The industrial application of thermophilic (eu)bacteria is hampered by the lack of genetic systems for these bacteria. We report here the first unequivocal transformation of a Gram-positive, thermophilic, anaerobic microorganism, Thermoanaerobacterium, with the kanamycin resistance-mediating plasmid pIKM1. The construct pIKM1 is based on the Escherichia coli–Clostridium acetobutylicum shuttle vector pIMP1 and contains the thermostable kanamycin cassette from S. faecalis plasmid pKD102. Using electrotransformation, plasmid pIKM1 mediated kanamycin resistance in Thermoanaerobacterium sp. strain JW/SL-YS485 up to 400 μg ml−1 at 48°C and 200 μg ml−1 at 60°C.
Several thermophilic anaerobic bacteria of the Gram-positive, low G+C subphylum Bacillus–Clostridium, including Thermoanaerobacter ethanolicus and Thermoanaerobacterium species are regarded as microorganisms with a high potential in industrial applications. The development of economically feasible industrial processes, however, is hampered by the lack of any developed genetic system for these organisms. To our knowledge no genetic system has been described for any Gram-positive thermophilic anaerobe. Little progress has been made in developing shuttle vectors for these potentially important organisms since the first reported equivocal evidence of transforming Clostridium thermohydrosulfuricum (renamed Thermoanaerobacter thermohydrosulfuricus) with the staphylococcal plasmid pUB110 and its derivative pGS13 . Even though Soutschek-Bauer et al. had evidence for increased kanamycin resistance upon PEG mediated transformation, plasmid DNA from their transformants did not yield distinct bands. Although in the last few years our understanding of the gene structure and codon usage in species of Thermoanaerobacter and Thermoanaerobacterium has been broadened by sequencing several genes cloned into Escherichia coli, to date, no cloned genes have been transformed and expressed in any of the thermophilic anaerobes. We failed to isolate an endogenous plasmid from thermophilic anaerobes despite reports of their existence . Since no convenient shuttle vector based on an endogenous plasmid was available for any of these organisms, we constructed a shuttle vector pIKM1 for this purpose. Plasmid pIKM1 (Fig. 1) contains the E. coli origin ColEH1, the Gram-positive origin of replication ORF2 and confers resistance to ampicillin, MLS (Macrolide, Lincoseamide, Streptogramin) and kanamycin. The plasmid was constructed by insertion of the thermostable kanamycin resistance cassette originating from S. faecalis plasmid pKD102  into the multiple cloning site of the E. coli–C. acetobutylicum shuttle vector pIMP1 . We report here the first unequivocal transformation of a Gram-positive anaerobic thermophile using the constructed plasmid pIKM1 containing the Gram-positive replicon from the Bacillus subtilis plasmid pIM13.
2Materials and methods
E. coli TG1 (supE, hsdΔ5, thi, Δ(lac-proAB), [F′,traD36, pro AB, lacIq ZΔM15]) was grown in LB media and selective media was supplemented with either ampicillin (100 μg ml−1) or kanamycin (50 μg ml−1). Thermoanaerobacterium sp. strain JW/SL-YS485 was grown either in prereduced mineral media  or in the same media but supplemented with 50–400 μg ml−1 kanamycin (selective media).
Plasmid pIKM1 was constructed as follows: using standard subcloning procedures , plasmid pKD102 was digested with EcoRI/PstI and the 1.5 kb band containing the kanamycin resistance cassette was isolated from an 1% agarose gel after electrophoresis. The DNA was subsequently ligated into EcoRI/PstI digested pIMP1 (4.8 kb) yielding plasmid pIKM1 (6.3 kb).
Cells were grown in four Hungate tubes with 10 ml of prereduced mineral medium at 60°C. Isonicotinic acid hydrazide (niacin) was added to cultures in early exponential phase at a final concentration of 4 μg ml−1 to weaken the cell wall . After the addition of niacin, cultures were allowed to grow for an additional two to four doubling times until they reached an OD of 0.6–0.8. Cells were harvested via centrifugation in closed Hungate tubes (3500×g; IEC centra-8 centrifuge) at room temperature and washed in sterile prereduced (Nitrogen flushed, 1 μmol ml−1 Na2S added) water. After the second centrifugation step, cells were resuspended into electroporation buffer (EP-buffer: 270 mM sucrose, 5 mM H2NaPO4) and incubated at 48°C until the beginning of autoplast (spheroplast) formation was observed using light microscopy. Cells were centrifuged in closed Hungate tubes and resuspended in 0.2 ml of either N2 flushed water or EP-buffer. Transformation was done via electroporation using a Biorad gene pulser. Resuspended cells (0.1 ml) were transferred within 10 min into prechilled (4°C) 0.1 cm electroporation cuvettes containing up to 5 μg of plasmid DNA. After a single electroporation pulse (1.25 kV, 400 Ω, 25 mF) with a time constant of 4–8 ms the mixture was immediately transferred into Hungate tubes with 5 ml of prereduced mineral Medium . The cells were allowed to recover at 48°C for 4 h. Dilutions of the electroporation mixture were then suspended into selective media containing 50 μg ml−1 kanamycin and aliquots were spread onto plates containing agar (1% w/v) solidified selective medium. Plates for controls of growth did not contain the antibiotic. The liquid cultures were incubated for 2 days at 48°C in a stationary incubator. The plates were incubated under the same conditions in anaerobic jars containing oxygen-free nitrogen as gas atmosphere.
