Application of COMPOCHIP Microarray to Investigate the Bacterial Communities of Different Composts

A microarray spotted with 369 different 16S rRNA gene probes specific to microorganisms involved in the degradation process of organic waste during composting was developed. The microarray was tested with pure cultures, and of the 30,258 individual probe-target hybridization reactions performed, there were only 188 false positive (0.62%) and 22 false negative signals (0.07%). Labeled target DNA was prepared by polymerase chain reaction amplification of 16S rRNA genes using a Cy5-labeled universal bacterial forward primer and a universal reverse primer. The COMPOCHIP microarray was applied to three different compost types (green compost, manure mix compost, and anaerobic digestate compost) of different maturity (2, 8, and 16 weeks), and differences in the microorganisms in the three compost types and maturity stages were observed. Multivariate analysis showed that the bacterial composition of the three composts was different at the beginning of the composting process and became more similar upon maturation. Certain probes (targeting Sphingobacterium, Actinomyces, Xylella/Xanthomonas/ Stenotrophomonas, Microbacterium, Verrucomicrobia, Planctomycetes, Low G + C and Alphaproteobacteria) were more influential in discriminating between different composts. Results from denaturing gradient gel electrophoresis supported those of microarray analysis. This study showed that the COMPOCHIP array is a suitable tool to study bacterial communities in composts

Inferring Carbon Sources from Gene Expression Profiles Using Metabolic Flux Models

Background: Bacteria have evolved the ability to efficiently and resourcefully adapt to changing environments. A key means by which they optimize their use of available nutrients is through adjustments in gene expression with consequent changes in enzyme activity. We report a new method for drawing environmental inferences from gene expression data. Our method prioritizes a list of candidate carbon sources for their compatibility with a gene expression profile using the framework of flux balance analysis to model the organism’s metabolic network. Principal Findings: For each of six gene expression profiles for Escherichia coli grown under differing nutrient conditions, we applied our method to prioritize a set of eighteen different candidate carbon sources. Our method ranked the correct carbon source as one of the top three candidates for five of the six expression sets when used with a genome-scale model. The correct candidate ranked fifth in the remaining case. Additional analyses show that these rankings are robust with respect to biological and measurement variation, and depend on specific gene expression, rather than general expression level. The gene expression profiles are highly adaptive: simulated production of biomass averaged 94.84% of maximum when the in silico carbon source matched the in vitro source of the expression profile, and 65.97% when it did not. Conclusions: Inferences about a microorganism’s nutrient environment can be made by integrating gene expression data into a metabolic framework. This work demonstrates that reaction flux limits for a model can be computed which are realistic in the sense that they affect in silico growth in a manner analogous to that in which a microorganism’s alteration of gene expression is adaptive to its nutrient environment.National Institute of Allergy and Infectious Diseases (U.S.) (grant HHSN 2722008000059C)National Institute of Allergy and Infectious Diseases (U.S.) (grant HHSN 26620040000IC)Bill & Melinda Gates Foundation (grant 18651010-37352-A

Whole community genome amplification (WCGA) leads to compositional bias in methane oxidizing communities as assessed by pmoA based microarray analyses and QPCR

Whole-genome amplification (WGA) using multiple displacement amplification (MDA) has recently been introduced to the field of environmental microbiology. The amplification of single-cell genomes or whole-community metagenomes decreases the minimum amount of DNA needed for subsequent molecular community analyses. The resolution of profiling methods of environmental microbial communities will increase substantially by the use of the whole-community genome amplification (WCGA) procedure, assuming that the original community composition is not affected qualitatively as well as quantitatively. The present study aims to test if WCGA introduces a bias when applied to aerobic proteobacterial methanotrophic communities. For this, first, we subjected samples from freshwater lake sediment to WCGA, and amplified using primers targeting the pmoA gene coding for the α-subunit of the methane monooxygenase enzyme. Second, we analysed community composition using a diagnostic microarray and quantitative PCR (QPCR) assays. These methods clearly demonstrated that the WCGA amplification introduced a bias. Thus, numbers of γ-proteobacterial methanotrophs ('type Ia') increased significantly while the α-proteobacterial methanotrophs ('type II') were not amplified by the WCGA procedure. It is hypothesized that this bias is caused by the differences in GC content, which may compromise the efficiency of the MDA reaction.

