Antonie Van Leeuwenhoek, 1994, 66(4), 319 - 26 Characterization of production and enzyme properties of an endo-beta-1,4- glucanase from Bacillus subtilis CK-2 isolated from compost soil; Aa K et al.; Bacillus subtilis CK-2, isolated from garden organic waste compost, was found to have high hydrolytic activity against carboxymethylcellulose (CMC) due to the secretion of an endo-beta-1,4- glucanase . Enzyme production was related to the sporulation process, and was regulated by the concentration of readily metabolizable carbohydrate in growth medium . Enzyme production did not require CMC or other cellulose containing materials . The endo-beta-1,4-glucanase activity was optimal at pH 5.6-5.8 and at 65 degrees C, and achieved thermal stability up to 55 degrees C . The activity was inhibited by Hg2+ . The purified enzyme gave a single band corresponding to a MW of 35.5 kDa on SDS-PAGE, while the Sephadex G-75 chromatography revealed a molecular weight of the active enzyme around 70 kDa, indicating a dimeric form of the active enzyme . The enzyme activity was irreversibly inhibited by SDS . Native PAGE and IEF revealed three different isoelectric forms of the enzyme, all with an identical N-terminal amino-acid sequence. DNA Res, 1994, 1(1), 1 - 14 Systematic sequencing of the 180 kilobase region of the Bacillus subtilis chromosome containing the replication origin; Ogasawara N et al.; We have determined a 180 kb contiguous sequence in the replication origin region of the Bacillus subtilis chromosome . Open reading frames (ORF) in this region were unambiguously identified from the determined sequence, using criteria characteristic for the B . subtilis gene structure, i.e., starting with an ATG, GTG or TTG codon preceded by sequences complementary to the 3' end of the 16S rRNA . Four rRNA gene sets, 7 individual tRNA genes and 1 scRNA gene were identified, occupying 20 kb in total . In the remaining 160 kb region, 158 ORFs were identified, suggesting that 1 ORF is coded on average by 1 kb of DNA of the B . subtilis genome . Among the 158 ORFs, the functions of 48 ORFs were assigned and those of 11 ORFs are suggested through significant similarities to known proteins present in data banks . However, the functions of more than half of the ORFs (63%) remain to be determined. Yi Chuan Xue Bao, 1994, 21(1), 74 - 80 {Cloning and sequencing of promoter and signal sequence coding regions from Bacillus subtilis}; Luo J et al.; Promoter and signal sequence coding regions from B . subtilis were cloned in E . coli using a bifunctional and signal sequence selection plasmid pGPB14 as a vector . The Sau3A digested chromosome DNA was ligated with BamHI digested pGPB14 . The ligated mixture was used to transform E . coli C600 . Ampicillin and erythromycin resistant clones were selected . Recombinant plasmids were isolated from double resistant transformants . Restriction analysis showed that inserts of various length had been cloned and these inserts rendered the E . coli cells resistant to different concentrations of ampicillin . The recombinant plasmids were transformed and showed the same secretion function in B . subtilis . The amount and localization of beta-lactamase were determined in transformed E . coli and B . subtilis . The results indicate that the beta-lactamase activities of E . coli mainly in periplasm while the enzyme produced by B . subtilis secreted extracellularly . 10 of the cloned fragments were sequenced by Sanger's dideoxy chain termination method . The sequencing data show that all fragments contain promoter, ribosome binding site and signal sequence coding region. Mol Microbiol, 1994 Jan, 11(1), 87 - 98 The citrulline biosynthetic operon, argC-F, and a ribose transport operon, rbs, from Bacillus subtilis are negatively regulated by Spo0A; O'Reilly M et al.; A method is described here that can be used to identify operons whose expression is controlled by any particular regulator protein . This method was used to identify operons whose expression is negatively regulated by Spo0A in Bacillus subtilis . Twenty-eight strains were identified, each of which contains an operon-lacZ transcriptional fusion, negatively regulated, either directly or indirectly, by Spo0A . In one of these strains (CSA8), the lacZ gene is fused to the argC-F operon positioned at 100 degrees on the B . subtilis chromosome . The regulated expression of this operon by Spo0A-P is mediated indirectly through the transition state regulator AbrB and is manifest only during growth on solid medium . In a second strain (CSA15), the lacZ gene is fused to an operon encoding a transport system which displays features characteristic of the ABC group of transporters, and which has a very high level of identity to the ribose transport system from Escherichia coli . Expression of the ribose transport operon is directed by a single SigA-type promoter . Transcription from this promoter is repressed by the phosphorylated form of Spo0A during the late-exponential/transition phase of the growth cycle and this control is not mediated through the transition-state regulator, AbrB. Gene, 1993 Dec 31, 137(2), 247 - 51 Identification of a promoter for the crystal protein-encoding gene cryIVB from Bacillus thuringiensis subsp . israelensis; Yoshisue H et al.; The cryIVB gene of Bacillus thuringiensis subsp . israelensis (Bti) codes for a 135-kDa insecticidal crystal protein, which is specifically toxic to dipteran larvae . We have identified a transcription start point (tsp) of cryIVB by a primer extension experiment . The promoter sequence alignment, together with the chronology of appearance of the transcript, suggested that cryIVB is transcribed by RNA polymerase containing sigma 35 (E sigma 35) . This was confirmed by investigation of cryIVB transcription in several Bacillus subtilis sporulation mutants . Unlike the lepidopteran-specific crystal protein-encoding genes {cryIA(a) and cryIB}, transcription of which is regulated by both sigma 35 and sigma 28, cryIVB transcription was controlled only by the sigma 35-dependent promoter at the midsporulation stage. Nucleic Acids Res, 1993 Dec 25, 21(25), 5916 - 20 Lead-catalyzed cleavage of ribonuclease P RNA as a probe for integrity of tertiary structure; Zito K et al.; Pb(2+)-catalyzed cleavage of RNA has been shown previously to be a useful probe for tertiary structure . In the present study, Pb2+ cleavage patterns were identified for ribonuclease P RNAs from three phylogenetically disparate organisms, Escherichia coli, Chromatium vinosum, Bacillus subtilis, and for E . coli RNase P RNAs that had been altered by deletions . Each of the native RNAs undergoes cleavage at several sites in the core structure that is common to all bacterial RNase P RNAs . All the cleavages occur in non-paired regions of the secondary structure models of the RNAs, in regions likely to be involved in tertiary interactions . Two cleavage sites occur at homologous positions in all the native RNAs, regardless of sequence variation, suggesting common tertiary structural features . The Pb2+ cleavage sites in four deletion mutants of E . coli RNase P RNA differed from the native pattern, indicating alterations in the tertiary structures of the mutant RNAs . This conclusion is consistent with previously characterized properties of the mutant RNAs . The Pb2+ cleavage assay is thus a useful probe to reveal alteration of tertiary structure in RNase P RNA. Gene, 1993 Dec 22, 136(1-2), 253 - 5 A counterselectable pACYC184-based lacZ alpha-complementing plasmid vector with novel multiple cloning sites; construction of chromosomal deletions in Klebsiella pneumoniae; Geissler S et al.; We have constructed a series of small, chloramphenicol-resistance-encoding, lacZ alpha-complementing vectors with novel multiple cloning sites, based on the pACYC184 replicon . The sacB gene of Bacillus subtilis, which is lethal to Gram- organisms in the presence of sucrose, was cloned into one of these, giving the counterselectable vector pSG335 . This was used to substitute a streptomycin-resistance-encoding cassette for the ntrBC genes in the Klebsiella pneumoniae chromosome. Gene, 1993 Dec 22, 136(1-2), 1 - 12 Analysis of the stabilization system of pSM19035-derived plasmid pBT233 in Bacillus subtilis; Ceglowski P et al.; The low-copy-number, 9.0-kb pSM19035-derived plasmid pBT233, is stably inherited in Bacillus subtilis . The complete nucleotide (nt) sequence of pBT233 has been determined . Analysis of the nt sequence revealed nine major open reading frames (orfs) . The repS, erm1 and erm2 genes have been assigned to three of these orfs, and given the gene order, repS-orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm1 . The organization of genes of the repS-orf gamma region resembles the organization of genes in the repE-orfI region of pAM beta 1 . Messenger RNA species of molecular weights corresponding to repS, orf alpha + orf beta, orf gamma, orf delta and orf epsilon + orf zeta were detected by Northern blotting . Proteins of 23.8, 81.3, 34.4, 10.7 and 32.4 kDa correspond to Orfs beta, gamma, delta, epsilon and zeta, respectively . Bands of radioactive proteins of 25, 81, 34, 10 and 32 kDa were detected using the T7 promoter-expression system . The orf beta and orf gamma encode proteins that share homology to site-specific recombinases and type-I topoisomerases, respectively . The orfs, delta, epsilon and zeta, encode proteins with unknown activity . Deletion of a 1.5-kb segment (nt 2999-4552) with coding capacity for orf beta, orf gamma and orf delta does not seem to affect plasmid maintenance . Removal of a 3.0-kb fragment (nt 4598-7689) with coding capacity for orf epsilon and orf zeta reduced plasmid segregational stability, but deletion of a 5.2-kb DNA segment (nt 2546-7826) abolished it.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Biol, 1993 Dec 20, 234(4), 1282 - 3 Crystallization and preliminary X-ray studies of wild type and catalytic-site mutant alpha-amylase from Bacillus subtilis; Mizuno H et al.; Recombinant alpha-amylase (EC3.2.1.1) from Bacillus subtilis has been crystallized by the hanging drop vapor diffusion method using polyethylene glycol as precipitant . Crystals of wild-type protein diffract to at least 2.2 A resolution, and belong to the space group P2(1)2(1)2(1) with a = 72.2 A, b = 74.9 A, c = 116.1 A with probably one molecule in the asymmetric unit . A catalytic site mutant created by site-directed mutagenesis has also been grown as isomorphous crystals with a = 72.6 A, b = 74.4 A, c = 116.7 A . Structural studies of both wild-type and mutant proteins will provide a basis for understanding the catalytic mechanism of alpha-amylase. J Biol Chem, 1993 Dec 5, 268(34), 25350 - 6 Chemotactic methyltransferase promotes adaptation to repellents in Bacillus subtilis; Kirsch ML et al.; Bacillus subtilis cheRB, which encodes the chemotactic methyltransferase, has been cloned and sequenced . CheRB is a polypeptide of 256 amino acids, with a predicted molecular mass of 28 kDa . A comparison of the predicted amino acid sequence of B . subtilis CheRB with that of Escherichia coli CheRE demonstrates that the two enzymes share 31% amino acid identity . The homology was functional in that the expression of cheBB in an E . coli cheRE null mutant made the bacteria Che+ . In contrast to cheRE null mutants which show a strong smooth swimming bias, cheRB null mutants were predominantly tumbly . They respond to the addition and subsequent removal of attractant . They also respond to the addition of repellent but do not adapt; they resume prestimulus bias on removal of repellent . Tethering analysis of a culture of a cheRB null mutant revealed two distinct subpopulations, each demonstrating unique behaviors . One showed a strong clockwise flagellar rotation bias, whereas the other was more random . The latter phenotype may be due to a deficiency of CheB and may reflect an interaction of CheB and CheR . Measurements of CheB activity in the cheR null mutant showed them to be only 20% of wild type levels . We conclude from this work that CheRB functions to promote adaptation to repellent stimuli in B . subtilis, whereas CheRE functions to promote adaptation to attractant stimuli in E . coli. J Biol Chem, 1993 Dec 5, 268(34), 25909 - 13 Fe.bleomycin cleaves a transfer RNA precursor and its "transfer DNA" analog at the same major site; Holmes CE et al.; Previously, Fe.bleomycin (BLM) has been shown to mediate RNA cleavage in a fashion more highly selective than that of DNA . Because RNAs often assume secondary and tertiary structures not commonly encountered with DNAs, it was not clear whether the greater selectivity of RNA cleavage was a consequence of differences in the mononucleotide constituents of RNA and DNA, or of the three-dimensional structures of the individual substrates . Accordingly, we prepared a "tDNA" identical in sequence with Bacillus subtilis tRNA(His) precursor, the latter of which is known to be a good substrate for Fe(II).BLM A2 and which undergoes oxidative cleavage predominantly at U35 . Remarkably, the tDNA underwent cleavage predominantly at T35 . At higher concentrations of Fe(II).BLM A2, the tDNA was extensively degraded, while the tRNA(His) precursor was not . Competition experiments suggested that this was not due to more efficient binding of Fe.BLM to the tDNA; in fact the tRNA precursor appeared to be bound more efficiently . The lesser cleavage of the tRNA(His) may be due to limitations in the facility of chemical transformation following Fe.BLM binding, or else to the formation of RNA lesions that do not lead directly to RNA strand scission. J Immunol Methods, 1993 Dec 3, 166(2), 271 - 80 Liposomes as vaccine carriers . Incorporation of soluble and particulate antigens in giant vesicles; Antimisiaris SG et al.; Giant