Journal of General Microbiology (1988), 134, 1491-1497.
Printed in Great Britain
1491
Cloning and Oxygen-regulated Expression of the Bacteriochlorophyll
Biosynthesis Genes bch E, B, A and C of Rhodobacter sphaeroides
By C . N E I L H U N T E R * A N D S H I R L E Y A . COOMBER
Department of Pure and Applied Biology, Imperial College of Science and Technology,
London SW7 ZBB, UK
(Receiued 24 November I987 ;revised I February 1988)
Four mutants of the photosynthetic bacterium Rhodobacter sphaeroides were isolated which were
incapable of photosynthetic growth due to inability to synthesize bacteriochlorophyll. A
Rb. sphaeroides gene bank was constructed in the mobilizable vector pSUP202 and was
transferred into these mutants using the helper plasmid pRK2073. Three clones that produced
photosynthetic transconjugants from one or more of the bch mutants were isolated and
characterized. These clones were used as probes to estimate levels of specific transcripts in cells
undergoing a 100-fold increase in bacteriochlorophyll content. The maximum level of
transcripts was observed at an early stage of photosynthetic membrane synthesis when only 7%
of the eventual level of pigment had been synthesized.
INTRODUCTION
The purple bacterium Rhodobacter sphaeroides provides a useful model system in which to
study the role of bacteriochlorophyll synthesis in photosynthetic membrane assembly, due to its
ability to grow aerobically in the dark as well as photosynthetically. This facultative property
has permitted the isolation of stable non-photosynthetic mutants that are unable to synthesize
bacteriochlorophyll ; such mutants excrete biosynthetic intermediates into the growth medium.
Several workers have proposed biosynthetic pathways based on these intermediates (Lascelles,
1966; Richards & Lascelles, 1969; Pudek & Richards, 1975; Jones, 1978; Rebeiz & Lascelles,
1982).
Further progresss depends upon the development of genetic tools for this organism. In this
regard, cosmid mobilizing techniques have already been used to investigate genes for the
carotenoid biosynthetic pathway in Rb. sphaeroides (Pemberton & Harding, 1986). Marrs and
coworkers have used chromosome and plasmid mobilizing techniques to map and isolate a 45 kb
gene cluster in Rb. capsulatus which contains genes for bacteriochlorophyll and carotenoid
biosynthesis, and reaction centre and light-harvesting polypeptides (Yen & Marrs 1976; Marrs,
1981; Taylor et al., 1983). A highly efficient mobilization system which employs plasmid
pRK2073 (Hunter &Turner, 1988)has been used here to transfer a bank of Rb. sphaeroides genes
into mutants unable to synthesize bacteriochlorophyll. In this way clones carrying genes
designated bch E, B, A and C according to the scheme outlined by Biel & Marrs (1983) have been
isolated. In view of the widespread use of Rb. sphaeroides as a model for bacteriochlorophyll
biosynthesis, the work described here seeks to provide a molecular genetic analysis of the
pathway.
It has been known for some time that bacteriochlorophyll synthesis and more generally
photosynthetic membrane assembly are repressed by oxygen (Cohen-Bazire et al., 1957). Under
conditions of high aeration, it is possible to repress cellular bacteriochlorophyll to less than 1%
of maximum. At the onset of low aeration, this repression is lifted and over a period of
approximately 20 h the cell elaborates a system of pigmented membranes which houses the
photosynthetic apparatus (Niederman et al., 1976). In the early stages of membrane assembly,
0001-4553
0 1988 SGM
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1492
C . N . HUNT E R AND S . A . COOMBER
Table 1. Bacterial strains and plasmids
Strain or plasmid
E. coli
DH5
SmlO
Rb. sphaeroides
NCIB 8253
N6
N5
N22
T127
Plasmids
pSUP202
pRK2073
pSCN6-1
pSCN5-1
pSCN22-1
Relevant characteristics
Source*
Reference
A derivative of DH1 (which is F- recA endA
gyrA thi-1 hsdR supE)
recA thi thr leu; RP4-2-Tc: :Mu integrated into
the chromosome; Tra+ KmR
V.S.
Hanahan (1985)
R. S.
Simon et al. (1983)
Wild-type
NTG mutant, bchE
NTG mutant, bchB
NTG mutant, bchA
Tn5 mutant, bchC
R. A. N.
ApR CmR TcR; Mob+ Tra-; ColEl replicon
SpR;Tra+ Mob+; ColEl replicon
ApR CmR TcS
ApR CmR TcS
ApR CmR TcS
R. S.
M. J. D.
This paper
This paper
This paper
This paper
Simon et al. (1983)
Leong et al. (1983)
This paper
This paper
This paper
* V. S., Dr V. Simanis, ICRF, London, UK; R. S., Dr R. Simon, University of Bielefeld, FRG; R. A. N.,
Professor R. A. Niederman, Rutgers University, NJ, USA; M.J.D., Dr M. J. Daniels, John Innes Research
Institute, UK.
there is a rapid rise in the level of transcripts specific for reaction centre and light-harvesting
apoproteins of Rb. sphaeroides (Hunter et al., 1987). In Rb. capsulatus the transcription of several
bch genes is influenced by oxygen (Biel & Marrs, 1983), but there has hitherto been no
information on the influence of oxygen on bch genes in Rb. sphaeroides. In this paper we describe
the use of the cloned probes for bch E, B, A and C to determine how the levels of specific
transcripts are affected by oxygen during derepression of photosynthetic membrane assembly.
