J Biol Chem, 2001 Aug 10, 276(32), 29819 - 25 Epub 2001 Jun 04. Roles of glucitol in the GutR-mediated transcription activation process in Bacillus subtilis: glucitol induces GutR to change its conformation and to bind ATP; Poon KK et al.; GutR is a 95-kDa glucitol-dependent transcription activator that mediates the expression of the Bacillus subtilis glucitol operon . Glucitol allows GutR to bind tightly to its binding site located upstream of the gut promoter . In this study, a second functional role of glucitol is identified . Glucitol induces GutR to change its conformation and triggers GutR to bind ATP efficiently . After sequential binding of glucitol and ATP to GutR, GutR adopts a new conformation by forming a compact structure that is resistant to trypsin digestion . Under this condition, the ATP.glucitiol.GutR complex can dissociate slowly from the gutR-binding site (t(12) = 274 min) . Interestingly, if ATP in the ATP.glucitiol.GutR complex is replaced by ADP, GutR adopts another conformation and can dissociate from the gutR-binding site even faster (t(12) = 82 min) . In all these GutR-DNA binding studies in the presence of different ligands (glucitol, ATP, or ADP), only the off-rate is affected . The vital role of ATP in the GutR-mediated transcription activation process is reflected by the poor transcription from the gut promoter with GutR(D285A) which has a mutation in the motif B of the putative ATP-binding site . A working model for this transcription activation process is presented. Eur J Biochem, 2001 Jun, 268(11), 3332 - 8 Paralogous gene analysis reveals a highly enantioselective 1,2-O-isopropylideneglycerol caprylate esterase of Bacillus subtilis; Droge MJ et al.; Carboxylesterase NP of Bacillus subtilis Thai I-8, characterized in 1992 as a very enantioselective (S)-naproxen esterase, was found to show no enantiopreference towards (S)-1,2-O-isopropylideneglycerol (IPG) esters . The ybfK gene was identified by the B . subtilis genome project as an unknown gene with homology to carboxylesterase NP . The purpose of the present study was to characterize the ybfK gene product in order to determine whether this paralogue of carboxylesterase NP had an altered or enhanced stereospecificity . The ybfK gene was cloned and expressed in B . subtilis using a combination of two strong promoters in a multicopy vector . The enzyme was purified from the cytoplasm of B . subtilis by means of anion exchange and hydrophobic interaction chromatography . The purified YbfK is an enzyme of 296 amino acids and shows an apparent molecular mass of 32 kDa (SDS/PAGE) . Comparison of the activities of YbfK and carboxylesterase NP towards caprylate esters of IPG revealed that YbfK produces (S)-IPG with 99.9% enantioselectivity . Therefore, we conclude that we have isolated a paralogue of carboxylesterase NP that can be used for the enantioselective production of (S)-IPG. Eur J Biochem, 2001 Jun, 268(11), 3180 - 9 The two-component regulatory system BacRS is associated with bacitracin 'self-resistance' of Bacillus licheniformis ATCC 10716; Neumuller AM et al.; Bacitracin is a peptide antibiotic produced by several Bacillus licheniformis strains that is most active against other Gram-positive microorganisms, but not against the producer strain itself . Recently, heterologous expression of the bacitracin resistance mediating BcrABC transporter in Bacillus subtilis and Escherichia coli was described . In this study we could determine that the transporter encoding bcrABC genes are localized about 3 kb downstream of the 44-kb bacitracin biosynthetic operon bacABC . Between the bac operon and the bcrABC genes two orfs, designated bacR and bacS, were identified . They code for proteins with high homology to regulator and sensor proteins of two-component systems . A disruption mutant of the bacRS genes was constructed . While the mutant displayed no effects on the bacitracin production it exhibited highly increased bacitracin sensitivity compared to the wild-type strain . Western blot analysis of the expression of BcrA, the ATP-binding cassette of the transporter, showed in the wild-type a moderate BcrA induction in late stationary cells that accumulate bacitracin, whereas in the bacRS mutant cells the BcrA expression was constitutive . A comparison of bacitracin stressed and nonstressed wild-type cells in Western blot analysis revealed increasing amounts of BcrA and a decrease in BacR in the stressed cells . From these findings we infer that BacR acts as a negative regulator for controlling the expression of the bcrABC transporter genes. Biosci Biotechnol Biochem, 2001 Apr, 65(4), 823 - 9 Incorporation of the whole chromosomal DNA in protoplast lysates into competent cells of Bacillus subtilis; Akamatsu T et al.; Competent cells of Bacillus subtilis AC870 (purB, leuB, trpC, ald-1) were transformed to Ade+, Trp+, or Ade+ Trp+ with DNA in protoplast lysates of B . subtilis AC819 (hisH, tet-1, rpsL, smo-1) . The cotransfer ratio of purB to trpC was constant at 7-9% (Ade+ Trp+/Trp+) or 3% (Ade+ Trp+/Ade+) at protoplast concentrations of 2.7 x 10(3) to approximately 2.7 x 10(6) per ml . The whole chromosomal DNA must be certainly incorporated into competent cells from the following reasons; (1) purB is opposite to trpC on the chromosome, (2) 2.7 x 10(3) protoplasts per ml is about 100 times lower than 3.2 x 10(5) competent cells per ml, and (3) the cotransfer ratio is constant at all the concentrations . Similar results were obtained with the cotransfer ratio of purA to trpC . The transformation requires several Com proteins including ComK. J Biol Chem, 2001 Aug 3, 276(31), 28927 - 