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Bacteriophytochromes (BphPs) are light-sensing regulatory proteins encoded by photosynthetic and nonphotosynthetic bacteria. This protein class has been characterized structurally, but its biological activities remain relatively unexplored. Two BphPs in the anoxygenic photosynthetic bacterium Rhodopseudomonas palustris, designated regulatory proteins RpBphP2 and RpBphP3, are configured as light-regulated histidine kinases, which initiate a signal transduction system that controls expression of genes for the low light harvesting 4 (LH4) antenna complex. In vitro, RpBphP2 and RpBphP3 respond to light quality by reversible photoconversion, a property that requires the light-absorbing chromophore biliverdin. In vivo, RpBphP2 and RpBphP3 are both required for the expression of the LH4 antenna complex under anaerobic conditions, but biliverdin requires oxygen for its synthesis by heme oxygenase. On further investigation, we found that the apo-bacteriophytochrome forms of RpBphP2 and RpBphP3 are necessary and sufficient to control LH4 expression in response to light intensity in conjunction with other signal transduction proteins. One possibility is that the system senses a reduced quinone pool generated when light energy is absorbed by bacteriochlorophyll. The biliverdin-bound forms of the BphPs have the additional property of being able to fine-tune LH4 expression in response to light quality. These observations support the concept that some bacteriophytochromes can function with or without a chromophore and may be involved in regulating physiological processes not directly related to light sensing.

Bacteriophytochromes (Bphs) are photoreceptors that help bacteria sense changes in light wavelength and intensity. Bphs contain a linear tetrapyrrole chromophore that, upon absorption of red or far-red light, undergoes a cis-trans isomerization that leads to a conformational change in the holoprotein. The conformation and type of Bph affects the expression of genes. The linear tetrapyrrole bound by Bphs is thought to come from O(2)-dependent cleavage of heme by a heme oxygenase. We have discovered that the absence of O(2) does not inhibit the normal function of two Bphs in the regulation of Rhodopseudomonas palustris light-harvesting complexes. These observations imply that: (i) a linear tetrapyrrole can be made anaerobically, either through anaerobic heme cleavage by a novel enzyme or directly from the heme precursor hydroxymethylbilane without ring cleavage; or (ii) that Bph-dependent signal transduction does not require a chromophore.

The bacterial genus Rhodopseudomonas is comprised of photosynthetic bacteria found widely distributed in aquatic sediments. Members of the genus catalyze hydrogen gas production, carbon dioxide sequestration, and biomass turnover. The genome sequence of Rhodopseudomonas palustris CGA009 revealed a surprising richness of metabolic versatility that would seem to explain its ability to live in a heterogeneous environment like sediment. However, there is considerable genotypic diversity among Rhodopseudomonas isolates. Here we report the complete genome sequences of four additional members of the genus isolated from a restricted geographical area. The sequences confirm that the isolates belong to a coherent taxonomic unit, but they also have significant differences. Whole genome alignments show that the circular chromosomes of the isolates consist of a collinear backbone with a moderate number of genomic rearrangements that impact local gene order and orientation. There are 3,319 genes, 70% of the genes in each genome, shared by four or more strains. Between 10% and 18% of the genes in each genome are strain specific. Some of these genes suggest specialized physiological traits, which we verified experimentally, that include expanded light harvesting, oxygen respiration, and nitrogen fixation capabilities, as well as anaerobic fermentation. Strain-specific adaptations include traits that may be useful in bioenergy applications. This work suggests that against a backdrop of metabolic versatility that is a defining characteristic of Rhodopseudomonas, different ecotypes have evolved to take advantage of physical and chemical conditions in sediment microenvironments that are too small for human observation.

Regulatory properties of bacteriophytochrome BphP1 were evaluated with respect to the photosynthesis gene transcription repressors PpsR1 and PpsR2 of Rhodopseudomonas palustris strain CGA009 in gene complementation, replacement and deletion experiments. The results indicate that 750 nm wavelength light activates BphP1 to antagonize repression of photosynthesis gene expression by PpsR2, but not PpsR1. It is suggested that an equilibrium exists between BphP1-active and -inactive conformations, with 750 nm light shifting the equilibrium to an active conformation, although a phenotypically detectable component of BphP1 is in the active conformation in the absence of illumination.

