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Cloning and transplantation of the Mesoplasma florum genome Vincent Baby, Fabien Labroussaa, Joëlle Brodeur, Dominick Matteau, Géraldine Gourgues, Carole Lartigue, and Sebastien Rodrigue ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00279 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 19, 2017
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Cloning and transplantation of the Mesoplasma florum genome
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Vincent Baby, Fabien Labroussaa, Joëlle Brodeur, Dominick Matteau, Géraldine
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Gourgues, Carole Lartigue* and Sébastien Rodrigue*
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*
Corresponding authors:
[email protected] and
[email protected] 8 9 10
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ABSTRACT
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Cloning and transplantation of bacterial genomes is a powerful method for the
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creation of engineered microorganisms. However, much remains to be understood about
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the molecular mechanisms and limitations of this approach. We report the whole-
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genome cloning of Mesoplasma florum in Saccharomyces cerevisiae, and use this
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model to investigate the impact of a bacterial chromosome in yeast cells. Our results
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indicate that the cloned M. florum genome is subjected to weak transcriptional activity,
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and causes no significant impact on yeast growth. We also report that the M. florum
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genome can be transplanted into Mycoplasma capricolum without any negative impact
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from the putative restriction enzyme encoding gene mfl307. Using whole-genome
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sequencing, we observed that a small number of mutations appeared in all M. florum
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transplants. Mutations also arose, albeit at a lower frequency, when the M. capricolum
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genome was transplanted into M. capricolum recipient cells. These observations suggest
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that genome transplantation is mutagenic, and that this phenomenon is magnified by the
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use of genome donor and recipient cell belonging to different species. No difference in
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efficiency was detected after three successive rounds of genome transplantation,
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suggesting that the observed mutations were not selected during the procedure. Taken
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together, our results provide a more accurate picture of the events taking place during
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bacterial genome cloning and transplantation.
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Keywords: whole-genome cloning, genome transplantation, Mesoplasma florum,
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Saccharomyces cerevisiae
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INTRODUCTION
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Synthetic genomics is a new biological discipline that is emerging due to the
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increased affordability and technological progress in DNA synthesis. This will allow
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entire genomes to be designed from scratch, which will in turn help unveil the
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fundamental rules of genome organization and global cell functioning. Because of the
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relative complexity of manipulating and building whole bacterial genomes, the original
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approach has been initiated with the handling and synthesis of some of the smallest
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bacterial genomes found in nature1. Mollicutes, and mycoplasmas in particular, are
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microorganisms that have lost much of their genetic material during evolution and
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therefore naturally possess small genomes (0.58 to 2.2 Mbp)2. The genome of
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Mycoplasma mycoides subsp. capri (M. mycoides) was the first to be cloned as an
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artificial circular chromosome in the yeast Saccharomyces cerevisae and reintroduced
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into a bacterial host from a different species by a transplantation procedure3. The
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resulting cell transplants adopted the genotype and phenotype conferred by the newly
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acquired genome, thereby changing the recipient species into another4 and allowing the
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synthetic genome designs to be brought to life. This breakthrough confirmed the
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feasibility of the “whole-genome cloning and transplantation” strategy for synthetic
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genomics3, and rapidly paved the way for the chemical synthesis of the M. mycoides
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genome5. This bacterium was also used as the foundation for the recent engineering of a
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minimal genome6.
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Among the 11 Mollicutes genomes currently cloned in yeast3,5,7,8, five have also
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been successfully back-transplanted from yeast3,4,8. A single recipient strain,
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Mycoplasma capricolum subsp. capricolum (M. capricolum), has been used so far. Using
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this recipient bacterium, Labroussaa and colleagues have recently reported a systematic
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investigation of phylogenetic compatibility in genome transplantation and demonstrated
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that genome transplantation efficiency is higher between closely related species than
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phylogenetically
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Prochlorococcus marinus9, Haemophilus influenza10 and Escherichia coli11 have been
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reportedly cloned in yeast but no subsequent studies have followed so far.
more
distant
species8.
Besides
Mollicutes,
the
genomes
of
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Mesoplasma florum is a small genome bacterium originally isolated from plants
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and insects12,13 . Although part of the Mollicutes class like mycoplasmas, M. florum has
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no known pathogenic potential to any organism, shows fast growth rates (doubling time
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of ~40 minutes)14, and does not require any sterol for growth in axenic media15. The M.
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florum genome is small (~800kb), and genetic manipulation tools have recently been
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developed16. In addition, M. florum is currently the only example of successful genome
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transplantation between two distinct bacterial genera and constitutes the most distant
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compatible genome to the universal recipient cell M. capricolum8. M. florum is thus an
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interesting model to study the requirements and limitations of the whole-genome cloning
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and transplantation approach. Nevertheless, while this procedure has been successful,
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many aspects remain poorly understood. What are the constraints for cloning bacterial
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chromosomes in yeast? Are genomes cloned in yeast transcriptionally active? Are they
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more or less susceptible to acquire mutations than the yeast genome? What are the
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factors explaining compatibility between the recipient cell and donor genome in
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transplantation? How can transplantation rates be increased? Could genome
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transplantation be broadly applicable to any species?
