scholarly journals Analysis of Microbial Gene Transcripts in Environmental Samples†

2005 ◽  
Vol 71 (7) ◽  
pp. 4121-4126 ◽  
Author(s):  
Rachel S. Poretsky ◽  
Nasreen Bano ◽  
Alison Buchan ◽  
Gary LeCleir ◽  
Jutta Kleikemper ◽  
...  
Keyword(s):  
Gene Expression ◽  
Microbial Communities ◽  
Environmental Samples ◽  
Sulfur Oxidation ◽  
Subtractive Hybridization ◽  
Functional Genes ◽  
New Approach ◽  
Total Rna ◽  
Gene Transcripts ◽  
Taxonomic Groups

ABSTRACT We analyzed gene expression in marine and freshwater bacterioplankton communities by the direct retrieval and analysis of microbial transcripts. Environmental mRNA, obtained from total RNA by subtractive hybridization of rRNA, was reverse transcribed, amplified with random primers, and cloned. Approximately 400 clones were analyzed, of which ∼80% were unambiguously mRNA derived. mRNAs appeared to be from diverse taxonomic groups, including both Bacteria (mainly α- and γ-Proteobacteria) and Archaea (mainly Euryarchaeota). Many transcripts could be linked to environmentally important processes such as sulfur oxidation (soxA), assimilation of C1 compounds (fdh1B), and acquisition of nitrogen via polyamine degradation (aphA). Environmental transcriptomics is a means of exploring functional gene expression within natural microbial communities without bias toward known sequences, and provides a new approach for obtaining community-specific variants of key functional genes.

The identification of bacterial gene expression differences using mRNA-based isothermal subtractive hybridization

1995 ◽  
Vol 41 (2) ◽  
pp. 152-156 ◽  
Author(s):  
Eric A. Utt ◽  
Jeffery P. Brousal ◽  
Lynne C. Kikuta-Oshima ◽  
Frederick D. Quinn
Keyword(s):  
Gene Expression ◽  
Listeria Monocytogenes ◽  
Subtractive Hybridization ◽  
Listeriolysin O ◽  
Bacterial Strains ◽  
Bacterial Gene ◽  
Total Rna ◽  
Bacterial Gene Expression ◽  
Hybridization Probes ◽  
The Difference

We describe a method for isolating and determining differences in gene expression between related bacterial strains. The method is based upon differences in mRNA expression. To demonstrate this procedure, cDNA generated from total RNA of Listeria monocytogenes serotype 1/2a was hybridized to total RNA from a Tn916 mutant of serogroup 1/2a (M3) that was deficient in the production of listeriolysin O, the product of the hly gene. The single-stranded cDNA fragments remaining after hybridization represent the difference in expressed genes between the two strains. These subtraction products were used as hybridization probes to identify the corresponding hly gene in a Southern hybridization.Key words: subtractive hybridization, Listeria monocytogenes, hemolysin, gene expression, isogenic.

scholarly journals Microarray-Based Analysis of Microbial Community RNAs by Whole-Community RNA Amplification

2006 ◽  
Vol 73 (2) ◽  
pp. 563-571 ◽  
Author(s):  
Haichun Gao ◽  
Zamin K. Yang ◽  
Terry J. Gentry ◽  
Liyou Wu ◽  
Christopher W. Schadt ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Environmental Samples ◽  
Bacterial Species ◽  
Shewanella Oneidensis ◽  
Rna Amplification ◽  
T7 Promoter ◽  
New Approach ◽  
Functional Activities ◽  
Amplification Method ◽  
Groundwater Samples

ABSTRACT A new approach, termed whole-community RNA amplification (WCRA), was developed to provide sufficient amounts of mRNAs from environmental samples for microarray analysis. This method employs fusion primers (six to nine random nucleotides with an attached T7 promoter) for the first-strand synthesis. The shortest primer (T7N6S) gave the best results in terms of the yield and representativeness of amplification. About 1,200- to 1,800-fold amplification was obtained with amounts of the RNA templates ranging from 10 to 100 ng, and very representative detection was obtained with 50 to 100 ng total RNA. Evaluation with a Shewanella oneidensis Δfur strain revealed that the amplification method which we developed could preserve the original abundance relationships of mRNAs. In addition, to determine whether representative detection of RNAs can be achieved with mixed community samples, amplification biases were evaluated with a mixture containing equal quantities of RNAs (100 ng each) from four bacterial species, and representative amplification was also obtained. Finally, the method which we developed was applied to the active microbial populations in a denitrifying fluidized bed reactor used for denitrification of contaminated groundwater and ethanol-stimulated groundwater samples for uranium reduction. The genes expressed were consistent with the expected functions of the bioreactor and groundwater system, suggesting that this approach is useful for analyzing the functional activities of microbial communities. This is one of the first demonstrations that microarray-based technology can be used to successfully detect the activities of microbial communities from real environmental samples in a high-throughput fashion.

