Journal of Microbiological Methods 90 (2012) 309–314
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Detection and validation of a small broad-host-range plasmid pBBR1MCS-2 for use in genetic manipulation of the extremely acidophilic Acidithiobacillus sp. Likai Hao 1, Xiangmei Liu ⁎, Huiyan Wang, Jianqun Lin, Xin Pang, Jianqiang Lin State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, PR China
a r t i c l e
i n f o
Article history: Received 28 April 2012 Received in revised form 5 June 2012 Accepted 5 June 2012 Available online 15 June 2012 Keywords: Acidithiobacillus Broad-host-range plasmid Conjugation Streptomycin resistance
a b s t r a c t An efficient genetic system for introducing genes into biomining microorganisms is essential not only to experimentally determine the functions of genes predicted based on bioinformatic analysis, but also for their genetic breeding. In this study, a small broad-host-range vector named pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups, was studied for the feasibility of its use in conjugative gene transfer into extremely acidophilic strains of Acidithiobacillus. To do this, a recombinant plasmid pBBR-tac-Sm, a derivative of pBBR1MCS-2, was constructed and the streptomycin resistant gene (Smr) was used as the reporter gene. Using conjugation, pBBR-tac-Sm was successfully transferred into three tested strains of Acidithiobacillus. Then we measured its transfer frequency, its stability in Acidithiobacillus cells, and the level of resistance to streptomycin of the transconjugants and compared this with the IncQ plasmid pJRD215 control. Our results indicate that pBBR1MCS-2 provides a new and useful tool in the genetic manipulation of Acidithiobacillus strains. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The extremely acidophilic, obligatory chemolithoautotrophic thiobacilli, such as Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans and Acidithiobacillus caldus, are widely spread in sulfide deposits, acid mine water and soil. They can obtain energy from the oxidation of reduced sulfur compounds or ferrous iron to support their autotrophic growth on carbon dioxide or carbonate (Rawlings, 2002, 2005; Dopson and Lindstrom, 1999; You et al., 2011). Since they possess the unique physiological characteristics and outstanding capacity to grow under pH 2.0, these bacteria do not only have widespread industrial applications in mineral leaching, and desulphurization of coal and oil, but also are interesting to study from a fundamental biological point of view (Rawlings, 2005) . Now the entire or partial genome sequences of some strains of the acidophilic thiobacilli are available in GenBank, such as At. ferrooxidans ATCC 23270, At. ferrooxidans ATCC 53993, At. caldus ATCC 51756 and At. caldus SM-1 (Valdes et al., 2008a, 2009; You et al., 2011). Bioinformaticbased analysis coupled with studies on genomics, metagenomics, comparative genomics, transcriptomics, etc., provides a valuable platform to
⁎ Corresponding author at: State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong Province, 250100, PR China. Tel.: +86 531 88365992; fax: +86 531 88565610. E-mail address:
[email protected] (X. Liu). 1 Present address: Environmental Analytical Microscopy & Center for Applied Geoscience, Eberhard Karls University Tuebingen, Sigwartstr. 10, 72076 Tuebingen, Germany. 0167-7012/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2012.06.003
search for genome-wide candidate genes encoding proteins involved in important metabolic pathways and many predicted regulatory models for iron and sulfur energy metabolism and central carbon metabolism have been constructed (Quantrini et al., 2006, 2009; Valdes et al., 2008b; Bonnefoy and Holmes, 2011; Bird et al., 2011). This knowledge greatly helps us to understand the physiological functions and roles of these microorganisms in bioleaching. However, there are still many hypothetical reactions and missing steps in these metabolic pathways. So, a convenient genetic system would be helpful not only for elucidating the functions of candidate genes involved in these pathways but also to facilitate the genetic breeding of Acidithiobacillus strains for industrial applications. The extremely