32 Epub 2001 May 30. Repression of bacteriophage phi 29 early promoter C2 by viral protein p6 is due to impairment of closed complex; Camacho A et al.; Bacillus subtilis phage phi 29 encodes a very abundant protein, p6, which is a non sequence-specific DNA-binding protein . Protein p6 has the potential to bind cooperatively to the phage genome, forming a nucleoprotein complex in which the DNA adopts a right-handed toroidal conformation winding around a protein core . The formation of this complex at the right end of the phage genome where the early promoter C2 is located affects local topology, which may contribute to the promoter repression, although the underlying molecular mechanism of this repression is not presently known . In this study, we analyzed the effect of the p6 nucleoprotein complex on the formation of transcription complexes at the C2 promoter . The results obtained indicate that the nucleoprotein complex does not occlude promoter C2 to RNA polymerase because both proteins can bind to the same DNA molecule . Protein p6 binds along the fragment including the sequence adjacent to the bound polymerase, altering the structure of the transcriptional complex and affecting specifically the stability of the closed complex . The findings presented might help to answer some of the open questions about the concerted molecular mechanisms of histone-like proteins as transcriptional silencers. Water Sci Technol, 2001, 43(4), 191 - 7 Photobiological effects of polychromatic medium pressure UV lamps; Kalisvaart BF; Ultraviolet (UV) light has become widely accepted for the disinfection of potable water, process water and wastewater as an alternative to chlorination . To avoid the failure of a UV disinfection system due to the recovery of micro-organisms, certain additional wavelengths in the UV area are emitted by newly developed UV lamps . To reduce the chance of microbial recovery after ultraviolet irradiation, damage must be inflicted in as many areas of the micro-organism as possible . The effective killing of micro-organisms by improved polychromatic medium pressure UV lamps is due to their exceptionally high UV energy output at specific wavelengths across a broad section of the UV spectrum . The combination of these properties results in several different lethal effects in small and large micro-organisms . Important biological molecules other than DNA are likely to be damaged, which helps to prevent the recovery of irradiated micro-organisms . Absorption line spectra of absorbing nucleotide bases, DNA and other biological molecules, including proteins and enzymes, show how effective UV light can be . Recent findings on the biological effects of short wavelengths on Bacillus subtilis, Cryptosporidium parvum and Escherichia coli confirm the effect of short wavelengths . Practical comparisons with conventional low pressure UV lamps at equal UV dosages show better killing rates from polychromatic medium pressure lamps, without formation of disinfection byproducts (DBPs). Curr Opin Drug Discov Devel, 2001 Mar, 4(2), 237 - 45 SMR-type multidrug resistance pumps; Chung YJ et al.; Multidrug resistance (MDR) efflux pumps in pathogenic microorganisms nullify the effects of antimicrobial drugs used in medicine . We have conducted phylogenetic analyses showing that these efflux pumps are associated with five superfamilies of transport systems . One of these, the drug/metabolite transporter (DMT) superfamily includes a family of small multidrug resistance (SMR)-conferring proteins that are discussed in detail in this review . A single microorganism such as Bacillus subtilis may possess multiple homologs of this family, and these homologs are believed to form both homo-oligomeric or hetero-oligomeric pumps, some of which export cationic drugs . The characteristics of some of these systems and the genes that encode them are described, with emphasis on the eight homologs encoded within the B subtilis genome . Anomalies and unanswered questions that provide impetus for future studies are presented. FEMS Microbiol Lett, 2001 May 30, 199(2), 221 - 7 Induction of sigma(B)-dependent general stress genes by amino acid starvation in a spo0H mutant of Bacillus subtilis; Eymann C et al.; Solely sigma(B)-dependent genes like gsiB and gspA are not significantly induced in amino acid-starved wild-type cells, since amino acid starvation does not trigger activation of sigma(B) . The general stress gene yvyD is subject to the control of both sigma(B) and sigma(H) therefore displaying induction in response to amino acid starvation at the sigma(H)-dependent promoter . Surprisingly, the proteins YvyD, GsiB and GspA were significantly induced in amino acid-starved cells of a strain lacking sigma(H) activity . Transcriptional studies provided evidence that sigma(B)-dependent transcription is indeed induced in a spo0H mutant during amino acid starvation and depends on RsbP but not on RsbU indicating that the stress signal transduction is not required for this induction . A similar phenomenon of sigma(B) activation was observed in amino acid-starved cells of a spo0A deletion mutant . The sigma(B)-dependent transcription in a spo0H mutant further needs an active RelA protein which is responsible for strong repression of house-keeping genes after amino acid starvation (stringent response) . Our data indicate that in the absence of sigma(H) and under conditions which provoke the stringent response, RsbP-dependent levels of active sigma(B) can more effectively compete for increased levels of free RNA polymerase core enzyme leading to the induction of the probably strongest sigma(B)-dependent