Aguilar G R, Soberón M
Departmento de Biologia Molecular de Plantas, Universidad Nacional Autonoma de México, Cuernavaca, Morelos, México.
Gene. 1996 Dec 5;182(1-2):129-35. doi: 10.1016/s0378-1119(96)00534-3.
In this paper we describe the sequence analysis of two Rhizobium etli genes (ccmA and ccmB) which are believed to participate in the transport of the haem moiety to the periplasm in other bacterial species. The characterized DNA region was isolated by complementation of a R. etli Tn5mob induced mutant (CFN4201) which was affected in the production of c-type cytochromes. Sequence analysis of this region identified three open reading frames, two were identified as the ccmA and ccmB genes. The predicted protein sequence of ccmA showed significant homology with ATP binding proteins of the ABC-type transporter systems, while ccmB encodes for a hydrophobic protein probably associated with the ccmA gene product. The Tn5mob insertion in CFN4201 strain was located in the carboxy terminus of CcmB. Restriction mapping of the EcoRI fragment containing the Tn5mob insertion showed that it involved a deletion of approximately 1.5 kb. Mutagenesis of the wild-type region with a miniMu transposon and complementation analysis showed that the mutation in ccmB, and not the deletion, was responsible for the phenotype of CFN4201 strain and that ccmA and ccmB are independent transcription units. We found that a region located downstream of ccmB is reiterated twice, one near the chromosomal ccmA-ccmB locus while the second in plasmid e. Finally, CFN4201 membranes had detectable levels of C1 apoprotein which did not contain bound haem. This data could suggest that haem binding to the apoprotein occurs after the translocation of the apo form of c1 to the membrane.
在本文中，我们描述了对两株费氏中华根瘤菌基因（ccmA和ccmB）的序列分析，据信这两个基因参与了血红素部分向其他细菌物种周质的转运。通过对一株费氏中华根瘤菌Tn5mob诱导突变体（CFN4201）进行互补，分离出了特征性DNA区域，该突变体在c型细胞色素的产生方面受到影响。对该区域的序列分析确定了三个开放阅读框，其中两个被鉴定为ccmA和ccmB基因。ccmA的预测蛋白质序列与ABC型转运系统的ATP结合蛋白具有显著同源性，而ccmB编码一种可能与ccmA基因产物相关的疏水蛋白。CFN4201菌株中的Tn5mob插入位于CcmB的羧基末端。对含有Tn5mob插入的EcoRI片段进行限制性图谱分析表明，它涉及约1.5 kb的缺失。用miniMu转座子对野生型区域进行诱变和互补分析表明，CFN4201菌株的表型是由ccmB中的突变而非缺失导致的，并且ccmA和ccmB是独立的转录单元。我们发现，位于ccmB下游的一个区域重复了两次，一次靠近染色体上的ccmA - ccmB位点，另一次在质粒e中。最后，CFN4201膜中可检测到不含结合血红素的C1脱辅基蛋白水平。这些数据可能表明，血红素与脱辅基蛋白的结合发生在c1脱辅基形式转运到膜之后。
