Bacillus subtilis

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 of the DNA hydrolysis have 5'-phosphate and 3'-hydroxyl ends . The enzyme could be activated in crude extracts by heat treatment or treatment with guanidine hydrochloride . The nuclease activity was inhibited by phosphate and by high concentrations of NaCl . A possible function for this endonuclease in bacterial transformation is discussed.

Zh Mikrobiol Epidemiol Immunobiol, 1977 Feb, (2), 39 - 42
{Use of transformation for studying the nature of formation of stable bacterial L forms}; Tartakovskii IS et al.; The authors obtained a stable variant of the L-forms of Bacillus subtilis capable of exponential growth of the minimal and synthetic medium . An electron-microscopic study of different stages of the L-form formation was carried out by the method of ultra-thin sections . A possibility was shown of the transfer of the L-form formation sign by the method of transformation . DNA isolation from the L-forms by soft lysis considerably facilitated and simplified the genetic analysis of the L-form formation by the transformation method.

Mutat Res, 1977 Feb, 42(2), 175 - 80
Mutation induction by the direct treatment of Bacillus subtilis deoxyribonucleic acid; Felkner IC; A procedure was developed to select for specific mutations obtained by means of transformation with DNA previously exposed to potentially dangerous chemical compounds . The 70% co-transformation of hisB and trpC genes in Bacillus subtilis provided a convenient opportunity to select for new mutations . When purified DNA from wild type bacteria was treated with N(OH) acetyl aminofluorene or Hoechst dye 37 507 and used to transform a recipient bearing of a trpC2 mutaion, a high proportion of the Trp+ transformants had new hisB mutations.

J Bacteriol, 1977 Feb, 129(2), 973 - 7
Isolation of acetyl esterase mutants of Bacillus subtilis 168; Higerd TB; Five mutants of Bacillus subtilis 168 defective in an intracellular esterase activity were identified . By polyacrylamide gel electrophoresis, four of the mutants were shown to lack esterase B activity, and the fifth lacked esterase A activity . All of the back-crossed esterase mutants were able to sporulate at wild-type frequency and produce exoprotease(s) and antibiotic(s) . No difference in motility could be attributed to the esterase mutation . PBS1 transduction analysis showed all the esterase B mutations to be linked to the hisA marker.

J Bacteriol, 1977 Feb, 129(2), 901 - 7
Media dependence of commitment in Bacillus subtilis; Cooney PH et al.; At some time during sporulation development, cells of Bacillus subtilis develop a commitment to continue sporulation even after addition of or dilution into a fresh nutrient . The extent of commitment was measured by the titer of spores produced at the time at which the original culture sporulated maximally . Since newly formed spores of B . subtilis soon germinate in the replenished medium, the measurement of their titer, especially of heat-resistant spores, gave low values . This problem was avoided by the germination-delaying effect of methyl anthranilate (1 mM) when added together with the fresh nutrients . In a given culture, the titer of committed cells was then independent of the method by which it was measured, i.e., by the phase-bright, octanol-resistant, or heat-resistant spore titer . The time of commitment depended on the type of nutrient added . Commitment occurred earlor casein hydrolysate . The rates at which non-metabolizable amino acid analogues or the 14C from an amino acid mixture were taken up by the cells increased toward the end of growth and later declined . This decline occurred slowly and was only weakly correlated with the commitment time of an analogous amino acid.

J Bacteriol, 1977 Feb, 129(2), 789 - 95
Some properties of a membrane-deoxyribonucleic acid complex isolated from Bacillus subtilis; Harmon JM et al.; Membrane-deoxyribonucleic acid (DNA) complexes were isolated from Bacillus subtilis by affinity for magnesium-Sarkosyl crystals . These complexes (M-bands) contained greater than 80% of the total cellular DNA; little of the remaining portion could be recovered in a secondary isolation . Isotopic labeling of the origin of replication showed this region of the chromosome to be closely associated with the cell membrane . Interruption of protein or DNA synthesis did not result in detachment of the chromosome from the membrane . Interruption of ribonucleic acid synthesis by rifampin resulted in a decreased ability to isolate DNA in the M-band . Analysis of attachment of the chromosome to membrane during the cell and replication cycles indicated that the chromosome is not released from the membrane at any time during the cell cycle.

J Bacteriol, 1977 Feb, 129(2), 678 - 89
Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state; DeCastro-Costa MR et al.; When the cell wall of Bacillus subtilis is removed by lysozyme and the resultant protoplasts are plated on hypertonic soft agar medium, each protoplast forms an L colony . L bodies from such L colonies again plate as L-colony-forming units (CFU) . However, if protoplasts or L bodies are "conditioned" by 1 h of incubation in 0.4% casein hydrolysate medium and then incubated in 25% gelatin medium for 1 h, 60 to 100% of the formerly naked cells give rist to bacillary colonies . The present experiments largely explain the mechanism responsible for the "heritable" persistence of the wall-less state in B . subtilis . It is shown that protoplasts produce a reversion inhibitory factor (RIF) which blocks reversion when the cell concentration exceeds 5 x 105 CFU/ml . This inhibitor is nondialyzable and sensitive to trypsin, heat, and detergent . Efficient reversion at 2 x 107 CFU/ml is obtained if the protoplasts are treated with trypsin after conditioning and chloramphenicol is incorporated into the gelatin reversion medium . In the presence of 500 mug of trypsin per ml, the requirement for gelatin is sharply reduced, and reversion occurs rapidly in liquid medium containing only 10% gelatin . Trypsin also stimulates reversion in L colonies growing on soft agar . Latent RIF is activated by beta-mercaptoethanol . This reagent blocks reversion of protoplast suspensions at densities of 5 x 105 CFU/ml . Comparison of the autolytic behavior of B . subtilis and of the RIF revealed that several or the properties of the two activities coincide: both are inhibited by high concentrations of gelatin, both are activated by beta-mercaptoethanol, and both have high affinity for cell wall . Going on the assumption that RIF is autolysin, models for protoplast reversion is suggested by the finding that mutants with altered teichoic acid show altered reversion behavior.

J Bacteriol, 1977 Feb, 129(2), 574 - 9
Metabolism of pyrimidine bases and nucleosides in Bacillus subtilis; Rima BK et al.; In Bacillus subtilis, uracil (Ura), uridine (Urd), and deoxyuridine (dUrd) are metabolized through pathways similar to those of enteric bacteria . Ura is probably converted to uridine 5'-monophosphate by uridine 5'-monophosphate pyrophosphorylase . More than 95% of dUrd added to cultures is converted to Ura and deoxyribose-1-phosphate . Although dUrd kinase activity is detectable in vitro, this enzyme does not seem to play an important role in the metabolism of dUrd . The metabolism of cytosine (Cyt), cytidine (Cyd), and deoxycytidine (dCyd) in B . subtilis appears to be different from that in enteric bacteria . Cytosine cannot be used by Ura-requiring mutants as pyrimidine source . dCyd is deaminated by dCyd-Cyd deaminase or phosphorylated to dCyd nucleotides by dCyd kinase . Cyd is deaminated by dCyd-Cyd deaminase of phosphorylated by Cyd kinase . This Cyd kinase activity has never been reported for B . subtilis.

J Virol, 1977 Feb, 21(2), 522 - 9
Temperate Bacillus subtilis bacteriophage phi 3T: chromosomal attachment site and comparison with temperate bacteriophages phi 105 and SPO2; Williams MT et al.; The temperate Bacillus subtilis bacteriophage phi 3T contains within its genome a locus, designated thyP3, that encodes for a protein with thymidylate synthetase activity . Bacteriophage phi 3T is different from the two previously characterized temperate phages, phi 105 and SPO2, in: heteroimmunity, response to bacteriophage antisera, endonuclease digestion pattern, induction in the presence of 6-(p-hydroxyphenylazo)-uracil, and effect on the lytic cycle of bacteriophage phi 1 . The mean burst size of phi 3T is 56 . The dose response curve with bacteriophage phi 3T DNA is linear for transfection and transformation to the Thy+ phenotype . The inserted prophage has been mapped by PBS1 transduction; it is between chromosomal markers ilvA8 and gltA in the terminus of the chromosome . Thus thyP3 maps at a site separate from, but between, the bacterial markers thyA and thyB when thyP3 is in the prophage state.

J Virol, 1977 Feb, 21(2), 493 - 6
Bacillus subtilis DNA polymerase III is required for the replication of DNA of bacteriophages SPP-1 and phi 105; Rowley SD et al.; The replication of the Bacillus subtilis bacteriophages SPP-1 and phi 105 is sensitive to 6-(p-hydroxyphenylazo)-uracil (HPUra), a selective inhibitor of replicative DNA synthesis of B . subtilis which acts specifically at the levels of a replication-specific polymerase, DNA polymerase III (pol III) . The origin of the HPUra-sensitive polymerase required for phage replication was examined by comparison of the drug sensitivity of phage development in a normosensitive host with that in a host carrying azp-12, a polC mutation that specifies production of an HPUra-resistant pol III . azp-12 specified HPUra-resistant phage host pol III . The host polIII requirement for SPP-1 replication also was confirmed by the demonstration that phage development was temperature sensitive in a host mutant carrying the polC mutation mut-1 (ts) . Examination of the pol III activity of crude and purified cell-free preparations derived from phage-infected cells did not indicate any detectable changes in the specific activity, purification behavior, or drug sensitivity of the enzyme.

J Biochem (Tokyo), 1977 Feb, 81(2), 467 - 76
Glutamate dehydrogenase from Bacillus subtilis PCI 219 . I . Purification and properties; Kimura K et al.; Bacillus subtilis PCI 219 has a single glutamate dehydrogenase (GDH) {EC 1.4.1.3} with dual coenzyme specificity {for NAD(H) and NADP(H)} . The enzyme was purified 800-fold from crude extracts of B . subtilis from the post-exponential phase of growth and showed one significant protein band on gel electrophoresis . This band was determined, by activity staining, to have all the GDH nucleotide specificities . Its molecular weight was estimated to be 250,000+/-20,000 by gel filtration, and 270,000+/-30,000 by zone centrifugation in a sucrose density gradient . Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that GDH has a subunit size of about 57,000 . The pI of GDH was found to bepH 3.7 by isoelectric focusing . GDH exhibited nonlinear kinetics in the reduction of NAD+, and in the reverse direction, the substrate, NH4+, was strongly inhibitory at high concentrations . Purine nucleotides did not affect the activity . The oxidative demination of glutamate was significantly inhibited by the metabolites oxaloacetate and citrate, which acted as allosteric effectors of this enzyme,inhibiting the reaction in one direction . The pH optimum of each of the activities of GDH and the stability of GDH are also reported.

Mol Gen Genet, 1977 Jan 18, 150(2), 147 - 59
Erythromycin resistant mutations in Bacillus subtilis cause temperature sensitive sporulation; Tipper DJ et al.; All of several hundred erythromycin resistant single site mutants of Bacillus subtilis W168 are temperature senstive for sporulation . The mutants and wild type cells grow vegetatively at essentially the same rates at both permissive (30 degrees C) and nonpermissive (47 degrees C) temperatures . In addition cellular protein synthesis, cell mass increases and cell viabilities are similar in mutant and wild type strains for several hours after the end of vegetative growth (47 degrees C) . in the mutants examined, the temperature sensitive periods begin when the sporulation process is approximately 40% completed, and end when the process is 90% completed . At nonpermissive temperatures, the mutants produce serine and metal proteases at 50% of the wild type rate, accumulate serine esterase at 16% of the wild type rate, and do not demonstrate a sporulation related increase in alkaline phosphatase activity . The eryR and spots phenotypes cotransform 100%, and cotransduce 100% using phage PBS1 . Revertants selected for ability to sporulate normally at 47 degrees C (spot), simultaneously regain parental sensitivity to erthromycin . No second site revertants are found . Ribosomes from eryR spots strains bind erythromycin at less than 1% of the wild type rate . A single 50S protein (L17) from mutant ribosomes shows an altered electrophoretic mobility . Ribosomes from spo+ revertants bind erythromycin like parental ribosomes and their proteins are electrophoretically identical to wild type . These data indicate that the L17 protein of the 50S ribosomal subunit from Bacillus subtilis may participate specifically in the sporulation process.

J Biol Chem, 1977 Jan 10, 252(1), 268 - 72
New types of RNA polymerase mutations causing temperature-sensitive sporulation in bacillus subtilis; Leighton T; A single site mutant of Bacillus subtilis with a streptovaricin-resistant RNA polymerase has been isolated; this mutation caused temperature-sensitive sporulation, but had no effect on vegetative growth . The mutant (ts710) temperature-sensitive period irreversibly affected the middle and late stages of sporulation . Mutant cells grown at the nonpermissive temperature exhibited abnormal serine protease accumulation, serine esterase accumulation, alkaline phosphatase accumulation, RNA polymerase template specificity changes, and pulse-labeled RNA synthesis profiles . The accumulation of metal protease was not affected at the nonpermissive temperature . Attempts to isolate single site mutants which were streptolydigin-resistant, and temperature-sensitive for sporulation, were unsuccessful.

Antonie Van Leeuwenhoek, 1977, 43(3-4), 305 - 16
A counting method for determining the burst size of defective phages from Bacillus subtilis; Steensma HY et al.; The defective phages of Bacillus subtilis cannot be counted by plating as they do not form plaques . In addition, counting under the electron microscope with latex spheres as an internal standard is not possible . The reliability of a method using Escherichia coli phage T4 as a substitute for the latex spheres has been tested and the results compared with those of other methods . Using this method, we determined the burst sizes of the defective phages PBS X, PBS Y and PBS Z under various conditions.

Folia Microbiol (Praha), 1977, 22(5), 346 - 52
Transformation in Bacillus subtilis . Initial stages of the transformation process and their energy dependence; Tichy P et al.; NaN3 was found to inhibit transformation but not the irreversible binding of donor 3H-DNA in competent cells of the original low-transformable strain Bacillus subtilis 168 trp2 . Addition of NaN3 to cells of two mutants Bacillus subtilis HT39 and HT46 with an increased transformability decreased substantially the irreversible binding of the donor DNA to the competent cells . The decreased irreversible binding of DNA is caused by an increased osmotic sensitivity of competent cells of the mutants HT39 and HT46 in the presence of NaN3, leading preferentially to lysis of the competent cells.

Folia Microbiol (Praha), 1977, 22(5), 329 - 38
Interaction of granaticin B with the transcription system of Bacillus subtilis; Weiser J et al.; The interaction of granaticin B, a quinone antibiotic produced by Streptomyces granaticolor, with some biologically important bivalent metal ions, DNA and ATP was demonstrated spectrophotometrically . The activity of isolated RNA polymerase was higher when the DNA of phage SP 50 served as template than with DNA isolated from Bacillus subtilis . Granaticin B inhibited in vitro RNA synthesis, similarly to certain other antibiotics (the inhibition was three times lower than that caused by actinomycin D or streptolydigin and slightly higher than that by epsilon-pyrromycinone) . The inhibitory effect was higher when the Mg2+ concentration in the reaction mixture was decreased . The inhibition was then proportional to the concentration of the DNA template . DNA-dependent RNA synthesis is thus inhibited in vitro by granaticin B but this does not appear to be the only site of action of this antibiotic in vivo.

Genetika, 1977, 13(8), 1489 - 91
{UV-mutagenesis in Bacillus subtilis . VII . Induction of auxotrophic mutants}; Lotareva OV et al.; An experimental testing of material from thin-layered, transparent in passing light, colonies which appear with some frequency after plating Bacillus subtilis cells on agar medium with limited enrichment, has shown that such colonies are formed by auxotrophic mutants . The growth requirements for many of them has been identified . The most of mutants can be reversed to original phenotype by UV-irradiation . The frequency of auxotrophs increases after UV-irradiation of suspension of original cells . The sensitivity of auxotrophic mutants to inactivating action of UV-light is near to that of original cells, hence the increase of the frequency of mutants with dose is a result of induction, but not of selection of preexisting spontaneous auxotrophic mutants . The frequency of induced auxotrophs, in contrast to that of suppressor revertants, badly give way to declining in the time of temporary inhibition of postradiation growth . In the case of Bac, subtilis, the system of induced auxotrophic mutants on the medium with limited enrichment is rather comfortable in use and can be recommended for studying UV-induced mutagenesis in structural genes as well as for testing mutagenic activities.

Genetika, 1977, 13(7), 1268 - 80
{W-reactivation and W-mutagenesis in UV-irradiated phage phil05 of Bacillus subtilis}; Kalinin VL et al.; The survival of UV-irradiated phage o105 on the lawns of several strains of Bacillus subtilis: wild type (strain 168) and 11 recombination-defficient mutants (recA1, recB2, recB3, recB19, recD27, recF15, recF18, recK4, recM13, recL16 and recO61) was investigated . All rec mutants have the phenotype Hcr+, i.e . normally host-cell reactivate UV-damaged phage . Small doses of UV-irradiation given to the wild type (rec+) cells increase the probability of survival of UV-irradiated o105 phage (W-reactivation) and significantly enhance the frequency of c-mutants (W-mutagenesis) . Maximal frequency of clear mutations in conditions of W-mutagenesis is 3-10(-3), i.e . is 100 times higher than the spontaneous background . Various rec mutations of host cells only diminish the level of W-reactivation but do not eliminate it completely . The most deficient in W-reactivation is recD27 mutant . Mutations recB2, B3, B19 and O61 have no effect on W-mutagenesis of UV-irradiated phage o105 and on UV-induction of o105, F15,F18 and L16 mutants . UV-irradiation of lysogenic cells of these mutants does not induce o105 prophage.

Zentralbl Bakteriol Parasitenkd Infektionskr Hyg, 1977, 132(4), 289 - 93
Antibiotic production by defective cytoplasmic membrane mutants of Bacillus subtilis; Perez-Silva G et al.; Eleven defective cytoplasmic membrane mutants of Bacillus subtilis were isolated . Their respective protoplasts presented different lytic behaviour when exposed to decreased concentrations of sucrose . Some relationship was found between the fragility of the protoplasts and the capacity of the mutants to produce antibiotic or to sporulate . The higher the fragility the lesser the capacity to produce antibiotic . The same applies to their amino acids requirements.

Genetika, 1977, 13(4), 735 - 6
{Effect of calcium and magnesium ions on different stages of genetic transformation in Bacillus subtilis}; Belov IS et al.; Optimal concentrations of magnesium and calcium ions under their combined effect on genetic transformation in Bacillus suttilis are 2 - 10(-2) M and 4 - 10(-2) M respectively . The same concentrations are optimal under the effect of each cation clone . Magnesium ions are efficient during irreversible DNA binding . In the presence of magnesium ions calcium ions stimulate more late stages of transformation . The greatest efficiency of transformation is shown in consecutive effect of magnesium ions at early stages of transformation and of calcium ions at late transformation stages . This suggests that magnesium and calcium ions stimulate the activity of nuclease, taking part at early and late transformation stages.

Genetika, 1977, 13(3), 490 - 5
{Analgos of riboflavin, lumiflavin and alloxazine derivatives . II . Effect of roseoflavin on 6,7-dimethyl-8-ribityllumazine and riboflavin synthetase synthesis and growth of Bacillus subtilis}; Stepanov AI et al.; The replacement of 8-CH3 group in the riboflavin molecule results in the formation of specific antimetabolites . They are rozeoflavin, 7-desmethylrozeoflavin, 8-amino (nor) riboflavin, 8-ribitylamino (nor) riboflavin . Effect of rozeoflavin and other riboflavin analogues on the growth and regulatory characteristics of Bacillus subtilis strains with different genetic state of riboflavin operon is studied . Roseoflavin at a concentration of 0.05 mkg/ml inhibits DRL synthesis in rib-b110 strain . An analogue inhibits the growth of auxotrophic and prototrophic strains at concentrations of 0.5 mkg/ml and 50 mkg/ml respectively . Riboflavin (1 mkg/ml) recovers the growth of bacteria . The curve of rozeoflavin regulation of DRL and riboflavin synthetase synthesis is shifted in 100 times in the direction of lesser concentrations as compared with riboflavin and 8 amino (nor) riboflavin . 180 mutants resistant to 100 mkg/ml of rozeoflavin were selected . 150 mutants over-synthetize riboflavin.

