Estimation of 16S rRNA gene copy number in several probioticLactobacillusstrains isolated from the gastrointestinal tract of chicken

ABSTRACT We investigated the effects of internal primer-template mismatches on the efficiency of PCR amplification using the 16S rRNA gene as the model template DNA. We observed that the presence of a single mismatch in the second half of the primer extension sequence can result in an underestimation of up to 1,000-fold of the gene copy number, depending on the primer and position of the mismatch.

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Multiple DNA Extractions Coupled with Stable-Isotope Probing of Anthracene-Degrading Bacteria in Contaminated Soil

Applied and Environmental Microbiology ◽

10.1128/aem.01942-10 ◽

2011 ◽

Vol 77 (9) ◽

pp. 2984-2991 ◽

Cited By ~ 29

Author(s):

Maiysha D. Jones ◽

David R. Singleton ◽

Wei Sun ◽

Michael D. Aitken

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Gene Copy Number ◽

Stable Isotope Probing ◽

Gene Copy ◽

Rrna Gene ◽

Content Type ◽

Dna Yield ◽

Degrading Bacteria ◽

Bacterial Groups

ABSTRACTIn many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the13C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-13C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the orderSphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from13C-enriched DNA and were designated “anthracene group 1.” Sequences clustering withVariovoraxspp., which were also highly represented, and sequences related to the genusPigmentiphagawere newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP.

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Development of a Real-Time PCR Assay for Detection and Quantification of Rhizobium leguminosarum Bacteria and Discrimination between Different Biovars in Zinc-Contaminated Soil

Applied and Environmental Microbiology ◽

10.1128/aem.02232-10 ◽

2011 ◽

Vol 77 (13) ◽

pp. 4626-4633 ◽

Cited By ~ 13

Author(s):

Catriona A. Macdonald ◽

Ian M. Clark ◽

Penny R. Hirsch ◽

Fang-Jie Zhao ◽

Steve P. McGrath

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Rhizobium Leguminosarum ◽

Gene Copy Number ◽

Gene Copy ◽

Most Probable Number ◽

Rrna Gene ◽

Probable Number ◽

Content Type ◽

Gene Copies

ABSTRACTPrimers were designed to target 16S rRNA andnodDgenes ofRhizobium leguminosarumfrom DNA extracted from two different soil types contaminated with Zn applied in sewage sludge. Numbers of rhizobia estimated using 16S rRNA gene copy number showed higher abundance than those estimated by bothnodDand the most-probable-number (MPN) enumeration method using a plant trap host. Both 16S rRNA gene copies and the MPN rhizobia declined with increased levels of Zn contamination, as did the abundance of the functional genenodD, providing compelling evidence of a toxic effect of Zn onR. leguminosarumpopulations in the soil. Regression analysis suggested the total Zn concentration in soil as a better predictor of rhizobial numbers than both NH4NO3-extractable and soil solution Zn.R. leguminosarumbv. viciaenodDgene copies were generally less sensitive to Zn thanR. leguminosarumbv. trifoliinodD.The latter were generally below detection limits at Zn levels of >250 mg kg−1. Although there were differences in the actual numbers estimated by each approach, the response to Zn was broadly similar across all methods. These differences were likely to result from the fact that the molecular approaches assess the potential for nodulation while the MPN approach assesses actual nodulation. The results demonstrate that the use of targeted gene probes for assessing environmental perturbations of indigenous soil rhizobial populations may be more sensitive than the conventional plant bioassay and MPN methods.

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GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring

Frontiers in Microbiology ◽

10.3389/fmicb.2021.787651 ◽

2022 ◽

Vol 12 ◽

Author(s):

Ilona A. Ruhl ◽

Andriy Sheremet ◽

Chantel C. Furgason ◽

Susanne Krause ◽

Robert M. Bowers ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Phylogenetic Trees ◽

Hot Spring ◽

Gc Content ◽

Gene Copy ◽

Marker Genes ◽

Rrna Gene ◽

Separate Species ◽

Distinct Subgroup

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria. We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2–79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 105 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45–77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria, representing a candidate order (Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O2, suggesting a microaerophilic lifestyle.

