JOURNAL OF BACTERIOLOGY, May 2007, p. 3793–3803 0021-9193/07/$08.00⫹0 doi:10.1128/JB.01764-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 189, No. 10
Error-Prone DNA Repair System in Enteroaggregative Escherichia coli Identified by Subtractive Hybridization䌤† Lucy M. Joo,1 Louissa R. Macfarlane-Smith,2 and Iruka N. Okeke1* Department of Biology, Haverford College, 370 Lancaster Ave., Haverford, Pennsylvania 19041,1 and Department of Biomedical Sciences, University of Bradford, West Yorkshire, United Kingdom2 Received 20 November 2006/Accepted 28 February 2007
Enteroaggregative Escherichia coli (EAEC) are etiologic agents of diarrhea. The EAEC category is heterogeneous, but most in-depth experimentation has focused on prototypical strain, 042. We hypothesized that 60A, another EAEC strain, might posses virulence or fitness genes that 042 does not have. Through subtractive hybridization we identified 60A-specific sequences, including loci present in other E. coli and phage DNA. One locus thus identified was impB, a LexA repressed error-prone DNA repair gene that has been identified in plasmids from other enteric organisms and which we detected in 21 of 34 EAEC strains. An isogenic 60A impB mutant showed decreased survival and mutagenesis after exposure to UV, as well as bile salt exposure, compared to the wild-type strain, and these phenotypes could be complemented in trans. The EAEC strain 60A imp operon differs structurally from previously described homologs. A cryptic gene, impC, present in other imp operons, is absent from 60A. In addition, transcription of impAB in strain 60A occurs from a promoter that is dissimilar to the previously described impC promoter but is still triggered by UV-mediated damage. In strain 60A the impAB and the aggregative adherence fimbriae I (AAF/I)-encoding genes are on the same large plasmid, and the 60A version of the operon is predominantly seen in AAF/I-positive EAEC. Supplementary imp SOS-inducible error-prone repair systems are common among EAEC even though they are absent in prototypical strain 042.
Enteroaggregative Escherichia coli (EAEC) are intestinal E. coli that demonstrate a characteristic “stacked brick” or aggregative pattern of adherence to eukaryotic cells and solid supports (44). EAEC isolates were originally associated with persistent diarrhea in children from developing countries (6). More recently, EAEC has been implicated as a leading diarrheal pathogen in people of all ages in both developing and industrialized nations (reviewed in references 27, 28, and 48). A tentative model of EAEC pathogenesis begins with aggregative adherence to the intestinal mucosa. EAEC strains then produce a copious in vivo biofilm associated with increased production of mucus by the bacteria and intestinal cells, followed by a cytokine-mediated inflammatory response with resultant mucosal toxicity and intestinal secretion (25, 43). Although this three-stage model highlights key phenotypes associated with EAEC pathogenesis, precise virulence mechanisms remain to be determined. A large part of the challenge associated with understanding of EAEC pathogenesis arises from the diversity among strains that show aggregative adherence. Several adhesins (different types of aggregative adherence and other fimbriae), toxins (Shigella enterotoxins, EAEC plasmid-encoded enterotoxin, alpha-hemolysin, and EAEC heat-stable toxin), iron acquisition systems, and other factors (mucinase, dispersin) have been proposed to contribute to EAEC pathogenesis, but none of
these factors have been found to be conserved among all EAEC strains (16, 27–29, 48, 49). Volunteer studies of EAEC have suggested that virulence in humans is similarly heterogeneous and arises from both bacterial and host factors (30, 37, 42). Most known EAEC virulence factors are encoded by genes on a partially conserved plasmid, pAA. Although it is probable that at least some EAEC strains are nonpathogenic, the current state of knowledge suggests that strains that carry the pAA plasmid, and some (including outbreak isolates) that do not, are pathogenic. It is possible that much of the variation among strains is due to differences in other mobile elements or the host chromosome, which do not appear to be conserved. EAEC strain 042 has a serotype of O44:H18 and carries an aggregative adherence (AA) plasmid with genes aaf (encoding type II aggregative adherence fimbriae [AAF/II]), shf (cryptic), pet (plasmid-encoded toxin), aggR (aggregative adherence regulator), aap, aat (dispersin, dispersin export system), and the chromosomal genes irp-2, chuA (iron acquisition), and pic/set1 (mucinase, Shigella enterotoxin 1) (16, 49). Multilocus enzyme electrophoresis (MLEE) has been used to sort strain 042 into the EAEC2 phylogenetic group. A number of virulence factors from this EAEC strain have been well studied, and the genome sequence of this strain has recently been completed (http: //www.sanger.ac.uk/Projects/Escherichia_Shigella/). The only other EAEC strain that has been studied at the molecular level in significant detail is strain 17-2, a Chilean isolate that was subsequently shown to be avirulent in adult volunteers (42). Very little is known about other potentially pathogenic EAEC strains, and recent reports suggest that studying them may uncover new virulence or fitness genes (19). Although one study reported that EAEC were frequently found in low pH sauces (1), there have been no reports of genes that may
* Corresponding author. Mailing address: Department of Biology, Haverford College, 370 Lancaster Ave., Haverford, PA 19041. Phone: (610) 896-1470. Fax: (610) 896-4963. E-mail:
[email protected]. † Supplemental material for this article may be found at http://jb .asm.org/. 䌤 Published ahead of print on 9 March 2007. 3793
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J. BACTERIOL. TABLE 1. Principal bacterial strains and plasmids used in this study
Strain or plasmid
E. coli strains 042 60A CFT073 DH5␣ TOP10 MC4100 60A⌬impB Plasmids pCR-Blunt pGEM-T pImp (pLR31) pINK1003 pINK1009 pINK1010 pINK655 pLMJ45 pLMJ50 pLR25::Cm pRS551 pTP223 pUC19 a
Description
O44:H18 Peruvian EAEC2 isolate; Cmr Tetr Mexican EAEC1 isolate Ampr Uropathogenic E. coli strain isolated from a case of pyelonephritis F⫺ 80dlacZ⌬M15 ⌬(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK⫺ mK⫹) phoA supE44 ⫺ thi-1 gyrA96 relA1 F⫺ mcrA (mrr-hsdRMS-mcrBC) 80lacZ⌬M15 lacX74 deoR recA1 araD139 ⌬(ara-leu)7697 galU galK rpsL (Strr) endA1 nupG F⫺ araD139 ⌬(argF-lac)U169 rpsL150 relA1 fblB3501 deoC1 ptsF25 rbsR Ampr Cmr; impB deletion mutant Kanr; blunt-end cloning vector Ampr; TA cloning vector Ampr; pWKS30 containing the 2.4-kb imp operon from Shigella flexneri 2a strain SA100 pLMJ50 subclone containing the disrupted impAB region in pUC19 60A impA promoter region fused to lacZ in pRS551 impAB genes from 60A cloned into pCR-Blunt Shigella enterotoxin 1 gene promoter described by Behrens et al. (4) fused to lacZ in pRS551 Kanr; impCAB operon cloned into pCR-Blunt Cmr; large plasmid from 60A⌬impB carrying the disrupted impB gene and aaf, aap, and aggR genes associated with aggregative adherence Cmr; pWKS30 containing the imp operon disrupted by a chloramphenicol cassette lacZ reporter gene fusion vector Tetr; ColE1 origin of replication; -Red plasmid Ampr; high-copy-number cloning vector
Source or reference
41 16 64 Invitrogen (53) Invitrogen 12 This study Invitrogen Promega 52 This study This study This study This study This study This study 52 58 39 Invitrogen
Cmr, chloramphenicol resistance; Tetr, tetracycline resistance; Ampr, ampicillin resistance; Strr, streptomycin resistance; Kanr, kanamycin resistance.
