Microbiology Papers in Press. Published January 7, 2015 as doi:10.1099/mic.0.000030
Microbiology Pyomelanin production in Penicillium chrysogenum is stimulated by L-Tyrosine --Manuscript Draft-Manuscript Number:
MIC-D-14-00089R1
Full Title:
Pyomelanin production in Penicillium chrysogenum is stimulated by L-Tyrosine
Short Title:
Pyomelanin production by Penicillium chrysogenum
Article Type:
Standard
Section/Category:
Physiology and Biochemistry
Corresponding Author:
Archana Vasanthakumar, PhD Harvard University Cambridge, MA UNITED STATES
First Author:
Archana Vasanthakumar, PhD
Order of Authors:
Archana Vasanthakumar, PhD Alice DeAraujo, MLA Joy Mazurek, MS Michael Schilling, PhD Ralph Mitchell, PhD
Abstract:
We isolated a strain of Penicillium chrysogenum from a tomb in Upper Egypt, which was capable of producing brown pigment in vitro when grown in a minimal salts medium containing tyrosine. We present evidence that this pigment is a pyomelanin, a compound that is known to assist in the survival of some microorganisms in adverse environments. We tested type strains of Penicillium chrysogenum, which were also able to produce this pigment under similar conditions. Inhibitors of the DHN and DOPA melanin pathways were unable to inhibit the formation of the pigment. FTIR analysis indicated that this brown pigment is similar to pyomelanin. Pyrolysis-GC/MS revealed the presence of phenolic compounds. Using LC/MS, homogentisic acid, the monomeric precursor of pyomelanin, was detected in supernatants of P. chrysogenum cultures growing in tyrosine medium but not in cultures lacking tyrosine. Partial regions of the genes encoding two enzymes in the homogentisic acid pathway of tyrosine degradation were amplified. Data from reverse-transcription PCR demonstrated that hmgA transcription was increased in cultures grown in tyrosine medium, suggesting that tyrosine induced the transcription.
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Title: Pyomelanin production in Penicillium chrysogenum is stimulated by L-Tyrosine
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Running title: Pyomelanin production by Penicillium chrysogenum
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Archana Vasanthakumar1*, Alice DeAraujo1, Joy Mazurek2, Michael Schilling2 and Ralph Mitchell1
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1
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University, 58 Oxford St. Cambridge, MA 02138, USA
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2
Laboratory of Applied Microbiology, School of Engineering and Applied Sciences, Harvard
Getty Conservation Institute, Getty Center Drive, Los Angeles CA 90049 USA
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*Corresponding author.
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Mailing address: School of Engineering and Applied Sciences, Harvard University, 58 Oxford
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Street, Cambridge, MA 02138 USA.
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Tel: 617-495-3307, Fax: 617-496-1471
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Email: [email protected]
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Contents Category: Physiology and Biochemistry
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Number of words in:
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Summary – 176
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Main text – 2807
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Five figures and three tables
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1
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Summary
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We isolated a strain of Penicillium chrysogenum from a tomb in Upper Egypt, which was capable of
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producing brown pigment in vitro when grown in a minimal salts medium containing tyrosine. We
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present evidence that this pigment is a pyomelanin, a compound that is known to assist in the
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survival of some microorganisms in adverse environments. We tested type strains of Penicillium
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chrysogenum, which were also able to produce this pigment under similar conditions. Inhibitors of
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the DHN and DOPA melanin pathways were unable to inhibit the formation of the pigment. FTIR
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analysis indicated that this brown pigment is similar to pyomelanin. Pyrolysis-GC/MS revealed the
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presence of phenolic compounds. Using LC/MS, homogentisic acid, the monomeric precursor of
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pyomelanin, was detected in supernatants of P. chrysogenum cultures growing in tyrosine medium
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but not in cultures lacking tyrosine. Partial regions of the genes encoding two enzymes in the
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homogentisic acid pathway of tyrosine degradation were amplified. Data from reverse-transcription
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PCR demonstrated that hmgA transcription was increased in cultures grown in tyrosine medium,
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suggesting that tyrosine induced the transcription.
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Keywords: Penicillium, brown pigment, pyomelanin, homogentisic acid, LC/MS, tyrosine, RT-PCR
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Introduction
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Microbial melanins are polymers that may aid in the survival of some microorganisms under
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adverse conditions. Different classes of melanins exist, including eumelanin, phaeomelanin,
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allomelanin and pyomelanin (Butler & Day, 1998; Eisenman & Casadevall, 2012).
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Dihydroxynapthalene melanin (DHN-melanin) and dihydroxyphenylalanin (DOPA) are two
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examples of melanins produced by a wide range of fungi (Butler & Day, 1998). In the former
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pathway, 1,3,6,8-tetrahydroxynaphthalene undergoes a series of reduction and dehydration reactions
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to form DHN-melanin. This melanin is responsible for the characteristic brown or black color of
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fungal spores (Bell & Wheeler, 1986). DOPA melanin, primarily studied in humans, has also been
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found in fungi. This pathway of melanin production relies on tyrosine oxidation (Butler & Day,
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1998).
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Chemically, melanins are heterogenous polymers consisting of phenolic compounds, usually
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containing carbohydrates and proteins in bound form. These complex mixtures are difficult to
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analyze (Hamilton & Gomez, 2002). Identification and analysis methods have traditionally been
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based on properties such as solubility in various solvents.
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Pyomelanin is a water-soluble melanin that is formed when homogentisic acid, an intermediate in
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the tyrosine degradation pathway, accumulates and subsequently, polymerizes (Figure 1). This
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extracellular melanin was first described in Pseudomonas aeruginosa (Yabuuchi & Ohyama, 1972).
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It has since been described in the filamentous fungus Aspergillus (Schmaler-Ripcke et al., 2009;
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Keller et al., 2011), the yeast Yarrowia (Carreira et al., 2001), the dimorphic fungus Sporothrix
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(Almeida-Paes et al., 2012) and many bacterial genera including Shewanella, Legionella, Vibrio,
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Alcaligenes, Hyphomonas, Streptomyces and Rhizobium (Arias-Barrau et al., 2004; Mendez et al.,
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2011; Turick et al., 2002; 2009).
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In microorganisms, several functions contributing to survival in adverse conditions have been
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ascribed to pyomelanin (Turick et al., 2009). A brown pigment in Legionella pneumophila was
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observed to confer protection from UV radiation (Steinert et al., 2001). Under conditions of low
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dissolved oxygen levels in the environment, pyomelanin, due to its ability to accelerate solid phase
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metal reduction, is hypothesized to aid in the survival of Shewanella oneidensis MR-1 (Turick et
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al., 2009). Pseudomonas aeruginosa strains in patients with chronic lung infections produce a
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brown pigment that helps them tolerate oxidative stress in vitro (Rodríguez-Rojas et al., 2009).
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Turick and colleagues suggested that pyomelanin was important in the life cycle of Shewanella
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algae, a facultative anaerobe, since this organism can use the pigment to accelerate the rate of
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dissimilatory iron mineral reduction in addition to using it as a terminal electron acceptor (Turick et
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al., 2002; 2003; 2008a). Recent research on Aspergillus fumigatus identified the production of
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pyomelanin (Schmaler-Ripcke et al., 2009). Deletion mutants, in which pyomelanin production was
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abolished, were more susceptible to reactive oxygen species (ROI). This finding suggests that
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pyomelanin protects Aspergillus from ROI.
