Strategies for cloning bacterial genomes in yeast
Large DNA molecules have been stably cloned in yeast by the addition of a yeast centromere (CEN
), which allows the molecules to be segregated along with the yeast chromosomes. Such molecules have been cloned both in the linear form (35
), by the addition of telomeres to the ends, and also as circles (37
). Since bacterial genomes are generally circular, it is easier to clone them in this form. This is also an advantage because circles can be readily separated from linear yeast chromosomes (16
We have taken three different approaches to the cloning of bacterial genomes in yeast (). The first approach is to insert a yeast vector into the bacterial genome prior to transformation of yeast (18
Figure 1. Three methods for cloning mycoplasma genomes in yeast. In order to be propagated by yeast (tan, with yellow nucleus) upon transformation, the bacterial genome (blue circle) must contain several yeast sequences (vector, red bar). (A) These can be incorporated (more ...)
In the second approach, the bacterial genome and a yeast vector are cotransformed into yeast (B). The linear vector contains terminal regions of homology to a site in the genome and is inserted by homologous recombination. Vector insertion is much more efficient if the genome contains a double-stranded break near the insertion point (41
). The second approach can also be done with multiple overlapping fragments (C) (16
The approach of A selects for vector insertion sites that do not interfere with bacterial viability. This method is favored for a genome that will be transplanted to produce a living cell. Another advantage is that vector insertion may not require knowledge of the genome’s sequence. However, this strategy requires that the bacterium be transformable so that the vector can be integrated into the genome. The approach of B does not require transformation or even cultivation of the organism, just intact genomic DNA. This method is favored for genetically intractable or uncultured organisms. These may include pathogens and environmental isolates. However, this strategy does not guarantee fitness or viability of the cloned genome. In addition, it may be difficult to find a suitable restriction site for vector insertion, and vector insertion may require some sequence knowledge. The approach of C is useful for assembling pieces of DNA, both natural and synthetic. The vector can be included as a separate fragment, or it can be included as a sequence within a larger fragment. In this way genomes can be constructed to study gene function and to create microbial cell factories.
We cloned the M. genitalium genome by all of the above approaches. We cloned the M. mycoides subspecies capri and the M. pneumoniae genomes separately in yeast, and the M. genitalium and M. mycoides genomes as two separate molecules in the same yeast cell, by the strategy depicted in A.
Construction of tri-shuttle vectors for cloning of mycoplasma genomes in yeast
To clone mycoplasma genomes by the strategy shown in A, we designed and constructed the vectors shown in A and B. These plasmids were intended to insert yeast centromeric vector sequences into a mycoplasma genome by transposition. The transposase was constructed outside of the set of IS256 inverted repeats, so that it would be lost after the initial transposition, preventing the inserted sequence from moving within the mycoplasma genome.
Figure 2. Yeast vector insertions in each of the M. genitalium, M. mycoides and M. pneumoniae genomes, using the approach shown in A. (A and B) The two shuttle vectors used. (C–E) The location of the vector insertion in each genome: mycoplasma markers (more ...)
The essential features of the pmycYACTn vector (Supplementary Table S1
) are: (i) a high copy origin from pUC19 and an ampicillin-resistance marker for propagation in E. coli
, (ii) the IS256 transposase and inverted repeats for transposition into a mycoplasma genome, (iii) tetM
markers, both expressed from spiralin promoters (42
), for selection and screening in E. coli
and mycoplasmas and (iv) an ARS
(autonomously replicating sequence) and a CEN
for replication and segregation in yeast, and HIS3
as a selectable yeast marker. The miniTn-Puro-JCVI-1.7 vector (Supplementary Table S2
) differs from pmycYACTn as follows: (i) it does not contain lacZ
and substitutes a puromycin resistance marker for tetM
and (ii) it contains a bacterial artificial chromosome (BAC) vector, for possible cloning in E. coli
Cloning mycoplasma genomes that contain an integrated yeast vector
strain MS5 (21
) was transformed with the vector pmycYACTn by electroporation (44
), and transformants were selected with tetracycline. A clone (YCpMgen16-2) was chosen for further analysis. Direct genomic sequencing (44
) using primers internal to the vector was performed to determine the site of vector insertion. This clone contained the sequence between and including the two IS256 inverted repeats of pmycYACTn, indicating that transposition had occurred, as designed (C). The transposase, pUC19 origin and ampicillin-resistance gene were lost during transposition. The vector inserted within the nonessential MG411 gene (44
), after the base pair corresponding to 514699 in the M. genitalium
G-37 sequence (GenBank accession L43967). Base pairs 514694–514699 were duplicated at the insertion site, a phenomenon which has been previously noted (44
strain GM12 (22
) was transformed with pmycYACTn using PEG (48
), and transformants were selected with tetracycline. All four clones were analyzed by direct genomic sequencing to locate the insertion site. Rather than the expected transposition, in all four cases we observed integration of the entire plasmid. In three clones, pmycYACTn was inserted by a crossover within or very close to the pUC origin. In the fourth clone the crossover occurred within the yeast HIS3
gene, rendering this genome unsuitable for transformation into yeast. All vector insertions were adjacent to an IS1296 element.
