Inter- and intraspecies variations of the 16S-23S rDNA intergenic spacer region of various streptococcal species

The 16S-23S rDNA intergenic spacer regions (ISR) of different streptococcal species and subspecies were amplified with primers derived from the highly conserved flanking regions of the 16S rRNA and 23S rRNA genes. The single sized amplicons showed a uniform pattern for S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis, S. phocae, S. uberis, S. parauberis, S. pyogenes and S. equi subsp. equi, respectively. The amplicons of S. equi subsp. zooepidemicus, S. porcinus and S. suis appeared with 3, 5 and 3 different sizes, respectively. ISR of selected strains of each species or subspecies investigated were sequenced and multiple aligned. This allowed a separation of ISR into regions, with 7 regions for S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis, S. phocae, S. pyogenes and S. suis, 8 regions for S. uberis and S. parauberis and mostly 9 regions for S. equi subsp. equi, S. equi subsp. zooepidemicus and S. porcinus. Region 4, encoding the transfer RNA for alanine (tRNA(Ala)), was present and identical for all isolates investigated. The size and sequence of ISR appears to be a unique marker for streptococci of various species and subspecies and could be used for bacterial identification. In addition the size and sequence variations of ISR of S. equi subsp. zooepidemicus, S. porcinus and S. suis allows a molecular typing of isolates of these species possibly useful in epidemiological aspects.     

Diversity and mobility of integrative and conjugative elements in bovine isolates of Streptococcus agalactiae, S. dysgalactiae subsp. dysgalactiae, and S. uberis

Bovine isolates of Streptococcus agalactiae (n = 76), Streptococcus dysgalactiae subsp. dysgalactiae (n = 32), and Streptococcus uberis (n = 101) were analyzed for the presence of different integrative and conjugative elements (ICEs) and their association with macrolide, lincosamide, and tetracycline resistance. The diversity of the isolates included in this study was demonstrated by multilocus sequence typing for S. agalactiae and pulsed-field gel electrophoresis for S. dysgalactiae and S. uberis. Most of the erythromycin-resistant strains carry an ermB gene. Five strains of S. uberis that are resistant to lincomycin but susceptible to erythromycin carry the lin(B) gene, and one has both linB and lnuD genes. In contrast to S. uberis, most of the S. agalactiae and S. dysgalactiae tetracycline-resistant isolates carry a tet(M) gene. A tet(S) gene was also detected in the three species. A Tn916-related element was detected in 30 to 50% of the tetracycline-resistant strains in the three species. Tetracycline resistance was successfully transferred by conjugation to an S. agalactiae strain. Most of the isolates carry an ICE integrated in the rplL gene. In addition, half of the S. agalactiae isolates have an ICE integrated in a tRNA lysine (tRNA(Lys)) gene. Such an element is also present in 20% of the isolates of S. dysgalactiae and S. uberis. A circular form of these ICEs was detected in all of the isolates tested, indicating that these genetic elements are mobile. These ICEs could thus also be a vehicle for horizontal gene transfer between streptococci of animal and/or human origin.     

Identification of streptococci isolated from various sources by determination of cfb gene and other CAMP-factor genes

In the present study, the CAMP-factor (cfb) gene of streptococci of serological group B (Streptococcus agalactiae) and the CAMP-factor (cfu) gene of S. uberis could be amplified by polymerase chain reaction. A cfb specific amplicon could be observed for all 128 phenotypically CAMP-positive S. agalactiae, for the phenotypically CAMP-negative S. agalactiae strain 74-360, and for 2 S. difficile reference strains. A cfu specific amplicon could be observed for all 7 phenotypically CAMP-positive S. uberis. Four S. agalactiae strains isolated from 4 cows with mastitis appeared to be phenotypically CAMP-negative and negative in the cfb gene PCR. The CAMP-positive and CAMP-negative isolates, including both S. difficile, could be identified as S. agalactiae by amplification of a S. agalactiae specific part of the V2 region of the 16S rRNA and a species-specific part of the 16S-23S rRNA intergenic spacer region. Amplification of an internal fragment of the cfb gene with a reduced annealing temperature yielded positive reactions not only for CAMP-positive S. agalactiae, but also for phenotypically CAMP-positive S. pyogenes (n = 4), S. canis (n = 28), and S. uberis (n = 7), indicating a close relation of the CAMP genes of these 4 species. The relation could be further demonstrated by sequencing the internal fragment of the CAMP-factor (cfg) gene of S. canis and comparing the sequence with those of S. agalactiae, S. pyogenes, and S. uberis.     

