Nauchnye Doki Vyss Shkoly Biol Nauki, 1990, (2), 113 - 9 {The surface membrane charge of bacteria and its role in serine proteinase secretion by Bacillus subtilis cells}; Artemov AV et al.; Univalent, bivalent and trivalent metal cations increase the fluorescence yield of 9-aminoacridine in the suspensions of chromatophores of the purple nonsulfur bacterium Rhodospirillum rubrum isolated thylakoid membranes and cells of cyanobacterium Anabaena variabilis, cells Bacillus subtilis . The active cation concentrations increase about in 10 times with the decrease of their valency by one . It points to the fact that the changes in 9-aminoacridine fluorescence serve for the monitoring of the surface charge of bacterial membranes . The negative surface charge of B . subtilis cells increases before the onset of the serine protease secretion . The metal cations stimulate the serine protease secretion by B . subtilis cells, the stimulating effect correlates with the action of cations on the 9-aminoacridine fluorescence yield . It is suggested that the surface charge of cytoplasmic membrane regulates the formation and release of serine protease by the cells of B . subtilis. Mol Biol (Mosk), 1990 Jan-Feb, 24(1), 256 - 61 {Unusual structure of the regulatory region of the riboflavin biosynthesis operon in Bacillus subtilis}; Mironov VN et al.; In vitro mutagenesis with methylhydroxylamine and nitrosomethylurea was used to obtain a number of Bacillus subtilis mutants impaired in flavin-dependent response . Mutants displayed varying degree of flavin-dependent repression of riboflavin synthase and of 6,7-dimethyl-8-ribityl-lumasine accumulation . Single nucleotide substitutions were detected by DNA sequencing in all of the mutants, affecting the 48 b.p . target area between the mRNA start and the AUG of the first gene. Ophthalmic Res, 1990, 22(2), 123 - 7 Changes in the antibacterial activity of melanin-bound drugs; Fukuda M et al.; Affinity to melanin and changes of antibacterial activity of melanin-bound drugs were examined in 11 drugs, all of which showed an affinity for melanin . The highest melanin-binding ratio was seen in aminoglycosides . Among these, sisomicin sulfate (SISO) had the highest binding ratio (95.5%) . The melanin-binding ratios seen in sulbenicillin sodium (SBPC), cephalosporins and fluoroquinolones were relatively low during the early phase of the reaction, but increased with time . The highest ratio seen in cefazolin sodium was 60.1% . Melanin-bound aminoglycosides showed a reduction in their antibacterial activity with both Bacillus subtilis and Escherichia coli . The reduction rate in the antibacterial activity of melanin-bound SISO against B . subtilis was 50.0% and that against E . coli was 43.0% . No changes in antibacterial activity were seen in the other 6 drugs bound to melanin. Arch Microbiol, 1990, 153(4), 355 - 9 The Bacillus subtilis menCD promoter is responsive to extracellular pH; Hill KF et al.; Activation kinetics of a Bacillus subtilis menaquinone biosynthetic gene promoter (the menCD promoter) were measured during growth and sporulation, with the aid of a menCD'-lacZ translational gene fusion . Transient maximal activation was seen shortly after the end of exponential growth in unbuffered complex medium containing a low glucose concentration . These activation kinetics were correlated with transient acidification of the medium under conditions permitting TCA cycle function during the post-exponential period, while mutations that blocked TCA cycle function (cit mutants) were associated with sustained acidification and promoter activation during this period . In cit+ strains, buffering of the medium to pH 5.7 caused sustained maximal activation, while buffering to pH 7.2 prevented enhancement of activation . The menCD promoter appears to be responsive to extracellular acidic pH. Microbios, 1990, 62(250), 29 - 35 Genetic transformation and cell morphology of Bacillus subtilis grown in Mg+(+)-limited chemostat culture; Sevinc MS et al.; The rate and frequency of genetic transformation of Bacillus subtilis grown in Mg+(+)-limited chemostat culture are dependent on the dilution rate (D) of the system and achieved maximum values at D = 0.23 h-1 . Mg+(+)-limitation induced a morphological change in the cells from their normal rod shape to extended helices . Although this change in shape was a transient phenomenon, under some conditions it persisted for several days and resulted in an apparent increase in the transformation frequency. Arch Microbiol, 1990, 153(3), 276 - 81 Rhizocticin A, an antifungal phosphono-oligopeptide of Bacillus subtilis ATCC 6633: biological properties; Kugler M et al.; Rhizocticin A, the main component of the antifungal, hydrophilic phosphono-oligopeptides of Bacillus subtilis ATCC 6633, was used for sensitivity testing and experiments into the molecular mechanism of the antibiotic action . Budding and filamentous fungi as well as the cultivated nematode Caenorhabditis elegans were found to be sensitive, whereas bacteria and the protozoon Paramecium caudatum were insensitive . Rhizoctonia solani was inhibited in agar dilution tests but not in diffusion tests . The antifungal effect of rhizocticin A was neutralized by a variety of amino acids and oligopeptides . Oligopeptide influence was mainly understood as transport antagonism, and it was concluded that the antibiotic enters the recipient cell via the peptide transport system . L- and D-cystine were also identified as potent, general antagonists of the oligopeptide transport . The rhizocticin-antagonism of four other amino acids was taken as a clue to the site of action . Provided that rhizocticin A is split by peptidases of the target cell into inactive L-arginine and toxic L-2-amino-5-phosphono-3-cis-pentenoic acid (L-APPA), the latter may interfere with the threonine or threonine-related metabolism. FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 271 - 6 Uptake of L-{14C}-alanine by glutaraldehyde-treated and untreated spores of Bacillus subtilis; Power EG et al.; The effect of glutaraldehyde on the uptake of L-alanine, and subsequent germination, in spores of Bacillus subtilis NCTC 8236 was examined . Germination was induced by single amino acids, D-glucose and phosphate buffer at 37 degrees C . L-alanine was the best germinant of all amino acids tested . Pretreatment of spores with low concentrations of acid and alkaline glutaraldehyde inhibited subsequent germination, complete inhibition being observed at concentrations of 0.1% (w/v) . This concentration also prevented the loss of heat resistance of spores placed in germination medium and exposed to 75 degrees C . Radioactive studies indicated that maximum uptake of L-alanine occurred after ca 30 min at 37 degrees C . Only 1.2% of available L-alanine was taken up during germination . Pretreatment of spores with glutaraldehyde did not interfere with L-alanine uptake at aldehyde concentrations up to 0.5% (w/v) . However, this was significantly reduced at a glutaraldehyde concentration of 1.0% (w/v) . Minimal differences were observed between acid and alkaline forms of the aldehyde . The results are discussed in terms of the mode of action of glutaraldehyde. Mutat Res, 1990 Jan, 235(1), 15 - 23 Isolation of a Bacillus subtilis mutant defective in constitutive O6-alkylguanine-DNA alkyltransferase; Morohoshi F et al.; A mutant of Bacillus subtilis defective in the constitutive activity of O6-alkylguanine-DNA alkyltransferase was isolated from a strain (ada-1) deficient in the adaptive response to DNA alkylation . Cells carrying the mutation dat-1 which was responsible for the defect in constitutive activity exhibited hypersensitivity for lethality and mutagenesis when challenged with methyl-nitroso compounds . The constitutive activity is independent of the adaptive response, and seems to function as a basal defense against environmental alkylating agents. J Bacteriol, 1990 Jan, 172(1), 86 - 93 The spoIIJ gene, which regulates early developmental steps in Bacillus subtilis, belongs to a class of environmentally responsive genes; Antoniewski C et al.; The Bacillus subtilis spoIIJ locus is defined by a Tn917 insertion which leads to an oligosporogenous phenotype . Here we show that this mutation severely decreases transcription of spoIIA, spoIIE, and spoIIG, three operons involved in asymmetric septation, the earliest morphological event of sporulation . A 14.3-kilobase region overlapping the site of the spoIIJ::Tn917 insertion was cloned and the exact location of the spoIIJ gene was defined with various integrative plasmids carrying subfragments of that region . DNA sequencing established that spoIIJ is a monocistronic locus encoding a 606-amino-acid polypeptide which contains a canonical "transmitter" domain, indicating that spoIIJ is a new member of the "sensor" class of signal-transducing systems in bacteria . Thus, spoIIj, which is transcribed during vegetative growth, presumably under the control of sigma H, encodes a protein that could interact with major regulators of early sporulation stages, such as SpoOA and/or SpoOF. J Bacteriol, 1990 Jan, 172(1), 80 - 5 Cloning and DNA sequence of the gene coding for the major sigma factor from Myxococcus xanthus; Inouye S; The gene for a sigma factor (rpoD) was cloned from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon starvation for nutrients . The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide of 708 amino acid residues (Mr = 80,391) was identified . Except for the amino-terminal sequence consisting of 100 residues, the M . xanthus sigma factor (sigma-80) showed extensive similarity with Escherichia coli sigma-70 as well as Bacillus subtilis sigma-43 . In particular, the carboxy-terminal sequence of 242 residues that is known to be required for promoter recognition and core recognition showed 78 and 72% amino acid sequence identity with the E . coli and B . subtilis sigma factors, respectively . The putative RpoD protein was detected at the position of an apparent molecular weight of 86,000 by Western blot (immunoblot) analysis by using antiserum against B . subtilis sigma-43, which agreed well with the position of a vegetative sigma factor of M . xanthus previously identified by Rudd and Zusman (K . Rudd and D . R . Zusman, J . Bacteriol . 151:89-105, 1982). J Bacteriol, 1990 Jan, 172(1), 7 - 14 Dramatic increase in negative superhelicity of plasmid DNA in the forespore compartment of sporulating cells of Bacillus subtilis; Nicholson WL et al.; Plasmid pUB110, isolated from vegetative cells of Bacillus subtilis, has an average of 34 negative supertwists (tau av = -34) . This value falls to -30 early in sporulation, and the plasmid in the mother cell compartment maintains a tau av of -30 . However, the plasmid within the developing forespore becomes much more negatively supercoiled, reaching a tau av of -47 in the dormant spore . This increased negative supercoiling in the forespore plasmid takes place in parallel with the synthesis of small, acid-soluble spore proteins, alpha and beta; and the plasmid from spores lacking small, acid-soluble proteins alpha and beta has a tau av of -40 . The large increase in negative supercoiling of spore plasmid was also observed with Bacillus megaterium and in B . subtilis containing a plasmid with an origin different from that of pUB110 . During spore germination plasmid pUB110 rapidly relaxed back to the tau av value characteristic of vegetative cells . It is possible that the observed changes in forespore plasmid topology are involved in modulating gene expression, DNA photochemistry, or both of these parameters in this compartment. J Bacteriol, 1990 Jan, 172(1), 488 - 90 Isolation and characterization of mutations in the gene encoding an endogenous Bacillus subtilis beta-galactosidase and its regulator; Errington J et al.; We have isolated mutations that appear to inactivate the gene (lacA) encoding an endogenous beta-galactosidase activity in Bacillus subtilis and in a closely linked negative regulatory element (lacR) . Both genes map to the hisA-thrA region . The lacA mutations may help to avoid some of the problems arising from the use of the Escherichia coli lacZ gene as a reporter gene in B . subtilis. J Bacteriol, 1990 Jan, 172(1), 473 - 6 Intracellular serine protease 1 of Bacillus subtilis is formed in vivo as an unprocessed, active protease in stationary cells; Sheehan SM et al.; Western immunoblots and assays of Bacillus subtilis extracts showed that intracellular serine protease 1 is produced in a form larger than previously reported, appears not to have undergone N-terminal processing, and is active in the presence or absence of calcium . No evidence for an inactive precursor form of the protease was found. J Bacteriol, 1990 Jan, 172(1), 411 - 8 Nucleotide sequence and cloning in Bacillus subtilis of the Bacillus stearothermophilus pleiotropic regulatory gene degT; Takagi M et al.; The regulatory gene (degT) from Bacillus stearothermophilus NCA1503 which enhanced production of extracellular alkaline protease (Apr) was cloned in Bacillus subtilis with pTB53 as a vector . When B . subtilis MT-2 (Npr- {deficiency of neutral protease} Apr+) was transformed with the recombinant plasmid, pDT145, the plasmid carrier produced about three times more alkaline protease than did the wild-type strain . In contrast, when B . subtilis DB104 (Npr- Apr-) was used as a host, the transformant with pDT145 could not exhibit any protease activity . After construction of the deletion plasmids, DNA sequencing was done . A large open reading frame was found, and nucleotide sequence analysis showed that the degT gene was composed of 1,116 bases (372 amino acid residues, molecular weight of 41,244) . A Shine-Dalgarno