liposomes (mean diameter 5.5 microns) composed of egg phosphatidylcholine or distearoyl phosphatidylcholine, phosphatidyl glycerol, cholesterol and triolein were prepared by a double emulsion technique . They were then mixed with model particulate (killed Bacillus subtilis, and killed Bacille Calmette-Guerin) and soluble (tetanus toxoid) vaccines and freeze-dried . Rehydration of the powder resulted in the generation of vesicles of similar mean diameter and diameter range, containing up to 27% (mean value) of the materials used for entrapment . Separation of entrapped from non-entrapped material was carried out by sucrose gradient centrifugation (B . subtilis and BCG) or centrifugation at 600 x g (toxoid) . Light microscopy of liposomes containing B . subtilis labelled with fluorescein isothiocyanate revealed the presence of bacteria in individual vesicles which, in separate studies, were also found to entrap latex particles (0.5 and 1.0 micron diameter) . Bacteria-containing liposomes could be freeze-dried in the presence of trehalose with most (83-87%) of the entrapped material recovered within the vesicles on reconstitution with saline . Liposomes were also shown to retain quantitatively their content of B . subtilis and, to a lesser extent, toxoid in the presence of mouse plasma at 37 degrees C and in situ after intramuscular injection into mice, for up to 24 h . Since liposomes are known (Gregoriadis, G . (1990) Immunol . Today 11, 89) to act as immunological adjuvants and vaccine carriers, giant vesicles containing microbes (live or attenuated if needed since the conditions of entrapment are mild) and, when appropriate, soluble antigens, could be used as multiple vaccines to ensure simultaneous presentation of antigens to immunocompetent cells. FEMS Microbiol Lett, 1993 Dec 1, 114(2), 207 - 14 In vitro assay for the Bacillus subtilis signal peptidase SipS: systems for efficient in vitro transcription-translation and processing of precursors of secreted proteins; Vehmaanpera J et al.; The signal peptidase (SPase) SipS of Bacillus subtilis is responsible for the processing of precursors of secreted proteins . It differs from the SPases of Gram-negative bacteria in structure and specificity . To assay the activity of SipS in vitro, two efficient transcription-translation systems for the synthesis of radio-labelled precursors were developed . The systems were completely derived from B . subtilis . Post-translational in vitro processing of pre-staphylokinase by SipS was demonstrated . SipS activity was stimulated in vitro by several non-ionic detergents, whereas it was not affected by a large variety of proteinase inhibitors . SipS shares the latter property with other SPases. J Bacteriol, 1993 Dec, 175(24), 7931 - 7 Stress-induced activation of the sigma B transcription factor of Bacillus subtilis; Boylan SA et al.; The alternative transcription factor sigma B of Bacillus subtilis is activated during the stationary growth phase by a regulatory network responsive to stationary-phase signals . On the basis of the results reported here, we propose that sigma B controls a general stress regulon that is induced when cells encounter a variety of growth-limiting conditions . Expression of genes controlled by sigma B, including the ctc gene and the sigB operon that codes for sigma B and its associated regulatory proteins, was dramatically induced in both the exponential and stationary phases by environmental challenges known to elicit a general stress response . After cells were subjected to salt stress, the increased expression of lacZ transcriptional fusions to the ctc and sigB genes was entirely dependent on sigma B, and primer extension experiments confirmed that the sigma B-dependent transcriptional start site was used during salt induction of sigB operon expression . Western blotting (immunoblotting) experiments measuring the levels of sigma B protein indicated that ethanol addition and heat stress also induced sigma B activity during logarithmic growth . Salt and ethanol induction during logarithmic growth required RsbV, the positive regulator of sigma B activity that is normally necessary for activity in stationary-phase cells . However, heat induction of sigma B activity was largely independent of RsbV, indicating that there are two distinct pathways by which these environmental signals are conveyed to the transcriptional apparatus. J Bacteriol, 1993 Dec, 175(24), 7887 - 900 A cytotoxic early gene of Bacillus subtilis bacteriophage SPO1; Wei P et al.; Some of the early genes of Bacillus subtilis bacteriophage SPO1 were hypothesized to function in the shutoff of host biosyntheses . Two of these genes, e3 and e22, were cloned and sequenced . E22 showed no similarity to any known protein, while E3, a highly acidic protein, showed significant similarity only to other similarly acidic proteins . Each gene was immediately downstream of a very active early promoter . Each was expressed actively during the first few minutes of infection and was then rapidly shut off and its RNA rapidly degraded . An e3 nonsense mutation severely retarded the degradation of e3 RNA . Expression of a plasmid-borne e3 gene, in either B . subtilis or Escherichia coli, resulted in the inhibition of host DNA, RNA, and protein syntheses and prevented colony formation . However, the e3 nonsense mutation caused no measurable decrease in either burst size or host shutoff during infection and, in fact, caused an increased burst size at high multiplicities of infection . We suggest that e3 is one of several genes involved in host shutoff, that its function is dispensable both for host shutoff and for phage multiplication, and that its shutoff function is not entirely specific to host activities. J Bacteriol, 1993 Dec, 175(23), 7604 - 16 Cloning and sequencing of the cell division gene pbpB, which encodes penicillin-binding protein 2B in Bacillus subtilis; Yanouri A et al.; The pbpB gene, which encodes penicillin-binding protein (PBP) 2B of Bacillus subtilis, has been cloned, sequenced, mapped, and mutagenized . The sequence of PBP 2B places it among the class B high-molecular-weight PBPs . It appears to contain three functional domains: an N-terminal domain homologous to the corresponding domain of other class B PBPs, a penicillin-binding domain, and a lengthy carboxy extension . The PBP has a noncleaved signal sequence at its N terminus that presumably serves as its anchor in the cell membrane . Previous studies led to the hypothesis that PBP 2B is required for both vegetative cell division and sporulation septation . Its sequence, map site, and mutant phenotype support this hypothesis . PBP 2B is homologous to PBP 3, the cell division protein encoded by pbpB of Escherichia coli . Moreover, both pbpB genes are located in the same relative position within a cluster of cell division and cell wall genes on their respective chromosomes . However, immediately adjacent to the B . subtilis pbpB gene is spoVD, which appears to be a sporulation-specific homolog of pbpB . Inactivation of SpoVD blocked synthesis of the cortical peptidoglycan in the spore, whereas carboxy truncation of PBP 2B caused cells to grow as filaments . Thus, it appears that a gene duplication has occurred in B . subtilis and that one PBP has evolved to serve a common role in septation during both vegetative growth and sporulation, whereas the other PBP serves a specialized role in sporulation. J Bacteriol, 1993 Dec, 175(23), 7581 - 93 Regions of the Bacillus subtilis ilv-leu operon involved in regulation by leucine; Grandoni JA et al.; The ilv-leu operon of Bacillus subtilis is regulated in part by transcription attenuation . The cis-acting elements required for regulation by leucine lie within a 683-bp fragment of DNA from the region upstream of ilvB, the first gene of the operon . This fragment contains the ilv-leu promoter and 482 bp of the ilv-leu leader region . Spontaneous mutations that lead to increased expression of the operon were shown to lie in an imperfect inverted repeat encoding the terminator stem within the leader region . Mutations within the inverted repeat of the terminator destroyed most of the leucine-mediated repression . The remaining leucine-mediated repression probably resulted from a decrease in transcription initiation . A systematic analysis of other deletions within the ilv-leu leader region identified a 40-bp region required for the derepression that occurred during leucine limitation . This region lies within a potential RNA stem-and-loop structure that is probably required for leucine-dependent control . Deletion analysis also suggested that alternate secondary structures proximal to the terminator are involved in allowing transcription to proceed beyond the terminator . Additional experiments suggested that attenuation of the ilv-leu operon is not dependent on coupling translation to transcription of the leader region . Our data support a model proposed by Grundy and Henkin (F . J . Grundy and T . M . Henkin, Cell 74:475-482, 1993) in which uncharged tRNA acts as a positive regulatory factor to increase gene expression during amino acid limitation. J Chemother, 1993 Dec, 5(6), 490 - 3 Brodimoprim concentrations in bronchial mucosa, bronchial secretions and middle ear effusions; Scaglione F et al.; Brodimoprim, a structural analogue of trimethoprim, is a long acting broad spectrum antibacterial agent characterized by a good pharmacokinetic profile, allowing once daily (OD) administration . The aim of this study was to investigate the penetration of brodimoprim into bronchial mucosa, bronchial secretion and middle ear effusion, in order to evaluate the efficacy of the antibiotic in respiratory tract infections . The study was performed in patients affected by chronic bronchitis having to undergo diagnostic bronchoscopy (n = 26), in patients affected by exacerbation of chronic bronchitis with purulent or mucopurulent secretions (n = 10), and in patients affected by otitis media with eardrum perforation (n = 28) . Patients were orally treated with 400 mg of brodimoprim (single dose) . Samples of serum, bronchial mucosa, bronchial secretion and middle ear effusion were collected in the separate series of patients above mentioned, at different times after drug administration . Brodimoprim determinations were performed by a microbiological method using Bacillus subtilis ATCC 6633 as test microorganism . Brodimoprim reached the highest concentration in serum 4 h after administration and was still detectable at 24th hour . In bronchial mucosa and in bronchial secretion the peaks were reached at 8th hour (9.7 +/- 5.3 mg/kg and 4.57 +/- 1 mg/l respectively) while in middle car effusion were reached at 4th hour (4.8 +/- 2.5 mg/l) . The drug was still detectable at antibacterial concentrations, both in infected fluids and in tissue samples, 24 hours after administration (4.3 +/- 1.8 mg/kg in bronchial mucosa; 3.5 +/- 0.66 mg/l in bronchial secretions; 3 +/- 0.6 mg/l in middle ear effusion).(ABSTRACT TRUNCATED AT 250 WORDS) Sheng Li Xue Bao, 1993 Dec, 45(6), 536 - 42 {Effect of nanhumycin on neuromuscular transmission and its mechanism}; Shi YL et al.; Nanhumycin (NHM) is a new polyether antibiotic, which suppresses the growth Bacillus subtilis and shows an anticoccidial effect . By means of intracellular recording technique, the effects of NHM on mouse phrenic nerve-diaphragm preparations were observed . The main results are as follows: (1)20 micrograms/ml NHM irreversibly blocked neuromuscular transmission in 1.5-2.0 h . (2) 2-20 micrograms/ml NHM evoked a drastic and spasmodic increase of the frequency of miniature endplate potentials (MEPPs) and a simultaneous increase of the mean quantal content of endplate potentials for 40-60 min, which was followed by complete blockage of EPPs . (3) NHM decreased the resting potential (RP) of muscle cells progressively and irreversibly . (4) NHM's effects on RP or MEPP disappeared in Na(+)-free physiological solution but persisted in the presence of tetrodotoxin (TTX) . All these results support the notion that NHM is a cation ionophore, Na+ carrier. Biol Mass Spectrom, 1993 Dec, 22(12), 712 - 20 Analysis of Bacitracin B using fast atom bombardment and tandem mass spectrometry; Tetler LW et al.; Bacitracin is a mixture of polypeptide antibiotics produced by the action of Bacillus subtilis and Bacillus licheniformis . The mixture has previously been shown to contain at least ten components, of which only the major component, Bacitracin A, and its oxidized counterpart, Bacitracin F, have been fully structurally evaluated . Using fast atom bombardment ionization with tandem mass spectrometry, three isobaric components of one of the minor constituents of the mixture, Bacitracin B, have been structurally characterized, delineating the three single substitution sites within Bacitracin A where Ile has been replaced by Val. J Gen Microbiol, 1993 Dec, 139 ( Pt 12), 3197 - 203 Effects of new mutations in the spoIIAB gene of Bacillus subtilis on the regulation of sigma F and sigma G activities; Foulger D et al.; The spoIIAB gene of Bacillus subtilis encodes an inhibitor of sigma F, a transcription factor that plays a crucial role in the establishment of prespore-specific gene expression during sporulation . The SpoIIAB protein can probably also inhibit a closely related sigma factor sigma G, which determines the later phase of prespore-specific transcription . We have isolated two new missense mutations in the spoIIAB gene . spoIIAB28 behaves like the previously described spoIIAB1 mutation, in that it mainly affects the activity of sigma G . In contrast, the spoIIAB22 mutation seems to be impaired mainly in its ability to inhibit sigma F . All three missense mutations are clustered in the N-terminal coding region of spoIIAB, suggesting that this region of the protein interacts with the sigma factors . The extreme N-terminal part of SpoIIAB may be specifically concerned with the regulation of sigma G activity. Antimicrob Agents Chemother, 1993 Dec, 37(12), 2693 - 8 Regional and systemic prophylaxis with teicoplanin in monolateral and bilateral total knee replacement procedures: study of pharmacokinetics and tissue penetration; de Lalla F et al.; Twenty-four patients undergoing monolateral or bilateral total knee replacement (TKR) procedures were randomized to receive teicoplanin (T) either systemically or regionally . Subjects scheduled for systemic prophylaxis and undergoing monolateral (six patients) or bilateral (five patients) TKR received a single 800-mg dose of T in 100 ml of saline as a 5-min infusion into a forearm vein 2.5 h before surgery . For regional prophylaxis, patients undergoing monolateral surgery (eight subjects) received 400 mg of T in 100 ml of saline as a 5-min infusion into a foot vein of the leg to be operated on immediately after the tourniquet was inflated . For the five patients scheduled for bilateral operation and regional prophylaxis, the administration of T was also repeated for the second knee operation . The tourniquet, as the standard TKR surgical technique, was inflated to 400 mm Hg (c . 