Efflux proteins MacAB confer resistance to arsenite and penicillin/macrolide-type antibiotics in Agrobacterium tumefaciens 5A

  • Kaixiang Shi
  • Min Cao
  • Chan Li
  • Jing Huang
  • Shixue Zheng
  • Gejiao WangEmail author
Original Paper


Antibiotic and arsenic (As) contaminations are worldwide public health problems. Previously, the bacterial ABC-type efflux protein MacAB reportedly conferred resistance to macrolide-type antibiotics but not to other metal(loid)s. In this study, the roles of MacAB for the co-resistance of different antibiotics and several metal(loid)s were analyzed in Agrobacterium tumefaciens 5A, a strain resistant to arsenite [As(III)] and several types of antibiotics. The macA and macB genes were cotranscribed, and macB was deleted in A. tumefaciens 5A and heterologously expressed in Escherichia coli AW3110 and E. coli S17-1. Compared to the wild-type strain 5A, the macB deletion strain reduced bacterial resistance levels to several macrolide-type and penicillin-type antibiotics but not to cephalosporin-type antibiotics. In addition, the macB deletion strain showed lower resistance to As(III) but not to arsenate [As(V)], antimonite [Sb(III)] and cadmium chloride [Cd(II)]. The mutant strain 5A-ΔmacB cells accumulated more As(III) than the cells of the wild-type. Furthermore, heterologous expression of MacAB in E. coli S17-1 showed that MacAB was essential for resistance to macrolide, several penicillin-type antibiotics and As(III) but not to As(V). Heterologous expression of MacAB in E. coli AW3110 reduced the cellular accumulation of As(III) but not of As(V), indicating that MacAB is responsible for the efflux of As(III). These results demonstrated that, in addition to macrolide-type antibiotics, MacAB also conferred resistance to penicillin-type antibiotics and As(III) by extruding them out of cells. This finding contributes to a better understanding of the bacterial resistance mechanisms of antibiotics and metal(loid)s.


Agrobacterium tumefaciens Antibiotics Arsenite MacAB Macrolide Penicillin 



The study was supported by the National Natural Science Foundation of China (31470226). We thank Dr. Timothy McDermott for providing A. tumefaciens 5A and the plasmid pJQ200SK and Dr. Christopher Rensing for providing E. coli AW3110.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflicts of interest.

Supplementary material

11274_2019_2689_MOESM1_ESM.docx (2.3 mb)
Supplementary file1 (DOCX 2312 kb)


