Bacillus subtilis

Rev Argent Microbiol, 1982, 14(3), 167 - 70
{Bacillus subtilis BSA 170 trp- ura-: a new nutritional mutant with absolute requirements for exogenous tryptophan and uracil for its growth}; Franco MA et al.; A new Bacillus subtilis mutant was prepared, with a double nutritional requirement for uracil and tryptophan . The mutant, designed Bacillus subtilis BSA 170 trp- ura-, was constructed by transformation method, acting B . subtilis strain PB 168 trp- as recipient and B . subtilis strain PB 3308 ura- as transforming DNA donor cells . The BSA 170 trp- ura- strain was selected by replication of transformed population on nutritionally selective media . Competence development induction and genetic markers transformability were tested . The new mutant was competent by Young and Spizizen's methodology . Furthermore, both markers, uracil and tryptophan, may be transformed when B . subtilis BSA 170 trp- ura- competent cells are treated with transforming DNA isolated from B . subtilis PB 19, prototroph . Transformation frequency rate for each marker alone was far larger than that reached for both taken together.

Rev Argent Microbiol, 1982, 14(2), 111 - 4
{Antimicrobial activity of the epicarp of the fruits of Paulownia fortunei and Paulownia tomentosa}; Cercos AP; One antimicrobial substance was obtained from the epicarp of the fruits of Paulownia fortunei and Paulownia tomentosa . Other parts of the fruits and leaves had no detectable antimicrobial activity . The substance was active in vitro for Staphylococcus aureus and Bacillus subtilis while it had lower activity for Saccharomyces carlsbergensis and the lowest was for Escherichia coli . The active material was extracted with organic solvents (ether, ethanol and acetone) . The activity "in vitro" was demonstrated by the method of dilution in nutrient agar media . The active substance looked as a resin and was insoluble in water at neutral or acid pH . It was very soluble in strong alkaline pH solution.

Mol Gen Genet, 1982, 188(3), 499 - 507
Initiation of recombination during transformation of Bacillus subtilis requires no extensive homologous sequences; van Randen J et al.; Lysates obtained shortly after entry of transforming DNA to Bacillus subtilis contain donor-recipient DNA complexes, in which the donor moiety is associated with the recipient DNA in an unstable way . The complexes could be artificially stabilized by crosslinking with 4,5',8-trimethylpsoralen . The unstable complexes dissociated upon helix-destabilizing treatments, such as heating at 70 degrees C, and CsCl gradient centrifugation at pH 11.2, but remained stable during CsCl gradient centrifugation at pH 10 . Donor-recipient DNA complexes were not formed after entry of heterologous pUB110 DNA . These observations suggest that base-pairing is involved in the unstable association . The donor moiety of the unstable complexes was completely, or almost completely, digestible by nuclease S1, indicating that the donor and recipient base-sequences are only paired over very short distances . The unstable donor-recipient DNA complexes are true recombination intermediates because (i) strain 7G224 (recE4) was impaired in the formation of the unstable complexes, and (ii) the unstable complexes were rapidly converted to stable complexes in recombination proficient strains, whereas their conversion was delayed in the recombination deficient strain 7G84 . Unstable complexes were also formed with Escherichia coli donor DNA, but to a lesser extent . Apparently a limited degree of base-sequence homology is sufficient to initiate recombination.

Z Allg Mikrobiol, 1982, 22(7), 495 - 502
{Activation of protein biosynthesis in outgrowing spores of Bacillus subtilis}; Wachlin G et al.; The programme of protein synthesis as an indicator for the control of gene expression was examined during outgrowth of Bacillus subtilis spores . At various stages of outgrowth cells of Bacillus subtilis were labelled with 35S-L-methionine . Extracted proteins were separated on two-dimensional gels according to O'Farrell (1975) . Three groups of proteins were synthesized during outgrowth: 1 . During all stages of outgrowth a great number of "vegetative genes" is expressed . The programme of protein synthesis of the outgrowing cell is very similar to that of a vegetative cell . 2 . Only a few proteins--probably the products of outgrowth-genes--are synthesized especially in outgrowing spores and turned off at different stages of outgrowth . 3 . The synthesis of a minor group of vegetative proteins is triggered during different stages of outgrowth . In contrast to earlier assumptions (comp . Torriani and Levinthal 1967, Hansen et al . 1970, Galizzi et al . 1976) we suggest that only a small portion of the genome is activated during outgrowth as a dependent sequence . These results are discussed on the basis of earlier concepts about the regulation of outgrowth as a developmentally regulated gene expression programme.

Mol Gen Genet, 1982, 188(2), 272 - 8
Diploid state of phenotypically recombinant progeny arising after protoplast fusion in Bacillus subtilis; Sanchez-Rivas C et al.; After fusion of Bacillus subtilis protoplasts the phenotypically recombinant clones isolated, whether immediately or as segregants of complementing diploid clones, have in common the following properties . They appear independently of the recN+ gene, most often as the result of apparently non-reciprocal recombination occurring in genetic intervals encompassing the origin and the terminus of replication . First indicated by reciprocal fusion crosses between o105-lysogenic and o105-sensitive strains, the diploidy of the recombinants was confirmed by studying the transforming activities of their DNA . These experiments establish heterozygosity at eight loci scattered on the chromosome map . By revealing the presence of the trpF+ allele in trpF7 recombinants, the results also strongly suggest that stable phenotypic recombinants may arise by genetic inactivation . Two possible genetic structures for these recombinants are discussed, one implying total inactivation of one recombinant chromosome, the other a segmentary inactivation of one unrecombined chromosome . Whatever the structure, genetic stability is not a reliable sign of haploidy in bacterial clones produced after protoplast fusion.

Mol Gen Genet, 1982, 187(3), 439 - 45
Transformation of Bacillus subtilis competent cells: identification of a protein involved in recombination; de Vos WM et al.; With the use of two-dimensional gel electrophoresis, the proteins present in a transformation-proficient B . subtilis strain were compared with those present in an isogenic, recombination-deficient strain carrying the recE4 mutation . One protein (molecular weight 45 kD, iso-electric point 5.4) was found to be virtually absent in the recE4 strain . This 45 kD protein is a prominent protein predominantly present in the competent fraction of a competent culture . The synthesis of the protein is substantially stimulated by irradiation with ultraviolet light or treatment with mitomycin C and, to a lesser extent, by treatment with nalidixic acid . Since the protein is also observed in a strain cured for SP beta and carrying non-inducible PBS X, it is unlikely that this protein is a gene product specified by one of these prophages usually present in B . subtilis strain 168 . Based on these results we conclude that the 45 kD protein is involved in recombination in B . subtilis.

Mol Gen Genet, 1982, 187(2), 297 - 301
Selection in Bacillus subtilis giving rise to strains with mutational alterations in a variety of ribosomal proteins; Dabbs ER; To facilitate mapping of ribosomal protein genes in Bacillus subtilis, a selection was devised which gave rise to strains with alterations in any one of a variety or ribosomal proteins . Alterations in eighteen ribosomal proteins were identified when eighty mutants were analysed . In addition, one strain showed a major assembly defect in the large ribosomal subunit resulting in the presence of a particle sedimenting at about 40S . Eighteen large subunit proteins were present on this particle in normal amounts, while twelve proteins were much reduced in amount or undetectable.

Z Allg Mikrobiol, 1982, 22(4), 255 - 60
The localization of SPO1 phage resistance in the genome of Bacillus subtilis as revealed by fusion of protoplasts; Mach F et al.; We localized the gene for resistance to phage SPO1 relatively to the markers pur B 34 and ura by means of the polyethylene-glycol induced fusion of bacterial protoplasts of three-fold auxotrophic Bacillus subtilis strains S3 and S13 . By this same method, the site of some auxotrophic markers was tentatively determined . The application of the protoplast fusion technique to exact genetic analysis will not be possible until the exo- and endogenous factors influencing cell wall regeneration are standardized . Fluctuations of this kind are very significant for the determination of genetic segregation.

Mol Gen Genet, 1982, 186(3), 399 - 404
Cloning and expression in Escherichia coli of the sucrase gene from Bacillus subtilis; Fouet A et al.; A recombinant cosmid carrying the sucrase gene (sacA) was obtained from a colony bank of E . coli harboring recombinant cosmids representative of the B . subtilis genome . It was shown that the sacA gene is located in a 2kb EcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment . A fragment of 2kb containing the gene was subcloned in both orientations in the bifunctional vector pHV33 and expression was further looked for in B . subtilis and E . coli . Complementation of a sacA mutation was observed in Rec+ and REc- strains of B . subtilis . Expression of sucrase was also demonstrated in E . coli, which is normally devoid of this activity, by SDS-polyacrylamide gel electrophoresis, specific immunoprecipitation and assay of the enzyme in crude extracts . The specific activity of the enzyme depended on the orientation of the inserted fragment . The saccharolytic activity was found to be cryptic in E . coli since the presence of the recombinant plasmids did not allow the transport of {U14C} sucrose and the growth of the cells . It was shown also that the recombinant cosmid contained part of the neighboring locus (sacP) which corresponds to a component of the PEP-dependent phosphotransferase system of sucrose transport of B . subtilis.

Mol Gen Genet, 1982, 186(3), 347 - 54
Revertants of a streptomycin-resistant, oligosporogenous mutant of Bacillus subtilis; Henkin TM et al.; Revertants of a streptomycin-resistant (Strr), oligosporogenous (Spo-) mutant of Bacillus subtilis were selected form the ability to sporulate . The revertants obtained fell into two phenotypic classes: Strs Spo+ (streptomycin-sensitive, sporeforming), which arose by reversion of the streptomycin resistance mutations of the parent strain; and Strr Spo+, which arose by the acquisition of additional mutations, some of which were shown to affect ribosomal proteins . Alterations of ribosomal proteins S4 and S16 in the 30S subunit and L18 inthe 50S subunit were detected in Strr Spo+ revertants by polyacrylamide gel electrophoresis . Streptomycin resistance of the parental strain and the Strr revertants was demonstrated to reside in the 30S ribosomal subunit . The second site mutations of the revertants depressed the level of streptomycin resistance in vivo and in the in vitro translation of phage SP01 messenger ribonucleic acid (mRNA) relative to the resistance exhibited by the Strr parental strain . The Strr parent grew slowly and sporulated at approximately 1% of the wild type level . The Strr revertants closely resembled the wild type strain with regard to growth and sporulation . The Strr revertants grew at rates intermediate between those of the Strr patent and wild type, and sporulated at wild type levels.

Microbios, 1982, 33(132), 73 - 80
Identification of taurine occurring sporulating cells of Bacillus subtilis; Nakashio S et al.; An unidentified ninhydrin-positive substance was detected in trichloroacetic acid extract from the sporulating cells of Bacillus subtilis NRRL B558 . The substance was isolated as the corresponding 2,4-dinitrophenyl (DNP) derivative, the structure of which was identified with a pyridinium salt of DNP-taurine, based on spectral and synthetic evidence . a pyridine molecule should be introduced into a taurine molecule during the isolation procedure and hence, the original amino acid was assigned to taurine . Taurine was accumulated into the cells from the medium during the early stages os sporulation, and thereafter metabolized or excreted.

Mol Gen Genet, 1982, 186(1), 118 - 21
Restriction and modification in Bacillus subtilis 168 . Regulation of hsrM(nonB) expression in spoOA mutants and effects on permissiveness for phi15 and phi105 phages; Fucik V et al.; Gene hsr M (nonB) of Bacillus subtilis 168, causing non-permissiveness to phage SP10 (Saito et al . 1979) and reduced plating efficiency of unmodified phage phi105, is responsible for non-permissiveness of B . subtilis 168 for phages phi15 and PZA . Upon transformation to sporulation deficiency (allele spoOA) B . subtilis 168 becomes permissive for phi15 and PZA and loses the ability to restrict phi105 . spoOA str-1 double transformants of B . subtilis 168, however, retain the restriction 168 and non-permissiveness for phi15 and PZA phages, in spite of their Spo- phenotype . Therefore it appears that a functional product of the spoOA gene is required for expression of gene hsrM in wild-type bacteria, but is not essential in streptomycin-resistant bacteria . Phage genomes (PZA) were trapped in spores of the restriction deficient strain with much higher efficiency than in the wild-type.

Prikl Biokhim Mikrobiol, 1982 Jan-Feb, 18(1), 71 - 5
{Optimization of protein hydrolysis by Bacillus subtilis alkaline proteinase}; Bogatkov SV et al.; Casein hydrolysis by alkaline proteinase from Bacillus subtilis str . 72 had been investigated . It has been shown that the hydrolytic rate depends on the substrate concentration and declines rapidly with time even at low protein concentrations . It has been suggested to use an equation describing the time dependence of the rate of casein proteolysis and characterizing the optimum time of protein hydrolysis, maximum rate of hydrolysis, and the maximum yield of the reaction products.

Mol Gen Genet, 1982, 185(2), 363 - 4
Study of the phenomenon of marker rescue in plasmid transformation and transduction of intact cells, protoplasts and different rec mutants of Bacillus subtilis; Prozorov AA et al.; Marker rescue in plasmid transformation of competent cells of different rec mutants of B . subtilis was studied . In most cases the value of marker rescue decreased proportionally to reduction of plasmid transformation efficiency (although there were certain exceptions) . Marker rescue was not observed either in plasmid transformation of protoplasts or in plasmid transduction of intact cells.

Mol Gen Genet, 1982, 185(2), 339 - 43
RNA polymerase subunit biosynthesis in Bacillus subtilis; Libby RT et al.; The relative rates of RNA polymerase biosynthesis in Bacillus subtilis has been examined under steady-state growth conditions . The synthesis of RNA polymerase subunits (alpha, beta, beta', omega) has been followed by subunit fractionation of immunoprecipitated {3H}-labelled samples on SDS-polyacrylamide gels . The stoichiometries of alpha:beta:beta':omega subunits have been determined from cultures pulse-labelled during steady-state growth . The results suggest that an unassembled pool of the alpha-subunit exists from which the holoenzyme is formed . Upon shift-up from acetate to glycerol containing medium, a rapid rise in the differential rate of core enzyme synthesis was observed, while the rate of synthesis of the alpha-subunit was not stimulated . During shift-down, a concomitant reduction in the rate of synthesis of all subunits occurred for the first 20 min after the shift; thereafter, a rate of synthesis characteristic of the new growth rate was established . As cultures enter sporulation, an immediate reduction in the rate of beta beta'-subunit synthesis was demonstrated.

Mol Gen Genet, 1982, 185(2), 329 - 33
Direct selection of complementing diploids from PEG-induced fusion of Bacillus subtilis protoplasts; Sanchez-Rivas C; A minimal medium containing horse serum is described on which Bacillus subtilis protoplasts revert to bacillary forms at high frequency (ca . 30%) . Used as a plating medium for a mixture of polyethyleneglycol-treated protoplasts from two complementary polyauxotrophic parental strains, it selects the prototrophic fusion products efficiently, and also allows isolation of various auxotrophic recombinants . These prototrophs and recombinants amount respectively to 1% and 10% of the regenerated bacteria . We confirm that two types of prototrophs can be isolated after fusion: stable recombinants and complementing diploids, the latter segregating into various types of recombinants . Based on easily recognized colonial aspects, an approximate estimation of the proportion of the two types becomes possible when a spoOA mutation has been introduced in one of the parents . At least 50% of the prototrophic fusion products are complementing diploids . Incidently, the data also settle a controversy by showing the dominance of spoOA mutations in heterozygotic bacteria.

Mol Gen Genet, 1982, 185(2), 239 - 44
Purification and characterization of 30S ribosomal proteins from Bacillus subtilis: correlation to Escherichia coli 30S proteins; Higo K et al.; Twenty proteins were isolated from the 30S ribosomal subunits of Bacillus subtilis and their amino acid compositions and amino-terminal amino acid sequences were determined . These results were compared with the data of Escherichia coli 30S ribosomal proteins and the structural correspondence of individual ribosomal proteins has been established between B . subtilis and E . coli . Post-translational modifications of amino-terminal amino acids of the ribosomal proteins which have been found in E . coli are almost absent in B . subtilis with the exception of acetylated forms of S9.

Mol Gen Genet, 1982, 185(1), 69 - 74
Isolation and characterization of the nucleoid of non-complementing diploids from protoplast fusion in Bacillus subtilis; Guillen N et al.; Nucleoids of non-complementing diploids (Ncd) from protoplast fusion of B . subtilis were isolated . Their purified DNA banded in neutral CsCl gradient as a single unimodal peak of buoyant density 1.711 g/cm3, a value which is similar to that of the DNA purified from the original parental strains, suggesting that methylation of bases is not a significant factor in chromosome inactivation . Nucleoids released from a Ncd clone give two peaks in a sucrose gradient with a characteristic S value for each nucleoid . That is in contrast to nucleoids from the haploid parents whose sedimental patterns show only one peak . Both nucleoid preparations from Ncd strains assayed for transformation activity show the fast sedimenting nucleoid devoid of transformation activity while the slow nucleoid was active in transformation for the alleles carried by the genome which is expressed in vivo . Both nucleoids of the Ncd strains are transcribed in vivo . The RNA associated with the inactive chromosome is synthesized by the RNA polymerase of the active one . This study provides evidence that inactivation of one parental genome in the Ncd strain may be related with the tertiary organization of its DNA.

Mol Gen Genet, 1982, 185(1), 165 - 8
The effect of chlorpromazine on transformation in Bacillus subtilis; Mooibroek H et al.; In the presence of the widely used tranquilizer, chlorpromazine, transforming DNA of Bacillus subtilis is photoinactivated by long-wave ultraviolet light . The loss of biological activity is predominantly caused by lack of binding of the DNA to recipient cells and the introduction of single-strand breaks in the treated DNA.

Int J Tissue React, 1982, 4(1), 31 - 40
Phagocytosis and killing of cefazolin-exposed Staphylococci and Bacilli by mouse peritoneal macrophages; Carlone NA et al.; The phagocytic and microbicidal capacity of proteose peptone-induced macrophages from Balb/c mice was assessed in vitro against Staphylococcus aureus and Bacillus subtilis in the presence of a subinhibitory concentration of cefazolin . Enhanced phagocytosis (50-60%) and a similar killing effect (99%) after two hours, incubation was noted towards both test organisms . Electron microscopy showed phagocytic activity on both control and antibiotic-treated strains . Since cefazolin does not penetrate macrophages, the peritoneal cell morphology and ultrastructure were unaffected by the drug.

Folia Microbiol (Praha), 1982, 27(2), 73 - 5
Variation of antimetabolite sensitivity with different carbon sources in Bacillus subtilis; Chaudhuri A et al.; The susceptibility of Bacillus subtilis to amino acid analogues was found to be markedly influenced by the carbon source used in the test media . Thialysine inhibited the bacterium with a greater number of carbon sources than the other two analogues tested . 5-Hydroxylysine was inhibitory with glycerol, lactose, D-xylose, L-arabinose and soluble starch while ethionine showed toxicity with lactose, D-xylose and L-arabinose . None of these analogues were toxic at the levels tested when D-galactose was used as carbon source . The bacterium was not susceptible to thialysine with glycerol, to 5-hydroxylysine with L-arabinose and to ethionine with lactose.

Z Allg Mikrobiol, 1982, 22(1), 69 - 71
Electron microscopic demonstration of negative surface charges on cytoplasmic membranes of Bacillus subtilis with protamine-ferritin; Mach F et al.; The application of a protamine-ferritin conjugate for labelling of isolated protoplast membranes of Bacillus subtilis S 13/1 is described . Contrary to Mycoplasma membranes which could only be labelled on the outer side of the membrane, ferritin was deposited on both membrane sides as a single layer without cluster formation.

Can J Microbiol, 1982 Jan, 28(1), 80 - 6
Effects of various inhibitory agents on sporulation of Bacillus subtilis; Takahashi I et al.; Electron microscopic examinations of Bacillus subtilis cells revealed that relatively high concentrations of carbon sources blocked sporulation at stage 0 in most cells . Both nalidixic acid and novobiocin blocked sporulation at stage 0 . The cells treated with acridine orange showed the morphology of stage IV 5 h after the end of exponential growth, but no further progression was observed . Mutants that are able to sporulate in the presence of these agents had the characteristic morphological changes observed in uninhibited cultures.

Int J Biochem, 1982, 14(1), 71 - 4
Transfer of phospholipids between mesosomes and protoplasts from Bacillus subtilis; Lemaresquier H et al.; 1 . Phospholipids were more intensively labelled from ammonium {1-14C}glycerophosphate in mesosomes than in protoplasts isolated from Bacillus subtilis . 2 . When mesosomes, containing phospholipids labelled from sodium-{32P} phosphate were incubated with non radioactive protoplasts, labelled phospholipids were recovered in protoplasts after incubation . 3 . This transfer of phospholipids from mesosomes toward protoplasts is time-dependent . 4 . Soluble proteins obtained from Bacillus subtilis after ammonium sulphate precipitation were separated on a Sephadex G-100 column . 5 . The two main fractions were able to accelerate the transfer of phospholipids from mesosomes toward protoplasts . 6 . The first peak stimulated more actively the transfer of phosphatidylethanolamine whereas the second one preferentially accelerated the transfer of phosphatidylglycerol and diphosphatidylglycerol.

Appl Environ Microbiol, 1982 Jan, 43(1), 177 - 84
Bacillus subtilis "rec assay" test with isogenic strains; Mazza G; The Bacillus subtilis "rec assay" test, widely used for the detection of DNA-damaging agents, was studied in detail, using an isogenic set of strains carrying different mutations in repair or recombination functions or both . recE4 and rec-45 mutations turned out to confer on the cells the highest sensitivity to known mutagens . The recE4 strain and its isogenic rec+ strain have been tested and validated by several rec assay procedures for preferential killing by known DNA-damaging agents . The use of purified spores of the tester strains offers advantages for standardization of the assay.

J Bacteriol, 1982 Jan, 149(1), 391 - 4
Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis; McDonald KO et al.; A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance . Recombinant molecules generated in vitro were introduced into a B . subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants . A single colony was isolated containing the recombinant plasmid pKO101 . This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.

J Bacteriol, 1982 Jan, 149(1), 329 - 37
Identification of cell wall subunits in bacillus subtilis and analysis of their segregation during growth; Schlaeppi JM et al.; Continuous as well as pulse-labeling and chase experiments with Bacillus subtilis demonstrated that the cell wall (both peptidoglycan and teichoic acid) is composed of a limited number of blocks which, once completed, segregate during subsequent growth without undergoing any mixing with newly synthesized blocks . This observation suggests that new wall material is inserted in a limited number of zones . Previously reported observations which suggested diffuse intercalation of new wall material are reinterpreted on the basis of our results . Experiments performed on different media showed that the number of segregation units per unit of cell length and thus the density of insertion zones increases with medium richness . This finding suggests analogies between the regulation of cell wall and DNA synthesis.

Antonie Van Leeuwenhoek, 1982, 48(4), 353 - 64
Secretion of beta-lactamase by Escherichia coli in vivo and in vitro: effect of cerulenin; Mantsala P et al.; The effect of cerulenin on the production of beta-lactamase and other periplasmic proteins was studied in Escherichia coli IA199 carrying plasmid pBR322 . Cerulenin (10 to 25 microgram/ml) had almost no effect on the growth rate of E . coli but it decreased the amount of beta-lactamase and other periplasmic proteins in shock fluid . Higher amounts of the antibiotic (40 to 100 micrograms/ml) decreased turbidity and almost completely prevented synthesis of beta-lactamase and other periplasmic proteins . Cerulenin decreased incorporation of L-{35S}methionine into membranes during growth as well . Spheroplasts secreted beta-lactamase into the external medium, but during a 3-h incubation in the presence of cerulenin (25 micrograms/ml) this secretion was prevented by more than 90% . Beta-Lactamase was secreted into the isolated membrane vesicles from E . coli IA199 . However, only 5% of the total amount of pre-beta-lactamase was secreted and processed by the membranes in vitro . Cerulenin did not prevent processing in vitro but the membranes prepared from the cells grown in the presence of cerulenin (25 micrograms/ml) did not catalyze processing of pre-beta-lactamase at all . Membrane preparations from Bacillus subtilis did not process pre-beta-lactamase either in the absence or in the presence of cerulenin.

EMBO J, 1982, 1(12), 1565 - 71
Intramolecular recombination during plasmid transformation of Bacillus subtilis competent cells; Michel B et al.; We have constructed plasmids carrying direct internal repeats 260-2000 bp long . Monomers of such plasmids transformed Bacillus subtilis competent cells . The efficiency of transformation varied with the square of the length of repeats . The transformed clones harbored either the entire transforming plasmid and the plasmid arising by recombination between the repeats, or only the latter plasmid . Internally-repeated plasmids linearized by in vitro cleavage with restriction endonuclease could transform, yielding clones which exclusively harbored a plasmid resulting from recombination between the repeats . When the transforming plasmid carried repeats which differed slightly, conversion of one repeat into the other could occur . The following model of plasmid transformation accounts for these data: (1) plasmid DNA is cleaved and rendered linear in contact with competent cells; (2) a linear, at least partially double-stranded plasmid molecule is introduced or formed by repair within the cell; (3) a circular viable plasmid is produced by recombination between repeats carried on this molecule; (4) alternatively, a viable plasmid is produced by repairing the cut within one of the repeats by DNA synthesis which uses the other repeat as a template.

Mol Gen Genet, 1982, 187(1), 138 - 47
Sequence homology and recombination between the genomes of morphologically dissimilar bacteriophages LP 52 and theta; Forstova J et al.; A restriction fragment map of Bacillus licheniformis temperate phage LP 52 DNA (molecular weight 38.5 X 10(6)) was established, using restriction endonucleases BamHI (8 target sites), BglI (10 sites,) BglII (13 sites) and EcoRI (22 sites) . The map is linear, with well-defined ends, without any signs of circular permutation . The DNA of a related phage, LP 51, produced identical restriction fragments . At least 62% DNA of LP 52 has been found homologous to the DNA of the recently discovered, morphologically quite dissimilar, phage I, as demonstrated by hybridization of electrophoretically separated restriction fragments of DNA . Under the same conditions, the DNAs of LP 52 and of the morphologically similar Bacillus subtilis phage phi 105 did not cross-hybridize . The homologous regions in the genomes of phages LP 52 and I have been shown to be colinear . Comparison of the cleavage maps of phages LP 52 and I has shown that, within the regions of homology, not a single restriction fragment and few restriction sites have been conserved during divergent evolution . Three major regions of heterology were defined; the longest one, covering the right-hand end of the map (73 +/- 2.75% up to 100% LP 52 genome length) appeared to contain genes coding for structural proteins of the virions; a shorter region at the left-hand end of the map (coordinates zero to 10.3 +/- 3.3% LP 52 genome length) and a very short central region (coordinates 41.8-43.9%) could be identified, the latter apparently containing a regulatory locus responsible for the heteroimmune behavior of the two phages . Recombinants between phages LP 52 and I were isolated . Mapping of recombinant genomes has indicated mutual substitution of allelic pieces of LP 52 and I DNAs upon strict conservation of overall genome length.

Gene, 1982 Jan, 17(1), 91 - 100
Construction and use of SPP1v, a viral cloning vector for Bacillus subtilis; Heilmann H et al.; A unique BamHI restriction site has been inserted into a nonessential region of the genome of a deletion mutant of phage SPP1 . Construction of this phage, designated SPP1 v, required the in vitro conversion of a BclI site to a BamHI site . SPP1 v has been used as a vector phage to clone BamHI, BglII and BclI-generated restriction fragments of DNA . A direct selection for recombinants has been developed . Transfection with SPP1 v requires intact, genomic-length molecules, and cleavage with BamHI destroys the transfecting ability of this DNA . Recombinants in which the BamHI site has been destroyed by ligation to Bg/II or BclI-generated fragments of DNA become resistant to BamHI digestion after ligation and are active in transfection . Cloning of DNA containing BamHI sites has been accomplished by using the enzyme Bst1503I to methylate BamHI sites before insertion, and so to protect them during the BamHI digestion used to select against vector molecules . The in vitro construction of SPP1 v generated XmaIII sites directly adjacent to, and on both sides of the inserted BamHI site . This permits precise excision of cloned DNA even when cloning destroys the BamHI insertion site . Restriction-enzyme generated fragments of DNA in the size range of 0 to 4 Md have been cloned, including a full-length copy of plasmid pUB110, almost the complete sequence of plasmid pBR322, and a sequence of DNA that carried the lambda cos site.

Z Allg Mikrobiol, 1982, 22(10), 711 - 6
{Cell division and macromolecular synthesis in growing spores of a temperature sensitive filamentous mutant of Bacillus subtilis}; Prosch S et al.; A temperature sensitive mutant of Bacillus subtilis SB 19, strain ts 33-6 was characterized . This strain grows at 46 degrees C (restrictive temperature) with reduced intensity without septation processes . Under restrictive conditions DNA- and RNA-synthesis are remarkably reduced . DNA, however, is synthesized continuously under restrictive conditions causing the formation of multinuclear filaments . Septation, induced at permissive temperature, is not prevented under restrictive conditions . That means that under restrictive conditions initiation of septation is blocked whereas formation of septa can be observed . Shift-up experiments have shown that the initiation of septation processes occurs at an early stage of cell cycle.

Z Allg Mikrobiol, 1982, 22(8), 529 - 34
{Effect of nalidixic acid on the activation of RNA synthesis in outgrowing spores of Bacillus subtilis}; Hecker M; Outgrowth of B . subtilis spores depends on the action of DNA gyrase (comp . Matsuda and Kameyama 1980) . Application of nalidixic acid (100 micrograms/ml) to dormant spores of Bacillus subtilis prevents the outgrowth . Application of nalidixic acid (100 micrograms/ml) during the early outgrowth phase (after a 20 min germination period) does not prevent, but only delay spore outgrowth . Germination of spores is not influenced . Nalidixic acid is an effective inhibitor of RNA synthesis in outgrowing spores, whereas vegetative cells are more resistant . Spores can grow out inspite of a remarkably reduced intensity of RNA synthesis . Nalidixic acid particularly inhibits the synthesis of stable RNA, probably that of ribosomal RNA . We suggest that DNA gyrase-catalyzed alterations in DNA structure are involved in the regulation of the gene expressional program of outgrowing B . subtilis spores.

Folia Microbiol (Praha), 1982, 27(5), 340 - 9
Production of extracellular enzymes in continuous culture; Fencl Z et al.; The production of bacterial enzymes in batch fermentations is compared with results obtained in continuous culture . When studying the production of alpha-amylase in Bacillus subtilis it was found that instability of the enzyme synthesis was due to nonhomogeneity of the population rather than to "the culture's history" (i.e . succession of several physiological states necessary for the enzyme production) . The plasmid contained in the production clone was found to be the factor responsible for the alpha-amylase production . Predominance of the production clone or of the nonproduction one depends on the cultivation conditions used . As compared with batch cultivation the continuous production yields higher enzyme concentrations under optimal conditions and the fermentor productivity may be four to five times higher.

Folia Microbiol (Praha), 1982, 27(5), 323 - 7
Comparison of growth rates of two clones of Bacillus subtilis, producer of alpha-amylase; Pazlarova J et al.; In a population of the productive Bacillus subtilis strain the production of alpha-amylase differentiates at the cell level . Individual cells in the population were analyzed and the production of isolates was tested . The mean specific activity of alpha-amylase in the productive group after the isolation was 5.0 U/mg dry substance and in the nonproductive group 1.5 U/mg dry substance . This ratio remained unchanged during long-term observations and after repeated transfers . In selected strains of both groups the specific growth rate was determined in a synthetic medium containing various amounts of casein hydrolyzate . The detected differences in the growth rate between the productive and the nonproductive clones are determined by amino acid concentration.

Folia Microbiol (Praha), 1982, 27(4), 222 - 7
Gluconimycin an inhibitor of pyrimidine synthesis in Bacillus subtilis; Dewedar A et al.; Pyrimidine synthesis in Bacillus subtilis PCI-219 cells was the primary site of action of the antibiotic gluconimycin as indicated by its arresting the activity of aspartate carbamoyltransferase as well as accumulation of aspartic acid in cultures fortified with gluconimycin . Microbial growth and viable counts were suppressed by the antibiotic whereas glycolysis and aerobic respiration were insignificantly affected . Gluconimycin failed to induce a lytic effect on cell protoplasts while considerable amounts of substances absorbing at 260 nm were released from microbial cells treated with gluconimycin.