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In this work, we describe the whole-genome cloning of M. florum in S. cerevisiae,
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and investigate the impact of carrying this additional bacterial chromosome for yeast
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cells. We also successfully transplanted the M. florum chromosome into M. capricolum, 4 ACS Paragon Plus Environment
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and analyzed the sequence of bacterial transplant genomes. Our findings provide a
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more accurate picture of the events that can affect bacterial genomes subjected to
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cloning and transplantation. Our work also constitutes a first step toward the definition of
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M. florum as a potential chassis of importance for systems biology and synthetic
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genomics.
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RESULTS
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Cloning of the M. florum genome in yeast
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Based on recently described M. florum vectors16, we generated pMfl-ACH, a
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plasmid that harbours a S. cerevisiae autonomous replicating sequence (ARS), a
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centromere and HIS3 nutritional marker along with the origin of replication (oriC) of the
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M. florum L1 genome (Fig. 1). Because of its extended homology to the chromosomal
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oriC locus, pMfl-ACH can integrate in a fraction of the population into the genome,
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resulting in the duplication of the dnaA gene16. After confirming the integration at the
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expected site (Fig. S1), a bacterial culture of M. florum L1-ACH was used to isolate
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intact chromosomes in agarose plugs and to transform S. cerevisiae VL6-48.
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Approximately 200 yeast colonies were obtained per transformation, and 20 out of 23
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clones (~85%) contained all targeted regions of the M. florum genome when analyzed by
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PCR (Fig. S2). After selecting one positive clone, we substituted one copy of the
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duplicated oriC locus for a URA3 cassette, which was in turn replaced by an HIS3
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nutritional marker (Fig. 1). This last modification allows the URA3 gene to be used for
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genetic manipulations, while also preventing potential recombination events between the
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two dnaA loci that could result in the loss of the cloned M. florum chromosome. A PCR
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screen was designed to amplify products of different sizes located at every ~100 kb of
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the M. florum genome, allowing easy analysis by gel electrophoresis (Fig. 2A). Every
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expected product was obtained, suggesting that the whole-bacterial genome was
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present in the yeast strain. This was further validated by PFGE using BssHII and RsrII
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restriction enzymes to digest the M. florum DNA, which cut the S. cerevisiae genome in
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small fragments and released the M. florum chromosome as a single band at the
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expected molecular weight of ~797kb (Fig. 2B). Following these validations of overall
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integrity, the cloned genome was also sequenced and was found to correspond to the 6 ACS Paragon Plus Environment
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expected sequence except for six SNP variants when compared to the M. florum
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reference sequence (RefSeq NC_006055.1) (Table S1). The selected clone was named
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S. cerevisiae VL6-MflL1-WT. Cryo-preserved aliquots made immediately before the
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Illumina sequencing of the VL6-MflL1-WT strain were used to inoculate fresh cultures for
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every subsequent experiments.
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Impact of the additional bacterial genome on yeast growth
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Little is known about the fundamental and practical implications of whole-genome
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cloning on either the yeast host or the residing bacterial chromosome. We first sought to
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measure if the growth rate of a S. cerevisiae strain carrying the M. florum genome was
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equivalent to a control strain containing p413gpd, a centromeric plasmid bearing the
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same replication elements and selection marker as pMfl-ACH. We did not observe any
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significant difference between the doubling time of a strain carrying the M. florum
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genome or the p413gpd control, either in S. cerevisiae VL6-48 or W303 (Fig. 3A and Fig.
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S3A). However, a minor difference in the cell cycle dynamics was noticed for the VL6-
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MflL1-WT strain, suggesting a slightly longer S phase (in which chromosome replication
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occurs) (Fig. 3B) and conversely shorter G1/G0 and G2/M stages. We carried the same
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experiments in S. cerevisiae W303-MflL1-WT but observed no significant difference in
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either the doubling time or the cell cycle dynamics (Fig. S3B). Nevertheless, since S.
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cerevisiae VL6-48 is widely used for whole-genome cloning projects1,3,5,6,10,17,18 and
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because of more stable auxotrophic mutations, this strain was used for every
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experiments that followed.
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Transcriptome profiling of yeast harbouring a bacterial genome 7 ACS Paragon Plus Environment
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We were next interested to determine if the M. florum chromosome cloned in
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yeast was actively transcribed, and if its presence had any impact on the expression of
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S. cerevisiae genes. We performed transcriptome profiling on the VL6-MflL1-WT and on
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VL6-48 carrying the p413gpd plasmid as a control (Fig. 4). We observed that only 0.51%
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of the mapped reads aligned throughout the M. florum sequence (Fig. 4A), while one
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copy of this additional chromosome represents ~6.2% of the VL6-MflL1-WT reference
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sequence. Using the p413gpd strain, the background transcription signal from
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misaligned sequences on the M. florum chromosome can be evaluated to ~0.05% of the
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total mapped reads. Globally, the M. florum chromosome showed ~12.6 times less read
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coverage per bp than the average of the 16 endogenous yeast chromosomes (Fig. 4B),
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reflecting a significantly lower transcriptional activity for the cloned bacterial genome.
