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PubMed2001

Changes in populations of rhizosphere bacteria associated with take-all disease of wheat.

Take-all, caused by Gaeumannomyces graminis var. tritici, is one of the most important fungal diseases of wheat worldwide. Knowing that microbe-based suppression of the disease occurs in monoculture wheat fields following severe outbreaks of take-all, we analyzed the changes in rhizosphere bacterial communities following infection by the take-all pathogen. Several bacterial populations were more abundant on diseased plants than on healthy plants, as indicated by higher counts on a Pseudomonas-selective medium and a higher fluorescence signal in terminal restriction fragment length polymorphism analyses of amplified 16S ribosomal DNA (rDNA). Amplified rDNA restriction analysis (ARDRA) of the most abundant cultured populations showed a shift in dominance from Pseudomonas to Chryseobacterium species in the rhizosphere of diseased plants. Fluorescence-tagged ARDRA of uncultured rhizosphere washes revealed an increase in ribotypes corresponding to several bacterial genera, including those subsequently identified by partial 16S sequencing as belonging to species of alpha-, beta-, and gamma-proteobacteria, sphingobacteria, and flavobacteria. The functional significance of some of these populations was investigated in vitro. Of those isolated, only a small subset of the most abundant Pseudomonas spp. and a phlD(+) Pseudomonas sp. showed any significant ability to inhibit G. graminis var. tritici directly. When cultured strains were mixed with the inhibitory phlD(+) Pseudomonas strain, the Chryseobacterium isolates showed the least capacity to inhibit this antagonist of the pathogen, indicating that increases in Chryseobacterium populations may facilitate the suppression of take-all by 2,4-diacetylphloroglucinol-producing phlD(+) pseudomonads.

Antibiosis↗
PubMed1998

Identification and sequencing of the groE operon and flanking genes of Lawsonia intracellularis: use in phylogeny.

Proliferative enteropathy (PE) is a complex of diseases of commercial importance to the pig industry. The obligate intracellular bacterium Lawsonia intracellularis is consistently associated with PE and pure cultures of this bacterium have been used to reproduce PE in pigs. In this study L. intracellularis bacteria were purified directly from PE-affected tissue. DNA extracted from purified bacteria was used to construct a partial genomic library which was screened using sera from L. intracellularis-immunized rabbits. Two seroreactive recombinant clones were identified, one of which expressed proteins of 10 and 60 kDa. The sequence of the insert from this clone, pISI-2, revealed ORFs with sequence similarity to the groES/EL operon of Escherichia coli, the 505 ribosomal proteins L21 and L27 of E. coli, a GTP-binding protein of Bacillus subtilis and a possible protoporphyrinogen oxidase, HemK, of E. coli. Primers designed from unique sequences from the pISI-2 insert amplified DNA from infected, but not non-infected, porcine ilea; the amplicon sequence obtained from tissue-cultured L. intracellularis was identical to the corresponding sequence in pISI-2, confirming the origin of the clone. The sequence of L. intracellularis GroEL and other GroEL sequences in the databases were used to construct a partial phylogenetic tree. Analysis of the GroEL sequence relationship suggested that L. intracellularis is not significantly related to other organisms whose GroEL sequences are held in the databases and supports previous data from 16S sequence analyses suggesting that L. intracellularis is a member of a novel group of enteric pathogens.

Amino Acid Sequence↗
PubMed1999

16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis.

The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species as Photobacterium damselae subsp. damselae (formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of P. damselae based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis.

Animals↗
PubMed1992

Phylogeny of cytoplasmic incompatibility micro-organisms in the parasitoid wasp genus Nasonia (Hymenoptera: Pteromalidae) based on 16S ribosomal DNA sequences.

Cytoplasmic incompatibility results in embryo mortality in diploids, or all male offspring in haplodiploids, when individuals carrying different cytoplasmic factors are crossed. Cytoplasmic factors have been identified as intracellular micro-organisms. Microbe-induced cytoplasmic incompatibility is found in many insect taxa and may play a role in reproductive isolation between populations. Such micro-organisms cause bidirectional incompatibility between species of the parasitoid wasp genus Nasonia. The phylogenetic relationship of cytoplasmic incompatibility microorganisms (CIM) of different Nasonia species was analysed using their 16S ribosomal DNA (rDNA) sequence. Two 16S rDNA operons were detected in the CIM of each Nasonia species. Sequence analysis indicates that the Nasonia CIM are closely related and belong to the alpha group of the Proteobacteria.

