Evaluation of a new fluorimetric DNA–DNA hybridization method

2011 ◽  
Vol 57 (3) ◽  
pp. 250-255 ◽  
Author(s):  
Jennifer Loveland-Curtze ◽  
Vanya I. Miteva ◽  
Jean E. Brenchley
Keyword(s):  
Dna Hybridization ◽  
Bacterial Species ◽  
Sybr Green ◽  
Sybr Green I ◽  
Rrna Gene ◽  
A New Technique ◽  
Thermal Cycler ◽  
Close Relatives ◽  
Taxonomic Groups ◽  
Species Descriptions

Standardized procedures must be followed when characterizing, officially describing, and validly naming novel bacteria. For species descriptions, DNA–DNA hybridization still is needed for whole-genome comparisons between close relatives, but many established hybridization methods have drawbacks, such as requiring labeled or large amounts of DNA. We evaluated a new technique based on the spectrophotometric method in which renaturation rates are used for calculating the degree of binding, which estimates relatedness. In this new approach, DNA is denatured and reassociated in a real-time PCR thermal cycler and the process monitored fluorimetrically using SYBR Green I dye that selectively binds to double-stranded DNA. We investigated the effects of different parameters on the renaturation rates, such as the quantities of DNA and SYBR Green I used. Then using this technique, we calculated the percent binding for pairs of selected bacterial species representing different taxonomic groups and compared our results with published values. We demonstrated that the SYBR Green I method is useful for describing new species and as a screening tool to quickly identify the relatedness of uncharacterized isolates with similar 16S rRNA gene sequences.

scholarly journals Novelty and Uniqueness Patterns of Rare Members of the Soil Biosphere

2008 ◽  
Vol 74 (17) ◽  
pp. 5422-5428 ◽  
Author(s):  
Mostafa S. Elshahed ◽  
Noha H. Youssef ◽  
Anne M. Spain ◽  
Cody Sheik ◽  
Fares Z. Najar ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Tall Grass ◽  
Data Set ◽  
Bacterial Phyla ◽  
Rare Biosphere ◽  
Soil Bacterial ◽  
Close Relatives ◽  
Taxonomic Groups

ABSTRACT Soil bacterial communities typically exhibit a distribution pattern in which most bacterial species are present in low abundance. Due to the relatively small size of most culture-independent sequencing surveys, a detailed phylogenetic analysis of rare members of the community is lacking. To gain access to the rarely sampled soil biosphere, we analyzed a data set of 13,001 near-full-length 16S rRNA gene clones derived from an undisturbed tall grass prairie soil in central Oklahoma. Rare members of the soil bacterial community (empirically defined at two different abundance cutoffs) represented 18.1 to 37.1% of the total number of clones in the data set and were, on average, less similar to their closest relatives in public databases when compared to more abundant members of the community. Detailed phylogenetic analyses indicated that members of the soil rare biosphere either belonged to novel bacterial lineages (members of five novel bacterial phyla identified in the data set, as well as members of multiple novel lineages within previously described phyla or candidate phyla), to lineages that are prevalent in other environments but rarely encountered in soil, or were close relatives to more abundant taxa in the data set. While a fraction of the rare community was closely related to more abundant taxonomic groups in the data set, a significant portion of the rare biosphere represented evolutionarily distinct lineages at various taxonomic cutoffs. We reason that these novelty and uniqueness patterns provide clues regarding the origins and potential ecological roles of members of the soil's rare biosphere.

scholarly journals Mucilaginibacter polysacchareus sp. nov., an exopolysaccharide-producing bacterial species isolated from the rhizoplane of the herb Angelica sinensis

2012 ◽  
Vol 62 (Pt_3) ◽  
pp. 632-637 ◽  
Author(s):  
Song-Ih Han ◽  
Hyo-Jin Lee ◽  
Hae-Ran Lee ◽  
Ki-Kwang Kim ◽  
Kyung-Sook Whang
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Hybridization ◽  
Gene Sequence ◽  
Novel Species ◽  
Bacterial Species ◽  
Angelica Sinensis ◽  
Rrna Gene ◽  
The 16S Rrna Gene ◽  
Sequence Similarities