50 kPa) in all 24 patients immediately before the beginning of surgery and kept in place for the duration of the operation . Samples of serum, bone, skin, synovia, and subcutaneous tissue were collected at timed intervals during surgery . They were microbiologically assayed for T by using Bacillus subtilis as the test organism . Overall, the mean T concentrations obtained with regional route prophylaxis were found to be 2 to 10 times higher than those achieved following systemic prophylaxis . Moreover, peak levels in different tissues after regional prophylaxis were significantly higher (P < 0.05) . None of the patients experienced adverse effects due to regional or systemic T administration; no prosthetic or wound infections were observed in the follow-up period (from 12 to 26 months). J Gen Microbiol, 1993 Dec, 139 ( Pt 12), 3177 - 84 Analysis of Bacillus subtilis 168 prophage-associated lytic enzymes; identification and characterization of CWLA-related prophage proteins; Foster SJ; The CWLA lytic amidase of Bacillus subtilis 168 was purified and antisera raised against the purified protein . No expression of cwlA could be demonstrated under any conditions by the use of the antisera and cwlA::lacZ fusion analysis . Two lytic enzymes of apparent molecular masses 34 and 30 kDa (as measured by renaturing SDS-PAGE) were found to be mitomycin C-inducible, the larger of which corresponds to a protein immunologically related to CWLA . Both of these inducible lysins were found to be encoded by prophage PBSX . Prophage SP beta was shown by renaturing SDS-PAGE to produce a 43 kDa lytic enzyme unrelated immunologically to CWLA . The smaller of the two PBSX enzymes was purified and found to be an N-acetylmuramyl-L-alanine amidase of 32 kDa (as measured by SDS-PAGE and Coomassie blue staining) which cross-reacts only weakly with the anti-CWLA sera . The potential origin of cwlA and its possible relationship to the other phage lytic enzymes are discussed. Biochem Mol Biol Int, 1993 Dec, 31(5), 955 - 66 Molecular cloning and physiological analysis of an invertase isoenzyme in Helianthus tissues; Venuat B et al.; A soluble acid invertase activity isolated from Helianthus tuberosus (Jerusalem artichoke) shoots and analyzed by immunochromatography using polyclonal yeast antibodies, represents around 5% of the total invertase activity . This invertase isoenzyme was also isolated from dormant tuber parenchyma . In these partially dormant tissues, the specific activity of this isoenzyme is low suggesting a partial inactivation of the invertase molecules . Polyacrylamide gel electrophoresis of immunopurified fractions yields similar levels of the 58 kDa polypeptide both in shoots and dormant tubers, but with much lower activity of the enzyme in the tubers . A cDNA library was constructed in pUEX 1 from poly (A)+ RNA extracted from Jerusalem artichoke tubers . This library was screened for invertase using (i) a Bacillus subtilis invertase DNA probe and (ii) anti-yeast invertase antibodies . A recombinant clone of approximately 1.8 kb size was selected by these two methods . Using Northern blots, a temporal sequence in the expression of invertase gene was observed during the breaking of dormancy with the main level after 8 weeks of cold treatment at 4 degrees C . A 2.5 kb transcript was detected, translation of which would yield a 97 kDa polypeptide representing the precursor of Jerusalem artichoke invertase. Biol Pharm Bull, 1993 Dec, 16(12), 1211 - 5 Effects of various esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, trypsin inhibitors, on the growth of Bacillus subtilis; Irisawa T et al.; Various aromatic esters of trans-4-guanidinomethylcyclohexanecarboxylic acid (GMCHA), trypsin inhibitors, strongly inhibited the growth of Bacillus subtilis 558 and their effects were markedly affected by the species of substitution on the phenyl nucleus of the GMCHA phenyl esters . 4-tert-Butylphenyl ester of GMCHA (GMCHA-OPhtBu), a representative of various GMCHA esters, dose-dependently inhibited the growth of B . subtilis and DNA, RNA and protein syntheses in the cells . The growth inhibition was preceded by suppressive effect of GMCHA-OPhtBu on DNA synthesis . These results suggested the possible involvement of a trypsin-like proteinase in DNA synthesis . A trypsin-like proteinase was partially purified from B . subtilis 558 by DEAE-cellulose column chromatography, ammonium sulfate fractionation and successive chromatographies on Sephadex G-200, phenyl-Sepharose CL-4B, L-arginine-Sepharose 4B and Sephadex G-200 columns . The properties were compared with those of proteinase In, which momentarily appears just before the onset of DNA synthesis and seems to participate in the initiation of DNA replication, and which was purified from E . coli K-12 IAM 1264 . The properties of the proteinase from B . subtilis 558 were similar to proteinase In, however, the molecular mass (110000) was different from that of the latter (66000) . Various GMCHA esters strongly inhibited the proteinase activity and the order of the effects was closely correlated with that on the cell growth . The proteinase was tentatively called subtilis proteinase In. Biochim Biophys Acta, 1993 Nov 28, 1158(3), 345 - 51 Purification and reconstitution of the methyl-accepting chemotaxis proteins from Bacillus subtilis; Hanlon DW et al.; The methyl-accepting chemotaxis proteins (MCPs) from Bacillus subtilis, designated as H1, H2, and H3, have been purified to near homogeneity . These purified MCPs were reconstituted into proteoliposome vesicles using a detergent dilution procedure . The ability of the reconstituted MCPs to be methylated in vitro strongly suggests that they are in a functionally active conformation . The MCPs of B . subtilis are considerably larger than those of Escherichia coli, with molecular weights of the purified proteins being 76, 86, and 97 kDa for H3, H2, and H1, respectively . Two-dimensional electrophoresis demonstrates that the isoelectric point of H1 and H2 is 5.1, while H3 is slightly more basic, having an isoelectric point of 5.3 . Immunoblot analysis using the cross reacting E . coli anti-Trg antibody reveals that maximal MCP expression occurs approx . 4 h after the onset of stationary phase, and remains relatively stable thereafter . However, the ability of the MCPs to be methylated in vivo is significantly reduced. FEBS Lett, 1993 Nov 22, 334(3), 296 - 300 Amino acid sequence and thermostability of xylanase A from Schizophyllum commune; Oku T et al.; The amino acid sequence (197 residues) of xylanase A from the fungus, Schizophyllum commune, was determined by automated analysis of peptides from proteolytic and acid cleavage . The sequence is similar to two Trichoderma xylanases (approximately 56% identical amino acids), but also shows at least 40% identities with xylanases from Bacillus subtilis, B . pumilus and B . circulans . The conserved regions of the enzyme contain only two glutamic acid residues which implicates their possible involvement in catalysis . The disulfide bond in xylanase A is not conserved in this family . In spite of this, the B . subtilis xylanase was found to be more thermostable than xylanase A. FEMS Microbiol Lett, 1993 Nov 15, 114(1), 47 - 52 Genetic mapping in Bacillus subtilis 168 of the aadK gene which encodes aminoglycoside 6-adenylyltransferase; Noguchi N et al.; Bacillus subtilis 168 has an aadK gene, which encodes aminoglycoside 6-adenylyltransferase, a streptomycin-modifying enzyme, on its chromosome . To characterize the aadK gene, we constructed a B . subtilis 168 strain that carried the chloramphenicol resistance gene near the aadK on the chromosome and an aadK deletion mutant using an integration technique . The aadK gene was mapped between azlB and pheA on the chromosome of B . subtilis 168 . The aadK deletion mutant was slightly more susceptible to streptomycin than the original strain . The result indicates that the aadK gene contributes low-level resistance to streptomycin in B . subtilis 168. Protein Eng, 1993 Nov, 6(8), 921 - 6 Identification of two amino acids contributing the high enzyme activity in the alkaline pH range of an alkaline endoglucanase from a Bacillus sp; Park JS et al.; An alkaline cellulase (beta-1,4-endoglucanase; NK1) from an alkalophilic Bacillus sp . shows great similarity in amino acid sequence to a neutral cellulase (BSC) from Bacillus subtilis, despite a considerable difference in their pH activity profiles . Multiple amino acid exchanges by site-directed mutagenesis, using BSC as the reference, were performed on the residues in region 5 of NK1, which was previously shown to be responsible for the high enzyme activity of this alkaline cellulase in a broad alkaline pH range . Two amino acid residues, Ser287 and Ala296, were identified as being responsible for the activity in the alkaline range . The double mutation, Ser287 to Asn and Ala296 to Ser, of NK1 made its pH activity profile almost the same as that of BSC . On the other hand, the pH activity profile in the acidic range was not significantly affected by various amino acid replacements including these two positions in region 5 . This observation, together with the information available on other endoglucanases, suggests that the above two amino acid substitutions caused a profound effect through rearrangement of the hydrogen bond network forming the substrate-binding site or the catalytic site. Plasmid, 1993 Nov, 30(3), 312 - 5 A useful cloning vector for Bacillus subtilis; Diderichsen B et al.; We have constructed plasmid pDN1050, a new small cloning vector for Bacillus subtilis . pDN1050 harbors the origin of replication of Staphylococcus aureus plasmid pUB110 and the chloramphenicol resistance gene of S . aureus plasmid pC194 . The plasmid is segregationally and structurally stable . Plasmid pDN1370, a low copy number mutant of pDN1050 was isolated and shown to harbor a mutation in the repA gene of the replication protein. J Appl Bacteriol, 1993 Nov, 75(5), 478 - 84 Role of cell-binding in the antibacterial mechanism of lactoferricin B; Bellamy WR et al.; The antibacterial cell-binding properties of lactoferricin B, a potent bactericidal peptide derived from bovine lactoferrin, were investigated for the first time . To facilitate measurements of binding the peptide was radiolabelled by reduction and treatment with iodo-{1-14C}acetamide . 14C-lactoferricin B bound rapidly to the surface of Escherichia coli and Bacillus subtilis . The rate of binding was consistent with the rapid rate of killing caused by this peptide . The extent of binding was reduced in the presence of Mg2+ or Ca2+ ions which act to reduce its antimicrobial effectiveness . The optimal pH for binding was strain-dependent and the killing effect was maximal near the optimal pH for cell binding with each strain tested . These observations indicate that direct interaction of lactoferricin B with the cell surface is necessary for its lethal effect . The number of peptide molecules bound (> 10(6) per cell) was more than would be expected for binding to specific protein receptors . Lactoferricin B inhibited bacterial uptake of 3H-proline with effectiveness similar to polymyxin B, a known membrane-disruptive agent . The cell-binding event appears to lead to a disruption of normal permeability functions of the cytoplasmic membrane. Appl Environ Microbiol, 1993 Nov, 59(11), 3894 - 8 Secretion of streptavidin from Bacillus subtilis; Nagarajan V et al.; Streptavidin is an extracellular tetrameric protein produced by Streptomyces avidinii . A series of hybrid gene fusions consisting of Bacillus signal peptide coding regions fused to the mature streptavidin sequence were constructed . B . subtilis strains harboring these plasmids accumulate a tetrameric streptavidin in the growth medium . The properties of the streptavidin produced by B . subtilis are similar to those of the streptavidin produced by S . avidinii . B . subtilis strains carrying the various fusions can be grown to a high cell density in a biotin-free medium . Thus, B . subtilis represents an alternate host system for the production of streptavidin. Mol Biol (Mosk), 1993 Nov-Dec, 27(6), 1218 - 29 {Evidence of the existence of hemolysin II from Bacillus cereus: cloning the genetic determinant of hemolysin II}; Sinev MA et al.; The hemolysin genetic determinant distinct from cereolysin AB genetic determinant (lecithinase and sphingomyelinase genes) has been cloned in Escherichia coli and Bacillus subtilis cells as an