Symbiotic Growth of a Thermophilic Sulfide-Oxidizing Photoautotroph and an Elemental Sulfur-Disproportionating Chemolithoautotroph and Cooperative Dissimilatory Oxidation of Sulfide to Sulfate.

A thermophilic filamentous anoxygenic photosynthetic bacterium, Chloroflexus aggregans, is widely distributed in neutral to slightly alkaline hot springs. Sulfide has been suggested as an electron donor for autotrophic growth in microbial mats dominated with C. aggregans, but remarkable photoautotrophic growth of isolated C. aggregans has not been observed with sulfide as the sole electron source. From the idea that sulfide is oxidized to elemental sulfur by C. aggregans and the accumulation of elemental sulfur may have an inhibitory effect for the growth, the effects of an elemental sulfur-disproportionating bacterium that consumes elemental sulfur was examined on the autotrophic growth of C. aggregans, strain NA9-6, isolated from Nakabusa hot spring. A sulfur-disproportionating bacterium, Caldimicrobium thiodismutans strain TF1, also isolated from Nakabusa hot spring was co-cultured with C. aggregans. C. aggregans and C. thiodismutans were successfully co-cultured in a medium containing thiosulfate as the sole electron source and bicarbonate as the sole carbon source. Quantitative conversion of thiosulfate to sulfate and a small transient accumulation of sulfide was observed in the co-culture. Then the electron source of the established co-culture was changed from thiosulfate to sulfide, and the growth of C. aggregans and C. thiodismutans was successfully observed with sulfide as the sole electron donor for the autotrophic growth of the co-culture. During the cultivation in the light, simultaneous consumption and accumulation of sulfide and sulfate, respectively, were observed, accompanied with the increase of cellular DNAs of both species. C. thiodismutans likely works as an elemental sulfur scavenger for C. aggregans, and C. aggregans seems to work as a sulfide scavenger for C. thiodismutans. These results suggest that C. aggregans grows autotrophically with sulfide as the electron donor in the co-culture with C. thiodismutans, and the consumption of elemental sulfur by C. thiodismutans enabled the continuous growth of the C. aggregans in the symbiotic system. This study shows a novel symbiotic relationship between a sulfide-oxidizing photoautotroph and an elemental sulfur-disproportionating chemolithoautotroph via cooperative dissimilatory sulfide oxidation to sulfate.

Hydrogen-dependent autotrophic growth in phototrophic and chemolithotrophic cultures of thermophilic bacteria, Chloroflexus aggregans and Chloroflexus aurantiacus, isolated from Nakabusa hot springs.

The genus Chloroflexus is a deeply branching group of thermophilic filamentous anoxygenic phototrophic bacteria. The bacteria in this genus have been shown to grow well heterotrophically under anaerobic photosynthetic and aerobic respiratory conditions. We examined autotrophic growth in new isolates of Chloroflexus strains from hot springs in Nakabusa, Japan. The isolates belonging to Chloroflexus aggregans (98.7% identity of 16S rRNA gene sequence to the respective type strain) and Chloroflexus aurantiacus (99.9% identity to the respective type strain) grew photoautotrophically under a 24% H2 atmosphere. We also observed chemolithotrophic growth of these isolates under 80% H2 and 5% O2 conditions in the dark. This is the first report showing that Chloroflexus grew under both photoautotrophic and chemolithotrophic conditions in addition to photoheterotrophic and aerobic chemoheterotrophic conditions.

Phylogenetic Diversity of Nitrogenase Reductase Genes and Possible Nitrogen-Fixing Bacteria in Thermophilic Chemosynthetic Microbial Communities in Nakabusa Hot Springs.