A GapC chimera retains the properties of the Streptococcus uberis wild-type GapC protein

The GapC products of Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis share considerable homology at the DNA and amino acid levels. The high similarity at the protein level suggests that one GapC protein might be used as a single antigen to protect dairy cows against infections with the contagious S. agalactiae and the environmental S. dysgalactiae and S. uberis strains. Despite their similarities, immunization with the S. dysgalactiae GapC did not protect dairy cows from a challenge with S. uberis, suggesting the presence of regions in GapC that are involved in species-specific protection. To produce a single antigen that can be used to protect against all streptococcal mastitis infections, we constructed a GapC chimeric protein using the S. uberis GapC product as the backbone followed by non-conserved peptide regions from the S. agalactiae and S. dysgalactiae GapC proteins. We report that the chimeric GapC protein retains the enzymatic activity of the S. uberis GapC protein. In addition, we fused the chimera to the OmpF and LipoF transport sequences of Escherichia coli and the GapC chimeras were present in membrane fractions of E. coli. These extracts could be the basis of an antigen preparation for use in mastitis vaccines.     

Diversity of lactic acid bacteria associated with fish and the fish farm environment, established by amplified rRNA gene restriction analysis

Lactic acid bacteria have become a major source of concern for aquaculture in recent decades. In addition to true pathogenic species of worldwide significance, such as Streptococcus iniae and Lactococcus garvieae, several species have been reported to produce occasional fish mortalities in limited geographic areas, and many unidentifiable or ill-defined isolates are regularly isolated from fish or fish products. To clarify the nature and prevalence of different fish-associated bacteria belonging to the lactic acid bacterium group, a collection of 57 isolates of different origins was studied and compared with a set of 22 type strains, using amplified rRNA gene restriction analysis (ARDRA). Twelve distinct clusters were delineated on the basis of ARDRA profiles and were confirmed by sequencing of sodA and 16S rRNA genes. These clusters included the following: Lactococcus raffinolactis, L. garvieae, Lactococcus l., S. iniae, S. dysgalactiae, S. parauberis, S. agalactiae, Carnobacterium spp., the Enterococcus "faecium" group, a heterogeneous Enterococcus-like cluster comprising indiscernible representatives of Vagococcus fluvialis or the recently recognized V. carniphilus, V. salmoninarum, and Aerococcus spp. Interestingly, the L. lactis and L. raffinolactis clusters appeared to include many commensals of fish, so opportunistic infections caused by these species cannot be disregarded. The significance for fish populations and fish food processing of three or four genetic clusters of uncertain or complex definition, namely, Aerococcus and Enterococcus clusters, should be established more accurately.     

Diversity of carbofuran-degrading soil bacteria and detection of plasmid-encoded sequences homologous to the mcd gene

Abstract: Fifty-five bacterial isolates, from English and French soils with different histories of carbofuran field treatment, which hydrolysed the N -methylcarbamate insecticide carbofuran to carbofuran 7-phenol were characterised phenotypically and genetically. The isolates were compared by using 125 physiological tests and morphological features, plasmid profiles and restriction fragment length polymorphism (RFLP) patterns of total DNA using the rRNA operon of Escherichia coli as a DNA probe. Cluster analysis of both phenotypic characters and RFLP patterns showed a high degree of diversity amongst the bacteria. Ten distinct plasmid profiles with 2–4 plasmids ranging in size from 84 to about 438 kb were visualised in 50 isolates. The majority of isolates had one of two types of plasmid profiles. Plasmid profiles and Eco RI restricted total DNA patterns were hybridised with an internal fragment of the carbofuran hydrolase ( mcd ) gene and 22 diverse soil isolates exhibited sequence homology with this gene probe. Our results indicate that sequences homologous to the mcd gene are located on a conserved Eco RI fragment (12 or 14 kb) of a plasmid (100, 105, 115 or 124 kb) found in diverse soil isolates from geographically distant areas. Thirty-three isolates did not exhibit detectable homology to the mcd gene probe and the hydrolase enzymes and genes in these isolates need further investigation.     