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Penicillium chrysogenum is a filamentous fungus of enormous medical significance, due to its
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ability to produce penicillin, the first antibiotic to be described (Fleming, 1929). It is a ubiquitous
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fungus and has been isolated from a range of different environments such as soil and caves
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(Alexopoulos, 1996). We recently isolated a fungus from a tomb in Upper Egypt, which was
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identified by ITS gene sequencing as a strain of Penicillium chrysogenum (Vasanthakumar et al.,
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2013). We describe in this report our data showing that both this isolate, as well as the type strain of
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Penicillium chrysogenum, produce a brown pigment when grown in a minimal salts medium
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containing tyrosine. We present evidence that this pigment is pyomelanin. We also describe the
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results of our investigation into the chemical and genetic pathways underlying the formation of this
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pigment by P. chrysogenum.
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Methods
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Isolation and growth of the fungal strains
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Swabs moistened with sterile deionized water were used to collect samples from the tomb. A
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dilution series of samples was spread onto microbiological growth media such as tryptic soy agar,
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potato dextrose agar and malt extract agar (Becton Dickinson, Franklin Lakes, NJ). Pure cultures of
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fungal isolates were obtained. Type strains of P. chrysogenum and A. niger were obtained from
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ATCC (P. chrysogenum strains Wisconsin 54-1255 ATCC® 28089 and NRRL 811, ATCC® 10107,
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Aspergillus niger ATCC® 10535).
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Identification of fungi by ITS sequencing
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DNA was extracted from fungal material using the UltraClean Microbial DNA extraction kit
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(MoBio Inc., Carlsbad, CA). The ITS region was amplified as previously described
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(Vasanthakumar et al. 2013), using the primers ITS1 and ITS4 (White, 1990) and amplicons were
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sequenced using ITS1 or ITS4 at the Dana-Farber/Harvard Cancer Center DNA Resource Core.
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Sequences were trimmed and edited using FinchTV version 1.5.0 (Geospiza Inc., Seattle, WA) and
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checked against GenBank and UNITE databases (Abarenkov et al., 2010). ITS sequences were
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submitted to GenBank.
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Pigment production
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Fungi were grown in a minimal salts medium ((NH4)2SO4 0.22 g/L, KH2PO4 1.20 g/L,
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MgSO4.7H2O 0.23 g/L, CaCl2.2H2O 0.25 g/L), containing 60% (w/v) sodium lactate (9 ml/L) and
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tyrosine (2g/L) as well as in growth medium without tyrosine. In addition to the isolated fungi, P.
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chrysogenum strains Wisconsin 54-1255 and NRRL 811 (Table 1) were also tested for pigment
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production. Inoculated strains were incubated at 37°C. Synthetic pyomelanin was generated by
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treating homogentisic acid with NaOH under aerobic conditions.
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To identify the putative pathway of melanin production, fungal cultures were grown on tyrosine
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medium amended with inhibitors of melanin synthesis. Briefly, 25 g/ml tricyclazole (an inhibitor
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of the DHN melanin pathway) (Liu et al. 2014) and 50 g/ml kojic acid (an inhibitor of the DOPA
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melanin pathway) were included in tyrosine media. Fungal cultures were observed every day for
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pigment production both in the fungal biomass as well as in the medium. The effect of sulcotrione, a
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known inhibitor of pyomelanin production (Almeida-Paes et al. 2012), was not tested in this study.
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However, the effect of including ascorbic acid, an antioxidant, which would presumably inhibit
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autooxidation of homogentisic acid, is reported.
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FTIR analysis
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The pigmented growth medium was filtered to exclude all fungal material. A representative sample
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particle was placed on a Diamond window, and analyzed by transmitted infrared beam with an
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aperture of approximately 100 x 100 microns, using a 15X objective. Each spectrum was the sum of
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200 scans at a resolution of 4 cm-1. Based on the initial analysis results of bulk material, extraction
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was done by placing a microdroplet of solvent on the sample, and analysis was performed on the
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resultant extracted dry solvent ring.
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Analysis of brown pigment using Pyrolysis-GC/MS
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The dialyzed, cell-free fungal growth medium was analyzed by (i) a Frontier Lab PY-2020D
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double-shot pyrolyzer with Agilent Technologies 5975C inert MSD/7890A GC; (ii) 320°C
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pyrolysis interface; (iii) 30 M x 0.25 mm x 0.25 µm J&W DB-5MS-UI column with Frontier Vent-
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Free adaptor (40 M effective column length); (iv) 1 ml/minute helium; (v) 320°C injector with 50:1
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split ratio; (vi) oven program: 2 min. at 40°C, 6°C/min. to 320°C, 9 min. isothermal; (vii) 320°C
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MS transfer line; (viii) 230°C source; (ix)150°C MS quad; (x) 10-600 amu scanned at 2.59 scans
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per second. Samples pyrolyzed at 550°C in 50 µl stainless steel.
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Detection of homogentisic acid using LC/MS
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The filtered growth medium was analyzed in an Agilent 6210 ESI-TOF LC/MS. Samples were run
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in negative ion mode for 20 minutes, using an Agilent Zorbax C-18 reverse phase column (3.5 m
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particle size, 2.1 x 100mm). A formula confirmation method was used in the Agilent MassHunter
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software to detect homogentisic acid (formula C8H8O4). Retention times and mass spectra were
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compared with those of the standard. Homogentisic acid (50 mM) and water were used as the
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standard and blank, respectively.
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Genetic analysis of enzymes in the tyrosine degradation pathway
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DNA was isolated from P. chrysogenum strains using MoBio Microbial DNA Isolation kit (MoBio,
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Inc. Carlsbad, CA). Primers were designed for genes encoding the enzymes, homogentisate
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dioxygenase (hmgA) and 4-hydroxyphenylpyruvate (hppD), based on the published genome
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sequence of AM920427 Penicillium chrysogenum Wisconsin 54-1255 (van den Berg et al., 2008,
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Table 2). These primers enabled amplification of partial regions of these genes using PCR. The
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conditions for PCR were as follows: an initial denaturation step at 94°C for 4 min, followed by 35
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cycles of denaturation at 94°C for 1 min, annealing at 52°C for 45 1 min and extension at 72°C for
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2 min, followed by a final extension step at 72°C for 7 min. Amplicons were sequenced in both
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directions using the forward and reverse primers at the Dana-Farber/Harvard Cancer Center DNA
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Resource Core.
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Transcription analysis of homogentisic acid dioxygenase (hmgA) and 4-
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hydroxyphenlypyruvate (hppD)
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Total RNA was extracted from fungi using RNAeasy Kit (Qiagen, Valencia, CA). DNAse (Qiagen,
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Valencia, CA) treatment was performed to exclude possible contamination by small amounts of
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lingering DNA. Reverse transcription-PCR was performed on the RNA using hmgA, hppD and ITS
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primers. Conditions for RT-PCR were as follows: 50°C for 30 min, 25-30 cycles of 94°C 30 s, 52
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°C 45s and 72° 120 s, followed by an extension cycle at 72°C for 7 min.
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Results
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Identification and description of Penicillium chrysogenum isolated in this study
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ITS sequencing confirmed the identity of the isolates as Penicillium chrysogenum, with 98-99%
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similarity to GenBank sequences (accession numbers JQ422624 and AM948960). Sequences
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obtained in this study were deposited in GenBank under the accession numbers KP280083 and
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KP280084. Penicillium chrysogenum colonies of the isolated strains contained white mycelia with
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olive green spores on rich media and grayish spores on minimal salts media.
