Photoactivated adenylyl cyclases attenuate sepsis-induced cardiomyopathy by suppressing macrophage-mediated inflammation.

Sepsis-induced myocardiopathy, characterized by innate immune cells infiltration and proinflammatory cytokines release, may lead to perfusion failure or even life-threatening cardiogenic shock. Macrophages-mediated inflammation has been shown to contribute to sepsis-induced myocardiopathy. In the current study, we introduced two photoactivated adenylyl cyclases (PACs), Beggiatoa sp. PAC (bPAC) and Beggiatoa sp. IS2 PAC (biPAC) into macrophages by transfection to detect the effects of light-induced regulation of macrophage pro-inflammatory response and LPS-induced sepsis-induced myocardiopathy. By this method, we uncovered that blue light-induced bPAC or biPAC activation considerably inhibited the production of pro-inflammatory cytokines IL-1 and TNF-α, both at mRNA and protein levels. Further, we assembled a GelMA-Macrophages-LED system, which consists of GelMA-a type of light crosslink hydrogel, gene modulated macrophages and wireless LED device, to allow light to regulate cardiac inflammation in situ with murine models of LPS-induced sepsis. Our results showed significant inhibition of leukocytes infiltration, especially macrophages and neutrophils, suppression of pro-inflammatory cytokines release, and alleviation of sepsis-induced cardiac dysfunction. Thus, our study may represent an emerging means to treat sepsis-induced myocardiopathy and other cardiovascular diseases by photo-activated regulating macrophage function.

Promoting bidirectional extracellular electron transfer of Shewanella oneidensis MR-1 for hexavalent chromium reduction via elevating intracellular cAMP level.

The bioreduction capacity of Cr(VI) by Shewanella is mainly governed by its bidirectional extracellular electron transfer (EET). However, the low bidirectional EET efficiency restricts its wider applications in remediation of the environments contaminated by Cr(VI). Cyclic adenosine 3',5'-monophosphate (cAMP) commonly exists in Shewanella strains and cAMP-cyclic adenosine 3',5'-monophosphate receptor protein (CRP) system regulates multiple bidirectional EET-related pathways. This inspires us to strengthen the bidirectional EET through elevating the intracellular cAMP level in Shewanella strains. In this study, an exogenous gene encoding adenylate cyclase from the soil bacterium Beggiatoa sp. PS is functionally expressed in Shewanella oneidensis MR-1 (the strain MR-1/pbPAC) and a MR-1 mutant lacking all endogenous adenylate cyclase encoding genes (the strain Δca/pbPAC). The engineered strains exhibit the enhanced bidirectional EET capacities in microbial electrochemical systems compared with their counterparts. Meanwhile, a three times more rapid reduction rate of Cr(VI) is achieved by the strain MR-1/pbPAC than the control in batch experiments. Furthermore, a higher Cr(VI) reduction efficiency is also achieved by the strain MR-1/pbPAC in the Cr(VI)-reducing biocathode experiments. Such a bidirectional enhancement is attributed to the improved production of cAMP-CRP complex, which upregulates the expression levels of the genes encoding the c-type cytochromes and flavins synthetic pathways. Specially, this strategy could be used as a broad-spectrum approach for the other Shewanella strains. Our results demonstrate that elevating the intracellular cAMP levels could be an efficient strategy to enhance the bidirectional EET of Shewanella strains and improve their pollutant transformation capacity.

Characterization and engineering of photoactivated adenylyl cyclases.

Cyclic nucleoside monophosphates (cNMP) serve as universal second messengers in signal transduction across prokaryotes and eukaryotes. As signaling often relies on transiently formed microdomains of elevated second messenger concentration, means to precisely perturb the spatiotemporal dynamics of cNMPs are uniquely poised for the interrogation of the underlying physiological processes. Optogenetics appears particularly suited as it affords light-dependent, accurate control in time and space of diverse cellular processes. Several sensory photoreceptors function as photoactivated adenylyl cyclases (PAC) and hence serve as light-regulated actuators for the control of intracellular levels of 3', 5'-cyclic adenosine monophosphate. To characterize PACs and to refine their properties, we devised a test bed for the facile analysis of these photoreceptors. Cyclase activity is monitored in bacterial cells via expression of a fluorescent reporter, and programmable illumination allows the rapid exploration of multiple lighting regimes. We thus probed two PACs responding to blue and red light, respectively, and observed significant dark activity for both. We next engineered derivatives of the red-light-sensitive PAC with altered responses to light, with one variant, denoted DdPAC, showing enhanced response to light. These PAC variants stand to enrich the optogenetic toolkit and thus facilitate the detailed analysis of cNMP metabolism and signaling.

