Real-Time PCR quantification of PAH-ring hydroxylating dioxygenase

(PAH-RHD α)genes from Gram positive and Gram negative

bacteria in soil and sediment samples

Aurélie Cébron ?,Marie-Paule Norini,Thierry Beguiristain,Corinne Leyval

Laboratoire des Interactions Microorganismes-Minéraux-Matière Organique dans les Sols UMR7137,Nancy Université,

CNRS,Facultédes Sciences,B.P .239,54506Vandoeuvre-les-Nancy Cedex,France

Received 10October 2007;received in revised form 8January 2008;accepted 18January 2008

Available online 2February 2008

Abstract

Real-Time PCR based assays were developed to quantify Gram positive (GP)and Gram negative (GN)bacterial populations that are capable of degrading the polycyclic aromatic hydrocarbons (PAH)in soil and sediment samples with contrasting contamination levels.These specific and sensitive Real-Time PCR assays were based on the quantification of the copy number of the gene that encodes the alpha subunit of the PAH-ring hydroxylating dioxygenases (PAH-RHD α),involved in the initial step of the aerobic metabolism of PAH.The PAH-RHD α-GP primer set was designed against the different allele types present in the data base (nar Aa,phd A/pdo A2,nidA /pdoA1,nid A3/fad A1)common to the Gram positive PAH degraders such as Rhodococcus ,Mycobacterium ,Nocardioides and Terrabacter strains.The PAH-RHD α-GN primer set was designed against the genes (nah Ac,nah A3,nag Ac,ndo B,ndo C2,pah Ac,pah A3,phn Ac,phn A1,bph Ac,bph A1,dnt Ac and arh A1)common to the Gram negative PAH degraders such as Pseudomonas ,Ralstonia ,Commamonas ,Burkholderia ,Sphingomonas ,Alcaligenes ,Polaromonas strains.The PCR clones for DNA extracted from soil and sediment samples using the designed primers showed 100%relatedness to the PAH-RHD αgenes targeted.Deduced from highly sensitive Real-Time PCR quantification,the ratio of PAH-RHD αgene relative to the 16S rRNA gene copy number showed that the PAH-bacterial degraders could represent up to 1%of the total bacterial community in the PAH-contaminated sites.This ratio highlighted a positive correlation between the PAH-bacterial biodegradation potential and the PAH-contamination level in the environmental samples studied.?2008Elsevier B.V .All rights reserved.

Keywords:PAH-bacterial degraders;PAH-ring hydroxylating dioxygenase alpha subunit (PAH-RHD α);Real-Time PCR;Soil;Sediment

1.Introduction

Polycyclic aromatic hydrocarbons (PAHs)are hydrophobic organic pollutants highly persistent in soil and sediment.Various microorganisms however,have been described to be able to catabolise PAH via either metabolism or cometabolism under both aerobic and anaerobic conditions (Cerniglia,1992;Juhasz and Naidu,2000).

The genes that encode the enzymes involved in the different metabolic steps of aerobic bacterial PAH-degradation pathways have been described in a wide range of Gram negative (GN)

bacterial and some Gram positive (GP)bacterial strains (Habe and Omori,2003).In these conditions,the initial step of the PAH metabolism commonly occurs via the incorporation of molecular oxygen into the aromatic nucleus by a multicomponent aromatic ring-hydroxylating-dioxygenase (RHD)enzyme system forming cis -dihydrodiol.In this system,the terminal dioxygenase is composed of large αand small βsubunits (Kauppi et al.,1998).The alpha subunit (RHD α)contains two conserved regions:the [Fe2-S2]Rieske centre and the mononuclear iron-containing catalytic domain.The most studied PAH dioxygenase (PAH-RHD α)is naphthalene 1,2dioxygenase from Pseudomonas putida NCIB 9816-4(Kauppi et al.,1998)where the alpha subunit is encoded by the nah Ac gene.Many GN bacteria possess PAH-RHD αgenes that encode similar enzymes despite their misleading

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?Corresponding author.

gene names,that were assigned on the basis of the substrate tested.Indeed,among the RHD α,the PAH-RHD αgenes (nah Ac,nah A3,nag Ac,ndo B,ndo C2,pah Ac,pah A3,phn Ac,phn A1,bph Ac,bph A1,dnt Ac and arh A1)form a tight monophyletic cluster (Habe and Omori,2003;Fig.1).These genes have been located on chromosomal or plasmid DNA and identified in bacterial strains belonging to the α-Proteo-bacteria (Sphingomonas ;Romine et al.,1999),β-Proteo-bacteria (Alcaligenes ,Burkholderia ,Commamonas ,Polaromo-nas ,Ralstonia ;Goyal and Zylstra,1996;Jeon et al.,2006;Laurie and Lloyd Jones,1999)and γ-Proteobacteria (Pseudomonas ;Simon et al.,1993).Less is known concerning the GP bacteria,although a recent report showed similar enzymatic dioxygenase systems that are encoded by phylogenetically distant genes (Habe and Omori,2003).GP bacteria possess PAH-RHD αalleles encoding for dioxygenases having different PAH-substrate specificity.These alleles can be classified into four clusters (Fig.1):the narA -like gene from Rhodococcus strains (Larkin et al.,1999),the nidA /pdoA1-like genes from Mycobacterium strains (Khan et al.,2001;Krivobok et al.,2003),the phd A/pdo A2-like genes from Nocardioides and Mycobacterium strains (Saito et al.,1999;Krivobok et al.,2003)and the nid A3/fad A1genes from Mycobacterium and Terrabacter strains (Kim et al.,2006;Zhou et al.,2006).The presence of different genes that encode similar enzymes in both GN and GP bacteria highlights the importance of paying attention to both of these groups

Fig.1.Phylogenetic neighbor-joining tree of ring hydroxylating dioxygenase alpha subunit (RHD α)amino acid sequences of reference strains taken from GenBank,where the Gram negative and Gram positive bacterial strains possessing a RHD specific to PAH and other aromatic compounds are shown.The 20Gram negative and the 15Gram positive bacterial PAH-RHD α,sequences that are highlighted in bold type have been used for the final amino acid sequence alignment performed for the PAH-RHD αGN and GP primer design.Amino acid sequences were aligned using BioEdit Sequence alignment Editor.The tree is based on protein distance matrix analysis and the neighbor-joining method (PROTDIST,NEIGHBOR,SEQBOOT,and CONSENSE)within Phylip 3.65software (Feselstein,1989).Bootstrap values greater than 60%derived from 100replicates are reported at the nodes.The bar represents 10%sequence divergence.

