Bacillus subtilis

Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9642 - 7
Identification and characterization of a negative regulator of FtsZ ring formation in Bacillus subtilis; Levin PA et al.; During the bacterial cell cycle, the tubulin-like cell-division protein FtsZ polymerizes into a ring structure that establishes the location of the nascent division site . We have identified a regulator of FtsZ ring formation in Bacillus subtilis . This protein, EzrA, modulates the frequency and position of FtsZ ring formation . The loss of ezrA resulted in cells with multiple FtsZ rings located at polar as well as medial sites . Moreover, the critical concentration of FtsZ required for ring formation was lower in ezrA null mutants than in wild-type cells . EzrA was associated with the cell membrane and also colocalized with FtsZ to the nascent septal site . We propose that EzrA interacts either with FtsZ or with one of its binding partners to promote depolymerization.

Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9545 - 50
Folding of a large ribozyme during transcription and the effect of the elongation factor NusA; Pan T et al.; We compared in vitro transcription-initiated folding of the ribozyme from Bacillus subtilis RNase P to refolding from the full-length, denatured state by monitoring the appearance of its catalytic activity . At 37 degrees C, Mg(2+)-initiated refolding of the wild type and a circularly permutate ribozyme takes minutes and is limited by a kinetic trap . Transcription by T7 RNA polymerase alters the folding pathway of both RNAs and introduces new kinetic traps . Transcription by the core Escherichia coli RNA polymerase yields the same result, in spite of its 4-fold-slower elongation rate . However, the presence of its elongation factor NusA accelerates more than 10-fold the transcription-initiated folding of the circularly, permutated ribozyme by E . coli RNA polymerase . The effect of NusA likely is caused by its enhancement of transcriptional pausing because NusA did not accelerate transcription-initiated folding using a mutant RNA polymerase that failed to pause or respond to NusA during ribozyme synthesis . We conclude that both transcription and specific pausing therein can alter RNA-folding pathways.

Curr Opin Biotechnol, 1999 Aug, 10(4), 376 - 81
Improving protein secretion by engineering components of the bacterial translocation machinery; Braun P et al.; The increased insight into the mechanism of bacterial protein translocation has resulted in new concepts for the production of heterologous proteins . The periplasm of gram-negative bacteria is revealed to have a role as a 'protein construction compartment', which can be used to fold complex proteins . Passage across the outer membrane, however, remains a challenge due to the high selectivity of the outer membrane translocase . In gram-positive bacteria, slow folding at the membrane-cell-wall interface can make heterologous proteins vulnerable to degradation by wall-associated proteases . The recent identification of thiol-disulfide oxidoreductases in Bacillus subtilis might open the possibility of secreting proteins containing multiple disulfide bonds from this host.

Mol Microbiol, 1999 Aug, 33(4), 886 - 94
The N- and C-terminal domains of MecA recognize different partners in the competence molecular switch; Persuh M et al.; ComK is a transcription factor required for the expression of competence genes in Bacillus subtilis . Binding to MecA targets ComK for degradation by the ClpCP protease . MecA therefore acts as an adapter protein recruiting a regulatory protein for proteolysis . However, when ComS is synthesized, ComK is released from binding by MecA and thereby protected from degradation . MecA binds to three protein partners during these processes: ComK, ClpC and ComS . Using limited proteolysis, we have defined N- and C-terminal structural domains of MecA and evaluated the interactions of these domains with the protein partners of MecA . Using surface plasmon resonance, we have determined that the N-terminal domain of MecA interacts with ComK and ComS and the C-terminal domain with ClpC . MecA is shown to exist as a dimer with dimerization sites on both the N- and C-terminal domains . The C-terminal domain stimulates the ATPase activity of ClpC and is degraded by the ClpCP protease, while the N-terminal domain is inactive in both of these assays . In vivo data were consistent with these findings, as comG-lacZ expression was decreased in a strain overproducing the N-terminal domain, indicating reduced ComK activity . We propose a model in which binding of ClpC to the C-terminal domain of MecA induces a conformational change enabling the N-terminal domain to bind ComK with enhanced affinity . MecA is widespread among Gram-positive organisms and may act generally as an adapter protein, targeting proteins for regulated degradation.

Eur J Biochem, 1999 Aug, 264(1), 200 - 10
NMR structure of active and inactive forms of the sterol-dependent antifungal antibiotic bacillomycin L; Volpon L et al.; The antifungal antibiotic lipopeptide bacillomycin L {cyclo-(L-Asp1-D-Tyr2-D-Asn3-L-Ser4-L-Gln5-D-Ser6++ +-L-Thr7-beta-amino fatty acid)} from Bacillus subtilis belongs to the iturinic family of antifungal agents and acts with a strict sterol-phospholipid dependence on biomembranes . This antibiotic has been analysed using solution NMR spectroscopy in its native active form and its inactive (L-Asp1, D-Tyr2) di-O-methylated form . The structures were calculated under NMR-derived restraints using molecular-dynamic simulated-annealing protocols starting from a random array of atoms . The structure of the native antibiotic is spread over different conformers in which two families are recognized . It was found that most structures have dihedral phi and psi angles defining a type-II' beta-turn including amino acids 5-8, in certain cases stabilized by a 8HN-5CO hydrogen bond, whereas a minority of structures adopt an inverse gamma-turn including amino acids 6-8, stabilized in all cases by an 8HN-6CO hydrogen bond . The di-O-methylation of L-Asp1 and D-Tyr2, an amino acid strictly conserved within the iturinic group of antibiotics, does not induce major differences in the NMR spectra and in the NMR structures . The results are discussed in relation to the specific loss of interaction with sterols when the native antifungal bacillomycin L is methylated on the conserved D-Tyr2 position.

Nucleic Acids Res, 1999 Sep 1, 27(17), 3577 - 82
Bacterial start site prediction; Hannenhalli SS et al.; With the growing number of completely sequenced bacterial genes, accurate gene prediction in bacterial genomes remains an important problem . Although the existing tools predict genes in bacterial genomes with high overall accuracy, their ability to pinpoint the translation start site remains unsatisfactory . In this paper, we present a novel approach to bacterial start site prediction that takes into account multiple features of a potential start site, viz., ribosome binding site (RBS) binding energy, distance of the RBS from the start codon, distance from the beginning of the maximal ORF to the start codon, the start codon itself and the coding/non-coding potential around the start site . Mixed integer programing was used to optimize the discriminatory system . The accuracy of this approach is up to 90%, compared to 70%, using the most common tools in fully automated mode (that is, without expert human post-processing of results) . The approach is evaluated using Bacillus subtilis, Escherichia coli and Pyrococcus furiosus . These three genomes cover a broad spectrum of bacterial genomes, since B.subtilis is a Gram-positive bacterium, E.coli is a Gram-negative bacterium and P . furiosus is an archaebacterium . A significant problem is generating a set of 'true' start sites for algorithm training, in the absence of experimental work . We found that sequence conservation between P . furiosus and the related Pyrococcus horikoshii clearly delimited the gene start in many cases, providing a sufficient training set.

Nucleic Acids Res, 1999 Sep 1, 27(17), 3567 - 76
Translation in Bacillus subtilis: roles and trends of initiation and termination, insights from a genome analysis; Rocha EP et al.; We analysed the Bacillus subtilis protein coding sequences termini, and compared it to other genomes . The analysis focused on signals, com-positional biases of nucleotides, oligonucleotides, codons and amino acids and mRNA secondary structure . AUG is the preferred start codon in all genomes, independent of their G+C content, and seems to induce less stable mRNA structures . However, it is not conserved between homologous genes neither is it preferred in highly expressed genes . In B.subtilis the ribosome binding site is very strong . We found that downstream boxes do not seem to exist either in Escherichia coli or in B.subtilis . UAA stop codon usage is correlated with the G+C content and is strongly selected in highly expressed genes . We found less stable mRNA structures at both termini, which we related to mRNA-ribosome and mRNA-release-factor interactions . This pattern seems to impose a peculiar A-rich nucleotide and codon usage bias in these regions . Finally the analysis of all proteins from B.subtilis revealed a similar amino acid bias near both termini of proteins consisting of over-representation of hydrophilic residues . This bias near the stop codon is partially release-factor specific.

J Bacteriol, 1999 Aug, 181(16), 4969 - 77
Sigma factor displacement from RNA polymerase during Bacillus subtilis sporulation; Ju J et al.; As Bacillus subtilis proceeds through sporulation, the principal vegetative cell sigma subunit (sigma(A)) persists in the cell but is replaced in the extractable RNA polymerase (RNAP) by sporulation-specific sigma factors . To explore how this holoenzyme changeover might occur, velocity centrifugation techniques were used in conjunction with Western blot analyses to monitor the associations of RNAP with sigma(A) and two mother cell sigma factors, sigma(E) and sigma(K), which successively replace sigma(A) on RNAP . Although the relative abundance of sigma(A) with respect to RNAP remained virtually unchanged during sporulation, the percentage of the detectable sigma(A) which cosedimented with RNAP fell from approximately 50% at the onset of sporulation (T(0)) to 2 to 8% by 3 h into the process (T(3)) . In a strain that failed to synthesize sigma(E), the first of the mother cell-specific sigma factors, approximately 40% of the sigma(A) remained associated with RNAP at T(3) . The level of sigma(A)-RNAP cosedimentation dropped to less than 10% in a strain which synthesized a sigma(E) variant (sigma(ECR119)) that could bind to RNAP but was unable to direct sigma(E)-dependent transcription . The E-sigma(E)-to-E-sigma(K) changeover was characterized by both the displacement of sigma(E) from RNAP and the disappearance of sigma(E) from the cell . Analyses of extracts from wild-type and mutant B . subtilis showed that the sigma(K) protein is required for the displacement of sigma(E) from RNAP and also confirmed that sigma(K) is needed for the loss of the sigma(E) protein . The results indicate that the successive appearance of mother cell sigma factors, but not necessarily their activities, is an important element in the displacement of preexisting sigma factors from RNAP . It suggests that competition for RNAP by consecutive sporulation sigma factors may be an important feature of the holoenzyme changeovers that occur during sporulation.

APMIS, 1999 Jul, 107(7), 624 - 30
Evaluation of an rDNA Listeria probe for Listeria monocytogenes typing; Jacquet C et al.; A Listeria monocytogenes DNA fragment, identified as part of the 23S rRNA gene and called B17, was used to type 266 L . monocytogenes strains and 43 strains of other Listeria species . Results were compared with those obtained: i) with pBA2 (which consists of a 2.3 kb Bacillus subtilis DNA fragment encoding 16S rRNA, inserted into the HindIII site of pBR322), a probe previously used for Listeria and L . monocytogenes ribotyping, and ii) with DNA macrorestriction profiles analysis . Twenty profiles were identified for L . monocytogenes using pB 17, three of which accounted for 87% of strains . This new rDNA probe had greater discriminatory power for serogroups 1/2 or 3 strains than for serogroup 4 strains . The number of varieties and the discrimination index were higher with this new probe than with pBA2, but DNA macrorestriction patterns analysis gave better discrimination between strains.

J Bacteriol, 1999 Aug, 181(16), 5060 - 7
Functional and transcriptional analyses of a fengycin synthetase gene, fenC, from Bacillus subtilis; Lin TP et al.; A 37-kb DNA fragment containing five fengycin synthetase genes, including fenC, fenD, fenE, fenA, and fenB, was cloned and sequenced . Among these genes, fenC encodes a fengycin synthetase 2,560 amino acids long with an estimated molecular mass of 287 kDa . This protein contains two amino acid activation modules, FenC1 and FenC2, which activate L-glutamic acid and L-ornithine, respectively . Primer extension, using mRNA isolated from the log-phase cells, identified a transcription start site located 86 nucleotides upstream from the initiation codon of fenC, implying that a promoter is located upstream from the start site . Primer extension using total RNA isolated from stationary-phase cells also identified a transcription start site located 61 nucleotides upstream from the initiation codon of fenC . Gene fusion studies demonstrated that in nHA medium, the cells transcribe the fengycin synthetase genes at two different stages of cell growth . The promoter is active during the log phase, and the activity reaches the highest level during the late log phase . The activity decreases sharply but is maintained at a low level for approximately 24 h after cells enter the early stationary phase . The results of this investigation also suggest that the transcription of fenC is positively regulated during the late log phase . Results presented herein provide further insight into fengycin synthesis by B . subtilis F29-3.

J Bacteriol, 1999 Aug, 181(16), 4995 - 5003
Regulation of the lic operon of Bacillus subtilis and characterization of potential phosphorylation sites of the LicR regulator protein by site-directed mutagenesis; Tobisch S et al.; The lic operon of Bacillus subtilis is required for the transport and degradation of oligomeric beta-glucosides, which are produced by extracellular enzymes on substrates such as lichenan or barley glucan . The lic operon is transcribed from a sigma(A)-dependent promoter and is inducible by lichenan, lichenan hydrolysate, and cellobiose . Induction of the operon requires a DNA sequence with dyad symmetry located immediately upstream of the licBCAH promoter . Expression of the lic operon is positively controlled by the LicR regulator protein, which contains two potential helix-turn-helix motifs, two phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domains (PRDs), and a domain similar to PTS enzyme IIA (EIIA) . The activity of LicR is stimulated by modification (probably phosphorylation) of both PRD-I and PRD-II by the general PTS components and is negatively regulated by modification (probably phosphorylation) of its EIIA domain by the specific EII(Lic) in the absence of oligomeric beta-glucosides . This was shown by the analysis of licR mutants affected in potential phosphorylation sites . Moreover, the lic operon is subject to carbon catabolite repression (CCR) . CCR takes place via a CcpA-dependent mechanism and a CcpA-independent mechanism in which the general PTS enzyme HPr is involved.

J Bacteriol, 1999 Aug, 181(16), 4986 - 94
Characterization of the yrbA gene of Bacillus subtilis, involved in resistance and germination of spores; Takamatsu H et al.; Insertional inactivation of the yrbA gene of Bacillus subtilis reduced the resistance of the mutant spores to lysozyme . The yrbA mutant spores lost their optical density at the same rate as the wild-type spores upon incubation with L-alanine but became only phase gray and did not swell . The response of the mutant spores to a combination of asparagine, glucose, fructose, and KCl was also extremely poor; in this medium yrbA spores exhibited only a small loss in optical density and gave a mixture of phase-bright, -gray, and -dark spores . Northern blot analysis of yrbA transcripts in various sig mutants indicated that yrbA was transcribed by RNA polymerase with sigma(E) beginning at 2 h after the start of sporulation . The yrbA promoter was localized by primer extension analysis, and the sequences of the -35 (TCATAAC) and -10 (CATATGT) regions were similar to the consensus sequences of genes recognized by sigma(E) . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins solubilized from intact yrbA mutant spores showed an alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some other minor changes . Electron microscopic examination of yrbA spores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the appearance of an inner coat layer-like structure around the outside of the coat . This abnormal coat structure was also observed on the outside of the developing forespores of the yrbA mutant . These results suggest that YrbA is involved in assembly of some coat proteins which have roles in both spore lysozyme resistance and germination.

J Bacteriol, 1999 Aug, 181(16), 4929 - 36
Identification of a gene in Staphylococcus xylosus encoding a novel glucose uptake protein; Fiegler H et al.; By transposon Tn917 mutagenesis, two mutants of Staphylococcus xylosus were isolated that showed higher levels of beta-galactosidase activity in the presence of glucose than the wild type . Both transposons integrated in a gene, designated glcU, encoding a protein involved in glucose uptake in S . xylosus, which is followed by a glucose dehydrogenase gene (gdh) . Glucose-mediated repression of beta-galactosidase, alpha-glucosidase, and beta-glucuronidase activities was partially relieved in the mutant strains, while repression by sucrose or fructose remained as strong as in the wild type . In addition to the pleiotropic regulatory effect, integration of the transposons into glcU reduced glucose dehydrogenase activity, suggesting cotranscription of glcU and gdh . Insertional inactivation of the gdh gene and deletion of the glcU gene without affecting gdh expression showed that loss of GlcU function is exclusively responsible for the regulatory defect . Reduced glucose repression is most likely the consequence of impaired glucose uptake in the glcU mutant strains . With cloned glcU, an Escherichia coli mutant deficient in glucose transport could grow with glucose as sole carbon source, provided a functional glucose kinase was present . Therefore, glucose is internalized by glcU in nonphosphorylated form . A gene from Bacillus subtilis, ycxE, that is homologous to glcU, could substitute for glcU in the E . coli glucose growth experiments and restored glucose repression in the S . xylosus glcU mutants . Three more proteins with high levels of similarity to GlcU and YcxE are currently in the databases . It appears that these proteins constitute a novel family whose members are involved in bacterial transport processes . GlcU and YcxE are the first examples whose specificity, glucose, has been determined.

J Bacteriol, 1999 Aug, 181(16), 4761 - 7
The icfG gene cluster of Synechocystis sp . strain PCC 6803 encodes an Rsb/Spo-like protein kinase, protein phosphatase, and two phosphoproteins; Shi L et al.; A set of open reading frames (ORFs) potentially encoding signal transduction proteins are clustered around icfG, a gene implicated in the regulation of carbon metabolism, in the genome of Synechocystis sp . strain PCC 6803 . slr1860 is the ORF for icfG, whose predicted product resembles the protein phosphatases SpoIIE, RsbU, and RsbX from Bacillus subtilis . Bracketing slr1860/icfG are (i) ORF slr1861, whose predicted product resembles the SpoIIAB, RsbT, and RsbW protein kinases from B . subtilis, and (ii) ORFs slr1856 and slr1859, whose predicted products resemble the respective phosphoprotein substrates for the B . subtilis protein kinases: SpoIIAA, RsbS, and RsbV . In order to determine whether the protein products encoded by these ORFs possessed the functional capabilities suggested by sequence comparisons, each was expressed in Escherichia coli as a histidine-tagged fusion protein and analyzed for its ability to participate in protein phosphorylation-dephosphorylation processes in vitro . It was observed that ORF slr1861 encoded an ATP-dependent protein kinase capable of phosphorylating Slr1856 and, albeit with noticeably lower efficiency, Slr1859 . Site-directed mutagenesis suggests that Slr1861 phosphorylated these proteins on Ser-54 and Ser-57, respectively . Slr1860 exhibited divalent metal ion-dependent protein-serine phosphatase activity . It catalyzed the dephosphorylation of Slr1856, but not Slr1859, in vitro.

Analyst, 1998 Dec, 123(12), 2737 - 41
Detection of residues of tetracycline antibiotics in pork and chicken meat: correlation between results of screening and confirmatory tests; De Wasch K et al.; Residues of the tetracycline group of antibiotics were quantified in pork and chicken muscle tissue that had previously been screened with a microbiological inhibition test and an immunological method . Pieces of frozen pork and chicken meat were screened on a pH 6 culture medium seeded with Bacillus subtilis . An aqueous extract of the inhibitor-positive samples was then screened with a group-specific commercial ELISA kit, able to detect levels of oxytetracycline, chlortetracycline, tetracycline and doxycycline corresponding with the European MRL or lower . The cut-off value of the ELISA was set at a B/B0 value of 75% . Finally, confirmation and quantification were performed using a validated HPLC method with fluorescence detection . The fluorescence was induced by complexation of the tetracyclines with the zirconium cation which is added post-column to the HPLC eluate . This fluorescence makes it possible to quantitate residues below one-half of the MRL . To gain additional qualitative information some samples were also analysed with LC-MS-MS . ELISA analysis demonstrated the presence of residues of tetracyclines in 12 out of 19 inhibitor-positive pork samples and in 19 out of 21 inhibitor-positive chicken samples . Doxycycline was detected with HPLC in 10 of these 12 pork samples and in 18 out of 19 chicken samples . The two other ELISA positive pork samples contained oxytetracycline, while no tetracyclines were found in one ELISA positive chicken meat sample . The correlation between the ELISA B/B0 values and the actual levels determined with the HPLC method was poor, whereas a better correlation was observed between the inhibition zones and the doxycycline levels . Our results indicate that an inhibition test with a medium at pH 6 and B . subtilis as test organism is well suited to screen pork and chicken muscle tissue for residues of tetracycline antibiotics . Since many positive samples contained doxycycline levels below the MRL, a confirmatory method is necessary to quantify the residues.

Biochemistry, 1999 Aug 3, 38(31), 10119 - 25
Decay of activated Bacillus subtilis pho response regulator, PhoP approximately P, involves the PhoR approximately P intermediate; Shi L et al.; PhoR of Bacillus subtilis is a histidine sensor-kinase belonging to the family of two-component signal transduction systems . PhoR is responsible for processing the phosphate-starvation signal and providing phosphate input to regulate the level of phosphorylated response regulator, PhoP, which activates/represses Pho regulon gene transcription . The catalytic domain of PhoR is sufficient for the low-phosphate inducible expression of Pho regulon genes since removing the N-terminal membrane-associated domain did not alter the kinetics of Pho induction, albeit the total level of induction was decreased (1) . In this study we showed that the complete B . subtilis PhoR protein produced in Escherichia coli can be reverse phosphorylated by PhoP-phosphate . We also used a C-terminal fragment of the PhoR protein, PhoR, to demonstrate that the phosphoryl group on phospho-PhoP was transferred back to PhoR in the reverse phosphorylation reaction or released as inorganic phosphate to the reaction mixture . The reverse phosphorylation of the PhoR protein likely occurs at the same histidine residue (His360) that is utilized for the autokinase reaction by the same protein . In the presence of ADP, the phosphoryl group is further transferred to ADP to form ATP . While the autokinase reaction, the forward phosphotransfer reaction from PhoR approximately P to PhoP, and the release of inorganic phosphate from PhoP approximately P in the presence of PhoR require Mg(2+), the reverse phosphotransfer from PhoP approximately P to PhoR does not . These results indicate that the energy levels of the phosphoryl groups on PhoP and PhoR are very similar . The reversible autokinase reaction and/or the reversible phosphotransfer reaction between PhoR approximately P and PhoP may have a role in PhoP approximately P decay thus influencing the PhoP approximately P concentration in the cell.

J Appl Microbiol, 1999 Jul, 87(1), 8 - 14
Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the alpha/beta-type protect spores against this DNA damage; Loshon CA et al.; Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed alpha-beta-) lacking the two major alpha/beta-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis . A recA mutation sensitized both wild-type and alpha-beta- spores to formaldehyde treatment, which caused significant expression of a recA-lacZ fusion when the treated spores germinated . Formaldehyde also caused protein-DNA cross-linking in both wild-type and alpha-beta- spores . These results indicate that: (i) formaldehyde kills B . subtilis spores at least in part by DNA damage and (b) alpha/beta-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1211 - 5
Relationship of Bacillus subtilis clades associated with strains 168 and W23: a proposal for Bacillus subtilis subsp . subtilis subsp . nov . and Bacillus subtilis subsp . spizizenii subsp . nov; Nakamura LK et al.; Earlier phylogenetic studies based on the inferred DNA sequences of the polC, rpoB and gyrA genes suggested that strains of the species Bacillus subtilis formed two clusters, indicating the presence two closely related taxa; one contained the laboratory strain 168 and the other the laboratory strain W23 . Significant sexual isolation was found between strain 168 and members of the group containing W23, but no sexual isolation was observed between strain 168 and other members of the 168 group . DNA reassociation between the two groups ranged from 58 to 69% and intragroup DNA relatedness ranged from 82 to 100% . Because group 168 strains were highly related to the B . subtilis type strain, they were considered to be bona fide members of the species . About 99.5% sequence identity was observed between the 16S rRNA genes of the 168 and W23 groups . Ribitol and anhydroribitol were principal cell wall constituents of the W23 but not of the 168 group . These observations revealed two closely related but genetically and phenotypically distinct groups within B . subtilis that correspond to two historically important strains . Subspecies distinction is proposed for the 168 and W23 groups, with the names Bacillus subtilis subsp . subtilis subsp . nov . and Bacillus subtilis subsp . spizizenii subsp . nov., respectively . The type strain of the former is NRRL NRS-744T and the latter NRRL B-23049T.

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1181 - 92
Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus; Kwok AY et al.; The phylogenetic relationships among 36 validly described species or subspecies within the genus Staphylococcus were investigated by cloning and sequencing their 60 kDa heat-shock protein (HSP60) genes using a set of universal degenerate HSP60 PCR primers . The cloned partial HSP60 DNA sequences from nine Staphylococcus aureus strains were highly conserved (97-100% DNA sequence similarity; mean 98%), indicating that the HSP60 gene of multiple isolates within the same species have little microheterogeneity . At the subspecies level, DNA sequence similarity among members of S . aureus, Staphylococcus schleiferi, Staphylococcus cohnii and Staphylococcus capitis ranged from 91 to 98% . At the interspecies level, sequence similarity among 23 distinct species of staphylococci ranged from 74 to 93% (mean 82%) . By comparison, the highest sequence similarity of Bacillus subtilis and Escherichia coli with members within the genus Staphylococcus was only 70 and 59%, respectively . Importantly, phylogenetic analysis based on the neighbour-joining distance method revealed remarkable concordance between the tree derived from partial HSP60 gene sequences and that based on genomic DNA-DNA hybridization, while 16S rRNA gene sequences correlated less well . The results demonstrate that DNA sequences from the highly conserved and ubiquitous HSP60 gene offer a convenient and accurate tool for species-specific identification and phylogenetic analysis of staphylococci.

J Biochem (Tokyo), 1999 Aug, 126(2), 333 - 9
Regulation of the Bacillus subtilis phosphotransacetylase gene; Shin BS et al.; The enzyme, phosphotransacetylase (Pta), catalyzes the conversion of acetyl coenzyme A to acetyl phosphate . The putative pta gene of Bacillus subtilis, which had been sequenced as part of the Genome Project, was cloned and overexpressed in Escherichia coli . We confirmed that the gene encodes Pta by measuring the enzymatic activity of the purified protein . Insertional mutagenesis of the pta gene resulted in complete loss of the Pta activity, indicating that B . subtilis contains only one kind of pta gene . Expression of a pta-lacZ fusion was induced in the presence of excess glucose in the growth medium, and the intact ccpA gene was required for this activation . The transcriptional start site of the pta gene was located at 37 nucleotides upstream of the pta start codon, and a cre (catabolite responsive element) sequence, a cis-acting element that is responsible for the catabolite repression of a number of carbon utilization genes in B . subtilis, was identified upstream of the tentative promoter site . Experiments involving oligonucleotide-directed mutagenesis showed that the cre sequence is involved in glucose-mediated transcriptional activation.

Nature, 1999 Jul 15, 400(6741), 289 - 93
Millisecond-timescale motions contribute to the function of the bacterial response regulator protein Spo0F; Feher VA et al.; Protein backbones and side chains display varying degrees of flexibility, which allows many slightly different but related conformational substates to occur . Such fluctuations are known to differ in both timescale and magnitude, from rotation of methyl groups (nanoseconds) to the flipping of buried tyrosine rings (seconds) . Because many mechanisms for protein function require conformational change, it has been proposed that some of these ground-state fluctuations are related to protein function . But exactly which aspects of motion are functionally relevant remains to be determined . Only a few examples so far exist where function can be correlated to structural fluctuations with known magnitude and timescale . As part of an investigation of the mechanism of action of the Bacillus subtilis response regulator SpoOF, we have explored the relationship between the motional characteristics and protein-protein interactions . Here we use a set of nuclear magnetic resonance 15N relaxation measurements to determine the relative timescales of SpoOF backbone fluctuations on the picosecond-to-millisecond timescale . We show that regions having motion on the millisecond timescale correlate with residues and surfaces that are known to be critical for protein-protein interactions.

J Bacteriol, 1999 Aug, 181(15), 4700 - 3
Three distinct phases of isoprene formation during growth and sporulation of Bacillus subtilis; Wagner WP et al.; During growth on a glucose-tryptone medium, Bacillus subtilis 6051 (Marburg strain) exhibited three phases of isoprene (2-methyl-1, 3-butadiene) formation, corresponding to (i) glucose catabolism and secretion of acetoin, (ii) catabolism of acetoin, and (iii) the early stages of sporulation . These results establish an experimental system for studying the biological role of isoprene formation.

J Bacteriol, 1999 Aug, 181(15), 4653 - 60
Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of transcription factor sigma(B); Scott JM et al.; sigma(B), the general stress response sigma factor of Bacillus subtilis, is activated when intracellular ATP levels fall or the bacterium experiences environmental stress . Stress activates sigma(B) by means of a collection of regulatory kinases and phosphatases (the Rsb proteins), which catalyze the release of sigma(B) from an anti-sigma factor inhibitor . By using the yeast dihybrid selection system to identify B . subtilis proteins that could interact with Rsb proteins and act as mediators of stress signaling, we isolated the GTP binding protein, Obg, as an interactor with several of these regulators (RsbT, RsbW, and RsbX) . B . subtilis depleted of Obg no longer activated sigma(B) in response to environmental stress, but it retained the ability to activate sigma(B) by the ATP responsive pathway . Stress pathway components activated sigma(B) in the absence of Obg if the pathway's most upstream effector (RsbT) was synthesized in excess to the inhibitor (RsbS) from which it is normally released after stress . Thus, the Rsb proteins can function in the absence of Obg but fail to be triggered by stress . The data demonstrate that Obg, or a process under its control, is necessary to induce the stress-dependent activation of sigma(B) and suggest that Obg may directly communicate with one or more sigma(B) regulators.

J Bacteriol, 1999 Aug, 181(15), 4605 - 10
Immunocytochemical localization of a calmodulinlike protein in Bacillus subtilis cells; Dominguez DC et al.; To determine possible functions of the calmodulinlike protein of Bacillus subtilis, the time course of its expression during sporulation and its cellular localization were studied . The protein was expressed in a constitutive manner from the end of logarithmic growth through 8 h of sporulation as determined by antibody cross-reactivity immunoblots and enzyme-linked immunosorbent assays (ELISAs) . In partially purified extracts, the immunopositive protein comigrated upon electrophoresis with a protein which selectively bound {(45)Ca}CaCl(2), ruthenium red, and Stains-all . Previous studies showed increased extractability of the calmodulinlike protein from B . subtilis cells when urea and 2-mercaptoethanol were used in breakage buffers, implying that the protein might be partially associated with the membrane fraction . This was confirmed by demonstrating that isolated membrane vesicles of B . subtilis also gave positive immunological tests with Western blotting and ELISAs . To more precisely locate the protein in cells, thin sections of late-log-phase cells, sporulating cells, and free spores were reacted first with bovine brain anticalmodulin specific antibodies and then with gold-conjugated secondary antibodies; the thin sections were examined by transmission electron microscopy . The calmodulinlike protein was found almost exclusively associated with the cell envelope of these fixed, sectioned cells . A possible function of the calmodulinlike protein in sensing calcium ions or regulating calcium ion transport is suggested.

J Bacteriol, 1999 Aug, 181(15), 4584 - 91
The Bacillus subtilis yaaH gene is transcribed by SigE RNA polymerase during sporulation, and its product is involved in germination of spores; Kodama T et al.; The expression of 21 novel genes located in the region from dnaA to abrB of the Bacillus subtilis chromosome was analyzed . One of the genes, yaaH, had a predicted promoter sequence conserved among SigE-dependent genes . Northern blot analysis revealed that yaaH mRNA was first detected from 2 h after the cessation of logarithmic growth (T(2)) of sporulation in wild-type cells and in spoIIIG (SigG(-)) and spoIVCB (SigK(-)) mutants but not in spoIIAC (SigF(-)) and spoIIGAB (SigE(-)) mutants . The transcription start point was determined by primer extension analysis; the -10 and -35 regions are very similar to the consensus sequences recognized by SigE-containing RNA polymerase . A YaaH-His tag fusion encoded by a plasmid with a predicted promoter for the yaaH gene was produced from T(2) of sporulation in a B . subtilis transformant and extracted from mature spores, indicating that the yaaH gene product is a spore protein . Inactivation of the yaaH gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme . The germination of yaaH mutant spores in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride was almost the same as that of wild-type spores, but the mutant spores were defective in L-alanine-stimulated germination . These results suggest that yaaH is a novel gene encoding a spore protein produced in the mother cell compartment from T(2) of sporulation and that it is required for the L-alanine-stimulated germination pathway.

J Bacteriol, 1999 Aug, 181(15), 4540 - 8
Mutational analysis and membrane topology of ComP, a quorum-sensing histidine kinase of Bacillus subtilis controlling competence development; Piazza F et al.; ComP is a sensor histidine kinase of Bacillus subtilis required for the signal transduction pathway that initiates the development of competence for genetic transformation . It is believed that ComP senses the presence of ComX, a modified extracellular peptide pheromone, and donates a phosphate to ComA, thereby activating this transcription factor for binding to the srfA promoter . In the present study, fusions to the Escherichia coli proteins PhoA and LacZ and analysis of its susceptibility to the protease kallikrein were used to probe the membrane topology of ComP . These data suggest that ComP contains six or eight membrane-spanning segments and two large extracytoplasmic loops in its N-terminal membrane-associated domain . Deletions were introduced involving the large extracellular loops to explore the role of the N-terminal domain of ComP in signal transduction . The absence of the second loop conferred a phenotype in which ComP was active in the absence of ComX . The implications of these data are discussed.

J Biol Chem, 1999 Jul 30, 274(31), 22114 - 21
Plant riboflavin biosynthesis . Cloning, chloroplast localization, expression, purification, and partial characterization of spinach lumazine synthase; Jordan DB et al.; Lumazine synthase, which catalyzes the penultimate step of riboflavin biosynthesis, has been cloned from three higher plants (spinach, tobacco, and arabidopsis) through functional complementation of an Escherichia coli auxotroph . Whereas the three plant proteins exhibit some structural similarities to known microbial homologs, they uniquely possess N-terminal polypeptide extensions that resemble typical chloroplast transit peptides . In vitro protein import assays with intact chloroplasts and immunolocalization experiments verify that higher plant lumazine synthase is synthesized in the cytosol as a larger molecular weight precursor protein, which is post-translationally imported into chloroplasts where it is proteolytically cleaved to its mature size . The authentic spinach enzyme is estimated to constitute <0.02% of the total chloroplast protein . Recombinant "mature" spinach lumazine synthase is expressed in E . coli at levels exceeding 30% of the total soluble protein and is readily purified to homogeneity using a simple two-step procedure . Apparent V(max) and K(m) values obtained with the purified plant protein are similar to those reported for microbial lumazine synthases . Electron microscopy and hydrodynamic studies reveal that native plant lumazine synthase is a hollow capsid-like structure comprised of 60 identical 16.5-kDa subunits, resembling its icosahedral counterparts in E . coli and Bacillus subtilis.

FEMS Microbiol Lett, 1999 Jul 1, 176(1), 45 - 50
Studies of the subtilin leader peptide as a translocation signal in Escherichia coli K12; Paul LK et al.; Subtilin is an antimicrobial peptide of the lantibiotic family that is produced by Gram-positive Bacillus subtilis, and its biosynthesis involves expression of presubtilin which consists of a leader segment and a mature segment . The leader segment is unlike a typical sec-type general secretion signal, and its ability to mediate translocation through a non-sec pathway has been previously studied by fusing the subtilin leader to an alkaline phosphatase reporter and expressing it in B . subtilis 168 {Izaguirre, G . and Hansen, J . N . (1997) Appl . Environ . Microbiol . 63, 3965-3971} . In this work, we have expressed the same subtilin leader-AP fusion in Gram-negative Escherichia coli, and found that the AP polypeptide is translocated into the periplasmic compartment and assembles into an enzymatically active form . The subtilin leader segment was not cleaved from this enzymatically active AP, which remained associated with the membrane . Conversion of the cells to spheroplasts followed by treatment with proteinase K showed that about 50% of the bound AP was sufficiently exposed on the surface of the spheroplasts to be inactivated by proteolytic cleavage.

Mol Microbiol, 1999 Aug, 33(3), 476 - 89
Mode of action of AraR, the key regulator of L-arabinose metabolism in Bacillus subtilis; Mota LJ et al.; The AraR protein is a negative regulator involved in L-arabinose-inducible expression of the Bacillus subtilis araABDLMNPQ-abfA metabolic operon and of the araE/araR genes that are organized as a divergent transcriptional unit . The two ara gene clusters are found at different positions in the bacterial chromosome . AraR was overproduced in Escherichia coli and purified to more than 95% homogeneity . AraR binds specifically to DNA fragments carrying the promoter region of the ara genes . DNase I protection assays showed that AraR binds to two sequences within the promoters of the araABDLMNPQ-abfA operon and the araE gene, and to one sequence in the araR promoter . The AraR target sequences are palindromic and share high identity, defining a 16 bp AraR consensus operator sequence showing half-symmetry, ATTTGTAC . Binding of AraR to DNA was inhibited by L-arabinose but not by other sugars . The two operator sites within the araABDLMNPQ-abfA operon and araE promoters are located on the same side of the DNA helix, and a pattern of enhanced and diminished DNase I cleavage was observed between them, but not in the araR promoter . Quantitative DNase I footprinting in DNA templates containing one, two or three AraR binding sites showed that the repressor binds cooperatively to the two operator sites within the metabolic operon and araE promoters but not to the site located in the araR promoter . These results are consistent with two modes for AraR transcriptional repression that might correlate with different physiological requirements: a high level of repression is achieved by DNA bending requiring two in-phase operator sequences (metabolic operon and araE transport gene), whereas binding to a single operator, which autoregulates araR expression, is 10-fold less effective.

Mol Microbiol, 1999 Aug, 33(3), 466 - 75
Characterization of a single-strand origin, ssoU, required for broad host range replication of rolling-circle plasmids; Kramer MG et al.; Single-stranded DNA (ssDNA) promoters are the key components of the single-strand origins (ssos) of replication of rolling-circle (RC) replicating plasmids . The recognition of this origin by the host RNA polymerase and the synthesis of a short primer RNA are critical for initiation of lagging-strand synthesis . This step is thought to be a limiting factor for the establishment of RC plasmids in a broad range of bacteria, because most of the ssos described are fully active only in their natural hosts . A special type of sso, the ssoU, is unique in the sense that it can be efficiently recognized in a number of different Gram-positive hosts . We have experimentally deduced the folded structure and characterized the ssDNA promoter present within the ssoU using P1 nuclease digestion and DNase I protection assays with the Bacillus subtilis and Staphylococcus aureus RNA polymerases . We have also identified the RNA products synthesized from this ssDNA promoter and mapped the initiation points of lagging-strand synthesis in vivo from ssoU-containing plasmids . Through gel mobility shift experiments, we have found that ssDNA containing the ssoU sequence can efficiently interact with the RNA polymerase from two different Gram-positive bacteria, S . aureus and B . subtilis . We have also realigned the narrow and broad host range sso sequences of RC plasmids, and found that they contain significant homology . Our data support the notion that the strength of the RNA polymerase-ssoU interaction may be the critical factor that confers the ability on the ssoU to be fully functional in a broad range of bacteria.

