Genome-Wide Transposon Mutagenesis Reveals a Role for pO157 Genes in Biofilm Development in Escherichia coli O157:H7 EDL933

Bacteria.The E. coli strains used in this study are shown in Table 1. A spontaneous nalidixic acid-resistant mutant of Escherichia coli O157:H7 strain EDL933 was selected to serve as a counterselection for these studies. The mutation in this mutant had no effect on biofilm formation or on the ability of EDL933 to colonize and persist in a sheep model of colonization (unpublished data). For all biofilm assays, the cultures were grown in Luria-Bertani (LB) broth for 24 h at 30°C under stationary conditions. For all other experiments, the cultures were grown overnight in LB broth at 37°C with shaking at 200 to 220 rpm. The temperature-sensitive E. coli strain harboring the pRedET plasmid (Gene Bridges GmbH, Dresden, Germany) was grown at 30°C. Antibiotic concentrations were 100 μg/ml for ampicillin, 50 μg/ml for kanamycin, and 20 μg/ml for nalidixic acid except where noted. All antibiotics were obtained from Sigma Chemical Co. (St. Louis, MO). The growth of individual mutants was assessed by measuring the growth endpoint optical density at 600 nm (OD₆₀₀) after 24 h of growth in 96-well plates and compared to that of wild-type EDL933.

Mutant Library Construction.Transposon mutagenesis was performed as described previously (22) with a few modifications. To obtain random mini-Tn5Km2 insertion mutants, the conjugal donor strain E. coli BW19795 containing the plasmid pUTmini-Tn5Km2 (supplied by David Holden, Imperial College, London, United Kingdom) was conjugated with E. coli O157:H7 EDL933. One milliliter of overnight culture of each strain was pelleted, washed three times with phosphate-buffered saline (PBS), resuspended in 5 ml of antibiotic-free LB broth, and incubated at 37°C with shaking until an OD₆₀₀ of 0.7 to 1.0 was reached. The BW19795 donor strain was then transferred to stationary conditions for 30 min to allow regeneration of pili. Two hundred microliters of each strain was combined, and the mating mixture was plated onto a sterile membrane placed on a stack of sterile filter paper. Once the liquid medium was removed by capillary action, the membrane was transferred to an LB agar plate cell side up. Following overnight incubation at 37°C, membranes were vortexed with LB medium, and the suspension was incubated with shaking for 1 h at 37°C and then plated in 100-μl aliquots on LB plates containing kanamycin plus nalidixic acid as a counterselection against the donor strain. Each plate yielded 300 to 400 colonies of kanamycin-resistant mutants. Colonies were picked into 96-well plates, and each was scored for ampicillin resistance. The ampicillin-sensitive mutants were rearrayed into 96-well plates and stored at −70°C for further analysis. A total of 11,000 independent mutants were obtained.

Assays for biofilm formation.The screen for the biofilm-negative phenotype was performed using a microtiter plate assay as described previously (16) with minor modifications. This assay is based on the ability of biofilm-forming bacteria to adhere to the wells of a 96-well microtiter plate, which are subsequently visualized by staining with crystal violet. Each plate of mini-Tn5 insertion mutants from an overnight LB culture was replica plated using a 96-prong replicator into fresh Costar 96-well, flat-bottom, nontreated polystyrene plates (Corning, Inc., New York, NY) containing 150 μl of LB broth per well. After 24 h of incubation at 30°C under stationary conditions, the plate was rinsed twice with water, and the adherent bacteria were stained with 0.01% crystal violet (175 μl/well) for 20 min. After staining, the plates were washed again twice with water. At this point, biofilms were visible as a violet ring on the side of each well and as a generalized staining of the well (Fig. 1). Mutants that lacked ring formation were scored as having a biofilm-negative phenotype. For growth assessment, a duplicate plate incubated overnight was briefly vigorously shaken on a microtiter plate shaker, and the OD₆₀₀ was measured and compared with that of the wild-type control strain. Strains with growth that differed from wild-type growth (P < 0.01) were not considered further, as were mutants that showed inconsistent biofilm formation. In each plate, E. coli strain BW19795 was used as a negative control, and a well containing medium only was used as a blank.

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FIG. 1.

Microtiter plate assay showing screening of mini-Tn5Km2 mutants for the biofilm-negative phenotype. Wells 4 and 9 contained the wild-type biofilm-positive cells, wells 5 and 10 contained the biofilm-negative control (BW19795), wells 3 and 6 contained biofilm-negative transposon mutants, and wells 1, 2, 7, and 8 contained biofilm-positive transposon mutants.

