Control of Escherichia coli Serotype O157:H7 Motility and Biofilm Formation by Salicylate and Decanoate: MarA/SoxS/Rob and pchE Interactions.

Prophage-encoded Escherichia coli O157:H7 transcription factor (TF), PchE, inhibits biofilm formation and attachment to cultured epithelial cells by reducing curli fimbriae expression and increasing flagella expression. To identify pchE regulators that might be used in intervention strategies to reduce environmental persistence or host infections, we performed a computational search of O157:H7 strain PA20 pchE promoter sequences for binding sites used by known TFs. A common site shared by MarA/SoxS/Rob TFs was identified and the typical MarA/Rob inducers, salicylate and decanoate, were tested for biofilm and motility effects. Sodium salicylate, a proven biofilm inhibitor, but not sodium decanoate, strongly reduced O157:H7 biofilms by a pchE-independent mechanism. Both salicylate and decanoate enhanced O157:H7 motility dependent on pchE using media and incubation temperatures optimum for culturing human epithelial cells. However, induction of pchE by salicylate did not activate the SOS response. MarA/SoxS/Rob inducers provide new potential agents for controlling O157:H7 interactions with the host and its persistence in the environment. IMPORTANCE There is a need to develop E. coli serotype O157:H7 nonantibiotic interventions that do not precipitate the release and activation of virulence factor-encoded prophage and transferrable genetic elements. One method is to stimulate existing regulatory pathways that repress bacterial persistence and virulence genes. Here we show that certain inducers of MarA and Rob have that ability, working through both pchE-dependent and pschE-independent pathways.

PchE Regulation of Escherichia coli O157:H7 Flagella, Controlling the Transition to Host Cell Attachment.

Shiga toxins and intimate adhesion controlled by the locus of enterocyte effacement are major enterohemorrhagic Escherichia coli (EHEC) virulence factors. Curli fimbriae also contribute to cell adhesion and are essential biofilm components. The transcriptional regulator PchE represses the expression of curli and their adhesion to HEp-2 cells. Past studies indicate that pchE also represses additional adhesins that contribute to HEp-2 cell attachment. In this study, we tested for pchE regulation of several tissue adhesins and their regulators. Three adhesin-encoding genes (eae, lpfA1, fliC) and four master regulators (csgD, stpA, ler, flhDC) were controlled by pchE. pchE over-expression strongly up-regulated fliC but the marked flagella induction reduced the attachment of O157:H7 clinical isolate PA20 to HEp-2 cells, indicating that flagella were blocking cell attachments rather than functioning as an adhesin. Chemotaxis, motor, structural, and regulatory genes in the flagellar operons were all increased by pchE expression, as was PA20 motility. This study identifies new members in the pchE regulon and shows that pchE stimulates flagellar motility while repressing cell adhesion, likely to support EHEC movement to the intestinal surface early in infection. However, induced or inappropriate pchE-dependent flagellar expression could block cell attachments later during disease progression.

Genome amplification and promoter mutation expand the range of csgD-dependent biofilm responses in an STEC population.

Expression of the major biofilm components of E. coli, curli fimbriae and cellulose, requires the CsgD transcription factor. A complex regulatory network allows environmental control of csgD transcription and biofilm formation. However, most clinical serotype O157 : H7 strains contain prophage insertions in the csgD regulator, mlrA, or mutations in other regulators that restrict csgD expression. These barriers can be circumvented by certain compensating mutations that restore higher csgD expression. One mechanism is via csgD promoter mutations that switch sigma factor utilization. Biofilm-forming variants utilizing RpoD rather than RpoS have been identified in glycerol freezer stocks of the non-biofilm-forming food-borne outbreak strain, ATCC 43894. In this study we used whole genome sequencing and RNA-seq to study genotypic and transcriptomic differences between those strains. In addition to defining the consequences of the csgD promoter switch and identifying new csgD-controlled genes, we discovered a region of genome amplification in our laboratory stock of 43894 (designated 43894OW) that contributed to the regulation of csgD-dependent properties.

Nonlabeled quantitative proteomic comparison identifies differences in acid resistance between Escherichia coli O157:H7 curli production variants.

