1. Physiology of acetogenic bacteria
Homoacetogens are a unique group of strictly anaerobic bacteria which live under anoxic conditions by a process often referred to as carbon dioxide respiration. Hexoses are oxidized, and the reducing equivalents gained during glycolysis and pyruvate oxidation are channeled to the Wood-Ljungdahl pathway, which serves as a sink for electrons thereby reducing two mol of CO2 to acetate. The Wood-Ljungdahl pathway also enables the organisms to grow on one carbon compounds such as methanol or H2 + CO2 (Fig. 1).
During autotrophic growth, the net ATP synthesis by substrate level phosphorylation is zero, and the question is: how is this pathway coupled to energy conservation? Our model organism Acetobacterium woodii is a Na+-dependent organism. During the operation of the Wood-Ljungdahl pathway a primary, electrogenic Na+ potential is generated by a yet to be identified enzyme. We are still seeking the enzyme responsible for the generation of the Na+ potential, but a number of data hint to the involvement of a methyltransferase in Na+ translocation. This is under investigation. The Na+ gradient is used as driving force for flagellar rotation and for ATP synthesis. The ATP synthase has been investigated in detail. It is a member of the F1FO class but it uses Na+ instead of H+ as coupling ion. This makes it a prime candidate for biochemical studies with respect to the mechanism of coupling of ion transport to ATP synthesis/hydrolysis. In contrast to any other known F1FO ATPase, the proteolipid oligomer, the membrane-buried rotor domain of the rotatory nanomachine, is a heterooligomer of 8- and 16-kDa, the first ever found in nature. We currently determine the ratio of the 8- and 16-kDa proteolipids in the oligomer and explore physiological functions of this unusual finding.
In the presence of alternative electron acceptors such as phenyl acrylates, the side chain of these compounds is used as a sink for electrons (Fig. 2).
This reduction is coupled to Na+-dependent energy conservation, and the process is referred to as caffeate respiration. Caffeate respiration by A. woodii is a sodium motive process, and we are currently in the process of identifying the components involved in electron transfer. In particular, the electron input modules (hydrogenase, NADH dehydrogenase), electron carriers, and the hypothetical caffeate reductase are under investigation.
Regulation of electron flow towards CO2 or alternative electron acceptors is a completely unexplored field in acetogens. We have developed the molecular tools to study regulation of electron as well as carbon flow and have just started to analyze gene expression and protein production under different experimental conditions.
Publications 1999 - 2004
Rahlfs, S., Müller, V. (1999) Sequence of subunit a of the Na+-translocating F1FO ATPase of Acetobacterium woodii: proposal for residues involved in Na+ binding. FEBS Lett. 453 : 35 - 40.
Rahlfs, S., Aufurth, S., Müller, V. (1999) The Na+-F1FO ATPase operon from Acetobacterium woodii. Operon structure and presence of multiple copies of atpE which encode proteolipids of 8 and 18 kDa. J. Biol. Chem. 274 : 33999 - 34004.
Aufurth, S., Schägger, H., Müller, V. (2000) Identification of subunits a, b, and c1 from Acetobacterium woodii Na+-F1FO-ATPase: subunits c1, c2, and c3 constitute a mixed c-oligomer. J. Biol. Chem. 275 : 33297 - 33301.
Müller, V., Aufurth, S., Rahlfs, S. (2001) The Na+-cycle in Acetobacterium woodii: identification and characterization of a Na+-translocating F1FO-ATPase with a mixed oligomer of 8 and 16-kDa proteolipids. Biochim. Biophys. Acta 1505 : 108 - 120.
Imkamp, F., Müller V. (2002) Chemiosmotic energy conservation with Na+ as the coupling ion during hydrogen-dependent caffeate reduction by Acetobacterium woodii. J. Bacteriol. 184 : 1947 - 1951.
Müller, V. (2003) Energy conservation in acetogenic bacteria. Appl. Environ. Microbiol. 69 : 6345 - 6353.
Müller, V., Imkamp, F., Rauwolf, A., Küsel, K., Drake, H.L. (2004) Molecular and cellular biology of acetogenic bacteria. In: Strict and facultative anaerobes: medical and environmental aspects (Nakano, M. M., Zuber, P., eds.). Horizon Press, UK.
