré dans un site en amont de pz@. Teadis que l ' M n du gène Kmr seul n'a qu'un
effet mineur sur la croissance des bactdries, la mutation ~ 1 8 cause
0 ~une réduction de
70% du taux de croissance. Le spectre d'absorption et I'anaiyse pigmentaire sur le CP
~ 1 8 0 "purifie ont rnontd que Bch a été effèctivernent remphde par Bph au site Be du
CP ~ 1 8 0 Une
~ . nouveiie composante d'absorption A 790 am a CtC identifiée dans le CP
mutant. En combinant l ' d y s e des propriétés de photo-décoioretion et La r e s s e r n b ~
h
une composante siniüain dans le spectre d'absorption du CP contenant trois mokules de
Bph de la souche sauvage de ChJoroflexlls aurantiacru, nous pmposons que cette
nouvelle composante correspond B la bande de baute dnergie hypsochrome siMe B 800
nm cians le CP sauvage.
Le chapitre IV décrit les efforts qui ont CtC fi d a m le but d'élucider La fonction
de la région N-terminale de la sous-unit6L de Rhodospirillum mbnan, région qui est très
wllsetvée. En se basant sur des comperaisolri de sdquemx, Bélanger et GiiigrrP (1988)
avaient indiqué que La région la plus conservée des sous-unités du CP est situCe dans la
région N-terminale de Le sous-unitt5 L, plutôt que d m les sites de liaiSn11des pigments. De
phs, ces deux auteurs avaient suggéré que cette région p o d Bavir de séquence de
ciblage B la membrane pour la sowunit6 L. Ana d'identifia le die de ces tésidus
conservés par strat6gie de compldmentation en trams, un mutant de ddlétion puf de h.
rubnun a d'abord Cte constniit. La possibii6 de restorer la photosynthèse chez ce mutant,
en compiémntant avec un plesmide contenant les gènes de i'opCron pfsauvage, nous a
permis de tester ks effets sur la photosynthèse des mutations de ces résidus co11servt5s
dans la région N-tenninale de la sous-unit6 L. Au total, IS mutations sur 12 nésidus
consemés d'acides uiMCs (certains mutants contiennent une combinaison de deux résidus
mutes ou plus), ainsi qu'une ddlétion de 17 résidus ont 6tC introduits dans cette région Les
résultats montrent que cette région N-terminale conservée est inchpensable i la
photosynthèse. Plusieurs résidus conse~éssont importants pour I'intdgration mrnbraaaire
de la sous-u&
L, ce qui c o n h m la suggestion hite par Beianger et Gingras (1988).
Cependant, mus ne savons pas encore pourquoi certains résidus qui tolèrent la
substitution par d'autres acides aminés sont conservés. Chaque résidu conservé peut etre
classe dans trois catdgories M i n t e s . La première catdgorie comprend les résidus très
sensiiles B la substitutioli. Ce sont l'acide glutamique en position 6, l'acide aspertique en
position 23, le tryptopheae en position 25, la proline en position 28 et la tyrosine en
position 30. La seande categork comprenâ les résidus qui tolèrent, B des degrés divers,
La substinaioa Ce sont Ics trois tésidus a
aspartique en position 23.
@ h en position 7.10 et 12, et un résidu d'acide
Les résidus classés deas la troisième categorie compmment
deux glycines en position 18 et 19, une tyrosine en position 9 a une valine en position 11.
La mutation de ces résidus n'a atE& ai le taux de croissance, ni I'activite photochimique
du CP, ni le contenu de la sous-unit6 L chez les mutants conespoiiAlurts. Les résultats
provenant de l'&ude & &w doubles mutants et d'un mutant quadruple suggèrent uu
interaction fonnant une structure en U entre les résidus 23D et 25W d'une part et 28P et
30Y d'autre part. Ces derniers résuttats porirraient indiquer que la structure secondaire est
impiiquée dam la formation d'un signai de rrcomiaissaace pour le ciblage B la mmbrane.
Les Ctudes réaüsés antdrieurement dans ce laboratoire avaient ddmontrt que, chez RF.
Rubmm, pl,et le gene en amont, GlI5, sont &par& par seulement 25 bases, mais sont
transcrits en deux mokules de mRNA distiactes. Afin de ddterminer la fonction de ces
deux gtnes, les régions de dClétion dans un des deux gènes ont Cte conçues de &on h
M e r d'intedirer dans a transcription de l'autre gène. Le mutant de ddldtion puh ainsi
produit s'est rév6ié incapable de croissance photosynthétique. Le mutant contenait une
quantitb de complexe B880 comparable ih celle du type sauvage, mais son CP ne possédait
que 11% de l'activitd photochimique du CP sauvage. Ce ddfaut du mutant a pu être
complément6 en tram par l'insertion d'un plasmide contenant le géne puh natif seukmnt.
Le mutant 6115 est capMe de photosynthèseymais sa croissance est ralentie (k taux de
croissance équivaut & 67% de celui du type sauvage). Ii a Ctd montré que la mutation de
G115 danecte que la quantite du complexe B880 (on retrouve 33% de La quantitd
mwée chez le type sawage); le contenu du CP n'était pas affect& Un pseudo-révertant
du mutant puh, capable de missancc photosynthctipue mais & uae vitesse beaucoup plus
lente que la souche sauvage, a CtC bié.L'analyse de séquence n'a pas détectd de mutation
dam ks gènes stwtwaux L et M cbez ce mutant. La deuxième mutation doit donc Ctre
locaüsCe dans un autre bcus. Étant d o a d que le pseudo-révertant montre une activitd
photochirnique du CP similsPe B cek du mutant d'originey ii semble que la deuxième
a
mutation supprime la ddficience observée au niveau du transfert e k c t r ~ a
chez
~ le
mutant d'origine.
XIV
Table of Contents
s-ary
Résumé
Table of Contents
Dedication
List of Figures
Chapter I
Introduction
1.
Structure and h c t i o n of bacterial photoreaction center
1. Structure
2. Function
3. Structure and fùnction relationship
II.
Organization and regdation of the expression of RC subunit
structural genes
1. Genomic organization of genes encoding RC subunits
2. Regulation of the expression of puf and puh operons
III.
Mutagenic studies of the interaction of the cofacton with
their protein environment
W.
The motivations for my thesis work
Chapter II
Cloning and sequence analysis of the Ectothiorhodosph
shuposhnikovii puf operon
Results
Discussion
Chapter III
Modified BB site in the photoreaction center of
Ectothiorhodospira shaposhnikovii
Results
Discussion
Chapter IV
Mutagenic saidy on the highly conserved N-terminai region of the
L polypeptide of photoreaction center in Rhodospirillum rubrun, 57
Results
Discussion
Chapter V
Construction and characterization of p h and G 115 deletion
mutants in Rhodospiriillwn rubrum
Resulîs
Discussion
Chapter VI
Overall discussionof thesis tesdts
1. The orgmhtion of the puf and p h opemns
1.1 puf opemn, conservation and variation
1.2 Why is the p h operon so fat away from the pqfopemn
2. The strategy of mutagenesis
3. The function of the cofactors in the B way
4. The bction of the H subunit in photoreactioncenter
5. Assembly of the RC
Chapter VI1
Conclusions
References
Appendix
Materials and methods
General materials and methods
Special techniques
ûediation
To my wife Hui Chen, my son Xinhao Zheog and my daughtcr Xinmei Zhang.
List of figum
Chapter 1
Figure 1.
Diagrammatic representation of the photoreaction center
structure with cytochrome b/cl wmplex and cyt c2of &S. viridis. 4
Figure 2.
Genetic fatures of the photosynthesis gene cluster
in Rb. capsuiatus
Chapter II
Figure 3.
Figure 4.
Restriction and physicai map and deletion strategy adopted
for the sequencing of bchZ and theptf genes
of Ect. shaposhnikovii
Sequence of the puf operon and partial sequence of gene bchZ
h m Ect. shapsnikovii
Figure 5.
Alignment of the C-temiinal region of the RC M subunit of Ect.
shaposhnikovii with those of other four cytocbromecontaining and three non cytwhrome-containing RCs.
Figure 6.
Alignment of the scqwnce of the four-heme cytochrome of Ect.
shupnikovii with those of 0 t h two photosynthetic bacteria.
Figure 7.
Gene arrangement in plasmid pZS-puf70 and the plasmids used
for making ORFQ' insertion mutation and for compIementation.
Figure 8.
Absorption spectra and the change of Atm of the chromatophores
of the wild type and ORFQ' mutant strains of Ec!. shponibvii. 34
Figure 9.
Phylogenetic trees of some photosynthetic bacteria b a d on the
pptide sepuences of L and M subunits or 16s rDNA sequence.
Chapter [II
Figure 10.
The plasmid and strategy used for the integration of ~l 8oHL
mutation in the chromosome of k t . shpshni&ovii.
Figure 11.
Photosynthetic growth of the wüd type and the two mutants
of Ect. shaposhnihvii.
Figure 12.
Absorption spectra and Al= changes of the chmmatophons
in different strains of k t . shaposlurihvii.
Figure 13.
Absorption spectra of the wild type and the ~ 1 8 0 ~
39
mutant RCs of Ect. shaposhnikovii.
Figure 14.
Light-minus-dark difference spectra of the wild type and
the M 18oHLRCSof Ect. shaposhnikovii.
Figure 15.
Absorption spectm of the pigment extracts from the wild type
and the M 180"' RCs.
Figure 16.
Cornparison of absorption spectra of the Ect. shaposhniko vii
~ 1 8 0 RC
" ~and the CR.aarantiucus RC.
Chapter IV
Figure 17.
Schematic view of the plasmids and the strategy used For
conswcting and cornplementing a Apuf mutant in Rr. rubrum.
61
Absorption spectra of the chromatophores from Rx mbrum
strains: the wild type. the puf deletion mutant and the mutant
complemented with the wild type puf operon.
63
The mutations in the N-terminal region oFthe L subunit
of Rs. rubmm RC.
65
Figure 20.
Photosynthetic growth of different Rs. mbmm strains
67
Figure 2 1.
Representative show of the absorption spectra of intact cells
of different Rs. rubrum strains.
68
Figure 18.
Figure 19.
Figure 22.
Relative levels of RC photochemical activity in the
chromatophores of different RÎ. nibrun mutant strains
compared with the wild type complement.
Figure 23.
Relative ievels of the L subunit in the chromatophores
of different RF. rubrum mutant strains compared with the
wild type complement.
Figure 24.
The relationship between the levles of the L subunit and
photochemical active RC in different RF. rubrum strains.
Chapter V
Figure 25.
Constructs used for making puh and G 1 15 substitution
mutants in Rs. rubrum,
Figure 26.
Photosynthetic growth of different Rî. mbmm strains:
the wild trype, the G115 deletion mutant, the p h deletion
mutant and its complement with the wild type puh gene.
Figure 27.
Absorption specha of the chromatophores h m different
fi mbmm siraim.
Figure 28.
Absorption change atl25O nm in the chromatophores of
different Rs. rtlbmm strains upon photooxidation of the
speciai pair.
Figure 29.
Constructs used to amplement 4puh mutant.
Chapter VI
Figure 30.
The orgmization of puf operon genes in dxerent
purple photosynthetic bactena
Figure 3 1.
Cornparison of the conservation of the N-tenninal region
between the L subunit and M subunit.
Appendix
Figure 32.
Stnicture of suicide plasmid pJQ2OO-SK.
iii
Figure 33.
Structure of wide-host-range plasmid pRK404.
iv
Figure 34.
Schematic show of the strategy useâ for cloning 16s rRNA
gene from Ect. shuposhnirbvii by PCR meâhod.
Figure 35.
htroduction of mutations by recombinant PCR method.
Chapter 1
Introduction
Photosynthesis is one of the most important life processes. Phototrophic
organisrns absorb energy fiom sunlight and convert it into utilizable chemical energy for
their growth. This in turn serves as energy source for almost dl other living things on this
pla.net.
Photosynthesis starts with light absorption usually by antenna, pigment-protein
surnplrxes. The excitation rnergy is thrn transferred to
ii photoreaction centar
(RC), a
complex of polypeptide chahs and chlorophyll and other prosthetic groups. It is in the
RC thnt charge separation takes place and leads
to the build-up of a proton gradient
across the membrane for genemting ATP. Its crucial role in photosynthesis makes the RC
a central focus for photosynthesis research. Because of their relative simplicity compared
with plants, the photosynthetic bacteria ofier irreplaceable models for the mechanism of
photosynthetic energy conversion and the regulation of expression of the photosynthesis
genes. With the detemination of the three-dimensional crystal structure of RCs in purple
photosynthetic bacteria, our understanding of the photosynthetic energy conversion has
achieved F a t progress. Based on this structural information, a great nurnber of mutated
RCs have ken engineered to study the interaction between the prosthetic groups and
their peptide environment. At the same tirne, genetic and rnolecular biological studies
have produced a wealth of information on the structure and organization of
photosynthetic genes and the mechanisms of their expression and regulation.
0
1.
Structure and fraction of bacterial photoreaction center
1. Structure
the^ are two known types of RCs in purple photosynthetic bacteria according to
whether they consist of three protein subunits, L, M and H, as in Rhodobacter cupsulutus
(Rb. capsulutus), Rhodobacter sphaeroides (Rb. sphaeroides) and Rhodospirilh
rtrbrum (Rs.rubrum) (Youvan el ul., 1984; Williams d cri., t 983; Williams et al., 1984;
Bélanger et al., 1988), or have another tightly bound four-heme cytochrome as a fourth
subunit. as in Rubrivivux gelatinoslrs (Ru. gelatinosirs), Rhodopseudomonas viridis (Rps.
viridis), Ectothiorhodospira shaposhnikovii (Ect. shaposhnikovii) and Chromatium
tepidum (Chr. tepidum) (Nagashima et al., 1994; Michel et al., 1986; Lefebvre et al.,
1984; Fathir et al., 1996). Except for the cytochrome subunit, the three dimensional
crystal structures of bacterial RCs of Rps. viridis and Rb. sphaeroides (Deisenhofer et
ul., 1985; Chang et ul., 1986; Allen et al., 1987) display an overall similarity. Such
structural sirnilarity
is believed to be applicable to the RCs of other purple
photosynthetic bacteria. Figure I shows the architecture of the RC from Rps. viridis. The
L and M subunits are two integral membrane proteins each possessing five
traasmembraw a helices. The closely associated L and M subunits, together with the
prosthetic groups Form the central part of the RC. The H subunit has a transmembrane
helk at its N-terminal region and its bulky globular domain anaches to L M complex
from the cytoplasmic side. Four-heme cytochrome associates with L M complex h m the
periplasmic side. Prosthetic groups in the L and M subunits include a specialized dimer
of bacûeriochlorophylls (P), two monomeric bactenochlorophylls (BA and Be), two
bacteriopheophytins (mA and
aB),two quinones (QA
and QB), one non-heme iron, and
Cyt.WC1
L
membrane
Figure 1. Diagrammatic representation of the photoreaction center structure
of Rps. vindis with cytochrome complex b/c and cytc2 (redrawn h m
Deisenhofer and Michel, 1991).
one carotenoid. The four hemes covalently bind to the cytochrome subunit, The crystal
structure reveals that both the L and M subunits and the prosthetic p u p s are arranged in
an approximate C2 symmetry, which appears to predict two potentiai pathways for photoinduced electron transfer across the RC.
2. Fuoction
RC is the seat where the photo-induced electron tnuisfrr initiates.
In native RC,
only the prosthetic groups in the L subunit (A pathway) are used as electron carriers, even
though the symmetric arrangement of the prosthetic groups in the two lines predicts two
potential pathways for electron tnuisfer. The photo-induced electron transfer starts with
the excitation of P to P* by accepting the excited energy from the antenna or directly
absorbing radiation energy. Fmm P* one electron flows to OAvia BA.The fùnction of BA
in the primary photochernicd reaction has long been controversial. The superexchange
mode! considers BA as a v h a l carrier of electron, but without a real population of p'BA(Michel-Beyerle
et
al.. 1988). With the introduction of the femtosecond technology, a
subpicosecond component between the 'P and
@A-
was evidenced (Arit et ai 1993,
Huber et ai., 1995; Holtzwarth and Muller 1996). This supports that BA is a real electron
carrier in the primary photochernical reaction. The reduced
@A
(OA-) transfers an
electron to the primary quinone A (Qd.In natural RC,the formation of the 'P
QAe
radical
pair has a quantum yield of almost 100% (Wraight and Claytoo, 1974). The reduced QA-
tramfers an electron to the secondary quinone, Qe. Oxidized 'P is then reduced by an
electron h m a tightly bound cytochrome subunit (Iike in Rps. vin'dis) or h m soluble
cytochrome c2 (me in Rb. sphaeroides)).A second round of electron transfer le& to the
second electron transfed to
QB
and the uptake of 2 protons h m solution to form
Q W .QBH2laves its binding site and passes the electnis to cytochrorne c2 on the
periplasmic side via a mernbranous cytochrome blc 1 cornplex. (Panon 1987, Feher et al.,
1989). The protons are r e l d into the puiplasm region. The net change h m tbis
cyclic electron flowing is the generation of a proton gradient across the plasma
membrane that is utilized to synthesize ATP.
in the four-subunit RCs, the cytochrome subunit serves as an electron doaor to
oxidized.'P in Ru. gefutinosus the removal of the cytochrome subunit h m the RC
preparation by detergent treatment does not prevent the ce-reduction of photwxidized P+
by the soluble cytochrome c. The mutant lacking the four-heme cytocbrome subunit is
still photosynthetic. These nsults suggest that the four-heme cytochmme subunit does
not have an indispensable role in the photosynthetic electmn transfer pathway
(Nagashirna et al., 19%, Fukushima et al., 1988; Matsuuni et al., 1988).
The H subunit binds no fiictors, therefore its fûnction in electron baasfer was not
clear until it was found that its residues were involveci in building-up o f a water chah
running h m the Qe to cytoplasm, which is believed to semes as a proton delivery
channel (Emler et al., 1994). New m a y ~ c t i o data
n h m crystals of th Rb.
sphaeroides RC with a higher resolution revealed more than one such chamels that
probgbly serve as the pathways for protons f h n aqueous phase to the QBpocket (Stowell
et al., 1997).
3.
Structure and function nlationship
The unidirectional electron transfer in RC ( A pathway ody) is a clear indication that
the properties of the factors in the two pathways are different. In the specid pair itself, it
was found that the oscillator strength of the high energy band is only 19% of the low
energy band, indicating that the charge transfer states in the two Bchls of the exciton are
nut eyuivalent (Mar et al.. 1993; Mar md Gingras, 1995). For two monomeric Bchis (BA
and Be), the low temperature absorption spectrum of carotenoid fiee mutant RC of Rb.
sphaeroides displays a split at the Qy band, indicating that these two pigments are
different in electronic structure. An unequivalence in two Bphe pigments is manifested
by the different peak positions of their Qx absorption bands: the Qx band of OApositions
at 546m but that ofQB is at 530nm (Kirmaier et al., 1985).
The L and M subunits possess a considerable sequence homology. However the
amino acid residues forming the binding pocket for the corresponding pigments in the
wo pathways (A and B) are generally different. Hitherto, al1 attempts to make symmetnc
RC mutants have resulted in deficiency in electron transfer (Robles et al., 1990; Taguchi
et ai., 1992; Stocker et al., 1992; Taguchi et al., 1995). Arnong the overall diflerences,
some residues might be of particular significance. For example, in Rb. sphaeroides RC,
L 16 1 histidine forms a hydrogen bond with the Zacetyl group of PL (BChl at L half)
(Allen et al., 1987), but there is no similar residue at the corresponding position to form a
hydrogen bond with Pu (Bchl at the M half). Such difierences must have an influence on
the propercies of the two Bchls in special pair (Rautter et al., 1995). Another exarnple is
the residue M208 tyrosine (M210in Rb. sphaeroides) that is conserved in al1 sequenced
purple bacteriai RCs, and is in the direct vicinity of P, BA and <DA. The role of this
nsidue is thought to tune the energy levels of intermediate states, particularly l o w e ~ g
the energy of the pfBA=
state (Finkele et al., 1990). This specdatioa is suppxteâ by both
theoretical calculation and mutagenesis experiments (Parson et al., 1990; Oray et al.,
1990; Finkele et al., 1990). Noticeably, there is no tyrosine residue in the correspondhg
position in the B lim. This difference might be an important factor that influences the
direction of the electron h.aasfer. A ciear manifmtion of the influence of the amino acid
residues on the direction of elecîmn transfet is the observation in a ~ 2 0 1 ' ~ / M 2 1 2 ~ ~
double mutant RC of Rb. cupsuIafus.The initial stage of charge separation in this mutant
yields 15% of electron flow hto the B line to mB,in addition to -15% rapid deactivation
to the ground state and leaving only
- 70% electron transfers to the A line (Heller et al.,
1995).
U. Organiution and regulrtion of the t s p ~ ~ iofo RC
i aubunit stmctural gcno
1. Cenomie orgaoization o f genes eacodiig RC rubunits
The genes coding for the L (p@) and M @uFi) subunits of RC are organized in
a puf operon in al1 snrdied puiple photosynthetic bacteria (Figure 2). Basically, the puf
operon also accommodates pufA and pufB coding for or and
subdts of the light
lwvesting wmplex 1 (LH-I) respectively (Yowm et al., 1984; Nqashima et al., 1994;
Bérard et al., 1986; Fathir et al., 1997; Kiley et al., 1987; Liebetanz et al., 1991). In the
bacteria with four-subunit RC, the fowheme cytochrorne subunit coâing gene
was found to immediately foiiow or few-base overlap withpufM(Wiessner et al., 1990;
Figure 2. Cenetic features of the pbotosynthesis gene cluster in Rb. capsulaius The genes in green color are
those whose products are involved in bacteriochlorophyll biosynthesis; the orange color marks the genes for
carotenoid biosynthesis; the red color marks the genes for reaction center and the blue refers to genes for light
harvesting complexes. Arrows denote mRNA produced from the superoperon (light) or the rigorously transcribed
puf, p h and pue operons (heavy). (afier Bauer and Bird, 1996)
Liebtairz et al., 1991; NagashUna et al., 1994; Fathir et al., 1997). The arrangement of
these genes is conserved in the noasulfur purple photosynthetic bactetia so fru studied.
