BACKGROUND
plant cell wall  polysaccharides i.e.
cellulose, hemicelluloses and pectins present in cereals, fruits and vegetables are an important part of the dietary fibers consumed by humans.
these polysaccharides are not digested in the upper digestive tract and reach the colon where they are degraded and fermented by the symbiotic intestinal microbiota.
it is estimated that the amount of pcw polysaccharides that reach the colon every day is between  <dig> and 25 g  <cit> , hemicelluloses representing up to 30 % of plant dry weight  <cit> .
two hemicellulosic polysaccharides that are widely consumed by humans consist primarily of glucuronoarabinoxylans  and arabinoxylans , and both belong to the xylan family of plant polysaccharides  <cit> .
structurally, xylans consist mostly of linear backbone of β- <dig> -d-xylopyranoside units which are commonly decorated with variable numbers 4-o-methyl-glucuronyl, acetyl, feruloyl, and arabinofuranosyl substituents.
depolymerization of xylans therefore requires the complementary action of enzymes acting on the xyl backbone i.e.
endo-β- <dig> -xylanase, β-xylosidase, and of enzymes debranching the substituents i.e.
α-l-arabinofuranosidase, α-glucuronidase, acetyl- and feruloyl-xylan esterases  <cit> .
in general, the enzymatic degradation of xylans leads to mixtures of oligosaccharides of different degree of polymerization and of various linkage compositions  <cit> .
xylans and some of their breakdown products i.e.
xylo-oligosaccharides  and arabinoxylan-oligosaccharides  have been shown to have a prebiotic effect and to provide health benefits, particularly in the management of obesity and related disorders  <cit> .
these health benefits are strongly correlated with changes in the composition of the gut microbiota: increase in bifidobacteria/lactobacilli as well as in bacteroides/prevotella and roseburia groups.
members of the bacteroides, prevotella and roseburia genera from the human colon  have been described for their xylanolytic activity .
these species can produce xos or axos of low degree of polymerization that can be utilized by glycolytic bacteria like bifidobacteria  <cit> .
hence the primary degraders of xylans play an important role in generating prebiotics in situ from pcw polysaccharides.
that is why it is essential to fully understand the xylanolytic enzyme systems of human gut bacteria, in order to identify which bacterial species play a key role in generating oligosaccharides from pcw polysaccharides that are beneficial to the host.
in a recent article, rogowski et al.
<cit>  highlighted the complexity of the xylanolytic enzyme system of bacteroides ovatus atcc 8483t.
here we present a combination of transcriptomics, proteomics and mutagenesis approaches to understand the xylanolytic function of another prevalent human gut symbiont, bacteroides xylanisolvens xb1at  <cit> , for which we previously showed that it displays a high xylan-degrading activity  <cit> .
indeed, the genome of this species harbors  <dig> genes encoding degradative carbohydrate-active enzymes  including glycoside hydrolases  and carbohydrate esterases  potentially active on xylans .
in addition, most of these genes were found to be clustered in genome loci called polysaccharide utilization loci .
pul function has been extensively dissected in bacteroides thetaiotaomicron vpi- <dig>  but needs to be better decrypted in other species of the bacteroides/prevotella genus involved in pcw degradation like b. xylanisolvens.
b. xylanisolvens xb1at genome was predicted to contain  <dig> puls by terrapon et al.
<cit> , and the pul numbering used hereafter is that given in the puldb database: http://www.cazy.org/puldb/.
RESULTS
transcriptomic and proteomic analyses of b. xylanisolvens xb1at grown on insoluble oat-spelt xylan
transcriptomic analysis
rna-seq data  were obtained from b. xylanisolvens xb1at grown on insoluble oat-spelt xylan  at mid- and late-log phase relative to glucose and/or xylose  .table  <dig> analyses performed from b. xylanisolvens xb1at cultures according to substrate and growth phasea
anot done
bdata that were discarded
transcriptional analysis of b. xylanisolvens xb1at grown on osx at mid-log phase revealed the strong expression  of several genes localized within two puls, i.e.
pul  <dig>  and pul  <dig> .
besides these two puls,  <dig> genes were also up-regulated  on osx relative to glucose and approximately half of these genes belonged to predicted puls or to gene clusters putatively involved in carbohydrate and energy metabolism.
interestingly, within these moderately induced puls,  <dig> puls  were also induced on citrus pectin  <cit> , whereas pul  <dig> and pul  <dig> were specifically induced on osx .fig.
<dig> polysaccharide utilization loci up- or down-regulated on insoluble oat-spelt xylan  or xylose, compared with glucose.
heatmap and gene organization of pul  <dig>  and pul  <dig> .
aheatmap based on log <dig> fold-change of gene expression on osx relative to glucose with b. xylanisolvens xb1at harvested at mid-log phase.
bheatmap based on log <dig> fold-change of gene expression on xylose relative to glucose with b. xylanisolvens xb1at harvested at late-log phase.
cpul predicted in bacteroides xylanisolvens xb1at  and presented below the heatmap.
the color code used for carbohydrate-active enzymes highlights the nature of the main functional module: glycoside hydrolase  or carbohydrate esterase .
pul marker genes, susc- and susd-like genes, are represented by purple and orange boxes, respectively, whilst the regulator genes appear in cyan.
other genes predicted as members of the puls are shown in grey.
genomic regions containing n stretches and/or unassigned genes are marked with a star.
dautomatic annotation of bacteroides xylanisolvens xb1at genome available at http://www.ncbi.nlm.nih.gov/genome/?term=xylanisolvens.
no log <dig> fold-change values were obtained  for orfs or fragments of orfs  not detected by automatic annotation because of sequence gaps  in the genomic region of interest; these orfs were manually annotated in this study
pul  <dig> is a cluster of  <dig> genes, including  <dig> genes with annotations consistent with xylan degradation, i.e.
