J Mol Biol, 1992 Jun 20, 225(4), 1065 - 73 Electron microscopy of decorated crystals for the determination of crystallographic rotation and translation parameters in large protein complexes; Bacher A et al.; The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits enclosing a core of 3 alpha subunits . The preparation of reconstituted hollow capsids consisting of 60 beta subunits and their crystallization in a hexagonal (space group P6(3)22) and in a monoclinic (space group C2) modification have been described . The rotational and translational parameters of the protein molecules in both crystal forms were studied by electron microscopy of freeze-etch replicas and by Patterson correlation techniques . Decoration with silver and image processing provided images with the positions of the 3-fold and 5-fold molecular axes being labelled by metal clusters . This allowed the unequivocal determination of the orientation and translational position of the protein molecules with respect to the crystallographic axes in the hexagonal modification . From inspection of the decoration images it was immediately obvious that the hexagonal crystal forms of alpha 3 beta 60 and of beta 60 are isomorphous . In the monoclinic crystals, a local icosahedral 2-fold coincides with the crystallographic 2-fold axis . The exact solution of the particle orientation was determined by interpretation of Patterson self-rotation functions for the icosahedral symmetry axes . Rotational and translational parameters for the monoclinic modification are given . A rational procedure for the efficient application of freeze-etching techniques in order to elucidate the packing in crystals of large proteins is described. Proc Natl Acad Sci U S A, 1992 Jun 15, 89(12), 5389 - 92 Adenylosuccinate lyase of Bacillus subtilis regulates the activity of the glutamyl-tRNA synthetase; Gendron N et al.; In Bacillus subtilis, the glutamyl-tRNA synthetase {L-glutamate:tRNA(Glu) ligase (AMP-forming), EC 6.1.1.17} is copurified with a polypeptide of M(r) 46,000 that influences its affinity for its substrates and increases its thermostability . The gene encoding this regulatory factor was cloned with the aid of a 41-mer oligonucleotide probe corresponding to the amino acid sequence of an NH2-terminal segment of this factor . The nucleotide sequence of this gene and the physical map of the 1475-base-pair fragment on which it was cloned are identical to those of purB, which encodes the adenylosuccinate lyase (adenylosuccinate AMP-lyase, EC 4.3.2.2), an enzyme involved in the de novo synthesis of purines . This gene complements the purB mutation of Escherichia coli JK268, and its presence on a multicopy plasmid behind the trc promoter in the purB- strain gives an adenylosuccinate lyase level comparable to that in wild-type B . subtilis . A complex between the adenylosuccinate lyase and the glutamyl-tRNA synthetase was detected by centrifugation on a density gradient . The interaction between these enzymes may play a role in the coordination of purine metabolism and protein biosynthesis. J Biol Chem, 1992 Jun 15, 267(17), 12055 - 60 Sequence and characterization of Bacillus subtilis CheW; Hanlon DW et al.; A Bacillus subtilis open reading frame (ORF) encoding a predicted polypeptide of 156 amino acids was subcloned and sequenced . The polypeptide was found to be homologous to CheW of Escherichia coli, sharing 28.6% amino acid identity . The ORF was verified by using a bacteriophage T7 expression system in E . coli . The gene was inactivated by insertion of a nonpolor chloramphenicol acetyltransferase cassette in its N-terminal region . In the absence of chemoeffectors, the mutant displayed a smooth swimming bias, with some tumbling . The CheW- mutant was defective on swarm plates but was complemented by a plasmid that expressed wild type CheW . Addition of attractant or repellent to the CheW- mutant resulted in transient smooth swimming or tumbling, respectively . However, capillary assays revealed that chemotaxis was substantially impaired in the mutant strain. FEMS Microbiol Lett, 1992 Jun 15, 72(3), 255 - 9 Electrochemical sterilization of bacteria absorbed on granular activated carbon; Matsunaga T et al.; The electrochemical sterilization of bacteria adsorbed on granular activated carbon (GAC) was demonstrated . The survival ratio of bacteria on GAC was dependent upon the applied potential . The survival ratio of Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Saccharomyces cerevisiae vs . saturated calomel electrode (SCE) was below 1% at 0.75, 0.8, 1.0 and 0.65 V, respectively . The electrochemical sterilization of tap water containing E . coli was carried out when 3.5 ml of the cell suspension (4.0 x 10(2) cells/ml) was incubated with 2.0 g of GAC for 5 h at 0.7 V vs . SCE . The number of E . coli adsorbed on GAC and the cell concentration in the water decreased rapidly . E . coli on GAC were electrochemically killed and the cell numbers did not increase. Mol Microbiol, 1992 Jun, 6(11), 1495 - 505 The spo0A gene is implicated in the maintenance of non-complementing diploids in Bacillus subtilis; Le Derout J et al.; Bacillus subtilis can exist in a diploid state in which two genetically distinct chromosomes co-exist in the same cell and yet only one of them is expressed, thereby determining the phenotype . Such cells are called non-complementing diploids (Ncds) . In this study, two types of experiments are reported which indicate that a previously known pleiotropic gene, spo0A, plays a role in the maintaining the diploid state, as follows . (i) When protoplasts of two Spo0A mutant strains were fused, the resulting products continued to segregate cells of both parental phenotypes for many more divisions than had been reported previously . (ii) When a stable Ncd (an Ncd in which the unexpressed markers are not spontaneously activated at a detectable level) harbouring a chloramphenicol acetyltransferase gene on the silent chromosome was transformed with spo0A null alleles the transformants often expressed chloramphenicol acetyltransferase activity . Together these results indicate that the spo0A gene is involved in maintenance of the diploid state in both unstable and stable Ncds. Appl Environ Microbiol, 1992 Jun, 58(6), 1930 - 9 Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA; Khanna M et al.; The equilibrium adsorption and binding of DNA from Bacillus subtilis on the clay mineral montmorillonite, the ability of bound DNA to transform competent cells, and the resistance of bound DNA to degradation by DNase I are reported . Maximum adsorption of DNA on the clay occurred after 90 min of contact and was followed by a plateau . Adsorption was pH dependent and was greatest at pH 1.0 (19.9 micrograms of DNA mg of clay-1) and least at pH 9.0 (10.7 micrograms of DNA mg of clay-1) . The transformation frequency increased as the pH at which the clay-DNA complexes were prepared increased, and there was no transformation by clay-DNA complexes prepared at pH 1 . After extensive washing with deionized distilled water (pH 5.5) or DNA buffer (pH 7.5), 21 and 28%, respectively, of the DNA remained bound . Bound DNA was capable of transforming competent cells (as was the desorbed DNA), indicating that adsorption, desorption, and binding did not alter the transforming ability of the DNA . Maximum transformation by bound DNA occurred at 37 degrees C (the other temperatures evaluated were 0, 25, and 45 degrees C) . DNA bound on montmorillonite was protected against degradation by DNase, supporting the concept that "cryptic genes" may persist in the environment when bound on particulates . The concentration of DNase required to inhibit transformation by bound DNA was higher than that required to inhibit transformation by comparable amounts of free DNA, and considerably more bound than free DNase was required to inhibit transformation by the same amount of free DNA . Similarly, when DNA and DNase were bound on the same or separate samples of montmorillonite, the bound DNA was protected from the activity of DNase. Mol Gen Genet, 1992 Jun, 233(3), 483 - 6 Riboflavin operon of Bacillus subtilis: unusual symmetric arrangement of the regulatory region; Kil YV et al.; Seventeen cis-dominant mutations leading to riboflavin overproduction in Bacillus subtilis were localized to the region between nucleotides +37 and +159 relative to the transcription initiation site of the riboflavin operon . This region displays an unusual structure for regulatory sequences . The main part of it represents clusters of A/T and G/C-rich sequences that symmetrically blank a short inverted repeat. J Bacteriol, 1992 Jun, 174(11), 3812 - 7 Mutations in the precursor region of a Bacillus subtilis sporulation sigma factor; Rong S et al.; Transcription from some sporulation-specific promoters of Bacillus subtilis is dependent on synthesis of pro-sigma E and its conversion to sigma E by proteolysis . Certain mutations in the precursor region of sigE, the gene encoding pro-sigma E, apparently allow the mutant sigE products to be active as sigma factors without being proteolysed in the normal way. J Bacteriol, 1992 Jun, 174(11), 3695 - 706 Activation of Bacillus subtilis transcription factor sigma B by a regulatory pathway responsive to stationary-phase signals; Boylan SA et al.; Alternative transcription factor sigma B of Bacillus subtilis controls a stationary-phase regulon induced under growth conditions that do not favor sporulation . Little is known about the metabolic signals and protein factors regulating the activity of sigma B . The operon containing the sigma B structural gene has the gene order orfV-orfW-sigB-rsbX, and operon expression is autoregulated positively by sigma B and negatively by the rsbX product (rsbX = regulator of sigma B) . To establish the roles of the orfV and orfW products, orfV and orfW null and missense mutations were constructed and tested for their effects on expression of the sigma B-dependent genes ctc and csbA . These mutations were tested in two contexts: in the first, the sigB operon was under control of its wild-type, sigma B-dependent promoter, and in the second, the sigB operon promoter was replaced by the inducible Pspac promoter . The principal findings are that (i) the orfV (now called rsbV) product is a positive regulator of sigma B-dependent gene expression; (ii) the orfW (now called rsbW) product is a negative regultor of such expression; (iii) sigma B is inactive during logarithmic growth unless the rsbW product is absent; (iv) the rsbX, rsbV, and rsbW products have a hierarchical order of action; and (v) both the rsbV and rsbW products appear to regulate sigma B activity posttranslationally . There are likely to be at least two routes by which information can enter the system to regulate sigma B: via the rsbX product, and via the rsbV and rsbW products. J Bacteriol, 1992 Jun, 174(11), 3570 - 6 Roles of rpoD, spoIIF, spoIIJ, spoIIN, and sin in regulation of Bacillus subtilis stage II sporulation-specific transcription; Louie P et al.; Bacillus subtilis strains containing defects in the sporulation gene spoIIF (kinA), spoIIJ (kinA), or spoIIN (ftsA) cannot transcribe the sigma E-dependent gene spoIID . Results presented here and by other workers demonstrate that the spoIIF, spoIIJ, and spoIIN gene products control spoIID transcription indirectly by coordinating the induction of the spoIIGAB, spoIIE, and spoIIAC operons, which are required for sigma E synthesis and processing . Sporulation competence and spoIIGAB, spoIIE, and spoIIAC transcription were restored in spoIIF, spoIIJ, and spoIIN mutants by introduction of crsA47, a mutation in the major vegetative sigma factor sigma A . crsA mutations are known to restore sporulation in certain spo0 mutants . crsA suppression of kinA and ftsA mutations was achieved through inhibition of the transcription of sin, a gene involved in the selection between several post-exponential-phase cell states . A deletion of sin restored sporulation competence in spoIIF, spoIIJ, or spoIIN mutant strains . A sin deletion was also able to restore sporulation competence in the crsA suppressible stage 0 mutant spo0K141. J Bacteriol, 1992 Jun, 174(11), 3561 - 9 Sin, a stage-specific repressor of cellular differentiation; Mandic-Mulec I et al.; Sin is a Bacillus subtilis DNA-binding protein which is essential for competence, motility, and autolysin production but also, if expressed on a multicopy plasmid, is inhibitory to sporulation and alkaline protease synthesis . We have now examined the physiological role of Sin in sporulation and found that this protein specifically represses three stage II sporulation genes (spoIIA, spoIIE, and spoIIG) but not the earlier-acting stage 0 sporulation genes . sin loss-of-function mutations cause higher expression of stage II genes and result in a higher frequency of sporulation, in general . Sin binds to the upstream promoter region of spoIIA in vitro and may thus gate entry into sporulation by directly repressing the transcription of stage II genes . In vivo levels of Sin increase rather than decrease at the time of stage II gene induction, suggesting that posttranslational modification may play a role in downregulation of negative Sin function. J Bacteriol, 1992 Jun, 174(11), 3522 - 31 celA from Bacillus lautus PL236 encodes a novel cellulose-binding endo-beta-1,4-glucanase; Hansen CK et al.; celA from the cellulolytic bacterium Bacillus lautus PL236 encodes EG-A, an endo-beta-1,4-glucanase . An open reading frame of 2,100 bp preceded by a ribosome-binding site encodes a protein with a molecular mass of 76,863 Da with a typical signal sequence . The NH2-terminal active domain of EG-A is not homologous to any reported cellulase or xylanase and may represent a new family of such enzymes . A 150-amino-acid COOH-terminal peptide is homologous to noncatalytic domains in several other cellulases (A . Meinke, N.R . Gilkes, D.G . Kilburn, R.C . Miller, Jr., and R.A.J . Warren, J . Bacteriol . 