Abstract
BackgroundH-NS is a DNA-binding protein with central roles in gene regulation and nucleoid structuring in Escherichia coli. There are over 60 genes that are influenced by H-NS many of which are involved in metabolism. To determine the significance of H-NS-regulated genes in metabolism and stress tolerance, an hns mutant of E. coli O157:H7 was generated (hns::nptI, FRIK47001P) and its growth, metabolism, and gastrointestinal passage compared to the parent strain (43895) and strain FRIK47001P harboring pSC0061 which contains a functional hns and 90-bp upstream of the open-reading frame.ResultsThe hns mutant grew slower and was non-motile in comparison to the parent strain. Carbon and nitrogen metabolism was significantly altered in the hns mutant, which was incapable of utilizing 42 carbon, and 19 nitrogen sources that the parent strain metabolized. Among the non-metabolized substrates were several amino acids, organic acids, and key metabolic intermediates (i.e., pyruvate) that limit carbon acquisition and energy generation. Growth studies determined that the parent strain grew in LB containing 14 to 15% bile or bile salts, while the hns mutant grew in 6.5 and 9% of these compounds, respectively. Conversely, log-phase cells of the hns mutant were significantly (p < 0.05) more acid tolerant than the parent strain and hns mutant complemented with pSC0061. In mouse passage studies, the parent strain was recovered at a higher frequency (p < 0.01) than the hns mutant regardless of whether log- or stationary-phase phase cells were orally administered.ConclusionThese results demonstrate that H-NS is a powerful regulator of carbon and nitrogen metabolism as well as tolerance to bile salts. It is likely that the metabolic impairments and/or the reduced bile tolerance of the E. coli O157:H7 hns mutant decreased its ability to survive passage through mice. Collectively, these results expand the influence of H-NS on carbon and nitrogen metabolism and highlight its role in the ability of O157:H7 strains to respond to changing nutrients and conditions encountered in the environment and its hosts.
Highlights
H-NS is a DNA-binding protein with central roles in gene regulation and nucleoid structuring in Escherichia coli
Generation and confirmation of the hns::nptI mutant A 695-bp fragment that includes the hns structural gene and upstream regulatory region was amplified by PCR from genomic DNA of E. coli O157:H7 ATCC 43895
Transconjugants resulting from the conjugation of pSC0063 from E. coli SM10 λpir to E. coli O157:H7 strain ATCC 43895 were kanamycin resistant, ampicillin sensitive, sucrose positive, and O157 positive
Summary
H-NS is a DNA-binding protein with central roles in gene regulation and nucleoid structuring in Escherichia coli. The changing conditions encountered by E. coli O157:H7 in the bovine intestinal tract and the environment require that it closely control metabolism and stress protection. H-NS is a cytoplasmic protein that is abundant in the E. coli nucleoid [6] It plays a role in the condensation of chromosomal DNA by binding to curved DNA and can influence the expression of many genes either directly or indirectly [7,8]. The involvement of H-NS in modulating genes responsive to environmental stimuli make it an important regulator of virulence in several pathogens including Shigella and Salmonella [12,13,16,17,18,19]
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