Characterization of the symbiotic bacterial partners in phototrophic consortia

Bacterial interactions play a major role in nature, but are poorly
understood, because of the lack of adequate model systems.
Phototrophic consortia represent the most highly developed type of
interspecific bacterial association due to the precise spatial
arrangement of phototrophic green sulfur bacteria (GSB) around a
heterotrophic central bacterium. Therefore, they are valuable model
systems for the study of symbiosis, signal transduction, and
coevolution between different bacteria. This thesis summarizes a
series of laboratory experiments with the objective of elucidating
the molecular, physiological and phylogenetical properties of the
two bacterial partners in the symbiotic phototrophic consortium
"Chlorochromatium aggregatum". The central bacterium of “C.
aggregatum” had been identified as a Betaproteobacterium, however,
it could not be characterized further due to the low amount of
consortia in enrichment cultures. In this work a suitable method
for enrichment and isolation of DNA of the central bacterium of "C.
aggregatum" has been established using cesium
chloride-bisbenzimidazole equilibrium density gradient
centrifugation (Chapter 3). In density gradients, genomic DNA of
the central bacterium of “C. aggregatum” formed a distinct band,
which could be detected by real-time PCR. Using this method, the
GC-content of the central bacterium was estimated to be 55.6%.
Furthermore, its precise phylogenetic position was determined and
it was shown to represent a novel and phylogenetically isolated
lineage of the Comamonadaceae within the -subgroup of the
Proteobacteria. Chapter 4 describes the detection of a new, highly
diverse subcluster of Betaproteobacteria, which contains several
central bacteria of phototrophic consortia. Genomic DNA of the
central bacterium of “C. aggregatum” was enriched several hundred
fold by employing a selective method for growth of consortia in a
monolayer biofilm followed by a purification of the central
bacterial genome by density gradient centrifugation. A combination
of molecular methods revealed that two rrn operons of the central
bacterium are arranged in a tandem fashion. This rare gene order
was exploited to screen various natural microbial communities. A
diverse and previously unknown subgroup of Betaproteobacteria was
discovered in the chemocline of Lake Dagow, Eastern Germany. All
16S rRNA gene sequences recovered are related to that of the
central bacterium of “C. aggregatum”. Phylogenetic analyses showed,
that the central, chemotrophic symbionts of phototrophic consortia
have a polyphyletic origin, just like their phototrophic
counterparts. This indictates that not only different GSB but also
different Betaproteobacteria have adapted to life in this type of
symbiosis. Chapter 5 focuses on the isolation of the epibiont of
“C. aggregatum” from a consortia enrichment culture and its
description as Chlorobium chlorochromatii strain CaD. It represents
a novel species within the genus Chlorobium and is characterized by
physiological properties typical for GSB. However, the symbiotic
strain differs from free-living GSB in the distribution of its
chlorosomes and the presence of a conspicuous additional structure
at the attachment-site to the central bacterium. Its capability to
grow in pure culture indicates that it is not obligately symbiotic.
The natural habitat of GSB and phototrophic consortia is the
chemocline of stratified lakes. Therefore, the physiological
response to oxygen exposure of the epibiont and the free-living GSB
Chlorobium limicola has been investigated (Chapter 6). It was shown
that GSB are able to survive oxygen exposure and have developed
several strategies for oxygen detoxification. Genome annotation
revealed the presence of several enzymes involved in oxygen
detoxification in all currently sequenced GSB genomes. Phylogenetic
analyses showed that most of these enzymes likely were present in
the common ancestor of this group. The activity of some of those
enzymes could be confirmed. Since carotenoids also act as
antioxidants, the carotenoid composition of the epibiont was
investigated. In contrast to all other GSB it lacks chlorobactene,
the major carotenoid in green-coloured GSB. In addition,
7,8-dihydro--carotene has been identified in the epibiont as a
novel carotenoid in nature. Substantial progress has been made in
the course of this study not only with the establishment of a
method facilitating genome sequencing of the central bacterium of
“C. aggregatum”, but also with the developement of a molecular
screening tool for central bacteria of phototrophic consortia. The
resulting sequences will enable the direct comparison of the
phylogeny of both bacterial partners in different phototrophic
consortia and hence will provide the unique opportunity to assess
for the first time the process of the coevolution of a
bacteria-bacteria-symbiosis.