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Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast
Carole Lartigue, Sanjay Vashee, Mikkel A. Algire, Ray-Yuan Chuang, Gwynedd a. Benders, Li Ma, Vladimir N. Noskov, Evgeniya A. Denisova, Daniel G. Gibson, Nacyra Assad-Garcia, Nina Alperovich, David W. Thomas, Chuck Merryman, Clyde A. Hutchison III, Hamilton O. Smith, J. Craig Venter, John I. Glass
Science, Vol. 325: 1693-6. 25 September 2009. (Link here)
Summary
In this paper, the authors describe a new method in which a genome from a bacteria strain (Mycoplasma mycoides) is transformed into yeast, modified, and re-inserted back into a bacterial strain (Mycoplasma capricolum); thus creating a new modified form of the original M. mycoides genome. This unique method allows the authors to modify the original genome in ways that would not be possible otherwise by involving the multitude of genetic tools available through yeast biology.
Before tackling the data presented in this paper, let us first take a closer look at some essential background information that must be understood in order to analyze the presented information. First the genome from M. mycoides was transformed with a vector containing several specific features. It contained: a tetracycline-resistance marker, a β-galactosidase gene, a yeast auxotrophic marker, a yeast centromere, and a yeast autonomously replicating sequence. These factors were important because they allowed for the transformation into yeast as a yeast centromeric plasmid (YCp).1 Additionally, the authors note that M. capricolum cells were used instead of previously used M. genitalium cells as the recipient of the altered yeast product because of their faster growth rate.1 Finally, two stains of yeast spheroplats, VL6-48N & W303a, were used and verified for completeness of transfer by PCR and CHEF gel electrophoresis.
Figure 1: Generation of Type III Restriction Enzyme Deletions
A) This panel illustrates the modifications applied to the M. myocides genome. In part i), the original genome (YCpMmyc1.1) is shown containing a nonessential Type III restriction endonuclease gene which was replaced with a linear knockout cassette containing two PCR products, CORE and tandem repeat sequence. This knockout cassette, containing a URA3 marker and SCEI endonuclease gene under the control of the Gal1 promoter, was incorporated into theYCpMmyc1.1 M. mycoides genome through transformation into yeast strain W303a by growth on (-)His (-)Ura plates. This new strain (YCpMmyc1.1-ΔtypeIII::URA3, ii), replaces the typeIIIres gene with the knockout cassette through homologous recombination of the 50bp sequence to the target sites. Finally with galactose induction and counter selection with 5-flouroorotic acid, the SceI endonuclease cleaves the segment creating a double-strand break which is followed by homologous recombination between two tandem repeat sequences creating a seamless deletion of the typeIIIres gene (iii). Thus two new M. mycoides YCp genomes were created, one with the URA3 cassette and the other with a seamless deletion of the typeIIIres gene.
B) This panel shows an agarose gel of the PCR products from panel A (i, ii, iii). PCR Primers P299 and P302, seen above the sequence in part A, i, were used to determine the presence or absence of the knockout cassette. As seen on the gel by differences in molecular weight, the changes to the genome in both yeast and post-transplant M. capricolum cells (lanes 2-7) showed successful alteration of the original genome in addition to sustained stability within the organism.
Table 1: Transplantation of M. mycoides YCp genomes from yeast into wild-type and RE(-) M. capricolum recipient cells
Here the number of tetracycline-resistant colonies obtained after transplantation of M. mycoides YCp genomes from yeast to M. capricolum recipient cells were noted as the average of at least three experiments. Initially, the original genome YCpMmyc1.1 was transplanted into wild-type M. capricolum cells with no success. To solve this, the authors deduced that a restriction endonuclease in the recipient cells degraded the unmethylated donor DNA, thus preventing successful transplantation. To solve this problem, the authors developed two methods: inactivation of the single restriction enzyme in M. capricolum by integrating a marker into the coding region of the gene [M. capricolum RE(-)], and by protecting the donor DNA by in vitro methylation. Both methods proved effective resulting in the conclusion that, "avoidance of the M. capricolum recipient restriction system is vital for successful transplantation of M. mycoides YCp genomes from yeast".1
Looking at the table, the data is separated in two groups depending on the strain of yeast. Both groups are adequately described in both the figure legend and supporting text (pg. 1694). For the first group (yeast strain VL6-48N), only the original genome YCpMmyc1.1 was used. Here various sources of treatment for the second methylation method was used in addition to testing in both M. capricolum RE(-) cells and wild-type cells. No colonies formed in the untreated wild-type cells or the mock-methylated wild-type cells for this strain indicating that methylation was indeed that cause of unsuccessful transplantation. For the second group (yeast strain W303a) the different YCp M. mycoides genomes were tested to show successful transplantation into M. capricolum RE (-) recipient cells. All three forms (i, ii, iii) proved successful. Also, the isolated YCpMmyc1.1-Δ500kb genome was used as a control because it maintains the YCp element and tetM yet lacks several presumed essential genes necessary for growth. As predicted, no colonies were formed.
Figure 2: Southern blot analysis of M. mycoides transplants
A) This panel verifies the recovered colonies as being M. mycoides by using a M. mycoides - specific IS1296 element as a probe.1 The genomic DNA of native wild-type M. capricolum recipient cells (lane 1) and native M. mycoides YCpMmyc1.1 cells (lane 2) were used as negative and positive controls respectively. Then transplanted genomic DNA from M. mycoides YCpMmyc1.1, YCpMmyc1.1-ΔtypeIII::URA3, and YCpMmyc1.1-ΔtypeIIIres (lanes 3-5 respectively) were probed and found to have the same molecular weight bands as the positive control, indicating that all transplanted forms were in fact M. mycoides.
