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Faculty of Science, Medicine and Health
2010
Essential biological processes of an emerging
pathogen: DNA replication, transcription, and cell
division in Acinetobacter spp.
Andrew Robinson
University of Wollongong, [email protected]
Anthony J. Brzoska
University of Newcastle
Kylie M. Turner
University of Technology, Sydney
Ryan Withers
University of Newcastle
Elizabeth J. Harry
University of Technology, Sydney
See next page for additional authors
Publication Details
Robinson, A., Brzoska, A., Turner, K., Withers, R., Harry, E., Lewis, P. & Dixon, N. E. (2010). Essential Biological Processes of an
Emerging Pathogen: DNA Replication, Transcription, and Cell Division in Acinetobacter spp.. Microbiology and Molecular Biology
Reviews, 74 (2), 273-297.
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[email protected]
Essential biological processes of an emerging pathogen: DNA replication,
transcription, and cell division in Acinetobacter spp.
Abstract
Species of the bacterial genus Acinetobacter are becoming increasingly important as a source of hospitalacquired infections (31, 185, 204). Acinetobacter spp. are ubiquitous nonmotile gammaproteobacteria,
typified by metabolic versatility and a capacity for natural transformation (172, 204). The species of most
clinical relevance is A. baumannii; however, pathogenic strains of A. lwoffi and A. baylyi have also been
described (38, 185, 215).
Keywords
CMMB
Disciplines
Life Sciences | Physical Sciences and Mathematics | Social and Behavioral Sciences
Publication Details
Robinson, A., Brzoska, A., Turner, K., Withers, R., Harry, E., Lewis, P. & Dixon, N. E. (2010). Essential
Biological Processes of an Emerging Pathogen: DNA Replication, Transcription, and Cell Division in
Acinetobacter spp.. Microbiology and Molecular Biology Reviews, 74 (2), 273-297.
Authors
Andrew Robinson, Anthony J. Brzoska, Kylie M. Turner, Ryan Withers, Elizabeth J. Harry, Peter J. Lewis, and
Nicholas E. Dixon
This journal article is available at Research Online: http://ro.uow.edu.au/scipapers/5059
M ic r o b io l o g y a n d M o l e c u l a r B io l o g y R e v ie w s , June 20 10, p. 2 7 3 -2 9 7
1 0 9 2 -2 1 7 2 /1 0 /$ 1 2 .0 0 d o i:1 0 .1 1 2 8 /M M B R .0 0 0 4 8 -0 9
Vol. 74, No. 2
Copyright © 2010, Am erican Society for Microbiology. All Rights Reserved.
Essential Biological Processes of an Emerging Pathogen: DNA
Replication, Transcription, and Cell Division in Acinetobacter spp.
Andrew Robinson,1 Anthony J. Brzoska,2 Kylie M. Turner,3 Ryan W ithers,2 Elizabeth J. H arry,3
Peter J. Lewis,2 and Nicholas E. Dixon1*
School o f Chemistry, University o f Wollongong, Wollongong, N ew South Wales, A u stralia1; School o f E nvironm ental and
L ife Sciences, University o f Newcastle, Newcastle, N ew South Wales, A ustralia2; and Institute fo r the Biotechnology o f
Infectious Diseases, University o f Technology, Sydney, N ew South Wales, A ustralia3
INTRODUCTION................................................................................................................................................................. 273
DNA REPLICATION...........................................................................................................................................................274
Initiation of Replication in Acinetobacter.................................................................................................................... 275
A Highly Diverged Pseudomonas-Type iji Subunit.................................................................................................... 277
An Unusual Primosome Complex: Unique Features of Acinetobacter DNA Primase...................................... 277
Highly Conserved Protein Interaction Modules within the p Sliding Clamp and SSB.................................278
CELL DIVISION.................................................................................................................................................................. 280
Assembly of FtsZ at M idcell..........................................................................................................................................282
Recruitment of Membrane-Associated Proteins....................................................................................................... 282
Peptidoglycan Recycling and Response to p-Lactam Antibiotics........................................................................ 284
RNA TRANSCRIPTION..................................................................................................................................................... 284
Architecture of Transcription Complexes and Suitability for Drug Design...................................................... 284
Alternative Sigma Factors and Their Regulation.................................................................................................... 287
Pathogenic Acinetobacter spp. Are Enriched in Transcription Factors...............................................................289
AN UNUSUAL DNA DAMAGE RESPONSE................................................................................................................ 289
PERSPECTIVES................................................................................................................................................................... 290
ACKNOWLEDGMENTS.................................................................................................................................................... 291
REFERENCES.......................................................................................................................................................................291
INTRODUCTION
negative pathogens, there is an u rgent need for new classes of
antibiotic com pounds with novel mechanism s o f action (245).
A ntibiotics act by inhibiting th e essen tial w orking p a rts o f
b a cterial cells, w ith th e m ost com m on targ ets being ribosom al
p ro tein synthesis, cell wall biosynthesis, an d p rim ary m etabolic
pathw ays. T h e processes o f ch ro m o so m al D N A replication,
tran scrip tio n an d cell division are relatively u n ex p lo red as drug
ta rg e ts. T h e y c o u ld , h o w ev er, be c o n sid e re d a ttra c tiv e ta r ­
gets fo r ra tio n a l desig n o f n o v el an tim ic ro b ia l c o m p o u n d s,
as th e y are (i) e ss e n tia l fo r th e p ro p a g a tio n o f b a c te ria l cells
a n d th ere fo re infection, (ii) conserved across all b a cterial sp e­
cies, an d (iii) in m any ways distinct from the equ iv alent p ro ­
cesses in eu karyotic cells (150). A cinetobacter rep re sen ts an
ap pealing system fo r studying these processes. In ad d ition to
th e ir d irect clinical relevance, A cinetobacter spp. show som e
sim ilarities to o th e r p ro b lem atic p a th o g en s, such as P seudo­
m onas aeruginosa, th a t are n o t sh ared by th e m ore thoroughly
stu d ied m o d el b acte riu m Escherichia coli (15). A t the sam e
tim e, th e A cinetobacter genus has diverged significantly from
o th e r g am m ap ro teo b acteria, as highlighted by a phylogenetic
tre e b ased on th e highly conserved sequences o f 16S rR N A
genes (Fig. 1) (116, 188, 213, 222, 265, 266). A s such, A cin eto ­
bacter sp. p ro tein s generally show considerable seq u ence di­
vergence from equivalent proteins in o th er organisms and can
therefore be used to highlight the m ost conserved aspects of
essential biological systems. O f course, these are the m ost attrac­
tive targets for the discovery or rational design of broad-spectrum
antibiotics.
C u rre n tly , c o m p le te g en o m e se q u e n c e s are av ailable fo r
Species o f the bacterial genus Acinetobacter are becom ing in ­
creasingly im portant as a source of hospital-acquired infections
(31, 185, 204). Acinetobacter spp. are ubiquitous nonm otile gamm aproteobacteria, typified by m etabolic versatility and a capacity
for natural transform ation (172, 204). T he species o f m ost clinical
relevance is A baumannii; however, pathogenic strains oL4. Iwoffi
and A baylyi have also been described (38, 185, 215).
A . baum annii is m ost com m only associated with pneum onia or
bacterem ia, although the incidence of soft tissue infections and
meningitis appears to be increasing (204). M ortality rates associ­
ated with A baum annii infection are a m atter o f som e contention,
as there is a tendency for A baum annii to infect patients w ho are
critically ill and already have a p o o r prognosis (204). It is clear,
however, th at patients with A . baum annii infections stay signifi­
cantly longer in hospitals than uninfected patients and represent
a significant burden on health care systems worldwide (204). Ex­
acerbating this, A . baum annii can be highly persistent within the
hospital setting, due in p a rt to its resistance to disinfectants (262)
and rem arkable insensitivity to desiccation (122). O f greatest con­
cern, however, is the rapid em ergence o f m ultidrug resistance
(65). A. baum annii strains th at are resistant to every antibiotic
com pound in clinical use, including recently approved drugs such
as tigecyclin, have now been rep o rted (65,187,203). In the face o f
the growing th reat posed by these and o th er intractable G ram -
* Corresponding author. Mailing address: School of Chemistry, U ni­
versity of W ollongong, W ollongong, NSW 2522, Australia. Phone:
61-2-42214346. Fax: 61-2-42214287. E-mail: nickd@ uow .edu.au.
273
274
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FIG . 1. Subbranch of a phylogenetic tree based on 16S rR N A sequences. 16S rR N A sequences encoded by the rrsA gene were from
gam m aproteobacteria with completely sequenced genomes and were filtered to remove identical sequences prior to tree construction. The tree was
constructed using the neighborhood-joining tree m ethod in G eneious (Biomatters, Auckland, New Z ealand), using the Jukes-Cantor genetic
distance m odel and employing the bootstrap m ethod with 10,000 replicates. The sequence of rrsA from the betaproteobacterium Nitrosospira
multiformis was included as an outgroup. Colored segm ents indicate the phylogenetic distribution of particular genetic characteristics: (i) dnaC,
holE, tus, amiC, and envC genes present on chromosome; (ii) long Pseudomonas-type i|i subunit of D N A polymerase III; (iii) short E. coli-type i|i
subunit of D N A polymerase III, m hA and dnaQ genes adjacent and transcribed in opposite directions, vacE gene present; (iv) m hA -dnaQ fusion
gene present and no homologs o ifts E ,fts X , sulA, or lexA on chromosome (includes Acinetobacter spp.); (v) m hA and dnaQ genes adjacent and
transcribed in same direction and amiC gene present.
seven A cin eto b a cter stra in s: th e highly tra n sfo rm a b le la b o ­
ra to ry stra in A . baylyi A D P 1 (15); a h u m a n b o d y louse
sym b iont, A . b a u m a n n ii S D F (248); a n d th e h u m a n p a th o ­
gens A . b a u m a n n ii stra in s A T C C 17978 (233), A Y E (248),
A C IC U (115), A B 0057 (4), a n d A B 307-0294 (4). A t le a st
e ig h t a d d itio n a l g en o m e se q u e n c e p ro je c ts are c u rre n tly in
p ro g re ss o r a t d ra ft assem bly stag e a t th e tim e o f w ritin g o f
this review . In a d d itio n , th e G e n o sc o p e g ro u p h as p ro d u c e d
a c o m p lete single-gen e d e le tio n lib rary fo r A. baylyi A D P 1 ,
allow ing th e esse n tia lity o f in d iv id u a l g en es to be assessed
(61). T h e se re so u rc e s p ro v id e a firm sta rtin g p o in t fo r th e
in v estig atio n o f e ss e n tia l b io lo g ical p ro c e sse s in th ese o r ­
ganism s. T h e g o a l o f th is review is to ex am in e th is re c en tly
av ailab le in fo rm a tio n , p rim a rily by e x tra p o la tin g know ledge
fro m w e ll-stu d ie d m o d e l o rg an ism s, in th e c o n te x t o f D N A
re p lic a tio n , tra n sc rip tio n , a n d cell division, w ith a view to ­
w a rd h ig h lig h tin g u n u su a l a sp ects a n d id e n tific a tio n o f c a n ­
d id a te enzym es a n d co m p lex es m o st su ita b le fo r fu tu re d ru g
d iscovery a n d design.
DNA REPLICATION
O u r know ledge o f b a cte rial D N A rep licatio n draws ex ten ­
sively from studies o f th e m o d el organism E. coli (16, 209, 226,
V ol . 74, 2010
242). O ver the last 5 decades, each o f th e individual D N A
replication p ro tein s have b een identified, for m o st th e ir th reedim ensional stru ctu res (o r those o f th e ir dom ains) have been
d eterm in ed , and m any p ro te in -p ro te in an d p ro te in -D N A in ­
teractions have been m a p p e d (T able 1).
In E. coli an d m any o th e r bacte ria, D N A rep licatio n is in i­
tia te d a t a single site on th e circular chrom osom e, th e origin o f
replication o r oiiC , w hich contains a series o f 9-bp sequence
rep eats know n as D n a A boxes (180). B inding o f m ultiple
D n aA m olecules to oiiC leads to sep ara tio n o f th e two te m ­
p late D N A stran d s at a n earb y A T -rich region. O ne m olecule
o f th e rep licativ e h elicase D n aB is lo a d e d o n to e a ch o f th e
re su ltin g single D N A stra n d s, a n d e a c h p ro c e e d s to u n w in d
th e p a re n ta l D N A d u p lex to c re a te re p lic a tio n fo rk s th a t
m ove aw ay fro m th e o rig in in o p p o site d ire c tio n s. D N A
p rim a se associates w ith D n aB an d constructs sh o rt R N A p rim ­
ers, o n to w hich the m u ltisu b u n it D N A polym erase III h oloenzyme (P ol III H E ) builds each new D N A strand. D N A syn th e­
sis by P ol III is carried o u t only in th e 5 '-to -3 ' direction; thus,
synthesis o f one stran d (the leading stran d ) is con tin u o u s while
synthesis o f the o th e r stra n d (the lagging stra n d ) is disco n tin ­
uous. T h e D naB helicase tran slo cates on th e lagging stra n d at
each fork. D N A replication co n tin u es bidirectionally aro u n d
the circular chrom osom e u n til th e two rep licatio n forks m e e t
in the term in u s region, lo cated approxim ately opp o site the
origin (182, 189). T his eventually yields tw o copies o f th e b ac­
terial chrom osom e, each contain in g one stra n d from th e p a ­
re n ta l chrom osom e an d one n ascen t strand.
