scholarly journals Multilocus sequence analysis of Ensifer and related taxa

2007 ◽  
Vol 57 (3) ◽  
pp. 489-503 ◽  
Author(s):  
Miet Martens ◽  
Manuel Delaere ◽  
Renata Coopman ◽  
Paul De Vos ◽  
Monique Gillis ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Housekeeping Genes ◽  
16S Rrna Genes ◽  
New Combination ◽  
Rrna Genes ◽  
Multilocus Sequence Analysis ◽  
Bootstrap Support ◽  
Rrna Gene ◽  
Separate Species

Multilocus sequence analysis (MLSA) was performed on representatives of Ensifer (including species previously assigned to the genus Sinorhizobium) and related taxa. Neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) phylogenies of dnaK, gltA, glnA, recA, thrC and 16S rRNA genes were compared. The data confirm that the potential for discrimination of Ensifer species is greater using MLSA of housekeeping genes than 16S rRNA genes. In incongruence-length difference tests, the 16S rRNA gene was found to be significantly incongruent with the other genes, indicating that this gene should not be used as a single indicator of relatedness in this group. Significant congruence was detected for dnaK, glnA and thrC. Analyses of concatenated sequences of dnaK, glnA and thrC genes yielded very similar NJ, MP and ML trees, with high bootstrap support. In addition, analysis of a concatenation of all six genes essentially produced the same result, levelling out potentially conflicting phylogenetic signals. This new evidence supports the proposal to unite Ensifer and Sinorhizobium in a single genus. Support for an alternative solution preserving the two genera is less strong. In view of the opinions expressed by the Judicial Commission, the name of the genus should be Ensifer, as proposed by Young [Young, J. M. (2003). Int J Syst Evol Microbiol 53, 2107–2110]. Data obtained previously and these new data indicate that Ensifer adhaerens and ‘Sinorhizobium morelense’ are not heterotypic synonyms, but represent separate species. However, transfer to the genus Ensifer is not possible at present because the species name is the subject of a pending Request for an Opinion, which would affect whether a novel species in the genus Ensifer or a new combination based on a basonym would be created.

scholarly journals Identification and classification of the genus Bacteroides by multilocus sequence analysis

Microbiology ◽  
2011 ◽  
Vol 157 (12) ◽  
pp. 3388-3397 ◽  
Author(s):  
Mitsuo Sakamoto ◽  
Moriya Ohkuma
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Sequence Similarity ◽  
Single Gene ◽  
Housekeeping Genes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Multilocus Sequence Analysis ◽  
Rrna Gene

Multilocus sequence analysis (MLSA) was performed on representative species of the genus Bacteroides. Internal fragments of the genes selected, dnaJ, gyrB, hsp60, recA, rpoB and 16S rRNA, were amplified by direct PCR and then sequenced from 38 Bacteroides strains representing 35 species. Neighbour-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP) phylogenies of the individual genes were compared. The data confirm that the potential for discrimination of Bacteroides species is greater using MLSA of housekeeping genes than 16S rRNA genes. Among the housekeeping genes analysed, gyrB was the most informative, followed by dnaJ. Analyses of concatenated sequences (4816 bp) of all six genes revealed robust phylogenetic relationships among different Bacteroides species when compared with the single-gene trees. The NJ, ML and MP trees were very similar, and almost fully resolved relationships of Bacteroides species were obtained, to our knowledge for the first time. In addition, analysis of a concatenation (2457 bp) of the dnaJ, gyrB and hsp60 genes produced essentially the same result. Ten distinct clades were recognized using the SplitsTree4 program. For the genus Bacteroides, we can define species as a group of strains that share at least 97.5 % gene sequence similarity based on the fragments of five protein-coding housekeeping genes and the 16S rRNA gene. This study demonstrates that MLSA of housekeeping genes is a valuable alternative technique for the identification and classification of species of the genus Bacteroides.

scholarly journals The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Ingo C. Starke ◽  
Wilfried Vahjen ◽  
Robert Pieper ◽  
Jürgen Zentek
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Dna Extraction ◽  
16S Rrna Genes ◽  
Read Length ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Variable Regions ◽  
Extraction Procedures ◽  
Primer Sets

In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2×105 sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis.

scholarly journals Molecular characterization of Mycobacterium intracellulare-related strains based on the sequence analysis of hsp65, internal transcribed spacer and 16S rRNA genes

