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Aquatic Toxicology 157 (2014) 120–133

Contents lists available at ScienceDirect

Aquatic Toxicology

j o ur na l ho me pag e: www.elsev ier .com/ locate /aquatox

he effect of cold stress on the proteome of the marine bacteriumseudomonas fluorescens BA3SM1 and its ability to copeith metal excess

sabelle Poiriera,∗, Lauriane Kuhnb,1, Christelle Caplatc,1, Philippe Hammannb,artine Bertranda

Microorganismes Métaux et Toxicité, Institut National des Sciences et Techniques de la Mer, Conservatoire National des Arts et Métiers, BP 324,0103 Cherbourg-Octeville Cedex, FrancePlateforme Protéomique Strasbourg Esplanade, CNRS FRC1589, Institut de Biologie Moléculaire et Cellulaire, 15 rue Descartes, 67084 Strasbourg Cedex,ranceUMR BOREA, Université de Caen Basse-Normandie, Esplanade de la Paix, BP 5186, 14032 Caen Cedex, France

r t i c l e i n f o

rticle history:eceived 10 May 2014eceived in revised form 6 August 2014ccepted 4 October 2014vailable online 17 October 2014

eywords:seudomonas fluorescens

a b s t r a c t

This study examined the effect of cold stress on the proteome and metal tolerance of Pseudomonas fluo-rescens BA3SM1, a marine strain isolated from tidal flat sediments. When cold stress (+10 ◦C for 36 h)was applied before moderate metal stress (0.4 mM Cd, 0.6 mM Cd, 1.5 mM Zn, and 1.5 mM Cu), growthdisturbances induced by metal, in comparison with respective controls, were reduced for Cd and Znwhile they were pronounced for Cu. This marine strain was able to respond to cold stress through a num-ber of changes in protein regulation. Analysis of the predicted differentially expressed protein functionsdemonstrated that some mechanisms developed under cold stress were similar to those developed in

arine strainold stressroteomicsetal biosorption

response to Cd, Zn, and Cu. Therefore, pre-cold stress could help this strain to better counteract toxicityof moderate concentrations of some metals. P. fluorescens BA3SM1 was able to remove up to 404.3 mgCd/g dry weight, 172.5 mg Zn/g dry weight, and 11.3 mg Cu/g dry weight and its metal biosorption abilityseemed to be related to the bacterial growth phase. Thus, P. fluorescens BA3SM1 appears as a promisingagent for bioremediation processes, even at low temperatures.

. Introduction

Environmental metal pollution is increasing as a result ofany anthropocentric activities (industrialization, fuel combus-

ion, smelting processes, roadway traffic) and can represent aerious threat to ecosystems and the safety of living organismsOyeyiola et al., 2013; Vrhovnik et al., 2013). Several studies haveemonstrated the efficiency of metal removal by bacteria (Cristanit al., 2012; Kamika and Momba, 2013). Among them, the genusseudomonas has been extensively studied because of its well-dapted metal resistance properties (Chien et al., 2013; Poirier et al.,013; Wasi et al., 2013).

In the marine environment, the tidal flat area is particularlyxposed to metal pollution via industrial and other human activ-ties. Furthermore, it is naturally subjected to wide ecological

∗ Corresponding author. Tel.: +33 2 33 88 73 34; fax: +33 2 33 88 73 39.E-mail address: isabelle.poirier@cnam.fr (I. Poirier).

1 Equal contribution of both authors.

ttp://dx.doi.org/10.1016/j.aquatox.2014.10.002166-445X/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

variations in salinity, temperature, water turbulence, and light.Consequently, bacteria living in this specific environment have toacclimatize to numerous stresses by developing various resistancestrategies (Poirier et al., 2013), thereby making them promisingagents for the study of bioremediation processes. In a previ-ous study, the metal resistant bacterium Pseudomonas fluorescensBA3SM1 (minimal inhibitory concentrations of 3 mM, 9 mM, and10 mM for Cd, Cu, and Zn, respectively) was isolated from tidal flatsediments collected in a moderately metal-contaminated site tothe west of Cherbourg’s port in northwestern France (Poirier et al.,2009). A proteomic analysis of the response of this marine strainto metal stress showed that it acclimatizes to metals by inducingdefense mechanisms such as cell aggregation/biofilm formation,modification of envelope properties, decrease in metal uptake,metal export, protection against oxidative stress, metal sequestra-tion, and over-synthesis of proteins inhibited by metal (Poirier et al.,

2013). To complete this first study, new experiments assessed themetal biosorption ability of the bacterium in order to confirm it as acandidate for use in the purification of metal-contaminated aque-ous systems. Also, its presence in marine sediment would permit

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reduction both in metal bioavailability in this environment, and,onsequently, in toxicity to other marine organisms.

Several studies have shown that the metal tolerance of aacterium and its performance in bioremediation processes are

nfluenced by physical factors such as temperature (Bhatti andmin, 2013; Zamil et al., 2008). Bioremediation processes of aque-us solutions or sediments could be subjected to low temperatureonditions. Thus, it is essential that bacterial strains used in theserocesses retain their ability to remove pollutants even at low tem-eratures. Consequently, the research objectives were (i) to studyhe proteomic response to cold stress in P. fluorescens BA3SM1 inrder to identify resistance mechanisms developed by this straint low temperatures; (ii) to assess the metal biosorption ability ofhe bacterium; (iii) to determine the effect of cold stress on metalolerance and metal biosorption ability of this strain.

. Materials and methods

.1. Bacterial strain and growth conditions

P. fluorescens BA3SM1 was isolated and identified as described in previous study (Poirier et al., 2009). It was deposited in the bacte-ial strain collection of the Institut Pasteur in France (CIP 110551).ong-term storage was at −80 ◦C in 25% glycerol. For all experi-ents, P. fluorescens BA3SM1 was cultivated in a nutrient broth

Biokar Diagnostics, Beauvais, France) and cultures (100 mL) werenoculated with a bacterial suspension from a 24 h-old pre-culturen nutrient broth at +20 ◦C, to obtain an initial cellular concentrationf 105 cells mL−1. The pre-culture allows reactivation of bacterialells and the conduction of experiments with cells all in the samehysiological and metabolic state. To reach the end of the exponen-ial growth phase, cultures were incubated at +20 ◦C with shaking150 rpm) for 20 h (non-cold-stressed cultures) or at +10 ◦C withhaking (150 rpm) for 36 h (cold-stressed cultures). To study theffect of metals on non-pre-cold-stressed and pre-cold-stressedells (growth kinetics and biosorption), metal supplemented andontrol cultures were inoculated with a bacterial suspension from

non-cold-stressed culture (cultures without pre-cold stress) andith a bacterial suspension from a cold-stressed culture (cul-

ures with pre-cold stress). The initial cellular concentration was05 cells mL−1. To the study proteome, non-cold-stressed and cold-tressed cultures were harvested at the end of the exponentialrowth phase. Culture conditions used in our study are schematizedn the Additional file 1.

Supplementary Additional file 1 related to this article cane found, in the online version, at http://dx.doi.org/10.1016/

.aquatox.2014.10.002.

.2. Growth kinetics

To determine the effect of metals on the growth kinetics ofon-pre-cold-stressed and pre-cold-stressed bacterial cells, theutrient broth was supplemented with Cd (0.4 mM, 0.6 mM, and

mM), Zn (1.5 mM and 6 mM), or Cu (1.5 mM and 6 mM), asescribed in our previous study (Poirier et al., 2013), which hadsed 0.4 mM Cd, 1.5 mM Zn, and 1.5 mM Cu metal concentrations.etal supplemented and control cultures were incubated at +20 ◦Cith shaking. Growth was monitored by absorbance measure-ents at 600 nm using an Apollo-1 microplate reader (Berthold

echnologies GmbG & Co. KG, Bad Wildbad, Germany), as also

escribed previously (Poirier et al., 2013). All experiments wereerformed in triplicate.