<dig> glycoside hydrolases of families gh <dig>  gh <dig>  gh <dig>  gh <dig>  gh <dig> and gh <dig> and a bimodular carbohydrate esterase of families ce <dig> and ce <dig>  in addition to two tandemly arranged susc/d-like genes encoding extracellular membrane proteins potentially involved in the sequestration/internalization of xylan or resulting oligosaccharides into the cell, one gene encoding a hybrid two component system  and two genes of unknown function .
pul  <dig> is predicted in a genomic locus containing sequence gaps  and therefore was not fully annotated; however partial gene sequences allowed us to improve manually the annotation of this pul .
pul  <dig> contains  <dig> genes encoding four gh proteins of families gh <dig>  gh <dig>  gh <dig> and gh <dig>  susc/d-like proteins, two putative sugar transporters, two regulatory proteins  and one unknown protein.
when comparing the rna-seq heatmaps of the two puls, one can notice that all the genes of pul  <dig> were up-regulated upon growth on osx, whereas only the expression of three genes of pul  <dig> were highly induced namely genes encoding two glycoside hydrolases  and one sugar transporter .
in order to confirm these rna-seq data and to get information on the expression of important genes in pul  <dig> , relative rt-qpcr was performed on a subset of pul  <dig> and pul  <dig> genes .
the results confirmed the over-expression of pul  <dig> genes upon growth on osx relative to glucose at mid-log phase.
in pul  <dig>  we could verify the absence of induction of bxy_ <dig> and bxy_ <dig> encoding susc- and susd-like proteins as well as of bxy_ <dig> encoding a putative abc transporter, and the over-expression of bxy_ <dig> encoding a gh <dig> endo-xylanase.
relative rt-qpcr was also carried out with b. xylanisolvens xb1at grown on osx at late-log phase.
as expected, gene over-expression was higher at mid-log phase than at late-log phase, except for two genes  in pul  <dig> and three genes in pul  <dig>  that were down-regulated at late-log phase .fig.
<dig> impact of growth substrate on gene expression in pul  <dig> and rpul  <dig>  relative expression measured by rt-qpcr of selected genes from pul  <dig> and rpul  <dig> on osx versus glucose at mid-log phase or osx versus xylose at late-log phase.
differences in gene expression between mid- and late-log phase were significant for all targeted genes
unfortunately, the rna-seq data of the bacterium grown on osx at late-log phase were not exploitable .
nevertheless, at late-log phase, transcriptomic analysis of the bacterium grown on xylose and glucose showed that  <dig> out of  <dig> puls predicted from genomic analysis   <cit>  were expressed similarly on both sugars.
three puls  were repressed on xylose relative to glucose, whereas one pul  was induced.
from their cazyme composition, only pul  <dig> and pul  <dig> could be predicted to target fructans.
overall, these data indicate that glucose and xylose can be used indifferently as reference condition when analyzing pul gene expression on complex polysaccharides, including xylans but excluding fructans .
proteomic analysis
to investigate beyond transcription, we examined protein production of b. xylanisolvens xb1at cultivated on osx relative to xylose at late-log phase, considering that the xylanase specific activity of the strain was approximately 50-fold higher on osx .
analyzing the water-soluble protein fraction of strain xb1at on 2d-gels revealed  <dig> proteins that were over-produced and  <dig> proteins that were produced to lower levels between the two culture conditions.
among the proteins over-produced on osx, we picked and analyzed  <dig> protein spots and identified eight spot regions onto the 2d-gels corresponding to  <dig> proteins encoded by pul  <dig> .
among them, the family gh <dig> endo-xylanase harboring two carbohydrate-binding modules  organized in tandem  was the most over-produced on osx compared with xylose .
the other family gh <dig> endo-xylanase of 40 kda  from pul  <dig>  previously characterized and called xyn10a  <cit>  could not be visualized on the 2d-gels.
nevertheless, zymogram analysis using osx as substrate and performed when the bacterium was grown on osx in comparison with glucose or xylose underlined one high activity band that was only cell-associated which may be xyn10a , and another high activity band in both cell-associated and extracellular protein fractions corresponding to the 83 kda gh <dig> protein  .
one can notice that no activity band corresponding to the expected molecular mass of the other cazymes encoded by pul  <dig> i.e.
gh5_ <dig>  and cbm6-harboring gh <dig>  which have been described as endo-xylanases in other studies  <cit>  could be detected on the zymogram.fig.
<dig> impact of growth substrate on the soluble proteome of b. xylanisolvens xb1at.
2-de analysis of the soluble proteome of strain xb1at grown on osx versus xylose underlined the over-production of protein spots as shown on the expanded views beside the osx 2d-gel
fig.
<dig> endo-xylanase profiles of b. xylanisolvens xb1at.
zymogram performed with osx as substrate using cell-associated proteins  or extracellular proteins  of b. xylanisolvens xb1at cultivated on glucose, xylose and osx
functional analysis of pul 43
operon organization
we further investigated the genetic organization of the pul  <dig> cluster.
this 33 kb region contains  <dig> orfs  that are present on the positive strand of the genome .
dna sequence analysis tools  predicted the presence of three operons and of one gene being transcribed independently .
this was partly validated by predictions compiling genomic and rna-seq data .
to underline the production of polycistronic transcripts, rt-pcr experiments were undertaken using rnas extracted from strain xb1at grown on osx.
detection of a rt-pcr product corresponding to the intergenic region # <dig> and the absence of amplification for the intergenic regions # <dig> and # <dig>  confirmed the first operon of two genes  .
detection of amplified fragments from intergenic regions # <dig> to # <dig> indicated that the  <dig> following genes  are also organized in operon .
the existence of two operons is in agreement with read coverage of pul  <dig> region since no read coverage is observed in the intergenic regions # <dig> and # <dig> .
in addition, the region with bxy_ <dig> and bxy_ <dig> shows much higher read coverage and contains two putative internal promoters and terminators, suggesting the existence of additional separate transcripts produced from operon  <dig> or post-transcriptional modifications.
our data also indicate that the two terminators predicted in operon  <dig> may be weak terminators resulting in a leak-through transcription of genes from the upstream promoter .
collectively, these data tend to show that pul  <dig> is organized in two operons, with the longest one  possibly leading to polycistronic transcripts of different sizes, as previously observed with cellulosome-encoding operons in clostridium cellulolyticum  <cit> .fig.
