Open AccessIsolation from recombinant Escherichia coli and characterization of CMP‐Kdo synthetase, involved in the expression of the capsular K5 polysaccharide (K‐CKS)
Author(s) -
Rosenow Carsten,
Roberts Ian S.,
Jann Klaus
Publication year - 1995
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1995.tb07352.x
Subject(s) - escherichia coli , bacterial capsule , biology , gene , recombinant dna , biochemistry , polysaccharide , bacterial outer membrane , microbiology and biotechnology , enterobacteriaceae , peptide sequence , gene expression , virulence
In Escherichia coli with group II capsules, the synthesis of capsular polysaccharide and its cellular expression are encoded by the kps gene cluster, which is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of E. coli with the group II capsular K5 polysaccharide, have been cloned and sequenced. Region 1 contains the kps E, D, U, C and S genes. In this communication we describe the overexpression of the kps D and kps U genes as well as the isolation of the KpsU protein from the recombinant bacteria by chloroform treatment. The purified KpsU protein exhibited CMP‐Kdo‐synthetase activity. The N‐terminal sequence and two internal peptide sequences of the isolated protein are in agreement with that previously predicted from the DNA sequence of the kps U gene. The kinetic data of the CMP‐Kdo‐synthetase participating in K5 capsule expression (K‐CMP‐Kdo‐synthetase) differ from those described for the CMP‐Kdo‐synthetase, participating in lipopolysaccharide synthesis (L‐CMP‐Kdo‐synthetase).