Mol Gen Genet, 1977 Nov 14, 156(2), 145 - 55 Transformation in Bacillus subtilis: biological and physical evidence for a novel DNA-intermediate in synchronously transforming cells; Buitenwerf J et al.; Competent B . subtilis cells exposed to transforming DNA in the presence of EDTA bind, but do not take up DNA . Rapid and almost synchronous uptake of the bound DNA is achieved by the addition of Mg2+ ions in excess of the EDTA . At 30 degrees and at 17 degrees comparable numbers of transformants are produced from cells pre-loaded with DNA at 30 degrees (after termination of uptake by the addition of DNA ase the samples were incubated at 37 degrees) . However, almost no transformants are produced when cells are exposed to DNA at 17 degrees, although binding does take place then . Because DNA is taken up at 17 degrees after having loaded the cells at 30 degrees, whereas no uptake occurs after binding at 17 degrees, it is suggested that binding of DNA to the cellular surface involves at least two steps . In DNA re-extracted from cells at 17 degrees, pre-loaded with DNA at 30 degrees, little recombinant type activity is present, indicating that integration is blocked at 17 degrees . However, physico-chemical analysis of the re-extracted DNA indicates that a complex between single-stranded donor DNA and the recipient chromosome is formed at 17 degrees . This complex has a higher buoyant density than donor-recipient complexes formed at 30 degrees. J Biol Chem, 1977 Nov 10, 252(21), 7525 - 9 Hydroxylaminolysis of penicillin binding componenets is enzymatically catalyzed; Kozarich JW et al.; The hydroxylaminolysis of the penicilloyl moiety from {14C}penicillin G binding component (PBC) complexes of the Bacillus subtilis D-alanine carboxypeptidase and of the mixture of PBC's of Staphylococcus aureus was inhibited by denaturation of the complexes by heat (55 degrees), detergent (1% sodium dodecyl sulfate), or trichloroacetic acid . The kinetics of inhibition by denaturation were comparable to those of the inhibition of {14C}penicillin G binding to the PBC's and of carboxypeptidase activity of the B . subtilis enzyme under identical denaturing conditions . These data establish that the hydroxylaminolysis is an enzymatically catalyzed process suggesting that penicillin G is bound to an enzymatically active site . Treatment of the denatured {14C}penicillin G-carboxypeptidase complex with sodium borohydride or at pH 12 resulted in the release of the penicilloyl moiety . These results are consistent with a carboxylic ester bond for the penicilloyl-PBC instead of a thiolester linkage as was initially presumed. J Biol Chem, 1977 Nov 10, 252(21), 7424 - 6 Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis . A novel iron-sulfur protein; Wong JY et al.; Glutamine phosphoribosylpyrophosphate amidotransferase, purifed to better than 98% purity from derepressed Bacillus subtilis, exists as a tetramer and as a dimer of apparently identical subunits with a molecular weight of 50,000 each . The enzyme contains 3 atoms of iron and 2 atoms of inorganic sulfide per subunit and has a yellow-brown color . The absorption spectrum is not altered by dithionite, but exposure to oxygen causes inactivation and partial bleaching of the visible spectrum . Thus, the Bacillus amidotransferase exhibits novel structural features and a new reaction type of proteins of the iron-sulfur group. Nature, 1977 Nov 3, 270(5632), 28 - 32 Purification of a positive regulatory subunit from phage SP01-modified RNA polymerase; Duffy JJ et al.; A phage-induced subunit has been purified from phage SP01-modified Bacillus subtilis RNA polymerase . This subunit binds in vitro to RNA polymerase core from uninfected B . subtilis thereby template-selective transcription and asymmetric synthesis of SP01 middle RNA. Prikl Biokhim Mikrobiol, 1977 Nov-Dec, 13(6), 819 - 28 {Study of lytic enzymes and prospects of their use}; Konovalov SA et al.; The paper reviews different procedures of preparation of lytic enzymes of microbiol origin, their properties and areas of application . It discusses the capacity of different microorganisms, actinomycetes and bacteria including (for instance, Actinomyces griseinus 11 and Bacillus subtilis 12), to produce lytic enzymes . The paper describes the conditions of disruption of yeast cells by lytic enzymes and demonstrates experimentally possible preparation of yeast lysates and protein isolates that can be used as food products. Proc Natl Acad Sci U S A, 1977 Nov, 74(11), 4752 - 6 Sensory electrophysiology of bacteria: relationship of the membrane potential to motility and chemotaxis in Bacillus subtilis; Miller JB et al.; The relationship of membrane potential to motility and chemotaxis of Bacillus subtilis has been tested by using the fluorescence of a cyanine dye as a probe of the potential . The dye fluorescence was found to be an indicator of membrane potential by correlation with triphenylmethylphosphonium ion distribution and with changes due to anaerobicity and ionophore addition . When the potential was sufficient for motility and constant over time, it was found that the absolute level of the potential did not affect the swimming behavior of the bacteria . Transient alteration of the membrane potential did, however, lead to changes in swimming behavior . Attractants were found to alter the swimming behavior of the bacteria without altering the membrane potential . Thus, change of the overall membrane potential of a normal B . subtilis is not required for chemotaxis, but such a change is sensed by the bacteria just as changing levels of attractants and repellents are sensed. Can J Microbiol, 1977 Nov, 23(11), 1585 - 93 The effect of Waring Blendor treatment on transformation in Bacillus subtilis; Weppner WA et al.; Treatment of competent Bacillus subtilis in a Waring Blendor for 10 s increases transformability of the culture about twofold while reducing the attachment of DNA to competent cells by 80% . The effectiveness of attached DNA in producing transformants is increased 10-fold by this treatment . The uptake of transforming DNA into a DNase-resistant state is progressively reduced by 70% during a 120-s blending treatment . Blending for 30-45 s diminishes transformability to about 10% of the original nonblended value without affecting the viable cell titer . No effect is produced by 30 s of blending on transformability if the irreversible uptake of DNA has been completed . Thus, the inhibition occurs at an early step in the transformation sequence . Treatment of the competent culture for 60 s or longer in the Waring Blendor reduces both the number of transformants obtained and the total number of viable cells. J Bacteriol, 1977 Nov, 132(2), 473 - 84 Bacillus subtilis mutant with temperature-sensitive net synthesis of phosphatidylethanolamine; Lindgren V et al.; Bacillus subtilis mutants with temperature-sensitive growth on complex media were screened for defects in phospholipid metabolism . One mutant was isolated that showed temperature-sensitive net synthesis of phosphatidylethanolamine . The mutant did not accumulate phosphatidylserine at the nonpermissive temperature . In the presence of hydroxylamine, wild-type B . subtilis accumulated phosphatidylserine at both 32 and 45 degrees C, whereas the mutant did only at 32 degrees C . In vitro phosphatidylethanolamine synthesis with bacterial membranes is no more temperature sensitive with mutant membranes than with wild-type membranes . The mutation probably affects the synthesis indirectly, possibly by altering a membrane protein . The mutant bacteria grew at the nonpermissive temperature, 45 degrees C, in a phosphate buffer-based minimal medium, although net synthesis of phosphatidylethanolamine was also temperature sensitive in this medium . One mutation caused both temperature-sensitive growth on complex media and temperature-sensitive net synthesis of phosphatidylethanolamine . The mutation is linked to aroD by transformation. J Bacteriol, 1977 Nov, 132(2), 419 - 25 Regulation of a common amidotransferase subunit; Kane JF; In Bacillus subtilis the trpX locus specifies a glutamine-binding protein designated subunit X, which forms a complex with subunit E to constitute the anthranilate synthase enzyme aggregate (EX) and subunit A to constitute the p-aminobenzoate synthase enzyme aggregate (AX) . Subunit X confers upon these enzyme complexes the ability to utilize glutamine as a substrate . The trpX locus has been examined to determine its map position and control . (i) The trpX locus was found to be cotransformed with the lysS and pabA loci . The results of three-factor transformation analyses suggest the following order of these markers: lysS-sul-trpX-pabA . (ii) Mutation to constitutivity of the tryptophan operon resulted in a 50- to 60-fold increase in the level of subunit X when the mutant contained functional trE and abA gene products; however, in the absence of subunit E there was only a 4- to 5-fold increase in the glutamine-binding protein . (iii) Formation of subunit X was derepressed under conditions that allow for the derepression of the trpE and/or pabA loci . (iv) Subunit X synthesis was derepressed to a greater extent in mutants that contain a functional trpE gene product than in mutants that contain a nonsense mutation in the trpE locus . These results are consistent with the hypothesis that the trpE and pabA gene products affect the expression and control of the trpX locus. Mol Gen Genet, 1977 Oct 24, 155(3), 329 - 38 Macromolecular synthesis in chromosome initiation mutants of Bacillus subtilis; Sargent MG; Inactivation of the dna B or dna D gene product in Bacillus subtilis stimulates RNA and protein synthesis . Strains containing ts dna B and D mutations have been constructed by introducing the mutations by transformation into a thymine requiring strain which does not lyse during thymine starvation . The consequences of inactivation of these gene products have been assessed by comparing RNA and protein synthesis during thymine starvation at the restrictive temperature with the recipient strain . In the ts+ strain, there is a doubling in rate of RNA synthesis during thymine starvation . In the ts dna B and D mutations at the restrictive temperature the rate of RNA synthesis increases four fold . By preincubating the mutants in the absence of thymine for one generation at the permissive temperature the two fold increase in rate of RNA synthesis associated with inactivation of the initiation complex can be demonstrated under conditions where the ts+ strain shows a decrease in rate of RNA synthesis . The rate of protein synthesis observed largely reflects the rate of RNA synthesis in all strains . Completion of the chromosome at the restrictive temperature has no significant effect on the rate of RNA synthesis . It is suggested that inactivation of the initiation complex after chromosome initiation could play an important role in control of RNA synthesis in relation to the cell cycle. Mol Gen Genet, 1977 Oct 24, 155(3), 241 - 7 Deletion mutants of temperate Bacillus subtilis bacteriophage phi105; Flock JI; Six deletion mutants of temperate Bacillus subtilis phage phi105 have been isolated on the basis of their increased resistance to chelating agents . The size and position of the deletions was determined by electronmicroscopy of DNA heteroduplexes . All deletions are located in a region about 55-70% from one end of the DNA molecule, in the right half of the known genetic map of the phage . The segment 55-65% does not contain any genes essential for lytic growth or lysogenization . A gene(s) for immunity is located in a segment 65-70% from the left end . By electronmicroscopy by partially denatured phi105 DNA two A-T rich regions have been localized in the right half of the molecule . One of these regions falls within the non-essential 55-65% DNA segment. Mol Gen Genet, 1977 Oct 20, 155(2), 179 - 83 Selective enrichment for genetic markers in DNA released by competent cultures of Bacillus subtilis; Crabb WD et al.; Deoxyribonucleic acid is released into the growth medium by Bacillus subtilis at the time of competence . This DNA is enriched for the genetic markers which have previously been demonstrated to be elevated in membrane-DNA preparations and more recently in cell wall-DNA complexes . Furthermore, the purA16/leu-8 relative marker enrichment varies with time, reaching its highest point at the time of maximal competence . Enrichment remains elevated for at least 60 min further in the competence regimen . Thr results suggest that certain genetic markers of the B . subtilis chromosome are preferentially more available to the external medium as the development of competence proceeds. Tijdschr Diergeneeskd, 1977 Oct 15, 102(20), 1173 - 86 Tissue distribution and residues of beta-lactam antibiotics in normal dairy cows; Nouws JF et al.; Tissue residues and concentrations of benzylpenicillin, cloxacillin, ampicillin, amoxycillin, cephapirin, and cephacetrile were determined in normal dairy cows after parenteral administration of several forms of these drugs . Assay methods included the Sarcina lutea Kidney Test of Van Schothorst, the Bacillus subtilis BGA Tests at pH 6.0 and 8.0, the Escherichia coli Test and a Sarcina lutea Test performed at pH 8.0 of the agar, and specific quantitative assay methods . The E . coli test method demonstrated an insensitivity for the beta-lactam antibiotic residues . Identical results in residue testing of meat and kidney were obtained with the B . subtilis BGA tests and S . lutea test at pH 8.0, and these test methods replicated each other . The S . lutea Kidney Test was very often positive at times after treatment when the antibiotics were no longer detected in the meat . The