2. Structure and function of the archaeal A1AO ATP synthase
Archaea are very different with respect to physiology but have in common that they employ chemiosmotic mechanisms for energy conservation. ATP synthases are key enzymes in energy metabolism of archaea, but their structure and function remains to be elucidated. We use methanogenic archaea as model organisms. Methanogenic archaea grow by the conversion of a relatively small number of substrates such as H2 + CO2, methanol or acetate.
The pathway of methane formation (Fig. 3) is coupled to the generation of a primary Na+-potential by the membrane-bound methyltetrahydromethanopterin-coenzyme M methyltransferase, and to the generation of a primary proton gradient by the heterodisulfide reductase reaction. Therefore, methanogens are unique since they produce two primary ion gradients at the same time. Both gradients contribute to ATP synthesis, but the molecular mechanisms are unknown. The genomic sequence revealed that most methanogens contain only one ATP synthase, the archaeal or A1AO ATP synthase. This enzyme is evolutionary closely related to V1VO ATPases from eukarya, but only distantly to F1FO ATPases from bacteria. However, its function is more similar to F1FO because it is used by the cells to synthesize ATP which is in sharp contrast to V1VO ATPases. The molecular basis for this difference is unknown and is explored in our group.
The proteolipid, one of the key components in ion transport in ATPases, was purified from various methanogens. Although most archaea contain a bacterial-type proteolipd (which was always believed to be the reason why the A1AO ATPases work as synthases, despite their structural relationship to V1VO ATPases), we found a duplicated, V1VO-type proteolipid in Methanobacterium thermoautotrophicum, a triplicated version of the bacterial proteolipid in Methanococcus jannaschii and Methanococcus maripaludis, and a proteolipid even thirteen-times the size in Methanopyrus kandleri. This finding led us to propose a new model for the evolution of structure and function of ATPases. Furthermore, the multiplied proteolipids may be useful in structural analyses.
The structure and function of A1AO ATPases (Fig. 4) is largely unknown. We have set up a heterologous expression system which enables us to overproduce the hydrophilic A1 domain and subcomplexes thereof in high amounts. The structure is being determined in the group of Dr. Grüber, Homburg. It turned out that the shape of the A1 molecule is different from V1 and F1. The subunit topology was determined by various biochemical means and revealed that subunit D is part of the central stalk whereas C and F are peripheral. Subunit C is most likely also part of the central stalk, located at the interface to the proteolipid ring in the membrane domain. We have purified an A1AO ATPase from the hyperthermophile Methanococcus jannaschii, the first archaeal ATP synthase that contains all the subunits deduced from the genomic sequence. The structure of this molecule, as determined by electron microscopy in the group of Dr. Grüber, shows two peripheral stalks and a central collar surrounding the central stalk. These new structural features are explored in detail.
We have just recently succeeded in overproducing an entire A1AO ATPase in a bacterial host. This system will be used in future studies to study structure and function of archaeal ATP synthases. One of the most important questions to solve is the ion specificity of the enzyme and the role of Na+ and H+ in ATP synthesis.
Publications 1999 - 2004
Ruppert, C., Kavermann, H., Wimmers, S., Schmid, R., Kellermann, J., Lottspeich, F., Huber, H., Stetter, K. O., Müller, V. (1999) The proteolipid of the A1AO ATP synthase from Methanococcus jannaschii has six predicted transmembrane helices but only two proton-translocating carboxyl groups. J. Biol. Chem. 274 : 25281 - 25284.
Schäfer, G., Engelhard, M., Müller, V. (1999) Bioenergetics of the Archaea. Microbiol. Mol. Biol. Rev. 63 : 570 - 620.
Ruppert, C., Schmid, R., Hedderich, R., Müller, V. (2001) Selective extraction of subunit D of the Na+-translocating methyltransferase and subunit c of the A1AO ATPase from the cytoplasmic membrane of methanogenic archaea by chloroform/methanol and characterization of subunit c of Methanothermobacter thermoautotrophicus as a 16-kDa proteolipid. FEMS Microbiol. Lett. 195 : 47 - 51.
