Bacterial γ-glutamyltranspeptidases: Comparison of Escherichia coli and Pseudomonasγ-glutamyltranspeptidase

Serpulina (Treponema) hyodysenteriae strain A-1 partially purified rRNA, labelled with photobiotin, was used as a non-radioactive probe to identify the rRNA gene restriction patterns of S. hyodysenteriae strains and other spirochetes. Sau3A restriction enzyme digests resulted in similar rRNA gene restriction patterns in S. hyodysenteriae strains from five different countries. Some S. hyodysenteriae strains could be differentiated by variations in their rRNA gene restriction patterns after cleavage of DNA by restriction enzymes SspI or BglII. S. innocens and Treponema succinifaciens, non-pathogenic pig intestinal spirochetes, had rRNA gene restriction patterns that differed markedly from the S. hyodysenteriae patterns, and from each other.     

Production of an inducible sucrase activity by Serpulina hyodysenteriae.

Strains of Serpulina hyodysenteriae and Serpulina innocens produced a cell-associated sucrase activity when grown in a medium containing sucrose. S. hyodysenteriae B204 sucrase activity cleaved sucrose and, to a lesser extent, raffinose and had a pH optimum of 5.7 to 6.2. This is the first report of an inducible enzyme produced by either S. hyodysenteriae or S. innocens.     

Detection of same sized 4.3 Kb extrachromosomal DNA elements in weakly beta-haemolytic human intestinal spirochaetes and Serpulina pilosicoli of swine origin

Agarose gel electrophoresis of total DNA from Italian strains of weakly beta-haemolytic human intestinal spirochaetes (w beta HIS) and porcine Serpulina pilosicoli reference strain P43/6/78 showed an extrachromosomal band having the same size and migrating at 4.3 Kb. The same results were observed after agarose gel electrophoresis of DNA obtained from the supernatant fluids of the spirochaetal cultures analysed. Swine Serpulina hyodysenteriae reference strain P18A was comparatively analysed and a 6.5 Kb extrachromosomal DNA element was found, as expected. Furthermore, S. hyodysenteriae reference strain P18A differed from all the other spirochaetes tested and had a higher number of flagella (8-12) at each cell end and was strongly beta-haemolytic. To the best of our knowledge, this is the first report on the detection of a band of extrachromosomal DNA having the same size in w beta HIS and S. pilosicoli from swine origin.     

Proposal to change the genus designation Serpula to Serpulina gen. nov. containing the species Serpulina hyodysenteriae comb. nov. and Serpulina innocens comb. nov.

The bacterial genus Serpula Stanton et al. 1991 is illegitimate due to the existence of a fungal genus Serpula Pers. ex S. F. Gray. Consequently, a new genus designation, Serpulina, is proposed for this spirochete genus. Serpula hyodysenteriae, the type species, and Serpula innocens Stanton et al. 1991, therefore, become Serpulina hyodysenteriae comb. nov. and Serpulina innocens comb. nov.     

A species-specific periplasmic flagellar protein of Serpulina (Treponema) hyodysenteriae.

We have previously reported that a 46-kDa protein present in an outer membrane protein preparation seemed to be a species-specific antigen of Serpulina hyodysenteriae (Z. S. Li, N. S. Jensen, M. Bélanger, M.-C. L'Espérance, and M. Jacques, J. Clin. Microbiol. 30:2941-2947, 1992). The objective of this study was to further characterize this antigen. A Western blot (immunoblot) analysis and immunogold labeling with a monospecific antiserum against this protein confirmed that the protein was present in all S. hyodysenteriae reference strains but not in the nonpathogenic organism Serpulina innocens. The immunogold labeling results also indicated that the protein was associated with the periplasmic flagella of S. hyodysenteriae. N-terminal amino acid sequencing confirmed that the protein was in fact a periplasmic flagellar sheath protein. The molecular mass of this protein, first estimated to be 46 kDa by Western blotting, was determined to be 44 kDa when the protein was evaluated more precisely by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein was glycosylated, as determined by glycoprotein staining and also by N-glycosidase F treatment. Five other periplasmic flagellar proteins of S. hyodysenteriae, which may have been the core proteins and had molecular masses of 39, 35, 32, 30, and 29 kDa, were antigenically related and cross-reacted with the periplasmic flagellar proteins of S. innocens. Finally, serum from a pig experimentally infected with S. hyodysenteriae recognized the 44-kDa periplasmic flagellar sheath protein. Our results suggest that the 44-kDa periplasmic flagellar sheath protein of S. hyodysenteriae is a species-specific glycoprotein antigen.     

