- Email: [email protected]
The aerobic respiratory NADH dehydrogenases in Escherichia coli
Abstracts
therefore analyzed the proteome of bacteria from acetate and pyruvate cultures in the exponential and stationary phase of growth. To our surprise H. modesticaldum expresses the entire pathway from acetate to butyrate when grown in the light on acetate + CO2, as well as in the stationary phase on pyruvate. References 1. L. Kimble, L. Mandelco, C. Woese, M. Madigan, Heliobacterium modesticaldum, sp. nov., a thermophilic heliobacterium of hot springs and volcanic soil, Arch Microbiol, 163 (1995) 259–267 2. W. Sattley, M. Madigan, W. Swingley, P. Cheung, K. Clocksin, A. Conrad, L. Dejesa, B. Honchak, D. Jung, L. Karbach, A. Kurdoglu, S. Lahiri, S. Mastrian, L. Page, H. Tayloe, Z. Wang, J. Raymond, M. Chen, R. Blankenship, J. Touchma, The genome of Heliobacterium modesticaldum, a phototrophic representative of the Firmicutes containing the simplest photosynthetic apparatus, J Bact, 190 (2008) 4687–4696. doi:10.1016/j.bbabio.2016.04.295
08.23 Investigation of cytochrome c dependent nitric oxide reductase (cNOR) from Paracoccus denitrificans Sinan Sabuncua, Josy Ter Beekb, Madeleine Stricklanda, Pia Ädelrothb, Frederic Melina, Petra Hellwiga a University of Strasbourg, UMR 7140, France b Stockholm University, Department of Biochemistry and Biophysics, Sweden E-mail address: [email protected] (S. Sabuncu) The Nitric oxide reductase (cNOR) has an important role in denitrification processes in bacteria and catalyzes the reaction of NO to N2O. It contains a low spin heme c, two b type hemes (low spin b, high spin b3) and one non-heme iron (FeB) [1]. This enzyme is related to the heme-copper oxidase superfamily of membrane proteins and it was suggested that cytochrome oxidases (COX) and NOR share a common ancestor [2]. A Ca2 + site was identified close to the hemes b and b3 which has a role in the electron transfer between these hemes and their conformation. In this study we work on wild type (WT) and mutant enzymes from P. denitrificans which are ligands of the Ca2 + site (E122A,Y74F and Y74S) [3]. The mid-point potentials of each heme was determined for wild type and mutant enzymes using UV/Vis potentiometric titrations and differential infrared spectroscopy was used to investigate changes in the protonation state of individual amino acids, secondary structure and environment of cofactors. Shifts in the mid-point potentials due to the single mutations at crucial residues are discussed. References 1. Y. Shiro, Structure and function of bacterial nitric oxide reductases: nitric oxide reductase, anaerobic enzymes, Biochimic. Biophys. Acta, 1817 (2012) 1907–1913 2. M. R. Cheesman, W. G. Zumft A. J., Thomson, The MCD and EPR of the heme centers of nitric oxide reductase from Pseudomonas stutzeri: evidence that the enzyme is structurally related to the heme-copper oxidases, Biochemistry-US, 37 (1998) 3994–4000 3. U. Flock, F. H. Thorndycroft, A. D. Matorin, D. J. Richardson, N. J. Watmough, P. Adelroth, Defining the proton entry point in the bacterial respiratory nitric-oxide reductase, J. Biol. Chem., 283 (2008) 3839–3845. doi:10.1016/j.bbabio.2016.04.296
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08.24 Cytochrome bd-type quinol oxidases: Structural variety even within the Gram-positive bacteria Junshi Sakamoto, Tsukasa Yoshida, Taichiro Hirose, Tomoichirou Kusumoto Kyushu Institute of Technology, Department of Bioscience and Bioinformatics, Kawazu 680-4, Iizuka, Fukuoka 820-8502, Japan E-mail address: [email protected] (J. Sakamoto) We have proposed two subfamilies of cytochrome bd quinol oxidase, S- and L-types [1]. They are different in the length of the Q loop, which is an important domain for quinol oxidation. We previously identified and purified cyt bd-type menaquinol oxidases from a thermophile Geobacillus thermodenitrificans K1041 and an amino acid-producing mesophile Corynebacterium glutamicum. These organisms are both Gram-positives and their cyts bd are both