JB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cirz, R. T.
Right arrow Articles by Romesberg, F. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cirz, R. T.
Right arrow Articles by Romesberg, F. E.
Journal of Bacteriology, October 2006, p. 7101-7110, Vol. 188, No. 20
0021-9193/06/$08.00+0     doi:10.1128/JB.00807-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Defining the Pseudomonas aeruginosa SOS Response and Its Role in the Global Response to the Antibiotic Ciprofloxacin

Ryan T. Cirz,1 Bryan M. O'Neill,1 Jennifer A. Hammond,2 Steven R. Head,2 and Floyd E. Romesberg1*

Department of Chemistry,1 DNA Microarray Core Facility, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 920372

Received 7 June 2006/ Accepted 7 August 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pseudomonas aeruginosa infections can be virtually impossible to eradicate, and the evolution of resistance during antibiotic therapy is a significant concern. In this study, we use DNA microarrays to characterize the global transcriptional response of P. aeruginosa to clinical-like doses of the antibiotic ciprofloxacin and also to determine the component that is regulated by LexA cleavage and the SOS response. We find that genes involved in virtually every facet of metabolism are down-regulated in response to ciprofloxacin. The LexA-controlled SOS regulon identified by microarray analysis includes only 15 genes but does include several genes that encode proteins involved in recombination and replication, including two inducible polymerases known to play a role in mutation and the evolution of antibiotic resistance in other organisms. The data suggest that the inhibition of LexA cleavage during therapy might help combat this pathogen by decreasing its ability to adapt and evolve resistance.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pseudomonas aeruginosa is a common opportunistic human pathogen which is particularly infamous for the high rates of illness and death it causes in patients with cystic fibrosis (18). Once established, P. aeruginosa infections can be controlled with some antibiotics, for example, the fluoroquinolones, but are virtually impossible to eradicate, at least in part due to the pathogen's ability to progress through a series of physiological changes that facilitate infection and persistence (18). Its ability to adapt to environmental stress, such as antibiotic therapy, may be related to the large size of its genome (6.3 Mb) and the large number of genes that encode transcriptional regulators.

One of the most important transcriptional responses to environmental stress in bacteria is the SOS response. In Escherichia coli (9) and Bacillus subtilis (2), it involves the controlled derepression of 43 and 33 genes, respectively, whose protein products facilitate the repair and/or tolerance of DNA damage. Transcription of these genes is induced by the single-stranded DNA (ssDNA) that results from stalled replication forks or direct damage to DNA (15). RecA forms filaments on the ssDNA that mediate recombinational repair and also bind and induce autocleavage of the SOS gene repressor, LexA, resulting in the transcription of the repressed genes. Interestingly, ciprofloxacin, the prototypical fluoroquinolone and an important antibiotic for treating P. aeruginosa infections, induces LexA cleavage and the SOS response in E. coli (11, 33).

In this study, we determined the global and SOS-mediated transcriptional response of P. aeruginosa PAO1 to clinical-like levels of ciprofloxacin. Experiments and controls were repeated in triplicate, which allowed us to identify changes in transcription with a confidence level of P ≤ 0.001. The data reveal a complex and coordinated LexA-independent response to ciprofloxacin that involves the down-regulation of metabolism, motility, and permeability. The LexA-mediated response is limited to the induction of 15 genes that appear to provide specialized DNA recombination and replication functions.

In addition to furthering our understanding of how the transcriptional response of P. aeruginosa contributes to its pathogenicity, we are interested in understanding the potential utility of LexA autoproteolysis inhibitors. For many bacteria, LexA is known to repress genes that regulate processes such as phage mobilization (17, 21, 34), resistance element transfer (3), toxin production (17, 21, 34, 38), mutation (14, 15, 26, 32), and the evolution of resistance (7, 8). For example, we recently demonstrated both in vivo and in vitro that the acquisition of the chromosomal mutations required for the evolution of ciprofloxacin resistance in E. coli requires the autoproteolysis activity of LexA and the subsequent induction of the error-prone SOS polymerases in both wild-type (7) and hypermutator strains (8). Thus, suitably designed inhibitors of LexA could be administered with different antibiotics to prevent the emergence of resistance. Identification of the SOS regulon in P. aeruginosa is expected to help define the broader utility of such drugs.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and growth. P. aeruginosa PAO1 was obtained from G. Sundin. Unless specified, solid medium was Lennox LB (28) plus 1.6% agar (LBA); liquid medium was Miller LB (28) (LB). For selection, antibiotics were used for E. coli and P. aeruginosa PAO1, respectively, as follows: streptomycin (Sm), 30 µg/ml and 250 µg/ml; gentamicin (Gm), 15 µg/ml and 50 µg/ml. Ciprofloxacin was obtained from MP Biomedicals (Aurora, Ohio) and used at the concentrations indicated below. All bacteria were grown aerobically at 37°C.