But in Chromutium vinosum, a sulfur purple bacterium, second pu@ and pufA wece found
dow~lstreampufi (Nagashima, GanBank No. AB0 11811). in Rb. capdatus, genetic
studies reveal the existence of the supcmperon that includes the puf operon genes and
other aeighboring genes involved in the biosynthesis of bacteriochlorophyll. The coupled
expnssion is believed to provide for balanceâ synthesis of pigment and sûucturai
polypeptide components during the transition h m aerobic to photosynthetic (mrobic)
growth conditions (Beatty, 1995). PufL and pujM are separated by only 12-13 bp (in Rb.
capsulatus, pufi and p u w even overlap). Such closely linked arrangement of pufi pujM
and pufC is thought to have benefit for maintahhg 1:1:1 stoichiometry of these subunits
in RC.
nie H subunit is encoded by puh, the only structural gene in the puh opemn. It
bas been detetmiaed in Rb. capsdatus that the p h operon is located around 45 kb away
h m and with a reversed transcription orientation to puf operon. In Rb* capsulatus, the
puh o p n seems to combiie with other opemns to compose a supemperon (Bauer et al.,
1991). But this seems not to be the case in Rs. rubrum (Wmd et ai., 1989)
in Rb. capsuIatus, genetic and sequence aaalysis dernonstnited that the genes
requinxi for synthesis of tbe pbotosystem are Linked within a 45
kb region of the
chromosome tenned the "photosynthesis gene cluster". This cluster contains the gents
for carotenoid and bacteriochiorophyll biosynthesis in the central region which in tum is
flanked by the puf and p h opennis (Hearst et al., ûeni3ank Accession no. 21 1166). This
"cluster" concept is likely applicable to other species (Yildiz et al., 1992).
2. Regulation o f the expression of pu/ and puh operons
The regulation of the photosynthetic apparatus in purple photosynthetic bacteria has
been intensively studied and is best understood in two Rhodobacter species. Rb.
capsuiatus and Rb. sphaeroides. Although there are some detailed differences, the main
pattern of regulation in these two species is çimilar. Three regulation circuits have been
recognized.
Global control on the expression of pbotosynthetic apparatus The expression of the
photosynthesis gene cluster is inhibited under aerobic conditions by an aerobic repressor
(Cd in Rb. capsularus and PpsR in Rb. sphaeroides). It inhibits the expression of
bacteriochlorophyll and carotenoid biosynthesis genes and also the light harvesting-II
genes (Penfold and Pemberton, 1994; Ponnampalam et al.. 1995: Gomelsky and Kaplan.
1995). The rninor inhibition by this repressor of puf and puh gene expression results from
the inhibition on the photosynthesis superoperons. The promoters regulated by C d
contain a conserved paiindrome sequence (TGT-N1 I -ACA) (Alberti et al., 1995; Ma et
al.. 1993; Ponnampalam et al., 1995) chat serve as the binding site for C d protein as
demonstrated by in vitro experiments (Ponnampdam and Bauer, 1997). nie binding of
C d to the palindrome sequence displays a redox-sensitive pattern: 4.5 fold higher
binding a f f i t y under oxidizing versus reducing conditions. No redox-sensitive structure
such as iron or iron-sulfùr clusters known fiom other redox responding DNA-binding
proteins has k e n identified in Cd. It is therefore unclear how this protein senses a
difference in the redox potentid to modi@ its binding affuiity to its target sequence. In
Rb. sphaeroides, Kaplan's group identified another ngulatory gene appA (Gomelsky and
Kaplan, 1995). AppA protein activates the expression of pigment-synthesis genes and
puc opetons (Gomelsky and Kaplan, 1995) and seems to intemct with PpsR in
regdation of photosynthesis gene expression (Oomclsky and Kaplan, 1997).
Anaerobk induction
The expression of W a n d puh opemas is highly activated under
anaerobic conditions. The activation depends on a signal transduction system wmposed
of membrane s p d g sensor kinase (Re@ in Rb. cupsulu~ur and RrB in Rb.
sphaeroides) (Mosley et al., 1994; Gomelsky and Kaplan, 1995) and a soluble response
regulator (RegA in Rb. capsulutus and PrrA in Rb. sphaeroides) (Sganga and Bauer,
1992; Eraso and Kaplan, 1994). Re@ and PmB are homologous histidine kinases. Re@
undergoes autophosphoryiation in anaerobic condition and is able to
transfer the
phosphate moiety to RegA at a consaved aspartate (Inoue et al., 1995). That RegB can
aot undergo autophosphorylation under aerobic condition implies that RegB may
hction as a redox sensor. But the mechanism that allows Re@ to monitor the redox
state is unclear. RegA fùnctions as a DNA-binding protein that ditectly affects the
expression of its target genes @u et al., 1998). Some other d t s indicate that RegA
may not only accept the signal delivered by RegB, but also b m other sensors, like SenC
which is involved in cytochrome c oxidase expression (Buggy anâ Bauer, 1995).
Lmt regahtion Although oxygen concentration is a major elernent in controlling the
expression of photosynthesis genes, light intensity also play a regulatocy d e . The
expression ofpujaid puh genes increases by around two folds in response to a reduction
in light intensity. One of the responsible genes has been identified as hvrA (Buggy et al.,
1994). The dimption of hvrA results in an inability to stimulate puf and puh expression
in response to a reduction in light intensity. HvrA contains a putative helix-tumhelix
DNA binding domain. DNA footprint experiments demonstrate that HvrA binds to the
puf and puh promoters. These data indicate that
HvrA hctions as a dim-iight
transcription activator. But the mechanism by which iight intensity influences HvrA
activity is unknown.
III. Interactions o f the prosthetic groups with their protein environment
The crystal structure of RC has revealed the protein environment of the prosthetic
groups and predicted a number of amino acid residues that might have important roles in
modiQing the properties of the prosthetic groups. These predictions have been tested by
mutagenesis studies to specify the functions of individual residues.
In RCs, the ~g
ions of Bchls are usually liganded with histidine. Substitution of
His with small residues like Gly (Goldsmith et al., 1996) and changes to residues with
liganding groups like Gln (Bylina and Youvan, 1988) do not cause the pigment exchange.
But the substitution of His with Leu or Phe brings about the replacement of Bchl by Bph.
The replacement of Bchl by Bph in special pair results in the soçalled heterodirner RCs
@chL-Bphu, or BphL-BchlM)(Bylina and Youvan, 1988; Schenck et al., 1990). The
mutant strains are incapable of photosynthetic growth (Bylina and Youvan, 1988). The
electron transfer in the heterodimer RCs stili flows through the A pathway but with much
lower efficiency (Bylina and Youvan, 1988; McDowell et al., 1991). In the absorption
spectra of heterodimer RCs, the 850 nm absorption peak attributable to the low-energy
singlet transition of the dimer is replaced by a brnad, diffuse band (Bylina d Youvan,
1988).
At two monomeric bacteriochiorophyll binding sites, the attempt to change Bchl
to Bph in Rb. coprrhtw and Rb. Jphoeroides encountered difficulty due to the instability
of the mutant RCs (Bylina et al., 1991; Wachtveitl, 1992), but was cewarding in Rps.
viridis (Arlt et al., 1996). The chromophore exchange at BA site lowers the energy level
of the electron transfer state P+/B~;,resulting in the repopulation of P+/@~-and f a e t
decay of P*. nie mutant strain bas ody S
O
O
h photosynthetic growth rate of the wild type.
The replacement of Bph at @Aby Bchl (so-called f3 type RCs) has been achieved
in Rb. cap sui^ and Rb. spheroides by replacing the conesponding Leu residues which
are located over the face of Bph with His (Heller et al., 1995; Kirmaier et al., 1991). In P
type RCs the kinetics and yield of initial electron transfer from
P* are largely
unpertwbed, but the quantum yield of P+/QI- is only 60-8û% of diat in the wild type.
M2 10 Tyrosine in Rb. sphaeroides (M208Yin other purple bactena) is conserved
in purple photosynthetic bacteria This residue is in the vicinity of P. BA and OA.
Theontical calculations suggcst that the interactions of M2lOY may lower the energy of
the state P%Â, thereby fiicilitating the electron transfer dong the A pathway (Ruson et
al., 1990). The mutation of this Tyr residue to Leu affects the ce11 growth in the low üght,
but the mutant M210F can grow uader both high and low light (Gray et al., 1990). In
these mutants the pcimary electmn transfer is much slower than that in the wild type,
indicating that this residue is important for the fast rate of the primary electron transfer
(Finkele et al., 1990). Interestingly, in mutant M210W RC, an altemative route for charge
sepnration has been established, that is the excited BAby illuminating at 799 nrn drives
ultrafiut transmembrane electron ûansfer without the involvement of P* (Van Bderode
et ai., 1997).
M.50Tryptophan is located within van der
Waals contact distance of both @A
and QA and its indole side chah is nearly coplanar with t&e QA. Out of m e n mutations of
this residue (Phe, Leu, Met, Val, Glu, and Arg), only the Phe mutant is capable of
gmwing photosyntheticaily (Coleman et al., 1990). The Phe mutation only slightiy affects
the
QA
binding, but the other six mutations cause a drastic decrease in
aanity. The electron-transfer rate of O,( to
QA
QA
binding
is impaired to different extents in the
different mutants. These resuits suggest that M250W may play dual fûnctions in tight
binding of the QA and the fast electron traasfer h m @A* to QA.
L229IIe cornes close to Qe as revealed by RC structures. It bas been changed to
al1 other amino acids except Pro a d Phe (Bylina and Youvan, 1987). in general,
hydrophobic residues of the moderate size are found to hinction kst at this position,
while the other mutants are either incapable of photosynthetic growth or of slower growth
rate. This indicates the importance of L229Ile residue pmbably in binding a d o r
orientating QB by the hydrophobic interaction.
L223Ser has been pndictedto be a possible pioton donor to the nduceâ QB.The
mutation of this residue to N a or Asa causes teduced protonation of Qs in the mutants.
The proton coupled second electron transfer rate in the mutant RCs is greatly reduced
(Bylina et al., 1989; Paddock et al., 1990; Paddock et al., 1995). RCs with His or Cys at
L223 do not bind QB.But the RCs with Thr, Asp or Gly at LU3 cetain a fast protonwupled second election trader rate. The RC with Gly at L223 bas a normal electron
traasfer rate, and a water molecule is suggested to replace the m*ssing Ser h y h x y l
group. Consistent with the crystal structure, this result implies that L223Ser fhctions as
a direct proton donor to the reduced Qe.
L2 12Glu is an acidic group close to the QB site. The substitution of Glu by Gln
causes a slower cytochrome turnover rate by a factor of >40 following the fast oxidation
of three cytochromes (Paddock et al., 1989). Further measurements on the electron
t~nsferrate and the proton uptake: rate demonstmted t h t the LZi2Giu residue piays the
role in the protonation of the reduced QeH (McPherson er al., 1990).
L2 13Asp is one of the residues important for protonation of Qe. Lts substitution
with Asn causes a drastic reduction of the cytochrome turnover rate f i e r a fast oxidation
of two cytochromes (Rongey et al., 1991). But a mechanistic interpretation is
cornplicated by potential electrostatic effects due to removal of a negative charge.
L 162Tyr is conserved in al\ purple bacteria with sequence data documented. In
Rb. sphaeroides, the roles of this residue in mediating the re-reduction of P+ by soluble
cytochrome c2 has been reveaied by mutagenesis studies. The replacement of Tyr by Phe,
Ser. Leu, Met, and Gly causes a slower photosynthetiç growth to various degrees, with
L162G the slowest. The fast phase of re-rcduction of P+ in the wild type is abolished in
al1 the mutants. dso with most severe in L162G mutant (Farchaus et al.. 1993). An
aliphatic substitution at LI62 slows cyt-cZ-RC association and dissociation rates
(Wachtveitl et al., 1993). It is not clear whether L162Tyr ody structurally facilitates the
electron transfer or, as an addition, directly mediates the electron itansfer.
H173Glu is near the QB binding site and with some o k residues is thought to
fonn a cluster of ionizable amino acid side chains with strong electronstatic interactions.
Ln a H173Gln mutant RC, both the fust and the second electron M e r are greatly
retarded (Takahashi and Wraight, 1996). The addition of azide (weak acid) c m reverse
most of these disruptions of events coupled to proton delivery to QBin the Hl 73Gl.nRC.
Because H l 73Glu is not in direct contact with Qe, its efict is thought to be electrostatic
(Takahashi and Wraight, 1996). Difference FTlR spectroscopie studies of H 173Glu+Gln
and H173Glu+Asp mutant RCs indicate that Hl73Glu is not a major contributor to
proton uptake upon Qe-formation, because compared to the ~ i l dtype RC there are no
new bands appearing in the carboxylic acids region in any of the mutant RCs (Nabedryk
et al., 1998). The H7 l3Glu was thought to remain ionized in the QB and Qe* States. The
stnictural change in the mutant RCs is suggested to contribute to the alterrd kinetics
observed in the mutant RCs (Takahoshi and Wraight, 1996; Nabedryk et al., 1998).
IV.
The motivations for my thesis work
The extensive genetic studies on the purple bacterid RC are featured by king
focused on nonsulfu purple photosynthetic bacteria. As a consequence, Iittle information
is available about the sulfur purple photosynthetic bacteria. The structure and function
studies by mutagenesis approaches have k e n largely directed to the prosthetic groups in
the active pathway for the electmn transfer. The importance of the prosthetic groups in
the silent pathway has not been Bdequately addressed. In another aspect, in overall
structure-fùnction studies, attention has been mostly given to the electron- and
H'-
tramfer related residues, while the residues that might play important roles in RC subunit
assembling and subunit-subunit interaction have not received adequate attention. It is also
worthwhile to point out that the most of the previous work has been aimed at the L-M
core cornplex, the H subunit has been relatively neglected. As a consequence, in contrast
to the rich idonnation about the subunit L and M. the exact function of the H subunit in
photosynthesis is not M y understood. Stimulateci by this situation and basod on the
pnvious work donc in this labmtoty, my thesis work was first directeci to investigate the
genetic organization of puf genes in a sulhir purple photosynthetic bactenum.
Ectothiorhodospiw sp. ATCC3 1751 and to make a cornparison with (hose in nonsulfur
of liiz work, ihe mutapnesis of the RC in this species
purple bacteria In t
h second put
was assignai to the
Be site, the monomenc Bchl in B
pathway, to understand how the
propcrties of this "silent" cofactor affects the RC fuaction. The third part of the work
was promoted by the findings h m this laboratory that the most coaserved sequence in
pu/ L and M is located in the Naterminal ngion of the L subunit instead of the prosthetic
groups binding pockets (Belanger et al., 1988). Some fatutes of chis region are
remllllscent of the matrix targeting signal of cytochrome cl and other mitochondrid
membrane pmteins, suggesting a fiinction in membrane integration of the L subunit
(Btlanger et al., 1988). But the exûeme sequence conservation seems beyond the
requirement oniy for membrane integration. A systematic mutagenesis was thetefore
conducted to investigate the hction of îhese conservai amino acid residues in this
region in RF. lubrum. The work in the last
of this thesis was aimed at clarifying tûc
physiologicd funciion of the H subunit and its upsûeam genc G 115, and also to cstabliq
a system for the finire mutagenesis studies of these two genes in RI. rubrum.
Chapter II
Cloning and sequence analysis of the Ectothiotlrodospira
shapushnikovii puf operoa.
Ectothiorhodospiraceae and Chromatiiocae are sulhit purple photosynîhetic
bacteria, located in the y subgroup of a four-branched evolutionary ûee based on 16s
rRNA sequence analysis (Woese, 1987). Chroniatiaceae accumulate sulfur granules
b i d e the celi whereas Ectothiorhodospiraceae fom them outside the ce11 (InhofZ 1984)
Ectoihiorhodospiia sp. ATCC 3 1751 is a sulfur purple photosynthetic bacterium
of undetewonedspecies of genus Ectothiorhodospim (Bognar et al., 1982). The disk-
like chromatophores of this species contain B800-850and B880 antennas. (Picorel et al.,
1984). The RC of this bacterium consists of the polypeptide subunits L, M, and H plus a
four heme c-type cytochrome. On a molar bais, the Ect. sp. RC contains four
bacteriochorophylls a, two bacteriopheophytins a, one h n atom and two menaquinone
molecules. The four-heme c cytochrome contains low (C552) and high potential (C555)
moieties (Lefebvre et al.. 1984; Lefebvre et al.. 1989)
In order to obtain an insight of the structure and the organization of photosynthesis
genes in this bacterium that rnight serve as a representetive of wlfùr purple
photosynthetic bacteria, we first identified Ect. sp. ATCC3 1751 as Ectothiorhodoospiru
shoposhnihvii based on 16s rRNA gene sequence analysis. From this species, the puf
operon genes w e n cloned and theù sequences were analyzed. The physicai gene
arrangement and the sequence of the pu/ operon and flanking regions are show to be
close to that of other known puple photosynthetic bacteria. Phylogenetic d y s i s of the
pf and of the 16s rRNA genes indicates that Ect. shapshnikovii
Clvonatium tepidun, aiso a mernber of the y subgroup
is closest to
Specks idenadion
Recently, Imhoff and Süling (19%) proposed a new classificationof species of
Ectothiorhodospira bssed on 16s rDNA sequence analysis. The availability of several
16s rDNA sequences affards a solution to the identification of Ectothiorhodhospira sp.
ATCC 3 1751. Its 16s rDNA was amplified by two rounds of PCR ushg two different
DNA polymereses to confum the sequence Wemnce of this bacterhm to other
Ectothiorhodospira species. The pair-wise sequence cornparison of the 16s rDNA with
known (Imhoff and Suling, 1996) 16s rDNA seqwnces is show in Table 1.
Tabk 1. 16s rDNA aquence Mentie khveen Ect sp ATCC31751 r h i n and other
specics of Ectothiurhodospim
Strain
Id~ntity(~)
Ect. shaposhnikovii ~ ~ M 2 4 3 ~
99.4
Eck v ~ ~ ~ t l o lDSM2111T
ata
98.9
Ecr. shaposhnikovi ~ ~ ~ 2 3 9 98.7
Eck murina DSM241T
96.5
Ect. hufoalhiliphia
96.4
Ect. h a ~ o a ~ k f i p h i ~1 93
i asT- ~ ~ ~ ~96.6
~
Ecr. marismortui D S M 80
~~
95.6
Ect. mnobiiisSDSM237
95.4
Caps
O
7
8
9
6
11
11
Il
Strain ATCC 31751 shares 99.39?!% sequence identity (without gaps) with the
Ectothiorhodhhospiru shqposhni&avii DM243
two
This value is cioser than that between
Eer. shriposhnibvii strallis ~sM239~
(originaily identified as Ecr, mobifis) and
DSM243 whose sequence alignment shows 99.24% identity and 8 gaps. We
conclude, accordiagly, that strains ATCC 31751 and DSM243
are very close if not
identicai.
Clonhg of thcpuf opemi genca
nie N-terminai region of the photoceaction center L polypeptide is highly
conservecl arnong purple photosynthetic bacteria (Bélanger et al*. 1988). We took
advmtage of this observation to prepace a probe to select clones of E. coli XL-1 Blue
transfomed with chimeric plasmids h a r b o ~ pf
g genes h m Ect* shaposhnikovii. This
probe was an 892-bp BamH1-HindIII fiagrnent of cbimeric plasmid pRR22 containhg
the beginning of pufi h m RF. mbmm (Bélanger et al., 1988). Southern blot of P d digested genomic DNA revealed a positive band at 2.6 kb. The DNA hgrnents at tbis
position were ncovered h m the agarose gel and ligated to pBluescript linearized with
Pst1 to produce a mini gene library. Colony hybridizstion led to the selection of clone
pZS26. B a d on sequencing, this 2.6 kb hgment encompassed ody p f l , M ad peit of
pu^, To obtain the entk sequence of the puf operon, chromosome w a h g was
proceeded in both âktions and two other clones, pZS21 and pZS25, were selected.
Figure 3 shows the arrangement of these t h e inserts, the restriction map and the
sequencing strategy. Taken togahet, the three inseris cover a range of appmximately 5.1
kb.
The inserts of chimenc plasrnids pZS21, pZS25 and pZS26 were subjected to
exonuclease LI1 digestion to generate nested deletion hgments that were subcloned and
sequenced. The sequence shown in Figure 4 was determined on both strands over their
entire length. From 5' to Y, the fmt identified open reading fiame (partial sequence
shown) was gene bchZ. The distd 366 bp of this ORF is 6 1% and 62.3% identical with
BchZ OF Rb. cup~wiutuswd of fi. rubrum (Béianger and Gingras, CienBank Access No.
AF018954). resjxctively. Between the TGA translation termination codon of the putative
bchZ gene and the end of the sequence of Figure 4. there are six open reading fiames in
the order ORF Q ', puf B.A.L.M.C. Each of these open reading -es
is preceded by a
Shine-Dalgarno sequence. They respectively encode a putative Q' pmtein. the
P and a
polypeptides of the B880 holochrome, the L and M polypeptides of the photoreaction
center and. k t , the four-heme c-type cytochrome. There are two possible ORFs (with SD
sequence preceded) detected downstream pujC, one is located from 478 1 -494 1 (ORFI),
the other is located reversed to pu/ operon. from 4378-4564 (ORF2). A Blast search using
the sequence downstream p u p did not hit any sigiificant homologous sequence.
The puf BA genes.