To understand the nature of a bacterial strain, it is necessary to be able to identify and measure the proteins expressed by the bacteria. In this research, the entire protein complements produced by Escherichia coli O157:H7 strain 43894OW and its naturally occurring curli producing variant 43894OR were compared to better understand the unique capabilities of these two closely related strains. A nonlabeled proteomic comparison was performed utilizing the spectra counting and peptide fractionation abilities of a quadrupole-time of flight analyzer mass spectrometer to identify and quantitate the proteins produced by the two strains. The process reliably identified and measured the concentration of 419 proteins from strains 43894OW and 43894OR within three separate biological replicates. From these two sets, 59 proteins were identified that were preferentially expressed in strain 43894OW compared to 43894OR and 14 proteins that were conversely preferentially expressed in 43894OR. A subset of the preferentially expressed proteins was assayed to determine whether their levels of gene transcription corresponded with the observed protein expression. From the resulting list of confirmed differentially expressed proteins, it was observed that the proteins contributing to acid survival--GadA and GadB--were overexpressed in 43894OW compared to 43894OR. The predicted enhanced acid resistance phenotype of 43894OW was confirmed by experimentation at pH 2.5. Additionally, a knockout mutation in the csgD genes of the 43894OR strain was constructed and suggested that CsgD had a repressive effect on acid survival in 43894OR.

Phage insertion in mlrA and variations in rpoS limit curli expression and biofilm formation in Escherichia coli serotype O157: H7.

Biofilm formation in Escherichia coli is a tightly controlled process requiring the expression of adhesive curli fibres and certain polysaccharides such as cellulose. The transcriptional regulator CsgD is central to biofilm formation, controlling the expression of the curli structural and export proteins and the diguanylate cyclase adrA, which indirectly activates cellulose production. CsgD itself is highly regulated by two sigma factors (RpoS and RpoD), multiple DNA-binding proteins, small regulatory RNAs and several GGDEF/EAL proteins acting through c-di-GMP. One such transcription factor MlrA binds the csgD promoter to enhance the RpoS-dependent transcription of csgD. Bacteriophage, often carrying the stx1 gene, utilize an insertion site in the proximal mlrA coding region of E. coli serotype O157 : H7 strains, and the loss of mlrA function would be expected to be the major factor contributing to poor curli and biofilm expression in that serotype. Using a bank of 55 strains of serotype O157 : H7, we investigated the consequences of bacteriophage insertion. Although curli/biofilm expression was restored in many of the prophage-bearing strains by a wild-type copy of mlrA on a multi-copy plasmid, more than half of the strains showed only partial or no complementation. Moreover, the two strains carrying an intact mlrA were found to be deficient in biofilm formation. However, RpoS mutations that attenuated or inactivated RpoS-dependent functions such as biofilm formation were found in >70 % of the strains, including the two strains with an intact mlrA. We conclude that bacteriophage interruption of mlrA and RpoS mutations provide major obstacles limiting curli expression and biofilm formation in most serotype O157 : H7 strains.

Peroxide resistance in Escherichia coli serotype O157 : H7 biofilms is regulated by both RpoS-dependent and -independent mechanisms.

In many Escherichia coli serotype O157 : H7 strains, defences against peroxide damage include the peroxiredoxin AhpCF and three catalases: KatG (catalase/peroxidase), KatE (catalase) and the plasmid-encoded KatP (catalase/peroxidase). AhpC and KatG basal expression is maintained by RpoS, and AhpC, KatG and KatP are all induced by OxyR/σ(70) in exponential phase. KatE is regulated by RpoS during stationary growth and is independent of OxyR. In a previous study we used mutant strains of ATCC 43895 (EDL933) with deletions of katG, ahpC, katE and katP in all possible combinations to characterize peroxide resistance during both exponential and 18-24 h growth in Luria-Bertani broth at 37 °C. In this study, we used triple deletion strains that isolated each catalase/peroxidase gene to investigate their role in the peroxide resistance of biofilm-forming variant 43895OR in 48 and 72 h biofilms. We also used quantitative real-time reverse transcriptase PCR and translational lacZ fusions to study gene expression. Peroxide resistance was greater (P<0.05) in biofilm cells than in planktonic cells, and full resistance required rpoS but not oxyR. In 72 h biofilms, katG and katE were the major protective genes. katG, ahpC and katE peroxide protection had both rpoS-dependent and rpoS-independent components, but katP protection was independent of rpoS. H(2)O(2) challenge induced (P<0.05) katG, ahpC and katP expression in biofilm cells, suggesting that peroxide induction of the OxyR-dependent resistance genes may contribute to the RpoS-independent protection in Shiga toxin-producing E. coli biofilms.

