J Bacteriol, 1976 Apr, 126(1), 384 - 99 Nuclear and cell division in Bacillus subtilis: cell development from spore germination; Iterson WV et al.; The changes in the morphology of the nucleoids and the mesosomes in Bacillus subtilis cells during synchronous outgrowth after spore germination were followed in large-scale three-dimensional cell reconstructions . Shortly after outgrowth of the cell begins in Spizizen medium with glucose, the mesosome becomes an elongated structure in close contact with a rounded nucleoid . When nuclear replication reaches full activity, the mesosome develops into a single, complicated versatile system, with tubules that traverse the cytoplasm and have elaborations in and near the nucleoplasm . Later the system may retract to form large rounded mesosomes; the tubules and strings of vesicles within these mesosomes probably have been collected from the cytoplasm . Shortly after the first cell division, both sister cells have two nucleoids, but with longer generation times induced by growth in media containing acetate instead of glucose; these sister cells have only one nucleoid each . In acetate-grown cells rounded nucleoids that have no contact with a mesosome may represent nucleoids in a temporary stage of rest . On the other hand, the nucleoids of cells growing in glucose-containing medium are always penetrated by mesosomal material, superficially or deeply . Since the mesosome appears capable of traversing the nuclear fibrils, and even reaching the last strands connecting the dividing nucleoids, it is suggested that this organelle may play a vital role in the Bacillus division cycle. J Bacteriol, 1976 Apr, 126(1), 294 - 9 Undermethylated transfer ribonucleic acid from a relaxed strain of Bacillus subtilis: construction of the strain and analysis of the transfer ribonucleic acid; Keisel N et al.; A strain of Bacillus subtilis is described from which undermethylated transfer ribonucleic acid (tRNA) can be obtained . The tRNA's from a methionine-limited culture were compared with those from a control culture with respect to general nucleoside composition, methylated components, and amino acid acceptor activity . The undermethylated tRNA's had the normal amounts of the four major nucleosides, pseudouridine, and 5-methyluridine (ribothymidine), but were deficient in methylated nucleosides other than 5-methyluridine . These methyl-deficient nucleosides can be fully remethylated in the presence of the appropriate methylases . Since the majority of the work characterizing undermethylated tRNA's has been done using Escherichia coli, the work with B . subtilis presents some interesting comparisons and offers an alternative substrate for methylase studies. J Bacteriol, 1976 Apr, 126(1), 108 - 21 Effect of deoxyribonucleic acid replication inhibitors on bacterial recombination; Canosi U et al.; Two inhibitors of replicative deoxyribonucleic acid (DNA) synthesis, nalidixic acid (NAL) and 6-(p-hydroxyphenylazo)-uracil (HPUra), showed different effects on genetic recombination and DNA repair in Bacillus subtilis . Previous work (Pedrini et al., 1972) showed that NAL does not interfere with the transformation process of B . subtilis . The results reported in this work demonstrated that the drug was also without effect on the transfection by SPP1 or SPO-1 phage DNA (a process that requires a recombination event) . The drug was also ineffective on the host cell reactivation of ultraviolet-irradiated SPP1 phage, as well as on transfection with ultraviolet-irradiated DNA of the same phage . HPUra instead markedly reduced the transformation process, as well as transfection, by SPO-1 DNA, but it did not affect the host cell reactivation of SPO-1 phage . In conclusion, whereas the NAL target seems to be specific for replicative DNA synthesis, the HPUra target (i.e., the DNA polymerase III of B . subtilis) seems to be involved also in recombination, but not in the excision repair process . The mutations conferring NAL and HPUra resistance used in this work were mapped by PBS-1 transduction. Eur J Biochem, 1976 Apr 1, 63(2), 391 - 8 {The structure of mycosubtilin, an antibiotic isolated from Bacillus subtilis (author's transl)}; Peypoux F et al.; Mycosubtilin, an antifungal agent isolated from Bacillus subtilis is a mixture of homologous lipopeptides essentially C54H83N14O16 and C55H85N15O16 . The differences in their structures was found in the lipid moiety which contains several beta-amino acids; the structure of these beta-amino acids was demonstrated by combined gas chromatography-mass spectrometry of the N-trifluoroacetyl n-butyl esters; a strong peak at m/e = 240 indicates a beta-amino group . The comparison of the derivatives of natural amino acids with synthetic 3-aminohexadecanoic and 3-aminoheptadecanoic acids indicates that natural beta-amino acids are a mixture of 3-amino-14-methylpentadecanoic acid (35%), 3-amino-14-methylhexadecanoic acid (59%), 3-aminohexadecanoic acid (6%) and a trace of C18 beta-amino acid . The peptide moiety contains 8 moles of amino acids, two of D-aspargine, two of L-asparagine, and one of L-glutamine, L-proline, D-serine and D-tyrosine . The peptide sequence was determinated by partial acid hydrolysis of mycosubtilin and isolation and structural determination of the peptides from the hydrolysates . Four liposoluble peptides and four hydrosoluble peptides were studied . The results gave the cyclic structure shown on Formula 1 for mycosubtilin. Can J Microbiol, 1976 Apr, 22(4), 592 - 7 Activities of alpha-ketoisovalerate, pyruvate, and alpha-ketoglutarate dehydrogenases in a mutant of Bacillus subtilis; Tu CL et al.; An acetate-requiring leaky mutant was induced from Bacillus subtilis 168, and activities of its three alpha-keto acid dehydrogenases were compared with the respectives activities of the parent strain . Both pyruvate and alpha-ketoisovalerate dehydrogenase activities in the mutant were consideralby lower, being only 10-17% of those of the parent, but alpha-ketoglutarate dehydrogenase activity was unchanged . These dehydrogenases are complexed composed of three enzymes: a carboxylase, a lipoic reductase-transacylase, and a dihydrolipoyl dehydrogenase . The carboxylase activity of the affected complexes was no different . Total dihydrolipoyl dehydrogenase activity was only one-third . Thus dihydrolipoyl dehydrogenase is the defective enzyme in the two dehydrogenase complexes; the activity remaining in the mutant is accounted for by the activity of the intact alpha-ketoglutarate dehydrogenase. Arch Microbiol, 1976 Apr 1, 107(3), 303 - 7 Influence of several nucleotides on the competence development of Bacillus subtilis; Urena MT et al.; The influence of adenosine-3',5'-cyclic monophosphate (cAMP) and other nucleotides on the competence development of Bacillus subtilis was studied . The stimulation of competence which can be achieved by exposing physiologically low-competent cells to supernatants from highly competent cultures can be inhibited with different cAMP doses . When the same cells were suspended in a minimal medium with cAMP, varying degrees of stimulatin- of competence were observed depending on the time of addition of the drug . This effect is not specific for cAMP . It appears to be correlated to an increase of the amount of DNA bound to the competent cells . cAMP activities were antagonized by equimolar doses of adenosine-triphosphate (ATP) and guanosine-triphosphate (GTP). Mol Gen Genet, 1976 Mar 30, 144(3), 323 - 31 A four-stranded DNA from Bacillus subtilis which may be an intermediate in genetic recombination; Kohnlein W et al.; DNA of Bacillus subtilis strain UVSS 19--8M, of high ultraviolet sensitivity, was isolated after cultivating in medium containing bromouracil . Isopycnic banding in CsC1 shows an unusual pattern with four bands, including an extra one halfway between those for hybrid and for DNA of this band, amounting to 15--25% of the total DNA mass in one preparation, was isolated and investigated . The characteristics found for this DNA are in agreement with a four-stranded DNA unit similar to one of the structures postulated by Holliday as intermediates during genetic recombination . UVSS 19--8M from which this DNA has been isolated is shown to be defective for transformation and transfection, and can be regarded as rec-. Mol Gen Genet, 1976 Mar 30, 144(3), 313 - 21 Mapping of the gene specifying DNA polymerase III of Bacillus subtilis; Love E et al.; polC, the gene specifying the structure of the replication-specific DNA polymerase III of B . subtilis, was mapped by exploiting azp-12, a mutation conferring resistance to azopyrimidine which determines a mutant, azopyrimidine-resistant enzyme . azp-12 was located in the area of the pyrA locus and is between spcB1 and recA1 . azp-12 was linked by transformation to four other mutations which influence the in vitro behaviour of DNA polymerase III--polC25, polC26, mut-1(ts), and DNAF133; the close linkage of these five mutations strongly suggests that they are alleles of the same gene. Mol Gen Genet, 1976 Mar 30, 144(3), 235 - 41 Thiostrepton-resistant mutants of Bacillus subtilis: localization of resistance to the 50S subunit; Pestka S et al.; A number of thiostrepton-resistant mutants of Bacillus subtilis were obtained . The thi mutations map proximally to strA . Effects of thiostrepton on polyphenylalanine synthesis with ribosomes of S-100 fractions from parent and mutant strains indicated that resistance was localized to the ribosomes . Furthermore, effects of thiostrepton on binding of {3H}GTP to ribosomes and 50S subunits from thiostrepton-sensitive and -resistant strains localized the site of resistance to the 50S subunit . In addition, revertants from thiostrepton-resistance to thiostrepton-sensitivity were obtained . Ribosomes and 50S subunits from these thiostrepton-sensitive revertants were sensitive to thiostrepton similar to parental sensitive B . subtilis. Mol Gen Genet, 1976 Mar 30, 144(3), 231 - 3 A micrococcin-resistant mutant of Bacillus subtilis: localization of resistance to the 50s subunit; Smith I et al.; The 50S subunit is the site of action of the antibiotic micrococcin . In addition, B . subtilis strain mic-1, which is resistant to micrococcin, contains altered 50S subunits. Eur J Biochem, 1976 Mar 16, 63(1), 53 - 63 Transcription from complementary deoxyribonucleic acid strands in various sporogenic and asporogenic mutants of Bacillus subtilis . Hybridization-competition studies on ribonucleic acid synthesized in vivo by a thermosensitive sporulation mutant (ts-4); Bonamy C et al.; The present paper describes an investigation, at the transcription level, in a thermosensitive sporulation mutant of Bacillus subtilis (ts-4) grown at the permissive (30 degrees C) or restrictive (42 degrees C) temperature where sporulation capacity is respectively expressed or arrested . These studies were carried out by analysing the ribonucleic acid from vegetative and stationary phase cells (t3 cells) grown under both conditions, by hybridization-competition experiments. Biochim Biophys Acta, 1976 Mar 11, 429(1), 191 - 7 Characterization of intracellular esterase A from Bacillus subtilis; Riefler JF 3rd et al.; Esterase A (EC 3.1.1.1) obtained by sonic disruption of Bacillus subtilis SR22 (spoA12, trpC2) was purified approximately 400-fold by differential chemical and heating precipitation, DEAE-cellulose chromatography, and Bio-Rad P-150 gel filtration chromatography, with an overall yield of 59% . The purified enzyme hydrolyzed both aliphatic and aromatic acetate esters at substrate concentrations of 0.25 M but did not hydrolyze amino acid esters . Aliphatic alcohols did not inhibit the hydrolysis of p-nitrophenyl acetate; the most potent inhibitors of esterase activity were mercuric chloride, diisopropylfluorophosphate, eserine, and sodium fluoride. J Biol Chem, 1976 Mar 10, 251(5), 1311 - 25 Purification and characterization of DNA polymerase III from Bacillus subtilis; Low RL et al.; DNA polymerase III from Bacillus subtilis has been purified about 4,500-fold . Disc gel electrophoresis of the purified fraction reveals a single major protein band which co-migrates with the polymerase activity . