Isr J Med Sci, 1984 Sep, 20(9), 768 - 72 A Spiroplasma tRNA gene cluster; Rogers MJ et al.; Using molecular cloning techniques, a clone containing a 7-kb insert of Spiroplasma species BC3 DNA that hybridized strongly to total labeled Spiroplasma tRNA was identified . Sequence analysis of a portion of this recombinant plasmid identified a cluster of tRNA genes . The gene order was as follows: tRNACys-tRNAArg-tRNAPro-tRNAAla-tRNAMet-tRNAIle and a portion of tRNASer All the genes encode the 3'-terminal CCA and have very high A + T and relatively long intergenic regions . An RNA polymerase promoter site was found upstream of the tRNACys gene . The tRNA gene cluster found in Spiroplasma can be compared with a similar Bacillus subtilis gene cluster, which raises interesting questions concerning gene organization and transcription. J Gen Microbiol, 1984 Sep, 130 ( Pt 9), 2403 - 14 Characterization of the replication terminus of the Bacillus subtilis chromosome; Monteiro MJ et al.; DNA in the terminal region of the chromosome of Bacillus subtilis was labelled by a procedure in which cells in sporulation inducing conditions were pulse-labelled with {3H}thymidine and then treated with p-hydroxyphenylazouracil, an inhibitor of DNA synthesis . The labelled DNA in isolated spores yielded a small number of restriction fragments . About 14 EcoRI fragments with a total length of 80 kb were labelled in a 2.5 min pulse . A fragment of 4.0 kb had the highest specific radioactivity in terminally labelled DNA from several strains . One of these strains lacked the 120 kb prophage of SP beta that is normally integrated close to the terminus . Loss of the 120 kb prophage did not affect the point of termination which must therefore be regarded as a specific 'stop' sequence . Labelled terminus DNA has been used to identify lambda (Charon 4A) clones containing sequences derived from the terminal region . The total length of the restriction fragments present was 150 kb and adds another 90 kb to the 150 kb region mapped previously . Only one group of these sequences was present in a B . subtilis strain (CU1695) carrying a deletion spanning from SP beta to the right of the terminus and gltA . This suggests that the terminator sequence found in the wild-type can be deleted but presumably this strain has an alternative mechanism of termination. J Bacteriol, 1984 Sep, 159(3), 811 - 9 Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein; Vasantha N et al.; The genes for alkaline protease (apr{BamP}) and neutral protease (npr{BamP}) from Bacillus amyloliquefaciens have been isolated and expressed in Bacillus subtilis . The DNA sequences of apr{BamP} and npr{BamP} revealed, in each case, the presence of a large open reading frame . The inferred amino acid sequence of either gene contained a signal sequence and an additional polypeptide sequence ('pro' sequence) preceding the mature protein . Based on DNA sequence, the start point of translation has been identified as amino acid residue - 107 for apr{BamP} and -221 for npr{BamP} . To demonstrate that the start point of translation of apr{BamP} in vivo is probably at codon -107, codon -103 (AAA) was changed to an ochre (TAA) by site-directed mutagenesis . Alkaline protease was produced from this ochre mutant derivative of apr{BamP} only when the host strain was Su+ . The presence of a pro sequence may be common to all of the secreted proteases from bacilli. J Bacteriol, 1984 Sep, 159(3), 1080 - 2 Revised restriction maps of Bacillus subtilis bacteriophage phi 105 DNA; Anaguchi H et al.; Physical mapping of Bacillus subtilis temperate phage phi 105 DNA was carried out by using restriction endonucleases EcoRI, SmaI, and KpnI, and a new revised EcoRI cleavage map is presented . In addition, the EcoRI cleavage maps of six specialized transducing phages carrying sporulation genes of B . subtilis were revised. Mycopathologia, 1984 Aug 30, 87(1-2), 43 - 9 {Antibacterial and genotoxic properties of 33 mycotoxins}; Boutibonnes P et al.; Most of the 33 fungal metabolites tested provoke: Bacterial growth inhibition of Bacillus thuringiensis similar to lethal effect of antibiotics . Positive response in the 'Rec' assay using strains of Bacillus subtilis; this fact shows that these toxins are DNA modifying agents . Enlargement of cell volume in the first bacteria species; this cell-abnormality induction resembles those obtained with mitomycin C . Correlation between elongation of cells (filamentation) and in vivo carcinogenicity of mycotoxins is discussed . The filamentation should be an expression of a perturbated DNA replication (S.O.S.-error prone repair) as the consequence of DNA damages induced by genotoxic agents (i.e . carcinogens). Biochem Biophys Res Commun, 1984 Aug 16, 122(3), 1104 - 9 Identification of the sporulation gene spoOA product of Bacillus subtilis; Kudoh J et al.; A 2.4-kilobase fragment of the Bacillus subtilis chromosome containing the wild-type spoOA gene derived from the phi 105dspoOA+-Bc-1 transducing phage was cloned onto plasmid pBR322 in Escherichia coli . A recombinant plasmid harboring the mutant spoOA12 allele on the 2.4-kilobase insert was also constructed from the phi 105dspoOA12-1 phage DNA and pBR322 . Protein products synthesized in response to plasmid DNA in a DNA-directed cell-free system derived from E . coli were analyzed by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis . A protein of approximately 27,500 daltons synthesized with the recombinant plasmid DNA harboring the wild-type spoOA gene as template was not formed with the recombinant plasmid DNA harboring the spoOA12 allele . Since the spoOA12 mutation is a nonsense mutation, we conclude that the 27.5-kilodalton protein is the product of the spoOA gene. Nucleic Acids Res, 1984 Aug 10, 12(15), 6307 - 23 Cloning the gyrA gene of Bacillus subtilis; Lampe MF et al.; We have isolated an eight kilobase fragment of Bacillus subtilis DNA by specific integration and excision of a plasmid containing a sequence adjacent to ribosomal operon rrn O . The genetic locus of the cloned fragment was verified by linkage of the integrated vector to nearby genetic markers using both transduction and transformation . Functional gyrA activity encoded by this fragment complements E . coli gyrA mutants . Recombination between the Bacillus sequences and the E . coli chromosome did not occur . The Bacillus wild type gyrA gene, which confers sensitivity to nalidixic acid, is dominant in E . coli as is the E . coli gene . The cloned DNA precisely defines the physical location of the gyrA mutation on the B . subtilis chromosome . Since an analogous fragment from a nalidixic acid resistant strain has also been isolated, and shown to transform B . subtilis to nalidixic acid resistance, both alleles have been cloned. J Biol Chem, 1984 Aug 10, 259(15), 9762 - 7 Utilization of a Bacillus subtilis sigma 37 promoter by Escherichia coli RNA polymerase in vivo; Wong SL et al.; The promoter region of Bacillus subtilis subtilisin E was found to be composed of two overlapping promoters with their transcription starting sites separated from each other by 15 base pairs (Wong, S.-L., Price, C . W., Goldfarb, D . S., and Doi, R . H . (1984) Proc . Natl . Acad . Sci . U.S.A . 81, 1184-1188) . At least one of the promoters is transcribed by a minor form of B . subtilis RNA polymerase with a sigma factor of 37,000 daltons . In vitro transcription analyses and in vivo studies with promoter probe plasmids pKO-1 and pCED-6 demonstrated that Escherichia coli RNA polymerase was able to initiate transcription from the subtilisin promoter cluster . S1 nuclease-mapping studies with both in vivo and in vitro transcribed RNA from E . coli and B . subtilis illustrate that E . coli can initiate transcription from both promoters with the same transcription start points as B . subtilis . The promoter strength of this promoter cluster in E . coli, as expressed in terms of galactokinase units, was 64 units and represents weak promoter activity in the E . coli system . These data indicate that either the single E . coli RNA polymerase is able to recognize the minor sigma 37 promoter or E . coli contains a hitherto unrecognized minor RNA polymerase holoenzyme which is capable of recognizing a B . subtilis sigma 37 promoter . On the other hand the B . subtilis RNA polymerase holoenzymes have been quite promoter-specific in our experiments to date. Zentralbl Bakteriol Mikrobiol Hyg {B}, 1984 Aug, 179(4), 365 - 80 {How many biological indicators have to be tested to get reliable information on their resistance?}; Spicher G et al.; Biological indicators are used in the efficacy test of microbicidal procedures . The indicators consist of an object carrying or holding micro-organisms which exhibit resistance to microbicidal agents . The biological indicators are exposed to the procedure to be tested and afterwards examined for viable germs . If test germs are still found to grow in the cultures, the microbicidal effect of the procedure is considered as insufficient . Biological indicators are suitable for such tests only if it is known how intensive the action of the microbicide has to be to destroy the test germs . The individuals of a germ population do not die at the same time under the action of a microbicide . This phenomenon can also be observed with germs simultaneously grown as a pure culture under identical conditions . Therefore, the biological indicators do not become sterile after one and the same period of action or dose of the microbicide but within a certain period or dose range . At the beginning of this transition range, sterile biological indicators will be found very rarely . With increasing period of action or dose, the frequency of indicators carrying viable germs decreases until, eventually, hardly any biological indicators with viable germs are detectable . When samples of identical biological indicators equal in size are exposed to one and the same period of action or dose of a microbicide, the number of indicators carrying viable germs will vary from one sample to another in the transition range . The number of biological indicators that has to be exposed to the resistance test per period of action and dose, respectively, in order to obtain reliable results, can be estimated only if the regularities are known by which the findings vary from one sample to another . Twelve different batches of biological indicators were employed to determine the variation of the resistance values obtained . Spores of Bacillus subtilis served as test germs . The batches differed in the number of spores per biological indicator . The microbicide used was saturated steam of 100 degrees C with a 9 min period of action . Forty-eight samples of five indicators each were taken per batch . The number of indicators carrying viable germs (n+) varied more or less and characteristic frequency distributions were observed (Table 2, columns 3 and 4) . Afterwards, the mean relative frequency of indicators with viable germs was calculated for the different batches (Q; Table 2, column 2).(ABSTRACT TRUNCATED AT 400 WORDS) J Appl Bacteriol, 1984 Aug, 57(1), 153 - 63 Emergence and development of resistance to antimicrobial chemicals and heat in spores of Bacillus subtilis; Gorman SP et al.; The emergence and development of chemical and thermal resistance in spores of Bacillus subtilis was examined . The chemicals studied were of the disinfectant type: glutaraldehyde, hypochlorite, hypochlorite-methanol and povidone-iodine . Growth and sporulation were followed by electron microscopy and resistance assigned to specific stages in relation to 45Ca and DPA accumulation . A sequential development of resistance was observed with thermal resistance appearing first at early Stage V corresponding to maturation of cortex and deposition of rudimentary spore coat material . Chemical resistance coincided with middle to late Stage V dependent on the chemical concerned . A progressive development of resistance was observed on prolonged incubation in sporulation medium and was affected by inclusion of lysozyme in the spore washing sequence. J Gen Microbiol, 1984 Aug, 130 ( Pt 8), 2115 - 21 Genetic and phenotypic characterization of a cluster of mutations in the spoVA locus of Bacillus subtilis; Errington J et al.; Twenty-nine mutants blocked during stage V of sporulation have been isolated following directed mutagenesis of the lys-1 region of the Bacillus subtilis 168 chromosome . All of a sample of eight mutants tested are unaffected in sporulation marker events up to stage IV but did not produce dipicolinic acid . They produced stable 'phase white' spores that were released from the mother cell, and were partially resistant to toluene and lysozyme but sensitive to chloroform and heat . Mutation spoV A89, known to be in the lys-1 region, showed similar phenotypic characteristics . Three-factor transformation crosses and recombination indices showed that the new mutations and spoV A89 lie in a single linkage group, which maps between lys-1 and another sporulation locus, spoIIA . The size of the spoV A locus is such that it probably contains several genes, and these may be contiguous with the cluster of genes included within the spoIIA locus. Eur J Biochem, 1984 Aug 1, 142(3), 565 - 70 Pressure dependence of thermolysin catalysis; Fukuda M et al.; A comparison of the pressure and temperature dependences of the catalytic reaction of thermolysin, a thermostable neutral protease from Bacillus thermoproteolyticus, with those of a non-thermostable neutral protease from Bacillus subtilis