Cell permeability, β-lactamase activity, and transport contribute to high level of resistance to ampicillin in Lysobacter enzymogenes

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Discovery of multidrug resistance (MDR) in environmental microorganisms provides unique resources for uncovering antibiotic resistomes, which could be vital to predict future emergence of MDR pathogens. Our previous studies indicated that Lysobacter sp. conferred intrinsic resistance to multiple antibiotics at high levels, especially ampicillin, the first broad-spectrum β-lactam antibiotics against both Gram-positive and Gram-negative bacteria. However, the underlying molecular mechanisms for resistance to ampicillin in Lysobacter enzymogenes strain C3 (LeC3) remain unknown. In this study, screening a Tn5 transposon mutant library of LeC3 recovered 12 mutants with decreased ampicillin resistance, and three mutants (i.e., tatC, lebla, and lpp) were selected for further characterization. Our results revealed that genes encoding β-lactamase (lebla) and twin-arginine translocation (tatC) system for β-lactamase transport played a pivotal role in conferring ampicillin resistance in L. enzymogenes. It was also demonstrated that the lpp gene was not only involved in resistance against β-lactams but also conferred resistance to multiple antibiotics in L. enzymogenes. Permeability assay results indicated that decreased MDR in the lpp mutant was in part due to its higher cellular permeability. Furthermore, our results showed that the difference of LeC3 and L. antibioticus strain LaATCC29479 in ampicillin susceptibility was partly due to their differences in cellular permeability, but not due to β-lactamase activities.

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  1. Allen HK, Donato J, Wang HH, Cloud-Hansen KA, Davies J, Handelsman J (2010) Call of the wild: antibiotic resistance genes in natural environments. Nat Rev Microbiol 8:251

  2. Balder R, Shaffer TL, Lafontaine ER (2013) Moraxella catarrhalis uses a twin-arginine translocation system to secrete the β-lactamase BRO-2. BMC Microbiol 13:140

  3. Baltz RH (2008) Renaissance in antibacterial discovery from actinomycetes. Curr Opin Pharmacol 8:557–563

  4. Berendonk TU, Manaia CM, Merlin C, Fatta-Kassinos D, Cytryn E, Walsh F, Bürgmann H, Sørum H, Norström M, Pons M-N (2015) Tackling antibiotic resistance: the environmental framework. Nat Rev Microbiol 13:310

  5. Berks BC (2015) The twin-arginine protein translocation pathway. Annu Rev Biochem 84:843–864

  6. Breton C, Snajdrova L, Jeanneau C, Koca J, Imberty A (2006) Structures and mechanisms of glycosyltransferases. Glycobiology 16:29r–37r

  7. Brown ED, Wright GD (2016) Antibacterial drug discovery in the resistance era. Nature 529(7586):336–343

  8. Charrel RN, Pagès J-M, De Micco P, Mallea M (1996) Prevalence of outer membrane porin alteration in beta-lactam-antibiotic-resistant Enterobacter aerogenes. Antimicrob Agents Chemother 40:2854–2858

  9. Christensen P, Cook FD (1978) Lysobacter, a new genus of non-fruiting, gliding bacteria with a high base ratio. Int J Syst Bacteriol 28:367–393

  10. Clavijo RI, Loui C, Andersen GL, Riley LW, Lu S (2006) Identification of genes associated with survival of Salmonella enterica serovar Enteritidis in chicken egg albumen. Appl Environ Microbiol 72:1055–1064

  11. Control and Prevention (2013) Antibiotic resistance threats in the United States, 2013. Centres for Disease Control and Prevention, US Department of Health

  12. de Bruijn I, Cheng X, de Jager V, Expósito RG, Watrous J, Patel N, Postma J, Dorrestein PC, Kobayashi D, Raaijmakers JM (2015) Comparative genomics and metabolic profiling of the genus Lysobacter. BMC Genomics 16:991

  13. de Kraker ME, Stewardson AJ, Harbarth S (2016) Will 10 million people die a year due to antimicrobial resistance by 2050? PLoS Med 13:e1002184

  14. Delcour AH (2009) Outer membrane permeability and antibiotic resistance. Bioch Biophys Acta 1794:808–816

  15. Doyle F, Fosker G, Nayler J, Smith H (1962) Derivatives of 6-aminopenicillanic acid. Part I α-aminobenzylpenicillin and some related compounds. J Chem Soc 272:1440–1444

  16. Dyer TF (2002) Rapid identification of EZ::TN™ transposon insertion sites in the genome of Neisseria genorrhoeae. Epic Forum 9:2

