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Twenty-nine OTUs changed in significantly in relative abundance with time
("BH” adjusted P-value $<$ 0.10 \citet{YBenjamini1995})). When SSU rRNA
gene abundances were combined at the taxonomic rank of "Class", only
Bacilli \textit{Bacilli} (decreased),
Flavobacteria \textit{Flavobacteria} (decreased),
Gammaproteobacteria \textit{Gammaproteobacteria} (decreased) and
Herpetosiphonales \textit{Herpetosiphonales}
(increased) significantly changed in relative abundance with time (P-value
$<$ 0.10, Figure~\ref{fig:time_class}). Of the 29 OTUs that changed in relative
abundance with time, 14 were found to have incorporated $^{13}$C into DNA
(Figure~\ref{fig:time} and below). Of the 14 OTUs that were found to have both
incorporated $^{13}$C into DNA and significantly changed in relative abundance
with time, OTUs that incorporated $^{13}$C from $^{13}$C-cellulose increased
with time whereas those that incorporated $^{13}$C from $^{13}$C-xylose
decreased over time and OTUs that responded to both substrates were found to
either have increased or decreased over time
(Figure~\ref{fig:time}~and~\ref{fig:babund}).
\subsection{OTUs that assimilated $^{13}$C into DNA} \label{responders}
% Fakesubsubsection:Within the first 7 days of incubation approximately 63\%
...
The phylogenetic types of putatively $^{13}$C labeled OTUs (i.e. responders)
changed with time (Figure~\ref{fig:l2fc}~and~\ref{fig:xyl_count}). On day 1,
Bacilli \textit{Bacilli} OTUs represented 84\% of xylose responders, and the majority
of these OTUs were closely related to cultivated representatives of the genus
\textit{Paenibacillus} (n $=$ XX, Table~\ref{tab:xyl}). For example, "OTU.57"
(Table\ref{tab:xyl}), annotated as \textit{Paenibacillus}, has a strong signal
of $^{13}$C incorporation from 13C-xylose into DNA at day 1, at its maximum
...
xylose responders (Figure~\ref{fig:xyl_count}) and were closely related to
cultivated representatives of the \textit{Flavobacteriales} and
\textit{Sphingobacteriales} (Table~\ref{tab:xyl}). For example, "OTU.14",
annotated as a Flavobacterium, has a strong signal for $^{13}$C labeling from
$^{13}$C-xylose at days 1 and 3 coinciding with its maximum relative abundance
in non-fractionated soil DNA. The relative abundance of "OTU.14" then declines
until day 14 and evidence of $^{13}$C labeling is not significant after day
3 (Figure X). Finally, on day 7, \textit{Actinobacteria} OTUs represented 53\%
of the xylose responders and these OTUs were closely related to cultivated
representatives of \textit{Micrococcales} (Table~\ref{tab:xyl}). For example,
"OTU.4", annotated as \textit{Agromyces}, has signal of $^{13}$C labeling on
days 1 through 7 with the strongest evidence $^{13}$C labeling at day 7, its
relative abundance in non-fractionated soil increases until day
3 and then declines gradually until day 30 and evidence of 13C labeling
declines after day 7 (Figure X). \textit{Proteobacteria} were also common
among xylose responders at day 7 where they comprised 40\% of xylose responder
...
(Table~\ref{tab:xyl}).
%Fakesubsubsection:Cellulose responders were
The phylogenetic types of cellulose responders did not change with time
to the
same extent as the unlike
phylogenetic types of xylose responders. Also, in
contrast to xylose responders, cellulose responders often belonged to
non-cultivated microbial clades. Both the relative abundance and the number of
cellulose responders increased over time peaking at days 14 and 30
(Figures~\ref{fig:l2fc}, \ref{fig:rspndr_count}, and \ref{fig:babund}). The
phylogenetic composition of cellulose responders changed little between days 14
and 30 (Table~\ref{tab:cell}). Cellulose responders belonged to the
...
OTUs), and \textit{Deltaproteobacteria} (6 OTUs).
