Chemosensory and photosensory perception in purple photosynthetic bacteria utilize common signal transduction components.

The chemotaxis gene cluster from the photosynthetic bacterium Rhodospirillum centenum contains five open reading frames (ORFs) that have significant sequence homology to chemotaxis genes from other bacteria. To elucidate the functions of each ORF, we have made various mutations in the gene cluster and analyzed their phenotypic defects. Deletion of the entire che operon (delta che), as well as nonpolar disruptions of cheAY, cheW, and cheR, resulted in a smooth-swimming phenotype, whereas disruption of cheB resulted in a locked tumbly phenotype. Each of these mutants was defective in chemotactic response. Interestingly, disruption of cheY resulted in a slight increase in the frequency of tumbling/reversal with no obvious defects in chemotactic response. In contrast to observations with Escherichia coli and several other bacteria, we found that all of the che mutant cells were capable of differentiating into hyperflagellated swarmer cells when plated on a solid agar surface. When viewed microscopically, the smooth-swimming che mutants exhibited active surface motility but were unable to respond to a step-down in light intensity. Both positive and negative phototactic responses were abolished in all che mutants, including the cheY mutant. These results indicate that eubacterial photosensory perception is mediated by light-generated signals that are transmitted through the chemotaxis signal transduction cascade.     

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.     

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.     

Phototaxis and other sensory phenomena in purple photosynthetic bacteria

Abstract: The mechanisms employed by purple photosynthetic bacteria to convert light to utilizable chemical energy have been a major focus of research over the past 50 years. Utilization of light by photosynthetic bacteria for other purposes, however, has received relatively little attention. The recent discovery of phototaxis by Rhodospirillum centenum provides new opportunities for biochemical and molecular biological analysis of sensory processes in purple bacteria.     

Photoactive yellow protein, bacteriophytochrome, and sensory rhodopsin in purple phototrophic bacteria.

The purple photosynthetic bacteria contain a large variety of sensory and regulatory proteins, and those responding to light are among the most interesting. These currently include bacteriophytochrome (Bph), sensory rhodopsin (SR), and photoactive yellow protein (PYP), which all appear to function as light sensors. We herein interpret new findings within the context of current knowledge. For greater detail, the reader is referred to comprehensive reviews on these topics. Of the three proteins, only PYP has been well-characterized in terms of structure and physical-chemical properties in the purple bacteria, although none have well-defined functions. New findings include a cluster of six genes in the Thermochromatium tepidum genome that encodes presumed sensory rhodopsin and phototaxis proteins. T. tepidum also has a gene for PYP fused to bacteriophytochrome and diguanylate cyclase domains. The genes for PYP and its biosynthetic enzymes are associated with those for gas vesicle formation in Rhodobacter species, suggesting that one function of PYP is to regulate cell buoyancy. The association of bacteriophytochrome genes with those for reaction centers and light-harvesting proteins in Rhodopseudomonas palustris suggests that the photosynthetic antenna as well as the reaction center are regulated by Bphs. Furthermore, Rc. centenum PPR is reversibly photobleached at 702 nm rather than red-shifted as in other phytochromes, suggesting that PPR senses the intensity of white light rather than light quality. PYP from Halorhodospira(aka Ectothiorhodospira)halophila is of special interest because it has become the structural prototype for the PAS domain, a motif that is found throughout the phylogenetic tree and which plays important roles in many signaling pathways. Thus, the structural and photochemical characterization of PYP, utilizing site-directed mutagenesis, provides insights into the mechanism of signal transduction.     

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.     

