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. H2
18O 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 H2
18O 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 H2
18O 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 H2
18O 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 DNAderived
community data, and dominant OTU pools didn’t mask rare OTUs as
much in RNA.
