Podcasts about chlorobium

Look out your window at that beautiful tree or shrub nearby. Now imagine it living in a hot springs at over 50C doing photosynthesis without oxygen as a byproduct but rather by excreting elemental sulfur. Kerry Vickers from the 2019 Hiram College Genetics course tells us about a microbial anaerobic thermophilic phototroph, Chlorobium tepidum strain TLS, that does just that.

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.

This study focuses on one brown-colored representative of the green sulfur bacteria, Chlorobium sp. BS-1, that survives by means of anoxygenic photosynthesis even at very low light intensities. This unusual representative of the green sulfur bacteria lives in the chemocline of the Black Sea, which is located at 80 to 120 m depth, offering only 0.0005 % (winter) to 0.002 % (summer) of surface irradiance (0.00075 to 0.0022 µmol Quanta m-2 s-1). The Black Sea represents the world’s biggest anoxic water body, and it is permanently stratified. An overview of the habitat Black Sea and the research on phyotosynthetic bacteria in the Black Sea chemocline gives the article Anoxygenic phototrophic bacteria in the Black Sea chemocline (pages 53 ff.) In the second article, Physiology and phylogeny of green sulfur bacteria forming a monospecific phototrophic assemblage at 100 m depth in the Black Sea (pages 75 ff.), it is shown that Chl. sp. BS-1 represents a novel phylotype in the marine cluster of green sulfur bacteria and is the only detectable phylotype of green sulfur bacteria in the Black Sea chemocline. It was shown that Chl. sp. BS-1 is the first organism known to date which fixes 14CO2 under laboratory conditions even at light intensities as low as 0.015 µmol Quanta m-2 s-1 which is much lower than the light intensity in any typical habitat of green sulfur bacteria. Therefore it is the best adapted species to extremely low light intensities documented. The major adaptive mechanism to extremely low light intensities might be a significant change in the secondary homologs of the main photosynthetic pigment, bacteriochlorophyll e (BChl e). The dominance of farnesyl esters and the presence of four unusual geranyl ester homologs of BChl e were revealed by HPLC analysis in cells shifted from 3 to 0.1 µmol Quanta m-2 s-1. Together with in situ light measurements and in situ BChl e concentrations, the growth experiments allowed for the calculation of doubling times and significance of the photosynthetic activity for the carbon and sulfur cycle in the Black Sea chemocline. With doubling times of a minimum of 3.1 years (for summer light intensity) and the contribution of only 0.002 - 0.01 % to total sulfide oxidation in the chemocline Chl. sp. BS-1 represents the slowest growing population of green sulfur bacteria known to date and does not contribute significantly to the carbon or sulfur cycle. The article Subfossil DNA sequences of green sulfur bacteria as indicators for past water column anoxia in the Black Sea (page 119 ff.) gives insight into the history of the strain Chl. sp. BS-1 and the green sulfur bacteria in the Black Sea during the last few thousand years. The Black Sea today is considered as closest contemporary analogue to past sulfidic oceans and its biogeography over several thousand years is well documented in its stratified sedimentary record. Since the 16S rRNA gene sequence of Chl. sp. BS-1 might be a useful indicator for past photic zone anoxia, the presence of its fingerprints in past periods of the Black Sea was investigated. 16S rRNA gene sequences of green sulfur bacteria from samples of Black Sea sediments up to 7 m below seafloor were amplified and sequenced. Nine green sulfur bacterial 16S rRNA gene sequences were identified. Surprisingly, not only green sulfur bacterial fingerprints were found but also closely related species clustering at the basis of the green sulfur bacterial subtree, together with not yet cultured species detected all over the world. The new cluster was called “deep-branching green sulfur bacteria” though it was not possible to enrich live organisms with medium for photosynthetic green sulfur bacteria. The chemocline strain Chl. sp. BS-1, found in Units III, II and I (>9000 years b.p. until today), and two other sequences, found in sediments of Unit IIb (between 8200 yr. b.p. and 5000 yr.b.p) only, were the only two sequences clustering with the marine green sulfur bacteria. The other green sulfur bacterial sequences clustered with freshwater or low-salt adapted species, indicating an allochthonous introduction of these sequences to the Black Sea sediments. A long-distance transport of green sulfur bacteria even through oxygenated water is possible since the group has protective mechanisms agains oxygen intoxication. In the article An obligately photosynthetic bacterial anaerobe from a deep-sea hydrothermal vent (pages 105 ff.) a strain is presented which was enriched from beneath a deep sea hydrothermal vent, Chlorobium bathyomarinum GSB1. It was shown to resist more than 16 days of oxygenated medium. Since this strain was isolated only from the vicinity of a vent and was shown to depend on photosynthesis, it might survive photosynthetically by exclusively using geothermal radiation. It might be the first green sulfur bacterium which is adapted to light intensities even lower than in the Black Sea chemocline. A lot of newly obtained sequences during this study evoked the necessity of a phylogenetic analysis. The article Biodiversity and Phylogeny of the family Chlorobiaceae based on comparison of multiple genetic regions (pages 145 ff.) discusses the phylogenetic system of the green sulfur bacteria, which is to date not satisfactory with respect to resolution of the group. A new system is presented which is based on green sulfur bacterial sequences of isolated strains and environmental sequences from the NCBI database (www.ncbi.nlm.nih.gov). The 16S rRNA gene phylogenetic tree revealed the need for a revised phylogenetic system of green sulfur bacteria and showed a close correlation of ecology, i.e. the habitat of the respective species, and phylogeny. Consequently, functional genes might reflect the adaptation to different habitats better than 16S rRNA gene sequences. Three different markers were used for an alternative phylogenetic analysis: ITS (16S-23S rRNA intergenic spacer region), bchG, and sigma factor A, respectively. Only sigma factor A showed a significantly higher resolution of the phylogenetic system of green sulfur bacteria and should be considered as more powerful tool than the16S rRNA gene to classify green sulfur bacteria.

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.

The specific Bchl a and c content of the vitamin B12-dependent Chlorobium limicola strain 1230 decreased strongly under vitamin B12 limitation. In comparison to a regularly grown culture (20 g vitamin B12/l) the specific Bchl c content of a B12-limited culture was reduced to 20% and the specific Bchl a content to 42%. By ultrathin sections it could be clearly demonstrated that B12-deficient cells contained no chlorosomes. After the addition of vitamin B12 to a deficient culture, chlorosomes were formed and the Bchl a and c content increased again to the level of regularly grown cells. The brown-colored Chlorobium phaeobacteroides strain 2430 (type strain) and the extremely low-light-adapted strain MN1 were compared with respect to the influence of light on the formation of chlorosomes and the Bchl e and carotenoid content. By ultrathin sections it could be demonstrated that strain MN1 produced two-fold larger chlorosomes. Chlorosome dimensions of strain MN1 decreased with increasing light intensities. The number of chlorosomes per cell in both strains did not change with different light intensities. Strain MN1 formed twice as much Bchl e as the type strain when grown at 30 or below 1 mol · m-2 · s-1. Under comparable light conditions strain MN1 formed 14–57% more carotenoids than the type strain. Low light intensities aaused the carotenoid content to increase by 25% in strain 2430 in comparison to high light intensity.