Genetic analysis of photosynthesis in Rhodospirillum centenum.

A genetic system has been developed for studying bacterial photosynthesis in the recently described nonsulfur purple photosynthetic bacterium Rhodospirillum centenum. Nonphotosynthetic mutants of R. centenum were obtained by enrichment for spontaneous mutations, by ethyl methanesulfonate mutagenesis coupled to penicillin selection on solid medium, and by Tn5 transposition mutagenesis with an IncP plasmid vector containing a temperature-sensitive origin of replication. In vivo and in vitro characterization of individual strains demonstrated that 38 strains contained mutations that blocked bacteriochlorophyll a biosynthesis at defined steps of the biosynthetic pathway. Collectively, these mutations were shown to block seven of eight steps of the pathway leading from protoporphyrin IX to bacteriochlorophyll a. Three mutants were isolated in which carotenoid biosynthesis was blocked early in the biosynthetic pathway; the mutants also exhibited pleiotropic effects on stability or assembly of the photosynthetic apparatus. Five mutants failed to assemble a functional reaction center complex, and seven mutants contained defects in electron transport as shown by an alteration in cytochromes. In addition, several regulatory mutants were isolated that acquired enhanced repression of bacteriochlorophyll in response to the presence of molecular oxygen. The phenotypes of these mutants are discussed in relation to those of similar mutants of Rhodobacter and other Rhodospirillum species of purple photosynthetic bacteria.     

Conservation of the photosynthesis gene cluster in Rhodospirillum centenum

Intraspecies and intergenus complementation analysis were utilized to demonstrate that photosynthesis genes are clustered in distantly related purple photosynthetic bacteria. Specifically, we show that the linkage order for genes involved in bacteriochlorophyll and carotenoid biosynthesis in Rhodospirillum centenum are arranged essentially as in Rhodobacter capsulatus and Rhodobacter sphaeroides. In addition, the location and relative distance observed between the puf and puh operons which encode for light harvesting and reaction-centre structural genes are also conserved between these species. Conservation of the photosynthesis gene cluster implies either that there are structural or regulatory constraints that limit rearrangement of the photosynthesis gene cluster or that there may have been lateral transfer of the photosynthesis gene cluster among different species of phototrophic bacteria.     

The effect of respiration on the phototactic behavior of the purple nonsulfur bacterium Rhodospirillum centenum

The effect of respiration on the positive phototactic movement of swarming agar colonies of the facultative phototroph Rhodospirillum centenum was studied. When the electron flow was blocked at the bc ₁ complex level by myxothiazol, the oriented movement of the colonies was totally blocked. Conversely, inhibition of respiration via the cytochrome c oxidase stimulated the phototactic response. No phototaxis was observed in a photosynthesis deficient mutant (YB707) lacking bacteriochlorophylls. Analyses of the respiratory activities as monitored by a oxygen microelectrode in single agar colonies during light/dark transitions showed a close functional correlation between the photosynthetic and respiratory apparatuses. The respiratory chain of Rsp. centenum was formed by two oxidative pathways: one branch leading to a cytochrome c oxidase inhibited by low cyanide concentrations and a second pathway formed by an oxidase less-sensitive to cyanide that also catalyzes the light-driven respiration. These results were interpreted to indicate that (1) there is a cyclic electron transport, and (2) photoinduced cyclic electron flow is required for the phototactic response of Rsp. centenum. Furthermore, under oxic conditions in the light, reducing equivalents may switch from photosynthetic to respiratory components so as to reduce both the membrane potential and the rate of locomotion. Received: 25 September 1996 / Accepted: 11 November 1996     

Photoresponses of the purple nonsulfur bacteria Rhodospirillum centenum and Rhodobacter sphaeroides.

