Proc Natl Acad Sci U S A, 1983 Jul, 80(13), 4074 - 8 Isolation and physical mapping of the gene encoding the major sigma factor of Bacillus subtilis RNA polymerase; Price CW et al.; At least four sigma factors separately bind the Bacillus subtilis RNA polymerase core (beta beta' alpha 2), each conferring a different promoter specificity on the holoenzyme in vitro . Using the Broome-Gilbert immunological screening, we isolated recombinant lambda phages that carry rpoD, the gene for the most abundant sigma factor, sigma 55 . These phages encode a 55,000-dalton protein whose size, immunological properties, and peptide map identify it as sigma 55 . All the phages have in common two adjacent 3.5-kilobase EcoRI fragments from the B . subtilis chromosome; most carry additional genomic DNA . Deletion analysis localized rpoD to a 1.6-kilobase region, suggested the direction of its transcription, and found two additional genes near rpoD, which code for proteins of 62,000 and 17,000 daltons. J Bacteriol, 1983 Jul, 155(1), 302 - 10 Teichoicase from Bacillus subtilis Marburg; Kusser W et al.; The properties of a teichoic acid degrading enzyme (teichoicase) isolated from Bacillus subtilis Marburg are described . The purified enzyme showed phosphodiesterase activity but not phosphomonoesterase activity, and it had an absolute substrate specificity for alpha-glucosylated glycerol teichoic acid, the endogenous cell wall teichoic acid of the enzyme-producing cell . The substrate was degraded by an exo-mechanism yielding the monomer alpha-D-glucose 1 leads to 2 (sn)glycero-3-phosphate . When B . subtilis Marburg was grown in a rich medium, enzyme activity was detected in extracts from sporulating cells . Teichoicase activity was present in a mutant blocked in stage II of the sporulation process but was absent in a mutant blocked in stage O . It was concluded that teichoicase is active on enzyme-producing cells since the reaction product could be detected in their culture supernatant . Attempts to demonstrate analogous enzyme activity in other Bacillus strains failed . The enzyme could be used for the rapid detection of alpha-glucosylated glycerol teichoic acid and for the controlled alteration of native bacterial cell surfaces exhibiting the appropriate structure. Biochem Biophys Res Commun, 1983 Jun 29, 113(3), 772 - 7 Acetylation of puromycin by Streptomyces alboniger the producing organism; Perez-Gonzalez JA et al.; Streptomyces alboniger produces the antibiotic puromycin and expresses an enzymic activity which acetylates the drug using acetyl CoA . The N-acetyl-puromycin formed is biologically inactive against protein synthesis in Bacillus subtilis (as assayed in vivo). Nucleic Acids Res, 1983 Jun 25, 11(12), 4157 - 75 Regions specifying transcriptional termination and pausing in the bacteriophage SP01 terminal repeat; Brennan SM et al.; We have determined the nucleotide sequences of four termination sites recognized by Bacillus subtilis RNA polymerase . These sites are located in the terminally repeated segment of the bacteriophage SP01 genome, where most early phage transcription occurs . The SP01 terminators have structures that are similar to those recognized by Escherichia coli RNA polymerase, containing a region of dyad symmetry followed by a stretch of HMU residues in the noncoding DNA strand (HMU is substituted for T in SP01 DNA) . We note that in a terminator that is only 60% efficient in vitro, there is a greater distance between these two conserved elements that exists in more efficient terminators . We also find that RNA polymerase molecules which elongate a transcript through a partial terminator often pause at this site. Biochim Biophys Acta, 1983 Jun 24, 740(2), 127 - 33 Similarity of genetic distance determined from DNA thermal denaturation profiles to standard estimates of bacteriophage relatedness; Ansevin AT et al.; High resolution thermal denaturation profiles of a series of potentially related bacteriophages of Bacillus subtilis contain a multitude of distinctive features that permit them to be divided into at least four groups . In addition, the profiles can be used to derive the quantitative parameter, genetic distance, defined by Soumpasis (Soumpasis, D . (1980) J . Theor . Biol . 86, 137-147) . Both the quantitative and the qualitative intercomparisons of profiles are in general agreement with the results of standard techniques for estimating genetic relatedness. J Dairy Sci, 1983 Jun, 66(6), 1228 - 31 Aerobic mesophilic and psychrotrophic sporeforming bacteria in buffalo milk; Shehata AE et al.; Seasonal variation of the population of aerobic sporeformers in raw milk was higher in summer than in other seasons . Least variation was in fall, but variation in winter and spring was similar . Aerobic mesophilic sporeformers in raw milk consisted mainly of Bacillus subtilis (42.5%) and Bacillus megaterium (34.8%), followed by Bacillus circulans (4.9%), Bacillus cereus (4.6%), Bacillus pumilus (2.9%), Bacillus polymyxa (2.8%), Bacillus licheniformis (1.9%), Bacillus badius (1.5%), Bacillus brevis (1.3%), Bacillus pulvifaciens (1.2%), Bacillus coagulans (1.1%), and Bacillus firmus (.5%) . Comparing these findings with those previously obtained for the same area reveal noticeable variations . The psychrotrophic Bacillus strains were cereus (42.6%), pumilus (31.9%), badius (12.8%), licheniformis (10.6%), and firmus (2.1%). J Gen Microbiol, 1983 Jun, 129 (Pt 6), 1945 - 58 Altered arrangement of proteins in the spore coat of a germination mutant of Bacillus subtilis; Jenkinson HF; Spores produced by a mutant of Bacillus subtilis were slow to develop their resistance properties during sporulation, and were slower to germinate than were wild-type spores . The coat protein composition of the mutant spores, as analysed by SDS-PAGE, was similar to that of the wild-type spores . However, one of the proteins (mol . wt 12000) which is normally present in the outer-most layers of mature wild-type spores and which is surface-exposed, was assembled abnormally into the coat of the mutant spores and not surface-exposed . The mutation responsible for this phenotype (spo-520) has been mapped between pheA and leuB on the B . subtilis chromosome, and was 47% cotransformable with leuB16 . This mutation, and three others closely linked to it, define a new sporulation locus, spoVIB, which is involved in spore coat assembly . The phenotype of the mutant(s) supports the contention that spore germination and resistance properties may be determined by the assembly of the coat. Appl Environ Microbiol, 1983 Jun, 45(6), 1949 - 52 Rapid assay for detection of microorganisms producing DNA-damaging metabolites; Mazza G; Microorganisms producing DNA-damaging metabolites (i.e., fungi and streptomycetes) were detected by the Bacillus subtilis rec assay with agar plugs from plates on which the microorganisms had been grown . This assay allowed rapid identification of aflatoxinogenic fungi and streptomycetes producing strong DNA-damaging metabolites . For screening programs, several media have to be used to grow the microorganisms to be tested. Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3287 - 91 A 64-kilodalton membrane protein of Bacillus subtilis covered by secreting ribosomes; Horiuchi S et al.; The complexed (ribosome-bearing) membrane fraction of Bacillus subtilis contains several proteins (CM-proteins) that are virtually absent from the ribosome-free fraction and hence might be components of the apparatus of protein secretion . We have determined, by trypsin digestion and by labeling with a nonpenetrating reagent (diazoiodosulfanilic acid), the accessibility of four of these proteins on the two surfaces of the membrane, as exposed either in protoplasts or in inverted membrane vesicles . The 68-kilodalton protein is a transmembrane protein and the 45-kilodalton protein faces only the external surface, whereas the 31-kilodalton protein is inaccessible from either side . Of particular interest is the 64-kilodalton protein: it can be digested by trypsin, and can bind antibody, on the cytoplasmic surface, but only after the ribosomes have been released . This protein is thus evidently a component of the apparatus of protein secretion, closely covered by secreting ribosomes . Whether the other CM-proteins are also involved in protein secretion is uncertain. J Virol, 1983 Jun, 46(3), 703 - 8 Resistance of bacteriophage H1 to restriction and modification by Bacillus subtilis R; Bron S et al.; H1, a 5-hydroxymethyluracil (HMU)-containing Bacillus subtilis bacteriophage, was neither restricted nor modified upon infection of B . subtilis R cells . In vitro, H1 DNA was not restricted by BsuR under standard conditions (200 mM salt), although the expected frequency of -GGCC- cleavage sites was approximately 250 . However, four specific sites were cleaved under nonstandard conditions (low salt or high pH) or in the presence of organic solvents, like dimethyl sulfoxide and glycerol . After the substitution of thymine for HMU by DNA cloning in B . subtilis, a BsuR cleavage site was restricted and modified under standard conditions . No additional sites were detected after shotgun-cloning of about 11% of the chromosome . The nucleotide sequence of a cleavage site was found to be 5' . .C-A-Hmu-A-A-C-Hmu-Hmu-Hmu-G-G-C-C-Hmu-A-G- . . .3', which shows the presence of a bona fide BsuR (GGCC) recognition sequence, flanked by (Hmu-A)-rich sequences . The results suggested that the resistance of H1 to restriction and modification by B . subtilis R was due to (i) a strong bias against the GGCC-recognition sequence and (ii) protection of the four remaining GGCC sites as a consequence of HMU-A base pairs flanking the sites. J Virol, 1983 Jun, 46(3), 690 - 702 In vitro synthesis of late bacteriophage phi 29 RNA; Holder RD et al.; A crude P-100 fraction prepared from Bacillus subtilis 21 min after infection with wild-type phage phi 29 supported the in vitro synthesis of late phi 29 RNA by added RNA polymerase . Synthesis of late RNA was also detected when purified phi 29 DNA was transcribed by RNA polymerase in the presence of an S-150 fraction obtained by lysis of phi 29-infected cells in the presence of 1 M NaCl . Late phi 29 RNA was not synthesized when either the P-100 or the S-150 fraction was prepared from cultures infected with phi 29 having a mutation in gene 4. J Bacteriol, 1983 Jun, 154(3), 1381 - 8 Proteins of ribosome-bearing and free-membrane domains in Bacillus subtilis; Marty-Mazars D et al.; In lysates of Bacillus subtilis a free-membrane fraction without ribosomes can be separated from the denser membrane-ribosome complexes . As determined by one-dimensional sodium dodecyl sulfate gel electrophoresis, these two fractions differ markedly in protein composition; at least six major bands (molecular weights, 130,000, 92,000, 68,000, 64,000, 45,000, and 31,000) are essentially unique to the complexed-membrane fraction (CM proteins), and two are unique to the free-membrane fraction . After growth was slowed, the proportion of the free-membrane fraction increased, but the composition of this fraction was the same, whereas after puromycin treatment, which abruptly increased the proportion of the free-membrane fraction, this fraction contained CM proteins . Thus, it appears that the two fractions recovered from growing cells represent topographically and functionally distinct domains . In addition, the effect of growth rate suggests that formation of the complexed domain is regulated at least roughly in parallel with the formation of ribosomes . The separation of these membrane fractions should facilitate the study of protein secretion, membrane topography, and morphogenesis in bacteria. J Bacteriol, 1983 Jun, 154(3), 1215 - 21 Localization and quantitation of proteins characteristic of the complexed membrane of Bacillus subtilis; Horiuchi S et al.; We prepared antibodies to four proteins (molecular weights, 68,000, 64,000, 45,000, and 31,000) that are characteristic of the complexed (ribosome-bearing) fraction of the membrane of Bacillus subtilis and found that these proteins are immunologically distinct . Quantitation by immunoprecipitation confirmed that the ribosome-free membrane fraction contains much lower concentrations of these four proteins than the complexed-membrane fraction . The 64-kilodalton protein appeared to be attached more loosely than the other proteins, since it was more readily extracted from the membrane . In addition, this protein was also present in the cytosol in an even greater amount than in the membrane . The 68-, 64-, and 31-kilodalton proteins are present in cells in stoichiometrically equivalent amounts. J Bacteriol, 1983 Jun, 154(3), 1184 - 94 Site-specific in vitro binding of plasmid pUB110 to Bacillus subtilis membrane fraction; Tanaka T et al.; The in vitro membrane binding of pSL103, a composite plasmid consisting of Staphylococcus aureus plasmid pUB110 and a Bacillus pumilus trpC+ DNA fragment, to the Bacillus subtilis membrane fraction was studied with a total lysate of B . subtilis cells . The binding reaction required a heat treatment at 45 degrees C and had an optimum KCl concentration of 60 mM . Nonradioactive pSL103, but not Escherichia coli plasmid pACYC184, competed with 3H-labeled pSL103 for binding to the membrane . By the use of 32P-labeled restriction fragments of pSL103 and pUB110, it has been found that only the pUB110 portion of pSL103 binds to the membrane and that there are four specific regions in pUB110 which bind to the membrane . Two of the four binding regions flank the replication origin . This in vitro binding was high-salt sensitive and apparently independent of the configurations of the plasmid . We have previously shown that the functional