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RpdD and RpoS ChIP-exo in Klebsiella pneumoniae

ABSTRACT: With increasingly concerning strains of antimicrobial resistant strains of the commensal, gram-negative bacteria Klebsiella pneumoniae emerging, there is a pressing need to better understand the pathogen and mechanisms behind its pathogenicity. This study investigated the regulatory mechanisms in strain MGH 78578 of two major sigma factors, the house-keeping sigma factor RpoD, and the general stress response sigma factor RpoS, in mid-exponential and early stationary phase using chromatin immunoprecipitation with exonuclease treatment (ChIP-exo) followed by deep sequencing. Combining ChIP-exo and transcriptome analysis allowed for the determination of sigma factor binding sites, binding motifs, and genes included in the phase-specific sigmulons. The number of genes included in the RpoS sigmulon was greater than in the RpoD sigmulon, with 1,833 and 1,690 genes included, respectively; however, a majority of sigmulon genes were found in all phase-specific sigmulons. Focussing on pathogenicity genes, 20 antimicrobial resistance genes (ARGs) and 155 virulence genes, only two ARGs were found exclusively in one phase-specific sigmulon, an oxacillin-hydrolysing class D beta-lactamase and chloramphenicol efflux MFS transporter CmlA5, which were found in the RpoD sigmulon in early stationary phase. Notably, six unnamed proteins that are or pertain to fimbrial proteins were found uniquely in the RpoS sigmulon in early stationary phase. From this, it can be hypothesised that early stationary phase might be an important phase for pathogenicity gene regulation. While there is little conservation in RpoS sigmulons from strain to strain, RpoS appears to have a consistent overarching role across strains, including a role as a regulator of pathogenicity genes.

ORGANISM(S): Klebsiella pneumoniae

PROVIDER: GSE226119 | GEO | 2025/02/24

REPOSITORIES: GEO

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Project description:RpoS, an alternative sigma factor, is critical for stress response in Escherichia coli. The RpoS regulon expression has been well characterized in rich media that support fast growth and high growth yields. In contrast, though RpoS levels are high in minimal media, how RpoS functions under such conditions has not been clearly resolved. In this study, we compared the global transcriptional profiles of wild type and an rpoS mutant of E. coli grown in glucose minimal media using microarray analyses. The expression of over 200 genes was altered by loss of RpoS in exponential and stationary phases, with only 48 genes common to both conditions. The nature of the RpoS-controlled regulon in minimal media was substantially different from that expressed in rich media. Specifically, the expression of many genes encoding regulatory factors (e.g., hfq, csrA and rpoE) and genes in metabolic pathways (e.g., lysA, lysC and hisD) were regulated by RpoS in minimal media. In early exponential phase, protein levels of RpoS in minimal media were much higher than that in LB media, which may at least partly account for the observed difference in the expression of RpoS-controlled genes. Expression of genes required for flagellar function and chemotaxis was elevated in the rpoS mutant. Western blot analyses show that the flagella sigma factor FliA was expressed much higher in rpoS mutants than in WT in all phase of growth. Consistent with this, the motility of rpoS mutants was enhanced relative to WT. In conclusion, RpoS and its controlled regulators form a complex regulatory network that mediates the expression of a large regulon in minimal media. Experiment Overall Design: A precise rpoS deletion mutant of MG1655 was constructed using the red recombinase method. Wild type and rpoS mutants were inoculated in triplicate into M63 glucose (0.2%) minimal media at a starting OD of 0.0001 and grown aerobically at 37oC. Cultures were harvested at OD600= 0.3 in exponential phase and at OD600= 1.5 in stationary phase. For RNA extraction, cultures were mixed directly with a boiling lysis buffer containing SDS and EDTA followed by acidic hot phenol (65C) to minimize RNA degradation. RNA samples were hybridized to Affymetrix E. coli Antisense Genome Array according to Affymetrix's standard protocols.

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RpdD and RpoS ChIP-exo in Klebsiella pneumoniae

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