Genetika, 1985 Oct, 21(10), 1618 - 26 {Integration of various plasmids into the Bacillus subtilis chromosome}; Khasanov FK et al.; Some features of integration of temperature-sensitive pE194, pGG10 and pGG20 plasmids into the Bacillus subtilis chromosome were studied . Several auxotrophic mutations were obtained using insertion of these plasmids into the chromosome . The sites of plasmids for illegitimate recombination were determined . It was shown that the integration into the Bac . subtilis chromosome is characteristic not only for the plasmid pE194 but is the property of Staphylococcus aureus plasmid pC194 and Escherichia coli pBR322 plasmid . The influence of different Bac . subtilis rec mutations on the frequency of integration was studied. J Bacteriol, 1985 Oct, 164(1), 288 - 93 Mechanism of bacteriophage conversion of lipase activity in Staphylococcus aureus; Lee CY et al.; Staphylococcus aureus PS54 harbors two temperate bacteriophages and manifests no lipase activity on egg yolk agar . Curing of one of the resident prophages (L54a) restores lipase activity . To study the mechanism of bacteriophage conversion, the prophage was cured, and the gene encoding lipase activity was cloned into pBR322 in Escherichia coli on a 2.9-kilobase DNA fragment of the chromosome . The fragment was subcloned into a shuttle vector and subsequently transformed into S . aureus and Bacillus subtilis . Lipase activity was expressed in all three genetic backgrounds . Transformation and transductional data indicated that conversion is due to insertional inactivation of the lipase gene . Hybridization analysis with probes made from converting-phage DNA and from the cloned fragment confirmed that the phage insertion site resides within the terminal 0.8 kilobase of the insert. Eur J Biochem, 1985 Oct 1, 152(1), 137 - 42 Cloning of DNA segments of phage 2C, which allows autonomous plasmid replication in Bacillus subtilis; Lannoy NN et al.; The chromosome of Bacillus subtilis phage 2C is a 100-MDa double-stranded DNA molecule, containing hydroxymethyluracil in place of thymine and carrying redundant ends each encompassing 10% of the genome . 2C DNA was cleaved with EcoRI and HindIII, and cloned in the shuttle plasmids pSC 540 and pCP 115, both containing segments originating from B . subtilis and Escherichia coli plasmids . These chimaerical plasmids, carrying the chloramphenicol resistance gene, were unable to replicate in B . subtilis; this ability was restored, however, after the insertion of viral DNA segments . Physical maps of the recombinant plasmids were made; a large deletion of the E . coli-derived segment of pSC 540 was observed (which paralleled a loss of replication in this host), whereas addition of 2C DNA segments in pCP 115 was not accompanied by deletion (replication in E . coli was conserved in this case) . Cloned viral segments mapped mostly, but not exclusively, within the redundant ends of 2C DNA . It is suggested that the thirteen recombinant clones carried the replication origin region of phage 2C DNA, and that these sequences originated within or close to the redundant extremities of the viral chromosome. J Gen Microbiol, 1985 Oct, 131 ( Pt 10), 2831 - 8 Molecular cloning of a Bacillus subtilis gene involved in spore outgrowth; Ferrari E et al.; A lambda Charon 4A derivative carrying the outB gene of Bacillus subtilis has been identified by transformation of a B . subtilis mutant temperature-sensitive in spore outgrowth . The cloned region is a single EcoRI fragment 14 kb in length . In addition to outB, the cloned DNA includes at least part of the amyE and aroI loci. J Bacteriol, 1985 Oct, 164(1), 155 - 64 Bacillus subtilis citB gene is regulated synergistically by glucose and glutamine; Rosenkrantz MS et al.; The activity of aconitase in Bacillus subtilis is greatly reduced in cells cultured in media containing rapidly metabolized carbon sources (e.g., glucose) . Thus, expression of this enzyme appears to be subject to a form of catabolite repression . Since the product of the citB gene of B . subtilis is required for aconitase activity, we cloned the wild-type allele of this gene and used this DNA as a probe for transcription of citB in cells grown in various media . The steady-state level of RNA that hybridized to this probe was about 10-fold higher in B . subtilis cells grown in citrate-glutamine medium than in cells grown in glucose-glutamine medium . This result correlates well with the steady-state levels of aconitase activity . Two transcripts were shown to initiate within the cloned DNA; the steady-state level of one of these transcripts varied in the same way as did aconitase activity when cells were grown in media containing different carbon sources . This is the first demonstration of regulation by the carbon source of the level of a vegatative-cell transcript in B . subtilis. Nucleic Acids Res, 1985 Sep 25, 13(18), 6403 - 21 Nucleotide sequence of the BsuRI restriction-modification system; Kiss A et al.; The genes of the 5'-GGCC specific BsuRI restriction-modification system of Bacillus subtilis have been cloned and expressed in E . coli and their nucleotide sequence has been determined . The restriction and modification genes code for polypeptides with calculated molecular weights of 66,314 and 49,642, respectively . Both enzymes are coded by the same DNA strand . The restriction gene is upstream of the methylase gene and the coding regions are separated by 780 bp . Analysis of the RNA transcripts by S1-nuclease mapping indicates that the restriction and modification genes are transcribed from different promoters . Comparison of the amino acid sequences revealed no homology between the BsuRI restriction and modification enzymes . There are, however, regions of homology between the BsuRI methylase and two other GGCC specific modification enzymes, the BspRI and SPR methylases. J Biol Chem, 1985 Sep 15, 260(20), 11252 - 5 Morphological transformation of Chinese hamster cells by acylpeptides, inhibitors of cAMP phosphodiesterase, produced by Bacillus subtilis; Hosono K et al.; Treatment of Chinese hamster ovary cells in vitro with acylpeptide, an inhibitor of adenosine 3':5'-cyclic monophosphate (cAMP) phosphodiesterase, produced by Bacillus subtilis C-756 converts the culture from one of randomly oriented, epithelial-like cells to one of elongated, fibroblast-like cells . This transformation is similar to the effect of N6,O2'-dibutyryl adenosine 3':5'-cyclic monophosphate (dibutyryl cAMP), but is manifested at a lower concentration of the inhibitor than with dibutyryl cAMP . The intracellular level of cAMP in the cells grown with the inhibitor is 3-4-fold higher than the level without the inhibitor . The transformation is reversible and, when reversed, the level of cAMP simultaneously decreases to the level without the inhibitor . The acylpeptide has cytolytic activity as well as inhibitory activity and the former activity is prevented by the addition of serum. FEBS Lett, 1985 Sep 2, 188(2), 209 - 14 Heat-shock proteins during growth and sporulation of Bacillus subtilis; Todd JA et al.; Four major heat-shock proteins (hsps) with apparent molecular masses of 84, 69, 32 and 22 kDa were detected in exponentially growing stationary phase and sporulating cells of Bacillus subtilis heat-shocked from 30 to 43 degrees C . The most abundant, hsp69, is probably analogous to the E . coli groEL protein . These proteins were transiently inducible by heat-shock . Partial purification of RNA polymerase revealed several other minor hsps . One of these, a 48 kDa polypeptide probably corresponds to sigma 43 . The synthesis of this polypeptide and at least two other proteins appeared to be under sporulation and heat-shock regulation and was affected by the SpoOA mutation. Can J Microbiol, 1985 Sep, 31(9), 875 - 7 Discontinuity of charge on cell wall poles of Bacillus subtilis; Sonnenfeld EM et al.; When cell wall poles of Bacillus subtilis were treated with dilute cationized ferritin, label was found only at discrete patches . Since cationized ferritin binds to negatively charged groups, the pole regions that retain label most likely represent localized surface sites of high electronegativity, indicating that the cell wall of B . subtilis is, at least, partially differentiated. J Gen Microbiol, 1985 Sep, 131 ( Pt 9), 2409 - 19 spoVIC, a new sporulation locus in Bacillus subtilis affecting spore coats, germination and the rate of sporulation; James W et al.; A mutation near cysB on the Bacillus subtilis chromosome marks a new sporulation locus, spoVIC . It causes spores to germinate more slowly than those of the wild-type under all conditions and, from indirect evidence, it does not appear to alter the affinity for the germinant L-alanine . The mutant spores have some deficiency of coat proteins (particularly the alkalisoluble coat protein, Mr = 12 000) and the spore coat layers are disorganized . The mutant strain grows normally and sporulates normally until stage II, after which its sporulation is delayed by about 2 h compared to that of the wild-type . This delay results in the prolonged synthesis of some coat proteins and the late synthesis of others . The abnormal coat may be the cause of the germination deficiency . A double mutant strain carrying the spoVIC610 mutation together with gerE36 sporulates slowly . Its spores have very little coat protein, are sensitive to heat, lysozyme and organic solvents, but germinate as well as the strain carrying the spoVIC mutation alone . The role of the spore coat in germination is discussed in the light of these findings. J Gen Microbiol, 1985 Sep, 131 ( Pt 9), 2269 - 79 Cloning and deletion analysis of a genomic segment of Bacillus subtilis coding for the sdhA, B, C (succinate dehydrogenase) and gerE (spore germination) loci; Hasnain S et al.; From a Bacillus subtilis gene bank constructed in Escherichia coli and based on a low copy number cloning vector we have isolated a hybrid plasmid, pSH1047, containing an 8.0 kb segment of B . subtilis DNA coding for the sdhA, B and C genes, which code for the component polypeptides of succinate dehydrogenase, and the gerE gene, which may code for or regulate a protease involved in producing spores which germinate normally . We report the restriction map of this segment and the analysis of deletion derivatives which allow us to correlate the physical and genetic maps of these chromosomal segments. Antibiot Med Biotekhnol, 1985 Sep, 30(9), 643 - 9 {Fusion of Bacillus subtilis and Bacillus licheniformis protoplasts . Interspecies recombination resulting from protoplast fusion}; Iaroslavtseva NG et al.; Recombinants between B . subtilis and B . licheniformis were prepared by fusion of the bacterial protoplasts . Genetically marked strains SB25 trp C hisH and 168 ade-met-leu- of B . subtilis and 1001 ura-thr- and 1001 met- of B . licheniformis were used as the parent strains . The recombinants were selected with the indirect method followed by analysis of their nutrient requirements and cultural and morphological features . All the hybrids acquired the specific properties of B . subtilis . Apparently, their formation was based on the whole chromosome of B . subtilis and recombination of separate fragments of B . licheniformis with it . Hybrids with prototrophic properties with respect to one, two or three markers of the initial strains were detected independent of the genotype of the B . subtilis parent strains . Moreover, the protoplast fusion resulted in formation of hybrids which were prototrophic with respect to the amino acid markers of B . subtilis and deficient with respect to homoserine and thiamine or only thiamine, whereas the initial strains were not auxotrophic with respect to homoserine and thiamine . Thi-Hom- and a number of the prototrophic recombinants were characterized by the capacity for increased synthesis of riboflavin lacking in the initial cultures . Homologous and heterologous transformation appeared to be possible in the recombinants of the Thi-Hom- phenotype, while transformation of the initial strain SB25 by the intergenocytic markers was possible in reciprocal crossings . It is concluded that contrary to transformation of isolated DNA, protoplast fusion may result in formation of interspecies recombinants of B . subtilis and B . licheniformis with respect to different operones of amino acid synthesis. Proc Natl Acad Sci U S A, 1985 Sep, 82(18), 6201 - 5 DNA-damage-inducible (din) loci are transcriptionally activated in competent Bacillus subtilis; Love PE et al.; DNA damage-inducible (din) operon fusions were generated in Bacillus subtilis by transpositional mutagenesis . These YB886(din::Tn917-lacZ) fusion isolates produced increased beta-galactosidase when exposed to mitomycin C, UV radiation, or ethyl methanesulfonate, indicating that the lacZ structural gene had inserted into host transcriptional units that are induced by a variety of DNA-damaging agents . One of the fusion strains was DNA-repair deficient and phenotypically resembled a UV-sensitive mutant of B . subtilis . Induction of beta-galactosidase also occurred in the competent subpopulation of each of the din fusion strains, independent of exposure to DNA-damaging agents . Both the DNA-damage-inducible and competence-inducible components of beta-galactosidase expression were abolished by the recE4 mutation, which inhibitS SOS-like (SOB) induction but does not interfere with the development of the competent state . The results indicate that gene expression is stimulated at specific loci within the B . subtilis chromosome both by DNA-damaging agents and by the development of competence and that this