qualitative and quantitative residue data from the renal cortex were higher than the data obtained from the muscle meat . The concentration relationship between renal cortex and muscle meat dependent on the formulation and type of drug used, and on the time of sampling after treatment . After treatment with products containing ampicillin trihydrate and procaine penicillin an unexpectedly long persistence of these drugs in the renal cortex was observed . It is suggested that, in the case of beta-lactam antibiotics, meat tests are more accurate indicators for the residue status of the carcass. Tijdschr Diergeneeskd, 1977 Oct 15, 102(20), 1187 - 96 Tissue distribution and residues of aminoglycoside antibiotics in normal dairy cows; Nouws JF et al.; The concentration and persistence of streptomycin, dihydostreptomycin (DHS), neomycin, and kanamycin in the body organs of normal dairy cows following intravenous (i.v.) and intramuscular (i.m.) injections were determined by qualitative and quantitative assay methods . The most sensitive qualitative procedure, i.e . Bacillus subtilis BGA at pH 8.0, failed to detect kanamycin in the meat of cows slaughtered 4 hours after i.m . and 6 hours after i.v . injections . This test, however, was positive for the kidneys of cows slaughtered 144 hours after the i.m . administration of neomycin . Low concentrations of neomycin were measured in the meat of cows slaughtered 2 hours and 7 hours after injection . Treatment with streptomycin i.v . resulted in significantly lower renal cortex drug levels than after the i.v . administration of other drugs . Injection of neomycin in a formulation containing benzylpenicillin procaine resulted in higher neomycin concentrations in the renal cortex compared to the levels of the drug found after i.v . injection of the drug . Results are discussed the possible role played by drug interaction on the persistence of aminoglycoside antibiotics in the kidneys after treatment with different dosage forms of these drugs. Eur J Biochem, 1977 Oct 3, 79(2), 363 - 73 Fructose transport in Bacillus subtilis; Gay P et al.; The transport of fructose in Bacillus subtilis was studied in various mutant strains lacking the following activities: ATP-dependent fructokinase (fruC), the fructose 1-phosphate kinase (fruB) the phosphofructokinase (pfk), the enzyme I of the phosphoenolpyruvate phosphotransferase system (the thermosensitive mutation ptsI1), and a transport activity (fruA) . Combinations of these mutations indicated that the transport of fructose in Bacillus subtilis is tightly coupled to its phosphorylation either in fructose 1-phosphate, identified in vivo and in vitro or in fructose 6-phosphate identified by indirect lines of evidence . These steps of fructose metabolism were shown to depend on the activity of the enzyme I of the phosphoenolpyruvate phosphotransferase systems . The fruA mutations affect the transport of fructose when the bacteria are submitted to catabolite repression . The mutations were localized on the chromosome of Bacillus subtilis in a cluster including the fruB gene . When grown in a medium supplemented by a mixture of potassium glutamate and succinate the fruA mutants are able to carry on the two vectorial metabolisms generating fructose 6-phosphate as well as fructose 1-phosphate . A negative search of strictly negative transport mutants in fruA strains indicated that more than two structural genes are involved in the transport of fructose. Biokhimiia, 1977 Oct, 42(10), 1910 - 8 {Isolation and some properties of phospholipase D from Bacillus subtilis G-22}; Garutskas RS et al.; A method of isolating highly purified phospholipase D from Bac . subtilis G-22 is described . It includes ammonium sulphate fractionation, thermal denaturation, chromatography on lipoprotein bound with sepharose 6B and AH-sepharose 4B . The enzyme is 130-fold purified, its yield exceeds 90.0%, its specific activity is 164 units per mg of protein . The homogeneity of the enzyme is demonstrated by polyacrylamide gel electrophoresis, ultracentrifugation, isoelectric focusing and N-terminal amino acid determination by means of dinitrophenylation and dancylation . Proline is found to be N-terminal amino acid . The molecular weight of the enzyme, as determined from gel filtration through Sephadex G-100, is 21500 +/- 300, its sedimentation constant is 1.4S, isoelectric point is at pH 4.2 . The molecular weight calculated from amino acid composition, is 21000--22000 . Polypeptide chain contains of 196--205 amino acid residues . Phospholipase D develops its maximal activity at pH 8.5 and does not contain free SH-groups . Benzylsulphofluoride does not inhibit the enzyme activity . Phospholipase D is activated by Cd2+, Co2+, Zn2+, Ca2+ and is inhibited by EDTA, pIi50 being about 2.6. J Bacteriol, 1977 Oct, 132(1), 73 - 9 Lipiarmycin-resistant ribonucleic acid polymerase mutants of Bacillus subtilis; Sonenshein AL et al.; Lipiarmycin inhibited the activity of deoxyribonucleic acid-dependent ribonucleic acid (RNA) polymerase in vitro . We showed that inhibition was due to interference by lipiarmycin with the activity of sigma-containing molecules of RNA polymerase . Transcription by core enzyme was relatively resistant to the drug, but addition of sigma led to highly drug-sensitive RNA synthesis . We isolated lipiarmycin-resistant mutants of Bacillus subtilis and characterized them genetically and biochemically . Drug-resistant mutants contained an altered RNA polymerase that was resistant to the drug in vitro . By separation and mixed reconstitution of core and sigma fractions of mutant and wild-type RNA polymerase, we showed that lipiarmycin resistance in one mutant strain was a property of the core fraction . Genetic mapping experiments indicated that at least two lpm mutants are located between loci determining rifampin resistance and streptolydigin resistance. J Bacteriol, 1977 Oct, 132(1), 262 - 9 Isolation and characterization of fusidic acid-resistant, sporulation-defective mutants of Bacillus subtilis; Kobayashi H et al.; Fusidic acid-resistant, sporulation-defective mutants were isolated from Bacillus subtilis 168 thy trp . About two-thirds of the fusidic acid-resistant (fusr) mutants were defective in sporulation ability and fell into three classes with respect to sporulation character . The representative mutants FUS426 and FUS429 were characterized in detail . FUS426 {fusr spo (Ts)}, a temperature-sensitive sporulation mutant, grew well at 30 and 42 degrees C but did not sporulate at 42 degrees C . FUS429 {fusr spo (Con)}, conditional sporulation mutant, grew and sporulated normally in the absence of fusidic acid, but its sporulation and growth rates decreased in the presence of fusidic acid, depending on the concentration of the drug . Although electron microscopic observation showed that both mutants were blocked at stage I of sporulation, the physiological analyses indicate that these mutants belong to the SpoOB class . Both mutants formed a thickened cell wall as compared with that of the parental strain . Genetic and in vitro protein synthesis analyses led to the conclusion that the sporulation-defective character of mutants FUS426 and FUS429 resulted from an alteration in elongation factor G caused by a single lesion in the fus locus . The possible role of elongation factor G in sporulation is discussed. J Bacteriol, 1977 Oct, 132(1), 13 - 22 Functional modifications of the translational system in Bacillus subtilis during sporulation; Chambliss GH et al.; Extracts of sporulating cells were found to be defective in vitro translation of phage SP01 ribonucleic acid (RNA) and vegetative Bacillus subtilis RNA . The activity of washed ribosomes from sporulating cells was very similar to that of washed ribosomes from vegetative cells in translating polyuridylic acid, SP01 RNA, and vegetative RNA . The S-150 fraction from either vegetative or sporulating cells grown in Difco sporulation medium contained an apparent inhibitor of protein synthesis . The crude initiation factor fraction from ribosomes of sporulating cells was defective in promoting the initiation factor-dependent translation of SP01 RNA . The crude initiation factor preparations from sporulating cells were as active as the corresponding preparations from vegetative cells in promoting the initiation factor-dependent translation of either phage Qbeta or phage T4 RNA by washed Escherichia coli ribosomes . The crude initiation factors from sporulating cells were perhaps more active than those from vegetative cells in promoting the initiation factor-dependent synthesis of phage T4 lysozyme by E . coli ribosomes . The crude initiation factor preparations from either vegetative or stationary-phase cells of an asporogenous mutant showed similar ability to promote the in vitro translation of SP01 RNA. J Virol, 1977 Oct, 24(1), 378 - 82 Order of the two major head protein genes of bacteriophage phi 29 of Bacillus subtilis; Mellado RP et al.; Bacteriophage phi 29 mutation sus8(22) has been mapped by two-factor crosses between markers sus8(769) and ts8(93) . Whe sus8(22) infects Bacillus subtilis su- proteins, HP1 (major head protein) and HP3 (fiber protein) are not synthesized; instead, a fragment with a molecular weight of 25,000 is produced . The tryptic peptides of the fragments overlap with corresponding peptides in protein HP1, but not with the peptides of protein HP3, showing that cistron 8 codes for the major head protein HP1. J Virol, 1977 Oct, 24(1), 194 - 200 Bacteriophage PMB12 conversion of the sporulation defect in RNA polymerase mutants of Bacillus subtilis; Bramucci MG et al.; The pseudotemperate phage PMB12 was isolated from soil on the basis of its ability to enhance the rate of sporulation of Bacillus subtilis 168 . PMB12 was subsequently shown to convert the sporulation defect in two genetically distinct classes of sporulation mutants . One class includes those rifampin-resistant mutants that are also spore-negative (mutated at the rif locus) . The other class includes a strain carrying the sporulation mutation spoCM-1 . The spoCM-1 mutation is linked to cysA15 by PBS1 transduction but is distinct from the rif locus . Several other sporulation mutants were not converted by PMB12 . PMB12 is related to phage PBS1 . However, PBS1 did not convert the above sporulation mutants . The replication of PBS2, a clear-plaquing derivative of PBS1, is rifampin insensitive, apparently due to a phage-induced rifampin-insensitive RNA polymerase . PMB12 replication is also rifampin insensitive. Appl Environ Microbiol, 1977 Oct, 34(4), 337 - 41 Mutation of an inosine-producing strain of Bacillus subtilis to DL-methionine sulfoxide resistance for guanosine production; Matsui H et al.; An inosine-producing strain of Bacillus subtilis was mutated to resistance against the antagonist of glutamine, DL-methionine sulfoxide . Among the mutants derived, guanosine producers were observed frequently . The best strain, 14119, produced 9.6 g of guanosine per liter at a weight yield of 12% from consumed sugar . Inosine production decreased concomitantly . When resistance was increased further by exposure to higher doses of DL-methionine sulfoxide, another strain, AG169, was obtained that did not excrete inosine but produced increased amounts of xanthosine . In these strains, the specific activity of 5'-nucleotidase was lower and that of inosine 5'-monophosphate (IMP) dehydrogenase was higher than the parent strain . It is speculated that the metabolic flow from IMP to xanthosine 5'-monophosphate proceeds more smoothly than that from IMP to inosine and yields more xanthosine and guanosine. J Bacteriol, 1977 Oct, 132(1), 282 - 93 Location and properties of glucose dehydrogenase in sporulating cells and spores of Bacillus subtilis; Fujita Y et al.; Late during sporulation, Bacillus subtilis produces glucose dehydrogenase (GlcDH; EC 1.1.1.47), which can react with D-glucose or 2-deoxy-D-glucose and can use nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a cofactor . This enzyme is found mainly in the forespore compartment and is present in spores; it is probably made exclusively in the forespore . The properties of GlcDH were determined both in crude cell extracts and after purification . The enzyme is stable at pH 6.5 but labile at pH 8 or higher; the pH optimum of enzyme activity is 8 . After inactivation at pH 8, the activity can be recovered in crude extracts, but not in solutions of the purified enzyme, by incubation with 3 M KCl and 5 mM NAD or NADP . As determined by gel filtration, enzymatically active GlcDH has a molecular weight of about 115,000 (if the enzyme is assumed to be globular) . GlcDH is distinct from a catabolite-repressible inositol dehydrogenase (EC 1.1.1.18), which can also react with D-glucose, requires specifically NAD as a cofactor, and has an electrophoretic mobility different from that of GlcDH. Biochim Biophys Acta, 1977 Sep 20, 478(2), 234 - 43 Studies on DNA repair in Bacillus subtilis . III . Identification of an exonuclease which enhances the priming activity of gamma-irradiated dna by "cleaning' damaged ends; Inoue T et al.; An enzyme which enhances the priming activity of gamma-irradiated DNA for type I DNA polymerase (EC 2.7.7.7) was identified and partially purified from extracts of Bacillus subtilis cells . The enzyme preferentially degraded gamma-irradiated DNA into acid-soluble materials . DNA preparations treated with heat, ultraviolet light, pancreatic DNAase (EC 3.1.4.5) or micrococcal DNAase (EC 3.1.4.7) were not susceptible to the enzyme . However, sonication rendered DNA susceptible to the enzyme to some extent . From these results, it is supposed that this enzyme may function by 'cleaning' damaged terminals produced by gamma-irradiation to serve as effective priming sites for repair synthesis by the type I DNA polymerase. J Med Chem, 1977 Sep, 20(9), 1186 - 9 Inhibitors of Bacillus subtilis DNA polymerase III . 