173:7126-7135, 1991) . Upstream of celA, a partial open reading frame encodes a 145-amino-acid peptide which also belongs to the family mentioned . Zymogram analysis of extracts from Escherichia coli and supernatants of Bacillus subtilis and B . megaterium, including protease-deficient mutants thereof, which express celA, revealed two active proteins, EG-A-L and EG-A-S, with Mrs of 74,000 and 57,000, respectively . The proportion of EG-A-L to EG-A-S depends on the extracellular proteolytic activity of the host organism, indicating that EG-A-S arises from posttranslational proteolytic modification of EG-A-L . Since EG-A-S has an NH2 terminus corresponding to the predicted NH2-terminal sequence of EG-A, processing appears to take place between the catalytic and noncatalytic domains described . EG-A-L and EG-A-S were purified to homogeneity and shown to have almost identical characteristics with respect to activity against soluble substrates and pH and temperature dependency . EG-A-L binds strongly to cellulose, in contrast to EG-A-S, and has higher activity against insoluble substrates than the latter . We conclude that the COOH-terminal 17,000-Mr peptide of EG-A-L constitutes a cellulose-binding domain. J Exp Med, 1992 Jun 1, 175(6), 1467 - 71 Listeriolysin O is a target of the immune response to Listeria monocytogenes; Bouwer HG et al.; The immunologic mechanism of protective immunity to the intracellular parasite Listeria monocytogenes (Lm) is not well understood, however, antilisterial immunity can be adoptively transferred with T lymphocytes from Lm-immune donors . The Lm-immune cells are believed to produce macrophage-activating lymphokines, which leads to the eventual macrophage-dependent eradication of the bacterium . Increasing evidence suggests that immunity to Lm resides exclusively within the CD8+ T cell subset . It is possible that the Lm-immune CD8+ T cells function to release sequestered Lm from nonprofessional phagocytes to awaiting activated macrophage populations . This study was conducted to determine if listeriolysin O (LLO), which is an essential determinant of Lm pathogenicity, is also a target of the antilisterial immune response . We have found that target cells infected with a LLO+ Lm strain are lysed by Lm-immune cytotoxic cells, whereas target cells infected with a LLO- Lm mutant, or pulsed with a heat-killed Lm preparation, are not lysed by the Lm-immune effector cells . We have used a Bacillus subtilis (Bs) construct that expresses the LLO gene product and found that target cells infected with the LLO+ Bs construct are lysed by antilisterial cytotoxic cells . The antilisterial cytotoxic response is targeted against LLO, in that we have also used a Bs construct that expresses the perfringolysin (PLO) gene product and found that target cells infected with the PLO+ Bs are not lysed by antilisterial cytotoxic effector cells . These data strongly suggest that LLO is a target antigen of antilisterial immunity and may represent the dominant target during the expression of the immune response to Lm. J Bacteriol, 1992 Jun, 174(11), 3684 - 94 Regulation and expression of the arsenic resistance operon from Staphylococcus aureus plasmid pI258; Ji G et al.; The arsenic resistance operon from Staphylococcus aureus plasmid pI258 was cloned and sequenced . The DNA sequence contains three genes in the order arsR, arsB, and arsC . The predicted amino acid sequences of the gene products are homologous with those of the products of the ars operons of plasmids pSX267 from Staphylococcus xylosus and R773 from Escherichia coli . The cloned staphylococcal ars operon confers resistances to arsenate, arsenite, and antimonite in S . aureus and Bacillus subtilis . The same operon was also expressed in E . coli and conferred resistance to arsenite but less resistance to arsenate and antimonite . Regulation of the pI258 ars operon was studied by using a translational arsB-blaZ fusion in S . aureus and a transcriptional arsB-luxAB fusion in E . coli . The ars operon was induced by arsenate {As(V)}, arsenite {As(III)}, and antimonite {Sb(III)}, to which the strains were resistant, plus Bi(III) in S . aureus . Only arsenate and arsenite induced the operon in E . coli . Northern (RNA) blot DNA-RNA hybridization analysis showed inducible synthesis of a full-length ars mRNA, about 2.1 kb in size, both in S . aureus and in E . coli . S . aureus ars proteins were expressed in E . coli from the T7 phage promoter under the control of the T7 RNA polymerase . Primer extension (reverse transcriptase) analysis showed that the ars mRNA started at the same position (nucleotides 17 and 18 upstream from the arsR ATG) both in S . aureus and in E . coli . An internal deletion mutation in arsB resulted in decreased resistance to arsenate and total loss of arsenite and antimonite resistances . Partial deletion of 56 bp from the 3' end of the arsC gene resulted in loss of resistance to arsenate; the determinant retained arsenite and antimonite resistances. J Bacteriol, 1992 Jun, 174(11), 3676 - 83 Expression and regulation of the antimonite, arsenite, and arsenate resistance operon of Staphylococcus xylosus plasmid pSX267; Rosenstein R et al.; The arsenate, arsenite, and antimonite resistance region of the Staphylococcus xylosus plasmid pSX267 was subcloned in Staphylococcus carnosus . The sequenced DNA region revealed three consecutive open reading frames, named arsR, arsB, and arsC . Expression studies in Escherichia coli with the bacteriophage T7 RNA polymerase-promoter system yielded three polypeptides with apparent molecular weights of 8,000, 35,000, and 15,000, which very likely correspond to ArsR, ArsB, and ArsC, respectively . ArsB was distinguished by its overall hydrophobic character, suggesting a membrane association . The arsenate, arsenite, and antimonite resistance was shown to be inducible by all three heavy metal ions . Inactivation of the first gene, arsR, resulted in constitutive expression of resistance . Similar results were obtained with transcriptional fusions of various portions of the ars genes with a lipase reporter gene, indicating a function of ArsR as a negative regulator of a putative promoter in front of arsR . The inactivation of arsR also resulted in reduction of resistance to arsenite and antimonite, while arsenate resistance was unaffected . The three ars genes conferred arsenite resistance in E . coli and arsenite as well as arsenate resistance in Bacillus subtilis. J Gen Microbiol, 1992 Jun, 138 ( Pt 6), 1089 - 95 Peptide carrier potentiality of Bacillus subtilis levansucrase; Petit-Glatron MF et al.; A synthetic oligodeoxynucleotide encoding the vasopressin peptide was ligated to the 3' terminal codon of sacB, the structural gene of levansucrase . This gene fusion was integrated into the chromosome of a Bacillus subtilis strain able to overproduce levansucrase . The extracellular production of the hybrid protein, consisting of the whole levansucrase primary sequence plus the nine amino acids of the vasopressin peptide added at the C-terminal end, represented 50-55% of that found for the wild-type levansucrase (20 mg l-1) . The purified hybrid protein displayed the same conformational stability, protease insensitivity and enzymic properties as the wild-type levansucrase . However, the rate and the yield of the unfolding-folding transition at the pH and temperature used for bacterial growth were lower in the case of the hybrid protein; the latter also required a higher iron concentration to be completely folded. J Gen Microbiol, 1992 Jun, 138 ( Pt 6), 1077 - 88 Ploidy of Bacillus subtilis exfusants: the haploid nature of cells forming colonies with biparental or prototrophic phenotypes; Hauser PM et al.; To investigate the relationship between DNA content and cell volume, we have attempted to repeat the construction of stable Bacillus subtilis diploid cells through protoplast fusion . Colonies with a biparental phenotype and those with a prototrophic phenotype were identified among exfusants of a cross between two polyauxotrophic strains . The ploidy of cells constituting such colonies was assessed by protoplast self-fusion, determination of the DNA to dry weight ratio of exponentially growing cells, and by quantitative DNA-DNA hybridization . Within the precision of these methods, all colonies were found to consist of haploid cells . A previously described non-complementing diploid was also found to be haploid . Therefore, the genetic evidence in favour of diploidy, based on continuing segregation of cells with a parental or recombinant phenotype, cannot be accounted for except by the maintenance of such cells as a minority population in mixed colonies through cross-feeding . Reconstruction experiments with mixtures of whole parental cells confirm that biparental colonies are indeed mixed colonies which arise either by sticking of parental cells or through coincidence, i.e . their plating within a distance of about 0.4 mm . The previously reported experimental results can be accounted for in the light of our results. Genetika, 1992 Jun, 28(6), 29 - 34 {Integration of the plasmid-cloned lysA gene of Bacillus subtilis into the chromosome of this microorganism}; Kal'cheva EO et al.; Integration of the Bacillus subtilis lysA gene cloned on pLP1 plasmid, into the 250 degrees region of the chromosome of this microorganism was performed . Significant differences in the level and character of their expression were shown between lysA gene integrated into the chromosome and plasmid-borne genes . It is suggested that the expression of the lysA gene could be under control of a certain cis-acting factor which is able to promote transcription of the lysA gene and mediate gene specific regulation. Genetika, 1992 Jun, 28(6), 22 - 8 {Ecologic ultraviolet--a real mutagenic factor for vegetative cells of Bacillus subtilis}; Lotareva OV et al.; The frequency of leu----Leu+ reversions represented mainly by suppressor mutations is increased in Bacillus subtilis uvr+ and uvr-1 cells after exposure to natural sunlight . Dependence of mutation yield on the time of exposure is linear (one hit kinetics) in case of the uvr-1 strain . In the uvr+ cells the yield of mutations is also linear, but only at short times of exposure, the curve bending and levelling off the plateau after 10-min cell illumination . It has been established in the experiments with optical filters that the mutagenic effect is related to wavelengths which correspond to the UVB zone of ecological UV . The mutagenesis caused by sunlight can be modified (weakened) by some post-irradiation treatments of bacteria, which also led to a decrease of mutations frequencies in B . subtilis uvr+ and uvr-1 cells after exposure to 254-nm UV . The data indicate that: 1) mutagenic influence of sunlight can be overcome only by the joint action of activities of the two cellular repair systems--photoreactivation and excision repair, 2) the real mutagenic effect of sunlight on such a non-photoreactivating organism as B . subtilis would not be enhanced with the increase of the UVB flow in sunlight spectrum. Mol Microbiol, 1992 Jun, 6(12), 1579 - 81 Amino acid sequences of several Bacillus subtilis proteins modified by apparent guanylylation; Mitchell C et al.; Bacillus subtilis cell extracts, prepared at different times during growth, contained several proteins that were apparently guanylylated in vitro with {alpha-32P}-GTP . Four of the proteins were partially purified and the N-terminal amino acid sequences (13 to 20 residues) were determined . One sequence had 84% identity to Bacillus stearothermophilus triosephosphate isomerase, two were 100% identical to the predicted sequences of the B . subtilis ptsI and ptsH genes while no identity was found for the fourth sequence . This apparent guanylylation occurred with proteins involved in glucose metabolism, although the significance is unknown. Hua Xi Yi Ke Da Xue Xue Bao, 1992 Jun, 23(2), 190 - 3 {The toxicity of herbicide Asulam}; Hu Y et al.; We conducted a series of toxicity tests and short-term mutagenic assays of Asulam and 40% (W/V) sodium Asulam . It was found that the LD50 of Asulam with acute oral toxicity was 30000 mg/kg for mice, and the LD50 of sodium Asulam for mice and rats were equal (8250 mg/kg) . The cumulative coefficient of sodium Asulam in Wistar rats was 9.42 . None died from sodium Asulam absorbed via skin . Negative results were obtained in Ames test, Bacillus subtilis repair test and the micronucleus test . There was no significant difference between the control group and the treated groups in the chromosomal aberration rates of spermatogonia and primary spermatocytes of mice testis . The results indicated that Asulam should be regarded as a substance of low toxicity and low accumulation . No mutagenicity was observed in our experiment. Protein Expr Purif, 1992 Jun, 3(3), 169 - 77 Protein expression from an Escherichia coli/Bacillus subtilis multifunctional shuttle plasmid with synthetic promoter sequences; Trumble WR et al.; A plasmid shuttle vector (pSP10) was designed and constructed to simplify screening of cloned DNA and to facilitate expression of the protein products . The plasmid contained the following features: (i) a selection gene, chloramphenicol acetyltransferase; (ii) an indicator gene encoding beta-galactosidase for visual identification of colonies containing DNA inserts; (iii) a cloning region immediately upstream from the indicator gene; (iv) origins of replication recognized by both Escherichia coli and Bacillus subtilis; and (v) a synthetic DNA expression control sequence, including -35 and -10 regions, ribosomal binding site, and transcriptional and translational start sites . The promoter region is a synthetic consensus sequence derived from published B . subtilis promoters . The plasmid has been shown to replicate actively in E . coli and B . subtilis and to confer chloramphenicol resistance to both hosts . DNA inserted at the cloning region inactivates the indicator gene, resulting in white colonies on 5'-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside plates . beta-Galactosidase has been expressed from pSP10 in both E . coli and B . subtilis . A comparison was made of the expression levels of beta-galactosidase from the same plasmid which had been modified to contain: (i) the synthetic control region, (ii) no promoter region, (iii) the synthetic control region cloned in the opposite orientation, or (iv) the tac promoter. Biosci Biotechnol Biochem, 1992 Jun, 56(6), 872 - 7 Nucleotide sequence of the gene for an alkaline endoglucanase from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis; Sumitomo N et al.; The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp . KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101 . The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2 . The protein deduced from ORF-1 was composed of 244 amino acids with an M(r) of 27,865 . Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M(r) of 91,040) . Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGCGGTTAACC) . The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E . coli and B . subtilis . One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms. Biosci Biotechnol Biochem, 1992 Jun, 56(6), 867 - 71 Maturation of an NH2-terminally extended thermostable alpha-amylase in Bacillus subtilis: a possible mechanism examined by in vitro experiments; Itoh Y et al.; An artificially inserted extra peptide (21 amino acid peptide) between the B . subtilis alpha-amylase signal peptide and the mature thermostable alpha-amylase was completely cleaved by B . subtilis alkaline protease in vitro . The cleavage to form a mature enzyme was observed between pH 7.5 and 10, but not between pH 6.0 and 6.5, although a similar protease activity toward Azocall was observed between pH 6.0 and 7.5 . To analyze the effects of pH on the cleavage, CD spectra at pH 6, 8, and 11 of the NH2-terminally extended thermostable alpha-amylase were analyzed and the results were compared with those of the mature form of the alpha-amylase . It is suggested that the cleavage of the NH2-terminally extended peptide is controlled by the secondary and tertiary structure of the precursor enzyme . Similar cleavage of different NH2-terminally extended peptides by the alkaline protease was also found in other hybrid thermostable alpha-amylases obtained. J Gen Microbiol, 1992 Jun, 138 ( Pt 6), 1067 - 76 Molecular cloning and sequencing of the upstream region of the major Bacillus subtilis autolysin gene: a modifier protein exhibiting sequence homology to the major autolysin and the spoIID product; Kuroda A et al.; The upstream region of the N-acetylmuramoyl-L-alanine amidase gene (cwlB; a major Bacillus subtilis autolysin) was cloned into Escherichia coli by chromosome walking . Sequencing of the region showed the presence of two open reading frames, one (designated as cwbA) which starts at a UUG codon and encodes a polypeptide of 705 amino acids with an M(r) of 76,725, and the other (designated as lppX), upstream of cwbA, comprising 102 amino acids and having a signal sequence characteristic of a lipoprotein . Purification of the CwbA protein and determination of its N-terminal amino acid sequence revealed that it contains a presumed signal peptide which is processed after Ala at position 25 from the N-terminal, and that the M(r) of the mature form is 75,000 . The amino acid sequences of the N-terminal and C-terminal regions of CwbA were found to be highly homologous with those of the cell wall binding domain of CwlB and the spoIID gene product, respectively . CwbA stimulated the major autolysin activity approximately threefold in vitro . These data indicate that CwbA is the modifier protein of the major autolysin reported by Herbold, D . R . & Glaser, L . (1975; Journal of Biological Chemistry 250, 1676-1682) . In-frame fusion between the lppX and lacZ genes demonstrated that lppX is translated in vivo and expressed during the exponential growth phase. J Bacteriol, 1992 Jun, 174(12), 3981 - 92 Cloning and characterization of the groESL operon from Bacillus subtilis; Li M et al.; The sequence of the 10 N-terminal amino acids of a Bacillus subtilis protein that cross-reacts with antibody to Escherichia coli GroEL was used to design a set of degenerate oligonucleotide probes . These probes identified a clone which carries almost the entire groESL operon from a B . subtilis subgenomic library . By chromosomal walking, an additional fragment carrying the 3' end of groESL and its flanking sequence was isolated . Sequence analysis revealed two open reading frames (ORFs) in the cloned DNA . The upstream ORF encodes a 10-kDa protein which has 47% amino acid identity with E . coli GroES . The downstream ORF encodes a 58-kDa protein which is 62% identical to E . coli GroEL . A 2.1-kb groESL mRNA from B . subtilis was detected independently by Northern (RNA) blot analyses with a groES- and a groEL-specific probe . This demonstrated that groES and groEL are in an operon . The groESL promoter was located by using a promoter-probing plasmid, and the apparent transcription start site was mapped by primer extension analysis . The same promoter is utilized under normal and heat shock conditions . This promoter has the same features as a typical sigma A promoter . A strain in which the groESL operon was under the control of the sucrose-inducible sacB promoter was created . With this strain, it was possible to show that both groES and groEL are essential genes under both normal and heat shock conditions. J Bacteriol, 1992 Jun, 174(12), 3928 - 35 Cloning and nucleotide sequence of the leucyl-tRNA synthetase gene of Bacillus subtilis; Vander Horn PB et al.; The leucyl-tRNA synthetase gene (leuS) of Bacillus subtilis was cloned and sequenced . A mutation in the gene, leuS1, increases the transcription and expression of the ilv-leu operion, permitting monitoring of leuS alleles . The leuS1 mutation was mapped to 270 degrees on the chromosome . Sequence analysis showed that the mutation is a single-base substitution, possibly in a monocistronic operon . The leader mRNA predicted by the sequence would contain a number of possible secondary structures and a T box, a sequence observed upstream of leader mRNA terminators of Bacillus tRNA synthetases and the B . subtilis ilv-leu operon . The DNA of the B . subtilis leuS open reading frame is 48% identical to the leuS gene of Escherichia coli and is predicted to encode a polypeptide with 46% identity to the leucyl-tRNA synthetase of E . coli. J Biol Chem, 1992 May 25, 267(15), 10225 - 31 Molecular cloning, sequencing, and physiological characterization of the qox operon from Bacillus subtilis encoding the aa3-600 quinol oxidase; Santana M et al.; Bacillus subtilis contains two aa3-type terminal oxidases (caa3-605 and aa3-600) catalyzing cytochrome c and quinol oxidation, respectively, with the concomitant reduction of O2 to H2O (Lauraeus, M., Haltia, T., Saraste, M., and Wikstrom, M . (1991) Eur . J . Biochem . 197, 699-705) . Previous studies characterized only the structural genes of caa3-605 oxidase . We isolated the genes coding for the four subunits of a B . subtilis terminal oxidase from a genomic DNA library . These genes, named qoxA to qoxD, are organized in an operon . Examination of the deduced amino acid sequence of Qox subunits showed that this oxidase is structurally related to the large family of mitochondrial-type aa3 terminal oxidases . In particular, the amino acid sequences are very similar to those of subunits of Escherichia coli bo quinol oxidase and B . subtilis caa3-605 cytochrome c oxidase . We produced, by in vitro mutagenesis, a mutation in the qox operon . From the phenotype of the mutant strain devoid of Qox protein, the study of expression of the qox operon in different growth conditions, and the analysis of the deduced amino acid sequence of the subunits, we concluded that Qox protein and aa3-600 quinol oxidase are the same protein . Although several terminal oxidases are found in B . subtilis, Qox oxidase (aa3-600) is predominant during the vegetative growth and its absence leads to important alterations of the phenotype of B . subtilis. Biochemistry, 1992 May 12, 31(18), 4413 - 25 Assignment of the aliphatic 1H and 13C resonances of the Bacillus subtilis glucose permease IIA domain using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy; Fairbrother WJ et al.; Nearly complete assignment of the aliphatic 1H and 13C resonances of the IIAglc domain of Bacillus subtilis has been achieved using a combination of double- and triple-resonance three-dimensional (3D) NMR experiments . A constant-time 3D triple-resonance HCA(CO)N experiment, which correlates the 1H alpha and 13C alpha chemical shifts of one residue with the amide 15N chemical shift of the following residue, was used to obtain sequence-specific assignments of the 13C alpha resonances . The 1H alpha and amide 15N chemical shifts had been sequentially assigned previously using principally 3D 1H-15N NOESY-HMQC and TOCSY-HMQC experiments {Fairbrother, W . J., Cavanagh, J., Dyson, H . J., Palmer, A . G., III, Sutrina, S . L., Reizer, J., Saier, M . H., Jr., & Wright, P . E . (1991) Biochemistry 30, 6896-6907} . The side-chain spin systems were identified using 3D HCCH-COSY and HCCH-TOCSY spectra and were assigned sequentially on the basis of their 1H alpha and 13C alpha chemical shifts . The 3D HCCH and HCA(CO)N experiments rely on large heteronuclear one-bond J couplings for coherence transfers and therefore offer a considerable advantage over conventional 1H-1H correlation experiments that rely on 1H-1H 3J couplings, which, for proteins the size of IIAglc (17.4 kDa), may be significantly smaller than the 1H line widths . The assignments reported herein are essential for the determination of the high-resolution solution structure of the IIAglc domain of B . subtilis using 3D and 4D heteronuclear edited NOESY experiments; these assignments have been used to analyze 3D 1H-15N NOESY-HMQC and 1H-13C NOESY-HSQC spectra and calculate a low-resolution structure {Fairbrother, W . J., Gippert, G . P., Reizer, J., Saier, M . H., Jr., & Wright, P . E . (1992) FEBS Lett . 296, 148-152}. Biochemistry, 1992 May 12, 31(18), 4394 - 406 Backbone dynamics of the Bacillus subtilis glucose permease IIA domain determined from 15N NMR relaxation measurements; Stone MJ et al.; The backbone dynamics of the uniformly 15N-labeled IIA domain of the glucose permease of Bacillus subtilis have been characterized using inverse-detected two-dimensional 1H-15N NMR spectroscopy . Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state (1H)-15N NOEs were measured, at a spectrometer proton frequency of 500 MHz, for 137 (91%) of the 151 protonated backbone nitrogens . These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and 15N exchange broadening contributions (Rex) for each residue, as well as the overall molecular rotational correlation time (tau m) . The T1 and T2 values for most residues were in the ranges 0.45-0.55 and 0.11-0.15 s, respectively; however, a small number of residues exhibited significantly slower relaxation . Similarly, (1H)-15N NOE values for most residues were in the range 0.72-0.80, but a few residues had much smaller positive NOEs and some exhibited negative NOEs . The molecular rotational correlation time was 6.24 +/- 0.01 ns; most residues had order parameters in the range 0.75-0.90 and tau e values of less than ca . 