  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 8:251–259CrossRefGoogle Scholar
  2. Amachawadi RG, Scott HM, Alvarado CA, Mainini TR, Vinasco J, Drouillard JS, Nagaraja TG (2013) Occurrence of the transferable copper resistance gene tcrB among fecal enterococci of U.S. feedlot cattle fed copper-supplemented diets. Appl Environ Microbiol 79:4369–4375CrossRefGoogle Scholar
  3. Baker-Austin C, Wright MS, Stepanauskas R, McArthur JV (2006) Co-selection of antibiotic and metal resistance. Trends Microbiol 14:176–182CrossRefGoogle Scholar
  4. Cai L, Liu G, Rensing C, Wang G (2009) Genes involved in arsenic transformation and resistance associated with different levels of arsenic-contaminated soils. BMC Microbiol 9:4CrossRefGoogle Scholar
  5. Carlin A, Shi W, Dey S, Rosen BP (1995) The ars operon of Escherichia coli confers arsenical and antimonial resistance. J Bacteriol 177:981–986CrossRefGoogle Scholar
  6. Carlin DJ et al (2015) Arsenic and environmental health: state of the science and future research opportunities. Environ Health Perspect 124:890–899CrossRefGoogle Scholar
  7. Chen F, Cao Y, Wei S, Li Y, Li X, Wang Q, Wang G (2015a) Regulation of arsenite oxidation by the phosphate two-component system PhoBR in Halomonas sp. HAL1. Front Microbiol 6:923PubMedPubMedCentralGoogle Scholar
  8. Chen S, Li X, Sun G, Zhang Y, Su J, Ye J (2015b) Heavy metal induced antibiotic resistance in bacterium LSJC7. Int J Mol Sci 16:23390–23404CrossRefGoogle Scholar
  9. Chen J, Yoshinaga M, Rosen BP (2019) The antibiotic action of methylarsenite is an emergent property of microbial communities. Mol Microbiol 111:487–494CrossRefGoogle Scholar
  10. Cheng J, Hicks DB, Krulwich TA (1996) The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline-cobalt/H+ and Na+(K+)/H+ exchange. Proc Natl Acad Sci USA 93:14446–14451CrossRefGoogle Scholar
  11. Escudero-Lourdes C (2016) Toxicity mechanisms of arsenic that are shared with neurodegenerative diseases and cognitive impairment: Role of oxidative stress and inflammatory responses. Neurotoxicology 53:223–235CrossRefGoogle Scholar
  12. Fan H, Su C, Wang Y, Yao J, Zhao K, Wang Y, Wang G (2008) Sedimentary arsenite-oxidizing and arsenate-reducing bacteria associated with high arsenic groundwater from Shanyin, Northwestern China. J Appl Microbiol 105:529–539CrossRefGoogle Scholar
  13. German N, Luthje F, Hao X, Ronn R, Rensing C (2016) Microbial virulence and interactions with metals. Prog Mol Biol Transl Sci 142:27–49CrossRefGoogle Scholar
  14. Guo X, Liu S, Wang Z, Zhang XX, Li M, Wu B (2014) Metagenomic profiles and antibiotic resistance genes in gut microbiota of mice exposed to arsenic and iron. Chemosphere 112:1–8CrossRefGoogle Scholar
  15. Henriques I, Tacao M, Leite L, Fidalgo C, Araujo S, Oliveira C, Alves A (2016) Co-selection of antibiotic and metal(loid) resistance in gram-negative epiphytic bacteria from contaminated salt marshes. Mar Pollut Bull 109:427–434CrossRefGoogle Scholar
  16. Huang JJ, Hu HY, Lu SQ, Li Y, Tang F, Lu Y, Wei B (2012) Monitoring and evaluation of antibiotic-resistant bacteria at a municipal wastewater treatment plant in China. Environ Int 42:31–36CrossRefGoogle Scholar
  17. Jacoby GA (2009) AmpC beta-lactamases. Clin Microbiol Rev 22:161–182CrossRefGoogle Scholar
  18. Kang YS, Heinemann J, Bothner B, Rensing C, McDermott TR (2012) Integrated co-regulation of bacterial arsenic and phosphorus metabolisms. Environ Microbiol 14:3097–3109CrossRefGoogle Scholar