Animals↗
PubMed2004

Molecular systematics of African 20-chromosome toads (Anura: Bufonidae).

Intrageneric lineages and the historical biogeography of toads, genus Bufo, are poorly resolved due to their conservative morphology, their highly conserved karyotypes (typically 2N = 22), and erratic patterns of interspecific hybridisation. Here, we use mitochondrial and nuclear DNA sequence data to reconstruct relationships of the 20-chromosome toads, a major component of the African bufonid fauna. Mitochondrial 12S and 16S sequences from 29 species revealed two independent transitions between phenotypically distinct savannah and forest adapted forms. Analyses of mitochondrial 12S, 16S, and ND2, along with nuclear ACTC and Rhodopsin sequences from 12 species greatly increased bootstrap, and likelihood support for internal branches including a basal split into two pan-African 20-chromosome clades. Hybridisation is a weak indicator of phylogenetic relationship as it occurs across these deeply divergent clades, between Bufo rangeri and Bufo gutturalis. These analyses suggest a secondary reversion to 22-chromosomes in Bufo pardalis, within the 20-chromosome group, although we could not reject an alternative hypothesis that this lineage forms a sister to all 2N = 20 toads. Other informally recognised 22-chromosome groups form independent phylogenetic lineages outside the 20-chromosome group, such as the Angusticeps and Vertebralis divisions. Bufo lindneri, from the Taitanus division, is closely related to Stephopaedes anotis, and these species should be considered congeners.

Africa↗
PubMed2004

Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases.

The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. Comparison of the bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, can be routinely used for identification of mycobacteria, and can lead to the recognition of novel pathogens and noncultured bacteria. Problems remain in that the sequences in some databases are not accurate, there is no consensus quantitative definition of genus or species based on 16S rRNA gene sequence data, the proliferation of species names based on minimal genetic and phenotypic differences raises communication difficulties, and microheterogeneity in 16S rRNA gene sequence within a species is common. Despite its accuracy, 16S rRNA gene sequence analysis lacks widespread use beyond the large and reference laboratories because of technical and cost considerations. Thus, a future challenge is to translate information from 16S rRNA gene sequencing into convenient biochemical testing schemes, making the accuracy of the genotypic identification available to the smaller and routine clinical microbiology laboratories.

Bacteria↗
PubMed2004

Widespread occurrence of a novel division of bacteria identified by 16S rRNA gene sequences originally found in deep marine sediments.

Phylogenetic analysis of 16S rRNA gene sequences from deep marine sediments identified a deeply branching clade, designated candidate division JS1. Primers for PCR amplification of partial 16S rRNA genes that target the JS1 division were developed and used to detect JS1 sequences in DNA extracted from various sedimentary environments, including, for the first time, coastal marine and brackish sediments.

Bacteria↗
PubMed2006

Genetic differentiation of geographically separated populations of the southern green stink bug Nezara viridula (Hemiptera: Pentatomidae).

Genetic variation in the southern green stink bug Nezara viridula (Linnaeus) from 11 geographically separated sampling locations (Slovenia, France, Greece, Italy, Madeira, Japan, Guadeloupe, Galapagos, California, Brazil and Botswana) was studied by sequencing 16S and 28S rDNA, cytochrome b and cytochrome c oxidase subunit I gene fragments and random amplified polymorphic DNA (RAPD) analysis. Sequencing revealed 11 distinct haplotypes clustering into lineages A, B and C. Lineage C was characteristic for a single analysed specimen from Botswana. Lineage B was detected in Japan, and it probably arose in Asia. Haplotypes of European and American specimens belonged to lineage A; specimens from France, Slovenia, Madeira and Brazil shared highly similar haplotypes (>99%) from subgroup A1, while all the specimens from Greece, California, Galapagos and Guadeloupe shared a haplotype from subgroup A2. RAPD data were more variable but consistent with mtDNA sequences, revealing the same clustering. They separated the Botswanian specimen from Japanese specimens and from a group of more closely related specimens from Europe and America. Sequence and RAPD results both support the African origin of N. viridula, followed by dispersal to Asia (lineage B) and, more recently, by expansion to Europe and America (lineage A). RAPD analysis revealed two highly supported subgroups in Japan, congruent with mtDNA lineages A2 and B, suggesting multiple colonization of Japan. Invariant sequences at the 28S rDNA combined with other results do not support the hypothesis that cryptic (sibling) species exist within the populations investigated in this study.

