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In this work we characterize the function of the flagellar protein FliL in Rhodobacter sphaeroides. Our results show that FliL is essential for motility in this bacterium and that in its absence flagellar rotation is highly impaired. A green fluorescent protein (GFP)-FliL fusion forms polar and lateral fluorescent foci that show different spatial dynamics. The presence of these foci is dependent on the expression of the flagellar genes controlled by the master regulator FleQ, suggesting that additional components of the flagellar regulon are required for the proper localization of GFP-FliL. Eight independent pseudorevertants were isolated from the fliL mutant strain. In each of these strains a single nucleotide change in motB was identified. The eight mutations affected only three residues located on the periplasmic side of MotB. Swimming of the suppressor mutants was not affected by the presence of the wild-type fliL allele. Pulldown and yeast two-hybrid assays showed that that the periplasmic domain of FliL is able to interact with itself but not with the periplasmic domain of MotB. From these results we propose that FliL could participate in the coupling of MotB with the flagellar rotor in an indirect fashion. doi: 10.1128/JB.00655-10

Molybdenum (Mo) is an important trace element that is toxic at high concentrations. To resolve the mechanisms underlying Mo toxicity, Rhodobacter capsulatus mutants tolerant to high Mo concentrations were isolated by random transposon Tn5 mutagenesis. The insertion sites of six independent isolates mapped within the same gene predicted to code for a permease of unknown function located in the cytoplasmic membrane. During growth under Mo-replete conditions, the wild-type strain accumulated considerably more Mo than the permease mutant. For mutants defective for the permease, the high-affinity molybdate importer ModABC, or both transporters, in vivo Mo-dependent nitrogenase (Mo-nitrogenase) activities at different Mo concentrations suggested that ModABC and the permease import molybdate in nanomolar and micromolar ranges, respectively. Like the permease mutants, a mutant defective for ATP sulfurylase tolerated high Mo concentrations, suggesting that ATP sulfurylase is the main target of Mo inhibition in R. capsulatus. Sulfate-dependent growth of a double mutant defective for the permease and the high-affinity sulfate importer CysTWA was reduced compared to those of the single mutants, implying that the permease plays an important role in sulfate uptake. In addition, permease mutants tolerated higher tungstate and vanadate concentrations than the wild type, suggesting that the permease acts as a general oxyanion importer. We propose to call this permease PerO (for oxyanion permease). It is the first reported bacterial molybdate transporter outside the ABC transporter family. doi: 10.1128/JB.00742-10

The unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 (Cyanothece 51142) is able to grow aerobically under nitrogen-fixing conditions with alternating light-dark cycles or continuous illumination. This study investigated the effects of carbon and nitrogen sources on Cyanothece 51142 metabolism via [sup.13]C-assisted metabolite analysis and biochemical measurements. Under continuous light (50 [micro]mol photons [m.sup.-2] [s.sup.-1]) and nitrogen-fixing conditions, we found that glycerol addition promoted aerobic biomass growth (by twofold) and nitrogenasedependent hydrogen production [up to 25 [micro]mol [H.sub.2] (mg chlorophyll) [sup.-1] [h.sup.-1]], but strongly reduced phototrophic C[O.sub.2] utilization. Under nitrogen-sufficient conditions, Cyanothece 51142 was able to metabolize glycerol photoheterotrophically, and the activity of light-dependent reactions (e.g. oxygen evolution) was not significantly reduced. In contrast, Synechocystis sp. PCC 6803 showed apparent mixotrophic metabolism under similar growth conditions. Isotopomer analysis also detected that Cyanothece 51142 was able to fix [CO.sub.2] via anaplerotic pathways, and to take up glucose and pyruvate for mixotrophic biomass synthesis. DOI 10.1099/mic.0.038232-0

The Calvin-Benson-Bassham cycle (Calvin cycle) catalyzes virtually all primary productivity on Earth and is the major sink for atmospheric C[O.sub.2]. A less appreciated function of C[O.sub.2] fixation is as an electron-accepting process. It is known that anoxygenic phototrophic bacteria require the Calvin cycle to accept electrons when growing with light as their sole energy source and organic substrates as their sole carbon source. However, it was unclear why and to what extent C[O.sub.2] fixation is required when the organic substrates are more oxidized than biomass. To address these questions we measured metabolic fluxes in the photosynthetic bacterium Rhodopseudomonas palustris grown with [sup.13]C-labeled acetate. R. palustris metabolized 22% of acetate provided to C[O.sub.2] and then fixed 68% of this C[O.sub.2] into cell material using the Calvin cycle. This Calvin cycle flux enabled R. palustris to reoxidize nearly half of the reduced cofactors generated during conversion of acetate to biomass, revealing that C[O.sub.2] fixation plays a major role in cofactor recycling. When [H.sub.2] production via nitrogenase was used as an alternative cofactor recycling mechanism, a similar amount of C[O.sub.2] was released from acetate, but only 12% of it was reassimilated by the Calvin cycle. These results underscore that N2 fixation and C[O.sub.2] fixation have electron-accepting roles separate from their better-known roles in ammonia production and biomass generation. Some nonphotosynthetic heterotrophic bacteria have Calvin cycle genes, and their potential to use CO2 fixation to recycle reduced cofactors deserves closer scrutiny. Calvin cycle | hydrogen gas metabolic flux analysis | nitrogenase | Rhodopseudomonas doi/ 10.1073/pnas.1006175107

We have investigated the adaptation of the light-harvesting system of the photosynthetic bacterium Phaeospirillum molischianum (DSM120) to very low light conditions. This strain is able to respond to changing light conditions by differentially modulating the expression of a family of puc operons that encode for peripheral light-harvesting complex (LH2) polypeptides. This modulation can result in a complete shift between the production of LH2 complexes absorbing maximally near 850 nm to those absorbing near 820 nm. In contradiction to prevailing wisdom, analysis of the LH2 rings found in the photosynthetic membranes during light adaptation are shown to have intermediate spectral and electrostatic properties. By chemical cross-linking and mass-spectrometry we show that individual LH2 rings and subunits can contain a mixture of polypeptides derived from the different operons. These observations show that polypeptide synthesis and insertion into the membrane are not strongly coupled to LH2 assembly. We show that the light-harvesting complexes resulting from this mixing could be important in maintaining photosynthetic efficiency during adaptation. chromatic adaptation | membrane protein | photosynthetic bacteria | Rhodospirillum molischianum www.pnas.org/cgi/doi/10.1073/pnas.0914854107

