J Biol Chem, 1980 Nov 10, 255(21), 10354 - 8 Inhibitor of uracil-DNA glycosylase induced by bacteriophage PBS2 . Purification and preliminary characterization; Cone R et al.; A PBS2 phage-coded inhibitor of uracil-DNA glycosylase activity from Bacillus subtilis has been purified extensively and characterized preliminary . The inhibitor has a relative S value of 1.44 +/- 0.08 measured by sedimentation in 15 to 40% glycerol density gradients . It is unusually stable to heating and to the presence of sodium dodecyl sulfate and/or 8 M urea . The inhibitor has no known cofactor requirement and is active in the presence of 10 mM EDTA . Inhibitor activity is sensitive to digestion with proteinase K, but is insensitive to DNase or RNase digestion . The purified inhibitor behaves anomalously during electrophoresis in poly-acrylamide gels containing sodium dodecyl sulfate; however, experiments designed to show that the inhibitor is a glycoprotein were negative . The inhibitor clearly contains a protein required for activity, however, the possibility that some other molecular component is part of the active inhibitor cannot be excluded. Biochim Biophys Acta, 1980 Nov 7, 620(2), 332 - 7 Cardiolipin, a major phospholipid of Gram-positive bacteria that is not readily extractable; Filgueiras MH et al.; Extraction of phospholipids from stationary phase grown cells of the Gram+ bacteria, Bacillus megaterium, Bacillus subtilis, Bacillus cereus and Micrococcus lysodeikticus was found to be incomplete with various commonly used extraction procedures . Phosphatidylglycerol and phosphatidylethanolamine were readily extracted but up to 95% of the cardiolipin appeared to be retained within the cell residue . Extraction of the cardiolipin could be slightly enhanced by increasing the temperature or the acidity of the extraction solutions but complete extraction was obtained only after lysozyme treatment of intact cells or cell residues remaining after extraction . In addition complete extraction could be observed in the case of cells harvested in the early logarithmic phase . Freeze-fracture electron microscopy was carried out on the cell residue remaining after extraction of all phospholipids except cardiolipin . A fracture plane through the plasma membrane could not be observed anymore . Instead fracture planes through lipid vesicles were observed . These vesicles reside within the remnants of the cytoplasm and consist most likely of the non-extracted cardiolipin. J Gen Microbiol, 1980 Nov, 121(1), 79 - 84 DNA binding and deoxyribonuclease activity in Bacillus subtilis during temperature-induced competence development; Espinosa M et al.; Rapid development of competence can be induced in cultures of Bacillus subtilis by incubation at 37 degrees C after previous growth at 42 degrees C . This temperature-induced competence was accompanied by an increase in DNA binding capacity and breakdown of donor DNA . Inhibition of protein synthesis prevented the rapid increase of competence . This is probably due to the inhibition of de novo synthesis of a constituent that enables the bacteria to bind DNA. J Gen Microbiol, 1980 Nov, 121(1), 69 - 78 Suppression of asporogeny in Bacillus subtilis . Allele-specific suppression of a mutation in the spoIIA locus; Yudkin MD et al.; From a strain carrying spoIIA69, a mutation giving rise to asporogeny, a revertant was isolated which sporulated at about 10(-2) of the wild-type frequency but in which the time-course of sporulation was much protracted . Genetic analysis of this revertant showed that it retained spoIIA69 but had acquired a secondary mutation sas . sas failed to suppress mutations in spoIID, spoIIE or spoIIG; it also failed to suppress another mutation in the spoIIA locus . sas is extremely closely linked (recombination frequency less than or equal to 1%) with the mutation spoIIA69 that it specifically suppresses . Strains carrying sas alone sporulated at a frequency at least two orders of magnitude below that in the spoIIA69 sas double mutant . It is suggested that spoIIA60 and sas lead to compensating amino acid changes in the protein specified by the spoIIA locus. J Gen Microbiol, 1980 Nov, 121(1), 263 - 6 Transfer of plasmids among bacilli; Dancer BN; Plasmids were transferred from Staphylococcus aureus to Bacillus subtilis and between B . subtilis and several other species of Bacillus by protoplast fusion and regeneration . The plasmids replicate and express themselves normally in all bacilli examined. J Gen Microbiol, 1980 Nov, 121(1), 139 - 49 Effects of ethanol and methanol on lipid metabolism in Bacillus subtilis; Rigomier D et al.; In Bacillus subtilis, the fatty acid moiety of the phospholipids was affected differently during growth in the presence of 1.1 M-methanol or 0.7 M-ethanol, though at these concentrations methanol and ethanol had the same effects on growth rate and completely inhibited sporulation . Synthesis of phosphatidylglycerol was also strongly inhibited and the amount of total cell phospholipids was reduced by 50% by both alcohols . The composition of fatty acids, especially the relative concentration of 12-methyltetradecanoic acid, was modified only by ethanol; in bacteria grown in the presence of methanol, changes in fatty acid composition were negligible . In non-sporulating mutants, synthesis of phosphatidylglycerol was much less affected than in the wild-type and synthesis of phosphatidylethanolamine was increased . In these strains, fatty acid composition was also modified by ethanol but unaffected by methanol. J Gen Microbiol, 1980 Nov, 121(1), 113 - 6 Statistical analysis of the patterns of spore formation in Bacillus subtilis; Dunn G; A description of the patterns of sporulation in chains of Bacillus subtilis cells has been obtained by fitting a statistical model to data obtained from a resuspended culture . The underlying assumptions of this model are: (a) resuspension causes an immediate response in the growing bacteria which is necessary for subsequent sporulation; (b) the probability of this response occurring is the same for all the bacteria present at the time of resuspension; (c) after resuspension some of the bacteria pass through a single round of cell division to produce a pair of sporulating bacteria, while the rest divide twice to produce four; and (d) the two sub-populations described in (c) differ in their ability to complete sporulation. Biokhimiia, 1980 Nov, 45(11), 2083 - 95 {Metalloproteinase from Bacillus subtilis: - "intracellular" and extracellular enzymes}; Shaginian KA et al.; "Intracellular" metalloproteinase was purified to homogeneity from Bacillus subtilis 103 crude cell extract, using affinity chromatography on bacitracin-Sepharose 4B . The degree of purification and the yield of the enzyme were about 260-fold and 3%, respectively . In its physico-chemical properties and the amino acid composition the enzyme is very similar, if not identical, to the extracellular metalloproteinase isolated from the culture filtrate of the same strain . Extracellular metalloproteinase-deficient mutant strain Bacillus subtilis SMY-512 does not produce the "intracellular" enzyme either . THe activity of "intracellular" metalloproteinase in the periplasmic space of the cells is about 70% of that in the cytoplasm, thus being indicative of a rather regular distribution of the enzyme throughout the cell compartment. Can J Microbiol, 1980 Nov, 26(11), 1328 - 33 Prophage-mediated production of a bacteriocinlike substance by SP beta lysogens of Bacillus subtilis; Hemphill HE et al.; Cultures of Bacillus subtilis lysogenic for the temperature bacteriophage SP beta release "betacin", a bacteriocinlike substance that inhibits B . subtilis strains which do not carry this phage . Production of betacin is blocked by mutations in the bet gene on the prophage and a second phage gene, tol, is apparently involved in making the lysogen itself tolerant to betacin . Mutations in a bacterial gene betR, located on the B . subtilis chromosome between metC and pyrD, render nonlysogens tolerant to betacin. Mikrobiologiia, 1980 Nov-Dec, 49(6), 876 - 9 {Effect of electro-immobilization on Bacillus subtilis}; Mogilevich NF et al.; The culture of Bacillus subtilis 21 was subjected to the action of nonuniform electric field, and the effect of the latter on the bacterial survival and biochemical activity was studied . The action of the field on the cells was shown to depend on the material of a load on which the culture was immobilized . The studied properties of Bac . subtilis 21 did not change when the culture was immobilized on cellulose fiber . About 50--60% of the cells died on silica gel under the action of field; the respiration activity and the rate of hexamethylene diamine destruction did not change . Almost all of the bacterial cells lost their viability upon electroimmobilization on ion-exchange resins . The destructive properties of the culture retained by the field exceed the activity of the control variants. J Gen Virol, 1980 Nov, 51(Pt 1), 125 - 35 Effects of calcium on the lytic cycle of Bacillus subtilis phage 41c; Landry EF et al.; The lytic cycle of Bacillus subtilis phage 41c required the presence of at least 10 mM-calcium . In the absence of this ion, the plaquing efficiency of the virus was reduced to less than 0.1 . Likewise, replacement of Ca2+ with other divalent ions (Ba2+, Sr2+, Mg2+, Mn2+) resulted in reduced efficiencies . Adsorption of 41c was Ca2+-dependent, requiring concentrations ranging from 0.1 to 10mM . Although more than 90% of the phage adsorbed at 0.1 mM-Ca2+, successful infection could only be achieved at higher Ca2+ levels . Sub-optimal concentrations of the ion resulted in the loss of 90% of infected centres within 1 min after the initiation of infection, indicating an early post-adsorption ion requirement . Penetration of experiments with 32P-labelled phage DNA indicated than an irreversible inhibition of injection was occurring in the majority of the phage-bacterium complexes . A third level of cation involvement became apparent when phage-bacterium complexes in which penetration had occurred exhibited a greatly reduced burst size . The post-penetration ionic requirement occurred early in the infection process since dilution of infected complexes into Ca2+-free medium at 2.5 min p.i . resulted in reduced phage yields . The requirement was dispensable after 6 min p.i., since infected complexes diluted into Ca2+-free medium at this time exhibited a normal one-step growth curve . Analysis of messenger RNA production by molecular DNA-RNA hybridization techniques indicated that transcriptional events were similar in the presence and absence of Ca2+ . At present, the identification of the third ion-dependent stage is unresolved. J Med Chem, 1980 Nov, 23(11), 1242 - 4 8-Hydroxyanthracyclinones from epsilon-rhodomycinone; Lin H et al.; epsilon-Rhodomycinone was converted into 8,9-dehydro-zeta-rhodomycinone, which gave a cis diol with osmium tetroxide and a pair of epimeric epoxides with m-chloroperbenzoic acid . Acid-catalyzed opening of the epoxides gave the corresponding trans diols . In contrast, acid treatment of the trimethyl ethers of these epoxides gave predominantly a lactone and an eta-rhodomycinone derivative, with only small amounts of the diols . None of the new rhodomycinones were active against Bacillus subtilis, but 8,9-dehydro-zeta-rhodomycinone was active in the induction of lytic phage in Escherichia coli. J Bacteriol, 1980 Nov, 144(2), 840 - 3 Postreplication repair of deoxyribonucleic acid and daughter strand exchange in uvr- mutants of Bacillus subtilis; Dodson LA et al.; The fate of pyrimidine dimers in deoxyribonucleic acid (DNA) newly synthesized by Bacillus subtilis after ultraviolet irradiation was monitored by use of a damage-specific endonuclease that introduces single-strand breaks adjacent to nearly all of the dimer sites . Two Uvr- strains, one defective in the initiation of dimer excision and the other defective in a function required for efficient dimer excision, were found to be similar to their wild-type parent in the kinetics and extent of converting low-molecular-weight DNA newly synthesized after ultraviolet irradiation to high molecular weight . In the Uvr- strains large molecules of newly synthesized DNA remained susceptible to nicking by the damage-specific endonuclease even after extended incubation in growth medium, whereas the enzyme-sensitive sites were rapidly removed from both preexisting and newly synthesized DNA in Uvr+ cells . Our results support the hypothesis that postreplication repair in bacteria includes recombination between dimer-containing parental DNA strands and newly synthesized strands. J Bacteriol, 1980 Nov, 144(2), 608 - 15 Capacity for postreplication repair correlated with transducibility in Rec- mutants of Bacillus subtilis; Dodson LA et al.; Bacillus subtilis strains deficient in transduction, transformation, or both were examined for the ability to remove pyrimidine dimers and to convert deoxyribonucleic acid newly synthesized after ultraviolet irradiation to high molecular weight . In one strain deficient in both recombination processes, short pieces of deoxyribonucleic acid synthesized after irradiation were not converted to high molecular weight . Two transformable strains deficient in transduction were also deficient in postreplication repair (i.e., joining of newly synthesized DNA fragments), whereas a nontransformable strain that was normal in transduction was proficient in postreplication repair . None of the transformable strains showed deficiencies in repair resynthesis or ligase activity . Our results suggest that some recombinational events may be common to transduction and postreplication