Grüber, G., Svergun, D. I., Coskun, Ü., Lemker, T., Koch, M. H. J., Schägger, H., Müller, V. (2001) Structural insights into the A1TPase from the archaeon Methanosarcina mazei Gö1. Biochemistry 40 : 1890 - 1896.
Lemker, T., Ruppert, C., Stöger, H., Wimmers, S., Müller, V. (2001) Overproduction of a functional A1ATPase from the archaeon Methanosarcina mazei Gö1 in Escherichia coli. Eur. J. Biochem. 268 : 3744 - 3750.
Grüber, G., Wieczorek, H., Harvey, W. R., Müller, V. (2001) Structure-function relationships of A1-, F1- and V1-ATPases. J. Exp. Biol. 204 : 2597 - 2605.
Coskun, Ü., Grüber, G., Koch, M. H. J., Godovac-Zimmermann, J., Lemker, T., Müller, V. (2002) Cross-talk in the A1 ATPase from Methanosarcina mazei Gö1 due to nucleotide binding. J. Biol. Chem. 279 : 17327 - 17333.
Lingl, A., Huber, H., Stetter, K. O., Mayer, F., Kellermann, J., Müller, V. (2003) Isolation of a complete A1AO ATPase synthase comprising nine subunits from the hyperthermophile Methanococcus jannaschii. Extremophiles 7 : 249 - 257.
Lemker, T., Grüber, G., Schmid, R., Müller, V. (2003) Defined subcomplexes of the A1 ATPase from the archaeon Methanosarcina mazei Gö1: biochemical properties and redox regulation. FEBS Lett. 544 : 206 - 209.
Müller, V., Grüber, G. (2003) ATP synthases: structure, function and evolution of unique energy converters. Cell. Mol. Life Sci. 60 : 474 - 494.
Müller, V. (2004) An exceptional variability in the motor of archaeal A1AO ATPases: from multimeric to monomeric rotors comprising 6 - 13 ion binding sites. J. Bioenerg. Biomembr. 36 : 115 - 125.
Coskun, Ü., Radermacher, M., Müller, V., Ruiz, T., Grüber, G. (2004) Three-dimensional organization of the archaeal A1-ATPase from Methanosarcina mazei Gö1. J. Biol. Chem. 279 : 22759 - 64.
Coskun, Ü., Chaban, Y. L., Lingl, A., Müller, V., Boekema, E. J., Grüber, G. (2004) Structure of and subunit arrangement in the A-type ATP synthase complex from the archaeon Methanococcus jannaschii visualized by electron microscopy. J. Biol. Chem., 279 : 38644 - 38648.
Cross, R. L., Müller, V. (2004) The evolution of A-, F-, and V-type ATP synthases and ATPases: reversals in function and changes in the H+/ATP coupling ratio. FEBS Lett. 576 : 1 - 4.
3. Salt adaptation and gene regulation in methanogenic archaea
Archaea have eukaryotic-like transcription machineries but bacterial-like regulators, and the basis for gene regulation is largely unknown. We study gene regulation in methanogenic archaea and use salt adaptation and salt stress response as the model system. Methanosarcina mazei Gö1 grows at salt concentrations ranging from micromolar to 0.8 M concentrations. To adjust turgor pressure it accumulates compatible solutes, i.e. N-acetyl-beta-lysine and glutamate as major compatible solutes by synthesis and glycine betaine by uptake (Fig. 5).
We have identified the enzymes catalyzing biosynthesis of N-acetyl-beta-lysine from lysine and an ABC-type transporter that mediates uptake of glycine betaine in a salt-activated manner. The encoding genes were identified, promotors have been mapped, and we are currently seeking for cis- and trans-acting elements involved in gene regulation.
Apart from accumulation of compatible solutes cells must export Na+ from the cytoplasm. This is achieved by a Na+/H+ antiporter. The gene was identified. The deduced protein has an unusual N-terminal PAS domain that is speculated to be involved in salt activation of the enzyme. This concept is currently tested by molecular and biochemical techniques.
DNA microarrays are employed to identify salt-induced genes on a genome wide scale. These studies revealed a large set of genes that are induced within the first 90 min after osmotic upshock. As the lag phase progresses, the number of induced genes declines, and the number of genes repressed increases. Currently, the function of individual genes/proteins identified by this technique is explored by biochemical and genetic means.
