May 2, 1994 - Mishra, A. K., P. Roy, and S. S. R. Mahapatra. 1983. Isolation of. Thiobacillus ... 456-461. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1994, 0099-2240/94/$04.00 + 0 Copyright (C 1994, American Society for Microbiology
p.
2653-2656
Vol. 60, No. 7
Expression of Heterogenous Arsenic Resistance Genes in the Obligately Autotrophic Biomining Bacterium Thiobacillus ferrooxidans JI-BIN PENG,* WANG-MING YAN, AND XUE-ZHEN BAO Institute of Microbiology, Shandong University, Jinan 250100, People's Republic of China Received 8 November 1993/Accepted 2 May 1994
Two arsenic-resistant plasmids were constructed and introduced into Thiobacillus ferrooxidans strains by conjugation. The plasmids with the replicon of wide-host-range plasmid RSF1O1O were stable in T.ferrooxidans. The arsenic resistance genes originating from the heterotroph were expressed in this obligately autotrophic bacterium, but the promoter derived from T. ferrooxidans showed no special function in its original host.
Thiobacillus ferrooxidans is a gram-negative, acidophilic, obligately chemolithotrophic bacterium. This organism derives its energy by oxidizing ferrous iron and reduced or partially reduced sulfur compounds and obtains its carbon by fixing carbon dioxide from the atmosphere (25). T. ferrooxidans is suited to growth in a mining environment and is used industrially to leach metals such as copper, uranium, and gold from mineral ores. However, the slow growth of this organism and its sensitivity to heavy metals such as As(III), Hg(II), and Ag(I) has limited its further use (3, 7, 8, 17, 24). To improve the bacterium genetically, considerable work has been done over the past 10 or more years in plasmid isolation (13, 20), vector construction (19), and gene cloning (1, 9, 11, 21). However, the gene transfer study of this bacterium progressed slowly (2, 21, 26). Kusano et al. (10) reported that they had introduced plasmids to T ferrooxidans by electrotransformation. With a mer determinant cloned from the chromosome of T ferrooxidans as the selective marker, only 1 of 30 T. ferrooxidans strains tested was transformed at a very low frequency and the expression of mer genes was poor. Recently, the conjugal transfer of plasmids from Escherichia coli to T. ferrooxidans has been demonstrated; genes originated from heterotrophic bacteria were expressed, and the cloning vector pJRD215 was stable in this obligately autotrophic bacterium (18). In this paper, we report the construction of arsenic-resistant plasmids and the expression of arsenic resistance genes in T ferrooxidans. The strains and plasmids used in this study are listed in Table 1. E. coli strains were cultured in Luria broth or on Luria agar at 37°C. Strains of T. ferrooxidans were cultured in 9K liquid medium (22) or on solid 2:2 medium. Solid 2:2 medium was prepared in four parts: Na2S203 5H20 (2 g) was added to 10 ml of H20 (solution A); FeSO4 * 7H20 (2 g) was added to 10 ml of H20 adjusted to pH 2 with 2 N H2SO4 (solution B); (NH4)2SO4 (4.5 g), KCl (0.15 g), and MgSO4- 7H20 (0.75 g) were dissolved in 500 ml of H20 adjusted to pH 4.6 with 2 N H2SO4 (solution C); and agar (6.0 g) was added to 480 ml of H20 (solution D). Solutions A and B were both filter sterilized, while solutions C and D were autoclaved. Solutions A, B, C, and D were mixed after solutions C and D had cooled to 45°C. When the mixture served as a mating medium, solution B was
