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Volume 161, Issue 10

An electron transfer flavoprotein is essential for viability and its depletion causes a rod-to-sphere change in Free

Abstract

Essential gene studies often reveal novel essential functions for genes with dispensable homologues in other species. This is the case with the widespread family of electron transfer flavoproteins (ETFs), which are required for the metabolism of specific substrates or for symbiotic nitrogen fixation in some bacteria. Despite these non-essential functions high-throughput screens have identified ETFs as putatively essential in several species. In this study, we constructed a conditional expression mutant of one of the ETFs in , and demonstrated that its expression is essential for growth on both complex media and a variety of single-carbon sources. We further demonstrated that the two subunits EtfA and EtfB interact with each other, and that cells depleted of ETF are non-viable and lack redox potential. These cells also transition from the short rods characteristic of to small spheres independently of MreB. The putative membrane partner ETF dehydrogenase also induced the same rod-to-sphere change. We propose that the ETF of is a novel antibacterial target.

  • Received:
  • Accepted:
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  • Published Online:
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2015-10-01
2024-04-25
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References

  1. Agnoli K., Schwager S., Uehlinger S., Vergunst A., Viteri D.F., Nguyen D.T., Sokol P.A., Carlier A., Eberl L. 2012; Exposing the third chromosome of Burkholderia cepacia complex strains as a virulence plasmid. Mol Microbiol 83:362–378 [View Article][PubMed]
     [Google Scholar]
  2. Anraku Y. 1988; Bacterial electron transport chains. Annu Rev Biochem 57:101–132 [View Article][PubMed]
     [Google Scholar]
  3. Battesti A., Bouveret E. 2012; The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. Methods 58:325–334 [View Article][PubMed]
     [Google Scholar]
  4. Baugh L., Gallagher L.A., Patrapuvich R., Clifton M.C., Gardberg A.S., Edwards T.E., Armour B., Begley D.W., Dieterich S.H., other authors. 2013; Combining functional and structural genomics to sample the essential Burkholderia structome. PLoS One 8:e53851 [View Article][PubMed]
     [Google Scholar]
  5. Bean G.J., Flickinger S.T., Westler W.M., McCully M.E., Sept D., Weibel D.B., Amann K.J. 2009; A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a low-affinity state in MreB. Biochemistry 48:4852–4857 [View Article][PubMed]
     [Google Scholar]
  6. Bharat A., Brown E.D. 2014; Phenotypic investigations of the depletion of EngA in Escherichia coli are consistent with a role in ribosome biogenesis. FEMS Microbiol Lett 353:26–32 [View Article][PubMed]
     [Google Scholar]
  7. Bloodworth R.A., Gislason A.S., Cardona S.T. 2013; Burkholderia cenocepacia conditional growth mutant library created by random promoter replacement of essential genes. Microbiology Open 2:243–258 [View Article][PubMed]
     [Google Scholar]
  8. Campbell T.L., Daigle D.M., Brown E.D. 2005; Characterization of the Bacillus subtilis GTPase YloQ and its role in ribosome function. Biochem J 389:843–852 [View Article][PubMed]
     [Google Scholar]
  9. Cardona S.T., Selin C., Gislason A.S. Genomic tools to profile antibiotic mode of action. Crit Rev Microbiol [View Article][PubMed]
     [Google Scholar]
  10. Castresana J. 2000; Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540–552 [View Article][PubMed]
     [Google Scholar]
  11. Chen D., Swenson R.P. 1994; Cloning, sequence analysis, and expression of the genes encoding the two subunits of the methylotrophic bacterium W3A1 electron transfer flavoprotein. J Biol Chem 269:32120–32130[PubMed]
     [Google Scholar]
  12. Christen B., Abeliuk E., Collier J.M., Kalogeraki V.S., Passarelli B., Coller J.A., Fero M.J., McAdams H.H., Shapiro L. 2011; The essential genome of a bacterium. Mol Syst Biol 7:528–535 [View Article][PubMed]