product of the initiation gene dna-1 is required in vivo both for replication initiation and the binding of a DNA region near the replication origin to the membrane . Unlike in vivo binding, which is high-salt resistant and dependent on the product of dna-1 gene (type-I binding), the in vitro binding reported in this paper was high-salt sensitive and independent of the dna-1 gene product (type-II binding). J Bacteriol, 1983 Jun, 154(3), 1077 - 87 Plasmid marker rescue transformation in Bacillus subtilis; Weinrauch Y et al.; We constructed an 18-megadalton plasmid (pBD221) carrying resistance determinants for kanamycin, chloramphenicol, and erythromycin, as well as the hisH determinant from the Bacillus licheniformis chromosome . This plasmid has a copy number of about one and can be stably maintained in Bacillus subtilis . Linear fragments of pBD221 DNA were used to transform competent cultures carrying mutant variants of the same plasmid . Rescue transformation did not proceed by recircularization and replication of the donor DNA . Rescue transformation exhibited first-order dependence on DNA concentration, and the concentration dependence curve was virtually identical to the curve obtained with chromosomal DNA . The donor DNA molecular weight dependence of plasmid marker rescue transformation obtained by using restriction fragments was not distinguishable from previously published data obtained by using fractionated sheared chromosomal DNA . Plasmid rescue transformation, like chromosomal transformation, was dependent on the recE, recA, recB, and recD gene products . Plasmid rescue transformation, like chromosomal transformation, proceeded with few exchanges . Linkage data obtained with the plasmid rescue system fit a quantitative model based on studies with chromosomal transformation . We conclude that plasmid marker rescue transformation probably proceeds by a mechanism similar to the mechanism used during the formation of chromosomal transformants and hence may be considered an appropriate general model for the study of transformational recombination. Antimicrob Agents Chemother, 1983 Jun, 23(6), 835 - 45 Roles of ribosomal binding, membrane potential, and electron transport in bacterial uptake of streptomycin and gentamicin; Bryan LE et al.; The effects of a set of conditions on aminoglycoside uptake were determined . Membrane vesicles either with a membrane potential (delta psi) of -125 mV (adequate to drive lysine uptake) or with succinate, lactate, or phenazine methosulfate did not accumulate gentamicin unless components of protein synthesis were included . Ribosomally resistant (rpsL) Escherichia coli cells demonstrated energy-dependent phase II uptake similar to that of a streptomycin-susceptible strain of E . coli when treated with 100 micrograms of puromycin per ml . Puromycin (100 micrograms/ml) also increased the uptake of the cationic compounds polyamine and arginine . These studies support a role of protein synthesis in aminoglycoside uptake and in the development of energy-dependent phase II . delta psi of cells did not increase either at the initiation of or during energy-dependent phase II, showing that energy-dependent phase II is not due to an elevation of delta psi . In a Bacillus subtilis system, significant streptomycin uptake requires a threshold value of delta psi which varies depending upon the concentration of streptomycin used . At 25 micrograms/ml, the uptake of streptomycin reached maximal levels after exceeding the threshold value, whereas at 100 micrograms/ml there was a gradual increase of the uptake to the maximal after the threshold value was exceeded . Several studies supported the view that electron transport has a specific role other than its requirement to produce the cellular delta psi . The uptake of gentamicin was stimulated to a greater extent by phenazine methosulfate-ascorbate than by the ionophore nigericin in strains of E . coli, although nigericin stimulated delta psi to a greater degree . Cells with 25% of the normal quinone concentration had delta psi values identical to cells with the normal quinone concentration, but the quinone-deficient cells had a significantly lower rate of gentamicin uptake . KCN prevented gentamicin uptake but did not prevent the development of delta psi . The effects of ubiquinone depletion in an E . coli strain were more evident on gentamicin uptake than on ATP-driven glutamine transport or proton motive force-driven proline transport, consistent with a specific requirement for quinones in aminoglycoside uptake . A detailed explanation of the mechanism of accumulation of streptomycin and gentamicin and a proposed mechanism for killing bacterial cells by these agents have been provided. Arch Microbiol, 1983 Jun, 134(3), 222 - 6 Activation and inactivation of synthesis of secondary wall polymers in Bacillus subtilis W23; Hancock IC; The progress of activation and inactivation of synthesis of the wall polymers, teichoic acid and teichuronic acid, in response to changes in the phosphate content of the growth medium has been examined using toluenised cells of B . subtilis W23 . Activation of teichoic acid synthesis from nucleotide precursors was independent of protein synthesis, but chloramphenicol prevented activation when DL-glycerol 3-phosphate and CTP replaced CDP-glycerol as one of the substrates of the reaction . Activation of teichuronic acid synthesis was dependent on synthesis of protein . Inactivation of synthesis of both polymers was slowed, but not prevented, by inhibition of protein synthesis . Evidence was obtained that a protein synthesised during phosphate starvation retards the activation of teichoic acid synthesis. Genetika, 1983 Jun, 19(6), 881 - 7 {Cloning of Bacillus subtilis 168 genes compensating for the defect in mutations for thymidine phosphorylase and uridine phosphorylase in Escherichia coli cells}; Maznitsa II et al.; On the basis of Bacillus subtilis DNA and vectors pBR325, pBR322, a collection of hybrid plasmids restoring the wild type in the double Escherichia coli mutant for thymidine phosphorylase (deoA) and uridine phosphorylase (udp) has been obtained . In spite of the fact that in Bac . subtilis, in contrast to E . coli, phosphorolytic cleavage of thymidine and uridine is catabolized by one enzyme-pyrimidine nucleoside phosphorylase (pdp gene), some plasmids caused the appearance of the activity similar to thymidine phosphorylase (deoA type) in E . coli cells . Plasmids of the deoA type as well as many of plasmids causing the appearance in E . coli cells of the activity of the pdp type were not able to transform cells of the Bac . subtilis pdp mutant, strain PC315, to the wild type . Moreover, on the basis of PC315 DNA, hybrid plasmids have been obtained giving the activity of the pdp type . The suggestion of the possible presence in Bac . subtilis 168 genome of non-expressible cryptic genes of the deoA and pdp type is discussed. J Antibiot (Tokyo), 1983 Jun, 36(6), 674 - 8 Acylpeptides, the inhibitors of cyclic adenosine 3',5'-monophosphate phosphodiesterase . II . Amino acid sequence and location of lactone linkage; Hosono K et al.; Bacillus subtilis C-756 produced three kinds of inhibitors of cyclic adenosine 3',5'-monophosphate (cAMP) phosphodiesterase . Each was an acylpeptide consisting of a beta-hydroxy fatty acid residue and heptapeptide . By the application of mass spectrometry, the amino acid sequence of peptide was determined to be beta-hydroxy fatty acid-Glu-Leu-Leu-Val-Asp-Leu-Leu in all three cases . Each had a lactone linkage between the carboxyl group of C-terminal leucine and the beta-hydroxyl group of the fatty acid moiety . The total structures of these inhibitors were thus established. J Antibiot (Tokyo), 1983 Jun, 36(6), 667 - 73 Acylpeptides, the inhibitors of cyclic adenosine 3',5'-monophosphate phosphodiesterase . I . Purification, physicochemical properties and structures of fatty acid residues; Hosono K et al.; An inhibitor of cyclic adenosine 3',5'-monophosphate (cAMP) phosphodiesterase was isolated from the culture filtrate of Bacillus subtilis C-756 isolated from soil . It was purified and finally separated into three fractions by reverse-phase HPLC . The respective fractions were designated as APD-I, -II and -III in the order eluted and the relative quantities of APD-I, -II and -III were approximately 10%, 40% and 50%, respectively . They were acylpeptides composed of beta-hydroxy fatty acid residues and heptapeptide . Though the amino acid compositions of the peptides were the same, the fatty acid residues were all different . APD-I contained a mixture of 3-hydroxy-11-methyldodecanoic acid (i-C13h3) and 3-hydroxy-10-methyldodecanoic acid (a-C13h3) . APD-II contained 3-hydroxytetradecanoic acid (n-C14h3) . APD-III contained a mixture of 3-hydroxy-13-methyltetradecanoic acid (i-C15h3) and 3-hydroxy-12-methyltetradecanoic acid (a-C15h3). J Bacteriol, 1983 Jun, 154(3), 1513 - 5 Construction and properties of an integrable plasmid for Bacillus subtilis; Ferrari FA et al.; A plasmid useful for locating the chromosomal site of cloned DNA fragments that lack intrinsic genetic activity, for insertional mutagenesis, for single-copy complementation, and for dominance studies was constructed . Some plasmids containing Bacillus subtilis DNA were only active in transformation when the tetracycline resistance determinant of the plasmid was inactivated . The results suggest that production of the tetracycline gene product is lethal to B . subtilis. J Bacteriol, 1983 Jun, 154(3), 1389 - 96 Identification of specific restriction fragments associated with a membrane subparticle from Bacillus subtilis; Sargent MG et al.; When lysates of Bacillus subtilis were treated with restriction endonucleases EcoRI or HindIII, almost all of the DNA was released from the major plasma membrane fraction that was sedimentable at low speed . However, a very small part of the released DNA, when centrifuged at high speed, appeared to be bound to small membrane fragments . On agarose gels, this material, prepared with either enzyme, contained only a small number of restriction fragments, and the DNA in the sample hybridized with 11 to 12 EcoRI or HindIII fragments of chromosomal DNA . This DNA was used after nick-translation to screen Charon 4A clone banks for phages containing membrane-bound fragments . One of these was studied in detail . Only a part (about 5 kilobases) of the region present in this clone is important in binding the DNA to the membrane subparticle. J Bacteriol, 1983 Jun, 154(3), 1088 - 97 Genetics of Bacillus subtilis chemotaxis: isolation and mapping of mutations and cloning of chemotaxis genes; Ordal GW et al.; We isolated a collection of chemotaxis mutants and characterized them for chemotactic phenotype and genotype . The mutations of most of these mutants mapped in the region between pyrD and thyA . However, the mutation in the gene specifying the chemotactic methyltransferase mapped very close to aroF . From a bank of phages containing Bacillus subtilis DNA we identified two lambda charon4 phages that contained genes specifying chemotactic functions . The inserted DNAs were removed by digestion with restriction endonuclease EcoRI and were found to share a 4.0-kilobase (kb) fragment . One of these DNAs also contained a 7.7-kb fragment, and the other also contained a 10.9-kb fragment . We identified mutants that were complemented by each fragment . The fragments were each ligated into plasmid pFH7 and were incorporated into lysogenic SP beta c2 or a deletion mutant of SP beta c2 in order to form transducing phages . The mutants in the collection containing mutations that mapped in the region between pyrD and thyA were tested for complementation by transducing phages containing the 4.0-kb fragment, the 7.7-kb fragment, the 4.0-kb fragment plus the 7.7-kb fragment, and the 10.9-kb fragment . A total of 24 mutants were complemented by the 4.0-kb fragment, 7 were complemented by the 7.7-kb fragment, 9 were complemented by the 4.0-kb fragment plus the 7.7-kb fragment, 15 were complemented by the 10.9-kb fragment, and 25 were complemented by none of the fragments. Nucleic Acids Res, 1983 May 25, 11(10), 3037 - 45 The nucleotide sequence of Bacillus subtilis tRNA genes; Yamada Y et al.; Clones carring Bacillus subtilis tRNA genes were isolated from a lambda 816 library . A recombinant phage lambda 816-BS83 which was hybridized effectively with unfractionated tRNA probes contained a 3-kb fragment . By a Southern's blot analysis, it was found that tRNA genes were located in Eco RI-Hinc II region of this fragment . Sequence determination revealed the presence of a cluster of four tRNA genes in this region . The gene organization was as follows: tDNALys-9bp-tDNAGlu-81bp-tDNAAsp-30bp-tDNAPhe . The RNA sequences expected from tDNALys and tDNAPhe were identical with the reported RNA sequences . Two tRNA genes, tDNALys and tDNAAsp encoded the CCA sequence of 3'-terminal region, but the other two, tDNAGlu and tDNAPhe did not . A promoter-like sequence which corresponds to the sigma 55-recognition site was found in a region about 100bp upstream from tDNALys. Eur J Biochem, 1983 May 16, 132(3), 589 - 93 Succinate dehydrogenase mutants of Bacillus subtilis lacking covalently bound flavin in the flavoprotein subunit; Hederstedt L; Succinate dehydrogenase consists of two unequal subunits; Fp and Ip . An FAD group is covalently linked to a histidyl residue in the Fp subunit . The mechanism by which flavin is attached to protein is not known . Covalently bound flavin was studied in wild-type and succinate-dehydrogenase-negative Bacillus subtilis . The Fp subunit of succinate dehydrogenase was found to be the only (major) flavinylated protein in the cell . Mutants lacking covalently bound flavin and still containing the Fp polypeptide are described . It is shown that the flavin is not essential for assembly and membrane binding of succinate dehydrogenase in B . subtilis. Antimicrob Agents Chemother, 1983 May, 23(5), 703 - 5 Lack of influence of commonly used drugs on bioassay indicator organisms; DiPiro JT et al.; Many commonly used pharmaceutical agents have been found to inhibit bacterial growth in vitro . Determinations of antimicrobial concentrations in sera of patients taking nonrecognized antibacterial agents could possibly be altered if bioassay systems are utilized for the determinations . We therefore attempted to determine the in vitro effect of commonly used drugs on bioassay indicator organisms . Fifty-one different agents (antihistamines, anticholinergics, central nervous system agents, cardiovascular agents, analgesics, steroids, muscle blockers, and other miscellaneous agents) were tested for inhibition or enhancement of the growth of Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus, and Klebsiella pneumoniae . None of the agents tested exhibited any effect on standard in vitro bioassay organisms . Nortriptyline hydrochloride inhibited the growth of B . subtilis and M . luteus at a concentration of 500 micrograms/ml (zones of inhibition, 14 and 13 mm, respectively), but no inhibition was observed with concentrations of 50 micrograms/ml or lower. J Antibiot (Tokyo), 1983 May, 36(5), 463 - 7 DC-52, a novel antitumor antibiotic . 