SSU-rRNA Gene Sequencing Survey of Benthic Microbial Eukaryotes from Guaymas Basin Hydrothermal Vent.

Microbial eukaryotes have important roles in marine food webs, but their diversity and activities in hydrothermal vent ecosystems are poorly characterized. In this study, we analyzed microbial eukaryotic communities associated with bacterial (Beggiatoa) mats in the 2,000 m deep-sea Guaymas Basin hydrothermal vent system using 18S rRNA gene high-throughput sequencing of the V4 region. We detected 6,954 distinct Operational Taxonomic Units (OTUs) across various mat systems. Of the sequences that aligned with known protistan phylotypes, most were affiliated with alveolates (especially dinoflagellates and ciliates) and cercozoans. OTU richness and community structure differed among sediment habitats (e.g. different mat types and cold sediments away from mats). Additionally, full-length 18S rRNA genes amplified and cloned from single cells revealed the identities of some of the most commonly encountered, active ciliates in this hydrothermal vent ecosystem. Observations and experiments were also conducted to demonstrate that ciliates were trophically active and ingesting fluorescent bacteria or Beggiatoa trichomes. Our work suggests that the active and diverse protistan community at the Guaymas Basin hydrothermal vent ecosystem likely consumes substantial amounts of bacterial biomass, and that the different habitats, often defined by distances of just a few 10s of cm, select for particular assemblages and levels of diversity.

Simultaneous Visualization of Enzymatic Activity in the Cytoplasm and at Polyphosphate Inclusions in Beggiatoa sp. Strain 35Flor Incubated with 18O-Labeled Water.

Here we report on a new nanoscale secondary ion mass spectrometry (nanoSIMS) approach based on enzyme-mediated oxygen isotope exchange, which combines the visualization of general metabolic activity in the cytoplasm with insights into the activity of enzymes related to polyphosphate (polyP) inclusions. The polyP-accumulating strain of the large sulfur bacterium Beggiatoa was used as a model organism. Beggiatoa cultures were grown under oxic and anoxic conditions when exposed to either low- or high-sulfide conditions, which are known to influence polyP metabolism in this strain. Subsequent incubation with 18O-labeled water led to high 18O enrichments above the natural background in the cytoplasm and polyP granules derived from enzymatically mediated oxygen isotope exchange. The relative importance of polyP under the different sulfide regimes became evident by an apparent continued metabolic activity at polyP inclusions under stressfully high sulfide concentrations, in contrast to a decreased general metabolic activity in the cytoplasm. This finding confirms the role of polyP as a critical component in bacterial stress response and maintenance of a survival metabolism.IMPORTANCE Microbial organisms exert a large influence on the environment as they directly affect the turnover of essential elements. This is particularly true for polyphosphate-accumulating large sulfur bacteria, which can either accumulate phosphate as polyphosphate or degrade it and release phosphate into the environment, depending on environmental conditions. This study presents a new approach to simultaneously visualize general metabolic activity and enzymatic activity at polyphosphate granules by incubation with 18O-labeled water as the only stable isotope tracer. For this purpose, the well-studied Beggiatoa sp. strain 35Flor was used as a model organism and was exposed to different stress regimes. General metabolic activity was strongly impaired during high-stress regimes. In contrast, intense intracellular polyP cycling was not restricted to favorable or stressful conditions, highlighting the importance of polyP for general cell physiology, especially during hostile conditions. The nanoSIMS approach adds a new tool to study microorganisms involved in phosphorus cycling in the environment together with the identification of general metabolic activity.

Photoactivation Mechanism of a Bacterial Light-Regulated Adenylyl Cyclase.