For each culture, the maximum growth rate (�max) was calcu-ated. The �max decrease in metal supplemented culture compared

ogy 157 (2014) 120–133 121

to the �max of the respective control was calculated and expressedin percentage.

2.3. 2D-PAGE analysis of soluble proteins

To study resistance mechanisms developed by cold-stressedcells, we examined differences in the proteome of cells coming fromnon-cold-stressed and cold-stressed cultures.

2.3.1. Preparation of protein extractsFor protein extraction, non-cold-stressed and cold-stressed cul-

tures were treated as described by Poirier et al. (2008). After celllysis with a French Press, the resulting homogenates were cen-trifuged for 1 h at 218,000 × g at +4 ◦C (Optima L-100 XP BeckmanCoulter, SW 40 Ti swing out rotor) to pellet down cell debris. Thesupernatants were purified, concentrated and stored as describedby Poirier et al. (2008). The protein concentration of the extractswas determined according to Bradford (1976).

2.3.2. 2D-electrophoresisIsoelectric focusing (IEF) was carried out as described by Poirier

et al. (2013). After IEF separation, strip treatment and seconddimension electrophoresis were carried out as described by Poirieret al. (2008). Polypeptides were visualized by gel staining withCoomassie Blue as done by Candiano et al. (2004). Gels werescanned using a GS-800 imaging densitometer (Bio-Rad) and ana-lyzed using the PDQuest software 7.3.0 version (Bio-Rad), which,after resizing and aligning the images, automatically detects andquantifies polypeptide spots. The detailed procedure of this analy-sis is described in our previous study (Poirier et al., 2013). For eachculture condition, four replicates were analyzed. The polypeptidespots of interest were excised and stored at −26 ◦C prior to massspectrometry analysis.

2.4. Protein identification by MALDI-TOF and LC–MS/MS

2.4.1. In-gel digestionThe in-gel digestion procedure was carried out as described by

Rabilloud et al. (2001). Preparation of the gel pieces before trypsindigestion was performed by a liquid handling robot (QuadZ215,Gilson International, France). Briefly, gel bands were washed suc-cessively with 100 �l of 25 mM NH4HCO3 and then with 100 �lof 50% acetonitrile (ACN) (3 min wash with shaking and theliquid was discarded before addition of the next solvent). Thishydrating/dehydrating cycle was repeated twice and the piecesof gel were dried for 20 min before reduction (10 mM DTT/25 mMNH4HCO3 buffer at +56 ◦C for 45 min) and alkylation (25 mMiodoacetamide/25 mM NH4HCO3 buffer for 45 min, room temper-ature). Afterwards, gel spots were again washed with 3 cyclesof 25 mM NH4HCO3/ACN alternately. Following a 20-min dryingstep, the gel pieces were rehydrated by three volumes of trypsin(Promega, V5111), 12.5 ng �l−1 in 25 mM NH4HCO3 buffer (freshlydiluted) and incubated overnight at room temperature. Trypticpeptides were extracted from gel by vigorous shaking for 30 minin 10 �L of 35% H2O/60% ACN/5% HCOOH and a 15-min sonicationstep at 180 W (Sonicator 88155, Fisher Bioblock Scientific, France)without cooling.

2.4.2. MALDI-TOF(/TOF) mass spectrometryMALDI mass measurement was carried out on an Auto-

flex III Smartbeam (Bruker-Daltonik GmbH, Bremen, Germany)matrix-assisted laser desorption/ionization time-of-flight massspectrometer (MALDI-TOF TOF) used in reflector positive mode. Aprespotted anchorchip target (PAC system from Bruker Daltonik,

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echnical note TN011) with 4-hydroxy-�-cyano-cinnamic acid (4-CCA) matrix was used to analyze tryptic digests.

.4.3. NanoLC–MS/MS mass spectrometryFor nanoLC–MS/MS analysis, peptides were transferred in glass

nserts, compatible with the LC autosampler system (nanoLC-3000, Dionex, US). The LC system was coupled to an ESI-Q-TOFass spectrometer (MicroTOFQ-II, Bruker, Germany). The method

onsisted of a 33-min run at a flow rate of 300 nL min−1 using a gra-ient prepared from two solvents: A (99.9% water: 0.1% formic acid)nd B (99.92% acetonitrile: 0.08% formic acid). The system included

300 �m × 5 mm PepMap C18 used for peptide preconcentrationnd a 75 �m × 150 mm C18 column used for peptide elution. TheOF analyzer was calibrated each day: data were acquired androcessed automatically using Hystar 2.8 and DataAnalysis 2.6 soft-ares.

.4.4. Database searchPeptide Mass Fingerprinting (PMF) and Peptide Fragment

ingerprinting (PFF) data were processed by Proteinscape 3.0 soft-are (Bruker Daltonik, Bremen, Germany), which allows MASCOTatabase searches (intranet Mascot 2.2 version, Matrix Science,ondon, UK) as well as further protein validation using the decoyrinciple. The following Mascot parameters were used: trypsinnzyme with maximal 1 missed cleavage; N-terminal proteincetylation, methionine oxidation and cysteine carbamidomethy-ation as variable modifications; 50 ppm as mass tolerance foreptide and 0.5 Da for fragment ions.

To assess the False Discovery Rate (FDR) for each sample, aCBI non-redundant database containing forward and randomized

equences was used. A global search without any taxonomy focusas first performed to assess potential contaminants, followed by

search using the restricted “Bacteria” taxonomy. ProteinScapearameters were set as follows: only peptides showing individualcores above the Mascot Identity threshold were taken into accountnd protein identifications were validated using a False Discoveryate of less than 1%.

.5. Metal biosorption by P. fluorescens BA3SM1

To study the metal biosorption ability of P. fluorescens BA3SM1,he nutrient broth was supplemented with Cd (0.4 mM or 0.6 mM),n (1.5 mM), or Cu (1.5 mM) as described in our previous studyPoirier et al., 2013). For each metal, two cultures were carriedut at +20 ◦C with shaking, the first one inoculated with a bacte-ial suspension from a non-cold-stressed culture (20 ◦C withoutre-cold stress) and the second inoculated with a bacterial sus-ension from a cold-stressed culture (20 ◦C with pre-cold stress);nd one culture was carried out at +10 ◦C with shaking after inoc-lation with a bacterial suspension from a cold-stressed culture10 ◦C). During the growth, 12 ml samples were removed fromhe cultures at successive times (28 h, 52 h and 144 h) and cen-rifuged at 70,000 × g (Optima L-100 XP Beckman Coulter, SW 40i swingout rotor) for 15 min at growth temperature. The pelletsere weighed, dried at +80 ◦C for 20 h, re-weighed, and digestedith 5 mL 65% HNO3 (Acros Organics). Sorbed metals were ana-

yzed by atomic absorption spectrometry in flame mode with aarian AAS 240FS instrument. The detection limit was defined as

he background plus three times the standard deviation of the blank3�). The detection limit was 0.011 mg L−1 for Zn, 0.005 mg L−1

or Cd, and 0.007 mg L−1 for Cu. At the same time, the cell con-entration was determined using a Thoma cell counting chamber.

ontrols without cells, treated in the same way, were done as aontrol for metal sedimentation and precipitation during centrifu-ation. All the experiments were repeated in triplicate. Results werexpressed in mg metal biosorbed/g dry weight. Since the drying at

ogy 157 (2014) 120–133

+80 ◦C for 20 h removed only 93% of the water contained in the pel-lets (compared to drying at +103 ◦C for 24 h), the dry weights werecorrected accordingly.