W ith the availability o f co m plete genom e sequences for
m ore th an 900 bacterial strains, we can now ex trap o late o u r
solid u n d erstan d in g o f E. coli D N A rep licatio n in to gen era
outside the Enterobacteriaceae, such as Acinetobacter. W hile
both are m em bers o f th e g a m m ap ro teo b acteria, th e A c in e to ­
bacter genus is relatively rem o te from E. coli (see th e p h y lo ­
genetic tree in Fig. 1). T h e sequences o f A cinetobacter D N A
replication p ro tein s have diverged significantly from th o se o f
o th e r organism s, in som e cases b eyond the d etectio n level o f
sta n d ard hom ology searches. W hile this in som e ways restricts
the level o f inform atio n th a t can be e x trap o lated from th e E.
coli system , it also provides stro n g indications o f w hich aspects
o f D N A replication are conserved an d w hich are not. A c in e to ­
bacter spp. display several pecu liarities am ong th e ir replication
com p onents, som e o f w hich are discussed in d etail below.
Som e o f these, such as th e fusion o f m h A an d dnaQ genes in
a single cistron as previously n o te d by B arbe an d colleagues
(15), are u nique to m em b ers o f A cinetobacter an d th e re la te d
genus Psychrobacter. O th ers highlight th e previously identified
relationship betw een the A cinetobacter an d P seudom onas g e n ­
e ra (15), w ith the tw o groups often displaying com m on p ro te in
featu res despite significant divergence a t th e D N A sequence
level.
Initiation of Replication in Acinetobacter
A ll A cinetobacter spp. fo r w hich genom e sequences have
b een co m p leted co ntain a single circular chrom osom e, w ith the
origin o f replication (oriC) lo cated b etw een th e rp m H and
dnaA loci (15). T his arran g e m e n t is also seen in Pseudom onas
spp. (270) an d contrasts w ith th e case fo r E . coli, w hose origin
m aps betw een the gidA (m n m G ) an d m io C genes, som e 40 kb
ESSENTIAL PRO CESSES IN ACINETOBACTER SPP.
275
from rp m H an d dnaA . F o r each A cinetobacter sp., th e rep lica­
tion in itia to r p ro te in D n a A is relatively w ell conserved (46%
id entity w ith E. coli D n a A [Table 1]), particu larly w ithin the
N -term in a l D n aB -in teractin g dom ain (dom ain I), th e C -term in al A A A + A T P ase dom ain (dom ain III), an d th e D N A -binding dom ain (dom ain IV ) (134). D o m ain II, identified as a
flexible lin k er in th e E. coli p ro tein , has diverged b eyond re c ­
o gnition in A cinetobacter D n aA pro tein s.
In itiatio n o f D N A rep licatio n in E. coli is reg u lated by a
p aralo g o f D n aA know n as H d a (138, 180). W hen b o u n d to a
D N A -asso ciated (3 sliding clam p, H d a causes th e active A T Pb o u n d form o f D n a A to hydrolyze its su b strate a n d th u s b e ­
com e inactive, p rev en tin g u n sch ed u led rein itiatio n o f rep lica­
tion (142). G en es encoding p ro te in s w ith m o d e rate hom ology
to E. coli H d a (30% sequence identity) are fo u n d w ithin the
A cinetobacter sp. genom es. A lth o u g h they are freq u ently a n ­
n o ta te d as “p utative ch ro m o so m al replication in itiato r, D n aA
type,” these p ro te in s likely p erfo rm a role analogous to th a t o f
H d a, i.e., suppression o f untim ely initiatio n o f D N A rep lica­
tion.
H istorically, th e study o f D N A rep licatio n has focused on
two m o d el organism s: th e G ram -negative E. coli (a gam m ap ro te o b ac teriu m ) an d th e G ram -positive Bacillus subtilis (a
firm icute). B oth th ese organism s utilize A A A + A T P ase fam ily
helicase lo a d e r p ro tein s to lo ad th e replicative D N A helicase
(D naB in E. coli an d D n a C in B. subtilis) o n to th e tem plate
D N A a t origins o f replication o r durin g replication re s ta rt after
fo rk blockage (59). In E. coli, helicase loading is c arried o u t by
D n aC , w hich com plexes an d th ereb y inactivates D n aB u n til its
activity is re q u ire d an d dissociates u p o n helicase loading (226).
In B. subtilis, th ree p ro tein s, D n aB , D n aD , an d D n a l, are used;
th e last is a hom olog o f E. coli D n aC (26, 117). N o hom ologs
o f E. coli D n aC o r B. subtilis D n aB , -D, o r -I are identifiable
w ithin th e A cinetobacter sp. genom es, w ith th e exception o f
p ro p h ag e-asso ciated D n aC hom ologs in A . baum annii strains
A Y E an d A C IC U . H om ology searches across those b acteria
w ith co m p leted genom e sequences show the phylogenetic dis­
trib u tio n o f D n aC to be restricted to th e Enterobacteriaceae
(Fig. 1). T h e D n aB , -D , an d -I helicase-loading p ro tein s are
sim ilarly restric te d to th e F im iicutes, w hich include th e genera
Bacillus, Staphylococcus, an d M ycoplasm a. T his im plies th a t
A cinetobacter spp., an d fo r th a t m a tte r all o th e r bacte ria o u t­
side th e Enterobacteriaceae an d th e F im iicutes, e ith e r are ca­
pable o f loading the replicative helicase w ith o u t ad ditional
lo a d e r p ro te in s o r have helicase lo ad ers w hose sequences are
n o t re la te d to the E. coli an d B. subtilis p ro tein s. In su p p o rt o f
th e fo rm er scenario, th e d naB (helicase) gene from the epsilo n p ro teo b acteriu m H elicobacter pylori has b een fo u n d to suc­
cessfully co m p lem en t tem p eratu re-sen sitiv e d n a C m u tations in
E. coli (236). T his suggests th a tH . pylori D naB n o t only can be
lo ad ed w ith o u t the activity o f D n aC in vivo b u t also can be
assem bled successfully in to an E. coli replisom e. F u rth e r evi­
dence com es from the o bservation th a t D naB p ro te in s from
P seudom onas spp. can be lo ad ed o n to th e origin o f a broadh ost-range plasm id in vitro, using only D n aA , i.e., w ith o u t
assistance from a lo a d e r p ro te in (35). Since th e E. coli D n aA
an d D n aC p ro tein s are structurally re la te d (58, 7 7 ,1 8 1 , 226), it
is possible th a t D n a A (o r a D n a A h om olog) loads D naB in
o th e r organism s, n o t ju st at oriC b u t also during replication
restart. F u rth e r w o rk is clearly re q u ire d to b e tte r u n d e rsta n d
276
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the process o f helicase loading in organism s outside th e Enterobacteriaceae and Firmicutes.
A Highly Diverged Pseudomonas-Type iji Subunit
M ost o f the 10 com p o n en ts o f th e P ol III H E com plex are
readily identifiable in A cinetobacter spp. b ased o n hom ology
searches w ith the E. coli p ro te in s (T able 1). N evertheless, such
searches could n o t locate a hom olog o f the holD gene, w hich
encodes th e iji subunit o f th e clam p lo ad e r com plex in E. coli
(7). A h e te ro d im e r fo rm ed b etw een th e x an d iji subunits binds
to th e clam p lo ad er com plex [(T/y)388'] o f D N A polym erase
III th ro u g h a region at th e N term in u s o f iji (96, 231). T he ijiy
com plex m ediates the han d o ff o f th e p rim ed tem p late m o le­
cule from the D n a G prim ase via single-stranded D N A -binding
p ro tein (SSB) to the clam p lo a d e r com plex ( t 3 8 8 ' ) , th u s re p ­
resenting a functional link b etw een the p rim osom e com plex
(D n aB -D n aG ) and P o l III H E w ithin th e replisom e (275).
O riginally th o u g h t to re p re se n t a sim ple “b rid g e” betw een the
clam p lo ad er com plex an d the x subunit, th e iji su b u n it has now
been show n to play a significant role in loading o f the (3 clam p
o n to its D N A tem p late in E. coli (7).
D u rin g th eir w o rk on th e P seudom onas replisom e, M c­
H en ry ’s group n o te d th a t P seudom onas spp. also lacked an
identifiable hom olog o f th e E . coli holD gene (120). T his group
found, how ever, th a t ad d itio n o f p urified ijiy com plex from E.
coli dram atically stim u lated th e activity o f a m inim al P seudo­
m onas replication com plex (ae-T88'-(3) in vitro. A s this stim u ­
lation w as n o t observed follow ing th e ad d itio n o f Pseudom onas
X subunit alone, this strongly suggested th e existence o f a
cryptic iji s u b u n it in P seu d o m o n a s, w hich th e g ro u p la te r
id e n tifie d an d c h a ra c te riz e d (121). T h e P seu d o m o n a s-ty p e iji
s u b u n it w as fo u n d to be c o n sid erab ly lo n g e r th a n its E. coli
c o u n te r p a rt (282 v ersu s 137 re sid u e s). D e sp ite th is, m u ltip le-seq u en ce alignm ents rev ealed th ree distinct conserved r e ­
gions corresponding to helices a l an d a 4 o f th e E. coli iji an d
a region at the N term in u s know n to in te ra ct w ith dom ain III
o f t h (7, 96).
W e have u sed h id d en M arkov m o d el (H M M )-b ased h o m o l­
ogy searches o f the A. baylyi A D P 1 genom e w ith th e pro g ram
H H search (234, 235) to rev eal a poorly conserved hom olog o f
P. aeruginosa iji (hypo th etical p ro te in A C IA D 1326, 17% se­
quence identity). H om ologs o f this putativ e iji p ro te in are
fo u n d w ithin each o f th e available A. baum annii genom e se­
quences (62% sequence iden tity w ith A. baylyi iji [Table 1]).
F rom m ultiple-sequence alignm ents, it ap p ears th a t the o p en
read in g fram e w ithin the A. baylyi gene sh o u ld begin 11 re si­
dues e a rlie r th a n in th e pub lish ed genom e an n o tatio n . U sing
these newly identified hom ologs as in p u t fo r H H search , it was
fu rth e r possible to d ete c t extrem ely w eak, b u t significant, h o ­
m ology betw een these p ro tein s an d th e E. coli iji su b u n it (for
the A. baylyi p ro tein , E value = 1.8 X I I P 4; 13% overall
sequence identity). T hese newly identified A cinetobacter iji p ro ­
teins m aintain the th re e conserved regions (Fig. 2A ) th a t w ere
previously identified w ithin th e E. coli an d P seudom onas iji
subunits (121). W hen th e conserved residues are m a p p e d o n to
the E. coli clam p lo ad er (y388'i|d an d ijiy stru ctu res (96, 231),
they fall w ithin th e N -term in a l clam p lo ad er-in teractin g region
and the x-contacting helices a l an d a 4 (Fig. 2B). W hen these
iji types are m ap p ed to a phylogenetic tre e fo r th e gam m apro-
ESSENTIAL PRO CESSES IN ACINETOBACTER SPP.
277
teo b ac teria (Fig. 1), a decisive split is seen, in w hich P seudo­
m onas, Acinetobacter, an d th e ir relatives use th e longer
P seudom onas-type iji subunit, while m em b ers o f th e Enterobacteriaceae, V ibrio,H aem ophilus, an d Shewanella have th e sh o rter
E. coli-type p ro tein . Interestingly, gene k n o ck o u t studies show
th a t th e holD gene is dispensable in E . coli (11) b u t is essential
in b o th A. baylyi A D P1 (61) an d P. aeruginosa PA01 (118)
(T able 1). T his m ight suggest th a t th e longer, P seudom onastype iji subunits have an additional, as-yet-unidentified function
in vivo.
An Unusual Primosome Complex: Unique Features of
Acinetobacter DNA Primase
D N A polym erase III c a n n o t synthesize new D N A stran d s de
novo an d first req u ires th e co n stru ctio n o f a sh o rt R N A p rim er
by the D n a G p rim ase (226); this occurs every second o r two
during O kazaki frag m en t synthesis on the lagging strands (67).
D u rin g this step, D n a G binds to the replicative helicase D naB ,
form ing a com plex know n as the p rim osom e. T his in teraction
fo rm ed by D n aB an d D n a G has b een show n to m o d u late the
activities o f b o th enzym es (39).
