Comparative Study of PCR-Based Approaches for the Genetic Characterization of Three Strains of Acidithiobacillus caldus Isolated from Different Sites in China

Comparative study of the genetic characteristics among three Acidithiobacillus caldus strains isolated from different typical environments in China was performed using a combination of molecular methods, namely sequencing analysis of PCR-amplified 16S rRNA genes and 16S-23S rRNA gene intergenic spacers (ITS), repetitive element PCR (rep-PCR), arbitrarily primed PCR (AP-PCR) fingerprinting and random amplified polymorphic DNA (RAPD). Both of the 16S rRNA gene and 16S-23S rRNA gene intergenic spacers sequences of the three strains exhibited small variations, with 99.9-100%, 99.7-100% identity respectively. In contrast, according to the analysis of bacterial diversity based on rep-PCR and AP-PCR fingerprinting, they produced highly discriminatory banding patterns, and the similarity values between them varied from 61.97% to 71.64%. RAPD analysis showed that banding profiles of their genomic DNA exhibited obvious differences from each other with 53.44-75% similarity. These results suggested that in contrast to 16S rRNA genes and 16S-23S rRNA gene intergenic spacers sequencing analysis, rep-PCR, AP-PCR fingerprinting and RAPD analysis possessed higher discriminatory power in identifying these closely related strains. And they could be used as rapid and highly discriminatory typing techniques in studying bacterial diversity, especially in differentiating bacteria within Acidithiobacillus caldus.

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Phylogenetic relationships within the family Halomonadaceae based on comparative 23S and 16S rRNA gene sequence analysis

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.013979-0 ◽

2010 ◽

Vol 60 (4) ◽

pp. 737-748 ◽

Cited By ~ 43

Author(s):

Rafael R. de la Haba ◽

David R. Arahal ◽

M. Carmen Márquez ◽

Antonio Ventosa

Keyword(s):

16S Rrna ◽

16S Rrna Genes ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Gene Sequences ◽

The Family ◽

23S Rrna Gene ◽

Group 2 ◽

Group 1

A phylogenetic study of the family Halomonadaceae was carried out based on complete 16S rRNA and 23S rRNA gene sequences. Several 16S rRNA genes of type strains were resequenced, and 28 new sequences of the 23S rRNA gene were obtained. Currently, the family includes nine genera (Carnimonas, Chromohalobacter, Cobetia, Halomonas, Halotalea, Kushneria, Modicisalibacter, Salinicola and Zymobacter). These genera are phylogenetically coherent except Halomonas, which is polyphyletic. This genus comprises two clearly distinguished clusters: group 1 includes Halomonas elongata (the type species) and the species Halomonas eurihalina, H. caseinilytica, H. halmophila, H. sabkhae, H. almeriensis, H. halophila, H. salina, H. organivorans, H. koreensis, H. maura and H. nitroreducens. Group 2 comprises the species Halomonas aquamarina, H. meridiana, H. axialensis, H. magadiensis, H. hydrothermalis, H. alkaliphila, H. venusta, H. boliviensis, H. neptunia, H. variabilis, H. sulfidaeris, H. subterranea, H. janggokensis, H. gomseomensis, H. arcis and H. subglaciescola. Halomonas salaria forms a cluster with Chromohalobacter salarius and the recently described genus Salinicola, and their taxonomic affiliation requires further study. More than 20 Halomonas species are phylogenetically not within the core constituted by the Halomonas sensu stricto cluster (group 1) or group 2 and, since their positions on the different phylogenetic trees are not stable, they cannot be recognized as additional groups either. In general, there is excellent agreement between the phylogenies based on the two rRNA gene sequences, but the 23S rRNA gene showed higher resolution in the differentiation of species of the family Halomonadaceae.

