Mining of unexplored habitats for novel chitinases—chiA as a helper gene proxy in metagenomics

Mariana Silvia Cretoiu,Anna Maria Kielak,Jan Dirk Van Elsas,Waleed Abu Al-Soud,Søren J Sørensen

doi:10.1007/s00253-012-4057-5

Mariana Silvia Cretoiu, Anna Maria Kielak + Show 3 more

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https://doi.org/10.1007/s00253-012-4057-5

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Abstract

The main objective of this study was to assess the abundance and diversity of chitin-degrading microbial communities in ten terrestrial and aquatic habitats in order to provide guidance to the subsequent exploration of such environments for novel chitinolytic enzymes. A combined protocol which encompassed (1) classical overall enzymatic assays, (2) chiA gene abundance measurement by qPCR, (3) chiA gene pyrosequencing, and (4) chiA gene-based PCR-DGGE was used. The chiA gene pyrosequencing is unprecedented, as it is the first massive parallel sequencing of this gene. The data obtained showed the existence across habitats of core bacterial communities responsible for chitin assimilation irrespective of ecosystem origin. Conversely, there were habitat-specific differences. In addition, a suite of sequences were obtained that are as yet unregistered in the chitinase database. In terms of chiA gene abundance and diversity, typical low-abundance/diversity versus high-abundance/diversity habitats was distinguished. From the combined data, we selected chitin-amended agricultural soil, the rhizosphere of the Arctic plant Oxyria digyna and the freshwater sponge Ephydatia fluviatilis as the most promising habitats for subsequent bioexploration. Thus, the screening strategy used is proposed as a guide for further metagenomics-based exploration of the selected habitats.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-012-4057-5) contains supplementary material, which is available to authorized users.

Highlights

Being one of the most abundant biopolymers in nature, chitin has been studied in many domains of science, from chemistry and biomedicine to synthetic material development (Agullo et al 2003; Muzzarelli 2009)
The aim of the study, i.e., the assessment of defined aspects of chitinolysis across habitats, was based on the premise that a better understanding of the ecology of chitinolysis will serve our subsequent exploration of habitats on the basis of educated guesses as to the occurrence of chitinases with unexplored features
We applied parallel function- and gene-based methodologies to evaluate the habitat specificity of family-18 chitinase genes using a combination of proxies for chiAspecific abundances and diversities compared with total bacterial ones

Summary

Introduction

Being one of the most abundant biopolymers in nature, chitin (polymer consisting of β-1,4-linked-N-acetyl-glucosamine) has been studied in many domains of science, from chemistry and biomedicine to synthetic material development (Agullo et al 2003; Muzzarelli 2009). Chitindegrading enzymes (chitinases) are present in many natural systems and they are widely distributed among all three domains of life (Li and Greene 2010; Gao et al 2003). They are responsible for hydrolysing the glycosidic bonds of chitin, releasing dimeric (chitobiose) and monomeric (N-acetylglucosamine, GlcNAc) compounds. Most relevant chitinases belong to the glycoside hydrolase families 18 and 19. The classification into these two groups is based on amino acid sequence similarities (Henrissat 1991; Henrissat and Davies 2000), substrate specificities and structures of the catalytic domains of the respective enzymes. Based on the structure of the catalytic domain and on the position of the hydrolysis site on the polysaccharide chain, chitinases may show either endo- or exoactivity (Henrissat and Davies 2000; Van Scheltinga et al 1994)

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