Fuchs T M, Deppisch H, Scarlato V, Gross R
Theodor-Boveri-Institut für Biowissenchaften, Lehrstuhl für Mikrobiologie, Universität Würzburg, Germany.
J Bacteriol. 1996 Aug;178(15):4445-52. doi: 10.1128/jb.178.15.4445-4452.1996.
Recently, a novel type of regulatory mutation causing differential effects on the expression of virulence genes due to a slight overexpression of the RNA polymerase alpha subunit (RpoA) was found in Bordetella pertussis (N. H. Carbonetti, T. M. Fuchs, A. A. Patamawenu, T. J. Irish, H. Deppisch, and R. Gross, J. Bacteriol. 176:7267-7273, 1994). To gather information on the molecular events behind this phenomenon, we isolated suppressor mutants of the RpoA-overexpressing strains after random mutagenesis. Genetic characterization of these suppressor strains revealed the existence of at least three distinct groups of dominant alleles. Mutations occurred either in the rpoA locus itself, in the bvg locus, or in unknown gene loci. One mutant of the latter group was further characterized. By the introduction of a cosmid library containing genomic B. pertussis DNA into this suppressor strain, we isolated a cosmid which suppressed the phenotype of the suppressor strain, thus restoring the negative effect on transcription of the ptx and cya toxin genes. Mutagenesis of the cosmid with Tn5 led to the identification of the gene locus responsible for this phenomenon. Its DNA sequence revealed the presence of an open reading frame (ORF) consisting of 2,373 bp coding for a hypothetical 86-kDa protein with extensive sequence similarities to ORFs with not yet identified functions of Escherichia coli, Haemophilus influenzae, and Neisseria meningitidis. The new gene, termed tex, for toxin expression, seems to be an essential factor for B. pertussis, as it cannot be deleted from the bacterial chromosome. All members of this new protein family show significant sequence similarities with the mannitol repressor protein MtlR and with the presumptive RNA-binding domains of the Pnp and ribosomal S1 proteins of E. coli in their N- and C-terminal parts, respectively. These sequence similarities and the fact that the tex gene was isolated by virtue of its effects on gene expression in B. pertussis indicate that the members of this new protein family may play an important role in the transcription machinery of prokaryotic organisms.
最近，在百日咳博德特氏菌中发现了一种新型调控突变，由于RNA聚合酶α亚基（RpoA）略有过表达，该突变对毒力基因的表达产生了不同影响（N. H. 卡尔博内蒂、T. M. 富克斯、A. A. 帕塔马韦努、T. J. 爱尔兰、H. 德皮施和R. 格罗斯，《细菌学杂志》176:7267 - 7273，1994年）。为了收集关于这一现象背后分子事件的信息，我们在随机诱变后分离了RpoA过表达菌株的抑制突变体。对这些抑制菌株的遗传特征分析揭示了至少三组不同的显性等位基因的存在。突变发生在rpoA基因座本身、bvg基因座或未知基因座。对后一组中的一个突变体进行了进一步表征。通过将含有百日咳博德特氏菌基因组DNA的黏粒文库导入该抑制菌株，我们分离出了一个黏粒，它抑制了抑制菌株的表型，从而恢复了对ptx和cya毒素基因转录的负面影响。用Tn5对该黏粒进行诱变导致鉴定出了负责这一现象的基因座。其DNA序列显示存在一个由2373 bp组成的开放阅读框（ORF），编码一种假定的86 kDa蛋白质，该蛋白质与大肠杆菌、流感嗜血杆菌和脑膜炎奈瑟菌中功能尚未确定的ORF具有广泛的序列相似性。这个新基因，称为tex（毒素表达之意），似乎是百日咳博德特氏菌的一个必需因子，因为它不能从细菌染色体中删除。这个新蛋白质家族的所有成员在其N端和C端部分分别与甘露醇阻遏蛋白MtlR以及大肠杆菌Pnp和核糖体S1蛋白的假定RNA结合结构域显示出显著的序列相似性。这些序列相似性以及tex基因因其对百日咳博德特氏菌基因表达的影响而被分离这一事实表明，这个新蛋白质家族的成员可能在原核生物的转录机制中发挥重要作用。
