Yael Helman

Genes encoding A-type flavoproteins are essential for photoreduction of O2 in cyanobacteria.

O(2) photoreduction by photosynthetic electron transfer, the Mehler reaction, was observed in all groups of oxygenic photosynthetic organisms, but the electron transport chain mediating this reaction remains unidentified. We provide the first evidence for the involvement of A-type flavoproteins that reduce O(2) directly to water in vitro. Synechocystis sp. strain PCC 6803 mutants defective in flv1 and flv3, encoding A-type flavoproteins, failed to exhibit O(2) photoreduction but performed normal photosynthesis and respiration. We show that the light-enhanced O(2) uptake was not due to respiration or photorespiration. After dark acclimation, photooxidation of P(700) was severely depressed in mutants Deltaflv1 and Deltaflv3 but recovered after light activation of CO(2) fixation, which gives P(700) an additional electron acceptor. Inhibition of CO(2) fixation prevented recovery but scarcely affected P(700) oxidation in the wild-type, where the Mehler reaction provides an alternative route for electrons. We conclude that the source of electrons for O(2) photoreduction is PSI and that the highly conserved A-type flavoproteins Flv1 and Flv3 are essential for this process in vivo. We propose that in cyanobacteria, contrary to eukaryotes, the Mehler reaction produces no reactive oxygen species and may be evolutionarily related to the response of anaerobic bacteria to O(2).

Fractionation of the Three Stable Oxygen Isotopes by Oxygen-Producing and Oxygen-Consuming Reactions in Photosynthetic Organisms

The triple isotope composition (δ17O and δ18O) of dissolved O2 in the ocean and in ice cores was recently used to assess the primary productivity over broad spatial and temporal scales. However, assessment of the productivity with the aid of this method must rely on accurate measurements of the 17O/16O versus 18O/16O relationship in each of the main oxygen-producing and -consuming reactions. Data obtained here showed that cleavage of water in photosystem II did not fractionate oxygen isotopes; the δ18O and δ17O of the O2 evolved were essentially identical to those of the substrate water. The fractionation slopes for the oxygenase reaction of Rubisco and respiration were identical (0.518 ± 0.001) and that of glycolate oxidation was 0.503 ± 0.002. There was a considerable difference in the slopes of O2 photoreduction (the Mehler reaction) in the cyanobacterium Synechocystis sp. strain PCC 6803 (0.497 ± 0.004) and that of pea (Pisum sativum) thylakoids (0.526 ± 0.001). These values provided clear and independent evidence that the mechanism of O2 photoreduction differs between higher plants and cyanobacteria. We used our method to assess the magnitude of O2 photoreduction in cyanobacterial cells maintained under conditions where photorespiration was negligible. It was found that electron flow to O2 can be as high as 40% that leaving photosystem II, whereas respiratory activity in the light is only 6%. The implications of our findings to the evaluation of specific O2-producing or -consuming reactions, in vivo, are discussed.

Passive Entry of CO2 and Its Energy-dependent Intracellular Conversion to HCO in Cyanobacteria Are Driven by a Photosystem I-generated ΔμH+

CO2 entry intoSynechococcus sp. PCC7942 cells was drastically inhibited by the water channel blocker p-chloromercuriphenylsulfonic acid suggesting that CO2 uptake is, for the most part, passive via aquaporins with subsequent energy-dependent conversion to HCO 3 − . Dependence of CO2uptake on photosynthetic electron transport via photosystem I (PSI) was confirmed by experiments with electron transport inhibitors, electron donors and acceptors, and a mutant lacking PSI activity. CO2 uptake was drastically inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and ammonia but substantially less so by the inhibitors of ATP formation arsenate and N, N,-dicyclohexylcarbodiimide (DCCD). Thus a ΔμH+ generated by photosynthetic PSI electron transport apparently serves as the direct source of energy for CO2 uptake. Under low light intensity, the rate of CO2 uptake by a high-CO2-requiring mutant ofSynechococcus sp. PCC7942, at a CO2concentration below its threshold for CO2 fixation, was higher than that of the wild type. At saturating light intensity, net CO2 uptake was similar in the wild type and in the mutant IL-3 suggesting common limitation by the rate of conversion of CO2 to HCO 3 − . These findings are consistent with a model postulating that electron transport-dependent formation of alkaline domains on the thylakoid membrane energizes intracellular conversion of CO2 to HCO 3 − .