Zentralbl Bakteriol Parasitenkd Infektionskr Hyg, 1977, 132(2), 109 - 16
Relation of lipids to the action of streptomycin on Bacillus subtilis and Escherichia coli and griseofulvin and fungizone on Aspergillus niger; Elwan SH et al.; An attempt was made to reveal the relation between the lipid content and components of B . subtilis and E . coli and the action of streptomycin, and those of A . niger and the action of griseofulvin and fungizone (Amphotericin B) . Total lipids were extracted in CO2 atmosphere, dried, and weighed . Lipid components were analyzed by thin-layer chromatography . Complements to the growth medium of A . niger with extracted total lipids and phospholipids were made to verify the obtained results . It has been found that the action of streptomycin and griseofulvin was not correlated with lipids . On the other hand, fungizone action was manifested by the decrease in total lipids, free sterols, sterol esters, and triglycerides . Supplementation studies gave evidence of the role of total lipids and phospholipids in protecting A . niger cells against fungizone . Supplementation of 1 g of phospholipid per litre medium raised the M.I.D . of fungizone from 2.5 up to 25 microgram/ml . It was suggested to keep the fat level controlled at a minimum if it is required to increase fungizone activity.

Clin Allergy, 1977 Jan, 7(1), 55 - 68
Diagnostic tests in the skin and serum of works sensitized to Bacillus subtilis enzymes; Belin LG et al.; Two allergen pools of commercial detergent enzymes were prepared as skin test reagents: (1) Carlsberg type, composed of three products containing subtilopeptidase A, and (2) BPN type, composed of two products containing subtilopeptidase B and alpha-amylase . In 100 non-exposed controls a reaction suggesting primary irritancy was found at protein concentrations greater than 1 microng/ml intradermally or 1 mg/ml by prick test . Intradermally at 10 microng/ml weals were accompained by less pronounced flare reactions than observed in specifically sensitized enzyme workers . At 100 micronh/ml the reactions were like strong specific reactions . Galse positive prick test reactions occurred irregularly at 10 mg/ml . In 100 sensitized enzyme workers, reactions were elicited at concentrations from 1-0 to 10(-5) microng/ml intradermally and from 1000 to 1 micron by prick test . Intradermal and prick tests correlated well (r=0-84, P less than 0-001) . Ratings of symptom severity upon exposure obtained from questionnaires were significantly correlated with skin test reactivity (P less than 0-01) . RAST performed on sera collected simultaneously also correlated significantly with symptom scores . PCA tests in monkeys were less sensitive . Standardized test reagents allow diagnostic skin testing by either intradermal or prick test in B . subtilis enzyme sensitive patients . A clear distinction between primary irritant reactions and true sensitization was made on the basis of the concentration required to elicit a reaction.

Z Allg Mikrobiol, 1977, 17(2), 153 - 61
{Relationship between aminoacyl-tRNA synthetases (AAA) and cell division in temperature-sensitive filamentous mutants of Bacillus subtilis SB 19 . III . Characterization of pre-incubation effect and effect of AAA-inhibitor produced by Agrostemma githago seedlings}; Suss J et al.; Further results on the correlations between the regulation of bacterial cell division and amino-acyl-tRNA synthetase are presented . Activity of aminoacyl-tRNA synthetases, extracted from a filamentous mutant of Bacillus subtilis SB 19, may be stimulated by preincubation of crude extracts . The mechanism of this stimulating effect has been studied by means of an inhibitor of amino-acyl-tRNA synthetases produced during the growth of Agrostemma githago-seedlings . According to preliminary results we suggest, this inhibitor can reduce the activity of subunits only, but not that of higher associates . Association of subunits to oligomers will be prevented by the inhibitor, too . Our results may be indicative of the assumption that the increase of enzyme activity during subunits with a low catalytic activity to functional oligomers . As to the verification of these hypotheses further work will still have to be done.

Biokhimiia, 1977 Jan, 42(1), 51 - 9
{Multiple forms of Bacillus subtilis subtilisin and effects of mutations on the distribution of its molecular forms}; Abramov ZT et al.; Using polyacrylamide-gel electrophoresis, isoelectric focusing and gel-filtration it was demonstrated that the auxotrophic mutant strains of Bac . subtilis A-50 and their prototrophic revertant strains produce multiple molecular forms of subtilisin . Three of them are the same as the corresponding molecular forms of subtilisin from the wild strain A-50 . In different mutant strains the relative amounts of the main three forms varies considerably resulting in the absence of certain forms in several strains . There is the additional minor form of subtilisin possessing high electrophoretic mobility in four prototrophic revertant strains and one Arg--auxotrophic strain of Bac . subtilis A-50 . It would be reasonable to suppose that different molecular forms of subtilisin derive from the product of its single structural gene as a result of post-translational modifications (limited proteolysis) . This enzyme and probably most, if not all secretory proteins may be synthesised as larger precursors and then specifically modified in the bacterial cell membranes . Thus, certain mutations, without affecting the structural gene of this secretory protein -- subtilisin -- have pronounced effects on this structural gene expression, varying the degree of its product modification and the amount of resulting secretory molecular forms of subtilisin.

Appl Environ Microbiol, 1977 Jan, 33(1), 52 - 8
Thermal resistance of Bacillus subtilis var . niger in a closed system; Peeler JT et al.; The heat resistance of Bacillus subtilis var . niger has been measured from 85 to 125 degrees C using moisture levels of percent relative humidity (%RH) less than or equal to 0.001 to 100 in a closed system . Five curves have been presented to characterize the thermal destruction, using thermal death times defined as F values at a given combination of three moisture and temperature conditions . Reductions of 99.99% (4-log10 cycles) of the initial population were estimated for the three moisture conditions . At 110 degrees C, the expected time for a 4-log10 reduction was 1.1 h at %RH = 100, 3.1 h at %RH less than or equal to 0.1 and 54 h at %RH = 10.7 . Goodness-of-fit tests to examine the adequacy of three polynomial models failed to indicate a trend . The linear model (from which estimates of D are obtained) was satisfactory for estimating the thermal death times (%RH less than or equal to 0.1) in the plate count range . The estimates based on observed thermal death times and D values for the %RH = 100 diverged so that D values generally gave a more conservative estimate over the temperature range 90 to 125 degrees C . Estimates of ZF and ZL ranged from 32.1 to 58.3 degrees C for the %RH less than or equal to 0.1 and 100 . A ZD value of 30.0 was obtained for data observed at %RH less than or equal to 0.1 . The ZF results were obtained from plotting observed log times to achieve a 99.99% reduction in the initial population versus temperature . Estimates of ZL and ZD were obtained by using linear estimates of L100 approximately equal to 4D and D values in a similar plot.

J Virol, 1977 Jan, 21(1), 54 - 60
Selective screening procedure for the isolation of heat- and cold-sensitive, DNA replication-deficient mutants of bacteriophage SPO1 and preliminary characterization of the mutants isolated; Glassberg J et al.; A procedure is described for the selective isolation of temperature-sensitive replication-deficient mutants of Bacillus subtilis phage SPO1 . A modification of the procedure permits the isolation of temperature-sensitive mutants in specific cistrons of interest . The applicability of these procedures to other viral systems is discussed . The mutations isolated were assigned to eight replication-deficient cistrons, with the cold-sensitive mutations showing a distribution strikingly different from that of the heat-sensitive mutations . As a preliminary to the identification of initiation-deficient mutants, the mutants were divided into three classes on the basis of their ability to synthesize DNA after a shift to nonpermissive temperature . We also report two incidental results: (i) the SPO1 dUMP hydroxymethylase, like the T4 dCMP hydroxymethylase, may be part of a multifunctional complex; and (ii) mutants were isolated that were replication positive but lysis deficient and failed to complement one of the replication-deficient mutants.

J Bacteriol, 1977 Jan, 129(1), 556 - 8
Bacillus subtilis bacteriophage SPbeta: localization of the prophage attachment site, and specialized transduction; Zahler SA et al.; The attachment site for the prophage of SPbeta lies between ilvA and kauA on the chromosome of Bacillus subtilis strain 168 . Specialized transduction of citK and kauA can be carried out by certain lysates of SPbeta.

J Bacteriol, 1977 Jan, 129(1), 550 - 3
Incorporation of 3,4-dihydroxybutyl-1-phosphonate, a glycerol 3-phosphate analogue, into the cell wall of Bacillus subtilis; Klein DA et al.; 3,4-Dihydroxybutyl-1-phosphonate, a bacteriostatic agent toward Bacillus subtilus 168 and a bactericidal agent toward strain W 23, is incorporated into cell walls and phospholipids of each strain.

J Bacteriol, 1977 Jan, 129(1), 492 - 500
Two-dimensional restriction analysis of the Bacillus subtilis genome: gene purification and ribosomal ribonucleic acid gene organization; Potter SS et al.; With two-dimensional restriction enzyme analysis we have been able to cleave the Bacillus subtilis genome and resolve the resulting deoxyribonucleic acid (DNA) segments into discrete bands on agarose gels . A general procedure for gene purification has been developed by coupling multidimensional restriction analysis with a biological assay for gene detection . The organization of ribosomal ribonucleic acid (rRNA) genes was studied by hybridizing 16S and 23S rRNA probes to the two-dimensional DNA banding patterns.

J Bacteriol, 1977 Jan, 129(1), 433 - 44
Bacillus subtilis ribonucleic acid polymerase mutants conditionally temperature sensitive at various stages of sporulation; Sumida-Yasumoto C et al.; Rifampin-resistant mutants of Bacillus subtilis that are conditionally temperature sensitive during sporulation have been isolated and characterized . The mutants can grow at the same rate as the wild type at the nonpermissive temperature but cannot sporulate . Depending on the mutation, they are blocked at either stage 0 to I, II, II to III, or IV of sporulation . The mutants showed an altered pattern of RNA synthesis after the stage at which they were blocked . The effect of rifampin on the activity of enzymes from mutant vegetative cells and sporulating cells was significantly different, suggesting that the RNA polymerase from sporulating cells was different from the RNA polymerase of vegetative cells . These results suggest that the conformation of the RNA polymerase core plays an important role in determining correct transcription during sporulation.

J Bacteriol, 1977 Jan, 129(1), 422 - 32
Two polypeptides associated with the ribonucleic acid polymerase core of Bacillus subtilis during sporulation; Fukuda R et al.; The ribonucleic acid (RNA) polymerase from log-phase and sporulating cells of Bacillus subtilis was analyzed to determine whether any structural changes occurred during sporulation . The elution pattern of RNA polymerase from a deoxyribonucleic acid (DNA)-cellulose column revealed that sporulating cells at stages III and IV contained a new RNA polymerase fraction in addition to the vegetative holoenzyme (alpha2betabeta'sigma) . Stage III cells contained the vegetative holoenzyme and a new enzyme with the composition alpha2betabeta'delta1; the molecular weight of delta1 was 28,000 . Stage IV cells contained the vegetative holoenzyme, the delta1-containing enzyme, and another enzyme with the composition alpha2betabeta'delta2 . The delta2 factor had a molecular weight of around 20,000 . The delta-containing enzymes have a higher affinity for the DNA-cellulose column and a higher specific activity on various templates than vegetative holoenzyme . The simultaneous appearance of these enzymes with vegetative holoenzymes in sporulating cells is consistent with the data found previously with DNA-RNA hybridization studies, which showed that sporulating cells contained both vegetative and sporulation messenger RNAs.

J Bacteriol, 1977 Jan, 129(1), 217 - 24
Role of sugar uptake and metabolic intermediates on catabolite repression in Bacillus subtilis; Lopez JM et al.; Many phosphorylated intermediates exert catabolite repression on the enzyme acetoin dehydrogenase in Bacillus subtilis . This was shown with strains that are blocked at different positions in central metabolism when they receive sugars that cannot be metabolized past enzymatic block(s) . In the case of sorbitol, transport events were not involved in catabolite repression, for this sugar cannot repress acetoin dehydrogenase in a strain lacking sorbitol dehydrogenase but otherwise able to take up sorbitol . The presence of glucose did not markedly influence the uptake of acetoin.

J Bacteriol, 1977 Jan, 129(1), 156 - 65
Amino acid chemoreceptors of Bacillus subtilis; Ordal GW et al.; Specificities of chemoreceptors for the 20 common amino acids, toward which Bacillus subtilis shows chemotaxis, were assessed by competition ("jamming") experiments using a modification of the traditional capillary assay, called the "sensitivity capillary assay." Many amino acids were sensed by at least two chemoreceptors . All the highest affinity chemoreceptors for the amino acids were distinct, except glutamate and aspartate, which may share one chemoreceptor, and tyrosine, for which the data could not be collected due to low solubility . The data suggest the hypothesis that each amino acid-chemoreceptor complex binds to a different signaler (from each amino acid-chemoreceptor complex binds to a different signaler (from which signals travel to the flagella to modify behavior appropriately), and that many of the signalers can also bind other attractant-chemoreceptor complexes as antagonists (no signals to flagella).

J Gen Microbiol, 1977 Jan, 98(1), 117 - 23
Excystment of axenically prepared cysts of Hartmanella culbertsoni; Kaushal DC et al.; Axenically prepared cysts of Hartmannella culbertsoni readily excysted in the presence of heat stable factors prepared from Escherichia coli, Klebsiella aerogenes, Staphylococcus aureus, Sarcina lutea, Bacillus subtilis, Bacillus megaterium and several fungi . Peptone, proteose peptone, tryptone or amino acids also promoted excystment . Crowding of the cysts and dilution of bacterial extracts adversely affected the excystment . Continual presence of the factors in the medium was essential for excystment.

Genetika, 1977, 13(5), 880 - 7
{Operon study of riboflavin biosynthesis in Bacillus subtilis . XII . The determination of the ATP:riboflavin-5'-phosphotransferase and riboflavinsynthetase content in the cells with varying genotypes}; Bresler SE et al.; Activities of riboflavinkinase and riboflavinsynthetase were measured in 15 strains of Bacillus subtilis with different genotype . The increased level of riboflavinkinase was observed in strains, resistant to lumiflavin or lumichrome . Specific activity of riboflavinkinase was found to be about 100 times lower than that of riboflavinsynthetase . The regulation of biosynthesis of these enzymes seems to proceed non-coordinately . This phenomenon can be the sequence of the existence of many operators, controlling the flavinogenesis in Bac . subtilis.

J Virol, 1977 Jan, 21(1), 84 - 95
SP-10 bacteriophage-specific nucleic acid and enzyme synthesis in Bacillus subtilis W23; Markewych O et al.; Bacillus subtilis W23 was infected with a clear-plaque variant of SP-10 phage, namely, SP-10c . Exogenous thymidine was not incorporated into phage DNA (even in the presence of deoxyadenosine), nor was there any transfer of thymidine nucleotides from bacterial to viral DNA . The lytic program was unaffected by concentrations of 5-fluorodeoxyuridine sufficient to reduce bacterial DNA synthesis by greater than 95% . Although these data are consistent with the interpretation that thymidine nucleotides are excluded from phage DNA, formic acid digests of SP-10c DNA contained what appeared to be the four conventional bases; however, adenine and thymine were not recovered in equimolar yields . DNA-RNA hybridization and hybridization competition experiments were done . Synthesis of host RNA started to wane moments postinfection and stopped completely by 36 min . SP-10c coded for discrete classes of early and late RNA . The possibility of discrete subclasses of early RNA exists . Replication of the bacterial genome appeared to terminate 12 min postinfection . Degradation of the host DNA to acid-soluble material started at 36 min and, by the end of the latent period, greater than 90% of the host chromosome was hydrolyzed . Four apparent phage-coded enzymes have been identified . A di- and triphosphatase degraded dUTP, dUDP, dTTP, and dTDP (and, to a lesser extent, dCDP and d CTP) to the corresponding monophosphates; the enzyme had no apparent activity on dATP and dGTP . SP10c also coded for a DNA-dependent DNA polymerase, lysozyme, and a nuclease that degrades native bacterial DNA . Judging from the dependence of enzyme synthesis on the time of addition of rifampin (an inhibitor of the initiation of RNA synthesis), messengers for the di- and triphosphatase, as well as the nuclease, are transcribed from promoters that start to function 6 min postinfection . Promoters for polymerase and lysozyme did not become functional until 8 and 16 min postinfection, respectively.

Cytobios, 1977, 20(79-80), 163 - 77
Inhibition of growth and cell wall morphogenesis of Bacillus subtilis by extracellular slime produced by Physarum flavicomum; Asgari M et al.; Slime secreted by microplasmodia of the myxomycete Physarum flavicomum inhibited the uptake of glucose and amino acids, as well as growth and cell division of the bacterium Bacillus subtilis . Morphological changes such as production of chains, swollen cells, and/or cell lysis, occurred coincident with these physiological inhibitory events . These phenomena were all dependent on the concentration of slime present in the growth medium . Electron microscopy revealed that the cell walls of slime-inhibited cells were undergoing degradation and the process was most pronounced in the swollen cells . Isolated cell walls of B . subtilis were also found to undergo degradation upon incubation with slime . Boiled slime did not exhibit lytic activity on native cell walls, but boiled cell walls were degraded by native slime . The inhibitory effect of slime seemed to be, at least in part, due to an inherent peptidase (protease) activity . B . subtilis eventually overcomes the inhibition exhibited by slime due to the production of an antagonist of slime.

Acta Biol Med Ger, 1977, 36(11-12), 1509 - 13
Intracellular proteinase of Bacillus subtilis; Stepanov VM et al.; An intracellular serine proteinase was isolated from Bacillus subtilis, strain A-50 . The molecular weight of the enzyme is 30000 +/- 1000, its amino acid composition is enriched in glutamic acid residues, the isoelectric point is 4.3, the N-terminal sequence Glu-Leu-Pro-Glu-Gly-Ile-Gln-Val-Ile-Lys-Ala-Pro-Glu-Leu-Xxx-Ala-Gln-Gly-Phe-Lys Gly-Ser-Asx-Ile-Lys-Ile-Ala-Val-Leu-Asx . The enzyme is structuraly homologous with secretory subtilisins.

Genetika, 1977, 13(11), 2023 - 8
{One of the classes of revertants of a rec H Bacillus subtilis mutant}; Lakomova NM et al.; A class of revertants of Bacillus subtilis mutant rec H, which completely restored the ability to transformation but without restoring the activity of ATP-dependent deoxyribonuclease, is isolated and studied . Reversions are located in the same chromosome region as the original mutation . The detection of such revertants points out the existence of more than one recombination pathway for Bac . subtilis transformation.

Genetika, 1977, 13(11), 1976 - 80
{Effect of EcoRI restrictase on the transforming DNA of different bacilli}; Prozorov AA et al.; The transforming activity of DNA from Bacillus subtilis 168, Bac . subtilis W23, Bac . subtilis NRS and Bac . aterrimus after the EcoRI restrictase treatment was studied . The auxotrophic strains of Bac . subtilis 168 were used as recipients in bacterial transformation . The transforming activity for different markers decreased up to 0.001--6 per cent from the initial level for different Bacilli DNAs . In some cases the sensitivity of the same marker from different Bacilli differed in more than 50 times . Bac . subtilis and Bac . aterrimus have demonstrated the maximal differences . Such differences can be used for the identification of close related Bacilli.

