Oxygen intervention in the regulation of gene expression: the photosynthetic bacterial paradigm.

The means by which oxygen intervenes in gene expression has been examined in considerable detail in the metabolically versatile bacterium Rhodobacter sphaeroides. Three regulatory systems are now known in this organism, which are used singly and in combination to modulate genes in response to changing oxygen availability. The outcome of these regulatory events is that the molecular machinery is present for the cell to obtain energy by means that are best suited to prevailing conditions, while at the same time maintaining cellular redox balance. Here, we explore the dangers associated with molecular oxygen relative to the various metabolisms used by R. sphaeroides, and then present the most recent findings regarding the features and operation of each of the three regulatory systems which collectively mediate oxygen control in this organism.

Role of the fnrL gene in photosystem gene expression and photosynthetic growth of Rhodobacter sphaeroides 2.4.1.

Anoxygenic photosynthetic growth of Rhodobacter sphaeroides 2.4.1 requires a functional fnrL gene, which encodes the anaerobic regulator, FnrL. Using transcriptional fusions to the puc operon in which the upstream FNR consensus-like sequence is either present or absent, we obtained results that suggest that FnrL has both a direct and an indirect role in puc operon expression. In addition to FnrL, several other factors, including the two-component Prr regulatory system and the transcriptional repressor PpsR, are known to mediate oxygen control of photosynthesis gene expression in this organism. Therefore, we examined the relationship between FnrL and these other regulatory elements. Our results indicate that while mutations of prr or ppsR can lead to an increase in expression of some photosynthesis genes under aerobic and anaerobic conditions, regardless of the presence or absence of FnrL, there remains an absolute requirement for a functional fnrL gene for photosynthetic growth. We examined the potential role(s) of FnrL in photosynthetic growth by considering several target genes which may be required for this growth mode.

Analysis of the fnrL gene and its function in Rhodobacter capsulatus.

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.

Control of hemA expression in Rhodobacter sphaeroides 2.4.1: regulation through alterations in the cellular redox state.

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.

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

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 chromsome 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.

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

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.

Shared operator recognition specificity between Trp repressor and the repressors of bacteriophage 434.

Trp repressor is the only DNA-binding regulatory protein having a helix-turn-helix motif that has been reported to engage its operator target by a mechanism termed indirect readout: the Trp repressor-DNA interface is replete with hydrogen bonds between amino acid residues and non-esterified oxygen atoms of the sugar-phosphate backbone, and contains numerous specifically positioned water molecules. In Escherichia coli mutants deleted for trpR, the immunity repressor of phage 434 led to an eightfold reduction in trp promoter utilization. The Cro434 repressor also inhibited transcription from the trp promoter. The 434 repressors, considered to interact directly with operator targets, carry recognition helices positioned near the N terminus of each protein. The DNA-recognizing elements of Trp repressor lie toward the C terminus. The trp operator thus appears to possess significant plasticity in terms of its ability to assume conformational states that allow complex formation with more than one class of regulatory protein.

Fragment tagging.

A tactic known as fragment tagging, which has proven to be exceptionally useful in expediting DNA cloning and plasmid construction schemes, is described. The advantage of fragment tagging is that it facilitates the isolation of specific plasmid DNA molecules present in small amounts within mixed pools of DNA. Four examples that illustrate several variations of the fragment tagging concept are presented.

A human hepatocellular carcinoma 3.0-kilobase DNA sequence transforms both rat liver cells and NIH3T3 fibroblasts and encodes a 52-kilodalton protein.

Neoplastic transformation of rat liver cells in vitro by DNA-mediated gene transfer with an oncogene, hhcM, derived from human (Mahlavu) hepatocellular carcinoma, is described and compared with that of NIH3T3 cells. hhcM was cloned in a neomycin-resistant simian virus 40 promoter vector (pNeor/S) and was designated pNrpM-1. BRL-1 or NIH3T3 cells, transfected with pNrpM-1 DNA, showed significant morphological changes, loss of contact inhibition, and anchorage-independent growth. They became highly tumorigenic in nude rats and nu/nu mice. Control BRL-1 and NIH3T3 cells, whether transfected with pNeor/S DNA or not, remained contact inhibited and nontumorigenic. Both the transformants and the tumor cells contained integrated hhcM DNA as shown by Southern blot hybridization. The complete nucleotide sequence of the hhcM 3.0-kilobase DNA was also determined, and it consisted of a possible open reading frame for a protein of 52 kilodaltons (467 amino acids). The high-level production of a slightly modified form of this 52-kilodalton protein in a bacterial expression system has been successfully achieved. The bacteria-produced protein was similar in electrophoretic behavior to the 52- to 53-kilodalton protein synthesized in a cell-free translation system using rabbit reticulocyte lysate programmed with hybrid-selected hhcM-specific mRNA from Mahlavu hepatocellular carcinoma cells.