Animals↗
PubMed2000

Interest of partial 16S rDNA gene sequences to resolve heterogeneities between Leptospira collections: application to L. meyeri.

This paper describes the advantage of using the first 330 bp (positions 46 to 375, Escherichia coli numbering) of the 16S rDNA gene for comparison of Leptospira isolates. Phylogenetic analysis conducted from the whole 16S rDNA sequences available in databanks as well as that conducted from the partial sequences yielded quite similar results, in accordance with data inferred from previous DNA-DNA relatedness studies. This tool was used for the comparison of Leptospira strains from different reference collections. Consistent results were obtained from the analysis of the polymorphism generated by pulsed-field gel electrophoresis. The study focused on different serovars of L. meyeri species, the classification of which has been controversial. The results revealed large collection heterogeneities, and suggest that the classification of the L. meyeri species should be revised.

Bacterial Typing Techniques↗
PubMed2004

Asaia bogorensis peritonitis identified by 16S ribosomal RNA sequence analysis in a patient receiving peritoneal dialysis.

Here the authors report a case of refractory peritonitis leading to multiple hospitalizations and the loss of peritoneal dialysis access in a patient on automated peritoneal dialysis, caused by Asaia bogorensis, a bacterium not previously described as a human pathogen. This organism was identified by sequence analysis of the 16S ribosomal RNA gene. Unusual microbial agents may cause peritonitis, and molecular microbiological techniques are important tools for identifying these agents.

Acetobacteraceae↗
PubMed2006

Bacterial genotyping by 16S rRNA mass cataloging.

BACKGROUND: It has recently been demonstrated that organism identifications can be recovered from mass spectra using various methods including base-specific fragmentation of nucleic acids. Because mass spectrometry is extremely rapid and widely available such techniques offer significant advantages in some applications. A key element in favor of mass spectrometric analysis of RNA fragmentation patterns is that a reference database for analysis of the results can be generated from sequence information. In contrast to hybridization approaches, the genetic affinity of any unknown isolate can in principle be determined within the context of all previously sequenced 16S rRNAs without prior knowledge of what the organism is. In contrast to the original RNase T1 cataloging method, when digestion products are analyzed by mass spectrometry, products with the same base composition cannot be distinguished. Hence, it is possible that organisms that are not closely related (having different underlying sequences) might be falsely identified by mass spectral coincidence. We present a convenient spectral coincidence function for expressing the degree of similarity (or distance) between any two mass-spectra. Trees constructed using this function are consistent with those produced by direct comparison of primary sequences, demonstrating that the inherent degeneracy in mass spectrometric analysis of RNA fragments does not preclude correct organism identification. RESULTS: Neighbor-joining trees for important bacterial pathogens were generated using distances based on mass spectrometric observables and the spectral coincidence function. These trees demonstrate that most pathogens will be readily distinguished using mass spectrometric analyses of RNA digestion products. A more detailed, genus-level analysis of pathogens and near relatives was also performed, and it was found that assignments of genetic affinity were consistent with those obtained by direct sequence comparisons. Finally, typical values of the coincidence between organisms were also examined with regard to phylogenetic level and sequence variability. CONCLUSION: Cluster analysis based on comparison of mass spectrometric observables using the spectral coincidence function is an extremely useful tool for determining the genetic affinity of an unknown bacterium. Additionally, fragmentation patterns can determine within hours if an unknown isolate is potentially a known pathogen among thousands of possible organisms, and if so, which one.

Bacteria↗
PubMed2005

Phylogenetic relationships of Xylella fastidiosa strains based on 16S-23S rDNA sequences.