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Reassessment of the phylogenetic relationships of Thiomonas cuprina

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.65537-0 ◽

2007 ◽

Vol 57 (11) ◽

pp. 2720-2724 ◽

Cited By ~ 22

Author(s):

Donovan P. Kelly ◽

Yoshihito Uchino ◽

Harald Huber ◽

Ricardo Amils ◽

Ann P. Wood

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Gene Sequence ◽

16S Rrna Genes ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Significant Similarity ◽

23S Rrna Gene ◽

The 16S Rrna Gene

The published sequence of the 16S rRNA gene of Thiomonas cuprina strain Hö5 (=DSM 5495T) (GenBank accession no. U67162) was found to be erroneous. The 16S rRNA genes from the type strain held by the DSMZ since 1990 (DSM 5495T =NBRC 102145T) and strain Hö5 maintained frozen in the Universität Regensburg for 23 years (=NBRC 102094) were sequenced and found to be identical, but to show no significant similarity to the U67162 sequence. This also casts some doubt on the previously published 5S and 23S rRNA gene sequences (GenBank accession nos U67171 and X75567). The correct 16S rRNA gene sequence showed 99.8 % identity to those from Thiomonas delicata NBRC 14566T and ‘Thiomonas arsenivorans’ DSM 16361. The properties of these three species are re-evaluated, and emended descriptions are provided for the genus Thiomonas and the species Thiomonas cuprina.

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rDNA analyses of planktonic heterocystous cyanobacteria, including members of the genera Anabaenopsis and Cyanospira The GenBank accession numbers of the 16S rDNA gene sequences reported in this paper are AY038032–AY038037.

Microbiology ◽

10.1099/00221287-148-2-481 ◽

2002 ◽

Vol 148 (2) ◽

pp. 481-496 ◽

Cited By ~ 76

Author(s):

Isabelle Iteman ◽

Rosmarie Rippka ◽

Nicole Tandeau de Marsac ◽

Michael Herdman

Keyword(s):

16S Rrna ◽

16S Rdna ◽

Genomic Sequence ◽

Sequence Similarity ◽

16S Rrna Genes ◽

Rrna Genes ◽

23S Rrna ◽

Phylogenetic Position ◽

Rrna Gene ◽

Heterocystous Cyanobacteria

The taxonomic coherence and phylogenetic relationships of 11 planktonic heterocystous cyanobacterial isolates were examined by investigating two areas of the rRNA operon, the 16S rRNA gene (rrnS) and the internal transcribed spacer (ITS) located between the 16S rRNA and 23S rRNA genes. The rrnS sequences were determined for five strains, including representatives of Anabaena flos-aquae, Aphanizomenon flos-aquae, Nodularia sp. and two alkaliphilic planktonic members of the genera Anabaenopsis and Cyanospira, whose phylogenetic position was previously unknown. Comparison of the data with those previously published for individual groups of planktonic heterocystous cyanobacteria showed that, with the exception of members assigned to the genus Cylindrospermopsis, all the planktonic strains form a distinct subclade within the monophyletic clade of heterocystous cyanobacteria. Within this subclade five different phylogenetic clusters were distinguished. The phylogenetic groupings of Anabaena and Aphanizomenon strains within three of these clusters were not always consistent with their generic or specific assignments based on classical morphological definitions, and the high degree of sequence similarity between strains of Anabaenopsis and Cyanospira suggests that they may be assignable to a single genus. Ribotyping and additional studies performed on PCR amplicons of the 16S rDNA or the ITS for the 11 planktonic heterocystous strains demonstrated that they all contain multiple rrn operons and ITS regions of variable size. Finally, evidence is provided for intra-genomic sequence heterogeneity of the 16S rRNA genes within most of the individual isolates.

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Mycoplasma anserisalpingitidis sp. nov., isolated from European domestic geese (Anser anser domesticus) with reproductive pathology

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijsem.0.004052 ◽

2020 ◽

Vol 70 (4) ◽

pp. 2369-2381 ◽

Cited By ~ 8

Author(s):

Dmitriy V. Volokhov ◽

Dénes Grózner ◽

Miklós Gyuranecz ◽

Naola Ferguson-Noel ◽

Yamei Gao ◽

...

Keyword(s):

16S Rrna ◽

Type Strain ◽

Type Species ◽

Novel Species ◽

16S Rrna Genes ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Content Type ◽