The vector in one of the clones may be able to transpose within the M. mycoides genome, since it is bounded on either side by an IS1296 inverted repeat. Since pmycYACTn did not transpose initially, it probably cannot move within the genome by IS256 transposition. We do not know the mechanism by which pmycYACTn inserted into the M. mycoides genome. The plasmid contains a functional transposon, since we saw evidence of its transposition into the M. genitalium genome. In addition, all four M. mycoides clones containing the vector have the correct sequence of the IS256 tranposase and its two inverted repeats. We have not found any published evidence for the functionality of IS256 in M. mycoides.
Clone YCpMmyc1.1 (D) was selected for cloning into yeast because it grew well and transplanted well into M. capricolum
). We later modified this genome in yeast, sequenced it and deposited the sequence in GenBank (accession CP001668). Base pairs 25040–35183 are pmycYACTn sequence. The insertion of pmycYACTn caused a duplication of several base pairs of the plasmid, and a deletion of several base pairs of M. mycoides
sequence as compared to the sequence of the other M. mycoides
clone that is available (GenBank accession CP001621).
strain M129-B170 (ATCC 29343) was transformed with MiniTn-Puro-JCVI-1.7 by electroporation (44
), and a pool of puromycin-resistant transformants was selected in liquid culture. Selection in liquid culture took several weeks less than plating the transformation mixture and then expanding the resulting individual colonies in liquid medium. When analyzed by restriction digestion, the pool of transformants gave discrete bands whose sizes summed to one M. pneumoniae
genome (M, label ‘Mp’). This indicated a predominant vector insertion site. Direct genomic sequencing with primers internal to the vector was unsuccessful. Thus, DNA from the pool was electroporated into E. coli
. Incubation of transformants with kanamycin selected circularized M. pneumoniae
fragments that contained the vector, which included BAC sequences. Plasmid DNA from one such transformant was isolated and sequenced. It contained insertion of the vector by transposition within MPN028, after the base pair corresponding to 33901 in the M. pneumoniae
M129 sequence (GenBank accession U00089), with a duplication of bp 33893–33901. This gene has been shown to be nonessential in M. genitalium
, but not in M. pneumoniae
. However, the vector insertion occurred near the 3′-end of the gene, which may not be disruptive to gene function (44
). The restriction map produced by vector insertion at this location matched our results. This genome was named YCpMpn028 (E).
Figure 3. Gel analysis of whole mycoplasma genome clones in yeast. The host yeast strain of each set of clones is marked above the gel. Clones were first assayed for completeness by multiplex PCR (B, D, G, I, L). Complete clones are marked with an asterisk, and (more ...)
In order to minimize breakage, genomes with yeast vector insertions from all three mycoplasmas were isolated in plugs using low melting-point agarose. Yeast spheroplasts were transformed with DNA from the plugs by a published method (26
). Cells were suspended in 1 M sorbitol and treated with zymolyase to remove the cell wall. DNA was recovered from the agarose plugs, sometimes in the presence of 6% PEG and 0.6 M NaCl (29
), then incubated with spheroplasts. After recovery in growth medium, cells were plated in selective medium.
All three genomes were transformed into the yeast strain VL6-48N, which has been developed for high transformation efficiency (25
). The YCpMgen16-2 and YCpMmyc1.1 genomes were also transformed into the commonly used strain W303a. In addition, we transformed the YCpMmyc1.1 genome into a recombination-deficient yeast strain. We speculated that this genome might be unstable in yeast, possibly due to recombination among the nearly identical 1.5-kb IS1296 copies. Yeast strains defective in the RAD54
gene have been shown to decrease the occurrence of a variety of recombination events in yeast artificial chromosomes (YACs) (49
). The rad54
mutant strain used (VL6-48-Δ54G) is nearly isogenic with VL6-48N.
