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, α, β, and γ. Sequences belonging to clusters α and β shared 58–97 % identity to known carboxypeptidase sequences from diverse species, whereas sequences in the cluster γ 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 α and β. 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.

Metagenomic technology is applied in studying the total genomes of complex microbial community in specific environment.It overcomes the limitations of conventional methods of microorganism cultivation,and provides powerful techniques to explore and utilize rich gene resources of unculturable microorganisms.Remarkable progress has been achieved in this research field.This paper reviewed the theory and application of metagenomic technology in screening novel functional enzymes from microorganisms.

为研究污水处理活性污泥微生物多样性,提取了活性污泥宏基因组DNA,并采用细菌通用引物27F和1492R扩增了上海污泥厂活性污泥细菌16S rDNA片段,构建了细菌16S rDNA克隆文库,并对该文库中的微生物群落进行了分析.共获得200条高质量序列并建立系统发育树,结果显示活性污泥主要的细菌类群为变形菌门(Proteobacteria)(91.9％)、厚壁菌门(Firmicures)(4.6％)、拟杆菌门(Bacteroidetes)(2％)、绿弯菌门(Chloroflexi)(0.5％)、硝化螺菌门(Nitrospirae)(1％).其中,明显的优势菌群为Alcaligenes feacalis(55％)、Pseudomonas aeruginosa(12.8％)和Stenotrophomonas(12.8％),优势菌的产酶能力在活性污泥中显示生态修复功能菌的作用.

The aim of this study was to investigate virulence and drug resistant mechanism of multidrug resistant Pseudomonas aeruginosa(PA) isolated from water sample of fishery pond,in order to provide scientific support for practical aquaculture disease treatment,effective management of aquaculture environment and aquatic food safety control.Antimicrobial susceptibility of PA isolates under this study were examined by using standard Kirby Bauer disk diffusion method according to the Clinical and Laboratory Standards Institute(USA,2010 Edition),showing distinct susceptibility to nine antimicrobial agents belonging to six drug classes tested.All PA isolates were resistant to trimethoprim and rifampin,of which,90% were to ampicilin and kanamycin,and 10% were to gentamycin.The genes involved in pathogenicity,intrinsic and transferred antibiotic resistance were detected by polymerase chain reaction(PCR).The toxA and exoU genes were absent from the PA isolates tested,50% of which were exoS+/exoU-infectious genetype,and none of which were exoS-/exoU+ cell toxic genetype that usually existed in clinical PA isolates.Subsequent analysis revealed that the multidrug efflux pumps including MexAB-OprM,MexXY-OprM and MexVW-OprM were present in all PA isolates,20% of which were positive for ampC gene,whereas the other four efflux pumps including MexEF-OprN,MexJK-OprM,MexCD-OprJ and MexGHI-OpmD were fully or partially deficient.In addition,the comINT gene of class Ⅰ-Ⅲ integrons was negative in the PA isolates,however,the functional genes(int,soj,pilS2,pilD)of conserved module structures of integrative conjugative elements(ICEs) were detected positive,suggesting possible transfer activity of the ICEs detected in the PA isolates.This study revealed the major mechanism under multidrug resistance of the PA isolates mediated by efflux pumps encoded by chromosome genes,and constituted the first evidence for mobile genetic element ICEs in PA isolates of environmental origin.