Assimilation of acetyl coenzyme A (acetyl-CoA) is an essential process in many bacteria that proceeds via the glyoxylate cycle or the ethylmalonyl-CoA pathway. In both assimilation strategies, one of the final products is malate that is formed by the condensation of acetyl-CoA with glyoxylate. In the glyoxylate cycle this reaction is catalyzed by malate synthase, whereas in the ethylmalonyl-CoA pathway the reaction is separated into two proteins: malyl-CoA lyase, a well-known enzyme catalyzing the Claisen condensation of acetyl-CoA with glyoxylate and yielding malyl-CoA, and an unidentified malyl-CoA thioesterase that hydrolyzes malyl-CoA into malate and CoA. In this study the roles of Mcl1 and Mcl2, two malyl-CoA lyase homologs in Rhodobacter sphaeroides, were investigated by gene inactivation and biochemical studies. Mcl1 is a true (3S)-malyl-CoA lyase operating in the ethylmalonyI-CoA pathway. Notably, Mcl1 is a promiscuous enzyme and catalyzes not only the condensation of acetyl-CoA and glyoxylate but also the cleavage of [beta]-methylmalyl-CoA into glyoxylate and propionyl-CoA during acetyl-CoA assimilation. In contrast, Mcl2 was shown to be the sought (3S)-malyl-CoA thioesterase in the ethylmalonyl-CoA pathway, which specifically hydrolyzes (3S)-malyl-CoA but does not use [beta]-methylmalyl-CoA or catalyze a lyase or condensation reaction. The identification of Mcl2 as thioesterase extends the enzyme functions of malyl-CoA lyase homologs that have been known only as 'Claisen condensation' enzymes so far. Mcll and Mcl2 are both related to malate synthase, an enzyme which catalyzes both a Claisen condensation and thioester hydrolysis reaction. doi: 10.1128/JB.01267-09

The photosynthetic growth of Synechocystis sp. strain PCC 6803 is hampered by exogenously added bacteriochlorophyllide a (Bchlide a) in a dose-dependent manner. The growth inhibition caused by Bchlide a, however, is relieved by an increased level of exogenously added chlorophyllide a (Chlide a). The results are explained by the competitive inhibition of chlorophyll synthase by Bchlide a, with inhibition constants ([K.sub.I]s) of 0.3 mM and 1.14 mM in the presence of sufficient geranylgeranyl pyrophosphate (GGPP) and phytyl pyrophosphate (PPP), respectively. Surprisingly, the bacteriochlorophyll synthase of Rhodobacter sphaeroides is inhibited competitively by Chlide a, with [K.sub.I]S of 0.54 mM and 0.77 mM in the presence of sufficient GGPP and PPP, respectively. Consistently, exogenously added Chlide a inhibits the metabolic conversion of exogenously added Bchlide a to bacteriochlorophyll a by an R. sphaeroides bchFNB-bchZ mutant that neither synthesizes nor metabolizes Chlide a. The metabolic inhibition by Chlide a, however, is relieved by the elevated level of Bchlide a. Thus, the chlorophyll synthase of Synechocystis sp. PCC 6803 and the bacteriochlorophyll synthase of R. sphaeroides, both of which perform ping-pong-type reactions, are inhibited by Bchlide a and Chlide a, respectively. Although neither inhibitor is catalyzed by the target enzyme, inhibitions in the competitive mode suggest a structural similarity between their active sites. doi: 10.1128/JB.01271-09

The phototrophic bacterium Chloroflexus aurantiacus uses a yet unsolved 3-hydroxypropionate cycle for autotrophic C[O.sub.2] fixation. It starts from acetyl-CoA, with acetyl-CoA and propionyl-CoA carboxylases acting as carboxylating enzymes. In a first cycle, (S)-malyl-CoA is formed from acetyI-CoA and 2 molecules of bicarbonate. (S)-Malyl-CoA cleavage releases the C[O.sub.2] fixation product glyoxylate and regenerates the starting molecule acetyl-CoA. Here we complete the missing steps devoted to glyoxylate assimilation. In a second cycle, glyoxylate is combined with propionyl-CoA, an intermediate of the first cycle, to form [beta]-methylmalyl-CoA. This condensation is followed by dehydration to mesaconyl-C1-CoA. An unprecedented CoA transferase catalyzes the intramolecular transfer of the CoA moiety to the C4 carboxyl group of mesaconate. Mesaconyl-C4-CoA then is hydrated by an enoyl-CoA hydratase to (S)-citramalyl-CoA. (S)-Citramalyl-CoA is cleaved into acetyl-CoA and pyruvate by a tri-functional lyase, which previously cleaved (S)-malyl-CoA and formed [beta]-methylmalyl-CoA. Thus, the enigmatic disproportionation of glyoxyiate and propionyl-CoA into acetyl-CoA and pyruvate is solved in an elegant and economic way requiring only 3 additional enzymes. The whole bicyclic pathway results in pyruvate formation from 3 molecules of bicarbonate and involves 19 steps but only 13 enzymes. Elements of the 3-hydroxypropionate cycle may be used for the assimilation of small organic molecules. The 3-hydroxypropionate cycle is compared with the Calvin-Benson-Bassham cycle and other autotrophic pathways. autotrophy | acetyl-CoA carboxylase | Calvin cycle doi/ 10.1073/pnas.0908356106

Horizontal gene transfer and selection are major forces driving microbial evolution. However, interactions between them are rarely studied. Phylogenetic analyses of purple bacterial carotenoid biosynthesis genes suggest two lineages: one producing spheroidenone and the other producing spirilloxanthin. Of the latter lineage, Rubrivivax gelatinosus S1 and Hoeflea phototrophica DFL-43 also or instead produce spheroidenone. Evolution of the spheroidenone pathway from that producing spirilloxanthin theoretically requires changes in the substrate specificity of upstream pathway enzymes and acquisition of a terminal ketolase (CrtA). In R. gelatinosus and likely also in H. phototrophica, CrtA was acquired from the Bacteroidetes, in which it functions as a hydroxylase. Estimation of nonsynonymous and synonymous mutations using several pairwise methods indicated positive selection upon both genes, consistent with their functional changes from hydroxylases to ketolases. Relaxed negative selection upon all other carotenoid biosynthetic genes in these organisms was also apparent, likely facilitating changes in their substrate specificities. Furthermore, all genes responsible for terminal carotenoid biosynthetic pathway steps were under reduced negative selection compared to those known to govern biosynthetic pathway specificity. Horizontal transfer of crtA into R. gelatinosus and H. phototrophica has therefore likely been promoted by (i) the apparent selective advantage of spheroidenone production relative to spirilloxanthin production, (ii) reduced negative selection upon other carotenoid biosynthetic genes, facilitating changes in their substrate specificities, and (iii) preexisting low enzyme substrate specificities due to relaxed negative selection. These results highlight the importance and complexity of selection acting upon both a horizontally transferred gene and the biochemical network into which it is integrating. doi: 10.1128/JB.01060-09

The phototrophic purple bacterium Rhodobacter capsulatus encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdate-binding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA-binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the R. capsulatus Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent mop-lacZ fusion in mutant strains defective for either mopA, mopB, or both suggested that MopB negatively modulates expression of the mop promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling mop gene expression.

