Cleavage A, binding B, and CAZy database classification C of purified enzymes. A Polysaccharide cleavage was quantified as nmol reducing sugar released per milligram enzyme per minute: . 160 red, 80–160 orange, 40–80 yellow, 20–40 green, , 20 gray. B Binding to insoluble polysaccharides was quantified as percentage enzyme bound to substrate: . 30% red, 20–30% orange, 15–20% yellow, 10–15% green, , 10% gray. C CAZy database classifications: glycoside hydrolases (GH), carbohydrate esterases (CE), polysaccharide lyases (PL), and carbohydrate binding domains (CBM). Among 56 purified CAZymes, only the 32 enzymes for which activities were found are shown. doi:10.1371/journal.pgen.1004773.g004 

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... galacturonic acid (Fig. S1F) and rhamnose (Fig. S1H), cell yield was low on RGI (Fig. 1B). C. phytofermentans grows well on galactan (Fig. 1C), xylans (Fig. 1F–G), mannans (Fig. 1H–I), xyloglucan (Fig. 1J), and starch (Fig. 1L). Limited growth on AGII (Fig. 1E) relative to galactan supports that C. phytofermentans cleaves b -1,4 galactan, but not the b -1,3 and b -1,6-galactose bonds in AGII. Poor growth on arabinan (Fig. 1D) is similar to arabinose (Fig. S1G), suggesting this sugar is transported or metabolized inefficiently. C. phytofermentans grows well on cellulose plates (Fig. S2) and solubilizes cellulosic substrates such as filter paper and raw corn stover (Fig. S3), but weak growth on carboxymethylcellulose (CMC) might result from either lack of a suitable endoglucanase or carboxymethyl side groups inhibiting its metabolism. C. phytofermentans shows diauxic growth on the mixed sugar polysaccharides galactomannan (Fig. 1I) and xyloglucan (Fig. 1J). For each of these substrates, one of the component sugars (galactose or glucose) supports faster growth than the other (mannose or xylose) (Fig. S1, Table S4). Growth on various mixtures of galactose/mannose (Fig. S4) and of glucose/xylose (Fig. S5) shows rapid metabolism of the preferred sugar followed by slower growth on the other one. However, in both cases when the favored sugar reached 75% of the total, the other sugar does not appear to be metabolized. Similar to some ruminal [19] and human gut microbes [20], C. phytofermentans often grows faster on polysaccharides than the constituent sugars (Table S4). When presented with mixtures of xylan and xylose, this bacterium shows diauxic growth with preferential metabolism of xylan (Fig. S6), which is surprising because xylan must be cleaved to xylose to be metabolized. Growth on polysaccharides could be energetically favorable if significant ATP is saved by simultaneous transport of multiple sugar units in a single oligosaccharide [21] or by intracellular phosphorolysis of oligosaccharides [22]. C. phytofermentans encodes at least a dozen phosphorylases [23] [24], which cleave oligosaccharides without using ATP. Although the mechanisms regulating sugar metabolism in C. phytofermentans are unknown, diauxic growth supports carbon catabolite repression prioritizes growth on preferred sugars and polysaccharides. We quantified mRNA expression by strand-specific RNA sequencing during log-phase growth on 8 polysaccharides, 3 monosaccharides, and raw corn stover as a complex biomass substrate. An average of 17.3 million mRNA reads were mapped per sample (Table S5), yielding expression (RPKM) values (Table S6) that were highly correlated (r 2 = 0.96–0.99) between duplicate cultures for all conditions (Fig. S7). The reads were also highly strand-specific (Fig. S8), which will facilitate their future use for de novo transcriptome assembly, gene annotation and detection of antisense transcription. The fraction of reads mapping to CAZymes during growth on glucose was 2.0%, but this increased greatly on polysaccharides, especially cellulose (11.9%) and stover (31.0%). We assessed which genes were significantly differentially expressed on each polysaccharide relative to glucose using DESeq [25] (Table S7). Expression of CAZyme genes on polysaccharides relative to glucose (Fig. 2) shows that between 15 (cellobiose) and 40 (stover) CAZymes were significantly up-regulated per treatment (Table S8) with a total of 92 CAZymes up-regulated on at least one polysaccharide. The differentially expressed CAZymes are putatively classified by the CAZy database as 67 glycoside hydrolases, 6 carbohydrate esterases, 4 polysaccharide lyases, 14 glycosyl transferases, and 2 CBM proteins. We analyzed the specificity of the CAZyme transcriptional response by K-means clustering the expression profiles of these genes (Fig. 3, Table S9). Cluster A consists six genes that were highly up-regulated on multiple substrates: the GH26 cphy1071 , the GH11 cphy2105 , two GH18 chitinases cphy1799 and cphy1800 [26], the GH9 cellulase cphy3367 [27] [28] and the GH48 cellulase cphy3368 [29]. Clusters B–F respond to specific polysaccharides such as homogalacturonan (clusters B,C), starch (cluster D), xylan (cluster E), and cellulose/arabinan (cluster F). C. phytofermentans thus perceives signals from individual polysaccharides and responds by up-regulating specific transcriptional regulons that enable it to tailor its complement of CAZymes to the polysaccharide substrate. A set of 56 CAZymes up-regulated on polysaccharides were His-tagged, overexpressed, purified, and their