6-(arylalkylamino)uracils and 6-anilinouracils; Brown NC et al.; 6-(Benzylamino)uracils and substituted 6-anilinouracils have been found to be potent inhibitors of Bacillus subtilis DNA polymerase III by a mechanism identical with that of 6-(phenylhydrazino)uracils . Higher phenylalkylamino homologues are progressively weaker inhibitors of the enzyme . Examination of the effects of substituents on the activity of 6-(benzylamino)uracils against wild-type and mutant enzymes and preliminary results for 6-anilinouracils have permitted further dissection of the mechanism of inhibition . The experimental results indicate that (1) the polymerase inhibitor binding site is compact, accommodating only small alterations in the distance between the uracil and phenyl rings, (2) the phenyl ring, which provides the major contribution to inhibitor-enzyme binding, adopts a specific active conformation, and (3) an enzyme site which interacts with substituents in the phenyl ring forms a part of the active site of DNA polymerase III. J Med Chem, 1977 Sep, 20(9), 1181 - 5 Inhibitors of Bacillus subtilis DNA polymerase III . Structure-activity relationships of 6-(phenylhydrazino)uracils; Wright GE et al.; 6-(Phenylhydrazino)uracils inhibit the replication-specific enzyme DNA polymerase III of Bacillus subtilis by forming a strong, reversible complex with template-primer DNA and enzyme . The phenyl ring interacts with a hydrophobic enzyme site which, on the basis of structure-activity relationships of substituted analogues, appears to possess the following characteristics: (1) planarity or near-planarity; (2) a finite capacity to accommodate bulky substituents; and (3) location near the domain of the enzyme active site . A mutant DNA polymerase III, derived from a mutant strain of B . subtilis selected for resistance to 6-(p-hydroxyphenylazo)pyrimidines, is resistant only to inhibitors bearing p-hydroxy or amino groups and is hypersensitive to inhibitors containing nonpolar substituents; these results suggest the existence of mutable, secondary regions of the binding site which interact with para substituents and, thus, influence the strength of the primary phenyl-enzyme interaction. J Antibiot (Tokyo), 1977 Sep, 30(9), 749 - 52 Kuwaitimycin, effect on synthesis of lipids in Bacillus subtillis cells; Shimi IR et al.; The effects exerted by kuwaitimycin on synthesis of lipids as well as some metabolic activities of Bacillus subtilis were studied . The antibiotic not only arrested the inocrporation of 14C-acetate into the microbial lipids but also altered the fatty acids pattern, contents of i-C 15, a-C 15, i-C 17 and a-C 17 WERE MARKEDLY REDUCED, CONCOMITANT WITH AN INCREASE IN THE CONtents of i-C 14 AND N-C 14 . Moreover, the rates of synthesis of phospholipids were decreased by the drug, especially that of phosphatidyl ethanolamine. J Gen Microbiol, 1977 Sep, 102(1), 69 - 80 New types of mutation affecting formation of alkaline phosphatase by Bacillus subtilis in sporulation conditions; Piggot PJ et al.; Mutations defining three new loci, sapA, sapB and phoS, were detected by their ability to overcome the phosphatase-negative phenotype of early-blocked asporogenous mutants in sporulation conditions . Synthesis of alkaline phosphatase by Bacillus subtilis is subject to 'vegetative' and 'sporulation' controls . The phoS mutations resulted in constitutive production of alkaline phosphatase and so could be altered in either the 'vegetative' or the 'sporulation' control system . The sapA and sapB mutations only affected alkaline phosphatase formation in sporulation conditions, and were considered to be sporulation specific . They rendered 'sporulation' alkaline phosphatase formation independent of all the spomutations tested, and so independent of the control of the dependent sequences of spo locus expression; as the enzyme was not formed constitutively, it remained subject to some other sporulation control . The sapA and phoS loci were placed between argC4 and metC3 on the genetic map; the sapB locus was located close to purB6 . The three loci mapped separately from all known spo loci. J Bacteriol, 1977 Sep, 131(3), 866 - 71 Specific alteration of the 30S ribosomal subunits of Bacillus subtilis during sporulation; Guha S et al.; Active 30S ribosomal subunits were isolated from vegetative and sporulating cells of Bacillus subtilis . Both subunits were able to function in polyuridylic acid of phage phie messenger ribonucleic acid-dependent protein synthesis in vitro . The sporulation 30S subunits were highly active in polyuridylic acid-dependent polyphenylalanine synthesis but showed a reduced activity in the presence of natural messenger ribonucleic acid as compared with their vegetative counter-parts . The reduced activity was independent of the source of 50S particles and initiation factors (vegetative or sporulation) . The alteration of the 30S sporulation subunits appears to be related to the sporulation process, since the same subunits isolated from stationary-phase cells of an asporogenic mutant did not show any impairment in protein synthesis in vitro. J Bacteriol, 1977 Sep, 131(3), 776 - 83 Modulation of deoxyribonucleic acid polymerase III level during the life cycle of Bacillus subtilis; Ciarrocchi G et al.; Deoxyribonucleic acid (DNA) polymerase III is not detectable in Bacillus subtilis spores; the enzyme activity appears 20 to 30 min after spore activation and rapidly increases just before the onset of the first round of DNA replication (30 min later); the level of polymerase III further increases and reaches its maximum (on a per-genome basis) when the cells enter the vegetative phase of growth; this level is six- to eightfold higher than the one observed during germination . In the stationary phase, the polymerase III drops to levels comparable to those found in germinating spores at the first round of replication . On the contrary, DNA polymerase I is present at appreciable levels in the dormant spore; it increases during vegetative growth by a factor of three and, during the stationary phase, reaches its maximum level which is sixfold higher than that observed in the spores . The block of protein synthesis during vegetative growth does not cause an appreciable reduction of the two enzymes (in absolute terms), showing that the regulation of their levels is probably not due to a balance between synthesis and breakdown . These results indicate that polymerase III is probably one of the factors controlling the initiation of DNA synthesis during spore germination. J Bacteriol, 1977 Sep, 131(3), 981 - 7 Alteration of the Bacillus subtilis glutamine synthetase results in overproduction of the enzyme; Dean DR et al.; A mutational leading to glutamine auxotrophy was located near a 5-fluorouracil resistance marker in the citB-thyA region of the Bacillus subtilis chromosome . This mutation resulted in a glutamine synthetase with altered kinetic and feedback properties . The specific activity of manganese-stimulated glutamine synthetase activity in crude extracts was 18-fold higher, and the magnesium-stimulated activity was about 30% that of the wild type . Quantitation of the enzyme by precipitation with antibody prepared against pure enzyme confirmed the presence of high enzyme levels in the mutant . This mutation is very closely linked (recombination index of 0.03) to another glutamine auxotroph containing enzyme with altered electrophoretic and heat sensitivity properties . Mutations in the structural gene for glutamine synthetase may result not only in altered catalytic and regulatory properties but also in altered production of the enzyme. J Gen Microbiol, 1977 Aug, 101(2), 299 - 306 Morphological stages of Bacillus subtilis sporulation and resistance to fusidic acid; Fortnagel P et al.; During spore development of Bacillus subtilis both protein synthesis and sporulation become resistant to the antibiotic fusidic acid . This resistance develops at the time when asymmetric prespore septa are formed . Simultaneously ribosomes lose their ability to bind fusidic acid, as demonstrated by their affinity chromatography with the immobilized drug . Mutants resistant to fusidic acid during growth are oligosporogenous; their sporulation development is blocked before septum formation . These results indicate that normal ribosomes are needed for prespore septation sporulation; only after septation can protein synthesis be maintained, throughout the development period, by fusidate resistant ribosomes. J Gen Microbiol, 1977 Aug, 101(2), 227 - 31 Inactivation of bacillus spores in dry systems at low and high temperatures; Molin G; A plot of the thermal resistance of Bacillus subtilis var . niger spores (log D value) against temperature was linear between 37 and 190 degrees C (z = 23 degrees C), provided that the relative humidity of the spore environment was kept below a certain critical level . The corresponding plot for Bacillus stearothermophilus spores was linear in the range 150 to 180 degrees C (z = 29 degrees C) but departed from linearity at lower temperatures (decreasing z value) . However, the z value of 29 degrees C was decreased to 23 degrees C if spores were dried before heat treatment . The straight line corresponding to this new z value was consistent with the inactivation rate at a lower temperature (60 degrees C) . The data indicate that bacterial spores which are treated in dry heat at an environmental relative humidity near zero are inactivated mainly by a drying process . By extrapolation of the thermal resistance plot obtained under these conditions for B . subtilis var . niger spores, the D value at 0 degrees C would be about 4 years. Biokhimiia, 1977 Aug, 42(8), 1478 - 86 {Effects of mutations in Bacillus subtilis genome decreasing the protease activity on the formation of subtilisin molecular forms}; Abramov ZT et al.; Multiple molecular forms of subtilisin--extracellular serine protease produced by the wild strain Bac . subtilis A-50 and its mutant strains with the protease activity decreased two-fold and more were studied . Six molecular forms of subtilisin were found on the whole when 33 mutant strains have been investigated under the experimental conditions . It is essential that both the wild and each of mutant strains under study produced not more than three out of these six forms . Three molecular forms of subtilisin from the mutant strains are similar to those found in the wild strain A-50, and have the molecular weight, of 27 000-30 000 . Three other forms of subtilisin were revealed only in the mutant strains, and had the molecular weight of about 20 000 . Apparently there is only one structural gene for subtilisin in Bac . subtilis genome . The appearence of multiple molecular forms of subtilisin may be due to the post-translational modifications (limited proteolysis) of the initial type of enzyme, i.e . pre-subtilisin . Probably, that certain mulations not affecting the structural gene can significantly change the expression of such gene by varying of the degree of product modifications. Cell, 1977 Aug, 11(4), 751 - 61 Cloned Bacillus subtilis DNA containing a gene that is activated early during sporulation; Segall J et al.; An endonuclease restriction fragment of Bacillus subtilis DNA has been identified that contains a gene whose transcription is activated early during the process of spore formation . This 4.4 kilobase (kb) DNA was detected by hybridizing electrophoretically separated Eco R1 restriction fragments with a radioactively labeled RNA of 0.4 kb from sporulating cells . The 4.4 kb B . subtilis DNA was then cloned and amplified in E . coli by insertion into the plasmid vector pMB9 . Using the cloned B . subtilis DNA as a hybridization probe, we were able to detect the 0.4 kb transcript in total RNA from pulse-labeled bacteria . In wild-type cells, the gene coding for the 0.4 kb RNA was turned on within the first 30 min of spore formation . Although transcribed normally in a mutant blocked at stage II of spore development, the gene for the 0.4 kb RNA was not turned on in six different mutants blocked at stage 0 of sporulation . We conclude that the cloned B . subtilis DNA contains a gene whose transcription is regulated by events occurring at the onset of spore development. J Virol, 1977 Aug, 23(2), 377 - 83 Fragmentation of Bacillus bacteriophage phi105 DNA by complementary single-stranded DNA in the cohesive ends of the molecule; Scher BM et al.; The structure of DNA from the temperate Bacillus subtilis phage phi105 was examined by using the restriction endonuclease EcoRI and by sedimentation analysis . The DNA contains six EcoRI cleavage sites . Although eight DNA fragments were identified in the EcoRI digests, the largest of these was shown to consist of the two fragments that carry the cohesive ends of the phage DNA . In neutral gradients, the majority of whole phi105 DNA sedimented as nicked circles and the remainder as oligomers . No unit-length linear structures were detected . The associated cohesive ends could be sealed by DNA ligase from Escherichia coli and could be cleaved by S1 nuclease . On the basis of these results and previously reported studies, it appears that, as isolated from phage particles, phi105 DNA is a circular molecule that is formed from the linear structure by the association of complementary single-stranded DNA. J Bacteriol, 1977 Aug, 131(2), 699 - 701 Isolation and characterization of four types of plasmids from Bacillus subtilis (natto); Tanaka T et al.; Covalently closed circular deoxyribonucleic acids were found in 10 