Genetika, 1977, 13(11), 2006 - 16
{Riboflavin biosynthesis operon of Bacillus subtilis . XIII . Genetic and biochemical study of mutants with regard to intermediate stages of biosynthesis}; Bresler SE et al.; New riboflavin dependent mutants of Bacillus subtilis accumulating different pteridines were studied . The data obtained show that the formation of ribityl side chain proceeds in a few steps at least on a part of riboflavin precursors . The oxidation of connected ribosyl into ribulose with subsequent restoration of it into ribityl proceeds at first . The corresponding genes are located on terminal part of riboflavin operon, as show the results of two-factor transformational crosses with different donors and recipients.

Biochimie, 1977, 59(3), 289 - 92
Presence of a third sucrose hydrolyzing enzyme in Bacillus subtilis: constitutive levanase synthesis by mutants of Bacillus subtilis Marburg 168; Kunst F et al.; A beta-D-fructofuranosidase -- called levanase -- capable of the hydrolysis of sucrose, inulin and levans has been identified in Bacillus subtilis Marburg . This enzyme can not be detected in strain 168 . However, sacL mutations -- mapped on the chromosome of strain 168 between the pheA and aroD reference markers -- lead to constitutive levanase synthesis . This synthesis is repressed by carbon sources such as glucose, glycerol or sucrose.

Prikl Biokhim Mikrobiol, 1977 Jan-Feb, 13(1), 55 - 60
{Properties of a preparation of lytic enzymes from a culture of Bacillus subtilis}; Kislukhina OV et al.; The effect of pH, temperature, various salts and other compounds on the activity of the preparation of lytic enzymes from the culture of Bacillus subtilis (lyzosubtilin) was investigated . The preparation was shown to be active in neutral solutions of low ionic strength at 30-50 degrees C . The salts of magnesium, manganese, copper, zinc, lead, mercury, aluminium and iron as well as Tris-buffer and Triton X-100 inhibited lyzosubtilin whereas lactic acid activated it . At 30 degrees C the preparation was stable within the pH range of 5 to 10 . Its incubation for 60 min at 60 degrees C resulted in the loss of 50% activity . Lyzosubtilin lyzed cells of gram-positive and gram-negative bacteria, yeast and fungi.

Biochimie, 1977, 59(1), 15 - 21
{Characterization and separation of exocellular gamma-D-glutamyl-(L)meso-diaminopimelate endopeptidase and LD-carboxypeptidase from Bacillus sphaericus 9602}; Vacheron MJ et al.; Two exocellular enzymes have been characterized in the culture media of sporulating Bacillus sphaericus 9602 : a gamma-D-glutamyl-(L) meso-diaminopimelate endopeptidase and a L-lysyl-D-alanine carboxypeptidase . These two enzymes and the corresponding membrane-bound peptidases found in Bacillus sphaericus and Bacillus subtilis strains have similar activities . Their separation is described . Both enzymes were precipitated between 25 and 65 per cent (NH4)2SO4 saturation and a first chromatography was carried out on a column of DEAE-cellulose . The separation was performed by chromatography on hydroxyapatite, each enzyme was finally filtered through Ultrogel AcA 34 . After separation, the endopeptidase activity and the carboxypeptidase activity increased respectively 93 and 11 fold . Both enzymes have a molecular weight near 200 000 . By gel electrophoresis at pH 8.5, they were shown to have different mobilities : the carboxypeptidase is more anionic than the endopeptidase.

J Bacteriol, 1977 Jan, 129(1), 151 - 5
Chemotaxis toward amino acids by Bacillus subtilis; Ordal GW et al.; Conditions for assaying chemotaxis in Bacillus subtilis are described . The chemotaxis medium we used afforded excellent motility for hours . In it, chemotaxis measured by capillary assays was insensitive to pH between 5.5 and 9, and to temperature between 28 degrees C and 42 degrees C . Chemotaxis was observed toward all 20 common amino acids, with thresholds varying from 3nM for alanine to 0.1 mM for glutamate, in the capillary assay, and from 0.1 muM for alanine to 0.32 mM for glutamate in the microscope assay.

Mol Gen Genet, 1976 Dec 31, 142(4), 277 - 87
Selective messenger translation by Bacillus subtilis ribosomes; Legualt-Demare L et al.; The in vitro B . subtilis protein synthesizing system is very restricted in its ability to translate E . coli phage messenger RNA's, specifically phage T4 RNA, even though it actively translates its proper mRNA species . In contrast, the E . coli system translates with similar efficiency mRNA from either source . The initiation factors from the two systems are functionally interchangeable . The 30S B . subtilis ribosomal subunit is responsible for the limited template specificity of the B . subtilis ribosomes . Although the efficiency of the T4RNA directed F Met-tRNA binding by B . subtilis ribosomes is less than that of SPOI RNA-directed binding, the most restrictive step in the translation of T4RNA by B . subtilis ribosomes appears to be at the level of the formation of the first peptide bond, as measured by F Metpuromycin formation.

Eur J Biochem, 1976 Dec 11, 71(2), 451 - 8
Isolation and characterization of hydrophobic proteins (H proteins) in the membrane fraction of Bacillus subtilis . Involvement in membrane biosynthesis and the formation of biochemically active membrane vesicles by combining H proteins with lipid; Kusaka I et al.; Cytoplasmic membranes of Bacillus subtilis, grown in complex medium containing glucose, were fractionated into three membrane subfractions {light band (1.155 - 1.158 g/cm3); medium band (1.181 - 1.183 g/cm3); heavy band (1.21 - 1.25 g/cm3)} by sucrose density gradient centrifugation . Among these subfractions, the light and medium bands consisted mainly of membranes but the heavy band consisted of an irregular arrangement or aggregate of small globular protein components of 5 - 8 nm in diameter . We named this H-protein . H-protein formed trilamellar unit membrane structure when combined with lipid . In pulse-labeling and pulse-chase experiments with radioactive leucine, it was found that H-protein consisted of the newest membrane protein synthesized in the cells and the label incorporated into H-protein was shifted into light and medium band of the membranes during the chase . Cytochromes were not found in H-protein . However, when H-protein was incubated with haem alpha and protohaem, these compounds were incorporated into the apoproteins of the cytochromes present in H-protein and form cytochromes a and b . Cytochromes were also formed in H-protein which were isolated from the cells grown in the presence of haemin (haemin-grown H protein) . Succinate dehydrogenase activity was increased about 4-fold by combining H-protein or haemin-grown H protein with lipid . H-protein had no cytochrome oxidase activity; however, haemin-grown H protein was found to have some of the activity and this was increased about 4-fold by combining the protein with lipid . Haemin-grown H protein was also found to form succinate: cytochrome c oxidoreductase when combined with lipid and vitamin K2 . On the other hand, succinate oxidase was required for the addition of lipid, vitamin K2 and cytochrome c . NADH oxidase was also found in haemin-grown H protein and was activated about 9-fold in constituted reaction systems . Vesicles formed by haemin-grown H protein and lipid, could accumulate alanine and proline by addition of NADH or reduced phenazine methosulfate . Alanine and proline was also accumulated into the vesicles when transport energy was supplied as a membrane potential introduced by K+-diffusion via valinomycin . These results would indicate that H-protein contains the apoprotein of cytochromes, and a carrier involved in the active transport of alanine and proline.

Eur J Biochem, 1976 Dec 11, 71(2), 493 - 508
Levansucrase of Bacillus subtilis . Characterization of a stabilized fructosyl-enzyme complex and identification of an aspartly residue as the binding site of the fructosyl group; Chambert R et al.; A covalently linked fructosyl-enzyme complex was isolated from a reaction mixture of enzyme and sucrose submitted to the quenching effect of a large decrease of the pH . The fructosyl-enzyme bond was shown to be stable under acidic and neutral conditions in the presence of high concentration of urea and of sodium dodecyl sulfate . This intermediate did not transfer at a measurable rate its fructosyl group to the usual fructosyl acceptors of the enzyme reaction under the usual conditions of enzyme activity . However stability measurements of the fructosyl-enzyme bond indicated a marked lability at pH values above 8.5 . The apparent rate constant of the hydrolytic reaction of this bond evaluated under the standard state of molar concentration of hydroxide ion was of the same order of magnitude as the apparent rate constant of the hydrolytic reaction of the transient fructosyl-enzyme postulated from the kinetic analysis of levansucrase . Furthermore, nucleophilic agents like imidazole enhanced the hydrolytic reaction of the fructosyl-enzyme bond . Identification of the fructosyl binding site on the enzyme was accomplished by proteolytic hydrolysis of the trapped complex . Peptic digestion followed by pronase digestion released a fructosyl-aspartate compound that we have isolated in a high state of purity . The lability of the fructosyl-aspartate bond under mild alkaline conditions suggested that the fructosyl was linked through an ester bond involving the beta-carboxyl of the aspartate residue . Treatment of the trapped complex with cyanogen bromide released only one fructosylated peptide . The apparent molecular weight of this peptide was estimated to be lower than 10000.

Am J Gastroenterol, 1976 Dec, 66(6), 540 - 5
Biliary excretion and concentration of cefazolin; Ram MD et al.; The excretion of cefazolin into the human biliary tract in health and disease was investigated in 34 patients undergoing surgical procedures . The patients included: I . Four controls . IIA . Eleven patients with cholelithiasis and/or cholecystitis and a radiological visualized gallbladder . IIB . Nine patients with cholethiasis and cholecystitis and a radiologically nonvisualized gallbladder . III . Five patients with obstructive jaundice . IV . Five patients with a T-tube in the common bile duct . Two dose regimes: 1 . A single dose of 500 mg and 2 . four doses each of 500 mg . given every six hours, were used . Samples of serum, gallbladder bile, common duct bile and gallbladder tissue were assayed for antibiotic activity by the cylinder plate method with Bacillus subtilis . Following administration of four doses of the antibiotic, the mean level of the drug in the gallbladder bile, in controls was 127.0 mug/ml . In the group with cholelithiasis and cholecystitis and a gallbladder that is visualized, a similar high level was noted (mean = 132.2 mug/ml.) . In the presence of a nonvisualized gallbladder or obstructive jaundice, the levels in bile were lower . Two hours following a single injection of the drug, the level in the common duct bile reaches a peak of 10 mug/ml and at eight hours falls to less than one mug/ml . In the absence of obstruction cefazolin reaches a significantly high level in bile and could be valuable in treatment of biliary infections.

Biokhimiia, 1976 Dec, 41(12), 2229 - 36
{Biospecific chromatography of Bacillus subtilis metalloendoproteinase and detection of multiple forms of the enzyme}; Vaganova TI et al.; Metalloendoproteinase was isolated from protosubtilin, i.e . a mixture of enzymes produced by Bacillus subtilis, during adsorption on activated carbon and a subsequent biospecific chromatography on DNP--hexamethylene diamine--Sepharose 4B and gramicidin S--Sepharose 4B . The molecular weight of the enzyme estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was found to be 36.000 . The preparation isolated contained four molecular forms of the enzyme, each splitting DNP-Gly-Gly-Val-Arg . It is shown that thermolysine, purified by biospecific chromatography, consists of three molecular forms . The amino acid composition of metalloendoproteinase was established.

J Gen Microbiol, 1976 Dec, 97(2), 297 - 301
Competence in continuous cultures of Bacillus subtilis: inhibition by arginine and reversal of the inhibition by Mn2+; Espinosa M et al.; Arginine inhibited the competence of Bacillus subtilis growing in a chemostat at a dilution rate of 0-277 h-1 . The biosynthesis of competence-stimulating activity was only partially repressed . The inhibitory effect may be due to an alteration in the cell's capacity for being stimulated to competence and/or in its ability to take up DNA irreversibly . MnSO4 at 10(-6) M restored competence immediately.

Eur J Biochem, 1976 Dec, 71(1), 77 - 83
Transcription in vitro of phi29 DNA and EcoRI fragments by Bacillus subtilis RNA polymerase; Inciarte MR et al.; EcoRI fragments A, B and C produced from linear phi29 DNA, but not D or E fragments, are transcribed by purified Bacillus subtilis RNA polymerase . The transcription of fragments A and C is initiated preferentially with GTP and to a lesser extent with ATP; the reverse happens in the case of fragment B . The dinucleotides GpU and GpA respectively, compete specifically with the incorporation of {gamma-32P}GTP directed by fragments A and C . The RNA synthesized in vitro by purified B . subtilis RNA polymerase is highly asymmetric . Most of the RNA synthesis directed by fragments A and C is early RNA . However, most of the RNA produced by fragment B is anti-late-RNA . Addition of crude extracts inhibit the transcription of fragment B but not that of fragments A and C.

Arch Microbiol, 1976 Dec 1, 111(1-2), 7 - 11
Proton-motive force and the motile behavior of Bacillus subtilis; De Jong MH et al.; Changes in the proton-motive force cause a transient change in the motile behavior of Bacillus subtilis cells . Both an increase and a decrease in the proton-motive force caused transient tumbling . Simultaneous decrease of proton-motive force and increase of attractant concentration lessens the response toward the attractant . A simultaneous increase of proton-motive force and increase of attractant concentration prolonges the response toward attractant . A hypothesis explaining the various effects is given.

Eur J Biochem, 1976 Dec, 71(1), 309 - 16
Isolation, subunit structure and properties of the ATP-dependent deoxyribonuclease of Bacillus subtilis . State of the protein in a mutant devoid of activity; Doly J et al.; A prodcedure was developed for the purification of the ATP-dependent deoxyribonuclease of Bacillus subtilis 168 . It comprises ammonium sulphate fractionation, Sephadex gel filtration, DEAE-cellulose chromatography and gel electrophoresis on a discontinuous polyacrylamide gradient . The enzyme has been obtained in a homogeneous state . Its molecular weight was estimated to be 270000 by disc electrophoresis . Dodecylsulfate-polyacrylamide gel electrophoresis showed the presence of five nonidentical subunits of the following molecular weights: 81000, 70000, 62000, 52500 and 42500 . These values give 308000 as the molecular weight of the native enzyme . The pH optimum of the purified enzyme is 9.6 . The optimal concentrations of Mg2+ and ATP for exonuclease activity on native B . subtilis DNA were determined . ATP-requirement for hydrolysis of single-stranded DNA is less strigent . The enzyme also possesses high DNA-dependent ATPase activity . The purification procedure was applied to extracts of a mutant devoid of activity for this enzyme (strain GSY 1290) . A protein was isolated which is very similar to the active DNAase as regards electrophoretic mobility, reaction with specific antisera and size of four of the subunits . One subunit is missing (Mr 70000) and is replaced by a smaller polypeptide (Mr 565000) . The latter results suggest that the mutant is affected in the genetic locus coding for the 70000-Mr subunit.

J Biol Chem, 1976 Nov 25, 251(22), 6974 - 80
Purification and properties of two aromatic aminotransferases in Bacillus subtilis; Weigent DA et al.; Two enzymes which transaminate tyrosine and phenylalanine in Bacillus subtilis were each purified over 200-fold and partially characterized . One of the enzymes, termed histidinol phosphate aminotransferase, is also active with imidazole acetyl phosphate as the amino group recipient . Previous studies have shown that mutants lacking this enzyme require histidine for growth . Mutants in the other enzyme termed aromatic aminotransferase are prototrophs . Neither enzyme is active on any other substrate involved in amino acid synthesis . The two enzymes can be distinguished by a number of criteria . Gel filtration analysis indicate the aromatic and histidinol phosphate aminotransferases have molecular weights of 63,500 and 33,000, respectively . Histidinol phosphate aminotransferase is heat-sensitive, whereas aromatic aminotransferase is relatively heat-stable, particularly in the presence of alpha-ketoglutarate . Both enzymes display typical Michaelis-Menten kinetics in their rates of reaction . The two enzymes have similar pH optima and employ a ping-pong mechanism of action . The Km values for various substrates suggest that histidinol phosphate aminotransferase is the predominant enzyme responsible for the transamaination reactions in the synthesis of tyrosine and phenylalanine . This enzyme has a 4-fold higher affinity for tyrosine and phenylalanine than does the aromatic aminotransferase . Competitive substrate inhibition was observed between tyrosine, phenylalanine, and histidinol phosphate for histidinol phosphate aminotransferase . The significance of the fact that an enzyme of histidine synthesis plays an important role in aromatic amino acid synthesis is discussed.

Mol Gen Genet, 1976 Nov 17, 148(3), 307 - 13
Discrimination of competent Bacillus subtilis with respect to ribonucleic acids; Soltyk A et al.; A study has been made of the affinity of double-stranded helical RNA for DNA receptors in competent Bacillus subtilis . In competition experiments, using homologous and heterologous DNA samples which had been sheared to molecular weights comparable to that of the RNA (about 2 x 10(6)), and which still exhibited appreciable competition in DNA uptake experiments, the replicative form of phage f2 RNA showed no evidence of affinity for receptor sites . A second double-stranded RNA preperation from a widely different source, a mycophage of Penicillium chrysogenum, behaved similarly to the f2 RNA . Transfer RNA and 23S ribosomal RNA, which reduce transformation frequencies in pneumococcus, did not compete for B . subtilis receptors . Lack of competition was not due to enzymatic degradation of the RNA, since the latter was recovered intact following exposure to competent cells . Under conditions where homologous native DNA undergoes normal uptake, there was virtually no uptake of native double-stranded RNA . The results are examined in the light of reports on transformation by RNA and DNA-RNA hybrids, and also in relation to the characterization of the specificity of cell-nucleic acid interactions.

Mol Gen Genet, 1976 Nov 17, 148(3), 281 - 5
Mapping of mutations affecting synthesis of exocellular enzymes in Bacillus subtilis . Identity of the sacUh, amyB and pap mutations; Steinmetz M et al.; The sacUh, amyB and pap mutations are identical with respect to their pleiotropic phenotype and their genetic location . Strains bearing these mutations overproduce several exocellular enzymes: alpha amylase, lavansucrase and proteases, they are poorly or not at all transformable and most of them are devoid of flagella . These mutations are tightly linked to the sacU- mutations by transformation and therefore lie between the hisA1 and gtaB290 markers . It is possible that the sacUh, amyB and pap mutations on one hand and the sacU- mutations on the other are two different classes of alterations of the same regulatory gene controlling the synthesis of some exocellular enzymes and several other cellular functions . Furthermore an amy- mutation, leading to the lack of alpha-amylase activity, was mapped between the lin2 and aroI906 markers which are not linked to the sacU locus.

Arch Microbiol, 1976 Nov 2, 110(23), 295 - 300
Morphology and anionic polymer content in the cell wall of a glycerol-requiring mutant of Bacillus subtilis; Wouters JT et al.; A glycerol-requiring mutant of Bacillus subtilis formed irregular spheres and showed disturbed septum formation, when subjected to growth limitation by the supply of glycerol . Under phosphate limitation the cells were also round and developed asymmetric septa . In magnesium-limited cultures the cells contained a thickened wall, as compared with that of the parent strain grown under the same conditions . Chemical analysis revealed the presence of teichoic acid as the major anionic polymer in the wall of the glycerol-, as well as the magnesium-limited cells of the glycerol-requiring B . subtilis mutant . Under phosphate limitation teichuronic acid was the only anionic polymer present in the wall . Thus, in this respect, there were no apparent differences between mutant organisms and the parent strain when grown under magnesium and phosphate limitation, respectively and the observed morphological deviations could not be correlated with an altered anionic polymer content of the wall.

Eur J Biochem, 1976 Nov 1, 70(1), 275 - 83
A novel endonuclease of human cells specific for single-stranded DNA; Pedrini AM et al.; We have fractionated from human aneuploid cell cultures three different enzyme fractions degrading single-stranded DNA . We have purified and characterized one of these DNases; this is an endonuclease working at alkaline pH (around 9.5) and requiring Mg2+ for its activity . The enzyme degrades denatured DNA over 100 times more efficiently than native DNA in optimal conditions . The termini produced by the enzyme have 5'P and 3'OH ends . The enzyme can attack, though at reduced rate, the supertwisted circular molecule of Simian virus 40 DNA, whereas it is inactive on the nicked circular molecule . The ultraviolet irradiation of DNA, whether native or denatured, does not affect its efficiency as substrate of the DNase . The properties of this endonuclease distinguish it from those of the other DNases described previously in mammalian cells; the denomination DNase VI is therefore proposed . Its properties are similar to those of DNases described in Ustilago maydis and Bacillus subtilis, for which an essential role in recombination seems likely.

