Podcasts about chlorochromatium

Matters Microbial #76:  Marvelous Multicellular Magnetotactic Microbes! January 29, 2025 Today, Dr. George Schaible, Postdoctoral Scholar at the University of California, Santa Barbara, joins the #QualityQuorum to discuss the exciting work he did during his PhD to unravel a fascinating topic:  multicellular magnetotactic microbes!   Host: Mark O. Martin Guest: George Schaible Subscribe: Apple Podcasts, Spotify Become a patron of Matters Microbial! Links for this episode What is a postdoctoral scholar in microbiology?  A previous #MattersMicrobial podcast about giant bacteria from Dr. Esther Angert.   A previous #MattersMicrobial podcast about magnetotactic bacteria from Dr. Arash Komeili. A previous #MattersMicrobial podcast about multicellularity in microbes from Dr Will Ratcliff.   The transformative Microbial Diversity Course at the Marine Biological Labs at Woods Hole, Massachusetts. An article describing the value of the Microbial Diversity Course.  I wrote this blog post on Chlorochromatium aggregatum consortium for Small Things Considered many years ago.   The “pink berry” consortium at Woods Hole. A research paper on genetic interactions within the pink berry consortium, coauthored by a former undergraduate researcher of mine, Dr. Danielle Campbell.  Yes, I am very proud. A strategy to easily enrich for magnetotactic bacteria from nature. Here is a video that informs and amuses.   An early report of multicellular magnetotactic microbes. The research article under discussion in today's podcast. A link to Dr. Roland Hatzenpichler's laboratory website (Dr. Hatzenpichler was the originator of this research, all the way back to his own attending the Microbial Diversity Course.). The deeply strange genome(s) of Achromatium. An introduction to nanoSIMS technology. An introduction to stable isotope probing. The laboratory website of Dr. Jean-Marie Volland, where Dr. Schaible works at UC Santa Barbara Intro music is by Reber Clark Send your questions and comments to mattersmicrobial@gmail.com

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.

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.

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.

>>Novel bacteriochlorophyll e structures and species-specific variability of pigment composition in green sulfur bacteria>Characterization and in situ carbon metabolism of phototrophic consortia>The significance of organic carbon compounds for in situ metabolism and chemotaxis of phototrophic consortia