Chemosynthetic microbial communities develop and form dense cell aggregates in slightly alkaline sulfidic hot springs in the temperature range of 70-86°C at Nakabusa, Japan. Nitrogenase activity has recently been detected in the microbial communities collected. To identify possible members capable of nitrogen fixation, we examined the diversities of 16S rRNA and nitrogenase reductase (NifH) gene sequences in four types of chemosynthetic communities with visually different colors and thicknesses. The results of a 16S rRNA gene analysis indicated that all four microbial communities had similar bacterial constituents; the phylum Aquificae was the dominant member, followed in abundance by Thermodesulfobacteria, Firmicutes, and Thermotogae. Most of the NifH sequences were related to sequences reported in hydrothermal vents and terrestrial hot springs. The results of a phylogenetic analysis of NifH sequences revealed diversity in this gene among the communities collected, distributed within 7 phylogenetic groups. NifH sequences affiliated with Aquificae (Hydrogenobacter/Thermocrinis) and Firmicutes (Caldicellulosiruptor) were abundant. At least two different energy metabolic pathways appeared to be related to nitrogen fixation in the communities analyzed; aerobic sulfur/hydrogen-oxidizing bacteria in Aquificae and fermentative bacteria in Firmicutes. The metabolic characteristics of these two dominant phyla differed from those previously inferred from nitrogenase activity assays on chemosynthetic communities, which were associated with hydrogen-dependent autotrophic sulfate reduction. These assays may correspond to the observed NifH sequences that are distantly related to the known species of Thermodesulfovibrio sp. (Nitrospirae) detected in the present study. The activities of nitrogen-fixing organisms in communities may depend on redox states as well as the availability of electron donors, acceptors, and carbon sources.

Nitrogenase Activity in Thermophilic Chemolithoautotrophic Bacteria in the Phylum Aquificae Isolated under Nitrogen-Fixing Conditions from Nakabusa Hot Springs.

The phylum Aquificae comprises chemolithoautotrophic thermophilic to hyperthermophilic bacteria, in which the nitrogenase reductase gene (nifH) has been reported. However, nitrogen-fixing activity has not yet been demonstrated in members of this deeply branching bacterial phylum. We isolated two thermophilic diazotrophic strains from chemosynthetic microbial communities in slightly alkaline hot springs (≥70°C) in Nakabusa, Nagano Prefecture, Japan. A phylogenetic analysis based on 16S rRNA genes identified these strains as members of the genus Hydrogenobacter within Aquificae. Their NifH sequences showed 96.5 and 97.4% amino acid sequence identities to that from Hydrogenobacter thermophilus TK-6. Nitrogenase activity, measured by acetylene reduction, was confirmed in both strains at 70°C. These novel strains grew under semi-aerobic conditions by using CO2 as the sole carbon source and N2 as the sole nitrogen source in media containing hydrogen and/or thiosulfate. To the best of our knowledge, this is the first demonstration of active nitrogen fixation in thermophilic bacteria at 70°C and in the phylum Aquificae.

Different Metabolomic Responses to Carbon Starvation between Light and Dark Conditions in the Purple Photosynthetic Bacterium, Rhodopseudomonas palustris.

Purple photosynthetic bacteria utilize light energy for growth. We previously demonstrated that light energy contributed to prolonging the survival of multiple purple bacteria under carbon-starved conditions. In order to clarify the effects of illumination on metabolic states under carbon-starved, non-growing conditions, we herein compared the metabolic profiles of starved cells in the light and dark using the purple bacterium, Rhodopseudomonas palustris. The metabolic profiles of starved cells in the light were markedly different from those in the dark. After starvation for 5 d in the light, cells showed increases in the amount of ATP and the NAD+/NADH ratio. Decreases in the amounts of most metabolites related to glycolysis and the TCA cycle in energy-rich starved cells suggest the active utilization of these metabolites for the modification of cellular components. Starvation in the dark induced the consumption of cellular compounds such as amino acids, indicating that the degradation of these cellular components produced ATP in order to maintain viability under energy-poor conditions. The present results suggest that intracellular energy levels alter survival strategies under carbon-starved conditions through metabolism.

Increase of Salt Tolerance in Carbon-Starved Cells of Rhodopseudomonas palustris Depending on Photosynthesis or Respiration.