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Production of brown pigment in microbiological growth medium amended with tyrosine
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Growth of P.chrysogenum isolates in tyrosine medium was apparent within 48 hours. However, the
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brown pigment was apparent only 10-15 days after inoculation. All Penicillium chrysogenum strains
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grown in a growth medium containing tyrosine produced a brown pigment whereas those grown in
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medium lacking tyrosine did not (Figure 2a, b). The pigment accumulated extracellularly. However,
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over time, the fungal cell walls appeared to also accumulate pigment, as observed using light
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microscopy (data not shown). It was not clear whether this pigment was pyomelanin or some other
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melanin.
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P. chrysogenum cultures grown on kojic acid-amended tyrosine medium were identical to cultures
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grown on tyrosine medium, indicating that kojic acid did not inhibit melanin production (Figure 2c).
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Cultures grown on tricyclazole-amended tyrosine medium, however, were tan –colored, rather than
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greenish, indicating that inhibition of DHN-melanin production led to a change in the pigments in
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the fungal biomass (data not shown). However, the extracellular pigment in the medium was not
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affected by tricyclazole (Figure 2d), indicating that the pathway for formation of this pigment was
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not inhibited by tricyclazole.
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Chemical analysis of brown pigment
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FTIR analysis demonstrated significant similarity between the Penicillium chrysogenum pigment
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and synthetic pyomelanin (Figure 3). However, since other humic acid polymers also show a similar
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FTIR spectrum, a more specific method, Pyrolysis-GC-MS (Py-GC/MS) was used to analyze the
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pigment. This method utilizes heat to break the complex phenolic compounds into smaller
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fragments. Our Py-GC/MS analysis demonstrated that the brown pigment was a complex
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heterogeneous polymer containing multiple ring structures. In particular, 4-methoxy benzeneacetic
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acid, 4-methoxy benzenepropanoic acid and other phenolic compounds were identified using
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information in the NIST library (Lindstrom & Mallard (Eds.) NIST Chemistry WebBook,
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retrieved September 4, 2013). Though Py-GC/MS was more successful in detecting phenolic
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compounds, the complexity of the sample made it difficult to separate other fungal products from
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the brown pigment (data not shown). In addition, the heterogeneity of the complex aromatic
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compounds made it difficult to obtain uniform results between samples. Therefore, an LC/MS
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technique was chosen so that the monomeric precursor, homogentisic acid, could be detected in the
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fungal cultures. The LC/MS analysis was successful in detecting homogentisic acid in P.
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chrysogenum cultures that were grown in tyrosine medium (Figure 4, Table 3). No homogentisic
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acid was detected when P. chrysogenum was grown in minimal salts medium lacking tyrosine
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(Table 3).
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Fungal cultures containing tyrosine and ascorbic acid (an antioxidant) were also tested for
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homogentisic acid, with the hypothesis that the antioxidant would inhibit the process of
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autooxidation to pyomelanin. Over time, the cultures containing ascorbic acid still contained
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detectable levels of homogentisic acid whereas cultures without the antioxidant contained very little
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homogentisic acid. It is likely that the homogentisic acid had oxidized to pyomelanin.
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Analysis of the genetic pathway for pyomelanin formation
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Partial hmgA and hppD genes were amplified in our isolates as well as in the type strain. The hmgA
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and hppD gene sequences in our isolate are at least 98% identical to those in the published genome
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of the type strain (Table 2). Reverse Transcription-PCR analysis of these genes demonstrated that in
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the presence of tyrosine, hmgA was expressed at a higher level (Figure 5). The difference in the
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transcription of hppD was not as obvious.
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Discussion
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In this study, we present evidence for the production of a water-soluble pigment, similar to
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pyomelanin, by Penicillium chrysogenum. Our data demonstrate that the production of this
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extracellular brown pigment is not inhibited by tricylazole or kojic acid, inhibitors of the DHN-
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melanin and DOPA melanin pathway, respectively, and can therefore, can be attributed to a
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different pathway. In the current study, we did not test the effect of sulcotrione, a known inhibitor
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of pyomelanin production (Almeida-Paes et al. 2012).
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The pyomelanin group of melanins is characterized by their extracellular nature and water solubility
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(Turick et al., 2010). The detection of homogentisic acid by LC/MS only in the P. chrysogenum
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cultures containing tyrosine but not in the cultures lacking tyrosine demonstrates that the production
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of homogentisic acid, a precursor of pyomelanin, is dependent on the presence of tyrosine. Though
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there is genomic information about the homogentisic acid pathway of tyrosine degradation (van den
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Berg et al., 2000), this is, to our knowledge, the first documented experimental evidence of
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pyomelanin production by a Penicillium chrysogenum.
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The pyomelanin itself was difficult to analyze because of its heterogeneous nature and the presence
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of complex phenolic structures. When Py-GC/MS was used, some aromatic compounds were
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identified that could have been derived from tyrosine degradation. However, it was not possible to
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conclusively identify the constituents of the brown pigment. Moreover, the polymeric nature of
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pyomelanin makes it extremely heterogeneous (Hunter & Newman, 2010). Therefore, its chemical
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composition can vary even between replicate tubes of fungal culture (begun from the same stock
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inoculum) grown in the same medium. However, the LC/MS method was successful in detecting
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homogentisic acid, the monomeric precursor of pyomelanin. The ability to detect hmgA was crucial
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in conclusively identifying the presence of pyomelanin.
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Since previously published genomic information was available, it was possible to design primers
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specific for the genes encoding the enzymes, homogentisate dioxygenase (hmgA) and 4-
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hydroxyphenylpyruvate dioxygenase (hppD). These two enzymes convert homogentisate to
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maleylacetoacetate and 4-hydroxyphenylpyruvate to homogentisate, respectively (Schmaler-Ripcke
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et al., 2009). Amplification of partial regions of these two genes revealed that they are 98-99%
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similar to the published sequences of Wis 54-1255. Using RT-PCR, it was possible to analyze the
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role of tyrosine in inducing transcription of the two genes. The increased transcription of hmgA in
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response to tyrosine indicates that tyrosine induces this response. This, in turn, suggests that the
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presence of tyrosine stimulates the tyrosine degradation pathway to be highly expressed. However,
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the cultures not exposed to tyrosine also demonstrated low levels of hmgA transcription, indicating
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that this gene is constitutively expressed in the fungus even in the absence of tyrosine. The
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difference observed in hppD transcription was subtle. This could be due to the fact that only partial
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regions of the gene were transcribed in this study.
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We demonstrated that Penicillium chrysogenum isolates from various environments were capable of
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producing brown pigment in vitro when tyrosine was included in the growth medium. This ability
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suggests that the homogentisic pathway of tyrosine degradation is active in all these isolates. It is
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possible that pyomelanin plays a role in survival of the fungus under adverse conditions (Turick et
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al., 2009). In fungi such as Sporothrix and Aspergillus fumigatus, this brown pigment is known to
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help protect against oxidative stress (Schmaler-Ripcke et al., 2009; Almeida-Paes et al., 2012). In
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bacteria such as Pseudomonas aeruginosa, and Burkholderia cenocepacia, this pigment performs
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functions of stress tolerance such as heavy metal binding (Turick et al., 2003; 2008b), or relief of
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oxidative stress (Keith et al., 2007). It is conceivable that the P. chrysogenum isolates found in
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tombs in Egypt are subjected to multiple stresses due to nutrient deficiency as well as lack of
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moisture. Pyomelanin might enable the fungus to tolerate these stresses. Future work will be
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required to determine whether this pigment enables Penicillium chrysogenum to be an ecologically
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important component of the microbiome in other stressed environments.