Mol Gen Genet, 1982, 186(3), 339 - 46
Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis; Moran CP Jr et al.; We have determined the nucleotide sequence of two Bacillus subtilis promoters (veg and tms) that are utilized by the principal form of B . subtilis RNA polymerase found in vegetative cells (sigma 55-RNA polymerase) and have compared our sequences to those of several previously reported Bacillus promoters . Hexanucleotide sequences centered approximately 35 (the "--35" region) and 10 (the "--10" region) base pairs upstream from the veg and tms transcription starting points (and separated by 17 base pairs) corresponded closely to the consensus hexanucleotides (TTGACA and TATAAT) attributed to Escherichia coli promoters . Conformity to the preferred --35 and --10 sequences may not be sufficient to promote efficient utilization by B . subtilis RNA polymerase, however, since three promoters (veg, tms and E . coli tac) that conform to these sequences and that are utilized efficiently by E . coli RNA polymerase were used with highly varied efficiencies by B . subtilis RNA polymerase . We have also analyzed mRNA sequences in DNA located downstream from eight B . subtilis chromosomal and phage promoters for nucleotide sequences that might signal the initiation of translation . In accordance with the rules of McLaughlin, Murray and Rabinowitz (1981), we observe mRNA nucleotide sequences with extensive complementarity to the 3' terminal region of B . subtilis 16S rRNA, followed by an initiation codon and an open reading frame.

J Bacteriol, 1982 Jan, 149(1), 79 - 91
Possible involvement of DNA-linked RNA in the initiation of Bacillus subtilis chromosome replication; Henckes G et al.; After thermal denaturation, an in vivo-labeled RNA was found in a temperature-sensitive initiation mutant of Bacillus subtilis (dna-37) associated with high-molecular-weight DNA . This RNA could be clearly distinguished from other RNA species by different techniques of separation, such as Sepharose 2B filtration, chromatography on nitrocellulose, and equilibrium centrifugation in density gradient . It was obtained even when HCHO was present during denaturation and chilling of nucleic acids and was still detected after a second denaturation as well as after incubation with proteinase K . Properties of the complex were not altered by prior treatment with RNase H . A control experiment using two samples of the complex treated either with pancreatic DNase or with pancreatic RNase, denatured together and centrifuged in the same density gradient, showed that no artifactual associations occur between the DNA and the RNA components of the complex . These results demonstrate that the DNA and RNA in the complex are associated by neither hydrogen bonds nor proteins, but are indicative of a DNA-RNA covalent linkage . In addition, during synchronous replication after a previous period at a nonpermissive temperature, DNA-linked RNA synthesis took place at specific times which coincided with the appearance of rifampin resistance of the first and the second replication cycles . A possible involvement of this RNA in the initiation of chromosome replication is discussed.

J Bacteriol, 1982 Jan, 149(1), 372 - 3
N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes; Kuhn H et al.; The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens . Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis.

J Bacteriol, 1982 Jan, 149(1), 378 - 80
Glutamine synthetase subunit mixing and regulation in Bacillus subtilis partial diploids; Gardner A et al.; A specialized transducing phage, SP beta c2 dglnA2, of Bacillus subtilis was used to construct partial diploids with various glutamine auxotrophs . The overproduction of manganese-stimulated glutamine synthetase no longer occurred in the diploids . The kinetics of heat inactivation of the enzyme extracted from two diploids suggests that there was subunit mixing.

J Biol Chem, 1981 Dec 25, 256(24), 13091 - 8
Purification, physical characterization, and NH2-terminal sequence of glutamine synthetase from the cyanobacterium Anabaena 7120; Orr J et al.; A procedure for the complete purification of glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) from the cyanobacterium (blue-green alga) Anabaena 7120 is described . The enzyme has structural characteristics in common with glutamine synthetases from other sources: a subunit molecular weight of approximately 50,000 and a native structure, determined by low dose exposure electron microscopy, consisting of a two-layered regular hexagon made up of 12 subunits . Sequence analysis suggests that the subunits are identical . There was no indication that the enzyme from cyanobacteria is adenylylated, a feature shared with the glutamine synthetase from Bacillus subtilis but not with the enzyme from Escherichia coli . NH2-terminal sequence analysis and the predicted conformation of the NH2-terminal regions showed definite homology among the enzymes from all three sources . The limited analysis suggested a stronger structural homology between the enzyme from Anabaena 7120 and that from E . coli.

J Clin Hosp Pharm, 1981 Dec, 6(4), 245 - 9
Quantitative structure activity relationships (QSAR) of lipophilic acids and related compounds on bacterial and mammalian cells; T'Ang A et al.; It is shown by means of regression analysis that the lipophilic character of the molecule as expressed by log P in octanol/water is very important in determining the relative activities of these compounds in all of the three systems examined . In addition, molecular weight is also important in some of the systems . In general, activity increases with increasing lipophilicity and molecular weight for the limited number of compounds studied . The role of degree of ionization of these acidic drugs may affect both penetration and intrinsic activity in different ways . The finding by Sheu et al., (1) that long-chain fatty acids inhibit gram-positive bacteria (Bacillus subtilis) but not gram-negative bacteria such as E . coli, due to the protective layer of lipopolysaccharide, is in agreement with the correlations obtained for many different series of antibacterial agents by Lien, Hansch & Anderson (2).

Biophys J, 1981 Dec, 36(3), 465 - 77
Fluorescence-determined preferential binding of quinacrine to DNA; Baldini G et al.; Quinacrine complexes with native DNA (Calf thymus, Micrococcus lysodeikticus, Escherichia coli, Bacillus subtilis, and Colstridium perfringens) and synthetic polynucleotides (poly(dA) . poly(dT), poly{d(A-T)} . poly{d(A-T)}, poly(dG) . poly(dC) and poly{d(G-C)} . poly{d(G-C)}) has been investigated in solution at 0.1 M NaCl, 0.05 M Tris HCl, 0.001 M EDTA, pH 7.5, at 20 degrees C . Fluorescence excitation spectra of complexes with dye concentration D = 5-30 microM and DNA phosphate concentration P = 400 microM have been examined from 300 to 500 nm, while collecting the emission above 520 nm . The amounts of free and bound quinacrine in the dye-DNA complexes have been determined by means of equilibrium dialysis experiments . Different affinities have been found for the various DNAs and their values have been examined with a model that assumes that the binding constants associated with alternating purine and pyrimidine sequences are larger than those relative to nonalternating ones . Among the alternating nearest neighbor base sequences, the Pyr(3'-5')Pur sequences, i.e., C-G, T-G, C-A and T-A seem to bind quinacrine stronger than the remaining sequences . In particular the three sites, where a G . C base pair is involved, are found to display higher affinities . Good agreement is found with recent calculations on the energetics of intercalation sites in DNA . The analysis of the equilibrium shows also that the strength of the excitation spectrum of bound dye depends strongly upon the ratio of bound quinacrine to DNA . This effect can be attributed to dye-dye energy transfer along DNA.

Int J Radiat Biol Relat Stud Phys Chem Med, 1981 Dec, 40(6), 613 - 22
Effects of oxygen and sulphydryl-containing compounds on irradiated transforming DNA . Part I . Actions of dithiothreitol; Held KD et al.; The actions and interactions of oxygen and the sulphydryl-containing compound dithiothreitol (DTT) upon the radiation sensitivity of the biological activity of purified Bacillus subtilis transforming DNA have been examined . It has previously been shown that the sensitivity of transforming DNA irradiated in dilute solution is less when irradiation is performed in 100 per cent O2 than when in 100 per cent N2, i.e . O2 protects transforming DNA with a dose-modifying factor of about 0.7 . DTT protects transforming DNA in a manner that is dependent on DTT concentration and on gassing conditions . In O2 the DTT protection can largely be attributed to the scavenging of .OH radicals by the DTT, but in anoxia DTT exerts a further protective effect which results in an increasing oxygen enhancement ratio (o.e.r.) with increasing DDT concentration to a maximum o.e.r . of about 14 at 2-5 mM DTT . This additional protective effect of DTT is attributable to hydrogen atom donation from DTT to DNA radicals, thus chemically repairing the DNA . Oxygen appears to block this chemical repair reaction.

Gene, 1981 Dec, 16(1-3), 199 - 206
Expression of Escherichia coli trp genes and the mouse dihydrofolate reductase gene cloned in Bacillus subtilis; Williams DM et al.; The mouse dihydrofolate reductase gene and a segment of the Escherichia coli trp operon are expressed in Bacillus subtilis when cloned in the "expression plasmid" pPL608 . The cloned mouse gene confers trimethoprim resistance on B . subtilis and the cloned trp fragment complements mutations in the B subtilis trpD, C and F genes Expression of both cloned fragments is dependent on a promoter present in the vector plasmid . The E . coli trp fragment is cloned in a HindIII site within a chloramphenicol acetyltransferase gene present on pPL608, and as a result, expression of the E . coli trpC gene product is inducible by chloramphenicol . The mouse gene is inserted at a PstI site preceding the chloramphenicol acetyltransferase gene and its expression is not chloramphenicol inducible . The replication functions and neomycin-resistance of pPL608 are derived from pUB110 . Accordingly, pPL608 is stably maintained at high copy number in B . subtilis.

Biochem J, 1981 Dec 1, 199(3), 795 - 805
Purification and characterization of methyl-accepting chemotaxis protein methyltransferase I in Bacillus subtilis; Ullah AH et al.; A methyltransferase that methylates one of the proteins involved in chemotactic adaptation to sensory stimuli in Bacillus subtilis was purified to homogeneity . The enzyme utilizes S-adenosylmethionine as donor for a methyl group that is transferred to a glutamate residue in a 69 000-mol.wt . membrane protein and also to a protein of 19 000 mol.wt . The molecular weights of the denatured enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and of the native enzyme by gel-filtration chromatography both show the protein to be a 44 000-mol.wt . monomer . Isoelectric focusing of the purified methyltransferase showed the protein to be a single species with isoelectric point pI 5.4 . On the basis of a molecular weight of 44 000, the molar absorption coefficient at 262 nm of the enzyme is 10.9 x 10(4) M-1 . cm-1 . The Km of the enzyme for S-adenosylmethionine is about 2 microM . The Ki for S-adenosylhomocysteine is about 0.2 microM . Ca2+ is a competitive inhibitor of methylation, with a Ki of 0.065 microM . The enzyme methylates membranes from the wild-type more efficiently than membranes isolated from a mutant strain defective in chemotaxis . The enzyme is unable to methylate Escherichia coli membranes.

Acta Pathol Microbiol Scand {C}, 1981 Dec, 89(6), 373 - 8
Enhancement of bacterial uptake and killing in lymphokine-activated human monocytes; Rasanen L et al.; Studies were undertaken to establish whether lymphokines affect bacteria in a direct and/or an indirect fashion via monocytes . Lymphokine-rich supernatants were generated by stimulating mononuclear cells with killed Bacillus subtilis bacteria . Various strains of bacteria were incubated in these culture supernatants and plated . Also, monocytes were incubated in the supernatants, after which their capacity to phagocytose and kill Staphylococcus albus was measured . It was found that the culture supernatants did not contain activities exerting a direct effect on bacteria . They did, however, contain factors activating monocytes . The uptake and the killing of staphylococci increased 91% and 68%, respectively, after three days' incubation in lymphokine-rich supernatants . The monocyte-activating principle eluted in Sephadex G-100 chromatography over a wide molecular weight range (greater than 68000 - less than 23000) was not inactivated at 56 degrees C and was reduced by L-fucose . It thus shared the characteristics of human MIF or MAF.

Infect Immun, 1981 Dec, 34(3), 712 - 7
Cell walls, peptidoglycans, and teichoic acids of gram-positive bacteria as polyclonal inducers and immunomodulators of proliferative and lymphokine responses of human B and T lymphocytes; Rasanen L et al.; In this study, the mitogenic and immunomodulating effects of bacterial cell wall preparations were investigated . Cell walls, peptidoglycans, and teichoic acids from Bacillus subtilis and Staphylococcus aureus Wood 46 activated both human T cells (supplemented with 10% monocytes) and B cells to proliferative and to produce leukocyte migration inhibitory factor . Similar results were obtained with adult and umbilical cord blood cells, suggesting that these bacterial preparations acted as mitogens . Cell walls and peptidoglycans had a modulating effect on purified protein derivative-induced and protein A-induced proliferation . In the presence of suboptimal concentrations of these stimulants, bacterial components enhanced the proliferative response . However, at optimal concentrations of purified protein derivative or protein A, bacterial components suppressed lymphocyte proliferation . Peptidoglycans solubilized by lysozyme activated B lymphocytes but not T cells . Solubilization had no effect on immunomodulating capacity.

Am J Hosp Pharm, 1981 Dec, 38(12), 1933 - 6
Comparison of an immunoassay and a microbiological assay for tobramycin serum concentrations; Fukuchi H et al.; An enzyme multiplied immunoassay technique (EMIT) and a microbiological assay were compared as methods for determining the concentration of tobramycin in human serum . Tobramycin sulfate in various concentrations (0.4-20.0 microgram/ml) was dissolved in lyophilized human serum . Tobramycin concentration was determined by EMIT assay and a microbiological agar diffusion assay using Bacillus subtilis ATCC 6633 as the test organism . Serum samples of 162 patients who received tobramycin 60 mg i.m . were also assayed by the two methods . The effect of six antibiotics on the determination of tobramycin concentration by EMIT assay was measured . To test whether freezing affects tobramycin concentration measured by EMIT assay, tobramycin in five concentrations in lyophilized human serum were frozen and analyzed at weekly intervals for seven weeks . The concentration of tobramycin determined by EMIT assay and microbiological assay in both lyophilized human serum and patient samples were highly correlated (r = 0.994 and 0.949) respectively) . Dibekacin, an aminoglycoside structurally similar to tobramycin, interfered with the determination of tobramycin concentration . No effect of freezing was observed . The results of this study show that EMIT assay is an acceptable method for routine laboratory analysis of tobramycin serum concentration . It is as accurate as the microbiological assay tested.

J Bacteriol, 1981 Dec, 148(3), 950 - 5
High-molecular-weight penicillin-binding proteins from membranes of bacilli; Waxman DJ et al.; Mixtures of high-molecular-weight, cephalosporin-sensitive penicillin-binding proteins (PBPs) can be purified from Bacillus subtilis membranes by cephalosporin affinity chromatography (G . Kleppe and J . L . Strominger, J . Biol . Chem . 254:4856-4862, 1979) . By appropriate modification of this technique, B . subtilis PBP 1 was purified to homogeneity, and a mixture of Bacillus stearothermophilus PBPs 1, 2, and 4 was isolated . {14C}penicillin-PBP complexes of high-molecular-weight PBPs purified from membranes of these two bacilli, after denaturation, were found to have chemical reactivities typical of the penicilloyl-serine derivative formed by D-alanine carboxypeptidase from B . stearothermophilus . Although enzymatic activity catalyzed by these and several other high-molecular-weight PBPs from gram-positive organisms has not been detected with cell wall-related substrates, a slow, enzymatic acylation of B . subtilis PBPs 1, 2ab, and 4 by {14C}-diacetyl-L-lysyl-D-alanyl-D-lactate was demonstrated . Further study is necessary to clarify the physiological relevance of the slow acylation by this analog of a natural cell wall biosynthetic intermediate.

J Bacteriol, 1981 Dec, 148(3), 1012 - 5
Defective specialized SP beta transducing bacteriophages of Bacillus subtilis that carry the sup-3 or sup-44 gene; Lipsky RH et al.; We isolated defective specialized transducing phages of SP beta that carry one of the extracistronic suppressors, sup-3 or sup-44 . Lysates containing these phages can be used in a simple spot test to determine whether an auxotrophic mutation can be suppressed . The sup-3 and sup-44 mutations are distinct, in that their suppression patterns differ for the markers hisA1, metC3, and thr-5; and they are not alleles.

J Bacteriol, 1981 Dec, 148(3), 869 - 76
Thiolation and 2-methylthio- modification of Bacillus subtilis transfer ribonucleic acids; Vold BS et al.; Six thionucleosides found in Bacillus subtilis transfer ribonucleic acids were investigated: N6-(delta 2-isopentenyl)-2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, 2-methylthioadenosine, N-{(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl}threonine, and one unknown (X1) . The presence of N-{(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl}threonine was demonstrated based on the affinity of the transfer ribonucleic acid containing it for an immunoadsorbent made with the antibody directed toward N-{9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl}-L-threonine . The existance of N-{(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl}threonine in two species of lysine transfer ribonucleic acids was also confirmed by high-resolution mass spectrometry . Four of these thionucleosides--N6-(delta 2-isopenenyl)-2-methylthioadenosine, 2-methylthioadenosine, 5-carboxymethylaminomethyl-2-thiouridine, and the unknown designated X1--occurred only in specific areas in the elution profile of an RPC-5 column and probably affect the chromatographic properties of the transfer ribonucleic acids containing them . In contrast with Escherichia coli, where 4-thiouridine is the most frequent type of sulfur-containing modification, approximately one-third of the sulfur groups in B . subtilis transfer ribonucleic acid are present as thiomethyl groups on the 2 position of an adenosine or modified adenosine residue.

J Bacteriol, 1981 Dec, 148(3), 1002 - 5
Regulation of glutamate dehydrogenase in Bacillus subtilis; Kane JF et al.; The activity of the nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase in Bacillus subtilis was influenced by the carbon source, but not the nitrogen source, in the growth medium . The highest specific activity for this enzyme was found when B . subtilis was grown in a minimal or rich medium that contained glutamate as the carbon source . It is proposed that glutamate dehydrogenase serves a catabolic function in the metabolism of glutamate, is induced by glutamate, and is subject to catabolite repression.

Nucleic Acids Res, 1981 Nov 25, 9(22), 5991 - 6000
Nucleotide sequences of two Bacillus subtilis promoters used by Bacillus subtilis sigma-28 RNA polymerase; Gilman MZ et al.; RNA polymerase holoenzymes from many bacterial species share a common promoter recognition specificity since they use the same promoter sites on a variety of templates . These promoters generally include sequences homologous to the average sequences -TTGACA- and -TATAATA-, located -35 and -10 base pairs, respectively, upstream of the transcriptional state site . We have isolated a minor form of B . subtilis RNA polymerase in which the normal sigma subunit (sigma 55) is replaced by a smaller polypeptide (sigma 28) and which is highly specific for a class of promoter sites not used by the sigma 55-holoenzyme . Sequencing of two B . subtilis promoter sites used by the sigma 28-holoenzyme reveals identical sequences at -35 and -10 base pairs from the start site, which are -CTAAA- and -CCGATAT-, respectively . These results confirm that sigma subunit plays a major direct role in promoter sequence recognition, and support a model in which sigma interacts sequentially with -35 and -10 regions, respectively.

Nucleic Acids Res, 1981 Nov 25, 9(22), 5979 - 90
Nucleotide sequence of a Bacillus subtilis promoter recognized by Bacillus subtilis RNA polymerase containing sigma 37; Moran CP Jr et al.; We report the nucleotide sequence of the promoter for a Bacillus subtilis gene (spoVC) whose transcription is controlled by a 37,000 dalton species of B . subtilis sigma factor known as sigma 37 but not by the principal- sigma factor of 55,000 daltons (sigma 55) . Using S1 nuclease mapping we show that the startpoint for sigma 37-directed transcription of the spoVC gene in vitro corresponded closely to the 5' terminus of in vivo synthesized spoVC RNA . The binding site for sigma 37-containing RNA polymerase extended from 43 bp to 51 bp (positions -43 to -51) upstream from the transcription startpoint to 22 bp (position +22) downstream from the startpoint . The nucleotide sequence of the spoVC promoter differed significantly from promoters whose recognition is controlled by sigma 55 but was similar to other sigma 37- controlled promoters in regions known to be important in promoter recognition . Our results are consistent with the hypothesis (Lee and Pero, J . Mol . Biol., in press) that sigma factors work by contacting specific bases in both the -35 and -10 regions of promoters.

Science, 1981 Nov 13, 214(4522), 797 - 8
Incorporation of 4-amino-5-hydroxymethylpyrimidine into thiamine by microorganisms; White RH; One possible route for the biosynthesis of the (4-amino-2-methyl-5-pyrimidyl)-methyl moiety of thiamine would involve the formation of a methyl group on the demethylated pyrimidine, 4-amino-5-hydroxymethylpyrimidine, before its incorporation into thiamine . This possibility was tested by preparing the 4-amino-5-hydroxymethylpyrimidine and feeding it to Escherichia coli . Bacillus subtilis, and Saccharomyces cerevisiae . Analysis of the thiamine produced by these organisms showed that 4-amino-5-hydroxymethylpyrimidine was readily incorporated into thiamine without the addition of a methyl group, and no evidence was found for the conversion of this pyrimidine into normal methylated pyrimidine . Substitution of the demethylated thiamine for thiamine had no effect on the growth rate or the yield of E . coli cells . Complete substitution of the thiamine with the (4-amino-5-pyrimidyl)-methyl moiety was possible in an E . coli pur I mutant . The extent of incorporation of the demethylated pyrimidine decreased in some organisms and increased in others by the addition of adenine to the growth medium; this difference led to a simple test to separate organisms that use 5-aminoimidazole ribonucleotide for the biosynthesis of thiamine pyrimidine from those that do not.

Biochemistry, 1981 Nov 10, 20(23), 6564 - 9
Changes in the association between Bacillus subtilis RNA polymerase core and two specificity-determining subunits during transcription; Chelm BK et al.; The Bacillus subtilis RNA polymerase sigma subunit and the phage SPO1-coded gene 28 protein are responsible for selective binding of RNA polymerase to early and middle SPO1 promoters, respectively . The association of the RNA polymerase core with each of these subunits weakens during the elongation of RNA chains . Similar changes are known to be an essential part of the Escherichia coli RNA polymerase sigma cycle.

J Biol Chem, 1981 Nov 10, 256(21), 11283 - 91
Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene; McLaughlin JR et al.; The base sequence of the ribosome binding site region of the Gram-positive Staphylococcus aureus beta-lactamase gene has been determined . The leader peptide sequence of 24 amino acids which precedes the NH2 terminus of extracellular S . aureus beta-lactamase has also been established . This initiation site possesses two unique features not observed for most initiation sites recognized by Escherichia coli ribosomes . A novel initiation codon, UUG, initiates protein synthesis with methionine; and a very strong Shine-Dalgarno complementarity containing five G-C base pairs precedes the UUG initiation codon . The strong Shine-Dalgarno complementarity may explain the reduced translational dependence on initiation factor IF-3 function that has been observed for the beta-lactamase mRNA and other mRNAs from Gram-positive bacteria . We suggest that this extent of complementarity between the mRNA and this extent of complementarity between the mRNA and the ribosome may be a requirement for efficient initiation by Bacillus subtilis and other Gram-positive ribosomes, and may provide the basis for the observed inability of the Gram-positive systems to translate most of the mRNAs from Gram-negative bacteria.

J Biol Chem, 1981 Nov 10, 256(21), 11272 - 83
Plasmid-directed expression of Staphylococcus aureus beta-lactamase by Bacillus subtilis in vitro; McLaughlin JR et al.; A plasmid carrying the Gram-positive Staphylococcus aureus PC1 beta-lactamase gene is active in directing a cell-free transcription and translation system from Bacillus subtilis . The major protein synthesized has been identified as the S . aureus beta-lactamase on the basis of peptide mapping . The protein is larger than the extracellular enzyme by about Mr = 3100 . Significant in vitro translation of the beta-lactamase mRNA occurs in the absence of the initiation factor fraction as is characteristic of translation of mRNAs of Gram-positive origin . The 1250 base transcript that encodes the beta-lactamase and leader sequence has been mapped on the plasmid molecule.

J Biochem (Tokyo), 1981 Nov, 90(5), 1545 - 8
Enzymic formation of difructose anhydride IV from bacterial levan; Tanaka K et al.; A novel enzyme for producing difructose anhydride IV (di-D-fructofuranose 2,6' : 6,2' dianhydride) from levan of Bacillus mesentericus was prepared from a culture of Arthrobacter ureafaciens, and the conditions and method to determine the enzymatic activity were determined . Levan of Bacillus subtilis also yielded the difructose dianhydride, but inulin, inulobiose, sucrose, levanbiose, and levantriose did not produce it on enzymic reaction under similar test conditions.

J Antibiot (Tokyo), 1981 Nov, 34(11), 1402 - 7
Fredericamycin A, a new antitumor antibiotic . II . Biological properties; Warnick-Pickle DJ et al.; Fredericamycin A is a novel antibiotic produced by a soil isolate of Streptomyces griseus (FCRC-48) . In vitro, fredericamycin A exhibits antibacterial, antifungal, and cytotoxic activities . In vivo, fredericamycin A exhibits very good antitumor activity against P388 mouse leukemia as well as the CD8F mammary tumor and marginal activity against B16 melanoma . Fredericamycin A failed to demonstrate any interaction with DNA and inhibited protein and RNA synthesis preferentially to DNA synthesis in Bacillus subtilis and P388 cells.

Prikl Biokhim Mikrobiol, 1981 Nov-Dec, 17(6), 870 - 4
{Azoalbumin hydrolysis by Bacillus subtilis 72 subtilisin}; Nesterenko EA et al.; The kinetic parameters of azoalbumin hydrolysis by alkaline proteinase from Bacillus subtilis were determined to be Km = 1.2 . 10(-3) M, kcat = 1.5 sec-1 according to the method of Lainuiwer-Berk and Km = 4 . 10(-3) M, kcat = 0.5 sec-1 from the analysis of the entire kinetic curve . It was found that pH optimum of subtilizin hydrolysis of various substrates and the shape of the curve depended on the substrate nature.

Can J Microbiol, 1981 Nov, 27(11), 1194 - 201
Inorganic cation transport and the effects on C4 dicarboxylate transport in Bacillus subtilis; Kay WW et al.; The complex interrelationships between the transport of inorganic cations and C4 dicarboxylate were examined using mutants defective in potassium transport and retention, divalent cation transport, or phosphate transport . The potassium transport system, studied using 86Rb+ as a K+ analogue, kinetically appeared as a single system (Km 200 microM for Rb+, Ki 50 microM for K+), the activity of which was only slightly reduced in K+ retention mutants . Divalent cation transport, studied using 54Mn2+, 60Co2+, and 45Ca2+, was more complex being represented by at least two systems, one with a high affinity for Mn2+ (Km 2.5 microM) and a more general one of low affinity (Km 1.3-10 mM) for Mg2+, Mn2+, Ca/2+, and Co2+ . Divalent cation transport was repressed by Mg2+, derepressed in K+ retention mutants, and defective in Co2+-resistant mutants . Phosphate was required for both divalent cation and succinate transport, and phosphate transport mutants (arsenate resistant) were found to be defective in both divalent cation and succinate transport . Divalent cations, especially Mg2+ and Co2+, decreased Km for succinate transport approximately 20-fold over that achieved with K+; neither cation was required stoichiometrically for succinate transport . The loss of divalent cation transport in cobalt-resistant mutants has been correlated with the loss of a 55,000 molecular weight membrane protein . Similarly, the loss of phosphate transport in arsenate-resistant mutants has been correlated with the loss of a 35,000 molecular weight membrane component.

J Bacteriol, 1981 Nov, 148(2), 624 - 8
Genetic mapping of a linked cluster of ribosomal ribonucleic acid genes in Bacillus subtilis; Wilson FE et al.; A ribosomal ribonucleic acid gene set consisting of genes for 16S, 23S, 5S, and 4S ribonucleic acid species has been genetically mapped to a position between the markers recG13 and abrB74 on the Bacillus subtilis chromosome and designated rrnA . A ribosomal mutation, ksgA, was found to be linked to rrnA . This places rrnA in a region of the chromosome where ribosome-related genes occur but that is not directly adjacent to the major cluster of ribosome-related markers.

J Bacteriol, 1981 Nov, 148(2), 527 - 33
Ferrisiderophore reductase activity associated with an aromatic biosynthetic enzyme complex in Bacillus subtilis; Gaines CG et al.; The cytoplasmic fractions obtained from Bacillus subtilis strains W168 and WB2802 catalyzed reductive release of iron from the ferric chelate of 2,3-dihydroxybenzoic acid (ferri-DHB), the ferrisiderophore produced by B . subtilis . Ferrisiderophore reductase activity may insert iron into metabolism . This activity required a reductant (reduced nicotinamide adenine dinucleotide phosphate was preferred), was oxygen sensitive, and was stimulated by flavin mononucleotide plus certain divalent cations . The cytoplasmic fractions also reduced 2,6-dichlorophenolindophenol; this reaction was stimulated by flavin mononucleotide plus a divalent cation . Ferri-DHB and 2,6-dichlorophenolindophenol reductase activities were copurified by phosphocellulose and diethylaminoethyl-cellulose chromatography . Nondenaturing polyacrylamide gel electrophoresis of the purified material revealed that both ferri-DHB and 2,6-dichlorophenolindophenol reductase activities were located in a protein band at Rf 0.75 . The chromatographic procedures purified a reductase known to be associated with two aromatic biosynthetic enzymes, chorismate synthase and dehydroquinate synthase . Therefore, a portion of the ferrisiderophore reductase activity in B . subtilis may be catalyzed by a reductase that also is essential for aromatic biosynthesis.

J Bacteriol, 1981 Nov, 148(2), 480 - 6
Sporulation in Bacillus subtilis is independent of membrane fatty acid composition; Boudreaux DP et al.; Growth and sporulation of a Bacillus subtilis mutant deficient in branched fatty acid synthesis (gene symbol bfmB) were examined . The mutant, which produces an acyl-coenzyme A:acyl carrier protein transacylase with reduced affinity for branched fatty acid primers, could grow in media containing any one of a wide range of low-molecular-weight fatty acids having branched, cyclic, saturated, or unsaturated carbon chains . The fatty acid composition of cellular lipids depended on the compound used to support growth . Cultures of the bfmB mutant grown in the presence of 3-methylcrotonate contained an unusually high fraction (73%) of straight-chain fatty acids in the cellular lipids . The mutant sporulated with any one of the precursors of branched fatty acids in the medium; isolated spores contained mainly this branched fatty acid and only 10% or less straight-chain fatty acids regardless of the straight-chain fatty acid content of vegetative cells . Exceptional were spores grown in the presence of cyclobutane-carboxylic acid, which contained 28% straight-chain fatty acids . The branched fatty acid composition of spores could be modified greatly by changing the supply of precursors in the medium.

J Bacteriol, 1981 Nov, 148(2), 443 - 9
Cell wall synthesis and initiation of deoxyribonucleic acid replication in Bacillus subtilis; Sandler N et al.; We have observed a connection between cell wall synthesis and the initiation of chromosome replication in Bacillus subtilis . Initiation of chromosome replication was prevented in synchronous cultures in the presence of the cell wall synthesis inhibitor vancomycin . When vancomycin was added to the cultures after initiation of chromosome replication, one round of replication was completed but no reinitiation occurred . Similar results were obtained when cell wall synthesis was inhibited by ristocetin, cycloserine, cloxacillin, or cephaloridine . When sucrose was added to the medium, initiation of deoxyribonucleic acid replication occurred in the presence of vancomycin, to an extent which allowed replication of no more than approximately one-half of the deoxyribonucleic acid of the culture . The same was found in cultures of spheroplasts of B . subtilis . However, initiation of chromosome replication in spheroplasts was completely insensitive to cloxacillin.