<dig> evidence of a multi-operon organization within pul  <dig>  a read coverage  within pul  <dig> region and read information for that region, colored by strand, are given for the three rna samples obtained with osx condition.
the total coverage of the genome in that region is extremely high, about 1000-4000× and was not scalable.
hence, the displayed reads were down-sampled in the window to ~50× depth.
the black bars just below the grey coverage histogram for each sample indicate regions where down-sampling was performed.
b gene organization of pul  <dig> with the position number of the neighboring gene regions used as target for the rt-pcr experiments.
the intergenic distance between two genes is given in the table.
c operons predicted from genomic data using the fgenesb tool .
d operons predicted from rna-seq data using rockhopper .
e operons experimentally validated by rt-pcr, by amplifying the intergenic regions between two consecutive genes.
f the detection of rt-pcr fragments is shown on the agarose gel and the expected sizes of each product are given in the table
mutagenesis of the sensor/regulator htcs gene
in order to determine the importance of the xylan utilization system encoded by pul  <dig>  an insertion mutation into the gene encoding the htcs  was created.
indeed, htcs mutants have proven to be useful in establishing the role of puls in polysaccharide degradation by bacteroides thetaiotaomicron  <cit> .
the growth of the mutant was compared to that of the wild type  strain on glucose, xylose, wheat arabinoxylan  and osx .
both strains showed a similar growth on glucose.
on xylose, the mutant growth rate and density were approximately half that displayed by the wt strain, which did not grow as well on this sugar source than on glucose.
the growth of the mutant was either strongly reduced on wax or abolished on osx in comparison to the wt strain.
the expression of six genes selected from the three operons in pul  <dig> and five genes in pul  <dig> was then analyzed in the mutant strain upon growth on wax at mid-log phase .
interestingly, four genes in pul  <dig>  were highly repressed in the htcs mutant compared to the wt strain, the level of the repression being similar to that of the six pul  <dig> genes.fig.
<dig> expression measured by rt-qpcr of pul  <dig> and rpul  <dig> selected genes in pul  <dig> htcs  mutant relative to b. xylanisolvens xb1at .
each strain was grown on wax and harvested at mid-log phase.
each bar represents the mean of three independent experiments
pul  <dig>  microsynteny with other bacteroides species
given the atypical transcriptional data obtained with pul  <dig> genes, the synteny with other bacteroides, although previously described  <cit> , was further analyzed.
related genomic organizations were identified in the genomes of all  <dig> strains belonging to b. xylanisolvens and b. ovatus species, thanks to highly conserved flanking genes.
all strains harbored a core of five genes, encoding three cazymes , one sugar transporter and an unknown protein.
however, genes surrounding this core show important rearrangements.
in particular, when considering the upstream region of the core, the  <dig> strains could be separated in two groups, as previously shown  <cit> :  group a harboring the complete xylanolytic pul dissected in b. ovatus atcc 8483t , and  group b only harboring the core of five genes which is a subset of pul-xyls with no pul-marker genes .
further inspection of the downstream region of this core allowed us to distinguish two subgroups for each group, depending on the presence and/or distance of a second unrelated pul: homologous region to experimentally validated pul  <dig> in b. ovatus atcc 8483t encoding two regulatory proteins anti-σ factor/ecf-σ, susc/d-like proteins and one unknown protein .
indeed, this downstream pul was either two gene distant , or very distant  from the core in the strains with a complete pul-xyls.
in strains with only the five gene core , the downstream pul was either absent  or only separated by one gene from the core .
this proximity in subgroup b <dig> combined with the presence of poly-n stretches in b. xylanisolvens xb1at led to the prediction of the chimeric pul  <dig>  which gathered the core and the downstream unrelated pul.
in addition, the core can be considered as a remnant of pul-xyls that is active on xylan in b. xylanisolvens xb1at and hence be referred to as rpul  <dig> fig.
<dig> evolution of the xylanolytic genomic region from pul-xyls in b. ovatus atcc 8483t to this work on b. xylanisolvens xb1at.
genomic regions are represented as horizontal black lines with encoded genes depicted by boxes, above or below to distinguish strands, with proportionality to intergenic distances and gene length.
genomic regions with poly-n stretches are shown in red and marked by a star indicating that the annotation of these regions may be incomplete.
missing gene models in b. xylanisolvens xb1at are depicted with dotted boxes.
whilst genes of unknown function are shown in grey, relevant gene functions are color-coded as follows:  glycoside hydrolase genes are represented in pink;  pul marker genes, susc- and susd-like genes, in purple and orange, respectively;  pul regulatory genes  and of four genes of one smaller pul of  <dig> genes .
pul  <dig> and pul  <dig> resemble two puls over-expressed by bacteroides ovatus atcc 8483t grown on xylans that were called pul-xyll  and pul-xyls    <cit> .
the proteomics results supported the up-regulation of several genes belonging to pul  <dig> on osx, and therefore confirmed the inducible nature of this xylan-utilization locus.
they also showed the important over-production of a gh <dig> endo-xylanase .
this enzyme has homologs in several bacteroides/prevotella species  <cit> .
one essential feature of this enzyme is that it presents a peculiar modular architecture consisting of a catalytic module disrupted by two cbm <dig>  it has been shown that these tandemly arranged cbms confer to the cognate gh <dig> enzyme a high binding capacity to xylan and it was suggested that such enzymes play a critical role in xylan degradation  <cit> .
to further decipher the functionalities of pul  <dig>  a htcs  insertion mutant was generated and the effect of the mutation on bacterial growth and on gene expression in pul  <dig> and pul  <dig> was analyzed.
the mutation had no effect on growth on glucose but had a substantial effect on growth on xylose.
growth reduction on xylose cannot be due to the repression of gene bxy_ <dig> encoding a sugar transporter  but must somehow be caused by a defect in xylose utilization by the bacterium.
furthermore, the htcs mutation had a dramatic effect on bacterial growth on insoluble osx and soluble wax.
it also strongly reduced the expression of both pul  <dig> and pul  <dig> genes when the bacterium was grown on wax.
in pul  <dig>  genes belonging to the two operons were repressed, indicating that the htcs protein regulates expression of the two operons.
in pul  <dig>  three genes  encoding cazymes  and one gene  encoding a sugar transporter were repressed while the gene encoding the susc-like protein  was not.
