High copy number mutants derived from Corynebacterium glutamicum cryptic plasmid pAM330 and copy number control

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Plasmids are crucial tools in genetic engineering, particularly in bacteria like Corynebacterium glutamicum.
• Controlling plasmid copy number is essential for efficient gene expression and strain development.
• The cryptic plasmid pAM330 from Brevibacterium lactofermentum is a basis for shuttle vectors.

Purpose of the Study:

• To investigate the genetic basis for high plasmid copy number in Corynebacterium glutamicum.
• To identify the regulatory mechanism controlling the replication of pAM330-derived plasmids.
• To characterize the role of a small RNA in plasmid copy number regulation.

Main Methods:

• Isolation and characterization of high copy number plasmid mutants (pVC7H1).
• Site-directed mutagenesis to introduce specific base changes (copA1, copA2).
• Genetic analysis and RNA-sequencing (RNA-Seq) to identify regulatory elements and transcripts.
• Determination of transcription start sites.

Main Results:

• A single base conversion (copA1) in the plasmid increased copy number from 11 to 112 per chromosome in C. glutamicum.
• A second mutation (copA2) further elevated copy number to over 300 per chromosome.
• A small RNA (sRNA1) was identified upstream of the repA gene, acting as a potential copy number regulator.
• sRNA1 likely functions via an antisense RNA mechanism, sequestering the ribosome binding site of repA mRNA.

Conclusions:

• Specific mutations in the plasmid's control region can dramatically enhance its copy number in C. glutamicum.
• A novel small RNA (sRNA1) plays a critical role in regulating plasmid replication.
• The secondary structure of sRNA1 is important for its function in plasmid copy number control.