Transcriptional Regulation Mechanism Of Carbaryl Degradation Gene Cluster In Pseudomonas Sp. XWY-1

Carbaryl(1-naphthyl-N-methyl carbamate)is one of the representative varieties of carbamate insecticides,which is widely used in the control of agricultural and forestry pests.Carbaryl is an inhibitor of acetylcholinesterase(an important enzyme in the signal transmission of the nervous system of the organism).Therefore,carbaryl resiudes in the environment pose a potential danger to humans and other non-target organisms.Based on this,the microbial degradation of carbaryl has always caught the sight of researchers.In 2016,the degradation pathway of carbaryl and the corresponding gene cluster mcb(mcbABCDEFGHIJKLMNOPQ)in Pseudomonas sp.C5 pp were reported by Trivedi et al.In our previous study,another carbaryl-degrading strain Pseudomonas sp.XWY-1 was isolated,which harbored the same carbaryl degradation pathway and gene cluster(mcb)as strain C5 pp.The functions of structural genes responsible for the carbaryl degradation have been identified in Pseudomonas sp.,but the regulatory mechanism of carbaryl degradation has not been investigated yet.In this study,strain XWY-1 was used to study the regulation mechanism of carbaryl degradation in Pseudomonas sp..The regulatory genes and mechanism of carbaryl degradation were identified.The main results obtained are as follows:1.Transcriptional analysis of gene clusters involved in carbaryl degradation and prediction of regulatory proteinsThe degradation pathway of carbaryl in Pseudomonas sp.strain XWY-1 include three parts:(i)the mcbABCDEF gene cluster is responsible for the upstream pathway(from carbaryl to salicylate),(ii)the mcbIJKLM gene cluster is responsible for the midstream pathway(from salicylate to gentisate),and(iii)the mcbOPQ gene cluster is responsible for the downstream pathway(from gentisate to pyruvate and fumarate).Real-Time quantitative PCR(RT-q PCR)showed that mcbA gene,responsible for the hydrolysis of carbaryl(generate1-naphthol),was constitutively expressed in strain XWY-1.mcbBCDEF,mcbIJKLM and mcbOPQ cluster belong to three different transcriptional units,and they were induced during the degradation of carbaryl.RAST and sequence alignment showed that there were three different regulatory proteins in the mcbgene cluster.They all belonged to the LysR-type transcriptional regulator and were named McbG,McbH and McbN,respectively.Bioinformatic analysis revealed that,McbG shared the highest similarity(28%)with the LysR family transcriptional regulator Pcp R(NCBI: protein accession number P52679.2)of the pentachlorophenol(PCP)-degrading strain Sphingobium chlorophenolicum ATCC 39723.McbH shared the highest similarity(69%)with the LysR transcriptional regulatory protein Nah R(NCBI: protein accession number P10183.2)of the naphthalene-degrading strain Pseudomonas putida.McbN showed the highest similarity(35%)with Gal R(NCBI: protein accession number Q88JX7.1)of the gallate-degrading strain Pseudomonas putida KT2440.2.McbG activates the mcbBCDEF gene cluster responsible for the upstream pathway of carbaryl degradationThe transcription start site(TSS)of mcbBCDEF was determined by 5′-rapid amplification of c DNA ends(5′-RACE).The “T” base at the 69 th position upstream of the translational start codon of mcbF was identified as the TSS.In addition,the-10 box and the-35 box were also predicted.The resualts of gene disruption and complementation indicated that the mcbG gene was indispensable for the degradation of 1-naphthol,and McbG activates the transcription of the mcbBCDEF gene cluster in response to 1-naphthol in strain XWY-1.McbG could bind to the promoter DNA of mcbBCDEF,and 1-naphthol could enhance this binding as an effector.McbG could bind to the 25 bp motif in the mcbBCDEF promoter area.The binding site is located between the-10 box and the transcription start site.The palindromic sequence TATCGATA in the motif is essential for McbG binding.3.McbH activates the mcbIJKLM gene cluster responsible for the midstream pathway of carbaryl degradationThe “A” base at the 31 st position upstream of mcbI was identified as the TSS of the mcbIJKLM cluster.The-10 box and the-35 box were predicted based on the identified TSS.The results of gene disruption and complementation revealed that McbH activates transcription of the mcbIJKLM gene cluster and thus affects salicylate degradation in strain XWY-1.McbH could bind to the mcbIJKLM promoter DNA,and salicylate could enhance its binding as an effector.The results of EMSA and DNase I footprinting showed that 13 bp motif(TGATACGTATTCA)in the promoter region of the mcbIJKLM gene cluster is critical for McbH binding to promoter DNA.4.McbN activates the mcbOPQ gene cluster responsible for the downstream pathway of carbaryl degradationThe TSS was determined by 5′-RACE.The “A” base at the 89 th position upstream of mcbO was identified as the TSS of mcbOPQ,but the-10 box and-35 box were not found.The results of gene disruption and complementation showed that McbN activates the transcription of mcbOPQ gene cluster and participates in the degradation of gentisate in strain XWY-1.McbN could bind to the promoter DNA of mcbOPQ,gentisate could enhance this binding as an effector.The results of EMSA and DNase I footprinting indicatied that the 12 bp DNA motif(GTCATGCCTTTG)plays an important role in sequence recognition of McbN.In summary,the transcriptional regulation mechanism of the carbaryl degradation gene cluster in strain XWY-1 was analyzed.The carbaryl hydrolase gene mcbA hydrolyzes carbaryl to 1-naphthol,was constitutively expressed.Subsequently,1-naphthol is an effector of McbG;McbG and 1-naphthol activate the transcription of the mcbBCDEF gene cluster,and then 1-naphthol is converted into salicylate.As an effector of the McbH,salicylate activates the transcription of the mcbIJKL gene cluster together with McbH,and then the salicylate is converted into gentisate.Then gentisate and McbN activate the transcription of the mcbOPQ gene cluster,and gentisate is further degraded into the TCA cycle.