EcoRI fragment (2.9 kb) of Bacillus cereus VKM-B771 chromosome DNA . The hemolytic product encoded by the cloned DNA fragment possessed all the properties of hemolysin II known to date: it was not inhibited by cholesterol, exhibited the Arrhenius effect, and had a relatively long (in comparison with cereolysin) lag period in erythrocyte lysis . The cloned DNA fragment was concluded to contain the gene of hemolysin II from B . cereus . In contrast to previous suggestions that hemolitic activity ascribed to hemolysin II is due to the combined action of sphingomyelinase and lecithinase, the results obtained present convincing evidence that hemolysin II is an independent B . cereus hemolytic factor different from cereolysin AB. J Gen Microbiol, 1993 Nov, 139 ( Pt 11), 2659 - 65 Biological activities of lipoteichoic acid and peptidoglycan-teichoic acid of Bacillus subtilis 168 (Marburg); Himanen JP et al.; To evaluate the suitability of Bacillus subtilis as a production host of heterologous proteins for pharmaceutical purposes, we assessed the biological activity of this bacterium and its major cell envelope components, lipoteichoic acid (LTA) and peptidoglycan-teichoic acid complex (PG-TA) in several eukaryotic effector assays . LTA and PG-TA were found to be non-toxic for mice and guinea-pigs in a short-term toxicity assay . PG-TA was weakly pyrogenic and weakly mitogenic . Both LTA and PG-TA acted as immunologic adjuvants in mice and when injected in mice, also caused an increase in the number of granulocyte-monocyte colony-forming cells in the bone marrow probably via stimulation of production of granulocyte-macrophage colony-stimulating factor. Mol Gen Genet, 1993 Nov, 241(3-4), 287 - 97 Integration of repeated sequences (pBR322) in the Bacillus subtilis 168 chromosome without affecting the genome structure; Itaya M; The Escherichia coli plasmid pBR322 sequence (4363 bp) was integrated at the met, pro, or leuB locus of the Bacillus subtilis chromosome without duplication of the flanking chromosomal regions . The integrated pBR322 was stably maintained as part of the chromosome regardless of its orientation or location . It was found that a DNA segment as large as 17 kb cloned in pBR322 can be readily transferred to the B . subtilis chromosome by transformation . It was demonstrated that a second pBR322 sequence could be effectively introduced at different regions of the chromosome by sequential transformation using chromosomal DNA isolated from a strain that had already acquired a pBR322 sequence at a different locus . Similarly, a third pBR322 sequence could be introduced . By this method, two or three pBR322 sequences can be incorporated at unlinked loci without affecting the overall structure of the B . subtilis genome. J Bacteriol, 1993 Nov, 175(22), 7348 - 55 Regulation of the Bacillus subtilis acetate kinase gene by CcpA; Grundy FJ et al.; The Bacillus subtilis gene encoding acetate kinase was identified on the basis of sequence similarity to the Escherichia coli ackA gene and to a second E . coli gene closely related to ackA . Insertional inactivation of this region of the B . subtilis chromosome resulted in the disappearance of acetate kinase enzyme activity in cell extracts . The ackA gene was mapped to a site close to the ccpA gene, at 263 degrees . The transcriptional start site for B . subtilis ackA was located 90 bp upstream from the start of the coding region, and expression was increased by growth in the presence of excess glucose . Growth of the AckA- mutant was inhibited by glucose, suggesting that acetate kinase is important for excretion of excess carbohydrate . The stimulation of ackA expression by glucose was blocked in a CcpA- mutant, indicating that CcpA, which is required for glucose repression of certain carbon source utilization genes, including amyE, may also be involved in activation of carbon excretion pathways . Two sequences resembling the amyO operator site were identified upstream of the ackA promoter; removal of this region resulted in loss of glucose activation of ackA expression. J Bacteriol, 1993 Nov, 175(22), 7341 - 7 Multilevel regulation of the sporulation transcription factor sigma K in Bacillus subtilis; Oke V et al.; Gene expression in the mother-cell compartment of the Bacillus subtilis sporangium is governed in part by the sporulation transcription factor sigma K . The production of sigma K is controlled at three levels: by a chromosomal rearrangement that generates the sigma K-coding sequence (sigK), by compartment-specific transcription of sigK, and by conversion of the inactive pro-protein product of sigK (pro-sigma K) to sigma K . To investigate the function of these multiple levels of regulation, we constructed a set of strains that bypass the chromosomal rearrangement, pro-protein processing, or both levels of control . Here we show that one of the functions of the chromosomal rearrangement and pro-protein processing is to prevent inappropriate production of sigma K under nonsporulation conditions . In the absence of both of these levels of control, a low level of sigma K-directed gene expression is observed during stationary phase after growth in rich medium . The appearance of sigma K under these conditions is probably due to a low level of sigma K-directed transcription from the sigK promoter in a positive feedback loop . We also report the construction of a strain that produces high levels of sigma K during growth . Using this strain, we demonstrate that the production of sigma K during growth is sufficient to induce a cascade of gene expression that closely mimics late events in the mother-cell line of gene expression. J Bacteriol, 1993 Nov, 175(22), 7150 - 9 Cloning and characterization of the RNA polymerase alpha-subunit operon of Chlamydia trachomatis; Tan M et al.; We have cloned the chlamydial operon that encodes the initiation factor IF1, the ribosomal proteins L36, S13, and S11, and the alpha subunit of RNA polymerase . The genes for S11 and alpha are closely linked in Escherichia coli, Bacillus subtilis, and plant chloroplast genomes, and this arrangement is conserved in Chlamydia spp . The S11 ribosomal protein gene potentially encodes a protein of 125 amino acids with 41 to 42% identity over its entire length to its E . coli and B . subtilis homologs; the gene encoding the alpha subunit specifies a protein of 322 amino acids with 25 to 30% identity over its entire length to its E . coli and B . subtilis homologs . In a T7-based expression system in E . coli, the chlamydial alpha gene directed the synthesis of a 36-kDa protein . Mapping of the chlamydial mRNA transcript by RNase protection studies and by a combination of reverse transcription and the polymerase chain reaction demonstrates that IF1, L36, S13, S11, and alpha are transcribed as a polycistronic transcript. J Bacteriol, 1993 Nov, 175(22), 7130 - 7 Characterization of the gene encoding an intracellular proteinase inhibitor of Bacillus subtilis and its role in regulation of the major intracellular proteinase; Shiga Y et al.; The gene (ipi) for an intracellular proteinase inhibitor (BsuPI) from Bacillus subtilis was cloned and found to encode a polypeptide consisting of 119 amino acids with no cysteine residues . The deduced amino acid sequence contained the N-terminal amino acid sequence of the inhibitor, which was chemically determined previously, and showed no significant homology to any other proteinase inhibitors . Analysis of the transcription initiation site and mRNA showed that the ipi gene formed an operon with an upstream open reading frame with an unknown function . The transcriptional control of ipi gene expression was demonstrated by Northern (RNA) blot analysis, and the time course of transcriptional enhancement roughly corresponded to the results observed at the protein level . Strains in which the ipi gene was disrupted or in which BsuPI was overexpressed constitutively sporulated normally . Analysis of the time course of production of the intracellular proteinase and proteinase inhibitor in these strains suggested that BsuPI directly regulated the major intracellular proteinase (ISP-1) activity in vivo. J Bacteriol, 1993 Nov, 175(21), 6842 - 9 Purification of an SOS repressor from Bacillus subtilis; Lovett CM Jr et al.; We have identified in Bacillus subtilis a DNA-binding protein that is functionally analogous to the Escherichia coli LexA protein . We show that the 23-kDa B . subtilis protein binds specifically to the consensus sequence 5'-GAACN4GTTC-3' located within the putative promoter regions of four distinct B . subtilis DNA damage-inducible genes: dinA, dinB, dinC, and recA . In RecA+ strains, the protein's specific DNA binding activity was abolished following treatment with mitomycin C; the decrease in DNA binding activity after DNA damage had a half-life of about 5 min and was followed by an increase in SOS gene expression . There was no detectable decrease in DNA binding activity in B . subtilis strains deficient in RecA (recA1, recA4) or otherwise deficient in SOS induction (recM13) following mitomycin C treatment . The addition of purified B . subtilis RecA protein, activated by single-stranded DNA and dATP, abolished the specific DNA binding activity in crude extracts of RecA+ strains and strains deficient in SOS induction . We purified the B . subtilis DNA-binding protein more than 4,000-fold, using an affinity resin in which a 199-bp DNA fragment containing the dinC promoter region was coupled to cellulose . We show that B . subtilis RecA inactivates the DNA binding activity of the purified B . subtilis protein in a reaction that requires single-stranded DNA and nucleoside triphosphate . By analogy with E . coli, our results indicate that the DNA-binding protein is the repressor of the B . subtilis SOS DNA repair system. J Bacteriol, 1993 Nov, 175(21), 6789 - 96 Alanine dehydrogenase (ald) is required for normal sporulation in Bacillus subtilis; Siranosian KJ et al.; The ski22::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ) . The ski22::Tn917lac insertion mutation was in ald, the gene encoding alanine dehydrogenase . Alanine dehydrogenase catalyzes the deamination of alanine to pyruvate and ammonia and is needed for growth when alanine is the sole carbon or nitrogen source . The sporulation defect caused by null mutations in ald was partly relieved by the addition of pyruvate at a high concentration, indicating that the normal role of alanine dehydrogenase in sporulation might be to generate pyruvate to provide an energy source for sporulation . The spoVN::Tn917 mutation was also found to be an allele of ald . Transcription of ald was induced very early during sporulation and by the addition of exogenous alanine during growth . Expression of ald was normal in all of the regulatory mutants tested, including spo0A, spo0K, comA, sigB, and sigD mutants . The only gene in which mutations affected expression of ald was ald itself . This regulation is probably related to the metabolism of alanine. Eur J Biochem, 1993 Nov 1, 217(3), 849 - 56 Conformations and conformational changes of four Phe-->Trp variants of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis studied by circular dichroism and fluorescence spectroscopy; Welfle H et al.; Circular dichroic spectra in the region 180-260 nm of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis and of four mutants with a Phe residue replaced by Trp, i.e . {F29W}HBsu, {F47W}HBsu, {F50W}HBsu and {F79W}HBsu, show minor differences only and demonstrate the general similarity of the conformations of these proteins . Fluorescence maxima at 315-320 nm and 330-335 nm indicate a more hydrophobic environment or a more effective stacking of Trp residues in mutants {F29W}HBsu and {F50W}HBsu in comparison to {F47W}HBsu and {F79W}HBsu, respectively . Unfolding of the mutants in high-ionic-strength buffers by increasing concentrations of urea results in a red shift of the fluorescence emission maxima to about 350 nm; the fluorescence intensities decrease strongly for {F29W}HBsu and {F50W}HBsu but show a small increase for {F47W}HBsu and {F79W}HBsu . The data suggest complex unfolding patterns with subtle differences between the single mutants . The circular dichroic spectra in the region 250-320 nm are dominated by the effects of the Trp residues and signal position-dependent differences in the environment of the Trp residues . The conformations of the mutant proteins depend on the ionic strength of the buffer and become more stable against unfolding by denaturants or increasing temperatures at higher ionic strength . At low ionic strength a pronounced protein-concentration dependence of the conformation of the mutants is seen. Mol Microbiol, 1993 Nov, 10(4), 771 - 9 Protein-nucleoside contacts in the interaction between the replication terminator protein of Bacillus subtilis and the DNA terminator; Langley DB et al.; The interaction between the DNA replication terminator, IRI, of Bacillus subtilis and its cognate replication terminator protein (RTP) has been examined by the technique of missing nucleoside interference (MNI) . IRI contains two adjacent binding sites (A and B) for RTP dimers . The B site is proximal to the replication fork arrest site . The present results have shown that nucleoside contacts with RTP in the two sites are very different . There are more extensive contacts of nucleosides in both strands of the B site with RTP compared with the A site . The data also strongly suggest that filling by RTP of the B site occurs first and is needed for subsequent co-operative filling of an overlapping A site . The A site alone binds RTP poorly . The findings are consistent with interaction occurring between RTP dimers bound to adjacent sites of IRI, which would explain why RTP bound to the B site alone cannot