For the quantitative biofilm assay (57), 12- by 75-mm polystyrene tubes (Fisher) were used. An overnight culture of each mutant, plus controls, was diluted 1:100 into 2 ml of LB broth and incubated at 30°C under stationary conditions for 24 h. The tubes were then rinsed twice with water and stained with 2.5 ml of 0.01% crystal violet for 20 min. After being washed three times with water, tubes were air dried and destained with 2.5 ml of 80% ethyl alcohol for 15 min. The tubes were vortexed, 100 μl was transferred to a new 96-well plate, and the optical density was measured at 595 nm using a Spectra MAX 190 spectrophotometer (Molecular Devices, Union City, CA). The optical density measurements were used as a measure of relative amounts of biofilms formed. All experiments were performed in triplicate.

Identification of mini-Tn5 insertion sites.The mini-Tn5 insertion sites in biofilm-negative mutants were mapped by sequencing the amplified genomic region at the site of mini-Tn5 insertion. The amplification of the region of the DNA at the site of transposon insertion was done using either single-primer PCR(25) or Y-Linker PCR(30). The PCR products were then sequenced and analyzed using BLAST against the E. coli O157: H7 EDL933 genome sequence.

Construction of deletion mutants.To generate deletion mutations, a one-step gene inactivation method adapted from that described by Datsenko and Wanner (13) was used. The temperature-sensitive plasmid pRedET (Gene Bridges, Dresden, Germany), encoding lambda red recombinase, was transformed into E. coli O157:H7 EDL933. The kanamycin resistance gene was amplified from pKD4 (13) using primers shown in Table 2. Each primer sequence contained target-homologous sequences as well as sequences for amplification of the kanamycin gene. The products of this reaction were electroporated (2,000 V, 129 Ω) using a BTX electrocell manipulator (model 600; Harvard Apparatus, Holliston, MA) into E. coli O157:H7 EDL933/pRedET that was previously induced with 0.4% l-arabinose for 1 h. The cells were incubated in SOC medium (20 g tryptone, 5 g yeast extract, 2 g MgCl₂·6H₂O, 2.5 g MgSO₄·7H₂O, and 3.6 g glucose per liter; pH 7.5) for 1 h and then plated on selective medium (LB supplemented with 25 μg/ml of kanamycin) at 37°C.

Confirmation of mutant constructions and determination of the locations of the kanamycin gene insertions were done by PCR. Primer F (which has homology within the kanamycin cassette) and primer R (which has homology immediately downstream of the gene sequences that were being replaced) were used to generate PCR products. To ensure curation of the temperature-sensitive pRedET plasmid, confirmed mutants were first grown at 42°C for 2 h and then plated on LB plates and incubated overnight at 37°C. The isolated colonies were picked and screened for kanamycin resistance and ampicillin sensitivity. All of the primers used are shown in Table 2.

Construction of plasmid pISM30 for genetic complementation.The ehxCABD operon was amplified using primers that also incorporated upstream KpnI and downstream XbaI restriction sites (Table 2). The PCR was done using LongAmpTaq DNA polymerase (New England Biolabs) with the following parameters: 2 min at 95°C; 30 cycles of 95°C for 30 s, 58°C for 7 min, and 72°C for 1 min; and 5 min of extension at 72°C. The fragments were digested with KpnI and XbaI, cloned into vector pBAD18, and then transformed into E. coli DH5α. One plasmid, designated pISM30, was confirmed by restriction digestion and was electroporated into E. coli O157:H7 EDL933 ΔehxD.

Cell culture.The T84 human colonic adenocarcinoma cells were maintained in 25-cm² (Falcon) tissue culture flasks as monolayers at 37°C with 5% CO₂. The cell cultures were grown in Dulbecco modified Eagle medium (DMEM)-F-12 medium (Invitrogen) supplemented with 2.5 mM l-glutamine, 5% fetal bovine serum, and gentamicin (50 μg/ml). The cells were passed every 7 days by treatment with 0.5% trypsin, and the medium was replaced every other day.

