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RESEARCH ARTICLE
Transcriptional Regulation and Adaptation to
a High-Fiber Environment in Bacillus subtilis
HH2 Isolated from Feces of the Giant Panda
Ziyao Zhou
1‡
, Xiaoxiao Zhou
2‡
, Jin Li
1‡
, Zhijun Zhong
1‡
, Wei Li
1
, Xuehan Liu
1
, Furui Liu
1
,
Huaiyi Su
1
, Yongjiu Luo
1
, Wuyang Gu
1
, Chengdong Wang
3
, Hemin Zhang
3
, Desheng Li
3
,
Tingmei He
3
, Hualin Fu
1
, Suizhong Cao
1
, Jinjiang Shi
1
, Guangneng Peng
1
*
1The Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary
Medicine, Sichuan Agricultural University, Ya'an, 625014, PR China, 2Chengdu Center for Animal Disease
Prevention and Control, Chengdu, 610041, PR China, 3Ya'an Bifengxia Base, China Conservation and
Research Center for the Giant Panda, Ya'an, 625007, PR China
‡These authors contributed equally to this work.
*pgn.sicau@163.com
Abstract
In the giant panda, adaptation to a high-fiber environment is a first step for the adequate
functioning of intestinal bacteria, as the high cellulose content of the gut due to the panda's
vegetarian appetite results in a harsh environment. As an excellent producer of several en-
zymes and vitamins, Bacillus subtilis imparts various advantages to animals. In our previous
study, we determined that several strains of B. subtilis isolated from pandas exhibited good
cellulose decomposition ability, and we hypothesized that this bacterial species can survive
in and adapt well to a high-fiber environment. To evaluate this hypothesis, we employed
RNA-Seq technology to analyze the differentially expressed genes of the selected strain B.
subtilis HH2, which demonstrates significant cellulose hydrolysis of different carbon
sources (cellulose and glucose). In addition, we used bioinformatics software and resources
to analyze the functions and pathways of differentially expressed genes. Interestingly, com-
parison of the cellulose and glucose groups revealed that the up-regulated genes were in-
volved in amino acid and lipid metabolism or transmembrane transport, both of which are
involved in cellulose utilization. Conversely, the down-regulated genes were involved in
non-essential functions for bacterial life, such as toxin and bacteriocin secretion, possibly to
conserve energy for environmental adaptation. The results indicate that B. subtilis HH2 trig-
gered a series of adaptive mechanisms at the transcriptional level, which suggests that this
bacterium could act as a probiotic for pandas fed a high-fiber diet, despite the fact that cellu-
lose is not a very suitable carbon source for this bacterial species. In this study, we present
a model to understand the dynamic organization of and interactions between various func-
tional and regulatory networks for unicellular organisms in a high-fiber environment.
PLOS ONE | DOI:10.1371/journal.pone.0116935 February 6, 2015 1/13
OPEN ACCESS
Citation: Zhou Z, Zhou X, Li J, Zhong Z, Li W, Liu X,
et al. (2015) Transcriptional Regulation and
Adaptation to a High-Fiber Environment in Bacillus
subtilis HH2 Isolated from Feces of the Giant Panda.
PLoS ONE 10(2): e0116935. doi:10.1371/journal.