cause replication fork arrest. Appl Microbiol Biotechnol, 1993 Nov, 40(2-3), 341 - 7 Extracellular production of human hepatitis B virus preS2 antigen as hybrid proteins with Bacillus subtilis alpha-amylases in high-salt-concentration media; Honda K et al.; To produce PreS2 antigen of human hepatitis B virus extra-cellularly in Bacillus subtilis, it was fused with the COOH-termini of B . subtilis alpha-amylases of 522 (Amy+), 467 (Amy+) and 443 (Amy-) amino acids . Among them, alpha-amylase-A467, which has 467 amino acids, was a relatively stable carrier when the cells were cultured in Luria-Bertani (LB) medium . The alpha-amylase-A443-PreS2 hybrid protein (Amy-) was quickly degraded . The alpha-amylase-A522-PreS2 hybrid was most efficiently produced when a B . subtilis transformant of a protease-super-deficient mutant was cultured in the presence of 0.5 M sodium sulphate . The production of A522-PreS2 hybrid protein under such conditions reached 5-10 mg/l and was eight, and 200-500 times higher than those obtained by the transformants of an alkaline/neutral protease-deficient mutant of B . subtilis, and a wild-type strain, in LB medium, respectively. Gene, 1993 Oct 29, 133(1), 47 - 53 An efficient expression and secretion system based on Bacillus subtilis phage phi 105 and its use for the production of B . cereus beta-lactamase I; Thornewell SJ et al.; A novel expression system based on the Bacillus subtilis bacteriophage phi 105 has been developed to permit the high-level synthesis and secretion of beta-lactamase I (BlaI) from Bacillus cereus . Shotgun insertion of a promoterless lacZ gene into the phage genome permitted the identification of a clone producing large amounts of beta-galactosidase (beta Gal), indicating the transcription of the reporter gene from a strong phage promoter . The insertion also blocked lysis of the host cell . Although the insertion in the original prophage was complex, plasmid vectors and prophage derivatives have been developed to facilitate the replacement of lacZ with other genes for expression . The new prophages contain two additional mutations: an ind mutation, which greatly enhances the normally poor transformability of phi 105 lysogens, and a cts mutation, which allows thermo-induction of phage development and protein production . Induction of a derivative prophage containing the blaI gene from B . cereus resulted in the production of up to 500 micrograms of secreted BlaI per ml of culture supernatant. Gene, 1993 Oct 29, 133(1), 135 - 40 Isolation and characterization of the Rickettsia prowazekii gene encoding the flavoprotein subunit of succinate dehydrogenase; Aliabadi Z et al.; The gene (sdhA) coding for the flavoprotein subunit (SdhA) of succinate dehydrogenase of the obligate intracellular parasitic bacterium, Rickettsia prowazekii, has been isolated using an oligodeoxyribonucleotide probe to the conserved flavin adenine dinucleotide (FAD)-binding region of characterized flavoproteins . Nucleotide (nt) sequence analysis revealed an open reading frame (ORF) of 1791 bp capable of encoding a protein of 596 amino acids (aa) with a deduced M(r) of 65,444 . The deduced aa sequence, when compared to the flavoprotein subunits of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Bos taurus, revealed 52.8, 34.0, 65.8 and 52.0% aa identity, respectively . R . prowazekii SdhA produced in E . coli minicells and analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) migrated as a protein of approximately 63 kDa, comparable to the size of the deduced protein . In addition, two proteins of approximately 12 and 41 kDa were also produced in the E . coli minicells . The production of these proteins resulted from additional translational starts within the SdhA coding sequence, suggesting differences between the translational start signals of E . coli and R . prowazekii . Despite the similarity of R . prowazekii SdhA to that of E . coli, the R . prowazekii SdhA did not complement an E . coli sdhA mutant . In addition, analysis of the nt sequence immediately upstream from R . prowazekii sdhA revealed that the rickettsial sdh gene organization differs from that of E . coli and B . subtilis. Gene, 1993 Oct 29, 133(1), 119 - 21 Cloning and mapping of the Bacillus subtilis locus homologous to Escherichia coli ent genes; Adams R et al.; Here, we report the cloning of a 3.5-kb HindIII fragment of chromosomal Bacillus subtilis DNA carrying at least two open reading frames exhibiting significant homology with entA and entE of Escherichia coli . This B . subtilis ent locus was mapped at about 41 degrees . Its inactivation did not cause any detectable phenotype. Biochemistry, 1993 Oct 26, 32(42), 11405 - 12 Site-directed mutagenesis of putative active-site amino acid residues of 3,2-trans-enoyl-CoA isomerase, conserved within the low-homology isomerase/hydratase enzyme family; Muller-Newen G et al.; During beta-oxidation of unsaturated fatty acids, mitochondrial 3,2-trans-enoyl-CoA isomerase (mECI) converts 3-cis- or 3-trans-enoyl-CoA intermediates into their 2-trans isomers . The cDNA-derived amino acid sequence of mECI shows weak but significant homologies to the peroxisomal trifunctional enzyme (pTFE), the alpha-subunit of the fatty acid degradation complex from Escherichia coli (FadB), the mitochondrial 2-enoyl-CoA hydratase (mECH), the naphthoate synthase encoded by the menB gene from Bacillus subtilis (MenB), and the 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp . (CBDH) . These proteins from the isomerase/hydratase enzyme family . Tyr-150, Arg-151, and Asp-211 of the mECI are the only amino acids with protic side chains conserved within the enzymes with isomerase activity (pTFE and FadB) . These amino acids are exchanged in the remaining enzymes of the isomerase/hydratase family . Glu-165 is conserved in all enzymes with isomerase and/or hydratase activity (pTFE, FadB, and mECH) . We argue that these amino acids are possibly involved in the proton transfer at the active site of mECI . To test this hypothesis, mECI was functionally expressed in E . coli . The recombinant enzyme (rmECI) exhibits the same specific activity as the enzyme from rat liver . Exchange of the candidate active-site amino acids by site-directed mutagenesis revealed that Tyr-150 is not involved in isomerase catalysis . The exchange of Arg-151 and Asp-211 leads to a reduced expression of the recombinant enzyme accompanied by a reduced specific activity . The replacement of Glu-165 by Gln leads to a strongly reduced enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Biol, 1993 Oct 20, 233(4), 695 - 704 Residues of the Bacillus subtilis phage phi 29 transcriptional activator required both to interact with RNA polymerase and to activate transcription; Mencia M et al.; Regulatory protein p4 from Bacillus subtilis phage phi 29 activates transcription from the viral late promoter, PA3, by stabilizing the binding of RNA polymerase to the DNA as a closed complex . Protein p4-induced DNA bending and direct contacts between p4 and RNA polymerase have been proposed to play a role in P(A3) activation . By site-directed mutagenesis at the carboxyl end of protein p4 we have identified residues that are critical both to interact with RNA polymerase and to activate transcription . Substitution of arginine 120 gives rise to a p4 derivative unable to activate transcription, that can bind to DNA and induce a normal DNA bending, but does not stimulate the binding of RNA polymerase to the promoter and cannot form complexes with RNA polymerase . Modification of the closely located residue leucine 117 had a similar but milder effect . The results obtained suggest that arginine 120 and leucine 117 form part of the activating domain of the protein, and show that direct contacts between protein p4 and RNA polymerase play a critical role in transcription activation . The p4-induced DNA bending is therefore necessary but not sufficient for the activation of the PA3 promoter. FEMS Microbiol Lett, 1993 Oct 15, 113(2), 201 - 3 Inducible resistance to zinc ions in Bacillus subtilis 168; Podlesek Z et al.; Chromosomally determined resistance to Zn2+ ions is a property of Bacillus subtilis 168 . Resistance is inducible and is not connected with Cd2+ resistance, characteristic of plasmid determined resistance in bacteria . A few proteins were shown to respond to inducible levels of the cation. J Biol Chem, 1993 Oct 5, 268(28), 20709 - 12 The p27 catalytic subunit of the apolipoprotein B mRNA editing enzyme is a cytidine deaminase; Navaratnam N et al.; The messenger RNA for apolipoprotein B undergoes a discrete and specific C to U editing of nucleotide 6666 . This generates a stop translation codon and defines the carboxyl terminus of apolipoprotein B48 . A 27-kDa rat intestinal protein that does not itself edit apolipoprotein B mRNA, but confers editing activity on chick intestinal extracts that do not have intrinsic editing activity, has recently been identified and its cDNA cloned (Teng, B., Burant, C . F., and Davidson, N . O . (1993) Science 260, 1816-1819) . Here we show that p27 is homologous in the zinc coordinating region of the active site to cytidine deaminases from Escherichia coli, Bacillus subtilis, yeast, and man and to deoxycytidylate deaminases from T2 and T4 bacteriophages and man . p27 expressed in Xenopus laevis oocyte extracts has cytidine deaminase activity and specifically confers editing activity on chick intestinal extracts . The homologous E . coli cytidine deaminase does not confer editing activity . The zinc-specific chelating agent o-phenanthroline abolishes p27 activity and site-specific apolipoprotein B mRNA editing in rat enterocyte editing extracts . We conclude that p27 is the catalytic subunit of the apolipoprotein B mRNA editing enzyme and is a zinc-containing cytidine deaminase. J Bacteriol, 1993 Oct, 175(20), 6717 - 20 Identification of an intracellular pyrimidine-specific endoribonuclease from Bacillus subtilis; Mathur S et al.; Two intracellular RNases which were easily separated by fractionation on strong anion- or cation-exchange resins were identified from Bacillus subtilis . One cleaved any phosphodiester bond, while the second cleaved only pyrimidine-N bonds . The enzyme with pyrimidine-N specificity was approximately 15 kDa, had a pH optimum of approximately 6.2, degraded C-C bonds approximately 10 times faster than U-U bonds, and was completely inactive against single-stranded DNA . The enzyme is called RNase C and may be the first reported broad-specificity endoribonuclease from B . subtilis. J Bacteriol, 1993 Oct, 175(19), 6348 - 53 The degA gene product accelerates degradation of Bacillus subtilis phosphoribosylpyrophosphate amidotransferase in Escherichia coli; Bussey LB et al.; A search for genes involved in the inactivation and degradation of enzymes in sporulating Bacillus subtilis led to identification of the B . subtilis degA gene, whose product stimulates degradation of B . subtilis glutamine phosphoribosylpyrophosphate amidotransferase in Escherichia coli cells . degA encodes a 36.7-kDa protein that has sequence similarity to several E . coli and B . subtilis regulatory proteins of the LacI class . B . subtilis degA::cat insertional inactivation mutants had no detectable defect in the inactivation or degradation of phosphoribosylpyrophosphate amidotransferase in glucose- or lysine-starved B . subtilis cells, however . We suggest that degA encodes either a novel protease or, more likely, a gene that stimulates production of such a protease. J Bacteriol, 1993 Oct, 175(19), 6321 - 7 Characterization of a new sporulation factor in Bacillus subtilis; Waldburger C et al.; We report the existence and partial purification of sporulation factor, which stimulates sporulation of Bacillus subtilis at low cell density . Proline or arginine is required for stimulation under the conditions of our assay . Sporulation factor is a small heat-stable substance produced by the cells during exponential growth phase . It is required in small amounts and is resistant to various proteolytic agents . Several spo mutants were tested for the ability to produce functional sporulation factor . All of these mutants produce factor and do not sporulate in the presence of factor from wild-type cells . Sporulation factor is not involved in the induction of alpha-amylase synthesis at the initiation of sporulation. J Bacteriol, 1993 Oct, 175(19), 6260 - 8 Molecular cloning of a sporulation-specific cell wall hydrolase gene of Bacillus subtilis; Kuroda A et al.; Southern hybridization analysis of Bacillus subtilis 168S chromosomal DNA with a Bacillus licheniformis cell wall hydrolase gene, cwlM, as a probe indicated the presence of a cwlM homolog in B . subtilis . DNA sequencing of the cwlM homologous region showed that a gene encoding a polypeptide of 255 amino acids with a molecular mass of 27,146 Da is located 625 bp upstream and in the opposite direction of spoVJ . The deduced amino acid sequence of this gene (tentatively designated as cwlC) showed an overall identity of 73% with that of cwlM and of 40% with the C-terminal half of the B . subtilis vegetative autolysin, CwlB . The construction of an in-frame cwlC-lacZ fusion gene in the B . subtilis chromosome indicated that cwlC is induced at 6 to 7 h after sporulation (t6 to t7) . The spoIIIC (sigma K) mutation and earlier sporulation mutations greatly reduced the expression of the cwlC-lacZ fusion gene . Northern hybridization analysis using oligonucleotide probes of the cwlC region indicated that a unique cwlC transcript appeared at t7.5 and t9 . Transcriptional start points determined by primer extension analysis suggested that the -10 region is very similar to the consensus sequence for the sigma K-dependent promoter . Insertional inactivation of the cwlC gene in