Fraser, C. M. et al. The minimal gene complement of Mycoplasma genitalium. Science 270, 397–399 (1995).
Google Scholar
Himmelreich, R. et al. Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res. 24, 4420–4449 (1996).
Google Scholar
Karr, J. R. et al. A whole-cell computational model predicts phenotype from genotype. Cell 150, 389–401 (2012).
Google Scholar
Lartigue, C. et al. Genome transplantation in bacteria: changing one species to another. Science 317, 632–638 (2007).
Google Scholar
Gibson, D. G. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52–56 (2010).
Google Scholar
Hutchison, C. A. III et al. Design and synthesis of a minimal bacterial genome. Science 351, aad6253–aad6253 (2016).
Google Scholar
Marcone, C. Molecular biology and pathogenicity of phytoplasmas. Ann. Appl. Biol. 165, 199–221 (2014).
Google Scholar
Taylor-Robinson, D. Antibiotic susceptibilities of mycoplasmas and treatment of mycoplasmal infections. J. Antimicrob. Chemother. 40, 622–630 (1997).
Google Scholar
Bébéar, C., Pereyre, S. & Peuchant, O. Mycoplasma pneumoniae: susceptibility and resistance to antibiotics. Future Microbiol. 6, 423–431 (2011).
Google Scholar
Wang, X. & Lutkenhaus, J. Characterization of the ftsZ gene from Mycoplasma pulmonis, an organism lacking a cell wall. J. Bacteriol. 178, 2314–2319 (1996).
Google Scholar
Perez-Casal, J., Prysliak, T., Maina, T., Suleman, M. & Jimbo, S. Status of the development of a vaccine against Mycoplasma bovis. Vaccine https://doi.org/10.1016/j.vaccine.2017.03.095 (2017).
Nicholas, R. A. J., Ayling, R. D. & McAuliffe, L. Vaccines for Mycoplasma diseases in animals and man. J. Comp. Pathol. 140, 85–96 (2009).
Google Scholar
Ishfaq, M. et al. Current status of vaccine research, development, and challenges of vaccines for Mycoplasma gallisepticum. Poult. Sci. 99, 4195–4202 (2020).
Google Scholar
Maes, D. et al. Perspectives for improvement of Mycoplasma hyopneumoniae vaccines in pigs. Vet. Res. https://doi.org/10.1186/s13567-021-00941-x (2021).
The MycoSynVac project. Engineering of Mycoplasma pneumoniae as a broad-spectrum animal vaccine. EU H2020 grant No 634942. https://www.mycosynvac.eu/ (2020).
Güell, M. et al. Transcriptome complexity in a genome-reduced bacterium. Science 326, 1268 (2009).
Google Scholar
Lluch-Senar, M. et al. Defining a minimal cell: essentiality of small ORFs and ncRNAs in a genome‐reduced bacterium. Mol. Syst. Biol. 11, 780 (2015).
Google Scholar
Junier, I., Unal, E. B., Yus, E., Llorens-Rico, V. & Serrano, L. Insights into the mechanisms of basal coordination of transcription using a genome-reduced bacterium. Cell Syst. 3, 1–11 (2016).
Llorens-Rico, V. et al. Bacterial antisense RNAs are mainly the product of transcriptional noise. Sci. Adv. 2, e1501363–e1501363 (2016).
Google Scholar
Trussart, M. et al. Defined chromosome structure in the genome-reduced bacterium Mycoplasma pneumoniae. Nat. Commun. 8, 14665 (2017).
Google Scholar
Yus, E., Yang, J., Sogues, A. & Serrano, L. A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants. Nat. Commun. 8, 368 (2017).
Google Scholar
Miravet-Verde, S. et al. Unraveling the hidden universe of small proteins in bacterial genomes. Mol. Syst. Biol. 15, e8290 (2019).
Google Scholar
Yus, E. et al. Determination of the gene regulatory network of a genome-reduced bacterium highlights alternative regulation independent of transcription factors. Cell Syst. 9, 143–158.e13 (2019).
Google Scholar
Montero-Blay, A., Piñero-Lambea, C., Miravet-Verde, S., Lluch-Senar, M. & Serrano, L. Inferring active metabolic pathways from proteomics and essentiality data. Cell Rep. 31, 107722 (2020).
Google Scholar
Kühner, S. et al. Proteome organization in a genome-reduced bacterium. Science 1235, 1235–1240 (2009).
Google Scholar
Yus, E. et al. Impact of genome reduction on bacterial metabolism and its regulation. Science 326, 1263–1268 (2009).