1 . Taxonomy, fermentation and biological activity; Tomita F et al.; A novel antitumor antibiotic, DC-52 was found in the culture broths of Actinomycete DO-52 . The producing organism was subsequently determined to be a new species and named Streptomyces melanovinaceus nov . sp . For the production of the antibiotic, soluble starch served as a good carbon source and soybean meal was a good nitrogen source tested . The antibiotic DC-52 is active against Bacillus subtilis, Staphylococcus aureus and Klebsiella pneumoniae, but not active against most Gram-negative bacteria . The antibiotic is also active against mouse leukemia P388. Genetika, 1983 May, 19(5), 737 - 43 {Role of the ribosomes in controlling cell differentiation and secondary metabolism in sporulating bacteria . II . The suppression of the phenotypic expression of ribosomal mutations (strA) as affected by RNA-polymerase mutations (rfm) in Bacillus subtilis}; Lukin AA et al.; Ribosomal mutants of Bacillus subtilis IG2 and IG7 resistant to 100 mkg/ml of streptomycin have been isolated . The strA80 and strA88 mutations reduce the level of sporulation as well as antibiotic and proteolytic activity of bacteria . RNA polymerase mutations rfm20 and rfm146 transferred by transformation into ribosomal mutants suppress strA80 and strA88 mutations . As a result, sporulation was restored . Both mutations were cotransformed with cysA gene and mapped within the cluster of ribosomal and RNA polymerase genes . It is proposed that the ribosomal strA80 and strA88 mutations influence transcription of gene (s) which regulate initiation of sporulation and that the RNA polymerase rfm20 and rfm146 mutations can restore sporulation . The results suggest that ribosomes are most likely to be not able to play a specific role in sporulation and secondary metabolism of Bac . subtilis. Biochem J, 1983 May 1, 211(2), 463 - 72 Wild-type and mutant forms of the pyruvate dehydrogenase multienzyme complex from Bacillus subtilis; Hodgson JA et al.; A simple procedure is described for the purification of the pyruvate dehydrogenase complex and dihydrolipoamide dehydrogenase from Bacillus subtilis . The method is rapid and applicable to small quantities of bacterial cells . The purified pyruvate dehydrogenase complex (s0(20),w = 73S) comprises multiple copies of four different types of polypeptide chain, with apparent Mr values of 59 500, 55 000, 42 500 and 36 000: these were identified as the polypeptide chains of the lipoate acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and the two types of subunit of the pyruvate decarboxylase (E1) components respectively . Pyruvate dehydrogenase complexes were also purified from two ace (acetate-requiring) mutants of B . subtilis . That from mutant 61142 was found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification . Subunit-exchange experiments demonstrated that the E2E3 subcomplex retained full enzymic activity, suggesting that the lesion was limited to the E1 component . Mutant 61141R elaborated a functional pyruvate dehydrogenase complex, but this also contained a defective E1 component, the Km for pyruvate being raised from 0.4 mM to 4.3 mM . The E1 component rapidly dissociated from the E2E3 subcomplex at low temperature (0-4 degrees C), leaving an E2E3 subcomplex which by subunit-exchange experiments was judged to retain full enzymic activity . These ace mutants provide interesting opportunities to analyse defects in the self-assembly and catalytic activity of the pyruvate dehydrogenase complex. Plasmid, 1983 May, 9(3), 273 - 85 Properties of thyP3-containing plasmids in Bacillus subtilis; Barstow DA et al.; A series of hybrid plasmid molecules which contain both antibiotic resistance genes and the thyP3 gene of the Bacillus subtilis bacteriophage phi 3T have been constructed . Monomeric or restriction enzyme-cleaved plasmid DNA is capable of transforming competent cells to thymine prototrophy only . However, multimeric plasmid DNA can transform competent cells to both thymine prototrophy and antibiotic resistance . Cells which have been transformed to thymine prototrophy only do not contain extrachromosomal plasmid DNA but instead contain the thyP3 gene integrated into the host chromosome; the antibiotic resistance genes, however, do not become integrated into the chromosome . Although the thyP3-containing plasmids have extensive DNA sequence homology with the B . subtilis chromosome, they can be stably maintained, extrachromosomally, even in recE4+ hosts, in complex broth, and in the absence of antibiotics. J Bacteriol, 1983 May, 154(2), 537 - 46 Timing and other features of the action of the ts1 division initiation gene product of Bacillus subtilis; Callister H et al.; The ts1 division initiation mutation of Bacillus subtilis 160 was transferred into a thymine-requiring strain of B . subtilis 168 . Aspects of the role and timing of the action of the ts1 gene product in relation to septum formation were studied by comparing the behavior of this new strain with that of the isogenic wild type after outgrowth of germinated spores . The ts1 gene product was shown to be required for the asymmetric division which occurs in the absence of chromosome replication, in addition to normal division septation . The time interval between completion of the action of the ts1 gene product and initiation of the first central division septum was estimated to be less than 4 min at 34 degrees C, and it is possible that an active ts1 gene product is required until the commencement of septal growth . Recovery of septa after transfer of outgrown spores (filaments) from the nonpermissive to the permissive temperature was also examined . During recovery, septa formed at sites which were discrete fractional lengths of the filaments, with the first septum located at the most polar of these sites . The data have been interpreted in terms of the formation of potential division sites at the nonpermissive temperature and the preferred utilization, upon recovery, of the most recently formed site . Recovery of septa at the permissive temperature occurred in the absence of DNA synthesis but was blocked completely by inhibitors of RNA and protein synthesis . It is possible that the only protein synthesis required for recovery of septa is that of the ts1 gene product itself. Antimicrob Agents Chemother, 1983 May, 23(5), 683 - 7 Automated fluorescence polarization immunoassay for monitoring streptomycin; Schwenzer KS et al.; The fluorescence polarization immunoassay technique has been used previously for the assay of vancomycin and the aminoglycoside antibiotics (gentamicin, tobramycin, and amikacin) . We extended this technique to assay streptomycin . Fluorescein-labeled streptomycin was used as the tracer, and antiserum specific for streptomycin was raised in rabbits by conventional procedures . Tracer, sample, and diluted antiserum were combined, and the polarization of tracer fluorescence was determined in a specially designed fluorometer (Abbott TDx) . Because of the instrument design, the possibility of fluorescent interferences was eliminated . The coefficient of variation within run was 4% (n = 5), and between run it was 5% (n = 5) . We compared the fluorescence polarization immunoassay technique (TDx streptomycin) with a conventional bioassay, using Bacillus subtilis for clinical specimens (n = 39) . A least-squares linear regression analysis gave a slope of 1.16, an intercept of 2.41 mg/liter, and a correlation coefficient of 0.885 . Repeat analyses by both techniques showed that the largest discrepancies could be explained by the imprecision of the bioassay. J Gen Microbiol, 1983 May, 129 (Pt 5), 1497 - 512 The mechanism of insertion of a segment of heterologous DNA into the chromosome of Bacillus subtilis; Young M; An Escherichia coli plasmid, p1949, that is derived from pMB9 and pC194, and unable to replicate in Bacillus subtilis, can give rise to stable CmR transformants of the latter species if it is inserted into the bacterial chromosome . A purified segment of the B . subtilis chromosome, with transforming activity against pheA1 and nic-38 recipients, was used to direct the insertion of p1949 into the B . subtilis chromosome . Insertion of the ligated DNA segments occurred in the region of the chromosome from which the purified phe-nic segment was derived . Many of the properties of the resulting CmR transformants of B . subtilis are consistent with the occurrence of a Campbell recombination mechanism leading to integration . However, certain of these properties are more easily explained if it is proposed that integration occurs by a reciprocal recombination event involving a linear ligation product . Evidence is presented suggesting that the inserted sequences may be tandemly duplicated . This may effectively vitiate the use of p1949 as a convenient means for complementation analysis of recessive mutations in B . subtilis. Gene, 1983 May-Jun, 22(2-3), 229 - 35 Secretion of interferon by Bacillus subtilis; Palva I et al.; Bacillus subtilis was transformed with a hybrid gene in which the sequence encoding the alpha-amylase signal peptide was joined by a linker to the sequence encoding mature human interferon alpha 2(IFN-alpha 2) . The hybrid preprotein was cleaved precisely following the last amino acid of the alpha-amylase signal sequence and was secreted at 0.5--1 mg per liter . IFN-alpha 2, preceded by either one or six amino acids, has the same specific antiviral activity as IFN-alpha 2 itself. J Virol, 1983 May, 46(2), 446 - 53 Restriction and modification in Bacillus subtilis: DNA methylation potential of the related bacteriophages Z, SPR, SP beta, phi 3T, and rho 11; Noyer-Weidner M et al.; The DNA methylation capacity and some other properties of the related temperate Bacillus subtilis phages Z, SPR, SP beta, phi 3T, and rho 11 are compared . With phage mutants affected in their methylation potential, we show that phage-coded methyltransferase genes are interchangeable among the phages studied . DNA/DNA hybridization experiments indicate that phage methyltransferase genes are structurally related, whereas no such relationship is observed to a bacterial gene, specifying a methyltransferase with the same specificity. J Bacteriol, 1983 May, 154(2), 988 - 91 Cloning of sporulation genes spo0A and spo0C of Bacillus subtilis onto rho 11 temperate bacteriophage; Ikeuchi T et al.; A HindIII fragment harboring the intact spo0C gene of Bacillus subtilis was cloned with rho 11 temperate bacteriophage as a vector . Transformation experiments with the DNA from rho 11 dspo0C+ specialized transducing phage showed that the spo0C gene resides on a 5.3-megadalton fragment generated by HindIII digestion . The 5.3-megadalton fragment also contains the intact spo0A gene, but not spoIIIA, spoIIIB, or spoIVB. J Bacteriol, 1983 May, 154(2), 864 - 9 Genetic analysis of a pleiotropic deletion mutation (delta igf) in Bacillus subtilis; Fujita Y et al.; A delta igf mutation of Bacillus subtilis (formerly called fdpAl) is a large deletion causing pleiotropic defects . The mapping of the delta igf deletion by phage PBS1 transduction revealed the following map order: sacA, thiC, hsrE, delta igf, ts199, purA . To analyze the pleiotropic nature of the delta igf mutation, mutants affected in each property of the pleiotropic mutation were isolated, and the mutations were mapped . iol and gnt mutants could not grow on inositol and gluconate, respectively, and fdp mutants were affected only in fructose-bisphosphatase . The map order from sacA to purA was as follows: sacA, thiC, hsrE, iol-6, gnt-4, fdp-74, hsrB, ts199, purA . The delta igf deletion covered loci from iol-6 to hsrB. J Bacteriol, 1983 May, 154(2), 831 - 7 Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus and Bacillus subtilis; Fujii M et al.; The structural gene for a thermostable protease from Bacillus stearothermophilus was cloned in plasmid pTB90 . It is expressed in both B . stearothermophilus and Bacillus subtilis . B . stearothermophilus carrying the recombinant plasmid produced about 15-fold more protease (310 U/mg of cell dry weight) than did the wild-type strain of B . stearothermophilus . Some properties of the proteases that have been purified from the transformants