Hemin-Deficient Mutants of Salmonella typhimurium

Abstract

Nine hemin-deficient mutants of Salmonella typhimurium LT2 were isolated as neomycin-resistant colonies. Five of these mutants could be stimulated by A-aminolevulinic acid (A-ALA), thus representing hemA mutants. Since S. typhimurium LT2 is not able to incorporate hemin, the identification of the mutants not stimulated by A-ALA was made on the basis of the simultaneous loss of catalase activity and cytochromes. The hemA gene was mapped by conjugation in the trp region, probably in the order purB-pyrD-hemA-trp; the episome FT71 trp does not carry the hemA gene. Transductional intercrosses by phage P22 indicate that hemA 11, 12, 13, and 37 are at very closely linked sites, whereas hemA14 is at a more distant site in the same or an adjacent gene. No joint transduction was detected between hemA and trp or pyrF. The loci affected in the other hemin-deficient mutants were linked in conjugation to the pro+ marker (frequency of linkage, 88 to 97%), but cotransduction of the two markers could not be obtained. The episome F lac hem purE, which originates from Escherichia coli K-12, could complement these hemin-deficient mutants of S. typhimurium LT2. As a result, the sequence of the markers on the chromosome of S. typhimurium LT2 is probably pro heme purE, analogous to the sequence found in E. coli K-12. Thus, the chromosome of S. typhimurium also possesses two hem regions, with a location similar to that described in E. coli K-12. Research of the last years has shown a great similarity of chromosomal structure between Salmonella typhimurium LT2 and Escherichia coli K-12, two organisms for which we possess wellelaborated chromosomal maps (3, 11). This is not surprising since the two organisms are closely related and probably originated from a common ancestor, or one species evolved first, with the other having originated from the first by evolution. Recently we described the position of the hem loci on the chromosome of E. coh K-12 (9). The

Electron‐Transport Chains of Escherichia coli

“…Colonies were selected which could grow on plates containing sodium succinate (0.2O/,, w/v) as sole carbon source, the amino acids (all a t 0.010/,, w/v) required by this strain, and 5-aminolaevulinic acid (25 pM) but not on plates deficient in this compound. One such mutant, designated A201, was chosen for further studies and the mutant allele shown to be 2-4O/, cotransducible with tryptophan, indicating that it corresponds to the hemA mutation described previously [12,13]. Strain A201 could not grow on glycerol or citric-acid-cycle metabolites unless supplemented with 6-aminolaevulinic acid : the concentration of 5-aminolaevulinic acid required to achieve growth rates equivalent to those of the parental strain on these substrates in batch culture was about 1 pM as previously described [12].…”

“…However, from these experiments it cannot be decided if these genes are concerned with the regulation of haem synthesis or with the synthesis of the apoprotein of a particular cytochrome. Methods for the isolation of 5-aminolaevulinic acid synthetase-deficient mutants [12,13] and mutants auxotrophic for haematin [ 14,151 have been reported for E . coli.…”

Use of Neomycin in the Isolation of Mutants Blocked in Energy Conservation in Escherichia coli

“…Selection for neomycin resistance has been used in the isolation of hemin-deficient mutants of Escherichia coli K-12 (13) and Salmonella typhimurium (12). It was postulated that respiratory-deficient cells incorporated less neomycin than normal cells (12). One explanation for this may be the inability of these mutants to generate sufficient energy to concentrate the antibiotic.…”

Uroporphyrin-accumulating mutant of Escherichia coli K-12

“…Mapping of hemA and hemB genes in Salmonella typhimurium LT2 showed that both have similar location to the corresponding genes in E. coli K-12 (30); this seems also to be true for at least a third hem gene in the two organisms (A. Sasarman et al, unpublished data). A relatively exhaustive mapping of hem genes in S. aureus showed that in this organism the hem loci were clustered in a single region of the chromosome (37).…”

“…Heme-deficient mutants of bacteria are partially resistant to aminoglycoside antibiotics, which were often used for their isolation (1,2,16,29,30,31,37). The mechanisms of this resistance is not yet well established, and a diminished uptake of the drug has been suggested (30). The resistance to aminoglycoside antibiotics seems to be a common property of several types of respiratory mutants of bacteria, for menaquinone-deficient mutants of Staphylococcus aureus are also resistant to them (32).…”