Google Scholar
Maier, T. et al. Quantification of mRNA and protein and integration with protein turnover in a bacterium. Mol. Syst. Biol. 7, 511 (2011).
Google Scholar
van Noort, V. et al. Cross-talk between phosphorylation and lysine acetylation in a genome-reduced bacterium. Mol. Syst. Biol. 8, 571 (2012).
Lluch-Senar, M. et al. Comprehensive methylome characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at single-base resolution. PLoS Genet. 9, e1003191 (2013).
Google Scholar
Wodke, J. A. H. et al. Dissecting the energy metabolism in Mycoplasma pneumoniae through genome-scale metabolic modeling. Mol. Syst. Biol. 9, 653 (2013).
Google Scholar
Lluch-Senar, M. et al. Comparative ‘-omics’ in Mycoplasma pneumoniae clinical isolates reveals key virulence factors. PLoS ONE 10, e0137354 (2015).
Google Scholar
Llorens-Rico, V., Lluch-Senar, M. & Serrano, L. Distinguishing between productive and abortive promoters using a random forest classifier in Mycoplasma pneumoniae. Nucleic Acids Res. 43, 3442–3453 (2015).
Google Scholar
Razin, S. The mycoplasmas. Microbiol. Mol. Biol. Rev. 42, 414–470 (1978).
Google Scholar
Razin, S. Molecular biology and genetics of mycoplasmas (Mollicutes). Microbiol. Rev. 49, 419–455 (1985).
Google Scholar
Dybvig, K. & Voelker, L. L. Molecular biology of mycoplasmas. Annu. Rev. Microbiol. 50, 25–57 (1996).
Google Scholar
Gupta, S., Bram, E. E. & Weiss, R. Genetically programmable pathogen sense and destroy. ACS Synth. Biol. 2, 715–723 (2013).
Google Scholar
Garrido, V. et al. Engineering a genome‐reduced bacterium to eliminate Staphylococcus aureus biofilms in vivo. Mol. Syst. Biol. 17, e10145 (2021).
Google Scholar
Simon, A. J. & Ellington, A. D. Recent advances in synthetic biosafety. F1000Res. 5, 2118 (2016).
Piñero-Lambea, C. et al. Mycoplasma pneumoniae genome editing based on oligo recombineering and Cas9-mediated counterselection. ACS Synth. Biol. 9, 1693–1704 (2020).
Google Scholar
Garcia-Morales, L. et al. A RAGE based strategy for the genome engineering of the human respiratory pathogen Mycoplasma pneumoniae. ACS Synth. Biol. 9, 2737–2748 (2020).
Google Scholar
Ruiz, E. et al. CReasPy-cloning: a method for simultaneous cloning and engineering of megabase-sized genomes in yeast using the CRISPR-Cas9 system. ACS Synth. Biol. 8, 2547–2557 (2019).
Tsarmpopoulos, I. et al. In-yeast engineering of a bacterial genome using CRISPR/Cas9. ACS Synth. Biol. 5, 104–109 (2016).
Google Scholar
Baby, V. et al. Cloning and transplantation of the Mesoplasma florum genome. ACS Synth. Biol. 7, 209–217 (2018).
Breton, M. et al. First report of a tetracycline-inducible gene expression system for mollicutes. Microbiology 156, 198–205 (2010).
Google Scholar
Mariscal, A. M., Gonzalez-Gonzalez, L., Querol, E. & Piñol, J. All-in-one construct for genome engineering using Cre-lox technology. DNA Res. 23, 263–270 (2016).
Google Scholar
de Sá-Nogueira, I. & Mota, L. J. Negative regulation of L-arabinose metabolism in Bacillus subtilis: characterization of the araR (araC) gene. J. Bacteriol. 179, 1598–1608 (1997).
Google Scholar
Lewis, M. et al. Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science 271, 1247–1254 (1996).
Google Scholar
Lewis, M. The lac repressor. C. R. Biol. 328, 521–548 (2005).
Google Scholar
Winstanley, C., Morgan, J. A., Pickup, R. W., Jones, J. G. & Saunders, J. R. Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system. Appl. Environ. Microbiol. 55, 771–777 (1989).
Google Scholar
Bakk, A. Transcriptional activation mechanisms of the PRM promoter of lambda phage. Biophys. Chem. 114, 229–234 (2005).
Google Scholar
Frey, J. Biological safety concepts of genetically modified live bacterial vaccines. Vaccine 25, 5598–5605 (2007).
