The complex physiology of Cellvibrio japonicus xylan degradation relies on a single cytoplasmic β‐xylosidase for xylo‐oligosaccharide utilization

Abstract

Lignocellulose degradation by microbes plays a central role in global carbon cycling, human gut metabolism and renewable energy technologies. While considerable effort has been put into understanding the biochemical aspects of lignocellulose degradation, much less work has been done to understand how these enzymes work in an in vivo context. Here, we report a systems level study of xylan degradation in the saprophytic bacterium Cellvibrio japonicus. Transcriptome analysis indicated seven genes that encode carbohydrate active enzymes were up-regulated during growth with xylan containing media. In-frame deletion analysis of these genes found that only gly43F is critical for utilization of xylo-oligosaccharides, xylan, and arabinoxylan. Heterologous expression of gly43F was sufficient for the utilization of xylo-oligosaccharides in Escherichia coli. Additional analysis found that the xyn11A, xyn11B, abf43L, abf43K, and abf51A gene products were critical for utilization of arabinoxylan. Furthermore, a predicted transporter (CJA_1315) was required for effective utilization of xylan substrates, and we propose this unannotated gene be called xntA (xylan transporter A). Our major findings are (i) C. japonicus employs both secreted and surface associated enzymes for xylan degradation, which differs from the strategy used for cellulose degradation, and (ii) a single cytoplasmic β-xylosidase is essential for the utilization of xylo-oligosaccharides.

Systems analysis of the glycoside hydrolase family 18 enzymes from Cellvibrio japonicus characterizes essential chitin degradation functions

“…S2). These results reinforce two features previously described for C. japonicus, specifically, that secreted enzymes are essential for recalcitrant polysaccharide degradation and that single CAZymes elicit major physiological effects (27,(45)(46)(47) (Fig. 3 and Fig.…”

“…In regards to the substratespecific response, C. japonicus seems to have two variations, one that is general (where diverse CAZyme genes are up-regulated), and one that is specific (where only substratespecific CAZymes genes are up-regulated). The response observed for cellulose is an example of the former (14), and the response for xylan is the latter (46). The regulatory network for the chitinolytic response appears to be analogous to the cellulose response, which is not surprising given the similarities of the substrates.…”

Two forms of phosphomannomutase in gammaproteobacteria: The overlooked membrane-bound form of AlgC is required for twitching motility ofLysobacter enzymogenes

“…aeruginosa strains PAO1 and PA14 (Supporting Information Figure S5), although in the former several SRA profiles showed expression of only the cytoplasmic fragment. Expression of “long” PMM genes was also detected in other Pseudomonas spp., as well as in representatives of other gammaproteobacterial orders, such as Cellvibrio japonicus (Blake et al ., 2018) and Methylobacter tundripaludum (Krause et al ., 2017). Finally, unpublished RNA-Seq data from the DOE Joint Genome Institute revealed robust expression of the full-length PMM gene of Methylococcus capsulatus , which is currently listed in GenBank in truncated form (Supporting Information Table S4).…”

High-throughput screening of environmental polysaccharide-degrading bacteria using biomass containment and complex insoluble substrates

“…The strategies used by microbes for efficient bioconversion of recalcitrant polysaccharides have gained interest for ecology and animal health studies along with biotechnological applications. In vivo studies are a useful tool to understand the degradation processes because they characterize physiologically relevant functions (Blake et al 2018;Gardner and Keating 2010;Monge et al 2018). However, it is difficult to study bacterial growth or enzyme activity using physiologically relevant substrates because of the insolubility of the substrates.…”

“…In order to evaluate if secreted enzymes are essential for polysaccharide degradation of a particular substrate, we previously generated a C. japonicus deletion mutant that no longer has a type II secretion system, as encoded by the general secretory pathway (gsp) operon (Nelson and Gardner 2015). This mutant was able to determine if secreted CAZymes were essential for the utilization of soluble polysaccharides (e.g., required for CMC but dispensable for xylan) (Blake et al 2018;Gardner and Keating 2010). The current challenge studying this biological process in C. japonicus or another saprophytic bacterium is that to obtain ecologically or industrially relevant data complex insoluble substrates need to be used.…”