Biotechnol Bioeng, 1999 Sep 20, 64(6), 750 - 4
Estimation of P-to-O ratio in Bacillus subtilis and its influence on maximum riboflavin yield; Sauer U et al.; Simultaneous growth and riboflavin overproduction were investigated using a previously developed stoichiometric model of Bacillus subtilis metabolism . A fit of model predictions to experimental data was used to obtain estimates of fundamental energetic parameters of B . subtilis . Although multiple solutions describe the experimental data, evidence for a P-to-O ratio of about 1(1/3) mole of ATP produced per atom of oxygen consumed in oxidative phosphorylation was provided by genomic analysis of electron transport components, because no homologue of the proton-translocating NADH dehydrogenase I was found in the B . subtilis genome database . These results allow us to devise a rational metabolic engineering strategy to improve riboflavin production . The potential influence of increased energy coupling in oxidative phosphorylation on riboflavin yield is discussed . Higher coupling is most significant under carbon-limiting conditions in slow-growing cells, that is, in fed-batch processes of industrial interest .

Mol Microbiol, 1999 Jul, 33(2), 415 - 28
Role of lon and ClpX in the post-translational regulation of a sigma subunit of RNA polymerase required for cellular differentiation in Bacillus subtilis; Liu J et al.; The RNA polymerase sigma subunit, sigmaH (Spo0H) of Bacillus subtilis, is essential for the transcription of genes that function in sporulation and genetic competence . Although spo0H is transcriptionally regulated by the key regulatory device that controls sporulation initiation, the Spo0 phosphorelay, there is considerable evidence implicating a mechanism of post-translational control that governs the activity and concentration of sigmaH . Post-translational control of spo0H is responsible for the reduced expression of genes requiring sigmaH under conditions of low environmental pH . It is also responsible for heightened sigmaH activity upon relief of acid stress and during nutritional depletion . In this study, the ATP-dependent proteases LonA and B and the regulatory ATPase ClpX were found to function in the post-translational control of sigmaH . Mutations in lonA and lonB result in elevated sigmaH protein concentrations in low-pH cultures . However, this is not sufficient to increase sigmaH-dependent transcription . Activation of sigmaH-dependent transcription upon raising medium pH and in cells undergoing sporulation requires clpX, as shown by measuring the expression of lacZ fusions that require sigmaH for transcription and by complementation of a clpX null mutation . A hypothesis is presented that low environmental pH results in the Lon-dependent degradation of sigmaH, but the activity of sigmaH in sporulating cells and in cultures at neutral pH is stimulated by a ClpX-dependent mechanism in response to nutritional stress.

Mol Microbiol, 1999 Jul, 33(2), 389 - 95
Alanine mutants of the Spo0F response regulator modifying specificity for sensor kinases in sporulation initiation; Jiang M et al.; Five single alanine substitution mutations in the Spo0F response regulator gave rise to mutant strains of Bacillus subtilis with seemingly normal sporulation that nevertheless rapidly segregated variants blocked in sporulation . The basis for this deregulated phenotype was postulated to be increased phosphorylation of the Spo0A transcription factor, resulting from enhanced phosphate input or decreased dephosphorylation of the phosphorelay . Strains bearing two of these Spo0F mutant proteins, Y13A and I17A, retained a requirement for KinA and KinB kinases in sporulation, whereas the remaining three, L66A, I90A and H101A, gave strains that sporulated well in the absence of both KinA and KinB . Sporulation of strains bearing L66A and H101A mutations was decreased in a mutant lacking KinA, KinB and KinC, but the strain bearing the I90A mutation required the further deletion of KinD to lower its sporulation frequency . The affected residues, L-66, I-90 and H-101, are involved in crucial hydrophobic contacts stabilizing the orientation of helix alpha4 of Spo0F . The data are consistent with the notion that these three mutations alter the conformation of the beta4-alpha4 loop of Spo0F that is known to contain residues critical for KinA:Spo0F recognition . As this loop has a propensity for multiple conformations, the spatial arrangement of this loop may play a critical role in kinase selection by Spo0F and might be altered by regulatory molecules interacting with Spo0F.

Mol Microbiol, 1999 Jul, 33(2), 377 - 88
Identification and characterization of a stress-responsive promoter in the macromolecular synthesis operon of Bacillus subtilis; Liao CT et al.; Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant . Induction of sigmaA has been observed exclusively in this mutant harbouring extra copies of the plasmid-borne Ts sigA gene transcriptionally controlled by the P1P2 promoters of the B . subtilis macromolecular synthesis (MMS; rpoD or sigA) operon . Investigation of the mechanisms leading to the induction has allowed us to identify a sigmaB-type promoter, P7, in the MMS operon for the first time . Therefore, at least seven promoters in total are responsible for the regulation of the B . subtilis MMS operon, including the four known sigmaA- and sigmaH-type promoters, as well as two incompletely defined promoters . The P7 promoter was activated in B . subtilis after the imposition of heat, ethanol and salt stresses, indicating that the MMS operon of B . subtilis is subjected to the control of general stress . The significant heat induction of P7 in B . subtilis DB1005 harbouring a plasmid-borne Ts sigA gene can be explained by a model of competition between sigmaA and sigmaB for core binding; very probably, the sigmaB factor binds more efficiently to core RNA polymerase under heat shock . This mechanism may provide a means for the expression of the B . subtilis MMS operon when sigmaA becomes defective in core binding.

Mol Microbiol, 1999 Jul, 33(1), 84 - 96
Selection of the midcell division site in Bacillus subtilis through MinD-dependent polar localization and activation of MinC; Marston AL et al.; Bacterial cell division commences with the assembly of the tubulin-like protein, FtsZ, at midcell to form a ring . Division site selection in rod-shaped bacteria is mediated by MinC and MinD, which form a division inhibitor . Bacillus subtilis DivIVA protein ensures that MinCD specifically inhibits division close to the cell poles, while allowing division at midcell . We have examined the localization of MinC protein and show that it is targeted to midcell and retained at the mature cell poles . This localization is reminiscent of the pattern previously described for MinD . Localization of MinC requires both early (FtsZ) and late (PbpB) division proteins, and it is completely dependent on MinD . The effects of a divIVA mutation on localization of MinC now suggest that the main role of DivIVA is to retain MinCD at the cell poles after division, rather than recruitment to nascent division sites . By overexpressing minC or minD, we show that both proteins are required to block division, but that only MinD needs to be in excess of wild-type levels . The results suggest a mechanism whereby MinD is required both to pilot MinC to the cell poles and to constitute a functional division inhibitor.

Mol Microbiol, 1999 Jul, 33(1), 33 - 40
Co-ordinating DNA replication with cell division in bacteria: a link between the early stages of a round of replication and mid-cell Z ring assembly; Harry EJ et al.; Spores of a thymine-requiring strain of Bacillus subtilis 168, which is also temperature sensitive for the initiation of chromosome replication, were germinated and allowed to grow out at the permissive temperature in a minimal medium containing no added thymine . Under these conditions, there was no or very limited progression into the elongation phase of the first round of replication . In a significant proportion of the outgrown cells, a Z ring formed precisely at mid-cell and over the centrally positioned nucleoid, leading eventually to the formation of a mature division septum . When initiation of the first round of replication was blocked through a temperature shift and with thymine present, the Z ring was positioned acentrally . The central Z ring that formed in the absence of thymine was blocked by the presence of a DNA polymerase III inhibitor . It is concluded that the very early stages of a round of replication (initiation plus possibly limited progression into the elongation phase) play a key role in the precise positioning of the Z ring at mid-cell and between replicating daughter chromosomes.

Mol Microbiol, 1999 Jul, 33(1), 8 - 17
Bacterial transcription factors involved in global regulation; Vicente M et al.; The presence of intricate global cell regulation mechanisms may be one reason for the exceptional environmental and evolutionary success of microbes . Promoters, the cis-acting signals, are responsive to several stimuli related to growth, stress and substrate specificity . Their response is mediated by a wide variety of trans-acting regulators that sense the environment and the physiological state of the cell and adjust the transcription of specific genes . One of the main transcriptional regulation webs operates in the transition from affluent to barren conditions, with sigmaS being the chief actor in a company of players that stage a competition for the sparsely available RNA polymerase molecules . In this role, sigmaS may be assisted by several factors, including nucleoid-related proteins and metabolites . In addition, the levels of sigmaS itself are regulated by mechanisms that include inactivation and degradation . Several transcription factors, belonging to different regulatory pathways, may operate in the same promoter . In such a case, the final transcriptional output depends both on the interplay of effectors and on the properties of the recruitment of the effector-RNA polymerase complex to the promoter . RNA polymerase itself is also capable of establishing selective interactions with activators and specific promoter regions through the carboxy-terminal domain of its alpha subunit (alphaCTD) . Transcriptional regulation controls pervade such crucial events in the life of bacterial cells as Escherichia coli cell division, Bacillus subtilis sporulation and Caulobacter crescentus differentiation . These examples suggest that bacteria have been particularly inventive in adapting gene expression regulation to survive under a diversity of environments and have done so by exploiting the malleable molecular mechanisms involved in transcription, developing complexities that may match those found in eukaryotic cells.

Comput Chem, 1999 Jun 15, 23(3-4), 333 - 40
Whole genome protein domain analysis using a new method for domain clustering; Gouzy J et al.; We present the outcome of a systematic analysis of protein domain shuffling in 17 completed microbial genomes . This analysis has been performed using MKDOM Version 2, a completely new version of the domain clustering program MKDOM based on PSI-BLAST recursive homology searches . It allows to delineate the most frequent protein domain building blocks, which domains are found specifically in Bacteria, Archaea or yeast, and which domains are shared between two or all three domains of life . The latter are good candidates as the basic protein building blocks underlying all forms of cellular life . Statistics of multi-domain proteins indicate that some organisms such as Bacillus subtilis or Mycobacterium tuberculosis contain an abnormally high number of large multi-domain proteins . We also provide examples of highly shuffled or circularly permutated domains . A WWW graphical interface has been made available to interactively browse domain arrangements of proteins in all 17 genomes, at http:@www.toulouse.inra.fr/prodomCG.html.

Phytother Res, 1999 Jun, 13(4), 296 - 9
Evaluation of the destructive effect of khaya gum on Bacillus subtilis spores during tableting; Odeku OA et al.; The destructive effect of khaya gum used as a binding agent in a paracetamol formulation on Bacillus subtilis spores during tableting has been investigated, in comparison with the effects of two standard binders-polyvinylpyrrolidone and gelatin . The destructive effect of khaya gum was generally similar to those of the standard binders . Significant (p < 0.001 in each case) inverse linear relationships of log % survival of the B . subtilis spores with compression pressure and with concentration of binder, were established . The effect of the binding agent was significantly dependent (p < 0.001 in each case) on both compression pressure and the binder concentration . However, there was no significant interaction (p > 0.05) between the destructive effects of compression pressure and binder concentration . The results suggest that khaya gum and the standard binders would be useful in the destruction of microorganisms during tableting.

J Bacteriol, 1999 Jul, 181(14), 4365 - 73
A region of sigmaK involved in promoter activation by GerE in Bacillus subtilis; Wade KH et al.; During endospore formation in Bacillus subtilis, the DNA binding protein GerE stimulates transcription from several promoters that are used by RNA polymerase containing sigmaK . GerE binds to a site on one of these promoters, cotX, that overlaps its -35 region . We tested the model that GerE interacts with sigmaK at the cotX promoter by seeking amino acid substitutions in sigmaK that interfered with GerE-dependent activation of the cotX promoter but which did not affect utilization of the sigmaK-dependent, GerE-independent promoter gerE . We identified two amino acid substitutions in sigmaK, E216K and H225Y, that decrease cotX promoter utilization but do not affect gerE promoter activity . Alanine substitutions at these positions had similar effects . We also examined the effects of the E216A and H225Y substitutions in sigmaK on transcription in vitro . We found that these substitutions specifically reduced utilization of the cotX promoter . These and other results suggest that the amino acid residues at positions 216 and 225 are required for GerE-dependent cotX promoter activity, that the histidine at position 225 of sigmaK may interact with GerE at the cotX promoter, and that this interaction may facilitate the initial binding of sigmaK RNA polymerase to the cotX promoter . We also found that the alanine substitutions at positions 216 and 225 of sigmaK had no effect on utilization of the GerE-dependent promoter cotD, which contains GerE binding sites that do not overlap with its -35 region.

J Bacteriol, 1999 Jul, 181(14), 4299 - 307
Interaction of Bacillus subtilis Fur (ferric uptake repressor) with the dhb operator in vitro and in vivo; Bsat N et al.; Bacillus subtilis contains three metalloregulatory proteins belonging to the ferric uptake repressor (Fur) family: Fur, Zur, and PerR . We have overproduced and purified Fur protein and analyzed its interaction with the operator region controlling the expression of the dihydroxybenzoate siderophore biosynthesis (dhb) operon . The purified protein binds with high affinity and selectivity to the dhb regulatory region . DNA binding does not require added iron, nor is binding reduced by dialysis of Fur against EDTA or treatment with Chelex . Fur selectively inhibits transcription from the dhb promoter by sigmaA RNA polymerase, even if Fur is added after RNA polymerase holoenzyme . Since neither DNA binding nor inhibition of transcription requires the addition of ferrous ion in vitro, the mechanism by which iron regulates Fur function in vivo is not obvious . Mutagenesis of the fur gene reveals that in vivo repression of the dhb operon by iron requires His97, a residue thought to be involved in iron sensing in other Fur homologs . Moreover, we identify His96 as a second likely iron ligand, since a His96Ala mutant mediates repression at 50 microM but not at 5 microM iron . Our data lead us to suggest that Fur is able to bind DNA independently of bound iron and that the in vivo role of iron is to counteract the effect of an inhibitory factor, perhaps another metal ion, that antagonizes this DNA-binding activity.

FEMS Microbiol Lett, 1999 Apr 1, 173(1), 127 - 31
Bacillus subtilis alpha-amylase: the rate limiting step of secretion is growth phase-independent; Haddaoui E et al.; When Bacillus subtilis alpha-amylase was expressed under the control of sacR in a degU32(Hy) strain, the production of exoenzyme occurred during both the exponential and stationary phases of growth . In each phase, pulse-chase experiments showed that the rate-limiting step of the secretion process was the release of the processed form of the protein in each physiological context . The rate of this event was slightly slower (t(1/2) = 3.2 min) during the stationary phase than during the exponential phase (t(1/2) = 2 min) . The effectors which possibly control the efficiency of the release stage, the level of PrsA or the calcium binding properties of the cell wall, remained unchanged throughout growth phases.

Biotechnol Bioeng, 1999 Jul 20, 64(2), 129 - 34
Metabolic fluxes, pools, and enzyme measurements suggest a tighter coupling of energetics and biosynthetic reactions associated with reduced pyruvate kinase flux; Goel A et al.; In this study, it is found that, for Bacillus subtilis, citrate-glucose cometabolism leads to zero acid production over a wide range of growth rates and nearly theoretical carbon yield . Experimental results are presented that point to pyruvate kinase (PYK) as a site of citrate-mediated glycolytic flux attenuation . First, the measured fluxes show that, compared with cultures grown on glucose, the PYK flux drops by more than tenfold when citrate is added . Second, relative to cultures metabolizing glucose, the phosphoenolpyruvate (PEP) pool elevates substantially, whereas the pyruvate pool drops, when citrate is present . Finally, our modeling results indicate that maximizing carbon yield corresponds to nearly eliminating pyruvate kinase (PYK) flux and that the pyruvate supplied by the PEP-consuming glucose transport system can supply the biosynthetic requirements . A literature review suggests some mechanisms for how PYK attenuation by citrate addition can occur . At this juncture, we hypothesize that direct PYK inhibition occurs which, in turn, also leads to phosphofructokinase inhibition via the elevated PEP pool . These two inhibition events combine to throttle glycolytic flux; minimize acid formation; and substantially increase cellular, product, and energetic yields .

Biotechnol Bioeng, 1999 Jun 20, 63(6), 737 - 49
Metabolic flux analysis of Escherichia coli expressing the Bacillus subtilis acetolactate synthase in batch and continuous cultures; Aristidou AA et al.; Metabolically engineered Escherichia coli expressing the B . subtilis acetolactate synthase has shown to be capable of reducing acetate accumulation . This reduction subsequently led to a significant enhancement in recombinant protein production . The main focus of this study is to systematically examine the effect of ALS in the metabolic patterns of E . coli in batch and continuous culture . The specific acetate production rate of a strain carrying the B . subtilis als gene is 75% lower than that of the control strain (host carrying the control plasmid pACYC184) in batch cultures . The ALS strain is further demonstrated to be capable of maintaining a reduced specific acetate production rate in continuous cultures at dilution rates ranging from 0.1 to 0.4 h-1 . In addition, this ALS strain is shown to have a higher ATP yield and lower maintenance coefficient . The metabolic flux analysis of carbon flux distribution of the central metabolic pathways and at the pyruvate branch point reveals that this strain has the ability to channel excess pyruvate to the much less toxic compound, acetoin .

Arch Biochem Biophys, 1999 Jul 15, 367(2), 317 - 21
Expression and processing of a bacterial endoglucanase in transgenic mice; Zhang JX et al.; The C6.5 endoglucanase from Bacillus subtilis catalyzes the hydrolyses of beta-glucans . This enzyme, which is also produced by many ruminant microbes, is not part of the normal digestive repertoire of monogastric animals . We have generated transgenic mice which express the C6.5 endoglucanase gene specifically in the pancreas with secretion of the enzyme into the small intestine . The secreted enzyme has a molecular mass of 55 kDa which is reduced by protease digestion to the principal forms of 37 and 35 kDa . These truncated forms are resistant to further protease degradation and exhibit enhanced specific activity compared to the native enzyme . These results encourage further investigation of the utility of this transgene for enhancing the digestive capability of monogastric animals .

Arch Biochem Biophys, 1999 Jul 15, 367(2), 297 - 302
Peptide aldehyde inhibitors of bacterial peptide deformylases; Durand DJ et al.; Bacterial peptide deformylases (PDF, EC 3.5.1.27) are metalloenzymes that cleave the N-formyl groups from N-blocked methionine polypeptides . Peptide aldehydes containing a methional or norleucinal inhibited recombinant peptide deformylase from gram-negative Escherichia coli and gram-positive Bacillus subtilis . The most potent inhibitor was calpeptin, N-CBZ-Leu-norleucinal, which was a competitive inhibitor of the zinc-containing metalloenzymes, E . coli and B . subtilis PDF with Ki values of 26.0 and 55.6 microM, respectively . Cobalt-substituted E . coli and B . subtilis deformylases were also inhibited by these aldehydes with Ki values for calpeptin of 9.5 and 12.4 microM, respectively . Distinct spectral changes were observed upon binding of calpeptin to the Co(II)-deformylases, consistent with the noncovalent binding of the inhibitor rather than the formation of a covalent complex . In contrast, the chelator 1,10-phenanthroline caused the time-dependent inhibition of B . subtilis Co(II)-PDF activity with the loss of the active site metal . The fact that calpeptin was nearly equipotent against deformylases from both gram-negative and gram-positive bacterial sources lends further support to the idea that a single deformylase inhibitor might have broad-spectrum antibacterial activity .

Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 7803 - 8
RNase P RNAs from some Archaea are catalytically active; Pannucci JA et al.; The RNA subunits of RNase Ps of Archaea and eukaryotes have been thought to depend fundamentally on protein for activity, unlike those of Bacteria that are capable of efficient catalysis in the absence of protein . Although the eukaryotic RNase P RNAs are quite different than those of Bacteria in both sequence and structure, the archaeal RNAs generally contain the sequences and structures of the bacterial, phylogenetically conserved catalytic core . A spectrum of archaeal RNase P RNAs were therefore tested for activity in a wide range of conditions . Many remain inactive in ionically extreme conditions, but catalytic activity could be detected from those of the methanobacteria, thermococci, and halobacteria . Chimeric holoenzymes, reconstituted from the Methanobacterium RNase P RNA and the Bacillus subtilis RNase P protein subunits, were functional at low ionic strength . The properties of the archaeal RNase P RNAs (high ionic-strength requirement, low affinity for substrate, and catalytic reconstitution by bacterial RNase P protein) are similar to synthetic RNase P RNAs that contain all of the catalytic core of the bacterial RNA but lack phylogenetically variable, stabilizing elements.

J Mol Biol, 1999 Jul 9, 290(2), 433 - 45
Role of metal ions in the hydrolysis reaction catalyzed by RNase P RNA from Bacillus subtilis; Warnecke JM et al.; Precursor tRNA (ptRNA) substrates carrying a single Rp or Sp-phosphorothioate modification at the RNase P cleavage site were used as tools to study the cleavage mechanism of RNase P RNA from Bacillus subtilis . Both the Sp and the Rp-diastereomer reduced the rate of processing at least 10(4)-fold under conditions where the chemical step is essentially rate-limiting . Neither the Rp nor the Sp-phosphorothioate modification affected ptRNA ground state binding to B . subtilis RNase P RNA . Processing of the Rp-diastereomeric ptRNA could be restored in the presence of Mn2+or Cd2+, demonstrating direct metal ion coordination to the pro -Rp oxygen during catalysis . With Cd2+, processing required the presence of another metal ion, such as Ca2+or Mg2+, to mediate substrate binding . This is in contrast to Escherichia coli RNase P RNA, which promotes cleavage of Rp-diastereomeric ptRNA in the presence of Cd2+as the sole divalent metal ion . Analysis of {Cd2+}-dependent processing of the Rp-diastereomeric substrate by B . subtilis RNase P RNA was consistent with the involvement of at least two metal ions in catalysis . The presence of two catalytic metal ion binding sites is also supported by the inhibition mode of Ca2+on cleavage of unmodified ptRNA . In the presence of an Sp-phosphorothioate modification at the scissile bond, neither Mn2+nor Cd2+were able to restore significant cleavage at this location . Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency . Unaffected ground state binding of the Sp-diastereomeric ptRNA but a >/=10(4)-fold reduced hydrolysis rate may indicate a crucial role of the pro -Sp oxygen in transition state stabilization or may be attributed to steric exclusion of catalytic metal ions . Based on our comparative analyses of B . subtilis and E . coli RNase P RNA, each representing the main structural subtypes of bacterial RNase P RNA, common features in terms of active site constraints and role of catalytic metal ions can now be formulated for bacterial RNase P RNAs . On the other hand, substantial and unexpected differences with respect to the overall metal ion requirements and tRNA binding modes have been observed for the two catalytic RNAs .

Appl Environ Microbiol, 1999 Jul, 65(7), 2934 - 41
Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtilis; Bolhuis A et al.; Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by Bacillus subtilis is frequently inefficient . In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins: Bacillus lichenifomis alpha-amylase (AmyL), Escherichia coli TEM beta-lactamase (Bla), human pancreatic alpha-amylase (HPA), and a lysozyme-specific single-chain antibody . The same expression and secretion signals were used for all four of these proteins . Notably, all identified bottlenecks relate to late stages in secretion, following translocation of the preproteins across the cytoplasmic membrane . These bottlenecks include processing by signal peptidase, passage through the cell wall, and degradation in the wall and growth medium . Strikingly, all translocated HPA was misfolded, its stability depending on the formation of disulfide bonds . This suggests that the disulfide bond oxidoreductases of B . subtilis cannot form the disulfide bonds in HPA correctly . As the secretion bottlenecks differed for each heterologous protein tested, it is anticipated that the efficient secretion of particular groups of heterologous proteins with the same secretion bottlenecks will require the engineering of specifically optimized host strains.

Protein Sci, 1999 Jun, 8(6), 1368 - 70
Selective association of protein molecules followed by mass spectrometry; Vis H et al.; Nanoflow electrospray mass spectrometry was used to monitor the formation of protein heterodimers of HU proteins from Bacillus stearothermophilus and Bacillus subtilis . This has enabled us to analyze both thermodynamic and kinetic features associated with the dissociation of homodimeric HU proteins . The results obtained correlate well with the kinetics of the protein dissociation process and the free energy difference between homo- and heterodimeric species anticipated from other studies . We suggest that this approach will have general applicability in studying protein association and dissociation under near-equilibrium conditions and will be relevant to a wide range of biological systems.

Protein Sci, 1999 Jun, 8(6), 1350 - 7
Formation of amyloid fibrils by peptides derived from the bacterial cold shock protein CspB; Gross M et al.; Three peptides covering the sequence regions corresponding to the first two (CspB-1), the first three (CspB-2), and the last two (CspB-3) beta-strands of CspB, the major cold shock protein of Bacillus subtilis, have been synthesized and analyzed for their conformations in solution and for their precipitation behavior . The peptides are nearly insoluble in water, but highly soluble in aqueous solutions containing 50% acetonitrile (pH 4.0) . Upon shifts of the solvent condition toward lower or higher acetonitrile concentrations, the peptides all form fibrils resembling those observed in amyloid associated diseases . These fibrils have been identified and characterized by electron microscopy, binding of the dye congo red, and X-ray fiber diffraction . Characterization of the peptides in solution by circular dichroism and NMR spectroscopy shows that the formation of these fibrils does not require specific preformed secondary structure in the solution state species . While the majority of the soluble fraction of each peptide is monomeric and unstructured, different types of structures including alpha-helical, beta-sheet, and random coil conformations are observed under conditions that eventually lead to fibril formation . We conclude that the absence of tertiary contacts under solution conditions where binding interactions between peptide units are still favorable is a crucial requirement for amyloid formation . Thus, fragmentation of a sequence, like partial chemical denaturation or mutation, can enhance the capacity of specific protein sequences to form such fibrils.

J Bacteriol, 1999 Jul, 181(13), 4114 - 7
ScoC regulates peptide transport and sporulation initiation in Bacillus subtilis; Koide A et al.; Oligopeptides are transported into Bacillus subtilis by two ABC transport systems, App and Opp . Transcription of the operon encoding the Opp system was found to occur during exponential growth, whereas the app operon was induced at the onset of stationary phase . Transcription of both operons was completely curtailed by overproduction of the ScoC regulator from a multicopy plasmid and was enhanced in strains with the scoC locus deleted . ScoC, a member of the MarR family of transcription regulators, is known from previous studies to be a negative regulator of sporulation and of protease production that acts by binding directly to the promoters of the genes it regulates . Since peptide transport is essential for inactivation of the negative regulation of sporulation by Rap phosphatases, the control of ScoC transcription repression activity plays a crucial role in the initiation of sporulation.

J Bacteriol, 1999 Jul, 181(13), 4081 - 8
sigmaK can negatively regulate sigE expression by two different mechanisms during sporulation of Bacillus subtilis; Zhang B et al.; Temporal and spatial gene regulation during Bacillus subtilis sporulation involves the activation and inactivation of multiple sigma subunits of RNA polymerase in a cascade . In the mother cell compartment of sporulating cells, expression of the sigE gene, encoding the earlier-acting sigma factor, sigmaE, is negatively regulated by the later-acting sigma factor, sigmaK . Here, it is shown that the negative feedback loop does not require SinR, an inhibitor of sigE transcription . Production of sigmaK about 1 h earlier than normal does affect Spo0A, which when phosphorylated is an activator of sigE transcription . A mutation in the spo0A gene, which bypasses the phosphorelay leading to the phosphorylation of Spo0A, diminished the negative effect of early sigmaK production on sigE expression early in sporulation . Also, early production of sigmaK reduced expression of other Spo0A-dependent genes but not expression of the Spo0A-independent ald gene . In contrast, both sigE and ald were overexpressed late in development of cells that fail to make sigmaK . The ald promoter, like the sigE promoter, is believed to be recognized by sigmaA RNA polymerase, suggesting that sigmaK may inhibit sigmaA activity late in sporulation . To exert this negative effect, sigmaK must be transcriptionally active . A mutant form of sigmaK that associates with core RNA polymerase, but does not direct transcription of a sigmaK-dependent gene, failed to negatively regulate expression of sigE or ald late in development . On the other hand, the negative effect of early sigmaK production on sigE expression early in sporulation did not require transcriptional activity of sigmaK RNA polymerase . These results demonstrate that sigmaK can negatively regulate sigE expression by two different mechanisms, one observed when sigmaK is produced earlier than normal, which does not require sigmaK to be transcriptionally active and affects Spo0A, and the other observed when sigmaK is produced at the normal time, which requires sigmaK RNA polymerase transcriptional activity . The latter mechanism facilitates the switch from sigmaE to sigmaK in the cascade controlling mother cell gene expression.

J Bacteriol, 1999 Jul, 181(13), 4071 - 5
Cloning and expression of cadD, a new cadmium resistance gene of Staphylococcus aureus; Crupper SS et al.; A cadmium resistance gene, designated cadD, has been identified in and cloned from the Staphylococcus aureus plasmid pRW001 . The gene is part of a two-component operon which contains the resistance gene cadD and an inactive regulatory gene, cadX* . A high degree of sequence similarity was observed between cadD and the cadB-like gene from S . lugdunensis, but no significant similarity was found with either cadA or cadB from the S . aureus plasmids pI258 and pII147 . The positive regulatory gene cadX* is identical to cadX from pLUG10 over a stretch of 78 codons beginning at the N terminus, but it is truncated at this point and inactive . Sequence analysis showed that the cadmium resistance operon resides on a 3,972-bp element that is flanked by direct repeats of IS257 . The expression of cadD in S . aureus and Bacillus subtilis resulted in low-level resistance to cadmium; in contrast, cadA and cadB from S . aureus induced higher level resistance . However, when the truncated version of cadX contained in pRW001 is complemented in trans with cadX from plasmid pLUG10, resistance increased approximately 10-fold suggesting that the cadmium resistance operons from pRW001 and pLUG10 are evolutionarily related . Moreover, the truncated version of cadX contained in pRW001 is nonfunctional and may have been generated by deletion during recombination to acquire the cadmium resistance element.

J Bacteriol, 1999 Jul, 181(13), 3956 - 66
Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation; Atrih A et al.; The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides . The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated . About 99% analyzed muropeptides in B . subtilis vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated . Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides . These two types of muropeptides are suggested to end glycan strands . An unexpected feature of B . subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides . This amount is, however, dependent on the composition of the growth media . Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total . B . subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides . The cross-linking index of the polymer changes with the growth phase . It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively . Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers . The total muropeptides with pentapeptide side chains accounts for almost 82% in the dacA mutant . This major low-molecular-weight PBP (DD-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.

J Bacteriol, 1999 Jul, 181(13), 3942 - 8
Expression of the sigmaB-dependent general stress regulon confers multiple stress resistance in Bacillus subtilis; Volker U et al.; The alternative sigma factor sigmaB of Bacillus subtilis is required for the induction of approximately 100 genes after the imposition of a whole range of stresses and energy limitation . In this study, we investigated the impact of a null mutation in sigB on the stress and starvation survival of B . subtilis . sigB mutants which failed to induce the regulon following stress displayed an at least 50- to 100-fold decrease in survival of severe heat (54 degrees C) or ethanol (9%) shock, salt (10%) stress, and acid (pH 4.3) stress, as well as freezing and desiccation, compared to the wild type . Preloading cells with sigmaB-dependent general stress proteins prior to growth-inhibiting stress conferred considerable protection against heat and salt . Exhaustion of glucose or phosphate induced the sigmaB response, but surprisingly, sigmaB did not seem to be required for starvation survival . Starved wild-type cells exhibited about 10-fold greater resistance to salt stress than exponentially growing cells . The data argue that the expression of sigmaB-dependent genes provides nonsporulated B . subtilis cells with a nonspecific multiple stress resistance that may be relevant for stress survival in the natural ecosystem.

Mol Microbiol, 1999 Jun, 32(6), 1183 - 97
Post-transcriptional regulation of the Bacillus subtilis dnaK operon; Homuth G et al.; The heptacistronic dnaK heat shock operon of Bacillus subtilis consists of the genes hrcA, grpE, dnaK, dnaJ, orf35, orf28 and orf50 . It is controlled by the CIRCE/HrcA operator/repressor system and specifies three primary transcripts, two of which are processed into three different products . We have analysed the regulatory consequences of this complex transcriptional organization in detail . First, the seven genes were heat induced to different extents at the mRNA level and can be classified into three groups by their induction factors . This differential induction was also reflected at the protein level . Secondly, the cellular amounts of the proteins HrcA, DnaK and DnaJ in B . subtilis differed drastically both under non-heat shock conditions and after thermal upshock . Thirdly, Northern blot analyses demonstrated that an mRNA-processing reaction generating products of differential stabilities plays an essential role during the regulation of gene expression . A crucial factor determining the low stability of two transcripts is the presence of the CIRCE element at their 5' ends . We demonstrate that CIRCE leads to the destabilization of mRNAs, but only if it is located in the immediate vicinity of a Shine-Dalgarno sequence . These results show that B . subtilis is using various, especially post-transcriptional, regulatory mechanisms to fine tune the expression of the individual genes of the heptacistronic dnaK operon.

Mol Microbiol, 1999 Jun, 32(6), 1173 - 82
Transcription- and translation-dependent changes in membrane dynamics in bacteria: testing the transertion model for domain formation; Binenbaum Z et al.; Cell cycle events have been proposed to be triggered by the formation of membrane domains in the process of coupled transcription, translation and insertion ('transertion') of nascent membrane and exported proteins . Disruption of domain structure should lead to changes in membrane dynamics . Membrane viscosity of Escherichia coli and Bacillus subtilis decreased after inhibition of protein synthesis by chloramphenicol or puromycin, or of RNA initiation by rifampicin, but not after inhibition of RNA elongation by streptolydigin or amino acid starvation of a stringent strain . The decrease caused by inhibitors of protein synthesis was prevented by streptolydigin if added simultaneously, but was not reversed if added later . The drug-induced decrease in membrane viscosity is energy dependent: it did not happen in KCN-treated cells . All treatments decreasing membrane viscosity also induced nucleoid compaction and fusion . Inhibition of macromolecular synthesis without membrane perturbation caused nucleoids to expand . Changes in membrane dynamics were also displayed during a nutritional shift-down transition that causes imbalance in macromolecular syntheses . The results are consistent with the transertion model, predicting dissipation of membrane domains by termination of protein synthesis or detachment of polysomes from DNA; domain structure is conserved if the transertion process is 'frozen'.

Biosci Biotechnol Biochem, 1999 May, 63(5), 792 - 8
Characterization of yrpC gene product of Bacillus subtilis IFO 3336 as glutamate racemase isozyme; Ashiuchi M et al.; Glr, the glutamate racemase of Bacillus subtilis (formerly Bacillus natto) IFO 3336 encoded by the glr gene, and YrpC, a protein encoded by the yrpC gene, which is located at a different locus from that of the glr gene in the B . subtilis genome, share a high sequence similarity . The yrpC gene complemented the D-glutamate auxotrophy of Escherichia coli WM335 cells defective in the glutamate racemase gene . Glutamate racemase activity was found in the extracts of E . coli WM335 clone cells harboring a plasmid, pYRPC1, carrying its gene . Thus, the yrpC gene encodes an isozyme of glutamate racemase of B . subtilis IFO 3336 . YrpC is mostly found in an inactive inclusion body in E . coli JM109/pYRPC1 cells . YrpC was solubilized readily, but glutamate racemase activity was only slightly restored . We purified YrpC from the extracts of E . coli JM109/pYRPC2 cells using a Glutathione S-transferase Gene Fusion System to characterize it . YrpC is a monomeric protein and contains no cofactors, like Glr . Enzymological properties of YrpC, such as the substrate specificity and optimum pH, are also similar to those of Glr . The thermostability of YrpC, however, is considerably lower than that of Glr . In addition, YrpC showed higher affinity and lower catalytic efficiency for L-glutamate than Glr . This is the first example showing the occurrence and properties of a glutamate racemase isozyme.

Microbiology, 1999 May, 145 ( Pt 5), 1069 - 78
Two genes from Bacillus subtilis under the sole control of the general stress transcription factor sigmaB; Akbar S et al.; The general stress response of Bacillus subtilis is triggered by a variety of environmental and metabolic stresses which activate the sigmaB transcription factor . Among the more than 100 genes controlled by sigmaB (the csb genes), the functions identified thus far include resistance to oxidative stress, resistance to protein denaturation and resistance to osmotic stress . To understand the breadth of functions in which csb genes participate, the transcriptional organization and predicted products of two such genes previously identified in a screen for sigmaB-dependent lacZ fusions were analysed . The csb-22::Tn917lacZ and csb-34::Tn917lacZ fusions are unusual among csb genes in that their expression appears to be completely dependent upon sigmaB . By plasmid-integration experiments, fusion analyses and site-directed mutagenesis, stress-inducible, sigmaB-dependent promoters for both these fusions were identified . The csb-34 fusion marked an ORF (yxcC or csbC) which by sequence analysis lay in a monocistronic transcriptional unit . This ORF encoded a predicted 461-residue product which had high identity with Class I sugar transporters of the major facilitator superfamily . It was speculated that the csbC product could serve either a nutritional or an osmotic protection function . In contrast, the csb-22 fusion identified an ORF (ywmG or csbD) which appeared to be the second gene of a two-gene operon . This ORF encoded a predicted 62-residue product which resembled a small Escherichia coli protein of unknown function . The sigmaB . dependent promoter lay immediately upstream from csbD and appeared to be an internal promoter for the operon.