The PpsR protein is a regulator of redox-dependent photosystem development in purple phototrophic bacteria. In contrast to most species, Rhodopseudomonas palustris contains two ppsR genes. We show that the inactivation of each of the R. palustris strain CGA009 ppsR genes results in an elevated level of formation of the photosystem under dark aerobic conditions. Absorption spectra of the two PpsR mutants revealed qualitative and quantitative differences in light-harvesting peak amplitude increases. A sequence difference in the helix-turn-helix DNA binding motif of PpsR2 (Arg 439 to Cys) between R. palustris strains CEA001 and CGA009 is shown to be a natural polymorphism that does not inactivate the repressor activity of the protein. To evaluate which photosynthesis genes are regulated by the two PpsR proteins, transcriptome profiles of the CGA009 and PpsR mutant strains were analyzed in microarray experiments. Transcription of most but not all photosystem genes was derepressed in the mutant strains to levels consistent with the in vivo absorption spectra, mathematical analyses of peak shapes and amplitudes, reaction center protein levels, and real-time PCR of selected mRNAs. Closely spaced PpsR binding motif repeats were identified 5' of genes that were derepressed in the transcriptome analysis of PpsR mutants. This work shows that both the PpsR1 and PpsR2 proteins from R. palustris strain CGA009 function as oxygen-responsive transcriptional repressors.

Rhodopseudomonas palustris is a purple nonsulfur anoxygenic phototrophic bacterium that is ubiquitous in soil and water. R. palustris is metabolically versatile with respect to energy generation and carbon and nitrogen metabolism. We have characterized and compared the baseline proteome of a R. palustris wild-type strain grown under six metabolic conditions. The methodology for proteome analysis involved protein fractionation by centrifugation, subsequent digestion with trypsin, and analysis of peptides by liquid chromatography coupled with tandem mass spectrometry. Using these methods, we identified 1664 proteins out of 4836 predicted proteins with conservative filtering constraints. A total of 107 novel hypothetical proteins and 218 conserved hypothetical proteins were detected. Qualitative analyses revealed over 311 proteins exhibiting marked differences between conditions, many of these being hypothetical or conserved hypothetical proteins showing strong correlations with different metabolic modes. For example, five proteins encoded by genes from a novel operon appeared only after anaerobic growth with no evidence of these proteins in extracts of aerobically grown cells. Proteins known to be associated with specialized growth states such as nitrogen fixation, photoautotrophic, or growth on benzoate, were observed to be up-regulated under those states.

Rhodopseudomonas palustris is among the most metabolically versatile bacteria known. It uses light, inorganic compounds, or organic compounds, for energy. It acquires carbon from many types of green plant-derived compounds or by carbon dioxide fixation, and it fixes nitrogen. Here we describe the genome sequence of R. palustris, which consists of a 5,459,213-base-pair (bp) circular chromosome with 4,836 predicted genes and a plasmid of 8,427 bp. The sequence reveals genes that confer a remarkably large number of options within a given type of metabolism, including three nitrogenases, five benzene ring cleavage pathways and four light harvesting 2 systems. R. palustris encodes 63 signal transduction histidine kinases and 79 response regulator receiver domains. Almost 15% of the genome is devoted to transport. This genome sequence is a starting point to use R. palustris as a model to explore how organisms integrate metabolic modules in response to environmental perturbations.

Segmental differences in stability within the polycistronic transcripts of the puf operon contribute to differential expression of photosynthesis genes in R. capsulatus. The comparatively stable 5' segment of these transcripts ends in a large intercistronic stem-loop structure. We show here that deletion of this RNA hairpin destabilizes the 5' puf mRNA segment but that its insertion at the 3' end of the puf operon transcripts fails to stabilize the labile 3' puf mRNA segment. Evidence is presented that decay of the 3' segment begins with endonucleolytic cleavage in which the intercistronic stem-loop structure does not participate. We conclude that this RNA hairpin is necessary but insufficient for the stability of mRNA upstream of it, and that it functions in message degradation solely as an mRNA decay terminator that protects upstream mRNA segments from degradation by 3' exoribonucleases.

