Microbiol Immunol, 1989, 33(5), 391 - 401 Presence of proteins derived from the vegetative cell membrane in the dormant spore coat of Bacillus subtilis; Fujita Y et al.; To confirm the presence of the outer spore membrane in dormant spore coats of Bacillus subtilis, the proteins from vegetative cell membrane and dormant spore coat fractions were compared by immunoblot assay with antibodies prepared against both preparations . The spore coat fraction contained at least 11 proteins antigenically identical to those in the vegetative cell membranes . Further, the cytochemical localization of the proteins derived from vegetative cell membrane in dormant spores was examined by an immunoelectron microscopy method with a colloidal gold-immunoglobulin G complex . The colloidal gold particles were observed in the coat region and around the core region of dormant spore . These results have provided evidence that some proteins from vegetative cell membrane remain in the dormant spore coat region of B . subtilis, although it is not clear whether the outer membrane persists as an intact functional entity or not. Microbios, 1989, 57(230), 49 - 63 Sporulation of bacterial species in the presence of metabisulphite; Oloyede OB et al.; The effect of metabisulphite on the sporulating ability of Bacillus subtilis E52 and B . cereus W18 was studied . Whereas metabisulphite concentrations of 500 and 600 micrograms ml-1 prevented sporulation of B . subtilis and B . cereus, respectively, lower concentrations caused reductions in their percentage sporulations . Glucose dehydrogenase (GDH) and alkaline phosphatase (ALP) activities of both sporulating bacteria were not detected at the stated metabisulphite concentrations . A relationship between the percentage sporulation of the bacteria and the activity of the enzymes in the presence of metabisulphite was exhibited . ALP activity may serve as an index of the effectiveness of the antisporulating activity of metabisulphite . Both enzymes are likely to be among the few targets for the antisporulating activity of metabisulphite. Prikl Biokhim Mikrobiol, 1989 Jan-Feb, 25(1), 15 - 21 {Enzymatic and conformational stability of polymeric complexes of alpha-amylase}; Iurchenko VS et al.; The enzymatic and conformational stability of Bacillus subtilis alpha-amylase and its polymeric complexes in acid media and subsequent renaturation in weakly alkaline media were investigated . The following parameters of alpha-amylase secondary structure were determined from circular dichroism spectra: helical units -25%, beta-structures -9%; beta-turns -13%; disordered conformations -53% . After complexation with polymethacrylic acid (PMAA) the alpha-amylase secondary structure did not change, and the tertiary structure underwent only small local changes . Complexation of alpha-amylase with linear and cross-linked PMAA led to an increase in both enzymatic and conformational stabilities in acid media . Purification of alpha-amylase using a biosorbent resulted in higher acid resistance of the free enzyme and of that in the complex with PMAA . Moreover, the degree of reversibility of the acid inactivation also increased. Biochimie, 1989 Jan, 71(1), 111 - 6 Methylation of the antifungal lipopeptide iturin A modifies its interaction with lipids; Harnois I et al.; Iturin A, extracted from the culture media of Bacillus subtilis, is an antifungal lipopeptide, the peptide cycle of which includes a D-Tyr residue in position 2 . The antibiotic strength of iturin A is related to a change in the permeability of the membrane cells which leads to a leakage of K+ from the intracellular medium . Methylation of the D-Tyr residue dramatically decreases the biological activity of iturin A . Using the intrinsic fluorescence of D-Tyr we have shown that both iturin A and O-methyl-tyrosine iturin A enter the lipid membranes . When dimyristoylphosphatidylcholine vesicles contain iturin A we observe a change in the order degree of the lipid phase and an increase in the transition temperature . The methylated derivative has no effect . Two model membranes have been used to study the permeability changes induced by iturin A and O-methyltyrosine iturin A . Studying ionic permeability we have found that the conductance of a planar lipid membrane increases very much less when the lipopeptide is methylated . On the other hand, the release of carboxyfluorescein trapped in lipid vesicles is less upon addition of O-methyltyrosine-iturin A . We conclude that the Tyr residue of the peptide cycle plays a role in determining the interactions of iturin A with lipid membrane. Arch Virol, 1989, 105(1-2), 137 - 40 Specialized transduction in Bacillus subtilis by the phages IG1, IG3, and IG4; Fernandes RM et al.; The temperate phages IG1, IG3, and IG4 of Bacillus subtilis were found to mediate specialized transduction of a lambda-like type . High frequency transducing lysates were also obtained . In many instances the transductants were shown to be heterogenotes, segregating the mutant alleles . They are presumably the result of transduction by addition . These findings are discussed together with data on other members of the group III of temperate bacteriophages aiming to a deeper understanding of that group, and of its evolutionary history. Mol Microbiol, 1989 Jan, 3(1), 23 - 8 Sequences required for regulation of arginine biosynthesis promoters are conserved between Bacillus subtilis and Escherichia coli; Smith MC et al.; The region required for regulation of a previously characterized arginine-regulatable promoter upstream from the argC gene in the argCAEBD-cpa-argF cluster of Bacillus subtilis was defined by integration of argC-lacZ translational fusions into the chromosome at a site distant from the arginine loci . Some sequence similarity was detected between the argC regulatory region and the well-characterized Escherichia coli arginine operators (ARG boxes) . This similarity was shown to be functional in vivo in that the B . subtilis repressor regulated the E . coli arginine genes, but the E . coli repressor, even when encoded by a multicopy plasmid, could not repress the B . subtilis argC promoter . In vitro binding studies using purified repressors on DNA fragments encoding operators from both E . coli and B . subtilis demonstrated interactions by both proteins. FEMS Microbiol Lett, 1989 Jan 1, 48(1), 87 - 92 Covalent modification of proteins in Bacillus subtilis during the process of sporulation, germination and outgrowth; Kohler E et al.; Cells of Bacillus subtilis 168+ were labeled with 32P-orthophosphate during the process of sporulation, germination and outgrowth . By two-dimensional gel electrophoresis, at least 30 protein species were found to be radioactively labeled; 30% of these were modified by phosphorylation . Significant changes in the protein phosphorylation pattern during growth and cellular differentiation could be demonstrated . Using gamma-32P-ATP evidence for an ATP-dependent protein kinase was also obtained . Under these conditions 4 proteins with a molecular mass of 109,600; 103,100; 73,300 and 32,200 Da were found to be phosphorylated. J Basic Microbiol, 1989, 29(2), 93 - 8 Stability and expression of the staphylococcal constitutive macrolide-lincosamide-streptogramin B resistance plasmid pE3692 in Staphylococcus aureus and Bacillus subtilis; Wieckiewicz J et al.; The constitutive MLS resistance plasmid pE3692 (2.4 kb) from S . aureus and pE3692-9 plasmid derived from pE3692 by in vitro deletion of the small HindIII-HindIII fragment Sta (0.3 kb) were introduced into S . aureus RN450 and B . subtilis UOT0277 strains . The pE3692 plasmid was completly stable in S . aureus RN450, however, in B . subtilis UOT0277 this plasmid was segregationally unstable . Deletion of the Sta t fragment from the pE3692 reduced plasmid stability considerably both in S . aureus and B . subtilis host strains . The lower expression of tylosin-resistance of the erm gene of the pE3692-9 plasmid in B . subtilis UOT0277 strain was also observed. Appl Environ Microbiol, 1989 Jan, 55(1), 252 - 3 Expression in Bacillus subtilis of the 51- and 42-kilodalton mosquitocidal toxin genes of Bacillus sphaericus; Baumann L et al.; A 3,080-base-pair KpnI-HindIII DNA fragment from Bacillus sphaericus 2362 coding for 51- and 42-kilodalton mosquitocidal proteins was cloned into Bacillus subtilis DB104 by using the vector pUB18 . In B . subtilis these proteins were not detected during vegetative growth but were expressed during sporulation at levels comparable to those found in B . sphaericus. Protein Eng, 1989 Jan, 2(5), 359 - 64 Effect of Glu-143 and His-231 substitutions on the catalytic activity and secretion of Bacillus subtilis neutral protease; Toma S et al.; On the basis of the homology with the Bacillus thermoproteolyticus zinc endopeptidase thermolysin, we hypothesized that Glu-143 and His-231 are the key residues for the catalytic activity of the Bacillus subtilis neutral protease . To test this possibility by site-directed mutagenesis, we substituted these two residues with Ala, Ser, Trp and Arg, and Leu, Val and Cys respectively . All these substitutions dramatically affected the amount of secreted mutant proteins, as determined by immunological methods, and their catalytic activities . No appreciable secretion was observed with the three Glu mutants Trp, Ser and Arg, whereas the Glu----Ala mutant enzyme was secreted at a level of a few hundred micrograms per litre of culture . The His mutants were all secreted at higher levels (in the order of a few milligrams per litre) and their residual catalytic activity could be determined using Z-Ala-Leu-Ala as substrate . Our results confirm the key role played by Glu-143 and His-231 in catalysis and moreover suggest the existence of a relationship between the catalytic activity of the enzyme and the extent of its secretion . In this context, we present data suggesting an autoproteolytic mechanism of cleavage of the precursor form of the enzyme, analogous to the one previously reported for the B . subtilis subtilisin. J Antibiot (Tokyo), 1989 Jan, 42(1), 84 - 93 Lactivicin, a naturally occurring non-beta-lactam antibiotic having beta-lactam-like action: biological activities and mode of action; Nozaki Y et al.; Lactivicin is moderately active against a wide range of Gram-negative bacteria and highly active against Gram-positive bacteria . It shows various biological activities commonly observed with beta-lactam antibiotics, such as higher activity against beta-lactam hypersensitive mutants than against their parents, sensitivity to beta-lactamases, inhibitory activity against beta-lactamases and ability to induce beta-lactamase activity . The primary lethal target of lactivicin in Escherichia coli is highly likely to be penicillin-binding protein (PBP) 1; lactivicin strongly lysed E . coli cells with induction of spheroplasts at its MIC, and showed high affinity for PBPs 1A and 1B . At concentrations above x 5 MIC, however, lactivicin dominantly exhibited secondary antibacterial action possibly owing to inhibition of crucial SH proteins engaged in the fundamental membrane functions . In contrast, against Bacillus subtilis, lactivicin showed the typical beta-lactam action under a wide range of concentrations . It showed high affinity for PBPs 1, 2 and 4, the possible lethal targets of beta-lactam antibiotics in this organism . In conclusion, lactivicin is the first non-beta-lactam antibiotic showing beta-lactam action through binding to PBPs. J Bacteriol, 1989 Jan, 171(1), 561 - 4 Evidence for an additional temporal class of gene expression in the forespore compartment of sporulating Bacillus subtilis; Panzer S et al.; We present evidence indicating that the previously studied, sporulation-induced gene 0.3 kb, which encodes a stable RNA present at late developmental stages, is transcribed in the forespore chamber of sporulating cells of Bacillus subtilis . Compartmentalized gene expression was demonstrated on the basis of subcellular fractionation experiments in which severalfold-higher levels of 0.3 kb-directed beta-galactosidase specific activity were observed in forespore extracts than in extracts from the mother cell and dependence studies in which 0.3 kb transcription was found to be blocked in mutants bearing mutations in spoIIIA, spoIIIE, and spoIIIG, genes which are known to govern forespore gene expression . Also, 0.3 kb transcription could be switched on during growth in cells in which transcription of the forespore regulatory gene spoIIIG was engineered to be activated in response to the lac inducer IPTG (isopropyl-beta-D-thiogalactopyranoside) . Although it is transcribed in the forespore, 0.3 kb is switched on at a later developmental stage than other previously studied forespore-expressed genes, and hence it appears to be representative of an additional temporal class of compartmentalized gene expression. J Bacteriol, 1989 Jan, 171(1), 120 - 3 Functional homology of chemotactic methylesterases from Bacillus subtilis and Escherichia coli; Nettleton DO et al.; The methylesterase enzyme from Bacillus subtilis was compared with that from Escherichia coli . Both enzymes were able to demethylate methyl-accepting chemotaxis proteins (MCPs) from the other organism and were similarly affected by variations in glycerol, magnesium ion, or pH . When attractants were added to a mixture of B . subtilis MCPs and E . coli methylesterase, the rate of demethylation was enhanced . Conversely, when attractants were added to a mixture of E . coli MCPs and B . subtilis methylesterase, the rate of demethylation was diminished . These effects are what would be expected if, in these in vitro systems, the MCPs determined