Strain construction. Primer sequences were designed based on the P. aeruginosa genome database (http://v2.pseudomonas.com) (35, 39). A lexA allelic exchange cassette was assembled containing ~800 bp of homology surrounding lexA, the lexA open reading frame, and the Gmr marker from vector pBBR1MCS-5 (22) using assembly PCR and the primers listed in Table S1 at the website http://www.scripps.edu/chem/romesberg/. The resulting cassette was cloned into vector pKNG101 (20) to create pRTC0021; the S125A mutation was then introduced using primers PA_lexA_S125A_QCF and PA_lexA_S125A_QCR and the QuikChange site-directed mutagenesis kit (Stratagene) to create vector pRTC0022.

pRTC0022 was transformed into E. coli strain SY-17 and introduced into P. aeruginosa by conjugative transfer with selection on M9 plus 0.2% citrate and Gm to select clones that integrated the allelic exchange cassette into the chromosome by either a single- or double-crossover event. Replica plating onto M9 plus 0.2% citrate containing either Gm or Sm identified clones containing the allelic exchange cassette and lacking the vector sequences due to a double-crossover event. Colonies were verified as Gmr and Sms, and the mutation was confirmed by sequencing.

Confirmation that the LexA(S125A) mutant is not cleaved in response to ciprofloxacin. For each strain, five clones were grown in LB for 18 h. Cultures were diluted 1:500 and grown to mid-log phase (optical density at 60 nm [OD600], ~0.4 to 0.5), and then ciprofloxacin was added to a final concentration of 1 µg/ml. At 0, 30, and 120 min following ciprofloxacin addition, cell aliquots were removed and stored at –20°C. During the experiment, the OD600 and viable CFU per ml were monitored for each of the cultures (see Fig. 1A and B, below). This protocol is identical to that used to prepare samples for the transcriptional studies (below). Whole-cell lysates were prepared by sonication in phosphate-buffered saline, and the soluble fraction was collected and normalized for total protein concentration (Bio-Rad protein assay). Samples were separated on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a 0.2-µm nitrocellulose membrane. Immunostaining was performed with a rabbit polyclonal antiserum to LexA (1:8,000; 2 h; kindly provided by J. Little) and horseradish peroxidase-linked anti-rabbit antibody (1:20,000; 1 h; Upstate Biotechnology), followed by detection with ECL Plus (GE Biosciences).


Figure 1
View larger version (19K):
[in this window]
[in a new window]
  		FIG. 1. Population kinetics of P. aeruginosa strains during microarray and whole-cell lysate sample preparation monitored by OD (A) and CFU/ml (B). Filled and open triangles represent PAO1 and the LexA(S125A) mutant, respectively. Time zero is defined as the point immediately following ciprofloxacin addition. As described in the text, samples for total RNA extraction and whole-cell lysates were collected at 0, 30, and 120 min post-ciprofloxacin addition. (C) Analysis of total full-length LexA measured by Western blotting with anti-LexA antibody.

 
Growth rate measurement. For each strain, three cultures were inoculated into 2 ml tryptic soy broth (Difco) and grown for 16 h with shaking, followed by continued growth after a 100-fold dilution in tryptic soy broth. At the indicated time points, the OD600 was measured and the number of viable CFU per ml was determined by dilution plating.

Sensitivity to UV light and MMS. Three independent cultures of each strain were grown overnight in LB. Appropriate dilutions were plated onto LBA, and UVC irradiations were performed using a G8T5 germicidal tube (Ushio America, Cypress, CA). UV fluences were determined using a UVX radiometer with a UVX-25 sensor (UV Products). After irradiation, plates were protected from light and incubated for 2 days before colonies were counted. To determine the methyl methanesulfonate (MMS; Aldrich) sensitivity, three independent cultures were grown overnight in LB. Appropriate dilutions were plated onto LBA containing MMS at the indicated concentrations and incubated for 2 days before colonies were counted.

MIC determination. For each strain, three independent cultures were grown for 25 h in LB containing no antibiotic. From each culture, ~105 CFU were used to inoculate LB containing increasing concentrations of ciprofloxacin in 96-well plates. Inoculations were done in duplicate to yield a total of six data points per strain. After 18 h of incubation, growth was measured by reading the OD650 in a Vmax Kinetic microplate reader (Molecular Devices, California). The MIC was defined as the lowest concentration of ciprofloxacin that prevented any detectable growth.