Nucleic Acids Res, 1976 Nov, 3(11), 3213 - 26
Specific cleavage of chromatin by restriction nucleases; Horz W et al.; Digestion of mouse and rat liver nuclei with a restriction nuclease from Bacillus subtilis (Bsu) is examined in continuation of previous work from this laboratory (Pfeiffer et al., 1975, Nature 258, 450) . The finding of more than 95% C in the 5'-termini of the DNA fragments generated during digestion with Bsu shows that the participation of endogenous nucleases in Bsu digestion is extremely small . The restriction nuclease Hae III, an isoschizomer of Bsu, yields identical degradation patterns . The patterns conform to what one expects from statistical calculations based on a nucleosome structure of chromatin with a region preferentially accessible to the nuclease of 40-50 nucleotide pairs per nucleosome . Integrity of the histones is maintained during digestion with restriction nucleases . Digestion of mouse liver nuclei with EcoRII shows that most if not all of the satellite DNA is organized in a nucleosome structure . Also in rat liver, much of the repetitive DNA appears to be present in nucleosomes.

Nucleic Acids Res, 1976 Nov, 3(11), 3087 - 99
A simplified procedure for the analysis of DNA polymerase III levels in Bacillus subtilis strains; Ciarrocchi G et al.; A simple and reproducible procedure is described which allows the fast and almost quantitative removal of DNA polymerases I and II from DNA polymerase III, in crude extracts of polA+ strains of Bacillus subtilis . The procedure entails streptomycin sulfate and ammonium sulfate fractionations; subsequent analysis of the partially purified preparation by G-200 chromatography, DEAE cellulose chromatography and density gradient sedimentation, shows that the ammonium sulfate fraction contains less than 5% of the total activity as DNA polymerase I and less than 2% as DNA polymerase II . The purification procedure, up to the ammonium sulfate step, was utilized for the analysis of the level of DNA polymerase III in several B . subtilis mutants, with results comparable to those obtained from the corresponding polA- strains following more cumbersome purification procedures . The M.W . of the purified form is of 227.000, somewhat greater than the published values . The early fractions of the purification have revealed the existence of a form with a M.W . of 426.000; the nature of this form, which has been observed in several instances and which is very unstable and short-lived, is under investigation.

Proc Natl Acad Sci U S A, 1976 Nov, 73(11), 4145 - 9
Expression of the thymidylate synthetase gene of the Bacillus subtilis bacteriophage Phi-3-T in Escherichia coli; Ehrlich SD et al.; The thymidylate synthetase gene of B . subtilis bacteriophage Phi-3-T, when cloned in plasmids pSC101 or pMB9 is expressed in E . coli . The promoter of the cloned gene is likely to originate in Phi-3-T . Rearrangements of hybrid plasmid sequences during the cloning have been noted . B . subtilis strains can be transformed with hybrid DNAs . The transformants contain sequences of Phi-3-T, but not those of plasmid vectors.

J Virol, 1976 Nov, 20(2), 535 - 8
Bacteriophage PBS2-induced inhibition of uracil-containing DNA degradation; Katz GE et al.; Degradation of uracil-containing DNA by Bacillus subtilis extracts and its inhibition after infection by the uracil-containing DNA phage PBS2 have been investigated to resolve differences between the published reports of Tomita and Takahashi (1975) and Friedberg et al . (1975, 1976) . The product of hydrolysis of PBS2 DNA, tritium labeled in its uracil and cytosine residues, is solely uracil and not deoxyuridine . The degrading activity is completely inhibited within 7 min after PBS2 infection, before any other known PBS2-induced protein is detectable . The production of the PBS2 inhibitor (a small, heat-stable protein) continues until 10 to 20 min postinfection.

Tijdschr Diergeneeskd, 1976 Nov 1, 101(21), 1194 - 8
{Antibiotic residues in organs and muscle tissues of broilers . I . Bacitracin, flavomycin, spiramycin and viriniamycin residues following administration of diets containing low levels of these antibiotics (author's transl)}; Mulder RW et al.; Two groups of broilers were fed two different feed mixtures . A feed containing a mixture of bacitracin, flavomycin, spiramycin and virginiamycin (20 ppm each) was administered to sixity broilers . Sixty other broilers were given a similar feed not containing any antibiotics . After slaughter, samples of kidney, liver and breast were examined for the presence of antibiotic residues . All samples were found to be negative for antibiotic residues . Four micro-organisms were used in performing the tests: Bacillus cereus Kiel, Bacillus subtilis 165, Bacillus subtilis BGA and Micrococcus luteus ATCC 9341.

J Virol, 1976 Nov, 20(2), 509 - 19
New temperate bacteriophage for Bacillus subtilis, rho 11; Dean DH et al.; A new temperate bacteriophage, rho11, isolated by J . Hoch, has been characterized . This new phage is very similar to the temperate phage phi3T in size (380 nm), host range, homoimmunity, DNA buoyant density (1.694 g/ml), antigenicity, and molecular weight (around 6.0 X 10(7)) as determined in gels . Like phi3T, rho11 converts thymine auxotrophs to prototrophy at high frequency (250 out of 250 tested) . Phage rho11 differs from phi3T in plaque morphology and in the endonuclease R-EcoRI digest pattern . Sixteen of the 20 rho11 DNA fragments have migration patterns corresponding to those of the 21 fragments of phi3T . The close similarities yet clear differences between these phages suggest that the two phages have a common ancestor.

Biochemistry, 1976 Oct 19, 15(21), 4675 - 80
Superactivation of neutral proteases: acylation with N-hydroxysuccinimide esters; Holmquist B et al.; A series N-hydroxysuccinimide esters of acylamino acids previously shown to acylate and thereby increase the activity of thermolysin by several orders of magnitude (Blumberg, S., and Vallee, B . L . (1975), Biochemistry 14, 2410) has been used to modify the related neutral proteases from Bacillus subtilis, Bacillus megaterium, and Aeromonas proteolytica . Each of these enzymes is activated to a level characteristic of the particular protein and the particular acyl group incorpporated when monitored with the substrate furylacryloyl-Gly-Leu-NH2 . Thus, for the modification of B . megaterium, B . subtilis, and A . proteolytica proteases with Ac-Trp-ONSu, kcat/Km increases 11-, 2.5-, and 18-fold whereas those of the Ac-Phe(4-DnpNH)-ONSu modified enzymes before and after deacylation with hydroxylamine indicate that from 1 to 2 residues are modified . The rate of removal of the Ac-Phe(4-DnpNH) label by 0.1 M hydroxylamine correlates directly with that of the return of native enzymatic activity, at a rate comparable with the rate of deacylation of O-acyltyrosine models . The competitive inhibitors Zn2+ and beta-phenyl-propionyl-Phe do not prevent activation indicating that modification occurs at a site(s) distinct from that at which inhibitors bind . The degree of activation depends also on the substrate employed, generally being greater for substrates which the native enzymes hydrolyze slowly . These data are interpreted to indicate the modification of a residue near the active site, but which serves as a subsite for substrate interaction.

Mol Gen Genet, 1976 Oct 18, 148(1), 9 - 17
On the identity of dnaP and dnaF genes of Bacillus subtilis; Attolini C et al.; The dnaP strains of Bacillus subtilis are altered in the initiation of DNA replication at high temperature (Riva et al., 1975) . Fine mapping of the gene shows that it is located very close to the dnaF gene described by Karamata and Gross (1970) and mapped by Love et al . (1976) in the polC region . The phenotype of both mutants is indistinguishable: the DNA synthesis stops at non permissive temperature after synthesizing an amount of DNA equivalent to the completion of the rounds of replication already initiated; at permissive temperature they are abnormally sensitive to MMS and are reduced in the ability to be transformed . Both mutants are to be considered as belonging to the dnaF locus . The dnaF gene is very close to the polC gene, which specifies the DNA polymerase III of B . subtilis . The DNA polymerase III of the dnaF mutants is not temperature sensitive in vitro, however, the level of this enzyme is lower by a factor of 4 or 5 in the dnaF mutants, at the permissive temperature . Following shift of dnaF cultures to the non permissive temperature, the level of DNA polymerase III activity specifically decreases further by a factor of at least 10 in the mutant, whereas the DNA polymerase I level is unaffected . The possible roles of the dnaF gene in the control of the cellular level of the DNA polymerase III, and the possibility of a regulatory role of DNA polymerase III in the initiation of DNA replication in bacteria are discussed.

Mol Gen Genet, 1976 Oct 18, 148(2), 159 - 64
Synthesis of RNA and protein in a mutant of Bacillus subtilis temperature sensitive during spore germination; Galizzi A et al.; Bacillus subtilis strain PB 2427 temperature sensitive in the synthesis of RNA during spore germination and outgrowth has been characterized to some extent . At non permissive temperature (46 degrees C) strain PB 2427 synthesizes stable and unstable RNA for 50 min from the beginning of germination and then stops . Most of the stable RNA is degraded to shorter molecules but can be identified as ribosomal RNA by hybridization-competition experiments . At non permissive temperature, in the presence of chloramphenicol, synthesis of RNA proceeds, though at a reduced rate, for at least 90 min . By hybridization-competition experiments it can also be shown that the RNA synthesized at 46 degrees C in the presence of chloramphenicol includes transcripts that are absent, from the RNA synthesized at 46 degrees C in the absence of drug . The RNA polymerase (holo and core) purified from vegatative cells of the mutant strain does not appear to have a greater heat-lability as compared with the enzyme purified from the parental strain . At non permissive temperature only six polypeptide chains with MW ranging from 47,000 to 78,000 daltons are synthesized by the germinating spores of the mutant.

J Biol Chem, 1976 Oct 10, 251(19), 5904 - 10
Structure of plant cell walls . Purification and characterization of a beta-1,4-galactanase which degrades a structural component of the primary cell walls of dicots; Labavitch JM et al.; Wild type Bacillus subtilis, when grown on a soybean arabinan-galactan, secretes a beta-1,4-galactanase which has been purified more than 200-fold from the culture fluid . Affinity chromatography was the most effective step in a purification procedure which resulted in a preparation that contained only a single 40,000 molecular weight protein band upon sodium dodecyl sulfate-disc gel electrophoresis . The purified galactanase digests a beta-1,4-galactan purified from citrus pectin and digests partially the isolated cell walls of suspension-cultured sycamore cells . The predominant product of the enzymic degradation of the substrates tested is a 4-linked tetragalactose . Evidence is presented to support the hypothesis that the galactanase attacks its substrates in both an exo- and endo-manner . The products obtained upon galactanase digestion of the soybean arabianin-galactan demonstrate that the earlier proposal concerning the structure of this polysaccharide must be incorrect.

J Biochem (Tokyo), 1976 Oct, 80(4), 755 - 66
RNA polymerase in vegetative cells of Bacillus subtilis . II . New polypeptide factors, FI and FII, stimulating in vitro RNA synthesis directed by phage M2 DNA; Ikeda JE et al.; Two polypeptides named FI and FII were isolated from vegetative cells of Bacillus subtilis Marburg . The molecular weights of FI and FII were 15,000 and 30,000 daltons, respectively . They were able to stimulate the transcription of phage M2 DNA in the presence of Mg2+ ions by RPase L1 and RPase L2 {RNA polymerase; EC 2.7.7.6} . Although both core- and holo-RPase L2 hardly exhibited transcription activity under these conditions, the factors could stimulate both activities up to the level of RPase L1 activity . The stimulation was much less marked when B . subtilis DNA was used as a template . These stimulatory functions were found to lie not in the chain elongation but in the initiation step of transcription, following the preinitiation step . To obtain stimulation by the factors, preincubation with RNA polymerase was necessary . FI stimulated RPase L1 or RPase L2 only when preincubated in the stimultaneous presence of FII, forming a complex, RPase L1(or L2)-FI-FII . On the other hand, FII alone could stimulate transcription, forming a complex . RPase L1 (or L2)-FII . In these complexes, the ratio of FI, FII, and RPase L1(or L2) was 1 : 1: 1 . Although the core-RPase L2 activity was inhibited by sigma subunits, it was not inhibited by was rather stimulated when the enzyme was present as a complex with FI and FII . Thus the complex, consisting of RPase L2 and the factors, resembled RPase L1 with respect to molecular weight, template specificity, the effect of sigma subunit, and sensitivity to rifampicin.

J Biochem (Tokyo), 1976 Oct, 80(4), 743 - 54
RNA polymerase in vegetative cells of Bacillus subtilis . I . Purification and properties of RNA polymerase L1 and L2; Ikeda JE et al.; Two forms of RNA polymerase {EC 2.7.7.6}, RPase L1 and RPase L2, isolated from a highly synchronized vegetative culture of Bacillus subtilis Marburg strain are described . RPase L1 is the major component (identical and the vegetative RNA polymerase already reported) and RPase L2 is a minor, new component corresponding to 3--5% of the total activity . The enzymes differed in their requirements for divalent ions, though the differences depended on the template DNA employed . PRase L1 is able to transcribe phage M2 DNA in the presence of Mg2+ ions and both B . subtilis DNA and phage M2 DNA in the presence of Mn2+ ions . On the other hand, RPase L2 activity can be detected only in the presence of 3 mM Mn2+ ions with all the templates . It is of interest that the transcription of phage M2 DNA by both enzymes stringently requires KC1 . It may be due to this ion dependence that RPase L2 has not been detected previously . RPase L2 consists of 1beta', 1beta gamma, 1sigma, and 2alpha subunits . The molecular weight of the beta gamma subunit (about 110,000) is close to the value reported for the beta subunit of RNA polymerase prepared from sporulating cells . However, RPase L2 as a whole molecule is different from the RNA polymerase of sporulating cells or spores in the following two respects: RPase L2 contains sigma subunit as a component essential for selective transcription, and it is resistant to 1 mug of rifampicin per ml . Elimination of the sigma subunit from RPase L2 greatly stimulates RNA synthesis by the enzyme . Conversely, the addition of sigma subunit to the core-enzyme is inhibitory.

Nucleic Acids Res, 1976 Oct, 3(10), 2851 - 60
5-methoxyuridine: a new minor constituent located in the first position of the anticodon of tRNAAla, tRNAThr, and tRNAVal from Bacillus subtilis; Murao K et al.; The sequences of the anticodon of tRNAAla, tRNAThr, and tRNAVal from Bacillus subtilis W 168 were N-G-C, N-G-U, and N-A-C, respectively . A new minor constituent, N, occupied the first position of the anticodon of each tRNA . N was indentified as 5-methoxyuridine (mo5U, Figure 1) by comparison of its UV absorption spectra, Rf values in thin-layer chromatography using several solvent systems and mass spectra with those of chemically synthesized specimen.

J Antibiot (Tokyo), 1976 Oct, 29(10), 1043 - 9
Characterization of iturin A in antibiotics from various strains of Bacillus subtilis; Besson F et al.; Iturin A, an antifungal lipopeptide characterized by the presence of homologous liposoluble beta-aminoacids was found to be the active component to bacillomycin B, bacillomycin R and eumycin . Iturin A was identified by thin-layer chromatography, aminoacid analysis and by characterization of liposoluble aminoacids and peptides . Another two preparations: the antibiotic of Raubitschek and the bacillomycin of Landy et al . contain components of the same structural type but they are different from iturin A.

J Virol, 1976 Oct, 20(1), 22 - 8
Prophage mutation causing heat inducibility of defective Bacillus subtilis bacteriophage PBSX; Buxton RS; A mutant of Bacillus subtilis 168 has been isolated in which the defective phage PBSX was heat inducible, whereas another phage, phi105, was not so induced . A culture of the mutant grown at 30 degrees C, when shifted to 45 degrees C, began to lyse after 45 min; cell viability began to decrease after 10 min . Heat-induced lysis of the mutant was prevented by chloramphenicol . DNA, RNA, protein, and peptidoglycan synthesis were normal at the nonpermissive temperature up to the time of lysis . The site of xhi-1479 mutation causing this phenotype was linked (50%) in phage PBS1-mediated transduction to the host marker metC and to another PBSX marker xtl and was thus thought to map within the PBSX prophage . The order of markers was argC-thiB-metA-xhi-metC . The xhi mutation was thus distinct from another mutation, tsi-23, causing a similar heat inducibility of PBSX (Siegel and Marmur, 1969), which was unlinked to the metC marker . tsi-23 is therefore thought to be a host mutation, and the available evidence for a scattered phage genome being the cause of the defective nature of PBSX is thus less tenable . It was shown that the mutant, besides carrying the xhi mutation, also carried another closely linked mutation, xki-1479, which caused the PBSX produced to have no killing activity on the sensitive strain W23 . The xki mutation was separated from xhi by recombination.

J Virol, 1976 Oct, 20(1), 188 - 95
Restriction and modification in Bacillus subtilis: inducibility of a DNA methylating activity in nonmodifying cells; Gunthert U et al.; The nonrestricting/nonmodifying strain Bacillus subtilis 222 (r-m-) can be induced to synthesize a DNA-modifying activity upon treatment with either mitomycin C (MC) or UV light . This is shown by the following facts . (i) Infection of MC-pretreated 222 cells with unmodified SPP1 phage yields about 3% modified phage that are resistant to restriction in B . subtilis R (r+m+) . The induced modifying activity causes the production of a small fraction of fully modified phage in a minority class of MC-treated host cells . (ii) The MC-pretreated host cells contain a DNA cytosine methylating activity: both bacterial and phage DNAs have elevated levels of 5-methylcytosine . (iii) The MC-induced methylation of SPP1 DNA takes place at the recognition nucleotide sequences of restriction endonuclease R from B . subtilis R . (iv) Crude extracts of MC-pretreated 222 cells have enhanced DNA methyltransferase activities, with a substrate specificity similar to that found in modification enzymes present in (constitutively) modifying strains.

J Bacteriol, 1976 Oct, 128(1), 80 - 5
Effect of netropsin on the derepression of enzymes during growth and sporulation of Bacillus subtilis; Keilman GR et al.; Netropsin, a polypeptide antibiotic which binds specifically to adenylate-thymidylate-rich regions of deoxyribonucleic acid, inhibitis sporulation at about stage II, but does not inhibit growth of Bacillys subtilis . An analysis of the sporulation-associated enzymes aconitase, alkaline phosphatase, and glucose dehydrogenase revealed that their rates of expression were not affected by the presence of the antibiotic . The derepression of histidase, a vegetatively induced enzyme was stimulated by netropsin . Oxygen utilization by the cells during sporulation was not effected nor was spore germination prevented by the drug . Netropsin, however, did prevent the formation of dipicolinic acid . These and earlier results suggest that netropsin may be affecting the transcription of only select sporulation genes that are particularly rich in adenylate-thymidylate base pairs.

J Bacteriol, 1976 Oct, 128(1), 8 - 14
Resporulation of outgrowing Bacillus subtilis spores; Keynan A et al.; Germinated spores of Bacillus subtilis were incubated in outgrowth medium and tested periodically for capacity to sporulate when suspended in sporulation medium . Concurrent measurements were made of deoxyribonucleic acid (DNA) content and numbers of cell division septa and nucleoids . Sporulation potential is shown to reach a peak at about 110 min at which time the chromosomes are probably well into the second round of replication . Experiments with nalidixic acid show that sporulation potential can be generated in the outgrowth medium even when DNA synthesis is largely prevented . Further experiments show that nalidixic acid apparently does not prevent the formation of DNA initiation complexes, which can subsequently function after resuspension in the sporulation medium . The results support those previously obtained with a temperature-sensitive DNA mutant which indicated that sporulation could only be induced at a specific stage of chromosome replication, and then only if the cells are in a state of nutritional "step-down".