These two genes code for polypeptides
P and a,predicted to have 52 and 61
amino acid iesidues, respectively. The a and f3 polypeptides of Ect. shaposhnikovii have
the typical putative hydrophobic a helix (21 residues for a and 24 for P) flanked on each
side by poladcharged sequences. The a polypeptide, at its position 5, has a conserved Trp
residue and, beginning at position 9, a conserved seqwnce Asp Pro Arg, followed by the
putative transmembrane a heh. Towards the end of this a heüx, there is a conserved
-G
G
T
A
C
C
C
C
C
T
T
C
A
T
G
G
C
T
A
G
C
C
C
C
G
C
G
C
G
G
A
C
G
T
A
T
C
T
C
G T P F H A S P G R T Y L V Q E V C N S L F D A L F H I L P L G T E L D U V D A
A
C
G
T
P
S
C
C
R
G
L
T
H
C
G
R
E
C
G
L
P
C
C
T
G
W
D
D
E
C
A
A
C
K
C
A
G
V
G
G
L
E
A
Q
A
C
T
G
~
V
E
A
C
C
Y
C
P
T
V
G
L
G
V
G
R
A
I
T
G
A
T
S
A
A
K
G
A
R
T
L
G
R
D
GCCATCGAGGCAGCTGCAAGGCGAGAAGGCGAGGAAAGCATCAGCGTCGACA~CAACGGCCCACGCGGATGTCGCC1CCCCGGCAGGGCGGCCTGMCAGACGACAGCCAGGGCAGGG
A I E A A A R R E G E E R X S V D R V N G P R G C G G R A G R P E Q T T A R A G
TGAACGAACTCTGCCnï;GcGCAGMTGGATTGAAGCAGTMC~ACAX~UWATTCGATWCWGCAWGMCW~CNa~~~ACGTCWC~CmT~TWAGT
ORFQ'
H T R R R T D S R P S G R R R L C H Q Y
ACGCGAGGCGATGTCGTCCTGACCTCCCGMGTCAGGGAGGTTATGGGCGATCGTGAAAGCGCTGTGACATGCTTCTGACAGGTTCATGTCTTCAGTCATAUIAGTTGACCGGCTCTTTA
A
R
R
C
R
P
D
L
P
K
S
G
R
L
W
A
X
V
K
A
L
4
MCGGTATGACAGGf~CAC~CGTCAGTTTCGTTGTGAGGCAATTTTTTCCATGACGGTCGTGACCCCCGAGTCAACGCGGTATTTGCCMTMCGCTCII~GGAGCGTATTTAAAT
mm u
CGTTCACGAAACCAMGGTTCCCTCTCTGGGCTCACCG~GACGACCCCATGGACTTTCACGGTGTCTTCATGACCAGCATGATGGGCTTCCTGGCCGTTGCTGCCGTTGCTCIICGTTCT
V D E T U G S L S G L T E D E A ~ E V H G V F ~ T S ~ ~ G F L A V A A V A H V L
GCCCTGWITmGGCGTCCCTGGGGCGTGGCTTGGTAAGACCTT~WTTCTm~ATTmCAGTACCA~C~TCaXC~GT~TWGTA~TCCCTWCATA~GA
A
W
~
W
R
P
W
G
V
A
W
*
TTTCAACIITGTGGCGTGTTTGGCTGCTGTTCCATCCCCGCAGAGCGCTGGTTGCCCTGTTCACAGTTCCTGGCGTGCTTGCAcn;CTCATCCACTTCIITCCTG~GAGCACCGILACCCTT
~
A
~
W
R
V
W
L
L
F
D
P
R
R
A
L
V
A
L
F
T
V
P
G
V
L
A
L
L
X
H
F
I
L
L
S
T
E
R
F
--*---"--a
CAILCTGGATGCACTCTCCGTCGGCTTGCGAAGCGACTCTGCAGGCTCAGGAAGGCGGTTCAGCACTGTCCT~TGATAGcGTCn;TAGCCGCAGGTG~GGGCGGOPGTCCGCAGTCATG
N
W
H
H
S
A
S
A
C
E
A
T
L
Q
A
Q
----------
E
G
G
S
A
L
----------------
S
*
---------------c
CCGGCTCCGCCCGGCATCCAAGGCCCTTCGCTMCAAGCGTGTTGAGAGTATAGGGCTAGCGCCGCMTCTCCGGCCTATGCCTTACTGACCACCGC;AGGTTCTAGGAATGTCCATGCTG
PJL
n
s
n
~
ANTTTGAGAAAACATACCGC01:ACGCGGGGGGACOTTGATCW~GACCTA~TGAC~CT~C~CTTTCTACWCWT~~~TCCTMCEG~~CMRTG
S F E K R Y R V R G G T L I G G D L r b F w v G P F Y v G F F G v L T A r F A L
TTCGGTACACTCCTGATCGTCTGGGGTGCGGCTATGGGCCCARCCTGGAACATCTGGCAWITCAGCATCAATCCCCCCGACCTGAGTTACGGCCTTGGCTTTCCACCCCTCACCGAGGGT
L G T L L I V U G A A ~ G P T W N X W Q I S X N P P D L S Y G L G F A P L T E O
GGGCTTTGGCAGATGATCACOATCTGTGCCTTGGGCGCATTC~TTCCTGGGCGCTGCGCCACGCTGIlAATTCCCCGCAAACTGGGTATGGGTCTGC:ATCTTCCCATCGCCTTCTCCGTG
G L W Q W I T X C A L G A F V S W A L R Q A E I A R K L G M G L H L P I A F S V
GCGGTCTTTGCCTATTTCACGCTGGTTGTCATTCCPCCGGTGCTGCTGGOPGCCTGGCGTCATGGTTTCCCCTACGGCATCIITGAGTCACC~GGACTGGGTGTC~CACCCCTTATCAG
A V F A Y F T L V V I R P V L ~ G A W G H G F P Y G I ~ S H L D Y V ~ N T G
Y
Q
TTCCTGCACTTCCACTACMCCCGGCCCACATGCTC;CCCATCGCCTTCTTCTTCACGACGGCTCTC(K:CCTGTCATTGCACGGTGGTCTCATCCTGTCCGCAACCMCCCCMGAAGCCT
F L H t H Y N O A H H L A I A F f F T T A L A L S L H G G L X L S A T ~ P K ~ G
GMCCGGTCMGACGCCT~TACGMGATACCTTCTTCCGTGACGTCGTCGCCTATTCCATCGGTGCCCTCGGCATCCACCGCCTGGGTCTGTTCCTTGCCCTTACCGCCGCCTTCTGG
E P V K T P E Y E D T F F R D V V G Y S I G A L G Z H R L G L F L A L S A A F U
AGTGCCGTCT~A~GTCATCAGCGGTCCCTTCTGGACGCAGAGCTGGCCGGAAT~GGAACTGGTGCCTTGACCTTCCTATCTGGGCTTMTCTCMGGCGACCIICAATGGCCGMTA
S A V C X V I S G P F W T Q S W P E W W N W U L D L P I W A *
~
~
A
C
~
G
M
C
A
T
A
T
T
T
M
C
C
G
G
G
P
G
C
A
G
G
T
C
A
G
C
A
G
A
C
C
T
E
G
Q N X F N R V Q V S R P E A G V P L P N G D R E R I G K P L L F Y W A G K L G N
CGCCCAGATCGGGCCGATCTACCTGGGCTGC;ACCGGATTGGCATCCATCCTGTTTGGTTTGATGGCMTCCTGATCATCGGTTTCMC~CTTGGCCCAGGTCMCTGG~ACGT~TCCA
A Q I C P I Y L G W T G L A S I L F G L H A ~ L I I G ~ N F L A Q V ~ W D V X
Y
~
Q
GTTCTTCCGTCAGCTGTTCTGGCTGGGACTGGAGCCGCCTCAGGCTCAGTACGGCCTCGGTATTCCTCCGCTGGCCGAGGCGGCTTGGT~GATGGCAGGTTTCTTCCTGACCACTTC2520
F
F
R
Q
L
F
W
L
G
L
E
P
P
Q
A
Q
Y
G
L
G
I
P
P
L
A
E
A
G
U
Y
L
~
A
G
F
~
L
T
T S
2640
CATCCTGCTGTGGTGGGTGCGGATGTATCGTCGTGCGCGTCTMCATACCTGTCCTCTCCCTATCTGATTCTTCCGTCC
I L L w w V R n Y R R A R A w N H G T n v A w A F A A A f F L Y L s L G F F R P
GATCCTGATGGGTGACTGGTCCGAAGCM;TGCCCTTCGGTATCTTCCCGCATCTTGACTGGACCACGGCATTCTCTCTGAAGTACGGCMCCTGTACTACAACCCGTTCCACATGCTCTG 2'160
I L H G D W S E A V P F G ~ P P ~ L D W T T A F S L K Y G N L Y Y N P F H ~ L C
C A T T G C C T T C C T C f T A T G G T T C T G C T G T T C ~ T T T G C C A T G C A T M C T T G T C A T G A C G M C A T C C G A C C T A C C T T C2060
I A F L Y G S A V L ~ A ~ H G A T V L A V G R Y G G D G E L E Q I T D R G T A S
CGACACAGCAATGC~CTGGCGCTGGAC~~TTCMCGCCACCATCGMTCCATCCATCGCTGGGGCTGGTGGTTCGCGGTTCTCGTACCGATCACCGGCGGCATTGGCATTCT
3000
D R A M L F W R W T U G F N A T U E S I H R Y C W W ~ A V L V P I T G G I G S L
GCTCACTGGCACGGTACTCGACAATTGGTATCTGTGGGCGATCGATCACGGCGFGGCGCCGTCCTATCCGATCGTACATCCGMCGTCGMCAXCGGCCATGCTTCAAGGGATCTCTCA
3120
L T G T V V D N W Y L W A I D ~ G V A P S Y P I V W P N V E D P A ~ L Q G ~ S Q
ATGAAAACGATGACGCGTAAAACGACGACAGTGGCGGCCCTTGCCGGTGCCG(=ACTCCTGATGACCGGCTGCGAACTGCCGATCGGGATCGAAACCUCACAGACCGGTTATCGCGGTCTG 3240
wP:
~
K
T
W
T
R
K
T
T
T
V
A
A
L
A
G
A
A
L
L
H
T
G
C
E
L
P
X
G
I
E
T
E
Q
T
G
Y
R
G
~
G G T A T G G A G C A G G T M C C A M ; C G C C A T C T G G I I A G C C A T G T G A C T G A G T C C C C T C C C C G C C A T C T A C G A G C 3360
G
~
E
Q
V
T
K
R
H
L
E
A
M
K
R
S
G
H
E
I
P
E
P
E
R
P
A
A
A
A
G
P
R
A
G
D
I
Y
E
N
G T G M G G T G C T G G G T G A C C T C A A C A T C T C C G A G T T C M C C G T C T G A T C T C A C C T C C G T M C T A C T T A C C C C C G A C 3400
V
K
V
L
G
D
L
N
I
S
E
F
N
R
L
M
N
A
I
T
A
W
V
A
P
E
E
G
C
H
Y
C
W
T
P
G
N
F
A
D
GIlAAATGTCTACACGAAGATTGTCTCCCGGCGCATGCTGGAGATGACCTACACCATCllACCAGGAATGGACCGCCCAC~GAGACCGGTGTGACCTGCTACACCTGTCACATGGGC
3600
E
N
V
Y
T
K
I
V
S
R
R
~
L
E
~
T
Y
T
I
N
Q
E
W
T
A
~
V
G
E
T
G
V
T
C
Y
T
C
H
I
I
C
MTCCGGTGCCGGAAARTATCTGGTATGAGGACCCGGCiCTTCAAGCAGCCCGGTGC~GCCGCCAGCC~ATGGGTAGAACCTGGTCCGGTACCMCGTGGCTTCCGCCTCGCTGCCG
3720
N P V P E N X W Y E D P G L K Q A G A F A A S R I G R T W S G T N V A S A S L P
M T G A T G T C T T C A C G C C C T T C C T G C A G G G T G A T G A G C C C 3040
N D V F T P F L Q G D E P K N I R V T P R P R C R W V T X R T T W W T S E W V H
CGTCTCATGATC~CTTCTCGGTGGCCCTGCGTGCCMCTGCACCACCTGCCACMCACGAA~CTTCCCGGClATGGCAGACCAGCCCTCCCCAGCCTGTCACGGCATGGCACGGCATC
3960
G
L
M
I
H
F
S
V
A
L
G
A
N
C
T
T
C
H
#
T
N
N
F
P
E
W
Q
T
S
P
P
Q
R
V
T
A
W
H
G
'
I
GAGATGGTTCGGGCACTGAACMTCAGTACCTGAATCCGCTGCAGCCCGAGTATCCGGATTACCGTCTGGGTCCCACGGOTGACGCGCCCAAGACCAACTGCGCCACCTGCCACAAC~4000
E ~ V R A L N N Q ~ L N P L ~ P E Y P ~ Y R L G P T G D A P K T N C A T C W N G
C T ~ G T T G C C G C T G G A T C G T ~ C A G A T G C T G A A G G A T T A T C C ~ M C T G M C C ~ G A A C C A C C ~ ~ W T C A K C C G C W T A C ~ C ~ G C ~ ~ T G C T W C T 4200
GCCaTGACG
L
Q
L
P
L
D
R
A
Q
~
L
R
D
Y
P
E
L
N
R
V
N
H
R
G
A
Q
----ad-----&
P
A
L
P
P
------------
A
L
L
L
K
L
P
V
T
C A G A T G C G G A G G C T G T C C A A C G G T M T T C T T C G T A C C T C T C G T T G A C C C A T M C C G C C C T C C C C T G T C T T M G 4320
Q M R R L S K G N S S Y L S A E '
GCGTCATGCCTTTGTGTATGTTGCTAGCCGCTTGMGCGC;TTTTCCGCTCTGGTAGCCTTGACGCTTWTGGCCCATGGGCTTGAGAGGMCAGCAGTCGTCCGAAATCCAGCGTTCGGG4440
Figure 4. Sequence of the puf operon and partial sequence of gene 6ckZ fmm Ert shqmsk~ikovi3 The deduced
peptide sequence is also shown. The sequences involveci in the possible formation of RNA stem-bop structure are markd
with broken lines above the nucleotide sequence.
motif Ala-X-X-W-His-X-X-X-bu (His-4 and HistQ) that has ken suggested to be
involved in the bactcriochlomphyll-bindiag pocket (Brunisholz and Zuber, 1992). The fl
polypeptide contains two such conserveci His-4 and His+4 sûetches, one starhg ai
position 17 and the other at position 35. In this case, the His-4 position is filled by Ala
and the Hi& position by an asornatic msidue such as Trp or Tyr.Oniy the latter stretch,
close to the C-terminus and situated on the periplasmic side of the membrane, has been
proposed to be involved in binding Bchl (Bnuiisholz and Zuber, 1992).
The p u p d intergenh region
Structural genes pujB and pufA are separatecl by 90 nucleotides. As in Ru.
gelatinosus (Nagashima et al., 1994), this intergenic noncoding region is much longer
than the 12-17 nuclwtides so fm reportcd for other purple photosynthetic bacteria
( B 6 d et al., 1986; Kiley et al., 1987; Liebetanz et al., 1991 ; Wiessner et al., 1990;
Youvan et al., 1984). Assuming that, in Ect. shaposhnikovii the B880 complex displays
the same 1:l molar stoichiometry of the p/ a polypeptides as in other photosynthetic
purpie bacteria, the translation of-
and pufA might be thought to be couplad, in spite
of the long distance between these genes. There are cases when the secondary and
tertiary structure of the mRNA ttarisctipt intercistronic region is such as to impede the
dissociation of the translation complex at the termination codon (Matteson et al., 1991).
interestingly,pufA is tenninated by a series of three stop codons, TGATGATAG.
As in the other purple photosynthetic bacteria, between pfA and pufi there is a long
(150 bp) noncodiag region. The transcript of this region is pdicted to contain two stem
and loop sccondary structures with respective fke energies of formation of -17.1
kcdmol and -30.4 kcal/mol. This b d of structure was dso found in other purple
bacteria (Bélanger and Gingras, 1988; N a g a s h et al., 1994; Wiessner et al.. 1990;
Youvan et al-, 1984). Presumably it plays the same pmtective role agairist the action of
exonuclease, thus conferring a greater stabüity to the @,A
mRNA (&lasco et al.,
1985; Chen et al., 1988).
The pufi M genes.
Pufi and pujM are respectively preceâed by 1 1 and 10 nucleotides center-tocenter becween the pdcted Shine-Dalgarno sequence ( N C GG A/G00)and the AUG
start
codon. The putative L polypeptide shown in Figure 4 contains 274 amino acid
residues for an Mrof 30,55 1. An alignment (not shown) of the L polypeptide predicted
sequence with those h m seven ocher bacterial species including Chromatium (Ch.)
tepidum, Rb. capsufattu, Rb. sphaeroides, Rr. rubmm,
&S.
vviriis, Rosebacter (Ro)
denirripaw (fomerly Erythrobacrr OCH I l # ) and R u gelutinosus confirms that the Ntemiinal region of subunit L is the most wnserved ammg these species. The first 31
amino acid residues o f this region are highly conserved, except for the consensus AaU
and ~~s~~ in the seven species king replaceci by Seru and
in Ect. sharpohnibvii.
The predicted M polypeptide is made of 325 amiw acids for an Mr of 34,570. It
has an extra stretch of 17 amiao acid residues at its C-tenninus compand to the
M
polypeptides h m RP. rubrun. Rb. sphaeroides or Rb. capdatus (Bélanger et al., 1988;
Williams et al., 1984; Youvan et al., 1984). An extra sûetch of approximately 20 amino
acid residues was also found in the M polypeptides of Chr. tepidum, R a denit@caanr,
Re. gelutinosus and Rps. viridis (Fathir et al., 1997; Liebetanz et al., 1991; Michel et
al., 1986; N a g a s b a et al., 1994) (Figure 5). InterestingIy, this extra stretch is found
only in M polypeptides h m RCs that include a tightly bound cytochrome as a fourth
subunit.
The puK gene.
In Ect. shaposhnikovii, pufM is not followed by ho-independent transcription
tennination signaIs like those predicted for RF. mbmm (Bélanger et al., 1988), it is
followed instead by gene p u t with an overlap of 4 bp. This is in line with the idea that
puf C is part of the same polycistmnic transcriptional unit as pu$Q',B,A,L,M. The
strongest evidence for that is the sequence similarity with puf C of Ru. gelatino~l~~,
and
Rps. viridis which are cotranscribed with pufB,A,L,M
(Nagashima et al.,
1994;
Wiessner et al., 19%). The mgion 18 bp domistrram of Uie TGA translation tennination
codon of pu^ encodes a mRNA putative stem and loop structure with a frrc energy of
-
formation of 26.7 kcaVmol. This structure which carries a rua of 3 Ts may serve as a
transcription terminator, as proposeci for a similar structure in Rps. viridis (Wiessner et
a * , 1990).
Pt& encodes a polypeptide of 378 amho acid residues with a M,of 40,256 Da,
close to die apparent moïecular weight of 39,100 estimated by SDS-PAGE (Lefebvre et
a', 1984). This polypeptide contains four motifs CysXXCysHis (highlighted in Figure 6)
that are presumably involved in the ligand binding of the four hemes. The aligament of
.
Ect Shaposhnikovii
Ru, g e l a t i n o s u s
Rps. v i r i d i s
Chr tepidum
Ro denitrificans
Rs rubrum
Rb. capsula t u s
Rb spha e r o i d e s
.
..
.
Figure 5.
AMgnment of the C-teroiinal regioa of the RC M palypcptide of Ect!
shapsknikovii
with tbose of other four cytocbromc-eontaiiing and t h m non
cytoclromc-eontaiiing RCs.
highlighted.
The consensus rcsidues (> -or = 50% identity) are
1
Ect. shaposhnikovii
Ru. gela t i n o s u s
Rps. v i r i d i s
.
E c t s h a p o s h n i k o v ii
Ru. gela t i n o s u s
Rps viridis
.
Ect ,shaposhni k o v i i
Ru. gela t i n o s u s
Rps. v i r f dis
50
MKTMTRKTTT VAALAGAALL MTGCELPIGI ETEQTGYRGL GMEQVTKRHL
.MALAVRIST LTVAVTAAAL LAGCERP.PV DAVQRGYRGT GMQHIVNPRT
...MKQLIVN SVATVALASL VAGCFEPPPA TTTQTGFRGL SMGEVLHPAT
51
100
E.AMKRSGME IPEPERPAAA AGPRAGDIYE N V W D L N I SEFNRLMNAI
L.AEQIPTQQ APVATPVADN SGPRANQVFQ NVKVLGHLSV AEFTRQMAAI:
VKAKKERDAQ YPPALAAVKA EGPPVSQVYK NVKVLGNLTE AEEZRTMTAI
101
TAWVAPEE
NEWVAPTE
TEWVSPQE
150
PGNFA DENVYTKXVS RRMLEMTYTI NQEWAHVGE
.ENLA DDSKYQKVVS RRMLEMTQKV NTQWTHHVAA
DENNLA SEAKYPYVVA RRMLEMTRAI NTNWTQHVAQ
.
151
TGVT
TGVT
TGVT
.
201
250
ASASLPN DVFTPFZQGD EPKNIRVTPR PRCRWVTI,.
RTTWWTS
...GLTSLPY DPFTTFLK.. EETNVRVYGT TALPTGTS,. ...KADfKQA
KYTAYSALNY DPFTMFL AN DKRQVRWPQ TALPLVGVSR GKERRPLSDA
Ect s h a p o s h n i k o v i i
Ru. gela t i n o s u s
Rps. v i r i d i s
E c t shaposhnik o v i i
Ru. gela t i n o s u s
Rps. v i r i d i s
.
Ect shaposhnikovii
Ru.gela t i n o s u s
Rps. v i r i d i s
Ect. s h a p o s h n i k o v i i
Ru. g e l a t i n o s u s
Rps. v i r i d i s
.
Ect s h a p o s h n i kovi i
Ru. gela t i n o s u s
Rps. v i r i d i s
200
GNPVPENIWY EDP..GFKQA GAFAASRMGR TWSGTNV..,
GNPVPKEIWF TAV. PQNKR ADFIGNLDGQ NQAAKVV. ,
GTPLPPYVRY LEPTLPLNNR ETPTHVERVE TRSGYVVRLA
.
.
...
...
.
251
EWVHGLMIHF SVAL
EKTYGLMMHF
Y A T F M S I SDSLGTN
300
TNNFPEW Q.TSPPQRVT AWHGIEMVRA
TNGE'GSW D. NAAPQRAT AWYGIRMARD
GKKSTPQRAI AWWGIRMVRD
301
LNNQYLNPLQ PEYPDYRLGP TGDAPKT
LNNNF'MEGLT KTFPAHRLGP TGDVAKI
LNMNYLAPLN ASLPASRLGR QGEAPQ
350
GLQLPL DRAQMLKDYP
GAYKPL YGAQMAKDYP
FGASRLKDYP
351
392
ELNRVNHRGA QPALPPALLL KLPVTQMRRL SKGNSSYLSA E*
GLKPAPAAAA ASAVEAA.,.
.,PVDAAASA APVATVATAA K*
ELGPIKAAAK *
Figure 6. Alignment o f the sequencea o f the four-lcw cytochrome of Eci s k ~ h a i k o v i i
with thore of two other photosyntbetk bideda.
The amino acid midues thought to k
involved in the Ligandhg of the hemes are highlightcd.
protein sequence of the homologous four-heme cytoctiromes of Rps. viridis (41%
identity) and Ru. gelatinow (44% identity) leaves no doubt about the close relationship
of these proteins.
In Rps. viridh, the four-berne cytochrome is located on the penplasmic side of the
ce11 membrane (Deisenhoff et al., 1985). That position is assured by its synthesis as a
precursor contalliing a signal peptide that is cleaved by signal peptiôase II (Weyer et al.,
1987a) leaving cysteine as the first amino acid in the mature cytochrome. This cysteine
covalently binds to fatty acids that an thought to anchor the cytochrome in the membrane
(Weyer et al., 1987b). The sequence of
eu''^ Met Thr Gly Cys in Ect. shaposhnihvii is
placed at a position corresponding to the peptidase II recognition sequence
eu'" Val
Ala Gly Cys of Rps. viridis. This suggests, by analogy, that the Ect. shqvoshnihvii
cytochrome is also synthesized as a precursor and that the proccssing of a 23 residue
putative signal peptide leaves an N-terminaicysteine in the mature cytochrome.
ORF Q9
This ORF is similarly psitioned as gene pt&
in Rb. capsuluttu and Rb.
sphueroides (Bauer and Marrs,1988; Kiley et al, 1987) and for that muon is designatsd
here as ORFQ'. Except for positional similarity, ORFQ' shares no cornmon traits with
pz@ that encodes a membrane proteh of 75
amim acid midues. InteteStitlgiy, at a
similar position and wïth a similar size as ORFQ',OWl in Ru gelutinosus (Nagashima
et al.. 1994) and ORFQ in Rs. mbmm (Lee and Colhi, 1993) were detected by
sequence analysis. The putative products of these t h ORFs would contain high net
positive charge and la& hyhphobic domains. They would dso be e ~ c h e din serine
and proline residues. But their sequences do not show any significant homology. A Blast
search using the ORFQ' amino acid sequence hits only one gene product in Oryza stivu
that is similar to a stress inducible protein in soybean, but the significant homology
region has only 9 residues.