response is under the control of the SOB regulatory system . Furthermore, they demonstrate that at the molecular level SOB induction and the development of competence are interrelated cellular events. J Bacteriol, 1985 Sep, 163(3), 895 - 9 Cellular location of origin and terminus of replication in Bacillus subtilis; Sonnenfeld EM et al.; The origin of replication of Bacillus subtilis 168 trp thy dna-1 (temperature-sensitive initiation mutant) was labeled with {3H}thymidine . Analysis of labeled cells by autoradiography revealed that most of the radioactivity was associated with cell pole areas . To label the terminus, cells that had initiated were treated with chloramphenicol to inhibit cell growth and division but to allow continued DNA synthesis . These cells were then labeled with {3H}thymidine at a time when chromosome replication was nearly complete . The distribution of radioactivity was similar to that observed in origin-labeled cells . In contrast, exponentially growing cells that were labeled for a brief time at the permissive temperature showed a random distribution of radioactivity . These data indicate that the origin and terminus of replication are located at cell poles. J Bacteriol, 1985 Sep, 163(3), 1167 - 71 Asymmetric distribution of charge on the cell wall of Bacillus subtilis; Sonnenfeld EM et al.; The cell wall of Bacillus subtilis is capable of binding different kinds of metal ions . The wall-ion complex appears to be dependent on both phosphoryl from teichoic acid and carboxylate from peptidoglycan . In the present study, cationized ferritin (CF) was used as a probe for charge distribution on the wall of B . subtilis 168 . Detergent-extracted cell walls bound CF only on the outer wall face . Completed cell poles bound CF, but septa did not . When the walls were permitted to autolyze briefly, binding of CF occurred on both faces . In contrast, limited hydrolysis of the walls by egg white lysozyme resulted in the penetration of CF into the wall matrix . When walls were made teichoic acid-free, CF-binding asymmetry was preserved, suggesting that carboxyl groups were oriented toward the surface . Walls with carboxylates chemically neutralized also retained charge asymmetry . Phosphate-free and carboxyl-modified walls bound CF only poorly or not at all . These results indicate that negative charges contributed by both phosphate and carboxyl are responsible for the binding of CF and that the observed asymmetry in the distribution of the label is due to the orientation of teichoic acid and muramyl peptides toward the outside of the cell wall, above the plane of the glycan strands. J Gen Microbiol, 1985 Sep, 131 ( Pt 9), 2421 - 30 Protease production during sporulation of germination mutants of Bacillus subtilis and the cloning of a functional gerE gene; James W et al.; Early in sporulation, cells of wild-type Bacillus subtilis produce three proteases (b, c and d) with monomeric Mr values of about 65 000, 53 000 and 43 500, and a further protease, e (Mr about 30 000) at the time of coat assembly . An additional protease, f (Mr about 15 000) appears transiently in sporangia at about the time of spore release . Three strains with defective spore coats were examined for alterations in sporulation proteases . A strain carrying the gerE36 mutation produces b, c and d normally, fails to produce e and accumulates f on or in its spores . A strain carrying the spoVIC610 mutation produces normal quantities of proteases b, c and d, but has a reduced amount of proteases e and f . A strain carrying both the gerE36 and the spoVIC610 mutations accumulates neither protease e nor f . The wild-type allele of the gerE gene was cloned in the vector, phage phi 105J9 . Complementation tests with the cloned gene showed that the gerE36 mutation is recessive to the wild-type allele. J Gen Virol, 1985 Sep, 66 ( Pt 9), 2029 - 32 Transcription of the Bacillus subtilis bacteriophage phi 3T in vitro; Kenny E et al.; The in vitro transcription pattern of Bg/II-digested phi 3T DNA is described . Eight Bg/II fragments that hybridized to in vitro transcription products were unequivocally identified . A further hybridizing region corresponding to a Bg/II triplet was also revealed, giving a total of nine to 11 Bg/II fragments . These represent 47 to 53% of the phi 3T genome . Transcription was shown to initiate within Bg/II fragments B, G, C, H, I, F and J, implying that all of these contain at least one promoter . The relevance of these data to the construction of a cloning vector based on phi 3T is discussed. J Bacteriol, 1985 Sep, 163(3), 824 - 31 Nucleotide sequence and promoter region for the neutral protease gene from Bacillus stearothermophilus; Takagi M et al.; The thermostable neutral protease gene nprT of Bacillus stearothermophilus was sequenced . The DNA sequence revealed only one large open reading frame, composed of 1,644 bases and 548 amino acid residues . A Shine-Dalgarno sequence was found 9 bases upstream from the translation start site (ATG), and the deduced amino acid sequence contained a signal sequence in its amino-terminal region . The sequence of the first 14 amino acids of purified extracellular protease completely matched that deduced from the DNA sequence starting at GTC (Val), 687 bases (229 amino acids) downstream from ATG . This suggests that the protease is translated as a longer polypeptide . The amino acid sequence of the extracellular form of this protease (319 amino acids) was highly homologous to that of the thermostable neutral protease from Bacillus thermoproteolyticus but less homologous to the thermolabile neutral protease from Bacillus subtilis . A promoter region determined by S1 nuclease mapping (TTTTCC for the -35 region and TATTTT for the -10 region) was different from the conserved promoter sequences recognized by the known or factors in bacilli . However, it was very homologous to the promoter sequence of the spo0B gene from B . subtilis . The guanine-plus-cytosine content of the coding region of the nprT gene was 58 mol%, while that of the third letter of the codons was much higher (72 mol%). J Bacteriol, 1985 Sep, 163(3), 1101 - 8 Thermoinducible transcription system for Bacillus subtilis that utilizes control elements from temperate phage phi 105; Osburne MS et al.; We describe a thermoinducible-expression system for Bacillus subtilis which utilized an early promoter-operator sequence from temperate phage phi 105 and the thermolabile prophage repressor from the phage variant phi 105 cts23 . The system operated at the transcriptional level to control expression in B . subtilis of the cat-86 gene derived from Bacillus pumilis . Details of the strategies used to isolate the early phage promoter are described . This promoter lay in close proximity to the prophage repressor gene on the phi 105 genome . The sequence of the early promoter differed from that of the vegetative B . subtilis consensus promoter by 1 base pair in both the -10 and -35 regions . We also present evidence that our phage-derived expression system could function in Escherichia coli to effect thermoinducible expression of the galK gene. J Bacteriol, 1985 Sep, 163(3), 957 - 64 Regulation of Bacillus subtilis glutamate synthase genes by the nitrogen source; Bohannon DE et al.; The wild-type alleles of the gltA292 and gltB1 mutations of Bacillus subtilis have been identified in banks of B . subtilis DNA cloned in phage lambda . These mutations are thought to define the genes for the two subunits of glutamate synthase . Sequences having transforming activity for each allele were subcloned in plasmids and used as hybridization probes for measurements of the rates of synthesis and steady-state levels of glt mRNAs under different growth conditions . For both gltA and gltB, the level of mRNA varied according to the nitrogen source in the growth medium, to an extent sufficient to explain the variation in glutamate synthase activity under the same conditions . Two start points for mRNA synthesis were detected within the cloned DNA, one of which corresponded to the gltA locus . The other start point appears to define a transcription unit, separate from gltA and gltB, within which mutations cause loss of glutamate synthase activity. Chem Phys Lipids, 1985 Aug 30, 38(1-2), 41 - 50 Phospholipid transfer proteins in microorganisms; Tai SP et al.; Phospholipid transfer activity has been demonstrated in cell lysates of Saccharomyces cerevisiae, Rhodopseudomonas sphaeroides and Bacillus subtilis, and proteins facilitating phospholipid transfer from the first two organisms have recently been purified . The phospholipid transfer protein from S . cerevisiae has mol . wt . 35 000 with a specificity of transfer for phosphatidylinositol and phosphatidylcholine . The purified phospholipid transfer protein from R . sphaeroides has mol . wt . 27 000 and, although it has the ability to transfer all phospholipid species tested it displays a preference for phosphatidylglycerol . The cellular levels of phospholipid transfer activity in both S . cerevisiae and R . sphaeroides are not strictly related to the level of subcellular membranes . However, in photosynthetically grown R . sphaeroides, the distribution of the activities between soluble and membrane-associated forms is correlated with the level of intracytoplasmic membrane (a postulated membrane substrate). FEBS Lett, 1985 Aug 19, 188(1), 123 - 6 Comparison of genomes of closely related phages phi 29, phi 15 and PZA using a rapid method of parallel physical mapping; Hostomsky Z et al.; The DNAs of phages phi 29, phi 15 and PZA of Bacillus subtilis were analysed with restriction enzymes EcoRI, HpaI and HindIII . A method was used which permits parallel physical mapping of all three phages, from both ends of their linear genomes . The method is based on transfer of partially digested DNA to DBM paper and sequential hybridization with labelled terminal fragments . It follows from the comparison of the physical maps that phages phi 29, phi 15 and PZA are closely related and that they probably have arisen from a common ancestor by accumulation of point mutations. Biochemistry, 1985 Aug 13, 24(17), 4645 - 50 Interaction of duramycin with artificial and natural membranes; Navarro J et al.; Duramycin is a polypeptide antibiotic (molecular weight 2012) obtained from culture filtrates of Streptomyces cinnamomeus forma azacoluta . In this work, we show that low concentrations of duramycin induced aggregation of lipid vesicles containing unsaturated phosphatidylethanolamine and unsaturated monogalactosyl diglyceride, and of sarcoplasmic reticulum vesicles from rabbit skeletal muscle . Furthermore, duramycin inhibited the ATP-dependent Ca2+ uptake in sarcoplasmic reticulum vesicles without affecting the hydrolysis of ATP or the permeability of Ca2+ . Also, duramycin only inhibited the bacteriorhodopsin proton pump reconstituted into phospholipid vesicles containing phosphatidylethanolamine . We have isolated a duramycin-resistant strain of Bacillus subtilis and have mapped the location of duramycin resistance . In this strain, the secretion of protons and influx of calcium were resistant to duramycin, and its lipid composition was profoundly different from that of the parent strain . No phosphatidylethanolamine was detected in the resistant strain . Our findings are consistent with the idea that duramycin recognizes a particular membrane conformation determined by the presence of phosphatidylethanolamine or monogalactosyl diglyceride. Nucleic Acids Res, 1985 Aug 12, 13(15), 5717 - 22 Nucleotide sequence of the Bacillus subtilis xylose isomerase gene: extensive homology between the Bacillus and Escherichia coli enzyme; Wilhelm M et al.; The xylose isomerase gene from Bacillus subtilis was cloned from a genomic BamH1 library by complementation of an isomerase defective Escherichia coli strain as previously described . The ATG initiation codon is preceded by a Shine-Dalgarno sequence and two hexamers being characteristic for the promoter region of Bacillus genes . The structural gene consists of 1320 base pairs, thus coding for a polypeptide chain of 440 amino acids with a molecular weight of 49 680 . The polypeptide primary structure shows over 50% homology to that of the E . coli xylose isomerase. Nucleic Acids Res, 1985 Aug 12, 13(15), 5441 - 55 Nucleotide sequence and mutational analysis of an immunity repressor gene from Bacillus subtilis temperate phage phi 105; Dhaese P et al.; We have identified and sequenced a bacteriophage phi 105 gene encoding an immunity repressor, the first to be characterized from a temperate phage infecting a Gram-positive host . Using superinfection immunity as an assay for repressor function, the phi 105 repressor gene was located within a 740-bp PvuII-HindIII subfragment near the left end of the phi 105 EcoRI-F fragment . We show that the repressor is specified by the 5'-proximal coding sequence of a translationally overlapping gene pair, transcribed from right to left on the conventional phi 105 map . Comparison of its amino acid sequence (146 residues) with that of a large number of Gram-negative bacterial and phage repressors revealed a putative DNA-binding region between positions 20 and 39 . The coding region is preceded by a strong Shine-Dalgarno sequence 5' AAAGGAG 3' . Deletion analysis of the 5'-flanking DNA allowed to identify transcriptional control elements . Their structure, 5' TTGTAT 3' at -35 and 5' TATAAT 3' at -10, strongly suggests that the phi 105 repressor gene is transcribed by the major vegetative form of B . subtilis RNA polymerase, as would be expected for an early phage gene. Chemioterapia, 1985 Aug, 