Anaerobic respiration of fumarate as a differential test betweenCampylobacter fetus andCampylobacter jejuni

The effect of formate and of various electron acceptors—fumarate, aspartate, or nitrate—on the growth of 36 catalase-producingCampylobacter strains was quantitatively investigated in a semisynthetic medium, under aerobic (5% oxygen, 10% carbon dioxide, 85% nitrogen) or anaerobic (10% carbon dioxide, 90% nitrogen) conditions. Under anaerobic conditions, 24 strains ofC. jejuni failed to grow, or grew poorly, in the presence of fumarate, whereas ten strains ofC. fetus subsp.fetus and two strains ofC. fetus subsp.venerealis grew abundantly, rather better than under aerobic conditions. The quantitative differences of growth yields were very significant between the two species with fumarate, but less pronounced with aspartate or nitrate. The activity of fumarate-reductase inC. fetus was substantiated by identification of relevant metabolites by gas liquid chromatography and by mass spectrometry. The anaerobic fumarate respiration in the genusCampylobacter has taxonomic implications.     

Identification of catalase-producingCampylobacter species based on biochemical characteristics and on cellular fatty acid composition

A total of 50 catalase-positive campylobacters from human and animal sources were studied. The nomenclatural type strains ofCampylobacter coli, C. jejuni, C. fetus, andC. fetus subsp.venerealis, a typical strain of the nalidixic acid-resistant thermophilic group, and various clinical isolates were characterized by bacteriological tests and by gas-liquid chromatographic analysis of their cellular fatty acids. The tests most useful in the differentiation of the various catalase-positive species were growth at 25 and 42°C, H₂S production, tolerance to nalidixic acid and to 2,3,5-triphenyltetrazolium chloride, and hippurate hydrolysis. The latter test was the only reliable means to differentiate betweenC. coli andC. jejuni. Differences between.C. fetus andC. jejuni/coli were confirmed by cellular fatty acid compositions. The bacteriological results indicated thatC. fetus andC. jejuni were distinct species, although within the thermophilic campylobacters there were several related taxa that included bothC. coli andC. jejuni strains with typicalC. coli and some thermophilic strains ofC. fetus subsp.fetus at the extremes.     

Organomineralization of cirratulid annelid tubes-fossil and recent examples

Summary The calcareous housing tubes of fossil species of the polychaete wormsDiplochaetetes andDodecaceria from the Tertiary of Lower California (Mexico) are composed, as well as the tubes of recent species ofDodecaceria, by micritic, peloidal lamellae. The wall ofDiplochaetetes spp. consists of only a few, that ofDodecaceria spp. of numerous lamellae. They form a stromatolitic fabric with intercalated lenses of fibrous calcite/aragonite. Histological investigation of the tubes ofDodecaceria showed that the tube formation is related to two processes. The initial process is weakly controlled by the worm itself (matrix mediated). The worm produces acidic organic mucus substances which are enriched between the soft tissue and the tube wall. The entire mucus inhibits the mineralization of the mucus for a certain time. The mineralization events of the mucus are responsible for the stromatolitic microfabric. Within the spaces between the primary lamellae non-specific EPS (extracellular polymeric substances)-rich mucus is enriched, which controls the formation of fibrous aragonitic crystals and peloidal fabrics. This is a very characteristic organomineralization process which is not controlled directly by the organism. The entire process is comparable with those seen in complex rigid modern microbialites from salt and alkaline lakes in Western Australia and Nevada.     