sequence was found nine bases upstream from the open reading frame . A B . subtilis strain carrying degT showed the following pleiotropic phenomena: (i) enhancement of production of extracellular enzymes such as alkaline protease and levansucrase, (ii) repression of autolysin activity, (iii) decrease of transformation efficiency for B . subtilis (competent cell procedure), (iv) altered control of sporulation, (v) loss of flagella, and (vi) abnormal cell division . When B . stearothermophilus SIC1 was transformed with the recombinant plasmid carrying degT, the transformants exhibited abnormal cell division . These phenomena are similar to those of the phenotypes of degSU(Hy) (hyperproduction), degQ(Hy), and degR mutants of B . subtilis . However, the amino acid sequence of the degT product (DegT) is different from those of the reported gene products . Furthermore, DegT includes a hydrophobic core region in the N-terminal portion (amino acid numbers 50 to 160), a consensus sequence for a DNA binding region (amino acid numbers 160 to 179), and a region homologous to transcription activator proteins (amino acid numbers 351 to 366) . We discuss the possibility that the membrane protein DegT functions as a sensor protein and transfers the signal of environmental stimuli to the regulatory region of target genes to activate or repress transcription of the genes. J Chem Technol Biotechnol, 1990, 48(3), 325 - 36 The effects of a biosurfactant on oxygen transfer in a cyclone column reactor; Sheppard JD et al.; A laboratory-scale cyclone column reactor was tested to determine how its oxygen transfer characteristics were affected by surfactants in the liquid medium . The volumetric oxygen transfer coefficient was greatly decreased by small quantities of the synthetic surfactants dodecyltrimethylammonium bromide and sodium dodecylsulfate, and the biosurfactant surfactin produced by Bacillus subtilis (ATCC 21332) . Since the gas holdup fraction was generally increased due to foaming, the effectiveness of the surfactants was probably due to an increase in the interfacial film resistance . B . subtilis was grown in the cyclone column to 0.6 g dm-3 with a significant level of surfactin produced while maintaining at least 75% oxygen saturation in the broth . Process optimization and scale-up of surfactin production will have to consider oxygen transfer as a key parameter. SAAS Bull Biochem Biotechnol, 1990 Jan, 3, 69 - 73 Illegitimate recombination in Bacillus subtilis: a site-specific mechanism in the formation of plasmid pKBT1; Hopkins KB et al.; The Bacillus subtilis plasmid pKBT1, the product of in vivo recE4-independent recombinal events, contains segments derived from pUB110 and the B . subtilis chromosome . To determine whether the pUB110 sequence is intact in PKBT1, two 1 kb fragments, each containing a site at which chromosomal and pUB110 sequences are joined, were cloned and sequenced . Sequencing data revealed that: 1) . An intact copy of pUB110 is present in pKBT1; 2) The apparent recombination sites were adjacent to the Bam HI-generated ends of pUB110 sequences; 3) pTL12-derived sequences from the original transforming DNA were limited to no more than 1 bp outside the Bgl II recognition sequence; 4) Recombination sites at both ends of pUB110 contain a 19 bp inverted repeat with 15 homologous nucleotides . These findings suggest a site-specific mechanism acting during in vivo formation of pKBT1. Biochim Biophys Acta, 1989 Dec 21, 999(3), 260 - 6 Purification and characterization of an insect alpha-amylase inhibitor/endochitinase from seeds of Job's Tears (Coix lachryma-jobi); Ary MB et al.; A protein inhibitor of locust gut alpha-amylase was purified from seeds of Job's Tears (Coix lachryma-jobi) using ammonium sulphate precipitation, affinity chromatography on Red Sepharose and reversed-phase HPLC . It consisted of two major isomers, each a dimer of two closely similar or identical subunits of Mr about 26,400, and associated by inter-chain disulphide bonds . These isomers also had closely similar amino acid compositions . The major isomer showed no inhibitory activity against amylases from other sources: human saliva, porcine pancreas, Bacillus subtilis, Aspergillus oryzae and barley malt . The manual DABITC/PITC method was used to determine about half of the amino acid sequence of the major isoform . This showed a high degree of homology with previously reported sequences of endochitinase enzymes from several species (tobacco, potato, barley, bean), and endochitinase activity was demonstrated by following the release of radioactivity from {3H}chitin . This novel combination of functions may be relevant to protection of the grain from insect feeding and fungal infection. Gene, 1989 Dec 21, 85(1), 187 - 92 Expression of the rtp gene of Bacillus subtilis is required for replication fork arrest at the chromosome terminus; Smith MT et al.; It was earlier proposed that clockwise replication fork arrest at the chromosome terminus in Bacillus subtilis is dependent upon expression of the rtp gene adjacent to the site of arrest, terC {Smith and Wake, J . Bacteriol . 170 (1988) 4083-4090} . A merodiploid strain of B . subtilis, in which rtp was placed under the control of the IPTG-inducible spac-1 promoter, was constructed . Replication fork arrest at terC, as monitored by the level of a forked DNA molecule of predicted dimensions, was shown to be dependent upon IPTG-induced expression of rtp in this strain . The very low concentration of IPTG needed to induce a substantial level of fork arrest suggests that relatively little RTP, the protein product of rtp, is needed for fork arrest at terC. Gene, 1989 Dec 21, 85(1), 177 - 86 The cloned polC gene of Bacillus subtilis: characterization of the azp12 mutation and controlled in vitro synthesis of active DNA polymerase III; Barnes MH et al.; Wild type (wt) Bacillus subtilis polC and polCazp12, a mutant derivative specifying a form of DNA polymerase III resistant to hydroxyphenylazopyrimidines, were cloned as genomic fragments approximating the length required to encode the entire polymerase . The cloned DNA fragments were subjected to restriction and partial sequence analysis to locate the 5' end of the polC-specific coding sequence and the azp12 mutation, which was identified as a T----G transversion specifying replacement of serine with alanine . The cloned wt and azp12-coding sequences were recloned in an Escherichia coli expression vector with their respective 5' ends under the control of the bacteriophage lambda PL promoter and cIts857-encoded repressor . In response to induction, the wt- and azp12-specific recombinant plasmids expressed active DNA polymerases indistinguishable from the native enzymes derived from the respective B . subtilis hosts. EMBO J, 1989 Dec 20, 8(13), 4307 - 14 Conformational alterations in the ermC transcript in vivo during induction; Mayford M et al.; ermC is an inducible antibiotic resistance gene from Staphylococcus aureus, one of several whose expression is regulated at the level of mRNA secondary structure . During induction of ermC, the inhibition of a ribosome active in translation of a short leader peptide by low levels of antibiotic belonging to the macrolide-lincosamide-streptogramin b family is believed to cause a rearrangement in mRNA secondary structure . The resultant conformational isomerization unmasks the methylase ribosome binding site and initiator Met codon, causing increased translation of the ermC transcript . Expression of ermC can also be demonstrated in Bacillus subtilis carrying plasmid pE194 . To probe the ermC transcript in vivo during induction, ermC was transferred to B . subtilis by transformation and the resultant transformants were treated with dimethyl sulfate which reacts with N-1 of adenine and N-3 of cytosine residues in a manner that is sensitive to secondary structure . The bases modified in vivo were detected by primer extension with reverse transcriptase using total cellular RNA as template and a complementary ermC-specific oligonucleotide as primer . Physical evidence was obtained for the secondary structural rearrangements predicted by the ermC regulatory model . Additionally, physical evidence was obtained demonstrating that during induction, the stalled ribosome protects codons 9 and 10 of the leader peptide from modification by dimethyl sulfate, in agreement with genetic data obtained previously that identified the integrity of codons 5-9 as critical for induction of ermC by erythromycin. Gene, 1989 Dec 14, 84(2), 279 - 86 Analysis of transcriptional control regions in the Streptomyces subtilisin-inhibitor-encoding gene; Taguchi S et al.; A transcript, of about 650 nucleotides (nt), from the Streptomyces subtilisin-inhibitor-encoding gene (ssi) was identified by Northern hybridization analysis in both the original strain, S . albogriseolus S-3253, and the transformant, S . lividans 66, carrying an expression plasmid with the cloned ssi gene, pJS1 . These results were quite consistent with the analysis of the major transcriptional start point (tsp; at nt 429) by primer extension experiments and the transcriptional end point (at nt 1065) by S1 nuclease mapping of the ssi gene . Deletion experiments on the 5'-flanking region of the major tsp suggested that two promoter sequences control the expression of ssi . The more proximal of these putative promoters appears to be homologous to the -45 to -25 region of the ctc promoter in Bacillus subtilis and includes a direct repeat in the -10 region. Nucleic Acids Res, 1989 Dec 11, 17(23), 9947 - 56 Sequence limits of DNA strands in the arrest replication fork at the Bacillus subtilis chromosome terminus; Williams NK et al.; The DNA sequence limits of the leading and lagging strands in the arrested clockwise replication fork at the terminus of the Bacillus subtilis chromosome have been investigated . On the basis of hybridization to synthetic oligonucleotides corresponding to known positions in the terminus region sequence it has been shown that neither the leading nor lagging strands, as they approach terC, traverse the distal inverted repeat, IRI . But a small fraction of the leading strands pass through the proximal inverted repeat, IRII . This is consistent with IRI being the functional inverted repeat in arresting the clockwise fork . But most of the forks appear to stop at least 100 nucleotides short of IRI, and at various positions extending over a distance of at least 100 nucleotides. J Theor Biol, 1989 Dec 7, 141(3), 391 - 402 The origin of the rotation of one end of a cell relative to the other end during growth of gram-positive rods; Koch AL; The Gram-positive rod wall elongates by an inside-to-outside mechanism of linking new peptidoglycan on the inside and the cracking, by autolysis, of old wall on the outside . During this process the peptidoglycan experiences stress in different directions in different levels of the wall . The stress that develops in a rod-shaped cell if the wall was uniform in physical properties throughout its thickness is twice as great in the hoop direction as in the axial direction . This leads to splitting in the direction of the longitudinal axis . However, the older, partially split, more peripheral wall is stressed in the direction of the elongating cell axis and thus favors circumferential cracks . It is suggested that these processes combine to form a system of helical cracks, grooves, or crevasses . The stable system of grooves would have the same handedness, fairly constant pitch and elongate as the cell grows . Their continuing development would result in the rotation of one end of the cell relative to the other even in cells with no spiral or apparent helical character . Such rotation has been experimentally observed with Bacillus subtilis . The proposed mechanism for rotation during growth may account, in part, for the formation of helical coils of bundles of filamentous organisms (macrofibers), the morphology of spirilla and vibroids, and for the shapes of some mutant and some antibiotic-treated organisms . Rotation due to generation of helical cracks as the result of the biophysics of the growth process as proposed here, is an alternative to the proposal by Mendelson (1976, Helical growth of Bacillus subtilis: a new model for cell growth . Proc . natn . Acad . Sci . U.S.A . 73, 1740-1744) that rotation is due to the laying down of nascent peptidoglycan in a helical pattern. Gene, 1989 Dec 7, 84(1), 127 - 33 Synthesis and refolding of human tissue-type plasminogen activator in Bacillus subtilis; Wang LF et al.; A 1.6 kb cDNA fragment encoding the mature part of the human tissue-type plasminogen activator (t-PA) was subcloned into a Bacillus subtilis dual plasmid expression system {Le Grice et al., Gene 55 (1987) 95-103} . Expression of the tPA gene in this vector was regulated by the inducible Escherichia coli lac elements, as well as a strong phage-T5-derived promoter and ribosome-binding site preceding the polylinker . The 5' end of the tPA gene corresponding to the N terminus of mature t-PA was fused in phase to the third codon present in the polylinker region of the expression vector, p602/22, to form p602-t-PA . B . subtilis containing p602-t-PA, when induced with isopropyl-beta-D-thiogalactopyranoside, produced large amounts of immunoreactive t-PA (approx . 