Intra-chromosomal heterogeneity between the four 16S rRNA gene copies in the genus Veillonella: implications for phylogeny and taxonomy

Among the seven species characterized within the genus Veillonella, three (Veillonella dispar, Veillonella parvula and Veillonella atypica) have so far been isolated from human flora and during infectious processes. Sequencing and analysis of 16S rDNA (rrs) has been described as the best method for identification of Veillonella strains at the species level since phenotypic characteristics are unable to differentiate between species. rrs sequencing for the three species isolated from humans showed more than 98 % identity between them. Four rrs copies were found in the reference strains and in all the clinical isolates studied. The sequences of each rrs were determined for the clinical strain ADV 360.1, and they showed a relatively high level of heterogeneity (1·43 %). In the majority of cases, polymorphic positions corresponded to nucleotides allowing differentiation between the three species isolated from humans. Moreover, variability observed between rrs copies was higher than that between 16S rDNA sequences of V. parvula and V. dispar. Phylogenetic analysis showed that polymorphism between rrs copies affected the position of strain ADV 360.1 in the tree. Variable positions occurred in stems and loops belonging to variable and hypervariable regions of the 16S rRNA secondary structure but did not change the overall structure of the 16S rRNA. PCR-RFLP experiments performed on 27 clinical isolates of Veillonella sp. suggested that inter-rrs heterogeneity occurs widely among the members of the genus Veillonella. These results, together with the lack of phenotypic criteria for species differentiation, give preliminary arguments for unification of V. dispar and V. parvula.

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Association between 16S rRNA gene mutations and susceptibility to amikacin in Mycobacterium avium Complex and Mycobacterium abscessus clinical isolates

Scientific Reports ◽

10.1038/s41598-021-85721-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Su-Young Kim ◽

Dae Hun Kim ◽

Seong Mi Moon ◽

Ju Yeun Song ◽

Hee Jae Huh ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Mycobacterium Avium ◽

Inhibitory Concentration ◽

Gene Mutations ◽

Mycobacterium Abscessus ◽

Clinical Isolates ◽

Rrna Gene ◽

High Level ◽

Culture Conversion

AbstractWe evaluated the association between 16S rRNA gene (rrs) mutations and susceptibility in clinical isolates of amikacin-resistant nontuberculous mycobacteria (NTM) in NTM-pulmonary disease (PD) patients. Susceptibility was retested for 134 amikacin-resistant isolates (minimum inhibitory concentration [MIC] ≥ 64 µg/ml) from 86 patients. Amikacin resistance was reconfirmed in 102 NTM isolates from 62 patients with either Mycobacterium avium complex-PD (MAC-PD) (n = 54) or M. abscessus-PD (n = 8). MICs and rrs mutations were evaluated for 318 single colonies from these isolates. For the 54 MAC-PD patients, rrs mutations were present in 34 isolates (63%), comprising all 31 isolates with amikacin MICs ≥ 128 µg/ml, but only three of 23 isolates with an MIC = 64 µg/ml. For the eight M. abscessus-PD patients, all amikacin-resistant (MIC ≥ 64 µg/ml) isolates had rrs mutations. In amikacin-resistant isolates, the A1408G mutation (n = 29) was most common. Two novel mutations, C1496T and T1498A, were also identified. The culture conversion rate did not differ by amikacin MIC. Overall, all high-level and 13% (3/23) of low-level amikacin-resistant MAC isolates had rrs mutations whereas mutations were present in all amikacin-resistant M. abscessus isolates. These findings are valuable for managing MAC- and M. abscessus-PD and suggest the importance of phenotypic and genotypic susceptibility testing.

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Phenotypic characterization of Astragalus glycyphyllos symbionts and their phylogeny based on the 16S rDNA sequences and RFLP of 16S rRNA gene

Antonie van Leeuwenhoek ◽

10.1007/s10482-014-0163-y ◽

2014 ◽

Vol 105 (6) ◽

pp. 1033-1048 ◽

Cited By ~ 12

Author(s):

Sebastian Gnat ◽

Magdalena Wójcik ◽

Sylwia Wdowiak-Wróbel ◽

Michał Kalita ◽

Aneta Ptaszyńska ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

16S Rdna ◽

Phenotypic Characterization ◽

Rrna Gene ◽

Rdna Sequences ◽

16S Rdna Sequences ◽

Astragalus Glycyphyllos Symbionts

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Cryptic haplotypes of "Candidatus Liberibacter africanus"