Light-regulated enzymes enable organisms to quickly respond to changing light conditions. We characterize a photoactivatable adenylyl cyclase (AC) from Beggiatoa sp. (bPAC) that translates a blue light signal into the production of the second messenger cyclic AMP. bPAC contains a BLUF photoreceptor domain that senses blue light using a flavin chromophore, linked to an AC domain. We present a dark state crystal structure of bPAC that closely resembles the recently published structure of the homologous OaPAC from Oscillatoria acuminata. To elucidate the structural mechanism of light-dependent AC activation by the BLUF domain, we determined the crystal structures of illuminated bPAC and of a pseudo-lit state variant. We use hydrogen-deuterium exchange measurements of secondary structure dynamics and hypothesis-driven point mutations to trace the activation pathway from the chromophore in the BLUF domain to the active site of the cyclase. The structural changes are relayed from the residues interacting with the excited chromophore through a conserved kink of the BLUF ß-sheet to a tongue-like extrusion of the AC domain that regulates active site opening and repositions catalytic residues. Our findings not only show the specific molecular pathway of photoactivation in BLUF-regulated ACs but also have implications for the general understanding of signaling in BLUF domains and of the activation of ACs.

Beggiatoa leptomitoformis sp. nov., the first freshwater member of the genus capable of chemolithoautotrophic growth.

A strain of filamentous sulfur bacteria was isolated from freshwater spring contaminated with residential and agricultural wastewater in Moscow region, Russia. According to the results of phylogenetic analysis, strain D-402T belonged to the genus Beggiatoa within the family Beggiatoaceae of the class Gammaproteobacteria. Within the genus Beggiatoa, strain D-402T was most closely related to Beggiatoa alba strains. Strain D-402T had a DNA G+C content 42.1 mol%. The DNA-DNA hybridization value between strain D-402T and Beggiatoa alba strain B15LD was 33 %. Predominant fatty acids were C18 : 1 (46.1 and 53.3 %), C16 : 0 (15.5 and 16.2 %) and C16 : 1 (32.9 and 25.4 %) for strains D-402T and B15LD, respectively. In contrast to known representatives of Beggiatoa, strain D-402T was capable of chemolithoautotrophic growth with sulfide and thiosulfate as electron donors. Oxidation of sulfide and thiosulfate was accompanied by deposition of sulfur globules within the cells. Strain D-402T was capable of heterotrophic growth. The strain was capable of using different organic compounds, sulfur compounds and hydrogen as electron donors. Based on these observations, strain D-402T is considered as a representative of a species Beggiatoa leptomitoformis sp. nov. of the genus Beggiatoa. The type strain is D-402T (=DSM 14946T=UNIQEM U 779T).

Oxidation of Molecular Hydrogen by a Chemolithoautotrophic Beggiatoa Strain.

UNLABELLED: A chemolithoautotrophic strain of the family Beggiatoaceae, Beggiatoa sp. strain 35Flor, was found to oxidize molecular hydrogen when grown in a medium with diffusional gradients of oxygen, sulfide, and hydrogen. Microsensor profiles and rate measurements suggested that the strain oxidized hydrogen aerobically when oxygen was available, while hydrogen consumption under anoxic conditions was presumably driven by sulfur respiration.Beggiatoa sp. 35Flor reached significantly higher biomass in hydrogen-supplemented oxygen-sulfide gradient media, but hydrogen did not support growth of the strain in the absence of reduced sulfur compounds. Nevertheless, hydrogen oxidation can provide Beggiatoa sp. 35Flor with energy for maintenance and assimilatory purposes and may support the disposal of internally stored sulfur to prevent physical damage resulting from excessive sulfur accumulation. Our knowledge about the exposure of natural populations of Beggiatoa ceae to hydrogen is very limited, but significant amounts of hydrogen could be provided by nitrogen fixation, fermentation, and geochemical processes in several of their typical habitats such as photosynthetic microbial mats and submarine sites of hydrothermal fluid flow. IMPORTANCE: Reduced sulfur compounds are certainly the main electron donors for chemolithoautotrophic Beggiatoa ceae, but the traditional focus on this topic has left other possible inorganic electron donors largely unexplored. In this paper, we provide evidence that hydrogen oxidation has the potential to strengthen the ecophysiological plasticity of Beggiatoa ceaein several ways. Moreover, we show that hydrogen oxidation by members of this family can significantly influence biogeochemical gradients and therefore should be considered in environmental studies.

Structure and function of natural sulphide-oxidizing microbial mats under dynamic input of light and chemical energy.

