Sulfide-dependent Photoautotrophy in the Filamentous Anoxygenic Phototrophic Bacterium, Chloroflexus aggregans.

Chloroflexus aggregans is a thermophilic filamentous anoxygenic phototrophic bacterium frequently found in microbial mats in natural hot springs. C. aggregans often thrives with cyanobacteria that engage in photosynthesis to provide it with an organic substrate; however, it sometimes appears as the dominant phototroph in microbial mats without cyanobacteria. This suggests that C. aggregans has the ability to grow photoautotrophically. However, photoautotrophic growth has not been observed in any cultured strains of C. aggregans. We herein attempted to isolate a photoautotrophic strain from C. aggregansdominated microbial mats in Nakabusa hot spring in Japan. Using an inorganic medium, we succeeded in isolating a new strain that we designated "ACA-12". A phylogenetic analysis based on 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer (ITS) region sequences revealed that strain ACA-12 was closely related to known C. aggregans strains. Strain ACA-12 showed sulfide consumption along with autotrophic growth under anaerobic light conditions. The deposited elemental sulfur particles observed by microscopy indicated that sulfide oxidation occurred, similar to that in photoautotrophic strains in the related species, C. aurantiacus. Moreover, we found that other strains of C. aggregans, including the type strain, also exhibited a slight photoautotrophic growing ability, whereas strain ACA-12 showed the fastest growth rate. This is the first demonstration of photoautotrophic growth with sulfide in C. aggregans. The present results strongly indicate that C. aggregans is associated with inorganic carbon incorporation using sulfide as an electron donor in hot spring microbial mats.

Chloroflexus islandicus sp. nov., a thermophilic filamentous anoxygenic phototrophic bacterium from a geyser.

A novel, thermophilic filamentous anoxygenic phototrophic bacterium, strain isl-2T, was isolated from the Strokkur Geyser, Iceland. Strain isl-2T formed unbranched multicellular filaments with gliding motility. The cells formed no spores and stained Gram-negative. The existence of pili was described in a species of the genus Chloroflexus for the first time, to our knowledge. Optimal growth occurred at a pH range of 7.5-7.7 and at a temperature of 55 °C. Strain isl-2T grew photoheterotrophically under anaerobic conditions in the light and chemoheterotrophically under aerobic conditions in the dark. The major cellular fatty acids were C18 : 1ω9, C16 : 0, C18 : 0 and C18 : 0-OH. The major quinone was menaquinone-10. The photosynthetic pigments were bacteriochlorophylls c and a as well as ß- and γ-carotenes. The results of phylogenetic analysis of the 16S rRNA gene sequences placed strain isl-2T into the genus Chloroflexus of the phylum Chloroflexi with Chloroflexus aggregans DSM 9485T as the closest relative (97.0 % identity). The whole-genome sequence of isl-2T was determined. Average nucleotide identity values obtained for isl-2T in comparison to available genomic sequences of other strains of members of the genus Chloroflexus were 81.4 % or less and digital DNA-DNA hybridisation values 22.8 % or less. The results of additional phylogenetic analysis of the PufLM and BchG amino acid sequences supported the separate position of the isl-2T phylotype from the phylotypes of other members of the genus Chloroflexus. On the basis of physiological and phylogenetic data as well as genomic data, it was suggested that isl-2T represents a novel species within the genus Chloroflexus, with the proposed name Chloroflexus islandicus sp. nov. The type strain of the species is isl-2T (=VKM B-2978T,=DSM 29225T,=JCM 30533T).

Filamentous anoxygenic phototrophic bacteria from cyanobacterial mats of Alla hot springs (Barguzin Valley, Russia).

Alkaline hydrotherms of the Baikal rift zone are unique systems to study the diversity of thermophilic bacteria. In this study, we present data on the phototrophic bacterial community of cyanobacterial mats from the alkaline Alla hot spring. Using a clonal analysis approach, this study evaluated the species diversity, the proportion of oxygenic and anoxygenic phototrophs and their distribution between various areas of the spring. Novel group-specific PCR primers were designed and applied to detect representatives of the Chloroflexus and Roseiflexus genera in mat samples. For the first time, the presence of Roseiflexus-like bacteria was detected in the Baikal rift zone.

Evaluating the annotation of protein-coding genes in bacterial genomes: Chloroflexus aurantiacus strain J-10-fl and Natrinema sp J7-2 as case studies.

Gene annotation plays a key role in subsequent biochemical and molecular biological studies of various organisms. There are some errors in the original annotation of sequenced genomes because of the lack of sufficient data, and these errors may propagate into other genomes. Therefore, genome annotation must be checked from time to time to evaluate newly accumulated data. In this study, we evaluated the gene density of 2606 bacteria or archaea, and identified 2 with extreme values, the minimum value (Chloroflexus aurantiacus strain J-10-fl) and maximum value (Natrinema sp J7-2), to conduct genome re-annotation. In the genome of C. aurantiacus strain J-10-fl, we identified 17 new genes with definite functions and eliminated 34 non-coding open-reading frames; in the genome of Natrinema sp J7-2, we eliminated 118 non-coding open reading frames. Our re-annotation procedure may provide a reference for improving the annotation of other bacterial genomes.