20 micrograms/ml) . As expected, t-PA was not secreted into the culture media, but was localized in intracellular inclusion bodies and was found to be enzymatically inactive . However, enzymatic activity could be regained following complete reduction followed by slow oxidation of the solubilized inclusion bodies . The recombinant t-PA (rt-PA) showed, after purification, a smaller molecular size than melanoma t-PA, probably due to lack of glycosylation in the Bacillus system . Like melanoma t-PA, rt-PA exhibited tremendous stimulation of plasminogen activation in the presence of fibrin . Our results illustrate that B . subtilis, when supplied with the proper transcriptional/translational regulatory elements, can be an effective system for expression of heterologous gene products. Biofactors, 1989 Dec, 2(2), 77 - 86 Non-redox roles for iron-sulfur clusters in enzymes; Switzer RL; In recent years a number of enzymes have been discovered which, contrary to prior expectations, contain FeS clusters but do not participate in redox reactions . In all cases but one, where the FeS cluster in these enzymes has been identified, it is a {4Fe-4S} cluster . In mammalian aconitase a single Fe atom of the {4Fe-4S} cluster participates in catalysis of hydration-dehydration reactions by direct ligation to the substrates . A number of hydrolyases containing FeS clusters have now been identified . In Bacillus subtilis glutamine phosphoribosyl-pyrophosphate amidotransferase the {4Fe-4S} cluster is essential for the active structure of the enzyme, but probably does not participate directly in catalysis . Rather, the cluster may serve as part of a mechanism of oxidative inactivation of the enzyme in vivo, which is followed by its intracellular degradation . The role played by a {4Fe-4S} cluster in Escherichia coli endonuclease III is at present completely unknown . Thus, a number of novel roles for FeS clusters in enzymology and protein structure have been discovered, and more novel findings must be anticipated. J Biochem (Tokyo), 1989 Dec, 106(6), 1110 - 3 Chemical modification of neutral protease from Bacillus subtilis var . amylosacchariticus with tetranitromethane: assignment of tyrosyl residues nitrated; Kobayashi R et al.; A neutral protease from Bacillus subtilis var . amylosacchariticus was modified with tetranitromethane (TNM) at pH 8.0 for 1 h at 25 degrees C, by which treatment the proteolytic activity toward casein was markedly reduced, whereas activity changes toward N-blocked peptide substrates were variable depending upon the substrate used . The modified enzyme was digested with a Staphylococcus aureus V8 protease at pH 7.9 and the resultant peptides were separated by HPLC . Two peptides which contain nitrotyrosyl residue(s) were purified . One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos . 153-159 of the neutral protease, and Tyr-158 was identified as PTH-nitrotyrosine . The other one was the amino-terminal peptide of residue Nos . 1-22, and Tyr-21 was shown to be nitrated . From a comparison with the active site structure of thermolysin, which is a zinc metalloprotease with a high sequence homology to B . subtilis neutral proteases, nitration of Tyr-158 was inferred to be closely related to the activity changes of the neutral protease from B . subtilis var . amylosacchariticus. Zentralbl Hyg Umweltmed, 1989 Dec, 189(3), 214 - 24 {UV-inactivation of microorganisms in water}; Sommer R et al.; UV-Inactivation of Escherichia coli, Bacillus subtilis spores, Staphylococcus-Phage A 994, Poliovirus type Mahoney and Rotavirus SA 11 was tested under controlled physical conditions . B . subtilis-spores were found to be the most resistant of these microorganisms, followed by Rotavirus, Bacteriophage and Poliovirus . E . coli required the lowest irradiation dose for inactivation . Causes and meaning of these dose-survival-reactions are discussed. J Bacteriol, 1989 Dec, 171(12), 6835 - 9 Cloning and expression of a Bacillus subtilis division initiation gene for which a homolog has not been identified in another organism; Harry EJ et al.; The Bacillus subtilis 168 division initiation genes defined by the temperature-sensitive mutations ts-1 and ts-12 were cloned into a 10.5-kilobase EcoRI fragment of DNA in the lambda EMBL4 vector . The two genes were separated by approximately 3 kilobases . The gene in which the ts-1 mutation resides was shown to be the same as the B . subtilis homolog of the Escherichia coli ftsZ gene . The other gene was named divIB . It showed no homology to any previously identified gene and coded for a protein of 30.1 kilodaltons which was probably membrane bound. J Bacteriol, 1989 Dec, 171(12), 6821 - 34 Nucleotide sequence and insertional inactivation of a Bacillus subtilis gene that affects cell division, sporulation, and temperature sensitivity; Beall B et al.; Located at 135 degrees on the Bacillus subtilis genetic map are several genes suspected to be involved in cell division and sporulation . Previously isolated mutations mapping at 135 degrees include the tms-12 mutation and mutations in the B . subtilis homologs of the Escherichia coli cell division genes ftsA and ftsZ . Previously, we cloned and sequenced the B . subtilis ftsA and ftsZ genes that are present on an 11-kilobase-pair EcoRI fragment and found that the gene products and organization of these two genes are conserved between the two bacterial species . We have since found that the mutation in the temperature-sensitive filamenting tms-12 mutant maps upstream of the ftsA gene on the same 11-kilobase-pair EcoRI fragment in a gene we designated dds . Sequence analysis of the dds gene and four other open reading frames upstream of ftsA revealed no significant homology to other known genes . It was found that the dds gene is not absolutely essential for viability since the dds gene could be insertionally inactivated . The dds null mutants grew slowly, were filamentous, and exhibited a reduced level of sporulation . Additionally, these mutants were extremely temperature sensitive and were unable to form colonies at 37 degrees C . Another insertion, which resulted in the elimination of 103 C-terminal residues, resulted in a temperature-sensitive phenotype less severe than that in the dds null mutant and similar to that in the known tms-12 mutant . The tms-12 mutation was cloned and sequenced, revealing a nonsense codon that was predicted to result in an amber fragment that was about 65% of the wild-type size (elimination of 93 C-terminal residues). Rev Fr Endod, 1989 Dec, 8(4), 29 - 34 {Efficacy of the glass bead sterilizer on endodontic instruments}; Canalda-Sahli C et al.; The time taken to achieve the sterilization of K files impregnated in a Bacillus subtilis culture was evaluated, placing the files in a glass bead sterilizer . The minimum time needed was 20 seconds at 240 degrees C and the previous cleaning of the file with a sterile wipe impregnated by alcohol reduced this time to 15 seconds. J Gen Microbiol, 1989 Dec, 135 ( Pt 12), 3431 - 45 Cloning and sequencing of the gerD gene of Bacillus subtilis; Yon JR et al.; A Tn917 insertion in the same region of the chromosome as gerD gave rise to a mutant (ger-97) with a germination phenotype similar to that of two gerD mutants which germinate abnormally in a range of germinants . The insertion and two gerD mutations were cotransformed with ribosomal protein genes rpoB, rpsE and rpsI . DNA cloned from one side of the insertion carried the 16S end of the ribosomal RNA operon rrnI . These data were consistent with the order rpoB-rpsE-rpsI-gerD/ger-97::Tn917-rrnI . Insertion into the wild-type chromosome of a plasmid carrying DNA adjacent to the insertion permitted the recovery of a 1.8 kb fragment of DNA which complemented ger-97::Tn917 and the gerD mutations . The DNA nucleotide sequence of the region of this fragment at which Tn917 had inserted revealed a 555 bp open reading frame, preceded by a ribosome-binding site and potential sigma E and sigma A promoter regions and encoding a predicted polypeptide of 21,117 Da . This polypeptide was largely hydrophilic but contained a hydrophobic region at the N-terminus resembling a signal peptide. Mol Microbiol, 1989 Dec, 3(12), 1805 - 12 Pseudo-allelic relationship between non-homologous genes concerned with biosynthesis of polyglycerol phosphate and polyribitol phosphate teichoic acids in Bacillus subtilis strains 168 and W23; Young M et al.; A 60 kbp region of the Bacillus subtilis chromosome encompassing the genes concerned with teichoic acid biosynthesis has been subjected to physical analysis . No homology was detected by Southern hybridization between DNA segments encoding the tag genes of strain 168, concerned with polyglycerol phosphate (poly(groP)) biosynthesis, and the tar genes of strain W23, concerned with polyribitol phosphate (poly-(rboP)) biosynthesis . Analysis of 168/W23 interstrain hybrids that incorporate poly(rboP) instead of poly-(groP) into their cell walls revealed that, in every case, integral substitution of the W23 tar genes for the 168 tag genes had occurred . Interstrain hybrids of the 'W23-like' type have inherited larger segments of W23 DNA than interstrain hybrids of the 'mixed' type . The tag and tar genes are located at equivalent positions on the chromosomes of strains 168 and W23, behaving, in genetic crosses, like an allelic pair . They provide the first example of a pseudo-allelic relationship between non-homologous genes in B . subtilis. J Radiat Res (Tokyo), 1989 Dec, 30(4), 338 - 51 Genotoxic action of sunlight upon Bacillus subtilis spores: monitoring studies at Tokyo, Japan; Munakata N; Samples of Bacillus subtilis spores dried on membrane filter were exposed to natural sunlight from solar-noon time at Tokyo . The survival and mutation induction of wild-type (UVR) and repair-deficient (UVS) spores were determined on 66 occasions since 1979 . Two of the values were considered to be useful in monitoring solar UV intensity; the inverse of the time (in minutes) of exposure to kill 63% of the UVS spores ("sporocidal index") and the induced mutation frequency at 60 minutes of exposure of the UVR spores ("mutagenic index") . Both values were varied greatly due to time of a year, weather and other conditions . Estimates of year-round changes under clear skies were obtained by connecting the maximum values attained in these years . In these curves, there are more than 7-fold differences in the genotoxicity between winter and summer months, with major increases observed in early spring and decreases through autumn . Using a series of UV cut-off filters, the wavelengths most effective for the sporocidal actions were estimated to be in the range of 308-325 nm, shorter wavelengths being effective when the genotoxicity was higher . Sunburn meter of Robertson-Berger type seems to respond to slightly longer wavelength components of the solar spectrum . However, a reasonable correlation was obtained between the reading of the meter and the sporocidal index. J Appl Bacteriol, 1989 Dec, 67(6), 619 - 28 Effect of heat and ultrasonic waves on the survival of two strains of Bacillus subtilis; Garcia ML et al.; The combined effect of ultrasonic (20 KHz, 150 W) and heat treatment on the survival of two strains of Bacillus subtilis in three suspending media (distilled water, glycerol and milk) has been studied . When spores suspended in water or milk were subjected to ultrasonic waves before heat treatments a little or no decrease of the heat resistance was observed . However, both sporicidal agents applied simultaneously (thermo-ultrasonication) decreased by 63% (B . subtilis, var . niger-40) and 74% (B . subtilis ATCC 6051) the decimal reduction times for the heat treatment when the spores were suspended in glycerol and by 79% and 40%, respectively when suspended in milk . The thermo-ultrasonication of spores in water markedly reduced the heat resistance of them (between 99.9% and 70%) in the range 70-95 degrees C but the effect of the thermo-ultrasonication significantly diminished as the temperature of the treatment was approached to the boiling point of the water. FEMS Microbiol Lett, 1989 Dec, 53(3), 327 - 32 Immunoelectron microscopy of Bacillus subtilis cells secreting human interferon alpha 1 or staphylokinase; Wagner B et al.; Post-embedding labelling techniques with colloidal gold-IgG or -protein A complexes were used to determine the subcellular location of IFN alpha 1 and staphylokinase secreted from Bacillus subtilis GB500 cells . Both proteins were present in the cytoplasma and the cell envelope pointing to a posttranslational mode of translocation across the cytoplasmic membrane . 5- to 10-fold higher concentrations of gold particles per 0.1 micron 2 were found on the cell envelope and clustering was observed suggesting preferential regions for secretion sites . Several control experiments ensured the specificity of the labelling data. Mol Gen Genet, 1989 Dec, 220(1), 1 - 7 Defects in the nutrient-dependent methylation of a membrane-associated protein in spo mutants of Bacillus subtilis; Golden KJ et al.; Methylation of a membrane-associated protein with an apparent molecular mass of 40,000 daltons has been observed in Bacillus subtilis . The methylation was nutrient dependent and occurred with a doubling time of 4 +/- 1 min . In wild-type strains, the half-life of turnover of the methyl group(s) was 17 +/- 6 min . Several isogenic strains of B . subtilis containing spo0 mutations (spo0A and spo0H) were found to be normal in glutamate-dependent methylation of the protein and turnover of the methyl group(s) . In strains containing spo0B and spo0E mutations, the methyl group(s) were incorporated in response to glutamate addition but turnover was not at a normal rate . The half-life of methyl group turnover was extended to 45 +/- 3 min in these strains . In a spo0K mutant and in spoIIJ and spoIIF mutants, the protein was not significantly methylated . The methylation of a 40,000 dalton protein was also found to be dependent on phosphate . This methylation was observed in wild-type and spo0A and spo0H strains with a doubling time of 4 +/- 1 min and a half-life of turnover of the methyl group(s) of 11 +/- 3 min . In strains containing spo0B, spo0E, and spo0F mutations, the phosphate-dependent incorporation of the methyl group(s) was normal (5 +/- 1 min) but the turnover half-life was extended to 46 +/- 8 min . It is not known whether the nitrogen-dependent and phosphate-dependent systems methylated the same protein.