5-Aminolevulinic acid availability and control of spectral complex formation in hemA and hemT mutants of Rhodobacter sphaeroides

Abstract

In the photosynthetic bacterium Rhodobacter sphaeroides, two genes, hemA and hemT, each encode a distinct 5-aminolevulinic acid (ALA) synthase isozyme (E. L. Neidle and S. Kaplan, J. Bacteriol. 175:2292Bacteriol. 175: -2303Bacteriol. 175: , 1993. This enzyme catalyzes the first and rate-limiting step in a branched pathway for tetrapyrrole formation, leading to the biosynthesis of hemes, bacteriochlorophylls, and corrinoids. In an attempt to determine the functions of hemA and hemT, mutant strains were constructed with specific chromosomal disruptions. These chromosomal disruptions allowed hemA and hemT to be precisely localized on the larger and smaller of two R. sphaeroides chromosomes, respectively. Mutants carrying a single hemA or hemT disruption grew well without the addition of ALA, whereas a mutant, HemAT1, in which hemA and hemT had both been inactivated required exogenous ALA for growth. The growth rates, ALA synthase enzyme levels, and the amounts of bacteriochlorophyllcontaining intracytoplasmic membrane spectral complexes of all strains were compared. Under photosynthetic growth conditions, the levels of bacteriochlorophyll, carotenoids, and B800-850 and B875 light-harvesting complexes were significantly lower in the Hem mutants than in the wild type. In the mutant strains, available bacteriochlorophyll appeared to be preferentially targeted to the B875 light-harvesting complex relative to the B800-850 complex. In strain HemAT1, the amount of B800-850 complex varied with the concentration of ALA added to the growth medium, and under conditions of ALA limitation, no B800-850 complexes could be detected. In the Hem mutants, there were aberrant transcript levels corresponding to the puc and puf operons encoding structural polypeptides of the B800-850 and B875 complexes. These results suggest that hemA and hemT expression is coupled to the genetic control of the R. sphaeroides photosynthetic apparatus.In the facultative photosynthetic bacterium Rhodobacter sphaeroides, 5-aminolevulinic acid (ALA), the first committed intermediate in tetrapyrrole biosynthesis, is formed from glycine and succinyl coenzyme A (succinyl-CoA) by ALA synthase (succinyl-CoA:glycine C-succinyl transferase [decarboxylating], EC 2.3.1.37). It has previously been shown that ALA formation regulates the production of hemes and bacteriochlorophylls (17). ALA was also shown to affect the transcriptional control of the cytochrome c2 gene, cycA (30). Despite key regulatory roles suggested for this metabolite, however, little is known about the mechanisms by which the formation of ALA is regulated or by which ALA-mediated regulation is exerted.In R. sphaeroides, each of two genes, hemA and hemT, encodes a distinct ALA synthase isozyme (26). Prior to the discovery of isozymes, many studies were interpreted on the assumption of a single ALA synthase. The validity of this assumption seemed to be supported by the isolation of a mutant R. sphaeroides strain, H-5, lacking ALA synthase activity (18). The H-5 and wild-type strains, however, differ...

Generalized approach to the regulation and integration of gene expression

“…This increased flow can result from extra copies of hemN encoding coproporphyrinogen III oxidase (Yeliseev and Kaplan, 1999) or after the addition of exogenous 5-aminolaevulinic acid (ALA) (Neidle and Kaplan, 1993a). This led us to suggest that increased cellular levels of certain porphyrin intermediate(s), e.g.…”

DNA Sequence Duplication in Rhodobacter sphaeroides 2.4.1: Evidence of an Ancient Partnership between Chromosomes I and II

“…The R. sphaeroides genome possesses a high degree of gene duplication (25,26). Studies involving the genetic and biochemical characterization of a number of gene duplications in R. sphaeroides have been conducted previously (8,13,14,28,29,30).Duplications can arise from single-gene duplications, duplication of short chromosomal fragments, duplication of an entire chromosome, or duplication of the whole genome; these events are thought to be major sources of evolutionary novelties (33). In order to uncover both the nature and the amount of exact DNA sequence duplication within and between the two chromosomes, we aligned the CI and CII DNA sequences to each other and also against their own sequences.…”

“…The R. sphaeroides genome possesses a high degree of gene duplication (25,26). Studies involving the genetic and biochemical characterization of a number of gene duplications in R. sphaeroides have been conducted previously (8,13,14,28,29,30).…”

Regulation of 5-aminolevulinic acid synthesis in Rhodobacter sphaeroides 2.4.1: the genetic basis of mutant H-5 auxotrophy

“…Vitamin B 12 , yet another tetrapyrrole, is required under all growth conditions. The synthesis of 5-aminolevulinic acid (ALA), the first committed precursor in the biosynthesis of all tetrapyrroles in R. sphaeroides, is catalyzed by ALA synthase, encoded in this organism by two genes, hemA and hemT (15,16,23). Whereas most bacteria examined and all plants synthesize ALA from glutamic acid in a series of steps involving a glutamyl-tRNA intermediate through the C5 pathway, R. sphaeroides and related bacteria synthesize ALA by the same reaction mechanism that occurs in yeast and animal cell mitochondria.…”