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polymerase yields a single, 166,000 dalton band . The hydrodynamic properties of the enzyme are ionic strength-dependent . The average values from determinations in high and low salt are 7.6 S for the sedimentation coefficient and 52 A for the Stokes radius . These two parameters indicate a molecular weight for the native enzyme of 160,000 . Therefore, the enzyme appears to contain a single, long, polypeptide chain . The enzyme has no endonuclease activity but does have single strand specific exonuclease activity . Hydrolysis is initiated exclusively from the 3' terminus yielding 5' mononucleotides, and a dinucleotide is the limit of digestion . The exonuclease activity has an ionic strength dependence of pH optimum similar to that of the polymerase but appears to be more fastidious in its divalent metal requirement . The mode of attack by the enzyme is strictly distributive . The activity of the exonuclease decreases markedly with increasing substrate size . Two opposing mechanisms account quantitatively for this effect--intrinsic competitive inhibition by interior substrate nucleotides and increasing accessibility of the substrate terminus to the enzyme with increasing chain length . The polymerase synthesizes DNA in the 5' leads to 3' direction and the apparent Km for each of the deoxyribonucleoside triphosphates is about 1 muM . The polymerase replicates RNA-primed, phiX174 DNA in the presence of Escherichia coli elongation Factors I and II . In contrast to polymerase III, B . subtilis DNA polymerase II has no detectable nuclease activity. J Antibiot (Tokyo), 1976 Mar, 29(3), 303 - 8 The mode of action of ASK-753 on Bacillus subtilis; Shimi IR et al.; The mode of action of ASK-753 on Bacillus subtilis was examined . Unlike proper sideromycin antibiotics ferrioxamine B failed to antagonize the antimicrobial effects of ASK-753 . The antibiotic could inhibit the biosynthesis of nucleic acids; effect on the RNA was more pronounced . ASK-753 affected the stability of prelabelled DNA of B, subtilis in growing or resting cultures; the effect on the latter was more pronounced . Lysis of B, subtilis protoplasts could be attained at 30 degrees C but not at 4 degrees C which excludes a possible detergent affect of the drug . The drug exerted a potent inhibiting influence on protein synthesis by arresting the activity of lysyl-tRNA synthetase and thus could prevent the incorporation of 14C-lysine. J Virol, 1976 Mar, 17(3), 718 - 26 Recombinational-type transfer of viral DNA during bacteriophage 2C replication in Bacillus subtilis; Hoet P et al.; The Bacillus subtilis phage 2C contains one molecule of double-stranded DNA of about 100 x 10(6) daltons in which thymine is replaced by hydroxymethyluracil; the two strands have different buoyant densities . Parental DNA, labeled with either {3H}uracil of {32P}phosphate, was quite effectively transferred to offspring phage, and the efficiency of transfer was the same for the two strands . Labeled nucleotide compositions of the H and L strands from parental and progeny virions were very close . These data exclude a degradation of the infecting DNA and reutilization of nucleotides . Upon infection of light unlabeled cells with heavy radioactive viruses, no DNA with either heavy or hybrid density was extracted from offspring phage . Instead, an heterogeneous population of DNA molecules of densities ranging from that of almost hybrid to that of fully light species was obtained . Shear degradation of such progeny DNA to fragments of decreasing molecular weight produced a progressive shift to the density of hybrid molecules . Denaturation of sheared DNA segments caused the appearance of labeled and heavy single-stranded segments . These findings indicate that 2C DNA replicates semiconservatively and then undergoes extensive genetic recombination with newly formed viral DNA molecules within the vegatative pool, thus mimicking a dispersive transfer of the infecting viral genome . The pieces of transferred parental DNA have an average size of 10 x 10(6) daltons. J Bacteriol, 1976 Mar, 125(3), 934 - 45 Effects of chloramine on Bacillus subtilis deoxyribonucleic acid; Lu Shih K et al.; The lesions induced in Bacillus subtilis deoxyribonucleic acid (DNA) after treating bacterial cells (in vivo) and bacterial DNA (in vitro) with chloramine were studied biologically and physically . Single-strand breaks and a few double-strand scissions (at higher chloramine doses) accompanied loss of DNA-transforming activity in both kinds of treatments . Chloramine was about three times more efficient in vitro than in vivo in inducing DNA single-strand breaks . DNA was slowly chlorinated; the subsequent efficiency of producing DNA breaks was high . Chlorination of cells also reduced activity of endonucleases in cells; however, chlorinated DNA of both treatments was sensitized to cleavage by endonucleases . The procedure of extracting DNA from cells treated with chloramine induced further DNA degradation . Both treatments introduced a small fraction of alkali-sensitive lesions in DNA . DNA chlorinated in vitro showed further reduction in transforming activity as well as further degradation after incubation at 50 C for 5 h whereas DNA extracted from chloramine-treated cells did not show such a heat sensitivity. J Bacteriol, 1976 Mar, 125(3), 880 - 6 In vitro synthesis of the unit that links teichoic acid to peptidoglycan; Hancock I et al.; The role of cytidine diphosphate (CDP)-glycerol in gram-positive bacteria whose walls lack poly(glycerol phosphate) was investigated . Membrane preparations from Staphylococcus aureus H, Bacillus subtilis W23, and Micrococcus sp . 2102 catalyzed the incorporation of glycerol phosphate residues from radioactive CDP-glycerol into a water-soluble polymer . In toluenized cells of Micrococcus sp . 2102, some of this product became linked to the wall . In each case, maximum incorporation of glycerol phosphate residues required the presence of the nucleotide precursors of wall teichoic acid and of uridine diphosphate-N-acetylglucosamine . In membrane preparations capable of synthesizing peptidoglycan, vancomycin caused a decrease in the incorporation of isotope from CDP-glycerol into polymer . Synthesis of the poly (glycerol phosphate) unit thus depended at an early stage on the concomitant synthesis of wall teichoic acid and later on the synthesis of peptidoglycan . It is concluded that CDP-glycerol is the biosynthetic precursor of the tri(glycerol phosphate) linkage unit between teichoic acid and peptidoglycan that has recently been characterized in S . aureus H. J Bacteriol, 1976 Mar, 125(3), 845 - 9 Adaptation of a stable L-form of Bacillus subtilis to minimal salts medium without osmotic stabilizers; Gilpin RW et al.; An L-form isolated from Bacillus subtilis 168 was adapted to growth in a 340 mOsm minimal salts medium without the addition of osmotically protective solutes . This L-form had no chemically detectable peptidoglycan residues on its surface, but 0.8% of the dry weight of washed membranes was hexosamine . The osmotic stability and susceptibility to bacitracin and vancomycin of the L-form adapted to growth in 340 mOsm osmotically unprotected medium was twice that of the L-form grown in 2,680 mOsm medium supplemented with 1.2 M NaCl. J Bacteriol, 1976 Mar, 125(3), 776 - 9 Order of expression of genes affecting septum location during sporulation of Bacillus subtilis; Dunn G et al.; The mean volumes of stationary-phase cells of wild-type and asporogenous mutants of Bacillus subtilis have been measured . Mutants blocked at stage 0 of sporulation either produced cells that had the same volume as the developing sporangium or they divided to produce cells of one-half this volume . The order of expression of the genes affected by the mutations in these strains was determined by biochemical characterization and by construction of double sporulation mutants . Mutants that produced small cells were blocked at an earlier stage of sporulation than those that produced large cells . It is suggested that the following dependent sequence must occur before the formation of the prespore spetum: (i) the initiation of sporulation, (ii) a signal to block the final central division site, and (iii) a signal to activate a polar septum site. J Bacteriol, 1976 Mar, 125(3), 755 - 61 Identification of coreplicating chromosomal sectors in Bacillus subtilis by nitrosoguanidine-induced comutation; Siccardi AG et al.; The simultaneous replication of four regions of dichotomously replicating chromosomes of Bacillus subtilis has been detected by means of nitrosoguanidine-induced comutation . The map distance between successive rounds of replication has been measured as one-half a replicative arm in cells growing exponentially in rich medium. J Bacteriol, 1976 Mar, 125(3), 1195 - 206 Bacteriophage SP50 as a marker for cell wall growth in Bacillus subtilis; Archibald AR et al.; When grown under conditions of phosphate limitation, Bacillus subtilis W23 lacked wall teichoic acid and did not adsorb phage SP50 . During transition from growth under conditions of phosphate limitation to those of potassium limitation, the bacteria developed an ability to adsorb phage which increased exponentially in relation to their content of wall teichoic acid . During transition in the reverse direction, the bacteria retained near-maximum phage-binding properties until their content of wall teichoic acid had fallen to a fairly low level . These observations suggest that newly incorporated wall material does not immediately appear at the cell surface in a structure to which phage can adsorb . Examination of the location of adsorbed phage particles showed that recently incorporated receptor material appeared at the cell surface first along the length of the cylindrical portion of the cell . The results are consistent with models of wall assembly in which newly synthesized wall material is intercalated at a large number of sites that are distributed along the length of the cell . This newly incorporated material may be located initially at a level underlying the surface of the cell and may become exposed at the surface only during subsequent growth . Incorporation of new material may also proceed rapidly into the developing septa, but new wall material is incorporated into existing polar caps more slowly, or perhaps not at all. J Bacteriol, 1976 Mar, 125(3), 1139 - 47 Layered distribution, according to age, within the cell wall of bacillus subtilis; Pooley HM; When soluble autolytic activity was added to growing cultures of a mutant possessing a reduced rate of cell wall turnover, there was a delay of more than one generation before solubilization of new cell wall began, in contrast to the immediate increase in the rate of solubilization of old cell wall . A similar delay was found before turnover of new cell wall occurred in the parent, in agreement with a previous report (Mauck et al., 1971) . When sodium lauryl sulfate-inactivated cell walls were prepared, the great bulk of the wall formed a uniformly susceptible substrate to added autolytic activity . The immediate solubilization of new wall eliminates insusceptibility to autolytic enzyme as an explanation for the failure to be turned over . There were, however, major differences in the rate of solubilization of wall of different ages . During solubilization of the initial 30% of the cell wall preparation, wall two generations old was solubilized at least seven times faster than wall one-half a generation old . This result is interpreted in terms of differences in accessibility . The cell wall is seen as consisting of a series of layers, the age of which increases with the distance from the membrane, such that wall newly synthesized on the membrane passes out through the thickness of the cell wall layer during subsequent growth and only becomes susceptible to turnover as it reaches the outer surface, largely in the form of a layer, more than one generation after incorporation. J Bacteriol, 1976 Mar, 125(3), 1127 - 38 Turnover and spreading of old wall during surface growth of Bacillus subtilis; Pooley HM; The steady-state concentration of cell wall turnover products in the medium of Bacillus subtilis 168 growing exponentially on a casein hydrolysate-supplemented medium is equivalent to an overall rate of turnover of less than 10% per generation . After transfer of a steady-labeled culture to nonradioactive medium, the rate of release of labeled turnover products increased exponentially for up to two generations . The rate of turnover finally attained by this culture reached an apparently first-order rate of about 50% per generation . The addition of soluble autolytic activity to growing cultures of a mutant possessing a reduced rate of wall turnover resulted in a marked stimulation in the rate of solubilization of the cell wall fraction . The increased rate of solubilization produced was proportional to the concentration of added enzyme and remained constant until less than 20% of the wall originally present was left . Autolytic activity added under these conditions was bound entirely to wall at least one generation old . The results are interpreted in terms of a model for cell wall growth in which wall two or more generations old covers a total surface area at least four times larger than that occupied at the time of synthesis, forming a shallow outer layer (overlying newer wall) from which all turnover takes place . The model is discussed in relation to previous attempts to determine the pattern of surface expansion in bacilli. J Bacteriol, 1976 Mar, 125(3), 1120 - 6 Bacteriophage resistance in Bacillus subtilis 168, W23, and interstrain transformants; Yasbin RE et al.; Strains of Bacillus subtilis 168 deficient in glucosylated teichoic acid vary in their resistance to bacteriophage infection . Although glucosylated teichoic acid is important for bacteriophage attachment, the results demonstrate that alternate receptor sites exist . Non-glucosylated cell wall mutants could be assigned to specific classes (gtaA, gtaB, gtaC) by their pattern of resistance to three closely related bacteriophages (phi25, phie, SP82) . In addition to glucosylation, the type of teichoic acid was also important for bacteriophage attachment . B . subtilis strains 168 and W23 have different teichoic acids in their cell walls and have varied susceptibilities to bacteriophage infection . Transfer of bacteriophage resistance from strain W23 into a derivative of strain 168 was accomplished . The resistant bacteria obtained were imparied in their ability to adsorb bacteriophage and in their capacity to be transfected by bacteriophage deoxyribonucleic acid. J Bacteriol, 1976 Mar, 125(3), 1074 - 9 Translocation in Bacillus subtilis: characterization of elongation factor G by peptidyl-{3H}puromycin synthesis; Aharonowitz Y et al.; This communication describes the characterization of elongation factor G from Bacillus subtilis by the translocation of "native" peptide donors . Translocation was followed by elongation factor G-dependent increase in the synthesis of peptidyl-{3H}puromycin using "washed" ribosomes carrying in vivo-bound peptidyl-transfer ribonucleic acid ("native" peptidyl-transfer