METHODS
Growth of cultures. Escherichia coli strains were grown in Luria media as described by Maniatis et al. (1982).
Rb. sphaeroides strains were grown in M22 medium (Sistrom, 1977) supplemented with sodium succinate
(4-6g F1),sodium glutamate (0-27g 1-I) and aspartic acid (0.04g 1-I). This is designated M22+. Where indicated,
0.1 % Casamino acids were used to supplement this medium.
Liquid cultures were shaken at 200 r.p.m. at 34 "C.
For E. coli, antibiotic concentrations were (pg ml-l) : ampicillin 50, tetracycline 10, chloramphenicol 25,
nalidixic acid 30, spectinomycin 50.
Strains and plasmids. These are summarized in Table 1.
Construction of the Rb. sphaeroides library in pSUP202. Rb. sphaeroides genomic DNA was prepared using the
method described by Hunter & Turner (1988). DNA (50 pg) was partially digested by TaqI and size-fractionated
on a NaCl gradient (1.5-5 M-NaCl in 10 mM-Tris pH 7.5,l mM-EDTA). Fragments in the range of 10-14 kb were
ligated into the CIaI site of pSUP202 which had been treated with calf intestinal alkaline phosphatase (BCL), and
the mixture was transformed into E. coli DH5. The gene bank consisted of 2500 clones of average insert size 11 kb
(f = 0.999; Maniatis et al., 1982). The library was subdivided into 40 sublibraries each representing approximately
50 clones, which were harvested and frozen in 25 % (v/v) glycerol in LB medium at - 80 "C.
Conjugation, and plasmid isolation and analysis. The techniques were as described in the accompanying paper
(Hunter & Turner, 1988).
Inductionof bacteriochlorophyllsynthesis in suspensions of Rb. sphaeroides under conditions of low aeration. This was
performed as described by Niederman et al. (1976).
Preparation and analysis of RNA. Total RNA was prepared from cells of Rb. sphaeroides harvested at various
times following pigment induction, using the method described by Hunter & Turner (1988). Northern blots were
made from formaldehyde denaturing gels as described by Maniatis et al. (1982). RNA dot and DNA hybridization
analyses were performed as described by Thomas (1983). Autoradiographs of blots were scanned on a Shimadzu
CS-930 dual-wavelength TLC scanner.
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Rb. sphaeroides bacteriochlorophyll genes
1
1
1
1
1
l
1
1
1
700
800
Wavelength (nm)
1
1493
lL,
, ,
1
900
600
700
Wavelength (nm)
f"6
800
Fig. 1. Absorption spectra of wild-type (WT) and bch strains. (a) Whole cells suspended in 80% (v/v)
glycerol to reduce light scattering, showingthe absence of bacteriochlorophyll-proteincomplexesin the
mutants. (b) Cell-free culture medium showing the presence of bacteriochlorophyll intermediates
excreted by the mutants.
Spectroscopic methods. Absorption spectra of bacteriochlorophyll intermediates were obtained on a PerkinElmer 554 spectrophotometer. Whole cells were suspended in 80% (v/v) glycerol to reduce light scattering.
Pigments were extracted from cells using acetone/methanol (7:2, v/v) and in some cases spectroscopy was
performed in pigment solubilized in diethyl ether.
Fluorescence emission spectra of acetone/methanol and ether cell extracts were obtained on a Perkin-Elmer
MPF-44A fluorescence spectrophotometer.
RESULTS A N D DISCUSSION
Characterization of bacteriochlorophyll mutants
A number of strains carrying lesions in bacteriochlorophyll biosynthesis were obtained
following chemical or transposon Tn5 mutagenesis of the wild-type. These are listed in Table 1.
Initially, mutants were isolated on the basis of an inability to grow photosynthetically (Psg-).
