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Manipulation of quorum sensing regulation in Pseudomonas fluorescens NCIMB 10586 to increase mupirocin production

  • Applied Genetics and Molecular Biotechnology
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Abstract

Transcription of the 74 kb Pseudomonas fluorescens mupirocin [pseudomonic acid (PA)] biosynthesis cluster depends on quorum sensing-dependent regulation via the LuxI/LuxR homologues MupI/MupR. To facilitate analysis of novel PAs from pathway mutants, we investigated factors that affect mup gene expression. First, the signal produced by MupI was identified as N-(3-oxodecanoyl)homoserine lactone, but exogenous addition of this molecule did not activate mupirocin production prematurely nor did expression of mupI in trans increase metabolite production. Second, we confirmed that mupX, encoding an amidase/hydrolase that can degrade N-acylhomoserine lactones, is also required for efficient expression, consistent with its occurrence in a regulatory module linked to unrelated genes in P. fluorescens. Third, and most significantly, mupR expression in trans to wild type and mutants can increase production of antibiotic and novel intermediates up to 17-fold.

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Acknowledgement

This work was supported by BBSRC grants P15257 and P07071, BBSRC/EPSRC grant E021611 employing JH, ES and AM, as well as EPSRC grant S78124 that employed JW. GC was supported by a University of Nottingham Studentship and work in PW’s laboratory was supported by BBSRC. DNA sequencing was performed by the JIF-funded Genomics Laboratory in the School of Biosciences (6/JIF13209). HPLC/LCMS equipment was funded by EPSRC grant EP/F066104). We thank Miguel Camara at the University of Nottingham for his input and comments and Ram Chhabra and Alex Truman for AHL synthesis.

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Authors and Affiliations

  1. School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

    Joanne Hothersall, Elton R. Stephens, Helen Cooper & Christopher M. Thomas

  2. School of Chemistry, University of Bristol, Cantock’s Close, Bristol, BS8 1TS, UK

    Annabel C. Murphy, Zafar Iqbal, Ji’en Wu, John Crosby, Christine L. Willis, Russell J. Cox & Thomas J. Simpson

  3. School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, UK

    Genevieve Campbell, Steve Atkinson & Paul Williams

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  1. Joanne Hothersall
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  2. Annabel C. Murphy
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  3. Zafar Iqbal
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  4. Genevieve Campbell
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Correspondence to Christopher M. Thomas.

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Fig. S1

Mupirocin 74 kb gene cluster organisation. Deduced open reading frame (ORF) functions are shown in the table. Polyketide synthase (PKS), ketosynthase (KS), acyl carrier protein (ACP), ketoreductase (KR), dehydratase (DH), thioesterase (TE), acyltransferase (AT), C-methyltransferase (MeT), 3-hydroxy-3-methylglutaric acid (HMG), phosphopantetheinyl transferase (PPTase), N-acyl homoserine lactone (N-AHL) (PDF 265 kb)

Fig. S2

Positive electrospray FT-ICR MS analysis of supernatant extracts from Escherichia coli with either expression plasmid pJH1 containing mupI or pJH10 with no insert revealed a peak at m/z 270.1699 (C14H24O4N1), which corresponds to 3-O-C10-HSL, present in E. coli (pJH1) but not E. coli (pJH10) (PDF 321 kb)

Fig. S3

Positive electrospray FT-ICR MS analysis of chemically synthesised 3-O-C10-HSL revealed a peak at m/z 270.1701 (PDF 195 kb)

Fig. S4

Positive electrospray FT-ICR MS analysis of solvent extracts from WT Pseudomonas fluorescens and mupI mutant revealed a peak at m/z 270.1699 corresponding to 3-O-C10-HSL in the WT sample but not the mupI mutant sample (PDF 225 kb)

Fig. S5

AHL degradation by Escherichia coli expressing mupX. Activity of E. coli (pSCCX; lanes 1 and 2), E. coli (pJH10) vector control (lanes 3 and 4) against a range of AHLs with acyl chains from C4 to C14 with or without a 3-oxo-substituent. The positive (lanes 5 and 6) and negative controls (lanes 7 and 8), respectively, contained AHLs only or the AHL reporters in the absence of AHLs. The E. coli pSB536 AHL biosensor did not respond to 3-oxo-C4-HSL (PDF 280 kb)

Table S1

Structural elucidation of PAs A7 (1) and B7 (2) (PDF 31 kb)

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Hothersall, J., Murphy, A.C., Iqbal, Z. et al. Manipulation of quorum sensing regulation in Pseudomonas fluorescens NCIMB 10586 to increase mupirocin production. Appl Microbiol Biotechnol 90, 1017–1026 (2011). https://doi.org/10.1007/s00253-011-3145-2

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