We have measured the photoresponse of two purple nonsulfur bacteria, Rhodobacter sphaeroides and Rhodospirillum centenum, under defined conditions in a light beam propagating at 90 degrees to the optical axis of the microscope. This beam presented cells with a steep gradient of intensity perpendicular to the direction of propagation and a shallow gradient in the direction of light propagation. R. centenum, a species that reverses to change direction, accumulated in the light beam, as expected for a "scotophobic" response, while R. sphaeroides, which stops rather than reverses, accumulated outside the light beam. We also compared the behavior of liquid-grown R. centenum, which swims by using a single polar flagellum, to that of surface-grown R. centenum, which swarms over agar by using many lateral flagella and has been shown to move as colonies toward specific wavelengths of light. When suspended in liquid medium, both liquid- and surface-grown R. centenum showed similar responses to the light gradient. In all cases, free-swimming cells responded to the steep gradient of intensity but not to the shallow gradient, indicating they cannot sense the direction of light propagation but only its intensity. In a control experiment, the known phototactic alga Chlamydamonas reinhardtii was shown to swim in the direction of light propagation.     

Biotype of the purple nonsulfur photosynthetic bacterium,Rhodospirillum centenum

Rhodospirillum centenum exhibited a number of general properties typically observed in nonsulfur purple bacteria, but also displayed a number of unusual characteristics that include: (1) conversion of the vibrioid/spiral cells to thick-walled cysts under certain growth conditions; (2) absence of O₂ repression of photopigment synthesis; (3) synthesis of “R-bodies”; and (4) swarming motility on agar surfaces that allows macroscopic observation of colony phototaxis. The unusual characteristics indicate that Rsp.centenum will prove to be a valuable experimental system for investigating various basic problems, especially in connection with photosensory phenomena and the regulation of photopigment synthesis by dioxygen and light. The present comparative study of 13 strains was undertaken to further define the Rsp. centenum biotype. Received: 3 August 1995 / Accepted: 1 November 1995     

Rhodospirillum centenum,sp. nov., a thermotolerant cyst-forming anoxygenic photosynthetic bacterium

A novel non-sulfur purple photosynthetic bacterium, designated Rhodospirillum centenum, was isolated from an enrichment culture designed to favor growth of anoxygenic photosynthetic N₂-fixing bacteria. R. centenum grows optimally at 40–42° C and has the capacity to produce cytoplasmic R bodies, refractile structures not observed hitherto in photosynthetic prokaryotes. The bacterium is also unusual among photosynthetic bacteria in that it forms desiccation-resistant cysts when grown aerobically in darkness with butyrate as the sole carbon source.     

A che-like signal transduction cascade involved in controlling flagella biosynthesis in Rhodospirillum centenum

Rhodospirillum centenum is a photosynthetic bacterium capable of undergoing swim cell to swarm cell differentiation that allows this species to be motile on both liquid and solid media. Previous experiments have demonstrated that the che1 operon is required for the control of chemotactic and phototactic behaviour of both swim and swarm cells. In this report, we analyse the function of a second che-like gene cluster in R. centenum, the che2 gene cluster. In-frame deletion mutants of cheW2, cheB2, cheR2, cheY2, and of the entire che2 operon, exhibit defects in swim and swarm cell motility. Analysis of these strains demonstrates that they are non-motile, and that the non-motile phenotype is resulting from reduced polar and lateral flagella synthesis. Additionally, mutations in mcp2, ORF204, cheA2 and ORF74 remain chemotacticly and phototacticly competent at both high and low growth temperatures. Mutations in these che2 genes result in elevated levels of flagellin proteins giving rise to a hyperflagellate phenotype. We propose a model in which R. centenum utilizes a che-like signal transduction pathway (che2) for regulating flagellum synthesis in order to optimize swim cell-swarm cell differentiation in response to changing environmental conditions.     