J Bacteriol, 1976 Oct, 128(1), 317 - 24
Postirradiation recovery dependent on the uvr-1 locus in Bacillus subtilis; Hadden CT; A mutant (uvr-1) of Bacillus subtilis that is deficient in excision of ultraviolet (UV)-induced pyrimidine dimers from deoxyribonucleic acid (DNA) shows a marked increase in ability to survive UV irradiation when plated on amino acid-supplemented agar medium compared with its survival ability when plated on nutrient plating medium, the effect is considered to be one of growth-dependent lethality . Irradiated stationary phase uvr-1 cells, incubated in liquid medium lacking amino acids required for growth, recover from this sensitivity to rich medium within 3 to 4 h after irradiation . Recovery is greatly reduced in the absence of glucose oiminated . Exponentially growing cells have a limited ability to recover from sensitivity to rich medium . Growth-dependent lethality can also occur in liquid medium . In nutrient broth the ability of irradiated stationary-phase uvr-1 cells to form colonies on defined agar medium decreases during postirradiation incubation, but treatmeth with chloramphenicol inhibits the loss of colony-forming ability . Recovery from sensitivity to rich media is inhibited by caffeine but not by 6-(p-hydroxyphenylazo)-uracil, and inhibitor of DNA replication . Alkaline sucrose gradient profiles show that conditions allowing recovery also favor maintaining intact DNA strands, whereas DNA strand breakage or degradation is associated with loss of viability . Recovery from sensitivity to rich medium has not been observed in the Ur+ parent or in strains carrying the mutations uvs-42 (another deficiency in dimer excision), recA1, or polA59 . A uvr-1 recA1 mutants shows a higher level of recovery than does the recA1 single mutant, but a much lower level than the uvr-1 single mutant . Apparently, both the uvr-1 defect and Rec+ and PoII+ functions are essential for recovery from sensitivity to rich medium . For optimal recovery, growth immediately after irradiation must be delayed . The process requires energy, apparently involves recombination, and probably results in rejoining of DNA strands in which incision but not excision has occurred.

J Bacteriol, 1976 Oct, 128(1), 221 - 7
Changes in deoxyribonucleic acid polymerase activities in synthesis of deoxyribonucleic acid during sporulation of Bacillus subtilis; Honjo M et al.; The deoxyribonucleic acid (DNA) polymerase activities in Bacillus subtilis strains Marburg 168 (thy-trp2) and D22, a DNA polymerase I-deficient mutant, were measured at various stages of sporulation . The DNA polymerase I activity, which had decreased after the exponential growth, began to increase at the early stage of sporulation, reached a maximum and then again decreased . The activity of neither DNA polymerase II nor III was observed to change so drastically as that of DNA polymerase I during sporulation . The incorporation of {3H}deoxythymidine 5'-triphosphate ({3H}dTTP) into Brij 58-treated permeable cells increased during sporulation . The stimulation of {3H}dTTP incorporation into the cells by irradiation with ultraviolet light was also observed to coincide with DNA polymerase I activity . In strain D22 the activities of DNA polymerase II and III were almost constant with time . Neither change of {3H}dTTP incorporation into Brij 58-treated cells nor stimulation of incorporation by irradiation with ultraviolet light was observed.

J Bacteriol, 1976 Oct, 128(1), 202 - 11
Regulation of histidinol phosphate aminotransferase synthesis by tryptophan in Bacillus subtilis; Weigent DA et al.; The effect of tryptophan on the synthesis of histidinol phosphate aminotransferase and prephenate dehydrogenase has been examined . The genes specifying two enzymes for tryptophan biosynthesis (anthranilate synthase and tryptophan synthase-B) were found to be derepressed in a temporal sequence according to their chromosomal location . The genes for histidinol phosphate aminotransferase and prephenate dehydrogenase were derepressed simultaneously approximately 8 min after tryptophan synthase-B . When excess tryptophan was added to a derepressed culture, the pattern of repression of trpE (anthranilate synthase), trpB (tryptophan synthase-B), hisH (histidinol phosphate aminotransferase), and tyrA (prephenate dehydrogenase) was found to be simultaneous . Methyl tryptophan-resistant mutants, which synthesize elevated levels of the tryptophan enzymes, also synthesized elevated levels of histidinol phosphate aminotransferase . Qualitatively similar data were obtained in a temperature-sensitive tryptophanyl-transferase ribonucleic acid synthetase mutant grown at elevated temperatures . The time at which messenger ribonucleic acid was synthesized for anthranilate synthase, tryptophan synthase-B, histidinol phosphate aminotransferase, and prephenate dehydrogenase in the presence of actinomycin D indicated that ordered enzyme synthesis was a result of ordered transcription of the corresponding portion of the genome . The effect of the drug rifampin on enzyme synthesis was also examined . The addition of this drug halted the transcription of anthranilate synthase very rapidly, but later regions of the tryptophan region continued to be transcribed . The transcription of the hisH and tyrA genes was also shut off rapidly after rifampin was added . The significance of these observations to the control of transcription of the hisH gene by tryptophan is discussed.

J Bacteriol, 1976 Oct, 128(1), 174 - 81
Pleiotropic, extragenic suppression of dna mutants in Bacillus subtilis; Siccardi AG et al.; Thermosensitive (dna) mutants of Bacillus subtilis defective in deoxyribonucleic acid replication can be divided into two groups on the basis of their ability to spontaneously yield secondary mutants with an HDS phenotype (thermoin-sensitivity and resistance to aryl-azo-pyrimidines) at frequencies higher than 10(-8) . Such a phenotype is due to alleles at the hds locus (mapping close to cysA), which act as extragenic pleiotropic suppressors . HDS suppressibility has been used as a screening tool to identify new dna strains among uncharacterized temperature-sensitive mutants.

Mol Biol (Mosk), 1976 Sep-Oct, 10(5), 1035 - 41
{DNA compact form in solution . VII . Transforming activity of precompact forms of DNA}; Akimenko NM et al.; Data on transforming activity of DNA in water-salt (0.3 M NaCl) solutions containing polyethylene glycol (PEG) are presented . Bacillus subtilis deficient mutant lys 42 and prototrophic strain SHgw were used as recipient and donor, respectively, for transformation experiments . It has been shown that when PEG concentrations were increased the relative experiments . It has been shown that when PEG concentrations were increased the relative transforming activity (RTA) at first (at PEG concentrations approximately 30-40 mg/ml) increased to the value of 200-250%, then began to decrease . At PEG concentrations approximately 100-125 mg/ml, when DNA molecules form compact particles, the RTA does not exceed 10% . The observed changes in RTA are interpreted as a result of conformational changes of double-stranded DNA molecules . The increase in the RTA at low PEG concentrations may be explained as an increase in transforming activity of the precompact form of DNA, while the decrease in RTA at PEG concentrations exceeding 60-70 mg/ml was believed to be connected with the fact that compact particles of DNA could not penetrate through the walls of bacterial cells.

Mikrobiologiia, 1976 Sep-Oct, 45(5), 864 - 8
{Inactivation of isogenous strains of Bacillus subtilis and their mixtures by ionizing radiation}; Samoilenko II et al.; Resistance to ionizing radiation of vegetative cells, spores and dehydrated cultures was studied with isogenous strains of Bacillus subtilis with defects in the repair systems . In vegetative cells of different classes of the recombination deficient mutants, DL90 was 31-35% of LD90 of the wild type cells . Differences in radiosensitivity of the vegetative cells were more pronounced cf . the spores and dehydrated cells: LD90 of rec- mutants was 55-88%; LD90 of uvr- mutants was 120% of LD90 of the parent bacterial cells.

Rev Bras Pesqui Med Biol, 1976 Sep, 9(4), 183 - 6
{Microbiological assay of some phenothiazine derivatives using different strains of Bacillus subtilis}; Fortes ZB et al.; This paper deals with the microbiological assay of chloropromazine, levomepromazine and promethazine, whose bacteriostatic power was measured in the diffusion test against four different strains of Bacillus subtilis . The four strains proved to be equally sensitive to solutions of phenothiazine derivatives containing more than 0.5 mg/ml . There was no difference between the four strains as referred to the median percentual error in the chlorpromazine determination . The median percentual error has been very high (around 20%) in the levomepromazine determination using Bacillus subtilis ATCC 9945 . When promethazine was used against the four strains of Bacillus subtilis a very high percentual error (around 20%) has been obtained, therefore, they are not recommended for the promethazine determination.

J Assoc Off Anal Chem, 1976 Sep, 59(5), 1122 - 24
Evaluation of two microbiological methods for detecting residual antibiotics in milk; Ouderkirk LA; Two methods described in the AOAC Official Methods of Analysis for the detection of penicillin residues in whole milk were evaluated to determine the capability of each method to detect residues of 12 antibiotics used in the dairy industry . The first method, a cylinder-plate method that uses Sarcina lutea as the test organism, detected levels of 1 ppm of 8 of the 12 antibiotics tested . The second method, using paper disks with Bacillus subtilis as the test organism, detected approximately 1 ppm of only 4 antibiotics . This disk method was unable to detect less than 40 ppm of 5 of the antibiotics tested . The data indicate that the S . lutea cylinder-plate technique is more sensitive to more antibiotics than the B . subtilis disk method and is far superior for screening purposes.

J Bacteriol, 1976 Sep, 127(3), 1502 - 18
Uptake and retention of metals by cell walls of Bacillus subtilis; Beveridge TJ et al.; Isolated walls of Bacillus subtilis Marburg, prepared in a manner which avoided metal contamination other than by the growth medium, were incubated in dilute metal solutions, separated by membrane filtration (0.22 mum), and monitored by atomic absorption to give uptake data for 18 metals . Substantial amounts of Mg2+, Fe3+, Cu2+, Na+, and K+ (amounts which were often visible as Au3+, and Ni2+ (the higher atomic-numbered elements also visible as electron scattering), and small amounts of Hg2+, Sr2+, Pb2+, and Ag+ were taken into the wall . Some (Li+, Ba2+, Co2+, and Al3+) were not absorbed . Most metals which had atomic numbers greater than 11 and which could be detected by electron microscopy appeared to diffusely stain thin sections of the wall . Magnesium, on the other hand, partitioned into the central region, and these sections of walls resisted ruthenium red staining, which was not true for the other metals . Areas of the walls also acted as nucleation sites for the growth of microscopic elemental gold crystals when incubated in solutions of auric chloride . Retention or displacement of the metals was estimated by a "chromatographic" method using the walls cross-linked by the carbodiimide reaction to adipic hydrazide agarose beads (which did not take up metal but reduced the metal binding capacity of the walls by ca . 1%) packed in a column . When a series of 12 metal solutions was passed through the column, it became evident that Mg2+, Ca2+, Fe3+, and Ni2+ were strongly bound to the walls and could be detected by both atomic absorption and by their electron-scattering power in thin sections, qhereas the other metals were fisplaced or replaced . Partial lysozyme digestion of the walls (causing a 28% loss of a {3H}diaminopimelic acid label) greatly diminished the Mg2+ retention but not that of Ca2+, Fe3+, or Ni2+, indicating that there are select sites for various cations.

J Bacteriol, 1976 Sep, 127(3), 1427 - 42
Autolytic enzyme-deficient mutants of Bacillus subtilis 168; Fein JE et al.; Mutants of Bacillus subtilis strain 168 have been isolated that are at least 90 to 95% deficient in the autolytic enzymes N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase . These mutants grow at normal rates as very long chains of unseparated cells . The length of the chains is directly related to the growth rates . They are nonmotile and have no flagella, but otherwise appear to have normal cell morphology . Their walls are fully sysceptible to enzymes formed by the wild type and have the same chemical composition as the latter . Cell wall preparations from the mutants lyse at about 10% of the rate of those from the isogenic wild type, with the correspondingly small liberation of both the amino groups of alanine at pH 8.0 and of reducing groups at pH 5.6 . Likewise, Microcococcus luteus walls at pH 5.6 and B . subtilis walls at pH 8 are lysed only very slowly by LiCl extracts made from the mutants as compared with rates obtained with wild-type extracts . Thus, the activity of both autolytic enzymes in the mutants is depressed . The frequencies of transformation, the isolation of revertants, and observations with a temperature-sensitive mutant all point to the likelihood that the pleiotropic, phenotypic properties of the strains are due to a single mutation . The mutants did not produce more protease or amylase than did the wild type . They sporulate and the spores germinate normally . The addition of antibiotics to exponentially growing cultures prevents wall synthesis but leads to less lysis than is obtained with the wild type . The bacteriophage PBSX can be induced in the mutants by treatment with mitomycin C.

J Bacteriol, 1976 Sep, 127(3), 1047 - 57
Genetic control of the glp system in Bacillus subtilis; Lindgren V et al.; In pleiotropic negative glycerol utilization mutants (GlpPI mutants) of Bacillus subitilis, glycerol kinase and sn-glycerol 3-phosphate (G3P) dehydrogenase are noninducible . GlpPI mutants also fail to take up exogenous {14C}G3P . To study the regulation of the glp system in B . subtilis phenotypically, Glp+ revertants were isolated from GlpPI mutants . Four classes of revertants were identified: phenotypically, wild type; R1 type, which contains an informational suppressor, R2 type, which produced G3P dehydrogenase constitutively; and R3 type, with a temperature-sensitive Glp phenotype producing G3P dehydrogenase constitutively at permissive temperature (32 degrees C) . The properties of the revertants indicate that the glpPI locus codes for a protein with a positive regulatory function.

Eur J Biochem, 1976 Aug 16, 67(2), 357 - 65
Reconstitution of reduced nicotinamide adenine dinucleotide oxidase activity with menadione in membrane vesicles from the menaquinone-deficient Bacillus subtilis aro D . Relation between electron transfer and active transport; Bisschop A et al.; Membrane vesicles from the menaquinone-deficient Bacillus subtilis aro D contain a low content of menaquinone and consequently oxidaze reduced nicotinamide adenine dinucleotide (NADH) at low rate . Supplementation of the membrane vesicles suspension with the menaquinone-analogue menadione, results in an incorporation of menadione in the membranes . The incorporated menadione increases with the external menadione concentration up to a maximum of 7 nmol of menadione bound per mg membrane protein . The NADH oxidase activity of the membrane vesicles increases linearly with the menadione content and a 35-fold stimulation is obtained in fully reconstituted membrane vesicles; this maximal NADH oxidase activity is about two-fold higher than the NADH oxidase activity in membrane vesicles from wild-type B.subtilis W23 . Supplementation of membrane vesicles from B.subtilis W23 with menadione also results in a stimulation of the NADH oxidase activity but only a stimulation of 1.6-fold is maximally obtained . The NADH oxidase activities in reconstituted B.subtilis aro D and B.subtilis W23 membrane vesicles are similarly affected by respiratory chain inhibitors, indicating that menadione occupies physiological sites of menaquinone . NADH and the non-physiological electron donor ascorbate + phenazine methosulphate are the best energy sources for active amino acid transport in membrane vesicles from B.subtilis W23 . Membrane vesicles from B.subtilis aro D accumulate amino acids in the presence of acorbate + phenazine methosulphate, but not with NADH . However, membrane vesicles from this mutant, reconstituted with menadione, demonstrate NADH-driven transport activity . This activity increases linearly with the NADH oxidase activity, but maximal transprt activities are reached under conditions where the NADH oxidase activity is not yet maximal . These results indicate that the rate of energy supply is the limiting factor for transport at low NADH oxidase activities and that the transport system itself becomes the limiting factor for transport at low NADH oxidase activities and that the transport system itself becomes the limiting factor under conditions of high NADH oxidase activities . Under energy-limiting conditions 135-235 molecules of NADH have to be oxidized in order to transport one molecule of amino acid . At all levels of energy supply a competition by the different amino acid transport systems for the available energy could not be observed . These observations indicate that only a fraction of the energy, generated by the respiratory chain, is used for the transport of an amino acid and that the bulk of the energy dissipates via other channels in the membrane vesicles.

Biotechnol Bioeng, 1976 Aug, 18(8), 1033 - 42
Immobilization of a cephalosporin acetylesterase by containment within an ultrafiltration device; Abbott BJ et al.; A cephalosporin acetylesterase produced by Bacillus subtilis catalyzes the deacetylation of 7-aminocephalosporanic acid (7-ACA) . Previous reports from our laboratory described the kinetic constants that characterize the reaction: Km = 2.8 X 10(-3)M, Kia acetate = 5 X 10(-2)M, and Kid deacetyl-7-ACA = 3.6 X 10(-2)M . These constants were used to predict the time course of the reaction using the following equation for dual competitive product inhibition . (see article) where St =mg/ml 7-ACA, At =mg/ml acetate, Dt =mg/ml deacetyl-7-ACA . The predicted time course closely matched the time course measured experimentally . The equation also was solved without the inhibition terms and the solution indicated that product inhibition caused about a 30% increase in the time required for complete (greater than 97%) hydrolysis of a 24 mg/ml 7-ACA solution . The esterase was immobilized by containment within an ultrafiltration device . With this technique the enzyme was reused 20 times over an 11 day span to deacetylate 7-ACA solutions containing 4 to 24 mg/ml 7-ACA . The specific activity after the 20th use was the same as the activity prior to the first use, indicating little enzyme inactiviation occurred.

Arch Microbiol, 1976 Aug, 109(1-2), 59 - 63
Some physiological functions of the L-leucine dehydrogenase in Bacillus subtilis; Obermeier N et al.; Mutants of Bacillus subtilis constitutive for L-leucine dehydrogenase synthesis were selected . Using these mutants we could determine two functional roles for the L-leucine dehydrogenase . This enzyme liberates ammonium ions from branched chain amino acids when supplied as the sole nitrogen source . Another function is to synthesize from L-isoleucine, L-leucine, and L-valine the branched chain alpha-keto acids which are precursors of branched chain fatty acid biosythesis . These results together with the inducibility of the enzyme suggest that the L-leucine dehydrogenase has primarily a catabolic rather than an anabolic function in the metabolism of Bacillus subtilis.

Arch Microbiol, 1976 Aug, 109(1-2), 195 - 7
Electron microscopy of isolated cell walls of Bacillus subtilis var . niger; Verwer RW et al.; Isolated cell walls of Bacillus subtilis have astriated appearance in the electron microscope . The structure persists when teichoic acids are removed . It is inferred that the structure bears on the arrangement of the peptidoglycan chains.

Arch Microbiol, 1976 Aug, 109(1-2), 181 - 6
{Relations between catabolite repression and sporulation in Bacillus subtilis (author's transl)}; Lopez JM et al.; Acetoin dehydrogenase can be catabolite repressed by numerous sources of carbon . The following results point out that the catabolite repression of this enzyme and the inhibition of sporulation are mediated by the same mechanism: 1 . Mutants, able to synthesize acetoin dehydrogenase in the presence of glucose, sporulate in glucose medium at a higher rate than the standard strain . 2 . The catabolite repressing effect of a compound and its ability to inhibit sporulation are in a direct relation to each other . 3 . The limitation of inorganic phosphate in the growth medium, which is known to favour sporulation, counteracts the catabolite repressing effect of glucose.

J Virol, 1976 Aug, 19(2), 518 - 32
Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: preliminary isolation and characterization of intermediate particles of the assembly pathway; Nelson RA et al.; Three classes of particles have been identified in restrictive phi 29 suppressor-sensitive (sus) mutant infections of Bacillus subtilis, including DNA-containing heads or phage, prohead, and empty heads . Pulse-chase labeling experiments indicate that the prohead, the first particle assembled in 14-infected cells, is converted to DNA-filled heads and phi 29 . In addition to the proteins Hd, P10, and F found in mature phi 29, the prohead contains a "core" protein P7 that exits as the prohead matures and appears to recycle during subsequent rounds of prohead assembly . Prohead-like structures accumulate in UV-irradiated cells and are present in restrictive infections with sus mutants of cistrons 9 and 16 . Empty heads are observed only when infection results in the formation of DNA-containing particles; this and other evidence indicates that the empty heads are probably not true intermediates . Phage phi 29 assembly apparently occurs by a single pathway in which neck and tail components interact to stabilize the completed DNA-containing head.