4(4), 310 - 2 Bacillus subtilis selectively stimulates the synthesis of membrane bound and secreted IgA; Fiorini G et al.; Peripheral blood lymphocyte subpopulations and IgA production in vitro have been studied in 30 elderly subjects . Fifteen patients were treated with Bacillus subtilis (Enterogermina, Midy) and 15 with a placebo preparation . At the end of the treatment, a significant increase was noticed in lymphocytes bearing membrane IgA and Ia+ cells in the group treated with Bacillus subtilis, but not in the controls . Similarly, a significantly enhanced spontaneous production of IgA in vitro was seen in the treated subjects . The relevance of these findings to the understanding of the mode of action of Bacillus subtilis and its usefulness in different immunodeficiency states is discussed. Appl Environ Microbiol, 1985 Aug, 50(2), 503 - 7 Production of cloned human leukocyte interferon by Bacillus subtilis: optimal production is connected with restrained growth; Meyer HP et al.; Bacillus subtilis, transformed with a plasmid containing the human alpha-2 (leukocyte) interferon gene, was cultivated in batch and continuous culture in a complex medium . In continuous culture with dissolved oxygen of less than 10% of air saturation, the extracellular interferon titer decreased sharply when the growth rate was lower or higher than the optimal one (mu = 0.14 h-1) . Thus, a relatively low growth rate was best for extracellular interferon production, and oxygen limitation enhanced interferon production . The mean output rate in batch culture after successful harvest was 20 X 10(6) IU/liter per h and the maximal output rate in continuous culture was 14 X 10(6) IU/liter per h. J Virol, 1985 Aug, 55(2), 513 - 5 Nucleotide sequence of the cohesive single-stranded ends of Bacillus subtilis temperate bacteriophage phi 105; Ellis DM et al.; The cohesive single-stranded ends of temperate Bacillus subtilis phage phi 105 were analyzed with the exonuclease activities of the Klenow fragment of DNA polymerase I and with exonuclease III and were found to be 3' extensions . Chemical sequencing of 3'-end-labeled fragments showed that the ends are 7-base extended 3' single strands and have the sequence: 5'-GCGCTCC-3' . 3'-CGCGAGG-5' J Bacteriol, 1985 Aug, 163(2), 774 - 7 Heat and UV light resistance of vegetative cells and spores of Bacillus subtilis Rec-mutants; Hanlin JH et al.; The heat and UV light resistance of spores and vegetative cells of Bacillus subtilis BD170 (rec+) were greater than those of B . subtilis BD224 (recE4) . Strain BD170 can repair DNA whereas BD224 is repair deficient due to the presence of the recE4 allele . Spores of a GSY Rec+ strain were more heat resistant than spores of GSY Rec- and Uvr- mutants . The overall level of heat and UV light resistance attained by spores may in part be determined by their ability to repair deoxyribonucleic acid after exposure to these two physical mutagens. J Bacteriol, 1985 Aug, 163(2), 648 - 53 Bacillus subtilis gene involved in cell division, sporulation, and exoenzyme secretion; Sadaie Y et al.; Bacillus subtilis strains carrying div-341 or sacU mutations, or both, have been characterized to reveal the roles of both genes in the initiation of sporulation, as well as in cell division and exoenzyme secretion . Both mutations were closely linked by transformation and caused the pleiotropic effects on sporulation and sporulation-associated events . Some sacU mutations (sacUh) resulted in hyperproduction of exoenzymes, reduced autolysis, and an ability to sporulate in the presence of excess nutrients . The div-341 mutation, on the other hand, resulted in filamentous growth at a higher temperature (45 degrees C) and showed spo0 properties at an intermediate permissive temperature (37 degrees C) in the usual sporulation medium . However, the div-341 strain sporulated better than wild-type strain at 37 degrees C in the presence of excess nutrients . Exoenzyme production and autolysis were reduced at 37 degrees C in the div-341 strain . A double mutant with sacUh32 and div-341 showed the complex phenotypes . It showed the sacUh32 property of autolysis and exoenyzme secretion . It showed the sacUh32 property of sporulation at 30 degrees C and the div-341 property at 37 degrees C . Slow growth and defective spore outgrowth of the div-341 strain at 37 degrees C were not observed in the double-mutant strain . Based on pleiotropic phenotypes and close linkages of both mutations, we discuss the relationship between the sacU and div-341 genes and their roles in sporulation, exoenzyme secretion, and cell division. J Bacteriol, 1985 Aug, 163(2), 543 - 51 Transcriptional control of synthesis of acid-soluble proteins in sporulating Bacillus subtilis; Johnson WC et al.; The major acid-soluble spore proteins (ASSPs) isolated from mature spores of Bacillus subtilis are designated alpha, beta, and gamma (about 60, 60, and 100 amino acids in length, respectively) . Alpha and beta are very similar, and gamma is very similar to a less predominant ASSP called delta (about 115 amino acids) . A minor and very basic ASSP called epsilon is the same size as alpha and beta but is unrelated antigenically . These and several minor ASSPs comprise at least three related families of sporulation-specific gene products . Expression of the alpha and beta genes, detectable as functional mRNA in vitro, coincides with the time of synthesis of all of the major ASSPs in vivo . This apparently coordinate expression is dependent on at least the spo0A, spoIIA, and spoIIIA loci, but not on the spoIVA or spoVA loci, consistent with the late stage of this expression (initiating at 3.5 h after the start of sporulation and peaking at 5 h after start of sporulation) . A few minor ASSPs may be asynchronously expressed. J Bacteriol, 1985 Aug, 163(2), 411 - 6 Genetic analysis of spo0A and spo0C mutants of Bacillus subtilis with a phi 105 prophage merodiploid system; Ikeuchi T et al.; An 8.0-kilobase chromosomal fragment of Bacillus subtilis which contained an intact spo0A gene was recloned onto temperate phage phi 105 from the rho 11dspo0A+-1 transducing phage . A specialized transducing phage, phi 105-dspo0A+-1, was constructed and used to transduce the spo0A12 mutant strain 1S9 . A Spo+ transductant which was a single lysogen of the phi 105dspo0A+-1 transducing phage was isolated . From competent cells of this Spo+ transductant was isolated a Spo- (Spo0A) strain which was immune to phi 105 . It was used to prepare a lysate of the phi 105dspo0A12 phage . Transduction of the spo0C9V recE4 strain with the phi 105dspo0A12 and phi 105dspo0A+-1 phages was carried out . The phi 105dspo0A+-1 phage gave rise to a large number of heat-resistant cells, but the phi 105dspo0A12 phage formed no heat-resistant cells . These results indicate that the spo0A12 and spo0C9V mutant genes do not complement each other in the ability to sporulate and that the spo0C9V mutation is located within the spo0A gene . Although the spo0C9V strain was completely asporogenous, the spo0C9V/spo0C9V diploid strain produced heat-resistant cells at a frequency of ca . 10(-3) in the sporulation medium . This result indicates that two copies of the spo0C9V mutant gene partially restore the ability of these cells to sporulate. J Bacteriol, 1985 Aug, 163(2), 610 - 4 Mutations that affect utilization of a promoter in stationary-phase Bacillus subtilis; Ray C et al.; Transcription of the ctc gene in Bacillus subtilis is activated only after exponentially growing cells enter stationary phase . The promoter of the ctc gene is utilized in vitro by two minor forms of RNA polymerase, E sigma 37 and E sigma 32, but not by the most abundant form of RNA polymerase, E sigma 55 . We have used the ctc promoter to direct transcription of the xylE gene on plasmid pLC4 and observed that xylE was expressed only in stationary-phase B . subtilis . We also have constructed a series of homologous plasmids that differ only by specific base substitutions in the ctc promoter . We observed that the base substitutions that affected utilization of the ctc promoter in vivo (xylE expression) were the same as those that we had previously shown to affect utilization of the promoter in vitro by E sigma 37 and E sigma 32 . We conclude that it is likely that the ctc promoter is utilized in vivo by E sigma 37 or E sigma 32. J Bacteriol, 1985 Aug, 163(2), 573 - 9 Determination of DNA sequences containing methylcytosine in Bacillus subtilis Marburg; Guha S; The methylcytosine-containing sequences in the DNA of Bacillus subtilis 168 Marburg (restriction-modification type BsuM) were determined by three different methods: (i) examination of in vivo-methylated DNA by restriction enzyme digestion and, whenever possible, analysis for methylcytosine at the 5' end; (ii) methylation in vitro of unmethylated DNA with B . subtilis DNA methyltransferase and determination of the methylated sites; and (iii) the methylatability of unmethylated DNA by B . subtilis methyltransferase after potential sites have been destroyed by digestion with restriction endonucleases . The results obtained by these methods, taken together, show that methylcytosine was present only within the sequence 5'-TCGA-3' . The presence of methylcytosine at the 5' end of the DNA fragments generated by restriction endonuclease AsuII digestion and the fact that in vivo-methylated DNA could not be digested by the enzyme XhoI showed that the recognition sequences of these two enzymes contained methylcytosine . As these two enzymes recognized a similar sequence containing a 5' pyrimidine (Py) and a 3' purine (Pu), 5'-PyTCGAPu-3', the possibility that methylcytosine is present in the complementary sequences 5'-TTCGAG-3' and 5'-CTCGAA-3' was postulated . This was verified by the methylation in vitro, with B . subtilis enzyme, of a 2.6-kilobase fragment of lambda DNA containing two such sites and devoid of AsuII or XhoI recognition sequences . By analyzing the methylatable sites, it was found that in one of the two PyTCGAPu sequences, cytosine was methylated in vitro in both DNA strands . It is concluded that the sequence 5'-PyTCGAPu-3' is methylated by the DNA methyltransferase (of cytosine) of B . subtilis Marburg. J Bacteriol, 1985 Aug, 163(2), 487 - 92 Cloning and sequencing of the beta-lactamase I gene of Bacillus cereus 5/B and its expression in Bacillus subtilis; Wang W et al.; The beta-lactamases of Bacillus cereus have attracted interest because they are secreted efficiently, because multiple enzymes are frequently present, and because their regulation has unusual features . beta-Lactamase I of strain 5/B is produced constitutively at a high level, and the exoenzyme appears to be several thousand daltons larger than the corresponding product of strain 569/H . We have cloned the gene for 5/B beta-lactamase I in Escherichia coli and B . subtilis and have sequenced the structural portion and the regulatory regions . The 5/B enzyme is produced at a low level in E . coli RR1(pRWY200) and remains cellbound . In B . subtilis it is formed in large amounts, and over 90% of it is released into the medium . There is a large degree of homology between the promoter and leader peptide regions of the 5/B and 569/H genes; both utilize UUG as the translation initiation codon (P . S . F . Mezes, R . W . Blacher, and J . O . Lampen, (J . Biol . Chem . 260:1218-1223, 1985) . Although there are significant differences in the peptide segment where processing would be expected to occur, the NH2 terminus of the major 5/B product from B . subtilis BD170(pRWY215) is His-44, which is the same as the NH2 terminus of the major 569/H product from B . subtilis BD170(pRWM5). J Bacteriol, 1985 Aug, 163(2), 445 - 53 Chromosomal-DNA amplification in Bacillus subtilis; Wilson CR et al.; Tetracycline-resistant (Tetr) mutants RAD1, RAD2, RAD6, and RAD7 were isolated from Bacillus subtilis BC92 after protoplasting, polyethylene glycol treatment, and regeneration on a medium containing tetracycline . The Tetr phenotype in RAD1, RAD2, and RAD6 was very stable with less than 5% loss of resistance after 30 generations of growth in the absence of selection . Of the four isolates, three contained amplified chromosomal DNA closely associated with the Tetr phenotype . The intensity of restriction fragments present in HindIII and EcoRI digests of chromosomal DNA from RAD1, RAD6, and RAD7 indicated the presence of tandemly duplicated DNA . Disparity in the size and number of amplified fragments suggested that the tandemly duplicated DNA is different in all three isolates . The sizes of the duplicated DNA present in RAD1, RAD6, and RAD7 were estimated to be 10, 19, and 20 kilobases, respectively . No amplified DNA was detected in RAD2 . Results of transductional-mapping studies with PBS1 showed that the tetracycline resistance (tet) loci of RAD1, RAD2, and RAD6 all mapped near the origin of chromosomal replication and close to the guaA locus . Amplified DNA characteristic of RAD1 and RAD6 was cotransduced with the tet locus . Cotransfer of amplified DNA with the guaA locus or other nearby loci in the absence of tet was not observed . In every case, loss of Tetr was accompanied by loss of amplified DNA . A possible explanation for the occurrence of the amplified DNA is presented. J Biol Chem, 1985 Jul 25, 260(15), 8721 - 5 Bovine mitochondrial ribosomes . Elongation factor specificity; Eberly SL et al.; The activity of bovine mitochondrial ribosomes with elongation factors from a variety of sources including the mitochondria of lower eukaryotes, chloroplasts, Gram-negative bacteria, Gram-positive bacteria, and the eukaryotic cell cytoplasm has been