Many bacterial species swim by employing ion-driven molecular motors that power the rotation of helical filaments. Signals are transmitted to the motor from the external environment via the chemotaxis pathway. In bidirectional motors, the binding of phosphorylated CheY (CheY-P) to the motor is presumed to instigate conformational changes that result in a different rotor-stator interface, resulting in rotation in the alternative direction. Controlling when this switch occurs enables bacteria to accumulate in areas favorable for their survival. Unlike most species that swim with bidirectional motors, Rhodobacter sphaeroides employs a single stop-start flagellar motor. Here, we asked, how does the binding of CheY-P stop the motor in R. sphaeroides--using a clutch or a brake? By applying external force with viscous flow or optical tweezers, we show that the R. sphaeroides motor is stopped using a brake. The motor stops at 27-28 discrete angles, locked in place by a relatively high torque, approximately 2-3 times its stall torque.

Formation of photosynthesis complexes in Rhodobacter sphaeroides is regulated in a redox- and light-dependent manner by the AppA/PpsR and PrrB/PrrA systems. While on the one hand, blue light is sensed by the flavin adenine dinucleotide-binding BLUF domain of AppA, on the other, light is absorbed by bacteriochlorophyll signals through PrrB/PrrA. We show that much smaller quantities initiate the AppA-mediated response to blue light than the bacteriochlorophyll-mediated response.

In the present study, we show in vitro binding of PrrA, a global regulator in Rhodobacter sphaeroides 2.4.1, to the PrrA site 2, within the RSP3361 locus. Specific binding, as shown by competition experiments, requires the phosphorylation of PrrA. The binding affinity of PrrA for site 2 was found to increase 4- to 10-fold when spermidine was added to the binding reaction. The presence of extracellular concentrations of spermidine in growing cultures of R. sphaeroides gave rise to a twofold increase in the expression of the photosynthesis genes pucB and pufB, as well as the RSP3361 gene, under aerobic growth conditions, as shown by the use of iacZ transcriptional fusions, and led to the production of light-harvesting spectral complexes. In addition, we show that negative supercoiling positively regulates the expression of the RSP3361 gene, as well as pucB. We show the importance of supercoiling through an evaluation of the regulation of gene expression in situ by supercoiling, in the case of the former gene, as well as using the DNA gyrase inhibitor novobiocin. We propose that polyamines and DNA supercoiling act synergistically to regulate expression of the RSP3361 gene, partly by affecting the affinity of PrrA binding to the PrrA site 2 within the RSP3361 gene.

Purple bacteria have thus far been considered to operate lightdriven cyclic electron transfer chains containing ubiquinone (UQ) as liposoluble electron and proton carrier. We show that in the purple [gamma]-proteobacterium Halorhodospira halophila, menaquinone-8 (MK-8) is the dominant quinone component and that it operates in the [Q.sub.B]-site of the photosynthetic reaction center (RC). The redox potentials of the photooxidized pigment in the RC and of the Rieske center of the bCl complex are significantly lower ([E.sub.m] = +270 mV and +110 mV, respectively) than those determined in other purple bacteria but resemble those determined for species containing MK as pool quinone. These results demonstrate that the photosynthetic cycle in H. halophila is based on MK and not on UQ. This finding together with the unusual organization of genes coding for the [bc.sub.1] complex in H. halophila suggests a specific scenario for the evolutionary transition of bioenergetic chains from the low-potential menaquinones to higher-potential UQ in the proteobacterial phylum, most probably induced by rising levels of dioxygen 2.5 billion years ago. This transition appears to necessarily proceed through bioenergetic ambivalence of the respective organisms, that is, to work both on MK-and on UQ-pools. The establishment of the corresponding low-and high-potential chains was accompanied by duplication and redox optimization of the [bc.sub.1] complex or at least of its crucial subunit oxidizing quinols from the pool, the Rieske protein. Evolutionary driving forces rationalizing the empirically observed redox tuning of the chain to the quinone pool are discussed. electron transport | evolution | photosynthesis

The identification of a universal biosignature that could be sensed remotely is critical to the prospects for success in the search for life elsewhere in the universe. A candidate universal biosignature is homochirality, which is likely to be a generic property of all biochemical life. Because of the optical activity of chiral molecules, it has been hypothesized that this unique characteristic may provide a suitable remote sensing probe using circular polarization spectroscopy. Here, we report the detection of circular polarization in light scattered by photosynthetic microbes. We show that the circular polarization appears to arise from circular dichroism of the strong electronic transitions of photosynthetic absorption bands. We conclude that circular polarization spectroscopy could provide a powerful remote sensing technique for generic life searches. homochirality | life detection | remote sensing

Expression of the flagellar genes in Rhodobacter sphaeroides is dependent on one of the four sigma-54 factors present in this bacterium and on the enhancer binding proteins (EBPs) FleQ and FleT. These proteins, in contrast to other well-characterized EBPs, carry out activation as a hetero-oligomeric complex. To further characterize the molecular properties of this complex we mapped the binding sites or upstream activation sequences (UASs) of six different flagellar promoters. In most cases the UASs were identified at approximately 100 bp upstream from the promoter. However, the activity of the divergent promoters flhAp-flgAp, which are separated by only 53 bp, is mainly dependent on a UAS located approximately 200 bp downstream from each promoter. Interestingly, a significant amount of activation mediated by the upstream or contralateral UAS was also detected, suggesting that the architecture of this region is important for the correct regulation of these promoters. Sequence analysis of the regions carrying the potential FleQ/FleT binding sites revealed a conserved motif. In vivo footprinting experiments with the motAp promoter allowed us to identify a protected region that overlaps with this motif. These results allow us to propose a consensus sequence that represents the binding site of the FleQ/ FleT activating complex.