2010 ◽  
Vol 59 (9) ◽  
pp. 1037-1043 ◽  
Author(s):  
Joo-Hee Park ◽  
Tae-Sun Shim ◽  
Seung-Ae Lee ◽  
Hyungki Lee ◽  
In-Kyung Lee ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Internal Transcribed Spacer ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Mycobacterium Intracellulare ◽  
Epidemiological Features ◽  
The 16S Rrna Gene

We investigated the molecular epidemiological features of 94 Mycobacterium intracellulare-related strains, isolated from Korean patients, using sequence analysis targeting 3 independent chronometer molecules, hsp65, the internal transcribed spacer 1 region and the 16S rRNA gene. By collective consideration of these three gene-based approaches, the 94 strains were divided into 5 groups (INT1, INT2, INT3, INT4 and INT5). The frequencies of genotype INT1, 2, 3, 4 and 5 in the 94 isolates were 57.4 % (54), 27.7 % (26), 6.4 % (6), 5.3 % (5) and 3.2 % (3), respectively. When correlations between genotypes and clinical parameters (age, sex, radiological type and the presence of a cavity) were analysed in 78 patients with non-tuberculous mycobacteria pulmonary diseases, no relationships were observed with respect to age, sex and radiological type, but genotype and the presence of a cavity tended to be related (P=0.051).

scholarly journals Multilocus Sequence Analysis of Clinical “Candidatus Neoehrlichia mikurensis” Strains from Europe

2015 ◽  
Vol 53 (10) ◽  
pp. 3126-3132 ◽  
Author(s):  
Anna Grankvist ◽  
Edward R. B. Moore ◽  
Liselott Svensson Stadler ◽  
Sona Pekova ◽  
Christian Bogdan ◽  
...  
Keyword(s):  
Czech Republic ◽  
Sequence Analysis ◽  
16S Rrna ◽  
The Other ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Multilocus Sequence Analysis ◽  
The Czech Republic ◽  
Content Type ◽  
Neoehrlichia Mikurensis

“CandidatusNeoehrlichia mikurensis” is the tick-borne agent of neoehrlichiosis, an infectious disease that primarily affects immunocompromised patients. So far, the genetic variability of “Ca. Neoehrlichia” has been studied only by comparing 16S rRNA genes andgroELoperon sequences. We describe the development and use of a multilocus sequence analysis (MLSA) protocol to characterize the genetic diversity of clinical “Ca. Neoehrlichia” strains in Europe and their relatedness to other species within theAnaplasmataceaefamily. Six genes were selected:ftsZ,clpB,gatB,lipA,groEL, and 16S rRNA. Each MLSA locus was amplified by real-time PCR, and the PCR products were sequenced. Phylogenetic trees of MLSA locus relatedness were constructed from aligned sequences. Blood samples from 12 patients with confirmed “Ca. Neoehrlichia” infection from Sweden (n= 9), the Czech Republic (n= 2), and Germany (n= 1) were analyzed with the MLSA protocol. Three of the Swedish strains exhibited identicallipAsequences, while thelipAsequences of the strains from the other nine patients were identical to each other. One of the Czech strains had one differing nucleotide in theclpBsequence from the sequences of the other 11 strains. All 12 strains had identical sequences for the genes 16S rRNA,ftsZ,gatB, andgroEL. According to the MLSA, among theAnaplasmataceae, “Ca. Neoehrlichia” is most closely related toEhrlichia ruminantium, less so toAnaplasma phagocytophilum, and least toWolbachiaendosymbionts. To conclude, three sequence types of infectious “Ca. Neoehrlichia” were identified: one in the west of Sweden, one in the Czech Republic, and one spread throughout Europe.

scholarly journals Vibrio salilacus sp. nov., a new member of the Anguillarum clade with six alleles of the 16S rRNA gene from a saline lake

2015 ◽  
Vol 65 (Pt_8) ◽  
pp. 2653-2660 ◽  
Author(s):  
Zhi-Ping Zhong ◽  
Ying Liu ◽  
Hong-Can Liu ◽  
Fang Wang ◽  
Yu-Guang Zhou ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Housekeeping Genes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Aerobic Bacterium ◽  
Vibrio Metschnikovii ◽  
The 16S Rrna Gene