10.1101/016410 ◽

2015 ◽

Author(s):

Warrick Nelson ◽

Sandrine Eveillard ◽

Marie-Pierre Dubrana ◽

Joseph Bové

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

16S Rdna ◽

Rrna Gene ◽

Gene Sequences ◽

16S Rrna Gene Sequences ◽

Rdna Sequences ◽

New Information ◽

Candidatus Liberibacter ◽

Citrus Leaves

“Candidatus Liberibacter africanus” (Laf) has long been recognised as a causal agent of the devastating citrus disease huanglongbing (HLB) or citrus greening. This species is currently restricted to Africa, the Arabian Peninsula and some Indian Ocean islands and vectored by the African citrus psyllid, Trioza erytreae. Blotchy mottle on citrus leaves is characteristic of the disease. Somewhat similar symptoms in the Rutaceous tree Calodendrum capensis (Cape Chestnut) resulted in the discovery of Laf outside commercial citrus crops in South Africa. This was classed as a subspecies of Laf (capensis, hence LafC). In subsequent surveys of both commercial citrus crops and Calodendrum, both natural and ornamental specimens, LafC was not found in the citrus crop, nor has Laf been found in C. capensis. HLB was reported from Madagascar in 1968 but no sequences from this source have so far been published. Until fairly recently, only the reference 16S rRNA gene sequences of Laf (L22533) and LafC (AF137368) had been deposited in GenBank. Both of these reference sequences contain a number of unresolved nucleotides. Resolving these nucleotide positions by aligning against more recently available sequences, it becomes evident that these unresolved positions represent one percentage point difference in similarity between Laf and LafC. The originally reported 97.4% similarity is therefore incorrect based on this new information. Recalculating the similarity on the full length 16S rDNA sequence results in 99.54% similarity, a value too high to justify a subspecies status. LafC should therefore be reduced to that of a haplotype of Laf. Further, the six 16S rRNA gene sequences currently available in GenBank identified as the species Laf separate into 2 haplotype groups. The 3 haplotypes of Laf are therefore LafA designated as the first accession sequenced (L22533), LafC for the former capensis subspecies and to recognise the prior use of this term, and LafB for the third haplotype not previously recognised. Thus the cryptic presence of 3 haplotypes is revealed by this review of the Laf 16S rDNA sequences.

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Phylogenetic Analysis of Nonthermophilic Members of the Kingdom Crenarchaeota and Their Diversity and Abundance in Soils

Applied and Environmental Microbiology ◽

10.1128/aem.64.11.4333-4339.1998 ◽

1998 ◽

Vol 64 (11) ◽

pp. 4333-4339 ◽

Cited By ~ 159

Author(s):

Daniel H. Buckley ◽

Joseph R. Graber ◽

Thomas M. Schmidt

Keyword(s):

16S Rrna ◽

16S Rdna ◽

Phylogenetic Analyses ◽

Pcr Primers ◽

Soil Samples ◽

Molecular Techniques ◽

Rrna Gene ◽

Rdna Sequences ◽

16S Rdna Sequences ◽

Long Term Ecological Research

ABSTRACT Within the last several years, molecular techniques have uncovered numerous 16S rRNA gene (rDNA) sequences which represent a unique and globally distributed lineage of the kingdom Crenarchaeotathat is phylogenetically distinct from currently characterized crenarchaeotal species. rDNA sequences of members of this novel crenarchaeotal group have been recovered from low- to moderate-temperature environments (−1.5 to 32°C), in contrast to the high-temperature environments (temperature, >80°C) required for growth of the currently recognized crenarchaeotal species. We determined the diversity and abundance of the nonthermophilic members of the Crenarchaeota in soil samples taken from cultivated and uncultivated fields located at the Kellogg Biological Station’s Long-Term Ecological Research site (Hickory Corners, Mich.). Clones were generated from 16S rDNA that was amplified by using broad-specificity archaeal PCR primers. Twelve crenarchaeotal sequences were identified, and the phylogenetic relationships between these sequences and previously described crenarchaeotal 16S rDNA sequences were determined. Phylogenetic analyses included nonthermophilic crenarchaeotal sequences found in public databases and revealed that the nonthermophilic Crenarchaeota group is composed of at least four distinct phylogenetic clusters. A 16S rRNA-targeted oligonucleotide probe specific for all known nonthermophilic crenarchaeotal sequences was designed and used to determine their abundance in soil samples. The nonthermophilicCrenarchaeota accounted for as much as 1.42% ± 0.42% of the 16S rRNA in the soils analyzed.

