Genetic Analysis, by Means of Transformation, of Histidine Linkage Groups in Bacillus Subtilis

Abstract

Within the past few years, new approaches have been employed in microorganisms and bacteriophage systems for the study of physiological genetics and fine structure analysis of genetic loci." 2 A detailed study of the correlation between genetic defects and enzymatic blocks in the histidine biosynthetic pathway was described recently in Salmonella,3 4 in which fine structure mapping of histidinerequiring mutants was constructed by means of transduction. This study, correlated with the study of enzymes involved in-the biosynthesis of histidine,4 suggested that the gene loci are linearly arranged in an order that corresponds to the sequence of the reactions they control.The use of DNA preparations, by means of transformation, for genetic analysis offers an opportunity to study the relationship between the molecular structure of DNA and the genetic map. Such a study was recently described in Pneumococcus.5The present investigation is concerned with the genetic study of linkage groups controlling the biosynthesis of histidine in Bacillus subtilis and their relationship to the indole locus, using as a tool the transformation phenomenon. Histidinerequiring (his-) mutants were isolated when spores of B. subtilis were heated to a high temperature (1550) in the dry state in a vacuum.6 Such mutants, as well as several others, were used in an attempt to analyze the genetic location of the sites involved. In addition, preliminary attempts were made to identify the corresponding biochemical blocks of such mutants.Materials and Methods.-The parent strain of all the mutants used was the indole-requiring strain 168 ind-of Bacillus subtilis, kindly provided by Dr. J. Spizizen, Western Reserve University. From this strain, nine his-mutants were induced by heat (mutants 12, 13, 16, (168 ind+/Sa) was isolated. Crude deoxyribonucleic acid (DNA), prepared from this strain, gave a high frequency of transformation from sulfanilamide sensitivity to sulfanilamide resistance.7 This DNA was used to transform the previously mentioned his-mutants to sulfanilamide resistance (strains 12/Sa, 13/Sa, etc.).Yeast extract (final concentration 0.002 per cent) was added to all minimal media.Bacterial stocks were maintained on potato agar slants8 stored at room temperature.Bacterial extracts for enzymatic study: Bacteria were grown for 7 to 8 hours in minimal medium" supplemented with 0.5 per cent glucose, 5 jg of L-histidine, and 5 j.g of L-tryptophan/ml. Harvesting the bacteria in the logarithmic phase was found to be critical for optimal enzymatic activity.9 The enzymatic reactions were carried out as described recently.'0 Biochemical methods: The cultures were grown in media indicated above and incubated with aeration for 72 hr at 37°. The cells were centrifuged and the supernatant fluids were tested for the presence of various metabolites. The Bratton-Marshall reaction" for detection of diazotizable 56

Genetic evidence that Neisseria gonorrhoeae produces specific receptors for transferrin and lactoferrin

“…To control for differences in competency, recipients were transformed with FA19 DNA. This allowed calculation of a recombination index (RI) (8,10). For example, the RI in a transformation cross between two TF-mutants = (TF+ transformants using mutant DNA/drugr transformants using mutant GONOCOCCAL TRANSFERRIN AND LACTOFERRIN RECEPTORS DNA) x (drugr transformants using FA19 DNA/TF+ transformants using FA19 DNA).…”

The Genetic Relationship and Phenotypic Expression of Mutations Endowing Pneumococcus with Resistance to Erythromycin

“…The analysis of genetic fine structure has been extended to deoxyribonucleic acid (DNA) transforming factors (Hotchkiss & Goodgal & Herriott, 1957), but also numerous mutable sites separable by recombination within each region (Ephrussi-Taylor, 1951 ;Ravin, 1960;Lacks & Hotchkiss, 1960;Ephrati-Elizur et al 1961; Rotheim & Ravin, to be published). Recombinable mutations borne by the same molecule of DNA can often be arranged in a linear order (Hotchkiss & Evans, 1958;Ravin, 1960;Lacks & Hotchkiss, 1960 ;Ephrati-Elizur et al 1961).…”

“…Recombinable mutations borne by the same molecule of DNA can often be arranged in a linear order (Hotchkiss & Evans, 1958;Ravin, 1960;Lacks & Hotchkiss, 1960 ;Ephrati-Elizur et al 1961).…”

Histidine biosynthesis genes in Lactococcus lactis subsp. lactis

“…In Staphylococcus aureus, six of the genes (hisE, -A, -B, -C, -D, and -G) are clustered, whereas in Streptomyces coelicolor, five (and possibly six) genes (hisD, -C, -B, -H, -A, and possibly -F) form an operon and two (hisIE and hisB) are independent (30,36). In Bacillus subtilis, the genes map in two locations, one grouping seven genes (hisA, -B, -D, -F, -G, -C, and -IE) and the other containing a single gene (hisH, corresponding to hisC in E. coli) (11,18,25,37). The hisI gene from a methanogenic archaebacterium is separated from the other his genes (6).…”