Microbiology, 1999 May, 145 ( Pt 5), 1055 - 67
Nucleotide sequence of the Bacillus subtilis temperate bacteriophage SPbetac2; Lazarevic V et al.; The Bacillus subtilis 168 chromosomal region extending from 184 degrees to 195 degrees, corresponding to prophage SPbeta, has been completely sequenced using DNA of the thermoinducible SPbetac2 mutant . This 134416 bp segment comprises 187 putative ORFs which, according to their orientation, were grouped into three clusters . Compared to its host, SPbetac2 is characterized by a lower G+C content, shorter mean ORF length, as well as a different usage of start codons . Nearly 75% of predicted ORFs do not share significant homologies to sequences in available databases . The only highly similar proteins to SPbetac2-encoded ones are host paralogues . SPbetac2 promoter regions contain SOS box consensus sequences and a repeated motif, designated SPbeta repeated element (SPBRE), that is absent from the host genome . Gene sspC, encoding the small acid-soluble protein C, that has been previously sequenced and mapped to the vicinity of the SPbeta region, was found to be part of the prophage.

Microbiology, 1999 May, 145 ( Pt 5), 1049 - 53
Assay for UDPglucose 6-dehydrogenase in phosphate-starved cells: gene tuaD of Bacillus subtilis 168 encodes the UDPglucose 6-dehydrogenase involved in teichuronic acid synthesis; Pagni M et al.; A novel assay permitting the detection of UDPglucose 6-dehydrogenase activity in cell-free extracts obtained from phosphate-starved cultures of Bacillus subtilis is described . The critical step, the separation of phosphate-starvation-induced exo-enzymes, phosphatases and phosphodiesterases from the cytoplasmic fraction containing the UDPglucose dehydrogenase, was achieved by protoplasting and removal of the periplasmic fraction by protoplast washing . Using this method, the following were unambiguously demonstrated: (i) the presence in the cytoplasm of an enzymic activity oxidizing UDPglucose to UDPglucuronic acid, and (ii) that detection of the activity in whole-cell-free extracts is prevented by the presence of 'periplasmic' enzymes catalysing the degradation of the sugar nucleotides . With this method, several B . subtilis 168 mutants unable to synthesize teichuronic acid were examined . Strains inactivated in gene tuaD, whose product shares homology with UDPglucose 6-dehydrogenase and GDPmannose 6-dehydrogenase from other organisms, were shown to lack UDPglucose 6-dehydrogenase activity . Anion exchange chromatography revealed that mutants deficient in tuaD lacked a cytoplasmic UDPglucuronate pool.

Microbiology, 1999 May, 145 ( Pt 5), 1043 - 8
Real-time monitoring of Bacillus subtilis endospore components by attenuated total reflection Fourier-transform infrared spectroscopy during germination; Cheung HY et al.; Chemical changes of particular Bacillus subtilis spore components were monitored by attenuated total reflection Fourier-transform infrared spectroscopy (ATR/FTIR) during spore germination on a ZnSe internal reflection element . Within minutes of the initiation of spore germination, significant changes in the amount of calcium dipicolinate (DPA-Ca) and proteins were noted in the wild-type strain . The changes in a germination mutant (strain 1G9, gerD) were similar to those in the wild-type strain, but the rates of change were slower . The changes in another germination mutant (strain 1G7, gerA) were very different from those in the first two strains: germination was slow and incomplete, and proteins and DPA-Ca remained unaltered throughout the course of the germination study . This technique thus offers a sensitive and non-destructive method for real-time monitoring of various cellular components during spore germination.

Microbiology, 1999 May, 145 ( Pt 5), 1033 - 41
Structural analysis of Bacillus megaterium KM spore peptidoglycan and its dynamics during germination; Atrih A et al.; The composition and structure of peptidoglycan from dormant spores of Bacillus megaterium KM and its dynamics during germination were investigated . Amino acid analysis and mass spectrometry identified 21 muropeptides resolved by reverse phase HPLC following digestion of peptidoglycan with Cellosyl . The basic structure of peptidoglycan in B . megaterium spores is similar to that of Bacillus subtilis: 44.2% of muramic acid residues are substituted with delta-lactam, 28.8% with single L-alanine, 25.1% with tetrapeptide and only 1.8% with tripeptide . The cross-linking index of the spore peptidoglycan, determined from muropeptides resolved by reverse phase HPLC, was 2.2 % per muramic acid . Spore peptidoglycan contains 2.9% of muropeptides with unsubstituted N-acetylmuramic acid . These muropeptides are likely to be intermediate products of delta-lactam formation . Analysis of muropeptide dynamics during germination revealed the activity of at least two hydrolytic enzymes, an N-acetylglucosaminidase and a lytic transglycosylase . A 4 M LiCl extract from 30 min germinated spores of B . megaterium KM caused 'germination-like' changes to permeabilized spores of B . megaterium and B . subtilis but not those of a B . subtilis cwlD mutant . Muropeptide analysis of the treated spores revealed the presence of products generated by the activity of a glucosaminidase.

Arch Microbiol, 1999 May-Jun, 171(6), 439 - 43
Analysis of the expression and function of the sigmaB-dependent general stress regulon of Bacillus subtilis during slow growth; Schweder T et al.; Glucose-limited continuous cultures were used to analyze sigmaB activity at decreasing growth rates . Expression of the sigmaB-dependent genes gsiB and ctc started to increase at a growth rate of 0.2 h-1, and both genes were induced approximately fivefold at a growth rate of 0.1 h-1 as compared to expression at the maximal growth rate . However, maximal sigmaB activity was only reached when the growth stopped as a result of the exhaustion of the carbon and energy source glucose . During glucose-limited growth, increased expression of the general stress regulon at growth rates below 0.2 h-1 did not provide wild-type cells with a growth advantage over sigB mutants . Instead, expression of the stress regulon seems to constitute a significant burden during glucose-limited growth, resulting in a selective growth advantage of the sigB mutant as compared to the wild-type at a growth rate of 0.08 h-1.

J Mol Biol, 1999 Jun 18, 289(4), 1003 - 16
Regulatory features of the trp operon and the crystal structure of the trp RNA-binding attenuation protein from Bacillus stearothermophilus; Chen X et al.; Characterization of both the cis and trans -acting regulatory elements indicates that the Bacillus stearothermophilustrp operon is regulated by an attenuation mechanism similar to that which controls the trp operon in Bacillus subtilis . Secondary structure predictions indicate that the leader region of the trp mRNA is capable of folding into terminator and anti- terminator RNA structures . B . stearothermophilus also encodes an RNA-binding protein with 77% sequence identity with the RNA-binding protein (TRAP) that regulates attenuation in B . subtilis . The X-ray structure of this protein has been determined in complex with L-tryptophan at 2.5 A resolution . Like the B . subtilis protein, B . stearothermophilus TRAP has 11 subunits arranged in a ring-like structure . The central cavities in these two structures have different sizes and opposite charge distributions, and packing within the B . stearothermophilus TRAP crystal form does not generate the head-to-head dimers seen in the B . subtilis protein, suggesting that neither of these properties is functionally important . However, the mode of L-tryptophan binding and the proposed RNA binding surfaces are similar, indicating that both proteins are activated by l -tryptophan and bind RNA in essentially the same way . As expected, the TRAP:RNA complex from B . stearothermophilus is significantly more thermostable than that from B . subtilis, with optimal binding occurring at 70 degrees C .

Prikl Biokhim Mikrobiol, 1999 Mar-Apr, 35(2), 150 - 4
{Serine protease of Bacillus subtilis R}; Grebeshova RN et al.; Properties of a protease preparation obtained by ethanol precipitation of a concentrate of the culture fluid of Bacillus subtilis R (1 : 4 v/v; 4 degrees C) grown under the conditions of deep cultivation were studied . The use of specific inhibitors, EDTA and phenylmethylsulfonyl fluoride, made it possible to show that the enzyme belongs to the group of serine proteases . The preparation exhibited high stability in alkaline medium and thermostability; it hydrolyzed protein substrates and retained catalytic properties in the presence of a multicomponent detergent system . The preparation is recommended for use in those branches of industry where proteolysis is required and in the production of detergents (as a biological additive).

Mikrobiologiia, 1999 Jan-Feb, 68(1), 45 - 50
{Cross-action of extracellular stress adaptation factors in microorganisms}; Nikolaev IuA et al.; The capacity of microorganisms from different taxa to adapt to stress conditions with the help of extracellular factors exhibiting similar mechanisms of action was demonstrated . The action of adaptation factors synthesized by the enteric bacterium Escherichia coli, the soil bacterium Bacillus subtilis, and the yeast Candida utilis was characterized in biological tests . It was demonstrated that the factor of accelerated adaptation to new media (FAANM) and the growth-rate-reducing factor (factor X(II)), which decreases the rate of exponential culture growth, were synthesized by all microorganisms tested . Antilysin (factor X(I)), which accelerates cell adaptation to N-ethylmaleimide, was not produced by C . utilis.

Genetika, 1999 Mar, 35(2), 409 - 11
{Analysis of an operator-like structure, regulating the activity of the ribC gene in Bacillus subtilis}; Kreneva RA et al.; A point mutation (C-->A substitution) in the -35 region of a putative promoter-operator site TTGCCG-17n-TACATT results in a more than 25-fold increase in the activity of ribC gene encoding the bifunctional enzyme--flavokinase/FAD-synthase--in Bacillus subtilis.

J Bacteriol, 1999 Jun, 181(12), 3632 - 43
A Bacillus subtilis secreted protein with a role in endospore coat assembly and function; Serrano M et al.; Bacterial endospores are encased in a complex protein coat, which confers protection against noxious chemicals and influences the germination response . In Bacillus subtilis, over 20 polypeptides are organized into an amorphous undercoat, a lamellar lightly staining inner structure, and an electron-dense outer coat . Here we report on the identification of a polypeptide of about 30 kDa required for proper coat assembly, which was extracted from spores of a gerE mutant . The N-terminal sequence of this polypeptide matched the deduced product of the tasA gene, after removal of a putative 27-residue signal peptide, and TasA was immunologically detected in material extracted from purified spores . Remarkably, deletion of tasA results in the production of asymmetric spores that accumulate misassembled material in one pole and have a greatly expanded undercoat and an altered outer coat structure . Moreover, we found that tasA and gerE mutations act synergistically to decrease the efficiency of spore germination . We show that tasA is the most distal member of a three-gene operon, which also encodes the type I signal peptidase SipW . Expression of the tasA operon is enhanced 2 h after the onset of sporulation, under the control of sigmaH . When tasA transcription is uncoupled from sipW expression, a presumptive TasA precursor accumulates, suggesting that its maturation depends on SipW . Mature TasA is found in supernatants of sporulating cultures and intracellularly from 2 h of sporulation onward . We suggest that, at an early stage of sporulation, TasA is secreted to the septal compartment . Later, after engulfment of the prespore by the mother cell, TasA acts from the septal-proximal pole of the spore membranes to nucleate the organization of the undercoat region . TasA is the first example of a polypeptide involved in coat assembly whose production is not mother cell specific but rather precedes its formation . Our results implicate secretion as a mechanism to target individual proteins to specific cellular locations during the assembly of the bacterial endospore coat.

Plasmid, 1999 May, 41(3), 179 - 86
Analysis of the requirement for a pUB110 mob region during Tn916-dependent mobilization; Showsh SA et al.; Tn916-dependent mobilization of nonconjugative plasmids pUB110 and its derivative pUB110Deltam was compared . Deleting a 787-bp fragment from the pUB110 mob region created plasmid pUB110Deltam . Deletion of the mob region of pUB110 rendered the plasmid nontransferable by the conjugative plasmids of Bacillus thuringiensis subsp . israelensis . During matings between Bacillus subtilis (Tn916) and B . thuringiensis subsp . israelensis, however, Tn916-dependent mobilization of plasmids pUB110 and pUB110Deltam was observed at a frequency of approximately 2 x 10(-6) transconjugants per donor . The results show that Tn916-mediated conjugal transfer of plasmids is a mob-independent event . Jaworski and Clewell (J . Bacteriol 177; 6644-6651) recently demonstrated the presence of an IncP-like nicking site in the oriT of Tn916 . These data suggest that a IncP-like nickling site is essential for Tn916-mediated plasmid transfer .

J Bacteriol, 1999 Jun, 181(12), 3860 - 3
Genotype, phenotype, and protein structure in a regulator of sporulation: effects of mutations in the spoIIAA gene of Bacillus subtilis; Barilla D et al.; SpoIIAA, a phosphorylatable protein, is essential to the regulation of sigmaF, the first sporulation-specific transcription factor of Bacillus subtilis . The solution structure of SpoIIAA has recently been published . Here we examine four mutant SpoIIAA proteins and correlate their properties with the phenotypes of the corresponding B . subtilis mutant strains . Two of the mutations severely disrupted the structure of the protein, a third greatly diminished the rate of its phosphorylation and abolished dephosphorylation, and the fourth left phosphorylation unaffected but reduced the rate of dephosphorylation about 10-fold.

J Bacteriol, 1999 Jun, 181(12), 3810 - 5
A role for a highly conserved protein of unknown function in regulation of Bacillus subtilis purA by the purine repressor; Rappu P et al.; Regulation of the purine biosynthetic gene purA was examined by using a transcriptional fusion to a luciferase reporter gene . Transcription was repressed about 10-fold by the addition of adenine and increased approximately 4.5-fold by the addition of guanosine . This regulation is mediated by a purine repressor (PurR) . In a purR mutant, basal expression was increased 10-fold, and there was no further stimulation by guanosine or repression by adenine . An open reading frame, yabJ, immediately downstream from purR was found to have a role in the repression of purA by adenine . Repression by adenine was perturbed in a purR+ yabJ mutant, although guanosine regulation was retained . Mutations in the PurR PRPP binding motif abolished guanosine regulation in the yabJ mutant . Thus, PRPP appears to be required for upregulation by guanosine . The amino acid sequence of YabJ is homologous to the YER057c/YjgF protein family of unknown function.

J Bacteriol, 1999 Jun, 181(12), 3792 - 802
An intracellular iron chelator pleiotropically suppresses enzymatic and growth defects of superoxide dismutase-deficient Escherichia coli; Maringanti S et al.; Mutants of Escherichia coli that lack cytoplasmic superoxide dismutase (SOD) exhibit auxotrophies for sulfur-containing, branched-chain, and aromatic amino acids and cannot catabolize nonfermentable carbon sources . A secondary-site mutation substantially relieved all of these growth defects . The requirement for fermentable carbon and the branched-chain auxotrophy occur because superoxide (O2-) leaches iron from the {4Fe-4S} clusters of a family of dehydratases, thereby inactivating them; the suppression of these phenotypes was mediated by the restoration of activity to these dehydratases, evidently without changing the intracellular concentration of O2- . Cloning, complementation, and sequence analysis identified the suppressor mutation to be in dapD, which encodes tetrahydrodipicolinate succinylase, an enzyme involved in diaminopimelate and lysine biosynthesis . A block in dapB, which encodes dihydrodipicolinate reductase in the same pathway, conferred similar protection . Genetic analysis indicated that the protection stems from the intracellular accumulation of tetrahydro- or dihydrodipicolinate . Heterologous expression in the SOD mutants of the dipicolinate synthase of Bacillus subtilis generated dipicolinate and similarly protected them . Dipicolinates are excellent iron chelators, and their accumulation in the cell triggered derepression of the Fur regulon and a large increase in the intracellular pool of free iron, presumably as a dipicolinate chelate . A fur mutation only partially relieved the auxotrophies, indicating that Fur derepression assists but is not sufficient for suppression . It seems plausible that the abundant internal iron permits efficient reactivation of superoxide-damaged iron-sulfur clusters . This result provides circumstantial evidence that the sulfur and aromatic auxotrophies of SOD mutants are also directly or indirectly linked to iron metabolism.

J Bacteriol, 1999 Jun, 181(12), 3666 - 73
Role in cell permeability of an essential two-component system in Staphylococcus aureus; Martin PK et al.; A temperature-sensitive lethal mutant of Staphylococcus aureus was found to harbor a mutation in the uncharacterized two-component histidine kinase (HK)-response regulator (RR) pair encoded by yycFG; orthologues of yycFG could be identified in the genomes of Bacillus subtilis and other gram-positive bacteria . Sequence analysis of the mutant revealed a point mutation resulting in a nonconservative change (Glu to Lys) in the regulator domain of the RR at position 63 . To confirm that this signal transduction system was essential, a disrupted copy of either the RR (yycF) or the HK (yycG) was constructed with a set of suicide vectors and used to generate tandem duplications in the chromosome . Resolution of the duplications, leaving an insertion in either the yycF or the yycG coding region, was achieved only in the presence of an additional wild-type copy of the two open reading frames . Phenotypic characterization of the conditional lethal mutant showed that at permissive growth conditions, the mutant was hypersusceptible to macrolide and lincosamide antibiotics, even in the presence of the ermB resistance determinant . Other mutant phenotypes, including hypersensitivity to unsaturated long-chain fatty acids and suppression of the conditional lethal phenotype by high sucrose and NaCl concentrations, suggest that the role of the two-component system includes the proper regulation of bacterial cell wall or membrane composition . The effects of this point mutation are strongly bactericidal at the nonpermissive temperature, indicating that this pathway provides an excellent target for the identification of novel antibiotics.

Biochim Biophys Acta, 1999 Jun 9, 1419(1), 97 - 104
Homeostasis of the membrane proton permeability in Bacillus subtilis grown at different temperatures; van de Vossenberg JL et al.; Bacillus subtilis was grown at its growth temperature limits and at various temperatures in between the lower and upper growth temperature boundary . Liposomes were made of the extracted membrane lipids derived from these cells . The headgroup composition of the cytoplasmic membrane lipids did not differ significantly at the lower (13 degrees C) and upper (50 degrees C) temperature boundary . The averaged lipid acyl chain length, degree of saturation, and ratio of iso- and anteiso-branched fatty acids increased with the temperature . At the temperature of growth, the membranes were in a liquid-crystalline phase, but liposomes derived from cells grown at 13 degrees C were almost threefold more viscous than those derived from 50 degrees C grown cells . The temperature dependence of the proton permeability of the liposomes was determined using the acid-pulse method with monitoring of the outside pH with the fluorescent probe pyranine . The proton permeability of each liposome preparation increased with the temperature . However, the proton permeability of the liposomes at the growth temperature of the cells from which the lipids were derived was almost constant . These data indicate that the growth temperature dependent variation in lipid acyl chain composition permits maintenance of the proton permeability of the cytoplasmic membrane . This 'homeo-proton permeability adaptation' precludes futile cycling of protons at higher growth temperatures and allows cells to sustain the proton motive force as a driving force for essential energy transducing processes.

Plasmid, 1999 May, 41(3), 274 - 81
Complete sequence of Bacillus subtilis plasmid p1414 and comparison with seven other plasmid types found in Russian soil isolates of Bacillus subtilis; Thorsted PB et al.; We determined the complete sequence of a cryptic 7949-bp plasmid isolated from naturally occurring Bacillus subtilis found in Russian soil from Moscow . We found 15 putative open reading frames (ORFs), all of which were preceded by a ribosome binding site . One encodes the gene (rep) which should be essential for vegetative rolling circle replication (RCR) . The putative double-stranded origin as well as a palT1-like single-stranded origin was also identified . The predicted product of another ORF showed similarity to a moblization protein while a third showed similarity to a ubiquitous family of small proteins whose members have so far been associated with stress response . We used fragments with these latter ORFs to probe representatives of seven other groups of cryptic RCR plasmids from geographically related B . subtilis isolates . All plasmids carried the mob function, suggesting a common ancestor for the rep/mob region but the putative hsp was present only on some of the plasmids . This suggests that the putative hsp gene is not an essential plasmid component and may therefore be present as a phenotypic marker-perhaps providing response to stress . This adds weight to the growing evidence that these small Bacillus plasmids may not be cryptic but may provide an adaptive advantage for the host in its natural environment .

J Ethnopharmacol, 1999 Mar, 64(3), 277 - 82
Antibacterial and antifungal activity of small protein of Indigofera oblongifolia leaves; Dahot MU; Four fractions from the leaves of Indigofera oblongifolia were obtained on Sephadex G-25 column chromatography . A single bands of these fractions were detected on Poly-acrylamide SDS gel electrophoresis . An antibacterial action of small protein peptide was tested against Escherichia coli, Klebsiella aerogenes, Kl . pneumoniae . Staphyllococcus aureus, and Bacillus subtilis . Peptide 3 showed strong inhibitory activity against B . subtilis and Aspergillus niger but clear zone of inhibition was also noted against A . fumigatus . Peptide 4 showed significant inhibitory zone against Kl . pneumoniae, S . aureus, B . subtilis . A . fumigatus, A . niger and A . flavus.

J Ethnopharmacol, 1999 Mar, 64(3), 241 - 8
Antimicrobial properties of alkamides present in flavouring plants traditionally used in Mesoamerica: affinin and capsaicin; Molina-Torres J et al.; The bioactive amides affinin and capsaicin isolated respectively from Heliopsis longipes roots and Capsicum spp fruits, were assayed for activity against Escherichia coli, Pseudomonas solanacearum, Bacillus subtilis and Saccharomyces cerevisicae suspension cultures . The alkamide affinin inhibited growth of E . coli and S . cerevisiae at concentrations as low as 25 microg/ml . Higher concentrations of affinin were necessary to inhibit growth of P . solanacearum and B . subtilis . However . high concentrations of capsaicin only retarded the growth of E . coli and P . solanacearum, whereas growth of B . subtilis was strongly inhibited and that of S . cerevisiae was initially enhanced . Results are discussed in relation to previous reports concerning crude extract and to the molecular structures of the bioactive compounds.

J Biotechnol, 1999 Apr 15, 69(2-3), 203 - 14
The isolation of strains of Saccharomyces cerevisiae showing altered plasmid stability characteristics by means of selective continuous culture; O'Kennedy RD et al.; A recombinant strain of Saccharomyces cerevisiae containing a plasmid-encoded lacZ gene from Escherichia coli was grown for 420 generations under selective conditions in glucose-limited continuous culture . A ura3-based auxotrophic system was used to apply selection in favour of plasmid-containing organisms . A similar strategy had previously proved successful at evolving clones of Bacillus subtilis, showing improved plasmid stability characteristics . In this study a series of clones were isolated which exhibited large variation in their ability to retain the recombinant plasmid . Clones showed both significantly increased and reduced capacity to maintain the recombinant plasmid . The probabilities of obtaining clones in either category were essentially equal so that selection was not seen to enrich for more stable clones . Periodic selection events appeared to exert a greater influence on the distribution of stability characteristics amongst clones than did the applied selective pressure . Alterations in plasmid retention characteristics could be associated with host or plasmid . The most stable clone isolated exhibited a approximately 30% improvement of its overall stability (sigma(N+)) and an 80% improvement in productivity, when compared to the parental strain CGpLG . This improved stability was associated with alterations in the plasmid genome.

Mol Microbiol, 1999 May, 32(4), 799 - 812
Mutational analysis of ComS: evidence for the interaction of ComS and MecA in the regulation of competence development in Bacillus subtilis; Ogura M et al.; The development of Bacillus subtilis genetic competence is a highly regulated adaptive response to stationary-phase stress . A key step in competence development is the activation of the transcriptional regulator ComK, which is required for the expression of genes encoding the products that function in DNA uptake . In log-phase cultures, ComK is trapped in a complex composed of MecA and ClpC, in which it is rendered inactive . The comS gene, contained within the srf operon, is induced in response to high culture cell density and nutritional stress . Its product functions to release active ComK from the complex, allowing ComK to stimulate the transcription initiation of its own gene as well as that of the late competence operons . Western analysis showed that ComS accumulates to maximal levels between T3 and T4, mirroring the pattern of competence cell development and late competence gene expression . Experiments to examine the target of ComS activity in vitro showed that ComS binds to MecA . This is further supported by coimmunoprecipitation using anti-MecA antiserum . To clarify the role of ComS in competence regulation, a system for evaluating the effect of comS and mutant derivatives on the expression of comG, one of the late competence operons, was constructed . comS mutations, created by alanine-scanning mutagenesis, that significantly reduced comG-lacZ expression were clustered within two regions, one at the N-terminus and the other at the C-terminus of ComS . ComSI13 --> A and ComSW43 --> A were selected for further analysis as representative mutants for both regions required for ComS activity . We observed that ComSI13 --> A showed significantly reduced affinity for MecA, whereas ComSW43 --> A showed near normal binding affinity for MecA . The results show that binding to MecA is critical for ComS function, but do not rule out the possibility that ComS possesses other activities.

Biochem J, 1999 Jun 15, 340 ( Pt 3), 753 - 8
Characterization of a novel spermidine/spermine acetyltransferase, BltD, from Bacillus subtilis; Woolridge DP et al.; Overexpression of the BltD gene in Bacillus subtilis causes acetylation of the polyamines spermidine and spermine . BltD is co-regulated with another gene, Blt, which encodes a multidrug export protein whose overexpression facilitates spermidine export {Woolridge, Vazquez-Laslop, Markham, Chevalier, Gerner and Neyfakh (1997) J . Biol . Chem . 272, 8864-8866} . Here we show that BltD acetylates both spermidine and spermine at primary propyl amine moieties, with spermine being the preferred substrate . In the presence of saturating concentrations of acetyl CoA, BltD rapidly acetylates spermine at both the N1 and N12 positions . The Km (app) values for spermine, spermidine and N1-acetylspermine are </=67, 200 and 1200 microM, respectively . Diamines ranging from 1, 3-diaminopropane to 1,12-diaminododecane, monoacetylputrescine and N8-acetylspermidine were not substrates for BltD . Putrescine (1, 4-diaminobutane) and N8-acetylspermidine were competitive inhibitors of spermidine acetylation by BltD, with Ki values of 0.25 and 5.76 mM, respectively . CoA competitively inhibited both spermidine and acetyl-CoA interactions with BltD . These data and other results indicate that the mechanism of spermidine and spermine acetylation by BltD is a random-order mechanism of bi-molecular kinetics.

Extremophiles, 1999 May, 3(2), 113 - 20
Sequence analysis and functional studies of a chromosomal region of alkaliphilic Bacillus firmus OF4 encoding an ABC-type transporter with similarity of sequence and Na+ exclusion capacity to the Bacillus subtilis NatAB transporter; Wei Y et al.; A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridization to a probe from a previously sequenced alkaliphile homolog of the natA gene from Bacillus subtilis . Sequence analysis of the entire fragment revealed that, as in B . subtilis, the natA gene was part of a putative gene locus encoding an ABC-type transporter . In the alkaliphile, the transporter involved three genes, designated natCAB, that are part of a larger operon of unknown function . This is in contrast to the two-gene natAB operon and to another homolog from B . subtilis, the yhaQP genes . Like natAB, however, the alkaliphile natCAB catalyzes Na+ extrusion as assessed in a mutant of Escherichia coli that is deficient in Na+ extrusion . The full 14.1-kb fragment of alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units . Among the 17 predicted ORFs apart from natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malK, an ATP-binding component that was unaccompanied by candidates for other mal transport genes but was able to complement a malK-deficient mutant of E . coli . No strong candidates for genes encoding a secondary Na+/H+ antiporter were found in the fragment, either from the sequence analysis or from analyses of complementation of E . coli mutants by subclones of the 14.1-kb piece . There were a total of 12 ORFs whose closest and significant homologs were genes from B . subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B . subtilis homologs.

J Med Chem, 1999 Jun 3, 42(11), 2035 - 40
6-Anilinouracil-based inhibitors of Bacillus subtilis DNA polymerase III: antipolymerase and antimicrobial structure-activity relationships based on substitution at uracil N3; Tarantino PM Jr et al.; 6-Anilinouracils (6-AUs) are dGTP analogues which selectively inhibit the DNA polymerase III of Bacillus subtilis and other Gram-positive bacteria . To enhance the potential of the 6-AUs as antimicrobial agents, a structure-activity relationship was developed involving substitutions of the uracil N3 position in two 6-AU platforms: 6-(3,4-trimethyleneanilino)uracil (TMAU) and 6-(3-ethyl-4-methylanilino)uracil (EMAU) . Series of N3-alkyl derivatives of both 6-AUs were synthesized and tested for their ability to inhibit purified B . subtilis DNA polymerase III and the growth of B . subtilis in culture . Alkyl groups ranging in size from ethyl to hexyl enhanced the capacity of both platforms to bind to the polymerase, and with the exception of hexyl, they also significantly enhanced their antimicrobial potency . N3 substitution of the EMAU platform with more hydrophilic hydroxyalkyl and methoxyalkyl groups marginally enhanced anti-polymerase III activity but enhanced antibacterial potency severalfold . In sum, the results of these studies indicate that the ring N3 of 6-anilinouracils can tolerate substituents of considerable size and structural variety and, thus, can be manipulated to significantly enhance the antibacterial potency of this novel class of polymerase III-specific inhibitors.

Trends Microbiol, 1999 May, 7(5), 201 - 7
When the going gets tough: survival strategies and environmental signaling networks in Bacillus subtilis; Msadek T; Regulatory pathways involving two-component histidine kinase/response regulator proteins of Bacillus subtilis are highly interconnected and form a signal transduction network controlling stationary-phase adaptive responses . These include chemotaxis and motility, degradative enzyme synthesis, antibiotic production, natural competence for DNA uptake, and sporulation . Many of these responses are mutually exclusive, with different control levels involving protein-environment, protein-protein and protein-DNA interactions, allowing the bacteria to adapt rapidly to environmental changes.

Mycopathologia, 1998-99, 143(3), 161 - 4
Viability studies on actinomycetes; Taddei A et al.; Eighty-nine Actinomycetes strains were tested for their viability, morphological and physiological characteristics after being kept under paraffin oil overlay and distilled water for a period between 10-30 years . Most of the studied strains belong to the "Lorenzo De Montemayor" collection . Almost all the recovered strains were 28-30 years old and had never been subcultured since the paraffin oil was overlaid . 71.4% of viable Streptomycetes strains had been kept on Sabouraud-dextrose agar and 28.6% were kept on Negroni and Bonfiglioli-medium . Streptomyces violaceusruber produced its characteristic pigment even after 28 years under these conditions . All of the recovered strains were tested for their biological activity, but only Streptomyces lavendulae showed growth-inhibition against Staphylococcus aureus and Bacillus subtilis.

Biochemistry, 1999 May 18, 38(20), 6460 - 70
Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 A resolution; Chiu HJ et al.; The crystal structure of Bacillus subtilis thiamin phosphate synthase complexed with the reaction products thiamin phosphate and pyrophosphate has been determined by multiwavelength anomalous diffraction phasing techniques and refined to 1.25 A resolution . Thiamin phosphate synthase is an alpha/beta protein with a triosephosphate isomerase fold . The active site is in a pocket formed primarily by the loop regions, residues 59-67 (A loop, joining alpha3 and beta2), residues 109-114 (B loop, joining alpha5 and beta4), and residues 151-168 (C loop, joining alpha7 and beta6) . The high-resolution structure of thiamin phosphate synthase complexed with its reaction products described here provides a detailed picture of the catalytically important interactions between the enzyme and the substrates . The structure and other mechanistic studies are consistent with a reaction mechanism involving the ionization of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate at the active site to give the pyrimidine carbocation . Trapping of the carbocation by the thiazole followed by product dissociation completes the reaction . The ionization step is catalyzed by orienting the C-O bond perpendicular to the plane of the pyrimidine, by hydrogen bonding between the C4' amino group and one of the terminal oxygen atoms of the pyrophosphate, and by extensive hydrogen bonding and electrostatic interactions between the pyrophosphate and the enzyme.

Biochemistry, 1999 May 18, 38(20), 6380 - 5
Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms; Charnock SJ et al.; The enzymatic formation of glycosidic bonds may be catalyzed by the transfer of the glycosyl moiety from an activated nucleotide-diphospho-sugar donor to a specific acceptor . SpsA is a glycosyltransferase implicated in the synthesis of the spore coat of Bacillus subtilis, whose homologues include cellulose synthase and many lipopolysaccharide and bacterial O-antigen synthases . The three-dimensional crystal structure of SpsA has been determined by conventional MIR techniques at a resolution of 1.5 A . It is a two-domain protein with a nucleotide-binding domain together with an acceptor binding domain which features a disordered loop spanning the active site . The structures of SpsA in complex with both Mg-UDP and Mn-UDP have also been determined at 2.0 and 1.7 A, respectively . These complexes, together with the sequence conservation, begin to shed light on the mechanism of this ubiquitous family of inverting glycosyltransferases.

J Bacteriol, 1999 Jun, 181(11), 3392 - 401
Role of SpoVG in asymmetric septation in Bacillus subtilis; Matsuno K et al.; Deletion of the citC gene, coding for isocitrate dehydrogenase, arrests sporulation of Bacillus subtilis at stage I after bipolar localization of the cell division protein FtsZ but before formation of the asymmetric septum . A spontaneous extragenic suppressor mutation that overcame the stage I block was found to map within the spoVG gene . The suppressing mutation and other spoVG loss-of-function mutations enabled citC mutant cells to form asymmetric septa and to activate the forespore-specific sigma factor sigmaF . However, little induction of mother cell-specific, sigmaE-dependent sporulation genes was observed in a citC spoVG double mutant, indicating that there is an additional defect(s) in compartmentalized gene expression in the citC mutant . These other defects could be partially overcome by reducing the synthesis of citrate, by buffering the medium, or by adding excess MnCl2 . Overexpression of the spoVG gene in wild-type cells significantly delayed sigmaF activation . Increased expression and stability of SpoVG in citC mutant cells may contribute to the citC mutant phenotype . Inactivation of the spoVG gene caused a population of otherwise wild-type cells to produce a small number of minicells during growth and caused sporulating cells to complete asymmetric septation more rapidly than normal . Unlike the case for inactivation of the cell division inhibitor gene minD, many of these minicells contained DNA and appeared only when the primary sporulation signal transduction pathway, the Spo0A phosphorelay, was active . These results suggest that SpoVG interferes with or is a negative regulator of the pathway leading to asymmetric septation.

J Bacteriol, 1999 Jun, 181(11), 3382 - 91
Metabolic imbalance and sporulation in an isocitrate dehydrogenase mutant of Bacillus subtilis; Matsuno K et al.; A Bacillus subtilis mutant with a deletion in the citC gene, encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, exhibited reduced growth yield in broth medium and had greatly reduced ability to sporulate compared to the wild type due to a block at stage I, i.e., a failure to form the polar division septum . In early stationary phase, mutant cells accumulated intracellular and extracellular concentrations of citrate and isocitrate that were at least 15-fold higher than in wild-type cells . The growth and sporulation defects of the mutant could be partially bypassed by deletion of the major citrate synthase gene (citZ), by raising the pH of the medium, or by supplementation of the medium with certain divalent cations, suggesting that abnormal accumulation of citrate affects survival of stationary-phase cells and sporulation by lowering extracellular pH and chelating metal ions . While these genetic and environmental alterations were not sufficient to allow the majority of the mutant cell population to pass the stage I block (lack of asymmetric septum formation), introduction of the sof-1 mutant form of the Spo0A transcription factor, when coupled with a reduction in citrate synthesis, restored sporulation gene expression and spore formation nearly to wild-type levels . Thus, the primary factor inhibiting sporulation in a citC mutant is abnormally high accumulation of citrate, but relief of this metabolic defect is not by itself sufficient to restore competence for sporulation.

J Bacteriol, 1999 Jun, 181(11), 3341 - 50
Isolation and characterization of mutations in Bacillus subtilis that allow spore germination in the novel germinant D-alanine; Paidhungat M et al.; Bacillus subtilis spores break their metabolic dormancy through a process called germination . Spore germination is triggered by specific molecules called germinants, which are thought to act by binding to and stimulating spore receptors . Three homologous operons, gerA, gerB, and gerK, were previously proposed to encode germinant receptors because inactivating mutations in those genes confer a germinant-specific defect in germination . To more definitely identify genes that encode germinant receptors, we isolated mutants whose spores germinated in the novel germinant D-alanine, because such mutants would likely contain gain-of-function mutations in genes that encoded preexisting germinant receptors . Three independent mutants were isolated, and in each case the mutant phenotype was shown to result from a single dominant mutation in the gerB operon . Two of the mutations altered the gerBA gene, whereas the third affected the gerBB gene . These results suggest that gerBA and gerBB encode components of the germinant receptor . Furthermore, genetic interactions between the wild-type gerB and the mutant gerBA and gerBB alleles suggested that the germinant receptor might be a complex containing GerBA, GerBB, and probably other proteins . Thus, we propose that the gerB operon encodes at least two components of a multicomponent germinant receptor.

Lett Appl Microbiol, 1999 May, 28(5), 363 - 7
Isolation, partial purification and characterization of a bacteriocin produced by a newly isolated Bacillus subtilis strain; Zheng G et al.; A wild type micro-organism producing antibacterial substances has been isolated from a Chinese fermented soybean seasoning and identified as Bacillus subtilis . A crude antibacterial preparation (CABP) was obtained by ammonium sulphate precipitation . Isoelectric focusing assay revealed at least four antimicrobial components in the CABP . However, in SDS-PAGE analysis, only one peptide band displayed antimicrobial activity against pathogenic Bacillus cereus and Listeria monocytogenes . This inhibitory peptide had a molecular weight of approximately 3.4 kDa and a pI value of approximately 4.7 . Results of this study suggest that at least one antimicrobial substance produced by this wild type strain of B . subtilis may be a new bacteriocin . Its sensitivity to gastric peptidases and activity against the food-borne pathogens make this bacteriocin potentially useful as an antimicrobial agent in foods.

Can J Microbiol, 1998 Dec, 44(12), 1186 - 92
pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis; Daxhelet G et al.; pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis . It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter . The cat gene is preceded by a Shine-Dalgarno sequence functional in E . coli but not in B . subtilis . Deleted pGR71 plasmids were obtained in B . subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety . These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B . subtilis resistance to high chloramphenicol concentrations . CAT expression peaked at the early postexponential phas of B . subtilis growth . The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety . N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids . The cat ribosome-binding site, functional in E . coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B . subtilis 16S rRNA, explaining the production of native enzyme.