Transcriptional analysis. P. aeruginosa genome arrays containing 25-mer probe sets for over 5,500 open reading frames from PAO1, 199 probe sets corresponding to 100 intergenic regions, and 117 probe sets from other P. aeruginosa strains were obtained from Affymetrix (Santa Clara, CA). A complete description and annotation for this P. aeruginosa genome array is available at http://www.affymetrix.com.

Sample preparation and data analysis. For each strain, five clones were inoculated in LB and grown for 18 h. Cultures were diluted 1:500 and grown to mid-log phase (OD600, ~0.4 to 0.5), at which point ciprofloxacin was added to a final concentration of 1 µg/ml. At 0, 30, and 120 min following ciprofloxacin addition, appropriate volumes from each of the five cultures per strain were pooled and added to 2 volumes of RNAprotect reagent (QIAGEN); cell pellets were stored at 4°C until RNA extraction. Total RNA was extracted using the RNeasy Mini kit (QIAGEN) at the end of the sample collection period. This procedure was repeated three independent times to generate three samples each just prior to and 120 min post-ciprofloxacin addition. Details of data analysis and reverse transcription-PCR validation have been provided along with our supplementary data sets via the internet (http://www.scripps.edu/chem/romesberg/).

Microarray accession numbers. Microarray data have been deposited at the National Center for Biotechnology Information's Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE5443.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
General characterization of the P. aeruginosa SOS response. We constructed a lexA(S125A) mutant of P. aeruginosa PAO1, where the catalytic serine of LexA has been replaced with alanine. We monitored the levels of full-length LexA in response to added ciprofloxacin (1 µg/ml ciprofloxacin) and showed that while the wild-type protein underwent cleavage, the mutant protein remained intact (Fig. 1).

No significant growth attenuation was observed with the lexA(S125A) strain relative to its isogenic parental strain (log phase doubling times of 45.0 ± 3.5 and 45.5 ± 4.5 min were observed for the LexA mutant and PAO1 strains, respectively [means ± standard deviations]), as was previously observed with analogous mutants of E. coli (15). Relative to the wild-type strain, we found that the lexA(S125A) mutant was hypersensitive to UV irradiation (Fig. 2A) but not to MMS (Fig. 2B) or ciprofloxacin [the ciprofloxacin MIC for both wild-type PAO1 and the lexA(S125A) strain was 0.125 µg/ml]. This suggests that the P. aeruginosa SOS response is important for repairing DNA damage associated with UV irradiation but not with MMS or ciprofloxacin.


Figure 2
View larger version (11K):
[in this window]
[in a new window]
  		FIG. 2. Killing kinetics of P. aeruginosa strains, as shown by survival following UV (A) or MMS (B) treatment. PAO1 and the LexA(S125A) mutant are represented by filled and open triangles, respectively.

 
Characterization of the P. aeruginosa transcriptional response to ciprofloxacin. Using DNA microarrays, we transcriptionally profiled mid-log-phase PAO1 at 30 and 120 min after exposure to suprainhibitory concentrations of ciprofloxacin (1 µg/ml; 8x MIC) (Fig. 1A and B). Only the magnitude of the observed changes was different, and so we focused the analysis on the 120-min data set. We observed that the levels of 196 transcripts increase at least twofold and the levels of 408 transcripts decrease at least twofold relative to levels immediately before the addition of the drug (Table 1; see also our supporting information at http://www.scripps.edu/chem/romesberg/).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Transcriptional response to 1 µg/ml ciprofloxacin

 
Sixty-four of the genes that are up-regulated in response to ciprofloxacin are in regulons that are likely controlled by LexA-like repressors (Table 2). For example, the autoregulated LexA-like Ser-Lys dyad repressor PtrR (25) is up-regulated by 5-fold, and seven genes thought to be directly or indirectly under its control are up-regulated by up to 100-fold. One of these genes, ptrB, acts to repress the type III secretion system (40). Thus, via induced cleavage of PtrR, exposure to ciprofloxacin results in a down-regulation of the type III secretion system. In addition, 35 nearby cryptic prophage genes spanning from PA0614 to PA0648 are strongly up-regulated. Another putative phage repressor that shares similarities with LexA and is highly homologous to PtrR is PA0906. PA0906 is divergently transcribed from a putative operon that spans PA0907 to PA0911. PA0906 and the five genes of this operon are up-regulated 20- to 80-fold after exposure to the drug. Overall, the data suggest that at least one-third of the positive transcriptional response to ciprofloxacin is controlled by LexA-like repressors.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Other damage-inducible Ser-Lys dyads and downstream targets

 
The number of genes that are down-regulated in response to ciprofloxacin is more than twice the number that are up-regulated. The down-regulated response appears to involve virtually every facet of cellular metabolism, including general metabolism, cell wall/capsule biosynthesis, DNA replication/repair, cell division, motility, and quorum sensing (Table 1; see also our supporting information at http://www.scripps.edu/chem/romesberg/). Changes observed were similar for all genes in a given operon, supporting the physiological significance of their regulation.