To shed some light on the function of ORFQ', we inactivated it by inserting an
aminuglycoside 3'-phosphotransferase Bene (Kmr) cassette at its Sphi site (Figure 7).
This insertion mutant had a similar gmwth rate and the sarne amount of the B800-850
antenna complex as the wild type. However, based on the absorption spectra in Figure 8
and the absorbance spectra of pure B800-850and B880 complexes (Picore1 et al. 1984),
the level of 8880 in the mutant was calculated to be only 20% of the wild type. The RC
level was estimated by measuring the absorption increase at 1250 nm instead of the
decrease at 890 nm in chromatophores. due to the photo oxidation of P to P'. The reason
for choosing
(that is smaller than Mew)
is to avoid the intetference by the high
absorption background fiom 8880 complex and a possible bleaching of 8880 complex
absorption caused by intense illumination. The results show in Figw 8 (lower)
indicated that the level of RC photochetnical activity in the mutant is around 19% of the
wild type, a similar situation as the 8880 complex, indicating the effect on expression of
whole puf operon by the insertion of the Kmr cassette. Because there is no transcriptional
termination structure down Stream of the kanamycin resistance gene, the polar effeft on
pufoperon expression by the insertion of kmtcassette is unlikely, although possible. The
above results indicate that unlike p j Q in Rb. capsuIatw (Bauer and M
al.,
1994; Fidai et al..
1995),
m , 198 8; Fidai et
ORFQ' is not essential for the biosynthesis of
bectenochlorophyli but may be involved in the expression of the puf operon. To test
Figure 7. Gene arrangement in phmid pZS-pufi0 and the plasmids uscd for m a h g ORFQ' Uscrtion mutation on
the chromosome by gene replacement [NQ-0RFQ9(Km)l and for complemenbtbn (pRK404-0RFQ'). The dotted line
means the removd of this region.
750
830
790
870
910
960
NANOMETERS
1 off
off
I
O
I
I
2.5
5.0
I
7.5
I
10 (min)
TIME
F i i r e 8. Absorption fi-
(upper) of the ebromitophotes of the wild
type rad ORFQ' mtitint imins and the rbrorptkii changer rt 1250 am
(iower) upon photw.id.tion of the r p h l pair in the cbrwrtopbam.
whether the effoct is cis or hum. the wild type ORFQ' on vector pRK404 was useci to
complement the Kinr insertion mutant of ORFQ'. Chmatophores h m teüacycline
cesistant transfo~11811tsshowd no dekctable testoration of the B880 cornplex. thetefore
suggesting that ORFQ' might not be a fiuns functioning element. The decreased puf
eqmssion could mult fiom the distuhance of pfpromoter ngion. ûther possibüities
are that ORFQ' i s transcriôed fiam a superopecon promoter that is not included in the
fiagrnent for wmplementation, or that the disruption had a polar effect on expression of
otherpufgenes.
Discussion
The putple photosynthetic bacteria can be grouped eccording to whether their RC
is composeci of the L, M and H submits, only, or wtiether it also contains a tetra-heme
cytochmme as a fouith subunit.
In those species of the second gmup for which the
complete sequence of the puf opemn is available, pufl patially overlaps with puFI,
suggesting that both genes belong to the same operon. This was, in hct, shown for Rps.
viridis and
Ru gelatinosus where put is a-transcribed as a polycistmnic unit with pu/
B,A, L,M (Nagashima et al., 1994; Wiessner et al., 1990). Another characteristic fature
of the RCs with a four-heme cytochrome is thrit theù M subunit is approximately 20
residues longer at its C-temiinal end than those without this cytochrome (Figure 5). There
is a 41% identity between the corresponding extensions of Ect. shaposhnibvii, Rps.
viridis and Ru. gelutinosus. Presumably, this extension is involveci
in the binding of
subunit M to the cytochrome as predicted by X-ray crystal structure of the Rps. viridis
RC (Deisenhofer et al., 1985). This would be in line with the truncation of the M subunit
as a consepuence of the evolutionary loss of the tetra-heme cytochrome.
Previous studies of the RC of Ecr. shaposhnibvii ATCC 3 1751 revealed that Qx
band of
is located at 540 nm (Lefebvre et al., 1984) rather than 546 am as found in
other purple bacteria RCs.Accordingly, L1O4 position is occupied by Gln in this species
rather than the consensus Glu in 0 t h puiple bacteria It has k e n demonstrated that the
replacement of GIU''~in wild type Rb. capdatus by ~ l n yielded
~ ' a~ mutant RC with
a Q, absorption band of @A at 540 MI rather than at 546 nm @ y l h et aï., 1988).
Glutamine was founci at a homologous psition in Chioroflexus awantiactls
(Ovchilinikov et al., 1988), a green gliding bacterium, with the same spectroscopie
outcome (Vasmel et al.. 1983). Crystailographic and spectroscopie studies of the Rps.
viridis and Rb. sphaeroides RCs indicate that
lu^'^ interacts with the cgketo group in
ring V of O*, thereby causing a red shift of its Qx absorption band (Bylina et al.. 1988;
Palaniappan et al.. 1993). There is no observable change of electron transfer kinetics in
mutant RC, suggesting that Glu at LI04 is Iikely protonated (Bylina et al., 1988),
because ionized Glu is expected to contribute electron expelling force to OAand rnay
affect its capacity of receiving electron.
Phylogenetic analysis.
The deduced amino acid sequences of subunits L and M
from 9 bacterial RCs were
compared pair-wise as recorded in Table 2 (next page). According to these data, the Ect.
shuposhnikovii RC (Land M amino acid sequences), while 60%42% similar to those of
other purple bacteria, is closea to that of Ru. gcfatinosus.These two sequences are closer
thon those of any other two species, except for the M polypeptide of Rb. capsufatusand
Rb. sphaeroidrs (77%). But the phylogenetic trees of subunit L and of the 16s rRNA,
built by the Neighbor Joining method (Figure 9), indicate that the species that shares the
nearest c o m o n ancestor with Ect. shaposhnikovii is Chr. tepidum, another member of
the y subgroup, rather than Ru. gelrilinosus. This apparent inconsistency may reflect a
different evolutionary Pace in individual species. A faster evolutionary pace in Chr.
tepidum relative to Ru. gelutinosus will increase the distance of the former but not of the
latter to Ect. shaposhnikovii, irrespective of their mbgroups in line with this, Chr.
tepidum is more distant h m other species than Ru. gelutinosus (Table 2).
Tabk 2. Sequcnce identity of polypeptide
L and M ktwcca dilltmmt rpcckr of
Above diagonal : L polypeptides. k l o w diagonai :M poiypeptides.
Ecr. sha Chr*tep. Ru. gel. fiRP.
rub. Ro. den Rb. cap* Rb. sph Rp. vir. CR our.
Ect. shaposhnikmii
79.6
81.5
70.2
742
65.5
67.3
65.5
43.2
80.3
69.7
74.1
63.9
67.2
67.2
41.5
71.8
77.8
66.1
68.9
65.8
43.2
69.0
69.7
69.7
59.3
43,8
66.8
70.7
64.4
43.8
78.4
58.5
43.2
58.5
40.7
Chr. tepiàum
69.5
Ru gelutinosus
73.4
71.1
As. rubrum
71.3
71.0
71.0
Ro. denitr~ycans
61.4
58.3
61.4
60.9
Rb. capsulatus
60.3
58.8
64.0
59.3
60.1
Rb. sphaeroides
61.2
62.7
66.3
64.3
62.7
77.0
Rps. viridis
60.6
59.7
62.0
59.6
51.1
50.2
50.2
C@. aaurantiacus
43.7
43.8
47.1
46.5
44.7
39.1
42.6
43.5
38.6
The phylogenetic tree of the M polypeptide shows several very close branches issuhg
h m the same node. These include species h m al1 three a,P, and y subgroups, namely
RF. rubrum, Rps. viridis, Ru gelatinosus, Ect. shqmshnihnivii and Chr. tepidum. This is
in line with a higher fkquency of second base replacements in the codons of the M
polypeptide, as shown for RF. rubrum (Bélanger et al., 1988). This can be rationalized by
assurming lesser b c t i o d constraints on the M polypeptide than on the L polypeptide or
the 16s rRNA.
Figure 9.
Phylogenetic trees of some photosynthetic bacteria fmm the
sequence of eitber their
RC L (A), M
(B) polypeptides or 165 rRNA
geae (C). PILEUP was used to create multi-sequence alignment by pair-wise
cornparison. DISTANCES was used to create the distance matrix and
GROWTREE to build the phylogenetic trees with the 'neighbor-joining'
rnethod.
Rb.
L polypeptide
M polypeptide
Chapter III
Modified BBsite in the photoreaction center of
Ectothiorhodospira shuposhnikovii
The photod-iven electron transfer in natural RCs flows only through one (A
pathway) of the two approximately C2 symmetric cofactor lines. This unidireçtionality
can be explained by structural differences between the two lines. Two monomeric
bacteriochlorophyll pigments (BA and Be) are spatially closest to the special pair, their
properties therefore play an important role in the primary photochernical process (Bylina
et al., 1990; 1-Meret al., 1995; Arlt el al., 1996).
The histidine residue that ligands the Mg* of Be in Rp. viridis and Rb.
sphaeroides is conserved in Ect. shaposhnikovii as histidine MI80 (Chapter 11). The
mutagenesis was designed to replace M l8OHis with Ml8OLeu in order to substitute Bchl
at Be site with Bph as has been demonstrated in number of similar snidies (Bylina et ai.,
1990; Arlt et al., 1996). Previous studies demonstrated that the replacement of Bph for
Bchl did not cause o substantial structural change in RC, but reduced the redox potential
of the corresponding site (Fajer et ai., 1975; Bylina et al., 1990; Heller et ai.. 1995; Arlt
et al.. 1996). EIectron flow between carriers can be afliected by many factors, such as the
redox potential diffetence, distance and spatially mutual orientation between two carriers.
In RCs. the prosthetic groups in two lines and the L and M subunits are m g e d in an
approximate C2 symrnetry, therefore the redox potential state could be the most
hdamental factor in determining the electron flow direction. A Bph at the Be site is
expected to have lower redox potential, and such a change might disturb the electron
transfer and thereby affect the RC Iùnction. This chapter reports the effort to construct a
~ 1 8 0 mutant
" ~ by a new strategy and preliminary chanrterization of the mutant RC in
Ect. shaposhnikmii.
Construction o f an ~ 1 8 0RC
" ~mutant
The in tram complementation of a nul1 background strain is a common strategy
For mutagenesis study.
In the present case, this wouid involve the selection of a puf-less
strain. Since Eci. rlupxhnikovii seems to be an obligate phototroph (Bogiiar ri al., 1982)
this amounts to having to select a lethal mutation. To overcorne this obstacle, the desired
mutation was introduced into the chromosome and selected with the help of a second
rnarker. The strategy is presented in Figure 10. First, nucleotides CAT coding for M 180
histidine were replaced by nucleotides CTT coding for leucine on plasmid pZS-pu10
(see Chapter II for information on this plasmid). The mutation was confmed by DNA
sequencing. Second, a kanamycin resistmce gene cassette (Km3 was introduced at the
Sphi site in ORFQ'. Insertion of Km'at this site, while reducing the expression of the puf
operon, has only margllial effect on photosynthetic growth (see Chapter 11). Md, the
whole insert was transferred to a suicide plasmid pJQ2OO-SK+ and the DNA was
introduced into Ect. shaposhnihvii cells by electroporation. The suicide plasmid amies a
conditional lethal gene sacB. The expression of this gene induced by sucrose has been
shown to be lethal in a wide range of Gram-negative bacteria, and has been successfbily
u d as a positive marker for selecting gene replacement mutants (Quardt and Hynes,
1993). The transformed cells were allowed to grow photosynthetically in culture medium
containing 5% sucmse and 25 )ig/ml kanamycin to select the mutant experienced gene
replacement. The survivors are supposed to k a mixture of single mutant (Kmronly) and
double mutant (Km'and M IBOH+L) (see Figure 10). To select the double mutants,
Chromosome DNA
I
CTT
Figure 10. The pîasmid and atrategy usai for the intcgntion of ~ 1 8 0 mutation
'~
in the
cbromowme of Ect s ~ a p k n i & o v i iThe
~ cross-over recornbination occurriog at the sites
indicated by two solid cross signs le& to a double mutant hcluding the insertion of Km'and
the replacement of CAT by CTI: Cross-over at the sites indicated by one solid cross sign at
the extmne left and one dotted cross sign will produce single mutant including the insertion
of Kmr only. Both types of mutants can grow on the selectioa medium containhg Km and
sucrose.
chmosomal DNA was exûacted from individual colonies and used as the template for
polymemse chah reaction (PCR) to amplie a 400 bp fragment containhg the Ml80
codon. Apart h m normal sized colonies, the selection dishes also containecl tiny
colonies. DNA sequencing data of PCR p d u c t s h m 20 large colonies and 8 tiny
colonies showed that only the tiny colonies were the double mutants carrying a CTT
(Leucine)substitution for residue Ml80 codon. This resdt indicated a direct relation
between the lower growth cornpetency and the ~ 1 8 0 "mutation. The numbers of the
double to the single integrants was in a ratio of about 1:s which is consistent with the
respective lengths dowastream the Ml 80 codon in the insert fragment and h m the Ml 80
codon to the Kmr insertion site, indicating a random recombination event.
Chamcterizatioa of tbe ( ~ m 3 ~ 1 mutant.
8 0 ~ ~
The diftierent size of the colonies incikates that the mutation of ~ 1 8 affecl~
0 ~
photosynthetic growth. Figure 1 I shows the photosynbiaic p w t h rates of mutants
(K~~
8oHL
M and
I Kmrcompand to the wild type. While the ( K I I ~ ' ) M Imutant
~ o ~ ~ grew
much more slowly than the other two sttallis, the growth rate was not much affected by
the sole insertion of the Kmr gene. The g e n d o n times were 4, 6 and 20 hom,
0~
respectively for parent strain (wild type), Km' siagle mutant and ( ~ c d ) ~ l 8double
mutant The slower growth was shared by ail ~ 1 8 mu0tan~
ts individdy slected,
indicating that the 2.3 h e s slower gn,wtb rate in ( m f ) ~ i 8 c0o~m~p a d to single
mutant (Kd)is due to the h¶180HL
mutation.
O
8
16
24
32
40
U
66
HOURS
Figurell. Photosynthetic gmwth of the wild type stnin and the two
mutant strains o f Ec&shaposhnikovil
The absorption spectrum of the chromatophore of the ( K ~ ~
8oHL
Mmutant
I was recorded
to compare with those of the Kmr mutant and the wild type as show in Figure 12a The
( ~ r n 9 ~ 1 8 0mutant
" ~ shows a similar spectrum as the Kmr mutant, but they are both
diffe~ntthan the wifd type, Le. with iess B880 wmplex absorption as has beai described
in Chapter II for the Km'mutant. In order to know whether the ~ 1 8 0 "mutation has any
s fict on RC stability in the cellular environment, the photochemical activity of RC in the
chromatophore of the ( K m ? ~ 1 8 0 ~
mutant
'
was compared with that in the Kmr mutant,
as indicated by the absorption increase at AIm in cesponse to illumination. The cesdts
shown in Figure 12b indicate that they have similar levels of RC photochemical activity,
implying that t h e ~ 1 8 mutation
0 ~ ~ did not duce the stability of the RC in the cell.
Isolation of the photoreaction center
The isolation rnethod (Lefebvre et ai. 1984), whiie very successfiil with the wild
type strain, encountereâ problems wben applieû to the ( ~ r n ? ~ 1 8 0 "mutant. nie main
difficulty was obtainiag eaough preparation to fie the RC of B880 antenna contaminant.
However, whenever enough preparation was obtained, we found the RC pure enough for
pigment analysis. Although les stable than the wild type
RC prepanitions, the
*
preparations of Ml goHLRC showed no appreciable degradation (no apparent change in
absorption spectca) &er ovemight storage at 4 OC in its elution buffer.
Spectmscopic analysis of RCs
Figure 13 presents tbe absorption spectm of ~ 1 8 0 " RC and the wild type RC.
The RC sampfe h m the colunm was eluted by 50 rnM [email protected]), contauiiag O. 1%
O
2
4
6
8
Minutes
Figure 12. Absorption s p c e h i (a) and absorption cbanges i t 1250 nm @)
of the cbromatophores in different stmins of Ect shapusknikovil. The
spectra are normalized to the protein concentration.
450
66û
7M)
8Sû
8Sû
Nanometers
Figure 13. Aborptba spectm of the wild type .id the ~ 1 8 0 "mutait RCs
of E a s&upd~ikovU.Thewild type RC sample is in SOmM T N C l (pH &O),
O. 1% Triton X-1ûû.The mutant RC sample is in 50 mM TrisCl (pH8.0),4.4%
Triton X-100, 200 niM NaCl (sec tes for explamtion). The specaa are
11omlindto qua1 abso~tionat 880 nm.
Triton X-100 and 200 m M NaCI. Because the eluted mutant ~ 1 8 0 ' ' RC was quite
diluteû and in a small arnount, to avoid the l o s of the sample, it was subjected to
concentration but not to didysis as for the wild type RC sampks (agakt 50 mM Tris-Cl
(pH8.0)-0.1% Triton X-100). It should be pointed out that al1 of the featwes shown here
in the sample aRer concentration also appeared in the diluted sample. The effect on the
spectra of RCs due to the different bufler solutions involved was that the scattering was
slightly higher in the Ml 80"' RC sample. This was due to a higher salt concentration and
probably also a higher Tnton X-100(the micelle of this detergent was likely retained in
the RC samples through the concentration pmcess). The spectra have been adjusted
coaside~gthe scattering effect.
Compared to the wild type, the mutant RC has a greatly decreased absorption at
800 nm and a slight decrease at the 600 m band accompanied by an incraseci absorption
in the 530-540 nm region. The Qyband of Bph at 750 nm in the wild type is s h i M to
762 m in the mutant and its peak height is increased by 30%. These changes Udicate the
replacement of Bchl (Be) by Bph in the ~ 1 8 RC.
0 Another
~
interesting fiture in the
absorption spectnim of the mutant RC is the appearance of a new component with a peak
at 790 m.
The light-minus*
differcnçc spectra in Figure 14 show a smailcr Iight-iiduced
A&io and &aO and a iarger
f a the ~ 1 8 mutant
p RC,king consistent with a
reduced absorption at 800 nm (one Bchl) and an hcreased absorption at 760 nm (thtee
.
Bphs). Photooridation of the special pair (HP'
will)
muse a blue shift of 800 nm band
to 786 nm with a slight increase of absorption (electrochromic effect) (Lefebvre et al.,
a
1984). which was reflected in the light-miaus-diirl ciifference spectta as a large positive
720
760
1
8
8
800
Mû
880
920
960
NANOMETERS
Fipi* 14. Ligtsiausdark dinercna r p e d m of the wihi type and the
~ 1 8 m0~ t ~
u iRCs
t~ of Eck s h ~ h n i I t o v üThe conditions of the samples are
the same as in Figure 13. The spectra are normabd to an qua1Mam.
band at 786 run as s h o w in Figure 14 for the wild type RC. For mutant RC, a small peak
would be expected because it has only one monomeric bacteriochlorophyll. However, the
peak in the wild type was replacd by a plateau in the mutant RC. This region (788-796
nm) corresponds to the new absorption component peak at 790 nm.This indicates that the
790 nm band experienced bleaching upon the photooxidation of the special pair and
baianced the electrochromic effect on BA by P'.
Pigment aiaiysis
The pigments were quantitatively extracted h m the lyophilized RC samples with
acetone-rnethanol(7/2 v/v) and assayed spectrophotometricatlyaccording to van der Rest
and Gingras (1974). The absorption spectra of the extracts from the wild type and the
~ 1 8 mutant
0 ~ RCs
~ were presented in Figure 15. Based on these spectra and applying
the calculation equations developed by van der Rest and Gingras (1974), the ratio of
bacteriochlorophyll to bacteriopheophytin was found to be 1:l in the mutant RC and
1.Tl in the wild type. These data are consistent with the effective replacement of Bchl
by Bphe at the Be site.
type
Figure 15. Absorption spectm of the pigment extracts
fmm the wild typa and the ~ 1 8 0 RCs.
'~
Discussion
A much slower growth rate in the double mutant (
~
~
1 than
8 in0the single
~
mutant (Kd) shows that mutation of ~ 1 8 0 is
" the
~ cause of that phenotype. The effccts
of this mutation could be either on the stability andor the primary photochemical
reaction of the RC. Because the same number of single mutant and double mutant cells
contain sllnilar amount of RC, which is Uidicated by similar photochemical activity
(Figure 12b), it is therefore unlikely that the ~ 1 8 caused
0 ~ the iastability of the RC in
the living cells. The replacement of Bchl by Bph does not cause an accountable structurai
change in the RCs, but causes a noticeable decrease of redox potential at the site (Fajer et
al., 1975; Bylina and Youvan, 1988; Bylina et al., 1990; Helier et al., 1995; Arlt et al.,
1996). Therefore, the ~ 1 8 0 mutation
~'
probably causes a change in the interaction
between the Be site and the special pair. Such a change rnight affect the primary
photochemical reaction presumably by reducing the efficiency of the electron üansfer in
the A pathway, a d o r diverthg elecûon flow to the B pathway. Apparently, the growth
of Cl' aurantiuctls, whose RC has Bph at Bs site, is not affectcd in this manner,
otherwise it would not have been selecteâ in the course of evolution. An explanation may
be found in the amino acids forming the Be binding pocket in these two strains.
The absorption spectmm of ~ 1 8 RC
0 takes
~ ~ a quite new look owing to the
replacement of the Bchl by Bph at Be site. The purity of the RC sample is indicated by a
complete bleaching of 880 nm band upon photo-oxidetion of P and also by the 1: 1 ratio
of Bph to Bchl shown by the pigment assay. It shouid k pointed out that the absorption
spectMn and the pigment assay was d e d out on a single preparation The purification
of mutant RC was difficuit, largely due to a much lower level of the mutant RC (2Ph)
cornpend with the wild type. In sewd other preparations, the RC samples were
contaminateci by the B880 antenna. But d l the new fatures amund 750-800 nm region
were ceproducible in these prepatatioas.
As far as we how, this is the first t h e that a Ml80 HL type mutant
isolated and its absorption spectnim obtained. The ~ 1 8 0 " mutation
RC was
has been
constructed in the plasmid-borne pufoperon to complement the puf nuli strain of Rb.
capsufatus (Bylina et al., 1990), but the RC could not be purifid and, thenfore, the
absorption spectra of the two preparatioas cannot be compared. The new and interesthg
featuces in the absorption spectnim of this ~ 1 8 0 RC
" ~find analogy in C f . aurantiucus
(Figure 16), a naniral occurring species whose RC has Bph at the Be site as a result of the
presence of leucine instead of histidine at the conespondhg position (Blankenship et al.,
1983). Similar to the red-shifted Qy band of Bph in our ~ 1 8 0 mutant
"
RC, in Cl.
uuran~iamRC the Qy band of Bph centers at 755 nrn, a 5 nrn red shift compareci to the
RCs with Bchl at Be site. This shift was consïderedto be the result of the interaction of
wo neigbbouring Bph molecules (Vasmel, 1986). The new absorption component at 790
nm in ~ 1 8 RC0 of~Ect.shaposhnikovii comsponds to ow in the Cfi; wantiactls RC
that has a peak at the same position (a shouider in Figure 15). It has been experimeatally
proved that this component is the bigh energy band of the special pair (Vasmel, 1986).
