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Non-lethal exposure to H2O2 boosts bacterial survival and evolvability against oxidative stress


Autoři: Alexandro Rodríguez-Rojas aff001;  Joshua Jay Kim aff001;  Paul Johnston aff001;  Olga Makarova aff001;  Murat Eravci aff004;  Christoph Weise aff004;  Regine Hengge aff005;  Jens Rolff aff001;  Paul R. Johnston aff001
Působiště autorů: Freie Universität Berlin, Institute of Biology, Evolutionary Biology, Berlin, Germany aff001;  Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany aff002;  Berlin Center for Genomics in Biodiversity Research, Berlin, Germany aff003;  Freie Universität Berlin, Institute of Chemistry and Biochemistry, Berlin, Germany aff004;  Humboldt-Universität zu Berlin, Institut für Biologie/Mikrobiologie, Berlin, Germany aff005
Vyšlo v časopise: Non-lethal exposure to H2O2 boosts bacterial survival and evolvability against oxidative stress. PLoS Genet 16(3): e32767. doi:10.1371/journal.pgen.1008649
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008649

Souhrn

Unicellular organisms have the prevalent challenge to survive under oxidative stress of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). ROS are present as by-products of photosynthesis and aerobic respiration. These reactive species are even employed by multicellular organisms as potent weapons against microbes. Although bacterial defences against lethal and sub-lethal oxidative stress have been studied in model bacteria, the role of fluctuating H2O2 concentrations remains unexplored. It is known that sub-lethal exposure of Escherichia coli to H2O2 results in enhanced survival upon subsequent exposure. Here we investigate the priming response to H2O2 at physiological concentrations. The basis and the duration of the response (memory) were also determined by time-lapse quantitative proteomics. We found that a low level of H2O2 induced several scavenging enzymes showing a long half-life, subsequently protecting cells from future exposure. We then asked if the phenotypic resistance against H2O2 alters the evolution of resistance against oxygen stress. Experimental evolution of H2O2 resistance revealed faster evolution and higher levels of resistance in primed cells. Several mutations were found to be associated with resistance in evolved populations affecting different loci but, counterintuitively, none of them was directly associated with scavenging systems. Our results have important implications for host colonisation and infections where microbes often encounter reactive oxygen species in gradients.

Klíčová slova:

Frameshift mutation – Gene expression – Mutagenesis – Oxidative stress – Pathogen motility – Polymerase chain reaction – Priming (psychology) – Reactive oxygen species


Zdroje

1. Hilker M, Schwachtje J, Baier M, Balazadeh S, Bäurle I, Geiselhardt S, et al. Priming and memory of stress responses in organisms lacking a nervous system. Biol Rev Camb Philos Soc. 2015; doi: 10.1111/brv.12215 26289992

2. Wittmann C, Chockley P, Singh SK, Pase L, Lieschke GJ, Grabher C, et al. Hydrogen Peroxide in Inflammation: Messenger, Guide, and Assassin. Adv Hematol. Hindawi Publishing Corporation; 2012;2012: 1–6. doi: 10.1155/2012/541471 22737171

3. Obata F, Fons CO, Gould AP. Early-life exposure to low-dose oxidants can increase longevity via microbiome remodelling in Drosophila. Nat Commun. Nature Publishing Group; 2018;9: 975. doi: 10.1038/s41467-018-03070-w 29515102

4. Levis NA, Pfennig DW. Evaluating ‘Plasticity-First’ Evolution in Nature: Key Criteria and Empirical Approaches. Trends Ecol Evol. 2016;31: 563–574. doi: 10.1016/j.tree.2016.03.012 27067134

5. Zheng M, Wang X, Templeton LJ, Smulski DR, LaRossa RA, Storz G. DNA microarray-mediated transcriptional profiling of the Escherichia coli response to hydrogen peroxide. J Bacteriol. 2001;183: 4562–70. doi: 10.1128/JB.183.15.4562-4570.2001 11443091

6. Zheng M, Aslund F, Storz G. Activation of the OxyR transcription factor by reversible disulfide bond formation. Science. 1998;279: 1718–21. Available: http://www.ncbi.nlm.nih.gov/pubmed/9497290 doi: 10.1126/science.279.5357.1718 9497290

7. Imlay J a. Cellular defenses against superoxide and hydrogen peroxide. Annu Rev Biochem. 2008;77: 755–76. doi: 10.1146/annurev.biochem.77.061606.161055 18173371

8. Ezraty B, Vergnes A, Banzhaf M, Duverger Y, Huguenot A, Brochado AR, et al. Fe-S cluster biosynthesis controls uptake of aminoglycosides in a ROS-less death pathway. Science. 2013;340: 1583–7. doi: 10.1126/science.1238328 23812717