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Acknowledgements
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This research was carried out as a collaborative agreement between the Getty Conservation Institute
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and Egypt’s Supreme Council of Antiquities and was supported by grant # 268237 from the Getty
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Conservation Institute to the Laboratory of Applied Microbiology, School of Engineering and
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Applied Sciences, Harvard University. The authors wish to thank Dr. Marc Mittelman, Dr. Federica
12
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Villa and Dr. Patricia Sanmartín for insightful discussions, Felix Wu for technical assistance and the
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anonymous reviewers for useful suggestions.
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Méndez, V., Agulló, L., González, M. & Seeger, M. (2011). The homogentisate and
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homoprotocatechuate central pathways are involved in 3- and 4-hydroxyphenylacetate degradation
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by Burkholderia xenovorans. LB400. PLoS One. 6, e17583. doi: 10.1371/journal.pone.0017583.
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Moyer, A.J., May, O.E. & Herrick, H.T. (1936). The production of gluconic acid by Penicillium
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chrysogenum. Zentralbl Bakteriol, II Abt 95, 311-324.
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Osler, W. (1904). Ochronosis, the pigmentation of cartilages, sclerotics and skin in alkaptonuria.
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Lancet 1, 10.
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O'Sullivan, C.Y. & Pirt, S.J. (1973). Penicillin production by lysine auxotrophs of Penicillium
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chrysogenum. J. Gen. Microbiol. 76, 65-75.
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Rodríguez-Rojas, A., Mena, A., Martín, S., Borrell, N., Oliver, A. & Blázquez, J. (2009).
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Inactivation of the hmgA gene of Pseudomonas aeruginosa leads to pyomelanin hyperproduction,
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stress resistance and increased persistence in chronic lung infection. Microbiology 155, 1050–1057.
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Schmaler-Ripcke, J., Sugareva, V., Gebhardt, P., Winkler, R., Kniemeyer, O., Heinekamp, T. &
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Brakhage, A.A. (2009). Production of pyomelanin, a second type of melanin, via the tyrosine
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degradation pathway in Aspergillus fumigatus. Appl. Environ. Microbiol. 75, 493–503. doi:
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10.1128/AEM.02077-08.
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Steinert, M., Flugel, M., Schuppler, M., Helbig, J.H., Supriyono, A., Proksch, P. & Luck, P.C.
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(2001). The Lly protein is essential for p-hydroxyphenylpyruvate dioxygenase activity in Legionella
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pneumophila. FEMS Microbiol Lett. 203, 41–47. doi: 10.1016/S0378-1097(01)00331-7.
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Turick, C.E., Tisa, L. S. & Caccavo, Jr. , F. (2002). Melanin production and use as a soluble
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electron shuttle for Fe(III) oxide reduction and as a terminal electron acceptor by Shewanella algae
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BrY. Appl. Environ. Microbiol. 68, 2436–2444.
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Turick, C.E., Caccavo, Jr., F. & Tisa, L. S. (2003). Electron transfer to Shewanella algae BrY to
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HFO is mediated by cell-associated melanin. FEMS Microbiol. Lett. 220, 99-104.
345
Turick, C.E., Caccavo, Jr. F. & Tisa, L.S. (2008a). Pyomelanin is Produced by Shewanella algae
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BrY and Affected by Exogenous Iron. Can. J. Microbiol. 54, 334-339.
347
Turick, C.E., Knox, A.S., Leverette, C. L. & Kritzas, Y.G. (2008b). In-situ uranium
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immobilization by microbial metabolites. J. Environ. Rad. 99, 890-899.
349
Turick, C. E., Beliaev, A., Ekechukwu, A.A., Poppy, T., Maloney, A. & Lowy, D.A. (2009). The
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role of 4-hydroxyphenylpyruvate dioxygenase in enhancement of solid-phase electron transfer by
351
Shewanella oneidensis MR-1. FEMS Microbiol. Ecol. 68, 223-235.
352
Turick, C. E., Knox, A. S., Becnel, J. M., Ekechukwu, A. A., & Milliken, C. E. (2010).
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Properties and Function of Pyomelanin. Biopolymers. 449-472. Magdy Elnashar (Ed.), InTech.
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http://www.intechopen.com/books/biopolymers/properties-and-function-of-pyomelanin.
355
van den Berg, M. A, Albang, R., Albermann, K., Badger, J.H., Daran, J.M., Driessen, A.J.,
356
Garcia-Estrada, C., Fedorova, N.D., Harris, D.M., Heijne, W.H., Joardar, V., Kiel, J.A.,
357
Kovalchuk, A., Martín, J.F., Nierman, W.C., Nijland, J.G., Pronk, J.T., Roubos, J.A., van der
358
Klei, I.J., van Peij, N. N., Veenhuis, M., von Döhren, H., Wagner, C., Wortman, J.,
359
Bovenberg, R.A. (2008). Genome sequencing and analysis of the filamentous fungus Penicillium
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chrysogenum. Nature Biotech. 26, 1161 – 1168. doi:10.1038/nbt.1498
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Vasanthakumar, A., DeAraujo, A., Mazurek, J., Schilling, M., & Mitchell, R. (2013).
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Microbiological survey for analysis of the brown spots on the walls of the tomb of King
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Tutankhamun. Int. Biodet. & Biodegr. 79, 56–63
364
White, T. J., Bruns, T., Lee, S. & Taylor, J. W. (1990). Amplification and direct sequencing of
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fungal ribosomal RNA genes for phylogenetics. In: Innis, M. A, Gelfand, D. H., Sninsky, J. J.,
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White, T. J., editors. PCR protocols: a guide to methods and applications. New York, N.Y:
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Academic Press, Inc.; pp. 315–322.
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Yabuuchi, E. & Ohyama, A. (1972). Characterization of “Pyomelanin”-Producing Strains of
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Pseudomonas aeruginosa IJSEM 22, 53-64. doi: 10.1099/00207713-22-2-53.
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Table and figure legends
371
Table 1. Isolates of Penicillium chrysogenum tested for brown pigment production.
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Table 2. Description of primers used in PCR amplification of genes for enzymes (hmgA and hppD)
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active in the pyomelanin pathway in Penicillium chrysogenum. GenBank accession numbers for the
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genes as well as the predicted proteins are included.
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Table 3. Retention time and abundance of homogentisic acid detected in supernatant of P.
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chrysogenum grown in MSM with or without Tyrosine. A formula confirmation program was used
377
in MassHunter software. The compound formula is C8H8O4 and the target mass is 168.04226.
378
Figure 1. Pathway for pyomelanin formation. When homogentisic acid accumulates, it autooxidizes
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and polymerizes to form pyomelanin. The enzymes HppD and HmgA catalyze the formation and
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breakdown of homogentisic acid, respectively.
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Figure 2. Growth of Penicillium chrysogenum in minimal salts medium a) without tyrosine or b)
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with tyrosine. Brown pigment is formed only when tyrosine is present. Inclusion of c) DOPA
17
383
melanin inhibitor kojic acid and d) DHN-melanin inhibitor tricyclazole, in the tyrosine medium did
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not inhibit extracellular brown pigment formation.
385
Figure 3. FTIR analysis of a) P. chrysogenum brown pigment, and b) synthetic pyomelanin
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(generated by treating homogentisic acid with NaOH under aerobic conditions).
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Figure 4. LC/MS detection of homogentisic acid in P. chrysogenum culture supernatant (bottom
388
panel). In the absence of tyrosine, no homogentisic acid was detected (middle panel). The top panel
389
demonstrates the retention time of homogentisic acid, which was used as the standard.
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Figure 5. Transcription of the genes hmgA and hppD in Penicillium chrysogenum grown with or
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without tyrosine. 12 ng of RNA was used in each RT-PCR reaction. A housekeeping gene for -
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actin was included as a control.