Google Scholar
Detmer, A. & Glenting, J. Live bacterial vaccines – A review and identification of potential hazards. Microb. Cell Fact. 5, 23 (2006).
Ding, C., Ma, J., Dong, Q. & Liu, Q. Live bacterial vaccine vector and delivery strategies of heterologous antigen: a review. Immunol. Lett. 197, 70–77 (2018).
Google Scholar
Reardon, S. Bacteria deliver gene therapies. Nature 558, 497–498 (2018).
Google Scholar
Baban, C. K., Cronin, M., O’Hanlon, D., O’Sullivan, G. C. & Tangney, M. Bacteria as vectors for gene therapy of cancer. Bioeng. Bugs 1, 385–394 (2010).
Google Scholar
FDA. Early clinical trials with live biotherapeutic products: chemistry, manufacturing, and control information. Guidance for Industry. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/early-clinical-trials-live-biotherapeutic-products-chemistry-manufacturing-and-control-information (2016).
Charbonneau, M. R., Isabella, V. M., Li, N. & Kurtz, C. B. Developing a new class of engineered live bacterial therapeutics to treat human diseases. Nat. Commun. 11, 1738 (2020).
Google Scholar
Ozdemir, T., Fedorec, A. J. H., Danino, T. & Barnes, C. P. Synthetic biology and engineered live biotherapeutics: toward increasing system complexity. Cell Syst. 7, 5–16 (2018).
Pich, O. Q., Burgos, R., Planell, R., Querol, E. & Piñol, J. Comparative analysis of antibiotic resistance gene markers in Mycoplasma genitalium: application to studies of the minimal gene complement. Microbiology 152, 519–527 (2006).
Google Scholar
Goodman, D. B., Church, G. M. & Kosuri, S. Causes and effects of N-terminal codon bias in bacterial genes. Science 342, 475–479 (2013).
Google Scholar
Montero-Blay, A., Miravet-Verde, S., Lluch-Senar, M., Piñero-Lambea, C. & Serrano, L. SynMyco transposon: engineering transposon vectors for efficient transformation of minimal genomes. DNA Res. 26, 327–339 (2019).
Google Scholar
Mota, L. J., Tavares, P. & de Sá-Nogueira, I. Mode of action of AraR, the key regulator of L-arabinose metabolism in Bacillus subtilis. Mol. Microbiol. 33, 476–489 (1999).
Google Scholar
Mota, L. J., Sarmento, L. M. & de Sá-Nogueira, I. Control of the arabinose regulon in Bacillus subtilis by AraR in vivo: crucial roles of operators, cooperativity, and DNA looping. J. Bacteriol. 183, 4190–4201 (2001).
Google Scholar
Sadler, J. R., Sasmor, H. & Betz, J. L. A perfectly symmetric lac operator binds the lac repressor very tightly. Proc. Natl Acad. Sci. USA 80, 6785–6789 (1983).
Google Scholar
Oehler, S., Amouyal, M., Kolkhof, P., von Wilcken-Bergmann, B. & Müller-Hill, B. Quality and position of the three lac operators of E. coli define efficiency of repression. EMBO J. 13, 3348–3355 (1994).
Google Scholar
Wilson, C. J., Zhan, H., Swint-Kruse, L. & Matthews, K. S. The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding. Cell. Mol. Life Sci. 64, 3–16 (2007).
Google Scholar
Meyer, B. J., Kleid, D. G. & Ptashne, M. Lambda repressor turns off transcription of its own gene. Proc. Natl Acad. Sci. USA 72, 4785–4789 (1975).
Google Scholar
Hawley, D. K. & McClure, W. R. The effect of a lambda repressor mutation on the activation of transcription initiation from the lambda PRM promoter. Cell 32, 327–333 (1983).
Google Scholar
Benson, N., Sugiono, P. & Youderian, P. DNA sequence determinants of lambda repressor binding. Challenge 29, 21–29 (1988).
Hillen, W. & Berens, C. Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Annu. Rev. Microbiol. 48, 345–369 (1994).
Google Scholar
Hinrichs, W. et al. Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance. Science 264, 418–420 (1994).
Google Scholar
Ramos, J. L. et al. The TetR family of transcriptional repressors. Microbiol. Mol. Biol. Rev. 69, 326–356 (2005).
Google Scholar
Lanzer, M. & Bujard, H. Promoters largely determine the efficiency of repressor action. Proc. Natl Acad. Sci. USA 85, 8973–8977 (1988).