ribonucleic acid) molecules as peptide donors . Such ribosomes were obtained from cell extracts by washing at a high salt concentration . The use of "native" peptide donors facilitated the study of translocation under conditions that are closer to the in vivo state than those in the methods previously employed. Can J Microbiol, 1976 Mar, 22(3), 359 - 63 Dry-heat inactivation of Bacillus subtilis var . niger spores with special reference to spore density; Molin G et al.; The dry-heat inactivation kinetics of Bacillus subtilis var . niger (ATCC9372) spores has been studied in the temperature range of 120-190 degrees C . The spores were applied to glass plates of a standardized area (3.24 cm2) . Spore preparations of five different spore densities were used (8.3 X 10(4), 7.4 X 10(6), 6.3 X 10(7), and 6.6 X 10(8) spores per sample, respectively) . The heat resistance of the spore was dependent on the number of spores per surface unit . Maximum resistance was observed when the concentration was 7.4 X 10(5) spores per sample . The D-values obtained at 160 degrees C from these samples were about twice as high as the D-values obtained from samples with a concentration of 6.3 X 10(7) or 6.6 X 10(8) spores per sample . The z-value was found to be independent of spore density . Thus, for the two concentrations 7.4 X 10(5) and 6.3 X 10(7) spores per sample, the z-value was found to be 22 degrees C and constant over the temperature range investigated. Mol Gen Genet, 1976 Feb 27, 144(1), 49 - 51 Gene locus of a 30s ribosomal protein S20 of Bacillus subtilis; Osawa S; The gene locus of the 30s ribosomal protein S20 of Bacillus subtilis was mapped in the str-region at the right side of S5 gene . The gene order was cysA-str(S12)-ery(50D)-{spc(S5); 50G}-S20-. Mol Gen Genet, 1976 Feb 27, 144(1), 39 - 42 Amino acid replacement in the protein S5 from a spectinomycin resistant mutant of Bacillus subtilis; Itoh T; Ribosomal protein S5 was isolated from wild type Bacillus subtilis ATCC 6633 and from a spectinomycin resistant mutant (BSPC 111) derived from spectinomycin sensitive to resistance is accomtrypsin and all the tryptic peptides were isolated by column- and paper-chromatography . By comparative amino acid analyses of the peptides, it was demonstrated that the S5 from the mutant differs from the wild type S5 by a replacement of one amino acid, namely lysine by isoleucine in the peptide T9 . The results are compared with E . coli spectinomycin resistant mutants. Biochim Biophys Acta, 1976 Feb 18, 425(1), 49 - 62 Base composition studies on mitochondrial 4 S RNA from rat liver and Morris hepatomas 5123D and 7777; Chia LL et al.; The major and modified base composition of mitochondrial 4 S RNA from rat liver and from Morris hepatomas 5123D and 7777 has been determined for 16 constituents using a chemical tritium-derivative method . The base composition of these mitochondrial 4 S RNA preparations was compared with the base composition of cytoplasmic and bacterial (Escherichia coli B and Bacillus subtilis) 4-S RNAs . The results of these studies are: 1 . When compared with cytoplasmic 4 S RNA, the liver and hepatoma mitochondrial 4-S RNAs are characterized by high (A + U)/(G + C) ratios and low overall degrees of base methylation and modification . 2 . The mammalian mitochondrial 4-S RNAs are qualitatively even more different from the bacterial 4-S RNAs than from their cytoplasmic counterparts . Thus, several modified constituents found in both cytoplasmic and mitochondrial 4 S RNA are absent from the bacterial 4-S RNAs . 3 . Mitochondrial 4S RNA from both hepatomas was found to be under-methylated and undermodified when compared with normal liver mitochondrial 4S RNA . This trend is more pronounced for the rapidly growing hepatoma 7777 (i.e., 17% undermethylation) than for the more slowly growing hepatoma 5123D (i.e., 8% undermethylation) . These findings are discussed in relationship to (1) results of other authors on composition of mitochondrial 4 S RNA, (2) special features of structure and biosynthesis of mitochondrial 4 S RNA, (3) the possible evolutionary origin of mitochondria and (4) the possible role played by aberrant mitochondrial 4 S RNA in altered mitochondrial protein synthesis in tumors. J Biol Chem, 1976 Feb 10, 251(3), 705 - 11 Amino acid sequence of flagellin of Bacillus subtilis 168 . III . Tryptic peptides, N-bromosuccinimide peptides, and the complete amino acid sequence; DeLange RJ et al.; Of the 28 expected tryptic peptides from Bacillus subtilis 168 flagellin, 24 were isolated and sequenced . Several overlapping tryptic peptides were also characterized . Studies were also performed on two peptides of 142 and 162 residues isolated after cleavage of the flagellin molecule at the single tyrosine residue (residue 142) with N-bromosuccinimide . These studies together with the previous data on the cyanogen bromide peptides and the tryptic peptides from maleylated flagellin permitted the complete amino acid sequence to be established: (see article) . The primary structure reveals no obvious regularities or major repetitions of homologous sequences . Hydrophobic residues are distributed randomly in the amino acid sequence . However, the distribution of charged residues is strikingly asymmetric . The NH2-terminal region (residues 1 to 101) possesses a net charge of 6 plus, the middle of the molecule (residues 102 to 203), a net charge of 9 minus, and the COOH terminal region (residues 204 to 304), a net charge of 4 minus. J Biol Chem, 1976 Feb 10, 251(3), 701 - 4 Amino acid sequence of flagellin of Bacillus subtilis 168 . II . Tryptic peptides from maleylated flagellin; Shaper JH et al.; Fifteen pure peptides, ranging in length from 2 to 141 residues, were isolated from the tryptic digest of maleylated flagellin . Studies of these peptides in conjunction with the data on the eight cyanogen bromide peptides described earlier (Chang, J . Y., DeLange, R . J., Shaper, J . H., and Glazer, A . N . (1976) J . Biol . Chem . 251, 695-700) established the order of three of the CNBr peptides at the NH2 terminus and of three at the COOH terminus of flagellin. J Biol Chem, 1976 Feb 10, 251(3), 695 - 700 Amino acid sequence of flagellin of Bacillus subtilis 168 . I . Cyanogen bromide peptides; Chang JY et al.; Sequence studies are presented on the cyanogen bromide peptides derived from the flagellin of Bacillus subtilis 168 . Eight unique CNBr peptides, ranging in length from 4 to 113 residues, were isolated in pure state . These peptides accounted for the amino acid composition of flagellin . The NH2-terminal methionyl residue of the protein reacted only partially with CNBr . Partial cleavage was observed at a Met-Glu bond (residues 22 to 23) of the protein. Eur J Biochem, 1976 Feb 2, 62(1), 1 - 6 The structure of teichoic acid from Bacillus subtilis var, niger WM as determined by C nuclear-magnetic-resonance spectroscopy; De Boer WR et al.; The walls of Bacillus subtilis var . niger WM, grown in a Mg2+ -limited chemostat culture (carbon source glucose, dilution rate = 0.2 h(-1), 37 degrees C, pH 7) contained 45% (w/w) teichoic acid, a polymer composed of glycerol, phosphate{ and glucose in the molar ratio 1.00:1.00:0.88, respectively . Alkaline hydrolysis of this teichoic acid yeilded 1-O-beta-glucosylglycerol phosphate (together with small amounts of glycerol phosphate0 and 13C nuclear magnetic resonance spectra of this hydrolysis product, and its derivative after alkaline phosphate treatment, confirmed that the monomeric unit was 1-O-beta-glucosylglycerol-3-phosphate . Assignment of the resonances in the spectrum of undergraded teichoic acid revealed that the polymer was a poly {(2,3) glycerol phosphate 1, glucosidically substituted on C-1 of glycerol with beta-glucose. Eur J Biochem, 1976 Feb 2, 62(1), 55 - 64 Levansucrase of Bacillus subtilis: kinetic and thermodynamic aspects of transfructosylation processes; Chambert R et al.; Simple kinetic considerations derived from the ping-pong mechanism previously proposed for levansucrase of Bacillus subtillis allowed us to predict an easily operated method to approach the kinetic studies of exchange and hydrolytic activities of this enzyme . The experimental kinetic pattern obtained from the study of both activities is in close agreement with those predicted by theoretical approach . The combination of kinetic results enabled us to determine with a good accuracy the values of the apparent rate constant of the step of fructosylation of the enzyme from the sucrose-enzyme Michaelis complex and the apparent rate constants of the steps of defructosylation of the fructosyl enzyme to water or to glucose . The standard free energy reaction coordinate diagram for the transfructosylation process from sucrose to water was constructed . We found that the high energy of the glycosidic linkage of sucrose is preserved in the fructosyl-enzyme intermediate . The temperature dependence studies of the rate constants of fructosylation and defructosylation of the enzyme show that the entropy of activation for the two steps of defructosylation of the fructosyl enzyme are nearly the same . However the enthalpy of activation for the transfructosylation step to water is greater than that to glucose . We attempted to explain this discrepancy . Furthermore comparison of mechanism and efficiency of enzymatic and acid catalysis of sucrose hydrolysis was developed. J Antibiot (Tokyo), 1976 Feb, 29(2), 121 - 4 De-esterification of cephalosporin para-nitrobenzyl esters by microbial enzymes; Brannon DR et al.; Bacteria and actinomycetes were screened for esterase enzymes capable of removing the para-nitrobenzyl ester from cephalosporins . An esterase preparation from Bacillus subtilis was used to prepare cephalexin and 7-ADCA from the corresponding para-nitrobenzyl esters. Cancer Res, 1976 Feb, 36(2 Pt 1), 445 - 51 DNA-attacking ability of carcinogenic mycotoxins in recombination-deficient mutant cells of Bacillus subtilis; Ueno Y et al.; Thirty mycotoxins and 5 chemically modified toxins were tested for DNA-attacking ability in the rec assay using the recombination-deficient mutant of Bacillus subtilis M45 (rec-) and the parent strain H17 (rec+) . Six Penicillium toxins (citrinin, penicillic acid, patulin, (-)-luteoskyrin, (+)-rugulosin, and PR-toxin), 5 Aspergillus toxins (aflatoxins B1 and G1, sterigmatocystin, O-acetylsterigmatocystin, and O-acetyldihydrosterigmatocystin), and 2 Fusarium toxins (zearalenone and zearalenol-b) were positive . Among these 13 compounds, the following 8 mycotoxins have been reported to be carcinogenic in animals: citrinin, penicillic acid, patulin, (-)-luteoskyrin, (+)-rugulosin, aflatoxins B1, and G1, sterigmatocystin . Correlation between the rec effect and in vivo carcinogenicity of mycotoxins is discussed. J Gen Microbiol, 1976 Feb, 92(2), 398 - 404 Genetic hybridization of the leu-ilv region in bacilli; Biswas GD et al.; Two auxotrophic strains of Bacillus subtilis 168 served as recipients for DNA extracted from various wild-type strains of B . subtilis and wild-type species of the genus Bacillus . Depending upon the DNA source, heterologous transformations of the linked try-his-tyr loci were either as efficient as those observed with donor DNA obtained from the wild type B . subtilis 168 strain or were undetectable . The order and relative distances of the three gene loci were the same for all active DNA preparations . Similar results were obtained in heterologous transformations of the linked leu-ilv loci, except that DNA preparations from the Bacillus globigii and B . subtilis var . niger species exhibited a reduced but detectable frequency of transformation . With the latter preparations a marked polarity of integration favouring the leu+ gene was observed, an effect not seen with homologous DNA . Six independent hybrid lines were obtained from transformation of B . subtilis leu- ilv- with DNA from B . subtilis var . niger leu+ ilv+ . DNA extracted from these lines fell into two classes on the basis of activity in transforming the parental recipient strain: (i) indistinguishable from homologous DNA, and (ii) intermediate between homologous DNA and DNA from the original donor strain . With either class, polarity of integration was no longer observed in the leu-ilv region . The intermediate type of hybrid demonstrates that at least some of the inefficiency of heterospecific transformation must be due to heterology in nucleotide sequence between the different species at the leu-ilv loci. Proc Natl Acad Sci U S A, 1976 Feb, 73(2), 331 - 5 Control of development: role of regulatory nucleotides synthesized by membranes of Bacillus subtilis in initiation of sporulation; Rhaese HJ et al.; A model explaining the mechanism of initiation of differentiation is presented . It is based on the finding that sporulation in B . subtilis can be induced by the same nutrient deficiencies that also induce the synthesis of highly phosphorylated nucleotides . Two of these nucleotides are synthesized by membrane vesicles . Synthesis can be inhibited by the same metabolites of glucose that also inhibit sporulation . It is concluded, therefore, that the plasma membrane synthesizes unusual nucleotides in response to nutrient deficiencies . By several as yet unknown steps, these nucleotides then cuase changes in the metabolism of the organism leading to the formation of spores . Both structure and mechanism of synthesis of adenosine hexaphosphate, pppAppp, have been elucidated by use of ATP analogues. J Bacteriol, 1976 Feb, 125(2), 556 - 64 Magnesium and anion requirements of rodB mutants of Bacillus subtilis; Rogers HJ et al.; rodB mutants of Bacillus subtilis have been found to require several hundred-fold more Mg2+ in a minimal