(ABSTRACT TRUNCATED AT 250 WORDS) Proc Natl Acad Sci U S A, 1989 Dec, 86(23), 9109 - 13 Genetic evidence that RNA polymerase associated with sigma A factor uses a sporulation-specific promoter in Bacillus subtilis; Kenney TJ et al.; The construction of allele-specific suppressor mutations has enabled us to demonstrate that a sporulation-specific transcription unit in Bacillus subtilis, the spoIIG operon, is transcribed by a form of RNA polymerase associated with sigma A, the principal sigma factor in vegetative cells . The spoIIG operon encodes sporulation-specific factor sigma E, and its transcription is directed from a promoter that is activated about 1 hr after the onset of endospore formation . This promoter contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by sigma A-associated RNA polymerase, but these sigma A-like recognition sequences are separated by 22 base pairs rather than the typical 17 or 18 base pairs . We have found that substitution of an arginyl residue for the glutamyl residue at position 196 of sigma A (Glu-196----Arg) suppresses the deleterious effect of a thymidine-to-cytidine base substitution at position -11 in the spoIIG promoter . This suppression was allele-specific, since it did not suppress the effects of base substitutions in other positions in the spoIIG promoter or the effects of a thymidine-to-guanosine change at -11 . These results support a model in which a form of RNA polymerase containing sigma A is utilized in an unusual manner to activate the transcription of the spoIIG operon well after the onset of endospore formation. J Bacteriol, 1989 Dec, 171(12), 6747 - 52 In vitro reconstitution of a hexagonal array with a surface layer protein synthesized by Bacillus subtilis harboring the surface layer protein gene from Bacillus brevis 47; Tsuboi A et al.; Bacillus brevis 47 contains two surface layer proteins, termed the outer wall protein and the middle wall protein (MWP), which form a hexagonal array in the cell wall . Introduction of the MWP structural gene into Bacillus subtilis by using a low-copy-number plasmid led to the synthesis of an immunoreactive polypeptide with a molecular mass almost the same as that of the MWP synthesized by B . brevis 47 . Biochemical analysis indicated that most of the MWP synthesized by B . subtilis was localized in the cytoplasmic fraction . This was further confirmed by using immunogold electron microscopy . The amino-terminal amino acid sequence of the MWP purified from the cytoplasm of B . subtilis indicated that the MWP was precursor with a signal peptide of 23 amino acid residues to the amino terminus of the mature protein . The precursor of the MWP possessed the ability to reassemble in vitro on the B . brevis 47 peptidoglycan layer, resulting in the formation of almost the same hexagonal arrays as with the mature MWP purified from B . brevis 47, judging from images averaged at a resolution of about 2.5 nm . Furthermore, a center-to-center distance of the hexagonal lattice on the envelope reconstituted by using the precursor MWP was calibrated as 18.3 nm, which was almost identical to the value of 17.8 nm obtained with the mature protein. Gene, 1989 Nov 30, 83(2), 215 - 23 Development of an inducible and enhancible expression and secretion system in Bacillus subtilis; Wong SL; A set of inducible secretion vectors has been developed in Bacillus subtilis based on the regulatory region and the signal peptide sequence of the sacB gene encoding an extracellular enzyme, levansucrase . The expression of the inserted foreign gene (bla) encoding TEM beta-lactamase (Bla), can be induced by the addition of sucrose to the medium . Either the installation of a sacQ expression cassette into the same secretion vector, or the use of a sacUh two-protease-deficient strain (WB30), can significantly enhance expression of the bla gene . However, the combined use of the sacQ-containing secretion vector and the WB30 strain results in no further increase in Bla activity . During development of the secretion vector, the nucleotide sequence around the signal peptidase cleavage site has been redesigned, so that unique restriction sites were installed to facilitate the insertion of foreign genes. Biochem Biophys Res Commun, 1989 Nov 15, 164(3), 1417 - 22 On the respective roles of the two proteins encoded by the Bacillus sphaericus 1593M toxin genes expressed in Escherichia coli and Bacillus subtilis; de la Torre F et al.; The 3.6 kb HindIII DNA fragment of B . sphaericus 1593M chromosomal DNA bears two genes encoding two polypeptides of 41.9 kDa (protein "42") and 51.4 kDa (protein "51") . DNA fragments carrying only one of these two genes when expressed in E . coli yield products that are inactive towards Culex larvae . The larvicidal activity is recovered when Triton X-100 treated E . coli cells containing each one of the two genes are incubated together . In E . coli these two polypeptides are acting synergistically . The protein "51" appears to be involved in the maturation of protein "42" for expression of the larvicidal activity . In B . subtilis however the toxicity is expressed by cells carrying only the gene coding for protein "42" . There is no need of the "51" gene product for the maturation of the "42" polypeptide, suggesting that the maturation is most likely accomplished by host enzymes. Biochem J, 1989 Nov 15, 264(1), 275 - 84 Electrochemical and spectroscopic characterization of the conversion of the 7Fe into the 8Fe form of ferredoxin III from Desulfovibrio africanus . Identification of a {4Fe-4S} cluster with one non-cysteine ligand; George SJ et al.; Desulfovibrio africanus ferredoxin III is a protein (Mr 6585) containing one {3Fe-4S}1+,0 and one {4Fe-4S}2+,1+ core cluster when aerobically isolated . The amino acid sequence contains only seven cysteine residues, the minimum required to ligand these two clusters . Cyclic voltammery by means of direct electrochemistry at a pyrolytic-graphite-'edge' electrode promoted by neomycin shows that, when reduced, the {3Fe-4S}0 centre reacts rapidly with Fe(II) ion to form a {4Fe-4S}2+ cluster . The latter, which can be reduced at a redox potential similar to that of the other {4Fe-4S} cluster, must include non-thiolate ligation . We propose that the carboxylate side chain of aspartic acid-14 is the most likely candidate, since this amino acid occupies the position of a cysteine residue in the sequence typical of an 8Fe ferredoxin . The magnetic properties at liquid-He temperature of this novel cluster, studied by low-temperature magnetic-c.d . and e.p.r . spectroscopy, are diamagnetic in the oxidized state and S = 3/2 in the one-electron-reduced state . This cluster provides a plausible model for the ligation states of the {4Fe-4S}1+ core in the S = 3/2 cluster of the iron protein of nitrogenase and in Bacillus subtilis glutamine:phosphoribosyl pyrophosphate amidotransferase. J Mol Biol, 1989 Nov 5, 210(1), 51 - 63 Regulation of Bacillus subtilis glutamine synthetase gene expression by the product of the glnR gene; Schreier HJ et al.; Transcription of the Bacillus subtilis gene coding of glutamine synthetase (glnA) is regulated by the nitrogen source . The glnA gene lies in an operon in which it is preceded by an open reading frame with the potential to encode a polypeptide of approximately 16,000 Mr . We have now shown that this open reading frame is utilized in vivo, that its product (GlnR) acts as a diffusible, negative regulator of gln transcription, and that GlnR is likely to be a DNA-binding protein . Certain mutations in glnR, including a large, in-frame deletion and a start codon mutation, led to high-level constitutivity of the operon; other mutations caused low-level constitutivity . These latter mutations, which affected the C terminus of GlnR, seemed to disrupt response to the nitrogen source without eliminating the ability of GlnR to bind to DNA . Wild-type GlnR by itself, however, did not impose nitrogen-dependent regulation; such regulation also required the product of glnA . A model is presented in which glutamine synthetase monitors the availability of nitrogen and imposes negative regulation by interaction with or modification of GlnR. J Biol Chem, 1989 Nov 5, 264(31), 18386 - 91 Spectroscopic characterization of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase; Onate YA et al.; The properties of the {4Fe-4S} cluster in glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis have been investigated using low temperature magnetic circular dichroism, electron paramagnetic resonance (EPR), and resonance Raman spectroscopies . The Raman spectra of the native enzyme in the Fe-S stretching region show a {4Fe-4S}2+ cluster that is structurally very similar to those in simple redox proteins . Photochemical reduction mediated by 5-deazaflavin with oxalate as the electron donor resulted in {4Fe-4S}+ clusters with a mixture of ground state spin multiplicities . Magnetic circular dichroism and EPR studies of samples ranging in concentration from 0.15 to 0.4 mM concur in finding S = 3/2 {4Fe-4S}+ clusters with predominantly axial and positive zero field splitting as the dominant species . The EPR studies also revealed minor contributions from S = 1/2 {4Fe-4S}+ centers and an S = 5/2 species . The latter becomes the dominant component in more concentrated samples (approximately 2 mM), and arguments are presented in favor of assignment to S = 5/2 {4Fe-4S}+ clusters rather than adventitiously bound high spin Fe(III) ions . The concentration-dependent spin state heterogeneity of the {4Fe-4S}+ cluster in glutamine phosphoribosylpyrophosphate amidotransferase is discussed in light of the magnetic and electronic properties of the {4Fe-4S}+ centers in other enzymes and proteins. Biochim Biophys Acta, 1989 Nov 2, 1009(2), 161 - 7 Purification and properties of the DNA-binding protein HPB12 from Bacillus subtilis nucleoid; Salti V et al.; We report the purification to homogeneity of a 12 KDa protein (HPB12) present in the nucleoids of Bacillus subtilis . From the purification data the abundance of the protein was estimated to about 20,000 monomers per cell . The HPB12 protein is heat-stable and acid-soluble and binds preferentially to supercoiled and linearized double-stranded DNAs . The binding of the protein to the supercoiled DNA occurs very rapidly and appears to be cooperative . Moreover, the complexes are extremely stable and do not dissociate after 90 min . These properties are consistent with a role of the HPB12 protein in the structure of the B . subtilis chromosome. J Bacteriol, 1989 Nov, 171(11), 5901 - 6 16S rRNA sequence indicates that plant-pathogenic mycoplasmalike organisms are evolutionarily distinct from animal mycoplasmas; Lim PO et al.; The plant-pathogenic mycoplasmalike organisms (MLOs) are so named because they lack cell walls . Many features that are essential to a definitive classification remain uncharacterized, because these organisms have resisted attempts at in vitro culturing . To establish the taxonomic position of the MLOs, the DNA region containing the 16S rRNA gene from a representative of the MLOs has been cloned and sequenced . Sequence comparisons indicate that the MLOs are related to Mycoplasma capricolum and that these two bacteria share their phylogenetic origin with Bacillus subtilis . The low G + C content of this gene and features of its deduced secondary structure further support this grouping . However, the presence of a single tRNAIle gene in the spacer between the 16S rRNA and 23S rRNA genes of the MLOs differentiates the MLOs from other representatives of the mycoplasmas, which indicates an early divergence in the evolution of the members of the class Mollicutes . The presence of certain characteristic oligonucleotides in the 16S rRNA sequence indicates that MLOs may be closely related to acholeplasmas. J Antibiot (Tokyo), 1989 Nov, 42(11), 1567 - 77 Maggiemycin and anhydromaggiemycin: two novel anthracyclinone antitumor antibiotics . Isolation, structures, partial synthesis and biological properties; Pandey RC et al.; Two new anthracyclinone antitumor antibiotics, maggiemycin (6, NSC-D344012) and anhydromaggiemycin (8) have been isolated from a culture of an unspeciated Streptomyces (ATCC No . 