Because there are not yet direct assays for most of the proteins required for differentiation of Rhodobacter capsulatus cytoplasmic membrane into photosynthetically competent intracytoplasmic membrane, a molecular inquiry into the mechanism and regulation of this process is difficult. We have, therefore, chosen to isolate R. capsulatus photosynthesis genes by creating in-frame fusions to lac'Z vectors, and selecting for those that direct appropriately regulated levels of beta-galactosidase in R. capsulatus. One lacZ fusion isolate was used to identify an open reading frame (ORF) of unknown function and flanking sequences that promoted initiation of transcription. The chromosomal copy of this ORF was mutated by insertion of a kanamycin-resistant cartridge into the cloned fragment and substitution for the chromosomal copy by homologous recombination. The phenotype of the resultant mutant cells showed that the ORF encodes 2-desacetyl-2-hydroxyethyl bacteriochlorophyllide a dehydrogenase, an enzyme that catalyzes the penultimate step in bacteriochlorophyll a biosynthesis. The nucleotide sequence of this bchC gene and its 5' regulatory region were determined. The deduced amino acid sequence shows that the bchC gene encodes a 33-kDa protein that is less hydrophobic than integral membrane proteins of R. capsulatus, although there are hydrophobic segments that could in principle interact with a lipid membrane. Results of S1 nuclease protection and primer extension experiments show that a 5' mRNA end is positioned within the cloned segment, and that this 5' end maps to sequences with significant sequence similarity to the previously characterized puf operon promoter region.

Rhodopseudomonas capsulata can grow in a number of alternative modes, including (i) photosynthetic, defined here as anaerobic growth with light as the energy source, and (ii) heterotrophic, referring to aerobic heterotrophic growth in darkness. The functions of citric acid cycle sequences in these growth modes were investigated using wild-type and appropriate mutant strains. Results of growth tests and O(2) utilization experiments showed that in the heterotrophic mode, energy conversion is dependent on operation of the classical citric acid cycle. Alpha-ketoglutarate dehydrogenase (KGD) activity in wild-type strain B10 is substantially higher in cells grown heterotrophically than in cells grown photosynthetically. Molecular oxygen, even at low concentration, appears to be important in regulation of KGD synthesis and, thus, in expression of citric acid cycle activity. Extracts of (photosynthetically grown) mutant strain KGD11 lack demonstrable KGD activity, and in contrast to the wild type, KGD11 is unable to grow heterotrophically on succinate, malate, or pyruvate owing to failure of the energy conversion function of the citric acid cycle. KGD11, however, grows well photosynthetically on malate or on CO(2) + H(2). The KGD activity level required to support the bioenergetic function of the citric acid cycle is evidently much higher than that necessary to satisfy biosynthetic demands; thus, a very low rate of succinyl-coenzyme A formation (needed for biosynthesis) in the mutant would suffice for growth under photosynthetic conditions. In wild-type R. capsulata, the alpha-ketoglutarate required for glutamate synthesis is ordinarily generated via citric acid cycle reactions, which include the conversions catalyzed by citrate synthase and isocitrate dehydrogenase. Mutants blocked in the former or both of these enzymes can grow photosynthetically if provided with an exogenous source of alpha-ketoglutarate or glutamate, but grow very poorly (if at all) as heterotrophs since the energy supply under these conditions depends on operation of the complete citric acid cycle.