Methyl directed DNA mismatch repair inVibrio cholerae

Mismatches in DNA occur either due to replication error or during recombination between homologous but non-identical DNA sequences or due to chemical modification of bases. The mismatch in DNA, if not repaired, result in high spontaneous mutation frequency. The repair has to be in the newly synthesized strand of the DNA molecule, otherwise the error will be fixed permanently. Three distinct mechanisms have been proposed for the repair of mismatches in DNA in prokaryotic cells and gene functions involved in these repair processes have been identified. The methyl-directed DNA mismatch repair has been examined inVibrio cholerae, a highly pathogenic gram negative bacterium and the causative agent of the diarrhoeal disease cholera. The DNA adenine methyltransferase encoding gene (dam) of this organism which is involved in strand discrimination during the repair process has been cloned and the complete nucleotide sequence has been determined.Vibrio cholerae dam gene codes for a 21.5 kDa protein and can substitute for theEscherichia coli enzyme. Overproduction ofVibrio cholerae Dam protein is neither hypermutable nor lethal both in Escherichia coli andVibrio cholerae. WhileEscherichia coli dam mutants are sensitive to 2-aminopurine,Vibrio cholerae 2-aminopurine sensitive mutants have been isolated with intact GATC methylation activity. The mutator genesmutS andmutL involved in the recognition of mismatch have been cloned, nucleotide sequence determined and their products characterized. Mutants ofmutS andmutL ofVibrio cholerae have been isolated and show high rate of spontaneous mutation frequency. ThemutU gene ofVibrio cholerae, the product of which is a DNA helicase II, codes for a 70 kDa protein. The deduced amino acid sequence of themutU gene hs all the consensus helicase motifs. The DNA cytosine methyltransferase encoding gene (dam) ofVibrio cholerae has also been cloned. Thedcm gene codes for a 53 kDa protein. This gene product might be involved in very short patch (VSP) repair of DNA mismatches. The vsr gene which is directly involved in VSP repair process codes for a 23 kDa protein. Using these information, the status of DNA mismatch repair inVibrio cholerae will be discussed.     

Chemotactic behavior of Campylobacter fetus subspecies towards cervical mucus,bovine placenta and selected substances and ion

The chemotaxis of C. fetus subsp. venerealis and C. fetus subsp. fetus was determined in the presence of bovine cervical mucus and bovine placental extract. Some reported substances and ion in those materials, such amino acids, ferrous iron, hormones, sugars and organic acids were also investigated. Bovine cervical mucus, bovine placenta extracts and some substances and ion of these materials namely L–fucose, L– aspartate, L–glutamate, L–serine, ferrous iron, fumarate, pyruvate and succinate were chemoattractants. The chemottraction was significantly larger in higher concentrations of the tested substances and ion and significant differences among tested strains were also observed. Meso-erythritol and hormones bovine placental lactogen, 17β-estradiol, and progesterone did not elicit chemotactical response. In conclusion, this chemotactic behavior may guide the C. fetus navigation in the bovine host''s genital tract and be an important cofactor of tissue tropism for this bacterium.     

Chemotaxis ofAeromonas hydrophila to the surface mucus of fish

Isolates ofAeromonas hydrophila from various sources show different chemotactic responses to mucus from the surface of freshwater fish. Some isolates were nonchemotactic to fish surface mucus. Isolates ofA. hydrophila from fish lesions had a significantly higher chemotactic index than isolates ofA. hydrophila from water. Maximum chemotactic responses occurred more often to diluted fish mucus than to undiluted samples. Fish which were experimentally stressed did not produce mucus that was more or less chemotactic than that of unstressed fish. Fish with red-sore lesions produced surface mucus which was not chemotactic toA. hydrophila. Differences between fish, for any isolate, were also not significant. The chemotactic substance(s) in fish mucus has a molecular weight of approximately 100,000 and did not appear to be labile when heated to 56°C.     

Genomic diversity of vibrios associated with the Brazilian coral Mussismilia hispida and its sympatric zoanthids (Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi)

