Project description:Electron transfer in respiratory chains generates the electrochemical potential that serves as the energy source for ATP synthesis, solute transport and motility. In eukaryotes and many bacteria, the respiratory chain consists of four electron transport complexes, known as complex I to IV. Respiratory chains of some prokaryotes differ in composition and organization. They can use a wide range of electron donors and acceptors and may have complexes performing the same catalytic reaction. The diversity and apparent redundancy of prokaryotic respiratory chains reflects the versatility and robustness of the organisms. Many of these alternative respiratory chain complexes are either unknown or their structures and mechanisms remain elusive. In this work we describe a 3.9 A cryo-EM structure of the alternative complex III (ACIII) from Rhodothermus marinus, which we demonstrated takes over the role of canonical respiratory complex III (bc1 complex), even though it is structurally unrelated. Our structure reveals that ACIII is an integral membrane protein complex of at least 7 subunits (ActABCDEFH). The periplasmic domain, with ActA, B, E and H, harbours four iron-sulphur clusters and six C-type hemes. The cofactors form two electron wires that converge on the putative quinol-binding site in subunit ActC. The two homologous subunits, ActC and ActF, each have two four-helix bundles in the membrane, with several conserved polar residues that delineate putative proton channels. ACIII meets all requirements for an energy-transducing machine that couples electron transfer from quinol to the oxygen reductase, to translocation of protons across the membrane.
Project description:Rhodothermus marinus, a marine aerobic thermophile, was first isolated from an intertidal hot spring in Iceland. In recent years, the R. marinus strain PRI 493 has been genetically modified, which opens up possibilities for targeted metabolic engineering of the species, such as of the carotenoid biosynthetic pathway. In this study, the carotenoids of the R. marinus type-strain DSM 4252T , strain DSM 4253, and strain PRI 493 were characterized. Bioreactor cultivations were used for pressurized liquid extraction and analyzed by ultra-high performance supercritical fluid chromatography with diode array and quadropole time-of-flight mass spectrometry detection (UHPSFC-DAD-QTOF/MS). Salinixanthin, a carotenoid originally found in Salinibacter ruber and previously detected in strain DSM 4253, was identified in all three R. marinus strains, both in the hydroxylated and nonhydroxylated form. Furthermore, an additional and structurally distinct carotenoid was detected in the three strains. MS/MS fragmentation implied that the mass difference between salinixanthin and the novel carotenoid structure corresponded to the absence of a 4-keto group on the ß-ionone ring. The study confirmed the lack of carotenoids for the strain SB-71 (ΔtrpBΔpurAcrtBI'::trpB) in which genes encoding two enzymes of the proposed pathway are partially deleted. Moreover, antioxidant capacity was detected in extracts of all the examined R. marinus strains and found to be 2-4 times lower for the knock-out strain SB-71. A gene cluster with 11 genes in two operons in the R. marinusDSM 4252T genome was identified and analyzed, in which several genes were matched with carotenoid biosynthetic pathway genes in other organisms.
Project description:This work presents an evaluation of batch, fed-batch, and sequential batch cultivation techniques for production of R. marinus DSM 16675 and its exopolysaccharides (EPSs) and carotenoids in a bioreactor, using lysogeny broth (LB) and marine broth (MB), respectively, in both cases supplemented with 10 g/L maltose. Batch cultivation using LB supplemented with maltose (LBmalt) resulted in higher cell density (OD620 = 6.6) than use of MBmalt (OD620 = 1.7). Sequential batch cultivation increased the cell density threefold (OD620 = 20) in LBmalt and eightfold (OD620 = 14) in MBmalt. In both single and sequential batches, the production of carotenoids and EPSs using LBmalt was detected in the exponential phase and stationary phase, respectively, while in MBmalt formation of both products was detectable in both the exponential and stationary phases of the culture. Heteropolymeric EPSs were produced with an overall volumetric productivity (QE) of 0.67 (mg/L h) in MBmalt and the polymer contained xylose. In LB, QE was lower (0.1 mg/L h) and xylose could not be detected in the composition of the produced EPSs. In conclusion, this study showed the importance of a process design and medium source for production of R. marinus DSM 16675 and its metabolites.