Isolation And Characterization Of3,5,6-Trichloro-2-Pyridinol-Degrading Ralstonia Sp. Strain T6and Its Degradation Characteristics And Mechanism

3,5,6-trichloro-2-pyridinol (TCP) is the main degradation product of the herbicide triclopyr and the insecticides chlorpyrifos and chlorpyrifos-methyl, and it is widespread in environment. Due to its lower sorption and greater persistence and water solubility, TCP has a much greater leaching potential than chlorpyrifos in soil. TCP is bad to human health, if swallowed, and TCP may cause serious eye damage, and it exhibits low-to-moderate toxicity to aquatic and terrestrial biota. Bioremediation is potentially a cost-effective and environment-friendly method for removal of contaminants compared to chemical and physical processes, but due to TCP’s antimicrobial properties, microbial degradation has rarely been reported. A few strains that can degrade low concentrations of TCP were isolated during the screening of chlorpyrifos or chlorpyrifos-methyl degraders. The study presented here reports on the isolation and characterization of Ralstonia sp. strain T6capable of degrading TCP as a sole source of carbon and energy from activated sludge by the enrichment culture technique, and reports on the characteristics and mechanism of TCP degradation by strain T6.A strain named T6was isolated from active sludge, which collected from a wastewater treatment system, by the enrichment culture technique. Isolate T6is a short, rod-shaped, gram-negative bacterium, and was preliminarily identified as Ralstonia sp. strain T6by analysis of its16S rRNA gene and phylogenetic tree.Strain T6effectively metabolized10mg/L TCP in1hour,100mg/L TCP in12hours and700mg/L TCP within80hours. The optimal pH and temperature for TCP degradation by strain T6are8and30℃respectively. K+accelerated the degradation of TCP by strain T6, while Zn2+, Co2+and Ni2+seriously inhibited the degradation of TCP. Fe3+, Ca2+, Mn2+and Mg2+had no significant influence on the degradation of TCP by strain T6. Surprisingly, Cu2+completely inhibited the degradation of TCP. A green metabolite in degradation of TCP by strain T6was putatively identified as3,6-dihydroxypyridine-2,5-dione by Silver nitrate titration method and LC-MS.A fragment of1330bp that is part of FADH2-utilizing monooxygenase gene tcpA was amplified from the genomic DNA of Ralstonia sp. strain T6with degenerate primers. Then the tcpA gene was disrupted by homologous recombination, yielding mutant T6-ΔtcpA, which could not degrade TCP, but could degrade the green metabolite3,6-dihydroxypyridine-2,5-dione. It was deduced that tcpA and its flanking genes are related with the initiation of TCP degradation by strain T6. The flanking sequences of tcpA gene were obtained by self-formed adaptor PCR (SEFA PCR), three genes from which constitute a tcp gene cluster, and respectively named tcpR, tcpX and tcpA. The genes of tcpR, tcpX and tcpA are respectively972bp,552bp and1554bp in length, and TcpR and TcpX are respectively highly homologous to LysR family transcriptional regulator and flavin redectase that can deliver H from NAD(P)H to FAD, producing FADH2. The complementation strain T6-AtcpA-com for the disrupted tcpA gene of mutant16-ΔtcpA could degrade TCP again, indicating that tcpA is the key gene at initiation of TCP degradation by strain T6, TcpA catalyzes TCP to3,6-dihydroxypyridine-2,5-dione. Additionally, FADH2is necessary to the catalytic reaction of TcpA and flavin reductase can transfer FAD to FADH2using NAD(P)H, it is deduced that TcpX could assist TcpA by providing FADH2and TcpR plays a regulatory role in TCP degradation by strain T6.A library of disable-degrade-green-metabolite mutants of strain T6was obtained by random mutagenesis. Four clones from6000mutants were selected, which could not degrade the green metabolite. The mutation genes and their flanking sequences were obtained by PCR amplification, four genes from the fragment of about5.9kb constitute a thp gene cluster, and respectively named thpR, thpl, thpJ and thpK. The genes of thpR, thpl, thpJ and thpK are respectively900bp,717bp,879bp and1251bp in length. The ThpI and ThpJ cell extracts from heterologous expression recombinants were assayed. ThpJ could degrade the green metabolite3,6-dihydroxypyridine-2,5-dione completely and transfer it to a new metabolite named5-amino-2,4,5-trioxopentanoic acid. ThpI could further degrade the metabolite of5-amino-2,4,5-trioxopentanoic acid. It is seen that ThpJ and Thpl collaboratively degrade the green metabolite3,6-dihydroxypyridine-2,5-dione in turn. Additionally, it is deduced that ThpR and ThpK respectively play a regulatory role and a transporter role in thp gene cluster by analysis of protein sequence homology. In summary, the protein products of tcp and thp gene clusters from strain T6collaboratively degrade TCP in sequence, tcp gene cluster is in charge of the generation of the green metabolite3,6-dihydroxypyridine-2,5-dione at initiation of TCP degradation and thp gene cluster further degrade3,6-dihydroxypyridine-2,5-dione.