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.

Mycoplasma pneumoniae Genome Editing Based on Oligo Recombineering and Cas9-Mediated Counterselection

“…Thus, researchers working with other Mycoplasma species may need to define their own regulatory regions or use a recently reported regulatory region that seems to be functional in all Mycoplasma species. 61 All plasmids generated in this work are available upon request. All oligonucleotides employed for plasmid assembly as well as details for vector construction are available in Table S7 .…”

Inferring Active Metabolic Pathways from Proteomics and Essentiality Data

“…For the generation of the metabolic map, we used genomics data from M. pneumoniae (Himmelreich et al, 1996) and M. agalactiae (Montero-Blay et al, 2019). This information was consistent with KEGG database (Kanehisa and Goto, 2000).…”

“…Furthermore, for each of the genes involved in carbohydrates, lipids, amino acids, nucleotides, and vitamins metabolism in M. agalactiae, it has been found an orthologous gene in the M. bovis genome evidencing their metabolic analogy (Table S1). For both bacteria, there are high-resolution essentiality studies available (Lluch-Senar et al, 2015;Montero-Blay et al, 2019).…”

LoxTnSeq: Random Transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions

“…Cells containing both lox sites were then transformed with the pMTnCreGm transposon, without lox sites and containing the cre recombinase and a gene encoding gentamicin resistance. The cre expression was controlled by the constitutive p438 promoter (Montero-Blay et al, 2019;Pich et al, 2006), and was introduced via transposon to ensure high levels of expression. In previous work we demonstrated that the action of the cre on a single active lox site (i.e.…”