Effect of genome size andrrn gene copy numbers of PCR amplification products of 16S rRNA genes from mixed bacterial species

Analysis of 16S ribosomal RNA (rRNA) genes provides a central means of taxonomic classification of bacterial species. Based on presumed sequence identity among species of the Bacillus cereus sensu lato group, the 16S rRNA genes of B. anthracis have been considered unsuitable for diagnosis of the anthrax pathogen. With the recent identification of a single nucleotide polymorphism in some 16S rRNA gene copies, specific identification of B. anthracis becomes feasible. Here, we designed and evaluated a set of in situ-, in vitro- and in silico-assays to assess the yet unknown 16S-state of B. anthracis from different perspectives. Using a combination of digital PCR, fluorescence in situ hybridization, long-read genome sequencing and bioinformatics we were able to detect and quantify a unique 16S rRNA gene allele of B. anthracis (16S-BA-allele). This allele was found in all available B. anthracis genomes and may facilitate differentiation of the pathogen from any close relative. Bioinformatics analysis of 959 B. anthracis genome data-sets inferred that abundances and genomic arrangements of the 16S-BA-allele and the entire rRNA operon copy-numbers differ considerably between strains. Expression ratios of 16S-BA-alleles were proportional to the respective genomic allele copy-numbers. The findings and experimental tools presented here provide detailed insights into the intra- and intergenomic diversity of 16S rRNA genes and may pave the way for improved identification of B. anthracis and other pathogens with diverse rRNA operons.

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Divergence and Redundancy of 16S rRNA Sequences in Genomes with Multiple rrn Operons

Journal of Bacteriology ◽

10.1128/jb.186.9.2629-2635.2004 ◽

2004 ◽

Vol 186 (9) ◽

pp. 2629-2635 ◽

Cited By ~ 407

Author(s):

Silvia G. Acinas ◽

Luisa A. Marcelino ◽

Vanja Klepac-Ceraj ◽

Martin F. Polz

Keyword(s):

16S Rrna ◽

Thermophilic Bacteria ◽

16S Rrna Genes ◽

Rrna Genes ◽

Bacterial Genomes ◽

16S Rrna Sequence ◽

Thermoanaerobacter Tengcongensis ◽

Copy Numbers ◽

Rrna Sequences ◽

16S Rrna Sequences

ABSTRACT The level of sequence heterogeneity among rrn operons within genomes determines the accuracy of diversity estimation by 16S rRNA-based methods. Furthermore, the occurrence of widespread horizontal gene transfer (HGT) between distantly related rrn operons casts doubt on reconstructions of phylogenetic relationships. For this study, patterns of distribution of rrn copy numbers, interoperonic divergence, and redundancy of 16S rRNA sequences were evaluated. Bacterial genomes display up to 15 operons and operon numbers up to 7 are commonly found, but ∼40% of the organisms analyzed have either one or two operons. Among the Archaea, a single operon appears to dominate and the highest number of operons is five. About 40% of sequences among 380 operons in 76 bacterial genomes with multiple operons were identical to at least one other 16S rRNA sequence in the same genome, and in 38% of the genomes all 16S rRNAs were invariant. For Archaea, the number of identical operons was only 25%, but only five genomes with 21 operons are currently available. These considerations suggest an upper bound of roughly threefold overestimation of bacterial diversity resulting from cloning and sequencing of 16S rRNA genes from the environment; however, the inclusion of genomes with a single rrn operon may lower this correction factor to ∼2.5. Divergence among operons appears to be small overall for both Bacteria and Archaea, with the vast majority of 16S rRNA sequences showing <1% nucleotide differences. Only five genomes with operons with a higher level of nucleotide divergence were detected, and Thermoanaerobacter tengcongensis exhibited the highest level of divergence (11.6%) noted to date. Overall, four of the five extreme cases of operon differences occurred among thermophilic bacteria, suggesting a much higher incidence of HGT in these bacteria than in other groups.

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The frequency of chimeric molecules as a consequence of PCR co-amplification of 16S rRNA genes from different bacterial species

Microbiology ◽

10.1099/13500872-142-5-1107 ◽

1996 ◽

Vol 142 (5) ◽

pp. 1107-1114 ◽

Cited By ~ 159

Author(s):

G. C. Y. Wang ◽

Y. Wang

Keyword(s):

16S Rrna ◽

Bacterial Species ◽

16S Rrna Genes ◽

Rrna Genes ◽

Chimeric Molecules

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

Epidemiology and Infection ◽

10.1017/s0950268899002101 ◽

1999 ◽

Vol 122 (2) ◽

pp. 323-328 ◽

Cited By ~ 18

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.

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The simplest and currently the most widely adopted method to obtain 16S rRNA genes from the environment is through the use of PCR. rRNA genes can be amplified directly from the total community DNA using rRNA-specific primers, and then cloned using standard methods (Giovannoni et al. 1990). By taking advantage of the highly conserved nature of rRNA, universal primers capable of annealing to rRNA genes from all three domains (Archaea, Bacteria, Eukarya) or primers designed to amplify rRNA genes from a particular group of organisms can be used. Following PCR amplification, the amplified products can be cloned. Commercially available kits exploit the fact that PCR-amplified products have an overhanging 3' deoxyadenosine residue at each end when certain DNA polymerases are used. This allows cloning of the product into a sequencing-ready vector containing a complementary deoxy-thymidine overhang, in many cases, without requiring the product to be purified or further modified. Alternatively, the PCR products can be cloned by "filling" overhanging residues followed by blunt-end ligation procedures.