Combinbg its bleaching pmperty upon the photo1oxidatioa of P aud the similar position
with the high energy band in Cfi. maantiacus RC, the 790 nm component in the Eer.
shaposhnihvii ~ 1 8 RC
0 is~ thought
~
to be a blue-shiM high eneqgy band from 800
nm in the wild type RC (Mar et al., 1993).
450 550 650
750
850 950
Nanometers
Figure 16. Cornparison of the m m temperature absorption spectra of
Ect. shaposhnikovii ~ 1 8 0 "RC (lower) and the Cf. aurantiacus RC
(upper) that was redrawn from Vasme1 (1 986).
In cases such as encountered hem, where the deletion of a gene is lethal to the
cell, it is impossible to obtain a nul1 background tiom which to express in
PULN
complementation. We circumvented this difficulty by dicectly replacing the native
chromomd puf by a puf carrying a b t e d mutation. As long as the intmduced
mutation is not lethai this sûategy is of general applicability. This strategy may have
advantage in studying gene regdation, because it will contain a cis-acting effect that is
not always guaranteed in in-tram complementation strategy. The main possible pitfidl
with this strategy is the influence from the second selection marker. Usually, that
influence depends on the insertion site and can probably be rninimized by testing
different sites.
Chapter IV
Mutagenic study on the higbly cooserved N-terminal region of
the L polypeptide of photoreaction center in RhodospiriUum
rubrun8
The L and M subunits of RCs are integrai membrane pmteins, each bas five
membrane spmnhg helices, 4th theù N-termini in the cytoplasmic side end their C-
tumini in the periplasmic side (kisenhofer et d., 1985; Allen et al., 1987). Theu
structurai genes @$L
and puM are organized in the pufoperon. By sequence analysis,
Bélanger et al. (1988) found that the most conserved @on was located in the Noterminal
ngioa of the L subunits, and not in the chromophore-binding pockeis. This discovery was
further supplemented and confirmed by incrrasing sequence data (Liebetanz et al., 1991;
Nagashima et al., 1994; Fathir et d,1997; Chapter Ii o f this thesis). In contrast to the
situation in the L polypeptide, the residues in the N-terminal region of the M subunits are
much l e s conserved, aithough their structural genes are thought to have evolved h m a
cornmon mcestor and although the IWO subunits have a substantial structural similacity.
Bdlanger et ai. (1988) also pointad out that some featms of the N-terminal
segment of the L subunit are rrrniniscait of the matrix targeting signal of cytochme cl
and other mitochondrial membrane proteias, suggesting a hction in the membrane
integration of the L peptide related to this segment. However, sequences of signal peptides
are featut.ed by a special structure block, that is a hydrophobic con (7-13 &dues) f l d e d
by several relatively hydrophilic midues that usually include one or more basic residws
near the N-termUIus, but not by the cornpletc conservation of the residues in the sequence.
Thecefore, the extreme conservation of the residues in the N-terminal region of the L
polypeptide seems beyond the muirement only for membrane integration. To investigete
the fùnction of this region, a systematic mutagenesis was conducted on the wnserved
residues of the N-terminal region of the L subunit of RI. mbmm, and the effects of the
mutations were tested by genetic complanentation of a puf nuil background Rr. mbmm
strain. The nsults show that most of the coasmed residues in this region have a h c t i o n
related to the membrane integration of the L subunit as proposeù by Bélanger et al. (1988).
But some conserved residues are tolerant to substitution.
Conatmdon of r puf deletion mutant
The m e g y employai in collst~ctinga puf deletion mutant in h. mbnun is
pcesented in Figure 17. The starting plasmid pZS-&O
contained a 3.0 kb Pd-Sa11
fragment on vector pUCl9, which contained the puf genes of Rr. rubrum. From this
plasmid, a 1.7 kb A@ m e n t was removed, which includes the 3'- part of puj8, ail of
pufA. pu/L and the 5'- part of pujiid. The gap was filled with a kanarnycin mistance gene
cassette (Km? denved h m pUC4-kapa. Restriction enzyme digestion c o n h e d the same
transcriptionai direction of the puf operon and Kmr. The whole insert was then moved into
the suicide plasmid pJQ2ûû-SKf. This chimenc plasmid (pJQ-pufi:KmA(Apai)) was used
to transfomi E coli S17-1. Plasmid DNA was transfemd into RF. r u b m S1 by conjugal
rnating with E. coli S17- 1 (pJQ-puj:KmA(Apai)) as a donor. After conjugation, the cells
were allowed to grow on CB medium supplemented with 50 p g h l of Km and 5% sucrose
to select the cells experiencing gene replacement (Quandt and Hynes, 1993). In addition,
the double crossover event was further distinguished h m the single crossover event by
checking the ceU's response to gentamicin (the resistance gene on the vector). Finslly, the
selectd clones are Kmrsucr and Gd,and incapable of photosynthetic growth (PS'). The
replacement of pufgenes by Km' in the mutant chromosome was M e r w h d by
Southem blot anaiysis. The mutant was designated as Apu$
Chiractedzationof the bpri mutant
--
Chcornosorne DNA
P
a
++
-II
A@
L
M
A N
lt-
Figure 17. &hematic v i e ~of the piaamidr and the sttategy useû for (a)
coastrpctiag and (b) romplementhgipuf dcktion mutant in 1Cn rubrum.
This mutant is incapable of photosynthetic gmwth. The absorption spectra of
chromatophons p r e p d h m the ceiis microacrobically p w n in the dark are presented
in Figure 18. The B88O antenna and the photoreaction caiter are expected to be absent
h m tbis mutant due to the deletion of pulB,A,LM. The shoulder et 750 nm is iikely due
the accumulation of bacteriopheophytin in the membrane, pmbably due to the lack of the
bacteriochlompbyii buiding protein, either as the nearest precrnsor or as the pcimary
product of degcaâation of Bchl molecules.
Complementation of Muf mutant
To test whether the Apuf mutant derived h m the wild type RF. rublum couiâ be in
trm complemented, the Apuf
mutant was t r a n s f o d with plasmid pK-pu135 harboring
the wild-type pfoperon on vector pRK404 (Figure 17b). The trarisformants were selected
on the tetracycline-contairing medium. The absorption specûum of the chromatophores
pnpareâ h m the complemented @uf mutant showed restoration of their photosynthetic
apparatus (Figure 18). To make sure that the complementation is in tram, insteaâ of the
result of beck ncombination between the wild type Mgenes on plasmid and the mutated
pufgenes on the chromosome, thc plasmid h m the transformants was checked by size and
eayme digestion pattern. The d t s codhmed that the restoration of photosynthetic
growth in & f t o PS+ was by in tram complementation
Mubtions ia N-terminal segment
In RF. rubrum, the first 30 arnino acid residues in the N-temiinus of the L subunit
possess the same sequace as the consensus sequence of 8 purple photosynthetic bacterial
420
520
620
720
820
920
Nanometen
Figure 18. Abaorptioa rpectra of chromatophoresofILr rubrun, atirlns: the wild
type,puf dektioo mutant and the mutant eompkmeated with the wild type puf
opmr Cells were grown microaerobically in the darlc.
species. This segment includes 7 charged axnino acid residues (3 negatively charged
residues, 4 positively charged nsidues), 20 nonpolar residues and 3 polar nsidues (Figure
19). To understand the funciion of these midues, a systemtic mutagenesis on this segment
was coaducted. B y using recombinant
PCR techniques (Higuichi, 1990) and degeneraîe
oligonucleotides, a pool of mutants was constructecl. AAcr nplacing the homologous wild
type region with the PCR products, the individual mutations were identifiecl by DNA
sequencing. In total, 15 different mutations were identifieci (Figure 19). 14 mutations
contain residue substitutions at single or multiple positions. They are LoEJK, L, 1 and Q;
L 7 W I ; LIOR12RJLL;L9YllV~HI;LIBGJV; L19G+V; L18G19GjW; L23D+Y;
L23D25WJNL; L28P30Y9RF and L23D25W28P30Y+NYRF. One mutation contains a
deletion of 17 amino acid residues fmm LA to L2O.
Pheootypic fatuns of the mubnb
The effect of mutation in N-terminal segment was evduated by in-tram
complementation in the Apuf mutant with the pRK-put35 containhg the wild type
M A L M as a reference. When micmaembically cultureâ in the âiuk, d l the mutants grew
with rates simüar to the wild-type construct. Their photosynthetic p w t h ability was
checked by growing the mutants on the soüd CB medium. in 10 days, four mutants did not
yield visible colonies; ~
6 ~ 2~3 ~ ~ ~ 2, ~5 .2 8 ~ ~ and
~ 3 LA4-20.
0 ' ~ The remahder did
grow up hto visible colonies with varied lag times, indicating the diffecent effects of the
mutations on the photosynthetic cornpetence. For the photosyntheticaily wmpetent mutants,
theù growth rates were M e r analyzeâ in liquid medium. Ceiis were taken h m cultures
grown in the dark to minimize the possibiity of including a second mutation possibly
20
1
Si
S1M3
RF. rubrunr (Sl)
K3
Pt
V3
N1
ALLSFERKYRVRGGTLICCDLFDFWVGPFY
Mutants
6K
6L
61
64
71
10L12L
9HllI
18V
19V
18V19V
23Y
23N25L
28R30F
23N25Y28R30F
A4-20
Figure 19. Tbe mutations in the N-terminal region of the L subunit of Rr. rubrum RC. The amino acid sequence of the Nterminal region of the L subunit of RF. mbmm, which is also the consensus sequence of 8 purple bacterial species is presented in
bold letters h m lefi to right dong the top of the figure. Above the sequence, the letter and the nwnber on its right side are the
variants and their Erequency o c c d in the sequences of 8 species. For each mutant, the mutated residues are listeâ at the
comsponding position below the wild type sequence. The deleted region is represented by a dotted line.
selected under the photosynthetic growth pressure. The results presented in Figure 20 show
that al1 mutants f d l y reached a growth rate similar to the wild-type construct. However,
mutants L 6 1 L6E1,L6EL (L6EK shown) and LIORLLIZRLtook longer to initiate grawth,
e.g., 5 days for LlORLL12RL and 11 days for L6EK. The lag in growth initiation in these
mutants was only observed when inoculated with the cells grown in the dark, but not with
cells gram in the light. M e r the light-cultured cells were submitted to the dark growth,
they again displayed a lag time in the next light gmwth.
The ultimately similar
photosynthetic growth rates in these mutants with long lag t h e are not due to reverted
mutation, because the photochernical activities of RC in these mutants were much lower
than the wild type complement (only 9?!for ~6~ and 30% for LlORL 12R1.).
When the cells of al1 c o l l ~ f ~ c were
t s microaerobicaily grown in the dark they
contained similar amount of B880 complex as representativeiy shown in Figure 21,
indicating a svnilar expression strength of the pf opeion in these mutants. But the
absorption at 800 nrn distinpuishes these mutants into two types (Figure 21). The mutants in
#
type 1 displayed a small 800 nm absorption shoulder similar to the wild-type complement.
This includes some photosynthetic mutants like ~ 7 " , L9Y?i11VI, LMGv, ~ 1 9 ~ ,
~ 1 8 ' ~ ~ 1L23Y
9 ~ and
~ , ~ 2 3 ~ ~ ~ 2 5 ~The
~ m~
uta2nts8in~type
~ 2~ showed
3 0 ~a lack
~ . of
the 800 nm absorption shoulder, indicating the absence or h t i c decrease of RC content in
these mutants,
Relative level o f photochemluIactMty of RC in mutaab
For the same reamn explained in Chapter II @age 32), the change of A i s o upon
photooxidation of the special jmir was useà to indicate the RC photochemical activity.
were grown in CB medium and monitored by measuring the density with
reading.
Figure 21. Aborpfian spectra of intact celb of different Ra. nrbrum stmim. The cells were grown rnicroaerobically in
the dark in CB medium. The density of the cell samples was adjusted to 0.3A, to take the absorption spectra.
Figure 22 shows the relative absorption change of Atm in the chromatophons of mutants
fe~pectedwith
LI8",
the wild type comtmct that was taken as l W ? . The mutants ~
9lV', ~
~
~19", ~ 1 8 ~ ~ and
~ 1 ~9 ' 2~ 3 ~ ~ 2 5 pssess
~ ~ 2~UNlar
8 ~level
~ of
~ 0 ~ ~
photochernical
activities
as
the
wiid-type
complement
(only
mutant
~ 2 3 ~ ~ ~ 2 s h~o w~ in~Figure
~ 222).8Mutants
~ ~ ~~ 2 33 0~ ~ ~~~ 62 L 5~O ~~~, ~ 96,
~
6 ~ 2~ 8 ~~~ ~ ,3and
0 'LM-20
~
show no photochemical activity. Mutants ~
~
~
7
~ 1 0 ~ and
~ ~23~'
~ 1 contain
2 ~ much
~
lower but varying levels of RC photochemical
activity: mund 60%. 30% and 50%, respectively.
L subunit level cornlates with RC photochernical activities in the mutants.
Because the mutations were locatecl in the L subunit, the decreased level of RC
photochemical activity codd be due to a change of the L subunit level in the mutants, By
running the chromatophore proteins on 15 % SDS-polyacrylamide gel, the L subunit can be
well separated Erom other protein bands to allow for quantification. The relative levels of
the L subunit in the mutants compared with the wild-type complement are presented in
Figure 23. in the mutants with undetectable photochemicai activîty of RC,w L subunit was
detected. In the mutants with full level of photachemical activity comparable with the wild-
type complement, the levels of the L subunit are also comparable with the wild-type
complement. For those mutants with decreased photochemical activity levels of varying
degrees, the L subunit Levels also shomd proportional variation.
The relatioaship between
the two parameters is show in Figure 24. That the relative levels of the
L subunits are
higher than the levels of RC activity indicates, in these mutants, some portion of the L
subunit does not assemble into a hctional RC.
,
,
1
Strains
Figum 22. Relative levek of RC photockmical activity in the chtomatophoier
of Rs. twbrum mutant sûains comparod with the wlld type complement The
cells were grown microaerobically in the da* in CB meâium. The RC photochernical
activity was measured as AA,,
due to photooxidation of the special pair. The
illumination perioâ was four minutes. The change of
in the chromatophores of
the wiîâ type cornpiement (WT) was taken as100%.
Strains
Figurc 23. Rclativc lcvles of thc L subunit in thc chromutophorcs o f Rr. rubrum
mutant strlu'nu compured with the nild typc complemcnt. The cclls were grown
rnicroaerobically in the dark. The L subunit lcvels wcrc obiained by scanning the densi' of
L subunit band on SDS-polyacry1;unidegel d e r staining with Coomassie Bdlimt Blue.
The wild type complement (WT) was taken as 100%.
20
40
60
80
100
Relative RC activity level (%)
Figure 24. The reiationship of the leveis of L subunit and
RC activity in diffemt RS. wbrum strains.
Discussion
To c a q out mutagenic studies on the highiy conserved N-terminal segment in the
L subunit, a puf deletion mutant was first constructed in RF. rubmm wild type strain S 1. The
success of in hum complementation in this mutant is in contrast to the nsults reported by
Hessncr et al. (1991) with puf deletion mutants derived h m a rifampicin resistant mutant of
RF. rubrum. These authors foutid that among three puf deletion mutants with different
deletion size, only one (P5) was complemented by plasmid-borne wild type puf genes The
success in PS was dependent on the deletion of a 1 kb BamHYWoI m e n t dowastnam of
pu^ that was intact in the other two mutants. &cause the fiagrnent used for
complementation contained this 1 kb region, the authors speculated that the 1 kb region
affkcîs complementation but only when it is in ci' relative to a sequence which is located
f.urther dowiistnem. In our study, the deletion region is within the pufoperon as the other
two consûucts reported by Hessner et al. (1991). But in contrast to the Mure in the
complementationof those two const~cts,the Apfrnutant derived h m the wild type
in this study clearly demonstrated that pvfdeletion wuld be in truns complemented without
the need to remove tbat 1 kb fhgment, therefore indica0irig that the 1 kb region did not
affect complementation in the mutant derived b m the wilii type suain.
The mutations in N-terminal region could sffkct the membranous insertion of the L
polypeptide or cause a rapid degradation if the mutations bring about a substantial structural
change a d o r sevenly deteriorate the h c t i o n of RCs. But two pieces informatioa suggest
thet these mutations mallily Scct insertion. First, it is impossible lbet the degradation is so
nist h some mutants as to tesult in an undetectabie RC photochernid activity and L
subunit. Second, near perfect photosynthesis fiuictioa must be possessed by the RCs in the
mutants like ~6~~and L8RI 10RI, because they can grow at a similar rate as the wild type in
the light with much l e s RC content. These RCs are most likely to bave a similar decay rate
as the wild type RC. The results prrscnted hem, therefore, generaily support the suggest of
BClanger et aï. (1988), that the consend residues in this segment are required for
membrane integration of the L polypeptide. But for each individual residue, the role is
differmt. It is interesthg to observe that L6E, a negatively charged residue, seems to play a
crucial d e in the membrane integration of the L subunit. The f'unction of this =sidue seems
not related to the photochernical reaction, otherwise one could not expect a normal growth
rate in the mutant ~6~~ that has only 904 RC of the wild type complement. Because
negatively charged residues are thought to not play a d e in topology determination (Nilsson
and Von Heijne, 1990), this residue is more likely involvecl in a crucial recognition reaction
for targeting the L subunit to the membrane. A linear relationship behwen the number of
positive charges and the RC level in mutants ~ 8 "and ~ 1 0 ~ is ~consistent
~ 1 with
2 ~the
observation of the Lep protein in E. coli (Nilsson and Von Heijne, IWO), indicating these
residues could be topological detetminants. An important clue was obtained h m two
double mutations and a quadruple suppression mutation.
Neither ~ u ' ~ ~ 2 5nor
"
~ 2 8 ~ contains
~ ~ 3a detectable
0 ~ ~ L subunit (and no RC activity and photosyntheticaily
incompetent). But the mutant ~ 2 3 ~ ~ ~ 2 5 possessed
~ ~ 2 a8similiir
~ ~phenotype
~ 3 0 as~the
wiid type complement. These results strongiy suggests the existence of an interaction
between these two pair of residws, and that such interaction might play a very important
role in the membiant integration of the L subunit. This speculation is supported by the
crystal sttucture of RCs of Rp- viridis and Rb- sphwroides. ih both, the N-terminal segment
o f the L subunit possesses a U-tum structure involving the above four residues. It might be
that not only the pcimary sequence but also the secondary structure in the N-terminal
segment play roles in targeting L subunit to membrane (Bird et al., 1990). intercstingly,
our results also reveai that some extremely conserved residues, as L9Y, LI IV, L18G and
L19G,can k changed to other residues without affécting the photosynthetic gmwth and the
RC activity. For the moment, it is not known the reasons for the conservation of these
residues.
Chapter V
Construction and characterizrition of puh and Cl15 deletion
mutants in Rhodospirülum rubrum
in purple bacterial photoreaction centers (RC), the H subiait binds to the L-M
proteins on the cytoplasmic side and also makcs contacts with the M subunit dong the H
trammembrane a helix. It contains three domains: a transmembrane a-helix located nesi.
the N-terminus presumably mchors the subunit in the membrane; a globular domain
caps the cytoplasmic side of the L and M complex; and the N-terminal mgion before the
helix is located in the periplasmic side (Deisenhofer et al., 1985).
In Rr. rubmm, the H subunit is encoded by the gene puh that is locateù sepamtely
h m the L and M stnictuai genes @ujL and pum. An open reading h
e G115 was
detected upstream ofpuh by sequence analysis (Mtatd and Gingras, 1990). Their studies
indicated that p h is transcribed in a single cistronic mRNA and that 01 15 is transcribed
in a 2.25 kb transcript (Bérard et al., 1989). G115 is homologous with a similarly
positioned ORF 16% in Rb. cupmiatus (Youvan et al., 1984) whose hction is related to
assembly of the light harvesting B880 complex (Bauer et al., 1991 ;Young et al., 1998).
In vitro -dies
indicated that the primary photochernical reaction (P*Q*+P+Q*?
is not affected in the H-less RC, but the elecmn transfer h m
a to Qe is greatly
reduced (Debus et al., 1986 ).This fhding is in line with the discovery of miter challis in
the RC ( E d e r et al., 1994; Stowell et al., 1997) iunning firom Q to cytoplasm, which
are believed to facilitate the protonation of raduced Qa. A numkr of residues in the H
subunit are involved in the build-up o f the wakr chain. The mutation of Hl73Glu (one of
these midues) to H l 730111mi9 shown to greatiy retard the nrst and the second electron
transfer to QB(Tachikashi aud Wraight, 19%).
The in vivo physiological f'wiction shdies of the H subunit in Rb. sphaeroides
(Sockeît et al., 1989) and Rb. cup~~luhcs
(Wong et al., 19%) came to a consistent
conclusion that the H subimit is necessary for photosyntheisis. But contmvemiai results
were obtained about the function of the H subunit in LH-1 complex assembly (Sockett et
al., 1989; Wong et al., 19%).
To identify the hction of puh and G 1 15 products and to pave the road for iùture
mutagenesis study on these two proteins, puh and G1IS deletion mutants of RP. lublvm
were individually constnicted and characterized. The muits cleady show that the
disruption of puh affects the RC content, but not B880 complex level. The disruption of
G115 gene greatly reduces the B880 complex level. The defect in photosynthesis in H-
les mutant seems more due to the fhctional disnubance in the H-less RC than the
decreaseâ RC content.
Construction o f puh and Cl15 deletion mutants
The strategies shown in Figure 25 were used to individually constmct the Kmr
substitution mutations ofpuh and G115. The plasrnid pS837 (Bérard and Gingras, 1990)
harboa the 3.7 kb Hindi11 Fragment ihat includes puh, G115 and o h r ORFs. The mRNA
structure studies (Bérard et al., 1989) indicated that the transcnpt of G115 might be
teninoted within puh. To avoid the disturbance with possible transcriptional structure for
both genes, the deletion was placed near the 3' end of the pirh gene for puh deletion
mutation, while the deletion in G 1 15 leaves around 500 bases upstream of puh. The gaps
lefi by deletion were filleci with a Kmr cassette. Two separate Kmr substitution mutations
in puh and (3115 were obtained by gene replacement strategy as has been described in
the previous Chapters, and the mutants were narned Apuh and AG 1 15, respectively.
