sRNA-mediated RNA processing regulates bacterial cell division.

Tight control of cell division is essential for survival of most organisms. For prokaryotes, the regulatory mechanisms involved in the control of cell division are mostly unknown. We show that the small non-coding sRNA StsR has an important role in controlling cell division and growth in the alpha-proteobacterium Rhodobacter sphaeroides. StsR is strongly induced by stress conditions and in stationary phase by the alternative sigma factors RpoHI/HII, thereby providing a regulatory link between cell division and environmental cues. Compared to the wild type, a mutant lacking StsR enters stationary phase later and more rapidly resumes growth after stationary phase. A target of StsR is UpsM, the most abundant sRNA in the exponential phase. It is derived from partial transcriptional termination within the 5' untranslated region of the mRNA of the division and cell wall (dcw) gene cluster. StsR binds to UpsM as well as to the 5' UTR of the dcw mRNA and the sRNA-sRNA and sRNA-mRNA interactions lead to a conformational change that triggers cleavage by the ribonuclease RNase E, affecting the level of dcw mRNAs and limiting growth. These findings provide interesting new insights into the role of sRNA-mediated regulation of cell division during the adaptation to environmental changes.

A single-component light sensor system allows highly tunable and direct activation of gene expression in bacterial cells.

Light-regulated modules offer unprecedented new ways to control cellular behaviour with precise spatial and temporal resolution. Among a variety of bacterial light-switchable gene expression systems, single-component systems consisting of single transcription factors would be more useful due to the advantages of speed, simplicity, and versatility. In the present study, we developed a single-component light-activated bacterial gene expression system (eLightOn) based on a novel LOV domain from Rhodobacter sphaeroides (RsLOV). The eLightOn system showed significant improvements over the existing single-component bacterial light-activated expression systems, with benefits including a high ON/OFF ratio of >500-fold, a high activation level, fast activation kinetics, and/or good adaptability. Additionally, the induction characteristics, including regulatory windows, activation kinetics and light sensitivities, were highly tunable by altering the expression level of LexRO. We demonstrated the usefulness of the eLightOn system in regulating cell division and swimming by controlling the expression of the FtsZ and CheZ genes, respectively, as well as constructing synthetic Boolean logic gates using light and arabinose as the two inputs. Taken together, our data indicate that the eLightOn system is a robust and highly tunable tool for quantitative and spatiotemporal control of bacterial gene expression.

Nitrite-reducing ability is related to growth inhibition by nitrite in Rhodobacter sphaeroides f. sp. denitrificans.

Growth inhibition of Rhodobacter sphaeroides f. sp. denitrificans IL106 by nitrite under anaerobic-light conditions became less pronounced when the gene encoding nitrite reductase was deleted. Growth of another deletion mutant of the genes encoding nitric oxide reductase was severely suppressed by nitrite. Our results suggest that nitrite reductase increases the sensitivity to nitrite through the production of nitric oxide.

An RpoHI-Dependent Response Promotes Outgrowth after Extended Stationary Phase in the Alphaproteobacterium Rhodobacter sphaeroides.

