Section of Infectious Diseases, Department of Medicine, Boston Medical Center, Boston University School of Medicine, Boston, Massachusetts 02118, USA
Correspondence
You-xun Zhang
yxzhang{at}bu.edu
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ABSTRACT |
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INTRODUCTION |
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One of the important mechanisms in the switch of gene expression in bacteria is the use of alternative factors to alter promoter selectivity of RNA polymerase (RNAP) (Ishihama, 2000
; Burgess & Anthony, 2001
; Gross et al., 1998
). The basic composition of chlamydial RNAP resembles that of other eubacterial RNAPs in containing subunits
2
'
(Stephens et al., 1998
). Three
genes, rpoD encoding
66, rpoN encoding
54 and rpsD (fliA or whiG) encoding
28, and several genes encoding the homologues to
regulators RsbW, RsbU and RsbV in Bacillus subtilis, exist in the chlamydial genome (Stephens et al., 1998
; Kalman et al., 1999
). Chlamydial
28 is homologous to the
28 family of bacterial
factors, including
28 in Escherichia coli and
B and
D in B. subtilis. Chlamydial RNAP containing
66 is capable of initiating transcription from both E. coli
70 consensus and non-consensus promoter sequences (Douglas & Hatch, 1995
; Shen et al., 2000
; Tan et al., 1996
; Hatch, 1999
). The major
factor rpoD transcripts were detected at all times post-infection (p.i.), consistent with their expected function in the expression of housekeeping genes (Douglas & Hatch, 2000
; Mathews et al., 1999
). In contrast, transcripts of the alternative
factors rpoN and rpsD were temporally expressed. Recently, Yu & Tan (2003)
demonstrated that recombinant
28, combined with chlamydial core RNAP, transcribes the late-stage-specific histone-like protein gene hctB from a promoter that resembles the E. coli
28 consensus recognition sequence. However, information on the conditions under which the chlamydial
28 is activated and the spectrum of promoter sequences recognized by
28 remains unclear. Homologues to
28 in other organisms regulate the expression of flagellar, sporulation, stress response, type III secretion and virulence components (Stephens, 1999
). Therefore, it is of great importance to identify the regulatory mechanism of gene expression in this medically important pathogen.
In the present work, we set out to characterize the biological role of the chlamydial alternative factor,
28. Our data show that the product of chlamydial rpsD is an E. coli
28 homologue. We also report for the first time that expression of rpsD is heat-responsive in C. trachomatis serovar F. These findings suggest that
28 may be involved in the transcription of a group of genes that are required for an adaptation response enabling the completion of the development cycle.
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METHODS |
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The plasmids and oligonucleotide primers used are listed in Table 1 and Table 2
, respectively. An expression vector pBAD24H was derived from pBAD24 (Guzman et al., 1995
) by insertion of a 5' terminal 6x His-tag and more cloning sites, and contained an arabinose-inducible/glucose-repressible ara gene promoter, PBAD. Primers for the coding region of chlamydial rpsD were designed according to the published sequence of C. trachomatis serovar D (Stephens et al., 1998
). The forward primer, S28pri-F, contained an NcoI site and the sequence downstream of the translation initiation codon. The reverse primer, S28pri-R, contained an SphI site, the stop codon TAA and its upstream sequence. Genomic DNA from purified EB of C. trachomatis F/IC-cal-13 was used as template for PCR. The PCR product of the chlamydial rpsD coding region was digested with NcoI and SphI, and cloned into pBADH. Using a similar strategy, the coding region of E. coli fliA was also cloned into pBADH. Oligonucleotide primers E28pri-F and E28pri-R were designed according to the published sequence of E. coli K-12 (Blattner et al., 1997
) and genomic DNA from E. coli DH5
was used as template for PCR. These constructs were transformed into E. coli TOP10. The transformants were analysed by restriction mapping and nucleotide sequencing. Resultant plasmids were designated as pS28H (containing chlamydial rpsD) and pES28H (containing E. coli fliA), respectively.