25 ps . Residues found to be more mobile than the average were concentrated in three areas: the N-terminal residues (1-13), which were observed to be highly disordered; the loop from P25 to D41, the apex of which is situated adjacent to the active site and may have a role in binding to other proteins; and the region from A146 to S149 . All mobile residues occurred in regions close to termini, in loops, or in irregular secondary structure. J Biol Chem, 1992 May 5, 267(13), 9158 - 69 Functional interactions between proteins of the phosphoenolpyruvate:sugar phosphotransferase systems of Bacillus subtilis and Escherichia coli; Reizer J et al.; Proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus subtilis were overexpressed, purified to near homogeneity, and characterized . The proteins isolated include Enzyme I, HPr, the glucose-specific IIA domain of the glucose-specific Enzyme II (IIAglc), and the mannitol-specific IIA protein, IIAmtl . Site specific mutant proteins of IIAglc and HPr were also overexpressed and purified, and their properties were compared with those of the wild type proteins . These proteins and their phosphorylated derivatives were characterized with respect to their immunological cross-reactivities employing the Western blot technique and in terms of their migratory behavior during sodium dodecyl sulfate-gel electrophoresis, nondenaturing gel electrophoresis, and isoelectric focusing . The interactions between homologous and heterologous Enzymes I and HPrs, between homologous and heterologous HPrs and the IIAglc proteins, and between homologous and heterologous IIAglc proteins and IIBCscr of B . subtilis as well as IICBglc of Escherichia coli were defined and compared kinetically . The mutant HPrs and IIAglc proteins were also characterized kinetically as PTS phosphocarrier proteins and/or as inhibitors of the phosphotransferase reactions of the PTS . These studies revealed that complexation of IIAglc with the mutant form of HPr in which serine 46 was replaced by aspartate (S46D) did not increase the rate of phosphoryl transfer from phospho Enzyme I to S46D HPr more than when IIAmtl was complexed to S46D HPr . These findings do not support a role for HPr(Ser-P) in the preferential utilization of one PTS carbohydrate relative to another . Functional analyses in E . coli established that IIAglc of B . subtilis can replace IIAglc of E . coli with respect both to sugar transport and to regulation of non-PTS permeases, catabolic enzymes, and adenylate cyclase . Site-specific mutations in histidyl residues 68 and 83 (H68A and H83A) inactivated IIAglc of B . subtilis with respect to phosphoryl transfer and its various regulatory roles. J Mol Biol, 1992 May 5, 225(1), 81 - 92 Identification of a gene in Bacillus subtilis bacteriophage SPP1 determining the amount of packaged DNA; Tavares P et al.; The virulent Bacillus subtilis bacteriophage SPP1 encapsidates its DNA by a headful mechanism . Analyzing phage missense mutants, which package less DNA than SPP1 wild-type but show no other affected properties, we have identified a gene whose product is involved in the sizing of phage DNA during maturation . Characterization of this gene and its product provides an experimental access to the poorly understood mechanism of DNA sizing in packaging . The gene (gene 6 or siz) was cloned and sequenced . An open reading frame (ORF) coding for a 57.3 kDa polypeptide was identified . All the single nucleotide substitutions present in different siz mutants affect the net charge of that protein . The gene was further characterized by assignment of several nonsense mutations (sus) to the ORF . Phages carrying the latter type of mutations could be complemented in trans when gene 6 is provided by a plasmid. J Biol Chem, 1992 May 5, 267(13), 9146 - 9 Mutational identification of an essential tryptophan in tryptophanyl-tRNA synthetase of Bacillus subtilis; Chow KC et al.; The strongly conserved single tryptophan residue, Trp92, in Bacillus subtilis tryptophanyl-tRNA synthetase has been mutagenized via site direction singly into Gln, Ala, and Phe . All three mutant enzymes were inactive toward the catalysis of tRNA tryptophanylation . The Trp92----Phe mutant has been subcloned into the high expression plasmid pKK223-3 to yield the recombinant plasmid pKSW-F92 . Growth of bacteria carrying the latter plasmid made possible the purification of the mutant TrpRS-F92 enzyme to homogeneity . This mutant enzyme was deficient in ultraviolet absorbance and fluorescence relative to the wild type enzyme and inactive in the partial reaction of Trp-activation as well as the overall reaction of tRNA tryptophanylation . Furthermore, unlike the wild type B . subtilis trpS gene, the mutant trpS-F92 gene upon transformation into Escherichia coli trpS 10343 failed to complement the temperature sensitive trpS mutation of the host cells . Trp92 therefore represents an essential residue both in vitro and in vivo for the function of the tryptophanyl-tRNA synthetase. Mol Microbiol, 1992 May, 6(10), 1345 - 9 The amino acid sequence of a Bacillus subtilis phosphoprotein that matches an orfY-tsr coding sequence; Mitchell C et al.; Bacillus subtilis contains a 30 kDa protein which was phosphorylated during late vegetative growth and sporulation . The sequence for the N-terminal 16 amino acids was found to be identical to the predicted sequence for the N-terminus of a small open reading frame, orfY, but diverged from the predicted sequence thereafter . The orfY region was resequenced and contained one less adenine residue than previously reported, resulting in an open reading frame from within orfY through the entire coding region for tsr which follows orfY . The predicted orfY-tsr amino acid sequence showed 24% identity to Escherichia coli fructose-1,6-bisphosphate aldolase . Two mutants in the tsr region had 2-5% of wild-type aldolase and the nucleotide sequences showed missense mutations . These results indicate that orfY-tsr encodes aldolase and should be renamed fba1. Mol Microbiol, 1992 May, 6(10), 1263 - 70 Inducible DNA repair and differentiation in Bacillus subtilis: interactions between global regulons; Yasbin RE et al.; The SOS response of Escherichia coli has become a paradigm for the study of inducible DNA repair and recombination processes in many different organisms . While these studies have demonstrated that the components of the SOS response appear to be highly conserved among bacterial species, as with most models, there are some significant variations . Perhaps the best example of this comes from an analysis of the SOS-like system of the developmental organism, Bacillus subtilis . Accordingly, the most striking difference is the complex developmental regulation of the SOS system as this organism differentiates into its competent state . In this review we have given an overview of the elements that comprise the SOS system of B . subtilis . Additionally, we have summarized our most recent findings regarding the regulation of this regulon . Using these results along with new findings from other laboratories we have provided provocative molecular models for the regulation of the B . subtilis SOS system in response to DNA damage and during competent cell formation. J Biochem (Tokyo), 1992 May, 111(5), 638 - 42 Dihydrofolate reductase from Bacillus subtilis and its artificial derivatives: expression, purification, and characterization; Iwakura M et al.; The Bacillus subtilis dihydrofolate reductase (DHFR) gene was expressed in Escherichia coli . The gene product was purified to homogeneity by Butyl-Toyopearl, Toyopearl HW55, and DEAE-Toyopearl column chromatographies, and its molecular properties were compared to those of E . coli DHFR . The specific enzyme activity of the B . subtilis DHFR was 240 units/mg under the standard assay conditions, being about four times higher than that of the E . coli DHFR . Km for coenzyme NADPH was 20.7 microM, a value about three times larger than that of E . coli, whereas Km (1.5 microM) for the substrate, dihydrofolate, was similar to that of E . coli DHFR . This seems to reflect the low homology of the amino acid sequence in residues 61-88 of the two DHFRs where one of the NADPH binding sites is located {Bystrof, C . & Kraut, J . (1991) Biochemistry 30, 2227-2239} . Similar to the E . coli DHFR {Iwakura, M . et al . (1992) J . Biochem . 111, 37-45}, the extension of amino acid sequences at the C-terminal end of the B . subtilis DHFR could be attained without loss of the enzyme function or decrease of the protein yield . Thus, the DHFR is useful as a carrier protein for expressing small polypeptides, such as leucine enkephalin, bradykinin, and somatostatin. Genetika, 1992 May, 28(5), 5 - 10 {Cloning and regulation of the expression of the lysA gene from Bacillus subtilis}; Kal'cheva EO et al.; Cloning of Bacillus subtilis DNA fragment with the lysA gene encoding diaminopimelatecarboxylase (EC 4.1.1.20) was done . The cloned gene in poorly expressed both in Escherichia coli and in Bacillus subtilis . Some DNA sequence distant from the lysA gene seems to be necessary for full gene expression, this sequence having been not cloned together with the lysA . The sequence in needed for regulation of the expression as well. Genetika, 1992 May, 28(5), 29 - 39 {Bacillus subtilis gene rec223: molecular cloning and proposed function of its protein product}; Gavrilova EV et al.; Chromosomal DNA fragment which complemented rec223 mutation of Bacillus subtilis was cloned . Introduction of one copy of the cloned gene into the cells of the rec mutant restored both normal activity for DNA damages repair after mitomycin C action and recombination proficiency . Using multicopy vector led to no formation of recombinants, which was probably connected with overproduction of rec223 gene protein product in Bacillus subtilis cells. Biochem J, 1992 May 1, 283 ( Pt 3), 649 - 52 Inhibition of bacterial protein synthesis by elongation-factor-Tu-binding antibiotics MDL 62,879 and efrotomycin; Landini P et al.; MDL 62,879 (formerly GE 2270 A) is a novel antibiotic active against Gram-positive bacteria by inhibiting protein synthesis . MDL 62,879 is not active against Gram-negative bacteria, but inhibits cell-free protein synthesis in extracts from Escherichia coli, and shows a high binding affinity for its elongation factor Tu (EF-Tu) . We prepared ribosomes and protein-synthesis elongation factors from three sources: E . coli, Bacillus subtilis, and a strain of B . subtilis selected for resistance to MDL 62,879 (strain G1674) . Homologous and heterologous reconstituted systems were used to compare the effects of MDL 62,879 and of efrotomycin, an EF-Tu inhibitor of the kirromycin class, which is inactive against both B . subtilis and E . coli . We showed that in cell-free protein synthesis: (a) E . coli was sensitive to both MDL 62,879 and efrotomycin; (b) B . subtilis was sensitive to MDL 62,879, but not to efrotomycin; (c) B . subtilis G1674 was resistant to both antibiotics . In the E . coli system and in the system from wild-type B . subtilis, inhibition by MDL 62,879 was reversed upon addition of purified EF-Tu from B . subtilis G1674 . This demonstrates that the antibiotic acts by inhibition of EF-Tu . In contrast, extracts from B . subtilis failed to restore activity in an efrotomycin-inhibited E . coli system . Dominance or resistance to MDL 62,879 and of sensitivity to efrotomycin in heterologous cell-free protein synthesis confirms that inhibition of EF-Tu by the two antibiotics is mediated by different mechanisms of action. Gene, 1992 May 1, 114(1), 121 - 6 Modular expression and secretion vectors for Bacillus subtilis; Nagarajan V et al.; A modular vector system has been developed for the extracellular production of heterologous proteins in Bacillus subtilis . This modular vector system consists of four secretion vectors which are based upon the genes encoding the Bacillus amyloliquefaciens extracellular alkaline protease, neutral protease, barnase and levansucrase . The modular vectors contain compatible restriction sites downstream from the signal peptide-coding region . Three reporter proteins (staphylococcal protein A, levansucrase and Escherichia coli alkaline phosphatase) that offer complementary advantages for cloning, genetic manipulations and media optimization have been fused to the various signal peptides . These secretion vectors function in E . coli and hence can be used to compare the mechanisms of protein secretion in E . coli and B . subtilis. EMBO J, 1992 May, 11(5), 1867 - 73 Histone H4-related osteogenic growth peptide (OGP): a novel circulating stimulator of osteoblastic activity; Bab I et al.; It has been established that regenerating marrow induces an osteogenic response in distant skeletal sites and that this activity is mediated by factors released into the circulation by the healing tissue . In the present study we have characterized one of these factors, a 14 amino acid peptide named osteogenic growth peptide (OGP) . Synthetic OGP, identical in structure to the native molecule, stimulates the proliferation and alkaline phosphatase activity of osteoblastic cells in vitro and increases bone mass in rats when injected in vivo . Immunoreactive OGP in high abundance is present physiologically in the serum, mainly in the form of an OGP-OGP binding protein complex . A marked increase in serum bound and unbound OGP accompanies the osteogenic phase of post-ablation marrow regeneration and associated systemic osteogenic response . Authentic OGP is identical to the C-terminus of histone H4 and shares a five residue motif with a T-cell receptor beta-chain V-region and the Bacillus subtilis outB locus . Since these latter proteins have not been implicated previously in the control of cell proliferation or differentiation, OGP may belong to a novel, heretofore unrecognized family of regulatory peptides . Perhaps more importantly, OGP appears to represent a new class of molecules involved in the systemic control of osteoblast proliferation and differentiation. J Bacteriol, 1992 May, 174(10), 3212 - 9 Transcriptional regulation of the ilv-leu operon of Bacillus subtilis; Grandoni JA et al.; We used primer extension and mutational analysis to identify a promoter upstream of ilvB, the first gene in the ilv-leu operon of Bacillus subtilis . Between the promoter and ilvB, there is a 482-bp leader region which contains a sequence that resembles a factor-independent transcription terminator . In in vitro transcription experiments, 90% of transcripts initiated at the ilvB promoter ended at a site near this terminator . Primer extension analysis of RNA synthesized in vivo showed that the steady-state level of mRNA upstream of the terminator was twofold higher from cells limited for leucine than it was from cells grown with excess leucine . mRNA downstream of the terminator was 14-fold higher in cells limited for leucine than in cells grown with excess leucine . Measurement