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Trichomes form genotype-specific microbial hotspots in the phyllosphere of tomato

Environmental Microbiome ◽

10.1186/s40793-020-00364-9 ◽

2020 ◽

Vol 15 (1) ◽

Author(s):

Peter Kusstatscher ◽

Wisnu Adi Wicaksono ◽

Alessandro Bergna ◽

Tomislav Cernava ◽

Nick Bergau ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Community Dynamics ◽

Statistical Tests ◽

Central Element ◽

Shannon Index ◽

Gene Copy ◽

Rrna Gene ◽

Core Microbiome ◽

Defense Strategies

Abstract Background The plant phyllosphere is a well-studied habitat characterized by low nutrient availability and high community dynamics. In contrast, plant trichomes, known for their production of a large number of metabolites, are a yet unexplored habitat for microbes. We analyzed the phyllosphere as well as trichomes of two tomato genotypes (Solanum lycopersicum LA4024, S. habrochaites LA1777) by targeting bacterial 16S rRNA gene fragments. Results Leaves, leaves without trichomes, and trichomes alone harbored similar abundances of bacteria (108–109 16S rRNA gene copy numbers per gram of sample). In contrast, bacterial diversity was found significantly increased in trichome samples (Shannon index: 4.4 vs. 2.5). Moreover, the community composition was significantly different when assessed with beta diversity analysis and corresponding statistical tests. At the bacterial class level, Alphaproteobacteria (23.6%) were significantly increased, whereas Bacilli (8.6%) were decreased in trichomes. The bacterial family Sphingomonadacea (8.4%) was identified as the most prominent, trichome-specific feature; Burkholderiaceae and Actinobacteriaceae showed similar patterns. Moreover, Sphingomonas was identified as a central element in the core microbiome of trichome samples, while distinct low-abundant bacterial families including Hymenobacteraceae and Alicyclobacillaceae were exclusively found in trichome samples. Niche preferences were statistically significant for both genotypes and genotype-specific enrichments were further observed. Conclusion Our results provide first evidence of a highly specific trichome microbiome in tomato and show the importance of micro-niches for the structure of bacterial communities on leaves. These findings provide further clues for breeding, plant pathology and protection as well as so far unexplored natural pathogen defense strategies.

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Influences of Infaunal Burrows on the Community Structure and Activity of Ammonia-Oxidizing Bacteria in Intertidal Sediments

Applied and Environmental Microbiology ◽

10.1128/aem.02073-06 ◽

2006 ◽

Vol 73 (4) ◽

pp. 1341-1348 ◽

Cited By ~ 38

Author(s):