D n aB consists o f tw o dom ains: a p rim ase-binding dom ain at
th e N term in u s an d an A T P -utilizing D N A helicase dom ain at
th e C term in u s (12). B acterial p rim ases co n tain th ree distinct
dom ains: a zinc-binding dom ain a t th e N term in u s, a cen tral
R N A polym erase (R N A P ) dom ain, an d a C -term in al helicase b inding dom ain th a t is structurally re la te d to the N -term inal
dom ain o f D n aB (51, 78, 191, 237, 241, 258). T h e stru ctu ral
basis fo r the p rim osom e in teractio n was rev ealed recently in
crystal stru ctu res o f th e com plex b etw een D n aB an d the Cterm in al dom ain o f D n a G from Geobacillus stearothermophilus
(12). D naB w as seen to form a 3-fold sym m etric hexam er,
m ed iated by th e fo rm atio n o f a ring o f N -term in a l prim ase b inding dom ains. T h e C -term in al helicase-binding dom ains o f
th re e D n a G m olecules w ere fo u n d to associate w ith this ring,
w ith D n a G residues form ing a n etw o rk o f hydrogen bonds,
w hich w ere la te r show n to play a role in m o d u latin g D naB
activity (12, 39).
In A cinetobacter spp., the enzym atic sites o f D naB and
D n a G are w ell conserved. T h e dom ains responsible for fo rm ­
ing th e p rim osom e com plex, how ever, have diverged signifi­
cantly. In fact, th e D n a G helicase-binding dom ain has diverged
so greatly th a t it b ears n o resem blance to any o th e r p ro te in in
th e c u rre n t seq u en ce databases, including D n a G p ro tein s from
m em b ers o f the closely re la te d genus Psychrobacter. T h erefo re,
it is n o t curren tly possible to m o d el the A cinetobacter p rim o ­
som e com plex b ased o n th e G. stearothermophilus stru cture. In
c o n trast, th e D n a G helicase-binding dom ains o f o th e r G ram negative organism s, nam ely, E. coli an d P. aeruginosa, do show
d etectab le sequence hom ology w ith th e G. stearothermophilus
dom ain. A s such, p rim osom e com plexes could conceivably be
m o d eled for these organism s using th e G. stearothermophilus
stru ctu re as a tem p late.
T h e D n a G p ro te in s o f A cinetobacter spp. an d Psychrobacter
spp. are fu rth e r com plicated by a large in sertio n (50 to 100
residues) b etw een th e N -term in a l zinc-binding dom ain and the
c en tral R N A polym erase dom ain. B ased on secondary stru c­
tu re an d d iso rd er p red ictio n s (155, 221), this in sertio n appears
to be d iso rd ered an d th u s pro b ab ly acts as a lin k er region.
278
RO BIN SO N E T AL.
E sch erich ia coli
P seu d o m on a s a eru gin osa
P seu d o m o n a s p u tid a
P seu d o m o n a s fluo rescen s
P seu d o m on a s sy rin g ae
A zo to b a cte r vin e la n dii
A cin e to b a cte r b aylyi
A cin e to b a cte r b a u m a n n ii
A cin e to b a cte r ra dioresisten s
P sych ro b a cter sp. PRwf-1
P sych ro b a cter arcticus
P sych ro b a cter cryo h a lo len tis
M ic r o b io l . M o l . B io l . R e v .
1-M T S [3 3 D WQHQ Q H g H T Q E ]S L G lR P G a
3 - E E Q E II I3 A F B G A E ]Q H T S E H P E 3 Q P L P
3- T E P i i l i l i l A Y ffls a B I o H v H E U P iilA E L P
S3-fflVfiIiiaT.iiElT.TV
ISB -H LRiiCTLE^AGL
138-fflL KliBlL HEIa g L
3- i e s a a a A y H s a [ U q 0 v s E H p H s e l p
i b s-W l k E E I l ii^ A g l
1 4 3 -0 L K !5 E 1 lE 0 A G L
134-fflT, R it UlT.fi^lA G T.
128-0W T 0H Q S 0 L SG
127 - H w a E H q ! S 3 k v g
1 35 -H w l i i l i oE IS Il s g
17 7 -HwQ s 0 l i 0 l s l
2 1 o -H w q S H tP 0 l s i
194-HWQEDHt P H L s i
3- N E S i:»:«:iiA Y n iT A B lo ta V N E lg P iilT E L P
3-D E A i i l i l i l A Yjj|G A B ]Q B V s E E p B V A L P
1 -M I G i i l i l i l N in iA T r c iG ia D V g ia P iilH V A V
i- m i g [iM ]n i H a t H g H d v ia ? S t q v c
1- m i g h e ] 3 k i H a t H g H d v E H p 0 g t v s
1-MAe i a d Q i H g l E J g H d i E H q S d r a t
1 3 -r r i R E E Q iH a m S g H n h E E q p s s e t
13-H R V [« ra a Q i HAM [J] g QN Q E E 0Q P S S E T
108-H T d H r a n
249-H e a u b ie e
233-H E l W 31 d e
233-H E lB E Ie e
238-H e l H d e
230- H e t u b i e e
1 9 5-H a e I 3 W e o
194-HK e C T S I d k
202-HQ e E H d k
246-H s q E H a d
279-HD e I3 C T t d
264-HD e E H t d
P T A 0A a S E I q q H c t
P AL0G e B E I r a B I r r
PQ R 0A d BEIk AitlR o
P QR0A DPIfflOAFTiRR
P ER0A d E E J k a E J rr
P AR0A eBCIraICTrr
P R 1 0 K rS D J tlE J q s
P L L 0 K RH3Q l H q d
P L L 0 K rB E Io fB Iy k
S D K0R q 0D 1 q l H n q
S S L 0R tH J ] e m 0 s g
S R L 0R t0 E 1 e m H sg
FIG . 2. Sequence conservation within the i]i subunit of D N A polymerase III. (A) M ultiple alignm ent of i|i amino acid sequences. Num bers
indicate the sequence position of the first residue in each alignm ent block; pairs of vertical lines denote discontinuities in the sequences. W hite
characters with blue shading indicate positions with homology across all sequences. W hite characters with orange shading indicate residues
conserved in 80% of sequences, while black characters with yellow shading indicate 60% conservation. The sequence alignm ent was produced using
the M U SCLE algorithm (72) within the Geneious software package (Biom atters). (B) Crystal structures of the E. coli -y3SS'vfi and
complexes.
Residues conserved in the A . baylyi i]i subunit are colored red and have side chains shown. (Produced with PyM OL [64] using data from Protein
D ata Bank entries 3GLI [231] and 1EM8 [96].)
In creased flexibility betw een the zinc-binding an d R N A poly­
m erase dom ains m ay have significant im plications for prim ase
activity, as (i) the zinc-binding dom ain is believed to act as an
anchor th a t recognizes prim ing sites in D N A an d regulates
R N A p rim er length an d (ii) prim ase zinc-binding dom ains
have been d em o n stra ted to function in trans w ith o th e r p ri­
m ase m olecules in E. coli (50). A th o ro u g h b iochem ical and
stru ctu ral investigation o f th ese u n u su al D n a G p ro te in s is r e ­
qu ired to fully elucidate the sim ilarities to an d differences from
the b etter-stu d ied E . coli enzym e.
Highly Conserved Protein Interaction Modules within the p
Sliding Clamp and SSB
In addition to binding th e P o l III 8 su b u n it durin g clam p
lo ading/unloading (127) an d th e a polym erase su b u n it (261),
greatly increasing its processivity, th e E. coli p sliding clam p
also binds to D N A polym erases I, II, IV , an d V; D N A ligase;
H d a; an d th e m ism atch re p a ir p ro tein s M utS an d M u tL (55,
159, 260). E ach o f th ese p ro te in s binds to a hydrophobic
groove (127) on th e p sliding clam p by w ay o f a p e n tam eric o r
ESSENTIAL PRO CESSES IN ACINETOBACTER SPP.
V ol . 74, 2010
[3-sliding clamp
279
B
a-subunit,DNA polymerase I
A cin e to b a c te r b a u m a n n ii
A cin e to b a c te r b aylyi
P se u d o m o n a s aeru g in o sa
E sc h e ric h ia co li
906- N R E S 0 I Ml i l H y l E B E E V
906- N R E T M I MI » H l 4 ME H E E V
931-SH D S g M ^ p G Q F A E
915- A E A I M Q A ^ m v i j A E E
8-subunit, DNA polymerase I
A cin e to b a c te r b a u m a n n ii
A cin e to b a c te r b aylyi
P se u d o m o n a s aeru g in o sa
E sc h e ric h ia co li
6 1 - 0 F N A L NK H H H l ^ OBlLA
33Q 0L A
61-0FN G L
63- 0 L E A G A S L S L F A e 0 r l
64-0 F S L C Q A M S L F A s S q t
Hda
A cin e to b a c te r b a u m a n n ii
A cin e to b a c te r b aylyi
P se u d o m o n a s aeru g in o sa
E sc h e ric h ia co li
E sc h e ric h ia c o li
S h ig e lla flexn e ri
Yersinia p e s tis
S a lm o n e lla en terica
Vibrio ch o lera e
P se u d o m o n a s a e ru g in o sa
H a em o p h ilu s in flu en za e
A cin e to b a c te r b a u m a n n ii
N eisseria m e n in g itid is
R ick e ttsia ty p h i
B a cillu s a n th ra c is
Liste ria m o n o c y to g e n e s
S ta p h y lo c o c c u s a ere u s
S tre p to c o c c u s p n e u m o n ia e
C lo strid iu m b o tu lin u m
L e p to sp ira in te rro g a n s
H e lico b a cte r p y lo ri
M y co b a cte riu m tu b ercu lo sis
C h la m yd ia tra c h o m a tis
1MR
1MR
1- M K P I
1-M N T P A
169169173167168156159184165143163169158147139141 172155148-
® 0 q H d | | e p q l EI
SO q 0 d h e p q l 0
• 0 P 0 S E 1LRD 0
S0S0PH Y LPD EI
MD Fi i i r o r a a
MD F E
DDDIPF
M D F [D D D I P F
MD :
□ 33
M D f [D D D I P F
D S f [D D D I P F
DGfE E E H E
A D L [D D D L P F
E D i !D D D I P F
SDLEEEH 33
D I sED D D L P F
D I sED D D L P F
D I sED D D L P F
D I sED D D L P F
P V d 0 g 0 E aa
DGg
a ta a
m
N I rr m
G G G [iliiJalP f J 3
Q Y a s in u a
FIG . 3. Highly conserved protein interaction m odules in the Acinetobacter replisome. (A) Structure of the E. coli fiS complex (127). The
solvent-accessible surface of p is shown in gray and red. R ed indicates residues that are conserved in A . baylyi. The 8 subunit is colored teal, and
side chains are shown for the p-binding consensus residues. (B) Portions of sequence alignments for D N A replication proteins containing p-binding
motifs. Boxes indicate p-binding motifs. Residues are highlighted as in Fig. 2. (C) Structure of E. coli exonuclease I bound to the C-term inal peptide
of SSB (162). The solvent-accessible surface of exonuclease I is shown in gray. The C-term inal peptide of SSB is shown in teal. (D ) Portion of a
sequence alignm ent for SSB proteins. In panels B and D , white characters with blue shading indicate positions with homology across all sequences,
white characters with orange shading indicate residues conserved in 80% of sequences, and black characters with yellow shading indicate 60%
conservation. Sequence alignm ents were produced using the M U SCLE algorithm (72) within the Geneious software package (Biom atters). (Panels
A and C were produced with PyM OL [64] using data from Protein D ata Bank entries IJQ L [127] and 3C94 [162], respectively.)
hexam eric p ep tid e m otif, w hich is o ften lo cated close to th e N
o r C term inus (55). In A cinetobacter spp., the residues co m ­
prising th e p ro tein-bin d in g groove o f th e (3 sliding clam p are
highly conserved (Fig. 3A ). V arian ts o f th e (3-binding m otifs
(Q xD/sL F o r QLxLxL) are also conserved w ithin sev eralH c/u etobacter p ro tein s, including the a subunits o f P ol III (IM D L F )
and H d a (Q L Q L D I) (Fig. 3B). In E. coli, a (3-binding m o tif
w ithin the P ol III 8 su b u n it (Q A M S L F ) is u tilized in the
loading o f th e (3 sliding clam p by th e clam p lo a d e r com plex
(Fig. 3B) (127). D esp ite sharing only w eak overall sequence
hom ology w ith the E. coli 8 (26% identity fo r th e A . baylyi
p ro tein ), this m o tif is also m ain ta in e d in A cinetobacter sp. 8
subunits (N SL SLF). T his suggests th a t despite significant se ­
quence divergence in 8 an d o th e r co m p o n en ts o f the clam p
lo a d e r com plex in A cinetobacter spp. (T able 1), loading o f the
(3 sliding clam p o n to D N A is likely to utilize a m echanism
sim ilar to th a t o f th e E. coli system.
T h e hydrophobic groove on th e (3 sliding clam p and its
binding m otifs in o th e r p ro te in s are strongly conserved across
b a cte rial species (27, 260). A n analogous m echanism is also
sh ared by th e P C N A sliding clam p o f arch aea an d eukaryotes
(209), although the consensus sequence recognized by P C N A
(QxxLxxFF) is different from th a t o f th e b a cte rial (3-binding
m otifs (55). T h e stro n g conservation o f this p ro tein -binding
m echanism across b acte ria, to g e th e r w ith th e essentiality o f the
in te ra c tio n s fo rm e d , m ak es th e p ro te in -b in d in g g roove o f
th e (3 sliding clam p (Fig. 3B ) an a ttra c tiv e ta rg e t fo r th e
ra tio n a l design o f n o v el a n tib io tic c o m p o u n d s. P rom isingly,
280
R O B IN S O N E T A L .
th e O ’D o n n ell group has recently described a sm all m olecule
th a t binds w ithin the (3 clam p binding groove an d is capable o f
disrupting key p ro tein -p ro te in in teractio ns in diverse b acte ria
w ith o u t inhibiting analogous in teractio n s in th e eukaryotic
P C N A system (87).