A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integration.

A novel cloning vector to aid in the construction of single copy ß-galactosidase reporter systems for gene expression studies in lactose metabolizing Escherichia coli strains, including STEC, is described. The plasmid allows construction of translational fusions of cloned gene promoters to a short segment of E. coli lacZ. A selectable spectinomycin resistance marker flanked by a short lacI segment is positioned 5' to the cloning site. PCR amplification using opposing primers complementary to the upstream lacI fragment and the downstream lacZ fragment generates a linear template suitable for integration using pRedET recombination. Integration of linear template derived from the recombinant plasmid into host strains replaces the entire native lacZ promoter and fuses the promoter of interest in-frame with the lacZ gene, thus simultaneously producing a single-copy, chromosomal reporter system and eliminating background lacZ expression. Studies comparing ahpC expression from a chromosomal fusion in the lac open with that on a plasmid in E. coli strain EDL933 are shown.

A method for correcting standard-based real-time PCR DNA quantitation when the standard's polymerase reaction efficiency is significantly different from that of the unknown's.

Standard-based real-time or quantitative polymerase chain reaction quantitation of an unknown sample's DNA concentration (i.e., [DNA](unk)) assumes that the concentration dependence of the standard and unknown reactions (related to reaction efficiency, E) are equivalent. In our work with background food-borne organisms which can interfere with pathogen detection, we have found that it is generally possible to achieve an acceptable E (1 ± 0.05) for standard solutions by optimizing the PCR conditions, template purity, primer sequence, and amplicon lengths. However, this is frequently not true for the solutions containing unknown amounts of target DNA inasmuch as cell extracts are more chemically complex than the standards which have been amplified (2(30)-fold) as well as undergone a purification process. When significant differences in E occur, it is not possible to accurately estimate unknown target DNA concentration from the standard solution's slope and intercept (from threshold cycle number, or C(T), versus Log[DNA] data). What is needed is a standard-mediated intercept which can be specifically coupled with an unknown solution's PCR concentration dependence. In this work, we develop a simple mathematical procedure to generate a new standard curve with a slope (∂C(T)/∂Log[Dilution](unk)) derived from at least three dilutions of the unknown target DNA solution ([DNA](unk)) and an intercept calculated from the unknown's C(T)s, DNA concentrations interpolated from the standard curve (i.e., the traditional estimate of [DNA](unk)), and ∂C(T)/∂Log[Dilution](unk). We were able to achieve this due to our discovery of the predictable way in which the observed and ideal C(T) versus Log[DNA] slopes and intercepts deviate from one another. This "correction" in the standard-based [DNA](unk) determination is typically 20-60% when the difference in the standard and unknown E is >0.1.

Cloning vectors for gene delivery, integration and expression in Campylobacter jejuni.

Campylobacter genetics research is negatively impacted by a shortage of molecular tools for expressing DNA elements. A previous technique coupled an antibiotic resistance gene and its promoter to a gene of interest, inserting this expression unit into a conserved chromosomal location. Here the authors describe two new plasmids for construction and gene integration utilizing aspects of the previous type of expression unit. pBlueKan+cysMPro allows for the assembly of amplified DNA targets behind a kanamycin resistance marker and a constitutively transcribed cysM promoter. Transfer of the transcription unit to plasmid pCJR01 adds flanking regions of Campylobacter rRNA homology for recombination into conserved rRNA regions. System utility was demonstrated by restoring function of a flaAB deletion (RM3194ΔflaAB::tet) with a flaA gene or flaA/flaB combination.