3. Results

3.1. P. fluorescens BA3SM1 growth kinetics

The effect of metals on the growth kinetics of non-pre-cold-stressed and pre-cold-stressed bacterial cells, cultivated in nutrientbroth at +20 ◦C, are presented in the Additional files 2 and 3.

Supplementary Additional files 2 and 3 related to this arti-cle can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox.2014.10.002.

When P. fluorescens BA3SM1 was exposed to cold stress beforegrowth at +20 ◦C in nutrient broth without metal (control withpre-cold stress), a slight increase in the lag phase (about 1.5-fold)and a decrease in the �max were observed in comparison with thecontrol without pre-cold stress (�max = 0.28 ± 0.03 h−1 with pre-cold stress instead of 0.47 ± 0.03 h−1 without pre-cold stress, i.e., a�max decrease of 40.4%). Since the pre-cold stress disrupts growthin nutrient broth at +20 ◦C without metal, the growth curves “metalwithout pre-cold stress” should be compared to the growth curves“control without pre-cold stress” and the growth curves “metalwith pre-cold stress”should be compared to the growth curves“control with pre-cold stress” to study and compare the effect ofmetals on growth of non-pre-cold-stressed and pre-cold-stressedcells. To make this analysis easier, Table 1 shows the effect of metalson the lag phase and �max of non-pre-cold-stressed and pre-cold-stressed cultures.

When a moderate metal stress (0.4 mM Cd, 0.6 mM Cd, 1.5 mMZn, and 1.5 mM Zn) was applied on non-pre-cold-stressed cells,growth disturbances were observed compared to respective con-trols (controls without pre-cold stress), e.g., an increase in the lagphase for Cd and a decrease in the �max for the three metals,strongly marked for Cd (decreases of 54.3% and 55.6% for 0.4 mMCd and 0.6 mM Cd, respectively; Wilcoxon test: p < 0.01) (Table 1;Additional file 2).

When a moderate metal stress was applied on pre-cold-stressedcells, growth disturbances, compared to respective controls (con-trols with pre-cold stress), were reduced for Cd (e.g., a �max

decrease of 30.8% instead of 54.3% with non-pre-cold-stressed cellsfor 0.4 mM Cd; and a �max decrease of 46.2% instead of 55.6% withnon-pre-cold-stressed cells for 0.6 mM Cd), disappeared for Zn andwere pronounced for Cu (a slight increase in the lag phase and a�max decrease of 14.3% instead of 4.5% with non-pre-cold-stressedcells) (Table 1; Additional file 2).

When a strong metal stress (1 mM Cd, 6 mM Zn and 6 mM Cu)was applied on non-pre-cold-stressed cells, a high increase in thelag phase and a high decrease in the �max (80.9%, 69.6% and 70.2%for Cd, Zn and Cu, respectively) were observed compared to respec-tive controls (Table 1; Additional file 3). These growth disturbanceswere pronounced when the metal stress was applied on pre-cold-stressed cells. No growth occurred for Zn and the lag phase wasdramatically increased for Cd and Cu (Table 1; Additional file 3).

3.2. Differential protein expression

When P. fluorescens BA3SM1 was cultivated in nutrient brothat +10 ◦C for 36 h (cold-stressed culture), major changes wereobserved in polypeptide profile, compared to the non-cold-stressed

culture (+20 ◦C for 20 h). The number of spots exhibiting a sig-nificant difference in intensity was 44 (Additional file 4). Fromthese spots, 42 up-regulated polypeptides and 14 down-regulatedpolypeptides were identified (Table 2). Sometimes, various proteins

I. Poirier et al. / Aquatic Toxicology 157 (2014) 120–133 123

Table 1Effect of Cd (0.4 mM, 0.6 mM, 1 mM), Zn (1.5 mM, 6 mM) and Cu (1.5 mM, 6 mM) on growth parameters (lag time and �max) of non-cold-stressed or pre-cold-stressed P.fluorescens BA3SM1 cells cultivated in nutrient broth at +20 ◦C.

Pre-cold stress Lag time (d) �max (h−1) �max decrease (%)

Control − 0.26 ± 0.02 0.46 ± 0.02 54.30.4 mM Cd − 0.30 ± 0.03 0.21 ± 0.03

Control + 0.32 ± 0.05 0.26 ± 0.01 30.80.4 mM Cd + 0.35 ± 0.03 0.18 ± 0.04

Control − 0.26 ± 0.02 0.45 ± 0.02 55.60.6 mM Cd − 0.43 ± 0.03 0.20 ± 0.02

Control + 0.40 ± 0.04 0.26 ± 0.03 46.20.6 mM Cd + 0.54 ± 0.05 0.14 ± 0.01

Control − 0.26 ± 0.02 0.47 ± 0.03 80.91 mM Cd − 0.90 ± 0.08 0.09 ± 0.01

Control + 0.37 ± 0.04 0.29 ± 0.03 65.51 mM Cd + 2.00 ± 0.50 0.10 ± 0.02

Control − 0.26 ± 0.02 0.51 ± 0.01 19.61.5 mM Zn − 0.26 ± 0.03 0.41 ± 0.01

Control + 0.35 ± 0.03 0.32 ± 0.02 0.01.5 mM Zn + 0.32 ± 0.02 0.32 ± 0.03

Control − 0.26 ± 0.02 0.46 ± 0.05 69.66 mM Zn − 3.00 ± 0.50 0.14 ± 0.03

Control + 0.39 ± 0.03 0.28 ± 0.03 100.06 mM Zn + no growth no growth

Control − 0.26 ± 0.02 0.44 ± 0.01 4.51.5 mM Cu − 0.26 ± 0.02 0.42 ± 0.02

Control + 0.35 ± 0.04 0.28 ± 0.03 14.31.5 mM Cu + 0.44 ± 0.02 0.24 ± 0.02

Control − 0.26 ± 0.02 0.47 ± 0.02 70.26 mM Cu − 2.00 ± 0.25 0.14 ± 0.04

Control + 0.38 ± 0.03 0.28 ± 0.02 85.76 mM Cu + 9.00 ± 1.00 0.04 ± 0.01

P a non-c maxic

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re-cold stress − : metal supplemented and control cultures were inoculated with

ontrol cultures were inoculated with a cold-stressed culture (+10 ◦C for 36 h). �max:ompared to the �max of the respective control, expressed in percentage.

ere identified from one spot. It was the case for SF4, SF7, SF13,F14, SF15, SF18, SF23, SF26 and SF44. These spots were analyzedy nanoLC–MS/MS, a very sensitive method allowing the identifi-ation of all proteins present in a mixture, while MALDI-TOF(/TOF)dentifies only the major ones. As it was difficult to know which pro-ein was the most responsible for the variation of the spot intensity,ll proteins were included in Table 2 (for example SF4a, SF4b andF4c for proteins identified from spot SF4).