We studied the interaction between phototrophic and chemolithoautotrophic sulphide-oxidizing microorganisms in natural microbial mats forming in sulphidic streams. The structure of these mats varied between two end-members: one characterized by a layer dominated by large sulphur-oxidizing bacteria (SOB; mostly Beggiatoa-like) on top of a cyanobacterial layer (B/C mats) and the other with an inverted structure (C/B mats). C/B mats formed where the availability of oxygen from the water column was limited (<5 µm). Aerobic chemolithotrophic activity of the SOB depended entirely on oxygen produced locally by cyanobacteria during high light conditions. In contrast, B/C mats formed at locations where oxygen in the water column was comparatively abundant (>45 µM) and continuously present. Here SOB were independent of the photosynthetic activity of cyanobacteria and outcompeted the cyanobacteria in the uppermost layer of the mat where energy sources for both functional groups were concentrated. Outcompetition of photosynthetic microbes in the presence of light was facilitated by the decoupling of aerobic chemolithotrophy and oxygenic phototrophy. Remarkably, the B/C mats conserved much less energy than the C/B mats, although similar amounts of light and chemical energy were available. Thus ecosystems do not necessarily develop towards optimal energy usage. Our data suggest that, when two independent sources of energy are available, the structure and activity of microbial communities is primarily determined by the continuous rather than the intermittent energy source, even if the time-integrated energy flux of the intermittent energy source is greater.

Barite encrustation of benthic sulfur-oxidizing bacteria at a marine cold seep.

Crusts and chimneys composed of authigenic barite are found at methane seeps and hydrothermal vents that expel fluids rich in barium. Microbial processes have not previously been associated with barite precipitation in marine cold seep settings. Here, we report on the precipitation of barite on filaments of sulfide-oxidizing bacteria at a brine seep in the Gulf of Mexico. Barite-mineralized bacterial filaments in the interiors of authigenic barite crusts resemble filamentous sulfide-oxidizing bacteria of the genus Beggiatoa. Clone library and iTag amplicon sequencing of the 16S rRNA gene show that the barite crusts that host these filaments also preserve DNA of Candidatus Maribeggiatoa, as well as sulfate-reducing bacteria. Isotopic analyses show that the sulfur and oxygen isotope compositions of barite have lower δ(34)S and δ(18)O values than many other marine barite crusts, which is consistent with barite precipitation in an environment in which sulfide oxidation was occurring. Laboratory experiments employing isolates of sulfide-oxidizing bacteria from Gulf of Mexico seep sediments showed that under low sulfate conditions, such as those encountered in brine fluids, sulfate generated by sulfide-oxidizing bacteria fosters rapid barite precipitation localized on cell biomass, leading to the encrustation of bacteria in a manner reminiscent of our observations of barite-mineralized Beggiatoa in the Gulf of Mexico. The precipitation of barite directly on filaments of sulfide-oxidizing bacteria, and not on other benthic substrates, suggests that sulfide oxidation plays a role in barite formation at certain marine brine seeps where sulfide is oxidized to sulfate in contact with barium-rich fluids, either prior to, or during, the mixing of those fluids with sulfate-containing seawater in the vicinity of the sediment/water interface. As with many other geochemical interfaces that foster mineral precipitation, both biological and abiological processes likely contribute to the precipitation of barite at marine brine seeps such as the one studied here.

Two new Beggiatoa species inhabiting marine mangrove sediments in the Caribbean.

Beggiatoaceae, giant sulphur-oxidizing bacteria, are well known to occur in cold and temperate waters, as well as hydrothermal vents, where they form dense mats on the floor. However, they have never been described in tropical marine mangroves. Here, we describe two new species of benthic Beggiatoaceae colonizing a marine mangrove adjacent to mangrove roots. We combined phylogenetic and lipid analysis with electron microscopy in order to describe these organisms. Furthermore, oxygen and sulphide measurements in and ex situ were performed in a mesocosm to characterize their environment. Based on this, two new species, Candidatus Maribeggiatoa sp. and Candidatus Isobeggiatoa sp. inhabiting tropical marine mangroves in Guadeloupe were identified. The species identified as Candidatus Maribeggiatoa group suggests that this genus could harbour a third cluster with organisms ranging from 60 to 120 µm in diameter. This is also the first description of an Isobeggiatoa species outside of Arctic and temperate waters. The multiphasic approach also gives information about the environment and indications for the metabolism of these bacteria. Our study shows the widespread occurrence of members of Beggiatoaceae family and provides new insight in their potential role in shallow-water marine sulphide-rich environments such as mangroves.