We observed significant and consistent changes in the operons encoding the subunits of ATP synthase (PA5553 to PA5561), which all decrease between 4- and 13-fold, and the subunits of NADH dehydrogenase complex I (PA2637 to PA2649), which all decrease between 2- and 5-fold. Similar decreases were observed in the response to acute H2O2 damage (30). In addition, while nrdA and nrdB, which encode the ribonucleotide reductase complex, are both up-regulated in response to ciprofloxacin four- to eightfold, two genes in a separate operon (PA5496 and PA5497) that are predicted to encode an alternate ribonucleotide reductase are down-regulated three- to fourfold. In addition, genes encoding many other proteins involved in metabolism were down-regulated after exposure to the drug (Table 1; see also our supporting information at the website http://www.scripps.edu/chem/romesberg/).

In addition to the decreased transcription of genes involved in general metabolism, decreases are also observed with genes involved in DNA metabolism. An operon containing genes that encode components of the replication machinery, including dnaA, dnaN, recF, and gyrB, is down-regulated ~4-fold in response to ciprofloxacin. In addition, genes encoding DNA polymerase I, the HolB subunit of DNA polymerase III, and the DNA binding protein HU are all down-regulated two- to fivefold. The recG and ruvABC genes, all encoding proteins thought to be important for repairing ciprofloxacin-induced damage (7, 11), are down-regulated, albeit less than twofold. In contrast, recA, recX, and recN are up-regulated 7- to 17-fold. Interestingly, the three genes encoding damage-inducible DNA polymerases, PA0923, PA0670, and PA0669, are up-regulated in response to ciprofloxacin. PA0923 encodes a dinB-like Y-family polymerase and is up-regulated fourfold in response to ciprofloxacin. PA0670 and PA0669 encode two polymerases recently shown to be involved in damage-induced mutagenesis in Caulobacter crescentus (16) and are up-regulated two- and sixfold, respectively. The overall pattern of expression in the DNA replication genes suggests a shift from the canonical DNA replication enzymes to the inducible polymerases in response to ciprofloxacin.

Nearly all of the major cell division and lipopolysaccharide genes are significantly down-regulated. Of particular note are the changes observed in the wbp region, which encodes the B-band lipopolysaccharide O antigen and spans from PA3141 to PA3160. Transcription of these genes decreased by two- to sixfold after exposure to ciprofloxacin. Another interesting trend is the down-regulation of 41 genes that encode proteins involved in motility. We also observe a two- to fourfold increase in transcription of two major efflux proteins (MexC and MexR) and a four- to fivefold decrease in transcription of three major membrane pore proteins (OprD, OprG, and OprI). These changes in mobility and permeability are consistent with the general trend toward reduced metabolic activity in response to antibiotic exposure.

Contribution of the P. aeruginosa SOS genes to the ciprofloxacin-induced transcriptional response. We next characterized the transcriptional response to ciprofloxacin in a lexA(S125A) PAO1 mutant under the same conditions as those used to characterize the wild-type strain. As expected, lexA, recA, recX, and recN are induced by ciprofloxacin in a LexA cleavage-dependent manner (Table 3). In addition, PA3413 and PA1045 are regulated by LexA. PA3413 is a probable homolog of E. coli yebG, which is LexA regulated in E. coli (29), but its biological function is not known. PA1045 appears to encode a DinG helicase (37), which is related to the mammalian XPD family of helicases, and it may play a role in transcription-coupled repair.


View this table:
[in this window]
[in a new window]
  		TABLE 3. LexA-regulated genes identified in the microarray-based whole-genome transcription assay

 
The induction of several hypothetical genes was found to depend on LexA cleavage: PA2288, PA3414, PA1044, PA0069, and PA0922. PA3414 is predicted to encode a protein of unknown function, but its location next to yebG (see above) suggests that these genes may be coordinatively transcribed. PA2288 encodes a hypothetical protein of no known function. However, it has recently been shown that mutation of wspF, which disrupts a signal cascade involved in biofilm formation, causes a mild induction of lexA and recA (each 1.6-fold) and a 4.0- and 2.8-fold increase in PA3414 and PA2288 expression, respectively, supporting the association of these genes with the SOS response (19). PA1044 encodes a hypothetical protein with no known function that is divergently transcribed from PA1045 (see above). PA0922 and PA0069 are predicted to encode a hypothetical protein and a photolyase-like protein, respectively.

