Michiels J, Van Soom T, D'hooghe I, Dombrecht B, Benhassine T, de Wilde P, Vanderleyden J
F. A. Janssens Laboratory of Genetics, K.U. Leuven, Heverlee, Belgium.
J Bacteriol. 1998 Apr;180(7):1729-40. doi: 10.1128/JB.180.7.1729-1740.1998.
The rpoN region of Rhizobium etli was isolated by using the Bradyrhizobium japonicum rpoN1 gene as a probe. Nucleotide sequence analysis of a 5,600-bp DNA fragment of this region revealed the presence of four complete open reading frames (ORFs), ORF258, rpoN, ORF191, and ptsN, coding for proteins of 258, 520, 191, and 154 amino acids, respectively. The gene product of ORF258 is homologous to members of the ATP-binding cassette-type permeases. ORF191 and ptsN are homologous to conserved ORFs found downstream from rpoN genes in other bacterial species. Unlike in most other microorganisms, rpoN and ORF191 are separated by approximately 1.6 kb. The R. etli rpoN gene was shown to control in free-living conditions the production of melanin, the activation of nifH, and the metabolism of C4-dicarboxylic acids and several nitrogen sources (ammonium, nitrate, alanine, and serine). Expression of the rpoN gene was negatively autoregulated and occurred independently of the nitrogen source. Inactivation of the ptsN gene resulted in a decrease of melanin synthesis and nifH expression. In a search for additional genes controlling the synthesis of melanin, an R. etli mutant carrying a Tn5 insertion in ptsA, a gene homologous to the Escherichia coli gene coding for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system, was obtained. The R. etli ptsA mutant also displayed reduced expression of nifH. The ptsN and ptsA mutants also displayed increased sensitivity to the toxic effects of malate and succinate. Growth of both mutants was inhibited by these C4-dicarboxylates at 20 mM at pH 7.0, while wild-type cells grow normally under these conditions. The effect of malate occurred independently of the nitrogen source used. Growth inhibition was decreased by lowering the pH of the growth medium. These results suggest that ptsN and ptsA are part of the same regulatory cascade, the inactivation of which renders the cells sensitive to toxic effects of elevated concentrations of malate or succinate.
用日本慢生根瘤菌rpoN1基因作为探针，分离出了费氏中华根瘤菌的rpoN区域。对该区域一个5600 bp的DNA片段进行核苷酸序列分析，发现存在四个完整的开放阅读框（ORF），即ORF258、rpoN、ORF191和ptsN，它们分别编码258、520、191和154个氨基酸的蛋白质。ORF258的基因产物与ATP结合盒式通透酶家族成员同源。ORF191和ptsN与其他细菌物种中rpoN基因下游发现的保守ORF同源。与大多数其他微生物不同，rpoN和ORF191相隔约1.6 kb。费氏中华根瘤菌的rpoN基因在自由生活条件下被证明可控制黑色素的产生、nifH的激活以及C4 - 二羧酸和几种氮源（铵、硝酸盐、丙氨酸和丝氨酸）的代谢。rpoN基因的表达受到负向自调控，且其发生与氮源无关。ptsN基因的失活导致黑色素合成和nifH表达减少。在寻找控制黑色素合成的其他基因时，获得了一株费氏中华根瘤菌突变体，该突变体在ptsA基因中插入了Tn5，ptsA基因与编码磷酸烯醇丙酮酸：糖磷酸转移酶系统酶I的大肠杆菌基因同源。费氏中华根瘤菌ptsA突变体的nifH表达也降低。ptsN和ptsA突变体对苹果酸和琥珀酸的毒性作用也表现出更高的敏感性。在pH 7.0时，20 mM的这些C4 - 二羧酸会抑制这两种突变体的生长，而野生型细胞在这些条件下正常生长。苹果酸的这种作用与所使用的氮源无关。通过降低生长培养基的pH值可减轻生长抑制。这些结果表明，ptsN和ptsA是同一调控级联的一部分，其失活使细胞对高浓度苹果酸或琥珀酸的毒性作用敏感。