Hisashi Satoh ◽

Yoshiyuki Nakamura ◽

Satoshi Okabe

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Gene Copy ◽

Sediment Surface ◽

Intertidal Sediment ◽

Rrna Gene ◽

Ammonia Oxidizing Bacteria ◽

Intertidal Sediments ◽

Burrow Wall ◽

Microelectrode Measurements

ABSTRACT Influences of infaunal burrows constructed by the polychaete (Tylorrhynchus heterochaetus) on O2 concentrations and community structures and abundances of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in intertidal sediments were analyzed by the combined use of a 16S rRNA gene-based molecular approach and microelectrodes. The microelectrode measurements performed in an experimental system developed in an aquarium showed direct evidence of O2 transport down to a depth of 350 mm of the sediment through a burrow. The 16S rRNA gene-cloning analysis revealed that the betaproteobacterial AOB communities in the sediment surface and the burrow walls were dominated by Nitrosomonas sp. strain Nm143-like sequences, and most of the clones in Nitrospira-like NOB clone libraries of the sediment surface and the burrow walls were related to the Nitrospira marina lineage. Furthermore, we investigated vertical distributions of AOB and NOB in the infaunal burrow walls and the bulk sediments by real-time quantitative PCR (Q-PCR) assay. The AOB and Nitrospira-like NOB-specific 16S rRNA gene copy numbers in the burrow walls were comparable with those in the sediment surfaces. These numbers in the burrow wall at a depth of 50 to 55 mm from the surface were, however, higher than those in the bulk sediment at the same depth. The microelectrode measurements showed higher NH4 + consumption activity at the burrow wall than those at the surrounding sediment. This result was consistent with the results of microcosm experiments showing that the consumption rates of NH4 + and total inorganic nitrogen increased with increasing infaunal density in the sediment. These results clearly demonstrated that the infaunal burrows stimulated O2 transport into the sediment in which otherwise reducing conditions prevailed, resulting in development of high NH4 + consumption capacity. Consequently, the infaunal burrow became an important site for NH4 + consumption in the intertidal sediment.

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Sequence analysis of 16S rRNA gene and 16S–23S rRNA gene intergenic spacer region for differentiation of probioticsLactobacillus strains isolated from the gastrointestinal tract of chicken

Annals of Microbiology ◽

10.1007/bf03179457 ◽

2008 ◽

Vol 58 (1) ◽

pp. 133-140 ◽

Cited By ~ 9

Author(s):

Chin Mei Lee ◽

Chin Chin Sieo ◽

Clemente Michael Vui Ling Wong ◽

Norhani Abdullah ◽

Yin Wan Ho

Keyword(s):

Gastrointestinal Tract ◽

Sequence Analysis ◽

16S Rrna ◽

16S Rrna Gene ◽

Intergenic Spacer ◽

23S Rrna ◽

Rrna Gene ◽

Intergenic Spacer Region ◽

23S Rrna Gene

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Trichomes form genotype-specific microbial hotspots in the phyllosphere of tomato

10.21203/rs.3.rs-25108/v1 ◽

2020 ◽

Author(s):

Peter Kusstatscher ◽

Wisnu Adi Wicaksono ◽

Alessandro Bergna ◽

Tomislav Cernava ◽

Nick Bergau ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Community Dynamics ◽

Statistical Tests ◽

Central Element ◽

Shannon Index ◽

Gene Copy ◽

Rrna Gene ◽

Core Microbiome ◽

Defense Strategies

Abstract Background The plant phyllosphere is a well-studied habitat characterized by low nutrient availability and high community dynamics. In contrast, plant trichomes, known for their production of a large number of metabolites, are a yet unexplored habitat for microbes. We analyzed the phyllosphere as well as trichomes of two tomato genotypes (Solanum lycopersicum LA4024, S. habrochaites LA1777) by targeting bacterial 16S rRNA gene fragments.Results Leaves, leaves without trichomes, and trichomes alone harbored similar abundances of bacteria (108- 109 16S rRNA gene copy numbers per gram of sample). In contrast, bacterial diversity was found significantly increased in trichome samples (Shannon index: 4.4 vs. 2.5). Moreover, the community composition was significantly different when assessed with beta diversity analysis and corresponding statistical tests. At the bacterial class level, Alphaproteobacteria (23.6%) were significantly increased, whereas Bacilli (8.6%) were decreased in trichomes. The bacterial family Sphingomonadacea (8.4%) was identified as the most prominent, trichome-specific taxa; Burkholderiaceae and Actinobacteria showed similar pattern. Moreover, Sphingomonas was identified as a central element in the core microbiome of trichome samples, while distinct low-abundant bacterial families including Hymenobacteraceae and Alicyclobacillaceae were exclusively found in trichome samples. Niche preferences were statistically significant for both genotypes and genotype-specific enrichments were further observed.Conclusion Our results provide first evidence of a highly specific trichome microbiome in tomato and show the importance of micro-niches for the structure of bacterial communities on leaves. These findings provide further clues for breeding, plant pathology and protection as well as so far unexplored natural pathogen defense strategies.

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