Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Quorum-sensing system in Acidithiobacillus ferrooxidans involved in its resistance to Cu2+

N. Wenbin1, Z. Dejuan2, L. Feifan3, Y. Lei1, C. Peng1, Y. Xiaoxuan1 and L. Hongyu1
1 MOE Key Laboratory of Arid and Grassland Ecology, School of Life Sciences, Lanzhou University, Lanzhou, China 2 Institute of Microbiology, School of Pharmaceutics, Lanzhou University, Lanzhou, China 3 School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, China

Keywords Acidithiobacillus ferrooxidans, brominated furanones, Cu2+ resistance, quorum sensing. Correspondence Li Hongyu, MOE Key Laboratory of Arid and Grassland Ecology, School of Life Sciences, Lanzhou University, Lanzhou, 730000, China. E-mail: nanwb06@lzu.edu.cn

Abstract Aims: The purpose of this study was to search for the relationship between quorum sensing (QS) and Cu2+ resistance in Acidithiobacillus ferrooxidans. Methods and Results: Resistance to Cu2+ of A. ferrooxidans signicantly decreased with the treatment dose of a synthetic QS blocker (5Z)-4-bromo-5(bromomethylene)-2(5H)-furanone (FUR). Relative differences in expression of the QS genes afeI, afeR and Cu2+ resistance-associated genes afe0329, afe0454 were examined in the presence of Cu2+ and or FUR compound. The expression of QS genes afeI and afeR increased signicantly with 50 mmol l)1 Cu2+ in the culture, while for samples treated with both 50 mmol l)1 Cu2+ and 001 lg ml)1 FUR compound, they showed little changes compared with control, and the expression of afe0329 and afe0454 genes increased slightly either. These results showed that QS system was positively related to the mechanism of Cu2+ resistance. Conclusions: QS system in A. ferrooxidans involved in its resistance to Cu2+. Signicance and Impact of the Study: The mechanisms of Cu2+ resistance in A. ferrooxidans could be revealed on a population level rather than on a single-cell level. Our work also provides useful data for further selection of A. ferrooxidans strains with suitable Cu2+ resistance that could probably increase the bioleaching efciency.

2010 1678: received 22 September 2010, revised and accepted 20 April 2011
doi:10.1111/j.1472-765X.2011.03066.x

Introduction Acidithiobacillus ferrooxidans is a chemolithoautotrophic bacterium that obtains its energy by oxidizing ferrous ions, elemental sulfur and partially oxidized sulfur compounds. This ability makes it widely used in biohydrometallurgy processes for recovery of metals such as copper, gold and uranium from ores by leaching (Leduc and Ferroni 1994; Jensen and Webb 1995). Heavy metal resistance of acidophilic bacteria is prerequisite for mineral dissolution in natural environments and industrial application. Garcia and da Silva (1991) suggested that appropriate resistance of A. ferrooxidans strains to metal ions would contribute to extractive hydrometallurgical operations, especially in industrial bioreactors, because it could increase the bioleaching efciency. Copper is an
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essential trace element for life, but high concentration of copper ion can cause serious damage to cells (Finney and OHalloran 2003). Although A. ferrooxidans can resist high concentrations of Cu2+ (Leduc et al. 1997), information regarding copper resistance in A. ferrooxidans is scarce. Now, A. ferrooxidans ATCC23270 has been sequenced; there are four genes afe0329, afe0454, afe0663 and afe1073 annotated to be related to copper homoeostasis and have been available in online database. Luo et al. (2008) have reported that the protein sequence encoded by afe0329 suggested a conserved domain of P1b3-type ATPase, which is a heavy metal pump, the protein AFE0454 that encoded by afe0454 was a putative copper resistance protein. However, afe0329 and afe0454 involving in the mechanisms of the Cu2+ resistance in A. ferrooxidans are many gaps in our knowledge.