Aims: A taxonomic survey of the vibrios associated with the Brazilian endemic coral Mussismilia hispida and the sympatric zoanthids (i.e. Palythoa caribaeorum, Palythoa variabilis and Zoanthus solanderi). Methods and Results: Mucus of 54 cnidarian specimens collected in three different places at São Sebastião in two consecutive years (i.e. 2005 and 2006) was used for taxonomic characterization of the cnidarian microbiota. Ninety‐eight of the 151 vibrio isolates fell within the vibrio core group according to partial 16S rDNA sequences. We performed the sequencing of recA and pyrH genes of all vibrio isolates. The most abundant taxa belonged to the vibrio core group (Vibrio harveyi, Vibrio rotiferianus, Vibrio campbellii and Vibrio alginolyticus), Vibrio mediterranei (=Vibrio shillonii) and Vibrio chagasii. With the exception of V. chagasii which was found only in the mucus of M. hispida, the other species appeared in different hosts with no evidence for the presence of host‐specific clones or species. Using rep‐PCR analysis, we observed a high genomic heterogeneity within the vibrios. Each vibrio isolate generated a different rep‐PCR fingerprint pattern. There was a complete agreement between the grouping based on rep‐PCR and concatenated sequences of pyrH, recA and 16S rDNA, but the pyrH gene has the highest discriminatory power for vibrio species identification. Conclusion: The vibrio core group is dominant in the mucus of these cnidarians. There is a tremendous diversity of vibrio lineages within the coral mucus. pyrH gene sequences permit a clear‐cut identification of vibrios. Significance and Impact of the Study: The taxonomic resolution provided by pyrH (but not recA) appears to be enough for identifying species of vibrios and for disclosing putative new taxa. The vibrio core group appears to be dominant in the mucus of the Brazilian cnidarians. The overrepresentation of these vibrios may reflect as yet unknown ecological functions in the coral holobiont.     

Unique features of the motility and structures in the flagellate polar region of Campylobacter jejuni and other species: an electron microscopic study

Similarly to Helicobacter pylori but unlike Vibrio cholerae O1/O139, Campylobacter jejuni is non‐motile at 20°C but highly motile at ≥37°C. The bacterium C. jejuni has one of the highest swimming speeds reported (>100 μm/s), especially at 42°C. Straight and spiral bacterial shapes share the same motility. C. jejuni has a unique structure in the flagellate polar region, which is characterized by a cup‐like structure (beneath the inner membrane), a funnel shape (opening onto the polar surface) and less dense space (cytoplasm). Other Campylobacter species (coli, fetus, and lari) have similar motility and flagellate polar structures, albeit with slight differences. This is especially true for Campylobacter fetus, which has a flagellum only at one pole and a cup‐like structure composed of two membranes.     

Genomic analysis of Campylobacter fetus subspecies: identification of candidate virulence determinants and diagnostic assay targets

Background  

Campylobacter fetus subspecies venerealis is the causative agent of bovine genital campylobacteriosis, asymptomatic in bulls the disease is spread to female cattle causing extensive reproductive loss. The microbiological and molecular differentiation of C. fetus subsp. venerealis from C. fetus subsp. fetus is extremely difficult. This study describes the analysis of the available C. fetus subsp. venerealis AZUL-94 strain genome (~75–80%) to identify elements exclusively found in C. fetus subsp. venerealis strains as potential diagnostic targets and the characterisation of subspecies virulence genes.     

New molecular microbiology approaches in the study of Campylobacter fetus

Campylobacter fetus infection is a substantial problem in herds of domestic cattle worldwide and a rising threat in human disease. Application of comparative and functional genomics approaches will be essential to understand the molecular basis of this pathogen's interactions with various hosts. Here we report recent progress in genome analyses of C. fetus ssp. fetus and C. fetus ssp. venerealis, and the development of molecular tools to determine the genetic basis of niche‐specific adaptations. Campylobacter research has been strengthened by the rapid advancements in imaging technology occurring throughout microbiology. To move forward in understanding the mechanisms underlying C. fetus virulence, current efforts focus on developing suitable in vitro models to reflect host‐ and tissue‐specific aspects of infection.     

The human gastric pathogen Helicobacter pylori has a gene encoding an enzyme first classified as a mucinase in Vibrio cholerae