S-type, but the oxidases are much different in subunit sizes, optical property, and operon configuration. The wavelength of absorption peak due to the D-type heme is different as much as 10 nm. Two genes for subunits I and II cydAB are followed by genes for ABC transporter cydDC in the C. glutamicum genome, while by short orfs in the G. thermodenitrificans genome [2]. Recently, we solved the atomic structure of cyt bd from the thermophile [3]. This entirely novel structure itself and related biochemical analyses provide colorful insights into the molecular diversity of cyt bd. Crystal structures have been solved recently for complexes I through V and terminal oxidases including A-, B-, C-types of hemeCu oxidases etc. The cyt bd structure is the final piece to fill the bioenergetic jigsaw puzzle and useful to design novel bactericidal drugs. References 1. A.M. Arutyunyan, J. Sakamoto, Y. Kabashima, Y. Inadome. V.B. Borisov, Optical and magneto-optical activity of cytochrome bd from Geobacillus thermodenitrificans. Biochim. Biophys. Acta 1817 (2012) 2087–2094 2. J. Sakamoto, E. Koga, T. Mizuta, C. Sato, S. Noguchi, N. Sone, Gene structure and quinol oxidase activity of a cytochrome bd-type oxidase from Bacillus stearothermophilus. Biochim. Biophys. Acta 1411 (1999) 147–158 3. S. Safarian, H. Müller, C. Rajendran, J. Preu, J.D. Langer, S. Ovchinnikov, T. Hirose, T. Kusumoto, J. Sakamoto, H. Michel, The bd oxidases possess a unique protein structure and an unexpected heme group arrangement. Science (2016) in press. doi:10.1016/j.bbabio.2016.04.297
08.25 The aerobic respiratory NADH dehydrogenases in Escherichia coli Johannes Schimpf, Ina Schweizer, Thorsten Friedrich Institut für Biochemie, Albert-Ludwigs Universität, Freiburg, Germany E-mail address: [email protected] (J. Schimpf)
The E. coli aerobic respiratory chain basically consists of the NADH:ubiquinone oxidoreductase (complex I), the alternative NADH dehydrogenase (NDH-II), the succinate:ubiquinone oxidoreductase (complex II) and the terminal quinol oxidases bo3, bd-I and bd-II [1]. NADH from catabolic processes is used by complex I and NDH-II to reduce ubiquinone to ubiquinol. Subsequently, the electrons are transferred to the terminal oxidases to reduce oxygen to water.
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Abstracts
Besides complex I and NDH-II, other NADH dehydrogenases such as WrbA, YhdH and QOR are discussed to be part of the E. coli aerobic respiratory chain [2]. To determine the number of NADH dehydrogenases involved in aerobic respiration, several deletion strains were generated from the parental strain E. coli BW25113. The mutants were characterized by the NADH/ferricyanide oxidoreductase activity, the NADH oxidase activity and their ability to perform proton translocation. Our results showed that 75% of the NADH oxidase activity derives from complex I and 25% from NDH-II. Deletion of complex I and NDH-II led to the lack of any NADH oxidase activity, while the deletion of the genes coding for either WrbA, YhdH or QOR did not alter this activity at all. From these data we conclude that WrbA, YhdH and QOR are no part of the aerobic E. coli respiratory chain. References 1. P.M.F. Sousa, M.A.M. Videira, A. Bohn, B.L. Hood, T.P. Conrads, L. F. Goulao, A.M.P. Melo, The aerobic respiratory chain of Escherichia coli: from genes to supercomplexes, Microbiol. 158 (2012) 2408–2418 2. M. Bekker, S. de Vries, A. Ter Beek, K.J. Hellingwerf, M.J. Texeira de Mattos, Respiration of Escherichia coli can be fully uncoupled via the nonelectrogenic terminal cytochrome bd-II oxidase, J. Bacteriol. 191 (2009) 5510–5517. doi:10.1016/j.bbabio.2016.04.298