this result highlights for the first time a cross-regulation between two puls, with the htcs regulator in pul  <dig> controlling directly or indirectly the expression of genes in pul  <dig>
the fact that several genes in pul  <dig> i.e.
bxy_ <dig>  bxy_ <dig>  bxy_ <dig> and bxy_ <dig> encoding susc/d-like proteins, an abc transporter and a gh <dig> protein, respectively were not over-expressed upon growth on osx suggests that osx is not the primary substrate of this pul and/or that the boundaries of this pul need to be redefined.
our analysis of the organization of pul  <dig> and its homologs in  <dig> strains of b. ovatus and b. xylanisolvens highlighted four subgroups of strains harboring different genetic organization around a core cluster of five genes common to all strains.
subgroups a <dig> and a <dig> possess pul-xyls described in b. ovatus  <cit> , whereas subgroups b <dig> and b <dig> only harbor the five gene core which was not predicted as a pul except in strain b. xylanisolvens xb1at.
we propose here that pul  <dig> is a pul chimera and the part corresponding to the five gene core was named rpul  <dig> as it may be a remnant of pul-xyls.
all strains from groups b <dig> only assigned to b. xylanisolvens species might then function as b. xylanisolvens xb1at.
apart from pul  <dig> and rpul  <dig>  the transcriptomics rna-seq profiles obtained with osx highlighted a low induction of  <dig> puls relative to the glucose condition; the same puls were also slightly up-regulated in another transcriptomic rna-seq study using citrus pectin  <cit> .
these cross-inductions between osx and citrus pectin could be due to the presence of contaminating polysaccharides in both substrates.
as a matter of fact, starch was detected in both substrates  and pul  <dig>  was induced on both substrates.
it could also be due to the presence of common oligosaccharide motifs in osx and pectin i.e.
arabinose-containing side chains targeted by pul  <dig> .
finally, cross-regulations among puls might occur as shown here between pul  <dig> and rpul  <dig>
from what is already known on pul functioning in bacteroides  <cit> , we suggest that pul  <dig> encodes proteins that bind to xylans , initiate their degradation at the surface of the bacterium , and allow the transfer of oligosaccharides to the periplasm , while rpul  <dig> encodes enzymes  completing the degradation of oligosaccharides in the periplasm, supported by enzymes encoded by pul  <dig>  rpul  <dig> may also be involved in the transport of the sugars from the periplasm to the cytoplasm via the sugar transporter encoded by bxy_ <dig>
rogowsky et al.
<cit>  proposed a model of xylan degradation by b. ovatus atcc 8483t that is different, especially because of the different cazyme composition of pul-xyll .
indeed, this pul would be dedicated to highly substituted glucuronoarabinoxylans  which require gh30- and gh98-encoding genes that are not present in pul  <dig> of b. xylanisolvens xb1at.
according to the rogowski model, pul-xyls  would act only on poorly decorated xylans  like osx.
it is interesting to note that it is the deletion of the small pul  and not of the large pul  that abolished the growth of b. ovatus on simple xylans  <cit> .
it occurs that pul-xyll of b. ovatus encodes an inactive gh <dig> in the so-called “core xylan utilization cluster” while pul-xyls harbors two genes encoding a gh <dig>  one of them being the peculiar cbm4-containing gh <dig>  <cit> .
contrary to b. ovatus atcc 8483t, the cbm4-containing gh <dig> of b. xylanisolvens xb1at is encoded by pul  <dig> and the other gh <dig> is encoded by rpul  <dig>  and through the pul  <dig> htcs mutation, we showed that both genomic regions, linked at the transcriptional level, are necessary for b. xylanisolvens to grow on xylans .
the cbm4-containing gh <dig> was also shown to hydrolyze xylan backbone of high degree of polymerization   <cit>  and to be located at the cell surface of b. ovatus atcc 8483t  <cit> , indicating that this enzyme initiates the process of xylan breakdown.
b. intestinalis dsm 17393t, b. cellulosilyticus dsm 14838t,  also harbor one gene encoding a cbm4-containing gh <dig> in their genomes although in puls with different organization than those of b. ovatus atcc 8483t and b. xylanisolvens xb1at .
interestingly, we found that two strains of b. xylanisolvens  that did not possess that gene did not grow on wax and had a very low growth on osx, contrary to b. xylanisolvens xb1at, b. ovatus atcc 8483t, b. intestinalis dsm 17393t and b. cellulosilyticus dsm 14838t .
from all these observations, we propose that this peculiar enzyme is crucial for xylan breakdown by human gut symbionts of the bacteroides genus and that it could be considered as a functional marker of simple xylan degradation in the human gut.
CONCLUSIONS
we have shown that the xylanolytic function of b. xylanisolvens xb1at involves one pul and one pul remnant that are linked at the transcriptomic level and that produce the necessary arsenal of cazymes and accessory proteins for an optimal utilization of xylans of low complexity.
this study, along with another recent study on b. ovatus atcc 8483t  <cit> , shows that b. xylanisolvens xb1at belongs to a group of strains that display pul functionalities and specificities regarding xylan utilization that are different from those displayed by other groups such as the one comprising b. ovatus atcc 8483t  <cit> .
these differences may provide ecological advantages of one group of strains over the others, depending on the availability of xylans in the diet but also on their structural complexity.
hence the nature of the xylan consumed in the diet and the bacterial species selected upon that diet may lead to different oligosaccharides with different prebiotic effects.
more studies are needed to better understand the function and ecology of pcw-degrading bacteria in the colon, including bacterial representatives of the firmicutes phylum, in order to better predict the impact of diet and dietary fibers on health.