revealed a distinct difference in Km values of these enzymes for 3-(2-furyl)acrylyl-blocked dipeptide and tripeptide substrates, but not for Kcat . Namely, the volume changes for the binding process (delta V) for these substrates and several competitive inhibitors were -20- -30 ml/mol for thermolysin and nearly 0 ml/mol for the non-thermostable neutral protease . The enthalpy and entropy changes for the binding process were negative for thermolysin, but positive for the latter enzyme . The activation volumes (delta V not equal to) for the kcat process were 25 -35 ml/mol for both proteases, and activation enthalpy and entropy showed no significant difference between the two enzymes . The characteristic difference in the pressure and the temperature dependences seen for the binding process is discussed in relation to the thermostability of the proteases. J Bacteriol, 1984 Aug, 159(2), 770 - 2 Order of ribosomal protein genes in the Rif cluster of Bacillus subtilis is identical to that of Escherichia coli; Dabbs ER; Mutants of Bacillus subtilis with electrophoretic variants of ribosomal protein L1, L5, L9, or L11 were used to determine the order of the genes for these proteins by transformation experiments . The proteins are homologous with Escherichia coli proteins L1, L10, L12, and L11, respectively; using the gene locus designations based on this correspondence, we determined the order of the loci to be cysA-rplK-rplA-rplJ-rplL-rpoB . The order of the last five loci was identical to that of E . coli. J Bacteriol, 1984 Aug, 159(2), 605 - 10 Voltage clamp effects on bacterial chemotaxis; Margolin Y et al.; To examine whether or not sensory signaling in bacteria is by way of fluctuations in membrane potential, we studied the effect of clamping the potential on bacterial chemotaxis . The potential was clamped by valinomycin, a K+ -specific ionophore, in the presence of K+ . Despite the clamped potential, sensory signaling did occur: both Escherichia coli and Bacillus subtilis cells were still excitable and adaptable under these conditions . It is concluded that signaling in the excitation and adaptation steps of chemotaxis is not by way of fluctuations in the membrane potential. Genetics, 1984 Aug, 107(4), 551 - 61 Identification of mutations associated with macrofiber formation in Bacillus subtilis; Saxe CL 3rd et al.; A search was made for the genes responsible for the production of helical macrofibers in the original collection of macrofiber-producing strains of B . subtilis . Two loci were identified: fibA, located between hisA and tag-1, and fibB, linked to cysB . fibA governs a short-lived division suppression phenomenon associated with the production of rudimentary fibers, whereas fibB appears to be responsible for a persistent division suppression and a more highly organized helical macrofiber . Both mutations are recovered from each of the original macrofiber-producing strains which also carried the div IV-B1 mutation responsible for minicell production . The latter mutation by itself is not sufficient, however, for the production of macrofibers . Other known mutations leading to division suppression that map in the same region are shown not to be allelic to fibA or fibB . Neither fib locus appears to be responsible for helix hand determination. Appl Environ Microbiol, 1984 Aug, 48(2), 280 - 4 Characterization of Bacillus subtilis DSM704 and its production of 1-deoxynojirimycin; Stein DC et al.; A Bacillus subtilis strain, DSM704, was characterized by genetic means, and its production of a human intestinal sucrase inhibitor, 1-deoxynojirimycin, was described . Synthesis of this compound is detected concomitant with the detection of heat-resistant spores . The amount of 1-deoxynojirimycin produced is highly dependent on the carbon source, with growth on substrates metabolized via glycolysis giving the greatest amount of production (up to 1 mg/ml) . 1-Deoxynojirimycin appears to be nonmetabolizable by the producing strain in that it cannot serve as a sole carbon or nitrogen source. Proc Natl Acad Sci U S A, 1984 Aug, 81(16), 5189 - 93 Generation of deletions in pneumococcal mal genes cloned in Bacillus subtilis; Lopez P et al.; The pneumococcal recombinant plasmid pLS70, which contains two strong promoters for transcription of the malM and malX genes, is unstable when transferred to Bacillus subtilis, and it gives rise to deleted derivatives . Analysis of proteins produced by the deleted plasmids and restriction mapping of 29 different deletions showed that stabilization in B . subtilis was accompanied by deletions affecting both promoters . Plasmids containing even a single strong promoter were at a selective disadvantage . Nucleotide sequences surrounding the deletions in 10 plasmids were determined . Six different deletions occurred between directly repeated sequences of 3-13 base pairs in length, presumably by a recombination mechanism involving short homologies . Four deletions occurred between sites not contained within repeated sequences . A weak but significant similarity of an 11-base sequence was found surrounding these deletions and the corresponding points of junction in the progenitor plasmids . It is suggested that this sequence may be the recognition site for a topoisomerase-like enzyme that can produce deletions. J Gen Microbiol, 1984 Aug, 130 ( Pt 8), 2165 - 7 Restriction enzyme analysis of Bacillus subtilis bacteriophage phi 105 DNA; Bugaichuk UD et al.; The recognition sites on phi 105 DNA for the restriction endonucleases EcoRI, Bg/II, SmaI, KpnI, SstI, SalI, XhoI, NcoI, PstI, HindIII, ClaI, EcoRV and MluI have been mapped . The sites for EcoRI are shown to be different from those published earlier . The DNA from phi 105 contains no recognition sites for the endonucleases BamHI and XbaI. J Gen Microbiol, 1984 Aug, 130 ( Pt 8), 2155 - 64 Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis; Jenkinson HF et al.; A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys . Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance . One type contained 26 kb and the other 32 kb of DNA . Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome . A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment . This phage, denoted phi 105 d(Cmrmet), transduced B . subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants. J Gen Microbiol, 1984 Aug, 130 ( Pt 8), 2147 - 53 Nucleotide sequence of sporulation locus spoIIA in Bacillus subtilis; Fort P et al.; We have determined a sequence of 2073 bp from two recombinant plasmids carrying the whole spoIIA locus from Bacillus subtilis, the expression of which is required for spore formation . The sequence contains three long open reading frames (ORFs), each of them being preceded by a ribosome binding site . These three putative proteins (mol . wts 13100, 16300 and 22200) are likely to be expressed and are probably encoded on the same mRNA . The stop codon of ORF1 overlaps with the start codon of ORF2 suggesting that there might be translational coupling between the two ORFs . Although some known promoter sequences were found, the only one upstream from the first open reading frame is about 260 bp from it. J Gen Microbiol, 1984 Aug, 130 ( Pt 8), 2137 - 45 Cloning of the Bacillus subtilis spoIIA and spoV A loci in phage phi 105DI:1t; Savva D et al.; A 6.95 kb HindIII-generated DNA fragment from Bacillus subtilis 168 was inserted into the DNA of phage phi 105DI:1t . The recombinant phage (phi 105DS1) contained DNA of 33.8 kb as compared with 35.2 kb for phi 105DI:1t and 39.2 kb for the wild-type phage . In the presence of helper phage, phi 105DS1 complemented both spoIIA and spoV A mutations in B . subtilis. J Gen Microbiol, 1984 Aug, 130 ( Pt 8), 2123 - 36 Use of integrational plasmid vectors to demonstrate the polycistronic nature of a transcriptional unit (spoIIA) required for sporulation of Bacillus subtilis; Piggot PJ et al.; Plasmids carrying different portions of the polycistronic spoIIA locus, and unable to replicate autonomously in Bacillus subtilis, were able to transform a Spo+ B . subtilis strain, BR151, for the plasmid-determined chloramphenicol resistance by Campbell-like insertion into the region of homology on the chromosome . Two such plasmids, pPP35 and pPP36, yielded Spo- transformants, indicating that the cloned regions of these plasmids were entirely within the chromosomal spoIIA transcriptional unit . The cloned regions overlapped the end of a known spoIIA cistron, so that the transcriptional unit was larger than this cistron, and was polycistronic . This is the first demonstration of such a polycistronic sporulation transcriptional unit . The DNA sequence of the region has now been determined (given in an accompanying paper) and suggests a transcriptional unit with three open reading frames . Two other plasmids yielded Spo+ transformants of BR151, and these define the outer limits of the transcriptional unit . The adjacent sporulation locus identified by the spoV A89 mutation was not part of the same transcriptional unit. Biochemistry, 1984 Jul 31, 23(16), 3659 - 62 Base pairing in Bacillus subtilis ribosomal 5S RNA as measured by ultraviolet absorption and Fourier-transform infrared spectrometry; Chang LH et al.; Ultraviolet (260 and 280 nm) and Fourier-transform infrared (FT-IR) spectra of Bacillus subtilis ribosomal 5S RNA have been acquired between 20 and 90 degrees C . In the presence of added Mg2+, the average UV melting midpoint, Tm, is 60 (A260) or 62 degrees C (A280), resolving into two components (Tm = 54 and 68 degrees C) . In the presence of 10 mM Mg2+, the normalized A260 increases by about 5%, and the average Tm increases to 70 degrees C (A260 or A280), resolving into components at 63 and 73 degrees C at 260 nm but not resolved at 280 nm . From the difference of the 5S RNA FT-IR spectra between 90 and 30 degrees C, the number of base pairs in B . subtilis 5S RNA was determined by the procedure outlined in the accompanying paper {Li, S.-J., Burkey, K . O., Luoma, G . A., Alben, J . O., & Marshall, A . G . (1984) Biochemistry (preceding paper in this issue)} . Addition of 10 mM Mg2+ increases the number of A-U pairs by 1 (from 11 to 12) and the number of G-C pairs by 2 (from 15 to 17) . FT-IR melting curve midpoints show that addition of Mg2+ increases the melting point for both A-U and G-C pairs in B . subtilis 5S RNA . The A-U pairs melt before G-C pairs (56 vs . 64 degrees C) in the absence of Mg2+, but both types of pairs melt at the same temperature (67 vs . 70 degrees C) in the presence of Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Biophys Res Commun, 1984 Jul 18, 122(1), 175 - 83 Cloning in Bacillus subtilis of an extremely thermostable alpha amylase: comparison with other cloned heatstable alpha amylases; Piggott RP et al.; A heatstable alpha amylase gene was shotgun cloned from Bacillus licheniformis RPO1 into Bacillus subtilis . Restriction endonuclease analysis of the recombinant plasmid revealed a map which was identical to a previously cloned alpha amylase from B . licheniformis FDO2 and very similar to the restriction map of a high temperature amylase from Bacillus coagulans . The thermostability and temperature optimum of the cloned alpha amylase was measureably different from those of the previously reported cloned alpha amylases. Nucleic Acids Res, 1984 Jul 11, 12(13), 5307 - 19 Length and structural effect of signal peptides derived from Bacillus subtilis alpha-amylase on secretion of Escherichia coli beta-lactamase in B . subtilis cells; Ohmura K et al.; The precursor of Bacillus subtilis alpha-amylase contains an NH2-terminal extension of 41 amino acid residues as the signal sequence . The E . coli beta-lactamase structural gene was fused with the DNA for the promoter and signal sequence regions . Activity of beta-lactamase was expressed and more than 95% of the activity was secreted into the culture medium . DNA fragments coding for short signal sequences 28, 31, and 33 amino acids from the initiator Met were prepared and fused with the beta-lactamase structural gene . The sequences of 31 and 33 amino acid residues with Ala COOH-terminal amino acid were able to secrete active beta-lactamase from B . subtilis cells . However beta-lactamase was not secreted into the culture medium by the shorter signal sequence of 28 amino acid residues, which was not cleaved . Molecular weight analysis of the extracellular and cell-bound beta-lactamase suggested that the signal peptide of B . subtilis alpha-amylase was the first 31 amino acids from the initiator Met . The significance of these results was discussed in relation to the predicted secondary structure of the signal sequences. J Biol Chem, 1984 Jul 10, 259(13), 8478 - 84 Glutamine nucleotide sequence of Saccharomyces cerevisiae ADE4 encoding phosphoribosylpyrophosphate amidotransferase; Mantsala P et al.; Saccharomyces cerevisiae gene ADE4 encoding glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) has been cloned by complementation of an ade4 auxotroph . The nucleotide sequence of ADE4 along with upstream and downstream flanking sequences was determined . The ADE4 structural gene consists of 1530 base pairs from which a 510-amino acid translation product, Mr = 56,691, was deduced . Yeast amidophosphoribosyltransferase is homologous to the enzymes from Escherichia coli and Bacillus subtilis . The active site cysteine residue in the bacterial amidophosphoribosyltransferases which functions in glutamine amide transfer is conserved in the yeast enzyme . Yeast amidophosphoribosyltransferase does not contain the