A second replicatio n p ro te in , th e sin g le-stran d ed D N A binding p ro tein (SSB), also in teracts w ith m any o th e r p ro tein s
by way o f a conserved p ep tid e m o tif (229). In ad d itio n to
interacting w ith the D n a G prim ase an d th e x su b u n it o f P ol III
H E during D N A replication, SSB also binds to a range o f D N A
rep a ir enzym es (9, 30, 8 6 ,1 0 0 ,1 3 5 ,1 4 8 ,1 6 2 , 225, 229, 243, 263,
275). T hese in teraction s are m e d ia ted by th e final six to nine
residues a t the SSB C term inus, w hich in E. coli has the se ­
quence M D F D D D IP F (229, 275). T h e crystal stru ctu re o f the
term in al SSB p ep tid e in com plex w ith exonuclease I (Fig. 3C)
w as recently d eterm in e d by K eck’s gro u p (162). T he stru ctu re
and ad d itio n al biochem ical studies (163) rev ealed a key role
fo r the final two residues o f SSB in binding to a hydrophobic
p o ck et in exonuclease I. W hile n o h igh-resolution stru ctu res
have been re p o rte d for any o th e r p ro te in s in com plex w ith the
SSB p ep tid e (230), it is a n ticip ated th a t its binding to o th e r
p ro tein s occurs in a sim ilar fashion; i.e., th e term in al residues
o f SSB will be in serted into specific pockets on th e p ro te in
surfaces.
T h e SSB C -term inal p ep tid e rep re se n ts a univ ersal system
fo r tying to g eth er processes tra n sa c ted o n single-stranded
D N A (229). E ven in genetically rem o te organism s, such as
A cinetobacter spp., the final six residues o f SSB are strongly
conserved (Fig. 3D ). T his conservation extends, in fact, to SSB
p ro tein s from a w ide range o f bacte rial species, indicating th a t
this system is probably u se d universally across all o f th e b ac­
teria (Fig. 3D ) (229).
T h e (3 clam p an d SSB con stitu te m ajo r p ro te in in teractio n
hubs in bacteria. E ach is know n to in te ra c t w ith at least 10
o th e r p ro tein s, form ing in teractio n s w ith different p a rtn e rs at
different tim es. In doing so, th e (3 clam p an d SSB tie to g e th e r
functional processes re q u ire d for genom e m ain ten an ce by r e ­
cruiting o th e r p ro tein s to th e ir sites o f action on double- and
single-stranded D N A , respectively. M any o f th ese in teractin g
p a rtn e rs are essential across a w ide range o f b acte ria (277). A t
the h e a rt o f these fun ctio n al linkages are sh o rt, highly c o n ­
served p ep tid e m otifs th a t could be exploited as the basis for
the design o f novel antibiotics. A t th e individual level, inh ib i­
tors o f (3 clam p an d SSB in teractio n s should be capable o f
sim ultaneously disrupting several functions critical to th e su r­
vival o f a b acterial cell. F u rth e rm o re , as these m odules show
strong sequence conservation across diverse b acte rial gen era,
inhibitors should also have b ro ad -sp ectru m activity. Finally,
and p erh ap s m ost crucially, th e fact th a t n u m ero u s p ro tein s
in teract at a single site o n each o f th ese hubs m ight h elp to
p reclude the o p p o rtu n ity fo r resistance to such inhibitors. T he
accum ulation o f p o in t m u tatio n s th a t decrease th e affinity o f
inhibitory com pounds for th e ir ta rg e t is a com m on th em e in
antibiotic resistance (168). Presum ably, in th e case o f p ro tein p ro tein in teractio n sites, any m u tatio n occurring o n one in te r­
acting p a rtn e r w ould n e e d to be c o m p en sated fo r w ith a co m ­
p lem en tary m u tatio n on th e o th e r p a rtn e r to m ain tain the
integrity o f the in teractio n site. F o r this to occur in the (3-clamp
and SSB p ro tein -b in d in g sites, it w ould necessitate th e unlikely
acquisition o f sim ultan eo u s co m p en sato ry m u tatio n s in the
M ic r o b io l . M o l . B io l . R e v .
binding sites o f every one o f th e m any p ro te in s th a t bind at
these sites. T hese p ro te in in teractio n hubs thus re p re se n t an
u n u tilized an d relatively u n ex p lo red reso u rce fo r th e future
discovery o f novel antibiotic drugs.
CELL DIVISION
C ell division is an app ealin g ta rg e t for the design o f an tib ac­
teria l drugs, as it is also essen tial fo r th e viability o f b acterial
p o p u latio n s and, as m any o f th e p ro te in s involved are external
to th e cytoplasm , it is a ta rg e t relatively accessible to attack
w ith sm all-m olecule inhibitors. In ad d itio n , th e m ajority o f
p ro tein s have little o r no hom ology to h u m an p ro teins. In
co n tra st to th e case fo r th e processes o f D N A replication and
tran scrip tio n , th e m ajority o f o u r know ledge on cell division
has em erg ed only over the p a st 10 to 15 years. M ost studies so
far have focused o n two ro d -sh ap ed organism s, B. subtilis and
E. coli, bo th o f w hich divide precisely at the cell cen ter. C u r­
rently, a t least 12 p ro te in s are know n to be involved in the cell
division process, an d th ese are d istrib u ted b etw een the cyto­
plasm , cell m em b ran es, an d periplasm ic space (76, 89, 103,
156, 259). F o r the m ajority o f these p ro te in s, th e ir exact b io ­
chem ical functions rem ain unknow n. T h ree-d im en sio n al stru c­
tu re s o f th e e n tire p ro tein s o r p a rts o f th em have b een solved
fo r m any, how ever, an d p ro te in -p ro te in in teractio n s are now
being d ete rm in ed an d ch aracterized (T able 2).
T h e process o f cell division involves th e ingrow th o f the cell
envelope layers to form a sep tu m a t th e cell c e n te r betw een
two newly rep licated chrom osom es. T his sep tu m th e n splits to
form tw o new d a u g h ter cells. T h e first step in cell division is the
assem bly o f th e tubulin-like p ro te in F tsZ in to a structure
know n as th e Z ring, precisely at m idcell (103). T his assem bly
involves Z ip A in E. coli (98) an d the m em b ran e-attach in g
p ro tein F tsA in b o th E. coli an d B. subtilis (54, 103, 255). T he
Z ring m arks th e fu tu re site o f cell division an d acts as a
“landing p a d ” fo r th e o th e r p ro tein s involved in the division
process. O nce in place, th e rem ain in g cell division p ro tein s are
rec ru ite d to th e Z ring, form ing a com plex collectively know n
as th e divisom e (103). In E. coli these later-assem bling p ro tein s
include F tsE , -X, -K, -Q , -L, -B, -W, -I, an d -N; A m iC ; and
EnvC (255, 259). H om ologs o f all o f th ese p ro te in s except FtsN
are fo u n d in B. subtilis.
H om ologs o f m o st o f th ese p ro tein s can also be fo u n d w ithin
th e genom es o f A cinetobacter spp., although in m o st cases th eir
sequences have diverged substantially from those o f th eir E.
coli co u n te rp a rts (T able 2). Interestingly how ever, no h o ­
m ologs could be fo u n d fo r F tsE , F tsX , o r SulA, w hich are
conserved in alm o st all o th e r g am m ap ro teo b acteria. F tsE and
-X to g e th e r form a m em b ran e-asso ciated A B C tra n sp o rte r o f
unknow n cellular function th a t is essen tial fo r th e survival o f E.
coli only u n d e r low -ionic-strength conditions (218). In B. su b ­
tilis this p ro te in com plex has recently b een linked to the in iti­
ation o f sp o ru latio n (85). SulA is an in h ib ito r o f F tsZ poly­
m erizatio n th a t is ind u ced as p a rt o f th e SOS response in E.
coli, w here it serves to tem p o rarily h a lt cell division in the
event o f D N A dam age (49, 112). A s discussed in a la te r sec­
tion, th e lack o f S ulA in A cinetobacter spp. has stro n g im pli­
cations fo r th e m echanism s underlying global stress responses
in th ese organism s.
ESSENTIAL PRO CESSES IN ACINETOBACTER SPP.
V o l . 74, 2 0 1 0
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282
R O B IN S O N E T A L .
Assembly of FtsZ at Midcell
F tsZ is the m ost conserved b a cte rial cell division p ro te in and
is an essential player in th e cell division process (103). H om ologs o f F tsZ have b een identified in alm o st all p ro k ary o tes
as w ell as in p la n t chloroplasts an d the m ito ch o n d ria o f low er
eukaryotes (165, 253). In E. coli, th e fts Z gene resides in the
dew cluster, from w hich ftsQ , -A, an d -Z are co tran scrib ed (63,
84, 271). A nalysis o f th e available A cinetobacter sp. genom es
suggests the sam e to be tru e fo r this genus, w ith ftsQ , A , an d
-Z being adjacen t to each o th e r in the chrom osom e. T he se­
quences o f F tsZ from A cinetobacter spp. are relatively well
conserved (T able 2), p articu larly w ithin the core dom ain,
w hich contains the G T P -b in d in g m o tif an d is im p o rta n t fo r the
polym erization o f F tsZ in to th e Z ring. A m ino acid residues
involved in G T P hydrolysis an d fo rm atio n o f th e active site
(190, 257) are also conserved.
A ssem bly o f F tsZ in to the Z ring at precisely m idcell r e ­
quires a balance am ong several p ro tein s th a t in te ra c t directly
w ith F tsZ to regulate its assem bly (3, 54, 156). T hese p ro tein s
e ith e r p ro m o te o r inhib it F tsZ p o lym erization an d are th o u g h t
to act to g e th e r to su p p o rt Z -rin g assem bly at the rig h t place
and tim e. In E. coli, F tsZ assem bly is p ro m o te d by F tsA , Z ipA ,
and Z a p A and -B an d is inh ib ited by M inC, C lpX , SulA, and
Slm A (3, 54, 156).
A t the C term in u s o f F tsZ is a nine-am ino-acid consensus
sequence, o ften re fe rre d to as th e C -term in al core dom ain,
w hich has been show n to be involved in in teractio n s w ith b o th
Z ip A an d F tsA (101, 165, 179, 267). In E. coli an d m ost o th e r
g am m ap ro teo b acteria, th e sequence o f this region is D IP A F L R K Q . T he Som ers gro u p has d ete rm in e d the crystal stru ctu re
o f the C -term inal core dom ain in com plex w ith Z ip A (Fig. 4A).
T he n o n ap ep tid e p ro tein -b in d in g m o tif o f F tsZ w as show n to
form an am phipathic helical stru ctu re in w hich residues 1374,
F377, an d L378 form th e p rim ary contacts w ith Z ipA . E arlie r
studies using alanine-scanning m utagenesis show ed th a t these
sam e th ree residues provide n early all o f th e binding stren g th
in the in teractio n w ith Z ip A in E . coli (179). R esid u es D373
and P375 o f E. coli F tsZ are also im p o rta n t in th e in teractio n
w ith Z ipA , how ever, p resu m ab ly setting th e C -term in al region
in the co rrect co nform atio n fo r binding (101).
By pro jectin g the n o n ap ep tid e m otifs from th e F tsZ p ro tein s
o f diverse bacteria o n to a helical w heel, it can be seen th a t
m ost m aintain an am p h ip ath ic helix stru ctu re w ith h y d ro p h o ­
bic residues at positions 2, 5, an d 6; A sp a t p o sitio n 1; and P ro
at position 3 (Fig. 4B). T he fact th a t this m o tif is conserved in
G ram -positive organism s (which lack Z ip A ) su p p o rts the idea
th a t the in teractio n o f F tsZ w ith o th e r p ro tein s, m ost notably
F tsA , also involves the C -term in al am p h ip ath ic helix. In te re st­
ingly, w hile hydrophobic residues are m ain tain ed at positions
2, 5, an d 6 o f the F tsZ C -term in al p ep tid es in A cinetobacter
spp. (S IQ D Y L K N Q ), th e conserved A sp an d P ro residues
fo u n d in o th e r organism s at positions 1 an d 3 have b een r e ­
placed w ith Ser and G in. It has previously b een show n th a t
site-specific m u tatio n o f th ese p ositions (D 373 an d P375) in E.
coli F tsZ results in d isru p ted binding to Z ip A (101). Im p o r­
tantly, P375 m u tan ts also show re d u c ed binding to F tsA (165).
T he u n u su al rep lacem e n t o f this resid u e w ith G in in A c in e to ­
bacter spp. m ight suggest a som ew hat different binding m ode
fo r F tsZ to b oth Z ip A an d F tsA in th ese organism s.
M ic r o b io l . M o l . B io l . R e v .