Relationships between arginine degradation, pH and survival in Lactobacillus sakei

Ribotyping and RAPD profiling of a collection of 18 Streptococcus parauberis strains isolated from diseased turbot in Galicia (NW Spain) was performed in order to analyze the possible genetic variability within this bacterial fish pathogen. In addition, the value of this technique for intraspecific classification and epidemiological studies was evaluated. Ribopatterns of DNA digested with three endonucleases and hybridized with a cDNA probe complementary to highly conserved sequences in the 16S and 23S rRNA genes showed a great homogeneity among the turbot isolates. Compared with ribotyping, RAPD appeared to be a reliable and fast technique for discriminating between isolates of S. parauberis on the basis of their farm of isolation and, therefore, represents a powerful tool for epidemiological studies of this fish pathogen.     

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 .     

Detection and cultivation of soil verrucomicrobia

Only one isolate each of the class "Spartobacteria" (subdivision 2 of the phylum Verrucomicrobia) and of subdivision 3 of Verrucomicrobia have previously been reported to grow in laboratory culture. Using media that had been used successfully in other studies to isolate members of diverse groups of soil bacteria, we generated a collection of over 1,200 isolates from soil from a pasture. An oligonucleotide probe that targets the 16S rRNA genes of verrucomicrobia was used to screen this collection, and 14 new verrucomicrobia were identified. Nine of these belonged to the class "Spartobacteria" and were related to "Chthoniobacter flavus." Five further isolates were members of subdivision 3 and were related to the only known isolate of this subdivision. The differences in the 16S rRNA gene sequences of the new isolates and previously described isolates, of up to 10%, indicated that the new isolates represent new species and genera. All but two of the verrucomicrobial isolates were from colonies that first became visible one or more months after inoculation of plates with soil, but subcultures grew more rapidly. Analysis of PCR-amplified 16S rRNA genes in the pasture soil showed that members of the class "Spartobacteria" were more numerous than members of subdivision 3. Isolates of subdivision 3 were only found on plates receiving an inoculum that yielded a mean of 29 colonies per plate, while members of the class "Spartobacteria" were only found on plates receiving a more dilute inoculum that resulted in a mean of five colonies per plate. This suggested that colony development by members of the class "Spartobacteria" was inhibited by other culturable bacteria.     

Riboprinting and 16S rRNA Gene Sequencing for Identification of Brewery Pediococcus Isolates

A total of 46 brewery and 15 ATCC Pediococcus isolates were ribotyped using a Qualicon RiboPrinter. Of these, 41 isolates were identified as Pediococcus damnosus using EcoRI digestion. Three ATCC reference strains had patterns similar to each other and matched 17 of the brewery isolates. Six other brewing isolates were similar to ATCC 25249. The other 18 P. damnosus brewery isolates had unique patterns. Of the remaining brewing isolates, one was identified as P. parvulus, two were identified as P. acidilactici, and two were identified as unique Pediococcus species. The use of alternate restriction endonucleases indicated that PstI and PvuII could further differentiate some strains having identical EcoRI profiles. An acid-resistant P. damnosus isolate could be distinguished from non-acid-resistant varieties of the same species using PstI instead of EcoRI. 16S rRNA gene sequence analysis was compared to riboprinting for identifying pediococci. The complete 16S rRNA gene was PCR amplified and sequenced from seven brewery isolates and three ATCC references with distinctive riboprint patterns. The 16S rRNA gene sequences from six different brewery P. damnosus isolates were homologous with a high degree of similarity to the GenBank reference strain but were identical to each other and one ATCC strain with the exception of 1 bp in one strain. A slime-producing, beer spoilage isolate had 16S rRNA gene sequence homology to the P. acidilactici reference strain, in agreement with the riboprint data. Although 16S rRNA gene sequencing correctly identified the genus and species of the test Pediococcus isolates, riboprinting proved to be a better method for subspecies differentiation.     

