The role of Aspergillus niger acrA in arsenic resistance and its use as the basis for an arsenic ...

43 Pages·2012·0.93 MB·English

AEM Accepts, published online ahead of print on 30 March 2012
Appl. Environ. Microbiol. doi:10.1128/AEM.07771-11
Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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2 The role of Aspergillus niger acrA in arsenic resistance and its
3 use as the basis for an arsenic biosensor
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5 Se-In Choe1, Fabrice N. Gravelat1, Qusai Al Abdallah1, Mark J. Lee1, Bernard
6 F. Gibbs2, and Donald C. Sheppard1*
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8 1Department of Microbiology and Immunology, McGill University, Montréal o
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9 (Québec), Canada
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11 2Endocrine Laboratory, McGill University, Montréal (Québec), Canada d
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14 Running Title: Aspergillus niger arsenic biosensor m
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17 *To whom correspondence should be addressed: :
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18 Donald Sheppard m
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19 3775 University, Lyman Duff, Rm D24 s
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20 Montréal, Québec, Canada. H3A 2B4 r
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21 Tel: (514) 398 - 1759 n

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22 Fax: (514) 398 - 7052 n
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23 [email protected] ry

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38 Abstract
39 Arsenic contamination of groundwater sources is a major issue worldwide since
40 exposure to high levels of arsenic has been linked to a variety of health problems.
41 Effective methods of detection are thus greatly needed as preventive measures. In an
42 effort to develop a fungal biosensor for arsenic, we first identified seven putative arsenic
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43 metabolism and transport genes in Aspergillus niger, a widely-used industrial organism
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44 that is generally regarded as safe (GRAS). Among the genes tested for RNA expression lo
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45 in response to arsenate, a putative plasma membrane arsenite efflux pump acrA, d

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46 displayed an over 200-fold increase in gene expression in response to arsenate. We m

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47 characterized the function of this A. niger protein in arsenic efflux by gene knockout and tt
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48 confirmed that AcrA was located at the cell membrane using an eGFP fusion construct. a
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49 Based on our observations, we developed a putative biosensor strain containing a .
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50 construct of the native promoter of acrA fused with egfp. We analyzed the fluorescence
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51 of this biosensor strain in the presence of arsenic using confocal microscopy and /

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52 spectrofluorimetry. The biosensor strain reliably detected both arsenite and arsenate in J
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53 the range of 1.8-180 μg/L, which encompass the threshold concentrations for drinking u
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54 water set by the World Health Organization (10-50 μg/L). 8
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68 INTRODUCTION
69 Arsenic is a ubiquitous toxic metalloid that contaminates both groundwater
70 sources (38, 43, 45) and soils (14) worldwide. Strikingly, more than 100 million people in
71 the world are at risk from consuming water contaminated with arsenic (20) and strategies
72 to detect and prevent this global problem are urgently required. D
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73 Arsenic has numerous valence states, but its two predominant forms in nature are n
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74 arsenate (V), present in more oxidized conditions, and arsenite (III), found in reducing d
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75 environments (40, 44, 46). Arsenate mimics phosphate and inhibits the production of f
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76 ATP (25). Moreover, arsenite binds to cellular enzymes containing –SH groups and thus
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77 disrupts their function (25). The long-term exposure to unsafe levels of arsenic leads to :
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78 arsenicosis, which is characterized by skin lesions, such as melanosis, leukomelanosis m
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79 and hyperkeratosis and ultimately death (32, 60). Arsenic was also one of the first s
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80 elements to be recognized as a carcinogen (40, 52) and chronic exposure has been r
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81 associated with bladder, lung and skin cancer (WHO, n

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82 http://www.who.int/topics/arsenic/en/). n
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83 The World Health Organization (WHO) has proposed guidelines for acceptable r
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84 concentrations in drinking water. Although these guidelines suggest a maximum 2
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85 exposure concentration of arsenic of 10 μg/L for drinking water, numerous countries 9

