Molecular cloning and analysis of genes for production of K5, K7, K12, and K92 capsular polysaccharides in Escherichia coli

Abstract

With a DNA fragment from within the region encoding the transport functions for K1 production as a hybridization probe in Southern blot experiments, homologous DNA sequences were detected in the DNA from Escherichia coli strains producing K5, K7, K92, and K100 capsular polysaccharides. No homology with the laboratory strain LE392 was detected. The same DNA probe was used to prescreen cosmid libraries in LE392 by colony hybridization, as a rapid method to isolate clones encoding the genes for K5, K7, K12, and K92 antigen production. Clones carrying sequences homologous to the probe that also produced capsular material were identified by using polyclonal and monoclonal antibodies raised against the K antigen in question and K antigen-specific phages. By restriction enzyme mapping of the appropriate cosmid clones it was possible to align the genes for the production of different K antigens in terms of common restriction endonuclease cleavage sites. A DNA fragment encoding the postulated transport functions for K7 antigen production could complement deletion mutations in the transport functions for K1 antigen production. Thus the transport to the cell surface of chemically distinct polysaccharides may be by a common process. Analysis in E. coli of the proteins produced by plasmids carrying the likely transport functions for K1, K5, and K7 antigen production revealed that each region coded for a similar polypeptide.

KpsC and KpsS are retaining 3-deoxy- d - manno -oct-2-ulosonic acid (Kdo) transferases involved in synthesis of bacterial capsules

“…The K. pneumoniae O2a transporter shows no specificity for the structure of the exported glycan (41). The properties of the CPS ABC transporters (6,12,13,42) suggest similarity to the latter model. KpsC has been shown to interact with the CPS polymerase and the export machinery (33), so it is possible that truncations of KpsC affect these interactions, and the proper coupling of synthesis and export, resulting in "unregulated" polymerase activity.…”

Sequence and expression of the Escherichia coli K1 neuC gene product

“…Three distinct regions have been identified in this gene cluster (18). The central region (region 2) is involved in polysaccharide polymerization and synthesis.…”

Cloning and expression in Escherichia coli of the nahA gene from Porphyromonas gingivalis indicates that -N-acetylhexosaminidase is an outer-membrane-associated lipoprotein

“…Radiolabelled probes were generated by extending hexadeoxynucleotide primers in the presence of [32P]dCTP using the Klenow fragment of DNA polymerase I (Feinberg & Vogelstein, 1983). Hybridization was performed as described previously (Roberts et al,, 1986), and the filters then washed each for 15 min at 65 "C in 2 x SSC 0.1 % SDS and then in 0.5 x SSC 0.1 YO SDS. pNahase assays.…”

“…Southern blots of restriction fragments were performed as described by Roberts et al (1986). Radiolabelled probes were generated by extending hexadeoxynucleotide primers in the presence of [32P]dCTP using the Klenow fragment of DNA polymerase I (Feinberg & Vogelstein, 1983).…”