Structural and Functional Analyses of Photosynthetic Regulatory Genes regA and regB from Rhodovulum sulfidophilum, Roseobacter denitrificans, and Rhodobacter capsulatus

Genes coding for putative RegA, RegB, and SenC homologues were identified and characterized in the purple nonsulfur photosynthetic bacteria Rhodovulum sulfidophilum and Roseobacter denitrificans, species that demonstrate weak or no oxygen repression of photosystem synthesis. This additional sequence information was then used to perform a comparative analysis with previously sequenced RegA, RegB, and SenC homologues obtained from Rhodobacter capsulatus and Rhodobacter sphaeroides. These are photosynthetic bacteria that exhibit a high level of oxygen repression of photosystem synthesis controlled by the RegA-RegB two-component regulatory system. The response regulator, RegA, exhibits a remarkable 78.7 to 84.2% overall sequence identity, with total conservation within a putative helix-turn-helix DNA-binding motif. The RegB sensor kinase homologues also exhibit a high level of sequence conservation (55.9 to 61.5%) although these additional species give significantly different responses to oxygen. A Rhodovulum sulfidophilum mutant lacking regA or regB was constructed. These mutants produced smaller amounts of photopigments under aerobic and anaerobic conditions, indicating that the RegA-RegB regulon controls photosynthetic gene expression in this bacterium as it does as in Rhodobacter species. Rhodobacter capsulatus regA- or regB-deficient mutants recovered the synthesis of a photosynthetic apparatus that still retained regulation by oxygen tension when complemented with reg genes from Rhodovulum sulfidophilum and Roseobacter denitrificans. These results suggest that differential expression of photosynthetic genes in response to aerobic and anaerobic growth conditions is not the result of altered redox sensing by the sensor kinase protein, RegB.     

Molecular genetic analysis suggesting interactions between AppA and PpsR in regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

The AppA protein plays an essential regulatory role in development of the photosynthetic apparatus in the anoxygenic phototrophic bacterium Rhodobacter sphaeroides 2.4.1 (M. Gomelsky and S. Kaplan, J. Bacteriol. 177:4609-4618, 1995). To gain additional insight into both the role and site of action of AppA in the regulatory network governing photosynthesis gene expression, we investigated the relationships between AppA and other known regulators of photosynthesis gene expression. We determined that AppA is dispensable for development of the photosynthetic apparatus in a ppsR null background, where PpsR is an aerobic repressor of genes involved in photopigment biosynthesis and puc operon expression. Moreover, all suppressors of an appA null mutation thus far isolated, showing improved photosynthetic growth, were found to contain mutations in the ppsR gene. Because ppsR gene expression in R. sphaeroides 2.4.1 appears to be largely independent of growth conditions, we suggest that regulation of repressor activity occurs predominately at the protein level. We have also found that PpsR functions as a repressor not only under aerobic but under anaerobic photosynthetic conditions and thereby is involved in regulating the abundance of the light harvesting complex II, depending on light intensity. It seems likely therefore, that PpsR responds to an integral signal (e.g., changes in redox potential) produced either by changes in oxygen tension or light intensity. The profile of the isolated suppressor mutations in PpsR is in accord with this proposition. We propose that AppA may be involved in a redox-dependent modulation of PpsR repressor activity.     

Autophosphorylation, phosphotransfer, and DNA-binding properties of the RegB/RegA two-component regulatory system in Rhodobacter capsulatus.

In the purple, photosynthetic bacterium, Rhodobacter capsulatus, the RegB/RegA two-component system is required for activation of several anaerobic processes, such as synthesis of the photosynthetic apparatus and assimilation of CO2 and N2. It is believed that RegB is an integral membrane histidine kinase that monitors the external environment. Under anaerobic growth conditions, it transduces a signal through phosphorylation of the response regulator, RegA, which then induces target gene expression. We used an in vitro assay to characterize the phosphorylation of wild-type RegA and a mutant variant (RegA*) that is responsible for abnormally high photosynthesis gene expression under both aerobic and anaerobic growth conditions. Phosphorylation assays indicate that phosphorylated RegA* (RegA* approximately P) is much more stable than RegA approximately P, indicating that it may be locked in a conformation that is resistant to dephosphorylation. DNase I footprint assays also indicate that unphosphorylated RegA* has a much higher affinity for specific DNA binding sites than the wild-type protein. Phosphorylation of RegA* increases DNA binding 2. 5-fold, whereas phosphorylation of RegA increases DNA binding more than 16-fold. Collectively, these results support the hypothesis that RegA* is a constitutively active variant that does not require phosphorylation to assume a structural conformation required to bind DNA.     

Binding of the bacteriophage T4 regA protein to mRNA targets: an initiator AUG is required.