  19. Kang Y-S, Shi Z, Bothner B, Wang G, McDermott TR (2015) Involvement of the Acr3 and DctA anti-porters in arsenite oxidation in Agrobacterium tumefaciens 5A. Environ Microbiol 17:1950–1962CrossRefGoogle Scholar
  20. Kashyap DR, Botero LM, Franck WL, Hassett DJ, McDermott TR (2006) Complex regulation of arsenite oxidation in Agrobacterium tumefaciens. J Bacteriol 188:1081–1088CrossRefGoogle Scholar
  21. Kobayashi N, Nishino K, Yamaguchi A (2001) Novel macrolide-specific ABC-type efflux transporter in Escherichia coli. J Bacteriol 183:5639–5644CrossRefGoogle Scholar
  22. Kruger MC, Bertin PN, Heipieper HJ, Arsene-Ploetze F (2013) Bacterial metabolism of environmental arsenic: mechanisms and biotechnological applications. Appl Microbiol Biotechnol 97:3827–3841CrossRefGoogle Scholar
  23. Li H, Li M, Huang Y, Rensing C, Wang G (2013) In silico analysis of bacterial arsenic islands reveals remarkable synteny and functional relatedness between arsenate and phosphate. Front Microbiol 4:347PubMedPubMedCentralGoogle Scholar
  24. Li X, Zhang L, Wang G (2014) Genomic evidence reveals the extreme diversity and wide distribution of the arsenic-related genes in Burkholderiales. PLoS One 9:e92236CrossRefGoogle Scholar
  25. Lin MF, Lin YY, Lan CY (2015) The role of the two-component system BaeSR in disposing chemicals through regulating transporter systems in Acinetobacter baumannii. PLoS One 10:e0132843CrossRefGoogle Scholar
  26. Link AJ, Phillips D, Church GM (1997) Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. J Bacteriol 179:6228–6237CrossRefGoogle Scholar
  27. Liu G et al (2012) A periplasmic arsenite-binding protein involved in regulating arsenite oxidation. Environ Microbiol 14:1624–1634CrossRefGoogle Scholar
  28. Lu S, Zgurskaya HI (2013) MacA, a periplasmic membrane fusion protein of the macrolide transporter MacAB-TolC, binds lipopolysaccharide core specifically and with high affinity. J Bacteriol 195:4865–4872CrossRefGoogle Scholar
  29. Macur RE, Jackson CR, Botero LM, McDermott TR, Inskeep WP (2004) Bacterial populations associated with the oxidation and reduction of arsenic in an unsaturated soil. Environ Sci Technol 38:104–111CrossRefGoogle Scholar
  30. Meng YL, Liu Z, Rosen BP (2004) As(III) and Sb(III) uptake by GlpF and efflux by ArsB in Escherichia coli. J Biol Chem 279:18334–18341CrossRefGoogle Scholar
  31. Nikaido H, Takatsuka Y (2009) Mechanisms of RND multidrug efflux pumps. Biochim Biophys Acta 1794:769–781CrossRefGoogle Scholar
  32. Pal C, Asiani K, Arya S, Rensing C, Stekel DJ, Larsson DGJ, Hobman JL (2017) Metal resistance and its association with antibiotic resistance. Adv Microb Physiol 70:261–313CrossRefGoogle Scholar
  33. Perron K, Caille O, Rossier C, Van Delden C, Dumas JL, Kohler T (2004) CzcR-CzcS, a two-component system involved in heavy metal and carbapenem resistance in Pseudomonas aeruginosa. J Biol Chem 279:8761–8768CrossRefGoogle Scholar
  34. Ren Y, Ren Y, Zhou Z, Guo X, Li Y, Feng L, Wang L (2010) Complete genome sequence of Enterobacter cloacae subsp. cloacae type strain ATCC 13047. J Bacteriol 192:2463–2464CrossRefGoogle Scholar
  35. Rosen BP, Ajees AA, McDermott TR (2011) Life and death with arsenic. Arsenic life: an analysis of the recent report "A bacterium that can grow by using arsenic instead of phosphorus". BioEssays 33:350–357CrossRefGoogle Scholar
  36. Sapkota A, Sapkota AR, Kucharski M, Burke J, McKenzie S, Walker P, Lawrence R (2008) Aquaculture practices and potential human health risks: current knowledge and future priorities. Environ Int 34:1215–1226CrossRefGoogle Scholar