Identification and genetic fingerprinting of Brachyspira species

Six Brachyspira type and reference strains, and 14 well characterized porcine field isolates representing all recognised porcine Brachyspira spp. were compared by different molecular methods. Sequence analysis of the 16S rRNA and the nox genes, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) were used in the study. In addition the isolates were analysed by five species-specific PCR systems. The topologies of the dendrograms obtained from each of the four typing systems were different. The B. pilosicoli isolates formed monophyletic clusters in all dendrograms, but with different sister lines. All five porcine Brachyspira species formed monophyletic clusters in the nox gene-based dendrogram only. All five PCR systems accurately identified their targets, except for the nox gene-based B. intermedia-specific system, by which it was not possible to identify one of the presumed B. intermedia isolates, and the other B. intermedia-specific system, based on the 23S rRNA gene, gave a positive reaction for one B. innocens isolate. In an extended study, 46 additional isolates and the original eight isolates with the phenotypes of B. hyodysenteriae or B. intermedia were compared by PFGE and PCR. The PFGE results indicated a high genetic diversity of isolates with the phenotype of B. intermedia. Thirty-three of 34 tested isolates could be identified by one or both of the two B. intermedia-specific PCR systems used, however, only 19 of the 34 isolates were positive in both systems.     

Reclassification of Treponema hyodysenteriae and Treponema innocens in a new genus, Serpula gen. nov., as Serpula hyodysenteriae comb. nov. and Serpula innocens comb. nov.

The intestinal anaerobic spirochetes Treponema hyodysenteriae B78T (T = type strain), B204, B169, and A-1, Treponema innocens B256T and 4/71, Treponema succinifaciens 6091T, and Treponema bryantii RUS-1T were compared by performing DNA-DNA reassociation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell proteins, restriction endonuclease analysis of DNA, and 16S rRNA sequence analysis. DNA-DNA relative reassociation experiments in which the S1 nuclease method was used showed that T. hyodysenteriae B78T and B204 had 93% sequence homology with each other and approximately 40% sequence homology with T. innocens B256T and 4/71. Both T. hyodysenteriae B78T and T. innocens B256T exhibited negligible levels of DNA homology (less than or equal to 5%) with T. succinifaciens 6091T. The results of comparisons of protein electrophoretic profiles corroborated the DNA-DNA reassociation results. We found high levels of similarity (greater than or equal to 96%) in electrophoretic profiles among T. hyodysenteriae strains, moderate levels of similarity (43 to 49%) between T. hyodysenteriae and T. innocens, and no detectable similarity between the profiles of either T. hyodysenteriae or T. innocens and those of T. succinifaciens, T. bryantii, and Escherichia coli. Restriction endonuclease analysis of DNA was not useful in assessing genetic relationships since there was heterogeneity even between strains of T. hyodysenteriae. Partial 16S rRNA sequences of the intestinal spirochetes were determined by using a modified Sanger method and were compared in order to evaluate the phylogenetic relationships among these and other spirochetes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic basis of macrolide and lincosamide resistance in Brachyspira (Serpulina) hyodysenteriae

Macrolide antibiotic resistance is widespread among Brachyspira hyodysenteriae (formerly Serpulina hyodysenteriae) isolates. The genetic basis of macrolide and lincosamide resistance in B. hyodysenteriae was elucidated. Resistance to tylosin, erythromycin and clindamycin in B. hyodysenteriae was associated with an A-->T transversion mutation in the nucleotide position homologous with position 2058 of the Escherichia coli 23S rRNA gene. The nucleotide sequences of the peptidyl transferase region of the 23S rDNA from seven macrolide and lincosamide resistant and seven susceptible strains of Brachyspira spp. were determined. None of the susceptible strains were mutated whereas all the resistant strains had a mutation in position 2058. Susceptible strains became resistant in vitro after subculturing on agar containing 4 micrograms ml-1 of tylosin. Sequencing of these strains revealed an A-->G transition mutation in position 2058.     

Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons

Abstract Multilocus enzyme electrophoresis (MEE) analysis and comparisons of nearly complete 16S rRNA gene sequences (1416 nucleotide positions) were used to evaluate phylogenetic relationships among Serpulina hyodysenteriae strain B78^T, S. innocens strain B256^T, Brachyspira aalborgi strain 513A^T, and eight uncharacterised strains of swine, avian, and human intestinal spirochaetes. From MEE analysis, nine strains could be assigned to five groups containing other intestinal spirochaetes ( genetic distances between groups = 0.6–0.9). Chicken spirochaete strain C1 and B. aalborgi 513A^T represented unique electrophoretic types and formed their own MEE groups. Despite MEE differences, the 11 strains had highly similar (96.3–99.9%) 16S rRNA sequences. These findings point out limitations of both MEE analysis and 16S rRNA sequence comparisons when used as solitary techniques for classifying intestinal spirochaetes related to Brachyspira/ Serpulina species.     

Molecular analysis of isolates of Streptococcus suis capsular type 2 by restriction-endonuclease-digested DNA separated on SDS-PAGE and by hybridization with an rDNA probe.

This study was undertaken to assess the discriminatory value of restriction endonuclease (RE) digestion patterns of Streptococcus suis chromosomal DNA using polyacrylamide gel electrophoresis (SDS-PAGE) and DNA-rDNA hybridization. For the RE digestion patterns, DNAs were digested separately with the enzymes BamHI and BglII and the resultant fragments were separated by SDS-PAGE. An Escherichia coli rDNA probe derived from pKK3535 was used for the hybridization. Twenty-three S. suis capsular type 2 isolates recovered from diseased and clinically healthy pigs, from a human case, and from a cow were compared in this study. The majority of isolates associated with septicaemia belonged to one restriction endonuclease analysis (REA) profile group. Isolates associated with pneumonia belonged either to the REA profile group of isolates associated with septicaemia or to a second REA profile group. The REA profiles of isolates from clinically healthy animals were more heterogeneous. The REA profile of the type 2 reference strain, S735, which was originally isolated from a pig, was very different from those of the porcine and bovine isolates but similar to the profile of the human isolate. The profiles obtained after rDNA hybridization were more homogeneous. Although different patterns were detected in the 23 isolates, there was no correlation between the source of the isolate and the patterns observed with this technique.     

Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae.

Serpulina hyodysenteriae B204 cells treated with mitomycin (20 microg of mitomycin/ml of culture broth) lysed and released bacteriophages. Bacteriophage particles, precipitated by using polyethylene glycol and purified by CsC1 density gradient ultracentrifugation, had a buoyant density of 1.375 g/cm3 and consisted of a head (45-nm diameter) and an ultrastructurally simple (noncontractile) tail (64 by 9 nm) composed of at least 13 proteins with molecular masses ranging between 13 and 101 kDa. The purified bacteriophage has been designated VSH-1 (VSH for virus of S. hyodysenteriae). VSH-1 was incapable of lytic growth on any of five intestinal spirochete strains, representing three Serpulina species. VSH-1 nucleic acid was determined to be approximately 7.5 kb in size and to be linear, double-stranded DNA based on differential staining with acridine orange, DNase I sensitivity, electrophoretic mobility, and contour length as measured by electron microscopy. Phage DNA digested by the restriction enzymes SspI, AseI, EcoRV, and AflII gave electrophoretic banding patterns nearly identical to those of digested chromosomal DNA from S. hyodysenteriae. Additionally, VSH-1 DNA fragments hybridized with probes complementary to S. hyodysenteriae chromosomal genes nox and flaA1. When purified bacteriophages induced from cultures of S. hyodysenteriae A203 (deltaflaA1 593-762::cat) were added to growing cells of strain A216 (deltanox 438-760::kan), transductants (Cmr Kmr) were obtained at a frequency of 1.5 x l0(-6) per phage particle (enumerated by electron microscopy). These findings indicate that induced VSH-1 virions package DNA of S. hyodysenteriae and are capable of transferring host genes between cells of that spirochete. To our knowledge, this is the first report of genetic transduction of a spirochete.     

Detection of bacteriophage VSH-1 svp38 gene in Brachyspira spirochetes

VSH-1 is a mitomycin C-inducible, non-lytic, phage-like agent that packages random 7.5-kb fragments of the Brachyspira hyodysenteriae genome. VSH-1 is the first recognized mechanism for gene transfer between B. hyodysenteriae cells. To analyze the distribution of VSH-1 among spirochetes, a 344-bp probe for gene svp38, encoding the VSH-1 major head protein, was amplified by polymerase chain reaction and used in Southern blot hybridizations with genomic DNA from various spirochete genera. The svp38 probe hybridized to a 40-kb SalI-SmaI fragment of the B. hyodysenteriae B78(T) chromosome, indicating VSH-1 DNA insertion into the chromosome at a unique site. Restriction endonuclease digested DNAs of 27 spirochete strains representing six Brachyspira species (B. hyodysenteriae, B. innocens, B. pilosicoli, B. murdochii, B. intermedia, B. alvinipulli) contained a single fragment hybridizing with the svp38 probe. DNAs from spirochete species of the genera Treponema, Spirochaeta, Borrelia, and Leptospira did not hybridize with the probe. VSH-1-like agents appear to be widely distributed among Brachyspira species and, as has been demonstrated for B. hyodysenteriae, may serve as useful gene transfer agents for those other species.     

