Metagenomic Analysis of an Aromatic Hydrocarbon Contaminated Soil using 1 Substrate-Induced ...

43 Pages·2015·2.36 MB·English

AEM Accepted Manuscript Posted Online 20 November 2015
Appl. Environ. Microbiol. doi:10.1128/AEM.03306-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1 Metagenomic Analysis of an Aromatic Hydrocarbon Contaminated Soil using
2 Substrate-Induced Gene Expression
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4 Matthew J. MEIERa*, E. Suzanne PATERSONa, Iain B. LAMBERTa#
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6 Department of Biology, Carleton University, Ottawa, Ontario, Canadaa. e
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7 Running Head: SIGEX Analysis of a Contaminated Soil Metagenome m

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8 # Address correspondence to Iain B. Lambert, [email protected]. p
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9 * Present address: Matt Meier, Mechanistic Studies Division of Health Canada, Ottawa, m
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10 Ontario, Canada. m
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11 ABSTRACT
12 Metagenomics allows the study of xenobiotic-degrading genes in a culture-
13 independent manner, but many of these studies are limited by the lack of genomic context for
14 metagenomic sequences. This study combined a phenotypic screen known as substrate-
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15 induced gene expression (SIGEX) with whole-metagenome shotgun sequencing. SIGEX is a o
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16 high throughput promoter-trap method that relies on transcriptional activation of a GFP lo
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17 reporter gene in response to an inducing compound, and subsequent fluorescence-activated e
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18 cell sorting, to isolate individual inducible clones from a metagenomic DNA library. We r
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19 describe a SIGEX procedure with improved library construction from fragmented h
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20 metagenomic DNA and improved flow cytometry sorting procedures. We used SIGEX to :
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21 interrogate an aromatic hydrocarbon (AH)-contaminated soil metagenome. The recovered m
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22 clones contained sequences with varying degrees of similarity to genes (or partial genes) s
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23 involved in aromatic metabolism; for example, various nahG (salicylate oxygenase) family o
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24 genes and their respective upstream nahR regulators. To obtain a broader context for the o
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25 recovered fragments, clones were mapped to contigs derived from de novo assembly of
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26 shogun-sequenced metagenomic DNA which, in most cases, contained complete operons m
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27 involved in aromatic metabolism, providing greater insight into the origin of the r

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28 metagenomic fragments. A comparable set of contigs was generated using a significantly less ,

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29 computationally intensive procedure in which assembly of shotgun-sequenced metagenomic 1
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30 DNA was directed by the SIGEX-recovered sequences. This methodology may have broad y

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31 applicability in identifying biologically relevant subsets of metagenomes (including both e
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32 novel and known sequences) that can be targeted computationally by in silico assembly and
33 prediction tools.
34
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35 INTRODUCTION
36 The massive influx of novel sequence data derived from Next Generation Sequencing
37 (NGS) technologies, both in the context of individual genomes (1) and metagenomes (2, 3),
38 has far outstripped efforts to link those sequences to specific organisms and biological
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39 functions (4). Techniques such as qPCR (5), microarrays (6), clone libraries (7), and stable o
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40 isotope probing (8) have been used successfully to identify organisms potentially involved in lo
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41 biodegradation. Substrate Induced Gene Expression (SIGEX) was proposed as a method for e
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42 uncovering novel catabolic operons from metagenomes (9–12). SIGEX is a promoter trap r
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43 method based on single-cell sorting of clones from a plasmid library using flow cytometry h
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44 (FCM), where metagenomic clones of interest are identified by the increased expression of a :
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45 downstream fluorescent reporter gene in the presence – but not in the absence – of an m
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46 inducing compound. SIGEX was initially perceived as having great potential for mining s
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47 genes from metagenomic samples in a high-throughput manner, without requiring prior o
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48 knowledge of the sequences being screened for (13–15). However, SIGEX, and o
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49 metagenomic promoter traps in general, have not lived up to this potential. In this study we
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50 address several issues, described below, that have limited the use of SIGEX, and demonstrate m
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51 that this method may be used to aid characterization of a metagenome derived from aromatic r

