Hiuga Saito

Preparation of transforming deoxyribonucleic acid by phenol treatment

Abstract Several procedures employing chloroform and phenol for extraction and deproteinization have been explored for the preparation of highly purified transforming DNA from Bacillus subtilis . The recovery, purity, transforming activity and the sedimentation coefficients of the preparations have been compared. The following procedure has been found to give the best results: lysis of cells with lysozyme (EC 3.2.1.17), followed by freezing and thawing, extraction of nucleic acids with a mixture of phenol and pH-9 buffer in the presence of sodium dodecyl sulfate, digestion of RNA with a mixture of pancreatic RNAase (EC 2.7.7.16) and RNAase T1 from Taka-diastase (EC 3.1.4.8), followed by treatment with phenol. When the digestion is carried out with pancreatic RNAase alone, it is followed by precipitation with isopropapol in order to eliminate RNA. The final DNA preparation has high transforming activity, especially for the joint transformation of linked markers.

Mapping of genes determining nonpermissiveness and host-specific restriction to bacteriophages in Bacillus subtilis Marburg

SummaryBacillus subtilis Marburg is nonpermissive for the multiplication of bacteriophages SP10 and ϕNR2. A permissive mutant was derived from the Marburg strain, and the genetic determinants of nonpermissiveness were analyzed by PBS1 transduction. The simultaneous presence of two genes as mutant alleles, nonA and nonB, was necessary for permissiveness. The gene nonA is linked very closely to rfm (cotransfer: 95%); nonB is located between dal and purB (cotransfer of nonB and purB6: 48%). The genetic determinant of host-specific restriction intrinsic to the Marburg strain (hsrM) was found to be identical or very closely linked to nonB. The segregation of nonB and hsrM has never been observed in the course of transduction analysis. The mutation, hsrM1, diminishes the restriction activity, but not the host-controlled modification.

Genetic analysis of two genes, dnaJ and dnaK, necessary for Escherichia coli and bacteriophage lambda DNA replication

SummaryWe show that a collection of 93 E. coli mutations which map between thr and leu and which block phage lambda DNA replication define two closely linked cistrons. Work published in the accompanying paper shows that these mutations also affect host DNA replication, so we designate them dnaJ and dnaK; the gene order is thr-dnaK-dnaJ-leu. Demonstration of two cistrons was possible with the isolation of lambda transducing phages carrying one or the other or both of the dna genes. These phages were employed in phage vs bacterial complementation studies which unambiguously show that dnaK and dnaJ are different cistrons.

Species specificity of the ribosomal RNA cistrons in bacteria

Abstract Hybridization of labelled ribosomal RNA of Bacillus subtilis Marburg strain with DNAs from various microorganisms was studied by means of a DNA-agar method involving ribonuclease digestion. The size and base ratio of the hybridized rRNA portion were analyzed. The B. subtilis rRNA was found to be highly complementary to the DNAs of Bacilli tested: the extent of the analogy seemed to be correlated with the gehetic relatedness and the guanine-cytosine content of the DNA. Appreciable cross-hybridization was also observed between B. subtilis rRNA and DNA from microorganisms of different families, e.g. the extents of hybridization with DNAs from Staphylococcus epidermidis, Alcaligenes faecalis, and Escherichia coli were 55, 33 and 20 %, respectively, of that with the homologous DNA. Competition experiments revealed similar extents of the analogy of rRNA in these bacteria. In addition, the heterologous hybridization between Staphylococcus DNA and labelled rRNA from B. subtilis was reduced by unlabelled rRNA from A. faecalis by the expected amount. The results suggest that analogous base sequences may occur at the same site(s) in the rRNAs from these bacteria. The majority of experiments were carried out with 23-S RNA, but similar results were also obtained with 16-S RNA. These results suggest the conservation of rRNA sites in the DNA of microorganisms.

Isolation and mapping of a new suppressor mutation of an early sporulation gene spo0F mutation in Bacillus subtilis

SummaryWe constructed an spo0F deletion (spo0FΔS) mutant of Bacillus subtilis by inserting a chromosomal segment carried by plasmid pUBSFΔS. This plasmid carries a 0.7-kilobase pair deletion that removes the spo0F promoter and a part of the structural gene. We used the spo0F deletion mutant to isolate a new intergenic suppressor of the spo0F phenotype, designated sof1. The sof1 suppressor completely restores the sporulation ability of all spo0F defective mutants tested, including spo0F77, spo0F221 and spo0FΔS. The sof1 suppressor maps to the left of lys1 on the B. subtilis chromosome, in a region rich in sporulation markers and distant from the spo0F locus.