the rate of demethylation . These data suggest that, although the enzymes are from evolutionarily divergent organisms and are different in size, they have considerable functional homology. Arch Microbiol, 1989, 152(3), 251 - 7 The preferential inhibition of Bacillus subtilis spore outgrowth by chloroquine; Smith KT et al.; Chloroquine inhibited the outgrowth of Bacillus subtilis spores at a 10-fold lower concentration than that required to prevent vegetative growth . Analysis of macromolecular synthesis in outgrowing spores and vegetative cells in the presence of chloroquine indicated that it acted preferentially on transcription . Differential sensitivity of outgrowing spores and vegetative cells to chloroquine was not due to changes in the specificity of the RNA-polymerase, since RNA-polymerase activity measured in permeabilized cells was not affected differently by the drug . The preferential inhibition of spore outgrowth was not evident at pH 8.0, at which the majority of chloroquine is in a monovalent, more lipophilic, form . In the presence of inhibitors affecting membrane potential, vegetative cells were as sensitive to chloroquine as outgrowing spores . Measurement of {14C}-chloroquine uptake showed that early outgrowing spores accumulated twice as much drug as resistant late outgrowing spores and seven times more than vegetative cells . Treatment of vegetative cells with metabolic inhibitors led them to accumulate chloroquine to the levels found in outgrowing spores . Therefore, the preferential inhibition of outgrowing spores by chloroquine is the result of increased uptake of the drug, reflecting differences in energy metabolism from vegetative cells. Gene, 1988 Dec 25, 74(1), 77 - 81 DNA methyltransferase of Bacillus subtilis Marburg: purification, properties and further evidence of specificity; Guha S; Bacillus subtilis Marburg strain displays DNA methyltransferase activity . This enzyme, M.BsuM, methylates cytosine in the sequence 5'-YTCGAR-3' (Y = pyrimidine; R = purine) . M.BsuM was purified from the exponentially growing cells of B . subtilis 168M . This enzyme (45 +/- 1 kDa) is monomeric and recognizes only double-stranded DNA . It is inhibited partially by Mg2+, Mn2+ ions and spermidine and almost totally by sodium dodecyl sulfate, urea and agarose . This enzyme methylates specifically the three methylatable sites of the plasmid pBM3 . Relaxation of specificity ('star' activity) was observed in the presence of organic solvents . A very low amount of M.BsuM was obtained in the standard Marburg strain . To obtain sufficient enzyme attempts are being made to clone the M.BsuM gene in Escherichia coli by using a constructed plasmid (pBM14) vector . Only one transformant containing a 3-kb insert and showing a low level of expression, was obtained. J Biol Chem, 1988 Dec 25, 263(36), 19592 - 6 Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis; Takagi H et al.; The specific activity of subtilisin E, an alkaline serine protease of Bacillus subtilis, was substantially increased by optimizing the amino acid residue at position 31 (Ile in the wild-type enzyme) in the vicinity of the catalytic triad of the enzyme . Eight uncharged amino acids (Cys, Ser, Thr, Gly, Ala, Val, Leu, and Phe) were introduced at this site, which is next to catalytic Asp32, using site-directed mutagenesis . Mutant enzymes were expressed in Escherichia coli and were prepared from the periplasmic space . Only the Val and Leu substitutions gave active enzyme, and the Leu31 mutant was found to have a greatly increased activity compared to the wild-type enzyme . The other six mutant enzymes showed a marked decrease in activity . This result indicates that a branched-chain amino acid at position 31 is essential for the expression of subtilisin activity and that the level of the activity depends on side chain structure . The purified Leu31 mutant enzyme was analyzed with respect to substrate specificity, heat stability, and optimal temperature . It was found that the Leu31 replacement caused a prominent 2-6-fold increase in catalytic efficiency (kcat/Km) due to a larger kcat for peptide substrates. Gene, 1988 Dec 20, 73(2), 537 - 43 Cloning and nucleotide sequence of the structural gene coding for Bacillus subtilis tryptophanyl-tRNA synthetase; Chow KC et al.; A 1.47-kb DNA fragment that carries the tryptophanyl-tRNA synthetase (TrpRS) gene of Bacillus subtilis has been cloned into the pUC8 plasmid . The recombinant plasmid, pTSQ2, conferred temperature-resistance to the temperature-sensitive trpS ts mutant of B . subtilis through chromosomal transformation, and to that of Escherichia coli through complementation . The pTSQ2 could be stably maintained in E . coli DH5 alpha, causing in the host cell a 200-fold amplification of TrpRS activity . The complete nucleotide sequence of the cloned fragment has been determined . A putative transcriptional promoter, a Shine-Dalgarno sequence, the 990-bp trpS gene proper, as well as a transcriptional terminator have been identified. EMBO J, 1988 Dec 20, 7(13), 4389 - 95 The arginine repressor is essential for plasmid-stabilizing site-specific recombination at the ColE1 cer locus; Stirling CJ et al.; The heritable stability in Escherichia coli of the multicopy plasmid ColE1 and its natural relatives requires that the plasmids be maintained in the monomeric state . Plasmid multimers, that arise through recA-dependent homologous recombination, are normally converted to monomers by a site-specific recombination system that acts at a specific plasmid site (cer in ColE1) . No plasmid functions that act at this site have been identified . In contrast, two unlinked E.coli genes that encode functions required for cer-mediated site-specific recombination have been identified . Here we describe the isolation and characterization of one such gene (xerA) and show it to be identical to the gene encoding the repressor of the arginine biosynthetic genes (argR) . The argR protein binds to cer DNA both in vivo and in vitro in the presence of arginine . We believe this binding is required to generate a higher order protein-DNA complex within the recombinational synapse . The argR gene of Bacillus subtilis complements an E.coli argR deficiency for cer-mediated recombination despite the two proteins having only 27% amino acid identity. Gene, 1988 Dec 15, 73(1), 215 - 26 A new cloning system for Bacillus subtilis comprising elements of phage, plasmid and transposon vectors; Poth H et al.; A new cloning system for Bacillus subtilis was devised which makes use of a combination of Tn917-containing phage SP beta derivatives and Tn917-containing Escherichia coli-B . subtilis shuttle plasmids . This system allows the initial cloning of genes in single copy, via 'prophage transformation', with a selection for complementation of mutational defects in B . subtilis hosts and permits subsequent transfer of the cloned material by homologous recombination to low-copy and high-copy vectors that replicate in both B . subtilis and E . coli . Because cloned sequences are adjacent to pB322-derived DNA in the recombinant phages, inserts can also be 'rescued' directly from the phage DNA after digestion with appropriate restriction enzymes, circularization of the fragments by ligation and transformation of an E . coli recipient . Two genomic libraries of B . subtilis chromosomal Sau3A-generated partial-digest fragments in the size ranges of 5-8 kb and 8-10 kb were constructed and screened for the complementation of mutations aroI906, cysA14, dal-1, glyB133, metC3, purA16, purB33, thrA5, trpC2 and recE4 . In all cases, specialized transducing phages carrying inserts that complemented the selected markers were recovered . Inserts complementing the dal-1 and trpC2 mutations could be transferred from recombinant phages to Tn917-containing plasmids by homologous recombination without in vitro subcloning . Another insert complementing the purB33 mutation was rescued directly into E . coli from a recombinant phage DNA. Gene, 1988 Dec 15, 73(1), 175 - 83 Analysis of the nucleotide sequence of the P1 operon of Mycoplasma pneumoniae; Inamine JM et al.; The attachment of virulent Mycoplasma pneumoniae to the ciliated epithelium of the respiratory tract involves a surface protein designated P1 . Our previous determination of the nucleotide sequence of the P1 attachment-protein gene revealed that it is flanked by open reading frames (ORFs) and there is no obvious ribosome-binding site (RBS) or transcription termination sequence in the adjacent regions . We extended this analysis by cloning and sequencing the 18-kb region containing the P1 gene . This study indicates that the P1 gene is transcribed as part of a larger polycistronic message . The P1 operon is composed of the P1 gene and two predicted genes, designated ORF-4 and ORF-6 . The gene order is ORF-4, P1, ORF-6 with intervening regions of 12 and 5 nt, respectively . ORF-4 and ORF-6 have respective coding capacities for proteins of Mr approximately equal to 28,000 and Mr approximately equal to 130,000 . Putative promoter and RBS sequences which correspond closely to those found in Escherichia coli and Bacillus subtilis, as well as a sequence indicative of a transcription terminator, have been found in the flanking sequences . The transcription start point has been determined by primer extension of M . pneumoniae RNA. J Biol Chem, 1988 Dec 5, 263(34), 18386 - 96 Biosynthesis of branched-chain fatty acids in Bacillus subtilis . A decarboxylase is essential for branched-chain fatty acid synthetase; Oku H et al.; Branched long-chain fatty acids of the iso and anteiso series are synthesized in many bacteria from the branched-chain alpha-keto acids of valine, leucine, and isoleucine after their decarboxylation followed by chain elongation . Two distinct branched-chain alpha-keto acid (BCKA) and pyruvate decarboxylases, which are considered to be responsible for primer synthesis, were detected in, and purified in homogenous form from Bacillus subtilis 168 strain by procedures including ammonium sulfate fractionation and chromatography on ion exchange, reversed-phase, and gel absorption columns . The chemical and catalytic properties of the two decarboxylases were studied in detail . The removal of BCKA decarboxylase, using chromatographic fractionation, from the fatty acid synthetase significantly reduced its activity . The synthetase activity was completely lost upon immunoprecipitation of the decarboxylase . The removal of pyruvate decarboxylase by the above two methods, however, did not affect any activity of the fatty acid synthetase . Thus, BCKA decarboxylase, but not pyruvate decarboxylase, is essential for the synthesis of branched-chain fatty acids . The very high affinity of BCKA decarboxylase toward branched-chain alpha-keto acids is responsible for its function in fatty acid synthesis. J Gen Microbiol, 1988 Dec, 134 ( Pt 12), 3259 - 68 Levansucrase of Bacillus subtilis: effects on the secretion process of single amino acid substitutions in the mature part of the protein; Benyahia F et al.; Substitutions of an aspartate or an arginine residue for the glycine residue at position 366 of the mature part of Bacillus subtilis levansucrase were obtained by mutagenesis . Quantitative estimation from immunoblot analysis showed that the two transient membrane forms of the modified proteins were present in the membrane at the same level as that of the wild-type protein . The proteolytic processing, which was previously shown to be the first step of the levansucrase secretion process, was not affected in these modified proteins . Results from pulse-chase experiments showed that the half-times for secretion of the modified levansucrases into the culture medium were nearly the same as that of the wild-type protein, but the amount of the modified proteins secreted was significantly reduced . Purified samples of the modified enzymes were subtilisin insensitive and possessed enzyme activities very similar to those of the wild-type enzyme . The results suggest that the 366 site probably belongs to a functional domain of the protein which could play an important role in the second step of the levansucrase secretion process. J Gen Microbiol, 1988 Dec, 134 ( Pt 12), 3249 - 57 New suppressor mutation sur0B of spo0B and spo0F mutations in Bacillus subtilis; Shoji K et al.; Two extragenic suppressor mutations, sur0B20 and sur0F1, which restore the sporulation of spo0B or spo0F mutants of Bacillus subtilis to the wild-type level, were obtained . These suppressor mutations were located in the spo0A gene . Their location is close to that of the sof-1 mutation, which suppresses spo0B, spo0E and spo0F mutations . However, spo0 strains bearing the sur0B20 mutation differed in several phenotypic characteristics from spo0 mutants bearing the sof-1 suppressor . Nucleotide sequence analysis revealed that the sur0B20 and sur0F1 mutations resulted in Glu14 to Val and Asn12 to Lys conversion, respectively, in the spo0A gene . This result indicates that sur0B20 is a new suppressor of spo0b and spo0F mutations, whereas sur0F1 is identical to sof-1. Biokhimiia, 1988 Dec, 53(12), 2042 - 50 {Changes in the redox potential and the number of accessible sulfhydryl groups in Escherichia coli and Bacillus subtilis cultures during transient processes}; Oktiabr'skii ON et al.; It was shown that changes in the redox potential can be due to the influence of compounds which alter the intracellular pH (acetate or propionate, protonophore carbonyl-cyanide-m-chlorophenyl hydrazone, permeate cation TPP+) . A correlation was found between the redox potential changes and the number of SH-groups in the medium and on cell surface . It was