Elsevier

Gene

Volume 506, Issue 2, 15 September 2012, Pages 274-282
Gene

Expressional and functional variation of horizontally acquired cellulases in the nematode Pristionchus pacificus

https://doi.org/10.1016/j.gene.2012.07.013Get rights and content

Abstract

Various whole genome-sequencing projects in the nematode phylum have revealed the widespread occurrence of horizontal gene transfer from different sources. Pristionchus pacificus was the first non-plant parasitic nematode that was found to contain cellulase genes in its genome and to have cellulolytic activity when grown in a monoxenic culture on Escherichia coli. The P. pacificus reference strain PS312 has seven cellulase genes, all of which were acquired by horizontal gene transfer. Previous phylogenomic studies indicated that the acquisition occurred at the base of the genus Pristionchus and was followed by rapid gene duplications and gene turnover. However, little was known about the protein domain architecture, gene expression and the functionality of individual proteins. Here, we analyzed the protein domain architecture, studied the expression at various developmentally stages and tried to induce cellulase gene expression by feeding nematodes with different polysaccharides. Only two of the encoded proteins, Ppa-CEL-2 and Ppa-CEL-3, have a carbohydrate-binding module. Interestingly, these were also the ones with developmental gene expression regulation. Ppa-cel-2 shows high expression in larval and adult stages but is only low expressed in eggs, while Ppa-cel-3 is highly upregulated in adult worms but hardly detectable in any other stage. Ppa-CEL-1, has a catalytic domain similar to Ppa-CEL-2 and Ppa-CEL-3, but lacks a carbohydrate-binding module. The other four cellulases have a very low transcriptional expression correlating with putative incompleteness of their catalytic domain. While, the expression of none of the genes is inducible by polysaccharides, zymographic studies and mass spectrometry indicate that Ppa-CEL-2 and Ppa-CEL-3 are the only two cellulases contributing to cellulase activity in carboxymethylcellulose. Thus, the cellulases of P. pacificus differ in their protein domain architecture, gene expression and functionality. These results indicate that horizontal gene transfer-acquired genes undergo rapid evolutionary changes that affect all aspects of their molecular biology.

Highlights

► Horizontally acquired cellulases in the nematode P. pacificus undergo rapid changes. ► Changes result in distinct protein architecture and different expression. ► Two of the seven genes are developmentally regulated. ► The enzymes encoded by these two genes show enzymatic activity.

Introduction

The phylum nematoda (roundworms) is one of the most diverse groups of animals, whose members occupy almost every ecological niche on earth (Hugot et al., 2001). Nematodes are found as free-living species in marine, fresh water and soil ecosystems and as parasites or pathogens of plants and all groups of animals. With this variety of lifestyles different kinds of adaptations to the environment are indispensable. Our understanding of the biology of nematodes was recently advanced by the beginning of whole genome sequencing projects in a number of nematode taxa. One surprising and unexpected finding was the occurrence of horizontal gene transfer (HGT) in various free-living, as well as animal and plant parasitic nematode groups (for review see Sommer and Streit, 2011).

In some cases, horizontally acquired genes represent a major adaptation for the nematode life style. Several nematodes are able to break down cellulose and other sugar polymers making use of secreted carbohydrate-active enzymes (CaZymes), a large group of enzymes that all possess specificity to different kinds of polysaccharides (Cantarel et al., 2009). In plant parasitic nematodes these enzymes are used to penetrate plant cell walls during infestation of the host plant (Curtis, 2007). During this process, cellulases (EC 3.2.1.4), xylanases (EC 3.2.1.8), pectate lyases (EC 4.2.2.2) and other cell wall-modifying enzymes are secreted by the worms through their esophagal glands (Curtis, 2007, Hussey et al., 2002, Smant et al., 1997). Genes encoding for these enzymes have been acquired multiple times independently in nematodes by HGT from different microorganisms (Danchin et al., 2010, Jones et al., 2005, Mayer et al., 2011). While e.g. the cellulases from the pine wood nematode Bursaphelenchus xylophilus belong to the glycoside hydrolase family 45 (GHF45), the root-knot nematode Meloidogyne incognita and the cyst nematode Heterodera glycines express cellulases of GHF5 (Kikuchi et al., 2004, Rosso et al., 1999, Smant et al., 1998). These plant parasitic nematodes received their set of CaZymes mostly from bacterial and fungal donors (Danchin et al., 2010, Jones et al., 2005).

