Unknown,Transcriptomics,Genomics,Proteomics

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Microorganisms with Novel Dissimilatory (Bi)Sulfite Reductase Genes are Widespread and Core Microbiota in Low-Sulfate Peatlands

ABSTRACT: Peatlands of the Lehstenbach catchment (Germany) house so far unidentified microorganisms with phylogenetically novel variants of the dissimilatory (bi)sulfite reductase genes dsrAB. These genes are characteristic for microorganisms that reduce sulfate, sulfite, or some organosulfonates for energy conservation, but can also be present in anaerobic syntrophs. However, nothing is currently known regarding the abundance, community dynamics, and biogeography of these dsrAB-carrying microorganisms in peatlands. To tackle these issues, soils from a Lehstenbach catchment site (Schlöppnerbrunnen II fen) from different depths were sampled at three time points over a six-year period to analyze the diversity and distribution of dsrAB-containing microorganisms by a newly developed functional gene microarray and quantitative PCR assays. Members of novel, uncultivated dsrAB lineages (approximately representing species-level groups) (i) dominated a temporally stable but spatially structured dsrAB community and (ii) represented ‘core’ members (up to 1-1.7% relative abundance) of the autochthonous microbial community in this fen. In addition, denaturing gradient gel electrophoresis (DGGE)- and clone library-based comparison of the dsrAB diversity in soils from a wet meadow, three bogs, and five fens of various geographic locations (distance ~1-400 km), identified one Syntrophobacter-related and nine novel dsrAB lineages to be widespread in low-sulfate peatlands. Signatures of biogeography in dsrB-DGGE data were not correlated with geographic distance but could largely be explained by soil pH and wetland type, implying that distribution of dsrAB-carrying microorganisms in wetlands on the scale of a few hundred kilometers is not limited by dispersal but determined by contemporary environmental conditions. 36 dsrAB clones for chip evaluation, 33 hybridizations of labeled dsrAB RNA from environmental peatsoil samples

ORGANISM(S): uncultured sulfate-reducing bacterium

SUBMITTER: Doris Steger

PROVIDER: E-GEOD-24582 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Lactococcus garvieae is a bacterial pathogen that affects different animal species and human. Despite the widespread distribution and emerging clinical significance of L. garvieae in both veterinary and human medicine, there is almost a complete lack of knowledge about the genetic content of this microorganism. In the present study the genomic content of L. garvieae CECT 4531 was analyzed by bioinformatic tools and microarray-based comparative genomic hybridizations (CGH) experiments, using Lactococcus lactis subsp. lactis IL1403 and Streptococcus pneumoniae TIGR4 as reference microorganisms. Results: The combination and integration of in silico analyses and in vitro (CGH) experiments performed between the reference microorganisms allowed establishing an inter-species hybridization framework with a detection threshold based on the sequence similarity ?70%. With this threshold value, 267 genes were identified as having an analogue in L. garvieae, most of which (n = 258) have been documented for the first time in this pathogen. Most of these genes are related to ribosomal, sugar metabolism or energy conversion systems. Some identified genes could be involved in the pathogenesis of L. garvieae infections. Conclusions: In this study a comparative analysis based on microarray interspecies hybridization and the use of bioinformatic tools were used for the first time to study the genetic content of L. garvieae CECT 4531. Towards this approach, we identified 267 potentially present genes in L. garvieae CECT 4531, some of which could be involved in the pathogenesis of L. garvieae infections, such as als or mycA. These results provide the first insight into the genome content of L. garvieae. Array-based comparative genome hybridization (CGH): The L. lactis subsp. lactis IL1403 and S. pneumoniae TIGR4 microarrays used for the CGH analysis were purchased from Eurogentec (Serain, Belgium). The L. lactis microarray contains 4608 spots: 2126 duplicated ORFs, 32 negative controls and 324 empty spots. The S. pneumoniae microarray contains 4608 spots: 2087 duplicated ORFs, 224 negative controls and 210 empty spots. The CGH experiments were performed by means of competitive hybridizations using DNA of L. lactis subsp. lactis IL1403 or S. pneumoniae TIGR4, depending on the array as positive controls. DNAs to be hybridized on the same array were labelled with Cy3-dUTP and Cy5-dUTP, respectively. For each microarray hybridization reaction, aliquots (1–2 µg) of labelled genomic DNAs of reference (labelled with Cy3) and test (labelled with Cy5) strains, were mixed in 45 µl EGT hybridization solution (Eurogentec, Serain, Belgium) and denatured at 65ºC for 2 min. The hybridization mixture was then loaded onto a microarray slide, covered with a coverslip and incubated at 38ºC overnight. Following hybridization, the slides were washed in 2 X SSC, 0.5% SDS for 5 min followed by 5 min in 1 X SSC, 0.25% SDS. Finally, slides were rinsed in 0.2 X SSC and dried by centrifugation. The results presented herein represent a compilation of sixteen separate CGH experiments: L. lactis subsp. lactis IL1403 arrays (reference microorganism) were hybridized with S. pneumoniae TIGR4 (test microorganism) (n=2); S. pneumoniae TIGR4 arrays (reference microorganism) were hybridized with L. lactis subsp. lactis IL1403 (test microorganism) (n=2); L. lactis subsp. lactis IL1403 arrays (reference microorganism) were hybridized with L.garvieae CECT 4531 (test microorganism) (n=8); S. pneumoniae TIGR4 arrays (reference microorganism) were hybridized with L.garvieae CECT 4531 (test microorganism) (n=4). Data acquisition and analysis: The microarray was scanned after hybridization using a Scanarray HT microarray scanner (Perkin-Elmer). The signal intensity of the two fluors was determined using ImaGene software (BioDiscovery, El Segundo, CA, USA). Microarray data were analysed using ImaGene software, Microsoft Excel and an in-house designed and built Microsoft Access database [30]. Gene calling was based on a signal-to-noise ratio (SNR) > 3 for each spot. After CGH experiments, a gene was considered to show a positive result when it was present in at least three of the four CGH assays. In the case of the L. garvieae CECT 4531 hybridizations with L. lactis subsp. lactis IL1403 arrays, it was necessary to perform a greater number of assays (n=8) due to the poor quality of one of the array batches used. Thus, the criteria chosen to determine a positive result in this case was when the gene was present in at least five of the eight CGH assays.

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Microorganisms with Novel Dissimilatory (Bi)Sulfite Reductase Genes are Widespread and Core Microbiota in Low-Sulfate Peatlands

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