Identification Of Bacteriostatic Active Protein Of Lysobacter Sp.SNNU513

Lysobacter sp. SNNU513, a new strain of Lysobacter genus, was isolated from the rhizosphere of medicinal plants Radix Polygalae. It has been deposited in China General Microbiology Culture Collection Center (CGMCC No.8375) and American Type Culture Collection (ATCC(?)BAA-2621TM). It is a gliding organism with a high G+C content, and extracelluar proteolytic activity to a variety of plant pathogens, thus shows a tremendous potential application value as a new biological control agent.The hydrolytic activities of the lysozymes are studied in most detail. In previous study, We separated and identified four kinds of antibacterial proteins using two-dimensional electrophoresis (2-DE) in combination with mass spectrometry from the culture medium of Lysobacter sp. SNNU513. Because there are many limitations of 2-DE electrophoresis technology in coverage, sensitivity and dynamic range, making it difficult to fully understand the extracellular activity proteins. It is better to develop a well coverage of the protein expression profile to obtain antimicrobial prpteins as much as possible. To fully excavate the potential antibacterial-related proteins, the whole genome of Lysobacter sp. SNNU513 was sequenced and annotated to make the preliminary genome-wide characteristics, after that the whole protein expression spectrum was built to determine the intracellular and extracellular proteomics. Based on the identification and comprehensive analysis of the whole bacterial proteins, our purpose was to screen out the potential and high application value of antimicrobial proteins, and to provide theoretical basis for its subsequent popularization and effective utilization.This research can be divided into two parts: 1. Genome sequencing and annotation of Lysobacter sp. SNNU513The draft genome of Lysobacter sp. SNNU513 was successfully built by the Illumina Miseq platform, yielding 4,043,014 total reads, providing with 143 fold sequencing deep of the genome. All reads and scaffolds were assembled using SOAPdenovo followed by an quality assement. The size of draft genome was 6,065,817bp with a 69.90% G+C contant, all reads were assembled into 917 total scaffolds. The genome was consisted of 3 rRNA,8tRNA and 5,051 total ORFs. The Glimmmer 3.0 software was used to do the prediction, predicted genes were subsequently homologily searched in Nr, GO, COG and KEGG database to give corresponding annotations. The COG annotation results showed that genes involved in energy production and conversion, amino acid transport and metabolism, the structure of the ribosome and biosynthesis of protein occupy a large proportion. The GO function classification showed that genes involved in cell process, metabolic process, binding and the catalytic activity hold an absolute advantage. KEGG metabolic pathway analysis indicated that most genes involved in various metabolic pathway, followed by the genes involved in environmental information processing and genetic information processing. According to the annotation,92 genes were identified to participate 12 completely two-component systems, as well as many encoding genes, which were involved in a variety of hydrolytic enzymes and polyketides compounds.2. The establishment of proteome profiling and its potential peptides identificationThe acquired draft genome of Lysobacter sp. SNNU513 from first part was used as reference database. Whole trypsin digested peptides from intracellular and secretory protein were separated and identified by using Shotgun LC-MS technology. A total of 13,246 peptides, corresponding to 1,696 total proteins were identified, which accounted for 30% of the genome prediction. Identified proteins were analyzed and annotationed by GO, COG and KEGG metabolic pathway. Potential peptides were futher analysed by bioinformatics software to give knowledge of the amino acids composition, cellular localization, physical and chemical parameters, spatial structure and other informations. The results showed that values of molecular weight from both the intracellular and secretory proteins were relatively concentrated:the former within 193.37kDa and the latter was small molecules below 50kDa. To isoelectric point, both distributed more widely (from 4 to 12). The identified proteins mainly involved in basic metabolism, transcription, translation, membrane synthesis, catalytic activity and cell membrane. Cell processing related proteins in two components occupied a large proportion. In addition,47 function unknown proteins were also identified, which showed good correlation with the genome analysis. Through literature search and protein sequence alignment,47 antibacterial related proteins were verified, which aligned 80% to the known proteins. Most of them were basic proteins. The biological characteristics of screened potential antibacterial proteins were subsequently analysed using bioinformatics software for amino acid composition, cell positioning, space structure prediction and other information.This study will provide a solid foundation for the further development and effective utilization of antibacterial related proteins from Lysobacter sp. SNNU513.