Dataset Information

Residue L193P Mutant of RpoS Affects Its Activity During Biofilm Formation in Salmonella Pullorum.

ABSTRACT: The role of alternative sigma factor RpoS in regulating biofilm formation may differ in various Salmonella Pullorum strains. In this study, the biofilm-forming ability of two Salmonella Pullorum strains S6702 and S11923-3 were compared. The biofilm forming ability of S11923-3 was much stronger than that of S6702. After knocking out the rpoS gene, S11923-3?rpoS had significantly reduced biofilm while S6702?rpoS demonstrated similar biofilm compared with each parent strain. The analysis of RpoS sequences indicated two amino acid substitutions (L193P and R293C) between S6702 and S11923-3 RpoS. A complementation study confirmed that the expression of S11923-3 RpoS rather than S6702 RpoS could restore the biofilm-forming ability of ?rpoS strains and the L193P mutation contributed to the restoration of the biofilm-forming ability. Further study indicated that RpoS with the L193P mutant had significantly improved expression level and binding activity to RNAP and csgD gene promoter, which increased the efficacy of the csgD gene promoter and biofilm-forming ability. Therefore, the L193P mutation of RpoS is critical for stronger biofilm formation of Salmonella Pullorum.

SUBMITTER: Feng Z

PROVIDER: S-EPMC7674402 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Residue L193P Mutant of RpoS Affects Its Activity During Biofilm Formation in Salmonella Pullorum.

Feng Zheng Z El Hag Muhanad M Qin Tao T Du Yinping Y Chen Sujuan S Peng Daxin D

Frontiers in veterinary science 20201105

The role of alternative sigma factor RpoS in regulating biofilm formation may differ in various Salmonella Pullorum strains. In this study, the biofilm-forming ability of two Salmonella Pullorum strains S6702 and S11923-3 were compared. The biofilm forming ability of S11923-3 was much stronger than that of S6702. After knocking out the rpoS gene, S11923-3ΔrpoS had significantly reduced biofilm while S6702ΔrpoS demonstrated similar biofilm compared with each par ...[more]

PMID: 33251260

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Project description:BackgroundSalmonella serovars Enteritidis and Gallinarum are closely related, but their host ranges are very different: the former is host-promiscuous and the latter can infect poultry only. Comparison of their genomic sequences reveals that Gallinarum has undergone much more extensive degradation than Enteritidis. This phenomenon has also been observed in other host restricted Salmonella serovars, such as Typhi and Paratyphi A. The serovar Gallinarum can be further split into two biovars: Gallinarum and Pullorum, which take poultry as their common host but cause distinct diseases, with the former eliciting typhoid and the latter being a dysentery agent. Genomic comparison of the two pathogens, with a focus on pseudogenes, would provide insights into the evolutionary processes that might have facilitated the formation of host-restricted Salmonella pathogens.Methodologies/principal findingsWe sequenced the complete genome of Pullorum strains and made comparison with Gallinarum and other Salmonella lineages. The gene contents of Gallinarum and Pullorum were highly similar, but their pseudogene compositions differed considerably. About one fourth of pseudogenes had the same inactivation mutations in Gallinarum and Pullorum but these genes remained intact in Enteritidis, suggesting that the ancestral Gallinarum may have already been restricted to poultry. On the other hand, the remaining pseudogenes were either in the same genes but with different inactivation sites or unique to Gallinarum or Pullorum, reflecting unnecessary functions in infecting poultry.ConclusionsOur results support the hypothesis that the divergence between Gallinarum and Pullorum was initiated and facilitated by host restriction. Formation of pseudogenes instead of gene deletion is the major form of genomic degradation. Given the short divergence history of Gallinarum and Pullorum, the effect of host restriction on genomic degradation is huge and rapid, and such effect seems to be continuing to work. The pseudogenes may reflect the unnecessary functions for Salmonella within the poultry host.

Project description:RpoS is a key stress-inducible sigma factor that regulates stress resistance genes in Escherichia coli, such as the katE gene encoding catalase HPII and the glg genes encoding glycogen synthesis proteins. Monitoring RpoS activity can provide information on the stress sensitivity of E. coli isolates in clinical settings because the RpoS in these isolates is often mutated. In the present study, we found a novel, missense point mutation at RpoS residue 128 in a clinical Shiga toxin-producing E. coli (STEC) isolate. This mutation caused RpoS dysfunction and increased stress sensitivity. A mutant rpoS was cloned from a clinical STEC that is vulnerable to cold temperature and oxidative stresses. Mutant RpoS protein expression was detected in the clinical isolate, and this RpoS was non-functional according to HPII activity and glycogen levels, which are positively regulated by RpoS and thus are used as indicators for RpoS function. A reporter assay with β-galactosidase indicated that the dysfunction occurred at the transcriptional level of genes regulated by RpoS. Furthermore, substitution analysis indicated that the hydrophobicity of the amino acid at residue 128 was critical for RpoS activity; the simulation analysis indicated that the amino acids of RNA polymerase (RNAP) that interact with RpoS residue 128 are hydrophobic, suggesting that this hydrophobic interaction is critical for RpoS activity. In addition, substitution of Ile128 to Pro128 abolished RpoS activity, possibly as a result of disruption of the secondsary structure around residue 128, indicating that the structure is also a crucial factor for RpoS activity. These results indicate that only one point mutation at a hydrophobic residue of the complex formed during transcription leads to a critical change in RpoS regulation. Moreover, we found that Ile128 is widely conserved among various bacteria: several bacterial strains have Met128 or Leu128, which are hydrophobic residues, and these strains had similar or higher RpoS activity than that observed with Ile128 in this study. These data indicate that the hydrophobicity of the amino acid at residue 128 is critical for RpoS activity and is consequently important for bacterial survival. Taken together, these findings may contribute to a deeper understanding of protein functional mechanisms and bacterial stress responses.

Dataset Information

Residue L193P Mutant of RpoS Affects Its Activity During Biofilm Formation in Salmonella Pullorum.

Publications

Residue L193P Mutant of RpoS Affects Its Activity During Biofilm Formation in <i>Salmonella</i> Pullorum.

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