shown also that the previously reported redox potential shifts during the transition of E . coli and B . subtilis cultures to the stationary phase under glucose or ammonium exhaustion are due to the increase in the number of SH-groups in the medium and on cell surface . A hypothesis is put forward, according to which the changes in intracellular pH play a trigger role, whereas those in the thiol: disulfide ratio inside and outside the cells are thought to amplify regulatory signals. Eur J Clin Microbiol Infect Dis, 1988 Dec, 7(6), 783 - 5 Nosocomial bacteremia caused by Bacillus species; Richard V et al.; During a six year period, 11 cases of bacteremia caused by Bacillus spp . were observed corresponding to 1% of all bacteremic episodes in our hospital . Most patients had cancer as underlying disease . All cases of positive blood cultures were associated with a clinical syndrome compatible with sepsis including high fever . None of the subsequent deaths could be related to the bacteremia caused by Bacillus spp . Four of eight cases of Bacillus subtilis bacteremia were associated with the absorption of an oral preparation containing Bacillus subtilis spores, which was administered empirically in some units of the hospital to reduce what was considered to be tube-feeding related diarrhea. J Bacteriol, 1988 Dec, 170(12), 5963 - 7 Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis; Francesconi SC et al.; Small, acid-soluble spore proteins SASP-alpha, SASP-beta, and SASP-gamma as well as a SASP-beta-lacZ gene fusion product were found only within the forespore compartment of sporulating Bacillus subtilis cells by using immunoelectron microscopy . The alpha/beta-type SASP were associated almost exclusively with the forespore nucleoid, while SASP-gamma was somewhat excluded from the nucleoid . These different locations of alpha/beta-type and gamma-type small, acid-soluble spore proteins within the forespore are consistent with the different roles for these two types of proteins in spore resistance to UV light. J Bacteriol, 1988 Dec, 170(12), 5935 - 8 Nucleotide sequence of the Bacillus subtilis phoR gene; Seki T et al.; The nucleotide sequence of phoR, the positive and negative regulatory gene for alkaline phosphatase and phosphodiesterase formation in Bacillus subtilis, was determined . The sequence data predicted an open reading frame of 1,740 base pairs (579 amino acids) which overlaps the 5 base pairs of the preceding phoP coding sequence . The deduced amino acid sequence was significantly homologous with that of the Escherichia coli phoR gene product, which is the sensory element for the pho regulon. J Bacteriol, 1988 Dec, 170(12), 5863 - 9 Characteristics of an RNA polymerase population isolated from Bacillus subtilis late in sporulation; Cummings CW et al.; The sigma-factor composition of Bacillus subtilis RNA polymerase alters during endospore formation . The best-documented change is the appearance of a major sporulation-specific sigma factor (sigma epsilon), which is an RNA polymerase subunit readily detected at 2 to 4 h into the 8-h sporulation process . To determine the nature of the RNA polymerase in differentiating cells after the period of sigma epsilon abundance, we isolated RNA polymerase from cells that were harvested at 6 h after the onset of sporulation . Highly purified fractions of RNA polymerase from these cells contained at least six proteins which cosedimented with core RNA polymerase (beta beta' alpha 2) during glycerol gradient centrifugation . Most of these proteins were in the size range of 20,000 to 29,000 daltons, although one 90,000-dalton protein was also evident . None of the putative RNA polymerase subunits were present in quantities similar to that observed for sigma epsilon during its period of prominence in the cell but instead resembled the minor vegetative-cell sigma factors in abundance . In vitro transcriptions using cloned B . subtilis DNAs as templates revealed at least two novel transcriptional activities in the enzyme that was isolated from cells at 6 h after the onset of sporulation but absent in an RNA polymerase preparation extracted from cells at 4 h after the onset of sporulation . One of these activities was reconstituted by the addition of a 25,000 to 27,000-dalton protein fraction to core RNA polymerase. J Bacteriol, 1988 Dec, 170(12), 5642 - 6 Induction of cat-86 by chloramphenicol and amino acid starvation in relaxed mutants of Bacillus subtilis; Ambulos NP Jr et al.; The chloramphenicol acetyltransferase gene cat-86 is induced through a mechanism that is a variation of classical attenuation . Induction results from the destabilization of an RNA stem-loop that normally sequesters the cat-86 ribosome-binding site . Destabilization of the stem-loop is due to the stalling of a ribosome in the leader region of cat-86 mRNA at a position that places the A site of the stalled ribosome at leader codon 6 . Two events can stall ribosomes at the correct location to induce cat-86 translation: addition of chloramphenicol to cells and starvation of cells for the amino acid specified by leader codon 6 . Induction by amino acid starvation is an anomaly because translation of the cat-86 coding sequence requires all 20 amino acids . To explain this apparent contradiction we postulated that amino acid starvation triggers intracellular proteolysis, thereby providing levels of the deprived amino acid sufficient for cat-86 translation . Here we show that a mutation in relA, the structural gene for stringent factor, blocks intracellular proteolysis that is normally triggered by amino acid starvation . The relA mutation also blocks induction of cat-86 by amino acid starvation, but the mutation does not interfere with chloramphenicol induction . Induction by amino acid starvation can be demonstrated in relA mutant cells if the depleted amino acid is restored at very low levels (e.g., 2 micrograms/ml) . A mutation in relC, which may be the gene for ribosomal protein L11, blocks induction of cat-86 by either chloramphenicol or amino acid starvation . We believe this effect is due to a structural alteration of the ribosome resulting from the relC mutation and not to the relaxed phenotype of the cells. J Bacteriol, 1988 Dec, 170(12), 5557 - 63 Gene encoding a minor extracellular protease in Bacillus subtilis; Sloma A et al.; The gene for a minor, extracellular protease has been identified in Bacillus subtilis . The gene (epr) encoded a primary product of 645 amino acids that was partially homologous to both subtilisin (Apr) and the major internal serine protease (ISP-1) of B . subtilis . Deletion analysis indicated that the C-terminal 240 amino acids of Epr were not necessary for activity . This C-terminal region exhibited several unusual features, including a high abundance of lysine residues and the presence of a partially homologous sequence of 44 amino acids that was directly repeated five times . The epr gene mapped near sacA and was not required for growth or sporulation. J Bacteriol, 1988 Dec, 170(12), 5522 - 8 Characterization of the tetracycline resistance gene of plasmid pT181 of Staphylococcus aureus; Mojumdar M et al.; Some genetic and biochemical properties of the tetracycline resistance element of the Staphylococcus aureus plasmid pT181 have been studied . Resequencing of a portion of the tetracycline resistance gene (tet) showed the presence of a single open reading frame of 1,299 nucleotides capable of encoding a polypeptide of 433 amino acids . Analysis of BAL 31 nuclease-generated deletion mutants of the tet gene showed the presence of two complementation groups within this region . Northern blot hybridizations demonstrated that the tet gene encodes a single mRNA, and its initiation site has been mapped by S1 nuclease protection experiments . We also identified an approximately 52,000-dalton tetracycline-inducible polypeptide in Bacillus subtilis minicells carrying pT181 . Induction of the tet gene by tetracycline resulted in a 4-fold increase in the levels of TET mRNA and at least a 15-fold increase in the amount of TET protein in B . subtilis minicells. J Biochem (Tokyo), 1988 Dec, 104(6), 980 - 4 Partial purification and properties of UDP-N-acetylmannosamine:N-acetylglucosaminyl pyrophosphorylundecaprenol N-acetylmannosaminyltransferase from Bacillus subtilis; Murazumi N et al.; An enzyme which catalyzes the conversion of GlcNAc-PP-undecaprenol into ManNAc(beta 1----4)GlcNAc-PP-undecaprenol, a key lipid intermediate in the de novo synthesis of various teichoic acids, was partially purified from the 20,000 x g supernatant fraction of Bacillus subtilis AHU 1035 cell homogenate . By means of ammonium sulfate precipitation, gel chromatography, and ion-exchange chromatography, the enzyme was purified about 70-fold, giving a preparation virtually free from substances obstructive to measurement of the N-acetylmannosaminyltransferase reaction . The enzyme was shown to be specific to UDP-ManNAc . The Km value for UDP-ManNAc was 4.4 microM, and the optimum pH was 7.3 . The enzyme required 10 mM MgCl2, 0.3 M KCl, 25% glycerol, and 0.1% Nonidet P-40 to function at full activity. J Bacteriol, 1988 Dec, 170(12), 5662 - 8 Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis; Nakano MM et al.; Surfactin is a lipopeptide antibiotic produced by the cells of Bacillus subtilis ATCC 21332 . A genetic locus responsible for surfactin production (sfp) was transferred from ATCC 21332 to JH642, a derivative of the standard B . subtilis 168 . To study the sfp locus at the molecular level, a Tn917 insertion mutant that was blocked in surfactin production (srf) was isolated . The srf::Tn917 mutation was found to be closely linked to sfp, and both loci mapped by PBS1 phage transduction to the chromosomal region between aroI and mtlB . These studies suggest that JH642, a strain which is not a producer of surfactin (genotypically sfp0), contains at least some of the genes encoding surfactin production . Expression of the srf gene(s) was examined in both sfp and sfp0 cells by assaying beta-galactosidase activity encoded by a promoterless lacZ gene that was fused to the srf::Tn917 insertion . In cells of both strains, srf-directed beta-galactosidase activity increased when cells entered the stationary phase of the growth curve, but the activity in sfp cells was higher than that in sfp0 cells . srf-lacZ expression was partially impaired by a mutation in spo0A . In sfp0 cells, this dependence on the spo0A gene product could be entirely bypassed by an abrB suppressor mutation . In the sfp cells, the abrB mutation could not restore the defect conferred by the spo0A mutation . These data suggest that the sfp locus, which is responsible for surfactin production, alters the transcriptional regulation of srf in JH642 cells. J Biol Chem, 1988 Nov 15, 263(32), 17050 - 4 Site-directed mutagenesis with the ptsH gene of Bacillus subtilis . Isolation and characterization of heat-stable proteins altered at the ATP-dependent regulatory phosphorylation site; Eisermann R et al.; The codon for Ser-46 of the ptsH gene of Bacillus subtilis was modified by site-directed mutagenesis to the codons for Ala, Thr, Tyr, and Asp . The mutant genes were overexpressed, three of the corresponding proteins were purified to homogeneity with the exception for the Asp derivative, which could not be detected, although the gene had the desired nucleotide sequence . The phosphotransferase activity of the altered proteins was determined to be 20-35% of wild type activity, which correlates well with the slow phosphorylation of heat-stable protein (HPr) by enzyme I and phosphoenolpyruvate . The ATP-dependent HPr kinase, which previously was shown to be involved in the regulation of carbohydrate uptake of Gram-positive bacteria by covalent phosphorylation of Ser-46 of HPr, is entirely inactive toward the OH group of Thr-46 and Tyr-46 proteins . In addition, we constructed a strain of B . subtilis, where the altered gene coding for the Ala-46 derivative of HPr was introduced into the bacterial chromosome . The physiological properties of this mutant are described. Ann Inst Pasteur Microbiol, 1988 Nov-Dec, 139(6), 645 - 54 Zonal turnover of cell poles of Bacillus subtilis; Kirchner G et al.; Turnover of cell walls of Bacillus subtilis occurs in three distinct phases: a lag phase, a relatively rapid phase persisting for 2-3 generations and a much slower phase continuing for several additional generations . A lectin probe revealed that cell pole material was lost during the slow phase of turnover and that the loss of wall occurred in zones, beginning at the cylinder-pole junction and continuing to the cell tip . This is in contrast to cell wall turnover in cylinders where turnover occurs randomly at many surface sites. Biochemistry, 1988 Nov 1, 27(22), 8453 - 7 Rapid attractant-induced changes in methylation of methyl-accepting chemotaxis proteins in Bacillus subtilis; Thoelke MS et al.; In Bacillus subtilis, addition of chemotactic attractant causes an immediate change in distribution of methyl groups on methyl-accepting chemotaxis proteins (MCPs), whereas in Escherichia coli, it causes changes that occur throughout the adaptation period . Thus, methylation changes in B . subtilis are probably related to excitation, not adaptation . If labeled cells are exposed to excess nonradioactive methionine, then attractant causes immediate 50% delabeling of the MCPs, suggesting that a flux of methyl groups through the MCPs occurs . Methanol is given off at a high rate during the adaptation period and probably reflects demethylation of some substance to bring about adaptation . The fact that many radioactive methyl groups are lost immediately from the MCPs but only slowly arise as methanol is consistent with the hypothesis that they are transferred from the MCPs to a carrier from which methanol arises . Demethylation of this carrier may cause adaptation. Mol Gen Mikrobiol Virusol, 1988 Nov, (11), 12 - 8 {Interplasmid illegitimate recombination in Bacillus subtilis cells}; Bashkirov VI et al.; The illegitimate recombination between S . aureus plasmids pE194 (or pGG20-the hybrid between pE194 and E . coli plasmid pBR322) and pBD17 (plasmid pUB110 without Hpa-II-C-fragment) in B . subtilis was studied . Plasmid cointegrates were generated with the frequency of 1-3.10(-8) . Among the 22 hybrids analysed 9 types of recombinants were found . Nucleotide sequences of all the parental plasmids were involved in intermolecular recombination . Nucleotide sequencing of recombinant DNA junctions has revealed that in 8 cases recombination occurred between short homologous regions (9-15 b.p.) . One of the recombinants resulted from nonhomologous recombination . The similarity between nucleotide sequences of recombination sites of two types of contegrates and those used for pE194 integration into the B . subtilis chromosome (Bashkirov et al . 