Bacteriophage T4 regA protein translationally represses the synthesis of a subset of early phage-induced proteins. The protein binds to the translation initiation site of at least two mRNAs and prevents formation of the initiation complex. We show here that the protein binds to the translation initiation sites of other regA-sensitive mRNAs. Analysis of mRNA binding by filtration and nuclease protection assays shows that AUG is necessary but not sufficient for specific binding of regA protein to its mRNA targets. Anticipating the need for large quantities of regA protein for structural studies to further define the regA protein-RNA ligand interaction, we also report cloning the regA gene into a T4 overexpression system. The expression of regA protein in uninfected E. coli is lethal, so in our system regA driven by a strong T7 promoter is sequestered in a T4 phage until 'induction' by phage infection is desired. We have replaced the regA sensitive wild-type ribosome binding site with a strong insensitive ribosome binding site at an optimal distance from the regA initiation codon for maximizing expression. We have obtained large amounts of regA protein.     

Interacting regulatory circuits involved in orderly control of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1

FnrL, the homolog of the global anaerobic regulator Fnr, is required for the induction of the photosynthetic apparatus in Rhodobacter sphaeroides 2.4.1. Thus, the precise role of FnrL in photosynthesis (PS) gene expression and its interaction(s) with other regulators of PS gene expression are of considerable importance to our understanding of the regulatory circuitry governing spectral complex formation. Using a CcoP and FnrL double mutant strain, we obtained results which suggested that FnrL is not involved in the transduction of the inhibitory signal, by which PS gene expression is "silenced," emanating from the cbb(3) oxidase encoded by the ccoNOQP operon under aerobic conditions. The dominant effect of the ccoP mutation in the FnrL mutant strain with respect to spectral complex formation under aerobic conditions and restoration of a PS-positive phenotype suggested that inactivation of the cbb(3) oxidase to some extent bypasses the requirement for FnrL in the formation of spectral complexes. Additional analyses revealed that anaerobic induction of the bchE, hemN, and hemZ genes, which are involved in the tetrapyrrole biosynthetic pathways, requires FnrL. Thus, FnrL appears to be involved at multiple loci involved in the regulation of PS gene expression. Additionally, bchE was also shown to be regulated by the PrrBA two-component system, in conjunction with hemN and hemZ. These and other results to be discussed permit us to more accurately describe the role of FnrL as well as the interactions between the FnrL, PrrBA, and other regulatory circuits in the regulation of PS gene expression.     

Requirement for terminal cytochromes in generation of the aerobic signal for the arc regulatory system in Escherichia coli: study utilizing deletions and lac fusions of cyo and cyd.

Escherichia coli has two terminal oxidases for its respiratory chain: cytochrome o (low O2 affinity) and cytochrome d (high O2 affinity). Expression of the cyo operon, encoding cytochrome o, is decreased by anaerobic growth, whereas expression of the cyd operon, encoding cytochrome d, is increased by anaerobic growth. We show by the use of lac gene fusion that the expressions of cyo and cyd are under the control of the two-component arc system. In a cyo+ cyd+ background, expression of phi(cyo-lac) is higher when the organism is grown aerobically than when it is grown anaerobically. A mutation in either the sensor gene arcB or the pleiotropic regulator gene arcA almost abolishes the anaerobic repression. In the same background, expression of phi(cyd-lac) is higher under anaerobic growth conditions than under aerobic growth conditions. A mutation in arcA or arcB lowers both the aerobic and anaerobic expressions, suggesting that ArcA plays an activating role instead of the typical repressing role. Under aerobic growth conditions, double deletions of cyo and cyd lower phi(cyo-lac) expression but enhance phi(cyd-lac) expression. The double deletions also prevent elevated aerobic induction of the lct operon (encoding L-lactate dehydrogenase), another target operon of the arc system. In contrast, these deletions do not circumvent aerobic repression of the nar operon (encoding the anaerobic respiratory enzyme nitrate reductase) under the control of the pleiotropic fnr gene product. It thus appears that ArcB senses the presence of O2 by level of an electron transport component in reduced form or that of an nonautoxidizable compound linked to the process by a redox reaction, whereas Fnr senses O2 by a different mechanism.     

Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12.

The anaerobic regulation of the gene encoding pyruvate formate-lyase from Escherichia coli was investigated. Expression of a pfl'-'lacZ protein fusion demonstrated that the gene is subject to a 12-fold anaerobic induction which can be stimulated a further 2-fold by the addition of pyruvate to the growth medium. Construction of a strain deleted for pfl verified that either pyruvate or a metabolite of glycolysis functions as an inducer of pfl gene expression. Complete anaerobic induction required the presence of a functional fnr gene product. However, the dependence was not absolute since a two- to threefold anaerobic induction could still be observed in an fnr mutant. These results could be confirmed immunologically by analyzing the levels of pyruvate formate-lyase protein present in cells grown under various conditions. It was also shown that pfl'-'lacZ expression was partially repressed by nitrate and that this repression was mediated by the narL gene product.     

