Spectroscopic insights into the decreased efficiency of chlorosomes containing bacteriochlorophyll f.

Chlorosomes are light-harvesting antenna complexes that occur in green photosynthetic bacteria which have only been shown naturally to contain bacteriochlorophyll (BChl) c, d, or e as the principal light-harvesting pigments. BChl f has long been thought to be an obvious fourth member of the so-called Chlorobium chlorophylls, because it possesses a C-7 formyl group like BChl e and lacks a methyl group at C-20 like BChl d. In organisms that synthesize BChl c or e, the bchU gene product encodes the enzyme that methylates the C-20 position of these molecules. A bchU null mutant of the green sulfur bacterium Chlorobaculum limnaeum strain 1677(T), which normally synthesizes BChl e, has recently been generated via insertional inactivation, and it produces chlorosomes containing BChl f [Vogl et al., 2012]. In this study, chlorosomes containing BChl f and monomeric BChl f in pyridine were characterized using a variety of spectroscopic techniques, including fluorescence emission and excitation spectroscopy, fluorescence lifetime and quantum yield determinations, and circular dichroism. These spectroscopic measurements, as well as Gaussian simulation of the data, show that chlorosomes containing BChl f are less efficient in energy transfer than those with BChl e. This can primarily be attributed to the decreased spectral overlap between the oligomeric BChl f (energy donor) fluorescence emission and the BChl a (energy acceptor) absorption in the chlorosome baseplate. This study allows us to hypothesize that, if they exist in nature, BChl f-containing organisms most likely live in rare high-light, anoxic conditions devoid of Chl a, d, or BChl e filtering. ABSTRACT REFERENCE: K. Vogl, M. Tank, G.S. Orf, R.E. Blankenship, D.A. Bryant, Bacteriochlorophyll f: properties of chlorosomes containing the "forbidden chlorophyll," Front. Microbiol. 3 (2012) 298.

Elucidation of the biosynthetic pathway for Okenone in Thiodictyon sp. CAD16 leads to the discovery of two novel carotene ketolases.

Okenone is a unique ketocarotenoid found in many purple sulfur bacteria; it is important because of its unique light absorption and photoprotection properties. Okenane, a compound formed by diagenetic reduction of okenone, is an important biomarker in geochemical analyses of sedimentary rocks. Despite its ecological and biogeochemical importance, the biochemical pathway for okenone synthesis has not yet been fully described. The genome sequence of an okenone-producing organism, Thiodictyon sp. strain CAD16, revealed four genes whose predicted proteins had strong sequence similarity to enzymes known to produce ψ-end group modifications of carotenoids in proteobacteria. These four genes encoded homologs of a 1,2-carotenoid hydratase (CrtC), an O-methyltransferase (CrtF), and two paralogs of carotenoid 3,4-desaturases (CrtD). Expression studies in lycopene- or neurosporene-producing strains of Escherichia coli confirmed the functions of crtC and crtF, but the crtD paralogs encoded enzymes with previously undescribed functions. One enzyme, CruS, was only distantly related to CrtD desaturases, was bifunctional, and performed a 3,4-desaturation and introduced a C-2 keto group into neurosporene derivatives in the presence of dioxygen. The enzyme encoded by the other crtD paralog also represents a new enzyme in carotenogenesis and was named cruO. CruO encodes the C-4/4' ketolase uniquely required for okenone biosynthesis. The identification of CruO and the demonstration of its biochemical activity complete the elucidation of the biosynthetic pathway for okenone and other related ketocarotenoids.

Comparison of Chloroflexus aurantiacus strain J-10-fl proteomes of cells grown chemoheterotrophically and photoheterotrophically.