Bacteria in natural environments are frequently exposed to nutrient starvation and survive against environmental stresses under non-growing conditions. In order to determine the energetic influence on survivability during starvation, changes in salt tolerance were investigated using the purple photosynthetic bacterium Rhodopseudomonas palustris after carbon starvation under photosynthetic conditions in comparison with anaerobic and aerobic dark conditions. Tolerance to a treatment with high concentration of salt (2.5 M NaCl for 1 h) was largely increased after starvation under anaerobically light and aerobically dark conditions. The starved cells under the conditions of photosynthesis or aerobic respiration contained high levels of cellular ATP, but starvation under the anaerobic dark conditions resulted in a decrease of cellular ATP contents. To observe the large increase of the salt tolerance, incubation of starved cells for more than 18 h under illumination was needed. These results suggest that the ATP-dependent rearrangement of cells induced salt tolerance.

Nitrogen Fixation in Thermophilic Chemosynthetic Microbial Communities Depending on Hydrogen, Sulfate, and Carbon Dioxide.

The activity of nitrogen fixation measured by acetylene reduction was examined in chemosynthetic microbial mats at 72-75°C in slightly-alkaline sulfidic hot springs in Nakabusa, Japan. Nitrogenase activity markedly varied from sampling to sampling. Nitrogenase activity did not correlate with methane production, but was detected in samples showing methane production levels less than the maximum amount, indicating a possible redox dependency of nitrogenase activity. Nitrogenase activity was not affected by 2-bromo-ethane sulfonate, an inhibitor of methanogenesis. However, it was inhibited by the addition of molybdate, an inhibitor of sulfate reduction and sulfur disproportionation, suggesting the involvement of sulfate-reducing or sulfur-disproportionating organisms. Nitrogenase activity was affected by different O2 concentrations in the gas phase, again supporting the hypothesis of a redox potential dependency, and was decreased by the dispersion of mats with a homogenizer. The loss of activity that occurred from dispersion was partially recovered by the addition of H2, sulfate, and carbon dioxide. These results suggested that the observed activity of nitrogen fixation was related to chemoautotrophic sulfate reducers, and fixation may be active in a limited range of ambient redox potential. Since thermophilic chemosynthetic communities may resemble ancient microbial communities before the appearance of photosynthesis, the present results may be useful when considering the ancient nitrogen cycle on earth.

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.

Differences in Survivability under Starvation Conditions Among Four Species of Purple Nonsulfur Phototrophic Bacteria.

Volume 29, no. 3, Page 326-328, 2014Page 327, Legend for Fig. 1 IncorrectFig. 1. Changes in viability (black line) and ATP levels (gray line) during carbon-starvation conditions. CorrectFig. 1. Changes in viability (gray line) and ATP levels (black line) during carbon-starvation conditions.

Phylogenetically Diverse Aerobic Anoxygenic Phototrophic Bacteria Isolated from Epilithic Biofilms in Tama River, Japan.

The diversity of aerobic anoxygenic phototrophic (AAP) bacteria in freshwater environments, particularly in rivers, has not been examined in as much detail as in ocean environments. In the present study, we investigated the phylogenetic and physiological diversities of AAP bacteria in biofilms that developed on submerged stones in a freshwater river using culture methods. The biofilms collected were homogenized and inoculated on solid media and incubated aerobically in the dark. Sixty-eight red-, pink-, yellow-, orange-, or brown-colored colonies were isolated, and, of these, 28 isolates contained the photosynthetic pigment, bacteriochlorophyll (BChl) a. Phylogenetic analyses based on 16S rRNA gene sequences showed that the isolates were classified into 14 groups in 8 operational taxonomic units (OTUs) and distributed in the orders Rhodospirillales, Rhodobacterales, and Sphingomonadales of Alphaproteobacteria and in Betaproteobacteria. Physiological analyses confirmed that none of the representative isolates from any of the groups grew under anaerobic phototrophic conditions. Seven isolates in 4 OTUs showed a 16S rRNA gene sequence identity of 98.0% or less with any established species, suggesting the presence of previously undescribed species of AAP bacteria. Six isolates in 2 other OTUs had the closest relatives, which have not been reported to be AAP bacteria. Physiological comparisons among the isolates revealed differences in preferences for nutrient concentrations, BChl contents, and light-harvesting proteins. These results suggest that diverse and previously unknown AAP bacteria inhabit river biofilms.