contribute to the fitness or survival of EAEC outside of human hosts. Furthermore, genetic analysis of other EAEC strains could assist in producing a genetic and therefore more robust definition of the category. We sought, through subtractive hybridization, to identify unique genetic loci in a relatively uncharacterized EAEC isolate. EAEC strain 60A was isolated from a child with diarrhea in Mexico and carries a pAA plasmid. Strain 60A shares with 042 pAA genes such as aap, aat, and aggR. Both 60A and 042 also carry the chromosomal irp-2 and pic/set1 loci, which encode iron utilization and toxin functions, respectively, and mark pathogenicity islands common among many EAEC strains (16). Strain 60A lacks the 042 plasmid-encoded enterotoxin, pet, and the cryptic shf gene. 60A also possesses AAF/I fimbrial genes, which have yet to be associated with disease, while 042 expresses AAF/II fimbriae, which have shown epidemiological association with diarrhea (10, 47). Strain 60A is phylogenetically classed with EAEC1 strains. In these respects, it is more like strain 17-2 than strain 042. However, unlike strain 17-2, 60A does not carry alpha-hemolysin genes but demonstrates pronounced in vitro virulence-associated phenotypes such as adherence, autoagglutination and biofilm formation, and therefore it was used as a subtractive hybridization tester with strain 042 as the driver. MATERIALS AND METHODS Bacterial strains and plasmids. Table 1 shows the strains used in the present study. Reference EAEC strain 042, whose genome sequence has recently been completed (http://www.sanger.ac.uk/Projects/Escherichia_Shigella/), was used as subtractive hybridization driver. The tester strain, 60A, shows a classic aggregative adherence pattern on HEp-2 cells in the definitive assay for EAEC and has
exceptional biofilm-forming capabilities (56, 62). Strain 60A belongs to one of three major MLEE-defined EAEC phylogenetic groups, EAEC1, whereas strain 042 belongs to the similarly defined EAEC2 group (16). Strain 60A demonstrates pronounced in vitro virulence-associated phenotypes such as adherence, autoagglutination, and biofilm formation (data not shown). In addition, compared to most other EAEC strains, we have determined that 60A has a limited antimicrobial resistance profile, making it amenable to genetic manipulation. Strain 60A was therefore used as a subtractive hybridization tester, and strain 042 was used as a driver. Thirty-four EAEC strains, including representatives from the major EAEC lineages identified by MLEE, as well as Shigella flexneri 2a strain 2425T, were used in a screen for the 60A-specific loci identified in the present study. E. coli K-12 strains TOP10 (Invitrogen) and DH5␣ were used as hosts for cloned genes, and promoter fusion constructs were evaluated in strain MC4100. Bacteria were cultured in Luria broth (LB) or LB agar. Antibiotics—ampicillin (100 g/ml), chloramphenicol (30 g/ml), tetracycline (25 g/ml), or neomycin (50 g/ml)—were added for selection when required. Strains were maintained in LB-glycerol (1:1) at ⫺70°C. Routine molecular biology procedures. Standard molecular biology procedures were used (54). DNA amplification was performed with 1 U of recombinant Taq polymerase enzyme, 2 mM MgCl2, PCR buffer (Invitrogen), and 1 M oligonucleotide primer in each reaction. All amplifications began with a 2-min hot start at 94°C, followed by 30 cycles of denaturing at 94°C for 30 s, annealing for 30 s at 5°C below primer annealing temperature, and extension at 72°C for 1 min for every kilobase of DNA. PCR templates were prepared with boiled bacterial colonies, plasmid, or genomic DNA. High-fidelity PCR for sequencing followed a similar protocol but used Pfx polymerase and magnesium sulfate (Invitrogen). Annealing temperatures were lowered by 2 to 3°C and the extension times were doubled for Pfx high-fidelity PCRs. Oligonucleotide primer sequences are listed in Table S1 in the supplemental material. Unless otherwise stated, ligations were performed by using Quick T4 ligase enzyme (NEB), and clones and plasmids were transformed into chemically competent E. coli K-12 DH5␣ or TOP10 cells. Transformation of large plasmids into Electromax DH5␣-E cells (Invitrogen) and all plasmids into EAEC strains was accomplished by electroporation using a Micropulser (Bio-Rad) according to the manufacturer’s instructions. Subtractive hybridization. Genomic DNA was isolated from 60A and 042 by using the Easy-DNA kit (Invitrogen) according to the manufacturer’s protocol.