The high excitation energy-transfer efficiency demanded in photosynthetic organisms relies on the optimal pigment-protein binding orientation in the individual protein complexes and also on the overall architecture of the photosystem. In green sulfur bacteria, the membrane-attached Fenna-Matthews-Olson (FMO) antenna protein functions as a 'wire' to connect the large peripheral chlorosome antenna complex with the reaction center (RC), which is embedded in the cytoplasmic membrane (CM). Energy collected by the chlorosome is funneled through the FMO to the RC. Although there has been considerable effort to understand the relationships between structure and function of the individual isolated complexes, the specific architecture for in vivo interactions of the FMO protein, the CM, and the chlorosome, ensuring highly efficient energy transfer, is still not established experimentally. Here, we describe a mass spectrometrybased method that probes solvent-exposed surfaces of the FMO by labeling solvent-exposed aspartic and glutamic acid residues. The locations and extents of labeling of FMO on the native membrane in comparison with it alone and on a chlorosome-depleted membrane reveal the orientation. The large differences in the modification of certain peptides show that the Bchl a #3 side of the FMO trimer interacts with the CM, which is consistent with recent theoretical predictions. Moreover, the results also provide direct experimental evidence to confirm the overall architecture of the photosystem from Chlorobaculum tepidum (C tepidum) and give information on the packing of the FMO protein in its native environment. chemical labeling | energy transfer | FMO protein | mass spectrometry | protein footprinting

The coordinated high-light response of genes encoding subunits of photosystem I (PSI) is achieved by the AT-rich region located just upstream of the core promoter in Synechocystis sp. strain PCC 6803. The upstream element enhances the basal promoter activity under low-light conditions, whereas this positive regulation is lost immediately after the shift to high-light conditions. In this study, we focused on a high-light regulatory 1 (HLR1) sequence included in the upstream element of every PSI gene examined. A gel mobility shift assay revealed that a response regulator RpaB binds to the HLR1 sequence in PSI promoters. Base substitution in the HLR1 sequence or decrease in copy number of the rpaB gene resulted in decrease in the promoter activity of PSI genes under low-light conditions. These observations suggest that RpaB acts as a transcriptional activator for PSI genes. It is likely that RpaB binds to the HLR1 sequence under low-light conditions and works for positive regulation of PSI genes and for negative regulation of high-light-inducible genes depending on the location of the HLR1 sequence within target promoters.

Photosynthetic organisms need defense systems against photooxidative stress caused by the generation of highly reactive singlet oxygen ([sup.1][O.sub.2]). Here we show that the alternative sigma factor Rpo[H.sub.II] is required for the expression of important defense factors and that deletion of rpo[H.sub.II] leads to increased sensitivity against exposure to [.sup.1][O.sub.2] and methylglyoxal in Rhodobacter sphaeroides. The gene encoding Rpo[H.sub.II] is controlled by RpoE, and thereby a sigma factor cascade is constituted. We provide the first in vivo study that identifies genes controlled by an Rpo[H.sub.II]-type sigma factor, which is widely distributed in the Alphaproteobacteria. Rpo[H.sub.II]-dependent genes encode oxidative-stress defense systems, including proteins for the degradation of methylglyoxal, detoxification of peroxides, [sup.1][O.sub.2] scavenging, and redox and iron homeostasis. Our experiments indicate that glutathione (GSH)-dependent mechanisms are involved in the defense against photooxidative stress in photosynthetic bacteria. Therefore, we conclude that systems pivotal for the organism's defense against photooxidative stress are strongly dependent on GSH and are specifically recognized by Rpo[H.sub.II] in R. sphaeroides.

In the facultatively phototrophic proteobacterium Rhodobacter sphaeroides, formation of the photosynthetic apparatus is oxygen dependent. When oxygen tension decreases, the response regulator PrrA of the global two-component PrrBA system is believed to directly activate transcription of the puf, pub, and puc operons, encoding structural proteins of the photosynthetic complexes, and to indirectly upregulate the photopigment biosynthesis genes bch and crt. Decreased oxygen also results in inactivation of the photosynthesis-specific repressor PpsR, bringing about derepression of the puc, bch, and crt operons. We uncovered a hierarchical relationship between these two regulatory systems, earlier thought to function independently. We also more accurately assessed the spectrum of gene targets of the PrrBA system. First, expression of the appA gene, encoding the PpsR antirepressor, is PrrA dependent, which establishes one level of hierarchical dominance of the PrrBA system over AppA-PpsR. Second, restoration of the appA transcript to the wild-type level is insufficient for rescuing phototrophic growth impairment of the prrA mutant, whereas inactivation of ppsR is sufficient. This suggests that in addition to controlling appA transcription, PrrA affects the activity of the AppA-PpsR system via an as yet unidentified mechanism(s). Third, PrrA directly activates several bch and crt genes, traditionally considered to be the PpsR targets. Therefore, in R. sphaeroides, the global PrrBA system regulates photosynthesis gene expression (i) by rigorous control over the photosynthesis-specific AppA-PpsR regulatory system and (ii) by extensive direct transcription activation of genes encoding structural proteins of photosynthetic complexes as well as genes encoding photopigment biosynthesis enzymes.

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. alphaproteobacteria | ecotype | genomes | photosynthesis | rhodopseudomonas

Quantitative information on the ecophysiology of individual microorganisms is generally limited because it is difficult to assign specific metabolic activities to identified single cells. Here, we develop and apply a method, Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy (HISH-SIMS), and show that it allows simultaneous phylogenetic identification and quantitation of metabolic activities of single microbial cells in the environment. Using HISH-SIMS, individual cells of the anaerobic, phototropic bacteria Chromatium okenii, Lamprocystis purpurea, and Chlorobium clathratiforme inhabiting the oligotrophic, meromictic Lake Cadagno were analyzed with respect to [H.sup.13]C[O.sub.3.sup.-] and [sup.15]N[H.sub.4.sup.+] assimilation. Metabolic rates were found to vary greatly between individual cells of the same species, showing that microbial populations in the environment are heterogeneous, being comprised of physiologically distinct individuals. Furthermore, C. okenii, the least abundant species representing [approximately equal to] 0.3% of the total cell number, contributed more than 40% of the total uptake of ammonium and 70% of the total uptake of carbon in the system, thereby emphasizing that numerically inconspicuous microbes can play a significant role in the nitrogen and carbon cycles in the environment. By introducing this quantification method for the ecophysiological roles of individual cells, our study opens a variety of possibilities of research in environmental microbiology, especially by increasing the ability to examine the ecophysiological roles of individual cells, including those of less abundant and less active microbes, and by the capacity to track not only nitrogen and carbon but also phosphorus, sulfur, and other biological element flows within microbial communities. anaerobic phototrophs | nanoSIMS

