Biological functions of nirS in Pseudomonas aeruginosa ATCC 9027 under aerobic conditions

  • Gang Zhou
  • Hong Peng
  • Ying-si Wang
  • Cai-ling Li
  • Peng-fei Shen
  • Xiao-mo Huang
  • Xiao-bao XieEmail author
  • Qing-shan ShiEmail author
Genetics and Molecular Biology of Industrial Organisms - Original Paper


Through our previous study, we found an up-regulation in the expression of nitrite reductase (nirS) in the isothiazolone-resistant strain of Pseudomonas aeruginosa. However, the definitive molecular role of nirS in ascribing the resistance remained elusive. In the present study, the nirS gene was deleted from the chromosome of P. aeruginosa ATCC 9027 and the resulting phenotypic changes of ΔnirS were studied alongside the wild-type (WT) strain under aerobic conditions. The results demonstrated a decline in the formations of biofilms but not planktonic growth by ΔnirS as compared to WT, especially in the presence of benzisothiazolinone (BIT). Meanwhile, the deletion of nirS impaired swimming motility of P. aeruginosa under the stress of BIT. To assess the influence of nirS on the transcriptome of P. aeruginosa, RNA-seq experiments comparing the ΔnirS with WT were also performed. A total of 694 genes were found to be differentially expressed in ΔnirS, of which 192 were up-regulated, while 502 were down-regulated. In addition, these differently expressed genes were noted to significantly enrich the carbon metabolism along with glyoxylate and dicarboxylate metabolisms. Meanwhile, results from RT-PCR suggested the contribution of mexEF-oprN to the development of BIT resistance by ΔnirS. Further, c-di-GMP was less in ΔnirS than in WT, as revealed by HPLC. Taken together, our results confirm that nirS of P. aeruginosa ATCC 9027 plays a role in BIT resistance along with biofilm formation and further affects several metabolic patterns under aerobic conditions.


Pseudomonas aeruginosa Nitrite reductase Isothiazolones Biofilm formation and dispersal RNA-seq technology 



This work was funded by the GDAS’ Project of Science and Technology Development (No. 2019GDASYL-0104006), the National Natural Science Foundation of China (Nos. 31770091 and 31500036), and Natural Science Foundation of Guangdong Province (No. 2015A030313713). We are grateful to Prof. Hai-hong Wang of South China Agricultural University for generously providing us the plasmids of pK18-GM and pSRK-GM.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

10295_2019_2232_MOESM1_ESM.xls (768 kb)
Additional file 1. Detection of differently expressed genes between WT and ΔnirS transcriptomes using RNA-seq technique. Differences with FDR ≤ 0.05 and log2FC absolute value ≥ 1 were set as the threshold for significant differences in gene expression. (XLS 767 kb)