9. Imlay JA. The molecular mechanisms and physiological consequences of oxidative stress: Lessons from a model bacterium. Nature Reviews Microbiology. 2013. pp. 443–454. doi: 10.1038/nrmicro3032 23712352

10. Imlay JA. Transcription Factors That Defend Bacteria Against Reactive Oxygen Species. Annu Rev Microbiol. Annual Reviews; 2015;69: 93–108. doi: 10.1146/annurev-micro-091014-104322 26070785

11. Imlay JA, Chin SM, Linn S. Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science (80-). 1988/04/29. 1988;240: 640–642. doi: 10.1126/science.2834821 2834821

12. Luo Y, Henle ES, Linn S. Nucleic Acids, Protein Synthesis, and Molecular Genetics: Oxidative Damage to DNA Constituents by Iron-mediated Fenton Reactions: THE DEOXYCYTIDINE FAMILY Oxidative Damage to DNA Constituents by Iron-mediated. J Biol Chem. 1996;271: 21167–76. doi: 10.1074/jbc.271.35.21167 8702887

13. Jang S, Imlay JA. Hydrogen peroxide inactivates the Escherichia coli Isc iron-sulphur assembly system, and OxyR induces the Suf system to compensate. Mol Microbiol. 2010;78: 1448–1467. doi: 10.1111/j.1365-2958.2010.07418.x 21143317

14. Imlay JA, Linn S. Bimodal pattern of killing of DNA-repair-defective or anoxically grown Escherichia coli by hydrogen peroxide. J Bacteriol. 1986;166: 519–27. Available: http://www.ncbi.nlm.nih.gov/pubmed/3516975 doi: 10.1128/jb.166.2.519-527.1986 3516975

15. Jenkins DE, Schultz JE, Matin A. Starvation-induced cross protection against heat or H2O2 challenge in Escherichia coli. J Bacteriol. 1988;170: 3910–3914. doi: 10.1128/jb.170.9.3910-3914.1988 3045081

16. Lange R, Hengge-Aronis R. Identification of a central regulator of stationary-phase gene expression in Escherichia coli. Mol Microbiol. 1991;5: 49–59. doi: 10.1111/j.1365-2958.1991.tb01825.x 1849609

17. Ritz C, Baty F, Streibig JC, Gerhard D. Dose-Response Analysis Using R. Xia Y, editor. PLoS One. Public Library of Science; 2015;10: e0146021. doi: 10.1371/journal.pone.0146021 26717316

18. Basan M, Hui S, Okano H, Zhang Z, Shen Y, Williamson JR, et al. Overflow metabolism in Escherichia coli results from efficient proteome allocation. Nature. Nature Publishing Group; 2015;528: 99–104. doi: 10.1038/nature15765 26632588

19. Halliwell B, Clement MV, Long LH. Hydrogen peroxide in the human body. FEBS Lett. No longer published by Elsevier; 2000;486: 10–13. doi: 10.1016/s0014-5793(00)02197-9 11108833

20. Foyer CH, Noctor G. Stress-triggered redox signalling: What’s in pROSpect? Plant Cell and Environment. Blackwell Publishing Ltd; 2016. pp. 951–964. doi: 10.1111/pce.12621 26264148

21. Ronin I, Katsowich N, Rosenshine I, Balaban NQ. A long-term epigenetic memory switch controls bacterial virulence bimodality. Elife. 2017;6. doi: 10.7554/eLife.19599 28178445

22. Veening J-W, Smits WK, Kuipers OP. Bistability, Epigenetics, and Bet-Hedging in Bacteria. Annu Rev Microbiol. Annual Reviews; 2008;62: 193–210. doi: 10.1146/annurev.micro.62.081307.163002 18537474

23. Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, et al. STRING v10: protein–protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 2015;43: D447–D452. doi: 10.1093/nar/gku1003 25352553

24. Arifuzzaman M, Maeda M, Itoh A, Nishikata K, Takita C, Saito R, et al. Large-scale identification of protein-protein interaction of Escherichia coli K-12. Genome Res. 2006;16: 686–691. doi: 10.1101/gr.4527806 16606699

25. Basineni SR, Madhugiri R, Kolmsee T, Hengge R, Klug G. The influence of Hfq and ribonucleases on the stability of the small non-coding RNA OxyS and its target rpoS in E. coli is growth phase dependent. RNA Biol. 2009/12/18. 2009;6: 584–594. 10082 [pii] doi: 10.4161/rna.6.5.10082 20016254