Bacteriochlorophylls (BChls) c, d, and e are the main light-harvesting pigments of green photosynthetic bacteria that self-assemble into nanostructures within the chlorosomes forming the most efficient antennas of photosynthetic organisms. All previous models of the chlorosomal antennae, which are quite controversially discussed because no single crystals could be grown so far from these organelles, involve a strong hydrogen-bonding interaction between the [3.sup.1] hydroxyl group and the [13.sup.1] carbonyl group. We have synthesized different self-assemblies of BChl c mimics having the same functional groups as the natural counterparts, that is, a hydroxyethyl substituent, a carbonyl group and a divalent metal atom ligated by a tetrapyrrole. These artificial BChl mimics have been shown by single crystal x-ray diffraction to form extended stacks that are packed by hydrophobic interactions and in the absence of hydrogen bonding. Time-resolved photoluminescence proves the ordered nature of the self-assembled stacks. FT-IR spectra show that on self-assembly the carbonyl frequency is shifted by [approximately equal to] 30 [cm.sup.-1] to lower wavenumbers. From the FT-IR data we can infer the proximal interactions between the BChls in the chlorosomes consistent with a single crystal x-ray structure that shows a weak electrostatic interaction between carbonyl groups and the central zinc atom. chlorosome model | crystal structure | FT-IR | light-harvesting | time-resolved fluorescence

The specific enzymes employed by Chlorobium tepidum for the anaerobic oxidation of thiosulfate, sulfide and elemental sulfur during anoxygenic photosynthesis are not well defined. In particular, it is unclear how C. tepidum completely oxidizes thiosulfate. A C. tepidum genomic region, encoding a putative quinone-interacting membrane-bound oxidoreductase (Qmo) complex (CT0866-0868), hypothetical proteins (CT0869-0875) and a sulfide : quinone oxidoreductase (SQR) homologue (CT0876), was analysed for its role in anaerobic sulfur oxidation. Transcripts of genes encoding the Qmo complex, which is similar to archaeal heterodisulfide reductases, were detected by RT-PCR only while sulfide or elemental sulfur were being oxidized, whereas the SQR homologue and CT0872 were expressed during thiosutfate oxidation and into early stationary phase. A mutant of C. tepidum was obtained in which the region between CT0868 and CT0876 was replaced by a transposon insertion resulting in the truncation or deletion of nine genes. This strain, C5, was completely defective for growth on thiosulfate as the sole electron donor in C. tepidum, but only slightly defective for growth on sulfide or thiosulfate plus sulfide. Strain C5 did not oxidize thiosulfate and also displayed a defect in acetate assimilation under all growth conditions. A gene of unknown function, CT0872, deleted in strain C5 that is conserved in chemolithotrophic sulfur-oxidizing bacteria and archaea is the most likely candidate for the thiosulfate oxidation phenotype observed in this strain. The defect in acetate assimilation may be explained by deletion of CT0874, which encodes a homologue of 3-oxoacyl acyl carrier protein synthase.

A series of Rhodobacter capsulatus AmtB variants were created and assessed for effects on ammonia transport, formation of AmtB-GlnK complexes, and regulation of nitrogenase activity and NifH ADP-ribosylation. Confirming previous reports, H193 and H342 were essential for ammonia transport and the replacement of aspartate 185 with glutamate reduced ammonia transport. Several amino acid residues, F131, D334, and D335, predicted to be critical for AmtB activity, are shown here for the first time by mutational analysis to be essential for transport. Alterations of the C-terminal tail reduced methylamine transport, prevented AmtB-GlnK complex formation, and abolished nitrogenase switch-off and NifH ADP-ribosylation. On the other hand, D185E, with a reduced level of transport, was capable of forming an ammonium-induced complex with GlnK and regulating nitrogenase. This reinforces the notions that ammonia transport is not sufficient for nitrogenase regulation and that formation of an AmtB-GlnK complex is necessary for these processes. However, some transport-incompetent AmtB variants, i.e., F131A, H193A, and H342A, form ammonium-induced complexes with GInK but fail to properly regulate nitrogenase. These results show that formation of an AmtB-GlnK complex is insufficient in itself for nitrogenase regulation and suggest that partial ammonia transport or occupation of the pore by ammonia is essential for this function.

Rhodobacter sphaeroides 2.4.1 is a facultative photosynthetic anaerobe that grows by anoxygenic photosynthesis under anaerobic-light conditions. Changes in energy generation pathways under photosynthetic and aerobic respiratory conditions are primarily controlled by oxygen tensions. In this study, we performed time series microarray analyses to investigate transcriptome dynamics during the transition from anaerobic photosynthesis to aerobic respiration. Major changes in gene expression profiles occurred in the initial 15 min after the shift from anaerobic-light to aerobic-dark conditions, with changes continuing to occur up to 4 hours postshift. Those genes whose expression levels changed significantly during the time series were grouped into three major classes by clustering analysis. Class I contained genes, such as that for the [aa.sub.3] cytochrome oxidase, whose expression levels increased after the shift. Class II contained genes, such as those for the photosynthetic apparatus and Calvin cycle enzymes, whose expression levels decreased after the shift. Class III contained genes whose expression levels temporarily increased during the time series. Many genes for metabolism and transport of carbohydrates or lipids were significantly induced early during the transition, suggesting that those endogenous compounds were initially utilized as carbon sources. Oxidation of those compounds might also be required for maintenance of redox homeostasis after exposure to oxygen. Genes for the repair of protein and sulfur groups and uptake of ferric iron were temporarily upregulated soon after the shift, suggesting they were involved in a response to oxidative stress. The flagellar-biosynthesis genes were expressed in a hierarchical manner at 15 to 60 min after the shift. Numerous transporters were induced at various time points, suggesting that the cellular composition went through significant changes during the transition from anaerobic photosynthesis to aerobic respiration. Analyses of these data make it clear that numerous regulatory activities come into play during the transition from one homeostatic state to another.