C om plexation o f F tsZ w ith F tsA , Z ip A , Z a p A , an d Z apB
rep re se n ts th e startin g p o in t o f th e cell division pathw ay in E.
coli. A s such, these in teractio n s m ed ia ted by the F tsZ C -term inal helix have b een view ed as attractive targ ets fo r the d e ­
sign o f novel antibiotics. W yeth, in p articu lar, has invested
considerable effort in the d ev elo p m en t o f antagonists o f the
F tsZ -Z ip A in teractio n an d has discovered several com pounds
th a t bind to Z ip A a t th e F tsZ in teractio n site (123, 124, 179,
223, 244, 247). Im p o rtan tly how ever, n o n e o f th ese have led to
a u seful a n tag o n ist o f the F tsZ -Z ip A in teractio n , d espite the
o p p o rtu n ities fo r stru ctu re-g u id ed design afforded by crystal
stru ctu res o f Z ip A in com plex w ith th ese ligands (1 2 3 ,124,223,
244, 247). T he difficulty in expanding th ese le a d com pounds
lies w ith th e fact th a t F tsZ binds in to a shallow , an d relatively
featureless, hydrophobic groove o n th e Z ip A surface (179).
T h e b ro a d in teractin g surface provides few o p p o rtu n ities to
in co rp o rate p o la r contacts b etw een Z ip A an d th e designed
ligands. T h e few attem p ts to do so have yielded little o r no
increase in binding stren g th (247). W yeth has since ab an d o n ed
its efforts to develop an F tsZ -Z ip A antagonist.
W y eth ’s inability to successfully ex ten d ap p aren tly prom ising
lead com pounds in to u seful inhibitors o f this in teractio n high­
lights th e difficulty associated w ith targ etin g p ro te in -p ro tein
in teractio n s using stru ctu re-aid ed design. T h e b ro a d and re la ­
tively shallow in teractio n surface o f Z ip A ultim ately decreases
th e utility o f this in teractio n as a targ et. F u rth e rm o re , analysis
o f th e C -term in al helices o f F tsZ p ro te in s from diverse b a c te ­
ria suggests only a vague re q u ire m e n t for hydrophobic residues
on the in teractin g face o f the helix (Fig. 4B). T his im plies th a t
th e re is significant variatio n in th e shape o f F tsZ -b in d ing sites
in p ro tein s from different b acteria, fu rth e r com plicating drug
design efforts. Im p o rtan tly , as th e binding site fo r F tsA w ithin
F tsZ overlaps significantly w ith the binding site for Z ip A , this
m ight im ply th a t th e F tsZ -F tsA in teractio n , also view ed as a
p o te n tia l drug targ et, could be equally difficult to target.
Recruitment of Membrane-Associated Proteins
Follow ing fo rm atio n o f th e Z ring, th e later-assem bling
m em b ran e-asso ciated cell division p ro te in s are re c ru ited to the
m idcell site an d drive the p rocess o f sep tu m fo rm ation (89,
254). T hese p ro te in s re p re se n t ap pealing targ ets fo r the devel­
o p m e n t o f novel an tibacterials, as regions essen tial for th eir
function are largely lo cated outsid e th e (in n er) cell m em brane,
m aking th em m ore accessible to inhibitors. T h e ir p o te n tia l as
an tib acterial targ ets is also increased because m o st o f these
p ro tein s are essen tial an d th e m ajority lack hom ologs in e u ­
karyotes (156). T h e core se t o f essen tial m em b ran e-associated
cell division p ro tein s have now b een id entified (103). H ow ever,
few biochem ical d ata are y et available fo r th ese p ro tein s, and
m any are still to be structurally d efined ow ing to th e in h eren t
difficulties associated w ith in vitro analyses o f m em b rane p ro ­
teins. A ll o f the core m em b ran e cell division p ro tein s are
identifiable in A cinetobacter spp. o n th e basis o f H M M -based
hom ology searches w ith E. coli p ro tein s (T able 2).
O f th e m em b ran e-asso ciated cell division p ro te in s in E. coli,
F tsQ in teracts w ith the g re ate st n u m b e r o f o th e r p ro tein s, w ith
tw o-hybrid studies an d co im m u n o p recip itatio n experim ents r e ­
vealing in teractio n s w ith F tsA , -K, -X, -I, -W, -B, -L, and -N
(28, 66, 69, 136). S ep arate regions w ithin th e periplasm ic po r-
ESSENTIAL PRO CESSES IN ACINETOBACTER SPP.
V ol . 74, 2010
283
A
ZipA surface
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- Acinetobacter baum annii
- Escherichia coli
- Salm onella enterica
- Shigella flexneri
- Yersinia pestis
- Vibrio cholerae
- Pseudom onas aeruginosa
- Neisseria meningitidis
- Rickettsia typhi
- Bacillus anthracis
- Clostridia botulinum
- Leptospira interrogans
- Helicobacter pylori
- Staphylococcus aureus
- Haemophilus influenzae
- Listeria m onocytogenes
- Mycobacterium tuberculosis
- Streptococcus pneum oniae
- Cam pylobacter jeju n i
- Porphyrom onas gingiva I is
eco pae cje vch rty cbo ype spn
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FIG . 4. The FtsZ -Z ipA interaction. (A) Diagram of the FtsZ C-term inal peptide in contact with the Z ipA surface (179). The solvent-accessible
surface of Z ipA is colored according to atom type: C, white; N, blue; O, red; S, yellow. N um bering indicates positions within the conserved
nine-residue motif. (Produced with PyM OL [64] using data from Protein D ata Bank entry 1F47 [179].) (B) Helical projection of the FtsZ
C-term inal peptide. L etters at each position indicate the residue occupying that position in different bacterial species.
tion o f FtsQ are re q u ire d fo r localization o f the p ro te in to the
division site an d re c ru itm en t o f dow nstream p ro te in s (37, 250).
T he periplasm ic region form s a m o d u lar tw o-dom ain stru ctu re
th a t crystallizes as a dim er y e t is m o n o m eric in solution (250).
L ocalization to th e Z ring req u ires in teractio n w ith u p stream
p ro tein s such as F tsK an d is m ed ia ted largely by th e a dom ain
(residues 58 to 125) (2 5 0 ). A cinetobacter FtsQ p ro tein s are only
m od erately conserved, sharing 26% overall seq u en ce identity
w ith E. coli F tsQ (T able 2). R esid u es previously show n to be
re q u ire d fo r localization o f E. coli F tsQ (V92, Q 108, V l l l , and
K113) (37, 90, 250) m ap to the o u te rm o st tip o f th e a dom ain
an d are conserved w ithin A cinetobacter F tsQ p ro te in s (equiv­
ale n t positions are L95, R 110, V 113, an d R 115).
Im m u n o p rec ip itatio n experim ents have d e m o n stra ted th a t
in E. coli th e th re e p ro te in s F tsL , -B, an d -Q assem ble away
from th e m idcell site an d arrive as a com plex (28). R e c ru itm en t
o f these p ro te in s by F tsQ involves th e (3 dom ain (residues 128
to 260), also w ithin th e p eriplasm ic p o rtio n (250). R esidues
re q u ire d for th ese in teractio n s m ap to the final two stran d s o f
a c e n tra l (3 sh eet, at the interface o f the crystallographic dim er.
284
R O B IN S O N E T A L .
T he corresponding positio n s in A cinetobacter F tsQ p ro tein s
are relatively w ell conserved, suggesting th e re c ru itm en t o f
FtsB and -L to share a m echanism w ith E. coli. F u rth e r studies
o f the stru ctu ral basis o f F tsQ in teractio n s are re q u ire d to
identify p o te n tia l targ ets fo r th e ra tio n a l design o f novel antibacterials. In the fu tu re, it m ight th e n be possible to design
com pounds th a t d isru p t th e localization o f F tsQ to th e Z ring
o r its re c ru itm en t o f dow nstream p ro te in s such as FtsB an d -L.
Peptidoglycan Recycling and Response to
p-Lactam Antibiotics
R e c e n t studies have rev ealed th a t A baylyi strains carrying
m utations in the peptidoglycan-recycling enzym e UDP-1Vacetylm uram ateiL -alanyl-y-D -glutam yl-m eso-diam inopim elate
ligase (m urein p ep tid e ligase; M pl) are hypersensitive to (3-lac tam antibiotics (91). In co n trast, m p l-deficient strain s o f E. coli
are no m ore sensitive to p-lactam co m p o u n d s th a n w ild-type
strains (91). D eletio n o f th e en tire m p l gene results in nonvi­
ability in A baylyi A D P 1 u n d e r th e grow th conditions u se d in
the hig h -th ro u g h p u t gene k n o ck o u t study (61). T h e equ iv alen t
gene is dispensable for grow th u n d e r sim ilar conditions in E.
coli (11, 170). Interestingly, an A baylyi strain carrying an m pl
m u tatio n is also sensitive to U V -in d u ced D N A dam age (36).
A side from tra n sp o rt p ro tein s, no p ro te in s involved in cell
envelope synthesis have b een linked w ith th e D N A dam age
response in E. coli (36).
B oth U V dam age an d p-lactam co m p o u n ds trig g er the
L exA -m ediated SOS resp o n se in E. coli (80 ,1 7 3 ). T his leads to
u p reg u latio n o f the cell division in h ib ito r SulA, leading to
tem p o rary a rrest o f cell division, allowing tim e fo r D N A re p air
processes to occur (143). A s discussed below , A cinetobacter
spp. lack n o t only SulA b u t also th e SOS reg u la to r p ro tein
LexA. T h e sensitivity o f ;u/>/-deficient A . baylyi to b o th (3-lac tam s an d U V irrad ia tio n m ight th e re fo re reflect an inability to
a rrest cell division in response to D N A dam age in A c in e to ­
bacter spp. It has previously been show n th a t A cinetobacter spp.
u n d erg o reductive division follow ing n u trie n t starvation; i.e., a
large bacilliform cell divides to yield sm aller coccoid cells
(119). I t is tem p tin g to specu late th a t reductive division re p ­
resents a g en eral stress response in Acinetobacter an d th a t the
observed p-lactam /U V sensitivity o f ;u/>/-deficient strains
m ight reflect a defect in rem o d elin g p eptidoglycan in the tr a n ­
sition to a coccoid m orphology. T his is su p p o rte d by th e id e n ­
tification o f m u tatio n s in o th e r peptidoglycan-recycling e n ­
zymes th a t lead to p-lactam sensitivity in Acinetobacter spp.
(91), although one can n o t ru le o u t m o re direct inhibition o f
each o f these enzym es by p -lactam com pounds.
I t has been suggested th a t if suitable inhibitors o f M pl can be
found, this m ight lead to novel antibiotics th a t are selective for
A cinetobacter spp. an d act to increase th e efficacy o f p-lactam
com pounds (91). T h e Jo in t C e n te r fo r S tru ctu ral G enom ics
has recently d eterm in e d th e crystal stru ctu re o f Psychrobacter
arcticum M pl (P ro tein D a ta B an k accession no. 3H N 7), h ig h ­
lighting th e previously n o te d relatio n sh ip w ith M urC enzym es
(177). P. arcticum M pl shows relatively high seq u en ce co n ser­
vation w ith A cinetobacter M pl p ro te in s (56% sequence id e n ­
tity). It m ight now be possible to rationally design inhibitors o f
A cinetobacter M pl p ro tein s b ased o n th e P. arcticum stru ctu re.
M ic r o b io l . M o l . B io l . R e v .
RNA TRANSCRIPTION
B acterial tran scrip tio n an d tra n scrip tio n al reg u lation have
been the subjects o f intense analysis since th e co n c u rren t iso­
lation o f D N A -d ep e n d e n t R N A polym erase (R N A P ) from E.
coli by fo u r sep arate lab o rato ries during th e early 1960s (114).
T h e process can be divided into th re e distinct phases: in itia­
tion, elong atio n , an d term in atio n (25). F irst, R N A P in com plex
w ith a p ro m o te r specificity co m p o n en t, o r a factor, recognizes
an d unw inds th e tem p late D N A a t a specific p ro m o te r se­
quence u p stre am o f th e gene to be tran scrib ed (25). R N A P
th e n begins synthesizing m R N A , a t first pro d u cin g a series o f
sh o rt abortive tran scrip ts before clearing th e p ro m o te r and
e n terin g a highly processive elong atio n stage. T ran scrip tio n is
c o m p leted e ith e r by intrinsic term in atio n at a specific sequence
in the tem p late D N A o r by R h o fa c to r-d e p e n d e n t term in atio n ,
w hich occurs at poo rly conserved R h o utilization sites (43).
E ach stage o f tran scrip tio n is highly reg u lated , e ith e r by
R N A P -asso ciated tran scrip tio n facto r p ro tein s o r by specific
R N A o r D N A sequences th a t in terac t w ith the R N A P tr a n ­
scription com plex (74). By specifically reg u latin g th e tran scrip ­
tion o f individual genes (or groups o f genes), th e cellular levels
o f each gene p ro d u c t can be re g u la ted a n d ad ju sted in r e ­
sponse to a v ariety o f cellu lar an d e n v iro n m en tal stim uli.
