Marcus Tank

A novel chlorophototrophic bacterium, strain BV2-C ^T , was isolated from a microbial mat (~40 °C) at Rupite hot springs, Bulgaria. Cells were rod-shaped, Gram-negative, with numerous fimbriae/pili, but non-motile. Optimal growth occurred under micro-oxic conditions in the light (20–50 µmol photons m ⁻² s ⁻¹ ) at 45 °C (range 35–50 °C) and pH 7.2 (range pH 5.5–9.0). Bacteriochlorophyll c , bacteriochlorophyll a _P , echinenone and γ -carotene were the major pigments in cells capable of producing chlorosomes and type-1 homodimeric reaction centres. Dense liquid culture appeared greenish-brown, with cells forming small clumps and aggregates. Strain BV2-C ^T grows photoheterotrophically using organic compounds as carbon source (i.e. a mixture of 20 proteinogenic amino acids or peptone) and thioglycolate as reduced sulphur source. Strain BV2-C ^T obligately required branched-chain amino acids ( l -isoleucine, l -leucine and l -valine) and l -lysine for growth. It is closely related to Chloracidobacterium thermophilum and ‘ Candidatus Chloracidobacterium aggregatum ’ with 16S rRNA gene sequence identities of 96.97 and 97.78%, respectively. The genome of strain BV2-C ^T comprises two circular chromosomes of 2,659,040 and 1,000,103 bp (total genome, 3.65 Mb) and G+C content of 59.9 mol%. Phenotypic, phylogenetic and genomic analyses demonstrated that Chloracidobacterium validum strain BV2-C ^T (=DSM 113832 ^T =JCM 39534 ^T ) represents a novel species of the genus ‘ Candidatus Chloracidobacterium’, which we hereby propose as Chloracidobacterium gen. nov. with Chloracidobacterium validum sp. nov. as the type species. Based on phylogenomic analyses, we also propose a new family, Chloroacidobacteriaceae fam. nov., and a new order, Chloracidobacteriales ord. nov., within the class Blastocatellia . We further propose the name ‘ Ca. C. aggregatum ’ for pro-valid publication with the genome sequence of strain E as nomenclatural type.

Scientific exploration of phototrophic bacteria over nearly 200 years has revealed large phylogenetic gaps between known phototrophic groups that limit understanding of how phototrophy evolved and diversified^1,2. Here, through Boreal Shield lake water incubations, we cultivated an anoxygenic phototrophic bacterium from a previously unknown order within the Chloroflexota phylum that represents a highly novel transition form in the evolution of photosynthesis. Unlike all other known phototrophs, this bacterium uses a type I reaction centre (RCI) for light energy conversion yet belongs to the same bacterial phylum as organisms that use a type II reaction centre (RCII) for phototrophy. Using physiological, phylogenomic and environmental metatranscriptomic data, we demonstrate active RCI-utilizing metabolism by the strain alongside usage of chlorosomes³ and bacteriochlorophylls⁴ related to those of RCII-utilizing Chloroflexota members. Despite using different reaction centres, our phylogenomic data provide strong evidence that RCI-utilizing and RCII-utilizing Chloroflexia members inherited phototrophy from a most recent common phototrophic ancestor. The Chloroflexota phylum preserves an evolutionary record of the use of contrasting phototrophic modes among genetically related bacteria, giving new context for exploring the diversification of phototrophy on Earth.

Strain MS-P2^T was isolated from microbial mats associated with Mushroom Spring, an alkaline siliceous hot spring in Yellowstone National Park, WY, USA. The isolate grows chemoheterotrophically by oxygen-dependent respiration, and light stimulates photoheterotrophic growth under strictly oxic conditions. Strain MS-P2^T synthesizes bacteriochlorophyll a and the carotenoid spirilloxanthin. However, photoautotrophic growth did not occur under oxic or anoxic conditions, suggesting that this strain should be classified as an aerobic anoxygenic phototrophic bacterium. Strain MS-P2^T cells are motile, curved rods about 0.5 to 1.0 μm wide and 1.0 to 1.5 μm long. The optimum growth temperature is 45–50 °C, and the optimum pH for growth is circum-neutral (pH 7.0–7.5). Sequence analysis of the 16S rRNA gene revealed that strain MS-P2^T is closely related to Elioraea species, members of the class Alphaproteobacteria, with a sequence identity of 96.58 to 98%. The genome of strain MS-P2^T is a single circular DNA molecule of 3,367,643 bp with a mol% guanine-plus-cytosine content of 70.6%. Based on phylogenetic, physiological, biochemical, and genomic characteristics, we propose this bacteriochlorophyll a-containing isolate is a new species belonging to the genus Elioraea, with the suggested name Elioraeatepida. The type-strain is strain MS-P2^T (= JCM33060^T = ATCC TSD-174^T).