of B . stearothermophilus and B . subtilis were examined . No significant difference was observed among the enzyme properties studied here despite the difference in host cells . We found that the protease, neutral in pH characteristics and with a molecular weight of 36,000, retained about 80% of its activity even after treatment of 65 degrees C for 30 min. J Bacteriol, 1983 May, 154(2), 946 - 54 Alkaline phosphatase secretion-negative mutant of Bacillus licheniformis 749/C; Kumar R et al.; An alkaline phosphatase secretion-blocked mutant of Bacillus licheniformis 749/C was isolated . This mutant had defects in the phoP and phoR regions of the chromosome . The selection procedure was based on the rationale that N-methyl-N'-nitro-N-nitrosoguanidine can induce mutations of closely linked multiple genes . The malate gene and the phoP and phoR genes are located at the 260-min position in the Bacillus subtilis chromosome; hence, the malate gene could be used as a marker for the mutation of the phoP and phoR regions of the chromosome . In a two-step selection procedure, strains defective in malate utilization were first selected with the cephalosporin C procedure . Second, these malate-defective strains were further screened in a dye medium to select strains with defects in alkaline phosphatase secretion . One stable mutant (B . licheniformis 749/cNM 105) had a total secretion block for alkaline phosphatase and had the following additional characteristics: (i) the amount of alkaline phosphatase synthesized was comparable to that in the wild type; (ii) the alkaline phosphatase was membrane bound; (iii) the mutant strain alkaline phosphatase, in contrast to that of the wild type, could not be extracted with MgCl2, although the amounts of protein extracted from each strain were comparable; (iv) the sodium dodecyl sulfate-polyacrylamide gel pattern of MgCl2-extracted proteins from the mutant strain was different from that of the wild-type proteins; (v) the mutant, unlike the wild type, could not use malate as a sole source of carbon; and (vi) the outside surface of the wall of the mutant cells contained an additional electron-dense layer that was not present on the wild-type cell wall surface. Biochem Biophys Res Commun, 1983 Apr 29, 112(2), 678 - 83 Nucleotide sequence of the promoter and NH2-terminal signal peptide region of Bacillus subtilis alpha-amylase gene cloned in pUB110; Ohmura K et al.; The nucleotide sequence of the promotor and NH2-terminal signal peptide region of the alpha-amylase gene derived from the alpha-amylase hyperproducing strain B . subtilis NA64 was determined . DNA sequences of the NH2-terminal region of the mature alpha-amylase, 41 amino acid residues of the signal peptide, a Shine-Dalgarno sequence (AGGAG), a potential RNA polymerase recognition site (TTGAAA), and a potential Pribnow box (AAGTAA) were identified . The DNA sequence was quite different from that of the alpha-amylase gene of B . amyloliquefaciens. Nature, 1983 Apr 28, 302(5911), 800 - 4 Two RNA polymerase sigma factors from Bacillus subtilis discriminate between overlapping promoters for a developmentally regulated gene; Johnson WC et al.; A developmentally regulated gene (spoVG) from the spore-forming bacterium Bacillus subtilis is expressed from two overlapping promoters, which direct transcription initiating from sites separated by 10 base pairs . Utilization of the upstream promoter is determined by an RNA polymerase sigma factor of molecular weight 37,000 (sigma 37) . We report the isolation of a 32,000-molecular weight species of sigma factor (sigma 32), which exclusively dictates transcription initiation from the downstream promoter, and suggest a model for the way in which sigma-specific recognition sequences are intermeshed within the spoVG transcription initiation region. Eur J Biochem, 1983 Apr 15, 132(1), 63 - 7 Comparison of the physical maps and redundant ends of the chromosomes of phages 2C, SP01, SP82 and phi e; Hoet P et al.; The physical map of 2C DNA (cf . following paper in this journal) was compared to the maps of SP01, SP82 and phi e (three other Bacillus subtilis phages containing hydroxymethyluracil in place of thymine in their DNA) . The overall organization of the four genomes was remarkably similar, as indicated by the topology of HaeIII and SalI cleavage segments . The proof was gathered for the presence in the four phage DNAs of large redundant ends carrying a single HaeIII recognition site . The location of the latter proved identical for 2C and SP01, but was shifted in the DNAs of SP82 and phi e . Since the redundant end components of these hydroxymethyluracil genomes are colinear, as shown by cross-hybridization studies, the shifting of the HaeIII cleavage site is presumably due to two base substitutions, suppressing an endonuclease recognition site and establishing a new site elsewhere . Relatedness between the genomes of this family of viruses was evaluated from the fraction of conserved restriction fragments . According to these calculations, 6% base substitutions have occurred within the four viral DNAs, in the course of evolution . However, specific segments of 2C DNA were not present in SP01 and phi e DNA, as shown by cross-hybridization with restriction fragments . These data indicate the occurrence of deletions, in addition to base substitutions, as evolutionary mechanisms prevailing in the genomes of this family of phages. Eur J Biochem, 1983 Apr 15, 132(1), 69 - 75 Physical map of phage 2 C DNA: evidence for the existence of large redundant ends; Coene M et al.; The chromosome of the Bacillus subtilis phage 2C, a linear molecule of double-stranded DNA of about 10(8) Da, in which thymine is completely replaced by hydroxymethyluracil, was cleaved by different endonucleases . In some cases restriction segments were much fewer than expected, suggesting a possible interference of the unusual base with the recognition mechanism of endonucleases . The physical map of 2C DNA was established by use of SalI and HaeIII restriction endonucleases, which yielded a limited number of fragments . The expected number of fragments was 240 for HaeIII and 23 for SalI; in reality, five segments were observed upon cleavage with HaeIII and four with SalI . The terminal fragments of the genome were first identified; the other fragments were ordered by hybridization and molecular weight determination of restriction fragments obtained by cleavage with the two endonucleases . In addition, hybridization of restriction fragments showed the presence of homologous regions at the ends of the 2C genome . The structure of these direct repetitive sequences was analyzed by cleavage with HaeIII and hybridization with EcoRI restriction fragments . Their size (9.2 MDa) was found to be about 1/11 of that of the whole chromosome. Nature, 1983 Apr 7, 302(5908), 543 - 5 Formation of proinsulin by immobilized Bacillus subtilis; Mosbach K et al.; There has been an increasing interest in the use of immobilized cells for the production of pharmaceuticals as well as for products such as high fructose syrup or ethanol . Some of these compounds are now produced on an industrial scale whereby the cells are used in a resting or growing state or in a nonviable form as natural carriers of the enzyme(s) involved in the synthesis . The advantages of immobilized cell technology should also apply to microorganisms modified by recombinant DNA techniques to produce a variety of eukaryotic proteins such as hormones . We describe here the properties of immobilized Bacillus subtilis cells carrying plasmids encoding rat proinsulin . Cell proliferation normally coupled to DNA replication is undesirable in immobilized cell systems as "clogging' of the system occurs due to cells growing outside the beads . Therefore, different ways were investigated to inhibit cell division while allowing continued protein synthesis . We found that the addition of certain antibiotics in the growth medium, such as novobiocin which inhibits DNA replication, fulfills these requirements, allowing proinsulin synthesis and excretion to take place over a period of several days. J Gen Microbiol, 1983 Apr, 129 (Pt 4), 917 - 22 Characterization of a pleiotropic succinate dehydrogenase-negative mutant of Bacillus subtilis; Magnusson K et al.; A succinate dehydrogenase-negative mutant of Bacillus subtilis is described which lacks all three subunits of the membrane-bound succinate dehydrogenase complex: flavoprotein, iron protein, and cytochrome b558 . The corresponding mutation is revertible and it maps at one extreme of the sdh region . The results presented suggest that the structural genes for the subunits of the succinate dehydrogenase complex are part of one operon. J Gen Microbiol, 1983 Apr, 129 (Pt 4), 1089 - 95 Alteration of membrane permeability in Bacillus subtilis by clofoctol; Yablonsky F; When Bacillus subtilis was treated with a bacteriostatic concentration of clofoctol {2-(2,4-dichlorobenzyl)-4-(tetramethyl-1,1,3,3-butyl)phenol}, UV-absorbing material was released . Electron micrographs showed evidence of physical alteration of the bacterial envelope . The uptake of {14C}glutamate was strongly inhibited by clofoctol, and preloaded glutamate was found to leak from the bacteria . Clofoctol caused an immediate and dramatic decrease in the amount of intracellular ATP . This was neither the consequence of the stimulation of an ATPase, nor of the inhibition of bacterial respiration . Both the proton gradient and the potential gradient across the cytoplasmic membrane collapsed and this inhibition of energy metabolism was sufficient to account for the inhibition of growth by clofoctol . At the same bacteriostatic concentration complete permeabilization of the bacteria occurred. Gene, 1983 Apr, 22(1), 47 - 57 Enhanced expression of mouse dihydrofolate reductase in Bacillus subtilis; Schoner RG et al.; pPL608-TR1 is a high-copy plasmid that permits phenotypic expression of the mouse dihydrofolate reductase (DHFR) gene in Bacillus subtilis . A plasmid mutation has been identified that increases expression of mouse DHFR more than ten-fold . The mutation is located in a 0.2-kb segment that intervenes between the DHFR gene and the 0.3-kb promoter fragment needed for transcriptional activation of DHFR . Nucleotide sequence analysis suggests that the effect of the mutation is to facilitate translation, initiated within the promoter fragment, through the 0.2-kb segment to the site of insertion of the DHFR-containing fragment . Additional promoter-containing fragments selected by their ability to promote expression of a plasmid gene located downstream from DHFR, the CAT gene, promote either high, intermediate or no phenotypic expression of DHFR . The results indicate that promoter fragments that allow phenotypic expression of the mouse DHFR gene contain two regulatory signals . One signal is essential to transcription of both DHFR and CAT and therefore functions as a promoter . The second signal may be necessary for translation of that portion of the mRNA specifying mouse DHFR. Gene, 1983 Apr, 22(1), 41 - 6 Insertion of foreign functioning DNA into the Bacillus subtilis chromosome; Prozorov AA et al.; Following the restriction and ligation of pBD12 plasmid and phi 105 phage DNA and subsequent transformation, the insertion of pBD12 DNA into the phi 105 prophage region of the Bacillus subtilis chromosome was achieved . The plasmid markers were linked with the region of prophage immunity and with the markers of the adjacent region of the B . subtilis chromosome . Their behaviour in transformation of cells and protoplasts of rec+ recipients and recE4 mutants was typical of chromosomal markers . The phenotypic expression (resistance to chloramphenicol and kanamycin) of plasmid genes inserted into the chromosome was preserved; however, the resistance levels were reduced. Mutat Res, 1983 Apr, 117(1-2), 55 - 65 Induction at high frequency of a unique phenotypic class of Bacillus subtilis mutants by methylxanthines; Sacks LE et al.; Caffeine and theophylline are mutagenic at high concentration in the B . subtilis multigene sporulation test for mutagens; caffeine is a stronger mutagen than theophylline in this test . An unusually high fraction of the mutant colonies appear to be phenotypically identical, as judged by colonial morphology and microscopic appearance of the vegetative cells . These mutants do not bring about the pH increase normally associated with sporulation of B . subtilis; such behavior is frequently associated with lack of a functional tricarboxylic acid (TCA) cycle, essential for normal sporulation of this species . Similar mutants have not been noted in the course of screening a variety of well-known mutagens, including acriflavine . Caffeine is maximally effective in inducing these mutants about 10 min after germination commences . Adenosine greatly reduces the ability of caffeine to induce these mutants. J Bacteriol, 1983 Apr, 154(1), 261 - 8 Effect of decoyinine on peptidoglycan synthesis and turnover in Bacillus subtilis; Uratani B et al.; The sporulation of Bacillus subtilis can be induced in the presence of amino acids and glucose by partially depriving the cells of guanine nucleotides . This can be achieved, e.g., by the addition of decoyinine, a specific inhibitor of GMP synthetase . To determine the effect of this and other inhibitors on cell wall synthesis, we measured in their presence the incorporation of acetylglucosamine into acid-precipitable material . The rate of wall synthesis decreased by 50% within 5 min after decoyinine addition; this decrease was prevented by the presence of guanosine . A comparison with the effects of other inhibitors of cell wall synthesis indicated that decoyinine inhibited the final portion of the cell wall biosynthetic pathway, i.e., after the steps inhibited by bacitracin or vancomycin . Decoyinine addition also prevented cellular autolysis and cell wall turnover . It is not known whether these two effects of decoyinine on cell wall synthesis are causally related. Biochem Genet, 1983 Apr, 21(3-4), 405 - 16 Human parotid size polymorphism (Ps): characterization of two allelic products, Ps 1 and 2, by limited proteolysis; Goodman PA et al.; The allelically determined human salivary proteins, Ps 1 and 2, were purified on sodium dodecyl sulfate gels, eluted, and compared by limited proteolysis with Streptomyces griseus protease VI, Bacillus subtilis protease VII, and Staphylococcus aureus protease V8 . Prior dansylation of the Ps isoproteins facilitated visualization of the peptides . Digestion patterns indicate considerable homology between the Ps isoproteins and support the conclusion {Azen, A . E., and Denniston, C . (1980) . Biochem . Genet . 