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.

Transcription of the glutamyl-tRNA reductase (hemA) gene in Salmonella typhimurium and Escherichia coli: role of the hemA P1 promoter and the arcA gene product

“…Similar to the previous experiments, the E. coli hemA promoter directed a lower level of lac expression during exponential growth than in stationary phase for cultures grown with ALA. We tested two other wild-type S. typhimurium strains; both had induction ratios for the E. coli hemA-lac fusion similar to those shown in Table 4. We also used an S. typhimurium strain which is permeable to heme (22) to determine whether heme is as effective as ALA in reducing the expression of hemA-lac. No substantial difference was seen between the induction behavior of strains grown with heme and that of strains grown with ALA (Table 5).…”

Isolation of a cytochrome-deficient mutant strain of Sporomusa sphaeroides not capable of oxidizing methyl groups

“…In contrast to this idea, addition of heme or 8-aminolevulinic acid to the growth medium did not result in reversion of the observed phenotypical defects of the mutant. However, this finding does not exclude a defect in heme synthesis, since it has to be kept in mind that Eschenchia coli or Salmonella typhimunum mutants defective in heme synthesis are unable to take up heme from the medium unless secondary mutations are introduced that allow heme to enter the cells (17,23).…”

The genes required for heme synthesis in Salmonella typhimurium include those encoding alternative functions for aerobic and anaerobic coproporphyrinogen oxidation

“…These mutants (hemA and hemL) are unable to take up heme and therefore cannot use heme provided in the medium to satisfy their ALA requirement. In previous studies, this problem was solved by using secondary mutations that permit heme to enter cells (31,41,42).…”