Function, expression, specificity, diversity and incompatibility of actinobacteriophage parABS systems

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Over 180 sequenced actinobacteriophages belong to Cluster A, sharing similar genome sequences and architectures.
• Cluster A phages are temperate, typically possessing an integration cassette with integrase and attP.
• A subset of these phages (approx. 20%) lacks integration cassettes, featuring a 1.4 kbp segment with partitioning genes (parA, parB) instead.

Purpose of the Study:

• To investigate the function and regulation of the parABS partitioning system in Cluster A actinobacteriophages.
• To understand the role of this system in establishing and maintaining lysogeny in Mycobacterium smegmatis.
• To characterize the interaction between phage partitioning proteins and their DNA binding sites.

Main Methods:

• Analysis of sequenced actinobacteriophage genomes to identify variations in genome organization.
• Prophage stability assays in Mycobacterium smegmatis infected with phage RedRock.
• Gene expression analysis of parAB genes during infection and lysogeny.
• DNA-protein binding studies to characterize the interaction of RedRock ParB with parS sites.

Main Results:

• Phage RedRock establishes stable lysogens in Mycobacterium smegmatis, with its prophage replicating at 2.4 copies/chromosome.
• The parABS system confers prophage maintenance, with parAB genes expressed upon infection and downregulated during lysogeny.
• RedRock ParB binds specifically to parS-L and parS-R sites, which consist of repeated 8 bp motifs, regulating gene expression.

Conclusions:

• The parABS partitioning system is crucial for stable lysogeny and maintenance of certain Cluster A actinobacteriophages.
• Regulation of parABS expression via ParB binding to parS sites ensures proper prophage establishment and stability.
• Actinobacteriophage parABS cassettes offer diverse and specific tools for mycobacterial genetics.