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Comparative Diversity of Ammonia Oxidizer 16S rRNA Gene Sequences in Native, Tilled, and Successional Soils

Applied and Environmental Microbiology ◽

10.1128/aem.65.7.2994-3000.1999 ◽

1999 ◽

Vol 65 (7) ◽

pp. 2994-3000 ◽

Cited By ~ 173

Author(s):

Mary Ann Bruns ◽

John R. Stephen ◽

George A. Kowalchuk ◽

James I. Prosser ◽

Eldor A. Paul

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

16S Rdna ◽

Ammonia Oxidizer ◽

Most Probable Number ◽

Rrna Gene ◽

Probable Number ◽

Rdna Sequences ◽

16S Rdna Sequences ◽

Native Soils

ABSTRACT Autotrophic ammonia oxidizer (AAO) populations in soils from native, tilled, and successional treatments at the Kellogg Biological Station Long-Term Ecological Research site in southwestern Michigan were compared to assess effects of disturbance on these bacteria. N fertilization effects on AAO populations were also evaluated with soils from fertilized microplots within the successional treatments. Population structures were characterized by PCR amplification of microbial community DNA with group-specific 16S rRNA gene (rDNA) primers, cloning of PCR products and clone hybridizations with group-specific probes, phylogenetic analysis of partial 16S rDNA sequences, and denaturing gradient gel electrophoresis (DGGE) analysis. Population sizes were estimated by using most-probable-number (MPN) media containing varied concentrations of ammonium sulfate. Tilled soils contained higher numbers than did native soils of culturable AAOs that were less sensitive to different ammonium concentrations in MPN media. Compared to sequences from native soils, partial 16S rDNA sequences from tilled soils were less diverse and grouped exclusively within Nitrosospira cluster 3. Native soils yielded sequences representing three different AAO clusters. Probes forNitrosospira cluster 3 hybridized with DGGE blots from tilled and fertilized successional soils but not with blots from native or unfertilized successional soils. Hybridization results thus suggested a positive association between the Nitrosospiracluster 3 subgroup and soils amended with inorganic N. DGGE patterns for soils sampled from replicated plots of each treatment were nearly identical for tilled and native soils in both sampling years, indicating spatial and temporal reproducibility based on treatment.

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Clover Proliferation Group (16SrVI) Subgroup A (16SrVI-A) Phytoplasma is a Probable Causal Agent of Dry Bean Phyllody Disease in Washington

Plant Disease ◽

10.1094/pdis.2004.88.4.429a ◽

2004 ◽

Vol 88 (4) ◽

pp. 429-429

Author(s):

