title: Insights into Yersinia pestis biofilm development: topology and co-interaction of Hms inner membrane proteins involved in exopolysaccharide production.
abstract: Primarily, three operons, hmsHFRS, hmsT and hmsP, are responsible for the development of a Yersinia pestis biofilm, which is essential for blockage-dependent transmission of plague from fleas to mammals. Here, using specific antibodies, a polymeric beta-1,6-N-acetyl-d-glucosamine-like polysaccharide was detected in the extracellular matrix of hmsHFRS-dependent Y. pestis biofilm. The production of this exopolysaccharide (EPS) was controlled by diguanylate cyclase HmsT and EAL domain phosphodiesterase HmsP, acting as positive and negative regulators respectively. Cellular compartmentalization of soluble segments of Hms inner membrane proteins, including the putative glycosyltransferase domain of HmsR, the diguanylate cyclase/GGDEF domain of HmsT and the phosphodiesterase/EAL domain of HmsP, was determined by a combination of topology prediction algorithms and construction of C-terminal translational fusions with beta-galactosidase and alkaline phosphatase. Multiple interactions of Hms inner membrane proteins were detected using bacterial cAMP based two-hybrid system. Biochemical analyses confirmed some of these protein-protein interactions. Our results indicate that synthesis and regulation of the Y. pestis biofilm EPS occurs in the cytoplasm by a proposed Hms enzymatic complex.
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky, USA. bobrov@pop.uky.edu
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Bobrov
authors.forename: Olga
authors.initials: O
authors.lastname: Kirillina
authors.forename: Stanislav
authors.initials: S
authors.lastname: Forman
authors.forename: Dietrich
authors.initials: D
authors.lastname: Mack
authors.forename: Robert D
authors.initials: RD
authors.lastname: Perry
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018441:Biofilms / Q000254:growth & development*; D015153:Blotting, Western; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D019761:Phosphorus-Oxygen Lyases / Q000378:metabolism; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020798:Two-Hybrid System Techniques; D015010:Yersinia pestis / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C122178:HMST protein, Yersinia pestis; D008565:Membrane Proteins; D011135:Polysaccharides, Bacterial; D010727:Phosphoric Diester Hydrolases; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase