DNA-Based Stable Isotope Probing.

Microbiomes on Earth are often considered the most heterogeneous biological entities, but their vital roles in driving global biogeochemical cycles often remain elusive. DNA-based stable isotope probing (DNA-SIP) provides a powerful means to establish a direct link between biogeochemical processes and the taxonomic identities of active microorganisms involved in the processes. Combined with high-throughput sequencing, it significantly aids in deciphering ecophysiological functions of active microorganisms at the level of microbial communities. DNA-SIP relies solely on the propagation of targeted microbial communities, during which the entire genomes of daughter cells are synthesized and increasingly 13C-labeled. This growth on 13C-labeled substrate in association with cell division provides solid evidence for the functional importance and metabolic potential of targeted microorganisms. The essential prerequisite for a successful DNA-SIP experiment is the identification, with confidence, of isotopically enriched 13C-DNA, of which the amount is generally too low to allow for the direct measurement of 13C atomic percent of nucleic acid. The 13C labeling can be readily identified in the fractionated DNA by quantification of functional genes specific to the known targeted microorganisms, and by high-throughput sequencing of the total microbial communities via 16S rRNA genes without prior knowledge of which microorganisms are 13C-labeled (i.e., highly enriched in the heavy fractions relative to 12C (natural isotope abundance) control treatments). In this chapter, the protocol for obtaining DNA highly enriched in heavy isotope is presented using diazotrophic methanotrophs in a paddy soil as a case study.

Targeted Metatranscriptomics of Soil Microbial Communities with Stable Isotope Probing.

Metatranscriptomics is a powerful tool for capturing gene expression patterns in microbial communities and investigating their responses to environmental conditions. Stable isotope probing (SIP) is a method to target specific functional groups of microorganisms in environmental samples. The combination of RNA-SIP with metatranscriptomic analysis enhances the detection and identification of mRNA from target microorganisms. In this chapter we provide a protocol for RNA-SIP, mRNA enrichment, and mRNA preparation for high-throughput sequencing using an example of targeting methanotrophs in rice field soil.

Novel s-triazine-degrading bacteria isolated from agricultural soils of central Chile for herbicide bioremediation

s-Triazine-degrading bacterial strains were isolated from long-term simazine-treated agricultural soils of central Chile. The number of culturable heterotrophic bacteria of these agricultural soils (7 x 10(6) CFU/g of dry soil) was not affected by simazine application on field. The simazine-degrading bacterial strains P51, P52 and C53 were isolated by enrichment in minimal medium using simazine as the sole nitrogen source. Resting cells of strains P51 and P52 degraded >80 percent of simazine within 48 hrs, whereas strain C53 was able to remove >60 percent of the herbicide. The atzA and atzD genes of the s-triazine upper and lower catabolic pathways were detected in strains P51 and C53, while only atzD gene was observed in strain P52. To compare the bacterial 16S rRNA gene sequence structure, ARDRA were performed using the restriction enzymes Msp1 and Hha1. ARDRA indicated that strain P52 was a different ribotype than C53 and P51 strains. For further characterization the novel isolates were identified by 16S rRNA gene sequencing. Strains C53 and P51 belong to the genus Stenotrophomonas and the strain P52 belongs to the genus Arthrobacter . s -Triazine-degrading bacterial strains isolated from contaminated soils could be used as biocatalysts for bioremediation of these herbicides.