 plant cell wall polysaccharides i.e. cellulose, hemicelluloses and pectins present in cereals, fruits and vegetables are an important part of the dietary fibers consumed by humans. these polysaccharides are not digested in the upper digestive tract and reach the colon where they are degraded and fermented by the symbiotic intestinal microbiota. it is estimated that the amount of pcw polysaccharides that reach the colon every day is between and 25 g, hemicelluloses representing up to 30 % of plant dry weight. two hemicellulosic polysaccharides that are widely consumed by humans consist primarily of glucuronoarabinoxylans and arabinoxylans, and both belong to the xylan family of plant polysaccharides. structurally, xylans consist mostly of linear backbone of β- -d-xylopyranoside units which are commonly decorated with variable numbers 4-o-methyl-glucuronyl, acetyl, feruloyl, and arabinofuranosyl substituents. depolymerization of xylans therefore requires the complementary action of enzymes acting on the xyl backbone i.e. endo-β- -xylanase, β-xylosidase, and of enzymes debranching the substituents i.e. α-l-arabinofuranosidase, α-glucuronidase, acetyl- and feruloyl-xylan esterases. in general, the enzymatic degradation of xylans leads to mixtures of oligosaccharides of different degree of polymerization and of various linkage compositions. xylans and some of their breakdown products i.e. xylo-oligosaccharides and arabinoxylan-oligosaccharides have been shown to have a prebiotic effect and to provide health benefits, particularly in the management of obesity and related disorders. these health benefits are strongly correlated with changes in the composition of the gut microbiota: increase in bifidobacteria/lactobacilli as well as in bacteroides/prevotella and roseburia groups. members of the bacteroides, prevotella and roseburia genera from the human colon have been described for their xylanolytic activity. these species can produce xos or axos of low degree of polymerization that can be utilized by glycolytic bacteria like bifidobacteria. hence the primary degraders of xylans play an important role in generating prebiotics in situ from pcw polysaccharides. that is why it is essential to fully understand the xylanolytic enzyme systems of human gut bacteria, in order to identify which bacterial species play a key role in generating oligosaccharides from pcw polysaccharides that are beneficial to the host. in a recent article, rogowski et al. highlighted the complexity of the xylanolytic enzyme system of bacteroides ovatus atcc 8483t. here we present a combination of transcriptomics, proteomics and mutagenesis approaches to understand the xylanolytic function of another prevalent human gut symbiont, bacteroides xylanisolvens xb1at, for which we previously showed that it displays a high xylan-degrading activity. indeed, the genome of this species harbors genes encoding degradative carbohydrate-active enzymes including glycoside hydrolases and carbohydrate esterases potentially active on xylans. in addition, most of these genes were found to be clustered in genome loci called polysaccharide utilization loci. pul function has been extensively dissected in bacteroides thetaiotaomicron vpi- but needs to be better decrypted in other species of the bacteroides/prevotella genus involved in pcw degradation like b. xylanisolvens. b. xylanisolvens xb1at genome was predicted to contain puls by terrapon et al., and the pul numbering used hereafter is that given in the puldb database: http://www.cazy.org/puldb/.  transcriptomic and proteomic analyses of b. xylanisolvens xb1at grown on insoluble oat-spelt xylan transcriptomic analysis rna-seq data were obtained from b. xylanisolvens xb1at grown on insoluble oat-spelt xylan at mid- and late-log phase relative to glucose and/or xylose.table analyses performed from b. xylanisolvens xb1at cultures according to substrate and growth phasea anot done bdata that were discarded transcriptional analysis of b. xylanisolvens xb1at grown on osx at mid-log phase revealed the strong expression of several genes localized within two puls, i.e. pul and pul. besides these two puls, genes were also up-regulated on osx relative to glucose and approximately half of these genes belonged to predicted puls or to gene clusters putatively involved in carbohydrate and energy metabolism. interestingly, within these moderately induced puls, puls were also induced on citrus pectin, whereas pul and pul were specifically induced on osx.fig. polysaccharide utilization loci up- or down-regulated on insoluble oat-spelt xylan or xylose, compared with glucose. heatmap and gene organization of pul and pul. aheatmap based on log fold-change of gene expression on osx relative to glucose with b. xylanisolvens xb1at harvested at mid-log phase. bheatmap based on log fold-change of gene expression on xylose relative to glucose with b. xylanisolvens xb1at harvested at late-log phase. cpul predicted in bacteroides xylanisolvens xb1at and presented below the heatmap. the color code used for carbohydrate-active enzymes highlights the nature of the main functional module: glycoside hydrolase or carbohydrate esterase. pul marker genes, susc- and susd-like genes, are represented by purple and orange boxes, respectively, whilst the regulator genes appear in cyan. other genes predicted as members of the puls are shown in grey. genomic regions containing n stretches and/or unassigned genes are marked with a star. dautomatic annotation of bacteroides xylanisolvens xb1at genome available at http://www.ncbi.nlm.nih.gov/genome/?term=xylanisolvens. no log fold-change values were obtained for orfs or fragments of orfs not detected by automatic annotation because of sequence gaps in the genomic region of interest; these orfs were manually annotated in this study pul is a cluster of genes, including genes with annotations consistent with xylan degradation, i.e. glycoside hydrolases of families gh gh gh gh gh and gh and a bimodular carbohydrate esterase of families ce and ce in addition to two tandemly arranged susc/d-like genes encoding extracellular membrane proteins potentially involved in the sequestration/internalization of xylan or resulting oligosaccharides into the cell, one gene encoding a hybrid two component system and two genes of unknown function. pul is predicted in a genomic locus containing sequence gaps and therefore was not fully annotated; however partial gene sequences allowed us to improve manually the annotation of this pul. pul contains genes encoding four gh proteins of families gh gh gh and gh susc/d-like proteins, two putative sugar transporters, two regulatory proteins and one unknown protein. when comparing the rna-seq heatmaps of the two puls, one can notice that all the genes of pul were up-regulated upon growth on osx, whereas only the expression of three genes of pul were highly induced namely genes encoding two glycoside hydrolases and one sugar transporter. in order to confirm these rna-seq data and to get information on the expression of important genes in pul, relative rt-qpcr was performed on a subset of pul and pul genes. the results confirmed the over-expression of pul genes upon growth on osx relative to glucose at mid-log phase. in pul we could verify the absence of induction of bxy_ and bxy_ encoding susc- and susd-like proteins as well as of bxy_ encoding a putative abc transporter, and the over-expression