growth medium than the wild type to achieve rapid growth . In the presence of all concentrations of Cl-, the organisms grow as deformed cocci, but with 10 mM Mg2+ and Br-, I-, or NO3- present they grow as rods . The morphology is then directly under the control of the concentration of both Mg2+ and anion . Originally, it was found that L-glutamic acid in the medium brought about the change from deformed spheres to rods . This amino acid will similarly function at a much lower concentration when the higher concentrations of Mg2+ and Cl- are also present . At a constant concentration of L-glutamate, the morphology can be controlled by varying the Mg2+ concentration . In the presence of Mg2+ and I-, the morphological change is temperature sensitive . At 30 C rods are formed and at 42 C deformed cocci are formed . The requirement of a rodB mutant for a high concentration of magnesium and the round morphology have been shown to be most probably due to a single mutation. J Bacteriol, 1976 Feb, 125(2), 489 - 500 Recombination-deficient mutants of Bacillus subtilis; Sadaie Y et al.; Two mutant strains of Bacillus subtilis Marburg, NIG43 and NIG45, were isolated . They showed high sensitivities to gamma rays, ultraviolet light (UV), and chemicals . Deficiencies in genetic recombination of these two mutants were shown by the experiments on their capacity in transformation . SPO2 transfection, and PBS1 phage transduction, as well as on their radiation and drug sensitivities and their Hcr+ capacity for UV-exposed phage M2 . Some of these characteristics were compared with those of the known strains possessing the recA1 or recB2 alleles . Mapping studies revealed that the mutation rec-43 of strain NIG43 lies in the region of chromosome replication origin . The order was purA dna-8132 rec-43 . Another mutation, rec-45, of strain NIG45 was found to be tightly linked to recA1 . The mutation rec-43 reduced mainly the frequency of PBS1 transduction . On the other hand, the mutation rec-45 reduced the frequency of recombination involved both in transformation and PBS1 transduction . The mutation rec-43 of strain NIG43 is conditional, but rec-45 of strain NIG45 is not . The UV impairment in cellular survival of strain NIG43 was gradually reverted at higher salt or sucrose concentrations, suggesting cellular possession of a mutated gene produce whose function is conditional . In contrast to several other recombination-deficient strains, SPO2 lysogens of strain NIG43 and NIG45 were not inducible, indicating involvement of rec-43+ or rec-45+ gene product in the development of SPO2 prophage to a vegetative form . The UV-induced deoxyribonucleic acid degradation in vegetative cells was higher in rec-43 and rec-45 strains. J Bacteriol, 1976 Feb, 125(2), 453 - 60 Repression of sporulation in Bacillus subtilis by L-malate; Ohne M et al.; L-Malate repressed sporulation in the wild-type strain of Bacillus subtilis . When 75 mM L-malate was added to the growth medium at the time of inoculation, the appearance of heat-resistant spores was delayed 6 to 8 h . The synthesis of extracellular serine protease, alkaline phosphatase, glucose dehydrogenase, and dipicolinic acid was similarly delayed . Sporulation was not repressed when malate was added to the culture at t4 or later . A mutant was selected for ability to sporulate in the presence of malate . This strain could also sporulate in the presence of glucose . The malate-resistant mutant grew poorly with malate as sole carbon source, although it possessed an intact citric acid cycle, and it showed increased levels of malic enzyme . This indicates a defect in the metabolism of malate in the mutant . A mutant lacking malate dehydrogenase activity was also able to sporulate in the presence of malate . A model for the regulation of sporulation by malate is presented and discussed . Citric acid cycle intermediates other than malate did not affect sporulation . In contrast to previous results, sporulation of certain citric acid cycle mutants could be greatly increased or completely restored by the addition of intermediates after the enzymatic block . The results indicate that the failure of citric acid cycle mutants to sporulate can be adequately explained by lack of energy and lack of glutamate. Can J Microbiol, 1976 Feb, 22(2), 322 - 3 Temperature-pH effect upon germination of bacterial spores; Ishida Y et al.; Using several kinds of criteria for the germination of bacterial spores, germination-pH curves were drawn for Bacillus subtilis spores observed at different temperatures . The experiments revealed that optimum pH for spore germination was markedly changed by changing the incubation temperature; the optimum pH for germination was 7.4 at 37 degrees C and 5.4 at 10 degrees C . A possible mechanism involved in this phenomenon is discussed. J Virol, 1976 Feb, 17(2), 492 - 502 Induction of prophage SPO2 in Bacillus subtilis: isolation of excised prophage DNA as a covently closed circle; Arwert F et al.; Bacillus subtilis tryC2, thyA, thyB, lysogenic for the phage DNA polymerase negative mutant SPO2 susL244, was induced under conditions preventing phage and bacterial DNA synthesis . The biological activity of DNA from induced cells and from uninduced controls was assayed by transformation and transfection, respectively . About 50% of the phage DNA biological activity in DNA extracted from induced cells was resistant to exposure to pH 11.8 TO 11.9 . This DNA was operationally defined as alkali-resistant phage DNA . Transforming bacterial DNA from uninduced or induced cells and transfecting DNA from uninduced cells were more than 95% inactivated after exposure to high pH . The alkali-resistant phage DNA was characterized by sucrose gradient centrifugation, by centrifugation in cesium chloride-propidium iodide, and by electron microscopy . It was found to consist of a majority of covalently closed circular DNA molecules . Length measurements of a few relaxed circular molecules indicate a molecular weight of these similar to that previously found for mature SPO2DNA . Attempts to isolate similar covalently closed circular phage DNA from induced bacteria lysogenic for SPO2 phage with a functional DNA polymerase gene were unsuccessful . The gene order in mature and prophage SPO2 was determined by rescue of single and double markers from the respective type of DNA . The data obtained show that prophage DNA is (genetically) permuted relative to mature DNA . The phage attachment site is suggested to be located between genes I and J. Eur J Biochem, 1976 Jan 15, 61(2), 487 - 92 An intracellular endonuclease of Bacillus subtilis specific for single-stranded DNA; Ciarrocchi G et al.; We have fractionated from extracts of Bacillus subtilis the DNase activity specific for single-stranded DNA; the activity separates in two main fractions on Sephadex G-200, a larger one (Mr greater than 400 000) and a smaller one (Mr approximately 30 000) . We have purified the smaller, more abundant fraction nearly 3000-fold . The purified enzyme has a pH optimum close to 8, is activated by Ca2+, and is inhibited by EDTA; the enzyme hydrolyses single-stranded DNA at a rate approximately 40 times greater than double-stranded DNA . The mode of action is endonucleolytic on both substrates, but the possiblility that the two activities may reside on different molecules is not ruled out . The products have 5'-P and 3'-OH ends . The enzyme is different from those purified from the culture media of the same organism in several respects; the latter are all extracellular enzymes, they are not specific for single-stranded DNA (except one) and have all an exonucleolytic mode of action. Biochemistry, 1976 Jan 13, 15(1), 101 - 7 Esterase activity of zinc neutral proteases; Holmquist B et al.; The hydrolysis of a series of depsipeptides demonstrates that the zinc neutral endopeptidases of bacteria are active esterases . Esters such as BzGly-OPhe-Ala, BzGly-OLeu-Ala, and FA-Gly-OLeu-NH2 are hydrolyzed at rates three- to eightfold slower than are their exact peptide analogues, when hydrolyzed by thermolysin, Bacillus subtilis neutral protease and the neutral protease from Aeromonas proteolytica . Ester hydrolysis by zinc neutral proteases follows the characteristic preference for hydrophobic amino acids adjacent to the site of cleavage, discerned from the hydrolysis of peptide substrates . Removal of zinc from thermolysin abolishes the esterase activity of the native enzyme . Among the metals examined, only Co2+ and Zn2+ restore esterase activity to any significant extent, Co2+ restoring 50% and Zn2+ 100% of the native thermolysin activity . The hydrolysis of esters and peptides by thermolysin does not differ with respect to either the binding or catalytic steps . Substrate specificity, pH-rate profiles, inhibitor, and deuterium isotope effects are identical for both types of substrates. J Immunol Methods, 1976, 11(1), 37 - 48 The antigen-binding capacity of serum IgG . An immunosorbent method applied to subtilisin as antigen; Jacoby B et al.; A method for estimating the antigen-binding capacity of serum IgG is presented . A Sepharose anti-human IgG immunosorbent is incubated with test serum and washed . The antigen-binding capacity of the bound specific IgG is determined by incubation with purified, inactivated 125I-subtilisin . The method was applied tp the sera of workers in the detergent industry exposed to enzymatic preparations from Bacillus subtilis . The sera of 33 out of 69 workers bound a detectable amount of subtilisin, the detection limit ranging from 8--34 ng/ml in different assays . The amount of antigen bound ranged from 42--9380 ng/ml . IgG to subtilisin was not detected in the sera of 61 subjects with no known exposure to subtilisin and who were not factory workers. Acta Otorhinolaryngol Belg, 1976, 30(6), 643 - 51 {Passage into normal salivary, lacrimal and nasal secretions of ampicillin and erythromycin administered intramuscularly}; Melon J et al.; The authors have measured, with the agar diffusion technique, the antibiotic concentrations in the serum as well as in the normal nasal, lachrymal and salivary secretions, after an intramuscular injection of 500 mg of ampicillin and of 200 mg of erythromycin . Bacillus subtilis is used as test germ for dosage of ampicillin and Sarcina lutea for the dosage of erythromycin . An important quantity of ampicillin (0.5 to 2.5 microgram/ml) passes into the nasal secretion . Its passage into the saliva is very irregular and is usually very slight . Bacillus subtilis is not suitable for systematically studying the ampicillin concentration in the lachrymal secretion, which displays a strong, inherent inhibitory effect on this micro-organism . Important concentrations of erythromycin are found in the three secretions: 0.5 to 3.25 microgram/ml in the nasal mucus and in the lachrymal secretion, 0.3 to 4.5 microgram/ml in saliva. Acta Biol, 1976, 27(2-3), 177 - 82 In vitro investigation of biological specimens by electron microscopy; Kalman E et al.; A microchamber was developed for the examination of biological specimens in nearly natural environments, in an electron microscope, at 70kV accelerating voltage. . The chamber can be supplied continually with the sample and with the reagents, which makes it suitable for the study of biochemical reactions, too . Temperature and vapour pressure in the chamber can be controlled and the thickness of the specimen can be varied . Transmission electron micrographs of biological specimens, such as human blood cells, bull gametes and Bacillus subtilis have been obtained . Mobility of microorganisms, which is regarded as a criterion of the wet state, has been observed. Vet Med (Praha), 1976, 21(8), 505 - 11 {Laboratory control of disinfectant effectiveness of sodium dichlorisocyanurate}; Skaloud J et al.; The level of active chlorine was determined to reach 59% in the first part of the trial; this corresponds to data asserted by the producer . In the solubility test, the preparation was classified as well-soluble . The determined sodium dichlorisocyanurate dilution coefficient suggests that increasing concentrations of the active substance do not imply any significant reduction of the time needed for exposure, and considering the value of the temperature coefficient (1.166) use can be considered as possible at different temperatures of environment, especially at lower temperature levels . During the effectiveness determinations on tissue carriers, 100% disinfecting effect was achieved at a 0.1% weight concentration per volume after five minutes of exposure against E . coli and Staph . aureus germs . For brief information, theeffectiveness of sodium dichlorisocyanurate on the spores of Bacillus subtilis was also examined . At the concentrations of 0.1% and 1% weight per volume, no favourable result was obtained even after 120 minutes of exposure . When buliding material was used as carriers (metallic sheets, bricks, wood, concrete) the total of 192 smears were prepared . Each of the carriers was adjusted in two positions--horizontal and vertical . 