J Gen Microbiol, 1981 Nov, 127, pt 1, 11 - 7
Inhibition of sporulation by DNA gyrase inhibitors; Vazquez-Ramos JM et al.; The effects of oxolinic acid and novobiocin - which respectively inhibit the A and B subunits of DNA gyrase, and therefore inhibit DNA synthesis - have examined in sporulating cultures of Bacillus subtilis . Although both inhibitors prevent sporulation, this is not due to inhibition of DNA synthesis . Instead, they affect protein synthesis generally, though weakly, but have very marked effects on the formation of individual enzymes . These effects are reproducible, but the type of enzyme that will be affected is not predictable . The results point to an involvement of DNA gyrase in the transcription of some genes . This is suggested as the reason for the effect of the inhibitors on spore formation, which they block mainly at Stage O-I.

J Gen Microbiol, 1981 Nov, 127, pt 1, 1 - 9
Oxolinic acid-resistant mutants of Bacillus subtilis; Vazquez-Ramos JM et al.; Two mutants of Bacillus subtilis have been independently selected for resistance to oxolinic acid, and inhibitor of DNA gyrase . The mutations, designated oxr-1 and oxr-2, map very close to one another but are clearly separated from mutations in the genes for DNA gyrase . Many of the phenotypic properties of the mutants differ from those of a strain containing the gyrA mutation described by other workers . In particular, the oxr strains are as sensitive as the wild-type to inhibition by nalidixic acid on solid medium . In addition, experiments with DNA synthesis in toluenized cells show that the enzyme of the gyrA mutant is resistant to oxolinic acid, whereas DNA synthesis in the oxr mutants is as sensitive as it is in wild-type preparations . It is concluded that resistance to oxolinic acid is not due to an alteration in the DNA gyrase, but is more probably the result of an impaired uptake of the inhibitor . Although growth of the mutants on agar plates is inhibited at high concentrations of oxolinic acid, lower concentrations (1-2 microgram ml-1) can be used to distinguish them from the wild-type . The oxr-1 and oxr-2 mutations define a new genetic locus and can be used as genetic markers in B . subtilis.

J Bacteriol, 1981 Nov, 148(2), 406 - 12
Synthesis of teichoic acid by Bacillus subtilis protoplasts; Bertram KC et al.; Protoplasts of Bacillus subtilis W23 readily synthesized ribitol teichoic acid from nucleotide precursors in the surrounding medium . With cytidine diphosphate-ribitol they made poly(ribitol phosphate), presumably attached to lipoteichoic acid carrier; when cytidine diphosphate-glycerol and uridine diphosphate-N-acetylglucosamine were also present a 10-fold increase in the rate of polymer synthesis occurred, and the product contained both the main chain and the linkage unit . Synthesis was inhibited by trypsin or p-chloromercuribenzenesulfonate in the medium, and we concluded that it occurred at the outer surface of the membrane . During synthesis, which was also achieved readily by whole cells after a brief period of wall lysis, the cytidine phosphate portion of the nucleotide precursors did not pass through the membrane . No evidence could be obtained for a transphosphorylation mechanism for the translocation process . It is suggested that reaction with exogenous substrates was due to temporary exposure of a protein component of the enzyme complex at the outer surface of the membrane during the normal biosynthetic cycle.

J Bacteriol, 1981 Nov, 148(2), 653 - 8
New class of Bacillus subtilis glutamine-requiring mutants; Reysset G; By using genetic analysis, the mutations of eight glutamine-requiring mutants isolated from Bacillus subtilis 168 were all shown to be linked to the thyA marker . A three-factor transduction analysis performed with one of the gln mutations indicated that the gene order in this region of the B . subtilis chromosome was gltA-thyA-gln . On the basis of recombination index values, two closely linked groups were identified . The mutations belonging to one group were assigned to the structural gene for glutamine synthetase, and those belonging to the other group might impair a regulatory locus . The residual glutamine synthetase activities and the cross-reacting materials of the mutants from both recombination groups supported these conclusions.

Biochemistry, 1981 Oct 13, 20(21), 5988 - 94
Antibacterial peptide from normal rabbit serum . 3 . Inhibition of microbial electron transport; Carroll SF et al.; The influence of the primary rabbit serum bactericide, PC-III, on the respiratory activity of Bacillus subtilis has been examined . Glucose- or lactate-dependent respiration by whole cells was rapidly and completely inhibited by concentrations of the bactericide producing significant cell death . Similar results were observed with membrane vesicles oxidizing NADH . In both cases, bactericide-induced inhibition of respiration was calcium dependent and blocked electron transport between cytochromes b and a . PC-III competed with oxidized Saccharomyces cytochrome c when the latter was used as an electron acceptor in cytochrome c reductase reactions catalyzed by B . subtilis membrane vesicles . Competitive inhibition by PC-III was also observed when reduced Saccharomyces cytochrome c was used as electron donor in the cytochrome c oxidase reaction . At an ionic strength of 0.13, PC-III exhibits a Ki of 25.9 and 102 nM for the reductase and oxidase complexes, respectively . Increasing the ionic strength to that producing optimal antibacterial action against whole cells (0.24) increased the Ki of PC-III for the reductase (75.4 nM), while the oxidase decreased (92.3 nM).

J Biol Chem, 1981 Oct 10, 256(19), 9966 - 72
A novel, highly modified, bacteriophage DNA in which thymine is partly replaced by a phosphoglucuronate moiety covalently bound to 5-(4',5'-dihydroxypentyl)uracil; Ehrlich M et al.; Bacteriophage SP-15, which infects Bacillus subtilis, contains a highly modified DNA in which 62% of its thymine residues are replaced by 5-(4',5'-dihydroxypentyl)uracil to which is attached a phosphoglucuronate via a phosphodiester linkage to one of the hydroxyl groups of the pentyl side chain . Glucose is also bound to this residue probably by glycosidic linkage to the other hydroxyl group of the pentyl side chain . In 0.3 M KOH at 37 degrees C, glucuronic acid 1-phosphate is slowly released from this DNA . After enzymatic or acid-induced dephosphorylation, this sugar was identified by chromatography in two thin layer chromatography systems, conversion to glucuronolactone under conditions known to lactonize glucuronic acid, and reaction in four colorimetric assays for hexuronic acids . Phage SP-15 DNA is the first DNA found to have a uronic acid moiety or a phosphate which is not part of the phosphodiester backbone . The glucuronic acid phosphate might be derived from uridine pyrophosphoglucuronic acid, whose glucuronic acid moiety is normally destined for synthesis of teichuronic acid in the host cell wall.

Lab Anim Sci, 1981 Oct, 31(5 Pt 1), 494 - 7
Evaluation of a chemical scrubber for the removal of airborne bacteria from recycled air; Jeszenka EV et al.; The effectiveness of a chemical scrubber utilizing a recycled aqueous media was compared to high efficiency particulate air filtration in its ability to remove bacteria from recycled air . Two bacteria, Staphylococcus epidermidis and Bacillus subtilis, which have different biologic and physical properties were used in the study . The scrubber, using an aqueous chlorine-dioxide solution, and high efficiency particulate air filtration were equally effective in removing both types of bacteria from recycled air . Viable bacteria could not be recovered from the processed air following either treatment . When the scrubber was used with the chlorine dioxide solution, sufficient chlorine dioxide residues remained in the scrubber processed air to affect bacterial counts in the isolator to which the processed air was returned . The bacteria differed in their susceptibility to low levels of chlorine dioxide in the return air which influenced the number of viable organisms recovered under similar conditions . The use of water without a chemical additive eliminated bacteria in recycled air immediately following the start of the unit but allowed their eventual build-up in recycled air to concentrations of approximately half that of untreated air . Overall, the use of a chemical scrubber for the removal of bacteria from recycled air appeared to be no more effective than high efficiency particulate air filtration especially if the scrubbing media is recycled.

Mutat Res, 1981 Oct, 83(3), 339 - 47
Mutagenesis during transformation of Bacillus subtilis . II . An increase in chemically-induced mutations during competency; Rudner R; During the development of competency in Bacillus subtilis there was an increased sensitivity to methyl methanesulfonate (MMS) treatments . The frequency of reverse mutation also increased among the MMS-revertible markers by a factor of 100 as compared to vegetative cultures . The frequency of 2-aminopurine(AP)-induced mutagenesis was the same in competent and noncompetent cultures . Studies with DNA-polymerase-deficient mutants showed a direct involvement of DNA polymerase I in promoting MMS and transformation-induced mutagenesis in competent cells.

Mutat Res, 1981 Oct, 83(3), 321 - 37
Mutagenesis during transformation of Bacillus subtilis . I . An increase in "selfing' resulting from hybrid donor DNAs; Rudner R; Reverse mutations increase when competent Bacillus subtilis cells are transformed with high concentrations of homologous "selfer' DNA . A high proportion of the mutants were also transformants of linked genes . A stimulation in the appearance of reversed mutations occurred when homoduplex and heteroduplex "selfer' DNAs were used as donors . Digestions of native and hybrid DNAs with nuclease S1 from Aspergillus oryzae resulted in the preferential decrease of mutations as compared to a much smaller inactivation of single marker transformation . Among various repair-deficient strains of B . subtilis, only poly A mutants showed a preferential effect of either suppressing or stimulating the frequency of reverse mutation induced by "selfer' DNA . The results are consistent with mutagenic errors occurring during gap-filling steps in the process of either mismatch repair or recombinational strand exchanges.

Can J Microbiol, 1981 Oct, 27(10), 991 - 7
The effect of magnesium of transfection and transformation in Bacillus subtilis; Lopez P et al.; The influence of Mg2+ on phage SPP1 DNA-mediated transfection as compared with chromosomal transformation was studied . A differential influence of this cation in both processes was detectable by analyzing the competence development and the kinetics of appearance of transformants and transfectants . Binding and uptake of DNA and release of acid-soluble products by competent cells in high- and low-Mg2+ media was measured for 3H-labelled SPP1 and 3H-labelled Bacillus subtilis DNAs . Phage SPP1 DNA was shown to be subjected to endonucleolytic cleavage after exposure to competent cells.

Res Commun Chem Pathol Pharmacol, 1981 Oct, 34(1), 161 - 4
DNA damaging effects of three sesquiterpene lactones in repair-deficient mutants of Bacillus subtilis; Jones DH et al.; Three sesquiterpene lactones (hymenoxon, helenalin, and tenulin) were tested for genotoxicity using six strains of Bacillus subtilis . Hymenoxon was found to produce lethal DNA damage in strains rec A8 and mc-1 while helenalin was lethal in strains mc-1 and rec E4 . Tenulin did not produce lethal DNA damage in any of the strains tested.

J Gen Virol, 1981 Oct, 56(Pt 2), 275 - 86
Effect of defective phages on the cell membrane of Bacillus subtilis and partial characterization of the phage protein involved in killing; Steensma HY; In order to investigate whether defective phages of Bacillus subtilis killed sensitive bacteria by a lysis from without mechanism, the minimal number of phages required for killing was determined . This figure was found to vary with the m.o.i., giving a value of 1 on extrapolation to an m.o.i . of 0 . This excluded lysis from without as the only killing mechanism, although it might play a role at high m.o.i.s . This was confirmed by experiments on leakage of ATP and u.v.-absorbing material, the uptake of oxygen and the effect of the phages on the membrane potential . Apart from a short, initial leakage of ATP, the cell membrane was not affected at low m.o.i.s . These results lead to the conclusion that at low m.o.i.s . the phages acted on a cytoplasmic component . Treatment of defective phages for 10 min at pH 2.5 resulted in breakdown of the phages without complete abolition of the killing activity . The active component, which was shown not to be DNA, could not be isolated from the mixture, but SDS gel electrophoresis of PBS X and a non-killing mutant of this phage suggested that a protein with a mol . wt . of 85000 was involved in killing.

Hoppe Seylers Z Physiol Chem, 1981 Oct, 362(10), 1323 - 9
{The use of alkaline protease from Bacillus subtilis, strain DY, for the hydrolysis of amino acid and peptide esters (author's transl)}; Aleksiev B et al.; We have studied the hydrolysis of methyl-, ethyl- and benzylesters of amino acids and peptides using alkaline protease from B . subtilis, strain DY . The hydrolysis with this enzyme gives high yields (usually over 90%) with high optical purity of the products . The proposed method is considerably better than the alkaline saponification or the hydrolysis of these esters with alpha-chymotrypsin . The method is not applicable in the case of substrates with free amino groups as well as with substrates which are insoluble under the conditions of the reaction.

J Bacteriol, 1981 Oct, 148(1), 91 - 100
Distribution of bacteriophage phi 3T homologous deoxyribonucleic acid sequences in Bacillus subtilis 168, related bacteriophages, and other Bacillus species; Stroynowski IT; The Bacillus subtilis 168 chromosome was found to share extensive homology with the genome of bacteriophage phi 3T . At least three different regions of the bacterial genome hydridized to ribonucleic acid complementary to phi 3T deoxyribonucleic acid (DNA) . The thymidylate synthetase gene, thyA, of B . subtilis and the sequences adjacent to it were shown to be homologous to the region in the phi 3T DNA containing the phage-encoded thymidylate synthetase gene, thyP3 . SP beta, a temperate bacteriophage known to be integrated into the B . subtilis 168 chromosome, was demonstrated to be closely related to phi 3T . Other regions of the bacterial genome were also found to hybridize to the phi 3T probe . The nature and location of these sequences in the bacterial and phage chromosomes were not identified . It was shown however, that they were not homologous to either the thyP3 gene or the DNA surrounding the thyP3 gene . The chromosomes of other Bacillus species were also screened for the presence of phi 3T homologous sequences, and the thyP3 gene was localized in the linear genomes of phages phi 3T and rho 11 by heteroduplex mapping . It is suggested that the presence of sequences of phage origin in the B . subtilis 168 chromosome might contribute to the restructuring and evolution of the viral and bacterial DNAs.

J Bacteriol, 1981 Oct, 148(1), 341 - 51
Identification of a new developmental locus in Bacillus subtilis by construction of a deletion mutation in a cloned gene under sporulation control; Rosenbluh A et al.; We removed by recombinant deoxyribonucleic acid (DNA) techniques a small DNA segment from within a cloned gene (the 0.4 kb gene) in which transcription in under sporulation control in Bacillus subtilis . These deletion mutation was introduced into the B . subtilis chromosome by transformation with cloned DNA . Competent cells bearing a mutation (tms-26) that is closely linked to the 0.4 kb gene were transformed with linearized plasmid DNA containing the truncated 0.4 kb gene and the wild-type allele of the tms locus . Selection for Tms+ transformants yielded oligosporogenous mutants of unusually dark-brown colony pigmentation . This phenotype was caused by a mutation which mapped at or very near the site of the 0.4 kg gene deletion, whose presence and position in chromosomal DNA was confirmed by Southern hybridization analysis . Phase-contrast microscopy and electron microscopy showed that the mutation, which we designated as spoVG, impaired sporulation at about the fifth stage; bacteria harboring the spoVG mutation proceeded normally through stage IV of development but frequently lysed thereafter, apparently as a result of disintegration of an immature spore cortex . This identifies the 0.4 kb gene (or DNA in its immediate vicinity) as a new sporulation locus and shows that its product functions at a late stage in development.

Allergy, 1981 Oct, 36(7), 513 - 6
Detergent enzymes and occupational safety . Observations on sensitization during Esperase production; Zachariae H et al.; During a 10-year survey of 667 workers producing the detergent enzyme Esperase, derived from an alkolophilic strain of Bacillus licheniformis, 31 were found to have been sensitized . All but one were at the same time sensitized to subtilisins, with which they also had been working . No distinction could be made between symptoms attributed to Esperase and symptoms attributed to enzymes derived from Bacillus subtilis (licheniformis) or other enzymes . Symptoms, when present, were mainly from the lower respiratory tract . Nine sensitized workers were symptom-free . Sensitization was by IgE antibodies and detected by the RAST test . Twenty-one sensitized workers were transferred for precautionary measures or left the company . All 26 sensitized workers, whom it was possible to follow and reinvestigate, were no longer RAST-positive . Ten workers remained in their jobs . No signs of deteriorating lung function or permanent lung damage were found . The study indicated that, when strictly enforced, recommended operating procedures for workers handling hitherto well-known detergent enzymes are sufficient for dealing with Esperase . The data suggest there is no significant risk of consumer sensitization.

J Bacteriol, 1981 Oct, 148(1), 101 - 8
Integration of the bacteriophage phi 3T-coded thymidylate synthetase gene into the Bacillus subtilis chromosome; Stroynowski IT; Transformation of Bacillus subtilis 168 Thy- auxotrophs with phi 3T deoxyribonucleic acid (DNA) to thymine independence was found to involve site-specific recombination of phi 3T DNA sequences with their homologous counterparts in the bacterial chromosome . During the transformation, the phage phi 3T-encoded thymidylate synthetase gene, thyP3, was shown to integrate at two genetically distinct sites in the B . Subtilis 168 chromosome . The first site was identified to be in the bacterial thymidylate synthetase gene, thyA . The second site was in a prophage (SPB) known to be carried in the host genome . The frequency of the integration of the thyP3 gene at each of the two loci and some of the parameters affecting this frequency were studied . The common origin of the thyP3 and thyA genes and their molecular evolution are also reported.

Biochemistry, 1981 Sep 29, 20(20), 5675 - 81
Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with oxygen: chemistry and regulation by ligands; Bernlohr DA et al.; The inactivation of glutamine phosphoribosylpyrophosphate amidotransferase by reaction of its iron-sulfur center with O2 is believed to be a physiologically important mode of regulation of this enzyme in Bacillus subtilis cells in the stationary phase of growth . Chemical and physical changes accompanying oxidation of the purified enzyme by O2 were studied . The iron of the 4Fe-4S center was oxidized to enzyme-bound high-spin Fe3+; the S2- was oxidized to a mixture of S0 bound as thiocystine and unidentified products . The oxidant appeared to be O2, rather than peroxide, superoxide, hydroxyl radical, or singlet oxygen . Gross physical changes in the oxidized enzyme were shown by its aggregation, decreased solubility, and altered circular dichroic spectrum . Experimental variables affecting the rate of oxidative inactivation were described; the most important of these was modulation of rates of inactivation by the allosteric inhibitors AMP, ADP, GMP, GDP and by the substrate P-Rib-PP . AMP was a potent stabilizer, whose effect was antagonized by P-Rib-PP . The other nucleotides, either acting singly or acting as synergistic pairs, were destabilizers and able to antagonize stabilization by AMP . The results are discussed in terms of the regulation of the stability of amidotransferase and its degradation in vivo.

Biochemistry, 1981 Sep 29, 20(20), 5669 - 74
Purification and properties of glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis; Wong JY et al.; A procedure for the rapid and efficient purification of glutamine phosphoribosylpyrophosphate amidotransferase to better than 98% homogeneity from depressed Bacillus subtilis cells is described . The molecular weight of the subunit was estimated to be about 50 000 . The purified enzyme exhibits microheterogeneity on electrophoresis on highly resolving polyacrylamide gels; it is suggested that this heterogeneity results from limited proteolytic modification of the native subunit . The native enzyme exists in equilibrium among tetrameric, dimeric, and monomeric forms . The influence of enzyme concentration and the presence of substrates and allosteric inhibitors on this equilibrium are described . There is no simple correlation between allosteric inhibition and stabilization of dimeric or tetrameric states . The amino acid composition of the amidotransferase is reported; presence of a 4Fe-4S center in the enzyme was described previously . Preparation of inactive apoprotein by treatment with 1,10-phenanthroline and general characteristics of the apoprotein are presented.

Biochemistry, 1981 Sep 29, 20(20), 5655 - 61
Use of isotope effects and pH studies to determine the chemical mechanism of Bacillus subtilis L-alanine dehydrogenase; Grimshaw CE et al.; Analysis of deuterium isotope effects with L-alanine-d4 and L-serine-d3, and of pH profiles with the same substrates, shows that L-alanine is sticky (that is, reacts to give products 1-7 times as fast as it dissociates) while L-serin is not . The pH profiles show the following: (1) NH3 and monoanionic amino acids are the substrates; (2) a cationic acid group on the enzyme (probably lysine) with a pK of 9.0-9.6 in E-NAD, but a pK well above 10 in E-NADH, must be protonated for activity and good binding of inhibitors and is probably important for maintaining the proper conformation of the enzyme; (3) A cationic acid group on the enzyme (probably histidine) with a pK around 7 in both E-NAD and E-NADA must be unprotonated for oxidation of amino acids but protonated for binding and reaction of pyruvate . This latter group is the acid-base catalyst for the chemical reaction . In E-NAD, it is so positioned that it can hydrogen bond to (and thus when protonated enhance the binding of) a D-hydroxy or a carbonyl group of an inhibitor, but its state of protonation does not affect the binding of L-lactate or propionate . In E-NADH, it is so placed that it can hydrogen bond to both D- and L-hydroxy groups, as well as in carbonyl groups . A chemical mechanism is postulated in which the dehydrogenation of L-alanine by NAD to produce iminopyruvate is followed by attack of water from the same side from which the hydride was removed . The catalytic histidine transfers a proton from the attacking water to the amino group of the resulting carbinolamine and then removes a proton from the hydroxyl group of the carbinolamine as ammonia is eliminated to give pyruvate.

Biochemistry, 1981 Sep 29, 20(20), 5650 - 5
Kinetic mechanism of Bacillus subtilis L-alanine dehydrogenase; Grimshaw CE et al.; L-Alanine dehydrogenase from Bacillus subtilis has a predominately ordered kinetic mechanism in which NAD adds before L-alanine, and ammonia, pyruvate, and NADH are released in that order . When pyruvate is varied at pH 9.35, levels of ammonia above 50 mM cause uncompetitive substrate inhibition and cause the slope replot to go through the origin . This pattern suggest that iminopyruvate (2% of pyruvate at this pH with 150 mM ammonia) can combine with E-NADH much more tightly than pyruvate does but reacts much more slowly because uptake of the required proton from solution is hindered . Isomerization of the initially formed E-NAD complex to a form which can productively bind L-alanine is the slowest step in the forward direction at pH 7.9, and substrate inhibition by L-alanine largely results from combination of the zwitterion in a nonproductive fashion with this initial E-NAD complex, with the result that the isomerization is prevented . All bimolecular rate constants approach diffusion-limited values at optimal states of protonation of enzyme and substrates except that for ammonia, suggesting that ammonia does not form a complex with E-NADH-pyruvate but reacts directly with it to give a bound carbinolamine.

J Biol Chem, 1981 Sep 10, 256(17), 9346 - 51
Restriction and modification in Bacillus subtilis: two DNA methyltransferases with BsuRI specificity . II . Catalytic properties, substrate specificity, and mode of action; Gunthert U et al.; The properties of two DNA methyltransferases, termed M . BsuRIa and M . BsuRIb, whose isolation was described in the preceding paper (Gunthert, U., Freund, M., and Trautner, T . A . (1981) J . Biol . Chem . 256, 9340-9345) were compared . Both enzymes recognize the same target sequence in double-stranded DNA, leading to methylation of the internal cytosine: 5'GGCC . The enzymes have identical reaction constants with their substrates, DNA (km = 2.7 nM for the 5' GGCC sequence), and S-adenosyl-L-methionine (km = 0.7 microM) . Initial rates of methyl group transfer were proportional to enzyme concentration over a range of 50-fold, indicating absence of aggregation . The enzymes are different in their ionic strength requirements using Tris-HCl, pH 8.4 . M . BsuRIa is most active at 100 mM, M . BsuRIb at 440 mM . As measured by incorporation kinetics and heat inactivation, M . BsuRIa is the more stable enzyme of the two . Equilibrium dialysis was used to study the mode of methyl group transfer to the DNA with either enzyme . The data indicate that initially S-adenosyl-L-methionine binds to methyltransferase . This complex attaches to either modified or nonmodified DNA . The methyl group will then be transferred to a nonmodified target sequence, leading to the dissociation of enzyme and S-adenosyl-L-homocysteine from the DNA.

J Biol Chem, 1981 Sep 10, 256(17), 9340 - 5
Restriction and modification in Bacillus subtilis: two DNA methyltransferases with BsuRI specificity . I . Purification and physical properties; Gunthert U et al.; Two S-adenosyl-L-methionine:DNA (cytosine 5)-methyltransferases, termed M.BsuRIa and M.BsuRIb, were purified 3,000- and 4,000-fold, respectively, from Bacillus subtilis strain OG3R (r+m+) by successive column chromatography . The molecular weights determined by gel filtration were 37,000 for M.BsuRIa and 40,000 for M.BsuRIb . The sedimentation coefficients s20,w were 3.55 for both enzymes as determined by glycerol gradient centrifugation, corresponding to molecular weights of 43,000 . Analysis of the two methyltransferases by agarose gel electrophoresis under native conditions, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed correspondence of the M.BsuRIa activity with one protein band at a molecular weight of 41,000, whereas M.BsuRIb activity was associated with two protein bands with molecular weights of 42,000 and 39,000, respectively.

Eur J Biochem, 1981 Sep, 119(1), 85 - 90
The biosynthesis of wall teichoic acid by toluenised cells of Bacillus subtilis W23; Hancock IC; Toluenised cells of Bacillus subtilis W23 synthesized the teichoic acid, poly(ribitol phosphate), from exogenous precursors . The synthesis was dependent on concomitant synthesis of the linkage unit that joins teichoic acid to peptidoglycan . Under conditions that reduced cell autolytic activity, a large proportion of the teichoic acid became linked to the cell wall, independently of peptidoglycan synthesis . The specific activity of the system was more than 30 times that of isolated membranes, so that activity could be measured readily in the cells from 2 ml of an exponential culture of bacteria.

Proc Natl Acad Sci U S A, 1981 Sep, 78(9), 5861 - 5
In vitro assembly of the Bacillus subtilis bacteriophage phi 29; Bjornsti MA et al.; In vitro assembly of the Bacillus subtilis bacteriophage phi 29 that approaches the efficiency of assembly in vivo has been demonstrated . Proheads, DNA, and gene 16 product (gp16) were essential for DNA encapsidation, and the average yield in extracts was 180 phage per prohead donor cell . The in vitro maturation was very similar to in vivo assembly in terms of yield, intermediates, and abortive structures . More that 30% of the proheads in the extract were converted to phage, and about 20% of DNA--protein extracted from phage could be repackaged . In vitro assembly was blocked by the addition of DNase I, EDTA, pyrophosphatase, or the ATP analogues adenosine 5'-{alpha, beta-methylene}triphosphate and adenosine 5'-{beta, gamma-methylene}triphosphate . Less than 1% of the proheads isolated in sucrose gradients can accept DNA--protein in packaging in vitro.

Cell, 1981 Sep, 25(3), 753 - 63
The energized membrane and cellular autolysis in Bacillus subtilis; Jolliffe LK et al.; Lysis of exponential cultures of B . subtilis follows the addition of reagents that dissipate either the electrical or pH gradients of cellular membranes . Stationary-phase cells or cultures that have been inhibited in division by macromolecular-synthesis inhibitors also lyse when uncoupling agents or ionophores are added to the growth medium . Autolysis occurs after brief starvation for a carbon source . Protoplasts are unaffected by azide or other lysis-inducing agents . Electron-donating agents, such as phenazine methosulfate and ascorbate, are effective in retarding autolysis . The addition of an oxidizable carbon source to starved and lysing cultures prevents their autolysis . These results suggest that cellular lysis in B . subtilis and energized membrane are tightly coupled . The fluorescence intensity and the wavelength of maximal fluorescence of 8-anilino-1-naphthalene sulfonic acid, when added to bacterial suspensions, appear to be qualitatively related to the rate of cell lysis . Analyses show that ATP limitations are probably not involved in the elicitation of lysis by ionophores, uncoupling agents or starvation . Measurements of protonmotive forces in the lysis-prone cells suggest that a threshold force of more than 85 mV may be required to maintain cellular integrity . Lipoteichoic acids, polyelectrolytes such as dextran sulfate or phospholipids do not modify the rate of cellular lysis when added to suspensions containing azide or other reagents that eliminate transmembrane protonmotive forces . We interpret the results to suggest that the in vivo control of autolysin activity in B . subtilis is related to the energized membrane

J Bacteriol, 1981 Sep, 147(3), 949 - 53
Ability of Bacillus subtilis protoplasts to repair irradiated bacteriophage deoxyribonucleic acid via acquired and natural enzymatic systems; Yasbin RE et al.; A novel form of "enzyme therapy" was achieved by utilizing protoplasts of Bacillus subtilis . Photoreactivating enzyme of Escherichia coli was successfully inserted into the protoplasts of B . subtilis treated with polyethylene glycol . This enzyme was used to photoreactivate ultraviolet-damaged bacteriophage deoxyribonucleic acid (DNA) . Furthermore, in polyethylene glycol-treated protoplasts, ultraviolet-irradiated transfecting bacteriophage DNA was shown to be a functional substrate for the host DNA excision repair system . Previous results (R . E . Yasbin, J . D . Fernwalt, and P . I . Fields, J . Bacteriol . 137:391-396, 1979) showed that ultraviolet-irradiated bacteriophage DNA could not be repaired via the excision repair system of competent cells . Therefore, the processing of bacteriophage DNA by protoplasts and by competent cells must be different . This sensitive protoplast assay can be used to identify and to isolate various types of DNA repair enzymes.

J Bacteriol, 1981 Sep, 147(3), 752 - 6
Transcriptional inhibition and production of guanosine polyphosphates in Bacillus subtilis; Price VL et al.; When exponentially growing cells of Bacillus subtilis were treated with rifampin or lipiarmycin, both inhibitors of the initiation of ribonucleic acid synthesis, large amounts of (p)ppGpp accumulated . This accumulation appears to be independent of the ribosome-dependent stringent factor reaction because both relA and relC mutants responded in a manner similar to that of the wild type . The possibility that ribonucleic acid polymerase is directly involved in (p)ppGpp metabolism is discussed.

J Bacteriol, 1981 Sep, 147(3), 1054 - 62
Effects of lipophilic cations on motility and other physiological properties of Bacillus subtilis; Zaritsky A et al.; Lipophilic cations (tetraphenylarsonium, tetraphenylphosphonium, and triphenylmethylphosphonium) caused a number of major changes in the physiology of Bacillus subtilis . Macromolecular synthesis was inhibited, adenosine 5'-triphosphate concentration increased, swimming speed was reduced, tumbling was suppressed, and the capacity to take up the cations was greatly enhanced; respiration was not significantly altered . The effects occurred at lipophilic cation concentrations in the range commonly employed for measurement of membrane potential . Neither the enhancement of cation uptake nor the motility inhibition was a consequence of alteration of membrane potential, since both effects were still seen in the presence of valinomycin, with the extent of 86Rb+ uptake indicating a constant potential . Because suppression of tumbling accompanied speed reduction, as has also been found when protonmotive force is reduced, it is likely that lipophilic cations are perturbing the process of conversion of proton energy into work, rather than simply causing structural damage.

J Bacteriol, 1981 Sep, 147(3), 1040 - 8
Coat protein synthesis during sporulation of Bacillus subtilis: immunological detection of soluble precursors to the 12,200-dalton spore coat protein; Goldman RC et al.; Antibody specific to the 12,200-dalton spore coat protein of Bacillus subtilis was used to detect the synthesis of cross-reacting material during sporulation . Cross-reacting protein was first detected by immunoprecipitation after 4 h of development and represented at least 1 to 2% of the total soluble protein synthesis at 5.5 h . A polypeptide of 21,000 daltons was detected in immunoprecipitates by gel electrophoresis . This polypeptide did not accumulate in sporulating cells and was rapidly turned over at the time of coat deposition . In contrast, a 32,000-dalton polypeptide reacted with antibody when unlabeled cell protein was denatured with sodium dodecyl sulfate, separated by gel electrophoresis, and transferred to nitrocellulose paper . This polypeptide was not detected during cell growth or the first 3.5 h of development but was found to accumulate in sporulating cells at 5.5 h . The lack of detection of this polypeptide by immunoprecipitation of undenatured protein indicates that the antigenic sites which cross-reacted with antibody to the 12,200-dalton protein sequence were not exposed unless the molecular conformation was altered . The 32,000-dalton protein may be a primary translation product which is proteolytically processed into mature spore coat protein via a 21,000-dalton intermediate.

