1887

Abstract

sp. strain ATCC 39006 produces the red-pigmented antibiotic prodigiosin. Prodigiosin biosynthesis is regulated by a complex hierarchy that includes the uncharacterized protein YgfX (DUF1434). The gene is co-transcribed with , an FAD assembly factor essential for the flavinylation and activation of the SdhA subunit of succinate dehydrogenase (SDH), a central enzyme in the tricarboxylic acid cycle and electron transport chain. The operon is highly conserved within the , suggesting that SdhE and YgfX function together. We performed an extensive mutagenesis to gain molecular insights into the uncharacterized protein YgfX, and have investigated the relationship between YgfX and SdhE. YgfX localized to the membrane, interacted with itself, forming dimers or larger multimers, and interacted with SdhE. The transmembrane helices of YgfX were critical for protein function and the formation of YgfX multimers. Site-directed mutagenesis of residues conserved in DUF1434 proteins revealed a periplasmic tryptophan and a cytoplasmic aspartate that were crucial for YgfX activity. Both of these amino acids were required for the formation of YgfX multimers and interactions with SdhE but not membrane localization. Multiple cell division proteins were identified as putative interaction partners of YgfX and overexpression of YgfX had effects on cell morphology. These findings represent an important step in understanding the function of DUF1434 proteins. In contrast to a recent report, we found no evidence that YgfX and SdhE form a toxin–antitoxin system. In summary, YgfX functions as a multimeric membrane-bound protein that interacts with SdhE, an important FAD assembly factor that controls SDH activity.

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2013-07-01
2020-01-27
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References

  1. Akiyama Y., Yoshihisa T., Ito K..( 1995;). FtsH, a membrane-bound ATPase, forms a complex in the cytoplasmic membrane of Escherichia coli. J Biol Chem270:23485–23490 [CrossRef][PubMed]
    [Google Scholar]
  2. Alteri C. J., Smith S. N., Mobley H. L. T..( 2009;). Fitness of Escherichia coli during urinary tract infection requires gluconeogenesis and the TCA cycle. PLoS Pathog5:e1000448 [CrossRef][PubMed]
    [Google Scholar]
  3. Baba T., Ara T., Hasegawa M., Takai Y., Okumura Y., Baba M., Datsenko K. A., Tomita M., Wanner B. L., Mori H..( 2006;). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol2:0008 [CrossRef][PubMed]
    [Google Scholar]
  4. Bardy S. L., Jarrell K. F..( 2003;). Cleavage of preflagellins by an aspartic acid signal peptidase is essential for flagellation in the archaeon Methanococcus voltae. Mol Microbiol50:1339–1347 [CrossRef][PubMed]
    [Google Scholar]
  5. Bowden S. D., Ramachandran V. K., Knudsen G. M., Hinton J. C. D., Thompson A. R..( 2010;). An incomplete TCA cycle increases survival of Salmonella Typhimurium during infection of resting and activated murine macrophages. PLoS ONE5:e13871 [CrossRef][PubMed]
    [Google Scholar]
  6. Cecchini G..( 2003;). Function and structure of complex II of the respiratory chain. Annu Rev Biochem72:77–109 [CrossRef][PubMed]
    [Google Scholar]
  7. Cecchini G., Schröder I., Gunsalus R. P., Maklashina E..( 2002;). Succinate dehydrogenase and fumarate reductase from Escherichia coli. Biochim Biophys Acta1553:140–157 [CrossRef][PubMed]