of mRNA degradation rates showed that the half-life of ilv-leu mRNA was the same when the cells were grown with or without leucine . These data demonstrate that the ilv-leu operon is regulated by transcription attenuation. J Bacteriol, 1992 May, 174(10), 3177 - 84 Characterization of bofA, a gene involved in intercompartmental regulation of pro-sigma K processing during sporulation in Bacillus subtilis; Ricca E et al.; Sporulating cells of the gram-positive bacterium Bacillus subtilis are partitioned into two cellular compartments called the mother cell and the forespore . Gene expression in the mother cell and the forespore is regulated differentially by the compartment-specific transcription factors sigma K and sigma G, respectively . Gene expression between the two compartments is also coordinated by a signal transduction pathway that couples the activation of sigma K (by processing of its inactive precursor pro-sigma K) in the mother cell to sigma G-directed gene expression in the forespore . To dissect the signal transduction pathway genetically, we previously isolated bypass of forespore mutations at loci called bofA and bofB that relieve the dependence of pro-sigma K processing on the action of sigma G . bofB mutations were previously shown to be allelic to the two-cistron sporulation operon spoIVF, which encodes the pro-sigma K-processing enzyme or its regulator . We now report that bofA mutations are located in a small open reading frame of 87 codons that encodes a putative integral membrane protein with three potential membrane-spanning domains . The possibility is discussed that BofA and the SpoIVF proteins form a heteromeric complex in the mother cell membrane that surrounds the forespore and that this complex mediates the intercompartmental coupling of pro-sigma K processing to events in the forespore. J Bacteriol, 1992 May, 174(10), 3171 - 6 Expression of the Bacillus subtilis dinR and recA genes after DNA damage and during competence; Raymond-Denise A et al.; The Bacillus subtilis dinR gene product is homologous to the LexA protein of Escherichia coli and regulates the expression of dinR and dinC . Using transcriptional fusions in the dinR and the recA genes, we have investigated the epistatic relationship between these two genes during the SOS response induced either by DNA damage or by competence . The results show that after DNA damage, induction of the expression of both recA and dinR is dependent on the activity of the DinR and RecA proteins . A RecA-dependent activity on DinR is proposed as the initial event in the induction of the SOS network . In contrast, the competence-related induction of dinR and recA appears to involve two distinct mechanisms . While one mechanism corresponds to the classical regulation of the SOS response, the other appears to involve an activating factor . Moreover, this factor is active in cells in which competence is prevented by a mutation in the regulatory gene comA. J Bacteriol, 1992 May, 174(10), 3161 - 70 Regulation of the sacPA operon of Bacillus subtilis: identification of phosphotransferase system components involved in SacT activity; Arnaud M et al.; The sacT gene which controls the sacPA operon of Bacillus subtilis encodes a polypeptide homologous to the B . subtilis SacY and the Escherichia coli BglG antiterminators . Expression of the sacT gene is shown to be constitutive . The DNA sequence upstream from sacP contains a palindromic sequence which functions as a transcriptional terminator . We have previously proposed that SacT acts as a transcriptional antiterminator, allowing transcription of the sacPA operon . In strains containing mutations inactivating ptsH or ptsI, the expression of sacPA and sacB is constitutive . In this work, we show that this constitutivity is due to a fully active SacY antiterminator . In the wild-type sacT+ strain or in the sacT30 mutant, SacT requires both enzyme I and HPr of the phosphotransferase system (PTS) for antitermination . It appears that the PTS exerts different effects on the sacB gene and the sacPA operon . The general proteins of the PTS are not required for the activity of SacY while they are necessary for SacT activity. J Bacteriol, 1992 May, 174(10), 3147 - 51 A cluster of nine tRNA genes between ribosomal gene operons in Bacillus subtilis; Green CJ et al.; A cluster of nine tRNA genes located in the 1-kb region between ribosomal operons rrnJ and rrnW in Bacillus subtilis has been cloned and sequenced . This cluster contains the genes for tRNA(UACVal), tRNA(UGUThr), tRNA(UUULys), tRNA(UAGLeu) . tRNA(GCCGly), tRNA(UAALeu), tRNA(ACGArg), tRNA(UGGPro), and tRNA(UGCAla) . The newly discovered tRNA gene cluster combines features of the 3'-end of trnI, a cluster of 6 tRNA genes between ribosomal operons rrnI and rrnH, and of the 5'-end of trnB, a cluster of 21 tRNA genes found immediately 3' to rrnB . Neither the tRNA(UAGLeu) gene nor its product has been found previously in B . subtilis . With the discovery of this new set of tRNA genes, a total of 60 such genes have now been found in B . subtilis . These known genes account for almost all of the tRNA hybridizing restriction fragments of the B . subtilis genome . The 60 known tRNA genes of B . subtilis code for only 28 different anticodons, compared with a total of 41 different anticodons for 78 tRNA genes in Escherichia coli . This may indicate that B . subtilis does not need as many anticodons because of more flexible translation rules, similar to the situation in Mycoplasma capricolum. J Bacteriol, 1992 May, 174(9), 3049 - 55 Catabolite repression of the xyl operon in Bacillus megaterium; Rygus T et al.; We characterized catabolite repression of the genes encoding xylose utilization in Bacillus megaterium . A transcriptional fusion of xylA encoding xylose isomerase to the spoVG-lacZ indicator gene on a plasmid with a temperature-sensitive origin of replication was constructed and efficiently used for single-copy replacement cloning in the B . megaterium chromosome starting from a single transformant . In the resulting strain, beta-galactosidase expression is 150-fold inducible by xylose and 14-fold repressed by glucose, showing that both regulatory effects occur at the level of transcription . Insertion of a kanamycin resistance gene into xylR encoding the xylose-dependent repressor leads to the loss of xylose-dependent regulation and to a small drop in the efficiency of glucose repression to eightfold . Deletion of 184 bp from the 5' part of the xylA reading frame reduces glucose repression to only twofold . A potential glucose-responsive element in this region is discussed on the basis of sequence similarities to other glucose-repressed genes in Bacillus subtilis . The sequence including the glucose-responsive element is also necessary for repression exerted by the carbon sources fructose and mannitol . Their efficiencies of repression correlate to the growth rate of B . megaterium, as is typical for catabolite repression . Glycerol, ribose, and arabinose exert only a basal twofold repression of the xyl operon, which is independent of the presence of the cis-active glucose-responsive element within the xylA reading frame. J Bacteriol, 1992 May, 174(9), 2771 - 8 Bacillus subtilis early sporulation genes kinA, spo0F, and spo0A are transcribed by the RNA polymerase containing sigma H; Predich M et al.; The Bacillus subtilis genes kinA (spoIIJ), spo0F, and spo0A encode components of the sporulation signal transduction pathway . Recent work has suggested that these genes are transcribed by a minor form of RNA polymerase, E sigma H (sigma H is the product of spo0H, another early sporulation gene) . We directly tested this hypothesis by performing in vitro transcription assays with reconstituted E sigma H and a set of plasmids containing the kinA, spo0F, and spo0A promoter regions . We were able to obtain distinct transcripts of the expected sizes with all three genes by using linearized or supercoiled templates . Furthermore, primer extension experiments indicate that the transcription start sites for the three genes in vitro and in vivo are the same . In addition, we measured steady-state levels of kinA, spo0F, and spo0A mRNAs during growth in sporulation medium; all of them were increased at or near the beginning of the stationary phase. J Inorg Biochem, 1992 May 1, 46(2), 119 - 27 Ligational behavior of N-substituted acid hydrazides towards transition metals and potentiation of their microbiocidal activity; Malhotra R et al.; New complexes of Cu(II), Ni(II), and Co(II) with 3-benzoyl-1-{2-N-(substituted-2'-thienyl methylmethylene/methylene)} prop-2-ene-1-oic acid hydrazides have been synthesized and characterized by elemental analysis, molecular weight determination, molar conductance, and magnetic moment and spectroscopic techniques . Conductance measurements indicate the nonionic nature of the complexes . From the spectroscopic studies, it has been concluded that the N-substituted acid hydrazides act as tridentate ligands forming an O-N-S conjugate system and coordinating with metal ions through oxygen of carbonyl group, nitrogen of azomethine, and sulphur of thiophene moiety . Octahedral geometry has been proposed for all the complexes . The ligands and their complexes were tested for in vitro growth inhibitory activity against phytopathogenic fungi viz . Alternaria alternata, Colletotrichum capsicum, Fusarium oxysporum, and Rhizoctonia solani at 28 degrees C; and bacteria viz . gram positive Bacillus subtilis and gram negative Escherichia coli at 37 degrees C by a two-fold serial dilution technique . In some cases an increase in the biocidal activity of the ligands as a consequence of coordination with metal ions was observed in terms of minimum inhibitory concentration (MIC) values . The trend of growth inhibition in the complexes was found to be in the order: Cu greater than Ni greater than Co. Mol Gen Mikrobiol Virusol, 1992 May-Jun, (5-6), 5 - 10 {Plasmids from bacilli related to BAcillus subtilis}; Kanapina ASh; The basic structural and functional properties of natural bacilli plasmids are analyzed in this review . Bacilli plasmids are mostly cryptic, but some are found to have selective markers . Small plasmids replicate by rolling-circle mechanism, however, the replication of large plasmids is likely to occur by the theta-mechanism . Plasmid structures involved in replication are analyzed . The bacilli plasmids are stable . They are promising material for vector construction. Mol Gen Mikrobiol Virusol, 1992 May-Jun, (5-6), 16 - 9 {Induction of the SOS-like system in Rec-mutants of Bacillus subtilis}; Suslov AV et al.; Influence of the recE1, recB2, recB3, recB19, recF15, recF18, recL16, recM13 and recM27 mutations of the induction of the SOS-like system component, i . e . the RecE protein of Bacillus subtilis was studied by RIA-dot-blot method in UV-irradiated or treated by nalidixic acid cells . These agents caused a significant increase in the wild type (rec+) cells but did not stimulate the RecE synthesis in the rec mutants tested . The two exceptions were recB2 and recF18 mutants treated by nalidixic acid . The tsi23 mutation caused thermoinduction of phi 105 bacteriophage in the rec+ genetic background while no prophage particles were induced in the recE, recF, recL, recM mutants . The data suggest that the genetic damage of several rec genes including recB, recE, recF, recL and recM can block induction of the SOS-like system of Bacillus subtilis. Chem Pharm Bull (Tokyo), 1992 May, 40(5), 1116 - 9 Isolation and structures of antibacterial binaphtho-alpha-pyrones, talaroderxines A and B, from Talaromyces derxii; Suzuki K et al.; New compounds designated talaroderxines A (1) and B (2) were isolated from a new heterothallic ascomycetous fungus, Talaromyces derxii, cultivated on rice . The structures of 1a and 1b were elucidated by means of spectroscopic examination and chemical reactions . Talaroderxines A (1a) and B (1b) are atropisomers of a 6,6'-binaphtho-alpha-pyrone derivative, and have strong antibacterial activity against Bacillus subtilis. Mol Microbiol, 1992 May, 6(9), 1105 - 14 The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo; Vellanoweth RL et al.; A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo . The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms . Both B . subtilis and E . coli exhibited similar spacing optima of 7-9 nucleotides . However, B . subtilis translated messages with spacings shorter than optimal much less efficiently than E . coli . In both organisms, AUG was the preferred initiation codon by two- to threefold . In E . coli GUG was slightly better than UUG while in B . subtilis UUG was better than GUG . The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms . B . subtilis was also much less able to tolerate secondary structure in the RBS than E . coli . While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity . The approach described provides a rapid and systematic means of elucidating such additional determinants. Appl Microbiol Biotechnol, 1992 May, 37(2), 211 - 5 The DNA sequence of gamma-glutamyltranspeptidase gene of Bacillus subtilis (natto) plasmid pUH1; Hara T et al.; The gamma-glutamyltranspeptidase (gamma-GTP) gene of Bacillus subtilis (natto) plasmid designated pUH1, which is responsible for polyglutamate production, has been cloned and the nucleotide sequence determined . The sequence contains a single open-reading frame stretching for 1260 bp with a relative molecular mass of 49,356 . Putative -35 and -10 sequences, TTCAAA and TATTAT, were observed as the consensus sequence for