Current Microbiology

Ruiz-Bravo, Alfonso ; Moreno, Encarnacion ; Sampedro, Antonio ; Jimenez-Valera, Maria

doi: 10.1007/PL00006798pmid: 10355112

An isogenic pair of Yersinia enterocolitica serotype O9 strains, with and without virulence plasmid, was used to study the plasmid role in the infection of BALB/c mice by oral, intraperitoneal, and intravenous routes. The plasmid-bearing strain, but not its plasmid-less derivative, caused enteric infection after challenge by all three routes. The virulence plasmid did not influence the peritoneal clearance of yersiniae, but only the plasmid-bearing yersiniae were able to move from the peritoneal cavity to the bloodstream, and thus they spread to spleen and liver. Moreover, plasmid-bearing yersiniae were able to move from the liver to the gallbladder, and they shed in bile into the intestine. Western blot analysis of antibody responses to chromosomally encoded outer membrane proteins revealed similar patterns with sera from mice challenged with each one of the two strains by intraperitoneal route. In contrast, only the plasmid-bearing strain elicited an antibody response to these antigens in mice challenged by oral route. Although mice experimentally infected with plasmid-bearing O9 yersiniae developed an enteric infection, irrespective of the inoculation route, differences between the first steps in infection by oral and parenteral routes may be important, especially when the infection model is used as an approach to study the yersinia-host interactions.

Gérard, Philippe ; Amine, Jamal ; Raval, Guy ; Petitdemange, Henri

doi: 10.1007/PL00006799pmid: 10355113

With PCR methods, the rubredoxin gene was systematically identified among 11 strains of Clostridium butyricum; this ubiquity means major functions in the metabolism of the Clostridia. The 11 PCR products allowed deduction of a sequence of 26 amino acids corresponding to positions 11–36 of the rubredoxin. They all contained the tyrosines at positions 11 and 13 and the phenylalanine at position 30 characteristic of the rubredoxin, but differed at positions 14–17, 20, 25, 29, and 31, allowing determination of three types of rubredoxins among these 11 strains of C. butyricum.

Champlin, Franklin R. ; Patterson, Charles E. ; Austin, Frank W. ; Ryals, Phillip E.

doi: 10.1007/PL00006800pmid: 10355114

The production of serotype A extracellular polysaccharide is thought to be associated with expression of an approximately 40-kDa lipoprotein (Plp-40) present on the outer surface of Pasteurella multocida strains of avian origin. The tendency of certain strains to undergo colonial dissociation concomitantly with serial passaging on laboratory growth media was exploited to derive two variant strains exhibiting the capsule-deficient phenotype from a heavily capsulated parental strain. Assessments of colonial consistency, iridescence, gentian violet binding, and hyaluronidase sensitivity were consistent with cellular observations indicating little or no capsulation of derivative strains. Fluorographic analysis of electrophoretically resolved cellular lipoproteins labeled with [3H]-palmitate revealed capsular loss occurred with a concomitant diminution of Plp-40 production in the variant strains. In contrast, a phenotypically stable strain that did not undergo colonial dissociation under identical conditions exhibited no decrease in Plp-40 content. This work provides a model system for investigating the role of extracellular polysaccharide in the cell surface physiology and pathogenicity of P. multocida. The present results strongly support the notion that Plp-40 is associated with serotype A capsular material and suggest coordinate regulation of their biosynthesis.

Hozbor, D. ; Rodriguez, M.E. ; Fernández, J. ; Lagares, A. ; Guiso, N. ; Yantorno, O.

doi: 10.1007/PL00006801pmid: 10355115

The aim of the study reported here was to investigate the production of Bordetella pertussis outer membrane vesicles (OMVs). Numerous vesicles released from cells grown in Stainer-Scholte liquid medium were observed. The formation of similar vesicle-like structures could also be artificially induced by sonication of concentrated bacterial suspensions. Immunoblot analysis showed that OMVs contain adenylate cyclase-hemolysin (AC-Hly), among other polypeptides, as well as the lipopolysaccharide (LPS). Experiments carried out employing purified AC-Hly and OMVs isolated from B. pertussis AC-Hly− showed that AC-Hly is an integral component of the vesicles. OMVs reported here contain several protective immunogens and might be considered a possible basic material for the development of acellular pertussis vaccines.

Ocaña, Virginia S. ; Pesce de Ruiz Holgado, Aída A. ; Nader-Macías, María E.

doi: 10.1007/PL00006802pmid: 10355116

Lactobacilli are believed to contribute to the control of the vaginal microflora by different mechanisms such as production of antagonistic substances like lactic acid, bacteriocins, and H2O2. This paper describes the selection of H2O2-generating lactobacilli among 35 hydrophobic isolates from the human vagina. Lactobacillus crispatus F117, which generated the highest H2O2 level, was chosen to study: (a) the kinetics of H2O2 production considering different culture conditions, and (b) the effect of this metabolite on the growth of urogenital tract pathogens. The levels of H2O2 in L. crispatus supernatant increased during its growth and were maximum at the early stationary phase (3.29 mmol H2O2L−1) under aerated conditions (agitated cultures). In nonagitated cultures there were no detectable levels of H2O2. L. crispatus F117 spent supernatant inhibited Staphylococcus aureus growth in plaque assay. Inhibition was due to H2O2 since catalase treatment of the supernatant suppressed inhibition. In mixed cultures performed with L. crispatus and S. aureus a significant decrease in pathogen growth was observed. The inhibitory effect depended on the initial inoculum of S. aureus. Further evaluation of the properties of L. crispatus F117 will be performed to consider its inclusion in a probiotic for local use in the vaginal tract.