the B . subtilis chromosome caused the disappearance of a 31-kDa protein lytic for Micrococcus cell walls, which is mainly located within the cytoplasmic and membrane fractions of cells at t9 . The CwlC protein hydrolyzed both B . subtilis vegetative cell walls and spore peptidoglycan. J Bacteriol, 1993 Oct, 175(19), 6203 - 11 Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: relationship between B . subtilis sfpo and Escherichia coli entD genes; Grossman TH et al.; In response to iron deprivation, Bacillus subtilis secretes a catecholic siderophore, 2,3-dihydroxybenzoyl glycine, which is similar to the precursor of the Escherichia coli siderophore enterobactin . We isolated two sets of B . subtilis DNA sequences that complemented the mutations of several E . coli siderophore-deficient (ent) mutants with defective enterobactin biosynthesis enzymes . One set contained DNA sequences that complemented only an entD mutation . The second set contained DNA sequences that complemented various combinations of entB, entE, entC, and entA mutations . The two sets of DNA sequences did not appear to overlap . AB . subtilis mutant containing an insertion in the region of the entD homolog grew much more poorly in low-iron medium and with markedly different kinetics . These data indicate that (i) at least five of the siderophore biosynthesis genes of B . subtilis can function in E . coli, (ii) the genetic organization of these siderophore genes in B . subtilis is similar to that in E . coli, and (iii) the B . subtilis entD homolog is required for efficient growth in low-iron medium . The nucleotide sequence of the B . subtilis DNA contained in plasmid pENTA22, a clone expressing the B . subtilis entD homolog, revealed the presence of at least two genes . One gene was identified as sfpo, a previously reported gene involved in the production of surfactin in B . subtilis and which is highly homologous to the E . coli entD gene . We present evidence that the E . coli entD and B . subtilis sfpo genes are interchangeable and that their products are members of a new family of proteins which function in the secretion of peptide molecules. J Biomol Struct Dyn, 1993 Oct, 11(2), 381 - 94 Conformational stability of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis, and of the four HBsu variants {F29W}, {F47W}, {F50W} and {F79W}; Welfle H et al.; From denaturation studies with urea a free energy delta GuH2O of unfolding of 49.8 kJ.mol-1 at 25C was calculated for the histone-like DNA-binding protein HBsu from Bacillus subtilis . Unfolding was monitored by circular dichroism measurements observing the changes of the molar mean residue ellipticity {theta} at 222 nm . For the calculation of delta Gu a two-state model of unfolding, i.e . the unfolding of native dimers into unfolded monomers, was applied . The validity of this model in high ionic strength buffer was proven by measurements at different protein concentrations yielding the same delta Gu values . Four HBsu variants, each carrying one single point mutation ({F29W}, {F47W}, {F50W} and {F79W}) were analysed with respect to their stability against unfolding at increasing temperatures and urea concentrations . The delta Gu values of mutants were calculated using the two-state model and show a reduced stability of the variants {F29W}, {F47W}, {F50W} and {F79W} in comparison to the wild type HBsu with delta delta Gu values of -9.2 kJ.mol-1, -7.5 kJ.mol-1, -5.9 kJ.mol-1, and -7.5 kJ.mol-1, respectively . Similar delta delta Gu values were obtained for the HBsu mutant proteins by thermal unfolding experiments. Curr Opin Genet Dev, 1993 Oct, 3(5), 775 - 82 Bacterial chromosome origins of replication; Marczynski GT et al.; Bacteria regulate chromosomal replication from one specific origin . We compare the regulatory requirements, DNA structures, and biochemical properties of the prototypic Escherichia coli origin with those of evolutionarily distant Bacillus subtilis and Caulobacter crescentus origins . The ubiquitous DnaA protein is a major regulator of all three bacterial origins . Unique features of these origins, however, may reflect specific regulatory requirements placed on them. Appl Environ Microbiol, 1993 Oct, 59(10), 3418 - 23 Binding of small, acid-soluble spore proteins to DNA plays a significant role in the resistance of Bacillus subtilis spores to hydrogen peroxide; Setlow B et al.; Dormant spores of Bacillus subtilis which lack the majority of the alpha/beta-type small, acid-soluble proteins (SASP) (termed alpha- beta- spores) that coat the DNA in wild-type spores are significantly more sensitive to hydrogen peroxide than are wild-type spores . Hydrogen peroxide treatment of alpha- beta- spores causes DNA strand breaks more readily than does comparable treatment of wild-type spores, and alpha- beta- spores, but not wild-type spores, which survive hydrogen peroxide treatment have acquired a significant number of mutations . The hydrogen peroxide resistance of wild-type spores appears to be acquired in at least two incremental steps during sporulation . The first increment is acquired at about the time of alpha/beta-type SASP synthesis, and the second increment is acquired approximately 2 h later, at about the time of dipicolinic acid accumulation . During sporulation of the alpha- beta- strain, only the second increment of hydrogen peroxide resistance is acquired . In contrast, sporulation mutants which accumulate alpha/beta-type SASP but progress no further in sporulation acquire only the first increment of hydrogen peroxide resistance . These findings strongly suggest that binding of alpha/beta-type SASP to DNA provides one increment of spore hydrogen peroxide resistance . Indeed, binding of alpha/beta-type SASP to DNA in vitro provides strong protection against cleavage of DNA by hydrogen peroxide. Anal Biochem, 1993 Oct, 214(1), 142 - 8 Purification of membrane proteins using a micropreparative gel electrophoresis apparatus: purification of subunits of the integral membrane protein Bacillus subtilis aa3-type quinol oxidase for low level amino acid sequence analysis; Baumann M et al.; We have developed a continuous elution micropreparative gel electrophoresis system in which small amounts of proteins, e.g., 1-10 micrograms, are continuously eluted from one-dimensional polyacrylamide gel columns in either native or denatured form . Depending on the electrophoretical parameters proteins are separated according to their size and/or net charge . The system is based on a simple modification of the Mini-Protean II 2D unit from Bio-Rad Inc . The apparatus is connected on-line to a high-performance liquid chromatograph allowing accurate delivery of the elution solvent and direct analysis of the separated samples by uv detection . The high resolution of the apparatus allows the purification of individual proteins, even from complex mixtures, in only one step, with overall recoveries of approximately 70% . In addition to standard proteins the system has been applied to the purification of the membrane-bound and thus highly hydrophobic subunits I and II from Bacillus subtilis aa3-type quinol oxidase . The hydrophobic character of these subunits has hindered their purification by other conventional methods . Isolated subunits were collected directly into a buffer suitable for proteolytic digestion and automated amino acid sequence analysis . The internal amino acid sequences determined could all be found in the DNA sequence recently reported by Santana et al . (J . Biol . Chem . 267, 10225-10231, 1992), thereby confirming the expression of such an oxidase. Mol Microbiol, 1993 Oct, 10(1), 193 - 201 Bacillus subtilis CtaA and CtaB function in haem A biosynthesis; Svensson B et al.; Haem A, a prosthetic group of many respiratory oxidases, is probably synthesized from haem B (protohaem IX) in a pathway in which haem O is an intermediate . Possible roles of the Bacillus subtilis ctaA and ctaB gene products in haem O and haem A synthesis were studied . Escherichia coli does not contain haem A . The ctaA gene on plasmids in E . coli resulted in haem A accumulation in membranes . The presence of ctaB together with ctaA increased the amount of haem A found in E . coli . Haem O was not detected in wild-type B . subtilis strains . A previously isolated B . subtilis ctaA deletion mutant was found to contain haem B and haem O, but not haem A . B . subtilis ctaB deletion mutants were constructed and found to lack both haem A and haem O . The results with E . coli and B . subtilis strongly suggest that the B . subtilis CtaA protein functions in haem A synthesis . It is tentatively suggested that if functions in the oxygenation/oxidation of the methyl side group of carbon 8 of haem O . B . subtilis CtaB, which is homologous to Saccharomyces cerevisiae COX10 and E . coli CyoE, also has a role in haem A synthesis and seems to be required for both cytochrome a and cytochrome o synthesis. Mol Microbiol, 1993 Oct, 10(1), 133 - 42 Isolation and characterization of the secE homologue gene of Bacillus subtilis; Jeong SM et al.; A 4.0 kb EcoRI fragment of Bacillus subtilis conferring thiostrepton resistance was cloned and characterized . By nucleotide sequencing of the relevant region, six open reading frames were established, which corresponded to a part of spo0H, a ribosomal protein gene (rpmG), an unidentified open reading frame (orfE), a transcription antiterminator gene nusG, and ribosomal protein genes rplK and rplA . The orfE-encoded 59-amino-acid polypeptide had a low, but significant, sequence similarity with the carboxy-terminal region of the Escherichia coli SecE protein . A cold-sensitive secE mutation of E . coli was complemented by the plasmid-borne orfE sequence . Furthermore, the normal processing of a proOmpA protein was observed when the secE cold-sensitive strain carried an orfE plasmid, indicating that orfE is the secE homologue of B . subtilis . The B . subtilis secE has only one transmembrane sequence compared to the three in E . coli. Mol Microbiol, 1993 Oct, 10(2), 397 - 405 The transcriptional organization of the Bacillus subtilis 168 chromosome region between the spoVAF and serA genetic loci; Azevedo V et al.; The genetic organization of the spoVAF-serA area of the Bacillus subtilis chromosome and its putative transcription map have been derived from analysis of the nucleotide sequence . In order to confirm this transcription map as regards size of transcripts and to determine growth conditions for their appearance, we undertook Northern hybridization analysis of total RNA from vegetatively growing and sporulating cells . Twenty-three distinct transcripts were thus identified, 14 of which were predicted from sequence analysis and nine of which were not predicted . Eight of the latter are homologous to open reading frames identified by sequence analysis but were not expected, since no obvious promoter or terminator was found in the sequence . The last unexpected transcript does not correspond to an ORF and might identify a novel gene . Three predicted transcripts were not detected . The transcripts were classified in four groups as (i) constitutive, (ii) regulated by nutritional depletion, (iii) specific for sporulation, and (iv) possibly regulated temporally . These studies demonstrate that systematic Northern analysis of a bacterial chromosome region is a useful complement to sequence analysis. Mol Microbiol, 1993 Oct, 10(2), 385 - 95 The organization of the Bacillus subtilis 168 chromosome region between the spoVA and serA genetic loci, based on sequence data; Sorokin A et al.; Three different lambda phage clones with overlapping inserts of Bacillus subtilis DNA, which cover the region from spoIIAA to serA, have been isolated . The nucleotide sequence of their inserts, starting after spoVAF and ending at serA, has been determined . A contiguous sequence of 35,354 bp was established, including previously analysed overlapping adjacent regions . Within the newly determined sequence 31 open reading frames (ORFs) with putative ribosome-binding sites have been found . Nine of them correspond to previously sequenced and characterized genes: spo-VAF, lysA, sipS, ribG, ribB, ribA, ribH, ribTD and dacB . Comparison of the amino acid sequences of the products encoded by the other ORFs to known proteins allowed putative functions to be assigned to seven of these ORFs . Among these are the following: (i) the ppiB gene, encoding a cytoplasmic peptidylprolyl isomerase; (ii) two pairs of signal-transducers, one homologous to phoR-phoP of B . subtilis, encoding regulators of phosphatase biosynthesis, and the second to the fecI-fecR of Escherichia coli, which is responsible for the regulation of the citrate-dependent iron (III) transport system; (iii) aroC and serA genes, involved in the biosynthesis of aromatic amino acids and serine, respectively, the function of which has been confirmed by constructing corresponding mutants with disrupted ORFs . The organization of putative operons has been postulated on the basis of the sequences of their transcription terminators, promoters and regulatory elements. Mol Microbiol, 1993 Oct, 10(2), 371 - 84 Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325 degrees to 333 degrees; Glaser P et al.; In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome the DNA region located between gerB (314 degrees) and sacXY (333 degrees) was assigned to the Institut Pasteur . In this paper we describe the cloning and sequencing of a segment of 97 kb of contiguous DNA . Ninety-two open reading frames were predicted to encode putative proteins among which only forty-two were found to display significant similarities to known proteins present in databanks, e.g . amino acid permeases, proteins involved in cell wall or antibiotic biosynthesis, various regulatory