Mechanismen der bakteriellen Interaktion in phototrophen Konsortien

Bacterial interactions play an important role in nature in which so-called consortia appear to represent the most developed type of bacterial interaction between non-related prokaryotes which maintain permanent cell-to-cell contact. A valuable model system for studying symbiosis is the phototrophic consortium “Chlorochromatium aggregatum”, which can be maintained in a laboratory culture. “C. aggregatum” consists of green sulfur bacteria; Chlorobium chlorochromatii also termed epibionts, which surround a central, Betaproteobacterium in a highly structured arrangement. This thesis summarizes laboratory experiments which had the objective to elucidate the molecular and physiological mechanisms of the bacterial interaction in the consortium “C. aggregatum”. Previously described putative symbiotic proteins which are unique to Chl. chlorochromatii compared to 11 genomes of free-living relatives and contain virulence factors, were localized within the consortium. Polyclonal antibodies were produced targeting the protein products of Cag_0614, Cag_0616 and Cag_1919. The expression of these three symbiotic proteins in the epibiont and the subsequent transfer to the central bacterium could be shown. Therefore proteins containing virulence factors are involved in the symbiotic relationship between two different bacteria. Metabolic coupling between the two partner bacteria in the consortium was investigated by tracking the flux of isotope-labelled carbon and nitrogen through the two partner organisms using nanoSIMS analysis. Measurements showed a simultaneous transfer of labelled carbon from the epibiont to the central bacterium, indicating the transfer of newly synthesized small organic matter instead of macromelcules. Depending on the nitrogen availability of the epibiont cell, the flux of carbon changed. Measurements also showed that consortia are not likely to grow solely on nitrogen gas, although the association with bacteria capable of nitrogen fixation was considered a competitive advantage for the central bacterium. The effect of zaragozic acid on consortia aggregates was tested, due to reports of its ability to inhibit biofilm formation of bacteria. The number of single epibionts and the number of disaggregated consortia in cultivations with zaragozic acid increased. Thus the possible inhibition of squalene synthase was theorized and the implications for the disruption of functional microdomains considered.Bakterielle Interaktionen spielen eine wichtige Rolle in der Umwelt. So genannte Konsortien stellen eine weit entwickelte bakterielle Interaktion zwischen nicht-verwandten Prokaryoten dar und stehen im permanenten Zell-Zell-Kontakt. Ein wertvolles Modellsystem zur Erforschung der Symbiose ist das im Labor kultivierbare phototrophe Konsortium “Chlorochromatium aggregatum”. “C. aggregatum” besteht aus dem grünen Schwefelbakterium Chlorobium chlorochromatii, auch als Epibiont bezeichnet, welche strukturiert um ein zentrales Betaproteobakterium angeordnet sind. Diese Arbeit fasst Laborexperimente zusammen, die die Erforschung der molekularen und physiologischen Mechanismen der bakteriellen Interaktion im Konsortium “C. aggregatum” zum Ziel hatte. Bereits beschriebene, putative Symbioseproteine sind, im Vergleich zu 11 weiteren Genomen von freilebenden Verwandten, einzigartig für Chl. chlorochromatii und enthalten Virulenzfaktoren. Polyklonale Antikörper wurden für die Lokalisierung von Cag_0614, Cag_0616 und Cag_1919 innerhalb des Konsortiums hergestellt. Die Expression der drei Symbioseproteine im Epibionten sowie der Transfer zum Zentralbakterium wurden gezeigt. Somit sind Proteine mit Virulenzfaktoren an der symbiotischen Beziehung zwischen zwei verschiedenen Bakterien beteiligt. Die metabolische Kopplung zwischen den beiden Partnerorganismen wurde untersucht, indem der Fluss von Isotop markiertem Kohlenstoff und Stickstoff mittels nanoSIMS Analyse verfolgt wurde. Die Messungen zeigten einen simultanen Transfer des markierten Kohlenstoffes vom Epibionten zum Zentralbakterium, welches auf den Transfer von neu synthetisierten kleinen organischen Komponenten, anstelle von Makromolekülen, hinweist. Abhängig von der Verfügbarkeit von Stickstoff für die Epibiontenzellen änderte sich dieser Kohlenstofffluss. Konsortien sind außerdem nicht in der Lage allein mit atmosphärischem Stickstoff zu wachsen, obwohl die Assoziation mit stickstofffixierenden Bakterien als ein kompetitiver Vorteil für das Zentralbakterium postuliert wurde. Da Berichten zur Folge Saragossasäure in der Lage ist die Bildung von Biofilmen zu inhibieren, wurde der Effekt dieser Säure auf die Stabilität der Konsortienaggregate getestet. Bei Zugabe von Saragossasäure stieg die Zahl der einzelnen Epibionten und disaggregierten Konsortien an. In Folge dessen wurde die Inhibierung einer Squalensynthase in Betracht gezogen und eine daraus resultierende Störung von funktionellen Mikrodomänen

