Zeilstra-Ryalls J H, Kaplan S
Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston 77225, USA.
J Bacteriol. 1996 Feb;178(4):985-93. doi: 10.1128/jb.178.4.985-993.1996.
Rhodobacter sphaeroides 2.4.1 has the ability to synthesize a variety of tetrapyrroles, reflecting the metabolic versatility of this organism and making it capable of aerobic, anaerobic, photosynthetic, and diazotrophic growth. The hemA and hemT genes encode isozymes that catalyze the formation of 5-aminolevulinic acid, the first step in the biosynthesis of all tetrapyrroles present in R. sphaeroides 2.4.1. As part of our studies of the regulation and expression of these genes, we developed a genetic selection that uses transposon mutagenesis to identify loci affecting the aerobic expression of the hemA gene. In developing this selection, we found that sequences constituting an open reading frame immediately upstream of hemA positively affect hemA transcription. Using a transposon-based selection for increased hemA expression in the absence of the upstream open reading frame, we isolated three independent mutants. We have determined that the transposon insertions in these strains map to three different loci located on chromosome 1. One of the transposition sites mapped in the vicinity of the recently identified R. sphaeroides 2.4.1 homolog of the anaerobic regulatory gene fnr. By marker rescue and DNA sequence analysis, we found that the transposition site was located between the first two genes of the cco operon in R. sphaeroides 2.4.1, which encodes a cytochrome c terminal oxidase. Examination of the phenotype of the mutant strain revealed that, in addition to increased aerobic expression of hemA, the transposition event also conferred an oxygen-insensitive development of the photosynthetic membranes. We propose that the insertion of the transposon in cells grown in the presence of high oxygen levels has led to the generation of a cellular redox state resembling either reduced oxygen or anaerobiosis, thereby resulting in increased expression of hemA, as well as the accumulation of spectral complex formation. Several models are presented to explain these findings.
球形红细菌2.4.1能够合成多种四吡咯，这反映了该生物体的代谢多样性，使其能够进行需氧、厌氧、光合和固氮生长。hemA和hemT基因编码同工酶，催化5-氨基乙酰丙酸的形成，这是球形红细菌2.4.1中所有四吡咯生物合成的第一步。作为我们对这些基因调控和表达研究的一部分，我们开发了一种遗传筛选方法，利用转座子诱变来鉴定影响hemA基因需氧表达的位点。在开发这种筛选方法时，我们发现hemA上游紧邻的一个开放阅读框序列对hemA转录有正向影响。利用基于转座子的筛选方法在没有上游开放阅读框的情况下提高hemA表达，我们分离出了三个独立的突变体。我们已经确定这些菌株中的转座子插入定位到位于染色体1上的三个不同位点。其中一个转座位点定位在最近鉴定的球形红细菌2.4.1厌氧调节基因fnr的同源物附近。通过标记拯救和DNA序列分析，我们发现转座位点位于球形红细菌2.4.1中cco操纵子的前两个基因之间，该操纵子编码一种细胞色素c末端氧化酶。对突变菌株表型的检查表明，除了hemA的需氧表达增加外，转座事件还赋予了光合膜对氧不敏感的发育。我们提出，在高氧水平下生长的细胞中转座子的插入导致了一种类似于还原氧或厌氧状态的细胞氧化还原状态的产生，从而导致hemA表达增加以及光谱复合物形成的积累。提出了几种模型来解释这些发现。
