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.

Listeria monocytogenes was examined for superoxide dismutase (SOD) activity. Two catalase-negative strains possessed at least twofold greater SOD activities than the catalase-positive L. monocytogenes strains examined. Growth conditions such as aeration and iron concentration influenced the specific activity of SOD obtained from cells cultured in defined media. L. monocytogenes SOD from crude extracts and after partial purification was analyzed by polyacrylamide gel electrophoresis. Iron was associated with the single band of SOD activity detected in the gels. SOD activity appeared to be primarily extracytoplasmic. Survival of organisms in a superoxide-generating medium was studied, with photoactivation of riboflavin used as the source of free radical formation. Virulent, catalase-positive L. monocytogenes strains were relatively resistant to killing in a pH 7 superoxidecontaining medium. An intact-cell assay for SOD was developed, which used the superoxide-generating system and employed the superoxide-dependent oxidation of sulfite, added to the medium, and inhibition of this oxidation by SOD. Maximal SOD activities of intact cells were observed when 100 to 400 ,g (dry weight) of viable Listeria cells per ml was added to the medium. A possible role for SOD in the pathogenesis of listeric infection is discussed. Facultative intracellular pathogens such as Listeria monocytogenes must possess means of overcoming the nonspecific immune responses mediated by phagocytic cells and various humoral factors. The sequence in phagocytic killing of bacteria includes the formation of a toxic superoxide (*02) free radical (5) which is later eliminated by the enzyme superoxide dismutase (SOD) (22). Indirect evidence also exists for a role of * 02 in leukocyte-mediated antimicrobial activity. Baehner et al. (6) showed that the nitroblue tetrazolium reduction by leukocytes is dependent on *°02 generated by phagocytic cell metabolism, and Curnutte et al. (11) observed that neutrophils obtained from patients with chronic granulomatous disease produced low or undetectable quantities *°2 SODs from Escherichia coli have been found to contain manganese (16) or iron (31). Yost and Fridovich (32) demonstrated that E. coli cells grown in iron-rich media are more resistant to killing by phagocytes than E. coli grown in irondeficient media. Increased resistance appeared to correlate with increased levels of iron-SOD rather than with catalase or other effects of iron.

Deoxyribonucleic acid (DNA)-binding proteins have been compared between wild-type Bacillus subtilis and five sporulation mutants blocked at different stage 0 loci. Extracts from exponentially growing cells have been fractionated for proteins binding to single-stranded calf thymus DNA-cellulose and doublestranded B. subtilis DNA-cellulose. In nutrient broth, stage 0 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 0 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 0 mutations affect proteins with affinity for single-stranded DNA. The results suggest that the products of stage 0 loci are functional and operative during vegetative growth.

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 T0, whereas at Ts new polypeptides appeared. Fewer changes in the profile of proteins with affinity for native DNA were observed between exponential phase and To; however, the dominant species in these eluates were clearly different.