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\section{Results}
% Fakesubsubsection:We tracked the flow of C from xylose
After adding
a nutrient and resource mixture containing
both cellulose and xylose the organic matter amendment to soil, we tracked the flow of
C from xylose or C from cellulose into microbial DNA over time using DNA-SIP
(Figure~\ref{fig:setup}).
We added 3 milligrams of the nutrient and resource
mixture (as C) per gram dry weight soil to experimental microcosms. This The amendment
comprised 25\% consisted of
total soil C and contained
fresh various plant biomass
components compounds including
sugars, cellulose, lignin,
and sugars found in hemicellulose, amino
acids. Xylose-C acids, and
cellulose-C made up 0.42 milligrams and 0.88 milligrams per
gram inorganic salts (see Supplemental Methods). The amendment was added
at 2.3 mg C g$^{-1}$ soil dry
soil, respectively, and these additions represented 3.5\% weight (d.w.), and
7.3\% this comprised 16\% of
the
total
C in the soil. The cellulose-C (0.88 mg C g$^{-1}$ soil
C, d.w.) and
xylose-C (0.42 mg C g$^{-1}$ soil d.w.) in the amendment comprised 6\% and 3\%
of the total C in the soil, respectively. The soil microbial community respired
65\% of the xylose within one day
(Figure~\ref{fig:13C}) (Figure S1) and 29\% of the added xylose
remained in the soil at day 30
(Figure~\ref{fig:13C}). (Figure S1). In contrast, cellulose-C declined
at a constant rate of approximately 18 $\mu$g C
g$^{-1}$ dry d $^{-1}$ g $^{-1}$ soil
d$^{-1}$ d.w.
and 40\% of added cellulose-C remained in the soil at day 30
(Figure~\ref{fig:13C}). (Figure S1).
\subsection{Soil microcosm microbial community changes with time}
% Fakesubsubsection:Changes in the soil microcosm microbial community structure
We assessed assimilation of $^{13}$C into microbial DNA by comparing the SSU
rRNA gene sequence composition of SIP density gradient fractions from
control treatments or
treatments
amended with $^{13}$C-xylose or
$^{13}$C-cellulose. We set up three types of microcosms. including a $^{13}$C label relative to the control treatment. All
microcosms
received treatments used the same
nutrient amendment which included xylose and
resource mixture, but, in two microcosm types
a $^{13}$C-labeled substrate (i.e. cellulose, but
$^{13}$C-xylose or
$^{13}$C-cellulose) $^{13}$C-cellulose was substituted for its unlabeled
equivalent. The equivalent in two amendments. A treatment without isotopically labeled
components served as the ``control''.
The In the control gradient density fractions
the majority of the variance in SSU rRNA gene composition
of density gradient fractions from the control treatment was represented by
fraction density (Figure~\ref{fig:ord}). DNA buoyant density correlates with
G$+$C content \citep{Buckley_2007} and therefore DNA G$+$C content strongly influences
variation in the SSU rRNA gene composition of density gradient fractions. For
the $^{13}$C-cellulose treatment, SSU rRNA gene composition in gradient
fractions deviated from control at high density
($>$
1.72 ($>$~1.72 g mL$^{-1}$) on days
14 and 30 (Figure~\ref{fig:ord}). For the $^{13}$C-xylose treatment, SSU rRNA
gene composition in density gradient fractions also deviated from control in
high density fractions, but in contrast to the $^{13}$C-cellulose treatment it
deviated from control on
days 1, 3, and
7 days~1,~3,~and~7 (Figure~\ref{fig:ord}). SSU rRNA gene
composition from $^{13}$C-cellulose treatment and $^{13}$C-xylose treatment
density fractions
were different differed at high density indicating different microorganisms
assimilated C from xylose
into DNA than
those C from cellulose
(Figure~\ref{fig:ord}). Further, the SSU rRNA gene sequence composition of high
density fractions from $^{13}$C-cellulose treatments at days 14 and 30 was
similar indicating similar microorganisms had $^{13}$C labeled DNA
from in
$^{13}$C-cellulose
treatments at days 14 and 30.