of bxy_ encoding a gh endo-xylanase. relative rt-qpcr was also carried out with b. xylanisolvens xb1at grown on osx at late-log phase. as expected, gene over-expression was higher at mid-log phase than at late-log phase, except for two genes in pul and three genes in pul that were down-regulated at late-log phase.fig. impact of growth substrate on gene expression in pul and rpul relative expression measured by rt-qpcr of selected genes from pul and rpul on osx versus glucose at mid-log phase or osx versus xylose at late-log phase. differences in gene expression between mid- and late-log phase were significant for all targeted genes unfortunately, the rna-seq data of the bacterium grown on osx at late-log phase were not exploitable. nevertheless, at late-log phase, transcriptomic analysis of the bacterium grown on xylose and glucose showed that out of puls predicted from genomic analysis were expressed similarly on both sugars. three puls were repressed on xylose relative to glucose, whereas one pul was induced. from their cazyme composition, only pul and pul could be predicted to target fructans. overall, these data indicate that glucose and xylose can be used indifferently as reference condition when analyzing pul gene expression on complex polysaccharides, including xylans but excluding fructans. proteomic analysis to investigate beyond transcription, we examined protein production of b. xylanisolvens xb1at cultivated on osx relative to xylose at late-log phase, considering that the xylanase specific activity of the strain was approximately 50-fold higher on osx. analyzing the water-soluble protein fraction of strain xb1at on 2d-gels revealed proteins that were over-produced and proteins that were produced to lower levels between the two culture conditions. among the proteins over-produced on osx, we picked and analyzed protein spots and identified eight spot regions onto the 2d-gels corresponding to proteins encoded by pul. among them, the family gh endo-xylanase harboring two carbohydrate-binding modules organized in tandem was the most over-produced on osx compared with xylose. the other family gh endo-xylanase of 40 kda from pul previously characterized and called xyn10a could not be visualized on the 2d-gels. nevertheless, zymogram analysis using osx as substrate and performed when the bacterium was grown on osx in comparison with glucose or xylose underlined one high activity band that was only cell-associated which may be xyn10a, and another high activity band in both cell-associated and extracellular protein fractions corresponding to the 83 kda gh protein. one can notice that no activity band corresponding to the expected molecular mass of the other cazymes encoded by pul i.e. gh5_ and cbm6-harboring gh which have been described as endo-xylanases in other studies could be detected on the zymogram.fig. impact of growth substrate on the soluble proteome of b. xylanisolvens xb1at. 2-de analysis of the soluble proteome of strain xb1at grown on osx versus xylose underlined the over-production of protein spots as shown on the expanded views beside the osx 2d-gel fig. endo-xylanase profiles of b. xylanisolvens xb1at. zymogram performed with osx as substrate using cell-associated proteins or extracellular proteins of b. xylanisolvens xb1at cultivated on glucose, xylose and osx functional analysis of pul 43 operon organization we further investigated the genetic organization of the pul cluster. this 33 kb region contains orfs that are present on the positive strand of the genome. dna sequence analysis tools predicted the presence of three operons and of one gene being transcribed independently. this was partly validated by predictions compiling genomic and rna-seq data. to underline the production of polycistronic transcripts, rt-pcr experiments were undertaken using rnas extracted from strain xb1at grown on osx. detection of a rt-pcr product corresponding to the intergenic region # and the absence of amplification for the intergenic regions # and # confirmed the first operon of two genes. detection of amplified fragments from intergenic regions # to # indicated that the following genes are also organized in operon. the existence of two operons is in agreement with read coverage of pul region since no read coverage is observed in the intergenic regions # and #. in addition, the region with bxy_ and bxy_ shows much higher read coverage and contains two putative internal promoters and terminators, suggesting the existence of additional separate transcripts produced from operon or post-transcriptional modifications. our data also indicate that the two terminators predicted in operon may be weak terminators resulting in a leak-through transcription of genes from the upstream promoter. collectively, these data tend to show that pul is organized in two operons, with the longest one possibly leading to polycistronic transcripts of different sizes, as previously observed with cellulosome-encoding operons in clostridium cellulolyticum.fig. evidence of a multi-operon organization within pul a read coverage within pul region and read information for that region, colored by strand, are given for the three rna samples obtained with osx condition. the total coverage of the genome in that region is extremely high, about 1000-4000× and was not scalable. hence, the displayed reads were down-sampled in the window to ~50× depth. the black bars just below the grey coverage histogram for each sample indicate regions where down-sampling was performed. b gene organization of pul with the position number of the neighboring gene regions used as target for the rt-pcr experiments. the intergenic distance between two genes is given in the table. c operons predicted from genomic data using the fgenesb tool. d operons predicted from rna-seq data using rockhopper. e operons experimentally validated by rt-pcr, by amplifying the intergenic regions between two consecutive genes. f the detection of rt-pcr fragments is shown on the agarose gel and the expected sizes of each product are given in the table mutagenesis of the sensor/regulator htcs gene in order to determine the importance of the xylan utilization system encoded by pul an insertion mutation into the gene encoding the htcs was created. indeed, htcs mutants have proven to be useful in establishing the role of puls in polysaccharide degradation by bacteroides thetaiotaomicron. the growth of the mutant was compared to that of the wild type strain on glucose, xylose, wheat arabinoxylan and osx. both strains showed a similar growth on glucose. on xylose, the mutant growth rate and density were approximately half that displayed by the wt strain, which did not grow as well on this sugar source than on glucose. the growth of the mutant was either strongly reduced on wax or abolished on osx in comparison to the wt strain. the expression of six genes selected from the three operons in pul and five genes in pul was then analyzed in the mutant strain upon growth on wax at mid-log phase. interestingly, four genes in pul were highly repressed in the htcs mutant compared to the wt strain, the level of the repression being similar to that of the six pul genes.fig. expression measured by rt-qpcr of pul and rpul selected genes in pul htcs mutant relative to b. xylanisolvens xb1at. each strain was grown on wax and harvested at mid-log phase. each bar represents the mean of three independent experiments pul