Related Experiment Videos
Pleiotropic consequences of mutations towards antibiotic-hypersensitivity in Serratia marcescens
Abstract:
Various mutants (oxas) were isolated from Serratia marcescens SM-6 by selecting for hypersensitivity towards oxacillin. All mutants found are highly pleiotropic and able to yield spontaneous revertants which behave like the wild-type. Mutant W 1421 mostly studied shows the following phenotypic properties not found in the wild-type: (1) The growth is hypersensitive to various antibiotics, detergents and dyes which differ remarkably in their chemical structure and antibacterial action-mechanism, (2) the cells can be easily solubilized by 0;05% Sodium-dodecyl-sulfate, (3) the cells allow the adsorption of the rough-mutant specific Salmonella phage 6SR; (4) strong cellular binding of crystal violet, (5) agglutination of the cells in 0.3% auramin solution and (6) reduced formation of red pigment. Strain W 1421 is assumed to be a lipopolysaccharide-defective mutant. The outer membrane of mutant W 1421 analyzed by Sodium-dodecylsulfate-polyacrylamide gel electrophoresis possesses a single protein less than that of the wild-type. Mutant W 1421 is further characterized by its low exolipase activity; exoprotease and exonuclease activities are as in the wild-type. This specific exoenzyme deficiency can be overcome either by backmutation to oxacillin-resistance or by growing mutant W 1421 in a medium supplemented with certain non-metabolizable polysaccharides, e.g. glycogen or pectin B. Both polysaccharides increase the exolipase activity of the wild-type too.
Insights
Researchers isolated oxacillin-hypersensitive mutants (oxas) from Serratia marcescens. Mutant W 1421, a lipopolysaccharide-defective strain, exhibits altered outer membrane proteins and reduced exolipase activity, which can be restored by specific polysaccharides.
Area of Science:
- Microbiology
- Bacterial Genetics
Background:
- Serratia marcescens is a Gram-negative bacterium with a complex outer membrane.
- Lipopolysaccharides (LPS) are crucial components of the Gram-negative outer membrane, influencing cell surface properties and interactions.
- Antibiotic resistance and hypersensitivity can arise from mutations affecting outer membrane composition.
Purpose of the Study:
- To isolate and characterize mutants of Serratia marcescens SM-6 exhibiting hypersensitivity to oxacillin.
- To investigate the phenotypic and molecular basis of oxacillin hypersensitivity in isolated mutants.
- To explore the role of lipopolysaccharides and outer membrane proteins in bacterial resistance and enzyme activity.
Main Methods:
- Isolation of oxacillin-hypersensitive mutants (oxas) through selection.
- Phenotypic characterization including antibiotic sensitivity, detergent solubility, phage adsorption, dye binding, and pigment formation.
- Sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for outer membrane protein analysis.
- Enzyme activity assays for exolipase, exoprotease, and exonuclease.
Main Results:
- Multiple pleiotropic oxacillin-hypersensitive mutants were obtained, all capable of reverting to wild-type characteristics.
- Mutant W 1421 displayed hypersensitivity to various agents, increased susceptibility to Sodium-dodecyl-sulfate, phage adsorption, crystal violet binding, and agglutination.
- SDS-PAGE revealed a deficiency in a single outer membrane protein in mutant W 1421, consistent with a lipopolysaccharide-defective phenotype.
- Mutant W 1421 exhibited significantly reduced exolipase activity, while exoprotease and exonuclease activities remained comparable to the wild-type.
Conclusions:
- Mutant W 1421 is a lipopolysaccharide-defective mutant of Serratia marcescens with altered outer membrane composition and reduced exolipase activity.
- The observed phenotypic changes in W 1421 are linked to its lipopolysaccharide defect.
- Exolipase activity can be restored in the mutant by backmutation or supplementation with specific non-metabolizable polysaccharides, suggesting a regulatory role for polysaccharides in enzyme secretion.