    [Google Scholar]
  8. Cole C., Barber J. D., Barton G. J..( 2008;). The Jpred 3 secondary structure prediction server. Nucleic Acids Res36:Web Server issueW197–W201 [CrossRef][PubMed]
    [Google Scholar]
  9. Cook G. M., Robson J. R., Frampton R. A., McKenzie J., Przybilski R., Fineran P. C., Arcus V. L..( 2013;). Ribonucleases in bacterial toxin–antitoxin systems. Biochem Biophys Acta1829:523–531
    [Google Scholar]
  10. de Boer P. A., Crossley R. E., Hand A. R., Rothfield L. I..( 1991;). The MinD protein is a membrane ATPase required for the correct placement of the Escherichia coli division site. EMBO J10:4371–4380[PubMed]
    [Google Scholar]
  11. Edmondson D. E., Newton-Vinson P..( 2001;). The covalent FAD of monoamine oxidase: structural and functional role and mechanism of the flavinylation reaction. Antioxid Redox Signal3:789–806 [CrossRef][PubMed]
    [Google Scholar]
  12. Fineran P. C., Everson L., Slater H., Salmond G. P..( 2005a;). A GntR family transcriptional regulator (PigT) controls gluconate-mediated repression and defines a new, independent pathway for regulation of the tripyrrole antibiotic, prodigiosin, in Serratia.. Microbiology151:3833–3845 [CrossRef][PubMed]
    [Google Scholar]
  13. Fineran P. C., Slater H., Everson L., Hughes K., Salmond G. P..( 2005b;). Biosynthesis of tripyrrole and beta-lactam secondary metabolites in Serratia: integration of quorum sensing with multiple new regulatory components in the control of prodigiosin and carbapenem antibiotic production. Mol Microbiol56:1495–1517 [CrossRef][PubMed]
    [Google Scholar]
  14. Fineran P. C., Williamson N. R., Lilley K. S., Salmond G. P..( 2007;). Virulence and prodigiosin antibiotic biosynthesis in Serratia are regulated pleiotropically by the GGDEF/EAL domain protein, PigX. J Bacteriol189:7653–7662 [CrossRef][PubMed]
    [Google Scholar]
  15. Fineran P. C., Blower T. R., Foulds I. J., Humphreys D. P., Lilley K. S., Salmond G. P. C..( 2009;). The phage abortive infection system, ToxIN, functions as a protein–RNA toxin–antitoxin pair. Proc Natl Acad Sci U S A106:894–899 [CrossRef][PubMed]
    [Google Scholar]
  16. Finn R. D., Mistry J., Tate J., Coggill P., Heger A., Pollington J. E., Gavin O. L., Gunasekaran P., Ceric G. et al.( 2010;). The Pfam protein families database. Nucleic Acids Res38:Database issueD211–D222 [CrossRef][PubMed]
    [Google Scholar]
  17. Gerdes K., Wagner E. G. H..( 2007;). RNA antitoxins. Curr Opin Microbiol10:117–124 [CrossRef][PubMed]
    [Google Scholar]
  18. Gerdes K., Christensen S. K., Løbner-Olesen A..( 2005;). Prokaryotic toxin–antitoxin stress response loci. Nat Rev Microbiol3:371–382 [CrossRef][PubMed]
    [Google Scholar]
  19. Gristwood T., Fineran P. C., Everson L., Williamson N. R., Salmond G. P..( 2009;). The PhoBR two-component system regulates antibiotic biosynthesis in Serratia in response to phosphate. BMC Microbiol9:112 [CrossRef][PubMed]
    [Google Scholar]
  20. Gristwood T., McNeil M. B., Clulow J. S., Salmond G. P. C., Fineran P. C..( 2011;). PigS and PigP regulate prodigiosin biosynthesis in Serratia via differential control of divergent operons, which include predicted transporters of sulfur-containing molecules. J Bacteriol193:1076–1085 [CrossRef][PubMed]
    [Google Scholar]