Isolation of Rhodospirillum centenum Mutants Defective in Phototactic Colony Motility by Transposon Mutagenesis

The purple photosynthetic bacterium Rhodospirillum centenum is capable of forming swarm colonies that rapidly migrate toward or away from light, depending on the wavelength of excitation. To identify components specific for photoperception, we conducted mini-Tn5-mediated mutagenesis and screened approximately 23,000 transposition events for mutants that failed to respond to either continuous illumination or to a step down in light intensity. A majority of the ca. 250 mutants identified lost the ability to form motile swarm cells on an agar surface. These cells appeared to contain defects in the synthesis or assembly of surface-induced lateral flagella. Another large fraction of mutants that were unresponsive to light were shown to be defective in the formation of a functional photosynthetic apparatus. Several photosensory mutants also were obtained with defects in the perception and transmission of light signals. Twelve mutants in this class were shown to contain disruptions in a chemotaxis operon, and five mutants contained disruptions of components unique to photoperception. It was shown that screening for photosensory defective R. centenum swarm colonies is an effective method for genetic dissection of the mechanism of light sensing in eubacteria.Behavioral change in response to alterations in the quality and quantity of light in the environment is a ubiquitous trait among motile photosynthetic bacteria. Three distinct types of responses to light have been described in the literature (14, 19, 36, 37). The scotophobic response (fear of darkness) is characterized by a tumbling, stop, or reversal that occurs when a swimming bacterium experiences a temporal, or spatial, step down in light intensity. Photokinesis describes an alteration in the rate of motility caused by differences in light intensity. A phototactic response, which has been studied most extensively in algae and cyanobacteria, involves an oriented movement of a cell toward or away from a light source (19). An important distinction is that the direction of irradiation is not relevant to scotophobic or photokinetic responses, whereas it is a critical determinant in phototaxis. Thus, phototactic organisms are uniquely capable of migrating towards a light source, irrespective of whether they are going up or down a gradient of light intensity (37).The various photosensory behaviors exhibited by anoxygenic photosynthetic bacteria have been studied mainly by physiological and biochemical tests, with little supporting genetic data (3, 4, 8, 9, 13, 16, 27, 38). The few genetic tests that have been undertaken have demonstrated that mutations which functionally impair the photosystem also disrupt the ability of cells to respond to light (3, 20). This indicates that a product of photosynthesis, such as the generation of proton motive force or photosynthesis-driven electron transfer, is most likely the signal that controls photosensory behavior, rather than direct absorption of light by a chromophore-containing receptor. This conclusion is supported by recent physiological studies which have shown that specific inhibitors of cyclic photosynthesis-driven electron transport inhibit photosensory behavior in Rhodobacter sphaeroides (13, 16) and Rhodospirillum centenum (38). By using a site-directed mutational approach, we have shown that the scotophobic and phototactic responses of the purple nonsulfur photosynthetic bacterium R. centenum involve components of the chemotaxis phosphorylation cascade (25, 26). This suggests that a sensor of photosynthetic activity may have features similar to that of chemoreceptors. However, which component of the photosynthesis electron transfer chain is being sensed and what is actually sensing alterations in electron transfer are unknown.To identify components responsible for prokaryotic behavioral responses to light, it is essential that techniques be developed for the isolation of mutants that are specifically defective in photosensory behavior. One of the reasons why screens for photosensory mutants have not been developed is the inherent difficulty of assaying for photosensory behavior. Until recently, screening for such mutants involved the onerous task of microscopically assaying individual cells from liquid-grown cultures for a response to a step up or down in light intensity. Since statistically meaningful results require that multiple cells be assayed, this “brute force” approach is infeasible. A significant advance in the isolation of prokaryotic photosensory mutants was recently provided by our observation that colonies of the purple photosynthetic bacterium R. centenum are capable of macroscopic phototactic motility (36, 37). Cells of R. centenum are dimorphic, existing in liquid medium as swim cells bearing a single polar flagellum or as hyperflagellated swarm cells on solid surfaces (36, 37). A unique feature of R. centenum swarming colonies is that they are capable of migrating rapidly (up to 75 mm/h) toward an infrared light source or away from a visible light source (36, 37). This behavior allows us to rapidly screen for mutants that are deficient in photosensory responses by simply assaying colonies for aberrant light-directed migration. In this study, we have utilized mini-Tn5-mediated mutagenesis to isolate numerous mutants that exhibit defects in light-directed motility. The phenotypes of specific classes of mutants provide some unique observations on photosensory behavior, as well as on the mechanism of swim cell to swarm cell differentiation.     

