Liesegang H, Lemke K, Siddiqui R A, Schlegel H G
Institut für Mikrobiologie, Universität Göttingen, Germany.
J Bacteriol. 1993 Feb;175(3):767-78. doi: 10.1128/jb.175.3.767-778.1993.
From pMOL28, one of the two heavy metal resistance plasmids of Alcaligenes eutrophus strain CH34, we cloned an EcoRI-PstI fragment into plasmid pVDZ'2. This hybrid plasmid conferred inducible nickel and cobalt resistance (cnr) in two distinct plasmid-free A. eutrophus hosts, strains AE104 and H16. Resistances were not expressed in Escherichia coli. The nucleotide sequence of the 8.5-kb EcoRI-PstI fragment (8,528 bp) revealed seven open reading frames; two of these, cnrB and cnrA, were assigned with respect to size and location to polypeptides expressed in E. coli under the control of the bacteriophage T7 promoter. The genes cnrC (44 kDa), cnrB (40 kDa), and cnrA (115.5 kDa) are probably structural genes; the gene loci cnrH (11.6 kDa), cnrR (tentatively assigned to open reading frame 1 [ORF]; 15.5 kDa), and cnrY (tentatively assigned to ORF0ab; ORF0a, 11.0 kDa; ORF0b, 10.3 kDa) are probably involved in the regulation of expression. ORF0ab and ORF1 exhibit a codon usage that is not typical for A. eutrophus. The 8.5-kb EcoRI-PstI fragment was mapped by Tn5 transposon insertion mutagenesis. Among 72 insertion mutants, the majority were nickel sensitive. The mutations located upstream of cnrC resulted in various phenotypic changes: (i) each mutation in one of the gene loci cnrYRH caused constitutivity, (ii) a mutation in cnrH resulted in different expression of cobalt and nickel resistance in the hosts H16 and AE104, and (iii) mutations in cnrY resulted in two- to fivefold-increased nickel resistance in both hosts. These genes are considered to be involved in the regulation of cnr. Comparison of cnr of pMOL28 with czc of pMOL30, the other large plasmid of CH34, revealed that the structural genes are arranged in the same order and determine proteins of similar molecular weights. The largest protein CnrA shares 46% amino acid similarity with CzcA (the largest protein of the czc operon). The other putative gene products, CnrB and CnrC, share 28 and 30% similarity, respectively, with the corresponding proteins of czc.
从嗜碱假单胞菌CH34菌株的两个重金属抗性质粒之一pMOL28中，我们将一个EcoRI - PstI片段克隆到质粒pVDZ'2中。这个杂种质粒在两个不同的无质粒嗜碱假单胞菌宿主菌株AE104和H16中赋予了可诱导的镍和钴抗性（cnr）。在大肠杆菌中不表达抗性。8.5 kb的EcoRI - PstI片段（8528 bp）的核苷酸序列揭示了7个开放阅读框；其中两个，cnrB和cnrA，根据大小和位置被指定为在噬菌体T7启动子控制下在大肠杆菌中表达多肽的基因。基因cnrC（44 kDa）、cnrB（40 kDa）和cnrA（115.5 kDa）可能是结构基因；基因位点cnrH（11.6 kDa）、cnrR（暂时指定为开放阅读框1 [ORF]；15.5 kDa）和cnrY（暂时指定为ORF0ab；ORF0a，11.0 kDa；ORF0b，10.3 kDa）可能参与表达调控。ORF0ab和ORF1表现出嗜碱假单胞菌不典型的密码子使用情况。通过Tn5转座子插入诱变对8.5 kb的EcoRI - PstI片段进行了定位。在72个插入突变体中，大多数对镍敏感。位于cnrC上游的突变导致了各种表型变化：（i）基因位点cnrYRH之一的每个突变导致组成型表达，（ii）cnrH中的一个突变导致宿主H16和AE104中钴和镍抗性的不同表达，以及（iii）cnrY中的突变导致两个宿主中镍抗性增加两到五倍。这些基因被认为参与了cnr的调控。将pMOL28的cnr与CH34的另一个大质粒pMOL30的czc进行比较，发现结构基因按相同顺序排列并决定分子量相似的蛋白质。最大的蛋白质CnrA与CzcA（czc操纵子的最大蛋白质）具有46%的氨基酸相似性。其他推定的基因产物CnrB和CnrC分别与czc的相应蛋白质具有28%和30%的相似性。