J Virol, 1976 Aug, 19(2), 501 - 17
Analysis of gene function of bacteriophage phi 29 of Bacillus subtilis: identification of cistrons essential for viral assembly; Hagen EW et al.; Restrictive infection of Bacillus subtilis by suppressor-sensitive (sus) mutants of phi 29 has been used to search for cistrons that function in viral assembly . The products of cistrons 7, 9, 10, and 16 are necessary for head morphogenesis . The neck upper collar protein P10 and the tail protein P9 must be present for DNA packaging to occur . The protein P7 must be present for phage-related particles to form . A prohead-like particle has been isolated during 16-restrictive infection . The particle is composed of the proteins Hd, P10, F, and P7 . P16 must function for DNA-filled particles to accumulate . A DNA-containing particle produced in the absence of the cistron 11 product may be an intermediate in the phi 29 assembly pathway . The protein P13 interacts with P9 and P11 to form a stable DNA-filled particle . The products of cistrons 2 and 3 are essential for viral DNA synthesis, and in their absence virus-related particles are not detected.

J Virol, 1976 Aug, 19(2), 495 - 500
Genetic analysis of bacteriophage phi 29 of Bacillus subtilis: integration and mapping of reference mutants of two collections; Mellado RP et al.; Reference mutants of Bacillus subtilis phage phi 29 of the Madrid and Minneapolis collections were employed to construct a genetic map . Suppressor-sensitive and temperature-sensitive mutants were assigned to 17 cistrons by quantitative complementation . Three-factor crosses were used to assign an unambiguous order for the 17 cistrons . Recombination frequencies determined by two-factor crosses were used to construct a linear genetic map of 24.4 recombination units . The genes were numbered sequentially from left to right (1 to 17) according to their relative map position.

J Virol, 1976 Aug, 19(2), 359 - 73
DNA strand specificity of temporal RNA classes produced during infection of Bacillus subtilis by SP82; Lawrie JM et al.; The DNA of the Bacillus subtilis bacteriophage SP82 has been separated into heavy (H) and light (L) fractions by centrifugation in buoyant density gradients in the presence of polyguanylic acid . Competition-hybridization experiments were performed with these separated fractions using RNAs isolated from cells labeled at intervals which account for 80% of the lytic cycle and unlabeled competitor RNAs isolated from phage-infected cells at 2-min intervals throughout infection . The analysis of temporal RNA classes were facilitated by use of a double reciprocal plot of the data . Five temporal classes binding to the H fraction and three binding to the L fraction were detected; the possible existence of an additional class transcribed from the H fraction is discussed . RNA synthesized in the presence of chloramphenicol contains two of the three classes produced from L-DNA and two of the five classes transcribed from H-DNA.

J Virol, 1976 Aug, 19(2), 338 - 45
Enzymatic degradation of uracil-containing DNA . II . Evidence for N-glycosidase and nuclease activities in unfractionated extracts of Bacillus subtilis; Duncan J et al.; Further studies have confirmed our earlier observations that in the presence of EDTA, degradation of phage PBS2 {3H}uracil-labeled DNA is effected by an N-glycosidase activity in extracts of Bacillus subtilis that removes free uracil from DNA . In addition, such extracts contain a nuclease activity that attacks PBS2 DNA in the presence of CaCl2 . The nuclease activity is not observed under conditions that inactivate N-glycosidase activity but does attack DNA that has been preincubated to remove uracil by N-glycosidase action . We therefore postulate that the nuclease requires N-glycosidase action to generate substrate for its activity, i.e., the nuclease appears to attack depyrimidinated sites rather than uracil sites in phage PBS2 DNA.

J Bacteriol, 1976 Aug, 127(2), 956 - 60
Cell wall assembly in Bacillus subtilis: development of bacteriophage-binding properties as a result of the pulsed incorporation of teichoic acid; Archibald AR; Addition of a pulse of excess phosphate to a phosphate-limited culture of Bacillus subtilis W23 resulted in the synthesis and incorporation of wall material that contained teichoic acid . Consequently, the bacteria regained the ability to bind phage SP50 although maximum phage-binding properties did not develop until approximately half a generation time after incorporation of teichoic acid had ceased . The present findings strongly support our earlier suggestion that newly synthesized receptor material is incorporated at the inner surface of the wall and becomes exposed at the outer surface only during subsequent growth.

J Bacteriol, 1976 Aug, 127(2), 934 - 40
Ethanol sensitivity of sporulation in Bacillus subtilis: a new tool for the analysis of the sporulation process; Bohin JP et al.; The growth rate of Bacillus subtilis is lowered but the final cell yield is unchanged when certain concentrations of ethanol are present in the culture medium . At the concentration allowing growth at half-maximal rate, practically no spores are formed . Blockage of spore formation generally occurs at stage 0-I . Sensitivity to ethanol of the capacity to form spores is limited, in a nonsynchronized culture, to a period of at most 45 min around t1 . Postexponential events such as excretion of certain enzymes and modification of ribonucleic acid polymerase are altered or suppressed in the presence of ethanol, possibly as the results of a physical change upon the cell membrane . In effect, ethanol is turning wild-type cells into phenocopies of spoO mutants.

J Bacteriol, 1976 Aug, 127(2), 817 - 28
Bacillus pumilus plasmid pPL10: properties and insertion into Bacillus subtilis 168 by transformation; Lovett PS et al.; Bacillus pumilus ATCC 12140 harbors 10 or more copies per chromosome of each two small plasmids . Variants of this strain were isolated which were sensitive to a killing activity produced by the plasmid-containing parent . Each of 24 such sensitive (S) variants tested lacked detectable levels of supercoiled deoxyribonucleic acid . Transduction of S variants to the Kill+ phenotype was performed using phage PBP1 propagated on a mutant of ATCC 12140, designated strain L10, that remained Kill+ but retained only a single plasmid species (plasmid pPL10; molecular weight, approximately 4.4 X 10(6), approximately 20 copies per chromosome; RHO = 1.698) . Resulting Kill+ transductants of S variants contained a single plasmid species having a size and copy number comparable to that of pPL10 . Transfer of pPL10 from strain L10 TO B . pumilus strain NRS 576 was accomplished by transduction with selection for the Kill+ phenotype . Strain NRS 576 naturally harbors about two copies per chromosome of a 28-million-dalton plasmid, pPL576 . In Kill + transductants of NRS 576, plasmids pPL10 and pPL576 stably coexisted at a ratio of about 11 molecules of pPL10 to 1 molecule of pPL576 . Therefore, pPL576 and pPL10 are compatible plasmids . B . subtilis 168 is naturally resistant to pPl10- determined killing activity . Plasmid pPl10 was therefore inserted into B-subtilis 168 by transformation, using an indirect selection procedure and a spoB mutant as recipient . The plasmid is stably maintained at an estimated 10 copies per chromosome in the spore- recipient and in spore+ transformants . pPL10 is sensitive to cleavage by the endonucleases Hind III and EcoR1.

J Bacteriol, 1976 Aug, 127(2), 803 - 11
Characterization of the N-acetylmuramic acid L-alanine amidase from Bacillus subtilis; Lindsay B et al.; The N-acetylmuramic acid L-alanine amidase from Bacillus subtilis W-23 has been purified to apparent homogeneity . The enzyme is a monomer of molecular weight 51,000, which binds extremely tightly to homologous cell walls but not to heterologous cell walls, even of the closely related strain B . subtilis ATCC 6051 . This difference in binding is only in part due to differences in teichoic acid between these two strains and to a large extent appears to represent differences in the arrangement of the peptidoglycan . A comparison of the amidase from B . subtilis W-23 and the enzyme previously purified from B . subtilis ATCC 6051 (Herbold and Glaser, 1975) shows that the two proteins, which cleave the same bond and are of the same size, do not cross-react immunologically and that the two enzymes are, therefore, not closely related in structure.

J Bacteriol, 1976 Aug, 127(2), 679 - 90
Bacillus subtilis mutant temperature sensitive in the synthesis of ribonucleic acid; Riva S et al.; A Bacillus subtilis temperature-sensitive mutant (PB1653) has been isolated in which the rate of ribonucleic acid (RNA) synthesis sharply decreases after shift to 45 degrees C . Both stable and unstable RNAs are affected by the mutation . The possibility that the block of transcription at high temperature could be due to a "stringent" effect, mediated by an increase in the concentration of "magic spot" nucleotides, has been ruled out . Treatment with chloramphenicol (or streptomycin) rapidly restores the rate of RNA synthesis at 45 degrees C . The synthesis of RNA in the mutant during the early phases of spore germination is not temperature sensitive . The phage-specific transcription during infection with SPP1 phage, at high temperature, is less affected than that of the bacterial chromosome . In vitro experiments indicate that, in the mutant at high temperature, RNA polymerase undergoes a change in template specificity . The rna-53 mutation has been located on the B . subtilis genetic map near the hisA locus.

Tijdschr Diergeneeskd, 1976 Aug 1, 101(15), 849 - 53
{Drug residues in poultry (author's transl)}; Jaartsveld FH et al.; The following microbiological tests: the Bacillus subtilis BGA (Bundes-Gesundheitsamt) test and Sarcina lutea test as well as the test bacteria Bacillus stearothermophilus var . calidolactis and E . coli were used to examine whether drug residues were detectable in broiler chickens during and after treatment with various drugs . With the exception of the occidiostat Esb3, residues were not found to be present using the two above tests . On the other hand, residues of a number of drugs were detected in some interior organs and faeces during treatment when the other test bacteria were used . Residues were no longer detected in the faeces, however, within twelve hours after treatment had been discontinued.

Mutat Res, 1976 Aug, 38(4), 271 - 86
The sporulation system of Bacillus subtilis as the basis of a multi-gene mutagen screening test; Macgregor JT et al.; The sporulation system of B . subtilis provides the basis of a simple and unique test for the detection of forward mutations in any of several hundreds genes in 28--45 separate operons scattered throughout the chromosome . Non-sporulating or oligosporogenous mutant colonies are easily identified by their lack of a brown pigment normally present in spore-forming colonies . N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate, acridine orange, acriflavin, nitrous acid, and UV irradiation are already known to produce sporulation mutants . This paper reports the dose dependence of sporulation mutant induction by 2-nitrosofluorene, ICR-191, nitrogen mustard, ethidium bromide and MNNG; mutagenesis is also demonstrated for aflatoxin B1 and 4-nitroquinoline-N-oxide . A mammalian liver enzyme metabolizing system was necessary for activation of aflatoxin B1 . Auramine-O and 4-nitro-o-phenylenediamine failed to give a significant mutagenic effect under the conditions employed . The wide variety of mutagen classes detected indicates the general applicability of the test . This test, based on many genes throughout the chromosome, may prove less apt to exclude rare mutagenic "hot-spots" than systems based on the detection of mutations in a restricted region of the chromosome.

J Bacteriol, 1976 Aug, 127(2), 739 - 46
Manganese requirement of phosphoglycerate phosphomutase and its consequences for growth and sporulation of Bacillus subtilis; Oh YK et al.; In the absence of manganese, rapidly metabolizable carbohydrates such as glucose or glycerol are not completely metabolized by Bacillus subtilis growing in a nutrient sporulation medium: 3-phosphoglyceric acid (3PGA) accumulates inside the cells, growth stops at a low cell titer, and normal sporulation remains suppressed (no prespore septa) . Upon the addition of manganese, 3PGA disappears, growth resumes, and normal sporulation takes place . These effects results from a specific manganese requirement of phosphoglycerate phosphomutase which catalyzes the interconversion of 3PGA and 2-phosphoglyceric acid (2PGA) . Other metal ions cannot replace manganese, for which the enzyme has an apparent Km of 0.22 mM.

Jpn J Microbiol, 1976 Aug, 20(4), 281 - 6
Purification and properties of glucose-6-phosphate dehydrogenase from Bacillus subtilis spores; Tanahashi T et al.; Glucose-6-phosphate dehydrogenase {D-glucose-6-phosphate: NADP oxidoreductase, EC . 1 . 1 . 1 . 49} obtained from spores of Bacillus subtilis PCI 219 strain was partially purified by filtration on Sephadex G-200, ammonium sulfate fractionation and chromatography on DEAE-Sephadex A-25 (about 54-fold) . The optimum pH for stability of this enzyme was about 6.3 and the optimum pH for the reaction about 8.3 . The apparent Km values of the enzyme were 5.7 X 10(-4) M for glucose-6-phosphate and 2.4 X 10(-4) M for nicotinamide adenine dinucleotide phosphate (NADP) . The isoelectric point was about pH 3.9 . The enzyme activity was unaffected by the addition of Mg++ or Ca++ . The inactive glucose-6-phosphate dehydrogenase obtained from the spores heated at 85 C for 30 min was not reactivated by the addition of ethylenediaminetetraacetic acid, dipicolinic acid or some salts unlike inactive glucose dehydrogenase.

Eur J Biochem, 1976 Jul 15, 66(3), 485 - 91
Phosphorylation of intracellular fructose in Bacillus subtilis mediated by phosphoenolpyruvate-1-fructose phosphotransferase; Delobbe A et al.; Intracellular fructose provided by the sorbitol pathway in Bacillus subtilis can be phosphorylated by the phosphenolpyruvate-1-fructose phosphotransferase which is known to mediate a vectorial metabolism . The fate of this intracellular fructose was studied using mutants lacking either the fructose 1-phosphate pathway or the fructose 6-phosphate pathway . It was shown that the phosphoenolpyruvate-dependent phosphorylation needs a prior exit of the sugar into the medium, this exit being probably catalysed by a transport system . A low affinitiy intracellular phosphenolpyruvate phosphotransferase system was found, which seems to be devoid of a physiological role.

Mol Gen Genet, 1976 Jul 5, 146(1), 85 - 7
Mapping the uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and ferrochelatase loci in Bacillus subtilis; Miczak A et al.; Three genes hemE, hemF, hemG taking part in the porphyrin biosynthesis of Baccillus subtilis were mapped by two- and three-factor transduction crosses . The gene hemE determines uroporphyrinogen decarboxylase (EC 4.1.1.37) the gene hemF coproporphyrinogen oxidase (EC 1.3.3.3) and the gene hemG ferrochelatase (EC 4.99.1.1) enzymes . The loci hemE, hemF, hemG, are not linked to hemA locus and located near the argC and metD loci.

Mikrobiologiia, 1976 JUL-AUG, 45(4), 733 - 5
{Effect of Ca2+ ions on late stages of genetic transformation in Bacillus subtilis}; Belova MM et al.; Calcium chloride stimulated later stages of genetic transformation after irreversible binding of DNA in the cells of Bacillus subtilis . The composition of a solid medium with CaCl2 for selection after transformation is presented.

Nord Vet Med, 1976 Jul-Aug, 28(7-8), 377 - 80
The problem of testing horse kidneys for the presence of antibiotics at meat inspection: how to avoid a false positive reaction; Korkeala H et al.; When 33 horse kidneys were tested for the presence of inhibitory substances by the Bacillus subtilis BGA method at pH 8 and the Micrococcus luteus ATCC 9341 method, 24 were positive and 9 negative . The pH of the seeded M . luteus test medium changed from pH 6.6 before incubation to 8.7 after 24 hours incubation at 30 degrees C . When the same 33 kidneys were tested by the B . subtilis BGA method, medium pH 6, and 15 of them also by the M . luteus method using a medium buffered to pH 6, all were negative . The cadmium concentration of the 33 horse kidneys was found to be 70.17 +/- 81.28 mg/kg wet weight (m +/- s, range 10.40-355.67) . The authors attributed the positive results to the presence of cadmium . The diffusion of cadmium from the horse kidney samples to the test media was found to differ at pH 6 and pH 8 . It is recommended that the testing of horse kidney samples for the presence of antibiotics and chemoterapeutic substances be done at substrate pH 6 to avoid false positive reactions.

Mol Biol Rep, 1976 Jul, 2(6), 439 - 43
Isolation of a DNA-protein complex containing a single DNA fragment which is at or near the replication origin of the Bacillus subtilis chromosome; Yamaguchi K et al.; A chromosomal fragment containing purA, the nearest marker from the replication origin of the Bacillus subtilis chromosome, was highly purified as a complex containing at least proteins and being solubilized easily during cell lysis . The complex had a markedly higher sedimentation rate (70-120S) than the bulk of the solubilized DNA (40S) . The electron microscopic observation showed the complex to be an aggregate of several DNA molecules with a local structure containing amorphous materials which stained black and bushes of RNA . This confirmed biochemical evidences suggesting that the complex is an intermolecular aggregate of the purA-DNA-protein-RNA complex.

J Virol, 1976 Jul, 19(1), 26 - 35
Behavior of a temperate bacteriophage in differentiating cells of Bacillus subtilis; Osburne MS et al.; During the first 6 hr of sporulation, infection of Bacillus subtilis by by phi105 wild type or the clear-plaque mutant phi105 c30 was nonproductive, but phage DNA was trapped inside developing spores . After infection with either wild-type or mutant phage at early times of sporulation (T1-T3), phage DNA entered the developing spores in a heat-stable form, which may represent integration of the phage DNA into the host chromosome . Phage DNA in carrier spores produced by infection at later times (T4-T6) was much more heat sensitive . Spore preparations containing either phi105 wild type or phi105 c30 carrier spores gave rise to a spontaneous burst of phage during outgrowth, although the fraction of carried wild-type phage that chose lysis over lysogeny at germination has not been determined . Heat induction of the thermoinducible lysogen 3610 (phi105 cts23) was also abortive during sporulation . Furthermore, induction neither prevented eventual spore formation nor resulted in the conversion of prophage DNA to the carrier state; during outgrowth, the previously induced lysogenic spores remained stable lysogens . However, if the sporulating lysogenic cells were plated immediately after induction, they did not form colonies at high efficiency, as though transfer to fresh medium allowed sufficient phage expression to kill the host.

Eur J Biochem, 1976 Jul 1, 66(2), 229 - 41
Bacillus subtilis phage phi29 . Characterization of gene products and functions; Carrascosa JL et al.; A total of 22 phi29-induced proteins have been resolved by slab gel electrophoresis; two of these proteins are the precursor and product fragment, respectively, in the synthesis of the neck appendage protein of the phage . The protein products of 10 out of the 17 cistrons detected in the genome of phage phi29 have been identified . Mutants in two other cistrons fail to synthesize two proteins . Mutants in six genes do not synthesize phage DNA . A cistron, probably involved in the final lysis of the infected bacteria, has been found . Mutants in this gene give place, under restrictive conditions, to delayed lysis and produce, after artificial lysis, a burst size similar or higher than that obtained after wild-type phage infection.

J Bacteriol, 1976 Jul, 127(1), 667 - 70
Uptake of branched-chain alpha-keto acids in Bacillus subtilis; Goldstein BJ et al.; Bacillus subtilis has a constitutive system for the uptake of alpha-keto-beta-methylvalerate, alpha-ketoisovalerate, and (probably) alpha-ketoisocaproate . A mutation, kauA1, which blocks the uptake of alpha-keto-beta-methylvalerate and alpha-ketoisovalerate, is located between metB and citK on the B . subtilis chromosome.