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In recent years, a variety of physiological processes of bacterial species such as pathogenesis, symbiosis, antibiotic production, motility, genetic competence and biolm formation were found to be regulated by quorum sensing (QS) (Miller and Bassler 2001). In Gram-negative bacteria, QS is often using acylated homoserine lactones (AHLs) as the signal molecules. These molecules could bind to their receptors on the surface of the cell membrane to form AHL-R complexes, and then, these complexes would activate transcription of AHL synthase, resulting in increased levels of AHLs. On the other hand, these complexes play important roles in a variety of physiological processes. Recently, AHL-like QS signal system containing afeI and afeR genes has been detected in A. ferrooxidans. Both afeI and afeR genes are respectively orthologs of luxI and luxR. The afeI gene encodes AHLs synthase that catalyzes the production of AHLs, and afeR gene encodes AfeR protein that is required for the response to exogenous AHLs (Farah et al. 2005; Rivas et al. 2007). However, little is known on whether QS is related to the Cu2+ resistance in A. ferrooxidans. The present study was attempted to correlate the QS in A. ferrooxidans and its resistance to copper ion. Synthetic (5Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone (FUR) was prepared as QS inhibitor. Halogenated furanone compounds showed efcient inhibitory effect to QS system; many marine organisms, such as the red alga Delisea pulchra, can produce a number of halogenated furanones, which acted as strong inhibitors by blocking QS system (Maneeld et al. 1999). Previous research revealed that halogenated furanones could inhibit many behaviours of Gram-negative bacteria, such as swarming (Gram et al. 1996; Ren et al. 2001), bioluminescence (Maneeld et al. 2000) and biolm formation (Ren et al. 2001; Hentzer et al. 2002). In this study, the effect of FUR compound on the Cu2+ resistance of A. ferrooxidans ATCC23270 was detected. To further illustrate the relationship between QS and Cu2+ resistance, we investigated both the impacts of Cu2+ and FUR compound on afeI, afeR, afe0329 and afe0454 expressions. Materials and methods Strains and primers Strains and primers used in this study were showed in Table 1. The strain of A. ferrooxidans ATCC23270 employed in this study was the type strain from ATCC, USA. This A. ferrooxidans strain was cultured in 9K medium (Silverman and Lundgren 1959). Real-time PCR primers were designed by Primer Premier 5.0 and then synthesized by TaKaRa (Dalian, China). Agrobacterium

Table 1 Strains and primers used in this study

Strains or primers Strains Acidithiobacillus ferrooxidans ATCC23270 Agrobacterium tumefaciens NTL4 (pZLR4) NTL4 (pTiC58DaccR) NTL4 Primers afeI afeR afe0329 afe0454 16S Description

Type strain from ATCC, USA. traG::lacZ, traR, AHL), Genr, Carr Trac, AHL+ Ti), AHL) 5-CGAATAATCTGTATGCGAAGGT-3 5-TGGTGCCAGTCCGTTGAGT-3 5-CCTTTGCTTGCCATTCTGCC-3 5-TTCGCCGTCTCCCGTCGTCT-3 5-CAGCAGGCGAATCAGTT-3 5-ACATGGCGACCTACACG-3 5-CGGCGACCGAAGAGGACTA-3 5-AAGCGGGTGGAGGCATT-3 5-TCCTTAGTTGCCAGCGGTTCG-3 5-GCGGCTTGGCTTCCCTCTGTA-3

tumefaciens NTL4 was selected as indicator incubated in LB medium. Inhibitory concentrations of Cu2+ detection The furanone compound (5Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone was prepared according to Manny et al. (1997) and stored in dichlormethane at 20 mg ml)1 at )20C. CuSO45H2O was of analytical grade and used for all analysis. Inhibitory concentrations (ICs) of metal are dened as the concentration that showed a signicant (P < 001) decrease in the percentage of ferrous iron oxidized when compared to an untreated control (Leduc et al. 1997). The ICs of Cu2+ were determined for each group of A. ferrooxidans in the absence or presence of FUR compound; the concentration of Cu2+ in 9K medium was from 20 to 200 mmol l)1. The cultures were divided into ve groups, and each was determined in triplicate by transferring 5% (v v) of log-phase cells into 10 ml of fresh 9K medium containing requisite amounts of the metals copper sulfate. The FUR compound concentrations of the four groups were 001, 005, 01 and 1 lg ml)1, respectively. The last group as control was absence of FUR compound. The cultures were incubated at 30C on a rotary shaker for 100 h. The concentrations of ferrous ion were determined by titrating 20 ml samples of the cultures with 001 mol l)1 K2Cr2O7. Ferrous ion oxidized was expressed as a percentage relative to an untreated control. A 100-h-long incubation time was chosen to ensure ferrous ion in the control being completely oxidized. The data were analysed with the statistics soft85