Cbaracterization o f Apuh and AG1 15 mutants
The Apuh mutant is incapable of photosynthetic growth (Figure 26). When
p w n in the dark, the Apuh mutant possessed a sllnilar amount of 8880 complex as the
wild type, as indicated by absorption spectra of the chromatophores (Figure 27). The
absence of absorption at 800 nm in the mutant indicates the lack or drastic decrease of
RC content. By measunhg the change of the absorption at 1250 nm due to photooxidation
of P to,'P the RC level in the A p h mutant was estimated to be about 11% o f the wild-
type (Figure 28). These data showed that dimption of the H structurai gene only affects
Figure 25. Coostructs useà for makhg puh aad Cl 15 substitution mutaib. a) the chromosomal arrangement of
p h , G11S and other hHo ORFs in 3.7 kb of HindIIl f m e n t ; b) disruption of puh by substitution of Km resistance
gene cassette for BstElVBstElI region; c) disniption of G11S by substitution of Km resistance gene cassette for
NotVApaI region.
Figure 26. Photoqaîhetic w w t b o f Rr rubrunr atmlas: the
wildtype, the Cl 15 deletion mutant, the puh dektion mutant and its
coaplcaent witb the wild type puh gcac. Cultures were grown
anaerobically in CB medium under saturating illum*nationand monitored
by meamring ce11 density with &60 reading.
Nanometers
Figure 27. Absorption rpectr8 o f the c h r w r t o p h o ~fma
~
different Ab. rubrum stmins. Chrotnatophorr samples were ptepered
h m ceiis grown micmaembically in the dark. The spectm were
n o d i z e d tu quai protein concentration. Apuh(pRKL580-) aad
Apuh@RK1580+) are two complements of Apuh mutant with 3.7 kb
Hindm hgment including wild type puh gene. Positive symbol
indicates paraile1 transcription of piJi with the lac pornoter in thc
.
vector pRK404,while the negative one means a c e v e d direction,
W ild type
O
2
off
n
4
6
8
Minutes
Figure 28. Absorption change at 1250 rn in the chromatophores of
different Rr. rubrum strains upon photooxidation of the special pair.
The chromatophores were prepared fiom cells grown in the dark
under microaerobic condition.
the RC, but not the B880 complex. The AGI 15 mutant is capable of photosynthetic
gmwth but at a reduced rate (67% of the wild-type) (Figure 26). The absorption
spectrum of the chromatophore From AG 1 15 mutant exhibits a substantial decrease of the
880 nm band and a big increase of the absorption near 765 nm (Figure 27). The decrease
of the 880 nrn band indicates the reduction of the B88O complex. Quantitatively, the
AG 1 15 mutant contains around 33% of the B880 cornplex of the wild type. The big 765
nm band mi@
be an indication of the degradation of bacteriochlorophyll due to the
inefficient assembly of the B880 complex in this mutant. It was found ihat the AG1 15
mutant has a similar level of photochemically active RC as the wild-type (Figure 28).
These results suggest that G115 is involved in the B880 complex assembly.
Compkmentationof the Apuh mutant
The conscnicts used to complement Q t i h mutant are shown in Figure 29. AAer
conjugation, the cells were allowed to grow in the light for 10 days in solid CB medium
before the colony size was checked. Except pRKl4(-), in which the promoter sequence
must have not k e n included, al1 other constructs could complement the Apuh mutant to
photosynthetic growth. The truncation at both the 5' and 3' regions, which deleted part of
ORF iî372 and G 115 gene, did not abolish the complementation by these constructs such
as pRK820(+), pRK820(-), and pRK34q-). This means that the H structural gene alone is
suficient to restore the photosynthetic growth in the A p h mutant. Comparing the
average size of the colonies, it was found that the conjugants havingpuh parallel with the
lac promotor on the vector pRK404 (Pt=) grew faster than those constructs with puh
reversal to the P .
This result is consistent with the observation in
I
I I
I
Hindlll NotI Sac11 Ddel
I
I
EcoRl
PS
HIndlll
Plac
+++
i
d-
Plasmids
pRKl580(+)
Plac
'
Sac11
Plac
Notl
EcoRI
Ecoiü
Plac
-.
Sac11
EcoRl
Plac
----+
Figure 29. Constructs used to complement Apuh deletion mutant. The numben represent the length of the upstream sequence of
puh. (+) indicates a parallel transcriptional direction o f p h with the lac promoter in the vector pRK404, while
direction. "PS" syrnbolizes photosynthetic growth. "+" means photosynthetic growth competent, while
growth incompetent. The more "+", the faster of the growth.
"-"
(0)
indicates a reversed
means photosynthetic
Rb. spheroides (Socken et al., 1989), and hdicates that a portion of p h mRNA might
be ûanscribed hm the Pi, promoter. The growth rate of the conjugant with pRK1580(+)
in liquid medium was measwed to be 12 hours per genecation, which is 50YI of the
p w t h rate of the wild-type.
The photochemically active RC in this wnjugant was
measured to be 81%of that in the wild-type.
photospthetic pseudottvertant of Apuk mutant
The Apuh mutant is not able to grow in the üght. But when the inocdated dish
was kept under iUwnination for around two weeks, some red colonies appearad on the
solid medium surface. The fkquency is amund lo4-10-'.
These cells maintaineci photosynthetic growth &er k i n g transferred into liquid
medium, with a growth rate of 27% of the wild type. Southem blot results demomted
that the Kmrcassette is still seated in puh as designeci. To determine whether the
restomtion of photosynthetic growth is due to the increase in RC content, the level of
photochemidly active RC was measured and compared with the parental stiain. It was
found that whea the pseudorevertant was grown in the light, it containcd 17% of the RC
activity of the wild type
also gmwn in the iight. But the RC activity in the
pseudorevertant cells grown micmaaobically in the da& was around 11% of the wild
type, similar to the RC activity in its parent Apuh mutant. The slight increase of the
relative RC activity in the light-grorni cells seems due to the growdi pressure on the RC
bction. These data indicate the second mutation seems to compensate for the hctional
defect of H-less RC, but not in assistame in the assembly or stabilizing of the H-less RC.
With the hope that the second mutation rnight be locateâ în L and M subunits,
the
sequence of L and M structurai genes in the suppression mutant was aaalyzed But the
resdts did not show auy change
in the sequence of these two genes. Thecefore, the
second mutation must be located at anoîhet locus.
Discussion
M d and Gingras (1989)demoll~tratedthat, in Rr. rubrum, p h was traascribed
as 0.95 and 1.15 kb pmducts, while the probe containing 0 1 15 quence detecteâ another
2.25 kb product. Because gene Gl 15 consists of 1440 bases, it is uniikely that this 2.25
kb transcript hatbrs puh mRNA. Therefore, the transcription temùaator of O115 is
likely located within the p h gene (Bérard and Gingras, 1989). Using the Stem-loop
program (GCG package) to search the stem-loop structure in the p h gene found one
started fiom 52 to 91 afler the stop codon of GllS gene with sequence of 5'CAGGTCGITCmACGCGmCTGG-3'. The free energy of the formation of the
corresponding structure in RNA was caiculated to be -16.7 k d m o l , and the seqwnce is
tailed by a T rich stretch (ATCTTCm, suggesting that this structure may fùnction as a
ho-independent transcription terminator of 0 115 gene. The puh deletion mutation and
the Gl 15 deletion mutation were designed to avoid the dismption of theu potential
transcriptional structure by lemhg reasonable length of upstream sequence for p h and
of domrstrram sequence for GllS. With two individdiy targeted mutations in RS.
rubmm, the work descriW hen distinguishes the different hctiions ofp h and GI 15.
The pJi p d u c t was demoastrated to be necessary for normal content and hction of
RC, but not for 8880 cornplex assembly. The dimption of the (3115gene causes a
dramatic decnast of the B880 complex. Even though the= is no wmplementation data
available for the 0 115 mutant, the mle of G115 p d u c t in the 8880 complex assembly is
supported by the redts of its homologous F16% in Rb. capsulatas (Bauer et al., 1991;
Young et al., 1998). The effcct ofpuh deletion in Rb. capsulatus (Wong et al., 1996) was
show to be different fiom the results in the present work. These authors observeci a
decrease in both the 8880 complex and the RC in the puh deletion mutant Such
difirence might be due to the différent strategies for mutant construction.
It is
notewotthy that in Rb. capsulattrs, the deletion in the puh pne starts 50 bases
downstream of the stop codon of FI 696 (Wong et al., l9%), while in the present study
the deletion siarts 528 bases downstrcm or the stop codon or G l i S. in the later cas*, the
transcription temination structure (the secondary structure might have a protective
function agiost the degradation by RNases) should be intact, which might help to stabilize
the G 115 mRNA, but it is not assured in the puh deletion mutant of R. cupsuIutus, since
the structure of FI696 mRNA is not clear.
Photosynthetic incompetence in the A p h mutant could be due to a much lower
content of the H-less RC resulting fiom a fmter degradation than the normal RC (Varga
et al., 1993), and/or the hampering of the proton transfer in the H-less RC simply due to
the destruction of the water chah. Because the ceIl with only around 10% RC of the wild
type is photosynthetic (
~ mutant
6 in
~ Chapter
~
IV), and also because the suppression
mutant contains a sirnilar level of photochemically active RC as the parent mutant, the
interference with proton transfer seerns to be a critical consequence in the H-less RC
leading to photosynthesis incompetence in the bpuh mutant.
In contrast to the results in this work, a photosynthesis-comptent revertant from
the puh deletion mutant was not obtained in Rb. sphaeroides (Socken et al., 1989). That
could be due to the deletion of bothpuh and its upstream gene (a G11S homology) in that
mutant, which affects both RC and 8880 complex. The success in this study may have
resulted h m the single gene targeting sûategy, which only affited the RC, but oot the
8880 complex.
Sequence analysis of G 1 15 (E3érard and Gingras, 1990) and cornparison with the
homologous Rb. capsdattrs FM% and pufC gene (LeBlanc and Beatty, 19%) pdcted
0 1 15 product to be an integral membrane protein. It seems uniikely that 0 115 product
tightly associates with B880 complex, for it was wt detected in the purifieci B880
complex (Picorel et ai.. 1984). Recently, Young et al. demonstrateci that ORF16%
protein enhances assembly of the light-harvesting 1 complex in Rb. capsulaus (Young et
al., 1998). It is likely that the Gll5 product plays a similar role as ORF16% protein in
facilitating 8880 complex assembly by a mechanism to be elucidated.
Chtpter VI
OveraU Discussion of Thesis Results
1.
puf operon, conserviîioa and variation
The cloning and sequence analysis show that the organization of the puf operon in
Ect. shaposhnibvii, a sulfut purple photosynthetic bacterium, is similar to that in nonsulfur photosynthetic bacteria, especially with regard to the genes encoding the subunits
of the RC and of the B880 complex. But the other ngions in the puf operon are variable
in different species. A general comprison of puf operons h m 8 different purple
photosynthetic bactena is s h o w in Figure 30. The variable regions are generally located
upstream of pufB or after p w . In three cases (Ecr. shupshnikovii, Chr. vinoswn and
Ru. gelutinosus,) a change located betweenpujB and pufA. These variations provide us an
opportunity to look at the relationship among these species fiom another angle in addition
to sequence cornparison.
In two Rhodobacter species (Rb. capsula~wand Rb. sphueroides)p$Q
and pufK
genes (Rb. capsulatus has homologous sequence with pufK of Rb. spharoides)are located
Unmediately upstream of pu@ gene (Bauer et al., 1988; Davis et al., 1988; Kiley et al.,
1987; Youvan et al., 1984). Another gene p u t is located downstrram of pujM (Youvan
et al., 1984; Lee et al., 1989).
These three genes are only found in diese two Rhodobacter
spcies. Therefoce, this fature clearly separate these two species from other ôteteria in
which the= is no homology in the cortesponding regions.
A small ORF was detected in the region immediately upsûeam of pu^ in three
specttes: fit.
shaposhnirtovii (ORFQ ', Chapter tI of this thesis), Rx mbmm [ORFI,
Rb. sphaeroides
Rb. capsulu~us
Ru. gelarinosus
ORFO '
Ect. shaposhnikovii
Pu@2
FufA2
Chr. vinomtm
Rs. rubrum
Ro. denifrflcans
Rps, viridis
Figure 30. The organization of pu/ operon gems in düferent purple photosyntbetic
bacteria The color filled rectangles represent the puf genes or ORFs. The same color
represents same gene or ORF (may have different names). The thickened red line in Rb.
c a p ~ ~ l ubetween
î ~ s P U T and pufB repesents the region having sequence homology with
p u in~ Rb. sphaeroides. The thickened purple line between pu@ and p f A in Ect.
shaposhnibvii and Chr. Vinosum represents the sequences that are homologous with
ORM in Ru. gelutinosus.
(Bélanger and Gingras, GenBank AF018954) or ORFQ,(Lee and Collins, 199311 and Ru
gelutinosus (ORF 1, Naphima et al., 1994). This type of ORF is different h m pu@ in
temis of
size and chernical properties of the pdicted pduct. Despite the diflcicuity in
hding homology in thek putative protein products, the three ORFs s h a n significant
homology in DNA sequence (4 1 % identity between Ect. shaposhnikovii and Rr. rubrtm
with one gap and 45%identiîy beîween Ect. shaposhnibvii and Ru gelc~tinoslcswith two
gaps). Theu putative products also have sirnilar size and chernical properties such as net
positive charge (rich in argiaine) and serine and proline richttess. Since w simüar ORFs
could be found in other species, this khd of ORF disthguished these three species h m
other species. However this situation is not in agreement with the phylogenetic
relationship besed on 16s rRNA gene sequence, in which RF. rubrtlm belongs to the a
subgroup, while Ecr. s ~ s h n i k o v i ibelongs to the y subgroup and Ru gelatinow
belongs to the P subgroup. nie discrrpancy tbat RF. r u b m is closer tO the species in the
P (Ru.gelutinosus) or y (Ed. Shaposhnihvii) subgroups than to the other species (îwo
Rhodobacter species, Ro. denitrijhns and Rps. viridis) in the a subgroup can be
interpreted as the lateral tmsfer of either photosynthesis geius or 16s rRNA genes
between their ancestors. Photosynthesis can be an additive hction to the rrceptor
bactczrum, whenas a foreign 16s rRNA, can not be expected as hctional as the native
one in the receptor bactenum. Thenfore, if the laterai gene transfer o c c d in
photosynthesis genes, it would have cbance to be preserved as a bemficial addition.
Laterai W e r o f 16s rRNA gene may oçcur bmv&n bacteria, but the fonign one
would k diIEcuIt to pnserve and pass dom.
Another interesting region is located betweenpujB and pufA. Usually, these two
genes are separated by only 12-14 bases, but in Ecr. skposhnibvii, a big spacer of 90 pb
was found in this region. The big spacer was also reported in Ch. Vinoswn (Nagasbima,
1998 GenBank access No. AB01 1811). In Ru. gelarinoms (Nagashima er al., 1994) an
ORF (ORFZ) is detected by SeQuence aaalysis in this region. The sequences of the spax
between pufB and pulA in these species share a sigaincant homology (38.5% ktween
Ect. shuposhnihvii and Chr. vinosum and 40% between Ect. sahpshnihvii and Ru.
gelutinosus with one srnall gap). These data cleady kdicate a common origination of the
spacer in these species, and implies that either a lateral gene transfer o c c d between
the species of the P subgroup and the y subgroup, or these two subgroups branched h m
the a subgroup before they branched h m each other.
lt is interesîing to see that Chr. v i n m m has two sets of p@, A. Each of them is
spaced by a long sequence (80 bp between pujB1 and pufA1 and 84 bp between pujB2
and ~ ~ $ 4 2The
) . duplication of these genes and then the separation of two set genes
indicate an active propeity of this region in the evolution, probably mediated by
transpsable elements.
13
Why is 8bepuh opcron no T i r away fmm thcpuf operoi
Considering the hction and the stoichiometryof the RC subunits, the location of
the H subunit structural gene is intriguing: 45 kb distant and transctiptiodly mversed
h m the pqfgenes* et least in Rb. cupst(ialu~.
This puzzle might be solved by the study of
the upstream gene ofp h , F16%
in Rb. capsuIatus (Youvan et al., 1984) or 0 115 in Rr.
m b m (Bérard and Gingras, 1990).
The F16% or 0 115 product seems to play a d e in assembling the B880 complex
(Bauer et al., 1991;Young et al., 1998; Chapter V of this thesis). Interestingiy, these hkro
genes, in
addition ta king homologous to each other, share a high homology with
enother photosynthetic gene pucC whose pmduct has been s h o w to be involved in the
assembly of the LHU (B8ûû-850) complex (LeBlanc and k t t y , 1993), indicating a gene
duplication in their evolutionary history. pucC immediately follows its target genes pucB
and pua¶ in the pue operon, while in Rb. capsulatus, FI696 is 45 kb away from pufA and
pun. This situation invokes the assumption that the cumnt locations of these genes
resulted fkom reorganization events during the course of evolution. The H structural gene
is located close to F16% or 0 1 15, which might k an indication that the H structural
gene is one of the components of that reorgsnllation event. It is possible that the
evoluti*onatycourse of these genes follows a defined sequence. 1speculate that originally,
the ancestral geaes coding for RC subunits, antenna subunits and their regdator (F16%
or G115) were linked together. Their duplication then gave rise to two sets of antenna
related genes. Later on, one set of such genes might have moved to th present locus and
evolved as puc opem. F W y , the other copy of ancestral genes of FI696 or 0 1 15 and
the H subunit separateci ûom the ancesmi genes of pujB,A,L
M,C to becorne located at
the present Locus. in RP. mbmm, t h e p c opemn might k Lost later on, or altemativeIy its
amestor received h m odicr photosynthetic bacterhm only a copy of photosynthesis
genes cluster without including the puc opemn, or else, the puc operon evolved after the
sepamtion of the ancestor of Rs. mbnun
2.
The stratcgr of mutageneah
In bactmal genrtics, wherever n suitable vector system is available, i n - t r m
complementation is the most common strategy to cany out mutagenesis studies. It has a
numkr of advantages. The oonvenience of this system is because the effects of
mutations can be tested in a ndi background mutant by complementatior The easy
retrîeval of the plasmid enables one to check the comtness of the engineered mutation
when it exists and to characterize the secondary spontaneous mutations in the gene, M e
avoiding the more lengthy procedure of cloning mutated genes h m the bacterial
chromosome. But, the dependence on a nul1 background strain limits the application to
those genes that are not i n d i s p d l e for ce11 suMvai. ûthecwise, conditionally Iethal
mutants (mostly temperature sensitive straias) are d l y screened to carry out the
mutagenesis study, but such mutants are not always easily obtainable. Another
shortcoming of this shategy is that in many cases the phenotype in the nuîi background
strain cannot k çompletely restod to that of the wild type, even though the gene
expression level could k higher h m the plasmid than that h m chromosome, probably
due to the dishubance of chtomosomal structure which
&ts
the expression
flanking genes or the existence of another replicon (Davis et al., 1988).
of the
The above mentioned shortcomings with in-tram complernentation can be
overcome by direct gme mutation on
the chromosome. This strategy has been
successfully used in out genetic engineering of the Be site of the RC in Ect.
shapahnibvii. Then are two important applications with this strategy. First, this
strategy can be used to study the function of indispensable gens, as long as the mutation
is not lethal. Second, because the mutation is d i d y brought onto the chromosome, the
cis-acting effect will be absent, or the interrupted cis-acting effect by the mutation can be
recognized. Theoretically, mutation on chromosome can be achieved by a gene
replacement strategy, however in practice the selection of the mutation is technically
almost impossible if there is no assistance h m a second selectable marker such as Kmr
used in the study of Chppter III. in this strategy, the interesting mutation is CO-selected
with the second marker (double mutation), the ratio of the double mutation to the single
second marker insertion is dependent on the lengths of the hoaidogous region
downstream (or upstreatn) of the interesting mutation site and the region betwœn the
i n t e d g mutation site and the sccond w k e r insertion site. The longer the former, the
higher frequency of the double mutation in the selected colony pool.
While greatly facilitating the selection, the introduction of the second selection
rnadcer on chromosome wuld have efféct9 on ceIl's physiologie properties, as
enaountered hem, the insertion of Kmrat SphI position caused a de-
in pfgene
expression by 80Yo and brought about a big problem in RC isolation. Testing d W î t
insertion positions for the second &et
shouid âelp to m h h h the effects. However, if
the second marker causes a noticeable slower gtowth of the mutant, then this d e r
could be removeci h m the chromosome by another round gene replacement expcriments.
By introducing to the mutant a wild type sequence wmsponding the second marker
dismpted region and under an appropriate growth pressure, it is possible to select off the
second marker and obtain a pure mutation (for example, only Ml 8 0 ~ ) .
3. The functioa of the prosthetic goups in the B w i y
Crurently available genetic and stnicnual information suggests that the L and M
encoding genes origlliate h m a gene duplication event foiiowed by divergent evolution. It is
possible that in a certain early period the primordial fonn of the RC was a homodimer
(Blankenship, 1992). even if it is not sure that such dimer had ever bctioned as an apparatus
for the energy conversion as in the cumnt RC. The electron flow in such a RC,if it happened,
should have no discrimination between the two iines of prosthetic groups. The highly efficient
and asymmetric electmn transfer pattem in cumnt RCs seems to be the selective redt of a '
long period of evolution.
Given that the Be and OBdo not f'unction as the electron amiers in the cumnt RCs,
an intriguing question is why they are still there (not lost during evolution). One of the
possibilities is chat the presence of these bacteriochiorins is a structural requisment for RC
hction. The absence of these bacteriochlorins might cause a severely structural distortion
and nsult in the hctio1181 failure in the RC. In addition, the electmnic structure of these
bacteriochlocins in the current RCs are n q u k d to mdce the A pathway ' ~ r f e c t for
' electron
tmder at the expense of theiu own silence. Of BB ead %, the fonna may play a more
important role because of its vicinity to the special pair*S b BB and BA are approximately
spatial symmetric to the special pair, the different nQx potential between these two
pigments seems to be the key factor in determiningthe direction of electrw transfer (Heller et
al., 1995). A number of mutant RCs containhg pigment exchanges between Bchl and Bph
have been engineered. These shdies hdicate that such pigment replacement docs not cause a
noticeable structural change in the mutant RCs, but b ~ g about
s
a substantial change of the
redox-potential at these sites (Bylina and Youvan, 1988; Arlt et al., 19%). Compand with
Bchl, Bph has a stronger tendency to accept ekctroas (Fajer et al., 1975). In this case, it is
possible that the substitution of Bchl with Bph at the Ba site might have significant effect on
the primary photochernical miction in the RC. A much slower gmwth rate in the ~ 1 8 0 ~ ~
mutant of Ect. shaposhinihvii seems to be an indication of such a consequence. in the Ect.
shaposhnikovii RCs, the pure absorption spectrum of the special pair was obtained by
ûapping the photochemically doubly ceduceci monobacteriochlorins in which the high energy
band was demonstrateci to be located at 800 nm (Mar et al., 1993). The shiA of the high
energy band in ~ 1 8 RC
0 to~790
~ nm indiates the interaction of the cofactor et the Be site
with the spcciai pitir ami the contribution to the hi&-energy band fomieti~n.CD spectrs
showed that the high-energy band had a positive rotatory strength (Vasmel, 1986; Mar aad
Gingras, 1995), ratber than a negatin one as opposed to the positive rotatory strength of the
low-energy band of the special pair. This f
a
t
u
t
eimplies that the high-energy band consists of
an additional cornponent that has nonexcitonic interaction with the special pair. The d t s
h m ~ 1 8 RC0 indicate
~
that the third wmponent appears to be the Bchl at Be site. Further
d i e s on Be site mutants and theù supprrssors will provide a deepet insight into the
electronic structure of the Be site and how it interacts with the speciai pair and presumably
influences electron transfer in RCs.
