1887

Abstract

The gold standard method for the creation of gene deletions in Staphylococcus aureus is homologous recombination using allelic exchange plasmids with a temperature-sensitive origin of replication. A knockout vector that contains regions of homology is first integrated into the chromosome of S. aureus by a single crossover event selected for at high temperatures (non-permissive for plasmid replication) and antibiotic selection. Next, the second crossover event is encouraged by growth without antibiotic selection at low temperature, leading at a certain frequency to the excision of the plasmid and the deletion of the gene of interest. To detect or encourage plasmid loss, either a beta-galactosidase screening method or, more typically, a counterselection step is used. We present here the adaptation of the counter-selectable marker pheS*, coding for a mutated subunit of the phenylalanine tRNA synthetase, for use in S. aureus . The PheS* protein variant allows for the incorporation of the toxic phenylalanine amino acid analogue para-chlorophenylalanine (PCPA) into proteins and the addition of 20–40 mM PCPA to rich media leads to drastic growth reduction for S. aureus and supplementing chemically defined medium with 2.5–5 mM PCPA leads to complete growth inhibition. Using the new allelic exchange plasmid pIMAY*, we delete the magnesium transporter gene mgtE in S. aureus USA300 LAC* (SAUSA300_0910/SAUSA300_RS04895) and RN4220 (SAOUHSC_00945) and demonstrate that cobalt toxicity in S. aureus is mainly mediated by the presence of MgtE. This new plasmid will aid the efficient and easy creation of gene knockouts in S. aureus .

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2019-04-03
2019-08-23
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References

  1. Thammavongsa V, Kim HK, Missiakas D, Schneewind O. Staphylococcal manipulation of host immune responses. Nat Rev Microbiol 2015;13:529–543 [CrossRef][PubMed]
    [Google Scholar]
  2. Chambers HF, Deleo FR. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol 2009;7:629–641 [CrossRef][PubMed]
    [Google Scholar]
  3. Arnaud M, Chastanet A, Débarbouillé M. New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria. Appl Environ Microbiol 2004;70:6887–6891 [CrossRef][PubMed]
    [Google Scholar]
  4. Bae T, Schneewind O. Allelic replacement in Staphylococcus aureus with inducible counter-selection. Plasmid 2006;55:58–63 [CrossRef][PubMed]
    [Google Scholar]
  5. Brückner R. Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus. FEMS Microbiol Lett 1997;151:1–8 [CrossRef][PubMed]
    [Google Scholar]
  6. Geiger T, Francois P, Liebeke M, Fraunholz M, Goerke C et al. The stringent response of Staphylococcus aureus and its impact on survival after phagocytosis through the induction of intracellular PSMs expression. PLoS Pathog 2012;8:e1003016 [CrossRef][PubMed]
    [Google Scholar]
  7. Monk IR, Shah IM, Xu M, Tan MW, Foster TJ. Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis. mBio 2012;3: [CrossRef][PubMed]
    [Google Scholar]
  8. D'Elia MA, Pereira MP, Chung YS, Zhao W, Chau A et al. Lesions in teichoic acid biosynthesis in Staphylococcus aureus lead to a lethal gain of function in the otherwise dispensable pathway. J Bacteriol 2006;188:4183–4189 [CrossRef][PubMed]
    [Google Scholar]