acidophilic bioleaching microorganisms of Acidithiobacillus are difficult to handle experimentally that their genetic system has been particularly challenging. Before there has been only one report each about introducing plasmids into strains At. ferrooxidans and At. caldus by electrotransformation (Kusano et al., 1992; Chen et al., 2010). The method of conjugation has been successfully developed in three species of Acidithiobacillus (Jin et al., 1992; Peng et al., 1994; Liu et al., 2007). With this method some plasmids from different incompatibility broad-host-range (bhr) groups, such as IncQ, IncP, and IncW, have been transferred from E. coli cells into different Acidithiobacillus strains. So far, the IncQ plasmids have been shown to be the best cloning vectors in Acidithiobacillus (Liu et al., 2000, 2007). By introducing and expressing heterologous genes on plasmids in Acidithiobacillus, the characteristics of the engineered strains have been improved, such as increased mercury resistance, or improved capacity for Fe 2 + or glucose metabolism (Chen et al., 2011; Liu et al., 2011; Tian et al., 2004). However, the
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plasmids are typically large in size and difficulty in genetic manipulation makes using the currently available IncQ vectors, such as plasmid pJRD215, extremely discommodious and laborious. So, more efficient vectors are undoubtedly essential and important to satisfy the increasing need for genetic studies of Acidithiobacillus. In this study, a small bhr plasmid pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups (Kovach et al., 1995), was studied for the feasibility of using the conjugative gene transfer system from E. coli into the extremely acidophilic At. caldus and At. thiooxidans. To characterize the functions of the new plasmid, the streptomycin resistant gene (Sm r) was used as a reporter gene and all experiments were carried out simultaneously and compared with an IncQ plasmid pJRD215 control. 2. Materials and methods 2.1. Strains, plasmids, media, and growth conditions The bacterial strains and plasmids used in this study are listed in Table 1. E. coli was grown in Luria broth or Luria agar at 37 °C. At. caldus MTH-04 (Liu et al., 2004), isolated from the acidic drainage of a hotspring in the Tengchong area, Yunnan Province of P. R. China, was grown at 40 °C in modified inorganic liquid Starkey-S0 or solid Starkey-Na2S2O3 medium (Starkey, 1925) as described previously (Jin et al., 1992). Sulfur, previously sterilized by intermittent steaming, was added aseptically at about 10 g/L at the time of inoculation. The solid Starkey-Na2S2O3 medium was prepared in two parts, the doublestrength basal salts (pH 4.8) and agar, which were separately autoclaved and combined after cooling to 50 °C. Sodium thiosulfate, previously sterilized by passage through a sterile Millipore filter, was added at the same time to a final concentration of 1% (wt/vol). At. thiooxidans ATCC 19377 was grown in the above media at 30 °C. The solid Starkey-Na2S2O3 medium was supplemented with 0.05% (wt/vol) yeast extract, when used as a mating medium for E. coli and Acidithiobacillus sp. Ampicillin (Ap) (50–100 μg/mL), kanamycin (Km) (50–100 μg/mL) or streptomycin Table 1 Bacterial strains and plasmids used in this study. Strain or plasmid Strains E. coli SM10 E. coli JM109 E.coli C600 At. caldus MTH-04 At. thiooxidans 19377 At. thiooxidans 12 Plasmids pBBR1MCS-2
Description
Source or reference
Thr leu hsd recA Kmr RP4-2-Tc::Mu
Wild type At. caldus
Simon et al., 1983 Laboratory stored Laboratory stored Liu et al., 2004
Standard ATCC wild type At. thiooxidans
ATCCa
Wild type At. thiooxidans
Laboratory stored
Kmr, pBBR1 replicon, mob+
Kovach et al., 1995 Laboratory stored Bagdasarian et al., 1983 Davison et al., 1987 Datta et al., 1971 This study This study
RecA1, supE44, endA1, hsdR17, gryA96, relA1, thi(lac-proAB), lacZ, lacIq, traD36 Integrated thr, leu, hsd
r
pUC19
Ap , ColE1 replicon, cloning vector
pMMB6
Apr, Smr, IncQ, mob+
pJRD215
Kmr, Smr, IncQ, mob+
RP4
Apr, Tcr, Kmr, IncP, tra+
pBBR-tac-Sm pUC-tac-Sm
pBBR1MCS-2 containing Smr gene with Ptac pUC19 containing Smr gene with Ptac
a
ATCC, American Type Culture Collection.
