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Updated: Jul 2, 2026
Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation
Published on: January 4, 2017
Bacterial conjugation in the cytoplasm of mouse cells
Yin Mei Lim1, Ad J C de Groof, Mrinal K Bhattacharjee
1Department of Genetics and Development, Columbia University, New York, NY 10032, USA.
Abstract:
Intracellular pathogenic organisms such as salmonellae and shigellae are able to evade the effects of many antibiotics because the drugs are not able to penetrate the plasma membrane. In addition, these bacteria may be able to transfer genes within cells while protected from the action of drugs. The primary mode by which virulence and antibiotic resistance genes are spread is bacterial conjugation. Salmonellae have been shown to be competent for conjugation in the vacuoles of cultured mammalian cells. We now show that the conjugation machinery is also functional in the mammalian cytosol. Specially constructed Escherichia coli strains expressing Shigella flexneri plasmid and chromosomal virulence factors for escape from vacuoles and synthesizing the invasin protein from Yersinia pseudotuberculosis to enhance cellular entry were able to enter 3T3 cells and escape from the phagocytic vacuole. One bacterial strain (the donor) of each pair to be introduced sequentially into mammalian cells had a conjugative plasmid. We found that this plasmid could be transferred at high frequency. Conjugation in the cytoplasm of cells may well be a general phenomenon.
Insights
Intracellular bacteria like Salmonella evade antibiotics by transferring genes via conjugation. This study shows bacterial conjugation machinery functions within the mammalian cell cytosol, not just vacuoles.
Area of Science:
- Microbiology
- Cell Biology
- Genetics
Background:
- Intracellular pathogens like Salmonella and Shigella evade antibiotics due to poor drug penetration of the host cell plasma membrane.
- Gene transfer, particularly via bacterial conjugation, is a primary mechanism for spreading virulence and antibiotic resistance genes.
- Salmonella has demonstrated competence for conjugation within mammalian cell vacuoles.
Purpose of the Study:
- To investigate if bacterial conjugation machinery is functional within the mammalian cell cytosol.
- To determine if gene transfer can occur between bacteria residing in the host cell cytoplasm.
Main Methods:
- Engineered Escherichia coli strains expressing virulence factors for vacuole escape and invasin for cellular entry.
- Introduction of engineered bacteria into 3T3 cells.
- Sequential introduction of bacterial strains, one containing a conjugative plasmid (donor).
- Assessment of plasmid transfer frequency within the host cell.
Main Results:
- Engineered E. coli strains successfully entered 3T3 cells and escaped the phagocytic vacuole.
- Conjugative plasmid transfer occurred at high frequency between bacteria within the mammalian cell cytosol.
- Demonstrated functionality of conjugation machinery in the mammalian cytosol.
Conclusions:
- Bacterial conjugation is functional within the mammalian cell cytosol, extending beyond previously observed vacuolar activity.
- Cytosolic conjugation may represent a general mechanism for gene transfer among intracellular bacteria.
- This finding has significant implications for understanding antibiotic resistance dissemination and developing novel therapeutic strategies.
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