FEMS Microbiol Lett, 1999 May 15, 174(2), 265 - 72
Identification of a new locus in Listeria monocytogenes involved in cellobiose-dependent repression of hly expression; Huillet E et al.; Expression of the PrfA-controlled virulence gene hly (encoding the pore-forming cytolysin listeriolysin) is down-regulated by readily metabolized carbon sources in Listeria monocytogenes . We isolated a Tn917-insertional mutant of L . monocytogenes (strain LO28), which expressed a hemolytic phenotype in the presence of cellobiose . Using hly fusions to luxAluxB genes, we show that hly expression was derepressed in the presence of cellobiose at the transcriptional level . Surprisingly, hly expression was still repressed by glucose, as observed for the parental strain . Genetic analysis of the Tn917-flanking regions indicated that the transposon had inserted in a non-coding region located between two genes in opposite orientations . These two newly identified genes were designated orfA and mdrL . The insertion occurred immediately upstream of orfA, likely into its promoter region . Transcriptional analysis of orfA and mdrL revealed that Tn917 had abolished orfA expression whereas it had activated expression of mdrL . orfA encodes a putative protein of 176 amino acids homologous to YfiO of Bacillus subtilis (28% identity), a protein of unknown function . mdrL codes for a putative protein of 398 amino acids homologous to Bmr and Blt of B . subtilis (21-24% identity), two members of the multidrug resistance efflux pump family . Our results indicate that we have identified a new locus which plays a crucial role in the cellobiose-dependent repression of hly expression.

Infect Immun, 1999 Jun, 67(6), 2964 - 8
Structures in Bacillus subtilis are recognized by CD14 in a lipopolysaccharide binding protein-dependent reaction; Fan X et al.; The CD14 molecule expressed on monocytes and macrophages is a high-affinity receptor for bacterial lipopolysaccharide (LPS) and hence an important component of the innate immune system . LPS binding protein (LBP) is required to facilitate the binding of LPS to CD14 in vitro and is necessary for the induction of an inflammatory response to LPS in vivo . Here we show that CD14 and LBP can also bind to lipoteichoic acid from the gram-positive bacterium Bacillus subtilis . Although CD14 does not interact with intact B . subtilis organisms, a brief exposure of the bacteria to serum converts them into a form which can bind to CD14 in an LBP-dependent reaction . When serum-pretreated B . subtilis organisms are incubated with the myelomonocytic cell line U937, which expresses CD14, the bacteria are rapidly phagocytosed . The phagocytosis is strictly dependent both on LBP and on CD14 . These in vitro results suggest that LBP plays a role in the innate response not only to gram-negative but also to gram-positive infections.

Eur J Biochem, 1999 Jun, 262(2), 299 - 307
Overexpression, purification and characterization of Mycobacterium bovis BCG alcohol dehydrogenase; Wilkin JM et al.; A previous study of the effect of zinc deprivation on Mycobacterium bovis BCG pointed out the potential importance of an alcohol dehydrogenase for maintaining the hydrophobic character of the cell envelope . In this report, the effect of the overexpression of the M . bovis BCG alcohol dehydrogenase (ADH) in Mycobacterium smegmatis and M . bovis BCG is described . The purification of the enzyme was performed to apparent homogeneity from overexpressing M . bovis BCG cells and its kinetic parameters were determined . The enzyme showed a strong preference for both aromatic and aliphatic aldehydes while the corresponding alcohols were processed 100-1000-fold less efficiently . The best kcat/Km values were found with benzaldehyde > 3-methoxybenzaldehyde > octanal > coniferaldehyde . A phylogenetic analysis clearly revealed that the M . bovis BCG ADH together with the ADHs from Bacillus subtilis and Helicobacter pylori formed a sister group of the class C medium-chain alcohol dehydrogenases, the plant cinnamyl alcohol dehydrogenases (CADs) . Comparison of the kinetic properties of our ADH with some related class C enzymes indicated that the mycobacterial enzyme substrate profile resembled that of the CADs involved in plant defence rather than those implicated in lignification . A possible role for the M . bovis BCG ADH in the biosynthesis of the lipids composing the mycobacterial cell envelope is proposed.

J Biol Chem, 1999 May 28, 274(22), 15953 - 8
Expression, abundance, and RNA polymerase binding properties of the delta factor of Bacillus subtilis; Lopez de Saro FJ et al.; The delta protein is a dispensable subunit of Bacillus subtilis RNA polymerase (RNAP) that has major effects on the biochemical properties of the purified enzyme . In the presence of delta, RNAP displays an increased specificity of transcription, a decreased affinity for nucleic acids, and an increased efficiency of RNA synthesis because of enhanced recycling . Despite these profound effects, a strain containing a deletion of the delta gene (rpoE) is viable and shows no major alterations in gene expression . Quantitative immunoblotting experiments demonstrate that delta is present in molar excess relative to RNAP in both vegetative cells and spores . Expression of rpoE initiates from a single, sigmaA-dependent promoter and is maximal in transition phase . A rpoE mutant strain has an altered morphology and is delayed in the exit from stationary phase . For biochemical analyses we have created derivatives of delta and sigmaA that can be radiolabeled with protein kinase A . Using electrophoretic mobility shift assays, we demonstrate that delta binds core RNAP with an apparent affinity of 2.5 x 10(6) M-1, but we are unable to demonstrate the formation of a ternary complex containing core enzyme, delta, and sigmaA.

J Biol Chem, 1999 May 28, 274(22), 15865 - 8
Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase mutants; Bolhuis A et al.; The type I signal peptidase SipS of Bacillus subtilis is of major importance for the processing of secretory precursor proteins . In the present studies, we have investigated possible mechanisms of thermal inactivation of five temperature-sensitive SipS mutants . The results demonstrate that two of these mutants, L74A and Y81A, are structurally stable but strongly impaired in catalytic activity at 48 degrees C, showing the (unprecedented) involvement of the conserved leucine 74 and tyrosine 81 residues in the catalytic reaction of type I signal peptidases . This conclusion is supported by the crystal structure of the homologous signal peptidase of Escherichia coli (Paetzel, M., Dalbey, R . E., and Strynadka, N . C . J . (1998) Nature 396, 186-190) . In contrast, the SipS mutant proteins R84A, R84H, and D146A were inactivated by proteolytic degradation, indicating that the conserved arginine 84 and aspartic acid 146 residues are required to obtain a protease-resistant conformation . The cell wall-bound protease WprA was shown to be involved in the degradation of SipS D146A, which is in accord with the fact that SipS has a large extracytoplasmic domain . As WprA was not involved in the degradation of the SipS mutant proteins R84A and R84H, we conclude that multiple proteases are responsible for the thermal inactivation of temperature-sensitive SipS mutants.

Gene, 1999 May 17, 232(1), 1 - 10
Regulation of four genes encoding small, acid-soluble spore proteins in Bacillus subtilis; Cabrera-Hernandez A et al.; Three genes (sspH, sspL, and tlp) encoding new, minor small, acid-soluble proteins (SASP) unique to spores of Bacillus subtilis are expressed only in the forespore compartment during sporulation of this organism . The sspH and sspL genes are monocistronic, whereas tlp is the second gene in an operon with a second small orf, which we have termed sspN . The sspH and sspL genes are recognized primarily by the forespore-specific sigma factor for RNA polymerase, sigmaG; the sspN-tlp operon is recognized equally well by sigmaG and the other forespore-specific sigma factor, sigmaF . Sequences centered 10 and 35nt upstream of the 5'-ends of sspH, sspL, and sspN mRNAs all show homology to -10 and -35 sequences recognized by sigmaF and sigmaG, which are generally quite similar . Mutations disrupting the sspH, sspL, sspN-tlp, or tlp loci cause a loss of the appropriate SASP from spores, but have no discernible effect on sporulation, spore properties, or spore germination.

Mol Biol Evol, 1999 Mar, 16(3), 332 - 46
Evolutionary instability of operon structures disclosed by sequence comparisons of complete microbial genomes; Itoh T et al.; Gene orders have been shown to be generally unstable by comprehensive analyses in several complete genomes . In this study, we examined instability of genome structures within operons, where functionally related genes are clustered . We compared gene orders of known operons obtained from Escherichia coli and Bacillus subtilis with corresponding those of operons in 11 complete genome sequences . We found that in many cases, gene orders within operons could be shuffled frequently during evolution, although several operon structures, such as ribosomal protein operons, were well conserved . This suggests that shuffling of a genome structure is virtually neutral in long-term evolution . Moreover, degrees of instability of the operon structures depended on the genomes examined . Variation in degrees of instability of the genome structures was likely to be related to differences in amounts of insertion sequences . Effects on transcription regulation are also discussed in association with operon destruction.

Genetika, 1999 Jan, 35(1), 46 - 9
{Simultaneous presence of three cryptic plasmids of various size in a soil strain of Bacillus subtilis}; Poluektova EU et al.; The Bacillus subtilis 1387 soil strain, which contains three cryptic plasmids simultaneously, was described . Two small plasmids (6.3 and 8.5 kb) were homologous to each other, and a large plasmid (30 kb) had no homology with them . The plasmids were separately transmitted into cells of the Bac . subtilis 168 strain, and some plasmid characteristics were analyzed.

Acta Crystallogr D Biol Crystallogr, 1999 Jun, 55 ( Pt 6), 1212 - 4
Crystallization and preliminary X-ray analysis of the 6-phospho-alpha-glucosidase from Bacillus subtilis; Varrot A et al.; 6-Phospho-alpha-glucosidase (GlvA) is the protein involved in the dissimilation of alpha-glycosides accumulated via a phosphoenolpyruvate-dependent maltose phosphotransferase system (PEP-PTS) in Bacillus subtilis . The purified enzyme has been crystallized in a form suitable for X-ray diffraction analysis . Thin rod-like crystals have been grown by the hanging-drop method in the presence of manganese and NAD . They diffract beyond 2.2 A using synchrotron radiation and belong to the space group I222 (or its enantiomorph) with unit-cell dimensions a = 83.26, b = 102.56, c = 145.31 A and contain a single molecule of GlvA in the asymmetric unit.

J Mol Biol, 1999 Apr 23, 288(1), 71 - 85
The Bacillus subtilis bacteriophage SPP1 G39P delivers and activates the G40P DNA helicase upon interacting with the G38P-bound replication origin; Ayora S et al.; Initiation of Bacillus subtilis bacteriophage SPP1 replication requires the phage-encoded genes 38, 39 and 40 products (G38P, G39P and G40P) . G39P, which does not bind DNA, interacts with the replisome organiser, G38P, in the absence of ATP and with the ATP-activated hexameric replication fork helicase, G40P . G38P, which specifically interacts with the phage replication origin (oriL) DNA, does not seem to form a stable complex with G40P in solution . G39P when complexed with G40P-ATP inactivates the single-stranded DNA binding, ATPase and unwinding activities of G40P, and such effects are reversed by increasing amounts of G38P . Unwinding of a forked substrate by G40P-ATP is increased about tenfold by the addition of G38P and G39P to the reaction mixture . The specific protein-protein interactions between oriL-bound G38P and the G39P-G40P-ATPgammaS complex are necessary for helicase delivery to the SPP1 replication origin . Formation of G38P-G39P heterodimers releases G40P-ATPgammaS from the unstable oriL-G38P-G39P-G40P-ATPgammaS intermediate . G40P-ATPgammaS binds to the origin region, the uncomplexed G38P fraction remains bound to oriL, and the G38P-G39P heterodimer is lost from the complex . We demonstrate that G39P is a component of an oligomeric nucleoprotein complex which plays an important role in the initiation of SPP1 replication .

J Mol Biol, 1999 Apr 23, 288(1), 29 - 39
Threonine phosphorylation of modulator protein RsbR governs its ability to regulate a serine kinase in the environmental stress signaling pathway of Bacillus subtilis; Gaidenko TA et al.; The sigmaB transcription factor of the bacterium Bacillus subtilis controls the synthesis of over 100 general stress proteins that are induced by growth-limiting conditions . Genetic evidence suggests that RsbR modulates the phosphorylation state of the RsbS antagonist in the signaling pathway that regulates sigmaB activity in response to environmental stresses that limit growth . According to the current model, the phosphorylated RsbS antagonist is unable to complex RsbT, which is then released to initiate a signaling cascade that ultimately activates sigmaB . Here, we show that the RsbR protein itself has no kinase activity but instead stimulates RsbS phosphorylation by the RsbT serine kinase in vitro . We further show that in addition to its previously known serine kinase activity directed toward the RsbS antagonist, purified RsbT also possesses a threonine kinase activity directed toward residues 171 and 205 of the RsbR modulator . Threonine residues 171 and 205 were each found to be important for RsbR function in vivo, and phosphorylation of these residues abolished the ability of RsbR to stimulate RsbT kinase activity in vitro . These results are consistent with a model in which RsbR modulates the kinase activity of RsbT directed toward its RsbS antagonist in vivo, either specifically in response to environmental signals or as part of a feedback mechanism to prevent continued signaling .

Genes Dev, 1999 May 1, 13(9), 1156 - 67
Septation, dephosphorylation, and the activation of sigmaF during sporulation in Bacillus subtilis; King N et al.; Cell-specific activation of transcription factor sigmaF during sporulation in Bacillus subtilis requires the formation of the polar septum and the activity of a serine phosphatase (SpoIIE) located in the septum . The SpoIIE phosphatase indirectly activates sigmaF by dephosphorylating a protein (SpoIIAA-P) in the pathway that controls the activity of the transcription factor . By use of a SpoIIE-GFP fusion protein in time-course and time-lapse experiments and by direct visualization of septa in living cells, we show that SpoIIE is present in the predivisional sporangium, where it often localizes near both cell poles in structures known as E-rings . We also present evidence consistent with the view that SpoIIE is present in both progeny cells after polar division . These findings are incompatible with a model for the control of sigmaF activity in which the phosphatase is simply sequestered to one cell . Instead, we conclude that the function of SpoIIE is subject to regulation, and we present evidence that this occurs in two stages . The first stage, which involves the phosphatase function of SpoIIE, depends on the cell division protein FtsZ and could correspond to the FtsZ-dependent assembly of SpoIIE into E-rings . The second stage occurs after the dephosphorylation of SpoIIAA-P and is dependent on the later-acting, cell-division protein DivIC . Evidence based on the use of modified and mutant forms of the phosphatase protein indicates that SpoIIE blocks the capacity of unphosphorylated SpoIIAA to activate sigmaF until formation of the polar septum is completed.

Mol Gen Genet, 1999 Apr, 261(3), 582 - 8
The Bacillus subtilis htpG gene is not involved in thermal stress management; Versteeg S et al.; To study the influence of the htpG gene on thermal stress management in Bacillus subtilis, two different kinds of htpG mutation were constructed . In one case, the gene was inactivated by insertion of a cat cassette in to the coding region; htpG was thus found to be non-essential . In the second case, the htpG gene was fused to a xylose-dependent promoter, allowing expression of the gene to be controlled . In the absence of HtpG protein, recovery of cells from a heat shock at 53 degrees C was retarded, and this delay could be eliminated by overproduction of HtpG . While htpG is not involved in the development of induced thermotolerance, DnaK and GroE proteins are absolutely required . Overproduction of class I heat-shock proteins prior to shifting cells to a lethal temperature is important but not sufficient for the development of intrinsic thermotolerance . It could be shown that the HtpG protein does not act as a cellular thermometer in B . subtilis.

Mol Gen Genet, 1999 Apr, 261(3), 567 - 73
Analysis of the Bacillus subtilis recO gene: RecO forms part of the RecFLOR function; Fernandez S et al.; The deduced protein product of the Bacillus subtilis gene yqfI, which is 255 residues long, shares homology (25% identity) with the Escherichia coli RecO protein . A null allele of yqfI, when present in an otherwise Rec+ B . subtilis strain, causes cells to become highly sensitive to DNA-damaging agents, and plasmid transformation (intramolecular recombination) is reduced by 25-fold while chromosomal transformation (intermolecular recombination) is only moderately affected (2.5-fold reduction) . Therefore, the yqfI gene was renamed recO and its null allele is referred to as recO1 . The recO1 mutation was introduced into recombination-deficient strains representative of the epistatic groups alpha (recF, recR and recL strains), beta (addA5 addB72), gamma (recH342) and epsilon (recU40) . The recO mutation did not affect the sensitivity of recF, recR or recL cells to DNA-damaging agents, increased the sensitivity of recU and addAB cells and abolished the DNA repair capacity of recH cells . The recO mutation did not affect intermolecular recombination in recF, recL, recH or recU cells, but reduced (by about 9-fold) the incidence of intermolecular recombination in addAB cells . The recO mutation did not affect intramolecular recombination in the addAB, recU, recF or recL cells, but reduced it by about 75-fold in recH cells . The defects caused by the recO1 mutation can be partially suppressed by a common suppressor of the recF, recL and recR phenotypes . We therefore assigned recO to epistatic group alpha and predict that the RecO protein acts at the same stage of recombination as the RecF, RecL and RecR proteins, in a RecFLOR complex.

Mol Gen Genet, 1999 Apr, 261(3), 558 - 66
Regulation of sigmaB-dependent transcription of sigB and asp23 in two different Staphylococcus aureus strains; Gertz S et al.; The alkaline shock protein Asp23 was identified as a sigmaB-dependent protein in Staphylococcus aureus . In Bacillus subtilis, the asp23 promoter from S . aureus is regulated like other sigmaB-dependent promoters, which are strongly induced by heat and ethanol stress . However, almost no induction of asp23 expression was found after heat or ethanol stress in S . aureus MA13 grown in a synthetic medium, where the basal expression level of asp23 is high . Under the same experimental conditions the sigmaB gene itself showed a similar expression pattern: it was highly expressed in synthetic medium but not induced by heat or ethanol stress . In contrast, sigmaB activity was increased by heat stress when the cells were grown in a complex medium . The constitutive expression of sigB and sigmaB-dependent stress genes in S . aureus MA13 grown in a synthetic medium is in a sharp contrast to the regulation of sigmaB activity in B . subtilis, and needs further investigation . A deletion of 11 bp in the rsbU gene, which encodes the phosphatase that acts on RsbV (the anti-anti-sigma factor), in S . aureus NCTC 8325-4 might be responsible for the failure of heat stress to activate sigmaB in complex medium, and thus reduce the initiation of transcription at sigmaB-dependent promoters in this strain.

Curr Opin Microbiol, 1999 Apr, 2(2), 142 - 7
Regulation by proteolysis: developmental switches; Gottesman S; The energy-dependent proteases originally defined in Escherichia coli have proven to have particularly important roles in bacterial developmental systems, including sporulation in Bacillus subtilis and cell cycle in Caulobacter . Degradation of key regulatory proteins participates, with regulation of synthesis and activity of the regulators, to ensure tight control and, where required, irreversible commitment of the cell to specific developmental pathways.

Curr Opin Microbiol, 1999 Apr, 2(2), 135 - 41
Anti-sigma factors; Helmann JD; Anti-sigma factors modulate the expression of numerous regulons controlled by alternative sigma factors . Anti-sigma factors are themselves regulated by either secretion from the cell (i.e . FlgM export through the hook-basal body), sequestration by an anti-anti-sigma (i.e . phosphorylation regulated partner-switching modules), or interaction with extracytoplasmic proteins or small molecule effectors (i.e . transmembrane regulators of extracytoplasmic function sigma factors) . Recent highlights include the genetic description of the opposed sigma/anti-sigma binding surfaces; the unexpected role of FlgM in holoenzyme destabilization and the finding that folding of FlgM is coupled to sigma28 binding; the first structure determination for an anti-sigma antagonist; and the detailed dissection of two complex partner-switching modules in Bacillus subtilis.

J Bacteriol, 1999 May, 181(10), 3242 - 5
Purification, kinetic properties, and intracellular concentration of SpoIIE, an integral membrane protein that regulates sporulation in Bacillus subtilis; Lucet I et al.; SpoIIE is a bifunctional protein which controls sigmaF activation and formation of the asymmetric septum in sporulating Bacillus subtilis . The spoIIE gene of B . subtilis has now been overexpressed in Escherichia coli, and SpoIIE has been purified by anion-exchange chromatography and affinity chromatography . Kinetic studies showed that the rate of dephosphorylation of SpoIIAA-P by purified SpoIIE in vitro was 100 times greater, on a molar basis, than the rate of phosphorylation of SpoIIAA by SpoIIAB . The intracellular concentrations of SpoIIE and SpoIIAB were measured by quantitative immunoblotting between 0 and 4 h after the beginning of sporulation . The facts that these concentrations were very similar at hour 2 and that SpoIIE could be readily detected before asymmetric septation suggest that SpoIIE activity may be strongly regulated.

J Bacteriol, 1999 May, 181(10), 3201 - 11
Septal localization of penicillin-binding protein 1 in Bacillus subtilis; Pedersen LB et al.; Previous studies have shown that Bacillus subtilis cells lacking penicillin-binding protein 1 (PBP1), encoded by ponA, have a reduced growth rate in a variety of growth media and are longer, thinner, and more bent than wild-type cells . It was also recently shown that cells lacking PBP1 require increased levels of divalent cations for growth and are either unable to grow or grow as filaments in media low in Mg2+, suggesting a possible involvement of PBP1 in septum formation under these conditions . Using epitope-tagging and immunofluorescence microscopy, we have now shown that PBP1 is localized at division sites in vegetative cells of B . subtilis . In addition, we have used fluorescence and electron microscopy to show that growing ponA mutant cells display a significant septation defect, and finally by immunofluorescence microscopy we have found that while FtsZ localizes normally in most ponA mutant cells, a significant proportion of ponA mutant cells display FtsZ rings with aberrant structure or improper localization, suggesting that lack of PBP1 affects FtsZ ring stability or assembly . These results provide strong evidence that PBP1 is localized to and has an important function in the division septum in B . subtilis . This is the first example of a high-molecular-weight class A PBP that is localized to the bacterial division septum.

J Bacteriol, 1999 May, 181(10), 3178 - 84
Peptidoglycan hydrolase LytF plays a role in cell separation with CwlF during vegetative growth of Bacillus subtilis; Ohnishi R et al.; Peptidoglycan hydrolase, LytF (CwlE), was determined to be identical to YhdD (deduced cell wall binding protein) by zymography after insertional inactivation of the yhdD gene . YhdD exhibits high sequence similarity with CwlF (PapQ, LytE) and p60 of Listeria monocytogenes . The N-terminal region of YhdD has a signal sequence followed by five tandem repeated regions containing polyserine residues . The C-terminal region corresponds to the catalytic domain, because a truncated protein without the N-terminal region retained cell wall hydrolase activity . The histidine-tagged LytF protein produced in Escherichia coli cells hydrolyzed the linkage of D-gamma-glutamyl-meso-diaminopimelic acid in murein peptides, indicating that it is a D,L-endopeptidase . Northern hybridization and primer extension analyses indicated that the lytF gene was transcribed by EsigmaD RNA polymerase . Disruption of lytF led to slightly filamentous cells, and a lytF cwlF double mutant exhibited extraordinary microfiber formation, which is similar to the cell morphology of the cwlF sigD mutant.

Biochim Biophys Acta, 1999 May 12, 1418(2), 307 - 19
A study on the interactions of surfactin with phospholipid vesicles; Grau A et al.; Surfactin, an acidic lipopeptide produced by various strains of Bacillus subtilis, behaves as a very powerful biosurfactant and posses several other interesting biological activities . By means of differential scanning calorimetry and X-ray diffraction the effect of surfactin on the phase transition properties of bilayers composed of different phospholipids, including lipids forming hexagonal-HII phases, has been studied . The interactions of surfactin with phosphatidylcholine and phosphatidylglycerol seem to be optimal in the case of myristoyl acyl chains, which have a similar length to the surfactin hydrocarbon tail . Data are shown that support formation of complexes of surfactin with phospholipids . The ionized form of surfactin seems to be more deeply inserted into negatively charged bilayers when Ca2+ is present, also supporting the formation of surfactin-Ca2+ complexes . In mixtures with dielaidoylphosphatidylethanolamine, a hexagonal-HII phase forming lipid, surfactin displays a bilayer stabilizing effect . Our results are compatible with the marked amphiphilic nature of surfactin and may contribute to explain some of its interesting biological actions; for instance the formation of ion-conducting pores in membranes.

Arch Pharm Res, 1994 Dec, 17(6), 438 - 42
Antimicrobial activity of Ganoderma lucidum extract alone and in combination with some antibiotics; Yoon SY et al.; Antimicrobial activity of GL (the aqueous extract from the carpophores of Ganoderma lucidum (FR)KARST) was tested in vitro against Gram positive and Gram negative bacteria by serial broth dilution method, and the antimicrobial activity was expressed by minimal inhibitory concentration (MIC) . Among fifteen species of bacteria tested, the antimicrobial activity of GL was the most potent against Micrococcus luteus (MIC, 0.75 mg/ml) . To investigate the effects of antimicrobial combinations of GL with four kinds of antibiotics (ampicillin, cefazolin, oxytetracycline and chloramphenicol), the fractional inhibitory concentration index (FICI) was determined by checkerboard assay for each strain . The antimicrobial combinations of GL with four antibiotics resulted in additive effect in most instances, synergism in two instances, and antagonism in two instances . Synergism was observed when GL was combined with cefazolin against Bacillus subtilis and Klebsiella oxytoca.

FEMS Microbiol Lett, 1999 May 1, 174(1), 201 - 6
Assembly of the CotSA coat protein into spores requires CotS in Bacillus subtilis; Takamatsu H et al.; The CotSA protein, encoded by cotSA (ytxN) of Bacillus subtilis, was detected from the cells at 5 h after the onset of sporulation (T5) and in the spore coat of wild-type cells, but not in cotE, cotS, gerE, or cotSA mutant spores . CotSA was also detected in the sporangium at T5 to T7 but not in the sporangium at T18 of cotS mutant cells, while the incorporation of CotS into the coat was not dependent upon CotSA . These results suggested that CotSA was synthesized simultaneously with CotS during T5 to T7 of sporulation and assembled into the coat dependent upon CotS.

FEMS Microbiol Lett, 1999 May 1, 174(1), 117 - 23
The Bacillus subtilis regulator protein SpoIIE shares functional and structural similarities with eukaryotic protein phosphatases 2C; Schroeter R et al.; Dephosphorylation of SpoIIAA-P by SpoIIE is strictly dependent on the presence of the bivalent metal ions Mn2+ or Mg2+ . Replacement by Ala of one of the four Asp residues, invariant in all representatives of protein phosphatase 2C, completely abolished the SpoIIE phosphatase activity in vitro, whilst replacement of the Asp residues by another acidic amino acid, Glu, had varying effects on the activities of the resulting mutated proteins . D610E and D795E exhibited some residual activity while D628E and D745E were without enzymatic activity . The results suggest that the functional model in which metal-associated water molecules are involved in the dephosphorylation reaction catalyzed by human protein phosphatase 2C alpha can also be applied to the bacterial protein phosphatase 2C-like protein.

FEMS Microbiol Lett, 1999 May 1, 174(1), 111 - 6
Characterisation of a new operon encoding a Zur-like protein and an associated ABC zinc permease in Listeria monocytogenes; Dalet K et al.; Metal ions uptake is mainly studied for iron, as it has often been implicated in bacterial virulence . Although Listeria monocytogenes virulence is expected to be controlled by the iron availability, little is known about such an uptake and its regulation . We describe the analysis of the first operon involved in metal ions uptake in L . monocytogenes . Its three ORFs encode respectively (1) an ABC protein, likely implicated in zinc uptake, (2) a hydrophobic membrane protein, generally associated with ABC proteins and (3) a ferric uptake regulator-like protein, that we named zinc uptake regulator, as it shows strong homologies with the zinc uptake regulator, a regulator of the zinc homeostasis in Bacillus subtilis . The expression of this operon is regulated by the zinc concentration.

Gene, 1999 Apr 29, 231(1-2), 187 - 93
Bacillus subtilis sequence-independent DNA-binding and DNA-bending protein Hbsu negatively controls its own synthesis; Fernandez S et al.; Transcription of the hbs gene under vegetative growth condition is subject to repression when cells enter in late exponential phase . We have determined the sites at which transcription of the hbs gene initiates in vitro . On a supercoiled template, transcription of the hbs gene is initiated by sigmaARNAP at two overlapping hbs promoters (P1 and P3) . We have demonstrated that highly purified Hbsu protein acts as a repressor of its own synthesis . The binding of the sequence-independent DNA-binding and DNA-bending Hbsu protein does not seem to exclude sigmaARNAP from the promoters . In this report we show that Hbsu, in vitro, does not repress transcription by a mere steric hindrance on sigmaARNAP binding.

Mol Microbiol, 1999 Apr, 32(2), 223 - 32
Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!
Fisher SH.
Nitrogen metabolism genes of Bacillus subtilis are regulated by the availability of rapidly metabolizable nitrogen sources, but not by any mechanism analogous to the two-component Ntr regulatory system found in enteric bacteria . Instead, at least three regulatory proteins independently control the expression of gene products involved in nitrogen metabolism in response to nutrient availability . Genes expressed at high levels during nitrogen-limited growth are controlled by two related proteins, GlnR and TnrA, which bind to similar DNA sequences under different nutritional conditions . The TnrA protein is active only during nitrogen limitation, whereas GlnR-dependent repression occurs in cells growing with excess nitrogen . Although the nitrogen signal regulating the activity of the GlnR and TnrA proteins is not known, the wild-type glutamine synthetase protein is required for the transduction of this signal to the GlnR and TnrA proteins . Examination of GlnR- and TnrA-regulated gene expression suggests that these proteins allow the cell to adapt to growth during nitrogen-limited conditions . A third regulatory protein, CodY, controls the expression of several genes involved in nitrogen metabolism, competence and acetate metabolism in response to growth rate . The highest levels of CodY-dependent repression occur in cells growing rapidly in a medium rich in amino acids, and this regulation is relieved during the transition to nutrient-limited growth . While the synthesis of amino acid degradative enzymes in B . subtilis is substrate inducible, their expression is generally not regulated in response to nitrogen availability by GlnR and TnrA . This pattern of regulation may reflect the fact that the catabolism of amino acids produced by proteolysis during sporulation and germination provides the cell with substrates for energy production and macromolecular synthesis . As a result, expression of amino acid degradative enzymes may be regulated to ensure that high levels of these enzymes are present in sporulating cells and in dormant spores.

Appl Biochem Biotechnol, 1998 Nov-Dec, 75(2-3), 193 - 204
Functional analysis of a hybrid endoglucanase of bacterial origin having a cellulose binding domain from a fungal exoglucanase; Kim H et al.; A cellulose binding domain (CBD) of an endo-beta-1,4-glucanase (Ben) from the bacterium Bacillus subtilis BSE616 was replaced with the CBD of exoglucanase I (TexI) from the fungus Trichoderma viride HK-75 . The resultant hybrid enzyme Ben'-CBDTexI, comprising the catalytic domain (Ben') of Ben and the CBD (CBDTexI) of TexI, was highly expressed at 20% of the total protein in Escherichia coli . The molecular mass of the hybrid enzyme was estimated to be ca . 38 kDa by SDS-PAGE, which was in good agreement with that calculated from 305 amino acids of Ben and 42 amino acids of CBDTexI . The hybrid enzyme exhibited almost the same activity as that of the original Ben toward soluble substrates, such as cellooligosaccharides . The hybrid enzyme showed higher binding ability and hydrolysis activity toward microcrystalline cellulose (Avicel), even though the length of the CBD of TexI was four times smaller than that of Ben . The hybrid enzyme was more resistant to tryptic digestion than the original Ben . The efficient binding ability of the hybrid enzyme to Avicel permitted purification of the enzyme using an Avicel-affinity column to the extent of ca . 90% purity.

Res Microbiol, 1999 Apr, 150(3), 167 - 77
Properties of maltose-inducible alpha-glucosidase MalL (sucrase-isomaltase-maltase) in Bacillus subtilis: evidence for its contribution to maltodextrin utilization; Schonert S et al.; Recently, we identified the maltose inducible alpha-glucosidase MalL of Bacillus subtilis . The malL gene encodes a 561-residue protein with amino acid identities to several alpha-glucosidases and is located in a nine-gene spanning gene cluster, which is presumably organized in an operon . MalL was overproduced, purified, and its enzymatic characteristics were described in more detail . This characterization of the enzyme showed a protein stable up to 37 degrees C after temperature treatment for 15 min and exhibiting an optimal reaction temperature of 42 degrees C . Various disaccharides such as sucrose, maltose, and isomaltose were hydrolyzed with different efficiencies . MalL also hydrolyzes longer maltodextrins from maltotriose up to maltohexaose, but not maltoheptaose, palatinose, isomaltotriose, or isomaltotetraose . MalL expression is subject to both maltose induction and carbon catabolite repression . In this article, we present data demonstrating that induction of MalL expression also occurs when starch, amylose, or glycogen are present in the growth medium . The hydrolysis of these substrates by alpha-amylase presumably leads to products which, when taken up into the cytoplasm, trigger the initiation of maltose operon transcription . Furthermore, MalL expression varies temporally, showing a second induction in the stationary growth phase.

J Biol Chem, 1999 May 7, 274(19), 13569 - 76
Bacillus subtilis histone-like protein, HBsu, is an integral component of a SRP-like particle that can bind the Alu domain of small cytoplasmic RNA; Nakamura K et al.; Small cytoplasmic RNA (scRNA) is metabolically stable and abundant in Bacillus subtilis cells . Consisting of 271 nucleotides, it is structurally homologous to mammalian signal recognition particle RNA . In contrast to 4.5 S RNA of Escherichia coli, B . subtilis scRNA contains an Alu domain in addition to the evolutionarily conserved S domain . In this study, we show that a 10-kDa protein in B . subtilis cell extracts has scRNA binding activity at the Alu domain . The in vitro binding selectivity of the 10-kDa protein shows that it recognizes the higher structure of the Alu domain of scRNA caused by five consecutive complementary sequences in the two loops . Purification and subsequent analyses demonstrated that the 10-kDa protein is HBsu, which was originally identified as a member of the histone-like protein family . By constructing a HBsu-deficient B . subtilis mutant, we showed that HBsu is essential for normal growth . Immunoprecipitating cell lysates using anti-HBsu antibody yielded scRNA . Moreover, the co-precipitation of HBsu with (His)6-tagged Ffh depended on the presence of scRNA, suggesting that HBsu, Ffh, and scRNA make a ternary complex and that scRNA serves as a functional unit for binding . These results demonstrated that HBsu is the third component of a signal recognition particle-like particle in B . subtilis that can bind the Alu domain of scRNA.

Biochem Biophys Res Commun, 1999 Apr 29, 258(1), 211 - 4
Depletion of Bacillus subtilis histone-like protein, HBsu, causes defective protein translocation and induces upregulation of small cytoplasmic RNA; Yamazaki T et al.; Small cytoplasmic RNA (scRNA) is a metabolically stable homologue of mammalian SRP RNA that contains an Alu-like domain . The Bacillus subtilis histone-like protein HBsu can bind this domain . We demonstrate here that repressing the level of HBsu results in slow growth and the accumulation of precursor of beta-lactamase fusion proteins having the signal sequence of alkaline protease, penicillin binding protein 5* (PBP5*) or CGTase . The degree of the translocation defect varied among the various signal sequences tested . A pulse-chase experiment showed that processing the alpha-amylase signal sequence is significantly inhibited in HBsu-depleted cells . Northern blot analysis indicated that repressing the HBsu gene induces scRNA upregulation, indicating that the defective translocation of presecretory proteins is not due to a reduced scRNA level . The data presented here suggest that HBsu plays a pivotal role in SRP function rather than simply stabilizing the other SRP components such as scRNA .

Microbiology, 1999 Apr, 145 ( Pt 4), 869 - 80
Identification and transcriptional analysis of new members of the sigmaB regulon in Bacillus subtilis; Petersohn A et al.; Bacillus subtilis responds to various stimuli (heat, ethanol and salt stress, energy starvation) with the induction of general stress proteins (GSPs) . Most of them belong to the stress and stationary-phase regulon controlled by the alternative sigma factor sigmaB . The majority of sigmaB-dependent proteins are thought to provide a precautionary general stress resistance in stressed or starved cells . In this report, the identification and transcriptional analysis of nine new members of the sigmaB regulon are described . The biochemical function was not determined for any of the proteins encoded by the nine new sigmaB-dependent stress genes, however, similarities to proteins in the databases allowed a distinction between proteins with putative (i-iv) and unknown (v) function . The putative functions of BmrU, YcdF, YdaD, YdaP, YhdN and YocK underline the suggested protective role of sigmaB-dependent GSPs and also elucidate new areas where sigmaB might play an important role . (i) The finding that the bmrUR operon is under sigmaB control indicates that the elimination of multidrug compounds might be a new function in multiple stress resistance . (ii) YcdF and YdaD resemble NAD(P)-dependent dehydrogenases . Both proteins could be involved in the generation of NAD(P)H and therefore in the maintenance of the intracellular redox balance under stress . (iii) The ydaP gene might belong to the increasing number of sigmaB-dependent genes whose orthologues are under the control of sigmas in Escherichia coli, indicating that both regulons may fulfil similar functions . (iv) YhdN shows weak similarities to potassium ion channel proteins and YocK shows resemblance to the DnaK suppressor protein DksA . (v) Three new sigmaB-dependent genes (ydaE, ydaG and yfkM) encoding proteins with still unknown functions were also described . Further analyses of corresponding mutants might allow a first prediction of their function within the framework of the general stress regulon.

J Bacteriol, 1999 May, 181(9), 2883 - 8
trans-acting factors affecting carbon catabolite repression of the hut operon in Bacillus subtilis; Zalieckas JM et al.; In Bacillus subtilis, CcpA-dependent carbon catabolite repression (CCR) mediated at several cis-acting carbon repression elements (cre) requires the seryl-phosphorylated form of both the HPr (ptsH) and Crh (crh) proteins . During growth in minimal medium, the ptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR . Examination of the CCR of the histidine utilization (hut) enzymes in cells grown in minimal medium showed that neither the ptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant . In contrast, the ptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium . The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine . CCR is partially relieved in B . subtilis mutants which synthesize low levels of active glutamine synthetase (glnA) . In addition, these glnA mutants grow more slowly than wild-type cells in glucose minimal medium . The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein . The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.