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Table 1_AVasanthakumar_pyomelanin Click here to download Table: Table 1_pyo.docx
A. Vasanthakumar et al.
Pyomelanin production by Peniillium chrysogenum
Table 1. Isolates of Penicillium chrysogenum tested for brown pigment production Isolate ID
Origin
Depository ID
Reference
Wis 541255
Cantaloupe
ATCC 28089/ NRRL 1951
O'Sullivan & Pirt, 1973
NRRL 811
Cheese
ATCC 10107/ NRRL 811
Moyer et al. 1936
T04C
Limestone wall – sealed tomb
N/A
Vasanthakumar et al. 2013
Fb
Limestone wall – open tomb N/A
Vasanthakumar et al. 2013
Table 2_AVasanthakumar_pyomelanin Click here to download Table: Table 2_pyo.docx
A. Vasanthakumar et al.
Pyomelanin production by Peniillium chrysogenum
Table 2. Description of primers used in PCR amplification of genes for enzymes (hmgA and hppD) active in the pyomelanin pathway in Penicillium chrysogenum. GenBank accession numbers for the genes as well as the predicted proteins are included. Gene
Primer Sequence
% identity GenBank of gene Accession #
% identity of predicted protein
GenBank Accession #
hmgA-F
GCGGGGATACATCTGTGAAT
99
AM920427.1
95
XP_002557732
hmgA-R
CCGACCATGTGAGCACTAAA
hppD-F
CAGTCAAGGACGTTGCCTTT
99
AM920427.1,
58
XP_002557734
hppD-R
TAGCCACCCTCATCGAAATC
XM_002557688
Table 3_AVasanthakumar_pyomelanin Click here to download Table: Table 3_pyo.docx
A. Vasanthakumar et al.
Pyomelanin production by Peniillium chrysogenum
Table 3. Retention time and abundance of homogentisic acid detected in supernatant of P. chrysogenum grown in MSM with or without Tyrosine. A formula confirmation program was used in MassHunter software. The compound formula is C8H8O4 and the target mass is 168.04226. Sample
Retention time Mass
Abundance
(minutes) Penicillium chrysogenum - Tyrosine
8.449
168.04235 109
Penicillium chrysogenum + Tyrosine 6.039
168.04235 2489666
Homogentisic acid
168.04269 661785
5.948
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Microbiology Pyomelanin production in Penicillium chrysogenum is stimulated by L-Tyrosine --Manuscript Draft-Manuscript Number:
MIC-D-14-00089R1
Full Title:
Pyomelanin production in Penicillium chrysogenum is stimulated by L-Tyrosine
Short Title:
Pyomelanin production by Penicillium chrysogenum
Article Type:
Standard
Section/Category:
Physiology and Biochemistry
Corresponding Author:
Archana Vasanthakumar, PhD Harvard University Cambridge, MA UNITED STATES
First Author:
Archana Vasanthakumar, PhD
Order of Authors:
Archana Vasanthakumar, PhD Alice DeAraujo, MLA Joy Mazurek, MS Michael Schilling, PhD Ralph Mitchell, PhD
Abstract:
We isolated a strain of Penicillium chrysogenum from a tomb in Upper Egypt, which was capable of producing brown pigment in vitro when grown in a minimal salts medium containing tyrosine. We present evidence that this pigment is a pyomelanin, a compound that is known to assist in the survival of some microorganisms in adverse environments. We tested type strains of Penicillium chrysogenum, which were also able to produce this pigment under similar conditions. Inhibitors of the DHN and DOPA melanin pathways were unable to inhibit the formation of the pigment. FTIR analysis indicated that this brown pigment is similar to pyomelanin. Pyrolysis-GC/MS revealed the presence of phenolic compounds. Using LC/MS, homogentisic acid, the monomeric precursor of pyomelanin, was detected in supernatants of P. chrysogenum cultures growing in tyrosine medium but not in cultures lacking tyrosine. Partial regions of the genes encoding two enzymes in the homogentisic acid pathway of tyrosine degradation were amplified. Data from reverse-transcription PCR demonstrated that hmgA transcription was increased in cultures grown in tyrosine medium, suggesting that tyrosine induced the transcription.
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Pyomelanin-paper-text-only_AVasanthakumar-Dec19.docx
1
Title: Pyomelanin production in Penicillium chrysogenum is stimulated by L-Tyrosine
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Running title: Pyomelanin production by Penicillium chrysogenum
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Archana Vasanthakumar1*, Alice DeAraujo1, Joy Mazurek2, Michael Schilling2 and Ralph Mitchell1
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1
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University, 58 Oxford St. Cambridge, MA 02138, USA
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2
Laboratory of Applied Microbiology, School of Engineering and Applied Sciences, Harvard
Getty Conservation Institute, Getty Center Drive, Los Angeles CA 90049 USA
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*Corresponding author.
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Mailing address: School of Engineering and Applied Sciences, Harvard University, 58 Oxford
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Street, Cambridge, MA 02138 USA.
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Tel: 617-495-3307, Fax: 617-496-1471
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Email: [email protected]
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Contents Category: Physiology and Biochemistry
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Number of words in:
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Summary – 176
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Main text – 2807
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Five figures and three tables
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1
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Summary
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We isolated a strain of Penicillium chrysogenum from a tomb in Upper Egypt, which was capable of
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producing brown pigment in vitro when grown in a minimal salts medium containing tyrosine. We
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present evidence that this pigment is a pyomelanin, a compound that is known to assist in the
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survival of some microorganisms in adverse environments. We tested type strains of Penicillium
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chrysogenum, which were also able to produce this pigment under similar conditions. Inhibitors of
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the DHN and DOPA melanin pathways were unable to inhibit the formation of the pigment. FTIR
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analysis indicated that this brown pigment is similar to pyomelanin. Pyrolysis-GC/MS revealed the
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presence of phenolic compounds. Using LC/MS, homogentisic acid, the monomeric precursor of
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pyomelanin, was detected in supernatants of P. chrysogenum cultures growing in tyrosine medium
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but not in cultures lacking tyrosine. Partial regions of the genes encoding two enzymes in the
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homogentisic acid pathway of tyrosine degradation were amplified. Data from reverse-transcription
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PCR demonstrated that hmgA transcription was increased in cultures grown in tyrosine medium,
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suggesting that tyrosine induced the transcription.
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Keywords: Penicillium, brown pigment, pyomelanin, homogentisic acid, LC/MS, tyrosine, RT-PCR
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Introduction
48 49
Microbial melanins are polymers that may aid in the survival of some microorganisms under
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adverse conditions. Different classes of melanins exist, including eumelanin, phaeomelanin,
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allomelanin and pyomelanin (Butler & Day, 1998; Eisenman & Casadevall, 2012).
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Dihydroxynapthalene melanin (DHN-melanin) and dihydroxyphenylalanin (DOPA) are two
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examples of melanins produced by a wide range of fungi (Butler & Day, 1998). In the former
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pathway, 1,3,6,8-tetrahydroxynaphthalene undergoes a series of reduction and dehydration reactions
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to form DHN-melanin. This melanin is responsible for the characteristic brown or black color of
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fungal spores (Bell & Wheeler, 1986). DOPA melanin, primarily studied in humans, has also been
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found in fungi. This pathway of melanin production relies on tyrosine oxidation (Butler & Day,
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1998).
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Chemically, melanins are heterogenous polymers consisting of phenolic compounds, usually
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containing carbohydrates and proteins in bound form. These complex mixtures are difficult to
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analyze (Hamilton & Gomez, 2002). Identification and analysis methods have traditionally been
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based on properties such as solubility in various solvents.