T h e A cinetobacter sp. genom es seq u en ced to d ate originate
from strains th a t occupy th re e distinct en v iro n m en tal niches: a
free-living soil organism , a h u m an body louse sym biont, an d a
series o f m u ltid ru g -resistan t an d drug-susceptible clinical iso­
lates (4 ,1 5 ,1 1 5 , 233, 248). T hus, as w ell as being able to assess
th e overall conservation o f tran scrip tio n c o m p o n en ts in A c in ­
etobacter spp. (T able 3), we are now in a p o sitio n to identify
accessory factors likely to be involved w ith gene regulation
during p ath o g en esis an d antibio tic-in d u ced stress.
Architecture of Transcription Complexes and
Suitability for Drug Design
T h e R N A P core contains five p ro te in subunits, assem bled in
th e o rd e r a —* a 2 —* a 2(3 —* a 2(3(3' —* a 2(3(3'co (24). T o initiate
tran scrip tio n (152), th e ~ 4 0 0 -k D a core m u st first associate
w ith a a facto r to form th e R N A P holoenzym e ( a 2(3(3'coo-). T he
arch itectu re o f th e R N A P com plex has b een elucidated
th ro u g h extensive biochem ical an d biophysical analyses in E.
coli (review ed in referen ce 24) an d crystal stru ctu res o f the
Therm us aquaticus R N A P core (276) an d Thermits thennophilus holoenzym e (251). T he overall topology o f R N A P resem ­
bles a “crab claw ,” in w hich th e large (3 an d (3' subunits com ­
prise th e “p in cers” o f the claw (276) (Fig. 5A ). In ad dition to
providing a stru ctu ral brace fo r R N A P , th e (3 an d (3' subunits
in terac t to create a cleft th a t op en s in to the m ain ch an n el o f
th e enzym e. D e e p inside th e base o f this cleft resides the
catalytically active c en te r, w hich is m ark e d by a stably b o u n d
M g2+ ion.
G en es encoding R N A P subunits are readily identifiable in
A cinetobacter spp. an d m ain tain a relatively high level o f se­
quence conservation (54 to 71% identify a t th e am ino acid
level) w ith equ iv alen t E . coli subunits (T able 3). W ithin the
id entified a , (3, (3', co, an d a subunits, key fu n ctio n al regions
(e.g., seq u en ce m otifs A to H o f th e (3' su b u n it [132]) are highly
conserved. T h e relatively high conservation o f R N A P subunits
V ol.
ESSENTIAL PRO CESSES IN ACINETOBACTER SPP.
4, 2010
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286
RO BIN SO N E T AL.
M ic r o b io l . M o l . B io l . R e v .
B
P'-subunit, RNA polymerase
(E
Thermus thermophilus
539-
Escherichia coli
2 6 4 - i a wg —
q g
t u m J T O i m n r A a jw M iiiM a jiw .w f a .M M .1
Acinetobacter baumannii
Acinetobacter baylyi
285
a™__________
Thermus thermophilus
l a
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G i: M t\ s EOT
Escherichia coli
Acinetobacter baumannii
Acinetobacter baylyi
389- K i;« d E l a y
E ia B o l a n i H M ti m c i d I m u
390- C T g a Eri r n m i n i a T O M M i l n a N E ltH lo u h »i i » h h h ; i i ia n ;i.f ;M
Region 2.1
Region 2.2
FIG . 5. A rchitecture of R N A polym erase and its interaction w ith a 70. (A) S tructure of the Thermus thermophilus R N A P holoenzym e
(251). The solvent-accessible surface is shown for R N A P core subunits (a jP P 'w ), and a 10 is shown in ribbon rep resen tatio n . (B) Prim ary
interaction surface betw een the coiled-coil region within the (!' subunit and conserved regions 2.1 and 2.2 of a 70. D ashed lines indicate hydrogen
bonds. (C) Sequence alignments for the coiled-coil region of the (!' subunit and conserved regions 2.1 and 2.2 of ct70. W hite characters with blue
shading indicate positions with homology across all sequences, while black characters with yellow shading indicate 60% conservation. Sequence
alignments were produced using the M U SCLE algorithm (72) within the Geneious software package (Biomatters). (Panels A and B were produced
with PyM OL [64] using data from Protein D ata Bank entry 1IW7 [251].)
despite th e relative g enetic rem o ten ess o f th e A cinetobacter
genus w ithin th e g am m ap ro teo b acteria (Fig. 1) highlights the
p o te n tia l utility o f R N A P as a ta rg e t for b ro ad -sp ectru m a n ti­
b acterial com pounds.
R N A P has been identified as th e ta rg e t fo r several classes o f
transcription inhibitors, including th e clinically im p o rta n t rifamycin fam ily (256). F ligh-resolution crystal stru ctu res o f
R N A P in com plex w ith m em b ers o f th e rifam ycin, streptolydigin, sorangicin, and lipiarm ycin in h ib ito r fam ilies have sh ed
light on th e ir m odes o f action (256). E ach o f th ese com pounds
w as fo u n d to bind at distinct locations w ithin th e D N A -binding
and secondary channels o f R N A P , away from the active site.
T his suggests th a t they b in d an d in terfe re w ith reg u lato ry sites
w ithin the enzym e, acting e ith e r to sterically block nucleoside
trip h o sp h ates (N T Ps) from e n terin g th e active site o r by allosteric effects. M ost com pounds th a t inh ib it R N A P activity ex­
hibit a b road-spectrum bactericid al effect. It has b een sug­
gested th a t R N A P is an u n d eru tilized reso u rce an d rem ain s a
prom ising ta rg e t for th e discovery o f new antibiotics (for r e ­
views, see references 42 an d 256).
W hile several antim icrobial co m pounds have b een fo u n d to
ta rg e t R N A P directly, very few ta rg e t th e highly conserved
p ro tein -p ro tein interactio n s th a t govern the fo rm atio n o f
higher tran scrip tio n com plexes. In vivo, th e R N A P core form s
a series o f tra n sien t com plexes w ith o th e r p ro te in cofactors:
a 70 an d o th e r a factors, an titerm in atio n factors (N usA , -B, -E,
an d -G ), tra n sc rip t cleavage factors (G re A an d -B), and re g u ­
lato ry factors (e.g., D ksA ) (152). U ltim ately, th ese in teractions
u n d e rp in th e reg u latio n o f gene expression an d th e tran scrip ­
tion o f rR N A an d th u s play im p o rta n t roles in cell p ro life ra ­
tion.
T h e p rim ary in teractio n surface b etw een a 70 an d th e R N A P
core has b een m a p p e d to th e coiled-coil m o tif o f th e (3' subunit
an d th e conserved region 2.2 o f a 70 (272). T h e stru c tu ral basis
fo r th e R N A P -ct70 in teractio n w as d e m o n stra te d by the crystal
stru ctu re o f th e T. them iophilus holoenzym e (251). T he in te r­
action surface (130) is relatively p o la r an d is fo rm ed prim arily
th ro u g h a n etw o rk o f side chain-side chain an d side chain-m ain
chain hydrogen b o nds (Fig. 5B). If novel antagonists o f this site
could be found, they should in terfe re w ith assem bly o f the
fu n ctio n al R N A P holoenzym e an d th u s inh ib it tran scription in
vivo. T he (3' an d a 70 residues th a t m ed iate this in teractio n in ft.
coli are iden tical in the A cinetobacter sp. p ro tein s (Fig. 5C) and
are strongly conserved am ong all o th e r b a cte ria (130).
U sing an enzym e-linked im m u n o so rb en t assay (E L ISA )b ased app ro ach , the L e o n etti group has screen ed a library o f
sm all m olecules for inhibitors o f th e assem bly o f a 70 o n to the
R N A P core (8). Several inhibitors o f holoenzym e assem bly
V ol . 74, 2010
w ere identified, w ith som e d em o n stratin g bactericid al activity,
particularly against G ram -positive organism s. Interestingly,
w hile E. coli p ro tein s w ere u sed fo r th e initial screen an d w ere
inhibited in vitro, no an tim icrobial activity w as o bserved fo r E.
coli o r its fellow G ram -negative b acte riu m P. aem ginosa. A c­
tivity w as seen, how ever, against an E. coli m u ta n t strain w ith
increased o u te r m em b ran e p erm eability, suggesting th a t the
lack o f activity against th e w ild-type b acte riu m stem s from
lim ited cellular u p tak e o f th e com pounds. N o n eth ele ss, this
w ork d em o n strates th e p o te n tia l fo r p ro te in -p ro tein in te ra c ­
tions w ithin transcriptio n com plexes as targ ets fo r a n tim icro ­
bial com pounds.
H ig h -th ro u g h p u t screening ap p ro ach es could be sim ilarly
u sed to identify antagonists o f o th e r R N A P -in teractin g p ro ­
teins such as N usA an d -G , bo th o f w hich are essen tial in E.
coli, P. aeruginosa, and A baylyi (T able 3). T rad itio n ally these
interactions w ould n o t be co n sid ered g o o d targ ets, as th e in ­
teractio n surfaces lack clear grooves, w ithin w hich an in h ib ito r
m ig h t form m o re extensive p ro te in c o n ta c ts. H o w ev er, r e ­
c e n t d ev e lo p m e n ts in a n tic a n c e r th e ra p ie s w ith sta p le d p e p ­
tid e an ta g o n ists o f N O T C H co m p lex fo rm a tio n in d ic a te th a t
(i) in te ra c tio n su rfaces lack in g d e e p clefts o r g ro o v es can be
ta rg e te d a n d (ii) p re v e n tio n o f th e fo rm a tio n o f active tr a n ­
scription com plexes rep re sen ts a p o ten tially valuable th e ra p e u ­
tic ap p ro ach (176).
T ran scrip tio n com plexes involved in rR N A synthesis, often
called an titerm in atio n com plexes, req u ire re cru itm en t o f many
p ro tein tran scrip tio n factors, such as N usA , -B, -E, an d -G, an d
also req u ire nucleic acid elem en ts o f th e R N A le a d e r o f rR N A
genes called boxB, -A, an d -C (202). A n a n titerm in atio n co m ­
plex c an n o t correctly form w ith o u t th e full co m p lem en t o f
p ro tein and R N A elem ents. T he boxA sequences are highly
conserved across the eu b ac te ria (consensus, 5 '-G C U C U U U [A
/G ][A /G ]A ; A cinetobacter consensus, 5 '-G A U C A U U A A G )
and are in teg ral to th e fo rm atio n o f th e N usB -N usE h e t­
ero d im er th a t binds R N A polym erase (93). M ore recently it
has been show n th a t N u sA binds boxC (consensus, 5 '-U G U G
U [U /G ][U /G ]; A cinetobacter consensus, 5 '-U G U G U G G ),
w hich is involved in a n titerm in atio n com plex fo rm atio n (10,
23). T argeting these pro tein -n u cleic acid in teractio n s m ay also
be a valid ap proach fo r th e d ev elo p m en t o f new antibiotics, as
preventing an titerm in atio n com plex fo rm atio n w ould severely
disrupt cell p roliferatio n . In d e e d rR N A gene dosage is closely
re la te d to th e ra te o f cell p ro life ra tio n , w ith fast-g ro w in g
species such as B. subtilis, E. coli, a n d Vibrio natriegens
c o n ta in in g 7 to 13 rR N A o p e ro n s p e r c h ro m o so m e , w hile
th e slow -grow ing o rg an ism M ycobacterium tuberculosis c o n ­
tain s a single rR N A gene (152). A cin eto b a c ter sp ecies c o n ­
ta in five to seven rR N A o p ero n s an d are know n to grow
rapidly, suggesting th a t rR N A synthesis m ay be a valid ta rg e t
fo r bacteriostatic o r bactericid al ag en t developm ent. It may,
fo r exam ple, be possible to design nucleic acid m im etic drugs,
such as p ep tid e nucleic acid (P N A ) o r lock ed nucleic acid
(L N A ) (217) to com petitively inh ib it an titerm in atio n com plex
form ation th ro u g h in teractio n w ith boxA o r C sequences. A l­
though cell p erm eability rem ain s a significant h u rd le to the
effective use o f PN A s an d L N A s as drugs, they can n e v e rth e ­
less be u sed to discover p h a rm aco p h o res fo r th e design o f
m ore cell-p erm ean t com pounds (217).
ESSENTIAL PRO CESSES IN ACINETOBACTER SPP.
287
Alternative Sigma Factors and Their Regulation
B acteria resp o n d to changing e n v iro n m en tal conditions by
(often dram atically) alterin g th e expression o f stress response
genes. T his reg u latio n is effected prim arily at th e level o f tr a n ­
scription, in p a rtic u la r th ro u g h th e differential use o f a factors
w ithin th e R N A P holoenzym e (167). D u rin g exp onential
grow th in rich m edia, a 70, the h o u sek eep in g a factor, directs
tran scrip tio n o f m ost essen tial genes. Follow ing ap p lication o f
a p a rticu lar cellular stress, an altern ativ e a facto r w ith specific
affinity for a certain p ro m o te r seq u en ce can rep lace a 70 and
thus d irect tran scrip tio n to w ard th e specific set o f genes r e ­
q u ired u n d e r th e stressed conditions. U n d e r n o rm al co n d i­
tions, these alternative a factors are rep ressed , e ith e r by rap id
d eg rad atio n o r by th e ir seq u estratio n by a n ti-a factors.