Single colonies of transformed Thermoanaerobacterium sp. strain JW/SL-YS485 were picked and transferred into 10 ml of selective medium (100 μg ml−1 kanamycin). After overnight growth 2 ml was transferred into serum bottles containing 100 ml of the selective medium (75 μg ml−1 kanamycin). Cells were harvested in 1.5 ml Eppendorf tubes and plasmid DNA was extracted via the modified isolation procedure of O'Sullivan and Klaenhammer . Ethidium bromide in 7.5 M ammonium hydroxide was included in the phenol precipitation step in order to improve the separation of protein from plasmid DNA. Plasmid DNA from 10 preparations was concentrated in a final volume of 50 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA).
3Results and discussion
Plasmid pIKM1 (Fig. 1) was constructed as described in Section 2 and was used to transform E. coli TG-1. Four clones were picked and resuspended in LB containing 50 μg ml−1 kanamycin. Restriction analysis from plasmid DNA extracted from these cultures verified the correct plasmid construct (Fig. 2, lanes 3 and 4). Thermoanaerobacterium sp. strain JW/SL-YS485 is resistant to 100 μg ml−1 ampicillin, 50 μg ml−1 chloramphenicol, but sensitive to kanamycin with a MIC of 25 μg ml−1. To transform Thermoanaerobacterium, the cells were converted to autoplasts as previously described  and prepared for electroporation as described in Section 2 (Fig. 3). In an attempt to optimize electroporation conditions individual parameters, including cell density (OD: 0.3–0.9), resistance, voltage (0.8–1.5 kV) and time constant (4–8) (200–400 Ω) were changed over a wide range; however, no consistent or significant increase in the transformation efficiency, which varied between 10 and 1000 transformants per μg plasmid DNA, was observed. Restriction analysis of plasmid DNA isolated from transformed Thermoanaerobacterium sp. according to O'Sullivan and Klaenhammer  yielded the characteristic banding pattern for plasmid pIKM1 (Fig. 2, lanes 7 and 8). To further demonstrate the transformation of Thermoanaerobacterium, plasmid DNA isolated from transformed Thermoanaerobacterium cells was used to retransform E. coli TG-1. Again, the restriction analysis of plasmid DNA isolated from retransformed E. coli TG-1 unequivocally showed the presence of the plasmid construct pIKM1 (Fig. 2, lanes 5 and 6).
Although the plasmids pIMP1 and pKD102 originated from mesophiles, the expression of the kanamycin resistance cassette during growth at 60°C (close to the maximal temperature 65°C for strain JW/SL-YS 485) demonstrated that the construct pIKM1 is stable in, and compatible with, a thermophilic host. The growth patterns for the wild-type and transformed Thermoanaerobacterium strain JW/SL-YS485 at different temperatures (48°C and 60°C) were similiar and only small differences were observed in the doubling times (Table 1). The plasmid was relatively stable as demonstrated by comparing the number of colony forming units (CFU) on selective (50 μg ml−1 kanamycin) and nonselective plates (absence of antibiotic). At least 20% of the transformed strain had retained the plasmid during growth in the absence of the selective pressure, i.e. after growth for at least 50 generations in the absence of kanamycin. The fact that pIKM1 confers kanamycin resistance and is stably replicated in Thermoanaerobacterium strain JW/SL-YS485, while retaining its capacity to transform E. coli, demonstrates its usefulness in developing a reliable system for transformation of foreign genes into thermophilic anaerobes. Future modification of pIKM1 including the incorporation of a strong promoter, ribosome binding region and a multicloning site should enhance its utility for the expression of cloned genes in Thermoanaerobacterium or closely related bacteria. It will also allow for augmentation of expression of endogenous activities which may be deficient.
Table 1. Growth of Thermoanaerobacterium wild-type JW/SL-YS485 and transformant YS485 pIKM1 in the presence and absence of kanamycin
Doubling times (min) in mineral medium+kanamycin (μg ml−1)
Average of three measurements with values within a variation of ±5%.
YS 485 (control
YS 485 (control)
YS 485 pIKM1
YS 485 pIKM1
We thank E.T. Papoutsakis for providing shuttle vector pIMP1. This study was supported by a grant to J.W. from the US Department of Energy (DE-FG05-95-ER-20199).