(Sm) (50–100 μg/mL) was added to the LB medium for E. coli, and Km (300 μg/mL) or Sm (300 μg/mL) was added to the Starkey-S0 liquid medium, whereas Km (50–100 μg/mL) or Sm (50–100 μg/mL) was added to the solid Starkey-Na2S2O3 medium for selection of Acidithiobacillus transconjugants. 2.2. Conjugation The conjugation experiments between E. coli cells and At. caldus or At. thiooxidans were performed by filter mating as described previously (Liu et al., 2007). The mating temperature for the cross was 37 °C between E. coli and At. caldus, and 30 °C between E. coli and At. thiooxidans. The incubations for the selection of transconjugants of At. caldus and At. thiooxidans were performed at 40 °C and 30 °C, respectively. The frequencies of plasmid transfer were calculated based on the number of transconjugants on selective plates divided by the number of recipients on nonselective plates (Tian et al., 2004). 2.3. Chemicals, enzymes, and DNA manipulations Ampicillin, kanamycin, and streptomycin were purchased from Sangon (Shanghai, China). Restriction enzymes, T4 DNA ligase, λ DNA/Hind III and DL2000 TM DNA markers were purchased from TaKaRa (Dalian, China). Plasmid DNA was prepared using a Plasmid Mini Kit I (OMEGA Bio-tek, USA). DNA was separated on agarose gels and purified using a Gel Extraction Kit (OMEGA Bio-tek, USA). Restriction endonuclease digestion, ligation, transformation, agarose gel electrophoresis, and other standard recombinant DNA techniques were performed according to standard procedures (Sambrook et al., 1989). 2.4. PCR PCR was performed using PrimeSTAR TM HS DNA polymerase from TaKaRa (Dalian, China) according to the manufacturer's recommendations. The primers used for PCR amplification were synthesized by Invitrogen Biotechnology Co. Ltd (Shanghai, China). In general, 50–100 ng of DNA was used in a 50 μL reaction volume containing 10 μL 5 × PrimeSTAR buffer (Mg 2 + plus), 4 μL dNTP mixture (2.5 mM each), 1 μM of each primer, and 0.5 μL of PrimeSTAR TM HS DNA polymerase (2.5 U/μL). Reactions were carried out in a DNA Thermal Cycler 480 from PERKIN ELMER with an initial denaturation at 94 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 60.4 °C for 30 s, and elongation at 72 °C for 2.5 min, and then a 10-min extension incubation at 72 °C. 2.5. Construction of plasmid pBBR-tac-Sm First, the 2088-bp DNA fragment carrying a Sm resistance gene with the tac promoter was amplified by PCR from plasmid pMMB6 (Bagdasarian et al., 1983) using primers F1 (5′-CCACAAGCTTATCGACTGCACGGT-3′) and F2 (5′-TAGTGGATCCTGTTTGGGGTCGTTTG-3′), based on the sequence of tac promoter (GenBank ID: K01728) and Sm resistance gene of plasmid RSF1010 (GenBank ID: NC001740), respectively. Hind III and BamH I sites were added to the primers. The amplified fragments were double digested with Hind III and BamH I, and then inserted into the Hind III/BamH I cloning sites of pBBR1MCS-2. The resulting plasmid was 7188 bps in size and designated as pBBR-tac-Sm. 2.6. DNA sequencing The inserted Hind III–BamH I fragment of pBBR-tac-Sm was subcloned into the Hind III/BamH I cloning sites of pUC19 to generate pUC19-tac-Sm, which was used for sequencing. Sequencing reactions were carried out using a 3730 DNA analyzer by Invitrogen Biotechnology Co. Ltd (Shanghai, China). The sequencing result of the
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inserted Hind III–BamH I DNA fragment was analyzed and verified by comparing its DNA sequence with that of the template plasmid pMMB6. 2.7. Expression determination of Sm r gene in strains of E. coli and Acidithiobacillus sp. The capacity to express the Sm r gene was determined by the growth of E. coli or Acidithiobacillus sp. carrying the corresponding plasmid in different concentrations of streptomycin. Briefly, E. coli cells were initially cultured overnight in LB broth supplemented with 50–100 μg/mL of Sm, and then 1% (v/v) of the fully grown cultures was inoculated into fresh LB broth, which had different concentrations of Sm added to it. The growth was monitored by measuring the optical density at 600 nm over 20 h. Cells of Acidithiobacillus sp. were initially cultured to stationary phase (5–7 d) in Starkey-S 0 with 300 μg/mL of Sm added, and then 1% (v/v) of the fully grown cultures was inoculated into fresh Starkey-S 0 medium, which had different concentrations of Sm added to it. The growth was monitored by measuring the optical density at 600 nm over 15 d. The solid sulfur was removed by low-speed centrifugation before detection. 