FEBS Lett, 1999 Apr 9, 448(2-3), 235 - 8
Fate of unstable Bacillus subtilis subgenome: re-integration and amplification in the main genome; Itaya M et al.; The plastic Bacillus subtilis genome was dissected into two physically separate genomes, the 3.9 Mb main genome and the 0.3 Mb subgenome . DNA replication of the main genome was initiated from the normal replication origin (oriC) and that of the subgenome was from a 7.2 kb oriN-containing fragment artificially inserted . When the 7.2 kb fragment was shortened to a 1.5 kb fragment that contains oriN but lacks the segregational function, the subgenome became unstable and was rapidly lost from the cell, producing inviable cells due to the loss of essential genes carried by the subgenome . Stable survivors were isolated in which the subgenome had re-integrated and multiplied in the main genome . These results suggest that a reduced genetic stability of the subgenome induces size variation of the B . subtilis genome.

J Bacteriol, 1999 May, 181(9), 2966 - 9
Phosphorylation of HPr and Crh by HprK, early steps in the catabolite repression signalling pathway for the Bacillus subtilis levanase operon; Martin-Verstraete I et al.; Carbon catabolite repression (CCR) of Bacillus subtilis catabolic genes is mediated by CcpA and in part by P-Ser-HPr . For certain operons, Crh, an HPr-like protein, is also implicated in CCR . In this study we demonstrated that in ptsH1 crh1 and hprK mutants, expression of the lev operon was completely relieved from CCR and that both P-Ser-HPr and P-Ser-Crh stimulated the binding of CcpA to the cre sequence of the lev operon.

J Bacteriol, 1999 May, 181(9), 2942 - 6
Synthesis of the sigmaD protein is not sufficient to trigger expression of motility functions in Bacillus subtilis; Yang DH et al.; The gene encoding sigmaD, sigD, is transcribed from two promoter regions, the fla/che promoter region in front of the fla/che operon and PsigD directly in front of sigD . If sigmaD is translated from transcripts originating from PsigD, the cell is unable to express motility functions but synthesizes autolysins . Therefore, one function of the additional promoter is to allow the cell to express autolysins without expressing motility functions as well.

J Bacteriol, 1999 May, 181(9), 2930 - 7
Ribosomal -1 frameshifting during decoding of Bacillus subtilis cdd occurs at the sequence CGA AAG; Mejlhede N et al.; During translation of the Bacillus subtilis cdd gene, encoding cytidine deaminase (CDA), a ribosomal -1 frameshift occurs near the stop codon, resulting in a CDA subunit extended by 13 amino acids . The frequency of the frameshift is approximately 16%, and it occurs both when the cdd gene is expressed from a multicopy plasmid in Escherichia coli and when it is expressed from the chromosomal copy in B . subtilis . As a result, heterotetrameric forms of the enzyme are formed in vivo along with the dominant homotetrameric species . The different forms have approximately the same specific activity . The cdd gene was cloned in pUC19 such that the lacZ' gene of the vector followed the cdd gene in the -1 reading frame immediately after the cdd stop codon . By using site-directed mutagenesis of the cdd-lacZ' fusion, it was shown that frameshifting occurred at the sequence CGA AAG, 9 bp upstream of the in-frame cdd stop codon, and that it was stimulated by a Shine-Dalgarno-like sequence located 14 bp upstream of the shift site . The possible function of this frameshift in gene expression is discussed.

J Bacteriol, 1999 May, 181(9), 2846 - 51
The Staphylococcus aureus rsbW (orf159) gene encodes an anti-sigma factor of SigB; Miyazaki E et al.; SigB, a newly discovered alternative sigma factor of Staphylococcus aureus, has been shown to play an important role in stress responses and the regulation of virulence factors . The rsbW (orf159) gene is immediately upstream of sigB . Its gene product is homologous to Bacillus subtilis RsbW which under appropriate conditions binds to B . subtilis SigB and functions as an anti-sigma factor or negative posttranslational regulator . To define the function of S . aureus RsbW, both the S . aureus SigB and RsbW proteins were expressed in Escherichia coli and purified . Cross-linking experiments with these purified proteins revealed that RsbW was capable of specific binding to SigB . In an in vitro transcription runoff assay, RsbW prevented SigB-directed transcription from the sar P3 promoter, a known SigB-dependent promoter, and the inhibitory activity of RsbW was found to be concentration dependent . We also identified SigB promoter consensus sequences upstream of the genes encoding alkaline shock protein 23 and coagulase and have demonstrated SigB and RsbW dependence for the promoters in vitro . These results show that RsbW is a protein sequestering anti-sigma factor of S . aureus SigB and suggest that SigB activity in S . aureus is regulated posttranslationally.

J Bacteriol, 1999 May, 181(9), 2710 - 8
Membrane-bound division proteins DivIB and DivIC of Bacillus subtilis function solely through their external domains in both vegetative and sporulation division; Katis VL et al.; The Bacillus subtilis membrane-bound division proteins, DivIB and DivIC, each contain a single transmembrane segment flanked by a short cytoplasmic N-terminal domain and a larger external C-terminal domain . Both proteins become localized at the division site prior to septation . Mutagenesis of both divIB and divIC was performed whereby the sequences encoding the cytoplasmic domains were replaced by the corresponding sequence of the other gene . Finally, the cytoplasmic-plus-transmembrane-encoding domain of each protein was replaced by a totally foreign sequence not involved in division, that encodes the N-terminal-plus-transmembrane domains of the Escherichia coli TolR protein . B . subtilis strains expressing the divIB and divIC hybrids, in the absence of the wild-type gene, were viable when grown under conditions in which the wild-type genes were found previously to be essential . Furthermore, these strains were able to sporulate to near normal levels . Thus, the cytoplasmic and transmembrane segments of DivIB and DivIC do not appear to have any specific functions other than to anchor these proteins correctly in the membrane . The implications of these findings are discussed.

Acta Crystallogr D Biol Crystallogr, 1999 May, 55(5), 1098 - 100
Crystallization and preliminary crystallographic studies of Sfp: a phosphopantetheinyl transferase of modular peptide synthetases; Mofid MR et al.; The Bacillus subtilis Sfp protein is required for the non-ribosomal biosynthesis of the lipoheptapeptide antibiotic surfactin . It converts seven peptidyl carrier protein (PCP) domains of the surfactin synthetase SfrA-(A-C) to their active holo-forms by 4'-phosphopantetheinylation . The B . subtilis sfp gene was overexpressed in Escherichia coli and its gene product was purified to homogeneity and crystallized . Well diffracting single crystals were obtained from Sfp as well as from a selenomethionyl derivative, using sodium formate as a precipitant . The crystals belong to the tetragonal space group P41212/P43212, with unit-cell parameters a = b = 65.3, c = 150.5 A . They diffract beyond 2.8 A and contain one molecule in the asymmetric unit.

Microbiology, 1999 Mar, 145 ( Pt 3), 613 - 9
Kinetics of the secretion of Bacillus subtilis levanase overproduced during the exponential phase of growth; Leloup L et al.; The Bacillus subtilis levanase structural gene sacC was expressed under the regulated control of sacR, the inducible levansucrase leader region, in a degU32(Hy) strain . In this genetic context, exocellular levanase is overproduced (0.5% of total protein) during the exponential phase of growth upon induction by sucrose at 37 degrees C and pH 7 . No precursor form that comprised a signal peptide was detected in pulse-chase experiments . The subsequent release of the cell-associated processed protein is a slow event (t(1,2) = 80+/-10 s) . The unfolding-folding transition of pure levanase monitored in vitro by the resistance to proteolysis was achieved within the same time range (t(1/2) = 50 s) under the same conditions of pH and temperature . Calcium ions, which modulate the rate and the yield of refolding, have a low affinity for the protein . Comparison of these results with those obtained previously with levansucrase and alpha-amylase overproduced in the same genetic and physiological context suggests that the precursor processing is more efficient in levanase and alpha-amylase than in levansucrase . This discrepancy could lie in information borne by the signal peptide sequence of these exoproteins . However, the rate of the ultimate stage of release of these three proteins, which includes the passage through the cell wall, is correlated with the rate of folding and appears to be independent of their molecular size.

Mol Microbiol, 1999 Apr, 32(1), 203 - 16
Two evolutionarily closely related ABC transporters mediate the uptake of choline for synthesis of the osmoprotectant glycine betaine in Bacillus subtilis; Kappes RM et al.; Biosynthesis of the compatible solute glycine betaine in Bacillus subtilis confers a considerable degree of osmotic tolerance and proceeds via a two-step oxidation process of choline, with glycine betaine aldehyde as the intermediate . We have exploited the sensitivity of B . subtilis strains defective in glycine betaine production against glycine betaine aldehyde to select for mutants resistant to this toxic intermediate . These strains were also defective in choline uptake, and genetic analysis proved that two mutations affecting different genetic loci (opuB and opuC) were required for these phenotypes . Molecular analysis allowed us to demonstrate that the opuB and opuC operons each encode a binding protein-dependent ABC transport system that consists of four components . The presumed binding proteins of both ABC transporters were shown to be lipoproteins . Kinetic analysis of {14C}-choline uptake via OpuB (K(m) = 1 microM; Vmax = 21 nmol min-1 mg-1 protein) and OpuC (K(m) = 38 microM; Vmax = 75 nmol min-1 mg-1 protein) revealed that each of these ABC transporters exhibits high affinity and substantial transport capacity . Western blotting experiments with a polyclonal antiserum cross-reacting with the presumed substrate-binding proteins from both the OpuB and OpuC transporter suggested that the expression of the opuB and opuC operons is regulated in response to increasing osmolality of the growth medium . Primer extension analysis confirmed the osmotic control of opuB and allowed the identification of the promoter of this operon . The opuB and opuC operons are located close to each other on the B . subtilis chromosome, and their high sequence identity strongly suggests that these systems have evolved from a duplication event of a primordial gene cluster . Despite the close relatedness of OpuB and OpuC, these systems exhibit a striking difference in substrate specificity for osmoprotectants that would not have been predicted readily for such closely related ABC transporters.

Mol Microbiol, 1999 Apr, 32(1), 41 - 50
Sigma M, an ECF RNA polymerase sigma factor of Bacillus subtilis 168, is essential for growth and survival in high concentrations of salt; Horsburgh MJ et al.; The Bacillus subtilis 168 genome encodes seven extracytoplasmic function (ECF) RNA polymerase sigma factors of unknown physiological function . The sigM(yhdM) gene, encoding an ECF sigma factor sigma M, is essential for growth and survival in nutrient broth (NB) containing 1.4 M NaCl . Strains insertionally inactivated in the sigM gene form aberrantly shaped cells, which swell and lyse spontaneously during growth in NB medium containing increased levels (0.35-0.7 M) of a wide range of different salts . The sigM gene was co-transcribed with the yhdL and yhdK genes with transcription initiating from two promoters, PA and PM . The transcript from PM was not detected in a sigM mutant, indicating that the expression of sigM was positively autoregulated . Expression of sigM was maximal during exponential growth and was increased by 50% in NB medium containing 0.7 M NaCl . The activity of sigma M is negatively regulated by the proteins encoded by the yhdL and yhdK genes.

Biochem J, 1999 May 1, 339 ( Pt 3), 713 - 20
Purification, characterization and cDNA cloning of an endo-exonuclease from the basidiomycete fungus Armillaria mellea; Healy V et al.; We have purified an endo-exonuclease from the fruiting body of the basidiomycete fungus Armillaria mellea by using an ethanol fractionation step, followed by two rounds of column chromatography . The enzyme had an apparent molecular mass of 17500 Da and was shown to exist as a monomer by gel-filtration analysis . The nuclease was active on both double-stranded and single-stranded DNA but not on RNA . It was optimally active at pH8.5 and also exhibited a significant degree of thermostability . Three bivalent metal ions, Mg2+, Co2+ and Mn2+, acted as cofactors in the catalysis . It was also inhibited by high salt concentrations: activity was completely abolished at 150 mM NaCl . The nuclease possessed both endonuclease activity on supercoiled DNA and a 3'-5' (but not a 5'-3') exonuclease activity . It generated 5'-phosphomonoesters on its products that, after a prolonged incubation, were hydrolysed to a mixture of free mononucleotides and small oligonucleotides ranging in size from two to eight bases . Elucidation of its N-terminal amino acid sequence permitted the cDNA cloning of the A . mellea nuclease via a PCR-based approach . Peptide mapping of the purified enzyme generated patterns consistent with the amino acid sequence coded for by the cloned cDNA . A BLAST search of the SwissProt database revealed that A . mellea nuclease shared significant amino acid similarity with two nucleases from Bacillus subtilis, suggesting that the three might constitute a distinct class of nucleolytic enzymes.

Mol Microbiol, 1999 Mar, 31(6), 1665 - 79
The replication checkpoint control in Bacillus subtilis: identification of a novel RTP-binding sequence essential for the replication fork arrest after induction of the stringent response; Autret S et al.; We have shown previously that induction of the stringent response in Bacillus subtilis resulted in the arrest of chromosomal replication between 100 and 200 kb either side of oriC at distinct stop sites, designated LSTer and RSTer, left and right stringent terminators respectively . This replication checkpoint was also shown to involve the RTP protein, normally active at the chromosomal terminus . In this study, we show that the replication block is absolutely dependent upon RelA, correlated with high levels of ppGpp, but that efficient arrest at STer sites also requires RTP . DNA-DNA hybridization data indicated that one or more such LSTer sites mapped to gene yxcC (-128 kb from oriC) . A 7.75 kb fragment containing this gene was cloned into a theta replicating plasmid, and plasmid replication arrest, requiring both RelA and RTP, was demonstrated . This effect was polar, with plasmid arrest only detected when the fragment was orientated in the same direction with respect to replication, as in the chromosome . This LSTer2 site was further mapped to a 3.65 kb fragment overlapping the next40 probe . Remarkably, this fragment contains a 17 bp sequence (B'-1) showing 76% identity with an RTP binding site (B sequence) present at the chromosomal terminus . This B'-1 sequence, located in the gene yxcC, efficiently binds RTP in vitro, as shown by DNA gel retardation studies and DNase I footprinting . Importantly, precise deletion of this sequence abolished the replication arrest . We propose that this modified B site is an essential constituent of the LSTer2 site . The differences between arrest at the normal chromosomal terminus and arrest at LSTer site are discussed.

Food Addit Contam, 1998 Aug-Sep, 15(6), 651 - 60
Evaluation of a modified EC Four Plate Method to detect antimicrobial drugs; Currie D et al.; This paper describes a modification of the EC Four Plate Method based on microbial growth inhibition of Bacillus subtilis on agar medium at pH 6.0, 7.2 and 8.0 and Micrococcus luteus at pH 8.0 developed to cope with large numbers of samples . The method's performance was evaluated by determining the Minimum Inhibitory Concentrations of 66 commonly used drugs and determining the between-assay variation of antimicrobial control standards . The modified method proved particularly sensitive for beta-lactams, tetracyclines, quinolones, marcrolides and lincosamides and least sensitive for anticoccidials and nitrofurans . The pH 6.0 and 7.2 plates were more sensitive for 39 of the 66 antimicrobials (59%) whereas the two pH 8.0 plates (B . subtilis, M . luteus) were the most sensitive for 27 (41%) . Muscle samples were taken from 1830 routine meat inspection investigations between 1994 and 1996 . Of the 38 (2%) positive meat inspection carcasses, the following antimicrobials were confirmed above the MRL: penicillin G (10), oxytetracycline (16), sulphadimidine and sulphadiazine in combination (4) and chlortetracycline (1) . The method as described is technically simple, cost effective, robust, suitable for large sample throughput and for frozen, thawed or fresh tissues . When all four plates are used the pattern of inhibition can reduce unnecessary confirmatory assays by indicating the antimicrobial group most likely to be present.

J Exp Med, 1999 Apr 19, 189(8), 1275 - 84
Microbial epitopes act as altered peptide ligands to prevent experimental autoimmune encephalomyelitis; Ruiz PJ et al.; Molecular mimicry refers to structural homologies between a self-protein and a microbial protein . A major epitope of myelin basic protein (MBP), p87-99 (VHFFKNIVTPRTP), induces experimental autoimmune encephalomyelitis (EAE) . VHFFK contains the major residues for binding of this self-molecule to T cell receptor (TCR) and to the major histocompatibility complex . Peptides from papilloma virus strains containing the motif VHFFK induce EAE . A peptide from human papilloma virus type 40 (HPV 40) containing VHFFR, and one from HPV 32 containing VHFFH, prevented EAE . A sequence from Bacillus subtilis (RKVVTDFFKNIPQRI) also prevented EAE . T cell lines, producing IL-4 and specific for these microbial peptides, suppressed EAE . Thus, microbial peptides, differing from the core motif of the self-antigen, MBPp87-99, function as altered peptide ligands, and behave as TCR antagonists, in the modulation of autoimmune disease.

Novartis Found Symp, 1999, 221, 167 - 79; discussion 179-82
pH tolerance in Bacillus: alkaliphiles versus non-alkaliphiles; Krulwich TA et al.; Monovalent cation/proton antiporters that catalyse electrogenic uptake of H+ in exchange for cytoplasmic K+ and/or Na+ are centrally involved in bacterial pH homeostasis under alkaline challenge . Systematic attempts have identified some, but not yet all, of the genes encoding such antiporters that participate in pH homeostasis in the neutrophilic Bacillus subtilis and the extremely alkaliphilic Bacillus firmus OF4 . In each organism there are at least three distinct antiporters involved in pH homeostasis . They differ in cation requirement, with pH homeostasis specifically utilizing Na+/H+ antiport in the alkaliphile and using either Na+ or K+/H+ antiport in B . subtilis . Some of the antiporters involved in pH homeostasis are constitutive and are in place to respond to sudden pH shifts, but there is also an inducible component . At least two sets of homologous antiporters (NhaC and Mrp/Pha) function in both alkaliphiles and neutrophiles . An additional antiporter of a different transport protein family, the Gram-positive tetracycline-metal/H+ antiporter, is important in pH homeostasis in B . subtilis but has not yet been shown to be present in any alkaliphile . There are also differences outside of the antiporters themselves that contribute to the greater capacity of the alkaliphiles for pH homeostasis, including cation re-entry capacity and possible surface properties.

J Biol Chem, 1999 Apr 23, 274(17), 12103 - 7
Overproduction and characterization of the Bacillus subtilis anti-sigma factor FlgM; Bertero MG et al.; FlgM is an anti-sigma factor of the flagellar-specific sigma (sigma) subunit of RNA polymerase in Bacillus subtilis, and it is responsible of the coupling of late flagellar gene expression to the completion of the hook-basal body structure . We have overproduced the protein in soluble form and characterized it . FlgM forms dimers as shown by gel exclusion chromatography and native polyacrylamide gel electrophoresis and interacts in vitro with the cognate sigmaD factor . The FlgM.sigmaD complex is a stable heterodimer as demonstrated by gel exclusion chromatography, chemical cross-linking, native polyacrylamide gel electrophoresis, and isoelectric focusing . sigmaD belongs to the group of sigma factors able to bind to the promoter sequence even in the absence of core RNA polymerase . The FlgM.sigmaD complex gave a shift in a DNA mobility shift assay with a probe containing a sigmaD-dependent promoter sequence . Limited proteolysis studies indicate the presence of two structural motifs, corresponding to the N- and C-terminal regions, respectively.

Microbiology, 1999 Jan, 145 ( Pt 1), 57 - 65
Bacillus subtilis 168 gene lytF encodes a gamma-D-glutamate-meso-diaminopimelate muropeptidase expressed by the alternative vegetative sigma factor, sigmaD; Margot P et al.; A gamma-D-glutamate-meso-diaminopimelate muropeptidase was detected in the vegetative growth phase of Bacillus subtilis 168 . It is encoded by the monocistronic lytF operon expressed by the alternative vegetative sigma factor, sigmaD . Sequence analysis of LytF revealed two domains, an organization common to exoproteins of B . subtilis as well as to those from other organisms . The N-terminal domain contains a fivefold-repeated motif attributed to cell wall binding, whilst the C-terminal domain is probably endowed with the catalytic activity . Overexpression of LytF allowed its purification and biochemical characterization . Inactivation of lytF led to the loss of the cell-wall-bound protein 49' (CWBP49') and of the corresponding lytic activity as revealed by renaturation gel assay . Native cell walls prepared from the multiple lytC lytD lytE lytF-deficient mutant did not exhibit any autolysis, whereas walls prepared from a strain endowed with LytF but not with the other three enzymes underwent a slight lysis . Analysis of degradation products of cell wall devoid of teichoic-acid-bound O-esterified D-alanine unambiguously confirmed that LytF cuts the gamma-D-glutamate-mesodiaminopimelate bond.

Nat Struct Biol, 1999 Apr, 6(4), 340 - 5
Crystal structure of brefeldin A esterase, a bacterial homolog of the mammalian hormone-sensitive lipase; Wei Y et al.; Brefeldin A esterase (BFAE), a detoxifying enzyme isolated from Bacillus subtilis, hydrolyzes and inactivates BFA, a potent fungal inhibitor of intracellular vesicle-dependent secretory transport and poliovirus RNA replication . We have solved the crystal structure of BFAE and we discovered that the previously reported amino acid sequence was in serious error due to frame shifts in the cDNA sequence . The correct sequence, inferred from the experimentally phased electron density map, revealed that BFAE is a homolog of the mammalian hormone sensitive lipase (HSL) . It is a canonical alpha/beta hydrolase with two insertions forming the substrate binding pocket . The enzyme contains a lipase-like catalytic triad, Ser 202, Asp 308 and His 338, consistent with mutational studies that implicate the homologous Ser 424, Asp 693 and His 723 in the catalytic triad in human HSL.

Mol Microbiol, 1999 Mar, 31(5), 1549 - 59
Mta, a global MerR-type regulator of the Bacillus subtilis multidrug-efflux transporters; Baranova NN et al.; Little is known about the natural functions of multidrug-efflux transporters expressed by bacteria . Although identified as membrane proteins actively extruding exogenous toxins from the cell, they may actually be involved in the transport of as yet unidentified specific natural substrates . The expression of two highly similar multidrug transporters of Bacillus subtilis, Bmr and Blt, is regulated by specific transcriptional activators, BmrR and BltR, respectively, which respond to different inducer molecules, thus suggesting distinct functions for the two transporters . Here, we describe an alternative mechanism of regulation, which involves a global transcriptional activator, Mta, a member of the MerR family of bacterial regulatory proteins . The individually expressed N-terminal DNA-binding domain of Mta interacts directly with the promoters of bmr and blt and induces transcription of these genes . Additionally, this domain stimulates the expression of the mta gene itself and at least one more gene, ydfK, which encodes a hypothetical membrane protein . These results and the similarity of Mta to the thiostrepton-induced protein TipA of Streptomyces lividans strongly suggest that Mta is an autogenously controlled global transcriptional regulator, whose activity is stimulated by an as yet unidentified inducer . This stimulation is mimicked by the removal of the C-terminal inducer-binding domain . The fact that both Bmr and Blt are controlled by this regulator demonstrates that some of their functions are either identical or, at least, related . Further analysis of Mta-mediated regulation may reveal the natural function of the system of multidrug transporters in B . subtilis and serve as a paradigm for similar systems in other bacteria.

Mol Microbiol, 1999 Mar, 31(5), 1407 - 15
The SpoIIE phosphatase, the sporulation septum and the establishment of forespore-specific transcription in Bacillus subtilis: a reassessment; Arigoni F et al.; Making a spore in Bacillus subtilis requires the formation of two cells, the forespore and the mother cell, which follow dissimilar patterns of gene expression . Cell specificity is first established in the forespore under the control of the sigma F factor, which is itself activated through the action of the SpoIIE serine phosphatase, an enzyme targeted to the septum between the two cells . Deletion of the 10 transmembrane segments of the SpoIIE protein leads to random distribution of SpoIIE in the cytoplasm . Activation of sigma F is slightly delayed and less efficient than in wild type, but it remains restricted to the forespore in a large proportion of cells and the bacteria sporulate with 30% efficiency . Overexpression of the complete SpoIIE protein in a divIC mutant leads to significant sigma F activity, indicating that the septum requirement for activating sigma F can be bypassed . In contradiction to current models, we propose that genetic asymmetry is not created by unequal distribution of SpoIIE within the sporangium, but by exclusion of an inhibitor of SpoIIE from the forespore . This putative inhibitor would be a cytoplasmic molecule that interacts with SpoIIE and shuts off its phosphatase activity until it disappears specifically from the forespore.

Mol Microbiol, 1999 Mar, 31(5), 1285 - 94
Control of sigma factor activity during Bacillus subtilis sporulation; Kroos L et al.; When starved, Bacillus subtilis undergoes asymmetric division to produce two cell types with different fates . The larger mother cell engulfs the smaller forespore, then nurtures it and, eventually, lyses to release a dormant, environmentally resistant spore . Driving these changes is a programme of transcriptional gene regulation . At the heart of the programme are sigma factors, which become active at different times, some only in one cell type or the other, and each directing RNA polymerase to transcribe a different set of genes . The activity of each sigma factor in the cascade is carefully regulated by multiple mechanisms . In some cases, novel proteins control both sigma factor activity and morphogenesis, co-ordinating the programme of gene expression with morphological change . These bifunctional proteins, as well as other proteins involved in sigma factor activation, and even precursors of sigma factors themselves, are targeted to critical locations, allowing the mother cell and forespore to communicate with each other and to co-ordinate their programmes of gene expression . This signalling can result in proteolytic sigma factor activation . Other mechanisms, such as an anti-sigma factor and, perhaps, proteolytic degradation, prevent sigma factors from becoming active in the wrong cell type . Accessory transcription factors modulate RNA polymerase activity at specific promoters . Negative feedback loops limit sigma factor production and facilitate the transition from one sigma factor to the next . Together, the mechanisms controlling sigma factor activity ensure that genes are expressed at the proper time and level in each cell type.

J Bacteriol, 1999 Apr, 181(8), 2631 - 3
Assembly requirements and role of CotH during spore coat formation in Bacillus subtilis; Zilhao R et al.; We report Western blot data showing that the 42.8-kDa product of the previously characterized cotH locus (8) is a structural component of the Bacillus subtilis spore coat . We show that the assembly of CotH requires both CotE and GerE . In agreement with these observations, the ultrastructural analysis of purified spores suggests that CotH is needed for proper formation of both inner and outer layers of the coat.

J Bacteriol, 1999 Apr, 181(8), 2394 - 402
mrp, a multigene, multifunctional locus in Bacillus subtilis with roles in resistance to cholate and to Na+ and in pH homeostasis; Ito M et al.; A 5.9-kb region of the Bacillus subtilis chromosome is transcribed as a single transcript that is predicted to encode seven membrane-spanning proteins . Homologues of the first gene of this operon, for which the designation mrp (multiple resistance and pH adaptation) is proposed here, have been suggested to encode an Na+/H+ antiporter or a K+/H+ antiporter . In the present studies of the B . subtilis mrp operon, both polar and nonpolar mutations in mrpA were generated . Growth of these mutants was completely inhibited by concentrations of added Na+ as low as 0.3 M at pH 7.0 and 0.03 M at pH 8.3; there was no comparable inhibition by added K+ . A null mutant that was constructed by full replacement of the mrp operon was even more Na+ sensitive . A double mutant with mutations in both mrpA and the multifunctional antiporter-encoding tetA(L) gene was no more sensitive than the mrpA mutants to Na+, consistent with a major role for mrpA in Na+ resistance . Expression of mrpA from an inducible promoter, upon insertion into the amyE locus, restored significant Na+ resistance in both the polar and nonpolar mrpA mutants but did not restore resistance in the null mutant . The mrpA disruption also resulted in an impairment of cytoplasmic pH regulation upon a sudden shift in external pH from 7.5 to 8.5 in the presence of Na+ and, to some extent, K+ in the range from 10 to 25 mM . By contrast, the mrpA tetA(L) double mutant, like the tetA(L) single mutant, completely lost its capacity for both Na+- and K+-dependent cytoplasmic pH regulation upon this kind of shift at cation concentrations ranging from 10 to 100 mM; thus, tetA(L) has a more pronounced involvement than mrpA in pH regulation . Measurements of Na+ efflux from the wild-type strain, the nonpolar mrpA mutant, and the complemented mutant indicated that inducible expression of mrpA increased the rate of protonophore- and cyanide-sensitive Na+ efflux over that in the wild-type in cells preloaded with 5 mM Na+ . The mrpA and null mutants showed no such efflux in that concentration range . This is consistent with MrpA encoding a secondary, proton motive force-energized Na+/H+ antiporter . Studies of a polar mutant that leads to loss of mrpFG and its complementation in trans by mrpF or mrpFG support a role for MrpF as an efflux system for Na+ and cholate . Part of the Na+ efflux capacity of the whole mrp operon products is attributable to mrpF . Neither mrpF nor mrpFG expression in trans enhanced the cholate or Na+ resistance of the null mutant . Thus, one or more other mrp gene products must be present, but not at stoichiometric levels, for stability, assembly, or function of both MrpF and MrpA expressed in trans . Also, phenotypic differences among the mrp mutants suggest that functions in addition to Na+ and cholate resistance and pH homeostasis will be found among the remaining mrp genes.

J Bacteriol, 1999 Apr, 181(8), 2373 - 8
Expression of a germination-specific amidase, SleB, of Bacilli in the forespore compartment of sporulating cells and its localization on the exterior side of the cortex in dormant spores; Moriyama R et al.; A germination-specific amidase of bacilli is a major spore-lytic enzyme that is synthesized with a putative signal sequence and hydrolyses spore cortex in situ . The sleB gene encoding this amidase in Bacillus subtilis and Bacillus cereus was expressed in the forespore compartment of sporulating cells under the control of sigmaG, as shown by Northern blot and primer extension analyses . The forespore-specific expression of B . subtilis sleB was further indicated by the forespore-specific accumulation of a SleB-green fluorescent protein fusion protein from which a putative secretion signal of SleB was deleted . Immunoelectron microscopy with anti-SleB antiserum and a colloidal gold-immunoglobulin G complex showed that the enzymes from both Bacillus species are located just inside the spore coat layer in the dormant spore, and in the dormant spore, the amidases appear exist in a mature form lacking a signal sequence . These results indicate that SleB is translocated across the forespore's inner membrane by a secretion signal peptide and is deposited in cortex layer synthesized between the forespore inner and outer membranes . The peripheral location of the spore-lytic enzymes in the dormant spore suggests that spore germination is initiated at the exterior of the cortex.

J Bacteriol, 1999 Apr, 181(8), 2346 - 50
Replacement of vegetative sigmaA by sporulation-specific sigmaF as a component of the RNA polymerase holoenzyme in sporulating Bacillus subtilis; Lord M et al.; Soon after asymmetric septation in sporulating Bacillus subtilis cells, sigmaF is liberated in the prespore from inhibition by SpoIIAB . To initiate transcription from its cognate promoters, sigmaF must compete with sigmaA, the housekeeping sigma factor in the predivisional cell, for binding to core RNA polymerase (E) . To estimate the relative affinity of E for sigmaA and sigmaF, we made separate mixtures of E with each of the two sigma factors, allowed reconstitution of the holoenzyme, and measured the concentration of free E remaining in each mixture . The affinity of E for sigmaF was found to be about 25-fold lower than that for sigmaA . We used quantitative Western blotting to estimate the concentrations of E, sigmaA, and sigmaF in sporulating cells . The cellular concentrations of E and sigmaA were both about 7.5 microM, and neither changed significantly during the first 3 h of sporulation . The concentration of sigmaF was extremely low at the beginning of sporulation, but it rose rapidly to a peak after about 2 h . At its peak, the concentration of sigmaF was some twofold higher than that of sigmaA . This difference in concentration cannot adequately account for the replacement of sigmaA holoenzyme by sigmaF holoenzyme in the prespore, and it seems that some further mechanism-perhaps the synthesis or activation of an anti-sigmaA factor-must be responsible for this replacement.

J Biol Chem, 1999 Apr 16, 274(16), 11092 - 100
CheY-dependent methylation of the asparagine receptor, McpB, during chemotaxis in Bacillus subtilis; Kirby JR et al.; For the Gram-positive organism Bacillus subtilis, chemotaxis to the attractant asparagine is mediated by the chemoreceptor McpB . In this study, we show that rapid net demethylation of B . subtilis McpB results in the immediate production of methanol, presumably due to the action of CheB . We also show that net demethylation of McpB occurs upon both addition and removal of asparagine . After each demethylation event, McpB is remethylated to nearly prestimulus levels . Both remethylation events are attributable to CheR using S-adenosylmethionine as a substrate . Therefore, no methyl transfer to an intermediate carrier need be postulated to occur during chemotaxis in B . subtilis as was previously suggested . Furthermore, we show that the remethylation of asparagine-bound McpB requires the response regulator, CheY-P, suggesting that CheY-P acts in a feedback mechanism to facilitate adaptation to positive stimuli during chemotaxis in B . subtilis . This hypothesis is supported by two observations: a cheRBCD mutant is capable of transient excitation and subsequent oscillations that bring the flagellar rotational bias below the prestimulus value in the tethered cell assay, and the cheRBCD mutant is capable of swarming in a Tryptone swarm plate.

Drug Dev Ind Pharm, 1999 Apr, 25(4), 523 - 8
Synthesis and properties of disodium tetraethyleneglycol-bis-(alpha-carboxybenzylpenicillin); Kim YT et al.; Disodium tetraethyleneglycol-bis-(alpha-carboxybenzylpenicillin) (TEG-carbenicillin), a tetraethyleneglycol (TEG) diester of carbenicillin, was synthesized to develop a carbenicillin prodrug with enhanced acid stability for oral administration . Antimicrobial activities of TEG-carbenicillin tested against gram-negative Escherichia coli (TG-1) and gram-positive Staphylococcus aureus (ATCC-12228) and Bacillus subtilis (NA-1) were comparable to that of carbenicillin . Stability of the beta-lactam ring of TEG-carbenicillin was determined by iodometry at pH 6.8, pH 4.5, and pH 2.0 at varied time intervals and was compared to that of carbenicillin . In 26 hr, both of the compounds were stable at pH 6.8 . At pH 4.5, about 41% of the carbenicillin was decomposed, while TEG-carbenicillin was not appreciably decomposed . At pH 2.0, carbenicillin was decomposed about 61% after 6 hr, while TEG-carbenicillin was decomposed about 21% during the same period.

Biochemistry, 1999 Apr 6, 38(14), 4252 - 8
Unexpected divergence of enzyme function and sequence: "N-acylamino acid racemase" is o-succinylbenzoate synthase; Palmer DR et al.; A protein identified as "N-acylamino acid racemase" from Amycolaptosis sp . is an inefficient enzyme (kcat/Km = 3.7 x 10(2) M-1 s-1) . Its sequence is 43% identical to that of an unidentified protein encoded by the Bacillus subtilis genome . Both proteins efficiently catalyze the o-succinylbenzoate synthase reaction in menaquinone biosynthesis (kcat/Km = 2.5 x 10(5) and 7.5 x 10(5) M-1 s-1, respectively), suggesting that this is their "correct" metabolic function . Their membership in the mechanistically diverse enolase superfamily provides an explanation for the catalytic promiscuity of the protein from Amycolaptosis . The adventitious promiscuity may provide an example of a protein poised for evolution of a new enzymatic function in the enolase superfamily . This study demonstrates that the correct assignment of function to new proteins in functional and structural genomics may require an understanding of the metabolism of the organism.

Biotechnol Bioeng, 1998 Apr 20-May 5, 58(2-3), 170 - 4
Multiple mechanisms controlling carbon metabolism in bacteria; Saier MH Jr; Catabolite repression is a universal phenomenon, found in virtually all living organisms . These organisms range from the simplest bacteria to higher fungi, plants, and animals . A mechanism involving cyclic AMP and its receptor protein (CRP) in Escherichia coli was established years ago, and this mechanism has been assumed by many to serve as the prototype for catabolite repression in all organisms . However, recent studies have shown that this mechanism is restricted to enteric bacteria and their close relatives . Cyclic AMP-independent mechanisms of catabolite repression occur in other bacteria, yeast, plants, and even E . coli . In fact, single-celled organisms such as E . coli, Bacillus subtilis, and Saccharomyces cerevisiae exhibit multiple mechanisms of catabolite repression, and most of these are cyclic AMP-independent . The mechanistic features of the best of such characterized processes are briefly reviewed, and references are provided that will allow the reader to delve more deeply into these subjects .

Mol Gen Mikrobiol Virusol, 1999, (1), 18 - 20
{Cloning and expression of a serine proteinase gene from Thermoactinomyces sp . in Bacillus subtilis cells}; Akimkina TV et al.; A gene coding for thermostable serine protease from Thermoactinomyces sp . K50 is cloned and expressed in Bacillus subtilis cells . Restriction map of cloned DNA fragment is determined . Thermostability and temperature optimum of proteolytic activity of the cloned gene product are lower than those of the natural proteinase of Thermoactinomyces sp . K50 . Serine protease, a product of cloned gene, is highly sensitive to proteolysis and its degradation can be prevented by Ca2+ ions.

Mol Gen Mikrobiol Virusol, 1999, (1), 12 - 7
{Expression of secreted guanyl-specific ribonuclease genes from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells}; Znamenskaia LV et al.; Plasmids with whole genes for ribonucleases from B . intermedius (binase) and B . pumilis (RNase Bp) assembled with the whole gene of barstar, a specific intracellular inhibitor, are constructed . The resultant plasmids pMZ55 and pMZ56 effectively express binase and RNase Bp genes in B . subtilis cells . A medium for maximum expression of RNase genes by recombinant strains is developed . The expression of binase and RNase Bp genes in B . subtilis cells is negatively regulated by exogenic inorganic phosphate.

Phytother Res, 1999 Feb, 13(1), 70 - 2
Studies on the antibacterial potential of Cryptostegia grandiflora R . Br . (Asclepiadaceae) extract; Mukherjee PK et al.; Different extracts of Cryptostegia grandiflora (Roxb) Rbr . leaves were investigated for their antibacterial potential against Pseudomonus cepacia NCIM-2106, Bacillus megatorium NCIM-2087, Staphylococcus aureus NCIM-2492, Escherichia coli NCIM-2345, Bacillus subtilis NCIM-2349 and Bacillus coagulans NCIM 2323 . Almost all the extracts produced significant antibacterial activity against all the microorganisms being tested and the effect so produced was comparable to the standard antibiotic, tetracycline hydrochloride . The petroleum ether (60 degrees-80 degrees C) extract showed maximum efficacy.