A Gram-stain-negative, catalase- and oxidase-positive, facultatively aerobic bacterium, strain DSG-S6T, was isolated from Dasugan Lake (salinity 3.1 %, w/w), China. Its taxonomic position was determined by using a polyphasic approach. Cells of strain DSG-S6T were non-spore-forming, slightly bent rods, and motile by means of a single polar flagellum. Growth occurred in the presence of 0–7.0  % (w/v) NaCl (optimum, 2.0  %), at 4–35 °C (optimum, 30 °C) and at pH 6.0–10.5 (optimum, pH 8.0–8.5). C16 : 0, C18 : 1ω7c and C16 : 1ω7c and/or C16 : 1ω6c were the major fatty acids. Six alleles of the 16S rRNA gene sharing 98.9–99.9  % similarity were detected in strain DSG-S6T, which showed highest 16S rRNA gene sequence similarity to Vibrio aestuarianus ATCC 35048T (97.7  %), then to Vibrio pacinii LMG 19999T (97.6  %) and Vibrio metschnikovii CIP 69.14T (96.8  %). Multilocus sequence analysis of four housekeeping genes and 16S rRNA genes clearly clustered it as a member of the Anguillarum clade. Mean DNA–DNA relatedness between strain DSG-S6T and V. aestuarianus NBRC 15629T, V. pacinii CGMCC 1.12557T and V. metschnikovii JCM 21189T was 20.6 ± 2.3, 38.1 ± 3.5 and 24.2 ± 2.8  %, respectively. The DNA G+C content was 46.8 mol% (T m). Based on the data, it is concluded that strain DSG-S6T represents a novel species of the genus Vibrio, for which the name Vibrio salilacus sp. nov. is proposed. The type strain is DSG-S6T ( = CGMCC 1.12427T = JCM 19265T).

scholarly journals Complete Deletion of the Fucose Operon in Haemophilus influenzae Is Associated with a Cluster in Multilocus Sequence Analysis-Based Phylogenetic Group II Related to Haemophilus haemolyticus: Implications for Identification and Typing

2015 ◽  
Vol 53 (12) ◽  
pp. 3773-3778 ◽  
Author(s):  
Camilla de Gier ◽  
Lea-Ann S. Kirkham ◽  
Niels Nørskov-Lauritsen
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Haemophilus Influenzae ◽  
Phylogenetic Group ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Multilocus Sequence Analysis ◽  
Content Type ◽  
Group Ii ◽  
Complete Deletion

Nonhemolytic variants ofHaemophilus haemolyticusare difficult to differentiate fromHaemophilus influenzaedespite a wide difference in pathogenic potential. A previous investigation characterized a challenging set of 60 clinical strains using multiple PCRs for marker genes and described strains that could not be unequivocally identified as either species. We have analyzed the same set of strains by multilocus sequence analysis (MLSA) and near-full-length 16S rRNA gene sequencing. MLSA unambiguously allocated all study strains to either of the two species, while identification by 16S rRNA sequence was inconclusive for three strains. Notably, the two methods yielded conflicting identifications for two strains. Most of the “fuzzy species” strains were identified asH. influenzaethat had undergone complete deletion of the fucose operon. Such strains, which are untypeable by theH. influenzaemultilocus sequence type (MLST) scheme, have sporadically been reported and predominantly belong to a single branch ofH. influenzaeMLSA phylogenetic group II. We also found evidence of interspecies recombination betweenH. influenzaeandH. haemolyticuswithin the 16S rRNA genes. Establishing an accurate method for rapid and inexpensive identification ofH. influenzaeis important for disease surveillance and treatment.

scholarly journals Sequence analysis of 12S rRNA and 16S rRNA mitochondrial genes in Iranian Afshari sheep

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Mohammad Mahmoodi ◽  
Kian Pahlevan Afshari ◽  
Hamid Reza Seyedabadi ◽  
Mehran Aboozari
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Pcr Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Conservation Strategies ◽  
12S Rrna ◽  
Rrna Gene ◽  
Salting Out ◽  
The Rich

