Cloning and Transplantation of the Mesoplasma florum Genome

Sep 11, 2017 - Abstract | Full Text HTML | PDF w/ Links | Hi-Res PDF · Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. co...
4 downloads 13 Views 1MB Size
Subscriber access provided by University of Sussex Library

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Synthetic Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

1

Cloning and transplantation of the Mesoplasma florum genome

2 3

Vincent Baby, Fabien Labroussaa, Joëlle Brodeur, Dominick Matteau, Géraldine

4

Gourgues, Carole Lartigue* and Sébastien Rodrigue*

5 6 7

*

Corresponding authors: [email protected] and [email protected]

8 9 10

1 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

11

ABSTRACT

12

Cloning and transplantation of bacterial genomes is a powerful method for the

13

creation of engineered microorganisms. However, much remains to be understood about

14

the molecular mechanisms and limitations of this approach. We report the whole-

15

genome cloning of Mesoplasma florum in Saccharomyces cerevisiae, and use this

16

model to investigate the impact of a bacterial chromosome in yeast cells. Our results

17

indicate that the cloned M. florum genome is subjected to weak transcriptional activity,

18

and causes no significant impact on yeast growth. We also report that the M. florum

19

genome can be transplanted into Mycoplasma capricolum without any negative impact

20

from the putative restriction enzyme encoding gene mfl307. Using whole-genome

21

sequencing, we observed that a small number of mutations appeared in all M. florum

22

transplants. Mutations also arose, albeit at a lower frequency, when the M. capricolum

23

genome was transplanted into M. capricolum recipient cells. These observations suggest

24

that genome transplantation is mutagenic, and that this phenomenon is magnified by the

25

use of genome donor and recipient cell belonging to different species. No difference in

26

efficiency was detected after three successive rounds of genome transplantation,

27

suggesting that the observed mutations were not selected during the procedure. Taken

28

together, our results provide a more accurate picture of the events taking place during

29

bacterial genome cloning and transplantation.

30

Keywords: whole-genome cloning, genome transplantation, Mesoplasma florum,

31

Saccharomyces cerevisiae

32

2 ACS Paragon Plus Environment

Page 2 of 41

Page 3 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

33 34

INTRODUCTION

35

Synthetic genomics is a new biological discipline that is emerging due to the

36

increased affordability and technological progress in DNA synthesis. This will allow

37

entire genomes to be designed from scratch, which will in turn help unveil the

38

fundamental rules of genome organization and global cell functioning. Because of the

39

relative complexity of manipulating and building whole bacterial genomes, the original

40

approach has been initiated with the handling and synthesis of some of the smallest

41

bacterial genomes found in nature1. Mollicutes, and mycoplasmas in particular, are

42

microorganisms that have lost much of their genetic material during evolution and

43

therefore naturally possess small genomes (0.58 to 2.2 Mbp)2. The genome of

44

Mycoplasma mycoides subsp. capri (M. mycoides) was the first to be cloned as an

45

artificial circular chromosome in the yeast Saccharomyces cerevisae and reintroduced

46

into a bacterial host from a different species by a transplantation procedure3. The

47

resulting cell transplants adopted the genotype and phenotype conferred by the newly

48

acquired genome, thereby changing the recipient species into another4 and allowing the

49

synthetic genome designs to be brought to life. This breakthrough confirmed the

50

feasibility of the “whole-genome cloning and transplantation” strategy for synthetic

51

genomics3, and rapidly paved the way for the chemical synthesis of the M. mycoides

52

genome5. This bacterium was also used as the foundation for the recent engineering of a

53

minimal genome6.