In E. coli there are three nonessential polymerases, each of which is LexA regulated: Pol II (encoded by polB), Pol IV (encoded by dinB), and Pol V (encoded by umuC and umuD). The P. aeruginosa genome encodes three nonessential polymerases, PA0923, PA0669, and PA0670. As mentioned above, transcription of PA0923, which encodes a polymerase that is highly homologous to E. coli dinB, is induced by ciprofloxacin; however, its induction is not LexA regulated. This agrees with recent findings in other organisms (6, 36) and suggests that a LexA-regulated dinB polymerase may be more the exception than the rule. In contrast, PA0669 and PA0670, which appear to be encoded in the same operon, are induced by ciprofloxacin in a LexA cleavage-dependent manner. PA0669 is predicted to encode an alternate alpha-subunit, and PA0670 is predicted to encode a Y-family polymerase. This operon resembles one that was recently found to play a role in damage-induced mutagenesis in the {alpha}-Proteobacteria Caulobacter crescentus (16), although a PAO1 mutant lacking this operon shows no signs of increased sensitivity to UV, MMS, or ciprofloxacin-mediated damage (R. T. Cirz and F. E. Romesberg, unpublished results). While we were able to detect sufficient levels of PA0670 in the microarray studies, the level of PA0669 was too low to observe a rigorous statistical difference between PAO1 and the LexA mutant directly (supporting information can be found at http://www.scripps.edu/chem/romesberg/). However, using real-time PCR we were able to detect PA0669 mRNA after ciprofloxacin treatment, but not in the LexA mutant, confirming that this gene is LexA regulated (see Table S2 in our supporting information at http://www.scripps.edu/chem/romesberg/).

Analysis of the P. aeruginosa LexA box and other potential SOS genes. By identifying the SOS regulon empirically, we were also able to identify a consensus binding sequence for LexA, CTG-TATAA-ATATA-CAG (bold residues are 100% conserved) (Table 3). The consensus is essentially the same as that in E. coli with the exception of position eight, where it is most frequently a dA in P. aeruginosa and a dT in E. coli.

We searched the Pseudomonas genome (35, 39) for other potential LexA binding sites using the sequence CTGN2TN7CAG with up to four mismatches in the central 10-bp region. In addition to the sites that regulate the 15 genes identified in our microarray studies, eight potential LexA binding sites were identified (see Table S4 in our supporting information at http://www.scripps.edu/chem/romesberg/). Four are positioned between 128 and 154 bp from a gene, and four are intragenic. The microarray data suggest that either these sites do not bind LexA in vivo or that they do not effectively regulate expression. Thus, the data suggest that the 15 genes identified experimentally represent the entire LexA regulon.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The global transcriptional response to ciprofloxacin was previously examined in P. aeruginosa PAO1 grown for 2.5 h in the presence of sub-MIC and MIC levels of the drug (5). Ciprofloxacin was found to induce changes in the transcription of >900 and >1,200 genes at sub-MIC and MIC levels, respectively (P ≤ 0.05). While this study identified the increased transcription of the cryptic prophage genes spanning from PA0614 to PA0648 (5), which we also observed with supra-MIC levels of the drug, it did not identify any consistent and significant changes in genes involved in metabolism or proliferation (see Table S3 in our supporting information at http://www.scripps.edu/chem/romesberg/). In fact, the present study reveals that the largest part of the response involves the down-regulation of genes that encode proteins involved in general metabolism and DNA replication/repair, as well as the down-regulation of genes involved in cell division, motility, quorum sensing, and cell permeability. These changes appear to be specific for higher, clinical-like levels of the drug and may contribute to the pathogen's survival during therapy, as has already been suggested for both planktonic and biofilm cells (1, 24). Indeed, this response is a reasonable survival strategy, considering that ciprofloxacin is more lethal to actively dividing cells than resting cells (11), and it may facilitate survival until a more specific response is orchestrated.

The SOS response is thought to be a major component of the bacterial response to stress and has been characterized thoroughly in E. coli, where it includes the derepression of 43 genes that orchestrate virtually the entire positive transcriptional response to UV irradiation (9). The only other globally characterized SOS response is that of B. subtilis, where 33 genes have LexA binding sites and are induced by UV irradiation and mitomycin C in a RecA-dependent manner. While the number of LexA-regulated genes in E. coli and B. subtilis is similar, only seven genes are common to both organisms.