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TABLE 2. EAEC strain 60A-specific fragments obtained when the strain was subtracted against EAEC 042 Subtracted clone
Fragment size (bp)
Gene identified by BLAST (GenBank accession no.)
pLMJ7 pLMJ8 pLMJ10 pLMJ18 pLMJ17 pLMJ24 pLMJ29
200 250 350 420 380 380 480
“F” from phage X174 (NC_001422) ydcD from E. coli K-12 (NC_000913) “D” from phage X174 (NC_001422) Ecs2800 from E. coli O157 Sakai (NC_002695) ygcG from E. coli K-12 (NC_000913) ymfE from E. coli K-12 (NC_000913) impB from Shigella flexneri (AF079316)
A PCR-Select subtractive hybridization kit (Clontech/BD Biosciences) was used to subtract RsaI-digested DNA with EAEC strain 60A as the tester and EAEC strain 042 as the driver according to the manufacturer’s instructions, which are essentially as described by Akopyants et al. (2). Briefly, two different PCR adaptors were ligated to different aliquots of the tester (strain 60A) DNA and then denatured and mixed with excess driver (strain 042) DNA, which did not have adaptors, in separate pools. The pools were combined, and a second round of driver subtraction was performed with more denatured 042 DNA. Unsubtracted complementary single strands of tester DNA were annealed and then amplified by PCR, using primer pairs complementary to the original adaptor sequences. PCR-amplified subtracted fragments were visualized on a 2% agarose gel, excised, and extracted by using a QIAquick gel extraction kit (QIAGEN). Gelextracted DNA was incubated at 72°C for 20 min in PCR mix with 0.5 U of Taq polymerase to replace deoxyadenosine overhangs that may have been destroyed during extraction and then ligated into the pGEM-T vector (Promega). The clones were sequenced by using a vector-priming M13F (Universal) oligonucleotide. The sequences obtained were then compared to the GenBank nucleotide database (www.ncbi.nlm.nih.gov) by a standard nucleotide-nucleotide BLAST search. The absence of subtracted sequences from strain 042 was confirmed by BLAST analysis of the strain 042 genome sequence assembly at http://www .sanger.ac.uk/cgi-bin/BLAST/submitblast/escherichia_shigella. The presence or absence of two subtracted targets in 34 EAEC strains was determined by PCR by using primer pairs specific for these loci (Table 2). Construction and transcomplementation of an impB mutant in EAEC strain 60A. A Shigella flexneri (SA100)-derived impB::cat deletion construct (pLR25::cat) (52) was kindly provided by Laura Runyen-Janecky of the University of Richmond. The disrupted impB insert was excised by Pst1 digestion, and allelic exchange of the linear fragment with impB in strain 60A was effected by using -Red-mediated recombination essentially as described by Murphy et al. (39, 40) and using the tetracycline-resistant, temperature-sensitive -Red construct pTP223. Briefly, 60A carrying pTP223 was grown up overnight at 30°C with shaking and diluted 50-fold in LB containing 1 mM IPTG (isopropyl--D-thiogalactopyranoside). The cells were grown at 30°C to a final optical density at 600 nm (OD600) of 0.5. They were then heat shocked at 42°C, pelleted, and resuspended in 20% glycerol plus 1 mM morpholinepropanesulfonic acid. Then, 100 l of cells was electroporated with the gel-purified DNA fragment, plated on LB agar plates containing chloramphenicol, and incubated overnight at 42°C. Chloramphenicol-resistant, tetracycline-sensitive mutant candidates were confirmed by PCR with three primer pairs. The disrupted region was also cloned from the mutant and sequenced. To complement the mutant, the impCAB operon was amplified from pIMP, a clone of the Shigella flexneri imp operon (52), using the primers impF and impR and the proofreading polymerase enzyme Pfx (Invitrogen). The resulting 2.4-kb product was cloned into the kanamycin- and neomycin-resistant vector pCR-Blunt, and the clone, pLMJ45, was verified by sequencing. For complementation, the pLMJ45 clone was introduced into 60A⌬impB by electroporation. UV irradiation survival and mutagenesis assay. Test strains wild-type 60A, wild-type 042, 60A⌬impB, and complemented mutant [60A⌬impB(pLMJ45)] were grown with shaking at 37°C, in LB with selective antibiotics where appropriate, to an OD600 of 0.7 to 0.8. The cultures were pelleted and resuspended in phosphate-buffered saline (PBS). Aliquots (3 ml) of the resulting suspensions containing 5 ⫻ 108 to 5 ⫻ 109 CFU/ml were subjected to UV exposure in glass beakers (diameter, 40 mm; liquid depth, 4 mm) using a UV cross-linker (Stratagene). Unexposed and exposed cells were appropriately diluted in 1% peptone, and then viable counts were determined on LB, as well as on plates containing 50 g of streptomycin/ml. Each experiment was performed in triplicate on at
Putative function of gene (degree of homology 关% identity兴; e-value)
Major coat protein (98; 1e⫺99) Unknown (100% Identity; 1e⫺127) Phage assembly (99; 1e⫺170) Unknown (99; 0.0) Unknown (99; 0.0) Unknown (99; 0.0) UV protection and mutation repair protein (99; 0.0)
least two separate occasions. Differences in survival were compared by using the chi-squared and Fisher exact tests. The Wilcoxon signed-rank test was used to estimate significance in mutagenesis experiments. UV mutagenesis assay on solid media. To minimize the fluctuation effect that is commonly seen when mutagenesis is measured by plating irradiated cells on antimicrobial-containing plates in the survival assay (36, 52), we used a method in which the plates were applied to a solid surface before exposure to UV (17). Test and control strains were grown overnight with shaking at 37°C, in LB with selective antibiotics where applicable. A portion (100 l) of overnight culture, representing approximately 109 bacteria, was plated on 20-ml Mueller-Hinton agar plates (Oxoid) in standard 9-cm petri dishes, followed by incubation at 37°C for 1 h. The inoculated plate was exposed to 20 J/m2 of UV in a UV cross-linker (Stratagene) and then incubated at 37°C for 30 min. Antibiotic disks were applied to the plates by using a disk dispenser (Oxoid) and then incubated overnight at 37°C. The antibiotic disks used in the present study included ampicillin (10 g), ciprofloxacin (10 g), tetracycline (30 g), and streptomycin (300 g) (Oxoid). The zones of inhibition were measured in millimeters, and squatter colonies visible inside the inhibition zone were counted by using a dissecting microscope at a magnification of ⫻20. Mutagenesis results were only deemed comparable when the inhibition zone diameters between exposed and unexposed strains did not differ by more than 2 mm. The experiment was performed in triplicate on two separate occasions, and data were analyzed by using the Wilcoxon signed-rank test. Bile salts survival and mutagenesis. Bacterial suspensions were diluted to 2 ⫻ 105 CFU/ml in PBS for controls or in 1% sodium deoxycholate (Sigma) with selecting antibiotics where necessary. At 24, 48, and 96 h of incubation at 37°C the survival was measured by obtaining viable counts on LB agar plates. Mutagenesis was measured by inoculating the surface of Mueller-Hinton plates with bacteria that had been incubated in PBS or 1% sodium deoxycholate for 24 h and then performing a solid medium mutagenesis assay as described above. Promoter fusion assays. Gene promoters were cloned into the BamHI and EcoRI sties of pRS551 to create a transcriptional fusion to lacZ as described by Simons et al. (58). Overnight bacterial cultures were subcultured into LB and grown to an OD600 of 0.6 to 0.8. The -galactosidase activity of culture lysates on the substrate ONPG (o-nitrophenyl--D-galactopyranoside; Sigma, St. Louis, MO) was measured in Miller units (38).