Ecological divergence of a novel group of Chloroflexus strains along a geothermal gradient.

Environmental gradients are expected to promote the diversification and coexistence of ecological specialists adapted to local conditions. Consistent with this view, genera of phototrophic microorganisms in alkaline geothermal systems generally appear to consist of anciently divergent populations which have specialized on different temperature habitats. At White Creek (Lower Geyser Basin, Yellowstone National Park), however, a novel, 16S rRNA-defined lineage of the filamentous anoxygenic phototroph Chloroflexus (OTU 10, phylum Chloroflexi) occupies a much wider thermal niche than other 16S rRNA-defined groups of phototrophic bacteria. This suggests that Chloroflexus OTU 10 is either an ecological generalist or, alternatively, a group of cryptic thermal specialists which have recently diverged. To distinguish between these alternatives, we first isolated laboratory strains of Chloroflexus OTU 10 from along the White Creek temperature gradient. These strains are identical for partial gene sequences encoding the 16S rRNA and malonyl coenzyme A (CoA) reductase. However, strains isolated from upstream and downstream samples could be distinguished based on sequence variation at pcs, which encodes the propionyl-CoA synthase of the 3-hydroxypropionate pathway of carbon fixation used by the genus Chloroflexus. We next demonstrated that strains have diverged in temperature range for growth. Specifically, we obtained evidence for a positive correlation between thermal niche breadth and temperature optimum, with strains isolated from lower temperatures exhibiting greater thermal specialization than the most thermotolerant strain. The study has implications for our understanding of both the process of niche diversification of microorganisms and how diversity is organized in these hot spring communities.

Complete genome sequence of the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus.

BACKGROUND: Chloroflexus aurantiacus is a thermophilic filamentous anoxygenic phototrophic (FAP) bacterium, and can grow phototrophically under anaerobic conditions or chemotrophically under aerobic and dark conditions. According to 16S rRNA analysis, Chloroflexi species are the earliest branching bacteria capable of photosynthesis, and Cfl. aurantiacus has been long regarded as a key organism to resolve the obscurity of the origin and early evolution of photosynthesis. Cfl. aurantiacus contains a chimeric photosystem that comprises some characters of green sulfur bacteria and purple photosynthetic bacteria, and also has some unique electron transport proteins compared to other photosynthetic bacteria. METHODS: The complete genomic sequence of Cfl. aurantiacus has been determined, analyzed and compared to the genomes of other photosynthetic bacteria. RESULTS: Abundant genomic evidence suggests that there have been numerous gene adaptations/replacements in Cfl. aurantiacus to facilitate life under both anaerobic and aerobic conditions, including duplicate genes and gene clusters for the alternative complex III (ACIII), auracyanin and NADH:quinone oxidoreductase; and several aerobic/anaerobic enzyme pairs in central carbon metabolism and tetrapyrroles and nucleic acids biosynthesis. Overall, genomic information is consistent with a high tolerance for oxygen that has been reported in the growth of Cfl. aurantiacus. Genes for the chimeric photosystem, photosynthetic electron transport chain, the 3-hydroxypropionate autotrophic carbon fixation cycle, CO2-anaplerotic pathways, glyoxylate cycle, and sulfur reduction pathway are present. The central carbon metabolism and sulfur assimilation pathways in Cfl. aurantiacus are discussed. Some features of the Cfl. aurantiacus genome are compared with those of the Roseiflexus castenholzii genome. Roseiflexus castenholzii is a recently characterized FAP bacterium and phylogenetically closely related to Cfl. aurantiacus. According to previous reports and the genomic information, perspectives of Cfl. aurantiacus in the evolution of photosynthesis are also discussed. CONCLUSIONS: The genomic analyses presented in this report, along with previous physiological, ecological and biochemical studies, indicate that the anoxygenic phototroph Cfl. aurantiacus has many interesting and certain unique features in its metabolic pathways. The complete genome may also shed light on possible evolutionary connections of photosynthesis.

Optoelectronic energy transfer at novel biohybrid interfaces using light harvesting complexes from Chloroflexus aurantiacus.

In nature, nanoscale supramolecular light harvesting complexes initiate the photosynthetic energy collection process at high quantum efficiencies. In this study, the distinctive antenna structure from Chloroflexus aurantiacusthe chlorosomeis assessed for potential exploitation in novel biohybrid optoelectronic devices. Electrochemical characterization of bacterial fragments containing intact chlorosomes with the photosynthetic apparatus show an increase in the charge storage density near the working electrode upon light stimulation and suggest that chlorosomes contribute approximately one-third of the overall photocurrent. Further, isolated chlorosomes (without additional photosynthetic components, e.g., reaction centers, biochemical mediators) produce a photocurrent (approximately 8-10 nA) under light saturation conditions. Correlative experiments indicate that the main chlorosome pigment, bacteriochlorophyll-c, contributes to the photocurrent via an oxidative mechanism. The results reported herein are the first to demonstrate that isolated chlorosomes (lipid-enclosed sacs of pigments) directly transduce light energy in an electrochemical manner, laying an alternative, biomimetic approach for designing photosensitized interfaces in biofuel cells and biomedical devices, such as bioenhanced retinal prosthetics.