08.26 Production and purification of the Escherichia coli cytochrome bd oxidase Alexander Theßeling, Jo Hoeser, Thorsten Friedrich Institute of Biochemistry, Albert-Ludwigs-Universität, Freiburg, Germany E-mail address: [email protected] (A. Theßeling) The conversion of energy in organisms is warranted by the respiratory chains. The cytochrome bd oxidase is one of the terminal oxidases in many prokaryotes and expressed under low oxygen conditions. It catalyzes the reduction of oxygen to water by coupling the reaction with the quinone pool. During this process, one proton per electron is translocated across the membrane [1]. Cytochrome bd shows little similarities to other terminal oxidases such as hemecopper oxidases, cytochrome c oxidase or alternative oxidases [2]. In Escherichia coli cytochrome bd oxidase consists of three different subunits, CydA, CydB and CydX [3,4]. The protein was homologously produced and purified by means of affinity-, and size exclusionchromatography. Different detergents were deployed for membrane extraction of the bd oxidase in order to obtain a pure and monodisperse preparation. References 1. V. B. Borisov,R. B. Gennis, J. Hemp, M. I. Verkhovsky, The cytochrome bd respiratory oxygen reductases. Biochim. Biophys. Acta 1807, (2011), 1398–1413 2. P. A. Cotter, V. Chepuri, R. B. Gennis, R. P. Gunsalus. Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product. J. Bacteriol. 172, (1990), 6333–6338 3. R. J. Allen, E. P. Brenner, C. E. VanOrsdel, J. J. Hobson, D. J. Hearn, M. R. Hemm. Conservation analysis of the CydX protein yields insights into small protein identification and evolution. BMC Genomics (2014), 15:946 4. J. Hoeser, S. Hong, G. Gehmann, R. B. Gennis,T. Friedrich. Subunit CydX of Escherichia coli cytochrome bd ubiquinol oxidase is essential for
assembly and stability of the di-heme active site. FEBS Lett. 588, (2014), 1537–1541. doi:10.1016/j.bbabio.2016.04.299
08.27 Solute transporters (DctA or DcuB) as structural coregulators in bacterial transporter/sensor complexes Gottfried Unden, Sebastian Wörner Institute for Microbiology and Wine Research, Johannes Gutenberg University, Mainz, Germany E-mail address: [email protected] (G. Unden) Free living bacteria use a large variety of sensors for responding to environmental stimuli and adapting of metabolism. The fumarate sensor kinase DcuS of Escherichia coli adapts metabolism for the utilization of external C4-dicarboxylates (or fumarate), including substrate transport and fumarate respiration. DcuS is part of a two-component system. It becomes auto-phosphorylated in the presence of fumarate, resulting in the activation of the target genes via the response regulator DcuR. The sensor kinase DcuS is membrane-integral by TM helices TM1 and TM2. Binding of fumarate to the extra-cytoplasmic binding domain triggers signal transduction across the membrane by piston-type displacement of TM2 [1] which induces the activity of the C-terminal kinase domain. In the functional state DcuS forms complexes with fumarate transporters DctA or DcuB [2,3]. The fumarate binding site for sensing by the DctA/DcuS or DcuB/DcuS sensor complexes is located within DcuS. The transporters DctA and DcuB are bifunctional proteins and serve in the sensor complexes as structural components for converting DcuS to the sensory competent state. Their role as co-regulators is independent of transport or substrate binding [3]. References 1. C. Monzel, G. Unden, Transmembrane signaling in the sensor kinase DcuS of E. coli. PNAS112 (2015) 11042–11047 2. A. Kleefeld, B. Ackermann, J. Bauer, J. Krämer, G. Unden, The fumarate/succinate antiporter DcuB of E. coli is a bifunctional protein with sites for regulation of DcuS dependent gene expression. J Biol Chem 284 (2009) 265–275 3. P. Steinmetz, S. Wörner, G. Unden, Differentiation of DctA and DcuS function in the DctA/DcuS sensor complex of E. coli. Molec Microbiol 94 (2014) 218–229. doi:10.1016/j.bbabio.2016.04.300
08.28 LUCA's informational core Madeline C. Weiss, Filipa L. Sousa, Natalia Mrnjavac, Sinje Neukirchen, Mayo Roettger, Shijulal Nelson-Sathi, William F. Martin Institute for Molecular Evolution, Heinrich-Heine University, Düsseldorf, Germany E-mail address: [email protected] (M.C. Weiss) Central to the studies of early evolution and the origin of life is the concept of a last common ancestor, or Luca and its genomic content. To date, most comparative genomic analysis of Luca's gene