Eur J Biochem, 1981 Sep 1, 118(3), 497 - 500
Control of synthesis of wall teichoic acid in phosphate-starved cultures of Bacillus subtilis W23; Cheah SC et al.; CDP-glycerol pyrophosphorylase, CDP-ribitol pyrophosphorylase and poly(ribitol phosphate) synthetase activities have been measured in cultures of Bacillus subtilis W23 as they became phosphate-starved either in batch culture or during changeover from potassium limitation to phosphate limitation in a chemostat . The results indicated that repression of synthesis of all three enzymes occurred at the onset of phosphate starvation and that this was accompanied by inhibition of inactivation of CDP-glycerol pyrophosphorylase and poly(ribitol phosphate) synthetase . These results show that the initial response to phosphate starvation involves more than inhibition of one enzyme as proposed by Glaser and Loewy {Glaser L . and Loewy, A . (1979) J . Biol . Chem . 254, 2184-2186} . Synthesis of both linkage unit and poly(ribitol phosphate) are inhibited independently.

J Virol, 1981 Sep, 39(3), 855 - 60
DNA gyrase inhibitors block development of Bacillus subtilis bacteriophage SP01; Alonso JC et al.; SP01 development was inhibited by nalidixic acid and novobiocin in the sensitive host Bacillus subtilis 168M . Inhibition by novobiocin was prevented by a Novr mutation in the cellular DNA gyrase gene . Nalidixic acid inhibition persisted in hosts carrying a Nalr gyrase, but could be overcome by phage mutation . We conclude that SP01 requires for its development subunit B of the host DNA gyrase, but replaces or modifies subunit A.

Cell, 1981 Sep, 25(3), 783 - 91
Promoter for a developmentally regulated gene in Bacillus subtilis; Moran CP Jr et al.; We have determined the nucleotide sequence of the promoter for aB . subtilis gene (the 0.4 kb gene) whose transcription is under developmental control . Transcription of the 0.4 kb gene is turned on at the onset of sporulation; this RNA synthesis depends on the products of the B . subtilis regulatory genes (the spoO loci) that control the initiation of development . Recognition of the 0.4 kb gene promoter in vitro is dictated by novel species of B . subtilis RNA polymerase sigma factor known as sigma 37 and sigma 29 but not by the principal B . subtilis sigma factor sigma 55 . Using S1 nuclease mapping, runoff transcription and dinucleotide priming, we have identified dual startpoints (separated by about 10 bp) for sigma37-directed transcription of the 0.4 kb gene These start-points correspond closely to the 5' termini of 0.4 kb RNA synthesized in vivo during the course of sporulation . Two forms of sigma37 containing RNA polymerase were distinguished that preferentially utilize either the upstream or the downstream startpoint in vitro . We investigated the requirements for sigma37-directed transcription by constructing in vitro deletion mutations that extend from the upstream direction into the 0.4 kb promoter region . One such deletion, which terminates 40 and 51 bp upstream from the transcription startpoints (thereby removing a highly AT-rich 26 bp sequence), completely eliminates transcription from the downstream startpoint but only partially inhibits transcription from the upstream startpoint . A second deletion that terminates at the upstream startpoint completely prevents transcription from both initiation sites . The implications of 0.4 kb gene promoter structure for developmentally regulated transcription of B, subtilis are discussed.

J Bacteriol, 1981 Sep, 147(3), 776 - 86
Cloning of the genes for penicillinase, penP and penI, of Bacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis, and Bacillus licheniformis; Imanaka T et al.; By using plasmid pMB9, penicillinase genes (penP and penI) from both the wild-type and constitutive strains of Bacillus licheniformis 9945A were cloned in EScherichia coli . When a low-copy-number plasmid was used, both wild-type and constitutive penicillinase genes could be transferred into Bacillus subtilis . However, when a high-copy-number plasmid was used, only the genes of the wild type could be transferred . These recombinant plasmids in B . subtilis could all be transferred by the protoplast transformation procedure into B . licheniformis . Transformants of E . coli were resistant to ampicillin (20 micrograms/ml) in spite of the low penicillinase activities (7 U/mg of cells) . However, transformants of B . subtilis and B . licheniformis were sensitive to ampicillin (20 micrograms/ml) even in high penicillinase activities (more than 10,000 U/mg of cells) . The secretion of penicillinase was rarely observed in E . coli . In contrast, penicillinases secreted from transformants of B . subtilis and B . licheniformis were around 30 and 60% of the total activities, respectively . We took advantage of the plasmids to permit the construction of hetero- and mero-polyploid structures in host cells, and we discuss a regulatory mechanism of penicillinase synthesis in B . licheniformis.

Boll Soc Ital Biol Sper, 1981 Aug 15, 57(15), 1601 - 7
{Comparative photodynamic effect of methylene blue in solution and immobilized on Escherichia coli and Bacillus subtilis}; Savino A et al.; The photodynamic activity of Methylene Bleu (MB) in homogeneous aqueous solution and in heterogeneous phase was compared in order to experiment the disinfection of waters and waste waters using the photosensitation technique . Insolubilized MB was obtained by covalent bonding on a styrene - 2% Divinylbenzene copolymer . E . coli and B . subtilis were used as gram-positive and gram-negative test microorganism . In the present experimental condition high inactivation towards both microorganism was obtained without substantial difference between homogeneous and heterogeneous phases . Therefore the photooxidation with immobilized BM seem to exalte the potential advantage of this technique in water disinfection for the remarkable bactericidal activity associated with the oossibility of the reuse of the photosensitizer.

Jpn J Antibiot, 1981 Aug, 34(8), 1158 - 72
{Clinical studies on tobramycin for infectious diseases following intravenous drip infusion (author's transl)}; Nakamura T et al.; An antibiotic drug of aminoglycoside group, tobramycin (TOB) for parenteral use was used to 18 hospitalized patients: 5 with cholecystitis, 10 with acute appendicitis and 3 others . TOB in a dose of 60-90 mg were administered before the operation, 9 cases were administered by intravenous drip infusion for 1-2 hours, 7 cases by intramuscularly and 2 cases by intravenously . The materials of A-bile, B-bile, wall of the gallbladder, the appendix, ascites and serum samples were taken during the operation . TOB concentration was measured by bioassay method with Bacillus subtilis ATCC 6633 strain . TOB concentration in B-bile and gallbladder wall were higher than those in the A-bile . TOB concentration in gallbladder wall and appendix were directly proportional to degree of pathological changes of the inflammation . For the therapeutic purpose, TOB were given to the 15 patients of the above 18 cases . TOB in a dose of 60-90 mg were administered by intravenous drip infusion for 1-2 hours, twice or 3 times a day for 3-18 days . Clinical response was excellent in 2 cases, good in 11 cases, fair in 1 case and poor in 1 case . No adverse effect was observed . Therefore, it was supposed that TOB could be used safety by intravenous drip infusion.

Eur J Biochem, 1981 Aug, 118(2), 323 - 7
Structure of bacillomycin D, a new antibiotic of the iturin group; Peypoux F et al.; Bacillomycin D is an antifungal agent extracted from the culture medium of a strain of Bacillus subtilis . It is a mixture of two homologous lipopeptides: the lipid moiety consists of 3-amino-12-methyltridecanoic acid or 3-amino-12-methyltetradecanoic acid; the peptide moiety contains one residue of each of the following seven amino acid: D-asparagine, L-aspartate, L-glutamate, L-proline, D-serine, L-threonine and D- tyrosine . The peptide sequence and the cyclic structure were determined by structural analysis of the peptides obtained by mild acid hydrolysis and by cleavage of the molecule with N-bromosuccinimide.

Proc Natl Acad Sci U S A, 1981 Aug, 78(8), 4912 - 6
Initiation factor-independent translation of mRNAs from Gram-positive bacteria; McLaughlin JR et al.; Initiation factor-independent translation of mRNA derived from bacillus phage phi29 DNA occurs with translation systems derived from Bacillus subtilis or Escherichia coli . This is in sharp contrast to the strict dependence on ribosome salt wash fraction of E . coli ribosomes for the translation of T7 and other mRNAs derived from Gram-negative organisms.

J Biochem (Tokyo), 1981 Aug, 90(2), 521 - 6
Structures of heterooligosaccharides synthesized by levansucrase; Tanaka T et al.; In the presence of excess amounts of various monosaccharides as acceptors, Bacillus subtilis levansucrase synthesized various heterooligosaccharides as a result of transfer of the fructosyl residue from sucrose . The saccharides produced in the presence of D-glucose, D-mannose, and D-xylose were all non-reducing disaccharides and were identified as beta-D-fructofuranosyl-alpha-D-glucopyranoside (sucrose), beta-D-fructo-furanosyl-alpha-D-mannopyranoside (mannosucrose), and beta-D-fructofuranosyl-alpha-D-xylopyranoside (xylsucrose), respectively . The saccharides produced in the presence of D-galactose were also non-reducing, but consisted of di-, tri-, and tetrasaccharides . The di- and trisaccharides were galsucrose and 6F-beta-D-fructofuranosyl-galsucrose . The saccharide synthesized in the presence of L-arabinose was a reducing saccharide and was determined to be 4-O-beta-D-fructofuranosyl-L-arabinose . In the presence of D-fructose, several reducing levan oligomers, levanbiose, levantriose, levantetraose, etc., were produced, which were not observed in the synthesis of levan from sucrose alone.

J Virol, 1981 Aug, 39(2), 536 - 47
Synthesis of deoxythymidylate and the unusual deoxynucleotide in mature DNA of Bacillus subtilis bacteriophage SP10 occurs by postreplicational modification of 5-hydroxymethyldeoxyuridylate; Witmer H; Mature DNA of Bacillus subtilis W23 phage SP10 contains a hypermodified nucleotide (YdTMP) that replaces ca . 20% of the DTMP . SP10 DNA was pulse-labeled for 1 min at 20 degrees C with 32Pi . Among the oxopyrimidine nucleotides, virtually all of the radioactivity was recovered as 5-hydroxymethyldeoxyuridylate (HMdUMP) . During the subsequent chase, radioactivity was lost from HMdUMP and recovered as YdTMP . At 37 degrees C, exogenous {6-3H}5-hydroxymethyldeoxyuridine (HMdUrd) was incorporated into SP10 DNA . Label administered as HMdUrd was phosphorylated to HMdUTP in the infected cells, but all radioactivity was recovered from SP10 DNA as YdTMP and dTMP . Two heat-sensitive mutants defective in hypermodification of SP10 DNA are described . In one mutant, HMdUMP replaces YdTMP in DNA . The other mutant generates a DNA containing a novel deoxynucleotide in place of YdTMP . The novel deoxynucleotide seems to consist of PPi esterified to the 5-hydroxymethyl function of HMdUMP (PP-HMdUMP) . Both mutants make normal amounts of dTMP . The data are discussed in terms of the following conclusions . (i) Both oxopyrimidine nucleotides in mature SP10 DNA are derived by postreplicative modification of HMdUMP in nascent DNA . (ii) PP-HMdUMP is an intermediate that facilitate formation of a putative exocyclic methylene intermediate which receives the hypermodification . It is also argued that PP-HMdUMP and the same exocyclic methylene intermediate could serve as intermediates in reductive modification to dTMP . (iii) YdTMP is not an intermediate in the formation of dTMP, and reductive modification proceeds independently of hypermodification.

Infect Immun, 1981 Aug, 33(2), 450 - 8
Plasmid-chromosomal transition of genes important in staphylococcal enterotoxin B expression; Dyer DW et al.; Experiments were performed to further elucidate the genetic mechanisms underlying the synthesis of staphylococcal enterotoxin B (SEB) . Our laboratory has previously shown that, in strains of Staphylococcus aureus which harbor a 1.15-megadalton plasmid (pentB or pSN2), the plasmid appears to be required for SEB synthesis; in other S . aureus strains, designated chromosomal SEB producers, this 1.15-megadalton plasmid is conspicuously absent . We report here than in both Bacillus subtilis minicells and a coupled translational assay, pSN2 codes for a polypeptide of 18,000 daltons . This product is not immunologically reactive with purified anti-SEB globulin . Nevertheless, pSN2 is necessary but not sufficient for SEB synthesis in strains which harbor the plasmid . Further, the data provide a reasonable link between plasmid-bearing and chromosomal SEB producers: transformational analysis indicates that both require functions specified (in plasmid-bearing strains) by pSN2 for SEB synthesis . The combined genetic and biochemical data suggest that pSN2 is not the reservoir for the SEB structural gene, but that the pSN2-specific functions required for SEB synthesis are regulatory in nature.

Zentralbl Bakteriol A, 1981 Aug, 249(3), 341 - 9
Increased serum antibacterial activity after turpentine-induced acute inflammation; Maxim PE et al.; An acute inflammatory response was induced in rabbits by an intramuscular injection of turpentine . After this injection serial serum samples were obtained and serum haptoglobin levels were monitored . In addition, increased antibacterial activity was measured using a viable plate and photometric growth assay . As early as 4 hrs after turpentine challenge an increase was noted in serum antibacterial activity towards Staphylococcus aureus . At 48 hrs pronounced killing activity was demonstrated against S . aureus and S . epidermidis but not against Escherichia coli or other gram negative bacilli . By five days another serum bactericidal activity was present against Bacillus subtilis . Enhanced serum antibacterial activity persisted for 2 weeks . Partial characterization of these factors indicates that they are probably beta lysins.

J Bacteriol, 1981 Aug, 147(2), 443 - 51
A cloned gene that is turned on at an intermediate stage of spore formation in Bacillus subtilis; Ollington JF et al.; Cells of Bacillus subtilis synthesize a relatively long-lived ribonucleic acid (RNA) of about 300 bases during the course of spore formation . This transcript does not appear until an intermediate stage (III or IV) of development but is the predominant sporulation-specific transcript among RNAs of discrete size in late (stages IV to VI) developing cells . Appearance of the 300-base RNA is under sporulation control as this transcript could not be detected in cells of an early-blocked sporulation mutant (Spo0A) . We have located the coding sequence for the 300-base RNA within a cloned chromosomal segment from the purA-cysA region that was previously shown to contain a cluster of genes that are actively transcribed during sporulation . The coding sequence for the 300-base RNA (designated as the 0.3 kb gene) mapped between a gene (veg) that was actively transcribed during growth and development and a gene (0.4 kb) that was turned on at the onset of sporulation . Although clustered within a small segment of the chromosome, the veg, 0.3 kb, and 0.4 kb transcription units exhibited, therefore, distinct patterns of temporally programmed gene expression . Models for the activation of the 0.3 kb gene at an intermediate stage of development are discussed.

J Bacteriol, 1981 Aug, 147(2), 432 - 42
Developmentally regulated transcription in a cloned segment of the Bacillus subtilis chromosome; Ollington JF et al.; We describe a model system for studying developmentally regulated transcription during spore formation in Bacillus subtilis . This model system is a cloned cluster of genes known as 0.4 kb, ctc, and veg from the purA-cysA region of the B . subtilis chromosome . Each gene exhibited a distinct pattern of transcription in cells growing in glucose medium and in cells deprived of nutrients in sporulation medium . The 0.4 kb gene was transcribed at a low level in growing cells but was actively transcribed during nutrient deprivation in sporulation medium . This ribonucleic acid (RNA) synthesis was dependent upon the products of five B . subtilis genes that are involved in the initiation of spore formation:spo0A, spo0A, spo0E, spo0F, and spo0H . A mutation in any one of these regulatory genes severely restricted transcription of the 0.4 kb sequence . Transcription of the ctc gene was also turned on by nutrient deprivation, but this RNA synthesis was not impaired in spo0 mutants . Although not under spo0 control, the ctc gene probably corresponds to a locus, spoVC, whose product is required at a late stage of sporulation . Finally, the veg gene was actively transcribed both in growing cells and in nutrient-deprived cells . Like ctc RNA synthesis, transcription of the veg gene was not dependent upon the spo0 gene products . We propose that the spo0A, spo0B, spo0E, spo0F, and spo0H gene products are components of a pathway(s) that senses nutrient deprivation in B . subtilis and translates this environmental signal into the transcriptional activation of a subset of developmental genes.

J Virol, 1981 Aug, 39(2), 407 - 12
Cloning and expression of bacteriophage SP02 DNAZ polymerase gene L in Bacillus subtilis, using the Staphylococcus aureus plasmid pC194; Rutberg L et al.; HindIII restriction endonuclease fragments of DNA from temperate Bacillus subtilis bacteriophage SP02 were cloned in B . subtilis by using the plasmid pC194 . Three hybrid plasmids which permit growth of the mutant SP02 susL244 in suppressor-negative bacteria were isolated . SP02 gene L is thought to code for a DNA polymerase essential for autonomous replication of SP02 DNA . Extracts of bacteria carrying one of these hybrid plasmids, pC194-96, had 10- to 30-fold increased DNA polymerase activity . The plasmid-induced DNA polymerase activity differed from that of the known B . subtilis DNA polymerases in several respects . The results of the experiments support the idea that phage SP02 codes for a new DNA polymerase.

J Bacteriol, 1981 Aug, 147(2), 535 - 42
Characterization of a membrane-associated cytidine diphosphate-diacylglycerol-dependent phosphatidylserine synthase in bacilli; Dutt A et al.; The synthesis of phosphatidylserine in two gram-positive aerobic bacteria has been partially characterized . We have located a cytidine 5'-diphospho-diacylglycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase) activity in the membrane fraction of Bacillus licheniformis and Bacillus subtilis . The activity was demonstrated to be membrane associated by differential centrifugation, sucrose gradient centrifugation, and detergent solubilization . The direct involvement of cytidine 5'-diphospho-diacylglycerol in the reaction was demonstrated by the conversion of the liponucleotide phosphatidyl moiety to phosphatidylserine . This activity is dependent on divalent metal ion (manganese being optimal) and is stimulated by nonionic detergent and its product phosphatidylserine . Based on studies with various combinations of products and substrates, the reaction appears to follow a sequential BiBi kinetic mechanism.

J Bacteriol, 1981 Aug, 147(2), 622 - 32
Cloning of an early sporulation gene in Bacillus subtilis; Dubnau E et al.; A 0.8-megadalton BglII restriction fragment of Bacillus licheniformis cloned into the BglII site of plasmid pBD64 can complement spo0H mutations of Bacillus subtilis . The clone was isolated by selecting for the Spo+ phenotype and antibiotic resistance, using the helper system described by Gryczan et al . (Mol . Gen . Genet . 177:459-467, 1980) . The insert is functional in both orientations and thus presumably has its own promoter . A deletion generated within the 0.8-megadalton insert by HindIII restriction and subsequent religation eliminates the ability of the cloned fragment to complement spo0H mutations . The cloned B . licheniformis deoxyribonucleic acid segment specifies the synthesis, in minicells, of a polypeptide of approximately 27,000 daltons . This protein is observed with both orientations, but not when the HindIII deletion is present in the cloned B . licheniformis chromosomal fragment . We have also demonstrated that ribonucleic acid complementary to the cloned B . licheniformis sporulation gene is transcribed in B . licheniformis both during vegetative growth and sporulation.

Can J Microbiol, 1981 Aug, 27(8), 795 - 800
Purification by affinity chromatography of a membrane dicarboxylate binding protein from Bacillus subtilis; Kay WW; Membrane vesicles of Bacillus subtilis W23 actively transport the C4 and C5 dicarboxylates of the tricarboxylate cycle by system(s) of relatively high affinity for their requisite substrates (Km 4-53 microM) . Glutamate and succinate binding activities were readily solubilized from membrane vesicles by nonionic detergents, particularly by Lubrol WX . From this extract, glutamate binding activity was highly enriched by affinity chromatography on phloroglucinol-expanded Sepharose-6B to which L-aspartate was coupled via divinylsulfone . Another protein (41000 molecular weight), which bound both L-glutamate and L-malate, was purified from affinity columns to which either L-glutamate or L-malate had been coupled via bisdiglycidyl ether . This protein bound labelled L-malate as well as L-glutamate with affinities similar to those seen with membrane vesicles (Kd's 8 microM L-malate and 52 microM L-glutamate).

J Biol Chem, 1981 Jul 25, 256(14), 7424 - 32
The role of the delta peptide of the Bacillus subtilis RNA polymerase in promoter selection; Achberger EC et al.; We have examined the effect of the delta subunit of the Bacillus subtilis RNA polymerase on the formation of closed, open, and stably initiated complexes with Hha I restriction fragments of phage SP82 DNA; the effect of delta on the transcription of these DNA fragments has also been investigated . In vitro, the holoenzyme (core-sigma-delta) bound to and transcribed the same regions of the phage genome that are transcribed in vivo early in infection . In the absence of the delta subunit, the polymerase (core-sigma) bound nonspecifically and transcribed regions of the genome other than those containing early phage genes . Addition of delta to preparations of core-sigma restored the pattern of binding and transcription observed with the holoenzyme . Similarly, delta-less preparations of two SP82-modified forms of polymerase (the enzyme isolated at 8 min after infection and the enzyme isolated 20 min after infection) bound nonspecifically and transcribed regions of the genome other than those containing "middle" and "late" genes . Addition of delta to these preparations resulted in patterns of binding and transcription expected for enzymes functioning a middle and late times of infection, respectively . Quantitation of polymerase-DNA complexes at various temperatures, NaCl concentrations, and polymerase-DNA ratios supported the conclusion that delta enhanced promoter selection.

Nucleic Acids Res, 1981 Jul 24, 9(14), 3545 - 54
Detection of high levels of polyadenylate-containing RNA in bacteria by the use of a single-step RNA isolation procedure; Gopalakrishna Y et al.; A new one-step procedure for the isolation of bacterial RNA, involving lysis by proteinase K in the presence of sodium dodecyl sulfate, is described . Pulse-labeled RNA isolated by this procedure for Bacillus brevis, Bacillus subtilis, and Escherichia coli B has been found to contain a substantial fraction (15-40%) of polyadenylated RNA as determined by adsorption to oligo(dT)-cellulose . This contrasts with RNA isolated by procedures involving phenol extraction, a process which appears to lead to the selective loss of polyadenylated RNA . The presence of polyadenylated RNA in E . coli was confirmed by an independent method which involved hybridization with {3H}polyuridylic acid . Using the proteinase K method for RNA isolation, it was possible to demonstrate the in vitro synthesis of polyadenylated RNA by toluene-treated cells of B . brevis, B . subtilis, and E . coli.

Tijdschr Diergeneeskd, 1981 Jul 15, 106(14), 136 - 42
Microbiological assay methods for sulfonamides in animal tissues, serum, and milk; Nouws JF; The sensitivity of microbiological assay methods for sulfonamides in muscle, kidney, serum, and milk was improved with Standard II Nahragar (Merck 7883), the medium being supplemented with trimethoprim, 1% sodium chloride, and 0.4% dextrose . The agar was seeded with 10(4) spores of Bacillus subtilis BGA per ml medium . The trimethoprim concentrations per ml agar were, at pH 6.20, pH 7.0, and pH 8.0 respectively 0.30, 0.15 and 0.07 microgram . The sulfonamide sensitivity of the test method depended on the pK-value of the drug tested and on the pH of the agar . For sulfonamides tested in muscle homogenate the optimum sensitivity of the bioassay methods was in the range of 0.32-0.63 microgram/ml; in kidney homogenate it was 0.32-0.63 microgram/ml; in serum 0.32-0.63 microgram/ml; and in milk 0.08-0.32 microgram/ml . The sensitivity for dapsone ranged in these substrates from 0.01 to 0.02 microgram/ml . The assay methods here developed may be used for both qualitative detection and quantitative determinations of sulfonamides in animal tissues, serum, and milk.

J Biol Chem, 1981 Jul 10, 256(13), 6866 - 75
Initiation of Bacillus subtilis sporulation by the stringent response to partial amino acid deprivation; Ochi K et al.; We have controlled the rates at which three different amino acids were available to auxotrophs of Bacillus subtilis by avoiding active transport of the respective substrate . The active transport of oxomethylvalerate, a precursor of isoleucine, was prevented by a kauA mutation, the uptake of L-aspartate was competed by 20 mM L-glutamate, and D-methionine was used instead of L-methionine . When in this way conditions of partial amino acid deprivation were achieved, a partial "stringent response" occurred which included the increase of ppGpp and pppGpp, and the decrease of GTP; such conditions initiated sporulation . In the corresponding relaxed (relA) mutants, the changes of guanine nucleotides were greatly reduced and no sporulation was observed at any substrate concentration; but addition of decoyinine produced a further decrease of GTP and caused sporulation.

Eur J Biochem, 1981 Jul, 117(3), 499 - 505
Purification, properties and assembly of the neck-appendage protein of the Bacillus subtilis phage phi 29; Villanueva N et al.; The purification of the neck appendage protein of phi 29, p12*, which is involved in the adsorption of the phage to Bacillus subtilis, is described . The purified native protein is in a dimeric form and can be assembled, in vitro, onto purified 12- particles that lack the neck appendages, suggesting that the incorporation of p12* to the rest of the phage structure is a self-assembly process . The assembly of protein p12* in vitro follows cooperative kinetics and it occurs with an efficiency of about 4%.

J Virol, 1981 Jul, 39(1), 318 - 20
High-frequency elimination of SP02 prophage from Bacillus subtilis by plasmid transformation; Marrero R et al.; Transformation of competent Bacillus subtilis lysogenic for SP02 with any of three plasmids (pCM194, pUB110, pAM77) generates drug-resistant transformants of which 5 to 20% have lost the infectivity and immunity associated with the SP02 prophage . Such cured derivatives can be again lysogenized with SP02 and again cured by introduction of a different plasmid . Elimination of the SP02 prophage was not detected when plasmids were introduced by PBS1 transduction or by transformation of protoplasts . Similarly, transformants of B . subtilis selected for chromosome markers retained the prophage . The phi 105 prophage was not eliminated from competent B . subtilis transformed with plasmids.

Mutat Res, 1981 Jul, 82(2), 263 - 8
Killing and mutagenic action of sunlight upon Bacillus subtilis spores: a dosimetric system; Munakata N; A method to monitor killing and mutagenic activity of sunlight was established by using wild-type spores (UVR) of Bacillus subtilis and mutant spores (UVS) sensitive to UV radiation . Samples exposed to radiations consisted of the spores spotted and dried on membrane filter . After the exposure, they were recovered as suspensions in water and assayed for colony-forming survival and frequency of reversion of an auxotrophic marker (hisB101) . In this system, the UVS spores were inactivated exponentially, and the 37% survival was attained with 2.0 Jm-2 of 254 nm or 2.5 X 10(3) Jm-2 of 313 nm radiation, and with 7 min (August) or 63 min (December) exposure to noon-time sunlight under a clear sky at Tsukiji (latitude 35 degrees 40' N) at sea level in Tokyo . The doubling of the spontaneous mutation frequency of the UVR spores was attained with 3.0 Jm-2 of 254 nm or 2.2 X 10(3) Jm-2 of 313 nm radiation, and with 32 min (August) or 136 min (December) of solar exposure . The results encourage the use of this B . subtilis spore system to determine the gene-damaging activity of the solar-UV radiation under a variety of environmental conditions.

Mutat Res, 1981 Jul, 82(2), 251 - 61
UV-induced mutation fixation in Bacillus subtilis; Filippov VD et al.; The influence was studied of a temporary specific inhibition of post-radiation macromolecular synthesis and of preliminary UV irradiation on the kinetics of accumulation of fixed mutations, that is mutations insensitive to MFD, in UV-irradiated B . subtilis cells . From experimental results it is deduced that the entry of a pre-mutagenic lesion into a round of replication, initiated before irradiation, is not a fixing event in UV mutagenesis . For performance of fixation, the proceeding of replication, initiated after irradiation, and protein synthesis are necessary . In irradiated cells incubated in medium with lowered concentration of nitrogen sources, the antimutagenic activity of a uvr+-dependent repair system competes with the process of fixation for pre-mutagenic lesions and reduces the efficiency of mutagenesis . The most efficient fixation and mutagenesis occur at high concentrations of nitrogen sources in post-radiation medium, when the manifestation of antimutagenic activity appears to be blocked . The possible nature of a process leading to mutation fixation is discussed.

Appl Environ Microbiol, 1981 Jul, 42(1), 79 - 82
Heat sensitization of bacterial spores after exposure to ethidium bromide, acriflavine, or daunomycin; Hanlin JH et al.; A 20-min exposure of 10(7) unmodified spores of either Bacillus subtilis NCTC 3610 (harvested from potato-dextrose agar plus manganese) or Bacillus megaterium ATCC 19213 (harvested from nutrient agar plus manganese) per ml to 5 microgram of ethidium bromide per ml did not kill the spores (recovered on TAM {thermoacidurans agar modified}-plus thymidine medium) . However, in both cases, the ability to survive various heat treatments was reduced after exposure of the spores to ethidium bromide . With B . subtilis, a 10-min heat treatment at 85 degrees C of unexposed spores resulted in an 85% survival rate, whereas only 50% of the ethidium bromide-exposed spores survived . With B . megaterium similar results were obtained at 75 degrees C; 77% of the unexposed spores survived, whereas only 31% of the ethidium bromide-exposed spores survived . Similarly, a 10-min exposure of B . subtilis spores to 0.005 microgram of acriflavine per ml did not kill unheated spores; however, the ability of the spores to survive exposure at 85 degrees C for 10 min was reduced to 40% . After exposure to 10 microgram of daunomycin per ml, the survival rate was 35% . Binding studies with ethidium bromide showed strong binding to spores, but as yet, the site of binding is unknown.

J Bacteriol, 1981 Jul, 147(1), 267 - 70
Sensory adaptation and deadaptation by Bacillus subtilis; Goldman DJ et al.; Cells of Bacillus subtilis, when tethered by using antiflagellar antibody, rotate briefly counterclockwise (swimming behavior) or clockwise (tumbling behavior) when amino acids are added or removed, respectively . "Dissociation constants" for attractant-binding site interactions, calculated from duration of the rotational response to addition of amino acids, agreed with those calculated for their removal and with previous values calculated from sensitivity capillary assays . The ratio of adaptation times for addition versus removal of attractant averaged 1.7, which differs greatly from the value of 50 for Escherichia coli.

J Bacteriol, 1981 Jul, 147(1), 259 - 61
Absence in Bacillus subtilis and Staphylococcus aureus of the sequence-specific deoxyribonucleic acid methylation that is conferred in Escherichia coli K-12 by the dam and dcm enzymes; Dreiseikelmann B et al.; Restriction analysis of plasmid pHV14 deoxyribonucleic acid isolated from Escherichia coli K-12, Bacillus subtilis, and staphylococcus aureus with restriction endonucleases MboI, Sau3AI, and EcoRII was used to study the methylation of those nucleotide sequences which in E . coli contain the major portions of N6-methyladenine and 5-methylcytosine . The results showed that neither B . subtilis nor S . aureus methylates deoxyribonucleic acid at the same sites and nucleotides which are recognized and methylated by dam and dcm enzymes in E . coli K-12.

J Bacteriol, 1981 Jul, 147(1), 206 - 16
Single-stranded fraction of deoxyribonucleic acid from Bacillus subtilis; Piwnicka M et al.; About 13% of the deoxyribonucleic acid (DNA) of various strains of Bacillus subtilis, independent of the stage of growth or competence for transformation, was rendered acid soluble by endonuclease S1 . In a pH 11.2 CsCl gradient, 4% of the untreated DNA banded at the density typical for single-stranded molecules, whereas 9% of the remaining DNA (main band) was sensitive to endonuclease S1 . Selective inhibition of DNA polymerase III, or of DNA-dependent ribonucleic acid polymerase, did not increase or abolish single-strandedness . The DNA purification procedure did affect the level of single-stranded DNA, indicating its binding to cell constituents containing ribonucleic acid, protein, and membranous material . The molecular weight of the single-stranded fraction resembled that of total denatured DNA, and its buoyant density in an alkaline CsCl gradient was centered partially at a density of 1.772 g/cm3 and partially at a density of 7.759 g/cm3 . Incubation of DNA under conditions leading to renaturation of its single-stranded fraction led to an increase in transforming activity for the purA16+ marker (close to the origin of replication) relative to leu-8+ and metC3+ markers (located in the middle of the chromosome), indicating this region is the main source of the single-stranded fraction.

