Deoxyribonucleic acid-binding proteins in vegetative Bacillus subtilis: alterations caused by stage O sporulation mutations.

Deoxyribonucleic acid (DNA)-binding proteins have been compared between wild-type Bacillus subtilis and five sporulation mutants blocked at different stage O loci. Extracts from exponentially growing cells have been fractionated for proteins binding to single-stranded calf thymus DNA-cellulose and double-stranded B. subtilis DNA-cellulose. In nutrient broth, stage O mutations cause an accumulation of proteins with affinity for double-stranded DNA. Suppression of the mutation with extragenic suppressors relieves the accumulation. In minimal glucose medium, the stage O mutations also cause accumulation of proteins with affinity for double-stranded DNA, but the species accumulated are different from those of nutrient broth-grown cells. In neither case did stage O mutations affect proteins with affinity for single-stranded DNA. The results suggest that the products of stage O loci are functional and operative during vegetative growth.

Developmental modulation of deoxyribonucleic acid-binding proteins of Bacillus subtilis during sporulation stages.

The isolation of deoxyribonucleic acid (DNA)-binding proteins from various stages of growth and sporulation of Bacillus subtilis is described. After adsorption and elution from phosphocellulose, the proteins were fractionated according to their ability to adsorb to denatured calf thymus DNA-cellulose or native B. subtilis DNA-cellulose. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and purification was monitored by a nitrocellulose filter binding assay. Approximately 1% of the proteins in the crude extract adsorbed to denatured calf thymus DNA-cellulose and 0.1% adsorbed to native B. subtilis DNA-cellulose. Each class of proteins varied qualitatively and quantitatively as sporulation proceeded. Several proteins from the exponential phase of growth that bound to denatured DNA were lost by T(0), whereas at T(5) new polypeptides appeared. Fewer changes in the profile of proteins with affinity for native DNA were observed between exponential phase and T(0); however, the dominant species in these eluates were clearly different.

Characterization of a bacteriophage-induced, host-specific lytic enzyme from lysates of Bacillus stearothermophilus infected with bacteriophage TP-8.

Phage TP-8 lysates of Bacillus stearothermophilus 4S or 4S(8) contain lytic activity exhibiting two pH optima, one at pH 6.5 and the other at pH 7.5. Using a variety of fractionation procedures, the two lytic activities could not be separated. At pH 7.5 the lytic enzyme is an endopeptidase which hydrolyzes the l-alanyl-d-glutamyl linkage in the peptide subunits of the cell wall peptidoglycan and at pH 6.5 it exhibits N-acetylmuramidase activity. Endopeptidase activity is inhibited by NaCl and neither lytic activity was significantly affected by divalent cations or ethylenediaminetetraacetic acid. Crude lysates contain 2.5 to 3.0 times more endopeptidase activity than N-acetylmuramidase activity. The ratio of the two lytic activities (endopeptidase/N-acetylmuramidase) changes to 1.3 to 1.7 during the course of purification, to 1.0 after isoelectric focusing, and 3.9 and 6.00 after exposure for 2 h at 60 and 65 C, respectively. We conclude that the two lytic activities may be associated with a single protein or a lytic enzyme complex composed of two enzymes. Lytic activity at pH 7.5 is more effective in solubilizing cells or cell walls than the lytic activity at pH 6.5. LiCl extracts of 4S and 4S(8) cells contain lytic activity exhibiting endopeptidase activity at pH 7.5 and N-acetylmuramidase activity at pH 6.5. Lytic activity in these LiCl extracts also has a number of other properties in common with those in lysates of phage TP-8. We proposed that the lytic enzyme(s) are not coded for by the phage genome but are part of the host autolytic system.

Phenotypes of pleiotropic-negative sporulation mutants of Bacillus subtilis.

The phenotypic properties of representatives of the five genetic classes of pleiotropic-negative sporulation mutants have been investigated. Protease production, alkaline and neutral proteases, was curtailed in spoA mutants, but the remainder of mutant classes produced both proteases, albeit at reduced levels. The spoA and spoB mutants plaqued phi2 and phi15 at high efficiency, but the efficiency of plating of these phages on spoE, spoF, and spoH mutants was drastically reduced. Antibiotic was produced by the spoH mutants and to a degree by some spoF mutants, but the other classes did not produce detectable activity. The spoA mutants were less responsive to catabolite repression of histidase synthesis by glucose than was the wild type. Severe catabolite repression could be induced in spoA mutants by amino acid limitation, suggesting that the relaxation of catabolite repression observed is not due to a defect in the mechanism of catabolite repression. Although others have shown a perturbation in cytochrome regulation in spoA and spoB mutants, the primary dehydrogenases, succinate dehydrogenase and reduced nicotinamide adenine dinucleotide dehydrogenase, leading to these cytochromes are unimpaired in all mutant classes. A comparison of the structural components of cell walls and membranes of spoA and the wild type is made. The pleiotropic phenotypes of these mutants are discussed.