Results shown in are summarized in . Clones were screened first for completeness by multiplex PCR. Amplicons were evenly spaced around the M. genitalium and M. pneumoniae genomes (A and K). Since we speculated that the most likely instability of the M. mycoides genome sequence in yeast might be homologous recombination among its copies of IS1296, one or more amplicons was located between each pair of these elements (F). We screened some M. mycoides genome clones with two additional primer sets. One amplicon lay within the HIS3 marker of the yeast vector, and an additional primer set amplified yeast rDNA as a positive control for the assay. Some or all clones that appeared complete by PCR were screened for size by restriction digestion and gel electrophoresis.
For M. genitalium, we examined 24 individual clones in strain VL6-48N. Of these, 22 appeared to be complete by PCR analysis (B). CHEF gel analysis showed two out of three of these to be the expected size (C). With strain W303a, five out of eight clones were complete by PCR analysis (D). Of these, four were of the expected size when examined by CHEF gel (E). For M. mycoides, we examined 48 clones in strain VL6-48N and found 20 to be complete by PCR (G). We obtained essentially the same PCR result when M. mycoides was transformed into the rad54 recombination-deficient strain (data not shown). Of the 20 complete clones in VL6-48N, six were examined by Southern blot of a CHEF gel and five appeared to be the correct size (blot not shown; restriction analysis of selected clones with subclones shown in H). A total of 8 out of 15 clones in strain W303a were complete by PCR (I), and all of these were the correct size by CHEF analysis (J). Transformations into W303a were carried out in the absence of the PEG and NaCl treatment of the plug. The same was true for transformation of strain VL6-48N with a pool of M. pneumoniae genomes containing a yeast vector. A total of 13 out of 20 transformants examined by PCR appeared to be complete (L). CHEF gel analysis showed five out of nine of these to be the expected size (M).
Cloning M. genitalium in yeast by homologous recombination
Vector insertion by homologous recombination (B and C) is much more efficient if the genome contains a double-stranded break near the insertion point. Such a break can be created by restriction digestion. The M. genitalium genome contains three single-cut restriction sites, two of which lie within its rRNA operon, which is essential for viability. The third lies at the 3′-end of a tRNA coding sequence. A vector was inserted by homologous recombination adjacent to this AscI site, since the cloning could be designed to preserve the integrity of the tRNA, which is probably essential.
The yeast cloning vector pARS-VN (30
) was used as template for PCR with a pair of primers, each containing 60 bp homologous to the region flanking the AscI insertion site in the M. genitalium
genome, and 20 bp of homology to the vector. Yeast strain VL6-48N spheroplasts were cotransformed with a mixture of linear vector DNA (140 ng) and M. genitalium
strain MS5 DNA (from about 107
cells). M. genitalium
DNA was isolated in agarose plugs to minimize breakage. Before transformation these plugs were melted and digested with agarase.
We tested the effect of a double-stranded break at the cloning site produced by AscI digestion of the M. genitalium genome (A versus C). To increase the fraction of intact genomes, broken DNA was removed from some of the plugs by CHEF gel electrophoresis.
Figure 4. Targeted insertion of a yeast vector using homologous recombination. A yeast vector insertion was attempted with and without a double-stranded break at the insertion point. (A) Intact genome and linear vector. (B) Overlapping genome fragments and vector (more ...)
AscI-digested DNA yielded 45 transformants. Of these, 21 were positive for all of 20 PCR amplicons tested. These 21 were examined by Southern blot of a CHEF gel and 15 appeared to be the correct size. Undigested M. genitalium
genomes yielded 50 transformants. Of these, seven were positive for all PCR amplicons. One of these was the correct size. This is consistent with findings that a double-stranded break near a site of homologous recombination increases the efficiency of this event by ~20-fold (41
Cloning M. genitalium by assembly of overlapping fragments in yeast
In C we show assembly of genomes from multiple pieces by homologous recombination in yeast. We have previously reported this strategy using pieces derived from E. coli
BAC clones. We assembled a synthetic M. genitalium
genome from six pieces, of which one was a vector (16
). We have also reported assembly of the genome from 25 pieces (17
). The same strategy could be used for assembling any complete set of overlapping genomic clones, natural or synthetic.
When we assembled a synthetic M. genitalium
genome from six pieces, we started with four quarter genomes (about 144 kb each) cloned as BACs. These quarter genomes were released from their BAC backbones by restriction digestion, yielding four overlapping linear fragments, as described previously (16
). Yeast was transformed with a mixture of 120 ng of each quarter genome and 10 ng of linear vector. The vector was produced by amplification of the plasmid pTARBAC3 (31
) in a manner analogous to our preparation of vector pARS-VN.