Escherichia coli serotype O157:H7 retention on solid surfaces and peroxide resistance is enhanced by dual-strain biofilm formation.

In a previous study we showed that an Escherichia coli O157:H7 strain that was unable to form biofilm was retained in large numbers in dual-strain biofilms formed with an E. coli O-:H4 companion strain. In this study we tested additional companion strains for their ability to retain E. coli O157:H7 strain 0475s. Companion strains producing biofilm that withstood aggressive washes were able to significantly increase serotype O157:H7 retention. Dual-strain biofilms with certain companion strains retained higher percentages of strain 0475s, and that ability was independent of biofilm total cell numbers. Tests with additional non-biofilm-forming E. coli O157:H7 strains showed that enhancement by companion strains was not unique to strain 0475s. Experiments using an E. coli companion strain with deletions of various curli and cellulose genes indicated that dual-strain biofilm formation was dependent on companion strain properties. Strain 0475s was not able to generate biofilm or persist on plastic when grown in broth with a biofilm-forming companion and separated by a 0.2 microm porous membrane, indicating a requirement for intimate contact with the companion strain. When dual-strain biofilms and planktonic cells were challenged with 5% H(2)O(2), strain 0475 showed greater survival in biofilms with certain companion strains compared to the corresponding planktonic cells. The results of this study indicate that non-biofilm-forming E. coli O157:H7 strains are retained on solid surfaces associated with biofilms generated by companion strains. However, properties other than biofilm mass enable certain companion strains to retain greater numbers of E. coli O157:H7.

Escherichia coli Serotype O157:H7 PA20R2R Complete Genome Sequence.

Escherichia coli serotype O157:H7 strain 20R2R is a derivative of clinical isolate PA20. Prophage excision from the coding region of a PA20 transcription factor restored RpoS-dependent biofilm formation in 20R2R, providing a model for O157:H7 stress adaptation when transitioning between clinical and environmental settings. We report here the complete 20R2R genome sequence.

The CsgA and Lpp proteins of an Escherichia coli O157:H7 strain affect HEp-2 cell invasion, motility, and biofilm formation.

In Escherichia coli O157:H7 strain ATCC 43895, a guanine-to-thymine transversion in the csgD promoter created strain 43895OR. Strain 43895OR produces an abundant extracellular matrix rich in curli fibers, forms biofilms on solid surfaces, invades cultured epithelial cells, and is more virulent in mice than strain 43895. In this study we compared the formic acid-soluble proteins expressed by strains 43895OR and 43895 using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and identified two differentially expressed proteins. A 17-kDa protein unique to strain 43895OR was identified from matrix-assisted laser desorption ionization-time of flight analysis combined with mass spectrometry (MS) and tandem MS (MS/MS) as the curli subunit encoded by csgA. A <10-kDa protein, more highly expressed in strain 43895, was identified as the Lpp lipoprotein. Mutants of strain 43895OR with disruption of lpp, csgA, or both lpp and csgA were created and tested for changes in phenotype and function. The results of this study show that both Lpp and CsgA contribute to the observed colony morphology, Congo red binding, motility, and biofilm formation. We also show that both CsgA and Lpp are required by strain 43895OR for the invasion of cultured HEp-2 cells. These studies suggest that in strain 43895OR, the murein lipoprotein Lpp indirectly regulates CsgA expression through the CpxAR system by a posttranscriptional mechanism.

KatP contributes to OxyR-regulated hydrogen peroxide resistance in Escherichia coli serotype O157 : H7.