In spite of the lack of resolution of Xylella fastidiosa phylogenetic relationships, parsimony analysis of the 16S-23S rDNA sequence from a wide range of hosts has been evaluated in this research. In order to establish an easier method for sequencing the spacer region completely, a new primer pair was designed. The sequences obtained revealed a higher level of variation than that found in 16S gene sequences, with similarity values ranging from 0.80 to 1.00. The cladogram constructed allowed the clustering of two major clades. From these results it has been possible to recognize the monophyletic grouping of some strains belonging to the same host, possibly representing only one infection process. However, for other hosts there is paraphyletic and polyphyletic grouping. This methodology followed from promising results regarding strain-host clustering. With the parsimony approach, hypothetical genealogical relationship among Xylella strains may be inferred.

DNA, Bacterial↗
PubMed2000

Mitochondrial sequence evolution in spiders: intraspecific variation in tRNAs lacking the TPsiC Arm.

Analyses of mitochondrial DNA sequences from three species of Habronattus jumping spiders (Chelicerata: Arachnida: Araneae) reveal unusual inferred tRNA secondary structures and gene arrangements, providing new information on tRNA evolution within chelicerate arthropods. Sequences from the protein-coding genes NADH dehydrogenase subunit 1 (ND1), cytochrome oxidase subunit I (COI), and subunit II (COII) were obtained, along with tRNA, tRNA, and large-subunit ribosomal RNA (16S) sequences; these revealed several peculiar features. First, inferred secondary structures of tRNA and, likely, tRNA, lack the TPsiC arm and the variable arm and therefore do not form standard cloverleaf structures. In place of these arms is a 5-6-nt T arm-variable loop (TV) replacement loop such as that originally described from nematode mitochondrial tRNAs. Intraspecific variation occurs in the acceptor stem sequences in both tRNAs. Second, while the proposed secondary structure of the 3' end of 16S is similar to that reported for insects, the sequence at the 5' end is extremely divergent, and the entire gene is truncated about 300 nt with respect to Drosophila yakuba. Third, initiation codons appear to consist of ATY (ATT and ATC) and TTG for ND1 and COII, respectively. Finally, Habronattus shares the same ND1-tRNA-16S gene arrangement as insects and crustaceans, thus illustrating variation in a tRNA gene arrangement previously proposed as a character distinguishing chelicerates from insects and crustaceans.

Amino Acid Sequence↗
PubMed1995

16S rRNA gene sequence of Neorickettsia helminthoeca and its phylogenetic alignment with members of the genus Ehrlichia.

Neorickettsia helminthoeca (tribe Ehrlichieae, family Rickettsiaceae) is the agent of salmon poisoning disease, which affects members of the family Canidae. This bacterium is unusual in that it is the only known obligately intracellular bacterium that is transmitted via a helminth vector. The nucleotide sequence of the N. helminthoeca 16S rRNA gene was determined and compared with the sequences of intracellular bacteria belonging to the alpha subgroup of the Proteobacteria. The N. helminthoeca sequence was most similar to the sequences of two Ehrlichia species, Ehrlichia risticii and Ehrlichia sennetsu (levels of sequence similarity, > 95%). All other members of the tribe Ehrlichieae, including members of the other Ehrlichia species, and the related species Cowdria ruminantium and Anaplasma marginale, were only distantly related phylogenetically (levels of sequence similarity, 84 to 86%). Our results corroborate the results of previous ultrastructural and Western blot (immunoblot) comparisons of N. helminthoeca with other ehrlichial species. The genus Ehrlichia is phylogenetically incoherent and can be separated into three identifiable clusters of species. Each cluster is closely associated with a species classified in another non-Ehrlichia bacterial genus. The close relationships among N. helminthoeca, E. risticii, and E. sennetsu and the striking differences between these organisms and other members of the tribe Ehrlichieae suggest that in the future, these organisms should be treated as members of a new bacterial genus separate from the genus Ehrlichia.

Animals↗
PubMed1984

The nucleotide sequence of the 16S ribosomal RNA gene of the archaebacterium Halococcus morrhua.