strains of Bacillus natto . The plasmids could be classified into four types on the basis of the molecular weights as well as the patterns in agarose gel electrophoresis after digestion with restriction endonucleases: (i) plasmids (seven were detected) with a molecular weight of 3.6 X 10(6); (ii) plasmids (two were detected) with a molecular weight of 4.0 X 10(6); (iii) plasmids (eight were detected) with a molecular weight of about 34 X 10(6); and (iv), a plasmid with an approximate molecular weight of 46 X 10(6) . Out of the 10 plasmid-carrying strains, 6 (IFO3009, IFO3013, IFO3335, IFO13169, IAM1143, and IAM1207) harbored both type 1 and 3 plasmids; 2 (IAM1114 and IAM1168) harbored both type 2 and 3 plasmids, and IFO3936 and IAM1163 carried type 1 and 4 plasmids, respectively. J Bacteriol, 1977 Aug, 131(2), 682 - 4 Conversion by trypsin of nonsense suppressor-produced isozymes of triosephosphate isomerase to isozymes resembling the wild type; Baptist JN et al.; Nonsense suppressor genes caused the synthesis of new triosephosphate isomerase isozymes in Bacillus subtilis . Incubation with trypsin produced a large decrease in the apparent molecular weight of one such isozyme and simultaneously changed the electrophoretic behavior such that it resembled that of the wild-type enzyme. J Bacteriol, 1977 Aug, 131(2), 382 - 8 Host cell reactivation of Bacillus subtilis bacteriophages; Ferrari E et al.; Host cell reactivation of ultraviolet-irradiated phage can be used as a probe of the bacterial repair system and to determine phage and cellular contributions to the repair process . Using the Bacillus subtilis phages SPP1, SP01, phie, and phi29, we found that the uvr-1 and polA functions are involved in the host cell reactivation of the four phages . SPP1 was the only phage whose reactivation was also decreased in recA, recD, and recF mutant cells . We studied variations of host cell reactivation for SPP1 during spore outgrowth; at high ultraviolet doses the activity of a spore repair system requiring deoxyribonucleic acid polymerase I became evident . The spore repair system was completely replaced by the vegetative one by 120 min of outgrowth. J Bacteriol, 1977 Aug, 131(2), 379 - 81 Porphyrin and corrinoid mutants of Bacillus subtilis; Miczak A; Porphyrin auxotrophs of Bacillus subtilis can be divided into two groups . Strains belonging to the first group (hemA, hemB, or hemC) are not able to synthesize or metabolize porphobilinogen . These strains require cysteine, cystine, and methionine, respectively . Traces of aminolevulinic acid, in a hemin-containing medium, can replace the cysteine requirement in a mutant lacking aminolevulinic acid synthetase . In bacteria belonging to the second group (hemE, hemF, or hemG), porphyrin biosynthesis is blocked at later steps, and the amino acids mentioned above are not required . It is of interest that both the activity of ribonucleotide reductase and the amount of vitamin B12 were significantly lower in the first group . The addition of vitamin B12 to the medium did not promote the growth of strains examined . We assume that porphobilinogen deaminase is essential for the synthesis of corrinoids. Biochemistry, 1977 Jul 12, 16(14), 3194 - 201 Partial purification and characterization of a uracil DNA N-glycosidase from Bacillus subtilis; Cone R et al.; A uracil specific DNA N-glycosidase activity has been partially purified from crude extracts of Bacillus subtilis . The enzyme has a molecular weight of approximately 24 000 with no subunit structure . It has no requirement for any known cofactors but is inhibited in the presence of Co2+, Fe2+, or Zn2+ . The enzyme is specific for uracil in single- and double-stranded deoxyribonucleopolymers and does not release free uracil from RNA or from poly(rU):poly(dA) . In addition, neither Udr, dUMP, nor dUTP is recognized as substrate . The enzyme will attack small poly(dU) oligomers but the minimum size recognized as substrate is (pU)4 . This enzyme may have a role in the repair (by base excision) or uracil in DNA arising either by incorporation during DNA synthesis or by deamination of cytosine in DNA. Ann Microbiol (Paris), 1977 Jul, 128B(1), 3 - 18 Development of bacteriophage phi29 in sporulating and non-sporulating cells of bacillus subtilis 168; Moreno F; Infection by bacteriophage phi29 of Bacillus subtilis 168 and of its asporogenous mutant spoOA-3NA has been studied in exponential and stationary phases . As first observed with phage phie infections, the burst-size decreases during the stationary phase much more rapidly in wild type than in mutant cells . In addition, the two strains are shown to differ even during growth in their response to phage phi29 infection . During a short period in the exponential phase, no phage production occurs when infected bacteria (whether spo+ or spo-) are incubated in their growth medium, but phage is produced when incubation takes place after transfer to fresh medium . From these and other unexpected findings it is concluded that any causal relation between sporulation and phage development must be considered with caution . Phage infection of spo+ cells at the end of the growth period does not affect the time required for mature spore formation. Mikrobiologiia, 1977 Jul-Aug, 46(4), 635 - 41 {Effect of different carbon, nitrogen and phosphorus sources on the biosynthesis of proteases with coagulase activity by Bacillus subtilis var, amyloliquefaciens}; Otroshko TA et al.; Proteolytic enzymes with coagulase activity were found in the cultural broth of Bacillus subtilis var . amyloliquefaciens 759 grown on chemically defined and natural media . The effect of various sources of carbon, nitrogen and phosphorus on biosynthesis of proteases with coagulase activity was studied; mineral and organic nitrogen sources were equally favourable for the growth and protease biosynthesis . The best sources of carbon for biosynthesis of the enzymes were glucose, sucrose, maltose, fructose, and sorbitol . Potassium salts of ortho-phosphoric acid were assimilated as a source of phosphorus. Eur J Biochem, 1977 Jul 1, 77(1), 61 - 7 {The structure of bacillomycin L, an antibiotic from Bacillus subtilis (author's transl)}; Besson F et al.; The structure of bacillomycin L, an antifungal agent isolated from the culture medium of a strain of Bacillus subtilis, has been investigated . The peptide moiety contains one mole each of D-aspartic acid, L-aspartic acid, L-glutamine, L-serine, D-serine, L-threonine, and D-tyrosine . The lipid moiety is a mixture of 3-amino-12-methyltridecanoic acid (46%), 3-amino-12-methyltetradecanoic acid (38%, 3-amino-14-methylpentadecanoic acid (11%), and two minor homologues . The peptide sequence and the cyclic structure were determined by structural analysis of the peptides obtained by mild acid hydrolysis . The molecular weight was determined by the thermoosmotic method; this showed that bacillomycin L has a monomeric structure which is given in Formula 1. J Biochem (Tokyo), 1977 Jul, 82(1), 1 - 8 Immunochemical studies on non-precipitating guinea pig antibody produced by administration of an excessive dose of Bacillus subtilis alpha-amylase; Mori N et al.; Administration of an excessive dose of Bacillus subtilis alpha-amylase {EC 3.2.1.1, alpha-1,4-glucan 4-glucanohydrolase} (BalphaA) induced the production of non-precipitating (non-ppt) IgG2 antibody in guinea pigs, whereas immunization with a normal dose produced precipitating (ppt) IgG1 and IgG2 antibodies . The non-ppt IgG2 antibody thus produced could be isolated from the coexisting ppt IgG2 antibody by means of the precipitin reaction at maximum precipitation . The non-ppt antibody was incapable of forming a precipitin arc with BalphaA in a conventional agar plate . In the presence of 4% polyethylene glycol (PEG), however, it formed a single arc which fused completely with those of the ppt IgG1 and IgG2 antibodies . The non-ppt antibody could not fix complement, but inhibited BalphaA activity, though with less efficiency than the ppt antibodies . These properties of the non-ppt IgG2 antibody may be due to a low affinity for BalphaA, since both gel filtration and precipitation of soluble antigen-antibody complexes with 20% PEG showed that the antibody was easily dissociable from BalphaA. J Bacteriol, 1977 Jul, 131(1), 374 - 7 Transformation of Escherichia coli and Bacillus subtilis with a hybrid plasmid molecule; Mahler I et al.; A hybrid molecule constructed from Escherichia coli plasmid pMB9 and a fragment of Bacillus subtilis 168 deoxyribonucleic acid functions in cells of leu-E . coli, converting them to leucine prototrophy, but fails to survive in strains of B . subtilis 168. Biull Eksp Biol Med, 1977 Jul, 84(7), 68 - 70 {Conduct of Bacillus subtilis transformation in mice under conditions of immunologic suppression of exogenous DNAase 1 activity}; Zhukov-Verezhnikov NN et al.; Bacillus subtilis transformation was conducted in the abdominal cavity of mice . The frequency of transformation was considerable decreased when bovine DNA-ase 1 (3-- 5microgram) was injected intraperitoneally to these animals . Immune rabbit gamma-globulins containing antibodies to bovine DNA-ase 1 inhibited in vivo the activity of DNA-ase 1, protected transforming DNA from the hydrolyzing effect of this enzyme . This model can be utilized in search for ways to preserve DNA injected into the animal organism for the purpose of genetical engineering. Prikl Biokhim Mikrobiol, 1977 Jul-Aug, 13(4), 577 - 80 {Denaturation of the alpha-amylase of Bacillus subtilis in an acid medium}; Kiseleva EM et al.; By fractionation of purine compounds (sorbtion on the cation exchange resin KU-2 (H+), extraction, precipitation of purine compounds as Ag-complexes) a "purine fraction" of the culture liquid epiphytic bacteria No . 703 isolated from barley overground organs was obtained . The presence of isopentenyl cytokinins was demonstrated by quality reactions of the purine fraction with aromatic amines and phenols as well as by the values of Rf and UV spectrum of individual compounds examined by paper chromatography of the purine fraction. Biochemistry, 1977 Jun 28, 16(13), 2880 - 4 Zinc is associated with the beta subunit of DNA-dependent RNA polymerase of Bacillus subtilis; Halling SM et al.; The Bacillus subtilis DNA-dependent RNA polymerase holoenzyme and core enzyme each contain approximately two atoms of zinc per molecule . When the dissociated subunits of the enzyme are passed through a blue dextran-Sepharose affinity column, only the beta subunit binds to the column . The total zinc content of the enzyme is tightly bound to the beta subunit . Dialysis studies suggest that the two zinc ions differ in the strength of their association with the beta subunit . The presence of zinc in beta is consistent with several other lines of evidence which indicate that this subunit is dirrectly involved in phosphodiester bond formation . The blue dextran-Sepharose column procedure should be useful in future studies of the dissociation and reassociation of the enzyme since the method is rapid and provides excellent recovery of the beta subunit as well as the alpha and beta' subunits of the RNA polymerase. J Biol Chem, 1977 Jun 25, 252(12), 4118 - 24 Incorporation of N-acetyl-D-glucosamine from UDP-N-acetyl-D-glucosamine by isolated membranes of Bacillus subtilis . Identification of undecaprenyl poly(N-acetylglucosaminyl pyrophosphate); Bettinger GE et al.; Membrane isolated from Bacillus subtilis strain 168 incorporated GlcNAc from UDP-GlcNAc directly onto undecaprenyl phosphate via transphosphorylation and subsequent transglucosylations . Chain lengths of 6, 4, and 1 units of GlcNAc were found . Approximately 80% of the isotope incorporated was extracted into chloroform:methanol (2:1 v/v), and could be distinguished from the undecaprenyl disaccharide cell wall intermediate by a different elution pattern on DEAE-cellulose (acetate form) . The GlcNAc-lipid(s) were eluted from a similar column in chloroform:methanol:water (10:10:3, v/v) with 6 mM NH4COOH indicating a pyrophosphate linkage between the lipid and the GlcNAc . The GlcNAc-lipid(s) were not degraded by conditions which completely deacylated {32P}glyceryl phospholipids, but were rapidly hydrolyzed by mild acid treatment (0.005 N HCl, 90 degrees) with the release of oligosaccharide phosphate (typical of sugars linked to undecaprenyl pyrophosphate) . Catalytic hydrogenation of the GlcNAc-lipid(s) resulted in the release of water-soluble sugar phosphate . Under these same conditions, undecaprenyl pyrophosphate and undecaprenyl disaccharide cell wall intermediate were similarly effected while {32P}glyceryl phospholipids remained intact . The formation of GlcNAc-lipid(s) in vitro was inhibited if membranes were prepared from cells previously treated with bacitracin . Thus, the GlcNAc-lipid(s) has the properties of undecaprenyl poly(N-acetylglucosaminyl pyrophosphate) and may represent a new synthetic role of the polyisoprenyl lipid in B . subtilis. Mol Gen Genet, 1977 Jun 8, 153(2), 219 - 25 DNA repair in Bacillus subtilis . II . Activation of the inducible system in competent bacteria; Yasbin RE; Competent B . subtilis are more UV sensitive than the non-competent population of the culture . This increased sensitivity is lose in mutants unable to induce the 'SOS system' (recA1,, recG13), in mutants defective in the induction of prophage PBSX (xin), and in late stage competent cells . Moreover, bacteriophage phi 105 produced from transfected cells are less restricted on strain YB880 than bacteriophage produced from infected cells . Therefore, competent cells (those capable of being transfected) have a DNA modification system, whereas the average log phase cell does not . These