Bacterial chitinase with phytopathogen control capacity from suppressive soil revealed by functional metagenomics

Karin Hjort 0 2 Ilaria Presti 0 2 Annelie Elvng 0 2 Flavia Marinelli 0 2 Sara Sjling 0 2 0 F. Marinelli The Protein Factory Research Center , Politecnico of Milano, ICRM CNR and University of Insubria , Varese 21100, Italy 1 ) School of Natural Sciences and Environmental Studies, Sdertrn University , 141 89 Huddinge, Sweden 2 K. Hjort Department of Medical Biochemistry and Microbiology, Uppsala University , Uppsala, Sweden Plant disease caused by fungal pathogens results in vast crop damage globally. Microbial communities of soil that is suppressive to fungal crop disease provide a source for the identification of novel enzymes functioning as bioshields against plant pathogens. In this study, we targeted chitindegrading enzymes of the uncultured bacterial community through a functional metagenomics approach, using a fosmid library of a suppressive soil metagenome. We identified a novel bacterial chitinase, Chi18H8, with antifungal activity against several important crop pathogens. Sequence analyses show that the chi18H8 gene encodes a 425-amino acid protein of 46 kDa with an N-terminal signal peptide, a catalytic domain with the conserved active site F175DGIDIDWE183, and a chitinase insertion domain. Chi18H8 was expressed (pGEX-6P-3 vector) in Escherichia coli and purified. Enzyme characterization shows that Chi18H8 has a prevalent chitobiosidase activity with a maximum activity at 35 C at pH lower than 6, suggesting a role as exochitinase on native chitin. To our knowledge, Chi18H8 is the first chitinase isolated from a metagenome library obtained in pure form and which has the potential to be used as a candidate agent for controlling fungal crop diseases. Furthermore, Chi18H8 may also answer to the demand for novel chitin-degrading enzymes for a broad range of other industrial processes and medical purposes. - Disease of plants caused by fungal pathogens contributes to extensive loss of crops important for food and energy production globally. Moreover, the norm of monoculture practice, further increases opportunities for the invasion of phytopathogens (Herrera-Estrella and Chet 1999). As a consequence, the use of synthetic fungicides, many which are toxic, is extensive and evidently results in costs for public and ecosystem health (Mullin et al. 2010). A more environmentally sustainable approach to the use of toxic chemicals is microbiological control of fungal disease employing bacteria that exhibit antifungal action (Herrera-Estrella and Chet 1999). There are soils that are naturally suppressive toward plant diseases and microorganisms in these soils are proposed to be involved in the suppressiveness (Borneman and Becker 2007; Steinberg et al. 2007). As a result, several bacterial species have been isolated and commercially introduced for biocontrol purposes (Steinberg et al. 2007 and references therein). However, given the inherent limitations in the use of living organisms, such as relatively short shelf life of the products or inconsistent performance in the field, their application is confined (Neeraja et al. 2010). Alternative solutions are formulations with enzymes possessing antiphytopathogenic activity. Promising candidates for this purpose are bacterial chitinases, as these degrade chitin, one of the main constituents of the fungal cell wall (Herrera-Estrella and Chet 1999; Zhang et al. 2001). Chitinolytic enzymes are also generally attractive for their potential use in a broad range of biotechnological applications, for example in biofuel production or bioconversion processes on shellfish waste to obtain valueadded products, such as chitosan and chitooligosaccharides for the pharmaceutical market (Bhattacharya et al. 2007; Li and Greene 2010). In an ecological perspective, bacterial chitinases are crucial in the global biogeochemical re-cycling of carbon and nitrogen through the hydrolyzation of chitin. After cellulose, chitin is the most abundant biopolymer in nature, widely distributed within exoskeletons invertebrates, fungal cell walls, marine diatoms, crustaceans, and zooplankton (Gooday 1990). It is otherwise rather resistant to degradation and would without bacterial chitinases be trapped in biomass as insoluble in nature (for reviews, see Karlsson and Stenlid 2009; Keyhani and Roseman 1999). Degradation of chitin enables bacterial utilization of the end products, chitobiose, and N -acetylglucosamine compounds, as an energy-, carbon-, and/or nitrogen source (Gooday 1990). Most of the bacterial chitinases are glycosyl hydrolases of family 18 (Henrissat and Bairoch 1993), which can be further classified into subfamilies A to C based mainly on amino acid sequence similarities of the catalytic domain and a conserved consensus motif of the catalytic site (Henrissat and Bairoch 1993; Karlsson and Stenlid 2009; Suzuki et al. 2002). Depending on the catalytic specificity as a result of enzyme structure, chitinases may show either endo- or exo-activity (Henrissat and Davies 2000). To date, bacterial chitinase genes have been identified, by conventional molecular screening approaches, in bacterial isolates or uncultured bacteria within both aquatic and soil environments (for example, Hobel et al. 2005; Ikeda et al. 2007; LeCleir et al. 2004; Metcalfe et al. 2002; Ramaiah et al. 2000; Terahara et al. 2009; Uchiyama and Watanabe 2006). Only a few studies, however, have used metagenomics tools to identify novel bacterial chitinase genes (Cottrell et al. 1999; LeCleir et al. 2007). The advantage of metagenome-based approaches is the complete access to the entire community genetic makeup without the need of microbial cultivation (for review, see Sjling et al. 2007). To our knowledge, none of these studies has yet resulted in the isolation and characterization of novel biologically active chitinases. As chitin degradation is such an important environmental function, we investigated, in a previous study, the effect of chitin amendment to a suppressive field soil on the bacterial community structure (Hjort et al. 2007). We could show using terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis analysesthat chitin amendment to the soil dramatically increased the relative abundances of known chitin-degrading genera, such as Oerskovia , Kitasatospora , and Streptomyces . These organisms became dominant also among the actively growing bacteria in the community (Hjort et al. 2007). Given that the soil bacterial community of the suppressive field responded to chitin amendment (Hjort et al. 2007), the active community contained taxa that typically are chitinolytic (Hjort et al. 2007) and that a number of isolates with antifungal and chitinase activity previously were obtained from this soil (Adesina et al. 2007), we sought to investigate this soil metagenome for novel chitinolytic enzymes with biocontrol capacity, suitable for more environmentally sustainable applications in agricultural processes. In this study, we used a ta (...truncated)