Jpn J Antibiot, 1981 Jun, 34(6), 980 - 93
{Clinical studies on dibekacin for infectious diseases following intramuscular and intravenous drip administration . Concentration in infected tissues and clinical responses (author's transl)}; Nakamura T et al.; An antibiotic drug of aminoglycoside group, dibekacin (DKB) for parental use was used in 48 patients hospitalized due to acute or subacute infection of abdominal organs: 36 appendicitis, 9 cholecystitis and 3 others . DKB in a dose of 100 mg was given intramuscularly in 38 cases, and in 10 cases was given intravenously by single or drip infusion before the operation . The materials of A-bile, B-bile, wall of gallbladder, the appendix wall, ascites with pus and serum were taken during the operation . DKB concentration was measured by bioassay method with Bacillus Subtilis ATCC 6633 strain . With a few marked exceptions, DKB concentration in B-bile were higher than those in A-bile . DKB concentrations in gallbladder wall and appendix wall were directly proportional to the degree of pathological changes of inflammation . DKB concentrations in infected tissues after intravenous drip infusion, they were higher relatively than those after intramuscular administration . DKB concentrations in serum after intravenous drip infusion reached to peak immediately the end of infusion, and in the infected tissue they reached to peak at the same time and stayed for a relatively long time, then they were declined slowly . For the therapeutic purpose, DKB was given to the 6 patients with acute peritonitis of the above cases . DKB in a dose of 100 mg were administered by intravenous drip infusion for 2 hours, twice in a day for 3 - 10 days . Clinical response was excellent in 2 cases, good in 3 cases, fair in 1 case and poor in none . No adverse effect was observed . Therefore, it was supposed that DKB could be used safely by intravenous drip infusion.

J Biochem (Tokyo), 1981 Jun, 89(6), 1681 - 91
Purification and properties of RNA polymerases from mother cells and forespores of sporulating cells of Bacillus subtilis; Nakayama T et al.; Bacillus subtilis sporulating cells at stage III were fractionated into mother cell and forespore fractions by means of a lysozyme-detergent method . Three forms of DNA-dependent RNA polymerase enzymes, termed M sigma, F sigma, and F delta, in addition to core enzyme (alpha 2, beta', and beta) have been purified from the cell fractions . Enzymes M sigma and F sigma are present in the mother cell and forespore, respectively, and contain sigma factor of 55,000 daltons in addition to the core subunits . On the other hand, enzyme F delta is present specifically in the forespore and contains delta 1 factor of 28,000 daltons instead of the sigma factor . The amount of RNA polymerase in the forespore is about twice that in the mother cell . The enzymes M sigma and F sigma also differed in their elution profiled from DEAE-cellulose columns and in their heat stabilities indicating that the two sigma-containing holoenzyme forms may be different in their structural properties . The enzyme F delta transcribed B . subtilis DNA about 1.6 times more actively than enzyme F sigma, and the enzymes M sigma and F sigma transcribed the DNA about 2.2 times more actively than did core enzyme.

Can J Microbiol, 1981 Jun, 27(6), 604 - 11
Studies on the association of the alpha and beta 2 subunits of the tryptophan synthase of Bacillus subtilis; Hoch SO et al.; Studies using Sephadex gel filtration indicated that the alpha and beta 2 components of the Bacillus subtilis tryptophan synthase associate to form complexes under the appropriate conditions of buffer, pH, and temperature . Monovalent cations, glycerol, and the cofactor, pyridoxal-5'-phosphate, were required to maintain and active beta 2 component, and in turn, affected the association of the alpha and beta 2 components . Under conditions that stabilized the individual components during their purification, the affinity of the subunits for each other was weak . Under the same buffer conditions, but at the higher pH of 7.8 which the enzymatic activities are assayed, the individual components readily associated . The substrate serine appeared to affect complex formation but there was no effect from the indole moiety . When the temperature was raised from 4 to 22-25 degrees C, complex formation was observed at both pH 6.6 and 7.8 . The results of these experiments are consistent with the formation of alpha beta 2 and alpha 2 beta 2 species as the associated tryptophan synthase complexes of B . subtilis.

J Bacteriol, 1981 Jun, 146(3), 972 - 82
Acid-soluble spore proteins of Bacillus subtilis; Johnson WC et al.; Acid-soluble spore proteins (ASSPs) comprise about 5% of the total protein of mature spores of different Bacillus subtilis strains . They consist of three abundant species, alpha, beta, and gamma, four less abundant species, and several minor species, alpha, beta, and gamma make up about 18, 18 and 36%, respectively, of the total ASSPs of strain 168, have molecular weights of 5,900, 5,9000, and 11,000, respectively, and resemble the major (A, C, and B) components of Bacillus megaterium ASSPs in several respects, including sensitivity to a specific B . megaterium spore endopeptidase . However, they have pI's of 6.58, 6.67, and 7.96, all lower than those of any of the B . megaterium ASSPs . Although strains varied in the proportions of different ASSPs, to overall patterns seen on gel electrophoresis are constant . ASSPs are located interior to the cortex, presumably in the spore cytoplasm, and are synthesized during sporulation and degraded during germination.

J Bacteriol, 1981 Jun, 146(3), 965 - 71
Comparison of various properties of low-molecular-weight proteins from dormant spores of several Bacillus species; Yuan K et al.; Several properties of the major proteins degraded during germination of spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis have been compared . All of the proteins had low molecular weights (6,000 to 13,000) and lacked cysteine, cystine, and tryptophan . The proteins could be subdivided into two groups: group I (B . megaterium A and C proteins, B . cereus A protein, and B . subtilis alpha and beta proteins) and group II (B . cereus and B . megaterium B proteins and B . subtilis gamma protein) . Species in group II had lower levels of (or lacked) the amino acids isoleucine, leucine, methionine, and proline . Similarly, proteins in each group were more closely related immunologically . However, antisera against a B . megaterium group I protein cross-reacted more strongly with the B . megaterium group II protein than with group I proteins from other spore species, whereas antisera against the B . megaterium group II protein cross-reacted most strongly with B . megaterium group I proteins . Analysis of the primary sequences at the amino termini and in the regions of the B . cereus and B . subtilis proteins cleaved by the B . megaterium spore protease revealed that the B . cereus A protein was most similar to the B . megaterium A and C proteins, and the B . cereus B protein and the B . subtilis gamma protein were most similar to the B . megaterium B protein . However, amino terminal sequences within one group of proteins varied considerably, whereas the spore protease cleavage sites were more highly conserved.

J Bacteriol, 1981 Jun, 146(3), 877 - 84
Cell wall metabolism in Bacillus subtilis subsp . niger: accumulation of wall polymers in the supernatant of chemostat cultures; de Boer W et al.; Cell wall polymers were measured both in the cells and in the cell-free medium of samples from steady-state chemostat cultures of Bacillus subtilis, growing at various rates under magnesium or phosphate limitation . The presence of both peptidoglycan and anionic wall polymers in the culture supernatant showed the occurrence of wall turnover in these cultures . Variable proportions of the total peptidoglycan present in the culture samples were found outside the cells in duplicate cultures, indicating that the rate of peptidoglycan turnover is variable in B . subtilis . Besides peptidoglycan, anionic wall polymers were detected in the culture supernatant: teichoic acid in magnesium-limited cultures and teichuronic acid in phosphate-limited cultures . In several samples, the ratio between the peptidoglycan and the anionic polymer concentrations was significantly lower in the extracellular fluid than in the walls . This divergency was attributed to the occurrence of direct secretion of anionic polymers after their synthesis.

J Bacteriol, 1981 Jun, 146(3), 1162 - 5
Cloning restriction fragments that promote expression of a gene in Bacillus subtilis; Williams DM et al.; Plasmid pPL603 (3.1 megadaltons) specifies neomycin resistance in Bacillus subtilis and contains a structural gene for chloramphenicol acetyltransferase . Cells harboring the plasmid cannot grow on solid media containing 10 microgram of chloramphenicol per ml . Cloning EcoRI (or EcoRI)-generated fragments of deoxyribonucleic acid from several sources into the single EcoRI site in plasmid pPL603, with subsequent selection of transformants of media containing 10 micrograms of chloramphenicol per ml, permits the identification of restriction fragments that promote expression of the chloramphenicol acetyltransferase gene.

J Bacteriol, 1981 Jun, 146(3), 1106 - 16
Germination properties of a spore coat-defective mutant of Bacillus subtilis; Moir A; The presence of the gerE36 mutation in strains of Bacillus subtilis 168 resulted in poor germination of their spores in a range of germinants, as measured by the fall in absorbance of spore suspensions . Although resistant to heat and organic solvents, spores were sensitive to lysozyme; electron microscopy revealed that their coat structure was incomplete . These spores responded to germinants by losing heat resistance and changing from phase bright to phase gray . The release of dipicolinic acid and the fall in absorbance of spore suspensions reached only 75 and 50% of wild-type levels, respectively, but followed the same time course as the loss of heat resistance . Although the germination response was incomplete, the concentration of L-alanine required to elicit it was the same for the mutant as for the wild type . The properties of mutant spores suggest that an intact spore coat is not required for the initial interaction between germinant and spore, but that the coat layers may contain molecules important in later stages of germination . In transduction with phage SPP1, the gerE36 mutation mapped between citF and ilvB and was 90% cotransduced with citF2 . The gerE mutation identifies the location of a gene important for the progress of late stages of spore formation.

J Bacteriol, 1981 Jun, 146(3), 867 - 76
Cell wall metabolism in Bacillus subtilis subsp . niger: effects of changes in phosphate supply to the culture; Kruyssen FJ et al.; Chemostat cultures of Bacillus subtilis subsp . niger WM were exposed to changes in the availability of phosphorus by means of a resuspension technique . Responses in wall metabolism were recorded by measuring the amounts of peptidoglycan and anionic polymers (teichoic or teichuronic acid) in the wall and extracellular fluid fractions . With respect to the wall composition, the effect of a change in orthophosphate supply was a complete shift in the nature of the anionic polymer fraction, the polymer originally present in the walls ("old" polymer) being replaced by the alternative ("new") anionic polymer . The peptidoglycan content of the walls remained constant . It was concluded that the incorporation of old polymer was completely blocked from the moment the orthophosphate supply was changed . However, from a measurement of the total amount of polymer in the whole culture during the course of the experiments, it was evident that synthesis of old polymer continued, but it was secreted . Synthesis of the new polymer started immediately, and it was incorporated exclusively into the wall . During adaption of the cells to the new environment, wall turnover continued in an identical fashion to that extant in steady-state cultures . It was concluded that the primary adaptive response to a change in orthophosphate supply occurred through a mechanism interacting with polymer incorporation and thus at the level of wall assembly at the membrane.

Experientia, 1981 May 15, 37(5), 470 - 2
Stimulation of sporulation by ppApp in a conditionally asporogenous rifampin-resistant mutant in Bacillus subtilis; Pun PP et al.; Addition of ppApp to Sterlini-Mandelstam medium stimulates sporulation of a conditionally asporogenous rifampin-resistant mutant of Bacillus subtilis to the same extent as the effect of 4 amino acids . Mutant cells sporulating in the presence of amino acids also produce 2 phosphorylated nucleotides one of which comigrated with ppApp on PEI thin layer chromatogram.

Prikl Biokhim Mikrobiol, 1981 May-Jun, 17(3), 441 - 7
{Chromatography of serine proteases on chitin and its derivatives}; Bendikene VG et al.; Chitin containing sorbents have been obtained for isolation and purification of serine proteases . Serine proteases from Bacillus subtilis have been purified 4-5 times and commercial preparations of trypsin and chymotrypsin 1.5-2 times by chromatography on nondeproteinized chitin . On the benzylated derivative of nondeproteinized chitin complete separation of trypsin and chymotrypsin has been achieved by chromatography of crude pancreatin . It has been shown that the protein moiety of chitin is important for preferential sorption of serine type proteases.

J Biochem (Tokyo), 1981 May, 89(5), 1581 - 7
Farnesyl pyrophosphate synthetase from Bacillus subtilis; Takahashi I et al.; Farnesyl pyrophosphate synthetase was detected in extracts of Bacillus subtilis and partially purified by Sephadex G-100, hydroxylapatite, and DEAE-Sephadex chromatography . The enzyme catalyzed the exclusive formation of all-trans farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate . Mg2+ was essential for the catalytic activity and Mn2+ was less effective . The enzyme was slightly activated by sulfhydryl reagents . This enzyme was markedly stimulated by K+, NH4+, or detergents such as Triton X-100 and Tween 80, unlike the known farnesyl pyrophosphate synthetases from eucaryotes . The molecular weight of the enzyme was estimated by gel filtration to be 67,000 . The Michaelis constants for dimethylallyl and geranyl pyrophosphate were 50 microM and 18 microM, respectively.

J Nat Prod, 1981 May-Jun, 44(3), 252 - 6
Antibiotic principle of Eupatorium capillifolium; Rao KV et al.; Ethanolic extracts of Eupatorium capillifolium (Lam) Small showed activity against Bacillus subtilis grown in a chemically defined medium but not in a complex natural medium . The active principle was isolated as a colorless crystalline solid . A study of its properties and mass spectral fragmentation pattern showed that it was costic acid, a sesquiterpenic acid previously isolated from Costus root oil . The observed inactivity of costic acid against B . subtilis in a complex medium was shown to be due to the presence in the medium of glutamic acid, which is capable of reversing 90% of the activity of costic acid.

Arch Microbiol, 1981 May, 129(3), 227 - 32
Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis; Srivastava OP et al.; Two proteases, designated I and II, have been isolated from sporulating cells of Bacillus subtilis . They were partially purified by ammonium sulfate fractionation, Sephadex chromatography and affinity columns . Protease I was found to be similar to an already characterized B . subtilis protease . Protease II is trypsin-like in its substrate specificity and is distinct from protease I in its pH optimum, pH stability, molecular weight, substrate specificity, heat stability and sensitivity to various inhibitors . While both enzymes were produced primarily during sporulation, they attained maximum levels of activity at different times . Distinct functions for these proteases in post exponential B . subtilis are likely.

Proc Natl Acad Sci U S A, 1981 May, 78(5), 2762 - 6
Heterogeneity of RNA polymerase in Bacillus subtilis: evidence for an additional sigma factor in vegetative cells; Wiggs JL et al.; Preparations of Bacillus subtilis RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from vegetatively growing cells contain small amounts of an activity (B . subtilis RNA polymerase holoenzyme II) that shows a unique promoter specificity with T7 bacteriophage DNA as compared with the normal B . subtilis holoenzyme (holoenzyme I) and lacks the normal sigma subunit {Jaehning, J . A., Wiggs, J . L . & Chamberlin, M . J . (1979) Proc . Natl . Acad . Sci . USA 76, 5470-5474} . By heparin-agarose chromatography we have obtained holoenzyme II fractions that have no detectable holoenzyme I activity as judged by their failure to utilize promoter sites for holoenzyme I on any template we have tested . These fractions are far more active with B . subtilis DNA than with T7 DNA or other heterologous templates . This high degree of specificity has allowed identification of plasmids containing cloned fragments of B . subtilis DNA that bear strong promoter sites for holoenzyme II . These promoter sites are not used at all by B . subtilis RNA polymerase holoenzyme I . The specificity of holoenzyme II is dictated by a peptide of Mr 28,000 as judged by copurification of the peptide with specific holoenzyme II activity and by reconstitution of the holoenzyme II promoter specificity when the isolated peptide is added to B . subtilis core polymerase . Hence the 28,000 Mr peptide appears to be a sigma factor that determines a promoter specificity distinct from that of RNA polymerase holoenzyme I and all other known bacterial RNA polymerases.

J Bacteriol, 1981 May, 146(2), 494 - 505
Cloning of sporulation gene spoOB of Bacillus subtilis and its genetic and biochemical analysis; Hirochika H et al.; A specialized transducing phage carrying a sporulation gene (spoOB) was constructed from Bacillus subtilis temperate phage rho 11 by in vitro and in vivo recombinations . Transformation experiments showed that the spoOB gene resides on a 1.4-megadalton fragment generated by EcoRI endonuclease treatment of the phage deoxyribonucleic acid (DNA) . Mutants of this phage which lost transducing activity were isolated and used for genetic complementation tests and the analysis of protein(s) coded by the 1.4-megadalton fragment . The spoOB locus was shown to be composed of one cistron . Sodium dodecyl sulfate-polyacrylamide gel analysis of proteins synthesized in ultraviolet-irradiated cells infected with these phages showed that the 1.4-megadalton fragment codes at least one protein, of molecular weight 39,000, which is synthesized in both vegetative and sporulating cells . A cleavage map of the phage DNA was constructed by use of restriction endonucleases, EcoRI, BamHI, and SalI, and the site of integration of the 1.4-megadalton fragment was determined . Expression and function of the spoOB gene are discussed.

Prikl Biokhim Mikrobiol, 1981 May-Jun, 17(3), 430 - 5
{Isolation and study of physico-chemical properties of alpha-amylase from Bacillus subtilis}; Kostareva IA et al.; By chromatography on ultragel ACA-54 alpha-amylase was isolated from the enzymic preparation amylosubtilin G10x . As compared to the initial preparation, the specific activity of the purified enzyme per mg increased 25-fold . The major physico-chemical characteristics of alpha-amylase were determined . The molecular weight of the enzyme measured by gel-chromatography and electrophoresis was estimated to be 49,000 . The isoelectric point determined by electrofocusing was found to be 5,2 . Irreversible acid inactivation of the enzyme in the range of pH 2-5 was investigated . The reaction was found to develop in at least two stages.

Prikl Biokhim Mikrobiol, 1981 May-Jun, 17(3), 415 - 21
{Relationship between morological variability of Bacillus subtilis R-623 and biosynthesis of hydrolytic enzymes}; Bakhmatova IB et al.; By synthetic sorbent chromatography the influence of Bacillus subtilis R-623 morphology on the qualitative and quantitative composition of alpha-amylase and proteases was studied . It was found that morphological variants of natural variability of Bac . subtilis R-623, alpha-amylase producer, differed in their cultural, morphological and physiological properties as well as in the amount of hydrolytic enzymes synthesized per unit of the cultural medium . Cells of P variant produced the highest quantity of alpha-amylase (314 U/lm) and cells of P and M variants synthesized the greatest amount of proteases (4.3 and 4.0 U/ml, respectively) . Quantitative variations of alpha-amylase and proteases were measured in the cultural medium of morphological variants of Bac . subtilis R-623 during cultivation . Qualitative composition of those enzymes was determined when their content in the cultural medium was at the highest level . R variant synthesized alpha-amylase and protease, P and S variants alpha-amylase and two proteases, and P and S variants alpha-amylase and two proteases, and M variant one protease.

J Bacteriol, 1981 May, 146(2), 819 - 22
Thiomethylation of tyrosine transfer ribonucleic acid is associated with initiation of sporulation in Bacillus subtilis: effect of phosphate concentration; Buu A et al.; The thiomethylation of Bacillus subtilis tyrosine transfer ribonucleic acid (tRNATyr) (i6A) has been shown to occur during the slowing-down of growth . The extent of this modification in stationary-phase cells grown in defined medium has been determined in parallel with the sporulation frequency . We observed that the presence of phosphate repressed sporulation and also inhibited the thiomethylation of tRNATyr (i6A) of B . subtilis W168 . These effects were partially eliminated by decreasing the glucose concentration until it was growth limiting . In the case of strain W23S, in which sporulation is insensitive to glucose repression, sporulation and tRNATyr thiomethylation were not inhibited by nonlimiting concentrations of phosphate . These results suggest that both sporulation and tRNATyr hyper-modification share some common regulatory process.

Nucleic Acids Res, 1981 Apr 24, 9(8), 1933 - 9
5-(carboxymethylaminomethyl)-2-thiouridine, a new modified nucleoside found at the first letter position of the anticodon; Yamada Y et al.; The structure of a modified uridine derivative which was detected at the first letter position of the anticodon of Bacillus subtilis tRNA1Lys was determined to be 5-(carboxymethylaminomethyl)-2-thiouridine . The determination was mainly based in this ultraviolet absorption spectra and mass spectrometric analysis of the trimethylsilyl derivative.

J Virol, 1981 Apr, 38(1), 15 - 9
Adsorption of bacteriophage phi 29 to Bacillus subtilis through the neck appendages of the viral particle; Villanueva N et al.; Phage phi 29 particles produced under restrictive conditions by mutants in gene 12 have normal amounts of all of the structural proteins except the appendage protein, p12*, which is missing . These particles are not infective and do not adsorb to Bacillus subtilis cells . By in vitro complementation of 12- particles with extracts containing protein p12* or with purified protein p12*, the defective particles could bind the appendage protein and become infective and able to adsorb to bacteria . Therefore, the neck appendages of phage phi 29, formed by protein p12*, are involved in the interaction of the phage with the cell wall receptors . Protein p12*, purified in its native state, competed with wild-type phage for adsorption to bacteria . Also, protein p12* could displace adsorbed phage from bacteria . Since the displaced phage was infective, protein p12* does not seem to be modified after phage adsorption.

J Bacteriol, 1981 Apr, 146(1), 7 - 9
Transformation of Bacillus thuringiensis subsp . galleria protoplasts by plasmid pBC16; Alikhanian SI et al.; Protoplasts of the entomopathogenic bacterium Bacillus thuringiensis subsp . galleria were transformed by plasmid pBC16 . The frequency of transformation was much lower than that of Bacillus subtilis . All isolated B . thuringiensis transformants were characterized by increased sensitivity to lysozyme as compared with the original strain.

Biokhimiia, 1981 Apr, 46(4), 603 - 11
{Purification and some properties of 1,3-1,4-beta-glucanase from Bacillus subtilis}; Firantene RK et al.; Using chromatography on cellulose, SE-Sephadex G-50 and gel filtration on acrylex P-60, 1.3 -- 1.4-beta-glucanase from Bac . subtilis, strain 103 was obtained and purified 142-fold . The specific activity of the purified enzyme was 18.5 units per mg of protein . The homogeneity of 1.3 -- 1.4-beta-glucanase was determined by gel filtration on acrylex P-60, polyacrylamide gel electrophoresis, isoelectrofocusing and ultracentrifugation . Using electrophoresis in Na-SDS and gel filtration on acrylex P-60, the molecular weight of the enzyme was found to be equal to 30 000 and 33 000, respectively . The isoelectric point for the enzyme lies at pH 5.4 . The enzyme does not contain tryptophane, free SH-groups or carbohydrates.

Eur J Biochem, 1981 Apr, 115(3), 511 - 7
Ligand-binding studies on light riboflavin synthase from Bacillus subtilis; Otto MK et al.; Light riboflavin synthase of Bacillus subtilis is a trimer of identical subunits . The enzyme catalyzes the transfer of a four-carbon moiety from one molecule of 6,7-dimethyl-8-ribityllumazine to a second molecule of this compound . Binding of substrate and product analogues to the enzyme was studied by analytical ultra-centrifugation and fluorescence titration . The ligands used in these experiments inhibit the enzyme activity competitively . Each enzyme subunit was shown to bind two molecules each of various analogues of the enzyme substrate, 6,7-dimethyl-8-ribityllumazine, at nonidentical sites . On the other hand, each subunit binds only one molecule of the product, riboflavin, or 5-nitroso-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, an analogue of the second product . The complex of the enzyme with the substrate analogue, 7-methyl-8-ribityllumazine, was studied by absorbance and difference absorbance measurements . The data suggest that binding of the lumazine to the donor site of the enzyme involves a nucleophilic attack at carbon 7 of the lumazine ring with formation of a covalent hydrate or a related structure.

J Bacteriol, 1981 Apr, 146(1), 337 - 44
Induction of citric acid cycle enzymes during initiation of sporulation by guanine nucleotide deprivation; Uratani-Wong B et al.; In Bacillus subtilis, conditions causing partial deprivation of guanine nucleotides initiated sporulation and caused the synthesis of citrate synthase, aconitase, and alpha-ketoglutarate dehydrogenase . Alpha-ketoglutarate dehydrogenase could also be induced by acetate, and the specific activity of this enzyme was elevated in mutants that had high intracellular acetyl coenzyme A concentrations because they lacked citrate synthase activity . After deprivation of guanine nucleotides, the intracellular concentration of acetyl coenzyme A also increased, which explained the induction of alpha-ketoglutarate dehydrogenase . Furthermore, the decreases in alpha-ketoglutarate and L-malate concentrations observed during this deprivation accounted for the observed increases in citrate synthase activity (which was repressed by alpha-ketoglutarate and malate) and aconitase activity (which was repressed by alpha-ketoglutarate).

J Bacteriol, 1981 Apr, 146(1), 430 - 2
Isolation of Bacillus subtilis genes from a charon 4A library; Ferrari E et al.; A library of Bacillus subtilis chromosomal deoxyribonucleic acid (DNA) was constructed, using lambda charon 4A as a cloning vector . Partially cleaved Bacillus subtilis DNA was prepared by partial methylation with EcoRI methylase, followed by complete EcoRI endonuclease digestion . More than 95% of the phage particles carried B . subtilis DNA inserts . When this library was screened for transforming activity, using competent cells, 70% of the genetic markers tested were found in a sample of 1,710 plaques . Cloned genetic loci were found to be about 100-fold more efficient in transforming activity than chromosomal DNA . Intact phage particles containing the pheA locus were found to be able to transform competent recipients with approximately the same efficiency as phage DNA . Transformation by intact particles was insensitive to deoxyribonuclease.

Arch Microbiol, 1981 Apr, 129(2), 160 - 4
Specificity of translation of spore polypeptides in bacillus in vitro systems; Wright W 3rd et al.; Cell free extracts prepared from exponentially growing Escherichia coli and Bacillus cereus as well as from Bacillus cereus at the end of exponential growth were optimized for various factors required for amino acid incorporation when programmed with Bacillus ribonucleic acid . All three preparations synthesized glutamine synthetase antigen when ribonucleic acid from a Bacillus subtilis strain that overproduces glutamine synthetase was added . The post exponential Bacillus cereus extract, however, was most active for the synthesis of Bacillus cereus sport coat antigen when supplemented with the appropriate ribonucleic acid . There appears to be some specificity in the translation of at least this sporulation messenger RNA.

J Bacteriol, 1981 Apr, 146(1), 305 - 11
Three deoxyribonucleic acid-dependent adenosine triphosphatases from Bacillus subtilis; Mazza G et al.; We have isolated from Bacillus subtilis three deoxyribonucleic acid (DNA)-dependent adenosine triphosphatases (ATPases) (gamma-phosphohydrolases) . The enzymes were extensively purified, and their physicochemical and functional properties were determined . The three enzymes (ATPases I, II, and III) were shown to be different by several criteria . ATPases II and III showed an absolute requirement for single-stranded DNA as a cofactor, whereas ATPase I had some residual activity also with double-stranded DNA . They required Mg2+ and had a pH optimum of 6.5 to 7 . Only adenosine 5'-triphosphate and deoxyadenosine 5'-triphosphate were hydrolyzed . The molecular weights of ATPases I, II, and III were 108,000, 115,000, and 148,000, respectively . Km values for adenosine 5'-triphosphate and DNA were also evaluated and shown to be different for each enzyme . All three enzymes formed physical complexes with single-stranded DNA . We present evidence that ATPases I and II might migrate along DNA during adenosine 5'-triphosphate hydrolysis . On the other hand, this effect was not observed with ATPase III, which exhibited the highest affinity for single-stranded DNA.

Eur J Biochem, 1981 Mar 16, 115(1), 175 - 81
Biochemical and genetic characterization of an auxotroph of Bacillus subtilis altered in the Acyl-CoA:acyl-carrier-protein transacylase; Boudreaux DP et al.; We have analyzed a mutation of Bacillus subtilis (bfmB) that results in an acyl-CoA:acyl-carrier-protein transacylase with low affinity for branched acyl-CoA substrates; it maps in the acf-hisH region of the chromosome . The aceA mutation, present in the parent of the bfmB mutant, causes a deficiency in pyruvate dehydrogenase and maps in the pycA-pyrA region . Strains carrying the bfmB mutation synthesize branched-chain fatty acids at a rate sufficient for normal growth only if branched acyl-CoA precursors are present in the medium . They grow well if the medium is supplemented with 0.1 mM 2-methylbutyrate, isobutyrate or isovalerate, or with 1.0 mM isoleucine or valine; leucine does not support growth . Growth supported by valine and isoleucine is inhibited by butyrate and other straight short-chain fatty acids at concentrations (0.1 mM) which do not inhibit growth of the standard strain; the inhibition is prevented by short branched fatty acids which are converted to long-chain fatty acids appearing as activity of B . subtilis is controlled by separate enzymatic sites for the acyl-CoA precursors of branched and straight-chain fatty acids . Whether these sites are contained in one or two enzymes is not known.

J Gen Microbiol, 1981 Mar, 123(Pt 1), 143 - 9
Involvement of menaquinone in the active accumulation of aminoglycosides by Bacillus subtilis; Taber HW et al.; Accumulation of aminoglycoside antibiotics by bacteria requires energy, and it appears that this must be derived from electron transport occurring within the cytoplasmic membrane . Dependence of aminoglycoside accumulation on cellular menaquinone content was examined using a menaquinone auxotroph of bacillus subtilis . This dependence manifested itself only when the menaquinone concentration was decreased to less than 10% of normal . The restricted aminoglycoside accumulation observed under these conditions was closely correlated with susceptibility to growth inhibition by the antibiotics . Evidence of saturation of the accumulation system was observed at low menaquinone concentrations, an effect not seen when menaquinone deficiency was relieved by supplying adequate shikimic acid (a menaquinone precursor) to the auxotroph . Lipophilic quinones may play two roles in aminoglycoside accumulation by bacteria: (i) as a binding site or part of a carrier complex: and (ii) as a crucial component of the electron transport system in maintaining the proton electrochemical gradient.

Prikl Biokhim Mikrobiol, 1981 Mar-Apr, 17(2), 233 - 7
{Concentration and purification of alkaline proteinase by ultrafiltration}; Shishkova EA et al.; The mode of ultrafiltration of the culture fluid of Bacillus subtilis, producer of alkaline proteinase, through semipermeable acetate cellulose membranes was examined . The ultrafiltration of the enzyme-containing solutions involved both concentration of the solution and enzyme purification from low molecular weight admixtures . The optimum mode of ultrafiltration of alkaline proteinase on UAM membranes "Vladipore" was developed.

Biokhimiia, 1981 Mar, 46(3), 512 - 9
{Isolation of DNA-membrane complexes from Bacillus subtilis}; Molchanov MI et al.; The DNA-membrane complexes were isolated from the membranes of Bacillus subtilis, disrupted by ultrasonication and subjected to chromatography on Sepharose 4B . The membrane-bound fraction of native 3H-DNA was found only in the freshly isolated material . The phospholipid and protein composition of this fraction was determined . It was assumed that the newly synthesized membrane-associated 3H-DNA from Bacillus subtilis is bound to the lipoprotein complexes enriched with phospholipids.

Ann Microbiol (Paris), 1981 Mar-Apr, 132(2), 119 - 28
{Serological relationships between serylprotease and esterase excreted by "Bacillus subtilis" during sporulation (author's transl)}; Karmazyn-Campelli C et al.; During the sporulating phase of Bacillus subtilis an extracellular serylprotease (SPE) and an esterase are excreted . The two enzymes follow the same kinetics of appearance in the culture medium and have some properties in common, in particular both enzymes hydrolyze benzoyltyrosine ethylester and are inhibited by phenylmethylsulfonyl fluoride . This raised the question of a possible structural relationship between the two proteins . A specific antiserum against the SPE was prepared . Using a radioimmunological assay, no reaction with the esterase could be detected . Asporogenous mutants (SpoOA and SpoOB) imparied in the production of both enzymes were analysed . Immunological assays with SPE-specific antiserum supported the hypothesis that the regulation of the biosynthesis of both enzymes would be affected by SpoOA and SpoOB mutations.