A w ide disparity is observed in th e n u m b e r o f a factors
en co d ed by bacteria; e.g., E. coli has 7 different a factors, B.
subtilis has 17, an d Streptomyces coelicolor has 63 (152). A lte r­
native a factors id entified in E. coli include a 28, a 38, a 32, a 54,
an d th e extracytoplasm ic function a factors (E C F s) cte and
a FecI (80, 175). O f th ese, only hom ologs o f a 32 an d a 54 are
identifiable in A cinetobacter spp., along w ith one to five E C F a
factors (T able 4).
a 32 (R p o H ) is th e p rim ary reg u la to r o f th e h e a t shock r e ­
sponse in bacteria. In E . coli, a te m p e ra tu re shift to >42°C
results in th e in duction o f a subset o f m o re th a n 20 <r32-regula te d h e a t shock genes, including th e m o lecu lar ch ap ero n es
D n a J a n d -K an d G ro E L an d -ES. T hese p ro tein s serve to
stabilize expressed p ro tein s in th e cell cytoplasm , p reventing
m isfolding an d aggregation, w hich are especially com m on d u r­
ing p erio d s o f h e a t stress. I t has been show n th a t th e strain A .
calcoaceticus 69-V resp o n d s to h e a t shock by u p reg u latin g a
sim ilar se t o f p ro tein s, including D n aK , G ro E L , an d H tp G
(18). T his suggests th a t th e a 32 regulon in A cinetobacter spp. is
pro b ab ly sim ilar to th a t in E. coli.
T h e a 54 fam ily is u n u su a l in th a t, unlike all o th e r a factors,
its m em b ers display n o sequence hom ology w ith th e h o u se ­
k eep in g facto r a 70. In E . coli, a 54 w as originally im plicated in
th e reg u latio n o f n itro g en m etabolism . H ow ever, m any ad d i­
tio n al reg u lato ry roles have since b een a ttrib u te d to this p ro ­
tein (211,219). W hile n o system atic a tte m p t has yet b een m ade
to ch aracterize th e a 54 regulon in A cinetobacter spp., a 54 has
b een show n to be re q u ire d fo r th e utilizatio n o f p h e n o l and
benzyl alk an o a tes as m etabolic su b strates in A baylyi (73,133).
T h e expression o f C om P, a pilin-like p ro te in essential for
tran sfo rm atio n al com petence in A . baylyi A D P 1 , is p re s e n t at
th e highest levels during late statio n ary phase an d also is likely
to be re g u lated by a 54 (210). It was recen tly fo u n d th a t genes
u n d e r th e c o n tro l o f the <r54-reg u lated Pu p ro m o te r from
P seudom onas pu tid a could be expressed exogenously in A bay­
lyi A D P 1 (111). E xpression p a tte rn s m irro re d those seen in its
n a tu ra l host, suggesting th a t th e re is a t least som e co m patibil­
ity b etw een th e a 54 reg u lato ry system s o f A cinetobacter spp.
an d P seudom onas spp.
E C F sigm a factors are extrem ely divergent, a n d in general
sequence analysis alone is insufficient to en ab le p red ictio n o f
th e ir function (106). E. coli contains two a factors w hich group
w ithin th e E C F subclass: cte an d a FecI. A nalysis o f th e genom e
sequence o f A . baylyi A D P 1 reveals th a t this organism contains
th ree genes w ith som e sim ilarity to E. coli a FecI, w hich is
RO BIN SO N E T AL.
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4. Escherichia coli alternative a factors and regulators and their homologs in Pseudomonas aeruginosa and Acinetobacter spp.
I
TABLE
288
V ol . 74, 2010
involved in a signaling cascade leading to th e cellular u p ta k e o f
iron, in the form o f ferric citrate (T able 4). In th e A . baylyi
A D P1 chrom osom e, all th re e o f these sequences reside u p ­
stream o f a gene th a t encodes a F ecR -like a n ti-a facto r an d a
m em brane rec e p to r p ro tein . In co n trast, A . baum annii strains
contain e ith e r n one o r only one o f these p ro te in s, suggesting
th a t alternative iro n u p tak e pathw ays are im p o rta n t in its
p athogenesis (279).
In E. coli, cte is indu ced follow ing h e a t shock an d durin g the
early stationary p hase an d reg u lates the expression o f aro u n d
200 genes, overseeing th e cell envelope stress response (57). It
is essential in E. coli, even u n d e r ap p aren tly n o n stressed co n ­
ditions, and loss o f cte activity has b een show n to resu lt in loss
o f cell envelope integrity (105). R em arkably, Salm onella enterica serovar T yphim urium cte , w hich shares 99% sequence
identity w ith E . coli cte , is n o n essen tial b u t plays a critical role
in virulence (113). T h e equ iv alen t p ro tein in P. aeruginosa,
A lgU , regulates genes involved w ith th e m ucoid phen o ty p e
associated w ith cystic fibrosis (211). C learly, th e re is m uch
variation in th e cellular functions re g u la ted by cte regulons in
different bacterial species, although all are linked by th e ir site
o f action at the cell envelope.
T h e scope o f the cte regulon in A cinetobacter spp. has y et to
be exam ined. Interestingly, how ever, it has b een n o ted th a t the
A deA B C m ultidrug efflux p u m p , w hich is associated w ith r e ­
sistance to several classes o f antibiotics in A . baum annii, is
likely to be tran scrib ed from an E C F -re g u late d p ro m o te r
(166). G iven th a t som e strain s o f A . baum annii do n o t contain
any d iscern ib le a FecI h o m o lo g s, it is highly likely th a t th e
adeABC operon is transcribed by RNAP-cte in Acinetobacter spp.
N o hom ologs o f the an ti-a facto r R seA /M u cA o r th e periplasmic p ro te in R seB /M ucB , w hich negatively regulate th e activity
o f a E (273), w ere fo u n d in th e p u b lish ed A cinetobacter sp.
genom es. T he genom e o f A . baylyi A D P 1 contains a second
hom olog o f a E (A C IA D 0935); th e function o f this p ro te in is
n o t yet clear (T able 4).
T h e absence o f a 28 (or R p o F ) from th e genom es o f A c in ­
etobacter spp. com es as n o surprise. T he a 28 facto r is a re g u ­
lato r o f flagellar biosynthesis (200), an d as m em b ers o f a nonm otile genus, Acinetobacter spp. do n o t carry genes for
flagellum assembly. Intriguingly, th e a 38 (o r R p o S ) sigm a fac­
to r, w hich is th o u g h t to be th e m a ster stress response re g u lato r
in g am m ap ro teo b acteria (107), is also m issing from A c in e to ­
bacter spp. T his a facto r con tro ls th e in duction o f a su b set o f
m ore th an 100 genes involved in stress resistance in E. coli,
including those w hose p ro d u cts resist oxidative stress, acidic
p H , eth an o l, and hyperosm olarity (107). A lth o u g h a 38 is u n ­
detectable in exponential-phase cultures, its levels reach 30%
o f th a t o f a 70 in statio n ary p h ase, allowing it to m ore effectively
com pete w ith a 70 fo r binding to core R N A P (129). T his is
fu rth e r en h an ced by th e co n c u rre n t in d u ctio n o f R sd , an a n ti-a
facto r th a t binds to a 70, p rev en tin g it from in teractin g w ith the
R N A P core (274). T he gene encoding R sd is sim ilarly ab sen t
from th e genom es o f A cinetobacter spp. T h e a p p a re n t lack o f a
a 38 system in Acinetobacter (an d th e re la te d genus Psy­
chrobacter) is u n iq u e w ithin g am m ap ro teo b acteria (excluding
sym biotic organism s w ith re d u ce d genom es). T h e p o ten tia l
im plications o f a lack o f a 38 in the global stress response and
ad ap ta tio n to antibiotics in these organism s are discussed
below.
ESSENTIAL PRO CESSES IN ACINETOBACTER SPP.
289
Pathogenic Acinetobacter spp. Are Enriched in
Transcription Factors
A re c e n t com parative genom ics study o f A cinetobacter
strains has rev ealed a se t o f 475 genes th a t are fo u n d in the
genom es o f all com pletely seq u en ced p ath o g en strains b u t n o t
in th e n o n p ath o g en ic A . baylyi A D P 1 genom e (4). T his set,
te rm e d th e p a n -A. baum annii accessory genes, includes 59 th a t
encode p ro te in s p re d ic te d to be tran scrip tio n factors b ased on
sequence hom ology. T h e L uxR , T e tR , M erR , A raC , M arR ,
G n tR , A crR , A rsR , B adM /R rf2, X R E , an d H xlR fam ilies o f
tran scrip tio n factors are re p re se n te d , suggesting th a t A cin eto ­
bacter path o g en esis involves a com plex tran scrip tio n al netw ork
th a t reg u lates th e expression o f p ro tein s w ith diverse b iochem ­
ical functions. F u rth e r investigation is re q u ire d to exam ine the
precise roles o f these tran scrip tio n factors in pathogenesis.
AN UNUSUAL DNA DAMAGE RESPONSE
S everal genes associated w ith D N A replication, tran scrip ­
tion, an d cell division are n o t fo u n d w ithin the genom es o f
A cinetobacter spp., despite being conserved in virtually all
o th e r g am m ap ro teo b acteria. In p articu lar, the e rro r-p ro n e
polym erase D inB (D N A polym erase II), th e stress response
facto r a 38, an d th e cell division p ro te in s SulA, F tsE , an d F tsX
a p p e a r to be u niquely m issing in A cinetobacter an d Psy­
chrobacter spp. (T able 5).
W hile a 38 is b est know n fo r its roles in statio n ary phase and
global stress response reg u latio n , its role in th e response to
D N A dam age in E. coli w as recently described (171). In the
sam e organism , dinB an d sulA are b o th in d u ced as p a rt o f the
SOS D N A dam age response m ed ia te d by th e tran scrip tio n al
reg u la to r LexA. T o g e th e r w ith th e fact th a t acin eto b acters also
lack LexA, th ese m issing genes indicate an u n u su al D N A d am ­
age response in A cinetobacter spp.
T h e SOS response has b een w ell ch aracterized in E. coli
(143). T h e response is trig g ere d by D N A dam age, som e classes
o f antibiotics, o r th e disru p tio n o r overexpression o f certain
genes (1 4 ,1 4 3 ,1 9 5 ). D ete c tio n o f one o f th ese triggers leads to
R e c A -d e p e n d e n t au toproteolysis o f LexA, a rep re sso r th a t
governs th e tran scrip tio n o f aro u n d 30 genes in E. coli (29).
W hile som e variatio n is seen am ong th e m em b ers o f the LexA
regulon in different species, secondary D N A polym erases (e.g.,
D inB an d U m u D ) an d cell division inhibitors, such as SulA,
are alm ost always u p re g u la te d (2, 44, 45, 56, 128, 143). P ro ­
m oters governing th e expression o f th ese p ro tein s are d e re ­
p ressed , an d th ere fo re u p reg u late d , u p o n cleavage o f LexA
(143). A s a result, cell division is tem p o rarily a rre ste d and
D N A dam age is re p a ire d w ith th e aid o f re p a ir an d reco m b i­
n atio n systems.
T h u s far, only two D N A dam age-inducible loci have been
id entified in A cinetobacter spp. (T able 5). N o t surprisingly,
D N A dam age in A baylyi A D P 1 (th en know n as A. calcoaceticus) w as fo u n d to resu lt in u p reg u latio n o f th e recA gene,
w hich is associated w ith D N A rep a ir an d reco m b in ation in
b acteria (216). U nlike in m ost o th e r organism s, how ever, this
regu latio n w as in d e p e n d e n t o f th e R e cA p ro te in , suggesting a
novel m echanism for th e D N A dam age response in this o rg an ­
ism. T h e second gene fo u n d to be u p re g u la te d follow ing D N A
dam age in A . baylyi A D P 1 is ddrR, w hich has n o identifiable
290
RO BIN SO N E T AL.
M ic r o b io l . M o l . B io l . R e v .
TABLE 5. D N A damage response proteins in Acinetobacter spp.
Protein
Function and comment
E. coli D N A damage proteins not found in
Acinetobacter spp.
D in B ................................................................................................................ D N A polymerase II; error-prone polymerase involved in SOS mutagenesis
ct38.................................................................................................................... Stationary phase/stress response ct factor
SulA ................................................................................................................. Cell division inhibitor; induced during SOS response
F tsE X ..............................................................................................................T ransporter of unknown function; induced during SOS response
L exA ................................................................................................................ Transcriptional regulator; primary regulator of SOS response
Recognized D N A damage response proteins
in Acinetobacter spp.
U m u C ..............................................................................................................D N A polymerase V subunit; truncated to UmuC* (first 64 residues of
Um uC) in A . baylyi ADP1
U m u D A b ........................................................................................................D N A polymerase V subunit; 50% longer than E. coli U m uD protein,
contains LexA-like protease recognition site, regulates expression of
D drR in response to D N A damage
D d r R ...............................................................................................................Unknown function; upregulated following induction of D N A damage in A.
baylyi ADP1, found only in Acinetobacter spp.