Under unfavorable growth conditions, bacteria enter stationary phase and can maintain cell viability over prolonged periods with no increase in cell number. To obtain insights into the regulatory mechanisms that allow bacteria to resume growth when conditions become favorable again (outgrowth), we performed global transcriptome analyses at different stages of growth for the alphaproteobacterium Rhodobacter sphaeroides The majority of genes were not differentially expressed across growth phases. After a short stationary phase (about 20 h after growth starts to slow down), only 7% of the genes showed altered expression (fold change of >1.6 or less than -1.6, corresponding to a log2 fold change of >0.65 or less than -0.65, respectively) compared to expression at exponential phase. Outgrowth induced a distinct response in gene expression which was strongly influenced by the length of the preceding stationary phase. After a long stationary phase (about 64 h after growth starts to slow down), a much larger number of genes (15.1%) was induced in outgrowth than after a short stationary phase (1.7%). Many of those genes are known members of the RpoHI/RpoHII regulons and have established functions in stress responses. A main effect of RpoHI on the transcriptome in outgrowth after a long stationary phase was confirmed. Growth experiments with mutant strains further support an important function in outgrowth after prolonged stationary phase for the RpoHI and RpoHII sigma factors.IMPORTANCE In natural environments, the growth of bacteria is limited mostly by lack of nutrients or other unfavorable conditions. It is important for bacterial populations to efficiently resume growth after being in stationary phase, which may last for long periods. Most previous studies on growth-phase-dependent gene expression did not address outgrowth after stationary phase. This study on growth-phase-dependent gene regulation in a model alphaproteobacterium reveals, for the first time, that the length of the stationary phase strongly impacts the transcriptome during outgrowth. The alternative sigma factors RpoHI and RpoHII, which are important regulators of stress responses in alphaproteobacteria, play a major role during outgrowth following prolonged stationary phase. These findings provide the first insight into the regulatory mechanisms enabling efficient outgrowth.

Influence of trehalose on high-temperature stability of the photosynthetic reaction centers.

The effect of heating at 65°C for 20 min on the absorption spectra and kinetics of the dark recombination of charges separated between photoactive bacteriochlorophyll and quinone acceptors was studied in dry films of bacterial photosynthetic reaction centers (RCs), RC films in polyvinyl alcohol, and trehalose. A pronounced protective effect of trehalose against pheophytinizaiton of molecules bacteriochlorophylls in RC structure and in maintaining their higher photochemical activity was found.

Connectivity of the intracytoplasmic membrane of Rhodobacter sphaeroides: a functional approach.

The photosynthetic apparatus in the bacterium Rhodobacter sphaeroides is mostly present in intracytoplasmic membrane invaginations. It has long been debated whether these invaginations remain in topological continuity with the cytoplasmic membrane, or form isolated chromatophore vesicles. This issue is revisited here by functional approaches. The ionophore gramicidin was used as a probe of the relative size of the electro-osmotic units in isolated chromatophores, spheroplasts, or intact cells. The decay of the membrane potential was monitored from the electrochromic shift of carotenoids. The half-time of the decay induced by a single channel in intact cells was about 6 ms, thus three orders of magnitude slower than in isolated chromatophores. In spheroplasts obtained by lysis of the cell wall, the single channel decay was still slower (~23 ms) and the sensitivity toward the gramicidin concentration was enhanced 1,000-fold with respect to isolated chromatophores. These results indicate that the area of the functional membrane in cells or spheroplasts is about three orders of magnitude larger than that of isolated chromatophores. Intracytoplasmic vesicles, if present, could contribute to at most 10% of the photosynthetic apparatus in intact cells of Rba. sphaeroides. Similar conclusions were obtained from the effect of a ∆pH-induced diffusion potential in intact cells. This caused a large electrochromic response of carotenoids, of similar amplitude as the light-induced change, indicating that most of the system is sensitive to a pH change of the external medium. A single internal membrane and periplasmic space may offer significant advantages concerning renewal of the photosynthetic apparatus and reallocation of the components shared with other bioenergetic pathways.

Electronic Structure and Dynamics of Higher-Lying Excited States in Light Harvesting Complex 1 from Rhodobacter sphaeroides.

Light harvesting in photosynthetic organisms involves efficient transfer of energy from peripheral antenna complexes to core antenna complexes, and ultimately to the reaction center where charge separation drives downstream photosynthetic processes. Antenna complexes contain many strongly coupled chromophores, which complicates analysis of their electronic structure. Two-dimensional electronic spectroscopy (2DES) provides information on energetic coupling and ultrafast energy transfer dynamics, making the technique well suited for the study of photosynthetic antennae. Here, we present 2DES results on excited state properties and dynamics of a core antenna complex, light harvesting complex 1 (LH1), embedded in the photosynthetic membrane of Rhodobacter sphaeroides. The experiment reveals weakly allowed higher-lying excited states in LH1 at 770 nm, which transfer energy to the strongly allowed states at 875 nm with a lifetime of 40 fs. The presence of higher-lying excited states is in agreement with effective Hamiltonians constructed using parameters from crystal structures and atomic force microscopy (AFM) studies. The energy transfer dynamics between the higher- and lower-lying excited states agree with Redfield theory calculations.

