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Vogl, Kajetan (2008): Chlorochromatium aggregatum: molecular basis of a bacterial symbiosis. Dissertation, LMU München: Faculty of Biology
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Abstract

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.