Shiga toxins and intimate adhesion controlled by the locus of enterocyte effacement are major enterohemorrhagic
(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
also represses additional adhesins that contribute to HEp-2 cell attachment. In this study, we tested for
regulation of several tissue adhesins and their regulators. Three adhesin-encoding genes (
,
,
) and four master regulators (
,
,
,
) were controlled by
.
over-expression strongly up-regulated
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
expression, as was PA20 motility. This study identifies new members in the
regulon and shows that
stimulates flagellar motility while repressing cell adhesion, likely to support EHEC movement to the intestinal surface early in infection. However, induced or inappropriate
-dependent flagellar expression could block cell attachments later during disease progression.
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+cysM
allows for the assembly of amplified DNA targets behind a kanamycin resistance marker and a constitutively transcribed
promoter. Transfer of the transcription unit to plasmid pCJR01 adds flanking regions of
rRNA homology for recombination into conserved rRNA regions. System utility was demonstrated by restoring function of a
deletion (RM3194Δ
::
) with a
gene or
combination.
In a previous study, we identified Congo red-binding and -nonbinding phase variants of Escherichia coli serotype O157:H7 strain ATCC 43895. The Congo red-binding variant, strain 43895OR, produced a ...dry, aggregative colony that was similar to the red, dry, and rough (rdar) phenotype characteristic of certain strains of SALMONELLA: In contrast, variant 43895OW produced a smooth and white colony morphology. In this study, we show that, similar to rdar strains of Salmonella enterica serovar Typhimurium, strain 43895OR forms large aggregates in broth cultures, firm pellicles at the air-medium interface on glass, and dense biofilms on glass and polystyrene. However, unlike S. enterica serovar Typhimurium, strain 43895OR does not stain positive for cellulose production. When strain 43895OR was fixed on agar, scanning electron microscopy showed cells expressing extracellular matrix (ECM) containing curli fibers. Strain 43895OW was devoid of any ECM or curli fibers on agar but showed expression of curli fibers during attachment to glass. Strain 43895OR produced >4-fold-larger amounts of biofilm than strain 43895OW on polystyrene, glass, stainless steel, and Teflon; formation was >3-fold higher in rich medium than in nutrient-limited medium. Biofilm-associated cells of both strains showed statistically greater resistance (P < 0.05) to hydrogen peroxide and quaternary ammonium sanitizer than their respective planktonic cells. This study shows that the rdar phenotype of E. coli O157:H7 strain 43895OR is important in multicellular growth, biofilm formation, and resistance to sanitizers. However, the lack of cellulose production by strain 43895OR indicates important differences in the ECM composition compared to that of SALMONELLA:
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
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
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
-independent mechanism. Both salicylate and decanoate enhanced O157:H7 motility dependent on
using media and incubation temperatures optimum for culturing human epithelial cells. However, induction of
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.
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
-dependent and
-independent pathways.
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.
Microbial Food Safety Research Unit, Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, PA, USA
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.
Correspondence Gaylen A. Uhlich gaylen.uhlich{at}ars.usda.gov
Abbreviations: Ahp, alkyl hydroperoxide proteins; AR, Amplex Red; CH, cumene hydroperoxide; CI, 95 % confidence interval; GT, generation time; HRP, horseradish peroxidase; LPD, lag phase duration
The biofilm life style helps bacteria resist oxidative stress, desiccation, antibiotic treatment, and starvation. Biofilm formation involves a complex regulatory gene network controlled by various ...environmental signals. It was previously shown that prophage insertions in mlrA and heterogeneous mutations in rpoS constituted major obstacles limiting biofilm formation and the expression of extracellular curli fibers in strains of Escherichia coli serotype O157:H7. The purpose of this study was to test strains from other important serotypes of Shiga toxin-producing E. coli (STEC) (O26, O45, O103, O111, O113, O121, and O145) for similar regulatory restrictions. In a small but diverse collection of biofilm-forming and non-forming strains, mlrA prophage insertions were identified in only 4 of the 19 strains (serotypes O103, O113, and O145). Only the STEC O103 and O113 strains could be complemented by a trans-copy of mlrA to restore curli production and Congo red (CR) dye affinity. RpoS mutations were found in 5 strains (4 serotypes), each with low CR affinity, and the defects were moderately restored by a wild-type copy of rpoS in 2 of the 3 strains attempted. Fourteen strains in this study showed no or weak biofilm formation, of which 9 could be explained by prophage insertions or rpoS mutations. However, each of the remaining five biofilm-deficient strains, as well as the two O145 strains that could not be complemented by mlrA , showed complete or nearly complete lack of motility. This study indicates that mlrA prophage insertions and rpoS mutations do limit biofilm and curli expression in the non-serotype O157:H7 STEC but prophage insertions may not be as common as in serotype O157:H7 strains. The results also suggest that lack of motility provides a third major factor limiting biofilm formation in the non-O157:H7 STEC. Understanding biofilm regulatory mechanisms will prove beneficial in reducing pathogen survival and enhancing food safety.
In a previous study, induction of the
serotype O157:H7 SOS response decreased
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
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
homolog,
, strongly repressed PA20
transcription (>7-fold) while the group 1 homologs,
and
, resulted in smaller reductions (<2.5-fold). However, SOS induction decreased rather than increased
expression (>6-fold) making group 1
, which are enhanced by the SOS response, the likely SOS-induced
repressors. Plasmid-based
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
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
, contributed to host cell adhesion in strain 20R2R. High levels of
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
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
homologs. This study has increased our understanding of the O157
genes at both host and environment temperatures, identifying
as a strong regulator of
and CsgD-dependent properties.
Biofilm formation in most Escherichia coli strains is dependent on curli fimbriae and cellulose, and the production of both varies widely among pathogenic strains. Curli and cellulose production by ...colonies growing on agar are often identified by their affinity for Congo red dye (CR). However, media composition and incubation temperature can affect dye affinity and impose limitations on red phenotype detection by this method. In this study, we compared different Shiga toxin-producing E. coli for CR affinity and biofilm formation under different media/temperature conditions. We found strain and serotype differences in CR affinities and biofilm formation, as well as temperature and media requirements for maximum CR binding. We also constructed strains with deletions of curli and/or cellulose genes to determine their contributions to the phenotypes and identified two O45 strains with a medium-dependent induction of cellulose.
Prophage insertions in Escherichia coli O157:H7 mlrA contribute to the low expression of curli fimbriae and biofilm observed in many clinical isolates. Varying levels of CsgD-dependent curli/biofilm ...expression are restored to strains bearing prophage insertions in mlrA by mutation of regulatory genes affecting csgD. Our previous study identified strong biofilm- and curli-producing variants in O157:H7 cultures that had lost the mlrA-imbedded prophage characteristic of the parent population, suggesting prophage excision as a mechanism for restoring biofilm properties. In this study, we compared genomic, transcriptomic and phenotypic properties of parent strain PA20 (stx1, stx2) and its prophage-cured variant, 20R2R (stx2), and confirmed the mechanism underlying the differences in biofilm formation.
Loss of the inserted Stx1 prophage from curli regulator, mlrA, restores CsgD-dependent Congo red affinity.
PDF