2.8. Stability of plasmids in Acidithiobacillus sp. Single colonies of Acidithiobacillus transconjugants on selective plates were inoculated into 20 mL of antibiotic-free Starkey-S 0 liquid medium. After growth to the late exponential phase at the proper temperature, 1% (v/v) of the cultures was transferred to 20 mL of fresh Starkey-S 0 medium and grown again to the late exponential phase. After five transfers (more than 50 generations), the cultures were diluted and plated on Starkey-Na2S2O3 solid medium with or without antibiotic, and incubated for ten days at the proper temperature. Plasmid stability was calculated as the ratio between the number of colonies observed in the presence and absence of antibiotic. 3. Results 3.1. Construction of plasmid pBBR-tac-Sm Plasmid pBBR1MCS-2, one of four derivatives of pBBR1MCS that were constructed from a 2.6 kb plasmid pBBR1 isolated from Bordetella bronchiseptica S87, was shown to replicate in many Gram-negative bacteria (Kovach et al., 1995) but not in strains in Acidithiobacillus. Its characteristics of a broad-host-range, small size (5144 bp) and compatibility with plasmids in IncQ, IncW, and IncP groups have made it a new promising candidate as vector in Acidithiobacillus. To test this possibility, a streptomycin resistant gene (Smr), which is used as a reporter gene, was introduced into pBBR1MCS-2. The resulting pBBR-tac-Sm was constructed (see Section 2.5) and is depicted in Fig. 1. The successful construction of pBBR-tac-Sm was analyzed on the agarose gel electrophoresis (Fig. 2) and the inserted fragment was verified by DNA sequencing.
Fig. 1. Construction of plasmid pBBR-tac-Sm.
growth of E. coli JM109 (pBBR-tac-Sm) was detected when the concentration of Sm was more than 2.0 mg/mL. These results showed that the Smr gene from pBBR-tac-Sm was efficiently expressed in E. coli JM109. The growth of E. coli JM109 (pJRD215) in LB broth with different concentrations of Sm was also measured as a control. The maximal cell concentrations versus Sm concentrations are plotted in Fig. 4. Cells of E. coli JM109 containing either pBBR-tac-Sm or pJRD215 could grow well when the concentration of Sm was less than 0.1 mg/mL. But E. coli JM109 (pBBR-tac-Sm) had greater resistance to different concentrations of Sm than E. coli JM109 (pJRD215) (Fig. 4). The Sm r gene from plasmid pJRD215 was under the control of the Tc r promoter of pBR322 (Davison et al., 1987). So the differences in resistance to Sm in E. coli JM109 containing pBBR-tac-Sm or pJRD215 might have two different causes: different promoters for the Sm r gene or different copy numbers of the plasmids in E. coli JM109.
3.2. Expression of the streptomycin resistant gene from pBBR-tac-Sm in E. coli JM109 The Smr gene from plasmid pBBR-tac-Sm is under the control of the Ptac promoter and its expression was measured in E. coli JM109. Plasmid pBBR-tac-Sm was transformed into E. coli JM109 producing E. coli JM109 (pBBR-tac-Sm). The cell growth of E. coli JM109 (pBBR-tac-Sm) in LB broth with different concentrations of Sm added was measured and compared (Fig. 3). Control E. coli JM109 cells could not grow in more than 40 μg/mL of Sm. However, less than 0.5 mg/mL of Sm had no influence on the cell growth of E. coli JM109 (pBBR-tac-Sm) and a slight inhibition was detected when the Sm concentration was increased to 1.0 mg/mL (Fig. 3). A more obvious inhibition on the cell
Fig. 2. Agarose gel electrophoresis of plasmid pBBR-tac-Sm digested with Hind III and BamHI. Lane 1: λ DNA/Hind III marker; Lane 2: pBBR1MCS-2 (Hind III + BamHI); Lane 3: pBBR-tac-Sm (EcoRI); Lane 4: pBBR-tac-Sm (EcoRI + BamHI); Lane 5: pBBR-tac-Sm (Hind III + BamHI); Lane 6: PCR product of Smr; Lane 7: DL2000 marker.
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C600 (RP4) as the recipient. Plasmid RP4 contains the tra gene and is transmissible, so it could be used as helper plasmid in the reverse transfers (Datta et al., 1971). When the transconjugants of At. thiooxidans 19377 (pBBR-tac-Sm), At. thiooxidans 12 (pBBR-tac-Sm) and At. caldus MTH-04 (pBBR-tac-Sm) were used as the donors, reversetransconjugants of E. coli C600 (pBBR-tac-Sm) were successfully obtained on LB selective plates containing Sm (100 μg/mL). The matings were performed on filter and confirmed by plasmid analysis (data not shown). No colonies grew on the selective Sm (100 μg/mL) plates when E. coli C600 was used as the control recipient. 3.4. Stability of pBBR-tac-Sm in Acidithiobacillus strains
Fig. 3. Growth curves of E. coli JM109 (pBBR-tac-Sm) grown with various concentrations of Sm (mg/mL). The data are presented as the means ± standard deviation for three independent experiments.
