A Highly Selective PCR Protocol for Detecting 16S rRNA Genes of the Genus Pseudomonas (Sensu Stricto) in Environmental Samples

1998 ◽  
Vol 64 (7) ◽  
pp. 2545-2553 ◽  
Author(s):  
Franco Widmer ◽  
Ramon J. Seidler ◽  
Patrick M. Gillevet ◽  
Lidia S. Watrud ◽  
George D. Di Giovanni
Keyword(s):  
16S Rrna ◽  
Environmental Samples ◽  
Restriction Analysis ◽  
Phylogenetic Analyses ◽  
Pcr Amplification ◽  
Sensu Stricto ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Human Pathogens ◽  
Soil Dna

ABSTRACT Pseudomonas species are plant, animal, and human pathogens; exhibit plant pathogen-suppressing properties useful in biological control; or express metabolic versatilities valued in biotechnology and bioremediation. Specific detection ofPseudomonas species in the environment may help us gain a more complete understanding of the ecological significance of these microorganisms. The objective of this study was to develop a PCR protocol for selective detection ofPseudomonas (sensu stricto) in environmental samples. Extensive database searches identified a highly selective PCR primer pair for amplification of Pseudomonas16S rRNA genes. A protocol that included PCR amplification and restriction analysis, a general cloning and sequencing strategy, and phylogenetic analyses was developed. The PCR protocol was validated by testing 50 target and 14 nontarget pure cultures, which confirmed the selectivity to 100%. Further validation used amplification of target sequences from purified bulk soil DNA followed by cloning of PCR products. Restriction analysis with HaeIII revealed eight different fragmentation patterns among 36 clones. Sequencing and phylogenetic analysis of 8 representative clones indicated that 91.7% of the products were derived from target organisms of the PCR protocol. Three patterns, representing only 8.3% of the 36 clones, were derived from non-Pseudomonas or chimeric PCR artifacts. Three patterns, representing 61.1% of the clones, clustered with sequences of confirmed Pseudomonas species, whereas two patterns, representing 30.6% of the clones, formed a novel phylogenetic cluster closely associated with Pseudomonas species. The results indicated that the Pseudomonas-selective PCR primers were highly specific and may represent a powerful tool forPseudomonas population structure analyses and taxonomic confirmations.

scholarly journals Detection of Verrucomicrobia in a Pasture Soil by PCR-Mediated Amplification of 16S rRNA Genes

1999 ◽  
Vol 65 (9) ◽  
pp. 4280-4284 ◽  
Author(s):  
Katrina A. O’Farrell ◽  
Peter H. Janssen
Keyword(s):  
16S Rrna ◽  
Restriction Analysis ◽  
Comparative Sequence Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Soil Dna ◽  
Probable Number ◽  
Pasture Soil

ABSTRACT Oligonucleotide primers were designed and used to amplify, by PCR, partial 16S rRNA genes of members of the bacterial divisionVerrucomicrobia in DNA extracted from a pasture soil. By applying most-probable-number theory to the assay, verrucomicrobia appeared to contribute some 0.2% of the soil DNA. Amplified ribosomal DNA restriction analysis of 53 cloned PCR-amplified partial 16S rRNA gene fragments and comparative sequence analysis of 21 nonchimeric partial 16S rRNA genes showed that these primers amplified only 16S rRNA genes of members of the Verrucomicrobia in DNA extracted from the soil.

scholarly journals Evaluation of 16S, map1 and pCS20 probes for detection of Cowdria and Ehrlichia species

1999 ◽  
Vol 122 (2) ◽  
pp. 323-328 ◽  
Author(s):  
M. T. E. P. ALLSOPP ◽  
C. M. HATTINGH ◽  
S. W. VOGEL ◽  
B. A. ALLSOPP
Keyword(s):  
16S Rrna ◽  
Ribosomal Rna ◽  
Pcr Amplification ◽  
16S Ribosomal Rna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Amblyomma Hebraeum ◽  
Ribosomal Rna Gene ◽  
Group Iii ◽  
16S Ribosomal Rna Gene

