Interspecies interaction and diversity of green sulfur bacteria

The following work is shedding light on the phylogenetic
classification on the family of the Chlorobiacea, the members of
which are showing signs of preadaptation to symbiosis. Symbioses
consisting of purely prokaryotic associations between
phylogenetically distinct bacterial species have been widely
documented. Only few are available as a laboratory culture to
elucidate the molecular basis of their interaction. One of these
few model organisms is the phototrophic consortium
“Chlorochromatium aggregatum”. It consists of 12-20 green sulfur
bacteria epibionts surrounding a central, Betaproteobacterium in a
highly ordered fashion. The phototrophic partner bacterium,
belonging to the green sulfur bacteria, is available in pure
culture and its physiology has been studied in detail. In this
work, novel insights into the physiology of the central bacterium
that was previously uncharacterized are provided. The family of the
Chlorobiaceae represents a phylogenetically coherent and isolated
group within the domain Bacteria. Green sulfur bacteria are
obligate photolithoautotrophs that require highly reducing
conditions for growth and can utilize only a very limited number of
carbon substrates. These bacteria thus inhabit a very narrow
ecologic niche. For the phylogenetic studies on green sulfur
bacteria, 323 16S rRNA gene sequences, including cultured species
as well as environmental sequences were analysed. By rarefaction
analysis and statistical projection, it was shown that the data
represent nearly the whole spectrum of green sulfur bacterial
species that can be found in the sampled habitats. Sequences of
cultured species, however, did not even cover half of the
biodiversity. In the 16S rDNA gene tree, different clusters were
found that in most cases correlated with physiological adaptations
of the included species. By combining all sampling sites of green
sulfur bacteria in a world map, large, unsampled areas were
revealed and it could be shown that in some regions, a non-random
distribution of GSB occurred. The wide dispersal of green sulfur
bacterial species can be seen in sequences that were found
ubiquitously all over the world. To imitate the phylogenetical
relationships of whole genome analyses, a concatenated tree was
constructed including 32 species and 3 different genetic regions,
the bchG gene, the sigA gene and the fmoA gene. Comparison with the
16S rRNA gene tree showed more genetic differences between species,
and led to a higher resolution and a more dependable phylogeny. A
distance matrix comparison showed that the fmoA gene sequence has
the highest correlation to the 16S rDNA of the sequences
investigated. Additionally, a dissimilarity matrix revealed that
the fmoA gene sequence provides the highest phylogenetic resolution
among the sequences investigated. Therefore, we showed that the
fmoA gene sequence is the most suitable among the sequences
investigated to support the 16S rDNA phylogeny of green sulfur
bacteria. To overcome the limitation of immobility, some green
sulfur bacteria have entered into a symbiosis with motile
Betaproteobacteria in a type of multicelllular association termed
phototrophic consortia. Recent genomic, transcriptomic, and
proteomic studies of "C. aggregatum" and its epibiont provided
insights into the molecular basis and the origin of the stable
association between the two very distantly related bacteria.
However, to date the possibility of a metabolic coupling between
the bacterial partners has not been investigated. The symbiotic
exchange of metabolites between the two species was therefore
investigated by tracking the flux of isotope-labeled CO2 through
the two partner organisms using NanoSIMS analysis and magnetic
capture, revealing a fast and simultaneous incorporation of labeled
carbon into both organisms. The transferred metabolites were
identified by isotopologue profiling for which the partner cells
were separated by cesium chloride density gradient centrifugation,
a method which identified amino acids as one group of substrates to
be transferred between the two partners. The addition of external
carbon substrates inhibited the transfer between the two partners,
suggesting that transporters are the means by which substrates are
exchanged. Genome sequencing revealed the central bacterium to be
an aerobic or microaerophilic chemoheterotrophic bacterium. The
existence of 32 PAS domains which are responsible for sensing
various signals indicate that the central bacterium is responsible
for the chemo- and phototactic responses of the consortium. The
central bacterium possesses all traits of an autonomous organism.
However, transcriptome analysis revealed the central bacterium to
be inactive in the dark although external carbon sources were
present. Thereby, a yet unexplained dependence on the epibiont is
revealed which indicates a complex metabolic coupling between the
two symbiotic partner organisms.