Cupriavidus necator JMP134(pJP4) harbors a catabolic plasmid, pJP4, which confers the ability to grow on chloroaromatic compounds. Repeated growth on 3-chlorobenzoate (3-CB) results in selection of a recombinant strain, which degrades 3-CB better but no longer grows on 2,4-dichlorophenoxyacetate (2,4-D). We have previously proposed that this phenotype is due to a double homologous recombination event between inverted repeats of the multicopies of this plasmid within the cell. One recombinant form of this plasmid (pJP4-F3) explains this phenotype, since it harbors two copies of the chlorocatechol degradation tfd gene clusters, which are essential to grow on 3-CB, but has lost the tfdA gene, encoding the first step in degradation of 2,4-D. The other recombinant plasmid (pJP4-FM) should harbor two copies of the tfdA gene but no copies of the tfd gene clusters. A molecular analysis using a multiplex PCR approach to distinguish the wild-type plasmid pJP4 from its two recombinant forms, was carried out. Expected PCR products confirming this recombination model were found and sequenced. Few recombinant plasmid forms in cultures grown in several carbon sources were detected. Kinetic studies indicated that cells containing the recombinant plasmid pJP4-FM were not selectable by sole carbon source growth pressure, whereas those cells harboring recombinant plasmid pJP4-F3 were selected upon growth on 3-CB. After 12 days of repeated growth on 3-CB, the complete plasmid population in C. necator JMP134 apparently corresponds to this form. However, wild-type plasmid forms could be recovered after growing this culture on 2,4-D, indicating that different plasmid forms can be found in C. necator JMP134 at the population level.

译文

:Cupriavidus necator JMP134(pJP4)带有分解代谢质粒pJP4,可赋予其在氯代芳香族化合物上生长的能力。在3-氯苯甲酸酯(3-CB)上重复生长导致选择了重组菌株,该菌株可更好地降解3-CB,但不再在2,4-二氯苯氧基乙酸酯(2,4-D)上生长。我们先前已经提出,该表型是由于该质粒在细胞内多拷贝的反向重复之间的双重同源重组事件引起的。该质粒的一种重组形式(pJP4-F3)解释了此表型,因为它具有两个副本的氯邻苯二酚降解tfd基因簇,这对于在3-CB上生长是必不可少的,但已经丢失了tfdA基因,编码了第一步。 2,4-D的降解。另一个重组质粒(pJP4-FM)应该包含tfdA基因的两个副本,但不包含tfd基因簇的副本。使用多重PCR方法进行了分子分析,以将野生型质粒pJP4与它的两种重组形式区分开。找到并证实了确认该重组模型的预期PCR产物。在几种碳源中培养的培养物中几乎没有检测到重组质粒形式。动力学研究表明,含有重组质粒pJP4-FM的细胞不能通过唯一的碳源生长压力进行选择,而带有重组质粒pJP4-F3的细胞则是在3-CB上生长时选择的。在3-CB上重复生长12天后,C。necator JMP134中的完整质粒群体显然对应于这种形式。但是,在2,4-D上培养此培养物后,可以回收野生型质粒形式,表明在种群水平上,在C. necator JMP134中可以找到不同的质粒形式。

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