proteins, proteins of several dehydrogenase families and enzymes II of the phosphotransferase system involved in sugar transport . Additional experiments led to the identification of the products of new B . subtilis genes, e.g . galactokinase and an operon involved in thiamine biosynthesis. Mol Microbiol, 1993 Oct, 10(2), 259 - 71 Identification of genes involved in utilization of acetate and acetoin in Bacillus subtilis; Grundy FJ et al.; The Bacillus subtilis ccpA gene has previously been shown to be involved in repression of amyE expression when cells are grown in excess glucose . The region of the B . subtilis chromosome downstream from ccpA was characterized to determine if additional genes involved in carbohydrate metabolism were present . Two open reading frames that exhibited sequence similarity to the Escherichia coli and B . subtilis motA and motB motility genes were found immediately downstream from ccpA; disruption of this region had no effect on growth, sporulation or motility . Two divergent transcriptional units containing the acsA and acuABC genes were also found in this region . The acsA gene encodes acetyl-CoA synthetase, and inactivation of this gene resulted in loss of the ability to utilize acetate as a carbon source for growth or sporulation . Disruption of the acuABC genes resulted in poor growth or sporulation on acetoin or butanediol . The acsA and acuABC promoter sequences were identified by primer extension, and are in close proximity . Two sequences resembling the amyO regulatory target site necessary for glucose repression of amyE were identified in the acsA-acuABC promoter regions. Phytochemistry, 1993 Oct, 34(3), 675 - 7 Structure-activity relationships of synthetic methyl ursolate glycosides; Takechi M et al.; From 15 synthetic methyl ursolate glycosides, the di- and tri-glycosides showed much higher haemolytic activity than the monoglycosides . The beta-gentiobioside and the beta-maltotrioside exhibited much stronger antibacterial (Staphylococcus aureus) or antifungal (Trichophyton mentagrophytes) activity than the other glycosides . However, none of them exhibited antibacterial activity against Bacillus subtilis. J Bacteriol, 1993 Oct, 175(20), 6512 - 7 The Bacillus subtilis ochre suppressor sup-3 is located in an operon of seven tRNA genes; Garrity DB et al.; Most Bacillus subtilis tRNA genes have been isolated from lambda libraries by use of probes that hybridize to tRNA or rRNA sequences . None of those genes map to the region of the sup-3 mutation . By cloning of the sup-3 allele, a cluster of seven tRNA genes (the trnS operon) that had not been isolated by other methods was identified . In principle, this approach could be used to isolate at least one more predicted tRNA-containing operon in this bacterium . The trnS operon was shown to contain tRNA genes for Asn (GUU), Ser (GCU), Glu (UUC), Gln (UUG), Lys (UUU), Leu (UAG), and Leu (GAG) . The sup-3 mutation was found to be a T-to-A transversion that changes the anticodon of the lysine tRNA from 5'-UUU-3' to 5'-UUA-3' . This result agrees with previous work that determined that the sup-3 mutation causes lysine to be inserted at ochre nonsense mutations. Gene, 1993 Sep 30, 132(1), 7 - 13 Autoregulation of the gene encoding the replication terminator protein of Bacillus subtilis; Ahn KS et al.; One of two putative sigma A promoters identified previously in the region immediately upstream from the rtp gene (encoding the replication terminator protein) {Smith and Wake, J . Bacteriol . 170 (1988) 4083-4090} has been shown by transcription start point (tsp) mapping to be the functional rtp promoter . In these tsp mapping experiments, it was observed that the level of mRNA from this promoter, Prtp, was increased by a factor of 30 in the absence of the replication terminator protein (RTP), consistent with the autoregulation of rtp at the level of transcription . In vitro transcription from Prtp by sigma A RNA polymerase has been shown to be specifically repressed by RTP . A Prtp-spoVG-lacZ fusion was inserted into the chromosome of a strain in which RTP production was inducible by IPTG . Addition of IPTG to cultures of the new strain lowered beta Gal production by a factor of at least four . It is concluded that rtp is autoregulated in vivo at the level of transcription. Biochemistry, 1993 Sep 28, 32(38), 10216 - 23 Symmetry and secondary structure of the replication terminator protein of Bacillus subtilis: sedimentation equilibrium and circular dichroic, infrared, and NMR spectroscopic studies; Kralicek AV et al.; We have used analytical ultracentrifugation in combination with a number of spectroscopic techniques to analyze the symmetry and secondary structure of the DNA-binding replication terminator protein (RTP) of Bacillus subtilis . Sedimentation equilibrium studies confirm that RTP is a dimer in solution under the conditions used for spectroscopic analysis, whereas the number of cross peaks displayed in 1H-15N HSQC NMR spectra of uniformly 15N-labeled RTP are consistent with the primary structure of the monomer . These two results in combination lead to the conclusion that RTP is a symmetric dimer in solution . Circular dichroic and Fourier-transform infrared spectra reveal, in contrast to the results obtained from a number of commonly used secondary structure prediction algorithms, that RTP contains 20-30% alpha-helical and 40-50% beta-sheet/beta-turn secondary structure and that the conformation of the protein remains unchanged over the pH range 5-8 . It is proposed on the basis of protein folding-class prediction algorithms, in combination with various physical properties of RTP, that it belongs to the alpha + beta protein-folding class. Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8600 - 3 Crystal structures of the monofunctional chorismate mutase from Bacillus subtilis and its complex with a transition state analog; Chook YM et al.; We have solved the structure of a chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5), the 1.9-A crystal structure of the monofunctional enzyme from Bacillus subtilis . The structure determination process was an unusual one, involving 12 monomers of the enzyme in the asymmetric unit . This structure was solved by the multiple isomorphous replacement method with partial structure phase combination and molecular averaging . The final model, which includes 1380 residues and 522 water molecules in an asymmetric unit, has been refined at 1.9 A and the current crystallographic R value is 0.201 . The B . subtilis chorismate mutase is a homotrimer, with beta-sheets from each monomer packing to form the core of a pseudo-alpha beta-barrel with helices on the outside of the trimer . In addition, the active sites have been located by using data from a complex with an endo-oxabicyclic inhibitor that mimics the transition state of the reaction . The structure of this complex has been refined to 2.2 A with a current R value of 0.182 for a model that includes 1388 residues, 12 inhibitor molecules, and 530 water molecules in the asymmetric unit . In each trimer, three equivalent active sites are located at the interfaces of two adjacent subunits. Biochemistry, 1993 Sep 7, 32(35), 9256 - 61 Purification and characterization of Bacillus subtilis CheY; Bischoff DS et al.; Amino acid sequence comparison suggests that numerous proteins are common to the signal transduction pathways controlling chemotaxis in Bacillus subtilis and Escherichia coli . However, previous work has indicated several differences between the two systems . We have undertaken a comparative study of the roles of the CheY protein in chemotaxis by B . subtilis and E . coli . Although CheY from the two species share only 36% amino acid sequence identity, purified B . subtilis CheY was phosphorylated in vitro by E . coli CheA, and dephosphorylation of CheY-P was enhanced by E . coli CheZ . Alteration of the putative site of phosphorylation in B . subtilis CheY, Asp54, eliminated chemotaxis in vivo, further confirming that phosphorylation is important for B . subtilis chemotaxis . Loss of CheY function resulted in tumbling behavior in B . subtilis . Introduction of positively charged residues in place of Asp10 of B . subtilis CheY abolished function, whereas the corresponding changes in E . coli CheY apparently result in constitutive activation . The B . subtilis CheY Asp10 mutant proteins also failed to cause tumbling in E . coli, consistent with a different interaction between CheY and the flagellar switch in the two species . Finally, B . subtilis adapted more rapidly to positive stimuli than negative stimuli, whereas the opposite is true of E . coli . We conclude that B . subtilis regulates its response to positive chemotactic stimuli by enhancing phosphorylation of chemotaxis proteins, whereas E . coli reduces phosphorylation in the same circumstance. Gene, 1993 Sep 6, 131(1), 97 - 102 Use of a gram- signal peptide for protein secretion by gram+ hosts: basic protease of Dichelobacter nodosus is produced and secreted by Bacillus subtilis; Wang LF et al.; The bprV gene, encoding the extracellular basic protease of the Gram- anaerobic bacterium Dichelobacter nodosus, was expressed and the protein secreted in Bacillus subtilis using the novel cloning/expression vector pNC3 {Wu et al., Gene 106 (1991) 103-107} . The pre- and pro-peptides were processed correctly in this heterologous system, and the 127-amino acid C-terminal extension region was also removed . The recombinant gene product was indistinguishable biochemically or immunochemically from the authentic protease and was able to form crystals upon dialysis, as was found for the authentic protease . This is the first example of the direct secretion of a Gram- extracellular enzyme in B . subtilis via its own signal peptide . The fact that this gene can be expressed and its product secreted in both Escherichia coli and B . subtilis provides a unique opportunity to study and compare the similarities and differences in protein secretion between Gram- and Gram+ organisms. J Biol Chem, 1993 Sep 5, 268(25), 18610 - 6 Chemotactic methylesterase promotes adaptation to high concentrations of attractant in Bacillus subtilis; Kirsch ML et al.; The Bacillus subtilis gene encoding CheB (cheBB), the chemotactic methylesterase, has been sequenced . The 39-kDa protein which resulted from the expression of cheBB, using a T7 expression system was consistent with the predicted open reading frame . CheBB shares 39.5% identity with Escherichia coli CheBE and can complement a cheBE null mutant . CheBB is required for removal of methyl groups from the receptors upon attractant stimulus and appears to play an important role in adaptation to the addition of attractants, whereas CheBE plays an important role in adaptation to the addition of repellents . Unlike the cheBE and cheRE mutants of E . coli, which show extreme flagellar rotational biases, the unstimulated cheBB mutant showed a normal (wild type) bias . Upon addition of attractant, the cheBB null mutant showed a counter-clockwise bias that was higher than for wild type and demonstrated only partial adaptation . In the capillary assay for the attractant azetidine-2-carboxylic acid, the mutant gave a wild type response at low concentrations but a very reduced response at high concentrations . We conclude that B . subtilis has an effective methylation-independent adaptation system but must utilize the methylation system for adaptation to high concentrations of attractant. J Biol Chem, 1993 Sep 5, 268(25), 18692 - 5 Cross-links in cell walls of Bacillus subtilis by rotational-echo double-resonance 15N NMR; Pan Y et al.; The cross-link index of peptidoglycan of intact cell walls of Bacillus subtilis grown in media containing L-{2-13C,15N}aspartic acid has been determined by rotational-echo double-resonance 15N NMR . A cross-link index of 72% decreased to 47% when the bacteria were exposed to the antibiotic cephalosporin C . The fraction of 15N label routed from aspartic acid in the media to glutamic acid in the peptidoglycan peptide stems more than doubled for the cephalosporin-exposed cells, indicating interference with general cell wall nitrogen metabolism by the antibiotic. J Hered, 1993 Sep-Oct, 84(5), 360 - 71 Genetic error, sex, and diploidy; Michod RE; Mathematical models and experiments on transformation are reported testing the hypothesis that sex and diploidy evolved as a DNA repair system . The models focus on the origin of diploidy and sex by studying selection between asexual haploids, sexual haploids, and diploids . Haploid cells are efficient replicators, while diploid cells are resistance to damage . A sexual haploid may combine the advantages of both: spending much of its life cycle in the haploid state, then temporarily fusing to become diploid, followed by splitting to the haploid state . During the diploid state DNA damage can be repaired, since there are two copies of the gene in the cell and one copy is presumed to be undamaged . Five basic rate parameters are employed: birth and death; genomic damage (for the haploids alone); and, for the sexual cell, fusion and splitting . Parameter space bifurcation diagrams for the equilibria are drawn, and solutions of the equations are described in terms of these diagrams . Each type of cell has a region of the parameter space that it occupies exclusively (given its initial presence in the competition) . The haploid wins in environments characterized by low damage . The diploid wins in environments characterized by high damage, low mortality, and abundant resources . In general, only a single type of cell occupies a given portion of the space . We find, however, that competitive coexistence of an asexual diploid and sexual haploid is possible in spite of the fact that they are competing for a