Hindawi Publishing Corporation International Journal of Microbiology Volume 2013, Article ID 901697, 5 pages http://dx.doi.org/10.1155/2013/901697 Research Article Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity Cory A. Leonard,1 Stacy D. Brown,2 and J. Russell Hayman1 1 Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, P.O. Box 70577, Johnson City, TN 37614, USA 2 Department of Pharmaceutical Sciences, Bill Gatton College of Pharmacy, East Tennessee State University, P.O. Box 70436, Johnson City, TN 37614, USA Correspondence should be addressed to J. Russell Hayman; [email protected] Received 30 April 2013; Revised 9 July 2013; Accepted 9 July 2013 Academic Editor: Joseph Falkinham Copyright © 2013 Cory A. Leonard et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin- resistant Staphylococcus aureus (MRSA) in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS). Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa,butnotKlebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae.BecausethegenomeofA. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system. 1. Introduction today are natural product compounds, or their derivatives, discovered during the “golden era” of antibiotic discovery Antibiotic resistant microorganisms became a medical con- from the 1940s through the 1960s [3]. More recent efforts to cern shortly after antibiotics became readily available in generate new antibiotics based on high-throughput, target- the 1940s. In fact, resistance has typically been identified focused screening of large libraries of synthetic compounds within four years of Food and Drug Administration approval have largely failed to produce new antibiotics [3, 4]. Gen- of antibacterial agents [1]. Currently, multidrug-resistant eration of novel antibiotic compounds may be possible by bacteria such as MRSA, coagulase negative Staphylococci, revealing previously untapped chemical diversity in known and Enterococci cause severe infections in both hospital natural products. For example, recent studies have shown and community settings [2]. Development of new drugs to that mutations in microbes can activate silent gene clusters, combattheincreasinghostofdrugresistantmicroorganisms regulating enzyme activity, and resulting in the production of is essential if we are to avoid the emergence of pathogens for uncharacterized compounds, some of which exhibit antimi- which there exist no effective antimicrobial therapies. crobial activity [5]. Most classes of antibiotics were developed from natural Aspergillus oryzae is a domesticated filamentous fungus products produced by fungi or filamentous bacteria. Sig- used in the food fermentation industry. Although A. oryzae nificantly, the majority of antibiotics still commonly used isknowntoproduceantibacterialcompounds,suchaskojic 2 International Journal of Microbiology ∘ acid and aspirochlorine, the fungus does not produce signifi- viable spores per well. The plates were incubated at 37 Cfor cant levels of these compounds under standard food industry four days with no shaking. or laboratory culture conditions. Recent work, however, utilizing manipulation of culture conditions, suggests that 2.3.2. Screening Assay of Supernatant Activity against MRSA. A. oryzae, in common with the closely related Aspergillus After four days of fungal culture, 100 LofA. oryzae culture flavus, has the potential to produce a broader range of supernatant was transferred from each well of the 96 well secondary metabolites than previously thought possible [6]. culture plates to wells in U-bottom 96 well plates containing Random mutagenesis might facilitate the production of 100 L fresh LB media (to provide nutrients that may have antibacterial compounds by altering expression of polyketide been depleted by fungal growth) and 10 Lof0.005OD600 synthase or nonribosomal peptide synthetase pathways, the MRSA suspended in sterile phosphate buffered saline (PBS). main pathways involved in fungal secondary metabolism Positive control wells for MRSA growth consisted of 200 L [7]. Additionally, such mutagenesis could modulate regula- LB media with no A. oryzae culture supernatant plus the tion of protein expression and production of antimicrobial described MRSA inoculum. Negative control wells consisted peptides. Filamentous fungi, including Aspergillus giganteus of 200 L LB media with no bacterial inoculum. The plates ∘ and Aspergillus clavatus,areknowntoproduceantimicro- were incubated overnight at 37 Cwithnoshakingand bial peptides with demonstrated antifungal or antibacterial MRSA growth in the presence of 3080 EMS-treated fungal activity [8]. It is our hypothesis that random mutagenesis of culture supernatants was visually evaluated. Images were A. oryzae may result in strains secreting novel antibacterial captured using an Epson Perfection 3200 Photo scanner. The compounds, or increased levels of known antibacterial com- remaining fungal culture supernatant volumes and associated ∘ pounds to detectable levels. To address this hypothesis, a bio- mycelia mats were stored at 4 C, tightly sealed in the original logical screening assay was employed to identify individual A. 96 well fungal culture plates. oryzae mutants that secrete factors inhibiting the growth of Methicillin-resistant Staphylococcus aureus (MRSA) in vitro. 2.3.3. Dilution/Isolation of EMS-Treated Putative Antibacterial A. oryzae. Fungal cultures that yielded supernatant samples 2. Materials and Methods with visible antibacterial activity, as determined by reduced bacterial growth in the screening assay, were diluted and 2.1. Bacterial and Fungal Strains. Methicillin-sensitive Staph- isolated. The corresponding fungal mats were retrieved from ∘ ∘ ylococcus aureus (ATCC 25922) and Pseudomonas aeruginosa 4 C storage and subcultured onto SAB plates at 37 C. When (ATCC 27853), as well as Methicillin-resistant Staphylococcus fungal colony growth was just visible, sterile wooden appli- aureus (MRSA), Klebsiella pneumonia,andProteus vulgaris cator sticks were used to transfer mycelia growth to gridded ∘ (clinical isolates), were generously provided by Dr. Donald SAB plates and cultures at 37 C. Colonies were observed for A. Ferguson, Jr. Ph.D., Director of the Clinical Microbi- morphological differences and retested by repeated fungal ology Laboratory, Quillen College of Medicine, East Ten- culture and screening assay. nessee State University. Aspergillus oryzae (FGSC#A815) was obtained from the Fungal Genetic Stock Center, University of 2.4. Characterization of Fungal Colony Size and Onset of Spore Missouri-Kansas City. Bacterial strains were maintained on ∘ Production. The single isolated antibacterial A. oryzae isolate blood agar plates at 37 CandA. oryzae was maintained on ∘ and the parental A. oryzae strain were inoculated onto SAB Sabourad dextrose agar (SAB) plates at 30 Cuntilscreening plates, 300 spores in 10 L sterile water at the plate center, A. oryzae culture supernatants for antibacterial activity (see ∘ and cultured at 37 Cfortwodays,thenmovedtoroom below). temperature for further culture. A Westover Scientific Stereo Microscope and Sony HQX Digital Still Camera were used 2.2. Ethyl Methanesulfonate (EMS) Mutagenesis of Aspergillus to observe colony size and spore formation at two through oryzae. A. oryzae spores (Fungal Genetics Stock Center), in seven days after inoculation. Colony diameter sizes were sterile water, were treated with zero (mock), 50, 100, 200, 400, −1 ∘ quantitated in cm. 800, 1600, 3200, or 6400 gmL EMS overnight at 37 C. Washed and diluted spores were then plated onto SAB plates ∘ 2.5. Characterization of Spore Production. The antibacterial and incubated at 37 C for 48 hours. The percentage of viable A. oryzae isolateandtheparentalA. oryzae strain were EMS-treated spores compared to mock treated spores was inoculated onto SAB plates, 3000 spores in 100 Lsterile determined by counting fungal colonies per plate. water spread to form lawns of fungal growth, and cultured ∘ at 37 C for two days then moved to room temperature 2.3. Screening A. oryzae Culture Supernatants for for further culture. At eight days after inoculation, fungal Antibacterial