The majority (85\%) of cellulose responders outside of the
\textit{Proteobacteria} shared $<$ 97\% SSU rRNA gene
sequence identity to
bacteria already cultivated in isolation. For example, most (70\%) of the
\textit{Verrucomicrobia} cellulose responders fell within a few unidentified
\textit{Spartobacteria} clades, and these shared $<$ 85\% SSU rRNA gene
sequence identity to any characterized isolate. The \textit{Spartobacteria} OTU
...
% Fakesubsubsection:Cellulose responders tended
Cellulose responders tended to have lower relative abundance in
non-fractionated soil DNA, demonstrated signal consistent with higher
$^{13}$C:12C $^{13}$C:$^{12}$C ratios in DNA upon $^{13}$C labeling, and lower estimated
\textit{rrn} copy number than xylose responders. In the non-fractionated soil
DNA, cellulose responders had significantly lower relative abundance (7e$^{-4}$
(s.d. 2e$^{-3}$)) than xylose responders (2e$^{-3}$ (s.d. 4e$^{-3}$))
(Figure~\ref{xyl_count}, (Figure~\ref{fig:xyl_count}, P-value $=$ 0.00028, Wilcoxon Rank Sum test). Six
of the ten most common OTUs observed in the non-fractionated soil DNA responded
to xylose, and, of the 10 most abundant $^{13}$C substrate responders in the
non-fractionated soil DNA 8 were xylose responders and 2 were cellulose
responders. However, $^{13}$C-xylose and $^{13}$C-cellulose responders included
OTUs at both high and low abundance (Figure~\ref{fig:shift}). Two
...
% Fakesubsubsection:Cellulose responders exhibited a greater shift in BD
DNA buoyant density increases as its ratio of $^{13}$C to $^{12}$C increases.
An organism that only assimilates C into DNA from
a $^{13}$C labeled
sources, source,
will have a greater DNA $^{13}$C:$^{12}$C than an organism utilizing a mixture
of $^{13}$C labeled and unlabeled C sources (see Supplemental~Note~1.8).
Therefore,
the specificity of C use can be evaluated by the change in DNA
buoyant density (BD) upon $^{13}$C labeling. In this
study, study we do not know the
absolute abundance of
OTUs OTU DNA across the density gradient as our SSU rRNA gene
sequence counts are compositional in nature hence we cannot assess absolute OTU
DNA buoyant density shifts due to $^{13}$C labeling. However, we can evaluate
relative C use specificity by quantifying and comparing the shift in the
relative abundance profile for an OTU along the density gradient in response to
$^{13}$C labeling. Specifically, in this study we calculated each OTU's
relative abundance density gradient profile
center of mass shift for
each every
label/control DNA density gradient pair (see supplemental methods for the
detailed calculation). We refer to this metric as $\Delta\hat{BD}$. This metric
indicates relative differences in DNA
13C:12C $^{13}$C:$^{12}$C and can be used to
compare DNA
13C:12C $^{13}$C:$^{13}$C between groups of responders. $\Delta\hat{BD}$
does not represent the true density shift for an OTU because it is based on
relative abundance and is therefore not directly comparable to literature
values for DNA density shifts due to isotopic labeling. Cellulose responder
$\Delta\hat{BD}$ (0.0163 g mL$^{-1}$ (s.d. 0.0094)) was significantly greater
than that of xylose responders (0.0097 g mL$^{-1}$ (s.d. 0.0094))
(Figure~\ref{fig:shift}, P-value $=$ 1.8610e$^{-6}$, Wilcoxon Rank Sum test).
% Fakesubsubsection:We predicted the rrn
We predicted the \textit{rrn} gene copy number for responders as described
by \citet{Kembel_2012}. The number of \textit{rrn} gene copies
a microorganism has is correlated to it's ability to increase growth
rapidly in response to nutrient influx \citep{Klappenbach_2000}.