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Pyomelanin is a water-soluble melanin that is formed when homogentisic acid, an intermediate in
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the tyrosine degradation pathway, accumulates and subsequently, polymerizes (Figure 1). This
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extracellular melanin was first described in Pseudomonas aeruginosa (Yabuuchi & Ohyama, 1972).
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It has since been described in the filamentous fungus Aspergillus (Schmaler-Ripcke et al., 2009;
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Keller et al., 2011), the yeast Yarrowia (Carreira et al., 2001), the dimorphic fungus Sporothrix
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(Almeida-Paes et al., 2012) and many bacterial genera including Shewanella, Legionella, Vibrio,
3
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Alcaligenes, Hyphomonas, Streptomyces and Rhizobium (Arias-Barrau et al., 2004; Mendez et al.,
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2011; Turick et al., 2002; 2009).
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In microorganisms, several functions contributing to survival in adverse conditions have been
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ascribed to pyomelanin (Turick et al., 2009). A brown pigment in Legionella pneumophila was
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observed to confer protection from UV radiation (Steinert et al., 2001). Under conditions of low
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dissolved oxygen levels in the environment, pyomelanin, due to its ability to accelerate solid phase
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metal reduction, is hypothesized to aid in the survival of Shewanella oneidensis MR-1 (Turick et
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al., 2009). Pseudomonas aeruginosa strains in patients with chronic lung infections produce a
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brown pigment that helps them tolerate oxidative stress in vitro (Rodríguez-Rojas et al., 2009).
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Turick and colleagues suggested that pyomelanin was important in the life cycle of Shewanella
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algae, a facultative anaerobe, since this organism can use the pigment to accelerate the rate of
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dissimilatory iron mineral reduction in addition to using it as a terminal electron acceptor (Turick et
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al., 2002; 2003; 2008a). Recent research on Aspergillus fumigatus identified the production of
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pyomelanin (Schmaler-Ripcke et al., 2009). Deletion mutants, in which pyomelanin production was
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abolished, were more susceptible to reactive oxygen species (ROI). This finding suggests that
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pyomelanin protects Aspergillus from ROI.
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Penicillium chrysogenum is a filamentous fungus of enormous medical significance, due to its
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ability to produce penicillin, the first antibiotic to be described (Fleming, 1929). It is a ubiquitous
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fungus and has been isolated from a range of different environments such as soil and caves
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(Alexopoulos, 1996). We recently isolated a fungus from a tomb in Upper Egypt, which was
89
identified by ITS gene sequencing as a strain of Penicillium chrysogenum (Vasanthakumar et al.,
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2013). We describe in this report our data showing that both this isolate, as well as the type strain of
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Penicillium chrysogenum, produce a brown pigment when grown in a minimal salts medium
4
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containing tyrosine. We present evidence that this pigment is pyomelanin. We also describe the
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results of our investigation into the chemical and genetic pathways underlying the formation of this
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pigment by P. chrysogenum.
95 96
Methods
97 98
Isolation and growth of the fungal strains
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Swabs moistened with sterile deionized water were used to collect samples from the tomb. A
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dilution series of samples was spread onto microbiological growth media such as tryptic soy agar,
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potato dextrose agar and malt extract agar (Becton Dickinson, Franklin Lakes, NJ). Pure cultures of
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fungal isolates were obtained. Type strains of P. chrysogenum and A. niger were obtained from
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ATCC (P. chrysogenum strains Wisconsin 54-1255 ATCC® 28089 and NRRL 811, ATCC® 10107,
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Aspergillus niger ATCC® 10535).
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Identification of fungi by ITS sequencing
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DNA was extracted from fungal material using the UltraClean Microbial DNA extraction kit
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(MoBio Inc., Carlsbad, CA). The ITS region was amplified as previously described
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(Vasanthakumar et al. 2013), using the primers ITS1 and ITS4 (White, 1990) and amplicons were
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sequenced using ITS1 or ITS4 at the Dana-Farber/Harvard Cancer Center DNA Resource Core.
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Sequences were trimmed and edited using FinchTV version 1.5.0 (Geospiza Inc., Seattle, WA) and
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checked against GenBank and UNITE databases (Abarenkov et al., 2010). ITS sequences were
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submitted to GenBank.
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Pigment production
114
Fungi were grown in a minimal salts medium ((NH4)2SO4 0.22 g/L, KH2PO4 1.20 g/L,
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MgSO4.7H2O 0.23 g/L, CaCl2.2H2O 0.25 g/L), containing 60% (w/v) sodium lactate (9 ml/L) and
5
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tyrosine (2g/L) as well as in growth medium without tyrosine. In addition to the isolated fungi, P.
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chrysogenum strains Wisconsin 54-1255 and NRRL 811 (Table 1) were also tested for pigment
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production. Inoculated strains were incubated at 37°C. Synthetic pyomelanin was generated by
119
treating homogentisic acid with NaOH under aerobic conditions.
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To identify the putative pathway of melanin production, fungal cultures were grown on tyrosine
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medium amended with inhibitors of melanin synthesis. Briefly, 25 g/ml tricyclazole (an inhibitor
122
of the DHN melanin pathway) (Liu et al. 2014) and 50 g/ml kojic acid (an inhibitor of the DOPA
123
melanin pathway) were included in tyrosine media. Fungal cultures were observed every day for
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pigment production both in the fungal biomass as well as in the medium. The effect of sulcotrione, a
125
known inhibitor of pyomelanin production (Almeida-Paes et al. 2012), was not tested in this study.
126
However, the effect of including ascorbic acid, an antioxidant, which would presumably inhibit
127
autooxidation of homogentisic acid, is reported.
128
FTIR analysis
129
The pigmented growth medium was filtered to exclude all fungal material. A representative sample
130
particle was placed on a Diamond window, and analyzed by transmitted infrared beam with an
131
aperture of approximately 100 x 100 microns, using a 15X objective. Each spectrum was the sum of
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200 scans at a resolution of 4 cm-1. Based on the initial analysis results of bulk material, extraction
133
was done by placing a microdroplet of solvent on the sample, and analysis was performed on the
134
resultant extracted dry solvent ring.
135
Analysis of brown pigment using Pyrolysis-GC/MS
136
The dialyzed, cell-free fungal growth medium was analyzed by (i) a Frontier Lab PY-2020D
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double-shot pyrolyzer with Agilent Technologies 5975C inert MSD/7890A GC; (ii) 320°C
138
pyrolysis interface; (iii) 30 M x 0.25 mm x 0.25 µm J&W DB-5MS-UI column with Frontier Vent-
6
139
Free adaptor (40 M effective column length); (iv) 1 ml/minute helium; (v) 320°C injector with 50:1
140
split ratio; (vi) oven program: 2 min. at 40°C, 6°C/min. to 320°C, 9 min. isothermal; (vii) 320°C
141
MS transfer line; (viii) 230°C source; (ix)150°C MS quad; (x) 10-600 amu scanned at 2.59 scans
142
per second. Samples pyrolyzed at 550°C in 50 µl stainless steel.
143
Detection of homogentisic acid using LC/MS
144
The filtered growth medium was analyzed in an Agilent 6210 ESI-TOF LC/MS. Samples were run
145
in negative ion mode for 20 minutes, using an Agilent Zorbax C-18 reverse phase column (3.5 m
146
particle size, 2.1 x 100mm). A formula confirmation method was used in the Agilent MassHunter
147
software to detect homogentisic acid (formula C8H8O4). Retention times and mass spectra were
148
compared with those of the standard. Homogentisic acid (50 mM) and water were used as the
149
standard and blank, respectively.
