Chlorochromatium aggregatum

The epibiont of the phototrophic consortium “Chlorochromatium aggregatum” was isolated in pure culture. This was the first time that a symbiotic green sulfur bacterium was isolated in pure culture indicating, that the symbiosis is not an obligate one with respect to the green sulfur bacterium. The phylogenetic affiliation revealed that the epibiont belongs to the genus Chlorobium, accordingly the isolate was named Chlorobium chlorochromatii strain CaD. The cells were gram-negative, nonmotile, rod-shaped, and contained chlorosomes. Strain CaD is obligately anaerobic and photolithoautotrophic, using sulfide as electron donor. Physiologically Chlorobium chlorochromatii exhibited no conspicuous differences to free-living green sulfur bacteria. The limited number of substrates photoassimilated was the same like in other green sulfur bacteria. The pH optimum was slightly shifted to the alkaline in contrast to free-living green sulfur bacteria, which probably represents an adaptation to the symbiotic association with the central bacterium. Photosynthetic pigments were bacteriochlorophylls a and c, and γ-carotene and OH-g-carotene glucoside laurate as dominant carotenoids. The unusual carotenoid composition for green sulfur bacteria indicates a different carotenoid biosynthesis in Chl. chlorochromatii in comparison to other green sulfur bacteria. 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 (≤ 94.6 % sequence homology). The pure culture of Chl. chlorochromatii enabled further studies on the molecular basis of the bacterial symbiosis of “C. aggregatum”. Suppression subtractive hybridization (SSH) against 16 free-living green sulfur bacteria revealed three different sequences unique to Chl. chlorochromatii. Dot blot analysis confirmed that these sequences are only present in Chl. chlorochromatii and did not occur in the free-living relatives. Based on the sequence information, the corresponding open reading frames in the genome sequence of Chl. chlorochromatii could be identified. Whereas the large ORF Cag0616 showed rather low similarity to a hemaglutinin, ORF Cag1920 codes for a putative calcium-binding hemolysin-type protein. The gene product of ORF Cag1919 is a putative RTX-like protein. Reverse transcriptase PCR of RNA isolated from free-living and symbiotic Chl. chlorochromatii demonstrated that all three ORFs are transcribed constitutively. The C-terminal amino acid sequence of Cag1919 comprises six repetitions of the consensus motif GGXGXD and is predicted to form a Ca2+ binding beta roll structure. The RTX-type protein is most likely involved in cell-cell-adhesion within the phototrophic consortium. 45Ca autoradiography exhibited calcium-binding proteins inthe membrane fraction of Chl. chlorochromatii in the free-living as well as the symbiotic state. On the other hand, Ca2+ binding proteins were absent in the cytoplasm of Chl. chlorochromatii and in both fractions of Chlorobaculum tepidum. The proteins detected by autoradiography were considerably smaller in size than predicted from the size of ORF Cag1919. The amino acid sequence of the RTX-type C-terminus coded by Cag1919 is similar to those of a considerable number of RTX-modules in various proteobacterial proteins, suggesting that this putative symbiosis gene has been acquired via horizontal gene transfer from a proteobacterium. An improved cultivation method to selectively grow intact consortia in a monolayer biofilm was the precondition for understanding the complex interaction between epibionts and the central bacterium on the morphological basis. Therefore detailed ultrastructural investigations combining high resolution analytical SEM, TEM, 3D reconstruction and image analysis were performed to provide a structural model for phototrophic consortia. The coherence of the consortia is most likely achieved by long carbohydrate chains of lipopolysaccharides which interconnect mainly the epibionts and to some extent the central bacterium. Numerous periplasmic tubules, formed from the outer membrane of the central bacterium are in direct contact to the epibionts, resulting in a common periplasmic space which is interpreted to be important for exchange of substances. In the epibionts the attachment site to the central bacterium is characterized by absence of chlorosomes and a single contact layer (epibiont contact layer, ECL) with a thickness of 17 nm attached to the inner side of the cytoplasmic membrane of each epibiont. The ECL is also observed in pure cultures of the epibiont, however, only in about 10-20% of the cells. A striking feature of the central bacterium is the occurrence of hexagonally packed flat crystals (central bacterium crystal, CBC) which are variable in size (up to 1 μm long) and in number (statistically, 1.5 per cell), and are formed by bilayers of subunits with a spacing of 9 nm. Deducing from serial sections, the CBC is interpreted to derive from accumulation of subunits on the inner side of the cytoplasmic membrane (or membranous invaginations), first forming a monolayer (central bacterium membrane layer; CML) and subsequently forming a bilayer of 35 nm, which can be freely orientated within the cytoplasm (CBC). Comparing structural details with published data, the CBC resembles a chemosensor

Genomic analysis reveals key aspects of prokaryotic symbiosis in the phototrophic consortium "<em>Chlorochromatium aggregatum</em>"

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

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 μm 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

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 β-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%

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

Photophysical Properties of the Excited States of Bacteriochlorophyll <i>f</i> in Solvents and in Chlorosomes

Bacteriochlorophyll <i>f</i> (BChl <i>f</i>) is a photosynthetic pigment predicted nearly 40 years ago as a fourth potential member of the <i>Chlorobium</i> chlorophyll family (BChl <i>c</i>, <i>d</i>, and <i>e</i>). However, this pigment still has not been found in a naturally occurring organism. BChl <i>c</i>, <i>d</i>, and <i>e</i> 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 <i>e</i>, two research groups produced mutants that synthesize BChl <i>f</i> 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 <i>f</i> with the application of ultrafast time-resolved absorption and fluorescence spectroscopies. The studies were performed on isolated BChl <i>f</i> in various solvents, at different temperatures, and on BChl <i>f</i>-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”

Abstract 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