the promoter recognized by the sigma 43 RNA polymerase of B . subtilis, and the ribosome binding site, the sequence of which was AACGAG, was complementary to the binding sequence of B . subtilis 16S rRNA except for one base . The amino acid sequence of the gene with the segment of putative protein C403 of staphylococcal plasmid pE194 indicates homology, whereas that with Escherichia coli and mammalian gamma-GTPs does not show any similarity at all. Biotechnol Prog, 1992 May-Jun, 8(3), 211 - 8 Physiological and genetic strategies for enhanced subtilisin production by Bacillus subtilis; Pierce JA et al.; Defined minimal media conditions were used to assess and subsequently enhance the production of subtilisin by genetically characterized Bacillus subtilis strains . Subtilisin production was initiated by the exhaustion or limitation of ammonium in batch and fed-batch cultures . Expression of the subtilisin gene (aprE) was monitored with a chromosomal aprE::lacZ gene fusion . The beta-galactosidase production driven by this fusion reflected subtilisin accumulation in the culture medium . Subtilisin gene expression was temporally extended in sporulation-deficient strains (spoIIG), relative to co-genic sporogenous strains, resulting in enhanced subtilisin production . Ammonium exhaustion not only triggered subtilisin production in asporogenous spoIIG mutants but also shifted carbon metabolism from acetate production to acetate uptake and resulted in the formation of multiple septa in a significant fraction of the cell population . Fed-batch culture techniques, employing the spoIIG strain, were investigated as a means to further extend subtilisin production . The constant provision of ammonium resulted in linear growth, with doubling times of 11 and 36 h in each of two independent experiments . At the lower growth rate, the responses elicited (subtilisin production, glucose metabolism, and morphological changes) during the feeding regime closely approximated the ammonium starvation response, while at the higher growth rate a partial starvation response was observed. J Biotechnol, 1992 May, 23(3), 231 - 40 Protein secretion in gram-positive bacteria; Freudl R; Gram-positive bacteria often secrete large amounts of proteins into the surrounding medium . This feature makes them attractive as hosts for the industrial production of extracellular enzymes . Compared to Escherichia coli, relatively little is known about the mechanism of protein secretion in these organisms . However, the recent identification of Bacillus subtilis genes whose gene products are highly homologous to some of the Sec (secretion) proteins of E . coli strongly suggests that important principles of protein translocation across the plasma membrane might be highly conserved . In contrast, the steps following the actual translocation event might be different in Gram-positive and Gram-negative bacteria . The scope of this review is to outline the recent progress that has been made in the elucidation of the secretion pathway in Gram-positive bacteria and to discuss potential applications in strain improvement for the industrial production of extracellular proteins. Antonie Van Leeuwenhoek, 1992 May, 61(4), 339 - 42 Bacteriophage phi 105clz induces the GroEL-homologue protein in Bacillus subtilis; Staples RR et al.; Using two-dimensional polyacrylamide gel electrophoresis, the GroEL homologue of Bacillus subtilis was shown to be induced upon infection with phi 105clz, a clear plaque mutant of the temperate bacteriophage phi 105 . Western blotting of one dimensional polyacrylamide gels also showed the induction of the GroEL homologue when cells were infected with phi 105clz. J Gen Microbiol, 1992 May, 138 ( Pt 5), 889 - 99 Evaluation of a ribosomal RNA gene probe for the identification of species and subspecies within the genus Staphylococcus; De Buyser ML et al.; To evaluate a 16S rRNA gene probe for the identification of staphylococcal species and subspecies, we have augmented previous studies involving 12 staphylococcal species by analysing the remaining 16 species currently classified in the genus Staphylococcus . HindIII- and EcoRI-restricted DNA of isolates from validly described species of Staphylococcus was probed with radiolabelled plasmid pBA2 containing 16S rDNA from Bacillus subtilis . The Dice coefficient was used to assess similarity between the 74 HindIII- and the 81 EcoRI-hybridization patterns obtained from a total of 271 isolates belonging to 31 staphylococcal taxa (28 species, of which three include two subspecies) . The use of HindIII yielded a better discrimination of the staphylococci than the use of EcoRI . All of the isolates belonging to the same species or subspecies, except S . hyicus isolates, were recovered as homogeneous clusters using their HindIII hybridization patterns . The phenotypically close taxa were clearly distinguished . Thus, the method presented in this study constitutes a powerful tool for the identification of taxa within the genus Staphylococcus. Mol Microbiol, 1992 May, 6(9), 1133 - 9 Isolation and characterization of the major vegetative RNA polymerase of Streptomyces coelicolor A3(2); renaturation of a sigma subunit using GroEL; Brown KL et al.; The promoter region of the agarase gene (dagA) of Streptomyces coelicolor A3(2) is complex; it consists of four distinct promoters with different -10 and -35 regions . We report the isolation of a form of RNA polymerase that mediates transcription in vitro from the dagAp4 promoter . The core components of this RNA polymerase are associated with a polypeptide of c . 66 kDa; holoenzyme reconstitution experiments show that the 66 kDa polypeptide functions as a sigma factor that directs transcription from the dagAp4 and Bacillus subtilis veg promoters in vitro . Alignment of the DNA sequences of these two promoters shows that they have bases in common in the -10 and -35 regions and that these sequences are similar to those observed for the major RNA polymerases of other bacteria . N-terminal amino acid sequence analysis of the 66 kDa polypeptide revealed it to be the product of the hrdB gene . Previous experiments showed that the predicted amino acid sequence of the hrdB gene product is very similar to the major sigma factors of other bacteria and suggested that disruption of the hrdB gene is lethal . These observations together lead to the conclusion that we have isolated the major RNA polymerase of Streptomyces coelicolor A3(2) . We have developed an improved protocol for the renaturation of sigma factors that have been isolated by preparative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) . This method involves renaturing the polypeptide in the presence of the bacterial chaperonin GroEL . We expect this protocol to find general application for renaturation of other polypeptides that have been subjected to SDS-PAGE. J Bacteriol, 1992 May, 174(10), 3300 - 10 Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis; Wetzstein M et al.; By using an internal part of the dnaK gene from Bacillus megaterium as a probe, a 5.2-kb HindIII fragment of chromosomal DNA of Bacillus subtilis was cloned . Downstream sequences were isolated by in vivo chromosome walking . Sequencing of 5,085 bp revealed four open reading frames in the order orf39-grpE-dnaK-dnaJ . orf39 encodes a 39-kDa polypeptide of unknown biological function with no noticeable homology to any other protein within the data bases . Alignment of the GrpE protein with those of three other bacterial species revealed a low overall homology, but a higher homology restricted to two regions which might be involved in interactions with other proteins . Alignment of the DnaK protein with six bacterial DnaK polypeptides revealed that a contiguous region of 24 amino acids is absent from the DnaK proteins of all known gram-positive species . Primer extension studies revealed three potential transcription start sites, two preceding orf39 (S1 and S2) and a third one in front of grpE (S3) . S2 and S3 were activated at a high temperature . Northern (RNA) analysis led to the detection of three mRNA species of 4.9, 2.6, and 1.5 kb . RNA dot blot experiments revealed an at-least-fivefold increase in the amount of specific mRNA from 0 to 5 min postinduction and then a rapid decrease . A transcriptional fusion between dnaK and the amyL reporter gene exhibited a slight increase in alpha-amylase activity after heat induction . A 9-bp inverted repeat was detected in front of the coding region of orf39 . This inverted repeat is present in a number of other heat shock operons in other microorganisms ranging from cyanobacteria to mycobacteria . The biological property of this inverted repeat as a putative key element in the induction of heat shock genes is discussed . The dnaK locus was mapped at about 223 degrees on the B . subtilis genetic map. J Bacteriol, 1992 May, 174(10), 3185 - 95 Interactions among mutations that cause altered timing of gene expression during sporulation in Bacillus subtilis; Ireton K et al.; The ski4::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but that are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ) . ski4::Tn917lac caused a small defect in sporulation, but in combination with a null mutation in kinA, it caused a much more severe defect . The insertion mutation was in an 87-amino-acid open reading frame (orf87 bofA) that controls the activation of a sigma factor, sigma K, at intermediate times during sporulation . The ski4 mutation caused the premature expression of cotA, a gene controlled by sigma K . An independent mutation that causes the premature activation of sigma K also caused a synthetic (synergistic) sporulation phenotype in combination with a null mutation in kinA, indicating that the defect was due to altered timing of gene expression directed by sigma K . Expression of ski4 was shown to be controlled by the sporulation-specific sigma factor sigma E. J Bacteriol, 1992 May, 174(9), 2943 - 50 Mutation and killing of Escherichia coli expressing a cloned Bacillus subtilis gene whose product alters DNA conformation; Setlow JK et al.; Expression of the Bacillus subtilis gene coding for SspC, a small, acid-soluble protein, caused both killing and mutation in a number of Escherichia coli B and K-12 strains . SspC was previously shown to bind E . coli DNA in vivo, and in vitro this protein binds DNA and converts it into an A-like conformation . Analysis of revertants of nonsense mutations showed that SspC caused single-base changes, and a greater proportion of these were at A-T base pairs . Mutation in the recA gene abolished the induction of mutations upon synthesis of SspC, but the killing was only slightly greater than in RecA+ cells . Mutations in the umuC and umuD genes eliminated most of the mutagenic effect of SspC but not the killing, while the lexA mutation increased mutagenesis but did not appreciably affect the killing . Since there was neither killing nor mutation of E . coli after synthesis of a mutant SspC which does not bind DNA, it appears likely that the binding of wild-type SspC to DNA, with the attendant conformational change, was responsible for the killing and mutation . A strain containing the B . subtilis gene that is constitutive for the RecA protein at 42 degrees C showed a lower frequency of mutation when that temperature was used to induce the RecA protein than when the temperature was 30 degrees C, where the RecA level is low, suggesting that at the elevated temperature the high RecA level could be inhibiting binding of the B . subtilis protein to DNA. J Mol Biol, 1992 Apr 20, 224(4), 967 - 79 Developmental regulation of transcription of the Bacillus subtilis ftsAZ operon; Gonzy-Treboul G et al.; The products of the ftsA and ftsZ genes play a major role in septum formation in Escherichia coli . Their homologues have been found in various bacterial species, such as Bacillus subtilis where they are involved in septation during vegetative growth as well as during sporulation, a developmental process that is initiated by the formation of an asymmetrically positioned septum . Transcription of the B . subtilis ftsAZ operon was studied during exponential growth and sporulation by monitoring beta-galactosidase synthesis in strains harboring fusions of the E . coli lacZ gene with various fragments of the ftsAZ regulatory region . Transcription of the ftsAZ operon was found to be controlled by three promoters which were mapped by primer extension and characterized by their temporal pattern of expression . Two of these promoters, P1 and P3, are dependent on sigma A, the major vegetative sigma factor, and are expressed mainly during growth . The third one, P2, is recognized by sigma H associated RNA polymerase and its activity increases three- to four-fold around the onset of sporulation . The post-exponential enhancement of P2-driven transcription is abolished in a spo0A mutant but partially restored in an abrB spo0A double mutant . After inactivation by oligonucleotide-directed mutagenesis mutated copies of P1 and P2 were introduced into the chromosome upstream from the ftsAZ operon . Transformants could be obtained only when ftsAZ transcription was controlled by a combination of two intact promoters, neither P1, P2 nor P3 being essential for viability . The sporulation efficiency was found to be dependent on the level of transcription of ftsAZ, the absence of P2 still allowing 30% of the normal sporulation rate . Therefore the post-exponential burst of synthesis of the FtsA and FtsZ proteins is not an absolute requirement for the successful completion of the asymmetric septum. Biochim