54

Among the 11 Mollicutes genomes currently cloned in yeast3,5,7,8, five have also

55

been successfully back-transplanted from yeast3,4,8. A single recipient strain,

56

Mycoplasma capricolum subsp. capricolum (M. capricolum), has been used so far. Using

3 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 41

57

this recipient bacterium, Labroussaa and colleagues have recently reported a systematic

58

investigation of phylogenetic compatibility in genome transplantation and demonstrated

59

that genome transplantation efficiency is higher between closely related species than

60

phylogenetically

61

Prochlorococcus marinus9, Haemophilus influenza10 and Escherichia coli11 have been

62

reportedly cloned in yeast but no subsequent studies have followed so far.

more

distant

species8.

Besides

Mollicutes,

the

genomes

of

63

Mesoplasma florum is a small genome bacterium originally isolated from plants

64

and insects12,13 . Although part of the Mollicutes class like mycoplasmas, M. florum has

65

no known pathogenic potential to any organism, shows fast growth rates (doubling time

66

of ~40 minutes)14, and does not require any sterol for growth in axenic media15. The M.

67

florum genome is small (~800kb), and genetic manipulation tools have recently been

68

developed16. In addition, M. florum is currently the only example of successful genome

69

transplantation between two distinct bacterial genera and constitutes the most distant

70

compatible genome to the universal recipient cell M. capricolum8. M. florum is thus an

71

interesting model to study the requirements and limitations of the whole-genome cloning

72

and transplantation approach. Nevertheless, while this procedure has been successful,

73

many aspects remain poorly understood. What are the constraints for cloning bacterial

74

chromosomes in yeast? Are genomes cloned in yeast transcriptionally active? Are they

75

more or less susceptible to acquire mutations than the yeast genome? What are the

76

factors explaining compatibility between the recipient cell and donor genome in

77

transplantation? How can transplantation rates be increased? Could genome

78

transplantation be broadly applicable to any species?

79

In this work, we describe the whole-genome cloning of M. florum in S. cerevisiae,

80

and investigate the impact of carrying this additional bacterial chromosome for yeast

81

cells. We also successfully transplanted the M. florum chromosome into M. capricolum, 4 ACS Paragon Plus Environment

Page 5 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

82

and analyzed the sequence of bacterial transplant genomes. Our findings provide a

83

more accurate picture of the events that can affect bacterial genomes subjected to

84

cloning and transplantation. Our work also constitutes a first step toward the definition of

85

M. florum as a potential chassis of importance for systems biology and synthetic

86

genomics.

87

5 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

88

RESULTS

89

Cloning of the M. florum genome in yeast

90

Based on recently described M. florum vectors16, we generated pMfl-ACH, a

91

plasmid that harbours a S. cerevisiae autonomous replicating sequence (ARS), a

92

centromere and HIS3 nutritional marker along with the origin of replication (oriC) of the

93

M. florum L1 genome (Fig. 1). Because of its extended homology to the chromosomal

94

oriC locus, pMfl-ACH can integrate in a fraction of the population into the genome,

95

resulting in the duplication of the dnaA gene16. After confirming the integration at the

96

expected site (Fig. S1), a bacterial culture of M. florum L1-ACH was used to isolate

97

intact chromosomes in agarose plugs and to transform S. cerevisiae VL6-48.

98

Approximately 200 yeast colonies were obtained per transformation, and 20 out of 23

99

clones (~85%) contained all targeted regions of the M. florum genome when analyzed by

100

PCR (Fig. S2). After selecting one positive clone, we substituted one copy of the

101

duplicated oriC locus for a URA3 cassette, which was in turn replaced by an HIS3

102

nutritional marker (Fig. 1). This last modification allows the URA3 gene to be used for

103

genetic manipulations, while also preventing potential recombination events between the

104

two dnaA loci that could result in the loss of the cloned M. florum chromosome. A PCR

105

screen was designed to amplify products of different sizes located at every ~100 kb of

106

the M. florum genome, allowing easy analysis by gel electrophoresis (Fig. 2A). Every

107

expected product was obtained, suggesting that the whole-bacterial genome was

108

present in the yeast strain. This was further validated by PFGE using BssHII and RsrII