Supplementary Additional file 4 related to this article cane found, in the online version, at http://dx.doi.org/10.1016/

.aquatox.2014.10.002.The up-regulated proteins were predicted to be involved in

arious biological activities, such as carbohydrate metabolism19.1%), TCA cycle (4.8%), amino acid metabolism (9.5%), lipid

etabolism (9.5%), nucleotide metabolism (7.1%), energy synthe-is (2.4%), cell envelope biosynthesis (2.4%), transport and binding9.5%), genetic information processing (9.5%), and other func-ions (anti-oxidative stress, electron transfer, denatured proteinydrolysis, etc.) (26.2%). The down-regulated proteins were pre-icted to be involved in motility/chemotaxis (7.1%), carbohydrateetabolism (7.1%), TCA cycle (7.1%), lipid metabolism (14.3%),

ucleic acid metabolism (7.1%), transport/binding proteins (7.1%),enetic information processing (7.1%) and other functions (electronransfer, phosphonoacetate hydrolysis, methylglyoxal degradation,rotein folding) (42.9%).

cold-stressed culture (+20 ◦C for 20 h). Pre-cold stress + : metal supplemented andmum growth rate. �max decrease (%): �max decrease in metal supplemented culture

3.3. Metal biosorption

The metal biosorption abilities of P. fluorescens BA3SM1 are pre-sented Fig. 1.

For 0.4 mM Cd, the maximum biosorption capacity (118.0 mgor 1.05 mmol Cd biosorbed/g dry weight; Fig. 1(A1)) was observedafter 28 h at +10 ◦C, corresponding to cells in exponential growthphase (Fig. 1(A2)). At the beginning of the stationary growth phase,the biosorption of Cd was about 10 mg Cd biosorbed/g dry weightfor all culture conditions. During the stationary growth phase, thebiosorption increased with time to reach, after 144 h, values nearto 20 mg Cd biosorbed/g dry weight for cultures at +20 ◦C withoutor with pre-cold stress and 12.2 mg Cd biosorbed/g dry weight forculture at +10 ◦C (Fig. 1(A1)).

For 0.6 mM Cd, after 28 h at +20 ◦C with pre-cold stress (expo-nential growth phase; Fig. 1(B2)) and 28 h at +10 ◦C (lag phase;Fig. 1(B2)), high biosorption values were observed, reaching404.3 mg or 3.60 mmol Cd/g dry weight at +10 ◦C (Fig. 1(B1)). As for0.4 mM Cd, biosorption was low at the beginning of the stationaryphase for all culture conditions and increased during this phase toreach, after 144 h, values near to 20 mg Cd biosorbed/g dry weight

◦

for cultures at +20 C without or with pre-cold stress and 12.2 mgCd biosorbed/g dry weight for culture at +10 ◦C (Fig. 1(B1)).

For 1.5 mM Zn, the results obtained were similar to thoseexposed for 0.4 mM Cd. The highest biosorption was observed for

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120–133Table 2P. fluorescens BA3SM1 polypeptides differentially expressed from cells growing at +10 ◦C for 36 h in nutrient broth (cold-stressed cells) compared to cells growing at +20 ◦C for 20 h in nutrient broth (non-cold-stressed cells).

Identified protein a

[strain/species]Acession number Mascot score Number of

matchedpeptides

Sequencecoverage (%)

TheoreticalMW (kDa)

Theorical pI Fold change c

(p-value) dError b

(ppm)Function Metals changing

regulation of theseproteins (Poirieret al., 2013)

Up-regulated polypeptides (42)Carbohydrate metabolismSF2 Putative glycogen debranching

protein [Pseudomonasfluorescens SBW25]

gi|229590818 147 8 27.0 82.2 5.05 8.43 (0.0021) 8.0 Glycogenmetabolism

SF3 Putative glucanotransferase[Pseudomonas fluorescensSBW25]

gi|229590816 230 4 37.0 76.8 5.22 2.47 (0.0004) 14.0 Glycogenmetabolism

SF16 Galactonate dehydratase[Pseudomonas fluorescensSBW25]

gi|229587610 215 16 48.7 42.4 5.24 3.57 (0.0008) 21.8 Galactosemetabolism

Cd, Zn, Cu

SF28 Putative sugar kinase[Pseudomonas fluorescensSBW25]

gi|229590178 272 3 15.0 33.6 5.39 2.57 (0.0014) 19.0 Sugarmetabolism

SF29 Ribulose-phosphate3-epimerase [Pseudomonasfluorescens Pf0-1]

gi|77461341 722 28 16.0 24.1 5.86 2.78 (0.0032) 42.9 Ribulosemetabolism

Zn

SF31 Putative carboxyvinyl-carboxyphosphonatephosphorylmutase[Pseudomonas fluorescensSBW25]

gi|229592034 94 1 39.0 32.1 5.51 1.50 (0.0007) 24.0 Phosphoryltransfer

Cu

SF4a TreA [Pseudomonas fluorescens] gi|11127691 460 17 13.5 63.1 5.34 2.11 (0.0003) 20.8 Trehalosemetabolism

SF4b Alpha,alpha-phosphotrehalase[Pseudomonas fluorescens Pf-5]

gi|70732257 272 13 13.9 63.5 5.65 2.11 (0.0003) 11.1 Trehalosemetabolism

TCA cycleSF44a Pyruvate dehydrogenase

[Pseudomonas fluorescens Pf-5]gi|70730964 276 11 16.4 62.5 5.55 2.75 (0.0007) 5.9 Acetyl CoA

biosynthe-sis

Cu

SF44b Malate:quinoneoxidoreductase [Pseudomonasfluorescens Pf0-1]

gi|77458329 223 7 16.6 59.7 6.06 2.75 (0.0007) 11.7 Pyruvate/oxaloacetatebiosynthe-sis

Cu

Amino acid metabolismSF4c Arginyl-tRNA synthetase

[Pseudomonas fluorescensPf0-1]

gi|77456627 193 4 16.4 63.6 5.37 2.11 (0.0003) 6.9 Proteinbiosynthe-sis

SF7a Aminotransferase, class III[Pseudomonas fluorescens Pf-5]

gi|70732209 105 2 5.8 52.0 5.58 1.62 (0.0014) 3.7 Aminogrouptransfer

Cd, Cu

SF13a Cysteinyl-tRNA synthetase[Pseudomonas fluorescensPf0-1]

gi|77459860 807 26 11.7 51.5 9.47 2.08 (0.0025) 27.2 Proteinbiosynthe-sis

Zn

SF30 Glycyl-tRNA synthetasesubunit alpha [Pseudomonasfluorescens Pf0-1]

gi|77456238 777 28 14.5 36.2 6.12 4.75 (0.0015) 38.8 Proteinbiosynthe-sis

Zn

Lipid metabolismSF9 Acetyl-CoA carboxylase (biotin

carboxylase subunit)[Pseudomonas fluorescensSBW25]

gi|229591325 179 10 33.0 69.1 5.33 1.88 (0.0009) 9.0 Fatty acidbiosynthe-sis

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125

SF14b Acyl-CoA dehydrogenasefamily protein [Pseudomonasfluorescens Pf-5]

gi|70732972 840 27 16.3 65.5 5.34 3.57 (0.0006)23.0 Fatty acidoxidation

Zn

SF15a Enoyl-CoAhydratase/isomerase FadB1x[Pseudomonas fluorescensSBW25]

gi|70730429 559 2 36.0 27.7 5.31 1.69 (0.0039)27.0 Fatty acidand phos-pholipidmetabolism