Biophys Acta, 1992 Apr 17, 1120(3), 281 - 8 Site-directed mutagenesis of active site residues in Bacillus subtilis alpha-amylase; Takase K et al.; Site-directed mutagenesis of Bacillus subtilis N7 alpha-amylase has been performed to evaluate the roles of the active site residues in catalysis and to prepare an inactive catalytic-site mutant that can form a stable complex with natural substrates . Mutation of Asp-176, Glu-208, and Asp-269 to their amide forms resulted in over a 15,000-fold reduction of its specific activity, but all the mutants retained considerable substrate-binding abilities as estimated by gel electrophoresis in the presence of soluble starch . Conversion of His-180 to Asn resulted in a 20-fold reduction of kcat with a 5-fold increase in Km for a maltopentaose derivative . The relative affinities for acarbose vs . maltopentaose were also compared between the mutants and wild-type enzyme . The results are consistent with the roles previously proposed in Taka-amylase A and porcine pancreatic alpha-amylase based on their X-ray crystallographic analyses, although different pairs had been assigned as catalytic residues for each enzyme . Analysis of the residual activity of the catalytic-site mutants by gel electrophoresis has suggested that it derived from the wild-type enzyme contaminating the mutant preparations, which could be removed by use of an acarbose affinity column; thus, these mutants are completely devoid of activity . The affinity-purified mutant proteins should be useful for elucidating the complete picture of the interaction of this enzyme with starch. Biochem Biophys Res Commun, 1992 Apr 15, 184(1), 277 - 82 Molecular cloning of a subtilisin J gene from Bacillus stearothermophilus and its expression in Bacillus subtilis; Jang JS et al.; The structural gene for a subtilisin J from Bacillus stearothermophilus NCIMB10278 was cloned in Bacillus subtilis using pZ124 as a vector, and its nucleotide sequence was determined . The nucleotide sequence revealed only one large open reading frame, composed of 1,143 base pairs and 381 amino acid residues . A Shine-Dalgarno sequence was found 8 bp upstream from the translation start site (GTG) . The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprised of 275 residues . The productivity of subtilisin in the culture broth of the Bacillus subtilis was about 46-fold higher than that of the Bacillus stearothermophilus . The amino acid sequence of the extracellular alkaline protease subtilisin J is highly homologous to that of subtilisin E and it shows 69% identity with subtilisin Carlsberg, 89% with subtilisin BPN' and 70% with subtilisin DY . Some properties of the subtilisin J that had been purified from the Bacillus subtilis were examined . The subtilisin J has alkaline pH characteristics and a molecular weight of 27,500 . It retains about 50% of its activity even after treatment at 60 degrees C for 30 min in the presence of 2 mM calcium chloride. J Biol Chem, 1992 Apr 15, 267(11), 7936 - 42 Avian glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing and activity are dependent upon essential cysteine residues; Zhou G et al.; Avian glutamine phosphoribosylpyrophosphate amidotransferase contains an NH2-terminal propetide-like sequence . NH2-terminal sequence analysis of immunoaffinity purified enzyme from chicken liver indicates that the propeptide is processed and the mature enzyme starts with Cys . Propeptide processing was investigated by site-directed mutagenesis using a system for expression in HeLa cells . Glutamine-dependent activity and processing were abolished by replacement of the conserved cysteine at position 1, whereas NH3-dependent activity was retained . Cys1 is thus inferred to have a role in glutamine-dependent activity and in propeptide processing . Inactive, insoluble enzymes in which the propeptide was not processed were obtained as a result of replacements of cysteines 415 and 488 . Cysteine residues at positions 415 and 488 are inferred to be ligands to an Fe-S cluster on the basis of sequence similarity to the enzyme from Bacillus subtilis . Mutation of Cys269 and Cys295 led to loss of enzyme activity and propeptide processing, although solubility was unchanged . The results suggest that incorporation of an Fe-S cluster is needed for native structure, resultant propeptide processing, and glutamine-dependent activity. J Bacteriol, 1992 Apr, 174(8), 2648 - 58 Spo0A controls the sigma A-dependent activation of Bacillus subtilis sporulation-specific transcription unit spoIIE; York K et al.; The spoIIE operon is a developmentally regulated transcription unit activated in the second hour of sporulation in Bacillus subtilis . Its promoter has an unusual structure, containing sequences which conform perfectly to the consensus for vegetative promoters recognized by sigma A-associated RNA polymerase (E sigma A), but with a spacing of 21 bp between the apparent -10 and -35 elements instead of the 17- or 18-bp spacing typical of promoters utilized by E sigma A . Mutations introduced into the apparent -10 element affected transcription in a manner consistent with its functioning as a polymerase recognition sequence . The deleterious effect of one -10 mutation was also suppressed in an allele-specific manner by a mutation in sigA known to suppress analogous -10 mutations in conventional vegetative promoters recognized by E sigma A . Similar suppression experiments failed to provide evidence for a direct interaction between E sigma A and the "-35-like" element, however, and DNase I protection experiments suggested instead that the Spo0A protein binds to a site overlapping this -35-like hexamer . Moreover, the effects of mutations within the -35-like hexamer on the binding of Spo0A in vitro paralleled their effects on transcription in vivo . We suggest that spoIIE belongs to a class of early-intermediate sporulation genes whose transcription by E sigma A is activated by the Spo0A protein. Carbohydr Res, 1992 Apr 6, 227, 215 - 25 Action patterns of amylolytic enzymes as determined by the {1-14C}malto-oligosaccharide mapping method; Pazur JH et al.; A valuable technique for oligosaccharide mapping, utilizing radioactive malto-oligosaccharides, multiple-ascent p.c., and radioautography, has been developed for identifying the action patterns of the glucoamylase isozymes, alpha-amylases, beta-amylase, glucosyltransferase, and glucanosyltransferase . The glucoamylase isozymes act by multi-chain mechanisms on malto-oligosaccharides and most likely on starch and glycogen . The alpha-amylases act endo-wise and randomly hydrolyze alpha-(1----4)- but not alpha-(1----6)-glucosidic bonds . These amylases may act by single-chain and/or multi-chain mechanisms, depending on the number of hydrolytic attacks per single encounter of the enzyme and the substrate . The beta-amylases hydrolyze malto-oligosaccharides by a multi-chain mechanism . A fungal glucosyltransferase from Aspergillus niger transfers glucose units by a single-chain mechanism from maltose to glucosyl acceptors to yield new gluco-oligosaccharides with alpha-(1----4) and alpha-(1----6) linkages . A novel type of transferase isolated from Bacillus subtilis acts by a multi-chain mechanism and transfers segments of 2 to 5 glucose residues from malto-oligosaccharides to acceptor co-substrates . An alpha-amylase from the same organism removes maltotriose units from the non-reducing ends of oligosaccharides by a multi-chain mechanism. Biochim Biophys Acta, 1992 Apr 6, 1130(3), 333 - 5 Nucleotide sequences of serine tRNAs from Bacillus subtilis; Matsugi J et al.; Three B . subitilis serine tRNAs were sequenced including modified nucleosides . All the serine tRNAs contained 1-methyl-adenosine in the D-loop . As other characteristic modified nucleosides, 5-methoxyuridine was found in the first letter of the anticodon in the tRNA(UGA). FEMS Microbiol Lett, 1992 Apr 1, 71(1), 23 - 7 Fine-structure mapping of cis-acting control sites in the lysC operon of Bacillus subtilis; Lu Y et al.; Mutations at the aecA locus of Bacillus subtilis lead to derepression of the lysC operon, which encodes aspartokinase II, and analysis of three independent aecA mutations has shown them to be nucleotide substitutions in the lysC leader region (Y . Lu, N.Y . Chen and H . Paulus (1991) J . Gen . Microbiol . 137, 1135-1141) . DNA sequence analysis of the lysC control region of nine other mutants with derepressed levels of aspartokinase II revealed each of the mutations to be associated with changes in one or a few nucleotide residues . The nucleotide substitutions were clustered at two sites in the lysC leader: in a region of imperfect dyad symmetry about 40 base pairs from the transcription start site, and in the open reading frame for a putative leader peptide, which starts about 40 residues further downstream . The effect of nucleotide substitutions at the two sites differed in that those at the upstream site gave twice the degree of derepression . A mutant with a small deletion in the leader peptide coding region potentially affecting RNA secondary structure also had a higher level of lysC derepression . These results suggest that the lysC leader region contains at least two cis-acting control sites that play important and perhaps independent roles in the repression of the lysC operon by lysine. Curr Opin Cell Biol, 1992 Apr, 4(2), 180 - 5 Cell cycle regulation in bacteria; Newton A et al.; Significant progress has been made in the study of ftsZ expression and the topology of FtsZ protein localization in Escherichia coli cells . Exciting results on the identification of new genes required for chromosome resolution and partitioning after the completion of DNA synthesis have also been reported . A recent area of study is asymmetric cell division and its role in differentiation in Bacillus subtilis and Caulobacter crescentus . Biochemical activities of bacterial cell division gene products are also beginning to be addressed. Mol Gen Genet, 1992 Apr, 232(3), 498 - 504 Expression of luciferase genes from different origins in Bacillus subtilis; Lampinen J et al.; A group of vectors for luciferase expression in Bacillus subtilis was constructed . So far, only bacterial luciferases have been expressed in Bacillus, but in this study we wanted also to express genes encoding eukaryotic luciferases to perform direct comparisons of the light levels produced by the two different systems in B . subtilis . The vectors constructed can replicate both in Escherichia coli and B . subtilis, and the luciferase expression is strictly regulated due to the dual plasmid system used . Nearly a 100-fold increase in light production compared to previous results was achieved when genes encoding bacterial luciferase were inserted into the constructs and transformed into B . subtilis . An additional tenfold increase in light production was obtained when luciferase genes from the North American firefly (Photinus pyralis) or a click beetle (Pyrophorus plagiophtalamus) were introduced in a similar fashion into B . subtilis . Measurement of the light emission was performed without disruption of bacterial cells in a real-time manner, which is a common feature when working with all of these constructions . Structures of the shuttle vector constructs and results from light emission measurements are presented. Mol Gen Genet, 1992 Apr, 232(3), 415 - 22 Regulation of the Bacillus subtilis W23 xylose utilization operon: interaction of the Xyl repressor with the xyl operator and the inducer xylose; Gartner D et al.; A crude protein extract of Bacillus subtilis W23 contains a sequence-specific DNA binding activity for the xyl operator as detected by the gel mobility shift assay . A xylR determinant encoded on a multicopy plasmid leads to increased expression of this binding activity . In situ footprinting analysis of the protein-DNA complex in a polyacrylamide gel shows that the xyl operator is sequence-specifically bound and protected from cleavage by copper-phenanthroline at 26 phosphodiester bonds on each strand . Quantitative competition assays for repressor binding reveal that a 25 bp synthetic xyl operator cloned into a polylinker is bound with the same affinity as the operator in the wild-type xyl regulatory region . This confirms that no additional sites in the wild-type sequence contribute to repressor binding . The xyl operator consists of ten palindromic base pairs flanking five central non-palindromic base pairs . A mutational analysis shows that the sequence of the central base pairs contributes to recognition by the repressor protein and that the spacing of the palindromic elements is crucial for repressor binding . An operator half site is not bound by the repressor . In vivo and in vitro induction studies suggest that, of several structurally similar sugars, xylose is the only molecular inducer of the Xyl repressor. Mol Gen Genet, 1992 Apr, 232(3), 359 - 66 Identification of the structural genes for N-acetylmuramoyl-L-alanine amidase and its modifier in Bacillus subtilis 168: inactivation of these genes by insertional mutagenesis has no effect on growth or cell separation; Margot P et al.; The region of the Bacillus subtilis 168 chromosome that contains the structural genes for the major vegetative cell autolysin, (N-acetyl-muramoyl-L-alanine amidase), and its modifier protein has been cloned . Insertional mutagenesis with integrative plasmids carrying small DNA fragments from this region has revealed that both genes are located on a 4 kb fragment; they are organised in one transcription unit, the modifier being transcribed first . Studies