Isolation of Haemin-requiring Mutants of Escherichia coli K12

Abstract

155Fifty-five haemin-requiring mutants were isolated from haemin-permeable mutants. According to their growth responses to haem precursors and their patterns of porphyrin accumulation, the 55 mutants fell into three groups which were judged to have defects in 5-aminolaevulinate dehydratase, ferrochelatase, and uroporphyrinogen I11 cosynthase or uroporphyrinogen decarboxylase. In mutants of the group deficient in 5-aminolaevulinate dehydratase, the mutations were adjacent to lac, and evidence is presented that the mutations were in hemB and were commonly deletions extending into proC. I N T R O D U C T I O NEscherichia coli K12 is impermeable to haemin under normal cultural conditions (SMrman et al., 1968), and genetic analysis of haem biosynthesis has depended on indirect methods. McConville & Charles (1975, 19798) described mutants of E. coli IS12 which are permeable to haemin. The present paper describes the isolation and study of haeminrequiring mutants derived from haemin-permeable mutants. M E T H O D SUnless stated otherwise, the methods were those used by McConville & Charles (1979~). Media. Glucose minimal medium (GM) was medium E of Vogel & Bonner (1956) supplemented with thiamin (5 mg 1-l) and glucose (5 g 1-l). Glucose minimal agar (GMA) was GM containing Difco Bacto agar (13 g P ) , autoclaved separately. When necessary, the glucose in GM was replaced by other sugars (2 g 1-l). Solutions of amino acids and other growth factors were sterilized by membrane filtration and aseptically added to minimal medium to give a final concentration of 40 mg l-l, except isoleucine and valine were added at 12 and 28 mgl-l, respectively, and 5-aminolaevulinic acid (5-ALA) at 1 mg 1-' . TNM contained (g 1-l): NaCl, 4; Difco Bacto nutrient broth, 8 ; MgS04. 7H20, 2.47. TNAM was TNM autoclaved with agar (13 g I-').Stock solutions of haemin (type 111, Sigma) and protoporphyrin IX (PP9; Calbiochem) were prepared by dissolving 0.2 g in 5 ml Tween 80 and slowly diluting to 50 ml with sterile distilled water; a few drops of alkali were added to give clear solutions. The procedure gave sterile solutions which were stored at -18°C and added aseptically to give concentrations of 25 mg 1-1 in TN media and 10 mg 1-1 in GM media.Genetical analysis. The transducing phage was Plkc; 12 min adsorption were allowed. Gene symbols and references to the E. culi linkage map follow Bachmann e f al. (1976). Details of the strains are given in Table 1.Assay ofporphyrins accumulated in cultures. Bacteria were inoculated into two 2 1 flasks containing glucose minimal medium (400 ml) plus sodium thiosulphate (10 mg 1-l) and 5-ALA (50 mg I-l); one flask contained haemin (10 mg 1-l). Proline was added if required. Incubation was for 48 h at 37 "C in an orbital incubator. Bacteria were then washed and resuspended in 10 ml of glass-distilled water, and a 1 ml sample was removed for drying and weighing. Porphyrins were extracted from the supernatant medium and bacteria by the method of Falk (1964). Coproporphyrin and protoporphyrin were extracted fr...

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).…”

Mutants of Escherichia coli K12 Permeable to Haemin

“…Unless stated otherwise, the methods were those used by McConville & Charles (1979). Glucose minimal medium (GM) was medium E of Vogel & Bonner (1956) supplemented with thiamin (5 mg 1-I) and glucose (5 g 1-I).…”

Molecular cloning of the 5-aminolevulinic acid dehydratase gene from Rhodobacter sphaeroides

“…It represents approximately 1 min on the E. coli chromosome * Corresponding author. (13). The growth cannot be stimulated either by the addition of 5-aminolevulinic acid or by porphobilinogen and hemin, as they do not pass through membranes (13).…”

“…(13). The growth cannot be stimulated either by the addition of 5-aminolevulinic acid or by porphobilinogen and hemin, as they do not pass through membranes (13). Growth of the bacteria was studied, and generation time was estimated to be 90 to 100 min.…”