Extracellular matrix production and calcium carbonate precipitation by coral cells in vitro

The evolution of multicellularity in animals required the production of extracellular matrices that serve to spatially organize cells according to function. In corals, three matrices are involved in spatial organization: (i) an organic ECM, which facilitates cell–cell and cell–substrate adhesion; (ii) a skeletal organic matrix (SOM), which facilitates controlled deposition of a calcium carbonate skeleton; and (iii) the calcium carbonate skeleton itself, which provides the structural support for the 3D organization of coral colonies. In this report, we examine the production of these three matrices by using an in vitro culturing system for coral cells. In this system, which significantly facilitates studies of coral cell physiology, we demonstrate in vitro excretion of ECM by primary (nondividing) tissue cultures of both soft (Xenia elongata) and hard (Montipora digitata) corals. There are structural differences between the ECM produced by X. elongata cell cultures and that of M. digitata, and ascorbic acid, a critical cofactor for proline hydroxylation, significantly increased the production of collagen in the ECM of the latter species. We further demonstrate in vitro production of SOM and extracellular mineralized particles in cell cultures of M. digitata. Inductively coupled plasma mass spectrometry analysis of Sr/Ca ratios revealed the particles to be aragonite. De novo calcification was confirmed by following the incorporation of 45Ca into acid labile macromolecules. Our results demonstrate the ability of isolated, differentiated coral cells to undergo fundamental processes required for multicellular organization.

Transition from Anaerobic to Aerobic Growth Conditions for the Sulfate-Reducing Bacterium Desulfovibrio oxyclinae Results in Flocculation

ABSTRACT A chemostat culture of the sulfate-reducing bacteriumDesulfovibrio oxyclinae isolated from the oxic layer of a hypersaline cyanobacterial mat was grown anaerobically and then subjected to gassing with 1% oxygen, both at a dilution rate of 0.05 h−1. The sulfate reduction rate under anaerobic conditions was 370 nmol of SO42− mg of protein−1 min−1. At the onset of aerobic gassing, sulfate reduction decreased by 40%, although viable cell numbers did not decrease. After 42 h, the sulfate reduction rate returned to the level observed in the anaerobic culture. At this stage the growth yield increased by 180% compared to the anaerobic culture to 4.4 g of protein per mol of sulfate reduced. Protein content per cell increased at the same time by 40%. The oxygen consumption rate per milligram of protein measured in washed cell suspensions increased by 80%, and the thiosulfate reduction rate of the same samples increased by 29% with lactate as the electron donor. These findings indicated possible oxygen-dependent enhancement of growth. After 140 h of growth under oxygen flux, formation of cell aggregates 0.1 to 3 mm in diameter was observed. Micrometer-sized aggregates were found to form earlier, during the first hours of exposure to oxygen. The respiration rate of D. oxyclinaewas sufficient to create anoxia inside clumps larger than 3 μm, while the levels of dissolved oxygen in the growth vessel were 0.7 ± 0.5 μM. Aggregation of sulfate-reducing bacteria was observed within a Microcoleus chthonoplastes-dominated layer of a cyanobacterial mat under daily exposure to oxygen concentrations of up to 900 μM. Desulfonema-like sulfate-reducing bacteria were also common in this environment along with other nonaggregated sulfate-reducing bacteria. Two-dimensional mapping of sulfate reduction showed heterogeneity of sulfate reduction activity in this oxic zone.

Genome sequence of the pattern forming Paenibacillus vortex bacterium reveals potential for thriving in complex environments

BackgroundThe pattern-forming bacterium Paenibacillus vortex is notable for its advanced social behavior, which is reflected in development of colonies with highly intricate architectures. Prior to this study, only two other Paenibacillus species (Paenibacillus sp. JDR-2 and Paenibacillus larvae) have been sequenced. However, no genomic data is available on the Paenibacillus species with pattern-forming and complex social motility. Here we report the de novo genome sequence of this Gram-positive, soil-dwelling, sporulating bacterium.ResultsThe complete P. vortex genome was sequenced by a hybrid approach using 454 Life Sciences and Illumina, achieving a total of 289× coverage, with 99.8% sequence identity between the two methods. The sequencing results were validated using a custom designed Agilent microarray expression chip which represented the coding and the non-coding regions. Analysis of the P. vortex genome revealed 6,437 open reading frames (ORFs) and 73 non-coding RNA genes. Comparative genomic analysis with 500 complete bacterial genomes revealed exceptionally high number of two-component system (TCS) genes, transcription factors (TFs), transport and defense related genes. Additionally, we have identified genes involved in the production of antimicrobial compounds and extracellular degrading enzymes.ConclusionsThese findings suggest that P. vortex has advanced faculties to perceive and react to a wide range of signaling molecules and environmental conditions, which could be associated with its ability to reconfigure and replicate complex colony architectures. Additionally, P. vortex is likely to serve as a rich source of genes important for agricultural, medical and industrial applications and it has the potential to advance the study of social microbiology within Gram-positive bacteria.