  1. 1.
    Arai H, Kodama T, Igarashi Y (1999) Effect of nitrogen oxides on expression of the nir and nor genes for denitrification in Pseudomonas aeruginosa. FEMS Microbiol Lett 170:19–24CrossRefGoogle Scholar
  2. 2.
    Barraud N, Hassett DJ, Hwang S-H, Rice SA, Kjelleberg S, Webb JS (2006) Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa. J Bacteriol 188:7344–7353CrossRefGoogle Scholar
  3. 3.
    Barraud N, Schleheck D, Klebensberger J, Webb JS, Hassett DJ, Rice SA, Kjelleberg S (2009) Nitric oxide signaling in Pseudomonas aeruginosa biofilms mediates phosphodiesterase activity, decreased cyclic Di-GMP levels, and enhanced dispersal. J Bacteriol 191:7333–7342CrossRefGoogle Scholar
  4. 4.
    Chapman JS (2003) Biocide resistance mechanisms. Int Biodeterior Biodegrad 51:133–138CrossRefGoogle Scholar
  5. 5.
    Chapman JS, Diehl MA (1995) Methylchloroisothiazolone-induced growth inhibition and lethality in Escherichia coli. J Appl Microbiol 78:134–141Google Scholar
  6. 6.
    Chen YC, Xie XB, Shi QS, Ouyang YS, Chen YB (2010) Species identification of industry spoilage microorganism and the resistance analysis. Microbiol China 37:1558–1565Google Scholar
  7. 7.
    Chuanchuen R, Beinlich K, Hoang TT, Becher A, Karkhoff-Schweizer RR, Schweizer HP (2001) Cross-resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps: exposure of a susceptible mutant strain to triclosan selects nfxB mutants overexpressing MexCD-OprJ. Antimicrob Agents Chemother 45:428–432CrossRefGoogle Scholar
  8. 8.
    Davies KJP, Lloyd D, Boddy L (1989) The effect of oxygen on denitrification in Paracoccus denitrificans and Pseudomonas aeruginosa. Microbiology 135:2445–2451CrossRefGoogle Scholar
  9. 9.
    De Kievit TR, Parkins MD, Gillis RJ, Srikumar R, Ceri H, Poole K, Iglewski BH, Storey DG (2001) Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 45:1761–1770CrossRefGoogle Scholar
  10. 10.
    Drenkard E (2003) Antimicrobial resistance of Pseudomonas aeruginosa biofilms. Microbes Infect 5:1213–1219CrossRefGoogle Scholar
  11. 11.
    Germ M, Yoshihara E, Yoneyama H, Nakae T (1999) Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa. Biochem Biophys Res Commun 261:452–455CrossRefGoogle Scholar
  12. 12.
    Hancock RE (1998) Resistance mechanisms in Pseudomonas aeruginosa and other non-fermentative gram-negative bacteria. Clin Infect Dis 27:S93–S99CrossRefGoogle Scholar
  13. 13.
    Huang CY, Hsieh SP, Kuo PA, Jane WN, Tu J, Wang YN, Ko CH (2009) Impact of disinfectant and nutrient concentration on growth and biofilm formation for a Pseudomonas strain and the mixed cultures from a fine papermachine system. Int Biodeterior Biodegrad 63:998–1007CrossRefGoogle Scholar
  14. 14.
    Huang YH, Lin JS, Ma JC, Wang HH (2016) Functional characterization of triclosan-resistant enoyl-acyl-carrier protein reductase (FabV) in Pseudomonas aeruginosa. Front Microbiol 7:1903Google Scholar
  15. 15.
    Su JJ, Liu BY, Liu CY (2001) Comparison of aerobic denitrification under high oxygen atmosphere by Thiosphaera pantotropha ATCC 35512 and Pseudomonas stutzeri SU2 newly isolated from the activated sludge of a piggery wastewater treatment system. J Appl Microbiol 90:457–462CrossRefGoogle Scholar
  16. 16.
    Köhler T, Michea-Hamzehpour M, Plesiat P, Kahr A-L, Pechere J-C (1997) Differential selection of multidrug efflux systems by quinolones in Pseudomonas aeruginosa. Antimicrob Agents Chemother 41:2540–2543CrossRefGoogle Scholar
  17. 17.
    Kohler T, Michea-Hamzehpour M, Henze U, Gotoh N, Curty LK, Pechere JC (1997) Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Mol Microbiol 23:345–354CrossRefGoogle Scholar
  18. 18.
    Krüger A, Grüning N-M, Wamelink MM, Kerick M, Kirpy A, Parkhomchuk D, Bluemlein K, Schweiger M-R, Soldatov A, Lehrach H (2011) The pentose phosphate pathway is a metabolic redox sensor and regulates transcription during the antioxidant response. Antioxid Redox Signal 15:311–324CrossRefGoogle Scholar
  19. 19.
    Lambert P (2002) Mechanisms of antibiotic resistance in Pseudomonas aeruginosa. J R Soc Med 95:22–26Google Scholar
  20. 20.
    Laopaiboon L, Smith RN, Hall SJ (2001) A study of the effect of isothiazolones on the performance and characteristics of a laboratory-scale rotating biological contactor. J Appl Microbiol 91:93–103CrossRefGoogle Scholar
  21. 21.
    Li XZ, Zhang L, Poole K (1998) Role of the multidrug efflux systems of Pseudomonas aeruginosa in organic solvent tolerance. J Bacteriol 180:2987–2991Google Scholar
  22. 22.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408CrossRefGoogle Scholar
  23. 23.
    Lyczak JB, Cannon CL, Pier GB (2000) Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2:1051–1060CrossRefGoogle Scholar
  24. 24.
    Lyczak JB, Cannon CL, Pier GB (2002) Lung infections associated with cystic fibrosis. Clin Microbiol Rev 15:194–222CrossRefGoogle Scholar
  25. 25.
    Ma JC, Wu YQ, Cao D, Zhang WB, Wang HH (2017) Only acyl carrier protein 1 (AcpP1) functions in Pseudomonas aeruginosa fatty acid synthesis. Front Microbiol 8:2186CrossRefGoogle Scholar
