Marcus Tank

Ether- and ester-bound iso-diabolic acid and other lipids in members of Acidobacteria subdivision 4

ABSTRACT Recently, iso-diabolic acid (13,16-dimethyl octacosanedioic acid) has been identified as a major membrane-spanning lipid of subdivisions 1 and 3 of the Acidobacteria, a highly diverse phylum within the Bacteria. This finding pointed to the Acidobacteria as a potential source for the bacterial glycerol dialkyl glycerol tetraethers that occur ubiquitously in peat, soil, lakes, and hot springs. Here, we examined the lipid composition of seven phylogenetically divergent strains of subdivision 4 of the Acidobacteria, a bacterial group that is commonly encountered in soil. Acid hydrolysis of total cell material released iso-diabolic acid derivatives in substantial quantities (11 to 48% of all fatty acids). In contrast to subdivisions 1 and 3 of the Acidobacteria, 6 out of the 7 species of subdivision 4 (excepting “Candidatus Chloracidobacterium thermophilum”) contained iso-diabolic acid ether bound to a glycerol in larger fractional abundance than iso-diabolic acid itself. This is in agreement with the analysis of intact polar lipids (IPLs) by high-performance liquid chromatography-mass spectrometry (HPLC-MS), which showed the dominance of mixed ether-ester glycerides. iso-Diabolic acid-containing IPLs were not identified, because these IPLs are not released with a Bligh-Dyer extraction, as observed before when studying lipid compositions of subdivisions 1 and 3 of the Acidobacteria. The presence of ether bonds in the membrane lipids does not seem to be an adaptation to temperature, because the five mesophilic isolates contained a larger amount of ether lipids than the thermophile “Ca. Chloracidobacterium thermophilum.” Furthermore, experiments with Pyrinomonas methylaliphatogenes did not reveal a major influence of growth temperature over the 50 to 69°C range.

Bacteriochlorophyll f: properties of chlorosomes containing the "forbidden chlorophyll"

The chlorosomes of green sulfur bacteria (GSB) are mainly assembled from one of three types of bacteriochlorophylls (BChls), BChls c, d, and e. By analogy to the relationship between BChl c and BChl d (20-desmethyl-BChl c), a fourth type of BChl, BChl f (20-desmethyl-BChl e), should exist but has not yet been observed in nature. The bchU gene (bacteriochlorophyllide C-20 methyltransferase) of the brown-colored green sulfur bacterium Chlorobaculum limnaeum was inactivated by conjugative transfer from Eshcerichia coli and homologous recombination of a suicide plasmid carrying a portion of the bchU. The resulting bchU mutant was greenish brown in color and synthesized BChl fF. The chlorosomes of the bchU mutant had similar size and polypeptide composition as those of the wild type (WT), but the Qy absorption band of the BChl f aggregates was blue-shifted 16 nm (705 nm vs. 721 nm for the WT). Fluorescence spectroscopy showed that energy transfer to the baseplate was much less efficient in chlorosomes containing BChl f than in WT chlorosomes containing BChl e. When cells were grown at high irradiance with tungsten or fluorescent light, the WT and bchU mutant had identical growth rates. However, the WT grew about 40% faster than the bchU mutant at low irradiance (10 μmol photons m−2 s-1). Less efficient energy transfer from BChl f aggregates to BChl a in the baseplate, the much slower growth of the strain producing BChl f relative to the WT, and competition from other phototrophs, may explain why BChl f is not observed naturally.

Nutrient requirements and growth physiology of the photoheterotrophic Acidobacterium, Chloracidobacterium thermophilum.

A novel thermophilic, microaerophilic, anoxygenic, and chlorophototrophic member of the phylum Acidobacteria, Chloracidobacterium thermophilum strain BT, was isolated from a cyanobacterial enrichment culture derived from microbial mats associated with Octopus Spring, Yellowstone National Park, Wyoming. C. thermophilum is strictly dependent on light and oxygen and grows optimally as a photoheterotroph at irradiance values between 20 and 50 μmol photons m-2 s-1. C. thermophilum is unable to synthesize branched-chain amino acids (AAs), l-lysine, and vitamin B12, which are required for growth. Although the organism lacks genes for autotrophic carbon fixation, bicarbonate is also required. Mixtures of other AAs and 2-oxoglutarate stimulate growth. As suggested from genomic sequence data, C. thermophilum requires a reduced sulfur source such as thioglycolate, cysteine, methionine, or thiosulfate. The organism can be grown in a defined medium at 51∘C (Topt; range 44–58∘C) in the pH range 5.5–9.5 (pHopt = ∼7.0). Using the defined growth medium and optimal conditions, it was possible to isolate new C. thermophilum strains directly from samples of hot spring mats in Yellowstone National Park, Wyoming. The new isolates differ from the type strain with respect to pigment composition, morphology in liquid culture, and temperature adaptation.