Cu

SF26b Short-chaindehydrogenase/reductase(SDR) [Pseudomonas syringaepv. syringae B728a]

gi|66045647 377 10 10.9 30.7 5.88 2.50 (0.0043)22.4 Lipidbiosynthe-sis

Zn

Nucleotide metabolismSF10 GMP synthase

(glutamine-hydrolyzing)(GuaA) [Pseudomonasfluorescens SBW25]

gi|229592427 340 1 54.0 58.8 5.46 2.90 (0.0007)13.0 Purinebiosynthe-sis

SF1 Carbamoyl phosphate synthaselarge subunit [Pseudomonasfluorescens Pf-5]

gi|70734326 199 1 24.0 118.5 5.14 2.24 (0.054) 9.0 Pyrimidinebiosynthe-sis

SF18b Adenylosuccinate synthetase[Pseudomonas fluorescensPf0-1]

gi|77456756 469 15 12.4 46.5 5.02 2.83 (0.0005)26.1 Purinebiosynthe-sis

Zn, Cu

Energy synthesisSF14a F0F1 ATP synthase subunit

alpha [Pseudomonas fluorescensPf0-1]

gi|77461949 1069 29 16.5 55.3 5.71 3.57 (0.0006)37.0 ATPbiosynthe-sis

Zn

Cell envelope biogenesisSF13c Phosphomannomutase

phosphoglucomutase (chain A)[Pseudomonas aeruginosa]

gi|20150647 87 4 15.6 50.1 7.50 2.08 (0.0025) 5.2 Alginateandlipopolysac-charidebiosynthe-sis

Zn

Transport/binding proteinsSF33 Putative amino acid transport

system, substrate-bindingprotein [Pseudomonasfluorescens Pf-5]

gi|229588843 112 2 33.0 27.6 6.19 2.00 (0.0008)14.0 Amino acidtransport

SF19 Putative bacterioferritin[Pseudomonas fluorescensSBW25]

gi|229588119 88 7 47.7 20.0 4.61 2.83 (0.0065) 9.4 Iron trans-port/bindingprotein

Cd, Zn, Cu

SF23b TonB-dependent copperreceptor [Pseudomonasfluorescens Pf0-1]

gi|77456824 461 17 14.7 77.0 5.23 4.27 (0.0022)13.4 Binding/transportof copper,iron, heme

Zn

SF27 Putative ABC transport system,substrate-binding protein[Pseudomonas fluorescensSBW25]

gi|229589540 100 5 29.0 38.6 5.91 1.58 (0.0011) 9.0 Transportofsubstrates

126

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et al.

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quatic Toxicology

157 (2014)

120–133Table 2 (Continued)

Identified protein a

[strain/species]Acession number Mascot score Number of

matchedpeptides

Sequencecoverage (%)

TheoreticalMW (kDa)

Theorical pI Fold change c

(p-value) dError b

(ppm)Function Metals changing

regulation of theseproteins (Poirieret al., 2013)

Genetic information processingSF15b DNA-binding response

regulator [Pseudomonasfluorescens Pf-5]

gi|70731532 100 4 20.7 24.7 5.41 1.69 (0.0039) 14.2 Replication/transcrip-tion

Cu

SF15c Two componenttranscriptional regulator[Pseudomonas fluorescensPf0-1]

gi|77460461 107 2 14.3 25.4 5.21 1.69 (0.0039) 13.3 Replication/transcrip-tion

Cu

SF25 elongation factor TU[Pseudomonas fluorescensSBW25]

gi|229592905 170 1 36.0 43.9 5.29 4.18 (0.0006) 35.0 Proteinbiosynthe-sis

Zn

SF35 RNA polymerase sigma factorAlgU [Pseudomonas fluorescensPf-5]

gi|70728827 120 8 44.0 22.3 5.30 2.00 (0.0034) 10.0 Transcription

Other proteinsSF6 Putative dehydrogenase

[Pseudomonas fluorescensSBW25]

gi|229592982 148 2 13.0 64.0 5.06 1.56 (0.0009) 7.0 Zn

SF7b HtrA protease (protease Do)[Pseudomonas fluorescens Pf-5]

gi|70728830 329 13 12.6 50.7 6.20 1.62 (0.0014) 4.6 Misfoldedproteinhydrolysis

Cd, Cu

SF11 Putative oxidoreductase[Pseudomonas fluorescensSBW25]

gi|229591981 88 8 29.9 37.0 5.42 1.65 (0.0013) 12.5 Electrontransfer

Cd, Cu

SF13b Aldehyde dehydrogenase(acceptor) [Pseudomonasfluorescens Pf0-1]

gi|77461755 239 10 16.1 53.1 5.68 2.08 (0.0025) 16.1 Intracellularaldehydeconcentra-tiondecrease

Zn

SF18a aldehyde dehydrogenase[Pseudomonas fluorescens Pf-5]

gi|70732774 482 17 16.5 55.0 5.42 2.83 (0.0005) 26.9 Intracellularaldehydeconcentra-tiondecrease

Zn, Cu

SF23a Putative Serine protein kinase,PrkA [Pseudomonas fluorescensPf0-1]

gi|77461360 1049 33 14.4 73.8 5.15 4.27 (0.0022) 32.2 Proteinphosphory-lation

Zn

SF26a Short chain dehydrogenase[Pseudomonas fluorescensPf0-1]

gi|77460304 426 16 13.6 26.5 5.10 2.50 (0.0043) 26.8 Zn

Hypothetical proteinsSF8 Hypothetical protein PFLU1203

[Pseudomonas fluorescensSBW25]

gi|229588740 107 1 18.0 50.2 5.03 2.67 (0.0052) 11.0 Zn

SF12 Hypothetical protein PFLU5582[Pseudomonas fluorescensSBW25]

gi|229592958 305 12 45.0 74.0 5.60 3.85 (0.0042) 18.0

SF34 Hypothetical proteinPfl01 2207 [Pseudomonasfluorescens Pf0-1]

gi|77458434 102 3 34.4 16.9 6.33 3.20 (0.0003) 13.9

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157 (2014)

120–133

127

SF43 Hypothetical proteinPfl01 1353 [Pseudomonasfluorescens Pf0-1]

gi|77457580 91 1 12.0 35.3 5.85 2.20 (0.0006)26.0 Cu

Down-regulated polypeptides (14)Motility/ChemotaxisT(-)SF3 Flagellin [Pseudomonas

fluorescens]gi|9843795 283 8 18.4 43.9 5.30 1.50 (0.0069)10.4 Flagella

biosynthe-sis

Zn, Cu

Carbohydrate metabolismT(-)SF18

Putative6-phosphogluconolactonase[Pseudomonas fluorescensSBW25]

gi|229592230 90 1 30.0 25.7 5.39 1.50 (0.0037)12.0 Pentosephosphatepathway

TCA cycleT(-)SF8 Succinyl-CoA synthetase alpha

chain [Pseudomonas fluorescensSBW25]

gi|229589341 119 1 33.0 30.3 5.89 2.02 (0.0004) 9.0 SuccinylCoAbiosynthe-sis

Cu (up)

Lipid metabolismT(-)SF7 Acetyl-CoA carboxylase

(carboxyl transferase subunitalpha) [Pseudomonasfluorescens SBW25]

gi|229588819 212 15 55.0 35.0 5.79 3.18 (0.0028)10.0 Fatty acidbiosynthe-sis

T(-)SF11

Acetyl-CoA carboxylase (biotincarboxyl carrier proteinsubunit) [Pseudomonasfluorescens Pf-5]

gi|70734174 148 5 21.1 16.1 4.81 1.64 (0.0003) 4.6 Fatty acidbiosynthe-sis

Cd

Nucleotide metabolismT(-)SF20

Putative deoxycytidinetriphosphate deaminase[Pseudomonas fluorescensSBW25]

gi|22958836 125 6 42.0 21.5 5.52 1.50 (0.0053) 9.0 Pyrimidinebiosynthe-sis

Transport/binding proteinsT(-)SF9 Putative ABC transport system,

exported protein [Pseudomonasfluorescens SBW25]

gi|229587937 84 5 32.0 30.0 6.04 1.50 (0.0007) 6.0 Transportof sub-strates/drugexport