26. González-Flecha B, Demple B. Role for the oxyS gene in regulation of intracellular hydrogen peroxide in Escherichia coli. J Bacteriol. American Society for Microbiology; 1999;181: 3833–6. Available: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=93864&tool=pmcentrez&rendertype=abstract 10368161

27. Zhang A, Altuvia S, Tiwari A, Argaman L, Hengge-Aronis R, Storz G. The OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF-I) protein. EMBO J. 1998;17: 6061–6068. doi: 10.1093/emboj/17.20.6061 9774349

28. Altuvia S, Weinstein-Fischer D, Zhang A, Postow L, Storz G. A small, stable RNA induced by oxidative stress: Role as a pleiotropic regulator and antimutator. Cell. Cell Press; 1997;90: 43–53. doi: 10.1016/s0092-8674(00)80312-8 9230301

29. Barshishat S, Elgrably‐Weiss M, Edelstein J, Georg J, Govindarajan S, Haviv M, et al. OxyS small RNA induces cell cycle arrest to allow DNA damage repair. EMBO J. 2018;37: 413–426. doi: 10.15252/embj.201797651 29237698

30. Willi J, Küpfer P, Eviquoz D, Fernandez G, Katz A, Leumann C, et al. Oxidative stress damages rRNA inside the ribosome and differentially affects the catalytic center. Nucleic Acids Res. Oxford University Press; 2018;46: 1945–1957. doi: 10.1093/nar/gkx1308 29309687

31. Aslund F, Zheng M, Beckwith J, Storz G. Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status. Proc Natl Acad Sci U S A. 1999;96: 6161–5. Available: http://www.ncbi.nlm.nih.gov/pubmed/10339558 doi: 10.1073/pnas.96.11.6161 10339558

32. Liu Y, Bauer SC, Imlay JA. The YaaA protein of the Escherichia coli OxyR regulon lessens hydrogen peroxide toxicity by diminishing the amount of intracellular unincorporated iron. J Bacteriol. American Society for Microbiology (ASM); 2011;193: 2186–96. doi: 10.1128/JB.00001-11 21378183

33. Ollagnier-De Choudens S, Sanakis Y, Hewitson KS, Roach P, Baldwin JE, Münck E, et al. Iron-sulfur center of biotin synthase and lipoate synthase. Biochemistry. 2000;39: 4165–73. Available: http://www.ncbi.nlm.nih.gov/pubmed/10747808 doi: 10.1021/bi992090u 10747808

34. Hong Y, Zeng J, Wang X, Drlica K, Zhao X. Post-stress bacterial cell death mediated by reactive oxygen species. Proc Natl Acad Sci U S A. National Academy of Sciences; 2019;116: 10064–10071. doi: 10.1073/pnas.1901730116 30948634

35. Beloin C, Roux A, Ghigo JM. Escherichia coli biofilms. Current Topics in Microbiology and Immunology. 2008. pp. 249–289. doi: 10.1007/978-3-540-75418-3_12 18453280

36. Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, Toyonaga H, et al. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res. 2006/06/14. 2005;12: 291–299. doi: 10.1093/dnares/dsi012 16769691

37. Pratt LA, Kolter R. Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili. Mol Microbiol. 1998;30: 285–293. doi: 10.1046/j.1365-2958.1998.01061.x 9791174

38. Schembri MA, Klemm P. Coordinate gene regulation by fimbriae-induced signal transduction. EMBO J. 2001;20: 3074–3081. doi: 10.1093/emboj/20.12.3074 11406584

39. Stentebjerg-Olesen B, Chakraborty T, Klemm P. FimE-catalyzed off-to-on inversion of the type 1 fimbrial phase switch and insertion sequence recruitment in an Escherichia coli K-12 fimB strain. FEMS Microbiol Lett. 2000;182: 319–325. doi: 10.1111/j.1574-6968.2000.tb08915.x 10620686

40. Avalos Vizcarra I, Hosseini V, Kollmannsberger P, Meier S, Weber SS, Arnoldini M, et al. How type 1 fimbriae help Escherichia coli to evade extracellular antibiotics. Sci Rep. Nature Publishing Group; 2016;6. doi: 10.1038/srep18109 26728082

41. Keith BR, Harris SL, Russell PW, Orndorff PE. Effect of type 1 piliation on in vitro killing of Escherichia coli by mouse peritoneal macrophages. Infect Immun. 1990;58: 3448–54. Available: http://www.ncbi.nlm.nih.gov/pubmed/1976116 1976116

42. Slauch JM. How does the oxidative burst of macrophages kill bacteria? Still an open question. Mol Microbiol. 2011;80: 580–583. doi: 10.1111/j.1365-2958.2011.07612.x 21375590