1987) was demonstrated . Possible mechanisms of illegitimate recombination are discussed. J Biochem (Tokyo), 1988 Nov, 104(5), 832 - 6 Amino acid sequence of a lysozyme (B-enzyme) from Bacillus subtilis YT-25; Kamei K et al.; The amino acid sequence of a lysozyme, (B-enzyme), from Bacillus subtilis YT-25 was determined by conventional methods . B-Enzyme comprised 117 amino acid residues and had a heterogeneous sequence in the amino-terminal region . The amino acid sequence of B-enzyme was different from those of all other lysozymes the sequences of which are known . However, the partial amino acid sequence of Ser(74) to Ser(97) of B-enzyme was homologous with that of the active-site region of hen egg-white lysozyme (Ser(36) to Ser(60}, which includes one of the catalytic amino acids, Asp(52) . It is interesting that B-enzyme has an amino acid sequence homologous with that of the gag protein p25 of the AIDS virus ARV-2. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Nov, 269(3), 314 - 22 Purification and characterization of the bacterial plasminogen activator staphylokinase secreted by a recombinant Bacillus subtilis; Gerlach D et al.; A gene coding for the bacterial plasminogen activator staphylokinase (SAK) was cloned from Staphylococcus aureus bacteriophage 42D into an exoprotease reduced mutant strain of Bacillus subtilis (1) . Yields of up to 50 mg SAK per litre of culture supernatant were obtained depending on the medium used . SAK purified by ion exchange chromatography and gel filtration had a specific activity of 16,000 units/mg protein . Isoelectric focusing of the purified SAK revealed heterogeneity with respect to the isoelectric points . Four different SAK proteins were identified among which the majority fraction had an IEP of 6.3 and a N-terminal amino acid sequence of NH2-Lys-Gly-Asp .. . This N-terminus was 10 amino acids downstream of the expected signal peptide cleavage site beyond AA 27 . It resulted most likely from a postsecretory proteolytic modification of the transiently appearing and correct processing product . In contrast to other plasminogen activators SAK was found to be resistant to proteolytic inactivation by plasmin. Mol Gen Genet, 1988 Nov, 214(3), 482 - 9 Functional analysis of the dna (Ts) mutants of Bacillus subtilis: plasmid pUB110 replication as a model system; Alonso JC et al.; We determined the effect of various Bacillus subtilis dna(Ts) mutations on pUB110 and chromosomal replication . Leading strand DNA synthesis of pUB110, starting by a nick at the plasmid replication origin (oriU), is performed by DNA polymerase III, since replication is blocked at non-permissive temperature in thermosensitive mutants dnaD, dnaF, dnaH and dnaN known to cause thermosensitivity of the various subunits of DNA polymerase III . When the lagging strand origin (oriL) is exposed, the DnaG protein (DNA primase) alone, or in association with unknown protein(s) binds asymmetrically to oriL to form a primer that is also extended by DNA polymerase III . In oriL- plasmids like pBT32, leading and lagging strand DNA syntheses are decoupled from each other . The DnaB protein, that is not required for pUB110 replication, may be associated with priming at a second unidentified lagging strand origin on pBT32 . At non-permissive temperature, the dnaC30 and dnaI2 mutations affect both pUB110 and chromosomal DNA synthesis. Mol Microbiol, 1988 Nov, 2(6), 813 - 9 Secretion and processing of the Bacillus subtilis endo-beta-1,3-1,4-glucanase in Escherichia coli; Gormley EP et al.; The endo-beta-1,3-1,4-glucanase enzyme of Bacillus subtilis C120, when synthesized in Escherichia coli, is located mainly in the cytoplasm, but enzyme activity is also detected in the periplasmic space and in the extracellular medium . The proportion recovered in the extracellular medium is not altered by changes in the levels of synthesis of the enzyme . Lysis of E . coli cells is ruled out as the cause of the secretion by the normal localization of beta-galactosidase, an intracellular protein . However, beta-lactamase, which is normally found in the periplasmic space, is detected in the extracellular medium of E . coli transformants containing beta-glucanase plasmids, suggesting that the presence of beta-glucanase in the cell alters the permeability of the outer membrane . The beta-glucanase proteins found in the extracellular medium, the periplasmic space and the cytoplasm have the same electrophoretic mobilities as the secreted enzyme of B . subtilis . Amino-terminal sequencing has shown that the beta-glucanase enzyme in the intracellular fraction of E . coli is processed at a site two amino acids distant from the processing site used in B . subtilis. Mol Microbiol, 1988 Nov, 2(6), 689 - 99 Structure of the gene for the transition state regulator, abrB: regulator synthesis is controlled by the spo0A sporulation gene in Bacillus subtilis; Perego M et al.; Sporulation begins coincidentally with the expression of several stationary-phase-associated gene products during the transition state of a culture from exponential to stationary phase . Mutations in the stage 0 sporulation genes prevent the expression of these gene products in addition to blocking sporulation . Suppressor mutations in the abrB gene, in a spo0 background, restore stationary-phase-associated gene expression but not sporulation . The nature of the abrB gene product was investigated by isolating and sequencing the abrB gene . The abrB gene coded for a 96-amino-acid protein (molecular weight 10773) and contained a helix-turn-helix structure common to DNA binding proteins . Analysis of expression of the abrB gene using lacZ transcription fusions and direct measurement of mRNA content by hybridization showed that the spo0A gene repressed transcription of the abrB gene . Primer extension analysis of abrB gene mRNA revealed two initiation sites . The downstream site was dramatically repressed in spo0A+ strains, while the upstream site appeared not to be regulated by spo0A . Five abrB mutant alleles were cloned and sequenced . One mutation, abrB4, resided within the structural gene and continued to overexpress abrB messenger RNA from both promoters . A promoter mutation, abrB15, reduced transcription from the downstream promoter but not the upstream promoter . Thus, the phenotype of abrB mutations results from inactivation of the abrB gene product or by prevention of its overexpression . The results suggest that the abrB gene codes for a regulator which controls several genes whose products are normally produced during the transition phase between active growth and sporulation . The level of this regulator is, in turn, controlled by the spo0A gene . The pleiotropic phenotypes of spo0A mutants result from uncontrolled overexpression of the abrB regulator. J Bacteriol, 1988 Nov, 170(11), 5102 - 9 Localization of Bacillus subtilis sacU(Hy) mutations to two linked genes with similarities to the conserved procaryotic family of two-component signalling systems; Henner DJ et al.; Mutations in the sacU region have a pleiotropic phenotype . Certain mutations designated sacU(Hy), for example, express degradative enzymes at high levels, are able to sporulate in the presence of glucose, have severely reduced transformation efficiencies, and are nonmotile . We isolated and sequenced the sacU gene region of Bacillus subtilis . Two open reading frames were found in the sacU region, and sacU(Hy) mutations were localized to both of these open reading frames . The two open reading frames have similarities to two widespread families of proteins that mediate responses to environmental stimuli. J Bacteriol, 1988 Nov, 170(11), 5086 - 92 Compartment-specific transcription in Bacillus subtilis: identification of the promoter for gdh; Rather PN et al.; Glucose dehydrogenase beings to accumulate in the forespore between 2 and 3 h after the onset of endospore formation in Bacillus subtilis . The promoter for the structural gene for glucose dehydrogenase (gdh) was shown to be located 800 base pairs upstream from the coding sequence by examining the effects of integrating plasmids into the gdh region of the chromosome . The location of the gdh promoter was confirmed by primer extension analysis and by the identification of two single-base substitutions in the gdh promoter that prevented its function . The results of cell fractionation experiments with a strain that contained a transcriptional fusion of the gdh promoter and lacZ indicated that the forespore-specific accumulation of glucose dehydrogenase during sporulation is probably due to forespore-specific transcription of gdh. Biochem J, 1988 Nov 1, 255(3), 833 - 41 Purification and properties of a 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli; Erfle JD et al.; A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite . The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan . The enzyme had a broad pH optimum with maximum activity at approx . pH 6.0 and a temperature optimum of 50 degrees C . The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B . succinogenes . The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis . The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively . The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%) . Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products . The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis . The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B . subtilis and the similarity with the 1,4-beta-glucanase in B . succinogenes. Mol Biol (Mosk), 1988 Nov-Dec, 22(6), 1658 - 66 {The use of phage lambda promotors for expressing genes of secreted proteins in Bacillus subtilis cells}; Sorokin AV et al.; At was shown with the help of promoterless alpha-amylase and staphylokinase genes that lambda PR and lambda PL promoters could be used in Bacillus subtilis . Promoters strength was compared to promoter of alpha-amylase gene, this enabled to order the promoters in a row: PAA greater than lambda PR greater than lambda PL . The lambda PR promoter region was controlled by temperature in E . coli cells only, but not in B . subtilis, therefore, the active lambda C1857 gene product was not produced in B . subtilis cells . The lambda PR promoter is used by B . subtilis at a later growth stage than PAA and the lambda PL promoter at a still later stage than lambda PR . The data enables lambda PR to be considered as quite useful for Bacilli. Mol Microbiol, 1988 Nov, 2(6), 735 - 41 Identification of the protein encoded by rodC, a cell division gene from Bacillus subtilis; Honeyman AL et al.; The rodC1 mutation of Bacillus subtilis is a temperature-sensitive marker which affects the orientation of the plane of cell division . We have cloned the rodC gene and have localized the site of the rodC1 lesion . To identify the rodC gene product, we have subjected several plasmid clones containing B . subtilis chromosomal DNA from the rodC region to maxicell analysis in Escherichia coli . A 68 kiloDalton protein has been identified as the rodC gene product . This is the initial cloning of a cell division gene and the identification of its product from B . subtilis . The rodC gene has also been implicated as being directly associated with the synthesis of glycerol teichoic acid. Genetics, 1988 Nov, 120(3), 625 - 35 Chromosomal organization of rRNA operons in Bacillus subtilis; Jarvis ED et al.; Integrative mapping with vectors containing ribosomal DNA sequences were used to complete the mapping of the 10 rRNA gene sets in the endospore forming bacterium Bacillus subtilis . Southern hybridizations allowed the assignment of nine operons to distinct BclI restriction fragments and their genetic locus identified by transductional crosses . Nine of the ten rRNA gene sets are located between 0 and 70 degrees on the genomic map . In the region surrounding cysA14, two sets of closely spaced tandem clusters are present . The first (rrnJ and rrnW) is located between purA16 and cysA14 closely linked to the latter; the second (rrnI, rrnH and rrnG) previously mapped within this area is located between attSPO2 and glpT6 . The operons at or near the origin of replication (rrnO,rrnA and rrnJ,rrnW) represent "hot spots" of plasmid insertion. Genes Dev, 1988 Nov, 2(11), 1381 - 8 The outB gene of Bacillus subtilis regulates its own transcription; Albertini AM et al.; The outB gene of Bacillus subtilis is under the control of two promoters (P1 and P2) . To study the regulation of expression from the P1 promoter we