Chlorochromatium aggregatum

Episode von Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 03/06 von Ludwig-Maximilians-Universität München · 18. Februar 2008

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.

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