A Cardiolipin-Deficient Mutant of Rhodobacter sphaeroides Has an Altered Cell Shape and Is Impaired in Biofilm Formation.

UNLABELLED: Cell shape has been suggested to play an important role in the regulation of bacterial attachment to surfaces and the formation of communities associated with surfaces. We found that a cardiolipin synthase (Δcls) mutant of the rod-shaped bacterium Rhodobacter sphaeroides--in which synthesis of the anionic, highly curved phospholipid cardiolipin (CL) is reduced by 90%--produces ellipsoid-shaped cells that are impaired in biofilm formation. Reducing the concentration of CL did not cause significant defects in R. sphaeroides cell growth, swimming motility, lipopolysaccharide and exopolysaccharide production, surface adhesion protein expression, and membrane permeability. Complementation of the CL-deficient mutant by ectopically expressing CL synthase restored cells to their rod shape and increased biofilm formation. Treating R. sphaeroides cells with a low concentration (10 µg/ml) of the small-molecule MreB inhibitor S-(3,4-dichlorobenzyl)isothiourea produced ellipsoid-shaped cells that had no obvious growth defect yet reduced R. sphaeroides biofilm formation. This study demonstrates that CL plays a role in R. sphaeroides cell shape determination, biofilm formation, and the ability of the bacterium to adapt to its environment. IMPORTANCE: Membrane composition plays a fundamental role in the adaptation of many bacteria to environmental stress. In this study, we build a new connection between the anionic phospholipid cardiolipin (CL) and cellular adaptation in Rhodobacter sphaeroides. We demonstrate that CL plays a role in the regulation of R. sphaeroides morphology and is important for the ability of this bacterium to form biofilms. This study correlates CL concentration, cell shape, and biofilm formation and provides the first example of how membrane composition in bacteria alters cell morphology and influences adaptation. This study also provides insight into the potential of phospholipid biosynthesis as a target for new chemical strategies designed to alter or prevent biofilm formation.

Fluorescence relaxation in intact cells of photosynthetic bacteria: donor and acceptor side limitations of reopening of the reaction center.

The dark relaxation of the yield of variable BChl fluorescence in the 10(-5)-10 s time range is measured after laser diode (808 nm) excitation of variable duration in intact cells of photosynthetic bacteria Rba. sphaeroides, Rsp. rubrum, and Rvx. gelatinosus under various treatments of redox agents, inhibitors, and temperature. The kinetics of the relaxation is complex and much wider extended than a monoexponential function. The longer is the excitation, the slower is the relaxation which is determined by the redox states, sizes, and accessibility of the pools of cytochrome [Formula: see text] and quinone for donor and acceptor side-limited bacterial strains, respectively. The kinetics of fluorescence decay reflects the opening kinetics of the closed RC. The relaxation is controlled preferentially by the rate of re-reduction of the oxidized dimer by mobile cytochrome [Formula: see text] in Rba. sphaeroides and Rsp. rubrum and by the rate constant of the [Formula: see text] interquinone electron transfer, (350 µs)(-1) and/or the quinol/quinone exchange at the acceptor side in Rvx. gelatinosus. The commonly used acceptor side inhibitors (e.g., terbutryn) demonstrate kinetically limited block of re-oxidation of the primary quinone. The observations are interpreted in frame of a minimum kinetic and energetic model of electron transfer reactions in bacterial RC of intact cells.

Positioning of chemosensory proteins and FtsZ through the Rhodobacter sphaeroides cell cycle.