I.-M. Lee ◽

K. D. Bottner ◽

P. N. Miklas ◽

M. A. Pastor-Corrales

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

16S Rdna ◽

Sequence Homology ◽

Rrna Gene ◽

16S Rrna Gene Sequences ◽

Dry Bean ◽

Rdna Sequences ◽

Bean Plants ◽

16S Rdna Sequences

During 2003, a new disease, dry bean phyllody (DBPh), was observed in the Columbia Basin of Washington in dry bean (Phaseolus vulgaris L.) cultivars of Andean origin grown in Mattawa and Paterson, WA that caused great reduction in dry bean production. Symptoms of DBPh became apparent during mid-to-late pod development and were characterized by leafy petals (phyllody) and aborted seed pods resembling thin, twisted, and corrugated leaf-like structures. Deformed sterile pods that were small, sickle-shaped, upright, and leathery were also observed. The infected plants generally exhibited chlorosis, stunting, or bud proliferation from leaf axils. Symptoms of DBPh were indicative of possible infection by phytoplasmas. Restriction fragment length polymorphism (RFLP) and phylogenetic analyses of amplified 16S rDNA sequences were used for phytoplasma identification. Four symptomatic bean plants were analyzed and tested positive for phytoplasma infection on the basis of results of initial polymerase chain reaction (PCR) and subsequent nested-PCR amplifications (2). RFLP analyses of 16S rDNA sequences with restriction enzymes, MseI, AluI, HhaI, RsaI, and HpaII indicated that the phytoplasma strains associated with DBPh belonged to the clover proliferation group (16SrVI) subgroup A (16SrVI-A) (2). This subgroup currently consists of three members, clover proliferation (CP; GenBank Accession No. AY500130), potato witches'-broom (PWB; GenBank Accession No. AY500818), and vinca virescence (VR; GenBank Accession No. AY500817), a strain of beet leafhopper-transmitted virescence agent (BLTVA) phytoplasmas (1,2). The taxonomic affiliations of the DBPh phytoplasma strains were confirmed by phylogenetic analysis of cloned 16S rRNA gene sequences (GenBank Accession Nos. DBPh2, AY496002; DBPh3, AY496003). Among the existing members of subgroup 16SrVI-A, the four DBPh strains were closely related to the VR strain with 99.7% 16S rDNA sequence homology and to the CP strain with 99.2% sequence homology. To gain further evidence on the role of 16SrVI-A phytoplasma strains in DBPh disease, a modified test of Koch's postulates was conducted. Infected tissue from one phytoplasma-positive dry bean sample was grafted onto three Pinto UI-114 bean seedlings in the greenhouse. Within 60 days, the bean seedlings exhibited corrugated leaf-like structures from aborted seedpods, a lack of flower formation, general chlorosis, and stunting similar to the original diseased plants. The lower leaves of the inoculated bean plants became epinastic and leathery. The transmitted phytoplasma was detected in each of the grafted symptomatic seedlings, and the RFLP patterns of its 16S rRNA gene sequences were identical to those of the phytoplasmas in the scions. A high correlation between the presence of disease symptoms and the presence of subgroup 16SrVI-A phytoplasmas in the bean plants suggests that these phytoplasmas play an etiological role in DBPh disease. To our knowledge, these findings provide the first confirmed case of phytoplasma-associated DBPh in the United States. References: (1) D. A. Golino et al. Plant Dis. 73:850, 1989. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

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Atypical 16S rRNA Gene Copies in Ochrobactrum intermedium Strains Reveal a Large Genomic Rearrangement by Recombination between rrn Copies

Journal of Bacteriology ◽

10.1128/jb.185.9.2901-2909.2003 ◽

2003 ◽

Vol 185 (9) ◽

pp. 2901-2909 ◽

Cited By ~ 43

Author(s):

Corinne Teyssier ◽

Hélène Marchandin ◽

Michèle Siméon De Buochberg ◽

Michel Ramuz ◽

Estelle Jumas-Bilak

Keyword(s):

16S Rrna ◽

16S Rdna ◽

Genomic Rearrangement ◽

Rrna Gene ◽

Stem Loop ◽

Large Genomic Rearrangement ◽

Rdna Sequences ◽

16S Rdna Sequences ◽

Ochrobactrum Intermedium

ABSTRACT Ochrobactrum intermedium is an opportunistic human pathogen belonging to the alpha 2 subgroup of proteobacteria. The 16S rDNA sequences of nine O. intermedium isolates from a collection of clinical and environmental isolates exhibited a 46-bp insertion at position 187, which was present in only one sequence among the 82 complete or partial 16S rDNA sequences of Ochrobactrum spp. available in data banks. Reverse transcription-PCR experiments showed that the 46-bp insertion remained in the 16S rRNA. The inserted sequence folded into a stem-loop structure, which took place in and prolonged helix H184 of the 16S rRNA molecule. Helix H184 has been described as conserved in length among eubacteria, suggesting the idiosyncratic character of the 46-bp insertion. Pulsed-field gel electrophoresis experiments showed that seven of the clinical isolates carrying the 46-bp insertion belonged to the same clone. Insertion and rrn copy numbers were determined by hybridization and I-CeuI digestion. In the set of clonal isolates, the loss of two insertion copies revealed the deletion of a large genomic fragment of 150 kb, which included one rrn copy; deletion occurred during the in vivo evolution of the clone. Determination of the rrn skeleton suggested that the large genomic rearrangement occurred during events involving homologous recombination between rrn copies. The loss of insertion copies suggested a phenomenon of concerted evolution among heterogeneous rrn copies.