The sequence of the 16S rRNA gene from the archaebacterium Halococcus morrhua was determined by the dideoxynucleotide sequencing method. It is 1475 nucleotides long. This is the second archaebacterial sequence to be determined and it provides sequence comparison evidence for the secondary structural elements confined to the RNAs of this kingdom and, also, support for controversial or additional base pairing in the eubacterial RNAs. Six structural features are localized that have varied during the evolution of the archaebacteria, eubacteria and eukaryotes. Moreover, although the secondary structures of both sequenced archaebacterial RNAs strongly resemble those of eubacteria, they contain sufficient eukaryotic-like structural characteristics to reinforce the view that they belong to a separate line of evolutionary descent.

Archaea↗
PubMed1985

Sequence of the 16S rRNA gene from the thermoacidophilic archaebacterium Sulfolobus solfataricus and its evolutionary implications.

The sequence of the small-subunit rRNA from the thermoacidophilic archaebacterium Sulfolobus solfataricus has been determined and compared with its counterparts from halophilic and methanogenic archaebacteria, eukaryotes, and eubacteria. The S. solfataricus sequence is specifically related to those of the other archaebacteria, to the exclusion of the eukaryotic and eubacterial sequences, when examined either by evolutionary distance matrix analyses or by the criterion of minimum change (maximum parsimony). The archaebacterial 16S rRNA sequences all conform to a common secondary structure, with the S. solfataricus structure containing a higher proportion of canonical base pairs and fewer helical irregularities than the rRNAs from the mesophilic archaebacteria. S. solfataricus is unusual in that its 16S rRNA-23S rRNA intergenic spacer lacks a tRNA gene.

Archaea↗
PubMed1995

Characterization of cilia-associated respiratory bacillus in rabbits and analysis of the 16S rRNA gene sequence.

The cilia-associated respiratory (CAR) bacillus is an unclassified, gram-negative bacterium that has been implicated as an etiologic agent of respiratory tract disease in laboratory rodents. A morphologically and antigenically similar organism has been identified in rabbits and is thought to be a related bacterium, although clinical signs of disease and histologic lesions are absent in infected rabbits. To compare the pathogenicity of rat- and rabbit-origin CAR bacillus isolates in rabbits, neonatal rabbits were experimentally infected with CAR bacillus isolates obtained from an infected rat and rabbit. Rabbits experimentally inoculated with rabbit-origin CAR bacillus had a nasal discharge, seroconverted and developed histologic lesions, whereas rabbits inoculated with rat-origin CAR bacillus seroconverted but did not have evidence of colonization of the respiratory tract. The CAR bacillus isolates were further examined at the genetic level by sequencing 1,261 base pairs of the 16S rRNA gene from six CAR bacillus isolates obtained from infected rabbits. A consensus sequence was obtained and compared with the analogous gene sequence data from rat-origin CAR bacillus isolates. Results indicated that these two organisms are distinctly different, with only 48.8% sequence homology. Comparison of the rabbit-origin 16S rRNA gene sequence with the database Genbank indicated that the organism is most closely related to members of the genus Helicobacter. Bacteria with the highest percentage of similarity with the rabbit-origin CAR bacillus were Helicobacter sp. strain Seymour and H. felis, with 91.1 and 90.8%, respectively. Findings of this study indicate that CAR bacillus isolates from rats and rabbits are host-specific and are different bacteria that belong to distinct genera.

Animals↗
PubMed1994

Phylogeny of spore-forming lactic acid bacteria based on 16S rRNA gene sequences.

The phylogeny of spore-forming lactic acid bacteria was investigated on the basis of 16S rRNA gene sequences. Sixteen strains were separated into three lines of descent; one consisted of 14 strains assigned to Sporolactobacillus spp. and Bacillus spp., and the other two each consisted of "Sporolactobacillus dextrus" and Bacillus coagulans. Strains of all the first lineage but one composed a cluster of similarity values of 97.2% and higher, and were represented by the type strain of S. inulinus. The cluster was further separated into five subclusters, four catalase negative and one positive. The definition of the genus Sporolactobacillus should be amended to accommodate catalase positive strains. Spore-forming lactic acid bacteria originated at different phylogenetic positions, and would have evolved convergently in the area of Bacillus.

Bacillus↗