  17. Ge Y, Lee JH, Hu B, Zhao YF (2018) Loss-of-function mutations in the Dpp and Opp permeases render Erwinia amylovora resistant to kasugamycin and blasticidin S. Mol Plant Microb Interact 31:823–832

  18. Giesler LJ, Yuen GY (1998) Evaluation of Stenotrophomonas maltophilia strain C3 for biocontrol of brown patch disease. Crop Prot 17:509–513

  19. Han Y, Wang Y, Yu Y, Chen H, Shen Y, Du L (2017) Indole-induced reversion of intrinsic multiantibiotic resistance in Lysobacter enzymogenes. Appl Environ Microbiol 83:e00995–e00917

  20. Hashizume H, Igarashi M, Hattori S, Hori M, Hamada M, Takeuchi T (2001) Tripropeptins, novel antimicrobial agents produced by Lysobacter sp. J Antibiot 54:1054–1059

  21. Hopwood DA (2007) How do antibiotic-producing bacteria ensure their self-resistance before antibiotic biosynthesis incapacitates them? Mol Microbiol 63:937–940

  22. Hsieh PC, Siegel SA, Rogers B, Davis D, Lewis K (1998) Bacteria lacking a multidrug pump: a sensitive tool for drug discovery. Proc Natl Acad Sci USA 95(12):6602–6606

  23. Jarosik GP, Hansen EJ (1995) Cloning and sequencing of the Haemophilus influenzae exbB and exbD genes. Gene 152:89–92

  24. Jekel PA, Weijer WJ, Beintema JJ (1983) Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein-sequence analysis. Anal Biochem 134:347–354

  25. Kaatz GW, Seo SM (1995) Inducible NorA-mediated multidrug resistance in Staphylococcus aureus. Antimicrob Agents Chemother 39(12):2650–2655

  26. King JD, Poon KKH, Webb NA, Anderson EM, McNally DJ, Brisson JR, Messner P, Garavito RM, Lam JS (2009) The structural basis for catalytic function of GMD and RMD, two closely related enzymes from the GDP-D-rhamnose biosynthesis pathway. FEBS J 276:2686–2700

  27. Koenning SR, Wrather JA (2010) Suppression of soybean yield potential in the continental United States by plant diseases from 2006 to 2009. Plant Health Prog.

  28. Kohanski MA, Dwyer DJ, Collins JJ (2010) How antibiotics kill bacteria: from targets to networks. Nat Rev Microbiol 8:423–435

  29. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176

  30. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

  31. Lee JH, Ancona V, Zhao Y (2018) Lon protease modulates virulence traits in Erwinia amylovora by direct monitoring of major regulators and indirectly through the Rcs and Gac-Csr regulatory systems. Mol Plant Pathol 19:827–840

  32. Leonard F, Markey B (2008) Meticillin-resistant Staphylococcus aureus in animals: a review. Vet J 175:27–36

  33. Lewis K (2012) Antibiotics: recover the lost art of drug discovery. Nature 485:439

  34. Mascher T (2013) Signaling diversity and evolution of extracytoplasmic function (ECF) σ factors. Curr Opin Microbiol 16:148–155

  35. Miclet E, Stoven V, Michels PA, Opperdoes FR, Lallemand JY, Duffieux F (2001) NMR spectroscopic analysis of the first two steps of the pentose-phosphate pathway elucidates the role of 6-phosphogluconolactonase. J Biol Chem 276:34840–34846

  36. Naas T, Oueslati S, Bonnin RA, Dabos ML, Zavala A, Dortet L, Retailleau P, Iorga BI (2017) Beta-lactamase database (BLDB)–structure and function. J Enzyme Inhib Med Chem 32:917–919

  37. Nicholas K, Nicholas HJ (1997) GeneDoc: a tool for editing and annotating multiple sequence alignments. Distributed by the author

  38. Nikaido H (2009) Multidrug resistance in bacteria. Ann Rrev Biochem 78:119–146

  39. O’Sullivan J, McCullough JE, Tymiak AA, Kirsch DR, Trejo WH, Principe PA (1988) Lysobactin, a novel antibacterial agent produced by Lysobacter sp. J Antibiot 41:1740–1744

  40. Palmer T, Berks BC (2012) The twin-arginine translocation (TAT) protein export pathway. Nat Rev Microbiol 10:483

  41. Park E, Rapoport TA (2012) Mechanisms of Sec61/SecY-mediated protein translocation across membranes. Ann Rrev Biophys 41:21–40

  42. Pearson MM, Hansen EJ (2007) Identification of gene products involved in biofilm production by Moraxella catarrhalis ETSU-9 in vitro. Infect Immun 75:4316–4325