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ware spss ver. 13.0 (Statistical Package for the Social Sciences, Chicago, IL) as well. Detection of the ferrous ions oxidation by Acidithiobacillus ferrooxidans Acidithiobacillus ferrooxidans ATCC23270 was cultured in fresh 9K media, and experiments were divided into four groups, and all carried out in 250-ml asks. The rst group was cultured in the presence of 50 mmol l)1 Cu2+, the second group was treated with 01 lg ml)1 FUR compound, the third group was treated with both 50 mmol l)1 Cu2+ and 01 lg ml)1 FUR compound while the last group cultured in 9K media without FUR compound and Cu2+ was selected as control. The cultures with inoculum concentration of 5% (v v) were transferred into the media, and then, the bacteria were incubated on a rotary shaker at 150 rev min)1 and 30C for about 78 h. Culture of 20 ml was sampled, and 20 ml 0K (9K medium without ferrous ions) medium was supplemented every 3 h. The samples were titrated with 001 mol l)1 K2Cr2O7 to determine the concentrations of ferrous ion in cultures, and then, the percentage of ferrous ions oxidized was employed as the index of A. ferrooxidans growth. Detection of the production of AHL by Acidithiobacillus ferrooxidans Extracts for bioassay and high-performance liquid chromatographyelectrospray ionization mass spectrometry (HPLCESIMS) were prepared from 500-ml culture. Bacterial cells were removed by centrifugation, and the supernatant was extracted twice with equal volume of dichloromethane (DCM) as described previously (Farah et al. 2005). Extracts were then dried over anhydrous magnesium sulfate ltered and then evaporated to dryness. The residues were dissolved in 1 ml HPLC-grade methanol and stored at )20C. For bioassay, Ag. tumefaciens NTL4 (pZLR4) was used as indicator; this reporter strain carrying the plasmid pZLR4 can respond to AHLs with N-acyl chain length from 6 to 14 carbon atoms and produce a blue colour in the presence of X-Gal (5-bromo-4-chloro3-indolyl-b-d-galactopyranoside). To assay, Ag. tumefaciens NTL4 (pZLR4) soft agar suspensions (07% agar) with 40 lg ml)1 X-Gal and Ag. tumefaciens NTL4 (pZLR4) were prepared (Chilton et al. 1974). The suspension was then overlayed onto plates containing AB minimal medium (15% agar) with glucose as carbon source was prepared. After the overlay solidied, patch onto the surface 5 ll DCM extracts obtained from the A. ferrooxidans media and incubate the plates at 28C for about 36 h. Besides, this strain was supplemented with 20 lg ml)1 gentamycin. Ag. tumefaciens NTL4 (pTiC58DaccR) con86