investigated . Bovine mitochondrial ribosomes are active with homologous mitochondrial elongation factor (EF)-G but display no activity with the mitochondrial or chloroplast translocases from the lower eukaryote Euglena gracilis, with Escherichia coli or Bacillus subtilis EF-G or with cytoplasmic EF-2 . In contrast to the results obtained with the translocases, E . coli EF-Tu, B . subtilis EF-Tu, and Euglena chloroplast EF-Tu all function to a significant extent on the mitochondrial ribosomes . Cytoplasmic EF-1 has barely detectable activity on the animal mitochondrial ribosomes . The polymerization of phenylalanine by these ribosomes is dependent on poly(U), displays a rather broad Mg2+ optimum around 12 mM, and proceeds most rapidly at low monovalent ion concentrations. Nucleic Acids Res, 1985 Jul 11, 13(13), 4953 - 69 Alkali-labile structures linked to the 5' ends of Bacillus subtilis short DNA chains; Yasuda H et al.; Alkali-labile portion covalently linked to the 5' ends of Bacillus subtilis short DNA chains, the putative primer RNA for discontinuous DNA synthesis, was isolated and analyzed using a temperature sensitive DNA polymerase I mutant, which accumulates nascent DNA fragments at a restrictive temperature . A novel oligonucleotide structure as well as mono- to triribonucleotide stretches were isolated at the 5' end of the short DNA chains . A structure for the novel oligonucleotide is proposed to be p5' X3' pp5' rN, where X represents unidentified nucleoside with a peculiar property . Possible metabolic relationship between these molecules and primer RNA has been discussed. J Biol Chem, 1985 Jul 5, 260(13), 8121 - 7 UV cross-linking of the Bacillus subtilis RNA polymerase to DNA in promoter and non-promoter complexes; Hilton MD et al.; Complexes between Bacillus subtilus RNA polymerase and 32P-labeled DNA were irradiated with UV light and digested with nuclease; electrophoresis and autoradiography were used to identify the polymerase subunits cross-linked to DNA . These experiments showed: 1) that cross-linkage of promoter complexes yielded predominantly the beta and sigma subunits; 2) that beta, beta', and sigma were detected in non-promoter complexes; 3) that addition of the delta subunit or high concentrations of NaCl decreased cross-linkage of all subunits, especially the cross-linkage of the sigma subunit in non-promoter complexes and the binding of polymerase at DNA ends; 4) that different patterns of cross-linkage were obtained at 0 degrees C (conditions favoring the formation of closed complexes) and 37 degrees C (conditions favoring the formation of open complexes); and 5) predominantly beta and possibly alpha were cross-linked by irradiation of core-DNA complexes whereas similar experiments with core-delta complexed to DNA showed the efficient cross-linkage of beta' and beta. Biochemistry, 1985 Jul 2, 24(14), 3672 - 7 Purification and characterization of a non-vitellogenin, estrogen-induced plasma protein from the American bullfrog Rana catesbeiana; Mitchell RO et al.; A non-vitellogenin, estrogen-induced protein has been detected for the first time in the plasma of male Rana catesbeiana . A greater than 90% purification of this plasma protein was achieved by salt fractionation with Mg(II) followed by ion-exchange chromatography on DEAE- and CM-cellulose . Immunoelectrophoretic analysis with various antisera showed no immunological cross-reactivity between this protein and vitellogenin . The molecular mass of the purified protein was determined to be 116 000 daltons by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and 105 000 daltons by analytical ultracentrifugation . Sedimentation studies indicate the protein is a nonaggregating spherical monomer with a sedimentation coefficient of 7.5 S . Amino acid analysis demonstrated a composition different from that of vitellogenin and lipovitellin A . Limited proteolysis with trypsin, chymotrypsin, and Bacillus subtilis protease revealed no common peptides on SDS-polyacrylamide gels . Phosphate analysis indicated that, on a molar basis, the non-vitellogen, estrogen-induced protein had less than or equal to 3% of the phosphate found in vitellogenin . Further studies of the structure, function, and metabolism of this protein may reveal information relating to the hormonal control of vitellogenesis. Kosm Biol Aviakosm Med, 1985 Jul-Aug, 19(4), 63 - 5 {Determining the Bacillus subtilis biomass increase coefficient in weightlessness}; Bergter F et al.; This paper presents the results of a microbiological experiment carried out by the Soviet and GDR scientists onboard Salyut-6 . The experiment was performed using a Bacillus subtilis suspension in the Jena unit . The purpose of the experiment was to study the time-course variations of the cell biomass increase in zero-g . The cell culture development was measured with respect to the utilization rate of glucose or casein hydrolyzate in the nutrient medium and the rate of protein accumulation in cells . It has been shown that the rate of biomass increment in zero-g lags behind the 1 g level . It can be concluded that the decreased metabolic activity of bacterial cells in zero-g is associated with changes in the cell population distribution and physicochemical parameters of the nutrient medium. J Gen Microbiol, 1985 Jul, 131 ( Pt 7), 1753 - 63 Effect of in vitro DNA rearrangement in the NH2-terminal region of the penicillinase gene from Bacillus licheniformis on the mode of expression in Bacillus subtilis; Imanaka T et al.; We have constructed secretion vector plasmids that have unique BglII sites within or near the signal sequence of Bacillus licheniformis penicillinase, and have also constructed penicillinase cartridges that lack either one, two or three of the processing sites for the membrane-bound, exo-large and exo-small enzymes . Each of these penicillinase cartridges was cloned on secretion vectors in Bacillus subtilis, and enzyme production was examined . The presence of both the signal sequence and the three host-specific processing sites on the secretion vector was required for an effective expression of the enzyme in B . subtilis . The presence of any of the processing sites on the cartridge reduced the accumulation of penicillinase in the culture medium . When a vector plasmid lacking part of the hydrophobic region of the signal sequence and lacking the three processing sites was used, total penicillinase production decreased and enzyme accumulation in the medium was extremely low, despite the complete or incomplete presence of the processing sites on the cartridge . Molecular mass determination of these extracellular penicillinases suggested the existence of a new cleavage site for the enzyme. J Appl Bacteriol, 1985 Jul, 59(1), 99 - 105 Effects of aqueous and alcoholic povidone-iodine on spores of Bacillus subtilis; Gorman SP et al.; Spores of Bacillus subtilis NCTC 10073 were examined for susceptibility to two proprietary brands of povidone-iodine: an aqueous solution, Betadine and an alcoholic solution, Videne . Spores were converted to ion-exchange (Ca, H) and coat-defective (SLS-, UME-, UMS-, UDT- and UDS-treated) forms . The resistance of these to povidone-iodine was compared and related to uptake . Effects on spore protoplasts and cortex in relation to hexosamine release were also examined . The degree of spore penetration and site of action of povidone-iodine is discussed. J Bacteriol, 1985 Jul, 163(1), 111 - 20 Integration of linear, heterologous DNA molecules into the Bacillus subtilis chromosome: mechanism and use in induction of predictable rearrangements; Niaudet B et al.; Linear DNA molecules composed of a central region nonhomologous with the Bacillus subtilis chromosome and two flanking regions homologous with the chromosome can integrate into the chromosome, provided that the homologous regions have the same relative orientation . The resulting chromosome can be maintained in a haploid or in a merodiploid cell together with a parental chromosome . This can most easily be explained by supposing that the integration occurs by crossing over at each homologous region and that a part of the chromosome between these regions is deleted and replaced by the central nonhomologous region of the integrating molecule . If no essential genes were replaced during that process a haploid cell would be obtained; if essential genes were replaced a merodiploid cell would be obtained . The use of appropriate linear molecules therefore should allow the induction of deletions, extending from a given chromosomal site in a predetermined direction, and defined duplications in the B . subtilis chromosome. J Virol, 1985 Jul, 55(1), 39 - 44 Deletion mutants of Bacillus subtilis bacteriophage SP beta; Spancake GA et al.; The central portion of the chromosome of temperate Bacillus subtilis bacteriophage SPB was found to contain a region in which large deletions occurred, sometimes at high frequency . Most of the deletions could be placed into one of three groups, del1, del3, and del4, which were missing 11.8, 14.2, and 14 kilobase pairs of DNA, respectively . The chromosomal positions of the three types of deletions overlapped and together defined a continuous region of 27 kilobase pairs surrounding the prophage attachment site attPSPB . The 27-kilobase-pair segment contained no functions required for lytic growth of the phage, but DNA within this region was used as a template for RNA synthesis at several stages in the life cycle of SPB . In addition the transcription of DNA during lytic infection was found to be initiated over a large portion of one-half of the viral chromosome (the arbitrary left half) . Subsequently, the synthesis of early RNA was terminated as late transcription continued on the opposite side of the chromosome. Mikrobiologiia, 1985 Jul-Aug, 54(4), 621 - 7 {Action of different types of gramicidin S derivatives on bacterial cells and protoplasts}; Bulgakova VG et al.; The work was concerned with studying the effect of gramicidin S derivatives with modified free amino groups of ornithine residues on bacterial cells and protoplasts . The substitution of the amino groups with neutral or carboxyl-containing groups eliminated or sharply decreased the antibacterial activity of gramicidin S, its binding to the cells, and the ability to change the permeability of the cytoplasmic membranes of the intact cells . However, the neutral derivatives and the derivative with acidic properties showed a considerable lytic activity when they were incubated with the protoplasts of Micrococcus lysodeikticus, Bacillus megaterium and Bacillus subtilis . Hence, these compounds preserved a certain membranotropic level . Those gramicidin S derivatives with modified ornithine amino groups which possessed basic properties were similar to gramicidin S in the antibiotic activity, the modified permeability of the membranes, the ability to bind with the cells, and the lytic action on the protoplasts. J Biol Chem, 1985 Jun 25, 260(12), 7178 - 85 A strong sequence homology exists between the major RNA polymerase sigma factors of Bacillus subtilis and Escherichia coli; Gitt MA et al.; The Bacillus subtilis rpoD gene has been sequenced and the primary structure of its product deduced . The molecular weight calculated for the sigma 43 is 42,828 . The DNA and protein sequences of sigma 43 exhibit extensive homology to the Escherichia coli rpoD gene and its sigma 70 product, especially in the C-terminal two-thirds of the sequence . Other proteins exhibiting partial homology with sigma 43 include the E . coli nusA protein, the E . coli htpR (heat-shock regulatory gene) product sigma 32, and specific DNA-binding proteins . No amino acid homology was found between sigma 43 and B . subtilis phage SP01 sigma gp28, phage T7 RNA polymerase, or E . coli DNA primase . The gene exhibits a strong ribosomal binding site and a typical rho-independent transcription terminator sequence . A typical transcription terminator sequence was not observed upstream from the sigma 43 gene . The sigma 43 gene may be part of an operon, resembling the situation found in the E . coli sigma operon. J Biol Chem, 1985 Jun 10, 260(11), 6518 - 21 Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation; Estell DA et al.; Site-directed mutagenesis can be employed to alter activity critical residues in proteins which are susceptible to chemical oxidation . Previous studies have implicated methionine 222 as a primary site for oxidative inactivation of subtilisin (Stauffer, C . E., and Etson, D . (1969) J . Biol . Chem . 