Bacterial adhesion assay.Quantitative adhesion assays were performed using monolayers of T84 cells grown on glass coverslips. The glass coverslips were treated with 1 N HCl for 10 min, washed three times with sterile water, and placed in six-well polystyrene tissue culture plates (Costar, Corning, Corning, NY). T84 cells (4 × 10⁵) were seeded onto the glass coverslips in each well and allowed to attach overnight. The monolayers were then washed with Hanks balanced salt solution and replenished with 1 ml of culture medium without antibiotics. An overnight culture of bacteria was diluted 1:20 in fresh LB and grown for another 2 h. One hundred microliters of this culture (approximately 4 × 10⁶ bacteria) was added to each well containing T84 monolayer cultures. Bacterial cultures were serially diluted and plated to enumerate bacteria added. The tissue culture plates were then incubated at 37°C with 5% CO₂ for 1.5 h. The coverslips were washed three times with PBS to remove nonadhered bacteria, and then the glass coverslip was transferred to a fresh six-well tissue culture plate. The T84 cells were then detached and lysed using 1 ml of 0.1% Triton X-100 for 15 min. In preliminary studies, this concentration of Triton X-100 had no effect on viability of E. coli O157:H7 EDL933. This solution was serially diluted in PBS and spread onto LB agar to enumerate the bacteria adhered to T84 cells. The percentage of adherence was calculated as number of bacteria adhered/number of bacteria added to the well × 100, and the relative percentage of adherence was calculated as percentage of adherence of mutant/percentage of adherence of wild type × 100. All experiments were done in triplicate. The paired Student t test was performed to identify statistical differences.

For microscopic analysis, bacteria were transformed with pISM31, a derivative of pMHE6 (20) expressing GFPuv (12). The T84 cells were seeded as described above and grown for 48 h until they were semiconfluent. Bacterial cultures were prepared, and the adherence assays were performed as described above. The plates were then incubated at 37°C with 5% CO₂ for 1.5 h. Following this, the coverslips were washed three times with PBS, and the cells were fixed using 4% paraformaldehyde in PBS for 10 min. The coverslips were washed twice with PBS, and treated with BSP buffer (250 mg bovine serum albumin and 100 mg saponin per 100 ml PBS) for 5 min, and then washed twice with BSP. The cells were stained for F-actin (54) with Alexa Fluor 546-labeled phalloidin (Invitrogen) (1:200 dilution in BSP) for 1 h, washed twice with BSP, and then mounted on a glass slide using mounting solution with DAPI (4′,6′-diamidino-2-phenylindole). Once the slides were dry, the coverslips were sealed using clear nail polish, and images were captured using green and red filters on an Olympus IX70 inverted fluorescence microscope equipped with a DP70 digital camera. The images were merged using ImageJ software (National Institutes of Health).

Identification of biofilm genes in E. coli O157:H7 EDL933.In previous studies, E. coli O157:H7 EDL933 was shown to form biofilms on inert surfaces (15). Since there have been no reports of studies performed on a global scale to identify biofilm-linked genes in E. coli O157:H7, we conducted a global mutational study using mini-Tn5Km2 to identify genes involved either directly or indirectly in biofilm formation. During the initial screen, 114 mutants (1.04% of the total library) had a biofilm-negative phenotype (Fig. 1). Following the confirmation of the phenotype, the growth of each strain was assessed. After elimination of mutants with inconsistent biofilm formation or a growth deficiency, 95 mutants that were biofilm negative (0.86% of the library) were studied further. For convenience, these mutants were designated biofilm-negative phenotype (Bnp) mutants.

The precise mini-Tn5 insertion sites of these Bnp mutants were identified by DNA sequencing. Our results indicated that there was only a single transposon insertion within the genome in each mutant. This was evident from the Y-Linker PCR, which showed amplification of only one fragment, and from the DNA sequence trace chromatograms, which gave a single sequence. The mini-Tn5 insertion sites in 19 of the Bnp mutants could not be identified due to a failure to amplify the region at the transposon insertion sites despite repeated attempts.

The 76 insertions that could be identified were distributed randomly throughout the genome of E. coli O157:H7 EDL933. In some cases, there were multiple insertions in the same gene coding region but at different locations. Fifty-one distinct genes/intergenic regions were identified (Table 3). Thirty-two insertions were in coding sequences of known function, and 19 were in hypothetical genes whose functions are yet to be assigned. Twenty-five insertions occurred in sequences having homology to E. coli K-12 sequences, and 19 occurred in sequences not shared with K-12 (O islands); three are on the pO157 plasmid, and five are phage carried. The functions of these genes vary; they include genes that encode structural components (ecpD, csgG, csgB, csgA, tolQ, waaL, and waaP), enzymes (yahF, yaiH, galU, cls, manC, wbdQ, fcl, aroC, relA, rfaC, and dsbA), regulators (Z2086, yihF, and hns), receptors (Z1178, Z0700, and Z3635), and hypothetical proteins. Three independent insertions were identified in the curli pilus operon (csgG, csgB, and csgA) and four independent insertions in the lipopolysaccharide (LPS) biosynthesis operon (waaL, waaP, waaD, and waaJ).