Methods Find Exp Clin Pharmacol, 1981 Mar-Apr, 3(2), 79 - 82
Comparison of gentamicin bioassay values utilizing three different indicator organisms; Flournoy DJ et al.; Gentamicin serum concentrations were monitored via bioassay with three indicators, to determine if assay results were influenced by the testing organism . When mean assay values, from seven samples, were compared to mean target values, there was no significant differences with Staphylococcus epidermidis, Bacillus subtilis or Klebsiella pneumoniae . However, levels from one of the seven samples yield dramatically lower concentrations, for all three indicators, than the target . This suggests the presence of a bioassay inhibitor substance . Additional testing of the organisms, versus serum without gentamicin, revealed Bacillus subtilis to be much more serum-sensitive than the others.

J Virol, 1981 Mar, 37(3), 1075 - 8
Early RNAs in SP82- and SP01-infected Bacillus subtilis may be processed; Downard JS et al.; Transcription of SP82 and SP01 DNAs in vitro by Bacillus subtilis RNA polymerase yielded mostly large RNA species, with many in excess of 1,500 bases in length, whereas most of the RNAs synthesized in vivo early in infection were much smaller . Addition of an extract from uninfected B . subtilis to reaction mixtures containing RNAs synthesized in vitro generated additional discrete RNAs whose mobilities on polyacrylamide gels matched the mobilities of some of the smaller RNAs synthesized in vivo.

J Bacteriol, 1981 Mar, 145(3), 1417 - 20
Tetracycline resistance genes from Bacillus plasmid pAB124 confer decreased accumulation of the antibiotic in Bacillus subtilis but not in Escherichia coli; Eccles S et al.; Expression of tetracycline resistance by genes originating in the Bacillus plasmid pAB124 was examined in both Bacillus subtilis and Escherichia coli host cells . Expression of resistance in B . subtilis by genes from pAB124 was inducible and associated with decreased accumulation of the antibiotic . A fragment of pAB124 carrying the genes coding for tetracycline resistance was cloned into the E . coli plasmid RSF2124 . The cloned fragment conferred a low level of resistance in E . coli, but this was not associated with decreased uptake of tetracycline and was not inducible.

J Bacteriol, 1981 Mar, 145(3), 1281 - 5
Analysis of Bacillus subtilis sporulation with spore-converting bacteriophage PMB12; Kinney DM et al.; Previous observations concerning the ability of the spore-converting bacteriophage PMB12 to cause sporulation in certain sporulation-deficient mutants of Bacillus subtilis 168 were extended to include a spoOK mutant and a mutant temperature sensitive for sporulation due to a ribosomal mutation . Mutants of PMB12 that were unable to induce sporulation in the spoOK mutant were isolated to determine whether PMB12-encoded products had to affect the sporulation-specific functions of both the transcription and the translation systems of B . subtilis to induce sporulation . A complementation assay for spore conversion was used to assign the spore conversion-negative PMB12 mutants to four groups . One group of mutants repressed the ability of wild-type PMB12 to induce sporulation . None of the spore conversion-negative PMB12 mutants could induce significant levels of sporulation in B . subtilis mutants that were temperature sensitive for sporulation due to mutations in the beta subunit of ribonucleic acid polymerase or the 30S ribosomal subunit . Our data suggest that PMB12 may have at least three genes for spore conversion . The products of these genes apparently interact with a host cell pathway that is expressed during the earliest stage of sporulation and is not dependent for expression upon sporulation-specific functions of the host cell's transcription and translation systems.

J Bacteriol, 1981 Mar, 145(3), 1222 - 31
Attachment of the main chain to the linkage unit in biosynthesis of teichoic acids; McArthur HA et al.; The main chain of teichoic acids can be assembled in cell-free membrane preparations by the transfer of residues from the appropriate nucleotide precursors to an incompletely characterized amphiphilic molecule, lipoteichoic acid carrier (LTC) . However, in the cell wall, the main chain is attached to peptidoglycan through a linkage unit which is synthesized independently . It is believed that, in these cell-free systems, lipid intermediates carrying linkage units are also able to accept residues directly from nucleotide precursors to build up the main chain . In this paper, we have shown that the main chain attached to LTC was transferred from LTC to lipids containing the linkage unit . Thus, in these systems, there appear to be two routes to the biosynthesis of teichoic acid-linkage unit complexes, one by direct assembly of the main chain on linkage unit lipids and the other by transfer of the preassembled main chain from LTC to the linkage unit . It was also shown that linkage unit lipids from different organisms were interchangeable and that these were used for polymer synthesis by Bacillus subtilis 3610, in which the teichoic acid is a poly(glycerol phosphate).

J Bacteriol, 1981 Mar, 145(3), 1177 - 88
Assimilation of single-stranded donor deoxyribonucleic acid fragments by nucleoids of competent cultures of Bacillus subtilis; van Randen J et al.; Lysates containing folded chromosomes of competent Bacillus subtilis were prepared . The chromosomes were supercoiled, as indicated by the biphasic response of their sedimentation rates to increasing concentrations of ethidium bromide . Limited incubation of the lysates with increasing concentrations of ribonucleases resulted in a gradual decrease in the sedimentation velocity of the deoxyribonucleic acid (DNA) until finally a constant S value was reached . Incubation with sonicated, 4,5',8-trimethylpsoralen-monoadducted, denatured, homologous donor DNA molecules at 37 degrees C and concomitant irradiation with long-wave ultraviolet light of the nucleoid-containing lysates resulted in the formation of complexes of the donor DNA molecules and the recipient chromosomes . This complex formation was stimulated when nucleoids were previously (i) unfolded by ribonuclease incubation, (ii) (partially) relaxed by X irradiation, or (iii) subjected to both treatments . Monoadducts were not essential . On the other hand, the complex-forming capacity of recipient chromosomes previously cross-linked by 4,5',8-trimethylpsoralen diadducts was greatly reduced, suggesting that strand separation of the recipient molecule was involved in the formation of the complex . None of these effects has been observed when heterologous (Escherichia coli) donor DNA has been used . When the same kind of experiments were carried out at 70 degrees C, donor-recipient DNA complexes were also formed and required strand separation and homology similar to donor-recipient complex formation at 37 degrees C . However, in contrast to what was found at 37 degrees C, unfolding plus relaxation of the nucleoids, as well as the absence of monoadducts in the donor DNA fragments, resulted in a decrease in complex formation . On the basis of these results, we assume that superhelicity can promote the in vitro assimilation of single-stranded donor DNA fragments by nucleoids of competents B . subtilis cells at 70 degrees C, but that at 37 degrees C a different mechanism is involved.

Eur J Biochem, 1981 Mar, 114(3), 493 - 9
Substrate specificity and adenosine triphosphatase activity of the ATP-dependent deoxyribonuclease of Bacillus subtilis; Doly J et al.; Studies on the specificity of the ATP-dependent DNase of Bacillus subtilis 168, carried out with pure enzyme at the optimal conditions for its action, have shown that the substrate is double-stranded linear DNA . Linear single-stranded DNA (separated strands of B . subtilis DNA and linear phage fd DNA) is not attacked, neither are there any circular forms (supercoiled or nicked simian virus 40 and circular single-stranded fd DNAs) . The double-stranded DNA can be completely hydrolysed, the limit products being, almost exclusively, mononucleotides . The presence of terminal phosphate residues in the substrate (either at the 3' or the 5' end) is not necessary for enzyme action . This DNase appears therefore to be an exonuclease processively liberating mononucleotides from both strands of the native linear DNA . ATP (indispensable for the DNase reaction) is also hydrolysed by the enzyme, to ADP and inorganic orthophosphate (Pi) in the presence of DNA . The apparent Km for ATP, in the ATPase reaction, is 0.15 mM . At high ATP concentrations, which inhibit the DNase activity, there is activation of the ATPase reaction . Three molecules of ATP are consumed for each DNA phosphodiester bond split, at optimal conditions for DNase activity.

Proc Natl Acad Sci U S A, 1981 Mar, 78(3), 1446 - 50
Nucleotide sequence at the termini of the DNA of Bacillus subtilis phage phi 29; Escarmis C et al.; Phage phi 29 DNA cannot be phosphorylated with polynucleotide kinase and {gamma-32P}ATP because of the presence of a viral protein covalently linked to the 5' termini . The 5' ends can, however, be made susceptible to phosphorylation by treatment with alkali and alkaline phosphatase . Restriction fragments Hpa II C and Hpa II F, corresponding to the right and left ends of phi 29 DNA, respectively, were labeled at the 5' ends with polynucleotide kinase and {gamma-32P}ATP or at the 3' ends with terminal transferase and {alpha-32P}ATP or {alpha-32P}cordycepin 5'-triphosphate . After a secondary cleavage of the labeled fragments, the sequence of the first 150-180 nucleotides at the termini of phi 29 DNA was determined by the method of Maxam and Gilbert . The ends of phi 29 DNA are flush, and a six-nucleotides-long inverted terminal repetition was found . The functional implications of the sequences determined are discussed.

J Virol, 1981 Mar, 37(3), 1099 - 102
Unusually infrequent cleavage with several endonucleases and physical map construction of Bacillus subtilis bacteriophage phi 1 DNA; Kawamura F et al.; HaeIII, BalI, StuI, BamHI, SlaI, and EcoRII did not cut the genome of Bacillus subtilis phage phi 1 at all, whereas ThaI, BglII, EcoRI, SalI, and Bsu1247I cut the genome once or twice . The physical map of the phi 1 genome was constructed with the latter restriction endonucleases.

J Gen Microbiol, 1981 Feb, 122(Pt 2), 345 - 9
Molecular origin of transducing DNA in bacteriophage SPP1; de Lencastre H et al.; Transducing particles produced by bacteriophage SPP1 infection of Bacillus subtilis were separated from plaque-forming units by CsCl density-gradient centrifugation . The density in CsCl of DNA isolated from such purified transducing particles was similar to that of bacterial DNA, indicating that the transducing particles probably contain DNA of exclusively bacterial origin . Bacterial DNA synthesized after infection of the donor culture was also encapsulated in the transducing particles . The number of transducing particles was at least 10 times higher than that of the transductants.

J Biochem (Tokyo), 1981 Feb, 89(2), 517 - 21
Disappearance of guanosine 5'-diphosphate 3'-diphosphate in Bacillus subtilis vegetative cells upon carbon source deprivation; Ikehara K et al.; Bacillus subtilis was grown in a nutrient medium, mNSMP, and a synthetic medium, mS6(C), in which spore formation was initiated after vegetative growth and exhaustion of carbon source or glucose . The amounts of intracellular phosphorylated compounds were analyzed at intervals by 2 M formic acid extraction and polyethyleneimine (PEI)-cellulose thin-layer chromatography followed by autoradiography . A hyperphosphorylated nucleotide, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), was accumulated in cells during vegetative growth in both mNSMP and mS6(C), and then the nucleotide was degraded upon initiation of sporulation in both cases . Furthermore, after the nucleotide had disappeared in cells cultivated in mS6(C) upon exhaustion of the carbon source, it could be reformed in the sporulating cells by addition of glucose to the medium . These results suggest that the ppGpp in vegetative cells may function in the regulation of B . subtilis sporulation.

J Biochem (Tokyo), 1981 Feb, 89(2), 511 - 6
Presence of guanosine 5'-diphosphate 3'-diphosphate in Bacillus subtilis vegetative cells; Ikehara K et al.; Extracts of Bacillus subtilis vegetative cells with 2 M formic acid contained a large amount of a hyperphosphorylated nucleotide ("spot 4" nucleotide) . The compound always comigrated with authentic guanosine 5'-diphosphate 3'-diphosphate (ppGpp) on two-dimensional polyethyleneimine (PEI)-cellulose thin-layer chromatography performed with three different solvent systems . Furthermore, all dephosphorylated 32P-labeled derivatives from the "spot 4" nucleotide comigrated on a one-dimensional PEI-cellulose plate with those from authentic ppGpp present in the same reaction mixture, when the compounds were hydrolyzed with snake venom phosphodiesterase or alkali . The level of the "spot 4" nucleotide (ppGpp) in the cell extracts was 0.14 nmol P/A660, corresponding to about one-third of the guanosine 5'-triphosphate (GTP) level and about 10% of the adenosine 5'-triphosphate (ATP) level . These results indicate that a "magic spot" nucleotide, ppGpp, is present at a high level in B . subtilis cells vegetatively growing in mNSMP.

J Virol, 1981 Feb, 37(2), 559 - 63
Phi W-14 DNA inhibits transfection of Bacillus subtilis by SPP1 DNA; Lopez P et al.; The DNA of bacteriophage phi W-14 is unusual in that half of the thymine residues are replaced with the hypermodified pyrimidine alpha-putrescinylthymine (Kropinski et al., Biochemistry 12:151-157, 1973) . Bacteriophage phi W-14 DNA and Bacillus subtilis DNA exhibited comparable competing abilities for the uptake of transfecting bacteriophage SPP1 DNA by competent cells of B . subtilis . B . subtilis DNA decreased transfection and uptake to the same extent, indicating that it merely competed with SPP1 DNA for uptake . Phi W-14 DNA, however, decreased transfection up to 30 times more effectively than it inhibited uptake . Phi W-14 DNA did not alter the kinetics of transfection . The degree of inhibition of transfection was dependent upon the time of addition of Phi W-14 DNA relative to the time of addition of SPP1 DNA . If failed to inhibit when added 30 min after SPP1 DNA . It had a fourfold-greater effect when added 10 min before, rather than simultaneously with, SPP1, but this enhancement was abolished by high concentrations of SPP1 DNA . The nature of the transfection process was not altered in those cells escaping inhibition by Phi W-14 DNA: two molecules of transfecting SPP1 DNA were required to form a transfectant with or without Phi W-14 DNA . Free putrescine did not affect transfection by SPP1 DNA . It was concluded that the putrescine groups covalently attached to phi W-14 DNA allowed this DNA to interfere with the transfection process at the intracellular level.

Cell, 1981 Feb, 23(2), 615 - 24
A sporulation-induced sigma-like regulatory protein from B . subtilis; Haldenwang WG et al.; We have isolated a sigma-like regulatory protein termed sigma 29 whose synthesis or association with Bacillus subtilis RNA polymerase was induced during spore formation . sigma 29 is a sporulation-specific component of RNA polymerase as it was absent in enzyme from an early-blocked sporulation mutant (SpoOA) . We have demonstrated specific RNA synthesis by sigma 29-RNA polymerase using as a DNA template a cloned cluster of vegetative and sporulation genes from the purA-cysA region of the B . subtilis chromosome . The pattern of gene recognition by sigma 29-RNA polymerase was distinct from that observed for RNA polymerases containing sigma 55 or sigma 37, species of sigma factor that are present in vegetative cells of B . subtilis . A reconstitution experiment in which purified sigma 29 was added to core RNA polymerase demonstrates that sigma 29 was directly responsible for the altered transcriptional specificity of sporulation RNA polymerase . We propose that sigma 29 is a regulatory protein that controls developmental gene transcription at an early stage of spore formation.

J Bacteriol, 1981 Feb, 145(2), 953 - 7
Purification of polypeptide P6 with a molecular weight of 6,000 from the vegetative chromosomes of Bacillus subtilis; Nakayama T et al.; Folded chromosomes were prepared as membrane-associated complexes from vegetative cells of Bacillus subtilis by stepwise sucrose gradient centrifugation . From nucleoids, a deoxyribonucleic acid-bound polypeptide with a molecular weight of 6,000 (P6) was purified by KCl-(NH4)2SO4 salting out, diethylaminoethyl cellulose column chromatography, and deoxyribonucleic acid cellulose column chromatography . The amino acid composition of polypeptide P6 was determined.

J Bacteriol, 1981 Feb, 145(2), 878 - 83
Transformation of a Bacillus subtilis L-form with bacteriophage deoxyribonucleic acid; White TB et al.; A stable L-form, sal-1, of Bacillus subtilis was transformed with deoxyribonucleic acid (DNA) from bacteriophages phi 25 and phi 29 to determine whether exogenous DNA can be introduced into this organism . The viral transformation (transfection) was successful with the use of polyethylene glycol . In the presence of the fusogen, bacteriophage phi 25 DNA initiated a single cycle of infection . When compared with transfection of competent cells of Bacillus subtilis, the appearance of viral particles was delayed and their production occurred over a longer time period . L-form cells were best able to support intracellular replication of phi 25 viral particles when in balanced growth in a rich medium . The addition of polyethylene glycol also induced infection of sal-1 with whole bacteriophage phi 25 particles which could not otherwise infect the L-form and enhanced infection by intact phi 29 particles . Primary recombination was shown to be required for polyethylene glycol-mediated phi 25 transfection, but not phi 29 transfection or for whole bacteriophage phi 25 infection mediated by polyethylene glycol . Successful transfection of sal-1 suggests that the L-form may be amenable to genetic modification with exogenous DNA.

J Bacteriol, 1981 Feb, 145(2), 832 - 9
Inactivation of transforming Bacillus subtilis deoxyribonucleic acid by monoadducts of 4,5',8-trimethylpsoralen; te Riele HP et al.; 4,5',8-Trimethylpsoralen (TMP) monoadducts inactive transforming deoxyribonucleic acid (DNA) in Bacillus subtilis . Contrary to TMP diadducts (TMP cross-links), which severely inhibit entry of donor DNA (G . Venema and U . Canosi, Mol . Gen . Genet . 179:1--11), TMP monoadducts have only a slight effect on entry . Since reextracted TMP-monoadduct-containing transforming DNA is a differentially repaired by Uvr- and Uvr+ recipients and cross-linkable to the recipient strand in the heteroduplex recombinant DNA molecules, the monoadducts can be integrated along with the donor DNA into the recipient chromosome.

J Bacteriol, 1981 Feb, 145(2), 768 - 74
Effect of glutamine on enzymes of nitrogen metabolism in Bacillus subtilis; Deshpande KL et al.; An earlier study of the regulation of glutamate synthase (GOGAT) in Bacillus subtilis (Deshpande et al., Bichem . Biophys . Res . Commun . 95:55--60, 1980) revealed an inverse relationship between the specific activity of this essential ammonia-assimilatory enzyme and the intracellular pool of glutamine: GOGAT activity decreased when the internal glutamine concentration reached or exceeded 2.5 mM . This finding prompted the present investigation of the intracellular events linking glutamine formation to the regulation of GOGAT . A growing culture of B . subtilis was shifted from glutamate plus NH+4 medium (high GOGAT activity) to glutamate medium (low GOGAT activity) . At various times after the shift, the intracellular concentrations of aspartate, glutamate, glutamine, alanine, and NH+4 and the activities of GOGAT and glutamine synthetase (GS) were measured . After 30 min, the only significant pool level change was an eightfold increase in glutamine, which paralleled a 2- to 3-fold increase in GS activity . Approximately 15 min after the glutamine pool reached its peak, GOGAT activity began to decrease and eventually declined 2.5-fold . In contrast, when B . subtilis was shifted from glutamate medium to glutamate plus NH+4 medium, there was a 1- to 2-h lag before the glutamine pool and GS activity approached a steady state . As a result, GOGAT activity was low until the concentration of glutamine dropped below 2.5 mM . We propose that glutamine is an important regulatory element in the control of GOGAT activity and that one form of GOGAT regulation involves enzyme inactivation . In addition, these results indicate that glutamine is neither a corepressor nor a feedback inhibitor of GS.

J Bacteriol, 1981 Feb, 145(2), 1079 - 81
Effect of a new pyrimidine analog on Bacillus subtilis growth; Raugei G et al.; 2-Amino-5-ethoxycarbonylpyrimidine-4(3H)-one, a pyrimidine analog, inhibited growth of Bacillus subtilis . Data were obtained which suggested that the analog interfered with the methylation process . A mutant resistant to the inhibitor was isolated, and the mutation was mapped.

J Bacteriol, 1981 Feb, 145(2), 1042 - 51
Characterization and mapping of temperature-sensitive division initiation mutations of Bacillus subtilis; Callister H et al.; Two temperature-sensitive, filamenting mutants of Bacillus subtilis (ts1 and ts12) have been shown to be defective in the initiation of septation . Recombination index mapping showed that these mutations mapped in two different but closely linked genes . A third proposed initiation mutation, tms-12, probably maps in the same gene as ts12 . Another proposed initiation mutation was not linked with these genes by transformation, indicating that there was a minimum of three genes involved in the initiation of division . PBS1 transduction mapping located these three genes close to the pyr cluster.

J Virol, 1981 Feb, 37(2), 574 - 9
Structure of Bacillus subtilis bacteriophage SPP1 DNA in relation to its transfection activity; Humphreys GO et al.; The availability of a detailed restriction map of SPP1 DNA allowed defined manipulations of such molecules . These were performed to investigate structural requirements for SPP1 transfection . (i) The transfection activity of SPP1 DNA was destroyed by degradation with restriction enzymes . Biological activity could be regenerated when transfection was performed with a combination of two different restriction endonuclease digests, provided that such digests generated widely overlapping DNA fragments . (ii) Unique DNA molecules were constructed from the natural population of circularly permuted SPP1 DNA molecules by using genetic engineering techniques . Such molecules had the same specific transfection activity as did the circularly permuted SPP1 DNA . These results are discussed in the context of current models of DNA processing in transfection.

J Bacteriol, 1981 Feb, 145(2), 760 - 7
Isolation and properties of a Bacillus subtilis mutant unable to produce fructose-bisphosphatase; Fujita Y et al.; A Bacillus subtilis mutation (gene symbol fdpA1), producing a deficiency of D-fructose-1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11, fructose-bisphosphatase), was isolated and genetically purified . An fdpA1-containing mutant did not produce cross-reacting material . It grew on any carbon source that allowed growth of the standard strain except myo-inositol and D-gluconate . Because the mutant could grow on D-fructose, glycerol, or L-malate as the sole carbon source, B . subtilis can produce fructose-6-phosphate and the derived cell wall precursors from these carbon sources in the absence of fructose-bisphosphatase . In other words, during gluconeogenesis B . subtilis must be able to bypass this reaction . Fructose-bisphosphatase is also not needed for the sporulation of B., subtilis . The fdpA1 mutation has the pleiotropic consequence that mutants carrying it cannot produce inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) under conditions that normally induce these enzymes.

J Mol Appl Genet, 1981, 1(3), 219 - 37
Construction and characterization of the host component of the Bacillus subtilis HV2 cloning system; Burke WF Jr et al.; A strain of Bacillus subtilis has been developed and tested for use as the host component of the B . subtilis HV2 system . The strain was constructed incorporating the following mutations: thyA, thyB, uvr-1, dal, spoOA delta 677, citD delta 20 (uvrC), strd . The strain is unable to grow in the absence of thymine, D-alanine, or streptomycin; unable to sporulate; and sensitive to UV radiation . Survival of the attenuated strain was severely restricted in nonpermissive environments, and the organism suffered a five-log decrease in viability within 24 h when dried at room temperature . The strain could be transformed with plasmid DNA using the protoplast transformation system and was transducible with PBS1 . The ability of the strain to serve as host for the temperate phages phi 3T and rho 11 and for the plasmid vectors pUB110 and pBD64 was unimpaired.

Mol Gen Genet, 1981, 183(3), 550 - 2
Physiological suppression of Bacillus subtilis conditioned sporulation phenotypes: RNA polymerase and ribosomal mutations; Wayne RR et al.; The temperature-sensitive sporulation phenotype (Spots) of Bacillus subtilis RNA polymerase, ribosomal and protein synthesis elongation factor G mutations can be corrected by supplementing the growth medium with carbohydrates such as ribose or glycerol, or with synthetic lipids such as Tween 40 . The data suggest that these mutations affect a single common aspect of developmental cell function . It is proposed that these lesions prevent sporulation by disturbing the regulation of sporulating cell metabolic balance.

Mol Gen Genet, 1981, 183(3), 544 - 9
Physiological suppression of the temperature-sensitive sporulation defect in a Bacillus subtilis RNA polymerase mutant; Wayne RR et al.; Five hundred putative RNA polymerase mutants of Bacillus subtilis were isolated by selecting for resistance to the RNA polymerase inhibitors rifampin (Rifr), streptovaricin (Strr) or streptolydigan (Stdr) . This collection was screened for mutants that were unable to sporulate at the non-permissive temperature of 46 degrees C, yet which sporulated well at 37 degrees C and had normal vegetative growth (Spots phenotype) . Nearly one half of the Rifr and one quarter of the Stvr mutants were Spots, whereas none of the Stdr mutants had this phenotype . The streptovaricin resistant strain stv84 was studied in detail . The stv84 mutation maps between cysA14 and strA39 on the B . subtilis chromosome, and the Stvr and Spots phenotypes cotransform at a frequency of 100% . The Spots phenotype of stv84 could be physiologically corrected by supplementing the growth medium with inhibitors of RNA synthesis such as rifampin or azauracil, with carbohydrates such as ribose, mannose or glycerol, or with lipids such as Tween 40 or fatty acids native to Bacillus subtilis membranes . A Spots phenotype resembling that of stv84 was produced in wild type B . subtilis by adding cerulenin, an inhibitor of fatty acid biosynthesis, to the growth medium . This cerulenin-induced sporulation defect was reversed by the same treatments that correct the temperature-sensitive genetic defect of stv84 . These data indicate that the Spots phenotype of strain stv84 is not due to an intrinsic inability of the mutant RNA polymerase to transcribe developmentally-specific genes at the nonpermissive temperature . Rather, the data suggest that the stv84 lesion causes a physiological imbalance which disrupts membrane structure or function in sporulating cells.

Mol Gen Genet, 1981, 183(3), 538 - 43
Macrolide and aminoglycoside antibiotic resistance mutations in the bacillus subtilis ribosome resulting in temperature-sensitive sporulation; Sharrock RA et al.; Mutants of Bacillus subtilis resistant to various macrolide antibiotics have been isolated and characterized with respect to their sporulation phenotype and the electrophoretic mobility of their ribosomal proteins (r-proteins) . Two types of major alterations of r-protein L17, one probably due to a small deletion, are found among mutants exhibiting high-level macrolide resistance . These mutants are all temperature-sensitive for sporulation (Spots) . Low-level resistance to some macrolides is found to be associated with minor alterations in r-protein L17 . These mutations do not cause a defective sporulation phenotype . All of the macrolide resistance mutations map at the same locus within the Str-Spc region of the B . subtilis chromosome . Hence, changes in a single ribosomal protein can result in different sporulation phenotypes . Mutants resistant to the aminoglycoside antibiotics neomycin and kanamycin have been isolated . Approximately 5% of these are Spots . Representative mutations, neo162 and kan25, cause concomitant drug resistance and sporulation temperature-sensitivity and map a single-site lesions in the Str-Spc region of the chromosome . Strains bearing neo162 or kan25 are equally cross-resistant to streptomycin or spectinomycin . These mutations define a new B . subtilis drug resistance locus at which mutation can cause defective sporulation.

Mol Gen Genet, 1981, 183(3), 532 - 7
Intergenic suppressors of temperature-sensitive sporulation in Bacillus subtilis are allele non-specific; Sharrock RA et al.; The Bacillus subtilis mutant cal1 carries a non-reverting mutation in ribosomal protein L17 (r-protein L17) that causes both resistance to the antibiotic chalcomycin (Calr) and temperature-sensitive sporulation (Spots) . Second-site suppressor (rev) mutations that relieve the Spots phenotype have been isolated from cal1 . Three suppressor mutations - rev4, rev10, rev11 - each increase the sporulation frequency of cal1 at the non-permissive temperature from 3% to 95% of the wild-type level . The cal1 rev strains remain resistant to chalcomycin and two-dimensional gel electrophoresis analysis indicates that they contain the same altered r-protein L17 as the original cal1 strain and no additional altered r-proteins . The three rev mutations have been mapped at a single locus between narA and sacA on the B . subtilis chromosome and recombination indexes for the rev mutations indicate that they are tightly linked to one another . Antibiotic resistance Spots mutations that cause temperature-sensitive sporulation have previously been isolated in RNA polymerase, in the 30S and 50S subunits of the ribosome, and in elongation factor G . The rev4, 10, and 11 suppressor mutations are non-specific in their action in that they restore significant levels of sporulation at the non-permissive temperature in all of the Spots strains that we have tested . This result suggests that Spots mutations in components of the B . subtilis transcription and translation systems share a common molecular basis for their sporulation-defective phenotypes.

Mol Gen Genet, 1981, 183(3), 478 - 83
Conditional lethal mutants of Bacillus subtilis dependent on kasugamycin for growth; Pai Y et al.; Mutants of Bacillus subtilis dependent on the antibiotic kasugamycin have been isolated and characterised . The mutant phenotype was the result of a kasugamycin resistance mutation mapping near leu, together with a mutation conferring dependence which mapped elsewhere on the chromosome . In some cases, the latter mutation caused spectinomycin dependence in a spectinomycin resistant strain . Four mutants had detectable alterations in ribosomal proteins, which were not, however, responsible for the phenotype . These alterations were in proteins BS3, BS7, BS9, and BL15 . Some mutants had defects in ribosomal subunit assembly, or altered cell morphology associated with the mutant phenotype.

Genetika, 1981, 17(11), 1936 - 44
{Instability of the auxotrophic markers in Bacillus thuringiensis}; Azizbekian RR et al.; Auxotrophic mutants of Bacillus thuringiensis have been isolated and compared with the mutants of Bacillus subtilis . It has been found that after spore formation and a prolonged storage, the considerable part of cells in a population of some strains display a genetically unstable additional requirement for arginine with high frequency . The correlation between tetracycline sensitivity and unstable requirement for arginine and other growth factors has been found . The possible reasons for instability of genetic markers in Bac . thuringiensis are discussed.

Genetika, 1981, 17(11), 1929 - 35
{"Marker augmentation" phenomenon in the plasmid transformation and transduction of Bacillus subtilis}; Bashkirov VI et al.; Transformation of Bacillus subtilis cells carrying pUB110 plasmid by DNA of homologous plasmid pBD12 results in the significant increase in the number of plasmid transformants . This phenomenon named "augmentation" was not observed when instead of intact cells, regenerating protoplasts were used, or if pBD12 DNA was introduced into the cells via transduction.

Genetika, 1981, 17(9), 1581 - 7
{Comparative study of chromosome and plasmid transformation in Bacillus subtilis: the effect of lysozyme and polyethylene glycol}; Glumova EF et al.; The influence of lysozyme and polyethyleneglycol (PEG) on chromosomal and plasmid transformation was comparatively investigated . Treatment of competent cell culture with low concentrations of lysozyme which do not affect cell viability stimulates both types of transformation . When transforming DNA was treated with definite concentrations of PEG, inhibition of transformation using chromosomal DNA occurred . The similar treatment, however, stimulated transformation by oligomeric forms of plasmid DNA . Possibly, PEG alters the DNA conformation rendering it less sensitive to nuclease degradation . This promotes plasmid transformation but reduces the frequency of transformation by chromosomal DNA, because the changes resulting from nuclease action further DNA integration into the genome of a recipient.

Microbiol Immunol, 1981, 25(6), 545 - 56
Protein synthesis in the isolated forespores from sporulating cells of Bacillus subtilis; Watabe K et al.; Developing forespores were isolated from Bacillus subtilis at different stages of sporulation and protein synthesis in the forespore compartment was examined . Pulse-labeling experiments indicated that {14C}phenylalanine was continuously incorporated into the sporangium throughout sporulation, and at t5 (early stage V of sporulation) 58% of the radioactivity was located in the forespore compartment . Significantly high incorporation of {14C}phenylalanine was observed when the isolated forespores at t5 were incubated with the corresponding mother-cell cytoplasmic fraction or an amino acid mixture . About 73% of the radioactivity incorporated into the isolated forespore at t5 was found in the cytoplasmic fraction and 26% in the membranous fraction . Analysis by sodium dodecyl sulfate-gel electrophoresis showed that the 14C-labeled cytoplasmic protein had a molecular weight of about 20,000, and that a protein having the same molecular weight was present in the t5 forespore as a slight protein band and also in the mature spore as a clear protein band . Gel electrophoresis also revealed that the 14C-labeled membranous-soluble protein (prepared by solubilization with detergents) had broad peaks with molecular weights of about 74,000, 33,000, 20,000, and 12,000.