pone.0116935
Academic Editor: Yung-Fu Chang, Cornell
University, UNITED STATES
Received: November 1, 2014
Accepted: December 16, 2014
Published: February 6, 2015
Copyright: © 2015 Zhou et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by the National
Natural Science Foundation of China (number
31272620), Sichuan Provincial Department of
Science and Technology Support Program (number
2011NZ0060) and the Program for Changjiang
Scholars and Innovative Research Team in University
(number IRT0848). The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Introduction
The intestinal microbiota, of which a major component is bacteria, greatly contributes to host
nutrition, metabolism, immunity and other characteristics [1–3]. However, the gut environ-
ment is not an ideal location for intestinal bacteria because most growth and reproduction
genes are inhibited [4]. For the giant panda (Ailuropoda melanoleuca), one of the most highly
endangered mammals, with only 2,500 to 3,000 individuals survived in western China [5], a
high-fiber vegetarian diet combined with a carnivore-like gastrointestinal system results in a
harsh gut environment, as soft bamboo shoots and stems are the major food sources of the
panda but this animal does not possess a rumen for fermentation [6,7]. Thus, adaptation to a
high-fiber environment is the first step for the intestinal microbiota of the giant panda to act as
a probiotic. Although several recent studies have established a framework for the community
composition and functions of the panda intestinal microbiota via metagenomics [8–10], it is
not yet well understood how a single organism can survive and adapt in the high-fiber gut
environment of the giant panda.
As a vital bacterial species of the mammalian intestinal microbiome, Bacillus subtilis imparts
various advantages to animals due to its excellent ability to produce several enzymes and vita-
mins [11,12]. In our previous study [13,14], we determined that several B.subtilis strains isolat-
ed from pandas demonstrated good cellulose decomposition capability as well as other
contributions; thus, we speculated that this bacterial species could help the panda in several
ways, including aiding in the digestion of bamboo in a high-fiber intestinal environment.
Previously, glucose had been recognized as the preferred carbon source for microbes [15],
while many bacterial species had been shown to not be able to use cellulose. Our previous
results proved that B.subtilis had ability to survive in a high-fiber environment, but the mecha-
nism is still unknown.
The literature has shown that the adaptation of B.subtilis in different environments occurs
mainly through transcriptional regulation [16]. The levels of most bacterial stress adaptation
molecules, such as the Hfq protein [17], small RNAs [18] and 16S rRNA [19], are determined
at the transcript level. Therefore, we employed RNA-Seq technology to compare the differen-
tially expressed genes (DEGs) of the selected B.subtilis HH2 strain when using cellulose and
glucose as primary carbon sources, and we then analyzed the functions and pathways of the
DEGs. In this study, we revealed major transcriptional reconfigurations in response to cellulose
adaptation as well as certain coordinated changes in the abundance of B.subtilis HH2. We
then produced a model to understand the dynamic organization of the interactions between
various functional and regulatory networks of unicellular organisms in the giant
panda intestine.
Materials and Methods
Bacterial strain and cultivation conditions
Glucose medium was modified from a previous study [20]: 70 mmol K
2
HPO
4
, 30 mmol
KH
2
PO
4
, 25 mmol (NH
4
)
2
SO
4
, 0.5 mmol MgSO
4
,10μmol MnSO
4
, 22 mg ferric ammonium
citrate, 8 g potassium glutamate, 6 g potassium succinate, 1% glucose, 0.5 mmol CaCl
2
,5μmol
MnCl
2
, and 1000 mL of ddH
2
O at pH = 7.2. The cellulose medium was formulated in the same
way as the glucose medium, except that the main carbon source was 1% sodium carboxymeth-
ylcellulose instead of 1% glucose.
B.subtilis HH2 from fresh feces that was collected from healthy pandas at the Ya'an
Bifengxia Base of the China Conservation and Research Center for the Giant Panda (CCRCGP)
and placed in sterile sampling bags in our previous study was isolated and identified. This
Adaptation to a High-Fiber Environment in Bacillus subtilis HH2
PLOS ONE | DOI:10.1371/journal.pone.0116935 February 6, 2015 2/13
Competing Interests: The authors have declared
that no competing interests exist.
strain has a good ability to digest cellulose; the diameter of its cellulose hydrolysis halo was
28.00±0.44 mm (S1 Fig.). The strain was grown in 100 mL of LB medium at 37°C in a shaker at
150 rpm for 24 h. After cultivation, 1% of the cells was inoculated into glucose and cellulose
media and grown at 37°C in a shaker at 150 rpm until OD
600
~1.
