Identification of Actinomyces, Propionibacteria, Lactobacilli and Bifidobacteria by Amplified 16S rDNA Restriction Analysis

Anaerobe ◽  
2001 ◽  
Vol 7 (2) ◽  
pp. 55-57 ◽  
Author(s):  
V. Hall ◽  
T. Lewis-Evans ◽  
B.I. Duerden
Keyword(s):  
16S Rdna ◽  
Restriction Analysis

scholarly journals Diversity of Free-Living and Attached Bacteria in Offshore Western Mediterranean Waters as Depicted by Analysis of Genes Encoding 16S rRNA

1999 ◽  
Vol 65 (2) ◽  
pp. 514-522 ◽  
Author(s):  
Silvia G. Acinas ◽  
Josefa Antón ◽  
Francisco Rodríguez-Valera
Keyword(s):  
16S Rdna ◽  
Restriction Analysis ◽  
Western Mediterranean ◽  
Bacterial Cells ◽  
Free Living ◽  
Attached Bacteria ◽  
Genes Encoding ◽  
Community Fingerprinting ◽  
Close Relatives ◽  
Alteromonas Macleodii

ABSTRACT In a previous study (S. G. Acinas, F. Rodrı́guez-Valera, and C. Pedrós-Alió, FEMS Microbiol. Ecol. 24:27–40, 1997), community fingerprinting by 16S rDNA restriction analysis applied to Mediterranean offshore waters showed that the free-living pelagic bacterial community was very different from the bacterial cells aggregated or attached to particles of more than about 8 μm. Here we have studied both assemblages at three depths (5, 50, and 400 m) by cloning and sequencing the 16S rDNA obtained from the same samples, and we have also studied the samples by scanning electron microscopy to detect morphology patterns. As expected, the sequences retrieved from the assemblages were very different. The subsample of attached bacteria contained very little diversity, with close relatives of a well-known species of marine bacteria, Alteromonas macleodii, representing the vast majority of the clones at every depth. On the other hand, the free-living assemblage was highly diverse and varied with depth. At 400 m, close relatives of cultivated γProteobacteria predominated, but as shown by other authors, near the surface most clones were related to phylotypes described only by sequence, in which the α Proteobacteria of the SAR11 cluster predominated. The new technique of rDNA internal spacer analysis has been utilized, confirming these results. Clones representative of the A. macleodii cluster have been completely sequenced, producing a picture that fits well with the idea that they could represent a genus with at least two species and with a characteristic depth distribution.

Phylogeny based on 16S rDNA andnifHsequences ofRalstonia taiwanensisstrains isolated from nitrogen-fixing nodules ofMimosa pudica, in India

2004 ◽  
Vol 50 (5) ◽  
pp. 313-322 ◽  
Author(s):  
Subhash Chandra Verma ◽  
Soumitra Paul Chowdhury ◽  
Anil Kumar Tripathi
Keyword(s):  
16S Rdna ◽  
South India ◽  
Restriction Analysis ◽  
Cross Reaction ◽  
North India ◽  
Mimosa Pudica ◽  
Free Living ◽  
South Indian ◽  
Indian Isolates ◽  
Dinitrogenase Reductase

Bacterial symbionts present in the indeterminate-type nitrogen (N)-fixing nodules of Mimosa pudica grown in North and South India showed maximum similarity to Ralstonia taiwanensis on the basis of carbon-source utilization patterns and 16S rDNA sequence. Isolates from the nodules of M. pudica from North India and South India showed identical ARDRA (Amplified Ribosomal DNA Restriction Analysis) patterns with Sau3AI and RsaI, but AluI revealed dimorphy between the North Indian and South Indian isolates. Alignment of 16S rDNA sequences revealed similarity of North Indian isolates with an R. taiwanensis strain isolated from M. pudica in Taiwan, whereas South Indian isolates showed closer relatedness with the isolates from Mimosa diplotricha. Alignment of nifH sequences from both North Indian and South Indian isolates with that of the related isolates revealed their closer affinity to α-rhizobia, suggesting that nif genes in the β-rhizobia might have been acquired from α-rhizobia via lateral transfer during co-occupancy of nodules by α-rhizobia and progenitors of R. taiwanensis, members of the β-subclass of Proteobacteria. Immunological cross-reaction of the bacteroid preparation of M. pudica nodules showed strong a positive signal with anti-dinitrogenase reductase antibody, whereas a weak positive cross-reaction was observed with free-living R. taiwanensis grown microaerobically in minimal medium with and without NH4Cl. In spite of the expression of dinitrogenase reductase under free-living conditions, acetylene reduction was not observed under N-free conditions even after prolonged incubation.Key words: symbiotic nitrogen fixation, Mimosa pudica, rhizobia, phylogeny, 16S rDNA, nifH, Ralstonia taiwanensis.