Link Type

In 1983, Mycoplasma sp. strain 1220 was isolated in Hungary from the phallus lymph of a gander with phallus inflammation. Between 1983 and 2017, Mycoplasma sp. 1220 was also identified and isolated from the respiratory tract, liver, ovary, testis, peritoneum and cloaca of diseased geese in several countries. Seventeen studied strains produced acid from glucose and fructose but did not hydrolyse arginine or urea, and all grew under aerobic, microaerophilic and anaerobic conditions at 35 to 37 ˚C in either SP4 or pleuropneumonia-like organism medium supplemented with glucose and serum. Colonies on agar showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma cellular morphology. Molecular characterization included analysis of the following genetic loci: 16S rRNA, 23S rRNA, 16S–23S rRNA ITS, rpoB, rpoC, rpoD, uvrA, parC, topA, dnaE, fusA and pyk. The genome was sequenced for type strain 1220T. The 16S rRNA gene sequences of studied strains of Mycoplasma sp. 1220 shared 99.02–99.19 % nucleotide similarity with M. anatis strains but demonstrated ≤95.00–96.70 % nucleotide similarity to the 16S rRNA genes of other species of the genus Mycoplasma . Phylogenetic, average nucleotide and amino acid identity analyses revealed that the novel species was most closely related to Mycoplasma anatis . Based on the genetic data, we propose a novel species of the genus Mycoplasma , for which the name Mycoplasma anserisalpingitidis sp. nov. is proposed with the type strain 1220T (=ATCC BAA-2147T=NCTC 13513T=DSM 23982T). The G+C content is 26.70 mol%, genome size is 959110 bp.

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Genetic diversity and phylogeny of rhizobia isolated from agroforestry legume species in southern Ethiopia

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.63534-0 ◽

2005 ◽

Vol 55 (4) ◽

pp. 1439-1452 ◽

Cited By ~ 63

Author(s):

Endalkachew Wolde-meskel ◽

Zewdu Terefework ◽

Åsa Frostegård ◽

Kristina Lindström

Keyword(s):

Genetic Diversity ◽

16S Rrna ◽

16S Rrna Gene ◽

Rrna Genes ◽

Partial Sequence ◽

23S Rrna ◽

Southern Ethiopia ◽

Rrna Gene ◽

Pcr Rflp ◽

23S Rrna Gene

The genetic diversity within 195 rhizobial strains isolated from root nodules of 18 agroforestry species (15 woody and three herbaceous legumes) growing in diverse ecoclimatic zones in southern Ethiopia was investigated by using PCR–RFLP of the ribosomal operon [16S rRNA gene, 23S rRNA gene and the internal transcribed spacer (ITS) region between the 16S rRNA and 23S rRNA genes] and 16S rRNA gene partial sequence (800 and 1350 bp) analyses. All of the isolates and the 28 reference strains could be differentiated by using these methods. The size of the ITS varied among test strains (500–1300 bp), and 58 strains contained double copies. UPGMA dendrograms generated from cluster analyses of the 16S and 23S rRNA gene PCR–RFLP data were in good agreement, and the combined distance matrices delineated 87 genotypes, indicating considerable genetic diversity among the isolates. Furthermore, partial sequence analysis of 67 representative strains revealed 46 16S rRNA gene sequence types, among which 12 were 100 % similar to those of previously described species and 34 were novel sequences with 94–99 % similarity to those of recognized species. The phylogenetic analyses suggested that strains indigenous to Ethiopia belonged to the genera Agrobacterium, Bradyrhizobium, Mesorhizobium, Methylobacterium, Rhizobium and Sinorhizobium. Many of the rhizobia isolated from previously uninvestigated indigenous woody legumes had novel 16S rRNA gene sequences and were phylogenetically diverse. This study clearly shows that the characterization of symbionts of unexplored legumes growing in previously unexplored biogeographical areas will reveal additional diversity.

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Species-Specific PCR for Identification of Common Contaminant Mollicutes in Cell Culture

Applied and Environmental Microbiology ◽

10.1128/aem.67.7.3195-3200.2001 ◽

2001 ◽

Vol 67 (7) ◽

pp. 3195-3200 ◽

Cited By ~ 46

Author(s):

Fanrong Kong ◽

Gregory James ◽

Susanna Gordon ◽

Anna Zelynski ◽

Gwendolyn L. Gilbert

Keyword(s):

16S Rrna ◽

Cell Cultures ◽

Bacterial Species ◽

Intergenic Spacer ◽

16S Rrna Genes ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Specific Pcr ◽

Species Specific

ABSTRACT Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5′ end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5′ end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.