Publications 1999 – 2004
Roeßler, M., Müller, V. (2001) Osmoadaptation in bacteria and archaea: common principles and differences. Environ. Microbiol. 3 : 743 - 754.
Roeßler, M., Pflüger, K., Flach, H., Lienard, T., Gottschalk, G., Müller, V. (2002) Identification of a salt-induced primary transporter for glycine betaine in the methanogen Methanosarcina mazei Gö1. Appl. Environ. Microbiol. 68 : 2133 - 2139.
Pflüger, K., Baumann, S., Gottschalk, G., Lin, W., Santos, H., Müller, V. (2003) Lysine-2,3-aminomutase and b-lysine acetyltransferase genes of methanogenic archaea are salt induced and are essential for the biosynthesis of NEpsilon-acetyl-b-lysine and growth at high salinity. Appl. Environ. Microbiol. 69 : 6047 - 6055.
Pflüger, K., Müller, V. (2004) Transport of compatible solutes in extremophiles. J. Bioenerg. Biomembr. 36 : 17 - 24.
Pflüger, K., Ehrenreich, A., Salmon, K., Gunsalus, R., Deppenmeier, U., Gottschalk, G., Müller, V. (2004) Identification of genes involved in salt adaptation in the archaeon Methanosarcina mazei using genomewide gene expression profiling. FEMS Microbiol. Lett. 277 : 79 - 83.
4. Role of chloride in Halobacillus halophilus
Chloride is an essential element for higher eukaryotes but there is only little information for a role of chloride in the physiology of prokaryotes. For the aerobic, endospore-forming, moderately halophilic bacterium Halobacillus halophilus we could recently demonstrate a chloride dependence for growth. This was the first report on a chloride-dependent bacterium. In addition, endospore germination, flagellation and thus motility was shown to be chloride dependent. We used the flagellar system as a model system and demonstrated chloride-induced expression of the gene encoding the structural protein of the flagellum, flagellin. In addition, the cellular level of flagellin was strictly chloride-dependent showing, for the first time, a role of chloride as a signal molecule involved in gene regulation (Fig. 6).
In our search for the essential function of chloride we analyzed, in collaboration with Prof. Santos, Oeiras, Portugal, the cellular pools of compatible solutes in this moderate halophile. The pool sizes changed with changing salt concentrations, as expected, but were also dependent on the anion used. In addition, uptake of the compatible solute glycine betaine was strictly chloride dependent. We have identified the genes of all major pathways for the biosynthesis of the different compatible solutes used by H. halophilus, and are currently studying their regulation. Major focus is on salt-dependent activation in general, and chloride-dependent gene activation in particular. The genome of H. halophilus is being sequenced by the group of Prof. Oesterhelt, Martinsried, Germany, and in collaboration with Prof. Oesterhelt we are exploring the mechanisms of salt adaptation and the role of chloride in this moderate halophile by bioinformatic approaches as well as functional genome analyses.
Publications 1999 – 2004
Dohrmann, A.-B., Müller, V. (1999) Chloride dependence of endospore germination in Halobacillus halophilus. Arch. Microbiol. 172 : 264 - 267.
Roeßler, M., Wanner, G., Müller, V. (2000) Motility and flagellum synthesis in Halobacillus halophilus are chloride dependent. J. Bacteriol. 182 : 532 - 535.
Roeßler, M., Müller, V. (2001) Chloride dependence of glycine betaine transport in Halobacillus halophilus. FEBS Lett. 489 : 125 - 128.
Roeßler, M., Müller, V. (2002) Chloride, a new environmental signal molecule invoved in gene regulation in a moderately halophilic bacterium, Halobacillus halophilus. J. Bacteriol. 184 : 6207 - 6215.
Roeßler, M., Sewald, X., Müller V. (2003) Chloride dependence of growth in bacteria. FEMS Microbiol. Lett. 225 : 161 - 165.
Müller, V., Oren. A. (2003) Metabolism of chloride in halophilic prokaryotes. Extremophiles 7 : 261 - 266.
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