Bacterial chemotaxis depends on signalling through large protein complexes. Each cell must inherit a complex on division, suggesting some co-ordination with cell division. In Escherichia coli the membrane-spanning chemosensory complexes are polar and new static complexes form at pre-cytokinetic sites, ensuring positioning at the new pole after division and suggesting a role for the bacterial cytoskeleton. Rhodobacter sphaeroides has both membrane-associated and cytoplasmic, chromosome-associated chemosensory complexes. We followed the relative positions of the two chemosensory complexes, FtsZ and MreB in aerobic and in photoheterotrophic R. sphaeroides cells using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which FtsZ extends circumferentially to form the Z-ring. Membrane-associated chemosensory proteins form a number of dynamic unit-clusters with mature clusters containing about 1000 CheW(3) proteins. Individual clusters diffuse randomly within the membrane, accumulating at new poles after division but not colocalizing with either FtsZ or MreB. The cytoplasmic complex colocalizes with FtsZ at midcells in new-born cells. Before cytokinesis one complex moves to a daughter cell, followed by the second moving to the other cell. These data indicate that two homologous complexes use different mechanisms to ensure partitioning, and neither complex utilizes FtsZ or MreB for positioning.

Assembly of photosynthetic apparatus in Rhodobacter sphaeroides as revealed by functional assessments at different growth phases and in synchronized and greening cells.

The development of photosynthetic membranes of intact cells of Rhodobacter sphaeroides was tracked by light-induced absorption spectroscopy and induction and relaxation of the bacteriochlorophyll fluorescence. Changes in membrane structure were induced by three methods: synchronization of cell growth, adjustment of different growth phases and transfer from aerobic to anaerobic conditions (greening) of the bacteria. While the production of the bacteriochlorophyll and carotenoid pigments and the activation of light harvesting and reaction center complexes showed cell-cycle independent and continuous increase with characteristic lag phases, the accumulation of phospholipids and membrane potential (electrochromism) exhibited stepwise increase controlled by cell division. Cells in the stationary phase of growth demonstrated closer packing and tighter energetic coupling of the photosynthetic units (PSU) than in their early logarithmic stage. The greening resulted in rapid (within 0-4 h) induction of BChl synthesis accompanied with a dominating role for the peripheral light harvesting system (up to LH2/LH1 ~2.5), significantly increased rate (~7·10(4) s(-1)) and yield (F v/F max ~0.7) of photochemistry and modest (~2.5-fold) decrease of the rate of electron transfer (~1.5·10(4) s(-1)). The results are discussed in frame of a model of sequential assembly of the PSU with emphasis on crowding the LH2 complexes resulting in an increase of the connectivity and yield of light capture on the one hand and increase of hindrance to diffusion of mobile redox agents on the other hand.

The lipidome of the photosynthetic bacterium Rhodobacter sphaeroides R26 is affected by cobalt and chromate ions stress.

A detailed characterization of membrane lipids of the photosynthetic bacterium Rhodobacter (R.) sphaeroides was accomplished by thin-layer chromatography coupled with matrix-assisted laser desorption ionization mass spectrometry. Such an approach allowed the identification of the main membrane lipids belonging to different classes, namely cardiolipins (CLs), phosphatidylethanolamines, phosphatidylglycerols (PGs), phosphatidylcholines, and sulfoquinovosyldiacylglycerols (SQDGs). Thus, the lipidomic profile of R. sphaeroides R26 grown in abiotic stressed conditions by exposure to bivalent cobalt cation and chromate oxyanion, was investigated. Compared to bacteria grown under control conditions, significant lipid alterations take place under both stress conditions; cobalt exposure stress results in the relative content increase of CLs and SQDGs, most likely compensating the decrease in PGs content, whereas chromate stress conditions result in the relative content decrease of both PGs and SQDGs, leaving CLs unaltered. For the first time, the response of R. sphaeroides to heavy metals as Co(2+) and CrO4 (2-) is reported and changes in membrane lipid profiles were rationalised.