RNA isolation and preparation
Total RNA was extracted using the hot phenol method [21]. In brief, cell pellets were resus-
pended and washed once in Buffer A (50 mM sodium acetate and 10 mM EDTA, pH = 5.2).
After collecting the cells by centrifugation, the pellets were resuspended in Buffer A containing
1% SDS and immediately added to hot phenol. After incubation at 65°C for 5 minutes followed
by centrifugation for 10 minutes at 4°C, the RNA-containing supernatants were transferred to
a new tube for ethanol precipitation, washed and then dissolved in DEPC-treated water. The
RNA was further purified with two phenol-chloroform treatments and then treated with RQ1
DNase (Promega) to remove DNA. The quality and quantity of the purified RNA were deter-
mined by measuring the absorbance at 260 nm/280 nm (A260/A280) using Smartspec Plus
(BioRad). The integrity of the RNA was further verified by 1.5% agarose gel electrophoresis.
cDNA library construction and sequencing
Ribosomal RNAs were removed from the RNA samples (10 μg) using a RiboMinus rRNA
depletion kit (Ambion), and the resulting samples were used to prepare directional RNA-Seq
libraries [22,23]. The purified mRNAs were then iron-fragmented at 95°C followed by end re-
pair and 5' adaptor ligation. Then, reverse transcription was performed using RT primers con-
taining a 3' adaptor sequence and a randomized hexamer. The cDNAs were purified and
amplified, and all 200-500-bp PCR products were purified, quantified and stored at -80°C until
they were used for sequencing.
For high-throughput sequencing, the libraries were prepared following the manufacturer's
instructions, and the Illumina GAIIx system was used to collect data from 80-nt single-end
sequencing (ABlife Inc.; Wuhan, China).
Alignment of reads to the genome
After obtaining the sequencing data, the raw data were screened, which included removal of
two-N-containing reads, removal of the sequence adaptor, and identification of clean reads
with lengths of more than 16 nt after removal of low-quality values. Because the 16S rRNA
gene of B.subtilis HH2 has maximal homology with Bacillus_subtilis_PY79 (CP006881.1)
when compared with all B.subtilis genomes in the NCBI database (before November 11, 2013),
we decided to use Bacillus_subtilis_PY79 as the reference genome for B.subtilis HH2. We uti-
lized Tophat [24] with 2-nt mismatches to align our sequencing data to the reference genome
of B.subtilis HH2 (ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/Bacillus_subtilis_PY79_
uid229877/). Using the RPKMs (reads per kilobase of a gene per million reads) [25], we
eliminated the deviations due to the lengths of different genes.
Analysis of differentially expressed genes
To perform differential gene expression analysis, we applied the software edgeR [26], which is
specifically used to analyze the differential expression of genes using RNA-Seq data. To deter-
mine whether a gene was differentially expressed, the analysis results were based on the fold
change (FC2orFC-2) and P-value (P0.01). To predict gene function and calculate the
Adaptation to a High-Fiber Environment in Bacillus subtilis HH2
PLOS ONE | DOI:10.1371/journal.pone.0116935 February 6, 2015 3/13
functional category distribution frequency, KEGG and Gene ontology (GO) analyses were em-
ployed using DAVID bioinformatics resources [27].
Results and Analysis
Cellulose is not a highly suitable carbon source for B.subtilis HH2
B.subtilis HH2 was first cultured in media supplemented with cellulose or glucose as the primary
carbon source. We found that the growth of HH2 was significantly inhibited in the cellulose me-
dium compared with the glucose medium (Fig. 1). In addition, when the bacterial culture reached
OD
600
~1, we observed both the cellulose- and glucose-grown bacterial samples under a light mi-
croscope. Bacterial spores were more apparent in the cellulose medium than in the glucose medi-
um (Fig. 2), which indicated that cellulose is not a suitable energy source for this bacterial strain.