Carotenoid Production by Corynebacterium: The Workhorse of Industrial Amino Acid Production as Host for Production of a Broad Spectrum of C40 and C50 Carotenoids

Corynebacterium glutamicum is used as a workhorse of industrial biotechnology for more than 60 years since its discovery as a natural glutamate producer in the 1950s. Nowadays, L-glutamate and L-lysine are being produced with this GRAS organism in the million-ton scale every year for the food and feed markets, respectively. Sequencing of the genome and establishment of a genetic toolbox boosted metabolic engineering of this host for a broad range of industrially relevant compounds ranging from bulk chemicals to high-value products. Carotenoids, the colourful representatives of terpenoids, are high-value compounds whose bio-based production is on the rise. Since C. glutamicum is a natural producer of the rare C50 carotenoid decaprenoxanthin, this organism is well suited to establish terpenoid-overproducing platform strains with the help of metabolic engineering strategies. In this work, the carotenogenic background of C. glutamicum and the metabolic engineering strategies for the generation of carotenoid-overproducing strains are depicted

Corynebacterium glutamicum as a platform strain for the production of a broad variety of terpenoids

Corynebacterium glutamicum is a natural carotenoid producing bacterium used in the million-ton-scale amino acid biotechnology that has been engineered for isoprenoid production1. The native membrane-bound carotenoid decaprenoxanthin is a rare C50 carotenoid. Volatile terpenoids such as valencene2 and patchoulol3 could be produced upon deletion of the first step of the specific carotenoid pathway and heterologous expression of the FPP synthase gene ispA from E. coli and terpene synthases from plant origin. However, these strains produced a yet unidentified carotenoid and only when all carotenoid biosynthetic genes were deleted, a colorless strain resulted. Expressing a codon optimized ADS from Artemisia annua in the white strain, amorphadiene, the volatile precursor for artemisinin was produced. For production of volatile terpenoids a dodecane overlay was used, a condition in which C. glutamicum benefits from its robust myco-membrane. Recently, we showed production of membrane-bound carotenoids with different length and/or cyclization status: bicyclic C50 sarcinaxanthin4, bicyclic C40 astaxanthin5, the linear lycopene6 and the linear C50 bisanhydrobacterioruberin7. This indicated that the C. glutamicum myco-membrane accepts these linear and bicyclic carotenoids. Please click Additional Files below to see the full abstract

Corynebacterium glutamicum CrtR and its orthologs in actinobacteria: conserved function and application as genetically encoded biosensor for detection of geranylgeranyl pyrophosphate