Gliding motility driven by individual cell-surface movements in a multicellular filamentous bacterium Chloroflexus aggregans.

Chloroflexus aggregans is an unbranched multicellular filamentous bacterium having the ability of gliding motility. The filament moves straightforward at a constant rate, ∼3 µm sec(-1) on solid surface and occasionally reverses the moving direction. In this study, we successfully detected movements of glass beads on the cell-surface along long axis of the filament indicating that the cell-surface movement was the direct force for gliding. Microscopic analyses found that the cell-surface movements were confined to a cell of the filament, and each cell independently moved and reversed the direction. To understand how the cellular movements determine the moving direction of the filament, we proposed a discrete-time stochastic model; sum of the directions of the cellular movements determines the moving direction of the filament only when the filament pauses, and after moving, the filament keeps the same directional movement until all the cells pause and/or move in the opposite direction. Monte Carlo simulation of this model showed that reversal frequency of longer filaments was relatively fixed to be low, but the frequency of shorter filaments varied widely. This simulation result appropriately explained the experimental observations. This study proposed the relevant mechanism adequately describing the motility of the multicellular filament in C. aggregans.

Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus.

PpsR is a major regulator of photosynthesis gene expression among all characterized purple photosynthetic bacteria. This transcription regulator has been extensively characterized in Rhodobacter (Rba.) capsulatus and Rba. sphaeroides which are members of the α-proteobacteria lineage. In this study, we have investigated the biochemical properties and mutational effects of a ppsR deletion strain in the ß-proteobacterium Rubrivivax (Rvi.) gelatinosus in order to reveal phylogenetically conserved mechanisms and species-specific characteristics. A deletion of the ppsR gene resulted in de-repression of photosystem synthesis showing that PpsR functions as a repressor of photosynthesis genes in this species. We also constructed a Rvi. gelatinosus PpsR mutant in which a conserved cysteine at position 436 was changed to an alanine to examine whether or not this residue is important for sensing redox, as reported in Rhodobacter species. Surprisingly, the Cys436 Ala mutant retained the ability to repress photosynthesis gene expression under aerobic conditions, suggesting that PpsR from Rvi. gelatinosus has different redox-responding characteristics. Furthermore, biochemical analyses demonstrated that Rvi. gelatinosus PpsR only shows redox-dependent binding to promoters with 9-bp spacing, but not 8-bp spacing, between two PpsR-recognition sequences. These results indicate that redox-dependent binding of PpsR requires appropriate cis configuration of PpsR target sequences in Rvi. gelatinosus. These results also indicate that PpsR homologs from different species regulate photosynthesis genes with altered biochemical properties.

Secreted protease mediates interspecies interaction and promotes cell aggregation of the photosynthetic bacterium Chloroflexus aggregans.

Interspecies interactions were studied in hot spring microbial mats where diverse species of bacterial cells are densely packed. The anoxygenic photosynthetic bacterium, Chloroflexus aggregans, has been widely found in the microbial mats as a major component in terrestrial hot springs in Japan at the temperature from 50 to 70°C. C. aggregans shows cellular motility to form a microbial mat-like dense cell aggregate. The aggregating ability of C. aggregans was affected by another bacterial species, strain BL55a (related to Bacillus licheniformis) isolated from the microbial mats containing C. aggregans. Cell aggregation rate of C. aggregans was promoted by the addition of culture supernatants of strain BL55a. Similar effects were also detected from other bacterial isolates, specifically Geobacillus sp. and Aeribacillus sp. Protease activity was detected from the culture supernatants from all of these isolates. The promoting effect of strain BL55a was suppressed by a serine protease inhibitor, phenylmethylsulfonyl fluoride. A purified serine protease, subtilisin obtained from B. licheniformis, showed a promoting effect on the cell aggregation. These results suggest that an extracellular protease, secreted from co-existing bacterial species promoted the aggregating motility of C. aggregans. This is the first report that exogenous protease affects bacterial cellular motility.

