Richard P. Silver

Coating the surface: a model for expression of capsular polysialic acid in Escherichia coli K1

Capsules are well‐studied components of the bacterial surface that modulate interactions between the cell and its environment. Generally composed of polysaccharide, they are key virulence determinants in invasive infections in humans and other animals. Genetic determinants involved in capsule expression have been isolated from a number of organisms, but perhaps the best characterized is the kps cluster of Escherichia coli K1. In this review, the current understanding of the functions of the kps gene products is summarized. Further, a proposed mechanistic model for capsule expression is presented and discussed. The model is based on the premise that the numerous components of the kps cluster form a hetero‐oligomeric complex responsible for synthesis and concurrent translocation of the capsular polysialic acid through sites of inner and outer membrane fusion. We view the ATP‐binding cassette (ABC) transporter, KpsMT, to be central to the functioning of the complex, interacting with the biosynthetic apparatus as well as the extracytoplasmic components of the cluster to co‐ordinate synthesis and translocation. The model provides the basis for additional experimentation and reflects emerging similarities among systems responsible for macromolecular export in Gram‐ negative bacteria.

Topological and mutational analysis of KpsM, the hydrophobic component of the ABC-transporter involved in the export of polysialic acid in Escherichia coli K1

The 17 kb kps gene cluster of Escherichia coli K1, which encodes the information required for synthesis, assembly and translocation of the polysialic acid capsule of E. coli K1, is divided into three functional regions. Region 3 contains two genes, kpsM and kpsT, essential for the transport of capsule polymer across the cytoplasmic membrane. The hydrophobicity profile of KpsM suggests that it is an integral membrane protein while KpsT contains a consensus ATP‐binding site. KpsM and KpsT belong to the ATP‐binding cassette (ABC) superfamily of membrane transporters. In this study, we investigate the topology of KpsM within the cytoplasmic membrane using β‐lactamase fusions and alkaline phosphatase sandwich fusions. Our analysis provides evidence for a model of KpsM having six membrane‐spanning regions, with the N‐ and C‐terminal domains facing the cytoplasm, and a short domain within the third periplasmic loop, which we refer to as the SV–SVI linker localizing in the membrane. Protease digestion studies are consistent with regions of KpsM exposed to the periplasmic space. In vivo cross‐linking studies provide support for dimerization of KpsM within the cytoplasmic membrane. Linker‐insertion and site‐directed mutagenesis define the N‐terminus, the first cytoplasmic loop, and the SV‐SVI linker as regions that are important for the function of KpsM in K1 polymer transport.

Evidence for Multimerization of Neu Proteins Involved in Polysialic Acid Synthesis in Escherichia coli K1 Using Improved LexA-Based Vectors

Recently, M. Dmitrova et al. (Mol. Gen. Genet. 257:205-212, 1998) described a LexA-based genetic system to monitor protein-protein interactions in an Escherichia coli background. However, the plasmids used in this system, pMS604 and pDP804, were not readily amenable for general use. In this report, we describe modifications of both plasmids that allow fragments of DNA to be fused to either vector in any reading frame. Homodimerization and heterodimerization of full-length proteins involved in polysialic acid synthesis in E. coli K1, as well as heterodimerization between a full-length protein and a protein fragment, demonstrate the usefulness of the modified plasmids for investigating bacterial protein-protein interactions in vivo.

ABC transporters and the export of capsular polysaccharides from gram-negative bacteria.

In this review, we discuss the kps cluster of Escherichia coli as the paradigm for the ABC capsular polysaccharide exporter (CPSE) family. Components of the cluster form a multimeric protein complex consisting of both biosynthetic and export machinery. We compare the Kps exporter with capsule export systems from other members of the CPSE family.

Use of LexA-based system to identify protein-protein interactions in vivo

Publisher Summary This chapter describes a system for analyzing both homo- and heterodimerization of full-length proteins and protein fragments in an Escherichia coli background. The characterization of protein-protein interactions can reveal much about the structure and function of individual proteins and protein complexes. Many methods designed to identify these interactions have been reported, including yeast two hybrid and plasmid-based bacterial systems. This system has been successful in determining interactions between proteins involved in the biosynthesis of the polysialic acid capsule of E. coli K1. These interactions include those between full-length proteins as well as those between full-length proteins and protein fragments. However, it is not necessary to restrict the systems use to studying only Escherichia coli protein interactions. Another feature of this system is that the detection of interaction between fusions is not disrupted by the tandem expression of unfused subunits in the host strain. Chloramphenicol acetyltransferase (CAT) fused to the wild-type LexA DNA binding domain(DBD) efficiently repressed lacZ transcription in SU101, even though this strain carries a transposon that expresses subunits of the same type I CAT. This data also illustrates that some multimeric proteins can be studied using this system, since CAT is a well-characterized homotrimer.

R-Plasmids in Pasteurella multocida.

Abstract Multiple drug-resistant strains of Pasteurella multocida were associated with a high incidence of fatal pneumonia in feedlot cattle. A representative strain, CAH160, resistant to tetracycline (Tc), streptomycin (Sm), and sulfonamide (Su) was studied. The minimal inhibitory concentration (MIC) of Tc was 32 μg/ml while Sm had an MIC of 256 μg/ml. Plasmid DNA was isolated from CAH160 by cesium chloride-ethidium bromide centrifugation. Agarose gel electrophoresis showed that at least three distinct species of plasmid DNA were present. DNA isolated from CAH160 was used to transform Escherichia coli K12 strain C600 r k − m k − . Transformants resistant to Tc; to Sm, Su; and to Tc, Sm, Su were obtained. Contour length measurements of plasmid DNA isolated from transformant cells showed that Tc resistance was associated with a 3-Mdal plasmid (pSR10), while Sm, Su resistance resided on a 2.7-Mdal molecule (pSR11). More than 20% of the transformants were resistant to Tc, Sm, Su and contained both plasmid species. In E. coli the MIC of Tc was 256 μg/ml and that of Sm was 64 μg/ml. The buoyant density of pSR10 was 1.699 g/cm 3 , while the density of pSR11 was 1.709 g/cm 3 .

Purification and characterization of KpsT, the ATP-binding component of the ABC-capsule exporter of Escherichia coli K1

The K1 capsule, an alpha(2,8)-linked polymer of sialic acid, is an important virulence determinant of invasive Escherichia coli. The 17-kb kps gene cluster of E. coli K1 encodes the information necessary for capsule expression at the cell surface. Two proteins, KpsM and KpsT, play a role in the transport of capsular polysaccharide across the cytoplasmic membrane, utilizing the energy from ATP hydrolysis. They belong to the ATP-binding cassette superfamily of transport proteins. In this study, we purified KpsT in its native form and show that the purified protein is able to bind ATP, undergo an ATP-dependent conformational change and hydrolyze ATP. Protease accessibility studies demonstrate the in vivo interaction between KpsM and KpsT.