omitted and 0.05% (wt/vol) sterile yeast extract was introduced. Streptomycin was added to 300 ,ug/ml in solid 2:2 medium to select transconjugants. Arsenic-resistant plasmids were constructed by standard protocols for DNA manipulation (12). The 14.8-kb arsenicresistant vector pSDRA1 was constructed by simply ligating the two longer EcoRI-SalI fragments of pJRD215 and pUM3 (Fig. 1). The other 16.5-kb arsenic-resistant plasmid, pSDRA21, containing a promoter cloned from the chromosome of T ferrooxidans Tf-52 was constructed as described below. The 4.3-kb HindlIl fragment of pUM3 containing arsenic resistance genes was cloned into the Hindlll site of pSDRF122 which contains the promoter of Tf-52, resulting in plasmid pSDRA2 (the direction of insertion was examined by electrophoresis of plasmids digested by PstI). Plasmid pSDRA21 was therefore constructed by fusion of the two longer EcoRI-SalI fragments of plasmids pSDRA2 and pJRD215 (Fig. 1). The arsenic-resistant ability of E. coli ED8654 carrying pSDRA1 or pSDRA21 was examined by growing the strains in Luria broth containing different concentrations of NaAsO2 with vigorous shaking (160 rpm) for 5 h and measuring the cell densities of the cultures (Fig. 2). Crosses between strains were conducted by filter mating, using a 1:1 mixture of donor and recipient cells that had been suspended in the basal salt solution of mating medium (about
TABLE 1. Bacterial strains and plasmids used in this study Strain or
plasmid
Phenotype
or
Source or reference
genotype
-
E. coli C600 ED8654
thr leu hsd supE supF hsdR metB lacY gal trp
12 16
Wild type Wild type
This study 6
Apr Tcr KMr IncP Tra+
4 John Davison Guangyong Ji 6 This study This study This study
T. ferrooxidans Tf-50 Tf-52 Plasmids RP4 pJRD215 pUM3 pSDRF122
* Corresponding author. Present address: Shanghai Institute of Cell Biology, Academia Sinica, 320 Yue Yang Road, Shanghai 200031, People's Republic of China. Phone: 0086-021-4315030 ext. 29. Fax: 0086-021-4331090.
2653
pSDRA1
Kmr Smr IncQ Mob' Apr Asr Apr TCr Kmr Smr Asr IncQ Mob+
pSDRA2 pSDRA21
Apr Asr Kmr Smr Asr IncQ Mob+
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NOTES EH
9
H
E FIG. 1. Construction of pSDRA1 and pSDRA21. Only the relevant restriction sites are shown. Abbreviations: E, EcoRI; H, HindIII; S, Sall; Apr, ampicillin resistance; As', arsenic resistance; Kmr, kanamycin resistance; Smr, streptomycin resistance; Tcr, tetracycline resistance; Tcs, tetracycline sensitivity; mob, plasmid mobilization functions; ori, origin of replication; PTf, promoter cloned from Tf-52.
2 x 10'0 cells per ml). After incubation at 30°C for 3 days, the filter was transferred to 3.0 ml of the basal solution of solid 2:2 medium and then diluted and plated on solid 2:2 medium with or without streptomycin. As a control for spontaneous mutation, the recipient strains were plated on the same selective plates. After culture at 30°C for 2 weeks, the colonies formed were counted, and plasmid transfer frequencies were calculated as the number of transconjugants divided by the number of recipients. The stability of the recombinant vectors in T. ferrooxidans strains was investigated as described below. Single colonies of T. ferrooxidans transconjugants on the selective plates were transferred into 9K liquid medium (20 ml); 1/1,000 of the fully grown cultures was transferred to 20 ml of fresh 9K liquid medium and was cultured at 30°C with vigorous shaking for 5 days. After five transfers (more than 50 generations) had been made, samples were diluted and plated on solid 2:2 medium with or without kanamycin (300 ,ug/ml) and cultured at 30°C. Two weeks later, the colonies formed were counted and the percentages of plasmid maintenance were calculated as the number of colonies on medium with kanamycin divided by the number of colonies on medium without kanamycin.
0
03
-4
be
8.0-N- As
*0
2
4
I
6
8
10
12
14
16
NaAsO. concentration (mM) FIG. 2. Growth of ED8654(pSDRA1) (O), ED8654(pSDRA21) (A), and ED8654 (0) in serial concentrations of NaAsO2. Values are the means of three independent experiments.
NOTES
VOL. 60, 1994
TABLE 2. Transfer frequencies and stabilities of the recombinant plasmids
Recipient strain
Plasmid
Transfer Transfer frequency"
2655
8.5r
~Plasmid
Q)
8.0
maintenancc (%k ) CJ2
Tf-50 Tf-50 Tf-52 Tf-52
pSDRA1 pSDRA21
pSDRA1 pSDRA21
5.4 1.8 2.9 6.7
X 10 5 X 10 x 10 4
86 81 85
x 10 5
10(
7.5
0
7.0
" Number of transconjugants/recipient colony after mating.