RESULTS Subtractive hybridization identifies 60A-specific genes that are absent in the prototypical EAEC strain 042 but found in other EAEC strains. PCR-based subtraction yielded seven 60A-specific DNA fragments. As shown in Table 2, we found an identical or nearly identical match to each subtracted sequence in the GenBank database using BLAST. Three subtracted sequences were virtually identical to the E. coli K-12 sequence derived from strain MG1655 (7), and two were identical to X174 phage sequences (55). One target identified, open reading frame (ORF) Ecs2800 from enterohemorrhagic E. coli O157 Sakai (26), and also present in uropathogenic E. coli strain CFT073 (64), is part of a cryptic island absent in E. coli K-12, as well as enterohemorrhagic E. coli O157 strain EDL933. The island carries an IS629 element and genes of
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J. BACTERIOL. TABLE 3. Prevalence of 60A-specific genes in diverse EAEC strains
Strain
Country of isolation
EAEC1 EAEC1 EAEC1 EAEC1 EAEC1 EAEC1 INT1 INT2 INT2 DAEC2 DAEC2 EAEC2 EAEC2 EAEC2 EAEC2 EAEC2 EAEC2 AADA AADA AADA AADA AADA AADA AADA ND ND ND ND ND ND ND ND INT3 INT1
Mexico Peru Peru Chile Thailand Thailand The Philippines Thailand Peru Thailand The Philippines Peru Peru Thailand Thailand Peru Thailand Thailand Peru Thailand Thailand Peru Thailand Thailand Peru Peru Peru Nigeria India United Kingdom United Kingdom United Kingdom Serbia Japan
AA 60A NA H191-1 AA H232-1 AA 17-2 AA 253-1 AA 6-1 AA DS65-R2 AA 501-1 AA H223-1 DAWC212-11 AA DS67-R2 AA H38-1 AA 042 AA 144-1 AA 44-1 AA H145-1 AA 309-1 AA 103-1 DA H92-1 AA 435-1 AA 199-1 AA H194-2 AA 278-1 AA 239-1 H133 MH46-2 10-H32-1 G02a BM369 D163 D165 D172 C1096 AA 101-1 a
Presence (⫹) or absence (⫺) of:
MLEE-determined phylogenetic groupa
CVD 432 probe
ORF Ecs2799
impB
⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫹
⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹
Based on Czeczulin et al. (13).
unknown function, one of which is homologous to an antirestriction gene and another that is homologous to the DNA repair protein-encoding gene radC. Using the O157-annotated ORF Ecs2799 as a marker for this island and CFT073 as positive control, we screened 34 EAEC strains and detected it in 9 strains (Table 3). The final strain 60A-specific subtracted clone was identical to impB, which is predicted to encode part of an error-prone repair system earlier described in plasmid pTP110 and in a large plasmid in Shigella flexneri (35, 52). As shown in Table 3, it was present in 21 of 34 EAEC strains screened. impB contributes to UV resistance in EAEC strain 60A. Since impB is common among EAEC strains, it could contribute to fitness by protecting strains that harbor the gene from lethal DNA damage when exposed to damaging agents. The survival of wild-type impB-positive 60A after UV irradiation was measured after exposure to 40 and 100 J/m2 of UV radiation. In this experiment, strain 60A showed a two-log better survival at 40 J/m2 than reference EAEC strain 042, which does not have the impB gene (P ⬍ 0.0001; Fig. 1). Although these data demonstrate an increased survival rate for strain 60A upon UV exposure compared to the wild-type strain 042, we sought to assess the specific contribution of impB to this resistance. We created an isogenic impB mutant in 60A and
examined the susceptibility of the wild-type strain and the impB mutant to UV. As shown in Fig. 1, compared to the wild-type strain, the impB mutant was UV sensitive when exposed to low and intermediate levels of UV irradiation (P ⬍
FIG. 1. Survival of EAEC strains 042 (impB negative, dotted bars) and 60A (impB-positive, dark bars), as well as the 60A⌬impB mutant (clear bars) and the mutant with impCAB genes supplied in trans (hatched bars) after UV exposure. Mid-logarithmic-phase bacteria were exposed to 0, 40, and 100 J of UV/m2. Error bars represent the standard deviations.