  21. Hao H.-X., Khalimonchuk O., Schraders M., Dephoure N., Bayley J. P., Kunst H., Devilee P., Cremers C. W., Schiffman J. D. et al.( 2009;). SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma. Science325:1139–1142 [CrossRef][PubMed]
    [Google Scholar]
  22. Hederstedt L..( 1983;). Succinate dehydrogenase mutants of Bacillus subtilis lacking covalently bound flavin in the flavoprotein subunit. Eur J Biochem132:589–593 [CrossRef][PubMed]
    [Google Scholar]
  23. Heuts D. P. H. M., Scrutton N. S., McIntire W. S., Fraaije M. W..( 2009;). What’s in a covalent bond? On the role and formation of covalently bound flavin cofactors. FEBS J276:3405–3427 [CrossRef][PubMed]
    [Google Scholar]
  24. Käll L., Krogh A., Sonnhammer E. L. L..( 2007;). Advantages of combined transmembrane topology and signal peptide prediction – the Phobius web server. Nucleic Acids Res35:Web Server issueW429–W432 [CrossRef][PubMed]
    [Google Scholar]
  25. Karimova G., Pidoux J., Ullmann A., Ladant D..( 1998;). A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A95:5752–5756 [CrossRef][PubMed]
    [Google Scholar]
  26. Kelley L. A., Sternberg M. J..( 2009;). Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc4:363–371 [CrossRef][PubMed]
    [Google Scholar]
  27. Kim J., Fuller J. H., Kuusk V., Cunane L., Chen Z. W., Mathews F. S., McIntire W. S..( 1995;). The cytochrome subunit is necessary for covalent FAD attachment to the flavoprotein subunit of p-cresol methylhydroxylase. J Biol Chem270:31202–31209 [CrossRef][PubMed]
    [Google Scholar]
  28. Kimelman A., Levy A., Sberro H., Kidron S., Leavitt A., Amitai G., Yoder-Himes D. R., Wurtzel O., Zhu Y. et al.( 2012;). A vast collection of microbial genes that are toxic to bacteria. Genome Res22:802–809 [CrossRef][PubMed]
    [Google Scholar]
  29. Krell T., Lacal J., Busch A., Silva-Jiménez H., Guazzaroni M.-E., Ramos J. L..( 2010;). Bacterial sensor kinases: diversity in the recognition of environmental signals. Annu Rev Microbiol64:539–559 [CrossRef][PubMed]
    [Google Scholar]
  30. Kruse T., Bork-Jensen J., Gerdes K..( 2005;). The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex. Mol Microbiol55:78–89 [CrossRef][PubMed]
    [Google Scholar]
  31. Langridge G. C., Phan M.-D., Turner D. J., Perkins T. T., Parts L., Haase J., Charles I., Maskell D. J., Peters S. E. et al.( 2009;). Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Res19:2308–2316 [CrossRef][PubMed]
    [Google Scholar]
  32. Larkin M. A., Blackshields G., Brown N. P., Chenna R., McGettigan P. A., McWilliam H., Valentin F., Wallace I. M., Wilm A. et al.( 2007;). Clustal W and Clustal X version 2.0. Bioinformatics23:2947–2948 [CrossRef][PubMed]
    [Google Scholar]
  33. Lim K., Doseeva V., Demirkan E. S., Pullalarevu S., Krajewski W., Galkin A., Howard A., Herzberg O..( 2005;). Crystal structure of the YgfY from Escherichia coli, a protein that may be involved in transcriptional regulation. Proteins58:759–763 [CrossRef][PubMed]
    [Google Scholar]
  34. Mascher T..( 2006;). Intramembrane-sensing histidine kinases: a new family of cell envelope stress sensors in Firmicutes bacteria. FEMS Microbiol Lett264:133–144 [CrossRef][PubMed]
    [Google Scholar]
  35. Mascher T., Helmann J. D., Unden G..( 2006;). Stimulus perception in bacterial signal-transducing histidine kinases. Microbiol Mol Biol Rev70:910–938 [CrossRef][PubMed]