Quarter 3 (B and D) was digested at the unique AscI site to produce a double-stranded break at the vector-insertion target. We obtained 65 transformants, which we first assayed for completeness by PCR with a set of six amplicons. One amplicon lay internal to each of the four quarter genome fragments, one lay within the HIS3 marker of the vector, and one amplified yeast rDNA as a positive control for the assay. Twenty-four clones were correct by this assay. Six out of 17 of these clones were correct when assayed by Southern blot. When we performed the same experiment with the uncleaved quarter we obtained two transformants, neither of which showed a complete M. genitalium genome.
We performed another experiment, using the BsmBI site in quarter 3. We obtained 73 transformants, 44 of which were correct as assayed by PCR. Five out of 28 of these clones examined by Southern blot were correct. With the quarter undigested we obtained two transformants, neither of which showed a complete M. genitalium
genome. These results agree with the known higher efficiency of homologous recombination at DNA ends (50
Construction of a diploid yeast strain carrying both M. genitalium and M. mycoides genomes
Some bacterial genomes consist of more than one chromosome. Thus, for the successful transplantation of these organisms’ genomes, it would be necessary to transplant more than one DNA molecule. If cloned in yeast, it would be convenient for all of these chromosomes to be cloned into the same yeast cell. This would also be useful if a bacterial chromosome were cloned as more than one molecule in yeast. This might be convenient for engineering or circumvention of a possible size barrier on individual chromosomes in yeast. To explore this we tested whether yeast could maintain two cloned bacterial genomes.
To produce a diploid strain carrying two mycoplasma genomes, we crossed two haploid strains (51
), each of which carried one of the genomes. We mated the W303a strain (mating type a) containing YCpMgen16-2 with the VL6-48N strain (mating type α) containing YCpMmyc1.1 (). The HIS3
marker in the M. genitalium
genome was replaced with a TRP
marker to allow selection of diploids carrying both genomes on medium lacking histidine and tryptophan. Plugs prepared from individual colonies of these diploid strains were analyzed by CHEF electrophoresis. Five out of five diploids contained both M. genitalium
and M. mycoides
genomes (one diploid shown in N).
The vector does not require an ARS for maintenance of the M. genitalium genome in yeast
For a clone to be propagated in yeast, it must contain sequences that can act as yeast replication origins. One such sequence can be included in the yeast cloning vector. However, one replication origin may not be enough to support propagation of an entire bacterial or archaeal genome. It has been extrapolated that one yeast origin may be sufficient to support the replication of 120–300 kb (52
). Yeast contains a replication origin on average every 30–40 kb (53–55
). Large eukaryotic sequences can be cloned in yeast without the addition of yeast replication origins; thus, they are assumed to contain sequences that can function as them. The frequency of such sequences in bacterial and archaeal genomes is unknown; the only bacterial ARS reported is from a Staphylococcus aureus
We relocated the yeast vector in the synthetic M. genitalium
genome from its original site within the RNaseP
gene to a new site in MG411 so as not to interrupt an essential gene (16
). The vector inserted in the new site did not contain an ARS. Because the M. genitalium
genome is AT-rich, it is likely to contain sequences that can function as ARS
in yeast. It is known that ARS-like sequences are frequent in eukaryotic AT-rich DNA (57
Stability of genomes cloned in yeast
A significant fraction of yeast transformants showed the presence of complete mycoplasma genomes. Data from five experiments showed that approximately half (range: 35–61%) of the clones were correct by the two assays used (; percentage of clones complete by PCR multiplied by percentage of those clones correct by size). PCR analysis of the remaining clones showed that most contained some mycoplasma DNA. We do not know how these incomplete clones arose. One or more genome fragments may transform yeast and be circularized in the process by yeast repair mechanisms. Alternatively, these incomplete clones may have arisen from recombination in yeast during the process of transformation with a complete mycoplasma genome.
Once we have identified a full-length clone, we have not observed genetic alterations unless under selection for a recombination event. To examine stability, we started with a glycerol stock of a small culture from a single colony of a re-streaked yeast transformant. This stock was streaked for single colonies on selective medium. Three to eight colonies from this plate (subclones) were individually selected and cultured to saturation in several milliliters of selective medium. DNA from each was isolated and clones were examined for size by restriction digestion followed by pulsed-field gel electrophoresis, and, in some cases, Southern blot. Three out of three YCpMmyc1.1 yeast clones were stable by this analysis (H). All 21 synthetic M. genitalium
yeast clones (16
) examined were also stable.