Study On Chemotaxis Of PAH-degrading Novosphingobium Bacteria

The study on biodegradation of the persistent organic pollutants （POPs） including PAHs has become a preceding subject in current environmental sciences. The bacteria belonging to the genus Novosphingobium generally have the characteristics of degrading aromatics, which makes them fine aromatics bioremediators. Previously, the researchers performed studies on the genes, metabolic pathways and regulatory systems involved in aromatic compounds biodegradation. In recent years, to solve the popular but thorny issues of contaminated environment remediation, mechanisms upon microbial uptake of hydrocarbon are important subjects, and understanding how microorganisms capture and transport PAHs is an important issue to realize the bioremediation of organic pollution. In this paper, the genus Novosphingobium is studied, focusing on how they capture the hydrophobic PAHs as the breakthrough, the chemotaxis, chemotaxis genes, chemotaxis protein expression and the relationship between chemotaxis and biodegradation of PAHs are studied using genomics, RT-qPCR and other techniques. The main results are as follows:1. We used Illumina sequencing to perform a whole-genome sequence of PAHs--biodegrading bacteria N. pentaromativorans F2isolated from the sediments of mangrove wetlands. The construction and bioinformatic analysis of detailed genome map showed that N. pentaromativorans F2genome size was5.12M,63.1%of GC content, the genome was assembled into33scaffolds, and5,291genes were obtained. The KEGG PATHWAY annotation revealed23genes assigned to bacterial chemotaxis pathway, consisting of5Methyl-accepting chemotaxis protein （MCP）,1sensor kinase CheA,1coupling protein CheW,2response regulator CheY,2MCP methylases CheB,2MCP methyltransferase CheR,1fusion protein cheBR,1chemotactic receptor glutamine deaminase CheD,3flagellar motor switch FliG/M/NY,2Flagellar stator MotA and3MotB. The model strain N.pentaromativorans US6-1which shares99.859%similarity with F2has22genes annotated to the bacterial chemotaxis pathway, containing CheX but lack fussion protein cheBR.2. The analysis of chemotaxis genes of the completed genome sequenced Novosphingobium distribution showed the bacteria belonging to genus Novosphingobium contain at least one "che" cluster at least, which forms a complete chemotaxis pathway, and the chemotactic system is actually functional. Strain F2, US6-1and PP1Y, shared a consistent order of chemotaxis genes in "che" cluster, which is differed from the strain AP12and Rr2-17that is slightly distant at the phylogenetic three, and it verified the phylogenetic relationship among them. Rr2-17has three "che"clusters, indicating its chemotaxis system is complex. The component analysis of the chemotaxis proteins showed that the genus Novosphingobium contains MCP, CheW, CheA, CheB, CheR and CheY, which is more similar to Bacillus other than E.coli in terms of forming mechanism.3. Domain architecture of chemotaxis proteins of strain F2and US6-1were visualized using Pfam and MiST. The results showed that both strains have the same chemotaxis protein domains architecture. The domains architecture analysis of sequenced bacterial chemotaxis genes CheA indicated that the chemotaxis systems of Novosphingobium belong to the Fla class. The crucial protein CheA in the chemotaxis system have two highly conserved domains, namely HATPase_c and CheW domains, which means CheA can be used as a biomarker to detect the presence of the chemotaxis bacteria in the environment.4. Drop and Swarm plate assays were used to study the chemotaxis of N. pentaromativorans F2, N. pentaromativorans US6-1, Novosphingobium indicum K13, Novosphingobium stygium DSM12445, Novosphingobium sp.C2AC, and plasmid mutant CP-US6-1of N. pentaromativorans US6-1toward aromatic compounds and intermediates. The results showed all these Novosphingobium bacteria show chemotaxis, but the chemotactic ability varies from each. The comparison between US6-1and its plasmid removed strain CP-US6-1showed CP-US6-1that loses the ability of degrading catechol also loses the chemotaxis to catechol, suggesting that the chemotaxis of US6-1is metabolic depended.5. According to gene sequences of chemotaxis proteins and transporters, the specific primers were designed, and RT-qPCR technique is used to study the real time expression of these proteins of N. pentaromativorans US6-1induced by Phe and Pyr, The results showed expression of chemotaxis protein and transporter were all up-regulation induced by Phe. Under the pressure of Pyr, the expression of chemotactic protein CheA and transport related protein MFS permease （NSU₀850） were down-regulation, while the Methyl-accepting chemotaxis protein MCPJ and MCP₁130were up-regulation. These results verified the chemotaxis of US6-1is metabolic depended and indicated MCP can directly sensor PAHs.