Expression‐based identification of genetic determinants of the bacterial symbiosis ‘Chlorochromatium aggregatum’

Abstract

SummaryThe phototrophic consortium 'Chlorochromatium aggregatum' is a highly structured association of green sulfur bacterial epibionts surrounding a central, motile bacterium and is the most specific symbiosis currently known between two phylogenetically distinct bacterial species. Genes and gene products potentially involved in the symbiotic interaction were identified on the genomic, transcriptomic and proteomic level. As compared with the 11 available genomes of free-living relatives, only 186 open reading frames were found to be unique to the epibiont genome. 2-D differential gel electrophoresis (2-D DIGE) of the soluble proteomes recovered 1612 protein spots of which 54 were detected exclusively in consortia but not in pure epibiont cultures. Using mass spectrometry analyses, the 13 most intense of the 54 spots could be attributed to the epibiont. Analyses of the membrane proteins of consortia, of consortia treated with cross-linkers and of pure cultures indicated that a branched chain amino acid ABCtransporter binding protein is only expressed in the symbiotic state of the epibiont. Furthermore, analyses of chlorosomes revealed that an uncharacterized 11 kDa epibiont protein is only expressed during symbiosis. This protein may be involved in the intracellular sorting of chlorosomes. Application of a novel prokaryotic cDNA suppression subtractive hybridization technique led to identification of 14 differentially regulated genes, and comparison of the transcriptomes of symbiotic and free-living epibionts indicated that 328 genes were differentially transcribed. The three approaches were mostly complementary and thereby yielded a first inventory of 352 genes that are likely to be involved in the bacterial interaction in 'C. aggregatum'. Notably, most of the regulated genes encoded components of central metabolic pathways whereas only very few (7.5%) of the unique 'symbiosis genes' turned out to be regulated under the experimental conditions tested. This pronounced regulation of central metabolic pathways may serve to fine-tune the symbiotic interaction in 'C. aggregatum' in response to environmental conditions.

Metabolic analysis of Chlorobium chlorochromatii CaD3 reveals clues of the symbiosis in ‘Chlorochromatium aggregatum’

“…From this initial reconstruction, only 382 reactions remained after manual curation (Supplementary Material 1). As mentioned above, several enzymes related to amino acid and nitrogen metabolism change their expression profile between free-living and symbiotic states (Wenter et al, 2010). To better understand the metabolic changes related to both states, we selected those reactions belonging to amino acid metabolism for further EFM analysis.…”

“…Experimental data showed that GS was present only in symbiosis (Wenter et al, 2010). Accordingly, the motifs obtained with ACoM-c software showed that the EFMs were clearly different in hotspots of NH 3 assimilation between symbiotic and free-living states (Figure 2).…”

“…chlorocromatii CaD3 changes between symbiosis and free-living states. Wenter et al (2010) and Overmann (2010) postulate that this bacterium in symbiosis is in a condition of low nitrogen availability, where the assimilation of ammonia becomes fundamental. The shift in the metabolic networks observed in our analysis supports this hypothesis.…”

“…These reactions were identified following the metabolic pathway classification of KEGG database. Then, based on the expression profiles published by Wenter et al (2010), we removed with YANA software 5 and 10 reactions not transcribed in symbiotic and free-living states respectively, thus generating two sub-networks: one with 94 reactions corresponding to the symbiotic state and the other with 89 reactions corresponding to the free-living state (see Supplementary Material 1, 2 and 3).…”

“…It showed that B350 genes of Chl. chlorochromatii CaD3 have differential expression patterns between both states (Wenter et al, 2010). A significant fraction of these correspond to genes involved in nitrogen and amino acid metabolism.…”