encodes a number of carbohydrate active enzymes (CAZymes) allowing for efficient degradation of cellulose and associated polysaccharides (Carbohydrate Active Enzyme database;
). These include (i) endo-β-glucanases, which cleave internal amorphous regions of the cellulose chain into shorter soluble oligosaccharides, (ii) exo-β-glucanases (cellodextrinases and cellobiohydrolases), which act in a possessive manner on reducing or nonreducing ends of the cellulose chain liberating shorter cellodextrins, and (iii) β-glucosidases (cellodextrin and cellobiose phosphorylases), which hydrolyze soluble cellodextrins ultimately into glucose
]. Other glycosidases that allow hydrolysis of lignocellulose include xylanases, lichenases, laminarinases, β-xylosidases, β-galactosidases, and β-mannosidases, while pectin processing is accomplished via pectin lyase, polygalacturonate hydrolase, and pectin methylesterase
]. These glycosidases may be secreted as free enzymes or may be assembled together into large, cell-surface anchored protein complexes (“cellulosomes”) allowing for the synergistic breakdown of cellulosic material. The cellulosome consists of a scaffoldin protein (CipA) which contains (i) a cellulose binding motifs (CBM) allowing for the binding of the scaffoldin to the cellulose fiber, (ii) nine type I cohesion domains with that mediate binding of various glycosyl hydrolases via their type I dockerin domains, and (iii) a type II dockerin domain which mediates binding to the type II cohesion domain found on the cell-surface anchoring proteins. The cell-surface anchoring proteins are in turn noncovalently bound to the peptidoglycan cell wall via C-terminal surface-layer homology (SLH) repeats
During growth on cellulose, the cellulosome is attached to the cell in early exponential phase, released during late exponential phase, and is found attached to cellulose during stationary phase
]. Cellulosome expression has been shown to be negatively regulated by cellobiose via a carbon catabolite repression mechanism
], and positively regulated through binding of cellulose and associated polysaccharides to anti-σ factors, allowing cellulosome expression using alternative σ factors
], suggesting that the cellulosome should not be expressed in cellobiose-grown cultures. The ability of C. thermocellum
to control scaffoldin and cellulase mRNA
] and protein
] levels in response to substrate type and growth rate has been extensively studied, and reveals that expression of cellulosomal enzymes is present in the absence of cellulose, albeit at lower levels. We detected expression of 7 cellulosomal structural proteins, 31 cellulosome-associated glycosidases, and 19 non-cellulosomal CAZymes on cellobiose using 2D-HPLC-MS/MS (
Additional file 3
Of the 8 encoded non-catalytic cellulosomal proteins, 7 were detected using the combined acquisition methods (shotgun and 4-plex). SdbA (Cthe_1307) was the most abundant anchoring protein, and scaffoldin CipA (Cthe_3077) was found in the top 50% of total proteins detected (RAI
0.42). OlpB, Orf2p, and OlpA located downstream of CipA (Cthe_3078-3080) were also detected, but at sequentially lower levels. Expression of cellulosomal anchoring proteins Cthe_0452 and Cthe0736 was also detected, but only during 4-plex acquisition. Microarray studies revealed that transcription of sdbA
was low compared to cipA, olpB, orf2p
, and olpA
], while nano-LC-ESI-MS revealed that SdbA was only expressed in cellobiose-grown cultures
]. This coincided with our high SdbA levels detected in cellobiose-grown cell-free extracts. On cellulose, Raman et al.
found no change in cipA
transcription and a 2-fold increase in orf2p
transcription in stationary phase
], while Dror et al.
observed an increase in transcription of orf2p
as well as cipA
with decreasing growth rate
]. Alternatively, Gold et al.
showed similar expression of Orf2p relative to CipA in both cellobiose and cellulose-grown samples and increased expression of OlpB in cellobiose-grown cultures
]. We, however, did not observe any statistically relevant changes of cellulosomal proteins on cellobiose during transition into stationary phase.