Differences in survivability under starvation conditions among four species of purple nonsulfur phototrophic bacteria.

Survivability under carbon-starvation conditions was investigated in four species of purple phototrophic bacteria: Rhodopseudomonas palustris, Rhodobacter sphaeroides, Rhodospirillum rubrum, and Rubrivivax gelatinosus. All these test organisms survived longer in the light than in the dark. ATP levels in the cultures were maintained in the light, which indicated that survivability was supported by photosynthesis. Survivability and tolerance against hypertonic stress in the dark was higher in Rhodopseudomonas palustris, which is widely distributed in natural environments including soils, than in the three other species.

Diversification of bacterial community composition along a temperature gradient at a thermal spring.

To better understand the biogeography and relationship between temperature and community structure within microbial mats, the bacterial diversity of mats at a slightly alkaline, sulfide-containing hot spring was explored. Microbial mats that developed at temperatures between 75-52°C were collected from an area of approximately 1 m(2) in Nakabusa, Nagano, Japan. Bacterial 16S rRNA genes from these samples were examined by terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis. T-RFLP profiles revealed 66 unique fragments (T-RFs). Based on total T-RFs observed in environmental profiles and clone libraries, a temperature effect on diversity was determined, with complexity in the community increasing as temperature decreased. The T-RF pattern indicated four distinct community assemblages related to temperature. Members of the Aquificales and particularly the sulfur-oxidizing bacterium Sulfurihydrogenibium were present at all temperatures and were the dominant component of mats taken at 75-67°C. Sulfide oxidation, which persisted throughout the temperature gradient, was the presumed dominant pathway of primary production above 67°C. As temperature decreased, successive additions of anoxygenic and oxygenic phototrophs increased primary productivity, allowing for diversification of the community.

Diversity of purple phototrophic bacteria, inferred from pufM gene, within epilithic biofilm in Tama River, Japan.

The diversity of purple phototrophic bacteria in algae-dominated biofilm of a streambed in Tama River, Japan was investigated. Clone library analysis of the pufM gene encoding a subunit of the photochemical reaction center of purple bacteria detected 18 operational taxonomic units (OTUs) in several classes of Proteobacteria. Most of the OTUs showed less than 85% identity to the PufM amino acid sequences of known phototrophic bacteria. These results suggest that phylogenetically divergent and unknown purple phototrophic bacteria are present in the epilithic biofilm of the river.

Production and consumption of hydrogen in hot spring microbial mats dominated by a filamentous anoxygenic photosynthetic bacterium.

Microbial mats containing the filamentous anoxygenic photosynthetic bacterium Chloroflexus aggregans develop at Nakabusa hot spring in Japan. Under anaerobic conditions in these mats, interspecies interaction between sulfate-reducing bacteria as sulfide producers and C. aggregans as a sulfide consumer has been proposed to constitute a sulfur cycle; however, the electron donor utilized for microbial sulfide production at Nakabusa remains to be identified. In order to determine this electron donor and its source, ex situ experimental incubation of mats was explored. In the presence of molybdate, which inhibits biological sulfate reduction, hydrogen gas was released from mat samples, indicating that this hydrogen is normally consumed as an electron donor by sulfate-reducing bacteria. Hydrogen production decreased under illumination, indicating that C. aggregans also functions as a hydrogen consumer. Small amounts of hydrogen may have also been consumed for sulfur reduction. Clone library analysis of 16S rRNA genes amplified from the mats indicated the existence of several species of hydrogen-producing fermentative bacteria. Among them, the most dominant fermenter, Fervidobacterium sp., was successfully isolated. This isolate produced hydrogen through the fermentation of organic carbon. Dispersion of microbial cells in the mats resulted in hydrogen production without the addition of molybdate, suggesting that simultaneous production and consumption of hydrogen in the mats requires dense packing of cells. We propose a cyclic electron flow within the microbial mats, i.e., electron flow occurs through three elements: S (elemental sulfur, sulfide, sulfate), C (carbon dioxide, organic carbon) and H (di-hydrogen, protons).