By directly comparing the response to ciprofloxacin of LexA-cleavable and uncleavable strains, we identified 15 P. aeruginosa genes that are induced by ciprofloxacin in a LexA cleavage-dependent manner. These genes appear to be controlled from nine LexA binding sites, with five of the sites controlling expression of divergent or polycistronic operons. The consensus binding site is the 16-nucleotide imperfect palindrome CTG-TATAA-ATATA-CAG (where the bold indicates absolutely conserved nucleotides). As expected, the SOS regulon includes lexA and recA. It also includes recN, recX, and probable yebG, dinG, and phl homologs, which are all commonly part of the SOS regulon in other bacteria. Also included is the polycistronic operon containing imuA/sulA, imuB, and dnaE2, as observed in several other SOS operons (12). The data suggests that, like other {gamma}-Proteobacteria, both DNA repair and induced mutation are central components of the LexA-regulated SOS response in P. aeruginosa. However, P. aeruginosa LexA appears to regulate only the recombinational repair proteins RecX and RecN and not the nucleotide excision repair proteins UvrA, UvrB, and UvrD, nor the recombinational repair proteins RuvA and RuvB, all of which are LexA regulated in both E. coli (9) and B. subtilis (2) and are also predicted to be LexA regulated in other {gamma}-Proteobacteria (13). RecX is thought to associate with RecA and cap filament extension (10), while RecN is thought to cooperate in some forms of recombination (31). Why these recombination proteins are regulated by LexA in P. aeruginosa, while ones common to other SOS regulons are not, is unclear but likely reflects the environment in which the pathogen has adapted to survive.

Induced mutation in E. coli is controlled by LexA cleavage-mediated derepression of polB, which encodes Pol II, dinB, which encodes Pol IV, and umuDC, which encodes the Pol V preprotein. P. aeruginosa does not have a umuDC homolog, and our data show that polB is not induced in response to ciprofloxacin and that dinB, while induced, is not repressed by LexA. Instead, P. aeruginosa appears to control induced mutation from the LexA-repressed imuA/sulA-imuB-dnaE2 operon. imuB and dnaE2 encode inducible polymerases, and their homologs are required for the majority of UV- and mitomycin C-induced mutations in the highly related organism Caulobacter crescentus (16). In addition, a dnaE2 homolog in Mycobacterium tuberculosis has been shown to be required for UV-induced mutation (4). The operon also appears in many other species and is predicted computationally to be universally LexA regulated (12). Interestingly, the presence of this operon has been correlated with the absence of a umuDC operon (12), suggesting that it may perform a similar function.

The SOS system may play an underappreciated role in the response to several commonly used antibiotics. In E. coli, the SOS response is induced by ciprofloxacin (11, 33), rifampin (7), ß-lactams (27), and trimethoprim (23). While the LexA regulon of P. aeruginosa is significantly smaller than that of E. coli, or B. subtilis, it appears to have retained control over induced mutation. Thus, it seems likely that the initial reduction in metabolism observed in P. aeruginosa provides the SOS response time to induce mutations that allow it to persist and eventually to evolve resistance, as has been observed in E. coli (7, 8) and M. tuberculosis (4). These results suggest that an inhibitor of LexA cleavage might have a profound and favorable effect on P. aeruginosa therapy.


   ACKNOWLEDGMENTS
 
This research was supported by the Office of Naval Research (award no. N00014-03-1-0126).