In contrast, Contrastingly, in the
$^{13}$C-xylose treatment the SSU rRNA gene composition
of the
$^{13}$C-xylose treatment high density gradient
fractions varied between
days
1, 3, and 7 days~1,~3,~and~7 indicating that different microbes
had $^{13}$C labeled DNA on these days. In the $^{13}$C-xylose treatment, the
SSU gene composition of high density fractions was similar to control on
days 14 and 30 days~14~and~30 (Figure~\ref{fig:ord}) indicating that
DNA $^{13}$C was no longer
contained $^{13}$C label
from $^{13}$C-xylose beyond day 14. detectable on these days for this treatment.
\subsection{Temporal dynamics DNA $^{13}$C incorporation of OTUs}
% Fakesubsubsection:We monitored the soil microbial community
...
Twenty-nine OTUs exhibited sufficient statistical evidence (adjusted P-value
$<$ 0.10) to conclude they changed in relative abundance over the
course of the experiment. When SSU rRNA gene abundances were combined at
the taxonomic rank of "class", the classes that changed in abundance
with
statistically significant evidence (P-value
$<$ 0.10) were the \textit{Bacilli} (decreased), \textit{Flavobacteria}
(decreased), \textit{Gammaproteobacteria} (decreased), and
\textit{Herpetosiphonales} (increased) (Figure~\ref{fig:time_class}). Of the 29
OTUs that
significantly changed in relative abundance over time, 14 putatively
incorporated $^{13}$C into DNA (Figure~\ref{fig:time}). OTUs that likely
assimilated $^{13}$C from $^{13}$C-cellulose into DNA tended to increase in
relative abundance with time whereas OTUs that assimilated $^{13}$C from
...
\subsection{OTUs that assimilated $^{13}$C into DNA} \label{responders}
% Fakesubsubsection:If an OTU exhibited
If an OTU exhibited strong evidence for
assimilating $^{13}$C
incorporation
into DNA, we refer to that OTU as a "responder" (see Supplemental Note 1.7.4
for our operational definition of "responder"). The SSU rRNA gene sequences
produced in this study could be distributed into 5,940 OTUs and we assessed the
evidence of $^{13}$C incorporation into DNA from $^{13}$C-cellulose and
$^{13}$C-xylose for each OTU. Forty-one OTUs responded to $^{13}$C-xylose,
55 OTUs responded to $^{13}$C-cellulose, and 8 OTUs
responded to both xylose and cellulose (Figure~\ref{fig:l2fc},
...
closely related to cultured representatives of the genus \textit{Paenibacillus}
(n $=$ XX, Table~\ref{tab:xyl}). For example, "OTU.57" (Table\ref{tab:xyl}),
annotated as \textit{Paenibacillus}, had a strong signal of $^{13}$C
incorporation assimilation from $^{13}$C-xylose into DNA at day 1 coinciding with its maximum
relative abundance in non-fractionated DNA. The relative abundance of "OTU.57"
declined until day 14 and did not appear to be $^{13}$C labeled after day
1 (Figure X). On day 3, \textit{Bacteroidetes} OTUs comprised 63\% of xylose
responders (Figure~\ref{fig:xyl_count}) and these OTUs were closely related to
cultured representatives of the \textit{Flavobacteriales} and
\textit{Sphingobacteriales} (Table~\ref{tab:xyl}). For example, ``OTU.14'',
annotated as a Flavobacterium, had a strong signal for $^{13}$C labeling
from in the
$^{13}$C-xylose
treatment at days 1 and 3 coinciding with its maximum relative
abundance in non-fractionated DNA. The relative abundance of ``OTU.14'' then
declined until day 14 and did not show evidence of $^{13}$C labeling beyond
day
3 day~3 (Figure X). Finally, on
day 7, day~7, \textit{Actinobacteria} OTUs represented
53\% of the xylose responders and these OTUs were closely related to cultured
representatives of \textit{Micrococcales} (Table~\ref{tab:xyl}). For example,
``OTU.4'', annotated as \textit{Agromyces}, had signal of $^{13}$C labeling
in
the $^{13}$C-xylose treatment on days 1, 3 and 7 with the strongest evidence of
$^{13}$C labeling at
day
7 day~7 and did not appear $^{13}$C labeled at
days 14 and 30. days~14
and~30. ``OTU.4'' relative abundance in non-fractionated DNA increased until
day 3 day~3 and then declined gradually until
day 30 day~30 (Figure X).
