Superstructure formation by RodZ hexamers of Shigella sonnei maintains the rod shape of bacilli.

The rod shape of bacilli is maintained by bacterial cytoskeletal protein MreB, an actin homolog that acts in concert with the inner membrane protein RodZ. We previously reported RodZ binds RNA to control the posttranscriptional regulation of invE (virB), which controls the type III secretion system essential for the virulence of Shigella. Here, we show that purified RodZ forms "superstructures" of high molecular mass that dissociate into a midsized "basal complex" in the presence of nonionic detergent, or to a monomer in the presence of dithiothreitol. We used mass spectrometry to show that the basal complex was a hexamer. Electrophoresis mobility shift assays combined with gel filtration detected the RNA-binding activity in fractions containing molecules larger than the basal hexamer. The superstructure was consistently detected with MreB in crude cell lysates of S. sonnei that were fractionated using gel filtration. Immunofluorescence microscopy using two different super-resolution settings showed that wild-type RodZ was distributed in cells as separate dots. Consistent with the superstructure comprising homohexamers, majority of the dots distributed among areas of discrete values. In addition, simultaneous immunodetection of MreB provided the first evidence of colocalization with RodZ as larger patch like signals. These findings indicate that native RodZ forms clusters of various sizes, which may correspond to a superstructure comprising multiple hexamers required for the RNA-binding activity.

[Phenomenology and molecular mechanisms of Shigella sonnei dissociation: its variability in model in vivo and in vitro systems]. / Fenomenologiia i molekuliarnye mekhanizmy dissotsiatsii Shigella sonnei: izmenchivost' v model'nykh sistemakh in vivo i in vitro.

The dynamics of the accumulation of phase II S. sonnei in the infectious process in germ-free rats, during cultivation and in subcultures was studied. The in vivo variability of S. sonnei showed a sharply defined phasic character of the process with the periods of the apparent absence of phase II, the increase of its occurrence and stabilization with the preservation of S-forms in minor amounts. Kanamycin-resistant phase II accumulated in vivo more rapidly than in vitro. Cultivation with sodium desoxycholate was found to accelerate the process of disassociation. The characteristic feature of all dissociants detected in all systems was their plasmid profile: in phase I, plasmids of 120 and 60 Md, as well as small plasmids, were detected; in phase II disassociants, plasmid with a molecular weight of 120 Md was absent. The restriction analysis of total plasmid DNA by means of restrictase EcoR1 showed the absence of 3 fragments in phase II while other 13 fragments were retained. These results open prospects for the molecular cloning of the gene antigen of phase II.

Bacterial antagonists of Shigellae.

Bacterial antagonism between a microorganisms and Shigella sonnei strains was studied in model experiments simulating conditions of the natural aquatic environment. In these studies surface waste samples from the river Vltava served as the experimental environment. To ensure bacteriologically defined conditions all water samples were heat-sterilized prior to antagonism testing. Consistently with the literature data and author's own observations the following bacterial species and genera were chosen as test organisms to be tested for antagonism against Shigella sonnei strains in water; E. coli, Citrobacter, Enterobacter, Klebsiella pneumoniae, Proteus, Pseudomonas aeruginosa and the fecal streptococci S. fecalis and S. faecium. Presence or absence of microbial antagonism against shigellae was determined in the experimental water medium contaminated with shigella-test organism mixtures of density ratios within the range 1 : 1 through 1 : 10(4). The highest degree of antagonism was observed with Pseudomonas aeruginosa that at density ratio 1 : 1 inhibited the Shigella sonnei growth in water within 42 hours of incubation. A similar degree of antagonism was also observed with Klebsiella pneumoniae at the density ratio 1 : 10(1) and with Enterobacter aerogenes at 1 : 10(2). At lower density ratios the antagonism exhibited by these two species was also present, but occurred much later, i.e. after 72 hours up to 5 days. The remaining test organisms used showed no antagonistic action Shigella sonnei strain in the model aquatic environment.

Outer membrane proteins of Shigella sonnei. I. Characterization of phase I, phase II and R-form Shigella sonnei.

The cell envelope of Shigella sonnei phase I, phase II and R-form was fractionated inot outer and cytoplasmic membrane components by means of sucrose density gradient centrifugation. The protein composition of the outer membrane has been analyzed by SDS-polyacrylamide gel electrophoresis. The outer membrane preparations contained 15-17 proteins. The major proteins of the outer membrane were of apparent molecular weights: 27,000, 28,000 and 31,000. Their amount varied depending on the structural defects of lipopolysaccharide.

Outer membrane proteins of Shigella sonnei. II. Comparative studies on virulent and avirulent strains of phase I.

The proteins of the outer membrane of virulent and avirulent Shigella sonnei phase I strains were characterized by SDS-polyarcylamide gel electrophoresis and their molecular weights determined. Significant quantitative differences were found in composition of the outer membrane proteins between virulent and avirulent strains. These differences were confirmed in electrofocusing experiments and disc-crossed immunoelectrophoresis.

[Immunoelectrophoretic analysis of the antigenic structure of Sh. sonnei]. / Immunoélektroforeticheskii analiz antigennoi struktury Sh. sonnei.

The authors studied the antigenic composition of 105 Sh. sonnei strains freshly isolated from patients suffering from acute dysentery and carriers. Immunophoregrams of pure S-and R-forms species were obtained. Up to 13 antigens differing by electrophoretic and diffusion mobility and immunological specificity were revealed among soluble Sh. sonnei antigens The position of common and specific antigens was determined on the immunophoregram. Along with the thermostable somatic O-antigen detected at the I phase of the S-forms, and two thermolabile O-antigen components at the II phase, and the R-forms, there was revealed a surface, relatively thermolabile, K-antigen of A-type capable of agglutinating live bacteria in the O-antiserum; position of the latter on the immunophoregram was also determined.