43. Wang S, Fleming RT, Westbrook EM, Matsumura P, McKay DB. Structure of the Escherichia coli FlhDC complex, a prokaryotic heteromeric regulator of transcription. J Mol Biol. 2006;355: 798–808. doi: 10.1016/j.jmb.2005.11.020 16337229

44. Barker CS, Prüss BM, Matsumura P. Increased motility of Escherichia coli by insertion sequence element integration into the regulatory region of the flhD operon. J Bacteriol. 2004;186: 7529–37. doi: 10.1128/JB.186.22.7529-7537.2004 15516564

45. Martínez-García E, Nikel PI, Chavarría M, de Lorenzo V. The metabolic cost of flagellar motion in Pseudomonas putida KT2440. Environ Microbiol. 2014;16: 291–303. doi: 10.1111/1462-2920.12309 24148021

46. Sarenko O, Klauck G, Wilke FM, Pfiffer V, Richter AM, Herbst S, et al. More than Enzymes That Make or Break Cyclic Di-GMP-Local Signaling in the Interactome of GGDEF/EAL Domain Proteins of Escherichia coli. doi: 10.1128/mBio.01639-17 29018125

47. Wang X, Kim Y, Ma Q, Hong SH, Pokusaeva K, Sturino JM, et al. Cryptic prophages help bacteria cope with adverse environments. Nat Commun. Nature Publishing Group; 2010;1: 147. doi: 10.1038/ncomms1146 21266997

48. Morgan C, Lewis PD. iMARS—Mutation analysis reporting software: An analysis of spontaneous cII mutation spectra. Mutat Res—Genet Toxicol Environ Mutagen. 2006;603: 15–26. doi: 10.1016/j.mrgentox.2005.09.010 16359913

49. Kino K, Sugiyama H. Possible cause of G·C→C·G transversion mutation by guanine oxidation product, imidazolone. Chem Biol. Cell Press; 2001;8: 369–378. doi: 10.1016/s1074-5521(01)00019-9 11325592

50. Rodríguez-Rojas A, Makarova O, Müller U, Rolff J. Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria. Matic I, editor. Public Library of Science; 2015;11: e1005546. doi: 10.1371/journal.pgen.1005546 26430769

51. Rodríguez-Rojas A, Makarova O, Rolff J. Antimicrobials, stress and mutagenesis. Zasloff M, editor. PLoS Pathog. Public Library of Science; 2014;10: e1004445. doi: 10.1371/journal.ppat.1004445 25299705

52. Jang S, Imlay JA. Micromolar intracellular hydrogen peroxide disrupts metabolism by damaging iron-sulfur enzymes. J Biol Chem. 2006/11/15. 2007;282: 929–937. M607646200 [pii] doi: 10.1074/jbc.M607646200 17102132

53. Galhardo RS, Hastings PJ, Rosenberg SM. Mutation as a Stress Response and the Regulation of Evolvability. Crit Rev Biochem Mol Biol. 2007;42: 399–435. doi: 10.1080/10409230701648502 17917874

54. Blázquez J, Couce A, Rodríguez-Beltrán J, Rodríguez-Rojas A, Blazquez J, Couce A, et al. Antimicrobials as promoters of genetic variation. Curr Opin Microbiol. 2012/08/15. 2012;15: 561–569. S1369-5274(12)00102-6 [pii] doi: 10.1016/j.mib.2012.07.007 22890188

55. Ram Y, Hadany L. THE EVOLUTION OF STRESS-INDUCED HYPERMUTATION IN ASEXUAL POPULATIONS. Evolution (N Y). 2012;66: 2315–2328. doi: 10.1111/j.1558-5646.2012.01576.x 22759304

56. Couce A, Rodríguez-Rojas A, Blázquez J. Bypass of genetic constraints during mutator evolution to antibiotic resistance. Proc Biol Sci. 2015;282: 20142698. doi: 10.1098/rspb.2014.2698 25716795

57. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000/06/01. 2000;97: 6640–6645. doi: 10.1073/pnas.120163297 120163297 [pii] 10829079

58. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2006/06/02. 2006;2: 2006 0008. msb4100050 [pii] doi: 10.1038/msb4100050 16738554

59. Thomason LC, Costantino N, Court DL. E. coli Genome Manipulation by P1 Transduction. Current Protocols in Molecular Biology. Hoboken, NJ, USA: John Wiley & Sons, Inc.; 2007. pp. 1.17.1–1.17.8. doi: 10.1002/0471142727.mb0117s79 18265391