109

restriction enzymes to digest the M. florum DNA, which cut the S. cerevisiae genome in

110

small fragments and released the M. florum chromosome as a single band at the

111

expected molecular weight of ~797kb (Fig. 2B). Following these validations of overall

112

integrity, the cloned genome was also sequenced and was found to correspond to the 6 ACS Paragon Plus Environment

Page 6 of 41

Page 7 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

113

expected sequence except for six SNP variants when compared to the M. florum

114

reference sequence (RefSeq NC_006055.1) (Table S1). The selected clone was named

115

S. cerevisiae VL6-MflL1-WT. Cryo-preserved aliquots made immediately before the

116

Illumina sequencing of the VL6-MflL1-WT strain were used to inoculate fresh cultures for

117

every subsequent experiments.

118 119

Impact of the additional bacterial genome on yeast growth

120

Little is known about the fundamental and practical implications of whole-genome

121

cloning on either the yeast host or the residing bacterial chromosome. We first sought to

122

measure if the growth rate of a S. cerevisiae strain carrying the M. florum genome was

123

equivalent to a control strain containing p413gpd, a centromeric plasmid bearing the

124

same replication elements and selection marker as pMfl-ACH. We did not observe any

125

significant difference between the doubling time of a strain carrying the M. florum

126

genome or the p413gpd control, either in S. cerevisiae VL6-48 or W303 (Fig. 3A and Fig.

127

S3A). However, a minor difference in the cell cycle dynamics was noticed for the VL6-

128

MflL1-WT strain, suggesting a slightly longer S phase (in which chromosome replication

129

occurs) (Fig. 3B) and conversely shorter G1/G0 and G2/M stages. We carried the same

130

experiments in S. cerevisiae W303-MflL1-WT but observed no significant difference in

131

either the doubling time or the cell cycle dynamics (Fig. S3B). Nevertheless, since S.

132

cerevisiae VL6-48 is widely used for whole-genome cloning projects1,3,5,6,10,17,18 and

133

because of more stable auxotrophic mutations, this strain was used for every

134

experiments that followed.

135 136

Transcriptome profiling of yeast harbouring a bacterial genome 7 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

137

We were next interested to determine if the M. florum chromosome cloned in

138

yeast was actively transcribed, and if its presence had any impact on the expression of

139

S. cerevisiae genes. We performed transcriptome profiling on the VL6-MflL1-WT and on

140

VL6-48 carrying the p413gpd plasmid as a control (Fig. 4). We observed that only 0.51%

141

of the mapped reads aligned throughout the M. florum sequence (Fig. 4A), while one

142

copy of this additional chromosome represents ~6.2% of the VL6-MflL1-WT reference

143

sequence. Using the p413gpd strain, the background transcription signal from

144

misaligned sequences on the M. florum chromosome can be evaluated to ~0.05% of the

145

total mapped reads. Globally, the M. florum chromosome showed ~12.6 times less read

146

coverage per bp than the average of the 16 endogenous yeast chromosomes (Fig. 4B),

147

reflecting a significantly lower transcriptional activity for the cloned bacterial genome.

148

The fragments per kilobase per million of mapped reads (FPKM) values of each gene

149

were also used to calculate Pearson correlation coefficients between the VL6-MflL1-WT

150

and p413gpd yeast strains (Fig. 4C bottom and Fig. S4). We observed a high correlation

151

between the FPKM values of the S. cerevisiae genes (average of 0.83) independently of

152

the strains and replicates. However, the FPKM values of the M. florum genes were

153

poorly correlated between the VL6-MflL1-WT replicates (Fig. 4C top; average of 0.09),

154

indicating that the low level of transcription is initiated inconsistently across the cloned

155

bacterial genome. We observed similar results when performing the correlation analysis

156

based on the read coverage in 1.5 kbp windows (Fig. S5).