     [Google Scholar]
  13. Commichau F.M., Pietack N., Stülke J. 2013; Essential genes in Bacillus subtilis: a re-evaluation after ten years. Mol Biosyst 9:1068–1075 [View Article][PubMed]
     [Google Scholar]
  14. Craig F.F., Coote J.G., Parton R., Freer J.H., Gilmour N.J. 1989; A plasmid which can be transferred between Escherichia coli and Pasteurella haemolytica by electroporation and conjugation. J Gen Microbiol 135:2885–2890[PubMed]
     [Google Scholar]
  15. Dalbey R.E., Wickner W. 1985; Leader peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane. J Biol Chem 260:15925–15931[PubMed]
     [Google Scholar]
  16. de Berardinis V., Vallenet D., Castelli V., Besnard M., Pinet A., Cruaud C., Samair S., Lechaplais C., Gyapay G., other authors. 2008; A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1. Mol Syst Biol 4:174–189 [View Article][PubMed]
     [Google Scholar]
  17. Donachie W.D., Begg K.J. 1989; Cell length, nucleoid separation, and cell division of rod-shaped and spherical cells of Escherichia coli. J Bacteriol 171:4633–4639[PubMed]
     [Google Scholar]
  18. Drevinek P., Mahenthiralingam E. 2010; Burkholderia cenocepacia in cystic fibrosis: epidemiology and molecular mechanisms of virulence. Clin Microbiol Infect 16:821–830 [View Article][PubMed]
     [Google Scholar]
  19. Edgar R.C. 2004; muscle: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797 [View Article][PubMed]
     [Google Scholar]
  20. Fang G., Rocha E., Danchin A. 2005; How essential are nonessential genes?. Mol Biol Evol 22:2147–2156 [View Article][PubMed]
     [Google Scholar]
  21. Figurski D.H., Helinski D.R. 1979; Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76:1648–1652 [View Article][PubMed]
     [Google Scholar]
  22. Flannagan R.S., Aubert D., Kooi C., Sokol P.A., Valvano M.A. 2007; Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo. Infect Immun 75:1679–1689 [View Article][PubMed]
     [Google Scholar]
  23. Flannagan R.S., Linn T., Valvano M.A. 2008; A system for the construction of targeted unmarked gene deletions in the genus Burkholderia. Environ Microbiol 10:1652–1660 [View Article][PubMed]
     [Google Scholar]
  24. Frerman F.E., Goodman S.I. 2001; Defects of electron transfer flavoprotein and electron transfer flavoprotein-ubiquinone oxidoreductase: glutaric acidemia type II. In The Metabolic and Molecular Bases of Inherited Disease, 2nd edn. pp. 2357–2365 Edited by Scriver C. R. New York: McGraw-Hill;
     [Google Scholar]
  25. Gerdes S., Edwards R., Kubal M., Fonstein M., Stevens R., Osterman A. 2006; Essential genes on metabolic maps. Curr Opin Biotechnol 17:448–456 [View Article][PubMed]
     [Google Scholar]
  26. Ghisla S., Thorpe C. 2004; Acyl-CoA dehydrogenases. A mechanistic overview. Eur J Biochem 271:494–508 [View Article][PubMed]
     [Google Scholar]
  27. Givskov M., Eberl L., Møller S., Poulsen L.K., Molin S. 1994; Responses to nutrient starvation in Pseudomonas putida KT2442: analysis of general cross-protection, cell shape, and macromolecular content. J Bacteriol 176:7–14[PubMed]
     [Google Scholar]
  28. Goswami M., Mangoli S.H., Jawali N. 2006; Involvement of reactive oxygen species in the action of ciprofloxacin against Escherichia coli. Antimicrob Agents Chemother 50:949–954 [View Article][PubMed]
     [Google Scholar]
  29. Griffin J.E., Gawronski J.D., Dejesus M.A., Ioerger T.R., Akerley B.J., Sassetti C.M. 2011; High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism. PLoS Pathog 7:e1002251 [View Article][PubMed]
     [Google Scholar]
  30. Guindon S., Dufayard J.F., Lefort V., Anisimova M., Hordijk W., Gascuel O. 2010; New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321 [View Article][PubMed]
     [Google Scholar]
  31. Hill N.S., Buske P.J., Shi Y., Levin P.A. 2013; A moonlighting enzyme links Escherichia coli cell size with central metabolism. PLoS Genet 9:e1003663 [View Article][PubMed]