Genetika, 1981, 17(7), 1211 - 9
{Role of the ribosomes in controlling cellular differentiation and secondary metabolism in sporulating bacteria . I . Sporogenesis, antibiotic formation and the proteolytic activity of streptomycin-resistant mutants}; Lukin AA et al.; A ribosomal mutant Bacillus subtilis IG1 resistant to 100 mkg/ml of streptomycin was isolated . The strA mutation is cotransduced with the cysA gene and, consequently, maps in the ribosomal cluster . The mutation does not influence cell division but does reduce a level of sporulation as well as its antibiotic and proteolytic activity . Involvement of ribosomes in the control of sporulation and secondary metabolism of spore forming bacteria is discussed.

Mol Gen Genet, 1981, 182(1), 99 - 105
Integration and excision of a plasmid in Bacillus subtilis; Galizzi A et al.; We have studied the behaviour in Bacillus subtilis of a plasmid (pPV21) carrying the thymidylate synthetase gene of phage phi3T (thyP3) . The plasmid can transform efficiently the competent cells of all the strains tested . Polyethylene glycol (PEG)-mediated protoplast transformation is efficient only for recE, recD or recF mutants . When present in recombination proficient strains, the plasmid can be integrated into the chromosome, primarily at the thyA locus . This has been shown by genetic mapping and by blot-hybridization . A second less efficient site is at (or near to) the attachment site of phage phi3T . Excision of the plasmid restores the EcoRI restriction pattern of the parental DNA, although with the loss of the defective thyA endogenotic allele and the retention of the thyP exogenotic gene.

Mol Gen Genet, 1981, 182(1), 39 - 43
Fate of plasmid DNA in transformation of Bacillus subtilis protoplasts; de Vos WM et al.; Polyethylene glycol-treated protoplasts of B . subtilis can be transformed by plasmid DNA at very high frequencies (Chang and Cohen 1979) . From analysis of plasmid mediated transformation of transformation-deficient mutants it appeared that mutants, reduced in the transformation by plasmid DNA in the competent state, were plasmid transformation-proficient when transformed as protoplasts . By means of CsCl-gradient centrifugation of re-extracted plasmid DNA it could be demonstrated that plasmid DNA enters the protoplasts in the double-stranded form . In addition, sucrose gradient centrifugation of the re-extracted plasmid DNA showed that the entered DNA is predominantly present as covalently closed circular DNA . The efficiency of plasmid transformation in protoplasts was found to be close to one (each plasmid molecule having entered into the protoplasts gives rise to a transformed cell) . This is in good agreement with the observation that little, if any, damage is done to this DNA during or after entry into protoplasts.

Mol Gen Genet, 1981, 181(4), 424 - 33
Plasmid transformation in Bacillus subtilis: fate of plasmid DNA; de Vos WM et al.; Only multimeric, and not monomeric forms of B . subtilis plasmids can transform B . subtilis cells (Canosi et al . 1978) . This finding prompted us to study the physico-chemical fate of plasmid DNA in transformation . Competent cells of B . subtilis were exposed to either unfractionated preparations or to preparations of multimeric plasmid DNA . Plasmid DNA was re-extracted from such cells and then analyzed by sedimentation and isopycnic centrifugation and also defined by its sensitivity to nuclease S1 degradation . No double-stranded plasmid DNA could be recovered from cells transformed with unfractionated plasmid preparations which contained predominantly monomeric covalently closed circular (CCC) DNA . Re-extracted plasmid DNA was single-stranded, had a molecular weight considerably smaller than monomer length DNA and had been subject to degradation to acid soluble products . However, when transformations were performed with multimeric DNA (constructed by in vitro ligation of linearized pC194 DNA), both double-stranded and partially double-stranded DNA could be recovered in addition to single-stranded DNA . We assume that plasmid DNA is converted to a single-stranded form in transformation, irrespective of its molecular structure . Double-stranded and partially double-stranded DNAs found in transformation with multimeric DNA would be the products of intramolecular annealing.

J Gen Virol, 1981 Jan, 52(Pt 1), 93 - 101
Adsorption of the defective phage PBS Z1 to Bacillus subtilis 168 Wt; Steensma HY; Three aspects of the adsorption of the defective phage PBS Z1 to Bacillus subtilis 168 Wt have been investigated . These are the kinetics, the number of receptors on the cell wall and the character of these receptors . The reaction constants for the binding of phages onto receptors, for the dissociation of the phage-receptor complex and for the transition from reversible to irreversible binding of the phages were calculated from adsorption curves obtained by an enzyme-linked immunosorbent assay (ELISA) . They were 1.8 x 10(-13), 6.7 x 10(-2) and 9.0 x 10(-3) respectively . The maximum number of phages adsorbed per cell was 2700, a number limited by the surface area of the cells . Apart from the receptors on the cell wall, receptors on the cell membrane were found . This was concluded from additional adsorption experiments with stable L-forms and contracted phages . Based on these results, together with data from the literature on bacteriocins, phage ghosts and yeast killer factors, a hypothesis concerning the first stage of killing by defective phages has been formulated.

Genetika, 1981, 17(5), 801 - 4
{Plasmid DNA transduction in Bacillus subtilis}; Surikov NN et al.; Transduction of Bacillus subtilis pUB110 plasmid by AR9 phage is described . Some aspects of this process are studied . Plasmid transduction depended on multiplicity of infection similar to cases of chromosomal markers transduction, though optimal multiplicity of infection was achieved using low number of phage particles . No cotransduction of plasmid and chromosomal markers was demonstrated . The transduction frequencies of plasmid and chromosomal markers increased after UV irradiation of phage suspensions within the range of definite doses.

Jpn J Antibiot, 1981 Jan, 34(1), 108 - 11
{In vitro experiment on combination effect of chromomycin A3 and amphotericin B (author's transl)}; Miyamura S et al.; The combination effect of chromomycin A3 and amphotericin B against HeLa cells and Bacillus subtilis ATCC 6633 by the agar plate diffusion and the serial dilution method was examined . As a result, evident synergistic effect was observed when the HeLa cell was used as the test cell.

Microbiol Immunol, 1981, 25(2), 113 - 26
Mutual relationship between antibiotics and resting spores of Bacillus subtilis: binding of cyclic polypeptide and aminoglycoside antibiotics to spores and their inhibitory effect on outgrowth and vegetative growth; Tochikubo K et al.; Not only cyclic polypeptide antibiotics such as polymyxin B, colistin and gramicidin S but also aminoglycoside antibiotics such as streptomycin, kanamycin, gentamicin and kanamycin derivatives combined with the resting spores of Bacillus subtilis and inhibited outgrowth or vegetative growth after germination . All the antibiotics other than gramicidin S were released from the resting spores and their inhibitory action was reversed by the addition of Ca2+ and Fe3+ . As the above antibiotics have free amino (or guanidine) groups in common, it was assumed that such groups play an important role in binding of the antibiotics to the resting spores . Moreover, it was shown that protamine and poly-L-lysine were also bound to the resting spores and were released from them by Ca2+ . On the other hand, free carboxyl groups had been demonstrated in the outermost surface of the resting spores in a previous study . Thus, we assume that the mode of binding of the antibiotics to the resting spores may be due to the formation of reinforced ionic bonds between amino (or guanidine) groups in the antibiotics and carboxyl groups on the spore surface.

Mol Gen Genet, 1981, 181(1), 63 - 8
Free sigma subunit of Bacillus subtilis RNA polymerase binds to DNA; Kudo T et al.; The affinity of Bacillus subtilis RNA polymerase sigma and delta subunits to DNA was examined by a non-denaturing polyacrylamide slab gel electrophoresis method which made it possible to resolve DNA-bound and free subunits . The results revealed that sigma subunit, but not delta subunit had a relatively high affinity for double stranded DNA . The sigma subunit was bound maximally to super-coiled pGR1-3 plasmid DNA at a mass ratio of sigma/DNA of 0.7 . With B . subtilis double stranded linear DNA one sigma subunit was bound per approximately 1,000 base pairs . The sigma-DNA complex was sufficiently stable for isolation by a molecular gel filtration column . The sigma subunit had much higher affinity for super-coiled than for linear pGR1-3 DNA or for linear double stranded or denatured DNA from B . subtilis, E . coli, and calf thymus . These results indicate that the free B . subtilis sigma subunit, in contrast to the E . coli sigma subunit, can bind by itself to DNA.

J Bacteriol, 1981 Jan, 145(1), 50 - 60
Cell wall turnover in batch and chemostat cultures of Bacillus subtilis; De Boer WR et al.; Wall turnover was studied in Bacillus subtilis . The loss of radioactively labeled wall polymers was followed during exponential growth in batch and chemostat cultures . Turnover kinetics were identical under all growth conditions; pulse-labeled wall material was lost with first-order kinetics, but only after exponential growth for 1 generation time after its incorporation . Similarly, continuously labeled cells showed an accelerating decrease in wall-bound radioactivity starting immediately after removal of the labeled precursor and also reached first-order kinetics after 1 generation time . A mathematical description was derived for these turnover kinetics, which embraced the concept of "spreading" of old wall chains (H . M . Pooley, J . Bacteriol . 125:1127-1138, 1976) . Using this description, we were able to calculate from our experimental data the rate of loss of wall polymers from cells and the fraction of the wall which was sensitive to turnover . We found that about 20% of the wall was lost per generation time and that this loss was affected by turnover activity located in the outer 20 to 45% of the wall; rather large variations were found with both quantities and also between duplicate cultures . These parameters were quite independent of the growth rate (the specific growth rate varied from 1.3 h-1 in broth cultures to 0.2 to 0.3 h-1 in chemostat cultures) and of the nature of the anionic polymer in the wall (which was teichoic acid in cultures with an excess of phosphate and teichuronic acid in phosphate-limited chemostat cultures) . Some implications of the observed wall turnover kinetics for models of wall growth in B . subtilis are discussed.

J Bacteriol, 1981 Jan, 145(1), 489 - 93
Induction of Bacillus subtilis sporulation by nucleosides: inosine appears to be sporogen; Sekar V et al.; Inosine completely reversed the selective inhibition of sporulation in Bacillus subtilis 168 caused bym-aminobenzeneboronic acid; guanosine and adenosine, but not xanthosine, partially reversed inhibition, whereas pyrimidine nucleosides were slightly effective . In addition, 0.005 to 0.025 mM inosine caused a four- to fivefold stimulation of sporulation of B . subtilis grown in minimal salts medium . Ultraviolet and infrared spectra and other physical and chemical properties of inosine were markedly similar to those of "sporogen," a previously described endogenous sporogenesis factor present in sporulating Bacillus species.

J Bacteriol, 1981 Jan, 145(1), 434 - 41
Postreplication repair of ultraviolet-irradiated transforming deoxyribonucleic acid in Bacillus subtilis; Hadden CT; Repair of ultraviolet-irradiated transforming deoxyriboinucleic acid (DNA) in several strains of Bacillus subtilis was studied in order to determine the effects of excision repair and postreplication repair on transformation . Two mutations that cause a Uvr- and phenotype (uvr-1 and uvr-42) were shown to have strikingly different effects on repair of ultraviolet-irradiated transforming DNA . Genetic and kinetic evidence is presented to show that integrated DNA was apparently repaired by both excision and postreplication repair in wild-type and in uvr-1 recipients, although the latter excise pyrimidine dimers very slowly . In uvr-42 mutants, which are defective in incision at pyrimidine dimers, dimer-containing DNA was integrated . Postreplication repair apparently saved uvr-42 recipient cells from the lethal effects of integrated dimers, but the recombination events accompanying postreplication repair greatly reduced the linkage between closely linked genetic markers in the donor DNA . Repair of transforming DNA in a recG recipient, which does excision repair but not postreplication repair, was nearly as efficient as in wild-type cells . However, in this recipient linkage was altered only slightly, if at all, compared with wild-type cells . The apparent reduction in size of integrated regions of ultraviolet-irradiation transforming DNA probably results mainly from postreplication repair of larger integrated regions.

J Bacteriol, 1981 Jan, 145(1), 328 - 32
Identification of Bacillus subtilis men mutants which lack O-succinylbenzoyl-coenzyme A synthetase and dihydroxynaphthoate synthase; Meganathan R et al.; Menaquinone (vitamin K2)-deficient mutants of Bacillus subtilis, whose growth requirement is satisfied by 1,4-dihydroxy-2-naphthoic acid but not by o-succinylbenzoic acid (OSB), have been analyzed for enzymatic defects . Complementation analysis of cell-free extracts of the mutants revealed that there are two groups, as already indicated by genetic analysis . The missing enzyme in each group was identified by complementation of the cell-free extracts with o-succinylbenzoyl-coenzyme A (CoA) synthetase and dihydroxynaphthoate synthase extracted from Mycobacterium phlei . Mutants found to lack dihydroxynaphthoate synthase, and which therefore complement with dihydroxynaphthoate synthase of M . phlei, were designated as menB; those lacking o-succinylbenzoyl-CoA synthetase, and therefore complementing with o-succinylbenzoyl-CoA synthetase, were designated as menE . The menB mutants RB413 (men-325) and RB415 (men-329), when incubated with {2,3-14C2}OSB, produced only the spirodilactone form of OSB in a reaction that was CoA and adenosine 5'-triphosphate dependent.

J Bacteriol, 1981 Jan, 145(1), 321 - 7
Menaquinone biosynthesis in Bacillus subtilis: isolation of men mutants and evidence for clustering of men genes; Taber HW et al.; Menaquinone (vitamin K2)-deficient mutants of Bacillus subtilis were selected by simultaneous resistance to two aminoglycoside antibiotics . These men mutants fell into two groups: group I, in which the nutritional requirement was satisfied either by o-succinylbenzoic acid or by 1,4-dihydroxy-2-naphthoic acid; and group II, comprising those capable of growing only when supplemented with 1,4-dihydroxy-2-naphthoic acid . The latter group could be further subdivided into two classes on the basis of syntrophy experiments, fine-structure genetic mapping, and in vitro complementation by cell-free extracts (Meganathan et al., J . Bacteriol., 145:328-332, 1981) . These subclasses of group II defined the menB and menE genes, whereas group I appeared to comprise mutations in the menC and menD genes . All of the men mutations tested, whether occurring in menB, menE, or menC,D, could be placed by genetic mapping with bacteriophage PBS1 between bioB and ald on the B . subtilis genome.

Mol Gen Genet, 1981, 182(2), 299 - 303
Cloning and expression of Bacillus subtilis phage SPP1 in E . coli . II . Expression of lambda/SPP1 hybrid phages in E . coli minicells; Amann EP et al.; In the preceding paper (Amann et al . 1981) we described the in vitro construction of hybrids between Escherichia coli phage lambda NM607 imm434 and B . subtilis phage SPP1 . These lambda/SPP1 hybrids have been used to infect minicells produced by E . coli strain DS410 . Analysis on polyacrylamide gels of 35S-methionine labeled proteins synthesized in infected minicells revealed the expression of both lambda and SPP1 genes . Infection of E . coli minicells carrying plasmid pGY101, which encodes and expresses the repressor gene of phage 434, results in the selective expression of the cloned SPP1 DNA . This has resulted in the assignment of 26 out of a total of 46 known SPP1 polypeptides (Mertens et al . 1979) to individual SPP1 DNA fragments . In addition, several lambda/SPP1 fusion peptides whose transcription either originates from lambda promoters or from promoters located on the inserted SPP1 fragment, were identified.

Mol Gen Genet, 1981, 182(2), 293 - 8
Cloning and expression of the Bacillus subtilis phage SPP1 in E . coli . I . Construction and characterization of lambda/SPP1 hybrids; Amann EP et al.; We have constructed lambda/SPP1 hybrid phages by in vitro ligation of EcoRI fragments of the Bacillus subtilis phage SPP1 DNA to a lambdoid bacteriophage vector . EcoRI digestion of SPP1 generated 15 DNA fragments of which 13 could be cloned . The SPP1 DNA of such hybrids was stably maintained and replicated in Escherichia coli, as indicated by marker rescue experiments in B . subtilis . EcoRI fragment 1 of SPP1 could not be cloned although subfragments of fragment 1 resulting from spontaneous deletions which occurred during the cloning regime were consistently obtained . A region within EcoRI fragment 1 responsible for its incompatibility with replication in E . coli was defined by these experiments.

Mol Gen Genet, 1981, 184(3), 405 - 9
Plasmid transformation in Bacillus subtilis . Alterations introduced into the recipient-homologous DNA of hybrid plasmids can be corrected in transformation; Iglesias A et al.; Various alterations (deletions, additions, inversions) were introduced into portions of pC194/B . subtilis or pC194/phi 105 hybrid plasmid molecules which are homologous to the DNA of recipients in transformation . These plasmids are stably maintained in transformations of recombination deficient cells . In transformations of recombination proficient cells, they can be corrected to those plasmid forms into which the alterations were originally introduced . This correction is most pronounced when transformations are performed with monomeric ccc forms of hybrid plasmids . It is suggested that correction is a consequence of mismatch repair occurring in the synapsis of homologous portions of plasmid and resident DNAs.

Mol Gen Genet, 1981, 184(3), 400 - 4
Plasmid transformation in Bacillus subtilis . The significance of partial homology between plasmid and recipient cell DNAs; Bensi G et al.; A series of hybrid plasmids consisting of pC194 or pUB112 and B . subtilis DNA were constructed . In contrast to plasmid pC194, purified monomeric forms of such plasmids were active in transformation, provided the recipient cells were recombination proficient . Similarly the monomers of pC194 derived plasmids, containing bacteriophage phi 105 DNA were able to transform phi 105 lysogenic but not nonlysogenic cells . From the results it is concluded that the presence of DNA/DNA homology between chromosomal DNA of the recipient cell and part of the hybrid plasmids used is a sufficient condition to endow monomeric plasmids with transforming activity.

Mol Gen Genet, 1981, 183(2), 234 - 7
Genetic and nucleotide sequence homologies in bacillus genomes; Tackney C et al.; The extent of divergence in the organization of the aromatic amino acid cluster among the heterogenetic strains of Bacillus subtilis has been examined by hybridizations to a trp homolog from B . pumilus and by marker survivals after restriction . The trp operon in the W23, 3610 and 168M genomes exhibit variations in the location of the ECoRI and HindIII cleavage sites consistent with the relative transforming activity of the surviving genes and the history of the strains.

Mol Gen Genet, 1981, 183(2), 227 - 33
Structure and function of the region of the replication origin of the Bacillus subtilis chromosome . II . Identification of the essential regions for inhibitory functions shown by the DNA segment containing the replication origin; Seiki M et al.; A BamHI restriction endonuclease fragment B7, which contains the replication origin of the Bacillus subtilis chromosome, showed inhibitory effects on cell growth and plasmid replication in Escherichia coli and Bacillus subtilis, when B7 was inserted into a composite plasmid pMS102' and introduced into these cells . In order to localize these properties in more limited regions within the B7 fragment, we developed a new and widely applicable method for deletion of DNA segments of various lengths from one or other end of a given region of the plasmid molecule . Using a set of deletions in the B7 fragments of pMS102'-B7, we determined the loci responsible for the inhibitory effects of B7 as described below . (1) Stickiness appearing in E . coli cells was caused by a segment residing in a region of approximately 2.2 kilobase pairs (kbp) overlapping the E19 and E22 fragments . (2) instability of the plasmid in E . coli was due to a segment localized in the 440 bp region of the E19-side terminal portion of the 2.2 kbp region . (3) The same 440 bp were also responsible for inhibition of the replication of the plasmid in B . subtilis . Hybridization of the cloned DNA fragments containing the 2.2 kbp region with the whole B . subtilis chromosome revealed that several regions of the chromosome are homologous to this characteristic sequence.

Mol Gen Genet, 1981, 183(2), 220 - 6
Structure and function of the region of the replication origin of the Bacillus subtilis chromosome . I . Isolation and characterization of plasmids containing the origin region; Seiki M et al.; A BamHI restriction endonuclease fragment, B7, which is replicated first among all other fragments derived from the Bacillus subtilis chromosome, was cloned in Escherichia coli using as vector a hybrid plasmid pMS102 that can replicate both in E . coli and B . subtilis . Digestion of pMS102 with BamHI produced two fragments and the smaller one was replaced by the B7 fragment . The cloned plasmid pMS102'-B7 exhibited some peculiar properties that were not observed with plasmids containing other fragments from the B . subtilis chromosome . (1) E . coli cells harboring this plasmid stuck to each other and to glass . This property was more apparent when cells were grown in poor media . (2) E . coli cells tended to lose the plasmid spontaneously when they were grown without the selective pressure favorable to the plasmid . (3) The frequency of transformation of B . subtilis by pMS102'-B7 was less than 1/1,000 of that by the vector plasmid pMS102' . The number of copies of pMS102'-B7 present in the transformants was also markedly reduced, although the pUB110 origin of replication on the vector was intact and should be functional in B . subtilis . This inhibitory effect of the B7 fragment on plasmid replication was confirmed more directly by developing a semi in vitro replication system using protoplasts . Both in E . coli and B . subtilis the B7 fragment affected replication of its own molecule but not that of the coexisting plasmid with an identical replication system . The implication of the function of the B7 fragment in the initiation of the B . subtilis chromosome will be discussed.

Mol Gen Genet, 1981, 183(1), 1 - 6
Chromosomal loci of genes controlling site-specific restriction endonucleases of Bacillus subtilis; Ikawa S et al.; We constructed transformation of B . subtilis 168 which acquired genes for site-specific restriction endonucleases . These endonucleases originated from various strains of B . subtilis and were classified into five groups based on the specificity of the sequences recognized by the enzymes . We examined the loci of genes for site-specific restriction endonucleases belonging to different groups: hsrE determined Endo . R . Bsu1231 (I), hsrB Endo.R.Bsu1247(I), hsrR Endo.R.BsuR and hsrC Endo.R.Bsu-1247(II) . One gene, hsrE, was located between sacA and purA by transduction crosses with phage PBS1, and another gene, hsrB, between hsrE and purA . Genes hsrR and hsrC had been suggested to be allelic or closely linked by previous studies with transformation . We located hsrR and hsrC between purB and tre . Our previous observation and this study show that B . subtilis 168 has at least three independent loci on the chromosome for four genes for site-specific restriction endonucleases in addition to the locus for the original restriction activity (Bsu168-specific restriction) of strain 168.

Zentralbl Bakteriol Naturwiss, 1981, 136(6), 524 - 32
Detection of different types of receptors for deoxyribonucleic acid in competent Bacillus subtilis; Lopez P et al.; Competent cells of Bacillus subtilis are able to bind but not to take up DNA in magnesium-free medium . Wall-membrane complexes, but not cell walls, bind homologous DNA . Binding of DNA to membrane vesicles suggests that DNA-receptor sites are located at the membrane level . On the basis of competition experiments with homologous and heterologous DNAs, in cells and in membrane vesicles, different types of DNA receptors are detected.

Genetika, 1981, 17(10), 1762 - 70
{Artificial Tn2551 transposon with replicative properties}; Khaikinson MIa et al.; A hybrid plasmid, pBE10 was constructed . It consists of DNAs of RSF2124 (ColE1 :: Tn3) plasmid and pUB110 plasmid of Staphylococcus aureus . The latter can be stably maintained in Bacillus subtilis . BamHI cleaved pUB110 was introduced into the BamHI site of transposon Tn3 and the resulting enlarged Tn3 (Tn2551) was transposed from pBE10 onto phage lambda and than to pMB9 (Tc) and RSF1010(Sm Su) plasmids . Restriction and heteroduplex analysis of pMB9 :: Tn2551(pBE21) and RSF1010 :: :: TN2551(pBE32) was carried out . Plasmids pBE10, pBE21 and pBE32 demonstrated some kind of molecular instability when introduced by transformation into Bacillus subtilis.

Mol Gen Genet, 1981, 182(3), 514 - 5
Bacillus subtilis bacteriophages SP beta c1 is a deletion mutant of SP beta; Fink PS et al.; The restriction fragment patterns of two mutants forms of the temperate Bacillus subtilis bacteriophage SP beta have been examined . The DNA of a heat-inducible mutant, SP beta c2, which has a molecular size of 128 kilobases (kb), yields the same restriction pattern as the wild type SP beta c+ DNA . The DNA of a clear-plaque mutant, SP beta c1, has a molecular size of 117 kb, and is deleted for an 11 kb region of phage DNA . Neither SP beta c1 nor SP beta c2 DNA is cleaved by the endonuclease HaeIII.

Mol Gen Genet, 1981, 181(4), 512 - 7
In vivo transcription of Bacillus subtilis bacteriophage SPP1; Montenegro MA et al.; The temporal program of SPP1 transcription was examined by hybridizing RNA extracted from infected B . subtilis cells, pulse-labelled at various times after infection, to restriction fragments of SPP1 DNA . RNA made early after infection hybridises to contiguous fragments in the left part of the SPP1 molecule, whereas hybridization to fragments in the right part of the chromosome is found late in infection . Viral gene transcription proceeds from right to left on the H-strand throughout the lytic cycle . At late times transcription occurs also from left to right using the L-strand as template . These assignments follow from the established 5'--3' polarity of the complementary (H- and L-) strains of SPP1 DNA and the determination of strand specificity in SPP1 transcription . Early and late transcriptions are also defined physiologically: protein synthesis and phage DNA replication must precede late transcription.

Mol Gen Genet, 1981, 181(4), 434 - 40
Plasmid transformation in Bacillus subtilis: effects of insertion of Bacillus subtilis DNA into plasmid pC194; Canosi U et al.; We have constructed a hybrid plasmid pBC1, which consists of plasmid pC194 with an insert of B . subtilis DNA as its HindIII restriction site . This plasmid is stably maintained in B . subtilis . In contrast with pC194, monomeric ccc forms of pBC1 are active in transformation . Transformations with these monomeric molecules of pBC1 have a stringent requirement for recombination proficiency, as defined by recE in the recipient cell . The extent of dependence of the transforming activity of oligomeric pBC1 DNA on the recombination proficiency of the recipient cell decreases with increasing oligomer size . A model of DNA processing during plasmid transformation of B . subtilis is presented.

Genetika, 1981, 17(5), 794 - 800
{Transformation and transduction in Bacillus subtilis strains with the BsuR restriction-modification system by modified and unmodified pUB110 plasmid DNA}; Belova TS et al.; Plasmid pUB110 DNA is restricted and modified during transformation of Bacillus subtilis cells possessing the BsuR system of restriction-modification . Restriction has comparatively little influence on the frequency of plasmid transformation only reducing it 20 times . The frequency of transduction of nonmodified plasmid DNA into modifying recipient cells, using phage Ar9 is also reduced a little . The frequency of transduction of chromosomal markers is much more lowered under the same experimental conditions.

J Virol, 1981 Jan, 37(1), 372 - 82
Analysis of bacteriophage SP82 major "early" in vitro transcripts; Panganiban AT et al.; A restriction map was constructed for the 17.5-kilobase SalI C fragment of SP82 DNA . Unfractionated SP82 DNA, the SalI C fragment, and restriction fragments derived from SalI-C were used as templates for in vitro synthesis by the Bacillus subtilis RNA polymerase, and the resulting transcripts were analyzed by gel electrophoresis . Comparison of the RNA species obtained from SalI-C with those produced from unfractionated DNA indicated that most of the RNAs and all of the major transcripts originate from the SalI C fragment; this fragment contains one copy of the terminally redundant portion of the genome . Seven major transcripts, a bidirectional terminator site, and 5 of the 13 minor transcripts were located within the 13-kilobase redundant region . The binding of polymerase to fragments of DNA produced by digestion of SalI-C with HpaII and HhaI was used to identify promoter-bearing regions within SalI-C.

J Virol, 1981 Jan, 37(1), 148 - 55
Restriction and modification of bacteriophage SP10 DNA by Bacillus subtilis Marburg 168: stabilization of SP10 DNA in restricting hosts preinfected with a heterologous phage, SP18; Witmer H et al.; SP10 phage cannot propagate in Bacillus subtilis Marburg 168 containing the wild-type allele of either gene nonA or gene nonB . The latter gene codes for the intrinsic cellular restriction activity . SP10 DNA was degraded in nonB+ derivatives of Marburg 168 . The degree of degradation depended upon the previous host in which SP10 was propagated . In the case of SP10 grown in B . subtilis W23 (a nonrestricting, nonmodifying bacterium), 90% of the phage DNA was hydrolyzed to acid solubles, and the residual acid-precipitable material was recovered as 0.5- to 1-megadalton fragments . In contrast, if SP10 was propagated in B . subtilis PS9W7 (a nonA nonB derivative of Marburg 168 that retains modifying activity), 40 to 50% of the input DNA was degraded to acid solubles, and most of the remainder was recovered as 15- to 20-megadalton fragments . In nonA+ nonB cells, SP10 DNA was conserved as unit-length molecules (ca . 80 megadalton) . Prior infection of nonB+ cells with SP18 protected superinfecting SP10 DNA, even when rifampin or chloramphenicol was added before the primary infection . The data are discussed in terms of the following conclusions . (i) The nonB gene product of B . subtilis Marburg 168 is required for restriction of SP10 DNA . (ii) Some sites on SP10 DNA are sensitive to both the restricting and modifying activities, whereas other sites are nonmodifiable even though they are sensitive to the restriction enzyme . (iii) In some manner, SP18 antagonizes the action of the nonB gene product.

J Bacteriol, 1981 Jan, 145(1), 422 - 8
Molecular cloning and expression of Bacillus licheniformis beta-lactamase gene in Escherichia coli and Bacillus subtilis; Gray O et al.; The chromosomal beta-lactamase (penicillinase, penP) gene from Bacillus licheniformis 749/C has been cloned in Escherichia coli . The locations of the target sites for various restriction enzymes on the 4.2-kilobase EcoRI fragment were determined . By matching the restriction mapping data with the potential nucleotide sequences of the penP gene deduced from known protein sequence, we established the exact position of the penP gene on the fragment . A bifunctional plasmid vector carrying the penP gene, plasmid pOG2165, was constructed which directs the synthesis of the heterologous beta-lactamase in both E . coli and Bacillus subtilis hosts . The protein synthesized in E . coli and B . subtilis is similar in size to the processed beta-lactamase made in B . licheniformis . Furthermore, the beta-lactamase made in B . subtilis is efficiently secreted by the host into the culture medium, indicating that B . subtilis is capable of carrying out the post-translational proteolytic cleavage(s) to convert the membrane-bound precursor enzyme into the soluble extracellular form.

Mol Gen Genet, 1981, 183(3), 422 - 7
Effects of the positively regulating product of gene 28 of the B . subtilis phage SPO1 on in vitro transcription; Giacomoni PU; Some of the properties of the RNA polymerase purified from SPO1-infected Bacillus subtilis have been compared with the properties of RNA polymerase from uninfected cells (core + sigma) . The two enzymes synthetize RNA from nonoverlapping regions on SPO1 DNA, and they lead to the retention of different restriction fragments of SPO1 DNA on cellulose-nitrate filters . The action of the positively regulating product of gene 28 of SPO1 (gp 28) has been analyzed . The isolated gp 28 has been shown to be unable to increase the dissociation rate of core + sigma from SPP1 DNA, while it efficiently blocks the initiation of RNA synthesis if it is added to performed complexes between core + sigma and SPP1 DNA in 0.2 M NaCl.

