Improved methods for handling Thermotoga cultures in an aerobic environment
The chance of obtaining Thermotoga
transformants on plates can be seriously compromised if plating efficiencies are low. Considering that Thermotoga
can tolerate brief exposures to oxygen, we simplified the overlay methods used by other groups (Kenneth Noll, University of Connecticut, private communication; [16
]) and developed an embedded growth method. Properly diluted liquid cultures were suspended in hot SVO containing 0.3% Gelrite, and the mixtures were allowed to solidify in Petri dishes. In this method, cells were embedded in the medium matrix, and their exposure to oxygen was reduced. Ten microliters of an overnight culture of T
. sp. RQ7 with a dilution factor of 104
formed 1256 colonies (Figure ), which equals to 1.26 × 109
colony forming units (CFU) per ml. By contrast, a surface culture, prepared by standard spreading in the same environment, would typically generate 7.56 × 103
(Figure ), about ten thousand times less. Given that T
cultures contain approximate 3.0 × 109
cells after growing in liquid SVO for 14 h at 70°C [17
], we estimate that the plating efficiency of our embedded method is close to 50%. This high efficiency enables us to select or screen a large number of single colonies while still enjoying the convenience of aerobic handling.
Figure 2 Single colonies formed by T. sp. RQ7 cells. (a) Embedded growth. Cells were mixed with hot SVO medium containing 0.3% Gelrite and were poured to Petri dishes before solidification. (b) Surface growth. Cells were spread evenly on the surface of freshly-made (more ...)
To facilitate the transfer of single colonies from solid to liquid media under aerobic conditions, we introduced a soft SVO medium by adding 0.075% Gelrite to liquid SVO. Gelrite prevents atmospheric oxygen from penetrating deep into the medium. To transfer cultures from solid to soft SVO, single colonies were picked up from plates by a loop and were pushed down to the bottoms of the test tubes containing soft SVO, where a local anaerobic environment has been created. After 12-24 h of incubation, cultures grown in soft SVO were then transferred to liquid SVO by a syringe. Although the introduction of soft SVO seemed to prolong the overall operation cycle, it ensured maximum viability of Thermotoga cells during the transfer, which eventually allowed us to isolate Thermotoga transformants for the first time (see below). Soft SVO may also serve as an excellent storage medium for Thermotoga. Cultures kept at the bench top for 2 months were still vital and exhibited no growth defects.
Kanamycin is a suitable selection marker for T. sp. RQ7 and T. maritima
To determine whether kanamycin is a suitable selection marker for Thermotoga
, one needs to know the sensitivity of Thermotoga
host strains. An initial study indicates that T. maritima
is sensitive to kanamycin [9
], but a more recent work states that it is highly resistant to the antibiotic [16
]. For T
. sp. RQ7, there are simply no related reports. We decided to clarify the discrepancy of the previous findings on T. maritima
and to determine the sensitivity of T
. sp. RQ7. Small discs of filter paper loaded with various amount of kanamycin were mounted on top of the SVO plates premixed with Thermotoga
cultures (Figure ). After two days of incubation, cells not affected by the antibiotic grew into a dense lawn, forming a visible background. Sensitive cells in close proximity to the paper discs were unable to grow, resulting in clear halos. The inhibition zones formed on T. maritima
plates (Figure ) revealed that this strain is indeed sensitive to kanamycin. A distinctive zone was visible even with the lowest concentration of kanamycin. T
. sp. RQ7 displayed a similar level of sensitivity to the drug. As for T
, slight inhibition was noticed when 100 μg of kanamycin was used, and a small inhibition zone was only apparent when 250 μg of the drug was used. Therefore, kanamycin may serve as a good selection marker for T
. sp. RQ7 and T. maritima
, but not for T
Figure 3 Sensitivity of Thermotoga spp. to kanamycin. (a) Sensitive cells formed inhibition zones surrounding the paper discs loaded with various amounts of the antibiotic, as indicated in the top left panel. Gas bubbles produced by the Thermotoga cells were clearly (more ...)
We next specified the selective levels of kanamycin in both liquid and solid media. The growth of T. maritima in liquid SVO was completely inhibited by 50 μg ml-1 kanamycin for at least 72 h (Figure ). However, spontaneous mutations sometimes caused the cultures to become resistant to the antibiotic, and a complete inhibition over a period of 72 h was only possible when the input amount was increased to 150 μg ml-1. Similar phenomena were also noticed with T. sp. RQ7. On SVO plates, spontaneous mutants of T. maritima and T. sp. RQ7 occasionally appeared after 48 h of incubation when up to 200 μg ml-1 kanamycin was added, but they rarely appeared when the antibiotic concentration was increased to 250 μg ml-1. Based on these observations, for the rest of the study, kanamycin was added at 150 μg ml-1 to liquid media and at 250 μg ml-1 to soft and solid media.