18:483} that there is an actual molecular weight difference between them . Further, the results suggest that this difference owes to an extension of the Ps 2 chain at one of its ends. J Appl Bacteriol, 1983 Apr, 54(2), 237 - 42 The effect of parabens on DNA, RNA and protein synthesis in Escherichia coli and Bacillus subtilis; Nes IF et al.; The effect of methyl, propyl and butyl esters of p-hydroxybenzoic acid on DNA and RNA synthesis has been tested in toluenized cells of Escherichia coli and Bacillus subtilis . Both RNA and DNA synthesis of these bacteria were inhibited . The inhibitory concentrations were higher than those previously reported for growth inhibition . Protein synthesis in cell-free extracts (S-30 fraction) of B . subtilis was even more sensitive to parabens than DNA and RNA synthesis, while protein synthesis in Esch . coli was largely unaffected. J Bacteriol, 1983 Apr, 154(1), 50 - 7 Replication origin region of Bacillus subtilis chromosome contains two rRNA operons; Ogasawara N et al.; The first replicating DNA fragment (BamHI-7) of the Bacillus subtilis chromosome contains two promoters for a rRNA operon . A map of restriction enzyme cleavage sites of the region of replication origin suggests the presence of a second rRNA operon in this region . Hybridization of rRNA genes (rDNA) with DNA fragments derived from the origin region by treatment with various enzymes clearly revealed two rRNA operons in this region, one at the B7-B3 junction and the other at the B5-B6 junction . The restriction enzyme cleavage sites surrounding the rRNA operons show that the operon at the B5-B6 junction corresponds to the rrnA operon . A novel operon at the B7-B3 junction was termed rrnO . Transformation by density-labeled fragments of the origin region showed that the first replicating marker, guaA, is located in the B3 fragment . From these results, a map was constructed for the first time to correlate the genetic markers with the physical structure of the replication origin region of the B . subtilis chromosome . The role of the rrnO operon in regulating the initiation of chromosomal replication is discussed, based on the fact that the promoter of the rrnO operon suppresses the replication of the plasmid carrying the promoter. J Biochem (Tokyo), 1983 Apr, 93(4), 1109 - 18 Incorporation of glutamic acid into protein by a soluble system; Hashizume S et al.; A heat-labile, non-dialyzable factor(s) in soluble fractions from Escherichia coli strains and Bacillus subtilis was found to incorporate the radioactivity of {14C}glutamic acid into 95 degrees C CCl3COOH-insoluble fraction . Incorporation catalyzed by a partially purified factor from E . coli B required ATP, Mg2+, tRNA, casein, carbonate, and 2-mercaptoethanol . A mixture of nineteen amino acids other than glutamic acid had no effect on the incorporation . Heparin, spermine and monovalent cations were inhibitory . Incorporation proceeded via glutamyl-tRNA . The incorporation from {14C}glutamyl-tRNA required Mg2+, casein, carbonate, and 2-mercaptoethanol, and there was no incorporation from {14C}aspartyl-tRNA . The reaction product was identified as protein . The incorporated moiety was the glutamyl moiety of glutamic acid and it retained a free alpha-amino group in the product protein . The incorporating factor of E . coli B was demonstrated to be glutamyl-tRNA synthetase. J Biol Chem, 1983 Mar 10, 258(5), 2843 - 51 Degradation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase in vivo; Ruppen ME et al.; Glutamine phosphoribosylpyrophosphate amidotransferase, the first enzyme of purine nucleotide biosynthesis, is inactivated and degraded in Bacillus subtilis during carbon, nitrogen, or amino acid starvation . Amidotransferase is stable in exponentially growing cells, and synthesis of the enzyme ceases prior to its inactivation at the end of exponential growth . Inactivation has been previously shown to result from reaction of an essential {4Fe-4S} center with oxygen . In this work, monospecific antibodies against amidotransferase have been used to demonstrate that inactivation is followed by proteolytic degradation in vivo and that the metabolic requirements for degradation differ from those for inactivation . Unlike inactivation, degradation is inhibited by addition of 10 mM KCN or antibiotic inhibitors of RNA and protein synthesis to glucose-starved cells . The cross-reactive material that accumulates when degradation is inhibited by chloramphenicol initially has native subunit molecular weight, but lower molecular weight polypeptides slowly accumulate . Degradation, but not inactivation, of amidotransferase is strongly inhibited during amino acid starvation of a relA strain . Degradation of amidotransferase is inhibited by pseudomonic acid, an antibiotic that blocks protein synthesis but permits a normal stringent response . This result indicates that both protein synthesis and normal relA gene function are required for degradation. J Gen Microbiol, 1983 Mar, 129 (Pt 3), 687 - 701 Bacillus subtilis strains carrying two non-tandem duplications of the trpE-ilvA and the purB-tre regions of the chromosome; Schneider AM et al.; Bacillus subtilis strains possessing the trpE30 marker (splitting of the trpE locus and a non-tandem duplication of chromosome segment Ib: purB-tre) when transformed or transduced to tryptophan independence mainly give rise to haploid cells with the genetic structure of strain 168 . However, among the Trp+ transformants or transductants about 10% are merodiploid carrying a non-tandem duplication of segment C (trpE-ilvA) while maintaining that of segment Ib . Linkage and segregation studies made it possible to determine their genetic structure, which can be represented by three different maps . In map a the copies of Ib are inverted repeats and one of them is flanked by two direct repeats of segment C; in map b two Ib-C segments are inverted repeats and in map c the copies of C are inverted repeats with one of them flanked by direct repeats of Ib . It is proposed that transition from map a to map b and then to map c, and vice versa, may occur by recombination between inverted repeats of either Ib or C . The merodiploids are unstable, recombination between direct repeats leading to haploid cells of 168-type structure . The models proposed for merodiploid formation call for fusion of two recipient chromosomes mediated by the donor segment and recombination between copies of a DNA sequence of the two chromosomes located in different regions . In the case of PBS-1 mediated transduction the greater length of the donor DNA segment makes it possible to obtain the merodiploids with a single recipient chromosome and this needs only a slight modification of the models . No trpE30+ merodiploids are found in transformation when the recipient carries a deletion of the SP beta prophage, or in transduction when both donor and recipient possess this deletion . These results indicate that the homologous sequences involved may be part of the SP beta prophage or that a sequence of bacterial DNA has a good homology with it. Appl Environ Microbiol, 1983 Mar, 45(3), 872 - 7 Bioassay and dose measurement in UV disinfection; Qualls RG et al.; A bioassay method was developed to measure the average intensity within a UV disinfection reactor . The survival of spores of Bacillus subtilis was determined as a function of UV dose to prepare a standard curve . Spores were added to unknown systems, and the survival rate was used to determine the average intensity . A modification was used for flow-through reactors by which spores were injected as a spike and collected at a known time after injection . A point source summation method for calculating intensity was verified by bioassay measurements in a simple cylinder . This calculation method was also applied to multiple-lamp reactors. Proc Natl Acad Sci U S A, 1983 Mar, 80(5), 1236 - 40 Structure of a Bacillus subtilis bacteriophage SPO1 gene encoding RNA polymerase sigma factor; Costanzo M et al.; Gene 28 of Bacillus subtilis bacteriophage SPO1 codes for a regulatory protein, a sigma factor known as sigma gp28, that binds to the bacterial core RNA polymerase to direct the recognition of phage middle gene promoters . middle promoters exhibit distinctive and conserved nucleotide sequences in two regions centered about 10 and 35 base pairs upstream from the start point of mRNA synthesis . Here we report the cloning of gene 28 and its complete nucleotide sequence . We infer that sigma gp28 is a 25,707-dalton protein of 220 amino acids . Neither the nucleotide sequence of gene 28 nor the inferred amino acid sequence of sigma gp28 exhibits extensive homology to the gene or protein sequence of Escherichia coli sigma factor. J Bacteriol, 1983 Mar, 153(3), 1521 - 7 Cell wall-DNA association in Bacillus subtilis; Doyle RJ et al.; Autolysis of cell walls of Bacillus subtilis 168 resulted in solubilization of wall-associated DNA . Most of the DNA was solubilized only in the later stages of autolysis . Solubilization of up to 70% of the wall by autolysins resulted in only 25 to 30% solubilization of wall-associated DNA . When the wall fragments remaining after 70% autolysis were analyzed by electron microscopy, it was observed that the preparations were highly enriched for completed septa, or poles . Partial autolysis at pH 5.2 or pH 8.6, both of which reflect hydrogen ion levels that permit either N-acetylglucosaminidase or N-acetylmuramyl-L-alanine amidase, but not both, to act, gave rise to enrichment of cell poles . When walls were incubated with subtilisin, DNase, or RNase, release of DNA (or DNA fragments) was accelerated . Density gradient centrifugation patterns of lysates of cells pulse-labeled with N-{3H}acetylglucosamine and then chased revealed that a small, but significant, proportion of the radioactivity sedimented to a density position equivalent to that of DNA-membrane complexes . Because the pulse-chase sequence enriched for radioactivity in cell poles, the results suggest that at least some molecules from polar cell walls have an affinity for DNA-membrane complexes . We suggest that DNA binds strongly, possibly via a DNA-membrane complex, to cell poles of B . subtilis . The results provide support for a view offered previously (Koch et al., FEMS Microbiol . Lett . 12:201-208, 1981) that some special structure in or very near the poles of gram-positive bacilli is involved in the segregation of DNA during cell division. J Bacteriol, 1983 Mar, 153(3), 1424 - 31 Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli; Gay P et al.; A clone bearing the structural gene sacB, coding for the exoenzyme levansucrase, was isolated from a library of Bacillus subtilis DNA that was cloned in phage lambda charon 4A on the basis of the transforming activity of the chimeric DNA . This lambda clone also was found to contain the sacR and smo loci . Subcloning the sacB-sacR region in plasmid pBR325 resulted in a clone which directed levansucrase synthesis in Escherichia coli . The nucleotide sequence coding for the secreted protein was localized on the physical map of the cloned DNA. J Bacteriol, 1983 Mar, 153(3), 1331 - 7 Changes in penicillin-binding proteins during sporulation of Bacillus subtilis; Sowell MO et al.; The penicillin-binding proteins (PBPs) of Bacillus subtilis were examined in samples collected at various times from sporulating cultures and compared with the PBPs in a presporulation sample . Large increases in vegetative PBPs 2B and 3 and the appearance of at least one new PBP (42,000 daltons) occurred at reproducible times during sporulation . In some strains a second new PBP (60,000 daltons) was also produced . By comparing the PBP activities in sporulating cells and two spo0 mutants we have classified these changes as sporulation-related events rather than the consequences of stationary-phase aging . The other vegetative PBPs (PBPs 1, 2A, 4, and 5) decreased during sporulation, but not in sufficient amount or at the appropriate time to account for the appearance of the new proteins . A possible connection between specific PBP changes and the penicillin-sensitive stages of sporulation is suggested. J Bacteriol, 1983 Mar, 153(3), 1133 - 7 Chromosomal localization of gut, fruC, and pfk mutations affecting genes involved in Bacillus subtilis D-glucitol catabolism; Gay P et al.; Mutations affecting the genes involved in B . subtilis D-glucitol catabolism were mapped either by PBS1-mediated transduction or DNA-mediated transformation . It was shown that the genes gutA and gutB coding for the D-glucitol permease and the D-glucitol dehydrogenase, respectively, and regulatory locus gutR are clustered in a gut operon localized between purB and dal close to the pha marker . A mutation affecting fructokinase