Microbiol Immunol, 1981, 25(7), 655 - 70
Mutual relationship between antibiotics and resting spores of Bacillus subtilis: morphological changes and macromolecular synthesis after germination of spores treated with cyclic polypeptide and aminoglycoside antibiotics; Hayakawa Y et al.; Morphological changes and synthesis of DNA, RNA, protein, and cell wall were investigated during germination of resting spores of Bacillus subtilis exposed transiently to the cyclic polypeptide antibiotics, polymyxin B and gramicidin S, and the aminoglycoside antibiotics, streptomycin, kanamycin, and gentamicin . Normal germinated spores showed breaks of the spore coat, a diminution in size and a fibrillar appearance of the cortex, a swelling core, a cell wall as thick as that of vegetable cells, some mesosomes and DNA fibrils . On the other hand, no breaks of the spore coat, a spore core with a slight swelling and irregular form, a thin cell wall, no demonstration of the nuclear material and no granularity in the cytoplasm were characteristic of the germinated spores derived from polymyxin B- and gramicidin S-treated resting spores . With gramicidin S-treated germinated spores a few vacuoles were formed in the cytoplasm . Both polymyxin B- and gramicidin S-treated germinated spores showed little or no synthesis of DNA, RNA, and protein . The vegetative cells derived from streptomycin-treated resting spores demonstrated several finely granular regions in the cytoplasm and a disorder of the fibrillar nucleoid, and their autolysis occurred early . Their DNA and RNA synthesis was normal, whereas protein synthesis was low . In spite of no occurrence of cell division and very low protein synthesis, the most striking characteristics of the outgrowing cells derived from kanamycin-treated resting spores were a markedly thickened cell wall and a continuous incorporation of labeled D-alanine suggesting cell wall synthesis; RNA synthesis was slightly lower and DNA synthesis was almost normal . The outgrowing cells from gentamicin-treated resting spores also revealed relatively thick cell walls and a very slight incorporation of labeled D-alanine . Their DNA and RNA synthesis was fairly low and protein synthesis was almost completely inhibited . These results coincide with the growth curves of individual antibiotic-treated resting spores.

Acta Microbiol Pol, 1981, 30(2), 195 - 201
Effect of microorganisms on the phytotoxicity of herbicides . VI . Modification of venzar activity by bacterial cultures; Balicka N et al.; Interactions between two bacterial strains and venzar were compared . It was found that the mechanism of interactions is various and causes the modification of herbicide phytotoxicity . Metabolites of Bacillus subtilis 72 interfered with herbicide by affecting physiological processes in plant tissues and enhancing its inhibitory influence . Arthrobacter sp . 18 strain decreased the phytoinhibitory effect of herbicide due to conjugation with the carrier from venzar.

Z Allg Mikrobiol, 1981, 21(1), 35 - 40
{Synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis}; Hecker M et al.; The synthesis of poly(A)-containing RNA in outgrowing spores of Bacillus subtilis was studied . A significant amount of RNA puls-labelled with 3H-uridine is polyadenylated . With the beginning of RNA synthesis in outgrowing spores labelled poly(A)-containing RNA was detected . The amount of poly(A)-RNA during the outgrowth and first cell division remains constant . Besides poly(A)-RNA the synthesis of tRNA and rRNA occurs . These results indicate a simultaneous activation of synthesis of tRNA, rRNA as well as of poly(A)-containing RNA during outgrowth of B . subtilis spores.

J Biol Chem, 1980 Dec 25, 255(24), 12034 - 6
The structure of BAcillus subtilis levansucrase at 3.8 A resolution; LeBrun E et al.; The tertiary structure of Bacillus subtilis levansucrase (EC 2.4.1.10) at 3.8 A resolution has been obtained from x-ray diffraction data by the method of multiple isomorphous replacements using three heavy atom derivatives . The crystals belong to space group P212121 with 1 molecule/crystallographic asymmetric unit . Unit cell dimensions are: a = 53.8 A, b = 67.7 A, and c = 125.6 A . Isomorphous heavy atom derivatives were prepared by soaking crystals in solutions containing K2Hg(CN)4, KAu(CN)2, and K2PtCl6 . Precession photographs were taken of 29 reciprocal lattice planes of the native compound and three derivatives . The intensities were measured with an automatic integrating microdensitometer . The heavy atoms were located by means of two-dimensional difference Patterson and Fourier projections . The heavy atom parameters were refined by a least squares method . In the subsequent phases of calculatin, a final mean figure of merit equal to 0.74 was obtained for the independent reflectins . A 3.8 A electron density map based on phases from three heavy atom derivatives has been calculated . The electron density map of levansucrase suggests that the molecule is a very elongated ellipsoid with overall dimensions of 26 X 32 X 117 A . A model of levansucrae is also presented.

J Biol Chem, 1980 Dec 25, 255(24), 11957 - 64
The interaction of Escherichia coli core RNA polymerase with specificity-determining subunits derived from unmodified and SP82-modified Bacillus subtilis RNA polymerase; Achberger EC et al.; the ability of the core isolated from Escherichia coli RNA polymerase to interact with specificity-determining subunits isolated from Bacillus subtilis RNA polymerase has been determined by measuring the transcription of "early" and "middle" genes of phage SP82 . Two specificity-determining subunits were tested: the sigma subunit and a 28,000 dalton (28 K) peptide isolated from a modified polymerase produced at approximately 8 min after infection of B . subtilis with SP82 . Earlier experiments (Spiegelman, G . B . and Whiteley, H . R . (1978) Biochem . Biophys . Res . Commun . 81, 1058-1065) demonstrated that sigma and the 28K peptide are required for the recognition of early and middle gene promoters, respectively, by the B . subtilis core assembly . The present investigation showed that E . coli core interacted more efficiently with the B . subtilis sigma than with the 28K peptide, as judged by the rate of RNA synthesis . Early RNA was produced by the E . coli and B . subtilis holoenzymes and by E . coli core supplenented with B . subtilis sigma and only minor differences were found in comparisons of transcripts by hybridization and by electrophoretic analysis . Measurements of template specificity, the formation of stable enzyme . DNA complexes, and the hybridization of transcripts to fragments of SP82 DNA produced by digestion with restriction endonuclease Hha indicated that E . coli core supplemented with the 28K-supplemented E . coli core with those synthesized by the modified polymerase extracted from B . subtilis 8 min after infection with SP82 suggest that both preparations recognized the same initiation and termination sequences.

J Biol Chem, 1980 Dec 10, 255(23), 11577 - 87
Linear, uncross-linked peptidoglycan secreted by penicillin-treated Bacillus subtilis . Isolation and characterization as a substrate for penicillin-sensitive D-alanine carboxypeptidases; Waxman DJ et al.; Incubation of growing Bacillus subtilis with penicillin G led to the secretion of a peptidoglycan-related polymer and a nonglycan-bound pentapeptide into the culture medium . The secreted polymer was isolated and characterized as a linear cell wall glycan strand substituted predominantly by uncross-linked pentapeptide side chains . Polymer formation and secretion are most likely the result of continued synthesis and elongation of nascent glycan strands in the absence of subsequent processing by peptidoglycan transpeptidase or D-alanine carboxypeptidase enzymes . The nonglycan-bound pentapeptide, L-Ala-D-iso-Glu-meso-diaminopimelic acid-D-Ala-D-Ala, was probably formed by an N-acetylmuramyl-L-alanine amidase active on the peptide side chains of The uncross-linked polymer . The uncross-linked peptidoglycan polymer was shown to be a good substrate for penicillin-sensitive D-alanine carboxypeptidases purified from membranes of B . subtilis, Bacillus stearothermophilus, and Escherichia coli . D-alanine release was not, however, coupled to the cross-linking of peptide side chains, suggesting that these enzymes do not function as peptidoglycan transpeptidases in vivo . No transpeptidase or D-alanine carboxypeptidase activity was detected in mixtures of high molecular weight penicillin-binding proteins from B . subtilis, B . stearothermophilus, or Staphylococcus aureus . Possible reasons for the inability to demonstrate these activities are discussed . In addition, an N-acetylmuramyl-L-alanine amidase activity which copurifies with penicillin-binding proteins from B . subtilis, S . aureus, and E . coli was partially characterized.

Biochim Biophys Acta, 1980 Dec 4, 616(2), 290 - 9
Inhibition of Bacillus subtilis aminopeptidase D by beta-lactam antibiotics; Starnes WL et al.; Penicillins and cephalosporins inhibit the hydrolysis of D-alanyl-beta-naphthylamide by aminopeptidase D (EC 3.4.11.-) from Bacillus subtilis . The inhibition is predominantly non-competitive, although the more effective inhibitors, i.e., those with the lower Ki values, seem to exhibit a tendency toward competitive kinetics, while penicillin V, the antibiotic with the largest Ki, exhibits a strong tendency toward uncompetitive kinetics . The removal of antibiotic from the enzyme by gel filtration on Sephadex G-25 restores 100% activity to the enzyme, and suggests that the inhibition does not derive from a covalent antibiotic-enzyme complex . The antibiotics which have been studied, with their respective Ki values (mM) and inhibition type are: methicillin (1.01 +/- 0.34, mixed non-competitive-competitive); cephaloridine (2.43 +/- 0.17, non-competitive); cloxacillin (3.19 +/- 1.29, mixed non-competitive-competitive); oxacillin (6.88 +/- 0.77, non-competitive); cephalothin (8.12 +/- 0.99, non-competitive); penicillin G (20.0 +/- 3.21, non-competitive) and penicillin V (32.1 +/- 4.90, mixed non-competitive-uncompetitive) . An empirical correlation exists between the Ki value and the freedom of rotation about the bond between the phenyl ring and the atom alpha to the ring in the variable side chain portion of the penicillin molecule.

J Hyg (Lond), 1980 Dec, 85(3), 359 - 69
Antibiotic residues in meat in the United Kingdom; an assessment of specific tests to detect and identify antibiotic residues; Smither R et al.; Investigations were conducted between 1977 and 1979 to assess the performance of microbiological tests for detecting and identifying residues of therapeutic-type antibacterial substances in meat and offal . Of the 5442 home-produced meat samples examined, 34 (0.63%) showed inhibitory activity in the screening test, which used Bacillus subtilis BGA and Micrococcus luteus as indicator organisms . Identification by electrophoretic and thin-layer chromatography/bio-autography techniques confirmed that only two of the 34 screen failures were due to true antibacterial residues: a pig sample contained a trace of penicillin and a horse sample contained a trace of an incompletely identified substance resembling a tetracycline . Twelve of the other 32 failures in the screen test were due to naturally produced inhibition and were, thus, falsely positive, whilst the remainder were shown to be negative . All of the 85 (8.7%) screen test failures from the 972 imported meat and offal samples tested were falsely positive . Additional samples from certain animals known to have been given antibiotic treatment were tested concurrently to give a more searching indication of screen and identification test efficacy.

Antimicrob Agents Chemother, 1980 Dec, 18(6), 858 - 62
Uptake of indolmycin in gram-positive bacteria; Werner RG; The antimicrobial activity of indolmycin correlates with the generation time of the investigated strains . Thus, in Staphylococcus aureus ATCC 13150 with a 37-min generation time, the minimal inhibitory concentration (MIC) was 0.6 microgram ml-1, and in Bacillus subtilis ATCC 27142 with a generation time of 23 min, the MIC reached 10.5 micrograms ml-1 . Competition experiments in staphylococci and B . subtilis with aromatic amino acids demonstrated that indolmycin uses the uptake systems that are responsible for tryptophan . When the Ki values of indolmycin for the uptake of the aromatic amino acids in staphylococci were compared, there was a significantly higher influence on the uptake of tryptophan with respect to phenylalanine and tyrosine . In addition, indolmycin low resistant mutants of S . aureus ATCC 13150 showed a 10- to 100-fold decrease in Km value for the uptake of tryptophan and a 10-fold decrease for tyrosine uptake . The Km value for phenylalanine remained unchanged . A significant correlation existed between the Ki values of indolmycin for the uptake of tryptophan in the wild-type strains of S . aureus and B . subtilis and the MIC against the corresponding strain . Low Ki values corresponded to low MIC . These results imply that, in addition to improvement of the antibiotic structure for target affinity, the tryptophan uptake system can be used as a test model for the structural evaluation of indolmycin with respect to an increased transport activity into bacterial cells.

Proc Natl Acad Sci U S A, 1980 Dec, 77(12), 7333 - 7
Replication of plasmids from Staphylococcus aureus in Escherichia coli; Goze A et al.; Plasmid pBR322 derives from plasmid ColE1 and does not replicate in Escherichia coli strains lacking DNA polymerase I . Hybrids between pBR322 and a plasmid isolated from Staphylococcus aureus, pC194, replicate in such E . coli strains, provided that the pC194 replication region is intact . Inactivation of the pBR322 replication region does not interfere with the replication of hybrids in E . coli . Hybrids between pBR322 and two other plasmids from S . aureus, pT127 and pUB112, and replicate at the restrictive temperature in E . coli having thermosensitive DNA polymerase I . Similar hybrids involving pC221 and pHV400, plasmids from S . aureus and Bacillus subtilis, respectively, do not replicate under such conditions . These results show that some plasmids from a Gram-positive bacterium, S . aureus, can replicate in a Gram-negative one, E . coli.

J Gen Microbiol, 1980 Dec, 121(Pt . 2), 487 - 90
Dissociation of an early event in sporulation from chromosome replication in Bacillus subtilis; Clarke S et al.; Synchronized populations of Bacillus subtilis are maximally inducible for sporulation about 15 min after chromosome replication has started . However, the induction of serine protease, one of the earliest marker events in sporulation, is not related to the state of chromosome replication.

Can J Microbiol, 1980 Dec, 26(12), 1386 - 91
Sporulation and regulation of homoserine dehydrogenase in Bacillus subtilis; Yeggy JP et al.; Homoserine dehydrogenase in dialyzed cell extracts of Bacillus subtilis 168 was studied, particularly with regard to inhibition, repression, and level of activity as a function of stage of development (growth and sporulation) . It was assayed in the "forward direction" using L-aspartic semialdehyde and NADPH as substrates . Of the potentials inhibitors tested, only cysteine and NADP were found to be effective . Both L- and D-cysteine were equally effective . Therefore, the physiological significance of cysteine as an inhibitor is somewhat questionable . Amino acids involved in repression of homoserine dehydrogenase included methionine, isoleucine, possibly threonine, and one or more unidentified components of Casamino acids . The specific activity of homoserine dehydrogenase was highest during the exponential phase of growth and declined steadily during the stationary phase of growth . The low specific activity during late sporulation may favor preferential funnelling of L-aspartic semialdehyde into the lysine pathway, where it is needed for synthesis of large amounts of dipicolinic acid and diaminopimelic acid.

Proc Natl Acad Sci U S A, 1980 Dec, 77(12), 7000 - 4
Novel RNA polymerase sigma factor from Bacillus subtilis; Haldenwang WG et al.; A modified form of Bacillus subtilis RNA polymerase (RNA nucleotidyltransferase) has been isolated that exhibits distinctive transcriptional specificity . This modified enzyme transcribes two cloned genes from the purA-cysA region of the B . subtilis chromosome whose expression in vivo is associated with the process of sporulation . Neither of these genes is transcribed by the usual form of B . subtilis RNA polymerase holoenzyme containing a sigma factor of 55,000 daltons (sigma 55) . The modified RNA polymerase lacks sigma 55 but contains a newly identified subunit of 37,000 daltons termed sigma 37 . A reconstitution experiment in which sigma 37 was added to core RNA polymerase strongly suggests that sigma 37 is responsible for the transcriptional specificity of the modified RNA polymerase . Sigma 37 apparently acts at the level of promoter recognition; this transcriptional determinant enabled core RNA polymerase to form stable binary and ternary ("initiation") complexes with endonuclease restriction fragments containing promoters for the cloned B . subtilis genes.

Gene, 1980 Dec, 12(1-2), 95 - 102
A site-specific recE4-independent intramolecular recombination between Bacillus subtilis and Staphylococcus aureus DNAs in hybrid plasmids; Fujii M et al.; A composite plasmid pLS253 was constructed from pLS103 {carrying the Bacillus subtilis leucine genes on B . subtilis (natto) plasmid pLS28} and pHV14 {a recombinant plasmid composed of pBR322 and the staphylococcal R-plasmid pC194} employing BamHI endonuclease, T4 DNA ligase, and B . subtilis transformation . All the Leu+ Cmr transformants tested harbored not only pLS253 but also two smaller plasmids designated as pLS251 and pLS252 . pLS253 DNA, when purified on an agarose gel, retained both Leu+ and Cmr transforming activities; however, in all the Leu+ Cmr transformants, the two smaller plasmids reappeared . pLS251 and pLS252 exhibited Leu+- or Cm4-transforming activity, respectively, and must have been derived from the pLS253 parent by an intramolecular recombination event, since the sum of the pLS251 and pLS252 DNAs represent the entire pLS253 genome . The recombination occurred between specific sites on the B . subtilis (natto) and Staphylococcus aureus plasmids . When the composite plasmid, pLS254, was constructed by BamHI cleavage of pLS251 and pLS252 followed by ligation, Leu+ Cmr transformants segregated two smaller plasmids which were indistinguishable from the original plasmids pLS103 and pHV14, respectively . They must have been derived from pLS254 through a reversal of the original recombination event . No intermolecular recombination between pLS251 and pLS252 DNA was detected . The recombination process was independent of recE function of the host cells, and its mechanism is discussed.

Gene, 1980 Dec, 12(1-2), 155 - 9
Properties of Bacillus subtilis 168 derivatives freed of their natural prophages; Yasbin RE et al.; An isogenic set of Bacillus subtilis 168 strains which are non-inducible for prophage PBSX and are cured of prophage SP beta has been constructed . By utilizing these strains, prophage SP beta has been shown to control the inducible DNA modification system which exists in this bacterium . However, neither the PBSX nor the SP beta prophages alter the ability of the bacterium to undergo genetic recombination, to repair damaged DNA or to sporulate . Prophageless B . subtilis would be a useful host for the phi 3T cloning vector, because of the absence of vector--prophage interactions.

J Bacteriol, 1980 Dec, 144(3), 957 - 66
Genetic transformation with cell wall-associated deoxyribonucleic acid in Bacillus subtilis; Doyle RJ et al.; Cell walls from bacillus subtilis 168 were prepared by conventional methods and found to contain deoxyribonucleic acid (DNA) . In transformation assays, after autolysis, it was found that two major regions of the chromosome were selectively enriched in the wall preparations . One region clustered around the replication origin and is represented by the markers purA16, ts8132, thiC5, sacA321, and hisA1 . The other region included the replication terminus with representative loci metB10, citK5, gltA292, and pyrA1 . All other (internal) loci which were examined showed no statistical enrichment . The two areas of enrichment were similar to but more extensive than those reported for membrane-DNA complexes . The wall preparations also contained protein and lipid, indicating a possible membrane involvement . Analyses of the cell walls revealed that the fatty acid composition of the membrane component was not typical of the for B . subtilis protoplast membranes or for lipoteichoic acids . In addition, radioiodination of cell wall autolysates, followed by gel electrophoresis and autoradiography, demonstrated the presence of proteins not readily detectable in bulk protoplast membranes or on the surfaces of intact cells . These data suggest that a unique component of the membrane and regions of the B . subtilis genome involved in DNA replication events are tightly associated with cell walls . The binding of DNA-membrane complexes to the "rigid" cell wall and the replication of the wall could be a mechanism by which the segregation of growing chromosomes occurs.

J Bacteriol, 1980 Dec, 144(3), 941 - 51
Biosynthesis and membrane binding of succinate dehydrogenase in Bacillus subtilis; Hederstedt L et al.; Antibodies specific for the Mr 65,000 (flavoprotein) and the Mr 28,000 subunits of the succinic dehydrogenase (SDH) of Bacillus subtilis were obtained . By using these antibodies it was shown that both subunits accumulated in the cytoplasm during 5-aminolevulinic acid starvation of a 5-aminolevulinic acid auxotroph . In the cytoplasm the subunits were not associated since they precipitated essentially independently of each other with subunit-specific antibody . In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the cytoplasmic subunits migrated identically with the corresponding subunits from the purified membrane-bound SDH complex . Cytoplasmic subunits were pulse-labeled with L-{35S}methionine during 5-aminolevulinic acid starvation . The labeled subunits bound to the membrane when heme synthesis was resumed and also when protein synthesis was blocked by chloramphenicol before readdition of 5-aminolevulinic acid . The experiments thus demonstrated a precursor relationship between cytoplasmic subunits and the subunits of the membrane-bound SDH complex . All SDH-negative mutants isolated so far carry mutations in the citF locus . None of the mutants was found to have either the Mr 65,000 or the Mr 28,000 SDH subunits in the membrane . Four citF mutants, however, contained both subunits in the cytoplasm . Three of these mutants lacked spectrally detectable cytochrome b558 . The respective mutations mapped at one end of the citF locus . These results strongly support our previous suggestion that cytochrome b558 is (part of) a membrane binding site for SDH in B . subtilis.

J Bacteriol, 1980 Dec, 144(3), 933 - 40
Cytochrome b reducible by succinate in an isolated succinate dehydrogenase-cytochrome b complex from Bacillus subtilis membranes; Hederstedt L; In previous work with membranes of Bacillus subtilis, the succinate dehydrogenase complex was isolated by immunoprecipitation of Triton X-100-solubilized membranes . The complex included a polypeptide with an apparent molecular weight of 19,000, probably attributable to apocytochrome . This paper reports the further characterization of this cytochrome and its relation to the respiratory chain of B . subtilis . The cytochrome was identified as cytochrome b, and its difference absorption spectra showed maxima at 426, 529, and 558 nm at room temperature . The oxidized cytochrome had an absorption maximum at 413 nm . The cytochrome was reduced by succinate in the isolated succinate dehydrogenase complex and in Triton X-100-solubilized membranes . In whole membranes cytochromes b, c, and a were reduced by succinate . In membranes from a mutant containing normal cytochromes but lacking succinate dehydrogenase no reduction of cytochrome was seen with succinate . It was concluded that the isolated succinate dehydrogenase-cytochrome b complex is a functional unit in the intact B . subtilis membrane . An accompanying paper describes cytochrome b as a structural unit involved in the membrane binding of succinate dehydrogenase.

J Bacteriol, 1980 Dec, 144(3), 1119 - 25
Enzyme changes during Bacillus subtilis sporulation caused by deprivation of guanine nucleotides; Vasantha N et al.; When sporulation is initiated by nutrient limitation, e.g., at the end of growth, certain biochemical processes occur in sequence . To determine which of these processes occur, even when the cells sporulate in the presence of a rapidly metabolizable carbon source, we induced sporulation of Bacillus subtilis by deprivation of guanine nucleotides, in a synthetic medium containing excess glucose, ammonium ions, and phosphate . The deprivation was produced either by decoyinine addition to a standard strain or by guanosin limitation of a guanine auxotroph . At 1 h after the onset of this deprivation, an extensive turnover of proteins began whose appearance was chloramphenicol sensitive . At least one enzyme (aspartate transcarbamylase) lost 70% of its activity within 15 min, indicating its rapid destruction . Whereas the magnitude of the above two changes was similar to that observed during sporulation at the end of growth in nutrient sporulation medium, protease (intracellular and extracellular) increased to less than one-tenth of the specific activity in nutrient sporulation medium, and alkaline phosphatase increased to less than one-half . However, glucose dehydrogenase, an enzyme made only in forespores, increased to the same specific activity under both conditions, presumably because the forespore compartment is protected from media (e.g., glucose) influences by the double membrane (two bilayers with opposite polarity).

Cancer Res, 1980 Dec, 40(12), 4381 - 4
Reduction of DNA transforming activity in culture by 6-mercaptopurine; Weigent DA et al.; The transforming activity of DNA isolated from 6-mercaptopurine-treated cultures of Bacillus subtilis strain UTH-8505 is markedly reduced when compared to that of DNA obtained from control cultures . The lower transforming activity appears to be a property of the isolated DNA; i.e., various treatments either to restore the activity or to indicate the presence of inhibitory substances that isolate with the DNA suggest a defect in the nucleic acid per se . The reduced transforming activity is not gene specific since the ability of DNA from 6-mercaptopurine-treated cultures to transform several mutants of different genetic loci is lowered . The dose-dependent effect is correlated with the extent of trichloroacetic acid-insoluble radioactivity associated with the DNA from 6-{35S}mercaptopurine-treated cultures . However, the level of apparent drug incorporation is low, being only 1 molecule equivalent to 6-mercaptopurine in 17,500 base residues of DNA having 40% of control transforming activity . The amount of 6-thioguanine incorporation possibly associated with the reduced transforming activity is even less, about one 6-thioguanine moiety per 100,000 base units . If base analog substitution accounts for the reduced transforming activity, exceedingly low levels of incorporation are sufficient to alter this biological property of B . subtilis DNA.

Gene, 1980 Dec, 12(1-2), 17 - 24
DNA modification induced during infection of Bacillus subtilis by phage phi 3T; Cregg JM et al.; The DNA of the Bacillus subtilis temperate phage phi 3T is not susceptible to cleavage by the restriction endonuclease HaeIII, although it is cut by many other restriction enzymes . The host DNA from uninfected cells is cut by HaeIII . We show that phi 3T DNA propagated in a restriction-modification-defective Escherichia coli cell can be digested by HaeIII . Thus, phi 3T DNA does contain the nucleotide recognition sequence of HaeIII . We suggest that this phage induces the modification of its own DNA . In support of this mechanism we show that extracts prepared from phi 3T-infected cells contain an activity which in the presence of S-adenosyl-L-methionine (SAM) can modify lambda DNA against cleavage by HaeIII . The same in vitro-modified DNA is still susceptible to cleavage by other restriction endonucleases.

Gene, 1980 Dec, 12(1-2), 147 - 54
DNA cloning in Bacillus subtilis . III . Efficiency of random-segment cloning and insertional inactivation vectors; Michel B et al.; Random segments of Bacillus amyloliquefaciens and yeast Saccharomyces cerevisiae DNA were used to determine two parameters pertinent to cloning in Bacillus subtilis, the yield of hybrids and the mean size of cloned segments . 10(3) to 10(4) hybrids/micrograms of DNA segments were obtained . Hybrids represented 11--18% of transformants . Mean m . wt . of cloned DNA segments was about 1 x 10(6), substantially lower than 3 x 10(6) found for donor DNAs after digestion with restriction endonucleases . We have cloned a B . amyloliquefaciens DNA segment which complemented a deficiency in B . subtilis hisH and E . coli hisC genes, which encode imidazolylacetolphosphate aminotransferase . The cloning efficiency for this gene was 10 transformed hosts/micrograms of donor DNA . Several B . subtilis insertional-inactivation cloning vectors were examined . One, pHV41, allows inactivation of the kanamycin-resistance (KmR) gene by insertion into its unique Bg/II site . In two other vectors, pHV11 and pHV23, insertion in their unique Kpn site inactivates the tetracycline-resistance (TcR) gene . pHV23 replicates both in E . coli and B . subtilis, and carries unique sites for seven restriction endonucleases (BamHI, EcoRI, HpaI, KpnI, PstI, SalI, XbaI) . This makes it one of the most versatile B . subtilis cloning vectors yet described.

Pharmazie, 1980 Dec, 35(12), 761 - 3
Solid-phase synthesis of a cyclic decapeptide, analog of the antibiotic polymyxin M; Salem EE; Pelargonoyl-cyclo{Dab-Thr-Dab-Dab(Pel)-Dab-D-Leu-Thr-Dab-Dab-Thr} . 5 HCl (5), analog of the polymyxin M, has been synthesized by the solid phase method . The intermediate linear Boc-protected decapeptide-resin (1) was assembled on the solid support by the stepwise addition of Boc-amino acids in the presence of DCC . For side-chain protection of diaminobutyric acid, Z-group was used . The peptide was cleaved from the resin in the form of methyl ester by triethylamine-catalyzed transesterification with methanol . Alkaline hydrolysis of the peptide ester with aqueous KOH (1 mol/l) afforded the Bocpenta-benzyloxycarbonyl-decapeptide acid (2) in 60% yield (based on Thr/resin) . After removal of the Boc-group, the linear peptide 3 was cyclized with DCC in high dilution in DMF (10(-3) mol/l) to give 4 in 51% yield . Hydrogenolysis of 4 in 80% formic acid afforded the pelargonyl cyclic decapeptide 5 in 74% yield . The synthetic peptide 5 retained the same activity of the natural polymyxin M against Bacillus subtilis and Staphylococcus aureus, whereas it showed only 3% of the activity of Escherichia coli.

J Bacteriol, 1980 Dec, 144(3), 891 - 7
Quantitative measurements of proton motive force and motility in Bacillus subtilis; Shioi JI et al.; The protein motive force of metabolizing Bacillus subtilis cells was only slightly affected by changes in the external pH between 5 and 8, although the electrical component and the chemical component of the proton motive force contributed differently at different external pH . The electrical component of the proton motive force was very small at pH 5, and the chemical component was almost negligible at pH 7.5 . At external pH values between 6 and 7.7, swimming speed of the cells stayed constant . Thus, either the electrical component or the chemical component of the proton motive force could drive the flagellar motor . When the proton motive force of valinomycin-treated cells was quantitatively decreased by increasing the external K+ concentration, the swimming speed of the cells changed in a unique way: the swimming speed was not affected until about--100 mV, then decreased linearly with further decrease in the proton motive force, and was almost zero at about--30 mV . The rotation rate of a flagellum, measured by a tethered cell, showed essentially the same characteristics . Thus, there are a threshold proton motive force and a saturating proton motive force for the rotation of the B . subtilis flagellar motor.

Biochim Biophys Acta, 1980 Nov 18, 602(3), 491 - 505
Influence of lipids with branched-chain fatty acids on the physical, morphological and functional properties of Escherichia coli cytoplasmic membrane; Legendre S et al.; Escherichia coli cells (unsaturated fatty acid auxotroph) have been adapted to grow on branched-chain fatty acids . Membrane vesicles were isolated from cells grown on a mixture of branched-chain fatty acids isolated from the lipids of Bacillus subtilis (E . coli (B . subtilis) membranes) and on a pure synthetic anti-isononadecanoic acid (E . coli (aC19) membranes) . We have shown, using wide-angle X-ray diffraction and differential scanning calorimetry, that the ordered state of the lipids is perturbed in the case of E . coli (B . subtilis) membranes but is unperturbed in the case of E . coli (aC19) membranes . The perturbation leads to the presence of a large wide-angle X-ray diffraction at 4.25--4.3 A, as opposed to the presence of a sharp 4.2 A reflection in unperturbed systems . We have shown, using freeze-fracture electron microscopy, that a protein segregation exists in the case of E . coli (aC19) membranes (at low temperature the integral membrane proteins aggregate in the membrane domains containing the disordered lipids); we do not observe such segregation in the case of E . coli (B . subtilis) membranes . We conclude that in cases where the branching of the fatty acids introduces a perturbation of the lipid order, the integral membrane proteins can still be accommodated in membrane domains containing the 'perturbed' ordered lipids . Finally, we have determined the rate of beta-galactoside transport in E . coli (aC19) and E . coli (B . subtilis) membranes as a function of temperature . We have shown that, in both cases, the Arrhenius representations display an increased slope in the region of the disorder-to-order transition . We conclude that such an increased slope may have different origins . In the case of E . coli (aC19) membranes, it is the result of the aggregation of the beta-galactoside carriers together with other integral membrane proteins which may lead to the inactivation of the carriers; in the case of E . coli (B . subtilis) membranes, it is the result of the partial immobilisation of the carriers embedded in a lipid environment, of which the fluidity, despite the perturbation of its lipid order, is still much less than that associated with lipids in a totally disordered state.

Biochim Biophys Acta, 1980 Nov 14, 610(1), 158 - 66
Template-independent poly(A) x poly(U) synthesizing activity of different forms of Bacillus subtilis RNA polymerase; Dooley MM et al.; Several, but not all, forms of bacillus subtilis RNA polymerase found in vegetative and sporulating cells can synthesize poly(A) x poly(U) in vitro . The vegetative delta-containing form of RNA polymerase (E delta) has little or no poly(A) x poly(U)-synthesizing activity, whereas RNA polymerase core (E) and sigma-containing core (E delta) both have significant activity . When purified vegetative delta factor was added to core, the core synthetic activity was reduced essentially to that of the vegetative enzyme E delta . When E sigma enzymes from vegetative and sporulating cells were compared for their salt sensitivity, it was found that the sporulation enzyme E sigma retained much more of its activity at 0.1 M KCl than the vegetative enzyme E sigma . Furthermore, when sporulation enzyme E delta 1 was compared with vegetative enzyme E sigma, it was found that the activity of the E sigma 1 form was much more resistant to high KCl concentrations than that of the vegetative E sigma form . These differences in enzyme activity, as affected by salt concentrations, suggest that the conformations of the sporulation E sigma and E delta 1 enzymes are different from that found in vegetative E sigma enzyme . These differences in conformation may be involved in selective gene expression during sporularion.

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