scholarly journals Metagenomic insights into the effects of submerged plants on functional potential of microbial communities in wetland sediments

2021 ◽  
Vol 3 (4) ◽  
pp. 405-415
Author(s):  
Binhao Wang ◽  
Xiafei Zheng ◽  
Hangjun Zhang ◽  
Xiaoli Yu ◽  
Yingli Lian ◽  
...  
Keyword(s):  
Microbial Communities ◽  
C:N Ratio ◽  
Sulfur Oxidation ◽  
Functional Genes ◽  
Submerged Plants ◽  
Wetland Ecosystems ◽  
Elemental Cycling ◽  
Methane Budget ◽  
S Cycling ◽  
Methane Metabolism

AbstractSubmerged plants in wetlands play important roles as ecosystem engineers to improve self-purification and promote elemental cycling. However, their effects on the functional capacity of microbial communities in wetland sediments remain poorly understood. Here, we provide detailed metagenomic insights into the biogeochemical potential of microbial communities in wetland sediments with and without submerged plants (i.e., Vallisneria natans). A large number of functional genes involved in carbon (C), nitrogen (N) and sulfur (S) cycling were detected in the wetland sediments. However, most functional genes showed higher abundance in sediments with submerged plants than in those without plants. Based on the comparison of annotated functional genes in the N and S cycling databases (i.e., NCycDB and SCycDB), we found that genes involved in nitrogen fixation (e.g., nifD/H/K/W), assimilatory nitrate reduction (e.g., nasA and nirA), denitrification (e.g., nirK/S and nosZ), assimilatory sulfate reduction (e.g., cysD/H/J/N/Q and sir), and sulfur oxidation (e.g., glpE, soeA, sqr and sseA) were significantly higher (corrected p < 0.05) in vegetated vs. unvegetated sediments. This could be mainly driven by environmental factors including total phosphorus, total nitrogen, and C:N ratio. The binning of metagenomes further revealed that some archaeal taxa could have the potential of methane metabolism including hydrogenotrophic, acetoclastic, and methylotrophic methanogenesis, which are crucial to the wetland methane budget and carbon cycling. This study opens a new avenue for linking submerged plants with microbial functions, and has further implications for understanding global carbon, nitrogen and sulfur cycling in wetland ecosystems.

scholarly journals Genome-reconstruction for eukaryotes from complex natural microbial communities

2017 ◽  
Author(s):  
Patrick T. West ◽  
Alexander J. Probst ◽  
Igor V. Grigoriev ◽  
Brian C. Thomas ◽  
Jillian F. Banfield
Keyword(s):  
Organic Carbon ◽  
Microbial Communities ◽  
Environmental Samples ◽  
Carbon Fixation ◽  
Sulfur Oxidation ◽  
Metabolic Potential ◽  
Microbial Eukaryotes ◽  
Sequence Identification ◽  
Ecosystem Studies ◽  
Almost All

AbstractMicrobial eukaryotes are integral components of natural microbial communities and their inclusion is critical for many ecosystem studies yet the majority of published metagenome analyses ignore eukaryotes. In order to include eukaryotes in environmental studies we propose a method to recover eukaryotic genomes from complex metagenomic samples. A key step for genome recovery is separation of eukaryotic and prokaryotic fragments. We developed a kmer-based strategy, EukRep, for eukaryotic sequence identification and applied it to environmental samples to show that it enables genome recovery, genome completeness evaluation and prediction of metabolic potential. We used this approach to test the effect of addition of organic carbon on a geyser-associated microbial community and detected a substantial change of the community metabolism, with selection against almost all candidate phyla bacteria and archaea and for eukaryotes. Near complete genomes were reconstructed for three fungi placed within the eurotiomycetes and an arthropod. While carbon fixation and sulfur oxidation were important functions in the geyser community prior to carbon addition, the organic carbon impacted community showed enrichment for secreted proteases, secreted lipases, cellulose targeting CAZymes, and methanol oxidation. We demonstrate the broader utility of EukRep by reconstructing and evaluating relatively high quality fungal, protist, and rotifer genomes from complex environmental samples. This approach opens the way for cultivation-independent analyses of whole microbial communities.