Extracellular DNA from Serpulina hyodysenteriae consists of 6.5 kbp random fragments of chromosomal DNA

Preparations of chromosomal DNA from a number of Serpulina hyodysenteriae strains have shown, using agarose gel electrophoresis, the presence of an additional band with a mobility similar to that of a 6.5 kbp linear DNA fragment. Analysis showed that this is not a plasmid but rather a form of extracellular DNA like that observed for Gram-negative bacteria. However, unlike the extracellular DNA from Gram-negative bacteria, which showed a similar band profile to that of the DNA from whole cells, that from S. hyodysenteriae consisted primarily of fragments of a fixed 6.5 kbp.     

Production and characterisation of a monoclonal antibody to Serpulina hyodysenteriae

Abstract A monoclonal antibody (mAb) directed against Serpulina hyodysenteriae , the causative agent of swine dysentery, was produced and characterised. The mAb (BJL/SH1) reacted in Western blots with a protein with a molecular mass of about 30 kDa in outer membrane preparations from a range of S. hyodysenteriae isolates of different serotypes. It did not react with preparations made from a variety of non- S. hyodysenteriae intestinal spirochaetes. Immunogold labelling was used to confirm the location of the reactive epitope on the cell outer membrane. The mAb agglutinated and produced fluorescence only with strains of S. hyodysenteriae , and should prove to be a useful reagent for identification of S. hyodysenteriae .     

Physical and genetic map of the Serpulina hyodysenteriae B78T chromosome.

A combined physical and genetic map of the Serpulina hyodysenteriae B78T genome was constructed by using pulsed-field gel electrophoresis and DNA blot hybridizations. The S. hyodysenteriae genome is a single circular chromosome about 3.2 Mb in size. The physical map of the chromosome was constructed with the restriction enzymes BssHII, EclXI, NotI, SalI, and SmaI. The physical map was used to constructed a linkage map for genes encoding rRNA, flagellum subunit proteins, DNA gyrase, NADH oxidase, and three distinct hemolysins. Several flaB2-related loci, encoding core flagellum subunit proteins, were detected and are dispersed around the chromosome. The rRNA gene organization in S. hyodysenteriae is unusual. S. hyodysenteriae has one gene each for 5S (rrf), 16S (rrs), and 23S (rrl) rRNAs. The rrf and rrl genes are closely linked (within 5 kb), while the rrs gene is about 860 kb from the other two rRNA genes. Using a probe for the S. hyodysenteriae gyrA gene, we identified a possible location for the chromosomal replication origin. The size and genetic organization of the S. hyodysenteriae chromosome are different from those of previously characterized spirochetes.     

Hybridization analysis of the gene encoding a hemolysin (suilysin) of Streptococcus suis type 2: evidence for the absence of the gene in some isolates

A hemolysin gene was cloned from a virulent strain of Streptococcus suis type 2 strain 1933. Analysis of the gene and its product revealed that it is identical to a previously reported hemolysin (suilysin) of S. suis type 2. Southern hybridization analysis of the digested total genomic DNA from S. suis with the cloned hemolysin DNA sequences as probe indicated that the hemolysin gene is present as a single copy on the genome. Genomic DNA of 63 isolates of S. suis encompassing all known serotypes were examined by DNA hybridization and polymerase chain reaction (PCR) studies for the presence of the hemolysin gene homolog. The results of both techniques were identical and demonstrated the absence of the hemolysin gene in some isolates. In DNA hybridization studies, three DNA probes derived from the hemolysin encoding gene were used. Results showed that sequences encoding the C-terminal 257 amino acid residues (Probe 1) were the most conserved and hybridized to a 1.2 kb fragment in 32 (51%) strains and a 4.0 kb fragment in 23 (36%) strains respectively. Thus, Probe 2 hybridized to the DNA of 55 (87%) of the isolates tested. The first probe (Probe 1) comprising almost the entire hemolysin gene and the third probe (Probe 3) which consisted of the N-terminal sequences hybridized only to a 4.0 kb fragment in 23 (36%) of the strains tested. Eight (13%) of the strains tested were hybridization and PCR negative. The hybridization of the C-terminal end sequences (Probe 2) to the 1.2 kb fragment in 32 (51%) of the strains and the lack of hybridization of the probes to eight (13%) strains may suggest the presence of different types of hemolysin molecule in S. suis strains.     