    [Google Scholar]
  36. Masuda H., Tan Q., Awano N., Wu K. P., Inouye M..( 2012a;). YeeU enhances the bundling of cytoskeletal polymers of MreB and FtsZ, antagonizing the CbtA (YeeV) toxicity in Escherichia coli.. Mol Microbiol84:979–989 [CrossRef][PubMed]
    [Google Scholar]
  37. Masuda H., Tan Q., Awano N., Yamaguchi Y., Inouye M..( 2012b;). A novel membrane-bound toxin for cell division, CptA (YgfX), inhibits polymerization of cytoskeleton proteins, FtsZ and MreB, in Escherichia coli.. FEMS Microbiol Lett328:174–181 [CrossRef][PubMed]
    [Google Scholar]
  38. McNeil M. B., Fineran P. C..( 2013;). Prokaryotic assembly factors for the attachment of flavin to complex II. Biochim Biophys Acta1827:637–647 [CrossRef][PubMed]
    [Google Scholar]
  39. McNeil M. B., Clulow J. S., Wilf N. M., Salmond G. P. C., Fineran P. C..( 2012;). SdhE is a conserved protein required for flavinylation of succinate dehydrogenase in bacteria. J Biol Chem287:18418–18428 [CrossRef][PubMed]
    [Google Scholar]
  40. Mercado-Lubo R., Gauger E. J., Leatham M. P., Conway T., Cohen P. S..( 2008;). A Salmonella enterica serovar typhimurium succinate dehydrogenase/fumarate reductase double mutant is avirulent and immunogenic in BALB/c mice. Infect Immun76:1128–1134 [CrossRef][PubMed]
    [Google Scholar]
  41. Mewies M., McIntire W. S., Scrutton N. S..( 1998;). Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: the current state of affairs. Protein Sci7:7–21 [CrossRef][PubMed]
    [Google Scholar]
  42. Miller J..( 1972;). Experiments in Molecular Genetics, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  43. Park S.-J., Tseng C.-P., Gunsalus R. P..( 1995;). Regulation of succinate dehydrogenase (sdhCDAB) operon expression in Escherichia coli in response to carbon supply and anaerobiosis: role of ArcA and Fnr. Mol Microbiol15:473–482 [CrossRef][PubMed]
    [Google Scholar]
  44. Park S. J., Chao G., Gunsalus R. P..( 1997;). Aerobic regulation of the sucABCD genes of Escherichia coli, which encode alpha-ketoglutarate dehydrogenase and succinyl coenzyme A synthetase: roles of ArcA, Fnr, and the upstream sdhCDAB promoter. J Bacteriol179:4138–4142[PubMed]
    [Google Scholar]
  45. Przybilski R., Richter C., Gristwood T., Clulow J. S., Vercoe R. B., Fineran P. C..( 2011;). Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum.. RNA Biol8:517–528 [CrossRef][PubMed]
    [Google Scholar]
  46. Robinson K. M., Rothery R. A., Weiner J. H., Lemire B. D..( 1994;). The covalent attachment of FAD to the flavoprotein of Saccharomyces cerevisiae succinate dehydrogenase is not necessary for import and assembly into mitochondria. Eur J Biochem222:983–990 [CrossRef][PubMed]
    [Google Scholar]
  47. Sambrook J., Fritsch E. F., Maniatis T..( 1989;). Molecular Cloning: a Laboratory Manual, , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press;
    [Google Scholar]
  48. Saraste M..( 1999;). Oxidative phosphorylation at the fin de siècle.. Science283:1488–1493 [CrossRef][PubMed]
    [Google Scholar]
  49. Sberro H., Leavitt A., Kiro R., Koh E., Peleg Y., Qimron U., Sorek R..( 2013;). Discovery of functional toxin/antitoxin systems in bacteria by shotgun cloning. Mol Cell50:136–148 [CrossRef][PubMed]
    [Google Scholar]