Gene-expression profiling using suppression-subtractive hybridization and cDNA microarray in rat mononuclear cells in response to welding-fume exposure

2004 ◽  
Vol 20 (1-5) ◽  
pp. 77-88 ◽  
Author(s):  
Kyung Taek Rim ◽  
Kun Koo Park ◽  
Jae Hyuck Sung ◽  
Yong Hyun Chung ◽  
Jeong Hee Han ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pulmonary Fibrosis ◽  
Suppression Subtractive Hybridization ◽  
Cdna Microarray ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Subtractive Hybridization ◽  
Welding Fumes ◽  
Welding Fume ◽  
Total Rna

Welders with radiographic pneumoconiosis abnormalities have shown a gradual clearing of the X-ray identified effects following removal from exposure. In some cases, the pulmonary fibrosis associated with welding fumes appears in a more severe form in welders. Accordingly, for the early detection of welding-fume-exposure-induced pulmonary fibrosis, the gene expression profiles of peripheral mononuclear cells from rats exposed to welding fumes were studied using suppression-subtractive hybridization (SSH) and a cDNA microarray. As such, Sprague-Dawley rats were exposed to a stainless steel arc welding fume for 2 h/day in an inhalation chamber with a 107.59 / 2.6 mg/m3 concentration of total suspended particulate (TSP) for 30 days. Thereafter, the total RNA was extracted from the peripheral blood mononuclear cells, the cDNA synthesized from the total RNA using the SMARTTM PCR cDNA method, and SSH performed to select the welding-fume-exposure-regulated genes. The cDNAs identified by the SSH were then cloned into a plasmid miniprep, sequenced and the sequences analysed using the NCBI BLAST programme. In the SSH cloned cDNA microarray analysis, five genes were found to increase their expression by 1.9-fold or more, including Rgs 14, which plays an important function in cellular signal transduction pathways; meanwhile 36 genes remained the same and 30 genes decreased their expression by more than 59%, including genes associated with the immune response, transcription factors and tyrosine kinases. Among the 5200 genes analysed, 256 genes (5.1%) were found to increase their gene expression, while 742 genes (15%) decreased their gene expression in response to the welding-fume exposure when tested using a commercial 5.0k DNA microarray. Therefore, unlike exposure to other toxic substances, prolonged welding-fume exposure was found to substantially downregulate many genes.

scholarly journals Habitat Elevation Shapes Microbial Community Composition and Alter the Metabolic Functions in Wild Sable (Martes zibellina) Guts

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 865
Author(s):  
Lantian Su ◽  
Xinxin Liu ◽  
Guangyao Jin ◽  
Yue Ma ◽  
Haoxin Tan ◽  
...  
Keyword(s):  
Microbial Community ◽  
Environmental Factors ◽  
Microbial Communities ◽  
Community Composition ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Functional Genes ◽  
Martes Zibellina ◽  
Clear Cutting ◽  
Metabolic Functions

In recent decades, wild sable (Carnivora Mustelidae Martes zibellina) habitats, which are often natural forests, have been squeezed by anthropogenic disturbances such as clear-cutting, tilling and grazing. Sables tend to live in sloped areas with relatively harsh conditions. Here, we determine effects of environmental factors on wild sable gut microbial communities between high and low altitude habitats using Illumina Miseq sequencing of bacterial 16S rRNA genes. Our results showed that despite wild sable gut microbial community diversity being resilient to many environmental factors, community composition was sensitive to altitude. Wild sable gut microbial communities were dominated by Firmicutes (relative abundance 38.23%), followed by Actinobacteria (30.29%), and Proteobacteria (28.15%). Altitude was negatively correlated with the abundance of Firmicutes, suggesting sable likely consume more vegetarian food in lower habitats where plant diversity, temperature and vegetation coverage were greater. In addition, our functional genes prediction and qPCR results demonstrated that energy/fat processing microorganisms and functional genes are enriched with increasing altitude, which likely enhanced metabolic functions and supported wild sables to survive in elevated habitats. Overall, our results improve the knowledge of the ecological impact of habitat change, providing insights into wild animal protection at the mountain area with hash climate conditions.

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