244, 5333-5338) . Because of uncertainties in predicting which amino acid would be the optimal substitute for methionine 222, we prepared all 19 amino acid substitutions at this site in the cloned subtilisin gene using a cassette mutagenesis method (Wells, J . A., Vasser, M., and Powers, D . P . (1985) Gene (Amst.), in press) . Mutant enzymes were expressed in Bacillus subtilis and were found to vary widely in specific activity . Mutants containing nonoxidizable amino acids (i.e . Ser, Ala, and Leu) were resistant to inactivation by 1 M H2O2, whereas methionine and cysteine enzymes were rapidly inactivated . These studies demonstrate the feasibility of improving oxidative stability in proteins by site-directed mutagenesis. Proc Natl Acad Sci U S A, 1985 Jun, 82(12), 4031 - 5 Secretory S complex of Bacillus subtilis forms a large, organized structure when released from ribosomes; Caulfield MP et al.; The S complex of Bacillus subtilis, a set of four proteins that appears to be involved in protein secretion, is shown to be attached to 70S ribosomes: antibody to its 64-kDa component can aggregate these ribosomes, and the complex can be chemically crosslinked to ribosomal proteins . Low Mg2+ or prolonged high-speed centrifugation in a sucrose gradient releases the S complex from the ribosomes, and it is recovered as an aggregate with an S value of 76 . Electron microscopy shows that these aggregates have a regular structure, somewhat resembling clathrin cages, with a diameter of about 45 nm . If these aggregates are physiological, their function would differ significantly from that of the signal recognition particle of eukaryotes. Mutat Res, 1985 Jun-Jul, 150(1-2), 127 - 32 Detection and chemical identification of natural bio-antimutagens . A case of the green tea factor; Kada T et al.; A bio-antimutagen, isolated from Japanese green tea (leaves of Camellia sinensis), reduced high spontaneous mutations due to altered DNA-polymerase III in a mutator strain of Bacillus subtilis . Chemical studies showed that the factor was epigallo-catechin-gallate (EGCg). J Virol, 1985 Jun, 54(3), 773 - 80 DNA packaging by the Bacillus subtilis defective bacteriophage PBSX; Anderson LM et al.; Defective bacteriophage PBSX, a resident of all Bacillus subtilis 168 chromosomes, packages fragments of DNA from all portions of the host chromosome when induced by mitomycin C . In this study, the physical process for DNA packaging of both chromosomal and plasmid DNAs was examined . Discrete 13-kilobase (kb) lengths of DNA were packaged by wild-type phage, and the process was DNase I resistant and probably occurred by a head-filling mechanism . Genetically engineered isogenic host strains having a chloramphenicol resistance determinant integrated as a genetic flag at two different regions of the chromosome were used to monitor the packaging of specific chromosomal regions . No dramatic selectivity for these regions could be documented . If the wild-type strain 168 contains autonomously replicating plasmids, especially pC194, the mitomycin C induces an increase in size of resident plasmid DNA, which is then packaged as 13-kb pieces into phage heads . In strain RB1144, which lacks substantial portions of the PBSX resident phage region, mitomycin C treatment did not affect the structure of resident plasmids . Induction of PBSX started rolling circle replication on plasmids, which then became packaged as 13-kb fragments . This alteration or cannibalization of plasmid replication resulting from mitomycin C treatment requires for its function some DNA within the prophage deletion of strain RB1144. J Bacteriol, 1985 Jun, 162(3), 960 - 71 Shape and fine structure of nucleoids observed on sections of ultrarapidly frozen and cryosubstituted bacteria; Hobot JA et al.; Very rapidly frozen cells of Escherichia coli and Bacillus subtilis were substituted at low temperature into acetone with 1% OsO4 and embedded in Epon . They showed ribosome-free spaces filled with globular and fibrillar material of up to 15 nm . The sizes of structures seen do not exclude DNA superstructures such as supercoils, aggregates, and nucleosomes . With the Feulgen analog osmium-ammines stain, DNA was localized within the ribosome-free space . The bulk of DNA, the nucleoid, is therefore a major part of, or identical to, the main ribosome-free space . The ribosome-free space would correspond directly to the light microscopy phase-contrast image of nucleoids in living bacteria . The shape of the ribosome-free space does not reflect intracellular salt concentrations, nor do the Feulgen-positive areas . The previously observed dependency on the salt concentration of the growth medium seems to be due to permeabilization induced by the chemical fixative at room temperature . The ribosome-free space is more cleft in appearance than the nucleoid obtained by fixation with OsO4 but more confined than its very dispersed form found after aldehyde fixation. J Bacteriol, 1985 Jun, 162(3), 1336 - 8 Genetic heterogeneity in the cysA-fus region of the Bacillus subtilis chromosome: identification of the hos gene; Matsuzaki S et al.; We identified a new gene, hos, which exerts different sporulation phenotypes in Bacillus subtilis strains with different genetic backgrounds . The hos+ gene showed normal sporulation in the genetic background of JH642 but showed temperature-sensitive sporulation in that of the Tano-oka W . The hos gene was mapped between cysA and rpoB. J Bacteriol, 1985 Jun, 162(3), 1302 - 3 Induction of penicillin-binding proteins under catabolite-repressed conditions; Buchanan CE; Decoyinine, an inhibitor of GMP synthetase, was used to induce sporulation under catabolite-repressed conditions in Bacillus subtilis . Sporulation-specific penicillin-binding proteins 4* and 5* were produced in abundance, and there was an increase in vegetative penicillin-binding proteins 2B and 3 . These results, which were completely blocked by addition of guanosine, suggest that synthesis of penicillin-binding proteins is neither catabolite repressed nor directly dependent on the stringent response. J Bacteriol, 1985 Jun, 162(3), 1280 - 4 Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety; Le Van Q et al.; We studied the incorporation of {1-13C}ribose and {1,3-13C2}glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis . The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor . The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate . C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement . Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi. J Bacteriol, 1985 Jun, 162(3), 1250 - 4 Ultrastructural localization of dipicolinic acid in dormant spores of Bacillus subtilis by immunoelectron microscopy with colloidal gold particles; Kozuka S et al.; The localization of dipicolinic acid in dormant spores of Bacillus subtilis was examined by an immunoelectron microscopy method with colloidal gold-immunoglobulin G complex . The colloidal gold particles were distributed mainly in the core regions of dormant spores and were not observed in those of germinated or autoclaved spores . This result clearly demonstrates that dipicolinic acid is localized in the cores of dormant spores. J Bacteriol, 1985 Jun, 162(3), 1238 - 43 Plasmid transduction by Bacillus subtilis bacteriophage SPP1: effects of DNA homology between plasmid and bacteriophage; Deichelbohrer I et al.; Any SPP1 DNA restriction fragment cloned into Bacillus subtilis plasmid pC194 or pUB110 increased the transduction frequency of the plasmid by SPP1 100- to 1,000-fold over the transduction level of the plasmid alone . This increment was observed irrespective of whether a fragment contained the SPP1 packaging origin (pac) . Furthermore, an SPP1 derivative into whose genome pC194 DNA had been integrated transduced pC194 DNA with a greatly enhanced frequency . Transduction enhancement mediated by DNA-DNA homology between plasmid and SPP1 was independent of the extent of homology (size range analyzed, 0.5 to 3.9 kilobases) and the recombination proficiency of donor or recipient. J Bacteriol, 1985 Jun, 162(3), 1203 - 11 Amplification of a chromosomal region in Bacillus subtilis; Albertini AM et al.; We report on the amplification in Bacillus subtilis of a defined DNA sequence after exposure of the bacteria to increasing levels of antibiotic . The experimental system consisted of transformation of competent cells with a plasmid (pRHA39) unable to replicate in the host and carrying the alpha-amylase gene derived from B . subtilis . Selection of transformants resistant to 5 micrograms of chloramphenicol per ml resulted in the isolation of strains with the plasmid integrated into the chromosome at the site of homology, by a Campbell type mechanism . Starting from such a nontandem duplication, amplification was achieved by growing the bacteria in increasing concentrations of chloramphenicol . By dilution, Southern blotting, and hybridization to a radioactive probe, we estimated a copy number of about 10 for the amplified sequence of samples grown in the presence of 50 micrograms of chloramphenicol per ml . No free plasmid could be detected in the amplified strains . The extent of the amplified region was the same for all transformants, and the endpoints appeared to be the same in all isolates . As a consequence of the amplification, there was a noticeable increase in amylase production, and the amount of enzyme produced correlated with gene dosage . The amplification did not occur in a recE genetic background. J Bacteriol, 1985 Jun, 162(3), 1106 - 10 Cadmium-resistant mutant of Bacillus subtilis 168 with reduced cadmium transport; Laddaga RA et al.; Cd2+ and Mn2+ accumulation was studied with wild-type Bacillus subtilis 168 and a Cd2+-resistant mutant . After 5 min of incubation in the presence of 0.1 microM 109Cd2+ or 54Mn2+, both strains accumulated comparable amounts of 54Mn2+, while the sensitive cells accumulated three times more 109Cd2+ than the Cd2+-resistant cells did . Both 54Mn2+ and 109Cd2+ uptake, which apparently occur by the same transport system, demonstrated cation specificity; 20 microM Mn2+ or Cd2+ (but not Zn2+) inhibited the uptake of 0.1 microM 109Cd2+ or 54Mn2+ . 54Mn2+ and 109Cd2+ uptake was energy dependent and temperature sensitive, but 109Cd2+ uptake in the Cd2+-resistant strain was only partially inhibited by an uncoupler or by a decrease in temperature . 109Cd2+ uptake in the sensitive strain followed Michaelis-Menten kinetics with a Km of 1.8 microM Cd2+ and a Vmax of 1.5 mumol/min X g (dry weight); 109Cd2+ uptake in the Cd2+-resistant strain was not saturable . The apparent Km value for the saturable component of 109Cd2+ uptake by the Cd2+-resistant strain was very similar to that of the sensitive strain, but the Vmax was 25 times lower than the Vmax for the sensitive strain . The Km and Vmax for 54Mn2+ uptake by both strains were very similar . Cd2+ inhibition of 54Mn2+ uptake had an apparent Ki of 3.4 and 21.5 microM Cd2+ for the sensitive and Cd2+-resistant strains, respectively . Mn2+ had an apparent Ki of 1.2 microM Mn2+ for inhibition of 109Cd2+ uptake by the sensitive strain, but the Cd2+-resistant strain had no defined Ki value for inhibition of Cd2+ uptake by Mn2+. J Bacteriol, 1985 Jun, 162(3), 1014 - 23 Analysis of plasmid deletional instability in Bacillus subtilis; Hahn J et al.; Using a model system, we have studied deletion formation in Bacillus subtilis . When the staphylococcal plasmids pSA2100 (7.1 kilobases) and pUB110 (4.5 kilobases) were ligated to one another at their unique XbaI sites and transformed into either rec+ or recE4 strains of B . subtilis, an intramolecular recombination event usually occurred . Two plasmids, one of 2.6 kilobases and the other of 9.0 kilobases, were consistently isolated and shown by restriction enzyme analysis to be derived by recombination occurring in the pSA2100-pUB110 cointegrate . Analysis of the sequence of the junctions of the recombinant plasmids and of the crossover regions of the parental plasmids suggested that a reciprocal, conservative, intramolecular recombination event had occurred between short 18-base-pair homologous sequences that were oriented as direct repeats and bounded by regions of dyad symmetry . Evidence is presented that the above illegitimate recombination event is biased to occur intramolecularly and that randomly chosen direct repeats of either 22 or 29 base pairs are not sufficient to support recombination . The recombination event occurs in recA1, recB2, recD3, recE5, recL16, recM13, polA59, polA13, uvr-22, uvr-13, and stb mutants of B . subtilis and does not require that the competent state be established. J Bacteriol, 1985 Jun, 162(3), 1180 - 5 Cloning and expression in Escherichia coli of sdhA, the structural gene for cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex; Magnusson K et al.; Bacillus subtilis cytochrome b558 is a transmembrane protein which anchors succinate dehydrogenase (SDH) to the cytoplasmic membrane and is reduced by succinate . The structural gene for this cytochrome was cloned and expressed in Escherichia coli . Random BamHI or BglII fragments of B . subtilis 168 DNA were cloned in the BamHI site of plasmid pHV32 . The derived plasmids were used to transform B . subtilis SDH mutants to chloramphenicol resistance by integration of the plasmid via DNA homology . Of some 3,000 transformants tested, 6 were SDH positive and had pHV32 integrated close to the sdh operon . Two plasmids, pKIM2 and pKIM4, with an insert of B . subtilis DNA of 5.7 and 3.4 kilobases, respectively, were generated by transforming E . coli with DNA from the SDH-positive transformants after cleavage with EcoRI or BglII and ligation . In E . coli carrying either of the two plasmids, about 4% of total membrane protein was B . subtilis cytochrome b558 . E . coli (pKIM2) also contained antigen which reacted with antibodies specific for the flavoprotein and the iron-sulfur protein subunit of B . subtilis SDH . Enzymatically active, membrane-bound B . subtilis SDH could not be demonstrated in E . coli (pKIM2) . The B . subtilis DNA insert in pKIM2 could transform B . subtilis sdhA (cytochrome b558), sdhB (flavoprotein), and sdhC (iron-sulfur protein) mutants to the wild type . The results suggest that pKIM2 carries the whole B . subtilis sdh operon . The data confirm the gene order and the proposed direction of transcription of the B . subtilis sdh operon . Most likely the sdh genes in E . coli(pKIM2) are controlled by their natural promoter. Proc Natl Acad Sci U S A, 1985 Jun, 82(12), 4189 - 92 Bacillus subtilis sigma factor sigma 29 is the product of the sporulation-essential gene spoIIG; Trempy JE et al.; Evidence is presented that the sporulation-essential locus spoIIG codes