Formaldehyde is an intermediate formed during the metabolism of methanol or other methylated compounds. Many Gram-negative bacteria generate formaldehyde from methanol via a periplasmic pyrroloquinoline quinone (PQQ)-dependent dehydrogenase in which the [alpha] subunit of an [[chi].sub.2][[beta].sub.2] tetramer has catalytic activity. The genome of the facultative formaldehyde-oxidizing bacterium Rhodobacter sphaeroides encodes XoxF, a homologue of the catalytic subunit of a proposed PQQ-containing dehydrogenase of Paracoccus denitrificans. R. sphaeroides xoxF is part of a gene cluster that encodes periplasmic c-type cytochromes, including Cycl, isocytochrome [c.sub.2] and CycB (a cyt [c.sub.553i] homologue), as well as adhl, a glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), and gfa, a homologue of a glutathione-formaldehyde activating enzyme (Gfa). To test the roles of XoxF, CycB and Gfa in formaldehyde metabolism by R. sphaeroides, we monitored photosynthetic growth with methanol as a source of formaldehyde and whole-cell methanol-dependent oxygen uptake. Our data show that R. sphaeroides cells lacking XoxF or CycB do not exhibit methanol-dependent oxygen uptake and lack the capacity to utilize methanol as a sole photosynthetic carbon source. These results suggest that both proteins are required for formaldehyde metabolism. R. sphaeroides Gfa is not essential to activate formaldehyde, as cells lacking gfa are capable of both methanol-dependent oxygen uptake and growth with methanol as a photosynthetic carbon source.

The Rhodobacter capsulatus genome contains three genes (olsA [plsC138], plsC316, and plsC3498) that are annotated as lysophosphatidic acid (1-acyl-sn-glycerol-3-phosphate) acyltransferase (AGPAT). Of these genes, olsA was previously shown to be an O-acyltransferase in the second step of ornithine lipid biosynthesis, which is important for optimal steady-state levels of c-type cytochromes (S. Aygun-Sunar, S. Mandaci, H.-G. Koch, I. V. J. Murray, H. Goldfine, and F. Daldal. Mol. Microbiol. 61:418-435, 2006). The roles of the remaining plsC316 and plsC3498 genes remained unknown. In this work, these genes were cloned, and chromosomal insertion-deletion mutations inactivating them were obtained to define their function. Characterization of these mutants indicated that, unlike the Escherichia coli plsC, neither plsC316 nor plsC3498 was essential in R. capsulatus. In contrast, no plsC316 olsA double mutant could be isolated, indicating that an intact copy of either olsA or plsC316 was required for R. capsulatus growth under the conditions tested. Compared to OlsA null mutants, PlsC316 null mutants contained ornithine lipid and had no c-type cytochrome-related phenotype. However, they exhibited slight growth impairment and highly altered total fatty acid and phospholipid profiles. Heterologous expression in an E. coli plsC(Ts) mutant of either R. capsulatus plsC316 or olsA gene products supported growth at a nonpermissive temperature, exhibited AGPAT activity in vitro, and restored phosphatidic acid biosynthesis. The more vigorous AGPAT activity displayed by PlsC316 suggested that plsC316 encodes the main AGPAT required for glycerophospholipid synthesis in R. capsulatus, while olsA acts as an alternative AGPAT that is specific for ornithine lipid synthesis. This study therefore revealed for the first time that some OlsA enzymes, like the enzyme of R. capsulatus, are bifunctional and involved in both membrane ornithine lipid and glycerophospholipid biosynthesis.

We have characterized open reading frame RSP0072, which is located within the flgG operon in Rhodobacter sphaeroides. The amino acid sequence analysis of this gene product showed the presence of a soluble lyric transglycosylase domain. The deletion of the N-terminal region (90 amino acids) of the product of RSP0072 yields a leaky nonmotile phenotype, as determined by swarm assays in soft agar. Electron micrographs revealed the lack of flagella in mutant cells. The purified wild-type protein showed lytic activity on extracts of Micrococcus luteus. In contrast, no iytic activity was observed when the residues E57 or E83 were replaced by alanine. Affinity blotting suggests that the protein encoded by RSP0072 interacts with the flagellar rod-scaffolding protein Fig J, which lacks the muramidase domain present in FlgJ of many bacteria. We propose that the product of RSP0072 is a flagellar muramidase that is exported to the periplasm via the Sec pathway, where it interacts with FlgJ to open a gap in the peptidoglycan layer for the subsequent penetration of the nascent flagellar structure.

Reaction center chlorophylls (Chls) in photosytems II and I were studied in the isolated thylakoid membranes of a cyanobacterium, marina. Mechanisms and the evolution of the Chl d based reaction centers using far red light are discussed.

The photosynthetic unit (PSU) of purple photosynthetic bacteria consists of a network of bacteriochlorophyll-protein complexes that absorb solar energy for eventual conversion to ATP. Because of its remarkable simplicity, the PSU can serve as a prototype for studies of cellular organelles. In the purple bacterium Rhodobacter sphaeroides the PSU forms spherical invaginations of the inner membrane, [approximately equal to] 70 nm in diameter, composed mostly of light-harvesting complexes, LH1 and LH2, and reaction centers (RCs). Atomic force microscopy studies of the intracytoplasmic membrane have revealed the overall spatial organization of the PSU. In the present study these atomic force microscopy data were used to construct three-dimensional models of an entire membrane vesicle at the atomic level by using the known structure of the LH2 complex and a structural model of the dimeric RC-LH1 complex. Two models depict vesicles consisting of 9 or 18 dimeric RC-LH1 complexes and 144 or 101 LH2 complexes, representing a total of 3,879 or 4,464 bacteriochlorophylls, respectively. The in silico reconstructions permit a detailed description of light absorption and electronic excitation migration, including computation of a 50-ps excitation lifetime and a 95% quantum efficiency for one of the model membranes, and demonstration of excitation sharing within the closely packed RC-LH1 dimer arrays. excitation transfer | network kinetics | photosynthetic light harvesting | quantum efficiency | systems biology

Haem [d.sub.1] is a unique tetrapyrrole molecule that serves as a prosthetic group of cytochrome [cd.sub.1], which reduces nitrite to nitric oxide during the process of denitrification. Very little information is available regarding the biosynthesis of haem [d.sub.1]. The extreme difficulty in studying the haem [d.sub.1] biosynthetic pathway can be partly attributed to the lack of a theoretical basis for experimental investigation. We report here a gene cluster encoding enzymes involved in the biosynthesis of haem [d.sub.1] in two heliobacterial species, Heliobacillus mobilis and Heliophilum fasciatum. The gene organization of the cluster is conserved between the two species, and contains a complete set of genes that lead to the biosynthesis of uroporphyrinogen III and genes thought to be involved in the late steps of haem [d.sub.1] biosynthesis. Detailed bioinformatics analysis of some of the proteins encoded in the gene cluster revealed important clues to the precise biochemical roles of the proteins in the biosynthesis of haem [d.sub.1], as well as the membrane transport and insertion of haem [d.sub.1] into an apocytochrome during the maturation of cytochrome [cd.sub.1].