  37. Serrato-Gamiño N, Salgado-Lora MG, Chávez-Moctezuma MP, Campos-García J, Cervantes C (2018) Analysis of the ars gene cluster from highly arsenic-resistant Burkholderia xenovorans LB400. World J Microbiol Biotechnol 34:142CrossRefGoogle Scholar
  38. Shi K, Fan X, Qiao Z, Han Y, McDermott TR, Wang Q, Wang G (2017) Arsenite oxidation regulator AioR regulates bacterial chemotaxis towards arsenite in Agrobacterium tumefaciens GW4. Sci Rep 7:43252CrossRefGoogle Scholar
  39. Shi K, Li C, Rensing C, Dai X, Fan X, Wang G (2018a) Efflux transporter ArsK is responsible for bacterial resistance to arsenite, antimonite, trivalent roxarsone, and methylarsenite. Appl Environ Microbiol 84:e01842CrossRefGoogle Scholar
  40. Shi K, Wang Q, Fan X, Wang G (2018b) Proteomics and genetic analyses reveal the effects of arsenite oxidation on metabolic pathways and the roles of AioR in Agrobacterium tumefaciens GW4. Environ Pollut 235:700–709CrossRefGoogle Scholar
  41. Sun W, Qian X, Gu J, Wang X, Zhang L, Guo A (2017) Mechanisms and effects of arsanilic acid on antibiotic resistance genes and microbial communities during pig manure digestion. Bioresour Technol 234:217–223CrossRefGoogle Scholar
  42. Tikhonova EB, Devroy VK, Lau SY, Zgurskaya HI (2007) Reconstitution of the Escherichia coli macrolide transporter: the periplasmic membrane fusion protein MacA stimulates the ATPase activity of MacB. Mol Microbiol 63:895–910CrossRefGoogle Scholar
  43. Turlin E, Heuck G, Simoes Brandao MI, Szili N, Mellin JR, Lange N, Wandersman C (2014) Protoporphyrin (PPIX) efflux by the MacAB-TolC pump in Escherichia coli. Microbiologyopen 3:849–859CrossRefGoogle Scholar
  44. Virdi JS, Sinha I, Rajendran P, Singh I (2001) Arsenite-induced multiple antibiotic resistance phenotype in environmental isolates of Yersinia enterocolitica. Curr Microbiol 43:144–146CrossRefGoogle Scholar
  45. Wang Q et al (2015a) Fate of arsenate following arsenite oxidation in Agrobacterium tumefaciens GW4. Environ Microbiol 17:1926–1940CrossRefGoogle Scholar
  46. Wang Q et al (2015b) Arsenite oxidase also functions as an antimonite oxidase. Appl Environ Microbiol 81:1959–1965CrossRefGoogle Scholar
  47. Wu S et al (2018) Signature arsenic detoxification pathways in Halomonas sp. strain GFAJ-1. MBio 9:e00515PubMedPubMedCentralGoogle Scholar
  48. Wysocki R, Bobrowicz P, Ulaszewski S (1997) The Saccharomyces cerevisiae ACR46 gene encodes a putative membrane protein involved in arsenite transport. J Biol Chem 272:30061–30066CrossRefGoogle Scholar
  49. Yamanaka H, Kobayashi H, Takahashi E, Okamoto K (2008) MacAB is involved in the secretion of Escherichia coli heat-stable enterotoxin II. J Bacteriol 190:7693–7698CrossRefGoogle Scholar
  50. Zhao Z et al (2017) Nutrients, heavy metals and microbial communities co-driven distribution of antibiotic resistance genes in adjacent environment of mariculture. Environ pollut 220:909–918CrossRefGoogle Scholar
  51. Zhu YG, Yoshinaga M, Zhao FJ, Rosen BP (2014) Earth abides arsenic biotransformations. Annu Rev Earth Planet Sci 42:443–467CrossRefGoogle Scholar
  52. Zhuang W, Liu H, Li J, Chen L, Wang G (2017) Regulation of class A beta-lactamase CzoA by CzoR and IscR in Comamonas testosteroni S44. Front Microbiol 8:2573CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Kaixiang Shi
    • 1
  • Min Cao
    • 1
  • Chan Li
    • 1
  • Jing Huang
    • 1
  • Shixue Zheng
    • 1
  • Gejiao Wang
    • 1
    Email author
  1. 1.State Key Laboratory of Agricultural Microbiology, College of Life Science and TechnologyHuazhong Agricultural UniversityWuhanPeople’s Republic of China

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