Femtosecond spectroscopy of the primary charge separation in reaction centers of Chloroflexus aurantiacus with selective excitation in the QY and Soret bands.

The primary charge separation and electron-transfer processes of photosynthesis occur in the reaction center (RC). Isolated RCs of the green filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus were studied at room temperature by using femtosecond transient absorption spectroscopy with selective excitation. Upon excitation in the Q(Y) absorbance band of the bacteriochlorophyll (BChl) dimer (P) at 865 nm, a 7.0 +/- 0.5 ps kinetic component was observed in the 538 nm region (Q(X) band of the bacteriopheophytin (BPheo)), 750 nm region (Q(Y) band of the BPheo), and 920 nm region (stimulated emission of the excited-state of P), indicating that this lifetime represents electron transfer from P to BPheo. The same time constant was also observed upon 740 nm or 800 nm excitation. A longer lifetime (300 +/- 30 ps), which was assigned to the time of reduction of the primary quinone, Q(A), was also observed. The transient absorption spectra and kinetics all indicate that only one electron-transfer branch is involved in primary charge separation under these excitation conditions. However, the transient absorption changes upon excitation in the Soret band at 390 nm reveal a more complex set of energy and electron-transfer processes. By comparison to studies on the RCs of the purple bacterium Rhodobacter sphaeroides, we discuss the possible mechanism of electron-transfer pathway dependence on excitation energy and propose a model of the Cf. aurantiacus RC that better explains the observed results.

[Biogeochemical processes in the algal-bacterial mats of the Urinskii alkaline hot spring].

The structure and production characteristics of microbial communities from the Urinskii alkaline hot spring (Buryat Republic, Russia) have been investigated. A distinctive characteristic of this hot spring is the lack of sulfide in the issuing water. The water temperature near the spring vents ranged from 69 to 38.5 degrees C and pH values ranged from 8.8 to 9.2. The total mineralization of water was less than 0.1 g/liter. Temperature has a profound effect on the species composition and biogeochemical processes occurring in the algal-bacterial mats of the Urinskii hot spring. The maximum diversity of the phototrophic community was observed at the temperatures 40 and 46 degrees C. A total of 12 species of cyanobacteria, 4 species of diatoms, and one species of thermophilic anoxygenic phototrophic bacteria, Chloroflexus aurantiacus, have been isolated from mat samples. At temperatures above 40 degrees C, the filamentous cyanobacterium Phormidium laminosum was predominant; its cell number and biomass concentration were 95.1 and 63.9%, respectively. At lower temperatures, the biomass concentrations of the cyanobacterium Oscillatoria limosa and diatoms increased (50.2 and 36.4%, respectively). The cyanobacterium Mastigocladus laminosus, which is normally found in neutral or slightly acidic hydrothermal systems, was detected in microbial communities. As the diatom concentration increases, so does the dry matter concentration in mats, while the content of organic matter decreases. The concentrations of proteins and carbohydrates reached their maximum levels at 45-50 degrees C. The maximum average rate of oxygenic photosynthesis (2.1 g C/m2 day), chlorophyll a content (343.4 mg/m2), and cell number of phototrophic microorganisms were observed at temperatures from 45 to 50 degrees C. The peak mass of bacterial mats (56.75 g/m2) occurred at a temperature of 65-60 degrees C. The maximum biomass concentration of phototrophs (414.63 x 10(-6) g/ml) and the peak rate of anoxygenic photosynthesis [0.42 g C/(m2 day)] were observed at a temperature of 35-40 degrees C.

Microbial diversity in natural environments: focusing on fundamental questions.

Interactions with Gijs Kuenen and other Dutch scientists have led my lab to fundamental insights into the composition, structure and function of a hot spring cyanobacterial mat community that should influence our thinking about all microbial communities. By focusing on the distribution of molecular sequence variants of predominant mat phototrophs, we have discovered that small-scale sequence variation can be ecologically meaningful. By applying novel cultivation approaches, we have been able to obtain genetically relevant community members and thus to test the hypothesis that closely related sequence variants arose via adaptive evolutionary radiation. By applying the analytical tools of organic geochemistry we have gained insight into the metabolisms of major phototrophic members of the mat community as well as interactions between phototrophic guilds. These observations challenge traditional paradigms about prokaryotic species and cause us to consider evolutionary ecology theory as we develop genome-based methods for high-resolution analysis of the species-like fundamental units comprising microbial communities, and for investigating how such units coordinate the physiological activities within guilds of the community.