Zeilstra-Ryalls J H, Kaplan S
Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston 77225, USA.
J Bacteriol. 1995 May;177(10):2760-8. doi: 10.1128/jb.177.10.2760-2768.1995.
Rhodobacter sphaeroides H-5 was isolated as a 5-aminolevulinic acid (ALA) auxotroph following treatment of wild-type cells with N-methyl-N-nitroso-N'-nitroguanidine (J. Lascelles and T. Altshuler, J. Bacteriol. 98:721-727, 1969). The existence in R. sphaeroides 2.4.1 of the genes hemA and hemT, each encoding the enzyme 5-aminolevulinic acid synthase (EC 2.3.1.37), raised questions as to the genetic basis for the ALA auxotrophy in mutant H-5. We therefore cloned both the hemA and hemT genes from mutant H-5. The hemA gene has been sequenced in its entirety and bears four base pair substitutions which encode three amino acid changes relative to the sequence of wild-type strain 2.4.1. Complementation analysis of an Escherichia coli ALA auxotroph has revealed that the loss of ALA synthase activity in the HemA mutant enzyme could be localized to two of the amino acid substitutions. On the other hand, the hemT gene from mutant H-5 was able to complement an E. coli mutant requiring ALA for growth. Complementation analyses were also carried out by introducing the cloned hemA or hemT gene of mutant H-5 or wild-type 2.4.1 in trans into H-5 and, in parallel, into our previously described HemA-HemT double mutant strain AT1 (E. L. Neidle and S. Kaplan, J. Bacteriol. 175:2304-2313, 1993). This analysis revealed that while the complementation pattern of mutant AT1 parallels that for the E. coli ALA auxotroph, mutant H-5 could only be complemented by the wild-type hemA gene. The ability of the hemT gene of either mutant H-5 or wild-type 2.4.1 to complement the ALA auxotrophy of mutant AT1 but not mutant H-5 was consistent with beta-galactosidase activities obtained with hemT-lacZ transcriptional fusions. We conclude that the ALA auxotrophy of mutant H-5 arises from (i) a nonfunctional HemA protein containing multiple missense substitutions and (ii) an inability of the normal hemT gene to be expressed in the mutant H-5 genetic background, i.e., an additional mutation of unknown origin is required for hemT expression. These studies bear directly on the regulation of the expression of the hemA and hemT genes of R. sphaeroides 2.4.1.
球形红细菌H-5是在用N-甲基-N-亚硝基-N'-硝基胍处理野生型细胞后作为5-氨基乙酰丙酸（ALA）营养缺陷型分离得到的（J. Lascelles和T. Altshuler，《细菌学杂志》98:721 - 727，1969年）。球形红细菌2.4.1中存在hemA和hemT基因，每个基因都编码5-氨基乙酰丙酸合酶（EC 2.3.1.37），这就引发了关于突变体H-5中ALA营养缺陷型的遗传基础的问题。因此，我们从突变体H-5中克隆了hemA和hemT基因。hemA基因已被完整测序，相对于野生型菌株2.4.1的序列，它有四个碱基对替换，编码三个氨基酸变化。对大肠杆菌ALA营养缺陷型的互补分析表明，HemA突变酶中ALA合酶活性的丧失可定位到两个氨基酸替换上。另一方面，突变体H-5的hemT基因能够互补需要ALA才能生长的大肠杆菌突变体。通过将突变体H-5或野生型2.4.1的克隆hemA或hemT基因反式导入H-5，并同时导入我们之前描述的HemA-HemT双突变体菌株AT1（E. L. Neidle和S. Kaplan，《细菌学杂志》175:2304 - 2313，1993年），也进行了互补分析。该分析表明，虽然突变体AT1的互补模式与大肠杆菌ALA营养缺陷型的互补模式相似，但突变体H-5只能被野生型hemA基因互补。突变体H-5或野生型2.4.1的hemT基因能够互补突变体AT1的ALA营养缺陷型，但不能互补突变体H-5，这与hemT - lacZ转录融合获得的β-半乳糖苷酶活性一致。我们得出结论，突变体H-5的ALA营养缺陷型源于（i）含有多个错义替换的无功能HemA蛋白，以及（ii）正常的hemT基因在突变体H-5遗传背景中无法表达，即hemT表达需要一个未知来源的额外突变。这些研究直接关系到球形红细菌2.4.1中hemA和hemT基因表达的调控。