Genetic information processingT(-)SF1 Cell division protein

[Pseudomonas fluorescensSBW25]

gi|229588495 196 8 42.0 42.1 5.09 2.10 (0.0009) 9.0 Celldivision

Other proteinsT(-)SF2 Putative reductase

[Pseudomonas fluorescensSBW25]

gi|229588917 113 1 33.0 37.8 5.46 1.60 (0.0002)11.0 Electrontransfer

T(-)SF10

Ferredoxin-NADP(+) reductase[Pseudomonas syringae pv.syringae B728a]

gi|66044635 88 8 23.2 29.6 5.32 1.85 (0.0041)29.8 Electrontransfer

Cd, Zn

128 I. Poirier et al. / Aquatic Toxicol

Tabl

e

2

(Con

tinu

ed) Iden

tifi

ed

pro

tein

a

[str

ain

/sp

ecie

s]A

cess

ion

nu

mbe

r

Mas

cot

scor

e

Nu

mbe

r

ofm

atch

edp

epti

des

Sequ

ence

cove

rage

(%)

Theo

reti

cal

MW

(kD

a)Th

eori

cal p

I

Fold

chan

gec

(p-v

alu

e)d

Erro

rb

(pp

m)

Fun

ctio

n

Met

als

chan

gin

gre

gula

tion

of

thes

ep

rote

ins

(Poi

rier

et

al.,

2013

)

T(-)

SF12

Phn

A

pro

tein

[Pse

udom

onas

fluor

esce

ns

Pf0-

1]gi

|774

6108

330

3

8

20.4

12.2

4.97

1.50

(0.0

087)

6.6

Phos

ph

onoa

ceta

teh

ydro

lysi

sC

d, C

u

T(-)

SF13

Lact

oylg

luta

thio

ne

lyas

e[P

seud

omon

as

fluor

esce

ns

Pf-5

]gi

|707

3075

792

2

24.0

19.5

5.31

1.63

(0.0

061)

15.0

Met

hyl

glyo

xal

deg

rad

a-ti

onT(

-)SF

17

Pep

tid

yl-p

roly

l cis

-tra

ns

isom

eras

e

B

[Pse

udom

onas

fluor

esce

ns

Pf-5

]

gi|7

0731

273

281

17

32.2

18.3

5.84

1.66

(0.0

008)

34.7

Prot

ein

fold

ing

Hyp

oth

etic

al

pro

tein

sT(

-)SF

16

Hyp

oth

etic

al

pro

tein

PFLU

6019

[Pse

udom

onas

fluor

esce

nsSB

W25

]

gi|2

2959

3384

119

14

48.0

18.4

5.1

1.67

(0.0

076)

13.0

aA

ll

the

pro

tein

iden

tifi

cati

ons

wer

e

vali

dat

ed

wit

h

Prot

ein

scap

e

3.0

soft

war

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wit

h

a

resu

ltin

g

FDR

<

1%.

bD

iffe

ren

ce

betw

een

theo

reti

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nd

mas

s

spec

trom

etry

mea

sure

d

MW

(val

ue

mu

st

be

low

er

than

50

pp

m).

cC

omp

ared

to

con

trol

(non

-col

d-s

tres

sed

cell

s).

dTh

e

stat

isti

cal s

ign

ifica

nce

of

the

spot

inte

nsi

ty

dif

fere

nce

betw

een

con

trol

and

cold

stre

ss

was

esta

blis

hed

usi

ng

Stu

den

t’s

t-te

st

(p

<

0.05

).

ogy 157 (2014) 120–133

cells in exponential growth phase (172.5 mg or 2.64 mmol Zn/g dryweight). The biosorption was low at the beginning of the stationarygrowth phase for all culture conditions and it increased during thisphase to reach a maximum value of 22.4 mg Zn/g dry weight after144 h at +20 ◦C with pre-cold stress (Fig. 1(C1)).

After 144 h, Cd and Zn biosorption appeared slightly lower at+10 ◦C than at +20 ◦C.

Cu biosorption remained low compared to other metals. Amaximum biosorption of 11.3 mg or 0.18 mmol Cu/g dry weight(Fig. 1(D1)) was obtained during the exponential growth phase(culture of 28 h at +10 ◦C; Fig. 1(D2)) while a constant biosorptionof about 1.5 mg Cu biosorbed/g dry weight was observed during thestationary growth phase for all culture conditions (Fig. 1(D1)).

Therefore, the metal biosorption ability of P. fluorescens BA3SM1seemed to be related to the bacterial growth phase and to the metalused.

4. Discussion

4.1. Influence of cold stress on the proteome of P. fluorescensBA3SM1 and its ability to tolerate metal contamination

When P. fluorescens BA3SM1 was exposed to cold stress beforegrowth at +20 ◦C in nutrient broth without metal (control withpre-cold stress), a decrease in the �max was observed compared tothe control without pre-cold stress. A decrease in growth rate undercold stress was also observed in Pseudomonas syringae pv. phaseoli-cola NPS3121 (Arvizu-Gómez et al., 2013) and Pseudomonas putida(Fonseca et al., 2013). This growth disturbance can be explainedby a down-regulation of some proteins involved in cell division, asevidenced by proteomic data. When cold stress was applied beforemoderate metal stress, growth disturbances induced by metal, incomparison with respective controls, were reduced for Cd and Znwhile they were pronounced for Cu. These results are in accordancewith other studies showing the influence of temperature on bacte-rial metal resistance (Andreazza et al., 2010; Zamil et al., 2008).

P. fluorescens BA3SM1 responded to cold stress by a numberof changes in protein regulation. Among the proteins differen-tially expressed under cold stress, eight were also differentiallyexpressed under Cd stress (0.4 mM), twelve under Zn stress(1.5 mM) and sixteen under Cu stress (1.5 mM) (Poirier et al.,2013;Table 2, last column). The distribution of up- and down-regulated proteins under cold and metal stress, according totheir function, is represented Fig. 2. Under cold stress, P. fluo-rescens BA3SM1 essentially carbohydrate metabolism, amino acidmetabolism, lipid metabolism, transport/binding proteins andgenetic information processing were predicted to be stimulater.These functional categories were also predicted to be strongly stim-ulated under metal stress. Cold stress was predicted to lead to adecrease in cell motility, a phenomenon also predicted to occurwith metals. In the following, we discuss the acclimatization strate-gies developed by P. fluorescens BA3SM1 under cold stress and theirpossible part on the metal tolerance of this strain.

4.1.1. Alginate biosynthesis/biofilm formationUnder cold stress, an increase in phosphomannomu-

tase/phosphoglucomutase and sigma factor AlgU (also called�E) biosynthesis indicates an increase in alginate and lipopolysac-charide (LPS) biosynthesis (Tart et al., 2005; Ye et al., 1994), twomajor molecules for biofilm formation and metal sequestration(Franc ois et al., 2012; Hou et al., 2013). Moreover, the down-

regulation of 6-phosphogluconolactonase (Pgl), a key enzymeinvolved in the pentose phosphate pathway, permits an increase inthe glucose-6-phosphate level which can be directly transformedinto fructose-6-phosphate, a precursor of alginate biosynthesis

I. Poirier et al. / Aquatic Toxicology 157 (2014) 120–133 129

Fig. 1. Effect of contact time (28 h, 52 h and 144 h) and culture conditions on biosorption of Cd (0.4 mM: (A1); 0.6 mM: (B1)), Zn (1.5 mM: (C1)) and Cu (1.5 mM: (D1)) byP. fluorescens BA3SM1. 20 ◦C without pre-cold stress: metal supplemented cultures were carried out at +20 ◦C in nutrient broth with shaking and inoculated with a non-cold-stressed culture; 20 ◦C with pre-cold stress: metal supplemented cultures were carried out at +20 ◦C in nutrient broth with shaking and inoculated with a cold-stressedc oth wt and ((

(ati2

ulture; 10 ◦C: metal supplemented cultures were carried out at +10 ◦C in nutrient brhree times and mean values and standard errors are shown. Graphs (A2), (B2), (C2)(A2): 0.4 mM Cd, (B2): 0.6 mM Cd, (C2): 1.5 mM Zn, (D2): 1.5 mM Cu).