Hypercyst mutants in Rhodospirillum centenum identify regulatory loci involved in cyst cell differentiation

Rhodospirillum centenum is a purple photosynthetic bacterium that forms resting cyst cells when starved for nutrients. In this study, we demonstrate that chalcone synthase gene (chsA) expression is developmentally regulated, with expression of chsA increasing up to 86-fold upon induction of the cyst developmental cycle. Screening for mini-Tn5-induced mutants that exhibit elevated chsA::lacZ expression has led to the isolation of a set of R. centenum mutants that display increased chsA gene expression concomitant with constitutive induction of the cyst developmental cycle. These "hypercyst" mutants have lost the ability to regulate cyst cell formation in response to nutrient availability. Sequence analysis indicates that the mini-Tn5-disrupted genes code for a variety of factors, including metabolic enzymes and a large set of potential regulatory factors, including four gene products with homology to histidine sensor kinases and three with homology to response regulators. Several of the disrupted genes also have sequence similarity to che-like signal transduction components.     

Involvement of a Che-like signal transduction cascade in regulating cyst cell development in Rhodospirillum centenum

Homologues of the E. coli chemotaxis (Che) signal transduction pathway are present in nearly all motile bacteria. Although E. coli contains only one Che cascade, many other bacteria are known to possess multiple sets of che genes. The role of multiple che-like gene clusters could potentially code for parallel Che-like signal transduction pathways that have distinctly different input and output functions. In this study, we describe a che-like gene cluster in Rhodospirillum centenum that controls a developmental cycle. In-frame deletion mutants of homologues of CheW (DeltacheW(3a)and DeltacheW(3b)), CheR (DeltacheR(3)), CheA (DeltacheA(3)) and a methyl-accepting chemotaxis protein (Deltamcp(3)) are defective in starvation-induced formation of heat and desiccation resistant cyst cells. In contrast, mutants of homologues of CheY (DeltacheY(3)), CheB (DeltacheB(3)), and a second input kinase designated as CheS (DeltacheS(3)) result in cells that are derepressed in the formation of cysts. A model of signal transduction is presented in which there are three distinct Che-like signal transduction cascades; one that is involved in chemotaxis, one that is involved in flagella biosynthesis and the third that is involved in cyst development.     

Regulation of carbon monoxide dehydrogenase and hydrogenase in Rhodospirillum rubrum: effects of CO and oxygen on synthesis and activity.

Exposure of the photosynthetic bacterium Rhodospirillum rubrum to carbon monoxide led to increased carbon monoxide dehydrogenase and hydrogenase activities due to de novo protein synthesis of both enzymes. Two-dimensional gels of [35S]methionine-pulse-labeled cells showed that induction of CO dehydrogenase synthesis was rapidly initiated (less than 5 min upon exposure to CO) and was inhibited by oxygen. Both CO dehydrogenase and the CO-induced hydrogenase were inactivated by oxygen in vivo and in vitro. In contrast to CO dehydrogenase, the CO-induced hydrogenase was 95% inactivated by heating at 70 degrees C for 5 min. Unlike other hydrogenases, this CO-induced hydrogenase was inhibited only 60% by a 100% CO gas phase.     

Component of the Rhodospirillum centenum photosensory apparatus with structural and functional similarity to methyl-accepting chemotaxis protein chemoreceptors