abilities to bind and cleave polysaccharides were quantified. The CAZy database classifies these enzymes putatively as 47 glycoside hydrolases, 4 polysaccharide lyases, and 4 carbohydrate esterases (Table S2); putative glycosyltransferases were not examined as they are not involved in polysaccharide catabolism [6]. Thirty-two enzymes have significant cleavage or binding activities (Fig. 4, Table S11). Some substrates such as b -1,4-galactan appear to be cut by a single, highly active enzyme, while multiple CAZymes from the same family degrade other substrates such as xylan (GH10), mannan (GH26), starch (GH13), and HG (PL9). CAZymes from multiple families together depolymerize substrates such as xyloglucan (GH2,5,12,31), glucomannan (GH5,GH9,GH26) and galactomannan (GH5,GH26). We found 15 CAZymes that bind insoluble polysaccharides, most commonly cellulose and mannan (Fig. 4B). Unexpectedly, no CAZyme bound corn stover, suggesting that partial digestion of raw biomass is needed to facilitate enzyme binding. Nine enzymes that bound substrates have carbohydrate binding modules (CBM), but some enzymes such as the cellulase Cphy1163 can bind their substrate without one. While CBM are known to discriminate between polysaccharides such as cellulose and mannan [30], we observed overlap with cellulase CBMs binding mannan and vice versa. Further, CBM from xylanases can bind cellulose and mannose, but with lower affinity, showing that CBM often bind a range of polysaccharides. Consistent with their cleavage activities, GH13 were the only enzymes to bind starch. Enzymes with CBM usually also have catalytic modules, but Cphy1713, a CAZyme with a CBM32 and no catalytic module, binds galactomannan. CBM32 are known to bind galactose and this protein may function similar to one in Yersinia that is proposed to bind oligosaccharides to prevent them from leaking out of the cell [31]. Thirty-two CAZy families have multiple members, which often have divergent cleavage activities and cellular localizations. Cphy1510 has the highest activity among the four GH10 active on xylan (Fig. 5A). Cphy3010, the GH10 with lowest activity, is the only one lacking a secretion signal, supporting it acts intracellularly on xylo-oligosaccharides while the other GH10 are extracellular. Members of the GH5 family act on a wide range of polysaccharides [32]. C. phytofermentans encodes 3 GH5 enzymes, among which one is active on galactomannan and two on xyloglucan (Fig. 5B). The GH5 Cphy1163 has no activity on either of these substrates, but is the most active on cellulose and glucomannan. The 3 GH26 also vary in substrate specificities (Fig. 5C); all the GH26 are similarly active on b -mannan, but only Cphy1071 has cellulase activity and it has lower activity on gluco- and galactomannan. Sequenced-based families are thus useful to make general substrate predictions for CAZymes, but experiments are needed to determine substrate range and catalytic efficiency. CAZymes mixtures can degrade polysaccharides more efficient- ly than individual enzymes. We assessed pairwise interactions between each CAZyme and a second enzyme on cellulose (Cphy3367), xylan (Cphy2105), glucomannan (Cphy1071), and homogalacturonan (Cphy1687) (Fig. 5D–G). Similar to results showing synergy between the GH9 Cphy3367 and a B. subtilis GH5 [33], we found that a mix of Cphy3367 and the GH5 Cphy1163 has higher activity on cellulose than either enzyme alone (Fig. 5D), supporting they have complementary roles in cellulolysis. CAZymes can also potentiate other enzymes that have no activity by themselves. For example, the xylanase Cphy2105 activates the putative xylosidases Cphy3009 and Cphy3207 on xylan (Fig. 5E). Similarly, Cphy1071 activates the putative mannosidase Cphy1719 on glucomannan (Fig. 5F). Activities of the GH28 Cphy2567 and Cphy3310 are enhanced by the carbohydrate esterase Cphy1687 (Fig. 5G), supporting this enzyme demethylesterifies homogalacturonan to facilitate its degradation. This carbohydrate esterase did not, however, increase cleavage by the PL9 enzymes that were the most active on homogalacturonan. Global correlations between CAZyme mRNA expression and cleavage activities were weak for all polysaccharides (Fig. S12), mostly because many CAZyme genes are up-regulated on substrates upon which they have no activity. CAZymes up- regulated on multiple substrates (Fig. 3, cluster A) may act as ‘carbon scouts’ [34] that degrade complex substrates into inducing molecules used to fine-tune the expression of hydrolytic enzymes. As described above, some CAZymes such as xylosidases (Fig. 5D) are inactive on intact xylan, but are potentiated by other xylanases. The GH18 Cphy1799 and Cphy1800 are the most highly-upregulated CAZymes on cellulose (Fig. S12), but are chitinases with no activity on cellulose or other plant substrates [26]. As such, the set of up-regulated CAZymes is useful to identify active enzymes, but strong up-regulation does not necessarily indicate activity on a given substrate. We assimilated our results into a model of C. phytofermentans polysaccharide catabolism that shows degradation by active CAZymes and uses mRNA expression profiles to predict how these substrates are transported and metabolized (Fig. 6). Unlike other clostridia that transport sugars with numerous phosphotransferase systems (PTS) [35] [36], C. ...