Browsing by Person "Fritz-Steuber, Julia"

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    Energy conservation in anaerobic Prevotella bryantii and Prevotella bivia : the role of membrane bound electron transfer complexes
    (2022) Schleicher, Lena; Fritz-Steuber, Julia
    Members of the family Prevotellaceae are Gram-negative, obligate anaerobic bacteria found in animal and human microbiomes, where they participate in the degradation of carbohydrates and peptides. Some Prevotella species are also opportunistic pathogens. In this study, growth requirements and central catabolic reactions of two different Prevotella strains were characterized. First, the energy conservation by Prevotella bryantii was analyzed. P. bryantii is a dominant species in the ruminal microbiome. It was demonstrated, that P. bryantii ferments glucose mainly to acetate and succinate. Furthermore, enzymatic and biochemical studies revealed that P. bryantii membranes harbor fully functional Na+ -translocating NADH:quinone oxidoreductase and quinol:fumarate reductase. It was shown, that electron transfer between these two enzymes occurs in native membranes. The enzymatic activities increased significantly by anoxic membrane preparations. Electron transfer in membrane vesicles was coupled to the build-up of a sodium motive force in P. bryantii. A respiratory chain composed of NQR and QFR in P. bryantii was proposed, which links succinate formation to NADH oxidation and SMF formation. Thus, P. bryantii does not rely solely on substrate-level phosphorylation for energy conservation, but gains additional energy utilizing the Na+-pump, NQR. This increases the overall yield of ATP per consumed glucose molecule. By gel electrophoresis and size exclusion chromatography, the existence of a supercomplex composed of NQR and QFR in P. bryantii membranes was demonstrated, which operates as sodium-translocating NADH:fumarate oxidoreductase. The understanding of the catabolic reactions in the rumen by the ruminal microbiota is important for the optimal nutrition of the ruminant. Our results indicate that P. bryantii plays an important role in the ruminal microbiota. P. bryantii extrudes mainly acetate and succinate as fermentative end-products into the rumen. The latter can be used by other organisms of the ruminal microbiome to metabolize propionate, which is an important nutrient for the ruminant since it enters the pathway of gluconeogenesis, yielding glucose. Prevotella bivia is considered to act as causative agent of human bacterial vaginosis. Growth of P. bivia on glucose was dependent on CO2 and resulted in the production of succinate, malate and acetate. With the help of optical spectroscopy and enzymatic measurements, the presence and activity of NQR and QFR in P. bivia were demonstrated. Electron transfer in membrane vesicles of P. bivia resulted in the build-up of a SMF. Similar to P. bryantii, P. bivia operates NQR and QFR for energy conservation in its membrane, resulting in succinate formation and SMF generation. P. bivia also exhibits high L-asparaginase and aspartate ammonia lyase activities in vitro, catalyzing the conversion of L-asparagine to fumarate and NH4+. These results were confirmed in vivo by growth experiments. Additional L-asparagine in the growth medium led to an elevated production of NH4+ and succinate from fumarate obtained during degradation of L-asparagine. At the same time an inhibitory effect of NH4+ on growth of P. bivia was observed. It is proposed, that amino acid degradation by P. bivia in microbial consortia associated with BV depends on the consumption of ammonium by Gardnerella vaginalis, another typical pathogen found in BV. At the same time, G. vaginalis could provide L-asparagine to P. bivia, strengthening their symbiotic relationship and triggering BV
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    Powerful proteins : structure and function of catalytic subunits of electrogenic NADH:quinone oxidoreductases
    (2013) Steffen, Wojtek; Fritz-Steuber, Julia
    Electrogenic NADH:quinone oxidoreductases are large, membrane-embedded enzyme complexes found in the respiratory chain of prokaryotes and the mitochondria of eukaryotes. They represent the first module of the oxidative phosphorylation system which converts the energy from nutrients into an electrochemical gradient by coupling redox reactions to the translocation of cations across membranes. A long chain of events, such as the synthesis of ATP, ion homeostasis, reactive oxygen species production and bacterial motility depend on the activity of these complexes. Complex I consists of up to 45 subunits and can be found in the inner mitochondrial membrane of eukaryotes and in prokaryotes, where it is called NDH I. We investigated the isolated, hydrophobic ND5 subunit, which shows homologies to cation/proton antiporters, from human or Yarrowia lipolytica complex I. In vivo and biochemical analyses provided data on the cation translocation function and the alteration of function by disease-associated mutations. Taken together with the recently published 3D structure of bacterial complex I, these data allowed us to demonstrate that the ND5 subunit could possibly act as an antiporter module of mitochondrial complex I. Sodium ion translocating NADH:quinone oxidoreductase (Na+-NQR) is an enzyme found in many pathogenic bacteria. It consists of six subunits (NqrA - NqrF) whose 3D structures and enzymatic mechanisms were not known in detail at the time this project was initiated. By using high-resolution X-ray structures and site-directed mutagenesis, combined with biochemical studies, we proposed a model for catalysis and substrate selectivity on the atomic level of the electron input module of the complex, the NADH oxidizing domain of subunit NqrF. Furthermore, we analyzed the binding of silver ions to a cysteine residue in the NADH binding pocket and found that it leads to the inhibition of the Na+-NQR and to the killing of Vibrio cholerae in the nanomolar range. Subunit NqrA forms part of the quinone reductase module. By the use of physicochemical and biochemical methods we identified the herbicide 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) as a quinone antagonist and inhibitor of the Na+-NQR complex and discovered two adjacent quinone binding sites on NqrA.