genes. FEMS Microbiol Lett, 2001 May 30, 199(2), 197 - 202 A novel diffusible substance can overcome the apparent AbrB repression of the Bacillus subtilis fatR promoter; Gustafsson MC et al.; In this work we present evidence for a novel diffusible extracellular factor that modulates gene expression in Bacillus subtilis . The factor was found when studying the regulation of the fatR-cyp102A3 operon . In a Spo0A(-) mutant expression of the fatR-cyp102A3 operon was almost abolished . The fatR-cyp102A3 expression defect of a Spo0A(-) mutant could be overcome either by a mutation in the abrB gene or by a diffusible substance excreted by wild-type, abrB mutant and abrB-spo0A double mutant strains. FEMS Microbiol Lett, 2001 May 30, 199(2), 177 - 9 Expression of chloramphenicol acetyltransferase in Bacillus subtilis under the control of a phytoplasma promoter; Palmano S et al.; A cloned putative promoter region upstream of the 16S rRNA gene of the western X-disease phytoplasma was inserted behind the promoterless chloramphenicol acetyltransferase gene of plasmid pPL603 . The DNA construct was used to transform Bacillus subtilis cells . The transformants were assayed for chloramphenicol acetyltransferase activity, showing that the phytoplasma promoter is efficiently expressed in a B . subtilis background. Acta Crystallogr D Biol Crystallogr, 2001 Jun, 57(Pt 6), 876 - 8 Epub 2001 May 25. Crystallization and preliminary crystallographic analysis of deoxyuridine 5'-triphosphate nucleotidohydrolase from Bacillus subtilis; Persson R et al.; Single crystals of purified homotrimeric deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) from Bacillus subtilis have been grown under several different conditions using vapour diffusion . X-ray diffraction data have been collected using synchrotron radiation from three crystal forms of the unliganded enzyme and from enzyme cocrystallized with a substrate analogue and inhibitor, dUDP, and a metal ion, Sr(2+) . The three crystal forms of unliganded enzyme belong to hexagonal (P6(3)), orthorhombic (P2(1)2(1)2) and cubic (P2(1)3) space groups and data have been recorded to 1.75, 1.90 and 2.50 A spacing, respectively . Crystals grown in the presence of dUDP and Sr(2+) belong to the orthorhombic space group P2(1)2(1)2(1) and data were measured to 1.90 A spacing . Solution of the hexagonal crystal form by molecular replacement using the dUTPase from feline immunodeficiency virus as a search model is in progress. Acta Crystallogr D Biol Crystallogr, 2001 Jun, 57(Pt 6), 806 - 12 Epub 2001 May 25. Stabilization of active-site loops in NH3-dependent NAD+ synthetase from Bacillus subtilis; Devedjiev Y et al.; The NH(3)-dependent NAD(+) synthetase (NADS) participates in the biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) by transforming nicotinic acid adenine dinucleotide (NaAD) to NAD(+) . The structural behavior of the active site, including stabilization of flexible loops 82-87 and 204-225, has been studied by determination of the crystal structures of complexes of NADS with natural substrates and a substrate analog . Both loops are stabilized independently of NaAD and solely from the ATP-binding site . Analysis of the binding contacts suggests that the minor loop 82-87 is stabilized primarily by a hydrogen bond with the adenine base of ATP . Formation of a coordination complex with Mg(2+) in the ATP-binding site may contribute to the stabilization of the major loop 204-225 . The major loop has a role in substrate recognition and stabilization, in addition to the protection of the reaction intermediate described previously . A second and novel Mg(2+) position has been observed closer to the NaAD-binding site in the structure crystallized at pH 7.5, where the enzyme is active . This could therefore be the catalytically active Mg(2+). J Biol Chem, 2001 Jul 27, 276(30), 28140 - 6 Epub 2001 May 24. Mutational analysis of catalytic sites of the cell wall lytic N-acetylmuramoyl-L-alanine amidases CwlC and CwlV; Shida T et al.; The Bacillus subtilis CwlC and the Bacillus polymyxa var . colistinus CwlV are the cell wall lytic N-acetylmuramoyl-l-alanine amidases in the CwlB (LytC) family . Deletion in the CwlC amidase from the C terminus to residue 177 did not change the amidase activity . However, when the deletion was extended slightly toward the N terminus, the amidase activity was entirely lost . Further, the N-terminal deletion mutant without the first 19 amino acids did not have the amidase activity . These results indicate that the N-terminal half (residues 1-176) of the CwlC amidase, the region homologous to the truncated CwlV (CwlVt), is a catalytic domain . Site-directed mutagenesis was performed on 20 highly conserved amino acid residues within the catalytic domain of CwlC . The amidase activity was lost completely on single amino acid substitutions at two residues (Glu-24 and Glu-141) . Similarly, the substitution of the two glutamic acid residues (E26Q and E142Q) of the truncated CwlV (CwlV1), which corresponded to Glu-24 and Glu-141 of CwlC, was critical to the amidase activity . The EDTA-treated CwlV1 did not have amidase activity . The amidase activity of the EDTA-treated CwlV1 was restored by the addition of Zn2+, Mn2+, and Co2+ but not by the addition of Mg2+ and Ca2+ . These results suggest that the amidases in the CwlB family are zinc amidases containing two glutamic acids as catalytic residues. Appl Environ Microbiol, 2001 Jun, 67(6), 2564 - 70 Enhanced detection of surface-associated bacteria in indoor environments by quantitative PCR; Buttner MP et al.; Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations . Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials . QPCR and culture analyses were used to quantitate Bacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method . The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative . The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores . The B . subtilis values obtained by the QPCR method were greater than those obtained by culture analysis . The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet . The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials . Inhibition of QPCR due solely to biological contamination of flooring materials was not evident . However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants . The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling methods. J Org Chem, 2001 Jun 1, 66(11), 3948 - 52 Enzyme-assisted preparation of isotope-labeled 1-deoxy-d-xylulose 5-phosphate; Hecht S et al.; Recombinant 1-deoxy-D-xylulose 5-phosphate synthase of Bacillus subtilis was used for the preparation of isotope-labeled 1-deoxy-D-xylulose 5-phosphate using isotope-labeled glucose and/or isotope-labeled pyruvate as starting materials . The simple one-pot methods described afford almost every conceivable isotopomer of 1-deoxy-D-xylulose 5-phosphate carrying (13)C or (14)C from commercially available precursors with an overall yield around 50%. J Org Chem, 2001 Jun 1, 66(11), 3811 - 9 19F NMR ligand perturbation studies on 6,7-bis(trifluoromethyl)-8-ribityllumazine-7-hydrates and the lumazine synthase complex of Bacillus subtilis . Site-directed mutagenesis changes the mechanism and the stereoselectivity of the catalyzed haloform-type reaction; Scheuring J et al.; The riboflavin synthase/lumazine synthase complex of Bacillus subtilis catalyzes the last two steps in riboflavin biosynthesis . The protein comprises a capsid of 60 beta subunits with lumazine synthase activity and a core of three alpha subunits with riboflavin synthase activity . The beta subunits catalyze the formation of 6,7-dimethyl-8-ribityllumazine (3) from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) and 3,4-dihydroxy-2-butanone 4-phosphate (2) . Complexes of recombinant lumazine synthase (beta(60) capsids) with 6-trifluoromethyl-7-oxo-8-ribityllumazine (10) as well as 7S- or 7R-6,7-bistrifluoromethyl-8-ribityllumazine hydrate (11) were studied by (19)F NMR spectroscopy . Despite the large molecular weight of approximately 960 kDa of the protein, spectra with separated signals of free and bound ligand could be obtained . An unusually large shift difference of 8 ppm was observed between the 7-trifluoromethyl signals of free and bound ligand for epimer B of 11 and the enzyme . The signal is sensitive to the replacement of amino acid residues F22 and H88 . Lumazine synthase catalyzes the elimination of the 7-trifluoromethyl group of R-diastereomer epimer A in a haloform-like reaction . The elimination reaction is also catalyzed by F22 mutants . The H88R mutant displays an opposite stereoselectivity for epimer B and a greatly enhanced reaction rate . From a model of the epimers in the active site of the protein, the main function of the side chain of F22 seems to be to keep the substrate ring in the correct position . H88 is in a position suited to act as proton acceptor in both the physiological as well as the haloform reaction . A different mechanism of the haloform-reaction is proposed in the case of the H88R mutant, initiated by hydrogen bonding of the 7-trifluorormethyl group and the guanidinium group of the arginine residue. J Bacteriol, 2001 Jun, 183(12), 3623 - 30 Analysis of promoter recognition in vivo directed by sigma(F) of Bacillus subtilis by using random-sequence oligonucleotides; Amaya E et al.; Formation of spores from vegetative bacteria by Bacillus subtilis is a primitive system of cell differentiation . Critical to spore formation is the action of a series of sporulation-specific RNA polymerase sigma factors . Of these, sigma(F) is the first to become active . Few genes have been identified that are transcribed by RNA polymerase containing sigma(F) (E-sigma(F)), and only two genes of known function are exclusively under the control of E-sigma(F), spoIIR and spoIIQ . In order to investigate the features of promoters that are recognized by E-sigma(F), we studied the effects of randomizing sequences for the -10 and -35 regions of the promoter for spoIIQ . The randomized promoter regions were cloned in front of a promoterless copy of lacZ in a vector designed for insertion by double crossover of single copies of the promoter-lacZ fusions into the amyE region of the B . subtilis chromosome . This system made it possible to test for transcription of lacZ by E-sigma(F) in vivo . The results indicate a weak sigma(F)-specific -10 consensus, GG/tNNANNNT, of which the ANNNT portion is common to all sporulation-associated sigma factors, as well as to sigma(A) . There was a rather stronger -35 consensus, GTATA/T, of which GNATA is also recognized by other sporulation-associated sigma factors . The looseness of the sigma(F) promoter requirement contrasts with the strict requirement for sigma(A)-directed promoters of B . subtilis . It suggests that additional, unknown, parameters may help determine the specificity of promoter recognition by E-sigma(F) in vivo. J Bacteriol, 2001 Jun, 183(12), 3574 - 81 Bacillus subtilis locus encoding a killer protein and its antidote; Adler E et al.; We have isolated mutations that block sporulation after formation of the polar septum in Bacillus subtilis . These mutations were mapped to the two genes of a new locus, spoIIS . Inactivation of the second