Acclimation of photosynthetic microorganisms to changing ambient CO2 concentration

Photosynthetic microorganisms can acclimate to a wide range of CO2 concentration, from as low as 0.001% to ≈10% CO2 (vol/vol in the air in equilibrium with their environment). Some can even grow in the presence of 40% CO2 (1). Acclimation to a limiting CO2 level, well below the Km(CO2) of their carboxylating enzyme, Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), is achieved by substantial physiological and structural changes at various cell levels (2–4). The most prominent of these is the induction of a CO2 concentrating mechanism (CCM), which raises the [CO2] in close proximity to Rubisco. The latter is for the most part located in pyrenoids or carboxysomes in eukaryotes and prokaryotes photosynthetic microorganisms, respectively (5). This CCM involves light energy-dependent inorganic carbon uptake and accumulation of HCO within the cell. In cyanobacteria, the accumulated HCO penetrates the carboxysomes where carbonic anhydrase (CA) catalyzes the formation of CO2 in the close vicinity of Rubisco. The elevated CO2 concentration is thus confined to the carboxysomes (3). This model is most likely also applicable to eukaryotic algae where pyrenoids, densely packed with Rubisco and also containing CA, may have the same function as carboxysomes (2–7). The elevated [CO2] in these bodies compensates for the relatively low affinity of Rubisco for CO2 and consequently also decreases photorespiration.

Lethal protein produced in response to competition between sibling bacterial colonies

Sibling Paenibacillus dendritiformis bacterial colonies grown on low-nutrient agar medium mutually inhibit growth through secretion of a lethal factor. Analysis of secretions reveals the presence of subtilisin (a protease) and a 12 kDa protein, termed sibling lethal factor (Slf). Purified subtilisin promotes the growth and expansion of P. dendritiformis colonies, whereas Slf is lethal and lyses P. dendritiformis cells in culture. Slf is encoded by a gene belonging to a large family of bacterial genes of unknown function, and the gene is predicted to encode a protein of approximately 20 kDa, termed dendritiformis sibling bacteriocin. The 20 kDa recombinant protein was produced and found to be inactive, but exposure to subtilisin resulted in cleavage to the active, 12 kDa form. The experimental results, combined with mathematical modeling, show that subtilisin serves to regulate growth of the colony. Below a threshold concentration, subtilisin promotes colony growth and expansion. However, once it exceeds a threshold, as occurs at the interface between competing colonies, Slf is then secreted into the medium to rapidly reduce cell density by lysis of the bacterial cells. The presence of genes encoding homologs of dendritiformis sibling bacteriocin in other bacterial species suggests that this mechanism for self-regulation of colony growth might not be limited to P. dendritiformis.

Surface-motility induction, attraction and hitchhiking between bacterial species promote dispersal on solid surfaces

The ability to move on solid surfaces provides ecological advantages for bacteria, yet many bacterial species lack this trait. We found that Xanthomonas spp. overcome this limitation by making use of proficient motile bacteria in their vicinity. Using X. perforans and Paenibacillus vortex as models, we show that X. perforans induces surface motility, attracts proficient motile bacteria and ‘rides’ them for dispersal. In addition, X. perforans was able to restore surface motility of strains that lost this mode of motility under multiple growth cycles in the lab. The described interaction occurred both on agar plates and tomato leaves and was observed between several xanthomonads and motile bacterial species. Thus, suggesting that this motility induction and hitchhiking strategy might be widespread and ecologically important. This study provides an example as to how bacteria can rely on the abilities of their neighboring species for their own benefit, signifying the importance of a communal organization for fitness.

Identification and characterization of a highly motile and antibiotic refractory subpopulation involved in the expansion of swarming colonies of Paenibacillus vortex.

Summary Bacteria often use sophisticated cooperative behaviours, such as the development of complex colonies, elaborate biofilms and advanced dispersal strategies, to cope with the harsh and variable conditions of natural habitats, including the presence of antibiotics. Paenibacillus vortex uses swarming motility and cell-to-cell communication to form complex, structured colonies. The modular organization of P. vortex colony has been found to facilitate its dispersal on agar surfaces. The current study reveals that the complex structure of the colony is generated by the coexistence and transition between two morphotypes – ‘builders’ and ‘explorers’ – with distinct functions in colony formation. Here, we focused on the explorers, which are highly motile and spearhead colonial expansion. Explorers are characterized by high expression levels of flagellar genes, such as flagellin (hag), motA, fliI, flgK and sigD, hyperflagellation, decrease in ATP (adenosine-5′-triphosphate) levels, and increased resistance to antibiotics. Their tolerance to many antibiotics gives them the advantage of translocation through antibiotics-containing areas. This work gives new insights on the importance of cell differentiation and task distribution in colony morphogenesis and adaptation to antibiotics.