therefore analyzed the proteome of bacteria from acetate and pyruvate cultures in the exponential and stationary phase of growth. To our surprise H. modesticaldum expresses the entire pathway from acetate to butyrate when grown in the light on acetate + CO2, as well as in the stationary phase on pyruvate. References 1. L. Kimble, L. Mandelco, C. Woese, M. Madigan, Heliobacterium modesticaldum, sp. nov., a thermophilic heliobacterium of hot springs and volcanic soil, Arch Microbiol, 163 (1995) 259–267 2. W. Sattley, M. Madigan, W. Swingley, P. Cheung, K. Clocksin, A. Conrad, L. Dejesa, B. Honchak, D. Jung, L. Karbach, A. Kurdoglu, S. Lahiri, S. Mastrian, L. Page, H. Tayloe, Z. Wang, J. Raymond, M. Chen, R. Blankenship, J. Touchma, The genome of Heliobacterium modesticaldum, a phototrophic representative of the Firmicutes containing the simplest photosynthetic apparatus, J Bact, 190 (2008) 4687–4696. doi:10.1016/j.bbabio.2016.04.295
08.23 Investigation of cytochrome c dependent nitric oxide reductase (cNOR) from Paracoccus denitrificans Sinan Sabuncua, Josy Ter Beekb, Madeleine Stricklanda, Pia Ädelrothb, Frederic Melina, Petra Hellwiga a University of Strasbourg, UMR 7140, France b Stockholm University, Department of Biochemistry and Biophysics, Sweden E-mail address: [email protected] (S. Sabuncu) The Nitric oxide reductase (cNOR) has an important role in denitrification processes in bacteria and catalyzes the reaction of NO to N2O. It contains a low spin heme c, two b type hemes (low spin b, high spin b3) and one non-heme iron (FeB) [1]. This enzyme is related to the heme-copper oxidase superfamily of membrane proteins and it was suggested that cytochrome oxidases (COX) and NOR share a common ancestor [2]. A Ca2 + site was identified close to the hemes b and b3 which has a role in the electron transfer between these hemes and their conformation. In this study we work on wild type (WT) and mutant enzymes from P. denitrificans which are ligands of the Ca2 + site (E122A,Y74F and Y74S) [3]. The mid-point potentials of each heme was determined for wild type and mutant enzymes using UV/Vis potentiometric titrations and differential infrared spectroscopy was used to investigate changes in the protonation state of individual amino acids, secondary structure and environment of cofactors. Shifts in the mid-point potentials due to the single mutations at crucial residues are discussed. References 1. Y. Shiro, Structure and function of bacterial nitric oxide reductases: nitric oxide reductase, anaerobic enzymes, Biochimic. Biophys. Acta, 1817 (2012) 1907–1913 2. M. R. Cheesman, W. G. Zumft A. J., Thomson, The MCD and EPR of the heme centers of nitric oxide reductase from Pseudomonas stutzeri: evidence that the enzyme is structurally related to the heme-copper oxidases, Biochemistry-US, 37 (1998) 3994–4000 3. U. Flock, F. H. Thorndycroft, A. D. Matorin, D. J. Richardson, N. J. Watmough, P. Adelroth, Defining the proton entry point in the bacterial respiratory nitric-oxide reductase, J. Biol. Chem., 283 (2008) 3839–3845. doi:10.1016/j.bbabio.2016.04.296
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08.24 Cytochrome bd-type quinol oxidases: Structural variety even within the Gram-positive bacteria Junshi Sakamoto, Tsukasa Yoshida, Taichiro Hirose, Tomoichirou Kusumoto Kyushu Institute of Technology, Department of Bioscience and Bioinformatics, Kawazu 680-4, Iizuka, Fukuoka 820-8502, Japan E-mail address: [email protected] (J. Sakamoto) We have proposed two subfamilies of cytochrome bd quinol oxidase, S- and L-types [1]. They are different in the length of the Q loop, which is an important domain for quinol oxidation. We previously identified and purified cyt bd-type menaquinol oxidases from a thermophile Geobacillus thermodenitrificans K1041 and an amino acid-producing mesophile Corynebacterium glutamicum. These organisms are both Gram-positives and their cyts bd are both S-type, but the oxidases are much different in