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52 hydrocarbon (AH) contaminated soil. ,

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53 Aromatic hydrocarbons (AHs) are common environmental chemicals that also 8

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54 encompass a wide variety of toxic and carcinogenic substances (16–18). They occur g
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55 naturally in soil and are formed as by-products of combustion. Although the structure of AHs s
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56 imparts a relatively long environmental half-life (19), most can be mineralized under both
57 oxic and anoxic conditions (20) by a variety of microbial species (21–23). Many studies have
58 examined AH degrading microbes and their functional genes through culture-based
59 techniques (19, 24, 25), which are limited, since most strains are resistant to culture using
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60 existing procedures (26). There is now a good understanding of the specific enzymes and
61 pathways (27) involved in aromatic degradation at contaminated sites, particularly regarding
62 the terminal dioxygenases that perform the first step in aerobic aromatic metabolism (28, 29).
63 However, biochemical characterization of degradation genes on a case-by-case basis cannot
64 keep pace with the discovery rate of ostensibly novel genes found using metagenomic D
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65 methods. Furthermore, the mechanisms by which many biodegradation genes (e.g., n
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66 polyaromatic hydrocarbon (PAH) degrading genes) are regulated are still unknown. d
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67 In this paper we have carried out a case study that combines SIGEX and NGS. We r
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68 have used a relatively simple metagenome derived from an enrichment culture of a PAH- h
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69 contaminated site to demonstrate the utility of a modified SIGEX protocol in recovering and :
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70 characterizing differentially regulated genes from a soil metagenome. We examine four m
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71 important factors that have limited the use of SIGEX: 1) the difficulty inherent to obtaining s
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72 high quality metagenomic DNA for cloning, 2) the potential lack of compatibility between o
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73 host transcriptional machinery and metagenomic DNA, 3) the difficulty associated with o
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74 measurement of gene expression changes between heterogeneous populations, and 4) the
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75 challenge of obtaining upstream and downstream sequences not contained on the SIGEX- m
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76 cloned metagenomic fragment. r

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77 METHODS 2
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78 Plasmids, Strains and Growth of Bacteria b
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79 Strains and plasmids used in this study are listed in Table 1. Antibiotics were added e
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80 from stocks to their appropriate final concentrations: ampicillin to 100 µg/mL,
81 chloramphenicol to 10 µg/mL, kanamycin to 10 µg/mL and erythromycin to 0.3 µg/mL.
82 Dilute LB (dLB) was made at 1:10 strength relative to LB (Lennox L broth, Invitrogen), and
83 if indicated, maltose was added to 2% (dLB/M). Dilute M9 media (dM9) contained 1X M9
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84 salts, 1% pyruvate, 0.1% casamino acids (Difco), and trace elements (30). The vector
85 pMMeb was used for metagenomic library creation. It is derived from the Bacillus-E. coli
86 shuttle vector pAD123, and carries a unique multi-cloning site (MCS) upstream of a
87 promotorless GFP (31).
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88 DNA Manipulations and Molecular Methods o
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89 Molecular methods were performed as described by Sambrook and Russell (30). For a
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90 routine plasmid isolation from E. coli, the Wizard® Plus SV kit or the Wizard® Midiprep kit d

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91 was used according to the manufacturer’s instructions (Promega). Restriction enzymes were m

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92 purchased from New England Biolabs (NEB). t
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93 Soil Samples and Treatments e
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94 Rock Bay (Victoria Harbour, British Columbia, Canada; samples donated by BC s
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95 Hydro and Transport Canada) was the location of various industrial activities since ca. 1862. r
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96 Runoff from a coal gasification plant, tannery, propane tank farm, concrete batch plant, and o
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97 an asphalt plant resulted in significant soil contamination consisting of coal gasification by- o
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98 products, with PAH concentrations ranging from 475 to 12,600 µg/g of soil (32). Soil was m
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99 homogenized before incubation in a 20% w/v bioslurry (in sterile milli-Q water) in duplicate r