previously deduced sequence required for binding of a {4Fe-4S} center indicating that a {4Fe-4S} center is an unlikely component of the yeast enzyme . Amidophosphoribosyltransferase was stable in growing and nongrowing cells and was not inactivated or degraded . Thus in the group, S . cerevisiae, E . coli, B . subtilis, the content of a {4Fe-4S} cluster in amidophosphoribosyltransferase correlates with a mechanism for oxygen-dependent inactivation of the enzyme . Northern blots demonstrate that ADE4 expression is transcriptionally regulated . The 5' end of the ADE4 mRNA was identified by nuclease S1 mapping. J Biol Chem, 1984 Jul 10, 259(13), 8619 - 25 Overlapping promoters transcribed by bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases; Wang PZ et al.; A 471-base pair HindIII DNA fragment of Bacillus subtilis contains two overlapping promoters which are recognized in vitro by sigma 55- and sigma 37-containing RNA polymerase holoenzymes from B . subtilis . In vitro transcript analyses and S1 nuclease mapping experiments with in vivo RNA indicate that both enzymes initiate transcription from the same putative +1 site . Physiological studies with the promoter-containing DNA fragment inserted into the expression probe plasmid pCED6 and quantitative S1 nuclease mapping experiments with RNA isolated from various stages of growth indicate that expression from these overlapping promoters is greater during the early stationary phase than during growth . We propose that the cryptic gene controlled by these promoters is transcribed by the sigma 55 enzyme during growth and by the sigma 37 enzyme during early stationary phase. J Mol Biol, 1984 Jul 5, 176(3), 333 - 48 A promoter whose utilization is temporally regulated during sporulation in Bacillus subtilis; Stephens MA et al.; The formation of endospores in the Gram-positive bacterium Bacillus subtilis proceeds according to a temporally ordered program of gene activation . To investigate timing mechanisms in sporulation gene expression, we have isolated and sequenced the promoter region for a B . subtilis gene known as 0.3 kb whose transcription is switched on at about stage III of development . The 5' terminus of the 0.3 kb mRNA was mapped by the S1 nuclease procedure to a position just upstream from its apparent ribosome binding site and initiation codon and just downstream from the transcription termination site for an adjacent gene . This information enabled us to construct a transcriptional fusion in which the 5' region of the 0.3 kb gene was joined to the lacZ gene of Escherichia coli . When introduced into cells of B . subtilis, the 0.3 kb-lacZ fusion caused the synthesis of a fusion-specified RNA that originated from within the 0.3 kb promoter region and extended into the adjacent E . coli DNA, and the induction of beta-galactosidase synthesis at the third to fourth hour of sporulation . Enzyme synthesis required the 0.3 kb promoter, since a deletion of the 5' region of the 0.3 kb gene in the transcription fusion eliminated the production of beta-galactosidase . Induction of the 0.3 kb-lacZ fusion was under developmental control, since the production of beta-galactosidase was blocked or substantially impaired by chromosomal mutations in the sporulation genes spoOB, spoIIA, spoIIE and spoIIIE, but not by a spoIIC mutation . We conclude that the 0.3 kb gene promoter is subject to a developmental clock, which delays its utilization until an intermediate stage of sporulation, and discuss models for how the timing of gene expression is regulated. Gene, 1984 Jul-Aug, 29(1-2), 135 - 43 Cloning and mapping of the dihydrofolate reductase gene of Bacillus subtilis; Myoda TT et al.; The structural gene for dihydrofolate reductase (dfrA) from the Bacillus subtilis 168 chromosome has been cloned, along with the thyB gene, on a 4.5-kb insert contained on chimeric plasmid pER1 . The presence of the dfrA gene on pER1 was demonstrated by showing that: (i) transformation of Escherichia coli strains RUE10(Thy-) and RUE11(Thy+) with pER1 resulted in a 60 to 130-fold increase in dihydrofolate reductase (DFRase) activity with a turnover number characteristic of that of B . subtilis and (ii) pER1-mediated transformation of trimethoprim-resistant E . coli strain D05, which overproduced a DFRase with a decreased affinity for trimethoprim, resulted in a 41-fold increase in DFRase activity with an affinity for trimethoprim similar to that of the B . subtilis enzyme . The dfrA gene was mapped to the 200 degrees region of the B . subtilis chromosome, and the gene order was established as thyB dfrA ilvA . Furthermore, the dfrA gene was shown to be linked closely (95-99% cotransformation) to the thyB gene. J Gen Microbiol, 1984 Jul, 130 ( Pt 7), 1613 - 21 Gene amplification in Bacillus subtilis; Young M; A strain of Bacillus subtilis that carries in its genome a staphylococcal chloramphenicol acetyltransferase gene (from pC194) responds to growth at different concentrations of chloramphenicol by an alteration in the number of copies per genome of the sequences encoding the gene . Growth at 20 micrograms chloramphenicol ml-1 results in a 15-fold amplification of the sequences, whereas growth in the absence of chloramphenicol results in their loss . The mechanism of in situ amplification probably has much in common with that involved in 'R factor transitioning' . The hybridization procedures that have been used for accurately determining the number of copies of the amplified DNA sequences are potentially useful for plasmid copy number determination . The findings reported here also provide a potentially useful alternative to more conventional cloning strategies that are based on autonomous plasmids in B . subtilis . The particular advantages that can be envisaged include enhanced stability of the cloned sequences and control of the number of copies that are present. Genetika, 1984 Jul, 20(7), 1061 - 6 {Cloning of the Bacillus subtilis DNA fragment containing the genes for lysine and riboflavin biosynthesis}; Okunev OV et al.; The SalI fragment of chromosomal DNA of Bacillus subtilis carrying the gene for lysine biosynthesis and the regulatory operator region (ribO) from the riboflavin gene was cloned into Escherichia coli cells . This fragment was shown to contain the gene coding for lysine synthesizing enzyme . Localization of this gene in Bac . subtili was determined . New plasmids pLRS33 and pLRB4 were constructed using pBR322; they carry a fragment homologous to pLP102 plasmid containing the operon for riboflavin biosynthesis. J Bacteriol, 1984 Jul, 159(1), 243 - 50 Regulation of glycerol uptake by the phosphoenolpyruvate-sugar phosphotransferase system in Bacillus subtilis; Reizer J et al.; Enteric bacteria have been previously shown to regulate the uptake of certain carbohydrates (lactose, maltose, and glycerol) by an allosteric mechanism involving the catalytic activities of the phosphoenolpyruvate-sugar phosphotransferase system . In the present studies, a ptsI mutant of Bacillus subtilis, possessing a thermosensitive enzyme I of the phosphotransferase system, was used to gain evidence for a similar regulatory mechanism in a gram-positive bacterium . Thermoinactivation of enzyme I resulted in the loss of methyl alpha-glucoside uptake activity and enhanced sensitivity of glycerol uptake to inhibition by sugar substrates of the phosphotransferase system . The concentration of the inhibiting sugar which half maximally blocked glycerol uptake was directly related to residual enzyme I activity . Each sugar substrate of the phosphotransferase system inhibited glycerol uptake provided that the enzyme II specific for that sugar was induced to a sufficiently high level . The results support the conclusion that the phosphotransferase system regulates glycerol uptake in B . subtilis and perhaps in other gram-positive bacteria. J Bacteriol, 1984 Jul, 159(1), 228 - 32 New mutation affecting the synthesis of some membrane proteins and sporulation in Bacillus subtilis; Matsuzaki S et al.; A new mutation, mpo, which affects the synthesis of some membrane proteins and sporulation in Bacillus subtilis was identified . The mpo mutation was tightly linked to the overproduction of membrane proteins MP32 and MP18 (molecular weights of 32,000 and 18,000, respectively) and the temperature-sensitive sporulation phenotype . Genetic analysis showed that the mpo mutation maps between the spoIIIB and lys loci. Gene, 1984 Jul-Aug, 29(1-2), 51 - 61 Restriction and modification in Bacillus subtilis: nucleotide sequence, functional organization and product of the DNA methyltransferase gene of bacteriophage SPR; Buhk HJ et al.; Bacillus subtilis phage SPR codes for a DNA methyltransferase (Mtase) which methylates the 5' cytosine in the sequence GGCC and both cytosines in the sequence CCGG . A 2126-bp fragment of SPR DNA containing the Mtase gene has been sequenced . This fragment has only one significant open reading frame of 1347 bp, which corresponds to the Mtase gene . Within the sequence the Mtase promoter has been defined by S1 mapping . The size of the SPR Mtase predicted from the deduced amino acid composition is 49.9 kDal . This is in agreement with both the Mr of the purified enzyme and with that of the SPR Mtase gene product identified here by minicell technique . Base changes leading to mutants affected in Mtase activity were localized within the Mtase gene. Gene, 1984 Jul-Aug, 29(1-2), 33 - 40 Cloning and expression of gene 2, required for the protein-primed initiation of the Bacillus subtilis phage phi 29 DNA replication; Blanco L et al.; A phi 29 DNA fragment containing gene 2, coding for a phi 29-specific DNA polymerase required for the formation of the terminal protein p3-dAMP initiation complex, the first step in phi 29 DNA replication, has been cloned in plasmid pPLc28 under the control of the pL promoter of bacteriophage lambda . Four polypeptides of Mr 68 000, 5800 and 3400 and less than 2000 were labelled with {35S}methionine after heat induction . The protein of Mr 68 000 had the size expected for protein p2 and it accounted for about 2% of the de novo synthesized protein . Protein p2 synthesized in Escherichia coli was shown to be stable and biologically active . Its enzymatic activity could be assayed by the in vitro formation of the protein p3-dAMP initiation complex when complemented with extracts from Bacillus subtilis infected with a phi 29sus2 mutant or with extracts from E . coli harbouring gene 3-containing recombinant plasmids . Moreover, protein p2-containing E . coli extracts could catalyze the initiation reaction in vitro when complemented with highly purified protein p3. Gene, 1984 Jul-Aug, 29(1-2), 21 - 6 New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments; Sullivan MA et al.; Two new shuttle vectors have been constructed by fusing the Escherichia coli plasmid pUC9 with the Staphylococcus aureus plasmids pU110 and pC194 . The resulting hybrids replicate in both E . coli and Bacillus subtilis and contain seven restriction sites within a part of the lacZ gene . Insertion of foreign DNA into those sites can be easily detected in E . coli and hybrid plasmids can subsequently be transformed into B . subtilis. Gene, 1984 Jul-Aug, 29(1-2), 11 - 9 Convergent transcription of the Escherichia coli hisG gene cloned in Bacillus subtilis stops in the vicinity of the attenuator; Ferretti L et al.; A 5300-bp DNA segment containing the promoter, the attenuator and the first gene (hisG) of the Escherichia coli his operon has been inserted into an interspecific E . coli-Bacillus subtilis plasmid vector, pHV14 . The resulting plasmid pPV48 restores the His+ phenotype to an E . coli hisG mutant, but fails to do so to a corresponding B . subtilis mutant . Experiments aimed at localizing the block to this heterologous expression in B . subtilis have shown that the enzymatic activity of the hisG+ gene product is neither detectable nor inhibited in crude extracts of B . subtilis cells harboring pPV48 . Furthermore, electron microscopic, Southern blot and S1 mapping analysis of the transcripts produced in vitro and in vivo by B . subtilis RNA polymerase indicate that the hisG+ region is transcribed, but that the transcripts initiate at sites different from the his promoter, converge towards, and terminate in the vicinity of the attenuator. Biochemistry, 1984 Jun 5, 23(12), 2600 - 6 In vitro methylation and demethylation of methyl-accepting chemotaxis proteins in Bacillus subtilis; Goldman DJ et al.; Bacillus subtilis responds to attractants by demethylating a group of integral membrane proteins referred to as methyl-accepting chemotaxis proteins (MCPs) . We have studied the methylation and demethylation of these proteins in an in vitro system, consisting of membrane vesicles, and purified methyltransferase and methylesterase . The chemoattractant aspartate was found to inhibit methylation and stimulate demethylation of MCPs . Escherichia coli radiolabeled membranes in the presence of B . subtilis enzyme do not respond to aspartate by an increase demethylation rate . We also report that B . subtilis MCPs are multiply methylated, demethylation resulting in slower migrating proteins on sodium dodecyl sulfate-polyacrylamide gels. Chemioterapia, 1984 Jun, 3(3), 152 - 5 Bacillus subtilis spores as a natural pro-host oral agent . Preliminary data in children; Novelli A et al.; The commercial preparation of Bacillus subtilis spores may be considered within the classification of biological response modifiers (BRM's) and included among exogenous natural substances . Recently