FEMS Microbiol Lett, 1999 Mar 15, 172(2), 187 - 96
Self-assembly product formation of the Bacillus stearothermophilus PV72/p6 S-layer protein SbsA in the course of autolysis of Bacillus subtilis; Howorka S et al.; In order to achieve high level expression and to study the release of a protein capable of self-assembly, the gene encoding the crystalline cell surface (S-layer) protein SbsA of Bacillus stearothermophilus PV72/p6, including its signal sequence, was cloned and expressed in Bacillus subtilis . To obtain high level expression, a tightly regulated, xylose-inducible, stably replicating multicopy-plasmid vector was constructed . After induction of expression, the S-layer protein made up about 15% of the total cellular protein content, which was comparable to the SbsA content of B . stearothermophilus PV72/p6 cells . During all growth stages, SbsA was poorly secreted to the ambient cellular environment by B . subtilis . Extraction of whole cells with guanidine hydrochloride showed that in late stationary growth phase cells 65% of the synthesised SbsA was retained in the peptidoglycan-containing layer, indicating that the rigid cell wall layer was a barrier for efficient SbsA secretion . Electron microscopic investigation revealed that SbsA release from the peptidoglycan-containing layer started in the late stationary growth phase at distinct sites at the cell surface leading to the formation of extracellular self-assembly products which did not adhere to the cell wall surface . In addition, intracellular sheet-like SbsA self-assembly products which followed the curvature of the cell became visible in partly lysed cells . Intracellularly formed self-assembly products remained intact even after complete lysis of the rigid cell envelope layer.

FEMS Microbiol Lett, 1999 Mar 15, 172(2), 153 - 8
A new gene, sigG, encoding a putative alternative sigma factor of Streptomyces coelicolor A3(2); Kormanec J et al.; An oligonucleotide probe encoding a peptide motif conserved in all sigma factors was used to isolate a new gene, sigG, from a Streptomyces coelicolor A3(2) genomic library . The deduced protein of 263 amino acids with an M(r) of 29,422 showed the greatest similarity to the previously identified sporulation sigma factor (sigma F) of Streptomyces coelicolor, and general stress response sigma factor (sigma B) of Bacillus subtilis, mostly in domains suggested to be involved in recognition of -10 and -35 promoter regions . Southern-blot hybridization with DNA from several Streptomyces spp . revealed the presence of a similar gene in all strains tested . Disruption of the S . coelicolor sigG gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin.

Eur J Biochem, 1999 Apr, 261(1), 25 - 32
Bacillus subtilis chorismate mutase is partially diffusion-controlled; Mattei P et al.; The effect of viscosogens on the enzyme-catalyzed rearrangement of chorismate to prephenate has been studied . The steady-state parameters kcat and kcat/Km for the monofunctional chorismate mutase from Bacillus subtilis (BsCM) decreased significantly with increasing concentrations of glycerol, whereas the 'sluggish' BsCM mutants C75A and C75S were insensitive to changes in microviscosity . The latter results rule out extraneous interactions of the viscosogen as an explanation for the effects observed with the wild-type enzyme . Additional control experiments show that neither viscosogen-induced shifts in the pH-dependence of the enzyme-catalyzed reaction nor small perturbations of the conformational equilibrium of chorismate can account for the observed effects . Instead, BsCM appears to be limited by substrate binding and product release at low and high substrate concentrations, respectively . Analysis of the kinetic data indicates that diffusive transition states are between 30 and 40% rate-determining in these concentration regimes; the chemical step must contribute to the remaining kinetic barrier . The relatively low value of the 'on' rates for chorismate and prephenate (approximately 2 x 106 m-1.s-1) probably reflects the need for a rare conformation of the enzyme, the ligand, or both for successful binding . Interestingly, the chorismate mutase domain of the bifunctional chorismate mutase-prephenate dehydratase from Escherichia coli, which has steady-state kinetic parameters comparable to those of BsCM but has a much less accessible active site, is insensitive to changes in viscosity and the reaction it catalyses is not diffusion-controlled.

Genetics, 1999 Apr, 151(4), 1239 - 44
Genetic and physical maps of the Bacillus subtilis chromosome; Rivolta C et al.; Sequencing of the complete Bacillus subtilis chromosome revealed the presence of approximately 4100 genes, 1000 of which were previously identified and mapped by classical genetic crosses . Comparison of these experimentally determined positions to those derived from the nucleotide sequence showed discrepancies reaching up to 24 degrees (approximately 280 kb) . The size of these discrepancies as a function of their position along the chromosome is not random but, apparently, reveals some periodicity . Our analyses demonstrate that the discrepancies can be accounted for by inaccurate positioning of the early reference markers with respect to which all subsequently identified loci were mapped by transduction and transformation . We conclude (i) that specific DNA sequences, such as recombination hotspots or presence of heterologous DNA, had no detectable effect on the results obtained by classical mapping, and (ii) that PBS1 transduction appears to be an accurate and unbiased mapping method in B . subtilis.

Biotechnol Bioeng, 1999 Feb 5, 62(3), 368 - 72
Kinetics of inactivation of Bacillus subtilis spores by continuous or intermittent ohmic and conventional heating; Cho HY et al.; Bacillus subtilis spores were suspended in 0.1% NaCl solution (ca . 10(7) CFU/mL) and treated by conventional or ohmic heating under identical temperature histories . Temperatures tested were in the range of 88 to 99 degrees C . Survival curves and calculated D values showed significantly higher lethality for spores by ohmic than conventional heating . The z or Ea values corresponding to the two heating methods, however, were not significantly different . Spores of B . subtilis were suspended in nutrient broth and treated with conventional and ohmic heating through a single- or a double-stage treatment . In case of double-stage treatment, heating was interrupted by a 20 min of incubation at 37 degrees C to induce a Tyndallization effect . Spore inactivation during double-stage treatment was greater for ohmic than conventional heating . The enhanced spore inactivation by ohmic, compared with conventional, heating resulted from a greater rate of spore death during the first stage of heating and greater decrease in count of viable spores immediately after the incubation period that intervened the heating process . Thus it is concluded that spore inactivation during ohmic heating was primarily due to the thermal effect but there was an additional killing effect caused by the electric current .

Biotechnol Bioeng, 1999 Jan 5, 62(1), 87 - 96
High-level secretory production of intact, biologically active staphylokinase from Bacillus subtilis; Ye R et al.; Staphylokinase is a promising blood-clot dissolving agent for the treatment of patients suffering from a heart attack . It would be desirable to produce this protein in large quantities for biochemical characterization and clinical trials . Production of intact, biologically active staphylokinase from bacterial expression systems has been a challenge because of N-terminal microheterogeneity, plasmid instability, or low-production yield . By using a seven-extracellular-protease deficient Bacillus subtilis strain, WB700, intact staphylokinase can be produced via secretion . However, native staphylokinase gene (sak) in a high-copy number plasmid was found to be unstable in B . subtilis . To optimize the production and the stability of the expression vectors, both the promoter and the signal sequence of sak were replaced by B . subtilis promoters (P43, a constitutively expressed promoter; Pamy, a stationary-phase promoter; and PsacB, a sucrose-inducible promoter) and the levansucrase-signal sequence, respectively . This overcame the plasmid instability problem . To enhance transcription from the sacB promoter, degQ encoding a transcriptional activator for sacB and other protease genes was also installed in the expression vector . The use of WB700 as the expression host allowed enhanced production of staphylokinase from the sucrose-inducible plasmid without causing any obvious degradation of staphylokinase . Both the P43 and PsacB (with DegQ) promoters worked well . Over 90% of staphylokinase synthesized can be secreted effectively . With the optimization of both the culture media and the fermentation conditions, production of staphylokinase reached a level of 337 mg/L, and staphylokinase could be purified to homogeneity by a simple three-step purification scheme . Secreted staphylokinase did not show any N-terminal heterogeneity . This presents an attractive system for the production of staphylokinase in both high quality and quantity .

Biotechnol Bioeng, 1998 Oct 5, 60(1), 1 - 9
Adaptive pole placement control algorithm for DO-control in beta-lactamase production; Sargantanis IG et al.; The dissolved oxygen (DO) is an important variable in aerobic fermentations and affects the cell growth and product formation . Dissolved oxygen control is difficult in batch fermentations because of the time-varying conditions, time delays, and the probe dynamics . Modeling of the various patterns of biological activity in fermentations and their impact on the DO process dynamics is essential to both achieve a satisfactory control and to track the aforementioned patterns . An adaptive pole placement algorithm with time-delay compensation was used for controlling the DO, coupled with system identification using recursively estimated autoregressive models with exogeneous inputs (ARX) . The flow rate of O2 in a constant flow rate gas inlet mixture is used as the manipulated variable . Supervision and coordination techniques are applied to improve the control performance . The control performance is affected by the accuracy of the model prediction and the selected time delay . The effect of DO level on the productivity of beta-lactamase using Bacillus subtilis under oxygen-limited conditions is investigated . Beta-lactamase stability is improved under prolonged growth conditions with low DO levels .

Biotechnol Bioeng, 1998 Jul 20, 59(2), 227 - 38
Metabolic capacity of Bacillus subtilis for the production of purine nucleosides, riboflavin, and folic acid; Sauer U et al.; We developed a stoichiometric model of Bacillus subtilis metabolism for quantitative analysis of theoretical growth and biochemicals production capacity . This work concentrated on biochemicals that are derived from the purine biosynthesis pathway; inosine, guanosine, riboflavin, and folic acid . These are examples of commercially relevant biochemicals for which Bacillus species are commonly used production hosts . Two previously unrecognized, but highly desirable properties of good producers of purine pathway-related biochemicals have been identified for optimally engineered product biosynthesis; high capacity for reoxidation of NADPH and high bioenergetic efficiency . Reoxidation of NADPH, through the transhydrogenase or otherwise, appears to be particularly important for growth on glucose, as deduced from the corresponding optimal carbon flux distribution . The importance of cellular energetics on optimal performance was quantitatively assessed by including a bioenergetic efficiency parameter as an unrestricted, ATP dissipating flux in the simulations . An estimate for the bioenergetic efficiency was generated by fitting the model to experimentally determined growth yields . The results show that the maximum theoretical yields of all products studied are limited by pathway stoichiometry at high bioenergetic efficiencies . Simulations with the estimated bioenergetic efficiency of B . subtilis, growing under glucose-limiting conditions, indicate that the yield of these biochemicals is primarily limited by energy and thus is very sensitive to the process conditions . The maximum yields that can reasonably be expected with B . subtilis on glucose were estimated to be 0.343, 0.160, and 0.161 (mol product/mol glucose) for purine nucleosides, riboflavin, and folic acid, respectively . Potential strategies for improving these maximum yields are discussed .

Mol Microbiol, 1999 Feb, 31(4), 1149 - 59
A vital stain for studying membrane dynamics in bacteria: a novel mechanism controlling septation during Bacillus subtilis sporulation; Pogliano J et al.; At the onset of sporulation in Bacillus subtilis, two potential division sites are assembled at each pole, one of which will be used to synthesize the asymmetrically positioned sporulation septum . Using the vital stain FM 4-64 to label the plasma membrane of living cells, we examined the fate of these potential division sites in wild-type cells and found that, immediately after the formation of the sporulation septum, a partial septum was frequently synthesized within the mother cell at the second potential division site . Using time-lapse deconvolution microscopy, we were able to watch these partial septa first appear and then disappear during sporulation . Septal dissolution was dependent on sigma E activity and was partially inhibited in mutants lacking the sigma E-controlled proteins SpoIID, SpoIIM and SpoIIP, which may play a role in mediating the degradation of septal peptidoglycan . Our results support a model in which sigma E inhibits division at the second potential division site by two distinct mechanisms: inhibition of septal biogenesis and the degradation of partial septa formed before sigma E activation.

Mol Microbiol, 1999 Feb, 31(4), 1075 - 85
Lipid modification of prelipoproteins is dispensable for growth but essential for efficient protein secretion in Bacillus subtilis: characterization of the Lgt gene; Leskela S et al.; We have identified and characterized the Igt gene of Bacillus subtilis . The prelipoprotein diacylglycerol transferase enzyme (Lgt) catalyses the first reaction in lipomodification of bacterial lipoproteins . Inactivation of Igt in B . subtilis by a nonsense mutation (prs-11 mutation) or by disruption was shown here to abolish lipomodification of prelipoproteins completely, as well as the cleavage of signal peptide . However, unlike in Gram-negative bacteria, the Igt mutants of B . subtilis were fully viable . In agreement with this observation, studies of two lipoproteins, PrsA and BlaP, indicated that non-lipomodified precursors of these proteins were functional and translocated across the cytoplasmic membrane . However, there was release of both precursors from cells, resulting in a reduced level of the cell-bound form . We have shown that the reduced level of the PrsA lipoprotein, a foldase involved in protein secretion, caused impaired protein secretion, a prominent phenotype of Igt mutants . There was no indication that non-lipomodified PrsA displayed reduced activity.

Eur J Biochem, 1999 Mar, 260(2), 499 - 504
Further improvement of the thermal stability of a partially stabilized Bacillus subtilis 3-isopropylmalate dehydrogenase variant by random and site-directed mutagenesis; Akanuma S et al.; A thermostabilized mutant of Bacillus subtilis 3-isopropylmalate dehydrogenase (IPMDH) obtained in a previous study contained a set of triple amino acid substitutions . To further improve the stability of the mutant, we used a random mutagenesis technique and identified two additional thermostabilizing substitutions, Thr22-->Lys and Met256-->Val, that separately endowed the protein with further stability . We introduced the two mutations into a single enzyme molecule, thus constructing a mutant with overall quintuple mutations . Other studies have suggested that an improved hydrophobic subunit interaction and a rigid type II beta-turn play important roles in enhancing the protein stability . Based on those observations, we successively introduced amino acid substitutions into the mutant with the quintuple mutations by site-directed mutagenesis: Glu253 at the subunit interface was replaced by Leu to increase the hydrophobic interaction between the subunits; Glu112, Ser113 and Ser115 that were involved in the formation of the turn were replaced by Pro, Gly and Glu, respectively, to make the turn more rigid . The thermal stability of the mutants was determined based on remaining activity after heat treatment and first-order rate constant of thermal unfolding, which showed gradual increases in thermal stability as more mutations were included.

Eur J Biochem, 1999 Mar, 260(2), 325 - 35
Interactions of the Streptomyces lividans initiator protein DnaA with its target; Majka J et al.; The Streptomyces lividans DnaA protein (73 kDa) consists, like other bacterial DnaA proteins, of four domains; it binds to 19 DnaA boxes in the complex oriC region . The S . lividans DnaA protein differs from others in that it contains an additional stretch of 120 predominantly acidic amino acids within domain II . Interactions between the DnaA protein and the two DnaA boxes derived from the promoter region of the S . lividans dnaA gene were analysed in vitro using three independent methods: Dnase-I-footprinting experiments, mobility-shift assay and surface plasmon resonance (SPR) . The Dnase-I-footprinting analysis showed that the wild-type DnaA protein binds to both DnaA boxes . Thus, as in Escherichia coli and Bacillus subtilis, the S . lividans dnaA gene may be autoregulated . SPR analysis showed that the affinity of the DnaA protein for a DNA fragment containing both DnaA boxes from the dnaA promoter region (KD = 1.25 nM) is 10 times higher than its affinity for the single 'strong' DnaA box (KD = 12.0 nM) . The mobility-shift assay suggests the presence of at least two classes of complex containing different numbers of bound DnaA molecules . The above data reveal that the DnaA protein binds to the two DnaA boxes in a cooperative manner . To deduce structural features of the Streptomyces domain II of DnaA protein, the amino acid DnaA sequences of three Streptomyces species were compared . However, according to the secondary structure prediction, Streptomyces domain II does not contain any common relevant secondary structural element(s) . It can be assumed that domain II of DnaA protein can play a role as a flexible protein spacer between the N-terminal domain I and the highly conserved C-terminal part of DnaA protein containing ATP-binding domain III and DNA-binding domain IV.

J Bacteriol, 1999 Apr, 181(7), 2118 - 23
Isolation of RNase H genes that are essential for growth of Bacillus subtilis 168; Itaya M et al.; Two genes encoding functional RNase H (EC 3.1.26.4) were isolated from a gram-positive bacterium, Bacillus subtilis 168 . Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a B . subtilis DNA library . One (28.2 kDa) revealed high similarity to Escherichia coli RNase HII, encoded by the rnhB gene . The other (33.9 kDa) was designated rnhC and encodes B . subtilis RNase HIII . The B . subtilis genome has an rnhA homologue, the product of which has not yet shown RNase H activity . Analyses of all three B . subtilis genes revealed that rnhB and rnhC cannot be simultaneously inactivated . This observation indicated that in B . subtilis both the rnhB and rnhC products are involved in certain essential cellular processes that are different from those suggested by E . coli rnh mutation studies . Sequence conservation between the rnhB and rnhC genes implies that both originated from a single ancestral RNase H gene . The roles of bacterial RNase H may be indicated by the single rnhC homologue in the small genome of Mycoplasma species.

J Bacteriol, 1999 Apr, 181(7), 2059 - 66
Role of bkdR, a transcriptional activator of the sigL-dependent isoleucine and valine degradation pathway in Bacillus subtilis; Debarbouille M et al.; A new gene, bkdR (formerly called yqiR), encoding a regulator with a central (catalytic) domain was found in Bacillus subtilis . This gene controls the utilization of isoleucine and valine as sole nitrogen sources . Seven genes, previously called yqiS, yqiT, yqiU, yqiV, bfmBAA, bfmBAB, and bfmBB and now referred to as ptb, bcd, buk, lpd, bkdA1, bkdA2, and bkdB, are located downstream from the bkdR gene in B . subtilis . The products of these genes are similar to phosphate butyryl coenzyme A transferase, leucine dehydrogenase, butyrate kinase, and four components of the branched-chain keto acid dehydrogenase complex: E3 (dihydrolipoamide dehydrogenase), E1alpha (dehydrogenase), E1beta (decarboxylase), and E2 (dihydrolipoamide acyltransferase) . Isoleucine and valine utilization was abolished in bcd and bkdR null mutants of B . subtilis . The seven genes appear to be organized as an operon, bkd, transcribed from a -12, -24 promoter . The expression of the bkd operon was induced by the presence of isoleucine or valine in the growth medium and depended upon the presence of the sigma factor SigL, a member of the sigma 54 family . Transcription of this operon was abolished in strains containing a null mutation in the regulatory gene bkdR . Deletion analysis showed that upstream activating sequences are involved in the expression of the bkd operon and are probably the target of bkdR . Transcription of the bkd operon is also negatively controlled by CodY, a global regulator of gene expression in response to nutritional conditions.

J Bacteriol, 1999 Apr, 181(7), 2017 - 25
Mutational analysis of the phoD promoter in Bacillus subtilis: implications for PhoP binding and promoter activation of Pho regulon promoters; Eder S et al.; The PhoP-PhoR two-component regulatory system controls the phosphate deficiency response in B . subtilis . A number of Pho regulon genes which require PhoP approximately P for activation or repression have been identified . The studies reported here were initiated to understand the PhoP-DNA interaction necessary for Pho promoter regulation . The regulatory region of phoD was characterized in detail using oligo-directed mutagenesis, DNase I footprinting, and in vivo transcription assays . These data reveal basic principles of PhoP binding relevant to PhoP's interaction with other Pho regulon promoters . Our results show that: (i) a dimer of PhoP approximately P is able to bind two consensus repeats in a stable fashion; (ii) PhoP binding is highly cooperative within the core promoter region, which is located from -66 to -17 on the coding strand and contains four TT(A/T/C)ACA-like repeats; (iii) specific bases comprising the TT(A/T/C)ACA consensus are essential for transcriptional activation, but the specific base pairs of the intervening sequences separating the consensus repeats are not important for either PhoP binding or promoter activation; (iv) the spacing between two consensus repeats within a putative dimer binding site in the core region is important for both PhoP binding and promoter activation; (v) the exact spacing between two dimer binding sites within the core region is important for promoter activation but less so for PhoP binding affinity, as long as the repeats are on the same face of the helix; and (vi) the 5' secondary binding region is important for coordinated PhoP binding to the core binding region, making it nearly essential for promoter activation.

Biochem Biophys Res Commun, 1999 Apr 2, 257(1), 106 - 10
A Bacillus-specific factor is needed to trigger the stress-activated phosphatase/kinase cascade of sigmaB induction; Scott JM et al.; The general stress regulon of Bacillus subtilis is controlled by the transcription factor sigmaB . Environmental stress activates sigmaB via a phosphatase/kinase cascade that triggers sigmaB's release from an anti sigma factor complex . To determine if the members of the phosphatase/kinase cascade are sufficient to detect environmental stress and activate sigmaB, we expressed sigmaB and its regulators in E . coli . In E . coli, as in B . subtilis, the intact collection of regulators silenced sigmaB, while allowing sigmaB to be active if the cascade's most upstream negative regulator was deleted . The regulators could not, however, activate sigmaB in response to ethanol treatment or heat shock . In other experiments, the GroEL and DnaK chaperones, known to be important in controlling stress sigma factors in E . coli, were found to be unimportant for sigmaB activity in B . subtilis . The findings argue that stress induction of sigmaB requires novel factors that are B . subtilis specific .

J Mol Biol, 1999 Apr 2, 287(3), 467 - 84
Inventory, assembly and analysis of Bacillus subtilis ABC transport systems; Quentin Y et al.; We have undertaken the inventory and assembly of the ATP binding cassette (ABC) transporter systems in the complete genome of Bacillus subtilis . We combined the identification of the three protein partners that compose an ABC transporter (nucleotide-binding domain, NBD; membrane spanning domain, MSD; and solute-binding protein, SBP) with constraints on the genetic organization . This strategy allowed the identification of 86 NBDs in 78 proteins, 103 MSD proteins and 37 SBPs . The analysis of transcriptional units allows the reconstruction of 59 ABC transporters, which include at least one NBD and one MSD . A particular class of five dimeric ATPases was not associated to MSD partners and is assumed to be involved either in macrolide resistance or regulation of translation elongation . In addition, we have detected five genes encoding ATPases without any gene coding for MSD protein in their neighborhood and 11 operons that encode only the membrane and solute-binding proteins . On the bases of similarities, three ATP-binding proteins are proposed to energize ten incomplete systems, suggesting that one ATPase may be recruited by more than one transporter . Finally, we estimate that the B . subtilis genome encodes for at least 78 ABC transporters that have been split in 38 importers and 40 extruders . The ABC systems have been further classified into 11 sub-families according to the tree obtained from the NBDs and the clustering of the MSDs and the SBPs . Comparisons with Escherichia coli show that the extruders are over-represented in B . subtilis, corresponding to an expansion of the sub-families of antibiotic and drug resistance systems .

Protein Sci, 1999 Mar, 8(3), 538 - 44
Crystal structure of thymidylate synthase A from Bacillus subtilis; Fox KM et al.; Thymidylate synthase (TS) converts dUMP to dTMP by reductive methylation, where 5,10-methylenetetrahydrofolate is the source of both the methylene group and reducing equivalents . The mechanism of this reaction has been extensively studied, mainly using the enzyme from Escherichia coli . Bacillus subtilis contains two genes for TSs, ThyA and ThyB . The ThyB enzyme is very similar to other bacterial TSs, but the ThyA enzyme is quite different, both in sequence and activity . In ThyA TS, the active site histidine is replaced by valine . In addition, the B . subtilis enzyme has a 2.4-fold greater k(cat) than the E . coli enzyme . The structure of B . subtilis thymidylate synthase in a ternary complex with 5-fluoro-dUMP and 5,10-methylenetetrahydrofolate has been determined to 2.5 A resolution . Overall, the structure of B . subtilis TS (ThyA) is similar to that of the E . coli enzyme . However, there are significant differences in the structures of two loops, the dimer interface and the details of the active site . The effects of the replacement of histidine by valine and a serine to glutamine substitution in the active site area, and the addition of a loop over the carboxy terminus may account for the differences in k(cat) found between the two enzymes.

Appl Microbiol Biotechnol, 1999 Feb, 51(2), 229 - 34
Bacterial growth in space flight: logistic growth curve parameters for Escherichia coli and Bacillus subtilis; Kacena MA et al.; Previous investigations have reported that bacterial suspension cultures grow to higher stationary concentrations in space flight than on Earth; however, none of these investigations included extensive ground controls under varied inertial conditions . This study includes extensive controls and cell-growth data taken at several times during lag phase, log phase, and stationary phase of Escherichia coli and Bacillus subtilis . The Marquardt-Levenberg, least-squares fitting algorithm was used to calculate kinetic growth parameters from the logistic bacterial growth equations for space-flight and control growth curves . Space-flight cultures grew to higher stationary-phase concentrations and had shorter lag-phase durations . Also, evidence was found for increased exponential growth rate in space.

Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1453 - 5
Bacillus subtilis regulatory protein GerE; Ducros VM et al.; GerE is the latest-acting of a series of factors which regulate gene expression in the mother cell during sporulation in Bacillus . The gene encoding GerE has been cloned from B . subtilis and overexpressed in Escherichia coli . Purified GerE has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant . The small plate-like crystals belong to the monoclinic space group C2 and diffract beyond 2.2 A resolution with a synchrotron radiation X-ray source.

Acta Crystallogr D Biol Crystallogr, 1999 Mar, 55 ( Pt 3), 691 - 3
Preliminary X-ray crystallographic analysis of a novel phytase from a Bacillus amyloliquefaciens strain; Ha NC et al.; A novel bacterial phytase from a Bacillus amyloliquefaciens strain was crystallized using the hanging-drop vapour-diffusion method . The amino-acid sequence of the enzyme does not show any homology to those of other known phytases or phosphatases, with the exception of a phytase from Bacillus subtilis . The enzyme exhibits a thermal stability which is strongly dependent on calcium ions . High-quality single crystals of the enzyme in the absence of calcium ions were obtained using a precipitant solution containing 20% 2-methyl-2, 4-pentanediol and 0.1 M MES (pH 6.5) . Native diffraction data to 2.0 A resolution were obtained from a flash-frozen crystal at 110 K using a rotating-anode X-ray source . The crystals belong to space group P212121 with unit-cell dimensions a = 50.4, b = 64.1, c = 104 . 2 A and contain one monomer per asymmetric unit . Structure determination using heavy-atom derivative crystals is in progress, along with an effort to crystallize the calcium ion bound form of the enzyme.

Acta Crystallogr D Biol Crystallogr, 1999 Mar, 55 ( Pt 3), 677 - 8
Cloning, crystallization and preliminary X-ray analysis of a nucleotide-diphospho-sugar transferase spsA from Bacillus subtilis; Charnock SJ et al.; Nucleotide-diphospho-sugar transferases represent, in terms of quantity, one of the most important groups of enzymes on Earth, yet little is known about their structure and mechanism . Such a transferase, the spsA gene product involved in the synthesis of the bacterial spore coat in Bacillus subtilis, has been cloned and over-expressed in an Escherichia coli expression system . Crystals have been grown, using PEG 8000 as a precipitant, in a form suitable for high-resolution X-ray analysis . They belong to space group C2221, with unit-cell dimensions a = 42.4, b = 142.0, c = 81.4 A and with one molecule of spsA in the asymmetric unit . The crystals diffract beyond 1.5 A using synchrotron radiation.

Extremophiles, 1999 Jan, 3(1), 29 - 34
Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp . strain C-125; Takami H et al.; The nucleotide sequences of three independent fragments (designated no . 3, 4, and 9; each 15-20 kb in size) of the genome of alkaliphilic Bacillus sp . C-125 cloned in a lambda phage vector have been determined . Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no . 3 and 11 ORFs were identified in no . 4 . Twenty ORFs were also identified in fragment no . 9 . All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases . The functions of 5 ORFs in fragment no . 3 and 3 ORFs in fragment no . 4 were suggested by their significant similarities to known proteins in the database . Among the 20 ORFs in fragment no . 9, the functions of 11 ORFs were similarly suggested . Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp . C-125 were conserved in the Bacillus subtilis genome . The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B . subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B . subtilis.

Zentralbl Hyg Umweltmed, 1999 Feb, 201(6), 541 - 53
Microbiological efficacy of superheated steam . I . Communication: results with spores of Bacillus subtilis and Bacillus stearothermophilus and with spore earth; Spicher G et al.; For the spores of Bacillus subtilis and Bacillus stearothermophilus as well as for spore earth (acc . DIN 58,946 Part 4 of August 1982), the dependence of resistance on the superheating of the steam used to kill germs was determined . A material (glass fibre fleece) was used as the germ carrier which does not superheat on contact with steam . The temperature of the saturated steam was 100 degrees C (B . subtilis) and 120 degrees C (B . stearothermophilus and spore earth) . The yardstick for the resistance of the spores or bioindicators was the exposure period of the saturated or superheated steam at which 50% of the treated test objects no longer showed any viable test germs . The spores of Bacillus subtilis were far more sensitive to superheating of steam and reacted far more than the spores of Bacillus stearothermophilus and the germs in the spore earth . When superheating by 4 Kelvin the spores of Bacillus subtilis were approximately 2.5 times more resistant than they were to saturated steam . The resistance of Bacillus stearothermophilus and spore earth was only slightly higher up to superheating by 10 Kelvin . The spores of Bacillus subtilis had the highest resistance during superheating by 29 Kelvin; they were 119 times more resistant than they were to saturated steam . The resistance maximum of the spores of Bacillus stearothermophilus was at an superheating by around 22 Kelvin . However, the spores were only 4.1 times more resistant than they were to saturated steam . When using steam to kill germs, we must expect superheated steam . This raises the question whether the spores of Bacillus stearothermophilus, with their weaker reaction to the superheating of steam, are suitable as test germs for sterilisation with steam in all cases.

Farmaco, 1998 Aug-Sep, 53(8-9), 623 - 5
Hydroxyamino sugar derivatives: sugar nitrones; Zosimo-Landolfo G et al.; We describe in this paper the preparation of 46 new sugar nitrone derivatives and their antibacterial activity against Escherichia coli and Bacillus subtilis.

FEMS Microbiol Rev, 1999 Jan, 23(1), 1 - 11
FtsH--a single-chain charonin?
Schumann W.
The ftsH gene encodes an ATP- and Zn(2+)-dependent metalloprotease with a molecular mass of about 70 kDa . It was first identified in Escherichia coli where it is also designated hflB, tolZ or mrsC, and seems to be present in most if not all bacteria . The FtsH protein is anchored to the cytoplasmic membrane via two transmembrane regions in such a way that the very short amino- and the long carboxy-termini are exposed into the cytoplasm . FtsH is member of the AAA family (ATPases associated with a variety of cellular activities) which are characterized by a module of about 200 amino acid residues in length containing an ATP-binding site . In Escherichia coli, FtsH forms a complex with a pair of periplasmically exposed membrane proteins, HflK and HflC . The E . coli enzyme is required for proteolytic degradation of some unstable proteins that include both soluble regulatory proteins such as sigma 32 (heat-shock sigma factor) and phage lambda CII (transcriptional activator), and membrane proteins including uncomplexed forms of SecY (forms the translocon together with SecE and SecG) and the a subunit of the F0 complex of the H(+)-ATPase . Its activity can be modulated by the HflKC proteins, by another membrane protein designated YccA which can transiently associate with both the FtsH and the HflKC proteins, or by small peptides such as CIII encoded by phage lambda (involved in lysogenization) or SpoVM (needed for sporulation) encoded by Bacillus subtilis . Besides being a protease, there is circumstantial evidence that FtsH also acts as a molecular chaperone . It influences protein assembly in and through the cytoplasmic membrane and associates with denatured alkaline phosphatase without degrading it . Therefore, FtsH may serve to maintain quality control of some cytoplasmic and membrane proteins . Such ATP-dependent proteases with intrinsic chaperone activity have been designated charonins.

Appl Microbiol Biotechnol, 1999 Jan, 51(1), 85 - 90
A novel mutation, of the Bacillus subtilis hut operon that relieves both catabolite repression and amino acid repression; Eda S et al.; A mutation, designated hutCR11, which resulted in high expression of the hut operon and release of the catabolite repression and amino-acid repression of hut expression, was isolated and determined to be a T-to-G transversion at position +30 (+1 indicates the transcription-initiation site) . In the hutCR11 mutant, levels of hutP mRNA were 5-fold higher than those in wild-type cells under conditions of non-induction and induction and 11-fold higher under conditions of catabolite repression and amino-acid repression . Mutation analysis showed that two types of base change (T-->A and T-->C) at position +30 did not cause high expression of the hut operon, indicating that this was specifically caused by the single base substitution (T-->G) at position +30 . The base substitution of A for T at position +30 also led to partial relief of both catabolite repression and amino-acid repression . These results indicate that the nucleotide sequence at +30 is important for regulation of both catabolite repression and amino-acid repression of the hut operon.

J Biol Chem, 1999 Mar 19, 274(12), 8322 - 7
Negative regulation by the Bacillus subtilis GerE protein; Ichikawa H et al.; GerE is a transcription factor produced in the mother cell compartment of sporulating Bacillus subtilis . It is a critical regulator of cot genes encoding proteins that form the spore coat late in development . Most cot genes, and the gerE gene, are transcribed by sigmaK RNA polymerase . Previously, it was shown that the GerE protein inhibits transcription in vitro of the sigK gene encoding sigmaK . Here, we show that GerE binds near the sigK transcriptional start site, to act as a repressor . A sigK-lacZ fusion containing the GerE-binding site in the promoter region was expressed at a 2-fold lower level during sporulation of wild-type cells than gerE mutant cells . Likewise, the level of SigK protein (i . e . pro-sigmaK and sigmaK) was lower in sporulating wild-type cells than in a gerE mutant . These results demonstrate that sigmaK-dependent transcription of gerE initiates a negative feedback loop in which GerE acts as a repressor to limit production of sigmaK . In addition, GerE directly represses transcription of particular cot genes . We show that GerE binds to two sites that span the -35 region of the cotD promoter . A low level of GerE activated transcription of cotD by sigmaK RNA polymerase in vitro, but a higher level of GerE repressed cotD transcription . The upstream GerE-binding site was required for activation but not for repression . These results suggest that a rising level of GerE in sporulating cells may first activate cotD transcription from the upstream site then repress transcription as the downstream site becomes occupied . Negative regulation by GerE, in addition to its positive effects on transcription, presumably ensures that sigmaK and spore coat proteins are synthesized at optimal levels to produce a germination-competent spore.

J Biol Chem, 1999 Mar 19, 274(12), 7901 - 6
Specific binding of the E2 subunit of pyruvate dehydrogenase to the upstream region of Bacillus thuringiensis protoxin genes; Walter T et al.; During sporulation, Bacillus thuringiensis produces inclusions comprised of different amounts of several related protoxins, each with a unique specificity profile for insect larvae . A major class of these genes designated cry1 have virtually identical dual overlapping promoters, but the upstream sequences differ . A gel retardation assay was used to purify a potential regulatory protein which bound with different affinities to these sequences in three cry1 genes . It was identified as the E2 subunit of pyruvate dehydrogenase . There was specific competition for binding by homologous gene sequences but not by pUC nor Bacillus subtilis DNA; calf thymus DNA competed at higher concentrations . The B . thuringiensis gene encoding E2 was cloned, and the purified glutathione S-transferase-E2 fusion protein footprinted to a consensus binding sequence within an inverted repeat and to a potential bend region, both sites 200-300 base pairs upstream of the promoters . Mutations of these sites in the cry1A gene resulted in decreased binding of the E2 protein and altered kinetics of expression of a fusion of this regulatory region with the lacZ gene . Recruitment of the E2 subunit as a transcription factor could couple the change in post exponential catabolism to the initiation of protoxin synthesis.

Biochemistry, 1999 Mar 9, 38(10), 2882 - 91
Microsecond folding of the cold shock protein measured by a pressure-jump technique; Jacob M et al.; A pressure-jump apparatus was employed in investigating the kinetics of protein unfolding and refolding . In the reaction cell, the pressure can be increased or decreased by 100-160 bar within 50-100 microseconds and then held constant . Thus, unfolding and refolding reactions in the time range from 70 microseconds to 70 s can be followed with this technique . Measurements are possible in the transition regions of thermally or denaturant-induced folding in a wide range of temperatures and solvent conditions . We used this pressure-jump method to determine the temperature dependence of the rate constants of unfolding and refolding of the cold shock protein of Bacillus subtilis and of three variants thereof with Phe --> Ala substitutions in the central beta-sheet region . For all variants, the change in heat capacity occurred in refolding between the unfolded and activated states, suggesting that the overall native-like character of the activated state of folding was not changed by the deletion of individual Phe side chains . The Phe27Ala mutation affected the rate of unfolding only; the Phe15Ala and Phe17Ala mutations changed the kinetics of both unfolding and refolding . Although the activated state of folding of the cold shock protein is overall native-like, individual side chains are still in a non-native environment.

J Bacteriol, 1999 Mar, 181(6), 1971 - 4
Construction and analysis of hybrid Escherichia coli-Bacillus subtilis dnaK genes; Mogk A et al.; The highly conserved DnaK chaperones consist of an N-terminal ATPase domain, a central substrate-binding domain, and a C-terminal domain whose function is not known . Since Bacillus subtilis dnaK was not able to complement an Escherichia coli dnaK null mutant, we performed domain element swap experiments to identify the regions responsible for this finding . It turned out that the B . subtilis DnaK protein needed approximately normal amounts of the cochaperone DnaJ to be functional in E . coli . The ATPase domain and the substrate-binding domain form a species-specific functional unit, while the C-terminal domains, although less conserved, are exchangeable . Deletion of the C-terminal domain in E . coli DnaK affected neither complementation of growth at high temperatures nor propagation of phage lambda but abolished degradation of sigma32.