150
Genetic analysis of enzymes in the tyrosine degradation pathway
151
DNA was isolated from P. chrysogenum strains using MoBio Microbial DNA Isolation kit (MoBio,
152
Inc. Carlsbad, CA). Primers were designed for genes encoding the enzymes, homogentisate
153
dioxygenase (hmgA) and 4-hydroxyphenylpyruvate (hppD), based on the published genome
154
sequence of AM920427 Penicillium chrysogenum Wisconsin 54-1255 (van den Berg et al., 2008,
155
Table 2). These primers enabled amplification of partial regions of these genes using PCR. The
156
conditions for PCR were as follows: an initial denaturation step at 94°C for 4 min, followed by 35
157
cycles of denaturation at 94°C for 1 min, annealing at 52°C for 45 1 min and extension at 72°C for
158
2 min, followed by a final extension step at 72°C for 7 min. Amplicons were sequenced in both
159
directions using the forward and reverse primers at the Dana-Farber/Harvard Cancer Center DNA
160
Resource Core.
7
161
Transcription analysis of homogentisic acid dioxygenase (hmgA) and 4-
162
hydroxyphenlypyruvate (hppD)
163
Total RNA was extracted from fungi using RNAeasy Kit (Qiagen, Valencia, CA). DNAse (Qiagen,
164
Valencia, CA) treatment was performed to exclude possible contamination by small amounts of
165
lingering DNA. Reverse transcription-PCR was performed on the RNA using hmgA, hppD and ITS
166
primers. Conditions for RT-PCR were as follows: 50°C for 30 min, 25-30 cycles of 94°C 30 s, 52
167
°C 45s and 72° 120 s, followed by an extension cycle at 72°C for 7 min.
168 169
Results
170
Identification and description of Penicillium chrysogenum isolated in this study
171
ITS sequencing confirmed the identity of the isolates as Penicillium chrysogenum, with 98-99%
172
similarity to GenBank sequences (accession numbers JQ422624 and AM948960). Sequences
173
obtained in this study were deposited in GenBank under the accession numbers KP280083 and
174
KP280084. Penicillium chrysogenum colonies of the isolated strains contained white mycelia with
175
olive green spores on rich media and grayish spores on minimal salts media.
176
Production of brown pigment in microbiological growth medium amended with tyrosine
177
Growth of P.chrysogenum isolates in tyrosine medium was apparent within 48 hours. However, the
178
brown pigment was apparent only 10-15 days after inoculation. All Penicillium chrysogenum strains
179
grown in a growth medium containing tyrosine produced a brown pigment whereas those grown in
180
medium lacking tyrosine did not (Figure 2a, b). The pigment accumulated extracellularly. However,
181
over time, the fungal cell walls appeared to also accumulate pigment, as observed using light
182
microscopy (data not shown). It was not clear whether this pigment was pyomelanin or some other
183
melanin.
8
184
P. chrysogenum cultures grown on kojic acid-amended tyrosine medium were identical to cultures
185
grown on tyrosine medium, indicating that kojic acid did not inhibit melanin production (Figure 2c).
186
Cultures grown on tricyclazole-amended tyrosine medium, however, were tan –colored, rather than
187
greenish, indicating that inhibition of DHN-melanin production led to a change in the pigments in
188
the fungal biomass (data not shown). However, the extracellular pigment in the medium was not
189
affected by tricyclazole (Figure 2d), indicating that the pathway for formation of this pigment was
190
not inhibited by tricyclazole.
191
Chemical analysis of brown pigment
192
FTIR analysis demonstrated significant similarity between the Penicillium chrysogenum pigment
193
and synthetic pyomelanin (Figure 3). However, since other humic acid polymers also show a similar
194
FTIR spectrum, a more specific method, Pyrolysis-GC-MS (Py-GC/MS) was used to analyze the
195
pigment. This method utilizes heat to break the complex phenolic compounds into smaller
196
fragments. Our Py-GC/MS analysis demonstrated that the brown pigment was a complex
197
heterogeneous polymer containing multiple ring structures. In particular, 4-methoxy benzeneacetic
198
acid, 4-methoxy benzenepropanoic acid and other phenolic compounds were identified using
199
information in the NIST library (Lindstrom & Mallard (Eds.) NIST Chemistry WebBook,
200
retrieved September 4, 2013). Though Py-GC/MS was more successful in detecting phenolic
201
compounds, the complexity of the sample made it difficult to separate other fungal products from
202
the brown pigment (data not shown). In addition, the heterogeneity of the complex aromatic
203
compounds made it difficult to obtain uniform results between samples. Therefore, an LC/MS
204
technique was chosen so that the monomeric precursor, homogentisic acid, could be detected in the
205
fungal cultures. The LC/MS analysis was successful in detecting homogentisic acid in P.
206
chrysogenum cultures that were grown in tyrosine medium (Figure 4, Table 3). No homogentisic
9
207
acid was detected when P. chrysogenum was grown in minimal salts medium lacking tyrosine
208
(Table 3).
209
Fungal cultures containing tyrosine and ascorbic acid (an antioxidant) were also tested for
210
homogentisic acid, with the hypothesis that the antioxidant would inhibit the process of
211
autooxidation to pyomelanin. Over time, the cultures containing ascorbic acid still contained
212
detectable levels of homogentisic acid whereas cultures without the antioxidant contained very little
213
homogentisic acid. It is likely that the homogentisic acid had oxidized to pyomelanin.
214
Analysis of the genetic pathway for pyomelanin formation
215
Partial hmgA and hppD genes were amplified in our isolates as well as in the type strain. The hmgA
216
and hppD gene sequences in our isolate are at least 98% identical to those in the published genome
217
of the type strain (Table 2). Reverse Transcription-PCR analysis of these genes demonstrated that in
218
the presence of tyrosine, hmgA was expressed at a higher level (Figure 5). The difference in the
219
transcription of hppD was not as obvious.
220 221
Discussion
222
In this study, we present evidence for the production of a water-soluble pigment, similar to
223
pyomelanin, by Penicillium chrysogenum. Our data demonstrate that the production of this
224
extracellular brown pigment is not inhibited by tricylazole or kojic acid, inhibitors of the DHN-
225
melanin and DOPA melanin pathway, respectively, and can therefore, can be attributed to a
226
different pathway. In the current study, we did not test the effect of sulcotrione, a known inhibitor
227
of pyomelanin production (Almeida-Paes et al. 2012).
228
The pyomelanin group of melanins is characterized by their extracellular nature and water solubility
229
(Turick et al., 2010). The detection of homogentisic acid by LC/MS only in the P. chrysogenum
10
230
cultures containing tyrosine but not in the cultures lacking tyrosine demonstrates that the production
231
of homogentisic acid, a precursor of pyomelanin, is dependent on the presence of tyrosine. Though
232
there is genomic information about the homogentisic acid pathway of tyrosine degradation (van den
233
Berg et al., 2000), this is, to our knowledge, the first documented experimental evidence of
234
pyomelanin production by a Penicillium chrysogenum.
235
The pyomelanin itself was difficult to analyze because of its heterogeneous nature and the presence
236
of complex phenolic structures. When Py-GC/MS was used, some aromatic compounds were
237
identified that could have been derived from tyrosine degradation. However, it was not possible to
238
conclusively identify the constituents of the brown pigment. Moreover, the polymeric nature of
239
pyomelanin makes it extremely heterogeneous (Hunter & Newman, 2010). Therefore, its chemical
240
composition can vary even between replicate tubes of fungal culture (begun from the same stock
241
inoculum) grown in the same medium. However, the LC/MS method was successful in detecting
242
homogentisic acid, the monomeric precursor of pyomelanin. The ability to detect hmgA was crucial
243
in conclusively identifying the presence of pyomelanin.