  26. 26.
    Morita Y, Tomida J, Kawamura Y (2014) Responses of Pseudomonas aeruginosa to antimicrobials. Front Microbiol 4:422CrossRefGoogle Scholar
  27. 27.
    O’Toole G, Kaplan HB, Kolter R (2000) Biofilm formation as microbial development. Annu Rev Microbiol 54:49–79CrossRefGoogle Scholar
  28. 28.
    Poole K (2011) Pseudomonas aeruginosa: resistance to the max. Front Microbiol 2:65CrossRefGoogle Scholar
  29. 29.
    Pucci MJ, Podos SD, Thanassi JA, Leggio MJ, Bradbury BJ, Deshpande M (2011) In vitro and in vivo profiles of ACH-702, an isothiazoloquinolone, against bacterial pathogens. Antimicrob Agents Chemother 55:2860–2871CrossRefGoogle Scholar
  30. 30.
    Römling U, Galperin MY, Gomelsky M (2013) Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 77:1–52CrossRefGoogle Scholar
  31. 31.
    Rashid MH, Kornberg A (2000) Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc Natl Acad Sci USA 97:4885–4890CrossRefGoogle Scholar
  32. 32.
    Rinaldo S, Giardina G, Castiglione N, Stelitano V, Cutruzzola F (2011) The catalytic mechanism of Pseudomonas aeruginosa cd(1) nitrite reductase. Biochem Soc T 39:195–200CrossRefGoogle Scholar
  33. 33.
    Robinson MD, Oshlack A (2010) A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11:R25–R25CrossRefGoogle Scholar
  34. 34.
    Roy AB, Petrova OE, Sauer K (2013) Extraction and quantification of cyclic di-GMP from P. aeruginosa. Bio Protoc 3:e828CrossRefGoogle Scholar
  35. 35.
    Ryan RP, Lucey J, O’Donovan K, McCarthy Y, Yang L, Tolker-Nielsen T, Dow JM (2009) HD-GYP domain proteins regulate biofilm formation and virulence in Pseudomonas aeruginosa. Environ Microbiol 11:1126–1136CrossRefGoogle Scholar
  36. 36.
    Sandoe JAT, Wysome J, West AP, Heritage J, Wilcox MH (2006) Measurement of ampicillin, vancomycin, linezolid and gentamicin activity against enterococcal biofilms. J Antimicrob Chemother 57:767–770CrossRefGoogle Scholar
  37. 37.
    Silvestrini MC, Galeotti CL, Gervais M, Schininà E, Barra D, Bossa F, Brunori M (1989) Nitrite reductase from Pseudomonas aeruginosa: sequence of the gene and the protein. FEBS Lett 254:33–38CrossRefGoogle Scholar
  38. 38.
    Singh PK, Schaefer AL, Parsek MR, Moninger TO, Welsh MJ, Greenberg E (2000) Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407:762–764CrossRefGoogle Scholar
  39. 39.
    Stepanović S, Vuković D, Dakić I, Savić B, Švabić-Vlahović M (2000) A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 40:175–179CrossRefGoogle Scholar
  40. 40.
    Su S, Panmanee W, Wilson JJ, Mahtani HK, Li Q, VanderWielen BD, Makris TM, Rogers M, McDaniel C, Lipscomb JD, Irvin RT, Schurr MJ, Lancaster JR, Kovall RA, Hassett DJ (2014) Catalase (KatA) plays a role in protection against anaerobic nitric oxide in Pseudomonas aeruginosa. PLoS One 9:e91813CrossRefGoogle Scholar
  41. 41.
    Tang QY, Feng MG (2007) DPS data processing system: experimental design, statistical analysis and data mining. Science Press, BeijingGoogle Scholar
  42. 42.
    Vicentini CB, Romagnoli C, Manfredini S, Rossi D, Mares D (2011) Pyrazolo [3,4-c] isothiazole and isothiazolo [4,3-d] isoxazole derivatives as antifungal agents. Pharm Biol 49:545–552CrossRefGoogle Scholar
  43. 43.
    Williams TM (2007) The mechanism of action of isothiazolone biocide. Power Plant Chem 9:14–22Google Scholar
  44. 44.
    Zhou G, Li LJ, Shi QS, Ouyang YS, Chen YB, Hu WF (2013) Effects of nutritional and environmental conditions on planktonic growth and biofilm formation for Citrobacter werkmanii BF-6. J Microbiol Biotechnol 23:1673–1682CrossRefGoogle Scholar
  45. 45.
    Zhou G, Li LJ, Shi QS, Ouyang YS, Chen YB, Hu WF (2014) Efficacy of metal ions and isothiazolones in inhibiting Enterobacter cloacae BF-17 biofilm formation. Can J Microbiol 60:5–14CrossRefGoogle Scholar
  46. 46.
    Zhou G, Shi QS, Huang XM, Xie XB (2016) Comparison of transcriptomes of wild-type and isothiazolone-resistant Pseudomonas aeruginosa by using RNA-seq. Mol Biol Rep 43:527–540CrossRefGoogle Scholar
  47. 47.
    Zhou G, Shi QS, Ouyang YS, Chen YB (2014) Involvement of outer membrane proteins and peroxide-sensor genes in Burkholderia cepacia resistance to isothiazolone. World J Microbiol Biotechnol 30:1251–1260CrossRefGoogle Scholar
  48. 48.
    Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61:533–616Google Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2019

Authors and Affiliations

  • Gang Zhou
    • 1
  • Hong Peng
    • 1
  • Ying-si Wang
    • 1
  • Cai-ling Li
    • 1
  • Peng-fei Shen
    • 1
  • Xiao-mo Huang
    • 1
  • Xiao-bao Xie
    • 1
    Email author
  • Qing-shan Shi
    • 1
    Email author
  1. 1.Guangdong Open Laboratory of Applied Microbiology, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, State Key Laboratory of Applied Microbiology Southern China, Guangdong Institute of MicrobiologyGuangdong Academy of SciencesGuangzhouPeople’s Republic of China

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