BioProject

As part of the Canadian MetaMicroBiome Library Open Resource, metagenomic DNA was isolated from representative Canadian soil samples and cloned into pJC8, which is a broad-host-range Gateway entry cosmid that replicates in diverse Proteobacteria. More...

As part of the Canadian MetaMicroBiome Library Open Resource, metagenomic DNA was isolated from representative Canadian soil samples and cloned into pJC8, which is a broad-host-range Gateway entry cosmid that replicates in diverse Proteobacteria. This publicly accessible collection of 13 metagenomic libraries, ranging from 4 ×104 to 9 × 106 clones, was generated for a functional screening comparison of multiple bacterial hosts. Selection of clones from one of the libraries, constructed from corn field soil DNA, was carried out for β-galactosidase activity in both Sinorhizobium meliloti (α-Proteobacteria) and Escherichia coli (γ-Proteobacteria) backgrounds. The results suggested that S. meliloti was superior to E. coli as a surrogate host, yielding a greater number of distinct clones following selection. One β-galactosidase, encoded by six overlapping Lac+ clones that were selected in both hosts, was identified as a member of glycoside hydrolase family 2. We were not able to identify ORFs encoding possible β-galactosidases in 19 other sequenced Lac+ clones that were only able to complement S. meliloti. Based on very low sequence similarity to known glycoside hydrolases, three ORFs were chosen for further examination. We subcloned Lac36W_ORF11, Lac161_ORF7 and Lac161_ORF10 from cosmids Lac36W and Lac161, and then expressed and purified the corresponding proteins. Biochemical analysis confirmed that all proteins had β-galactosidase activity. Lac36W_ORF11 and Lac161_ORF7 had conserved domains, but there were no similarities to known glycoside hydrolases. This suggested that these two new β-galactosidases might have other functions. Lac161_ORF10 had neither conserved domains nor similarity to known and described glycoside hydrolases. Bioinformatic and structural modeling implied that the ORF10 protein represented a novel enzyme family with a five-bladed propeller glycoside hydrolase domain. These data suggested that all three β-galactosidases isolated by complementation of S. meliloti were members of novel glycoside hydrolase families. Taxonomic analysis implied that the cloned metagenomic DNA in the cosmids likely originated from Xanthomonas (Lac36W) and Verrucomicrobia (Lac161). By discovering founding members of three novel β-galactosidase enzyme families, this work reinforces the value of functional metagenomics for isolating novel genes that would not be predicted from DNA sequence analysis alone. Less...