Metagenomic analyses reveal phylogenetic diversity of carboxypeptidase gene sequences in activated sludge of a wastewater treatment plant in Shanghai, China

Hao Jin 0 Bailin Li 0 Xu Peng 0 Lanming Chen 0 0 X. Peng Department of Biology, Copenhagen University , Ole Maales Vej 5, 2200 N Copenhagen, Denmark 1 ) Key Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation ( Shanghai ) , China Ministry of Agriculture, Engineering Centre for Quality Control and Risk Assessment of Aquatic Products, College of Food Science and Technology, Shanghai Ocean University , 999 Hu Cheng Huan Road, Shanghai 201306, People's Republic of China Activated sludge of wastewater treatment plants carries a diverse microflora. However, up to 80-90 % of microorganisms in activated sludge cannot be cultured by current laboratory techniques, leaving an enzyme reservoir largely unexplored. In this study, we investigated carboxypeptidase diversity in activated sludge of a wastewater treatment plant in Shanghai, China, by a culture-independent metagenomic approach. Three sets of consensus degenerate hybrid oligonucleotide primers (CODEHOPs) targeting conserved domains of public carboxypeptidases have been designed to amplify carboxypeptidase gene sequences in the metagenomic DNA of activated sludge by PCR. The desired amplicons were evaluated by carboxypeptidase sequence clone libraries and phylogenetic analyses. We uncovered a significant diversity of carboxypeptidases present in the activated sludge. Deduced carboxypeptidase amino acid sequences (127-208 amino acids) were classified into three distinct clusters, , , and . Sequences belonging to clusters and shared 58-97 % identity to known carboxypeptidase sequences from diverse species, whereas sequences in the cluster were remarkably less related to public carboxypeptidase homologous in the GenBank database, strongly suggesting that novel carboxypeptidase families or microbial niches exist in the activated sludge. We also observed numerous carboxypeptidase sequences that were much closer to those from representative strains present in industrial and sewage treatment and bioremediation. Thermostable and halotolerant carboxypeptidase sequences were also detected in clusters and . Coexistence of various carboxypeptidases is evidence of a diverse microflora in the activated sludge, a feature suggesting a valuable gene resource to be further explored for biotechnology application. - Activated sludge is used for treating sewage and industrial wastewaters, and it is the most common biological wastewater treatment technology used in industrialized countries (Kim et al. 2010). Sewage and industrial wastewater contain high amounts of complex organic matter which is mainly in the form of proteins and lipids (Raunkjr et al. 1994), and often also nitrogen (as ammonium and organic nitrogen) and phosphorus. The biomass that reduces the complex content of the sewage is the activated sludge, biological flocs mainly composed of saprotrophic bacteria, and some protozoa and small metazoans (Wiln et al. 2008). Microorganisms not only take up some small molecules for intracellular metabolism but also enzymatically hydrolyze a large fraction of the organic matter through a series of hydrolytic reactions to smaller units, which can be taken up by the bacterial cell uptake system (Gessesse et al. 2003). Previous research indicated that activated sludge is a good source for studying and discovering proteases (Gessesse et al. 2003), which play a vital role in the extracellular catabolism of organic matter in activated sludge. As one important group of proteases, carboxypeptidases (EC number 3.4.16 3.4.18) hydrolyze a peptide bond of amino acid residues at the carboxyl-terminal (C-terminal) end of a protein or peptide. They have diverse functions ranging from catabolism to protein post-translational modification and regulation of biological processes (Neurath and Walsh 1976). Based on their active sites of catalytic reactions, carboxypeptidases are usually classified into metallo-carboxypeptidases (EC number 3.4.17), serine carboxypeptidases (EC number 3.4.16), and cysteine carboxypeptidase (or thiol carboxypeptidases) (EC number 3.4.18). However, up to 8090 % of the microorganisms detected in activated sludge by 16S rRNA genes based on molecular taxonomic studies cannot be cultured using standard cultivation techniques (Amann et al. 1995), leaving a potentially valuable resource largely unexplored. Recently, metagenomic technology has been used successfully to obtain microbial products from uncultivated microorganisms in various environments (Amann et al. 1995; Chu et al. 2008; Lee et al. 2008). Genes encoding novel enzymes, such as cellulases, chitinases, amylases, and lipases, have been screened from metagenomic libraries (Cottrell et al. 1999; Ferrer et al. 2005; Hong et al. 2007). Nevertheless, little if anything has been done to systematically investigate carboxypeptidases in microbial communities of complex environments. In this study, we focused on the diversity of carboxypeptidases in activated sludge of wastewater treatment systems in Shanghai, China, by a culture-independent metagenomic approach. Based on conserved domains including active site signature sequences of known carboxypeptidases in the GenBank database, a set of CODEHOPs was designed to amplify carboxypeptidase gene sequences in the metagenomic DNA of activated sludge by polymerase chain reaction (PCR). Our results provide the first evidence of a substantial level of carboxypeptidase sequence diversity in the microbial community of activated sludge in industrial and municipal wastewater treatment. Bacterial strains, plasmids and culture conditions E s c h e r i c h i a c o l i TOP10 [g enotype: F mcr A (mrr -hsd RMS-mcr BC) 80 lac Z M15 lac X74 rec A1 ara D139 (ara -leu )7697 gal U gal K rps L (StrR) end A1nup G] (TianGen Biotech, Beijing, China) was used as a host strain for transformation of recombinant plasmids in the cloning. Plasmid pGM-T (TianGen) was employed as a cloning vector for PCR products. E. coli was routinely grown in LuriaBertani (LB) medium (Sambrook and Russell 2001) aerobically at 37 C, while recombinant E. coli strains were grown in LB medium in both liquid broth and agar plates supplemented with amplicillin (100 g/ml). Extraction of metagenomic DNA from activated sludge Samples of activated sludge were collected from a wastewater treatment plant located in Lingang, Pudong District, Shanghai, China, where industrial and municipal wastewater was treated. Extraction of metagenomic DNA was carried out immediately after samples had been transported on ice to the laboratory in Shanghai Ocean University (Shanghai, China). Metagenomic DNA was isolated according to the method of in situ lysis of microorganisms described by Zhou et al. (1996) with some modifications. Briefly, a sample (50 g) of wet activated sludge and 135 ml DNA extraction buffer [100 mmol/l Tris-HC1, 100 mmol/l EDTA, 1.5 mol/l NaC1 and 1 % (w/v) hexadecyltrimethylammonium bromide, pH 8.0; SigmaAldrich, MO, USA)] were (...truncated)