Project description:The viral metagenome within an activated sludge microbial assemblage was sampled using culture-dependent and culture-independent methods and compared to the diversity of activated sludge bacterial taxa. A total of 70 unique cultured bacterial isolates, 24 cultured bacteriophages, 829 bacterial metagenomic clones of 16S rRNA genes, and 1,161 viral metagenomic clones were subjected to a phylogenetic analysis.
Project description:A metagenomic library was prepared using pCC2FOS vector containing about 3.0 Gbp of community DNA from the microbial assemblage of activated sludge. Screening of a part of the un-amplified library resulted in the finding of 1 unique lipolytic clone capable of hydrolyzing tributyrin, in which an esterase gene was identified. This esterase/lipase gene consists of 834 bp and encodes a polypeptide (designated EstAS) of 277 amino acid residuals with a molecular mass of 31 kDa. Sequence analysis indicated that it showed 33% and 31% amino acid identity to esterase/lipase from Gemmata obscuriglobus UQM 2246 (ZP_02733109) and Yarrowia lipolytica CLIB122 (XP_504639), respectively; and several conserved regions were identified, including the putative active site, HSMGG, a catalytic triad (Ser92, His125 and Asp216) and a LHYFRG conserved motif. The EstAS was overexpressed, purified and shown to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (< or = C8). This EstAS had optimal temperature and pH at 35 degrees C and 9.0, respectively, by hydrolysis of p-NP hexanoate. It also exhibited the same level of stability over wide temperature and pH ranges and in the presence of metal ions or detergents. The high level of stability of esterase EstAS with its unique substrate specificities make itself highly useful for biotechnological applications.
Project description:Metagenome-assembled genomes (MAGs) are microbial genomes reconstructed from metagenomic data and can be assigned to known taxa or lead to uncovering novel ones. MAGs can provide insights into how microbes interact with the environment. Here, we performed genome-resolved metagenomics on sequencing data from four studies using sequencing batch reactors at microcosm (~25 mL) and mesocosm (~4 L) scales inoculated with sludge from full-scale wastewater treatment plants. These studies investigated how microbial communities in such plants respond to two environmental disturbances: the presence of toxic 3-chloroaniline and changes in organic loading rate. We report 839 non-redundant MAGs with at least 50% completeness and 10% contamination (MIMAG medium-quality criteria). From these, 399 are of putative high-quality, while sixty-seven meet the MIMAG high-quality criteria. MAGs in this catalogue represent the microbial communities in sixty-eight laboratory-scale reactors used for the disturbance experiments, and in the full-scale wastewater treatment plant which provided the source sludge. This dataset can aid meta-studies aimed at understanding the responses of microbial communities to disturbances, particularly as ecosystems confront rapid environmental changes.
Project description:A new esterase gene, est6, was discovered in an activated sludge metagenomic library. The 729-bp gene encodes a 242-amino acid protein (designated Est6) with a molecular mass of 26.1 kDa. Est6 shared only a moderate identity to a putative hydrolase with the highest BLASTP analysis score. Most of the closely related proteins are uncharacterized and are predicted from genome sequencing data of microorganisms or metagenomic DNA sequences. The phylogenetic analysis of Est6 showed that the protein was assigned to family VI esterases/lipases. The catalytic triad of Est6 was predicted to be Ser135, Asp188, and His219, with Ser135 in a typically conserved pentapeptide (GFSQG) of family VI members, which was further confirmed by site-directed mutagenesis. The est6 gene was overexpressed successfully in its soluble form in Escherichia coli and then purified to its tag-free form and homogeneity by affinity chromatography. The purified Est6 in pH 8.0 buffer was active as a monomer. The optimal conditions for Est6 activity were at a temperature of 45 °C and pH of 8.0 when using p-nitrophenyl acetate as a substrate. The enzyme was stable over wide temperature and pH ranges, and it exhibited activity in the presence of organic solvents, metal cations, or detergents. Furthermore, the enzyme showed significant regioselectivity in the spectrophotometric analysis. In conclusion, Est6 might have the potential for applications in biotechnological processes.