encodes 73 glycosidases containing a type I dockerin, 65 of which have been detected and characterized at the protein level
]. 2D-HPLC-MS/MS of exponential phase cell-free extracts detected 31 cellulosomal glycosidases (
Additional file 3
), 19 of which were in the top 90th
percentile of total proteins detected (RAI
0.1). In addition to high RAI levels of CelS, a cellulosomal subunit shown to be highly expressed
], XynC, CelA, XynA/U, CelG, and glycosidase Cthe_0821 were also detected in high amounts. Other characterized cellulosomal glycosidases detected included CelB, XynZ, XghA, CelR, CelK, and CelV. Proteomic analysis has shown that exoglucanases CelS and CelK, and endoglucanase CelJ are higher in cellulose versus cellobiose-grown cultures, while hemicellulases (XynZ, XynC, XynA/U, XghA, Cthe_0032) and endoglucanases belonging to family GH5 (CelB, CelG, Cthe_2193) and GH8 (CelA) were more abundant in cellobiose versus cellulose-grown cultures
]. This agrees with our relative protein abundance profiles exhibiting high xylanase, GH5 family glycosidase, and CelA expression, and lower CelK and CelJ expression in exponential cellobiose-grown cell-free extracts. Interestingly, despite the presence xylanases, sequence homology-based annotation has not revealed the presence of xylose reductase, xylitol dehydrogenase, xylose isomerase, or xylulokinase required for xylose utilization. This suggests that, in the absence of cellulose, C. thermocellum
may be predisposed to expressing xylanases, which typically degrade hemicellulosomal xylans, exposing buried cellulose fibres.
With the exception of a 2-fold increase in cellulosomal glycosidases Cthe_0821, Cthe_2761, and Cthe_0745, and a 1.6-fold decrease in XynD (Cthe_0625), no other statistically significant changes were observed in detected cellulosomal cellulases during transition from exponential to stationary phase. While this contradicted high variability in transcription of cellulosomal glycosidases of cellulose-grown cells
], lack of variability in our experiment may have been attributed to differences in growth substrate used. In fact, Dror et al.
found negligible changes in transcription of celB
, and celF
between exponential and stationary phase cellobiose-grown cultures
]. Alternatively, our processing method, which included several wash steps prior to lysing the cells, may have imposed bias and variability by potentially washing off weakly bound cellulosomal glycosidases.
In addition to cellulosomal glycosidases, 35 non-cellulosomal CAZymes that do not have a dockerin domain are encoded in the genome. Of the 19 non-cellulosomal CAZymes detected in exponential phase cell-free extracts using 2D-HPLC-MS/MS, half had RAI ratios in the top 90% (RAI
0.1) of total peptides detected. Not surprisingly, the most abundant CAZyme cellobiose phosphorylase Cthe_0275 (glycosyltransferase family 36), which is involved in intracellular phosphorylytic cleavage of cellobiose, fell within the top 25% of detected proteins. Cellobiose phosphorylase Cthe_2989 was also found in high amounts (RAI
0.23), whereas glycosyltransferase Cthe_1221, a putative cyclic β-1,2 glucan synthetase, was detected in the bottom 10% of all proteins detected (Figure
). CelI, an endo-1,4-β-glucanase (Cthe_0040) was not detected, consistent with growth on cellobiose. Other highly abundant non-cellulosomal CAZymes include amidohydrolase (Cthe_1777), glucoamylase (Cthe_1787), xylanase A precursor (Cthe_1911), α-N-arabinofuranosidase (Cthe_2548), CelC (Cthe_2807), and several less characterized glycosidases (Cthe_3163, Cthe_1911, Cthe_2989). While Raman et al.
report decreased transcription of glycosyltransferases involved in intracellular phosphorolytic cleavage of cellodextrin and cellobiose (family 36), and increased transcription of a number of other CAZymes in response to decreased substrate availability in stationary phase
], we saw no statistically significant changes in CAZyme expression with two exceptions: LicA (Cthe_2809) increased in stationary phase, consistent with reports by Newcomb and Wu
] and Raman et al.
], and acetyl xylan esterase (Cthe_3063) also increased contradicting previously reported microarray data
]. CelC expression (Cthe_2807), which is negatively regulated by the co-transcribed LacI family transcriptional regulator GlyR3 (Cthe_2808), has consistently been shown to increase in the presence of laminaribiose
] and in stationary phase on cellulose
] and cellobiose
]. While CelC expression was shown to have an overall increase in stationary phase among biological replicates, deviation between replicates makes it difficult to tell if this is simply an articat. Finally, of the 7 membrane-associated RsgI-like anti-σI
factors proposed to activate expression of different glycosidases in the presence of cellulose and other polysaccharides, three have been detected (Cthe_0059, Cthe_0267, and Cthe_2521). The binding of a particular polysaccharide to corresponding anti-σI
factor N-terminal carbohydrate binding domains is proposed to promote the C-terminal release of putative alternative σI
-factors (SigI) encoded upstream of these anti-σI
factors, allowing for expression of select glycosidases, some of which (ex. CelA) are encoded downstream of the anti-σI
factors that regulate their expression
Figure 2 Relative abundance indexes and changes in protein expression levels of protein involved in glycolysis, glycogen metabolism, and pentose phosphate pathway. Relative abundance indexes (values 1 and 2), changes in protein expression ratios (value 3), and (more ...)