Escherichia coli K-12 defends itself against peroxide-mediated oxidative damage using two catalases, KatG and KatE, and the peroxiredoxin, alkyl hydroperoxide reductase, encoded by ahpC. In E. coli O157 : H7 strain ATCC 43895 (EDL933), plasmid pO157 carries an additional catalase-peroxidase gene, katP. KatP has been shown to be a functional catalase-peroxidase. However, deletion of pO157 does not alter the peroxide resistance of strain EDL933, leaving the physiological role of katP unclear. To examine the individual roles of peroxide-resistance genes in E. coli O157 : H7, mutant strains of ATCC 43895 were constructed bearing individual deletions of katG, katE, katP and ahpC, as well as double, triple and quadruple deletions encompassing all possible gene combinations thereof. The wild-type and all 15 mutant strains were compared for differences in aerobic growth, ability to scavenge exogenous H(2)O(2) and resistance to exogenous peroxides. Although KatG scavenged the most exogenous H(2)O(2), KatP scavenged statistically greater amounts than either KatE or AhpC during exponential growth. However, katG and ahpC together were sufficient for full peroxide resistance in disc diffusion assays. Strains with only katG or ahpC were the only triple deletion strains with significantly shorter generation times than the quadruple deletion strain. ahpC was the only gene that could allow rapid transition from lag phase to exponential phase in a triple deletion strain. Gene expression studies revealed that katP is an OxyR-regulated gene, but its expression is suppressed in stationary phase by RpoS. These studies indicate that pO157-borne katP contributes to the complex gene network protecting strain 43895 from peroxide-mediated oxidative damage in an OxyR-dependent manner.

Whole-Genome Sequence of Escherichia coli Serotype O157:H7 Strain ATCC 43888.

Escherichia coli serotype O157:H7 strain ATCC 43888 is a Shiga toxin-deficient human fecal isolate. Due to its reduced toxicity and its availability from a curated culture collection, the strain has been used extensively in applied research studies. Here, we report the Illumina-corrected PacBio whole-genome sequence of E. coli O157:H7 strain ATCC 43888.

Pch Genes Control Biofilm and Cell Adhesion in a Clinical Serotype O157:H7 Isolate.

In a previous study, induction of the Escherichia coli serotype O157:H7 SOS response decreased csgD expression in the clinical isolate PA20 at 30°C but strongly induced genes in the horizontally transferred-DNA regions (HTR), including many known virulence regulators. To determine the role of HTR regulators in the control of csgD and curli, specific regulators were plasmid-expressed in the wild-type and mutant strains of PA20 and its biofilm-forming derivative, 20R2R. At 30°C, plasmid over-expression of the O157:H7 group 3 perC homolog, pchE, strongly repressed PA20 csgD transcription (>7-fold) while the group 1 homologs, pchA and pchB, resulted in smaller reductions (<2.5-fold). However, SOS induction decreased rather than increased pchE expression (>6-fold) making group 1 pch, which are enhanced by the SOS response, the likely SOS-induced csgD repressors. Plasmid-based pchE over-expression also reduced 20R2R biofilm formation (>6-fold) and the curli-dependent, Congo red affinity of both PA20 and 20R2R. However, to properly appreciate the regulatory direction, expression patterns, and environmental consequences of these and other CsgD-controlled functions, a better understanding of natural pchE regulation will be required. The effects of HTR regulators on PA20 and 20R2R adhesion to HEp-2 cell at host temperature were also studied. Under conditions where prophage genes were not induced, curli, rather than espA, contributed to host cell adhesion in strain 20R2R. High levels of pchE expression in trans reduced curli-dependent cell adherence (>2-fold) to both 20R2R and the clinical isolate PA20, providing a host-adapting adhesion control mechanism. Expression of pchE was also repressed by induction of the SOS response at 37°C, providing a mechanism by which curli expression might complement EspA-dependent intimate adhesion initiated by the group1 pch homologs. This study has increased our understanding of the O157 pch genes at both host and environment temperatures, identifying pchE as a strong regulator of csgD and CsgD-dependent properties.

Sulfamethoxazole - Trimethoprim represses csgD but maintains virulence genes at 30°C in a clinical Escherichia coli O157:H7 isolate.