Three exopolysaccharide-producing bacteria, designated strains DRP28T, DRP29 and DRP31, were isolated from the rhizoplane of Angelica sinensis from the Geumsan, Republic of Korea. Cells were straight rods, Gram reaction-negative, aerobic, non-motile, and catalase- and oxidase- positive. Flexirubin-type pigments were absent. Phylogenetic analysis of the 16S rRNA gene indicated that these bacteria belong to the genus Mucilaginibacter in the phylum Bacteroidetes. 16S rRNA gene sequence similarities to strains of recognized species of the genus Mucilaginibacter were 93.8–97.4 %. The major fatty acids were iso-C15 : 0 and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). The strains contained MK-7 as the major isoprenoid quinone. Strains DRP28T, DRP29 and DRP31 formed a single, distinct genomospecies with DNA G+C contents of 41.9–42.7 mol% and DNA hybridization values of 82.6–86.8 %; the strains exhibited DNA–DNA hybridization values of only 20.4–41.3 % with related species of the genus Mucilaginibacter. On the basis of evidence presented in this study, strains DRP28T, DRP29 and DRP31 were considered to represent a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter polysacchareus sp. nov. is proposed. The type strain is DRP28T ( = KACC 15075T  = NBRC 107757T).

scholarly journals Proposal of Lentzea deserti (Okoro et al. 2010) Nouioui et al. 2018 as a later heterotypic synonym of Lentzea atacamensis (Okoro et al. 2010) Nouioui et al. 2018 and an emended description of Lentzea atacamensis

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246533
Author(s):  
Mo Ping ◽  
Zhao Yun-Lin ◽  
Liu Jun ◽  
Gao Jian ◽  
Xu Zheng-Gang
Keyword(s):  
Dna Hybridization ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Gene Sequence Analysis ◽  
Emended Description ◽  
Relationship Of ◽  
Type Strains ◽  
The 16S Rrna Gene

The taxonomic relationship of Lentzea atacamensis and Lentzea deserti were re-evaluated using comparative genome analysis. The 16S rRNA gene sequence analysis indicated that the type strains of L. atacamensis and L. deserti shared 99.7% sequence similarity. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the genomes of two type strains were 88.6% and 98.8%, respectively, greater than the two recognized thresholds values of 70% dDDH and 95–96% ANI for bacterial species delineation. These results suggested that L. atacamensis and L. deserti should share the same taxonomic position. And this conclusion was further supported by similar phenotypic and chemotaxonomic features between them. Therefore, we propose that L. deserti is a later heterotypic synonym of L. atacamensis.

scholarly journals Microbacterium azadirachtae sp. nov., a plant-growth-promoting actinobacterium isolated from the rhizoplane of neem seedlings

2010 ◽  
Vol 60 (7) ◽  
pp. 1687-1692 ◽  
Author(s):  
Munusamy Madhaiyan ◽  
Selvaraj Poonguzhali ◽  
Jung-Sook Lee ◽  
Keun-Chul Lee ◽  
Venkatakrishnan Sivaraj Saravanan ◽  
...  
Keyword(s):  
Dna Hybridization ◽  
Genetic Characterization ◽  
Novel Species ◽  
Botanical Garden ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Light Yellow ◽  
Plant Growth Promoting ◽  
Growth Promoting ◽  
Close Relatives

