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

Abstract

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.

Molecular characterization of the plasmid‐encoded eps gene cluster essential for exopolysaccharide biosynthesis in Lactococcus lactis

“…All genes are transcribed as a single polycistronic mRNA of Ϸ12 kb. Similarly, large transcripts encompassing all biosynthetic genes for polysaccharide production were also reported for the ams gene cluster from Erwinia amylovora and the cps gene cluster from Klebsiella pneumoniae K2 (Bugert and Geider, 1995;Arakawa et al, 1995), and postulated for the eps gene cluster of Pseudomonas solanacearum (Huang and Schell, 1995). This suggests a co-ordinate control of the genes involved in the biosynthesis of these polysaccharides.…”

A Novel NDP-6-deoxyhexosyl-4-ulose Reductase in the Pathway for the Synthesis of Thymidine Diphosphate-d-fucose

“…This low GϩC content suggests that the gene cluster was transferred from another species. A lower GϩC content has been found in many gene clusters involved in the synthesis of various bacterial polysaccharides (34,37,41,42). In particular, the region containing ORF14-ORF19 has the lowest GϩC content (27.0%), and the discrepancy of the GϩC content within the gene cluster suggests that the fcd and dTDP-L-rhamnose synthetic genes have different origins.…”

RobA‐lnduced Multiple Antibiotic Resistance Largely Depends on the Activation of the AcrAB Efflux

“…DNA preparation, ligation, and cell transformation were performed as outlined by Sambrook et al (18) and modified in our laboratory (2). Restriction endonucleases were purchased from Takara Shuzo Co., Kyoto, Japan, and a deletion kit and a ligation kit were supplied by Nippon Gene Co., Tokyo.…”