J Bacteriol, 1999 Mar, 181(6), 1939 - 43
SodA and manganese are essential for resistance to oxidative stress in growing and sporulating cells of Bacillus subtilis; Inaoka T et al.; We constructed a sodA-disrupted mutant of Bacillus subtilis 168, BK1, by homologous recombination . The mutant was not able to grow in minimal medium without Mn(II) . The spore-forming ability of strain BK1 was significantly lower in Mn(II)-depleted medium than that of the wild-type strain . These deleterious effects caused by the sodA mutation were reversed when an excess of Mn(II) was used to supplement the medium . Moreover, the growth inhibition by superoxide generators in strain BK1 and its parent strain was also reversed by the supplementation with excess Mn(II) . We therefore estimated the Mn-dependent superoxide-scavenging activity in BK1 cells . Whereas BK1 cells have no detectable superoxide dismutase (Sod) on native gel, the superoxide-scavenging activity in crude extracts of BK1 cells grown in Mn(II)-supplemented LB medium (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl per liter) was significantly detected by the modified Sod assay method without using EDTA . The results obtained suggest that Mn, as a free ion or a complex with some cellular component, can catalyze the elimination of superoxide and that both SodA and Mn(II) are involved not only in the superoxide resistance of vegetative cells but also in sporulation.

J Bacteriol, 1999 Mar, 181(6), 1820 - 6
Differential dependence of levansucrase and alpha-amylase secretion on SecA (Div) during the exponential phase of growth of Bacillus subtilis; Leloup L et al.; SecA, the translocation ATPase of the preprotein translocase, accounts for 0.25% of the total protein in a degU32(Hy) Bacillus subtilis strain in logarithmic phase . The SecA level remained constant irrespective of the demand for exoprotein production but dropped about 12-fold during the late stationary phase . Modulation of the level of functional SecA during the exponential phase of growth affected differently the secretion of levansucrase and alpha-amylase overexpressed under the control of the sacB leader region . The level of SecA was reduced in the presence of sodium azide and in the div341 thermosensitive mutant at nonpermissive temperatures . Overproduction of SecA was obtained with a multicopy plasmid bearing secA . The gradual decrease of the SecA level reduced the yield of secreted levansucrase with a concomitant accumulation of unprocessed precursor in the cells, while an increase in the SecA level resulted in an elevation of the production of exocellular levansucrase . In contrast, alpha-amylase secretion was almost unaffected by high concentrations of sodium azide or by very low levels of SecA . Secretion defects were apparent only under conditions of strong SecA deprivation of the cell . These data demonstrate that the alpha-amylase and levansucrase precursors markedly differ in their dependency on SecA for secretion . It is suggested that these precursors differ in their binding affinities for SecA.

J Bacteriol, 1999 Mar, 181(6), 1786 - 92
Functional identification of the product of the Bacillus subtilis yvaL gene as a SecG homologue; van Wely KH et al.; Protein export in Escherichia coli is mediated by translocase, a multisubunit membrane protein complex with SecA as the peripheral subunit and the SecY, SecE, and SecG proteins as the integral membrane domain . In the gram-positive bacterium Bacillus subtilis, SecA, SecY, and SecE have been identified through genetic analysis . Sequence comparison of the Bacillus chromosome identified a potential homologue of SecG, termed YvaL . A chromosomal disruption of the yvaL gene results in mild cold sensitivity and causes a beta-lactamase secretion defect . The cold sensitivity is exacerbated by overexpression of the secretory protein alpha-amylase, whereas growth and beta-lactamase secretion are restored by coexpression of yvaL or the E . coli secG gene . These results indicate that the yvaL gene codes for a protein that is functionally homologous to SecG.

J Bacteriol, 1999 Mar, 181(6), 1719 - 27
Identification and characterization of a DeoR-specific operator sequence essential for induction of dra-nupC-pdp operon expression in Bacillus subtilis; Zeng X et al.; The deoR gene located just upstream the dra-nupC-pdp operon of Bacillus subtilis encodes the DeoR repressor protein that negatively regulates the expression of the operon at the level of transcription . The control region upstream of the operon was mapped by the use of transcriptional lacZ fusions . It was shown that all of the cis-acting elements, which were necessary for full DeoR regulation of the operon, were included in a 141-bp sequence just upstream of dra . The increased copy number of this control region resulted in titration of the DeoR molecules of the cell . By using mutagenic PCR and site-directed mutagenesis techniques, a palindromic sequence located from position -60 to position -43 relative to the transcription start point was identified as a part of the operator site for the binding of DeoR . Furthermore, it was shown that a direct repeat of five nucleotides, which was identical to the 3' half of the palindrome and was located between the -10 and -35 regions of the dra promoter, might function as a half binding site involved in cooperative binding of DeoR to the regulatory region . Binding of DeoR protein to the operator DNA was confirmed by a gel electrophoresis mobility shift assay . Moreover, deoxyribose-5-phosphate was shown to be a likely candidate for the true inducer of the dra-nupC-pdp expression.

Gene, 1999 Mar 4, 228(1-2), 123 - 32
DnaA proteins of Escherichia coli and Bacillus subtilis: coordinate actions with single-stranded DNA-binding protein and interspecies inhibition during open complex formation at the replication origins; Krause M et al.; DnaA-mediated unwinding of the AT-rich region in the replication origins of Escherichia coli and Bacillus subtilis was analysed in vitro with and without single-stranded DNA-binding protein (SSB) . In the presence of SSB, the unwound region was larger by a defined number of base pairs . Although the overall structure of the origins is very different, the size and structure of the unwound region were similar . The unwinding reaction at oriC of one organism was inhibited by DnaA protein of the other bacterium . Similarly, hybrid DnaA proteins with swapped DNA-binding domains were inactive and inhibitory to 'open complex' formation at both origins . We suggest that the inhibition is due to inactive mixed complexes.

J Protein Chem, 1999 Jan, 18(1), 21 - 7
Bacillus intermedius glutamyl endopeptidase . Molecular cloning and nucleotide sequence of the structural gene; Rebrikov DV et al.; The glutamyl endopeptidase gene of Bacillus intermedius was cloned from a genomic library expressed in Bacillus subtilis and sequenced (EMBL accession number Y15136) . The encoded preproenzyme contains 303 amino acid residues; the mature 23-kDa enzyme consists of 215 residues . The mature enzyme reveals 38% of identical residues when aligned with the glutamyl endopeptidase from Bacillus licheniformis, whereas only five invariant residues were found among all known glutamyl endopeptidases . The amino acid residues that form the catalytic triad (H47, D98, and S171) as well as H186 participating in the binding of the substrate carboxyl group were identified . It seems that the structural elements responsible for the function of glutamyl endopeptidases from various sources are highly variable.

Arch Biochem Biophys, 1999 Mar 15, 363(2), 273 - 82
Molecular approaches to probe differential NADH activation of phosphoribulokinase isozymes from Rhodobacter sphaeroides; Novak JS et al.; The cbbPI and cbbPII genes from Rhodobacter sphaeroides, encoding highly similar phosphoribulokinase (PRK) isozymes, PRK I and PRK II, respectively, exhibited differential allosteric activation by NADH . The two cbbP genes were cloned into expression vectors and homogeneous recombinant protein prepared . PRK II was found to be inherently less stable than PRK I; however, the addition of substrate ATP resulted in the complete protection of both isozymes to a 15-min incubation at 50 degrees C . The relative molecular masses for both octameric isozymes were determined to be approximately 230,000; however, the protective effect of ATP was in accordance with aggregation of monomers to a molecular mass of approximately 750,000 . While PRK I exhibited a nearly absolute dependence upon NADH for activity, PRK II retained substantial activity in the absence of NADH . PRK chimeras were thus constructed to facilitate elucidation of the basis for the differential effect of NADH, with advantage taken of the relative sequence identity of about 90% between the two isozymes . Chimeras were constructed either by in vivo homologous recombination, using the sacB gene from Bacillus subtilis as a conditionally lethal marker, or by using convenient restriction sites to combine different parts of the two cbbP genes . The PRK chimeras generated contained either the amino-terminal domain of PRK II and the carboxy-terminal domain of PRK I or the opposite configuration . Subsequent analyses of the chimeras pointed to particular regions and residue(s) as likely being important for NADH activation .

Microbiol Mol Biol Rev, 1999 Mar, 63(1), 1 - 20
Bacillus subtilis spore coat; Driks A; In response to starvation, bacilli and clostridia undergo a specialized program of development that results in the production of a highly resistant dormant cell type known as the spore . A proteinacious shell, called the coat, encases the spore and plays a major role in spore survival . The coat is composed of over 25 polypeptide species, organized into several morphologically distinct layers . The mechanisms that guide coat assembly have been largely unknown until recently . We now know that proper formation of the coat relies on the genetic program that guides the synthesis of spore components during development as well as on morphogenetic proteins dedicated to coat assembly . Over 20 structural and morphogenetic genes have been cloned . In this review, we consider the contributions of the known coat and morphogenetic proteins to coat function and assembly . We present a model that describes how morphogenetic proteins direct coat assembly to the specific subcellular site of the nascent spore surface and how they establish the coat layers . We also discuss the importance of posttranslational processing of coat proteins in coat morphogenesis . Finally, we review some of the major outstanding questions in the field.

Curr Opin Microbiol, 1998 Dec, 1(6), 630 - 5
Cell cycle and sporulation in Bacillus subtilis; Levin PA et al.; Recent work on cell division and chromosome orientation and partitioning in Bacillus subtilis has provided insights into cell cycle regulation during growth and development . The cell cycle is an integral part of development and entrance into sporulation is modulated by signals that transmit the status of DNA integrity, chromosome replication and segregation . In addition, B . subtilis modifies cell division and DNA segregation to establish cell-type-specific gene expression during sporulation.

J Mol Biol, 1999 Mar 12, 286(5), 1325 - 35
Site-directed mutants of RTP of Bacillus subtilis and the mechanism of replication fork arrest; Duggin IG et al.; DNA replication fork arrest during the termination phase of chromosome replication in Bacillus subtilis is brought about by the replication terminator protein (RTP) bound to specific DNA terminator sequences (Ter sites) distributed throughout the terminus region . An attractive suggestion by others was that crucial to the functioning of the RTP-Ter complex is a specific interaction between RTP positioned on the DNA and the helicase associated with the approaching replication fork . In support of this was the behaviour of two site-directed mutants of RTP . They appeared to bind Ter DNA normally but were ineffective in fork arrest as ascertained by in vitro Escherichia coli DnaB helicase and replication assays . We describe here a system for assessing the fork-arrest behaviour of RTP mutants in a bona fide in vivo assay in B . subtilis . One of the previously studied mutants, RTP.Y33N, was non-functional in fork arrest in vivo, as predicted . But through extensive analyses, this RTP mutant was shown to be severely defective in binding to Ter DNA, contrary to expectation . Taken in conjunction with recent findings on the other mutant (RTP.E30K), it is concluded that there is as yet no substantive evidence from the behaviour of RTP mutants to support the RTP-helicase interaction model for fork arrest . In an extension of the present work on RTP.Y33N, we determined the dissociation rates of complexes formed by wild-type (wt) RTP and another RTP mutant with various terminator sequences . The functional wtRTP-TerI complex was quite stable (half-life of 182 minutes), reminiscent of the great stability of the E . coli Tus-Ter complex . More significant were the exceptional stabilities of complexes comprising wtRTP and an RTP double-mutant (E39K.R42Q) bound to some particular terminator sequences . From the measurement of in vivo fork-arrest activities of the various complexes, it is concluded that the stability (half-life) of the whole RTP-Ter complex is not the overriding determinant of arrest, and that the RTP-Ter complex must be actively disrupted, or RTP removed, by the action of the approaching replication fork .

Appl Environ Microbiol, 1999 Mar, 65(3), 1316 - 9
Heat resistance of native and demineralized spores of Bacillus subtilis sporulated at different temperatures; Palop A et al.; Demineralization reduced heat resistance of B . subtilis spores, but the pattern and magnitude of the reduction depended on sporulation temperature and on heating menstruum pH . The differences in heat resistance of native spores caused by sporulation temperature almost disappeared after demineralization . Demineralized spores were still susceptible to the heat-sensitizing effect of acidic pH.

Biochem Biophys Res Commun, 1999 Feb 16, 255(2), 444 - 50
A bacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells; Ailor E et al.; The production of an antibody single chain fragment (scFv) in insect cells was accompanied by the formation of an insoluble intracellular precursor even with the inclusion of the bee melittin signal peptide . The presence of the precursor polypeptide suggests a limitation in the processing of the signal peptide so a baculovirus containing a signal peptidase from Bacillus subtilis (SipS) was constructed for expression studies . When the wild type SipS was coexpressed with scFv, preprocessed scFv fragments were no longer detected in insect cell lysates . Conversely, coexpression of scFv alone or with an inactive mutant SipS resulted in at least 30% of the intracellular polypeptide in an unprocessed form at 3 days post infection . Production of scFv in the medium was also enhanced in the presence of SipS; however, low secretion levels indicate the presence of a post-processing bottleneck .

J Bacteriol, 1999 Mar, 181(5), 1664 - 72
Secretion, localization, and antibacterial activity of TasA, a Bacillus subtilis spore-associated protein; Stover AG et al.; The synthesis and subcellular localization of the proteins that comprise the Bacillus subtilis spore are under a variety of complex controls . To better understand these controls, we have identified and characterized a 31-kDa sporulation protein, called TasA, which is secreted into the culture medium early in sporulation and is also incorporated into the spore . TasA synthesis begins approximately 30 min after the onset of sporulation and requires the sporulation transcription factor genes spo0H and spo0A . The first 81 nucleotides of tasA encode a 27-amino-acid sequence that resembles a signal peptide and which is missing from TasA isolated from a sporulating cell lysate . In B . subtilis cells unable to synthesize the signal peptidase SipW, TasA is not secreted, nor is it incorporated into spores . Cells unable to produce SipW produce a 34-kDa form of TasA, consistent with a failure to remove the N-terminal 27 amino acids . In cells engineered to express sipW and tasA during exponential growth, TasA migrates as a 31-kDa species and is secreted into the culture medium . These results indicate that SipW plays a crucial role in the export of TasA out of the cell and its incorporation into spores . Although TasA is dispensable for sporulation under laboratory conditions, we find that TasA has a broad-spectrum antibacterial activity . We discuss the possibility that during the beginning of sporulation as well as later, during germination, TasA inhibits other organisms in the environment, thus conferring a competitive advantage to the spore.

J Bacteriol, 1999 Mar, 181(5), 1429 - 35
Analysis of 4-phosphopantetheinylation of polyhydroxybutyrate synthase from Ralstonia eutropha: generation of beta-alanine auxotrophic Tn5 mutants and cloning of the panD gene region; Hoppensack A et al.; The postulated posttranslational modification of the polyhydroxybutyrate (PHA) synthase from Ralstonia eutropha by 4-phosphopantetheine was investigated . Four beta-alanine auxotrophic Tn5-induced mutants of R . eutropha HF39 were isolated, and two insertions were mapped in an open reading frame with strong similarity to the panD gene from Escherichia coli, encoding L-aspartate-1-decarboxylase (EC 4.1.1.15), whereas two other insertions were mapped in an open reading frame (ORF) with strong similarity to the NAD(P)+ transhydrogenase (EC 1.6.1.1) alpha 1 subunit, encoded by the pntAA gene from Escherichia coli . The panD gene was cloned by complementation of the panD mutant of R . eutropha Q20 . DNA sequencing of the panD gene region (3,312 bp) revealed an ORF of 365 bp, encoding a protein with 63 and 67% amino acid sequence similarity to PanD from E . coli and Bacillus subtilis, respectively . Subcloning of only this ORF into vectors pBBR1MCS-3 and pBluescript KS- led to complementation of the panD mutants of R . eutropha and E . coli SJ16, respectively . panD-encoded L-aspartate-1-decarboxylase was further confirmed by an enzymatic assay . Upstream of panD, an ORF with strong similarity to pntAA from E . coli, encoding NAD(P)+ transhydrogenase subunit alpha 1 was found; downstream of panD, two ORFs with strong similarity to pntAB and pntB, encoding subunits alpha 2 and beta of the NAD(P)+ transhydrogenase, respectively, were identified . Thus, a hitherto undetermined organization of pan and pnt genes was found in R . eutropha . Labeling experiments using one of the R . eutropha panD mutants and {2-14C}beta-alanine provided no evidence that R . eutropha PHA synthase is covalently modified by posttranslational attachment of 4-phosphopantetheine, nor did the E . coli panD mutant exhibit detectable labeling of functional PHA synthase from R . eutropha.

J Bacteriol, 1999 Mar, 181(5), 1403 - 8
Mutational analysis of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing; Li S et al.; Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is a member of an N-terminal nucleophile hydrolase enzyme superfamily, several of which undergo autocatalytic propeptide processing to generate the mature active enzyme . A series of mutations was analyzed to determine whether amino acid residues required for catalysis are also used for propeptide processing . Propeptide cleavage was strongly inhibited by replacement of the cysteine nucleophile and two residues of an oxyanion hole that are required for glutaminase function . However, significant propeptide processing was retained in a deletion mutant with multiple defects in catalysis that was devoid of enzyme activity . Intermolecular processing of noncleaved mutant enzyme subunits by active wild-type enzyme subunits was not detected in hetero-oligomers obtained from a coexpression experiment . While direct in vitro evidence for autocatalytic propeptide cleavage was not obtained, the results indicate that some but not all of the amino acid residues that have a role in catalysis are also needed for propeptide processing.

Genes Dev, 1999 Feb 15, 13(4), 394 - 9
Transient gene asymmetry during sporulation and establishment of cell specificity in Bacillus subtilis; Frandsen N et al.; Sporulation in Bacillus subtilis is initiated by an asymmetric division generating two cells of different size and fate . During a short interval, the smaller forespore harbors only 30% of the chromosome until the remaining part is translocated across the septum . We demonstrate that moving the gene for sigmaF, the forespore-specific transcription factor, in the trapped region of the chromosome is sufficient to produce spores in the absence of the essential activators SpoIIAA and SpoIIE . We propose that transient genetic asymmetry is the device that releases SpoIIE phosphatase activity in the forespore and establishes cell specificity.

Antimicrob Agents Chemother, 1999 Mar, 43(3), 655 - 60
Esterases in serum-containing growth media counteract chloramphenicol acetyltransferase activity in vitro; Sohaskey CD et al.; The spirochete Borrelia burgdorferi was unexpectedly found to be as susceptible to diacetyl chloramphenicol, the product of the enzyme chloramphenicol acetyltransferase, as it was to chloramphenicol itself . The susceptibilities of Escherichia coli and Bacillus subtilis, as well as that of B . burgdorferi, to diacetyl chloramphenicol were then assayed in different media . All three species were susceptible to diacetyl chloramphenicol when growth media were supplemented with rabbit serum or, to a lesser extent, human serum . Susceptibility of E . coli and B . subtilis to diacetyl chloramphenicol was not observed in the absence of serum, when horse serum was used, or when the rabbit or human serum was heated first . In the presence of 10% rabbit serum, a strain of E . coli bearing the chloramphenicol acetyltransferase (cat) gene had a fourfold-lower resistance to chloramphenicol than in the absence of serum . A plate bioassay for chloramphenicol activity showed the conversion by rabbit, mouse, and human sera but not bacterial cell extracts or heated serum of diacetyl chloramphenicol to an inhibitory compound . Deacetylation of acetyl chloramphenicol by serum components was demonstrated by using fluorescent substrates and thin-layer chromatography . These studies indicate that esterases of serum can convert diacetyl chloramphenicol back to an active antibiotic, and thus, in vitro findings may not accurately reflect the level of chloramphenicol resistance by cat-bearing bacteria in vivo.

Protein Sci, 1999 Feb, 8(2), 394 - 403
Cloning, overexpression, purification, and physicochemical characterization of a cold shock protein homolog from the hyperthermophilic bacterium Thermotoga maritima; Welker C et al.; Thermotoga maritima (Tm) expresses a 7 kDa monomeric protein whose 18 N-terminal amino acids show 81% identity to N-terminal sequences of cold shock proteins (Csps) from Bacillus caldolyticus and Bacillus stearothermophilus . There were only trace amounts of the protein in Thermotoga cells grown at 80 degrees C . Therefore, to perform physicochemical experiments, the gene was cloned in Escherichia coli . A DNA probe was produced by PCR from genomic Tm DNA with degenerated primers developed from the known N-terminus of TmCsp and the known C-terminus of CspB from Bacillus subtilis . Southern blot analysis of genomic Tm DNA allowed to produce a partial gene library, which was used as a template for PCRs with gene- and vector-specific primers to identify the complete DNA sequence . As reported for other csp genes, the 5' untranslated region of the mRNA was anomalously long; it contained the putative Shine-Dalgarno sequence . The coding part of the gene contained 198 bp, i.e., 66 amino acids . The sequence showed 61% identity to CspB from B . caldolyticus and high similarity to all other known Csps . Computer-based homology modeling allowed the conclusion that TmCsp represents a beta-barrel similar to CspB from B . subtilis and CspA from E . coli . As indicated by spectroscopic analysis, analytical gel permeation chromatography, and mass spectrometry, overexpression of the recombinant protein yielded authentic TmCsp with a molecular weight of 7,474 Da . This was in agreement with the results of analytical ultracentrifugation confirming the monomeric state of the protein . The temperature-induced equilibrium transition at 87 degrees C exceeds the maximum growth temperature of Tm and represents the maximal Tm-value reported for Csps so far.

Mol Microbiol, 1999 Feb, 31(3), 995 - 1006
Regulation of the activity of the Bacillus subtilis antiterminator LicT by multiple PEP-dependent, enzyme I- and HPr-catalysed phosphorylation; Lindner C et al.; The transcriptional antiterminator LicT regulates the induction and carbon catabolite repression of the Bacillus subtilis bglPH operon . LicT is inactive in mutants affected in one of the two general components of the phosphoenolpyruvate (PEP):glycose phosphotransferase system, enzyme I or histidine-containing protein (HPr) . We demonstrate that LicT becomes phosphorylated in the presence of PEP, enzyme I and HPr . The phosphoryl group transfer between HPr and LicT is reversible . Phosphorylation of LicT with PEP, enzyme I and HPr led to the appearance of three additional LicT bands on polyacrylamide-urea gels . These bands probably correspond to one-, two- and threefold phosphorylated LicT . After phosphorylation of LicT with {32P}-PEP, enzyme I and HPr, proteolytic digestion of {32P}-P-LicT, separation of the peptides by reverse-phase chromatography, mass spectrometry and N-terminal sequencing of radiolabelled peptides, three histidyl residues were found to be phosphorylated in LicT . These three histidyl residues (His-159, His-207 and His-269) are conserved in most members of the BglG/SacY family of transcriptional antiterminators . Phosphorylation of LicT in the presence of serylphosphorylated HPr (P-Ser-HPr) was much slower compared with its phosphorylation in the presence of HPr . The slower phosphorylation in the presence of P-Ser-HPr leading to reduced LicT activity is presumed to play a role in a recently described LicT-mediated CcpA-independent carbon catabolite repression mechanism operative for the bglPH operon.

Mol Microbiol, 1999 Feb, 31(3), 949 - 58
A 'gram-negative-type' DNA polymerase III is essential for replication of the linear chromosome of Streptomyces coelicolor A3(2); Flett F et al.; The Streptomyces coelicolor dnaE gene, encoding the catalytic alpha-subunit of DNA polymerase III (pol III) was isolated by genetic complementation of a temperature-sensitive DNA replication mutant, S . coelicolor ts-38 . The deduced protein sequence (1179 residues) is highly similar to the Escherichia coli-type pol III alpha-subunit, rather than to the PolC-type alpha-subunit that is known to be essential for replication in the 'low G + C' Gram-positive bacteria such as Bacillus subtilis . The dnaE gene is able to restore replication to a 'slow stop' mutant (ts-38) and a 'fast stop' mutant (ts-114); the dnaE gene of ts-38 carries a single amino acid substitution (Glu-802 to Lys), and the mutation in ts-114 has been mapped between codons 697 and 1062 of dnaE . Mutant ts-38 is considered to be defective in assembly of the multisubunit pol III holoenzyme and, hence, in initiation of replication, whereas ts-114 is defective in chain elongation . This study provides the first evidence that a DnaE-type pol III is essential for replication in a Gram-positive bacterium . In addition, the complementation studies suggest that the C-terminal 117 residues are not essential for DnaE function in S . coelicolor . When integrated at a distant site on the chromosome, a fragment containing the 3' half of dnaE(codons 697-1179) is capable of rescuing ts-38 (but not ts-114) at the restrictive temperature; it was demonstrated that homogenotization was responsible for this phenomenon.

Mol Microbiol, 1999 Feb, 31(3), 795 - 805
Teichuronic acid operon of Bacillus subtilis 168; Soldo B et al.; Sequence analysis reveals that the Bacillus subtilis 168 tuaABCDEFGH operon encodes enzymes required for the polymerization of teichuronic acid as well as for the synthesis of one of its precursors, the UDP-glucuronate . Mutants deficient in any of the tua genes, grown in batch cultures under conditions of phosphate limitation, were characterized by reduced amounts of uronate in their cell walls . The teichuronic acid operon belongs to the Pho regulon, as phosphate limitation induces its transcription . Placing the tuaABCDEFGH operon under the control of the inducible Pspac promoter allowed its constitutive expression independently of the phosphate concentration in the medium; the level of uronic acid in cell walls was dependent on the concentration of the inducer . Apparently, owing to an interdependence between teichoic and teichuronic acid incorporation into the cell wall, in examined growth conditions, the balance between the two polymers is maintained in order to insure a constant level of the wall negative charge.

Bioseparation, 1998, 7(3), 159 - 65
Alginate beads as an affinity material for alpha amylases; Sardar M et al.; Calcium-alginate beads were found to bind a variety of enzymes in a nonspecific fashion . However, alpha amylases from porcine pancreas, Bacillus subtilis (BAN 240L) and wheat germ bound at a significant level and B . subtilis and wheat germ amylases could be eluted with 1M maltose . The wheat germ alpha amylase could be purified 45 fold with 70% recovery . The SDS-PAGE pattern showed significant purification by this single step strategy.

Biochemistry, 1999 Feb 23, 38(8), 2287 - 94
Effects of site-directed mutagenesis of protolytic residues in subunit I of Bacillus subtilis aa3-600 quinol oxidase . Role of lysine 304 in proton translocation; Villani G et al.; Various protolytic residues in subunit I of aa3-600 quinol oxidase of the aerobic Gram-positive Bacillus subtilis were mutagenized to nonpolar residues . Two of the mutations, Y284F and K304L, impaired the bioenergetic function of the microorganism . The Y284F mutation suppressed the electron-transfer activity of quinol oxidase and altered its interaction with CO and H2O2, thus showing destruction of the binuclear domain as observed for the bo3 quinol oxidase of Escherichia coli . The K304L mutation did not alter significantly the redox activity of the oxidase and its interaction with CO and H2O2 but suppressed the proton pumping activity of the enzyme . These results show that the K304 residue, which is invariantly conserved (as K or R) in practically all the sequences of the heme-copper oxidases so far available (around 100), is essential for the proton pumping activity of the oxidase.

J AOAC Int, 1999 Jan-Feb, 82(1), 151 - 9
Sporicidal testing of commercial germicides using a chemical standard and a calibrated bioindicator; Danielson JW et al.; The AOAC sporicidal method uses as a standard the resistance of spores on carriers to 2.5N HCl . This resistance is variable at exposure times ranging from 2 to 20 min . The method described in this paper uses a glutaraldehyde standard and distinguishes various levels of sporicidal activity in the presence of 1-5% glutaraldehyde by using appropriate spore strains, spore preparations, and spore levels . The resistances of 2 Bacillus subtilis 19659 spore preparations cultured in 10% Columbia broth plus manganese and nutrient agar plus minerals, as well as that of B . subtilis var . niger cultured on Lab-Lemco agar, were tested . T-soy broth was a better recovery medium than fluid thioglycollate or modified fluid thioglycollate for B . subtilis 19659 spores exposed to HCl . Sporicidal tests were done on B . subtilis 19659 spores with 2 types of spore preparations . A commercial glutaraldehyde germicide was used for comparison of the sporicidal activity of the glutaraldehyde standard . Two strains of B . subtilis spores and 4 levels of spores (20,000-80,000, 100,000-400,000, 500,000-800,000, and 1,000,000 and up) were removed from check penicylinders from the same batches used for sporicidal tests . B . subtilis var . niger spores were the most resistant to HCl, while B . subtilis 19659 spores were more resistant to glutaraldehyde . Sporicidal activities of a commercial germicide containing 2.5% glutaraldehyde with additives and another containing 5% glutaraldehyde in phosphate buffer were similar . Both totally destroyed high levels of B . subtilis 19659 spores cultured in 10% Columbia broth plus manganese . Results indicate that use of a glutaraldehyde standard, calibrated numbers of spores on penicylinders (bioindicators), and appropriate spore strains and preparations can reduce the variability of sporicidal testing of commercial germicides.

Mol Microbiol, 1999 Jan, 31(2), 651 - 63
Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis; Ingham CJ et al.; The expression and activity of transcription termination factor Rho and the requirement for transcription elongation factors NusA and NusG was investigated in Bacillus subtilis . Rho was present at < 5% of the level found in Escherichia coli, but Rho factors from these two bacteria had similar properties as RNA-activated ATPases and in vitro termination of transcription on the lambda tR1 terminator . The B . subtilis rho gene was autoregulated at the level of transcription; autoregulation required sequences within the rho mRNA leader region and gene . To date, the B . subtilis rho is the only gene from a Gram-positive bacterium found to be regulated by Rho . Rho was not involved in bulk mRNA decay in B . subtilis . The E . coli elongation factors NusA and NusG target Rho, and the importance of these proteins in B . subtilis was examined by gene disruption . The B . subtilis NusG was inessential for both the viability and the autoregulation of Rho, whereas NusA was essential, and the requirement for NusA was independent of Rho . This contrasts with E . coli in which NusG is essential but NusA becomes dispensable if Rho terminates transcription less efficiently.

Mol Microbiol, 1999 Jan, 31(2), 597 - 607
The Spo0A sof mutations reveal regions of the regulatory domain that interact with a sensor kinase and RNA polymerase; Cervin MA et al.; Spo0A is a two-domain response regulator required for the initiation of sporulation in Bacillus subtilis . Spo0A is activated by phosphorylation of its regulatory domain by a multicomponent phosphorelay . To define the role of the regulatory domain in the activation of Spo0A, we have characterized four of the sof mutations in vitro . The sof mutations were identified previously as suppressors of the sporulation-negative phenotype resulting from a deletion of the gene for one of the phosphorelay components, spo0F . Like wild-type Spo0A, the transcription stimulation properties of all of the Sof proteins were dependent upon phosphorylation . Sof mutants from two classes were improved substrates for direct phosphorylation by the KinA sensor kinase, providing an explanation for their suppression properties . Two other Sof proteins showed a phosphorylation-dependent enhancement of the stability of the Sof approximately P-RNA polymerase-DNA complex . One of these mutants, Sof114, increased the stability of the Sof114 approximately P-RNAP-DNA complex without increasing its own affinity for the spoIIG promoter . A comparison of the location of the sof mutations with mutations in CheY suggests that phosphorylation of Spo0A results in the exposure of a region in the regulatory domain that interacts with RNA polymerase, thereby contributing to the signal transduction mechanism.

Mol Microbiol, 1999 Jan, 31(2), 533 - 43
Ecs, an ABC transporter of Bacillus subtilis: dual signal transduction functions affecting expression of secreted proteins as well as their secretion; Leskela S et al.; ecs is a three-cistron operon of Bacillus subtilis, encoding proteins with similarity to the ATPase (EcsA) and hydrophobic components (EcsB) of ABC transporters . The ecsA26 point mutation was shown to cause a strong processing defect of a secreted alpha-amylase precursor (preAmyQ) and of three other exoproteins . Northern analysis of the level of amyQ mRNA showed that ecsA26 also decreases amyQ transcription . This effect too was pleiotropic, as judged by a drastic decrease in the expression from an exoprotease promoter of a reporter protein . A knockout mutation of the ecsB cistron caused a processing defect similar to ecsA26 but, unlike ecsA26, did not affect amyQ transcription . These was also no defect in transcription in the ecsA ecsB double mutant . Thus, an intact ecsB product was required for the downregulation of amyQ by the mutant ecsA . These results suggest a dual regulatory function for Ecs, in which Ecs, possibly as part of a signal transduction mechanism, regulates some component(s) of the protein secretion apparatus as well as secretory protein transcription in a co-ordinated fashion.

Biochemistry, 1999 Feb 9, 38(6), 1873 - 83
Identification of adenosine functional groups involved in substrate binding by the ribonuclease P ribozyme; Siew D et al.; The RNA component of bacterial ribonuclease P (RNase P) binds to substrate pre-tRNAs with high affinity and catalyzes site-specific phosphodiester bond hydrolysis to generate the mature tRNA 5' end . Herein we describe the use of biotinylated pre-tRNA substrates to isolate RNase P ribozyme-substrate complexes for nucleotide analogue interference mapping of ribozyme base functional groups involved in substrate recognition . By using a series of adenosine base analogues tagged with phosphorothioate substitutions, we identify specific chemical groups involved in substrate binding . Only 10 adenosines in the Escherichia coli ribozyme show significant sensitivity to interference: A65, A66, A136, A232-234, A248, A249, A334, and A347 . Most of these adenosine positions are universally conserved among all bacterial RNase P RNAs; however, not all conserved adenosines are sensitive to analogue substitution . Importantly, all but one of the sensitive nucleotides are located at positions of intermolecular cross-linking between the ribozyme and the substrate . One site of interference that did not correlate with available structural data involved A136 in J11/12 . To confirm the generality of the results, we repeated the interference analysis of J11/12 in the Bacillus subtilis RNase P ribozyme, which differs significantly in overall secondary structure . Notably, the B . subtilis ribozyme shows an identical interference pattern at the position (A191) that is homologous to A136 . Furthermore, mutation of A136 in the E . coli ribozyme gives rise to a measurable increase in the equilibrium binding constant for the ribozyme-substrate interaction, while mutation of a nearby conserved nucleotide (A132) that is not sensitive to analogue incorporation does not . These results strongly support direct participation of nucleotides in the P4, P11, J5/15, and J18/2 regions of ribozyme structure in pre-tRNA binding and implicate an additional region, J11/12, as involved in substrate recognition . In aggregate, the interference results provide a detailed chemical picture of how the conserved nucleotides adjacent to the pre-tRNA substrate contribute to substrate binding and provide a framework for subsequent identification of the specific roles of these chemical groups in substrate recognition.

Cell, 1999 Feb 5, 96(3), 353 - 62
Structural basis of multidrug recognition by BmrR, a transcription activator of a multidrug transporter; Zheleznova EE et al.; Multidrug-efflux transporters demonstrate an unusual ability to recognize multiple structurally dissimilar toxins . A comparable ability to bind diverse hydrophobic cationic drugs is characteristic of the Bacillus subtilis transcription regulator BmrR, which upon drug binding activates expression of the multidrug transporter Bmr . Crystal structures of the multidrug-binding domain of BmrR (2.7 A resolution) and of its complex with the drug tetraphenylphosphonium (2.8 A resolution) revealed a drug-induced unfolding and relocation of an alpha helix, which exposes an internal drug-binding pocket . Tetraphenylphosphonium binding is mediated by stacking and van der Waals contacts with multiple hydrophobic residues of the pocket and by an electrostatic interaction between the positively charged drug and a buried glutamate residue, which is the key to cation selectivity . Similar binding principles may be used by other multidrug-binding proteins.

Protein Expr Purif, 1999 Feb, 15(1), 69 - 76
Expression, purification, and characterization of recombinant forms of membrane-bound cytochrome c-550nm from Bacillus subtilis; David PS et al.; Bacillus subtilis expresses a cytochrome c-550nm that participates in respiratory electron transfer and is an integral membrane protein . Analysis of the B . subtilis cytochrome c-550nm amino acid sequence predicts a single N-terminal transmembrane helix attached to a water-soluble heme binding domain {C . von Wachenfeldt and L . Hederstedt (1990) J . Biol . Chem . 265, 13939-13948} . We have purified cytochrome c-550nm from wild-type B . subtilis and B . subtilis transformed with the shuttle vector pHP13 containing the gene for B . subtilis cytochrome c-550nm (cccA) . In B . subtilis transformed with pHP13/cccA there is better than eightfold more membrane-bound cytochrome c-550nm than in wild-type B . subtilis . The overexpressed cytochrome c-550nm can be purified by chromatography on hydroxylapatite and Q-Sepharose media . A six-histidine tag has been added to the C-terminus of cytochrome c-550nm from B . subtilis as a further aid for purification . This strain produces cytochrome c-550nm to a level fourfold greater than wild type and allows for one-step purification using metal affinity chromatography . UV-Vis spectroscopy detects no change in the heme C spectrum due to the addition of six histidines . Neither form of B . subtilis cytochrome c-550nm is stable in its reduced state in aerated buffer, unless EDTA is added . The two forms, wild-type and his-tagged, of cytochromes c have similar midpoint redox potentials of 195 and 185 mV, respectively, and are equally good substrates for B . subtilis cytochrome c oxidase . We conclude that the addition of the histidine tag eases the purification of cytochrome c-550nm from B . subtilis plasma membranes and that the additional metal binding site does not compromise the stability or functional properties of the protein .

J Mol Biol, 1999 Feb 26, 286(3), 721 - 31
Pathway modulation, circular permutation and rapid RNA folding under kinetic control; Pan T et al.; The thermodynamics and folding kinetics of a circularly permuted construct of the ribozyme from Bacillus subtilis RNase P are analyzed and compared with the folding properties of the wild-type ribozyme using optical spectroscopy and catalytic activity . The folding of the wild-type ribozyme is slow due to the rearrangement of kinetically trapped species containing misfolded structures . To test whether any misfolded structure arises from interactions between the two independently folding domains of the RNase P RNA, a circular permuted form was created where one of the two phosphodiester bonds connecting these domains is broken . This construct folds approximately 15-fold faster (t1/2 approximately nine seconds) than the wild-type ribozyme at 37 degreesC . While the complete folding of both domains is kinetically indistinguishable in the wild-type ribozyme, one domain folds much faster than the other domain in the circularly permuted construct . Hence, the major kinetic trap in the folding of the wild-type RNase P RNA involves interdomain interactions . This kinetic trap is avoidable at 37 degreesC in the circularly permuted RNA . However, at temperatures below 30 degreesC or when refolding begins from an equilibrium intermediate stabilized by submillimolar concentrations of Mg2+, a subpopulation containing an interdomain misfold still forms . These results indicate that the folding pathway of this large RNA is highly malleable and can be under kinetic control .

