16S rRNA gene-based analysis of microbial community by whole-genome amplification and minigel-single-strand conformation polymorphism technique

ABSTRACT Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5′ end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR–single-stranded DNA approach for microbial community analysis.

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Bacterial Community Dynamics during Production of Registered Designation of Origin Salers Cheese as Evaluated by 16S rRNA Gene Single-Strand Conformation Polymorphism Analysis

Applied and Environmental Microbiology ◽

10.1128/aem.69.7.3840-3848.2003 ◽

2003 ◽

Vol 69 (7) ◽

pp. 3840-3848 ◽

Cited By ~ 102

Author(s):

Frédérique Duthoit ◽

Jean-Jacques Godon ◽

Marie-Christine Montel

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Single Strand Conformation Polymorphism ◽

Single Strand ◽

Rrna Gene ◽

Polymorphism Analysis ◽

Gram Positive ◽

Leuconostoc Pseudomesenteroides ◽

The 16S Rrna Gene ◽

Conformation Polymorphism

ABSTRACT Microbial dynamics during processing and ripening of traditional cheeses such as registered designation of origin Salers cheese, an artisanal cheese produced in France, play an important role in the elaboration of sensory qualities. The aim of the present study was to obtain a picture of the dynamics of the microbial ecosystem of RDO Salers cheese by using culture-independent methods. This included DNA extraction, PCR, and single-strand conformation polymorphism (SSCP) analysis. Bacterial and high-GC% gram-positive bacterial primers were used to amplify V2 or V3 regions of the 16S rRNA gene. SSCP patterns revealed changes during the manufacturing of the cheese. Patterns of the ecosystems of cheeses that were provided by three farmers were also quite different. Cloning and sequencing of the 16S rRNA gene revealed sequences related to lactic acid bacteria (Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lactobacillus plantarum, and Lactobacillus pentosus), which were predominant during manufacturing and ripening. Bacteria belonging to the high-GC% gram-positive group (essentially corynebacteria) were found by using specific primers. The present molecular approach can effectively describe the ecosystem of artisanal dairy products.

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Identification of Mycobacterium Species by Multiple-Fluorescence PCR-Single-Strand Conformation Polymorphism Analysis of the 16S rRNA Gene

Journal of Clinical Microbiology ◽

10.1128/jcm.39.9.3085-3091.2001 ◽

2001 ◽

Vol 39 (9) ◽

pp. 3085-3091 ◽

Cited By ~ 25

Author(s):

L. M. Gillman ◽

J. Gunton ◽

C. Y. Turenne ◽

J. Wolfe ◽

A. M. Kabani

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Single Strand Conformation Polymorphism ◽

Single Strand ◽

Rrna Gene ◽

Polymorphism Analysis ◽

Mycobacterium Species ◽

Conformation Polymorphism Analysis ◽

The 16S Rrna Gene ◽

Conformation Polymorphism

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Molecular identification of bacteria by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

Journal of Clinical Microbiology ◽

10.1128/jcm.33.10.2601-2606.1995 ◽

1995 ◽

Vol 33 (10) ◽

pp. 2601-2606 ◽

Cited By ~ 43

Author(s):

M N Widjojoatmodjo ◽

A C Fluit ◽

J Verhoef

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Molecular Identification ◽

Single Strand Conformation Polymorphism ◽

Rrna Gene ◽

Polymorphism Analysis ◽

Conformation Polymorphism Analysis ◽

Identification Of Bacteria ◽

The 16S Rrna Gene ◽

Conformation Polymorphism

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Effect of Field Inoculation with Sinorhizobium meliloti L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (Medicago sativa) and a Non-Target Plant (Chenopodium album)—Linking of 16S rRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the Diversity of Cultivated Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.66.8.3556-3565.2000 ◽

2000 ◽

Vol 66 (8) ◽

pp. 3556-3565 ◽

Cited By ~ 102

Author(s):