have constructed a set of multicopy plasmids carrying different portions of the outB region and analyzed the transcripts present in vivo by RNase protection experiments . The data indicate that the product of gene outB regulates its own transcription from the P1 promoter . We also constructed an outB-lacZ fusion in an insertional plasmid . The plasmid was inserted into the chromosome adjacent to or distal from the outB gene . Assays of beta-galactosidase activity and RNase protection experiments are in accordance with a model implying that the product of gene outB regulates the initiation of transcription from the P1 promoter acting in the cis configuration. Gene, 1988 Oct 30, 70(2), 351 - 61 Characterization of signal-sequence-coding regions selected from the Bacillus subtilis chromosome; Smith H et al.; Signal-sequence-coding regions for protein export were selected from chromosomal Bacillus subtilis DNA . The number of different signals obtained was higher than expected on the basis of known exported proteins in B . subtilis . Most of the selected regions showed the characteristics of typical signal sequences, including a basic N-terminal region followed by a hydrophobic core and a potential signal-peptidase cleavage site . The signal-coding regions were functionally interchangeable between the B . licheniformis alpha-amylase and Escherichia coli TEM beta-lactamase genes . In addition to the signal-sequence-coding regions, the nature of the host cells, and the mature parts of the reporter proteins contributed to the amounts of protein secreted. J Mol Biol, 1988 Oct 20, 203(4), 1045 - 70 Heavy riboflavin synthase from Bacillus subtilis . Crystal structure analysis of the icosahedral beta 60 capsid at 3.3 A resolution; Ladenstein R et al.; Geometric features as well as possible functional properties of the substrate binding sites at the pentamer interfaces are described . Ligand binding at the pentamer interface regions increases the stability of the beta 60 capsid considerably and influences the reassembly of isolated beta-subunits. J Mol Biol, 1988 Oct 20, 203(4), 905 - 15 Erythromycin-induced stabilization of ermA messenger RNA in Staphylococcus aureus and Bacillus subtilis; Sandler P et al.; Erythromycin-induced stabilization of ermA mRNA was studied in Staphylococcus aureus, its original host background, and in Bacillus subtilis, subcloned on plasmid vectors . By RNA blot analysis it was shown that 40 nM-erythromycin specifically increased the chemical half-life of ermA mRNA from 2.5 to 17.5 minutes whereas the half-life of cat-86 mRNA was not increased by erythromycin . While expression of ermA has been shown to be induced by erythromycin at the level of translation, our studies with three ermA constitutive mutants demonstrated that mRNA stabilization in growing cells occurred independently of induced gene expression, suggesting that the stabilized mRNA was not functional for protein synthesis . Studies of ermA/lacZ fusions demonstrated that the 5' end of the mRNA was sufficient to confer stabilization . Translation of specific amino acid codons in a leader peptide located at the extreme 5' end of the mRNA was required for the erythromycin-induced stabilization as a frameshift mutation introduced into the leader peptide determinant abolished stabilization . By S1 mapping, no differences were detected in the length of the 5' or 3' end of ermA mRNA with the addition of erythromycin, indicating that the stabilized transcript was not processed at its ends. J Biol Chem, 1988 Oct 15, 263(29), 14654 - 60 Cloning and nucleotide sequence of the Bacillus subtilis hom gene coding for homoserine dehydrogenase . Structural and evolutionary relationships with Escherichia coli aspartokinases-homoserine dehydrogenases I and II; Parsot C et al.; The Bacillus subtilis hom gene, encoding homoserine dehydrogenase (L-homoserine:NADP+ oxidoreductase, EC 1.1.1.3) has been cloned and its nucleotide sequence determined . The B . subtilis enzyme expressed in Escherichia coli is sensitive by inhibition by threonine and allows complementation of a strain lacking homoserine dehydrogenases I and II . Nucleotide sequence analysis indicates that the hom stop codon overlaps the start codon of thrC (threonine synthase) suggesting that these genes, as well as thrB (homoserine kinase) located downstream from thrC, belong to the same transcription unit . The deduced amino acid sequence of the B . subtilis homoserine dehydrogenase shows extensive similarity with the C-terminal part of E . coli aspartokinases-homoserine dehydrogenases I and II; this similarity starts at the exact point where the similarity between E . coli or B . subtilis aspartokinases and E . coli aspartokinases-homoserine dehydrogenases stops . These data suggest that the E . coli bifunctional polypeptide could have resulted from the direct fusion of ancestral aspartokinase and homoserine dehydrogenase . The B . subtilis homoserine dehydrogenase has a C-terminal extension of about 100 residues (relative to the E . coli enzymes) that could be involved in the regulation of the enzyme activity. Nucleic Acids Res, 1988 Oct 11, 16(19), 9127 - 45 Functional analysis of the leading strand replication origin of plasmid pUB110 in Bacillus subtilis; Alonso JC et al.; Supercoiled plasmid DNA is the substrate for initiation of pUB110 replication, and - by inference - for binding of its initiator protein (RepU) to the plasmid replication origin (oriU) in vivo . No hairpin structure is required for RepU-oriU recognition . RepH (the pC194 replication initiation protein) failed to initiate replication in trans at oriU . The nucleotides that determine the specificity of the replication initiation process are located within oriU but termination is unefficient . Therefore the segment that forms the full recognition signal for termination is probably located 3' of the oriU recognition sequence . Two overlapping domains, one for initiation and one required for termination, compose the leading strand replication origin of plasmid pUB110. J Biol Chem, 1988 Oct 5, 263(28), 14480 - 4 Transcriptional analysis of Bacillus subtilis rRNA-tRNA operons . I . The tRNA gene cluster of rrnB has an internal promoter; Vold BS et al.; Although the sequence and organization of many Bacillus subtilis tRNA genes are known, primary transcripts from these regions have not been previously analyzed . In this paper, S1 nuclease mapping, S1-type mapping, and Northern analyses were applied to the end of the 23 S rRNA, the 5 S rRNA, and the 21 tRNA genes of B . subtilis operon rrnB . Primary transcripts from the 5 S rRNA and tRNA genes up to approximately 600-800 nucleotides long were observed with S1-type mapping . The presence of discrete bands of processing intermediates indicated preferred processing points within the initial transcript . S1 nuclease mapping delineated a start point for transcription between the second and third tRNA genes . The -10 sequence was within the 37-base pair spacer region between tRNA genes, and the -35 sequence was within the structural gene for the upstream tRNA . Precursors from this region were evident during midexponential growth and two sporulation stages . Thus, in addition to promotion from the rRNA promoters, 19 of the 21 downstream tRNA genes are also under the control of an internal tRNA gene promoter . The accompanying paper (Vold, B . S., Green, C . J., Narasimhan, N., Strem, M., and Hansen, J . N . (1988) J . Biol . Chem . 263, 14485-14490) investigates the minor 5 S rRNA and 16 tRNA genes of another rRNA-tRNA gene set and emphasizes unique promoter elements in that system as well as a potentially unique rRNA processing scheme. J Biol Chem, 1988 Oct 5, 263(28), 14485 - 90 Transcriptional analysis of Bacillus subtilis rRNA-tRNA operons . II . Unique properties of an operon containing a minor 5 S rRNA gene; Vold BS et al.; This is part of a series of two papers on gene regulation in Bacillus subtilis rRNA-tRNA operons that contain large clusters of tRNA genes . The preceding paper (Vold, B.S., Okamoto, K., Murphy, B.J., and Green, C.J . (1988) J . Biol . Chem . 263, 14480-14484) investigates the rrnB operon containing 21 tRNA genes, and this paper investigates a B . subtilis rRNA-tRNA operon containing 16 tRNA genes and a minor 5 S rRNA . Hybridization studies suggest this minor 5 S rRNA occurs as a single copy in the B . subtilis 168 genome . S1 nuclease mapping indicates that this minor 5 S rRNA gene has its own promoter . No promoters have been found immediately 5' to any of the major 5 S rRNA species in B . subtilis rRNA operons . S1 mapping of the spacer region between the 23 S and minor 5 S rRNA revealed that the maturation of the 23 S rRNA in this operon may arise from an unusual processing mechanism . S1 nuclease mapping experiments suggest the existence of a promoter element immediately upstream of the last gene, for tRNA(Leu CAA), in the operon . A precursor leucine tRNA resulting from transcription of this last tRNA gene was observed in Northern hybridizations, and the amounts of this precursor increased during sporulation . A single terminator-like element is located just upstream of this last tRNA gene; however, S1 nuclease mapping experiments suggest that some read-through transcription occurs . Thus, all 16 tRNA genes are under control of the upstream 16 S rRNA promoters and the minor 5 S rRNA promoter . However, the last tRNA gene is primarily under the control of its own unique promoter. J Biol Chem, 1988 Oct 5, 263(28), 14390 - 6 Processing of a multimeric tRNA precursor from Bacillus subtilis by the RNA component of RNase P; Vold BS et al.; Processing of multimeric precursor tRNAs from Bacillus subtilis by the catalytic RNA component of RNase P was studied in vitro . Previous studies on processing by either Escherichia coli or B . subtilis RNase P-RNA utilized monomeric or dimeric substrates . In the experiments described here, a multimeric precursor tRNA containing six complete tRNA sequences and the partial sequence of a seventh were used . One species did not encode the 3'-terminal CCA sequence and the partial tRNA lacked 3' nucleotides and could form only a 3-base pair instead of a 7-base paired aminoacyl stem . Two species had the potential for forming extended base-paired aminoacyl stems . Processing was studied under varied ionic conditions . Chemical sequencing of the products showed that the RNase P-RNA cleavage produced the proper mature 5' termini for all of the six complete tRNA species, but no 5'-cleavage of the partial species was observed . At suboptimal ionic concentrations, the two species capable of forming extended base-paired aminoacyl stems were not observed . Thus, encoding of the 3'-CCA in a tRNA species is not critical for processing, but the formation of an aminoacyl stem with more than 3 base pairs is necessary . Particularly noteworthy was the observation that all species of the multimeric precursor could be processed at significantly lower ionic conditions than monomeric precursors used previously by ourselves and others . However, a single precursor species produced from the multimeric precursor could also be processed at the same lower ionic conditions as the multimeric precursor . This demonstrates that precursor tRNA species can differ widely in their ionic requirements for processing and that, to a large extent, the optimal conditions of MgCl2 or NH4Cl are a function of the substrate which is used. Int J Food Microbiol, 1988 Oct, 7(2), 161 - 8 Physiology of Sporolactobacillus strains isolated from different habitats and the indication of in vitro antagonism against Bacillus species; Holzapfel WH et al.; In an ecological study only low numbers of Sporolactobacillus were found in habitats such as the faeces of herbivores, the rumen of cattle and the final waste water of an abattoir . Their presence in the final waste water of an abattoir indicates their possible association with food, and, more specifically, with meat . Differences were found in some physiological characteristics . One isolate (L2404) differed from the authentic Sporolactobacillus ATCC 15538 by its inability to ferment inulin, its growth in presence of 6.5% NaCl and in 0.2% tellurite, by the isomer(s) of lactic acid produced and the mol% G + G in the DNA . One Sporolactobacillus isolate (L2407) showed antagonism against Bacillus cereus, Bacillus cereus var, mycoides, Bacillus megaterium and Bacillus subtilis. Biochem Int, 1988 Oct, 17(4), 655 - 64 Nucleotide sequence of maize chloroplast rpS11 with conserved amino acid sequence between eukaryotes, bacteria and plastids; Markmann-Mulisch U et al.; Nucleotide sequence of a 721 base pair segment of maize chloroplast DNA, encoding the putative chloroplast ribosomal protein S11 at physical map position 33.1-33.5 Kbp, is described . A Shine-Dalgarno sequence and computer-derived stem-loop structures of dyad symmetry are present in the spacer region between rpS11 and its 5' upstream gene rpL36 . The deduced amino acid sequence of maize chloroplast S11 shows 69%, 66%, 62%, 57%, 48% and 45% sequence identity to the corresponding sequences of tobacco, spinach, pea, liverwort, Escherichia coli and Bacillus subtilis, respectively, and 41% sequence identity to three eukaryotic cytoplasmic ribosomal proteins, S14 of Chinese hamster and of human and rp59 of yeast . Maize chloroplast r-protein S11 is larger than the other published S11s of plants and bacteria, due to the apparent tandem introduction of a short sequence stretch of internal homology. Mol Gen Genet, 1988 Oct, 214(2), 321 - 4 Further studies on recombination in diploid clones from Bacillus subtilis protoplast fusion; Sanchez-Rivas C et al.; Diploid prototrophs