Bacterial cell identification in differential interference contrast microscopy images.

BACKGROUND: Microscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding. RESULTS: We have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides. CONCLUSIONS: Results demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins.

Cobalt binding in the photosynthetic bacterium R. sphaeroides by X-ray absorption spectroscopy.

Cobalt is an important oligoelement required for bacteria; if present in high concentration, exhibits toxic effects that, depending on the microorganism under investigation, may even result in growth inhibition. The photosynthetic bacterium Rhodobacter (R.) sphaeroides tolerates high cobalt concentration and bioaccumulates Co(2+) ion, mostly on the cellular surface. Very little is known on the chemical fate of the bioaccumulated cobalt, thus an X-ray absorption spectroscopy investigation was conducted on R. sphaeroides cells to gain structural insights into the Co(2+) binding to cellular components. X-ray absorption near-edge spectroscopy and extended X-ray absorption fine structure measurements were performed on R. sphaeroides samples containing whole cells and cell-free fractions obtained from cultures exposed to 5 mM Co(2+). An octahedral coordination geometry was found for the cobalt ion, with six oxygen-ligand atoms in the first shell. In the soluble portion of the cell, cobalt was found bound to carboxylate groups, while a mixed pattern containing equivalent amount of two sulfur and two carbon atoms was found in the cell envelope fraction, suggesting the presence of carboxylate and sulfonate metal-binding functional groups, the latter arising from sulfolipids of the cell envelope.

Changes in morphology, cell wall composition and soluble proteome in Rhodobacter sphaeroides cells exposed to chromate.

The response of the carotenoidless Rhodobacter sphaeroides mutant R26 to chromate stress under photosynthetic conditions is investigated by biochemical and spectroscopic measurements, proteomic analysis and cell imaging. Cell cultures were found able to reduce chromate within 3-4 days. Chromate induces marked changes in the cellular dimension and morphology, as revealed by atomic force microscopy, along with compositional changes in the cell wall revealed by infrared spectroscopy. These effects are accompanied by significant changes in the level of several proteins: 15 proteins were found up-regulated and 15 down-regulated. The protein content found in chromate exposed cells is in good agreement with the biochemical, spectroscopic and microscopic results. Moreover at the present stage no specific chromate-reductase could be found in the soluble proteome, indicating that detoxification of the pollutant proceeds via aspecific reductants.

Light harvesting, energy transfer and electron cycling of a native photosynthetic membrane adsorbed onto a gold surface.

Photosynthetic membranes comprise a network of light harvesting and reaction center pigment-protein complexes responsible for the primary photoconversion reactions: light absorption, energy transfer and electron cycling. The structural organization of membranes of the purple bacterial species Rb. sphaeroides has been elucidated in most detail by means of polarized light spectroscopy and atomic force microscopy. Here we report a functional characterization of native and untreated membranes of the same species adsorbed onto a gold surface. Employing fluorescence confocal spectroscopy and light-induced electrochemistry we show that adsorbed membranes maintain their energy and electron transferring functionality. Gold-adsorbed membranes are shown to generate a steady high photocurrent of 10 microA/cm(2) for several minutes and to maintain activity for up to three days while continuously illuminated. The surface-adsorbed membranes exhibit a remarkable functionality under aerobic conditions, even when exposed to light intensities well above that of direct solar irradiation. The component at the interface of light harvesting and electron cycling, the LH1 complex, displays exceptional stability, likely contributing to the robustness of the membranes. Peripheral light harvesting LH2 complexes show a light intensity dependent decoupling from photoconversion. LH2 can act as a reversible switch at low-light, an increased emitter at medium light and photobleaches at high light.

Photosynthetic vesicle architecture and constraints on efficient energy harvesting.

