Gene Silencing Through CRISPR Interference in Mycoplasmas

Overview

Journal Microorganisms

Specialty Microbiology

Date 2022 Jun 24

PMID 35744677

Authors

Daria V Evsyutina

Gleb Y Fisunov

Olga V Pobeguts

Sergey I Kovalchuk

Vadim M Govorun

Affiliations

Soon will be listed here.

Abstract

Mycoplasmas are pathogenic, genome-reduced bacteria. The development of such fields of science as system and synthetic biology is closely associated with them. Despite intensive research of different representatives of this genus, genetic manipulations remain challenging in mycoplasmas. Here we demonstrate a single-plasmid transposon-based CRISPRi system for the repression of gene expression in mycoplasmas. We show that selected expression determinants provide a level of dCas9 that does not lead to a significant slow-down of mycoplasma growth. For the first time we describe the proteomic response of genome-reduced bacteria to the expression of exogenous . The functionality of the resulting vector is confirmed by targeting the three genes coding transcription factors-, essential , , and histone-like protein in . As a result, the expression level of each gene was decreased tenfold and influenced the mRNA level of predicted targets of transcription factors. To illustrate the versatility of this vector, we performed a knockdown of metabolic genes in a representative member of another cluster of the Mycoplasma genus-. The developed CRISPRi system is a powerful tool to discover the functioning of genes that are essential, decipher regulatory networks and that can help to identify novel drug targets to control Mycoplasma infections.

Citing Articles

BAC-browser: the tool for synthetic biology.

Semashko T, Fisunov G, Shevelev G, Govorun V BMC Bioinformatics. 2025; 26(1):27.

PMID: 39849360 PMC: 11758742. DOI: 10.1186/s12859-025-06049-9.

WhiA transcription factor provides feedback loop between translation and energy production in a genome-reduced bacterium.

Fisunov G, Tsvetkov V, Tsoy E, Evsyutina D, Vedyaykin A, Garanina I Front Microbiol. 2025; 15:1504418.

PMID: 39764455 PMC: 11701221. DOI: 10.3389/fmicb.2024.1504418.

A toolbox for manipulating the genome of the major goat pathogen, subsp. .

Gourgues G, Manso-Silvan L, Chamberland C, Sirand-Pugnet P, Thiaucourt F, Blanchard A Microbiology (Reading). 2024; 170(1).

PMID: 38193814 PMC: 10866025. DOI: 10.1099/mic.0.001423.

Gene editing tools for mycoplasmas: references and future directions for efficient genome manipulation.

Zhao G, Lu D, Li M, Wang Y Front Microbiol. 2023; 14:1191812.

PMID: 37275127 PMC: 10232828. DOI: 10.3389/fmicb.2023.1191812.

Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System.

Ipoutcha T, Rideau F, Gourgues G, Arfi Y, Lartigue C, Blanchard A Appl Environ Microbiol. 2022; 88(17):e0099622.

PMID: 36000854 PMC: 9469718. DOI: 10.1128/aem.00996-22.

References

Mariscal A, Kakizawa S, Hsu J, Tanaka K, Gonzalez-Gonzalez L, Broto A . Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells. ACS Synth Biol. 2018; 7(6):1538-1552. DOI: 10.1021/acssynbio.8b00028. View

Alvarez R, Blaylock M, Baseman J . Surface localized glyceraldehyde-3-phosphate dehydrogenase of Mycoplasma genitalium binds mucin. Mol Microbiol. 2003; 48(5):1417-25. DOI: 10.1046/j.1365-2958.2003.03518.x. View

Yavlovich A, Rechnitzer H, Rottem S . Alpha-enolase resides on the cell surface of Mycoplasma fermentans and binds plasminogen. Infect Immun. 2007; 75(12):5716-9. PMC: 2168360. DOI: 10.1128/IAI.01049-07. View

Rideau F, Le Roy C, Sagne E, Renaudin H, Pereyre S, Henrich B . Random transposon insertion in the Mycoplasma hominis minimal genome. Sci Rep. 2019; 9(1):13554. PMC: 6753208. DOI: 10.1038/s41598-019-49919-y. View