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0.0001). This phenotype could be restored by complementation with the Shigella flexneri impCAB genes in trans (Fig. 1). Effect of impB on UV-induced mutagenesis. Error-prone repair proteins confer elevated mutagenesis rates. The genes encoding them are generally repressed by LexA until they are needed for repair as a result of DNA damage. We sought to determine the contribution of impB to UV-induced mutagenesis in EAEC. Wild-type EAEC strain 60A (impB positive) was tested in parallel with the survival assay, as were the 60A⌬impB mutant and the transcomplemented strain, 60A⌬impB(pLMJ45), bearing an impCAB operon from Shigella. Mutagenesis was measured as the CFU showing streptomycin resistance, a phenotype that arises due to single point mutations in the ribosomal protein S12 (rpsL) gene or 16S rRNA and is indicative of error-prone repair. As shown in Fig. 2A, although there was no detectable difference in strains not exposed to UV, the impB mutant was significantly less likely than the wild-type strain to generate streptomycin-resistant colonies after UV treatment (P ⬍ 0.03), and this phenotype could be complemented in trans. The range of datum points obtained by this method was very large, occasionally spanning more than 2 logs (Fig. 2A). This is evident in similar studies of this nature and consistent with a fluctuation effect, which is unavoidable in experiments where the test bacteria are exposed to UV in liquid suspension (33, 36, 52). We therefore elected to use the method of Denamur et al. (17) to further assess mutagenesis since the E. coli cells are exposed to UV after plating, and the antibiotic is applied as a disk. Mutants arising from UV-mediated errorprone repair are fixed in their position on the plate, obviating the need for postexposure fluctuation, and are measured as squatter colonies within the zone of inhibition (Fig. 2B). We examined mutation to resistance as measured by the number of squatter colonies within the zones of streptomycin (300-g) and ciprofloxacin (10-g) disks. Resistance to these agents can be conferred by one or multiple point mutations, respectively. Since 60A is naturally ampicillin resistant and 042 is tetracycline resistant and the appearance of resistance of E. coli to either typically requires the acquisition of horizontally acquired DNA, we used ampicillin (10-g) and tetracycline (30-g) disks as controls. In all control zones, we found fewer than 10 squatter colonies when the strain was susceptible and an undisturbed lawn in resistant strains, even after exposure to 20 J of UV/m2. In contrast, strain 60A produced large numbers of squatter colonies around the streptomycin and ciprofloxacin disks after UV exposure, which was significantly more than were present at baseline (P ⬍ 0.03) (Fig. 2B, C, and D). The impB-negative EAEC strain 042 produced much fewer squatter colonies after UV exposure, which was significantly more than the baseline with streptomycin (P ⱕ 0.03) but not with ciprofloxacin (P ⬎ 0.05). Exposure to 20 J of UV/m2 did not significantly increase the appearance of squatter colonies in
FIG. 2. (A) Proportion of survivors that have acquired resistance to streptomycin, with or without exposure to 40 J of UV/m2. Horizontal bars show medians, and vertical bars represent the range of datum points generated. (B) Squatter colonies within streptomycin inhibition zones counted on plates surface inoculated with EAEC strain 60A, its impB deletion mutant, and the mutant complemented with the Shigella impCAB genes. The plates had not (top) or had (bottom) been exposed
to 20 J of UV/m2. (C and D) Mutagenesis in response to streptomycin (C) and ciprofloxacin (D) resistance as measured by the number of squatter colonies within inhibition zones around streptomycin (300g) and ciprofloxacin (10-g) disks, respectively, in experiments performed as described for panel B. Diamonds represent datum points, and median values are depicted as horizontal bars.
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FIG. 3. (A and B) Survival (A) and mutagenesis (B) of EAEC strain 042 (impB negative), EAEC strain 60A (impB negative), the 60A⌬impB strain and its impCAB transcomplement (pLMJ45) in 1% sodium deoxycholate. For panel A, shaded, hatched, and clear bars indicate the proportions of the inocula surviving after 1, 2, and 4 days, respectively. In panel B, squatter colonies within 300-g streptomycin disks from inocula incubated in PBS and 1% sodium deoxycholate (SDC). Diamonds represent datum points, and median values are depicted as horizontal bars.
streptomycin or ciprofloxacin zones for the 60A⌬impB isogenic mutant (P ⬎ 0.05), and the mutant phenotype could be significantly complemented in trans (P ⱕ 0.03). Our data imply that impB confers not only UV resistance to killing but also UVinduced mutagenesis in EAEC strain 60A, suggesting that error-prone repair may contribute to UV survival. imp-mediated survival and mutagenesis is induced by bile salts. Unlike enteropathogenic and enterohemorrhagic E. coli strains, EAEC strains are considerably heterogeneous, even within MLEE-determined clonal groups (16). These strains also show an as-yet-unexplained greater propensity to harbor antimicrobial resistance genes (21, 47, 63). We hypothesized that the imp genes could have contributed to this heterogeneity. Our hypothesis would be supported if EAEC strains are commonly exposed to DNA damage that could lead to LexA derepression and consequent imp gene upregulation, enhancing the mutagenesis and survival of repaired genomes. Since LexA derepression also upregulates other error-prone polymerases, the potential for genomic innovation in SOS-re-