taining the trac Ti plasmid that can constitutively synthesize AHLs was used as the positive control. On the other hand, Ag. tumefaciens NTL4 lacks the Ti plasmid was used as the negative control which cannot produce AHLs detectable with the assay system. HPLCESIMS was used to further determine the AHLs produced by A. ferrooxidans ATCC23270 cultured in the presence or absence of the FUR compound. The identication was carried out by comparison with AHL standard based on: ESIMS product ions ([M+H]+), the HPLC retention times and relative intensities. The AHL standard N-tetradecanoyl-dl-homoserine lactone (AHL-C14) was purchased from Fluka. Total RNA extraction and cDNA synthesis Total RNA of different treated samples was extracted and puried from 500 ml of culture using the EASYspin bacterial rapid RNA extraction kit (Biomed, Beijing, China). The cDNA was synthesized using the obtained RNA as the template with PrimeScript RT reagent kit (TaKaRa). Quantitative real-time RT-PCR detection The relative abundances of QS genes afeI, afeR and Cu2+ resistance-associated genes afe0329, afe0454 in Cu2+- and FUR-grown cells were determined by real-time PCR with the SYBR Premix Ex Taq II kit (TaKaRa). Each real-time PCR mixture (nal volume 20 ll) contained 10 ll SYBR Premix Ex Taq II, 16 ll of 10 mmol l)1 primer, 2 ll of cDNA template and 64 ll of ddH2O. The real-time PCR was carried out with the CFX Manager detection system (Bio-Rad, Hercules, CA): one cycle of 95C for 2 min and then 40 cycles of 95C for 10 s, 60C for 30 s. Melting curves of the amplicons were obtained through raising the temperature from 60 to 95C by 05C per 3 s while monitoring uorescence at the ending cycle. The transcript levels of afeI, afeR afe0329 and afe0454 were determined from the threshold values, which were rst normalized with endogenous control (16S rRNA) gene expression, and then normalized with control cultured without any Cu2+ or FUR compound. The relative expression ratios were calculated using a mathematical model, which has an efciency correction for the real-time RT-PCR efciency of the individual transcripts, this method is in accordance with Pfaf (2001). Results The effect of FUR on ICs of Cu2+ in Acidithiobacillus ferrooxidans ATCC23270 The Cu2+ resistance of A. ferrooxidans ATCC23270 strain was studied in the absence or presence of FUR com-

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pound. Data in our test indicated that resistances of this strain to Cu2+ showed different sensitivity to FUR compound in a dose-dependent manner (Table 2). Acidithiobacillus ferrooxidans ATCC23270 could tolerate high concentration of Cu2+ about to a level of 160 mmol l)1, the ICs of Cu2+ in the absence of FUR compound were 180 mmol l)1, and these results were similar to those described by Dopson et al. (2003). When this strain was treated with 001 lg ml)1 FUR compound, it still showed a high resistance to Cu2+, for it could still be grown in the presence of 140 mmol l)1 Cu2+. When the concentration of FUR compound increased to 005 lg ml)1, A. ferrooxidans showed more sensitive to Cu2+, for it could not resist Cu2+ higher than 60 mmol l)1. As the concentration of FUR compound increased to a higher level of 01 lg ml)1, this strain could still resist 20 mmol l)1 of Cu2+, the ICs of Cu2+ reduced to 40 mmol l)1. However, the growth of this strain with or without Cu2+ was both inhibited completely in the presence of 1 mmol l)1 FUR compound. The effects of Cu2+ and or FUR compound on ferrous ions oxidation by Acidithiobacillus ferrooxidans The effects of Cu2+ and FUR compound on growth of A. ferrooxidans ATCC23270 were assessed in 9K medium by monitoring the percentage of ferrous ion oxidized. The results showed that the growth of A. ferrooxidans ATCC23270 was inhibited more or less in the presence of Cu2+ and FUR compound. During the growth of A. ferrooxidans ATCC23270, an extension in the lag phase was observed when supplemented with Cu2+ or FUR compound (Fig. 1). The lag phase lasted for about 27 h in the presence of 50 mmol l)1 Cu2+, which was 6 h longer than the control without Cu2+. In contrast, the lag phase became 15 h longer in the presence of 01 lg ml)1 FUR

Percentage of ferrous ion oxidized (%)

100 90 80 70 60 50 40 30 20 10 0 0 6 12 18 24 30 36 42 48 54 60 66 72 78 Culture time (h)

Figure 1 Effect of Cu2+ and FUR compound on ferrous ion oxidized percentage of Acidithiobacillus ferrooxidans ATCC23270. ( ) Without Cu2+ or FUR compound; (s) with 50 mmol l)1 Cu2+ only; ( ) with 01 lg ml)1 FUR compound only; (4) with both 50 mmol l)1 Cu2+ and 01 lg ml)1 FUR compound.