A panel of 16S ribosomal RNA gene probes has been developed for the study of the epidemiology of heartwater; five of these detect different cowdria genotypes, one detects five distinct genotypes; one detects any Group III Ehrlichia species other than Cowdria and one detects any Group II Ehrlichia species. These probes have been used on PCR-amplified rickettsial 16S rRNA genes from over 200 Amblyomma hebraeum ticks. Control ticks were laboratory-reared and either uninfected or fed on sheep experimentally infected with different cowdria isolates, field ticks were collected from animals in heartwater-endemic areas. All tick-derived DNA samples were also examined by PCR amplification and probing for two other cowdria genes (map1 and pCS20) which have previously been used for heartwater epidemiology. This paper describes the first direct comparison of all currently available DNA probes for heartwater-associated organisms.

scholarly journals Respiration of 13C-Labeled Substrates Added to Soil in the Field and Subsequent 16S rRNA Gene Analysis of 13C-Labeled Soil DNA

2003 ◽  
Vol 69 (3) ◽  
pp. 1614-1622 ◽  
Author(s):  
P. Padmanabhan ◽  
S. Padmanabhan ◽  
C. DeRito ◽  
A. Gray ◽  
D. Gannon ◽  
...  
Keyword(s):  
Organic Matter ◽  
Soil Organic Matter ◽  
16S Rrna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gas Chromatography Mass Spectrometry ◽  
Rrna Gene ◽  
Labeled Compounds ◽  
Field Soil ◽  
Soil Dna

ABSTRACT Our goal was to develop a field soil biodegradation assay using 13C-labeled compounds and identify the active microorganisms by analyzing 16S rRNA genes in soil-derived 13C-labeled DNA. Our biodegradation approach sought to minimize microbiological artifacts caused by physical and/or nutritional disturbance of soil associated with sampling and laboratory incubation. The new field-based assay involved the release of 13C-labeled compounds (glucose, phenol, caffeine, and naphthalene) to soil plots, installation of open-bottom glass chambers that covered the soil, and analysis of samples of headspace gases for 13CO2 respiration by gas chromatography/mass spectrometry (GC/MS). We verified that the GC/MS procedure was capable of assessing respiration of the four substrates added (50 ppm) to 5 g of soil in sealed laboratory incubations. Next, we determined background levels of 13CO2 emitted from naturally occurring soil organic matter to chambers inserted into our field soil test plots. We found that the conservative tracer, SF6, that was injected into the headspace rapidly diffused out of the soil chamber and thus would be of little value for computing the efficiency of retaining respired 13CO2. Field respiration assays using all four compounds were completed. Background respiration from soil organic matter interfered with the documentation of in situ respiration of the slowly metabolized (caffeine) and sparingly soluble (naphthalene) compounds. Nonetheless, transient peaks of 13CO2 released in excess of background were found in glucose- and phenol-treated soil within 8 h. Cesium-chloride separation of 13C-labeled soil DNA was followed by PCR amplification and sequencing of 16S rRNA genes from microbial populations involved with 13C-substrate metabolism. A total of 29 full sequences revealed that active populations included relatives of Arthrobacter, Pseudomonas, Acinetobacter, Massilia, Flavobacterium, and Pedobacter spp. for glucose; Pseudomonas, Pantoea, Acinetobacter, Enterobacter, Stenotrophomonas, and Alcaligenes spp. for phenol; Pseudomonas, Acinetobacter, and Variovorax spp. for naphthalene; and Acinetobacter, Enterobacter, Stenotrophomonas, and Pantoea spp. for caffeine.

scholarly journals Vitellibacter echinoideorum sp. nov., isolated from a sea urchin (Tripneustes gratilla)

2015 ◽  
Vol 65 (Pt_7) ◽  
pp. 2320-2325 ◽  
Author(s):  
Shih-Yao Lin ◽  
Asif Hameed ◽  
Cheng-Zhe Wen ◽  
You-Cheng Liu ◽  
Yi-Han Hsu ◽  
...  
Keyword(s):  
16S Rrna ◽  
Sea Urchins ◽  
Sequence Similarity ◽  
Phylogenetic Analyses ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
The Novel ◽  
Moderate Amount ◽  
Polar Lipid Profile