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A number of G-less cassette-based plasmids were constructed for in vitro transcription analysis. Plasmid pMT504 (gift of Dr Ming Tan, University of California, Irvine, CA, USA) (Tan & Engel, 1996), which contained a longer promoterless G-less cassette and a shorter G-less cassette under the control of the promoter of chlamydial rrn P1, was used for cloning the P1 and P3 promoters of tuf (encoding translation elongation factor EF-Tu) (Shen et al., 2000
). The P1tuf and P3tuf PCR products were generated using primer pairs Ptufpri-F/P1tufpri-R and PtufpriF/P3tufpriR (Table 2
), digested with EcoRI and EcoRV and inserted into EcoRI and EcoRV sites of pMT504. The resultant plasmids were designated as pGP1tuf (containing chlamydial P1tuf and P1rrn) and pGP3tuf (containing P3tuf and P1rrn), respectively. Plasmid pGL was derived from pMT504 by removing the chlamydial rRNA P1 promoter and introducing a PacI site by inverse PCR using primers Gpri-F and Gpri-R. Oligonucleotide pairs, fliCpri-U and fliCpri-L, containing the promoter region of E. coli fliC (Blattner et al., 1997
) were annealed and cloned into PacI and SacI sites of pGL, resulting in pGLC (Fig. 1
). The promoter region of groE (Tan et al., 1996
) was amplified by PCR using GroEpri-F and GroEpri-R (Table 2
) and inserted into EcoRI and EcoRV sites of pGLC, resulting in pGPgroE (containing PgroE and PfliC). Oligonucleotide pairs Psig28-U and Psig28-L (Table 2
), containing the putative promoter P1 of chlamydial rpsD, were annealed and cloned into EcoRI and EcoRV sites of pGLC, resulting in pGPS1. The DNA sequences of all cloned genes were confirmed by restriction mapping and nucleotide sequencing.
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Primer extension was carried out as described previously (Shen et al., 2000). Synthetic oligonucleotide primers were complemented to chlamydial rpsD, covering 3858 bp downstream sequence of the GTG translation initiation codon (fliA-pe1, Table 2
) and of the G-less region of pMT504 (Gless-pe1, Table 2
) by PCR. The Gel Doc system with molecular analysis software Quantity One (Bio-Rad) was used for calculation of integrated volumes of signals, which were proportional to the levels of the transcripts.
Total RNA was reverse-transcribed using AMV reverse transcriptase (Promega) and the resulting cDNA was amplified by PCR using Taq DNA polymerase (USB). Primers P1s28-F and S28pri-R2 were expected to generate a 447 bp fragment from C. trachomatis rpsD mRNA. A 302 bp fragment of 16S rRNA was amplified using primers 16sRT-F and 16sRT-R (Table 2) by PCR and used as the internal standard for relative comparison of gene expression at each time point p.i.
Western blot analysis.
Levels of 28 expression at different time points during the developmental cycle were measured by Western blot analysis (Sambrook & Russell, 2001
). Briefly, infected cells were collected by centrifugation of aliquots of suspension culture at 2800 r.p.m. (Centra GP8R; IEC) for 10 min at 35 °C. Cells were gently resuspended in 0·1 M PBS (pH 7·4) and sonicated briefly to break cell membranes. Chlamydial EB and RB were collected by microcentrifuge at 15 000 r.p.m. for 20 min, quickly resuspended in distilled water, followed by the immediate addition of 2x loading buffer and boiled for 10 min. After centrifugation for 10 min at 15 000 r.p.m. at 4 °C, the supernatant fluids were carefully collected. Uninfected cells served as negative controls. Following separation by SDS-PAGE, proteins were transferred onto Immobilon-P (Millipore). Anti-RpsD, a polyclonal antibody directed against the putative chlamydial
28 of C. trachomatis was generously provided by Dr Thomas P. Hatch (University of Tennessee, USA) and was used to detect chlamydial
28. GP-45, an mAb against chlamydial EF-Tu (Zhang et al., 1994
) was used to probe EF-Tu in companion samples or its dilutions. Blots were developed by using mouse anti-rabbit immunoglobulin G-horseradish peroxidase (Sigma) and a SuperSignal Chemiluminescent Detection kit (Pierce).
