Cloning of the Rhodobacter capsulatus hemA gene

Biel, Susan W.; Wright, Maureen S.; Biel, A J

doi:10.1128/jb.170.9.4382-4384.1988

Cited by 31 publications

(23 citation statements)

References 13 publications

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“…In the case of the hemA gene, we have demonstrated that the presence of the FNR binding sequence correlates with control of hemA gene expression by FnrL. Because the only known hemA gene in Rhodobacter capsulatus has no evident FNR binding consensus in its upstream sequences (2,11), the mechanism of regulation of the hemA gene between these two organisms is apparently different. Further, the presence of a second gene, hemT, in R. sphaeroides 2.4.1 implies that the mechanism by which ALA synthesis is regulated in these two organisms must be completely different and could have implications as to the regulation of other gene products such as cytochromes and the polypeptides of the photosynthetic complexes.…”

Section: Discussionmentioning

confidence: 93%

Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene

1995

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In Rhodobacter sphaeroides 2.4.1, the cellular requirements for 5-aminolevulinic acid (ALA) are in part regulated by the level of ALA synthase activity, which is encoded by the hemA and hemT genes. Under standard growth conditions, only the hemA gene is transcribed, and the level of ALA synthase activity varies in response to oxygen tension. The presence of an FNR consensus sequence upstream of hemA suggested that oxygen regulation of hemA expression could be mediated, in part, through a homolog of the fnr gene. Two independent studies, one detailed here, identified a region of the R. sphaeroides 2.4.1 genome containing extensive homology to the fix region of the symbiotic nitrogen-fixing bacteria Rhizobium meliloti and Bradyrhizobium japonicum. Within this region that maps to 443 kbp on chromosome I, we have identified an fnr homolog (fnrL), as well as a gene that codes for an anaerobic coproporphyrinogen III oxidase, the second such gene identified in this organism. We also present an analysis of the role of fnrL in the physiology of R. sphaeroides 2.4.1 through the construction and characterization of fnrL-null strains. Our results further show that fnrL is essential for both photosynthetic and anaerobic-dark growth with dimethyl sulfoxide. Analysis of hemA expression, with hemA::lacZ transcriptional fusions, suggests that FnrL is an activator of hemA under anaerobic conditions. On the other hand, the open reading frame immediately upstream of hemA appears to be an activator of hemA transcription regardless of either the presence or the absence of oxygen or FnrL. Given the lack of hemT expression under these conditions, we consider FnrL regulation of hemA expression to be a major factor in bringing about changes in the level of ALA synthase activity in response to changes in oxygen tension.

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Section: Discussionmentioning

confidence: 93%

Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene

1995

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“…Although a single hemA gene is found in other bacteria, including Rhodobacter capsulatus (3,12), two homologous ALA synthase-encoding genes have been maintained in R. sphaeroides. In this bacterium, there are several homologous genes, each located on a different chromosome (13,33,34).…”

Section: Discussionmentioning

confidence: 99%

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

Neidle

Kaplan

1993

J Bacteriol

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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...

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“…Our initial interest in the identification of an fnrL homolog in R. capsulatus stemmed from a distinction with respect to the hemA genes of R. capsulatus (6,23) and R. sphaeroides (38). Both genes code for ALA synthase, which catalyzes the synthesis of ALA, the first step in the biosynthesis of all tetrapyrroles in these organisms.…”

Section: Discussionmentioning

confidence: 99%

Analysis of the fnrL gene and its function in Rhodobacter capsulatus

et al. 1997

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The fnr gene encodes a regulatory protein involved in the response to oxygen in a variety of bacterial genera. For example, it was previously shown that the anoxygenic, photosynthetic bacterium Rhodobacter sphaeroides requires the fnrL gene for growth under anaerobic, photosynthetic conditions. Additionally, the FnrL protein in R. sphaeroides is required for anaerobic growth in the dark with an alternative electron acceptor, but it is not essential for aerobic growth. In this study, the fnrL locus from Rhodobacter capsulatus was cloned and sequenced. Surprisingly, an R. capsulatus strain with the fnrL gene deleted grows like the wild type under either photosynthetic or aerobic conditions but does not grow anaerobically with alternative electron acceptors such as dimethyl sulfoxide (DMSO) or trimethylamine oxide. It is demonstrated that the c-type cytochrome induced upon anaerobic growth on DMSO is not synthesized in the R. capsulatus fnrL mutant. In contrast to wild-type strains, R. sphaeroides and R. capsulatus fnrL mutants do not synthesize the anaerobically, DMSO-induced reductase. Mechanisms that explain the basis for FnrL function in both organisms are discussed.Rhodobacter capsulatus is a purple nonsulfur bacterium capable of both aerobic and anaerobic respiration, as well as photosynthetic growth. Development of the photosynthetic apparatus in this organism is induced following the lowering of oxygen tension, and several recent reviews outline the current models of regulation of the R. capsulatus photosystem genes (4,5,29). A key regulator in this process is the product of the crtJ gene, which appears to encode a repressor of certain photosystem genes under aerobic conditions (42). Another major pathway is the reg two-component regulatory system. The RegA protein (43) and its cognate histidine kinase, RegB (35), appear to control activation of certain photosystem genes under anaerobic conditions (reviewed in reference 5).Genes corresponding to both crtJ (18, 41) and the reg genes (15, 16, 33) have been identified in the closely related photosynthetic bacterium Rhodobacter sphaeroides. Also present in R. sphaeroides is the anaerobic regulatory protein, FnrL (58). FnrL is considered to be the R. sphaeroides homolog of the Fnr protein of Escherichia coli, which acts as a redox-responsive transcriptional regulator that activates genes whose products are involved in anaerobic respiration and represses other genes required for aerobic respiration in that organism. Homologs of Fnr have been identified in several gram-negative bacteria (reviewed in reference 46), with specific differences attributed to members of the FNR regulons identified for each organism. While these differences reflect the unique characteristics of the various bacteria, it appears that all of the genes under positive control of Fnr or its homologs are involved in metabolisms active under conditions of reduced oxygen availability (46).R. sphaeroides fnrL mutants are incapable of photosynthetic growth (58), suggesting that FnrL is involved, di...

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Cloning of the Rhodobacter capsulatus hemA gene

Cited by 31 publications

References 13 publications

Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene

Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene

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

Analysis of the fnrL gene and its function in Rhodobacter capsulatus

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