University of Birmingham Antimicrobial properties of Pseudomonas strains producing the antibiotic mupirocin Matthijs, Sandra; Vander Wauven, Corinne; Cornu, Bertrand; Ye, Lumeng; Cornelis, Pierre; Thomas, Christopher; Ongena, Marc DOI: 10.1016/j.resmic.2014.09.009 License: Other (please specify with Rights Statement) Document Version Peer reviewed version Citation for published version (Harvard): Matthijs, S, Vander Wauven, C, Cornu, B, Ye, L, Cornelis, P, Thomas, C & Ongena, M 2014, 'Antimicrobial properties of Pseudomonas strains producing the antibiotic mupirocin', Research in Microbiology, vol. 165, no. 8, pp. 695-704. https://doi.org/10.1016/j.resmic.2014.09.009 Link to publication on Research at Birmingham portal Publisher Rights Statement: NOTICE: this is the author’s version of a work that was accepted for publication. 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Ongena, Antimicrobial properties of Pseudomonas strains producing the antibiotic mupirocin, Research in Microbiology (2014), doi: 10.1016/j.resmic.2014.09.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 ACCEPTED MANUSCRIPT 1 Antimicrobial properties of Pseudomonas strains producing the antibiotic mupirocin 2 Sandra Matthijs a*, Corinne Vander Wauven a, Bertrand Cornu a, Ye Lumeng b, Pierre 3 Cornelis b, Christopher M. Thomas c, Marc Ongena d 4 5 aInstitut de Recherches Microbiologiques – Wiame, Campus du CERIA, 1 avenue Emile 6 Gryson, bât 4B, B-1070 Bruxelles, Belgium 7 bDepartment of Bioengineering Sciences, Research Group of Microbiology and Vlaams 8 Instituut voor Biotechnologie, Vrije Universiteit Brussel, Brussels, Belgium 9 cSchool of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK 10 dWalloon Center for Industrial Biology, University of Liège/Gembloux Agro-Bio Tech, 11 Gembloux, Belgium. MANUSCRIPT 12 13 Sandra Matthijs: [email protected], correspondence and reprints 14 Corinne Vander Wauven: [email protected] 15 Bertrand Cornu: [email protected] 16 Ye Lumeng: [email protected] 17 Pierre Cornelis: [email protected] 18 Christopher M. Thomas: [email protected] 19 Marc Ongena: [email protected] 20 2 ACCEPTED MANUSCRIPT 21 Abstract 22 Mupirocin is a polyketide antibiotic with broad antibacterial activity. It was isolated 23 and characterized about 40 years ago from Pseudomonas fluorescens NCIMB 10586. To 24 study the phylogenetic distribution of mupirocin producing strains in the genus Pseudomonas 25 a large collection of Pseudomonas strains of worldwide origin, consisting of 117 26 Pseudomonas type strains and 461 strains isolated from different biological origins, was 27 screened by PCR for the mmpD gene of the mupirocin gene cluster. Five mmpD + strains from 28 different geographic and biological origin were identified. They all produced mupirocin and 29 were strongly antagonistic against Staphylococcus aureus . Phylogenetic analysis showed that 30 mupirocin production is limited to a single species. 31 Inactivation of mupirocin production leads to complete loss of in vitro antagonism 32 against S. aureus , except on certain iron-reduced media where the siderophore pyoverdine is 33 responsible for the in vitro antagonism of a mupirocin-negativeMANUSCRIPT mutant. In addition to 34 mupirocin some of the strains produced lipopeptides of the massetolide group. These 35 lipopeptides do not play a role in the observed in vitro antagonism of the mupirocin producing 36 strains against S. aureus . 37 38 Key words : mupirocin; massetolide; pyoverdine, Pseudomonas fluorescens NCIMB 10586; 39 Pseudomonas sp. W2Aug9 40 ACCEPTED 3 ACCEPTED MANUSCRIPT 41 1. Introduction 42 Mupirocin is a polyketide antibiotic which has been isolated and characterized from 43 the soil bacterium P. fluorescens NCIMB 10586 [1]. In fact mupirocin is a mixture of four 44 pseudomonic acids (A-D). The basic structure of mupirocin comprises a monic acid (a 45 heptaketide) containing a pyran ring, attached to 9-hydroxynonanoic acid via an ester linkage 46 [2]. Mupirocin has a broad spectrum activity against both Gram-positive and Gram-negative 47 bacteria, although most Gram-negative bacteria tested are less susceptible than Gram-positive 48 bacteria [3]. The antibiotic acts through the inhibition of bacterial isoleucyl-tRNA synthetase 49 [4]. Mupirocin is currently used topically for the treatment of skin infections, impetigo and for 50 the decolonization of patients with nasal carriage of Staphylococccus [5]. 51 Although the biosynthetic pathway and the antimicrobial activity of mupirocin have 52 been extensively studied [6], the strain producing this antibiotic is not well characterized. The 53 taxonomic position of strain P. fluorescens NCIMB 10586 is not known since its 16S rRNA 54 gene nor any other housekeeping gene have been MANUSCRIPTsequenced yet. It is also not known whether 55 the ability to produce mupirocin is widespread throughout the genus Pseudomonas or limited 56 to only a few species. Besides P. fluorescens NCIMB 10586, another two mupirocin 57 producing strains, strain D7 and G11 isolated from groundwater sediment samples, have been 58 reported [7]. Both strains were allocated to the P. fluorescens group [7] based on their 16S 59 rRNA sequence. 60 The aim of this study was dual; first to assess the distribution of mupirocin producing 61 Pseudomonas spp.ACCEPTED throughout the genus Pseudomonas and to subsequently study their 62 diversity. Therefore a specific mupirocin primer set was developed based on the mmpD gene. 63 This multifunctional gene is involved in the synthesis of the backbone of monic acid. A large 64 collection of Pseudomonas type strains and partially identified environmental strains 65 representing a high phylogenetic diversity was screened using the mupirocin primer set. 4 ACCEPTED MANUSCRIPT 66 Mupirocin production was confirmed through isolation and identification of the antibiotic and 67 in vitro antagonism tests against Staphylococcus aureus . The second part of the work focused 68 on the mupirocin producing strains themselves whereby it was investigated whether the 69 strains produce additional well-known antimicrobial metabolites and extracellular enzymes to 70 gain insight in their antimicrobial potential. The respective role of the antimicrobial 71 compounds in the in vitro antagonism against S. aureus was further investigated via 72 transposon mutagenesis. 73 74 2. Material and Methods 75 2.1. Bacterial strains 76 A total of 578 strains and isolates from different biological origins were studied. These 77 included 117 type strains of the genus Pseudomonas (listed in Supplementary Table S1) and a 78 collection of 198 Pseudomonas isolates from the River Woluwe, which were isolated from the 79 source (site W2) and the mouth of the river (siteMANUSCRIPT W15) (Supplementary Table S2 in [8]). In 80 addition, a collection of 48 Pseudomonas non type strains obtained from different culture 81 collections (Supplementary Table S2) were included together with 215 strains and isolates 82 received from different laboratories (Supplementary Table S3). 83 84 2.2. Growth conditions 85 The Pseudomonas strains (Table 1) were routinely grown at 28°C on an in house -1 -1 -1 86 medium, mediumACCEPTED 853, composed of 10 g l bacto tryptone, 5 g l yeast extract, 5 g l NaCl, 1 -1 -1 -1 87 g l glucose, 0.7 g l K2HPO 4 and 0.3 g l KH 2PO 4.