J Bacteriol, 1976 Jul, 127(1), 433 - 9
Segregation of Bacillus subtilis chromosomes radioactively labeled during the first round of replication after germination of spores; Wake RG; Spores of Bacillus subtilis W23 thy his were allowed to incorporate {3H}thymine for short periods of time either continuously from, or soon after, the start of the first round of replication after germination . They were then transferred to nonradioactive medium to allow growth into microcolonies (up to 12 cells), which were autoradiographed . The relative numbers of various types (major versus minor) of grain clusters associated with individual microcolonies throughout the populations were scored . Analysis of the results showed clearly that, in the majority of spores at least, only one chromosome was undergoing replication soon after the start of deoxyribonucleic acid synthesis . Furthermore, under the conditions used, no evidence for initiation of replication of a second chromosome within 25 min after the first could be obtained . Accepting that B . subtilis spores are essentially homogenous in deoxyribonucleic acid content, the results support the conclusion that the spore contains only one copy of the chromosome, not two.

J Bacteriol, 1976 Jul, 127(1), 268 - 80
Study of tyrosine transfer ribonucleic acid modification in relation to sporulation in Bacillus subtilis; Menichi B et al.; A reversal in the relative amounts of the two major species of tyrosine transfer ribonucleic acid (tRNATyr) (I and II) has been previously observed by others during the development of Bacillus subtilis . These species have been purified by benzoylated diethylaminoethyl-cellulose chromatography and were shown to differ by the modification of an adenosine residue (species I contains i6A and species II ms2i6A) . As suggested by competitive hybridization assays, they might possess the same nucleotide sequence . A tRNATyr species lacking isopentenyl and methylthio moieties was not detected . The structural difference between species I and II was shown to be important for ribosome binding but not for charging . The extent of alteration during growth was studied in parallel with physiological events . Like sporulation, tRNATyr change is iron dependent . Moreover, when sporulation is prevented by an excess of glucose, the tRNATyr change is delayed as is the synthesis of enzymatic systems required for the onset of sporulation . tRNATyr change also demands unceasing protein synthesis.

J Bacteriol, 1976 Jul, 127(1), 258 - 67
Modified nucleosides of Bacillus subtilis transfer ribonucleic acids; Vold B; An analysis of the kinds and amounts of minor nucleosides of transfer ribonucleic acids (tRNA's) from Bacillus subtilis 168 trpC2 is presented . Identification and quantitation were accomplished using ion exclusion chromatography, thin-layer and paper chromatography, and ultraviolet absorption properties . Nucleosides and their amount in moles per 80 residues are as follows: guanosine (25.7), cytidine (22.0), adenosine (15.2), uridine (13.1), 5-methyluridine (0.98), pseudouridine (1.54), 1-methyladenosine (0.15), N6-methyladenosine (0.01), 7-methyladenosine (0.10), 2-methyladenosine (0.03), 7-methylguanosine (0.20), N2-methylguanosine (0.14), 1-methylguanosine (0.14), a methylated pyrimidine (0.17), a methylated derivative of N6-(delta 2-isopentenyl)adenosine (0.02), ribose methylated nucleosides (0.02), 4-thiouridine (0.12), 2-thio-5-(N-methylaminomethyl) (0.09), and an unknown thionucleoside (0.12) . Although the composition is similar to that of Escherichia coli in the proportion of major nucleosides, the content of pseudouridine and 5-methyluridine, and the degree of base and ribose methylation, the composition is more similar to that of the tRNA's of yeast and higher organisms in its lower degree of thiolation, the presence of significant amounts of 1-methyladenosine, and the low levels of 2-methyladenosine and 6-methyladenosine . Therefore, the nucleoside composition of B . subtilis presents some different aspects from those usually given as characteristic for bacterial tRNA's . It is not known whether these differences are due to variation between bacterial species in general or related to the process of differentiation.

Mutat Res, 1976 Jul, 40(3), 177 - 84
Studies on selenium-related compounds . V . Cytogenetic effect and reactivity with DNA; Nakamuro K et al.; Five selenium compounds, Na2Se04, H2Se04, Na2Se03, H2Se03 and Se02, were tested for their capacity to induce chromosome aberrations in cultured human leukocytes and for their reactivity with DNA by a rec-assay system and inactivation of transforming activity in Bacillus subtilis . Chromosome-breaking activity was significantly higher for the compounds with four-valent than with six-valent selenium, the efficiency being in the decreasing order H2S03 greater than Na2Se03 greater than Se02 greater than H2Se04 greater than Na2Se04 . Rec assay using B . subtilis with different recombination capacities suggested that damage to DNA was produced by selenites but not by selenates . The reactivity of selenites with DNA was also indicated by a significant loss of transformation of the tryptophan marker of B . subtilis DNA treated with H2Se03 and Se02.

Biochim Biophys Acta, 1976 Jun 18, 435(2), 109 - 18
Purification and characterization of tRNAMet-f, tRNAPhe and tRNATyr2 from Baccillus subtilis; Raettig R et al.; Three tRNAs specific for methionine, phenylalanine and tyrosine were isolated from the total tRNA of Bacillus subtilis by chromatographic procedures using BD-cellulose and reversed-phase (5) chromatography . The acceptor activities of the purified tRNAs are 1160, 1260 and 1320 pmoles per A260nm unit for tRNAMetf, tRNAPhe and tRNATyr2 respectively . In tRNAMetf and tRNAPhe ribothymidine, pseudouridine and dihydrouridine are present, in addition, in tRNAPhe 7-methyguanosine and a 2'-O-methylated nucleoside were found . The modified nucleosides of tRNATyr2 are ribothymidine, pseudouridine, dihydrouridine, 4-thiouridine and 1-methyladenosine . The results suggest the presence of 2-methylthio-N6(delta 2-isopentenyl)adenosine in tRNAPhe and tRNATyr2 . The thermal denaturation profiles of the three tRAN species are presented.

Biochim Biophys Acta, 1976 Jun 18, 435(2), 128 - 31
Activity of stringent protein in ribosomes of Escherichia coli during the growth cycle; Ramagopal S; To investigate whether the stringent protein in Escherichia coli was lost from ribosomes at certain phase of growth as in other organisms (Bacillus subtilis, mouse embryo), cells growing at various phases of the growth cycle were harvested and ribosomes tested for activity . The results showed that in E . coli, stringent protein was associated with the ribosomes throughout the growth cycle . The peak activity for the synthesis of guanosine tetra- and pentaphosphates appeared around midlogarithmic phase.

Mol Gen Genet, 1976 Jun 15, 145(3), 293 - 302
Macromolecular synthesis after a nutritional shift-up of Bacillus subtilis; Murakami S et al.; Effects of amino acids of macromolecular synthesis in Bacillus subtilis were studied . Two mutants, CRK4001 and NIG45, that were selected as slow growers in rich media were proved to be useful to analyse early events occurring after addition of amino acids to exponentially growing cells in a glucose-salts medium (nutritional shift-up) . In a wild type strain, the rate of stable RNA (sRNA: essentially ribosomal RNA) synthesis increased 2.3 fold shortly after the shift-up to the rate characteristic of the post-shift steady state growth . In contrast,sRNA synthesis in the mutant strains responded to the shift-up in two steps . Thus, shortly after the shift the rate of sRNA synthesis increased 2.2 fold as in the wild type but this increased level was maintained temporarily for 60 min and suddenly decreased to the post-shift steady state rate (1.4 fold increase) . On the other hand, rates of DNA synthesis increased some 30 min after the shift directly to the post-shift steady state rates in all strains . Ratios of an origin to a terminus marker (purA/metB) began to increase exponentially to reach maximum values at 60 min after the shift, indicating that initiation of DNA replication occurred at frequencies characteristic of respective post-shift growth rates soon after the shift . These results revealed that the initial increase in the rate of sRNA synthesis and the frequency of initation of DNA replication after nutritional shift are not related to each other and are independently affected by amino acids . In concert with these findings, the increase in sRNA synthesis was not affected by inhibition of DNA synthesis for the first 60 min after the shift, while it was completely prevented by puromycin and choramphenicol . Protein synthesis for 10 min after the shift was sufficient to fully change the sRNA synthesis rate by amino acids.

Science, 1976 Jun 11, 192(4244), 1141 - 3
Chloramine mutagenesis in Bacillus subtilis; Shih KL et al.; Chloramine (which occurs widely as a by-product of sanitary chlorination of water supplies) is shown to be a weak mutagen, when reversion of trpC to trpC in Bacillus subtilis is used as an assay . Some DNA-repair mutants appear to be more sensitive to chloramine, suggesting the involvement of DNA targets in bactericide . The influence of plating media on survival of cells treated with chloramine suggests a bacterial repair system acting upon potentially lethal lesions induced by chloramine.

J Biol Chem, 1976 Jun 10, 251(11), 3480 - 8
Nucleotide sequence of 5 S ribosomal RNA precursor from Bacillus subtilis; Sogin ML et al.; The complete nucleotide sequence of a 179-nucleotide precursor (p5A) of 5 S ribosomal RNA from Bacillus subtilis is presented . In addition to the 116-nucleotide mature segment, the p5A molecule contains 21 additional nucleotides at its 5' end and 42 at its 3' terminus . Structural features within the p5A molecule which possibly interact with a specific maturation endonuclease (RNase M5) are identifiable . These include 2-fold rotational symmetry about the cleavage sites, which may approximately place the p5A molecule on the RNase M5 surface, and translational (positional) symmetry, which may precisely orient substrate phosphodiester bonds with respect to catalytic amino acids . Certain features of the 3' -terminal, precursor-specific portion of p5A possibly are involved in the termination of DNA transcription.

J Antibiot (Tokyo), 1976 Jun, 29(6), 611 - 7
U-43,120; a new antitumor antibiotic . I . Production, biological activity, microbiological assay and taxonomy of the producing microorganism; Hanka LJ et al.; A new antitumor antibiotic, U-43,120 was discovered . It is produced by fermentation of a new species of Streptomyces, designated Streptomyces paulus DIETZ sp . n . Its antimicrobial activity is limited to bacteria . A microbiological assay with Bacillus subtilis was developed that can be detect concentrations of 1 approximately 2 mug/ml of the drug in fermentation liquors . U-43,120 was active in vivo against P-388 leukemia in mice.

J Bacteriol, 1976 Jun, 126(3), 1342 - 3
Appearance of spore coat protein in the cell extracts of Bacillus subtilis asporogenic mutants; Uchida A et al.; By use of the antigen-antibody techniques we have studied whether asporogenic mutants of Bacillus subtilis can synthesize the spore coat protein . Antibody specific to spore coat protein was prepared and used to demonstrate that the spore coat protein was synthesized at the early stage of sporulation . We report here that asporogenic mutants synthesize the spore coat protein.

J Bacteriol, 1976 Jun, 126(3), 1285 - 96
Cellular organization of Bacillus subtilis: sodium dodecyl sulfate-induced cell partitioning into zebra structures; Mendelson NH et al.; Cells of Bacillus subtilis heated in high concentrations of sodium dodecyl sulfate (5%) and then washed free of detergent with a hot salt solution (80 C) become structurally reorganized into regions of densely compacted cytoplasm (termed zebras) and regions of sparsely filled material (termed spaces) . Size distribution studies of zebras indicate that division-suppressed mutants and wild-type cells both yield zebras of comparable length . Similarly the lengths of zebras found in populations emerging from spores are uniform in one-, two-, three-, and four-zebra-containing cells . In contrast, the length of spaces is slightly larger than that of zebras and is unusually large in two-zebra-containing cells . The locations of zebras and spaces along cell length have been studied in spore out-growth populations . A statistical procedure developed previously in genome location investigations was used to analyze the location of zebras along cell length . The data indicate that as cells elongate, new sites arise where the cell contents are strongly bound to the cell surface . Within filament populations produced by division-suppressed mutants there is a linear relationship of mean filament length and zebra number per filament . These data indicate that cytoplasm in filaments with no obvious structural compartmentalizations may be organized into units associated with particular regions of cell surface . The attachment of cell contents to the cell surface may involve deoxyribonucleic acid . Zebra-containing cells digested with proteolytic enzyme and ribonuclease are converted to cells that contain a crystalline-like granule fixed at the location of each zebra . Exposure to deoxyribonuclease mobilizes these granules within the cell wall.

J Bacteriol, 1976 Jun, 126(3), 1037 - 41
Repair deficiency, mutator activity, and thermal prophage inducibility in dna-8132 strains of Bacillus subtilis; Sadaie Y et al.; A ts mutation, dna-8132 (Hara and Yoshikawa, 1973), in the region of chromosome replication origin of Bacillus subtilis was found to cause pleiotropic effects at a permissive temperature (30 C) . Strains carrying this mutation were lethan at 48 C but exhibited higher spontaneous mutation frequency and a lower capacity for repairing radiation damages at 30C . Introduction of the polA59 (Gass et al., 1971) mutation further enhanced the repair deficiency and the mutator activity . These results suggest that the dna-8132 gene product may be directly involved in chromosome replication and repair . SPO2 lysogens carrying this mutation produced mature phages upon a temperature shift from 30 to 48 C . Phage production at nonpermissive temperature suggests that there are few defects in the precursors of deoxyribonucleic acid synthesis in the mutant.

Proc Natl Acad Sci U S A, 1976 Jun, 73(6), 2151 - 5
Fusion of bacterial protoplasts; Schaeffer P et al.; Prototrophic Bacillus subtilis cells can be formed in the presence of DNase as a result of cell fusion occurring in mixed populations of protoplasts derived from two parental strains which are both nutritionally-complementing and polyauxotrophic . No prototrophs ever appear from mixed nonprotoplasted bacteria, or from the auxotrophic parental protoplasts plated separately . The frequency of prototroph formation, which is appreciable only when the mixed protoplasts are exposed to polyethylene glycol treatment, may exceed 1 X 10(-4) of the total protoplast population initially present, which is 1 to 4 X 10(-3) of those protoplasts which reverted to the bacillary form . It is strongly dependent on the number and chromosomal location of the markers used in the selection of the prototrophs, and it is unaffected when either one of the parental strains bears the phage phi105 in the inducible prophage state . No auxotrophic bacteria, parental or otherwise, were found as segregants from repeatedly isolated protrotrophic clones growing in a nonselective medium . Unselected markers segregate among the selected recombinants . It is concluded that the observed formation of prototropic bacteria is due to protoplast fusion, a process which does not induce prophage development, and that the only stable products of the resulting diploid state are haploid recombinants.

Proc Natl Acad Sci U S A, 1976 Jun, 73(6), 1816 - 20
Altered penicillin-binding components in penicillin-resistant mutants of Bacillus subtilis; Buchanan CE et al.; Penicillin- (cloxacillin-) resistant mutants of Bacillus subtilis were isolated in a stepwise fashion and the five penicillin-binding components (PBCs) in each were examined to determine which of the proteins, if any, corresponds to the penicillin killing site . PBCs II and V were previously eliminated as the likely penicillin targett . In the present work, PBC IV showed no change in sensitivity to cloxacillin in any of the resistant mutants isolated . PBC I did not change until the fifth-step mutant, in which it could not be detected by penicillin binding . Since PBC I did not bind penicillins that are lethal for this mutant, it also cannot be the lethal target . PBC II showed increased resistance to cloxacillin in three discrete steps, i.e., in mutants 1, 4, and 5, accompanied by changes in its electrophoretic mobility . However, the sensitivity of PBC II to penicillin G changed very little . Correspondingly, the cloxacillin-resistant mutants were unaltered in their sensitivity to penicillin G in vivo . Thus, of the five PBCs found in B . subtilis, PBC II is the most likely target for killing by penicillins.

J Hyg (Lond), 1976 Jun, 76(3), 355 - 66
A source isolator for infected patients; Babb JR et al.; A plastic, mechanically ventilated source isolator with filters in the air effluent was designed to enable infected patients to be nursed and treated in a general ward or to be transported without risk to staff or other contacts . Two models of isolator were developed . Their potential value was tested by the challenge of heavy dispersal, inside the isolator, of bacteria (a) from patients with burns, during the change of dressings, (b) from contaminated bedding during simulated bed-making, and (c) from the dispersal of a suspension of Bacillus subtilis var . globigii . Sampling of air by slit samplers outside the isolator and, in comparable control patients, from the air of the room in which dressings were changed, showed consistently lower counts of bacteria and of Staph . aureus during dressings when the isolator was used; on removal of the isolator canopy there was, in some experiments, a considerable increase in airborne bacteria, due to residual bacteria in the isolator of to the re-dispersal of bacteria which settled on the patient and his bedding during the dressing . Simultaneous sampling with slit samplers inside and outside the isolator during and after bed-making or dispersal of B . subtilis var . globigii showed an almost complete protection of the air outside the isolator against contamination by bacteria released inside the isolator.

J Virol, 1976 Jun, 18(3), 839 - 47
Genetic and physiological studies of abortive infections of hydroxymethyluracil-containing bacteriophages in lysogens of temperate Bacillus subtilis bacteriophage SPO2; Friedman SO et al.; Wild-type bacteriophage phie and IS (interference-sensitive) mutants of the related phage SP82G did not productively infect strains of Bacillus subtilis that were lysogenic for temperate phage SPO2 . In these abortive infections, the sensitive phages adsorbed to and penetrated the nonpermissive host, phage-directed macromolecular syntheses were initiated, but both viral and bacterial nucleic acid production abruptly stopped about 15 min after addition of the phages . The cessation of RNA and DNA synthesis was preceded or coincident with a reduction in oxygen utilization by the infected cultures . Genetic studies of both phie and SP82G suggest sensitivity to SPO2-mediated abortive infection was controlled by a single gene . A mutant of SPO2, SPO2ehp4-, lysogens of which no longer interfere with the development of SP82GIs, was also isolated . The discovery of this ehp- variant suggests the normal SPO2 prophage synthesized a substance that alters cell physiology in some manner detrimental to SP82GIs development . Since SPO2ehp4- grew on and lysogenized bacteria sensitive to wild-type SPO2, the product of the eph gene was apparently not an essential function of this temperate phage.Overall, these observations exhibit remarkable similarities to the inhibition of T4rII mutants by the product of the rex gene of phage lambda.

Cancer Res, 1976 Jun, 36(6), 2040 - 5
Aflatoxin B1 metabolism to aflatoxicol and derivatives lethal to Bacillus subtilis GSY 1057 by rainbow trout (Salmo gairdneri) liver; Schoenhard GL et al.; Aflatoxicol, R0, was isolated from Mt . Shasta strain rainbow trout (Salmo gairdneri), and liver homogenates were incubated with aflatoxin B1 . Its identity was confirmed by mass, infrared, and ultraviolet spectrometry . The structure was identical to one of the diastereomers prepared by chemical reduction of aflatoxin B1 . Aflatoxicol was apparently formed by a reduced nicotinamide adenine dinucleotide phosphate-dependent soluble enzyme of the 105,000 x g supernatant from rainbow trout . Aflatoxicol was not lethal in phosphate buffer to Bacillus subtilis GSY 1057 (metB4, hisA1, uvr-1) nor were aflatoxins B1, Q1, and B2 . In the presence of reduced nicotinamide adenine dinucleotide phosphate and trout liver microsomes, aflatoxicol reduced the viability of B . subtilis . Aflatoxin B2, which lacks the vinyl ether present in the other compounds, could not be activated . The product of aflatoxin B1 activation by trout liver microsomes was sought after incubation of 14C-labeled aflatoxin B1 . The radioactivity was found in unaltered aflatoxin B1 and in three extremely polar metabolites . The quantity of the new metabolites and the level of microbial lethality was reduced by addition of cytosine and cysteine to the incubation medium . The vinyl ether configuration was a structural requirement for activation, and this finding and the nature of the enzymatic reaction were consistent with the hypothesis that the compounds were metabolized to highly reactive and unstable electrophilic products which bound to nucleophiles such as cytosine and were lethal to B . subtilis . The formation of aflatoxicol as the major product of trout liver metabolism is of great significance considering that it could be activated to a lethal compound and that rainbow trout are one of the most sensitive species to aflatoxin B1-induced carcinoma.

