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Relationship between mutant amidases of Pseudomonas aeruginosa and hydroxyurea as an inhibitor
Abstract:
Hydroxyurea inhibited growth of Pseudomonas aeruginosa strain AI 3 on media containing either acetanilide (N-phenyl acetamide) or acetamide as sole carbon sources. Mutants resistant to hydroxyurea inhibition of growth on acetanilide (OUCH strains) and acetamide (AmOUCH strains) displayed altered growth properties on various amide media compared with the parent strain AI3. AI3 amidase, which catalyses the initial step in the metabolism of acetanilide and acetamide, was inhibited by hydroxyurea in a time-dependent reaction that was slowly reversible at pH 7.2. Compared with AI3 amidase, amidases from the OUCH mutants were much less sensitive to inhibition by hydroxyurea and showed altered substrate specificities and pH/activity profiles; amidases from the AmOUCH mutants were more sensitive to hydroxyurea inhibition but showed increased activity towards acetamide. Association of resistance to hydroxyurea inhibition with a mutation in the amidase structural gene of strain OUCH 4 was confirmed by transduction.
Insights
Hydroxyurea inhibits Pseudomonas aeruginosa growth by targeting amidase. Resistant mutants reveal altered amidase properties, indicating mutations in the amidase structural gene confer resistance.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Pseudomonas aeruginosa utilizes acetanilide and acetamide as carbon sources.
- Hydroxyurea is known to inhibit bacterial growth.
Purpose of the Study:
- To investigate the effect of hydroxyurea on Pseudomonas aeruginosa growth using acetanilide and acetamide as carbon sources.
- To characterize hydroxyurea-resistant mutants and their amidase enzymes.
Main Methods:
- Growth inhibition assays with hydroxyurea.
- Isolation and characterization of hydroxyurea-resistant mutants (OUCH and AmOUCH strains).
- Enzyme inhibition studies and characterization of amidase activity and substrate specificity.
Main Results:
- Hydroxyurea inhibited growth of Pseudomonas aeruginosa on acetanilide and acetamide.
- Resistant mutants (OUCH and AmOUCH) exhibited altered growth and amidase properties.
- AI3 amidase was inhibited by hydroxyurea; mutant amidases showed reduced sensitivity or altered activity.
- Transduction confirmed mutation in the amidase structural gene for resistance.
Conclusions:
- Hydroxyurea targets the amidase enzyme in Pseudomonas aeruginosa.
- Mutations in the amidase structural gene confer resistance to hydroxyurea.
- Altered amidase properties in resistant mutants provide insights into enzyme function and metabolism.