data support the hypothesis that the development of competence is correlated with the activation of derepression of the "SOS" system in B . subtilis. Mol Gen Genet, 1977 Jun 8, 153(2), 211 - 8 DNA repair in Bacillus subtilis . I . The presence of an inducible system; Yasbin RE; Following UV irradiation of Bacillus subtilis there is a coordinate induction of: 1) a new protein, 2) a W-reactivation system, 3) a DNA modification system, and 4) prophages . These functions are induced following UV irradiation of repair proficient bacteria and mutants deficient in excision repair (UVR-1) and DNA polymerase I activity (polA5) . However, they are not induced, or are impaired in their ability to be induced in bacteria containing the recA1 and the recG13 mutations . This inducible system is compared to the SOS system observed in E . coli. Mol Gen Genet, 1977 Jun 8, 153(2), 129 - 33 Expression of an excision repair gene in transformation of Bacillus subtilis; Tanooka H et al.; DNA of Bacillus subtilis proficient in excision repair (hcr+) was introduced into Angiografinpurified competent cells of an excision repair-deficient strains UVS-1 (hcr-1) . The hcr+ gene was found to affect the UV-survival curve of the cells, giving rise to a UV-resistant component . However, a considerable number of colonies of the UV-resistant component consisted of cells that were not transformed to hcr+ as judged by their sensitivity to mitomycin C (MC), UV, and by their ability to reactivate UV-irradiated M2 phages . This suggests that the hcr gene may be expressed without integration . The recA function of B . subtilis was necessary for expression of UV resistance to occur . When DNA-treated cells were selected for met+ recombinants, the UV-resistant component was again found on the UV-survival curve and about half of the colonies of the UV-resistant component consisted of Hcr- cells . This result was explained by an integration-segregation model for hcr+ and met+ genes . The effect of the hcr+ gene was seen even when DNA was added after cells were irradiated with UV, although this effect was gradually diminished by delaying the time of DNA addition . A complementation effect was found between two excision repair mutations residing in two distant loci, using hcr-114 DNA as a donor and hcr-1 cells as a recipient. Nucleic Acids Res, 1977 Jun, 4(6), 1769 - 82 Methylation of an adenosine in the D-loop of specific transfer RNAs from yeast by a procaryotic tRNA (adenine-1) methyltransferase; Raettig R et al.; tRNA (adenine-1) methyltransferase occurs in Bacillus subtilis . Eucaryotic tRNAThr and tRNATyr from yeast in which 1-methyladenosine (m1A) is already present in the TpsiC loop, can be methylated in vitro with S-adenosylmethionine and B . subtilis extracts . Each of the specific tRNAs accepts 1 mol of methyl groups per mol tRNA . The enzyme transforms into m1A the 3'-terminal adenylic acid residue of the dihydrouridine loop, a new position for a modified adenosine residue in tRNA . Both tRNAs have the sequence Py-A-A-G-G-C-m2(2)G in the D-loop and D-stem region . Other tRNAs with the same sequence in this region also serve as substrates for the tRNA (adenine-1) methyltransferase. J Virol, 1977 Jun, 22(3), 835 - 8 Metabolism of uracil-containing DNA: degradation of bacteriophage PBS2 DNA in Bacillus subtilis; Duncan BK et al.; When Bacillus subtilis is infected by the uracil-containing DNA phage PBS2, the parental DNA labeled with radioactive uracil and cytosine remains acid insoluble . If the synthesis of the phage-induced uracil-DNA N-glycosidase inhibitor is prevented, the parental DNA is completely degraded to acid-soluble products beginning at about 6 min after infection . The host N-glycosidase probably initiates the degradation pathway, with nucleases being responsible for the remaining degradation of the DNA. J Bacteriol, 1977 Jun, 130(3), 1224 - 33 Altered accumulation of a membrane protein unique to a membrane-deoxyribonucleic acid complex in a dna initiation mutant of Bacillus subtilis; Harmon JM et al.; Membrane-deoxyribonucleic acid complexes (M-bands) have been isolated from Bacillus subtilis by their affinity for crystals of Mg2+-Sarkosyl . The membrane proteins of these complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Comparison of the membrane protein composition of M-band and unfractionated membrane revealed three protein components of 125,000 (mac-1), 57,000 (mac-2), and 42,000 (mac-3) daltons unique to M-band membrane . Growth of a temperature-sensitive dna initiation mutant at the restrictive temperature resulted in an accumulation in the membrane of mac-2 . This accumulation did not begin, however, until cell growth had nearly ceased, some 3 to 4 h after the cessation of deoxyribonucleic acid synthesis . Upon return of the mutant to the permissive temperature, mac-2 did not begin to return to normal levels until after the first round of deoxyribonucleic acid synthesis . A protein of 30,000 daltons, common to both M-band and whole membrane, was found to disappear from the membrane when the mutant was grown at the restrictive temperature . This disappearance is the result of increased degradation or removal from the membrane followed by a decreased rate of synthesis or insertion. J Bacteriol, 1977 Jun, 130(3), 1055 - 63 Biosynthesis of wall polymers in Bacillus subtilis; Wyke AW et al.; Preparations of membrane plus wall derived from Bacillus subtilis W23 were used to study the in vitro synthesis of peptidoglycan and teichoic acid and their linkage to the preexisting cell wall . The teichoic acid synthesis showed an ordered requirement for the incorporation of N-acetylglucosamine from uridine 5'-diphosphate (UDP)-N-acetylglucosamine followed by addition of glycerol phosphate from cytidine 5'-diphosphate (CDP)-glycerol and finally by addition of ribitol phosphate from CDP-ribitol . UDP-N-acetylglucosamine was not only required for the synthesis of the teichoic acid, but N-acetylglucosamine residues formed an integral part of the linkage unit attaching polyribitol phosphate to the cell wall . Synthesis of the teichoic acid was exquisitely sensitive to the antibiotic tunicamycin, and this was shown to be due to the inhibition of incorporation of N-acetylglucosamine units from UDP-N-acetylglucosamine. J Bacteriol, 1977 Jun, 130(3), 1000 - 9 Mode of degradation of precursor-specific ribonucleic acid fragments by Bacillus subtilis; Schroeder E et al.; A precursor of 5S ribosomal ribonucleic acid (rRNA) from Bacillus subtilis was cleaved by ribonuclease (RNase) M5 in cell-free extracts from B . subtilis to yield the mature 5S rRNA plus RNA fragments derived from both termini of the precursor . The released, mature 5S rRNA was stable in these extracts; however, as occurred in vivo, the precursor-specific fragments were rapidly and completely destroyed . Such destruction was not observed in the presence of partially purified RNase M5, so fragment scavenging was not effected by the maturation nuclease itself . The selective destruction of the precursor-specific fragments was shown to occur through a 3'-exonucleolytic process with the release of nucleoside 5'-monophosphates; the responsible activity therefore had the character of RNAse II . Consideration of the primary and probable secondary structures of the precursor-specific fragments and mature 5S rRNA suggested that involvement of 3' termini in tight secondary structure may confer protection against the scavenging activity. Arch Microbiol, 1977 May 13, 113(1-2), 153 - 8 The effect of amino acids on the motile behavior of Bacillus subtilis; de Jong MH et al.; Constant levels of amino acids enhanced the velocity of Bacillus subtilis 60015 cells about 2-fold and stimulated the response in motility assays . The stimulation of velocity did not occur via the receptors for chemotaxis . Cysteine and methionine, general inhibitors of chemotaxis, both completely inhibited the smooth response in a temporal gradient of attractant . After methionine starvation B . subtilis 60015 showed no measurable response in a temporal gradient of attractant, this in contrast to the effect observed with some other bacteria . Addition of methionine to starved cells restored the response toward attractant . Revertants of B . subtilis 60015 for methionine requirement could not be starved and showed a normal behavior toward temporal gradients of attractant. J Biol Chem, 1977 May 10, 252(9), 2934 - 9 Kinetic evidence for an acyl-enzyme intermediate in D-alanine carboxypeptidases of Bacillus subtilis and Bacillus stearothermophilus; Nishino T et al.; The kinetics of hydrolysis and transpeptidation of the synthetic substrate diacetyl-L-lysyl-D-alanyl-D-alanine and of the natural substrate UDP-acetylmuramyl pentapeptide and related compounds catalyzed by the D-alanine carboxypeptidases of Bacillus subtilis and Bacillus stearothermophilus in the presence of the nucleophiles hydroxylamine or glycine have been examined . These kinetic data suggest that an acyl-enzyme intermediate is formed in the first step of the reaction and that the transpeptidation is the consequence of the partitioning of this intermediate between water and the nucleophile in the second step. Biochim Biophys Acta, 1977 May 3, 476(1), 76 - 87 Initiation of protein synthesis in bacillus subtilis in the presence of trimethoprim or aminopterin; Arnold HH; Initiation of protein synthesis has been studied in the presence of the tetrahydrofolic acid analogues trimethoprim or aminopterin in Bacillus subtilis . This bacterium can grow in the presence of the inhibitors, when the medium is supplemented with the low molecular weight products of tetrahydrofolate-dependent pathways . In an attempt to show whether formylation of initiator tRNA is a prerequisite for the iniation of protein synthesis in procaryotic cells, the amount of N-formylmethionine in tRNA and in protein has been determined . The level of formylation of methionyl-tRNA was found to be 70% in control cells and approximately 2% in inhibitor-treated cells . The content of formyl groups in protein has also been found to be drastically reduced . Trimethoprim or aminopterin did not alter the amount of tRNAMet nor the degree of aminoacylation of tRNAMet in vivo . These results indicate that in B . subtilis inititation of protein synthesis is possible without prior formylation of initiator tRNA. Zh Mikrobiol Epidemiol Immunobiol, 1977 May, (5), 33 - 7 {Heterologous transformation in Bacillus subtilis . 1 . Transmission of DNA of the R1drd19 plasmid}; Naumov LS et al.; Bac . subtilis 168 (BD-25) cells were infected with DNA of plasmide R1drd19 isolated from E . coli strain; transformants resistant to streptomycin (500 microgram/ml) and kanamycin (40 microgram/ml) appeared with the frequency of 2.10(-6) . These transformants retained resistance to the mentioned antibiotics stably . A satellite DNA peak was revealed in centrifugation in the density gradient of cesium chloride with ethidium bromide . It was possible to infect cells of Bac . subtilis 168 (BD-25) with plasmide DNA isolated from the transformants . Plasmide transduction with the aid of phages AR9 and PBSI multiplied on the transformant strains was also effected . Physico-chemical analysis of the transformed plasmide DNA was conducted; its molecular weight was determined. Mikrobiologiia, 1977 May-Jun, 46(3), 539 - 46 {Comparative study of the multiplicity of exoenzymes produced by different strains of Bacillus subtilis}; Strongin AIa et al.; Molecular forms of two exoenzymes, subtilisin and alpha-amylase, produced by mutants of Bacillus subtilis were studied . The degree of the post-translational modification of subtilisin was less pronounced for P and M mutants than for R mutants . Some of the P and M mutants produced subtilisin having higher molecular weight and hydrophobicity as compared to the R mutants . This form of subtilisin may be the primary product of translation of the structural gene and therefore a precursor of subtilisin . Its appearance outside the cell may be due to the alteration in the cell surface structures in the P and M mutants and abnormal post-translational modification . The P and M mutants produced also differing exocellular proteins as compared to the R mutants, e.g . alpha-amylase forms with the higher isoelectric points. Mikrobiologiia, 1977 May-Jun, 46(3), 450 - 5 {Amylase formation in a periodic and continuous culture of Bacillus subtilis}; Pazlarova Ia et al.; The dynamics of alpha-amylase production by two strains of Bacillus subtilis (A32 and A32.6) was studied under periodic and continuous conditions of cultivation on a chemically defined medium and on a natural medium . In the periodic culture, the highest activity of the enzyme was found during the stationary growth phase . In the conditions of chemostat, as the dilution rate from 0.05 to 0.15 hr-1, the activity of amylase remained at the same, high level for a certain lapse of time and then decreased . The rate of the decrease of alpha-amylase activity depended on the dilution rate. J Biochem (Tokyo), 1977 May, 81(5), 1571 - 4 Alteration in two enzymatically active forms of valyl-tRNA synthetase during the sporulation of Bacillus subtilis; Ohyama K et al.; Two enzymatically active forms of valyl-tRNA synthetase {EC 6.1.1.9} were found in the cells of Bacillus subtilis . The aminoacylation activities of the two forms were altered during the sporulation of B . subtilis . The tRNA'S acylated by these enzymes were analyzed by methylated albumin-Kieselguhr column chromatography. J Biochem (Tokyo), 1977 May, 81(5), 1187 - 92 Hydrolysis of phenyl beta-maltoside catalyzed by saccharifying alpha-amylase from Bacillus subtilis; Ishikura K et al.; 1 . The hydrolytic reaction of phenyl beta-maltoside catalyzed by saccharifying alpha-amylase {EC 3.2.1.1} of Bacillus subtilis was studied at 25 degrees C and pH 5.4, by measuring the total reducing power and the amount of phenol liberated, and by thin layer chromatography . 