Characterizing the diversity of active bacteria in soil by comprehensive stable isotope probing of DNA and RNA with H₂¹⁸O

Abstract

Current limitations in culture-based methods have lead to a reliance on culture-independent approaches, based principally on the comparative analysis of primary semantides such as ribosomal gene sequences. DNA can be remarkably stable in some environments, so its presence does not indicate live bacteria, but extracted ribosomal RNA (rRNA) has previously been viewed as an indicator of active cells. Stable isotope probing (SIP) involves the incorporation of heavy isotopes into newly synthesized nucleic acids, and can be used to separate newly synthesized from existing DNA or rRNA. H218O is currently the only potential universal bacterial substrate suitable for SIP of entire bacterial communities. The aim of our work was to compare soil bacterial community composition as revealed by total versus SIP-labeled DNA and rRNA. Soil was supplemented with H218O and after 38 days the DNA and RNA were co-extracted. Heavy nucleic acids were separated out by CsCl and CsTFA density centrifugation. The 16S rRNA gene pools were characterized by DGGE and pyrosequencing, and the sequence results analyzed using mothur. The majority of DNA (∽60%) and RNA (∽75%) from the microcosms incubated with H218O were labeled by the isotope. The analysis indicated that total and active members of the same type of nucleic acid represented similar community structures, which suggested that most dominant OTUs in the total nucleic acid extracts contained active members. It also supported that H218O was an effective universal label for SIP for both DNA and RNA. DNA and RNA-derived diversity was dissimilar. RNA from this soil more comprehensively recovered bacterial richness than DNA because the most abundant OTUs were less numerous in RNA than DNA-derived community data, and dominant OTU pools didn't mask rare OTUs as much in RNA.

Bacteria utilizing plant‐derived carbon in the rhizosphere of Triticum aestivum change in different depths of an arable soil

“…Crossfeeding in natural food‐webs can complicate the interpretation of SIP data and time‐resolved SIP analyses can help to overcome this caveat (Coyotzi et al ., ). In our present analyses, microbes with a higher 13 C enrichment were considered more likely to be primary consumers of root exudates, however, the simultaneous presence of slow‐growing primary consumers can also not be excluded (Haichar et al ., ; Rettedal and Brözel, ).…”

Degradation of fuel components

“…The analysis indicated that total and active members of the communities represented by the light and heavy DNA fractions, respectively, were very similar to each other. This similarity between labelled and unlabelled nucleic acids is in agreement with previous studies [213,214].…”

Cultivable bacterial diversity associated with bromeliad roots from ironstone outcrops in central Brazil

“…We observed the presence of Bacillus cereus and B. thuringiensis within the same cluster at the phylogenetic tree, consistent with reports by Ohba (2011) and Liu et al (2015) that B. thuringiensis is allocated into the Bacillus cereus group together with the two other species, Bacillus anthracis and Bacillus mycoides. Bacillus species are known to form aerobic spores and survive in arid regions, which comprise a range of soil depths, altitudes, and other climatic conditions (Rettedal and Brozel, 2015). The bromeliad species from which the strains were isolated occur in a dry environment exposed to high thermal amplitudes and dehydration, the ironstone outcrops, which could favor the presence of Bacillus species instead of other bacterial species.…”