Chloroflexus aggregans is a metabolically versatile, thermophilic, anoxygenic phototrophic member of the phylum Chloroflexota (formerly Chloroflexi), which can grow photoheterotrophically, photoautotrophically, chemoheterotrophically, and chemoautotrophically. In hot spring-associated microbial mats, C. aggregans co-exists with oxygenic cyanobacteria under dynamic micro-environmental conditions. To elucidate the predominant growth modes of C. aggregans, relative transcription levels of energy metabolism- and CO₂ fixation-related genes were studied in Nakabusa Hot Springs microbial mats over a diel cycle and correlated with microscale in situ measurements of O₂ and light. Metatranscriptomic analyses indicated two periods with different modes of energy metabolism of C. aggregans: (1) phototrophy around midday and (2) chemotrophy in the early morning hours. During midday, C. aggregans mainly employed photoheterotrophy when the microbial mats were hyperoxic (400–800 µmol L⁻¹ O₂). In the early morning hours, relative transcription peaks of genes encoding uptake hydrogenase, key enzymes for carbon fixation, respiratory complexes as well as enzymes for TCA cycle and acetate uptake suggest an aerobic chemomixotrophic lifestyle. This is the first in situ study of the versatile energy metabolism of C. aggregans based on gene transcription patterns. The results provide novel insights into the metabolic flexibility of these filamentous anoxygenic phototrophs that thrive under dynamic environmental conditions.

A novel thermophilic phototrophic purple sulphur bacterium was isolated from microbial mats (56 °C) at Nakabusa hot springs, Nagano prefecture, Japan. Cells were motile, rod-shaped, stain Gram-negative and stored sulphur globules intracellularly. Bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series were the major pigments. Dense liquid cultures were red in colour. Strain No.7^T was able to grow photoautotrophically using sulfide, thiosulfate, sulfite and hydrogen (in the presence of sulfide) as electron donors and bicarbonate as the sole carbon source. Optimum growth occurred under anaerobic conditions in the light at 50 °C (range, 40–56 °C) and pH 7.2 (range, pH 7–8). Major fatty acids were C_16 : 0 (46.8 %), C_16 : 1 ω7c (19.9 %), C_18 : 1 ω7c (21.1 %), C_14 : 0 (4.6 %) and C_18 : 0 (2.4 %). The polar lipid profile showed phosphatidylglycerol and unidentified aminophospholipids to be the major lipids. The only quinone detected was ubiquinone-8. 16S rRNA gene sequence comparisons indicated that the novel bacterium is only distantly related to Thermochromatium tepidum with a nucleotide identity of 90.4 %. The phylogenetic analysis supported the high novelty of strain No.7^T with a long-branching phylogenetic position within the Chromatiaceae next to Thermochromatium tepidum . The genome comprised a circular chromosome of 2.99 Mbp (2 989 870 bp), included no plasmids and had a DNA G+C content of 61.2 mol%. Polyphasic taxonomic analyses of the isolate suggested strain No.7^T is a novel genus within the Chromatiaceae . The proposed genus name of the second truly thermophilic purple sulphur bacterium is Caldichromatium gen. nov. with the type species Caldichromatium japonicum sp. nov. (DSM 110881=JCM 39101).

Scientific exploration of phototrophic bacteria over nearly 200 years has revealed large phylogenetic gaps between known phototrophic groups that limit understanding of how phototrophy evolved and diversified. Through Boreal Shield lake water incubations, we cultivated an anoxygenic phototrophic bacterium from a previously unknown order within the Chloroflexota phylum that represents a highly novel transition form in the evolution of photosynthesis. Unlike all other known phototrophs, this bacterium uses a Type I reaction center (RCI) for light energy conversion yet belongs to the same bacterial phylum as organisms that use a Type II reaction center (RCII) for phototrophy. Using physiological, phylogenomic, and environmental metatranscriptomic data, we demonstrate active RCI-utilizing metabolism by the strain alongside usage of chlorosomes and bacteriochlorophylls related to those of RCII-utilizing Chloroflexota members. Despite using different reaction centers, our phylogenomic data provide strong evidence that RCI- and RCII-utilizing Chloroflexia members inherited phototrophy from a most recent common phototrophic ancestor that used RCI, RCII, or both reaction center classes, substantially revising our view of the diversity and evolution of phototrophic life. The Chloroflexota phylum preserves an evolutionary record of interaction between RCI and RCII among anoxygenic phototrophs that gives new context for exploring the origins of phototrophy on Earth.