we decided to study the effect of a long-term B . subtilis spores oral treatment in children suffering from recurrent infectious diseases of the respiratory tract . Fifty-three children 5-9 years old have been studied . The clinical valuative parameter was the number of days of absence from school during a 4-month period . In another group of 12 diseased children, mean age 5.5 yrs we recently initiated a laboratory immunological evaluation of peripheral lymphomonocytes in relation to an oral treatment with B . subtilis spores for at least 2 months . Our results show that B . subtilis spore therapy significantly reduced the frequency of respiratory tract infections in the group of treated children . In addition, preliminary immunological laboratory evaluation demonstrated a complete return to the normal lymphomonocyte status after at least 2 months of treatment with B . subtilis spores. Int J Radiat Biol Relat Stud Phys Chem Med, 1984 Jun, 45(6), 615 - 26 Effects of oxygen and sulphydryl-containing compounds on irradiated transforming DNA . II . Glutathione, cysteine and cysteamine; Held KD et al.; This paper extends our earlier observations on the effects of the sulphydryl (SH)-containing compound dithiothreitol (DTT) on the radiation response of Bacillus subtilis transforming DNA to three other SH-containing compounds-cysteamine, cysteine and glutathione (GSH) . In general, all four compounds protect transforming DNA in a manner which is dependent on gassing conditions . In O2, the protection is consistent with the scavenging of OH radicals by the SH compounds, but in N2 there is additional protection which may be due to hydrogen atom donation from the SH compound to radiation-induced DNA lesions, a process which is blocked by O2 . This additional protection in N2 results in an increase in the ratio of inactivation in the absence and presence of oxygen with increasing SH concentration to a maximum followed by a decrease at high SH concentrations . The maximum value of the ratio and the SH concentration at which it occurs depend on the SH compound . In particular, GSH appears to be significantly less efficient in the hydrogen-donation repair reaction with transforming DNA than are the other three SH compounds . Furthermore, on the basis of our results, we postulate the existence of a damage fixation process which occurs in the absence of O2, in competition with damage repair by SH compounds, and that this anoxic damage fixation occurs at a rate not less than 300 s-1 . We also demonstrate here that the damage fixing reaction of O2 with transforming DNA radicals proceeds 200-fold faster than the competing repair reaction by hydrogen-donation from DTT. Proc Natl Acad Sci U S A, 1984 Jun, 81(11), 3562 - 6 Twisted states of Bacillus subtilis macrofibers reflect structural states of the cell wall; Mendelson NH et al.; Static and dynamic studies of helical Bacillus subtilis macrofibers reveal that a spectrum of twisted states exists ranging from tight left-handed structures with twist equal to approximately equal to 40 left turns per mm to tight right-handed structures with twist equal to 57 right turns per mm . In the lytic-deficient strain FJ7 , twist varies as a function of growth temperature above or below 39 degrees C, where there is zero twist . The relationship between the temperature (below 39 degrees C) at which right-hand structures are produced to the time it takes for them to begin the inversion process in which they become left-handed following transfer to 48 degrees C reveals that structures with less twist are more rapidly converted to left-handedness than are those with higher values of twist . The initial response of live macrofibers to digestion by lysozyme consists of "relaxation" motions in which the twist of both left- and right-handed structures changes towards the right-hand end of the spectrum . The rate of relaxation is approximately equal to 5-fold higher at the left-hand end than at the right-hand end . These findings suggest that cell wall polymers can assume a range of structural states during helical growth and that these determine the quantitative aspects of macrofiber shape as well as the sensitivity of walls to attack by lysozyme. Proc Natl Acad Sci U S A, 1984 Jun, 81(11), 3457 - 60 Selective expression of a plasmid cat gene at a late stage of Bacillus subtilis sporulation; Mongkolsuk S et al.; The cat-86 gene in plasmid pPL603 specifies chloramphenicol acetyltransferase (CAT) and is selectively expressed in Bacillus subtilis at a stage in sporulation in which internal spores are first observed (approximately T8) . The gene is unexpressed in vegetatively growing cells . cat-86 expression and spore formation are both blocked when cells are grown in excess glucose . cat-86 expression at T8 is due to selective transcription of the gene, since cat-86 mRNA is undetectable in vegetatively growing cells but is readily demonstrated in sporulating cells . The transcription start site for cat-86 mRNA from sporulating cells is within a 203-base-pair restriction fragment designated P1, which is located upstream from the cat coding region on pPL603 . Deletion of P1 from pPL603 eliminates the sporulation -associated expression of cat-86 . Host sporulation genes, whose function is absolutely required for cat-86 expression at T8, include six early sporulation, spo0 , genes and spoIIE . Therefore, pPL603 provides a novel system in which the in vivo expression of a known, plasmid-linked gene is dependent on sporulation-specific changes in B . subtilis. J Bacteriol, 1984 Jun, 158(3), 990 - 6 N-acetylmannosaminyl(1----4)N-acetylglucosamine, a linkage unit between glycerol teichoic acid and peptidoglycan in cell walls of several Bacillus strains; Kaya S et al.; The structure of teichoic acid-glycopeptide complexes isolated from lysozyme digests of cell walls of Bacillus subtilis (four strains) and Bacillus licheniformis (one strain) was studied to obtain information on the structural relationship between glycerol teichoic acids and their linkage saccharides . Each preparation of the complexes contained equimolar amounts of muramic acid 6-phosphate and mannosamine in addition to glycopeptide components and glycerol teichoic acid components characteristic of the strain . Upon treatment with 47% hydrogen fluoride, these preparations gave, in common, a hexosamine-containing disaccharide, which was identified as N- acetylmannosaminyl (1----4) N-acetylglucosamine, along with large amounts of glycosylglycerols presumed to be the dephosphorylated repeating units of teichoic acid chains . The glycosylglycerol obtained from each bacterial strain was identified as follows: B . subtilis AHU 1392, glucosyl alpha (1----2)glycerol; B . subtilis AHU 1235, glucosyl beta(1----2) glycerol; B . subtilis AHU 1035 and AHU 1037, glucosyl alpha (1----6)galactosyl alpha (1----1 or 3)glycerol; B . licheniformis AHU 1371, galactosyl alpha (1----2)glycerol . By means of Smith degradation, the galactose residues in the teichoic acid-glycopeptide complexes from B . subtilis AHU 1035 and AHU 1037 and B . licheniformis AHU 1371 were shown to be involved in the backbone chains of the teichoic acid moieties . Thus, the glycerol teichoic acids in the cell walls of five bacterial strains seem to be joined to peptidoglycan through a common linkage disaccharide, N- acetylmannosaminyl (1----4)N-acetylglucosamine, irrespective of the structural diversity in the glycosidic branches and backbone chains. J Bacteriol, 1984 Jun, 158(3), 884 - 9 Effect of purine and pyrimidine limitations on RNA synthesis in Bacillus subtilis; Vasantha N et al.; The effects of varying the intracellular levels of GTP or UTP on the rate of RNA synthesis in Bacillus subtilis were studied . The levels of these nucleotides were manipulated by pyrimidine limitation in a pyr auxotroph, by purine limitation in a pur auxotroph, or by the addition of decoyinine , which specifically inhibits GMP synthesis . Decreased levels of UTP and GTP were accompanied by dramatically decreased synthesis and accumulation of stable RNAs (tRNA and rRNA), but mRNA synthesis was less affected . However, sporulation was initiated only when the intracellular level of GTP decreased. J Bacteriol, 1984 Jun, 158(3), 1182 - 7 Helical macrofiber formation in Bacillus subtilis: inhibition by penicillin G; Zaritsky A et al.; The folding process required for helical macrofiber formation after the outgrowth of Bacillus subtilis spores was found to be blocked by very low concentrations of penicillin G (1 to 3 ng/ml) . Under such conditions, growth and septation without cell separation resulted in characteristic disorganized multicellular structures . Higher concentrations (4 and 10 ng/ml) were needed to inhibit spore outgrowth and vegetative growth, respectively. Virology, 1984 Jun, 135(2), 555 - 60 Ribonucleoside triphosphate concentration-dependent termination of bacteriophage SP01 transcription in vitro by Bacillus subtilis RNA polymerase; Brennan SM; Several sites specifying transcription termination in the bacteriophage SP01 terminal repeat have recently been located and characterized . Some of these were identified as partial terminators . Further characterization of three of the partial terminators leads to the conclusion that they are not sites of inefficient transcriptional termination by Bacillus subtilis RNA polymerase . Rather, these are sites where termination is either completely efficient or fails to occur at all, depending upon the ribonucleoside triphosphate (rNTP) concentration in the reaction mixture . The threshold rNTP concentration, above which termination will not occur, is the same for two of the terminators studied here and different for the third. J Bacteriol, 1984 Jun, 158(3), 784 - 90 Regulatory regions that control expression of two chloramphenicol-inducible cat genes cloned in Bacillus subtilis; Duvall EJ et al.; Plasmid pPL603 is a promoter cloning vector for Bacillus subtilis and consists of a 1.1-kilobase fragment of Bacillus pumilus DNA inserted between the EcoRI and BamHI sites of pUB110 . The gene cat-86, specifying chloramphenicol-inducible chloramphenicol acetyltransferase, is located on the 1.1-kilobase cloned DNA . When pPL603 is present in B . subtilis, cat-86 is unexpressed during vegetative growth but expressed during sporulation . The regulation of cat-86 in pPL603 is due to sequences within two restriction fragments, designated P1 and R1, that precede the main coding portion of the gene . The P1 fragment promotes transcription of cat-86 only during sporulation, whereas the adjacent R1 fragment lacks promoter function but contains sequences essential to chloramphenicol inducibility . A second B . pumilus gene, cat-66, was cloned in B . subtilis and is expressed throughout the vegetative growth and sporulation cycle . The cat-66 coding region is preceded by two adjacent restriction fragments designated as P2 and R2 . P1 and P2 are identical in size and share 95% conservation of base sequence . R1 and R2 are also identical in size and share 91% conservation of base sequence . Fragment substitution experiments demonstrate that R2 can functionally replace R1 . The substitution of P2 for P1 promotes cat-86 expression throughout vegetative growth and sporulation . Analysis of a derivative of pPL603 in which P2 has replaced P1 demonstrates that P2 promotes transcription of cat-86 during vegetative growth and that P2 contains the start site for transcription of cat-86 . Thus, P1 and P2 differ strikingly in vegetative promoter function, yet they differ by single-base substitutions at only 11 positions of 203. J Bacteriol, 1984 Jun, 158(3), 1054 - 60 Molecular cloning of a major cell wall protein gene from protein-producing Bacillus brevis 47 and its expression in Escherichia coli and Bacillus subtilis; Tsukagoshi N et al.; Bacillus brevis 47 contains two major cell wall proteins . Each protein forms a hexagonal array in the cell wall . A 4.8-kilobase HindIII fragment of B . brevis 47 DNA cloned into Escherichia coli with pBR322 as a vector directed the synthesis of polypeptides cross-reactive with antibody to the middle wall protein . A 700-base-pair BamHI-HpaI fragment was shown to be the essential region for the synthesis of immunoreactive polypeptides . Furthermore, this fragment appeared to contain the promoter activity . The 3.5-kilobase BamHI fragment covering the essential region as well as its downstream sequence was subcloned into the corresponding restriction site of pUB110 by using Bacillus subtilis as the cloning host . Both E . coli and B . subtilis carrying the cloned DNA synthesized several immunoreactive polypeptides which were mainly found in the cytoplasm . B . subtilis secreted polypeptides cross-reactive with antibody to the middle wall protein . These extracellular polypeptides were degraded upon prolonged culture. J Gen Microbiol, 1984 Jun, 130 ( Pt 6), 1577 - 86 The isolation of lambda phage carrying DNA from the histidine and isoleucine-valine regions of the Bacillus subtilis chromosome; Walton DA et al.; From a lambda gtWES library of the chromosome of Bacillus subtilis, phages carrying DNA from the hisA and ilv-leu regions were isolated . They were identified by their ability to form complementing plaques on hisB, ilvC or leuB mutants of Escherichia coli K12 under selective conditions and in the presence of a helper phage . The his phages complemented E . coli his A, B or D mutations and could transform seven mutations in the hisA region of the B . subtilis chromosome; each carried a single EcoR1 insert of about 8.2 kb . Phages complementing E . coli ilvC or leuB mutations and carrying the equivalent B . subtilis genes ilvC and leuC transformed a range of mutations in the B . subtilis ilv-leu region . The distribution of genetic markers carried by the phages suggests that the entire ilv-leu cluster from az1A through leuD is covered in the collection of phages obtained and is carried in three EcoR1 restriction fragments of approximately 6.7, 4.7 and 2.85 kb. Gene, 1984 Jun, 28(3), 301 - 10 Transfer and expression of recombinant plasmids carrying pneumococcal mal genes in Bacillus subtilis; Espinosa M et al.; The pneumococcal mal recombinant plasmid pLS70, which carries two strong promoters for transcription, could not be transferred and maintained intact in Bacillus subtilis . Although it could be established at low frequency, pLS70 was unstable and was rapidly replaced by deleted forms of the plasmid . A deleted derivative plasmid, pLS69, could be transferred at high frequency and maintained intact . In pLS69 the deletion reduces function of both the malM (amylomaltase) and malX (X-fragment) promoters . This mutant mal plasmid still codes for an intact amylomaltase, and the enzyme is produced in both S . pneumoniae and B . subtilis . The amylomaltase, which is inducible by maltose in S . pneumoniae, is synthesized constitutively in B . subtilis and is localized in the cytosol . Although pLS69 enables S . pneumoniae to grow with maltose, the plasmid did not enhance the ability of B . subtilis to use this sugar, presumably because the latter does not transport free maltose into the cell . Minicells of B . subtilis containing pLS69 synthesized the amylomaltase polypeptide but no X-fragment . In S . pneumoniae carrying pLS69, production of the X-fragment is also reduced more than the amylomaltase, when compared to cells carrying pLS70, which produce equal amounts of the two proteins . Inasmuch as the down promoter mutation leaves unchanged both structural genes, their ribosome-binding sites and -10 and -35 promoter sequences, the unequal effect is attributed to differential reduction in AT composition proximal to the promoters . Vector proteins were revealed in minicells as several bands, all located in the cytosol except for an Mr 35000 polypeptide located in the membrane. J Bacteriol, 1984 Jun, 158(3), 967 - 71 Expression of the Bacillus subtilis glutamine synthetase gene in Escherichia coli; Gardner AL et al.; The structural gene for glutamine synthetase (glnA) in Bacillus subtilis ( glnAB ) cloned in the lambda vector phage Charon 4A was used to transduce a lysogenic glutamine auxotrophic Escherichia coli strain to prototrophy . The defective E . coli gene ( glnAE ) was still present in the transductant since it could be transduced . In addition, curing of the prototroph resulted in the restoration of glutamine auxotrophy . Proteins in crude extracts of the transductant were examined by a "Western blotting" procedure for the presence of B . subtilis or E . coli glutamine synthetase antigen; only the former was detected . Growth of the strain in media without glutamine was not curtailed even when the bacteriophage lambda pL and pRM promoters were hyperrepressed . The specific activities and patterns of derepression of glutamine synthetase in the transductant were similar to those of B . subtilis, with no evidence for adenylylation . The information necessary for regulation of glnAB must be closely linked to the gene and appears to function in E . coli. Genetika, 1984 Jun, 20(6), 943 - 8 {Cloning of the purA16 locus in Rec+ cells of Bacillus subtilis}; Poluektova EU et al.; A portion of purA16 chromosomal locus of Bacillus subtilis was cloned into Rec+ cells of this microorganism with pBD12 plasmid (carrying chloramphenicol and kanamycin resistance determinants) serving as a vector . The hybrid plasmids were stably maintained in cells grown on media supplemented with antibiotics and were lost from cells in the absence of drugs . The cloned fragment could incorporate into the chromosome some with a frequency of 10(-2) per cell per generation . A clone carrying the hybrid plasmid inserted into the chromosome was detected. J Biol Chem, 1984 May 25, 259(10), 6364 - 8 Isolation and characterization of pteroylpolyglutamate hydrolase from rat intestinal mucosa; Elsenhans B et al.; Pteroylpolyglutamate hydrolase was isolated from rat intestinal mucosa and purified with the aid of affinity chromatography . The affinity ligand was poly-gamma-glutamic acid (Mr approximately 12,000) derived from Bacillus subtilis . The specific enzymatic activity was increased 2,000-fold over the 100,000 X g supernatant of the mucosal homogenate with a yield of 20% . Sephadex G-200 gel filtration yielded an estimated molecular mass of 80,000 daltons . The isoelectric point was pH 8.2 . The pH optimum in acetate buffer containing 1 mM zinc was 4.5 . The KM values for pteroylheptaglutamate and pteroyltriglutamate were 0.21 and 0.67 microM, respectively . Polyanionic compounds, poly-gamma-glutamic acid, dextran sulfate, and heparin were noncompetitive inhibitors . Studies of the time course of hydrolysis of synthetic {3H}pteroylheptaglutamate by three separate techniques demonstrated the appearance of {3H}pteroylmonoglutamate, synchronous with substrate cleavage . Intermediate pteroyloligoglutamates were not detected . An endopeptidase-like mode of hydrolysis was further established by identification of a hexaglutamyl peptide as the other reaction product. J Biol Chem, 1984 May 25, 259(10), 6681 - 5 Overproduction and purification of a bacteriophage SPO1-encoded RNA polymerase sigma factor; Costanzo M et al.; Gene 28 of bacteriophage SPO1 encodes an RNA polymerase sigma factor sigma gp28, which replaces the host Bacillus subtilis sigma subunit sigma 55, to alter the promoter recognition specificity of RNA polymerase . A fragment of SPO1 DNA containing gene 28 was placed under the control of the PL promoter of bacteriophage lambda in an Escherichia coli expression vector . When transcription of gene 28 was induced by derepression of PL, the sigma gp28 synthesized constituted several per cent of total cellular protein . Sigma gp28 purified from these cells was able to confer specificity for SPO1 middle gene promoters upon B . subtilis core RNA polymerase, and also enabled E . coli core RNA polymerase to recognize and initiate transcription from an SPO1 middle gene promoter. J Mol Biol, 1984 May 25, 175(3), 285 - 97 Promoter recognition by sigma-37 RNA polymerase from Bacillus subtilis; Tatti KM et al.; Bacillus subtilis possesses at least five different forms of RNA polymerase holoenzyme which are distinguished by their sigma subunit and their promoter recognition specificity . Sigma-37 RNA polymerase, a minor form of RNA polymerase, recognizes a class of promoters, which includes promoters for genes transcribed early during endospore formation . We have used site-directed bisulfite mutagenesis to construct a series of single and multiple base substitutions in a promoter recognized by sigma-37 RNA polymerase . In vitro transcription analysis of this series of mutant promoters demonstrated that single base substitutions at positions -36, -16, -15 and -14 most dramatically reduced the efficiency of promoter utilization by sigma-37 RNA polymerase . These results support a model in which sigma-37 RNA polymerase recognizes its cognate promoters by interacting with a sequence of nucleotides near the -10 region and the -35 region of the promoter--a sequence not recognized by B . subtilis sigma-55 RNA polymerase or Escherichia coli RNA polymerase. Biochem J, 1984 May 15, 220(1), 117 - 23 Quasi-elastic light scattering studies on dormant and germinating Bacillus subtilis spores; Harding SE et al.; Spores of Bacillus subtilis in suspension, both dormant and germinating, have been examined by light-scattering methods, both integrated intensity and correlation versions . When intensity of scatter at constant volume was plotted against angle, curves possessing a maximum at about 20 degrees were regularly obtained but without any noticeable features at higher angles . This indicated polydispersity and/or asymmetry among the population . Curves of the intensity correlation function for both dormant and germinating spores at different angles, theta, did not superimpose at the lower angles when plotted appropriately, but did so for theta greater than 35 degrees . This was considered to arise from asymmetry of the spores . By using the high-angle data the apparent diffusion coefficient was determined for both dormant spores and for germinating spores from 1 min after germinant addition . No appreciable difference was observed, from which volume changes greater than 6% during germination could be excluded . The occurrence of germination was confirmed by both absorbance and phase-contrast-microscopy observations. Eur J Biochem, 1984 May 15, 141(1), 83 - 9 The primary structure of teichuronic acid in Bacillus subtilis AHU 1031; Yoneyama T et al.; Structural studies were carried out on the acidic polysaccharide fraction obtained from lysozyme digest of the cell walls of Bacillus subtilis AHU 1031 . The polysaccharide fraction contained N- acetylmannosaminuronic acid ( ManNAcA ), N-acetylglucosamine (GlcNAc), glucose, glycerol and phosphorus in a molar ratio of 2:2:4:1:1, together with glycopeptide components . The results of analyses involving Smith degradation, chromium trioxide oxidation, methylation and proton magnetic resonance spectroscopy led to the conclusion that the backbone chain of the polysaccharide has the repeating unit----6)Glc(alpha 1----3/4) ManNAcA (beta 1----4)GlcNAc(beta 1---- . About 50% of the N-acetylglucosamine residues in the backbone chain seem to be substituted at C-3 by the glycosidic branches, glycerol phospho-6-glucose, while the other half seem to be substituted by glucose. Appl Environ Microbiol, 1984 May, 47(5), 1039 - 46 Characterization of the cellulolytic activity of a Bacillus isolate; Robson LM et al.; A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis . When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose . Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity . The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production . Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000 . Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity . Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose . Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates . In contrast to T . reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable. Gene, 1984 May, 28(2), 171 - 6 Constitutive variants of the pC194 cat gene exhibit DNA alterations in the vicinity of the ribosome binding site sequence; Ambulos NP Jr et al.; The chloramphenicol-inducible regulation of the expression of cat genes from two Gram-positive bacteria, Staphylococcus aureus and Bacillus pumilus has been suggested to result from the presence of inverted repeat sequences that span the ribosome-binding site (RBS) for cat . In support of this hypothesis, we demonstrate that two derivatives of the pC194 cat gene which are constitutively expressed in Bacillus subtilis are deleted for all or a major portion of the inverted-repeat sequences. J Gen Microbiol, 1984 May, 130 ( Pt 5), 1263 - 9 Use of temperature-sensitive mutants to study gene expression of two closely linked sporulation loci in Bacillus subtilis; Lamont IL et al.; The spoOB and spoIVF loci are contiguous on the chromosome of Bacillus subtilis, so that genes in these loci may be parts of a single polycistronic operon . Temperature-sensitive strains having mutations in these loci were isolated, and temperature-shift experiments were carried out to investigate expression of the genes . The temperature-sensitive periods of spoOB mutants extended from the beginning of sporulation until the end of the stage II . The temperature-sensitive periods of spoIVF strains were during stage IV of sporulation . Therefore, although the spoOB and spoIVF loci are contiguous on the chromosome it is unlikely that genes in them are parts of a single polycistronic operon. J Gen Microbiol, 1984 May, 130 ( Pt 5), 1253 - 61 Identification of a new sporulation locus, spoIIIF, in Bacillus subtilis; Lamont IL et al.; We have isolated a mutant of Bacillus subtilis, strain 590, which is blocked at stage III of sporulation . The spo mutation which is carried by this strain is linked to pheA by transformation and defines a previously unidentified locus, spoIIIF . The spoIIIF locus is contiguous with the spoVB locus, in which a mutation causes a block at stage V of sporulation . We also give a detailed genetic map of the pheA region of the chromosome. J Gen Microbiol, 1984 May, 130 ( Pt 5), 1247 - 52 Bacillus subtilis 168 mutants resistant to arginine hydroxamate in the presence of ornithine or citrulline; Baumberg S et al.; Mutations in Bacillus subtilis 168 have been isolated that confer resistance to arginine hydroxamate in the presence, but not absence, of ornithine . Seven such Ahor mutants have been studied in detail . In common with certain classes of Ahr mutant (resistant to arginine hydroxamate in the absence of arginine precursors) described previously, these Ahor mutants showed little or no inducibility of enzymes of arginine catabolism . Mutants that showed no inducibility were unable to utilize arginine or ornithine as sole nitrogen source . The only biosynthetic enzyme to show any consistent differences in activity from the parent was ornithine carbamoyltransferase, whose level was slightly elevated in cells