scholarly journals Succession of Microbial Communities during Hot Composting as Detected by PCR–Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes

2000 ◽  
Vol 66 (3) ◽  
pp. 930-936 ◽  
Author(s):  
Sabine Peters ◽  
Stefanie Koschinsky ◽  
Frank Schwieger ◽  
Christoph C. Tebbe
Keyword(s):  
16S Rrna ◽  
18S Rrna ◽  
Single Strand Conformation Polymorphism ◽  
Small Subunit ◽  
Ssu Rdna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Small Subunit Rrna ◽  
Genetic Profiles ◽  
Conformation Polymorphism

ABSTRACT A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4–V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8–V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of γ-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.

scholarly journals Monitoring Bacterial Communities in Raw Milk and Cheese by Culture-Dependent and -Independent 16S rRNA Gene-Based Analyses

2007 ◽  
Vol 73 (6) ◽  
pp. 1882-1891 ◽  
Author(s):  
Céline Delbès ◽  
Leila Ali-Mandjee ◽  
Marie-Christine Montel
Keyword(s):  
16S Rrna ◽  
Culture Media ◽  
Raw Milk ◽  
Single Strand Conformation Polymorphism ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Plate Count ◽  
Rrna Gene ◽  
Dual Culture ◽  
Culture Dependent

ABSTRACT The diversity and dynamics of bacterial populations in Saint-Nectaire, a raw-milk, semihard cheese, were investigated using a dual culture-dependent and direct molecular approach combining single-strand conformation polymorphism (SSCP) fingerprinting and sequencing of 16S rRNA genes. The dominant clones, among 125 16S rRNA genes isolated from milk, belonged to members of the Firmicutes (58% of the total clones) affiliated mainly with the orders Clostridiales and the Lactobacillales, followed by the phyla Proteobacteria (21.6%), Actinobacteria (16.8%), and Bacteroidetes (4%). Sequencing the 16S rRNA genes of 126 milk isolates collected from four culture media revealed the presence of 36 different species showing a wider diversity in the Gammaproteobacteria phylum and Staphylococcus genus than that found among clones. In cheese, a total of 21 species were obtained from 170 isolates, with dominant species belonging to the Lactobacillales and subdominant species affiliated with the Actinobacteria, Bacteroidetes (Chryseobacterium sp.), or Gammaproteobacteria (Stenotrophomonas sp.). Fingerprinting DNA isolated from milk by SSCP analysis yielded complex patterns, whereas analyzing DNA isolated from cheese resulted in patterns composed of a single peak which corresponded to that of lactic acid bacteria. SSCP fingerprinting of mixtures of all colonies harvested from plate count agar supplemented with crystal violet and vancomycin showed good potential for monitoring the subdominant Proteobacteria and Bacteroidetes (Flavobacteria) organisms in milk and cheese. Likewise, analyzing culturable subcommunities from cheese-ripening bacterial medium permitted assessment of the diversity of halotolerant Actinobacteria and Staphylococcus organisms. Direct and culture-dependent approaches produced complementary information, thus generating a more accurate view of milk and cheese microbial ecology.

scholarly journals Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length Heterogeneity

1998 ◽  
Vol 64 (11) ◽  
pp. 4522-4529 ◽  
Author(s):  
Marcelino Suzuki ◽  
Michael S. Rappé ◽  
Stephen J. Giovannoni
Keyword(s):  
Clone Library ◽  
Fluorescence Emission ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Length Variation ◽  
Small Subunit Rrna ◽  
Gene Frequencies ◽  
Rdna Sequences

ABSTRACT Marine bacterioplankton diversity was examined by quantifying natural length variation in the 5′ domain of small-subunit (SSU) rRNA genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast. This new technique, length heterogeneity analysis by PCR (LH-PCR), determines the relative proportions of amplicons originating from different organisms by measuring the fluorescence emission of a labeled primer used in the amplification reaction. Relationships between the sizes of amplicons and gene phylogeny were predicted by an analysis of 366 SSU rDNA sequences from cultivated marine bacteria and from bacterial genes cloned directly from environmental samples. LH-PCR was used to compare the distribution of bacterioplankton SSU rDNAs from a coastal water sample with that of an SSU rDNA clone library prepared from the same sample and also to examine the distribution of genes in the PCR products from which the clone library was prepared. The analysis revealed that the relative frequencies of genes amplified from natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product molecules can skew gene frequencies when PCR product concentrations exceed threshold values.

scholarly journals Pig Manure Contamination Marker Selection Based on the Influence of Biological Treatment on the Dominant Fecal Microbial Groups

2009 ◽  
Vol 75 (15) ◽  
pp. 4967-4974 ◽  
Author(s):  
Romain Marti ◽  
Patrick Dabert ◽  
Anne-Marie Pourcher
Keyword(s):  
16S Rrna ◽  
Intestinal Tract ◽  
Single Strand Conformation Polymorphism ◽  
Land Application ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Pig Manure ◽  
Aerobic Treatment ◽  
Potential Marker