A novel and mild isolation procedure of chlorosomes from the green sulfur bacterium Chlorobaculum tepidum.

In this article, we developed a new and mild procedure for the isolation of chlorosomes from a green sulfur bacterium Chlorobaculum tepidum. In this procedure, Fenna-Matthews-Olson (FMO) protein was released by long cold treatment (6°C) of cells under the presence of a chaotrope (2 M NaSCN) and 0.6 M sucrose. Chlorosomes were released by an osmotic shock of the cold-treated cells after the formation of spheroplasts without mechanical disruption. Chlorosomes were finally purified by a sucrose step-wise density gradient centrifugation. We obtained two samples with different density (20 and 23% sucrose band, respectively) and compared them by SDS-PAGE, absorption spectroscopy at 80 K, fluorescence and CD spectroscopy at room temperature. Cells whose absorption maximum was longer than 750 nm yielded higher amount of the 20% sucrose fraction than those having an absorption maximum shorter than 750 nm.

Sulfur-metabolizing bacterial populations in microbial mats of the Nakabusa hot spring, Japan.

At the Nakabusa hot spring, Japan, dense olive-green microbial mats develop in regions where the slightly alkaline, sulfidic effluent has cooled to 65°C. The microbial community of such mats was analyzed by focusing on the diversity, as well as the in situ distribution and function of bacteria involved in sulfur cycling. Analyses of 16S rRNA and functional genes (aprA, pufM) suggested the importance of three thermophilic bacterial groups: aerobic chemolithotrophic sulfide-oxidizing species of the genus Sulfurihydrogenibium (Aquificae), anaerobic sulfate-reducing species of the genera Thermodesulfobacterium/Thermodesulfatator, and filamentous anoxygenic photosynthetic species of the genus Chloroflexus. A new oligonucleotide probe specific for Sulfurihydrogenibium was designed and optimized for catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). In situ hybridizations of thin mat sections showed a heterogeneous vertical distribution of Sulfurihydrogenibium and Chloroflexus. Sulfurihydrogenibium dominated near the mat surface (50% of the total mat biovolume), while Chloroflexus dominated in deeper layers (up to 64% of the total mat biovolume). Physiological experiments monitoring in vitro changes of sulfide concentration indicated slight sulfide production by sulfate-reducing bacteria under anoxic-dark conditions, sulfide consumption by photosynthetic bacteria under anoxic-light conditions and strong sulfide oxidation by chemolithotrophic members of Aquificae under oxic-dark condition. We therefore propose that Sulfurihydrogenibium spp. act as highly efficient scavengers of oxygen from the spring water, thus creating a favorable, anoxic environment for Chloroflexus and Thermodesulfobacterium/Thermodesulfatator in deeper layers.

Cytochrome c4 can be involved in the photosynthetic electron transfer system in the purple bacterium Rubrivivax gelatinosus.

Three periplasmic electron carriers, HiPIP and two cytochromes c8 with low- and high-midpoint potentials, are present in the purple photosynthetic bacterium Rubrivivax gelatinosus. Comparison of the growth rates of mutants lacking one, two, or all three electron carrier proteins showed that HiPIP is the main electron donor to the photochemical reaction center and that high-potential cytochrome c8 plays a subsidiary role in the electron donation in photosynthetically growing cells. However, the triple deletion mutant was still capable of photosynthetic growth, indicating that another electron donor could be present. A new soluble cytochrome c, which can reduce the photooxidized reaction center in vitro, was purified. Based on amino acid sequence comparisons to known cytochromes, this cytochrome was identified as a diheme cytochrome c of the family of cytochromes c4. The quadruple mutant lacking this cytochrome and three other electron carriers showed about three times slower growth than the triple mutant under photosynthetic growth conditions. In conclusion, cytochrome c4 can function as a physiological electron carrier in the photosynthetic electron transport chain in R. gelatinosus.