Profile of the RNA-Seq data
After cultivation, we extracted total RNA from the bacteria to analyze the transcriptional
regulation in the different carbon environments via next generation sequencing (NGS). We ob-
tained 20,231,277 and 24,807,479 fragments from the cellulose and glucose groups, respective-
ly. After screening, 14,733,930 and 20,066,967 clean reads were generated from the two groups,
respectively. We then analyzed whether these reads matched the reported reference genome in
the GenBank database using blastn (E-value1e-5). We found that more than 75% of the reads
could be mapped to the reference genome, of which approximately 50% were uniquely mapped
Fig 1. The growth curves of B.subtilis HH2 exposed to different carbon sources. B.subtilis HH2 was cultured in cellulose or glucose medium following
1% inoculation at 37°C in a shaker at 150 rpm; the OD
600
was measured every hour. Each graph represents the mean of three independent biological
replicates grown on three different days. The error bars represent the standard deviations (SDs) of the optical densityat each time point.
doi:10.1371/journal.pone.0116935.g001
Adaptation to a High-Fiber Environment in Bacillus subtilis HH2
PLOS ONE | DOI:10.1371/journal.pone.0116935 February 6, 2015 4/13
reads, representing a combined sequence coverage of 250X (Table 1). In this study, the detected
expressed genes (mapped reads number10) comprised 81.67% (3,279/4,278) of the reads for
the cellulose group and 92.69% (3,779/4,278) of the reads for the glucose group. The total num-
ber of detected genes in the two samples was 4,134, which accounted for 96.63% of the B.subti-
lis genes, indicating that the gene detection in this study largely reached saturation.
The correlation coefficient (R
2
) for the expression of the same gene in the two samples was
0.739, which demonstrated that parts of genes exhibited changes in expression levels. When
comparing the cellulose group with the glucose group, the number of significantly down-regu-
lated genes (by more than 10 times) was 164, which was significantly greater than the number
of up-regulated genes (23 genes), illustrating that these organisms may significantly reduce the
expression of some genes and lose partial function as a way to cope with the impacts of an ex-
treme environment [28]. According to GO functional analysis, 23 gene-function clusters were
enriched among the up-regulated genes when comparing the cellulose group to the glucose
group, and 22 clusters among the down-regulated genes were enriched (S1 Table). Interesting-
ly, among the clusters with the top 10 enrichment scores, the functions of the up-regulated
genes (clusters) were mainly associated with cellulose utilization, whereas most of the down-
regulated genes were associated with non-essential functions for bacterial life, indicating a re-
duction in energy consumption to permit environmental adaptation (Table 2).
Carbon metabolism gene expression is significantly altered in both
groups
Generally, carbon metabolism-related gene cluster expression is altered in bacteria in conjunc-
tion with changes in nutritional factors. We found that the most significantly up-regulated
Fig 2. Bacteria under the light microscope. (A) B.subtilis HH2 was cultured in glucose medium until OD
600
~1. (B) B.subtilis HH2 was cultured in cellulose
medium until OD
600
~1.
doi:10.1371/journal.pone.0116935.g002
Table 1. Mapping of clean reads in the B.subtilis genome.
Sample Input reads Total mapped Unique mapped Multiple mapped
Cellulose 14,733,930 11,293,808 (76.65%) 5,314,734 (47.06%) 5,979,074 (52.94%)
Glucose 20,066,967 16,316,254 (81.31%) 10,742,645 (65.84%) 5,573,609 (34.16%)
doi:10.1371/journal.pone.0116935.t001
Adaptation to a High-Fiber Environment in Bacillus subtilis HH2
PLOS ONE | DOI:10.1371/journal.pone.0116935 February 6, 2015 5/13
clusters in the cellulose group were involved in the metabolism of amino acids, lipids, galactose,
and ketone bodies, which indicates that when a directly used carbon source (usually beta-D-
glucose) in the environment is limiting, an organism may consume other intracellular sub-
stances for supplementation. Cellulase component genes, such as the beta-glucanase gene
U712_19750, were significantly highly expressed in the cellulose group compared to the glu-
cose group. Similar observations were noted for U712_19485 and U712_19495, genes that en-
code two subunits of the lichenin-specific phosphotransferase enzyme, which has been
confirmed to be involved in the digestion of cellobiose, a secondary product of cellulose cataly-
sis [29]. To further utilize cellobiose, various forms of glucosidase (such as U712_19480 and
U712_03600) are also up-regulated to create the final product beta-D-glucose.