A CheW homologue is required for Myxococcus xanthus fruiting body development,social gliding motility,and fibril biogenesis

In bacteria with multiple sets of chemotaxis genes, the deletion of homologous genes or even different genes in the same operon can result in disparate phenotypes. Myxococcus xanthus is a bacterium with multiple sets of chemotaxis genes and/or homologues. It was shown previously that difA and difE, encoding homologues of the methyl-accepting chemoreceptor protein (MCP) and the CheA kinase, respectively, are required for M. xanthus social gliding (S) motility and development. Both difA and difE mutants were also defective in the biogenesis of the cell surface appendages known as extracellular matrix fibrils. In this study, we investigated the roles of the CheW homologue encoded by difC, a gene at the same locus as difA and difE. We showed that difC mutations resulted in defects in M. xanthus developmental aggregation, sporulation, and S motility. We demonstrated that difC is indispensable for wild-type cellular cohesion and fibril biogenesis but not for pilus production. We further illustrated the ectopic complementation of a difC in-frame deletion by a wild-type difC. The identical phenotypes of difA, difC, and difE mutants are consistent and supportive of the hypothesis that the Dif chemotaxis homologues constitute a chemotaxis-like signal transduction pathway that regulates M. xanthus fibril biogenesis and S motility.     

A new set of chemotaxis homologues is essential for Myxococcus xanthus social motility

Myxococcus xanthus cells aggregate and develop into multicellular fruiting bodies in response to starvation. A new M. xanthus locus, designated dif for defective in fruiting, was identified by the characterization of a mutant defective in fruiting body formation. Molecular cloning, DNA sequencing and sequence analysis indicate that the dif locus encodes a new set of chemotaxis homologues of the bacterial chemotaxis proteins MCPs (methyl-accepting chemotaxis proteins), CheW, CheY and CheA. The dif genes are distinct genetically and functionally from the previously identified M. xanthus frz chemotaxis genes, suggesting that multiple chemotaxis-like systems are required for the developmental process of M. xanthus fruiting body formation. Genetic analysis and phenotypical characterization indicate that the M. xanthus dif locus is required for social (S) motility. This is the first report of a M. xanthus chemotaxis-like signal transduction pathway that could regulate or co-ordinate the movement of M. xanthus cells to bring about S motility.     

Attenuated effect of oxygen on photopigment synthesis in Rhodospirillum centenum.

Rhodospirillum centenum resembles typical nonsulfur photosynthetic bacteria in a number of respects, including its ability to grow either anaerobically as a phototroph or aerobically as a heterotroph. We demonstrate, however, that R. centenum is unusual in its ability to synthesize a functional photosynthetic apparatus regardless of the presence of molecular oxygen. Aerobically expressed photopigments were shown to be functionally active, as demonstrated by the ability of heterotrophically grown cells to grow photosynthetically, without a lag, when suddenly placed under anaerobic conditions. An R. centenum mutant that has acquired the ability to repress synthesis of photopigments in the presence of oxygen was also characterized. Both the wild type and the oxygen-repressed mutant of R. centenum were found to exhibit high light intensity repression of photopigment biosynthesis. The latter result suggests that R. centenum contains separate regulatory circuits for controlling synthesis of its photochemical apparatus by light intensity and oxygen.     

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.     

Light-induced behavioral responses (`phototaxis') in prokaryotes

Light-induced sensory responses are among the oldest scientific observations on bacterial behavior. Various types of response have been characterized physiologically in detail. However, the molecular basis of this type of response is only slowly emerging. In many of these systems photosynthetic pigments absorb the light. This then generates a signal via electron transport, feeding into a canonical chemotaxis signal transduction pathway. Nevertheless, several examples have been identified in which dedicated photoreceptor proteins do play a role. The intrinsic complexity of some of these signal transduction systems is overwhelming, in part because of the significant apparent redundancy. The genomics information that is now available for several model organisms (in particular Rhodobacter sphaeroides and Synechocystis sp. PCC6803) facilitates obtaining an increasingly detailed view of the molecular basis of the partial reactions that jointly form the basis of this type of elementary behavioral response. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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.     