Phylogenetic relationships and genetic variation in Iranian Afshari sheep breed were analyzed using 12S rRNA and 16S rRNA gene sequences. The genomic DNA was isolated by salting out method and amplified 12S rRNA and 16S rRNA genes using PCR method. PCR amplification of 12S and 16S rRNA generated PCR amplicons at 859 and 1053 bp lengths, respectively. Sequence analysis was performed using BioEdit software. Phylogenetic tree was constructed using MEGA software. Phylogenetic analysis of haplotype in the combination with the sheep from GenBank showed that Iranian Afshari sheep made a close to the Australian sheep cluster. This study was found informative for establishing relationships between breeds from different parts of the world. This study may facilitate the future researchers and breeders for better understanding the genetic interactions and breed differentiation for devising future breeding and conservation strategies to preserve the rich animal genetic reservoir of the country.

scholarly journals Optimization of Multilocus Sequence Analysis for Identification of Species in the Genus Vibrio

2014 ◽  
Vol 80 (17) ◽  
pp. 5359-5365 ◽  
Author(s):  
Michael W. Gabriel ◽  
George Y. Matsui ◽  
Robert Friedman ◽  
Charles R. Lovell
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Multilocus Sequence Analysis ◽  
Bootstrap Support ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Accurate Identification ◽  
Content Type

ABSTRACTMultilocus sequence analysis (MLSA) is an important method for identification of taxa that are not well differentiated by 16S rRNA gene sequences alone. In this procedure, concatenated sequences of selected genes are constructed and then analyzed. The effects that the number and the order of genes used in MLSA have on reconstruction of phylogenetic relationships were examined. TherecA,rpoA,gapA, 16S rRNA gene,gyrB, andftsZsequences from 56 species of the genusVibriowere used to construct molecular phylogenies, and these were evaluated individually and using various gene combinations. Phylogenies from two-gene sequences employingrecAandrpoAin both possible gene orders were different. The addition of thegapAgene sequence, producing all six possible concatenated sequences, reduced the differences in phylogenies to degrees of statistical (bootstrap) support for some nodes. The overall statistical support for the phylogenetic tree, assayed on the basis of a reliability score (calculated from the number of nodes having bootstrap values of ≥80 divided by the total number of nodes) increased with increasing numbers of genes used, up to a maximum of four. No further improvement was observed from addition of the fifth gene sequence (ftsZ), and addition of the sixth gene (gyrB) resulted in lower proportions of strongly supported nodes. Reductions in the numbers of strongly supported nodes were also observed when maximum parsimony was employed for tree construction. Use of a small number of gene sequences in MLSA resulted in accurate identification ofVibriospecies.

scholarly journals Molecular characterization of Pseudomonas aeruginosa isolates from Sudanese patients: A cross-sectional study

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 1135
Author(s):  
Reem H. Amoon ◽  
Amna H. Abdallha ◽  
Ahmed Osman Sharif ◽  
Ehssan H. Moglad ◽  
Hisham N. Altyb ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sequence Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Bacterial Disease ◽  
Clinical Specimens

Background:16S rRNA gene sequence analysis is a robust tool for characterization of new pathogens in clinical specimens with suspected bacterial disease. The aim of this study was to characterizePseudomonas aeruginosaisolated from clinical specimens by sequencing the 16S rRNA gene.Methods:Forty bacterial isolates were obtained from different clinical specimens (wound, urine and sputum) using enrichment selective media and biochemical tests to characterize and identify the bacteria asP. aeruginosa.DNA was extracted fromP. aeruginosausing the Chelex method. A universal primer was used to amplify 16S rRNA genes by a conventional PCR technique. The amplified PCR products were sequenced, and the sequences were viewed by Finch TV program version 1.4.0. The identity and similarity of the nucleotide sequence of the isolated strains was detected by comparing them with published sequences using BLASTn. Phylogenetic trees were constructed using Phylogeny.fr software.Results:Sequence analysis by BLASTn displayed high similarity and identity withP. aeruginosafrom China KX461910, Australia JN609194 and with otherP. aeruginosaisolates from the GenBank database.Conclusions:Our observation of isolates from different origin sites, further show the utility of 16s rRNA PCR amplification. This reveals the high specify of the primers and accuracy of the PCR. Thus, 16S rRNA sequencing can be used to identify genetically atypicalP. aeruginosaisolates from different origins.

Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

2015 ◽  
Vol 41 (1) ◽  
pp. 51-58
Author(s):  
Mohammad Shamimul Alam ◽  
Hawa Jahan ◽  
Rowshan Ara Begum ◽  
Reza M Shahjahan
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fish Larva ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Valuable Insight ◽  
Multiple Sequence ◽  
Pcr Rflp ◽  
Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

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