repair but not to transformation, emphasizing the difference between these two pathways for genetic exchange. Mol Biol (Mosk), 1980 Nov-Dec, 14(6), 1342 - 53 {Guanosine polyphosphate concentration and stable RNA synthesis in Bacillus subtilis following suppression of protein synthesis}; Belitskii BR et al.; Amount of guanosine-5'-triphosphate, 3'-diphosphate (pppGpp) and guanosine-5'-diphosphate, 3'-diphosphate (ppGpp) in the cells of b . subtilis increased several times during starvation for lysine or after treatment with serine hydroxamate (analog of serine) or norvaline (analog of leucine), or in the presence of trimethoprim, which induced deficiency of methionine and leucine . In exponentially growing cells the concentration of pppGpp was found to be 10-20 pmol/A600 . When serine hydroxamate or trimethoprim were added, concentration of pppGpp increased to 500-800 pmol/A600 and then slowly diminished . Elimination of lysine or addition to the culture medium of norvaline caused slight transitory accumulation of pppGpp (150 pmol/A600) . The amount of another nucleotide ppGpp was always 2-3 times lower than one of pppGpp . Accumulation of (p)ppGpp in rel+ cells was accompanied by cessation of stable RNA synthesis . Under conditions described above rel- cells continued RNA synthesis and did not accumulate (p)ppGpp . In the rel+ cells treated with serine hydroxamate synthesis of stable RNA resumed and the amount of (p)ppGpp decreased after addition of serine or tetracycline and chloramphenicol . The half-life period for pppGpp in the presence of chloramphenicol was determined to be 30-40 seconds . Thus, during aminoacyl-tRNA deficiency rel+ cells of B . subtilis accumulate (p)ppGpp, which are believed to participate in negative regulation of RNA synthesis . Slight accumulation of pppGpp without concomitant inhibition of stable RNA synthesis was observed after treatment of growing cells with chloramphenicol. Nucleic Acids Res, 1980 Oct 10, 8(19), 4517 - 20 Nucleotide sequence of non-initiator methionine tRNA from Bacillus subtilis; Yamada Y et al.; Non-initiator methionine tRNA (tRNAMet) was purified from Bacillus subtilis W 168 by a consecutive use of several column chromatographic systems . The nucleotide sequence was determined to be p-G-G-C-G-G-U-G-U-A-G-C-U-C-A-G-C-G-G-C-D-A-G-A-G-C-G-U-A-C-G-G-U-U-C-A-U-m6A-C-C-C -G-U-G-A-G-G(m7G)-U(D)-C-G-G-G-G-G-T-psi-C-G-A-U-C-C-C-C-U-C-C-G-C-C-G-C-U-A-C- C-A-OH . The nucleosides of G46 and U47 were partially modified to m7G and D, respectively . The nucleotide sequence shows a unique feature that the position adjacent to 3'-end of the anticodon C-A-U is occupied by m6A, not by t6A, although the tRNAMet belongs to a groups of tRNAs which recognize codons starting with A. J Antibiot (Tokyo), 1980 Oct, 33(10), 1146 - 9 Characterization of a new antibiotic of iturin group: bacillomycin D; Peypoux F et al.; The characterization of an antibiotic isolated from a strain of Bacillus subtilis revealed that this compound is a new antifungal antibiotic of the iturin group . It contains a lipid moiety which is a mixture of 3-amino 12-methyl tridecanoic acid (40%) and 3-amino 12-methyl tetradecanoic acid (60%) and a peptide moiety: L-Asp1, D-Asp1, L-Glu1, L-Pro1, D-Ser1, L-Thr1 and D-Tyr1 . These two moieties are joined by a threonyl-beta-aminoacid linkage. Biokhimiia, 1980 Oct, 45(10), 1788 - 96 {Characteristics of intracellular proteolysis in the cells of Bacillus subtilis}; Belitskii BR et al.; The rate of total protein degradation down to acid-soluble products in the B . subtilis cells growing on a minimal medium is about 4--5% per hour . Under amino acid deficiency the rate of proteolysis depends on the allelic state of the relA gene, so that in the rel+ cells it increases two-fold, while in the rel- cells it remains low . Elimination of NH4+, PO43- and Mg2+ from the culture medium or an addition of NaN3 (8 mM) or 2,4-dinitrophenol (2 mM) results in 1.5--2.0-fold stimulation of proteolysis independently of the relA gene . In all cases studied the rate of proteolysis decreases after addition of chloramphenicol (100 micrograms/ml) . It is proposed that chloramphenicol decreases the intracellular concentration of ppGpp, which is believed to exert pleiotropic alterations of cellular metabolism under condition of growth limitation . Quite different is the case of degradation of anomalous proteins synthesized in the presence of the lysine analog--S-2-aminoethylcystein . Degradation of anomalous proteins proceeds very rapidly (about 70% per hour); chloramphenicol (100 micrograms/ml) decreases the rate of proteolysis only two-fold . It was found that tetracycline (100 micrograms/ml) effectively inhibits the degradation of anomalous proteins . This activity of tetracycline was not observed in the presence of 50 mM of Mg2+ and seems to be dependent on the capacity of the antibiotic to form complexes with bivalent cations . These results reveal common features of control of proteolysis in the cells of B . subtilis and E . coli. Int J Pept Protein Res, 1980 Oct, 16(4), 327 - 38 Thermolysin and Bacillus subtilis neutral protease . Conformation and stability of two homologous neutral metalloendopeptidases; Grandi C et al.; A comparative study on thermolysin from Bacillus thermoproteolyticus and neutral protease from Bacillus subtilis involving (far- and near-ultraviolet circular dichroism (CD) and immunological techniques is reported . These enzymes are homologous metalloendopeptidases, having similar size, kinetic behaviour, substrate specificity and susceptibility to inhibitors . The far-ultraviolet CD spectrum of each protein shows a minimum at 208 and a shoulder near 220 nm; differences in the extent of ellipticity, however, have been observed . Estimates of secondary structure obtained by quantitation of the far-ultraviolet CD spectra indicated a higher helicity of neutral protease relative to thermolysin . In the presence of ethylenediaminetetraacetic acid, which removes calcium and the functional zinc ion from the metalloenzymes, neutral protease is immediately denatured, whereas thermolysin maintains a globular structure, although thermolabile . On the other hand, the zinc-specific chelating agent tetraethylenepentamine does not have measurable effects on the conformation and conformational stability of either protein . Marked higher stability to temperature and guanidine hydrochloride were observed for thermolysin as compared with neutral protease, as indicated by monitoring conformational transitions with CD measurements at 220 nm . Antisera prepared in rabbits using thermolysin as immunogen do not cross-react with neutral protease, indicating differences of surface structure between the two proteins . On the basis and limitations of the techniques employed, it is proposed that the two sequentially and functionally homologous metalloendopeptidases may have similar conformations at specific regions (active and binding sites, at least) of the polypeptide chain essential for biological function, while some variability in the structure of other regions may be tolerated. Proc Natl Acad Sci U S A, 1980 Oct, 77(10), 5644 - 8 Recognition of local nucleotide conformation in contrast to sequence by a rRNA processing endonuclease; Stahl DA et al.; RNase M5 of Bacillus subtilis cleaves twice in a double-helical region of a 179-nucleotide precursor of 5S rRNA to yield mature 5S rRNA (116 nucleotides) plus fragments (21 and 42 nucleotides) derived from both termini . Previous experiments had shown that the major recognition elements for the highly specific RNase M5 are in the mature domain of the precursor . However, one precursor residue, a G adjacent to the 5' cleavage site, significantly enhances the rate of its own cleavage as well as that of the 3' precursor fragment, so it must be an important component of the features recognized by the enzyme . This G residue is opposed in the helical substrate region to a C residue, which is at the 3' terminus of the mature domain, presenting the question of whether RNase M5 specifically contacts the cleavage site on the basis of nucleotide sequence (the G residue per se) or on the basis of more general aspects of helical conformation . We tested these alternatives by fabricating partially synthetic test substrates for RNase M5 . Experiments were performed on 5' and 3' half-molecules derived from mature 5S rRNA . The 3'-terminal C was removed by periodate oxidation and beta elimination and replaced in a T4 RNA ligase condensation with each of the four mononucleoside bisphosphates . Artificial "precursor" segments containing each of the four nucleotides adjacent to the 5' cleavage site were added to the 5' terminus of the 5S rRNA half-molecule . We then annealed the modified half-molecules to yield test substrates containing all permutations of complementary in contrast to noncomplementary nucleotides at the cleavage site . The susceptibilities of these test substrates show that conformation, not sequence, is the important feature in the locale of the cleaved bonds. J Bacteriol, 1980 Oct, 144(1), 473 - 5 Involvement of deoxyribonucleic acid polymerase III in W-reactivation in Bacillus subtilis; Fields PI et al.; 6-(p-Hydroxyphenylazo)-uracil, a purine analog that is known to specifically inhibit deoxyribonucleic acid polymerase III in gram-positive organisms, inhibited W-reactivation in Bacillus subtilis . On the other hand, W-reactivation proceeded normally in the presence of 6-(p-hydroxyphenylazo)-uracil when a strain possessing a resistant deoxyribonucleic acid polymerase III was used as the host. J Bacteriol, 1980 Oct, 144(1), 238 - 46 Effects of carbon source and growth rate on cell wall composition of Bacillus subtilis subsp . niger; Kruyssen FJ et al.; A study was made to determine whether factors other than the availability of phosphorus were involved in the regulation of synthesis of teichoic and teichuronic acids in Bacillus subtilis subsp . niger WM . First, the nature of the carbon source was varied while the dilution rate was maintained at about 0.3 h-1 . Irrespective of whether the carbon source was glucose, glycerol, galactose, or malate, teichoic acid was the main anionic wall polymer whenever phosphorus was present in excess of the growth requirement, and teichuronic acid predominated in the walls of phosphate-limited cells . The effect of growth rate was studied by varying the dilution rate . However, only under phosphate limitation did the wall composition change with the growth rate: walls prepared from cells grown at dilution rates above 0.5 h-1 contained teichoic as well as teichuronic acid, despite the culture still being phosphate limited . The wall content of the cells did not vary with the nature of the growth limitation, but a correlation was observed between the growth rate and wall content . No indications were obtained that the composition of the peptidoglycan of B . subtilis subsp . niger WM was phenotypically variable. Gene, 1980 Oct, 11(1-2), 157 - 62 A physical map of the genome of the Bacillus subtilis temperate phage rho 11; Mizukami T et al.; A cleavage map of Bacillus subtilis temperate phage rho 11 was constructed with restriction endonucleases SalI, BamHI and BglII, which cut the genome into 6, 7 and 21 fragments, respectively . The molecular weight of the rho 11 genome was calculated to be 78 x 10(6) . Among other endonucleases tested, PvuII, EcoRI and XbaI cleaved the genome into more than 25 fragments, while HaeIII, StuI, BalI and BamNx did not cut the genome at all . The rho 11-coded thymidylate synthetase gene, thyP11, was found to be located in the SalI-D fragment, which was in the central region of the genome. J Gen Microbiol, 1980 Oct, 120(Pt 2), 539 - 43 Characterization of Bacillus subtilis mutants temperature-sensitive in the synthesis of ribonucleic acid; Mazza G et al.; Two mutants of Bacillus subtilis temperature-sensitive in RNA synthesis were isolated . One mutation (rna-20) was demonstrated to be an allele of a previously identified gene (Riva et al., 1976) . The other mutation (rna-16) identified a different gene and was mapped near aroI . The rna-16 mutation at the permissive temperature affected the spore outgrowth process . Purified RNA polymerase from rna-16 did not show any temperature sensitivity or structural defect. J Biol Chem, 1980 Sep 25, 255(18), 8819 - 30 Specificity of promoter site utilization in vitro by bacterial RNA polymerases on Bacillus phage phi 29 DNA . Transcription mapping with exonuclease III; Davison BL et al.; Bacillus subtilis RNA polymerase holoenzyme transcribes phi 29 DNA in vitro producing five major RNA species defined by characteristic electrophoretic mobilities . In addition to these products, Escherichia coli RNA polymerase transcribes phi 29 DNA to yield three RNA species not detected when transcribing with the B . subtilis enzyme under the same optimal reaction conditions for RNA synthesis . Transcriptional analysis of purified restriction fragments and exonuclease III-digested DNA established locations of six promoter and three termination sites defining the eight transcripts . The transcription map shows that E . coli RNA polymerase initiates transcription at three sites not efficiently utilized by the B . subtilis enzyme . However, initiation by the B . subtilis polymerase from at least two of these sites could be detected at E:DNA ratios greater than 10 in the absence of competing promoters . These results indicate that differences between the two polymerases in promoter utilization are not explained by specificity of promoter binding, but represent differences in responding to promoter strength . Transcription of phi 29 DNA and T7 DNA by E . coli core polymerase