Surprisingly, the non-plant parasitic diplogastrid nematode P. pacificus was also found to possess cellulase genes (Dieterich et al., 2008). P. pacificus is a cosmopolitan nematode living in a close necromenic relationship with scarab beetles (Herrmann et al., 2007, Herrmann et al., 2010). The worms infest their beetle host as dauer larva and only resume development once the beetle has died to feed on the microorganisms growing on the beetle carcass (Weller et al., 2010). P. pacificus has been developed as a model system in evolutionary biology with special emphasis on evo–devo, ecology and population genetics (Sommer, 2009, Sommer et al., 1996). This species has fully developed genetic and genomic tools, including forward and reverse genetics and DNA-mediated transformation (Hong and Sommer, 2006, Schlager et al., 2009). P. pacificus was one of the first nematodes to have its genome fully sequenced after Caenorhabditis elegans, with transcriptome and proteome data being available (Borchert et al., 2010, Dieterich et al., 2008).

The P. pacificus cellulases belong to GHF5 and are unrelated to the ones found in any of the plant parasitic nematodes, suggesting a recent and independent HGT-event with slime molds as potential donors (Mayer et al., 2011). A phylogenetic transcriptomic study by 454 sequencing indicated that all seven tested Pristionchus species contain cellulases in their genome that are strictly associated with cellulase activity (Mayer et al., 2011). This study also showed that the original acquisition of cellulase genes in Pristionchus was followed by multiple gene duplications in association with high gene turnover (Mayer et al., 2011). Together, these results clearly supported the integration of cellulases into the biology of Pristionchus nematodes, which is a basic criterion for successful HGT (Blaxter, 2007, Mayer et al., 2011). However, questions about the functionality and the ecological role of the individual cellulase genes in non-plant parasitic nematodes, specifically in Pristionchus have remained unanswered so far (Scholl and Bird, 2011).

Here, we present a first detailed study on the functionality of all seven predicted P. pacificus cellulases in order to find out which of them contribute to enzymatic activity in vivo and to hypothesize their role in the necromenic lifestyle of P. pacificus. We describe the gene structure and protein domain architecture for all genes and analyze their expression profile at six distinct developmental time points. We test, if cellulase gene expression can be induced by carbohydrate application on the culture plate and we identify the active cellulase proteins from semi-native gels by mass spectrometry. All these experiments indicate that only two cellulase genes of P. pacificus are expressed and developmentally regulated and show strong association with enzymatic activity.

Section snippets

Nematodes fed with different substrates

NGM-plates were prepared 2 days before usage by mixing a 1% (w/v) solution (or suspension respectively) of the carbohydrate substrates with an OP50-culture (LB-Medium) leading to a 0.5% suspension/solution to feed the worms. 20 μl of these solutions were spotted on each assay plate. All experiments were performed with the P. pacificus wild-type strain PS312 originally isolated from Pasadena, California (Sommer et al., 1996). P. pacificus worms at mostly young J4 were distributed homogeneously

Domain architecture of P. pacificus cellulase enzymes

P. pacificus cellulases belong to the glycoside hydrolase family 5 (GHF5). Phylogenetic sequence comparison of the conserved carbohydrate binding regions obtained from 454 transcriptomic data had indicated that these domains are more closely related to each other than to the ones of its sister species Pristionchus sp. 11 (Mayer et al., 2011), (Fig. 1A). Unfortunately, the 454 sequencing data of all seven sequenced Pristionchus species did not generate complete sequence data for the catalytic

Discussion

We have performed a structure-function analysis of the seven cellulases of P. pacificus in order to increase our understanding of the evolutionary patterns associated with HGT-acquired genes. The P. pacificus cellulases are the result of one HGT event that is independent of the acquisition of cellulases found in other nematodes. HGT was followed by gene duplications, rapid gene turnover and sequence diversification (Dieterich and Sommer, 2009, Dieterich et al., 2008, Mayer et al., 2011). In

Acknowledgments

We want to thank Dr. Andrew Spiers for the wrinkly spreader strains used in this study and the group of Prof. Boris Macek from the Proteome Center at the University of Tubingen for help with the mass spectrometry. We thank Drs. Etienne Danchin, Adrian Streit, Gabriel Markov and members of the Sommer lab for continuous discussions and Dr. Christian Rödelsperger and Amit Sinha for help with data analysis. This work was funded by the Max Planck Society.

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