scholarly journals Polymorphism of the Polyketide Synthase Gene phlD in Biocontrol Fluorescent Pseudomonads Producing 2,4-Diacetylphloroglucinol and Comparison of PhlD with Plant Polyketide Synthases

2001 ◽  
Vol 14 (5) ◽  
pp. 639-652 ◽  
Author(s):  
Alban Ramette ◽  
Yvan Moënne-Loccoz ◽  
Geneviève Défago
Keyword(s):  
16S Rdna ◽  
Polyketide Synthase ◽  
Restriction Analysis ◽  
Polyketide Synthases ◽  
Fluorescent Pseudomonads ◽  
Phylogenetic Groups ◽  
Gram Positive ◽  
Restriction Patterns ◽  
Gram Positive Bacteria ◽  
Rdna Sequencing

Many biocontrol fluorescent pseudomonads can protect plants from soilborne fungal pathogens through production of the antifungal secondary metabolite 2,4-diacetylphloroglucinol (Phl). One of the phl biosynthetic genes, phlD, encodes a polyketide synthase similar to plant chalcone synthases. Here, restriction analysis of phlD from 39 Phl+ biocontrol fluorescent pseudomonads yielded seven different banding patterns. The gene was sequenced in seven strains, representing the different restriction patterns. Cluster analysis of phlD restriction data or phlD sequences indicated that phlD polymorphism was high, and two main clusters were obtained when predicted PhlD sequences were compared. When the seven PhlD sequences were studied with those of other procaryotic polyketide synthases (gram-positive bacteria) and plant chalcone synthases, however, Phl+ pseudomonads, gram-positive bacteria, and plants clustered separately. Yet, sequence analysis of active site regions for PhlD and plant chalcone synthases revealed that PhlD can be considered a member of the chalcone synthase family, which may be interpreted as convergent evolution of key enzymes involved in secondary metabolism. For the 39 Phl+ pseudomonads, a relationship was found among phlD restriction patterns, phylogenetic groups defined by 16S rDNA restriction analysis (confirmed by 16S rDNA sequencing), and production levels of Phl in vitro.

scholarly journals Two Groups of Phytoplasmas from Japan Distinguished on the Basis of Amplification and Restriction Analysis of 16S rDNA

Plant Disease ◽  
1997 ◽  
Vol 81 (3) ◽  
pp. 301-305 ◽  
Author(s):  
Seiichi Okuda ◽  
James P. Prince ◽  
Robert E. Davis ◽  
Ellen L. Dally ◽  
Ing-Ming Lee ◽  
...  
Keyword(s):  
16S Rdna ◽  
Restriction Analysis ◽  
Pcr Amplification ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Rrna Gene ◽  
Gene Group ◽  
Beet Leafhopper ◽  
Pcr Products ◽  
Elm Yellows

Phytoplasmas (mycoplasmalike organisms, MLOs) associated with mitsuba (Japanese hone-wort) witches'-broom (JHW), garland chrysanthemum witches'-broom (GCW), eggplant dwarf (ED), tomato yellows (TY), marguerite yellows (MY), gentian witches'-broom (GW), and tsu-wabuki witches'-broom (TW) in Japan were investigated by polymerase chain reaction (PCR) amplification of DNA and restriction enzyme analysis of PCR products. The phytoplasmas could be separated into two groups, one containing strains JHW, GCW, ED, TY, and MY, and the other containing strains GW and TW, corresponding to two groups previously recognized on the basis of transmission by Macrosteles striifrons and Scleroracus flavopictus, respectively. The strains transmitted by M. striifrons were classified in 16S rRNA gene group 16SrI, which contains aster yellows and related phytoplasma strains. Strains GW and TW were classified in group 16SrIII, which contains phytoplasmas associated with peach X-disease, clover yellow edge, and related phytoplasmas. Digestion of amplified 16S rDNA with HpaII indicated that strains GW and TW were affiliated with subgroup 16SrIII-B, which contains clover yellow edge phytoplasma. All seven strains were distinguished from other phytoplasmas, including those associated with clover proliferation, ash yellows, elm yellows, and beet leafhopper-transmitted virescence in North America, and Malaysian periwinkle yellows and sweet potato witches'-broom in Asia.