gene, spoIISB, decreases sporulation efficiency by 4 orders of magnitude . Inactivation of the first gene, spoIISA, has no effect on sporulation but it fully restores sporulation of a spoIISB null mutant, indicating that SpoIISB is required only to counteract the negative effect of SpoIISA on sporulation . An internal promoter ensures the synthesis of an excess of SpoIISB over SpoIISA during exponential growth and sporulation . In the absence of SpoIISB, the sporulating cells show lethal damage of their envelope shortly after asymmetric septation, a defect that can be corrected by synthesizing SpoIISB only in the mother cell . However, forced synthesis of SpoIISA in exponentially growing cells or in the forespore leads to the same type of morphological damage and to cell death . In both cases protection against the killing effect of SpoIISA can be provided by simultaneous synthesis of SpoIISB . The spoIIS locus is unique to B . subtilis, and since it is completely dispensable for sporulation its physiological role remains elusive. J Biomol NMR, 2001 Apr, 19(4), 293 - 304 NMR studies of the sporulation protein SpoIIAA: implications for the regulation of the transcription factor sigmaF in Bacillus subtilis; Kovacs H et al.; SpoIIAA participates in a four-component mechanism for phosphorylation-dependent transcription control at the outset of sporulation . We report the refinement of the solution structure of SpoIIAA by using the automated iterative NOE assignment method ARIA . To complement the structural data, the protein dynamics were determined by measuring the T1, T2 and NOE of the backbone 15N-nuclei . The refined structure permits a discussion of the structural features that are important for the function of SpoIIAA in the regulation of the sporulation sigma factor sigmaF, and for homologous regulatory pathways present in B . subtilis and in other bacilli. Dtsch Tierarztl Wochenschr, 2001 Apr, 108(4), 168 - 71 Pharmacokinetics, intramuscular bioavailability and tissue residue profiles of ceftazidime in a rabbit model; Abd el-Aty AM et al.; This study investigated the disposition kinetics and plasma availability of ceftazidime in rabbits after single intravenous (i.v.) and intramuscular (i.m.) injections of 50 mg kg-1 b.wt . Tissue residue profiles were studied after repeated intramuscular injections of 50 mg kg-1 b . wt, twice daily for five consecutive days . A microbiological assay with Bacillus subtilis as the test organism was used to measure its concentrations in plasma and tissues . The plasma concentration-vs-time curves were best described by a two compartment open model . The decline in plasma drug concentration was biexponential with half-lives of 0.258 h for t1/2 alpha, 2.22 h for t1/2 beta, for distribution and elimination phases, respectively, following i.v . injection . After intramuscular injection of ceftazidime at the same dose, it was detected in plasma at 5 min and reached its minimum level 12 h post-injection . The peak plasma concentration (Cmax) 66.3 micrograms.ml-1 was attained at 0.779 h (Tmax) . The elimination half-life (T1/2el,) was 2.12 h, the mean residence time (MRT) was 3.06 h and the systemic bioavailability was 96.6% . In vitro protein binding percent of ceftazidime in rabbit's plasma was ranged from 13.3 to 21.6% . The limit of quantification (LOQ) for the assay was 0.01 microgram.ml-1 in plasma and tissues . The tissue level concentrations were highest in the kidneys, and decreased in the following order: liver > heart > muscles and plasma . No ceftazidime residues were detected in tissues and plasma after 72 h . It is concluded that tissue kinetics is an important tool in predicting and controlling drug residues in edible tissues of food producing animal. Arch Biochem Biophys, 2000 Dec 15, 384(2), 351 - 60 Expression, purification, and characterization of BioI: a carbon-carbon bond cleaving cytochrome P450 involved in biotin biosynthesis in Bacillus subtilis; Stok JE et al.; Pimelic acid formation for biotin biosynthesis in Bacillus subtilis has been proposed to involve a cytochrome P450 encoded by the gene bioI . We have subcloned biol and overexpressed the encoded protein, Biol . A purification protocol was developed utilizing ion exchange, gel filtration, and hydroxyapatite chromatography . Investigation of the purified BioI by UV-visible spectroscopy revealed spectral properties characteristic of a cytochrome P450 enzyme . BioI copurifies with acylated Escherichia coli acyl carrier protein (ACP), suggesting that in vivo a fatty acid substrate may be presented to BioI as an acyl-ACP . A combination of electrospray mass spectrometry of the intact acyl-ACP and GCMS indicated a range of fatty acids were bound to the ACP . A catalytically active system has been established employing E . coli flavodoxin reductase and a novel, heterologous flavodoxin as the redox partners for BioI . In this system, BioI cleaves a carbon-carbon bond of an acyl-ACP to generate a pimeloyl-ACP equivalent, from which pimelic acid is isolated after base-catalyzed saponification . A range of free fatty acids have also been explored as potential alternative substrates for BioI, with C16 binding most tightly to the enzyme . These fatty acids are also metabolized to dicarboxylic acids, but with less regiospecificity than is observed with acyl-ACPs . A possible mechanism for this transformation is discussed . These results strongly support the proposed role for BioI in biotin biosynthesis . In addition, the production of pimeloyl-ACP explains the ability of BioI to function as a pimeloyl CoA source in E . coli, which, unlike B . subtilis, is unable to utilize free pimelic acid for biotin production. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 1997 Dec, 14(4), 359 - 62 {Study on immobilized cells for producing alpha-amylase by using polyvinyl alcohol as the carrier (I)}; Liu Z et al.; The method of immobilization of Bacillus subtilis by using polyvingyl alcohol(PVA) as the carrier of immobilized cells was studied . The effects of conditions of immobilization on the structure and mechanical strength of PVA immobilized cells and the ability for producing alpha-amylase of the immobilized cells were investigated . The comparative test of fermentation by using the immobilized cells with PVA as carrier and the immobilized cells with sodium glyinate as carrier and free cells was carried out in the same condition . The results showed that Bacillus substilis cells were immobilized efficently by PVA and the immobilized cells could normally grow and reproduce and produce alpha-amylase . In the same condition, the activity of alpha-amylase produced boy the immobilized cells was 28% higher than that produced by free cells and 11% higher than that produced by the cells immobilized with sodium glyinate as carrier. J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 439 - 44 Phosphotransfer functions mutated Bacillus subtilis HPr-like protein Crh carrying a histidine in the active site; Darbon E et al.; The Bacillus subtilis protein Crh exhibits strong similarity to HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) . HPr phosphorylated at His-15 can transfer its phosphoryl group to several EIIAs of the PTS for sugar transport and phosphorylation . In addition, it phosphorylates and activates transcriptional regulators containing PTS regulation domains (PRDs) . In Gram-positive bacteria, it also controls the enzyme glycerol kinase . Since in Crh the active site His-15 of HPr is replaced with a glutamine, Crh was not able to carry out the catalytic and regulatory functions mediated by P approximately His-HPr . However, when Gln-15 of Crh was replaced with a histidine, Crh gained most of the catalytic and regulatory functions exerted by HPr . To allow CrhQ15H to efficiently phosphorylate and activate the PRD-containing antiterminator LicT, which controls the expression of the bgIS gene and the bgIPH operon, it was sufficient to express the crhQ15H allele under control of the spac promoter in monocopy . By contrast, to phosphorylate and activate glycerol kinase and to allow a ptsH deletion strain (devoid of HPr) to slowly grow on the non-PTS substrate glycerol and to efficiently utilize the PTS sugars glucose and mannitol, the crhQ15H allele had to be expressed from a multicopy plasmid. J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 433 - 8 Catabolite regulation of the cytochrome c550-encoding Bacillus subtilis cccA gene; Monedero V et al.; In Gram-positive bacteria, catabolite control protein A (CcpA)-mediated catabolite repression or activation regulates not only the expression of a great number of catabolic operons, but also the synthesis of enzymes of central metabolic pathways . We found that a constituent of the Bacillus subtilis respiratory chain, the small cytochrome c550 encoded by the cccA gene, was also submitted to catabolite repression . Similar to most catabolite-repressed genes and operons, the Bacillus subtilis cccA gene contains a potential catabolite response element cre, an operator site recognized by CcpA . The presumed cre overlaps the -35 region of the cccA promoter . Strains carrying a cccA'-IacZ fusion formed blue colonies when grown on rich solid medium, whereas white colonies were obtained when glucose was present . beta-Galactosidase assays with cells grown in rich medium confirmed the repressive effect of glucose on cccA'-lacZ expression . Introduction of a ccpA or hprK mutation or of a mutation affecting the presumed cccA cre relieved the repressive effect of glucose during late log phase . An additional glucose repression mechanism was activated during stationary phase, which was not relieved by the ccpA, hprK or cre mutations . An interaction of the repressor/corepressor complex (CcpA/seryl-phosphorylated HPr (P-Ser-HPr)) with the cccA cre could be demonstrated by gel shift experiments . By contrast, a DNA fragment carrying mutations in the presumed cccA cre was barely shifted by the CcpA/P-Ser-HPr complex . In footprinting experiments, the region corresponding to the presumed cccA cre was specifically protected in the presence of the CcpA/P-Ser-HPr complex. J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 429 - 32 Evidence for a dimerisation state of the Bacillus subtilis catabolite repression HPr-like protein, Crh; Penin F et al.; The Bacillus subtilis catabolite repression HPr (Crh) exhibits 45% sequence identity when compared to histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system . We report here that Crh preparations contain a mixture of monomers and homodimers, whereas HPr is known to be monomeric in solution . The dissociation rate of dimers is very slow (t1/2 of about 10 hours), and the percentage of dimers in Crh preparations increases with rising temperature or protein concentration . However, at temperatures above 25 degrees C and a protein concentration of 10 mg/ml, Crh dimers slowly aggregate . Typically, NMR spectra recorded at 25 degrees C showed the coexistence of both forms of Crh, while in Crh solutions kept at 35 degrees C, almost exclusively Crh monomers could be detected . Circular dichroism analysis revealed that the monomeric and dimeric forms of Crh are well folded and exhibit the same overall structure . The physiological significance