4. The functian of the A aubuait in the photosynthttic mction
The H subunit is a basic component of the purple bacterial RC. The importance
of this subunit in photosynthesis is manifesteci by the obsewation that H subunit deletion
mutants are photosynthesis incompetent (Sockett et al., 1989; Wong et al., 19%; Chapter
V o f this thesis). An apparent effect of H subunit deletion is the drastic reduction of RC
content, which seems to result fiom a faster degradation of the H-lee RC (Varga and
Kaplan, 1993). However this reduction alone cannot account for the photosynthesis
incornpetence, because the cells with 100/o RC relative to the wild type can grow
nomially (for example, ~
6in Chapter
~ , IV). The key effect of the H subunit depletion
on photosynthetc growth is probably the fhctional disruption in proton transfer, which
dects electron transfer in the H-las RC. It has been shown that a Rb. sphaeroides RC
preparation deprived of its H subunit in vitro has almost the same kinetics of electron
transfer h m P* to Qa as the intact RC, but that the electron transfer from QA to QB is
greatiy reduced. Recotl~t~ction
of the L and M corc complex with the H subunit restored
the wild type electton transfer h m QA to Qe. (Debus et ai., 1986). The chromatophote
of the R m b m Apuh also shows that the RC is active in the primary photochernical
&on
with respect to the formation of,'P even though at a much lower level. Because
the globular domain of the H subunit intecacts with the L and M complex
fnmi the
cytoplasmic side, close to two quinone biiding sites, the interference with
to
QA
QB
electron traasfer in H less RC could be explained as a structurai disturbance of the two
quinone binding pockets. But the high-resolution crystal structure of Rb. sphaeroides RC
reveals water chains from the QBsite to the cytoplasm (Ermler et al*, 1994; Stoweli et al.,
1998). The fiuction o f the chahs is suggested as a pathway for the protonation of
reduced QB.Many arnino ecid residues in H subunit ere predicted to be involved in the
build-up of the wter chah (Ermler et al*,1994; Stoweil et ai., 1998).
S.
Assembly of the RC
In contrast to the intensive studies on the structure and fiuiction of RCs, the
mechanisms underlying the assembly of the subunits into a fllnctiormi RC are poorly
understood in purple photosynthetic bacteria The crystal structures of RCs do not
indicate a clear fiction of the N-terminal region of the L subunit. However hi@
sequena conservation in this region strongly suggests an important role, most likely in
the membrane integration of the L subunit (Bélanger and Gingras, 1989), which is
evidenced by the results in Chapter IV.
The RC assembly can be viewed in two steps: the membrane integration of
individual subunits and then theu mutual adjustment (interaction) to nach an optimal
state. For integration of membranous proteins in pmkaryotic systems,
a SRP (signal
recognition particle) dependent mechanism has been discovend in addition to the Sec
system.
By using a genetically engineered cytoplasmic
peptidase (Lep) in E.c&
membrane protein leader
de Gier et ai. (1996) found that depletion of the Ecoli SRP
strongly affectecl the insertion of Lep into the cytoplasm membrane. In vitro experiments
demonstrated that the E-coli SRP and its meptor are associated with nascent membrane
and presecretory proteins. The SRP receptor can be efficiently cross-linked to nascent
polypeptides of different Iength, indicating that it is positioned near the nascent chain exit
site on the E.coli ribosome (Valent
et
al, 1997). By conducting an elegant genetic
screening system, Ulbrandt et al. identified a set of b e r membrane proteins whose
insertion requires Ecoli SRP (ülbraidt et al., 1997). These findings provideci very strong
evidence for the existence of a co-translational translocation mechsnism in E.c& anci
other proiraryotic cells. in purple photosynthetic bacteria, it is not clear whether this
mechanism is used for the membrane targeting of the subunits L and M of RC. But
sequence anaîysis of their N terminal regions provides a due discussed below.
The RC L and M subunits ate very similar in both sequence and three dimeasional
structure. Theu structurai gents are located within the same puf opeton and are separated
by oniy 12-13 bases (in Rb. cupsuIut11~they even overlap by 5 bases). Such gene
o r g h t i o n is considered to favor the uniOadiLIg-reloading translation pattern for
polycistmnic mRNA. In contrast to an extreme c o d o n in the N-teminal region of
L subunit, intcnstingly, the cocfesponding region of
the
M subunit is much less
conserveci. (Figure 31). These facts wggest that membrane locabtion of the L and M
subunits could use a CO-translationaltranslocation mechanism. To fit this mechanism, the
non-polar cluster (iicluding the involvement of polar or charged residues) in N-terminal
region of bit L polypeptide may W o n as the signai for SRP recognition to bring the
nascent L polypeptide chah ont0 the l d o n site on tbe membrane. 'Ibe following
translation event will continue on the membrane. Due to the short intergenk sequence
between pufL and puw, the ribosome at the end of the translation of the L polypeptide
will seat on the Shine-Dalgamo sequence of puyMc The same ribosome that has been
membrane localid will start the translation o f p u t without hpping off the tenninator
of p@.
in this hypothetid mode!, M subunit does not necessarily have to contain a
SRP recognition for membrane targeting.
The studies in yeast iadicate, in addition to the hydrophobie core, the
conformation of the signal sequence might be also important for SRP recognition (Bird et
al., 1990). This view may also be true for the N-segment of L subunit. There is a U-tum
structure ia N-tennulal region of L subunit preceding the tirst transmembrane helix. The
four amino acid residues (L23,25, 28 and 30) are located within this structure. The
suppressive effact in the quadruple mutations to recover the L subunit and RC contents in
the two parental double mutants apparently indicates an i n t e d o n among these residues
that is important for membrane targeting of L subunit, a d o r for the assembly of
fiinctional RC.
Chapter VU
Conclusions
In this Ph.D. thesis research work, several aspects of the reaction center in purple
photosynthetic bacterial have been investigated. This work provides some important
information to this research field.
Sequence analysis indicates that the puf operon of Ect. shuposhnikovii, a sulFur
purple bacteriurn. contains five basic pufgenes, puf B,A,L.M and C. These genes are
similar to hosr in nonsuifiur purpir bacteria, in both sequence and organization.
The comparative studies on the other ORFs or genes in puf operons can be an
alternative way to look at the phylogenetic relationship among these purple
photosynthetic bacteria The analysis of ORFQ' and its homologs suggests the occurrence
of latenl tmnsfer of photosynthesis genes between the ancestors of Rs. nrbrum and Ect.
shaposhnikovii or Ru. gelarinosus.
The stnitegy used in constructing the Be site mutation in the Ect. shuposhniikovii
RC is a new approach in mutagenesis study that could be of gened application to study
the hction of other indispensable genes.
The pmperty of the cofactor ai the Be site is important for RC hction. A hi&
redox potential at this site seems an essential requirement for a hctionally escient RC.
The 790 nrn band in the M 1 8 0 ~RC
' ~ absorption spectrurn is o blue-shified high energy
band h m 800 nm in the wild type RC. This demonstrates the interaction between Be
and the special pair and indicates that Be could be the third component contributhg to
high energy band. This study will stimulate further investigations on the d e s of the B
side prosthetic groups in RC fuilction, especially how the cofactor at the Be site interacts
with the special pair to influence the properties of the special pair and contribute to the
unidirectional and efficient electron transfer in native RCs,
Rs. mbmm puf deletion mutation derived fiom the wild type strain can be in
tram complemented. It is not necessary to cemove 1 kb kgment dowmtream ofpufM in
the pifdeletion mutation for a successf'ul complementation as pmiously reported.
N-terminai region of the L subunit of RC is essential for photosynthesis. Most of
the conserveci residues in this region are important for membrane integration of the L
polypeptide. An acidic amino acid residue (L6E)seems to be iavolved in a ncognition
process that could be crucial for the targeting of the L polypeptide to the cytoplesmic
membrane. Four interactive residues (L23D.L2SW, L28P and L30Y) play crucial roles
seemingly in a recognition process for membrane targeting of the L polypeptide. This
work has laid a basis for M e r studies on the membrane assembly OP the L and M
subunits.
The H subunit of RC and the G115 product have àifferent f'unction, the fppner is
necessary for RC assembly or stability but more important for its proper huiction, while
the latter is involved in the assembly of the B880 cornplex.
Refemnces
Alberti, M., Burke, D.H.and Hearst, J.E. (1995) In Anoxygenic Photosynthetic bacteria
Eds. Blaakenship, RE., Madigan, M. and Bauer, C.E. Amsterdam: Kluwer Academic
Publishing. Pp. 1083-106.
Allen JP, Feher O, Yeates TO, Komiya H and Rees DE (1987) P m . Natl. Acad. Sci.
U.S.A. 84: 5730-34.
Arlt T, Dohse B, Schmidt S, Wachtveitl J, Laussennair E, Zinth W and Oesterhelt D
(1996) Biochemistry 35: 923594.
ArlfT. Schimidt, S., Kaiser, W., Lautemser, C., Meyer, M., Scheer, H. and Zinth, W.
(1993) P m . Natl. Acad. Sci. USA 90: 11757-61.
Bauer, C.E., Young, D.A. and Marrs, B.L. (1988) J. Biol. Chem. 263:4820.
Bauer, C. E., and B. L. M m . (1988) Proc. Nad. Acaâ. Sci. U.S.A. 85:7074-78.
Bauer, C.E., Buggy, Ill., Yang, 2.and Marrs, B.L. (1991) 228:433-44.
k t t y , J.T. (1995) in Anoxygenic Photosynthetic bacteria Eds. Blankeaship, RE., Madigan, M.
and Bauer, C.E.Amsterdam: Kluwer Academic Publishing. Pp. 1209-19.
Bélanger, 0. and G. Gingras. (1988) 1. Biol. Chem.263:7639-45.
Bélanger, G.,J. Bérarâ, P. Corriveau, and G. Gingras. (1988) 1. Biol. Chem. 263:7632-8.
Bélanger, 0. and Gingras, G. (1997) CienBank Access No. AFO 18954.
Belasco, J. O., J. T.Beatty, C. W. Adams, A. von Gabain, and S. N. Cohen.
(1985) Ce11 4&171-81.
Bérarâ, L,O. Bélanger, P. Comveau, and 0.Gingras. 1986. J. Biol. Chem.261:82-7.
Bérard, J., 0.and Ghgras, G. J. Biol. Chem. (1989) 264: 10897-903.
Bérard, l.and Gingras, G. (1990) Biochem. Ce11 Biol. 69:122-3 1.
Bernstein, H. D., M. A. Pori@ K. Smb, P. J. Hoben, S. Bremer, and P. Walter. (1989)
Nature 340:4824.
Bird, P,Gething, M.
J O y
and Sambrook, J. (1990) J. Biol. Chem.265:8420-25.
Blankenship RE, Feick R, Bnice BD, Kirmaier C, Holten D and Fuller RC (1983) J.
Cellular Biochem. 22(4): 25 1-6 1.
fhrmann, B. J., and D.M.Engelman. (1992) A m . Rev. Biophys. Biomol. Struct.
2 13223-42.
Bognar A, Libman DM and Newman EB (1982) J. Bacteriol. 12: 706-13.
Bninisholz, R. A., V. Wiemken, F. Suter, R. Bachofea, and H. Zuber. (1984) Hoppe
Seyler's 2.Physiol. Chem.365689-70 1.
Brunisholz, R A., and H. Zuber. (1992) J. Photochem. Photobiol. B: Biol. 15:1 13-40.
Buggy, JJ. and Bauer, C.E.(1995) 1. Bactenol. 3 7736958-65.
Buggy,J.J. Sganga, M. W. Bauer, CE. (1994) J. Bacteriol. l75:6 1-9.
Bylina, E. J. and Youvan, D. C.(1987) 2.Nahuforsch. Teil C 42:769-774.
Bylina, E. J., Kinnaier, C., McDowell, L., Holten, D. and Youvm, D. C. (1988)
Nature 336: 182-4.
Bylina, E. J., Ismail, R V. M. and Youvan, D. C. (1989) BioiTecbnology 7:69-74.
Bylina, E.J., kolaczkowski, S.V., Noms, J.R. and Youvan D.C. (1990) Biocheniistry
Chang CH, Tiede 4 Tang J, Smith U,Noms SR and Schiffer M (1986) FEBS Lett. 205:
82-6.
Chen, C. Y., L T.&a~y,S. N.Cohen, and J. G.Belasco. (1988) Ce11 52:6û9-19.
Clayton, R K (1978) Biochim. Biophys. Acîa 504:255-64.
Coleman, W. J., Byliaa, E. L and Youvan, D. C. (1990) in Current Research in Photosynthesis,
(Maltscheffsicy, M. Ed.) pp. 149-52. Dordrecht: Kiuwer.
Davis, J., Donohue, TJ. and Kaplan, S. (1988) J. Bacteriol. 1îO:3îO.
Debus, RJ., Feher, 0.and Okamum, M.Y. (1986) Biochemistry 25:227687.
De Gier, J. W., P. Mansoumia, Q.A. Vaient, O. J. Phillips, J. Luirink, and G. Von Heijne.
(1996)FEBS Letters 399(3):307-9.
üeisenhofer J, Epp O, Miki K, Huber R and Michel H (1985) N a m 318: 618-24.
Deisenhofer, J. and Michel, H. (1989) EMBO J.8:2149-70.
Deisenhofer, J. and Michel, H. (1991) Annu. Rev. Ce11 Biol. 79-23.
Demgon, J. M., P. Comveau, and G. Gingras. (1990) Plasmid 23,225-36.
Ditta, G., T. Schmidhauser, E. Yacobson, P. Lu, X-W. Liang, D. Finlay, D. Guiney, and
D.R Helinski. (1985) Plkmid 13:149-53.
Donohue, T.J.,Hoger, J.H. and Kaplan, S. (1986) J. Bacteriol. 16895361.
Du, S., Bird, T.H.and Bauer, C.E.(1998) J. Biol. Chem. 273:18509-13
Eraso, J. and Kaplan, S. (1994) J. Bacteriol. 176:32-43.
Errnier, U., Fritzsch, G., Buchanan,S.K.and Michel, H. (1994) Stnicture 2:925-36.
Fajer, Je,Brune, D., Davis, M.S.,Fonnan, A. and Spaulding, L.D.(1975) Roc. NA.
Acad. Sci. USA 72,4956-60.
Farchaus, J.W., Wachtveitl, J., Mathis, P. and Oesterhelt, D. (1993) Biochemistry
32:10885-93.
Fathir, I., K.Ta&
K. Yoza, A. Kojima, M. Kobayashi, 2.Y. Wang, F. Lottspeicj, and
T.Nozawa. 1997. Photosynthesis Research. 51:7 1-82.
Feher, G., Ailen, J. P. Okamwa, M. Y.and Lubitz, W. (1989) Nature 339: 111-16.
Fidai, S., S. B. Hinchigeri, T.J. Borgfioad, and W. R Richards. (1994)
Je
Bacteriol. 17637244-51.
Fidai, S., I. A. Dahl, and W. R Richards. (1995) FEBS Leâters. 372(23):264-8.
and Zînth, W. (1990)Biochemistry 29:8S17-21.
FinkeIe, U., Leutenvaser, CC.
Ful<ushime, A., Matsu-
IL,Shimada, K. and Satoh, T.(1988) Biochim. Biophys. Acta
93:399405.
Goldsmith, J. O., King, B. and Boxer, S. 0.(1996) Biochemistry 35:2421-2428.
Gomelsky, M. and Kaplan, S. (1995) Miaobiology Ml :1805-19.
a
Gomelsky, M. and Kaplan, S. (1995) J. Bacteriol. 177:4609-18.
Gomelsky, M. and Kaplan, S. (1997) 3. Bacteriol. l79:128-34.
Gray, KA., Farchaus, J.W., Wachhreitl, J., Bieton, J. and Oestethelt, D. (1990) EMBO J.
9:2W 1-70,
Hearst et al., EMBO accession numbet Z 11166.
Heller, B.A., Holten, D.and Kirmaier, C. (1995) Science 269:940-5.
Heller, B.A., Holten, D. and Kimiaier, C. (1995) Biochanistry 34:5294.
Hessner, M. J., P. J. Wejksnora, and M. L. Collins. (1991) J. Bacteriol. 1735712-22
Higuchi, R (1990) Recombinant PCR p. 177-183. In Innis, M. A.; D.H. Gelfand; J. J.
Sninsky and T. J. White (eds.), PCR protocols: A guide to methods and applications.
Academic Press, San Diego
Holtmarth, A.R. and Muller M.G. (1996) Biochemistry 35:11820-3 1.
Huber, H., Meyer, M., Nagele, T., Hartl, I., Scheer, H., Zinth, W. and Wachtveitl, J.
(1 995) Chem. Phys. l97:297-306.
imhoff, I. F. (1984) ïnt. I. Systematic Bacteriol. 34,338-9.
Imhoff, J. F., and J. Suling. (1 996) Arch. of Micfobiology l65(2): 106-13
houe, K., Kouadio, J K , Mosley, C.S. and Bauer, C.E.(1 995) Biocheinistry 34:39 1-6.
Kiley, P. J., Donohue, T. J., Havelka, W. A. aiid Kaplan, S. (1987) J. Bactenol. 169~74250.
Kimiaier, C., Holten, D., and Parson,W.W. (1985) Biochim. Biophys. Acta 8 10: 49-61
Kirmaier, C., Gad, D., DeBey, R, Holten, D. and Scbeck, C. C. (1991) Science 251:922
L a e d , U. K. (1970) Nature 227: 680-5.
LeBlanc, H.N. and Begny J.T. (1993) J. Gen. Micmbiol. 139: 101-9.
LeBlanc, H.N.and Beaîty J.T. (L996)J. Bacteriol. 178: 480 1-6.
Lee., MC.,Dehoff, B.S., Donohue, TJ., Gurnport, RI. and Kaplan, S. (1989) J. Biol. Chem.
264: 19354-65,
Lee, I.Y.and Collins, M.LR (1993) Cumnt Microbiology 27:85-90.
Lefebvre, S, Picorel, R, Cloutier, Y. and Gingras, G. (1984) Biochemistry 23: 5379-88.
Lefebvre S, Picorel, R and Gingras G (1989) FEMS Micmbiol. Letters 65: 247-52.
Liebeîanz, EL,Homberger, U.and Dnws. G. (1991) Mol. Microbiol. 5:145968
Ma, D.,Cook, DN.,O'Brien, DA.and Hearst, J.E. (1993) 1. k t e n o l . 175:2037-45.
McDowell, L. M., Gad, D., Khaier, C., Holten, D.and Scheck, C.C. (1991)
Biochemistry 30:83 15-8322.
Macf~lane,J., and Muller, M. (1 995) Eur. J. Biochem. 233(3):7667 1.
Mar, T.,Vadeboncoeur, C. and Gingras, G. (1983) Biochim. Biophys. Acta 724:3 17-22.
Mar, T. and Gingras, 0. (1990) Biochm. Biophys. Acta 1017: 112-7.
Mar, T-and Gingras G (1 991) Biochim. Biophys. Acta 1056: 190-4.
Mar,T., Picorel, R. and O. Gingras. (1993) Biochemistry 32: 1466-70.
Mar,Teand Gingras, G. (1995) Biochemistry 34: 907 1-8.
Matteson, R. J., S. J. Biswas, and D. A Steege. (1991) Nucleic Acids Res. l9:3499-506.
Matsuura, K., and K. Shimnda. (1990) In M.Baltschewsky (Ed.) Current Research in
Photosynthesis. Vol. 1. P. 193-1%. Kluwer Acaâemic Publishers ,Netherlands
Matsuura, K.,Fukushima, A., Sbimida, K and Satoh, T. (1988) FEBS lett. 237:21-5.
McPhemn, P. H., Okarnura, M. Y. and Fehet, 0. (1990) Biochim. Biophys. Acta 1016:289-292.
Michel, H., K. A. Weyer, HeGruenberg, 1. Dunget, D. Oesterhelt, and FeLottspeich. (1986)
EMBO J. S:l149-S8e
Mosley, C.S., Suzuki, J.Y.and Bauer, C.E. (1994) J. Bacteriol. 176:7566-73.
Nabedryâ, E., Breton, J., Okamurq M.Y. and Pddock, M.L.(1998) Biochemistry 37:
14457-62
Nagashna, K. V. P., Matsuma, K., Ohyama, S. and Sh2699477-84,
K. (1994) 1. Biol. Chem.
i)
Nagashirna, K.V. P., Shimada. K. and Matsuura, K. (1996) FEBS Letters 385:209-13
Nilsson, I., and G. Von Heijne. (1990) Ce11 62:1135-41.
Ochman, H., Medhora, M. Mo, Gama,D. and Hartl, D. L. (1990) Amplification of
flanking sequence by inverse PCR, p. 219-27. In (InnisM. A.; D.H.Gelfaad; I. J.
Sninsky and TJ.White (eds.), PCR protocol :A Guide to Methods and Applications1990. Academic Press,San Diego
Ovchinnikov Yu& Abdulaev, N.G., Zolotarev, AS., Shmuktet, B.E., Zzargmv, A.A.,
Kutuzov, M.A., Telezhinskaya, I.N. and Levina, N.B.(1 988) FEBS lett. 23 1:237-42.
Paddock, M. L., Feher, G., Okamura, M. Y. (1990) Biophys. J. 57569a.
Paddock, M. L., Feher, G., Okamura, M. Y. (1995) Biochemistry 34:1574î-l57SO.
Paddock, M. L., Rongey, S. H., Feher, O., Okamura, M. Y. (1989) Pmc. Natl. Acad. Sci.
U.S.A. 86:6602-6606.
Paianiappan, V.,P.C.Martin, V. Chynwat,
Am. Chem,Soc. 115: 12035-49.
H.A. Frank, and D. F. Bocian. (1993) J.
Parson, W.W.,Chu, Z.T.and Warshel, A. (1990) Biochun. Biophys. Acta 1012: 25 1
Parson W.W.and Warshel, A. J. (1987) J. Am. Chem. Soc. 109:615263.
Peafold, R J. aad Pemberton, J.M. (1994) J. Bacteriol. 176:2869-76.
Picorel, R., S. Lefebvre, and O. Gingras. (1984) Eur. J. Biochem. l42:3OS-ll.
Poritz, M. A., K. Stmb, and P. Walter. (1988) Cell55(1):4-6.
Ponnampaiam, S.N.,Buggy, JJ. and Bauer, C.E. (1995) J. Bacteriol. 177:299O-î.
Ponnampalam, S. and Bauer, C.E.(1997) 1. Biol. Chem. 272: 18391-6.
Quandt, J. and Hynes, M. F. (1993) Gene 127:15-2 1.