     [Google Scholar]
  32. Juhas M., Eberl L., Church G.M. 2012; Essential genes as antimicrobial targets and cornerstones of synthetic biology. Trends Biotechnol 30:601–607 [View Article][PubMed]
     [Google Scholar]
  33. Klein B.A., Tenorio E.L., Lazinski D.W., Camilli A., Duncan M.J., Hu L.T. 2012; Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis. BMC Genomics 13:578–595 [View Article][PubMed]
     [Google Scholar]
  34. Langridge G.C., Phan M.D., Turner D.J., Perkins T.T., Parts L., Haase J., Charles I., Maskell D.J., Peters S.E., other authors. 2009; Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Res 19:2308–2316 [View Article][PubMed]
     [Google Scholar]
  35. Law R.J., Hamlin J.N.R., Sivro A., McCorrister S.J., Cardama G.A., Cardona S.T. 2008; A functional phenylacetic acid catabolic pathway is required for full pathogenicity of Burkholderia cenocepacia in the Caenorhabditis elegans host model. J Bacteriol 190:7209–7218 [View Article][PubMed]
     [Google Scholar]
  36. Lehmann E.L. 1998 Nonparametrics: Statistical Methods Based on Ranks Upper Saddle River, NJ: Prentice-Hall;
     [Google Scholar]
  37. Leitão J.H., Sousa S.A., Cunha M.V., Salgado M.J., Melo-Cristino J., Barreto M.C., Sá-Correia I. 2008; Variation of the antimicrobial susceptibility profiles of Burkholderia cepacia complex clonal isolates obtained from chronically infected cystic fibrosis patients: a five-year survey in the major Portuguese treatment center. Eur J Clin Microbiol Infect Dis 27:1101–1111 [View Article][PubMed]
     [Google Scholar]
  38. Li F., Hinderberger J., Seedorf H., Zhang J., Buckel W., Thauer R.K. 2008; Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri. J Bacteriol 190:843–850 [View Article][PubMed]
     [Google Scholar]
  39. Liu H., Chen C., Zhang H., Kaur J., Goldman Y.E., Cooperman B.S. 2011; The conserved protein EF4 (LepA) modulates the elongation cycle of protein synthesis. Proc Natl Acad Sci U S A 108:16223–16228 [View Article][PubMed]
     [Google Scholar]
  40. Mahenthiralingam E., Coenye T., Chung J.W., Speert D.P., Govan J.R., Taylor P., Vandamme P. 2000; Diagnostically and experimentally useful panel of strains from the Burkholderia cepacia complex. J Clin Microbiol 38:910–913[PubMed]
     [Google Scholar]
  41. Maravić A., Skočibušić M., Sprung M., Samanić I., Puizina J., Pavela-Vrančić M. 2012; Occurrence and antibiotic susceptibility profiles of Burkholderia cepacia complex in coastal marine environment. Int J Environ Health Res 22:531–542 [View Article][PubMed]
     [Google Scholar]
  42. Matsuoka H., Hirooka K., Fujita Y. 2007; Organization and function of the YsiA regulon of Bacillus subtilis involved in fatty acid degradation. J Biol Chem 282:5180–5194 [View Article][PubMed]
     [Google Scholar]
  43. Miller V.L., Mekalanos J.J. 1988; A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170:2575–2583[PubMed]
     [Google Scholar]
  44. Monahan L.G., Hajduk I.V., Blaber S.P., Charles I.G., Harry E.J. 2014; Coordinating bacterial cell division with nutrient availability: a role for glycolysis. MBio 5:e00935-14 [View Article][PubMed]
     [Google Scholar]
  45. Moule M.G., Hemsley C.M., Seet Q., Guerra-Assunção J.A., Lim J., Sarkar-Tyson M., Clark T.G., Tan P.B., Titball R.W., other authors. 2014; Genome-wide saturation mutagenesis of Burkholderia pseudomallei K96243 predicts essential genes and novel targets for antimicrobial development. MBio 5:e00926-13 [View Article][PubMed]
     [Google Scholar]
  46. Nagata T., Fukuda R., Koike I., Kogure K., Kirchman D.L. 1998; Degradation by bacteria of membrane and soluble protein in seawater. Aquat Microb Ecol 14:29–37 [View Article]
     [Google Scholar]
  47. Orsburn B.C., Melville S.B., Popham D.L. 2010; EtfA catalyses the formation of dipicolinic acid in Clostridium perfringens. Mol Microbiol 75:178–186 [View Article][PubMed]
     [Google Scholar]