with either B . subtilis or E . coli sigma subunits results in formation of transcripts identical with those produced by E . coli holoenzyme, suggesting that core polymerase contains elements important in determining relative promoter strength . The efficiency of rifampicin-resistant complex formation on phi 29 and T7 promoters is also dependent upon the source of core polymerase. J Biol Chem, 1980 Sep 10, 255(17), 8216 - 20 Deoxyadenosine/deoxycytidine kinase from Bacillus subtilis . Purification, characterization, and physiological function; Mollgaard H; Three different deoxyribonucleoside kinases with specificities toward thymidine, deoxyguanosine, and deoxyadenosine/deoxycytidine, respectively, are identified in Bacillus subtilis . The deoxyadenosin/deoxycytidine kinase is purified 950-fold employing blue Sepharose CL-6B column chromatography . The two deoxyribonucleoside kinase activities copurify and are present in the same band after polyacrylamide gel electrophoresis . The molecular weight is determined by gel filtration to be 47,000 . Cytidine, adenosine, arabinosylcytosine, and arabinosyladenine are substrates for the enzyme . The activities toward these substrates are less than 20% of the activities obtained with deoxyadenosin and deoxycytidine . The deoxycytidine and deoxyadenosine saturation curves are hyperbolic with Km values for both nucleosides around 5 microM . The maximal velocities for the two deoxyribonucleosides are nearly identical with GTP as phosphate donor . GTP is the best donor showing hyperbolic saturation curves and Km values around 150 microM depending on the deoxyribonucleoside concentration . dATP and dCTP are inhibitors when GTP is the phosphate donor . They may both act as phosphate donors themselves . A divalent metal ion is required, Mg2+ giving the highest activity . A spontaneous mutant, selected as resistant to 5-fluorodeoxycytidine, lacks both deoxycytidine and deoxyadenosine kinase activity, while it retains normal activities toward deoxyguanosine, deoxyuridine, and thymidine. J Antibiot (Tokyo), 1980 Sep, 33(9), 940 - 5 Tetrocarcins, novel antitumor antibiotics . I . Producing organism, fermentation and antimicrobial activity; Tomita F et al.; A novel antitumor antibiotic complex has been obtained from the culture broth of Actinomycete strain DO-11 (KY11091) isolated from a soil sample collected in Sendai, Miyagi, Japan . On the taxonomic studies the producing organism is described as Micromonospora chalcea KY11091 . For the production of the antibiotics, soluble starch served as a good carbon source and yeast extract was the best nitrogen source tested . The antibiotic complex designated as tetrocarcins is active against Gram-positive bacteria, but is not active against Gram-negative bacteria . Tetrocarcin A showed bacteriocidal activity against Bacillus subtilis. J Gen Microbiol, 1980 Sep, 120(1), 259 - 63 Two erythromycin-resistance plasmids of diverse origin and their effect on sporulation in Bacillus subtilis; Mahler I et al.; A Bacillus subtilis plasmid capable of producing phenotypic erythromycin resistance was compared with an Eryr staphylococcal plasmid . The two plasmids did not interfere with the sporulation process in B . subtilis, in contrast to chromosomal erythromycin mutations. J Biochem (Tokyo), 1980 Sep, 88(3), 847 - 57 Studies on the enzymatic reduction of C-nitroso compounds . I . Distribution of c-Nitrosoreductase activity in animal tissues and partial purification of the enzyme from porcine liver; Horie S et al.; The subcellular distribution of NADH-p-nitrosophenol (p-NSP) reductase activity at pH 6.0 in porcine liver was studied by spectrophotometric assay . About two-thirds of the activity was found in the cytosol fraction and the pH optimum of this fraction was about 5.5 . The activity at pH 5.8 of cytosol fractions from various tissues of rats, quails, frogs, carp, and scallops was also studied . All these fractions showed more or less NADH-p-NSP reductase activity but the activity of NADH-aldehyde reductase (alcohol dehydrogenase {EC 1.1.1.1}) was detected only in these from liver and a few other tissues . Supernatants from sonicated cells of Bacillus subtilis and Escherichia coli also showed C-nitrosoreductase activity but were devoid of aldehyde reductase activity . The major C-nitrosoreductase of porcine liver cytosol was purified 20- to 30-fold by fractionation with ammonium sulfate, gel filtration, and ion-exchange chromatography . The pH optimum of this preparation was 5.5 and activity was strongly inhibited by p-chloromercuribenzoate (p-CMB) . The enzyme preparation was stable at 5 degrees C for at least a week in the presence of NADH at pH 8.4 . High concentrations of ammonium sulfate also stabilized the enzyme . An equilibrium between monomeric and dimeric forms of the enzyme was found and the molecular weight was estimated to be about 83,000 and 160,000 daltons for the monomeric and dimeric forms, respecively . The enzyme utilized NADH almost specifically and 2 mol of NADH were consumed per mol of p-NSP reduced to p-aminophenol . Nitrosobenzene and aldehydes could also serve as the electron acceptor . The aldehyde reductase activity became concentrated roughly in parallel with the C-nitrosoreductase activity during the course of the purification and these two activities could not be separated even after further purification by 5'-AMP-Sepharose affinity chromatography . N-Nitroso compounds were not affected by this enzyme. Biokhimiia, 1980 Sep, 45(9), 1609 - 18 {Study of membrane potential of Bacillus subtilis and Escherichia coli cells by the penetration ions methods}; Grinius LL et al.; Using the penetrating ions of tetraphenylphosphonium (TPP+) and tetraphenylborone (TPB-), the membrane potential of the Bacillus subtilis and Escherichia coli cells was shown that the TPP+ absorption by the cells is an energy-coupled process . The TPB- anions are released from the cells after addition of an energy substrate . The value of the membrane potential calculated from the distribution pattern of the penetrating ions in the cells and the incubation medium lies within the interval of --100--150 mV (intracellular negative electric potential) . The value of the membrane potential strongly depends on pH of the incubation medium; our attempts to measure the membrane potential in the E . coli cells at ph 6.0 were unsuccessful; however, at pH 8.5 it was found to be equal to --100 mV . Treatment of the cells with nigericin partially prevents the decrease of the membrane potential in an acidic medium and increases the potential in neutral and alkaline media . The formation of the membrane potential is suppressed by valinomycin and gramicidine, as well as by the oxidative phosphorylation uncouplers; the inhibiting effect of valinomycin requires the presence of K+ in the incubation medium . The membrane potential of the B . subtilis cells is insensitive to the effect of cyanide in the absence of arsenate . It is concluded that the membrane potential of B . subtilis and E . coli is formed both via respiration and by hydrolysis of intracellular ATP. J Antibiot (Tokyo), 1980 Sep, 33(9), 946 - 50 Tetrocarcins, novel antitumor antibiotics . II . Isolation, characterization and antitumor activity; Tamaoki T et al.; Novel antitumor antibiotics, tetrocarcin complex composed of three components (A, B and C) were isolated from the culture broth of Micromonospora chalcea KY11091 through various procedures . Tetrocarcins showed marked activity against experimental tumors such as mouse sarcoma 180 and mouse leukemia P388 . Multiple injections showed better therapeutic effect against tumors . Mode of action of tetrocarcin A against Bacillus subtilis was found to be through the inhibition of RNA synthesis. J Bacteriol, 1980 Sep, 143(3), 1345 - 52 Transcription of exogenous and endogenous deoxyribonucleic acid templates in cold-shocked Bacillus subtilis; Kuhl SJ et al.; Ribonucleic acid (RNA) synthesis was examined in cold-shocked Bacillus subtilis cells . The cells were grown to mid-log stage, harvested, and cold shocked . RNA synthesis was monitored by the incorporation of {3H}uridine triphosphate or {alpha 32P}adenosine triphosphate into trichloroacetic acid-precipitable material in the presence of all four nucleoside triphosphates . The inhibition of RNA synthesis in cold-shocked cells by lipiarmycin, ethidium bromide, rifampin . or streptolydigin was analyzed using mutant or wild-type cells . Also examined were the effects of temperature, salt concentration, and the addition of polyamines or highly phosphorylated nucleotides . In ultraviolet-irradiated and cold-shocked cells, RNA wynthesis decreased to low levels . The addition of exogenous phi 29 or TSP-1 template to these cells caused a 13- to 20-fold increase in RNA synthesis, as monitored by trichloroacetic acid-precipitable counts . RNA synthesized in the presence of phi 29 deoxyribonucleic acid (DNA) hybridizes mainly to EcoRI fragments A and C of phi 29 DBA, These two fragments direct transcription by purified RNA polymerase in vitro and hybridize to early phi 29 DNA produced in vivo . Our results with TSP-1 DNA in this system indicated that the RNA produced hybridizes to the same fragments as early RNA produced in vivo . Plasmic pUB110 DNA was not transcribed in this system. J Biol Chem, 1980 Aug 25, 255(16), 7507 - 9 RNase P of Bacillus subtilis has a RNA component; Gardiner K et al.; Ribonuclease P from Bacillus subtilis cleaves a Gln-Leu tRNA dimeric precursor from bacteriophage T4-infected Escherichia coli, yielding products identical with those generated by the E . coli RNase P . Using this tRNA dimer as an assay substrate, the RNase of P of B . subtilis was shown to consist of at least two components, one of which bands in CsCl equilibrium buoyant density centrifugation at 1.7 g/ml, characteristic of a protein x nucleic acid complex . Both this component and a second, retrieved from the low density (less than 1.4 g/ml) regions of CsCl gradients, are required for RNase P activity . Enzyme activity is abolished by treating the component of density 1.7 g/ml with insoluble RNase A prior to assay . These observations suggest that the RNase P of B . subtilis, like that of E . coli, contain a RNA component essential for activity . That this RNA component is of functional importance, and not an artifact of isolation procedures, is supported by the fact that it is observed in these two phylogenetically disparate organisms. Biochim Biophys Acta, 1980 Aug 14, 600(3), 844 - 52 Control of membrane potential by external H+ concentration in Bacillus subtilis as determined by an ion-selective electrode; Hosoi S et al.; The membrane potential of intact bacteria was monitored by measuring the tetraphenylphosphonium ion distribution across the membrane using poly--(vinyl chloride) matrix-type electrode selective to tetraphenylphosphonimum ion . It was found that the tetraphenylphosphonium ion was not countertransported against H+ movement . The membrane potential of Bacillus subtilis was estimated to be 80-120 mV inside-negative at external pH 7 . The effect of the external pH on the membrane potential was studied . It varied from 30 to 40 mV/decade change in the external {H+} in the pH region of greater than 6.5, increasing pH making it more inside-negative . The addition of carbonyl cyanide m-chlorophenylhydrazone depolarized the membrane, and the membrane potential approached the H+ equilibrium potential . The addition of N,N'-dicyclohexylcarbodiimide did not abolish the pH dependence of the membrane potential . Increasing the external {K+} did not affect the pH dependence . CN- partially depolarized the membrane . A parallel conductance model for membrane potential could explain the results qualitatively. Biokhimiia, 1980 Aug, 45(8), 1524 - 33 {Isolation and characterization of the lytic enzyme from Bacillus subtilis 797}; Borovikova VP et al.; The isolation procedure and some properties of the lytic enzyme produced by Bacillus subtilis 797 and capable of hydrolyzing the E . coli K-12 cells are described . Using hydrophobic chromatography on DNP-hexamethylene diamine Sepharose 4B and ion-exchange chromatography on DEAE-cellulose, a highly purified enzyme preparation with mol . weight of 28000, pI 8.2 has been obtained . The amino acid composition of the enzyme has been determined: Asp--37, Thr--17, Ser--34, Glu--15, Pro--14, Gly--17, Ala--36, Val--28, Met--4, Ile--11, Leu--17, Tyr--9, Phe--4, Lys--18, His--5, Arg--4 . The enzyme is inhibited by a specific inhibitor of serine proteinases--benzylsulfonylfluoride, and is insensitive to EDTA and iodoacetic acid . The lytic enzyme has a proteolytic activity and splits the peptide substrate of bacterial serine proteinases--p-nitroanilide benzyloxycarbonyl-L-analyl-L-alanyl-L-leucine; the maxima for both activities lie within the pH range of 7.8-8.5 . The lytic protease has the highest stability at pH 6-10 . In some of its properties the enzyme is similar to serine proteinase from Bac . subtilis, i . e . subtilisins. J Gen Microbiol, 1980 Aug, 119(Pt 2), 493 - 504 The sodium effect of Bacillus subtilis growth on aspartate; Whiteman P et al.; aspH mutants of Bacillus subtilis have a constitutive aspartase activity and grow well on aspartate as sole carbon source . aspH aspT mutants, which are deficient in high affinity aspartate transport as a result of the aspT mutation, grow as well as aspH mutants in medium containing high concentrations of aspartate and Na+ . This Na+ effect is not due to an enhancement of aspartate transport but is the result of increased cellular metabolism . The ability to grow rapidly in sodium aspartate is induced by prior growth in the presence of Na+ . In potassium