Peters, Verena ; Janssen, Peter H. ; Conrad, Ralf

doi: 10.1007/PL00006803pmid: N/A

The homoacetogenic bacteria Acetobacterium woodii, A. carbinolicum, Sporomusa ovata, and Eubacterium limosum, the methanogenic archaeon Methanobacterium formicicum, and the sulfate-reducing bacterium Desulfotomaculum orientis all produced formate as an intermediate when they were growing chemolithoautotrophically with H2 and CO2 as sources of energy, electrons, and carbon. The sulfate-reducing bacterium Desulfovibrio vulgaris grew chemolithoheterotrophically with H2 and CO2 using acetate as carbon source, but also produced formate when growth was limited by sulfate. All these bacteria were also able to grow on formate as energy source. Formate accumulated transiently while H2 was consumed. The maximum formate concentrations measured in cultures of A. woodii and A. carbinolicum were proportional to the initial H2 partial pressure, giving a ratio of about 0.5 mM formate per 10 kPa H2. The methanogen Methanobacterium bryantii, on the other hand, was unable to grow on formate and did not produce formate during chemolithoautotrophic growth on H2. The results indicate that the ability to utilize formate, that is, to possess a formate dehydrogenase, was the precondition for the production of formate during chemolithotrophic growth on H2.

Cordier, Hélène ; Lacombe, Christian ; Karst, Francis ; Bergès, Thierry

doi: 10.1007/PL00006804pmid: 10355117

The wild-type ERG19 gene of the yeast Saccharomyces cerevisiae encoding mevalonate diphosphate decarboxylase (MVD) and the mutated recessive erg19-34 allele leading to a decrease of sterol production and to a thermosensitive phenotype have been characterized [2]. The mutated erg19-34 allele bears a single amino acid leucine79-to-proline (L79P) substitution. It was shown that this mutation does not affect the level of production of the enzyme. We performed a two-hybrid assay to show that the yeast Saccharomyces cerevisiae MVD forms homodimers in vivo and that the single point mutation drastically impairs the oligomerization of the protein, thereby explaining the deficiency of MVD activity observed in the temperature-sensitive strain.

Inselman,, Amy L. ; Gathman, Allen C. ; Lilly, Walt W.

doi: 10.1007/PL00006805pmid: N/A

Vacuole-mediated proteolysis is important to sustained growth of filamentous wood-decaying fungi such as Schizophyllum commune. Demonstrating that specific proteases are vacuole associated has been difficult in these organisms due to the lack of specific markers for vacuolar compartments. We used 5-(and 6-)-carboxy-2′, 7′-dichlorofluorescein diacetate (carboxy-DCFDA) and a proprietary vacuolar membrane marker for yeast (MDY-64; Molecular Probes) for in situ fluorescent labeling of the vacuoles of S. commune mycelia grown on microscope slides. MDY-64 labels numerous small vesicles in S. commune mycelia in addition to larger vacuolar structures. In contrast, carboxy-DCFDA apparently is taken up by a subset of the MDY-64-labeled vesicles, accumulating primarily in larger vacuoles. Staining of mycelia with carboxy-DCFDA shows a transition from mostly cytoplasmic fluorescence in apical cells with little vacuolar fluorescence to nearly complete sequestration of the stain in vacuoles of older cells. In penultimate cells, both cytoplasm and vacuolar structures fluoresce. Vacuoles stained with carboxy-DCFDA typically were spherical and ranged in size from 0.4 μm to 3.2 μm in diameter with a mean of 1.8 um. Occasionally, in penultimate cells, tubular structures which stained with carboxy-DCFDA were found. ScPrB, a principal enzyme of nitrogen-limitation induced autolysis in S. commune, copurified in sucrose density gradients with carboxy-DCFDA and acid phosphatase, demonstrating its vacuolar localization.

Merzouki, Mohamed ; Bernet, Nicolas ; Delgenès, Jean-Philippe ; Moletta, René; Benlemlih, Mohamed

doi: 10.1007/PL00006806pmid: 10355118

This paper studies the phosphate uptake by pure cultures of Aeromonas hydrophila, Klebsiella oxytoca, Agrobacterium tumefaciens, and Aquaspirillum dispar in the presence of both nitrate and oxygen. It is shown that species were able to respire both electron acceptors for phosphate accumulation. A. tumefaciens and A. dispar accumulated overall phosphate both in oxic and anoxic culture conditions, whereas A. hydrophila and K. oxytoca eliminated overall phosphate only in oxic conditions. A. dispar was able to remove phosphate by reducing oxygen and nitrate simultaneously with the production of dinitrogen gas. The anoxic denitrification observed in the cultures of adapted and nonadapted cells to nitrate showed that only A. dispar have a denitrification rate superior when the cells were adapted to nitrate.

Clark, Marta A. ; Baumann, Paul ; Moran, Nancy A.

doi: 10.1007/PL00006807pmid: N/A

Buchnera are prokaryotic endosymbionts found in most aphids. One of their functions is the synthesis of the essential amino acid tryptophan for the aphid host. In Buchnera from some aphids that have a long development time, trpEG, which encodes the first enzyme of the tryptophan biosynthetic pathway (anthranilate synthase), is found as one copy on the endosymbiont chromosome and is located between hsIU and fpr. In Buchnera from Schizaphis graminum, which has a short development time, trpEG is amplified on plasmids. We have cloned and sequenced a 4.1-kb DNA fragment from Buchnera of S. graminum and have found the gene order hsIU-ibp-fpr-yjeA-kdtB. The proximity of hsIU and fpr is consistent with the excision, in an endosymbiont ancestor, of trpEG from a location between these two genes, with the excision either followed or preceded by acquisition of ibp.