J Mol Biol, 1999 Feb 26, 286(3), 683 - 93
Effect of mutations in the "extended -10" motif of three Bacillus subtilis sigmaA-RNA polymerase-dependent promoters; Camacho A et al.; The "extended -10" motif described originally in Escherichia coli promoters occurs frequently in other bacterial promoters . Most Bacillus subtilis bacteriophage o29 promoters contain this motif . To analyse the influence of the motif on sigmaA-RNA polymerase transcription, the 5'-TG-3' dinucleotide was changed to 5'-AC-3' in three o29 promoters . This change impaired RNA polymerase binding to the promoters; the yields of closed and open complexes were reduced independently of other differences inherent to each promoter . The mutation abolished transcription in vitro from a promoter lacking the consensus sequence at the -35 hexamer . In contrast, at other promoters with a -35 consensus sequence, the yield of run off transcription was not reduced by the mutation . Indeed an apparent interference phenomenon at high polymerase/DNA ratios was relieved . These results indicate that the extended -10 motif provides contact points for sigmaA-RNA polymerase with a role restricted to the first steps of transcription .

Chem Biol, 1999 Feb, 6(2), 99 - 110
Prodigious substrate specificity of AAC(6')-APH(2"), an aminoglycoside antibiotic resistance determinant in enterococci and staphylococci; Daigle DM et al.; BACKGROUND: High-level gentamicin resistance in enterococci and staphylococci is conferred by AAC(6')-APH(2"), an enzyme with 6'-N-acetyltransferase and 2"-O-phosphotransferase activities . The presence of this enzyme in pathogenic gram-positive bacteria prevents the successful use of gentamicin C and most other aminoglycosides as therapeutic agents . RESULTS: In an effort to understand the mechanism of aminoglycoside modification, we expressed AAC(6')-APH(2") in Bacillus subtilis . The purified enzyme is monomeric with a molecular mass of 57 kDa and displays both the expected aminoglycoside N-acetyltransferase and O-phosphotransferase activities . Structure-function analysis with various aminoglycosides substrates reveals an enzyme with broad specificity in both enzymatic activities, accounting for AAC(6')-APH(2")'s dramatic negative impact on clinical aminoglycoside therapy . Both lividomycin A and paromomycin, aminoglycosides lacking a 6'-amino group, were acetylated by AAC(6')-APH(2") . The infrared spectrum of the product of paromomycin acetylation yielded a signal consistent with O-acetylation . Mass spectral and nuclear magnetic resonance analysis of the products of neomycin phosphorylation indicated that phosphoryl transfer occurred primarily at the 3'-OH of the 6-aminohexose ring A, and that some diphosphorylated material was also present with phosphates at the 3'-OH and the 3"'-OH of ring D, both unprecedented observations for this enzyme . Furthermore, the phosphorylation site of lividomycin A was determined to be the 5"-OH of the pentose ring C . CONCLUSIONS: The bifunctional AAC(6')-APH(2") has the capacity to inactivate virtually all clinically important aminoglycosides through N- and O-acetylation and phosphorylation of hydroxyl groups . The extremely broad substrate specificity of this enzyme will impact on future development of aminoglycosides and presents a significant challenge for antibiotic design.

Vet Q, 1999 Jan, 21(1), 21 - 7
Suitability of the Charm HVS and a microbiological multiplate system for detection of residues in raw milk at EU maximum residue levels; Nouws JF et al.; In this paper we assessed the suitability of the Charm HVS and a newly developed microbiological multiplate system as post-screening tests to confirm the presence of residues in raw milk at or near the maximum permissible residue level (MRL) . The multiplate system is composed of Bacillus stearothermophilus var . calidolactis plate at pH 8.0 for detection of beta-lactam antibiotics and tylosin, Bacillus cereus plate at pH 6.0 for detection of tetracyclines, Micrococcus luteus plate at pH 8.0 for detection of macrolides, Bacillus subtilis BGA plate at pH 8.0 for detection of aminoglycosides, trimethoprim-containing plate seeded with B . subtilis BGA at pH 7.0 for detection of sulphonamides, Escherichia coli plate at pH 6.0 for detection of quinolone and polymyxin, and Staphylococcus epidermidis plate at pH 6.0 for detection of novobiocin . For each test plate an action level is proposed in such a way that residues can be detected in raw bulk tank milk at levels near or below the established EU MRLs of beta-lactam antibiotics, tetracyclines, aminoglycosides, macrolides, sulphonamides, colistin, and quinolones . The Charm HVS test used to confirm the presence of tetracycline and macrolide residues gave false-positive results near the EU MRLs . The multiplate system gave valid results . Based on data for raw bulk tank milk samples and the proposed action level for each test plate for suspected samples, we demonstrated that the multiplate system is a reliable post-screening method that can be performed easily and cheaply in microbiological laboratories.

J Protein Chem, 1998 Nov, 17(8), 855 - 66
A model of the quaternary structure of enolases, based on structural and evolutionary analysis of the octameric enolase from Bacillus subtilis; Brown CK et al.; Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa . Since B . subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B . subtilis is octameric . The pl for B . subtilis enolase is 6.1, the pH optimum (pHo) for activity is 8.1-8.2, and the Km for 2-PGA is approximately 0.67 mM . Using the dimeric Calpha structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima . This arrangement allowed identification of helix J of one dimer (residues 86-96) and the loop between helix L and strand 1 (HL-S1 loop) of another dimer as possible subunit interaction regions . Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL-S1 loop is truncated by 4-6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers . From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL-S1 loop may play a critical role in octamer formation of enolases . Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.

Mol Microbiol, 1998 Dec, 30(5), 943 - 53
Pho signal transduction network reveals direct transcriptional regulation of one two-component system by another two-component regulator: Bacillus subtilis PhoP directly regulates production of ResD; Birkey SM et al.; The Bacillus subtilis ResD-ResE two-component system is responsible for the regulation of a number of genes involved in cytochrome c biogenesis and haem A biosynthesis, and it is required for anaerobic respiration in this organism . We reported previously that the operon encoding these regulatory proteins, the resABCDE operon, is induced under several conditions, one of which is phosphate starvation . We report here that this transcription requires the PhoP-PhoR two-component system, whereas other induction conditions do not . The PhoPP response regulator directly binds to and is essential for transcriptional activation of the resABCDE operon as well as being involved in repression of the internal resDE promoter during phosphate-limited growth . The concentration of ResD in various phoP mutant strains corroborates the role of PhoP in the production of ResD . These interactions result in a regulatory network that ties together the cellular functions of respiration/energy production and phosphate starvation . Significantly, this represents the first evidence for direct involvement of one two-component system in transcription of a second two-component system.

Mol Microbiol, 1998 Dec, 30(5), 923 - 32
Bacillus subtilis tetA(L) gene expression: evidence for regulation by translational reinitiation; Stasinopoulos SJ et al.; The tetA(L) gene of Bacillus subtilis encodes a transmembrane protein that can function as a Tc-metal/H+ antiporter, conferring low-level resistance to tetracycline . The TetA(L) coding sequence is preceded by a leader region that contains a 20-amino-acid open reading frame and an appropriately spaced ribosome binding site . Expression of the gene is induced by addition of tetracycline, which is thought to act by binding to ribosomes that translate the tetA(L) leader peptide coding sequence . Here we demonstrate that induction of tetA(L) expression includes minor transcriptional and major translational components . Deletion and point mutations of the tetA(L) leader region were constructed to probe the mechanism of translational induction . To account for the observed mutant phenotypes, we propose that tetA(L) expression is regulated by a translational reinitiation mechanism.

Pharmazie, 1999 Jan, 54(1), 26 - 30
Synthesis and biological activity of new diarylalkenes; Golebiewski WM et al.; Condensation of 5-nitro, 3-chloro-, and 5-chlorosalicylic acid with formaldehyde afforded dimeric disalicylmethanes which were O-methylated with dimethyl sulfate and oxidized with chromium(VI) oxide to give the diarylketones 10, 11, 12 . Wittig reaction with ylides obtained by deprotonation of alkyltriphenylphosphonium salts with sodium bis (trimethylsilyl)amide yielded a series of diarylalkenes . Some of the obtained compounds showed high antimicrobial activity in vitro against Bacillus subtilis and Mycobacterium smegmatis.

Mol Microbiol, 1999 Jan, 31(1), 361 - 71
Identification of target promoters for the Bacillus subtilis extracytoplasmic function sigma factor, sigma W; Huang X et al.; The Bacillus subtilis sigW gene encodes an extracytoplasmic function (ECF) sigma factor that is expressed in early stationary phase from a sigW-dependent autoregulatory promoter, PW . Using a consensus-based search procedure, we have identified 15 operons preceded by promoters similar in sequence to PW . At least 14 of these promoters are dependent on sigma W both in vivo and in vitro as judged by lacZ reporter fusions, run-off transcription assays and nucleotide resolution start site mapping . We conclude that sigma W controls a regulon of more than 30 genes, many of which encode membrane proteins of unknown function . The sigma W regulon includes a penicillin binding protein (PBP4*) and a co-transcribed amino acid racemase (RacX), homologues of signal peptide peptidase (YteI), flotillin (YuaG), ABC transporters (YknXYZ), non-haem bromoperoxidase (YdjP), epoxide hydrolase (YfhM) and three small peptides with structural similarities to bacteriocin precursor polypeptides . We suggest that sigma W activates a large stationary-phase regulon that functions in detoxification, production of anti-microbial compounds or both.

Mol Microbiol, 1999 Jan, 31(1), 271 - 80
ComEA is a DNA receptor for transformation of competent Bacillus subtilis; Provvedi R et al.; Competent cells of Bacillus subtilis efficiently bind and internalize DNA . ComEA and the seven proteins encoded by the comG operon are required in vivo for the binding step . We show here that ComEA, a bitopic membrane protein, is itself capable of high-affinity DNA binding . A domain necessary for DNA binding is located at the C-terminus of ComEA . Proteins with similar 60-80 amino acid residue domains are widespread among bacteria and higher organisms . ComEA shows a marked preference for double-stranded DNA and can bind to oligomers as small as 22 bp in length . DNA binding by ComEA exhibits no apparent base sequence specificity . Using a membrane vesicle DNA-binding assay system we show that in the absence of cell wall, ComEA is still required for DNA binding, whereas the requirement for the ComG proteins is bypassed . We conclude that the ComG proteins are needed in vivo to provide access of the binding domain of ComEA to exogenous DNA . Possible specific roles for the ComG proteins are discussed.

Mol Microbiol, 1999 Jan, 31(1), 211 - 22
The cytoplasmic kinase domain of PhoR is sufficient for the low phosphate-inducible expression of pho regulon genes in Bacillus subtilis; Shi L et al.; PhoP-PhoR, one of three two-component systems known to be required to regulate the pho regulon in Bacillus subtilis, directly regulates the alkaline phosphatase genes that are used as pho reporters . Biochemical studies showed that B . subtilis PhoR, purified from Escherichia coli, was autophosphorylated in vitro in the presence of ATP . Phosphorylated PhoR showed stability under basic conditions but not acidic conditions, indicating that the phosphorylation probably occurs on a conserved histidine residue . Phospho-PhoR phosphorylated its cognate response regulator, PhoP in vitro . B . subtilis phoR was placed in the Bacillus chromosome under the control of the Pspac promoter, which is IPTG inducible . The wild-type phoR, under either native promoter or Pspac promoter with IPTG induction, resulted in a similar level of alkaline phosphatase production . Under high phosphate conditions, strains containing wild-type phoR, or phoR mutant gene products that lacked either the periplasmic domain, or both N-terminal transmembrane PhoR mutant gene products that lacked either the periplasmic domain, or both N-terminal transmembrane PhoR sequences or various extended N-terminal sequences, showed no significant APase production . Under phosphate starvation conditions, in the presence of IPTG, all strains containing mutated phoR genes showed alkaline phosphatase induction patterns similar to that of the wild-type strain, although the fully induced level was lower in the mutants . The decrease in total alkaline phosphatase production in these mutant strains can be compensated completely or partially by increasing the copy number of the mutant phoR gene . These in vivo results suggest that the C-terminal kinase domain of PhoR is sufficient for the induction of alkaline phosphatase expression under phosphate-limited conditions, and that the regulation for repression of APase under phosphate-replete conditions remains intact.

Mol Microbiol, 1999 Jan, 31(1), 185 - 96
ClpE, a novel member of the HSP100 family, is involved in cell division and virulence of Listeria monocytogenes; Nair S et al.; We identified, in the facultative intracellular pathogen Listeria monocytogenes, a previously unknown Clp ATPase, unique among the HSP100 proteins because of the presence of a short N-terminal region with a potential zinc finger motif . This protein of 726 amino acids is highly homologous to ClpE of Bacillus subtilis, and is a member of a new subfamily of HSP100/Clp ATPases . The clpE gene is transcribed as a monocistronic mRNA from a typical consensus sigma A promoter . clpE is not stimulated by various stresses, but is upregulated in a clpC mutant . This is the first example of cross-regulation between Clp ATPases . By constructing a clpE mutant of L . monocytogenes, we found that ClpE is required for prolonged survival at 42 degrees C and is involved in the virulence of this pathogen . A double mutant deficient in both ClpE and ClpC was avirulent in a mouse model and completely eliminated in the liver . Electron microscopy studies did not show any morphological alterations in clpE or clpC mutants . In the clpE-clpC double mutant, however, cell division was affected, indicating that ClpE acts synergistically with ClpC in cell septation . These results show that the Clp chaperones play a crucial role in both cell division and virulence of L . monocytogenes.

J Mol Biol, 1999 Feb 19, 286(2), 307 - 14
Phosphorylation of either crh or HPr mediates binding of CcpA to the bacillus subtilis xyn cre and catabolite repression of the xyn operon; Galinier A et al.; Carbon catabolite repression (CCR) of several Bacillus subtilis catabolic genes is mediated by ATP-dependent phosphorylation of Ser46 of the histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP): sugar phosphotransferase system . A recently discovered HPr-like protein of B . subtilis, Crh, cannot be phosphorylated by PEP and enzyme I but becomes phosphorylated at Ser46 by the ATP-dependent, metabolite-activated HPr kinase . Genetic data suggested that Crh is also implicated in CCR . We here demonstrate that in a ptsH1 crh1 mutant, in which Ser46 of both HPr and Crh is replaced with an alanyl residue, expression of the beta-xylosidase-encoding xynB gene was completely relieved from CCR . No effect on CCR could be observed in strains carrying the crh1 allele, suggesting that under the experimental conditions P-Ser-HPr can substitute for P-Ser-Crh in CCR . By contrast, a ptsH1 mutant was slightly relieved from CCR of xynB, indicating that P-Ser-Crh can substitute only partly for P-Ser-HPr . Mapping experiments allowed us to identify the xyn promoter and a catabolite responsive element (cre) located 229 bp downstream of the transcription start point . Using DNase I footprinting experiments, we could demonstrate that similar to P-Ser-HPr, P-Ser-Crh stimulates binding of CcpA to the xyn cre . Fructose 1,6-bisphosphate was found to strongly enhance binding of the P-Ser-HPr/CcpA and P-Ser-Crh/CcpA complexes to the xyn cre, but had no effect on binding of CcpA alone .

J Bacteriol, 1999 Feb, 181(4), 1348 - 51
The lethal effect of a benzamide derivative, 3-methoxybenzamide, can be suppressed by mutations within a cell division gene, ftsZ, in Bacillus subtilis; Ohashi Y et al.; 3-Methoxybenzamide (3-MBA), which is known to be an inhibitor of ADP-ribosyltransferase, inhibits cell division in Bacillus subtilis, leading to filamentation and eventually lysis of cells . Our genetic analysis of 3-MBA-resistant mutants indicated that the primary target of the drug is the cell division system involving FtsZ function during both vegetative growth and sporulation.

Am J Gastroenterol, 1999 Jan, 94(1), 139 - 43
In vitro evaluation of integrity and sterilization of single-use argon beam plasma coagulation probes; Roach SK et al.; OBJECTIVE: Argon plasma coagulation probes (APC) are currently marketed in the United States as single-use items, and may constitute a significant per-procedure expense . It is unknown whether these probes can be sterilized after endoscopic use and if electrical integrity can be maintained after reprocessing . METHODS: Ten probes (2.3 mm diameter, 220 cm length) manufactured by ERBE Inc., (Marietta, GA) were studied using the ERBE APC 300 at 60 watts . Baseline coagulation depth was measured by coagulating a piece of beefsteak for 60 s . Probes were contaminated with 10(6) Bacillus subtilis spores, cultured, and manually cleaned . Culturing involved introducing 10 cc sterile water through the probes; water was filtered, plated onto blood agar, and incubated for 48 h . After ethylene oxide (ETO), the probes were cultured to determine sterilization . Finally, the per-procedure cost of each probe was assessed . RESULTS: Ten of 10 probes completed 10 testing sessions . One probe split at the proximal end but remained functionally intact . Electrical integrity remained intact for all 10 sessions . All probes grew too numerous to count colonies of B . subtilis after inoculation and no B . subtilis was detected after ETO sterilization . Assuming 10 uses clinically, a total per-procedure equipment cost would approximate $24.00, whereas per-procedure probe cost would equal $42.66 if only five uses were obtained in vivo . CONCLUSIONS: The combination of manual cleaning and ETO sterilization consistently sterilized APC probes . Ninety percent of the probes showed no sign of physical deterioration and 100% maintained their electrical activity after 10 uses . APC probes can potentially be safely and effectively reused up to 10 times, and a significant procedural savings is possible with reuse.

Gene, 1999 Feb 4, 227(1), 101 - 10
GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis; Lewis PJ et al.; We report the development of a series of plasmid vectors for the construction of fusions to mutants of the intrinsically fluorescent green fluorescent protein, GFPmut1 (Cormack et al., 1996 . Gene 173, 33-38) and GFPuv (Crameri et al., 1996 . Nature Biotechnology 14, 315-319) . Both N- and C-terminal fusions can be produced, and their expression can be finely controlled from the inducible Pxyl promoter following double crossover integration into the amyE locus of the Bacillus subtilis chromosome . Other vectors designed for single crossover insertion into the chromosome allow downstream genes to be placed under inducible control . We also show that fusions to GFPmut1 and GFPuv can be co-localized within the cell by virtue of their different excitation spectra.

Protein Eng, 1998 Dec, 11(12), 1205 - 10
Random mutagenesis into the conserved Gly154 of subtilisin E: isolation and characterization of the revertant enzymes; Takagi H et al.; We analyzed the role played by the conserved Gly154, a constituent of the P1 substrate-binding pocket of Bacillus subtilis subtilisin E, in the catalytic properties of the protease . Using an Escherichia coli expression system, the termination codon at position 154 in subtilisin E was first introduced to abolish the catalytic activity through truncation of the C-terminus from amino acid residues 154-275 . We then attempted to obtain revertants with substitutions of various amino acids at position 154 by the polymerase chain reaction using a mixture of oligonucleotides . In addition to the Gly residue (wild-type), six amino acid substitutions (Ala, Arg, Leu, Phe, Pro and Thr) gave caseinolytic activity . When assayed with synthetic peptide substrates, most of the revertants showed a considerable decrease in specific activity and a P1 specificity similar to that of the wild-type enzyme . An Ala154 mutant purified from the periplasmic space in E . coli, however, resulted in an up to 2.3-fold preference for Val rather than Pro as a P2 substrate relative to the wild-type . Further, a significant 2-10-fold increase in the catalytic efficiency occurred in the Gly127Ala plus Gly154Ala combination variant, relative to the single Gly127Ala variant, without any change in the restricted specificity . The kinetic data and molecular modeling analysis demonstrate the important role of position 154 in the catalytic efficiency as well as in the substrate specificity of subtilisin E.

J Mol Evol, 1999 Feb, 48(2), 197 - 208
Molecular phylogeny of phi29-like phages and their evolutionary relatedness to other protein-primed replicating phages and other phages hosted by gram-positive bacteria; Pecenkova T et al.; The phi29-like phage genus of Podoviridae family contains phages B103, BS32, GA-1, M2, Nf, phi15, phi29, and PZA that all infect Bacillus subtilis . They have very similar morphology and their genomes consist of linear double-stranded DNA of approximately 20 kb . The nucleotide sequences of individual genomes or their parts determined thus far show that these phages evolved from a common ancestor . A terminal protein (TP) that is covalently bound to the DNA 5'-end primes DNA replication of these phages . The same mechanism of DNA replication is used by the Cp-1 related phages (also members of the Podoviridae family) and by the phage PRD1 (member of the Tectoviridae family) . Based on the complete or partial genomic sequence data of these phages it was possible to analyze the evolutionary relationship within the phi29-like phage genus as well as to other protein-primed replicating phages . Noncoding regions containing origins of replication were used in the analysis, as well as amino acid sequences of DNA polymerases, and with the phi29-like phages also amino acid sequences of the terminal proteins and of the gene 17 protein product, an accessory component of bacteriophage DNA replicating machinery . Included in the analysis are also results of a comparison of these phage DNAs with the prophages present in the Bacillus subtilis genome . Based on this complex analysis we define and describe in more detail the evolutionary branches of phi29-like phages, one branch consisting of phages BS32, phi15, phi29, and PZA, the second branch composed of phages B103, M2, and Nf, and the third branch having phage GA-1 as its sole member . In addition, amino acid sequences of holins, proteins involved in phage lysis were used to extend the evolutionary study to other phages infecting Gram-positive bacteria . The analysis based on the amino acid sequences of holins showed several weak points in present bacteriophage classification.

J Mol Biol, 1999 Feb 5, 285(5), 1911 - 5
The prosequence of thermolysin acts as an intramolecular chaperone when expressed in trans with the mature sequence in Escherichia coli; Marie-Claire C et al.; The zinc metalloendopeptidase, thermolysin (EC 3.4.24.27) produced by Bacillus thermoproteolyticus serves as a model of important physiological enzymes such as neprilysin, angiotensin converting enzyme and endothelin converting enzyme . Thermolysin is synthesised as a pre-proenzyme, with an N-terminal prosequence of 204 residues and a mature sequence of 316 residues . The prosequence facilitates the folding of the denatured mature sequence in vitro and the cleavage of the peptide bond linking the pro and mature sequences occurs by an autocatalytic, intramolecular process . With the aim to study the role of the prosequence in vivo and to produce active mutants for structural studies, the mature sequence of thermolysin has now been expressed in Escherichia coli, either alone or with the prosequence as an independent polypeptide, i.e . in trans form . In addition, the mature sequence of an inactive mutant in which Glu143 involved in the catalytic process was replaced by Ala has also been expressed in trans with the prosequence . The results show that the pro-sequence is required to obtain active thermolysin and that a covalent link with the mature sequence is not necessary for the correct folding of the protease in vivo . Moreover, when expressed in E . coli (in trans with the prosequence), the yield of correctly folded E143A mutant was similar to that of the wild-type protease, whereas no mature enzyme was detected when it was expressed as a pre-proenzyme in Bacillus subtilis . These results demonstrate that the thermolysin prosequence acts as an intramolecular chaperone in vivo and open the way to structural studies of catalytic site mutants produced in large quantities in E . coli .

Appl Environ Microbiol, 1999 Feb, 65(2), 560 - 8
High-affinity transport of choline-O-sulfate and its use as a compatible solute in bacillus subtilis
Nau-Wagner G, Boch J, Le Good JA, Bremer E.
We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus subtilis, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake . The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine . Growth experiments with a set of B . subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O-sulfate was specifically acquired from the environment via OpuC . Competition experiments demonstrated that choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a Ki of approximately 4 &mgr;M . Uptake studies with {1, 2-dimethyl-14C}choline-O-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O-sulfate, with a Km value of 4 +/- 1 &mgr;M and a maximum rate of transport (Vmax) of 54 +/- 3 nmol/min . mg of protein in cells grown in minimal medium with 0.4 M NaCl . Growth studies utilizing a B . subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance 13C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B . subtilis . In contrast, the osmoprotective effect of acetylcholine for B . subtilis is dependent on its biotransformation into glycine betaine . Choline-O-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for B . subtilis . OpuC mediates the efficient transport not only of glycine betaine and choline-O-sulfate but also of carnitine, crotonobetaine, and gamma-butyrobetaine (R . Kappes and E . Bremer, Microbiology 144:83-90, 1998) . Thus, our data underscore its crucial role in the acquisition of a variety of osmoprotectants from the environment by B . subtilis.

Appl Environ Microbiol, 1999 Feb, 65(2), 506 - 13
Staphylokinase as a plasminogen activator component in recombinant fusion proteins; Szarka SJ et al.; The plasminogen activator staphylokinase (SAK) is a promising thrombolytic agent for treatment of myocardial infarction . It can specifically stimulate the thrombolysis of both erythrocyte-rich and platelet-rich clots . However, SAK lacks fibrin-binding and thrombin inhibitor activities, two functions which would supplement and potentially improve its thrombolytic potency . Creating a recombinant fusion protein is one approach for combining protein domains with complementary functions . To evaluate SAK for use in a translational fusion protein, both N- and C-terminal fusions to SAK were constructed by using hirudin as a fusion partner . Recombinant fusion proteins were secreted from Bacillus subtilis and purified from culture supernatants . The rate of plasminogen activation by SAK was not altered by the presence of an additional N- or C-terminal protein sequence . However, cleavage at N-terminal lysines within SAK rendered the N-terminal fusion unstable in the presence of plasmin . The results of site-directed mutagenesis of lysine 10 and lysine 11 in SAK suggested that a plasmin-resistant variant cannot be created without interfering with the plasmin processing necessary for activation of SAK . Although putative plasmin cleavage sites are located at the C-terminal end of SAK at lysine 135 and lysine 136, these sites were resistant to plasmin cleavage in vitro . Therefore, C-terminal fusions represent stable configurations for developing improved thrombolytic agents based on SAK as the plasminogen activator component.

Biochem Cell Biol, 1998, 76(2-3), 359 - 67
The structure and function of HPr; Waygood EB; Histidine-containing phosphocarrier protein, HPr, was one of the early protein tertiary structures determined by two-dimensional 1H-NMR . Tertiary structures for HPrs from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus have been obtained by 1H NMR and the overall folding pattern of HPr is highly conserved, a betaalpha betabeta alphabeta alpha arrangement of three alpha-helices overlaying a four-stranded beta-sheet . High-resolution structures for HPrs from E . coli and B . subtilis have been obtained using 15N- and 13C-labeled proteins . The first application of NMR to the understanding of the structure and function of HPr was to describe the phosphohistidine isomer, Ndelta1-P-histidine in S . aureus phospho-HPr, and the unusual pKas of the His-15 side chain . The pKa values for the His-15 imidazole from more recent studies are 5.4 for HPr and 7.8 for phospho-HPr from E . coli, for example . A consensus description of the active site is proposed for HPr and phospho-HPr . In HPr, His-15 has a defined conformation and N-caps helix A, and is thus affected by the helix dipole . His-15 undergoes a small conformational change upon phosphorylation, a movement to allow the phosphoryl group to be positioned such that it forms hydrogen bonds with the main chain amide nitrogens of residue 16 (not conserved) and Arg-17 . Interactions between residue 12 side chain (not conserved: asparagine, serine, and threonine) and His-15, and between the Arg-17 guanidinium group and the phosphoryl group, are either weak or transitory.

J Bacteriol, 1999 Feb, 181(3), 1045 - 8
Genetic transfer of large DNA inserts to designated loci of the Bacillus subtilis 168 genome; Itaya M; It was found that contiguous DNA segments of up to 50 kb can be transferred between Bacillus subtilis genomes when a sufficient length of the flanking genomic region is provided for homologous recombination, although the efficiency of transfer was reduced as the insert size increased . Inserts were translocated to different loci, where appropriate integration sites were created.

J Biol Chem, 1999 Feb 5, 274(6), 3407 - 13
The family of cold shock proteins of Bacillus subtilis . Stability and dynamics in vitro and in vivo; Schindler T et al.; Bacillus subtilis possesses three homologous small cold shock proteins (CSPs; CspB, CspC, CspD, sequence identity >72%) . They share a similar beta-sheet structure, as shown by circular dichroism, and have a very low conformational stability, with CspC being the least stable . Similar to CspB, CspC and CspD unfold and refold extremely fast in a N <==> U two-state reaction with average lifetimes of only 100-150 ms for the native state and 1-6 ms for the unfolded states at 25 degreesC . As a consequence of their low stability and low kinetic protection against unfolding, all three cold shock proteins are rapidly degraded by proteases in vitro . Analysis of the CSP stabilities in vivo by pulse-chase experiments revealed that CspB and CspD are stable during logarithmic growth at 37 degreesC as well as after cold shock . The cellular half-life of CspC is shortened at 37 degreesC, but under cold shock conditions CspC becomes stable . The proteolytic susceptibility of the CSPs in vitro was strongly reduced in the presence of a nucleic acid ligand, suggesting that the observed stabilization of CSPs in vivo is mediated by binding to their substrate mRNA at 37 degreesC and, in particular, under cold shock conditions.

Biochim Biophys Acta, 1998 Dec 8, 1429(1), 284 - 91
Cloning and bacterial expression of adenosine-5'-triphosphate sulfurylase from the enteric protozoan parasite Entamoeba histolytica; Nozaki T et al.; A gene encoding adenosine-5'-triphosphate sulfurylase (AS) was cloned from the enteric protozoan parasite Entamoeba histolytica by polymerase chain reaction using degenerate oligonucleotide primers corresponding to conserved regions of the protein from a variety of organisms . The deduced amino acid sequence of E . histolytica AS revealed a calculated molecular mass of 47925 Da and an unusual basic pI of 9.38 . The amebic protein sequence showed 23-48% identities with AS from bacteria, yeasts, fungi, plants, and animals with the highest identities being to Synechocystis sp . and Bacillus subtilis (48 and 44%, respectively) . Four conserved blocks including putative sulfate-binding and phosphate-binding regions were highly conserved in the E . histolytica AS . The upstream region of the AS gene contained three conserved elements reported for other E . histolytica genes . A recombinant E . histolytica AS revealed enzymatic activity, measured in both the forward and reverse directions . Expression of the E . histolytica AS complemented cysteine auxotrophy of the AS-deficient Escherichia coli strains . Genomic hybridization revealed that the AS gene exists as a single copy gene . In the literature, this is the first description of an AS gene in Protozoa.

FEMS Microbiol Lett, 1999 Jan 1, 170(1), 41 - 9
Expression and secretion of Bacillus polymyxa neopullulanase in Saccharomyces cerevisiae; Yebra MJ et al.; We have isolated the gene encoding the neopullulanase enzyme from Bacillus polymyxa CECT 155 . It consists of an open reading frame of 1545 bp that could code for a protein of 515 amino acids . This open reading frame was expressed in Bacillus subtilis and the corresponding transformants produced extracellular neopullulanase . The neopullulanase gene was also expressed in Saccharomyces cerevisiae placing it under the control of the yeast actin gene (ACT1) promoter . Clones containing the intact neopullulanase gene, including its own bacterial signal sequence, gave rise to the synthesis of active, but intracellular, enzyme by S . cerevisiae transformants . When sequences specifying the signal sequence and leader region of the yeast mating pheromone alpha-factor (MF alpha 1) were fused upstream of the gene encoding the neopullulanase enzyme, the enzyme was secreted by S . cerevisiae . The secreted protein presented the same biochemical properties and the same apparent molecular mass as the Bacillus polymyxa original enzyme . The predicted amino acid sequence of the neopullulanase protein contained sequence motifs conserved among amylolytic enzymes . Northern blot analysis indicated that the transcription of the neopullulanase gene in B . polymyxa was induced by the presence of the substrate, pullulan, in the culture, and was repressed by glucose.

J Mol Biol, 1999 Jan 29, 285(4), 1789 - 800
The bacterial SecY/E translocation complex forms channel-like structures similar to those of the eukaryotic Sec61p complex; Meyer TH et al.; The SecYEG complex is a major component of the protein translocation apparatus in the cytoplasmic membrane of bacteria . We have purified a translocationally active complex of the two subunits, SecY and SecE, from Bacillus subtilis . As demonstrated by electron microscopy, SecY/E forms ring structures in detergent solution and in intact lipid bilayers, often with a quasi-pentagonal appearance in projection . The particles represent oligomeric assemblies of the SecY/E complex and are similar to those formed by the eukaryotic Sec61p complex . We propose that these SecY/E rings represent protein-conducting channels and that the two essential membrane components SecY and SecE are sufficient for their formation .

Arch Microbiol, 1999 Jan, 171(2), 135 - 8
Cold shock proteins CspB and CspC are major stationary-phase-induced proteins in Bacillus subtilis; Graumann PL et al.; Shortly after the transition from exponential growth to stationary phase, the pattern of protein synthesis in Bacillus subtilis changes markedly . Among the most profoundly induced proteins are two homologous small acidic proteins, CspB and CspC, which are also major cold-shock-induced proteins . The third cold shock protein (CSP) in B . subtilis, CspD, is not induced following entry into stationary phase . Deletion of both cspB and cspC genes has been previously shown to lead to lysis of cells during stationary phase . These findings reveal that CSPs in B . subtilis are induced under several stress conditions, and that an increase in the synthesis of CspB and CspC is needed for efficient adaptation to stationary phase . Enhanced synthesis of CspB occurs through a combination of transcriptional and post-transcriptional activation, indicating a mechanism similar to that mediating cold shock induction of CSPs . Induction of CSPs in bacteria may be triggered by a common signal, the inactivation of ribosomes, occurring under both cold shock and stationary-phase conditions.

Mol Gen Genet, 1998 Dec, 260(5), 487 - 91
Mutational analysis of the nucleoid-associated protein HBsu of Bacillus subtilis; Kohler P et al.; The essential nucleoid-associated protein HBsu of Bacillus subtilis comprises 92 residues, 20% of which are basic amino acids . To investigate the role of the residues located within the DNA-binding arm, the arginine residues R58 and R61 were changed to leucine, while lysine residues K80 and K86 were replaced by alanine . All altered proteins exhibited a reduction in DNA binding capacity, ranging from 10% to 30% of HBsu wild type DNA-binding ability . To investigate the physiological effect of these mutations in B . subtilis, the indigenous hbs gene was replaced by the mutated genes . B . subtilis strain PK20, which carries the HBsu mutation R58L which exhibits the lowest DNA binding ability in vitro, showed the strongest retardation of growth compared to the wild type . Furthermore, PK20 cells displayed an increased rate of cell lysis, diminished sporulation efficiency and a reduced level of negatively supercoiled DNA . These observations suggest that the DNA binding ability of HBsu DNA is important for growth and differentiation and influences DNA topology.

Biochimie, 1998 Oct, 80(10), 821 - 36
Cooperative coupling and role of heme a in the proton pump of heme-copper oxidases; Papa S et al.; In the last few years, evidence has accumulated supporting the applicability of the cooperative model of proton pumps in cytochrome systems, vectorial Bohr mechanisms, to heme-copper oxidases . The vectorial Bohr mechanism is based on short- and long-range protonmotive cooperative effects linked to redox transitions of the metal centers . The crystal structure of oxidized and reduced bovine-heart cytochrome c oxidase reveals, upon reduction, the occurrence of long-range conformational changes in subunit I of the oxidase . Analysis of the crystal structure of cytochrome c oxidase shows the existence of hydrogen-bonded networks of amino acid residues which could undergo redox-linked pK shifts resulting in transmembrane proton translocation . Our group has identified four proteolytic groups undergoing reversible redox-linked pK shifts . Two groups result in being linked to redox transitions of heme a3 . One group is apparently linked to CuB . The fourth group is linked to oxido-reduction of heme a . We have shown that the proton transfer resulting from the redox Bohr effects linked to heme a and CuB in the bovine oxidase displays membrane vectorial asymmetry, i.e., protons are taken up from the inner aqueous space (N), upon reduction, and released in the external space (P), upon oxidation of the metals . This direction of proton uptake and release is just what is expected from the vectorial Bohr mechanism . The group linked to heme a, which can transfer up to 0.9 H+/e- at pHs around neutrality, can provide the major contribution to the proton pump . It is proposed that translocation of pumped protons, linked to electron flow through heme a, utilizes a channel (channel D) which extends from a conserved aspartate at the N entrance to a conserved glutamate located between heme a and the binuclear center . The carboxylic group of this glutamic acid, after having delivered, upon electron flow through heme a, pumped protons towards the P phase, once reprotonated from the N phase, moves to deliver, subsequently, to the binuclear center chemical protons consumed in the conversion of the peroxy to ferryl and of the latter to the oxy intermediate in the redox cycle . Site-directed mutagenesis of protolytic residues in subunit I of the aa3-600 quinol oxidase of Bacillus subtilis to non-polar residues revealed that the conserved Lys 304 is critical for the proton pumping activity of the oxidase . Crystal structures of cytochrome c oxidase show that this lysine is at the N entrance of a channel which translocates the protons consumed for the production of the peroxy intermediate . Inhibition of this pathway, by replacement of the lysine, short-circuits protons from channel D to the binuclear center, where they are utilized in the chemistry of oxygen reduction.

Annu Rev Microbiol, 1998, 52, 165 - 90
Anaerobic growth of a "strict aerobe" (Bacillus subtilis); Nakano MM et al.; There was a long-held belief that the gram-positive soil bacterium Bacillus subtilis is a strict aerobe . But recent studies have shown that B . subtilis will grow anaerobically, either by using nitrate or nitrite as a terminal electron acceptor, or by fermentation . How B . subtilis alters its metabolic activity according to the availability of oxygen and alternative electron acceptors is but one focus of study . A two-component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, occupies an early stage in the regulatory pathway governing anaerobic respiration . One of the essential roles of ResD and ResE in anaerobic gene regulation is induction of fnr transcription upon oxygen limitation . FNR is a transcriptional activator for anaerobically induced genes, including those for respiratory nitrate reductase, narGHJI.B . subtilis has two distinct nitrate reductases, one for the assimilation of nitrate nitrogen and the other for nitrate respiration . In contrast, one nitrite reductase functions both in nitrite nitrogen assimilation and nitrite respiration . Unlike many anaerobes, which use pyruvate formate lyase, B . subtilis can carry out fermentation in the absence of external electron acceptors wherein pyruvate dehydrogenase is utilized to metabolize pyruvate.

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