Recent Advances in Marine Biotechnology, Vol. 8 ◽

10.1201/9781482279986-14 ◽

2003 ◽

pp. 221-221

Keyword(s):

16S Rrna ◽

Dna Polymerases ◽

Pcr Amplification ◽

16S Rrna Genes ◽

Rrna Genes ◽

Universal Primers ◽

Standard Methods ◽

Pcr Products ◽

Total Community ◽

Total Community Dna

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Quantitative Assessment of Ammonia-Oxidizing Bacterial Communities in the Epiphyton of Submerged Macrophytes in Shallow Lakes

Applied and Environmental Microbiology ◽

10.1128/aem.01917-09 ◽

2010 ◽

Vol 76 (6) ◽

pp. 1813-1821 ◽

Cited By ~ 18

Author(s):

M. Coci ◽

G. W. Nicol ◽

G. N. Pilloni ◽

M. Schmid ◽

M. P. Kamst-van Agterveld ◽

...

Keyword(s):

16S Rrna ◽

Shallow Lakes ◽

Submerged Macrophytes ◽

16S Rrna Genes ◽

Rrna Genes ◽

Gene Copy ◽

Potamogeton Pectinatus ◽

Rrna Gene ◽

Ammonia Oxidizing Bacteria ◽

Potential Nitrification

ABSTRACT In addition to the benthic and pelagic habitats, the epiphytic compartment of submerged macrophytes in shallow freshwater lakes offers a niche to bacterial ammonia-oxidizing communities. However, the diversity, numbers, and activity of epiphytic ammonia-oxidizing bacteria have long been overlooked. In the present study, we analyzed quantitatively the epiphytic communities of three shallow lakes by a potential nitrification assay and by quantitative PCR of 16S rRNA genes. On the basis of the m2 of the lake surface, the gene copy numbers of epiphytic ammonia oxidizers were not significantly different from those in the benthic and pelagic compartments. The potential ammonia-oxidizing activities measured in the epiphytic compartment were also not significantly different from the activities determined in the benthic compartment. No potential ammonia-oxidizing activities were observed in the pelagic compartment. No activity was detected in the epiphyton of Chara aspera, the dominant submerged macrophyte in Lake Nuldernauw in The Netherlands. The presence of ammonia-oxidizing bacterial cells in the epiphyton of Potamogeton pectinatus was also demonstrated by fluorescent in situ hybridization microscopy images. By comparing the community composition as assessed by the 16S rRNA gene PCR-denaturing gradient gel electrophoresis approach, it was concluded that the epiphytic ammonia-oxidizing communities consisted of cells that were also present in the benthic and pelagic compartments. Of the environmental parameters examined, only the water retention time, the Kjeldahl nitrogen content, and the total phosphorus content correlated with potential ammonia-oxidizing activities. None of these parameters correlated with the numbers of gene copies related to ammonia-oxidizing betaproteobacteria.

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

Applied and Environmental Microbiology ◽

10.1128/aem.68.10.5064-5081.2002 ◽

2002 ◽

Vol 68 (10) ◽

pp. 5064-5081 ◽

Cited By ~ 469

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).

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

Applied and Environmental Microbiology ◽

10.1128/aem.66.6.2365-2371.2000 ◽

2000 ◽

Vol 66 (6) ◽

pp. 2365-2371 ◽

Cited By ~ 106

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.

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High-resolution microbiome analysis enabled by linking of 16S rRNA gene sequences with adjacent genomic contexts

Microbial Genomics ◽

10.1099/mgen.0.000624 ◽

2021 ◽

Vol 7 (9) ◽

Author(s):

Žana Kapustina ◽

Justina Medžiūnė ◽

Gediminas Alzbutas ◽

Irmantas Rokaitis ◽

Karolis Matjošaitis ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

16S Rrna Sequencing ◽

Gene Copy ◽

Rrna Gene ◽

Gene Sequences ◽

Copy Numbers ◽

Rrna Sequencing ◽

Metagenome Sequencing ◽

Gene Copy Numbers

Sequence-based characterization of bacterial communities has long been a hostage of limitations of both 16S rRNA gene and whole metagenome sequencing. Neither approach is universally applicable, and the main efforts to resolve constraints have been devoted to improvement of computational prediction tools. Here, we present semi-targeted 16S rRNA sequencing (st16S-seq), a method designed for sequencing V1–V2 regions of the 16S rRNA gene along with the genomic locus upstream of the gene. By in silico analysis of 13 570 bacterial genome assemblies, we show that genome-linked 16S rRNA sequencing is superior to individual hypervariable regions or full-length gene sequences in terms of classification accuracy and identification of gene copy numbers. Using mock communities and soil samples we experimentally validate st16S-seq and benchmark it against the established microbial classification techniques. We show that st16S-seq delivers accurate estimation of 16S rRNA gene copy numbers, enables taxonomic resolution at the species level and closely approximates community structures obtainable by whole metagenome sequencing.

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