ARTICLE IN PRESS Systematic and Applied Microbiology 32 (2009) 56–64 www.elsevier.de/syapm Isolation of two Pseudomonas strains producing pseudomonic acid A Eva Fritza, Agnes Feketeb, Jutta Lintelmannb, Philipe Schmitt-Kopplinb, Rainer U. Meckenstocka,Ã aInstitute of Groundwater Ecology, Helmholtz Zentrum Mu¨nchen, German Research Center for Environmental Health, Ingolsta¨dter Landstrasse 1, 85764 Neuherberg, Germany bInstitute of Ecological Chemistry, Helmholtz Zentrum Mu¨nchen, German Research Center for Environmental Health, Ingolsta¨dter Landstrasse 1, 85764 Neuherberg, Germany Received 16 June 2008 Abstract Two novel Pseudomonas strains were isolated from groundwater sediment samples. The strains showed resistance against the antibiotics tetracycline, cephalothin, nisin, vancomycin, nalidixic acid, erythromycin, lincomycin, and penicillin and grew at temperatures between 15 and 37 1C and pH values from 4 to 10 with a maximum at pH 7 to 10. The 16S ribosomal RNA gene sequences and the substrate spectrum of the isolates revealed that the two strains belonged to the Pseudomonas fluorescens group. The supernatants of both strains had an antibiotic effect against Gram-positive bacteria and one Gram-negative strain. The effective substance was produced under standard cultivation conditions without special inducer molecules or special medium composition. The antibiotically active compound was identified as pseudomonic acid A by off-line high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The measurement on ultra performance liquid chromatography (UPLC, UV–vis detection) confirmed the determination of pseudomonic acid A which was produced by both strains at 1.7–3.5 mg/l. Our findings indicate that the ability to produce the antibiotic pseudomonic acid A (Mupirocin) is more spread among the pseudomonads then anticipated from the only producer known so far. r 2008 Elsevier GmbH. All rights reserved. Keywords: Mupirocin; Pseudomonas fluorescens; Environmental isolates; FT-ICR-MS; UPLC; HPLC; Antibiotic Introduction Pseudomonas fluorescens strain NCIB 10586 produces the antibiotic pseudomonic acid that was introduced to The ability of Pseudomonas strains to inhibit other the market in 1986 as mupirocin for human use. The bacteria was well known already at the end of the 19th effective substance pseudomonic acid A was described century [2], and turned out to play an important role [13] and later it could be shown that derivatives of especially in the case of bacterial biocontrol in the pseudomonic acid are produced simultaneously by the rhizosphere of plants [31]. As the only known isolate, same organism [5,6,12]. The most important and major form is pseudomonic acid A also known as mupirocin ÃCorresponding author. Tel.:+49 89 3187 2561; fax:+49 89 3187 3361. and used as a topical antibiotic [34]. This polyketide E-mail address: [email protected] antibiotic targets isoleucyl-tRNA synthase, where it (R.U. Meckenstock). competes with isoleucine for the binding site and inhibits 0723-2020/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2008.11.001 ARTICLE IN PRESS E. Fritz et al. / Systematic and Applied Microbiology 32 (2009) 56–64 57 the protein synthesis of sensitive bacterial strains. It is from a pellet of 5 ml culture. Full length 16S rRNA also useful against methicillin-resistant Staphylococcus genes were PCR-amplified using primers Ba27f aureus (MRSA). For all antibiotics known so far, (sequence: AGA GTT TGA TCM TGG CTC AG) bacteria developed a resistance and thus also mupirocin and 907r (sequence: CCG TCA ATT CMT TTR AGT resistant strains of MRSA were detected [7]. The TT) [21,35]. The PCR mixture (50 ml) consisted of 5.0 ml resistance mechanism is given by a mutation of the 10 Â PCR-buffer (Fermentas), 3.0 ml 25 mM MgCl2, isoleucyl-tRNA synthetase or by an additional new 0.5 ml20mg/ml BSA, 0.5 ml 10 mM dNTPs, 0.5 ml50mM isoleucyl-tRNA synthetase [7]. The producer strain f-primer and 0.5 ml50mM r-primer, 0.25 ml5U/mlMBI Pseudomonas fluorescens NCIB10586 has two different Taq, and 1 ml DNA-template. The temperature profile isoleucyl-tRNA synthases and one enzyme is highly for the thermocycler Mastercyclers Ep Gradient resistant against the produced pseudomonic acid A (Eppendorf, Hamburg, Germany) was as follows: [17,36]. 