aspartate, the addition of arginine, citrulline, ornithine, delta 1-pyrroline-5-carboxylase or proline instead of Na+ also allows rapid growth; but in a mutant deficient in ornithine--oxo-acid aminotransferase, only pyrroline-carboxylate or proline can replace Na+ . The amino acid pool of cells growing slowly in potassium aspartate contains proline at a low concentration which increases upon addition of proline (but not Na+) to the medium . Thus, Na+ addition does not increase the synthesis of proline, but proline or pyrroline-carboxylate acts similarly to Na+ either in preventing some inhibitory effect (by aspartate or the accumulating NH4+) or in overcoming some deficiency (e.g . in further proline metabolism. J Bacteriol, 1980 Aug, 143(2), 879 - 86 Transductional selection of cloned bacteriophage phi 105 and SP02 deoxyribonucleic acids in Bacillus subtilis; Marrero R et al.; The Bacillus subtilis temperate bacteriophages phi 105 and SP02 are incapable of transduction of the small, multicopy drug resistance plasmids pUB110 and pCM194 . Cloning endonuclease-generated fragments of phi 105 or SP02 DNA into each of the plasmids renders the chimeric derivatives susceptible to transduction specifically by the phage whose deoxyribonucleic acid is present in the chimera . The majority of phage deoxyribonucleic acid fragments identified that render plasmids transducible by phi 105 or SP02 appear to be internal fragments, not fragments containing the cohesive ends . However, the highest overall transduction frequency was observed in SP02-mediated transduction of a derivative of pUB110 containing a 1.6-megadalton EcoRI fragment that likely contains the SP02 cohesive ends (plasmid pPL1010) . The transducing activity present in a phi 105 transducing lysate had a buoyant density slightly greater than infectious particles, whereas the majority of transducing particles in an SP02(pPL1010) transducing lysate had a buoyant density slightly less than infectious particles . Although no detectable change in plasmid structure resulted from transduction by phi 105 or SP02, deoxyribonucleic acid isolated from a purified SP02(pPL1010) transducing lysate contained no detectable monomeric pPL1010, but did contain a form of pPL1010 of higher molecular weight than the monomer. J Bacteriol, 1980 Aug, 143(2), 1036 - 8 Highly specific labeling of the Bacillus subtilis chromosome terminus; Adams RT et al.; By making use of the sporulation process, the terminus region of the Bacillus subtilis chromosome has been labeled with {3H}thymine in a highly specific manner . The result achieved supports the view that B . subtilis spores contain only completed chromosomes. J Bacteriol, 1980 Aug, 143(2), 1033 - 5 Specific labeling of the Bacillus subtilis chromosome terminus; Sargent MG; The deoxyribonucleic acid labeled by a procedure described previously for labeling the chromosomal terminus of B . subtilis 168 was substantially enriched for sequences homologous to bacteriophages SP beta and phi 3T, which integrate in the terminal region. Pharmazie, 1980 Aug, 35(8), 463 - 5 Synthesis of newer N-furylidene and N-acetophenonylidene-5-chlorodiphenylamine-2-carboxylic acid hydrazides as antiviral and antibacterial agents; Bahadur S et al.; Five N-furylidene and fifteen N-acetophenonylidene-5-chlorodiphenylamine-2-carboxylic acid hydrazides were synthesized by the condensation of 5-chlorodiphenylamine-2-carboxylic acid hydrazides with furfuraldehyde and appropriate acetophenones . All the N-acetophenonylidenes thus synthesized were screened for their antiviral activity against animal viruses (Ranikhet disease virus and vaccinia virus in a stationary culture of chorioallantoic membrane of chick embryo) and against plant virus, namely gomphrena mosaic virus (GMV) in vitro as well as in vivo . Whereas five N-furylidenes were tested against gomphrena mosaic virus only in vitro and in vivo for their antiviral activity . Maximum significant protection was observed to the extent of 20% against Ranikhet disease virus . The majority of these compounds showed highly significant antiviral activity up to the extent of 96% in vitro and 89% in vivo against gomphrena mosaic virus . These compounds were also screened for their antibacterial activity against two micro-organisms; Bacillus subtilis and Staphylococcus aureus . Almost all of them were found to exhibit significant inhibition against B . subtilis while only a fewer showed significant inhibition against S . aureus. J Biochem (Tokyo), 1980 Aug, 88(2), 317 - 26 Fractionation and biochemical properties of the mother cell and forespore functions from sporulating cells of Bacillus subtilis; Nakayama T et al.; A lysozyme-detergent method was developed for the fractionation of sporulating cells of B . subtilis 168 wild type into mother cell and forespore fractions . The method is very mild and is reproducible with optimum concentrations of Brij-58, deoxycholic acid and sucrose . The results were confirmed by application of the method to temperature sensitive mutants . Ts-1 and Ts-3 . The amounts of proteins, and the activities of protease, alkaline phosphatase and glucose dehydrogenase were about 55, 56, 91, and 40%, respectively, in the mother cell fraction, and about 45, 44, 9, and 60%, respectively, in the forespore fraction, taking the totals for the combined fractions as 100% . Slab gel electrophoretic patterns indicated that many species of proteins with different molecular weights were present in the two fractions . Pulse-labeling with {3H}UTP was carried out in vivo at stage III, and 35.2 and 64.8% of the {3H}UMP incorporated into RNAs were distributed in the mother cell and forespore fractions, respectively . The results indicate that more RNA synthesis occurs in the forespores than in the mother cells of sporulating cells. Am Rev Respir Dis, 1980 Aug, 122(2), 339 - 48 Familial hypersensitivity pneumonitis induced by Bacillus subtilis; Johnson CL et al.; Six members of one family developed symptoms consistent with hypersensitivity pneumonitis after exposure to wood dust generated during the remodeling of a bathroom in their house . A detailed microbiologic investigation of the house and surrounding areas resulted in the isolation of 2-Bacillus species . The vegetative cell and spore extracts of these organisms were used for extensive in vivo and in vitro laboratory tests . All symtomatic members of the family demonstrated positive bronchoprovocation responses to vegetative cell extracts of B . subtilis . Two patients with clinical disease exhibited immediate positive skin tests to similar extracts . Positive lymphoproliferative responses to the vegetative cell extract of B . subtilis were observed in 4 of 5 symptomatic patients and in 1 additional patient tested with the spore form of B . subtilis . It is postulated that the abrupt appearance of this family epidemic depended on the special circumstance of continued exposure to high concentrations of B . subtilis organisms within the household . B . subtilis should be added to the list of antigens causing hypersensitivity penumonitis . Prompt recognition and elimination of this new causal agent could prevent irreversible lung damage in susceptible patients. Gene, 1980 Aug, 10(3), 283 - 9 Direct fractionation of genes by preparative electrophoresis of Bacillus subtilis DNA; Bott KF et al.; Discontinuous electrophoresis through agarose has been shown to be a satisfactory method for preparation of biologically active restriction fragments from milligram quantities of DNA . The DNA is obtained in sufficient quantity for: (1) direct use in genetic transformation, (2) the production of multiple-dimensional restriction analyses, or (3) use as a high-resolution hybridization probe. Gene, 1980 Aug, 10(3), 227 - 35 Expression of thymidylate synthetase activity in Bacillus subtilis upon integration of a cloned gene from Escherichia coli; Rubin EM et al.; The gene from Escherichia coli encoding thymidylate synthetase was cloned in the plasmid pBR322 . The resulting chimeric plasmid, pER2, was effective in transforming both E . coli and Bacillus subtilis to thymine prototrophy . Uncloned linear E . coli chromosomal DNA was unable to transform thymine-requiring strains of B . subtilis to thymine independence . Linearization of the chimeric plasmid, pER2, with restriction enzymes markedly diminished its ability to transform B . subtilis auxotrophs . The Thy+ transformants derived from the transformation of B . subtilis with pER2 DNA did not contain detectable extrachromosomal DNA as demonstrated by Southern hybridization patterns and centrifugation in CsCl gradients of DNA isolated from B . subtilis colonies transformed with the chimeric plasmid . We conclude that the DNA from the chimeric plasmid was integrated into the chromosome of B . subtilis, demonstrating that extensive homology is not required for the integration of foreign DNA . This is the first reported case of a gene from a Gram-negative bacterium functioning in a Gram-positive organism. J Biol Chem, 1980 Jul 10, 255(13), 6007 - 10 Evidence for a tetranuclear iron-sulfur center in glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis; Averill BA et al.; Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is an unusual enzyme because it contains an essential iron-sulfur center but does not catalyze an obvious oxidation-reduction reaction . In this communication, results of revised sulfide analyses, iron-sulfur cluster displacement studies, and Mossbauer spectroscopy are presented that lead to the conclusion that the native enzyme contains a tetranuclear {4Fe-4S} center in a diamagnetic state. C R Seances Acad Sci D, 1980 Jul 7, 291(1), 67 - 70 {Formation of stable diploid bacteria by fusion of protoplasts of Bacillus subtilis and the effect of rec- mutations on the fusion products formed}; Levi-Meyrueis C et al.; A minority among the prototrophic clones formed when protoplasts from two polyauxotrophic strains of B . subtilis are fused has been shown to be diploid, carrying the parental deficient alleles in their DNA, and generally segregating auxotrophs when grown in nutrient medium . This is true whether the strains being fused are Rec+ or Rec-, since the rec- mutations used hardly affect recombinations occurring between chromosomes in diploid Bacteria. J Gen Microbiol, 1980 Jul, 119(1), 277 - 9 Sensitivity of synchronous cultures of Bacillus subtilis to lysozyme; Edwards C; Rates of lysis by lysozyme (expressed as the decrease in A550 min-1) of synchronously dividing cells of Bacillus subtilis rose in concert with the stepwise increase in cell numbers . Asynchronously growing cells showed no periodicities in sensitivity; rates of lysis reflected the exponential increase in culture density. J Bacteriol, 1980 Jul, 143(1), 471 - 80 Chemical basis for selectivity of metal ions by the Bacillus subtilis cell wall; Doyle RJ et al.; The use of equilibrium dialysis techniques established that isolated cell walls of Bacillus subtilis possess selective affinities for several cations . The binding of these cations to the cell wall was influenced by the presence of various functional groups in the peptidoglycan matrix . Selective chemical modification of the free carboxyl and amino groups showed that when amino groups were replaced by neutral, bulky, or negatively charged groups, the sites available for cation complexing generally increased . Introduction of positive charges into the wall resulted in a marked decrease in the numbers of metal binding sites and usually a decrease in the apparent association constants . Both teichoic acid and peptidoglycan contribute to the sites available for interaction with metals . Hill plots of equilibrium dialysis data suggest that metal binding to cell walls involves negative cooperativity . Competition between various metals for binding sites suggested that the cations complex with identical sites on the cell walls . When the hydrogen ion concentration was increased, the affinity of the walls for metals decreased, but the numbers of metal binding sites remained constant, suggesting that cations and protons also compete for the same sites. Biokhimiia, 1980 Jul, 45(7), 1319 - 28 {Substrate specificity of Bacillus subtilis intracellular serine protease . Hydrolysis of insulin beta-chain, native ribonuclease A and p-nitroanilide peptide substrates}; Markarian AN et al.; Intracellular serine protease, termed ISP-103, was isolated from Bacillus subtilis, strain 103 . The substrate specificity of the enzyme was compared to that of secretory subtilisins . Similar to subtilisins, ISP-103 cleaves a single peptide bond Ala20-Ser21 within the native pancreatic ribonuclease A, which results in the accumulation of trypsin-sensitive ribonuclease S, consisting of a non-covalently bound S-peptide (20 amino acid residues) and S-protein (104 amino acid residues) . The enzyme hydrolyzes a single peptide bond Leu15-Tyr16 of the B-chain of oxidized bovine insulin, in contrast to the subtilisins cleaving four additional bonds . ISP prefers Leu rather than Phe in the P1 binding site of the rho-nitroanilide peptide substrates and shows a more strict dependence of the activity on the presence of the hydrophobic residues in the P2 and P3 sites . The data obtained indicate that the substrate specificity of ISP, being within the borders of subtilisin specificity, is nevertheless much more restricted. J Med Chem, 1980 Jul, 23(7), 809 - 11 Simple beta-lactam compounds derived from 6-aminopenicillanic acid; Sheehan JC et al.; As part of a general program of structural modification in beta-lactam antibiotics, we have synthesized several simple penicillins from 6-aminopenicillanic acid where the C-3 carboxyl group has been replaced