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RcsB contributes to the distinct stress fitness among Escherichia coli O157:H7 curli variants of the 1993 hamburger-associated outbreak strains.

ABSTRACT: Curli are adhesive fimbriae of Enterobactericaeae and are involved in surface attachment, cell aggregation, and biofilm formation. We reported previously that curli-producing (C(+)) variants of E. coli O157:H7 (EcO157) were much more acid sensitive than their corresponding curli-deficient (C(-)) variants; however, this difference was not linked to the curli fimbriae per se. Here, we investigated the underlying molecular basis of this phenotypic divergence. We identified large deletions in the rcsB gene of C(+) variants isolated from the 1993 U.S. hamburger-associated outbreak strains. rcsB encodes the response regulator of the RcsCDB two-component signal transduction system, which regulates curli biogenesis negatively but acid resistance positively. Further comparison of stress fitness revealed that C(+) variants were also significantly more sensitive to heat shock but were resistant to osmotic stress and oxidative damage, similar to C(-) variants. Transcriptomics analysis uncovered a large number of differentially expressed genes between the curli variants, characterized by enhanced expression in C(+) variants of genes related to biofilm formation, virulence, catabolic activity, and nutrient uptake but marked decreases in transcription of genes related to various types of stress resistance. Supplying C(+) variants with a functional rcsB restored resistance to heat shock and acid challenge in cells but blocked curli production, confirming that inactivation of RcsB in C(+) variants was the basis of fitness segregation within the EcO157 population. This study provides an example of how genome instability of EcO157 promotes intrapopulation diversification, generating subpopulations carrying an array of distinct phenotypes that may confer the pathogen with survival advantages in diverse environments.

SUBMITTER: Carter MQ

PROVIDER: S-EPMC3485712 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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RcsB contributes to the distinct stress fitness among Escherichia coli O157:H7 curli variants of the 1993 hamburger-associated outbreak strains.

Carter Michelle Q MQ Parker Craig T CT Louie Jacqueline W JW Huynh Steven S Fagerquist Clifton K CK Mandrell Robert E RE

Applied and environmental microbiology 20120824 21

Curli are adhesive fimbriae of Enterobactericaeae and are involved in surface attachment, cell aggregation, and biofilm formation. We reported previously that curli-producing (C(+)) variants of E. coli O157:H7 (EcO157) were much more acid sensitive than their corresponding curli-deficient (C(-)) variants; however, this difference was not linked to the curli fimbriae per se. Here, we investigated the underlying molecular basis of this phenotypic divergence. We identified large deletions in the rc ...[more]