Frank Schwieger ◽

Christoph C. Tebbe

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Bacterial Diversity ◽

Sinorhizobium Meliloti ◽

Chenopodium Album ◽

Single Strand Conformation Polymorphism ◽

Rrna Gene ◽

Field Plots ◽

Target Plant ◽

Conformation Polymorphism

ABSTRACT Fourteen weeks after field release of luciferase gene-taggedSinorhizobium meliloti L33 in field plots seeded withMedicago sativa, we found that the inoculant also occurred in bulk soil from noninoculated control plots. In rhizospheres of M. sativa plants, S. meliloti L33 could be detected in noninoculated plots 12 weeks after inoculation, indicating that growth in the rhizosphere preceded spread into bulk soil. To determine whether inoculation affected bacterial diversity, 1,119 bacteria were isolated from the rhizospheres of M. sativa and Chenopodium album, which was the dominant weed in the field plots. Amplified ribosomal DNA restriction analysis (ARDRA) revealed plant-specific fragment size frequencies. Dominant ARDRA groups were identified by 16S rRNA gene nucleotide sequencing. Database comparisons indicated that the rhizospheres contained members of the Proteobacteria (α, β, and γ subgroups), members of the Cytophaga-Flavobacterium group, and gram-positive bacteria with high G+C DNA contents. The levels of many groups were affected by the plant species and, in the case ofM. sativa, by inoculation. The most abundant isolates were related to Variovorax sp., Arthrobacter ramosus, and Acinetobacter calcoaceticus. In the rhizosphere of M. sativa, inoculation reduced the numbers of cells of A. calcoaceticus and members of the genus Pseudomonas and increased the number of rhizobia. Cultivation-independent PCR–single-strand conformation polymorphism (SSCP) profiles of a 16S rRNA gene region confirmed the existence of plant-specific rhizosphere communities and the effect of the inoculant. All dominant ARDRA groups except Variovoraxspecies could be detected. On the other hand, the SSCP profiles revealed products which could not be assigned to the dominant cultured isolates, indicating that the bacterial diversity was greater than the diversity suggested by cultivation.

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The diversity of active microbial groups in an activated sludge process treating painting process wastewater

E3S Web of Conferences ◽

10.1051/e3sconf/202014801002 ◽

2020 ◽

Vol 148 ◽

pp. 01002

Author(s):

Herto Dwi Ariesyady ◽

Mentari Rizki Mayanda ◽

Tsukasa Ito

Keyword(s):

Wastewater Treatment ◽

Microbial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Activated Sludge ◽

Biological Treatment ◽

Rrna Gene ◽

Activated Sludge Process ◽

Painting Process ◽

Process Wastewater

Activated sludge process is one of the wastewater treatment method that is applied for many wastewater types including painting process wastewater of automotive industry. This wastewater is well-known to have high heavy metals concentration which could deteriorate water environment if appropriate performance of the wastewater treatment could not be achieved. In this study, we monitored microbial community diversity in a Painting Biological Treatment (PBT) system. We applied a combination of cultivation and genotypic biological methods based on 16S rRNA gene sequence analysis to identify the diversity of active microbial community. The results showed that active microbes that could grow in this activated sludge system were dominated by Gram-negative bacteria. Based on 16S rRNA gene sequencing analysis, it was revealed that their microbial diversity has close association with Bacterium strain E286, Isosphaera pallida, Lycinibacillus fusiformis, Microbacterium sp., Orchobactrum sp., Pseudomonas guariconensis, Pseudomonas sp. strain MR84, Pseudomonas sp. MC 54, Serpens sp., Stenotrophomonas acidaminiphila, and Xylella fastidiosa with similarity of 86 – 99%. This findings reflects that microbial community in a Painting Biological Treatment (PBT) system using activated sludge process could adapt with xenobiotics in the wastewater and has a wide range of diversity indicating a complex metabolism mechanism in the treatment process.

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