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adequately evaluate the PAH-degradation potential of whole bacterial communities.

The enumeration of PAH-degrading bacterial populations by traditional culture-dependent methods can be time consuming and only reflects a small percentage of the community.Therefore, quantification through Real-Time PCR,using PCR primers that specifically target the functional PAH-RHDαgenes,could be a more accurate method for estimating the biodegradation potential of a PAH-degrading bacterial consortium in contaminated environments.Many authors have designed and used highly specific PCR primers that target nah Ac-or pah Ac-like genes from Pseudomonas species(Lloyd Jones et al.,1999;Wilson et al.,1999;Ferrero et al.,2002),phn Ac-like genes from Bur-kholderia(Laurie and Lloyd Jones,1999),nag Ac-like genes from Ralstonia(Widada et al.,2002;Dionisi et al.,2004),and nid A-or pdo A1-like genes found in Mycobacterium species(Brezna et al., 2003;Johnsen et al.,2006).These primers have been used extensively for PCR based applications such as general PCR, competitive PCR(Laurie and Lloyd Jones,2000;Baldwin et al., 2003;Dionisi et al.,2004)and even Real-Time PCR(Nyyss?nen et al.,2006;Park and Crowley,2006;Johnsen et al.,2006,2007). However,the high specificity of these primers for a limited range of bacterial taxa does not allow for more inclusive targeting of all PAH degraders;indeed,they restrict detection to the most studied genes.As multiple sets of primers are required to detect many of the PAH degraders,their use in routine Real-Time PCR assays would not be suitable.Other studies developed unique sets of degenerate primers that target the highly conserved Rieske center common to most of the aromatic-compounds-RHDαgenes from a large range of GP and GN bacteria(Hamann et al.,1999; Chadhain et al.,2006).These primers are inadequate for quantification of PAH degraders through Real-Time PCR due to their lack of specificity for PAH dioxygenase,as they also target the polychorobiphenyl-,benzene-,toluene-,xylene-dioxygenases and some sequences that are not closely related to any RHDαgene (Chadhain et al.,2006).In a recent study,Zhou et al.(2006) developed two sets of nested PCR primers that target the majority of GN and GP PAH-RHDαgenes.These have been successfully used in nested PCR on bacterial isolates(Zhou et al.,2006,in press),although they have yet to be tested on DNA from environmental samples.Despite their specificity,these primers could not be used in Real-Time PCR quantification because a nested PCR step is required for effective amplification of a single size PCR product.

Following on from this work,the aim of this study was to design new degenerate primers that could specifically amplify the PAH-RHDαgenes in environmental soil and sediment DNA samples to be used to quantify the PAH-degrading bacteria using Real-Time PCR assays.As the PAH-RHDαgenes possessed by GN and GP bacteria do not belong to a monophyletic cluster,our strategy was to develop two Real-Time PCR assays,one against GN-and one against GP-PAH degraders.The main outcome of this study showed that the newly designed primers successfully targeted the majority of the previously described PAH-RHDα

Table1

Bacterial strains used in this study that possess a ring hydroxylating dioxygenase system(RHD)

Reference strains PAH degradation

described a Amplification efficiency with

the PAH-RHDαPCR primer sets b

Cloned genes to generate standard

plasmids for Real-Time PCR calibration GN GP

Bukholderia sp.JS150++?GN-phn Ac

Comamonas testosterone GZ42++?

Novosphingomonas aromaticivorans F199++?

Pseudomonas fluorescens LP6a++?

Pseudomonas putida G7++?GN-nah Ac

P.putida ATCC17484++?

P.putida NCIB9816-4++?16S rDNA

P.putida NCIB9816++?

Rastonia sp.U2++?

Comamonas testosterone B-356c???

P.putida F1???

Sphingomonas paucimobilis ATCC29837???

Mycobacterium vanbaalnii PYR-1+?+16S rDNA GP-nid A3 Mycobacterium sp.6PY1+?+

Rhodococcus opacus R7+?+GP-nar Aa

Rhodococcus sp.NCIMB12038+?+

Mycobacterium austroafricanum ATCC33464???

Rhodococcus globerulus P6???

Rhodococcus opacus BIE-20???

Rhodococcus sp.RHA1???

Some of these strains have previously been described for PAH degradation.They originated from the Centre de Ressources Biologiques de l'Institut Pasteur(CIP)or the institutes'private culture collections.The right column specify the standard plasmids(two standards for each of the three target genes)that have been generated by cloning the PCR amplicons of16S rRNA,PAH-RHDαGN and PAH-RHDαGP genes and then that have been used for Real-Time PCR calibration curves.

a+:strains that can use at least one PAH as sole carbon source,?:strains that have never been described as PAH degraders.

b Amplification efficiency,?:no amplification,+right size PCR product amplification.

c New phylogenetic affiliation,strain closely relate

d to Pandora

e pneumenusa based on16S rDNA gene sequence analysis.