grown in the presence of ornithine or citrulline . PBS1 transduction crosses showed that two of the ahor mutations map at the ahrA locus, while a third (unique in its resistance to arginine hydroxamate in the presence of citrulline) mapped at a hitherto undescribed locus closely linked to metC, designated ahrD. J Bacteriol, 1984 May, 158(2), 746 - 8 Degradation of aspartate transcarbamylase in Bacillus subtilis is deficient in rel mutants but is not mediated by guanosine polyphosphates; Bond RW et al.; Degradation of aspartate transcarbamylase in growing and starved Bacillus subtilis was deficient in relA and relC mutants, but these effects were not correlated with differences in the intracellular level of guanosine polyphosphates. J Bacteriol, 1984 May, 158(2), 411 - 8 Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation; Stahl ML et al.; The entire subtilisin structural gene from Bacillus subtilis I168 has been cloned, and its nucleotide sequence has been determined . When expressed on a high-copy-number shuttle vector, a fivefold increase in serine protease activity was observed . The DNA sequence of the gene is 80% homologous to the Bacillus amyloliquefaciens subtilisin structural gene, and the translated mature coding sequence is 85% homologous to the published protein sequence of subtilisin BPN' . The chloramphenicol resistance determinant of a plasmid integrated at the subtilisin locus was mapped by PBS1 transduction and was found to be linked to glyB (83%) and argC (60%), but not with metC or purB . The chromosomal locus containing the wild-type subtilisin allele was replaced with an in vitro-derived allele of the gene (delta apr-684) that contained a 684-base-pair deletion . The technique used for introducing the deletion is a variation of the gene replacement methods used in Saccharomyces cerevisiae and Escherichia coli . When used in B . subtilis, deletion mutants could be directly screened among the transformants . Physiological characterization of the delta apr-684 mutation revealed no discernable effect on the formation of heat-resistant endospores, but strains carrying the mutation produced only 10% of wild-type serine protease activity . A model is presented that outlines the pathway for plasmid integration and deletion formation in B . subtilis. J Bacteriol, 1984 May, 158(2), 507 - 12 Cloning of sporulation gene spoIIG in Bacillus subtilis; Ayaki H et al.; Two specialized transducing phages carrying a sporulation gene, spoIIG , of Bacillus subtilis were constructed from B . subtilis temperate phages p11 and phi 105 by the "prophage transformation" method . Restriction enzyme analysis and transformation experiments showed that the spoIIG gene was present on a 6.2 X 10(6)-dalton (6.2-Md) EcoRI fragment in both transducing phage genomes . Further analysis showed that spoIIG + transforming activity resides on a 2.25-Md EcoRI-BamHI fragment within the 6.2-Md EcoRI fragment . The 2.25-Md fragment was subcloned into the region between the EcoRI and BamHI sites of pUB110, and deletion plasmids lacking PstI or HindIII fragments within the 2.25-Md fragment were constructed . The recombinant plasmid carrying the intact spoIIG gene restored sporulation of strain HU1002 ( spoIIG41 recE4 ) to a frequency of 10(4) spores per ml and inhibited sporulation of strain 4309 ( spo + recE4 ) to a level of 10(3) spores per ml. Anal Biochem, 1984 May 1, 138(2), 465 - 71 Preparative-scale isolation and purification of procaryotic and eucaryotic ribosomal 5 S RNA: Bacillus subtilis, Neurospora crassa, and wheat germ; Li SJ et al.; Ribosomal 5 S RNA from three different organisms has been isolated in high yield and purity . Without prior isolation of ribosomes, a presoak in buffer followed by phenol extraction, DE-32 ion-exchange chromatography, and Sephadex G-75 gel-permeation chromatography yields at least 5-10 mg of electrophoretically homogeneous 5 S RNA from 100 g of cells . Ribonuclease activity is eliminated by various combinations of low temperature, sodium dodecyl sulfate, phenol, and bentonite . High-molecular-weight contaminants are suppressed by either 65 degrees C heat treatment or lowered sodium dodecyl sulfate concentration . For the eucaryotes, 5.8 S RNA contamination is reduced either by low temperature in the initial solubilization or by postponing 65 degrees C heat treatment until after the phenol extraction step. J Bacteriol, 1984 May, 158(2), 543 - 50 Post-transcriptional regulation of chloramphenicol acetyl transferase; Byeon WH et al.; The +1 site for initiation of inducible chloramphenicol acetyl transferase (CAT) mRNA encoded by plasmid pC194 was determined experimentally by using {alpha-32P}ATP-labeled runoff transcripts partially digested with T1 RNase . By partial digestion of the in vitro transcripts with S1, T1, and cobra venom nucleases as probes of mRNA conformation, single- and double-stranded regions, respectively, were also identified . Thus, a prominent inverted complementary repeat sequence was demonstrated spanning the +14 to +50 positions, which contain the complementary sequences CCUCC and GGAGG (the Shine and Dalgarno sequence for synthesis of CAT) symmetrically apposed and paired as part of a perfect 12-base-pair inverted complementary repeat sequence (-19.5 kcal {ca . -81.7 kJ} per mol) . The CAT mRNA was stable to digestion by T1 RNase at the four guanosine residues in the Shine and Dalgarno sequence GGAGG , even at 60 degrees C, suggesting that nascent CAT mRNA allows ribosomes to initiate protein synthesis inefficiently and that induction involves post-transcriptional unmasking of the Shine and Dalgarno sequence . Consistent with this model of regulation, we found that cells carrying pC194 , induced with chloramphenicol, contain about the same concentration of pulse-labeled CAT-specific RNA as do uninduced cells . Induction of CAT synthesis by the non- acetylatable chloramphenicol analog fluorothiamphenicol was tested by using minicells of Bacillus subtilis carrying pC194 as well as minicells containing the cloned pC194 derivatives in which parts of the CAT structural gene were deleted in vitro with BAL 31 exonuclease . Optimal induction of both full-length (active) and deleted (inactive) CAT required similar concentrations of fluorothiamphenicol, whereas induction by chloramphenicol required a higher concentration for the wild-type full-length (active) CAT than for the (inactive) deleted CAT . Because synthesis of deleted CAT was inducible, we infer that CAT plays no direct role in regulating its own synthesis. J Gen Microbiol, 1984 May, 130 ( Pt 5), 1285 - 91 Cloning and expression of a Bacillus subtilis Endo-1,3-1,4-beta-D-glucanase gene in Escherichia coli K12; Hinchliffe E; EcoRI fragments of DNA from Bacillus subtilis NCIB 8565, a high producer of an endo-1,3-1,4-beta-D-glucanase, were 'shot-gun' cloned in the plasmid vector pBR325 . A 3.5 kb insert, carrying single restriction sites for AvaI, BglII, ClaI, PvuI and PvuII, was shown to direct the synthesis of beta-glucanase in Escherichia coli K12 . Enzyme activity was demonstrated in extracellular fractions of E . coli harbouring the beta-glucanase gene; however, the largest proportion (greater than 50%) of total enzyme activity was periplasmic in location . beta-Glucanase activity and cellular location were independent of the orientation of the 3.5 kb fragment in pBR325. FEBS Lett, 1984 Apr 9, 169(1), 40 - 4 The interaction of Bacillus protoplasts with sonicated phosphatidylcholine liposomes; Urbaneja MA et al.; When protoplasts from Bacillus subtilis are incubated with sonicated liposomes made from egg-yolk phosphatidylcholine, this phospholipid is incorporated into the protoplast membranes . Biochemical, fluorescence and ultrastructural data suggest that incorporation occurs through membrane fusion. Can J Microbiol, 1984 Apr, 30(4), 423 - 9 A catabolite-resistance mutation is localized in the rpo operon of Bacillus subtilis; Sun DX et al.; By transformation analysis, a mutation (crsE1), which makes Bacillus subtilis cells able to sporulate in the presence of relatively high concentrations of glucose and other carbon sources, was mapped in the rpoBC operon . The effect of crsE1 mutation can be suppressed by another mutation in the same operon, rfm11, which confers resistance to rifamycin . Mutants carrying stv or std mutations, which are also located in the rpoBC operon, showed partial resistance to catabolites in sporulation . It appears therefore that a change in the structure or synthesis of RNA polymerase may alter the response of cells to the inhibitory effect of catabolites on sporulation. J Bacteriol, 1984 Apr, 158(1), 55 - 62 Synthesis of oxaloacetate in Bacillus subtilis mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex; Fisher SH et al.; Bacillus subtilis mutants deficient in the 2-ketoglutarate dehydrogenase enzymatic complex required aspartate for growth at wild-type rates on carbon sources for which synthesis of the degradative enzymes is sensitive to catabolite repression (e.g., poor carbon sources), but did not require aspartate for growth on carbon sources which exert catabolite repression (e.g., good carbon sources) . Measurement of metabolite pools in a mutant lacking the 2-ketoglutarate dehydrogenase active complex showed that the aspartate requirement for growth on poor carbon sources resulted from a deficiency in intracellular oxaloacetate pools even through pyruvate carboxylase was present at levels corresponding to those in wild-type cells . The oxaloacetate deficiency most likely resulted from the inability of the mutant to regenerate oxaloacetate from citrate due to the enzymatic block in the tricarboxylic acid cycle . Mutants in the enzymes of the dicarboxylic acid half of the citric acid cycle similarly required aspartate for wild-type growth in minimal medium . These results suggested that the complete turning of the tricarboxylic acid cycle is involved in the maintainance of oxaloacetate levels in B . subtilis . The ability of the mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex to grow at wild-type rates on media containing good carbon sources in the absence of exogenous aspartate is not understood. J Bacteriol, 1984 Apr, 158(1), 386 - 8 New chloramphenicol resistance locus in Bacillus subtilis; Anderson LM et al.; A spontaneously occurring, noninducible, chloramphenicol-resistant mutant of Bacillus subtilis 168 has a mutation (cam-2) which maps in the ribosomal protein region of the chromosome near dal . Its presence does not confer dependence on chloramphenicol . Ribosomes of the cam-2 strain remained sensitive to chloramphenicol in in vitro protein synthesis . No chloramphenicol acetyltransferase activity could be detected. J Gen Microbiol, 1984 Apr, 130 ( Pt 4), 757 - 60 Cloning of sporulation gene spoIIC in Bacillus subtilis; Anaguchi H et al.; Specialized transducing phages rho 11spoIIC and phi 105spoIIC, carrying the Bacillus subtilis sporulation gene spoIIC, were constructed by the prophage transformation method . An EcoRI fragment (2.4 MDal) carrying the spoIIC gene was isolated from the phi 105spoIIC genome and recloned into the EcoRI site of plasmid pUB110 . The recombinant plasmids corrected the sporulation defect of a Spo- Rec- host, but slightly inhibited the sporulation of a Spo+ Rec- host. J Appl Bacteriol, 1984 Apr, 56(2), 295 - 303 Hypochlorite effects on spores and spore forms of Bacillus subtilis and on a spore lytic enzyme; Gorman SP et al.; Spores of Bacillus subtilis NCTC 10073 were converted to ion-exchange (Ca, H) forms and coat-defective (urea-mercaptoethanol, urea-dithiothreitol-sodium lauryl sulphate) forms . The resistance of these to sodium hypochlorite (1000 parts/10(6) free chlorine) was compared and related to uptake from which the assumed monolayer capacities were calculated . Hypochlorite effects on spore protoplasts and cortical fragments were also examined in relation to DPA and hexosamine release . A spore lytic enzyme was extracted and examined in respect of hypochlorite activity . The results are discussed in terms of the mechanism and site of action of hypochlorite on the bacterial spore. J Bacteriol, 1984 Apr, 158(1), 169 - 79 Insertion and fate of the cell wall in Bacillus subtilis; Mobley HL et al.; Cell wall assembly was studied in autolysin-deficient and -sufficient strains of Bacillus subtilis . Two independent probes, one for peptidoglycan and the other for surface-accessible teichoic acid, were employed to monitor cell surface changes during growth . Cell walls were specifically labeled with N-acetyl-D-{3H}glucosamine, and after growth, autoradiographs were prepared for both cell types . The locations of silver grains revealed that label was progressively lost from numerous sites on the cell cylinders, whereas label was retained on the cell poles, even after several generations . In the autolysin-deficient and chain-forming strain, it was found that the distance between densely labeled poles approximately doubled after each generation of growth . In the autolysin-sufficient strain, it was found that the numbers of labeled cell poles remained nearly constant for several generations, supporting the premise that completed septa and poles are largely conserved during growth . Fluorescein-conjugated concanavalin A was also used to determine the distribution of alpha-D-glucosylated teichoic acid on the surfaces of growing cells . Strains with temperature-sensitive phosphoglucomutase were used because in these mutants, glycosylation of cell wall teichoic acids can be controlled by temperature shifts . When the bacteria were grown at 45 degrees C, which stops the glucosylation of teichoic acid, the cells gradually lost their ability to bind concanavalin A on their cylindrical surfaces, but they retained concanavalin A-reactive sites on their poles . Discrete areas on the cylinder, defined by the binding of fluorescent concanavalin A, were absent when the synthesis of glucosylated teichoic acid was inhibited during growth for several generations at the nonpermissive temperature . When the mutant was shifted from a nonpermissive to a permissive temperature, all areas of the cylinder became able to bind the labeled concanavalin A after about one-half generation . Old cell poles were able to bind the lectin after nearly one generation at the permissive temperature, showing that new wall synthesis does occur in the cell poles, although it occurs slowly . These data, based on both qualitative and quantitative experiments, support a model for cell wall assembly in B . subtilis, in which cylinders elongate by inside-to-outside growth, with degradation of the stress-bearing old wall in wild-type organisms . Loss of wall material, by turnover, from many sites on the cylinder may be necessary for intercalation of new wall and normal length extension . Poles tend to retain their wall components during division and are turned over much more slowly. Antibiotiki, 1984 Apr, 29(4), 253 - 7 {Effect of gramicidin S and its derivatives on protoplasts of Bacillus subtilis}; Petrykina ZM et al.; The lysis of Bacillus subtilis protoplasts by gramicidin S, a membrane active antibiotic, and its derivatives was studied according to free amino groups of the ornithine residue . The initial antibiotic and guanylgramicidin , a positive charge-preserving derivative, had a high lytic activity . Succinylgramicidin , a gramicidin S derivative with acid properties, and carbomoylgramicidin , a neutral derivative, actively lysed B . subtilis protoplasts suspended in 1/15 M phosphate buffer solution with sucrose . No lytic activity of succinylgramicidin was observed with respect to B . subtilis protoplasts suspended in an aqueous solution of sucrose . Comparative study on the sensitivity of the protoplasts of Micrococcus lysodeikticus, B . megaterium and B . subtilis to the lytic action of gramicidin S and its derivatives showed in the main a similar character of their interaction with the membranes of the protoplasts of the taxonomically close species (B . megaterium and B . subtilis) . It is likely that the specificity of the action of the above substances on the protoplasts of M . lysodeikticus, i . e . a complicated character of the dependence of the lytic action of gramicidin S on its concentration, manifestation of the lytic activity of the neutral and acid derivatives in the presence of phosphates or other salts and in sucrose aqueous solution was mainly defined by the properties of the micrococcal membranes. J Bacteriol, 1984 Apr, 158(1), 379 - 82 2-Ketoglutarate and the regulation of aconitase and histidase formation in Bacillus subtilis; Fisher SH et al.; In contrast to wild-type cells, the Bacillus subtilis mutant SF109 that lacks the active 2-ketoglutarate dehydrogenase enzymatic complex is unable to increase the specific activity of two enzymes subject to glucose catabolite repression, aconitase and histidase, during limitation of growth by glucose . Examination of the intracellular metabolite pools in the mutant and wild-type cells grown in excess and limiting glucose medium showed that the complete derepression of aconitase and histidase could be correlated with the decrease in the intracellular concentration of 2-ketoglutarate . The complete repression of aconitase that occurred in wild-type and mutant cells could be correlated with a high intracellular concentration of 2-ketoglutarate. Biochem Biophys Res Commun, 1984 Mar 30, 119(3), 1191 - 7 Retrohydroxamate ferrichrome, a biomimetic analogue of ferrichrome; Emery T et al.; A new synthetic analogue of ferrichrome, retrohydroxamate ferrichrome, has been examined for biological activity . Although spectroscopic evidence indicates that the analogue is a weaker Fe(III) chelator than ferrichrome, retrohydroxamate ferrichrome is indistinguishable from ferrichrome in its growth factor activity for Arthrobacter flavescens, and in its potency in antagonizing the antibiotic activity of albomyhcin against Bacillus subtilis . It is as active as ferrichrome as a siderophore for the fungus, Ustaligo sphaerogena . In contrast, desmethylretrohydroxamate ferrichrome shows no significant biological activity. Biochim Biophys Acta, 1984 Mar 27, 793(1), 86 - 94 Products of phosphatidylglycerol turnover in two Bacillus strains with and without lipoteichoic acid in the cells; Koga Y et al.; In order to understand the phosphatidylglycerol turnover mechanism, especially the differential turnover of diacylated and unacylated glycerol moieties of the lipid, products of phosphatidylglycerol metabolism were surveyed in vivo in Bacillus subtilis W23 and an alkalophile, Bacillus sp . strain A007 . When cells of B . subtilis W23 labeled with radioactive glycerol were chased, lipoteichoic acid accumulated 90% of the radioactivity lost from the unacylated glycerol moiety of phosphatidylglycerol . Also, lipids other that phosphatidylglycerol, except diacylglycerol, and glycerol and glycerophosphate incorporated much less radioactivity . The {32P}phosphoryl group was also transferred from phosphatidylglycerol to lipoteichoic acid almost quantitatively in B . subtilis W23 . A unique metabolism of phosphatidylglycerol was found in Bacillus sp . strain A007 which lacked phosphoglycolipid and lipoteichoic acid, that is, the turnover of phosphatidylglycerol of this organism was less extensive compared with that of B . subtilis W23, and both glycerol moieties of the lipid were metabolized at an identical rate . These results suggested that the major reaction involved in the turnover of phosphatidylglycerol was the transfer of glycerophosphate residue to lipoteichoic acid in a bacterium which possessed lipoteichoic acid and that several minor reactions also were involved in phosphatidylglycerol turnover. J Biol Chem, 1984 Mar 25, 259(6), 3694 - 702 Two large clusters with thirty-seven transfer RNA genes adjacent to ribosomal RNA gene sets in Bacillus subtilis . Sequence and organization of trrnD and trrnE gene clusters; Wawrousek EF et al.; Sequences of two large tRNA gene clusters (trrnD and trrnE) in Bacillus subtilis 168 revealed 16 and 21 tRNA genes, respectively, as identified by anticodon assignments . Each cluster contains upstream flanking 23 and 5 S rRNA sequences . The 23-5 S intergenic space in trrnE corresponds exactly to the analogous space in trrnB, which was previously sequenced (Wawrousek, E.F., and Hansen, J.N . (1983) J.Biol . Chem . 258, 291-298) . The 5 S rRNA genes in trrnB and trrnE are B . subtilis major species; but trrnD possesses a minor species (Raue, H.A., and Planta, R . J . (1977) Mol . Gen . Genet . 156, 185-193) gene with a putative promoter that may allow differential expression with respect to the upstream rRNA gene set . Most of the tRNA genes are probably expressed as large transcriptional units, except for a LeuTTG tRNA in the trrnD cluster that appears to constitute its own operon with putative promoter and terminator sequences . Although all the amino acids are represented among the tRNA anticodons, there are few repeats of amino acid types within clusters; trrnD with 16 tRNA genes has anticodons corresponding to 15 amino acids . About two-thirds of the tRNA genes encode a 3'-terminal-CCA, and these are intermingled with those that do not, with no apparent pattern. Biochim Biophys Acta, 1984 Mar 22, 798(1), 88 - 95 Catabolite repression of inositol dehydrogenase and gluconate kinase syntheses in Bacillus subtilis; Nihashi J et al.; The regulation of induction of inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) was studied in Bacillus subtilis . Inositol dehydrogenase is induced by myo-inositol and gluconate kinase is induced by D-gluconate . Both inductions were strongly repressed by rapidly metabolizable carbohydrates such as D-glucose, D-mannose, D-fructose and glycerol (D-glucose had the strongest repressive effect) but they were weakly repressed by slowly metabolizable carbohydrates . Although each carbohydrate exerted a stronger effect on the induction of inositol dehydrogenase than that of gluconate kinase, it showed a similar tendency with respect to the degree of repression of each induction . This catabolite repression could not be diminished by addition of cyclic AMP to medium . In addition, non-metabolizable D-glucose analogues had no or weak repressive effects . On the assumption that rapidly metabolizable carbohydrates might be metabolized to repress both inductions, it was investigated whether several mutants blocked in the Embden-Meyerhof pathway could produce metabolite(s) (repressor) to repress them . A phosphoglycerate kinase (EC 2.7.2.3) deficient mutant could produce the repressor from D-glucose, D-mannose, D-fructose and glycerol but other mutants could not produce it from carbohydrates unable to be metabolized in each mutant . Thus, catabolite repression of both enzyme inductions seemed to be under similar regulation . The identification of the possible repressor of the induction of in of inositol dehydrogenase and gluconate kinase in vivo was discussed. Biochem Biophys Res Commun, 1984 Mar 15, 119(2), 795 - 800 Characterization of the precursor form of the exocellular levansucrase from Bacillus subtilis; Fouet A et al.; Expression of the cloned levansucrase gene (sacB) was demonstrated in E . coli minicells by assay of the enzyme in crude extracts, SDS-polyacrylamide gel electrophoresis and immunoblotting . The existence of a precursor form of the enzyme of MW 53000 was also demonstrated and confirmed by the DNA sequence corresponding to the NH2 terminal region of the protein. Eur J Biochem, 1984 Mar 15, 139(3), 593 - 603 Changes in membrane-associated proteins during sporulation in Bacillus subtilis; Rhaese HJ et al.; Membrane proteins from vegetative and sporulating cells of Bacillus subtilis were separated by the two-dimensional gel electrophoresis system using isoelectric focusing and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (O'Farrell technique) . Membrane proteins were isolated according to published procedures . The gels were stained with Coomassie blue . Three different concentrations of proteins were analyzed to detect even minor constituents . Over two hundred different membrane proteins were identified in vegetative cells by their isoelectric point (pI) and molecular weight (Mr) . Analysis of membrane proteins from cells harvested during and at the end of logarithmic growth (A600 approximately equal to 0.8; T0) and every hour thereafter until T4 showed that in the wild-type strain 55 proteins are degraded mostly at the beginning or sporulation . Many others (76 proteins) are newly synthesized during sporulation . About 16 proteins are synthesized at times during sporulation but again degraded within 1 h or less . Others (uncertain proteins, 65) are degraded and resynthesized again . This observation is in agreement with experiments previously published by Andreoli et al . {Andreoli, A . J., Kao, M., Chui, R., Cabrera, J., and Wong, S . K . S (1981) in Sporulation and Germination (Levinson, H . S., Sonenshein, A . L., and Tipper, D . J., eds) pp . 168-173, American Society for Microbiology, Washington} using Bacillus cereus . Experiments with the early blocked asporogenous mutant JH 649 (spoOF) showed that few proteins (40%) are degraded and even fewer (30%) are newly synthesized between A600 approximately equal to 0.8 and T4 . Protease inhibitors (phenylmethylsulfonyl fluoride, EDTA, o-phenanthroline) have no effect on the protein patterns . The experiments presented here show that proteins involved in differentiation in B . subtilis can be identified by the two-dimensional gel electrophoresis system and with the aid of asporogenous mutants . In order to assure that no cytoplasmic proteins are contaminating the membrane preparations, several cytoplasmic enzyme activities have been measured . Their concentration was found to be always below 0.005% of total protein, which is below the level of detection by Coomassie blue staining. Nucleic Acids Res, 1984 Mar 12, 12(5), 2351 - 65 Overproduction and purification of the connector protein of Bacillus subtilis phage phi 29; Ibanez C et al.; A phi 29 DNA fragment containing genes 10 and 11, coding for the connector protein and the lower collar protein, respectively, has been cloned in the pBR322 derivative plasmid pKC30 under the control of the PL promoter of phage lambda . Two polypeptides with the electrophoretic mobility of proteins p10 and p11 were labelled with 35S-methionine after heat induction . The proteins were characterized as p10 and p11 by radioimmunoassay and they represented about 10% and 7%, respectively, of the total E . coli protein after 4 hours of induction . These proteins represent less than 1% of the B . subtilis protein in phi 29-infected cells . Protein p10 has been highly purified from the E . coli cells carrying the recombinant plasmid . Antibodies raised against the purified protein p10 reacted with the connector protein produced in phi 29-infected B . subtilis. Sangyo Igaku, 1984 Mar, 26(2), 147 - 54 {Mutagenicity of organic rubber additives}; Ueno S et al.; The DNA-damaging activities of organic rubber additives such as rubber vulcanizing agents, vulcanization accelerators and rubber anti-oxidants were investigated by the rec-assay using spores of Bacillus subtilis strains H 17 and M 45 . For metabolic activation, 9,000 X g supernatant solutions of the liver homogenate of Sprague-Dawley male rats previously treated with aroclor 1,254 were used . Spore rec-assays were carried out at the dose of 1 mg/disk, and the ratio of inhibition zones for M 45 to that for H 17 was calculated . Samples showing a ratio of more than 1.2 were judged positive . In order to know the DNA-damaging capacity of positive samples, the dose-response curves were prepared by carrying out the assays at various doses, and minimal inhibition concentration (MIC) for H 17 and M 45 was obtained from these curves by extrapolation . Then indices of DNA damagenicity were calculated through division of the MIC obtained with H 17 by that with M 45 . The 0.005 micrograms/disk of mitomycin C and at 4 micrograms/disk of Trp-P1 were used as positive control, and the 50 micrograms/disk of kanamycin as negative control . The results obtained are as follows: Among 20 tested samples, p-quinone dioxime, bis-morpholine disulfide used as rubber vulcanizing agents and hexamethylenetetramine, zinc butylxanthate used as vulcanization accelerators gave positive results . It was considered that the action of hexamethylenetetramine against DNA was due to the electrophilic state of this material . Furthermore, we supposed that DNA-damaging activity of p-quinone dioxime was concerned with free hydroxyl groups of this compound.