Schmidt M, Arnold W, Niemann A, Kleickmann A, Pühler A
Universität Bielefeld, Lehrstuhl für Genetik, Germany.
Gene. 1992 Dec 1;122(1):35-43. doi: 10.1016/0378-1119(92)90029-o.
Interspecific complementation of a Xanthomonas campestris pv. campestris phosphomannose isomerase (PMI) mutant was used to isolate a cosmid from a genomic library of Rhizobium meliloti 2011 carrying the pmi gene of this strain. Subcloning experiments localized the coding region to a 2.0-kb SalI-ClaI fragment. Nucleotide sequence analysis of this fragment indicated the presence of two open reading frames (ORFs), coding for 18- and 43-kDa polypeptides. The analysis of the gene function by gene disruption experiments showed that ORF2 codes for pmi. A comparison of the deduced amino acid sequence with the corresponding sequences of the Pseudomonas aeruginosa and Escherichia coli PMIs revealed no significant homology, indicating that the isolated gene encodes a new type of PMI. The construction of a pmi-deficient mutant of R. meliloti using the sacB-sacR cassette technique showed that the loss of PMI activity does not affect the symbiotic properties of this strain.
利用野油菜黄单胞菌野油菜致病变种（Xanthomonas campestris pv. campestris）磷酸甘露糖异构酶（PMI）突变体的种间互补作用，从苜蓿根瘤菌（Rhizobium meliloti）2011基因组文库中分离出一个携带该菌株pmi基因的黏粒。亚克隆实验将编码区定位到一个2.0 kb的SalI-ClaI片段。对该片段的核苷酸序列分析表明存在两个开放阅读框（ORF），分别编码18 kDa和43 kDa的多肽。通过基因破坏实验对基因功能进行分析，结果表明ORF2编码pmi。将推导的氨基酸序列与铜绿假单胞菌（Pseudomonas aeruginosa）和大肠杆菌（Escherichia coli）的PMI相应序列进行比较，未发现显著同源性，这表明分离出的基因编码一种新型的PMI。利用sacB-sacR盒技术构建苜蓿根瘤菌的pmi缺陷突变体，结果表明PMI活性的丧失并不影响该菌株的共生特性。