activity (fruC) was mapped near the gut markers . The fruC gene does not belong to the operon . A mutation affecting phosphofructokinase activity (pfk) was mapped between the leuA and aroG markers. Rev Can Biol Exp, 1983 Mar, 42(1), 83 - 6 Effect of ozone on DNA-repair deficient mutants of Bacillus subtilis; Song JM et al.; The wild type strains and DNA-repair deficient mutant strains of B . subtilis were exposed to ozone . The results show that the lethal effect of ozone is greatly influenced by factors such as ozone concentration, treatment time, growth phase, and treatment of medium . The recA and recC loci seem to be most important and perhaps the polA and uvrA loci may also be required, but the recB and recD loci are not required for the repair of DNA-damase induced by ozone in B . subtilis. Eur J Biochem, 1983 Mar 1, 131(1), 97 - 103 Structural and functional properties of cytochrome c oxidase from Bacillus subtilis W23; De Vrij W et al.; The terminal component of the electron transport chain, cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase) was purified from Bacillus subtilis W23 . The enzyme was solubilized with alkyglucosides and purified to homogeneity by cytochrome c affinity chromatography . The enzyme showed absorption maxima at 414 nm and 598 nm in the oxidized form and at 443 nm and 601 nm in the reduced form . Upon reaction with carbon monoxide of the reduced purified enzyme the absorption maxima shifted to 431 nm and 598 nm . Sodium dodecylsulfate polyacrylamide gel electrophoresis indicated that the purified enzyme is composed out of three subunits with apparent molecular weights of 57 000, 37 000 and 21 000 . This is the first report on a bacterial aa3-type oxidase containing three subunits . The functional properties of the enzyme are comparable with those of the other bacterial cytochrome c oxidases . The reaction catalyzed by this oxidase was strongly inhibited by cyanide, azide and monovalent salts . Furthermore a strong dependence of cytochrome c oxidase activity on negatively charged phospholipids was observed . Crossed immunoelectrophoresis experiments strongly indicated a transmembranal localization of cytochrome c oxidase. J Gen Microbiol, 1983 Feb, 129 (Pt 2), 293 - 302 SpoVH and spoVJ--new sporulation loci in Bacillus subtilis 168; Hill SH; Three sporulation mutants have been isolated which produce spores with an atypical resistance phenotype, i.e . they are sensitive to organic solvents and heat but resistant to lysozyme . All three mutants produced serine protease, alkaline phosphatase and glucose dehydrogenase which are biochemical marker events for stages I, II and III . Two of the three mutants produced dipicolinic acid, a late marker, but the third was defective in its production . Heat-resistance was not restored to any of the mutants by the provision of exogenous dipicolinate . Gel electrophoresis showed that the mutant spores had similar patterns of spore coat proteins to the wild-type and electron microscopy revealed no significant structural differences . The three mutations responsible for the phenotypes of the mutant spores lie in the phe-argA region of the Bacillus subtilis chromosome . Recombination index values indicate that the mutations are in three separate genes . They define at least two new sporulation loci, designated spoVH and spoVJ. Virology, 1983 Feb, 125(1), 18 - 30 Assembly of the tail protein of the Bacillus subtilis phage phi 29; Garcia JA et al.; By in vitro complementation we have determined that gene 13 product functions during phi 29 morphogenesis after the formation of 11- particles, specifically in the functional assembly of the tail protein, p9 . Protein p9 from 8- but not from 8-13- extracts assembles in vitro into either 11-13- or 12-13- particles . The action of gene 13 product on p9 for its correct assembly has to take place in vivo; no complementation of 12-13- and 9- lysates occurs in vitro . Protein p9, isolated from phi 29-infected cells, copurifies with the 13+ activity and it is present both in 13+ and 13- extracts as an aggregate with dimensions similar to those of the tail assembled in mature phage. Proc Natl Acad Sci U S A, 1983 Feb, 80(3), 658 - 62 Early sporulation gene spo0F: nucleotide sequence and analysis of gene product; Shimotsu H et al.; We have determined the sequence of a 1,162-base-pair DNA fragment containing a spo0F gene which is required for an early stage of sporulation in Bacillus subtilis . The sequence has only one long open reading frame consisting of 173 codons, which has been confirmed to be the spo0F cistron by DNA-mediated transformation and in vitro transcription . In UV-irradiated "maxicells" containing pUBSF13, the plasmid that carries cloned spo0F gene, we have observed the synthesis of a 20-kilodalton polypeptide that is absent from cells carrying a vector plasmid pUB110 . The molecular weight of this protein is in agreement with the calculated molecular weight of the spo0F gene product (Mr, 19,065) . The putative promoter sequences of spo0F gene were 5' T-A-T-A-A-T 3' at -10 and 5' T-T-G-A-T-T 3' at -35 . An octamer sequence, 5' A-A-A-G-G-A-G-G 3', situated 8 base pairs prior to the initiation codon was found to be perfectly complementary with the 3' end of 16S ribosomal RNA . This result offers additional evidence for the proposal by Rabinowitz's group that an extensive mRNA-rRNA interaction is a requirement for efficient translation by B . subtilis ribosomes. Chem Biol Interact, 1983 Feb, 43(2), 175 - 97 The mode of action of cytotoxic and antitumor 1-nitroacridines . III . In vivo interstrand cross-linking of DNA of mammalian or bacterial cells by 1-nitroacridines; Konopa J et al.; To define a critical lesion in presumable target DNA cause in vivo by the antitumor and cytotoxic 1-nitroacridines, Ehrlich ascites tumor (Eat) cells implanted into mice, HeLa cells grown in monolayer culture or Bacillus subtilis SB 1058 strain cells were exposed to Ledakrin {Nitracrine; 1-nitro-9-(3'-dimethylamino-n-propylamino)acridine}, its non-antitumor congeners, or mitomycin C tested for comparison; total intracellular DNA was isolated from control or treated cells and denatured by heat, alkali or formamide, after which the chemically-induced fraction of interstrand cross-linked DNA molecules was assessed by thermal denaturation-renaturation curve analysis, hydroxylapatite column chromatography, or partitioning in a Dextran T500-polyethylene glycol 6000 biphasic system . Ledakrin, as compared to mitomycin C, was a very effective cross-linking agent, inducing one covalent cross-link per approx . 20 X 10(3) (B . subtilis), 56 X 10(3) (HeLa) or 80 X 10(3) (Eat) DNA base pairs . The first cross-links were introduced in B . subtilis cell genomes at minimal bactericidal concentrations of Ledakrin of mitomycin C . Ledakrin failed to induce discernible cross-linking of bihelical DNA when complexed with in cell-free system . Unlike the other two 1-nitroacridines which cross-linked DNA of cultured HeLa or B . subtilis cells, the non-antitumor 2-, 3- or 4-nitroacridines did not cause such effect and diminished cross-linking by Ledakrin or mitomycin C . Hence, upon metabolic activation in mammalian or bacterial cell Ledakrin and, most probably other 1-nitroacridines, become very effective DNA cross-linking agents and such effects appear to be responsible for the antitumor and potent cytotoxic activities of 1-nitroacridines. Chem Biol Interact, 1983 Feb, 43(2), 151 - 73 The mode of action of cytotoxic and antitumor 1-nitroacridines . II . In vivo enzyme-mediated covalent binding of a 1-nitroacridine derivative, Ledakrin or Nitracrine, with dna and other macromolecules of mammalian or bacterial cells; Pawlak JW et al.; Earlier evidence, in Part I of this paper, has shown that cytotoxic and antitumor 1-nitroacridines did not primarily exert their potent inhibitory effects on cultured mammalian cells by physicochemical binding with DNA, although it undoubtedly occurred (Chem.-Biol . Interact., 43 (1983) 131) . As a result it was investigated (i) whether 9-14C- or 1'-14C-labeled derivatives of their representative, 1-nitro-9-/3'-dimethylamino-n-propylamino/acridine (Ledakrin or Nitracrine), were capable of covalent binding with nucleic acids and other suitable macromolecules in target cells in vivo and/or (ii) whether activation of the agent in the cell was a necessary prerequisite for such binding . Using the criteria of resistance to exhaustive extractions with trichloroacetic acid and/or organic solvents, {14C}Ledakrin was found to bind covalently, with relatively little discrimination, with: (i) intracellular macromolecules, including DNA, of cultured tumor HeLa cells (370-2500 DNA base pairs/one Ledakrin molecule; (ii) experimental animal tumor Ehrlich ascites (Eat) cells in vivo (650-5880 DNA base pairs/one Ledakrin molecule); (iii) bacterial Bacillus subtilis SB 1058 cells (7000-33 000 Ledakrin links/one cell genome); (iv) NADPH-fortified rat liver homogenates in vitro (25.6 nmol/mg microsomal protein under air) . These results far exceed the common levels reported for alkylating agents or chemical carcinogens . Unlike {ethyl-14C}quinacrine, compared in vitro, covalent macromolecules binding with Ledakrin in vitro, and most probably in vivo, can be equated to NADPH-dependent activation(s) by oxidoreductase systems and the presence of DNA alone was not satisfactory in itself to attain Ledakrin binding . Fractionation of the enzymatic digest of 14C-associated DNA, isolated from Eat cells exposed in vivo to {9-14C}Ledakrin, by Sephadex LH-20 column chromatography followed by mass spectrometry analyses of modified nucleosides, indicated that both mono- and dinucleosidical Ledakrin metabolites were the products of an in vivo reaction . This implied that the lethal reaction of the drug could be its cross-linking of the target macromolecules and/or its monofunctional attack on vitally important cellular components. J Bacteriol, 1983 Feb, 153(2), 937 - 49 Regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase inactivation in vivo; Bernlohr DA et al.; Glutamine phosphoribosylpyrophosphate amidotransferase is stable in growing cells, but is inactivated in an oxygen-dependent process at various rates in starving or antibiotic-treated cells . On the basis of studies of the purified enzyme, we suggested (D.A . Bernlohr and R.L . Switzer, Biochemistry 20:5675-5681, 1981) that the inactivation in vivo was regulated by substrate stabilization and a competition between stabilizing (AMP) and destabilizing (GMP, GDP, and ADP) nucleotides . This proposal was tested by measuring the intracellular levels of these metabolites under cultural conditions in which the stability of the amidotransferase varied . The results established that the stability of amidotransferase in vivo cannot be explained by the simple interactions observed in vitro . Metabolite levels associated with stability of the enzyme in growing cells did not confer stability under other conditions, such as ammonia starvation or refeeding of glucose-starved cells . The data suggest that a previously unrecognized event, possibly a covalent modification of amidotransferase, is required to mark the enzyme for oxygen-dependent inactivation. J Bacteriol, 1983 Feb, 153(2), 756 - 62 Adaptive response of Bacillus subtilis to N-methyl-N'-nitro-N-nitrosoguanidine; Hadden CT et al.; Cell extracts of Bacillus subtilis contain a methyltransferase that appears to remove the O6-methyl group from O6-methylguanine in DNA in situ . This reaction proceeds in a stoichiometric fashion, as in Escherichia coli . However, the basal level of the enzyme (approximately 240 molecules per cell) is significantly higher in B . subtilis than in E . coli . In addition, the methyltransferase level increases by an order of magnitude as a result of de novo protein synthesis after adaptive treatment with a low concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as in E . coli . Concomitant with adaptation, B . subtilis cells become more resistant to both killing and mutagenesis by a challenge dose of N-methyl-N'-nitro-N-nitrosoguanidine . We present evidence to support the hypothesis that the majority of N-methyl-N'-nitro-N-nitrosoguanidine-induced mutations in B . subtilis are of the guanine-to-adenine transition type. J Appl Bacteriol, 1983 Feb, 54(1), 91 - 9 Death, injury and revival of chemically treated Bacillus subtilis spores; Gorman SP et al.; The resistance of spores of Bacillus subtilis NCTC 10073 to glutaraldehyde, sodium hypochlorite and povidone-iodine was compared . Revival of treated spores was examined by use of defined germination media and conditions, protein denaturing agents, ultrasonics and heat . Revival, obtained after treatment with each of the three chemical agents, originated under different sets of conditions and was of two recognizably distinct types . The results, including the evidence of electron microscopy, are discussed in terms of chemical-spore reactivity and the implications on their use and suitability as chemical sterilizers. Proc Natl Acad Sci U S A, 1983 Feb, 80(3), 785 - 9 Isolation of a developmental gene of Bacillus subtilis and its expression in Escherichia coli; Vasantha N et al.; Glucose dehydrogenase of Bacillus subtilis is a developmental enzyme that is not found in growing (vegetative) cells but is synthesized after the differentiation process that leads to the production of endospores has started . We have isolated the gene coding for this enzyme from a lambda Charon 4A phage library of B . subtilis DNA . It is transcribed and translated in vegetative cells of the nondifferentiating organism Escherichia coli into enzymatically active glucose dehydrogenase that has the same physicochemical properties