Upgrading a microplate reader for photobiology and all-optical experiments.

Automation can vastly reduce the cost of experimental labor and thus facilitate high experimental throughput, but little off-the-shelf hardware for the automation of illumination experiments is commercially available. Here, we use inexpensive open-source electronics to add programmable illumination capabilities to a multimode microplate reader. We deploy this setup to characterize light-triggered phenomena in three different sensory photoreceptors. First, we study the photoactivation of Arabidopsis thaliana phytochrome B by light of different wavelengths. Second, we investigate the dark-state recovery kinetics of the Synechocystis sp. blue-light sensor Slr1694 at multiple temperatures and imidazole concentrations; while the kinetics of the W91F mutant of Slr1694 are strongly accelerated by imidazole, the wild-type protein is hardly affected. Third, we determine the light response of the Beggiatoa sp. photoactivatable adenylate cyclase bPAC in Chinese hamster ovary cells. bPAC is activated by blue light in dose-dependent manner with a half-maximal intensity of 0.58 mW cm(-2); intracellular cAMP spikes generated upon bPAC activation decay with a half time of about 5 minutes after light switch-off. Taken together, we present a setup which is easily assembled and which thus offers a facile approach to conducting illumination experiments at high throughput, reproducibility and fidelity.

Absorption and emission spectroscopic characterization of photo-dynamics of photoactivated adenylyl cyclase mutant bPAC-Y7F of Beggiatoa sp.

The photoactivated cyclase bPAC of the microbial mats bacterium Beggiatoa sp. consists of a BLUF domain and an adenylyl cyclase domain. It has strong activity of photo-induced cyclic adenylyl monophosphate (cAMP) formation and is therefore an important optogenetic tool in neuroscience applications. The SUMO-bPAC-Y7F mutant where Tyr-7 is replaced by Phe-7 in the BLUF domain has lost the typical BLUF domain photo-cycle dynamics. Instead, the investigated SUMO-bPAC-Y7F mutant consisted of three protein conformations with different triplet based photo-dynamics: (i) reversible flavin quinone (Fl) cofactor reduction to flavin semiquinone (FlH), (ii) reversible violet/near ultraviolet absorbing flavin photoproduct (FlA) formation, and (iii) irreversible red absorbing flavin photoproduct (FlC) formation. Absorption and emission spectroscopic measurements on SUMO-bPAC-Y7F were carried out before, during and after light exposure. Flavin photo-dynamics schemes are developed for the SUMO-bPAC-Y7F fractions performing photo-induced FlH, FlA, and FlC formation. Quantitative parameters of the flavin cofactor excitation, relaxation and recovery dynamics in SUMO-bPAC-Y7F are determined.

Key residues for the light regulation of the blue light-activated adenylyl cyclase from Beggiatoa sp.

Photoactivated adenylyl cyclases are powerful tools for optogenetics and for investigating signal transduction mechanisms in biological photoreceptors. Because of its large increase in enzyme activity in the light, the BLUF (blue light sensor using flavin adenine dinucleotide)-activated adenylyl cyclase (bPAC) from Beggiatoa sp. is a highly attractive model system for studying BLUF domain signaling. In this report, we studied the influence of site-directed mutations within the BLUF domain on the light regulation of the cyclase domain and determined key elements for signal transduction and color tuning. Photoactivation of the cyclase domain is accomplished via strand ß5 of the BLUF domain and involves the formation of helical structures in the cyclase domain as assigned by vibrational spectroscopy. In agreement with earlier studies, we observed severely impaired signaling in mutations directly on strand ß5 as well as in mutations affecting the hydrogen bond network around the flavin. Moreover, we identified a bPAC mutant with red-shifted absorbance and a decreased dark activity that is highly valuable for long-term optogenetic experiments. Additionally, we discovered a mutant that forms a stable neutral flavin semiquinone radical in the BLUF domain and surprisingly exhibits an inversion of light activation.

Horizontal gene transfer and the rock record: comparative genomics of phylogenetically distant bacteria that induce wrinkle structure formation in modern sediments.