Transformation of Thermotoga-E. coli shuttle vector pDH10
Because most bacteria become competent after a short electric pulse, electroporation was attempted to introduce pDH10 to Thermotoga. Electric pulses of various strengths were applied to both T. sp. RQ7 and T. maritima in the presence of 4 μg of plasmid DNA, and the transformants were selected with embedded growth. When an electric pulse of 2.0 kV was employed, five T. sp. RQ7 and one T. maritima transformants were obtained. A pulse of 1.8 kV resulted in eight T. sp. RQ7 and no T. maritima transformants. When the voltage dropped to 1.5 kV, no transformants were available with either species. These results suggest that the optimal voltage for Thermotoga is around 1.8 to 2.0 kV. In the control experiment, T. sp. RQ7 and T. maritima cells were treated with a pulse of 1.8 kV in the absence of DNA, and no spontaneous mutants were found.
All transformants (designated as RQ7/pDH10 or Tm/pDH10 hereafter) displayed visible growth after a 24 h incubation in soft SVO. Three RQ7/pDH10 strains (#5, #6, and #13) and the single Tm/pDH10 strain were propagated in liquid SVO for extraction of plasmid and genomic DNA. On agarose gels, no pDH10 DNA could be detected from the plasmid extract of any strain, even though pRQ7 was clearly visible from the three RQ7/pDH10 samples, indicating that the extraction procedure was successful. Plasmid and genomic DNA extracted from an equal amount of each transformant culture was then subject to PCR analysis. A fragment of 778 bp, corresponding to the size of the kan gene, was obtained from each plasmid extract (lanes are labeled in bold in Figure ) but was missing from the genomic DNA of RQ7/pDH10 #5 and #6. The PCR products were gel-purified and were subject to restriction digestion with AgeI, which is expected to cleave the kan gene into two fragments of 208 and 570 bp. Indeed, the digestion reactions released these expected fragments from every sample (Figure ), indicating that the PCR products were authentic kan genes.
Figure 4 Detection of the transformed kan gene. PCR products of the kan gene were obtained from the plasmid extracts (P, labeled in bold) or the genomic DNA preparations (G) of the recombinant strains. Three RQ7/pDH10 and one Tm/pDH10 transformants, all obtained (more ...)
Figure 5 Restriction digestions of the kan gene. PCR products of the kan gene were prepared from the plasmid extracts of DH5a/pDH10 (lanes 1 and 4), RQ7/pDH10 (lanes 2 and 5), and Tm/pDH10 (lanes 3 and 6). Lanes 1-3 represent the PCR products of the kan gene, (more ...)
For validation and comparison purposes, pDH10 was also introduced to T. sp. RQ7 and T. maritima through liposome-mediated transformation. Four T. sp. RQ7 and five T. maritima transformants were obtained from 1 μg of plasmid DNA, as opposed to zero colonies from the samples treated with liposomes containing no DNA. All transformants grew well in both soft and liquid selective media, and the presence of the kan gene was also confirmed by PCR.
Transformed DNA was stably maintained in Thermotoga
To determine the stability of the transformed DNA, liquid cultures of RQ7/pDH10 and Tm/pDH10 were transferred every 12 h, for six consecutive times, to fresh media in the presence or absence of kanamycin. Cultures from each transfer cycle were tested with both SVO (no kanamycin) and SVO+Kan plates. The number of colonies on a plain SVO plate represents the quantity of total viable cells in a sample, whereas the number from a SVO+Kan plate defines the abundance of resistant cells. Surprisingly, by the end of the experiment, ~100% of both RQ7/pDH10 and Tm/pDH10 still had the transformed DNA, even without the selective pressure (Table ). The kan gene was confirmed by PCR from the plasmid preparations of all strains after each transfer.
Percentage of Thermotoga colonies resistant to kanamycin after six transfers*
Incorporation of pRQ7 increased the stability of pUC19 derivatives in E. coli
The stable maintenance of the transformed DNA in Thermotoga motivated us to test the stability of pDH10 in E. coli under non-selective conditions. The parent vector pKT1, a derivative of pUC19, was used as the control. Interestingly, pDH10 was much more stable in E. coli than pKT1 was. The parent vector pKT1 was eliminated by ~90% of the host cells after a single transfer and was completely lost from the population after three transfers (Table ). By contrast, pDH10 was eliminated at a much slower rate. It was carried by ~90% of the cells after three transfers and ~32% of the cells after six transfers. Intact pDH10 was obtained from the resistant colonies. It is noteworthy to mention that similar results were also obtained when pDH1 and pUC19 were compared in the same way. These data demonstrate that the insertion of the pRQ7 sequence somehow enhances the stability of pUC family vectors. We next compared the copy numbers of pDH10 and pKT1 in their E. coli hosts. The two vectors were prepared from the same amount of cells and were digested by XbaI and EcoRI. These double digestions released the pRQ7 sequence from pDH10 (Figure &). The abundances of the shared vector backbone of pDH10 and pKT1 were comparable on an agarose gel (indicated by the arrow in Figure ), suggesting that the copy numbers of two vectors were similar. Therefore, the dramatically improved stability of pDH10 is not caused by an increase in copy number.
Percentage of E. coli colonies resistant to ampicillin during consecutive transfers*
Figure 6 Comparison of the copy numbers of pDH10 and pKT1 in E. coli. Plasmid DNA was extracted from the same amount of recombinant cells and was digested by XbaI and EcoRI. The arrow indicates the shared sequence of the two vectors. Analyzed with a 0.8% agarose (more ...)