6.5
Since plasmid pJRD215 was stable in T. ferrooxidans (18), arsenic-resistant vectors based on pJRD215 were constructed as described above. The two plasmids constructed, pSDRAI and pSDRA21, both contain two antibiotic resistance markers (kanamycin and streptomycin resistance genes), arsenic resistance genes derived from plasmid R773 (15), and the widehost-range replicon and mobilization functions of RSF10IO (5). The main difference between pSDRA1 and pSDRA21 lies in the promoter of arsenic resistance genes. In pSDRA1, the promoter of arsenic resistance genes is derived from promoter P1 of pBR322 (23), while in pSDRA21, it is derived from the promoter cloned from the chromosome of T. ferrooxidans Tf-52 (6). To introduce the two arsenic-resistant plasmids into T. ferrooxidans strains, crosses between ED8654(RP4, pSDRAI) or ED8654(RP4, pSDRA21) and Tf-50 or Tf-52 were conducted by filter mating as described above. The donor strains ED8654(RP4, pSDRA1) and ED8654(RP4, pSDRA21) were obtained by mating between C600(RP4) and ED8654 (pSDRA1) or ED8654(pSDRA21) and subsequently selecting on Luria agar containing ampicillin (50 Lg/ml) and streptomycin (50 [Lg/ml). Transfer frequencies of pSDRAI and pSDRA21 to Tf-50 and Tf-52 were at the level of 10-5 (Table 2). The stability of vectors in T. ferrooxidans strains was determined by checking for kanamycin resistance as described above. More than 80% of T. ferrooxidans cells carried the recombinant plasmids after being cultured for 50 generations without selective pressure (Table 2). After 50 generations, 36 kanamycin-resistant colonies of Tf-50(pSDRA1) were cultured in 9K medium to fully grown cultures and then the cultures were diluted 10 times and streaked on solid medium, which was described by Mishra et al. (14), containing 15 mM NaAsO2. All kanamycin-resistant clones grew on the medium in a week, while the original strain did not. The expression of arsenic resistance genes in T. ferrooxidans was investigated by examining the arsenic-resistant abilities of the transconjugants as well as of the original strains. Figure 3 shows the growth of T. ferrooxidans transconjugants and the original strains in 9K liquid medium in the presence of serial concentrations of NaAsO2. Strains were cultured at rest at 30°C for 4 days, with a 5% (vol/vol) inoculum. The cell number data were obtained by direct microscopic count. It is shown that the T ferrooxidans strains containing pSDRA1 or pSDRA21 were more resistant to NaAsO, than the original strains (Fig. 3), and the plasmid-mediated arsenic resistance in T. ferrooxidans (Fig. 3) was similar to that in E. coli (Fig. 2). However, because the intrinsic arsenic-resistant ability of T ferrooxidans was much greater than that of E. coli, the arsenicresistant ability of T. ferrooxidans carrying pSDRA1 or pSDRA21 was also greater than that of E. coli carrying the plasmids. As shown in Fig. 3, the intrinsic arsenic-resistant ability of Tf-50 is less than that of Tf-52, but Tf-50(pSDRA1) was more resistant to arsenic than Tf-52(pSDRA1). A similar
0
2
4
6
8 10 12 14 16 18 20
NaAsO 2 concentration (mM) FIG. 3. Growth of the T. ferrooxidlans transconjugants and the original strains in serial concentrations of NaAsO2. Values are the means of three independent experiments. Symbols: A, Tf-50); A, Tf-52; O, Tf-50(pSDRA21); *. Tf-52(pSDRA21); 0. Tf-50(pSDRA1); *, Tf-52(pSDRAl).