Anderson et al., 1987). The down-regulation of flagellin synthesis

nd the up-regulation of carbamoyl phosphate synthase facili-ate the development of biofilms, cell organizations reported toncrease the metal tolerance (Chien et al., 2013; Garavaglia et al.,012; Kumar et al., 2012; Lira-Silva et al., 2013).

ith shaking and inoculated with a cold-stressed culture. Experiments were repeatedD2) indicate the cell concentration of each culture at the various time points tested

4.1.2. The involvement of carbohydrate and protein metabolism

Cold stress triggered an increase in the synthesis of sev-

eral enzymes involved in carbohydrate and protein metabolism.The strengthening of these two areas of metabolism is fre-quently reported in studies relating to bacterial response to low

130 I. Poirier et al. / Aquatic Toxicol

454035302520151050

Motility/chemotaxis

Carbohydrate metabolism

TCA cycle

Amino acid metabolism

Lipid metabolism

Nucleic acid metabolism

Energy synthesis

Cell envelope biogenesis

Transport/binding proteins

Metal resistance proteins

Genetic information processing

Regulator

Other function

0.4 mM Cd

1.5 mM Zn

1.5 mM Cu

cold stress

(A)

454035302520151050

Motility/chemotaxis

Carbohydrate metabolism

TCA cycle

Amino acid metabolism

Lipid metabolism

Nucleic acid metabolism

Energy synthesis

Cell envelope biogenesis

Transport/binding proteins

Metal resistance proteins

Genetic information processing

Regulator

Other function

0.4 mM Cd

1.5 mM Zn

1.5 mM Cu

cold stress

(B)

Fig. 2. Distribution of P. fluorescens BA3SM1 up- (A) and down- (B) regulated pro-teins under cold and metal stress. Black: 0.4 mM Cd; dark grey: 1.5 mM Zn; lightgrey: 1.5 mM Cu; white: cold stress (+10 ◦C for 36 h). This distribution is based onftm

tTpp(iw3Srm

4

ommbCfpf(mCeFd

unction of proteins. The horizontal axis represents the percentage of the differen-ially expressed proteins in each category. Up- and down-regulated proteins under

etal stress were determined in a previous study (Poirier et al., 2013).

emperatures (Phadtare and Inouye, 2004; Weinberg et al., 2005).emperature changes are known to stimulate the use of alternateathways of carbohydrate metabolism (e.g., pentose phosphateathway) to rapidly generate energy to overcome cold stressSardesai and Babu, 2001). In our study, an up-regulation of proteinsnvolved in glycogen, galactose, ribulose and trehalose metabolism

as observed. As galactonate dehydratase and ribulose-phosphate-epimerase can be the target of metals (Sobota and Imlay, 2011;zumito, 1981), their overproduction enables bacteria to quicklyeplace them under metal stress, in order to maintain carbohydrateetabolism.

.1.3. The upholding of the membrane integrityIt is well known that temperature changes induce modifications

n membrane fluidity. To maintain exchanges with the environ-ent, the bacterial cell has to modify its fatty acid composition toaintain fluidity. Phospholipids are the main components of mem-

ranes, and their melting point varies with fatty acid composition.ertain modifications are known to decrease the melting point of

atty acids and to improve bacterial acclimatization to low tem-eratures: chain length reduction, the synthesis of branched-chainatty acids (BCFA), and notably, anteiso- and unsaturated fatty acidsUFA) (Haque and Russell, 2004; Russell, 1997). In our study, these

odifications were highlighted by the up-regulation of acetyl-

oA carboxylase (Nikolau et al., 2003), biotin carboxylase (Matht al., 2012; Ting et al., 2010) and enoyl-CoA hydratase/isomeraseadB1x (Okuyama et al., 1998). The up-regulation of acyl-CoAehydrogenase is in accordance with previous studies showing

ogy 157 (2014) 120–133

an up-regulation of proteins involved in fatty acid degradation bybacteria at low temperatures, e.g., Sphingopyxis alaskensis (Tinget al., 2010) and Moraxella catarrhalis (Spaniol et al., 2013). Theacetyl-CoA end-product of fatty acid degradation can enter the TCAcycle as a specific component of the energy generation process.Consequently, it is likely that free fatty acids are used by P. fluores-cens BA3SM1 for efficient energy generation under cold stress, assuggested by Ting et al. (2010).

4.1.4. Protection against oxidative stressSeveral studies have shown that cold stress typically induces

an antioxidant response (Mykytczuk et al., 2013; Shi et al., 2013).Consistent with these observations, proteomic data revealed thatseveral proteins involved in oxidative stress resistance were differ-entially expressed under cold stress, e.g., aldehyde dehydrogenase(Mailloux et al., 2011), oxidoreductase (Kaakoush et al., 2008),serine protein kinase PrKA (Liu et al., 2007), ABC transport sys-tem and lactoylglutathione lyase (glyoxalase I). The up-regulationof an ABC transport system and the down-regulation of lactoyl-glutathione lyase seem to confirm the implication of glutathioneagainst oxidative stress caused by low temperatures. Indeed, arecent study has demonstrated glutathione import via an ABCtransport system (Vergauwen et al., 2013) and a down-regulationof lactoylglutathione lyase permits an increase in glutathionebioavailability (Deponte, 2013). Metals are known to trigger reac-tive oxygen species (ROS) production which can lead to cellulardamage (Bertrand and Poirier, 2005). Consequently, cold stressenables bacteria to be better armed to combat oxidative stressgenerated by metals.

4.1.5. The “Multi-stress protein” biosynthesisSome proteins, up-regulated under cold stress, have also been

reported to be over-expressed when facing other stresses, e.g., GMPsynthase (glutamine-hydrolyzing), protease and sigma factor AlgU.An over-production of GMP synthase is observed in response to acidstress in Listeria monocytogenes (Melo et al., 2013) and to acid andoxidative stress in Lactobacillus lactis (Rallu et al., 2000). HtrA pro-tease is reported to prevent the accumulation of misfolded proteinsunder a number of stress conditions such as temperature and highlight stress (Miranda et al., 2013), metal stress (Poirier et al., 2013),alkylating agents (Seol et al., 1991) and antibiotics (Kaldalu et al.,2004). Sigma factor AlgU is a main regulator of the bacterial stressresponse (Lai and Wong, 2013). The up-regulation of these “multi-stress proteins” under cold stress is undoubtedly an advantage tocombat effectively metal toxicity.