In contrast, the expression of glucose metabolism-related genes was significantly decreased
in the cellulose group compared to the glucose group, particularly at the start of glucose metab-
olism. In addition, the expression of the UDP-glucose 6-dehydrogenase tuaD (U712_17840), a
necessary enzyme in the pentose phosphate pathway [30], was decreased by more than 16-fold.
Furthermore, at the beginning of glucose metabolism, the expression of gluconate kinase
(U712_20270) was decreased 9-fold in the cellulose group compared to the glucose group; and
at the same time, the downstream production of glucose 6-phosphate, which is the substrate
for the pentose phosphate pathway, was decreased [31]. S2 Table shows the differential expres-
sion of various genes that are important for carbon metabolism in the two samples.
The expression of most non-essential genes is decreased in the
cellulose group to reduce energy consumption
Apart from glucose metabolism genes, the down-regulated genes in the cellulose group were
involved in chemotaxis, secretion of toxins and bacteriocins, motility, polymer compound
Table 2. GO term analysis of DEGs (top 10 enrichment scores).
Differentially expressed gene cluster Description Enrichment Score
Down-regulated Cluster 1 Toxin, peptide, antibiotic and bacteriocin metabolic processes 3.96
Down-regulated Cluster 2 Chemotaxis, taxis and locomotor behavior 2.59
Down-regulated Cluster 3 Flagellar assembly and motility 2.29
Down-regulated Cluster 4 Membrane and transmembrane 2.00
Down-regulated Cluster 5 Flagellar assembly and bacterial flagellum protein export 1.98
Down-regulated Cluster 6 Cellular macromolecular complex assembly 1.28
Down-regulated Cluster 7 Anion transport 1.14
Down-regulated Cluster 8 ABC transporters 0.79
Down-regulated Cluster 9 Cell wall biogenesis/degradation 0.78
Down-regulated Cluster 10 Metal-binding 0.68
Up-regulated Cluster 1 Amino acid metabolism 1.44
Up-regulated Cluster 2 Transmembrane 1.23
Up-regulated Cluster 3 Carbohydrate transport 1.23
Up-regulated Cluster 4 Oxidoreductase and electron carrier activity 1.02
Up-regulated Cluster 5 Cell wall macromolecule catabolic processes 0.74
Up-regulated Cluster 6 Protein transport and localization 0.72
Up-regulated Cluster 7 Calcium ion binding and substrate binding 0.56
Up-regulated Cluster 8 Aminoglycan and polysaccharide catabolic processes 0.55
Up-regulated Cluster 9 Amino acid transmembrane transporter activity 0.52
Up-regulated Cluster 10 Sporulation 0.50
doi:10.1371/journal.pone.0116935.t002
Adaptation to a High-Fiber Environment in Bacillus subtilis HH2
PLOS ONE | DOI:10.1371/journal.pone.0116935 February 6, 2015 6/13
assembly, and complex protein assembly. The expression of the subtilosin-A assembly gene
U712_18825 was sharply decreased by ~187-fold. In addition to the decreased expression of
genes involved in the assembly of complex substances, the down-regulation of certain non-es-
sential genes reduced the energy consumption of the cell. For example, multiple vital genes in
the flagellar assembly gene cluster (Fig. 3) were significantly decreased in the cellulose group.