B) This panel verifies the removal of typeIIIres gene from the modified M. mycoides transplants. All lanes were probed with the typeIIIres gene sequence for this Southern blot. Again genomic DNA from native wild-type M. capricolum recipient cells (lane 1) and native M. mycoides YCpMmyc1.1 cells (lane 2) were used as negative and positive controls respectively. This time, as expected, the typeIIIres gene is absent in the M. mycoides ΔtypeIII::URA3 and ΔtypeIIIres genomes while it is present in the native M. mycoides YCpMmyc1.1 genome. This supports data from Fig. 1B, signifying the deletion of the Type III restriction gene from the modified genomes.
C) Finally this panel verifies the removal of the typeIIIres gene by sequencing the YCpMmyc1.1-ΔtypeIIIres M. mycoides transplant genome. The deleted region is shown with an overlap between the typeIIImod and typeIIIres genes shown in blue. While data is not shown, the authors sequenced the entire genome to ensure no mistakes were made. This panel serves as the final step in validating that their modified constructs (i, ii, and iii, from Fig 1A) were indeed successful.
Figure 3: Moving a bacterial genome into yeast, engineering it, and installing it back into a bacterium by genome transplantation.1
In this figure the authors summarize the method described in this paper. First a yeast vector is transformed into a bacterial genome, it is then isolated and put into yeast. Here the multitude of genetic methods for yeast can be used to modify the bacterial genome. This newly modified genome is then methylated (if necessary) and transplanted back into a recipient cell - creating a new engineered bacterium. At this point the cycle is complete, however a new modification can be started from this stage, in which the same steps would follow.
Critique:
The goal of this paper sought to describe a new method that would allow for the construction of a bacterium by using the genetic tools for yeast to produce a new strain of bacterium that could not be created otherwise. While I feel that the data and figures presented support and indeed reach this goal, there are still some areas in which the overall strength of the paper could be improved. First in Figure 1B, a negative control would be greatly helpful. As it stands now, without a negative control, the primers cannot be validated as specific probes for the YCpMmyc1.1 genome. By adding this, the authors could then clearly state that engineered genomes are in fact specific to the PCR products. Secondly, Table 1 also lacks the use of controls to clarify that indeed methylation was the principal cause. While a negative control is present for the W303a strain, it is lacking for the VL6-48N strain. If this were added does colony growth again not grow? Additionally, by testing the W303a strain with the methylation treatments in the wild-type M. capricolum cells, do they achieve the same results? This could greatly improve their argument because in Figure 3, the authors claim that methylation of the donor DNA is all that is necessary to protect it, while in Table 1 they have no evidence supporting this for their engineered genomes, only for the original native YCpMmyc1.1 genome. Finally, a major portion of the paper hinges on the resolution of the host DNA. While Lartigue et al. addresses this issue in the text of the paper, there is no evidence or method describing how exactly this process was carried out. This weakens the authors’ argument because there is no assurance that the entire host DNA was eliminated from the bacterium before transplantation.
Future Experiments:
This paper presents a new unique method to engineer "a bacterial cell by altering its genome outside of its native cellular environment".1 Using this proposed method, several new experiments could be preformed to either confirm the claim here or further knowledge about this unique method. One example would be to attempt transplanting the modified bacterial genome clone in yeast back into the original cell line. By modifying and creating a new strain of say M. mycoides and re-transplanting this back into M. mycoides, further knowledge would be gained about proposed pathogenicity and biology of mycoplasmas. To do this, the method described here would be followed. This experiment could prove to be useful in gene therapy as well as helping to better understand how mycoplasmas cause diseases like ruminants.1 Another example as hinted by Lartigue et al. would be to try this process in other species. Does it hold true for other species of bacterium? Yeast? To test this, other species like Escherichia coli could be implemented to see if they too follow this method. In this case, genomic DNA would be isolated from E. coli and transformed into yeast. If successful this would prove to be very effective because like yeast, E. coli is a very well known model system. Finally, one large unanswered question is whether or not methylation is the sole cause of ineffective transplantation? This question, coming from Table 1, points to effectiveness of methylation treatments for wild-type M. capricolum compared to M. capricolum RE (-) recipient cells on colony growth. Looking at the data, there is a large difference in number of colonies/plugs between these two methods. Why is this? This disparity suggests that other factors could be involved that causes for more effective transplantation. To test this, one way would be to perform either RT-PCR or an RNA microarray on the colonies tested in Table 1 (in addition to the new factors described in the section above), in order to see how gene expression changes depending on treatment type. Ideally, expression should be relatively equal no matter which methylation method was used. However if different levels of expression resulted, this would allow the method proposed here by Lartigue et al. to be improved. While, it is still unclear as to the usefulness of this new method, its cleaver design will surely lead to many unanswered questions.
References:
1) Lartigue, C, Vashee, S, Algire, MA, Chuang, RY, Benders, GA, Ma, L, Noskov, VN, Denisova, EA, Gibson, DG, Assad-Garcia, N, Alperovich, N, Thomas, DW, Merryman, C, Hutchison, CA 3rd, Smith, HO, Venter, JC, Glass, JI. 2009 September. Creating bacterial strains from genomes that have been cloned and engineered in yeast. Science; 325 (5948): 1693-1696. PubMed.
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