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Epibacterium ulvae gen. nov., sp. nov., epibiotic bacteria isolated from the surface of a marine alga

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.042838-0 ◽

2013 ◽

Vol 63 (Pt_5) ◽

pp. 1589-1596 ◽

Cited By ~ 20

Author(s):

Anahit Penesyan ◽

Sven Breider ◽

Peter Schumann ◽

Brian J. Tindall ◽

Suhelen Egan ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Type Species ◽

Marine Alga ◽

16S Rrna Genes ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Content Type ◽

Link Type

Two Gram-reaction-negative, rod-shaped, motile bacteria, designated strains U82 and U95T, were isolated from the marine alga Ulva australis collected at Sharks Point, Clovelly, a rocky intertidal zone near Sydney, Australia. Both strains were oxidase- and catalase-positive, formed brown- to black-pigmented colonies and required NaCl for growth. Phylogenetic analysis based on nearly complete 16S rRNA gene sequences revealed that these strains belong to the Roseobacter clade within the Alphaproteobacteria . The 16S rRNA genes of both strains were identical across the sequenced 1326 nt, but showed differences in the intergenic spacer region (ITS) between the 16S and the 23S rRNA genes. At the genomic level the DNA G+C contents of strains U82 and U95T were identical (52.6 mol%) and they had a DNA–DNA hybridization value of 83.7 %, suggesting that these strains belong to the same species. The closest described phylogenetic neighbour to strains U82 and U95T was Thalassobius aestuarii DSM 15283T with 95.8 % 16S rRNA gene sequence similarity. Other close relatives include further species of the genera Thalassobius and Shimia . Strains U82 and U95T were negative for bacteriochlorophyll a production, showed antibacterial activity towards other marine bacteria, were resistant to the antibiotics gentamicin and spectinomycin and were unable to hydrolyse starch or gelatin. The major fatty acids (>1 %) were 18 : 1ω7c, 16 : 0, 18 : 2, 10 : 0 3-OH, 12 : 0, 20 : 1 2-OH and 18 : 0. The polar lipid pattern indicated the presence of phosphatidylglycerol, phosphatidylcholine, two unidentified aminolipids and four unidentified phospholipids. Both strains produced ubiquinone 10 (Q-10) as the sole respiratory lipoquinone. Based on their phenotypic and phylogenetic characteristics, it is suggested that strains U82 and U95T are members of a novel species within a new genus for which the name Epibacterium ulvae gen. nov., sp. nov. is proposed. The type strain of the type species is U95T ( = DSM 24752T = LMG 26464T).

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‘Candidatus Mycoplasma haemoalbiventris’ and tick-borne pathogens screening in white-eared opossums (Didelphis albiventris) from Curitiba and Foz do Iguaçu Cities, Paraná State, southern Brazil

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612021072 ◽

2021 ◽

Vol 30 (4) ◽

Author(s):

Renata Prestes Antonangelo de Oliveira ◽

Flávia Carolina Meira Collere ◽

Larissa Dantas Roeder Ferrari ◽

Vanessa dos Santos Coradi ◽

Nathália de Albuquerque Soares ◽

...

Keyword(s):

16S Rrna ◽

Clinical Signs ◽

16S Rrna Genes ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Southern Brazil ◽

Specific Pcr ◽

Gapdh Gene ◽

Didelphis Albiventris

Abstract Hemoplasmas are epierythrocytic bacteria that infect mammals. ‘Candidatus Mycoplasma haemoalbiventris’ was detected in white-eared opossums (Didelphis albiventris) from southern and central-western Brazil. The present study aimed at: i) screening opossums for tick-borne (TBP) pathogens (Piroplasmida and Anaplasmataceae) and ii) detecting and characterizing hemoplasma species infecting opossums from Curitiba and Foz do Iguaçu cities in the Paraná State, southern Brazil. Thirty blood samples from white-eared opossums were evaluated by PCR assays. Animals were not infested by ectoparasites. The mammalian endogenous gapdh gene was consistently amplified in all samples. All opossums tested negative for Theileria/Babesia spp. and Ehrlichia/Anaplasma spp. by PCR based on 18S rRNA and 16S rRNA genes, respectively. A genus-specific PCR assay based on the 16S rRNA gene of hemoplasmas showed that three/13 (23.08%; CI 95%: 8.18-50.26%) opossums from Foz do Iguaçu were positive for hemotropic Mycoplasma sp. All opossums from Curitiba tested negative for hemoplasmas. Sequencing of both the 16S and 23S rRNA genes revealed that the animals were infected by ‘Ca. M. haemoalbiventris’. Although ‘Ca. M. haemoalbiventris’ is prevalent in opossums in Brazil, clinical signs associated with its infection and its putative vectors remain unknown.