The Rhodobacter sphaeroides intracytoplasmic membrane (ICM) is an inducible membrane that is dedicated to the major events of bacterial photosynthesis, including harvesting light energy, separating primary charges, and transporting electrons. In this study, multichromatographic methods coupled with Fourier transform ion cyclotron resonance mass spectrometry, combined with subcellular fractionation, was used to test the hypothesis that the photosynthetic membrane of R. sphaeroides 2.4.1 contains a significant number of heretofore unidentified proteins in addition to the integral membrane pigment-protein complexes, including light-harvesting complexes 1 and 2, the photochemical reaction center, and the cytoehrome [bc.sub.1] complex described previously. Purified ICM vesicles are shown to be enriched in several abundant, newly identified membrane proteins, including a protein of unknown function (AffyChip designation RSP1760) and a possible alkane hydroxylase (RSPI467). When the genes encoding these proteins are mutated, specific photosynthetic phenotypes are noted, illustrating the potential new insights into solar energy utilization to be gained by this proteomic blueprint of the ICM. In addition, proteins necessary for other cellular functions, such as ATP synthesis, respiration, solute transport, protein translocation, and other physiological processes, were also identified to be in association with the ICM. This study is the first to provide a more global view of the protein composition of a photosynthetic membrane from any source. This protein blueprint also provides insights into potential mechanisms for the assembly of the pigment- protein complexes of the photosynthetic apparatus, the formation of the lipid bilayer that houses these integral membrane proteins, and the possible functional interactions of ICM proteins with activities that reside in domains outside this specialized bioenergetic membrane.

The highly toxic oxyanion tellurite (Te[O.sub.3.sup.2-]) is a well known pro-oxidant in mammalian and bacterial cells. This work examines the effects of tellurite on the redox state of the electron transport chain of the facultative phototroph Rbodobacter capsulatus, in relation to the role of the thiol:disulfide oxidoreductase DsbB. Under steady-state respiration, the addition of tellurite (2.5 mM) to membrane fragments generated an extrareduction of the cytochrome pool (c- and b-type hemes); further, in plasma membranes exposed to tellurite (0.25 to 2.5 mM) and subjected to a series of flashes of light, the rate of the Q[H.sub.2]:cytochrome c (Cyt c) oxidoreductase activity was enhanced. The effect of tellurite was blocked by the antibiotics antimycin A and/or myxothiazol, specific inhibitors of the Q[H.sub.2]:Cyt c oxidoreductase, and, most interestingly, the membrane-associated thiol:disulfide oxidoreductase DsbB was required to mediate the redox unbalance produced by the oxyanion. Indeed, this phenomenon was absent from R. capsulatus MD22, a DsbB-deficient mutant, whereas the tellurite effect was present in membranes from MD22/[pDsbB.sup.WT], in which the mutant gene was complemented to regain the wild-type DsbB phenotype. These findings were taken as evidence that the membrane-bound thiol:disulfide oxidoreductase DsbB acts as an 'electron conduit' between the hydrophilic metalloid and the lipid-embedded Q pool, so that in habitats contaminated with subinhibitory amounts of [Te.sup.IV], the metalloid is likely to function as a disposal for the excess reducing power at the Q-pool level of facultative phototrophic bacteria.

Purple aerobic anoxygenic phototrophs (AAPs) are the only organisms known to capture light energy to enhance growth only in the presence of oxygen but do not produce oxygen. The highly adaptive AAPs compose more than 10% of the microbial community in some euphotic upper ocean waters and are potentially major contributors to the fixation of the greenhouse gas C[O.sub.2]. We present the complete genomic sequence and feature analysis of the AAP Roseobacter denitrificans, which reveal clues to its physiology. The genome lacks genes that code for known photosynthetic carbon fixation pathways, and most notably missing are genes for the Calvin cycle enzymes ribulose bisphosphate carboxylase (RuBisCO) and phosphoribulokinase. Phyiogenetic evidence implies that this absence could be due to a gene loss from a RuBisCO-containing [alpha]-proteobacterial ancestor. We describe the potential importance of mixotrophic rather than autotrophie C[O.sub.2] fixation pathways in these organisms and suggest that these pathways function to fix C[O.sub.2] for the formation of cellular components but do not permit autotrophic growth. While some genes that code for the redox-dependent regulation of photosynthetic machinery are present, many light sensors and transcriptional regulatory motifs found in purple photosynthetic bacteria are absent.

The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic C[O.sub.2] fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to [beta]-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for succinyl-CoA:D-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction D-citramalate + succinyl-CoA [right arrow] D-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also accepted D-malate and itaconate instead of D-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for D-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route.

In this article, a mechanism of arsenite [As(III)] resistance through methylation and subsequent volatization is described. Heterologous expression of arsM from Rhodopseudomonas palustris was shown to confer As(III) resistance to an arsenic-sensitive strain of Escherichia coli. ArsM catalyzes the formation of a number of methylated intermediates from As(III), with trimethylarsine as the end product. The net result is loss of arsenic, from both the medium and the cells. Because ArsM homologues are widespread in nature, this microbial-mediated transformation is proposed to have an important impact on the global arsenic cycle. As(III) | ArsM | methylation

Bacteria in anoxic environments typically convert aromatic compounds derived from pollutants or green plants to benzoyl-CoA, and then to the [C.sub.7] dicarboxylic acid derivative 3-hydroxypimelyl-CoA. Inspection of the recently completed genome sequence of the purple nonsulfur phototroph Rhodopseudomonas palustris revealed one predicted cluster of genes for the [beta]-oxidation of dicarboxylic acids. These genes, annotated as pimFABCDE, are predicted to encode acyl-CoA ligase, enoyl-CoA hydratase, acyl-CoA dehydrogenase and acyl-CoA transferase enzymes, which should allow the conversion of odd-chain dicarboxylic acids to glutaryl-CoA, and even-chain dicarboxylic acids to succinyl-CoA. A mutant strain that was deleted in the pim gene cluster grew at about half the rate of the wild-type parent when benzoate or pimelate was supplied as the sole carbon source. The mutant grew five times more slowly than the wild-type on the [C.sub.14] dicarboxylic acid tetradecanedioate. The mutant was unimpaired in growth on the Ca-fatty acid caprylate. The acyl-CoA ligase predicted to be encoded by the pimA gene was purified, and found to be active with [C.sub.7]-[C.sub.14] dicarboxylic and fatty acids. The expression of a pimA--lacZ chromosomal gene fusion increased twofold when cells were grown in the presence of straight-chain [C.sub.7]-[C.sub.14] dicarboxylic and fatty acids. These results suggest that the [beta]-oxidation enzymes encoded by the pim gene cluster are active with medium-chain-length dicarboxylic acids, including pimelate. However, the finding that the pim operon deletion mutant is still able to grow on dicarboxylic acids, albeit at a slower rate, indicates that R. palustris has additional genes that can also specify the degradation of these compounds.