microsynteny with other bacteroides species given the atypical transcriptional data obtained with pul genes, the synteny with other bacteroides, although previously described, was further analyzed. related genomic organizations were identified in the genomes of all strains belonging to b. xylanisolvens and b. ovatus species, thanks to highly conserved flanking genes. all strains harbored a core of five genes, encoding three cazymes, one sugar transporter and an unknown protein. however, genes surrounding this core show important rearrangements. in particular, when considering the upstream region of the core, the strains could be separated in two groups, as previously shown: group a harboring the complete xylanolytic pul dissected in b. ovatus atcc 8483t, and group b only harboring the core of five genes which is a subset of pul-xyls with no pul-marker genes. further inspection of the downstream region of this core allowed us to distinguish two subgroups for each group, depending on the presence and/or distance of a second unrelated pul: homologous region to experimentally validated pul in b. ovatus atcc 8483t encoding two regulatory proteins anti-σ factor/ecf-σ, susc/d-like proteins and one unknown protein. indeed, this downstream pul was either two gene distant, or very distant from the core in the strains with a complete pul-xyls. in strains with only the five gene core, the downstream pul was either absent or only separated by one gene from the core. this proximity in subgroup b combined with the presence of poly-n stretches in b. xylanisolvens xb1at led to the prediction of the chimeric pul which gathered the core and the downstream unrelated pul. in addition, the core can be considered as a remnant of pul-xyls that is active on xylan in b. xylanisolvens xb1at and hence be referred to as rpul fig. evolution of the xylanolytic genomic region from pul-xyls in b. ovatus atcc 8483t to this work on b. xylanisolvens xb1at. genomic regions are represented as horizontal black lines with encoded genes depicted by boxes, above or below to distinguish strands, with proportionality to intergenic distances and gene length. genomic regions with poly-n stretches are shown in red and marked by a star indicating that the annotation of these regions may be incomplete. missing gene models in b. xylanisolvens xb1at are depicted with dotted boxes. whilst genes of unknown function are shown in grey, relevant gene functions are color-coded as follows: glycoside hydrolase genes are represented in pink; pul marker genes, susc- and susd-like genes, in purple and orange, respectively; pul regulatory genes and of four genes of one smaller pul of genes. pul and pul resemble two puls over-expressed by bacteroides ovatus atcc 8483t grown on xylans that were called pul-xyll and pul-xyls. the proteomics results supported the up-regulation of several genes belonging to pul on osx, and therefore confirmed the inducible nature of this xylan-utilization locus. they also showed the important over-production of a gh endo-xylanase. this enzyme has homologs in several bacteroides/prevotella species. one essential feature of this enzyme is that it presents a peculiar modular architecture consisting of a catalytic module disrupted by two cbm it has been shown that these tandemly arranged cbms confer to the cognate gh enzyme a high binding capacity to xylan and it was suggested that such enzymes play a critical role in xylan degradation. to further decipher the functionalities of pul a htcs insertion mutant was generated and the effect of the mutation on bacterial growth and on gene expression in pul and pul was analyzed. the mutation had no effect on growth on glucose but had a substantial effect on growth on xylose. growth reduction on xylose cannot be due to the repression of gene bxy_ encoding a sugar transporter but must somehow be caused by a defect in xylose utilization by the bacterium. furthermore, the htcs mutation had a dramatic effect on bacterial growth on insoluble osx and soluble wax. it also strongly reduced the expression of both pul and pul genes when the bacterium was grown on wax. in pul genes belonging to the two operons were repressed, indicating that the htcs protein regulates expression of the two operons. in pul three genes encoding cazymes and one gene encoding a sugar transporter were repressed while the gene encoding the susc-like protein was not. this result highlights for the first time a cross-regulation between two puls, with the htcs regulator in pul controlling directly or indirectly the expression of genes in pul the fact that several genes in pul i.e. bxy_ bxy_ bxy_ and bxy_ encoding susc/d-like proteins, an abc transporter and a gh protein, respectively were not over-expressed upon growth on osx suggests that osx is not the primary substrate of this pul and/or that the boundaries of this pul need to be redefined. our analysis of the organization of pul and its homologs in strains of b. ovatus and b. xylanisolvens highlighted four subgroups of strains harboring different genetic organization around a core cluster of five genes common to all strains. subgroups a and a possess pul-xyls described in b. ovatus, whereas subgroups b and b only harbor the five gene core which was not predicted as a pul except in strain b. xylanisolvens xb1at. we propose here that pul is a pul chimera and the part corresponding to the five gene core was named rpul as it may be a remnant of pul-xyls. all strains from groups b only assigned to b. xylanisolvens species might then function as b. xylanisolvens xb1at. apart from pul and rpul the transcriptomics rna-seq profiles obtained with osx highlighted a low induction of puls relative to the glucose condition; the same puls were also slightly up-regulated in another transcriptomic rna-seq study using citrus pectin. these cross-inductions between osx and citrus pectin could be due to the presence of contaminating polysaccharides in both substrates. as a matter of fact, starch was detected in both substrates and pul was induced on both substrates. it could also be due to the presence of common oligosaccharide motifs in osx and pectin i.e. arabinose-containing side chains targeted by pul. finally, cross-regulations among puls might occur as shown here between pul and rpul from what is already known on pul functioning in bacteroides, we suggest that pul encodes proteins that bind to xylans, initiate their degradation at the surface of the bacterium, and allow the transfer of oligosaccharides to the periplasm, while rpul encodes enzymes completing the degradation of oligosaccharides in the periplasm, supported by enzymes encoded by pul rpul may also be involved in the transport of the sugars from the periplasm to the cytoplasm via the sugar transporter encoded by bxy_ rogowsky et al. proposed a model of xylan degradation by b. ovatus atcc 8483t that is different, especially because of the different cazyme composition of pul-xyll. indeed, this pul would be dedicated to highly substituted glucuronoarabinoxylans which require gh30- and gh98-encoding genes that are not present in pul of b. xylanisolvens xb1at. according to the rogowski model, pul-xyls would act only on poorly decorated xylans like osx. it is interesting to note that it is the deletion of the small pul and not of the large pul that abolished the growth of b. ovatus on simple xylans. it occurs that pul-xyll of b. ovatus encodes an inactive