Lewis T A, Cortese M S, Sebat J L, Green T L, Lee C H, Crawford R L
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington 05405, USA.
Environ Microbiol. 2000 Aug;2(4):407-16. doi: 10.1046/j.1462-2920.2000.00122.x.
A spontaneous mutant of Pseudomonas stutzeri strain KC lacked the carbon tetrachloride (CCl4) transformation ability of wild-type KC. Analysis of restriction digests separated by pulsed-field gel electrophoresis (PFGE) indicated that the mutant strain CTN1 differed from strain KC by deletion of approximately 170 kb of chromosomal DNA. CTN1 did not produce pyridine-2,6-bis(thiocarboxylic acid) (PDTC), the agent determined to be responsible for CCl4 dechlorination in cultures of strain KC. Cosmids from a genomic library of strain KC containing DNA from within the deleted region were identified by hybridization with a 148 kb genomic Spel fragment absent in strain CTN1. Several cosmids identified in this manner were further screened for complementation of the PDTC biosynthesis-negative (Pdt -) phenotype. One cosmid (pT31) complemented the Pdt- phenotype of CTN1 and conferred CCl4 transformation activity and PDTC production upon other pseudomonads. Southern analysis showed that none of three other P. stutzeri strains representing three genomovars contained DNA that would hybridize with the 25,746 bp insert of pT31. Transposon mutagenesis of pT31 identified open reading frames (ORFs) whose disruption affected the ability to make PDTC in the strain CTN1 background. These data describe the pdt locus of strain KC as residing in a non-essential region of the chromosome subject to spontaneous deletion. The pdt locus is necessary for PDTC biosynthesis in strain KC and is sufficient for PDTC biosynthesis by other pseudomonads but is not a common feature of P. stutzeri strains.
施氏假单胞菌菌株KC的一个自发突变体丧失了野生型KC的四氯化碳（CCl4）转化能力。通过脉冲场凝胶电泳（PFGE）分离的限制性酶切分析表明，突变菌株CTN1与菌株KC的差异在于染色体DNA缺失了约170 kb。CTN1不产生吡啶-2,6-双（硫代羧酸）（PDTC），该物质被确定为菌株KC培养物中CCl4脱氯的原因。通过与菌株CTN1中不存在的148 kb基因组Spel片段杂交，鉴定出来自菌株KC基因组文库中包含缺失区域内DNA的黏粒。以这种方式鉴定出的几个黏粒进一步筛选其对PDTC生物合成阴性（Pdt-）表型的互补作用。一个黏粒（pT31）互补了CTN1的Pdt-表型，并赋予其他假单胞菌CCl4转化活性和PDTC产生能力。Southern分析表明，代表三个基因组变种的其他三株施氏假单胞菌中没有一株含有能与pT31的25,746 bp插入片段杂交的DNA。对pT31进行转座子诱变，鉴定出了一些开放阅读框（ORF），其破坏影响了在菌株CTN1背景下产生PDTC的能力。这些数据表明，菌株KC的pdt基因座位于染色体的一个非必需区域，易发生自发缺失。pdt基因座对于菌株KC中PDTC的生物合成是必需的，对于其他假单胞菌的PDTC生物合成是足够的，但不是施氏假单胞菌菌株的共同特征。