as the enzyme produced in B . subtilis during sporulation . Subcloning of the lambda DNA insert into pBR322 plasmid derivatives showed that the glucose dehydrogenase gene was transcribed in E . coli from a promoter within the B . subtilis genome. Biochem J, 1983 Feb 1, 209(2), 561 - 4 The purification of a novel amylase from Bacillus subtilis and its inhibition by wheat proteins; Orlando AR et al.; A new alpha-amylase (EC 3.2.1.1) from Bacillus subtilis was purified by affinity chromatography . The molecular weight of the purified enzyme, estimated from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was 93000, which is very different from the molecular weights of two well-characterized amylases from B . subtilis . Electrofocusing showed an isoelectric point of 5 . Amylase shows a broad maximum of activity between pH 6 and 7; maximal inhibition of enzyme by wheat-protein alpha-amylase inhibitors is displayed at pH 7. J Bacteriol, 1983 Feb, 153(2), 813 - 21 Formation of competent Bacillus subtilis cells; Sadaie Y et al.; The process of competent cell formation for transformation has been studied with early-stationary-phase (T1) cells of Bacillus subtilis which had been grown in an enriched Spizizen minimal medium and transferred to a second synthetic medium . Rifampin, chloramphenicol, and tunicamycin were strong inhibitors of competent cell formation, as well as vegetative growth . After formation, competent cells were no longer sensitive to the above agents . Methicillin and an inhibitor of chromosomal replication, hydroxyphenylazouracil, did not inhibit the development of competence . A D-alanine-requiring mutant strain developed competence even in the absence of D-alanine in the second medium . A T1-stage culture showed the activity of extracellular serine protease which is necessary for sporulation . Competent cell formation was completely blocked by 0.7 M ethanol, which is a specific inhibitor of early events during sporulation, including forespore septum formation . Competent cells were formed even in media which supported sporulation . The development of competence was also studied with spo0 mutants at 10 different loci . Most spo0 mutations repressed the development of competence except for spo0C, spo0G, and spo0J . These results suggest that competent cells are formed from early sporulating cells with the synthesis of cell wall materials and by factors whose genes are activated by the supply of nutrients . It is suggested that common steps are involved both in forespore septation and in competent cell formation. J Gen Microbiol, 1983 Feb, 129 (Pt 2), 465 - 78 The bactericidal action of beta-lactam antibiotics on an autolysin-deficient strain of Bacillus subtilis; Rogers HJ et al.; An autolysin-deficient mutant of Bacillus subtilis was completely tolerant to 5 h incubation with 50-100 micrograms cycloserine ml-1 whereas the wild-type was rapidly lysed and killed by 12 micrograms ml-1 . Lysis also did not occur when low concentrations of beta-lactams were added to exponentially growing cultures of the mutant, but over 90% of the bacteria were killed within 90-120 min . Protein, lipid and peptidoglycan synthesis as well as growth were inhibited after about 60 min . At this time, but not earlier, small amounts of these three cell components appeared in culture supernatants . Earlier, at about 20-30 min, the intracellular pools of amino acids started to decline rapidly and there was a temporary apparent increase in the rate of lipid synthesis . Neither of the latter phenomena occurred with cycloserine, with which protein and lipid synthesis declined only slowly and the rate of peptidoglycan synthesis was 80% inhibited within 30 min . Only occasional cells with damaged walls were seen 30-90 min after addition of either beta-lactams or cycloserine to the cultures . It thus seems unlikely that wall hydrolysis or penetration by residual autolysins in the mutant are responsible for mass cell death caused by the beta-lactams. J Biol Chem, 1983 Jan 25, 258(2), 753 - 9 The monomeric glutamyl-tRNA synthetase from Bacillus subtilis 168 and its regulatory factor . Their purification, characterization, and the study of their interaction; Proulx M et al.; The glutamyl-tRNA synthetase from Bacillus subtilis has been purified to homogeneity . It is a monomer of Mr = 65,500 whose NH2-terminal sequence is Met-Asn-Glu-Val-Arg-Val-Arg-Tyr-Ser-Pro-Ser-Pro-Thr-Gly-His-Leu . The number of tryptic peptides indicates the absence of a significant amount of sequence duplication . Under certain conditions, this monomeric enzyme is co-purified with a polypeptide beta of Mr = 46,000, which increases the affinity of the enzyme about 10-fold for glutamate and for ATP, and stabilizes it against heat inactivation . gamma-Globulins prepared against the monomeric enzyme can inhibit completely the glutamyl-tRNA synthetase activity of a B . subtilis extract and precipitate from this extract both the monomeric enzyme and the regulatory factor beta . These anti-alpha immunoglobulins do nt precipitate pure beta . These results show that the glutamyl-tRNA synthetase of B . subtilis has a structure similar to that of the Escherichia coli enzyme (Lapointe, J., and Soll, D . (1972) J . Biol . Chem . 247, 4966-4974) and indicate that the beta factor has a function in the regulation of glutamyl-tRNA biosynthesis in vivo. Nucleic Acids Res, 1983 Jan 25, 11(2), 237 - 49 Nucleotide sequence of the amylase gene from Bacillus subtilis; Yang M et al.; The gene coding for amylase (EC.3.2.1.1) has been isolated and sequenced from Bacillus subtilis by cloning in lambda Charon4A and pBR322 . The entire coding sequence and large preceding and following regions, comprising the presumed transcriptional and translational regulatory regions, were sequenced . The coding sequence shows a large open reading frame with a translated molecular weight of 72,800 and a presumed signal sequence of approximately thirty-two amino acids . When the intact gene is present in Escherichia coli, it confers the ability to degrade starch, indicating that the gene is expressed in a functional state. J Biol Chem, 1983 Jan 25, 258(2), 1007 - 13 Amino acid sequence of alpha-amylase from Bacillus amyloliquefaciens deduced from the nucleotide sequence of the cloned gene; Takkinen K et al.; We have isolated by molecular cloning the gene coding for the alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1.) from Bacillus amyloliquefaciens and determined its complete nucleotide sequence . The gene cloned in the plasmid pUB110 using Bacillus subtilis as a host, was contained in a 2.3-kilobase insert . Starting from an ATG initiator codon, an open reading frame comprising a total of 514 amino acids (1542 base pairs) was found within the cloned DNA fragment . The gene region encoding the COOH terminus of alpha-amylase was located by direct COOH-terminal analysis of the purified exoenzyme . The NH2-terminal portion of the gene encodes a 31 amino acid-long signal peptide (Palva, L., Pettersson, R . F., Kalkkinen, N., Lehtovaara, P., Sarvas, M., Soderlund, H., Takkinen, K., and Kaariainen, L . (1981) Gene 15, 43-51) . Since the signal peptide is correctly cleaved in the new host, as shown here by direct NH2-terminal sequence analysis, the exoamylase consists of 483 amino acid residues, corresponding to a molecular weight of 54,778 . The reading frame used to deduce the amino acid sequence was found to be correct by comparison with partial amino acid sequence data published previously (Detera, S . D., and Friedberg, F . (1979) Int . J . Peptide Protein Res . 14, 364-372; Chung, H., and Friedberg, F . (1980) Biochem . J . 185, 387-395) . Several differences between the sequence presented here and the partial ones published previously, however, were found . The nucleotide sequences both 5' and 3' to the alpha-amylase gene revealed palindromic structures including a stretch of six T-residues, suggesting transcription termination signals on both sides of the gene . Thus, it appears that alpha-amylase is translated from a monocistronic mRNA. J Mol Biol, 1983 Jan 15, 163(2), 147 - 69 Analysis of the 5'-termini of nascent DNA chains synthesized in permeable cells of Bacillus subtilis; Banfalvi G et al.; The nascent DNA synthesized by permeable cells of Bacillus subtilis in the presence of 5'-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP has been isolated and characterized . The newly synthesized DNA was isolated free from other cellular nucleic acids by affinity chromatography on thiol-substituted agarose . The number average chain length of the nascent DNA synthesized in one minute at 25 degrees C was 33 nucleotide residues, due to the chain-terminating action of 2',3'-dideoxyATP . Several lines of evidence indicated that at least 90% of the DNA thus isolated carried a terminally phosphorylated RNA moiety at its 5'-end: (1) the nascent DNA was resistant to exonucleolytic degradation by spleen phosphodiesterase unless first hydrolyzed by strong alkali or ribonuclease; (2) the 5'-termini of nascent DNA could not be phosphorylated by polynucleotide kinase unless first treated with alkaline phosphatase or subjected to hydrolysis by strong alkali or ribonuclease; (3) alkaline hydrolysis of nascent DNA labeled with 32P at the 5'-end released unlabeled DNA with a free 5'-terminus and 32P-labeled ribonucleoside 3',5'-bisphosphates; (4) ribonuclease degradation of similarly labeled material produced an unlabeled DNA-containing polynucleotide fraction and 32P-labeled ribo-oligonucleotides; (5) chromatography on dihydroxyboryl cellulose showed that the RNA moiety lacked a 3'-terminal cis-diol grouping (even after treatment with alkaline phosphatase) unless first subjected to the 3'-exonucleolytic action of bacteriophage T4 DNA polymerase . The sequence of the ribonucleotide chains was elucidated by end-group labeling with polynucleotide kinase and digestion with various ribonucleases . The ribonucleotide moiety was primarily three and four residues in length with the predominant sequence (pp)pApG(pC)1-2pDNA . The possibility that it represents a primer for discontinuous DNA synthesis is discussed. J Biol Chem, 1983 Jan 10, 258(1), 291 - 8 Structure and organization of a cluster of sic tRNA genes in the space between tandem ribosomal RNA gene sets in Bacillus subtilis; Wawrousek EF et al.; Hybridization of Southern blots of EcoRI digests of total Bacillus subtilis DNA with ribosomal RNA and transfer RNA probes provides evidence of highly clustered tRNA and rRNA genes, with several tRNA clusters being located in spaces which span between tandem ribosomal RNA gene sets . Clones containing these tRNA clusters were isolated from a Charon 4A library . One of them, denoted trrnB, was partially sequenced . A cluster of six tRNA genes was found, with anticodon assignments of Asn, Thr, Gly, Arg, Pro, Ala . This cluster is closely flanked on both sides by ribosomal RNA gene sets, which were identified by sequencing upward through the 5 S rRNA gene, across a space, and into the 23 S rRNA gene, and also sequencing downward into the 16 S rRNA gene . The tRNA gene cluster appears to be organized into at least two transcriptional units separated by an attenuator region . These transcriptional units may be components of the flanking ribosomal RNA operons . The putative promoter region of the downstream 16 S rRNA is organized differently from Escherichia coli; it is smaller and seems less complex . This gene organization provides insight into possible mechanisms for coordinate and differential control of transfer and ribosomal RNA gene expression. Zentralbl Mikrobiol, 1983, 138(1), 17 - 23 Phosphate-solubilizing potentiality of the microorganisms capable of utilizing aluminium phosphate as a sole phosphate source; Banik S et al.; Eight bacteria, each of the genus Bacillus, two actinomycetes, each of the genus Streptomyces, and six fungi, one each of the genus Penicillium and Chaetomium and four of the genus Aspergillus, were isolated on AlPO4-sucrose agar from a typical Indian lateritic soil (Typic Ochragualf) . All of them were capable of solubilizing Ca3(PO4)2 to a higher degree than AlPO4 . Bacillus subtilis (B-7655), LAB4, Bacillus sp., LAB5, Penicillium sp., LAF2, and Aspergillus spp., LAF3 and LAF4, were solubilizing Ca3(PO4)2 very efficiently, but AlPO4 to a lesser degree . Bacillus spp., LAB1, LAB2, LAB5, LAB6 and LAB7, Chaetomium nigricolor, LAF1, and Aspergillus spp., LAF5 and LAF6, were unable to bring detectable amounts of soluble phosphorus to solution from AlPO4 . Except Chaetomium, all the other organisms produced free aliphatic organic acid in detectable amounts . The organic acids produced were oxalic, succinic, citric, and 2-keto gluconic acid . 