were obtained from protoplast fusion of Bacillus subtilis strains . They are unstable but upon further cultivation they stabilize retaining diploidy but are genetically inactive . It has been suggested that recombination between the parental chromosomes is involved in the production of stable prototrophs and recombinants . In this work the occurrence of this recombination was searched for by determining genetic linkages in transformation experiments . In prototrophs two alleles: hisH2 and trpE8 carried originally on each parental chromosome, were shown to be 48% co-transformable in a stable clone whereas they were only cotransformed in 10% of the unstable colonies . For Trp- recombinants (the most frequent type of a Leu- Met- Thr- x Ade- Ura- Trp- fusion pair) lysed protoplasts were used as donor DNA for the transformations . High values of co-transfer for Ura+ Met+ were obtained . These results confirm the occurrence of recombination in stable diploid clones, prototrophs or recombinants. Biochimie, 1988 Oct, 70(10), 1401 - 9 Improved purification and properties of glucose dehydrogenase from Bacillus subtilis; Karmali A et al.; Glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was purified about 5240-fold, using an aqueous two-phase system and triazine-dye affinity chromatography . The specific activity of the purified preparation was about 460 units/mg of protein with a final recovery of enzyme activity of about 75% . The affinity column could be regenerated and reused again several times . The purified enzyme appeared to be homogeneous when analyzed both on SDS-PAGE and native PAGE . The protein band on native PAGE coincided with the activity stain . ATP acts apparently as a competitive inhibitor for this enzyme with respect to NAD and protects the enzyme from dissociation into partially inactive dimers . In the absence of either glycerol or ATP, the enzyme dissociates into partially inactive dimers. Proc Natl Acad Sci U S A, 1988 Oct, 85(20), 7452 - 6 Origin-specific DNA-binding membrane-associated protein may be involved in repression of initiation of DNA replication in Bacillus subtilis; Laffan JJ et al.; Previous binding studies with labeled double-stranded Bacillus subtilis DNA fragments to a protein blot of renatured Bacillus membrane proteins showed selective binding of two adjacent origin fragments to a 64-kDa protein . The selective binding of the 64-kDa protein could be blocked by prior incubation of the blots with a specific polyclonal antibody . An in vitro replication system derived from a B . subtilis DNA-membrane complex showed initiation activity without addition of exogenous enzymes or template . When the complex was first incubated with the 64-kDa antibody or with its Fab fragments, initiation activity was enhanced . Antibodies to several other Bacillus membrane proteins as well as nonspecific antibodies did not show any significant stimulatory effect . A heavy-density-label experiment indicated that the complex initiated multiple rounds of replication in the presence of the 64-kDa antibody but not in its absence . The 64-kDa antibody plus an initiation inhibitor (streptovaricin) showed only repair and elongation activity . The 64-kDa protein may act in vivo as a repressor/regulator of initiation activity. J Bacteriol, 1988 Oct, 170(10), 4855 - 64 Cloning and characterization of Bacillus subtilis homologs of Escherichia coli cell division genes ftsZ and ftsA; Beall B et al.; The Bacillus subtilis homolog of the Escherichia coli ftsZ gene was isolated by screening a B . subtilis genomic library with anti-E . coli FtsZ antiserum . DNA sequence analysis of a 4-kilobase region revealed three open reading frames . One of these coded for a protein that was about 50% homologous to the E . coli FtsZ protein . The open reading frame just upstream of ftsZ coded for a protein that was 34% homologous to the E . coli FtsA protein . The open reading frames flanking these two B . subtilis genes showed no relationship to those found in E . coli . Expression of the B . subtilis ftsZ and ftsA genes in E . coli was lethal, since neither of these genes could be cloned on plasmid vectors unless promoter sequences were first removed . Cloning the B . subtilis ftsZ gene under the control of the lac promoter resulted in an IPTGs phenotype that could be suppressed by overproduction of E . coli FtsZ . These genes mapped at 135 degrees on the B . subtilis genetic map near previously identified cell division mutations. J Bacteriol, 1988 Oct, 170(10), 4791 - 7 Cloning and characterization of a Bacillus subtilis transcription unit involved in ATP-dependent DNase synthesis; Kooistra J et al.; By insertional mutagenesis, 36 transformation-deficient, mitomycin C-sensitive Bacillus subtilis mutants were isolated, 16 of which were ATP-dependent DNase (ADD) deficient . PBS1 transduction showed that the mutations were closely linked to the metD marker and weakly linked to the glyB marker . With the aid of one of the mutants, a transcription unit involved in ADD synthesis was cloned . The chromosomal location of the transcription unit was established at the restriction site level by determining the presence or absence of ADD in transformants of wild-type cells obtained with various DNA fragments inserted in pUC derivatives . The transcription unit complemented a mutant in which the add transcription unit had been deleted. Mutat Res, 1988 Oct, 206(2), 171 - 6 Erythrosine, a permitted food dye, is mutagenic in the Bacillus subtilis multigene sporulation assay; Lakdawalla AA et al.; Erythrosine increased the yield of sporulation minus mutants of Bacillus subtilis excision repair-proficient strain 168 by approximately equal to 400% at a concentration of 1 mg/ml under ambient light conditions . This mutagenic response was dose-dependent (0-1 mg/ml) . Significantly, the food dye did not mutate the corresponding repair-deficient B . subtilis, hcr-9 (exc-) . A decrease in the mutagenicity of erythrosine to B . subtilis strain 168 was apparent on metabolism by rat liver S9 mixture or by rat caecal cell-free extracts . Erythrosine did not exhibit differential toxicity to B . subtilis excision repair-proficient (168) and -deficient (hcr-9) strains, although it was highly toxic to both strains . This indicated non-involvement of excision repair in the dye-mediated toxic reactions. Proc Natl Acad Sci U S A, 1988 Oct, 85(20), 7637 - 41 Two developmental genes encoding sigma factor homologs are arranged in tandem in Bacillus subtilis; Masuda ES et al.; The sporulation-essential gene spoIIG of the Gram-positive bacterium Bacillus subtilis encodes the sporulation-specific sigma factor sigma 29(sigma E) . We report here the initial characterization of a gene, referred to as ORF3, located immediately downstream of the spoIIG gene . The results indicate that ORF3 encodes a sigma homolog, whose expression is highly regulated during development . Analysis of the ORF3 nucleotide sequence reveals an open reading frame encoding a polypeptide of 260 amino acid residues (molecular mass of 30.1 kDa) . Its predicted amino acid sequence shows significant similarity to that of other RNA polymerase sigma factor sequences . S1 nuclease mapping experiments indicate that ORF3 is initially cotranscribed with spoIIG from about 1 to 4 hr into the sporulation process and that later on ORF3 is transcribed independently from a new site located between spoIIG and ORF3 . The role of ORF3 was investigated by constructing a deletion mutation in its structural gene . The mutant exhibits normal growth but is unable to produce heat-resistant spores . We propose that the ORF3 gene product is a sigma factor or a related peptide essential for sporulation at a late stage of development. Gene, 1988 Sep 15, 69(1), 59 - 69 Construction of a new shuttle expression vector for Bacillus subtilis and Escherichia coli by using a polycistronic system; Nagami Y et al.; A shuttle vector has been constructed by fusing the Bacillus subtilis trimethoprim-resistance-carrying (TpR) plasmid pNC601 with the Escherichia coli plasmid pBR322 . The resultant plasmid pNBL1 can replicate in both B . subtilis and E . coli, conferring Tp resistance on both cells and ampicillin resistance (ApR) on E . coli . The B . subtilis dihydrofolate reductase operon (dfr) on pNC601 and therefore on pNBL1 consists of the thymidylate synthase B gene (thyB) and the TpR-dihydrofolate reductase gene lacking the C-terminal seven codons (designated as drfA' as compared with the complete dfrA gene) . A direct-expression vector pNBL3 has been constructed by inserting synthetic oligodeoxynucleotides containing a Bacillus ribosome-binding site (RBS) and the ATG codon downstream from dfrA' on pNBL1 . When the E . coli lacZ gene was placed downstream from the dfrA' gene in pNBL3, efficient synthesis of beta-galactosidase was observed in both cells, showing that the polycistronic expression system is suitable for directing expression of heterologous genes . Translational efficiency of the lacZ gene on pNBL3 was further examined in B . subtilis by changing the sequence upstream from lacZ . Unlike the results previously reported {Sprengel et al., Nucleic Acids Res . 13 (1985) 893-909}, when RBS was present, the high level of lacZ expression was preserved irrespective of spacing between the stop codon of the upstream dfrA' gene and the start codon of the downstream lacZ gene . However, in the absence of RBS, the spacing between both genes affected lacZ expression . That is, translational coupling of dfrA'-lacZ was observed, although the translational efficiency was very low. Gene, 1988 Sep 15, 69(1), 39 - 47 Expression and secretion of human atrial natriuretic alpha-factor in Bacillus subtilis using the subtilisin signal peptide; Wang LF et al.; Using the signal peptide of the Bacillus subtilis subtilisin gene (aprE) and a synthetic cDNA corresponding to the mature region of the human atrial natriuretic alpha-factor (hANF), we have constructed a secretion vector . B . subtilis cells, when transformed with this vector, secrete immunoreactive hANF peptides into the medium at about 500 micrograms/liter . The hANF is the first human gene product to be secreted from B . subtilis using this signal peptide . We have used promoters active during vegetative growth or sporulation and hosts deficient in several extracellular proteases but some proteolysis of the secretion products still occurs . In addition, both cell growth and sporulation are adversely affected by hANF production . Possible explanations for this observation are inefficient secretion of the atrial hormone or toxicity of the precursor or mature peptide. J Biol Chem, 1988 Sep 15, 263(26), 13252 - 7 Purification and characterization of a repressor for the Bacillus subtilis gnt operon; Miwa Y et al.; The GntR protein is a negative regulator involved in gluconate-inducible expression of the Bacillus subtilis gnt operon which is responsible for gluconate metabolism . The GntR protein has been purified to homogeneity from an overproducing Escherichia coli strain harboring a gntR gene-carrying plasmid . The total amino acid composition and the NH2-terminal amino acid sequence of the purified protein were essentially the same as those deduced from the nucleotide sequence of the gntR gene . Molecular weight determination by gel filtration revealed that the purified protein is in a highly polymerized form, but it likely exists as a dimer when highly diluted . The purified GntR protein was found to be specifically bound to DNA fragments carrying the promoter of the gnt operon in an electrophoretic mobility shift assay . This binding was specifically inhibited by the addition of gluconate or glucono-delta-lactone . The purified protein repressed in vitro transcription from the promoter of the gnt operon . This repression was suppressed by gluconate or glucono-delta-lactone . These results indicate that the GntR protein is a repressor for the gnt operon and that gluconate and glucono-delta-lactone are inducers for this operon. Biochim Biophys Acta, 1988 Sep 7, 950(3), 441 - 4 Nucleotide sequence homology of the tetracycline-resistance determinant naturally maintained in Bacillus subtilis Marburg 168 chromosome and the tetracycline-resistance gene of B . subtilis plasmid pNS1981; Sakaguchi R et al.; The nucleotide sequence (1579 bp) of tetracycline-resistance determinant and flanking regions of the cloned 5.1 kb DNA fragment from Bacillus subtilis GSY908 chromosome (Sakaguchi, R . and Shishido, K . (1988) Biochim . Biophys . Acta 949, 49-57) were determined and compared with those of the B . subtilis tetracycline-resistance plasmid pNS1981 . The tetracycline-resistance structural (tet) genes of the B . subtilis GSY908 chromosome (tetBS908) and pNS1981 (tetpNS1981) were found to be highly homologous (80% identical) . Both tet genes were composed of 1374 bp and 458 amino-acid residues initiating from a GTG codon preceded by a ribosome-binding site (RBS-2) . Upstream from tetBS908 there exists a short open reading frame (20 amino acids) initiating from a ATG codon preceded by its own RBS (RBS-1) . This leader sequence was also highly homologous to that of tetpNS1981 except for a deletion of one bp between the RBS-1 and the ATG codon. Mikrobiologiia, 1988 Sep-Oct, 57(5), 745 - 50 {Stimulating effect of pancreatic DNase on Bacillus subtilis growth as a function of the physiologic properties of the inoculation culture}; Kupriianova FG et al.; The stimulating effect of pancreatic DNAse on Bacillus subtilis growth was studied in relation to the content of "slowly growing" cells in the inoculation culture in the phase of decelerated growth . Three cell fractions of B . subtilis were obtained