by a hydroxy or an acetoxy group and the C-6 side chain has been substituted by bromine or hydrogen . Some of the compounds exhibit mild activity against the Gram-positive strain Bacillus subtilis. Gene, 1980 Jul, 10(2), 131 - 6 Cloning vehicles for the homologous Bacillus subtilis host-vector system; Tanaka T et al.; A series of Bacillus subtilis plasmids was constructed which carry either the leu region or both the leu and the dihydrofolate reductase (DHFR) regions of the B . subtilis chromosome . The DHFR-coding gene was derived from a trimethoprim resistant (Tmpr) B . subtilis strain, and cells harboring the DHFR plasmid showed resistance to trimethoprim (Tmp) . One such leu+tmpr plasmid, pTL12, was found to be useful for cloning DNA fragments at the BamHI, EcoRI, BglII and XmaI sites . It was also shown that insertion of DNA fragments at the BamHI and XmaI sites of pTL12 inactivated the leuA gene function (insertional inactivation) but not tmpr, indicating that cells carrying recombinant plasmids can be detected easily by selecting Leu-Tmpr colonies . Combination of B . subtilis 168 and plasmid pTL12 should serve as an efficient homologous cloning system in B . subtilis. Proc Natl Acad Sci U S A, 1980 Jul, 77(7), 3903 - 7 Posttranscriptional regulation of an erythromycin resistance protein specified by plasmic pE194; Shivakumar AG et al.; Induction of the synthesis of a plasmid-encoded polypeptide (E3) by erythromycin is known to be required for the inducible expression of resistance to the macrolide-lincosamide-streptogramin B group of antibiotics in Bacillus subtilis strains carrying the plasmid pE194 . This resistance is mediated by a specific N6-dimethylation of adenine in the 23S rRNA of the large ribosomal subunit . We show in this report that E3 induction is regulated posttranscriptionally in the sense that it can occur when RNA synthesis is blocked and that induction is accompanied by an increase in the functional half-life of E3 mRNA but not of the mRNA species that code for the remaining four known pE194 polypeptides . The induction of E3 is subject to feedback regulation and involves the ribosome . Modification of the erythromycin binding site on the ribosome by methylation or by mutation interferes with induction. Biochem J, 1980 Jun 15, 188(3), 705 - 13 Enzymic determination of branched-chain amino acids and 2-oxoacids in rat tissues . Transfer of 2-oxoacids from skeletal muscle to liver in vivo; Livesey G et al.; 1 . A procedure is described for the purification of leucine dehydrogenase (EC 1.4.1.9) from Bacillus subtilis . 2 . The preparation is suitable for the quantitative assay of branched-chain amino acids and their 2-oxoacid analogues . 3 . The content of total branched-chain 2-oxoacids in freeze-clamped liver, kidney, heart or mammary gland of fed rats is less than 5 nmol/g fresh wt . Higher amounts are present in skeletal muscle and arterial blood (25 +/- 4 nmol per g fresh wt., and 33 +/- 6 nmol per ml respectively; means +/- S.D . of 3 and 11 animals respectively) . The values are not significantly affected by starvation for 24 h . 4 . Arteriovenous difference measurements show that considerable amounts of branched-chain 2-oxoacids are released by skeletal muscle into the circulation and similar amounts are removed by the liver (about 1 mmol/24 h in a 400 g rat). J Antibiot (Tokyo), 1980 Jun, 33(6), 620 - 4 Penicillin-binding proteins in Streptomyces cacaoi . The effects on penicillin-binding proteins and the antibacterial activities of beta-lactams; Ogawara H et al.; Penicillin-binding proteins (PBPs) in membrane of Streptomyces cacaoi were investigated by sodium dodecylsulfate-polyacrylamide gel electrophoresis-fluorography . At the same time, eleven beta-lactams were examined on the affinities for these PBPs and the antibacterial activities against S . cacaoi, comparing with those in Bacillus subtilis reported in the preceding paper . The affinity patterns of beta-lactams for PBPs both in S . cacaoi and B . subtilis were similar in many points . Here again, the grouping of beta-lactams based on the affinity for PBP-2 (M . W., 91,000) was in accord with that based on the antibacterial activity . These results suggest that among PBPs detected in S . cacaoi, PBP-2 is the most likely target of killing by these beta-lactam antibiotics. J Gen Microbiol, 1980 Jun, 118(2), 471 - 8 Action of uncouplers of oxidative phosphorylation as chemotactic repellents of Bacillus subtilis; Ordal GW et al.; Bacillus subtilis alternately swims smoothly and tumbles; when administered repellent it only tumbles, but later resumes normal swimming and tumbling . Repellents of B . subtilis include membrane-active agents like uncouplers of oxidative phosphorylation and local anaesthetics and have previously been found to act in a fundamentally different way compared with attractants . It has been suggested previously that uncouplers act as repellents as a result of their ability to depolarize the membrane and that depolarization might effect flagellar function by causing a flux of Ca2+ into the cell . However, we found that there is no correlation between membrane depolarization and chemotaxis and no detectable flux of Ca2+ following tactic stimulation by uncouplers . Experiments with analogues of the uncoupler pentachlorophenol, all of which are weaker acids than pentachlorophenol, indicated that the anionic form of the uncoupler is the potent form and we propose that it binds to a certain membrane protein to cause release into the cytoplasm of the substance (ion, metabolite or protein) that controls tumbling frequency . Adaptation is assumed to occur when this excess is removed by active transport or metabolism. J Antibiot (Tokyo), 1980 Jun, 33(6), 614 - 9 Penicillin-binding proteins in Bacillus subtilis . The effects on penicillin-binding proteins and the antibacterial activities of beta-lactams; Horikawa S et al.; Several beta-lactams were investigated on the affinity for the penicillin-binding proteins (PBPs) and the antibacterial activity in Bacillus subtilis . The beta-lactams such as ampicillin, PS-5, methicillin and SCE-963, which had high affinities for PBP-2 showed strong antibacterial activities and the beta-lactams such as cephamycin C, Y-G19Z-GG and Y-G19Z-G, which had high affinities for PBP-1 but low affinities for PBP-2, showed weak antibacterial activities . Clavulanic acid and nocardicin A, which had almost no affinities for all the PBPs detected, showed very low antibacterial activities . These results suggest that PBP-2 in Bacillus subtilis is the lethal target of these beta-lactam antibiotics. Proc Natl Acad Sci U S A, 1980 Jun, 77(6), 3553 - 7 Biparental products of bacterial protoplast fusion showing unequal parental chromosome expression; Hotchkiss RD et al.; Efficiently regenerated single colonies from mixed multiply auxotrophic Bacillus subtilis protoplasts, fused with polyethylene glycol, reveal colonies carrying each of the parent types (biparentals) and recombinant colonies . The latter appear in high yields (up to 1% of certain recombinant classes); the yield of biparentals may be as large as 10%, in the range of the indicated physical fusion events . Many of the biparentals are diploids although, contrary to expectation, they are not complementing prototrophs, but show precisely the phenotype of one (either one) of the parent strains . Extensive pedigree analysis and subcloning of diploid lines show that they can propagate with varying stability on the appropriate parental selective medium to reproduce diploid progeny, parental segregants, and late-appearing recombinants, including some prototrophs . Thus, the principal product of intertype protoplast fusion is a diploid carrying two chromosomes, only one of which is expressed in each particular clone . The extinction of one parental genome is especially well demonstrated when it includes suppression of a normally dominant antibiotic sensitivity marker . In transformation experiments, DNA made from a selected diploid clone was able to transfer several of the unexpressed genes . The structural or topological character of DNA associated with the chromosome extinction remains unexplained. J Biochem (Tokyo), 1980 Jun, 87(6), 1619 - 24 Cooperative effects of daunorubicin and actinomycin D on the sporulation of Bacillus subtilis; Nakayama T et al.; Actinomycin D allowed 3.6% of spore formation and 4.9% of dipicolinic acid synthesis of control cultures when added at 0.4 microM at late stage (T6.5) during sporulation process of Bacillus subtilis although the drug inhibited sporulation almost completely when added at log phase and early stages . Daunorubin added at log phase, on the other hand, did not inhibit either growth and sporulation of the bacteria up to a concentration of 2.9 microM . The joint use of both antibiotics at late stages decreased the levels of spore formation and the synthesis of dipicolinic acid allowed by the use of actinomycin D alone to 0.9% and 2.1% respectively, and also inhibited the synthesis of spore coat protein in a cooperative manner. J Virol, 1980 Jun, 34(3), 789 - 91 Expression of superinfection immunity to bacteriophage phi 105 by Bacillus subtilis cells carrying a plasmic chimera of pUB110 and EcoRI fragment F of phi 105 DNA; Cully DF et al.; A 2.1-megadalton, EcoRI-generated fragment of Bacillus subtilis phage phi 105 DNA was cloned into plasmid pUB110 . The hybrid plasmid produces a biologically active product which renders B . subtilis immune to infection by phi 105. J Clin Pathol, 1980 Jun, 33(6), 575 - 80 Comparison of methods available for assay of chloramphenicol in clinical specimens; de Louvois J et al.; Eight methods for the assay of chloramphenicol in clinical samples were compared with our own modification of a plate diffusion technique using Sarcina lutea and yeast extract agar . Six of the eight methods were less sensitive than originally reported, and five of them were considered unsuitable for use in clinical microbiology practice . The remaining three methods together with the S . lutea/yeast extract modification were used to assay chloramphenicol in 20 samples of serum . Twenty samples of cerebrospinal fluid were also assayed by the S . lutea/yeast extract method . Our results indicate that only the Bacillus subtilis (sensitivity 6x0 mg/l) and the S . lutea (sensitivity 2x5 mg/l) diffusion methods are suitable for use with clinical samples in routine practice . The problems of chloramphenicol toxicity, appropriate dosage regimens, and the need for assay of the drugs are considered. Can J Microbiol, 1980 Jun, 26(6), 690 - 7 Slow growing Bacillus subtilis: deoxyribonucleic acid replication in turbidostat cultures; Shay LK et al.; Comparisons of slowly growing Bacillus subtilis cultures grown in batch culture, a chemostat, and a turbidostat were made . Cultures in a turbidostat were found to grow optimally and to be in balanced growth . The duration of deoxyribonucleic acid (DNA) replication, or C period, was estimated by means of an autoradiographic analysis, a genetic analysis, and determinations of DNA per cell . These measurements were performed on cells growing at different slow rates . The results showed that the C period increased with increasing generation times, but the increase in duration of the C period was not proportional to the increase in generation time. J Bacteriol, 1980 Jun, 142(3), 1045 - 8 In vitro translation of messenger ribonucleic acid from sporulating and nonsporulating strains of Bacillus subtilis; Arnaud M et al.; An Escherichia coli translation system supplemented with ribonucleic acid from sporulating Bacillus subtilis produces unique polypeptides which are missing among translation products of ribonucleic acid from six early sporulation mutants. Biochim Biophys Acta, 1980 May 28, 618(2), 202 - 13 Incorporation of hydrogen atoms from deuterated water and stereospecifically deuterium-labeled nicotin amide nucleotides into fatty acids with the Escherichia coli fatty acid synthetase system; Saito K et al.; The mechanism of hydrogen incorporation into fatty acids was investigated with intact Escherichia coli cells, a crude enzyme preparation and purified reductases of fatty acid synthetase system . The distributions of deuterium atoms incorporated into fatty acids from 2H2O and stereospecifically deuterium-labeled NADPH or NADH were determined by mass spectrometry . When E . coli was grown in 2H2O, almost every hydrogen atom of cellular fatty acids was incorporated from the medium . When fatty acids were synthesized from acetyl-CoA, malonyl-CoA and NADPH in the presence of a crude enzyme preparation of either E . coli or Bacillus subtilis, almost every hydrogen atom was also incorporated from the medium . In contrast to these results, purified beta-ketoacyl acyl carrier reductase directly transferred the HB hydrogen of NADPH to beta-ketoacyl acyl carrier protein, and purified enoyl acyl carrier protein reductase also transferred the HB hydrogen of NADPH and NADH directly to enoyl acyl carrier protein . In the crude enzyme preparation of E . coli, we found high activities which exchanged the HB hydrogen of NADPH with the deuterium of 2h2o . the conflicting results of the origin of hydrogen atoms of fatty acids mentioned above are explained by the presence of enzymes, which catalyzed the rapid exchange of NADPH with the deterium of 2H2O prior to the reaction of fatty acid synthetase. J Biol Chem, 1980 May 25, 255(10), 4539 - 43 Heptaprenyl pyrophosphate synthetase from Bacillus subtilis; Takahashi I et al.; Heptaprenyl pyrophosphate synthetase was detected in partially purified extracts of Bacillus subtilis . The enzyme catalyzed the synthesis of all-trans C35 prenyl pyrophosphate from isopentenyl pyrophosphate and farnesyl or geranylgeranyl pyrophosphate, but it did not catalyze a reaction between isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate . The enzyme reaction proceeded with an elimination of 2-pro-R hydrogen of isopentenyl pyrophosphate without accumulation of any prenyl pyrophosphate shorter than C35 . The molecular weight of the enzyme was estimated by gel filtration to be 45,000 . Michaelis constants for isopentenyl, farnesyl, and geranylgeranyl pyrophosphate were 12.8, 13.3, and 8.3 microM, respectively. J Biol Chem, 1980 May 10, 255(9), 3964 - 76 Sequence of active site peptides from the penicillin-sensitive D-alanine carboxypeptidase of Bacillus subtilis . Mechanism of penicillin action and sequence homology to beta-lactamases; Waxman DJ et al.; It has been proposed that penicillin and other beta-lactam antibiotics are substrate analogs which inactivate certain essential enzymes of bacterial cell wall biosynthesis by acylating a catalytic site amino acid residue (Tipper, D.J., and Strominger, J.L . (1965) Proc . Natl . Acad . Sci . U.S.A . 