39235) . Bioautography against Bacillus subtilis was used for the preliminary detection of these anthracyclinones . Structures were proposed based on their UV-visible, IR, 1H NMR, 13C NMR spectra, electron impact (EI) and high-resolution EI-MS and confirmed by partial synthesis and a direct correlation with epsilon-rhodomycinone . Both the anthracyclinones are active against KB, P388 and L1210 murine tumor cell lines; however, anhydromaggiemycin was more active than maggiemycin . A number of related anthracyclinones have also been prepared and their biological activity has been determined . The structure-activity relationship of these new anthracyclinones is also discussed. Plasmid, 1989 Nov, 22(3), 281 - 6 Identification and nucleotide sequence of the minimal replicon of the low-copy-number plasmid pBS2; Darabi A et al.; The plasmid pBS2 has a low copy number and is endogenous to Bacillus subtilis . The replication of this plasmid depends on the function of most of the host's dna genes including dnaB, which is unique to B . subtilis and is required for both the initiation of chromosome replication and the DNA-membrane association . We have identified the region that is essential for the replication of pBS2 and determined the complete 2279-bp nucleotide sequence of this region . In this region, there are two stretches of sequence homologous to the 18-bp consensus sequence which commonly appears at the origin of replication of plasmids pUB110 and pC194 . The entire region contains six sizable open reading frames . Two of them are probably translated . One open reading frame, designated ORF A, coding for 269 amino acids, has significant homology, in terms of amino acid sequence, with the open reading frame of the gene for the Rep U protein of plasmid pUB110 . The similarities between pBS2 and other plasmids suggest that the pBS2 may also replicate as a rolling circle, which appears to be the salient feature of a mechanism of replication that is common to small plasmids in gram-positive bacteria. Appl Environ Microbiol, 1989 Nov, 55(11), 2949 - 53 Comparison of ozone inactivation, in flowing water, of hepatitis A virus, poliovirus 1, and indicator organisms; Herbold K et al.; In steadily flowing water at 20 degrees C and pH 7, five organisms had the following order of resistance to ozone (at constant levels of ozone): poliovirus 1 (PV1) less than Escherichia coli less than hepatitis A virus (HAV) less than Legionella pneumophila serogroup 6 less than Bacillus subtilis spores . The tests were repeated at 10 degrees C with HAV, PV1, and E . coli . Ozone inactivation of HAV and E . coli was faster at 10 degrees C than at 20 degrees C . At 20 degrees C, 0.25 to 0.38 mg of O3 per liter was required for complete inactivation of HAV but only 0.13 mg of O3 per liter was required for complete inactivation of PV1. J Gen Microbiol, 1989 Nov, 135 ( Pt 11), 2931 - 40 Chromosomal location of the Bacillus subtilis aspartokinase II gene and nucleotide sequence of the adjacent genes homologous to uvrC and trx of Escherichia coli; Chen NY et al.; The aspartokinase II (ask) operon of Bacillus subtilis consists of two in-phase overlapping genes that encode the two subunits of the lysine-sensitive isoenzyme of aspartokinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) . Transduction mapping of the ask operon, inactivated by recombinational insertion of a cat marker, indicates a chromosomal location (about 253 degrees) between leuA and aroG . ask is thus remote from aecB, eliminating aecB as a possible locus for the structural gene of aspartokinase II, but close to aecA and uvrB . The nucleotide sequence of a 2 kb DNA fragment just upstream of the ask operon was determined and found to contain two open reading frames . The deduced amino acid sequence of the distal reading frame exhibits extensive homology with Escherichia coli thioredoxin and that of the proximal one, which overlaps with the ask promoter, is homologous to the deduced product of the E . coli uvrC gene . Insertional mutagenesis of the proximal open reading frame led to a mitomycin-sensitive phenotype, consistent with a role in DNA repair . In conjunction with the data of M . Petricek, L . Rutberg & L . Hederstedt {FEMS Microbiology Letters 61, 85-88} our results define the nucleotide sequence of an 8.8 kb segment of the B . subtilis chromosome near 253 degrees and the following order of genes: trx-uvrB-ask-orfX-sdhC-sdhA-sdhB-orfY++ +-gerE. Proc Natl Acad Sci U S A, 1989 Nov, 86(22), 8660 - 4 Overreplication of the origin region in the dnaB37 mutant of Bacillus subtilis: postinitiation control of chromosomal replication; Henckes G et al.; When the Bacillus subtilis dnaB37 mutant, defective in initiation, is returned to permissive temperature after accumulation of initiation proteins at 45 degrees C, we have shown, by extensive DNA.DNA hybridization analysis, that the origin region is replicated in excess (approximately 2-fold) . However, this replication is limited to a region of about 120-175 kilobases on either side of the origin . This has been confirmed by autoradiographic analysis of the overreplicated region . During the second round of synchronized replication at 30 degrees C, replication in fact appears to resume from the stalled forks on either side of the origin . We propose that in B . subtilis, in addition to a first level of control at the origin, a second level of control exists downstream of the origin in order to limit overreplication of the chromosome . These two controls might normally be tightly coupled . We suggest that the second level of control is exerted through the reversible inhibition of replisome movement at specific regions on either side of the origin. Proc Natl Acad Sci U S A, 1989 Nov, 86(21), 8457 - 61 AbrB, a regulator of gene expression in Bacillus, interacts with the transcription initiation regions of a sporulation gene and an antibiotic biosynthesis gene; Robertson JB et al.; The abrB gene of Bacillus subtilis is believed to encode a repressor that controls the expression of genes involved in starvation-induced processes such as sporulation and the production of antibiotics and degradative enzymes . Two such genes, spoVG, a sporulation gene of B . subtilis, and tycA, which encodes tyrocidine synthetase I of the tyrocidine biosynthetic pathway in Bacillus brevis, are negatively regulated by abrB in B . subtilis . To examine the role of abrB in the repression of gene transcription, the AbrB protein was purified and then tested for its ability to bind to spoVG and tycA promoter DNA . In a gel mobility shift experiment, AbrB was found to bind to a DNA fragment containing the sequence from -95 to +61 of spoVG . AbrB protein exhibited reduced affinity for DNA of two mutant forms of the spoVG promoter that had been shown to be insensitive to abrB-dependent repression in vivo . These studies showed that an upstream A + T-rich sequence from -37 to -95 was required for optimal AbrB binding . AbrB protein was also observed to bind to the tycA gene within a region between the transcription start site and the tycA coding sequence as well as to a region containing the putative tycA promoter . These findings reinforce the hypothesis that AbrB represses gene expression through its direct interaction with the transcription initiation regions of genes under its control. J Bacteriol, 1989 Nov, 171(11), 6043 - 51 Molecular cloning and characterization of comC, a late competence gene of Bacillus subtilis; Mohan S et al.; comC is a Bacillus subtilis gene required for the development of genetic competence . We have cloned a fragment from the B . subtilis chromosome that carries comC and contains all the information required to complement a Tn917lac insertion in comC . Genetic tests further localized comC to a 2.0-kilobase HindIII fragment . Northern (RNA) blotting experiments revealed that an 800-base-pair comC-specific transcript appeared at the time of transition from exponential to stationary phase during growth through the competence regimen . The DNA sequence of the comC region revealed two open reading frames (ORFs), transcribed in the same direction . The upstream ORF encoded a protein with apparent sequence similarity to the folC gene of Escherichia coli . Insertion of a chloramphenicol resistance determinant into this ORF and integration of the disrupted construct into the bacterial chromosome by replacement did not result in competence deficiency . The downstream ORF, which contained the Tn917lac insertion that resulted in a lack of competence, is therefore the comC gene . The predicted protein product of comC consisted of 248 amino acid residues and was quite hydrophobic . The comC gene product was not required for the expression of any other com genes tested, and this fact, together with the marked hydrophobicity of ComC, suggests that it may be a component of the DNA-processing apparatus of competent cells. Genetika, 1989 Nov, 25(11), 1937 - 45 {Mutants of Bacillus subtilis resistant to lysine analog S-2 aminoethyl-L-cysteine}; Shevchenko TN et al.; AECR mutants of Bacillus subtilis were obtained and analyzed . The mutants were characterized by derepression of aspartokinase II and diaminopimelate decarboxylase synthesis and the synthesis of the precursor of lysine--DAP . According to genetic mapping data, aec mutations are localized in some B . subtilis chromosomal regions; they are linked to the thr5, leuA8, lys21 markers. Appl Environ Microbiol, 1989 Nov, 55(11), 2976 - 84 Physicochemical interaction of Escherichia coli cell envelopes and Bacillus subtilis cell walls with two clays and ability of the composite to immobilize heavy metals from solution; Walker SG et al.; Isolated Escherichia coli K-12 cell envelopes or Bacillus subtilis 168 cell walls were reacted with smectite or kaolinite clay in distilled deionized water (pH 6.0); unbound envelopes or walls were separated by sucrose density gradient centrifugation, and the extent of adsorption was calculated . At saturation, both clays adsorbed approximately 1.0 mg (dry weight) of envelopes or walls per mg (dry weight) of clay . Clays showed a preference for edge-on orientation with both walls and envelopes, which was indicative of an aluminum polynuclear bridging mechanism between the wall or envelope surface and the clay edge . The addition of heavy metals increased the incidence of planar surface orientations, which suggested that multivalent metal cation bridging was coming into play and was of increasing importance . The metal-binding capacity of isolated envelopes, walls, clays, and envelope-clay or wall-clay mixtures was determined by atomic absorption spectroscopy after exposure to aqueous 5.0 mM Ag+, Cu2+, Cd2+, Ni2+, Pb2+, Zn2+, and Cr3+ nitrate salt solutions at pHs determined by the buffering capacity of wall, envelope, clay, or composite system . The order of metal uptake was walls greater than envelopes greater than smectite clay greater than kaolinite clay for the individual components, and walls plus smectite greater than walls plus kaolinite greater than envelopes plus smectite greater than envelopes plus kaolinite for the mixtures . On a dry-weight basis, the envelope-clay and wall-clay mixtures bound 20 to 90% less metal than equal amounts of the individual components did.(ABSTRACT TRUNCATED AT 250 WORDS) Mikrobiol Zh, 1989 Nov-Dec, 51(6), 34 - 9 {The effect of specific inducers on the biosynthesis of lytic enzymes by a strain of Streptomyces recifensis var . lyticus}; Kilochek TP et al.; The effect of specific inductors at different stages of Streptomyces recifensis var . lyticus 2435 cultivation was studied . It is shown that introduction of killed bacterial cells into inoculum not only influences the level of accumulation of lytic enzymes by the strain S . recifensis var . lyticus 2435 but also determines the qualitative composition of the synthesized complex . The yield of bacteriological glycosidases grows with introduction of the Micrococcus lysodeikticus cells into the inoculum or of dissolvable chitin into the enzymic medium . The possibility to preserve the initial level of the lytic activity in the producer during re-inoculations by introducing the Bacillus subtilis cells into the cultivation medium has been studied. Lipids, 1989 Nov, 24(11), 940 - 4 Fatty acid and beta-amino acid syntheses in strains of Bacillus subtilis producing iturinic antibiotics; Hourdou ML et al.; Iturinic antibiotics, produced by different strains of Bacillus subtilis, contain long-chain beta-amino acids (beta-AA) . The regulation of the synthesis of fatty acids (FA) and beta-AA was studied by modifying the culture medium . Addition of possible precursors, branched-chain alpha-amino acids, to the medium affected the FA and beta-AA compositions . According to this, the B . subtilis strains can be divided into two groups . The first contains the producers of mycosubtilin and bacillomycin F which synthesize a high level of iso C16 chains; the second contains the producers of bacillomycin D, bacillomycin L and iturin which synthesize a high level of n carbon chains . The incorporation of radioactive sodium acetate into FA and beta-AA showed rapid FA synthesis followed by a second synthetic step . Although the detailed mechanism has not yet been elucidated, this second step, corresponding to the beta-AA synthesis, seemed to be a key step in determining the alkyl chain of beta-AA. FEMS Microbiol Lett, 1989 Nov, 53(1-2), 59 - 63 The expression of a highly expressed Bacillus subtilis gene is not reduced by introduction of multiple codons normally not present in such genes; Loshon CA et al.; Four serine or threonine codons were introduced into a highly expressed Bacillus subtilis gene . The introduced codons were ones either common in highly expressed B . subtilis genes, or never used in such genes . Strikingly, the level and rate of expression of the modified genes containing either type of extra codons was identical . This suggests that in B . subtilis codon usage patterns may play little or no role in effecting the level of gene expression. Genes Dev, 1989 Nov, 3(11), 1735 - 44 Temporal and spatial control of the mother-cell regulatory gene spoIIID of Bacillus subtilis; Kunkel B et al.; Gene expression during endospore formation in Bacillus subtilis is compartmentalized between the mother-cell and forespore chambers of the sporangium, which follow separate pathways of cellular differentiation . The earliest acting regulatory gene so far identified in the mother-cell line of gene expression is spoIIID, whose product is required for the transcription of the composite gene (sigK) encoding the mother-cell RNA polymerase sigma-factor sigma K and for the chromosomal rearrangement that gives rise to the composite gene . Here we report the nucleotide sequence of spoIIID and studies on the temporal, spatial, and genetic control of its expression during sporulation . We show that the deduced spoIIID gene product, a 93-residue-long polypeptide, is a previously identified transcription factor that is known to activate the promoter for the sigK gene in vitro . Expression of spoIIID is largely confined to the mother-cell chamber of the sporangium and is turned on at, or shortly before, the time (hour 3 of sporulation) that the mother-cell chromosome is rearranged and transcription of the sigK gene commences . This gene expression depends strongly on the sporulation sigma-factor sigma E and partially on the spoIIID gene product, itself . We conclude that the timing and compartmentalization of the rearrangement and transcription of the sigK gene and, hence, of subsequent gene activation in the mother cell, are, in part, direct consequences of the temporal and spatial control of spoIIID gene expression. J Appl Bacteriol, 1989 Nov, 67(5), 497 - 504 Repression of sporulation: isolation and characterization of repression-resistant mutants of Bacillus subtilis; Bhaduri S et al.; The sporulation of Bacillus subtilis B34 was repressed by 24 h if glutamine or ammonium chloride but not glutamate was added at late log phase (70 h) when glucose and glutamate were nearly exhausted . Glutamine-resistant mutants were isolated by selective heat treatment during the delay period induced by glutamine . Glutamine-resistant mutants showed cross resistance not only against ammonium chloride but also against glucose just as glucose-resistant mutants showed resistance against nitrogenous catabolites. J Bacteriol, 1989 Nov, 171(11), 6375 - 8 Structural similarity among Escherichia coli FtsW and RodA proteins and Bacillus subtilis SpoVE protein, which function in cell division, cell elongation, and spore formation, respectively; Ikeda M et al.; The Escherichia coli cell division gene ftsW (2 min) was cloned and sequenced . It encodes a hydrophobic protein(s) with 414 and/or 384 amino acid residues . The deduced amino acid sequence and the hydropathy profile of the protein showed high homology with those of the E . coli RodA protein functioning in determination of the cell shape and the Bacillus subtilis SpoVE protein functioning in spore formation . Probably similar functional membrane proteins are involved in these three cell cycle process. J Bacteriol, 1989 Nov, 171(11), 6187 - 96 Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory proteins Spo0A and Spo0F of Bacillus subtilis; Perego M et al.; The kinA (spoIIJ) locus contains a single gene which codes for a protein of 69,170 daltons showing strong homology to the transmitter kinases of two component regulatory systems . The purified kinase autophosphorylates in the presence of ATP and mediates the transfer of phosphate to the Spo0A and Spo0F sporulation regulatory proteins . Spo0F protein was a much better phosphoreceptor for this kinase than Spo0A protein in vitro . Mutants with deletion mutations in the kinA gene were delayed in their sporulation . They produced about a third as many spores as the wild type in 24 h, but after 72 h on solid medium, the level of spores approximated that found for the wild-type strain . Such mutations had no effect on the regulation of the abrB gene or on the timing of subtilisin expression and therefore did not impair the repression function of the Spo0A protein . Placement of the kinA locus on a multicopy vector suppressed the sporulation-defective phenotype of spo0B, spo0E, and spo0F mutations but not of spo0A mutations . The results suggest that the spo0B-, spo0E-, and spo0F-dependent pathway of activation (phosphorylation) of the Spo0A regulator may be by-passed through the kinA gene product if it is present at sufficiently high intracellular concentration . The results suggest that multiple kinases exist for the Spo0A protein. J Bacteriol, 1989 Nov, 171(11), 5933 - 9 Role of sigma H in expression of the fumarase gene (citG) in vegetative cells of Bacillus subtilis 168; Price VA et al.; The fumarase gene (citG) of Bacillus subtilis is transcribed from two promoter regions, citGp1 and citGp2 (P1 and P2); the P2 promoter is used by the E sigma H form of RNA polymerase . In order to study the role of P1 and P2 in citG expression, the promoter region and various deletion derivatives that effectively separate P1 and P2 were fused to the Escherichia coli beta-galactosidase gene (lacZ) and introduced into the chromosome in single copy at the amyE locus . P1 functioned to provide a relatively low and stable basal level of fumarase activity throughout growth . In contrast, P2 activity was found to vary over at least a 50-fold range and was responsible for regulating fumarase activity during growth and sporulation in a rich medium and in response to changes in carbon source . To further investigate the role of sigma H in fumarase regulation, citGp2-lacZ fusions were introduced into a strain in which the expression of the chromosomal spoOH gene was under the control of the isopropylthiogalactopyranoside-inducible spac promoter . Induction of pspac did not lead to P2 induction, suggesting that citG expression is not regulated at the level of spoOH transcription. J Bacteriol, 1989 Nov, 171(11), 5928 - 32 Sigma H-directed transcription of citG in Bacillus subtilis; Tatti KM et al.; The RNA polymerase sigma factor sigma H is essential for the onset of endospore formation in Bacillus subtilis . sigma H also is required for several additional stationary-phase-specific responses, including the normal expression of several genes that are required for the development of competence for DNA uptake . It is necessary to identify the genes that are transcribed by sigma H RNA polymerase (E sigma H) in order to understand the role of this sigma factor during the transition from exponential growth to stationary phase . Feavers et al . (Mol . Gen . Genet . 211:465-471, 1988) proposed that citG, the structural gene for fumarase, is transcribed from two promoters, one of which (citGp2 {P2}) may be used by E sigma H . It is likely that the citGp2 promoter is used by E sigma H because we found that this promoter was used accurately in vitro by E sigma H and directed expression of xylE in vivo . This xylE expression was dependent on spo0H, the structural gene for sigma H, and was independent of the citGp1 promoter . Comparison of the nucleotide sequences of several sigma H-dependent promoters showed that these sequences were similar at two regions approximately 10 and 35 base pairs upstream from the start points of transcription . These sequences may signal recognition of these promoters by E sigma H . Primer extension analyses were used to examine transcription from three sigma H-dependent promoters during growth and sporulation . The citGp2 promoter appeared to be active during the middle and late stages of exponential growth, whereas activation of the spoIIA promoter was delayed until after the end of exponential growth . Evidently, promoters used by E sigma H can display different temporal patterns of expression. Appl Environ Microbiol, 1989 Nov, 55(11), 3026 - 8 Enzyme activities and antibiotic susceptibility of colonial variants of Bacillus subtilis and Bacillus licheniformis; Carlisle GE et al.; A nonmucoid colonial variant of a mucoid Bacillus subtilis strain produced less amylase activity and a transparent colonial variant of a B . licheniformis strain produced less protease activity compared with their parents . Antibiotic susceptibility patterns of the colonial variants differed, and increased resistance to beta-lactam antibiotics was correlated with increased production of extracellular beta-lactamase. J Bacteriol, 1989 Nov, 171(11), 5803 - 11 Mechanism of erythromycin-induced ermC mRNA stability in Bacillus subtilis; Bechhofer DH et al.; In Bacillus subtilis, the ermC gene encodes an mRNA that is unusually stable (40-min half-life) in the presence of erythromycin, an inducer of ermC gene expression . A requirement for this induced mRNA stability is a ribosome stalled in the ermC leader region . This property of ermC mRNA was used to study the decay of mRNA in B . subtilis . Using constructs in which the ribosome stall site was internal rather than at the 5' end of the message, we show that ribosome stalling provides stability to sequences downstream but not upstream of the ribosome stall site . Our results indicate that ermC mRNA is degraded by a ribonucleolytic activity that begins at the 5' end and degrades the message in a 5'-to-3' direction. Gene, 1989 Oct 30, 82(2), 291 - 300 Differential screening of a cDNA library with cDNA probes amplified in a heterologous host: isolation of murine GRP78 (BiP) and other serum-regulated low-abundance mRNAs; Parfett CL et al.; A novel method was used to screen differentially a cDNA library for clones representing serum-regulated mRNA species of low abundance . To increase the amount of probe available for screening, the cDNA probe was cloned and amplified . Two separate cDNA 'probe' libraries were constructed in the Escherichia coli plasmid vector pDE613, using poly(A)+mRNA from murine cells at 0 and 16 h after stimulation of a G0 population . Radiolabelled plasmid DNA from each library was hybridized sequentially to colony blots of the third 'target' library, constructed with mRNA from serum-stimulated cells in the Bacillus subtilis vector pBD214 . Differential screening of the target cDNA library with the two probe libraries identified novel murine cDNA clones, some representing cytoplasmic poly(A)+RNA species of low (0.01%) abundance, accumulating after serum stimulation of a quiescent mouse embryo fibroblast population . One cDNA clone was found to correspond to mitochondrial 16S rRNA and a second was identified as the murine equivalent of previously described cDNA clones for the hamster 78-kDa glucose-regulated protein (GRP78) and the rat immunoglobulin heavy-chain-binding protein . GRP78 mRNA has not previously been recognized as a serum-inducible message. FEMS Microbiol Lett, 1989 Oct 15, 52(3), 285 - 90 Determination and evolutionary significance of nucleotide sequences near to the 3'-end of 16S ribosomal RNA of mycobacteria; Estrada-G IC et al.; The nucleotide sequence at the 3'-end of 16Sr-RNA (nucleotides 1305-1508) was determined, by the primer extension method, for Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium vaccae, in addition to Mycobacterium leprae . No differences in nucleotide sequence were detected, indicating that this region of 16SrRNA is highly conserved among mycobacteria . The nucleotide sequence common to the four above-mentioned mycobacteria differs from that reported for species of other genera . For example, for helix 39 (nucleotides 1408-1491) the mycobacterial sequence has 58% similarity with the Escherichia coli sequence, 74% similarity with the Bacillus subtilis sequence and 93% similarity with the Streptomyces sequence . The observations support the assignment of M . leprae to the genus Mycobacterium. J Immunol, 1989 Oct 15, 143(8), 2706 - 13 Incorporation of bacterial peptidoglycan constituents into macrophage lipids during phagocytosis; Polanski M et al.; It has previously been established that several glycopeptides of peptidoglycan origin are formed as a result of processing of Bacillus subtilis cell walls by the macrophage-like cell line RAW264 . Although the formation of these glycopeptides could account for the humoral immune responses characteristic of bacterial peptidoglycans, their formation does not account for the cellular-mediated immune responses observed for water-in-oil emulsions of peptidoglycan or for lipophilic derivatives of glycopeptide fragments thereof . Therefore, the processing of peptidoglycan by macrophages was reexamined to establish whether the lipophilic derivative of any peptidoglycan-derived glycopeptide was formed . The experiments were performed by incubating B . subtilis cell walls radiolabeled in muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid residues in the presence of the macrophage-like cell line RAW264 . The crude lipid fraction derived from the macrophages was further fractionated and analyzed, revealing the presence of two lipophilic glycopeptides that contained glucosamine, muramic acid, and alanine of bacterial origin. FEBS Lett, 1989 Oct 9, 256(1-2), 195 - 9 EPR characterization of soluble fragments of succinate dehydrogenase from mutant strains of Bacillus subtilis; Maguire JJ et al.; Succinate dehydrogenase is a membrane-bound metallo-flavo-enzyme containing a bi- (S-1), a tri- (S-3) and a tetranuclear (S-2) iron-sulfur cluster . The catalytic portion of the enzyme contains two distinct subunits designated Fp and Ip . Using concentrated extracts from mutant strains of Bacillus subtilis it was demonstrated, by using low temperature EPR, that cluster S-2 can be assembled in a soluble succinate dehydrogenase . In a mutant with a truncated Ip subunit which lacks 7 of the 11 conserved cysteine residues, cluster S-1 lacked the spin relaxation properties attributable to an adjacent cluster S-2 . These data are consistent with a model where one or more cysteine residues from the middle set of 4 conserved cysteines in the Ip subunit are ligands to the tetranuclear cluster. J Gen Microbiol, 1989 Oct, 135 ( Pt 10), 2651 - 4 Partial characterization of Mycobacterium fortuitum and Mycobacterium smegmatis auxotrophs by syntrophism using Bacillus subtilis; Subramanyam VR et al.; Syntrophism (cross-feeding) could be demonstrated between mutants of Mycobacterium fortuitum and Mycobacterium smegmatis, and previously characterized mutants of Bacillus subtilis, auxotrophic for arginine, histidine, lysine or phenylalanine . Based on this cross-feeding data, the possible site of blockage in the biosynthetic pathways of the mutants could be