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Isolation and characterization of hemin-permeable, envelope-defective mutants of Salmonella typhimurium
Abstract:
From Salmonella typhimurium LT2 hemA (delta-aminolevulinic acid requiring) 15 mutants were isolated which grew on the hydrophobic compound hemin. All had increased sensitivity to antibiotics such as vancomycin, bacitracin, novobiocin, erythromycin, rifampin, and oleandomycin, and were considered to be envelope mutants (Env-). Half the mutants were rough , based on altered bacteriophage sensitivity and deoxycholate sensitivity, whereas the remainder were smooth; three of the smooth mutants were studied in detail. They gave increased uptake of gentian violet but no increase in leakage of a periplasmic protein, RNase I. The total membranes and fractions from sucrose gradient centrifugations representing inner and outer membranes of the wild type and three mutants were examined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focussing - PAGE (IEF-PAGE). The major outer membrane proteins (molecular weights (MW)33 000, 34 000, 35 000, and 36 000) showed no or very little alterations in the Env- mutants. In SA1926 (env-52) one protein spot at MW 48 000, proven to be an outer membrane protein, was missing, whereas a new spot appeared nearby, and other proteins in this area of the gel were reduced. An Env+ transductant selected from this strain had the wild-type protein pattern restored. The two other Env- mutants had similar but not identical changes in protein composition.
Insights
Salmonella typhimurium envelope mutants (Env-) requiring delta-aminolevulinic acid showed increased antibiotic sensitivity and altered outer membrane protein profiles. One mutant lacked a specific outer membrane protein, which was restored in a complemented strain.
Area of Science:
- Microbiology
- Bacterial genetics
- Membrane biology
Background:
- Salmonella typhimurium LT2 hemA mutants require delta-aminolevulinic acid for growth.
- These mutants were investigated for alterations in cell envelope properties.
Purpose of the Study:
- To characterize Salmonella typhimurium hemA mutants with altered growth requirements.
- To investigate the cell envelope properties, including antibiotic sensitivity and outer membrane protein composition, of these mutants.
Main Methods:
- Isolation and characterization of Salmonella typhimurium hemA mutants.
- Antibiotic sensitivity testing.
- Bacteriophage and deoxycholate sensitivity assays.
- Analysis of membrane proteins using SDS-PAGE and IEF-PAGE.
Main Results:
- Fifteen hemA mutants were isolated, all exhibiting increased sensitivity to multiple antibiotics and classified as envelope mutants (Env-).
- Half the mutants were rough, and half were smooth; smooth mutants showed increased gentian violet uptake but not increased RNase I leakage.
- SDS-PAGE and IEF-PAGE revealed alterations in outer membrane protein composition in some mutants, including the absence of a 48,000 MW protein in one strain (SA1926).
- Complementation of SA1926 restored the wild-type protein pattern.
Conclusions:
- Salmonella typhimurium hemA mutants can exhibit pleiotropic envelope defects, including increased antibiotic sensitivity and altered outer membrane protein profiles.
- Specific alterations in outer membrane proteins may be associated with the hemA mutation and associated envelope changes.