Mateos J, Tilmes V, Madrigal P, Severing E, Richter R, Rijkenberg C . Divergence of regulatory networks governed by the orthologous transcription factors FLC and PEP1 in Brassicaceae species. Proc Natl Acad Sci U S A. 2017; 114(51):E11037-E11046. PMC: 5754749. DOI: 10.1073/pnas.1618075114. View

Hosie A, Poole P . Bacterial ABC transporters of amino acids. Res Microbiol. 2001; 152(3-4):259-70. DOI: 10.1016/s0923-2508(01)01197-4. View

Zhang X, Smits A, van Tilburg G, Ovaa H, Huber W, Vermeulen M . Proteome-wide identification of ubiquitin interactions using UbIA-MS. Nat Protoc. 2018; 13(3):530-550. DOI: 10.1038/nprot.2017.147. View

Razin S . The mycoplasmas. Microbiol Rev. 1978; 42(2):414-70. PMC: 281436. DOI: 10.1128/mr.42.2.414-470.1978. View

Schafer H, Turgay K . Spx, a versatile regulator of the Bacillus subtilis stress response. Curr Genet. 2019; 65(4):871-876. DOI: 10.1007/s00294-019-00950-6. View

10.

Pflaum K, Tulman E, Beaudet J, Canter J, Geary S . Variable Lipoprotein Hemagglutinin A Gene () Expression in Variant Mycoplasma gallisepticum Strains . Infect Immun. 2018; 86(11). PMC: 6204691. DOI: 10.1128/IAI.00524-18. View

11.

Peters J, Colavin A, Shi H, Czarny T, Larson M, Wong S . A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria. Cell. 2016; 165(6):1493-1506. PMC: 4894308. DOI: 10.1016/j.cell.2016.05.003. View

12.

Ipoutcha T, Tsarmpopoulos I, Talenton V, Gaspin C, Moisan A, Walker C . Multiple Origins and Specific Evolution of CRISPR/Cas9 Systems in Minimal Bacteria (). Front Microbiol. 2019; 10:2701. PMC: 6882279. DOI: 10.3389/fmicb.2019.02701. View

13.

Maglennon G, Cook B, Matthews D, Deeney A, Bosse J, Langford P . Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae. Vet Res. 2013; 44:63. PMC: 3765554. DOI: 10.1186/1297-9716-44-63. View

14.

Fisunov G, Evsyutina D, Semashko T, Arzamasov A, Manuvera V, Letarov A . Binding site of MraZ transcription factor in Mollicutes. Biochimie. 2016; 125:59-65. DOI: 10.1016/j.biochi.2016.02.016. View

15.

Fisunov G, Garanina I, Evsyutina D, Semashko T, Nikitina A, Govorun V . Reconstruction of Transcription Control Networks in Mollicutes by High-Throughput Identification of Promoters. Front Microbiol. 2016; 7:1977. PMC: 5138195. DOI: 10.3389/fmicb.2016.01977. View

16.

Glass J, Assad-Garcia N, Alperovich N, Yooseph S, Lewis M, Maruf M . Essential genes of a minimal bacterium. Proc Natl Acad Sci U S A. 2006; 103(2):425-30. PMC: 1324956. DOI: 10.1073/pnas.0510013103. View

17.

Hutchison 3rd C, Chuang R, Noskov V, Assad-Garcia N, Deerinck T, Ellisman M . Design and synthesis of a minimal bacterial genome. Science. 2016; 351(6280):aad6253. DOI: 10.1126/science.aad6253. View

18.

Pereyre S, Sirand-Pugnet P, Beven L, Charron A, Renaudin H, Barre A . Life on arginine for Mycoplasma hominis: clues from its minimal genome and comparison with other human urogenital mycoplasmas. PLoS Genet. 2009; 5(10):e1000677. PMC: 2751442. DOI: 10.1371/journal.pgen.1000677. View

19.

Semashko T, Evsyutina D, Ladygina V, Zubov A, Rakovskaya I, Kovalchuk S . Data on proteome of cultivated with arginine or thymidine as a carbon source. Data Brief. 2020; 31:106034. PMC: 7390850. DOI: 10.1016/j.dib.2020.106034. View

20.

Fisunov G, Pobeguts O, Ladygina V, Zubov A, Galyamina M, Kovalchuk S . Thymidine utilisation pathway is a novel phenotypic switch of . J Med Microbiol. 2022; 71(1). PMC: 8895549. DOI: 10.1099/jmm.0.001468. View