The switch complex ArlCDE connects the chemotaxis system and the archaellum

Cells require a sensory system and a motility structure to achieve directed movement. Bacteria and archaea possess rotating filamentous motility structures that work in concert with the sensory chemotaxis system. This allows microorganisms to move along chemical gradients. The central response regulator protein CheY can bind to the motor of the motility structure, the flagellum in bacteria, and the archaellum in archaea. Both motility structures have a fundamentally different protein composition and structural organization. Yet, both systems receive input from the chemotaxis system. So far, it was unknown how the signal is transferred from the archaeal CheY to the archaellum motor to initiate motor switching. We applied a fluorescent microscopy approach in the model euryarchaeon Haloferax volcanii and shed light on the sequence order in which signals are transferred from the chemotaxis system to the archaellum. Our findings indicate that the euryarchaeal-specific ArlCDE are part of the archaellum motor and that they directly receive input from the chemotaxis system via the adaptor protein CheF. Hence, ArlCDE are an important feature of the archaellum of euryarchaea, are essential for signal transduction during chemotaxis and represent the archaeal switch complex.     

Identification of Archaea-specific chemotaxis proteins which interact with the flagellar apparatus

Background  

Archaea share with bacteria the ability to bias their movement towards more favorable locations, a process known as taxis. Two molecular systems drive this process: the motility apparatus and the chemotaxis signal transduction system. The first consists of the flagellum, the flagellar motor, and its switch, which allows cells to reverse the rotation of flagella. The second targets the flagellar motor switch in order to modulate the switching frequency in response to external stimuli. While the signal transduction system is conserved throughout archaea and bacteria, the archaeal flagellar apparatus is different from the bacterial one. The proteins constituting the flagellar motor and its switch in archaea have not yet been identified, and the connection between the bacterial-like chemotaxis signal transduction system and the archaeal motility apparatus is unknown.     

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.     

Rhodospirillum centenum utilizes separate motor and switch components to control lateral and polar flagellum rotation

Rhodospirillum centenum is a purple photosynthetic bacterium that is capable of differentiating from vibrioid swimming cells that contain a single polar flagellum into rod-shaped swarming cells that have a polar flagellum plus numerous lateral flagella. Microscopic studies have demonstrated that the polar flagellum is constitutively present and that the lateral flagella are found only when the cells are grown on solidified or viscous medium. In this study, we demonstrated that R. centenum contains two sets of motor and switch genes, one set for the lateral flagella and the other for the polar flagellum. Electron microscopic analysis indicated that polar and lateral flagellum-specific FliG, FliM, and FliN switch proteins are necessary for assembly of the respective flagella. In contrast, separate polar and lateral MotA and MotB motor subunits are shown to be required for motility but are not needed for the synthesis of polar and lateral flagella. Phylogenetic analysis indicates that the polar and lateral FliG, FliM, and FliN switch proteins are closely related and most likely arose as a gene duplication event. However, phylogenetic analysis of the MotA and MotB motor subunits suggests that the polar flagellum may have obtained a set of motor genes through a lateral transfer event.     

The Relation of Signal Transduction to the Sensitivity and Dynamic Range of Bacterial Chemotaxis

Complex networks of interacting molecular components of living cells are responsible for many important processes, such as signal processing and transduction. An important challenge is to understand how the individual properties of these molecular interactions and biochemical transformations determine the system-level properties of biological functions. Here, we address the issue of the accuracy of signal transduction performed by a bacterial chemotaxis system. The chemotaxis sensitivity of bacteria to a chemoattractant gradient has been measured experimentally from bacterial aggregation in a chemoattractant-containing capillary. The observed precision of the chemotaxis depended on environmental conditions such as the concentration and molecular makeup of the chemoattractant. In a quantitative model, we derived the chemotactic response function, which is essential to describing the signal transduction process involved in bacterial chemotaxis. In the presence of a gradient, an analytical solution is derived that reveals connections between the chemotaxis sensitivity and the characteristics of the signaling system, such as reaction rates. These biochemical parameters are integrated into two system-level parameters: one characterizes the efficiency of gradient sensing, and the other is related to the dynamic range of chemotaxis. Thus, our approach explains how a particular signal transduction property affects the system-level performance of bacterial chemotaxis. We further show that the two parameters can be derived from published experimental data from a capillary assay, which successfully characterizes the performance of bacterial chemotaxis.