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

Abstract

In the genome of Klebsiella pneumoniae NTUH-K2044, nine fimbrial gene clusters were identified. Besides type 1 and type 3 fimbriae, the others are novel and were named Kpa, Kpb, Kpc, Kpd, Kpe, Kpf and Kpg fimbriae. Prevalence analysis among 105 K. pneumoniae clinical isolates revealed that the kpc genes were highly associated with the K1 serotype isolates. Induced expression of the recombinant kpcABCD genes in Escherichia coli resulted in Kpc fimbriation and increased biofilm formation. A putative site-specific recombinase encoding gene kpcI and a 302 bp intergenic DNA flanked by 11 bp inverted repeats, namely kpcS, were identified in the upstream region of the kpcABCD genes. Using LacZ as the reporter, a dramatic difference in promoter activity of kpcS in two different orientations was observed and accordingly assigned as ON and OFF phase. kpcI expression was found to be able to invert kpcS in trans from phase ON to OFF and vice versa. Using the two-plasmid system, expression of kpcA, encoding the major component of the Kpc fimbriae, could be observed upon the induced expression of kpcI. These results indicate that KpcI is involved in the regulation of Kpc fimbriation in a phase-variable manner.

FimK regulation on the expression of type 1 fimbriae in Klebsiella pneumoniae CG43S3

“…As described previously (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 20 h. After removing planktonic cells, the wells were washed once with distilled water to remove unattached cells. Crystal violet (0.1 % w/v; Sigma-Aldrich) was used to stain the attached cells for 30 min.…”

“…For complementation analysis or protein overexpression, the coding region from pyT-fimB, pyT-fimK, pyT-fimK E245A , pyT-HTH fimK , pyT-EIL fimK , pyT-AIL fimK or pyT-mrkJ was subcloned into the broad-hostrange vector pRK415 (Keen et al, 1988) or expression vectors pETQ31, pETQ33 (Wu et al, 2010), pQE31 (Qiagen) or pET30a (Novagen). This resulted in low-copy-number complementation plasmids pRK415-fimK, pRK415-fimK E245A , pRK415-HTH fimK and pRK415-EIL fimK and overexpression clones pETQ33-fimB, pETQ31-EIL fimK , pETQ31-AIL fimK , pQE31-fimK, pETQ33-EIL fimK , pETQ33-AIL fimK , pETQ33-mrkJ and pET30a-mrkJ.…”

More than One Way To Control Hair Growth: Regulatory Mechanisms in Enterobacteria That Affect Fimbriae Assembled by the Chaperone/Usher Pathway

“…pneumoniae Kpc fimbriae. A recently identified fimbrial type from Klebsiella pneumoniae NTUH-K2044, termed Kpc fimbriae, has also been shown to be regulated by an invertible DNA element (91). Genes encoding these fimbriae were shown to be predominantly found in K1 serotypes which, interestingly, are significantly associated with the formation of liver abscesses in diabetic patients in Southeast Asia.…”

“…Genes encoding these fimbriae were shown to be predominantly found in K1 serotypes which, interestingly, are significantly associated with the formation of liver abscesses in diabetic patients in Southeast Asia. Inversion of a 302-bp promoter-containing region between two 11-bp inverted repeats upstream of the major subunit gene is mediated by the recombinase KpcI (91). The inversion of the switch (kpcS) by recombinant KpcI in an E. coli background could be mediated from either off-to-on or on-to-off orientations (91).…”

“…Inversion of a 302-bp promoter-containing region between two 11-bp inverted repeats upstream of the major subunit gene is mediated by the recombinase KpcI (91). The inversion of the switch (kpcS) by recombinant KpcI in an E. coli background could be mediated from either off-to-on or on-to-off orientations (91). One of the inverted repeats lies within the KpcI reading frame, so when kpcS is in the on orientation, a portion of the predicted amino acid sequence changes (91).…”