150 A.Cébron et al./Journal of Microbiological Methods73(2008)148–159

genes and were efficiently used for Real-Time PCR assays on various environmental DNA.2.Materials and methods

2.1.Bacterial strains and culture media

The reference bacterial strains used in this study are listed in Table 1.The strains were grown in liquid LB broth (Lennox,Athena Enzyme systems)culture medium and incubated overnight at 28°C prior to total DNA extraction.2.2.Experimental sites and samples

Table 2describes the geographical location of soil and sediment samples and also lists characteristics of the samples.Three aged PAH-contaminated soil samples (Homécourt:H,Neuves Maisons:NM,and Flémalle:T5R,Table 2)were collected on three former industrial coking plant sites (industries had been present on these sites for over 50years during the 20th century).Three weakly contaminated agricultural soil samples (La Bouzule,B,and Feucherolles:F and F GWUS )were selected (Table 2),and finally,two sediment samples from the Moselle River drainage basin (North-East part of France)were collected:Z5and Z12(Table 2).Sediment samples were stored as slurry and soil samples were sieved (b 2mm)and stored as field moist,at ?20°C prior to DNA extraction.Analyses of the samples characteristics (Table 2)were performed at LAS,INRA laboratory (Arras,France)and PAH analyses were carried out using Gas chromatography-Mass spectrometry (GC-MS,HP5890series II GC coupled to an HP5972A mass spectro-meter).Quantifications of 16priority PAHs from the EPA list were carried out by a registered analysis laboratory according to the NF T 90115AFNOR method.

2.3.DNA extraction and quantification

DNA from GN and GP reference strains was extracted according to the manufacturer's recommendation,using the FastDNA kit (Bio101Systems,Q-BIOgene)and the master pure Gram+DNA purification kit (Epicentre,Tebu-bio),respectively.Total DNA from 0.5g of soil samples and from 0.8g sediment samples (wet weight)were extracted using a bead beating protocol described by Norini et al.(in prep)and a slightly modified method of Corgiéet al.(2004).Samples were mixed with glass beads,800μL of extraction buffer (100mM Tris,100mM EDTA,100mM NaCl,1%[wt/vol]polyvinyl-polypyrrolidone,2%[wt/vol]sodium dodecyl sulfate,pH 8.0)and 40μL of 6%CTAB in 5mM CaCl 2.After bead beating on a horizontal grinder Retsch (Roucaire Instruments Scientifiques,France),DNA was extracted using phenol –chloroform –isoamyl alcohol (25/24/1)and washed twice with chloroform –isoamyl alcohol (24/1).DNA precipitation was carried out using isopropanol and then dissolved in 100μL Tris –HCl buffer (10mM,pH 8.0).For two soil samples (H and NM),DNA was extracted in triplicate and then analysed separately.

DNA concentrations in extracts were determined using Hoechst 33258DNA quantification Kit and VersaFluor fluorometer (Bio-Rad)and with Quant-iTPicoGreen dsDNA quantification kit (Invitrogen)for samples with DNA concen-tration below 10ng μL ?1.2.4.Primer design

Two sets of primers were designed to separately target the dioxygenase genes specific for the GP and GN PAH-degrading bacteria due to the large divergence in their phylogeny (Fig.1).All PAH-RHD αamino acid sequences that were available from GenBank on the 22nd December 2005were aligned using

Table 2

Description,properties and PAH-contamination levels of the soil and sediment samples studied

Geographical location

Texture Organic matter

(%)

C:N pH PAH (mg kg ?1)Sand (%)

Silt (%)Clay (%)2–3rings 4rings 5–6

rings Total 16

PAH Feucherolles

Experimental agricultural soil,West of Paris,Yvelines (78),France (plot numbers are 201and 205,“Qualiagro ”experimental site,INRA-Veolia Environment)

6.90.05

0.180.13

0.36

Feucherolles +GWUS a F GWUS

778152011 6.80.040.240.210.49La Bouzule B Agricultural soil,East of Nancy,

Meurthe &Moselle (54),France

125731397.50.380.65 1.20 2.23Homécourt H Former coking plant soil,North of Metz,

Meurthe &Moselle (54),France

66241018459.599514695683032Neuves Maisons NM Former coking plant soil,South of Nancy,Meurthe &Moselle (54),France 61261312267.1271580

368

1219Flémalle T5R Former coking plant soil,south of Liège,Belgium

(site referred as F01in Joubert et al.,2007)

48411127nd 7.5130516878293821Moselle Z5River sediment,upstream Epinal,

V osges (88),France

9352215 5.60.77000.77Fensch Z12River sediment from a tributary of the Moselle,

North of Metz,Meurthe &Moselle (54),France

7.180

138

229

GWUS:soil amended every two years since 1995with a mix of with green waste and urban sludge compost.

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BioEdit Sequence alignment Editor and compared using a similarity matrix to eliminate100%identical sequences.Thus, 20and15protein sequences were kept for representative PAH-RHDαof GN and GP bacteria,respectively(Fig.1).

The CoDeHOP program(Rose et al.,1998)was used to design a series of oligonucleotides from which the two sets of degenerate primers(PAH-RHDαGN and PAH-RHDαGP,see Table3)were selected.These primers consisted of a long5'consensus clamp region(italicized in the primer sequences,see Table3)and a short 3'degenerate core region with no more than3degenerate bases representing a total degeneracy up to16per primer.The primer sets targeted a short fragment in the C-terminal catalytic domain of the PAH-RHDα(Table3).

2.5.PCR and Real-Time PCR conditions

The amplification efficiency for the two PAH-RHDαprimer sets was estimated with a gradient of annealing temperature (from52to62°C)against DNA from strains of pure bacterial cultures(Table1).Both general and Real-Time PCR experi-ments were conducted in an iCycler iQ(Bio-Rad),associated with iCycler Optical System Interface software(version2.3; Bio-Rad).General PCR reactions were performed in50μL reaction volumes containing1×PCR buffer(Invitrogen) supplemented with1.5mM MgCl2,200μM of each dNTP (Fermentas),0.2μM of each primer(MWG-Biotech),1.25U of Taq DNA Polymerase recombinant(Invitrogen),and1μL of template DNA.Real-Time PCR were performed in25μL reaction volumes containing1×iQ SYBR Green Supermix (Bio-Rad),0.4μM of each primers,0.9μgμL?1of bovine serum albumine(BSA),0.5μL of dimethyl sulfoxide(DMSO), 0.1μL of T4bacteriophage gene32Product(QBiogene)and 1μL of template DNA(10times dilution series of plasmid standard and environmental samples DNA)or distilled water (negative control).Environmental DNA samples were used at a concentration of1–5ngμL?1.To avoid PCR amplification problems due to the presence of inhibitors,some samples were diluted10to100times in Tris–HCl buffer(10mM,pH8.0).

