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Sharma S.,Indian Institute of Technology Delhi | Mehta R.,Indian Institute of Technology Delhi | Gupta R.,Indian Institute of Technology Delhi | Schloter M.,Research Unit for Environmental Genomics
Journal of Microbiological Methods | Year: 2012

An improved protocol for extraction of prokaryotic mRNA from soil samples was developed by modifying the extraction procedure to obtain higher yields of mRNA and to reduce co-extraction of humic acids. The modified protocol was found to be more robust and efficient compared to the original protocol by Griffiths et al. (2000) without compromising with the quality and quantity of RNA. © 2012 Elsevier B.V.


Giebler J.,Helmholtz Center for Environmental Research | Wick Y.L.,Helmholtz Center for Environmental Research | Schloter M.,Research Unit for Environmental Genomics | Harms H.,Helmholtz Center for Environmental Research | Chatzinotas A.,Helmholtz Center for Environmental Research
Applied and Environmental Microbiology | Year: 2013

Sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses revealed multiple alkB gene copies/cell in soil bacterial isolates and an apparently high genetic mobility among various phylogenetic groups. Identifying alkane degraders by alkB terminal restriction fragments (T-RFs) and sequences is strongly biased, as the phylogenetic trees based on 16S rRNA and alkB gene sequences were highly inconsistent. © 2013, American Society for Microbiology.


Schulz S.,TU Munich | Schulz S.,Research Unit for Environmental Genomics | Giebler J.,Helmholtz Center for Environmental Research | Chatzinotas A.,Helmholtz Center for Environmental Research | And 5 more authors.
ISME Journal | Year: 2012

Alkanes are major constituents of plant-derived waxy materials. In this study, we investigated the abundance, community structure and activity of bacteria harbouring the alkane monooxygenase gene alkB, which catalyses a major step in the pathway of aerobic alkane degradation in the litter layer, the litter-soil interface and in bulk soil at three time points during the degradation of maize and pea plant litter (2, 8 and 30 weeks) to improve our understanding about drivers for microbial performance in different soil compartments. Soil cores of different soil textures (sandy and silty) were taken from an agricultural field and incubated at constant laboratory conditions. The abundance of alkB genes and transcripts (by qPCR) as well as the community structure (by terminal restriction fragment polymorphism fingerprinting) were measured in combination with the concentrations and composition of alkanes. The results obtained indicate a clear response pattern of all investigated biotic and abiotic parameters depending on the applied litter material, the type of soil used, the time point of sampling and the soil compartment studied. As expected the distribution of alkanes of different chain length formed a steep gradient from the litter layer to the bulk soil. Mainly in the two upper soil compartments community structure and abundance patterns of alkB were driven by the applied litter type and its degradation. Surprisingly, the differences between the compartments in one soil were more pronounced than the differences between similar compartments in the two soils studied. This indicates the necessity for analysing processes in different soil compartments to improve our mechanistic understanding of the dynamics of distinct functional groups of microbes. © 2012 International Society for Microbial Ecology All rights reserved.


Pereira e Silva M.C.,University of Groningen | Schloter-Hai B.,Research Unit for Environmental Genomics | Schloter M.,Research Unit for Environmental Genomics | van Elsas J.D.,University of Groningen | Salles J.F.,University of Groningen
PLoS ONE | Year: 2013

Background:Despite the fact that the fixation of nitrogen is one of the most significant nutrient processes in the terrestrial ecosystem, a thorough study of the spatial and temporal patterns in the abundance and distribution of N-fixing communities has been missing so far.Methodology/Principal Findings:In order to understand the dynamics of diazotrophic communities and their resilience to external changes, we quantified the abundance and characterized the bacterial community structures based on the nifH gene, using real-time PCR, PCR-DGGE and 454-pyrosequencing, across four representative Dutch soils during one growing season. In general, higher nifH gene copy numbers were observed in soils with higher pH than in those with lower pH, but lower numbers were related to increased nitrate and ammonium levels. Results from nifH gene pyrosequencing confirmed the observed PCR-DGGE patterns, which indicated that the N fixers are highly dynamic across time, shifting around 60%. Forward selection on CCA analysis identified N availability as the main driver of these variations, as well as of the evenness of the communities, leading to very unequal communities. Moreover, deep sequencing of the nifH gene revealed that sandy soils (B and D) had the lowest percentage of shared OTUs across time, compared with clayey soils (G and K), indicating the presence of a community under constant change. Cosmopolitan nifH species (present throughout the season) were affiliated with Bradyrhizobium, Azospirillum and Methylocistis, whereas other species increased their abundances progressively over time, when appropriate conditions were met, as was notably the case for Paenibacilus and Burkholderia.Conclusions:Our study provides the first in-depth pyrosequencing analysis of the N-fixing community at both spatial and temporal scales, providing insights into the cosmopolitan and specific portions of the nitrogen fixing bacterial communities in soil. © 2013 Pereira e Silva et al.

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