Microbacterium strain AI-S262T was isolated from the rhizoplane of neem seedlings in the Botanical garden of Tamilnadu Agricultural University, Coimbatore, India, and subjected to phenotypic, chemotaxonomic and genetic characterization. Cells of this strain were Gram-stain-positive, motile, non-spore-forming, short rods and formed light-yellow-pigmented colonies on nutrient agar. Strain AI-S262T contained MK-12 and MK-13 as the main respiratory quinones, anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0 as the predominant fatty acids, peptidoglycan-type B2β with glycolyl residues, and had a DNA G+C content of 69.5 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed 98.0–98.6 % pair-wise similarity with respect to close relatives in the genus Microbacterium. DNA–DNA hybridization experiments revealed a low level of DNA–DNA relatedness (less than 39%) between strain AI-S262T and its closest relatives. Data from DNA–DNA hybridization and phenotypic analyses supported the conclusion that strain AI-S262T represents a novel species in the genus Microbacterium, for which the name Microbacterium azadirachtae sp. nov. is proposed. The type strain is AI-S262T (=JCM 15681T =LMG 24772T =KCTC 19668T).

Reclassification of Herbaspirillum putei as a later heterotypic synonym of Herbaspirillum huttiense, with the description of H. huttiense subsp. huttiense subsp. nov. and H. huttiense subsp. putei subsp. nov., comb. nov., and description of Herbaspirillum aquaticum sp. nov.

2010 ◽  
Vol 60 (6) ◽  
pp. 1418-1426 ◽  
Author(s):  
Anatoly P. Dobritsa ◽  
M. C. S. Reddy ◽  
Mansour Samadpour
Keyword(s):  
Dna Hybridization ◽  
Type Strain ◽  
Novel Species ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Phenotypic Characteristics ◽  
Phylogenetic Relatedness ◽  
The 16S Rrna Gene

Resequencing of the 16S rRNA gene of the type strain of Herbaspirillum putei Ding and Yokota 2004 revealed 99.9 % sequence similarity to that of the type strain of Herbaspirillum huttiense (Leifson 1962) Ding and Yokota 2004. This high phylogenetic relatedness of H. putei and H. huttiense was confirmed by the results of DNA–DNA hybridization between H. huttiense DSM 10281T and H. putei ATCC BAA-806T (reassociation value 96 %). Therefore, it is proposed to reclassify the type strain of H. putei as a strain of H. huttiense. However, the genome of the type strain of H. putei is about 0.9 Mb larger than that of the H. huttiense type strain. This results in a decrease in the reassociation value in the reciprocal DNA–DNA hybridization to 72 %, a level slightly above the threshold for delineating bacterial species. These data and distinctive phenotypic characteristics indicate that the name Herbaspirillum putei is a later heterotypic synonym of Herbaspirillum huttiense and permit the description of two novel subspecies, Herbaspirillum huttiense subsp. huttiense subsp. nov. (type strain ATCC 14670T =JCM 21423T =DSM 10281T) and Herbaspirillum huttiense subsp. putei subsp. nov., comb. nov. (type strain 7-2T =JCM 21495T =ATCC BAA-806T). Three bacterial strains, IEH 4430T, IEH 4515 and IEH 8757, isolated from water were found to be the closest relatives of these strains. Strain IEH 8757 was classified as a strain of H. huttiense subsp. putei. Studies of genotypic and phenotypic features of strains IEH 4430T and IEH 4515 showed that the strains represent a novel species, which is most closely related to H. huttiense and for which the name Herbaspirillum aquaticum sp. nov. is proposed (type strain IEH 4430T =DSM 21191T =ATCC BAA-1628T).

scholarly journals Vibrio comitans sp. nov., Vibrio rarus sp. nov. and Vibrio inusitatus sp. nov., from the gut of the abalones Haliotis discus discus, H. gigantea, H. madaka and H. rufescens

2007 ◽  
Vol 57 (5) ◽  
pp. 916-922 ◽  
Author(s):  
Tomoo Sawabe ◽  
Yusuke Fujimura ◽  
Kentaro Niwa ◽  
Hideaki Aono
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
Dna Hybridization ◽  
Type Strain ◽  
Sequence Similarity ◽  
Phylogenetic Analyses ◽  
Bacterial Species ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Haliotis Discus