Chloracidobacterium thermophilum is a microaerophilic, anoxygenic member of the green chlorophototrophic bacteria. This bacterium is the first characterized oxygen-requiring chlorophototroph with chlorosomes, the FMO protein, and homodimeric type-1 reaction centers (RCs). The RCs of C. thermophilum are also unique because they contain three types of chlorophylls, bacteriochlorophyll a_P esterified with phytol, Chl a_PD esterified with Δ2,6-phytadienol, and Zn-BChl a_P′ esterified with phytol, in the approximate molar ratio 32:24:4. The light-induced difference spectrum of these RCs had a bleaching maximum at 839 nm and also revealed an electrochromic bandshift that is probably derived from a BChl a molecule near P840⁺. The F_X [4Fe–4S] cluster had a midpoint potential of ca. − 581 mV, and the spectroscopic properties of the P⁺ F _X ⁻ spin-polarized radical pair were very similar to those of reaction centers of heliobacteria and green sulfur bacteria. The data further indicate that electron transfer occurs directly from A₀⁻ to F_X, as occurs in other homodimeric type-1 RCs. Washing experiments with isolated membranes suggested that the PscB subunit of these reaction centers is more tightly bound than PshB in heliobacteria. Thus, the reaction centers of C. thermophilum have some properties that resemble other homodimeric reaction centers but also have specific properties that are more similar to those of Photosystem I. These differences probably contribute to protection of the electron transfer chain from oxygen, contributing to the oxygen tolerance of this microaerophile.

In this study we present evidence for a novel, thermophilic bacterium with dissimilatory sulfur metabolism, tentatively named “Candidatus Thermonerobacter thiotrophicus,” which is affiliated with the Bacteroides/Ignavibacteria/Chlorobi and which we predict to be a sulfate reducer. Dissimilatory sulfate reduction (DSR) is an important and ancient metabolic process for energy conservation with global importance for geochemical sulfur and carbon cycling. Characterized sulfate-reducing microorganisms (SRM) are found in a limited number of bacterial and archaeal phyla. However, based on highly diverse environmental dsrAB sequences, a variety of uncultivated and unidentified SRM must exist. The recent development of high-throughput sequencing methods allows the phylogenetic identification of some of these uncultured SRM. In this study, we identified a novel putative SRM inhabiting hot spring microbial mats that is a member of the OPB56 clade (“Ca. Kapabacteria”) within the Bacteroidetes/Chlorobi superphylum. Partial genomes for this new organism were retrieved from metagenomes from three different hot springs in Yellowstone National Park, United States, and Japan. Supporting the prediction of a sulfate-reducing metabolism for this organism during period of anoxia, diel metatranscriptomic analyses indicate highest relative transcript levels in situ for all DSR-related genes at night. The presence of terminal oxidases, which are transcribed during the day, further suggests that these organisms might also perform aerobic respiration. The relative phylogenetic proximity to the sulfur-oxidizing, chlorophototrophic Chlorobi further raises new questions about the evolution of dissimilatory sulfur metabolism.

Phototrophic microbial mats are assemblages of vertically layered microbial populations dominated by photosynthetic microorganisms. In order to elucidate the vertical distribution and diversity of phototrophic microorganisms in a hot spring-associated microbial mat in Nakabusa (Japan), we analyzed the 16S rRNA gene amplicon sequences of the microbial mat separated into five depth horizons, and correlated them with microsensor measurements of O2 and spectral scalar irradiance. A stable core community and high diversity of phototrophic organisms dominated by the filamentous anoxygenic phototrophs, Roseiflexus castenholzii and Chloroflexus aggregans were identified together with the spectral signatures of bacteriochlorophylls (BChls) a and c absorption in all mat layers. In the upper mat layers, a high abundance of cyanobacteria (Thermosynechococcus sp.) correlated with strong spectral signatures of chlorophyll a and phycobiliprotein absorption near the surface in a zone of high O2 concentrations during the day. Deeper mat layers were dominated by uncultured chemotrophic Chlorobi such as the novel putatively sulfate-reducing “Ca. Thermonerobacter sp.”, which showed increasing abundance with depth correlating with low O2 in these layers enabling anaerobic metabolism. Oxygen tolerance and requirements for the novel phototroph “Ca. Chloroanaerofilum sp.” and the uncultured chemotrophic Armatimonadetes member type OS-L detected in Nakabusa hot springs, Japan appeared to differ from previously suggested lifestyles for close relatives identified in hot springs in Yellowstone National Park, USA. The present study identified various microenvironmental gradients and niche differentiation enabling the co-existence of diverse chlorophototrophs in metabolically diverse communities in hot springs.

The production of secondary metabolites by a new isolate of the purple sulfur bacterium Allochromatium vinosum, which had shown antibiotic activities during a preliminary study, revealed the production of several metabolites. Growth conditions suitable for the production of one of the compounds shown in the metabolite profile were established and compound 1 was purified. The molecular formula of compound 1 (C₂₀H₂₈O₂) was determined by high resolution mass spectra, and its chemical structure by means of spectroscopic methods. The evaluation of these data revealed that the structure of the compound was identical to dehydroabietic acid, a compound known to be characteristically produced by conifer trees, but so far not known from bacteria, except cyanobacteria. The purified substance showed weak antibiotic activities against Bacillus subtilis and Staphylococcus lentus with IC₅₀ values of 70.5 µM (±2.9) and 57.0 µM (±3.3), respectively.