using the stepwise separation of the population in terms of buoyant density in the phase of decelerated growth . In contrast to fractions II and III, fraction I contained cells with decelerated growth, competent, permeable to exogenous DNAase I, and sensitive to the action of this enzyme . The faster growth of bacterial cells in fraction I was shown to be associated with the shorter lag period of these cells having a longer generation time. Mikrobiologiia, 1988 Sep-Oct, 57(5), 740 - 4 {Cultivation of the producer of alpha-amylase Bacillus subtilis under batch and continuous conditions}; Lirova SA et al.; The growth of Bacillus subtilis FU-79 and its production of alpha-amylase (EC 3.2.1.1) were studied under the conditions of batch and continuous cultivation in a semisynthetic medium . The enzyme activity fell down abruptly upon a pulse addition of either glucose or yeast extract to the chemostat culture, and remained at a low level for the following ten generations . Apparently, a double limitation of the culture growth (viz., with residual glucose and with yeast extract components) is required for the activity of alpha-amylase to be high. Plasmid, 1988 Sep, 20(2), 148 - 54 Use of a versatile lacZ vector to analyze the upstream region of the Bacillus subtilis spoOF gene; Lewandoski M et al.; We have constructed a versatile vector, pIS112, in which lacZ translational fusions can be made in Escherichia coli and then analyzed in Bacillus subtilis in three contexts, without recloning: in multicopy during propagation of the plasmid, in single copy integrated via a Campbell-type mechanism into the wild-type locus of the cloned fragment, or in single copy integrated into a heterologous locus . Upstream regions are reconstituted in the integration into the wild-type locus, but not into the heterologous locus, allowing the identification of upstream regulatory sequences . We have used this vector to analyze the expression of the early sporulation gene, spoOF, which, during early stationary phase, is induced 10-fold from a basal vegetative level . When a region, -50 to -150 bp relative to the transcriptional start site, is removed in the spoOF-lacZ gene, stationary phase induction of beta-galactosidase is lost . The same deletion in the upstream region of the functional spoOF gene results in cells which sporulate very poorly, although they are not blocked at the onset of sporulation, as in an spoOF null mutant . This suggests induction of spoOF expression during the beginning of stationary phase is necessary for wild-type sporulation. Mol Gen Mikrobiol Virusol, 1988 Sep, (9), 39 - 43 {Reduced frequency of homologous recombination in Bacillus subtilis mutants resistant to novobiocin}; Barabanshchikov BI et al.; B . subtilis mutants resistant to novobiocin that were selected after treatment of the strain SB25 by nitrosoguanidine acquired the slow growth, increased UV-sensitivity, the low frequency of homologous recombination in transduction and transformation . The mutant strains are characterized by the low activity of the double stranded DNA-dependent ATP-ase peculiar for cells with impaired B-subunit of DNA-gyrase. Anal Biochem, 1988 Sep, 173(2), 440 - 4 Precipitation and recovery of proteins from culture supernatants using zinc; Zaworski PG et al.; A convenient method for the concentration of proteins from culture supernatants is described . Heterologous gene products expressed and secreted into the culture medium by Saccharomyces cerevisiae, Chinese hamster ovary cells (CHO), and Bacillus subtilis were precipitated out of solution by the addition of millimolar concentrations of zinc chloride . Porcine urokinase secreted by S . cerevisiae, the tissue-type plasminogen activator analog FK2P secreted by CHO cells, and human interleukin-1 beta secreted by B . subtilis were all precipitated by zinc ions . Both urokinase and FK2P were precipitated with as low as 2 mM zinc, with recovery of activity in the precipitate approaching 100%. J Bacteriol, 1988 Sep, 170(9), 3978 - 82 Efficiency of homologous DNA recombination varies along the Bacillus subtilis chromosome; Vagner V et al.; Structures consisting of a genetic marker (erythromycin or kanamycin resistance, thymidylate synthetase) flanked by 3.4-kilobase direct repeats (pBR322 sequences) were inserted in 12 different locations of the Bacillus subtilis chromosome . Recombination between the repeats was followed by the loss of the genetic marker . Recombination frequencies found in different locations varied from 1.2 X 10(-5) to 40 X 10(-5) per cell generation . Such differences were highly significant (P less than 0.001). EMBO J, 1988 Sep, 7(9), 2911 - 7 Regulation of initiation of the chromosomal replication by DnaA-boxes in the origin region of the Bacillus subtilis chromosome; Moriya S et al.; A gene homologous to the Escherichia coli dnaA gene and two flanking 'regulatory' regions which contain nine and four DnaA-boxes respectively, are located in the replication origin region of the Bacillus subtilis chromosome . Attempts to isolate an autonomously replicating fragment from these 'regulatory' regions in order to identify oriC have been unsuccessful because the DnaA-box-containing regions strongly inhibited plasmid transformation particularly when inserted into a high-copy number plasmid pUB110 . Using two plasmids differing in copy number, the two regions were subdivided into three regions, A, B and C, each containing five, four and four DnaA-boxes respectively, which differed in level of inhibition of transformation . Region C is downstream of the 'dnaA' gene and inhibits transformation in high-copy but not in low-copy number plasmids . When a part of the DnaA-boxes was deleted from the incompatible plasmids, they became transformable and produced slow-growing transformants in which the initiation frequency of chromosomal replication was selectively reduced . Fast-growing revertants were found containing the same number of plasmids as the parent but with single base changes in the DnaA-boxes . These mutations were in the most highly conserved bases of the DnaA-box sequence . This indicates that a sequence-specific interaction of the DnaA-box, probably with the B . subtilis DnaA protein is responsible for the observed incompatibility and thus appears to be involved in control of initiation frequency of the chromosomal replication. J Bacteriol, 1988 Sep, 170(9), 4083 - 90 DNA sequence requirements for replication fork arrest at terC in Bacillus subtilis; Smith MT et al.; The replication terminus, terC, of Bacillus subtilis is the chromosomal site at which movement of the clockwise replication fork is blocked . The effect of deletion or modification of DNA sequences on either side of terC (defined by the sequence location of the arrested clockwise fork junction) has been investigated . Deletion of sequences ahead of terC to within 250 base pairs (bp) had no effect on fork arrest, whereas removal of a further 130 bp abolished it . The 250-bp segment immediately ahead of terC encompassed the previously identified inverted repeat region as well as potential promoters for the transcription of an adjoining open reading frame (ORF) . Deletion of DNA from the other side of terC up to 80 bp from it also abolished fork arrest . This deletion removed the bulk of the ORF . Disruption of this ORF by the insertion of 4 bp also abolished fork arrest . A model for clockwise fork arrest at terC, implicating both the inverted repeat region and the protein product of the ORF, is proposed. J Virol, 1988 Sep, 62(9), 3455 - 62 TF1, the bacteriophage SPO1-encoded type II DNA-binding protein, is essential for viral multiplication; Sayre MH et al.; The lytic Bacillus subtilis bacteriophage SPO1 encodes an abundant, 99-amino-acid type II DNA-binding protein, transcription factor 1 (TF1) . TF1 is special in this family of procaryotic chromatin-forming proteins in its preference for hydroxymethyluracil-containing DNA, such as SPO1 DNA, and in binding with high affinity to specific sites in the SPO1 chromosome . We constructed recessive null alleles of the TF1 gene and introduced them into SPO1 chromosomes . Segregation analysis with partially diploid phage heterozygous for TF1 showed that phage bearing only these null alleles was inviable . Deletion of the nine C-proximal amino acids of TF1 prohibited phage multiplication in vivo and abolished its site-specific DNA-binding activity in vitro. J Bacteriol, 1988 Sep, 170(9), 4194 - 208 Complete sequence and transcriptional analysis of the spo0F region of the Bacillus subtilis chromosome; Trach K et al.; The total sequence of a 6,314-base-pair BglII fragment of the Bacillus subtilis chromosome containing the spo0F locus has been accomplished . Several genes of interest have been identified on this DNA fragment . The ctrA locus was recognized as coding for CTP synthetase by comparison of its deduced sequence with that of Escherichia coli CTP synthetase . A total of 53% of the residues are identical between the enzymes from these organisms . The spo0F locus was followed immediately by a locus, tsr, required for RNA synthesis in this organism . Temperature-sensitive mutations within the tsr locus have been identified, but strains with deletions of the locus are nonviable . It was concluded that tsr codes for an unknown essential component of the RNA synthesis machinery . The tsr gene was followed by another open reading frame which could code for a protein of 19,975 Mr . This gene was translated in vivo, but deletion-insertion mutations within the gene had no phenotype . The gene was cotranscribed with the tsr gene, although about 50% of the transcripts terminated between the two genes . The rev-4 mutation which reverts the sporulation-defective phenotype of erythromycin-resistant mutants was located to a partial open reading frame at the end of the fragment . Disruption of this open reading frame by deletion-insertion mutation did not result in a discernible phenotype . S1 protection experiments located the start sites of transcription for several of the genes on this fragment . The spo0F gene was found to be monocistronic . Regulation of the identified genes was investigated by using beta-galactosidase gene fusions. FEBS Lett, 1988 Aug 29, 236(2), 437 - 40 Calcium channel blockers inhibit bacterial chemotaxis; Matsushita T et al.; The effect of several Ca2+ channel blockers, which inhibit the voltage-dependent Ca2+ uptake in Bacillus subtilis, on chemotactic behaviour of the bacterium was studied . Nitrendipine, verapamil, LaCl3 and omega-conotoxin were tested and these blockers inhibited chemotactic behaviour in the bacterium toward L-alanine . Among these blockers, omega-conotoxin was the most effective inhibitor of chemotaxis . EGTA was also as effective as omega-conotoxin . In contrast, these blockers, did not inhibit the motility and the growth of the bacterium . These results suggest that internal Ca2+ plays an important role in the sensory system of bacterial chemotaxis. J Biol Chem, 1988 Aug 25, 263(24), 11743 - 9 Effect of polyadenine-containing curved DNA on promoter utilization in Bacillus subtilis; McAllister CF et al.; The effect of DNA upstream of the -35 region on promoter function was examined using two promoters isolated from the Bacillus subtilis bacteriophage SP82 . The affinity of RNA polymerase for the two promoters in vitro differed significantly . For each promoter the nucleotide sequence of the upstream DNA was characterized by the presence of successive runs of adenines with a 10-11-base pair periodicity . DNA fragments with the polyadenine-containing upstream DNA displayed aberrant electrophoretic mobilities when analyzed on polyacrylamide gels indicative of curved DNA . A series of mutant promoters in which the upstream DNA was deleted or altered was constructed . The curved DNA upstream of the -35 region was required for efficient RNA polymerase binding . Decreased in vitro transcription observed when the upstream DNA was deleted could be partially restored if the template was negatively supercoiled . Measurements of chloramphenicol acetyltransferase specific activity from B . subtilis strains carrying transcriptional fusions indicate that the curved upstream DNA stimulated transcription from the promoter with the weaker affinity for RNA polymerase . The curved DNA reduced the in vivo activity of the promoter with the strong affinity for RNA polymerase . One function of the curved upstream DNA may be to provide RNA polymerase-promoter interactions that facilitate open complex formation. J Biol Chem, 1988 Aug 25, 263(24), 11617 - 20 Ionic conditions for the cleavage of the tRNA-like structure of turnip yellow mosaic virus by the catalytic RNA of RNase P; Green CJ et al.; The 3'-end of the RNA genome of turnip yellow mosaic virus can form a pseudoknotted tRNA-like structure that can be recognized by several tRNA-specific enzymes . We have found that the catalytic RNA component of Bacillus subtilis RNase P can cleave this structure in unusually low ionic strength buffers at a site analogous to the 5'-end of an aminoacyl stem of a tRNA . Most other precursors can only be processed under low ionic strength conditions if the RNase P holoenzyme is used; processing by the catalytic RNA component alone requires a higher ionic strength buffer . The cleavage of the turnip yellow mosaic virus tRNA-like structure demonstrates the importance of the substrate in determining the optimal buffer conditions for this reaction and also shows that high ionic strength buffers are not always necessary for cleavage by the catalytic RNA. J Mol Biol, 1988 Aug 20, 202(4), 697 - 709 Nucleotides that contribute to the identity of Escherichia coli