single resource (nucleotide building blocks) . Sex can increase from rarity if matings occur with asexual cells . Only sex can cope with both high mortality and high damage . We then turn to natural bacterial transformation as a model system for the experimental study of sex . Natural transformation in distributed widely, but apparently sparsely, in all bacterial groups . A very preliminary phylogenetic analysis of the bacilli and related species indicates that transformation is probably not a diversifying force in bacterial evolution . However, it is difficult to be sure because of the ambiguity surrounding negative data . Experiments with the bacterium Bacillus subtilis indicate that transformation frequencies respond adaptively to DNA damage if homologous donor DNA is used . Several specific hypotheses for this response are considered . Recent work in other labs on the evolution of transformation is discussed from the point of view of the hypothesis that transformation functions in DNA repair. FEMS Microbiol Lett, 1993 Sep 1, 112(2), 135 - 40 Bacillus subtilis mutant deficient in the major autolytic amidase and glucosaminidase is impaired in motility; Rashid MH et al.; The purified autolytic endo-beta-N-acetylglucosaminidase of Bacillus subtilis AC327 was cleaved with cyanogen bromide, and the N-terminal amino acid sequence of one of the peptide fragments was determined . Then, a DNA fragment containing a part of the glucosaminidase gene was cloned into Escherichia coli JM109 using synthetic oligonucleotides as probes whose sequences had been deduced from the N-terminal amino acid sequence . Zymographic analysis showed that the resultant glucosaminidase-deficient strain lacked a 35-kDa lytic band in addition to a 90-kDa lytic one corresponding to the glucosaminidase . A double mutant strain deficient in the major two autolysins (amidase and glucosaminidase) exhibited greatly impaired motility on a swarm plate whereas the single mutant strains were motile. J Bacteriol, 1993 Sep, 175(17), 5690 - 6 Proton motive force may regulate cell wall-associated enzymes of Bacillus subtilis; Kemper MA et al.; Bacterial metabolism excretes protons during normal metabolic processes . The protons may be recycled by chemiosmosis, diffuse through the wall into the medium, or bind to cell surface constituents . Calculations by Koch (J . Theor . Biol . 120:73-84, 1986) have suggested that the cell wall of gram-positive bacteria may serve as a reservoir of protons during growth and metabolism, causing the wall to have a relatively low pH . That the cell wall may possess a pH lower than the surrounding medium has now been tested in Bacillus subtilis by several independent experiments . When cultures of B . subtilis were treated with the proton conductors azide and carbonylcyanide m-chlorophenylhydrazone, the cells bound larger amounts of positively charged probes, including the chromium (Cr3+) and uranyl (UO2(2+) ions and were readily agglutinated by cationized ferritin . In contrast, the same proton conductors caused a decrease in the binding of the negatively charged probe chromate (CrO4(2-)) . Finally, when levansucrase was induced in cultures by the addition of sucrose, the enzyme was inactive as it traversed the wall during the first 0.7 to 1.0 generation of growth . The composite interpretation of the foregoing observations suggests that the wall is positively charged during metabolism, thereby decreasing its ability to complex with cations while increasing its ability to bind with anions . This may be one reason why some enzymes, such as autolysins, are unable to hydrolyze their substrata until they reach the wall periphery or are in the medium. J Bacteriol, 1993 Sep, 175(17), 5428 - 37 Metalloregulation in Bacillus subtilis: isolation and characterization of two genes differentially repressed by metal ions; Chen L et al.; We have cloned two metal-regulated genes (mrgA and mrgC) from Bacillus subtilis by using transposon Tn917-lacZ . Both were isolated as iron-repressible gene fusions, but the metal specificity and sensitivity of gene repression are distinct . Transcription of mrgA-lacZ is induced at the end of logarithmic-phase growth in minimal medium, and this induction is prevented by excess manganese, iron, cobalt, or copper . Limitation for metal ions is sufficient for mrgA-lacZ induction, since resuspension in medium lacking both manganese and iron rapidly induces transcription . Transcription of mrgC-lacZ is also induced by iron deprivation but is not repressed by added manganese or other metal ions . Expression of mrgC-lacZ and a 2,3-dihydroxybenzoic acid-based siderophore is repressed in parallel by iron, and in both cases, only iron effects repression . We have cloned and sequenced the promoter and regulatory regions of both mrgA and mrgC . Both genes are preceded by a predicted sigma A-dependent promoter element with overlapping sequences similar to the iron box consensus element for recognition by the Escherichia coli ferric uptake regulator protein (Fur) . Mutation of the putative iron box for gene mrgC leads to partial derepression in iron-replete medium. J Bacteriol, 1993 Sep, 175(18), 6010 - 7 Bacillus subtilis alkA gene encoding inducible 3-methyladenine DNA glycosylase is adjacent to the ada operon; Morohoshi F et al.; In Bacillus subtilis, the adaptive response to DNA alkylation depends on the ada operon, which consists of the adaA and adaB genes, which encode methylphosphotriester DNA methyltransferase (AdaA protein) and O6-methylguanine DNA methyltransferase (AdaB protein), respectively . A structural gene (alkA) that encodes 3-methyladenine DNA glycosylase was found upstream of the ada operon, but in the opposite orientation . This cluster of genes was mapped at about 235 kb from the SfiI recognition site near the origin of replication in the physical map of the B . subtilis chromosome . Disruption of the alkA gene sensitized cells to N-propyl-N'-nitro-N-nitrosoguanidine, while its overproduction rendered cells highly resistant to N-propyl-N'-nitro-N-nitrosoguanidine, indicating that lethal DNA damage produced by bulky alkylating agents was effectively counteracted by AlkA glycosylase . Transcription of the alkA gene was induced by treating adaA+ cells with methylating agents concurrent with transcription of the ada operon . This was accomplished by using methylated AdaA protein bound to a 30-bp segment in the middle of the 100-bp sequence between the transcriptional start sites of the alkA gene and ada operon . Thus, in this organism, the adaptive response to DNA alkylation is achieved by autologous activation of a divergent regulon composed of the genes for a DNA glycosylase and two species of DNA alkyltransferase. J Bacteriol, 1993 Sep, 175(17), 5697 - 700 Chloramphenicol acetyltransferase, a cytoplasmic protein is incompatible for export from Bacillus subtilis; Chen MW et al.; Bacillus subtilis cells expressing a hybrid protein (Lvsss-Cat) consisting of the B . amyloliquefaciens levansucrase signal peptide fused to B . pumilus chloramphenicol acetyltransferase (Cat) are unable to export Cat protein into the growth medium . A series of tripartite protein fusions was constructed by inserting various lengths of the Cat sequences between the levansucrase signal peptide and staphylococcal protein A or Escherichia coli alkaline phosphatase . Biochemical characterization of the various Cat protein fusions revealed that multiple regions in the Cat protein were causing the export defect. J Bacteriol, 1993 Sep, 175(17), 5636 - 41 Molecular and phenotypic characterization of promoter-proximal mutations in the spoIIA locus of Bacillus subtilis; Challoner-Courtney IJ et al.; Eight mutations lying within the promoter-proximal one-fifth of the spoIIA locus of Bacillus subtilis were studied . Two of these mutations (spoIIAA42 and spoIIAA69) were previously characterized at the DNA level, five more (spo-562, spo-565, spo-567, spo-568, and spo-569) were isolated in our laboratory several years ago but not fully characterized, and the eight (an in-frame deletion confined to spoIIAA, the first gene in the spoIIA operon) was constructed for this study . DNA sequencing showed that spo-569 was a transitions in the -35 region of the spoIIA promoter; the remaining point mutations were all G:C to A:T transitions in spoIIAA, with spo-565 having two transitions, one of which was identical to that in spo-562 . All the spoIIAA mutations except spo-562 led to the replacement of Gly residues . The incidence of sporulation, the rate of synthesis of sporulation-associated alkaline phosphatase, and the rate of expression of the forespore-specific genes gpr and spoIIIG were determined for isogenic strains carrying the eight mutations . All the mutations except spoIIAA42 and spo-569 (which were slightly leaky) made the strains asporogenous, and all except spo-562 and spo-569 abolished the synthesis of alkaline phosphatase and the expression of gpr and spoIIIG . spo-562 allowed alkaline phosphatase synthesis and gpr and spoIIIG expression to occur at about 15% of the wild-type rates but with normal kinetics . spo-59 allowed appreciable gpr and spoIIIG expression during exponential growth; we attribute this expression to transcription by RNA polymerase containing sigma G and suggest that a spo-569 strain makes insufficient SpoIIAB to inhibit sigma G in growing cells. J Bacteriol, 1993 Sep, 175(17), 5611 - 6 Mutation in the plasmid pUB110 Rep protein affects termination of rolling circle replication; Bidnenko VE et al.; We isolated a mutant of plasmid pUB110 that has the following properties in Bacillus subtilis: (i) it is toxic for recA and add cells, particularly at elevated temperature; (ii) it has a copy number threefold higher than that of the parental plasmid, and the extra copies are present as multimers; and (iii) it can efficiently complement replication of a cmp- satellite plasmid, despite being cmp+ . All these properties are due to a single change in the plasmid replication protein, i.e., Gly at position 148 to Glu . These properties of the mutant Rep protein reflect a diminished ability to terminate rolling circle replication . We propose that the Rep protein may have a diminished affinity for the plasmid origin; alternatively, it may be impaired for recognition of the plasmid conformations which distinguish initiation and termination. J Biochem (Tokyo), 1993 Sep, 114(3), 385 - 8 Signal transduction and sporulation in Bacillus subtilis: heterologous phosphorylation of Spo0A, a sporulation initiation gene product; Asayama M et al.; Spo0A is both a positive and a negative transcriptional regulator which plays a very important role in sporulation initiation in Bacillus subtilis . Its N-terminal amino acid sequence is homologous to that of regulator proteins of the two-component regulatory systems involved in signal transduction in bacteria . Phosphorylation of Spo0A through phosphorelay has been reported by Burbulys et al . (1991) . In this study, we found that (i) Spo0A is phosphorylated effectively with phospho-EnvZ* (N-terminal truncated EnvZ), which is a heterologous osmotic sensor protein in Escherichia coli, and (ii) a phosphorylation deficient mutant of Spo0A protein is completely defective in initiating sporulation . These results suggest that Spo0A phosphorylation may be an essential event in signal transduction of sporulation in B . subtilis and the signal transduction mechanism has a common feature in Gram-positive and Gram-negative bacteria. Genetika, 1993 Sep, 29(9), 1579 - 83 {Cloning of the Bacillus subtilis chromosome fragment suppressing the Escherichia coli sbc B mutation}; Dzhanga AA et al.; A . 3.54 kb chromosomal fragment of Bacillus subtilis was cloned on the plasmid pBluescript SK(+) . The fragment suppresses the repair deficiency of the sbc B15 mutation of Escherichia coli . It is shown that the B . subtilis gene of exonuclease! is expressed in the E . coli cells. J Nat Prod, 1993 Sep, 56(9), 1613 - 7 Isolation and structure elucidation of 34-sulfatobastadin 13, an inhibitor of the endothelin A receptor, from a marine sponge of the genus Ianthella; Gulavita NK et al.; 34-Sulfatobastadin 13 {1} was isolated from the sponge Ianthella sp . Its structure was elucidated by nmr techniques and chemical transformation to bastadin 13 {2} . Compound 1 weakly inhibited binding to the endothelin A receptor (ETA), while compound 2 inhibited growth of Bacillus subtilis. J Gen Microbiol, 1993 Sep, 139 ( Pt 9), 2047 - 54 Catabolite repression of beta-glucanase synthesis in Bacillus subtilis; Kruger S et al.; beta-Glucanase synthesis in Bacillus subtilis was repressed by glucose and other substrates of glycolysis . Experiments with different pts mutants showed that the phosphoenolpyruvate: sugar phosphotransferase system is not involved in carbon catabolite repression of beta-glucanase synthesis . Carbon catabolite repression of beta-glucanase synthesis was completely abolished in a ccpA mutant . An operator structure similar to those upstream of amyE and the xyl operon was found and was shown by site-directed mutagenesis to be the target for carbon catabolite repression of beta-glucanase synthesis . The presence of this operator on a multi-copy plasmid resulted in a reduced repression of both beta-glucanase and alpha-amylase synthesis . It seems likely that the gene encoding these enzymes are part of one regulon with respect to catabolite repression. J Gen Microbiol, 1993 Sep, 139 ( Pt 9), 2041 - 5 Temporal activation of beta-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool; Stulke J et al.; beta-Glucanase synthesis was temporally activated in Bacillus subtilis at the onset of stationary phase . This regulation was dependent upon a drop in the GTP concentration in response to nutrient limitation . The Spo0A and Abr