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86 continue to use a cutoff of 50 μg/L (20). In many parts of the world, arsenic g
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87 concentrations far exceed both of these limits, particularly in West Bengal and s
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88 Bangladesh (5). There are currently two widely used methods of arsenic detection in
89 drinking water: laboratory-based analytical methods and field-based testing methods (5).
90 The laboratory-based analytical methods require highly trained personnel and expensive
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91 analytical machinery, such as Inductively Coupled Plasma Mass Spectroscopy (ICPMS)
92 and Atomic Absorption Spectroscopy. Further, the delay in turnaround time between
93 specimen collection and result availability limits their use in day-to-day use. Field-based
94 testing methods are largely chemical colorimetric assays, such as the Gutzeit method,
95 which generate toxic arsine gas by reducing arsenic with a strong acid (20). These tests D
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96 require the use of hazardous chemicals and can generate toxic by-products. Biosensors n
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97 and bioreporters are beginning to emerge as safe, alternate methods to detect d
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98 environmental pollutants such as arsenic (22, 29, 50). Although several arsenic f
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99 biosensors have been reported, these traditionally rely on bacterial reporter systems (7),
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100 which have a relatively narrow tolerance to variation in culture systems, transportation :
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101 and storage. No commercial biosensors are currently in use in affected countries. One m
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102 approach to developing a field-use friendly biosensor is the use of a more robust host s
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103 strain, such as fungi. r
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104 Filamentous fungi are highly adaptable organisms that can grow in extreme n

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105 environmental conditions, such as in contaminated areas with potentially toxic chemical n
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106 compounds and elements (10). This intrinsic resistance, combined with the long-term r
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107 stability of resting fungal spores suggests they may serve as useful organisms for the 2
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108 development of biodetection or bioremediation strategies. Arsenic hyper-resistant 9

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109 filamentous fungi, such as Aspergillus sp. P37, have been isolated from nature, and some g
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110 biochemical pathways of arsenic detoxification have been characterized in these strains s
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111 (14-17, 54, 58). However, the genetics of arsenic metabolism still remain unknown in
112 filamentous fungi (14, 59).
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113 Aspergillus niger is a filamentous fungus commonly associated with wood and is
114 routinely used for the industrial production of enzymes and organic acids (6, 19). This
115 organism has been observed to survive and propagate under concentrations of arsenic as
116 high as 300 mg/L (18). A. niger is efficient in bioleaching of several heavy metals (19),
117 possibly due to its ability to produce large amounts of oxalic acids in culture. This D
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118 organism also produces catalase, which allows the fungus to protect itself against n
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119 environmental stress, including arsenic (11). The intrinsic arsenic resistance of A. niger d
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120 coupled with the availability of genomic sequence data led us to select this organism as a f
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121 model system to examine arsenic resistance and develop an arsenic biosensor system in
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122 filamentous fungi. :
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123 Although the mechanisms of arsenic detoxification remain unstudied in m
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124 filamentous fungi, several pathways mediating detoxification of arsenic have been s
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125 elucidated in the yeast Saccharomyces cerevisiae. One mechanism of resistance is r
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126 mediated by the products of three contiguous genes, ACR1, ACR2 and ACR3 (9, 15, 37). n

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127 ACR1 encodes a transcriptional regulator, ACR2 encodes an arsenate reductase that n
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128 converts arsenate into arsenite and ACR3 encodes a plasma membrane arsenite efflux r
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129 transporter that pumps arsenite out of the cell, allowing resistance (9, 15, 26, 37). In 2
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130 addition, conjugation of arsenite with glutathione (GSH) to form As(GS) and subsequent 9
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131 transport into vacuoles by Ycf1p for detoxification purposes has been proposed to g
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132 increase arsenic resistance in S. cerevisiae (14, 26). s
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133 We therefore used bioinformatics to identify putative genes involved in the
134 arsenic homeostasis of A. niger and developed a hypothetical model of A. niger arsenic
135 metabolism and transport based on S. cerevisiae. To begin to validate this model, we
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136 characterized the function of one of these genes, acrA, in detail. This gene is the putative
137 orthologue of the S. cerevisiae ACR3 gene, which encodes an arsenite efflux pump (66).
138 Consistent with its role as an efflux pump, an AcrA-eGFP fusion localized to the plasma
139 membrane in A. niger, and disruption of A. niger acrA was associated with increased
140 accumulation of and sensitivity to arsenic species. Moreover, we discovered that the D
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141 expression of the acrA gene was rapidly and highly induced in the presence of very low n
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142 concentrations of arsenate. Based on this observation, we tested a strain containing the d
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143 construct of the native promoter of acrA fused with egfp as a potential biosensor for f
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144 arsenic and confirmed the ability of this strain to detect levels of arsenic at and below the
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145 WHO thresholds for arsenic levels in drinking water in the developed and developing :
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146 worlds (24). m
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148 MATERIALS AND METHODS r
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149 Fungal strain culture conditions. Aspergillus niger strains were propagated on Potato n