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100 BioFlo 110 bioreactors. To enrich for organisms involved in aerobic degradation of ,
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101 aromatics, the slurry was agitated and aerated constantly for 90 days at a pH of 6.5 – 8.0, kept 1
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102 at 25 °C. Samples of the bioslurry, taken every 15 days beginning on day 0, were stored at - y

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103 80 °C (with glycerol to 15%). Days 0-60 from this bioslurry showed a significant decrease or e
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104 elimination of aromatic compounds, including complete elimination of naphthalene,
105 acenaphthene, fluorene and phenanthrene, and an overall 60% decrease, by mass, of priority
106 PAHs ((32); Supplementary Figure S1).
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107 Canadian Forces Base (CFB) Petawawa was the source of an explosive-contaminated
108 sandy soil, from the anti-tank firing position of a firing range, donated by Dr. Sylvie Brochu
109 (Defence Scientist, Life Cycle of Munitions Group, Energetic Materials Section, Defence
110 Research and Development Canada [DRDC], Valcartier, Québec). The 2.5 cm of topsoil was
111 collected with an acetone-rinsed stainless steel scoop; samples were stored immediately in D
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112 polyethylene bags, and kept in the dark at 4 °C until use in bioslurry experiments done as n
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113 described for Rock Bay soil, above. d
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114 Metagenomic DNA Isolation r
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115 The isolation of DNA from soils is an ongoing technical challenge for metagenomics t
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116 studies. In this study, the high levels of hydrocarbon contamination in the soil resulted in /a
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117 reduced yield of high-quality DNA. Therefore, in order to obtain sufficient quantities of
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118 DNA for cloning and sequencing, 5 time points (days 0, 15, 30, 45, and 60) from the Rock m
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119 Bay bioslurry glycerol stocks were combined from duplicate reactors for a total of 10 samples g
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120 each containing 25 mL of slurry. Following centrifugation at 4500 × g for 10 minutes, a n