  50. Schiffer M., Chang C. H., Stevens F. J..( 1992;). The functions of tryptophan residues in membrane proteins. Protein Eng5:213–214 [CrossRef][PubMed]
    [Google Scholar]
  51. Sevin E. W., Barloy-Hubler F..( 2007;). RASTA-Bacteria: a web-based tool for identifying toxin–antitoxin loci in prokaryotes. Genome Biol8:R155 [CrossRef][PubMed]
    [Google Scholar]
  52. Shi J., Blundell T. L., Mizuguchi K..( 2001;). FUGUE: sequence–structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. J Mol Biol310:243–257 [CrossRef][PubMed]
    [Google Scholar]
  53. Slater H., Crow M., Everson L., Salmond G. P..( 2003;). Phosphate availability regulates biosynthesis of two antibiotics, prodigiosin and carbapenem, in Serratia via both quorum-sensing-dependent and -independent pathways. Mol Microbiol47:303–320 [CrossRef][PubMed]
    [Google Scholar]
  54. Smeitink J., van den Heuvel L., DiMauro S..( 2001;). The genetics and pathology of oxidative phosphorylation. Nat Rev Genet2:342–352 [CrossRef][PubMed]
    [Google Scholar]
  55. Tchawa Yimga M., Leatham M. P., Allen J. H., Laux D. C., Conway T., Cohen P. S..( 2006;). Role of gluconeogenesis and the tricarboxylic acid cycle in the virulence of Salmonella enterica serovar Typhimurium in BALB/c mice. Infect Immun74:1130–1140 [CrossRef][PubMed]
    [Google Scholar]
  56. Thomson N. R., Crow M. A., McGowan S. J., Cox A., Salmond G. P..( 2000;). Biosynthesis of carbapenem antibiotic and prodigiosin pigment in Serratia is under quorum sensing control. Mol Microbiol36:539–556 [CrossRef][PubMed]
    [Google Scholar]
  57. Vasudevan S. G., Tang P., Dixon N. E., Poole R. K..( 1995;). Distribution of the flavohaemoglobin, HMP, between periplasm and cytoplasm in Escherichia coli.. FEMS Microbiol Lett125:219–224 [CrossRef][PubMed]
    [Google Scholar]
  58. Wang X., Lord D. M., Cheng H.-Y., Osbourne D. O., Hong S. H., Sanchez-Torres V., Quiroga C., Zheng K., Herrmann T. et al.( 2012;). A new type V toxin–antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS. Nat Chem Biol8:855–861 [CrossRef][PubMed]
    [Google Scholar]
  59. Williamson N. R., Simonsen H. T., Ahmed R. A., Goldet G., Slater H., Woodley L., Leeper F. J., Salmond G. P..( 2005;). Biosynthesis of the red antibiotic, prodigiosin, in Serratia: identification of a novel 2-methyl-3-n-amyl-pyrrole (MAP) assembly pathway, definition of the terminal condensing enzyme, and implications for undecylprodigiosin biosynthesis in Streptomyces.. Mol Microbiol56:971–989 [CrossRef][PubMed]
    [Google Scholar]
  60. Williamson N. R., Fineran P. C., Leeper F. J., Salmond G. P..( 2006;). The biosynthesis and regulation of bacterial prodiginines. Nat Rev Microbiol4:887–899 [CrossRef][PubMed]
    [Google Scholar]
  61. Williamson N. R., Fineran P. C., Ogawa W., Woodley L. R., Salmond G. P..( 2008;). Integrated regulation involving quorum sensing, a two-component system, a GGDEF/EAL domain protein and a post-transcriptional regulator controls swarming and RhlA-dependent surfactant biosynthesis in Serratia.. Environ Microbiol10:1202–1217 [CrossRef][PubMed]
    [Google Scholar]
  62. Yankovskaya V., Horsefield R., Törnroth S., Luna-Chavez C., Miyoshi H., Léger C., Byrne B., Cecchini G., Iwata S..( 2003;). Architecture of succinate dehydrogenase and reactive oxygen species generation. Science299:700–704 [CrossRef][PubMed]
    [Google Scholar]
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