3.3. Conjugative transfer of plasmid pBBR-tac-Sm from E. coli to Acidithiobacillus strains It was shown that plasmid pBBR1 could be transmitted by conjugation to other bacteria using the RK2 transfer functions in trans (Antoine and Locht, 1992). So, plasmid pBBR-tac-Sm was first transformed into E. coli SM10, which contains the helper plasmid RP4 integrated within the chromosome (Simon et al., 1983). E. coli SM10 (pBBR-tac-Sm) was used as the donor to conjugate with three recipients of Acidithiobacillus by filter mating as described in Materials and methods section. The transconjugants of Acidithiobacillus were obtained on solid Starkey-Na2S2O3 medium with both Sm (100 μg/mL) and Km (100 μg/mL) added. The results indicated that plasmid pBBR-tac-Sm could mobilize from E. coli SM10 into At. thiooxidans 19377, At. thiooxidans 12 and At. caldus MTH-04. The transfer frequency of pBBRtac-Sm to different strains of Acidithiobacillus varied from 10− 7 to 10− 5, which was about 10 times lower than the control plasmid pJRD215 (Table 2). The successful transfers of plasmid pBBR-tac-Sm into Acidithiobacillus were confirmed by reverse conjugative transfer experiments. Since pBBR-tac-Sm is not self-transmissible, its reverse transfers from selected transconjugants of Acidithiobacillus were performed using E. coli
To test the stability of plasmid pBBR-tac-Sm in Acidithiobacillus strains, we repeatedly subcultured the transconjugants containing pBBR-tac-Sm in liquid medium and counted total c.f.u. on StarkeyNa2S2O3 agar plates with or without antibiotic as described in Materials and methods section. After being cultured for about 50 generations without selective pressure, more than 60% of the tested cells still contained the pBBR-tac-Sm (Table 3). The results indicated that pBBRtac-Sm was relatively stable in the three strains of Acidithiobacillus. Although it was less stable than plasmid pJRD215 (IncQ) (Table 3), plasmid pBBR-tac-Sm was more stable as a vector than plasmid pUFR034 (IncW) and pJB3Km1 (IncP) in Acidithiobacillus (Liu et al., 2000). 3.5. Streptomycin resistance of Acidithiobacillus strains containing pBBRtac-Sm Both kanamycin and streptomycin resistance selectable markers have been shown to be useful in Acidithiobacillus. Wild type strains of At. caldus, At. thiooxidans 19377 and 12 showed obvious inhibition of their growth when the Sm concentration was increased to 0.5 mg/mL (data not shown). When pBBR-tac-Sm or pJRD215 was present, all strains could tolerate Sm to a certain extent. The Sm resistances of the three Acidithiobacillus strains containing plasmids pBBR-tac-Sm or pJRD215 grown in liquid Starkey-S0 medium with various concentrations of Sm were investigated by comparing their maximal cell concentrations (Fig. 5). As shown in Fig. 5, the cells with pBBR-tac-Sm were more tolerant to Sm compared with pJRD215 in all three Acidithiobacillus strains. The results indicated that the expression level of the Smr gene from plasmid pBBR-tac-Sm was higher than from plasmid pJRD215. So, plasmid pBBR-tac-Sm was demonstrated to serve as a good vector for expressing foreign genes in the extremely acidophilic strains of Acidithiobacillus. 4. Discussion Conjugation, one of the main gene transfer processes among bacteria, involves the transmission of genetic material from one bacterium to another and requires cell-to-cell contact. Based on compatible mating media, which were originally designed by Jin et al. (1992) and Peng et al. (1994), conditions that permit conjugation between strains of E. coli and Acidithiobacillus sp. were feasible in spite of their great differences in Physiology. Some plasmids from the three incompatibility groups, IncQ, IncP, and IncW, were successfully
Table 2 The transfer frequency of pBBR-tac-Sm and pJRD215 into different strains of Acidithiobacillus. Recipient
The transfer frequency of pBBR-tac-Sm
Fig. 4. The maximal cell concentrations of E. coli JM109 (pBBR-tac-Sm) and E. coli JM109 (pJRD215) grown with various concentrations of streptomycin. The data are presented as the means ± standard deviation for three independent experiments.