The effect ofspo0 mutations on the expression ofspo0A- andspo0F-lacZ fusions

SummaryWe have constructedspo0A-lacZ andspo0F-lacZ fusions with a temperate phage vector and have investigated howspo0 gene products are involved in the expression of each of these genes. The expression ofspo0A-lacZ andspo0F-lacZ was stimulated at about the time of cessation of vegetative growth in Spo+ cells. This stimulation ofspo0A-lacZ was impaired by mutations in thespo0B, D, E, F orH genes but was not affected by mutations in thespo0J orK genes. Similar results were obtained with thespo0F-lacZ fusion. The effect of thespo0A mutation onspo0A-lacZ expression was characteristic: thespo0A-directed β-galactosidase activity found during vegetative growth was significantly enhanced in thespo0A mutant. This result suggests thatspo0A gene expression is autoregulated being repressed by its own gene product. Another remarkable observation was the effect of thesof-1 mutation, which is known to be aspo0A allele; it suppressed the sporulation deficiency ofspo0B, spo0D andspo0F mutants. Thespo0A-lacZ stimulation, which is impaired by any one of thesespo0 mutations, was restored by the additionalsof-1 mutation.

Dissection of the expression signals of the spo0A gene of Bacillus subtilis: glucose represses sporulation-specific expression

The expression of the spo0A-lacZ fusion gene was partially repressed in the presence of an excess of glucose. Expression was restored either by the mutation sigA47(crsA47) or by addition of decoyinine, an inhibitor of GMP synthetase, to the medium. By constructing a lacZ fusion with a smaller fragment of the spo)A gene, we observed a beta-galactosidase profile in which expression was completely repressed by an excess of glucose. This expression was restored by the addition of decoyinine. These results indicate that the expression of the spo0A gene is regulated by at least two different mechanisms, one sensitive to glucose, the other not. Furthermore, the glucose-sensitive regulation was shown to reside at the transcriptional level. It is likely that the reduced expression of the spo0A gene in the presence of glucose at an early stage of sporulation causes the repression of sporulation.

A specialized transducing phage constructed from Bacillus subtilis phage φ105

Chromosomal DNA of Bacillus subtilis 168 (trpC2) prepared from defective phage P BSX was digested by restriction endonuclease Eco RI and ligated in vitro with DNA fragments of page phi 105C digested by the same endonuclease. The ligated DNA was used to transform a competent culture of B. subtilis (trpC2 lys3 metB10) which was lysogenic for phi 105, and transformants of the auxotroph markers were selected. The bacterial DNA ligated to the phage DNA fragments could be integrated into the prophage genome by transformation. The transformants in toto were treated with mitomycin C and the lysate was used to transduce B. subtilis (trpC2 lys3 metB10). Among metB+ transductants, one clone appeared to be a double lysogen carrying both plaque forming and metB+ transducing phage genomes. The latter defective phage was designated phi 105dmetB. Physical mapping of these phages was carried out by agarose gel electrophoresis of the restriction endonuclease digests and also by electron microscopic analysis of heteroduplex DNA. These results indicate that two adjacent fragments Eco RI-G and E of phi 105 DNA had been substituted with a foreign fragment Eco RI-M in phi 105dmetB DNA. Transformation experiments showed that the metB+ gene resided on the fragment Eco RI-M. This fragment was found to have a BamHI-sensitive site. The transforming activity for the metB marker, however, was not affected by the treatmment with BamHI.

Mapping by Interspecies Transformation Experiments of Several Ribosomal Protein Genes Near the Replication Origin of Bacillus subtilis Chromosome

SummaryBacillus subtilis 168 was transformed with DNAs from B. amyloliquefaciens K or B. licheniformis IAM 11054. These two species show a considerable difference in ribosomal proteins from B. subtilis. Analyses of the transformants indicated that the genes for 16 proteins, S3, S5, S8, S12, S17, S19, BL4, BL5, BL6, BL8, BL14, BL16, BL17, BL22, BL23 and BL25 are located in the cysA-str-spc region on B. subtilis chromosome. The genes for 10 proteins, S4, S6, S13, S16, S20, BL15, BL18, BL20, BL24 and BL28 could not be found in this region in the prsent experiments.

High-frequency transduction of pBR322 by cytosine-substituted T4 bacteriophage: Evidence for encapsulation and transfer of head-to-tail plasmid concatemers

Abstract Transduction of plasmid pBR322 by cytosine-substituted T4 phages has been studied. Three T4 phage mutants which substitute cytosine for all of hydroxymethylcytosine residues in the DNA, were shown to transduce pBR322 at frequencies of 2 × 10−2 to 4 × 10−3 transductants per singly infected cell. Also, three T4 phage strains which partially substitute cytosine for hydroxymethylcytosine, transduced pBR322 at frequencies of 2 × 10−3 to 2 × 10−4. The transduction frequencies of pBR322 we attained are at least 10-fold higher than those reported by G. G. Wilson, K. Young, and G. J. Edlin (1979, Nature (London)280, 80–82). We found that multiplicity of infection in preparation of the transducing phage is the most important factor affecting the frequency of pBR322 transduction. When a lysate made at a multiplicity of infection ranging from 0.5 to 0.05 was used as the donor phage, transduction frequency of pBR322 was 10- to 40-fold higher than that of high-m.o.i. lysate. The transduction frequency was not affected by either restriction systems or amber suppressors of the recipient cells. However, no pBR322-containing transductant was obtained when either recA or polA mutants were used as the recipients. DNA from T4dC phage containing pBR322-transducing particles was analyzed on agarose gel electrophoresis after cleavage with restriction endonucleases. It was suggested that the pBR322 DNA in the T4dC phage particles exists as head-to-tail concatemers.