J. BACTERIOL.
sponding EAEC strains is high. DNA-damaging agents that could be encountered in the wild include UV light, as well as DNA-damaging antimicrobials, which are increasingly used clinically (15). A given EAEC strain would be expected to encounter these conditions occasionally, or even rarely, but there is reason to believe that they could lead to significant genetic alterations. If LexA-repressed genes were routinely upregulated in the intestinal tract, EAEC strains, which are long-term colonizers, would be exposed to considerable errorprone repair and its mutagenic consequences. Studies in E. coli and Salmonella enterica serotype Typhimurium have shown that the SOS system is activated by bile salts, which are abundant in the human intestine (5, 51). As shown in Fig. 3A, the recovery of strain 042, an impB-negative EAEC strain, decreases significantly after incubation at 37°C in the presence of low amounts (1%) of sodium deoxycholate, one of the most abundant bile salts (P ⱕ 0.0001). Although almost half of a similarly treated 60A inoculum can be recovered after 1 day, the numbers do not decrease significantly thereafter (P ⬎ 0.05). In contrast, survival of the 60A⌬impB mutant in the presence of sodium deoxycholate is drastically diminished (P ⱕ 0.0001 [chi-squared analysis for linear and logarithmic trends]) but remains at wild-type levels upon complementation in trans (Fig. 4A). Deletion of impB also reduced the appearance of squatter colonies within streptomycin disk zones when the inocula had been exposed to 1% sodium deoxycholate for 24 h (P ⱕ 0.03; Fig. 3B) EAEC strains possess intact umuDC operons. An impCAB operon, which is a plasmid-borne homolog of the LexA-repressed umuDC operon, has been described in Shigella. As shown in Fig. 1 to 3, this operon can complement the EAEC 60A impB deletion in trans. Shigella strains have a nonfunctional chromosomal umuDC operon, due to sequence polymorphism within the Shigella LexA box of umuDC, that compromises SOS-induced transcription of those genes. In Shigella, this defect has been naturally complemented by plasmid-borne impCAB genes (52). Thus, impCAB genes provide compensation for a chromosomal defect in these strains. We sequenced the umuDC operon from EAEC strain 60A and retrieved the 042 sequence of these genes from the Sanger genome project (http://www.sanger.ac.uk/Projects/Escherichia _Shigella/). The region encompassing ⫺35, ⫺10, and LexA binding sites was entirely conserved in strains 042 and 60A and is identical to the E. coli K-12 sequence, and the 2.4-kb operons are 97% identical to that of E. coli K-12. The 60A imp genes therefore represent a supplementary error-prone repair system. Strain 60A carries a modified but functional imp operon on its aggregative adherence plasmid. Electroporation of total plasmid preparation from the impB::cat mutant 60A⌬impB into E. coli DH5␣ and selection on chloramphenicol plates revealed that the region marked by the impB::cat replacement was located on a single large plasmid, pLMJ50 (approximately 100 kb in size). The 480-bp subtracted product that contained most of the impB gene from strain 60A was 99% identical to impB from Shigella. However, multiple primer pairs designed to match different regions in and around impC and impA from Shigella and avian pathogenic E. coli failed to amplify these genes in EAEC strain 60A, suggesting that although the impB gene is highly conserved, its context was less conserved. We
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FIG. 4. impAB operon from EAEC strain 60A. (A) Schematic diagram of the imp operons from strain 60A (the present study) and Shigella flexneri (52). (B) CLUSTAL W multiple alignment of ImpA sequences from Shigella and EAEC strain 60A with the E. coli UmuD protein. A required A-G/C-G cleavage site at UmuD positions 24 and 25 is boxed. A serine residue predicted to be a nucleophile (UmuD position 60) and a lysine residue required for activation (UmuDC position 97) are shaded. Other residues conserved within the UmuD family but absent in related RecA autocleavable proteins are indicated in boldface.
created pINK1003, a clone of the regions flanking the impB::cat region of mutant 60A⌬impB using the inserted chloramphenicol acetyltransferase (cat) gene as a selective tool. We sequenced the region immediately flanking the impB gene then designed primers impAupEcoRI and 60AimpR2 (Table S1 in the supplemental material), complementary to this flank and used these primers to amplify the entire imp operon and its immediate flanking sequence from wild-type strain 60A. As shown in Fig. 4, the sequence of the imp operon from 60A (GenBank accession no. EF100676) differs considerably from imp operons that have been reported from other enteric plasmids (31, 35, 52). The impB gene is conserved with other imp operons, but impC, whose function is not known, is entirely absent from strain 60A. All ImpA residues predicted to be essential for function (11, 20, 45, 50, 57) are conserved in strain 60A. However, the predicted N-terminal 18 amino acids of ImpA, which are believed to be cleaved off from the active from of the mature protein, are not conserved between impA alleles or with chromosomal homolog UmuD (Fig. 4). We evaluated the ability of the cloned 60AimpAB operon, in plasmid pINK1010, to complement the 60A⌬impB mutation in trans. As shown below (see Fig. 6A), the cloned 60A impAB operon was able to restore UV protection to the wild-type strain in a manner comparable to the clone of impCAB from Shigella flexneri. Thus, this variant imp operon is indeed functional (see Fig. 6). At the 3⬘ end of the imp operon, downstream of impB, lie stb and par genes, which are similarly adjacent to impB (and its homolog samB) from Salmonella plasmids R64 and pYQ122 and avian pathogenic E. coli plasmid pAPEC-O2-R (31, 46). Upstream of impA in strain 60A is a divergent ORF predicted to encode a resolvase (ORF1 in Fig. 5). The gene shows no similarity with any GenBank database entries at the DNA level. Translated BLAST (tBLASTX) analysis demonstrated that predicted residues 26 to 197 of ORF1 showed 78% iden-
FIG. 5. UV survival of wild-type strain 60A and the impB mutant after exposure to UV doses ranging between 10 and 50 J/m2. The mutant was complemented in trans by the cloned imp genes from strain 60A (A) and a Shigella strain (B).