A Gram-stain-negative, aerobic, rod-shaped, yellow-pigment-producing bacterium (designated strain CC-CZW007T) was isolated from seafood samples (sea urchins) at Penghu Island in Taiwan. Strain CC-CZW007T grew optimally at pH 7.0 and 30 °C in the presence of 3 % (w/v) NaCl. The novel strain shared highest 16S rRNA gene sequence similarity to Vitellibacter vladivostokensis JCM 11732T (96.8 %), Vitellibacter soesokkakensis KCTC 32536T (96.4 %), Vitellibacter nionensis KCTC 32420T (95.8 %) and Vitellibacter aestuarii JCM 15496T (95.6 %) and lower sequence similarity to members of other genera. Phylogenetic analyses based on 16S rRNA genes revealed a distinct taxonomic position attained by strain CC-CZW007T with respect to other species of the genus Vitellibacter. The major fatty acids were iso-C15 : 0 and iso-C17 : 0 3-OH. The polar lipid profile was composed of major amounts of phosphatidylethanolamine, unidentified lipids and aminolipids; a moderate amount of aminophospholipid was also detected. The DNA G+C content was 34.7 mol%. The predominant quinone system was menaquinone (MK-6). On the basis of polyphasic taxonomic evidence presented here, strain CC-CZW007T is proposed to represent a novel species within the genus Vitellibacter, for which the name Vitellibacter echinoideorum sp. nov. is proposed. The type strain is CC-CZW007T ( = BCRC 80886T = JCM 30378T).

scholarly journals Genetic and Biochemical Diversity among Isolates ofPaenibacillus alvei Cultured from Australian Honeybee (Apis mellifera) Colonies

2000 ◽  
Vol 66 (3) ◽  
pp. 1098-1106 ◽  
Author(s):  
Steven P. Djordjevic ◽  
Wendy A. Forbes ◽  
Lisa A. Smith ◽  
Michael A. Hornitzky
Keyword(s):  
Apis Mellifera ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Restriction Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Whole Cell ◽  
Restriction Profiles ◽  
Dna Profiles

ABSTRACT Twenty-five unique CfoI-generated whole-cell DNA profiles were identified in a study of 30 Paenibacillus alvei isolates cultured from honey and diseased larvae collected from honeybee (Apis mellifera) colonies in geographically diverse areas in Australia. The fingerprint patterns were highly variable and readily discernible from one another, which highlighted the potential of this method for tracing the movement of isolates in epidemiological studies. 16S rRNA gene fragments (length, 1,416 bp) for all 30 isolates were enzymatically amplified by PCR and subjected to restriction analysis with DraI, HinfI,CfoI, AluI, FokI, andRsaI. With each enzyme the restriction profiles of the 16S rRNA genes from all 30 isolates were identical (one restriction fragment length polymorphism [RFLP] was observed in theHinfI profile of the 16S rRNA gene from isolate 17), which confirmed that the isolates belonged to the same species. The restriction profiles generated by using DraI,FokI, and HinfI differentiated P. alvei from the phylogenetically closely related speciesPaenibacillus macerans and Paenibacillus macquariensis. Alveolysin gene fragments (length, 1,555 bp) were enzymatically amplified from some of the P. alvei isolates (19 of 30 isolates), and RFLP were detected by using the enzymesCfoI, Sau3AI, and RsaI. Extrachromosomal DNA ranging in size from 1 to 10 kb was detected in 17 of 30 (57%) P. alvei whole-cell DNA profiles. Extensive biochemical heterogeneity was observed among the 28 P. alvei isolates examined with the API 50CHB system. All of these isolates were catalase, oxidase, and Voges-Proskauer positive and nitrate negative, and all produced acid when glycerol, esculin, and maltose were added. The isolates produced variable results for 16 of the 49 biochemical tests; negative reactions were recorded in the remaining 30 assays. The genetic and biochemical heterogeneity inP. alvei isolates may be a reflection of adaptation to the special habitats in which they originated.

scholarly journals Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

2002 ◽  
Vol 68 (10) ◽  
pp. 5064-5081 ◽  
Author(s):  
Alexander Loy ◽  
Angelika Lehner ◽  
Natuschka Lee ◽  
Justyna Adamczyk ◽  
Harald Meier ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Amplification ◽  
Oligonucleotide Microarray ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Sulfate Reducing ◽  
Specific Pcr

ABSTRACT For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).

scholarly journals Prey Range Characterization, Ribotyping, and Diversity of Soil and Rhizosphere Bdellovibriospp. Isolated on Phytopathogenic Bacteria