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121 combined total of 3.0 g sediment was collected and rinsed with one volume of wash buffer v
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122 (10 mM EDTA, 50 mM Tris, 50 mM phosphate buffer, pH 8.0). DNA was extracted using
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123 the MO-BIO PowerMax® Soil DNA Isolation Kit, EtOH precipitated, and resuspended in 1 1
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124 mL of 2 mM Tris (pH 8.0). 2
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125 Preparation of Metagenomic Libraries b
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126 Metagenomic insert DNA was prepared by partial digestion of 5 µg of DNA with e
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127 Sau3AI. Purified DNA was run on an agarose gel, and showed elimination of the high-
128 molecular-weight fraction and production of fragments from 0-12 kb with approximately
129 even density (Figure 1A, Supplementary Figure S2). The desired size range was isolated
130 from the gel using GeneClean, then purified by phenol/chloroform extraction and EtOH
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131 precipitation. Remaining contaminants were eliminated by precipitation with polyethylene
132 glycol (PEG) as follows: DNA dissolved in 100 µL of 10 mM Tris pH 8.0 was mixed with
133 35% PEG8000 / 30 mM MgCl , vortexed, and centrifuged at room temperature at 21000 x g
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134 for 45 min. Pelleted DNA was rinsed with 70% EtOH. Size-fractionated metagenomic DNA
135 was resuspended in 10 mM Tris (pH 8.0) and ligated using T4 DNA ligase (Invitrogen) to D
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136 100 ng vector DNA digested by BamHI (5:1 insert:vector molar ratio) for 18 h at 4 °C. n
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137 Ligated DNA was purified using the PureLink™ PCR kit (Invitrogen). d
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138 To transform ligated metagenomic DNA libraries, electrocompetent E. coli was r
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139 prepared using cells grown in salt-free LB (1% tryptone, 0.5% yeast extract, no NaCl) but h
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140 otherwise prepared according to Reference 30. Transformations were carried out in an E. coli :
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141 Gene Pulser set at 2.5 kV using a 2 mm gap electroporation cuvette (Fisher). Following m
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142 recovery in 1 mL S.O.C. medium (Invitrogen), 4 mL LB/Amp was added, and the cultures s
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143 were incubated at 37 °C, with shaking at 220 rpm, for 16 h. Cultures from individual o
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144 transformations were pooled and resuspended in LB/Amp with 25% glycerol in a volume o
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145 1/10th the original culture volume, and stored at -80°C until use; this comprised the library
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146 stock. m
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147 Flow Cytometry Analysis 1
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148 The BD Biosciences FACSAria II flow cytometer was fitted with a quartz cuvette 0
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149 flow cell operated at 70 PSI using the 70 µm nozzle, resulting in sheath flow velocities of ~6 b
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150 m/s at the point of interrogation. The light source was a non-polarized 488 nm laser u
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151 (Coherent Sapphire solid state) operated at 13 mW (33). Side scattered light was filtered by a t
152 530/20 bandpass filter for analysis of GFP fluorescence. Parameters were acquired using
153 logarithmic amplification over 5 decades, with FSC and SSC thresholds of 200.
154 Identification of bacterial populations and sorting is described in Supplementary Methods and
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155 shown in Supplementary Figure S3. Plots and histograms were created using Flowing
156 Software (Perttu Terho, http://www.flowingsoftware.com).
157 SIGEX Induction Protocol and Screening Libraries for Individual Clones
158 To obtain individual clones from a library, a scheme similar to differential
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159 fluorescence induction (DFI, (34, 35)) was employed for cell sorting (Figure 1A)). The entire w
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160 metagenomic library was propagated in a single liquid culture for each induction experiment a
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161 (see below for details), and the top 1% of GFP-expressing cells was collected from the total d

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162 library in the presence of inducing compounds and recultured. This subset of the library m

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163 (GFP positive in the presence of inducer) was then applied to the cytometer in the absence of t
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164 inducer, and cells exhibiting the least GFP expression (bottom 10%) were collected and re- /a
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165 cultured. Finally, this subset of the library was again induced and the cells expressing GFP at
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166 the highest levels (top 0.1% and 1%, separately) were sorted and plated for analysis. Putative m
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167 inducible clones were confirmed as inducible using flow cytometric analysis of individual g
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168 clones grown in monoculture with and without inducer as well as measurement of GFP in n

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169 microtitre plates (Supplementary Methods). v
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170 Prior to FCM analysis, a 100 µL aliquot of the library was inoculated into 5 mL of e
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171 media supplemented with appropriate antibiotics. When using frozen stocks, a 1 h recovery 5
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172 period in 1 mL S.O.C. was used. Cultures were then rinsed in phosphate buffered saline 0
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173 (PBS) 3 times before resuspension in media. For samples requiring induction, the inducing b
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174 compounds were added at the following concentrations: 100 µM of salicylate (≥99.5%, u
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175 Sigma), benzoate (≥99.0%, Sigma), naphthalene (≥99.0%, BDH Chem. Ltd.), catechol t
176 (≥99.0%, Sigma), phenol (Ultra-Pure Buffer Saturated, Invitrogen), and phenylacetic acid
177 (Eastman Kodak, lot #574); 10 µM fluoranthene (Moltox), pyrene (Moltox), and
178 phenanthrene (>98.0%, Aldrich). When defined mixtures of compounds were used as
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179 inducers, they were added at an equimolar concentration for a total of 100 µM for AHs and
180 10 µM for PAHs. Negative controls used a culture of the strain containing an empty vector
181 treated with media (and solvent if applicable). Cultures were grown at 37 °C (shaking at 220
182 rpm) for 18 h. Cells were harvested at 10,000 × g for 3 min, washed 3 times in one volume
183 of PBS, and resuspended in 1 mL PBS. This suspension was then used to make dilutions at D
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184 densities resulting in approximately 3,000 events per second on the flow cytometer. n
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185 DNA Sequencing e
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186 Seven µg of metagenomic DNA (concentrated and purified by PEG precipitation) was m