2 . The enzyme hydrolyzed phenyl beta-maltoside at the glucosidic linkage between the two glucose residues to form D-glucose and phenyl beta-D-glucoside . Besides these products, maltose, maltotriose, and phenyl beta-maltotrioside were also observed as reaction products . The identification of phenyl beta-maltotrioside is described in detail . The formation of these products was attributed to the transglycosylation reaction of the enzyme . The time course of reaction as followed by reducing power measurement showed an induction period of several minutes. Appl Environ Microbiol, 1977 May, 33(5), 1170 - 6 Effect of combined heat and radiation on microbial destruction; Fisher DA et al.; A series of experiments at several levels of relative humidity and radiation dose rates was carried out using spores of Bacillus subtilis var . niger to evaluate the effect of heat alone, radiation alone, and a combination of heat and radiation . Combined heat and radiation treatment of microorganisms yields a destruction rate greater than the additive rates of the independence agents . The synergistic mechanism shows a proportional dependency on radiation dose rate an Arrhenius dependency on temperature, and a dependency on relative humidity . Maximum synergism occurs under conditions where heat and radiation individually destroy microorganisms at approximately equal rates . Larger synergistic advantage is possible at low relative humidities rather than at high relative humidities. J Gen Microbiol, 1977 May, 100(1), 177 - 88 Arginine hydroxamate-resistant mutants of Bacillus subtilis with altered control of arginine metabolism; Harwood CR et al.; Arginine hydroxamate inhibits the growth of Bacillus subtilis . From a large number of mutants isolated as resistant to this arginine analogue, 29 were chosen for further investigation . Most of these shared diminished ability to utilize arginine, citrulline and/or ornithine as sole nitrogen source . All 29 had reduced levels of the catabolic enzymes arginase and ornithine aminotransferase under various conditions in which these enzymes are induced in the parent . In some circumstances, five of the mutants also showed elevated levels of the biosynthetic enzyme ornithine carbamoyltransferase . On the basis of these data, the 29 mutants were divided into six phenotypic classes; in four of these, control of ornithine carbamoyltransferase was the same as in the wild type, while in the other two it was altered . It is suggested that the isolates carry regulatory mutations, and that certain of these may affect simultaneously the formation of arginine catabolic and biosynthetic enzymes . The implication of the latter is that in B . subtilis, as in yeast, controls of the catabolic and biosynthetic pathways are connected . Single representatives of five of the phenotypic classes carry mutations conferring arginine hydroxamate resistance linked to cysA by transduction with phage PBSI; this did not appear to be true for a representative of the sixth class. J Bacteriol, 1977 May, 130(2), 610 - 9 Relation between cell wall turnover and cell growth in Bacillus subtilis; Glaser L et al.; The kinetics of cell wall turnover in Bacillus subtilis have been examined in detail . After pulse labeling of the peptidoglycan with N-acetylglucosamine, the newly formed peptidoglycan is stable for approximately three-quarters of a generation and is then degraded by a process that follows first-order kinetics . Deprivation of an auxotroph of amino acids required for protein synthesis results in a cessation of turnover . If a period of amino acid starvation occurs during the lag phase of turnover, then the initiation of turnover is delayed for a period of time equivalent to the starvation period . During amino acid starvation, new cell wall peptidoglycan is synthesized and added to preexisting cell wall . This peptidoglycan after resumption of growth is also subject to degradation (turnover) . It is suggested that cell wall turnover is dependent on cell growth and elongation . Several possible control mechanisms for cell wall autolytic enzymes are discussed in light of these observations. Vet Med (Praha), 1977 Apr, 22(4), 249 - 54 {Pathogenicity of aerobically sporulating microorganisms: Bacillus subtilis}; Jankova B et al.; Bioassays were made on white mice and rats to find the potential pathogenicity of B . subtilis and its metabolites . The peroral and intraperitoneal application of several strains of B . subtilis to white mice did not cause any changes either in the behaviour or health condition of the animals until the first to seventh day after test . B . subtilis cultures or cellular suspensions on the one hand and non-cellular filtrates of cultures represented by the B . subtilis metabolism products on the other hand were given to the animals . No changes in the health condition of the mice and rats were observed when feed artificially infected with B . subtilis was fed . Dissection did not reveal any macroscopic changes on the organs of the abdominal and thoracic cavity . Cultures from the individual body organs (liver, spleen, kidneys, parts of the small and large intestines, stomach) perorally infected with B . subtilis showed the presence of the microbe only in parts of the digestive tracts. Proc Natl Acad Sci U S A, 1977 Apr, 74(4), 1680 - 2 Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis; Ehrlich SD; One S . aureus plasmid coding for tetracycline resistance, pT127, and four plasmids (pC194, pC221, pC223, and pUB112) coding for chloramphenicol resistance have been introduced by transformation into B, subtilis . The plasmids replicate in--and confer antibiotic resistance upon--their new host . These experiments show that the potential for genetic exchange between diverse bacterial species is greater than has been commonly assumed. J Bacteriol, 1977 Apr, 130(1), 538 - 9 Initiation and termination of chromosome replication at 45 degree C in a temperature-sensitive deoxyribonucleic acid initiation mutant of Bacillus subtilis 168, TsB134; Burnett L et al.; Deoxyribose nucleic acid transfer experiments showed that upon shifting Bacillus subtilis TsB134 to 45 degree C, initiation of new rounds of replication was effectively blocked and the majority of existing rounds terminated. J Bacteriol, 1977 Apr, 130(1), 200 - 4 Classification of Bacillus subtilis flagellins; Simon MI et al.; Purified flagellins derived from 16 strains of Bacillus subtilis were classified into at least five distinct groups on the basis of their reaction with antiflagellar filament antibody and antiflagellin antibody . This classification was in good accord with that derived independently on the basis of amino acid analyses of the flagellins . Flagellar antigenicity appears to provide a useful typological character in classifying B . subtilis strains. Eur J Biochem, 1977 Apr 1, 74(2), 313 - 8 Thymidine uptake in bacteria: the effect of purine nucleosides; Vyacheslavov LG et al.; The kinetics of thymidine uptake by Escherichia coli and Bacillus subtilis cells in the presence of adenine and guanine nucleosides was investigated . The initial concentration of thymidine in the growth medium was 0.35 microng/ml while the initial concentration of purine nucleosides ranged from 25 to 250 microng/ml . Adenine nucleosides when present at a concentration more than 50 microng/ml strongly inhibit thymidine uptake by the bacteria . The duration of the inhibition depends on the initial concentration of adenine nucleoside in the growth medium . At an initial concentration of deoxyadenosine (or adenosine) of 250 microng/ml the time of inhibition of thymidine uptake was about 60 min . During this period thymidine is almost completely preserved from the action of bacterial thymidine phosphorylase . Guanine nucleosides (guanosine or deoxyguanosine) do not markedly inhibit thymidine uptake by bacteria even at a concentration of 250 microng/ml . It is shown that they do protect thymidine from the phosphorolytic action of the thymidine phosphorylase although much less effectively than adenine nucleosides . It is suggested that some areas in the bacterial membrane where thymidine phosphorylase is located are not available to guanine nucleosides. Mol Gen Genet, 1977 Mar 28, 152(1), 65 - 9 Restriction and modification in Bacillus species: genetic transformation of bacteria with DNA from different species, part I; Uozumi T et al.; Host specific restriction was detected in 13 Bacillus strains, when 63 strains of Bacillus subtilis and 15 other Bacillus strains were tested with phage phi 105C . These 13 strains were classified into 8 groups (M,H,C,N,E,F,G,P) by the type of restriction . M-type strains (B . subtilis Marburg 168, its derivatives, and two other strains) showed relatively weak restriction, restricting phi 105C from other groups of Bacillus by ratios of 10(-1) to 10(-3) . Strains of groups H,C,N,E,F,G, and P restricted phi 105C from other groups by ratios of 10(-2) to 10(-8) . It was confirmed with some of the strains that type-specific modification was endowed only by the last host . Furthermore, we isolated one restriction deficient mutant of B . subtilis Marburg 168-YS11, which had also lost its modification phenotype. Eur J Biochem, 1977 Mar 15, 74(1), 149 - 54 Purification and comparative properties of the delta and sigma subunits of RNA polymerase from Bacillus subtilis; Tjian R et al.; Bacillus subtilis delta protein is a 21 500-Mr polypeptide that can be isolated in association with RNA polymerase holoenzyme from uninfected bacteria and with modified forms of RNA polymerase from cells infected with phage SP01 {Pero, J., Nelson, J . and Fox, T . (1975) Proc . Natl Acad . Sci . U.S.A . 72,1589} . Although no function has been assigned to delta protein in uninfected cells, this host polypeptide enhances the specificity of transcription by phage-modified forms of RNA polymerase that contain SP01-coded regulatory subunits . This report describes the purification of delta and sigma proteins from uninfected B . subtilis and examines the comparative effects of these polypeptides on transcription by core RNA polymerase . Purified sigma polypeptide was found to stimulate the transcription of phage DNA while having little effect on RNA synthesis with the synthetic DNA poly(dA-dT) as template . In contrast, purified delta protein markedly depressed the transcription of poly(dA-dT) while having little effect on enzyme activity with phage DNA as template . The inhibitory effect of delta protein on poly (dA-dT) transcription was strongly dependent on the presence of KC1 in the RNA synthesis reaction mixture. J Biol Chem, 1977 Mar 10, 252(5), 1571 - 5 Inactivation of bacterial D-amino acid transaminases by the olefinic amino acid D-vinylglycine; Soper TS et al.; D-Vinylglycine (2-amino-3-butenoate) functions as a transamination substrate and irreversible inactivator of the homogeneous pyridoxal phosphate-dependent D-amino acid transaminases from Bacillus subtilis and Bacillus sphaericus . In the absence of alpha-ketoglutarate as co-substrate, vinyl-glycine causes little if any inactivation of either enzyme; in the presence of excess alpha-ketoglutarate, both enzymes are inactivated with pseudo-first order kinetics . The limiting rate constant for inactivation of the B . sphaericus enzyme is 1.9 min-1, for the B . subilis enzyme it is 0.36 min-1 . The number of catalytic events before inactivation is about 450 for the B . sphaericus enzyme and about 800 for the B . subtilis enzyme; that is, about 0.2% inactivation in each catalytic cycle for the former enzyme and 0.15% for the latter . Comparisons are made with the L-aspartate amino-transferase from pig heart which is inactivated completely in one catalytic cycle and the L-alanine aminotransferase which is not inactivated in many cycles . Comparisons are also made between the likely mode of D-transaminase inactivation produced by vinylglycine and the mode of inactivation induced by beta-chloro-D-alanine. J Biol Chem, 1977 Mar 10, 252(5), 1647 - 53 Endonuclease V of Escherichia coli; Gates FT 3rd et al.; A small endodeoxyribonuclease )2.3 S) that is active on single-stranded DNA has been extensively purified from Escherichia coli so as to be free of other known DNases . It has an alkaline pH optimum (9.5), requires Mg2+, and makes 3'-hydroxy and 5'-phosphate termini . The nuclease nicks duplex DNA, particularly if treated with OsO4, irradiated with ultraviolet light, or exposed to pH 5 . The uracil-containing duplex DNA from the Bacillus subtilis phage PBS-2 is an especially good substrate; it is made acid-soluble by levels of the enzyme which fail to produce any acid-soluble material in other single-stranded or duplex DNAs . Neither RNA nor RNA-DNA hybrid are degraded by the enzyme . The enzyme specificity suggests that it might act at abnormal regions in DNA, so that its in vivo function could be to initiate an excision repair sequence . Its high activity on uracil-containing DNA could imply that the enzyme provides an alternative mechanism for excising uracil residues from DNA to the pathway utilizing uracil-DNA N-glycosidase . We suggest that this enzyme be designated as endonuclease V of E . coli. Gene, 1977 Mar, 1(2), 169 - 80 Effect of site-specific endonuclease digestion on the thyP3 gene of bacteriophage phi 3T and the thyP11 gene of bacteriophage rho11; Graham RS et al.; phi 3T and rho11 are closely related bacteriophages of Bacillus subtilis which can "convent" thymine auxotrophs to thymine prototrophs upon infection or transfection . The