Ecological and molecular analysis of the rhizospheric methanotroph community in tropical rice soil: effect of crop phenology and land use history

To study the effect of crop phenology and cultivation practices on methanotrophic communities, two tropical rice fields located in the upper Gangetic plain of India with similar soil type and different cropping history were selected. A laboratory incubation experiment for the enumeration of methanotrophs and for the measurement of CH4 oxidation potential was conducted on a parallel basis. The methanotroph population size was found to be significantly higher in the Banaras Hindu University (BHU), Varanasi soil than the Indian Institute of Vegetable Research (IIVR), Varanasi soil. The population size increased with the age of the plant for both the sites. The CH4 oxidation potential was higher with the BHU soil compared to the IIVR soil. The CH4 oxidation rate increased significantly from tillering to flowering to grain-filling stages, and finally there was no significant difference between the grain-filling and the grain-maturation stages. A diagnostic microarray targeting the pmoA gene and a 16S rRNA denaturing gradient gel electrophoresis (DGGE)-based approach were applied to assess the diversity of the methanotrophic community for the two sites. A broad diversity of methanotrophs was detected at both sites, including type I and type II methanotrophs of the genera Methylobacter, Methylomonas, Methylosarcina, Methylosphaera, Methylomicrobium and Methylocystis. Type II methanotrophs were found in higher abundance as compared to type I methanotrophs at both the sites. DGGE analysis indicated that the methanotroph community in BHU soil was more or less stable, while little variation was found in IIVR soil during crop growth

Ecological and molecular analyses of the rhizospheric methanotroph community in tropical rice soil: effect of crop phenology and land-use history

To study the effect of crop phenology and cultivation practices on methanotrophic communities, two tropical rice fields located in the upper Gangetic plain of India with similar soil type and different cropping history were selected. A laboratory incubation experiment for the enumeration of methanotrophs and for the measurement of CH4 oxidation potential was conducted on a parallel basis. The methanotroph population size was found to be significantly higher in the Banaras Hindu University (BHU), Varanasi soil than the Indian Institute of Vegetable Research (IIVR), Varanasi soil. The population size increased with the age of the plant for both the sites. The CH4 oxidation potential was higher with the BHU soil compared to the IIVR soil. The CH4 oxidation rate increased significantly from tillering to flowering to grain-filling stages, and finally there was no significant difference between the grain-filling and the grain-maturation stages. A diagnostic microarray targeting the pmoA gene and a 16S rRNA denaturing gradient gel electrophoresis (DGGE)-based approach were applied to assess the diversity of the methanotrophic community for the two sites. A broad diversity of methanotrophs was detected at both sites, including type I and type II methanotrophs of the genera Methylobacter, Methylomonas, Methylosarcina, Methylosphaera, Methylomicrobium and Methylocystis. Type II methanotrophs were found in higher abundance as compared to type I methanotrophs at both the sites. DGGE analysis indicated that the methanotroph community in BHU soil was more or less stable, while little variation was found in IIVR soil during crop growth

The Gpr1-regulated Sur7 family protein Sfp2 is required for hyphal growth and cell wall stability in the mycoparasite <em>Trichoderma atroviride</em>.