for both sigma 29 and a structurally related protein, P31 . This demonstrates that at least one specific Bacillus subtilis RNA polymerase binding protein provides a critical function in endospore formation . spoIIG-specific RNA is present in B . subtilis cultures that are synthesizing P31 and sigma 29 and is absent in those that are not . A monoclonal antibody specific for an antigenic determinant on P31/sigma 29 detected crossreacting proteins (P25/P21) but not P31 or sigma 29 in a Spo- B . subtilis strain with a mutation at the spoIIG locus (spoIIG41) . The appearance of P25 and P21 occurs in this mutant at a time when P31 and sigma 29 would normally appear and suggests that they are homologous proteins . Transformation of the spoIIG41 strain with plasmid DNA carrying the structural gene for spoIIG complements the Spo- phenotype and results in the synthesis of P31, sigma 29, P25, and P21 at the appropriate times during sporulation . In Escherichia coli, the cloned spoIIG sequence encoded a protein that reacted with the anti-P31/sigma 29 monoclonal antibody and had the electrophoretic mobility of authentic P31. Biochim Biophys Acta, 1985 May 28, 815(3), 405 - 9 Pore-forming properties of iturin A, a lipopeptide antibiotic; Maget-Dana R et al.; The addition of iturin A, a lipopeptide antibiotic extracted from Bacillus subtilis, to a bimolecular lipid membrane (BLM) increases dramatically its electrical conductance . For very low concentration of iturin A, discrete conductance steps are observed which are assigned to the formation of conducting pores . The characteristics of these pores depend on the lipid content of the BLM and they change with time . Cholesterol considerably increases the lifetimes of open states . The pores are slightly anion versus cation selective . These first observations unable us to briefly discuss the pore-forming properties of lipopeptides. J Biol Chem, 1985 May 25, 260(10), 5950 - 5 Nucleotide sequence and transcription of a bacteriophage 29 early promoter; Dobinson KF et al.; We have studied the in vitro and in vivo transcription of a promoter for Bacillus subtilis RNA polymerase on bacteriophage phi 29 DNA . The promoter is identified as an early promoter as it is transcribed in vitro by uninfected B . subtilis sigma 55-containing RNA polymerase; is transcribed in vivo at both 7 min after infection and in the presence of chloramphenicol; and is transcribed right to left on the standard phi 29 map . The nucleotide sequence of the promoter and the initiation site for RNA synthesis are reported . We have also examined the kinetics of RNA synthesis initiation using a single round run-off transcription assay . The overall rate of initiation was found to be 1.6 X 10(6) M-1 s-1 while the rate of conversion of unstable to stable polymerase-promoter complexes was 0.049 s-1 . These values are comparable to those for similar promoters for Escherichia coli RNA polymerase. J Biol Chem, 1985 May 10, 260(9), 5415 - 9 Ion dependence of the Bacillus subtilis RNase P reaction; Gardiner KJ et al.; The properties of the Bacillus subtilis RNase P are characterized with regard to the types and concentrations of monovalent and divalent ions required to potentiate precursor tRNA cleavage by the protein-RNA holoenzyme and the catalytic RNA alone . The ionic dependence of the RNase P RNA-catalyzed reaction in part seems due to a requirement for ion shielding between substrate and catalytic RNAs . The RNase P protein, which binds to RNA nonspecifically and tightly, likely serves, in part, as a cation screen . However, the character of the ion dependence of the RNA catalysis, the inhibition by high SO2-4 concentration, and potentiation by solvents suggest that RNA conformational transition may be involved in the reaction . It is proposed that the reason for catalysis by RNA in the RNase P reaction may be a requirement for fluidity in the structure of the catalyst, so that it can accommodate many tRNA substrates, which vary in their structural details. Nucleic Acids Res, 1985 May 10, 13(9), 3083 - 100 Overproduction and purification of protein P6 of Bacillus subtilis phage phi 29: role in the initiation of DNA replication; Pastrana R et al.; A phi 29 DNA fragment containing gene 6, required for DNA replication, has been cloned in plasmid pPLc28 under the control of the PL promoter of phage lambda . A polypeptide with an electrophoretic mobility close to that of p6 was labelled with 35S-methionine after heat induction . This protein, representing about 4% of the total E . coli protein after 1 h of induction, was obtained in a highly purified form . The protein was characterized as p6 by amino acid analysis and NH2-and COOH-terminal sequence determination . Protein p6 has an apparent molecular weight of 23,600, suggesting that the native form of the protein is a dimer . The purified protein p6 stimulated the protein-primed initiation of phi 29 DNA replication when added to purified proteins p2 (phi 29-coded DNA polymerase) and p3 (terminal protein). J Biol Chem, 1985 May 10, 260(9), 5753 - 9 Modification and processing of internalized signal sequences of prolipoprotein in Escherichia coli and in Bacillus subtilis; Hayashi S et al.; We have cloned the Escherichia coli lipoprotein structural gene (lpp) into a shuttle vector and studied its expression in both E . coli and in Bacillus subtilis . Using in vitro gene fusion techniques, the lpp gene was placed under the control of the promoter for the erythromycin-resistance (ery) gene . This fusion gene directed the synthesis of Braun's prolipoprotein which can be subsequently processed into the mature lipoprotein . In addition to the prolipoprotein, two ery-lpp hybrid proteins containing a 45- and a 22-amino acid extension preceding the NH2 terminus of prolipoprotein, respectively, are also synthesized in E . coli . The synthesis of these three proteins appears to involve the utilization of three distinct translation initiation sites . In B . subtilis, only two proteins are synthesized, the hybrid protein with a 45-amino acid extension and the prolipoprotein . In both E . coli and B . subtilis, the precursor forms of the hybrid proteins are lipid-modified, and they are processed to mature lipoprotein in vivo . These results indicate that internalized signal sequence containing the prolipoprotein modification and processing site (Leu-Ala-Glys-Cys) can function normally and permit the modification of hybrid proteins to lipid-modified precursors which can be subsequently processed by the globomycin-sensitive prolipoprotein signal peptidase. J Biol Chem, 1985 May 10, 260(9), 5554 - 62 Characterization by electron paramagnetic resonance and studies on subunit location and assembly of the iron-sulfur clusters of Bacillus subtilis succinate dehydrogenase; Hederstedt L et al.; Succinate dehydrogenase is a conserved membrane-bound enzyme consisting of two nonidentical subunits: a flavo iron-sulfur protein (Fp) subunit, containing a covalently bound flavin, and an iron-sulfur protein (Ip) subunit . Bacillus subtilis succinate dehydrogenase in wild type bacteria and 12 well characterized succinate dehydrogenase-defective mutants were examined by low temperature EPR spectroscopy to characterize the enzyme and study subunit location and biosynthesis of its iron-sulfur clusters . The wild type B . subtilis enzyme contains iron-sulfur clusters which are analogous to clusters S-1 and S-3 of bovine heart succinate dehydrogenase but with slightly different EPR characteristics . Spins from cluster S-2 were not detectable as in the case of the intact form of bovine heart succinate dehydrogenase . However, dithionite reduction of the B . subtilis enzyme greatly enhanced spin relaxation of the ferredoxin-type cluster S-1, indicating the presence of the cluster S-2 . Iron-sulfur cluster S-1 was found to be assembled in soluble succinate dehydrogenase subunits in the cytoplasm, but only if full-length Fp polypeptides and relatively large fragments of Ip polypeptides were present . Cluster S-1 was not detected in mutants with soluble mutated Fp polypeptides or in a mutant totally lacking Ip subunit polypeptide . Iron-sulfur clusters S-1, S-2, and S-3 were assembled also when the covalently bound flavin in the Fp subunit was absent . Clusters S-1 and S-3 in the membrane-bound flavin-deficient succinate dehydrogenase were not reduced by succinate but could be reduced by electron transfer from NADH dehydrogenase via the menaquinone pool. EMBO J, 1985 May, 4(5), 1345 - 9 Site-specific DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein; Greene JR et al.; The bacteriophage SP01 genome encodes a virus-specific type II DNA-binding protein, TF1 . The bacterial proteins of this ubiquitous and evolutionarily conserved class are thought to bind non-specifically to DNA . In contrast, the experiments described here demonstrate that TF1 binds to specific sites in SP01 DNA . Several of these sites have been characterized by DNase I 'footprinting' and four of them have been shown to overlap strong phage promoters for Bacillus subtilis RNA polymerase holoenzyme . We speculate on the possible structural basis of site-selective DNA binding by a protein of this class. Virology, 1985 May, 143(1), 16 - 22 Bacteriophage SPO1 DNA polymerase and the activity of viral gene 31; De Antoni GL et al.; Bacteriophage SPO1 DNA-negative (D0) mutants were tested for the induction of viral DNA polymerase during Bacillus subtilis infection . Extracts from SPO1-infected bacteria exhibited enzymatic activity when representative mutants of seven out of the nine known D0 genes were employed . This activity was undetectable in cells infected with mutants in genes 28 and 31 . The product of gene 28 (gp28) is known to be responsible for turning on SPO1 middle gene expression . Results show that nonsense mutation in gene 31 leads to the absence of a single polypeptide of 100-105 kDa and that phage DNA synthesis "in vivo" directly depends on gp31 activity . Based on these data it is proposed that SPO1 gene 31 codes for the viral DNA polymerase. J Gen Microbiol, 1985 May, 131 ( Pt 5), 1091 - 105 Nucleotide sequence and complementation analysis of a polycistronic sporulation operon, spoVA, in Bacillus subtilis; Fort P et al.; We have determined the nucleotide sequence of a 3706 bp stretch of Bacillus subtilis chromosomal DNA that complements all known spoVA mutations . The sequence contains five consecutive large open reading frames capable of encoding proteins of molecular weights ranging from approximately 15000 to 36000 . Analysis using integrational plasmids suggests that the region is likely to be transcribed as a single mRNA . A novel form of complementation analysis, based on derivatives of bacteriophage phi 105 carrying the cloned spoVA locus, has been used to define four distinct complementation groups among the eight previously characterized spoVA mutations . The spoVA locus is the largest polycistronic sporulation operon yet characterized. EMBO J, 1985 May, 4(5), 1333 - 8 Stabilized non-complementing diploids (Ncd) from fused protoplast products of B . subtilis; Guillen N et al.; Non-complementing diploids (Ncd) displaying the parental phenotype can be selected from polyethylene glycol (PEG)-treated fused polyauxotrophic protoplasts of Bacillus subtilis . These bacteria carry the two parental genomes, but only one of them is phenotypically expressed, the other being replicated but not expressed . Cellular cloning and DNA-DNA in situ hybridization led to the discovery of non-complementing diploid cells which at first sight could have been considered as parental haploids . The new class of stabilized Ncd (10(-7) segregants) can be obtained either directly after the primary fusion event or from segregating Ncd after further growth . The totally inactive chromosome of a stable Ncd can be activated after PEG-induced self fusion . DNA-mediated transformation studies using crude stable Ncd lysates as DNA donors show low frequencies for the genetic markers from the 'silent' chromosome . Contrary to the unstable Ncd situation, however, these frequencies remain low even with purified donor DNA . The differences in the transformation properties of the non-expressed markers are correlated to Ncd clone stability . These facts suggest that chromosome inactivation in PEG-induced fusion involves at least a two-stage process . The first would be reversible and the second irreversible, thus preserving the inactive chromosome state. Appl Environ Microbiol, 1985 May, 49(5), 1084 - 9 Molecular cloning of Bacillus sphaericus penicillin V amidase gene and its expression in Escherichia coli and Bacillus subtilis; Olsson A et al.; The Bacillus sphaericus gene coding for penicillin V amidase, which catalyzes the hydrolysis of penicillin V to yield 6-aminopenicillanic acid and phenoxyacetic acid, has been isolated by molecular cloning in Escherichia coli . The gene is contained within a 2.2-kilobase HindIII-PstI fragment and is expressed when transferred into E . coli and Bacillus subtilis . The expression in B . subtilis carrying the recombinant plasmid is approximately two times higher than in the original B . sphaericus strain . A comparison of the purified enzyme from B . sphaericus and the expressed gene product in E . coli minicells suggests that the native enzyme consists of four identical subunits, each with a molecular weight of 35,000. J Clin Microbiol, 1985 May, 21(5), 826 - 9 Simplified procedure for producing Bacillus subtilis spores for the Guthrie phenylketonuria and other microbiological screening tests; Jinks DC et al.; Bacillus subtilis ATCC 6051 and ATCC 6633 spores used in bacterial