effect of endonuclease digestion on the ability of both bacteriophage and prophage DNA from phi eT and rho11 to transform for thymine prototrophy was determined . All of the endonucleases tested: BamHI, Bg/II, BsuRI, EcoRI, HindII+ III, and HpaII reduced the efficiency of thyP transformation to an equal extent in prophage and bacteriophage DNA . Only HpaII completely abolished thyP transformation . The reduction in transformation with BamHI, Bg/II, BsuRI, EcoRI, and HpaII fragments is size related . The thyP transforming fragments generated by these endonucleases are potentially clonable. Gene, 1977 Mar, 1(2), 153 - 67 Transformation of Bacillus subtilis and Escherichia coli by a hybrid plasmid pCD1; Duncan CH et al.; The gene thyP3 from Bacillus subtilis bacteriophage phi 3T was cloned in the plasmid pMB9 . The resulting chimeric plasmid, pCD1, is effective in transforming both Escherichia coli and Bacillus subtilis to thymine prototrophy . The activity of the thyP3 gene product, thymidylate synthetase, was assayed and found to be 9 times greater in a transformed strain of Escherichia coli than in a phi 3T lysogen of Bacillus subtilis . The physical location of restriction sites has been determined for two related plasmids pCD1 and pCD2 . Hybridization studies clearly indicate that the plasmid gene responsible for Thy+ transformation is the gene from the bacteriophage phi 3T . The lack of restriction in this transformation process is consistent with our previous studies using bacterial DNA in heterospecific exchanges indicating that the nucleotide sequence surrounding the gene is the dominant factor in determining interspecific transformation. Nucleic Acids Res, 1977 Mar, 4(3), 603 - 23 RNA polymerase from Bacillus subtilis: isolation of core and holo enzyme by DNA-cellulose chromatography; Plevan P et al.; A new procedure for the purification of B . subtilis RNA polymerase, based on mild lysis of cells, low speed centrifugation, gel filtration, DEAE-Sephadex chromatography and affinity chromatography on DNA-cellulose, yields three forms of enzyme referred here as enzyme A, B and C . As revealed by SDS gel electrophoresis, enzyme A has the subunit structure of core polymerase plus some small polypeptides . Its catalytic properties are similar to those of core polymerase . Enzyme B has the composition of core polymerase . Both enzymes A and B can be stimulated by the addition of beta factor . Enzyme C has the holo-enzyme composition . The pattern of sensitivity of the three forms of enzyme towards KCl are very different: enzymes A and B, even at low concentration of salt, are inhibited with all the DNA templates tested, whereas enzyme C shows a pattern of stimulation specific for each DNA tested . The transcripts of the three enzymes on phage SPP1 DNA template have been analyzed by hybridization to the separated strands . Only enzyme C selectively transcribed the H strands. Proc Natl Acad Sci U S A, 1977 Mar, 74(3), 1125 - 9 Studies on the control of development: isolation of Bacillus subtilis mutants blocked early in sporulation and defective in synthesis of highly phosphorylated nucleotides; Rhaese HJ et al.; To test our model on the mechanism of initiation of differentiation in Bacillus subtilis, we tested early blocked (stage 0) sporulation mutants for their ability to synthesize highly phosphorylated nucleotides . We also isolated early blocked asporogenous mutants with the aid of the intercalating drug tilorone . Among all mutants tested we found that the spo0F-bearing strain was unable to synthesize adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp . A revertant of this mutant regained the ability to both sporulate and synthesize pppAppp . Ribosomes of the asporogenous mutant isolated at T2 (2 hr after the end of logarithmic growth) of sporulation, in contrast to the wild type, do not synthesize adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp, or adenosine 3'(2')-diphosphate 5'-triphosphate, pppApp, but synthesize guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp . This behavior is characteristic of ribosomes from vegetative, not sporulating, cells . Ribosomes from the sporogenous revertant behave like those of the wild type . The results suggest that the spo0F mutation may be a mutation in the structural gene for pppAppp synthetase . The inability to synthesize pppAppp in this strain also prevents the formation of "sporulation-specific ribosomes," i.e., ribosomes that synthetize ppApp and pppApp . The present experiments suggest that the nucleotide pppAppp participates in the initiation of sporulation by triggering a sequencies of events required for the production of heat-resistant spores. Proc Natl Acad Sci U S A, 1977 Mar, 74(3), 1009 - 12 Isolation of the penicillin-binding peptide from D-alanine carboxypeptidase of Bacillus subtilis; Georgopapadakou N et al.; The D-alanine carboxypeptidase of B . subtilis is a membrane-bound enzyme which is inhibited by penicillins and binds them covalently . The enzyme has been labeled with {14C}- or {35S}penicillin . After tryptic or Pronase digestion of the labeled, denatured, reduced, and carboxymethylated enzyme, a radioactive peptide was isolated in each case . The amino acid compositions of these two peptides are reported . The Pronase peptide was a subset of the tryptic peptide . Neither contained a cysteine residue and the only amino acid in the Pronase peptide to which the penicillin could be bound was a serine residue. J Virol, 1977 Mar, 21(3), 924 - 31 Effect of antibiotics on certain aspects of bacteriophage SP-15 development in Bacillus subtilis W23; Dosmar M et al.; Bacillus subtilis W23 was infected with bacteriophage SP-15 . Two waves of phage-specific RNA synthesis were observed . Wave I was prereplicative, and wave II was coincident with replication of the viral genome . To determine the temporal appearance of general classes of phage-coded messengers and proteins, we studied the dependence of lysozyme synthesis, phage production, and DNA synthesis on time of addition of transcriptional and translational inhibitors . Lysozyme synthesis started to become refractile to a variety of transcriptional inhibitors (rifampin, streptolydigin, and actinomycin D) between 20 and 22 min postinfection and was completely refractile by 30 min . Nevertheless, functional enzyme did not appear until 45 to 47 min postinfection; lysozyme was maximal by 65 min . Rna isolated from SP-15 phage-infected cells was used to program the cell-free synthesis of lysozyme . The messenger was synthesized exclusively between 20 and 30 min postinfection . Lysozyme messengers were stable . The data imply that lysozyme messengers were present 52 min prior to their translation . Progeny virus formation remained sensitive to transcriptional inhibitors until 40 to 50 min postinfection, and sensitivity to chloramphenicol lasted 65 min . The first progeny viruses appeared at 75 min . Again, an unusually long lag between completion of functional messengers and their translation was evident . The aforementioned data indicated that transcription of lysozyme messengers and, at least, some messengers, whose products are essential for phage production, are uniquely associated with waves I and II of RNA synthesis, respectively . However, messengers whose products are essential for normal amounts of DNA synthesis were apparently synthesized during both waves; transcription of these messengers was transiently repressed (using the term broadly) between 30 and 40 min postinfection . Judging from the dependence of DNA synthesis on time of chloramphenicol addition, proteins essential for normal amounts of DNA synthesis were also synthesized in two discrete waves, each yielding sufficient protein for half-maximal levels of DNA synthesis . An hiatus in the synthesis of the proteins in question was evident between 45 and 65 min postinfection; evidence cited in this paper indicates that this hiatus did not result from messenger depletion, which, in turn, implied some type of translational-level control . This latter conclusion is substantiated by the lysozyme synthesis that occurred during the same interval when synthesis of certain proteins for DNA replication was transiently repressed. J Virol, 1977 Mar, 21(3), 1223 - 7 Adsorption of bacteriophages phi 29 and 22a to protoplasts of Bacillus subtilis 168; Jacobson ED et al.; Adsorption of bacteriophages phi 29 and 22a to protoplasts of Bacillus subtilis 168 is described . The number of binding sites on bacilli and protoplasts is determined for each phage . Bacilli and protoplasts possess roughly the same number of sites per unit area for phi 29, i.e., approximately 700 sites per bacillus . There are also approximately 700 sites per bacillus for 22a, but only about one-third as many sites per unit area on the protoplast surface . A model for phi 29 adsorption is proposed. J Bacteriol, 1977 Mar, 129(3), 1639 - 41 Inhibition of iron uptake and deoxyribonucleic acid synthesis by Desferal in a mutant strain of Bacillus subtilis; Arceneaux JE et al.; In the Bacillus subtilis mutant 1D-4, the hydroxamate Desferal inhibited growth, iron uptake, and deoxyribonucleic acid synthesis but did not quantitatively affect synthesis of ribonucleic acid and protein. J Bacteriol, 1977 Mar, 129(3), 1487 - 94 Isolation and characterization of four plasmids from Bacillus subtilis; Tanaka T et al.; Nineteen Bacillus subtilis isolates obtained from type culture collections were examined for the presence of covalently closed circular duplex deoxyribonucleic acid molecules by the technique of cesium chloride-ethidium bromide density gradient centrifugation . Four of the 19 strains tested carried covalently closed circular molecules . Two of these strains (IFO3022, IFO3215) harbored a similar plasmid with a molecular weight of 5.4 X 10(6) . The other two strains (IAM1232, IAM1261) carried 4.9 C 10(6)-and 5.3 X 10(6)-dalton plasmids, respectively . These plasmid-harboring strains did not show phenotypic traits such as antibiotic resistance orbacteriocin production . The plasmid deoxyribonucleic acids were digested by three restriction endonucleases, EcoRI, HindIII, and BamNI, and were classified into three different types from their electrophoretic patterns in agarose gels. J Bacteriol, 1977 Mar, 129(3), 1440 - 7 Sodium effect of growth on aspartate and genetic analysis of a Bacillus subtilis mutant with high aspartase activity; Iijima T et al.; Most strains of Bacillus subtilis, dervied from the 168 (Marburg) strain, grow slowly on aspartate as sole carbon source . We isolated a mutant (aspH) that grows rapidly on aspartate because it produces aspartase constitutively . Thus, aspartase is needed for rapid growth on aspartate, whereas aspartate-alpha-ketoglutarate aminotransferase is not needed, as was demonstrated by a mutant lacking that enzyme activity . By two--and three-factor crosses using PBSl transduction, the aspH mutation was located between the aroD and the lys markers of the genetic map . Although sodium ions do not affect growth on glucose or L-malate, they specifically stimulate growth on aspartate in both the parent and the aspH mutant strains . Enzyme activities of crude aspartase and fumarase and of purified aspartase do not increase in the presence of sodium . These results show that stimulation by sodium involves some reaction other than the enzymes catabolizing aspartate . The ease of purification from the aspH strain and the stability of aspartase suggest that the B . subtilis enzyme is particularly useful for aspartate determinations. J Bacteriol, 1977 Mar, 129(3), 1313 - 9 Transitory germinative excision repair in Bacillus subtilis; Wang TC et al.; Bacillus subtilis strains UVSSP-42-1 (hcr42 ssp1) and UVSSP-1-1 (hcr1 ssp1) are ultraviolet (UV) radiation sensitive both as dormant spores and as vegetative cells . These strains are unable to excise cyclobutane-type dimers from the deoxyribonucleic acid (DNA) of irradiated vegetative cells and fail to remove spore photoproduct from the DNA of irradiated spores either by excision (controlled by gene hcr) or by spore repair (controlled by gene ssp1) . When irradiated soon after spore germination, these strains excise dimers, but not spore photoproduct, from their DNA . This process, termed germinative excision repair, functions only transiently in the germination phase and is responsible for the high UV resistance of germinated spores and for their temporary capacity to host cell reactivate irradiated phages infecting them . The recA1 mutation confers higher UV sensitivity to the germinated spores, but does not interfere with dimer removal by germinative excision repair. J Bacteriol, 1977 Mar, 129(3), 1215 - 21 Selective inhibition of Bacillus subtilis sporulation by acridine orange and promethazine; Burke WF Jr et al.; Two structurally similar compounds were found to inhibit sporulation in Bacillus subtilis 168 . A dye, acridine orange, and an antischizophrenic drug, promethazine, blocked spore formation at concentrations subinhibitory to vegetative growth, while allowing synthesis of serine protease, antibiotic, and certain catabolite-repressed enzymes . The sporulation process was sensitive to promethazine through T2, whereas acridine orange was inhibitory until T4 . The drug-treated cells were able to support the replication of phages phie and phi29, although the lytic cycles were altered slightly . The selective inhibition of sporulation by these compounds may be related to the affinity of some sporulation-specific genes to intercalating compounds. J Bacteriol, 1977 Mar, 129(3), 1198 - 207 Synthesis of cell envelope components by anucleate cells (minicells) of Bacillus subtilis; Mertens G et al.; Minicells produced by Bacillus subtilis