Wrinkle structures are sedimentary features that are produced primarily through the trapping and binding of siliciclastic sediments by mat-forming micro-organisms. Wrinkle structures and related sedimentary structures in the rock record are commonly interpreted to represent the stabilizing influence of cyanobacteria on sediments because cyanobacteria are known to produce similar textures and structures in modern tidal flat settings. However, other extant bacteria such as filamentous representatives of the family Beggiatoaceae can also interact with sediments to produce sedimentary features that morphologically resemble many of those associated with cyanobacteria-dominated mats. While Beggiatoa spp. and cyanobacteria are metabolically and phylogenetically distant, genomic analyses show that the two groups share hundreds of homologous genes, likely as the result of horizontal gene transfer. The comparative genomics results described here suggest that some horizontally transferred genes may code for phenotypic traits such as filament formation, chemotaxis, and the production of extracellular polymeric substances that potentially underlie the similar biostabilizing influences of these organisms on sediments. We suggest that the ecological utility of certain basic life modes such as the construction of mats and biofilms, coupled with the lateral mobility of genes in the microbial world, introduces an element of uncertainty into the inference of specific phylogenetic origins from gross morphological features preserved in the ancient rock record.

Polysulfides as intermediates in the oxidation of sulfide to sulfate by Beggiatoa spp.

Zero-valent sulfur is a key intermediate in the microbial oxidation of sulfide to sulfate. Many sulfide-oxidizing bacteria produce and store large amounts of sulfur intra- or extracellularly. It is still not understood how the stored sulfur is metabolized, as the most stable form of S(0) under standard biological conditions, orthorhombic α-sulfur, is most likely inaccessible to bacterial enzymes. Here we analyzed the speciation of sulfur in single cells of living sulfide-oxidizing bacteria via Raman spectroscopy. Our results showed that under various ecological and physiological conditions, all three investigated Beggiatoa strains stored sulfur as a combination of cyclooctasulfur (S8) and inorganic polysulfides (Sn(2-)). Linear sulfur chains were detected during both the oxidation and reduction of stored sulfur, suggesting that Sn(2-) species represent a universal pool of bioavailable sulfur. Formation of polysulfides due to the cleavage of sulfur rings could occur biologically by thiol-containing enzymes or chemically by the strong nucleophile HS(-) as Beggiatoa migrates vertically between oxic and sulfidic zones in the environment. Most Beggiatoa spp. thus far studied can oxidize sulfur further to sulfate. Our results suggest that the ratio of produced sulfur and sulfate varies depending on the sulfide flux. Almost all of the sulfide was oxidized directly to sulfate under low-sulfide-flux conditions, whereas only 50% was oxidized to sulfate under high-sulfide-flux conditions leading to S(0) deposition. With Raman spectroscopy we could show that sulfate accumulated in Beggiatoa filaments, reaching intracellular concentrations of 0.72 to 1.73 M.

Close association of active nitrifiers with Beggiatoa mats covering deep-sea hydrothermal sediments.

Hydrothermal sediments in the Guaymas Basin are covered by microbial mats that are dominated by nitrate-respiring and sulphide-oxidizing Beggiatoa. The presence of these mats strongly correlates with sulphide- and ammonium-rich fluids venting from the subsurface. Because ammonium and oxygen form opposed gradients at the sediment surface, we hypothesized that nitrification is an active process in these Beggiatoa mats. Using biogeochemical and molecular methods, we measured nitrification and determined the diversity and abundance of nitrifiers. Nitrification rates ranged from 74 to 605 µmol N l(-1) mat day(-1), which exceeded those previously measured in hydrothermal plumes and other deep-sea habitats. Diversity and abundance analyses of archaeal and bacterial ammonia monooxygenase subunit A genes, archaeal 16S ribosomal RNA pyrotags and fluorescence in situ hybridization confirmed that ammonia- and nitrite-oxidizing microorganisms were associated with Beggiatoa mats. Intriguingly, we observed cells of bacterial and potential thaumarchaeotal ammonia oxidizers attached to narrow, Beggiatoa-like filaments. Such a close spatial coupling of nitrification and nitrate respiration in mats of large sulphur bacteria is novel and may facilitate mat-internal cycling of nitrogen, thereby reducing loss of bioavailable nitrogen in deep-sea sediments.

Sulfide oxidation, nitrate respiration, carbon acquisition, and electron transport pathways suggested by the draft genome of a single orange Guaymas Basin Beggiatoa (Cand. Maribeggiatoa) sp. filament.

