Table_1_Resistance-Nodulation-Division Efflux Pump, LexABC, Contributes to Self-Resistance of the Phenazine Di-N-Oxide Natural Product Myxin in Lysobacter antibioticus.docx

Antibiotic-producing microorganisms have developed several self-resistance mechanisms to protect them from autotoxicity. Transporters belonging to the resistance- nodulation-division (RND) superfamily commonly confer multidrug resistance in Gram-negative bacteria. Phenazines are heterocyclic, nitrogen-containing and redox-active compounds that exhibit diverse activities. We previously identified six phenazines from Lysobacter antibioticus OH13, a soil bacterium emerging as a potential biocontrol agent. Among these phenazines, myxin, a di-N-oxide phenazine, exhibited potent activity against a variety of microorganisms. In this study, we identified a novel RND efflux pump gene cluster, designated lexABC, which is located far away in the genome from the myxin biosynthesis gene cluster. We found a putative LysR-type transcriptional regulator encoding gene lexR, which was adjacent to lexABC. Deletion of lexABC or lexR gene resulted in significant increasing susceptibility of strains to myxin and loss of myxin production. The results demonstrated that LexABC pump conferred resistance against myxin. The myxin produced at lower concentrations in these mutants was derivatized by deoxidation and O-methylation. Furthermore, we found that the abolishment of myxin with deletion of LaPhzB, which is an essential gene in myxin biosynthesis, resulted in significant downregulation of the lexABC. However, exogenous supplementation with myxin to LaPhzB mutant could efficiently induce the expression of lexABC genes. Moreover, lexR mutation also led to decreased expression of lexABC, which indicates that LexR potentially positively modulated the expression of lexABC. Our findings reveal a resistance mechanism against myxin of L. antibioticus, which coordinates regulatory pathways to protect itself from autotoxicity.

产抗生素微生物已进化出多种自我耐药机制，以抵御自身产生的自毒作用。隶属于耐药-结节分化（resistance-nodulation-division, RND）超家族的转运蛋白，通常可赋予革兰氏阴性菌多重耐药性。吩嗪类化合物是一类含氮杂环氧化还原活性物质，具有多样的生物活性。我们此前从抗生素溶杆菌（Lysobacter antibioticus）OH13——一种具有潜在生防应用前景的土壤细菌——中分离得到6种吩嗪类化合物。其中，粘菌素（myxin，一种二N-氧化吩嗪）对多种微生物展现出强效抑制活性。本研究中，我们鉴定得到一个全新的RND外排泵基因簇，命名为lexABC，该基因簇与粘菌素生物合成基因簇在基因组中相距较远。我们同时发现了一个毗邻lexABC的假定LysR型转录调节因子编码基因lexR。敲除lexABC或lexR基因会显著提升菌株对粘菌素的敏感性，同时使菌株丧失粘菌素的合成能力。实验结果表明，LexABC外排泵可介导菌株对粘菌素的耐药性。上述突变体中合成的低浓度粘菌素均发生了脱氧与O-甲基化修饰。进一步研究发现，敲除粘菌素生物合成必需基因LaPhzB以阻断粘菌素合成后，lexABC的表达量显著下调；但向LaPhzB突变株中外源添加粘菌素，可有效诱导lexABC基因的表达。此外，lexR突变同样会降低lexABC的表达水平，这提示LexR可能正向调控lexABC的转录。本研究揭示了抗生素溶杆菌抵御自身合成粘菌素的耐药机制，该机制通过协调调控通路避免菌株遭受自毒伤害。