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JOO ET AL. TABLE 4. imp operon and AAF types of different EAEC strains
EAEC MLEE-determined phylogenetic group (no. of strains)
impB-negative strains (fimbrial type)
No. of impB-positive strains
EAEC1 (6)
NA H191-1
5
Intercluster (5)
DS65-R2 AA 501-1 AA H223-1 C1096 DA WC212-11 042 (AAF/II)
1
DAEC2 (2) EAEC2 (6) AA/DA (7) Undetermined (8)
AA 103-1 AA 435-1 (AAF/II) AA 199-1 (AAF/II) AA 239-1 H133 (AAF/II) G02a
tity and 86% similarity to a predicted member of the serine recombinase family from plant pathogen Erwinia pyrifoliae (protein database accession no. AAN04546). We screened EAEC strains from the Shigella and 60A versions of the 5⬘ imp operon region. All impB-positive strains identified in the study harbored one or other of these two versions of the operons. We detected the 60A impAB version in 12 EAEC strains and the Shigella impCAB version in 9 strains. Consistent with the location of both versions of the operon on plasmids, there was no association of imp operon type with host strain phylogeny as determined previously by MLEE (16). Interestingly, 9 of the 12 strains bearing the 60Atype impAB operon harbor the aggA structural gene for AAF/I fimbriae (Table 4). Strains that had the Shigella-type impCAB operon were negative for this gene. Three of the strains examined in the present study are known to harbor the aafA structural gene for AAF/II fimbriae. Two of these, including reference strain 042, lacked impB. It therefore appeared that there was some linkage disequilibrium between the fimbrial and imp genes. We isolated pLMJ50, the disrupted impAB-bearing plasmid, from the 60A⌬impB mutant (60A harbors up to six separate plasmids) and transferred it to a plasmidless E. coli strain. The resulting chloramphenicol-resistant transformant carried the aagA gene on a single 100-kb plasmid, confirming that the impAB and aagA genes localize to the same mobile element. This plasmid, pLMJ50, was also shown to bear genes encoding the antiaggregative factor, aap, and the aggregative adherence activator, aggR. Thus, in strain 60A, the impAB operon is located on the aggregative adherence plasmid. The 60A impAB promoter region is activated by DNA damage. Since the impAB upstream region differed significantly from that of impCAB from Shigella, we constructed and evaluated a promoter fusion, pINK1009, with lacZ placed downstream of the impA operon. For control purposes, we constructed a similar fusion of the EAEC Shigella enterotoxin 1 gene promoter, pINK655, which is the only EAEC promoter that has been characterized in detail (4). We then assayed the -galactosidase activity from these fusions after exposure to no
1 5 3 6
Strains with 60A-type impAB operon (fimbrial type)
60A (AAF/I) 17-2 (AAF/I) AA 253-1 (AAF/I) AA 6-1
Strains with Shigella-type impCAB operon (fimbrial type)
AA H232-1
AA 101-1 (AAF/II)
AA AA AA AA AA
DS67-R2 (AAF/I) H38-1 (AAF/I) H145-1 (AAF/I) 309-1 (AAF/I) H194-2 (AAF/I)
BM369 (AAF/I) MH46-2 10-H32-1
AA 144-1 AA 44-1 DA H92-1 AA 278-1 D163 D165 D172
UV or to 20 or 40 J of UV/m2. The Miller unit of -galactosidase activity incorporates an OD600 correction factor for cell density. Since this factor measures total count and UV treatment leads to a decline in viable cells (Fig. 1 and 5), we also determined the viable counts for each sample at the time of enzyme assay. As shown in Fig. 6, a comparable decline in viable count with increasing UV dose was seen in E. coli MC4100 cultures harboring the vector alone (Fig. 6A), the impA promoter fusion (Fig. 6B), or the set1 promoter fusion (Fig. 6C). However, only with the impA promoter fusion was there a concomitant increase in -galactosidase activity, in spite of the reduction in cell viability. Similar results were seen in a 60A⌬impB background (Fig. 6D to F). Although the set1 promoter was much stronger overall, its activity was not appreciably influenced by UV. DISCUSSION The genome sequence of EAEC strain 042 has recently been completed. Identification and characterization of genes in less well studied EAEC strains will improve the general understanding of this heterogeneous category of pathogens and might contribute toward a sensitive and specific test that could identify EAEC strains not detected by the currently used CVD 432 probe (3, 48). With these goals in mind, we subtracted the genome of EAEC strain 60A from that of prototypical strain 042. The subtraction was nonexhaustive and the number of subtractive clones we obtained is small but similar to numbers obtained by other workers using our methodology, due to acknowledged limitations of PCR-based suppression subtractive hybridization (14, 34). We were able to identify and amplify seven 60A-specific sequences, three of which are present in the E. coli K-12 genome. Four of the identified targets were on mobile elements or likely to have been acquired through recent horizontal transfer events, a finding consistent with emerging genomic information that suggests that differences between E. coli genomes arise largely due to horizontal trans-
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FIG. 6. The lacZ fusion vector pRS551 (used as a negative control in plots A and D) was used to assay impAB promoter activity. The activity of LacZ expressed from promoter fusions of 60A impA (B and E) and set1 promoters (C and F) was measured with or without exposure to UV. Bars represent LacZ activity in Miller units, and viable counts of bacteria are plotted as line graphs. Graphs A to C (black bars) show data obtained in an E. coli MC4100 background, whereas the data in graphs D to F (white bars) were obtained in a 60A⌬impB background.
