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Identification of two genes, kpsM and kpsT, in region 3 of the polysialic acid gene cluster of Escherichia coli K1.

ABSTRACT: The polysialic acid capsule of Escherichia coli K1, a causative agent of neonatal septicemia and meningitis, is an essential virulence determinant. The 17-kb kps gene cluster, which is divided into three functionally distinct regions, encodes proteins necessary for polymer synthesis and expression at the cell surface. The central region, 2, encodes products required for synthesis, activation, and polymerization of sialic acid, while flanking regions, 1 and 3, are thought to be involved in polymer assembly and transport. In this study, we identified two genes in region 3, kpsM and kpsT, which encode proteins with predicted sizes of 29.6 and 24.9 kDa, respectively. The hydrophobicity profile of KpsM suggests that it is an integral membrane protein, while KpsT contains a consensus ATP-binding domain. KpsM and KpsT belong to a family of prokaryotic and eukaryotic proteins involved with a variety of biological processes, including membrane transport. A previously described kpsT chromosomal mutant that accumulates intracellular polysialic acid was characterized and could be complemented in trans. Results of site-directed mutagenesis of the putative ATP-binding domain of KpsT are consistent with the view that KpsT is a nucleotide-binding protein. KpsM and KpsT have significant similarity to BexB and BexA, two proteins that are essential for polysaccharide capsule expression in Haemophilus influenzae type b. We propose that KpsM and KpsT constitute a system for transport of polysialic acid across the cytoplasmic membrane.

SUBMITTER: Pavelka MS

PROVIDER: S-EPMC208135 | biostudies-other | 1991 Aug

REPOSITORIES: biostudies-other

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Identification of two genes, kpsM and kpsT, in region 3 of the polysialic acid gene cluster of Escherichia coli K1.

Pavelka M S MS Wright L F LF Silver R P RP

Journal of bacteriology 19910801 15

The polysialic acid capsule of Escherichia coli K1, a causative agent of neonatal septicemia and meningitis, is an essential virulence determinant. The 17-kb kps gene cluster, which is divided into three functionally distinct regions, encodes proteins necessary for polymer synthesis and expression at the cell surface. The central region, 2, encodes products required for synthesis, activation, and polymerization of sialic acid, while flanking regions, 1 and 3, are thought to be involved in polyme ...[more]

PMID: 1856162

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Project description:The K1 capsular polysaccharide, a polymer of sialic acid, is an important virulence determinant of extraintestinal pathogenic Escherichia coli. The genes responsible for the synthesis and expression of the polysialic acid capsule of E. coli K1 are located on the 17-kb kps gene cluster, which is functionally divided into three regions. Central region 2 encodes proteins necessary for the synthesis, activation, and polymerization of sialic acid, while flanking regions 1 and 3 are involved in polymer transport to the cell surface. In this study, we identified two genes at the proximal end of region 2, neuD and neuB, which encode proteins with predicted sizes of 22.7 and 38.7 kDa, respectively. Several observations suggest that the neuB gene encodes sialic acid synthase. EV24, a neuB chromosomal mutant that expresses a capsule when provided exogenous sialic acid, could be complemented in trans by the cloned neuB gene. In addition, NeuB has significant sequence similarity to the product of the cpsB gene of Neisseria meningitidis group B, which is postulated to encode sialic acid synthase. We also present data indicating that neuD has an essential role in K1 polymer production. Cells harboring pSR426, which contains all of region 2 but lacks region 1 and 3 genes, produce an intracellular polymer. In contrast, no polymer accumulated in cells carrying a derivative of pSR426 lacking a functional neuD gene. Unlike strains with mutations in neuB, however, neuD mutants are not complemented by exogenous sialic acid, suggesting that NeuD is not involved in sialic acid synthesis. Additionally, cells harboring a mutation in neuD accumulated sialic acid and CMP-sialic acid. We also found no significant differences between the endogenous and exogenous sialyltransferase activities of a neuD mutant and the wild-type organism. NeuD shows significant similarity to a family of bacterial acetyltransferases, leading to the theory that NeuD is an acetyltransferase which may exert its influences through modification of other region 2 proteins.