R ecA .................................................................................................................Recom bination protein A; upregulated following induction of D N A
damage in A . baylyi ADP1, required for D N A damage-inducible
regulation of D drR
sequence hom ologs in th e c u rre n t sequence d atab ases an d is o f
unknow n function (102). R eg u latio n o f ddrR w as fo u n d to be
R ecA d ep e n d e n t and ap p ears to involve an u n u su al U m u D
(D N A polym erase V ) p ro te in , U m u D A b , th a t contains a
LexA-like dom ain at th e N term inus. D isru p tio n o f this novel
reg u lato r led to a decrease in the expression o f ddrR, suggest­
ing th a t it acts as an activator ra th e r th a n a rep resso r, in
co n trast to LexA. In A . baylyi A D P 1 , th e u m u D A b gene form s
p a rt o f an u n u su al u m u D C o p e ro n in w hich th e u m u C gene has
b een tru n cated , ap p aren tly as a resu lt o f tran sp o so n activity
(102). u m uD A b an d th e tru n c a te d u m u C gene are constitutively expressed from this o p ero n . A . baum annii strains each
a p p ear to contain an u m u D A b -h ke gene in an o p e ro n w ith a
full-length u m u C gene.
T h e <T38-m ed iated stress an d L exA -m ediated SOS responses
are activated n o t only by D N A dam age b u t also by com m on
drug classes: (3-lactams, quinolones, an d folate biosynthesis
inhibitors (143, 184). A ctivation o f these resp o n ses leads to
u p reg u latio n o f m utatio n -in d u cin g e rro r-p ro n e D N A poly­
m erases an d in creased ra tes o f la te ra l gene tra n sfe r (80). T he
a 38 an d SOS responses are now th o u g h t to play a significant
role in the evolution o f antibiotic resistance in b acte ria by
increasing the scope for adaptive evolution th ro u g h in creased
rates o f m u tatio n and gene tran sfer (75, 80, 81, 143). T his sets
up an a p p a re n t p arad o x for som e A cinetobacter spp.: they lack
the a 38 an d L exA stress responses y et have acq u ired resistance
to all antibiotics in clinical use. In fact, it has b een show n th a t
unlike m ost o th e r bacte ria, A . baylyi A D P 1 does n o t show an
induction o f m utagenesis following irrad ia tio n w ith U V light
(19). T his suggests e ith e r th a t alte rn ate u n c h aracterized stress
response m echanism s su p p o rt th e acquisition o f dru g resis­
tance d eterm in an ts in A cinetobacter spp. o r th a t th e pathw ays
leading to m u tatio n an d /o r la te ra l gene tran sfe r are constitutively enabled. T he high level o f transform ability associated
w ith the lab o rato ry strain A . baylyi A D P 1 could be tak en as
evidence fo r the la tte r scenario; how ever, high-level n a tu ral
com petence has n o t yet been d em o n stra te d fo r any o th er A c in ­
etobacter strain. G iven the ra p id em ergence o f d ru g resistance
in A cinetobacter spp. an d th e ir increasing significance as p a th o ­
gens, it is im p o rtan t to develop an u n d e rstan d in g o f antibioticin d u ced stress resp o n ses in th ese organism s, as they are clearly
different from those th a t have b een stu d ied to date.
PERSPECTIVES
T his review exam ines th ree essen tial biological systems
w ithin A cinetobacter spp., i.e., D N A replication, cell division,
an d R N A tran scrip tio n , w ith a view to fu tu re ex ploitation o f
these system s as targ ets fo r ra tio n a l design o f novel a n tim icro­
b ial com pounds. O f course, th ese are n o t the only p o ten tial
targ ets fo r drug design in A cinetobacter spp. R ib o so m al p ro tein
synthesis, fo r exam ple, has n o t b een co v ered h ere, y et it is one
o f th e m o st com m on targ ets fo r a n tib acterial com pounds.
C o m p ared w ith rep licatio n , cell division, an d tran scrip tion p ro ­
teins, rib o so m al p ro tein s an d rR N A m olecules are very highly
conserved across th e b acte ria an d in fact even w ithin the e u ­
karyotes an d archaea. A cinetobacter spp. show little variation
w ithin these co m p o n en ts relative to o th er, b e tte r-stu d ied b ac­
teria. P rim ary m etabolism an d cell w all stru ctu res, w hich are
also com m on drug targ ets, are n o t covered h ere , as in fo rm a­
tion a b o u t th ese system s is difficult to ex tract from genom e
sequence info rm atio n . O u r analysis has, how ever, d em o n ­
stra te d th e m erits o f using genom e sequence d ata to ex trap o ­
late in fo rm atio n a b o u t fu n d a m e n ta l biological processes from
m o d el organism s to clinically relevant, b u t m ore poo rly u n d e r­
stood, bacteria. T his analysis has allow ed us to identify highly
conserved aspects o f D N A rep licatio n , cell division, an d tr a n ­
scription in these organism s, w hich could have p o te n tia l as
targ ets for th e ra tio n a l design o f novel antim icrobials. W e
recognize, o f course, th a t a g re a t d eal m o re w o rk is re q u ire d to
assess th e actual dru g susceptibilities o f these systems, even
b efore th e challenges o f developing inhibitors th a t are n o n ­
toxic to hum ans, are available at th e site o f infection, are
capable o f being tak en u p by bacterial p ath o g en s, an d th en
exhibit a bacterio static effect can be addressed.
W hile research in to th e fu n d am en ta l aspects o f A cineto-
ESSEN TIA L PROCESSES IN ACINETOBACTER SPP.
V o l. 74, 2010
bacter biology is still in its infancy, it is already clear th a t th ese
b acteria have m any un u su al attrib u tes. H e re w e have bro u g h t
to th e surface som e p ecu liar characteristics o f th eir D N A re p ­
lication m achinery. T h e se observations imply th a t ch ro m o ­
som al replication in th ese organism s m ight involve subtle
m echanistic differences from E. coli and o th e r bacteria. W ithin
th e cell division p ro tein s, in te rru p tio n of th e gene encoding th e
peptidoglycan-recycling enzym e M pl has been show n to p ro ­
duce a |3-lactam -sensitive p h en o ty p e in A . baylyi A D P 1. Such
readily observable phenotypes are ra re for knockouts o f p e p ­
tidoglycan-recycling genes (199). A lth o u g h th e reaso n for this
is currently unknow n, it does suggest th a t A baylyi A D P1 m ight
prove to be a useful m odel organism for studying p ep tid o g ly ­
can recycling in G ram -negative b acteria.
R N A P an d o th e r co m p o n en ts o f th e basic tran scrip tio n m a ­
chinery in A cinetobacter spp. are sim ilar to th o se fo u n d in o th er
bacteria. S om e variation is seen , how ever, in th e u se of tra n ­
scriptional regulators. A cinetobacter spp. a p p e ar to contain a
som ew hat m inim ized se t o f altern ativ e a factors, with som e
strains containing only a E, a 32, a 54, and o n e E C F -ty p e a fac­
tor. Interestingly, Acinetobacter spp. also lack m any o th e r ca­
nonical stress response p ro tein s, including th e D N A dam age
response reg u lato r LexA. T h ey do, how ever, contain a large
nu m b er o f p u tativ e D N A -binding rep resso rs and activators,
m any o f which are of unknow n function. T h e num bers and
types o f th ese transcrip tio n al regulators vary am ong A cin eto ­
bacter species, suggesting th a t they play specific roles in a d a p ­
tatio n o f each species to its en v iro n m en t. T h is is evident in th e
fact th a t the genom es of p ath o g en ic A cinetobacter spp. are
highly enriched for genes encoding tran scrip tio n al regulators.
F u rth e r research into tran scrip tio n al stress responses in indiv id u A A cinetobacter species is vital, as such responses are likely
to play a cen tral role in th e dev elo p m en t o f antibiotic resis­
tan ce in th ese organism s, w hich is p e rh ap s th e fo rem o st p ro b ­
lem associated with tre a tin g A cinetobacter sp. infections in th e
clinical setting.
A lready it is clear th a t A cinetobacter spp. have diverged
significantly from o th e r g am m ap ro teo b acteria over tim e and
have developed u n iq u e solutions for coping w ith evolutionary
pressures. W hile this p resen ts challenges for conducting A c in ­
etobacter research, th e trem en d o u s utility o f A baylyi A D P1 as
a laboratory organism provides an exciting o p p o rtu n ity to u n ­
d erstan d m any processes and pathw ays in th ese organism s,
thereby increasing o u r u n d e rsta n d in g o f bacte rial evolution in
th e context o f a group o f b a c te ria th a t are still em erging as
hum an pathogens.
ACKNOWLEDGMENTS
Our research in these areas was supported by a project grant (to
P.J.L., E.J.H., and N.E.D.) from the Australian National Health and
Medical Research Council and by an Australian Research Council
Australian Professorial Fellowship to N.E.D.
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V o l . 74, 2 0 1 0
Andrew Robinson obtained a B.Sc. in Biolog­
ical Chemistry with First Class Honors from
Macquarie University (Sydney, Australia) in
2003. He then commenced postgraduate stud­
ies in the laboratory of Associate Professor
Bridget Mabbutt at Macquarie, receiving his
Ph.D. in 2008. His graduate studies focused
on the structures of integron-associated
proteins and their roles in adaptation in bac­
teria. He joined the laboratory of Professor
Nick Dixon at the University of W ollongong
in 2007, where he is characterizing structures and functions of D N A
replication proteins from Acinetobacter spp. He has a keen interest in
the evolution of bacterial proteins and the developm ent of antibiotic
resistance.
Anthony J. Brzoska graduated with First
Class H onors from the University of Tech­
nology, Sydney, in 2001, before commencing
a Ph.D. in the laboratory of Dr. Neville
Firth and Professor Ron Skurray at the U ni­
versity of Sydney in 2003. His Ph.D. studies
focused on the molecular and functional
analysis of par, an active partitioning system
from the staphylococcal multiresistance
plasmid pSK41. In 2007, he joined the lab­
oratory of Associate Professor P eter Lewis
at the University of Newcastle, where his current work focuses on the
determ ination of novel protein-protein interactions in transcription
and D N A replication in Acinetobacter spp. and Staphylococcus aureus.
Kylie M. Turner undertook her postgradu­
ate studies at the University of Sydney, A us­
tralia, in the laboratory of Dr. Lesley W right
and D r. Julianne Djordjevic and received
her Ph.D. in 2007. The focus of her graduate
studies was the biochemical and structural
characterization of the cryptococcal viru­
lence factor phospholipase B. She com­
menced her postdoctoral work in Associate
Professor Elizabeth H arry’s laboratory at
the Institute for the Biotechnology of Infec­
tious Diseases at the University o f Technology, Sydney, in 2007, where
her research focused on the identification of novel cell division pro­
teins in Acinetobacter spp.. Bacillus subtilis, and Staphylococcus aureus.
She has a strong interest in the discovery of novel antimicrobials.
Ryan Withers obtained a bachelor’s degree
in biotechnology at the University of New­
castle in 2007. He is currently undertaking
Ph.D. studies in m olecular microbiology in
Peter Lewis’ laboratory, focusing on charac­
terization of transcription factors in A cineto­
bacter baylyi.
ESSENTIAL PRO CESSES IN ACINETOBACTER SPP.
297
Elizabeth J. Harry received her Ph.D. in the
laboratory of Professor Gerry W ake from
the University of Sydney in Australia. H er
postdoctoral work was in Professor Richard
Losick’s laboratory at H arvard University.
She was an A ustralian Research Council
Research Fellow at the University of Sydney
until 2004, when she was appointed an As­
sociate Professor and principal investigator
at the Institute for the Biotechnology of In­
fectious Diseases (IBID ) at the University
of Technology, Sydney. An enduring focus of her research is on the
mechanism of bacterial cell division and its regulation. A more recent
interest is the discovery and m echanism of action of antibacterials.
Peter J. Lewis obtained a B.Sc.(Hons) in
Biochemistry at the University of Sheffield,
U nited Kingdom, in 1986. Following a move
to Australia, he gained a Ph.D. in Biochem ­
istry at the University of Sydney in 1991. He
returned to the U nited Kingdom in 1992 as
an SERC/NATO postdoctoral fellow with
Professor Jeff E rrington, working on the
regulation of sporulation, chromosome seg­
regation, and subcellular protein localiza­
tion in Bacillus subtilis. In 2000 he returned
to Australia to establish an independent research group at the U ni­
versity of Newcastle. Currently, his research program focuses on bac­
terial transcription regulation, transcription complex composition,
structure, and subcellular dynamics.
Nicholas E. Dixon was awarded a Ph.D. in
biochemistry by the University of Q ueens­
land, Australia, in 1978, and carried out
postdoctoral studies in chemistry at the
Australian National University (ANU),
Canberra, and held Fulbright and C.J. M ar­
tin Fellowships at Stanford University,
where he worked with Professor A rthur
Kornberg. He returned to Australia as a
Q ueen Elizabeth II Fellow and was ap­
pointed to the faculty of the Research
School of Chemistry, A N U , in 1986. In 2006, he moved to the U ni­
versity of W ollongong, Australia, where he is currently an Australian
Research Council Professorial Fellow in biological chemistry. His in­
terests are in mechanistic and structural aspects of bacterial D N A
replication.