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.