SynMyco transposon: engineering transposon vectors for efficient transformation of minimal genomes

Abstract

Mycoplasmas are important model organisms for Systems and Synthetic Biology, and are pathogenic to a wide variety of species. Despite their relevance, many of the tools established for genome editing in other microorganisms are not available for Mycoplasmas. The Tn4001 transposon is the reference tool to work with these bacteria, but the transformation efficiencies (TEs) reported for the different species vary substantially. Here, we explore the mechanisms underlying these differences in four Mycoplasma species, Mycoplasma agalactiae, Mycoplasma feriruminatoris, Mycoplasma gallisepticum and Mycoplasma pneumoniae, selected for being representative members of each cluster of the Mycoplasma genus. We found that regulatory regions (RRs) driving the expression of the transposase and the antibiotic resistance marker have a major impact on the TEs. We then designed a synthetic RR termed SynMyco RR to control the expression of the key transposon vector elements. Using this synthetic RR, we were able to increase the TE for M. gallisepticum, M. feriruminatoris and M. agalactiae by 30-, 980- and 1036-fold, respectively. Finally, to illustrate the potential of this new transposon, we performed the first essentiality study in M. agalactiae, basing our study on more than 199,000 genome insertions.

Keywords: Mycoplasma; essentiality; regulatory region; transformation efficiency; transposon.

Figure 1

Screening of transposon TEs across the mycoplasmal landscape. (A) Phylogenetic tree of 21 selected Mycoplasma species in which three main clusters (pneumoniae, spiroplasma and hominis) can be identified using the maximum composite likelihood method. The tree is drawn to scale with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. (B) Bar plot showing the average of gentamicin resistant CFUs (in logarithmic scale) obtained for each of the indicated strains when using the pMTnGm vector (n = 3). (C) Bar plot showing the average of chloramphenicol resistant CFUs (in logarithmic scale) obtained for each of the indicated strains when using the pMTnCm vector. For the statistical analysis, for those species in which no mutants were detected the number of CFU was set to 49, the maximum value below the limit of detection. One-tailed t-test P-values are indicated with one asterisk (*) when P < 0.05 for TE obtained with pMTnGm vector compared to the TE obtained with pMTnCm vector.

Figure 2

Analysis of RRs found in Mycoplasma species. (A) Sequence alignment of the EF-Tu RRs of 10 selected Mycoplasma species. Four main domains are highlighted in boxes: the -35 box, Pribnow box, the RBS sequence and the Translation Starting Codon. In addition, the experimentally determined transcriptional start site (TSS) is also shown for the M. pneumoniae RR. (B) Bar plot representing the fraction of RBS-positive genes normalized by the total number of genes per genome in 21 different Mycoplasma species. (C) Sequence of the SynMyco RR. The boxes highlight the same domains shown in panel A, plus the extended Pribnow domain, and the expected TSS inferred from the one experimentally determined in M. pneumoniae.

Figure 3

Comparison between the transposon TE obtained with pMTnGm and pMTnGm-SynMyco in four different Mycoplasma species. (A) Scheme of the key modules of the pMTnGm transposon. (B) Scheme of the key modules of the pMTnGm-SynMyco transposon. For both (A) and (B) the abbreviations that appears in the figure are: Tnp for transposase coding gene, Amp for ampicillin resistance coding gene, IR for inverted repeats, ColE1 ori for ColE1 origin of replication and GmR for gentamicin resistance coding gene. (C) Bar plot representing the average CFUs of M. pneumoniae resistant to gentamicin (in logarithmic scale) obtained for three independent transformation replicates carried out with either pMTnGm (left side of each panel) or pMTnGm-SynMyco (right side of each panel). For each group of bars, the average of TE (CFU resistant to gentamicin / total CFU viable after transformation) is displayed on top. The fold change in TE is indicated in arrows connecting both sides of each panel. One-tailed t-test p-values are indicated with one asterisk (*) when P < 0.05 for TE obtained with pMTnGm-SynMyco vector compared to the TE obtained with pMTnGm vector. Similar bar plots are shown in (D) for M. gallisepticum, (E) for M. feriruminatoris and (F) for M. agalactiae.

Figure 4

Comparison of the SynMyco RR efficiency versus the native regulatory regions of pMTnGm transposon. (A) Scheme of the key modules of (i) pMTnGm-SynMyco+SynMyco RR-Venus+SynMycoRR-mCherry transposon, (ii) pMTnGm-SynMyco+SynMycoRR-Venus+Gm RR-mCherry transposon and (iii) pMTnGm-SynMyco+SynMyco-Venus+TnpRR-mCherry transposon. The abbreviations that appear in the figure are: Tnp for transposase coding gene, Amp for ampicillin resistance coding gene, IR for inverted repeats, ColE1 ori for ColE1 origin of replication, GmR for gentamicin resistance coding gene, Venus for Venus protein coding gene and mCherry as mCherry protein coding gene. (B) Bar plot representing the ratio obtained in M. pneumoniae for mCherry/Venus fluorescence using transposons represented in panel A. While Venus reporter is always under control of SynMyco RR, the mCherry gene is controlled by SynMyco RR in the left bar, Gm RR in the central bar and Tnp RR in the right bar. On top of each bar is represented the average ratio of mCherry/Venus fluorescence obtained for each of the constructs in three replicates. One-tailed t-test p-values are indicated with one asterisk (*) when P < 0.05 for the ratio of mCherry/Venus under the control of SynMyco RR versus either Gm RR or Tnp RR. Similar bar plots are shown in (C) for M. gallisepticum, (D) for M. feriruminatoris and (E) for M. agalactiae.

Figure 5

Essentiality study in M. agalactiae using the pMTnGm-SynMyco transposon and a comparison with previous studies in M. pneumoniae. (A) Genome disruption profile for M. pneumoniae. The y-axis represents the logarithmic average of total reads covering a window of 1,000 bp (x-axis). (B) Genome disruption profile for M. agalactiae representing the same information as in the previous panel. (C) Insertion density by gene distribution in M. pneumoniae and M. agalactiae as indicated. The x-axis represents the percentage of bp in a gene that is disrupted and the y-axis the frequency of densities in the distribution. To better compare M. pneumoniae and M. agalactiae transposon insertion distributions, we standardized both distributions using min-max scaling. (D) Box-plot representing the statistical comparison of specific subsets of genes expected to be essential (E) and non-essential (NE) in M. pneumoniae and M. agalactiae as indicated. The asterisk represents P-value < 0.05 (3.62 e⁻⁴¹ and 1.20 e⁻²⁰) when comparing density of insertions of E and NE coding genes in M. pneumoniae and M. agalactiae respectively.