Photosynthetic chromatophore vesicles found in some purple bacteria constitute one of the simplest light-harvesting systems in nature. The overall architecture of chromatophore vesicles and the structural integration of vesicle function remain poorly understood despite structural information being available on individual constituent proteins. An all-atom structural model for an entire chromatophore vesicle is presented, which improves upon earlier models by taking into account the stoichiometry of core and antenna complexes determined by the absorption spectrum of intact vesicles in Rhodobacter sphaeroides, as well as the well-established curvature-inducing properties of the dimeric core complex. The absorption spectrum of low-light-adapted vesicles is shown to correspond to a light-harvesting-complex 2 to reaction center ratio of 3:1. A structural model for a vesicle consistent with this stoichiometry is developed and used in the computation of excitonic properties. Considered also is the packing density of antenna and core complexes that is high enough for efficient energy transfer and low enough for quinone diffusion from reaction centers to cytochrome bc(1) complexes.

Dependence of catalytic activity on driving force in solution assays and protein film voltammetry: insights from the comparison of nitrate reductase mutants.

Rhodobacter sphaeroides periplasmic nitrate reductase (Rs NapAB) is one of the enzymes whose assays give odd results: in spectrophotometric assays with methyl viologen as the electron donor, the activity increases as the reaction progresses, whereas the driving force provided by the soluble redox partner decreases; in protein film voltammetry (PFV), whereby the enzyme directly exchanges electrons with an electrode, the activity of NapAB decreases at large overpotential, whereas a monotonic increase is expected [Elliott, S. J., et al. (2002) Biochim. Biophys. Acta 1555, 54-59]. The relations between these phenomena and the catalytic mechanism are still debated. By studying NapAB mutants, we found that the peculiar dependences of electrochemical and solution activities on driving force are greatly affected by substituting certain amino acids that are located in the vicinity of the active site (M153, Q384, R392); this led us to establish and discuss the relation between the experimental parameters of the electrochemical and spectrophotometric assays: we show that the rate of reduction of the enzyme (which depends on the electrode potential or on the concentration of reduced MV) modulates the activity of the enzyme, but the "solution potential" does not. Our results also support the view that the complex profiles of activity versus potential are fingerprints of the active site chemistry, rather than direct consequences of changes in the redox states of relays that are remote from the active site.

Absorbance changes accompanying the fast fluorescence induction in the purple bacterium Rhodobacter sphaeroides.

The authors present a study of the fluorescence and absorbance transients occurring in whole cells of purple nonsulfur bacterium Rhodobacter sphaeroides on the millisecond timescale under pulsed actinic illumination. The fluorescence induction curve is interpreted in terms of combination of effects of redox changes in the reaction center and the membrane potential. The results of this study support the view that the membrane potential act predominantly to increase the fluorescence yield. Advantages of the pulsed actinic illumination for study of the operation of the electron transport chain in vivo are discussed.

The native architecture of a photosynthetic membrane.

In photosynthesis, the harvesting of solar energy and its subsequent conversion into a stable charge separation are dependent upon an interconnected macromolecular network of membrane-associated chlorophyll-protein complexes. Although the detailed structure of each complex has been determined, the size and organization of this network are unknown. Here we show the use of atomic force microscopy to directly reveal a native bacterial photosynthetic membrane. This first view of any multi-component membrane shows the relative positions and associations of the photosynthetic complexes and reveals crucial new features of the organization of the network: we found that the membrane is divided into specialized domains each with a different network organization and in which one type of complex predominates. Two types of organization were found for the peripheral light-harvesting LH2 complex. In the first, groups of 10-20 molecules of LH2 form light-capture domains that interconnect linear arrays of dimers of core reaction centre (RC)-light-harvesting 1 (RC-LH1-PufX) complexes; in the second they were found outside these arrays in larger clusters. The LH1 complex is ideally positioned to function as an energy collection hub, temporarily storing it before transfer to the RC where photochemistry occurs: the elegant economy of the photosynthetic membrane is demonstrated by the close packing of these linear arrays, which are often only separated by narrow 'energy conduits' of LH2 just two or three complexes wide.