In addition, the expression of nearly every structural protein gene within the type III secretion
system (T3SS) of the flagellar assembly gene cluster was down-regulated. Furthermore, the ex-
pression of a series of flagellar genes was decreased, indicating that motility and infection abili-
ty were decreased during nutritional deficiency. Given the inhibition of various non-essential
metabolic genes as well as of genes involved in non-essential functions in the cellulose group, it
is apparent that B.subtilis HH2 triggers a series of mechanisms that conserve energy to be used
for adaptation to a high-fiber environment.
Two-component systems (TCSs) and ATP-binding cassette (ABC)
transporters are important regulatory systems for high-fiber environment
adaption
When bacteria experience stress, they signal to the organism that an appropriate physiological
response is required. TCSs and ABC transporters have been proposed to be units that partici-
pate in the common physiological process of signal transduction and substance transport
[32,33]. In B.subtilis, the yts,yvc and yxd gene clusters encode an ABC transporter from sub-
family 9 and a coupled TCS from the OmpR family [32], and the transcription factor of this
system can activate transcription by binding DNA and interacting with RNA polymerase [34].
We found that the expression of each gene in the three clusters, as well as those of the corre-
sponding ABC transporters, was decreased by more than half in our simulated intestinal envi-
ronment. Given the involved signaling cascade, the reduced expression of the OmpR family
may regulate the inhibition of many genes. Furthermore, the expression of several zinc-binding
lipoproteins and zinc metalloproteases was decreased in the cellulose group, which could nega-
tively affect the expression of many enzymes, storage proteins, transcription factors, and pro-
teins involved in replication [35]. Therefore, TCSs and ABC transporters both regulate energy
conservation in response to a high-fiber environment.
Conversely, TCSs and ABC transporters are also widely involved in nutrient absorption. In B.
subtilis, ATP-driven uptake systems prefer primary carbon and energy sources [36]. Several ki-
nases in TCS pathways involved in the metabolism of some amino acids were observed to be
up-regulated in cellulose medium, e.g., GlnT (U712_01235) and YesM (U712_03510), which in-
dicates changes in the expression of carbon metabolism genes. Several high-affinity ABC trans-
porters for different sugars catalyze the transport of sugar-oligomers to an even greater degree
than peptide transporters, which permits organisms to thrive in nutrient-poor environments
[37,38]. In this study, the expression of many oligosaccharide and polyol transporters involved in
cellulose hydrolyzate transport were increased in the cellulose group; for example, the expression
of the gene U712_03520, which encodes the putative ABC transporter substrate-binding protein
yesO, was increased 11.26-fold compared to the glucose group. It is likely that the expression of
many peptide- and calcium-binding ABC transporter protein genes (such as U712_06760,
U712_06745 and U712_08240) was enhanced to improve the transport of cellulose hydrolyzates.
Structural membrane and transporter proteins are key factors for
adaptation to stress
As the first organelle to experience pressure and the key organelle for adaptation to a harsh
environment, the cell membrane makes vital contributions to combat the effects of environmental
Adaptation to a High-Fiber Environment in Bacillus subtilis HH2
PLOS ONE | DOI:10.1371/journal.pone.0116935 February 6, 2015 7/13
Fig 3. KEGG analysis of flagellar assembly (bsu02040) in B.subtilis.Yellow boxes indicate significantly down-regulated genes in the cellulose group,
and gray boxes indicate up-regulated genes (none in this figure). Green indicates a group of proteins.
doi:10.1371/journal.pone.0116935.g003
Adaptation to a High-Fiber Environment in Bacillus subtilis HH2
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aggression, such as initiating the secretion of several proteins and exchanging intracellular and ex-
tracellular substances [38]. In this study, membrane and transmembrane genes were enriched in
both the up- and down-regulated gene clusters according to GO analysis (S1 Table); however, on
the individual level, the specifics of this enrichment were completely different. The up-regulated
clusters primarily consisted of transporter proteins, including carbohydrate transporters, sugar
transporters, and symporters, indicating that cellulose hydrolyzate may require a greater amount
of energy to be delivered into the cell. In contrast, structural proteins, such as plasma membrane,
cell membrane, and intrinsic membrane proteins, were primarily down-regulated. The down-
regulation of structural membrane genes increases the permeability of the cell membrane, allow-
ing the easy transport of cellulose hydrolyzates into the bacteria. Therefore, with a series of
adjustments, the cell membrane could become more adapted to cellulose medium.