Overexpression of rpsD genes in E. coli and purification of the recombinant proteins.
E. coli YK4104 (Table 1) carrying plasmids pS28H or pES28H was grown at 30 °C in LB containing 100 µg ampicillin ml-1, induced with 0·002 % L-arabinose in the exponential growth phase (OD595=0·50·7) for 2 h and then harvested by centrifugation. Three grams of cell pellet were resuspended in 20 ml buffer A (50 mM Tris/HCl, pH 8·0, 1 mM EDTA, 5 mM DTT, 1 mM PMSF). The 6x His-tagged recombinant protein was purified by metal-chelate affinity chromatography as described previously (Zhang et al., 1997
) and dialysed against buffer B (20 mM Tris/HCl, pH 8·0, 0·2 M NaCl, 0·5 mM DTT, 0·1 mM EDTA, pH 8·0, 5 % glycerol), followed by a HiTrap HP Q Column (Amersham Pharmacia Biotech). After washing the column with buffer B, the proteins were eluted with a linear gradient of NaCl (0·21 M) in buffer B. Purified protein fractions were monitored by SDS-PAGE, pooled, dialysed against a storage buffer (10 mM Tris/HCl, pH 8·0 at 4 °C, 10 mM MgCl2 0·1 mM EDTA, 0·2 M NaCl, 50 % glycerol, 1 mM DTT, 0·1 % Triton X-100) and stored at -20 °C until use.
In vitro transcription analysis.
An RNAP holoenzyme containing chlamydial 28 was reconstituted by adding 1 unit E. coli core RNAP (Epicentre Technologies) to 4·0 pmol recombinant chlamydial
28 in protein dilution buffer (10 mM Tris/HCl, pH 8·0, 10 mM NaCl, 1 mM DTT, 0·1 mM EDTA, pH 8·0, 0·4 mg bovine serum albumin ml-1, 0·1 % Triton X-100) and incubating on ice for 15 min. Similarly, a holoenzyme containing E. coli
28 was reconstituted using E. coli core and recombinant
28. E. coli RNAP holoenzyme saturated with
70 (E
70) was obtained from Epicentre Technologies. In vitro transcription was performed in a 10 µl reaction volume containing 1 µg G-less supercoiled template plasmid, 2 µl holoenzyme, 400 µM ATP, 400 µM UTP, 1·2 µM CTP, 0·20 µM [
-32P]CTP, 100 µM 3'-O-methylguanosine 5'-triphosphate (Amersham Pharmacia Biotech) and 20 U RNase inhibitor, following the conditions described by Tan & Engel (1996)
. One volume of gel loading buffer containing 95 % (v/v) formamide, 0·025 % (w/v) xylene cyanol, 0·025 % (w/v) bromophenol blue and 0·5 mM EDTA (pH 8·0), was then added to the mixture to stop the reaction. The products were run on a 6 % polyacrylamide/8 M urea gel as described by Sambrook & Russell (2001)
and gels were subjected to autoradiography. Transcripts were quantified by determining the integrated volume of signal using the Gel Doc system with molecular analysis software Quantity One (Bio-Rad).
Genetic complementation.
E. coli K-12 strain YK410 is wild-type for motility and chemotaxis, while its isogenic fliA- mutant YK4104 (Table 1) lacks this phenotype (Komeda et al., 1980
). YK4104 was used as host for expression of the putative chlamydial
28 and E. coli
28. Plasmids pLF28 (carrying chlamydial rpsD), pLE28 (carrying E. coli fliA) and pLC3 (the vector control) were transformed into YK4104, respectively. Motility of the transformants was tested on 0·35 % agar (10 g Bacto tryptone, 3 g Bacto agar, 5 g NaCl in 1 l water) plates using swarm assays. Swarm assays were performed by stabbing fresh bacteria onto a semisolid agar plate and incubating the plates at 30 °C for 8 h. Motility was assessed by examining the circular swarm formed by the growing motile bacterial cells.