of the slow Crh monomer/dimer equilibrium remains enigmatic. Environ Health Perspect, 2001 May, 109 Suppl 2, 325 - 32 Biological control of Fusarium moniliforme in maize; Bacon CW et al.; Fusarium moniliforme Sheldon, a biological species of the mating populations within the (italic)Gibberella fujikuroi species complex, i.e., population A {= G . moniliformis (Sheld.) Wineland}, is an example of a facultative fungal endophyte . During the biotrophic endophytic association with maize, as well as during saprophytic growth, F . moniliforme produces the fumonisins . The fungus is transmitted vertically and horizontally to the next generation of plants via clonal infection of seeds and plant debris . Horizontal infection is the manner by which this fungus is spread contagiously and through which infection occurs from the outside that can be reduced by application of certain fungicides . The endophytic phase is vertically transmitted . This type infection is important because it is not controlled by seed applications of fungicides, and it remains the reservoir from which infection and toxin biosynthesis takes place in each generation of plants . Thus, vertical transmission of this fungus is just as important as horizontal transmission . A biological control system using an endophytic bacterium, Bacillus subtilis, has been developed that shows great promise for reducing mycotoxin accumulation during the endophytic (vertical transmission) growth phase . Because this bacterium occupies the identical ecological niche within the plant, it is considered an ecological homologue to F . moniliforme, and the inhibitory mechanism, regardless of the mode of action, operates on the competitive exclusion principle . In addition to this bacterium, an isolate of a species of the fungus Trichoderma shows promise in the postharvest control of the growth and toxin accumulation from F . moniliforme on corn in storage. Mol Microbiol, 2001 May, 40(3), 634 - 44 NucA is required for DNA cleavage during transformation of Bacillus subtilis; Provvedi R et al.; We have re-examined the roles of nucA and nin, in the transformation of Bacillus subtilis as conflicting accounts have been presented concerning the importance of these genes for transformation . The present report demonstrates that nucA deficiency lowers the rate of DNA transport and that NucA is needed for the double-strand cleavage of transforming DNA, probably acting directly as an endonuclease . A relative paucity of DNA termini, resulting from the absence of this endonuclease activity, most probably accounts for the decreased transport rate . NucA is a bitopic integral membrane protein, with its C-terminus external to the membrane where it is appropriately located to effect the cleavage of bound transforming DNA . We have also investigated the roles of the known competence genes in the DNA processing that accompanies transformation in B . subtilis . The genes that are required for DNA transport (comEA, comEC and comFA) are also required for the degradation of the non-transforming strand that accompanies internalization, but comEC and comFA are not needed for the double-strand cleavage that occurs external to the cell membrane. Eur J Biochem, 2001 May, 268(10), 3069 - 74 Generation of a proton potential by succinate dehydrogenase of Bacillus subtilis functioning as a fumarate reductase; Schnorpfeil M et al.; The membrane fraction of Bacillus subtilis catalyzes the reduction of fumarate to succinate by NADH . The activity is inhibited by low concentrations of 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO), an inhibitor of succinate: quinone reductase . In sdh or aro mutant strains, which lack succinate dehydrogenase or menaquinone, respectively, the activity of fumarate reduction by NADH was missing . In resting cells fumarate reduction required glycerol or glucose as the electron donor, which presumably supply NADH for fumarate reduction . Thus in the bacteria, fumarate reduction by NADH is catalyzed by an electron transport chain consisting of NADH dehydrogenase (NADH:menaquinone reductase), menaquinone, and succinate dehydrogenase operating in the reverse direction (menaquinol:fumarate reductase) . Poor anaerobic growth of B . subtilis was observed when fumarate was present . The fumarate reduction catalyzed by the bacteria in the presence of glycerol or glucose was not inhibited by the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or by membrane disruption, in contrast to succinate oxidation by O2 . Fumarate reduction caused the uptake by the bacteria of the tetraphenyphosphonium cation (TPP+) which was released after fumarate had been consumed . TPP+ uptake was prevented by the presence of CCCP or HOQNO, but not by N,N'-dicyclohexylcarbodiimide, an inhibitor of ATP synthase . From the TPP+ uptake the electrochemical potential generated by fumarate reduction was calculated (Deltapsi = -132 mV) which was comparable to that generated by glucose oxidation with O2 (Deltapsi = -120 mV) . The Deltapsi generated by fumarate reduction is suggested to stem from menaquinol:fumarate reductase functioning in a redox half-loop. Food Addit Contam, 2001 May, 18(5), 385 - 93 Inhibition tests for detection and presumptive identification of tetracyclines, beta-lactam antibiotics and quinolones in poultry meat; Okerman L et al.; A combination of three plates, seeded with strains of Micrococcus luteus, Bacillus cereus or Escherichia coli, can be used for detection of residues of beta-lactam antibiotics, tetracyclines and fluoroquinolones . The sensitivity of each plate is optimal for only one of these groups, resulting in detection