Unique communities of anoxygenic phototrophic bacteria in saline lakes of Salar de Atacama (Chile): evidence for a new phylogenetic lineage of phototrophic Gammaproteobacteria from pufLM gene analyses

Phototrophic bacteria are important primary producers of salt lakes in the Salar de Atacama and at times form visible mass developments within and on top of the lake sediments. The communities of phototrophic bacteria from two of these lakes were characterized by molecular genetic approaches using key genes for the biosynthesis of the photosynthetic apparatus in phototrophic purple bacteria (pufLM) and in green sulfur bacteria (fmoA). Terminal restriction fragment length polymorphism of the pufLM genes indicated high variability of the community composition between the two lakes and subsamples thereof. The communities were characterized by the dominance of a novel, so far undescribed lineage of pufLM containing bacteria and the presence of representatives related to known halophilic Chromatiaceae and Ectothiorhodospiraceae. In addition, the presence of BChl b-containing anoxygenic phototrophic bacteria and of aerobic anoxygenic bacteria was indicated. Green sulfur bacteria were not detected in the environmental samples, although a bacterium related to Prosthecochloris indicum was identified in an enrichment culture. This is the first comprehensive description of phototrophic bacterial communities in a salt lake of South America made possible only due to the application of the functional pufLM genes.

Photophysical properties of the excited states of bacteriochlorophyll f in solvents and in chlorosomes.

Bacteriochlorophyll f (BChl f) is a photosynthetic pigment predicted nearly 40 years ago as a fourth potential member of the Chlorobium chlorophyll family (BChl c, d, and e). However, this pigment still has not been found in a naturally occurring organism. BChl c, d, and e are utilized by anoxygenic green photosynthetic bacteria for assembly of chlorosomes--large light-harvesting complexes that allow those organisms to survive in habitats with extremely low light intensities. Recently, using genetic methods on two different strains of Chlorobaculum limnaeum that naturally produce BChl e, two research groups produced mutants that synthesize BChl f and assemble it into chlorosomes. In this study, we present detailed investigations on spectral and dynamic characteristics of singlet excited and triplet states of BChl f with the application of ultrafast time-resolved absorption and fluorescence spectroscopies. The studies were performed on isolated BChl f in various solvents, at different temperatures, and on BChl f-containing chlorosomes in order to uncover any unusual or unfavorable properties that stand behind the lack of appearance of this pigment in natural environments.

Structure of Light-Harvesting Aggregates in Individual Chlorosomes

Among all photosynthetic organisms, green bacteria have evolved one of the most efficient light-harvesting antenna, the chlorosome, that contains hundreds of thousands of bacteriochlorophyll molecules, allowing these bacteria to grow photosynthetically by absorbing only a few photons per bacteriochlorophyll molecule per day. In contrast to other photosynthetic light-harvesting antenna systems, for which a protein scaffold imposes the proper positioning of the chromophores with respect to each other, in chlorosomes, this is accomplished solely by self-assembly. This has aroused enormous interest in the structure-function relations of these assemblies, as they can serve as blueprints for artificial light harvesting systems. In spite of these efforts, conclusive structural information is not available yet, reflecting the sample heterogeneity inherent to the natural system. Here we combine mutagenesis, polarization-resolved single-particle fluorescence-excitation spectroscopy, cryo-electron microscopy, and theoretical modeling to study the chlorosomes of the green sulfur bacterium Chlorobaculum tepidum. We demonstrate that only the combination of these techniques yields unambiguous information on the structure of the bacteriochlorophyll aggregates within the chlorosomes. Moreover, we provide a quantitative estimate of the curvature variation of these aggregates that explains ongoing debates concerning the chlorosome structure.

The Dark Side of the Mushroom Spring Microbial Mat: Life in the Shadow of Chlorophototrophs. I. Microbial Diversity Based on 16S rRNA Gene Amplicons and Metagenomic Sequencing

Microbial-mat communities in the effluent channels of Octopus and Mushroom Springs within the Lower Geyser Basin at Yellowstone National Park have been studied for nearly 50 years. The emphasis has mostly focused on the chlorophototrophic bacterial organisms of the phyla Cyanobacteria and Chloroflexi. In contrast, the diversity and metabolic functions of the heterotrophic community in the microoxic/anoxic region of the mat are not well understood. In this study we analyzed the orange-colored undermat of the microbial community of Mushroom Spring using metagenomic and rRNA-amplicon (iTag) analyses. Our analyses disclosed a highly diverse community exhibiting a high degree of unevenness, strongly dominated by a single taxon, the filamentous anoxygenic phototroph, Roseiflexus spp. The second most abundant organisms belonged to the Thermotogae, which have been hypothesized to be a major source of H2 from fermentation that could enable photomixotrophic metabolism by Chloroflexus and Roseiflexus spp. Other abundant organisms include two members of the Armatimonadetes (OP10); Thermocrinis sp.; and phototrophic and heterotrophic members of the Chloroflexi. Further, an Atribacteria (OP9/JS1) member; a sulfate-reducing Thermodesulfovibrio sp.; a Planctomycetes member; a member of the EM3 group tentatively affiliated with the Thermotogae, as well as a putative member of the Arminicenantes (OP8) represented ≥1% of the reads. Archaea were not abundant in the iTag analysis, and no metagenomic bin representing an archaeon was identified. A high microdiversity of 16S rRNA gene sequences was identified for the dominant taxon, Roseiflexus spp. Previous studies demonstrated that highly similar Synechococcus variants in the upper layer of the mats represent ecological species populations with specific ecological adaptations. This study suggests that similar putative ecotypes specifically adapted to different niches occur within the undermat community, particularly for Roseiflexus spp.