The amplifications were carried out with the following temperature profiles:step one heated to95°C(5min),followed by30cycles of3steps for general PCR and50cycles of4steps for Real-Time PCR.These steps were30s of denaturation at 95°C,30s at the primers specific annealing temperature(56°C, 57°C and54°C for16S rDNA amplification using the universal primers968F/1401R(Felske et al.,1998),PAH-RHDαGN and PAH-RHDαGP primer sets,respectively),30s of elongation at72°C and during Real-Time PCR alone the SYBR Green I signal intensities were measured during a10s step at80°C to dissociate the primer's dimers.The final step consisted of7min at72°C.At the end of the Real-Time PCR a melting curve analysis was performed by a final step that consists of the measurement of the SYBR Green I signal intensities during a0.5°C temperature increment every10s from51°C to95°C.

2.6.Standards for Real-Time PCR calibration

Purified16S rDNA,PAH-RHDαGN and PAH-RHDαGP PCR products(QIAquick PCR purification kit,QIAGEN)from the reference strains(Table1)were cloned into pCR4vector, using the TOPO TA cloning method(Invitrogen).After plasmid purification(QIAprep Spin Miniprep kit,Qiagen)and linear-ization using restriction enzymes(Bcl I or Bgl I,Euromedex),to prevent erroneous calibration curve due to coiled DNA(Suzuki et al.,2000),the plasmid DNA concentration was quantified using Hoechst33258DNA quantification Kit and VersaFluor fluorometer(Bio-Rad).The copy number of standard plasmids was calculated according to plasmid(3957bp)plus insert lengths and assuming a molecular mass of660Da for a base pair.Standard DNA stock solutions of109copies of plasmid μL?1were prepared.

2.7.Real-Time PCR assays

Standard ranges for Real-Time PCR assays quantitative calibration were created by producing a ten times dilution series from108to101target gene copiesμL?1.At the end of the Real-Time PCR runs,the threshold line was manually defined within the logarithmic increase phase of the acquired fluorescence data.The Ct values(number of cycles where the fluorescence

Table3

Characteristics of PCR primer sets used in this study

Primer Position Target gene Sequence5'→3'Amplicon

size(bp)Annealing

temperature(°C)

Reference

968R16S rDNA AAC GCG AAG AAC CTT AC43356Felske et al.

(1998) 1401F CGG TGT GTA CAA GAC CC

PAH-RHDαGN F610a Gram negative PAH-RHDαGAG ATG CAT ACC AC G TKG GTT GGA30657This study PAH-RHDαGN R916a AGC TGT TGT TCG GGA AG A YWG TGC MGT T

PAH-RHDαGP F641b Gram positive PAH-RHDαCGG CGC CGA C AA YTT YGT NGG292c54This study PAH-RHDαGP R933b GGG GAA CAC GGT G CC RTG DAT RAA

Lambda7131F Bacteriophage lambda DNA GAT GAG TTC GTG TCC GTA CAA CT50060Sanger et al.

(1982) Lambda7630R GGT TAT CGA AAT CAG CCA CAG CG

a Numbers indicate the position of forward(F)and reverse(R)primers on nah Ac gene of Pseudomonas putida9816-4,GenBank accession no[AF491307].

b Numbers indicate the position of forward(F)and reverse(R)primers on nid A gene of Mycobacterium vanbaalenii PYR-1,GenBank accession no[AF249301].

c The predicte

d amplicon siz

e is292bp for nid A-like genes,289bp for pdo A2-like genes and286bp for both nid A3-and nar A-like genes.

152 A.Cébron et al./Journal of Microbiological Methods73(2008)148–159

data cross the threshold line)were determined for all assays and initial target gene copy number in environmental samples was deduced from the standard curves (Fig.2).

Real-Time PCR was first carried out on ten times dilutions of environmental DNA to determine the optimal concentration for further quantification,and then quantifications were performed in triplicate Real-Time PCR runs.

The presence of PCR inhibitors was evaluated by mixing 1μL of environmental DNA with 1μL of 105copies of the lambda-standard plasmid (500bp PCR product from bacter-iophage lambda DNA (Table 3),cloned as described above for other standards)and then by comparing PCR amplification results with the ten times dilution range curve of standard as described by Beller et al.(2002).When recovery of lambda DNA was below 100%,the quantification data was corrected using the corresponding efficiency factor.

2.8.Analysis of PCR products:melting curve,electrophoresis,cloning and sequencing

Purity of amplified PCR products was checked by:i)the observation of a single melting peak during melting curve analysis following the Real-Time PCR assays,and ii)the presence of a unique band of the expected size on 1%molecular grade agarose (Euromedex)or 2%small DNA Ms-8agarose (Euromedex)gel electrophoresis,and further visualization under a UV GelDoc transilluminator (Bio-Rad)after staining with 0.5μg mL ?1ethidium bromide.

PAH-RHD αamplicons from soil and sediment DNA samples were cloned and sequenced.Briefly,purified PCR products (QIAquick PCR Purification Kit,Qiagen)were cloned into pCR 2.1vector,using the TA cloning kit method (Invitrogen).Eighteen and 20clones from the PAH-RHD αGN and GP amplicon libraries were respectively randomly chosen for sequencing (MWG-Biotech).The amino acids sequences were compared with

GenBank databases using BlastP tool.The PAH-RHD αGN and GP fragment sequences are available from GenBank under the respective accession numbers EU359798–EU359815and EU359778–EU359797.2.9.Statistical analysis

Univariate Kruskal –Wallis analysis of variance (ANOVA),followed by the pair wise multiple comparison test (Student –Newman –Keuls method),was performed using StatView software (JMP),with mean differences significant at the 0.05level.The statistical tests were carried out to highlight the variability in the contribution of independent triplicate DNA extraction from the H and NM soil samples,and to highlight the difference between the two soils,treating the results from the triplicate DNA extractions as replicate samples.3.Results

