Tadako Iijima

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.

Chromosomal loci of genes controlling site-specific restriction endonucleases of Bacillus subtilis.

SummaryWe constructed transformants of B. subtilis 168 which acquired genes for site-specific restriction endonucleases. These endonucleases originated from various strains of B. subtilis and were classified into five groups based on the specificity of the sequences recognized by the enzymes. We examined the loci of genes for site-specific restriction endonucleases belonging to different groups: hsrE determined Endo.R.Bsu1231(I), hsrB Endo.R.Bsu1247(I), hsrR Endo.R.BsuR and hsrC Endo.R.Bsu1247(II). One gene, hsrE, was located between sacA and purA by transduction crosses with phage PBS1, and another gene, hsrB, between hsrE and purA.Genes hsrR and hsrC had been suggested to be allelic or closely linked by previous studies with transformation. We located hsrR and hsrC between purB and tre.Our previous observation and this study show that B. subtilis 168 has at least three independent loci on the chromosome for four genes for site-specific restriction endonucleases in addition to the locus for the original restriction activity (Bsu168-specific restriction) of strain 168.

Evidence for a Low Affinity but High Velocity Aspartate Transport System Needed for Rapid Growth of Bacillus subtilis on Aspartate as Sole Carbon Source

Mutants (aspT) of Bacillus subtilis which lack the high affinity transport of L-aspartate and L-glutamate have been isolated by their resistance to DL-threo-β-hydroxyaspartate. They transport low concentrations (100 μM) of all three amino acids at a greatly reduced rate but they can still grow at the normal rate on high concentrations of aspartate (25 mM) as sole carbon source. The aspT mutation has been mapped between argC and glpK, glpD. In glucose/citrate medium, an aspB mutant, auxotrophic for aspartate, requires much lower aspartate concentrations for growth than an aspB aspT double mutant. These results demonstrate that a low affinity aspartate transport system is still present in aspT mutant strains. This was also shown by the fact that both aspH and aspH aspT mutants, which carry the gene (aspH) for high aspartase production, grew at the same rate in media containing high concentrations (25 mM) of L-aspartate as sole carbon source. The high affinity aspartate transport system (K m = 67 μM) alone can satisfy the growth requirements of aspartate auxotrophs in media containing glucose or some other carbon source. However, the maximum rate (V max) of this transport system is too low to allow rapid growth on aspartate as sole carbon source. For such rapid uptake the low affinity aspartate transport system is needed. Membrane vesicles, energized by glycerophosphate, exhibit only the active high affinity transport which is saturated at about 100 μM-L-aspartate (K m = 66 μM). Apparently, the proton motive force, which energizes the high affinity aspartate (and glutamate) transport, is not (directly) required for the low affinity aspartate transport.