subunit sizes, optical property, and operon configuration. The wavelength of absorption peak due to the D-type heme is different as much as 10 nm. Two genes for subunits I and II cydAB are followed by genes for ABC transporter cydDC in the C. glutamicum genome, while by short orfs in the G. thermodenitrificans genome [2]. Recently, we solved the atomic structure of cyt bd from the thermophile [3]. This entirely novel structure itself and related biochemical analyses provide colorful insights into the molecular diversity of cyt bd. Crystal structures have been solved recently for complexes I through V and terminal oxidases including A-, B-, C-types of hemeCu oxidases etc. The cyt bd structure is the final piece to fill the bioenergetic jigsaw puzzle and useful to design novel bactericidal drugs. References 1. A.M. Arutyunyan, J. Sakamoto, Y. Kabashima, Y. Inadome. V.B. Borisov, Optical and magneto-optical activity of cytochrome bd from Geobacillus thermodenitrificans. Biochim. Biophys. Acta 1817 (2012) 2087–2094 2. J. Sakamoto, E. Koga, T. Mizuta, C. Sato, S. Noguchi, N. Sone, Gene structure and quinol oxidase activity of a cytochrome bd-type oxidase from Bacillus stearothermophilus. Biochim. Biophys. Acta 1411 (1999) 147–158 3. S. Safarian, H. Müller, C. Rajendran, J. Preu, J.D. Langer, S. Ovchinnikov, T. Hirose, T. Kusumoto, J. Sakamoto, H. Michel, The bd oxidases possess a unique protein structure and an unexpected heme group arrangement. Science (2016) in press. doi:10.1016/j.bbabio.2016.04.297
08.25 The aerobic respiratory NADH dehydrogenases in Escherichia coli Johannes Schimpf, Ina Schweizer, Thorsten Friedrich Institut für Biochemie, Albert-Ludwigs Universität, Freiburg, Germany E-mail address: [email protected] (J. Schimpf)
The E. coli aerobic respiratory chain basically consists of the NADH:ubiquinone oxidoreductase (complex I), the alternative NADH dehydrogenase (NDH-II), the succinate:ubiquinone oxidoreductase (complex II) and the terminal quinol oxidases bo3, bd-I and bd-II [1]. NADH from catabolic processes is used by complex I and NDH-II to reduce ubiquinone to ubiquinol. Subsequently, the electrons are transferred to the terminal oxidases to reduce oxygen to water.
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Besides complex I and NDH-II, other NADH dehydrogenases such as WrbA, YhdH and QOR are discussed to be part of the E. coli aerobic respiratory chain [2]. To determine the number of NADH dehydrogenases involved in aerobic respiration, several deletion strains were generated from the parental strain E. coli BW25113. The mutants were characterized by the NADH/ferricyanide oxidoreductase activity, the NADH oxidase activity and their ability to perform proton translocation. Our results showed that 75% of the NADH oxidase activity derives from complex I and 25% from NDH-II. Deletion of complex I and NDH-II led to the lack of any NADH oxidase activity, while the deletion of the genes coding for either WrbA, YhdH or QOR did not alter this activity at all. From these data we conclude that WrbA, YhdH and QOR are no part of the aerobic E. coli respiratory chain. References 1. P.M.F. Sousa, M.A.M. Videira, A. Bohn, B.L. Hood, T.P. Conrads, L. F. Goulao, A.M.P. Melo, The aerobic respiratory chain of Escherichia coli: from genes to supercomplexes, Microbiol. 158 (2012) 2408–2418 2. M. Bekker, S. de Vries, A. Ter Beek, K.J. Hellingwerf, M.J. Texeira de Mattos, Respiration of Escherichia coli can be fully uncoupled via the nonelectrogenic terminal cytochrome bd-II oxidase, J. Bacteriol. 191 (2009) 5510–5517. doi:10.1016/j.bbabio.2016.04.298