Nine alginolytic, facultatively anaerobic, non-motile bacteria were isolated from the guts of the abalones Haliotis discus discus, H. gigantea, H. madaka and H. rufescens. Phylogenetic analyses based on 16S rRNA gene sequences indicated that these bacteria were closely related to Vibrio superstes G3-29T (98.6–99.3 % sequence similarity). DNA–DNA hybridization and phylogenetic analysis based on the gapA gene demonstrated that six strains constituted one bacterial species, two strains represented a second species and one strain represented a third species. The three novel bacterial species were different from all currently known vibrios. The names Vibrio comitans sp. nov. (type strain GHG2-1T=LMG 23416T=NBRC 102076T; DNA G+C content 45.0–48.0 mol%), Vibrio inusitatus sp. nov. (type strain RW14T=LMG 23434T=NBRC 102082T; DNA G+C content 43.1–43.7 mol%) and Vibrio rarus sp. nov. (type strain RW22T=LMG 23674T=NBRC 102084T; DNA G+C content 43.8 mol%) are proposed to encompass these new taxa. Several phenotypic features were revealed that discriminate V. comitans, V. rarus and V. inusitatus from other Vibrio species.

scholarly journals Quantitative Detection of Corynebacterium casei in Cheese by Real-Time PCR

2006 ◽  
Vol 72 (11) ◽  
pp. 6972-6979 ◽  
Author(s):  
Christophe Monnet ◽  
Karine Correia ◽  
Anne-Sophie Sarthou ◽  
Françoise Irlinger
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Sybr Green ◽  
Sybr Green I ◽  
Quantitative Detection ◽  
Rrna Gene ◽  
Polymorphism Analysis

ABSTRACT The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 105 to 1010 CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 105 CFU equivalents of C. casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C. casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses.

scholarly journals Kordia zhangzhouensis sp. nov., isolated from surface freshwater

2015 ◽  
Vol 65 (Pt_10) ◽  
pp. 3379-3383 ◽  
Author(s):  
Juan Du ◽  
Yang Liu ◽  
Qiliang Lai ◽  
Chunming Dong ◽  
Yanrong Xie ◽  
...  
Keyword(s):  
Dna Hybridization ◽  
Type Strain ◽  
Novel Species ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Gram Stain ◽  
16S Rrna Gene Sequences ◽  
Optimum Ph ◽  
Pr China ◽  
Type Strains

An aerobic, Gram-stain-negative, rod-shaped and non-motile bacterium, JS14SB-1T, was isolated from the surface freshwater of the Jiulong River, PR China. Strain JS14SB-1T grew at 15–38 °C (optimum, 28–35 °C), at pH 6.0–9.0 (optimum pH 7.0) and in the presence of 1.0–7.0 % (w/v) NaCl [optimum 3.0–5.0 % (w/v)]. Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that strain JS14SB-1T was affiliated to the genus Kordia, sharing low similarities (95.1–97.1 %) to all type strains of species of this genus. The digital DNA–DNA hybridization (DDH) value between strain JS14SB-1T and the closely related strain Kordia jejudonensis SSK3-3T was 20.70 ± 2.33 % and far below the 70 % DDH value taken as the gold standard for delineation of bacterial species. The major fatty acids were identified as iso-C15 : 0 and iso-C17 : 0 3-OH. The polar lipids were phosphatidylethanolamine, glycolipid, aminolipid, several unidentified phospholipids and lipids. The predominant menaquinone was MK-6. The G+C content of the genomic DNA was 33.8 mol%. Based on the phenotypic, phylogenetic and chemotaxonomic distinctiveness, strain JS14SB-1T is considered to represent a novel species of the genus Kordia, for which the name Kordia zhangzhouensis sp. nov. is proposed; the type strain is JS14SB-1T ( = MCCC 1A00726T = KCTC 42140T).

Sign in / Sign up

Export Citation Format

Share Document