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150 Dextrose Agar plates (PDA–0.4% potato starch, 2% dextrose, 1.5% agar) and incubated n
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151 at 37°C. Other media used were Yeast Peptone Dextrose (YPD–1% yeast extract, 2% ry

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152 peptone, 2% glucose) and Sabouraud (1% enzymatic digest of casein, 2% dextrose). All 2
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153 media were purchased from GIBCO (Invitrogen, USA). 9

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154 Nucleic and proteic sequences comparison. The A. niger genomic sequences were g
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155 obtained from Central Aspergillus Data Repository (CADRE) (www.cadre- s
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156 genomes.org.uk/Aspergillus_niger/). The putative arsenic homeostasis genes in
157 Aspergillus niger were found by comparing gene and protein sequences with those of S.
158 cerevisiae (12, 35, 39, 49, 61, 62, 66) using nucleotide-BLAST and protein-BLAST from
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159 the National Center for Biotechnology Information
160 (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
161 Identification and phylogenetic analysis of A. niger arsenic transporter. Select
162 arsenic transporter amino acid sequences found in literature (1, 13), and highly
163 homologous sequences to A. niger AcrA identified through BLAST searches (3) were D
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164 aligned with A. niger AcrA using ClustalW Multiple Sequence Alignment (MSA) tool n
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165 (34). Gonnet substitution matrix and Neighbor-Joining clustering model were used in d
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166 ClustalW MSA sequence alignment, and results were exported and formatted to analyze f
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167 phylogenetic relationships and build a cladogram using TreeVector (42). To ensure that
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168 only sequences within the functional domain related to arsenic transport were analyzed, :
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169 each candidate sequence was queried to identify conserved domain regions using NCBI m
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170 Conserved Domain Database (CDD) query protocol (36). CDD domain cluster, s
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171 TIGRFAMs, and InterPro matches were further drilled and cross-referenced to assess and r
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172 verify classification of A. niger AcrA (4, 28). n

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173 Isolation of DNA and RNA. Conidia of A. niger were inoculated into 20 mL of YPD n
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174 broth and incubated with shaking (200 rpm) at 37°C for 16-18 hours. Subsequently, ry

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175 mycelia were harvested by filtration through a P5 Whatman paper (GE Healthcare, USA) 2
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176 then ground to fine powder under liquid nitrogen in order to break the fungal cell wall. 9

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177 Samples were then processed for the extraction of RNA or DNA. DNA extraction was g
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178 conducted by resuspending the ground hyphae in 500 µL of DNA extraction buffer (0.7 s
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179 M NaCl, 0.1 M Na (SO ), 0.05 M EDTA, 1% SDS, 0.1 M Tris-HCl pH 7.5). Following
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180 resuspension, DNA was extracted with phenol-chloroform and precipitated with ethanol.
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181 RNA extraction was conducted according to the manufacturer’s instructions of the
182 Nucleospin RNA Plant Mini Kit (Macherey-Nagel, Germany).
183 Stimulation and quantification of expression of putative genes involved in arsenic
184 homeostasis. Conidia were inoculated into 20 mL of YPD broth and subsequently
185 incubated (200 rpm, 37°C) for 16 hours. The resulting mycelia were then stimulated by D
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186 incubation in YPD containing different concentrations of arsenate (Potassium arsenate, n
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187 monobasic; KH AsO ; Sigma-Aldrich, USA). RNA was harvested and extracted at d
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188 different time points of culture and then analyzed using real-time RT-PCR. The priming fr
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189 oligonucleotides for the genes of interest were designed using Primer-BLAST from the
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190 National Center for Biotechnology Information :
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191 (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primers used for each gene are m
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192 shown in Table S1. The synthesis of cDNA from 1 µg of RNA was performed in 20 µL s
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193 using Quantitect Reverse Transcriptase (QIAGEN, Mississauga, Canada) with random r
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194 primers according to manufacturer’s recommendations. Subsequently, 0.2 µL of the n

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195 synthesized cDNA was analyzed in real-time RT-PCR with SYBR Green Quantitect n
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196 System (Fermentas, Canada), using an ABI 7000 thermocycler (Applied Biosystems, ry

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197 Streetsville, Canada). The fungal gene expression was normalized to Aspergillus niger 2
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198 TEF1 (transcription elongation factor 1) expression, which was used as the endogenous 9