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RESULTS |
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Expression of rpsD transcript I was further examined by an RT-PCR assay of total RNA extracted from normal and heat-shocked cells. 16S rRNA was used as the internal standard for relative comparison of gene expression. As shown in Fig. 2(c), the levels of control transcript were almost equal at each time point p.i., enabling direct comparison of rpsD transcript I levels between normal and heat-shocked cells. Consistent with the result of primer extension analysis, rpsD transcript I apparently increased on temperature upshifting from 35 to 42 °C at both 16 and 30 h p.i., but the most significant change occurred at 16 h p.i. The finding that
28 was upregulated as a result of heat shock suggested that it might play a role in the chlamydial stress response.
Expression of chlamydial 28 levels during the developmental cycle
The levels of 28 protein in infected cells harvested at different time points p.i. were measured by Western blot analysis using anti-RpsD. We examined the amounts of sequentially appearing protein in an equal number of infected cells (Fig. 3
a). A single immunoreactive band that migrated at approximately 31 kDa (the predicated relative molecular mass of the chlamydial putative
28 is 28·9 kDa) was observed in lysates of infected cells harvested after 24 h p.i.; the band persisted at subsequent time points, but was not present in lysates harvested at 16 h p.i. and earlier (Fig. 3a
). The slow migration of
28 seen on SDS-PAGE was similar to E. coli
28, which has an anomalous electrophoretic motility because of its acidic nature (Kundu et al., 1997
; Liu & Matsumura, 1995
). In a separate experiment shown in Fig. 3(b)
, we normalized
28 levels against the level of EF-Tu, which is essential for protein biosynthesis and constitutively expressed (Nicholson et al., 2003
), to compensate for the increase in numbers of chlamydiae as the infection progressed. Whereas the ratio of
28 to EF-Tu appeared nearly constant at 1628 h p.i., it declined after 28 h p.i. A common observation made in these experiments was that, relative to EF-Tu, the level of
28 decreased after 28 h p.i. and remained detectable at later times p.i.
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A fixed amount (1 µg) of supercoiled DNA template pGLC, which contains the fliC promoter from E. coli, and the 2 µl of reconstituted RNAP holoenzyme (1 unit of E. coli core enzyme reconstituted with increasing amounts of recombinant chlamydial 28) were used in each reaction of the in vitro transcription assay. As shown in Fig. 5
(a), E
28 was able to initiate transcription from PfliC and generated a single transcript of 130 bp. This represents the
28-driven transcription for the following reasons. First, recombinant
28 was expressed in E. coli YK4104, which lacks the functional
28. Thus, the potential artefact caused by contamination of E. coli
28 could be excluded. Second, E. coli core RNAP provided no signal in the absence of
28 (lane 1). Third, the level of transcription increased with increasing amounts of
28 (Fig. 5b
). Fourth, there was no 130 nt product when vector control pGL was used (data not shown). Finally, there was no transcription when E
70 was used (Fig. 6
).
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To examine the specificity of recombinant chlamydial 28 on promoter recognition, templates containing four chlamydial promoters with high similarity to
70 consensus sequences were tested using the in vitro transcription assay consisting of either E
70 or E
28, or both, and compared with templates containing PfliC (Fig. 6a
). Templates pGP1tuf, pGP3tuf, pMT504 and pGPgroE are predicted to produce the following
70-dependent transcripts: pMT504, 130 nt (from P1rrn); pGP1tuf, 130 and 152 nt (from P1rnn and P1tuf); pGP3tuf, 130 and 152 nt (from P1rnn and P3tuf); and pGPgroE, 130 nt (from PgroE). In addition, templates pGLC and pGPgroE are predicted to produce 130 nt transcripts from their fliC promoters in the presence of E
28. When the reconstituted RNAPs were tested individually, transcripts of the predicted size were produced, including the 130 nt
28-dependent transcripts when
28 was present (Fig. 6b
). Most significantly, the pGPgroE template, which contains the
70 consensus-like groE promoter and the E. coli
28-dependent fliC promoter, yielded the 152 nt
70 transcript and the 130 nt
28 transcript when
70 and
28 were simultaneously present in the in vitro transcription reaction (Fig. 6c
). This analysis further confirmed that E
28 and E
70 only recognize their own promoter sequences in the in vitro transcription assay, providing evidence that there is no cross-activity on promoter recognition between chlamydial housekeeping
factor
66 and the alternative
factor
28.