compound than that in the presence of 50 mmol l)1 Cu2+. As to the group treated with both 50 mmol l)1 Cu2+ and 01 lg ml)1 FUR compound, no ferrous ion oxidation was observed, and the growth of A. ferrooxidans ATCC23270 was completely inhibited. Effect of FUR compound on production of AHLs by Acidithiobacillus ferrooxidans For the bioassays using Ag. tumefaciens NTL4 (pZLR4), the positive control Ag. tumefaciens NTL4 (pTiC58DaccR) appeared on a blue colour expectedly, so did the A. ferrooxidans ATCC23270 extracted samples (Fig. 2a). However, when treated A. ferrooxidans ATCC23270 with 001 lg ml)1 FUR compound, extracts from the supernatant failed to induce Ag. tumefaciens NTL4 (pZLR4) to produce a blue zone (Fig. 2b). These results demonstrated that A. ferrooxidans ATCC23270 could produce long chain AHLs, and FUR compound probably has an inhibitory effect on production of long chain AHLs. To further determine the AHLs, the extracts from A. ferrooxidans ATCC23270 cultured in the presence or absence of the FUR compound were analysed by HPLCESIMS. For extracts from A. ferrooxidans ATCC23270 cultured in the absence of FUR compound, a visible peak at the same retention time as standard AHL-C14 was shown, but for extracts from A. ferrooxidans ATCC23270 cultured in the presence of 001 lg ml)1 FUR compound showed no peak at the same retention time. ESIMS analysis illustrated that standards of AHL-C14 had an m z 312 (M+H) ion peak (Figs S1 and S2). These results indicated that AHL-C14 could be synthesized by A. ferrooxidans
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Table 2 Inhibitory concentrations (ICs) of Cu2+ in the presence of FUR compound

Cu2+ (mmol l)1) FUR concentration (lg ml)1) 0 001 005 01 1 Tolerated 160 140 60 20 0 ICs 180 160 80 40 0

AHL, acylated homoserine lactone; tolerated, concentration that shows no signicant differences in the percentage of ferrous iron oxidized when compared to a control; ICs, the concentration that shows a signicant (P < 001) decrease in the percentage of ferrous iron oxidized when compared to a control.

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(a)

(b)
Figure 2 Effect of FUR compound on the bioassay of extract of Acidithiobacillus ferrooxidans ATCC23270. (A) Agrobacterium tumefaciens NTL4 (pTiC58DaccR) which can constitutively synthesize long-chain acylated homoserine lactones (AHLs) was used as the positive control; (B) Ag. tumefaciens NTL4 which cannot produce AHLs was used as the negative control; (C) extract from A. ferrooxidans ATCC23270; (D) extract from ATCC23270 treated with 001 lg ml)1 FUR compound.