Bacterial diversity associated with subalpine fir (Abies lasiocarpa) ectomycorrhizae following wildfire and salvage-logging in central British Columbia

2002 ◽  
Vol 48 (7) ◽  
pp. 611-625 ◽  
Author(s):  
Madhukar B Khetmalas ◽  
Keith N Egger ◽  
Hugues B Massicotte ◽  
Linda E Tackaberry ◽  
M Jill Clapperton
Keyword(s):  
Bacterial Diversity ◽  
16S Rdna ◽  
Bacterial Communities ◽  
Restriction Analysis ◽  
Rrna Gene ◽  
Abies Lasiocarpa ◽  
Gram Positive ◽  
Salvage Logging ◽  
Gram Positive Bacteria ◽  
Significant Difference

To assess the effect of fire and salvage logging on the diversity of mycorrhizal–bacterial communities, bacteria associated with Cenococcum, Thelephora, Tomentella, Russulaceae, and E-strain ectomycorrhizae (ECM) of Abies lasiocarpa seedlings were characterized using two approaches. First, bacteria were isolated and characterized by Biolog©, gas chromatography fatty acid methyl ester (GC-FAME), and amplified 16S rDNA restriction analysis (ARDRA). The bacterial communities retrieved from ECM from both sites were dominated by Proteobacteria (groups gamma and beta). Pseudomonas was the most common genus isolated, followed by Variovorax, Burkholderia, and Xanthomonas. Gram-positive isolates (mostly high-G+C Gram-positive bacteria) were more frequently retrieved on the burned-salvaged site, many commonly associated with the two ascomycete ECM, Cenococcum and E-strain. Pseudomonas species were retrieved more frequently from Thelephora. Although actinomycetes were isolated from all sites, almost no actinomycetes or other Gram-positive bacteria were isolated from either Thelephora or Tomentella. Second, amplified 16S rRNA gene sequences were amplified directly from root tips and then cloned into the plasmid vector pAMP1, followed by restriction analysis. This technique distinguished more genotypes than isolates retrieved by culturing methods, but generally, results were similar in that the largest proportion of the bacteria were putatively Gram-negative; putative Gram-positive bacteria were fewer and most were from the burned–salvaged site. Direct cloning resulted in many patterns that did not match any identified isolates, suggesting that a large proportion of clones were unique or not culturable by the methods used. Analysis for both protocols showed no significant difference in bacterial diversity between the burned–salvaged and unburned sites. Key words: rhizosphere bacteria, ARDRA, 16S rDNA, Biolog©, GC-FAME.

scholarly journals Novel Diagnostic Algorithm for Identification of Mycobacteria Using Genus-Specific Amplification of the 16S-23S rRNA Gene Spacer and Restriction Endonucleases

2000 ◽  
Vol 38 (3) ◽  
pp. 1094-1104 ◽  
Author(s):  
Andreas Roth ◽  
Udo Reischl ◽  
Anna Streubel ◽  
Ludmila Naumann ◽  
Reiner M. Kroppenstedt ◽  
...  
Keyword(s):  
16S Rdna ◽  
Restriction Analysis ◽  
Diagnostic Algorithm ◽  
Species Level ◽  
23S Rrna ◽  
Rrna Gene ◽  
Correct Identification ◽  
Mycobacterium Fortuitum ◽  
Mycobacterium Chelonae ◽  
Rdna Sequencing

A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence,HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found inMycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium aviumcomplex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, orAvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus,M. chelonae, Mycobacterium farcinogenes,Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S rDNA sequencing results, as was shown by including strains with unsettled taxonomy. Since this approach recognized significant subspecific genotypes while identification of a broad spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, this method can be used by both reference (or research) and routine laboratories.

scholarly journals First-tier detection of intragenomic 16S rRNA gene variation in culturable endophytic bacteria from cacao seeds