Psg- mutants were grown under oxygen-limited heterotrophic conditions which promote the
excretion of biosynthetic intermediates of bacteriochlorophyll into the growth medium;
absorption spectra are shown in Fig. 1. Following extraction of cell pellets by acetone/methanol
and diethyl ether a variety of absorption and fluorescence emission spectra were obtained and
the results (Table 2) were compared with similar work on bacteriochlorophyll-lessmutants of
Rb. sphaeroides (Lascelles, 1966; Richards & Lascelles, 1969). We conclude that N6 excretes
magnesium protoporphyrin monomethyl ester, N5 excretes magnesium divinyl phaeoporphyrin
a5 monomethyl ester, N22 excretes 2-desvinyl-2-hydroxyethylchlorophyllide a, and T 127
excretes 2-desacetyl-2-hydroxyethylbacteriochlorophyllide a. However, further work is needed
in order to see if a range of spectrally similar pigments are present within mutant N5 for
example, since our methods do not provide a means to resolve monovinyl or divinyl derivatives
of protochlorophyllide. Further analysis by HPLC indicates that mono- and divinyl species may
be excreted by mutant N5 (B. White, W. T. Griffiths, S. A. Coomber & C. N. Hunter,
unpublished results). The scheme of Pudek & Richards (1975) predicts that disruption of the
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1494
C . N . HUNTER A N D S . A . COOMBER
Table 2. Major absorption and fluorescence emission peaks of acetonelmethanol and diethyl ether
extracts of whole cells of bch mutants
Absorption (nm)
Solvent
c-*-,
Mutant
Fluorescence emission (nm)
A
f
Acetone/ Diethyl
methanol ether
7
Solvent
Excitation
wavelength
(nm)
Acetone/
methanol
Diethyl
ether
I
N6
584
588
423
590
592
N5
624
622
450
629
625
N22
656
656
427
668
660
T127
704
708
370
718
718
1
Possible bacteriochlorophyll
intermediate
Mg protoporphyrin monomethyl ester
Mg divinyl phaeoporphyrin as
monomethyl ester
2-desvinyl-2-hydroxyethylchorophyllide a
2-desacetyl-2-hydroxyethylbacteriochlorophyllidea
gene encoding reduction of protochlorophyllide to chlorophyllide may yield such a mixture of
intermediates. For the present, the mutants have been assigned to the linear pathway proposed
by Jones (1978). The genes are allocated to the stages of this pathway according to the scheme of
Biel & Marrs (1983) although they provide no designation for the gene encoding
protochlorophyllide reduction which appears in Fig. 2 as bchL. Another area of uncertainty in
bacteriochlorophyll and chlorophyll synthesis includes the number of steps and genes involved
in the cyclization reactions that lead to the formation of the E ring of mono- or divinyl
magnesium protoporphyrin monomethyl ester.
Isolation and characterization of pSUP202 recombinant clones carrying bch genes
Following transfer of the Rb. sphaeroides gene library to the mutants N5, N6, N22 and T127,
newly photosynthetic recombinant clones were isolated. Direct recovery of the bch genes from
the recombinants was not attempted because of the instability of pSUP202 in Rb. sphaeroides
(Hunter, 1988). Instead the clones were isolated by repeated subdivision and transfer of the gene
library. Each of the 40 sublibraries (see Methods) was transferred to mutants N5, N6 and N22
using the helper plasmid pRK2073, followed by selection for photosynthetic growth. On
average, three sublibraries out of 40 gave 20-30 photosynthetic transconjugants. These
transconjugants have fully restored levels of photosynthetic apparatus, are stable in aerobic
culture and no detectable pigment is excreted into the growth medium. Clones which produced
photosynthetic growth were analysed by restriction mapping (Figure 3). These plasmids, which
were named pSCN6-1, pSCN5-1 and pSCN22-1, have inserts of 11.3, 12.1 and 10.5 kb
respectively, and complement mutants N6, N5 and N22 respectively. No overlaps in
complementation were noted between the three plasmids and the three mutants, although
pSN22-1 also restores mutant T127 to photosynthetic growth. Moreover, pSCN5-1 and pSCN6-1
overlap and share 6 kb in common. None of these clones overlap with pJW1, which contains a
12.2 kb BamHI fragment carrying puf genes A, B, L and M encoding subunits of the LHl and
reaction centre complexes. Pemberton & Harding (1986) have already shown that several
carotenoid (crt) genes map closely together in Rb. sphaeroides. Work is currently being
undertaken to link pSCN6-1, pSCN5-1 and pSCN22-1 with the puf and puh genes of the
photosynthetic cluster as found in Rb. capsulatus (Taylor et al., 1983).