tRNA(Phe); McClain WH et al.; A series of sequence variants of amber suppressor genes of tRNA(Phe) were synthesized in vitro and cloned in Escherichia coli to examine the contributions of individual nucleotides to identity for amino acid acceptance . Three different but complementary types of tRNA variants were constructed . The first involved the substitution of base-pairs on the cloverleaf stem regions of the E . coli tRNA(Phe) . The second type of variant involved total gene synthesis based on wild-type tRNA(Phe) sequences found in Bacillus subtilis and in Halobacterium volcanii . In the third type of variant, the identity of E . coli tRNALys was changed to that of tRNA(Phe) . The nucleotides which are important for tRNA(Phe) identity in E . coli are located on the corner of the L-shaped tRNA molecule, where the dihydrouridine loop interacts with the T loop, and extend to the interior opening of the anticodon stem and the adjoining variable loop . The nucleotide sequence on the dihydrouridine stem region, which joins the corner and stem regions, was not successfully studied though it may contribute to tRNA(Phe) identity . The fourth nucleotide from the 3' end of tRNA(Phe) has some importance for identity. J Biol Chem, 1988 Aug 15, 263(23), 11548 - 53 NH2-terminal processing of Bacillus subtilis alpha-amylase; Takase K et al.; Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by removal of the NH2-terminal 41-amino acid sequence . To study the mechanism of this processing, the extracellular forms of alpha-amylase were analyzed for B . subtilis N7 alpha-amylase cloned and expressed in B . subtilis . The major form (form N34) isolated from log phase cultures in L-broth had an NH2 terminus corresponding to position 34 from the initiator Met but appeared to be microheterogeneous, as judged by native gel electrophoresis . The major forms from stationary phase cultures had NH2 termini at positions 40 (form N40) or 42 (form N42) and were homogeneous . The conversion of the larger to smaller forms could be achieved in culture supernatants or partially purified samples . The process N34----N40 was inhibited by EDTA; N40----N42 was facilitated by Ca2+ . Phenylmethylsulfonyl fluoride inhibited the former but not the latter process . These results suggest that the signal peptidase cleavage site 30 decreases 35 is -Ala-Ala-Ala-Ser-Ala-Glu-Thr- (arrow or further upstream) and that proteolytic maturation occurs after secretion, which involves at least two different processing enzymes. Biochem J, 1988 Aug 15, 254(1), 269 - 75 A bacterial factor induces changes in cysteine proteinase forms in the cellular slime mould Dictyostelium discoideum; North MJ; The electrophoretic pattern of cysteine proteinases in axenically grown myxamoebae of Dictyostelium discoideum can be altered by the addition of either Gram-negative (Klebsiella aerogenes, Escherichia coli) or Gram-positive (Micrococcus lysodeikticus, Bacillus subtilis) bacteria to the culture . No changes occurred, however, if either yeast or latex beads were used in place of bacteria . The changes involved the simultaneous loss of proteinases characteristic of the axenic cells (the A-forms) and the acquisition of those found in cells which have been grown on bacteria (the B-forms) . Using K . aerogenes the conversion was complete within 4 h . Extracellular proteinase activity was unaffected during this period . After the D . discoideum cells had been lysed, no equivalent change in proteinase band pattern could be produced either by prolonged incubation of cell extracts or by treatment with proteinases . An identical conversion could be induced in cultures of myxamoebae by a factor, cysteine proteinase converting factor (CPCF), present in the 15,000 g supernatant of a sonicated suspension of K . aerogenes . CPCF was macromolecular, as demonstrated by both ultrafiltration and gel filtration, acid-precipitable, but was soluble in ethanol or alkali . Its activity was unaffected by treatment with trypsin . The results suggested that CPCF might be a component of the bacterial cell wall, and since its activity was affected by lysozyme treatment, peptidoglycan is implicated . The results can be interpreted in terms of a novel nutrient-dependent post-translational change which affected most of the cysteine proteinases present in D . discoideum myxamoebae. Gene, 1988 Aug 15, 68(1), 11 - 22 Cloning and characterization of the gene for a methanol-utilising alcohol dehydrogenase from Bacillus stearothermophilus; Dowds BC et al.; The cloning and characterization of the alcohol dehydrogenase (ADH) gene (adh) from Bacillus stearothermophilus strain DSM2334, an obligate aerobe, are described . The clone directed the synthesis of ADH as judged on Western blots, activity gels and tetrazolium plates . It specified an enzyme that oxidised methanol as well as ethanol . The enzyme was found to be encoded by a single gene in B . stearothermophilus which did not cross-hybridize to adh clones from Escherichia coli, yeast or maize . The cloned gene was expressed in E . coli but activity was not detected in Bacillus subtilis, despite stable maintenance of the recombinant plasmid in this host . The gene is catabolite-repressed in DSM2334. J Biol Chem, 1988 Aug 5, 263(22), 10894 - 902 Detection of pur operon-attenuated mRNA and accumulated degradation intermediates in Bacillus subtilis; Ebbole DJ et al.; Transcription of the Bacillus subtilis pur operon is regulated independently by adenine and guanine nucleotides (Ebbole, D . J., and Zalkin, H . (1987) J . Biol . Chem . 262, 8274-8287) . Guanine nucleotides regulate transcription by a termination-antitermination mechanism in a 242-nucleotide, 5'-untranslated mRNA leader region . We have identified an apparently intact, terminated transcript of approximately 200 nucleotides in length, having a half-life of about 0.7 min . The terminated transcript is degraded in a series of discrete steps resulting in the accumulation of stable intermediates in vivo . We have used Northern blot analysis, primer extension, and nuclease S1 mapping to align the degradation intermediates with the nucleotide sequence and assign secondary structures that may contribute to the stability of the intermediates . Degradation is initiated by endonucleolytic cleavage of the approximately 200-nucleotide terminated transcript generating approximately 93- and approximately 97-nucleotide 5' and 3' moieties, respectively . The approximately 93-nucleotide 5' and approximately 97-nucleotide 3' intermediates are further degraded to approximately 88 and approximately 58 nucleotides, respectively . The 5'-end of pur operon mRNA and the attenuated transcript are degraded by different pathways. J Mol Biol, 1988 Aug 5, 202(3), 527 - 35 Single amino acid changes in the Bacillus thuringiensis var . israelensis delta-endotoxin affect the toxicity and expression of the protein; Ward ES et al.; Site-directed mutagenesis has been used to change individual amino acids of the larvicidal 27,000 Mr delta-endotoxin of Bacillus thuringiensis var . israelensis . Basic and acidic residues have been systematically replaced by alanine, and the resulting mutant polypeptides analysed for cytolytic and larvicidal activity, and binding to phosphatidyl choline liposomes . Replacement of residues at positions 154, 163, 164, 213 and 225 results in proteins which accumulate as inclusions in recombinant Bacillus subtilis cells similar to the wild-type, but have considerably reduced in-vitro and in-vivo toxicity . One mutant (Glu45 to Ala45) results in a protein that has reduced activity in vitro, but retains wild-type larvicidal toxicity . In addition, seven other mutations of charged residues result in proteins which form small or no inclusions in recombinant cells, despite being produced at levels similar to the wild-type in six out of seven cases . In most instances, the toxicity of these aberrantly expressed proteins is considerably less than the wild-type, although one (Lys124 to Ala124) results in a polypeptide with approximately threefold increased activity in vitro . A secondary structural model is proposed to explain these observations. Biochim Biophys Acta, 1988 Aug 4, 943(1), 13 - 8 Large decreases in membrane phosphatidylethanolamine and diphosphatidylglycerol upon mutation to duramycin resistance do not change the protonophore resistance of Bacillus subtilis; Dunkley EA Jr et al.; Duramycin-resistant mutant strains were selected from wild-type Bacillus subtilis (BD99) and its protonophore-resistant mutant derivative, strain AG1A3 . Analyses of the membranes of the duramycin-resistant mutants showed that they had little or no phosphatidylethanolamine and diphosphatidylglycerol as determined by chemical detection after thin-layer chromatography . Small amounts of these phospholipids must remain in the mutant strains, however, because during studies of incorporation of exogenous, radioactive fatty acids, label associated with palmitoleic acid was found in chromatographic positions that corresponded to the expected positions of phosphatidylethanolamine and diphosphatidylglycerol . The duramycin-resistant strains both showed elevated levels of phosphatidylglycerol and aminoacyl(lysyl)phosphatidylglycerol . The duramycin-resistant derivative of protonophore-resistant AG1A3 (AG1A3-DR4), but not that of the wild type, also showed a decreased content of neutral relative to polar lipid in the membrane . The composition of neutral lipid in that strain was higher in free fatty acids and lower in 1,2-diacylglycerol than its parent strain . AG1A3-DR4 also contained appreciable levels of lysophosphatidylethanolamine and somewhat elevated diglycosyldiacylglycerol relative to the other strains in the study . The protonophore resistance of AG1A3 was unaltered by mutation to duramycin resistance . Nor was there any change in the efficacy of exogenous palmitoleic acid in diminishing the protonophore resistance of AG1A3-DR4 . This phenomenon persists upon dramatic reduction in the content of phosphatidylethanolamine and diphosphatidylglycerol even though those phospholipids are normally the preferred sites of incorporation of the exogenous unsaturated fatty acids that mediate the effect. J Antibiot (Tokyo), 1988 Aug, 41(8), 1066 - 73 Metabolic products of microorganisms . 249 . Tetracenomycins B3 and D3, key intermediates of the elloramycin and tetracenomycin C biosynthesis; Rohr J et al.; Tetracenomycins B3 and D3, besides tetracenomycin D (D1), were produced by a blocked mutant of the elloramycin producer Streptomyces olivaceus TU 2353 . The compounds were isolated as red powders, and their structures were elucidated by comparing their physicochemical data with those of the known tetracenomycins A2, B1, B2, D and E . Tetracenomycin B3 (2), the main compound, and tetracenomycin D (3) were antibiotically inactive against Gram-positive and Gram-negative bacteria, whereas tetracenomycin D3 (1) showed a moderate activity against Bacillus subtilis and Arthrobacter aurescens . Tetracenomycin B3 (2) is the key intermediate where the biosynthesis of the elloramycins branches off from the line leading to tetracenomycin C (5) as the final product of the tetracenomycin biosynthesis branch. J Gen Microbiol, 1988 Aug, 134 ( Pt 8), 2333 - 44 Mutants of Bacillus subtilis defective in protein export; Kontinen VP et al.; We have isolated a set of strains with mutations (designated prs) that decrease secretion of alpha-amylase and have a pleiotropic effect on secretion of other exoproteins . The seven mutants were selected in a strain of Bacillus subtilis which overproduces alpha-amylase due to the presence of an alpha-amylase gene on a multicopy plasmid . The mutations were mapped to four different chromosomal loci . The phenotype of the mutants, especially their pleiotropic effects and the accumulation of alpha-amylase precursor, indicated that they have defects in the mechanism of protein export . Double mutants with certain pairwise combinations of mutations in different loci had additive effects on secretion, suggesting that these prs genes encode different components of the secretion pathway. J Gen Microbiol, 1988 Aug, 134 ( Pt 8), 2095 - 101 The isolation of strains of Bacillus subtilis showing improved plasmid stability characteristics by means of selective chemostat culture; Fleming G et al.; A pUB110-derived plasmid encoding chloramphenicol resistance, kanamycin resistance and high-temperature alpha-amylase showed a high degree of segregational instability when inserted into Bacillus subtilis . In an attempt to obtain stable derivatives, the organism was grown in chemostat culture in the presence of chlorampheniol . It was periodically found necessary to increase the concentration of chloramphenicol in the medium feed in order to avoid plasmid loss . Strains were isolated after 19 and 160 generations, which showed high levels of plasmid stability . This characteristic appeared to be genotypic . No detectable difference in plasmid copy number was found between the original and the improved strains . The stability characteristics resided in the host, rather than in the plasmid . Stable isolates possessed elevated MICs for both chloramphenicol and kanamycin . Their maximum specific growth rates were higher than that of the original strain, and similar to that of the plasmid-free parent strain. Tohoku J Exp Med, 1988 Aug, 155(4), 385 - 6 Activity of agricultural chemicals to modify mitomycin C induced growth inhibition of Bacillus subtilis in the rec assay; Yamamoto M et al.; Rec assay of agricultural chemicals, MCPA-E, MCPA, CNP and PCNB was conducted . It was found that MCPA-E and MCPA accelerated the growth-inhibition originall