Eur J Biochem, 1976 May 17, 65(1), 213 - 23
Isolation of a strong suppressor of nonsense mutations in Bacillus subtilis; Mellado RP et al.; By treatment of Bacillus subtilis MO-101-P spoA- met thr- su- with ethyl methanesulfonate, a strong suppressor strain of nonsense mutations, B . subtilis MO-101-P spoA- {met-}+thr- su+44, was isolated . This strain does not suppress phage phi 29 mutant susB47, selected on a B . subtilis strain containing the su+3 suppressor isolated by Georgopoulos . A revertant from this mutant, susB610, was isolated, being suppressed by both the su+3 and su+44 suppressor strains . The efficiency of suppression by strain su+44 is about 50% . The experiments shown in this paper suggest that strain su+44 contains an amber and strain su+3 an ochre suppressor.

Eur J Biochem, 1976 May 17, 65(1), 3 - 12
Exo-beta-N-acetylmuramidase--a novel hexosaminidase . Production by Bacillus subtilis B, purification and characterization; del Rio LA et al.; Exo-beta-N-acetylmuramidase, or beta-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-D-glucoside acetamidodeoxyglucohydrolase, is produced by Bacillus subtilis B, growing in a succinate/peptone/salts medium, at the end of exponential growth and occurs partly in the medium and partly bound to the cells . A lysozyme digest of Micrococcus lysodeikticus cell walls, O-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 leads to 4)-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-D-glucose and O-{2-acetamide-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl}-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose in decreasing order of efficiency, induce the enzyme but O-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose does not do so . The enzyme was purified from the growth medium, after removal of the cells by continuous centrifugation, by ammonium sulphate precipitation, continuous filtration through XM-300 membranes (to remove the high-molecular-weight material which renders the enzyme sedimentable in low-ionic-strength solutions), diafiltration through PM-30 membranes and ion-exchange chromatography on DEAE-Sephadex and CM-Sephadex . Two peaks of activity were obtained . Peak A was purified 1800-fold and was homogenous on polyacrylamide disc gel electrophoresis . A second heterogeneous fraction (peak B) was also collected . Exo-beta-N-acetylmuramidase is most stable at pH 8.0 and has a molecular weight of about 90000 . The results of studies on its ability to attack several synthetic and natural substrates are given . The Km and V values for 4-methylumbelliferyl-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucose and O-{2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl}-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose are respectively 0.19 and 0.65 mM and 1.50 and 16.29 mumol min(-1) mg(-1) . From these results and those of inhibition studies it is concluded that the enzyme is specific for substrates with non-reducing N-acetylmuramic acid end groups . Possible roles for this enzyme are discussed.

Biochemistry, 1976 May 4, 15(9), 1838 - 43
Evidence that both growing DNA chains at a replication fork are synthesized discontinuously; Sternglanz R et al.; Escherichia coli, Bacillus subtilis, and T7-infected E . coli have been labeled with short pulses of {3H} thymidine, and the labeled DNA has been examined by sedimentation in alkaline sucrose . In all three systems, the great majority of the DNA labeled by a short pulse is found in the form of small DNA chains of 10S, the so-called Okazaki pieces . The B . subtilis and T7 nascent DNA fragments hybridize with equal efficiency to the separated strands of B . subtilis and T7 DNA, respectively . The results suggest that both growing DNA chains at a given replication fork are synthesized discontinuously in the case of E . coli, B . subtilis, and T7 . We have found that the method used to terminate the pulse affects the size distribution of the labeled DNA; some methods allow joining of nascent DNA fragments after termination of the pulse . Previous reports of discontinuous DNA synthesis on only one growing DNA chain and continuous synthesis on the other DNA chain are probably due to preferential joining of Okazaki pieces on the DNA chain growing in the overall 5' leads to 3' direction.

J Bacteriol, 1976 May, 126(2), 914 - 8
Cortex content of asporogenous mutants of Bacillus subtilis; Imae Y et al.; A method for the measurement of muramic lactam, which is specifically located in the cortical peptidoglycan of bacterial spores, was developed as a quantitative assay method for spore cortex content . During sporulation of Bacillus subtilis 168, muramic lactam (i.e., spore cortex) began to appear at state IV of sporulation and continued to increase over most of the late stages of sporulation . Spore cortex contents of various spo mutants of B . subitils were surveyed . Cortex was not detected in mutants in which sporulation was blocked earlier than stage II sporulation . Spores of spo IV mutant had about 40% of the cortex content of the wild-type spores . One spo III mutant had a low amount of cortex, but four others had none.

Cell, 1976 May, 8(1), 103 - 14
The program of protein synthesis during sporulation in Bacillus subtilis; Linn T et al.; The program of protein synthesis was examined during sporulation in Bacillus subtilis as an index of the control of gene expression . At various stages of growth and spore formation, cells of B . subtilis were pulse-labeled with (35)S-methionine . Protein was extracted from the radioactively labeled bacteria and then subjected to high resolution one-dimensional and two-dimensional slab gel electrophoresis . We report that sporulating cells restricted or "turned off" the synthesis of certain polypeptides characteristic of the vegetative phase of growth . In certain cases, this "turn off" was prevented in a mutant (SpoOa-5NA) blocked at the first stage of spore formation . Sporulating bacteria also elaborated new polypeptide species that could not be detected in vegetatively growing cells or in cells of the asporogenous mutant SpoOa-5NA in sporulation medium . The synthesis of these sporulation-specific proteins was "turned off" in a temporally defined sequence throughout the period of spore formation . Spore coat protein, for example, was first synthesized at 4 hr after the onset of sporulation, the time at which refractile prespores appeared . Certain sporulation-specific polypeptides including the coat protein were among the most actively produced polypeptides in sporulating cells.

Nucleic Acids Res, 1976 May, 3(5), 1249 - 62
Changes in transfer ribonucleic acids of Bacillus subtilis during different growth phases; Singhal RP et al.; The transfer ribonucleic acids (tRNAs) of B . subtilis at different growth phases are examined for changes in the composition and the methylation of minor constituents . The composition of the tRNAs indicates about equal amounts of adenosine and uridine, and of guanosine and cytidine . About 3-4 residues are present as modified bases in the average tRNA molecule . The net composition of tRNAs appears to remain unaltered during different growth phases . In vitro methylation of tRNAs indicates lack of methyl groups in both exponentially growing cells and spores . In vivo methylation studies show tRNA methylation occurs during the stationary phase in the absence of net tRNA synthesis . Thus, both in vitro and in vivo methylation indicate that the tRNAs in exponentially growing cells do not contain their full complement of modified bases . More complete modification is noted in tRNAs from stationary cells or spores . Hence, tRNA mofifications in general are preserved with fidelity even in the dormant spore but the possibility is left open that specific modifications of selected isoacceptors of tRNAs may occur.

Proc Natl Acad Sci U S A, 1976 May, 73(5), 1740 - 4
Helical growth of Bacillus subtilis: a new model of cell growth; Mendelson NH; A multiple mutant of Bacillus subtilis that grows in an unusual double-helix morphology was studied . Construction of models led to the assumption that cell surface elongation must proceed in a helical path in this mutant . The observation that all newly formed double-helix clones propagated, after spore outgrowth in fluid culture, consisted of closed-circular structures suggested that double-helix structures are tension-registered forms.

J Bacteriol, 1976 May, 126(2), 928 - 36
Use of temperature-sensitive mutants to study gene expression during sporulation in Bacillus subtilis; Young M; Two temperature-sensitive sporulation mutants have been characterized . One mutant, which is blocked at stage II, has a short temperature-sensitive period that occurs at about the time when the spore septum is formed . Cells can escape the sporulation block, if incubated for a short period at the permissive temperature, but are prevented from doing so by inhibitors of transcription and translation; this suggests that the product of the defective gene is a protein and that the messenger ribonucleic acid which codes for this protein is short-lived . The other mutant is blocked at stage IV to V and has a long temperature-sensitive period that starts during stage III and precedes the stage at which the mutational defect is phenotypically expressed . The behavior of this mutant in temperature-shift experiments suggests that synthesis of the product of the defective gene commences long before it assumes its physiological function.

J Bacteriol, 1976 May, 126(2), 706 - 11
Control of tumbling in bacterial chemotaxis by divalent cation; Ordal GW; Chemotaxis is migration of organisms to higher concentrations of attractant or lower concentrations of repellent . Understanding the switch than controls whether the flagella rotate counterclockwise for swimming or clockwise for tumbling (thrashing about without making much forward progress) is central to understanding chemotaxis of peritrichous bacteria, since chemotaxis results from selective suppression of tumbles . Depletion of divalent cation by chelating agents in the presence of A23187, an ionophore that conveys divalent cation across membrane, causes incessant tumbling in Bacillus subtilis . Small additions of MgCl2 prevent this tumbling . In this tumbling condition, the bacteria which normally swim extensively when given attractant, do not respond even to 10 mM alanine, a strong attractant . MnCl2, by contrast to others potentiated by the ionophore . Permanent cations, including tetraphenylarsonium ion and triphenylmethylphosphonium ion, cause permanent swimming, even in the presence of A23187 and chelating agents . We propose that divalent cation, probably Mg2+ ion, binds to the switch to cause swimming and that, in the absence of divalent cation at the switch, the bacterium tumbles.

J Bacteriol, 1976 May, 126(2), 609 - 18
Differences in the genetic structure of Bacillus subitilis strains carrying the trpE26 mutation and strain 168; Trowsdale J et al.; It was previously shown that in strains of Bacillus subtilis bearing the trpE26 mutation a chromosome segment (from trpD to ilvA) is translocated to a position near the thr region . Further PBS1-mediated transduction data have now revealed that these strains also possess an inversion of part of the chromosome from the origin of replication, down to the tre locus on one side and the cysB locus on the other . These data concern evidence of linkage of tre-12- to markers in the translocation (hisH2, tyrA1, and metB3) as well as linkage of the cysB3 marker to thi-86, gly-133, and catA . They explain the previously observed absence of linkage of markers in the translocated segment to cysB3 . The model proposed for the formation of merodiploids in trpE26 strains, which calls for the fusion of two genetic elements, is not incompatible with this new finding.

J Bacteriol, 1976 May, 126(2), 699 - 705
An enzyme common to histidine and aromatic amino acid biosynthesis in Bacillus subtilis; Nester EW et al.; Two transaminases exist for tyrosine and phenylalanine synthesis in Bacillus subtilis . One enzyme is also responsible for the transamination of imidazole acetol phosphate to histidinol phosphate, an obligatory reaction in the synthesis of histidine . The gene involved in the synthesis of this enzyme lies in the middle of a cluster of genes, all of which are concerned with the synthesis of the aromatic amino acids . The other gene has not yet been mapped . Mutants have been isolated that lack one or the other enzyme activity . These mutants are prototrophic for tyrosine and phenylalanine . However, both classes of mutants are more sensitive than the wild-type strain to the phenylalanine analogue, fluorophenylalanine, suggesting that each of these mutants synthesizes less phenylalanine than does the wild-type strain . The two enzymes can be separated from one another by ion-exchange chromatography and glycerol-gradient centrifugation . The significance of the observation that an enzyme of histidine synthesis also plays a role in the synthesis of the aromatic acids is considered in light of cross-pathways regulation between the two pathways.

Biochim Biophys Acta, 1976 Apr 23, 428(2), 516 - 24
Control of teichoic and teichuronic acid biosynthesis in Bacillus subtilis 168trp . Evidence for repression of enzyme synthesis and inhibition of enzyme activity; Rosenberger RF; Phosphate starvation induced teichuronic acid synthesis in cells of Bacillus subtilis 168trp-which had previously been grown with excess phosphate . This induction was prevented when protein systhesis was inhibited immediately prior to phosphate starvation and under these conditions cells continued to form teichoic acid . The converse was true when phosphate was added to cells previously grown in a phosphate-limited chemostat . The increase in teichoic acid synthesis normally following phosphate addition was prevented by chloramphenicol or amino acid starvation and cells continued to make teichuronic acid . This suggestion that repression of enzyme synthesis is involved in controlling the type of wall polymer made was supported by the low levels of UDP-glucose dehydrogenase found in cells grown with excess phosphate and of CDP-glycerol pyrophosphorylase in phosphate-limited cells . The greater amounts of teichoic acid made under phosphate limitation and of teichuronic acid with excess phosphate when protein synthesis was also inhibited indicated that modulation of enzyme activity occurs . Glycerol starvation of a glycerol-requiring mutant did not derepress teichuronic acid synthesis, indicating that glycerol-containing imtermediates do not act as repressors.

Eur J Biochem, 1976 Apr 15, 64(1), 243 - 7
Role of calcium ions in the thermostability of thermolysin and Bacillus subtilis var . amylosacchariticus neutral protease; Tajima M et al.; The stabilizing effect of calcium ions on thermolysin and Bacillus subtilis var . amylosacchariticus neutral protease has been investigated . Calcium and zinc ions were removed from the proteases by gel filtration over Sephadex G-25 equilibrated with metal chelating agents . Using these enzymes with different metal content, heat inactivation kinetics were studied at various temperatures . Removal of calcium ions caused a sharp decrease in thermostability and diminished the values of the activation enthalpy (deltaH*) and entropy (deltaS*) for heat inactivation . There was little difference in stability between thermolysin containing 0.3 g-atom/mol and B . subtilis neutral protease containing 1.4 g-atoms/mol . Calcium binding isotherms of the proteases were obtained by equilibrium gel chromatography with various concentrations of free calcium ions . Thermolysin had four independent calcium binding sites with an identical intrinsic binding constant (K) of 2.0 X 10(4) M-1 . B . subtilis neutral protease had four independent sites . The K value for three sites was 1.1 X 10(5) M-1 and the binding constant for the other site was 1.5 X 10(3) M-1 . There was little difference in total free energy change for calcium binding between these proteases . From these results it is concluded that the stabilizing effect of calcium on these enzymes is almost equal, and the extra thermal stability of thermolysin is likely to come from its polypeptide chain structure.

Eur J Biochem, 1976 Apr 15, 64(1), 205 - 13
Studies on the control of development . Correlation of initiucleotides in Bacillus subtilis; Rhaese HJ et al.; Unusual highly phosphorylated nucleotides are found in sporulating cells of Bacillus subtilis . Adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp (highly phosphorylated nucleotide I), and adenosine 3(2')-dephosphate 5'-triphosphate, pppApp (highllls are starved for carbon and nitrogen sources . These nucleotides are correlated with sporulation because only ribosomes from sporulating but not vegetative cells are able to synthesize ppApp and pppApp in vitro . Two other nucleotides, adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp (highly phosphorylated nucleotide IV), and a nucleotide with a tentative structure of ppZpUp (highly phosphorylated nucleotide III), where Z is an undetermined sugar, also seem to be involved in regulation of sporulation, especially initiation of sporulation . Sporulation can be initiated even in the presence of amino acids, salts, vitamins etc . in logarithmically growing or stationary-phase cells when carbon sources, i.g . glucose, are used up or artifically removed from the medium . A drastic increase in spore titer is observed 4--5 h later . Also, carbon starvation causes accumulation of the highly phosphorylated nucleotides pppAppp and ppZpUp . On the other hand, sporulation is prevented under the same conditions when excess glucose is maintained in the medium . Correlated with this inhibition of sporulation is the inhibition of formation of highly phosphorylated nucleotides, pppAppp and ppZpUp . Since synthesis of these nucleotides is closely related to sporulation, we anticipate that these substances can cause initiation of development in B . subtilis . Further evidence for our hypothesis on initiation of sporulation by highly phosphorylated nucleotides is that phosphate starvation also causes sporulation with prior accumulation of pppAppp and ppZpUp . Apparently, as long as phosphate is present to synthesize phosphorylated metabolites of glucose, formation of highly phosphorylated nucleotides is repressed . Derepression occurs when either lack of glucose or phosphate or both prevents synthesis of phosphorylated metabolites of glucose allowing synthesis of highly phosphorylated nucleotides . These nucleotides, representing the signal 'lack of glucose or phosphate', then somehow cause changes in gene activity, initiating the complex process of sporulation . Whether or not pppAppp alone or together wtih ppZpUp or even further substances (nucleotides, proteins etc.) is necessary for the above described processes will be answered with the help of suitable mutants lacking the ability to synthesize either one or both regulatory nucleotides . Guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp, are not involved in regulation of devlopment as is shown by using a normally sporulating mutant of B . subtilis, unable to synthesize these nucleotides.

Biochim Biophys Acta, 1976 Apr 15, 432(1), 37 - 48
Inhibition of Bacillus subtilis DNA polymerase III by arylhydrazinopyrimidines . Novel properties of 2-thiouracil derivatives; Wright GE et al.; 6-(p-Tolylhydrazino)-uracil, 6-(p-tolylhydrazino)-isocytosine and 6-(p-tolylhydrazino)-2-thiouracil were synthesized and compared with respect to their chemical properties, their activity as inhibitors of DNA polymerase III of Bacillus subtilis, and their capacity to induce the formation of a complex between polymerase III and template DNA . As expected from earlier studies of analogous hydroxyphenylhydrazino compounds, the effects of the uracil derivative were reversed specifically by dGTP and those of the isocytosine derivative were reversed specifically by dATP . In contrast, reversal of the effects of the thiouracil derivative required both dGTP and dATP . The unique capacity of the 2-thiouracil analog to mimic either purine deoxyribonucleotide appears to reside in its ability to undergo tautomerism between the 2-thione and 2-thiol forms, which can pair with, respectively, template cytosine and thymine.

J Bacteriol, 1976 Apr, 126(1), 520 - 3
Characteristics of Bacillus stearothermophilus mutants blocked in catabolic function; Rowe JJ et al.; With polyacrylamide disc gel electrophoresis and specific staining, it was demonstrated that one mutation involving the alcohol dehydrogenase of a double mutant of Bacillus stearothermophilus 1503 apparently prevented enzyme synthesis, and another lesion in the same organism resulted in synthesis of an inactive form of aconitase . Some properties of the double mutant and two fumarase mutants are discussed in relation to similar mutants derived from Bacillus subtilis.

J Bacteriol, 1976 Apr, 126(1), 72 - 9
Recognition sites for chemotactic repellents of Bacillus subtilis; Ordal GW; Repellents of Bacillus subtilis include many membrane-active compounds, such as uncouplers of oxidative phosphorylation, local anesthetics, chlorpromazine (a central nervous system depressant), and tetraphenylboron (a lipophilic anion) . Normally, bacteria swim smoothly, and occasionally tumble, but addition of repellent causes all bacteria to tumble, then later resume original frequency of swimming and tumbling (adaptation) . Bacteria adapted to repellent can then be tested to determine the minimum concentration (threshold) of the same or different repellents that causes tumbling . The results indicate that repellents act at (saturable) recognition sites, which differ for chemically different species . An implication is that uncouplers of oxidative phosphorylation affect cell properties by interaction at specific locations.

J Bacteriol, 1976 Apr, 126(1), 429 - 38
Purification and properties of a manganese-stimulated endonuclease from Bacillus subtilis; Scher B et al.; An endonuclease stimulated by manganese or calcium ions was isolated from Bacillus subtilis . This enzyme attacked double- or single-stranded deoxyribonucleic acid from a variety of sources, including B . subtilis, and was purified from the material released into the medium during protoplast formation . The enzyme appeared as a single peak after glycerol gradient centrifugation and comprised approximately 30 to 35% of the protein in the most purified preparations, as estimated by gel electrophoresis . It had a molecular weight of about 46,000 . The mode of action of the enzyme was endonucleolytic, and circular deoxyribonucleic acid was readily cleaved . The enzyme introduced a limited number of both double- and single-strand breaks into native deoxyribonucleic acid, generally yielding products of 1 X 10(6) daltons or more in size . The reasons for this limitation of cleavage were not clear . The activity of the enzyme was inhibited by low levels of Cu2+, Co2+, Hg2+, and Zn2+ . It was also inhibited by high concentrations of NaCl . A role for this enzyme in bacterial transormation is suggested.

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