Sporulation is a last-resort response to pressure and is suppressed until
alternative responses prove inadequate
When alternative responses prove inadequate to relieve stress, sporulation is the fate chosen by
most Bacillus species [39]. We observed increased sporulation in the cellulose group, both
under the light microscope (Fig. 1) and at the transcriptional level (U712_12580, encoding the
sporulation inhibitor Sda, was decreased more than 16-fold). However, among the seven stages
(0-VI) of sporulation protein genes (clusters), which are proteins that determine the sporula-
tion form, only stage II and stage III sporulation proteins were observed to be significantly up-
regulated in the cellulose group. In contrast, the expression of proteins involved in the five
other stages of sporulation was not greatly changed. Therefore, cellulose remains an acceptable
carbon source for B.subtilis HH2, and this strain can adapt well to a high-fiber environment
through a series of alterations in transcriptional regulation.
A regulatory model of B.subtilis HH2 in a high-fiber environment
As described above, we revealed major transcriptional reconfigurations in response to cellulose
adaptation as well as certain coordinated changes in the abundance of B.subtilis HH2 in a
high-fiber environment. To summarize these adaptation mechanisms, we propose a regulatory
model of B.subtilis HH2 for high-fiber environmental adaptation and cellulose digestion. In
this model, utilization of and adaptation to cellulose require at least four functional classes of
proteins, including (i) membrane proteins and membrane-associated signal channels, (ii) en-
zymes that catalyze cellulose hydrolysis, (iii) proteins encoded by operons that decrease cellular
energy and nutrient consumption, and (iv) proteins involved in sporulation. The cellular deg-
radation of and adaptation to cellulose consist of five steps. First, when bacteria are grown on a
medium with cellulose as a primary carbon source, ion channel-coupled receptors in the cell
membrane are stimulated by cellulose and send signals to the associated transduction systems.
As a result, cellulase components are expressed, secreted, assembled and transported to the cell
surface, which hydrolyzes the cellulose. Cellobiose and glucan, both products of cellulose
hydrolyzation, are transported into the cell through channels for hydrolyzates and ABC trans-
porters for further utilization. Simultaneously, upon pressure signals, bacteria partially reduce
the synthesis of non-essential proteins to save energy. The expression of sporulation genes also
partially increases to address potential continuing harsh pressures.
Discussion
B.subtilis is widely used as a probiotic and food additive with mammalian applications due to
its excellent ability to secrete a variety of antimicrobial substances that maintain the intestinal
microflora balance [40] and improve the digestibility of foraged foods in the gastrointestinal
Adaptation to a High-Fiber Environment in Bacillus subtilis HH2
PLOS ONE | DOI:10.1371/journal.pone.0116935 February 6, 2015 9/13
tract [41]. A number of studies have examined B.subtilis resistance [16,19], but there is still a
relative dearth of research on environmental cellulose adaptation mechanisms. A high-fiber
environment is most characteristic of the herbivorous animal gut environment, particularly for
the giant panda, which has no rumen for the fermentation of vegetation. In addition to the ge-
nomic potential of the intestinal microbiota, understanding bacterial adaptation mechanisms
for a cellulose environment will help us to better clarify the interactions between intestinal bac-
teria and their panda host.
