A Streamlined Approach for Fluorescence Labelling of Low-Copy-Number Plasmids for Determination of Conjugation Frequency by Flow Cytometry
Bacterial conjugation plays a major role in the dissemination of antibiotic resistance and virulence traits through horizontal transfer of plasmids. Robust measurement of conjugation frequency of plasmids between bacterial strains and species is therefore important for understanding the transfer dyn...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-03-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/11/4/878 |
| _version_ | 1797604246257926144 |
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| author | Qin Qi Muhammad Kamruzzaman Jonathan R. Iredell |
| author_facet | Qin Qi Muhammad Kamruzzaman Jonathan R. Iredell |
| author_sort | Qin Qi |
| collection | DOAJ |
| description | Bacterial conjugation plays a major role in the dissemination of antibiotic resistance and virulence traits through horizontal transfer of plasmids. Robust measurement of conjugation frequency of plasmids between bacterial strains and species is therefore important for understanding the transfer dynamics and epidemiology of conjugative plasmids. In this study, we present a streamlined experimental approach for fluorescence labelling of low-copy-number conjugative plasmids that allows plasmid transfer frequency during filter mating to be measured by flow cytometry. A blue fluorescent protein gene is inserted into a conjugative plasmid of interest using a simple homologous recombineering procedure. A small non-conjugative plasmid, which carries a red fluorescent protein gene with a toxin–antitoxin system that functions as a plasmid stability module, is used to label the recipient bacterial strain. This offers the dual advantage of circumventing chromosomal modifications of recipient strains and ensuring that the red fluorescent protein gene-bearing plasmid can be stably maintained in recipient cells in an antibiotic-free environment during conjugation. A strong constitutive promoter allows the two fluorescent protein genes to be strongly and constitutively expressed from the plasmids, thus allowing flow cytometers to clearly distinguish between donor, recipient, and transconjugant populations in a conjugation mix for monitoring conjugation frequencies more precisely over time. |
| first_indexed | 2024-03-11T04:43:37Z |
| format | Article |
| id | doaj.art-77e56f6ca49f4ea4a82c088996a587d7 |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-11T04:43:37Z |
| publishDate | 2023-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-77e56f6ca49f4ea4a82c088996a587d72023-11-17T20:31:49ZengMDPI AGMicroorganisms2076-26072023-03-0111487810.3390/microorganisms11040878A Streamlined Approach for Fluorescence Labelling of Low-Copy-Number Plasmids for Determination of Conjugation Frequency by Flow CytometryQin Qi0Muhammad Kamruzzaman1Jonathan R. Iredell2Centre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, The University of Sydney, Westmead, Sydney, NSW 2145, AustraliaCentre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, The University of Sydney, Westmead, Sydney, NSW 2145, AustraliaCentre for Infectious Diseases and Microbiology, The Westmead Institute for Medical Research, The University of Sydney, Westmead, Sydney, NSW 2145, AustraliaBacterial conjugation plays a major role in the dissemination of antibiotic resistance and virulence traits through horizontal transfer of plasmids. Robust measurement of conjugation frequency of plasmids between bacterial strains and species is therefore important for understanding the transfer dynamics and epidemiology of conjugative plasmids. In this study, we present a streamlined experimental approach for fluorescence labelling of low-copy-number conjugative plasmids that allows plasmid transfer frequency during filter mating to be measured by flow cytometry. A blue fluorescent protein gene is inserted into a conjugative plasmid of interest using a simple homologous recombineering procedure. A small non-conjugative plasmid, which carries a red fluorescent protein gene with a toxin–antitoxin system that functions as a plasmid stability module, is used to label the recipient bacterial strain. This offers the dual advantage of circumventing chromosomal modifications of recipient strains and ensuring that the red fluorescent protein gene-bearing plasmid can be stably maintained in recipient cells in an antibiotic-free environment during conjugation. A strong constitutive promoter allows the two fluorescent protein genes to be strongly and constitutively expressed from the plasmids, thus allowing flow cytometers to clearly distinguish between donor, recipient, and transconjugant populations in a conjugation mix for monitoring conjugation frequencies more precisely over time.https://www.mdpi.com/2076-2607/11/4/878plasmidconjugationfluorescence labellingflow cytometryhorizontal gene transfer |
| spellingShingle | Qin Qi Muhammad Kamruzzaman Jonathan R. Iredell A Streamlined Approach for Fluorescence Labelling of Low-Copy-Number Plasmids for Determination of Conjugation Frequency by Flow Cytometry Microorganisms plasmid conjugation fluorescence labelling flow cytometry horizontal gene transfer |
| title | A Streamlined Approach for Fluorescence Labelling of Low-Copy-Number Plasmids for Determination of Conjugation Frequency by Flow Cytometry |
| title_full | A Streamlined Approach for Fluorescence Labelling of Low-Copy-Number Plasmids for Determination of Conjugation Frequency by Flow Cytometry |
| title_fullStr | A Streamlined Approach for Fluorescence Labelling of Low-Copy-Number Plasmids for Determination of Conjugation Frequency by Flow Cytometry |
| title_full_unstemmed | A Streamlined Approach for Fluorescence Labelling of Low-Copy-Number Plasmids for Determination of Conjugation Frequency by Flow Cytometry |
| title_short | A Streamlined Approach for Fluorescence Labelling of Low-Copy-Number Plasmids for Determination of Conjugation Frequency by Flow Cytometry |
| title_sort | streamlined approach for fluorescence labelling of low copy number plasmids for determination of conjugation frequency by flow cytometry |
| topic | plasmid conjugation fluorescence labelling flow cytometry horizontal gene transfer |
| url | https://www.mdpi.com/2076-2607/11/4/878 |
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