  48. Ortega X.P., Cardona S.T., Brown A.R., Loutet S.A., Flannagan R.S., Campopiano D.J., Govan J.R., Valvano M.A. 2007; A putative gene cluster for aminoarabinose biosynthesis is essential for Burkholderia cenocepacia viability. J Bacteriol 189:3639–3644 [View Article][PubMed]
     [Google Scholar]
  49. Roberts D.L., Salazar D., Fulmer J.P., Frerman F.E., Kim J.J. 1999; Crystal structure of Paracoccus denitrificans electron transfer flavoprotein: structural and electrostatic analysis of a conserved flavin binding domain. Biochemistry 38:1977–1989 [View Article][PubMed]
     [Google Scholar]
  50. Roggo C., Coronado E., Moreno-Forero S.K., Harshman K., Weber J., van der Meer J.R. 2013; Genome-wide transposon insertion scanning of environmental survival functions in the polycyclic aromatic hydrocarbon degrading bacterium Sphingomonas wittichii RW1. Environ Microbiol 15:2681–2695[PubMed]
     [Google Scholar]
  51. Rushton L., Sass A., Baldwin A., Dowson C.G., Donoghue D., Mahenthiralingam E. 2013; Key role for efflux in the preservative susceptibility and adaptive resistance of Burkholderia cepacia complex bacteria. Antimicrob Agents Chemother 57:2972–2980 [View Article][PubMed]
     [Google Scholar]
  52. Sargent M.G. 1975; Control of cell length in Bacillus subtilis. J Bacteriol 123:7–19[PubMed]
     [Google Scholar]
  53. Scott J.D., Ludwig R.A. 2004; Azorhizobium caulinodans electron-transferring flavoprotein N electrochemically couples pyruvate dehydrogenase complex activity to N2 fixation. Microbiology 150:117–126 [View Article][PubMed]
     [Google Scholar]
  54. St John A.C., Jakubas K., Beim D. 1979; Degradation of proteins in steady-state cultures of Escherichia coli. Biochim Biophys Acta 586:537–544 [View Article][PubMed]
     [Google Scholar]
  55. Toogood H.S., Leys D., Scrutton N.S. 2007; Dynamics driving function: new insights from electron transferring flavoproteins and partner complexes. FEBS J 274:5481–5504 [View Article][PubMed]
     [Google Scholar]
  56. Tracy B.S., Edwards K.K., Eisenstark A. 2002; Carbon and nitrogen substrate utilization by archival Salmonella typhimurium LT2 cells. BMC Evol Biol 2:14–20 [View Article][PubMed]
     [Google Scholar]
  57. Tsai M.H., Saier M.H. Jr 1995; Phylogenetic characterization of the ubiquitous electron transfer flavoprotein families ETF-alpha and ETF-beta. Res Microbiol 146:397–404 [View Article][PubMed]
     [Google Scholar]
  58. Vandamme P., Dawyndt P. 2011; Classification and identification of the Burkholderia cepacia complex: past, present and future. Syst Appl Microbiol 34:87–95 [View Article][PubMed]
     [Google Scholar]
  59. Walt A., Kahn M.L. 2002; The fixA and fixB genes are necessary for anaerobic carnitine reduction in Escherichia coli. J Bacteriol 184:4044–4047 [View Article][PubMed]
     [Google Scholar]
  60. Weart R.B., Lee A.H., Chien A.C., Haeusser D.P., Hill N.S., Levin P.A. 2007; A metabolic sensor governing cell size in bacteria. Cell 130:335–347 [View Article][PubMed]
     [Google Scholar]
  61. Weidenhaupt M., Rossi P., Beck C., Fischer H.M., Hennecke H. 1996; Bradyrhizobium japonicum possesses two discrete sets of electron transfer flavoprotein genes: fixA, fixB and etfS, etfL. Arch Microbiol 165:169–178[PubMed]
     [Google Scholar]
  62. Winsor G.L., Khaira B., Van Rossum T., Lo R., Whiteside M.D., Brinkman F.S. 2008; The Burkholderia Genome Database: facilitating flexible queries and comparative analyses. Bioinformatics 24:2803–2804 [View Article][PubMed]
     [Google Scholar]
  63. Young K.D. 2010; Bacterial shape: two-dimensional questions and possibilities. Annu Rev Microbiol 64:223–240 [View Article][PubMed]
     [Google Scholar]
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An electron transfer flavoprotein is essential for viability and its depletion causes a rod-to-sphere change in Burkholderia cenocepacia
Microbiology 161, 1909 (2015); https://doi.org/10.1099/mic.0.000156
/content/journal/micro/10.1099/mic.0.000156
/content/journal/micro/10.1099/mic.0.000156
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