Evidence for Temporal Regulation of the Two Pseudomonas cellulosa Xylanases Belonging to Glycoside Hydrolase Family 11

Abstract

Pseudomonas cellulosa is a highly efficient xylan-degrading bacterium. Genes encoding five xylanases, and several accessory enzymes, which remove the various side chains that decorate the xylan backbone, have been isolated from the pseudomonad and characterized. The xylanase genes consist of xyn10A, xyn10B, xyn10C, xyn10D, and xyn11A, which encode Xyn10A, Xyn10B, Xyn10C, Xyn10D, and Xyn11A, respectively. In this study a sixth xylanase gene, xyn11B, was isolated which encodes a 357-residue modular enzyme, designated Xyn11B, comprising a glycoside hydrolase family 11 catalytic domain appended to a C-terminal X-14 module, a homologue of which binds to xylan. Localization studies showed that the two xylanases with glycoside hydrolase family (GH) 11 catalytic modules, Xyn11A and Xyn11B, are secreted into the culture medium, whereas Xyn10C is membrane bound. xyn10C, xyn10D, xyn11A, and xyn11B were all abundantly expressed when the bacterium was cultured on xylan or ␤-glucan but not on medium containing mannan, whereas glucose repressed transcription of these genes. Although all of the xylanase genes were induced by the same polysaccharides, temporal regulation of xyn11A and xyn11B was apparent on xylan-containing media. Transcription of xyn11A occurred earlier than transcription of xyn11B, which is consistent with the predicted mode of action of the encoded enzymes. Xyn11A, but not Xyn11B, exhibits xylan esterase activity, and the removal of acetate side chains is required for xylanases to hydrolyze the xylan backbone. A transposon mutant of P. cellulosa in which xyn11A and xyn11B were inactive displayed greatly reduced extracellular but normal cell-associated xylanase activity, and its growth rate on medium containing xylan was indistinguishable from wild-type P. cellulosa. Based on the data presented here, we propose a model for xylan degradation by P. cellulosa in which the GH11 enzymes convert decorated xylans into substituted xylooligosaccharides, which are then hydrolyzed to their constituent sugars by the combined action of cell-associated GH10 xylanases and side chain-cleaving enzymes.

Insights into Plant Cell Wall Degradation from the Genome Sequence of the Soil Bacterium Cellvibrio japonicus

“…These small polymers are then hydrolyzed by enzymes on the bacterial cell surface that target small saccharide polymers (29,35) to generate mono-and disaccharides. This model of temporal and spatial regulation has been shown for enzymes that catalyze mannan and xylan degradation in C. japonicus (17) and for cellulose degradation in S. degradans (38).…”

“…The signal peptides on the CBM-containing enzymes are generally cleaved by type I signal peptidases and are likely secreted into the extracellular milieu by a type II secretion system (CJA_3333-CJA_3323), similar to the secretion of pectate lyases by the plant pathogen Erwinia chrysanthemi (27). While it is possible that some of these proteins may interact with the cell membrane through hydrophobic and/or ionic interactions, where analyzed in vivo, the majority of the CBM-containing enzymes appear to be present in the culture medium and thus do not associate with the outer envelope of C. japonicus (7,17,22,23,26). Interestingly, ϳ33% of the exported C. japonicus plant cell wall-degrading enzymes contain signal peptides that are cleaved by type II signal peptidases and are predicted to be lipoproteins.…”

“…The catalytic modules of the C. japonicus glycoside hydrolases belong to 31 different families, with the largest families being GH5, GH13, and GH43 (15,17, and 14 enzymes, respectively) (see Table S2 in the supplemental material). GH5 contains an extensive repertoire of endo-acting enzymes that hydrolyze plant structural polysaccharides, which is consistent with the observation that C. japonicus can target these polymers as major nutrients (7,22,23,26).…”

The Cellvibrio japonicus Mannanase CjMan26C Displays a Unique exo-Mode of Action That Is Conferred by Subtle Changes to the Distal Region of the Active Site

“…Indeed, this hierarchical pattern of plant cell wall degradation is a common theme in this organism. Thus, xylan, the other major hemicellulosic polysaccharide, is initially degraded by enzymes containing cellulose-targeted CBMs, and the processing of the resultant xylooligosaccharides into their constituent sugars occurs on the surface or in the periplasm of the bacterium (45,46). Indeed, this pattern of polymer degradation parallels the human intestine, where endo-acting enzymes generate oligopeptides and oligosaccharides that are further hydrolyzed on the surface of the epithelium into monomeric species.…”

The Use of Forced Protein Evolution to Investigate and Improve Stability of Family 10 Xylanases

“…An elegant example of this variation in stability is found in the glycoside hydrolase family (GH) 1 10 xylanases expressed by the mesophilic bacteria Cellvibrio japonicus and Cellvibrio mixtus. C. japonicus expresses three GH10 xylanases; two are secreted into the extracellular environment (CjXyn10A and CjXyn10C), whereas CjXyn10D is translocated into the periplasm (2). C. mixtus also produces a periplasmic GH10 xylanase, CmXyn10B, which displays 90% sequence identity with CjXyn10D (1).…”