54, 1133-1141) . A key prediction of this hypothesis, that the penicilloyl moiety and an acyl moiety derived from substrate both bind to the same active site residue, has been examined . D-Alanine carboxypeptidase, a penicillin-sensitive membrane enzyme, was purified from Bacillus subtilis and labeled covalently at the antibiotic binding site with {14C}penicillin G or with the cephalosporin {14C}cefoxitin . Alternatively, an acyl moiety derived from the depsipeptide substrate {14C}diacetyl L-Lys-D-Ala-D-lactate was trapped at the catalytic site in near-stoichiometric amounts by rapid denaturation of an acyl-enzyme intermediate . Radiolabeled peptides were purified from a pepsin digest of each of the 14C-labeled D-alanine carboxypeptidases and their amino acid sequences determined . Antibiotic- and substrate-labeled peptic peptides had the same sequence: Tyr-Ser-Lys-Asn-Ala-Asp-Lys-Arg-Leu-Pro-Ile-Ala-Ser-Met . Acyl moieties derived from antibiotic and from substrate were shown to be bound covalently in ester linkage to the identical amino acid residue, a serine at the penultimate position of the peptic peptide . These studies establish that beta-lactam antibiotics are indeed active site-directed acylating agents . Additional amino acid sequence data were obtained by isolating and sequencing {14C}penicilloyl peptides after digestion of {14C}penicilloyl D-alanine carboxypeptidase with either trypsin or cyanogen bromide and by NH2-terminal sequencing of the uncleaved protein . The sequence of the NH2-terminal 64 amino acids was thus determined and the active site serine then identified as residue 36 . A computer search for homologous proteins indicated significant sequence homology between the active site of D-alanine carboxypeptidase and the NH2-terminal portion of beta-lactamases . Maximum homology was obtained when the active site serine of D-alanine carboxypeptidase was aligned correctly with a serine likely to be involved in beta-lactamase catalysis . These findings provide strong evidence that penicillin-sensitive D-alanine carboxypeptidases and penicillin-inactivating beta-lactamases are related evolutionarily. J Bacteriol, 1980 May, 142(2), 724 - 5 Double mutations induced in Escherichia coli by ultraviolet light; Kubitschek HE; Double mutations to azide resistance and to bacteriophage T5 resistance of genes separated by more than 50 kilobases were induced in Escherichia coli WP2s in chemostat cultures by exposure to a single low dose of ultraviolet light . Frequencies of induced double mutations were three orders of magnitude greater than would be predicted by chance . Reversions from azide resistance and phage resistance occurred independently, showing that that the double mutation was not due to pleiotropic effects of a single gene mutation . These results support earlier findings which show that low doses of ultraviolet light induce multiple gene mutations in Bacillus subtilis over a similarly broad range. Rev Argent Microbiol, 1980 May-Aug, 12(2), 52 - 8 {Production on alkaline protease by Bacillus subtilis NRRL 3411}; Massucco AE et al.; The aim of this work was to study the influence of medium composition and the effect of the operative conditions on alkaline protease production . Several nitrogen sources (in amount of 10 g/l) were tested and compared with bacto peptone Difco: Casein was the best . The addition of "Tween 80" 0,5% resulted in a marked increase in yields of the alkaline protease . The maximum alkaline protease production (3.342.290 UAPAM/g) was achieved using the following medium: Lactose 20 g/l; casein 10 g/l; NaCl 1,5 g/l; MgSO4.7H2O 0,15 g/l; CaCl2.2H2O 0,06 g/l; MnCl2.4H2O 0,01 g/l; "Tween 80" 5,0 g/l; EDTA 1,5 X 10(-4)M; KH2PO4 1,5 g/l; K2H PO4 1,5 g/l; Na2SO4 1,5 g/l . It was found that a volumetric relation of 0,1 ml, vol . of medium/vol . of flask at 25 degrees C of temperature was the best process condition. Int J Pept Protein Res, 1980 May, 15(5), 430 - 40 Fluorescence properties of neutral protease from Bacillus subtilis; Grandi C et al.; The fluorescence properties of neutral protease from B . subtilis have been investigated under a variety of conditions and the results compared with those previously reported for the homologous metalloendopeptidase thermolysin (Fontana et al., 1977) . In the pH range 5-9 neutral protease displayed a quite unusual fluorescence emission spectrum with a maximum near 320 nm, when excitation was at 295 nm . At this wavelength the protein fluorescence is due to tryptophan residues only, which, considering their blue-shift emission, appear rather buried in the hydrophobic protein interior . Specific removal of the functional zinc ion from the enzyme with tetraethylenepentamine does not lead to alteration of the microenvironment around tryptophan residues . On the other hand, removal of both zinc and clacium with ethylenediaminetetraacetic acid brings these residues in full contact with the aqueous solvent medium . Fluorescence quenching measurements were also used to determine the exposure of tryptophan residues in the native enzyme as well as in the presence of chelating agents and protein denaturants . Melting profile experiments carried out by monitoring the fluorescence intensity at 320 nm indicated a cooperative transition at 60-70 degrees . Temperature effects were also determined under conditions perturbed with respect to pH, guanidine hydrochloride and chelating agents . The results reveal differences in the fluorescence properties of the tryptophanyl residues of B . subtilis neutral protease relative to those of thermolysin, which are interpretable considering the location of these residues in the sequences of the two homologous proteins. Eur J Biochem, 1980 May, 106(2), 579 - 91 Purification and DNA-binding properties of RNA polymerase from Bacillus subtilis; Giacomoni PU; Four RNA-polymerizing activities having different subunit composition can be purified from uninfected and from SPO1-infected Bacillus subtilis . Lysozyme and sodium deoxycholate are used for lysing the cells . Polymin P is used for precipitating nucleic acids and DEAE-cellulose chromatography allows separation of enzymatic activity from the residual Polymin P . After these common steps, one can purify core + sigma + delta by chromatography on single-stranded DNA-agarose followed by gel filtration while pure core + sigma can be obtained by chromatography on double-stranded DNA cellulose . Core + delta is obtained by high-salt sucrose/glycerol gradient centrifugation . The host enzyme modified by the product of gene 28 of phage SPO1 can be purified from SPO1 infected cells by chromatography on DNA cellulose (or CNA agarose) followed by chromatography on phosphocellulose . The pH and salt dependance of the initial rate of RNA synthesis of core + sigma has been investigated using SPO1 and SPP1 DNA as templates . The optimum pH for the initial rate of transcription is 8.2 at 30 degrees C in 50 mM N,N-bis(2-hydroxyethyl)glycine buffer, and the optimum Na+ concentration is between 0.1 and 0.15 M . The kinetics of formation and of dissociation of non-filterable complexes between SPP1 DNA and core + sigma have been analyzed at different cationic concentrations . The value of the rate constant of dissociation in 0.1 M NaCl at 30 degrees C is kd = 2.16 x 10(-4) S-1 . The value of the rate constant of association, under the same conditions, is ka = 5.5 x 10(8) M-1 S-1; this value is compatible with a diffusion-controlled reaction for promoter selection. Biull Eksp Biol Med, 1980 May, 89(5), 601 - 3 {Factors increasing the effectiveness of Bacillus subtilis fusion}; Arbuzov VN et al.; The factors potentiating the efficacy of fusion of protoplasts of Bacillus subtilis were studied . A 50-fold increase in the yield of primary prototrophs was provided by 90-minute preincubation of protoplasts in a liquid hyperbaric medium and 10-minute treatment with trypsin . Increased efficacy of fusion of protoplasts depended on their regeneration percentage. Proc Natl Acad Sci U S A, 1980 May, 77(5), 2834 - 8 DNA-membrane association is necessary for initiation of chromosomal and plasmid replication in Bacillus subtilis; Winston S et al.; We examined the effect of the inhibition of initiation of DNA replication on the membrane association of the chromosomal origin of replication of Bacillus subtilis and the Staphylococcus aureus-Bacillus pumilus chimeric plasmid pSL103, using temperature-sensitive mutants of B . subtilis that have specifically affected initiation . Inhibition of initiation of the chromosome and pSL103 in the initiation mutant dna-1 results in a decrease in the membrane association of both a marker near the chromosomal origin, purA16, and the plasmid pSL103 . The membrane association of both purA16 and pSL103 can be recovered by allowing initiation to resume at the permissive temperature . In another initiation mutant, dnaB19, only the initiation and membrane association of the host chromosome are affected at the nonpermissive temperature, whereas both initiation and membrane association are not affected in the plasmid pSL103 . In experiments in vitro, DNA containing the purA16 marker and pSL103 DNA molecules are both selectively released during incubation of purified DNA-membrane complexes prepared from dna-1 cells at the nonpermissive temperature . On the other hand, only purA16 DNA is released in vitro from the DNA-membrane complex prepared from dnaB19 cells . This consistent coupling between initiation and membrane association indicates that DNA-membrane association is critical for the initiation of the B . subtilis chromosome and the plasmid pSL103. J Biochem (Tokyo), 1980 May, 87(5), 1261 - 9 Nucleotide sequence of initiator tRNA from Bacillus subtilis; Yamada Y et al.; Initiator methionine tRNA, tRNAfMet, was purified from Bacillus subtilis W168 by the use of two column chromatographies on DEAE-Sephadex A-50 and BD-cellulose . The nucleotide sequence was determined to be pC-G-C-G-G-G-G-U-G-G-A-G-C-A-G-U-U-C-G-G-D-A-G-C-U-C-G-U-C-G-G-G-C-U-C-A-U-A-A-C-C-C-G-A-A-G-G-U-C-G-C-A-G-G-T-psi-C-A-A-A-U-C-C-U-G-C-C-C-C-C-G-C-A-A-C-C-AOH . This TRNAfMet exhibited a melting temperature 8 degrees C lower than that of E . coli tRNAfMet in the presence of 0.01 M magnesium acetate. Genetics, 1980 Apr, 94(4), 809 - 23 Genetic mapping of a group of temperature-sensitive dna initiation mutants in Bacillus subtilis; Imada S et al.; Recombination frequencies among temperature-sensitive dna mutants from various laboratories were analyzed, and eleven dna mutants were found to be closely linked . They are classified as group B dna mutants, since these are closely linked with dnaB19, originally isolated and approximately mapped near leuA8 by KARAMATA and GROSS (1970) . However, the dnaB19 mutation itself has relatively high recombination frequencies with the other mutations, thus, we propose to subdivide the dnaB group into two subgroups--dnaBI, including ten mutants (dna-1, dna-3, dna-5, dna-17, dna-27, dna-51, dna-60, dna-62, dna-103 and dna-134) and dnaBII, including dnaB19 . The map order of dnaB and markers in the vicinity was determined to be argA-citH-citC-phoP-PhoR-polA-dnaBI-dnaBII-citF-leuA-pheA. J Gen Microbiol, 1980 Apr, 117(2), 347 - 55 Characterization of bacteriophage SPP1 transducing particles; de Lencastre H et al.; Bacillus subtilis lysates produced by virulent bacteriophage SPP1 retained their transducing ability upon purification from contaminating PBSX particles . The buoyant density in CsC1 of the transducing activity was indistinguishable from that of the SPP1 plaque-forming units and the sedimentation behaviour in sucrose gradients of purified transducing particles was the same as that of SPP1 phage particles . Further, high concentrations of anti-SPP1 serum inactivated transducing particles and SPPl plaque-forming units at the same rate . The transduction process was resistant to DNAase treatment, but was enhanced by temperatures that did not allow transformation . It was concluded that particles of the size, shape, density and serum-sensitivity characteristic of SPP1, but carrying bacterial DNA, are vectors in a true transduction process . Cell survival upon SPP1 infection is discussed. J Biochem (Tokyo), 1980 Apr, 87(4), 1185 - 201 The primary structure of Bacillus subtilis acidic ribonsomal protein B-19 . Isolation and characterization of peptides and the complete amino acid sequence; Itoh T et al.; The complete primary structure of Bacillus subtilis acidic protein B-L9, functionally equivalent to protein L7/L12 from E . coli, has been determined . B-L9 is composed of 122 residues and has the amino