A B

ATCC23270, which was consistent with the result reported by Farah et al. (2005), and FUR compound could reduce the production of AHL-C14. Quantication of afeI, afeR, afe0329 and afe0454 genes by real-time PCR The effects of the FUR compound and Cu2+ metal ions on the transcription levels of the A. ferrooxidans afeI, afeR, afe0329 and afe0454 genes were determined by quantitative real-time RT-PCR (Fig. 3). The specicity of PCR amplication was checked by examining melting curve for Tm, and PCR amplications of four genes were symmetry and lack of nonspecic peaks (data not shown). When A. ferrooxidans ATCC23270 was treated with 001 lg ml)1 FUR compound, afe0329 and afe0454 showed no signicant difference from the control group, whereas the expression of afeI and afeR genes signicantly decreased to 7 and 3%, respectively (Fig. 3a). For 50 mmol l)1 Cu2+ treated cell samples, A. ferrooxidans ATCC23270 expressed both afe0329 and afe0454 genes at a higher level compared with the control, and it was very interesting that the expressions of QS genes afeI and afeR also increased signicantly by 41- and 26-fold, respectively (Fig. 3b). While for samples treated with both 50 mmol l)1 Cu2+ and 001 lg ml)1 FUR compound, the expressions of QS genes afeI and afeR showed little changes compared with the control, the expression of afe0329 and afe0454 genes also only increased slightly (Fig. 3c). Discussion To determine the effect of FUR compound on Cu2+ resistance of A. ferrooxidans ATCC23270 strain, ICs of Cu2+ were studied in the absence or presence of FUR compound. Data showed that the resistance of A. ferrooxidans to Cu2+ signicantly decreased in the presence of 005 lg ml)1 FUR compound. This suggested that the
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concentration of FUR compound above 005 lg ml)1 had an efcient inhibitory effect on the resistance of A. ferrooxidans to Cu2+. As most heavy metals are toxic to many physiological functions (Nies 1999), the lag phase of the strains would extend in the presence of these soluble metal ions. The lag phase of A. ferrooxidans ATCC23270 extended in our study probably resulted from biological toxicity of Cu2+. This extension indicated an adaptation of A. ferrooxidans to copper ion. Furthermore, no iron oxidation was detected in the presence of both Cu2+ and FUR compound. Acidithiobacillus ferrooxidans could not tolerate 50 mmol l)1 Cu2+ any more when 01 lg ml)1 FUR compound was added. We know that halogenated furanones are widely used as efcient blockers in QS studies (Defoirdt et al. 2007; Kim et al. 2008), so we assume that the decrease of ICs of Cu2+ in the presence of FUR compound may be caused by the inhibition of QS. To determine whether FUR compound used in this study is an efcient blocker to QS in A. ferrooxidans, production of AHLs was detected. Previous study had reported that extracts obtained from A. ferrooxidans irongrown cells contained diverse AHLs with chain length of C8C16 (Farah et al. 2005). Our results of AHLs detection revealed that A. ferrooxidans ATCC23270 produced AHLs with long acyl chain that was accorded with previous report, and FUR compound used in this study could signicantly reduce the production of AHLs by blocking the QS system in A. ferrooxidans. To correlate the QS in A. ferrooxidans and its resistance to Cu2+, the effects of FUR compound on expression of QS-associated genes and Cu2+ resistance associated genes were determined, respectively. Acidithiobacillus ferrooxidans strain possesses a QS system that contains two function genes afeI and afeR. Both afeI and afeR genes are orthologs of luxI and luxR, respectively. The afeI gene encodes AHL synthase that catalyzes the production of AHLs, and afeR gene encodes AfeR protein that is

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(a)

20

15

10

05

00

afeI

afeR

afe0329

afe0454

(b) 60 50 Relative expression ratio 40 30 20 10 0 (c)

afeI

afeR

afe0329

afe0454

20

15

10

05

00

afeI

afeR

afe0329

afe0454

Figure 3 Effect of Cu2+ or FUR on expression of afeI, afeR, afe0329 and afe0454 genes in Acidithiobacillus ferrooxidans ATCC23270. (a) ( ) Control, ( ) treated with 001 lg ml)1 FUR compound; (b) ( ) control, ( ) treated with 50 mmol l)1 Cu2+; (c) ( ) control, ( ) treated with both 50 mmol l)1 Cu2+ and 001 lg ml)1 FUR compound. Relative levels of mRNA were normalized for gene expression of the endogenous control (16S rRNA), and then A. ferrooxidans ATCC23270 grown without any Cu2+ or FUR compound was dened as 10. The relative levels of afeI, afeR, afe0329 and afe0454 genes expression in different treated samples were compared. The data were expressed as the average of triplicates the standard deviation.