2-Keto gluconic acid, singly and in combination with succinic or citric acid, accounted for higher solubilization . Amount of free organic acids in the growth medium was not directly correlated with phosphate solubilization. Jpn J Antibiot, 1983 Jan, 36(1), 55 - 70 {Clinical studies on gentamicin for infectious diseases following intravenous drip infusion}; Nakamura T et al.; An antibiotic drug of aminoglycoside group, gentamicin (GM) for parenteral use was used to 14 hospitalized patients; 5 with acute or subacute cholecystitis, 6 with acute peritonitis (4 cases were due to acute appendicitis, a case was torsion of right ovarian cyst and a case was cecal CROHN's disease), 1 with fistula ani and abscess, and 2 with localized peritonitis after gastrectomy due to gastric ulcer . GM in a dose of 60 mg were administered by intravenous drip infusion for 1 to 2 hours, twice a day for 4 to 12 days . To the cases of biliary tract infection, GM was treated for preoperative chemotherapy and to the other cases GM was treated for postoperative chemotherapy . Clinical response was excellent in 7 cases, good in 6 cases, fair in 1 case and poor in none . No adverse effect was observed . The organisms were isolated in 7 cases, 7 were Escherichia coli, 2 were Klebsiella pneumoniae and 3 were Bacteroides fragilis . The MICs for GM were 0.78--1.56 micrograms/ml in 10(8) and 10(6) cells/ml, except B . fragilis . Before the operation of above cases, GM in a dose of 60 mg (a case was 40 mg) were administered by intravenous drip infusion for 1 to 2 hours in 7 cases (3 biliary tract infection, 2 acute peritonitis and 2 gastric ulcer) and 7 cases by intramuscularly . The materials of common duct bile, gall bladder bile, gall bladder wall, the appendix and other tissues, ascites and serum samples were taken during the operation . GM concentration was measured by bioassay method with Bacillus subtilis ATCC 6633 as test organism . GM concentrations in bile and gall bladder wall after intravenous drip infusion were higher than those after intramuscular administration . In the appendicitis with localized peritonitis, GM concentration in the appendix wall with catarrhal appendicitis was 0.90 microgram/g after intramuscular administration . In the cases with diffuse peritonitis and catarrhal appendicitis, GM concentrations in appendixes were 1.18 micrograms/g and 1.37 micrograms/g after intravenous drip infusion . Therefore, it was supposed that GM could be used safety and usefully by intravenous drip infusion than that by intramuscular administration. Boll Ist Sieroter Milan, 1983, 62(6), 509 - 16 Immunomodulation by Bacillus subtilis spores; Benedettini G et al.; This study was undertaken in order to investigate the immunomodulatory effect of heat-killed Bacillus subtilis spores as well as of one soluble and one insoluble fraction obtained after their mechanical breakage . The results demonstrate that heat-killed spores, the soluble and the insoluble fraction enhance the primary PFC response to T-dependent antigens such as SRBC and HRBC, but not to T-independent antigens such as Ficoll-TNP . However, the same doses capable of enhancing the production of antibodies against T-dependent antigens were unable to modify the host CMI, evaluated in terms of contact sensitivity to oxazolone. Microbiol Immunol, 1983, 27(12), 1005 - 19 Sporulation promoting factor in vegetative cells of Bacillus subtilis; Hachisuka Y et al.; A simple experimental system for detection of sporulation promoting factors was presented . This system showed that there was a sporulation promoting factor in the vegetative cells of Bacillus subtilis cultivated on nutrient agar for 9 hr (at stage T0) . The factor was partially purified from the sonicate of vegetative cells by ethanol fractionation, gel filtration, chromatography and preparative gel electrophoresis, and it was identified as manganese-containing protein. Mol Gen Genet, 1983, 192(3), 386 - 90 Mapping of the genes for Bacillus subtilis ribosomal proteins S6 and S16: comparison of the chromosomal distribution of ribosomal protein genes in this bacterium with the distribution in Escherichia coli; Dabbs ER; Using mutants of Bacillus subtilis with alterations in the small subunit ribosomal proteins S6 and S16, the corresponding genes were mapped . rpsF was located between purA and cysA, close to the origin of replication . rpsP was located near pyrD, at about 135 degrees on the linkage map . The distribution on the chromosome of the ribosomal protein gene loci so far identified in B . subtilis was compared with the distribution of the genes for the same proteins in Escherichia coli . Considered in terms of relative distance from the origin and terminus of chromosomal replication, the order was the same in both species with the sole exception of protein S6. Mol Gen Genet, 1983, 192(3), 330 - 4 Isolation and mapping of a new suppressor mutation of an early sporulation gene spoOF mutation in Bacillus subtilis; Kawamura F et al.; We constructed an spoOF deletion (spoOF delta S) mutant of Bacillus subtilis by inserting a chromosomal segment carried by plasmid pUBSF delta S . This plasmid carries a 0.7-kilobase pair deletion that removes the spoOF promoter and a part of the structural gene . We used the spoOF deletion mutant to isolate a new intergenic suppressor of the spoOF phenotype, designated sof1 . The sof1 suppressor completely restores the sporulation ability of all spoOF defective mutants tested, including spoOF77, spoOF221 and spoOF delta S . The sof1 suppressor maps to the left of lys1 on the B . subtilis chromosome, in a region rich in sporulation markers and distant from the spoOF locus. Microbiol Immunol, 1983, 27(9), 733 - 48 Effect of disruption by sonication under different conditions on the activity of glucose dehydrogenase from resting spores of Bacillus subtilis; Tochikubo K et al.; The activity of glucose dehydrogenase present in resting spores of Bacillus subtilis varied strikingly with the conditions for disrupting the spores by sonic treatment, namely, the time and strength of sonication, and the type and pH of the solution used for suspending the spores . When the resting spores were sonicated for 30 min at a current of 1.45 A in 100 mM phosphate buffer in the range of pH 6.0 to 6.6 or in deionized water, the enzyme activity of the former suspension was approximately 10 times higher than that of the latter suspension . However, the enzyme activity of the latter was markedly stimulated in the presence of sodium chloride . The glucose dehydrogenase from resting spores disrupted in 100 mM phosphate buffer (pH 6.6) was a salt-independent, active enzyme with a molecular weight of about 120,000, whereas the enzyme from resting spores disrupted in deionized water was a salt-dependent, inactive one with a molecular weight of about 55,000 . A high concentration of dipicolinic acid strongly inhibited activation by a salt of inactive glucose dehydrogenase from resting spores in deionized water, suggesting one of its several important roles in vivo. Microbios, 1983, 38(153-154), 205 - 16 Influence of temperature-induced competence in the genetic transformation of Bacillus subtilis; Lopez P et al.; Competence of Bacillus subtilis can be induced or repressed by decreasing or raising the temperature of incubation from 42 degrees C to 37 degrees C or vice-versa . Concomitantly with changes in transformability of the cultures, changes in the cell ability to bind 3H-transforming DNA and to degrade the donor DNA was discovered . These alterations reflect changes in the processing of donor DNA rather than at the level of integration of the transforming DNA, since phage SPP1 DNA transfection was affected to the same extent as transformation . Induction of competence development by temperature shift from 42 degrees C to 37 degrees C requires continuous synthesis of protein(s) involved in the binding of donor DNA to the competent cells, whereas the nucleolytic activity related to DNA entry into the cells is already synthesized at the moment of the induction of competence. Mol Gen Genet, 1983, 192(1-2), 149 - 54 Plasmid transformation in Bacillus subtilis . Effects of the insertion of Bacillus subtilis rRNA genes into plasmids; Iglesias A et al.; Two HindIII generated DNA fragments of 3.0 and 2.3 Kb derived from rRNA genes of B . subtilis were cloned in E . coli with pBR322 . The 3.0 Kb fragment could be subcloned in B . subtilis using pC194 . However, only the multimeric, but not the monomeric derivatives of this hybrid plasmid were active in transformation of B . subtilis cells . The 2.3 Kb fragment could neither be subcloned in pC194 nor in pPL603, using both cell and protoplast transformation . We attribute the nonclonability of the 2.3 Kb fragment in B . subtilis to the presence of strong promoter activity in this fragment . Direct proof for the presence of a strong promoter, which is apparently responsible for the transcription of the rRNA gene, was obtained in experiments with B . subtilis and E . coli promoter search plasmids. Mol Gen Genet, 1983, 192(1-2), 124 - 30 Arrangement of loci within the principal cluster of ribosomal protein genes of Bacillus subtilis; Dabbs ER; Strains of Bacillus subtilis harboring mutations causing altered migration of one or more of ten ribosomal proteins were used to map the corresponding gene loci . The loci of the genes for proteins BL1, BL5, BL9, and BL11 mapped between cysA and rpoB . The locus of the gene for protein S7 was very close to the gene for protein S12 (rpsL) . Those for proteins S8, BL6, BL7, BL8, and BL23 were between rpsL and rpsE (the gene for protein S5) . Where the order of loci could be deduced, it was strikingly similar to that of Escherichia coli. Microbiol Immunol, 1983, 27(7), 565 - 74 Two active forms of valyl-tRNA synthetase from Bacillus subtilis: alteration related to the early stages of sporulation; Ohyama K et al.; Two enzymatically active forms, E-I and E-II, of valyl-tRNA synthetase {EC 6.1.1.9} from cells at various stages in the life cycle of Bacillus subtilis 168 LTT, germinated cells, vegetative cells (t-0.5), sporulating cells (t0, t1, t3, and t4), forespores and mature spores, were analyzed by hydroxyapatite column chromatography . The E-II activity was detected in the main fraction of valyl-tRNA synthetase during the life cycle of B . subtilis 168 LTT . The high activity of E-II at t0 decreased rapidly in the stationary and sporulating phases . On the other hand, the E-I activity increased in the early sporulating stage and was about twofold higher at t3 than at t0 . After t3, this activity also decreased rapidly and was not detected in forespores and mature spores . The relative amount of E-I at t0 was 3.4% of the total valyl-tRNA synthetase activity eluted from the hydroxyapatite column, 12.9% at t1 and 29.2% at t3, but it was less than 10% at t4 and in germinated cells . The alteration in E-I and E-II activities was also observed in cells of B . subtilis NIG 1121 (spo+), W23 and 168W, but not in any asporogenous mutant strain studied . These results show that the alteration in the valyl-tRNA synthetase activity appears only during the early stages of sporulation and is closely related to the sporulation of B . subtilis. Mol Gen Genet, 1983, 191(1), 138 - 44 {Genetic analysis of sacB, the structural gene of a secreted enzyme, levansucrase of Bacillus subtilis Marburg}; Steinmetz M et al.; The structural gene sacB encoding B . subtilis levansucrase, a secreted enzyme, expresses in E . coli . E . coli hosts of the sacB gene are poisoned by sucrose . This property allowed a powerful selection of mutants affected in the cloned gene . The plasmidic mutations were readily introduced in the B . subtilis chromosome . Using a collection of plasmids bearing various deletions extending in sacB we developed a technique of deletion mapping based on plasmid integration in the chromosome of B . subtilis . A generalization of this technique is discussed. Mol Gen Genet, 1983, 190(3), 481 - 6 The genetics and specificity of the constitutive excision repair system of Bacillus subtilis; Friedman BM et al.; An isogenic set of DNA repair-proficient and -deficient strains of B . subtilis, cured of all prophages, were constructed and analyzed for their sensitivities to selected mutagens . The results demonstrated that the lethal damage caused by ultraviolet (UV) radiation and by 4-nitroquinoline-1-oxide (4NQO) were repaired by the bacterial excision and/or recombination repair systems . In contrast, the lethal damages caused by ethyl methane sulfonate (EMS) and methyl methane sulfonate (MMS) were removed from the DNA by the recombination repair system of the bacteria, and not by the excision repair system . Significantly, the bacteria required both a functional recombination repair system and a functional excision repair system in order to remove the DNA damage caused by the bifunctional alkylating agent mitomycin C (MC). Mol Gen Genet, 1983, 190(3), 475 - 80 DNA repair in B . subtilis: an inducible dimer specific W-reactivation system; Fields PI et al.; The W-reactivation system of Bacillus subtilis repairs pyrimidine dimers in bacteriophage DNA . This inducible repair system can be activated by treatment of the bacteria with UV, alkylating agents, cross-linking agents and gamma radiation . However, bacteriophage treated with agents other than those that cause pyrimidine dimers were not repaired by this unique form of W-reactivation . In contrast, the W-reactivation system of Escherichia coli repairs a variety of damages in the bacteriophage DNA. Mol Gen Genet, 1983, 190(3), 384 - 9 Expression of antibiotic resistance genes from Escherichia coli in Bacillus subtilis; Kreft J et al.; Bifunctional recombinant plasmids were constructed, comprised of the E . coli vectors pBR322, pBR325 and pACYC184 and different plasmids from Gram-positive bacteria, e.g . pBSU161-1 of B . subtilis and pUB110 and pC221 of S . aureus . The beta-lactamase (bla) gene and the chloramphenicol acetyltransferase (cat) gene from the E . coli plasmids were not transcribed and therefore not expressed in B . subtilis . However, tetracycline resistance from the E . coli plasmids was expressed in B . subtilis . Transcription of the tetracycline resistance gene(s) started in B . subtilis at or near the original E . coli promoter, the sequence of which is almost identical with the sequence recognized by sigma 55 of B . subtilis RNA polymerase. Jpn J Clin Oncol, 1983, 13 Suppl 1, 127 - 31 Microwave food processing for bioclean room patients; Fujita S et al.; In order to establish an efficient way to prepare diets for patients in bioclean rooms, sterilization and temperature increase of food and drinks were studied by means of a specially designed microwave ove