Mutations affecting glutamine synthetase activity in Salmonella typhimurium

Area of Science:

• Microbiology
• Molecular Biology
• Enzymology

Background:

• Glutamine synthetase (GS) is a critical enzyme in nitrogen metabolism.
• Altered GS activity can lead to auxotrophy and affect bacterial growth.
• Salmonella typhimurium serves as a model organism for studying bacterial genetics and metabolism.

Purpose of the Study:

• To devise a positive selection method for isolating Salmonella typhimurium mutants with altered glutamine synthetase activity.
• To characterize the biochemical and genetic properties of these mutants.
• To investigate the relationship between GS activity, adenylylation state, and enzyme structure.

Main Methods:

• Positive selection using D-histidine as the sole histidine source to identify auxotrophic mutants.
• Biochemical assays to measure glutamine synthetase activity and response to divalent cations.
• Enzyme characterization, including heat lability studies.
• Genetic mapping of mutations to the Salmonella chromosome.

Main Results:

• Isolation of two types of glutamine-requiring mutants: those with significantly reduced GS activity (<10% of parent) and those with altered GS response to divalent cations.
• Mutants with altered GS activity showed enzyme properties mimicking highly adenylylated, presumed inactive, wild-type enzyme.
• One mutant exhibited a heat-labile GS, indicating a structural alteration.
• Mutations were mapped to the glutamine synthetase structural gene (glnA) at approximately 125 minutes on the Salmonella chromosome.
• Mutants retained normal glutamate dehydrogenase activity.

Conclusions:

• A novel positive selection strategy effectively isolated Salmonella typhimurium mutants with defective glutamine synthetase.
• The identified mutants display diverse alterations in GS activity, adenylylation state, and structural integrity.
• These findings suggest that mutations primarily affect the glnA gene, providing insights into GS regulation and function in vivo.