We thank J. Little for providing the LexA antibody. We thank A. Smania for providing vector pKNG101 and technical advice and G. Sundin for providing strain PAO1.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 784-7290. Fax: (858) 784-7472. E-mail: floyd{at}scripps.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Anderl, J. N., J. Zahller, F. Roe, and P. S. Stewart. 2003. Role of nutrient limitation and stationary-phase existence in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob. Agents Chemother. 47:1251-1256.[Abstract/Free Full Text]
  2. Au, N., E. Kuester-Shoeck, V. Mandava, L. E. Bothwell, S. P. Canny, K. Chachu, S. A. Colavito, S. N. Fuller, E. S. Groban, L. A. Hensley, T. C. O'Brien, A. Shah, J. T. Tierney, L. L. Tomm, T. M. O'Gara, A. I. Goranov, A. D. Grossman, and C. M. Lovett. 2005. Genetic composition of the Bacillus subtilis SOS system. J. Bacteriol. 187:7655-7666.[Abstract/Free Full Text]
  3. Beaber, J. W., B. Hochhut, and M. K. Waldor. 2004. SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature 427:72-74.[CrossRef][Medline]
  4. Boshoff, H. I., M. B. Reed, C. E. Barry III, and V. Mizrahi. 2003. DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113:183-193.[CrossRef][Medline]
  5. Brazas, M. D., and R. E. W. Hancock. 2005. Ciprofloxacin induction of a susceptibility determinant in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 49:3222-3227.[Abstract/Free Full Text]
  6. Brooks, P. C., F. Movahedzadeh, and E. O. Davis. 2001. Identification of some DNA damage-inducible genes of Mycobacterium tuberculosis: apparent lack of correlation with LexA binding. J. Bacteriol. 183:4459-4467.[Abstract/Free Full Text]
  7. Cirz, R. T., J. K. Chin, D. R. Andes, V. D. Crecy-Lagard, W. A. Craig, and F. E. Romesberg. 2005. Inhibition of mutation and combating the evolution of antibiotic resistance. PLoS Biol. 3:e176.[CrossRef][Medline]
  8. Cirz, R. T., and F. E. Romesberg. 2006. Induction and inhibition of ciprofloxacin resistance-conferring mutations in hypermutator bacteria. Antimicrob. Agents Chemother. 50:220-225.[Abstract/Free Full Text]
  9. Courcelle, J., A. Khodursky, B. Peter, P. O. Brown, and P. C. Hanawalt. 2001. Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics 158:41-64.[Abstract/Free Full Text]
  10. Drees, J. C., S. L. Lusetti, S. Chitteni-Pattu, R. B. Inman, and M. M. Cox. 2004. A RecA filament capping mechanism for RecX protein. Mol. Cell 15:789-798.[CrossRef][Medline]
  11. Drlica, K., and X. Zhao. 1997. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev. 61:377-392.[Abstract]
  12. Erill, I., S. Campoy, G. Mazon, and J. Barbe. 2006. Dispersal and regulation of an adaptive mutagenesis cassette in the Bacteria domain. Nucleic Acids Res. 34:66-77.[Abstract/Free Full Text]
  13. Erill, I., M. Escribano, S. Campoy, and J. Barbe. 2003. In silico analysis reveals substantial variability in the gene contents of the gamma proteobacteria LexA-regulon. Bioinformatics 19:2225-2236.[Abstract/Free Full Text]
  14. Foster, P. L. 2000. Adaptive mutation: implications for evolution. Bioessays 22:1067-1074.[CrossRef][Medline]
  15. Friedberg, E. C., G. C. Walker, and W. Siede. 1995. DNA repair and mutagenesis. ASM Press, Washington, D.C.
  16. Galhardo, R. S., R. P. Rocha, M. V. Marques, and C. F. M. Menck. 2005. An SOS-regulated operon involved in damage-inducible mutagenesis in Caulobacter crescentus. Nucleic Acids Res. 33:2603-2614.[Abstract/Free Full Text]
  17. Goerke, C., J. Koller, and C. Wolz. 2006. Ciprofloxacin and trimethoprim cause phage induction and virulence modulation in Staphylococcus aureus. Antimicrob. Agents Chemother. 50:171-177.[Abstract/Free Full Text]
  18. Govan, J. R., and V. Deretic. 1996. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol. Rev. 60:539-574.[Abstract/Free Full Text]
  19. Hickman, J. W., D. F. Tifrea, and C. S. Harwood. 2005. A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels. Proc. Natl. Acad. Sci. USA 102:14422-14427.[Abstract/Free Full Text]
  20. Kaniga, K., I. Delor, and G. R. Cornelis. 1991. A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene 109:137-141.[CrossRef][Medline]
  21. Koudelka, A. P., L. A. Hufnagel, and G. B. Koudelka. 2004. Purification and characterization of the repressor of the shiga toxin-encoding bacteriophage 933W: DNA binding, gene regulation, and autocleavage. J. Bacteriol. 186:7659-7669.[Abstract/Free Full Text]