4.1.6. Increased ion and substrate uptakeUnder cold stress, the up-regulation of proteins involved in

uptake of substrates, e.g., amino acid transport system and ABCtransport system, suggests the transport, into the cell, of essen-tial solutes for bacterial metabolism. Indeed, the accumulation ofsolutes such as glutamate, aspartate, trehalose and glycine-betaineis a common response of many bacteria for preventing damagescaused by low temperatures (Kandror et al., 2002; Shivaji andPrakash, 2010). Several studies have shown the implication of ABCtransport systems in stress adaptation of microorganisms, e.g., acidstress in Synechocystis sp. PCC6803 (Tahara et al., 2012), osmoticstress in Salmonella enterica (Frossard et al., 2012) and cold stressin Bacillus subtilis (Hoffmann and Bremer, 2011). The ABC trans-port system can serve to accumulate protectants against stress. Forexample, the uptake of glycine betaine via an ABC transport systemwas observed in B. subtilis subjected to cold stress (Hoffmann and

Bremer, 2011). Furthermore, an increase in amino acid uptake isinteresting to combat metal toxicity. Indeed, this will allow a rapidmetallothioneins (MTs) synthesis, as prokaryotic MTs are particu-larly rich in histidine, glutamate and cysteine (Blindauer, 2011).

oxicol

purict2mu

osiChCecapett6fippi

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4

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I. Poirier et al. / Aquatic T

Cold stress induced the up-regulation of TonB-dependent cop-er receptor and bacterioferritin, two proteins involved in Feptake and storage (Madeira et al., 2011; Yasmin et al., 2011). Thisesult is in accordance with a previous study showing an increasen the expression levels of genes involved in iron acquisition in M.atarrhalis (Spaniol et al., 2013). Some Fe-containing molecules arehe target of metals (Macomber and Imlay, 2009; Xu and Imlay,012) and an increase in Fe uptake and storage during cold stressay help P. fluorescens BA3SM1 to replace quickly vital molecules

nder metal stress.Consequently, P. fluorescens BA3SM1 is able to develop vari-

us resistance strategies to maintain its metabolism under coldtress and some of them probably help this strain to combat tox-city of moderate metal concentrations. Nevertheless, for 1.5 mMu, pre-cold stress penalized cell growth. In a previous study, weave shown that P. fluorescens BA3SM1 was able to counteractu toxicity by limiting Cu uptake and activating its efflux (Poiriert al., 2013). Consequently, the up-regulation of TonB-dependentopper receptor, which allows Cu uptake (Madeira et al., 2011),nd the down-regulation of an ABC transport system-exportedrotein, which may be used by the cell for Cu efflux (Szczypkat al., 1994), probably penalize the bacterial cell to combat Cuoxicity. Furthermore, because Cu is capable of generating an oxida-ive stress (Rensing and Grass, 2003), the down-regulation of-phosphogluconolactonase decreases the ability of our strain toght effectively against oxidative stress induced by Cu. Indeed, 6-hosphogluconolactonase is a key enzyme involved in the pentosehosphate pathway, one of the main antioxidant cellular systems

n the sense that is a major source of NADPH (Riganti et al., 2012).For high metal concentrations, growth of pre-cold-stressed cells

as more affected than that of non-pre-cold-stressed cells, com-ared to respective controls. Therefore, resistance mechanismseveloped under cold stress prove inefficient to counteract dele-erious effects induced by high metal concentrations, and in thisase, pre-cold stress becomes even probably a handicap because iteads to multiple stresses in mesophilic bacteria (Moreno and Rojo,013).

.2. Metal biosorption ability of P. fluorescens BA3SM1

At +10 ◦C, metal biosorption ability of P. fluorescens BA3SM1 waselated to the bacterial growth phase. Indeed, it appeared more sub-tantial for cells in lag phase and exponential growth phase than forells in stationary phase. At +20 ◦C, only one biosorption measure-ent was performed during the exponential growth phase and this

ne confirmed a high biosorption during this growth phase com-ared to stationary phase. The high biosorption observed during the

ag phase and the exponential growth phase are in accordance with recent report showing that a rapid metal biosorption occurs whenacterial cells are inoculated in a metal contaminated mediumWang and Sun, 2013). A decrease in biosorption at the beginningf the stationary growth phase could be explained by the devel-pment of various resistance mechanisms during the exponentialrowth phase such as active efflux of metal, extracellular seques-ration and modification of the properties and fluidity of the cellnvelope. Indeed, our previous study has shown an up-regulationf proteins involved in these resistance mechanisms in P. fluores-ens BA3SM1 cells cultivated in metal supplemented nutrient brothnd harvested during the exponential growth phase (Poirier et al.,013). An increase in Cd and Zn biosorption during the stationaryhase could be explained by an increase in EPS production. Indeed,

recent study has shown a high correlation between the tolerancef a Pseudomonas strain to Cd and the metal biosorption ability ofhe EPS produced by this bacterium during the stationary growthhase (Chien et al., 2013).

ogy 157 (2014) 120–133 131

The low Cu biosorption observed in our study seems to showthat P. fluorescens BA3SM1 develops resistance strategies to keepCu outside the cell and avoid its adsorption on cells. An extracellularCu sequestration through the synthesis of specific proteins releasedin the environment could be proposed. Indeed, an extracellular Cu-binding protein was highlighted in Vibrio alginolyticus (Harwoodand Gordon, 1994).

After 144 h, Cd and Zn biosorption appeared lower at +10 ◦C thanat +20 ◦C. This result is in agreement with the study of Zhou et al.(2013) whereas Zamil et al. (2008) showed a better biosorption ofHg and Cd at +10 ◦C than at +30 ◦C in P. fluorescens BM07.

Several factors affect metal biosorption, such as temperature(Ahemad and Kibret, 2013) and pH (Huang and Liu, 2013). Con-sequently, it is difficult to compare our results with the previousstudies. According to data in the literature, Cd biosorption isbetween 0.097 �g/g dry weight for Serratia marcescens (Cristaniet al., 2012) and 135.3 mg/g dry weight for Rhizobium legu-minosarum bv. viciae (Abd-Alla et al., 2012), Zn biosorption isbetween 0.72 mg/g dry weight for Lactobacillus acidophilus WC0203(Leonardi et al., 2013) and 32.6 mg/g dry weight for P. putida(Li et al., 2014), and Cu biosorption is between 15.72 mg/g dryweight for P. putida (Fang et al., 2011) and 408 mg/g dry weight forMicrococcus luteus (Puyen et al., 2012). Consequently, P. fluorescensBA3SM1 appears to be a promising agent for removing Cd and Znin polluted environments since it allows an efficient biosorption ofthese two metals compared to other strains, especially during thelag phase and exponential growth phase.

Concluding remarks

The marine bacterium P. fluorescens BA3SM1, cultivated innutrient broth, is able to develop various resistance strategies tomaintain its metabolism under cold stress. Some of these strate-gies were also set up under metal stress. Consequently, pre-coldstress seems to be a good means to help this strain to combattoxicity of some metals at least to moderate concentrations. Thisstrain has shown good abilities to remove metals, even at low tem-peratures, and particularly during the lag phase and exponentialgrowth phase. This property is very interesting as most bacteria inthe temperate regions are mesophiles and relatively inactive in thewinter, so, P. fluorescens BA3SM1 appears as a promising agent forbioremediation processes. Moreover, its presence in marine sed-iment certainly permits a reduction both in metal bioavailabilityin this environment, and, consequently, in toxicity to other marineorganisms.

In this study, biosorption ability of P. fluorescens BA3SM1 wasevaluated in a rich medium, probably improving the establishmentof metal resistance mechanisms. Future experiments will be carriedout in a minimal culture medium to approximate environmentalconditions.

Acknowledgements

This work was financially supported by the Syndicat Mixte duCotentin Cherbourg-Octeville (AVAL agreement/exercise 2012) andthe Région Basse-Normandie France (Grant 2012 PCM 43).

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