The high frequency of prophage insertions in the mlrA gene of clinical serotype O157:H7 isolates renders such strains deficient in csgD-dependent biofilm formation but prophage induction may restore certain mlrA properties. In this study we used transcriptomics to study the effect of high and low sulfamethoxazole-trimethoprim (SMX-TM) concentrations on prophage induction, biofilm regulation, and virulence gene expression in strain PA20 under environmental conditions following 5-hour and 12-hour exposures in broth or on agar. SMX-TM at a sub-lethal concentration induced strong RecA expression resulting in concentration- and time-dependent major transcriptional shifts with emphasis on up-regulation of genes within horizontally-transferred chromosomal regions (HTR). Neither high or low levels of SMX-TM stimulated csgD expression at either time point, but both levels resulted in slight repression. Full expression of Ler-dependent genes paralleled expression of group 1 pch homologues in the presence of high glrA. Finally, stx2 expression, which is strongly dependent on prophage induction, was enhanced at 12 hours but repressed at five hours, in spite of early SOS initiation by the high SMX-TM concentration. Our findings indicate that, similar to host conditions, exposure to environmental conditions increased the expression of virulence genes in a clinical isolate but genes involved in the protective biofilm response were repressed.

Whole-Genome Sequence of Escherichia coli Serotype O157:H7 Strain B6914-ARS.

Escherichia coli serotype O157:H7 strain B6914-MS1 is an isolate from the Centers for Disease Control and Prevention that is missing both Shiga toxin genes and has been used extensively in applied research studies. Here we report the genome sequence of strain B6914-ARS, a B6914-MS1 clone that has unique biofilm properties.

Whole-Genome Sequence of Escherichia coli Serotype O157:H7 Strain PA20.

Escherichia coli serotype O157:H7 strain PA20 is a Pennsylvania Department of Health clinical isolate. It has been used to study biofilm formation in O157:H7 clinical isolates, where the high incidence of prophage insertions in the mlrA transcription factor disrupts traditional csgD biofilm regulation. Here, we report the complete PA20 genome sequence.

Inactivation of the Crp/Fnr family of regulatory genes in Listeria monocytogenes strain F2365 does not alter its heat resistance at 60 degrees C.

A surprising facet of the Listeria monocytogenes genome is the presence of 15 genes that code for regulators in the Crp/ Fnr family and include the virulence regulator PrfA. The genes under the transcriptional control of these regulators are currently undetermined, with the exception of some genes controlled by the major virulence regulator PrfA. Using 12 strains of L. monocytogenes, each with an inserted gene cassette that interrupts and renders nonfunctional a different L. monocytogenes strain F2365 Crp/Fnr regulator, we heat challenged each strain at 60 degrees C with an immersed-coil heating apparatus, modeled the survivor data to calculate the underlying mean and mode of the heat resistance distribution for each strain, and compared the thermal tolerance of each mutant to the wild-type strain to determine if any of the Crp/Fnr mutants demonstrated altered heat tolerance. All 12 of the Crp/Fnr mutant strains tested had heat resistance characteristics similar to the wild-type strain (P > 0.05), indicating that mutations in these Crp/Fnr genes neither increased nor decreased the sensitivity of L. monocytogenes strain F2365 to mild heat.

Whole genome comparisons of serotype 4b and 1/2a strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species.

The genomes of three strains of Listeria monocytogenes that have been associated with food-borne illness in the USA were subjected to whole genome comparative analysis. A total of 51, 97 and 69 strain-specific genes were identified in L.monocytogenes strains F2365 (serotype 4b, cheese isolate), F6854 (serotype 1/2a, frankfurter isolate) and H7858 (serotype 4b, meat isolate), respectively. Eighty-three genes were restricted to serotype 1/2a and 51 to serotype 4b strains. These strain- and serotype-specific genes probably contribute to observed differences in pathogenicity, and the ability of the organisms to survive and grow in their respective environmental niches. The serotype 1/2a-specific genes include an operon that encodes the rhamnose biosynthetic pathway that is associated with teichoic acid biosynthesis, as well as operons for five glycosyl transferases and an adenine-specific DNA methyltransferase. A total of 8603 and 105 050 high quality single nucleotide polymorphisms (SNPs) were found on the draft genome sequences of strain H7858 and strain F6854, respectively, when compared with strain F2365. Whole genome comparative analyses revealed that the L.monocytogenes genomes are essentially syntenic, with the majority of genomic differences consisting of phage insertions, transposable elements and SNPs.