Rauttcr, J., Lendzian, F., Schulz, C,, Fetsch, A., Kuhn, M., Lin, X., Williams, J.C., Allen,
J.P. and Lubitz, W. (1995) Biochernistry 34: 8130-43.
RoMes, S. J., Breton, J. and Yowan, D.C. (1990) Science 248: 1402-5.
Romisch, K., Webb, J., Hen, J., Rehn, S., Frank, R,Vingron, M.and Dobbusteh, B.
(1989) Naîure 340:478-82.
Rongey, S. H., Paddock, M. L., Mcphemn, P. H., Feher, O. and OLamura, M. Y. (1991)
Biophys. J.59:142.
Sambrook J, Fritsch EF and Maniahis T (1989) Molecular cloning: a laboratory manual,
2nd ed Cold S p ~ Harbor
g
Laboratory, Cold Spring Harbor, N.Y.
Scheck, C.C.,Gaul, D., Steffkn, M., Boxer, S. G., McDowell, L., Kimiaier, C. and
Holten, D. (1990) in Reaction Center of Photosynthetic Bacteria (Michel-Ekyerle, M. E.,
Ed.) pp 229-239, Springer-Verlag, Berlin Heidelberg.
Sganga, M.W. and Bauer, CI.(1992) Ce11 68:945-54.
Simon, R., U. Pnefer, and A. Puhler. (1983) Bio/Technology 1:37-45.
Sockett, RE.,Donohue, T.J. Varga, A.R and Kaplan, S. (1989) L Bacteriol. 171:436-46.
Stocker, J.W., Taguchi A.K.W.,Murchison, H.A., Woodbury, N. W. and Boxer, S.G.
(1992) Biochemistry 31: 10356-62.
Stowell, M.H.B.,McPhillips, T.M.,Rees, D.C, Soltis, S.M.,Abresch, E. and Feher, G.
(1997) Science 276~812-16.
Taguchi A.K.W., Stocker, J. W., Alden, R G., Causgrove, T.P.,Peloquh, LM.,Boxer,
S.G.and Woodbury, N. W. (1 992) Biochemistry 3 1:10345-55.
Taguchi A.K.W.,Eastman,LE., Gallo, D.M.,Sheagley, E., Xiao, W and Woodbury, N.
W. (1996) Biochemistry 35: 3175-86.
Takahashi, E. and Wraight, C.A. (1996) Roc. Natl. Acad. Sci. USA. 93:2640-5.
Ulbraadt, N. D., J. A. Newitt, and H. D. Bernstein. (1997) Cell88(2):187-96.
Valent, Q.A., I. W. L. De Gier, G. Von Heijne, D. A. Kendall, C. M. Tenôagenjongman,
B. Oudega, and J. LuUinL. (1997) Mol. Micmbiol. 25(1):534.
Van Bredemde, M.E.,Jones M.R, Van Mourik, F., Van stokhun, I.H.M.and Van
Grondelle, R (1997) Biochemistry 36:68SS-6 1.
van der Rest M and Gingras G (1974). 1. Biol. Chem. 249: 6446-53.
Varga, A.R and Kaplan, S. (1993) JeBiol. Chem.268,19842-50.
Vaanel, H, R F. Meiburg, E. I. M. Krsmer, L. L Vos, and L Amm, (1983)
Biochim. Biophys. Acta 724:333-9.
Vasmel, H. (1986) The photosynthekic membrane of green bacteria Ph.D. thesis, The
State University, RA leiden, The Netherlands.
Vieze, J. Williams, J. C. Allen, J. P. and Hoff,A. J. (19%) Biochim. Biophys. Acta
1276: 221-28
Von Heijne, G. (1986) EMBO J o d 5(1l):3021-7.
Wachtveitl, J. (1992) PkD. Thesis, Uaiversity of Munich, Munich, Gennany.
Wachtveitl, J., Farchaus, J. W., Mathis, P. and Oesterhelt, D. (1993) Biochemisüy 32:1089410904.
Weyer, K. A., F. Lottspeich, H. Gruenberg, F. Lang, D. Oesterhelt, and H.
Michel. (1987a) EMBO L6:2 197-202.
Weyer, K. A., W. Shaffer, F. httspeich, and H. Michel. (198%) Biochemistry 26:2909-14.
Wiessner, C., 1. Dunger, and H. Michel. 1990.1. Bacteriol. 172:2877-87.
Williams, J. C., L. A. Steiner, R. C. Ogden, M. 1. Simon, and G. Feher. (1983). Proc. Nat.
Acad. Sci. U.S.A. 80:6505-9.
Williams, J. C., L. A. Steiner, G. Feher, and M. 1. Simon. (1984) Proc. Nat. Acad. Sci.
U.S.A. 8 1(23):7303-7.
Woese, C.R 1987. Bacterial Evolution. Microbiol. Rev. 51221-7 1.
Wong, D.K.H., CoLiins, WJ., Harmer, A., Lilbum, T-G. and Beatty, J.T.(19%) J.
Bacteriol. 178,2334-2.
Yil&, F.H.,Gest, H. and Bauer, C.E. (1992) Mol. Microbiol. 6:2683-9 1.
Young, C.S., Reyes, RC. and Beatty, J.T.(1998) J. Bacteriol. l8O:lîS9-65.
Young, C.S. and Beatty,J.T.(1998) J. Bacteriol. 18O:4742-5.
Yowan, D.C., E. J. Byüna, M. Alberti, H. Begush, and J. E. Hearst.1984. Cell37949-57.
Zhang W,R
-eadn
ig
C and Deissemth RB (1992) Trends in Genetics 8 (10): 332.
e
Appendix
Materials and methods
Genenl materiah and methods
Bacterial itmins, gmwtl conditionr
E.coli strains Top10 (Invitmgen) and XL1-blue (Stmtagene) were mutinely
used as cloaing hosts E. coli strein S17-1 was used as the donor in conjugation
experiments to deliver the mobilizable plasmid DNA into meptor photosynthetic
bacterhi strain. E.coli strains wen culhired in LB medium at 37OC with appropriate
antibiotics. Ect shaposhnikovii ATCC 31751 Smr Hgr(Demgon et al., 1990) and its
mutants wece grown anaerobically in the light at 37 'C in the medium described by
Bognar et al. (1982). When appropriate, Ect. shpshnikovii cells were grown at 37 O C
on solid medium containing 1.5 % Difco agar in a GasPak anaerobic chamber (BBL
Micmbiology Systems, Cockeysville, Md.) at 50 cm h m a 150-Watt photoflood lamp.
Rhodospillumm rubrwn strains were culairrd in Cohen-Bazire (CB) medium (Cohen-
BaPR et al., 1975) at 30°C for photosynthetic gmwth or in nch CB medium (CB
medium supplemented with 0.5% tryptoae and 0.5% yeast extnict) for micmaerobicai
growth in the dark by using Gas Pack micmaerobic chamber.
Moleculir cioning techniques
Standard techniques were used for plasmid isolation, restriction endonuclease
digestion, ligation, transformation, and hybrihtion (Shamb m k et al., 1989).
Restriction endonucleases were usually pwhased h m Pbarmacia Biotech. The Erasea-Base System h m Promega Corp. (Madison, W[) was employed to generate nested
deletions in the insnts. Sequencing was done on the double-strandd plasmid DNA
using the l7 DNA Sequencing Kit, or, when necessary, the T7 Deaza DNA Sequencing
iii
Kit (Pharmacia Biotech), following the aan&turerys instructions. 3 S ~ or
a'%d
- ~ ~ ~
adCTP was fiom Amershem and 32~ud~TP
was h m ICN.Large d e pnparation
of Ect. shapshnikovii and Rr. rubmm chromosod DNA foliowed thq method as
describeci by Bérard et al. (1986). The minipnparation of chromosod DNA followed
the method of Zhang et al. (1992). but omitting the use of lysozyme and proteinase K.
PCR was conducted in 100 pl solution containjng 100 ng chromosomal DNA or 10 ng
plasmid DNA, 0.1 m g / d bovine senun albumin, 200 pmole of each primer, 20 p o l e
of each âNTP, 1 x DNA polymerase buffer and 5 Units of Taq DNA polymerase
(Pharmacia Biotech) or Vent DNA polymerase (Bio lab, New England). The PCR
products were cloned Uito vector pGEM-T (Promega Corp. Madison WI.).
Plasmids
Plasmids pUC19 (Phamtacia Biotoch) and pBlueScript (Stratagene) were
mutinely used as clonhg vectors. Suicide plasmid pJQ200-SK+ ,a gift h m Dt. Hynes (
Quandt and Hynes, 1993), was wd to carry the mutated gene for gene replacement in
photosynthetic bacteria The structure of this plasmid is show Figun 32. pRK404, a
wild-range-host plasmid (Diîîa et d . 1985) used to carry the gene to transfonn
photosynthetic bacteria, bas the structure show in Figure 33.
Figure 32. Structure of suicide plasmid plQ-200-SK. MCS. multiple cloning site;
Plac, lac promoter, oriV, replication ongin element; oci T, onginai site of gene e a ~ f e r
(mobilization); trd, encodes a product necessary for gene mobilhtion; sacB. denved
h m B. subtilis. Its high expression induced by sucrose is toxic'toa wide range of Oram
negative bacteria, therefore used as a market to select gene replacement event h m the
insertion mutation; Gmt, gentomicin resistance gene used to furthex- conficm the gene
replacement event*
Figure 33. Structure of pRK4W.
TC^, tetrecycline resistance gene. The definitions
for other symbols are the same as described for pJQ-200SK plasmid.
Sequence andysb
Sequence anaiysis wss perfomed using the program in GCG package (Genetic
Cornputer Gtoup, Wisconsin,version 8.0).
Bacterial genetic manipuhtion
Plasmid DNA was mobilizcd into Rs. mbrum through diparental conjugation
between E. d i S17-1 as donor and RF. mbrum as feceptor by usbg a nIter matiag
method as described by Davis et al. (1988). The gene replacement mutants were selected
in the dark on CB cich medium containhg 5% sucrose and 50 pglml kaaamycin.
Plasrnid DNA was introduced into Ect. shapshnikovii by electroporation on E. coli
pulser (Bio-Rad). A potential field of 10 kvlcm was applied with a t 01 4.5 m. The
selection of the gene replacement mutant was p e r f o d in the light on the medium with
5% sucrose and 50 pg/mi kanmycin.
P n p i d o n of chmmatophore
Chromatophores were pre@
follow the method described by Van der Rest
and Gingras (1974). The cells were harvested by centrifuge and the ce11 pellet was
resuspended in 50 m M Tns-Cl, pH 8.0. Cells were broken by sonication and the crude
chmatophoes weie first collected in the supernatant d e r centrifugation at 15,000 rpm
(Sorvall 34 rotor) for 10 minutes. Then chmmatophore was pelletecl by
untracentrifiigation at 33,000 rpm (Backrnan 4 5 4 rotor or 40 rotor) for 90 minutes. The
chmatophore pellet was washed once ami tinally nsuspended in the same Tris buffer.
vii
Purification o f reaction center
The photoreaction center of Ect. Shaposhnikovii was isolated and purified
according to the method of Lefebvre et al. (1984). The cells were cultureci in Ibliter
cylindrical botties (around 35 cm in diameter) cornpletely filled with medium and
stoppered with rubber stoppers. Illumination was provided by t h e 150-W photoflood
lamps at JO cm From the center of the culture bonies. Two lamps were used for the fm
16 h-20 h aAer inoculation. and the third lamp was tumed on for the rest of the growth
period. AAer three days (wild type) or seven days of culture, the cells were harvested by
centrifugation for 20 minutes at 4,200 rpm (Sorvall H G 4 rotor). The bacteria were
ground with 1.5-2 times their weight of levigated alumina (Fisher Scientific Co.) with a
mortar and pestle for around one half hour. Then the bacteria were resuspended in
distilled water and centrifuged at 5,000 rpm (Sorvall GSA rotor) for 20 minutes to
remove most of the alumina and ce11 debris. The supematant was centrifùged at 15,000
rpm (Sorvall SS-34 rotor) For 20 minutes to remove the rest of the alumina and ce11
debris. The supematant fiom this second centrifugation was then subjected to
ultracentrifugation at 33,000 rpm for 90 minutes. The pellet was homopnized in
distilled water and recentrifuged under the sarne conditions as the first one. The new
pellet was resuspended in distilled water to a fmd Asa of 75.
The following chromatography was performed at 4 O C , in darkness if possible.
To the chromatophore suspension was added an equal volume of 100 rnM phosphate
b a e r (pH 7.5) containhg 0.9% (wh) lauryldimethylamine N-oxide (LDAO), after
testing different concentrations of this detergent. This mixture was incubated at 40 O C
for 1 hour in âarkness, with occasional stimng to solubilize the RC h m
viii
for 1 hour in darkness, with occasional stimng to solubilize the RC h m
chromatophore. Solubilization was stopped by dilution with a suficient volume of cold
50 m M phosphate (pH 7.5) to brhg the final detergent concentration to 0.1%. The
solution was centnfuged for 2 hours at 40,000 rpm (Beckrnan 45-Ti rotor). To the
supematant was added ammonium sulfate to 226 g/L, (final concentration). The
pwcipitair: w u rrmoved by centrifugation at 10,000 rpm for 30 minutes (Sorvdl GSA
rotor). To the supematant, more ammonium sulfate was added to a fud concentration of
305 g/L,,and the solution was centrifuged as above. The precipitate was collected by
filtration on Whatman No. 1 filter paper in a Buchner funnel. The precipitate was
resuspended in 50 mM Tris-Cl buffer (pH 8.0) and didyzed ovemight against the same
buffer containing 0.1 % Triton X-100.The dispersion was centrifuged for 20 minutes at
10.000 rpm and filtered as above to remove any particulste material. The sarnple was
then applied to a two-third filled 1.6 i< 30 cm DM-Sephacyl column equilibrated with
10 mM Tris-Cl (pH &O), containing 0.1% (w/v) Triton X-100. AAer loaâing, the
column was washed with 50 mi of the same b a e r and then washed with 150 ml of the
same buff'er containing 120 mM NaCl until the elutant was colorless. The RC was then
eluted with the sarne buffer containing 175 m M NaCl. For the wild type RC, the elutant
was dialyzed against 50 m M Tris-Cl (pH8.O) containing 0.1% triton X-100. For the
mutant RC,due to its low arnount and low concentration, the elutant did not go through
didysis to avoid any m e r loss, but underwent concentration by using Centriprep
(Amicon inc.). Mer concentration, the sarnple was supplemented with 1M Tris-Cl@H
8.0) to a h a l concentration to 50 mM.
The pigments fiam the purified RC were exttacted and analyzed as described by
van der Rest and Gingras (1974). The RC samples (2 ml of samples with hoof amund
0.2) were h t lyophilizeâ and then extracted thm times with 0.2 ml each of
acetone/methanol (7:2) solution. The thne extracts were combined and the absorption
spectra were taken. B a d on the spectra, the amounts of &hi and Bph in the extracts
were calculatecl according to the equations developed by van der Rest and Gingras
(1 974).
Spectroscopie analysis
Absorption spectra and light-induced absorption change were recorded with a
Cary 2300 spectrophotometer at room temperature in 1c m path-length ce11 as described
in Mar and Gingras (1 99 1). The chrornatophore samples received the actinic light of an
Oriel 250W tungsten illuminator filtered by a Baird Atomic broadband interference filter
centered at 930nm. Appropriate light filters pmtected the photo-detector.
Proteicli u u y
Protein concentration was detedned by using the melhod of Bradford (1974),
with bovine s e m albumin as standard. Analytic sodium ddecyl sulfate
polyrcrylarnide gel electrophoresis (SDS-PAGE)was conducteâ by h
g the methoci of
Laemmli (1970), on EC175 system (E-C Apparatus Corporation, St. Peterbwg, Florida).
The pmtein samples were precipitated by 5% ûichloroacetic acid, and the pellets were
dissolved in lx SDS loading bu&r (62.5 mM Tris-CI (pH6.8) containhg 7% sumwe,
2% SDS and 4% 2-mercaptoetnanol) and incubated at 37 O C for 2 hom before king
loaded ont0 the gel. After king stajned with Coo~issieBrilliant BLue, the proteins
bands were quantined by scanning on Ulûascan XL Enhanced LasCr Densitometer
(LKB,Brornma).
Special techniques ia each cbapter
Cbapter II
Inverse PCR to amplify the 16s rRNA gene ia Ect. shaposhnikovii
The 16s rRNA gene of Ect. shaposhnikovii was cloned by PCR method. Two runs of
PCR were perfomed to ampli@ the fragments covering whole gene as illustrated in Figure
34. The primen used in the fmt run of PCR were derived fiom the regions that are located
close to two ends of 16s rRNA gene and completely conserved as observed in 1 I species of
several families. The sequences of the two primers are as follows,
P l : 5'-
GAGTTTGATCCTGGCTCAGA-3'; P2 : 5'-ACTTCTGGTGCAGCCGACTC-3'.The k t
nin PCR
produced an 1.4 kb fragment covering most of the 16s rRNA gene, and left two
small end regions unknown. The inverse PCR strategy (Ochrnan et al., 1990) was used to
clone the two end regions. The primers used in the inverse PCR were derived fiom the
product of the fint run PCR and have the sequence shown here,
P3 : 5'-
GAAGGCCTG'ITACCGTTCGACTTG-3'; P4 : 5'-CATTGCTGCGGTGAATACGTT-3'.
These two primers are located near the two ends of the fi-agment with divergent direction. The
template for inverse PCR was prepared as described below. First, the Ect. shaposhnikovii
chromosome DNA was completely digested independently with several restriction enzymes
that do not cut the frrigment of the first run PCR, for example, EcoRI, BglII and BamHI. The
digested products undenvent self-ligation and then served as the template for the PCR
amplification. From BglII treated template, PCR produced a fkgment of 8 5 0 - 9 0 bp. Afier
cloning this fragment to PCR cloning vector, sequencing was conducted to obtain the
sequences of the two ends of 16s rRNA gene of Ect. shaposhnikovii.
xii
Step 1.
Pl
+
16s rRNA mat
chromosome DNA
-
Step 2.
+
+ ~ d l ~
chromosome DNA
Figure jJ. 165 rRNA gent w u amplukd by PCR method inciudiig one nu of invcm
PCR (see test for exphnation).
xiii
Cbapter III
Construction of CAT +CCT mutation on chromosomal DNA
The sequence of the oligonucleotide used for mutation on Ml 80 was as following:
5' GGTATCTT'CCCGCTTC'ITGACTGG-3'.The mutagenesis was conducted on
plasmid pZS-puf70 (see Chapter 11 for the information on this plasmid). Mutagenesis
was carrird out by usinp U.S.E. Mutigenesis f i t (Pharmacia Biotech) foliowing the
manufacture's instructions. The mutation was confirmed by sequencing. The strategies
employed to bnng the mutation to the chromosome are schematized in Figure 10. The
transformants that expenenced gene replacement including both Kmrinsertion and M 180
(CATJ CCT) mutation were selected on growth medium containing 5% sucrose and 25
p g h l Km. The mutation (CAT+CCT) was identified by sequencing the PCR hgment
amplified from the chromosomal DN A of individual colonies.
The Ect. shaposhnik~viichromosomal DNA from single colony used as template
for PCR was a mini preparation made principally by the method of Zhang et uL (1 992),
but omitting the use of lysozyme and proteinase K. The DNA was finaily dissolved in 15
pl TE b a e r and directly used for PCR. The two primen employed to ampli@ the 400 bp
fmgment containing the Ml80 codon have the following sequences: Pl, 5'-
CAATCTTCCTGTATCTGAGCCTTG-3'; P2, 5'-GACTACCGTGCCAGTGAGCAG
C AGAATG-3'.
xiv
Cbapter IV
Recombinant PCR for Mutagenesis
Recombinant PCR method (Higuchi, 1990) was used to introduce mutations into
the N-terminal region of the L polypeptide as illustrated in Figure 35.
To a specific point, it can be changed to several other bases or kept unchanged by
using degenerate primer. As illustnited in Figure 34, more than one mutation at different
positions cm be introduced in a single mutant, either using one primer having more than
one mutation or combining the different mutations fiom two primea. By this strategy, a
great number of mutations can be introduced into the interesting regions with a
reasonable number of primers. In manipulation, two PCR products were First amplified
by using two pairs of primen ( P M 3 and P2-P4). Part of P3 and P4 share sequence
homology. AAer purification, these two PCR products were then put together and
underwent denature and annealing. mer tbat, five cycle PCR was run to elongatc the
annealed partial duplicates without involving P l and P2 primers. When the elongation
reaction was done, Pl and P2 primers were added to the reaction mixture and another 25
cycle PCR was run to ampliS. the murated fragments.
Primer PI and P2 contain XhoI and HindIII cutting sequence (highîighted)
respectively for the replacement of the mutated region for the wild type sequence on the
plasmid pBS-puJS (see Figure 1% for the structure of this plasmid). Mer idencifying the
mutation by seqwncing in individual clone, the 3.5 kb hgment were cloned into pRK404
to W o n n E. coli S17-I strain.
The sequences of the primen used in this mutagenesis study are given below:
@
P 1. 5'-TACTCTGC-CC-3
';
P2.5'-CC AG AAGG~TG-~ ';
P3. S'XAGCGTCCCGCCGAGGACGAGATAT-3 ';
P4. 5'-TG AAA(G,T)AA(A,T) ATATCGTGTCCGCGGC-3';
P5.5'-TGt\AAGAAAA(A.ïjATCGT(G,A)TCCGCGGCGGGACGCTGATC-3';
P6. S'-CGAACAGATCC(A,C)CG(C,G,A)CGATCAGCGTCCCCCGCGGA3';
P7. 5'-CGGCGGOOATCTGTTCAACTTCTCGTGTGGGCCC3';
P8.5'-CCG AAGAAGCCC AC A(A,G) AAAAGCGCCCCACC-3';
P9.5'-GCTCAGm(CA)(A,T)AAGAAAATATC-3';
P 10.5'-GATATMTCTT(T,A)(G,T)AAAACTGAGC3';
P 11. S'-GGATCTGlTC(A,T)(A,G) CTTCTGGGTGG-3';
P12. 5'-CCACCCAGAAG(T,C)(T,A)GAACAGATCC-3';
P 13.5'-TGGCGTTGCTCCTG~CGAC~CTGGGTGG-3'
P14.5'-GAAGTCGAACAGGAGC AACGCC ATAGCTG-3'.
P3 (ml)
I
Denature and annealing
Fbpre 34. Introductionof mrtrtions by recombinant PCR mcthod (see test for
esplaoation)
ml and m2 with the bump aymbolize the mutationa.
Acknowleâgments
1 would like to thank my supe~sorDr. Gabriel Gingras for his consistent concerning,
patient and skillful puidance of my research work. His encouragement aad fkiendship
accompanied my sut-year life in Moneeal and will continue to accompany my fiiture life.
Tbanks also go to aii my colleagws used to work in Dr. Gingras's lab for the fiendship we
shared with each other. Special thanks go to Madam Renpei Zheng for her help with my
experiments and my daily life.