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187 used to create two TruSeq gDNA libraries for whole-metagenome shotgun sequencing t
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188 (performed by Genome Québec, Montréal, Québec). The libraries had average insert sizes of /a
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189 255 bp and 447 bp (Supplementary Figure S4). Two lanes of Illumina sequencing (HiSeq
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190 2000) were obtained from the 255 bp insert library, and one lane from the 447 bp library, m
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191 using 100 bp paired-end reads. g
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192 Clones recovered using SIGEX were end-sequenced by BioBasic (Mississauga, N
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193 Canada) using the primers GfpSeq (5’-GTTGCATCACCTTCACCCTCTCCACTGACAG- e
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194 3’) and pADLeft (5′-ACCTGACGTCTAAGAACCCATTATT-3′), which anneal upstream of e
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195 the GFP and downstream of the MCS, respectively, on pMMeb; subsequent reads were 5
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196 obtained by primer walking. The Sanger reads were aligned manually using BioEdit and 0
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197 vector sequence was removed. Any regions of overlap between reads were used to build a b
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198 consensus sequence for each SIGEX-recovered clone. These sequences were used to query u
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199 the NCBI nr protein database using tBLASTx (36) with a cutoff of 1e-5. BLAST results t
200 were analyzed with Epos BlastViewer and Geneious.
201 MG-RAST (37) was used to determine functional and taxonomic relationships
202 (http://metagenomics.anl.gov/). We uploaded the FASTQ files obtained from Illumina
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203 sequencing to the MG-RAST server, as well as several metagenomic assemblies in FASTA
204 format. FASTQ reads were processed using the recommended MG-RAST pipeline
205 configuration to ensure comparison to other datasets could be performed. Coverage of the
206 assembled sequence was computed for each contig from the IDBA-UD output file using the
207 formula: coverage = (number of reads on contig × 100 bp) / (contig length in bp). SEED D
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208 subsystem annotations were used to identify any features annotated with ‘Metabolism of n
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209 aromatics’; these reads were assembled using IDBA-UD to obtain contigs putatively d
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210 containing only aromatic-metabolizing genes.
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211 Assembly and Analysis of Illumina Sequence Data h
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212 De Novo Assembly /a
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213 Figure 1B summarizes the bioinformatics workflow used to complement the sequence .
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214 data obtained from Sanger sequencing of the SIGEX-isolated metagenome fragments. IDBA-
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215 UD (38) was run using a kmer size iterated from 20 to 100 with a step size of 1, following a /

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216 pre-correction step using a kmer size of 60. PRICE was used to assemble metagenomic
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217 sequences directly flanking known subsets of the metagenome using paired-end information v
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218 (39). This tool can expand an initial set of user-specified sequences using NGS read mapping b
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219 and local assembly. We used 20 cycles of PRICE, using default parameters, to expand the 1
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220 SIGEX clone sequences (Sanger reads). A second subset of the metagenome, the
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221 ‘Metabolism of aromatics’ reads (as annotated by the MG-RAST pipeline and pre-assembled
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222 using IDBA-UD in order to collapse overlapping sequences), was also expanded using 20 g
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223 cycles of PRICE. s
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224 Mapping SIGEX Clones to Metagenomic Contigs
225 A local BLAST database composed of assembled contigs was used to determine the
226 sequence similarity and overlap shared between Sanger-sequenced SIGEX clones and NGS
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