inferred. Mol Gen Mikrobiol Virusol, 1989 Oct, (10), 38 - 40 {The effect of various rec-mutations on the frequency of the Tn917 transposition in Bacillus subtilis cells}; Gavrilova EV et al.; The effect of 11 rec-genes on the transposition frequency of Tn917 has been studied . Transposition frequencies in RecP, RecF15, RecB3 mutants differed from the ones in the control strains . The collection of mitomycin-sensitive mutants has been tested for transposition proficiency . The mms315 mutation decreasing the transposition frequency possess the properties of the rec mutation too. FEMS Microbiol Lett, 1989 Oct 1, 52(1-2), 85 - 7 The structural gene for aspartokinase II in Bacillus subtilis is closely linked to the sdh operon; Petricek M et al.; The aecA and aecB loci map at 250 and 290 degrees, respectively, on the Bacillus subtilis chromosomal genetic map . The aecB locus has been proposed as the structural gene for aspartokinase II . From DNA sequence analyses and comparisons to the sequence of the aspartokinase II gene, it can be concluded that the structural gene for aspartokinase II is located close to sdh at 250 degrees and cannot be aecB . A detailed map over 7 kbp in the 250 degree region is presented. J Bacteriol, 1989 Oct, 171(10), 5376 - 85 Cloning and characterization of a cluster of linked Bacillus subtilis late competence mutations; Albano M et al.; We characterized a segment of chromosomal DNA from Bacillus subtilis that was required for the development of genetic competence . The chromosomal DNA was cloned from a group of genetically linked and phenotypically similar Tn917lac insertion mutants deficient in competence . This cluster of mutations defined the comG locus . Chromosomal DNA flanking each of the six insertions was cloned . Restriction maps of the cloned plasmids revealed that their chromosomal inserts consisted of overlapping fragments . These data, together with Southern blots of chromosomal DNA from the comG mutants, showed that the six Tn917lac comG insertions occurred in the following order: comG12, comG39, comG412, comG107, comG56, and comG210 . Expression of the comG Tn917lac insertions was from a promoter located upstream from the first insertion, comG12 . This was determined genetically and by low-resolution S1 nuclease mapping of the 3' terminus . The comG region spanned about 5 kilobase pairs, based on low-resolution S1 nuclease mapping of the transcription terminator and Northern blotting . The comG12 mutation had a partial epistatic effect on the expression of one other com locus, comE, but none of the other comG mutations affected expression of this or any other com gene tested . Based on these conclusions, and on its size and phenotype, the comG locus must be organized as a polycistronic operon that is subject to competence-specific regulation. J Gen Microbiol, 1989 Oct, 135 ( Pt 10), 2723 - 33 Biochemical analysis of the Bacillus subtilis 1604 spore germination response; Venkatasubramanian P et al.; Germination at 37 degrees C of spores of Bacillus subtilis 1604 in the L-alanine and potassium phosphate (ALA) and the glucose, fructose, L-asparagine, potassium chloride (GFAK) germinant systems was triggered following heat activation at 70 degrees C for 1 h . In these conditions, 50% of the spore population became committed to germinate after exposure for 10 min and 14 min to ALA and GFAK, respectively, at which time 38% and 30% losses of OD600 had taken place . Dipicolinic acid (DPA) release, loss of heat resistance and release of soluble hexosamine-containing fragments occurred after commitment and were closely associated with loss of refractility in both the ALA and GFAK pathways . Net ATP synthesis could not be detected until 3-4 min after initiation of germination in both ALA and GFAK, by which time greater than 20% of the spore population was committed to germinate . The ALA and GFAK germination pathways were greater than 99% inhibited by 3 and 1 mM-HgCl2, respectively, as measured by OD600 loss . Reversible post-commitment HgCl2-sensitive sites were present in the ALA and GFAK pathways which were 50% inhibited by 0.125 mM and 0.05 mM-HgCl2, respectively . A pre-commitment HgCl2-sensitive site was identified in the ALA pathway which was 55% inhibited by 6 mM-HgCl2 . At 3 mM-HgCl2, 70% of the spore population became committed to germinate in the ALA pathway, whereas less than 5% OD600 loss occurred . In this system, loss of heat resistance was associated with commitment, whereas OD600 loss and DPA release were identified as post-commitment events . The ALA and GFAK pathways were insensitive to a variety of metabolic inhibitors . Protease inhibitors had different effects on the ALA and GFAK pathways: phenylmethanesulphonyl fluoride (PMSF) solely inhibited ALA germination at a pre-commitment site and had little effect on GFAK germination, whereas N alpha-p-tosyl-L-arginine methyl ester (TAME) inhibited both the ALA and GFAK pathways at pre- and post-commitment sites . These results are discussed in relation to a recently proposed model for the triggering of Bacillus megaterium KM spore germination. Mol Gen Genet, 1989 Oct, 219(1-2), 129 - 36 In vivo selected promoter and ribosome binding site up-mutations: demonstration that the Escherichia coli bla promoter and a Shine-Dalgarno region with low complementarity to the 16 S ribosomal RNA function in Bacillus subtilis; Hung A et al.; We have constructed a plasmid, pQS1, in which a mouse dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP:oxidoreductase; EC 1.5.1.3; DHFR) cDNA is inserted in the unique PstI site of a gram-positive/gram-negative shuttle vector derived from pBR322 . The cDNA is expressed under the control of the bla promoter, which, like most gram-negative bacterial genes, is considered not to be expressed in Bacillus subtilis, and its coding sequence is translated from a polycistronic message . We have selected in vivo and studied, in Escherichia coli and B . subtilis, expression mutants with promoter and ribosome binding site sequence mutations . One promoter mutation changes the third nucleotide of the -35 region from a C to a G . As expected, this substitution results in increased transcriptional activity in E . coli . In B . subtilis, this mutation induces the accumulation not only of a low but significant amount of dhfr mRNA but also of DHFR, demonstrating that binding strengths with a free energy as low as -9.4 kcal/mol are sufficient to promote ribosome binding in B . subtilis . The association of the promoter mutation (C-G) with a mutation which creates a strong B . subtilis ribosome binding site (-21 kcal/mol) results in the accumulation of a large amount of dhfr mRNA . This demonstrates the importance of having an efficient ribosome binding site in the evaluation of promoter function: for example, with this strong ribosome binding site we can show that the wild-type bla promoter is recognized by the B . subtilis transcription machinery. J Clin Pharm Ther, 1989 Oct, 14(5), 373 - 80 Inactivation of B . subtilis spores and E . coli endotoxin by ethylene oxide; Pedersen MR et al.; Exposure of Bacillus subtilis spores to ethylene oxide (EO) showed correlation between the killing rate and the EO concentration, when the temperature was kept at 55 degrees C and the relative humidity at 100% . The co-efficient of dilution was calculated to be 0.9 . The effect of EO on Escherichia coli endotoxin was investigated by the chromogenic Limulus Amebocyte Lysate (LAL) test . A solution of endotoxin was dried on glass tubes and exposed to 450 or 900 mg EO/l during 1-46 h under the same conditions as the spore inactivation . The LAL activity of the endotoxin was reduced to about 30% . The EO-treated endotoxin was tested in the rabbit pyrogen test . The summed temperature increase for three rabbits was 0.9 degrees C, while the same assay using untreated test pieces showed an increment of 3.7 degrees C . Administration of the same quantity of EO-treated and untreated endotoxin to the rabbits, as adjusted by the LAL-test, produced the same temperature increment . The addition of polymyxin B (PB) to an endotoxin solution reduced the LAL activity by 75% . Had the endotoxin been exposed to EO, thereby reducing the LAL activity by 70%, addition of PB further reduced the activity by 99% . The reaction of EO on the endotoxin reduced the LAL activity as well as the pyrogenic response and increased the affinity to PB. Zentralbl Hyg Umweltmed, 1989 Oct, 189(1), 37 - 49 Disinfection with gaseous formaldehyde . Fifth Part: Influence of albumin, mucin and blood on the bactericidal and sporicidal effectiveness; Casella ML et al.; The influence of 0.1% peptone, 0.2% albumin, 1% mucin solutions and whole human blood on the inactivation of Staphylococcus aureus ATCC 6538 and spores of Bacillus subtilis var . niger DSM 675 was determined . The bactericidal and sporicidal effectiveness of 0.75, 1.5 and 3.2 mg HCHO l-1 air at temperatures of 35, 40, 45 degrees C and a relative humidity (RH) of 90% decreased in the following order of loading substances: peptone, albumin, mucin and blood . The calculated D-values of the microorganisms suspended and dried in 0.1% petone and in 0.2% albumin after exposure to 3.2 mg HCHO l-1 air at 40 degrees C and a relative humidity of approximately 90% were in both suspensions 1.9 min for S . aureus and 6.1 and 7.3 min respectively for B . subtilis spores . Under the same exposure conditions but with an addition of 1% mucin D-values of 2.5 min and 7.7 min for S . aureus and B . subtilis spores respectively were found . In the presence of blood, D-values of 12.3 and 18.3 min were obtained under the same conditions for S . aureus and B . subtilis spores respectively . Thus the suspension in blood caused a 2-fold increase in D-value compared to the other substances . The nature of the anticoagulant in the whole blood did not cause much difference in the inactivation of B.subtilis spores . Decreasing concentrations of blood caused an increase in sensitivity of B.subtilis spores to gaseous formaldehyde, whereby diluting blood 1:6 reduced the D-value from 23.9 min to only 7.1 min. J Bacteriol, 1989 Oct, 171(10), 5747 - 9 Timing of spoII gene expression relative to septum formation during sporulation of Bacillus subtilis; Gholamhoseinian A et al.; spoII mutants formed heat-resistant spores when transformed with spo+ DNa near the start of sporulation . Many of the spores formed remained genetically spoII . It is deduced from this result and previous epistasis experiments that the spoII loci are transcribed before the spore septum is formed. J Bacteriol, 1989 Oct, 171(10), 5362 - 75 Sequence and transcription mapping of Bacillus subtilis competence genes comB and comA, one of which is related to a family of bacterial regulatory determinants; Weinrauch Y et al.; The complete nucleotide sequences of the comA and comB loci of Bacillus subtilis were determined . The products of these genes are required for the development of competence in B . subtilis and for the expression of late-expressing competence genes . The major 5' termini of both the comA and comB transcripts were determined . The inferred promoters of both comA and comB contained sequences that were similar to those found at the -10 and -35 regions of promoters that are used by sigma A-RNA polymerase, the primary form of this enzyme in vegetative cells . The comB gene was located approximately 3 kilobase pairs upstream of the comA gene and encoded a 409-amino-acid protein with a predicted molecular weight of 46,693 . The comA locus contained two open reading frames (ORFs) and comB contained one ORF . The predicted amino acid sequence of the comA ORF1 gene product consisted of 214 amino acids, with an aggregate molecular weight of 24,132 . The ORF1 product was required for competence, while ORF2, which was cotranscribed with ORF1 and encoded a predicted protein of 126 amino acids, was not . The predicted protein sequence of the comA ORF1 gene product was found to be similar to that of several members of the effector class of procaryotic signal transducers . The C-terminal portion of the predicted comA sequence contained a possible helix-turn-helix motif, which is characteristic of DNA-binding proteins . comA ORF1 was cloned on a multicopy plasmid and was shown to complement the competence-deficient phenotype caused by the comA124 insertion of Tn917lac . Also, the presence of comA ORF1 in multiple copies interfered with sporulation . Anti-peptide antibodies raised to the predicted product of comA ORF1 reacted strongly with a single protein band of about 24,000 daltons in immunoblots . The possible roles of multiple signal transduction systems in triggering the development of competence are discussed. J Bacteriol, 1989 Oct, 171(10), 5354 - 61 Cloning and characterization of the regulatory Bacillus subtilis competence genes comA and comB; Guillen N et al.; comA and comB are Bacillus subtilis competence genes that are identified by insertions of Tn917lac . They are classified as early genes because of their expression throughout growth; the expression of late com genes increases sharply during the transition to the stationary phase . The comA and comB determinants were cloned, and the 5' and 3' termini of their transcripts were localized by low-resolution S1 nuclease protection experiments . comA and comB were found by Southern blotting to be localized near one another, but they were nevertheless apparently transcribed independently . Epistatic relationships among the com genes were explored by using the beta-galactosidase expressed from transcriptional fusions as a marker . Late com genes were found to be dependent on the products of comA, comB, and sin for their expression . The sin gene is a transcriptional regulator that is required for the development of competence (N . K . Gaur, E . Dubnau, and I . Smith, J . Bacteriol . 