The putative promoter P1 of chlamydial rpsD was also tested using the in vitro transcription assay. pGPS1 was used as a template in which chlamydial P1rpsD served as the test promoter and PfliC served as the control promoter. Consistent with our observations above, both chlamydial E28 and E. coli E
28 initiated transcription from PfliC; however, there were no detectable transcripts generated with P1rpsD (data not known). This suggested that the putative P1 of the rpsD, although it bears some similarity to the E. coli
28 consensus (Fig. 2a
), may not be recognized by chlamydial
28 or that an additional molecule(s) is required for the efficient transcription.
Expression of chlamydial 28 was unable to restore motility to an E. coli fliA- mutant
The similarity of promoter specificity and selectivity between chlamydial 28 and E. coli
28 prompted us to perform complementation experiments in an E. coli fliA- mutant, which is non-motile because it does not express several
28-dependent genes required for motility, with the corresponding cloned rpsD gene from C. trachomatis. Plasmids pLF28, containing the coding region of chlamydial rpsD, pLE28, containing the coding region of E. coli fliA, and pLC3, the vector control, were transformed into E. coli fliA strain YK4104, respectively. Expression of recombinant chlamydial
28 and E. coli
28 in YK4104 under arabinose induction were confirmed by Western blot analysis (data not shown), indicating that the system was functional. On semisolid agar, wild-type strain YK410 formed a migrating ring of bacteria indicative of flagellar activity, whereas strain YK4104 lacking functional
28 failed to form a migrating ring. E. coli YK4104 harbouring pLF28 was unable to form a migrating ring on both arabinose-containing and arabinose-free semisolid agar. In contrast, E. coli YK4104 harbouring pLE28 was able to complement the fliA defect and to restore motility on arabinose-containing semisolid agar (Fig. 7
a and b), but not on arabinose-free semisolid agar. In addition, there was no observable effect of plasmid pLC3 on the motility of the tested bacterium (data not shown). These results indicated that, unlike E. coli
28, the expression of chlamydial
28 could not restore the defect in motility of E. coli mutant.
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DISCUSSION |
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At this point, it is not clear how chlamydial 28 binds to RNAP core enzyme and how it interacts with the regulatory proteins affecting
28 activation. In E. coli and Salmonella, while fliA is itself transcriptionally regulated (Liu & Matsumura, 1996
; Ikebse et al., 1999
), it is also post-translationally controlled by anti-
factor FlgM, which binds
28 and blocks transcription initiation (Hughes & Mathee, 1998
), and by protease digestion (Tomoyasu et al., 2002
). Chlamydial
28 is also homologous to B. subtilis
B. The activity of
B is inhibited when it interacts with anti-
factor RsbW, the activity of which is controlled by RsbV and RsbU. Homologues of all of these
regulators are encoded by the chlamydial genome. Therefore,
28 may be present but not functional under a given environmental condition, if it is bound to the anti-
factor RsbW.
Our results also showed that expression of chlamydial rpsD was induced by heat shock. The chlamydial genome lacks genes with homology to the heat-shock regulator H (
32) and
E (
24) of E. coli. The sequences located upstream of known heat-shock-regulated genes, dnaK and groE, contain chlamydial
66 promoters, which are homologous with the consensus of
70 in E. coli (Tan et al., 1996
). An alternative regulatory mechanism used by chlamydiae involves repression: the HrcA repressor acts at a cis-acting regulatory element (controlling inverted repeat of chaperone expression, CIRCE) to repress the transcription of dnaK and groE (Wilson & Tan, 2002
). However, no obvious CIRCE is present upstream of other heat-induced genes based on sequence homology searches. At present, regulatory mechanisms that respond to universal stresses in addition to heat shock, such as nutrition deprivation, phase of growth, high osmolarity and acid stress, that chlamydiae may encounter during infection are unidentified. They are mediated by
B in B. subtilis (Vicente et al., 1999
; Dufour et al., 1996
) and
S in E. coli (Hengge-Aronis, 1999
). Under stress conditions or upon entering into stationary phase, they are synthesized and activated and can then express numerous genes required to protect cells from stress (Vicente et al., 1999
; Helmann et al., 2001
; Yura & Nakahigashi, 1999
). Our findings, along with the homology between chlamydial
28 and
B of B. subtilis, suggest that the function of chlamydial
28 may be associated with adaptation to stress, which may provide enhanced resistance to changing environments. On the basis of in vitro transcription analysis,
28 may activate a set of genes in a mechanism different from that of
66 because
28 was unable to transcribe from the well-defined
66-recognized promoters, including heat-responsive PgroE. Recently, chlamydial
28 was shown to bind chlamydial putative RbsW, a homologue of the stress-responsive
B regulator in B. subtilis (A. L. Douglas & T. P. Hatch, personal communication), suggesting that interaction of chlamydial
28 and its putative regulators may be involved in chlamydial
28-specific transcription. The possibility that the interplay of
28 with other
factors or additional regulatory factors may mediate rpsD activation in response to environmental stresses remains to be examined.