of derivatives in which either the amidase or the modifier or both proteins are inactivated have revealed that amidase-deficient strains are not affected in growth, cell separation, transformability or sporulation . Observed phenotypic differences were altered kinetics of, cell wall turn-over and a reduced rate of, autolysis of native cell wall preparations . A residual amidase activity, about 3% of that of the wild-type strain, was found in strains devoid of the major amidase . A new, distinct cell wall-bound protein, designated CWBP49', with the same molecular weight as the amidase, was identified in mutants devoid of the latter enzyme. EMBO J, 1992 Apr, 11(4), 1317 - 26 Induction of DNA amplification in the Bacillus subtilis chromosome; Petit MA et al.; A system allowing the induction of DNA amplification in Bacillus subtilis was developed, based on a thermosensitive plasmid, pE194, stably integrated in the bacterial chromosome . An amplification unit, comprising an antibiotic resistance marker flanked by directly repeated sequences, was placed next to the integrated plasmid . Activation of pE194 replication led to DNA amplification . Two different amplification processes appeared to take place: one increased the copy number of all sequences in the vicinity of the integrated plasmid and was possibly of the onion skin type, while the other increased the copy number of the amplification unit only and generated long arrays of amplification units . These arrays were purified and shown to consist mainly of directly repeated amplification units but to also contain non-linear regions, such as replication forks and recombination intermediates . They were attached to the chromosome at one end only, and were, in general, not stably inherited, which suggests that they are early amplification intermediates . Longer arrays were detected before the shorter ones during amplification . When the parental amplification unit contained repeats which differed by a restriction site the arrays which derived thereof contained in a majority of cases only a single type of repeat . We propose that the amplified DNA is generated by rolling circle replication, and that such a process might underlie a number of amplification events. J Bacteriol, 1992 Apr, 174(8), 2474 - 7 Identification of proteins phosphorylated by ATP during sporulation of Bacillus subtilis; Mitchell C et al.; Protein phosphorylation in Bacillus subtilis was assayed in vitro by using extracts prepared from cells at various times during growth and sporulation . At least six proteins were labeled in vitro by using {gamma-32P}ATP and extracts of vegetative cells . In extracts prepared at the end of exponential growth and during stationary phase, 12 to 13 proteins were labeled . Seven of the phosphoproteins were purified by fast-performance liquid chromatography and polyacrylamide gel electrophoresis, blotted to Immobilon membranes, and subjected to partial protein sequencing . One of the sequences had sequence homology (greater than 45%) to elongation factor G from several bacterial species, and four sequences matched the predicted amino-terminal sequences of the outB, orfY-tsr, orfU, and ptsH genes. J Bacteriol, 1992 Apr, 174(7), 2398 - 403 Impaired cell division and sporulation of a Bacillus subtilis strain with the ftsA gene deleted; Beall B et al.; The ftsZ and ftsA genes of Bacillus subtilis are organized in a simple operon expressed from promoter sequences immediately upstream of ftsA . The promoter-distal ftsZ gene is an essential septation gene . In this report, it is shown that the promoter-proximal ftsA gene can be deleted in a previously constructed strain in which the essential gene, ftsZ, is under the control of the inducible spac promoter . Absence of the ftsA gene product resulted in a very filamentous morphology indicating an important role for ftsA in cell division . Also, growth was severely impaired, and viability and sporulation were reduced . The defective sporulation phenotype correlated with a deficiency in the processing of pro-sigma E to its active form. J Bacteriol, 1992 Apr, 174(7), 2281 - 7 Cloning, characterization, and inactivation of the Bacillus brevis lon gene; Ito K et al.; A gene of Bacillus brevis HPD31 analogous to the Escherichia coli lon gene has been cloned and characterized . The cloned gene (B . brevis lon gene) encodes a polypeptide of 779 amino acids with a molecular weight of 87,400 which resembles E . coli protease La, the lon gene product . Fifty-two percent of the amino acid residues of the two polypeptides were identical . The ATP-binding sequences found in E . coli protease La were highly conserved . The promoter of the B . brevis lon gene resembled that recognized by the major RNA polymerase of Bacillus subtilis and did not contain sequences homologous to the E . coli heat shock promoters . The B . brevis lon gene was inactivated by insertion of the neomycin resistance gene . A mutant B . brevis carrying the inactivated lon gene showed diminished ability for the degradation of abnormal polypeptides synthesized in the presence of puromycin. J Bacteriol, 1992 Apr, 174(7), 2059 - 64 The mtrAB operon of Bacillus subtilis encodes GTP cyclohydrolase I (MtrA), an enzyme involved in folic acid biosynthesis, and MtrB, a regulator of tryptophan biosynthesis; Babitzke P et al.; mtrA of Bacillus subtilis was shown to be the structural gene for GTP cyclohydrolase I, an enzyme essential for folic acid biosynthesis . mtrA is the first gene in a bicistronic operon that includes mtrB, a gene involved in transcriptional attenuation control of the trp genes . mtrA of B . subtilis encodes a 20-kDa polypeptide that is 50% identical to rat GTP cyclohydrolase I . Increased GTP cyclohydrolase I activity was readily detected in crude extracts of B . subtilis and Escherichia coli in which MtrA was overproduced . Biochemical evidence indicating that MtrA catalyzes dihydroneopterin triphosphate and formic acid formation from guanosine triphosphate is presented . It was also shown that mtrB of B . subtilis encodes a 6-kDa polypeptide . Expression of mtrB is sufficient for transcriptional attenuation control of the B . subtilis trp gene cluster in Escherichia coli . Known interrelationships between genes involved in folic acid and aromatic amino acid biosynthesis in B . subtilis are described. ASAIO J, 1992 Apr-Jun, 38(2), 116 - 9 Sterilization of a small caliber vascular graft with a polyexpoxy compound; Chan-Myers HB et al.; Sterilization of tissue based medical devices via cold sterilization processes has been limited to formaldehyde, glutaraldehyde, and mixtures of the same with alcohols and surfactants . The authors report the sterilization of a small caliber vascular graft with a combination of diglycidyl ether and ethanol . The sterilant contains 1-4% diglycidyl ether and 10-20% ethanol as an aqueous solution . Sterilization is achieved after exposure of the graft to the sterilant solution for a period of 7 days at an elevated temperature (30 degrees - 40 degrees C) . The biologic indicator selected for efficacy studies was Bacillus subtilis niger ATCC 9372 (endospores) . The grafts were inoculated with a concentrated endospore suspension and immersed in the sterilant solution for increasing time periods . After extensive rinsing over membrane filters to remove any residual sterilant, the grafts and filters were cultured in tryptic soy broth . D10 values were calculated using a fraction-negative, most probable number technique . Additionally, many representative bacteria and fungi were tested and found to be susceptible to the new sterilant developed . The diglycidyl ether/alcohol sterilant developed was found to be efficacious for sterilization of the tissue based vascular grafts tested. J Bacteriol, 1992 Apr, 174(7), 2185 - 92 Small cytoplasmic RNA of Bacillus subtilis: functional relationship with human signal recognition particle 7S RNA and Escherichia coli 4.5S RNA; Nakamura K et al.; Small cytoplasmic RNA (scRNA; 271 nucleotides) is an abundant and stable RNA of the gram-positive bacterium Bacillus subtilis . To investigate the function of scRNA in B . subtilis cells, we developed a strain that is dependent on isopropyl-beta-D-thiogalactopyranoside for scRNA synthesis by fusing the chromosomal scr locus with the spac-1 promoter by homologous recombination . Depletion of the inducer leads to a loss of scRNA synthesis, defects in protein synthesis and production of alpha-amylase and beta-lactamase, and eventual cell death . The loss of the scRNA gene in B . subtilis can be complemented by the introduction of human signal recognition particle 7S RNA, which is considered to be involved in protein transport, or Escherichia coli 4.5S RNA . These results provide further evidence for a functional relationship between B . subtilis scRNA, human signal recognition particle 7S RNA, and E . coli 4.5S RNA. J Biotechnol, 1992 Apr, 23(2), 225 - 9 Secretion of correctly processed and folded pancreatic secretory trypsin inhibitor by Bacillus subtilis; Nakayama A et al.; We constructed a plasmid, designated pNPP126, containing a DNA sequence encoding a fusion protein composed of Bacillus amyloliquefaciens neutral protease prepeptide (signal peptide) and human pancreatic secretory trypsin inhibitor (hPSTI), where the mature hPSTI is accurately fused to the 3'-terminal of the prepeptide coding region . It was observed that the strain Bacillus subtilis MT600 harboring pNPP126 could secrete a trypsin inhibitory activity into the culture medium . The N-terminal amino acid sequence, the amino acid composition and the stoichiometry of the purified hPSTI produced by B . subtilis were the same as those of natural hPSTI, indicating that the transformant B . subtilis MT600 (pNPP126) could efficiently secrete the correctly processed and folded hPSTI into the culture medium. Mol Microbiol, 1992 Apr, 6(8), 981 - 90 Secretion of the Escherichia coli outer membrane proteins OmpA and OmpF in Bacillus subtilis is blocked at an early intracellular step; Puohiniemi R et al.; When the genes coding for the outer membrane (OM) proteins OmpA and OmpF of Escherichia coli are fused to a signal sequence of a bacillar exoenzyme and expressed in Bacillus subtilis they remain cell-bound and the signal sequence is not cleaved . To identify the step of arrest in the export of these proteins we studied their accessibility to protease applied to intact protoplasts; they remained resistant indicating fully intracellular localization . Both proteins appeared associated with the cell membranes in sedimentation and flotation centrifugation experiments . However, OmpA and OmpF proteins synthesized in B . subtilis without a signal sequence were similarly associated with membranes in centrifugation experiments whereas electron microscopy showed the presence of intracytoplasmic inclusion bodies not obviously attached to the cytoplasmic membrane . We conclude that OmpA and OmpF proteins even when provided with a functional signal sequence do not enter the export pathway in B . subtilis, probably owing to lack of a specific export component in B . subtilis. Proc Natl Acad Sci U S A, 1992 Mar 15, 89(6), 2499 - 503 Structure of the histidine-containing phosphocarrier protein HPr from Bacillus subtilis at 2.0-A resolution; Herzberg O et al.; The crystal structure of the histidine-containing phosphocarrier protein (HPr) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) from Bacillus subtilis has been determined at 2.0-A resolution and refined to a crystallographic residual error R-factor of 0.150 . The secondary-structure folding topology of the molecule is that of an open-face beta-sandwich formed by four antiparallel beta-strands packed against three alpha-helices . The active-site histidine, His-15, caps the N terminus of the first helix, suggesting that the helix dipole plays a role in stabilizing the phosphorylated state of the histidine . A sulfate anion located between His-15 and the neighboring Arg-17 has been identified in the electron-density map . Association of this negatively charged species with the two key catalytic residues implies that the crystal structure resembles the phosphorylated state of the protein . A model of the phosphorylated form of the molecule is proposed, in which the negatively charged phosphoryl group interacts with two main-chain nitrogen atoms of the following helix and with the guanidinium group of Arg-17 . It is also proposed that the phosphoryl transfer from HPr to the IIA domain of the glucose permease involves Arg-17 switching between two salt bridges: one with the phosphorylated histidyl of HPr and the other with two aspartyl residues associated with the active site of the IIA domain of glucose permease, which are accessible upon complex formation. Biochim Biophys Acta, 1992 Mar 24, 1130(2), 229 - 31 spoVG sequence of Bacillus megaterium and Bacillus subtilis; Hudspeth DS et al.; We have sequenced the stage V sporulation specific gene spoVG in both Bacillus megaterium and Bacillus subtilis . The open reading frames encode polypeptides of 96 and 97 residues, respectively, and have an 88.6% amino acid identity . Both genes have putative rho-independent terminators . No significant amino acid or nucleotide homology of either gene was found when compared with sequences contained in either the Genbank or EMBL data bases. FEBS Lett, 1992 Mar 23, 300(1), 56 - 62 Sequence-specific resonance assignment and conformational analysis of subtilin by 2D NMR; Chan WC et al.; Subtilin, a 32-amino acid peptide with potent antimicrobial activity, has been isolated from Bacillus sub