\textit{Proteobacteria} were also common among xylose responders at
day 7 day~7 where
they comprised 40\% of xylose responder OTUs. Notably, \textit{Proteobacteria}
represented the majority (6 of 8) of OTUs that responded to both cellulose and
xylose.
%Fakesubsubsection:Cellulose responders were
The phylogenetic composition of cellulose responders did not change with time
unlike the phylogenetic composition
of xylose responders. Also, in contrast to
xylose responders, cellulose responders often were not closely related ($<$
95\% 97\% SSU rRNA gene sequence identity) to cultured isolates. 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}). Cellulose responders belonged to the \textit{Proteobacteria}
(46\%), \textit{Verrucomicrobia} (16\%), \textit{Planctomycetes} (16\%),
\textit{Chloroflexi} (8\%), \textit{Bacteroidetes} (8\%),
\textit{Actinobacteria} (3\%), and \textit{Melainabacteria} (1 OTU)
(Table~\ref{tab:cell}). The majority (86\%) of cellulose responders in the
\textit{Proteobacteria} were closely related ($>$ 97\% identity) to bacteria
already cultured in isolation, including representatives of the genera:
\textit{Cellvibrio}, \textit{Devosia}, \textit{Rhizobium}, and
\textit{Sorangium}, which are all known for their ability to degrade cellulose
(Table~\ref{tab:cell}). Proteobacterial cellulose responders belonged to
\textit{Alpha-}
(13 OTUs), (13~OTUs), \textit{Beta-}
(4 OTUs), (4~OTUs), \textit{Gamma-}
(5 OTUs), (5~OTUs),
and \textit{Deltaproteobacteria}
(6 OTUs). (6~OTUs).
% Fakesubsubsection:The majority (85\%) of cellulose
The majority (85\%) of cellulose responders outside of the
...
sequence identity to any characterized isolate. The \textit{Spartobacteria} OTU
``OTU.2192'' exemplified many cellulose responders (Figure X). ``OTU.2192''
gradually increased in non-fractionated DNA relative abundance with time and
evidence for $^{13}$C labeling of ``OTU.2192''
from in the $^{13}$C-cellulose
treatment increased gradually over time with the strongest evidence at
days 14 and
30 days~14
and~30 (Figure X). \textit{Choloflexi} cellulose responders predominantly
belonged to an unidentified clade within the \textit{Herpetosiphonales} and
these they shared $<$ 89\% SSU rRNA gene sequence identity to any characterized
isolate. Characteristic of
other \textit{Chloroflexi} cellulose responders,
"OTU.64" increased in relative abundance over 30 days and evidence for $^{13}$C
labeling of ``OTU.64''
in the $^{13}$C-cellulose treatment peaked days 14 and
30 (Figure X). Cellulose responders found within the \textit{Bacteroidetes}
fell within the \textit{Cytophagales} contrasting with \textit{Bacteroidetes}
xylose responders that fell instead within the \textit{Flavobacteria} or
\textit{Sphingobacteriales}. \textit{Bacteroidetes} cellulose responders
included one OTU that shared 100\% SSU rRNA gene sequence identity to species
of \textit{Sporocytophaga}, a genus that includes known cellulose degraders.