  9. Redder P, Linder P. New range of vectors with a stringent 5-fluoroorotic acid-based counterselection system for generating mutants by allelic replacement in Staphylococcus aureus. Appl Environ Microbiol 2012;78:3846–3854 [CrossRef][PubMed]
    [Google Scholar]
  10. Chen W, Zhang Y, Yeo WS, Bae T, Ji Q. Rapid and Efficient Genome Editing in Staphylococcus aureus by Using an Engineered CRISPR/Cas9 System. J Am Chem Soc 2017;139:3790–3795 [CrossRef][PubMed]
    [Google Scholar]
  11. Liu Q, Jiang Y, Shao L, Yang P, Sun B et al. CRISPR/Cas9-based efficient genome editing in Staphylococcus aureus. Acta Biochim Biophys Sin 2017;49:764–770 [CrossRef][PubMed]
    [Google Scholar]
  12. Penewit K, Holmes EA, McLean K, Ren M, Waalkes A et al. Efficient and scalable precision genome editing in Staphylococcus aureus through conditional recombineering and CRISPR/Cas9-mediated counterselection. mBio 2018;9: [CrossRef][PubMed]
    [Google Scholar]
  13. Yao J, Zhong J, Fang Y, Geisinger E, Novick RP et al. Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing. RNA 2006;12:1271–1281 [CrossRef][PubMed]
    [Google Scholar]
  14. Kato F, Sugai M. A simple method of markerless gene deletion in Staphylococcus aureus. J Microbiol Methods 2011;87:76–81 [CrossRef][PubMed]
    [Google Scholar]
  15. Sun F, Cho H, Jeong DW, Li C, He C et al. Aureusimines in Staphylococcus aureus are not involved in virulence. PLoS One 2010;5:e15703 [CrossRef][PubMed]
    [Google Scholar]
  16. Leibig M, Krismer B, Kolb M, Friede A, Götz F et al. Marker removal in staphylococci via Cre recombinase and different lox sites. Appl Environ Microbiol 2008;74:1316–1323 [CrossRef][PubMed]
    [Google Scholar]
  17. Pagels M, Fuchs S, Pané-Farré J, Kohler C, Menschner L et al. Redox sensing by a Rex-family repressor is involved in the regulation of anaerobic gene expression in Staphylococcus aureus. Mol Microbiol 2010;76:1142–1161 [CrossRef][PubMed]
    [Google Scholar]
  18. Monk IR, Tree JJ, Howden BP, Stinear TP, Foster TJ. Complete bypass of restriction systems for major Staphylococcus aureus lineages. mBio 2015;6:e0030800315 [CrossRef][PubMed]
    [Google Scholar]
  19. Ibba M, Kast P, Hennecke H. Substrate specificity is determined by amino acid binding pocket size in Escherichia coli phenylalanyl-tRNA synthetase. Biochemistry 1994;33:7107–7112 [CrossRef][PubMed]
    [Google Scholar]
  20. Xie Z, Okinaga T, Qi F, Zhang Z, Merritt J. Cloning-independent and counterselectable markerless mutagenesis system in Streptococcus mutans. Appl Environ Microbiol 2011;77:8025–8033 [CrossRef][PubMed]
    [Google Scholar]
  21. Gurung I, Berry JL, Hall AMJ, Pelicic V. Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology. Nucleic Acids Res 2017;45:e40 [CrossRef][PubMed]
    [Google Scholar]
  22. Kristich CJ, Chandler JR, Dunny GM. Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis. Plasmid 2007;57:131–144 [CrossRef][PubMed]
    [Google Scholar]
  23. Kino Y, Nakayama-Imaohji H, Fujita M, Tada A, Yoneda S et al. Counterselection employing mutated pheS for markerless genetic deletion in Bacteroides species. Anaerobe 2016;42:81–88 [CrossRef][PubMed]
    [Google Scholar]
  24. Zhou C, Shi L, Ye B, Feng H, Zhang J et al. pheS (*), an effective host-genotype-independent counter-selectable marker for marker-free chromosome deletion in Bacillus amyloliquefaciens. Appl Microbiol Biotechnol 2017;101:217–227 [CrossRef][PubMed]
    [Google Scholar]