The human bacterial pathogen Helicobacter pylori has been suggested to be the causative agent of the most common chronic infection of man. Since its first isolation in 1982, H. pylori has been associated with gastric and duodenal ulcer disease, and more recently, gastric cancer. The proteolytic digestion of gastric mucus by this microorganism has been suggested as an important mechanism by which its pathogenicity is at least partly exerted. Here we report the detection of protease activity in H. pylori total-cell and supernatant extracts. On the basis that zinc metalloproteases are common microbial pathogenicity factors, we identified a single protein in H. pylori protein extracts with antibodies to the Pseudomonas aeruginosa elastase (a secreted zinc metallo-protease). This same protein was identified by pooled serum from patients infected with H. pylori. We used the functional and immunological relationship between the P. aeruginosa elastase and the Vibrio cholerae haemagglutinin/protease (HAP) to clone the H. pylori hap gene, which was over 99% similar to the V. cholerae hap gene in the coding region. A 4 kb DNA fragment containing the entire cloned gene was highly unstable in Escherichia coli and Bacillus subtilis cloning vectors. We also demonstrated that a hap-like gene sequence is present in ail nine Helicobacter species so far discovered. The V. cholerae HAP was first classified on the basis of its mucinase activity.     

Separation of Cell Types of Vibrio fetus

Separation of the coccoid form of Vibrio fetus from cultures containing both the coccoid and vibrioid cell types has been accomplished using rate-zonal centrifugation.     

Morphological Differences in Flagella in Campylobacter fetus Subsp. intestinalis and C. fetus Subsp. jejuni

Intact flagella were isolated from human pathogenic strains of Campylobacter, C. fetus subsp. intestinalis and C. fetus subsp. jejuni, by the method of DePamphilis and Adler and examined by electron microscopy. The isolated flagella were composed of a filament, a hook, a basal body, and a large disk associated with the end of the hook region covering the basal body. The width of the hook was approximately 28 nm, somewhat greater than that of the filament (20 nm in diameter). The hook region of C. fetus subsp. intestinalis was curved, but it was straight in C. fetus subsp. jejuni. The structure of the basal body of the two subspecies was similar to that reported for other gram-negative bacteria. The large disk detached from the flagella showed concentrically arranged circular structures. This structure was more clearly observed in the disk of C. fetus subsp. jejuni than in C. fetus subsp. intestinalis. Observations of thin-sectioned profiles at the attachment site of the flagellum revealed that the large disk is located on the inner side of the outer membrane. The role of the large disk in bacterial movement is not clear, but it is assumed that it acts as an organ to protect the flagellar insertion site from vigorous rotation of the polar end inflicted during bacterial movement.     

Transformation ofVibrio vulnificus by electroporation

Vibrio vulnificus is a pathogenic, estuarine bacterium associated with primary septicemia and severe wound infections. Previous studies have reported limited success in the introduction of plasmids intoV. vulnificus by conjugation, and there have been no reports of successful electroporation procedures. We report here on the introduction of recombinant plasmids into four strains ofV. vulnificus by electroporation. In contrast to the complex medium that others have used for the electroporation ofVibrio cholerae, the efficiency of transformation ofV. vulnificus was greatly improved by the use of a defined growth medium containing glucose. The addition of glycine betaine further enhanced transformant yield.     

Comparative <Emphasis Type="Italic">in silico</Emphasis> analysis of chemotaxis system of <Emphasis Type="Italic">Campylobacter fetus</Emphasis>

Chemoreceptor and chemotaxis signal transduction cascade genes of C. fetus subsp. fetus 82-40 show high level of similarity to that in C. jejuni and appears to include sixteen diverse transducer-like protein (tlp) genes that appear similar to nine of the twelve tlp genes in the C. jejuni NCTC 11168 with a percent identity ranging from 15 to 50%. Sixteen putative C. fetus 82-40 tlp genes belong to three classes: A, B, and C, as well as an aerotaxis gene, based on their predicted structure. C. fetus subsp. fetus 82-40 chemoreceptor and chemotaxis signal transduction pathway genes have close phylogenetic relationship of chemotaxis genes between Campylobacteraceae and Helicobacteraceae.     