Influence of oxygen on mRNA levels for bch E, B, A and C
Cohen-Bazire et al. (1957) first showed that oxygen represses bacteriochlorophyll biosynthesis
in purple non-sulphur bacteria. A number of groups have shown that oxygen represses the levels
of mRNA for LH2, LH1 and reaction centre complexes, in both Rb. capsulatus and Rb.
sphaeroides (Clark et al., 1984; Zhu & Kaplan, 1985; Klug et al., 1985; Zhu & Hearst, 1986; Zhu
et al., 1986; Hunter et al., 1987). The transcription of several genes for bacteriochlorophyll
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--
Rb. sphaeroides bacteriochlorophyll genes
1495
Uroporphyrinogen I11
b-Aminolaevulinic acid
Porphobilinogen
Coproporphyrinogen 111
Protoporphyrinogen IX
Protoporphyrin IX -Mg-protoporph
bchD
yrin IX +
bchH
N5
N6
Mg-protoporphyrin IX monomethyl e s t e r 4 Mg-2,4-divinylpheoporphyrina, MME ~ P r o t o c h l o r o p h y l l i d e +
bchE
bchB
bchL
N22
Chlorophyllide a +2-Desvinyl-2-hydroxyethyl chlorophyllide a
bchF
bchA
T127
2-Desacetyl-2-hydroxyethyl bacteriochlorophyllide a 4Bacteriochlorophyllide aBacteriochlorophyll
bchC
bchG
Fig. 2. The bacteriochlorophyll biosynthetic pathway showing the positions of lesions in mutant
strains.
PBEHP
P B
1
P
P P
1 1 1
B
P
E
E
E H
H
I I (
I
P E B P B E H P
1
I
P
pSCN6-1
P P
3
pSCN5-I
pSCN22-1
1 kb
U
Fig. 3. Restrictionmaps of insert DNA from clones pSCN6-1, pSCN5-1 and pSCN22-1. Dotted areas
indicate regions of overlap between pSCN6-1 and pSCN5-1. B, BumHI; E, EcoRI; H, HindIII; P, PsfI.
biosynthesis has been shown to be regulated by oxygen in Rb. capsulutus (Biel & Marrs, 1983).
Also, the levels of mRNA for the bch genes of Rb. capsulutus are affected by light and oxygen
(Clark et ul., 1984; Zhu & Hearst, 1986; Zhu et al., 1986).
Total mRNA was prepared from Rb. sphaeroides cultures at various stages of pigmentation
after the oxygen concentration was lowered; over the time course the level of cellular
bacteriochlorophyll increased 100-fold. A series of dot blots were probed with the plasmids
pSCN6-1, pSCN5-1 and pSCN22-1 (Fig. 4). The results show that the maximum level of
transcripts is achieved 1 h after the lowering of oxygen concentration and represents a two- to
threefold increase. The timing of this increase can be compared with a recent study on the
induction of mRNA for reaction centre and light-harvesting polypeptides (Hunter et ul., 1987):
the maximum levels of transcripts for reaction centre and LH1 complexes were achieved 2 h
after the start of induction, and for LH2, which binds much of the bacteriochlorophyll, a gradual
increase was observed over the 6 h of the experiment. Thus, it can be concluded that the rise in
mRNA for the bacteriochlorophyll biosynthesis pathway within 1 h is a primary event in
photosynthetic membrane assembly. It is interesting that the products of this pathway are
needed to stabilize the polypeptide components of photosynthetic complexes in Rb. capsulatus
(Dierstein, 1983).
Northern blots probed with these plasmids did not reveal bands representing bch transcripts
with sufficient clarity for densitometry. We attribute this to the low abundance of these
transcripts, in comparison with those encoding structural proteins. Nevertheless, the sizes of
these transcripts could be measured. pSCN5-1 encoded five transcripts of 1.4, 1.0,0.8,0.73 and
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1496
C . N . H U N T E R AND S . A . COOMBER
6
5
5
4
.C(
8
3
&
2
& 2
1
1
2
3
Time (h)
4
5
6
Fig. 4. Graph showing the change in levels of mRNA for bch E, B, A and C during semi-aerobic
induction of photosynthetic complexes in the dark. These levels are expressed as the height of the peaks
obtained from densitometry scans of RNA dot blots. The following plasmids were used as probes:
pSCN6-1 (-),
pSCN5-1 (- - - - -), pSCN22-1 (--.--.-). The level of cellular bacteriochlorophyllis
denoted by (.......).
0.43 kb, pSCN6-1 encoded four transcripts of 1.95, 1.45, 1.07 and 0.84 kb, and pSCN22-1
encoded four transcripts of 1.4, 1.26, 1-06and 0.8 kb.The levels of all these transcripts increased
two- to threefold when oxygen concentration was lowered. These studies show that oxygen
tension affects the amount of transcripts encoded by clones carrying bch genes although they do
not differentiate between increase in transcription or decrease in degradation of these mRNA
species. Work is currently underway to position the bch genes and transcripts on the clones
obtained so far, and to clone genes for the remainder of the pathway.
This work was funded by the Science and Engineering Research Council of Great Britain. The authors would
like to thank Dr W. R. Richards, Dr W. T. Griffiths and Professor 0. T. G. Jones for discussions on
bacteriochlorophyll mutant identification.
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