fer (18, 64). Two loci were associated with a X174-like bacteriophage, and a third was associated with a putative antirestriction genomic island. Within and outside the EAEC category, the distribution of the island did not correlate with phylogeny or pathotype, a finding indicative that close phylo-
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genetic or pathotypic relationships cannot predict the distribution of some gene clusters or pathotypes and illustrating the genetic fluidity of the species (16, 65). The impB gene, which we also found to be present in EAEC strain 60A and absent in prototypical EAEC strain 042, has been previously reported from plasmids in select E. coli, Shigella flexneri, and Salmonella strains (22, 31, 52). Related to the imp genes are similar error-prone repair systems, borne on plasmids or other mobile elements, that have been described in Salmonella enterica serovar Typhimurium (samAB, mucAB), Providencia rettgeri (rumAB) Pseudomonas syringae, and Pseudomonas putida (rulAB) (9, 32, 46). As with these other plasmidborne systems, the impAB genes are homologous to umuDC, chromosomal E. coli genes that encode an error-prone DNA polymerase, PolV (22). PolV is one of three core but nonessential DNA polymerases encoded by chromosomal E. coli genes, all of which are transcriptionally repressed by LexA (23). PolV plays an important role in translesion repair, has an error rate of 10⫺2 to 10⫺4, and is not present in detectable levels when uninduced (23, 59, 60). Single-stranded DNA, produced when DNA is damaged, interacts with RecA, the first step in the SOS response. The resulting RecA nucleoprotein filament causes autoproteolysis of LexA and consequent derepression of genes encoding PolII (polB), PolIV (dinB), and PolV, as well as other genes involved in DNA repair and the inhibition of cell division. RecA also induces the autoproteolysis of UmuD, an essential step in the formation of the active UmuD⬘2C PolV enzyme. Plasmid-borne umuDC homologs are believed to encode similarly functioning proteins and, in the case of Shigella, effectively complement a mutation that results in a defect in the umuDC promoter (20, 52). The 60A-type imp operon is structurally more similar to other umuDC operons than is the previously described impCAB operon (35, 52) in that it is composed of two genes, impA and impB, that are homologous to umuD and umuC, respectively. In the present study, it was possible to demonstrate that although prototypical EAEC strain 042 does not have the gene, the majority of EAEC strains are impB positive. Both strains 042 and 60A harbor intact umuDC operons, and strain 042, which lacks the imp genes, demonstrates UV survival and mutagenesis comparable to E. coli K-12. EAEC strain 60A is more resistant to UV and showed an eightfold increased tendency to mutation to streptomycin resistance when exposed to UV light. Deleting the impB gene, and complementing it in trans, demonstrated that UV-mediated survival and mutagenesis in strain 60A can be attributed to this gene. imp-driven mutagenesis is relevant to survival and mutagenesis in other enteric bacteria. Homologs of these genes are found on plasmids that have been isolated from Shigella, Salmonella, avian pathogenic E. coli, Pseudomonas syringae, and Serratia marcescens, as well as a chromosomal integrative element, R391, originally identified in Providencia rettgeri (8, 20). Phage-borne homologues are also widely disseminated among the Enterobacteriaceae (13). Some E. coli strains carry truncated and therefore nonfunctional imp operons. For example, the virulence plasmid of enteropathogenic E. coli strain B171-8 lacks impC and impA, as well as 281 bp from the 5⬘ end of impB (61). Sequence analysis showed that all components of the operon known to be required for activity are present in the 60A imp operon but that the impC gene and the upstream sequence
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that has previously reported for this system (35, 52) were absent and altered, respectively. We have shown that, in spite of these polymorphisms, the imp genes contribute to UV survival in strain 60A. Furthermore, experiments with an impAB promoter fusion have demonstrated that the 60A imp operon is UV inducible and therefore likely under the SOS response system control. The imp operon could provide a selective advantage in the EAEC strains harboring this gene through impB-mediated repair after exposure to DNA-damaging agents. rulAB, a plasmid-borne impAB homolog described in the plant pathogen Pseudomonas syringae is activated by UV in vitro but is also upregulated when bacteria carrying this operon infect plants, which are exposed to UV-B radiation (32). This presents a scenario whereby rulAB-mediated survival and mutagenesis is perceptively relevant to the lifestyle and evolution of P. syringae. In another example, Yeiser et al. (66) have also demonstrated that error-prone polymerases contribute to evolutionary fitness of E. coli and that the loss of any core error-prone polymerases reduces strain competitiveness. Our data suggest that imp-positive EAEC strains exposed to DNA-damaging agents are more likely to survive and to experience genetic alterations than strains that lack the gene. Were UV radiation the principal means for imp operon activation, such opportunities for genomic innovation could be uncommon in an organism that inhabits mammalian intestines. We have, however, demonstrated that EAEC strains are likely exposed repeatedly to impAB error-prone repair from bile salt induction. Prieto et al. (51) recently reported that bile can cause DNA damage and thereby induce the SOS response in Salmonella a finding that corroborates an earlier report by Bernstein et al. (5). Prieto et al. (51) went on to show that the bile salt sodium deoxycholate is sufficient to produce this effect. We have demonstrated that bile salts induce error-prone repair in imp-positive E. coli by showing that survival and mutagenesis of EAEC strain 60A in the presence of sodium deoxycholate, one of the most abundant bile salts, is greater than its isogenic mutant and 042, an impAB-negative strain. Since EAEC strains are long-term intestinal colonizers, these are conditions that could be replicated in vivo. In addition, quinolone and other DNA-damaging antimicrobials can induce the SOS response and consequently derepress impAB. This is of particular concern because EAEC are often resistant to multiple antimicrobials and the fluoroquinolones are recommended for treating EAEC infections when an antimicrobial is indicated (24). Following the observation that antibiotic-induced in vivo LexA derepression enhanced mutation to ciprofloxacin, Cirz et al. (15) proposed that interfering with LexA cleavage could be a potentially viable strategy for preventing the emergence of clinical resistance. Such a strategy would be especially beneficial for EAEC control because many EAEC strains possess impAB or impCAB, encoding a supplementary error-prone polymerase. ACKNOWLEDGMENTS This study was supported by National Science Foundation grant RUI 0516591 and through a Branco Weiss Fellowship from the Society in Science, ETHZ, Zurich, Switzerland, to I.N.O. L.M.J. was an undergraduate Howard Hughes Medical Institute multicultural scholar at Haverford College. We thank Laura Runyen-Janecky at the University of Richmond for plasmids, Kenan Murphy for the -Red recombination system, and
J. BACTERIOL. Amanda Muir, Justin Dorff, and Jessica Kim for technical assistance. Validation of subtracted targets for this study was greatly assisted by access to in-process sequence data produced by the Escherichia coli and Shigella spp. comparative Sequencing Group at the Sanger Institute, which can be accessed at http://www.sanger.ac.uk/Projects /Escherichia_Shigella/. REFERENCES 1. Adachi, J. A., J. J. Mathewson, Z. D. Jiang, C. D. Ericsson, and H. L. DuPont. 2002. Enteric pathogens in Mexican sauces of popular restaurants in Guadalajara, Mexico, and Houston, Texas. Ann. Intern. Med. 136:884–887. 2. Akopyants, N. S., A. Fradkov, L. Diatchenko, J. E. Hill, P. D. Siebert, S. A. Lukyanov, E. D. Sverdlov, and D. E. Berg. 1998. PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc. Natl. Acad. Sci. USA 95:13108–13113. 3. Baudry, B., S. J. Savarino, P. Vial, J. B. Kaper, and M. M. Levine. 1990. 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