100% effectiveness against the germs of S . aureus was obtained at the concentration of 0.35% active chlorine (0.6% wt . per active chlorine concentration as ls carriers in the Chloramin B comparative test (28% act . chlorine) . The comparison of effectiveness on the basis of the same concentrations of active chlorine in both chemicals revealed that sodium dichlorisocyanurate is repeatedly effective at a 0.020% active chlorine concentration . In Chloramin B the active concentration was 0.13% active chlorine. Nahrung, 1976, 20(10), 883 - 7 Determination of relationship between heating value and the thermal resistance of microorganisms in canned meat; Wojciechowski J et al.; Meat was sterilized with gamma-radiation in an aluminum foil bag and then contaminated with a determined quantity of Bacillus subtilis spores . The bag was then placed in the centre of a can filled with meat . After autoclave sterilization, the whole contents of the aluminum foil bags were examined for the quantity of survived bacteria . This procedure enables the determination of thermal resistance of some bacteria on the condition that appears during typical heating processes . Moreover from all the critical areas all surviving microorganisms can be determined. Antonie Van Leeuwenhoek, 1976, 42(4), 387 - 95 Formation of dry-heat resistant Bacillus subtilis var . niger spores as influenced by the composition of the sporulation medium; Molin G et al.; Bacillus subtilis var . niger spores were produced on 20 different media . The spore yield from each medium and the dry-heat resistance at 160 C of the different spore populations were determined . The yield varied with a factor of 10(6) and the variation in D 160-value was about 10-fold (less than 20 S-190 S) . A "synthetic" medium producing a high yield of spores with high dry-heat resistance was formulated . The concentrations of glucose, sucrose and calcium were found to be critical. Antonie Van Leeuwenhoek, 1976, 42(4), 365 - 86 Nuclear and cell division in Bacillus subtilis . Antibiotic-induced morphological changes; van Iterson W et al.; Incubation of Bacillus subtilis after outgrowth from spores in the presence of four different antibiotics in two different concentrations, showed that septation can occur without termination of nuclear division . Septation is then only partially uncoupled from the normal division cycle . Observations on location and development of mesosomes in the presence of the antibiotics, made in three-dimensional cell reconstructions, suggest that the mesosome plays a role in the normal coordination between nuclear and cell division, and may explain the partial independence between these two processes in B . subtilis. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg, 1976, 131(7), 661 - 4 Bactericidal effect of acrylic acid esters; Ibrahim I et al.; The antibacterial effect of p-Cl, p-Br, p-CH3, O-NO2, M-NO2, and p-NO2 substitutions of dimethylaminoethyl-alpha-phenyl-cinnamate on Bacillus subtilis, Sarcina lutea, and Escherichia coli was studied . The compounds were found to be more active against B . subtilis and S . lutea than against E . coli . The NO2 group in the para position was found to have the highest activity compared with the meta and ortho positions. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg, 1976, 131(3), 565 - 8 Characterization and microbiological evaluation of the red compounds, associated with red rot idsease of sugar cane; Badr El-Din SM et al.; The red coloured compounds, isolated from sugar cane tissues infected with red rot, were phenolic and included a mixture of flavonoid compounds . Two of these compounds were purified and partially characterized . Neither the mixture of the coloured compounds nor any of its individual compounds exhibited in vitro activity against the causal organism Physalospora tucumanensis . One of the purified compounds showed in vitro activity against Bacillus subtilis . The role of the red coloured compounds in the mechanism of disease resistance is discussed. Genetika, 1976, 12(11), 115 - 20 {UV-mutagenesis in Bacillus subtilis . VI . Mutagenesis under conditions of experimental delay of post-radiation DNA replication}; Filippov VD et al.; To test a suggestion that mutation frequency decline (MFD) in UV-irradiated bacteria starved by nitrogen sources is caused by long delay of the first postradiation cycle of DNA replication, we used a strain 1201-05 of Bacillus subtilis 168 (ade6 met5tsdnaC) which does not synthesize DNA at 42 degrees C . In a suspension of UV-irradiated cells of this strain which is incubated at 42 degrees in liquid deprived by nitrogen sources, a decrease of frequencies of Ade+ and Met+ revertants is observed . This shows that the mechanism responsible for MFD normally operates at higher temperature . Such a level of decline is not observed in a suspension incubated in nutritional medium when the DNA replication is blocked by non-permissive temperature for the same period as in starved cells . On the basis of these facts it is concluded that MFD in suspension of bacteria starved by nitrogen sources can not be caused only by delay of postradiation replication of DNA. Prep Biochem, 1976, 6(6), 479 - 88 Comparison of various procedures for removing proteins and nucleic acids from cell walls of Bacillus subtilis; Brown WC et al.; Several procedures were used in an attempt to prepare clean cell walls from Bacillus subtilis . The results indicate that protein and nucleic acids are tightly bound to the walls . The cleanest wall preparations were found following trichloroacetic acid extraction at 60 degrees or by extraction with 0.1N NaOH under a nitrogen atmosphere for 10 hrs . Protein denaturants, such as sodium dodecyl sulfate and concentrated guanidine hydrochloride were relatively ineffective in removing proteins and nucleic acids from the cell walls . Cell wall-bound DNA was biologically active in transformation assays. Genetika, 1976, 12(8), 124 - 30 {Operon for riboflavin synthesis in Bacillus subtilis . XI . Determination of the type of regulation using a test for dominance of operator-constitutive and regulator-constitutive mutations}; Bresler SE et al.; The strain GSY 468 of Bacillus subtilis, giving persistent unstable merozogotes was used as recipient in transformation . Diploid cells bearing a regulator constitutive or operator constitutive mutation were obtained, and their phenotype was studied . The operator constitutive mutation behaves as a dominant one, but the regulator constitutive is recessive . Hence the regulation type in riboflavin operon is negative . The product of the regulator gene is obviously a repressor protein. Genetika, 1976, 12(7), 74 - 9 {Mutants of Bacillus subtilis--producer of alkaline proteinase, sporulating in the presence of high concentrations of glucose}; Dobrzhanskaia EO et al.; Glur mutants of Bacillus subtilis A-50 capable of sporulating in the medium containing 5% glucose are isolated . The mutants are divided into three groups . The first group comprises glur mutants whose level of proteolytic activity is lower than that of the initial strain A-50 . The productivity of mutants of the 2nd group is equal to the level of alkaline proteinase synthesis characteristics of the original strain . In mutants of the third group the level of alkaline protease synthesis exceeds the productivity of the initial strain . There was no correlation between the efficiency of spore formation in the mutants obtained and the level of alkaline protease activity . The mutants of different groups differ in their ability to grow on different sugars. Prikl Biokhim Mikrobiol, 1976 Jan-Feb, 12(1), 59 - 67 {Effect of addition of bases and amino acids on inosine biosynthesis by mutants of Bacillus subtilis}; Kazarinova LA et al.; The relationship between the inosine biosynthesis by the Bacillus subtilis mutants VNII-genetika-265 and VNII-genetika-21 and the concentrations of adenine, guanine, uracile and amino acids was investigated . Adenine and guanine in high concentrations inhibited the inosine biosynthesis in both mutants . The mutant Gene-21 showed an increased requirement for adenine as compared with the mutant Gene-265 . In the presence of uracile the pattern of the relationship between the inosine growth and biosynthesis and adenine in the mutane Gene-265 was similar to that in the mutant Gene-21 . Excessive concentrations of histidine and tyrosine inhibited the inosine biosynthesis. Genetika, 1976, 12(6), 167 - 70 {Temperature-dependent mutants of Bacillus subtilis A-50 with a diminished level of alkaline proteinase synthesis}; Dobrzhanskaia EO et al.; Temperature-sensitive proteolytically inactive mutants of Bacillus subtilis A-50 are obtained . The optimal ability to synthetise the enzyme both in the original strain and in mutants is expressed at 37 degrees C . The level of proteolytic activity of selected prot(ts) mutants depends both on the temperature of growth and on the temperature at which the activity of subtilisin is analysed . The optima of functioning ts-proteinases are shifted in mutants as compared with the original strain . The ability to form spores at 37 degrees C and 45 degrees C are considerably reduced as compared with this process at 28 degrees C . The results of disc electrophoresis in polyacrylamide gel indicate the presence of structural alteration of proteinases in the mutants obtained. Z Allg Mikrobiol, 1976, 19(5), 369 - 75 {Excretion of organic compounds bei microorganisms in presence of diphenylamine}; Phai LD et al.; Diphenylamine (DPA) causes an increased leakage of organic compounds with resting cells of Candida guilliermondii, Bacillus subtilis, Escherichia coli, and Staphylococcus aureus already after a treatment of 1 h . The leakage depends both on the concentration of DPA and the duration of the treatment . Moreover, DPA influences the composition of the leaked ninhydrine-positive compounds . After 20 h of treatment an increased release of mannan and ribose is observed in addition to the stronger leakage of ninhydrine positive compounds . With regard to the relation between structure and activity of DPA, the activity of 21 compounds was investigated . The two apolar phenyl groups as well as the secondary amino group being able to form ionic bounds are essential for maximal activity . A disturbance of the function of the cell membrane caused by DPA is discussed. Int Arch Allergy Appl Immunol, 1976, 51(5), 529 - 43 Influence of detergent on aerosol allergic sensitization with enzymes of Bacillus subtilis; Markham RJ et al.; An anionic detergent, sodiumdodecylbenzenesulphonate, had an adjuvant effect upon aerosol allergic sensitization with subtilopeptidase A, a proteolytic enzyme of Bacillus subtilis . Both local and systemic antibody responses were accelerated and prolonged by use of the detergent . This adjuvant effect was only seen when an inactive form of the enzyme was used . The detergent, however, was able to potentiate early clinical responses upon initial exposure to the active enzyme. Biochimie, 1976, 58(1-2), 99 - 108 Increased nitrate reductase A activity as a sign of membrane alteration in early blocked asporogenous mutants of Bacillus subtilis; Bohin JP et al.; Nitrate reductase (Nar) activity, and its regulation, have been studied in B . subtilis and Spo0 mutants derived from it . The mutants are blocked at the stage zero of sporulation . The only Nar detected was the membrane-bound Nar A, which has been solubilized and purified . The enzyme itself, and its regulation, seem to be the same in Spo+ and Spo0 strains . Under all conditions tested, however, the mutants were hyperproducers of Nar A . Whether produced by a Spo+ or a Spo0 strain, the purified enzyme has the same Km on nitrate, and the same heat inactivation kinetics . In situ in membrane vesicles of a Spo+ strain, it displays the same Km and its thermoinactivation is exponential . In mutant vesicles, however, two Km's are observed, one normal and one five times higher, and thermoinactivation follows an initial period of activation . The higher Km disappears after heat activation . The Spo0 mutation studied seems to result in a modification of the membrane, such that insertion of Nar A in the modified membrane confers to the enzyme new allotopic properties . Additional and abnormal enzyme-binding sites may be created as a result of the mutation and these may be normalized during heat activation. Folia Microbiol (Praha), 1976, 21(2), 100 - 6 D-Glucosamine as inhibitor of early processes of transformation in Bacillus subtilis 168 trp2; Tichy P et al.; Glucosamine added to a transformation medium (TM2) after a 30-min cultivation of cells exhibited the highest inhibitory effect on the transformation process in Bacillus subtilis 168 trp2 . The recipient culture was least sensitive to glucosamine added after 50 min . Glucosamine had no inhibitory effect when added 10 min after the transformation DNA. Biochimie, 1976, 58(4), 435 - 41 The phosphoenolpyruvate : methyl-alpha-D-glucoside phosphotransferase system in Bacillus subtilis Marburg 168 : purification and identification of the phosphocarrier protein (HPr); Marquet M et al.; The phosphocarrier protein (HPr) of the phosphoenol pyruvate : alpha-methyl-D-glucoside-phosphotransferase system (PTS) has been purified from Bacillus subtilis Marburg 168 . The molecular weight is about 8300 . HPr contains 1 histidine residue . Phophoenzyme I appears to be an intermediate in the initial phosphoryl transfer from phosphoenolpyruvate (PEP) to HPr . Phospho-HPr is isolated and characterized as a component of the complete system. Folia Microbiol (Praha), 1976, 21(1), 36 - 42 Effect of sodium arsenite on the biosynthesis of mitomycins by Streptomyces caespitosus and mode of action of mitomycin C on Bacillus subtilis NRRL B-543; Abou-Zeid AA et al.; Addition of different concentrations of sodium arsenite to the fermentation medium used for the production of mitomycin antibiotics by Streptomyces caespitosus hindered the biosynthesis of mitomycins and led to the accumulation of 2-oxoglutarate, pyruvate and acetone . Mitomycin C isolated and purified using thin-layer chromatography in low concentration of about 0.1 mug/ml did not affect the RNA, DNA and protein biosynthesis of the growing Bacillus subtilis, while at 10 mug/ml mitomycin C markedly affected RNA, DNA and protein biosynthesis. J Bacteriol, 1976 Jan, 