Acta Microbiologica 22. (1975)
2. szám - Berek, I.–Miczák, A.-Kiss, I.–Ivánovics, G.–Durkó, I.: Genetic and Biochemical Analysis of Haemin Dependent Mutants of Bacillus subtilis
Acta microbiol. Acad. Sei. hung. 22, 157—167, 1975 GENETIC AND BIOCHEMICAL ANALYSIS OF HAEMIN DEPENDENT MUTANTS OF BACILLUS SUBTILIS I. Berek, A. Miczák, I. Kiss, G. Ivánovics and Irene Durkó Institute of Microbiology and Institute of Brain Research, University Medical School, Szeged (Received May 21, 1974) Summary. Numerous porphyrin auxotrophic mutants have been isolated from the 168 trpC2 strain of Bacillus subtilis by selection with streptomycin. Some of them could be supplemented with ALA while the majority grew only in the presence of haemin. Among the latter strains, the syntropism test allowed to distinguish two groups different in phenotype, viz., feeders accumulating ALA and non-feeders accumulating instead of ALA other porphyrin intermediates. On the basis of transductional studies, feeders and non-feeders could be divided into two and four groups, respectively. Biochemical investigation revealed that, with one exception, one enzyme of the porphyrin biosynthesis was coordinated to each hem locus. The following genes were identified: hemB —>- ALA-dehydrase; hemC y PBG-deaminase; hemE —*- uroporphyrinogen decarboxvlase; hemF * coproporphyrinogen oxidase; hemG —► protoporphyrin-iron-chelatase. Haemin dependent mutants can be isolated from the 168 trpC2 strain of Bacillus subtilis by different methods [1]. Selection on plates containing streptomycin was found to be the method most effective for the isolation of haemin auxotrophs [2]. Some of the strains deficient in porphyrin biosynthesis can grow in the presence of d-amino-laevulinic acid (ALA) and probably have a defect of ALA-synthetase; the requirement of the majoriy of auxotrophs can be met only with haemin. In the present study, a number of haemin requiring mutants have been isolated by selection with streptomycin [2] and their genetical and biochemical characterization has been attempted. The aim of these studies was to reveal any genetical heterogeneity among the haemin auxotrophs and to determine the number of the different hem loci in the isolated auxotrophs. The biochemical studies have been carried out to determine the enzyme deficiencies of the auxotrophs. Materials and methods Strains. Haemin dependent auxotrophs were isolated from B. subtilis strain 168 trpC2. Twenty-one of the one-hundred and ninety-eight haemin dependent isolates proved to be ALA auxotrophs. The strain hemAl was used as reference strain [3]. Media. A yeast extract peptone medium (YP) was used routinely as complete medium [4]. The chemically defined medium designated GGM [1] was supplemented with 50 ,ug/ml tryptophan and 2 fig/ml ALA was added for hemAl mutants. HCA medium [2] was used for cultivating haemin auxotrophs; it consisted of GGM medium supplemented with 50 fig tryptophan, 2.5 fig haemin, 50 fig cysteine and 0.5 mg bovine albumin (Calbiochem) per ml of medium. YPB (blood extract medium): citrated ox blood was washed in saline and the red blood cells Acta Microbiologica Academiae Scientiarum Hungaricae 22, 1975