acid composition: Asp3, ASN3, Thr4, Ser3, Glu22, Gln1, Pro3, Gly11, Ala21, Val14, Ile9, Leu12, Phe2, Lys13, and Arg1 . The molecular weight of B-L9 is 12,633 . The amino acid sequence was determined by a combination of automated Edman degradation of the intact protein in a modified Beckman sequenator, and micro dansyl-Edman degradation of the peptides obtained from digestions with trypsin, thermolysin, Staphylococcus aureus protease, chymotrypsin and pepsin . A comparison of protein B-L9 from B . subtilis with E-L12 from E . coli shows a relatively high degree of homology. Biochemistry, 1980 Apr 1, 19(7), 1271 - 5 On the mechanism of 2'-deoxyuridylate hydroxymethylase; Kunitani MG et al.; dUMP hydroxymethylase from SP01-infected Bacillus subtilis has been purified 160-fold by chromatography on DEAE-cellulose and ethylagarose . The enzyme catalyzes exchange of the 5-hydrogen of dUMP for protons of water in the presence or absence of the cofactor CH2-H4folate . Upon treatment with FdUMP and CH2-H4folate, an isolable covalent complex is formed which is believed to be structurally similar to a steady-state intermediate of the normal reaction . The FdUMP-CH2-H4folate-dUMP hydroxymethylase complex is stable toward denaturation with sodium dodecyl sulfate and shows a subunit molecular weight of 46 000 . By analogy with chemical models and studies of dTMP synthetase, a mechanism is proposed for the reaction catalyzed by dUMP hydroxymethylase. Mol Gen Genet, 1980 Apr, 178(1), 21 - 5 Stimulation of Bacillus subtilis transformation by spermidine; Clark PO et al.; Addition of spermidine in millimolar concentrations to Bacillus subtilis cells curing competence development increases transformability . The spermidine must be added at least 30 min before DNA for maximum stimulation . An incubation period of about 30 minutes is also required for the maximum uptake of labeled spermidine . The amount of DNA initially attached and the rate of DNA uptake are increased to the same extent as transformation . The rate of protein synthesis is also equivalently increased . These observations are consistent with an increase in the number of competent cells in the cell population; this increase is mediated by a spermidine-stimulated protein synthesis. Gene, 1980 Apr, 9(1-2), 115 - 26 A specialized transducing phage constructed from Bacillus subtilis phage phi 105; Iijima T et al.; Chromosomal DNA of Bacillus subtilis 168 (trpC2) prepared from defective phage P BSX was digested by restriction endonuclease Eco RI and ligated in vitro with DNA fragments of page phi 105C digested by the same endonuclease . The ligated DNA was used to transform a competent culture of B . subtilis (trpC2 lys3 metB10) which was lysogenic for phi 105, and transformants of the auxotroph markers were selected . The bacterial DNA ligated to the phage DNA fragments could be integrated into the prophage genome by transformation . The transformants in toto were treated with mitomycin C and the lysate was used to transduce B . subtilis (trpC2 lys3 metB10) . Among metB+ transductants, one clone appeared to be a double lysogen carrying both plaque forming and metB+ transducing phage genomes . The latter defective phage was designated phi 105dmetB . Physical mapping of these phages was carried out by agarose gel electrophoresis of the restriction endonuclease digests and also by electron microscopic analysis of heteroduplex DNA . These results indicate that two adjacent fragments Eco RI-G and E of phi 105 DNA had been substituted with a foreign fragment Eco RI-M in phi 105dmetB DNA . Transformation experiments showed that the metB+ gene resided on the fragment Eco RI-M . This fragment was found to have a BamHI-sensitive site . The transforming activity for the metB marker, however, was not affected by the treatmment with BamHI. J Virol, 1980 Apr, 34(1), 187 - 99 Structure of replicating DNA molecules of Bacillus subtilis bacteriophage phi 29; Inciarte MR et al.; We isolated phi 29 DNA replicative intermediates from extracts of phage-infected Bacillus subtilis, pulsed-labeled with {3H}thymidine, by velocity sedimentation in neutral sucrose followed by CsCl equilibrium density gradient centrifugation . During a chase, the DNA with a higher sedimentation coefficient in neutral sucrose and a lower sedimentation rate in alkaline sucrose than that of viral phi 29 DNA was converted into mature DNA . The material with a density higher than that of mature phi 29 DNA consisted of replicative intermediates, as analyzed with an electron microscope . We found two major types of molecules . One consisted of unit-length duplex DNA with one single-stranded branch at a random position . The length of the single-stranded branches was similar to that of one of the double-stranded regions . The other type of molecules was unit-length DNA with one double-stranded region and one single-stranded region extending a variable distance from one end . Partial denaturation of the latter molecules showed that replication was initiated with a similar frequency from either DNA end . These findings suggest that phi 29 DNA replication occurs by a mechanism of strand displacement and that replication starts non-simultaneously from either DNA end, as in the case of adenovirus. J Bacteriol, 1980 Apr, 142(1), 90 - 8 Mapping a cloned gene under sporulation control by inserttion of a drug resistance marker into the Bacillus subtilis chromosome; Haldenwang WG et al.; A segment of Bacillus subtilis deoxyribonucleic acid (DNA) previously cloned in Escherichia coli contains a gene (the 0.4-kilobase {kb} gene) whose transcription is activated at an early stage of spore development . To map the genetic location of the 0.4-kb gene, we constructed a hybrid plasmid that inserts a chloramphenicol resistance determinant into the B . subtilis chromosome by recombination at a site of homology between cloned B . subtilis DNA and the chromosome . This hybrid plasmid (p1949-2) was constructed from the E . coli plasmid pMB9, the B . sultilis chloramphenicol resistance plasmid pCM194 (whose replication function was inactivated), and B . subtilis DNA from the vicinity of the 0.4-kb gene . Transformation of B . subtilis cells to drug resistance by p1949-2 was dependent upon the B . subtilis RecE+ phenotype and resulted in specific and predictable changes in the pattern of endonuclease restriction sites in the 0.4-kb gene region of the chromosome . Chloramphenicol resistance in cells transformed by p1949-2 was mapped to the purA-cysA region of the B . subtilis chromosome, a region . In addition, DNA adjacent to the 0.4-kb gene was shown to contain the wild-type allele of genetic marker (tms-26) from that region. J Bacteriol, 1980 Apr, 142(1), 355 - 8 Methylation of ribosomal proteins in Bacillus subtilis; Mardones E et al.; We measured the methylation of ribosomal proteins from the 30S and 50S subunits of Bacillus subtilis after growing the cells in the presence of {1-14C}methionine and {methyl-3H}methionine . Two-dimensional polyacrylamide gel electrophoretic analysis revealed a preferential methylation of the 50S ribosomal proteins . Proteins L11 and L16, and possibly L9, L10, L18, and L20, were methylated . On the other hand, only two possibly methylated proteins were found on the 30S subunit . A comparison of these results with those for Escherichia coli suggests a common methylation pattern for the bacterial ribosomal proteins. J Bacteriol, 1980 Apr, 142(1), 339 - 43 Membrane association of a Staphylococcus aureus plasmid in Bacillus subtilis; Winston S et al.; The Staphylococcus aureus plasmid pUB110 was found to be enriched in deoxyribonucleic acid-membrane complexes isolated from Bacillus subtilis containing pUB110. J Bacteriol, 1980 Apr, 142(1), 331 - 4 Identification of a sporulation locus in cloned Bacillus subtilis deoxyribonucleic acid; Moran CP Jr et al.; A cloned deoxyribonucleic acid from the purA-cysA region of the Bacillus subtilis chromosome was shown to contain the spoVC locus, a gene whose product is required for sporulation . This is the first demonstration of a spo locus in cloned B . subtilis deoxyribonucleic acid. J Bacteriol, 1980 Apr, 142(1), 254 - 61 Cell division of cycle of Bacillus subtilis: evidence of variability in period D; Holmes M et al.; In Bacillus subtilis the deoxyribonucleic acid content and the extent of cell division during inhibition of chromosome replication increased as a function of the average cell mass, independent of the growth rate . At each growth rate, mass, deoxyribonucleic acid, and residual division varied in different cultures . The variation is consistent with a large variability in the D period . At growth rates higher than 1.5 doublings per h at 37 degrees C, the change in D accounts for the growth rate dependence of the mass and deoxyribonucleic acid content. J Bacteriol, 1980 Apr, 142(1), 229 - 35 Interactions of homologous and heterologous deoxyribonucleic acids and competent Bacillus subtilis cells; Lopez P et al.; Glucosylated and nonglucosylated bacteriophage T4 deoxyribonucleic acids (DNAs) are able to bind to competent cells of Bacillus subtilis, although the former does so in a rather unstable fashion, probably because of the glucosylation . Several heterologous DNAs compete with homologous DNA for the same receptors in binding and in transformation . A different pattern in competition for DNA binding was observed for homologous and T4 glucosylated DNAs in intact cells as compared with protoplasts or membrane vesicles . The results are consistent with the existence of two types of receptor sites on the membrane of competent B . subtilis cells. Can J Microbiol, 1980 Apr, 26(4), 420 - 6 Ultrastructural analysis of the effect of netropsin on sporulation of Bacillus subtilis; Beaman BL et al.; An electron microscopic analysis of Bacillus subtilis cells revealed that netropsin blocked sporulation ultrastructurally at stages 0-I . These observations are consistent with previous results which indicated that cells were not committed to sporulation in the presence of the drug . Further, the addition of netropsin up to stage III of sporulation prevented the normal sharp increase in dihydrodipicolinate synthase activity which results in dipicolinic acid accumulation of stage IV . The addition of netropsin after stage III had much less effect on the synthesis of dihydrodipicolinate synthase and on sporulation . Thus, morphological events in sporulation are blocked early whereas a sporulation-associated enzyme may be affected at later stages . These data indicate than netropsin affects the expression of sporulation-associated genes in a differential manner. J Bacteriol, 1980 Apr, 142(1), 315 - 8 New cryptic plasmid of Bacillus subtilis and restriction analysis of other plasmids found by general screening; Uozumi T et al.; A new cryptic plasmid, pTA1030 (4.5 megadaltons, copy number 16), was characterized by restriction analysis, together with some other plasmids of Bacillus subtilis. J Bacteriol, 1980 Apr, 142(1), 366 - 8 Inhibitory effect of novobiocin on ribonucleic acid synthesis during germination of Bacillus subtilis spores; Matsuda M et al.; Novobiocin inhibited ribonculeic acid synthesis during germination of Bacillus subtilis spores . Transcription of certain kinds of genes probably required a preceding conformational change in deoxyribonucleic acid. Rev Can Biol, 1980 Mar, 39(1), 1 - 8 {Regulation of transcription by elements of the translation system in Escherichia coli}; Lapointe J; The study of the structure of the operons tryptophan, phenylalanine and histidine has indicated that the transcription of these operons is controlled by the level of aminoacylation of the corresponding tRNAs which modulates the termination of transcription at an attenuator site . The derepression of the level of two enzymes responsible for the biosynthesis of glutamate and glutamine in Escherichia coli, following a decrease of the level of aminoacylation of tRNAglutamate in vivo, suggests that attenuator sites are involved in the control of the transcription of the two operons coding for these enzymes . The fact that tRNAglutamate is involved in this regulation whereas tRNAglutamine is not, supports the model of an evolution of the gene for the glutaminyl-tRNA synthetase of E . coli from the gene of a primitive glutamyl-tRNA synthetase similar to that of Bacillus subtilis. Z Ernahrungswiss, 1980 Mar, 19(1), 21 - 3 Fibrinolytic activity of an enzyme produced by Bacillus subtilis; Fayek KI et al.; Proteolytic activity of a fibrinolytic enzyme isolated from B . subtilis was tested on fibrin plates plasminogen-free and plasminogen-rich . Results showed that the enzyme is not a plasminogen activator . Inhibitory effect of human serum and plasma was also tested . The plasma and not serum showed a slight inhibitory effect on proteolytic activity of this enzyme. J Virol, 1980 Mar, 33(3), 945 - 53 Inhibition by lipiarmycin of bacteriophage growth in Bacillus subtilis; Osburne MS et al.; We have used lipiarmycin, a specific inhibitor of initiation of transcription, to study the role of host RNA polymerase in the transcription programs of various phages of Bacillus subtilis . Unlike rifampin, lipiarmycin preferentially inhibits transcription dependent on the sigma subunit of RNA polymerase because it inactivates holoenzyme at a much greater rate than it does core enzyme . With phage SP01, addition of lipiarmycin at a middle-to-late time of infection did not inhibit phage production even though phage production was sensitive to addition of rifampin at that time . This result is consistent with the notion that unmodified host RNA polymerase holoenzyme becomes dispensable after transcription of early classes of SP01 