157

The presence of a cloned bacterial genome can potentially create a burden or a

158

stress that could affect gene expression in the yeast host. Transcriptome profiling

159

identified 195 differentially expressed yeast genes and no M. florum genes from a total

160

set of 7072 genes between the VL6-MflL1-WT and p413gpd strains (Table S2).

161

However, we could not establish any clear link between them based on gene ontology

8 ACS Paragon Plus Environment

Page 8 of 41

Page 9 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

162

(GO), either at the biological process or molecular function levels. Since only 23 out of

163

the 195 differentially expressed genes are of unknown function, this suggests that the

164

absence of explicit relationship is likely not imputable to a lack of functional annotation in

165

the gene set. Taken together, these results support the idea that the cloned M. florum

166

genome is subjected to weak and randomly distributed transcriptional activity when

167

hosted in yeast. Although some genes encoded by the yeast chromosomes (Table S2)

168

were differentially expressed in the presence of the cloned M. florum genome, those

169

changes were not reflected in the yeast’s growth or cell cycle and the affected genes

170

were not enriched for a particular function.

171

Transplantation of the M. florum genome into M. capricolum

172

M. florum was previously successfully transplanted into recipient M. capricolum

173

cells, but at a lower efficiency than other Mollicutes8. A well-described barrier to

174

transplantation is the presence of genes encoding restriction enzymes that can cleave

175

unmethylated DNA obtained from yeast when introduced in a recipient bacterium3. For

176

this reason, a restriction deficient M. capricolum strain was used as the recipient cell

177

during genome transplantation. A single gene, mfl307, encodes a predicted restriction

178

endonuclease in M. florum, and was deleted to create S. cerevisiae VL6-MflL1-ΔRE (Fig.

179

S6). This strain was compared to the unmodified cloned M. florum chromosome to

180

investigate the impact of restriction enzyme encoding genes present in the donor

181

genome. Between 1 and 24 colonies were obtained per transplantation of genomes

182

isolated from yeast, without any significant difference between the unmodified and

183

restriction deficient mutant (transplantation rates of 21.15 ± 5.11 and 25.64 ± 3.12

184

transplants per µg of gDNA respectively). The colonies showed the typical M. florum

185

morphology and appeared after the expected incubation time for this species.

9 ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

186

We next performed DNA sequencing to confirm the genotype conversion of the

187

transplants and quantify the number of mutations that might be acquired during this

188

procedure. The genome of 30 transplants obtained from VL6-MflL1-WT and VL6-MflL1-

189

ΔRE (15 each; see Table S3 for a more detailed description) were analyzed. All

190

transplants were found to carry between 1 and 5 variants that were not detected by

191

sequencing when the M. florum chromosome was hosted in yeast just prior to the

192

transplantation assays (Table 1 and Table S1). Surprisingly, most variants were present

193

in all the other transplants from a same genome transplantation experiment, raising the

194

possibility that only a few cells acquired the M. florum genome during the procedure,

195

divided and then yielded multiple isogenic colonies on solid medium. We then wondered

196

if the presence of mutations and the high frequency of isogenic transplants could be the

197

result of interspecies genome transplantation into M. capricolum. We therefore

198

performed transplantation experiments using M. capricolum both as the genome donor

199

(using chromosomal DNA isolated from a bacterial culture) and recipient cell. In this

200

context, the transplantation rates reached ~240 transplants per µg of gDNA,

201

approximately 80 fold more than what was obtained when the M. florum genomic DNA

202

isolated from a bacterial culture was used8. Out of 23 clones sequenced from four

203

independent experiments, an average of 1.0 mutation per transplant was detected for M.

204

capricolum (Table S4), while this number reached 2.1 for M. florum obtained from yeast

205

to bacteria transplantations (Table S1). Taking genome size into account, the M. florum

206

transplants had 2.7 times more mutations per base pair than the M. capricolum

207

transplants, a statistically significant difference according to both the Mann-Whitney (p

208