125(1), 74 - 83 Evolution of the transcription complex during sporulation of Bacillus subtilis; Brevet J; Ribonucleic acid polymerase activity in partially purified extract of cells of Bacillus subtilis harvested at different times (t-1, to, t1, and t2) was studied by zone centrifugation . During the course of sporulation, vegetative sigma-factor activity decreased and the transcription complex lost some of its affinity for active sigma factor . The complex underwent a two-stage change in sedimentation value, from 14.5S in vegetative growth phase to a 13S species very early in sporulation to a 16S species at later times . Two SpoO mutants have been studied by zone centrifugation . One strain, a rifampin-resistant (RfmR) mutant, failed to show any modification of the transcription complex, whereas the other, a Rfms strain, underwent a partial evolution of the transcription complex after to. J Bacteriol, 1976 Jan, 125(1), 166 - 73 Analysis of autolysins in temperature-sensitive morphological mutants of Bacillus subtilis; Brown WC et al.; The content and distribution of autolysin were measured in temperature-sensitive morphological mutants of Bacillus subtilis . Strains RUB1000 and RUB1012 grew as rods at 30 C . At 45 C the mutants contained disproportionately less teichoic acid than peptidoglycan and grew as irregular spheres . The amount of enzyme that could be extracted from rods was at least 31 times the amount extracted from spheres . The rate of autolysis of cell walls was 7- to 28-fold greater in rods than in spheres . The low activity found associated with the cell walls of spheres was not compensated for by larger amounts of autolytic activity in the cytoplasm . No activity was found in the growth medium at either temperature . The failure of the mutant cells to autolyze was due to low amidase activity and relatively resistant cell walls . Revertants of RUB1012 were isolated that had 13, 23, and 55% of the normal proportions of teichoic acid when grown at the nonpermissive temperature . Cell walls from the revertants were as sensitive to added amidase as the wild-type strain . None of the revertant strains regained the wild-type ability to produce more amidase at 45 C . However, the deficiency in autolysin observed with RUB1012 was partially restored in revertants containing higher proportions of teichoic acid. Biochimie, 1976, 58(5), 533 - 41 Isolation and properties of a cyclic guanosine-monophosphate sensitive intracellular ribonuclease from Bacillus subtilis; Kerjan P et al.; A ribonuclease was isolated and completely purified from sporulating cells of Bacillus subtilis . This RNase has a M.W . of about 150,000 daltons . It hydrolyzes single stranded RNA and single stranded synthetic polynucleotides yielding nucleoside 5'-monophosphates . The enzyme is an exonuclease which degrades polynucleotides from the 3'-end in the direction of the 5'-terminal . The RNase activity is strikingly inhibited by cGMP and to a lesser extent by cAMP . This inhibition (Ki = 0.1 mM) is of a non competitive nature . It appeared that in addition to the inhibition site, the enzyme contains a high affinity binding site for the two cyclic mononucleotides (K (cAMP) = 8.3 x 10-8; K (cGMP) = 2.5 x 10-7) . The RNase activity is also strongly inhibited by spermidine . This inhibition appeared to be due to the polyamine binding with the RNA, thus lowering the affinity of the substrate for the active site of the enzyme . This RNase may play a role in vivo in selective degradation of newly synthesized mRNA during sporulation. Folia Microbiol (Praha), 1976, 21(2), 117 - 24 Biochemical characterization and visualization of plasma membrane-DNA-protein complexes from Bacillus subtilis; Hochmannova J et al.; Subcellular fractions containing plasma membranes with bound DNA and associated proteins were isolated from two different cultures of Bacillus subtilis 168 growing exponentially at different rates . Differences in the contents of individual between the dissociated complexes, established electrophoretically, can be explained by dynamic binding of the proteins to DNA, resulting in a control of DNA, resulting in a control of DNA replication . Electron microphotographs of isolated complexes display, in addition to unit membranes, associated filamentous structures in different arragement . Patterns obtained after treating the complexes with nucleases suggest a polydeoxyribonucleotide character of the filamentous structures. C R Seances Soc Biol Fil, 1976, 170(4), 769 - 71 {The effect of somatic antigen from Bacillus subtilis on migration of mouse macrophages}; Lallouette P et al.; The migration of mice peritoneal macrophages has been studied on agar plates . The migration of the macrophages from mice treated by the intra peritoneal route by a somatic antigen of Bacillus subtilis, was 37% less than the migration of the macrophage from control mice . The presence of the antigen in the gel did not modify the migration of macrophages in either treated or control mice . This confirms the non specific character of the activity of this antigen. C R Seances Soc Biol Fil, 1976, 170(4), 765 - 8 {Reversal of the depressive power of cyclophosphamide on the anti-infectious defense of the mouse by means of a somatic antigen from Bacillus subtilis}; Lallouette P et al.; In the experimental conditions reported the cyclophosphamide increases the pathogenic effect of Escherichia coli in mice . Treating the animals with a somatic antigen of Bacillus subtilis, reverses the aggravating effect of cyclophosphamide on the experimental infection . Similar results are obtained through the parenteral and the rectal routes . This antigen does not limit either the leucopenient effect of cyclophosphamide nor its blocking effect on the synthesis of sheep red blood cells antibody in mice. Adv Enzymol Relat Areas Mol Biol, 1976, 44, 165 - 85 Bacillus subtilis RNA polymerase and its modification in sporulating and phage-infected bacteria; Losick R et al.; Bacillus subtilis RNA polymerase holoenzyme consists of the subunits beta', beta, sigma, alpha, delta, and omega . In sporulating bacteria and in bacteria infected with phages SP01 and SP82, this enzyme undergoes changes in subunit composition and transcriptional specificity that could play a regulatory role in gene transcription . Sporulating bacteria may contain a specific component that inhibits the activity of the sigma subunit of polymerase probably by interfering with the binding of sigma-polypeptide to core enzyme . The hypothetical inhibitor may be metabolically unstable, since its activity is rapidly depleted from sporulating cells in the presence of chloramphenicol . Inhibition of sigma-polypeptide activity may restrict the transcription of phage DNA an infected sporulating cells . Although lacking the sigma-subunit, RNA polymerase purified from sporulating cells contains sporulation-specific subunits of 85,000 and 27,000 daltons . In SP01-infected bacteria, the sigma-subunit is replaced by phage-induced subunits . Purified enzyme containing the protein product of SP01 regulatory gene 28 directs the transcription of phage middle genes in vitro, while enzyme containing phage-induced polypeptides V and VI preferentially copies late genes . Accurate transcription of middle and late genes in vitro requires the host delta-subunit of polymerase (or high ionic strength) but not sigma-subunit . Phage PBS2 induces an entirely new multisubunit RNA polymerase that specifically transcribes PBS2 DNA in vitro . This enzyme is synthesized de novo after infection and does not arise by modification of the B . subtilis holoenzyme. Appl Environ Microbiol, 1976 Jan, 31(1), 108 - 18 Purification and some properties of an extracellular maltase from Bacillus subtilis; Wang LH et al.; Bacillus subtilis P-11, capable of producing extracellular maltase, was isolated from soil . Maximum enzyme production was obtained on a medium containing 2.0% methyl-alpha-D-glucose, 0.5% phytone, and 0.2% yeast extract . After the removal of cells, extracellular maltase was precipitated by ammonium sulfate (85% saturation) . The enzyme was purified by using the following procedures: Sephadex G-200 column chromatography, diethylaminoethyl-Sephadex A-50 ion-exchange column chromatography, and a second Sephadex G-200 column chromatography . A highly purified maltase without amylase or proteinase activities was obtained . Some properties of the extracellular maltase were determined: optimum pH, 6.0; optimum temperature, 45 C, when the incubation time was 30 min; pH stability, within 5.5 to 6.5; heat stability, stable up to 45 C; isoelectric point, pH 6.0 (by gel-isoelectric focusing); molecular weight, 33,000 (by gel filtration with Sephadex G-200); substrate specificity: the relative rates of hydrolysis of maltose, maltotriose, isomaltose, and maltotetraose were 100:15:14:4, respectively, and there was no activity toward alkyl or aryl-alpha-D-glucosides, amylose, or other higher polymers . Transglucosylase activity was present . Glucose and tris(hydroxymethyl)aminomethane were competitive inhibitors with Ki values of 4.54 and 75.08 mM, respectively; cysteine was a noncompetitive inhibitor . Michaelis constants were 5 mM for maltose, 1 mM for maltoriose, and 10 mM for isomaltose . A plot of pKm (-log Km) versus pH revealed two deflection points, one each at 5.5 and 6.5; these probably corresponded to an imidazole group of a histidine residue in or near the active center; this assumption was supported by the strong inhibition of enzyme activity by rose bengal. Mol Gen Genet, 1975 Dec 30, 143(1), 13 - 23 Restriction and modification in B . subtilis . Purification and general properties of a restriction endonuclease from strain R; Bron S et al.; All Bacillus subtilis R-type strains showing the phenomena of restriction and modification contain an endonuclease that inactivates in vitro the biological activity of a variety of DNAs lacking R-specific modification, such as transfecting SPPI, SPO2 and phi105 DNA, and transforming B . subtilis 168-type DNA . The corresponding DNAs carrying R-specific modification are resistant to the enzyme . The enzyme has been purified approximately 400-fold and is essentially free from contaminating double strand-directed unspecific exo- or endonuclease activity . Only Mg2+ is required as cofactor . The substrate DNAs are cleaved at specific sites . The double-stranded fragments produced from SPP1 DNA (molecular weight 2.5 x 10(7)) have an average molecular weight of about 3 x 10(5). J Biol Chem, 1975 Dec 25, 250(24), 9256 - 61 RNA polymerase from sporulating Bacillus subtilis . Purification and properties of a modified form of the enzyme containing two sporulation polypeptides; Linn T et al.; A new form of DNA-dependent RNA polymerase termed enzyme III has been purified from sporulating cells of Bacillus subtilis . In addition to the subunits of core RNA polymerase (beta', beta, alpha, and omega), enzyme III contains sporulation-specific polypeptides of 85,000 (P85) and 27,000 (P27) daltons . P85 corresponds to an RNA polymerase-binding protein previously identified by precipitation of RNA polymerase from crude extracts of sporulating cells with antibody directed against core enzyme . Both P85 and P27 co-purified with RNA polymerase highly purified by gel filtration, DEAE-cellulose chromatography, phosphocellulose chromatography, and glycerol gradient centrifugation . Enzyme III bound more tightly to phosphocellulose and sedimented more rapidly during zone centrifugation than did RNA polymerase lacking the sporulation polypeptides . RNA polymerase containing P85 and P27 transcribed B . subtilis DNA about 4.5 times more actively than did core RNA polymerase, although both enzymes exhibited similar activities with poly(dA-dT) and phage phie DNA as templates . Enzyme III and core RNA polymerase also differed in their response to increasing concentrations of Mg2+ and KCl. Mol Gen Genet, 1975 Dec 23, 142(1), 45 - 55 Gene expression of bacteriophage SPPI . I . Phage directed protein synthesis; Esche H et al.; A total of 23 phage specific proteins (including four head and six tail proteins) could be identified after SDS polyacrylamide gel electrophoresis of extracts from phage SPP1 infected Bacillus subtilis cells . The total molecular weight of the proteins amounts to approximately 1.9 X 10(6) daltons, equivalent to the majority of the coding capacity of SPP1 DNA . It can thus be assumed that almost all SPP1 coded proteins have been identified . Protein assignments to phage cistrons were made by analysis of extracts from nonpermissive cells infected with sus-mutants . The SPP1 specified proteins can be subdivided into three groups on the basis of the time of their synthesis during the latent period . Host protein synthesis is not significantly affected by SPP1 infection . Normal expression of host genes appears to be essential for SPP1 growth. Biochim Biophys Acta, 1975 Dec 18, 410(2), 354 - 60 Endo-arabinanase from Bacillus subtilis F-11; Kaji A et al.; An arabinanase was purified from the culture fluid of Bacillus subtilis F-11 . The process was as follows: salting out by (NH4)2SO4, repeated chromatography on hydroxy apatite and gel filtration on Sepharose-6B . The purified enzyme was demonstrated to be homogeneous by disc electrophoresis . The enzyme was found to be active on arabinan and 1,5-arabinan, but inactive on phenyl alpha-L-arabinofuranoside, p-nitrophenyl beta-D-galactopyranoside, arabinoxylan, gum arabic . The enzyme released arabinose, arabinobiose, arabinotriose and higher oligosaccharides during the course of hydrolysis of 1,5-arabinan . The end products were found to be arabinose and arabinobiose after 144 h of hydrolysis. Biochem J, 1975 Dec, 152(3), 517 - 22 Stringent control of ribonucleic acid synthesis in Bacillus subtilis treated with granaticin; Ogilvie A et al.; The antibiotic granaticin interferes in Bacillus subtilis with the charging process of tRNALeu causing both the arrest of protein synthesis and bacteriostasis {A . Ogilvie, K . Wiebauer & W . Kersten (1975) Biochem . J . 