Chloroflexus aurantiacus J-10-fl is a thermophilic green bacterium, a filamentous anoxygenic phototroph, and the model organism of the phylum Chloroflexi. We applied high-throughput, liquid chromatography-mass spectrometry in a global quantitative proteomics investigation of C. aurantiacus cells grown under oxic (chemoorganoheterotrophically) and anoxic (photoorganoheterotrophically) redox states. Our global analysis identified 13,524 high-confidence peptides that matched to 1,286 annotated proteins, 242 of which were either uniquely identified or significantly increased in abundance under photoheterotrophic culture condition. Fifty-four of the 242 proteins are previously characterized photosynthesis-related proteins, including chlorosome proteins, proteins involved in the bacteriochlorophyll biosynthesis, 3-hydroxypropionate (3-OHP) CO(2) fixation pathway, and components of electron transport chains. The remaining 188 proteins have not previously been reported. Of these, five proteins were found to be encoded by genes from a novel operon and observed only in photoheterotrophically grown cells. These proteins candidates may prove useful in further deciphering the phototrophic physiology of C. aurantiacus and other filamentous anoxygenic phototrophs.

Ultrastructural characterization of the prokaryotic symbiosis in "Chlorochromatium aggregatum".

The phototrophic consortium "Chlorochromatium aggregatum" currently represents the most highly developed interspecific association of bacteria and consists of green sulfur bacteria, so-called epibionts, surrounding a central, motile, chemotrophic bacterium. In order to identify subcellular structures characteristic of this symbiosis, consortia were studied by a combination of high-resolution analytical scanning electron microscopy, transmission electron microscopy, and three-dimensional reconstruction and image analyses. Epibionts are interconnected and to a lesser extent are also connected with the central bacterium, by electron-dense, hair-like filaments. In addition, numerous periplasmic tubules extend from the outer membrane of the central bacterium and are in direct contact with the outer membrane of the epibionts. In each epibiont cell, the attachment site to the central bacterium is characterized by the absence of chlorosomes and an additional 17-nm-thick layer (epibiont contact layer [ECL]) attached to the inner side of the cytoplasmic membrane. The ECL is only occasionally observed in pure cultures of the epibiont, where it occurs in about 10 to 20% of the free-living cells. A striking feature of the central bacterium is the presence of one or two hexagonally packed flat crystals (central bacterium crystal [CBC]) per cell. The CBC reaches 1 microm in length, is 35 nm thick, and consists of bilayers of subunits with a spacing of 9 nm. A detailed model for consortia is presented, summarizing our conclusions regarding (i) cohesion of the cells, (ii) common periplasmic space between the central bacterium and the epibiont, (iii) ECL as a symbiosis-specific structure, and (iv) formation of the interior paracrystalline structures, central bacterium membrane layer, and CBC.

Identification and analysis of four candidate symbiosis genes from 'Chlorochromatium aggregatum', a highly developed bacterial symbiosis.

The consortium 'Chlorochromatium aggregatum' currently represents the most highly developed interspecific association between prokaryotes. It consists of green sulfur bacteria, so-called epibionts, which surround a central, motile, chemotrophic bacterium. Four putative symbiosis genes of the epibiont were recovered by suppression subtractive hybridization and bioinformatics approaches. These genes are transcribed constitutively and do not occur in the free-living relatives of the epibiont. The haemagglutinin-like putative gene products of open reading frames (ORFs) Cag0614 and Cag0616 are unusually large and contain repetitive regions and RGD tripeptides. Cag0616 harbours two betagamma-crystalline Greek key motifs. Cag1920 codes for a putative haemolysin whereas the gene product of Cag1919 is a putative RTX-like protein. Based on detailed analyses of Cag1919, the C-terminal amino acid sequence comprises six repetitions of the motif GGXGXD predicted to form a Ca(2+)-binding beta roll. Intact 'C. aggregatum' consortia disaggregated upon the addition of EGTA or pyrophosphate, but stayed intact in the presence of various lectine-binding sugars or proteolytic enzymes. Unlike other RTX toxins, a gene product of Cag1919 could not be detected by (45)Ca(2+) autoradiography, indicating a low abundance of the corresponding protein in the cells. The RTX-type C-terminus coded by Cag1919 exhibited a significant similarity to RTX modules of various proteobacterial proteins, suggesting that this putative symbiosis gene has been acquired via horizontal gene transfer from a proteobacterium.

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.