Plasmid pO157 genes involved in biofilm formation.In our mutational analysis we identified three independent insertions of mini-Tn5km2 in pO157, one in the type V secreted serine protease espP (7) and two in the enterohemolysin operon ehxCABD (3, 4) (one in ehxB and one between ehxC and ehxA). We confirmed the role of pO157 in biofilm formation by testing strain ISM1230, a derivative of EDL933 lacking the plasmid. This strain failed to make biofilms (Fig. 2). To confirm the linkage between specific genes and the biofilm phenotype, deletion mutants were constructed for three of the genes that were biofilm negative during screening, i.e., espP, ehxB, and ehxA. The deletion mutants were then compared for their growth and biofilm phenotype using a quantitative tube assay with the corresponding mini-Tn5km2 Bnp mutants (Bnp6, Bnp18, and Bnp44, respectively) (Fig. 2). Two of the deletion mutants (the ΔehxB and ΔehxA mutants) showed no difference in biofilm formation compared to the wild type, but their corresponding mini-Tn5km2 Bnp mutants, Bnp18 and Bnp44, were deficient in biofilm formation. The other deletion-insertion mutant (the ΔespP mutant) behaved in manner similar to that of its corresponding mini-Tn5km2 Bnp mutant (Bnp6). One explanation for the inconsistency in ehxCABD mutations is that the insertion of the transposon caused polarity effects on downstream expression of ehxD (8) that would not occur with the deletion mutations. All deletions were constructed in frame, and the kanamycin marker used in the deletion reaction lacks a transcriptional stop signal, allowing for continued transcription and expression of downstream genes. Thus, these results indicate that ehxD, and not ehxA or ehxB, may be the critical element in biofilm formation. To test this hypothesis, we constructed a ΔehxD mutant and tested it for the biofilm phenotype. Our quantitative assay showed that the deletion of ehxD caused a negative biofilm phenotype (Fig. 2). To further confirm this linkage, we constructed a plasmid for genetic complementation by cloning the ehxCABD operon under control of its native promoter into plasmid pBAD18. When this plasmid was transformed in the ΔehxD mutant (ΔehxD+ pISM30), the biofilm phenotype was restored to levels comparable to those for the wild type (Fig. 2).

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FIG. 2.

Comparison of biofilm formation on polystyrene by E. coli O157:H7 EDL933 transposon insertion and deletion mutants along with wild-type (WT) and negative-control (BW19795) strains. Data represent means plus standard errors for three replicates. Bnp6, espP::Tn5; Bnp18, ehxB::Tn5; Bnp44, Tn5 inserted at the ehxC-ehxA junction; ΔehxD+ehx, ΔehxD complemented with pISM30. *, significantly different from the wild-type control (P < 0.01).

Role of pO157 genes in adherence to T84 cells.To analyze the role of ehxCABD in adherence to T84 cells, wild-type E. coli O157:H7 EDL933, the wild type without pO157, and the Bnp6, Bnp18, Bnp44, ΔespP, ΔehxA, ΔehxB, ΔehxD, ΔehxD+pISM30 mutants were tested for adherence to T84 cells. Both microscopic and quantitative analyses were done. For microscopic analysis, all of the bacterial strains were transformed with a green fluorescent protein-expressing plasmid, pISM31. As shown in Fig. 3, wild-type E. coli O157:H7 EDL933 and the ΔehxA, ΔehxB, and ΔehxD+pISM30 mutants adhered to T84 cells, while E. coli O157:H7 EDL933 without pO157 and the Bnp6, Bnp18, Bnp44, ΔespP, and ΔehxD mutants failed to adhere. The quantitative analysis also showed that there is no significant difference in relative adherence to T84 cells between wild-type E. coli O157:H7 EDL933 and the ΔehxA, ΔehxB, and ΔehxD+pISM30 mutants but that there was a significant reduction in the relative adherence of E. coli O157:H7 EDL933 without pO157, and the Bnp6, Bnp18, Bnp44, ΔespP, and ΔehxD mutants (Fig. 4). For this experiment, the percentage of wild-type bacteria adhering to the T84 monolayers was 32.6%. Strains with less than 4% adherence were considered negative.

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FIG. 3.

Fluorescence microscopic pictures of GFPuv, expressing wild-type E. coli O157:H7 EDL933 and transposon insertion and deletion mutants, adhering to T84 cells. All pictures are merged images of DAPI (blue), F-actin (orange), and GFPuv-expressing bacterium (green) staining.

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FIG. 4.

Adherence of wild-type E. coli O157:H7 EDL933 and transposon insertion and deletion mutants to T84 cells shown quantitatively. Data represent means plus standard errors for three replicates. *, significantly different from the wild-type control (P < 0.01).