inhibition screening assays for genetic metabolism defects in newborn infants were produced by using liquid synthetic replacement sporulation media . These media allowed a high degree of sporulation, as judged by direct cell counts . Sporulation took place within 23 to 27 h with these media . Also, a more rational procedure for selecting the most sensitive clones of these organisms to the various inhibitors used in the microbiological screening assays is presented. J Bacteriol, 1985 May, 162(2), 521 - 8 Secretion of staphylococcal nuclease by Bacillus subtilis; Kovacevic S et al.; The staphylococcal nuclease (nuc) gene from Staphylococcus aureus has been cloned and expressed in Bacillus subtilis . The nuclease protein was expressed either from its own promoter and translation start signals, or from a combination of a B . subtilis promoter, ribosome binding site, and a signal peptide sequence . Greater than 80% of the active gene product was secreted into the medium, whereas, when a signal peptide sequence was absent, as little as 4% of the nuclease activity was found in the culture medium . Intracellular (or cell-bound) nuclease, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, was shown to have the molecular weight of the predicted precursor protein with the signal peptide . Levels of nuclease reached 50 mg per liter in the culture medium, depending on the growth medium and the strain used . These findings indicate the prospective use of nuclease as a model system for studying secretion of heterologous proteins in B . subtilis. Proc Natl Acad Sci U S A, 1985 May, 82(9), 2647 - 51 Characterization of the spo0A locus and its deduced product; Ferrari FA et al.; The highly pleiotropic stage 0 sporulation locus of Bacillus subtilis, spo0A, has been cloned in bacteriophage lambda, subcloned in plasmids, and sequenced . The locus was found to code for a protein of 29,691 Da . Analysis of the in vivo transcripts from this region by nuclease S1 protection experiments located the start and stop of transcription of the locus . The transcription start site was preceded by a promoter resembling sigma 37-dependent promoters . Two mutations originally assigned to a second locus, spo0C, in this region because of their weakly pleiotropic phenotypes were cloned and sequenced . The mutations were found to be different missense alterations in the same base of the 10th codon preceding the carboxyl end of the Spo0A protein . These results, along with the finding that mutations in the spo0A gene product {Hoch, J . A., Trach, K., Kawamura, F . & Saito, H . (1985) J . Bacteriol . 161, 552-555} suppress the requirement for spo0B, spo0E, and spo0F gene products in transcription from sigma 28-dependent promoters, suggest that the Spo0A protein interacts directly with the transcription machinery to effect the initiation of sporulation . The deduced amino acid sequence of the Spo0A protein was highly related to that of the OmpR regulatory protein of Escherichia coli. J Gen Microbiol, 1985 May, 131 ( Pt 5), 1259 - 62 The genome of Bacillus subtilis phage SPP1: structure of an early promoter; Tailor R et al.; The strongest of five 'early' promoters of Bacillus subtilis phage SPP1 was localized in a DNA restriction fragment by analysis of RNA polymerase binding and R-loop formation . The nucleotide sequence of the promoter region was established . The signal structures identified were similar to those recognized by the sigma 55 RNA polymerase of B . subtilis . The promoter precedes an open reading frame with 51 codons . A protein with the Mr predicted from the nucleotide sequence was identified in minicells. J Bacteriol, 1985 May, 162(2), 756 - 62 Identification of three complementation units in the gerA spore germination locus of Bacillus subtilis; Zuberi AR et al.; The gerA locus, mutations in which affect the germination response of spores to L-alanine and related amino acids, is contained within a 6-kilobase region of DNA cloned in phage and plasmid vectors . Fragments from this region, subcloned in the shuttle vector pHV33, were introduced into Bacillus subtilis, and their ability to complement chromosomal gerA mutations in a recE4 background was examined . Although the plasmids were somewhat unstable, it was possible to score complementation within spore-containing colonies on nutrient agar by their ability to reduce 2,3,5-triphenyltetrazolium chloride in an overlay . These studies have assigned the 10 gerA mutations tested to three complementation groups . An analysis of Tn1000 insertions into the cloned DNA of two relatively stable plasmids that together encompass the entire gerA region has identified more precisely the location and extent of the complementation units; recombination studies and in vitro mutagenesis were used to further delineate the extents of two of the units . The evidence suggests that the three complementation units are adjacent and that they are probably capable of separate transcription. Can J Microbiol, 1985 May, 31(5), 429 - 35 Suppressor mutations for crs mutants of Bacillus subtilis; Sun D et al.; Mutants of Bacillus subtilis which carried suppressor mutations for catabolite-resistance gene crsA47 were isolated from methylmethanesulfonate-treated cultures of GLU-47 (crsA47) . The suppressor mutation, sca19, suppressed resistance of crsA47 mutant to glucose and other inhibitors of sporulation . Moreover, the suppressor mutation could restore the rate of growth and the level of IMP dehydrogenase and alkaline phosphatase of crsA47 mutant to the wild-type level . The scal19 mutation was also able to suppress catabolite resistance of other crs mutants . The map position of the sca19 mutation indicated that this mutation was an intergenic suppressor for the crs mutants . It was also found that an erythromycin-resistance mutation, eryl, could suppress the catabolite resistance of some of the crs mutants . Our results were discussed in relation to the importance of a proper state of metabolic activities and membrane functions during the initiation of sporulation. Proc Natl Acad Sci U S A, 1985 May, 82(10), 3375 - 9 Regulation of expression from the glnA promoter of Bacillus subtilis requires the glnA gene product; Schreier HJ et al.; Expression of the cloned glnA gene {coding for glutamine synthetase (EC 6.3.1.2)} of Bacillus subtilis was 10-fold higher in an Escherichia coli strain grown under nitrogen-limiting conditions than in the same strain under nitrogen-excess conditions . Mutations in the E . coli glnA, glnB, glnD, glnE, glnF, glnG, and glnL genes had no effect on the observed regulation . To test whether sequences within the B . subtilis DNA (3.2 kilobase pairs) were responsible for the observed regulation, a plasmid carrying a transcriptional fusion of the B . subtilis glnA promoter with E . coli lacZ was constructed . beta-Galactosidase levels coded for by this plasmid were found to be negatively regulated in trans by a plasmid carrying the entire B . subtilis glnA gene . Analysis of various deletion plasmids showed that the 1.4-kilobase-pair region encoding glutamine synthetase was necessary for the observed regulation of beta-galactosidase . Plasmids coding for 67% or more of the glutamine synthetase polypeptide gave at least partial repression, but a plasmid carrying 30% of the structural gene, as well as a plasmid carrying a deletion internal to glnA, gave no repression . DNA downstream from glnA (to within 130 base pairs of the end of the gene) was not required for the observed regulation . These results suggest that the glnA gene of B . subtilis is autoregulated, supporting the model for glnA control proposed by Dean et al . {Dean, D . R., Hoch, J . A . & Aronson, A . I . (1977) J . Bacteriol . 131, 981-987}. Biochem Biophys Res Commun, 1985 Apr 30, 128(2), 601 - 6 Stable hyper-production of Escherichia coli beta-lactamase by Bacillus subtilis grown on a 0.5 M succinate-medium using a B . subtilis alpha-amylase secretion vector; Nakamura K et al.; Extracellular production of Escherichia coli beta-lactamase by Bacillus subtilis, using a B . subtilis secretion vector constructed from its own alpha-amylase gene, was promoted when the cells were grown on LG-medium containing 0.5 M succinate under poor aeration conditions . The amount of the enzyme secreted was 50 to 60 times as large as that obtained by cultivation of the cells on LG-medium under good aeration conditions . The effect of protease-deficient mutant of B . subtilis on the enzyme production by B . subtilis was significant while that of protease inhibitors was negligible or inexistent. Virology, 1985 Apr 15, 142(1), 78 - 97 Cloning and mapping of the SPO1 genome; Curran JF et al.; Many of the XbaI, EcoRI, KpnI, and BglII fragments of bacteriophage SPO1, accounting for about 65% of the genomic sequences, were cloned in Bacillus subtilis . Four of the EcoRI fragments were specifically refractory to cloning in both Escherichia coli and B . subtilis, probably because of expression of deleterious genes carried on the SPO1 fragments . To permit complete identification of the regions cloned, the SPO1 restriction map has been extended to include the XbaI fragments and the previously unmapped KpnI fragments . Markers for 26 of the 39 known genes have been located on specific cloned fragments, permitting more precise determination of the positions of most of the genes . One cloned SPO1 fragment was inhibitory to SPO1 development. Nucleic Acids Res, 1985 Apr 11, 13(7), 2267 - 79 Structure and function of the region of the replication origin of the Bacillus subtilis chromosome . IV . Transcription of the oriC region and expression of DNA gyrase genes and other open reading frames; Ogasawara N et al.; We have determined nucleotide sequence of some 10,000 base pairs (bp) in the oriC region of the Bacillus subtilis chromosome . Initiation sites of transcription from this region were determined in vivo by the S1-mapping method . Five major initiation sites were found in the leader sequences of five open reading frames (ORF) deduced from the nucleotide sequence . The sixth site is located inside the ORF323("recF") . Putative promoters were found for each transcript . Function of these promoters was demonstrated in Escherichia coli by the Maxi-cell method using appropriate fragments cloned in pBR vectors . Based on these results, genes in 10,000 bp oriC region are divided into 4 transcriptional units . GyrB composes one unit with two other ORFs, while gyrA constitutes a single unit by itself . The promoters for ORF446("dnaA") and ORF378("dnaN") are located within the putative signal sequences for oriC . Transcription from these promoters is dependent on a dna-initiation gene, dnaB. Nucleic Acids Res, 1985 Apr 11, 13(7), 2251 - 65 Structure and function of the region of the replication origin of the Bacillus subtilis chromosome . III . Nucleotide sequence of some 10,000 base pairs in the origin region; Moriya S et al.; Approximately 10,000 nucleotides were sequenced in the oriC region of the Bacillus subtilis chromosome . The first replicating DNA strands are hybridized with a SalI-EcoRI fragment (nucleotide #1206-2954) in one direction (left to right) and an EcoRI-PstI fragment (#2949-4233) in the other . Seven open reading frames (ORF) accompanied with Shine-Dalgarno (SD) sequences were identified . ORF638 and ORF821 were identified as gyrB and gyrA genes respectively based on genetic evidences and amino acid sequence data . Comparison of amino acid sequences revealed that ORF44, ORF446, ORF378 and ORF323 are homologous with rpmH, dnaA, dnaN and recF of Escherichia coli, respectively . Thus, the organization of the ORFs from ORF44 to ORF638 resembles the organization of genes in the rpmH-gyrB region of the E . coli chromosome . Two non-coding regions characteristic for oriC signals were found near the site of initiation of the first replicating DNA . They are composed of repeating sequences whose consensus sequence TTAT(C/A)CACA is identical to that of 4 repeating sequences in the oriC of E . coli. Photodermatol, 1985 Apr, 2(2), 101 - 6 Screening for drug photosensitization activity by measuring the variations in oxygen consumption of Bacillus subtilis; Beani JC et al.; An original method is described for detecting the photosensitizing ability of a compound . The principle of this method is based on the analysis of variations in the consumption of oxygen by Bacillus subtilis (measured with Warburg's apparatus or an oxygenometric cell), induced by irradiation of the test compound added to the bacterial culture medium . This methodology was applied to 7 substances: 5 known photosensitizers (8-MOP, chlorpromazine, 5-fluorouracil, Vitamin A acid and benzoyl peroxide) and 2 products without any photoactive properties (aspirin and erythromycin) . The comparison of results obtained with the method of photo-patch tests and the analysis of the photophysical properties of the compounds confirm the reliability, reproducibility and the quantitative nature of this method. Biol Chem Hoppe Seyler, 1985 Apr, 366(4), 421 - 30 Determination of the complete amino-acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisins BPN', Carlsberg and amylosacchariticus; Nedkov P et al.; The complete amino-acid sequence of subtilisin DY, an extracellular alkaline proteinase produced by Bacillus subtilis strain DY was determined . This included automated sequence analysis of the whole molecule and its large fragments such as tryptic peptides obtained from the inactivated enzyme, peptides generated by cyanogen bromide, by o-iodosobenzoic acid and by hydroxylamine . The peptides were isolated by gel filtration and by reversed-phase high performance liquid chromatography . The amino-acid sequence of subtilisin DY was determined by overlapping the isolated peptides . It consists of 274 amino-acid residues, like that of subtilisin Carlsberg . By comparison with the structures of the subtilisins Carlsberg, amylosacchariticus and BPN' 32, 80 and 82 amino-acid substitutions were found, which are caused by 37, 102 and 106 nucleotide mutations, respectively . It was found also that 62.5% of the amino-acid residues in the molecules of these four subtilisins are identical with respect to kind and position of the residue, which suggests that these molecules have had a common ancestral precursor . The amino-acid replacement analysis of the four subtilisins leads to the conclusion that they have evolved almost independently. Biochem Int, 1985 Apr, 10(4), 663 - 71 The nucleotide sequence of proline tRNAmo5UGG from Bacillus subtilis; Hasegawa T et al.; The nucleotide sequence of a proline tRNA from Bacillus subtilis W168 was determined to be pC-G-G-G-A-A-G-U-A-G-C-U-C-A-G- C-U-U-G-G-D-A-G-A-G-C-A-C-A-U-G-G-psi-U-mo5U-G-G-m1G-A-C-C-A-U-G-G -G-m7G-U-C-G-C-A-G-G-T-psi-C-G-A-A-U-C-C-U-G-U-C-U-U-C-C-C-G-A-C-C- AOH, by the analysis of the unlabeled preparation and by post-labeling technique . This tRNAPro contained 5-methoxyuridine (mo5U) which is specifically distributed in bacillaceae at the wobble position of the anticodon. Can J Microbiol, 1985 Apr, 31(4), 367 - 70 Bacteristatic activity of phenanthrolines against Escherichia coli and Bacillus subtilis; Sharrock P; Using optical turbidimetry to measure the growth of Escherichia coli and Bacillus subtilis, we determined the mean lethal dose (LD50) values for various phenanthrolines . The dimethyl-substituted compounds are found to be more toxic to bacteria, with doses near 5 micrograms/mL reducing the number of viable cells by 50% over a 24-h period . 