94 1C for 5 min, 94 1C for 30 s, 20–25 cycles (depending Because of a fast rupture of the epoxide ring, on DNA concentration) at 52 1C for 30 s, 70 1C for 60 s, mupirocin is probably not stable in the environment followed by an elongation cycle at 70 1C for 5 min at the over longer time frames [8]. end. Direct sequencing using amplicon primers (Ba27f, In times of rising resistances of pathogens, new Ba519r, Ba1492r) was performed on an ABI 3730 strategies seem to be necessary to find new and effective sequencer (Applied Biosystems) using the Big antibiotics. We, therefore, performed a systematic study Dye-terminator v3.1 chemistry as specified by the manu- of antibiotic producers in groundwater. A high through- facturer. The 16S rRNA gene sequence reads were put cultivation study with an artificial freshwater manually assembled and checked for quality using the medium (AFM) led to several bacterial cultures SeqMan II software module (Lasergene 6 suite, DNAs- which were tested for antibiotic production. Here, we tar, Madison, USA). For phylogenetic analysis, the 16S present two novel pseudomonic acid A producing rRNA gene sequences were integrated into an ARB strains and characterised them indicating that the ability database of 16S rRNA gene sequences by the use of the to produce pseudomonic acid is spread more among the alignment tool of the ARB software package (http:// pseudomonads. www.arb-home.de). Phylogenetic analyses based on nucleotide sequences were performed and verified by applying maximum parsimony and neighbour-joining Material and methods methods by use of the respective tools in the ARB software package. The 16S gene sequences of the two new antibiotic- Cultivation of microorganisms producing strains were submitted to GeneBank with the accession numbers EU680857 and EU680856. Two microbial strains, D7 and G11, were isolated from sediment of a pristine aquifer located at Scheyern, Germany with serial dilution in microtiter plates. Growth temperature/pH determination Cultivation was performed in nutrient broth medium (NB, peptone 5 g/l, meat extract 5 g/l, pH 7) or LB The optimal growth temperature for the strains was medium (10 g tryptone /l, 5 g yeast extract /l, 10 g NaCl/l). tested for agar and liquid cultures with NB at The cultures were kept by streaking on NB agar plates temperatures between 4 and 60 1C. The optimal pH every weak and stored at 4 1C. For growth in liquid conditions for strains were determined with AFW cultures 50 ml of medium in an Erlenmeyer flask were medium set to pH from 3 to 11 with 2 M NaOH or inoculated with cells of an overnight culture to an 2 M HCL. OD595 nm of 0.01 and shaken at 200 rpm. The same cultivation was used for the test strain Bacillus subtilis subspecies subtilis (DSMZ 10) at 30 1C (see below). For Test for catalase, oxidase activity, and growth on solidified medium, the strains were streaked metabolic properties out on the agar plates and incubated at the given temperature. The test for catalase activity was performed with a 3% aqueous solution of H2O2. A grown colony was Identification of antibiotic-producing picked from an agar plate and mixed with the solution. bacterial strains Frothing indicated catalase activity. A solution of 0.1 g of citric acid and 1.0 g 4-amino- DNA extraction for sequencing of the unknown N,N-dimethylanilin-dihydrochlorid was prepared and a bacterial strains was done with the Fast DNAs Spin soaked filter spread with a grown colony. A blue for Soil Kit (MP Biomedicals, Eschwege, Germany) colouring showed the existence of cytochrom c-oxidase. ARTICLE IN PRESS 58 E. Fritz et al. / Systematic and Applied Microbiology 32 (2009) 56–64 To test for metabolic properties of D7 and G11, the solvent strength gradient 0.1% formic acid (A) and standard test kit apis 20NE from Biome´rieux (Marcy 100% acetonitrile containing 0.06% formic acid (B) was l’Etoile, France) was used. used. The following gradient profile was used: 0–15 min from 0% to 100% B, 15–25 min 100% B, 25–30 min Identification of antibiotic compounds from 100% to 0% B, and 30–45 min 0% B. The flow rate was 1 ml/min causing pressure drop on the column For identification of the antibacterial active com- between 95 and 160 bar. The injection volume was 20 ml. pounds, strains D7 and G11 were grown in 50 ml of NB The eluent was fractioned from 0 until 30 min at the rate medium or 50 ml of AFM [3]. AFM as a mineral of 0.5 min/tube with Gilson FC203B fraction Collector medium was chosen to have less disturbing components (Limburg, Germany). in the chromatographic analysis. Nevertheless, both media were compared for antibiotic production, because Fourier transform ion cyclotron resonance mass a better bacterial growth was found in the rich medium. spectrometry (FT-ICR-MS) analysis After 18 h of growth, the cells were separated from the supernatant by centrifugation at 4800 rpm in a High-resolution mass spectra were acquired on a FT- Megafuge 1.0R centrifuge (Fisher Scientific, Schwerte, ICR-MS (Bruker, Bremen, Germany), equipped with a Germany). The supernatant was filtered through a 12 T superconducting magnet and an Apollo II ESI 0.22 mm filter (Millipore, Schwalmbach, Germany), source. Samples containing 75% methanol were infused frozen at À80 1C and lyophilised in a freeze drier (VirTis with the micro-electrospray source at a flow rate of Company, New York, USA).