152, 511-515} . A concomitant inhibition of RNA synthesis is observed . This inhibition was studied with mutant strains of B . subtilis . 2 . Granaticin inhibits protein and RNA synthesis in stringently controlled B . subtilis (rel+) to about the same extent . In a relaxed mutant strain (rel-) of B . subtilis, protein synthesis is also inhibited, but the accumulation of RNA continues after the addition of the drug . 3 . Chloramphenicol, which is known to abolish the stringent control mechanism, added simultaneously with granaticin, allows the synthesis of RNA to proceed in the stringent strain . 4 . Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) accumulate in granaticin-treated stringently controlled B . subtilis but not in the rel- mutant . 5 . It is concluded that the inhibition of RNA synthesis granaticin can adequately be explained as a stringent response caused by the interference by the drug with leucyl-tRNA synthetase. Biochem J, 1975 Dec, 152(3), 511 - 5 Inhibition of leucyl-transfer ribonucleic acid synthetasymol; Ogilvie A et al.; The bacteriostatic effect of low concentrations of the antibiotic granaticin on Bacillus subtilis is relieved by the addition leucine to the growth medium . In cells treated with granaticin, aminoacylation of leucine tRNA is specifically decreased, but the content of free leucine is not . It is concluded that granaticin interferes with the charging process of leucine tRNA in B . subtilis leading to leucine auxotrophy. Proc Natl Acad Sci U S A, 1975 Dec, 72(12), 4886 - 90 Synthesis of specific functional messenger RNA in vitro by phage-SP01-modified RNA polymerase of Bacillus subtilis; Swanton M et al.; RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was purified from rifampicin-resistant Bacillus subtilis, from both uninfected cells and cells infected with bacteriophage SP01 . The enzyme from infected cells lacked all traces of the sigma subunit, contained several polypeptides absent from the enzyme made in uninfected cells, and had an altered template specificity in a transcription assay . A cell-free protein synthesizing system from Escherichia coli, when poisoned with rifampicin, was completely dependent on addition of either of these RNA polymerase preparations for DNA-dependent protein synthesis . Under these conditions, the SP01-modified RNA polymerase preferentially stimulated the synthesis of functional mRNA for the phage enzyme dCMP deaminase (deoxycytidylate aminohydrolase, EC 3.5.4.12), whereas unmodified B . subtilis RNA polymerase could stimulate synthesis of this mRNA in small quantity and only after prolonged incubation . This mRNA belongs to a class of phage transcripts (m) which cannot be transcribed in vivo in the absence of phage-specific protein synthesis. J Bacteriol, 1975 Dec, 124(3), 1429 - 38 Heterologous deoxyribonucleic acid uptake and complexing with cellular constituents in competent Bacillus subtilis; Soltyk A et al.; With competent cultures of Bacillus subtilis the uptake of Escherichia coli deoxyribonucleic acid (DNA) is about 50% that for homologous DNA . Uptake of phage T6 DNA, if any, is of the order of 7%, while nonglucosylated phage T6 (T6) DNA is taken up almost as effectively as homologous DNA . Both T6 and T4 DNA interfere only minimally with uptake of homologous DNA; by contrast, T6 DNA competes with homologous DNA as effectively as the latter itself . These results indicate that the glucose residues in the T-even phage DNA, located in the large groove of the DNA helix, reduce affinity for cellular receptors, leading to low binding of T6 DNA . The latter DNA is considerably less degraded by extracellular nucleases than homologous DNA, thus excluding enzymatic hydrolysis as the source of poor uptake . Affinity of DNA for competent cells was also evaluated by the formation, and detection in a CsCl density gradient, of complexes of DNA with cellular constituent(s) . Such comlexes, similar to those previously observed with transforming DNA, are formed by E . coli DNA and T6 DNA; in reconstruction experiments the denatured forms of these same DNA samples form complexes when added to the cells before lysis . T6 DNA, on the other hand, does not form such a complex . The possible role of such complexes in transport of DNA to the cell interior is discussed. J Bacteriol, 1975 Dec, 124(3), 1236 - 9 Identification of poly-gamma-glutamyl chain lengths in folates of Bacillus subtilis; Hintze DN et al.; Bacillus subtilis strains 168 met ile leu and 23 thy contain folates which differ from one another in the number of glutamyl residues . The folate species were identified by reductive cleavage to the corresponding p-aminobenzoylglutamyl poly-gamma-glutamates and chromatography on diethylaminoethyl-cellulose . Pteroyltriglutamate is the predominant folate type, accounting for 86 to 88% of the total . Pteroyltetraglutamate is the only other type present in appreciable quantities, accounting for 5 to 6% of the total folates . Pteroyldiglutamate and pteroylpentaglutamate are present in small amounts, accounting for 1 to 3% and 1% of the total folates, respectively . Strain 168 met ile leu contains a very small amount of pteroylmonoglutamate (less than 0.5% of the total folates), but the other strain contains none. J Bacteriol, 1975 Dec, 124(3), 1062 - 6 Isolation of 30S and 50S active ribosomal subunits of Bacillus subtilis, Marburg strain; Guha S et al.; Active 30S and 50S ribosomal subunits were isolated from Bacillus subtilis . These subunits were able to perform not only protein synthesis in the presence of artificial or natural messenger ribonucleic acid but also the specific functions characteristic of each of the subunits . Thus the 30S subunits alone are able to bind formyl-methionyl-transfer ribonucleic acid, and the 50S subunits carry the peptidyl transferase activity. J Biol Chem, 1975 Nov 25, 250(22), 8824 - 8 Antibody directed against Bacillus subtilis rho factor purified by sodium dodecyl sulfate slab gel electrophoresis . Effect on transcription by RNA polymerase in crude extracts of vegetative and sporulating cells; Tjian R et al.; Antibody directed against rho factor from vegetative Bacillus subtilis was prepared by immunizing a rabbit with denaturated rho polypeptide isolated by electrophoresis of partially purified DNA-dependent RNA polymerase on a sodium dodecyl sulfate-polyacrylamide slab gel . Antiserum to rho reacted specifically with native rho polypeptide but not with core RNA polymerase as judged by complement fixation and by an immunodiffusion assay . Anti-rho antibody also inhibited the ability of rho to stimulate transcription of phage phie DNA but failed to inhibit transcription of poly(dA-dT) by core enzyme . Specific antibody was also raised against a mixture of the beta and beta' subunits of RNA polymerase purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The effect of the anti-rho gamma-globulin on the transcription of phage phie DNA by RNA polymerase in crude extracts of vegetative and sporulating cells was examined . Anti-rho antibody markedly inhibited the transcription of phage DNA by RNA polymerase partially purified from vegetative bacteria by ammonium sulfate fractionation but had little effect on transcription of the phage DNA template by enzyme from sporulating cells . Addition of purified rho to a vegetative extract that had been depleted of rho by treatment with the anti-rho antibody restored active transcription of phage DNA . However, addition of purified rho to an antibody-treated extract of sporulating cells had little effect on phie RNA synthesis . These findings suggest that sporulating cells contain a component that interferes with the activity of the rho subunit of RNA polymerase. J Biol Chem, 1975 Nov 25, 250(22), 8804 - 11 Bacillus subtilis deoxyuridinetriphosphatase and its bacteriophage PBS2-induced inhibitor; Price AR et al.; Extracts of Bacillus subtilis contain a deoxyuridinetriphosphatase (dUTPase) activity with a molecular weight of approximately 48,000 . The enzyme is maximally active at pH 8.5, being stimulated by Mg2+ and inhibited by EDTA . The enzyme is specific for dUTP among all the natural nucleotides tested, with an apparent Km for dUTP of 2 muM . Bacteriophage PBS2, whose DNA contains uracil instead of thymine, induces upon infection of B . subtilis a new 83,000-dalton protein which inhibits the host's dUTPase . The inhibitor acts immediately and reversibly in vitro to inhibit dUMP production from dUTP . The inhibitor's action is maximal in dUTPase assays performed at pH 6 to 7, and is minimal at pH 9.7 . The inhibitor seems to form a higher molecular weight complex with the B . subtilis dUTPase . Increasing the pH of the medium for PBS2 infection from pH 7 to pH 8.85 caused a dramatic decrease in the synthesis of phage DNA and progeny phage . The newly synthesized DNA had an altered thymine/uracil ratio, being increased from less than 0.03 to greater than 1.0 . We propose that infection at high pH prevents the PBS2-induced dUTPase inhibitor from blocking the B . subtilis dUTPase activity, thereby allowing the degradation of dUTP and the synthesis of dTTP (both of which are DNA polymerase substrates), so that thymine replaces some of the uracil normally found in PBS2 DNA. J Biol Chem, 1975 Nov 25, 250(22), 8664 - 9 Purification and properties of Bacillus subtilis aspartate transcarbamylase; Brabson JS et al.; Aspartate transcarbamylase from Bacillus subtilis has been purified to apparent homogeneity . A subunit molecular weight of 33,500 +/- 1,000 was obtained from electrophoresis in polyarcylamide gels containing sodium dodecyl sulfate and from sedimentation equilibrium analysis of the protein dissolved in 6 M guanidine hydrochloride . The molecular weight of the native enzyme was determined to be 102,000 +/- 2,000 by sedimentation velocity and sedimentation equilibrium analysis . Aspartate transcarbamylase thus appears to be a trimeric protein; cross-linking with dimethyl suberimidate and electrophoretic analysis confirmed this structure . B . subtilis aspartate transcarbamylase has an amino acid composition quite similar to that of the catalytic subunit from Escherichia coli aspartate transcarbamylase; only the content of four amino acids is substantially different . The denaturated enzyme has one free sulfhydryl group . Aspartate transcarbamylase exhibited Michaelis-Menten kinetics and was neither inhibited nor activated by nucleotides . Several anions stimulated activity 2- to 5-fold . Immunochemical studies indicated very little similarity between B . subtilis and E . coli aspartate transcarbamylase or E . coli aspartate transcarbamylase catalytic subunit. Can J Microbiol, 1975 Nov, 21(11), 1866 - 76 The function of slime from Physarum flavicomum in the control of cell division; Henney HR Jr et al.; A haploid cell of the myxomycete Physarum flavicomum undergoes cytokinesis, producing a large population of cells . However, after syngamy, cytokinesis no longer occurs but karyokinesis does and subsequent growth results in the formation of a diploid syncytial plasmodium . Slime, which is produced by the plasmodium but not the haploid cells, was aseptically isolated and purified, and tested for its effect as a cytokinetic regulator . Slime (a viscous, high molecular weight, acidic glycoprotein) affected cytokinesis of the haploid myxamoebae growing in pure culture in soluble media, and the effect was concentration dependent . In simple media, a slime concentration of about 6 10(-5) mug protein per cell suppressed cytokinesis about 50%, unequally inhibited the synthesis of protein, RNA, and DNA, but stimulated respiration . The biological activity of slime was not species specific and it also affected the bacterium Bacillus subtilis by inhibiting cytokinesis, stimulating oxygen uptake, and producing an aberrant cell morphology . Slime was inactivated by heat, fragmentation, and incubation with dithiothreitol, mercaptoethanol, and the proteolytic enzyme papain (EC 3.4.22.2) . The inhibitory effect of slime on cell division of haploid cells could not be achieved using mucin or various polyanions . The possible role of slime in the production of the diploid syncytium is discussed. Biochim Biophys Acta, 1975 Nov 3, 406(4), 564 - 74 Control of membrane protein synthesis in Bacillus subtilis; Sargent MG; In synchronous cultures of Bacillus subtilis 168/S grown on succinate as a sole carbon source (mean generation time 115 min), chromosome initiation occurs at the beginning of the cell cycle but the rate of membrane protein synthesis doubles in mid-cycle more or less coincident with nuclear segregation . In glucose-grown cultures, the doubling in rate of membrane protein synthesis occurs at about the same time as nuclear segregation and DNA initiation at the beginning of the cycle . Control of the rate of membrane synthesis by the chromosome has been demonstrated by inhibiting DNA synthesis using thymine starvation and showing that membrane protein synthesis continues at a constant rate, whereas the rate of cytoplasmic protein synthesis almost doubles . I suggest that the replication of a region at or close to the chromosome terminus is required to allow the doubling in rate of membrane synthesis. J Biochem (Tokyo), 1975 Nov, 78(5), 897 - 903 Structures of multi-branched dextrins produced by saccharifyiing alpha-amylase from starch; Umeki K et al.; From the digest of beta-limit dextrin (prepared from glutinous rice starch) with saccharifying alpha-amylase of Bacillus subtilis {EC 3.2.1.1} (BSA), two extensibely branched dextrins consisting of nine (No . 