CU403 (divIVB1) are capable of mucopeptide biosynthesis as shown by the incorporation of L-alanine, D-alanine, and N-acetylglucosamine into trichloroacetic acid-precipitable material, which can be degraded to trichloroacetic acid-soluble material by lysozyme digestion . Incorporation of the precursors is sensitive to vancomycin and D-cycloserine and insensitive to chloramphenicol . Penicillin inhibits the incorporation of D- and L-alanine N-acetylglucosamine at concentrations in excess of 10 mug of penicillin per ml; however, minicells are insensitive to penicillin-induced lysis . The material synthesized in minicells from N-acetylglucosamine is not subject to turnover during a subsequent 6-h incubation period . {2-3H}glycerol is converted to a cold trichloroacetic acid-precipitable form by minicells . This synthesis is not inhibited by vancomycin, penicillin, D-cycloserine, or chloramphenicol . Fractionation of the material synthesized from glycerol into hot trichloroacetic acid-soluble material and chloroform/methanol-extractable material indicates that minicells convert glycerol into teichoic acid and lipid. Eur J Biochem, 1977 Mar 1, 73(2), 393 - 400 Comparative analysis of three guinea pig satellite DNA's by restriction nucleases; Altenburger W et al.; The structures of guinea pig satellite DNAs I, II, and III have been analyzed by digestion with seven restriction nucleases . From the cleavage patterns it is obvious that the long-range periodicities in these three satellites differ rather characteristically Satellite I is fairly resistant to six nucleases and gives only a number of weak discrete bands which do not show a simple regularity . By the restriction nuclease from Arthrobacter luteus, however, it is cleaved extensively and yields very heterogeneous breakdown products . This is consistent with the high extent of divergence previously found for this satellite, e . g . by sequence analysis . Satellite II is almost completely resistant to all nucleases, indicative of a high degree of sequence homogeneity of this satellite . Satellite III is completely broken by the restriction nuclease from Bacillus subtilis into fragments which form a novel, highly regular series of bands in gel electrophoresis . The patterns show that the satellite is composed of tandem repeats ofapproximately 215 nucleotide pairs length, each repeat unit containing two cleavage sites for this nuclease . The data are consistent with the assumption that 30--40% of all cleavage sites have been eliminated by a random process . Satellite III DNA yields weak degradation patterns of the same periodicity with a number of other restriction nucleases . Cleavage sites for these nuclease are clustered on separatesmall segments of the satellite DNA . In this respect, the satellite is similar to others, notably the mouse satellite DNA . The three guinea pig satellites are examples of more general types of satellite structures also found in othe organisms . Similarities and differences to other satellites are discussed with special consideration to theories on the evolution of this class of DNA. J Biol Chem, 1977 Feb 25, 252(4), 1350 - 7 Partial purification and properties of a ribosomal RNA maturation endonuclease from Bacillus subtilis; Sogin ML et al.; Data are presented on the partial purification and properties of a 5 S ribosomal RNA maturation nuclease, termed RNase M5, from Bacillus subtillis 168 . RNase M5 specifically cleaves 21 and 42 nucleotides, respectively, from the 5' and 3' termini of a 5 S rRNA precursor to yield the mature (116 nucleotides) 5 S rRNA . The cleavage is endonucleolytic with the formation of 5'-phosphoryl and 3'-hydroxyl groups . Enzyme action requires divalent cations, which may be furnished by either certain metals or by polyamines . The activity is separable into two components both of which are required for activity . It appears that the same nuclease excises the 5'- and 3'-terminal segments since preparations lose the capacity to modify the two termini with an identical first order thermal decay rate . Certain features of the rRNA precursor which may be involved in cognitive interaction with RNase M5 are discussed. Biochim Biophys Acta, 1977 Feb 16, 474(4), 562 - 77 Ribosomal RNA genes in Bacillus subtilis . Evidence for a cotranscription mechanism; Zingales B et al.; The analysis of the transcriptional mechanism of the ribosomal RNA genes in Bacillus subtilis was undertaken by a study of the rRNA chain elongation in the presence of rifampicin . The residual RNA synthesis after the addition of rifampicin and {3H} uridine to exponentially growing cells has shown that 56% of the radioactivity incorporated into total RNA belongs to the unstable fraction and 44% to the fraction containing mature rRNA and tRNA . Such study allowed an estimation of the half-life of messenger RNAs as being approximately 2 min . The analysis of the transcription pattern of the ribosomal RNA genes, as measured by the amount of radioactivity found in the ribosomal subunits, was complicated by a contamination of the 30 S subunits by 50 S subunits . A contamination of approximately 15% was estimated by polyacrylamide gel electrophoresis and competitive hybridization . The ratios of incorporated radioactivity at zero time when drug and label were concomitantly added ranged between 5.4-6.0, after correction for this contamination . The decay of the 23 S rRNA followed a straight line which became parabolic in its final portion . These results, and theoretical considerations on the lag of rifampicin action and on the variance of the specific activity of the nucleotide pool at the very early times of the experimental observation, favor the interpretation that the 16 and 23 S rRNA genes in B . subtilis belong to the same transcriptional unit, being cotranscribed, in that order, by the same molecule of RNA polymerase . The transcriptional times of the 16 and 23 S rRNA genes were estimated as being 30 and 60 s, respectively. Mol Gen Genet, 1977 Feb 15, 150(3), 309 - 16 Characterization of a combined DNA initiation and cell division mutant of Bacillus subtilis; Travis SL et al.; The temperature-sensitive mutation in Bacillus subtilis 168-134ts, a conditional lethal DNA initiation mutant, was transferred to the minicell producing strain, CU 403 div IV-B1, to study he relationship of DNA synthesis to cell division . Markers in the combined mutant were verified by transduction . DNA replication kinetics, genome location by autoradiography, and clonal analysis of cell division patterns during spore outgrowths were investigated . Growth of the double mutant at the restrictive temperature results in an impressive reduction of the percentage cell length covered by DNA grain clusters (60.2% at 30 degrees C compared to 8.6% after 2 h at 45 degress C) . The probability of a minicell producing division in double mutant clones is essentially the same at 30 degrees C and during the initial 2-3 h growth at 45 degrees C at which time lysis begins . Residual division at 45 degrees C is attributable to processes initiated at 30 degrees C . The CU 403 div IV-B1, 134ts, double mutant divides about 25% as frequently relative to growth as do wild type CU 403 clones when incubated at permissive temperature . This is approximately 15% greater division suppression than previously found in the CU 403 div IV-B1 mutant strain, and is presumably due to interactions of the mutant gene products both of which affect DNA. Eur J Biochem, 1977 Feb 15, 73(1), 57 - 72 Assembly of Bacillus subtilis phage phe29 . 2 . Mutants in the cistrons coding for the non-structural proteins; Jimenez F et al.; The effect on phage morphogenesis of sus mutations in the cistrons coding for nonstructural proteins has been studied . Mutants in three cistrons analyzed that are involved in phage DNA synthesis, as well as in cistron 16 which codes for a late nonstructural protein, produce prolate capsids which are more rounded at the corners than complete phage heads and have an internal core; they contain the head proteins, the upper collar protein and protein p7, not present in mature phage particles . Mutants in cistron 7 do not produce capsids nor other phage-related structures; this result and the presence of p7 in phage capsids suggest an essential role in capsid assembly for this protein . The protein product of cistron 13 is probably needed for a stable DNA encapsulation since mutants in this cistron produce mainly DNA-free complete phage particles and only about 10% of uninfective DNA-containing complete phage . Cistron 15 codes for a late, partially dispensable, nonstructural protein which is present in the DNA-free capsids produced after infection with the delayed-lysis mutant sus14(1242), used as the wild-type control, or with mutants in cistrons 9, 11,12 and 13 . Proteins p15 and p16 are probably involved in the encapsulation of viral DNA in a prohead. Eur J Biochem, 1977 Feb 15, 73(1), 39 - 55 Assembly of Bacillus subtilis phage phi29 . 1 . Mutants in the cistrons coding for the structural proteins; Camacho A et al.; The effect of mutations in the cistrons coding for the phage structural proteins has been studied by analyzing the phage-related structures accumulated after restrictive infection . Infection with susmutants in cistron 8, lacking both the major head and the fiber protein, does not produce any phage-related structure, suggesting a single route for the assembly of phage phi29; infection with ts mutants in this cistron produces isometric particles . Mutants is cistron 9, coding for the tail protein, TP1, produce DNA-free prolate heads with an internal core; these particles are abortive and contain the head proteins HPO, HP1 and HP3, the upper collar protein NP2 and the nonstructural proteins p7, p15 and p16 . Mutants in cistron 10, coding for the upper collar protein, NP2, produce DNA-free isometric heads also with an internal core; they contain the head proteins and the nonstructural protein p7, suggesting that this protein forms the internal core . Mutants in cistrons 11 and 12, coding for the lower collar protein, NP3, and the neck appendages, NP1, respectively, give rise to the formation of DNA-containing normal capsids and DNA-free prolate particles, more rounded at the corners than the normal capsids and with an internal core; the DNA-containing 11-particles are formed by the head proteins and the upper collar protein; the DNA-free 11-particles contain, besides these proteins, the nonstructural protein p7 and a small amount of proteins p15 and 16 . The DNA-containing 12-particles have all the normal phage structural proteins except the neck appendages, formed by protein NP1; the DNA-free particles are similar to the DNA-free 11-particles . After restricitive infection mutant sus14(1241) has a delayed lysis phenotype and produces a phage burst higher than normal, after artificial lysis . It produces DNA-containing particles, identical to wild-type phage, which have all the normal phage structural proteins, and DNA-free prolate particles, more rounded at the corners than the final phage particles and with an internal core; the last particles contain the same proteins as the DNA-free 11 or 12-particles . These particles could represent a prohead state, ready for DNA encapsulation . None of the DNA-containing particles have the nonstructural proteins p7, p15 or p16, suggesting that these proteins are released from the proheads upon DNA encapsulation. Biochem J, 1977 Feb 15, 162(2), 359 - 65 Binding of magnesium ions to cell walls of Bacillus subtilis W23 containing teichoic acid or teichuronic acid; Heckels JE et al.; When grown in a chemostat under various nutritional conditions, cells of Bacillus subtilis W23 produce walls containing teichoic acid or teichuronic acid . The binding of Mg2+ to these walls and to the isolated anionic polymers in solution was measured by equilibrium dialysis . In solution the ribitol teichoic acid bound Mg2+ in the molar ratio Mg2+/P=1:1 with an apparent association constant (Kassoc.) of 0.61 X 10(3)M-1, and the teichuronic acid bound Mg2+ in the ratio Mg2+/CO2-=1.1, Kassoc.=0.3 X 10(3)M-1 . Cell walls containing teichuronic acid exhibited closely similar binding properties to those containing teichoic acid; in both cases Mg2+ was bound in the ratio Mg/P or Mg/CO2- of 0.5:1 and with a greater affinity than displayed by the isolated polymers in solution . It was concluded that Mg2+ ions are bound bivalently between anionic centres in the walls and that the incorporation of teichoic acid or teichuronic acid into the walls gives rise to similar ion-binding and charged properties . The results are discussed in relation to the possible functions of anionic polymers in cell walls. Biochemistry, 1977 Feb 8, 16(3), 403 - 10 Isolation, characterization, and activation of the magnesium dependent endodeoxyribonuclease from Bacillus subtilis; Burke WF Jr et al.; A major endodeoxyribonulcease was isolated from a mutant of the transformable Bacillus subtilis 168 . The magnesium-dependent endonuclease was purified approximately 750-fold to electrophoretic homogeneity . The enzyme had a molecular weight of about 31 000, as determined by gel filtration and polyacrylamide gel electrophoresis . The protein appears to be composed of two subunits . The nuclease was dependent on magnesium or maganese ions for hydrolytic activity . The purified nuclease degraded DNA from several species of Bacillus, as well as Escherichia coli DNA, alkylated, depurinated, and thymine-dimer containing B . subtilis DNA, and hydroxymethyluracil-containing phage DNA . The enzyme also hydrolyzed single-stranded DNA, although native DNA was the preferred substrate . However, the nuclease was unable to degrade ribosomal RNA . The cleavage products