Superoxide dismutase from the thermophilic anoxygenic photosynthetic bacterium Chloroflexus aurantiacus was cloned, purified, and characterized. This protein is in the manganese- and iron-containing family of superoxide dismutases and is able to use both manganese and iron catalytically. This appears to be the only soluble superoxide dismutase in C. aurantiacus. Iron and manganese cofactors were identified by using electron paramagnetic resonance spectroscopy and were quantified by atomic absorption spectroscopy. By metal enrichment of growth media and by performing metal fidelity studies, the enzyme was found to be most efficient with manganese incorporated, yet up to 30% of the activity was retained with iron. Assimilation of iron or manganese ions into superoxide dismutase was also found to be affected by the growth conditions. This enzyme was also found to be remarkably thermostable and was resistant to [H.sub.2][O.sub.2] at concentrations up to 80 mm. Reactive oxygen defense mechanisms have not been previously characterized in the organisms belonging to the phylum Chloroflexi. These systems are of interest in C. aurantiacus since this bacterium lives in a hyperoxic environment and is subject to high UV radiation fluxes.

Induction of biosynthesis of the photosystem in anoxygenic photosynthetic bacteria occurs when the oxygen concentration drops. Control of this induction takes place primarily at the transcriptional level, with photosynthesis genes expressed preferentially under anaerobic conditions. Here, we report analysis of the transcriptional control of two photosynthesis promoters, pucBA and crtI, by the PpsR factor in Rubrivivax gelatinosus. This was accomplished by analyzing the photosystem production in the wild type and in the PPSRK (ppsR::Km) mutant grown under anaerobic and semiaerobic conditions and by assessing the [beta]-galactosidase activity of lacZ transcriptionally fused to promoters possessing the putative PpsR-binding consensus sequences. It was found that under semiaerobic conditions, inactivation of the ppsR gene resulted in overproduction of carotenoid and bacteriochlorophyll pigments, while the production of LH2 was drastically reduced. The [beta]-galactosidase activity showed that, in contrast to what has been found previously for Rhodobacter species, PpsR acts in R. gelatinosus as an aerobic repressor of the crtI gene while it acts as an activator for the expression of pucBA. Inspection of the putative PpsR-binding consensus sequences revealed significant differences that may explain the different levels of expression of the two genes studied.

The photosynthetic bacterium Rhodobacter capsulatus contains two [NiFe] hydrogenases: an energy-generating hydrogenase, HupSL, and a regulatory hydrogenase, HupUV. The synthesis of HupSL is specifically activated by [H.sub.2] through a signal transduction cascade comprising three proteins: the [H.sub.2]-sensing HupUV protein, the histidine kinase HupT, and the transcriptional regulator HupR. Whereas a phosphotransfer between HupT and HupR was previously demonstrated, interaction between HupUV and HupT was only hypothesized based on in vivo analyses of mutant phenotypes. To visualize the in vitro interaction between HupUV and HupT proteins, a six-His ([His.sub.6])-HupU fusion protein and the HupV protein were coproduced by using a homologous expression system. The two proteins copurified as a [His.sub.6]-HupUHupV complex present in dimeric and tetrameric forms, both of which had [H.sub.2] uptake activity. We demonstrated that HupT and HupUV interact and form stable complexes that could be separated on a native gel. Interaction was also monitored with surface plasmon resonance technology and was shown to be insensitive to salt concentration and pH changes, suggesting that the interactions involve hydrophobic residues. As expected, [H.sub.2] affects the interaction between HupUV and HupT, leading to a weakening of the interaction, which is independent of the phosphate status of HupT. Several forms of HupT were tested for their ability to interact with HupUV and to complement hupT mutants. Strong interaction with HupUV was obtained with the isolated PAS domain of HupT and with inactive HupT mutated in the phosphorylable histidine residue, but only the wild-type HupT protein was able to restore normal [H.sub.2] regulation.

Research has been conducted on tetranuclear manganese cluster of the oxygen-evolving complex. The reactivity of nitric oxide (super). with the oxygen-evolving complex in Synechococcus elongatus and spinach has been investigated via the use of flash-induced oxygen evolution measurements and electron paramagnetic resonance spectroscopy, and the results are reported.

Photosystem II (PSII) is a multisubunit membrane protein complex performing light-induced electron transfer and water-splitting reactions, leading to the formation of molecular oxygen. The first crystal structure of PSII from a thermophilic cyanobacterium Thermosynechococcus elongatus was reported recently [Zouni, A., Witt, H. T., Kern, J., Fromme, P., Krauss, N., Saenger, W. & Orth, P. (2001) Nature 409, 739-743)] at 3.8-[Angstrom] resolution. To analyze the PSII structure in more detail, we have obtained the crystal structure of PSII from another thermophilic cyanobacterium, Thermosynechococcus vulcanus, at 3.7-[Angstrom] resolution. The present structure was built on the basis of the sequences of PSII large subunits D1, D2, CP47, and CP43; extrinsic 33- and 12-kDa proteins and cytochrome c550; and several low molecular mass subunits, among which the structure of the 12-kDa protein was not reported previously. This yielded much information concerning the molecular interactions within this large protein complex. We also show the arrangement of chlorophylls and cofactors, including two [beta]-carotenes recently identified in a region close to the reaction center, which provided important clues to the secondary electron transfer pathways around the reaction center. Furthermore, possible ligands for the Mn-cluster were determined. In particular, the C terminus of D1 polypeptide was shown to be connected to the Mn cluster directly. The structural information obtained here provides important insights into the mechanism of PSII reactions.

The synergy between two oxygen sensing systems, Prr and PrrB, is involved in the response of Rhodobacter sphaeroides to oxygen. Data indicate that Prr causes a negaive response to atmospheric oxygen, whereas the PrrB causes positive response to low oxygen concentrations. The activity of cbb(sub)3 cytochrome oxidase influence bacterium's tactic behavior.

Research indicates that in Rhodovulum sulfidophilum cytochrome c(sub)2 is not the dominant electron donor to reaction center as a soluble cytochrome, c-549, donates electron to the reaction center-bound triheme cytochrome. This process is different from those of other purple bacteria.