Applied Microbiology and Biotechnology

, Volume 87, Issue 5, pp 1895–1905

All genomic mutations in the antimicrobial surfactant-resistant mutant, Escherichia coli OW66, are involved in cell resistance to surfactant

  • Kunihiro Nakata
  • Myo Myoung Koh
  • Tetsuaki Tsuchido
  • Yoshinobu Matsumura
Applied Microbial and Cell Physiology

DOI: 10.1007/s00253-010-2638-8

Cite this article as:
Nakata, K., Koh, M.M., Tsuchido, T. et al. Appl Microbiol Biotechnol (2010) 87: 1895. doi:10.1007/s00253-010-2638-8

Abstract

The spontaneous antimicrobial surfactant-resistant mutant, Escherichia coli OW66, has been isolated, and its physiological properties have been characterized in our previous paper (Ishikawa et al., J Appl Microbiol 92:261–268, 2002b). This report revealed that strain OW66 had seven mutations in their chromosomal DNA by comparative genomic hybridization microarray, and that their alternative functions were involved in cell resistance to antimicrobial surfactants. These mutations were located in oppB, ydcR, IVR(vacJ-yfdC), rpoN, rpoB, rpoC, and soxR. Furthermore, seven of the single-mutated isogenic strains and seven of the six-mutated isogenic strains were constructed from strains OW6 (NBRC106482) and OW66, respectively, through homologous recombination, and their resistances to an antimicrobial surfactant were measured using the minimum inhibitory concentration method. These results revealed that all six-mutated strains were more sensitive than strain OW66, and that the soxR66 mutation was independently involved in antimicrobial surfactant resistance of E. coli cells. Expression of soxR66 and soxS was increased in both strains OW66 and OW6-soxR66 without the surfactant treatment by the quantitative real time-polymerase chain reaction analysis, compared with strain OW6. Two-dimensional polyacrylamide gel electrophoresis analysis also revealed that some proteins in the soxRS regulon, including Mn-SOD, were overexpressed in both strains OW66 and OW6-soxR66. These results indicate that the soxR66 mutation leads to the constitutive expression of the soxRS regulon, resulting in the acquired resistance of E. coli cells to an antimicrobial surfactant.

Keywords

Spontaneous mutant Quaternary ammonium compound Antimicrobial surfactant Cetyltrimethylammonium bromide soxRS regulon Comparative genomic hybridization microarray 

Supplementary material

253_2010_2638_MOESM1_ESM.pdf (93 kb)
ESM 1Bacterial strains and plasmids used in this study (PDF 93 kb)
253_2010_2638_MOESM2_ESM.pdf (76 kb)
ESM 2Method for the recombinant plasmid construction (PDF 75 kb)
253_2010_2638_Fig2_ESM.gif (124 kb)
ESM 3

Comparative genomic hybridization microarray analysis. Oligonucleotide probes designed from the MG1655 chromosomal DNA spotted on the array. The fluorescene intensities for strains OW6 and OW66 are indicated in (a) and (b), respectively. Ratios of spot intensities between strain OW6 and strain OW66 and 18 candidates of mutation regions were illustrated in (c) and (d), respectively. Numbers are indicated as a nucleotide sequence position in MG1655 chromosomal DNA (accession no. U00096 in GenBank) (GIF 123 kb)

253_2010_2638_MOESM3_ESM.tif (27.2 mb)
High resolution image (TIFF 27867 kb)
253_2010_2638_Fig3_ESM.gif (254 kb)
ESM 4

Intracellular protein profilings of strain OW6 (a) and OW66 (b) were analyzed with 2D polyacrylamide gel electrophoresis (PAGE). The first dimension (horizontal) was generated with IEF (IPG strip, pH 4–7, 11 cm), and the second dimension was carried out with sodium dodecyl sulfate-PAGE (15% SDS-PAGE). Seventeen upward and 32 downward pointing arrows indicate upregulated and downregulated proteins in strain OW66, respectively, comparing with strains OW6 and OW6-soxR66 (see Fig. 1) (GIF 253 kb)

253_2010_2638_MOESM4_ESM.tif (6.1 mb)
High resolution image (TIFF 6225 kb)

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Kunihiro Nakata
    • 1
  • Myo Myoung Koh
    • 1
  • Tetsuaki Tsuchido
    • 1
    • 2
  • Yoshinobu Matsumura
    • 1
    • 2
  1. 1.Department of Life Science and BiotechnologyKansai UniversityOsakaJapan
  2. 2.Organization for Research and Development of Innovative Science and TechnologyKansai UniversityOsakaJapan

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