  43. Price K (1970) Structure-activity relationships of semisynthetic penicillins. Adv Appl Microbiol 11:17–75

  44. Qian GL, Wang YL, Liu YR, Xu FF, He YW, Du LC, Venturi V, Fan JQ, Hu BS, Liu FQ (2013) Lysobacter enzymogenes uses two distinct cell-cell signaling systems for differential regulation of secondary-metabolite biosynthesis and colony morphology. Appl Environ Microbiol 79:6604–6616

  45. Raja A, LaBonte J, Lebbos J, Kirkpatrick P (2003) Daptomycin. Nat Rev Drug Discov 2:943–944

  46. Roca I, Akova M, Baquero F, Carlet J, Cavaleri M, Coenen S, Cohen J, Findlay D, Gyssens I, Heure O (2015) The global threat of antimicrobial resistance: science for intervention. New Microb New Infect 6:22–29

  47. Rodrigues L, Viveiros M, Ainsa J (2015) Measuring efflux and permeability in mycobacteria, Mycobacteria protocols, 3rd edn. Springer, New York, pp 227–240

  48. Shallcross LJ, Howard SJ, Fowler T, Davies SC (2015) Tackling the threat of antimicrobial resistance: from policy to sustainable action. Philos Trans R Soc B 370:20140082

  49. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539

  50. Smith SG, Mahon V, Lambert MA, Fagan RP (2007) A molecular Swiss army knife: OmpA structure, function and expression. FEMS Microbiol Lett 273:1–11

  51. Stanley NR, Findlay K, Berks BC, Palmer T (2001) Escherichia coli strains blocked in TAT-dependent protein export exhibit pleiotropic defects in the cell envelope. J Bacteriol 183:139–144

  52. Tahlan K, Ahn SK, Sing A, Bodnaruk TD, Willems AR, Davidson AR, Nodwell JR (2007) Initiation of actinorhodin export in Streptomyces coelicolor. Mol Microbiol 63:951–961

  53. Trafford J, Maclaren D, Lillicrap D, Barnes R, Houston J, Knox R (1962) Ampicillin. A broad-spectrum penicillin. Lancet:987–990

  54. Venugopal AA, Johnson S (2011) Fidaxomicin: a novel macrocyclic antibiotic approved for treatment of Clostridium difficile infection. Clin Infect Dis 54:568–574

  55. Vianney A, Muller MM, Clavel T, Lazzaroni JC, Portalier R, Webster RE (1996) Characterization of the Tol-pal region of Escherichia coli K-12: translational control of tolR expression by TolQ and identification of a new open reading frame downstream of pal encoding a periplasmic protein. J Bacteriol 178:4031–4038

  56. Vontigerstrom RG (1980) Extracellular nucleases of Lysobacter enzymogenes-production of the enzymes and purification and characterization of an endonuclease. Can J Microbiol 26(9):1029–1037

  57. Wang Y, Qian GL, Li YY, Wang YS, Wang YL, Wright S, Li YZ, Shen YM, Liu FQ, Du LC (2013) Biosynthetic mechanism for sunscreens of the biocontrol agent Lysobacter enzymogenes. PLoS One 8:e0066633

  58. Wilson BA, Salyers AA (2011) Bacterial pathogenesis: a molecular approach, 3rd edn. ASM Press, Washington, DC

  59. Yu M, Zhao Y (2019) Comparative resistomic analyses of Lysobacter species with high intrinsic multidrug resistance. J Global Antimicrobial Res.

  60. Zhang W, Li YY, Qian GL, Wang Y, Chen HT, Li YZ, Liu FQ, Shen YM, Du LC (2011) Identification and characterization of the anti-methicillin-resistant Staphylococcus aureus WAP-8294A2 biosynthetic gene cluster from Lysobacter enzymogenes OH11. Antimicrob Agents Chemother 55:5581–5589

  61. Zhang W, Huffman J, Li S, Shen Y, Du L (2017) Unusual acylation of chloramphenicol in Lysobacter enzymogenes, a biocontrol agent with intrinsic resistance to multiple antibiotics. BMC Biotechnol 17:59

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Correspondence to Youfu Zhao.

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Yu, M., Zhao, Y. Cell permeability, β-lactamase activity, and transport contribute to high level of resistance to ampicillin in Lysobacter enzymogenes. Appl Microbiol Biotechnol 104, 1149–1161 (2020) doi:10.1007/s00253-019-10266-7

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  • Lysobacter
  • Intrinsic resistance
  • Ampicillin
  • β-Lactamase
  • Tat transport system