Characterization of Candida krusei strains from spontaneously fermented maize dough by profiles of assimilation, chromosome profile, polymerase chain reaction and restriction endonuclease analysis

Several isolates of Candida krusei from indigenous spontaneously fermented maize dough have been characterized for the purpose of selecting appropriate starter cultures and methods for their subspecifies typing. The present work describes the occurrence of C. krusei in Ghanaian fermented maize dough. For detailed pheno- and genotyping, 48 representative isolates were selected and comparison was made with clinical isolates of C. krusei and reference cultures. The techniques applied included the assimilation of carbon compounds by the API ID 32 C kit, determination of chromosome profile by pulse field gel electrophoresis, polymerase chain reaction (PCR) profiles, restriction endonuclease analysis (REA) and Southern blot hybridization. For the 48 isolates tested, 82% had the same assimilation profiles, being able to assimilate N-acetyl-glucosamine, DL-lactate, glycerol and to ferment glucose. Chromosome and PCR profiles, REA and Southern blot hybridization techniques all had a high discriminatory power and revealed DNA polymorphism, which allowed for discrimination among the strains and hence subspecific typing. On the basis of PCR and REA profiles, isolates were grouped into clusters. Southern blot hybridization appeared to be the most sensitive with respect to strain specificity. Our results demonstrated that the three methods, PCR, REA and Southern blot hybridization, were suitable tools, easy to analyse, fast (with regard to PCR) and reliable methods for the typing of C. krusei isolates to species and below species level. Based on the use of these techniques, we demonstrated that several strains of C. krusei were involved in the fermentation of maize dough from the onset and remain dominant throughout the fermentation.     

Differentiation of Coxiella burnetii isolates by analysis of restriction-endonuclease-digested DNA separated by SDS-PAGE.

Thirty-two isolates of Coxiella burnetii collected from various hosts ranging from arthropods to man were compared by restriction endonuclease (RE) digestion patterns of chromosomal DNA using SDS-PAGE. SDS-PAGE provided better DNA fragment separation than agarose gel electrophoresis and enabled the differentiation of these isolates into six distinct groups on the basis of DNA restriction fingerprints. Two groups of chronic disease isolates could be distinguished, each having unique RE digestion patterns of chromosomal DNA. Three similar but distinct RE digestion patterns were seen among the group of acute disease isolates. Three additional isolates included in this study exhibited a unique RE digestion pattern and also had a unique plasmid type, designated QpDG. DNA-DNA hybridization on selected isolates quantified the relatedness between several groups and supported the classification of these groups as distinct strains.     

Characterization of Lactobacillus sake isolates from dry-cured sausages by restriction fragment length polymorphism analysis of the 16S rRNA gene

Lactobacillus sake strains originally isolated from dry-fermented sausages were characterized by phenotypic and genotypic methods, including DNA-DNA hybridization, restriction fragment length polymorphism (RFLP), and 16S rDNA sequencing analysis, in order to establish their taxonomic position and relation to well defined reference species. Initially, isolates of Lact. sake showing a characteristic phenotype (melibiose-positive, maltose- and arabinose-negative) were identified by DNA-DNA hybridization. Subsequently, RFLP studies using Eco RI and Hin dIII as restriction enzymes, and cDNA from Escherichia coli or 16S rDNA from Lact. sake strains as probes, showed distinct polymorphism levels. Thus, Eco RI-digested DNA probed with cDNA from E. coli disclosed the presence of a unique cluster for the meat isolates tested, allowing their differentiation from the reference type strain. When Hin dIII-digested DNA was hybridized with the cDNA probe, strain-specific patterns were obtained, showing a higher discrimination power. Considerable strain differentiation was also observed when Eco RI and Hin dIII digests were hybridized with 16S rDNA probes. Finally, sequence analysis of the 16S rDNA from one isolate also revealed a certain degree of genetic variability with respect to the reference strain of Lact. sake .