Rodríguez-Herva José J, García Vanina, Hurtado Ana, Segura Ana, Ramos Juan L
Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, E-18008 Granada, Spain.
Environ Microbiol. 2007 Jun;9(6):1550-61. doi: 10.1111/j.1462-2920.2007.01276.x.
Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of > 1% (v/v) toluene in the culture medium. A set of multidrug efflux pumps have been found to play a major role in the tolerance of this bacterium to organic solvents (Rojas et al., J Bacteriol 183: 3967-3973). In the course of studies of the mechanisms underlying solvent tolerance in DOT-T1E, we isolated a spontaneous solvent-sensitive mutant derivative which had lost the genes encoding the TtgGHI efflux pump, the most important extrusion element in quantitative terms. Genomic comparisons between the mutant and its parental strain by microarray analysis revealed that in addition to the ttgVW-ttgGHI gene cluster, another group of genes, highly similar to those found in the Tn4653A and ISPpu12 transposable elements of the TOL plasmid pWW0 from P. putida mt-2, were also absent from this strain. Further analysis demonstrated that strain DOT-T1E harboured a large plasmid (named pGRT1) that was lost from the solvent-sensitive mutant. Mapping analysis revealed that the ttgVW-ttgGHI genes and the Tn4653A-like transposon are borne by the pGRT1 plasmid. Plasmid pGRT1 is highly stable and its frequency of loss is below 10(-8) per cell per generation under a variety of growth conditions, including nutritional and physical stresses. The pGRT1 plasmid is self-transmissible, and its acquisition by the toluene-sensitive P. putida KT2440 and Pseudomonas aeruginosa PAO1 increased the recipient's tolerance to toluene up to levels similar to those exhibited by P. putida DOT-T1E. We discuss the importance and potential benefits of this plasmid for the development of bacteria with enhanced solvent tolerance, and its potential impact for bioremediation and whole-cell biotransformations.
恶臭假单胞菌DOT-T1E是一种耐溶剂菌株，能够在培养基中甲苯含量大于1%（v/v）的情况下生长。已发现一组多药外排泵在该细菌对有机溶剂的耐受性中起主要作用（罗哈斯等人，《细菌学杂志》183：3967 - 3973）。在研究DOT-T1E耐溶剂机制的过程中，我们分离出了一个自发的溶剂敏感突变衍生物，它失去了编码TtgGHI外排泵的基因，从数量上来说这是最重要的排出元件。通过微阵列分析对突变体及其亲本菌株进行基因组比较发现，除了ttgVW - ttgGHI基因簇外，另一组与恶臭假单胞菌mt - 2的TOL质粒pWW0的Tn4653A和ISPpu12转座元件中发现的基因高度相似的基因，在该菌株中也不存在。进一步分析表明，菌株DOT-T1E含有一个大质粒（命名为pGRT1），该质粒在溶剂敏感突变体中丢失。图谱分析表明，ttgVW - ttgGHI基因和Tn4653A样转座子由pGRT1质粒携带。质粒pGRT1高度稳定，在包括营养和物理应激在内的各种生长条件下，其每细胞每代的丢失频率低于10^(-8)。pGRT1质粒是自我传递的，甲苯敏感的恶臭假单胞菌KT2440和铜绿假单胞菌PAO1获得该质粒后，其对甲苯的耐受性提高到与恶臭假单胞菌DOT-T1E相似的水平。我们讨论了该质粒对于开发具有增强溶剂耐受性的细菌的重要性和潜在益处，以及其对生物修复和全细胞生物转化的潜在影响。
