Ethanolamine metabolism through two genetically distinct loci enables Klebsiella pneumoniae to bypass nutritional competition in the gut
Fig 5
K. pneumoniae eut short locus expression is regulated in an NtrC-dependent manner.
(A) (i) Shown are the putative NtrC and RpoN binding sites present in the promoter region upstream of the eut short locus. The conserved NtrC and RpoN binding elements are shown, with the conserved -12(GC) and the -24 (GG) RpoN binding sites capitalized. NtrC binding site selected for site-directed mutagenesis (SDM) highlighted within box. (ii) Proposed binding of NtrC and RpoN when EA is present as a nitrogen source. (B-C) GFP kinetic assay of the WT and the ntrC- mutant strain carrying plasmid with gfp transcriptional fusion to either (B) the eutS promoter (euts΄-gfp+) or (C) eutL promoter (eutL΄-gfp+). Strains were grown in M9 MM + 0.4% Gly and 2.5 mM EA (experimental) or M9 MM + 0.4% Gly & 10 mM NH4Cl (control). (D) qRT-PCR measuring transcript abundance of the eut short (eat) and long (eutBL, eutCL) locus in the WT and the ntrC- isogenic mutant background. Strains were grown in M9 MM + 0.4% Gly and 2.5 mM EA (experimental) or M9 MM + 0.4% Gly & 10 mM NH4Cl (control). KPPR1S gyrA-specific primers were used as housekeeping gene for 2-ΔΔCT analysis. Mann-Whitney U test was performed comparing eut loci transcript abundance in the WT and ntrC- strain background. (E) GFP kinetic assay of WT strain carrying plasmid with gfp fused to either the WT eutS promoter region (eutS΄-gfp+) or the eutS promoter with the putative NtrC binding element mutated through SDM. Strains were grown in M9 MM + 0.4% Gly and 2.5 mM EA (experimental) or M9 MM + 0.4% Gly & 10 mM NH4Cl (control). All strains grown with EA as a nutrient source included 200nM B12. Mean ± SEM for ≥ 3 independent experiments is shown. *, P ≤ 0.05; ns, not significant.