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    Stressful environments : motility and catecholamine response in Vibrio cholerae
    (2014) Halang, Petra; Fritz-Steuber, Julia
    The human pathogen Vibrio cholerae is able to inhabit a variety of environments. These include especially aquatic ecosystems, but the human intestine as well. V. cholerae is thus tolerant to a wide range of salinity and pH. Motility is achieved by a sodium driven polar flagellum. The affinity for Na+ to run the flagellum is determined by the stator complex PomAB, which is embedded in the cell membrane within the flagellar motor. A critical aminoacid residue for the binding of Na+ is aspartate 23 within the transmembrane helix of PomB. A mutation of this aminoacid residue leads to an immotile phenotype of V. cholerae. It was thus of interest to investigate if other polar or acidic aminoacid residues within PomB are important for the passage of Na+. Two potential candidates are serine at position 26 and aspartate at position 42 of PomB, both aminoacid residues are conserved within sodium driven flagellar stator complexes. To characterize the pathway of Na+ through the PomAB channel, the influence of chloride salts (Na+ and K+) and the pH on the motility of V. cholerae was studied. Motility decreased at elevated pH but increased if a chaotropic chloride salt was added, which excludes a direct Na+ and H+ competition in the process of binding to the conserved PomB D23 residue. Cells expressing the PomB S26A/T or D42N variants lost motility at low Na+ concentrations but regained motility in the presence of 170mM chloride. The swimming speeds of individual cells were also analyzed and revealed that S26 located within the membrane helix of PomB is required to promote very fast swimming of V. cholerae. Loss of hypermotility was observed with the S26T variant of PomB which was partially restored by lowering the pH of the external medium. Modification of PomA and PomB by N,N’-dicyclohexylcarbodiimide indicates the presence of protonated carboxyl groups in the hydrophobic regions of the two proteins. Na+ did not protect PomA and PomB from this modification. It could be demonstrated that the motility of V. cholerae is influenced by the pH and osmolality of the medium and thus, the aminoacid residues – S26 and D42 together with D23 – of PomB have a function in the passage of Na+ into the cell. The H+ rather than the Na+ concentration determines the efficiency of the motor, indicating the presence of a catalytical important hydrogen bond network in the motor channel. It is proposed that D23, S26 and D42 of PomB are part of an ion-conducting pathway formed by the PomAB stator complex. As mentioned above, V. cholerae is a pathogen which settles the human intestine. As other pathogens are able to respond specifically to the stress associated mammalian hormones epinephrine and norepinephrine it was of an interest to investigate the influence of these hormones on growth and motility of V. cholerae. The response to epinephrine and norepinephrine is mediated by the QseC sensor protein. The genome of V. cholerae comprises a gene which is homolog to qseC from E. coli. Growth and swarming of V. cholerae was enhanced in the presence of 0.1mM epinephrine or norepinephrine. qRT-PCR experiments revealed increased expression of the genes encoding the putative sensor kinase qseC and pomB, a component of the flagellar motor complex under the influence of catecholates. HPLC measurements of bacterial supernatant revealed that norepinephrine is completely degraded or metabolized after 48 h in the presence of V. cholerae, concomitant with the appearance of another, unidentified compound. On the other hand, V. cholerae seemed to stabilize epinephrine. After 48 h, 0.46% of the epinephrine added at the beginning of the growth experiment was retained. Again, a yet unidentified compound was detected. The experiments conducted in this work strongly indicate the presence of a catecholate receptor in V. cholerae.