Isolation, DNA sequence analysis, and mutagenesis of a proline dehydrogenase gene (putA) from Bradyrhizobium japonicum.
Isolation, DNA sequence analysis, and mutagenesis of a proline dehydrogenase gene (putA) from Bradyrhizobium japonicum.
复制标题
日本慢生根瘤菌脯氨酸脱氢酶基因 (putA) 的分离、DNA 序列分析和诱变。
DOI：
10.1128/aem.62.1.221-229.1996
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发表时间：
1996
影响因子：
4.4
通讯作者：
Kohl,DH
中科院分区：
文献类型：
--
作者：
Straub,PF;Reynolds,PH;Althomsons,S;Mett,V;Zhu,Y;Shearer,G;Kohl,DH
本文报道了大豆慢生根瘤菌（Bradyrhizobium japonicum）脯氨酸脱氢酶（putA）基因的克隆和序列分析。鉴定了编码1，016个氨基酸的开放阅读框。B japonicum基因编码具有脯氨酸脱氢酶和吡咯啉-5-羧酸（P5 C）脱氢酶活性的双功能蛋白质，如在大肠杆菌和鼠伤寒沙门氏菌中一样。将这些蛋白质的序列与其它脯氨酸和P5 C脱氢酶序列进行比较，鉴定了脯氨酸脱氢酶和P5 C脱氢酶催化结构域。在脯氨酸脱氢结构域中，观察到了与B. japonicum，日本E. coli、S.鼠伤寒沙门氏菌、酿酒酵母put 1和黑腹果蝇slgA。在P5 C脱氢酶结构域中，观察到B. japonicum，日本E. coli、S.鼠伤寒沙门氏菌、枯草芽孢杆菌ipa 76 d和S.酿酒酵母PUT 2。在P5 C脱氢酶结构域中观察到半醛脱氢酶的共有催化位点。这表明该结构域的底物可能是开链γ-谷氨酰半醛，而不是其环化形式P5 C。与从E. coli、S.鼠伤寒沙门氏菌和K.肺炎杆菌、B. japonicum putA基因似乎不是具有脯氨酸转运体基因（putP）的操纵子的一部分。此外，B. japonicum基因缺失了E. coli和革兰氏阳性菌S.鼠伤寒基因。通过插入抗生素抗性基因盒破坏该基因，然后将其重组到细菌染色体中。分离出缺乏putA活性的共生活性突变株。利用这个脯氨酸脱氢酶克隆，我们将检验putA在共生固氮菌B中的假说。japonicumBacteroid在转录上受干旱和其它胁迫的调节。
We report here the cloning and sequencing of the gene for proline dehydrogenase (putA) of Bradyrhizobium japonicum. An open reading frame coding for 1,016 amino acids was identified. The B. japonicum gene codes for a bifunctional protein with proline dehydrogenase and pyrroline-5-carboxylate (P5C) dehydrogenase activities, as it does in Escherichia coli and Salmonella typhimurium. Comparison of the sequences of these proteins with other proline and P5C dehydrogenase sequences identified proline dehydrogenase and P5C dehydrogenase catalytic domains. Within the proline dehydrogenation domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Saccharomyces cerevisiae put1, and Drosophila melanogaster slgA. Within the P5C dehydrogenase domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Bacillus subtilis ipa76d, and S. cerevisiae put2. A consensus catalytic site for semialdehyde dehydrogenase was observed in the P5C dehydrogenase domain. This suggests that the substrate for this domain may be the open-chain gamma-glutamylsemialdehyde, not its cyclized form, P5C. Unlike the gene isolated from E. coli, S. typhimurium, and K. pneumoniae, the B. japonicum putA gene does not appear to be part of an operon with the proline porter gene (putP). Additionally, the B. japonicum gene lacks the putative C-terminal regulatory domain present in the E. coli and S. typhimurium genes. The gene was disrupted by insertion of antibiotic resistance gene cassettes, which were then recombined into the bacterial chromosome. Symbiotically active mutant strains that were devoid of putA activity were isolated. With this proline dehydrogenase clone, we will test the hypothesis that putA in symbiotic nitrogen-fixing B. japonicum bacteroids is transcriptionally regulated by drought and other stresses.