Larraín-Linton Juanita, De la Iglesia Rodrigo, Melo Francisco, González Bernardo
Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile.
J Bacteriol. 2006 Oct;188(19):6793-801. doi: 10.1128/JB.00869-06.
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.
食酸戴尔福特菌JMP134(pJP4)含有一个分解代谢性质粒pJP4，该质粒赋予其在氯代芳香族化合物上生长的能力。在3-氯苯甲酸(3-CB)上反复传代表现出一种重组菌株，该菌株能更好地降解3-CB，但不再能在2,4-二氯苯氧乙酸(2,4-D)上生长。我们之前提出，这种表型是由于细胞内该质粒多拷贝的反向重复序列之间发生了双同源重组事件。这种质粒的一种重组形式(pJP4-F3)解释了这种表型，因为它含有两个氯儿茶酚降解tfd基因簇拷贝，这对在3-CB上生长至关重要，但失去了编码2,4-D降解第一步的tfdA基因。另一种重组质粒(pJP4-FM)应该含有两个tfdA基因拷贝，但没有tfd基因簇拷贝。使用多重PCR方法对野生型质粒pJP4及其两种重组形式进行了分子分析。发现了确认这种重组模型的预期PCR产物并进行了测序。在几种碳源中生长的培养物中检测到的重组质粒形式很少。动力学研究表明，含有重组质粒pJP4-FM的细胞不能通过单一碳源生长压力进行选择，而含有重组质粒pJP4-F3的细胞在以3-CB为碳源生长时被选择。在3-CB上反复传代12天后，食酸戴尔福特菌JMP134中的完整质粒群体显然对应于这种形式。然而，在以2,4-D为碳源培养该菌株后，可以回收野生型质粒形式，这表明在群体水平上食酸戴尔福特菌JMP134中可以发现不同的质粒形式。
