RmpA2, an Activator of Capsule Biosynthesis inKlebsiella pneumoniaeCG43, Regulates K2cpsGene Expression at the Transcriptional Level

Abstract

The rmpA2 gene, which encodes an activator for capsular polysaccharide (CPS) synthesis, was isolated from a 200-kb virulence plasmid of Klebsiella pneumoniae CG43. Based on the sequence homology with LuxR at the carboxyl-terminal DNA-binding motif, we hypothesized that RmpA2 exerts its effect by activating the expression of cps genes that are responsible for CPS biosynthesis. Two luxAB transcriptional fusions, each containing a putative promoter region of the K. pneumoniae K2 cps genes, were constructed and were found to be activated in the presence of multicopy rmpA2. The activation is likely due to direct binding of RmpA2 to the cps gene promoter through its C-terminal DNA binding motif. Moreover, the loss of colony mucoidy in a K. pneumoniae strain deficient in RcsB, a regulator for cps gene expression, could be recovered by complementing the strain with a multicopy plasmid carrying rmpA2. The CPS production in Lon protease-deficient K. pneumoniae significantly increased, and the effect was accompanied by an increase of RmpA2 stability. The expression of the rmpA2 gene was negatively autoregulated and could be activated when the organism was grown in M9 minimal medium. An IS3 element located upstream of the rmpA2 was required for the full activation of the rmpA2 promoter. In summary, our results suggest that the enhancement of K2 CPS synthesis in K. pneumoniae CG43 by RmpA2 can be attributed to its transcriptional activation of K2 cps genes, and the expression level of rmpA2 is autoregulated and under the control of Lon protease.

Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae , an urgent threat to public health

“…Several genetic loci have been identified as virulence factors in K. pneumoniae on the basis of murine models of infection. These include gene clusters associated with the synthesis of siderophore systems yersiniabactin, aerobactin, colibactin, salmochelin (40)(41)(42)(43), or microcin (44); the "regulators of mucoid phenotype" rmpA and rmpA2, which can up-regulate capsule production (45,46); an allantoinase gene cluster (47); the ferric uptake operon kfuABC (48); and the two-component regulator kvgAS (49). Most of these genes were detected only in KpI, except for kfuABC (found in all KpII-B, 75% of KpIII, and 20% of KpI) and allantoinase (found in 50% of KpII-B and 5% of KpI).…”

PecS regulates the urate-responsive expression of type 1 fimbriae in Klebsiella pneumoniae CG43

“…Specific gene deletion was introduced to the chromosome of K. pneumoniae CG43S3 by using an allelic-exchange strategy essentially as described previously (Lai et al, 2003). In brief, the DNA fragments of 1 kb flanking both ends of cpxAR, pecS and pecM were amplified using PCR with the primer sets WCC138/WCC139 and WCC140/WCC141, WCC111/WCC112 and WCC113/WCC114, and WCC117/WCC118 and WCC119/WCC114, respectively.…”

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

“…The amplified DNA fragments were individually cloned into suicide vector pKAS46 (Skorupski & Taylor, 1996). The resulting plasmid was transformed into E. coli S17-1lpir and then mobilized by conjugating to the streptomycin-resistant strain, K. pneumoniae CG43S3 (Lai et al, 2003). Several kanamycin-resistant transconjugants, with the plasmid integrated into the chromosome by homologous recombination, were selected from the M9 agar plates supplemented with kanamycin and propagated in 2 ml LB broth overnight.…”

“…Specific gene deletion was introduced into the chromosome of K. pneumoniae CG43S3 using an allelic-exchange strategy (Lai et al, 2003). Briefly, DNA fragments of 1 kb flanking both ends of mrkA, fimK, EIL fimK and HTH fimK DNA were amplified by PCR with primer sets SL0141/ SL0142 and SL0143/SL0158, K1/K2 and K3/K4, WCC32/WCC33 and WCC34/WCC35, and WCC127/CC128 and WC133/WCC130, respectively.…”