genes, even though host core enzyme is required for synthesis of all classes of phage RNA . SP01-modified forms of RNA polymerase, which lack sigma subunit but contain phage-coded polypeptides and are able to transcribe middle and late genes, were resistant to lipiarmycin in vitro . For phage phi 105, phage development was sensitive to both lipiarmycin and rifampin in wild-type cells and resistant to both drugs in resistant mutant cells, leading to the conclusion that the activity of host holoenzyme was required for phage RNA synthesis . Growth of phage PBS2, which was resistant to rifampin, was sensitive to the addition of lipiarmycin at early times of infection of a wild-type host strain . In a lipiarmycin-resistant mutant host, PBS2 growth was resistant to lipiarmycin . This result suggests that host holoenzyme plays a previously unanticipated role in transcription of PBS2 genes. J Bacteriol, 1980 Mar, 141(3), 1447 - 9 Catabolite repression of enzyme synthesis does not prevent sporulation; Lopez JM et al.; In the presence of excess glucose, a decrease of guanine nucleotides in Bacillus subtilis initiated sporulation but did not prevent catabolite repression of three enzymes . Therefore, the ultimate mechanism(s) repressing enzyme synthesis differs from that suppressing sporulation. J Bacteriol, 1980 Mar, 141(3), 1432 - 4 Identification of flagellin associated with the Bacillus subtilis folded chromosome; Guerout-Fleury AM et al.; We have analyzed the nature and contents of a major protein, P36, in the nucleoid of the Bacillus subtilis wild type and an isogenic mutant devoid of flagella . It appears that deoxyribonucleic acid-P36 complex is flagellin present as membrane-associated flagella. J Bacteriol, 1980 Mar, 141(3), 1331 - 9 Bacillus subtilis deoxyribonucleic acid gyrase; Sugino A et al.; Bacillus subtilis 168 was shown to contain a deoxyribonucleic acid (DNA) gyrase activity which closely resembled those of the enzymes isolated from Escherichia coli and Micrococcus luteus in its enzymatic requirements, substrate specificity, and sensitivity to several antibiotics . The enzyme was purified from the wild type and nalidixic acid-resistant and novobiocin-resistant mutants of B . subtilis and was functionally characterized in vitro . The genetic loci nalA and novA but not novB were shown to code for portions of the functional gyrase . Enzyme from the antibiotic-resistant mutants was resistant to the drug in vitro . The most striking observation was the remarkable similarity between the B . subtilis enzyme and other DNA gyrases, especially with respect to the oxolinic acid-induced DNA cleavage in the presence of sodium dodecyl sulfate . All of the enzymes appeared to possess the same specificity of cutting sites regardless of the source or type of DNA used . This result implies that gyrase binding to DNA is highly specific. J Bacteriol, 1980 Mar, 141(3), 1178 - 82 Purification and characterization of a kanamycin nucleotidyltransferase from plasmid pUB110-carrying cells of Bacillus subtilis; Sadaie Y et al.; The nucleotidyltransferase encoded by plasmid pUB110 was purified to greater than 95% purity with a 33% yield . The enzyme is a monomeric protein with a molecular weight of 34,000 . The optimum pH for activity is 5, and the optimum MgCl2 concentration for activity is 18 mM . The enzyme, which is synthesized constitutively, is stable for several weeks at 4 degrees C . This enzyme would appear to be a good model gene product for the development of a pUB110 deoxyribonucleic acid-dependent in vitro protein-synthesizing system from Bacillus subtilis. Can J Microbiol, 1980 Mar, 26(3), 330 - 7 Helical growth and macrofiber formation of Bacillus subtilis 168 autolytic enzyme deficient mutants; Fein JE; Two poorly lytic, chain-forming mutants of Bacillus subtilis 168, strains FJ3 and FJ6, each 90-95% deficient in the production of N-acetylmuramoyl-l-alanine amidase and endo-beta-N-acethyglucosaminidase, grew helically under a variety of cultural condtions . The structures formed ranged in complexity from double-standed helices to complex aggregates of entangled and interwoven single chains and multistransded helical fibres . Factors favoring this type of helical growth were investigated . Occasional tight single-standed corkscrew like forms were detected in the mutant cultrues . Two other poorly lytic mutant strains of Bacillus were also found to have helical growth capacity . These results have been interpreted as support for the recently proposed (1976) tension restricted helical growth model of Mendelson. J Bacteriol, 1980 Mar, 141(3), 1199 - 208 Extracellular proteases modify cell wall turnover in Bacillus subtilis; Jolliffe LK et al.; The rate of turnover of peptidoglycan in exponentially growing cultures of Bacillus subtilis was observed to be sensitive to extracellular protease . In protease-deficient mutants the rates of cell wall turnover were greater than that of wild-type strain 168, whereas hyperprotease-producing strains exhibited decreased rates of peptidoglycan turnover . The rate of peptidogylcan turnover in a protease-deficient strain was decreased when the mutant was grown in the presence of a hyperprotease-producing strain . The addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to cultures of hyperprotease-producing strains increased their rates of cell wall turnover . Isolated cell walls of all protease mutants contained autolysin levels equal to or greater than that of wild-type strain 168 . The presence of filaments, or cells with incomplete septa, was observed in hyperprotease-producing strains or when a protease-deficient strain was grown in the presence of subtilisin . The results suggest that the turnover of cell walls in B . subtilis may be regulated by extracellular proteases. J Biol Chem, 1980 Feb 25, 255(4), 1521 - 5 Effect of 3,4-dihydroxybutyl-1-phosphonate on phosphoglyceride and lipoteichoic acid synthesis in Bacillus subtilis; Deutsch RM et al.; 3,4-Dihydroxybutyl-1-phosphonate (CH2OHCHOHCH2CH2PO3HPO3H2), an analogue of glycerol 3-phosphate, preferentially inhibits the rate of synthesis and accumulation of phosphatidylglycerol in Bacillus subtilis W23 and 168 . The rate of phosphatidylethanolamine synthesis is only slightly inhibited, whereas that of lysylphosphatidylglycerol is somewhat stimulated . As expected, decreased phosphatidylglycerol synthesis results in the inhibition of the formation of the putative lipoteichoic acid precursor, sn-glycero-1-phospho-beta-gentiobiosyldiacyglycerol and of lipoteichoic acid itself. Pharmazie, 1980 Feb, 35(2), 78 - 80 Synthesis of 4-(3-benzazolymethyl)aminodiphenyl ethers and other related products as potential antimicrobial agents; Varma RS et al.; 4-Aminodiphenyl ethers (2) obtained by reducing the corresponding nitro analogue (1), have been incorporated into benzazoles, phthalimides and 4-quinazolone in the presence of 40% formalin to furnish a series of 4-(3-benzazolymethyl)aminodiphenyl ethers (3) under the conditions of the Mannich reaction . In an alternate procedure, 3 were obtained on refluxing the N-hydroxymethyl derivatives of benzazoles etc . with 2 in methanol . A few amine exchange reactions were also performed and the stability of a few representative compounds was studied in different acidic media . The antibacterial spectrum of all the synthesized compounds was determined against Bacillus subtilis as well as Bacillus pumilus by the agar diffusion technique. J Gen Virol, 1980 Feb, 46(2), 427 - 37 Selection of Bacillus subtilis 168 mutants with deletions of the PBSX prophage; Buxton RS; Heat-resistant derivatives of a Bacillus subtilis 168 strain carrying an xhi mutation, which causes heat-sensitive induction of the PBSX prophage, have been isolated and screened for the acquisition of auxotrophy . Two classes of auxotrophs were isolated, namely Pro- and Pro-Met-; they lacked the ability to produce PBSX, as shown by their resistance to mitomycin C-induced lysis . The proline and methionine requirements and the resistance to mitomycin C were shown to segregate together in phage PBS1-mediated transduction crosses and to be linked to thiB, which is known to be co-transducible with the PBSX prophage . It was therefore proposed that these strains had deletions which removed all or part of the PBSX prophage together with adjacent bacterial DNA encoding the pro(AB) and metC genes . The met mutation was shown to be metC in PBS1 transduction crosses; this gene is known to be co-transducible with the PBSX prophage . The proline requirement was probably due to the deletion of a pro gene which was demonstrated to lie between the PBSX prophage and metC and which was 90% co-transducible with metC . These deletions have been transduced into a strain which was cured of phage SP beta, another bacteriophage carried by B . subtilis 168 . No phage particles could be seen in mitomycin C-induced cultures of such strains . The PBSX-deletion strains grew with the same generation time as the PBSX+ parent in L-broth (27 min at 35 degrees C) but they were slower in minimal medium (e.g . 72 min as against 51 min in the PBSX+ strain) . Besides being resistant to mitomycin C-induced lysis, the deletion strains were also resistant to lysis induced by thymine starvation of thymine auxotrophs and the loss of viability of these strains after thymine starvation was 100-fold less than in the PBSX+ parent . The deletion strains had not, however, lost the bacterial autolytic enzymes, since they were still susceptible to lysis when placed under semi-anaerobic conditions. J Gen Microbiol, 1980 Feb, 116(2), 545 - 7 Crude lysates of Staphylococcus aureus can transform Bacillus subtilis; Feitelson JS et al.; Plasmids can be transferred from Staphylococcus aureus to Bacillus subtilis by crude lysates prepared with penicillin or lysostaphin . These lysates mediate drug-resistance plasmid transformation in competent B . subtilis at an efficiency paralleling that of purified DNA. J Gen Microbiol, 1980 Feb, 116(2), 511 - 4 A procedure for isolating high quality DNA from spores of Bacillus subtilis 168; Sargent MG; A method of isolating DNA from spores of Bacillus subtilis is described . The DNA has approximately the same efficiency in genetic transformation as DNA isolated from vegetative cells and gives a restriction pattern similar to that from vegetative DNA. J Bacteriol, 1980 Feb, 141(2), 993 - 5 Regulation of a glutamine amidotransferase subunit from Bacillus subtilis by a cloned trpE gene from Bacillus pumilus; Kane JF et al.; The influence of a cloned trpE (trpE+p) gene from Bacillus pumilus on the expression of the gat locus in Bacillus subtilis was examined . The trpE gene was regulated by tryptophan and the mtr locus, which specifies the presumed aporepressor . The specific activity of subunit G varied directly with the level of subunit Ep, and the heterologous EpG complex that was formed was stable to gel filtration. J Bacteriol, 1980 Feb, 141(2), 968 - 70 Bacillus subtilis deoxyribonuclease activity specific for single-stranded deoxyribonucleic acid: cellular site and variations during germination and sporulation; Cobianchi F et al.; The endonuclease of Bacillus subtilis specific for single-stranded deoxyribonucleic acid is absent in spores, appears during germination only after the start of deoxyribonucleic acid synthesis, and is located almost exclusively in the periplasm. J Bacteriol, 1980 Feb, 141(2), 876 - 87 Sites of metal deposition in the cell wall of Bacillus subtilis; Beveridge TJ et al.; Amine and carboxyl groups of the cell wall of Bacillus subtilis were chemically modified individually to neutralize their electrochemical charge for determination of their contribution to the metal uptake process . Mild alkali treatment removed ca . 94% of the constituent teichoic acid (expressed as inorganic phosphorus) and allowed estimation of metal interaction with phosphodiester bonds . Chemical modifications of amine functions did not reduce the metal uptake values as compared to native walls, whereas extraction of teichoic acid caused a stoichiometric reduction in levels . In contrast, alteration of carboxyl groups severely limited metal deposition of most of the metals tested . X-ray diffraction and electron microscopy suggested, in this case, that the form and structure of the metal deposit could be different from that found in native walls . The observations suggest that carboxyl groups provide the major site of metal deposition in the B . subtilis wall. J Bacteriol, 1980 Feb, 141(2), 793 - 805 Temporal dissociation of late events in Bacillus subtilis sporulation from expression of genes that determine them; Jenkinson HF et al.; During sporulation in replacement medium, resistance to toluene to heating at 65 degrees C, to lysozyme, and to heating at 80 degrees C appeared in sequence between 4 and 8 h after the induction of sporulation (i.e., between t4 and t8) . The addition of sufficient chloramphenicol at t4.5 to prevent protein synthesis nevertheless allowed the emergence of all of these types of resistance except lysozyme resistance . The numbers of spores with these types of resistance (lysozyme resistance again excepted) increased about fourfold when phenylmethylsulfonyl fluoride (an inhibitor of serine protease activity) was also present . Thus, the observed increases in resistance in the 2 h after the addition of chloramphenicol resulted from the utilization of preformed protein elements . Dipicolinate did not seem to be a determining factor in the development of any of these forms of resistance . Electron micrographs showed that inhibition of protein synthesis did not prevent deposition of the outer layers of the spores . Lysozyme resistance developed diffe