The periplasmic nuclease of Vibrio vulnificus, Vvn, has been purified to homogeneity by a one step purification procedure using chromatography on a SP Sepharose column. The purified enzyme showed different mobilities on reducing and non-reducing SDS-PAGE, suggesting that disulfide bonds are involved in the maintenance of a stable tertiary conformation of the protein. Vvn randomly cleaved single and double stranded DNA and RNA, and possessed endonucleolytic activity. The enzyme exhibited an optimal activity between pH 8.0 and pH 10.0, and the optimal temperatures for the DNase and RNase activity were 40 oC ¡V 60 oC and 40 oC ¡V 50 oC, respectively. The enzymatic activity was inhibited by EDTA and EGTA, indicating that Vvn was a metalloenzyme. The DNase and RNase activity of Vvn had different requirements for divalent cations. Chemical modification studies on Vvn revealed the involvement of lysine, arginine, tryptophan and carboxylate residues in the catalytic activity of the enzyme. The extents of inactivation of the DNase and RNase activity of Vvn by modification of the carboxylate group with EDC were different. Substrate DNA and RNA protected the DNase and RNase activity of Vvn from inactivation by PLP, PGO, NBS and EDC which modified lysine, arginine, tryptophan and the carboxylate group. Mg2+ could not protect the DNase and RNase activity of Vvn against the inactivation by PLP and PGO. Whereas Mg2+ protection was observed in NBS- and EDC-mediated inactivation of the DNase but not the RNase activity of Vvn . From these results, it is postulate that there may be two distinct but overlapping active sites, for the DNase and RNase activity, respectively.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 119-136).

Thesis (M.S.)--University of North Carolina at Wilmington, 2004. / Includes bibliographical references (leaves : [25]-27).

The Type 2 Secretion System (T2SS) is responsible for the transport of toxins and enzymes across the outer membrane of many Gram-negative bacteria. A crucial component of the T2SS is a large pore, composed of a multimer of EpsD, named the secretin. This pore inserts in the outer membrane with the assistance of a pilotin (EpsS) or assembly factors (EpsAB), both of which are present within the genome of Vibrio cholerae. The goal of this study was to determine whether or not both assembly mechanisms operate on the same secretin assembly in V. cholerae. Protease deficient mutants generated from an insertion transposon library in V. cholerae epsAB were analyzed. The transposon was found to disrupt the operon encoding VC1702 and epsS. Mutant strains of V. cholerae were constructed or obtained that are deficient in epsA, epsB, epsC, and epsS. Double mutants were constructed that were deficient in epsA and epsS or epsB and epsS. These mutants were tested for assembly of the secretin and secretion of lipase, protease, and cholera toxin. The epsA and epsB mutants have slightly reduced levels of secretion and secretin assembly, while the levels in the pilotin mutant are drastically reduced. The double mutants had little to no assembly, and secretion was reduced to the levels of the control mutant epsC. In an attempt to restore function epsAB was over-expressed in all strains. It successfully complemented the epsA and epsB mutants, and restored levels of secretion to epsS levels in the double mutant, epsAS. In a similar manner to epsAB complementation, epsS was over-expressed. It was found to require the preceding gene VC1702 to complement. The operon, encoding both epsS and VC1702, could complement both epsA and epsS mutations and over-expression increased secretin assembly and secretion to levels greater than wild-type levels. Lastly, a phylogenomic analysis demonstrates that the EpsAB protein complex is found in most orders of the gamma proteobacteria and is ancestral. The pilotins appear to be a late acquisition as they are only found in the family Enterobacteriales.

Bibliography : leaves 147-185. / xi, 185, [6] leaves [16] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1990

Corrigenda pasted onto back end-paper. / Bibliography: leaves 167-189. / 191, [229] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / In this study the wbh region responsible for O-antigen biosynthesis was isolated and partially characterised. The operon appears to be made up of genes that were acquired from other bacteria. The presence of IS1358 indicates that it may have played a role in the acquisition or rearrangement of the polysaccharide biosynthesis genes in V. anguillarum 01. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1999

xiv, 165, xxix leaves : graphs, tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1975

Typescript (photocopy) / 132 leaves, [2] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1982

xiii, 161, xxxiii leaves : ill., tables ; 26 cm / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1975

Corrigenda pasted onto back end-paper. / Bibliography: leaves 167-189. / 191, [229] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / In this study the wbh region responsible for O-antigen biosynthesis was isolated and partially characterised. The operon appears to be made up of genes that were acquired from other bacteria. The presence of IS1358 indicates that it may have played a role in the acquisition or rearrangement of the polysaccharide biosynthesis genes in V. anguillarum 01. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1999