Methods of introduction of foreign DNA into mycobacteria

Area of Science:

• Microbiology
• Molecular Biology
• Genetic Engineering

Background:

• Mycobacterial species possess complex genetic systems, including cryptic genes that regulate important cellular functions.
• Understanding genetic determinants of steroid biotransformation and drug resistance is crucial for developing novel therapeutic strategies.

Purpose of the Study:

• To introduce plasmid DNA into mycobacterial cells using triparental conjugation and electrotransformation.
• To investigate the activation of a chromosomal cryptic kanamycin resistance (KmR) gene in *Mycobacterium fortuitum* mutants.
• To generate a collection of mutants for studying the genetic basis of steroid biotransformation and drug resistance.

Main Methods:

• Triparental conjugation was employed for plasmid DNA transfer.
• Electrotransformation was utilized as an alternative method for DNA introduction.
• Shuttle plasmid pMY10 and integration vector pUS 903 were used in *M. fortuitum* mutants.

Main Results:

• Successful introduction of plasmid DNA into *M. fortuitum* was achieved via both methods.
• Introduction of shuttle plasmid pMY10 led to the activation of the chromosomal cryptic KmR gene.
• The integration vector pUS 903 facilitated the generation of a mutant collection.

Conclusions:

• Both triparental conjugation and electrotransformation are effective for genetic manipulation of mycobacteria.
• Activation of the cryptic KmR gene demonstrates the utility of shuttle plasmids for genetic studies.
• The generated mutants provide valuable tools for elucidating the genetic control of steroid biotransformation and drug resistance in mycobacteria.