CTP limitation increases expression of CTP synthase in Lactococcus lactis

Area of Science:

• Microbiology
• Molecular Biology
• Gene Regulation

Background:

• CTP synthase, encoded by the pyrG gene, is crucial for converting UTP to CTP.
• Cytidine limitation in Lactococcus lactis leads to a significant decrease in the CTP pool.
• This decrease impacts central cellular metabolism, inhibiting RNA and protein synthesis.

Purpose of the Study:

• To investigate the regulation mechanism of the L. lactis pyrG gene.
• To understand how CTP levels influence pyrG gene expression.
• To elucidate the role of the pyrG leader sequence in gene regulation.

Main Methods:

• Construction of a Lactococcus lactis pyrG mutant requiring cytidine.
• Utilizing a pyrG-lacLM transcriptional fusion to monitor pyrG gene expression via beta-galactosidase activity.
• Analyzing the effects of cytidine limitation on CTP pool size and pyrG expression levels.

Main Results:

• A 10-fold decrease in CTP pool size immediately increased L. lactis pyrG gene expression.
• PyrG expression reached a level 37-fold higher than the uninduced level.
• PyrG expression is solely dependent on cellular CTP concentration, not UTP, GTP, or ATP levels.
• A potential terminator structure in the pyrG leader sequence was identified as essential for regulation.

Conclusions:

• A novel attenuator mechanism regulates L. lactis pyrG expression based on CTP concentration.
• At normal CTP levels, a terminator forms in the pyrG leader, reducing CTP synthase expression.
• At low CTP levels, RNA polymerase pausing allows antiterminator formation, enabling transcription.
• This model suggests CTP regulation of pyrG in L. lactis, and potentially other Gram-positive bacteria, does not involve trans-acting proteins.