A Panoply of Phototrophs: An Overview of the Thermophilic Chlorophototrophs of the Microbial Mats of Alkaline Siliceous Hot Springs in Yellowstone National Park, WY, USA

Chlorophototrophs are organisms that can synthesize chlorophylls or bacteriochlorophylls, and they use these molecules to harvest and convert light energy into stored chemical potential energy. Some of these organisms also perform photosynthesis, in which light provides the energy (ATP) and reducing power (NAD(P)H or reduced ferredoxin) required for inorganic carbon (Ci) fixation. Over the past decade, we have studied the chlorophototrophs found in two alkaline siliceous hot springs in Yellowstone National Park, WY, USA. The microbial mats that occur at temperatures of 40–73 °C in Mushroom and Octopus Springs have proven to contain a surprisingly diverse array of chlorophototrophs. These include members of six of the seven bacterial phyla known to have members capable of synthesizing (bacterio)-chlorophylls: Acidobacteria, Cyanobacteria, Chlorobi, Chloroflexi, Firmicutes, and Proteobacteria. More than 16 chlorophototrophs have now been associated with these microbial mats, and this does not include the many ecotypes of these organisms that occur within these communities. In this chapter we will briefly describe the panoply of phototrophic organisms that occur in these mat communities and will provide an introduction to their morphological appearance and other basic properties. Metagenomic analyses have revealed several novel organisms, e.g., Chloracidobacterium thermophilum, “Candidatus Thermochlorobacter aerophilum,” “Candidatus Chloranaerofilum corporosum,” “Candidatus Roseovibrio tepidum,” and “Candidatus Roseilinea gracile,” which were hitherto unknown to microbiologists because they escaped isolation by classical, culture-based methods. However, by combining molecular methods, in situ physiological observations, metabolic reconstruction, and enrichment techniques, we are now making remarkable progress toward the isolation of these chlorophototrophic organisms.

Thiohalocapsa marina sp. nov., from an Indian marine aquaculture pond.

A spherical-shaped, phototrophic, purple sulfur bacterium was isolated in pure culture from anoxic sediment in a marine aquaculture pond near Bheemli (India). Strain JA142T is Gram-negative and non-motile. It has a requirement for NaCl (optimum of 2% and maximum of 6% w/v NaCl). Intracellular photosynthetic membranes are of the vesicular type. In vivo absorption spectra indicate the presence of bacteriochlorophyll a and carotenoids of the okenone series as photosynthetic pigments. Phylogenetic analysis on the basis of 16S rRNA gene sequences showed that strain JA142T is related to halophilic purple sulfur bacteria of the genera Thiohalocapsa and Halochromatium, with the highest sequence similarity to Thiohalocapsa halophila DSM 6210T (97.5%). Morphological and physiological characteristics differentiate strain JA142T from other species of the genera Halochromatium and Thiohalocapsa. Strain JA142T is sufficiently different from Thiohalocapsa halophila based on 16S rRNA gene sequence analysis and morphological and physiological characteristics to allow the proposal of a novel species, Thiohalocapsa marina sp. nov., with the type strain JA142T (=JCM 14780T=DSM 19078T).

Glycolipid analyses of light-harvesting chlorosomes from envelope protein mutants of Chlorobaculum tepidum

Chlorosomes are large and efficient light-harvesting organelles in green photosynthetic bacteria, and they characteristically contain large numbers of bacteriochlorophyll c, d, or e molecules. Self-aggregated bacteriochlorophyll pigments are surrounded by a monolayer envelope membrane comprised of glycolipids and Csm proteins. Here, we analyzed glycolipid compositions of chlorosomes from the green sulfur bacterium Chlorobaculum tepidum mutants lacking one, two, or three Csm proteins by HPLC equipped with an evaporative light-scattering detector. The ratio of monogalactosyldiacylglyceride (MGDG) to rhamnosylgalactosyldiacylglyceride (RGDG) was smaller in chlorosomes from mutants lacking two or three proteins in CsmC/D/H motif family than in chlorosomes from the wild-type, whereas chlorosomes lacking CsmIJ showed relatively less RGDG than MGDG. The results suggest that the CsmC, CsmD, CsmH, and other chlorosome proteins are involved in organizing MGDG and RGDG and thereby affect the size and shape of the chlorosome.