Google Scholar
Topp, S. et al. Synthetic riboswitches that induce gene expression in diverse bacterial species. Appl. Environ. Microbiol. 76, 7881–7884 (2010).
Google Scholar
Topp, S. & Gallivan, J. P. Riboswitches in unexpected places–A synthetic riboswitch in a protein coding region. RNA 14, 2498–2503 (2008).
Google Scholar
Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011).
Google Scholar
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Google Scholar
Ashihara, H., Kato, M. & Crozier, A. Distribution, biosynthesis and catabolism of methylxanthines in plants. Handb. Exp. Pharmacol. 200, 11–31 (2011).
Google Scholar
Rocha, E. P. C. & Blanchard, A. Genomic repeats, genome plasticity and the dynamics of Mycoplasma evolution. Nucleic Acids Res. 30, 2031–2042 (2002).
Google Scholar
Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).
Google Scholar
Hutchison, C. A. III et al. Global transposon mutagenesis and a minimal Mycoplasma genome. Science 286, 2165–2169 (1999).
Google Scholar
Gaspari, E. et al. Model-driven design allows growth of Mycoplasma pneumoniae on serum-free media. 6, 33 (2020).
Carvalho, F. M. et al. DNA repair in reduced genome: the Mycoplasma model. Gene 360, 111–119 (2005).
Google Scholar
Zakeri, B. & Carr, P. A. The limits of synthetic biology. Trends Biotechnol. 33, 57–58 (2015).
Google Scholar
Department of Health and Human Services. National Institutes of Health (NIH). NIH guidelines for research involving recombinant or synthetic nucleic acid molecules. NIH Guidel. 2, 142 (2019).
IDRC – International Development Research Centre. Development of a novel vaccine for contagious caprine pleuropneumonia based on a fast-growing Mycoplasma feriruminatoris chassis. https://www.idrc.ca/en/project/development-novel-vaccine-contagious-caprine-pleuropneumonia-based-fast-growing-mycoplasma (2021).
Jores, J. et al. Removal of a subset of non-essential genes fully attenuates a highly virulent mycoplasma strain. Front. Microbiol. 0, 664 (2019).
Torres, L., Krüger, A., Csibra, E., Gianni, E. & Pinheiro, V. B. Synthetic biology approaches to biological containment: pre-emptively tackling potential risks. Essays Biochem 60, 393–410 (2016).
Google Scholar
Schmidt, M. & de Lorenzo, V. Synthetic bugs on the loose: containment options for deeply engineered (micro)organisms. Curr. Opin. Biotechnol. 38, 90–96 (2016).
Google Scholar
Moe-Behrens, G. H. G., Davis, R. & Haynes, K. A. Preparing synthetic biology for the world. Front. Microbiol. 4, 5 (2013).
Google Scholar
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
Google Scholar
Paetzold, B., Carolis, C., Ferrar, T., Serrano, L. & Lluch-Senar, M. In situ overlap and sequence synthesis during DNA assembly. ACS Synth. Biol. 2, 750–755 (2013).
Google Scholar
Calisto, B. et al. The EAGR box structure: a motif involved in mycoplasma motility. Mol. Microbiol. 86, 382–393 (2012).
Google Scholar
Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).
Zimmermann, C. U. & Herrmann, R. Synthesis of a small, cysteine-rich, 29 amino acids long peptide in Mycoplasma pneumoniae. FEMS Microbiol. Lett. 253, 315–321 (2005).
Green, M. R. & Sambrook, J. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2012).
Sprouffske, K. & Wagner, A. Growthcurver: an R package for obtaining interpretable metrics from microbial growth curves. BMC Bioinforma. 17, 1–4 (2016).
Orth, J. D., Thiele, I. & Palsson, B. O. What is flux balance analysis? Nat. Biotechnol. 28, 245–248 (2010).
Ebrahim, A., Lerman, J. A., Palsson, B. O. & Hyduke, D. R. COBRApy: COnstraints-based reconstruction and analysis for Python. BMC Syst. Biol. 7, 1–6 (2013). 2013 71.
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Google Scholar
Garrison, E. & Marth, G. Haplotype-based variant detection from short-read sequencing. Preprint at arXiv:1207.3907v2 [q-bio.GN] (2012).
Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff. Fly 6, 80–92 (2012).
Google Scholar
Broto, A., Gaspari, E., Miravet-Verde, S., dos Santos, V. A. P. M. & Isalan, M. A genetic toolkit and gene switches to limit Mycoplasma growth for biosafety applications (this paper). Zenodo https://doi.org/10.5281/zenodo.6323207 (2022).