Characterization of Vibrios Diversity in the Mucus of the Polychaete Myxicola infundibulum (Annellida,Polichaeta)

Vibrios are among the most abundant culturable microbes in aquatic environments. They can be either free-living in the water column or associated with several marine organisms as mutualists, saprophytes, or parasites. In the present study we analysed vibrios abundance and diversity in the mucus of the polychaete Myxicola infundibulum, complementing culture-based with molecular methods. Vibrios reached 4.6?×?10³ CFU mL^?1 thus representing a conspicuous component of the heterotrophic culturable bacteria. In addition, luminous vibrios accounted for about 60 % of the total culturable vibrios in the mucus. The isolates were assigned to: Vibrio gigantis, Vibrio fischeri, Vibrio jasicida, Vibrio crassostreae, Vibrio kanaloae, and Vibrio xuii. Two Vibrio isolates (MI-13 and MI-15) may belong to a new species. We also tested the ability of the Vibrio isolates to grow on M. infundibulum mucus as the sole carbon source. All strains showed appreciable growth in the presence of mucus, leading us to conclude that this matrix, which is abundant and covers the animal entirely, may represent a microcosm and a food source for some bacteria, playing a crucial role in the structuring of a mucus-associated beneficial microbial community. Moreover, the trophic relationship between vibrios and M. infundibulum mucus could be enhanced by the protection that mucus offers to vibrios. The results of this study represent a contribution to the growing evidence for complex and dynamic invertebrate-microbe associations present in nature and highlight the importance of exploring relationships that Vibrio species establish with marine invertebrates.     

Molecular mechanisms of Campylobacter fetus surface layer protein expression

Cells of the Gram-negative bacteria Campylobacter fetus are covered by monomolecular arrays of surface layer proteins (SLPs) critical for both persistence in their natural hosts and for virulence. For C. fetus cells, expression of SLPs essentially eliminates C3b binding and their antigenic variation thwarts host immunological defences. Each cell possesses multiple partially homologous and highly conserved SLP gene cassettes, tightly clustered in the genome, that encode SLPs of 97–149 kDa. These attach non-covalently via a conserved N-terminus to the cell wall lipopolysaccharide. Recent studies indicate that C. fetus reassorts a single promoter, controlling SLP expression, and one, or more, complete open reading frame strictly by DNA inversion, and that rearrangement is independent of the distance between sites of inversion. In contrast to previously reported programmed DNA inversion systems, inversion in C. fetus is recA- dependent. These rearrangements permit variation in protein expression from the family of SLP genes and suggest an expanding paradigm of programmed DNA rearrangements among microorganisms.     

Construction of shuttle vectors for cloning inVibrio cholerae andEscherichia coli

Starting from a naturally occurring cryptic plasmid pVC540 ofVibrio cholerae non-OI. strain 1095, a number of plasmid vectors have been constructed for cloning genes inVibrio cholerae by introducing antibiotic resistance markers containing a set of unique cloning sites. The constructs pVC810 and pVE920 have the origins of bothVibrio cholerae andEscherichia coli replicons and are stable in both organisms in the absence of selective pressure. These plasmids can serve as shuttle vectors betweenEscherichia coli andVibrio cholerae. The plasmid vectors reported here along with the demonstration of transformation inVibrio cholerae by plasmid DNA will facilitate genetic analysis of this important human pathogen.     

Evidence for two newVibrio anguillarum K antigens

Surface polysaccharides from five strains ofVibrio anguillarum were studied by means of immunoelectrophoretic procedures. The study suggested existence of two new K antigens, displaying cross-reactivity, in strains derived from diseased feral fish. The importance of a detailed serologic characterization of isolates for ecologic and epidemiologic studies ofV. anguillarum is considered.     

Diagnosis of trichomoniasis in pregnant cows by culture of cervical-vaginal mucus

The objective of this study was to evaluate the effectiveness of Cervical-Vaginal Mucus (CVM) cultures for isolating $\text{Trichomonas fetus}$ from pregnant cows. Cervical-vaginal mucus was cultured from sixty-eight cows averaging 98 days of gestation. $\text{Trichomonas fetus}$ was isolated from 7 of 36 cows in groups exposed to bulls found to be infected at the end of the breeding season. The remaining 32 cows which were bred to bulls found not to be infected at the end of the breeding season were negative. Seven of the cows serviced by infected bulls aborted. Three of these had been positive and 4 negative on culture. The incidence of abortion was determined by examination 81 days after the previous examination and CVM culture. During the same interval there were no abortions in the 32 cows served by non-infected bulls. Culture of CVM identified infected groups of cows which had a high risk for abortion.