3.1.Specificity and efficiency of the P AH-RHD αprimer sets The specificity of the two newly designed sets of primers was tested on DNA from pure GP and GN bacterial strains (Table 1)possessing PAH-RHD αgenes,other aromatic-compounds-RHD αgenes (e.g.toluene dioxygenase,biphenyl dioxygenase,see Fig.1)and on DNA extracted from soil and sediment samples that had contrasting levels of PAH contam-ination (Table 2).These new primer sets were found to be highly specific to the target bacteria and to the PAH-RHD αgenes.Visualization of PCR products on agarose gel showed a single band of the expected size only for the expected PAH-degrading bacterial strains without unspecific or cross-reaction amplifica-tions (Table 1).Furthermore,cloning,sequencing and phylo-genetic analysis of PCR products was achieved to highlight primers specificity on environmental DNA but not to analyse the bacterial diversity in the samples.Therefore,18GN and 20GP PAH-RHD αclones from 5and 4gene libraries,respectively,were found to be closely related to the dioxygenase genes targeted by respective primer pairs (Table 4).Clone sequences from the gene libraries using the GN primers were found in three clusters (Table 4)out of the four identified in the general PAH-RHD αGN phylogeny (Fig.1).Fifty percent of the PAH-RHD αGN clones were closely related to nag Ac genes from Ralstonia U2and Polaromonas naphthalenivorans Cj2or to pah Ac gene from Comamonas testosteroni ,44%of the clones were related to nah Ac/pah A3genes from Pseudomonas species,and 6%of the clones were closely related to phn Ac genes from Burkholderia sp.Eh1-1,(Table 4).No bph A1-like gene sequence from Sphingomonas strains (Fig.1)was recovered in spite of good amplification of bph A1f gene from the reference strain Novosphingobium aromaticivorans F199(Table 1).Clone sequences from the gene libraries performed with the GP primers were found in a tied cluster represented by 95%of the clone sequences that had 94to 100%amino acid sequence identity (Table 4);these clones were closely related to fad A1and nid A3genes from Terrabacter sp.HH4and My-cobacterium vanbaalenii PYR-1,respectively (Table 4).

Only

Fig.2.Standard curves of the 16S rRNA,PAH-RHD αGN and PAH-RHD αGP from Real-Time PCR amplification assays obtained by plotting the logarithm of the gene copy number (equivalent to the plasmid copy number)vs.the Ct values that express the number of cycles required to elevate the fluorescence signal above the threshold line.Both standards were run together to generate three unique mean standard curves for each of the three gene assays (see Table 1for description).

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A.Cébron et al./Journal of Microbiological Methods 73(2008)148–159

one sequence representing5%of GP clones were found to be closely related to pdo A2gene from Mycobacterium sp.6PY1 (Table4).No nid A-like sequences were recovered from environmental samples even though cloned PCR products for M.vanbaalenii PYR-1,obtained with the PAH-RHDαGP primers,were positive for nid A and nid A3genes.

3.2.Accuracy and efficiency of Real-Time PCR assays

Six plasmid standards were developed from pure bacterial DNA listed in Table1and used to produce the three calibration curves for the three Real-Time PCR assays.Two plasmid standards dilution series(101to108gene copiesμL?1)were tested for each of the three gene quantification assays(Fig.2).The amplification efficiencies were similar for both standards of16S rRNA,PAH-RHDαGN and PAH-RHDαGP(Fig.2).The standard curves were linear(R2N0.99)on7orders of magnitude from108to101gene copiesμL?1for all standards,except the16S rDNA assay for which the detection limit was102gene copiesμL?1(Fig.2).

Due to the presence of PCR inhibitor substances(e.g.humic acids)in environmental DNA,data was corrected using the bacteriophage lambda internal standard during Real-Time PCR assays as described by Beller et al.(2002).The variation in the internalλstandard recovery corresponded to various PCR inhibition percentages ranging from0%to70%(Fig.3)for the different soil and sediment DNA samples.Such wide variations in DNA amplification efficiency are probably due to inter-ference from sample impurities and Taq polymerase(Porteous et al.,1997).

We have deliberately expressed our Real-Time quantification data as gene copy numbers per gram of soil or sediment dry weight (dw)(Fig.3),and not in terms of quantity of DNA extracted

Table4

Identities of amino acid sequences deduced from nucleotidic sequences of cloned PCR products obtained from PCR with the PAH-RHDαGN and GP primers on DNA from environmental soil and sediment samples(refer to Table2for names and location)

PAH-RHDαprimers Environmental

DNA sample

Clone name Nucleotidic sequence

accession number

Amino acid

sequence identity

Closest relative gene,bacteria and corresponding amino acid

sequence accession number

GN B GN.Bouzule-5EU359806100%pah Ac,Comamonas testosteroni[AAF72976]

GN.Bouzule-7EU35980796%pah Ac,C.testosteroni[AAF72976]

GN.Bouzule-8EU35980898%nah Ac,Pseudomonas putida[AAA25902]

GN.Bouzule-11EU35980997%nah Ac,P.putida[AAA25902]

H GN.Hom-3EU35979894%nah Ac,Pseudomonas fluorescens[AAL07262]

GN.Hom-5EU35979991%pah Ac,C.testosteroni[AAF72976]

GN.Hom-6EU35980090%pah Ac,C.testosteroni[AAF72976]

GN.Hom-7EU359801100%pah A3,Pseudomonas aeruginosa[BAA12240] NM GN.NM-2EU35980298%nah Ac,P.putida[AAA25902]

GN.NM-5EU359803100%pah A3,Pseudomonas aeruginosa[BAA12240]

GN.NM-6EU359804100%pah A3,Pseudomonas aeruginosa[BAA12240]

GN.NM-9EU35980596%nah Ac,P.putida[AAA25902]

T5R GN.T5R-4EU35981094%phn Ac,Burkholderia sp.Eh1-1[AAQ84686]