Henke NA, Austermeier S, Grothaus IL, et al. Corynebacterium glutamicum CrtR and its orthologs in actinobacteria: conserved function and application as genetically encoded biosensor for detection of geranylgeranyl pyrophosphate. International Journal of Molecular Sciences. 2020;21(15): 5482.Carotenoid biosynthesis in Corynebacteriumglutamicum is controlled by the MarR-type regulator CrtR, which represses transcription of the promoter of the crt operon (PcrtE) and of its own gene (PcrtR). Geranylgeranyl pyrophosphate (GGPP), and to a lesser extent other isoprenoid pyrophosphates, interfere with the binding of CrtR to its target DNA in vitro, suggesting they act as inducers of carotenoid biosynthesis. CrtR homologs are encoded in the genomes of many other actinobacteria. In order to determine if and to what extent the function of CrtR, as a metabolite-dependent transcriptional repressor of carotenoid biosynthesis genes responding to GGPP, is conserved among actinobacteria, five CrtR orthologs were characterized in more detail. EMSA assays showed that the CrtR orthologs from Corynebacteriumcallunae, Acidipropionibacteriumjensenii, Paenarthrobacternicotinovorans, Micrococcusluteus and Pseudarthrobacterchlorophenolicus bound to the intergenic region between their own gene and the divergently oriented gene, and that GGPP inhibited these interactions. In turn, the CrtR protein from C. glutamicum bound to DNA regions upstream of the orthologous crtR genes that contained a 15 bp DNA sequence motif conserved between the tested bacteria. Moreover, the CrtR orthologs functioned in C. glutamicum in vivo at least partially, as they complemented the defects in the pigmentation and expression of a PcrtE_gfpuv transcriptional fusion that were observed in a crtR deletion mutant to varying degrees. Subsequently, the utility of the PcrtE_gfpuv transcriptional fusion and chromosomally encoded CrtR from C. glutamicum as genetically encoded biosensor for GGPP was studied. Combined FACS and LC-MS analysis demonstrated a correlation between the sensor fluorescent signal and the intracellular GGPP concentration, and allowed us to monitor intracellular GGPP concentrations during growth and differentiate between strains engineered to accumulate GGPP at different concentrations

Production of the marine carotenoid astaxanthin by metabolically engineered Corynebacterium glutamicum

Henke NA, Heider S, Peters-Wendisch P, Wendisch VF. Production of the marine carotenoid astaxanthin by metabolically engineered Corynebacterium glutamicum. Marine Drugs. 2016;14(7): 124.Astaxanthin, a red C40 carotenoid, is one of the most abundant marine carotenoids. It is currently used as a food and feed additive in a hundred-ton scale and is furthermore an attractive component for pharmaceutical and cosmetic applications with antioxidant activities. Corynebacterium glutamicum, which naturally synthesizes the yellow C50 carotenoid decaprenoxanthin, is an industrially relevant microorganism used in the million-ton amino acid production. In this work, engineering of a genome-reduced C. glutamicum with optimized precursor supply for astaxanthin production is described. This involved expression of heterologous genes encoding for lycopene cyclase CrtY, β-carotene ketolase CrtW, and hydroxylase CrtZ. For balanced expression of crtW and crtZ their translation initiation rates were varied in a systematic approach using different ribosome binding sites, spacing, and translational start codons. Furthermore, β-carotene ketolases and hydroxylases from different marine bacteria were tested with regard to efficient astaxanthin production in C. glutamicum. In shaking flasks, the C. glutamicum strains developed here overproduced astaxanthin with volumetric productivities up to 0.4 mg·L−1·h−1 which are competitive with current algae-based production. Since C. glutamicum can grow to high cell densities of up to 100 g cell dry weight (CDW)·L−1, the recombinant strains developed here are a starting point for astaxanthin production by C. glutamicum

Isoprenoid pyrophosphate-dependent transcriptional regulation of carotenogenesis in Corynebacterium glutamicum

Henke NA, Heider S, Hannibal S, Wendisch VF, Peters-Wendisch P. Isoprenoid pyrophosphate-dependent transcriptional regulation of carotenogenesis in Corynebacterium glutamicum. Frontiers in Microbiology. 2017;8: 633.Corynebacterium glutamicum is a natural producer of the C50 carotenoid decaprenoxanthin. The crtEcg0722crtBIYEb operon comprises most of its genes for terpenoid biosynthesis. The MarR-type regulator encoded upstream and in divergent orientation of the carotenoid biosynthesis operon has not yet been characterized. This regulator, named CrtR in this study, is encoded in many actinobacterial genomes co-occurring with terpenoid biosynthesis genes. CrtR was shown to repress the crt operon of C. glutamicum since DNA microarray experiments revealed that transcript levels of crt operon genes were increased 10 to 70-fold in its absence. Transcriptional fusions of a promoter-less gfp gene with the crt operon and crtR promoters confirmed that CrtR represses its own gene and the crt operon. Gel mobility shift assays with purified His-tagged CrtR showed that CrtR binds to a region overlapping with the −10 and −35 promoter sequences of the crt operon. Isoprenoid pyrophosphates interfered with binding of CrtR to its target DNA, a so far unknown mechanism for regulation of carotenogenesis. The molecular details of protein-ligand interactions remain to be studied. Decaprenoxanthin synthesis by C. glutamicum wild type was enhanced 10 to 30-fold upon deletion of crtR and was decreased 5 to 6-fold as result of crtR overexpression. Moreover, deletion of crtR was shown as metabolic engineering strategy to improve production of native and non-native carotenoids including lycopene, β-carotene, C.p. 450 and sarcinaxanthin