Isolation, characterization, and distribution of denitrifying toluene degraders from a variety of habitats.

Enrichments capable of toluene degradation under O2-free denitrifying conditions were established with diverse inocula including agricultural soils, compost, aquifer material, and contaminated soil samples from different geographic regions of the world. Successful enrichment was strongly dependent on the initial use of relatively low toluene concentrations, typically 5 ppm. From the enrichments showing positive activity for toluene degradation, 10 bacterial isolates were obtained. Fingerprints generated by PCR-amplified DNA, with repetitive extragenic palindromic sequence primers, showed that eight of these isolates were different. Under aerobic conditions, all eight isolates degraded toluene, five degraded ethylbenzene, three consumed benzene, and one degraded chlorobenzene, meta-Xylene was the only other substrate used anaerobically and was used by only one isolate. All isolates were motile gram-negative rods, produced N2 from denitrification, and did not hydrolyze starch. All strains but one fixed nitrogen as judged by ethylene production from acetylene, but only four strains hybridized to the nifHDK genes. All strains appeared to have heme nitrite reductase since their DNA hybridized to the heme (nirS) but not to the Cu (nirU) genes. Five strains hybridized to a toluene ortho-hydroxylase catabolic probe, and two of those also hybridized to a toluene meta-hydroxylase probe. Partial sequences of the 16S rRNA genes of all isolates showed substantial similarity to 16S rRNA sequences of Azoarcus sp. Physiological, morphological, fatty acid, and 16S rRNA analyses indicated that these strains were closely related to each other and that they belong to the genus Azoarcus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic Fingerprinting of Brevibacterium linens by Pulsed-Field Gel Electrophoresis and Ribotyping

Members of Brevibacterium linens display physiological features that are relevant for cheese production. The genomes of five B. linens strains deposited on culture collections were compared by examining large restriction fragments on pulsed-field gel electrophoresis and detection of polymorphism at the level of 16S rRNA genes. Pulsed-field analysis with the endonucleases DraI and AsnI showed a characteristic restriction profile for each strain and allowed the calculation of genome sizes ranging between 3.2 and 3.9 Mbp. No linear genomic elements were detected. Polymorphisms at the level of 16S rRNA genes were revealed by hybridization with an oligonucleotide probe complementary to a universal domain of the 16S genes. An EcoRI fragment of 1.4 kb was identified as common to all strains under study. According to the number of positive bands detected by the probe, at least four rRNA operons must be present on the genome of the B. linens strains here studied. Received: 13 January 2000 / Accepted: 9 February 2000     

Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences.

A fluorescently labeled version of a population-specific oligonucleotide hybridization probe was used to monitor the enrichment and isolation of a sulfate-reducing bacterium from a multispecies anaerobic bioreactor. The organism was originally identified as a molecular isolate that was phylogenetically related to Desulfovibrio vulgaris by amplification and sequencing of part of its 16S rRNA sequence. The sequence, in turn, was used to design a population-specific probe. The anaerobic medium used for the organism's enrichment and isolation was based on the physiological properties of the its closest relatives as identified by sequence comparisons. Of 30 isolates examined, only 3 hybridized with the probe. Nearly complete 16S rRNA sequences determined for each of these three isolates (i) had no mismatches with the probe target site, (ii) were identical to the amplified partial sequence of about 500 nucleotides and to one another in all other positions, and (iii) were 93.9% similar to that of D. vulgaris. In addition, one isolate chosen for further study (strain PT-2) had a substrate specificity comparable to that of D. vulgaris. These results confirmed that polymerase chain reaction amplification of 16S rRNA sequences from environmental samples can be accurate and can also provide phylogenetic information from which aspects of a population's physiology can be inferred.     

Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes

Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in Escherichia coli DSM 30083(T). The use of helpers enhanced the fluorescence signal of all six probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes available for E. coli. The temperature of dissociation and the mismatch discrimination of probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with oligonucleotide probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.     

Ribotyping of Pseudomonas aeruginosa isolates from patients and water springs and genome fingerprinting of variants concerning mucoidy

Abstract Thirty-one strains of Pseudomonas aeruginosa , isolated from water springs, clinical isolates (some of which were from cystic fibrosis (CF) patients), and two type cultures, were characterized by ribotyping. After restriction of chromosomal DNA of the different isolates with Eco RI and hybridization of Southern transfer blots with 2-acetylaminofluorene labelled Escherichia coli 16S + 23S rRNA probe, eleven different ribopatterns were obtained, representing variations of a dominant profile. This largely predominant pattern included both type cultures, all six isolates from water springs, 33% of the nine CF isolates and 43% of fourteen other clinical isolates most of them from nosocomial infections. When the genomic macrorestriction fingerprints of three mucoid CF isolates, with Ase I, Dra I or Bfr I were compared with those of their spontaneous variants, concerning mucoidy, no differences were detected.     

Biochemical and phylogenetic analyses of psychrophilic isolates belonging to the Arthrobacter subgroup and description of Arthrobacter psychrolactophilus, sp. nov.

During our work on psychrophilic microorganisms we obtained a large collection of new isolates. In order to identify six of these, we examined their growth properties, cell wall compositions, and their 16S rRNA gene sequences. The results showed that all of the isolates are gram-positive, aerobic, contain lysine in their cell walls, and belong to the high mol% G+C Arthrobacter subgroup. Phylogenetic analysis of the 16S rRNA genes grouped five isolates obtained from a small geographical region into a monophyletic clade. Isolate B7 had a 16S rRNA sequence that was 94.3% similar to that of Arthrobacter polychromogenes and 94.4% similar to that of Arthrobacter oxydans. Primary characteristics that distinguish isolate B7 from the Arthrobacter type strain (Arthrobacter globiformis) and A. polychromogenes include lack of growth at 37 degrees C, growth at 0-5 degrees C, the ability to use lactose as a sole carbon source, and the absence of blue pigments. Because of these differences, isolate B7 was chosen as a type strain representing a new Arthrobacter species, Arthrobacter psychrolactophilus. The sixth isolate, LV7, differed from the other five because it did not have the rod/ coccus morphological cycle and was most closely related to Arthrobacter agilis.     

Unlabeled Helper Oligonucleotides Increase the In Situ Accessibility to 16S rRNA of Fluorescently Labeled Oligonucleotide Probes

Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in Escherichia coli DSM 30083^T. The use of helpers enhanced the fluorescence signal of all six probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes available for E. coli. The temperature of dissociation and the mismatch discrimination of probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with oligonucleotide probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.     

A very large repeating unit of mouse DNA containing the 18S, 28S and 5.8S rRNA genes

The organization of the 18S, 28S and 5.8S rRNA genes in the mouse has been elucidated by mapping with restriction endonucleases Eco RI, Hind III and Bam HI. Ribosomal DNA fragments were detected in electrophoretically fractionated digests of total nuclear DNA by in situ hybridization with radioiodinated rRNAs or with complementary RNA synthesized directly on rRNA templates. A map of the rDNA which includes 13 restriction sites was constructed from the sizes of rDNA fragments and their labeling by different probes The map indicates that the rRNA genes lie within remarkably large units of reiterated DNA, at least 44,000 base pairs long. At least two, and possibly four, classes of repeating unit can be distinguished, the heterogeneity probably residing in the very large nontranscribed spacer region. The 5.8S rRNA gene lies in the transcribed region between the 18S and 28S genes.