2000 ◽  
Vol 66 (6) ◽  
pp. 2365-2371 ◽  
Author(s):  
E. Jurkevitch ◽  
D. Minz ◽  
Barak Ramati ◽  
Gili Barel
Keyword(s):  
16S Rrna ◽  
Common Bean ◽  
Pcr Amplification ◽  
Erwinia Carotovora ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Range Analysis ◽  
Pcr Product ◽  
Utilization Patterns ◽  
Range Characterization

ABSTRACT Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp.carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A. tumefaciens was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 × 102 to 6 × 103 and 2.8 × 102 to 2.3 × 104 PFU g−1. A prey range analysis of five soil and rhizosphereBdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of theBdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to theBdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered withBdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.

scholarly journals 16S rRNA Phylogenetic Investigation of the Candidate Division “Korarchaeota”

2006 ◽  
Vol 72 (7) ◽  
pp. 5077-5082 ◽  
Author(s):  
Thomas A. Auchtung ◽  
Cristina D. Takacs-Vesbach ◽  
Colleen M. Cavanaugh
Keyword(s):  
16S Rrna ◽  
Hot Springs ◽  
Phylogenetic Analyses ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Environmental Distribution ◽  
Candidate Division ◽  
High Level ◽  
The 16S Rrna Gene

ABSTRACT The environmental distribution and phylogeny of “Korarchaeota,” a proposed ancient archaeal division, was investigated by using the 16S rRNA gene framework. Korarchaeota-specific primers were designed based on previously published sequences and used to screen a variety of environments. Korarchaeota 16S rRNA genes were amplified exclusively from high temperature Yellowstone National Park hot springs and a 9°N East Pacific Rise deep-sea hydrothermal vent. Phylogenetic analyses of these and all available sequences suggest that Korarchaeota exhibit a high level of endemicity.

scholarly journals Low genetic diversity among Francisella-like endosymbionts within different genotypes of Hyalomma dromedarii ticks infesting camels in Saudi Arabia

2020 ◽  
Vol 13 (7) ◽  
pp. 1462-1472
Author(s):  
Haitham Elbir ◽  
Faisal Almathen ◽  
Ayman Elnahas
Keyword(s):  
Genetic Diversity ◽  
Saudi Arabia ◽  
Population Structure ◽  
16S Rrna ◽  
Sequence Similarity ◽  
Phylogenetic Analyses ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Hyalomma Dromedarii ◽  
Low Genetic Diversity

Background and Aim: Hyalomma dromedarii ticks are vectors of disease agents and hosts of Francisella-like endosymbionts (FLEs). Knowledge about intraspecific genetic variation among H. dromedarii and its Francisella species is limited. The aims of this study were to investigate whether certain H. dromedarii genotypes are specialized in carrying specific Francisella species genotypes and scrutinize the population structure of H. dromedarii ticks in Saudi Arabia. Materials and Methods: We collected 151 H. dromedarii ticks from 33 camels from 13 locations in Saudi Arabia. The second internal transcribed spacer (ITS2), cytochrome c oxidase subunit-1(COI), and 16S rRNA genes were used for single-and multi-locus sequence typing and phylogenetic analyses. H. dromedarii-borne Francisella was screened using the tul4 gene and 16S rRNA Francisella-specific primers followed by amplicon Sanger sequencing. Results: Single-locus typing of ticks using ITS2, 16S rRNA, and COI genes yielded 1, 10, and 31 sequence types (ST), respectively, with pairwise sequence similarity of 100% for ITS2, 99.18-99.86% for COI, and 99.50-99.75% for 16S rRNA. COI sequence analysis indicated a lack of strict geographical structuration, as ST15 was found in both Saudi Arabia and Kenya. In contrast, multilocus sequence typing resolved 148 H. dromedarii ticks into 39 genotypes of ticks and three genotypes of FLEs. The ST2-FLE genotype was carried by the tick genotype ST35, while the ST1-FLE genotype and 41.89% of the ST3-FLE genotype were carried by the tick genotype ST32. Accordingly, there appeared to be no specialization of certain tick genotypes to harbor-specific FLE genotypes. Conclusion: For the 1st time, we have provided an overview of the population structure of H. dromedarii ticks and FLE strains. We found a low level of genetic diversity among FLEs and non-specialized circulation of FLEs among H. dromedarii ticks.

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