required for the response to AHLs (Farah et al. 2005). Our results showed that the expression levels of afeI and afeR genes were repressed in the presence of 001 lg ml)1 FUR compound, whereas expressions of afe0329 and afe0454 genes showed little changes. FUR compound has a similar structure to AHLs, and it disrupts QS by competitively binding to the AHL receptor site of R protein family (Givskov et al. 1996), but FUR-R complex could not activate transcription of afeI gene. It explained that FUR compound could inhibit the transcription of QS gene. However, the expressions of Cu2+ resistance-associated genes afe0329 and afe0454 were not affected by FUR compound. The two genes afe0329 and afe0454 of A. ferrooxidans ATCC23270 showed intensive response to copper stress, the transcription levels of both afe0329 and afe0454 genes signicantly increased when we treated A. ferrooxidans with 50 mmol l)1 Cu2+. When external Cu2+ concentration increases, all of the Cu2+ resistance-related genes would be induced to eliminate Cu2+ out of the cell, this phenomenon accords to the annotation that the two genes were involved in copper homoeostasis in A. ferrooxidans. Furthermore, we noticed that the expressions of afeI and afeR genes were also up-regulated. Possibly, copper ions could induce QS genes expression to a higher level which could help A. ferrooxidans survive at high concentration of copper ions stress in some special environment. As we know, a variety of physiological processes in bacterial are regulated by QS, so QS system probably has a relationship with the Cu2+ resistance mechanism. When we treated A. ferrooxidans with both 50 mmol l)1 Cu2+ and 001 lg ml)1 FUR compound, there was no increase any more in expressions of afeI and afeR genes, expressions of afe0329 and afe0454 gene increased slightly as well. The increase tendency of afe0329 and afe0454 genes expressions was signicantly repressed in the presence of FUR which might be resulted from the repressed QS genes afeI and afeR genes expressions. These results indicated that QS system was positively related to the mechanism of Cu2+ resistance. It further supported that FUR compound could inhibit the production of AHLs in A. ferrooxidans, and the QS signal system blocked (Maneeld et al. 2002), as a result of that the Cu2+ resistance of A. ferrooxidans was reduced. Together with our data on quantitative real-time RT-PCR of the QS-associated genes afeI and afeR, Cu2+ could induce the transcription of both genes, it can be concluded that QS in A. ferrooxidans was involved in its resistance to Cu2+. Most QS-controlled processes are unproductive when undertaken by an individual bacterium acting alone but become benecial when carried out simultaneously by a large number of cells (Waters and Bassler 2005). Previous
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studies on metal ions resistance mechanisms of bacteria mainly focused on individual response (Dopson et al. 2003); our results indicated that the mechanism of Cu2+ resistance in A. ferrooxidans probably was a population function, and QS signal system might play an important role in Cu2+ resistance mechanism. Notably, heavy metal resistance is a complex process that may be regulated by diverse mechanisms, and how the QS signal system regulates heavy metal resistance needs further investigation. Such work may offer an approach for selecting or adapting A. ferrooxidans strains with appropriate resistance to metal ions, for it probably increases the bioleaching efciency. Acknowledgements This work was supported by the Scientic and Technological Project of Gansu Province, China (grant no. 2GS064-A43-019-02), Scientic and Technological Project of Lanzhou City, Gansu Province, China (grant no. 0302-24), Fund Project of the Science and Technology Innovative Team of Gansu Province (grant no. 098TTCA013) and International Cooperation Projects of Gansu Province, China (grant no. 0708WCGA150). We gratefully acknowledge Dr B. Johnson for providing A. ferrooxidans-type strain A. ferrooxidans ATCC23270, Prof Stephen K. Farrand, University of Illinois at UrbanaChampaign for providing Agrobacterium tumefaciens strain NTL4. We also gratefully acknowledge Prof Baohua Chen, School of Chemistry and Chemical Engineering, Lanzhou University, for providing synthetic (5Z)-4bromo-5-(bromomethylene)-2(5H)-furanone. References
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1 The extracts from A. ferrooxidans ATCC23270 cultured in the absence of the FUR compound were analyzed by HPLC-ESIMS. Figure S2 The extracts from A. ferrooxidans ATCC23270 cultured in the presence of 0.01 lg ml)1 FUR compound were analyzed by HPLC-ESIMS. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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