08.26 Production and purification of the Escherichia coli cytochrome bd oxidase Alexander Theßeling, Jo Hoeser, Thorsten Friedrich Institute of Biochemistry, Albert-Ludwigs-Universität, Freiburg, Germany E-mail address: [email protected] (A. Theßeling) The conversion of energy in organisms is warranted by the respiratory chains. The cytochrome bd oxidase is one of the terminal oxidases in many prokaryotes and expressed under low oxygen conditions. It catalyzes the reduction of oxygen to water by coupling the reaction with the quinone pool. During this process, one proton per electron is translocated across the membrane [1]. Cytochrome bd shows little similarities to other terminal oxidases such as hemecopper oxidases, cytochrome c oxidase or alternative oxidases [2]. In Escherichia coli cytochrome bd oxidase consists of three different subunits, CydA, CydB and CydX [3,4]. The protein was homologously produced and purified by means of affinity-, and size exclusionchromatography. Different detergents were deployed for membrane extraction of the bd oxidase in order to obtain a pure and monodisperse preparation. References 1. V. B. Borisov,R. B. Gennis, J. Hemp, M. I. Verkhovsky, The cytochrome bd respiratory oxygen reductases. Biochim. Biophys. Acta 1807, (2011), 1398–1413 2. P. A. Cotter, V. Chepuri, R. B. Gennis, R. P. Gunsalus. Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product. J. Bacteriol. 172, (1990), 6333–6338 3. R. J. Allen, E. P. Brenner, C. E. VanOrsdel, J. J. Hobson, D. J. Hearn, M. R. Hemm. Conservation analysis of the CydX protein yields insights into small protein identification and evolution. BMC Genomics (2014), 15:946 4. J. Hoeser, S. Hong, G. Gehmann, R. B. Gennis,T. Friedrich. Subunit CydX of Escherichia coli cytochrome bd ubiquinol oxidase is essential for
assembly and stability of the di-heme active site. FEBS Lett. 588, (2014), 1537–1541. doi:10.1016/j.bbabio.2016.04.299
08.27 Solute transporters (DctA or DcuB) as structural coregulators in bacterial transporter/sensor complexes Gottfried Unden, Sebastian Wörner Institute for Microbiology and Wine Research, Johannes Gutenberg University, Mainz, Germany E-mail address: [email protected] (G. Unden) Free living bacteria use a large variety of sensors for responding to environmental stimuli and adapting of metabolism. The fumarate sensor kinase DcuS of Escherichia coli adapts metabolism for the utilization of external C4-dicarboxylates (or fumarate), including substrate transport and fumarate respiration. DcuS is part of a two-component system. It becomes auto-phosphorylated in the presence of fumarate, resulting in the activation of the target genes via the response regulator DcuR. The sensor kinase DcuS is membrane-integral by TM helices TM1 and TM2. Binding of fumarate to the extra-cytoplasmic binding domain triggers signal transduction across the membrane by piston-type displacement of TM2 [1] which induces the activity of the C-terminal kinase domain. In the functional state DcuS forms complexes with fumarate transporters DctA or DcuB [2,3]. The fumarate binding site for sensing by the DctA/DcuS or DcuB/DcuS sensor complexes is located within DcuS. The transporters DctA and DcuB are bifunctional proteins and serve in the sensor complexes as structural components for converting DcuS to the sensory competent state. Their role as co-regulators is independent of transport or substrate binding [3]. References 1. C. Monzel, G. Unden, Transmembrane signaling in the sensor kinase DcuS of E. coli. PNAS112 (2015) 11042–11047 2. A. Kleefeld, B. Ackermann, J. Bauer, J. Krämer, G. Unden, The fumarate/succinate antiporter DcuB of E. coli is a bifunctional protein with sites for regulation of DcuS dependent gene expression. J Biol Chem 284 (2009) 265–275 3. P. Steinmetz, S. Wörner, G. Unden, Differentiation of DctA and DcuS function in the DctA/DcuS sensor complex of E. coli. Molec Microbiol 94 (2014) 218–229. doi:10.1016/j.bbabio.2016.04.300
08.28 LUCA's informational core Madeline C. Weiss, Filipa L. Sousa, Natalia Mrnjavac, Sinje Neukirchen, Mayo Roettger, Shijulal Nelson-Sathi, William F. Martin Institute for Molecular Evolution, Heinrich-Heine University, Düsseldorf, Germany E-mail address: [email protected] (M.C. Weiss) Central to the studies of early evolution and the origin of life is the concept of a last common ancestor, or Luca and its genomic content. To date, most comparative genomic analysis of Luca's gene