GN.T5R-5EU359811100%nag Ac,Polaromonas naphthalenivorans[AAZ93388]

GN.T5R-6EU359812100%nag Ac,P.naphthalenivorans[AAZ93388] Z12GN.Z12-4EU35981398%nag Ac,Ralstonia U2[AAD12610]

GN.Z12-5EU359814100%nag Ac,P.naphthalenivorans[AAZ93388]

GN.Z12-6EU359815100%nag Ac,P.naphthalenivorans[AAZ93388]

GP H GP.Hom-1EU35977894%nid A3,Mycobacterium vanbaalenii PYR-1[AAY85176] GP.Hom-2EU359779100%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.Hom-7EU35978096%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.Hom-8EU35978198%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.Hom-9EU35978298%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.Hom-10EU35978398%nid A3,M.vanbaalenii PYR-1[AAY85176] NM GP.NM-1EU359784100%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.NM-2EU359785100%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.NM-3EU359786100%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.NM-6EU359787100%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.NM-7EU359788100%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.NM-23EU35978994%pdo A2,Mycobacterium sp.6PY1[CAD38643] T5R GP.T5R-2EU359793100%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.T5R-3EU359794100%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.T5R-4EU35979598%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.T5R-5EU35979698%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.T5R-13EU35979798%nid A3,M.vanbaalenii PYR-1[AAY85176] Z12GP.Z12-2EU35979098%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.Z12-3EU35979198%nid A3,M.vanbaalenii PYR-1[AAY85176]

GP.Z12-4EU35979298%nid A3,M.vanbaalenii PYR-1[AAY85176]

Closest relative sequences from BastP analysis were taken from GenBank and are named by their accession numbers.Fragments of255and220nucleotides corresponding to85and73amino acid fragments for GN and GP clones were used for the analysis,respectively.

154 A.Cébron et al./Journal of Microbiological Methods73(2008)148–159

because the method used allows the co-extraction of a significant proportion of fungal DNA (data not shown).Data shown represents the mean value from three runs of Real-Time PCR and has a variation coefficient (CV =standard deviation value×100/mean value)ranging from 5.5–93.9%that corre-sponds to less than 0.2Log errors on the quantification values.The Real-Time PCR assays that were developed in the present study could therefore highlight a difference between two samples for quantification data that was separated by a factor four at least.In order to test the effects of sub-sampling and DNA extraction steps on the quantification data,DNA extraction was performed in triplicate for two highly PAH-contaminated soils H and NM (see Table 2).Among the three DNA extractions from the H soil,statistical analysis (ANOV A)showed that 16S rRNA and PAH-RHD αgene copy numbers were not significantly different (P N 0.05;Fig.3)while the quantification data from the triplicate DNA extracts from the NM soil was significantly different (P b 0.05,Fig.3).These differences may be due to bacterial biomass heterogeneity in the triplicate soil sub-samples or uneven losses of DNA during the extraction step.

3.3.Total bacteria and P AH degraders in various soil and sediment samples

For the various soil samples,16S rRNA gene copy number ranged from 4.9×108(±2.5×108)to 4.2×109(±1.2×109)

copies g ?1soil dw (Fig.3).These variations were not correlated to the PAH-contamination level (Table 2).Statistical analysis performed on the two PAH-contaminated soils H and NM,showed that the data from the quantification of 16S rDNA was not significantly different (P N 0.05,Fig.3).A higher bacterial number was quantified in the contaminated Z12sediment sample showing 10times more 16S rRNA gene copies g ?1sediment dw than in the uncontaminated Z5sediment sample (Fig.3).

In the highly PAH-contaminated samples (H,NM,T5R and Z12),the PAH-RHD αgenes represented 4.4×104(±2.4×104)to 4.7×107(±2.4×107)copies g ?1soil or sediment dw (Fig.3).Among the two PAH-contaminated soils H and NM,data of PAH-RHD αgene quantification was significantly different (P b 0.05,Fig.3),with a higher number of PAH-RHD αGP and GN genes in H and NM soils,respectively.Interestingly the PAH-RHD αgenes were either not detected or were in low quantity for samples that were only weakly contaminated (Fig.3).

For easier comparison of the functional populations among the samples,the PAH-RHD αgene levels were normalized as a percentage of the entire community,according to 16S rRNA gene copy number (Fig.3).The DNA extractions of H and NM soils,in triplicate,highlight that the ratios of PAH-RHD αgenes to 16S rRNA genes have relatively low standard deviation values (Fig.3).These ratios have variation coefficients (CV)that are between 18.7%and 50.8%.In highly

PAH-

Fig.3.Real-Time PCR quantification results:A)the 16S rRNA,PAH-RHD αGN and GP gene copy number for the 8soil and sediment samples studied.Error bars indicate standard deviation of the three independent PCR runs.Results have been corrected by the level of PCR amplification inhibition determined with lambda-standard recovery (see Material and methods),percentage of inhibition was 70,4.8,65.5,0,0,15.7and 9.9%for F,B,H,NM,T5R,Z5and Z12samples respectively.DNA extraction was carried out in triplicate for the H and NM soils and these replicates have been analysed separately (H1,H2,H3,NM1,NM2and NM3).Different lowercase letters indicate significant differences (P b 0.05)for a gene between the DNA extraction replicates within a soil.Asterisks indicate significant differences for a gene between the two soils H and NM (mean of three replicates).B)The ratio of PAH-RHD αGN and GP gene copy numbers relative to 16S rRNA gene copy numbers is indicated for each sample indicating the percentage of PAH degraders relative to the total bacterial population.Standard deviations of mean data from three independent DNA extractions are shown.

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contaminated samples(H,NM,T5R and Z12),the ratios of PAH-RHDαgenes to16S rRNA genes ranged from0.001to 0.922%and were higher than for the weakly contaminated samples,that were often below the detection limit of the Real-Time PCR assays(Fig.3).