244
Since previously published genomic information was available, it was possible to design primers
245
specific for the genes encoding the enzymes, homogentisate dioxygenase (hmgA) and 4-
246
hydroxyphenylpyruvate dioxygenase (hppD). These two enzymes convert homogentisate to
247
maleylacetoacetate and 4-hydroxyphenylpyruvate to homogentisate, respectively (Schmaler-Ripcke
248
et al., 2009). Amplification of partial regions of these two genes revealed that they are 98-99%
249
similar to the published sequences of Wis 54-1255. Using RT-PCR, it was possible to analyze the
250
role of tyrosine in inducing transcription of the two genes. The increased transcription of hmgA in
251
response to tyrosine indicates that tyrosine induces this response. This, in turn, suggests that the
252
presence of tyrosine stimulates the tyrosine degradation pathway to be highly expressed. However,
11
253
the cultures not exposed to tyrosine also demonstrated low levels of hmgA transcription, indicating
254
that this gene is constitutively expressed in the fungus even in the absence of tyrosine. The
255
difference observed in hppD transcription was subtle. This could be due to the fact that only partial
256
regions of the gene were transcribed in this study.
257
We demonstrated that Penicillium chrysogenum isolates from various environments were capable of
258
producing brown pigment in vitro when tyrosine was included in the growth medium. This ability
259
suggests that the homogentisic pathway of tyrosine degradation is active in all these isolates. It is
260
possible that pyomelanin plays a role in survival of the fungus under adverse conditions (Turick et
261
al., 2009). In fungi such as Sporothrix and Aspergillus fumigatus, this brown pigment is known to
262
help protect against oxidative stress (Schmaler-Ripcke et al., 2009; Almeida-Paes et al., 2012). In
263
bacteria such as Pseudomonas aeruginosa, and Burkholderia cenocepacia, this pigment performs
264
functions of stress tolerance such as heavy metal binding (Turick et al., 2003; 2008b), or relief of
265
oxidative stress (Keith et al., 2007). It is conceivable that the P. chrysogenum isolates found in
266
tombs in Egypt are subjected to multiple stresses due to nutrient deficiency as well as lack of
267
moisture. Pyomelanin might enable the fungus to tolerate these stresses. Future work will be
268
required to determine whether this pigment enables Penicillium chrysogenum to be an ecologically
269
important component of the microbiome in other stressed environments.
270
Acknowledgements
271
This research was carried out as a collaborative agreement between the Getty Conservation Institute
272
and Egypt’s Supreme Council of Antiquities and was supported by grant # 268237 from the Getty
273
Conservation Institute to the Laboratory of Applied Microbiology, School of Engineering and
274
Applied Sciences, Harvard University. The authors wish to thank Dr. Marc Mittelman, Dr. Federica
12
275
Villa and Dr. Patricia Sanmartín for insightful discussions, Felix Wu for technical assistance and the
276
anonymous reviewers for useful suggestions.
277
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Table and figure legends
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Table 1. Isolates of Penicillium chrysogenum tested for brown pigment production.
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Table 2. Description of primers used in PCR amplification of genes for enzymes (hmgA and hppD)
373
active in the pyomelanin pathway in Penicillium chrysogenum. GenBank accession numbers for the
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genes as well as the predicted proteins are included.
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Table 3. Retention time and abundance of homogentisic acid detected in supernatant of P.
376
chrysogenum grown in MSM with or without Tyrosine. A formula confirmation program was used
377
in MassHunter software. The compound formula is C8H8O4 and the target mass is 168.04226.
378
Figure 1. Pathway for pyomelanin formation. When homogentisic acid accumulates, it autooxidizes
379
and polymerizes to form pyomelanin. The enzymes HppD and HmgA catalyze the formation and
380
breakdown of homogentisic acid, respectively.
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Figure 2. Growth of Penicillium chrysogenum in minimal salts medium a) without tyrosine or b)
382
with tyrosine. Brown pigment is formed only when tyrosine is present. Inclusion of c) DOPA
17
383
melanin inhibitor kojic acid and d) DHN-melanin inhibitor tricyclazole, in the tyrosine medium did
384
not inhibit extracellular brown pigment formation.
385
Figure 3. FTIR analysis of a) P. chrysogenum brown pigment, and b) synthetic pyomelanin
386
(generated by treating homogentisic acid with NaOH under aerobic conditions).
387
Figure 4. LC/MS detection of homogentisic acid in P. chrysogenum culture supernatant (bottom
388
panel). In the absence of tyrosine, no homogentisic acid was detected (middle panel). The top panel
389
demonstrates the retention time of homogentisic acid, which was used as the standard.
390
Figure 5. Transcription of the genes hmgA and hppD in Penicillium chrysogenum grown with or
391
without tyrosine. 12 ng of RNA was used in each RT-PCR reaction. A housekeeping gene for -
392
actin was included as a control.
18
Table 1_AVasanthakumar_pyomelanin Click here to download Table: Table 1_pyo.docx
A. Vasanthakumar et al.
Pyomelanin production by Peniillium chrysogenum
Table 1. Isolates of Penicillium chrysogenum tested for brown pigment production Isolate ID
Origin
Depository ID
Reference
Wis 541255
Cantaloupe
ATCC 28089/ NRRL 1951
O'Sullivan & Pirt, 1973
NRRL 811
Cheese
ATCC 10107/ NRRL 811
Moyer et al. 1936
T04C
Limestone wall – sealed tomb
N/A
Vasanthakumar et al. 2013
Fb
Limestone wall – open tomb N/A
Vasanthakumar et al. 2013
Table 2_AVasanthakumar_pyomelanin Click here to download Table: Table 2_pyo.docx
A. Vasanthakumar et al.
Pyomelanin production by Peniillium chrysogenum
Table 2. Description of primers used in PCR amplification of genes for enzymes (hmgA and hppD) active in the pyomelanin pathway in Penicillium chrysogenum. GenBank accession numbers for the genes as well as the predicted proteins are included. Gene
Primer Sequence
% identity GenBank of gene Accession #
% identity of predicted protein
GenBank Accession #
hmgA-F
GCGGGGATACATCTGTGAAT
99
AM920427.1
95
XP_002557732
hmgA-R
CCGACCATGTGAGCACTAAA
hppD-F
CAGTCAAGGACGTTGCCTTT
99
AM920427.1,
58
XP_002557734
hppD-R
TAGCCACCCTCATCGAAATC
XM_002557688
Table 3_AVasanthakumar_pyomelanin Click here to download Table: Table 3_pyo.docx
A. Vasanthakumar et al.
Pyomelanin production by Peniillium chrysogenum
Table 3. Retention time and abundance of homogentisic acid detected in supernatant of P. chrysogenum grown in MSM with or without Tyrosine. A formula confirmation program was used in MassHunter software. The compound formula is C8H8O4 and the target mass is 168.04226. Sample
Retention time Mass
Abundance
(minutes) Penicillium chrysogenum - Tyrosine
8.449
168.04235 109
Penicillium chrysogenum + Tyrosine 6.039
168.04235 2489666
Homogentisic acid
168.04269 661785
5.948
Figure1_AVasanthakumar_pyomelanin Click here to download high resolution image
Figure 2_AVasanthakumar_pyomelanin Click here to download high resolution image
Figure 3_AVasanthakumar_pyomelanin Click here to download high resolution image
Figure 4_AVasanthakumar_pyomelanin Click here to download high resolution image
Figure 5_AVasanthakumar_pyomelanin Click here to download high resolution image