gh in the so-called “core xylan utilization cluster” while pul-xyls harbors two genes encoding a gh one of them being the peculiar cbm4-containing gh. contrary to b. ovatus atcc 8483t, the cbm4-containing gh of b. xylanisolvens xb1at is encoded by pul and the other gh is encoded by rpul and through the pul htcs mutation, we showed that both genomic regions, linked at the transcriptional level, are necessary for b. xylanisolvens to grow on xylans. the cbm4-containing gh was also shown to hydrolyze xylan backbone of high degree of polymerization and to be located at the cell surface of b. ovatus atcc 8483t, indicating that this enzyme initiates the process of xylan breakdown. b. intestinalis dsm 17393t, b. cellulosilyticus dsm 14838t, also harbor one gene encoding a cbm4-containing gh in their genomes although in puls with different organization than those of b. ovatus atcc 8483t and b. xylanisolvens xb1at. interestingly, we found that two strains of b. xylanisolvens that did not possess that gene did not grow on wax and had a very low growth on osx, contrary to b. xylanisolvens xb1at, b. ovatus atcc 8483t, b. intestinalis dsm 17393t and b. cellulosilyticus dsm 14838t. from all these observations, we propose that this peculiar enzyme is crucial for xylan breakdown by human gut symbionts of the bacteroides genus and that it could be considered as a functional marker of simple xylan degradation in the human gut.  we have shown that the xylanolytic function of b. xylanisolvens xb1at involves one pul and one pul remnant that are linked at the transcriptomic level and that produce the necessary arsenal of cazymes and accessory proteins for an optimal utilization of xylans of low complexity. this study, along with another recent study on b. ovatus atcc 8483t, shows that b. xylanisolvens xb1at belongs to a group of strains that display pul functionalities and specificities regarding xylan utilization that are different from those displayed by other groups such as the one comprising b. ovatus atcc 8483t. these differences may provide ecological advantages of one group of strains over the others, depending on the availability of xylans in the diet but also on their structural complexity. hence the nature of the xylan consumed in the diet and the bacterial species selected upon that diet may lead to different oligosaccharides with different prebiotic effects. more studies are needed to better understand the function and ecology of pcw-degrading bacteria in the colon, including bacterial representatives of the firmicutes phylum, in order to better predict the impact of diet and dietary fibers on health. 
xylanolytic bacteria are the first degraders of these complex polysaccharides and they release breakdown products that can have beneficial effects on human health. our results also suggest that b. xylanisolvens xb1at has specialized in the degradation of xylans of low complexity. this study shows that xylan degradation by b. xylanisolvens xb1at is orchestrated by one pul and one pul remnant that are linked at the transcriptional level. coupled to studies on other xylanolytic bacteroides species, our data emphasize the importance of one peculiar cbm4-containing gh endo-xylanase in xylan breakdown and that this modular enzyme may be used as a functional marker of xylan degradation in the human gut. xylan degradationhuman gutbacteroidespolysaccharide-utilization locuscazymesrna-seqproteomicsmutagenesiseuropean union's seventh framework program fp/2007/2013322820henrissat bernard issue-copyright-statement© the author 2016 proteomic analyses supported the up-regulation of several genes belonging to pul and showed the important over-production of a cbm4-containing gh endo-xylanase. we also show that pul is organized in two operons and that the knockout of the pul sensor/regulator htcs gene blocked the growth of the mutant on insoluble osx and soluble wheat arabinoxylan. xylans are not degraded by human digestive enzymes in the upper digestive tract and therefore reach the colon where they are subjected to extensive degradation by some members of the symbiotic microbiota. the mutation not only repressed gene expression in the pul operons but also repressed gene expression in rpul transcriptomic analyses of b. xylanisolvens xb1at grown on insoluble oat-spelt xylan at mid- and late-log phases highlighted genes in a polysaccharide utilization locus, hereafter called pul and genes in a fragmentary remnant of another pul, hereafter referred to as rpul which were highly overexpressed on osx relative to glucose. in order to understand better how these bacteria metabolize xylans in the colon, this study was undertaken to investigate xylan breakdown by the prominent human gut symbiont bacteroides xylanisolvens xb1at. plant cell wall polysaccharides and especially xylans constitute an important part of human diet.
 plant cell wall polysaccharides and especially xylans constitute an important part of human diet. xylans are not degraded by human digestive enzymes in the upper digestive tract and therefore reach the colon where they are subjected to extensive degradation by some members of the symbiotic microbiota. xylanolytic bacteria are the first degraders of these complex polysaccharides and they release breakdown products that can have beneficial effects on human health. in order to understand better how these bacteria metabolize xylans in the colon, this study was undertaken to investigate xylan breakdown by the prominent human gut symbiont bacteroides xylanisolvens xb1at.  transcriptomic analyses of b. xylanisolvens xb1at grown on insoluble oat-spelt xylan at mid- and late-log phases highlighted genes in a polysaccharide utilization locus, hereafter called pul and genes in a fragmentary remnant of another pul, hereafter referred to as rpul which were highly overexpressed on osx relative to glucose. proteomic analyses supported the up-regulation of several genes belonging to pul and showed the important over-production of a cbm4-containing gh endo-xylanase. we also show that pul is organized in two operons and that the knockout of the pul sensor/regulator htcs gene blocked the growth of the mutant on insoluble osx and soluble wheat arabinoxylan. the mutation not only repressed gene expression in the pul operons but also repressed gene expression in rpul  this study shows that xylan degradation by b. xylanisolvens xb1at is orchestrated by one pul and one pul remnant that are linked at the transcriptional level. coupled to studies on other xylanolytic bacteroides species, our data emphasize the importance of one peculiar cbm4-containing gh endo-xylanase in xylan breakdown and that this modular enzyme may be used as a functional marker of xylan degradation in the human gut. our results also suggest that b. xylanisolvens xb1at has specialized in the degradation of xylans of low complexity. this functional feature may provide a niche to all xylanolytic bacteria harboring similar puls. further functional and ecological studies on fibrolytic bacteroides species are needed to better understand their role in dietary fiber degradation and their impact on intestinal health. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords xylan degradationhuman gutbacteroidespolysaccharide-utilization locuscazymesrna-seqproteomicsmutagenesiseuropean union's seventh framework program fp/2007/2013322820henrissat bernard issue-copyright-statement© the author 2016 