ABSTRACT The objective of this study was to identify a microbial marker for pig manure contamination. We quantified the persistence of four dominant bacterial groups from the pig intestinal tract throughout manure handling at 10 livestock operations (including aerobic digestion) by using molecular typing. The partial 16S rRNA genes of Bacteroides-Prevotella, Eubacterium-Clostridiaceae, Bacillus-Streptococcus-Lactobacillus (BSL), and Bifidobacterium group isolates were amplified and analyzed by capillary electrophoresis single-strand conformation polymorphism. The most dominant bacterial populations were identified by cloning and sequencing their 16S rRNA genes. The results showed that Bifidobacterium spp. and, to a lesser extent, members of the BSL group, were less affected by the aerobic treatment than either Eubacterium-Clostridiaceae or Bacteroides-Prevotella. Two Bifidobacterium species found in raw manure were still present in manure during land application, suggesting that they can survive outside the pig intestinal tract and also survive aerobic treatment. The 16S-23S rRNA internal transcribed spacer of one species, Bifidobacterium thermacidophilum subsp. porcinum, was sequenced, and a specific pair of primers was designed for its detection in the environment. With this nested PCR assay, this potential marker was not detected in samples from 30 bovine, 30 poultry, and 28 human fecal samples or in 15 urban wastewater effluents. As it was detected in runoff waters after spreading of pig manure, we propose this marker as a suitable microbial indicator of pig manure contamination.

scholarly journals In Situ Accessibility of Small-Subunit rRNA of Members of the Domains Bacteria, Archaea, and Eucarya to Cy3-Labeled Oligonucleotide Probes

2003 ◽  
Vol 69 (3) ◽  
pp. 1748-1758 ◽  
Author(s):  
Sebastian Behrens ◽  
Caroline Rühland ◽  
João Inácio ◽  
Harald Huber ◽  
Á. Fonseca ◽  
...  
Keyword(s):  
16S Rrna ◽  
18S Rrna ◽  
Small Subunit ◽  
Oligonucleotide Probes ◽  
Consensus Model ◽  
Class Iii ◽  
Small Subunit Rrna ◽  
Data Set ◽  
Secondary Structure Models

ABSTRACT Low accessibility of the rRNA is together with cell wall impermeability and low cellular ribosome content a frequent reason for failure of whole-cell fluorescence hybridization with fluorescently labeled oligonucleotide probes. In this study we compare accessibility data for the 16S rRNA of Escherichia coli (gamma Proteobacteria, Bacteria) with the phylogenetically distantly related organisms Pirellula sp. strain 1 (Planctomycetes, Bacteria) and Metallosphaera sedula (Crenarchaeota, Archaea) and the 18S rRNA accessibility of Saccharomyces cerevisiae (Eucarya). For a total of 537 Cy3-labeled probes, the signal intensities of hybridized cells were quantified under standardized conditions by flow cytometry. The relative probe-conferred fluorescence intensities are shown on color-coded small-subunit rRNA secondary-structure models. For Pirellula sp., most of the probes belong to class II and III (72% of the whole data set), whereas most of the probes targeting sites on M. sedula were grouped into class V and VI (46% of the whole data set). For E. coli, 45% of all probes of the data set belong to class III and IV. A consensus model for the accessibility of the small-subunit rRNA to oligonucleotide probes is proposed which uses 60 homolog target sites of the three prokaryotic 16S rRNA molecules. In general, open regions were localized around helices 13 and 14 including target positions 285 to 338, whereas helix 22 (positions 585 to 656) and the 3′ half of helix 47 (positions 1320 to 1345) were generally inaccessible. Finally, the 16S rRNA consensus model was compared to data on the in situ accessibility of the 18S rRNA of S. cerevisiae.

scholarly journals Airborne Bacterial and Eukaryotic Community Structure across the United Kingdom Revealed by High-Throughput Sequencing

Atmosphere ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 802
Author(s):  
Hokyung Song ◽  
Ian Crawford ◽  
Jonathan Lloyd ◽  
Clare Robinson ◽  
Christopher Boothman ◽  
...  
Keyword(s):  
16S Rrna ◽  
High Throughput ◽  
18S Rrna ◽  
High Throughput Sequencing ◽  
Gene Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Biological Aerosols ◽  
Rrna Gene Sequencing

Primary biological aerosols often include allergenic and pathogenic microorganisms posing potential risks to human health. Moreover, there are airborne plant and animal pathogens that may have ecological and economic impact. In this study, we used high-throughput sequencing techniques (Illumina, MiSeq) targeting the 16S rRNA genes of bacteria and the 18S rRNA genes of eukaryotes, to characterize airborne primary biological aerosols. We used a filtration system on the UK Facility for Airborne Atmospheric Measurements (FAAM) research aircraft to sample a range of primary biological aerosols across southern England overflying surface measurement sites from Chilbolton to Weybourne. We identified 30 to 60 bacterial operational taxonomic units (OTUs) and 108 to 224 eukaryotic OTUs per sample. Moreover, 16S rRNA gene sequencing identified significant numbers of genera that have not been found in atmospheric samples previously or only been described in limited number of atmospheric field studies, which are rather old or published in local journals. This includes the genera Gordonia, Lautropia, and Psychroglaciecola. Some of the bacterial genera found in this study include potential human pathogens, for example, Gordonia, Sphingomonas, Chryseobacterium, Morganella, Fusobacterium, and Streptococcus. 18S rRNA gene sequencing showed Cladosporium to be the major genus in all of the samples, which is a well-known allergen and often found in the atmosphere. There were also genetic signatures of potentially allergenic taxa; for example, Pleosporales, Phoma, and Brassicales. Although there was no significant clustering of bacterial and eukaryotic communities depending on the sampling location, we found meteorological factors explaining significant variations in the community composition. The findings in this study support the application of DNA-based sequencing technologies for atmospheric science studies in combination with complementary spectroscopic and microscopic techniques for improved identification of primary biological aerosols.

Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

2015 ◽  
Vol 41 (1) ◽  
pp. 51-58
Author(s):  
Mohammad Shamimul Alam ◽  
Hawa Jahan ◽  
Rowshan Ara Begum ◽  
Reza M Shahjahan
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fish Larva ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Valuable Insight ◽  
Multiple Sequence ◽  
Pcr Rflp ◽  
Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

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