  22. Kovach, M. E., P. H. Elzer, D. S. Hill, G. T. Robertson, M. A. Farris, R. M. Roop II, and K. M. Peterson. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175-176.[CrossRef][Medline]
  23. Lewin, C. S., and S. G. Amyes. 1991. The role of the SOS response in bacteria exposed to zidovudine or trimethoprim. J. Med. Microbiol. 34:329.[Abstract]
  24. Lewis, K. 2005. Persister cells and the riddle of biofilm survival. Biochemistry (Moscow) 70:267-274.[CrossRef][Medline]
  25. Matsui, H., Y. Sano, H. Ishihara, and T. Shinomiya. 1993. Regulation of pyocin genes in Pseudomonas aeruginosa by positive (ptrN) and negative (ptrR) regulatory genes. J. Bacteriol. 175:1257-1263.[Abstract/Free Full Text]
  26. McKenzie, G. J., R. S. Harris, P. L. Lee, and S. M. Rosenberg. 2000. The SOS response regulates adaptive mutation. Proc. Natl. Acad. Sci. USA 97:6646-6651.[Abstract/Free Full Text]
  27. Miller, C., L. E. Thomsen, C. Gaggero, R. Mosseri, H. Ingmer, and S. N. Cohen. 2004. SOS response induction by ß-lactams and bacterial defense against antibiotic lethality. Science 305:1629-1631.[Abstract/Free Full Text]
  28. Miller, J. H. 1992. A short course in bacterial genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  29. Oh, T. J., and I. G. Kim. 1999. Identification of genetic factors altering the SOS induction of DNA damage-inducible yebG gene in Escherichia coli. FEMS Microbiol. Lett. 177:271-277.[CrossRef][Medline]
  30. Palma, M., D. DeLuca, S. Worgall, and L. E. N. Quadri. 2004. Transcriptome analysis of the response of Pseudomonas aeruginosa to hydrogen peroxide. J. Bacteriol. 186:248-252.[Abstract/Free Full Text]
  31. Picksley, S. M., P. V. Attfield, and R. G. Lloyd. 1984. Repair of DNA double-strand breaks in Escherichia coli K12 requires a functional recN product. Mol. Gen. Genet. 195:267-274.[CrossRef][Medline]
  32. Ponder, R. G., N. C. Fonville, and S. M. Rosenberg. 2005. A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation. Mol. Cell 19:791-804.[CrossRef][Medline]
  33. Power, E. G., and I. Phillips. 1992. Induction of the SOS gene (umuC) by 4-quinolone antibacterial drugs. J. Med. Microbiol. 36:78-82.[Abstract]
  34. Quinones, M., H. H. Kimsey, and M. K. Waldor. 2005. LexA cleavage is required for CTX prophage induction. Mol. Cell 17:291-300.[CrossRef][Medline]
  35. Stover, K. C., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. L. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K.-S. Wong, Z. Wu, I. Paulsen, J. Reizer, M. H. Saier, R. E. W. Hancock, S. Lory, and M. V. Olson. 2000. Complete genome sequence of Pseudomonas aerguinosa PAO1: an opportunistic pathogen. Nature 406:959-964.[CrossRef][Medline]
  36. Tegova, R., A. Tover, K. Tarassova, M. Tark, and M. Kivisaar. 2004. Involvement of error-prone DNA polymerase IV in stationary-phase mutagenesis in Pseudomonas putida. J. Bacteriol. 186:2735-2744.[Abstract/Free Full Text]
  37. Voloshin, O. N., F. Vanevski, P. P. Khil, and R. D. Camerini-Otero. 2003. Characterization of the DNA damage-inducible helicase DinG from Escherichia coli. J. Biol. Chem. 278:28284-28293.[Abstract/Free Full Text]
  38. Waldor, M. K., and D. I. Friedman. 2005. Phage regulator circuits and virulence gene expression. Curr. Opin. Microbiol. 8:459-465.[CrossRef][Medline]
  39. Winsor, G. L., R. Lo, S. J. Sui, K. S. Ung, S. Huang, D. Cheng, W. K. Ching, R. E. Hancock, and F. S. Brinkman. 2005. Pseudomonas aeruginosa Genome Database and PseudoCAP: facilitating community-based, continually updated, genome annotation. Nucleic Acids Res. 33:D338-D343.[Abstract/Free Full Text]
  40. Wu, W., and S. Jin. 2005. PtrB of Pseudomonas aeruginosa suppresses the type III secretion system under the stress of DNA damage. J. Bacteriol. 187:6058-6068.[Abstract/Free Full Text]

Journal of Bacteriology, October 2006, p. 7101-7110, Vol. 188, No. 20
0021-9193/06/$08.00+0     doi:10.1128/JB.00807-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cirz, R. T.
Right arrow Articles by Romesberg, F. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cirz, R. T.
Right arrow Articles by Romesberg, F. E.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Appl. Environ. Microbiol. Infect. Immun. Eukaryot. Cell
Mol. Cell. Biol. J. Virol. Microbiol. Mol. Biol. Rev.
ALL ASM JOURNALS