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

ABSTRACT: 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.

ORGANISM(S): Escherichia coli Escherichia coli O157:H7

PROVIDER: GSE39439 | GEO | 2012/08/31

SECONDARY ACCESSION(S): PRJNA170911

REPOSITORIES: GEO

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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:Dental caries is closely associated with the virulence of Streptococcus mutans (S. mutans). The stress adaptation of S. mutans in the fluctuating oral environment is critical to the virulence expression of this bacterium. Here we used whole-genome microarrays to profile the dynamic transcriptomic responses of S. mutans during physiological heat stress. We also evaluated the phenotypic changes, including initial biofilm formation, acid production and ATP turnover of S. mutans during heat stress. We found that S. mutans responded to heat stress in a distinct pattern, which featured a differential transcription of 885 genes in total and 114 “core” transcripts throughout the six post-exposure time points investigated (5 min, 10 min, 15 min, 30 min, 45 min and 60 min). Further gene ontology analysis showed that transcriptional changes of genes involved in transcriptional regulation and cellular homeostasis were critical for heat stress responses of S. mutans. In addition to those highly conserved heat-shock proteins, we observed differential expression of multiple transcriptional regulators. The expression of genes involved in sugar transport, soluble glucans biosynthesis (gtfC) and binding (gbpC) were upregulated, whereas genes involved in ABC transporters, insoluble glucans biosynthesis (gtfB) and binding (gbpB), which are critical for biofilm skeleton, were either down-regulated or unchanged. These results together with enhanced glycolytic activity, attenuated sucrose-dependent initial attachment and impaired biofilm architecture indicate metabolic adaptations by this bacterium to compensate the extra energy demand for a better fitness under adverse conditions. We used time series microarrays to detect the dynamic changes of S. mutans under heat stress. Mid-logarithmic phase cell cultures of S. mutans (OD600nm = 0.5) were incubated at 42℃ for 5 min, 10 min, 15 min, 30 min, 45 min and 60 min, which were compared with the S. mutans cultured at 37℃. We applied three biological replicates at each time point.

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

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