Function And Crosstalk Of RND Efflux Pumps TtgABC And TtgGHI In Pseudomonas Putida B6-2 (DSM 28064)

Microbial remediation owing to its low cost and no secondary pollution is becoming a promising method to remove polycyclic aromatic hydrocarbon pollutants.With the development of genome sequencing technology,more and more PAH-degrading strains with application potential have been sequenced.It was notable that PAH-degrading bacteria often harbor efflux pump genes in their genomes.However,their specific roles in degradation process have not been clarified.Pseudomonas putida strain B6-2 was previously isolated as a PAH-degrader and its genome was completely sequenced.There are two bioinformatically predited Resistance-Nodulation-Division?RND?efflux pumps,ttgABC and ttgGHI.Herein,the physiological function of them and the cross-regulation mechanism between them were systematically studied.Firstly,we investigated the roles of ttgABC and ttgGHI in organic solvent tolerance and antibiotic resistance.The disruption of genes ttgABC or ttgGHI resulted in a defect in organic solvent tolerance,which strongly suggests that both ttgABC and ttgGHI play critical roles in the organic solvent tolerance of strain B6-2.TtgABC contributed to antibiotic resistance.The mechanism of multidrug resistance and organic solvent tolerance of strain B6-2 was revealed.Secondly,we emphatically studied the roles of them during biphenyl?BP?degradation and the underlying mechanism.It was found that mutant strain?ttgGHI did not maintain its ability in long-term BP degradation,which was relevant to the loss of cell viability.The expression level of ttgGHI increased significantly during BP degradation.BP,2-hydroxybiphenyl,3-hydroxybiphenyl,2,3-dihydroxybiphenyl?2,3-DHBP?,and catechol were inducers of ttgGHI expression.2,3-OHBP was determined to be a substrate of pump TtgGHI using resting-cell assay.In addition,TtgGHI could enhance 2,3-OHBP tolerance by pumping it out of cell.The mutant strain?ttgV increased cell viability and prolonged BP degradation.These results indicate that the pump TtgGHI of strain B6-2 benefits the BP degradation process by pumping out toxic metabolites such as 2,3-OHBP.This study provides insights into the versatile physiological functions of the widely distributed efflux pumps in the biodegradation of PAHs.The findings also provide its application prospect in the process of PAH degradation.Thirdly,the cross-regulation mechanism between ttgABC and ttgGHI were studied.Based on the results of?-galactosidase activity assay and RT-qPCR assay with strains?ttgW and ttgW-overexpressed mutant,it indicates that TtgW is involved in repressing the expression of ttgABC,which affected organic solvent tolerance and antibiotic resistance mediated by TtgABC.TtgW was expressed and purified in vitro.EMSAs and DNase I footprinting assays were used to identify the DNA binding site of TtgW,which was the same as TtgR.The expression of ttgABC was co-modulated by both TtgR and TtgW.TtgW acted as a repressor of TtgABC pump primarily under conditions of TtgGHI being upregulated.Gene ttgW coexists with the ttgGHIV gene cluster but had no function observed on it,which may be a highly specialized genetic unit to inhibit the expression of the efflux pump ttgABC when the efflux pump ttgGHI was induced.The regulatory mechanism was clarified for microorganisms coordinating multiple efflux pumps through a single regulatory protein.The ttgV mutation was easily found in the ttgABC-null strain.The sequencing revealed ISPpu14 or IS1394a insertion mutation and some site-mutations.In vitro expression experiments showed that glutamate at the 127_th position of TtgV is essential for the tetramer formation.In summary,this systematic work about the function and crosstalk mechanism of efflux pumps ttgABC and ttgGHI in strain B6-2 has shed light on the multiple physiological functions of efflux pumps and enriched the understanding of microbial gene expression regulation.