6, Fig . 1) and ten (No 7, Fig.1) glucose units were isolated by paper chromatography . Structural analysis using various enzymes revealed that No . 6 and No . 7 were both mixtures of four triply branched dextrins . They had structures which were built up with 63-alpha-glucosylmaltotriose and/or 62-alpha-glucosylmaltose as a linking unit . However, the branching configuration and the minimum alpha-1, 4-glucosidic linkages existing between two branches followed one of the three structures shown below: (see article). Mutat Res, 1975 Nov, 30(2), 177 - 90 Repair of UV-induced DNA damage in recombination-deficient strains of Bacillus subtilis; Shibata T et al.; To clarify the role of gene products for genetic recombination which might be concerned in excision repair, the repair of DNA lesions induced by ultraviolet (UV) irradiation was examined under non-growing conditions with a variety of recombination deficient (Rec-) mutants of Bacillus subtilis . The extent of repair was estimated by the recovery of transforming activity of DNA extracted from the cells during the post-irradiation incubation period (the assay method was termed as marker-repair experiment) . The marker repair seemed to be accomplished by the excision repair as assumed from the effects of inhibitors . Among Rec- mutants tested, a mutant strain UVS80TH (rec-80) exhibited a normal level of marker repair activity, whereas strains GSY1025 (recA1), GSY1028 (recB2) and GSY908 (rec-4) exhibited reduced marker repair activities . These results and the data on UV sensitivity of the mutants indicate that (1) the products of all these Rec genes are related to UV resistance of the cell viability and are factors functioning through mechanisms other than excision repair, (2) the products of recA, recB and rec-4 genes display some roles in maintenance of a level of excision repair activity, and (3) the product of the rec-80 gene does not participate at all in excision repair. Hoppe Seylers Z Physiol Chem, 1975 Nov, 356(11), 1679 - 84 Effects of kryptopyrrole on porphyrin synthesis in Bacillus subtilis 168; Durko I et al.; We investigated the effects of exogenous 5-aminolaevulinate and kryptopyrrole, separately and together, on the porphyrin synthesis of Bacillus subtilis 168 . It was confirmed that 5-aminolaevulinate increases the porphyrin content of the culture fluid in the way previously described . In comparison with the basic activity of the bacteria, kryptopyrrole enhances the amount of extracellular copro III, uro III and 5-carboxyl porphyrins . The combination of 5-aminolaevulinate and kryptopyrrole enhances the porphyrin synthesis; moreover, a more pronounced increased in porphyrin synthesis occurs if the kryptopyrrole solution used is rich in its oxidation products . In this latter case, the amount of 5,6,7-carboxylic porphyrins increases markedly along with uro III and particularly copro III, while suburoporphyrins also makes their appearance . An hypothesis is advanced to explain tentatively the increase in the porphyrin synthesis provoked by kryptopyrrole. Genetics, 1975 Nov, 81(3), 437 - 58 Induction and transmission of a merodiploid condition near the terminal area of the chromosome of Bacillus subtilis; Jamet-Vierny C et al.; A small fraction (about 0.5%) of the transformants for a particular marker of B . subtilis (ilvA4; most probably a deletion) were found to be relatively unstable merodiploids . They possess a redundancy of the metB-ilvA chromosome segment . When their DNA is used as donor in transformation a merodiploid condition for the whole of this segment is created in all ilvA4+ transformants . For several of the duplicated loci both copies often are of recipient strain origin . Markers originally belonging to different copies of the diploidized region can be cotransferred in PBS1-mediated transduction . The data are well in agreement with the hypothesis that the merodiploids carry a tandem duplication . An alternative hypothesis which does not call for integration of the exogenote within the recipient chromosome was also considered . Models are proposed for interpreting the segregation of the merodiploids, the transmission of the diploid state and its generation during transformation of the ilvA4 marker by wild-type DNA. Proc Natl Acad Sci U S A, 1975 Nov, 72(11), 4341 - 5 Evidence of homologous relationship between thermolysin and neutral protease A of Bacillus subtilis; Levy PL et al.; A comparison of the partial amino-acid sequence of neutral protease A from Bacillus subtilis with the structure of thermolysin (EC 3.4.24.4) from Bacillus thermoproteolyticus reveals that these two proteins are homologous . Of 171 residues placed in neutral protease (54% of the sequence), 83 residues (49%) occur in identical positions in thermolysin, and include nine of the 13 residues previously identified as components of the active site of thermolysin . This similarity provides support for the hypothesis that the two enzymes have similar three-dimensional structures and a common mechanism of action . Since these enzymes differ markedly in their resistance to heat inactivation, a comparison of their structures may eventually provide a chemical basis for explaining the differences in their thermal stability. Infect Immun, 1975 Nov, 12(5), 1189 - 94 Use of Bacillus subtilis minicells to demonstrate an antigenic relationship between the poles and lateral cylindrical regions of rod-shaped cells; Coyne SI et al.; Purified minicells produced by Bacillus subtilis div IV-B1 mutants were used to immunize rabbits . Immune serum was obtained that agglutinated minicells and was able to form precipitin lines when reacted with minicell antigens in double-diffusion or immunoelectrophoretic procedures . Antiminicell serum agglutinated rod-shaped B . subtilis cells to long filaments produced by growth of a cell division-defective mutant at restrictive temperature . These findings indicate that minicells are immunogens capable of eliciting the production of antibodies that cross-react with the lateral, cylindrical regions of B . subtilis rods . It appears, therefore, that poles share a common antigen(s) with cylindrical regions of the cell. J Virol, 1975 Nov, 16(5), 1282 - 95 Morphogenesis of bacteriophage phi29 of Bacillus subtilis: cleavage and assembly of the neck appendage protein; Tosi ME et al.; Each of the 12 neck appendages of the Bacillus subtilis bacteriophage phi29 consists of a single protein molecule with a molecular weight of about 75,000, and on the mature virion the appendages are assembled to the lower of two collars . The appendage protein is cleaved from a precursor protein, P(J), with a molecular weight of about 88,000 . This cleavage is independent of neck assembly, occurring during infection by mutants that cannot synthesize the proteins of the upper and lower collars of the neck . The cleaved form of the appendage protein is efficiently complemented in vitro to particles lacking appendages . Thus, cleavage of the appendage precursor protein apparently does not occur in situ on the maturing virus. J Bacteriol, 1975 Nov, 124(2), 977 - 84 Deoxyribonucleic acid-binding proteins in vegetative Bacillus subtilis: alterations caused by stage O sporulation mutations; Brehm SP et al.; Deoxyribonucleic acid (DNA)-binding proteins have been compared between wild-type Bacillus subtilis and five sporulation mutants blocked at different stage O loci . Extracts from exponentially growing cells have been fractionated for proteins binding to single-stranded calf thymus DNA-cellulose and double-stranded B . subtilis DNA-cellulose . In nutrient broth, stage O mutations cause an accumulation of proteins with affinity for double-stranded DNA . Suppression of the mutation with extragenic suppressors relieves the accumulation . In minimal glucose medium, the stage O mutations also cause accumulation of proteins with affinity for double-stranded DNA, but the species accumulated are different from those of nutrient broth-grown cells . In neither case did stage O mutations affect proteins with affinity for single-stranded DNA . The results suggest that the products of stage O loci are functional and operative during vegetative growth. J Bacteriol, 1975 Nov, 124(2), 1030 - 3 Association of the replication terminus of the Bacillus subtilis chromosome to the cell membrane; Yamaguchi K et al.; A chromosomal segment containing several genetic markers, from metB to thyA, near the replication terminus is associated with the membranous structure of Bacillus subtilis, but markers adjacent to this region, lys, ura, and metC, are not. J Bacteriol, 1975 Nov, 124(2), 668 - 78 Peptidoglycan synthesis in L-phase variants of Bacillus licheniformis and Bacillus subtilis; Ward JB; Stable L-phase variants isolated from Bacillus licheniformis and Bacillus subtilis, when grown in osmotically stabilized media, do not synthesize peptidoglycan but have been found to accumulate the nucleotide precursors of this polymer . The enzymes involved in the synthesis of these precursors and the later membrane-bound stages of peptidoglycan synthesis have been investigated, and the L-phase variants have been shown to contain lesions, which provide a rational explanation for the absence of peptidoglycan and for the nature of the precursor accumulated . The majority of the L-phase variants contained a single enzymic defect, but two strains were isolated with double lesions . Five out of seven strains examined accumulated uridine 5'-diphosphate (UDP)-MurAc-L-ala-D-glu and were unable to synthesize diaminopimelic acid as a consequence of a defect in aspartate-beta-semialdehyde dehydrogenase activity . Two strains were deficient in UDP-MurAc: L-alanine ligase and accumulated UDP-MurAc . One strain accumulated the complete nucleotide precursor UDP-MurAc-L-ala-D-glu-mA2pm-D-ala-D-ala and was deficient in phospho-N-acetylmuramyl pentapeptide translocase . A second strain also had this lesion, together with defective aspartate-beta-semialdehyde dehydrogenase activity . The other enzymes of peptidoglycan synthesis were present in the L-phase variants, with activities similar to those found in the parent bacilli grown under identical conditions . Membrane preparations from certain of the L-phase variants were also capable of synthesizing the secondary polymers poly(glycerol phosphate) teichoic acid and teichuronic acid and also a polymer of N-acetylglucosamine. J Bacteriol, 1975 Nov, 124(2), 613 - 22 Dicarboxylic acid transport in membrane vesicles from Bacillus subtilis; Bisschop A et al.; Membrane vesicles isolated from Bacillus subtilis W23 catalyze active transport of the C4 dicarboxylic acids L-malate, fumarate, and succinate under aerobic conditions in the presence of the electron donor reduced beta-nicotinamide adenine dinucleotide or the non-physiological electron donor system ascorbate-phenazine methosulfate . The dicarboxylic acids are accumulated in unmodified form . Inhibitors of the respiratory chain, sulfhydryl reagents, and uncoupling agents inhibit the accumulation of the dicarboxylic acids . The affinity constants for transport of L-malate, fumarate, and succinate are 13.5, 7.5, and 4.3 muM, respectively; these values are severalfold lower than those reported previously for whole cells . Active transport of these dicarboxylic acids occurs via one highly specific transport system as is indicated by the following observations . (i) Each dicarboxylic acid inhibits the transport of the other two dicarboxylic acids competitively . (ii) The affinity constants determined for the inhibitory action are very similar to those determined for the transport process . (iii) Each dicarboxylic acid exchanges rapidly with a previously accumulated dicarboxylic acid . (iv) Other metabolically and structurally related compounds do not inhibit transport of these dicarboxylic acids significantly, except for L-aspartate and L-glutamate . However, transport of these dicarboxylic amino acids is mediated by independent system because membrane vesicles from B . subtilis 60346, lacking functional dicarboxylic amino acid transport activity, accumulate the C4 dicarboxylic acids at even higher rates than vesicles from B . subtilis W 23 . (v) A constant ratio exists between the initial rates of transport of L-malate, fumarate, and succinate in all membrane vesicle preparations isolated from cells grown on various media . This high-affinity dicarboxylic acid transport system seems to be present constitutively in B . subtilis W23. Prikl Biokhim Mikrobiol, 1975 Nov-Dec, 11(6), 842 - 7 {Thermo- and pH-stability of soluble and silichrome-80 immobilized proteinase from Bacillus subtilis}; Kalashinikova AM et al.; The stability to heating and pH changing of two proteinases of Bacillus subtilis was studied . The preparations were: protosubtilin G10x with an activity of 21500 units/g and proteinase immobilized on silochrome C-80 with an activity of 3880 units/g . For protosubtilin G10x the pH optimum was 7.0-7.2 . For the immobilized preparation 96-100% activity was found at pH 5.5-10.0 . The difference between the two proteinase preparations was more distinct with respect to the temperature minimum . The initial preparation-protosubtilin G10x was more stable to low temperature and showed maximum activity at 40 degrees; the immobilized preparation was less active at low temperatures and showed maximum activity at 60-70 degrees . Protosubtilin G10x was more sensitive to pH changes, especially in the acid zone (pH 4.5) . It was significantly activated at 30-40 degrees and was unstable at 50