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The fragments per kilobase per million of mapped reads (FPKM) values of each gene
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were also used to calculate Pearson correlation coefficients between the VL6-MflL1-WT
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and p413gpd yeast strains (Fig. 4C bottom and Fig. S4). We observed a high correlation
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between the FPKM values of the S. cerevisiae genes (average of 0.83) independently of
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the strains and replicates. However, the FPKM values of the M. florum genes were
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poorly correlated between the VL6-MflL1-WT replicates (Fig. 4C top; average of 0.09),
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indicating that the low level of transcription is initiated inconsistently across the cloned
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bacterial genome. We observed similar results when performing the correlation analysis
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based on the read coverage in 1.5 kbp windows (Fig. S5).
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The presence of a cloned bacterial genome can potentially create a burden or a
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stress that could affect gene expression in the yeast host. Transcriptome profiling
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identified 195 differentially expressed yeast genes and no M. florum genes from a total
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set of 7072 genes between the VL6-MflL1-WT and p413gpd strains (Table S2).
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However, we could not establish any clear link between them based on gene ontology
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(GO), either at the biological process or molecular function levels. Since only 23 out of
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the 195 differentially expressed genes are of unknown function, this suggests that the
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absence of explicit relationship is likely not imputable to a lack of functional annotation in
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the gene set. Taken together, these results support the idea that the cloned M. florum
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genome is subjected to weak and randomly distributed transcriptional activity when
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hosted in yeast. Although some genes encoded by the yeast chromosomes (Table S2)
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were differentially expressed in the presence of the cloned M. florum genome, those
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changes were not reflected in the yeast’s growth or cell cycle and the affected genes
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were not enriched for a particular function.
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Transplantation of the M. florum genome into M. capricolum
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M. florum was previously successfully transplanted into recipient M. capricolum
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cells, but at a lower efficiency than other Mollicutes8. A well-described barrier to
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transplantation is the presence of genes encoding restriction enzymes that can cleave
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unmethylated DNA obtained from yeast when introduced in a recipient bacterium3. For
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this reason, a restriction deficient M. capricolum strain was used as the recipient cell
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during genome transplantation. A single gene, mfl307, encodes a predicted restriction
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endonuclease in M. florum, and was deleted to create S. cerevisiae VL6-MflL1-ΔRE (Fig.
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S6). This strain was compared to the unmodified cloned M. florum chromosome to
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investigate the impact of restriction enzyme encoding genes present in the donor
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genome. Between 1 and 24 colonies were obtained per transplantation of genomes
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isolated from yeast, without any significant difference between the unmodified and
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restriction deficient mutant (transplantation rates of 21.15 ± 5.11 and 25.64 ± 3.12
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transplants per µg of gDNA respectively). The colonies showed the typical M. florum
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morphology and appeared after the expected incubation time for this species.
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We next performed DNA sequencing to confirm the genotype conversion of the
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transplants and quantify the number of mutations that might be acquired during this
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procedure. The genome of 30 transplants obtained from VL6-MflL1-WT and VL6-MflL1-
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ΔRE (15 each; see Table S3 for a more detailed description) were analyzed. All
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transplants were found to carry between 1 and 5 variants that were not detected by
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sequencing when the M. florum chromosome was hosted in yeast just prior to the
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transplantation assays (Table 1 and Table S1). Surprisingly, most variants were present
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in all the other transplants from a same genome transplantation experiment, raising the
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possibility that only a few cells acquired the M. florum genome during the procedure,
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divided and then yielded multiple isogenic colonies on solid medium. We then wondered
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if the presence of mutations and the high frequency of isogenic transplants could be the
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result of interspecies genome transplantation into M. capricolum. We therefore
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performed transplantation experiments using M. capricolum both as the genome donor
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(using chromosomal DNA isolated from a bacterial culture) and recipient cell. In this
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context, the transplantation rates reached ~240 transplants per µg of gDNA,
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approximately 80 fold more than what was obtained when the M. florum genomic DNA
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isolated from a bacterial culture was used8. Out of 23 clones sequenced from four
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independent experiments, an average of 1.0 mutation per transplant was detected for M.
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capricolum (Table S4), while this number reached 2.1 for M. florum obtained from yeast
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to bacteria transplantations (Table S1). Taking genome size into account, the M. florum
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transplants had 2.7 times more mutations per base pair than the M. capricolum
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transplants, a statistically significant difference according to both the Mann-Whitney (p
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