60. Navarro JA, Roncel M, De la Rosa FF, De la Rosa MA. Light-driven hydrogen peroxide production as a way to solar energy conversion. Bioelectrochemistry Bioenerg. Elsevier; 1987;18: 71–78. doi: 10.1016/0302-4598(87)85009-2

61. Sprouffske K, Wagner A. Growthcurver: an R package for obtaining interpretable metrics from microbial growth curves. BMC Bioinformatics. BioMed Central; 2016;17: 172. doi: 10.1186/s12859-016-1016-7 27094401

62. Rappsilber J, Mann M, Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc. 2007;2: 1896–906. doi: 10.1038/nprot.2007.261 17703201

63. Tyanova S, Temu T, Cox J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc. Nature Publishing Group; 2016;11: 2301–2319. doi: 10.1038/nprot.2016.136 27809316

64. Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods. 2016;13: 731–40. doi: 10.1038/nmeth.3901 27348712

65. Bachmair A, Finley D, Varshavsky A. In vivo half-life of a protein is a function of its amino-terminal residue. Science (80-). 1986;234: 179–186. doi: 10.1126/science.3018930 3018930

66. Ciechanover A, Schwartz AL. How are substrates recognized by the ubiquitin-mediated proteolytic system? Trends Biochem Sci. 1989;14: 483–8. doi: 10.1016/0968-0004(89)90180-1 2696178

67. Tobias JW, Shrader TE, Rocap G, Varshavsky A. The N-end rule in bacteria. Science. 1991;254: 1374–7. doi: 10.1126/science.1962196 1962196

68. Guruprasad K, Reddy B V, Pandit MW. Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng. 1990;4: 155–61. doi: 10.1093/protein/4.2.155 2075190

69. Wilkins MR, Gasteiger E, Bairoch A, Sanchez J-C, Williams KL, Appel RD, et al. Protein Identification and Analysis Tools in the ExPASy Server. 2-D Proteome Analysis Protocols. New Jersey: Humana Press; 1999. pp. 531–552. doi: 10.1385/1-59259-584-7:531

70. Humbard MA, Surkov S, De Donatis GM, Jenkins LM, Maurizi MR. The N-degradome of Escherichia coli: limited proteolysis in vivo generates a large pool of proteins bearing N-degrons. J Biol Chem. American Society for Biochemistry and Molecular Biology; 2013;288: 28913–24. doi: 10.1074/jbc.M113.492108 23960079

71. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004;26: 509–15. Available: http://www.ncbi.nlm.nih.gov/pubmed/15127793 doi: 10.1023/b:bile.0000019559.84305.47 15127793

72. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002;30: e36. Available: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=113859&tool=pmcentrez&rendertype=abstract doi: 10.1093/nar/30.9.e36 11972351

73. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55: 611–22. doi: 10.1373/clinchem.2008.112797 19246619

74. Seemann T. Snippy: fast bacterial variant calling from NGS reads [Internet]. 2015. Available: https://github.com/tseemann/snippy

75. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25: 1754–1760. doi: 10.1093/bioinformatics/btp324 19451168

76. Garrison E, Marth G. Haplotype-based variant detection from short-read sequencing. 2012; Available: https://arxiv.org/pdf/1207.3907.pdf

77. Deatherage DE, Barrick JE. Identification of Mutations in Laboratory-Evolved Microbes from Next-Generation Sequencing Data Using breseq. Methods in molecular biology (Clifton, NJ). 2014. pp. 165–188. doi: 10.1007/978-1-4939-0554-6_12 24838886

78. Sanders LH, Sudhakaran J, Sutton MD. The GO system prevents ROS-induced mutagenesis and killing in Pseudomonas aeruginosa. FEMS Microbiol Lett. 2009/03/18. 2009;294: 89–96. FML1550 [pii] doi: 10.1111/j.1574-6968.2009.01550.x 19291074

79. Rodriguez-Rojas A, Blazquez J, Rodríguez-Rojas A, Blázquez J. The Pseudomonas aeruginosa pfpI gene plays an antimutator role and provides general stress protection. J Bacteriol. 2008/11/26. American Society for Microbiology; 2009;191: 844–850. JB.01081-08 [pii] doi: 10.1128/JB.01081-08 19028889

80. Fahrner KA, Berg HC. Mutations That Stimulate flhDC Expression in Escherichia coli K-12. J Bacteriol. 2015;197: 3087–96. doi: 10.1128/JB.00455-15 26170415

81. R Development Core Team. RStudio Team (2015). RStudio: Integrated Development for R. [Internet]. Boston: MA URL http://www.rstudio.com/; 2015. Available: http://www.rstudio.com


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