Mycoparasites, e.g. fungi feeding on other fungi, are prominent within the genus Trichoderma and represent a promising alternative to chemical fungicides for plant disease control. We previously showed that the seven-transmembrane receptor Gprl regulates mycelia! growth and asexual development and governs mycoparasitism-related processes in Trichoderma atroviride. We now describe the identification of genes being targeted by Gpr1 under mycoparasitic conditions. The identified gene set includes a candidate, sfp2, encoding a protein of the fungal-specific Sur7 superfamily, whose upregulation in T. atroviride upon interaction with a fungal prey is dependent on Gpr1. Sur7 family proteins are typical residents of membrane microdomains such as the membrane compartment of Canl (MCC)/eisosome in yeast. We found that GFP-labeled Gpr1 and Sfp2 proteins show partly overlapping localization patterns in T. atroviride hyphae, which may point to shared functions and potential interaction during signal perception and endocytosis. Deletion of sfp2 caused heavily altered colony morphology, defects in polarized growth, cell wall integrity and endocytosis, and significantly reduced mycoparasitic activity, whereas sfp2 overexpression enhanced full overgrowth and killing of the prey. Transcriptional activation of a chitinase specific for hyphal growth and network formation and strong downregulation of chitin synthase-encoding genes were observed in Delta sfp2. Taken together, these findings imply crucial functions of Sfp2 in hyphal morphogenesis of T. atroviride and its interaction with prey fungi

Environmental distribution and abundance of the facultative methanotroph Methylocella

Methylocella spp. are facultative methanotrophs, which are able to grow not only on methane but also on multicarbon substrates such as acetate, pyruvate or malate. Methylocella spp. were previously thought to be restricted to acidic soils such as peatlands, in which they may have a key role in methane oxidation. There is little information on the abundance and distribution of Methylocella spp. in the environment. New primers were designed, and a real-time quantitative PCR method was developed and validated targeting Methylocella mmoX (encoding the alpha-subunit of the soluble methane monooxygenase) that allowed the quantification of Methylocella spp. in environmental samples. We also developed and validated specific PCR assays, which target 16S rRNA genes of known Methylocella spp. These were used to investigate the distribution of Methylocella spp. in a variety of environmental samples. It was revealed that Methylocella species are widely distributed in nature and not restricted to acidic environments. The ISME Journal (2011) 5, 1061-1066; doi:10.1038/ismej.2010.190; published online 16 December 2010 Subject Category: microbial ecology and functional diversity of natural habitat

One millimetre makes the difference : high-resolution analysis of methane-oxidizing bacteria and their specific activity at the oxic–anoxic interface in a flooded paddy soil

Aerobic methane-oxidizing bacteria (MOB) use a restricted substrate range, yet >30 species-equivalent operational taxonomical units (OTUs) are found in one paddy soil. How these OTUs physically share their microhabitat is unknown. Here we highly resolved the vertical distribution of MOB and their activity. Using microcosms and cryosectioning, we sub-sampled the top 3-mm of a water-saturated soil at near in situ conditions in 100-μm steps. We assessed the community structure and activity using the particulate methane monooxygenase gene pmoA as a functional and phylogenetic marker by terminal restriction fragment length polymorphism (t-RFLP), a pmoA-specific diagnostic microarray, and cloning and sequencing. pmoA genes and transcripts were quantified using competitive reverse transcriptase PCR combined with t-RFLP. Only a subset of the methanotroph community was active. Oxygen microprofiles showed that 89% of total respiration was confined to a 0.67-mm-thick zone immediately above the oxic-anoxic interface, most probably driven by methane oxidation. In this zone, a Methylobacter-affiliated OTU was highly active with up to 18 pmoA transcripts per cell and seemed to be adapted to oxygen and methane concentrations in the micromolar range. Analysis of transcripts with a pmoA-specific microarray found a Methylosarcina-affiliated OTU associated with the surface zone. High oxygen but only nanomolar methane concentrations at the surface suggested an adaptation of this OTU to oligotrophic conditions. No transcripts of type II methanotrophs (Methylosinus, Methylocystis) were found, which indicated that this group was represented by resting stages only. Hence, different OTUs within a single guild shared the same microenvironment and exploited different niches. © 2012 International Society for Microbial Ecology All rights reserved