Involvement of rcsB in Klebsiella K2 capsule synthesis in Escherichia coli K-12

Wacharotayankun, R; Arakawa, Yoshichika; Ohta, Michio; Hasegawa, Takaaki; Mori, Masashi; Horii, Toshinobu; Kato, Naoya

doi:10.1128/jb.174.3.1063-1067.1992

Cited by 30 publications

(17 citation statements)

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“…Sequence analysis of a pathogenicity island carried by pLVPK has revealed a locus named rmpA2, which has been reported to enhance the colony mucoidy of various serotypes of K. pneumoniae (34). Introduction of multicopy rmpA2 could confer on E. coli HB101 the ability to produce Klebsiella K2 capsule in the presence of K2 cps gene cluster, and the transcription of a long-strand mRNA from K2 cps operon was simultaneously increased (1,33). These findings indicated that RmpA2 functions as a trans-acting activator for the CPS biosynthesis.…”

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

2003

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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.

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

2003

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“…Recently, we succeeded with the cloning and expression of the Klebsiella cps cluster in E. coli HB101 (1). Moreover, we subsequently found that the Klebsiella cps cluster requires the functions of both rmpA2 (41) and rcsB (3,37) for production of Klebsiella K2 CPS in E. coli K-12 (40). In the present report, we show the genomic organization of a region which contains the cps genes of K. pneumoniae Chedid.…”

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confidence: 79%

Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid

et al. 1995

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The genomic organization of the chromosomal cps region that is responsible for capsular polysaccharide synthesis in Klebsiella pneumoniae Chedid (O1:K2) was investigated. Deletion analyses and Southern hybridization studies suggested that the central region of the cloned 29-kb BamHI fragment is indispensable for K2 capsular polysaccharide synthesis. The 24,329-bp nucleotide sequence of the Klebsiella cps region was determined and deposited in the EMBL and GenBank databases through DDBJ and assigned accession number D21242. Nineteen possible open reading frames (ORFs) were identified in the sequenced area. Among them, 13 ORFs are very close to each other. Six of the 19 ORFs show considerable nucleotide sequence similarities to Salmonella typhimurium cpsG, cpsB, rfbP, and orf2.8, Escherichia coli gnd, and Haemophilus influenzae bexD, respectively. Moreover, the deduced amino acid sequence of the ORF10 product demonstrated a highly hydrophobic profile and showed putative membrane topology similarity to Rickettsia prowazekii ATP/ADP translocase. Nucleotide sequences similar to the 54 -dependent promoter, as well as the usual ؊35 and ؊10 sequences, were identified just upstream of ORF3, which is the first ORF in the polycistronic structure. Furthermore, a sequence (GGGCGGTAGCGT) found just downstream of the 54 -dependent promoter-like sequence was generally conserved among gene clusters implicated in cell surface polysaccharide synthesis, such as Salmonella rfb and viaB and E. coli kpsMT and rfaQPG. A possible transcriptional terminator with a hairpin loop structure found just downstream of ORF15 that is a homolog of E. coli gnd. K2 capsular polysaccharide biosynthesis in E. coli K-12 depends on cpsB (mannose-1-phosphate guanyltransferase gene), and Klebsiella cpsB, found in the downstream region of the polycistronic structure, was able to complement cpsB of E. coli. Results of transposon insertion and promoter-cloning analyses were consistent with the results of nucleotide sequence analysis.

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“…The involvement of RcsB in Klebsiella K2 capsule biosynthesis of the recombinant E. coli K-12 has been demonstrated (44). Previous studies have found that RcsB must interact with RcsA to form a heterodimer in order to bind specifically to the cps promoter for transcription initiation (21).…”

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confidence: 99%

RmpA Regulation of Capsular Polysaccharide Biosynthesis inKlebsiella pneumoniaeCG43

et al. 2010

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Sequence analysis of the large virulence plasmid pLVPK in Klebsiella pneumoniae CG43 revealed the presence of another mucoid factor encoding gene rmpA besides rmpA2. Promoter activity measurement indicated that the deletion of rmpA reduced K2 capsular polysaccharide (CPS) biosynthesis, resulting in decreased colony mucoidy and virulence in mice. Introduction of a multicopy plasmid carrying rmpA restored CPS production in the rmpA or rmpA2 mutant but not in the rcsB mutant. Transformation of the rmpA deletion mutant with an rcsB-carrying plasmid also failed to enhance CPS production, suggesting that a cooperation of RmpA with RcsB is required for regulatory activity. This was further corroborated by the demonstration of in vivo interaction between RmpA and RcsB using two-hybrid analysis and coimmunoprecipitation analysis. A putative Fur binding box was only found at the 5 noncoding region of rmpA. The promoter activity analysis indicated that the deletion of fur increased the rmpA promoter activity. Using electrophoretic mobility shift assay, we further demonstrated that Fur exerts its regulatory activity by binding directly to the promoter. As a result, the fur deletion mutant exhibited an increase in colony mucoidy, CPS production, and virulence in mice. In summary, our results suggested that RmpA activates CPS biosynthesis in K. pneumoniae CG43 via an RcsB-dependent manner. The expression of rmpA is regulated by the availability of iron and is negatively controlled by Fur.

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Involvement of rcsB in Klebsiella K2 capsule synthesis in Escherichia coli K-12

Cited by 30 publications

References 30 publications

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

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

Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid

RmpA Regulation of Capsular Polysaccharide Biosynthesis inKlebsiella pneumoniaeCG43

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