Additionally, \textit{Bacillus~subtilis} mutants constructed with one to
ten \textit{rrn} gene copies had progressively shorter doubling times as
\textit{rrn} gene copy number increased \citep{yano_multiple_2013}
suggesting that in some microorganisms \textit{rrn} gene copy number is
proportional to growth rate. The estimated \textit{rrn} gene
copy number of cellulose responders (X) was significantly lower than that
of xylose responders (X) (Figures~\ref{fig:shift} and \ref{fig:copy};
P = 1.878e$^{-9}$). Furthermore, we observed that estimated \textit{rrn}
gene copy number for xylose responders was inversely related to the day of
first response (P = 2.02e$^{-15}$, Figure~\ref{fig:copy}).
% Fakesubsubsection:We assessed phylogenetic
We assessed phylogenetic clustering of $^{13}$C-responsive OTUs with the
Nearest Taxon Index (NTI) and the Net Relatedness Index (NRI)
\citep{Webb2000}. We also quantified the average clade depth of cellulose and
xylose responders with the consenTRAIT metric \citep{Martiny2013}. Briefly,
the
NRI and NTI evaluate phylogenetic clustering against a null model for the
distribution of a trait in a phylogeny. The NRI and NTI values are z-scores and
thus the greater the magnitude of the NRI/NTI, the stronger the evidence for
clustering (positive values) or overdispersion (negative values). NRI assesses
overall clustering whereas the NTI assesses terminal clustering. An NRI of
1.96, for instance, would signify overall phylogenetic clustering with
a corresponding P-value of 0.05 \citep{Evans2014a}. The consenTRAIT metric is
a measure of the average clade depth for a trait in a phylogenetic tree. NRI
values indicate that cellulose responders clustered phylogenetically (NRI:
4.49) while xylose responders are overdispersed (NRI: -1.33). NTI values show
that both cellulose and xylose responders are terminally clustered (NTI: 1.43
and 2.69, respectively). The consenTRAIT clade depth for xylose and cellulose
responders was 0.012 and 0.028 SSU rRNA gene sequence dissimilarity,
respectively. As reference, the average clade depth is 0.017 SSU rRNA gene
sequence dissimilarity for arabinose utilization (another five C sugar found in
hemicellulose) and was 0.013 and 0.034 SSU rRNA gene sequence dissimilarity for
glucosidase and cellulase activity of isolates in culture, respectively
\citep{Martiny2013,Berlemont2013}.
diff --git a/bibliography/biblio.bib b/bibliography/biblio.bib
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...
@article{yano_multiple_2013,
eprinttype = {pmid},
eprint = {23970567},
title = {Multiple {rRNA} operons are essential for efficient cell growth and sporulation as well as outgrowth in Bacillus subtilis},
issn = {1350-0872, 1465-2080},
url = {http://mic.sgmjournals.org/content/early/2013/08/22/mic.0.067025-0},
doi = {10.1099/mic.0.067025-0},
timestamp = {2015-06-18 10:44:09},
journal = {Microbiology},
shortjournal = {Microbiology},
author = {Yano, Koichi and Wada, Tetsuya and Suzuki, Shota and Tagami, Kazumi and Matsumoto, Takashi and Shiwa, Yuh and Ishige, Taichiro and Kawaguchi, Yasuhiro and Masuda, Kenta and Akanuma, Genki and Nanamiya, Hideaki and Niki, Hironori and Yoshikawa, Hirofumi and Kawamura, Fujio},
urldate = {2015-06-18},
date = {2013-08-22},
year = {2013},
pages = {mic.0.067025--0},
langid = {english},
keywords = {\ensuremath{<}it\ensuremath{>}Bacillus subtilis\ensuremath{<}/it\ensuremath{>},\ensuremath{<}it\ensuremath{>}rrn\ensuremath{<}/it\ensuremath{>} operon,rRNA},
file = {Snapshot:/home/chuck/.zotero/zotero/is3tm1mp.default/zotero/storage/U566GS5J/mic.0.html:text/html}
}
@article{Aoyagi2015,
title = {Ultra-high-sensitivity stable-isotope probing of {rRNA} by high-throughput sequencing of isopycnic centrifugation gradients},
volume = {7},