(ABSTRACT TRUNCATED AT 250 WORDS) J Biochem (Tokyo), 1984 Mar, 95(3), 895 - 7 Accumulation of relA gene-independent ppGpp in Bacillus subtilis vegetative cells upon temperature shift-down; Ikehara K et al.; Both Bacillus subtilis BR16S (rel+) and BR16R (relA-) cells accumulated ppGpp after a temperature shift-down from 37 to 0 degree C . This indicates that a ppGpp accumulation system is present in B . subtilis vegetative cells, which is induced upon cold-shock treatment and is mediated by a relA gene-independent product. Proc Natl Acad Sci U S A, 1984 Mar, 81(6), 1639 - 43 Purification in a functional form of the terminal protein of Bacillus subtilis phage phi 29; Prieto I et al.; Phage phi 29 terminal protein, p3, essentially pure, was isolated in a denatured form from viral particles, and anti-p3 antiserum was obtained . A radioimmunoassay to detect and quantitate protein p3 was developed . By using this assay, native protein p3 was highly purified from Escherichia coli cells harboring a gene 3-containing recombinant plasmid . After three purification steps, the protein was more than 96% pure; its amino acid composition was very similar to that deduced from the nucleotide sequence of gene 3 . The purified protein was active in the formation of the covalent p3-dAMP initiation complex when supplemented with extracts of B . subtilis infected with a sus mutant of phi 29 in gene 3 . No DNA polymerase or ATPase activities were present in the final preparation of protein p3. J Bacteriol, 1984 Mar, 157(3), 942 - 4 Isolation of Bacillus subtilis mutants pleiotropically insensitive to glucose catabolite repression; Fisher SH et al.; A pleiotropic mutant of Bacillus subtilis was isolated which overproduced in the presence of glucose several enzymes whose synthesis is subject to glucose catabolite repression . Examination of intracellular metabolites suggested that the mutation may have resulted in a defect in glycolysis, increasing phosphoenolpyruvate and decreasing pyruvate, 2-ketoglutarate, and oxaloacetate. J Bacteriol, 1984 Mar, 157(3), 931 - 3 Expression of Bacillus megaterium and Bacillus subtilis small acid-soluble spore protein genes during stationary-phase growth of asporogenous B . subtilis mutants; Mason JM et al.; The small acid-soluble spore proteins alpha and beta were not detected during stationary-phase growth of asporogenous Bacillus subtilis mutants blocked in stages 0, II, or III, but mutants blocked in stages IV or V accumulated nearly wild-type levels of these small acid-soluble spore proteins . Similar results were obtained when production of Bacillus megaterium C protein (also a small acid-soluble spore protein), as well as alpha and beta, were monitored in these mutants containing a recombinant plasmid carrying the B . megaterium C protein gene . The only exception was a spo0H mutant which synthesized a small amount of C protein, but no alpha or beta. J Bacteriol, 1984 Mar, 157(3), 733 - 8 Transformation in Bacillus subtilis: a 75,000-dalton protein complex is involved in binding and entry of donor DNA; Smith H et al.; A 75,000-dalton protein complex involved in DNA binding during transformation was purified from membranes of competent Bacillus subtilis cells . Previous results (Smith et al., J . Bacteriol . 156:101-108, 1983) showed that the complex contained two polypeptides, polypeptide a (molecular weight, 18,000; isoelectric point, 5.0) and polypeptide b (molecular weight, 17,000; isoelectric point, 4.7) in approximately equal amounts . In the present experiments the two polypeptides were extracted from two-dimensional gels and studied separately and in combination with respect to DNA binding and nuclease activities . For DNA binding the interaction of both polypeptides was required . DNA binding occurred efficiently in the presence of EDTA . Nuclease activity was restricted to polypeptide b . The nucleolytic properties of b were identical to those of the native 75,000-dalton complex . Polypeptide a affected b by reducing its nuclease activity . Analysis of the nuclease subunit b on DNA-containing polyacrylamide gels revealed nuclease activities at four different molecular weight positions . These activities were identical to the major competence-specific nuclease activities which were previously implicated in the entry of donor DNA during transformation (Mulder and Venema, J . Bacteriol . 152:166-174, 1982) . These results indicate that the 75,000-dalton protein complex is composed of two different competence-specific polypeptides involved in both binding and entry of donor DNA . The possible roles of the two polypeptides in the transformation of B . subtilis are discussed. Prikl Biokhim Mikrobiol, 1984 Mar-Apr, 20(2), 200 - 7 {Purification and properties of intracellular nuclease of a competent strain of Bacillus subtilis}; Belov IS et al.; A new nuclease was isolated from a membrane-nucleoproteid complex (MNC) of the cell lysate of the competent strain Bacillus subtilis SB25 his2trp2 and purified 674-fold . It differs from the known exonucleases of Bacillus subtilis by the place of "attacking" the phosphodiester bond, by the absence of the specificity towards the end groups of denatured DNA and by a high pH optimum . Isolation and purification were performed as follows: cell destroying by lysozyme and osmotic shock, isolating the MNC by centrifugation, treating the MNC with pancreatic RNase and dialysis with Ca+2 ions against Tris-acetate buffer, chromatography on CM-and phosphocellulose . The nuclease of the partially purified preparation was activated by Ca+2 ions with the optimal concentration 0.5-1.0 mM . It hydrolysed denatured DNA and RNA with the pH optimum at 10.0-10.5 . The main products of the denatured DNA hydrolysis were mono- and dinucleotides with 5'-end phosphate . The enzyme is insensitive to the end hydroxyl and phosphate groups of denatured DNA and was completely inhibited by 1.0 mM EDTA. J Virol, 1984 Mar, 49(3), 806 - 12 Characterization of proteins induced by mitomycin C treatment of Bacillus subtilis; Mauel C et al.; A total of 26 polypeptides have been resolved by gel electrophoresis of purified phage PBSX, 3 of which belong to the head and the remainder to the tail . After mitomycin C treatment, synthesis of 11 additional proteins which are not found in the assembled phage particle was demonstrated, all but 4 being under the control of the phage repressor . Existence of a prehead and of a precursor of the main capsid protein (molecular weight, 35,000) suggested phage head maturation which is accompanied by cleavage of the precursor (molecular weight, 36,500) . The role of induced proteins related and unrelated to PBSX is discussed . Finally, the estimated phage genome mass of 4 X 10(7) daltons exceeded by more than four times its head capacity, which could explain the defectiveness of the phage. J Bacteriol, 1984 Mar, 157(3), 965 - 7 Promoter-probe plasmid for Bacillus subtilis; Donnelly CE et al.; We have constructed a promoter-probe expression vector for Bacillus subtilis . This plasmid, pCED6, can be used to fuse various DNA sequences to the structural gene of Escherichia coli beta-galactosidase, permitting analysis of the promoter activity of such sequences . pCED6 replicates and confers drug resistances in both E . coli and B . subtilis. J Bacteriol, 1984 Mar, 157(3), 718 - 26 Use of chromosomal integration in the establishment and expression of blaZ, a Staphylococcus aureus beta-lactamase gene, in Bacillus subtilis; Saunders CW et al.; With several different vectors, attempts were made to establish blaZ, a Staphylococcus aureus beta-lactamase gene, in Bacillus subtilis . Stable establishment of blaZ in B . subtilis was achieved by use of a vector that allowed the integration of a single copy of the gene into the chromosome of that host . blaZ was expressed in the heterologous host since B . subtilis strains carrying integrated blaZ produced beta-lactamase and were more resistant to ampicillin than was wild-type B . subtilis . blaZ was stably inherited in such strains, as no loss of the ability to produce beta-lactamase was observed after growth in nonselective liquid medium or on solid medium . In contrast, a blaZ-containing restriction fragment could not be established in B . subtilis with either pUB110- or pC194-based vectors . Similarly, a pC194-based shuttle vector (pGX318) containing the 5' end of blaZ (including the promoter and the coding region for the signal sequence and the first few amino acids of the mature protein) was unable to transform B . subtilis . Two derivatives of pGX318 that could be stably established in B . subtilis were isolated . The structures of these derivatives suggested that inactivation of the blaZ promoter was associated with the acquisition of the ability to be established. Nucleic Acids Res, 1984 Feb 24, 12(4), 1943 - 60 In vitro transcription of the Bacillus subtilis phage phi 29 DNA by Bacillus subtilis and Escherichia coli RNA polymerases; Sogo JM et al.; The Escherichia coli RNA polymerase bound to phage phi 29 DNA has been visualized by electron microscopy . Thirteen specific binding sites have been observed at 1.7,2.6,5.5,10.4,13.7,25.2,25.7,26.3,33.5,59.5,69.2,91.7 and 99.6 DNA length units and they have been named A1,A1I,A1II,A1III,A1IV,A2,A2I, A3, A4,B1,B1I,C1 and C2, respectively . The binding sites A1,A2,A3,B1,C1 and C2 coincide with those found with Bacillus subtilis RNA polymerase . The transcription of phage phi 29 DNA with B . subtilis or E . coli RNA polymerases has been studied . With the B . subtilis RNA polymerase eight transcripts were found, starting at positions corresponding to the binding sites A1, A1III, A2,A3,B1I,B2,C1 and C2, respectively . With the E . coli RNA polymerase the same transcripts were found and a new one starting at position corresponding to the A4 binding site . The RNAs starting at binding sites A1,A1III,A2,B1I, B2,C1 and C2 are transcribed from right to left, as expected for early RNA . The RNAs which initiate at positions A3 and A4 are transcribed from left to right and probably correspond to late RNAs. Biochemistry, 1984 Feb 14, 23(4), 675 - 80 Purification and characterization of chemotactic methylesterase from Bacillus subtilis; Goldman DJ et al.; By utilization of methanol evolution as an assay, a protein methylesterase from Bacillus subtilis has been purified . A 1200-fold purification has been achieved by CM-Bio-Gel A, hydroxylapatite, and Bio-Gel P-60 column chromatography . Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate the enzyme is a monomer of 41 000 in molecular weight . The enzyme is stabilized and activated by aqueous glycerol solutions . Methyl-accepting chemotaxis proteins (MCPs) serve as substrates for the enzyme . The enzyme requires divalent cation for activity, with maximum activity obtained at 1.1 mM Mg2+ . The enzyme is most active at pH 7.5 and at 28 degrees C . Methylesterase has an apparent Km for methylated MCPs of about 10 nM. J Biol Chem, 1984 Feb 10, 259(3), 1483 - 90 Synthesis of peptidoglycan by high molecular weight penicillin-binding proteins of Bacillus subtilis and Bacillus stearothermophilus; Jackson GE et al.; The high molecular weight penicillin-binding proteins (PBP(s) ) Bacillus subtilis PBPs 1, 2, and 4 and Bacillus stearothermophilus PBPs 1-4 were shown to catalyze peptidoglycan synthesis from the undecaprenol-containing lipid intermediate substrate in two assay systems . In a filter paper assay system, high levels of substrate polymerization occurred when reaction mixtures were incubated on Whatman 3MM filter paper . The pH optimum for peptidoglycan synthesis was 7.5 for B . subtilis PBPs 1, 2, and 4 and 8.5 for B . stearothermophilus PBPs 1-4 . Polymerization was Mg2+-independent and was unaffected by sulfhydryl reagents . Reconstitution with membrane lipids or addition of detergent (optimal concentration, 0.1%) was necessary for synthesis to occur . Bacitracin, penicillin, and cephalothin did not affect polymerization while vancomycin, ristocetin, moenomycin