4.Discussion

Previously,many specific PCR primers were designed directly on the nucleotidic sequences,with either no or low degeneracy,and targeted specifically for each type of PAH-dioxygenase genes (Laurie and Lloyd Jones,1999;Lloyd Jones et al.,1999;Wilson et al.,1999;Ferrero et al.,2002;Widada et al.,2002;Baldwin et al., 2003;Brezna et al.,2003;Dionisi et al.,2004;Johnsen et al., 2006).Other studies have developed degenerate primers to target a broader range of PAH-dioxygenase genes,however either the primers have failed to amplify some genes(Ferrero et al.,2002)or they lacked in specificity,targeting a broad range of aromatic-dioxygenases(Hamann et al.,1999;Chadhain et al.,2006).More recently,Zhou et al.(2006,in press)have designed nested PCR primers however,this approach is not recommended for Real-Time PCR quantification.

In this context,our aim was to fill the void from the lack of PAH-RHDαtargeting primers and to develop Real-Time PCR quantification assays for environmental application.The main outcome of this study was the designing of two sets of PCR primers targeting a broad range of GN and GP specific PAH-dioxygenase genes that can be used for accurate Real-Time PCR quantification of PAH-degrading bacteria directly on DNA from soil and sediment samples.The novel aspect of this primer design was the use of the CoDeHOP program(Rose et al.,1998) that designs amino acid sequence based primers,this strategy allowed the amplification of evolutionarily divergent and potentially novel genes,as successfully demonstrated for other functional genes(Achour et al.,2007).

The newly designed PAH-RHDαGN primer set targeted for the first time such a large variety of GN PAH degraders belonging to Pseudomonas,Polaromonas and Ralstonia,Co-mamonas,and Burkholderia genera that possess nah Ac-, nagAc-,pah Ac-and phn Ac-like genes,respectively.This was demonstrated by carrying out specificity tests on DNA extracted from pure cultures(Table1)and on DNA extracted from environmental samples(Table4).The newly designed primers were not biased towards nah Ac-like genes belonging to Pseu-domonas-like strains as has previously been found with other primer sets(Baldwin et al.,2003;Nyyss?nen et al.,2006;Park and Crowley,2006).As almost all GP PAH degraders possess at least2PAH-dioxygenase genes(Khan et al.,2001;Brezna et al., 2003;Kim et al.,2006;Zhou et al.,2006),the newly designed PAH-RHDαGP primer set was designed to target the different alleles found in GP bacterial strains(Fig.1).Cloning and sequencing of the PCR amplicons using the PAH-RHDαGP primer set confirmed that both alleles(nid A and nid A3)from M. vanbaalenii PYR-1were amplified(data not shown).However, according to soil and sediment samples clone libraries results,it would appear that the nid A3-like gene cluster was preferentially detected(Table4).To explain this finding,we can hypothesise:(i)That in environmental DNA samples,the PAH-RHDαGP primers preferentially amplify the nid A3-like genes or(ii)That this gene is actually more represented amongst the in situ GP bacterial population than in cultivated isolates,as supported by the results of Zhou et al.(2006)who found that the nid A3allele was present in nearly all their Gram positive isolates contrary to the other allele.The nid A3-like alleles were shown to be involved and specialized in the degradation of a broader range of PAH molecules,mainly of high molecular weight(HMW)-PAH such as fluoranthene,pyrene and benzo[a]pyrene(Kanaly and Harayama,2000;Baldwin et al.,2003;Kim et al.,2006;Zhou et al.,2006).The biogeography of PAH degraders is unclear yet but it would seem that GP PAH degraders dominate in older PAH-polluted sites(Uyttebroek et al.,2006)like the former coking plant sites studied here(Table2).As hypothesised by Leys et al.(2005)the majority of initial PAH degradation could be done by GN r-strategists,whereas K-strategists GP bacteria could outcompete for the biodegradation of more persistent HMW-PAH.GP PAH degraders may experience advantages by increasing the PAH bioavailability in aged-contaminated soils thanks to biofilm formation directly on hydrophobic pollutants (Bastiaens et al.,2000;Johnsen and Karlson,2004).

The Real-Time PCR assays developed in the present study were highly specific and allowed accurate amplification and quantification of PAH-RHDαgenes directly from environmen-tal soil and sediment samples.Results were consistent among the triplicate PCR runs.As discussed previously,the SYBR Green detection system is a good compromise between sensitivity and specificity(Stubner,2002)and the use of a fourth step for fluorescence acquisition at80°C during PCR cycle avoids any fluorescence signal from primer dimers (Lopez-Gutiérrez et al.,2004).The detection limit for16S rRNA gene quantification is102gene copies per PCR assay due to difficulty of completely removing all background from PCR reagents(Smith et al.,2006).However,the detection limits for PAH-RHDαgenes were lower:101gene copies per assay corresponding to N2×103gene copies g?1soil dw.Depending on the study,some authors obtained similar detection limits (Park and Crowley,2006)or a higher one(2–3×102gene copies per PCR assay;Baldwin et al.,2003).

For the first time,the Real-Time PCR assays developed in this study allow the quantification of the whole PAH-degrading bacterial community present in environmental samples:(i)without prior activation with a supply of fresh PAH substrate,(ii)without the use of a laboratory microcosm,(iii)in various soil and sediment samples that had differing levels of PAH contamination and pollution history,and(iv)with a good congruence between PAH-RHDαgene copy number and PAH-contamination level.

The total bacterial community was estimated as16S rRNA gene copy number that ranged from4.9×108to4.2×109copies g?1soil dw(Fig.3).These values are of the same order of magnitude as in previously published studies performed on uncontaminated meadow and agricultural soil(Marlowe et al.,2002;Kolb et al., 2005)and polluted motorway site(Johnsen et al.,2006).