PMID: 22923406

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Project description:Curli are adhesive fimbriae of Enterobactericaeae and are involved in surface attachment, cell aggregation and biofilm formation. We previously reported that natural curli variants of E. coli O157:H7 (EcO157) displayed distinct acid resistance; however, this difference was not linked to the curli fimbriae per se. Here, we investigated the underlying molecular basis of this phenotypic divergence between the curli variants. Among curli-producing (C+) variants isolated from the 1993 U.S. hamburger-associated outbreaks strains, we identified large deletions in the rcsB gene that encodes the response regulator of RcsCDB two-component signal transduction system of rcsB ,. Further comparison of stress fitness revealed that C+ variants were also significantly more sensitive to heat shock, but remained similar resistance to osmotic stress and oxidative damage as curli-deficient (C-) variants. Transcriptomics analysis uncovered a large number of differentially expressed genes between the curli variants, characterized by the enhanced expression of genes related to biofilm formation, virulence, catabolic activity and nutrients uptake, but marked decrease in transcription of genes related to various stress resistance in C+ variants. Supplying C+ variants with a functional rcsB restored cells resistance to heat shock and acid challenge, but blocked the curli production, confirming that inactivation of RcsB in C+ variants was the basis of fitness segregation within the EcO157population. This study provides an example of how genome instability of EcO157promotes the intra-population diversification, generating sub-populations carrying an array of distinct phenotypes that may confer the pathogen survival advantages in host and nonhost environments. Three replicates for isolates RM6607R, RM6607W, RM6608R, RM6608W independently grown from single colonies in LB broth and incubated at 28M-BM-0C overnight on a shaker (150 rpm). The cells were collected by centrifugation, washed once in LBNS broth, and inoculated in 25ml of LBNS broth with a concentration equivalent to 0.001OD600 ml-1. The cultures were incubated at 28M-BM-0C for 24 h with aeration (150 rpm). At the end of incubation, ice-cold phenol-ethanol (5%:95%) solution was immediately added to culture at a final concentration of 20% (volume), and the mixture was incubated on ice for 30 min. The cells were collected by centrifugation at 4M-BM-0C and the pellets were stored at -80M-BM-0C until RNA extraction. The cells were collected by centrifugation, washed once in LBNS broth, and inoculated in 25ml of LBNS broth with a concentration equivalent to 0.001OD600 ml-1. The cultures were incubated at 28M-BM-0C for 24 h with aeration (150 rpm). At the end of incubation, ice-cold phenol-ethanol (5%:95%) solution was immediately added to culture at a final concentration of 20% (volume), and the mixture was incubated on ice for 30 min. The cells were collected by centrifugation at 4M-BM-0C and the pellets were stored at -80M-BM-0C until RNA extraction. RNA was isolated using Promega SV Total RNA kit. A type 2 gene expression experimental design was used, with fluorescently labeled genomic DNA as a reference channel in each experiment as described by Lucchini, S., et al. 2005. Infect Immun 73:88-102.

Project description:BackgroundEscherichia coli O157:H7 (O157) strain 86-24, linked to a 1986 disease outbreak, displays curli- and biofilm-negative phenotypes that are correlated with the lack of Congo red (CR) binding and formation of white colonies (CR-) on a CR-containing medium. However, on a CR medium this strain produces red isolates (CR+) capable of producing curli fimbriae and biofilms.ResultsTo identify genes controlling differential expression of curli fimbriae and biofilm formation, the RNA-Seq profile of a CR+ isolate was compared to the CR- parental isolate. Of the 242 genes expressed differentially in the CR+ isolate, 201 genes encoded proteins of known functions while the remaining 41 encoded hypothetical proteins. Among the genes with known functions, 149 were down- and 52 were up-regulated. Some of the upregulated genes were linked to biofilm formation through biosynthesis of curli fimbriae and flagella. The genes encoding transcriptional regulators, such as CsgD, QseB, YkgK, YdeH, Bdm, CspD, BssR and FlhDC, which modulate biofilm formation, were significantly altered in their expression. Several genes of the envelope stress (cpxP), heat shock (rpoH, htpX, degP), oxidative stress (ahpC, katE), nutrient limitation stress (phoB-phoR and pst) response pathways, and amino acid metabolism were downregulated in the CR+ isolate. Many genes mediating acid resistance and colanic acid biosynthesis, which influence biofilm formation directly or indirectly, were also down-regulated. Comparative genomics of CR+ and CR- isolates revealed the presence of a short duplicated sequence in the rcsB gene of the CR+ isolate. The alignment of the amino acid sequences of RcsB of the two isolates showed truncation of RcsB in the CR+ isolate at the insertion site of the duplicated sequence. Complementation of CR+ isolate with rcsB of the CR- parent restored parental phenotypes to the CR+ isolate.ConclusionsThe results of this study indicate that RcsB is a global regulator affecting bacterial survival in growth-restrictive environments through upregulation of genes promoting biofilm formation while downregulating certain metabolic functions. Understanding whether rcsB inactivation enhances persistence and survival of O157 in carrier animals and the environment would be important in developing strategies for controlling this bacterial pathogen in these niches.

Dataset Information

RcsB contributes to the distinct stress fitness among Escherichia coli O157:H7 curli variants of the 1993 hamburger-associated outbreak strains.

Publications

RcsB contributes to the distinct stress fitness among Escherichia coli O157:H7 curli variants of the 1993 hamburger-associated outbreak strains.

Similar Datasets

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