The bacteriophage T4 regA gene: primary sequence of a translational repressor

The regA gene product of bacteriophage T4 is an autogenously controlled translational regulatory protein that plays a role in differential inhibition (translational repression) of a subpopulation of T4-encoded "early" mRNA species. The structural gene for this polypeptide maps within a cluster of phage DNA replication genes, (genes 45-44-62-regA-43-42), all but one of which (gene 43) are under regA-mediated translational control. We have cloned the T4 regA gene, determined its nucleotide sequence, and identified the amino-terminal residues of a plasmid-encoded, hyperproduced regA protein. The results suggest that the T4 regA gene product is a 122 amino acid polypeptide that is mildly basic and hydrophilic in character; these features are consistent with known properties of regA protein derived from T4-infected cells. Computer-assisted analyses of the nucleotide sequences of the regA gene and its three upstream neighbors (genes 45, 44, and 62) suggest the existence of three translational initiation units in this four-gene cluster; one for gene 45, one for genes 44, 62 and regA, and one that serves only the regA gene. The analyses also suggest that the gene 44-62 translational unit harbors a stable RNA structure that obligates translational coupling of these two genes.     

Translational repression: biological activity of plasmid-encoded bacteriophage T4 RegA protein

The RegA protein of bacteriophage T4 is a translational repressor that regulates expression of several phage early mRNAs. We have cloned wild-type and mutant alleles of the T4 regA gene under control of the heat-inducible, plasmid-borne leftward promoter (PL) of phage lambda. Expression of the cloned regA+ gene resulted in the synthesis of a protein that closely resembled phage-encoded RegA protein in biological properties. It repressed its own synthesis (autogenous translational control) as well as the synthesis of specific T4-encoded proteins that are known from other studies to be under RegA-mediated translational control. Cloned mutant alleles of regA exhibited derepressed synthesis of the mutant regA gene products and were ineffective in trans against RegA-sensitive mRNA targets. The effects of plasmid-encoded RegA proteins were also demonstrated in experiments using two compatible plasmids in uninfected Escherichia coli. The two-plasmid assays confirm the sensitivities of several cloned T4 genes to RegA-mediated translational repression and are well-suited for genetic analysis of RegA target sites. Repression specificity in this system was demonstrated by using wild-type and operator-constitutive translational initiation sites of T4 rIIB fused to lacZ. The results show that no additional T4 products are required for RegA-mediated translational repression. Additional evidence is provided for the proposal that uridine-rich mRNA sequences are preferred targets for the repressor. Surprisingly, plasmid-generated RegA protein represses the synthesis of some E. coli proteins and appears to enhance selectively the synthesis of others. The RegA protein may have multiple functions, and its binding sites are not restricted to phage mRNAs.     

The PuhB protein of Rhodobacter capsulatus functions in photosynthetic reaction center assembly with a secondary effect on light-harvesting complex 1

The core of the photosynthetic apparatus of purple photosynthetic bacteria such as Rhodobacter capsulatus consists of a reaction center (RC) intimately associated with light-harvesting complex 1 (LH1) and the PufX polypeptide. The abundance of the RC and LH1 components was previously shown to depend on the product of the puhB gene (formerly known as orf214). We report here that disruption of puhB diminishes RC assembly, with an indirect effect on LH1 assembly, and reduces the amount of PufX. Under semiaerobic growth conditions, the core complex was present at a reduced level in puhB mutants. After transfer of semiaerobically grown cultures to photosynthetic (anaerobic illuminated) conditions, the RC/LH1 complex became only slightly more abundant, and the amount of PufX increased as cells began photosynthetic growth. We discovered that the photosynthetic growth of puhB disruption strains of R. capsulatus starts after a long lag period, which is due to physiological adaptation rather than secondary mutations. Using a hybrid protein expression system, we determined that the three predicted transmembrane segments of PuhB are capable of spanning a cell membrane and that the second transmembrane segment could mediate self-association of PuhB. We discuss the possible function of PuhB as a dimeric RC assembly factor.     

Germ-soma differentiation in volvox.

Volvox carteri is a spherical green alga with a predominantly asexual mode of reproduction and a complete germ-soma division of labor. Its somatic cells are specialized for motility, incapable of dividing, and programmed to die when only a few days old, whereas its gonidia (asexual reproductive cells) are nonmotile, specialized for growth and reproduction, and potentially immortal. When a gonidium is less than 2 days old it divides to produce a juvenile spheroid containing all of the somatic cells and gonidia that will be present in an adult of the next generation. The first visible step in germ-soma differentiation is a set of asymmetric cleavage divisions in the embryo that set apart small somatic initials from their large gonidial-initial sister cells. Three types of genes have been found to play key roles in germ-soma specification. First a set of gls genes act in the embryos to shift cell-division planes, resulting in the asymmetric divisions that set apart the large-small sister-cell pairs. Then a set of lag genes act in the large cells to prevent somatic differentiation, while the regA gene acts in the small cells to prevent reproductive development. An inducible transposon was used to tag and recover some of these and other developmentally important genes. The glsA gene encodes a chaperone-like protein that, like another chaperone that is one of its putative binding partners, is associated with the cell division apparatus, although how this leads to asymmetric division remains to be elucidated. The regA gene encodes a somatic-cell-specific nuclear protein that appears to function by repressing genes required for chloroplast biogenesis, thereby preventing somatic cells from growing enough to reproduce. Somatic-cell-specific expression of regA is controlled by three intronic enhancers.