A near-complete draft genome has been obtained for a single vacuolated orange Beggiatoa (Cand. Maribeggiatoa) filament from a Guaymas Basin seafloor microbial mat, the third relatively complete sequence for the Beggiatoaceae. Possible pathways for sulfide oxidation; nitrate respiration; inorganic carbon fixation by both Type II RuBisCO and the reductive tricarboxylic acid cycle; acetate and possibly formate uptake; and energy-generating electron transport via both oxidative phosphorylation and the Rnf complex are discussed here. A role in nitrite reduction is suggested for an abundant orange cytochrome produced by the Guaymas strain; this has a possible homolog in Beggiatoa (Cand. Isobeggiatoa) sp. PS, isolated from marine harbor sediment, but not Beggiatoa alba B18LD, isolated from a freshwater rice field ditch. Inferred phylogenies for the Calvin-Benson-Bassham (CBB) cycle and the reductive (rTCA) and oxidative (TCA) tricarboxylic acid cycles suggest that genes encoding succinate dehydrogenase and enzymes for carboxylation and/or decarboxylation steps (including RuBisCO) may have been introduced to (or exported from) one or more of the three genomes by horizontal transfer, sometimes by different routes. Sequences from the two marine strains are generally more similar to each other than to sequences from the freshwater strain, except in the case of RuBisCO: only the Guaymas strain encodes a Type II enzyme, which (where studied) discriminates less against oxygen than do Type I RuBisCOs. Genes subject to horizontal transfer may represent key steps for adaptation to factors such as oxygen and carbon dioxide concentration, organic carbon availability, and environmental variability.

Mobile elements in a single-filament orange Guaymas Basin Beggiatoa ("Candidatus Maribeggiatoa") sp. draft genome: evidence for genetic exchange with cyanobacteria.

The draft genome sequence of a single orange Beggiatoa ("Candidatus Maribeggiatoa") filament collected from a microbial mat at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) shows evidence of extensive genetic exchange with cyanobacteria, in particular for sensory and signal transduction genes. A putative homing endonuclease gene and group I intron within the 23S rRNA gene; several group II catalytic introns; GyrB and DnaE inteins, also encoding homing endonucleases; multiple copies of sequences similar to the fdxN excision elements XisH and XisI (required for heterocyst differentiation in some cyanobacteria); and multiple sequences related to an open reading frame (ORF) (00024_0693) of unknown function all have close non-Beggiatoaceae matches with cyanobacterial sequences. Sequences similar to the uncharacterized ORF and Xis elements are found in other Beggiatoaceae genomes, a variety of cyanobacteria, and a few phylogenetically dispersed pleiomorphic or filamentous bacteria. We speculate that elements shared among filamentous bacterial species may have been exchanged in microbial mats and that some of them may be involved in cell differentiation.

Why orange Guaymas Basin Beggiatoa spp. are orange: single-filament-genome-enabled identification of an abundant octaheme cytochrome with hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities.

Orange, white, and yellow vacuolated Beggiatoaceae filaments are visually dominant members of microbial mats found near sea floor hydrothermal vents and cold seeps, with orange filaments typically concentrated toward the mat centers. No marine vacuolate Beggiatoaceae are yet in pure culture, but evidence to date suggests they are nitrate-reducing, sulfide-oxidizing bacteria. The nearly complete genome sequence of a single orange Beggiatoa ("Candidatus Maribeggiatoa") filament from a microbial mat sample collected in 2008 at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) was recently obtained. From this sequence, the gene encoding an abundant soluble orange-pigmented protein in Guaymas Basin mat samples (collected in 2009) was identified by microcapillary reverse-phase high-performance liquid chromatography (HPLC) nano-electrospray tandem mass spectrometry (µLC-MS-MS) of a pigmented band excised from a denaturing polyacrylamide gel. The predicted protein sequence is related to a large group of octaheme cytochromes whose few characterized representatives are hydroxylamine or hydrazine oxidases. The protein was partially purified and shown by in vitro assays to have hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities. From what is known of Beggiatoaceae physiology, nitrite reduction is the most likely in vivo role of the octaheme protein, but future experiments are required to confirm this tentative conclusion. Thus, while present-day genomic and proteomic techniques have allowed precise identification of an abundant mat protein, and its potential activities could be assayed, proof of its physiological role remains elusive in the absence of a pure culture that can be genetically manipulated.