Genomic analysis reveals key aspects of prokaryotic symbiosis in the phototrophic consortium "<em>Chlorochromatium aggregatum</em>"

BACKGROUND: ‘Chlorochromatium aggregatum’ is a phototrophic consortium, a symbiosis that may represent the highest degree of mutual interdependence between two unrelated bacteria not associated with a eukaryotic host. ‘Chlorochromatium aggregatum’ is a motile, barrel-shaped aggregate formed from a single cell of ‘Candidatus Symbiobacter mobilis”, a polarly flagellated, non-pigmented, heterotrophic bacterium, which is surrounded by approximately 15 epibiont cells of Chlorobium chlorochromatii, a non-motile photolithoautotrophic green sulfur bacterium. RESULTS: We analyzed the complete genome sequences of both organisms to understand the basis for this symbiosis. Chl. chlorochromatii has acquired relatively few symbiosis-specific genes; most acquired genes are predicted to modify the cell wall or function in cell-cell adhesion. In striking contrast, ‘Ca. S. mobilis’ appears to have undergone massive gene loss, is probably no longer capable of independent growth, and thus may only reproduce when consortia divide. A detailed model for the energetic and metabolic bases of the dependency of ‘Ca. S. mobilis’ on Chl. chlorochromatii is described. CONCLUSIONS: Genomic analyses suggest that three types of interactions lead to a highly sophisticated relationship between these two organisms. Firstly, extensive metabolic exchange, involving carbon, nitrogen, and sulfur sources as well as vitamins, occurs from the epibiont to the central bacterium. Secondly, ‘Ca. S. mobilis’ can sense and move towards light and sulfide, resources that only directly benefit the epibiont. Thirdly, electron cycling mechanisms, particularly those mediated by quinones and potentially involving shared protonmotive force, could provide an important basis for energy exchange in this and other symbiotic relationships

Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies

The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of osteogenesis imperfecta (OI) and the Ehlers-Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen-related disease models. Our studies reveal a high degree of intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention

Patchoulol production with metabolically engineered Corynebacterium glutamicum

Henke NA, Wichmann J, Baier T, et al. Patchoulol production with metabolically engineered Corynebacterium glutamicum. Genes. 2018;9(4): 219.Patchoulol is a sesquiterpene alcohol and an important natural product for the perfume industry. Corynebacterium glutamicum is the prominent host for the fermentative production of amino acids with an average annual production volume of ~6 million tons. Due to its robustness and well established large-scale fermentation, C. glutamicum has been engineered for the production of a number of value-added compounds including terpenoids. Both C40 and C50 carotenoids, including the industrially relevant astaxanthin, and short-chain terpenes such as the sesquiterpene valencene can be produced with this organism. In this study, systematic metabolic engineering enabled construction of a patchoulol producing C. glutamicum strain by applying the following strategies: (i) construction of a farnesyl pyrophosphate-producing platform strain by combining genomic deletions with heterologous expression of ispA from Escherichia coli; (ii) prevention of carotenoid-like byproduct formation; (iii) overproduction of limiting enzymes from the 2-c-methyl-d-erythritol 4-phosphate (MEP)-pathway to increase precursor supply; and (iv) heterologous expression of the plant patchoulol synthase gene PcPS from Pogostemon cablin. Additionally, a proof of principle liter-scale fermentation with a two-phase organic overlay-culture medium system for terpenoid capture was performed. To the best of our knowledge, the patchoulol titers demonstrated here are the highest reported to date with up to 60 mg L−1 and volumetric productivities of up to 18 mg L−1 d−1