Project description:The kps locus for biosynthesis of the capsular polysialic acid virulence factor in Escherichia coli K1 contains at least two convergently transcribed operons, designated region 1 and regions 2 plus 3. On the basis of DNA sequence analysis, kpsF appeared to be a good candidate for the first gene of region 1 (M. J. Cieslewicz, S. M. Steenbergen, and E. R. Vimr, J. Bacteriol. 175:8018-8023, 1993). A preliminary indication that kpsF is required for capsule production is the capsule-negative phenotype of an aph T insertion in the chromosomal copy of kpsF. The present communication describes the isolation and phenotypic characterization of this mutant. Although transcription through kpsF was required for capsule production, complementation analysis failed to indicate a clear requirement for the KpsF polypeptide. However, since E. coli contains at least two other open reading frames that could code for homologs of KpsF, the apparent dispensability of KpsF remains provisional. DNA sequence analysis of 1,100 bp upstream from the kpsF translational start site did not reveal any open reading frames longer than 174 nucleotides, consistent with kpsF being the first gene of region 1. Since kpsF appeared to be the first gene of a region whose gene products are required for polysialic acid transport and because capsule production is known to be thermoregulated, primer extension analyses were carried out with total RNA isolated from cells grown at permissive (37 degrees C) and nonpermissive (20 degrees C) temperatures. The results revealed a potentially complex kpsF promoter-like region that was transcriptionally silent at the nonpermissive temperature, suggesting that thermoregulation of region 1 may be exerted through variations in kpsF expression. Additional evidence supporting this conclusion was obtained by demonstrating the effects of temperature on expression of the gene kpsE, immediately downstream of kpsF. Chloramphenicol acetyltransferase assays were carried out with constructs containing the kpsF 5' untranslated region fused to a promoterless cat cassette, providing further evidence that kpsF is thermoregulated. Although the function of KpsF is unclear, primary structure analysis indicated two motifs commonly observed in regulatory proteins and homology with glucosamine synthase from Rhizobium meliloti.

Project description:Reviewing the genetics underlying the arms race between bacteria and bacteriophages can offer an interesting insight into the development of bacterial resistance and phage co-evolution. This study shows how the natural development of resistances to the K1F bacteriophage, a phage which targets the K1 capsule of pathogenic Escherichia coli, can come about through insertion sequences (IS). Of the K1F resistant mutants isolated, two were of particular interest. The first of these showed full resistance to K1F and was found to have disruptions to kpsE, the product of which is involved in polysialic acid translocation. The second, after showing an initial susceptibility to K1F which then developed to full resistance, had disruptions to neuC, a gene involved in one of the early steps of polysialic acid biosynthesis. Both of these mutations came with a fitness cost and produced considerable phenotypic differences in the completeness and location of the K1 capsule when compared with the wild type. Sequential treatment of these two K1F resistant mutants with T7 resulted in the production of a variety of isolates, many of which showed a renewed susceptibility to K1F, indicating that these insertion sequence mutations are reversible, as well as one isolate that developed resistance to both phages. IMPORTANCE Bacteriophages have many potential uses in industry and the clinical environment as an antibacterial control measure. One of their uses, phage therapy, is an appealing alternative to antibiotics due to their high specificity. However, as with the rise in antimicrobial resistance (AMR), it is critical to improve our understanding of how resistance develops against these viral agents. In the same way as bacteria will evolve and mutate antibiotic receptors so they can no longer be recognized, resistance to bacteriophages can come about via mutations to phage receptors, preventing phage binding and infection. We have shown that Escherichia coli will become resistant to the K1F bacteriophage via insertion element reshufflings causing null mutations to elements of the polysialic acid biosynthetic cluster. Exposure to the T7 bacteriophage then resulted in further changes in the position of these IS elements, further altering their resistance and sensitivity profiles.

Dataset Information

Identification of two genes, kpsM and kpsT, in region 3 of the polysialic acid gene cluster of Escherichia coli K1.

Publications

Identification of two genes, kpsM and kpsT, in region 3 of the polysialic acid gene cluster of Escherichia coli K1.

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