Broad-host-range plasmid vectors were constructed for expression of heterologous genes in the photosynthetic bacterium Rhodobacter capsulatus. These plasmids utilize an RK2-derived replicon for maintenance and conjugative transfer and the R. capsulatus rxcA promoter to obtain transcription of genes within appropriately positioned DNA fragments. The expression vectors were used to obtain synthesis of endoglucanase and exoglucanase in R. capsulatus from cellulase genes present on exogenously derived DNA fragments. The cellulase genes were expressed either by use of their native translation initiation signals or by in-frame fusion with the rxcA B870 beta gene translation initiation signals to form a hybrid protein. The level of cellulase gene expression was found to be modulated in response to the extent of aeration of plasmid host cultures.

Photosynthetic organisms exhibit a variety of responses to changes in light intensity, including differential biosynthesis of chlorophyll-protein complexes. Cultures of Rhodobacter capsulatus grown anaerobically with a low intensity of light (2 W/m2) contained about four times as much B800-850 light-harvesting complex as cells grown under high light intensity (140 W/m2). The mRNA transcripts encoding B800-850 beta and alpha peptides were analyzed by Northern blot (RNA blot), S1 nuclease protection, and capping with guanylyl transferase. It was found that the steady-state levels of B800-850 mRNAs in high-light-grown cultures were about four times as great as in cells grown under low light intensity. Therefore, the lesser amounts of mature B800-850 peptide gene products found in cells grown with high light intensity are the result of a posttranscriptional regulatory process. It was also found that there are two polycistronic messages encoding the B800-850 peptides. These messages share a common 3' terminus but differ in their 5'-end segments as a result of transcription initiation at two discrete sites. Moreover, the half-lives of B800-850 mRNAs were about 10 min in cells grown with high light and approximately 19 min in cultures grown with low light. It is concluded that there must be more frequent initiations of transcription of B800-850 genes in cells grown with high light than in those grown with low light, and that the relative amounts of B800-850 complexes under these conditions are controlled by a translational or posttranslational mechanism.

We report that the light-harvesting and reaction center genes in the rxcA locus of R. capsulata are contained within a single operon and that their differential expression results predominantly from marked segmental differences in stability within the polycistronic rxcA transcript. The 3' portion of this transcript is rapidly degraded to give rise to either of two slowly decaying mRNA remnants, both of which encode only the light-harvesting polypeptides. The greater stability of these remnants accounts for nearly all of the difference between the concentrations of the light-harvesting and reaction center proteins. The unstable 3' portion of the transcript is delimited by two alternative stem-and-loop structures, which apparently act as barriers to 3' exoribonucleases and thereby protect the upstream RNA segment. When a DNA fragment containing the rxcA locus was fused to a plasmid promoter and transcribed in E. coli, the long precursor transcript was processed to two short messages of greater stability, as in R. capsulata.

The homology of Rhodopseudomonas capsulata DNA segments carrying photosynthesis genes with sequences present in total DNA from certain other photosynthetic and non-photosynthetic bacterial species was determined by hybridization. R. capsulata DNA fragments that carry loci for production of peptide components of the photosynthetic reaction center and light-harvesting I antenna complex were found to hybridize to DNA from some photosynthetic species. However, fragments that carry carotenoid or bacteriochlorophyll biosynthesis genes showed either weak or undetectable heterospecific hybridization under the conditions employed.

We report data indicating that the Rhodobacter capsulatus puf operon promoter and the site for its oxygen regulation are located more than 700 base pairs upstream from the previously identified puf genes and have identified the nucleotide sequences that constitute these control signals. A model is proposed in which a polycistronic transcript at least 3.4 kilobases in length is initiated near the O2-regulated promoter and is processed posttranscriptionally by endonucleolytic cleavage at multiple sites, yielding discrete mRNA segments that are degraded at different rates. A newly identified gene (pufQ), which includes a hydrophobic domain having some similarity to domains of the products of the pufL and pufM genes, begins 313 nucleotides into the puf transcript and is located entirely within the most rapidly degraded segment of the transcript. A previously identified puf transcript segment encoding structural proteins for photosynthetic membrane complexes persists after degradation of the most 5' region of the transcript and is itself subject to segmentally specific degradation. Our results suggest a model in which differential expression of the multiple genes encoded by the puf operon is at least in part attributable to major differences in the rates of decay of the various segments of puf mRNA.