We further demonstrated that the holoenzyme containing chlamydial 28 was able to direct E. coli RNAP to initiate transcription from a
28-dependent promoter, PfliC. The overall amino acid identity between chlamydial
28 and E. coli
28 is 34·7 %. The ability of chlamydial
28 to function with core E. coli RNAP in vitro is consistent with the high homology within regions 2.1, 2.2 and 3.2, which are implicated in core-binding (Burgess & Anthony, 2001
; Lonetto et al., 1992
). The conserved amino acid sequence at subregions 2.4 and 4.2 that are known to be involved in the recognition of promoter -10 and -35 sequences (Lonetto et al., 1992
), respectively, may account for the similarity of promoter recognition between chlamydial E
28 and E. coli E
28. Although transcripts of PfliC were initiated from the same start sites using chlamydial and E. coli E
28, the transcription signal initiated by chlamydial holoenzyme was weaker. Sequence analysis of chlamydial
28 also revealed some important differences from the E. coli homologue. For instance, they lack homology in the N-terminal region 1.2 sequence, which can affect promoter binding, open complex and initiation complex formation, and the transition from abortive transcription to elongation (Nicole & Dombroski, 2001
). The differences between the gene product of C. trachomatis rpsD and E. coli fliA may allow chlamydial
28 to recognize promoters with more divergent sequences. Complementation studies showed that the expression of chlamydial
28 was unable to restore motility in E. coli lacking its own functional
28. Possible explanations for this include the inability of chlamydial
28 to transcribe all the
28-dependent genes that are required for motility in E. coli, or expression of chlamydial
28 might indirectly affect multicellular processes that are required for swarming motility (Fraser & Hughes, 1999
).
Our observation that chlamydial rpsD expresses two steady-state transcripts, which were differentially regulated upon heat shock, suggested that each of them might be derived from different promoters or regulated by different mechanisms. In E. coli, the fliA operon belongs to class 2 in the transcriptional hierarchy of flagellar genes and can be transcribed by E70 in the presence of activator FlhD/C, and by E
28 (Macnab, 1996
; Liu & Matsumura, 1996
). It would be interesting to determine if the transcription of rpsD itself is dependent upon
28 and regulated by other transcription factors. The putative promoter P1 of chlamydial rpsD was not recognized by holoenzymes containing either chlamydial
28 or E. coli
28 and
70 in vitro using the G-less plasmid, which is designed to test the initiation of transcription at a specific site. Corroboration experiments are needed to clarify the activities of the putative promoters using alternative approaches.
The findings of this study suggest that chlamydial 28 may participate in transcription of a group of genes that are involved in the adaptive response of chlamydiae to environmental conditions. Having a relatively small genome, Chlamydia species may employ a complex strategy for expression of developmental genes through the participation of
factors and their regulators (Mathews et al., 1999
; Douglas & Hatch, 2000
; Nicholson et al., 2003
). Such a regulatory strategy may reflect the ability of Chlamydia to respond to stressful conditions with economy and efficiency.
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ACKNOWLEDGEMENTS |
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Received 15 August 2003;
revised 19 September 2003;
accepted 19 September 2003.
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