Genomic analysis reveals key aspects of prokaryotic symbiosis in the phototrophic consortium "Chlorochromatium aggregatum".

BACKGROUND: 'Chlorochromatium aggregatum' is a phototrophic consortium, a symbiosis that may represent the highest degree of mutual interdependence between two unrelated bacteria not associated with a eukaryotic host. 'Chlorochromatium aggregatum' is a motile, barrel-shaped aggregate formed from a single cell of 'Candidatus Symbiobacter mobilis", a polarly flagellated, non-pigmented, heterotrophic bacterium, which is surrounded by approximately 15 epibiont cells of Chlorobium chlorochromatii, a non-motile photolithoautotrophic green sulfur bacterium. RESULTS: We analyzed the complete genome sequences of both organisms to understand the basis for this symbiosis. Chl. chlorochromatii has acquired relatively few symbiosis-specific genes; most acquired genes are predicted to modify the cell wall or function in cell-cell adhesion. In striking contrast, 'Ca. S. mobilis' appears to have undergone massive gene loss, is probably no longer capable of independent growth, and thus may only reproduce when consortia divide. A detailed model for the energetic and metabolic bases of the dependency of 'Ca. S. mobilis' on Chl. chlorochromatii is described. CONCLUSIONS: Genomic analyses suggest that three types of interactions lead to a highly sophisticated relationship between these two organisms. Firstly, extensive metabolic exchange, involving carbon, nitrogen, and sulfur sources as well as vitamins, occurs from the epibiont to the central bacterium. Secondly, 'Ca. S. mobilis' can sense and move towards light and sulfide, resources that only directly benefit the epibiont. Thirdly, electron cycling mechanisms, particularly those mediated by quinones and potentially involving shared protonmotive force, could provide an important basis for energy exchange in this and other symbiotic relationships.

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 f(F). The chlorosomes of the bchU mutant had similar size and polypeptide composition as those of the wild type (WT), but the Q(y) 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.

Complete Genome of Ignavibacterium album, a Metabolically Versatile, Flagellated, Facultative Anaerobe from the Phylum Chlorobi.

Prior to the recent discovery of Ignavibacterium album (I. album), anaerobic photoautotrophic green sulfur bacteria (GSB) were the only members of the bacterial phylum Chlorobi that had been grown axenically. In contrast to GSB, sequence analysis of the 3.7-Mbp genome of I. album shows that this recently described member of the phylum Chlorobi is a chemoheterotroph with a versatile metabolism. I. album lacks genes for photosynthesis and sulfur oxidation but has a full set of genes for flagella and chemotaxis. The occurrence of genes for multiple electron transfer complexes suggests that I. album is capable of organoheterotrophy under both oxic and anoxic conditions. The occurrence of genes encoding enzymes for CO(2) fixation as well as other enzymes of the reductive TCA cycle suggests that mixotrophy may be possible under certain growth conditions. However, known biosynthetic pathways for several amino acids are incomplete; this suggests that I. album is dependent upon on exogenous sources of these metabolites or employs novel biosynthetic pathways. Comparisons of I. album and other members of the phylum Chlorobi suggest that the physiology of the ancestors of this phylum might have been quite different from that of modern GSB.

Heterotrophic symbionts of phototrophic consortia: members of a novel diverse cluster of Betaproteobacteria characterized by a tandem rrn operon structure.

