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

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. Overall design: Three replicates for isolates RM6607R, RM6607W, RM6608R, RM6608W independently grown from single colonies in LB broth and incubated at 28°C 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 28°C 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 4°C and the pellets were stored at -80°C 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 28°C 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 4°C and the pellets were stored at -80°C 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.