2,9-Dimethyl phenanthroline is the most potent compound against B . subtilis, being six times more effective than against E . coli . Bipyridine is the least toxic substance and is twice as effective against E . coli as it is against B . subtilis . Evidence is presented to show copper ions enhance the antibacterial action of phenanthrolines and may be required for activity. J Gen Microbiol, 1985 Apr, 131 ( Pt 4), 959 - 62 Effects of transition mutations in the regulatory locus spoIIA on the incidence of sporulation in Bacillus subtilis; Yudkin MD et al.; We have determined the changes in DNA sequence corresponding to three mutations in the promoter-proximal open reading frame of spoIIA, a locus that regulates sporulation in Bacillus subtilis . All three mutations prevent the synthesis of two sporulation-associated enzymes, but they differ in their effects on spore incidence . We now find that mutation spo-42, which allows spores to be produced at a low incidence, is a transition that changes Gly95 to Asp in the protein encoded by the open reading frame . Mutation spo-69, which blocks sporulation entirely, consists of two transitions: these change Gly62 to Asp and Ala1 16 to Thr . Mutation sas-1, which partially suppresses spo-69, is also a transition: this changes residue 62 (which had become Asp as a result of the spo-69 mutation) to Asn. Proc Natl Acad Sci U S A, 1985 Apr, 82(7), 2163 - 7 Biomechanics of bacterial walls: studies of bacterial thread made from Bacillus subtilis; Thwaites JJ et al.; Bacterial threads of up to 1 m in length have been produced from filaments of separation-suppressed mutants of Bacillus subtilis . Individual threads may contain 20,000 cellular filaments in parallel alignment . The tensile properties of bacterial threads have been examined by using conventional textile engineering techniques . The kinetics of elongation at constant load are indicative of a viscoelastic material . Both Young's modulus and breaking stress are highly dependent upon relative humidity . By extrapolation to 100% relative humidity, it appears that cell walls may be able to bear only internal osmotic pressures of about 2 atmospheres (2.03 X 105(5) Pa) in living cells . Similarly, the strength of wall material limits the amount of cell-surface charge permissible to only a small fraction of that known to be carried by the negatively charged wall polymers. J Bacteriol, 1985 Apr, 162(1), 176 - 82 Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector; Ulmanen I et al.; Expression levels of Bacillus amyloliquefaciens alpha-amylase, Escherichia coli TEM-beta-lactamase, and Semliki Forest virus glycoprotein E1 genes were compared in Bacillus subtilis . All three model genes were expressed by using a secretion vector, constructed by joining the B . amyloliquefaciens alpha-amylase promoter and signal sequence with plasmid pUB110 (I . Palva, M . Sarvas, P . Lehtovaara, M . Sibakov, and L.Kaariainen, Proc . Natl . Acad . Sci . U.S.A . 79:5582-5586, 1982) . When transformed B . subtilis cells were grown to early stationary phase, the amount of beta-lactamase in the culture medium was ca . 10% and that of E1 was ca . 0.01% of the amount of alpha-amylase . The amounts of specific, full-length transcripts of the cloned genes were estimated by Northern blot hybridization to be roughly equal . The half-lives of these transcripts in B . subtilis were also similar . Pulse-chase experiments with {35S}methionine showed that alpha-amylase and beta-lactamase were translated and secreted at comparable rates but that beta-lactamase was degraded during the chase periods . In transformed minicells from B . subtilis, the products of alpha-amylase, beta-lactamase, and E1 genes accumulated at similar rates . We conclude that the expression of the three genes cloned in the secretion vector was similar at the levels of transcription and translation in B . subtilis . In the case of beta-lactamase, the low-yield could be explained by proteolytic degradation of the secreted product by B . subtilis exoproteases, whereas with E1 we could not determine whether the low yield was due to proteolytic degradation, inefficient secretion, or both. J Bacteriol, 1985 Apr, 162(1), 78 - 84 Genetic and physical organization of the cloned gyrA and gyrB genes of Bacillus subtilis; Lampe MF et al.; An 8-kilobase fragment already known to contain the gyrA gene of Bacillus subtilis was shown to encode the gyrB gene as well . Plasmids containing this fragment can rescue both B . subtilis gyrA and gyrB mutants and complement Escherichia coli gyrA mutants . Deletion analysis has indicated the gene locations on the cloned fragment . Under low-stringency conditions the cloned E . coli gyrA and gyrB genes each hybridized to the appropriate subfragments, confirming the assignment of the gene locations on the cloned DNA . In E . coli maxicells, proteins of 67,000 (gyrA) and 77,000 (gyrB) Mr were synthesized . Analysis of proteins encoded by various subfragments indicated the direction of transcription . Although the gyrA and gyrB genes are located adjacent to each other on the chromosome, they may be transcribed independently since expression of gyrA protein is not dependent upon the gyrB gene in maxicells. J Bacteriol, 1985 Apr, 162(1), 42 - 6 Involvement of autolysin in cellular lysis of Bacillus subtilis induced by short- and medium-chain fatty acids; Tsuchido T et al.; The addition of saturated C6, C8, C10, and C12 fatty acids appeared to lyse actively growing cells of Bacillus subtilis 168, as judged by a decrease in the optical density of the culture . Of these fatty acids, dodecanoic acid was the most effective, with 50% lysis occurring in about 30 min at a concentration of 0.5 mM . These conditions also decreased the amount of peptidoglycan estimated by the incorporated radioactivity of N-acetyl-D-{1-14C}glucosamine . At concentrations above 1 mM, however, bacterial lysis was not extensive . Dodecanoic acid did not affect autolysis of the cell wall . The lytic action of dodecanoic acid was greatly diminished in cells in which protein synthesis was inhibited and in an autolytic enzyme-deficient mutant . The results suggest that fatty acid-induced lysis of B . subtilis 168 is due to the induction of autolysis by an autolytic enzyme rather than massive solubilization of the cell membrane by the detergent-like action of the fatty acids. Biochem Biophys Res Commun, 1985 Mar 29, 127(3), 713 - 9 Bacillus subtilis contains multiple forms of somatostatin-like material; LeRoith D et al.; Extracts of B . subtilis contain somatostatin-like immunoactivity (1-20 pg per g wet weight cells) . Two major forms were detected, one with reactivity in both N- and C-terminal immunoassays similar to somatostatin-28 and a second form reactive only in the C-terminal specific immunoassay similar to somatostatin-14 . Both forms were active in a bioassay and the bioactivity was neutralized in the presence of antibody to the central, biologically active part of somatostatin-14 . Preconditioned medium contained no detectable somatostatin whereas conditioned medium had 80-380 pg per liter. J Biol Chem, 1985 Mar 25, 260(6), 3368 - 72 Bacillus subtilis dnaE encodes a protein homologous to DNA primase of Escherichia coli; Wang LF et al.; Bacillus subtilis dnaE encodes a protein essential for DNA replication and is tightly linked to rpoD, the gene for the major sigma factor of RNA polymerase . We have now determined the 1809-base pair sequence of the dnaE coding region, which precedes rpoD and is transcribed in the same counterclockwise direction on the chromosome . From the DNA sequence, we found that the dnaE protein comprised 603 amino acids with a calculated molecular mass of 68,428 daltons . This protein had significant and extensive regions of homology with Escherichia coli DNA primase, the polymerase that synthesizes short RNA primers during discontinuous DNA replication . Features of the coding and flanking regions that may modulate dnaE expression include a relatively weak ribosomal binding site (delta G' = -13.8 kcal), the use of uncommon codons in the reading frame, and no obvious promoter sequence for either dnaE or rpoD . Together, these results suggest that dnaE codes for B . subtilis DNA primase and, in light of the similarities to the organization of the E . coli sigma operon, that expression of dnaE may be coregulated with rpoD in B . subtilis. J Biol Chem, 1985 Mar 25, 260(6), 3305 - 13 Purification of a RecA protein analogue from Bacillus subtilis; Lovett CM Jr et al.; We have identified in Bacillus subtilis an analogue of the Escherichia coli RecA protein . Its activities suggest that it has a corresponding role in general genetic recombination and in regulation of SOS (DNA repair) functions . The B . subtilis protein (B . subtilis Rec) has a Mr of 42,000 and cross-reacts with antisera raised against E . coli RecA protein . Its level is significantly reduced in the recombination-deficient recE4 mutant . B . subtilis Rec is induced 10- to 20-fold in rec+ strains following treatment with mitomycin C, whereas it is not induced in the recombination-deficient mutants recE4, recE45, and recA1 . We have purified B . subtilis Rec about 2000-fold to near homogeneity and we describe its activities . It catalyzes DNA-dependent hydrolysis of dATP at a rate comparable to that of E . coli RecA protein . However, B . subtilis Rec has a negligible ATPase activity, although ATP effectively inhibits dATP hydrolysis . In the presence of dATP, B . subtilis Rec catalyzes DNA strand transfer, assayed by the conversion of phi X174 linear duplex DNA and homologous circular single-stranded DNA to replicative form II (circular double-stranded DNA with a discontinuity in one strand) . ATP does not support strand transfer by this protein . B . subtilis Rec catalyzes proteolytic cleavage of E . coli LexA repressor in a reaction that requires single-stranded DNA and nucleoside triphosphate . This result suggests that an SOS regulatory system like the E . coli system is present in B . subtilis . The B . subtilis enzyme does not promote any detectable cleavage of the E . coli bacteriophage lambda repressor. Nature, 1985 Mar 14-20, 314(6007), 190 - 2 Utilization of one promoter by two forms of RNA polymerase from Bacillus subtilis; Tatti KM et al.; Bacillus subtilis possesses several forms of RNA polymerase, each differing in its sigma subunit and its specificity of promoter recognition . The sequential appearance of sigma subunits, which change the promoter recognition specificity of RNA polymerase, may have a key role in controlling the temporal pattern of gene expression required for endospore development in B . subtilis . Several genes that are expressed over relatively long periods of time during the developmental cycle are transcribed by more than one form of RNA polymerase, which initiate transcription from either tandem or overlapping promoter . The promoter region for the ctc gene is interesting because transcription is initiated at or near the same position by both sigma 37 RNA polymerase (E sigma 37), a minor form in growing cells, and sigma 29 RNA polymerase (E sigma 29), a form which appears approximately 2 h after the initiation of sproulation . Here we report that several base substitutions in the ctc promoter differentially affect the utilization of the promoter by E sigma 37 or E sigma 29. Tsitol Genet, 1985 Mar-Apr, 19(2), 141 - 4 {Biological activity of thiophosphamide-alkylated DNA in the transformation system of Bacillus subtilis}; Kitam OE et al.; DNA treated with thiophosphamide was studied for changes in its biological activity in transformation system of Bac . subtilis . DNA alkylation by this three-functional alkylating agent is shown to be followed by the reduction of transforming