Phototrophic consortia represent the most highly developed type of interspecific association of bacteria and consist of green sulfur bacterial epibionts attached around a central colourless rod-shaped bacterium. Based on 16S rRNA gene sequencing, the central bacterium of the consortium 'Chlorochromatium aggregatum' was recently shown to represent a novel and phylogenetically isolated lineage of the Comamonadaceae within the beta-subgroup of the Proteobacteria. To date, 19 types of phototrophic consortia are distinguished based on the different 16S rRNA gene sequences of their epibionts, but the diversity and phylogenetic relationships of the heterotrophic partner bacteria are still unknown. We developed an approach based on the specific rrn (ribosomal RNA) operon structure of the central bacterium of 'C. aggregatum' to recover 16S rRNA gene sequences of other central bacteria and their close relatives from natural consortia populations. Genomic DNA of the central bacterium of 'C. aggregatum' was first enriched several hundred-fold by employing a selective method for growth of consortia in a monolayer biofilm followed by a purification of the genome of the central bacterium by cesium chloride-bisbenzimidazole equilibrium density gradient centrifugation. A combination of inverse PCR, cloning and sequencing revealed that two rrn operons of the central bacterium are arranged in a tandem fashion and are separated by an unusually short intergenic region of 195 base pairs. This rare gene order was exploited to screen various natural microbial communities by PCR. We discovered a diverse and previously unknown subgroup of Betaproteobacteria in the chemoclines of freshwater lakes. This group was absent in other freshwater and soil samples. All the 16S rRNA gene sequences recovered are related to that of the central bacterium of 'C. aggregatum'. Fluorescence in situ hybridization indicated that two of these sequences originated from central bacteria of different phototrophic consortia, which, however, were only distantly related to the central bacterium of 'C. aggregatum'. Based on a detailed phylogenetic analysis, these central bacterial symbionts of phototrophic consortia have a polyphyletic origin.

Chlorobium chlorochromatii sp. nov., a symbiotic green sulfur bacterium isolated from the phototrophic consortium "Chlorochromatium aggregatum".

A symbiotic green sulfur bacterium, strain CaD, was isolated from an enrichment culture of the phototrophic consortium "Chlorochromatium aggregatum". The capability of the epibiont to grow in pure culture indicates that it is not obligately symbiotic. Cells are Gram-negative, nonmotile, rod-shaped and contain chlorosomes. Strain CaD is obligately anaerobic and photolithoautotrophic, using sulfide as electron donor. Acetate and peptone are photoassimilated in the presence of sulfide and hydrogencarbonate. Photosynthetic pigments contain bacteriochlorophylls a and c, and gamma-carotene and OH-gamma-carotene glucoside laurate as the dominant carotenoids. In cells from pure cultures, chlorosomes are equally distributed along the inner face of the cytoplasmic membrane. In contrast, the distribution of the chlorosomes in symbiotic epibiont cells is uneven, with chlorosomes being entirely absent at the site of attachment to the central bacterium. The symbiotic epibiont cells display a conspicuous additional layered structure at the attachment site. The G + C content of genomic DNA of strain CaD is 46.7 mol%. On the basis of 16S rRNA sequence comparison, the strain is distantly related to Chlorobium species within the green sulfur bacteria phylum (

Molecular characterization of the nonphotosynthetic partner bacterium in the consortium "Chlorochromatium aggregatum".

Phototrophic consortia represent valuable model systems for the study of signal transduction and coevolution between different bacteria. The phototrophic consortium "Chlorochromatium aggregatum" consists of a colorless central rod-shaped bacterium surrounded by about 20 green-pigmented epibionts. Although the epibiont was identified as a member of the green sulfur bacteria, and recently isolated and characterized in pure culture, the central colorless bacterium has been identified as a member of the beta-Proteobacteria but so far could not be characterized further. In the present study, "C. aggregatum" was enriched chemotactically, and the 16S rRNA gene sequence of the central bacterium was elucidated. Based on the sequence information, fluorescence in situ hybridization probes targeting four different regions of the 16S rRNA were designed and shown to hybridize exclusively to cells of the central bacterium. Phylogenetic analyses of the 1,437-bp-long sequence revealed that the central bacterium of "C. aggregatum" represents a so far isolated phylogenetic lineage related to Rhodoferax spp., Polaromonas vacuolata, and Variovorax paradoxus within the family Comamonadaceae. The majority of relatives of this lineage are not yet cultured and were found in low-temperature aquatic environments or aquatic environments containing xenobiotica or hydrocarbons. In CsCl-bisbenzimidazole equilibrium density gradients, genomic DNA of the central bacterium of "Chlorochromatium aggregatum" formed a distinct band which could be detected by quantitative PCR using specific primers. Using this method, the G+C content of the central bacterium was determined to be 55.6 mol%.