砷氧化根癌农杆菌砷抗性与抗生素抗性的共调控机制

Arsenides is used as feed additive supplement and can cause cross contaminate with antibiotics in the environment. In the mean time, Arsenic (As) can be used as drug and faces drug tolerance challenges as antibiotic does. So far, the correlated mechanism between bacterial As resistance and antibiotic resistance is still not clear. Previously, we have systematically analyzed arsenite resistant and oxidation regulation mechanisms in an arsenite-oxidizing bacterium Agrobacterium tumefaciens 5A and found three component system AioXRS and phosphate regulation two component systems PhoBR. Meanwhile, experiments of gene knock out，complementation and bacterial one-hybrid system showed an arsenite oxidation regulator AioR is possibly to co-regulate both the As oxidation and the resistance of ampicillin etc. Based on these preliminary results, we predict the presence of co-regulation mechanisms between bacterial As resistance and certain antibiotics resistance. To confirm this hypothesis, we will conduct comprehensive analyses using gene knock out and complementation, protein in vivo and in vitro interactions, report gene assay, over-expression and biochemistry tests etc. We will determine whether the arsenic related regulators regulate the resistance to ampicillin or tetracycline etc.,or whether antibiotic regulators regulate As resistance. Finally, we will establish the co-resistance correlation,the regulated genes, the active or negative regulation models, and the metabolic pathways. The results of this project will provide a new perspective and will make significant contributions for better understanding the molecular regulation mechanisms between As resistance and antibiotic resistance and the interaction among bacteria, arsenic and antibiotics.

砷化物作为饲料添加剂等与抗生素在环境中会产生交叉污染，同时砷作为药物和抗生素一样面临着抗药性的挑战。细菌砷抗性与抗生素抗性相关机制尚未明确。我们前期对砷氧化型根癌农杆菌5A的砷抗性和氧化调控进行了系统研究，发现了调控砷氧化的AioXRS三组份系统和PhoBR磷酸调控双组份系统。同时我们发现该菌加砷增强了对氨苄青霉素等的抗性，砷调控基因aioR的突变、互补和细菌单杂交等预示砷氧化的调控和抗生素抗性的调控相关联。在此基础上，我们将进行基因敲除与互补、蛋白质体内/体外互作、报告基因表达、超表达、生化测定、比较基因组等综合分析，确定砷调控蛋白是否调控青霉素类或四环素类等的抗性，或抗生素调控子是否调控砷的抗性；进而阐明抗性的相关性、调控的基因、启动子被激活或抑制的机理、代谢途径等，建立调控模型。通过该研究，揭示根癌农杆菌砷与抗生素抗性的共调控机制，提供对细菌-砷-抗生素互作的新认识。具有重要价值。

为了阐明细菌对砷/锑（As/Sb）与抗生素共抗性和共调控的机制，我们在根癌农杆菌5A发现了大环内酯类抗生素外排系统MacAB-TolC的编码基因，反转录PCR表明外排基因macA与macB共转录。macB的缺失导致细菌对如红霉素及如青霉素G、氨苄青霉素和苯唑西林抗性的下降，但是对头孢菌素类抗生素无效果。此外，macB的缺失同样导致细菌对As(III)抗性的下降。把MacAB在大肠杆菌AW3110异源表达后，同样提高了AW3110对红霉素、青霉素G及As(III)的抗性。此外，MacAB的表达降低了细菌胞内As(III)的积累。以上结果表明MacAB系统赋予根癌农杆菌5A对红霉素、青霉素及As(III)的协同外排抗性。根癌农杆菌5A中砷氧化酶调控蛋白AioR可以在体内和体外与MacA基因相互作用，AioR同时调控MacAB和砷抗性，其可能是二者共抗性的关键。.此外，我们发现睾丸酮丛毛单胞菌S44中Fe-S簇合成调控蛋白IscR正调控锑(III)和头孢菌素的抗性，进一步发现LysR家族的调控蛋白（命名为CzoR）位于A类型的抗生素抗性的β-内酰胺酶czoA基因前面，在czoR 和czoA 基因之间存在一个IscR结合位点。体外酶活实验证实CzoA可以水解头孢氨苄和头孢唑林等头孢菌素类抗生素。通过EMSA 和DNA 足迹实验证实了两个调控因子CzoR 和IscR 都可以与czoA启动子区域结合，并确定了其具体的结合位点。对czoR和iscR基因进行了敲除和互补，发现其突变株对头孢氨苄和头孢唑林的抗性都发生了下降，其互补株表型都得到了有效恢复。我们还发现iscR突变株对头孢氨苄和头孢唑林的抗性强于ΔczoR，说明CzoR 在对头孢菌素抗性的调控中发挥的作用强于IscR. 荧光定量实验发现突变iscR基因后czoR 基因表达会下调，但突变czoR基因并不影响iscR的表达。报告基因融合实验发现在iscR突变株中，CzoA表达量下调，但在czoR 突变株中，czoA不表达，说明IscR 调控CzoA表达依赖于CzoR的存在。整体结论是CzoR正调控CzoA的表达，而IscR通过调控CzoR的表达加强了CzoR对czoA基因的调控。本课题的研究结果丰富了细菌对砷锑和抗生素抗性的共调控和共外排机理。