At. caldus MTH-04 At. thiooxidans 19377 At. thiooxidans 12
a
pJRD215 −7
2.53 ± 0.78 × 10 1.24 ± 0.36 × 10− 7 4.46 ± 0.63 × 10− 5
9.61 ± 0.54 × 10− 6 3.73 ± 0.25 × 10− 6 8.72 ± 0.91 × 10− 5
a The data are presented as the means ± standard deviation for at least three independent experiments.
L. Hao et al. / Journal of Microbiological Methods 90 (2012) 309–314 Table 3 The stability of pBBR-tac-Sm and pJRD215 in different strains of Acidithiobacillus. Strain
At. caldus MTH-04 At. thiooxidans 19377 At. thiooxidans 12
The stability of (%)
a
pBBR-tac-Sm
pJRD215
63 ± 7 71 ± 5 67 ± 9
79 ± 5 74 ± 8 71 ± 6
a The data are presented as the means ± standard deviation for at least three independent experiments.
transferred by conjugation into Acidithiobacillus strains (Jin et al., 1992; Liu et al., 2000, 2007). In this study, we have measured and confirmed the successful transfer of pBBR-tac-Sm, a derivative of pBBR1MCS-2, from strains of E. coli into At. caldus and At. thiooxidans. Except for the three tested strains of Acidithiobacillus, pBBR1MCS-2 and its derivatives have been proven to be successfully transferred and stably maintained in many other strains of At. thiooxidans and At. caldus stored in our laboratory, even including strains of At. ferrooxidans (Wang et al., 2012). So, pBBR1MCS-2 should not be strain specific and would no doubt be widely used as a new vector in the genetic manipulation of all strains of Acidithiobacillus. Compared with the widely used IncQ vector pJRD215, pBBR1MCS-2 had a slightly lower but workable transfer frequency from E. coli SM10 into Acidithiobacillus strains under the double selectable markers (Sm and Km) and a similar stability in the tested strains of Acidithiobacillus. However, its small size made pBBR1MCS-2 much easier to use in all the genetic manipulations and the Smr gene from pBBR1MCS-2 was efficiently expressed in both strains of E. coli and Acidithiobacillus. The Acidithiobacillus transconjugants containing plasmid pBBR-tac-Sm had the same greater resistance to Sm as E. coli JM109. This high tolerance to Sm might be due high levels of expression from the Ptac promoter, and the high plasmid copy number. It was shown that there were about 30 to 40 copies of pBBR1CM in E. coli MM294 (Antoine and Locht, 1992), while RSF1010 (origin of pJRD215) was only about 10–12 copies per chromosome in E. coli (Frey et al., 1992). So, the copy numbers of pBBR1MCS-2 and pJRD215 in Acidithiobacillus strains need further study. Moreover, pBBR1MCS-2 can be directly selected for its recombinant plasmid molecules in E. coli via disruption of the LacZα peptide, which makes it more convenient for the manipulation of recombinant DNA compared with the available bhr vectors used in Acidithiobacillus
Fig. 5. The maximal cell concentrations of Acidithiobacillus strains carrying plasmids pBBR-tac-Sm or pJRD215 grown in liquid Starkey-S0 medium with various concentrations of streptomycin. The data are presented as the means ± standard deviation for three independent experiments.
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strains. Although of unknown classification, pBBR1MCS-2 is compatible with other bhr plasmids of the IncQ, IncW and IncP groups, making it particularly useful in expressing foreign genes together with these vectors in the same Acidithiobacillus strain. By overexpressing some relative genes in Acidithiobacillus strains, the genetic characteristic of these biomining bacteria could be improved, such as increased capacity for Fe 2 + or sulfur metabolism. This will greatly facilitate the study on the genetic improvement of these biomining bacteria for industrial applications. So, pBBR1MCS-2 is no doubt a new and useful tool in the genetic manipulation of Acidithiobacillus strains. Acknowledgments This work was supported by grants from the National Natural Science Foundation (30970025), the National Basic Research Program (2010CB630902), the Natural Science Foundation (ZR2010CM004) and Key Scientific and Technological Project (2010GSF10626) of Shandong Province, P. R. China. 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