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Intragenomic and intraspecific heterogeneity in rrs may surpass interspecific variability in a natural population of Veillonella

Microbiology ◽

10.1099/mic.0.038224-0 ◽

2010 ◽

Vol 156 (7) ◽

pp. 2080-2091 ◽

Cited By ~ 28

Author(s):

Anne-Laure Michon ◽

Fabien Aujoulat ◽

Laurent Roudière ◽

Olivier Soulier ◽

Isabelle Zorgniotti ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Gene Diversity ◽

Natural Populations ◽

Variable Region ◽

Housekeeping Gene ◽

Single Copy ◽

Population Based ◽

Rrna Gene ◽

Gene Copies

As well as intraspecific heterogeneity, intragenomic heterogeneity between 16S rRNA gene copies has been described for a range of bacteria. Due to the wide use of 16S rRNA gene sequence analysis for taxonomy, identification and metagenomics, evaluating the extent of these heterogeneities in natural populations is an essential prerequisite. We investigated inter- and intragenomic 16S rRNA gene heterogeneity of the variable region V3 in a population of 149 clinical isolates of Veillonella spp. of human origin and in 13 type or reference Veillonella strains using PCR-temporal temperature gel electrophoresis (TTGE). 16S rRNA gene diversity was high in the studied population, as 45 different banding patterns were observed. Intragenomic heterogeneity was demonstrated for 110 (74 %) isolates and 8 (61.5 %) type or reference strains displaying two or three different gene copies. Polymorphic nucleotide positions accounted for 0.5–2.5 % of the sequence and were scattered in helices H16 and H17 of the rRNA molecule. Some of them changed the secondary structure of H17. Phylotaxonomic structure of the population based on the single-copy housekeeping gene rpoB was compared with TTGE patterns. The intragenomic V3 heterogeneity, as well as recombination events between strains or isolates of different rpoB clades, impaired the 16S rRNA-based identification for some Veillonella species. Such approaches should be conducted in other bacterial populations to optimize the interpretation of 16S rRNA gene sequences in taxonomy and/or diversity studies.

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Molecular Diversity Assessment of Plant Growth Promoting Rhizobacteria Using Denaturing Gradient Gel Electrophoresis (DGGE) of 16s rRNA Gene

International Journal of Applied Sciences and Biotechnology ◽

10.3126/ijasbt.v5i1.17029 ◽

2017 ◽

Vol 5 (1) ◽

pp. 72-80

Author(s):

Umesh Prasad Shrivastava

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

16S Rdna ◽

Denaturing Gradient Gel Electrophoresis ◽

Uttar Pradesh ◽

Plant Growth Promoting Rhizobacteria ◽

Melting Behavior ◽

Rrna Gene ◽

Dgge Profile ◽

Diversity Assessment

The rhizobacteria were isolated from rhizosphere of rice plant of different fields of 4 districts of Nepal and 5 districts of Bihar and Uttar Pradesh, adjoining states of India with Nepal. The DGGE analysis was performed for diversity analysis. For the construction of dendrogram, 16S rRNA gene was amplified by two different sets of primers. The DGGE ladder consisting of PCR amplified products of nine pure bacterial cultures were obtained. The first DGGE ladder was prepared by 400 bp fragment of 16S rDNA with GC clamp and the second DGGE ladder was prepared with 200 bp fragment of 16S rDNA with GC clamp. The perpendicular DGGE of these amplicons based on their melting behavior clearly demonstrated separation of different isolates. The 16S rDNA fragment amplified with primer set of V2-V3 regions with GC clamp showed separation between 40-60% of denaturant. The DGGE profile based on primer set F352T and 519r for various bacteria present in soil samples of 5 districts of India and 4 districts of Nepal revealed that the number of bands which might be specific for diazotrophic isolates varied from 2 to 11. The dendrogram constructed based on DGGE profile of various samples of 5 districts of India and 4 districts of Nepal showed that all the samples could be clustered in nine groups with 58-96% similarity to each other. Among all these 37 samples, only Var-4 and Var-5 showed 100% similarity, no other samples from any site showed 100% similarity. Int. J. Appl. Sci. Biotechnol. Vol 5(1): 72-80

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