  25. Wang YC, Yuan LS, Tao HX, Jiang W, Liu CJ. pheS* as a counter-selectable marker for marker-free genetic manipulations in Bacillus anthracis. J Microbiol Methods 2018;151:35–38 [CrossRef][PubMed]
    [Google Scholar]
  26. Geissendörfer M, Hillen W. Regulated expression of heterologous genes in Bacillus subtilis using the Tn10 encoded tet regulatory elements. Appl Microbiol Biotechnol 1990;33:657–663 [CrossRef][PubMed]
    [Google Scholar]
  27. Helle L, Kull M, Mayer S, Marincola G, Zelder ME et al. Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus. Microbiology 2011;157:3314–3323 [CrossRef][PubMed]
    [Google Scholar]
  28. Payandeh J, Pfoh R, Pai EF. The structure and regulation of magnesium selective ion channels. Biochim Biophys Acta 2013;1828:2778–2792 [CrossRef][PubMed]
    [Google Scholar]
  29. Tomita A, Zhang M, Jin F, Zhuang W, Takeda H et al. ATP-dependent modulation of MgtE in Mg2+ homeostasis. Nat Commun 2017;8:148 [CrossRef][PubMed]
    [Google Scholar]
  30. Schuster CF, Bellows LE, Tosi T, Campeotto I, Corrigan RM et al. The second messenger c-di-AMP inhibits the osmolyte uptake system OpuC in Staphylococcus aureus. Sci Signal 2016;9:ra81 [CrossRef][PubMed]
    [Google Scholar]
  31. Price MN, Arkin AP. PaperBLAST: Text Mining Papers for Information about Homologs. mSystems 2017;2: [CrossRef][PubMed]
    [Google Scholar]
  32. Armitano J, Redder P, Guimarães VA, Linder P. An Essential Factor for High Mg2+ Tolerance of Staphylococcus aureus. Front Microbiol 2016;7:7 [CrossRef][PubMed]
    [Google Scholar]
  33. Nelson DL, Kennedy EP. Magnesium transport in Escherichia coli. Inhibition by cobaltous ion. J Biol Chem 1971;246:3042–3049[PubMed]
    [Google Scholar]
  34. Snavely MD, Florer JB, Miller CG, Maguire ME. Magnesium transport in Salmonella typhimurium: 28Mg2+ transport by the CorA, MgtA, and MgtB systems. J Bacteriol 1989;171:4761–4766 [CrossRef][PubMed]
    [Google Scholar]
  35. Hattori M, Iwase N, Furuya N, Tanaka Y, Tsukazaki T et al. Mg(2+)-dependent gating of bacterial MgtE channel underlies Mg(2+) homeostasis. EMBO J 2009;28:3602–3612 [CrossRef][PubMed]
    [Google Scholar]
  36. Kreiswirth BN, Löfdahl S, Betley MJ, O'Reilly M, Schlievert PM et al. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 1983;305:709–712 [CrossRef][PubMed]
    [Google Scholar]
  37. Boles BR, Thoendel M, Roth AJ, Horswill AR. Identification of genes involved in polysaccharide-independent Staphylococcus aureus biofilm formation. PLoS One 2010;5:e10146 [CrossRef][PubMed]
    [Google Scholar]
  38. Schuster CF, Bertram R. Fluorescence based primer extension technique to determine transcriptional starting points and cleavage sites of RNases in vivo. J Vis Exp 2014;92:e52134 [CrossRef][PubMed]
    [Google Scholar]
  39. Bowman L, Zeden MS, Schuster CF, Kaever V, Gründling A. New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus. J Biol Chem 2016;291:26970–26986 [CrossRef][PubMed]
    [Google Scholar]
  40. Gründling A, Schneewind O. Genes required for glycolipid synthesis and lipoteichoic acid anchoring in Staphylococcus aureus. J Bacteriol 2007;189:2521–2530 [CrossRef][PubMed]
    [Google Scholar]
  41. Zeden MS, Schuster CF, Bowman L, Zhong Q, Williams HD et al. Cyclic di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions. J Biol Chem 2018;293:3180–3200 [CrossRef][PubMed]
    [Google Scholar]
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