168:860-869, 1986). J Bacteriol, 1989 Oct, 171(10), 5347 - 53 Cloning and characterization of srfB, a regulatory gene involved in surfactin production and competence in Bacillus subtilis; Nakano MM et al.; A Tn917 insertion mutation srfB impairs the production of the lipopeptide antibiotic surfactin in Bacillus subtilis . srfB is located between aroG and ald in the B . subtilis genome, as determined by phage PBS1 transduction mapping, and is not linked to the previously described surfactin loci sfp or srfA . A srfB mutant was found to be also deficient in the establishment of competence . SP beta phage-mediated complementation analysis showed that both competence and surfactin production were restored in the srfB mutant by a single DNA fragment of 1.5 kilobase pairs . The sequence of the complementing DNA revealed that the srfB gene is comA, an early competence gene which codes for a product similar to that of the activator class of bacterial two-component regulatory systems . The srfB mutation impaired the expression of a srfA-lacZ fusion, suggesting that surfactin production is positively regulated at the transcriptional level by the srfB (comA) gene product. J Bacteriol, 1989 Oct, 171(10), 5322 - 4 Bacillus subtilis mutant allele sup-3 causes lysine insertion at ochre codons: use of sup-3 in studies of translational attenuation; Mulbry WW et al.; The mutation sup-3 in Bacillus subtilis suppresses ochre (TAA) mutations at each of three codons in the 5' end of the cat-86 coding sequence . The suppressor is shown to insert lysine at ochre codons . The efficiency of suppression by sup-3 is about 15%, as determined by changing a cat-86 Lys codon (codon 12) to an ochre codon and measuring the level of CAT in the suppressor-containing strain . The results obtained are discussed in light of previous observations that ochre mutations at cat leader codons 2 and 3 can be phenotypically suppressed by sup-3, whereas ochre mutations at leader codons 4 and 5 cannot . Translation of the cat leader is essential to inducible expression of cat . Our data support the interpretation that the nature of amino acids 2 through 5 of the leader peptide contributes to determining whether chloramphenicol can stall a ribosome in the leader, which in turn leads to induction of cat expression. Nucleic Acids Res, 1989 Sep 25, 17(18), 7469 - 86 Cloning and analysis of the spc ribosomal protein operon of Bacillus subtilis: comparison with the spc operon of Escherichia coli; Henkin TM et al.; A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned E . coli rplE (L5) DNA as a hybridization probe . DNA sequence analysis of the B . subtilis cloned DNA indicated a high degree of conservation of spc operon ribosomal protein genes between B . subtilis and E . coli . This fragment contains DNA homologous to the promoter-proximal region of the spc operon, including coding sequences for ribosomal proteins L14, L24, L5, S14, and part of S8; the organization of B . subtilis genes in this region is identical to that found in E . coli . A region homologous to the E . coli L16, L29 and S17 genes, the last genes of the S10 operon, was located upstream from the gene for L14, the first gene in the spc operon . Although the ribosomal protein coding sequences showed 40-60% amino acid identity with E . coli sequences, we failed to find sequences which would form a structure resembling the E . coli target site for the S8 translational repressor, located near the beginning of the L5 coding region in E . coli, in this region or elsewhere in the B . subtilis spc DNA. J Biol Chem, 1989 Sep 5, 264(25), 14784 - 91 Purification and in vitro DNA-binding specificity of the Bacillus subtilis phage phi 105 repressor; Van Kaer L et al.; The Bacillus subtilis phage phi 105 repressor, a lambda repressor-like transcriptional regulatory protein, was overproduced in Escherichia coli and purified to near homogeneity in order to examine its in vitro DNA-binding properties . The active form of repressor appears to be a tetramer . DNase I protection experiments demonstrate that repressor can specifically bind to six distinct sites, all located within the phi 105 EcoRI-F immunity region (immF) . Three of these sites had been identified earlier as functional operators by genetic analysis . They share a common 14-base pair, asymmetric "core" sequence, 5'-GACGGAAATACAAG-3', termed OR . The three additional sites show significant homology with OR . For an individual binding site, hydroxyl-radical footprinting reveals symmetrical repressor-DNA interactions established via one side of the helix . Dimethyl sulfate protection indicates that guanines at the conserved OR base pair positions 1, 3, and 4 may participate in sequence-specific interactions with repressor in agreement with a previously proposed recognition model . However, since the OR sequence is not symmetrical with respect to this GNCG motif, at present it remains difficult to completely understand the molecular basis of this interaction. Food Chem Toxicol, 1989 Sep, 27(9), 599 - 606 Subchronic toxicity studies in dogs and in utero rats fed diets containing Bacillus stearothermophilus alpha-amylase from a natural or recombinant DNA host; MacKenzie KM et al.; Beagle dogs and Fischer 344 rats were fed diets containing 0, 36 or 72 units Bacillus stearothermophilus alpha-amylase (Bsa)/g food or of Bacillus subtilis alpha-amylase (cBsa)/g food . The dogs (four/sex/group) received treated diets for 13 wk . For the rat studies, the parental (F0) generation (12 males and 24 females/group for the Bsa study, and 26 rats/sex/group for the cBsa study) received treated diets for 13 or 4 wk, respectively, before breeding and through weaning of the F1 pups; 20 F1 rats/sex/group received treated diets for at least 13 wk (from weaning until necropsy) . There were no treatment-related antemortem observations, reproductive effects or ophthalmic, haematological, macroscopic or microscopic findings in treated dogs or rats, and no differences were noted in body weights for dogs or parental rats . Mean body weights of F1 pups from F0 rats exposed to 72 units cBsa/g were significantly lower than those of the control animals on lactation day 28 . This effect may have been related to the slight reduction in body weights and significant reduction in food consumption (gestation days 14-20) of the F0 dams . However, this did not continue into the growth phase for the F1 generation . In the Bsa studies, there were no treatment-related effects in clinical pathology values, and organ-weight data did not correlate with macroscopic or microscopic findings . Male dogs given cBsa had significantly lower albumin (36 units/g), calcium (36 and 72 units/g) and inorganic phosphorus (72 units/g) values compared with those of the control males; there were no treatment-related changes in blood chemistry values in rats . Based on the results of these studies, the no-observable-effect level for alpha-amylase fed to dogs or rats is 36 units/g food. Gene, 1989 Sep 1, 81(1), 159 - 63 Investigation into the nature of a Bacillus promoter cloned into a promoter-probe plasmid; Teixeira AV et al.; The alpha-amylase-coding gene (amy) of Bacillus amyloliquefaciens NCP1 was cloned into the Bacillus subtilis promoter probe vector pPL603b.1, using a BglII digest of chromosomal DNA . The resulting plasmid, pVC102, was shown to have a BglII site within the insert . It was determined that this was the result of the fortuitous co-cloning of 2.88-kb and 0.92-kb BglII fragments separated in NCP1 DNA by approx . 3 kb . Unexpectedly, this co-cloning was readily repeated . Subcloning showed that while the 2.88-kb amy-bearing fragment was sufficient for amylase production, it might not have been capable of promoting sufficient levels of chloramphenicol resistance under the conditions used in the cloning experiments . The promoter on the 0.92-kb BglII fragment was more efficient, although its sequence differed from the canonical promoter sequence recognised by B . subtilis RNA polymerase E.sigma 43 . As other promoter-bearing fragments from NCP1 DNA operated equally efficiently when cloned into pPL603b.1, the reason for the repeated co-cloning of the 2.88-kb and 0.92-kb NCPI BglII fragments may well be due to structural parameters, whereby certain nucleotide sequences are more readily cloned than others. Mol Gen Genet, 1989 Sep, 218(3), 565 - 71 Primary structure of the tms and prs genes of Bacillus subtilis; Nilsson D et al.; The nucleotide sequence was determined of a 3211 nucleotide pair EcoRI-PvuII DNA fragment containing the tms and prs genes as well as a part of the ctc gene of Bacillus subtilis . The prs gene encodes phosphoribosylpyrophosphate (PRPP) synthetase, whereas the functioning of the tms and ctc gene products remains to be established . The prs gene contains an open reading frame of 317 codons resulting in a subunit Mr of 34828 . An open reading frame comprising the tms gene contained 456 codons resulting in a putative translation product with an Mr of 49,554 . Comparison of the deduced B . subtilis PRPP synthetase amino acid sequence with PRPP synthetases from Escherichia coli and rat liver showed extensive similarity . The deduced Tms amino acid sequence was found to be 43% similar to the deduced amino acid sequence of ecourfl, a gene of E . coli with unknown function. J Bacteriol, 1989 Sep, 171(9), 4979 - 86 Structure and expression of the cytochrome aa3 regulatory gene ctaA of Bacillus subtilis; Mueller JP et al.; Mutations that define the ctaA gene of Bacillus subtilis block cytochrome aa3 formation and sporulation . We have recently described the isolation and initial characterization of the ctaA locus . Analysis of in vivo mRNA transcripts by RNase protection experiments located the 5' and 3' termini of the ctaA transcript, confirming a monocistronic structure . By using a nuclease protection assay, an increase in the abundance of steady-state ctaA mRNA was observed during the initiation of sporulation, followed by a decrease during subsequent stages . Transcripts originating from the ctaA gene were most abundant 2.0 h after the end of exponential growth . This pattern of ctaA mRNA accumulation was confirmed by coupling the transcription of the ctaA gene to lacZ in an integrative plasmid vector . Expression of ctaA was not repressed by glucose and was independent of the spoOA and spoOH (sigH) gene products . Postexponential expression was found to be dependent on the product of the strC gene . The expression of ctaA appears to be regulated in a growth stage-specific manner . The transcriptional start site, identified by high-resolution S1 nuclease protection experiments, was preceded by a single sigma A-dependent promoter sequence. J Bacteriol, 1989 Sep, 171(9), 4967 - 78 Isolation and sequence of ctaA, a gene required for cytochrome aa3 biosynthesis and sporulation in Bacillus subtilis; Mueller JP et al.; Cytochrome aa3 is one of two terminal oxidase complexes in the Bacillus subtilis electron transport chain . A novel genetic strategy was devised which permitted the isolation of B . subtilis mutants lacking cytochrome aa3 by selection for streptomycin-resistant clones which failed to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine . Two mutations were studied intensively . Spectroscopic examination showed that each mutant lacked cytochrome aa3; they were also asporogenous and unable to grow on lactate as the sole carbon and energy source . These mutations were mapped to a locus designated ctaA, located at 127 degrees between pyrD and metC on the B . subtilis chromosome . Both ctaA mutations were closely linked by transformation to the pycA locus . The ctaA locus and a portion of the pycA locus were cloned from a B . subtilis integration library constructed in Escherichia coli . A recombinant plasmid containing a 4.0-kilobase insert of B . subtilis DNA could transform both ctaA mutants to CtaA+ . Gene disruption and complementation experiments with subcloned fragments revealed that the ctaA locus consisted of a single transcriptional unit about 1.35 kilobase pairs in size . The nucleotide sequence of the ctaA transcriptional unit contains a single open reading frame capable of coding for a protein with a predicted molecular weight of 34,065 . The predicted protein is extremely hydrophobic, with several probable membrane-spanning domains . No sequence similiarity was found between ctaA and the highly conserved procaryotic and mitochondrial oxidase polypeptides . Cloning and sequence analysis of two ctaA mutations revealed that one allele is a nonsense mutation in the carboxy terminus and the other is a missense mutation in the amino terminus; this indicates that the pleiotropic phenotype conferred by each mutation was caused by loss of CtaA or of its activity . Genetic evidence suggests that the ctaA gene product is required as an accessory protein in the genetic expression, posttranslational biogenesis, or both, of the cytochrome aa3 complex and during an early stage of sporogenesis. J Bacteriol, 1989 Sep, 171(9), 4718 - 27 Positive regulation of glutamate biosynthesis in Bacillus subtilis; Bohannon DE et al.; Nitrogen source regulation of glutamate synthase activity in Bacillus subtilis occurs at the level of transcription of the gltA and gltB genes, which encode the two subunits of the enzyme . We show here that transcription of gltA requires the product of gltC, a gene whose transcription is divergent from that of gltA and whose transcriptional control sequences overlap those of gltA . gltC mutants had decreased, aberrantly regulated levels of glutamate synthase activity and decreased gl