Through phenotype experiment of B.subtilis HH2 isolated from pandas and grown on dif-
ferent carbon sources, we demonstrated that cellulose is not a very suitable carbon source for B.
subtilis HH2. However based on our transcriptional pathway analysis, we found that this bacte-
rium can adapt well via a series of regulatory networks and that the differentially expressed
genes clustered into two main categories: cellulose utilization and high-fiber environment ad-
aptation. For cellulose utilization, strain HH2 not only increased the expression of cellulase but
also of a series of enzyme components that hydrolyze cellulose, as well as some ABC transport-
ers, which served as support. Whereas it has been shown that organisms can selectively express
some genes highly for stress adaptation [39,42], in this study, it was observed that many genes
(clusters), such as several protein kinases, were up-regulated but that the expression of most
non-essential genes were down-regulated to conserve energy.
Interestingly, the expression of the Hfq protein (U712_09100), several sporulation kinases
and genes of proteins involved in sporulation was decreased in the cellulose group. The Hfq
protein is an RNA-binding protein associated with small regulatory RNAs (sRNAs) and has
many functions in pressure adaptation [17]. Sporulation is the final adaptation by the genus
Bacillus to relieve stress, and the decrease in the expression of these genes indicates that cellu-
lose may be an acceptable carbon source for B.subtilis HH2.
As an intestinal probiotic, B.subtilis HH2’s cellulose utilization ability could aid pandas in
digesting bamboo. The bacterial metabolite substances, such as polypeptides and lipids, which
could be digested by the host were produced in cellulose decomposing. In addition to its nutri-
tional effects, HH2 also contributes to maintaining the intestinal microflora balance in the
host, since the substances for antimicrobial effect and immune stimulation were continuous
producing in a high cellulose environment. Based on the gene expression data, we found that
although HH2 decreasing some antimicrobial peptides expression in the cellulose medium
compared to that in glucose medium may reduce its antimicrobial functions, most of bacitracin
expression could still be detected in the cellulose group. Such as flagellin (U712_17730) and a
series of surfactin components, which are involved in immune stimulation and resistance to
pathogen colonization [40], were detected more than 2,000 reads. Thus, we believe that B.sub-
tilis HH2 can still exert most probiotic functions both in nutritional and antimicrobial effects
in a high-fiber environment within animals.
In summary, this study revealed major changes in transcriptional regulations in response
to cellulose adaptation of B.subtilis HH2 on different carbon sources, as detected by RNA-
Seq. These results demonstrate that this bacterium could play part of functions as a probiotic
for pandas in a high-fiber environment, although cellulose is not a very suitable carbon
source for this strain. We also demonstrated a model for understanding the dynamic organi-
zation and interactions of the various functional and regulatory networks for unicellular or-
ganisms in a high-fiber environment. As a well-characterized bacterium and a Gram-positive
laboratory model, the transcriptional regulation of B.subtilis HH2 in a high-fiber environ-
ment will be a reference for other intestinal bacteria. Therefore, these results represent an im-
portant contribution to the research on the protection by the intestinal microbiota of
the panda.
Adaptation to a High-Fiber Environment in Bacillus subtilis HH2
PLOS ONE | DOI:10.1371/journal.pone.0116935 February 6, 2015 10 / 13
Supporting Information
S1 Fig. Cellulose hydrolysis halos of B.subtilis HH2. This strain has a good ability to digest
cellulose; the diameter of its cellulose hydrolysis halo was 28.00±0.44 mm.
(TIF)
S1 Table. GO term analysis of differentially expressed genes (DEGs).
(XLSX)
S2 Table. The expression levels of selected important carbon-metabolism genes.
(DOCX)
Author Contributions
Conceived and designed the experiments: ZYZ XZ ZJZ GP. Performed the experiments: ZYZ
XZ JL WL. Analyzed the data: ZYZ XZ WL XL. Contributed reagents/materials/analysis tools:
FL HS YL WG CW HZ DL TH. Wrote the paper: ZYZ XZ HF SC JS GP. Sample collect permis-
sion: HZ DL.
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