\subsection{Characteristics of cellulose and xylose responders}
% Fakesubsubsection:Cellulose responders tended
Cellulose responders, relative to xylose responders, tended to have lower
relative abundance in non-fractionated DNA, demonstrated signal consistent with
higher
atom \% $^{13}$C
incorporation per unit DNA upon $^{13}$C labeling, in labeled DNA, and have lower estimated \textit{rrn} copy number. In the
non-fractionated DNA, cellulose responders had lower relative abundance
(7e$^{-4}$ (s.d. 2e$^{-3}$)) than xylose responders (2e$^{-3}$ (s.d.
4e$^{-3}$)) (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 DNA
responded to xylose, and, eight of the ten most abundant responders to xylose
or cellulose in the non-fractionated DNA were xylose responders. However,
xylose and cellulose responders included OTUs at both high and low abundance
(Figure~\ref{fig:shift}).
% Fakesubsubsection:DNA buoyant density increases as the amount
DNA buoyant density
(BD) increases
as in proportion to the atom \% $^{13}$C of the
DNA.
Hence, the
amount extent of $^{13}$C
per unit incorporation into DNA
increases. can be evaluated as the
change in BD in enriched treatments relative to control. We calculated for each
OTU its mean BD weighted by relative abundance to determine its “center of
mass” within a given density gradient. We then quantified for each OTU the
difference in center of mass between control gradients and gradients from
13C-xylose or 13C-cellulose treatments (see supplemental methods for the
detailed calculation).
Therefore, the amount of $^{13}$C per unit DNA can be evaluated by the change
in DNA buoyant density (BD) upon $^{13}$C labeling. In this study, we found the
density weighted average for each OTU in each gradient (i.e. mean density
...
of an OTU's relative abundance profile across a density gradient. We then
quantified the difference in each OTU's center of mass between control
gradients and corresponding $^{13}$C labeled gradients (see supplemental
methods for the detailed calculation).
We refer to the change in center of mass position for an OTU in response to
$^{13}$C labeling as $\Delta\hat{BD}$. $\Delta\hat{BD}$ can
indicate be used to compare
relative differences in $^{13}$C
incorporation per unit
DNA labeling between
OTUs although it does OTUs. $\Delta\hat{BD}$
values, however, are not
represent directly comparable to the
true density shift BD changes observed for
an
OTU DNA
from pure cultures which generate uniformly isotopically labeled molecules, in
part because
it $\Delta\hat{BD}$ is based on relative abundance
in density
gradient fractions (and not DNA concentration) and in part because all members
of an OTU may not
responding identically uniformly respond to the isotopic label.
$\Delta\hat{BD}$ 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)) (s.d.~0.0094)) was greater than that of
xylose responders (0.0097 g mL$^{-1}$
(s.d. 0.0094)) (s.d.~0.0094)) (Figure~\ref{fig:shift},
P-value $=$ 1.8610e$^{-6}$, Wilcoxon Rank Sum test).
% Fakesubsubsection:We predicted the rrn
...
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 although statistical support for xylose responder
overdispersion is not strong (NRI: -1.33). NTI values show that both cellulose overall and
xylose responders clustered at the tips of the phylogeny
(NTI: 1.43 and
2.69, respectively). (NRI:~4.49,
NTI:~1.43) while xylose responders cluster terminally (NRI:~-1.33, NTI:~2.69).
The consenTRAIT clade depth for xylose and cellulose responders
was 0.012 and 0.028 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 was~0.013
and~0.034 SSU rRNA gene sequence dissimilarity for glucosidase and cellulase
activity of isolates in culture, respectively
\citep{Martiny2013,Berlemont2013}. These results indicate xylose responders
form terminal clusters dispersed throughout the phylogeny while cellulose
responders form deep clades of terminally clustered OTUs.