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7452
Author(s):  
Cleiziane Bispo da Silva ◽  
Hellen Ribeiro Martins dos Santos ◽  
Phellippe Arthur Santos Marbach ◽  
Jorge Teodoro de Souza ◽  
Valter Cruz-Magalhães ◽  
...  
Keyword(s):  
16S Rdna ◽  
Endophytic Bacteria ◽  
Restriction Analysis ◽  
Natural Populations ◽  
Amplicon Sequencing ◽  
Limiting Factor ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Restriction Profiles ◽  
Chi Square Analysis

Background Intragenomic variability in 16S rDNA is a limiting factor for taxonomic and diversity characterization of Bacteria, and studies on its occurrence in natural/environmental populations are scarce. In this work, direct DNA amplicon sequencing coupled with frequent-cutter restriction analysis allowed detection of intragenomic 16S rDNA variation in culturable endophytic bacteria from cacao seeds in a fast and attractive manner. Methods Total genomic DNA from 65 bacterial strains was extracted and the 16S rDNA hyper variable V5–V9 regions were amplified for enzyme digestion and direct Sanger-type sequencing. The resulting electropherograms were visually inspected and compared to the corresponding AluI-restriction profiles, as well as to complete genome sequences in databases. Restriction analysis were employed to substitute the need of amplicon cloning and re-sequencing. A specifically improved polyacrylamide-gradient electrophoresis allowed to resolve 5-bp differences in restriction fragment sizes. Chi-square analysis on 2 × 2 contingency table tested for the independence between the ‘number of AluI bands’ and ‘type of eletropherogram’. Results Two types of electropherograms were obtained: unique template, with single peaks per base (clean chromatograms), and heterogeneous template, with various levels of multiple peaks per base (mixed chromatograms). Statistics revealed significant interaction between number of restriction fragments and type of electropherogram for the same amplicons: clean or mixed ones associated to ≤5 or ≥6 bands, respectively. The mixed-template pattern combined with the AluI-restriction profiles indicated a high proportion of 49% of the culturable endophytes from a tropical environment showing evidence of intragenomic 16S rDNA heterogeneity. Conclusion The approach presented here was useful for a rapid, first-tier detection of intragenomic variation in culturable isolates, which can be applied in studies of other natural populations; a preliminary view of intragenomic heterogeneity levels can complement culture-dependent and -independent methods. Consequences of these findings in taxonomic and diversity studies in complex bacterial communities are discussed.

scholarly journals Paenibacillus cineris sp. nov. and Paenibacillus cookii sp. nov., from Antarctic volcanic soils and a gelatin-processing plant

2004 ◽  
Vol 54 (4) ◽  
pp. 1071-1076 ◽  
Author(s):  
Niall A. Logan ◽  
Elke De Clerck ◽  
Liesbeth Lebbe ◽  
An Verhelst ◽  
Johan Goris ◽  
...  
Keyword(s):  
16S Rdna ◽  
Restriction Analysis ◽  
Sequence Similarity ◽  
Major Fatty Acid ◽  
Rdna Sequence ◽  
Processing Plant ◽  
Sequence Comparisons ◽  
16S Rdna Sequence ◽  
Dna Reassociation ◽  
Sds Page

Seven strains of aerobic, endospore-forming bacteria were found in soil taken from an active fumarole on Lucifer Hill, Candlemas Island, South Sandwich archipelago, Antarctica, and four strains were from soil of an inactive fumarole at the foot of the hill. Amplified rDNA restriction analysis, 16S rDNA sequence comparisons, SDS-PAGE and routine phenotypic tests support the proposal of two novel species of Paenibacillus, Paenibacillus cineris sp. nov. and Paenibacillus cookii sp. nov., the type strains of which are LMG 18439T (=CIP 108109T) and LMG 18419T (=CIP 108110T), respectively. A further strain, isolated from a gelatin-production process, showed more than 99 % 16S rDNA sequence similarity to the proposed P. cookii type strain and, although the gelatin isolate was atypical when compared with the fumarole isolates by repeated element primed-PCR, SDS-PAGE and phenotypic analyses, it was shown by DNA–DNA reassociation studies to belong to the same species. Strains of P. cookii produce spreading growth with motile microcolonies. Both species produce swollen sporangia that are typical for the genus, they both show 97·6 % 16S rDNA sequence similarity to Paenibacillus azoreducens, they have 51·5–51·6 mol% G+C in their DNA and their major fatty acid is anteiso-C15 : 0; however, fatty acids C16 : 0 and anteiso-C17 : 0 represent, respectively, 18 and 10 % of the total in P. cineris, but 11 and 20 % in P. cookii.

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