result was obtained by measuring arsenic-resistant abilities of strains on solid medium, which was described by Mishra et al. (14), containing NaAsO2. The original strains Tf-50 and Tf-52 could not grow on solid medium when the NaAsO2 concentrations were higher than 6 and 10 mM, respectively; when carrying pSDRA1, Tf-50 was resistant to up to 26 mM NaAsO2 on solid medium, while Tf-52 was resistant up to only 18 mM. The question of why there is a difference between the two strains in the increase in arsenic resistance is still unsolved. Figure 4 shows the growth rates of T. ferrooxidans transconjugants and the original strains in the presence of 10 mM NaAsO,. The strains were cultured in 9K liquid medium with vigorous shaking (160 rpm), with a 2.5% inoculum. Cells were counted by direct microscopic count. In this experiment, the difference in levels of arsenic resistance between the T. ferrooxidans transconjugants and the original strains resulted in the different lengths of their lag periods. As shown in Fig. 3 and 4, the promoter cloned from the chromosome of Tf-52 showed
8.5-4-
8.0-
7.00
6.5 6.0
0
-, 1 2 3 4 5 6
7
8
9 10
Time (days) FIG. 4. Growth of T fetrrooxidanis transconjugants and the original strains in the presence of 1) mM NaAsO.. Values are the means of three independent experiments. Symbols: A, Tf-50; A, Tf-52; D, Tf-50(pSDRA21); M, Tf-52(pSDRA2l); 0, Tf-50(pSDRAI); 0, Tf52(pSDRA1).
2656
APPL. ENVIRON. MICROBIOL.
NOTES
no special function in its original host. This was an unexpected result. As described above, the arsenic resistance genes originated from a heterotrophic bacterium were expressed in the obligately autotrophic T. ferrooxidans. The promoter cloned from T. ferrooxidans was equally inefficient in T. ferrooxidans and in E. coli. This indicates there might be no great difference in the gene expression systems of autotrophic T. ferrooxidans and heterotrophic E. coli. Since the biological leaching of arsenopyrite-pyrite ores has been limited by the sensitivity of the organisms involved to the arsenic which is released in the process, the arsenic-resistant strains constructed would be useful in the recovery of metals such as gold from arsenopyrite-pyrite ores. The methods presented in this paper have implications for the genetic improvement of the biomining bacterium. We thank Guangyong Ji and John Davison for providing plasmids. Gratitude is also expressed to Jo Quam and Paul Quam for their kind editorial assistance. This work was supported by the National Natural Science Foundation of the People's Republic of China. REFERENCES 1. Barros, M. E. C., D. E. Rawlings, and D. R. Woods. 1985. Cloning and expression of the Thiobacillus ferrooxidans glutamine synthetase gene in Escherichia coli. J. Bacteriol. 164:1386-1389. 2. Barros, M. E. C., D. E. Rawlings, and D. R. Woods. 1985. Production and regeneration of Thiobacillus ferrooxidans spheroplasts. Appl. Environ. Microbiol. 50:721-723. 3. Brierley, C. L. 1978. Bacterial leaching. Crit. Rev. Microbiol. 6:207-262. 4. Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, and M. H. Richmond. 1971. Properties of an R factor from Pseudomonas aeruginosa. J. Bacteriol. 108:1244-1249. 5. Davison, J., M. Heusterspreute, N. Chevalier, V. Ha-Thi, and F. Brunel. 1987. Vectors with restriction site banks V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites. Gene 51:275-280. 6. Han, T., and W.-M. Yan. 1993. The cloning and restriction analysis of a promoter of Thiobacillus ferrooxidans. Shandong Daxue Xuebao Ziran Kexue Ban 28:474-481. 7. Hoffman, L. E., and J. L. Hendrix. 1976. Inhibition of Thiobacillus ferrooxidans by soluble silver. Biotechnol. Bioeng. 18:1161-1165. 8. Imai, K., T. Sugio, T. Tsuchida, and T. Tano. 1975. Effect of heavy metal ions on the growth and iron-oxidizing activity of Thiobacillus ferrooxidans. Agric. Biol. Chem. 39:1349-1354. 9. Kusano, T., K. Sugawara, C. Inoue, and N. Suzuki. 1991. Molecular cloning and expression of Thiobacillus ferrooxidans chromosomal ribulose biphosphate carboxylase genes in Escherichia coli. Curr. Microbiol. 22:35-41. 10. Kusano, T., K. Sugawara, C. Inoue, T. Takeshima, M. Numata, and T. Shiratori. 1992. Electrotransformation of Thiobacillus
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