Photosynthetic bacteria respond to alterations in light conditions by migrating to locations that allows optimal use of light as an energy source. Studies have indicated that photosynthesis-driven electron transport functions as an attractant signal for motility among purple photosynthetic bacteria. However, it is unclear just how the motility-based signal transduction system monitors electron flow through photosynthesis-driven electron transport. Recently, we have demonstrated that the purple photosynthetic bacterium Rhodospirillum centenum is capable of rapidly moving swarm cell colonies toward infrared light as well as away from visible light. Light-driven colony motility of R. centenum has allowed us to perform genetic dissection of the signaling pathway that affects photosynthesis-driven motility. In this study, we have undertaken sequence and mutational analyses of one of the components of a signal transduction pathway, Ptr, which appears responsible for transmitting a signal from the photosynthesis-driven electron transport chain to the chemotaxis signal transduction cascade. Mutational analysis demonstrates that cells disrupted for ptr are defective in altering motility in response to light, as well as defective in light-dependent release of methanol. We present a model which proposes that Ptr senses the redox state of a component in the photosynthetic cyclic electron transport chain and that Ptr is responsible for transmitting a signal to the chemotaxis machinery to induce a photosynthesis-dependent motility response.     

A novel pathway for L-citramalate synthesis in Rhodospirillum rubrum.

When [14C]propionate was incubated with a cell-free extract of Rhodospirillum rubrum in the presence of glyoxylate, ATP, CoA, Mg2+, and Mn2+, radioactivity was incorporated into mesaconate (MSA) as well as into beta-methylmalate (MMA) and citramalate (CMA). MSA was suggested to be an intermediate of the conversion of MMA to CMA based on the following observations. (i) When non-labeled MSA was added to the CMA-forming reaction system, radioactivity was trapped in MSA. (ii) When MSA was incubated with the cell-free extract, CMA was formed. (iii) The alpha-carboxyl group of CMA was shown to be derived from the beta-carboxyl group of MMA, implying that CMA was formed from MMA via MSA through successive dehydration and hydration. From the results of Sephadex G-10 column chromatography of the reaction products, beta-methylmalyl-CoA and mesaconyl-CoA were presumed to be involved in the reaction. A new CMA-forming metabolic pathway is proposed as follows: erythro-beta-methylamalyl-CoA leads to mesaconyl-CoA leads to MSA leads to L-CMA.     

The photoactivated PYP domain of Rhodospirillum centenum Ppr accelerates the recovery of the bacteriophytochrome domain after white light illumination

Ppr from the purple phototrophic bacterium, Rhodospirillum centenum (also known as Rhodocista centenaria), is a hybrid of photoactive yellow protein (PYP), bacteriophytochrome (Bph), and histidine kinase (HK) domains. The holo-Ppr (containing both chromophores) exhibits characteristic absorption maxima at 435 nm due to the PYP domain and at 400, 642, and 701 nm due to the Bph domain. Illumination of the Ppr with white light causes a bleach of both PYP and Bph absorbance; weak blue light primarily bleaches the PYP, and red light activates only the Bph. When excited by blue light, the PYP domain in Ppr recovers with biphasic kinetics at 445 nm (32% with a lifetime of 3.8 min and the remainder with a lifetime of 46 min); white light primarily results in fast recovery, whereas the 130-residue PYP construct shows only the faster kinetics in both blue and white light. Furthermore, there is a slight red shift of the ground state Bph when the PYP is activated; thus, both spectroscopy and kinetics suggest interdomain communication. When Ppr is illuminated with red light, the recovery of the Bph domain to the dark state is significantly slower than that of PYP and is biphasic (57% of the 701 nm decay has a lifetime of 17 min and the remainder a lifetime of 50 min). However, when illuminated with white light or red followed by blue light, the Bph domain in Ppr recovers to the dark-adapted state in a triphasic fashion, where the fastest phase is similar to that of the fast phase of the PYP domain (in white light, 25% of the 701 nm recovery has a lifetime of approximately 1 min) and the slower phases are like the recovery after red light alone. Apo-holo-Ppr (with the biliverdin chromophore only) recovers with biphasic kinetics similar to those of the slower phases of holo-Ppr when activated by either red or white light. We conclude that the photoactivated PYP domain in Ppr accelerates recovery of the activated Bph domain. Phytochromes can be reversibly switched between Pr and Pfr forms by red and far-red light, but the consequence of a bleaching phytochrome is that it cannot be photoreversed by far-red light. We thus postulate that the function of the PYP domain in Ppr is to act as a blue light switch to reverse the effects of red light on the Bph.