The PAH-RHDαgene quantification data found here is close to values that are found using culture-dependent methods for example Most Probable Number(MPN)quantification where

156 A.Cébron et al./Journal of Microbiological Methods73(2008)148–159

PAH-degrading bacteria can be detected around1×106to 2×107cells per g soil dw(Joner et al.,2002;Johnsen and Karlson,2005).Few studies have quantified the PAH-degrading GN bacteria by targeting nah Ac-like genes and found results close to the PAH-RHDαGN gene copy numbers quantified here

(3.9×105to1.0×107g?1soil or sediment dw,Fig.3).Indeed,in

a soil from Finland containing1430mg kg?1of total PAH, Nyyss?nen et al.(2006)detected4.5×106nah Ac gene copies g dw soil?1by Real-Time PCR.Similarly,Stapleton and Sayler (1998)enumerated5.5×107PAH-degrading cells g?1sedi-ment,in a contaminated aquifer by DNA probe hybridization. However,other authors found lower proportions of these GN PAH degraders representing only2×104cells g?1soil in microcosm(Johnsen et al.,2007)and7×103copies g?1soil dw in petroleum hydrocarbon-contaminated soil(Tuomi et al., 2004)by quantification of nah Ac-like genes in Real-Time PCR and Replicate Limiting Dilution-PCR approaches,respectively. Finally,in some cases the nah-or phn-like genes could not be reliably quantified(Johnsen et al.,2006).The quantification data of GP PAH-degrading bacteria is rare.Johnsen et al.(2006) found that nid A-like genes,detected using pdo1primers,were abundant in samples close to the pavement at a motorway site, and represent4.6×103to3.6×105cells g?1soil,values close to those described here ranging from4.4×104to4.7×107PAH-RHDαGP gene copies g?1soil or sediment dw.Moreover,in a microcosm these nid A-like genes became dominant after 30days of incubation of a pristine soil that was primed with bioremediated soil and artificially polluted with phenanthrene, fluoranthene and pyrene(Johnsen et al.,2007).The originality of Real-Time PCR assays developed was capable of quantifying the whole PAH-degrading bacterial community by targeting both GN and GP PAH-degrading populations.

As DNA recovery efficiency could vary among different samples and among the same soil sample,our data was expressed as a percentage of PAH-RHDαgene copy number to16S rRNA gene copy number(Fig.3).These ratios allow an easier comparison of various samples(Fig.3).We deliberately did not assume a mean value for16S rRNA and PAH-RHDαgene copy numbers per bacterial cell,because(i)bacteria can possess1to15 copies of16S rRNA gene(Klappenbach et al.,2000)and(ii)PAH-RHDαgene copy number can vary between bacterial genera depending on plasmid or chromosomal location and on the presence of several gene alleles.For example,P.putida KT2440 and Mycobacterium species contains7and1–2complete ribosomal operons,respectively(Leys et al.,2005;Nyyss?nen et al.,2006)and P.putida G7strain seems to have10–100plasmid copies holding the PAH-RHD gene operons per cell(Park and Crowley,2006).The ratios of PAH-RHDαgenes to16S rRNA genes ranged between0.001and0.922%for the highly PAH-contaminated samples(Fig.3),values that were at least ten times higher than in uncontaminated samples.The comparison with other studies remains complicated due to the variety in analytical methods used.Similar results were found by MPN-PCR method where Mycobacterium species represented0.1–10%of total soil bacteria(Uyttebroek et al.,2006).Lower ratios however,were obtained,e.g.nah Ac genes represented less than0.01%of total bacteria in petroleum hydrocarbon-contaminated soils(Tuomi et al.,2004)and nid A-like gene represented less than0.003%in a soil close to a motorway pavement(Johnsen et al.,2006).

The ratios of PAH-RHDαgene copy number to16S rRNA gene copy number appear to be good indicators of the PAH-degradation potential of the bacterial community present in an environmental sample.A positive correlation was found between the percentage of PAH-RHDαgene number and the level of PAH contamination (log(%of PAH-RHDαGN+GP relative to16S rRNA gene copy number)vs.log(16PAH concentration),R2=0.82)and it can reasonably be expected that selection pressures exerted by aged PAH contamination would enrich populations that are able to degrade PAH in these environments.Even if PAH-degradation activities are usually low in aged PAH-contaminated samples probably a result of low PAH bioavailability and the toxic effects of multi-pollution(presence of heavy metals),the maintenance of a high proportion of GN and/or GP PAH degraders seems to be favoured with time.Similarly,some studies have also shown positive relationships between the PAH-degradation potential expressed as abundance of PAH-dioxygenase gene and the PAH pollution/biodegradation activity(Dionisi et al.,2004;Tuomi et al., 2004;Johnsen and Karlson,2005).On the contrary,Johnsen et al. (2006)found that the density of pdo1-like genes was only slightly correlated to the total PAH concentration(R2=0.375).The specific contribution of the GN vs.the GP PAH-degrading population to PAH-degradation activity was not assessed by targeting DNA but the analysis of mRNA by Real-Time Reverse-Transcription PCR would be the next step towards understanding the different factors that influence not only population density but also functional gene activity in soils.

5.Conclusions

This study describes the design of new PCR primer sets and the development of Real-Time PCR quantification assays that are demonstrated to be highly sensitive,specific and accurate.The genes encoding the PAH-RHDαfrom most of the known GP and GN PAH-degrading bacteria were quantified in environmental DNA samples.These results showed that the PAH-biodegradation potential(the percentage of GN and GP PAH-RHDαgenes rela-tive to16S rRNA gene copy number)was positively correlated to the level of PAH contamination in soil and sediment samples.The Real-Time PCR assays developed in the present study could be largely implied on environmental samples and used as bio-indicators of PAH-degrading bacterial population density for diagnostic on contaminated medium or for remediation treatment monitoring.

Acknowledgements

We would like to acknowledge all colleagues(C.Cerniglia, J.Davies,J.Foght,B.Hofer,R.Parales,M.Sylvestre,P. Williams,J.Willison and G.Zylstra)who sent reference bacterial strains.We are grateful to P.Billard for his precious advice on CoDeHOP software.We thank A.Joubert,S.Houot and I.Auvray,for providing some of the sediment and soil samples from the research projects AquaTerra,Qualiagro and PRST-CPER-ZABM,respectively.This work was supported by

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the GISFI(http://www.gisfi.prd.fr).We thank J.V ohra for the valuable English editing on the latest version of the manuscript. References

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