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199 control. g
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200 Disruption of the acrA gene. All PCR reactions were prepared using KAPA HiFi s
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201 HotStart ReadyMix according to the manufacturer’s instructions (KAPA Biosystems,
202 Boston, USA). The Aspergillus fumigatus split marker transformation protocol previously
203 developed in our laboratory for the generation of homologous integrants was adapted for
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204 use in A. niger (55). This technique relies on co-transformation of A. niger protoplasts
205 with DNA fragments, each containing half of the marker (hph encoding hygromycin
206 phosphotransferase) fused to one flanking sequence of the gene to delete. Successful
207 homologous integration of these DNA fragments regenerates the hygromycin marker to
208 permit selection. Fragments were generated using a modified GATEWAY system D
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209 cloning approach. Briefly, the amplification of the upstream and downstream flanking n
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210 sequences was performed using the priming oligonucleotides AcrA Gates 1-2 and 3-4 d
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211 respectively (Table S1). These primers were created using Primer3 (53). Subsequently, f
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212 the amplified flanking sequences were cloned into pENTR-D-TOPO vector (Invitrogen,
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213 Canada) and transformed into DH5α Escherichia coli cells. The plasmids were then :
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214 isolated from putative transformants and analyzed using restriction enzymes to verify if m
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215 the fragments were inserted in the correct orientation. The flanking sequence was then s
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216 transferred from the pENTR-D-TOPO plasmid to the attR-ccdB modified pAN7.1, which r
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217 contains the hph resistance cassette (27) using LR clonase (Invitrogen, Canada). n

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218 Consequently, E. coli DH5α cells were transformed and selected on ampicillin plates. n
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219 The insertions in the plasmids containing the correct products allowed amplification by y

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220 PCR with the long range PCR enzyme (Fermentas, Canada) of one flanking sequence 2
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221 fused with half of the marker cassette. Aspergillus niger protoplasts were then produced

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222 from young hyphae after digesting the cell wall for 3 hours at 30°C (60 rpm) with g
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223 protoplasting solution containing Driselase® from Basidiomycetes sp. and Lysing st
224 Enzymes from Trichoderma harzianum (Sigma-Aldrich, Canada). Subsequently, these
225 protoplasts were co-transformed with 5 μg of each DNA fragment (63). Transformants
226 were selected on PDA plates with 250 μg/mL of hygromycin. The deletion of the acrA
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227 open reading frame was confirmed by long range PCR with primers AcrA Gate 1, AcrA
228 Gate 4, AcrA-RT sense and AcrA-RT antisense, as well as by real-time RT-PCR using
229 primers AcrA-RT sense and AcrA-RT antisense (Table S1). The isolation of a pure clone
230 was done by single spore purification.
231 Construction of the acrA-complemented strain. To verify that any phenotypes D
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232 observed in the acrA null mutant strain were due to specific deletion of acrA, we n
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233 complemented the acrA null mutant with a wildtype allele of acrA. We used the same d
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234 GATEWAY cloning technique as the one used for the gene deletion in order to replace f
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235 the hph cassette by the acrA ORF (open reading frame) fused to the ble cassette (coding
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236 for resistance to phleomycin). Variations were as follows. Priming oligonucleotides AcrA :
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237 Gates 1 and 5 were used to amplify the upstream flanking sequence and the ORF while m
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238 the downstream flanking sequence was amplified by AcrA Gate 3 and Gate 4 (Table S1). s
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239 Cloned sequences were transferred from pENTR-D-TOPO to a modified p402 vector, r
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240 which contains the ble resistance cassette (27). Transformants were selected on PDA n

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241 plates with 150 μg/mL of phleomycin. The re-insertion of the acrA gene was confirmed n
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242 by long range PCR and real-time RT-PCR. r
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243 Construction of the AcrA-GFP over-expression strain. The GFP encoding gene (egfp) 2
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244 was amplified by PCR using the primers GFP-F and GFP-R (Table S1) and using the 9

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245 plasmid p123 (57) as a template. After PCR amplification, the PCR product was cloned g
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246 into the plasmid pEYFPC (31) using NcoI and NotI. The resulting plasmid was s
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247 designated pGFP and contains egfp under the expression of the A. nidulans gpdA
248 promoter. Afterwards, the 1278bp AcrA-encoding gene (acrA) was amplified by PCR
249 using the primers acrA-F and acrA-R (Table S1). The PCR product was cloned into
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