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Phylogenetic Ecology of the Freshwater Actinobacteria acI Lineage

Applied and Environmental Microbiology ◽

10.1128/aem.00794-07 ◽

2007 ◽

Vol 73 (22) ◽

pp. 7169-7176 ◽

Cited By ~ 141

Author(s):

Ryan J. Newton ◽

Stuart E. Jones ◽

Matthew R. Helmus ◽

Katherine D. McMahon

Keyword(s):

16S Rrna ◽

Community Composition ◽

Strong Predictor ◽

Regional Scale ◽

16S Rrna Genes ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Data Set ◽

Biogeographic Patterns

ABSTRACT The acI lineage of freshwater Actinobacteria is a cosmopolitan and often numerically dominant member of lake bacterial communities. We conducted a survey of acI 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions from 18 Wisconsin lakes and used standard nonphylogenetic and phylogenetic statistical approaches to investigate the factors that determine acI community composition at the local scale (within lakes) and at the regional scale (across lakes). Phylogenetic reconstruction of 434 acI 16S rRNA genes revealed a well-defined and highly resolved phylogeny. Eleven previously unrecognized monophyletic clades, each with ≥97.9% within-clade 16S rRNA gene sequence identity, were identified. Clade community similarity positively correlated with lake environmental similarity but not with geographic distance, implying that the lakes represent a single biotic region containing environmental filters for communities that have similar compositions. Phylogenetically disparate clades within the acI lineage were most abundant at the regional scale, and local communities were comprised of more closely related clades. Lake pH was a strong predictor of the community composition, but only when lakes with a pH below 6 were included in the data set. In the remaining lakes (pH above 6) biogeographic patterns in the landscape were instead a predictor of the observed acI community structure. The nonrandom distribution of the newly defined acI clades suggests potential ecophysiological differences between the clades, with acI clades AI, BII, and BIII preferring acidic lakes and acI clades AII, AVI, and BI preferring more alkaline lakes.

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Discordant 16S and 23S rRNA Gene Phylogenies for the Genus Helicobacter: Implications for Phylogenetic Inference and Systematics

Journal of Bacteriology ◽

10.1128/jb.187.17.6106-6118.2005 ◽

2005 ◽

Vol 187 (17) ◽

pp. 6106-6118 ◽

Cited By ~ 91

Author(s):

Floyd E. Dewhirst ◽

Zeli Shen ◽

Michael S. Scimeca ◽

Lauren N. Stokes ◽

Tahani Boumenna ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Gene Sequence ◽

Sequence Data ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Rrna Gene Sequence ◽

23S Rrna Gene ◽

23S Rrna Genes

ABSTRACT Analysis of 16S rRNA gene sequences has become the primary method for determining prokaryotic phylogeny. Phylogeny is currently the basis for prokaryotic systematics. Therefore, the validity of 16S rRNA gene-based phylogenetic analyses is of fundamental importance for prokaryotic systematics. Discrepancies between 16S rRNA gene analyses and DNA-DNA hybridization and phenotypic analyses have been noted in the genus Helicobacter. To clarify these discrepancies, we sequenced the 23S rRNA genes for 55 helicobacter strains representing 41 taxa (>2,700 bases per sequence). Phylogenetic-tree construction using neighbor-joining, parsimony, and maximum likelihood methods for 23S rRNA gene sequence data yielded stable trees which were consistent with other phenotypic and genotypic methods. The 16S rRNA gene sequence-derived trees were discordant with the 23S rRNA gene trees and other data. Discrepant 16S rRNA gene sequence data for the helicobacters are consistent with the horizontal transfer of 16S rRNA gene fragments and the creation of mosaic molecules with loss of phylogenetic information. These results suggest that taxonomic decisions must be supported by other phylogenetically informative macromolecules, such as the 23S rRNA gene, when 16S rRNA gene-derived phylogeny is discordant with other credible phenotypic and genotypic methods. This study found Wolinella succinogenes to branch with the unsheathed-flagellum cluster of helicobacters by 23S rRNA gene analyses and whole-genome comparisons. This study also found intervening sequences (IVSs) in the 23S rRNA genes of strains of 12 Helicobacter species. IVSs were found in helices 10, 25, and 45, as well as between helices 31′ and 27′. Simultaneous insertion of IVSs at three sites was found in H. mesocricetorum.

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