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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

Cultures ofBacillus subtilisrelease transforming DNA during the early exponential and stationary phases of growth. The pattern of release of transforming DNA was followed by measuring transformation in a system consisting of a non-transformable DNA donor and a differently marked transformable recipient. Transformation in this system seems to be at least as efficient as that induced by purified DNA. Fluorescence microscopy revealed that released DNA remained bound extracellular to intact cells. The release of DNA during early exponential growth seemed to be correlated with the cells' proneness to lysis; both DNA release and cell lysis were inhibited by chloramphenicol. In stationary cells, the release of DNA was neither correlated with a similar proneness to lysis nor inhibited by chloramphenicol.

Chromosomes in spores of a thymineless mutant of Bacillus subtilis strain 168 were shown to have a replication fork, unlike chromosomes in spores of the thy+ strain which are in a complete form. As a consequence the number of replication forks in germinating cultures is higher in the thystrain than in the thy+ one. Chromosome replication time (C) in the thy+ strain was found to be about 53 min for growth rates from 20 to 60 min. In the thystrain, C was about 75 min at high growth rates and increased with decreasing growth rate when the thymine concentration was not limiting. With limiting thymine concentrations in the medium replication velocity decreased independently of growth rate.Studies on chromosome replication in Escherichia coli and Bacillus subtilis have furnished considerable evidence that the regulation of the cell chromosome content as a function of growth rate is carried out only by varying the frequency of initiation of new cycles of replication (9, 11). Chromosome replication time is constant over a range of cell doubling time from 22 to 53 min (7). At slower growth rates, replication time increases with decreasing growth rates (3,4,8). It was recently shown (12) that in a thymineless mutant of E. coli suboptimal concentrations of thymine in the medium strongly affect the chromosome replication velocity but have almost no effect on the growth rate.In a recent study on the release of transforming deoxyribonucleic acid (DNA) by germinating spores of B. subtilis (5), it was observed that a significant amount of DNA was released by a thymineless mutant, whereas almost no extracellular DNA was detected in the culture of a thymine prototroph. Since DNA released was shown to be highly correlated with chromosome replication (5), the pattern of chromosome replication in thymineless and thymine-independent strains of B. subtilis has been studied. The present communication shows that the velocity of chromosome replication in a thymineless mutant is slower than that found in the independent strain and varies linearly with the growth rate. The velocity of replication in the thymine prototroph is independent of the growth rate. MATERIALS AND METHODSBacterial strains. Strains 168TT (thy trp-2) [The symbols indicate mutations to the following requirements: thy (thymine), trp (tryptophan), his (histidine), pur (adenine), leu (leucine), met (methionine), arg (arginine).] and 31 (irp-2 hisB5) were used as DNA donors. Strains BD25 (31) (purA16 leu-8 try-2 hisB5 metB5) and BD25 (26) (purA16 argC4 hisA8 metB5) were the DNA recipients in transformation experiments. Strain 168T (trp-2) was used for the preparation of synchronized DNA.Growth conditions. The media used for germination and transformation experiments have been as described previously (5). Spores were collected after 4 to 5 days of growth on Schaeffer's medium supplemented with 20 Ag of thymine per ml. Unless stated otherwise the growth medium M consisted of minimal medium (17), supplemented with 0.5% glucose, 50 ug of glutamic acid per ml, 150 ug of asparag...

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