Homologous Response Regulators KvgA, KvhA and KvhR Regulate the Synthesis of Capsular Polysaccharide in Klebsiella pneumoniae CG43 in a Coordinated Manner

Abstract

On the basis of phenotypic analysis, the Klebsiella pneumoniae CG43 derived mutants with deletions of the gene encoding respectively the response regulators KvgA, KvhA, and KvhR were classified into two groups. Group I bacteria carrying either kvgA- or kvhR- exhibited less mucoidy, lower level of capsular polysaccharide (CPS) synthesis and higher LD50 than the parental strain. No apparent change of the group II, including kvhA- and kvhA- kvhR- mutants, was observed. However, the mucoidy of kvhA- kvhR- mutant was found to be diminished after introducing into a kvhA- expressing plasmid. Via promoter-lacZ fusion analysis, kvhA deletion was found to reduce kvhR expression. A regulatory role of KvhA for the expression of kvhR was supported further by EMSA showing a specific binding of KvhA to the putative promoter of kvhR. The promoter activity measurement and EMSA also revealed that KvgA acted as an autoregulator and an activator for the expression of kvhAS and kvhR. In addition, deletion of kvgA suppressed slightly the promoter activity of the cps-orf16-17, and the expression of all three cps transcripts orf1-2, orf3-15, and orf16-17 were reduced in the kvhR- mutant. These suggest that the three homologous response regulators interact to control, in coordination, the bacterial cps expression.

RmpA Regulation of Capsular Polysaccharide Biosynthesis inKlebsiella pneumoniaeCG43

“…To investigate whether the CPS-deficient phenotype of ⌬rmpA mutant was a result of altered expression of the cps genes, three reporter plasmids, pOrf12 (P orf1-2 ::lacZ), pOrf315 (P orf3-15 ::lacZ), and pOrf1617 (P orf16-17 ::lacZ), each carrying a lacZ transcriptional fused to the putative promoter region of the K2 cps gene cluster (25), were used to transform K. pneumoniae strains CG43S3⌬lacZ, CG43S3⌬rmpA⌬lacZ, CG43S3⌬rmpA2⌬lacZ, or CG43S3⌬rcsB⌬lacZ individually. The promoter activity measurements shown in Fig.…”

“…The promoter reporter plasmids were individually mobilized into K. pneumoniae strains by conjugation from E. coli S17-1pir. The ␤-galactosidase activity was measured as described previously (25). In brief, the bacteria was grown to the log phase in LB medium (OD 600 of 0.7) or M9-glucose medium (OD 600 of 0.5), and 100 l of the culture was mixed with 900 l of Z buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , 50 mM ␤-mercaptoethanol), 17 l of 0.1% sodium dodecyl sulfate (SDS), and 35 l of chloroform, followed by vigorous shaking.…”

Fur-dependent MrkHI regulation of type 3 fimbriae in Klebsiella pneumoniae CG43

“…The ability of bacteria to form biofilm was analysed as described previously, with a minor modification (Lin et al, 2006;Wu et al, 2010). Bacteria diluted 1 : 100 in LB broth supplemented with appropriate antibiotics were inoculated into each well of a 96-well microtitre dish (Orange Scientific) and statically incubated at 37 uC for 48 h. Planktonic cells were removed, and the wells were washed once with distilled water to remove unattached cells.…”

Regulation of the Klebsiella pneumoniae Kpc fimbriae by the site-specific recombinase KpcI

“…kpcS DNA was PCR-amplified from K. pneumoniae NTUH-K2044 by primers YCY001 and YCY002, and cloned into the promoter reporter plasmid placZ15 (Lin et al, 2006) to yield plasmids pSY003 and pSY004, containing kpcS without the two inverted repeats in opposite orientations. pSY003 and pSY004 were introduced into the K. pneumoniae NTUH-K2044 DlacZ strain CCW01, which was constructed by using the gene replacement vector pKOV (Link et al, 1997).…”

“…One-tenth of an overnight culture of the bacteria carrying either plasmid was subcultured in LB broth to OD 600~0 .6-0.7. The b-galactosidase activity assay was carried out as described by Lin et al (2006).…”