limits (LOD) lower than the corresponding maximum residue limits (MRL) and in distinct inhibition patterns typical for each antibiotic family . Beta-lactam antibiotics such as penicillin G, ampicillin and amoxicillin give only inhibition zones on the plate with M . luteus . Tetracyclines are detected up to the MRL level with B . cereus, and fluoroquinolones with E . coli . The LODs of the antibiotics tested were as follows: penicillin G (PENG) 0.9 ng, ampicillin (AMPI) 0.6 ng and amoxicillin (AMOX) 1.0 ng on the plate with M . luteus; tetracycline (TET) 4 ng, oxytetracycline (OXY) 3 ng, doxycycline (DOX) 0.6 ng, and chlortetracycline (CHL) 0.3 ng on the plate with B . cereus; enrofloxacin (ENRX) 1.5 ng, ciprofloxacin (CIPX) 0.5 ng and flumequine (FLUM) 1.5 ng on the plate with E . coli . The combination of plates enables the laboratory to select appropriate chromatographic techniques for identification and quantification of the residues . On the other hand, the three groups can also be detected on one plate seeded with Bacillus subtilis, although the limits of detection are higher: PENG 0.4 ng, AMPI and AMOX 3 ng, TET 5 ng, OXY 8 ng, DOX 1 ng, CHL 0.5 ng, ENRX 4 ng, CIPX 10 ng and FLUM 4 ng . The test was applied to 228 broiler fillets and to 27 turkey thighs, originating from different poultry slaughterhouses . Nineteen broiler fillets contained inhibiting substances . The positive results of the inhibition tests were confirmed with a chromatographic technique . Doxycycline residues were found in 16 samples and amoxicillin in two. Arch Microbiol, 2001 Mar, 175(3), 234 - 7 Growth-rate regulation of the Bacillus subtilis accBC operon encoding subunits of acetyl-CoA carboxylase, the first enzyme of fatty acid synthesis; Marini PE et al.; Acetyl-CoA carboxylase catalyses the synthesis of malonyl-CoA, the first intermediate in fatty acid (hence phospholipid) synthesis . The accBC operon from Bacillus subtilis codes for two subunits of acetyl-CoA carboxylase, biotin carboxyl-carrier and biotin carboxylase . In this work, the 5'-end of the accBC mRNA was determined by primer-extension, and transcriptional fusion analysis of the accBC promoter was carried out under a variety of growth conditions . A direct correlation between the levels of transcription of the accBC genes and the rate of cellular growth is reported . Consistent results were also obtained in nutritional upshift and downshift experiments. FEBS Lett, 2001 May 11, 496(2-3), 117 - 20 Mrp-dependent Na(+)/H(+) antiporters of Bacillus exhibit characteristics that are unanticipated for completely secondary active transporters; Ito M et al.; The Na(+)/H(+) antiport activity encoded by the seven-gene mrp operons of Bacillus subtilis and alkaliphilic Bacillus pseudofirmus OF4 were cloned into a low copy plasmid, were expressed in several Escherichia coli mutant strains and compared side-by-side with similarly cloned nhaA, a major secondary antiporter from E . coli . All three antiporter systems exhibited electron donor-dependent antiport in a fluorescence-based vesicle assay, with NhaA being the most active . In whole cells of the same antiporter-deficient strain from which the vesicles were made, E . coli KNabc, Mrp-mediated Na(+) exclusion was significantly more protonophore-resistant than that conferred by NhaA . The Mrp systems were also more efficacious than NhaA: in supporting anaerobic Na(+) resistance in wild type and a terminal oxidase mutant strain of E . coli (SBS2115); and in increasing non-fermentative growth of an NADH dehydrogenase-minus E . coli mutant (ANN0222) . The results suggest the possibility that the Mrp systems may have both secondary and primary energization capacities. Anal Biochem, 2001 May 15, 292(2), 272 - 9 Nonradioactive assay for cellular dimethylallyl diphosphate; Fisher AJ et al.; A sensitive, nonradioactive method was developed to measure cellular levels of dimethylallyl diphosphate (DMAPP), a central intermediate of isoprenoid metabolism in nature . The assay is based on the hydrolysis of DMAPP in acid to the volatile hydrocarbon isoprene (2-methyl-1,3-butadiene), with subsequent analysis of isoprene by headspace gas chromatography with reduction gas detection . In the assay, cell samples are directly acidified with 4 M H(2)SO(4) in sealed reaction vials . Therefore, there is no need to extract metabolites, purify them, and keep them stable prior to analysis, and degradative enzymatic activities are destroyed . DMAPP levels of 23 +/- 4 nmol (g fresh weight)(-1) {ca . 85 nmol (g dry weight)(-1)} and 80 +/- 14 nmol (g fresh weight)(-1) {ca . 296 nmol (g dry weight)(-1)} were measured in dark- and light-adapted leaves of Populus deltoides (Eastern cottonwood), respectively . Evidence is presented to show that DMAPP is the major leaf metabolite giving rise to isoprene following acid hydrolysis . DMAPP levels in Bacillus subtilis and Saccharomyces cerevisiae were determined to be 40.8 +/- 16.7 pmol (OD(600))(-1) {ca . 638 pmol (mg dry weight)(-1)} and 6.3 +/- 3.7 pmol (OD(600))(-1) {ca . 139 pmol (mg dry weight)(-1)}, respectively . The method should be suitable for any cell or tissue type and isolated cellular organelles . Infect Immun, 2001 Jun, 69(6), 3924 - 32 Characterization of the groESL operon in Listeria monocytogenes: utilization of two reporter systems (gfp and hly) for evaluating in vivo expression; Gahan CG et al.; The ability of intracellular pathogens to sense and adapt to the hostile environment of the host is an important factor governing virulence . We have sequenced the operon encoding the major heat shock proteins GroES and GroEL in the gram-positive food-borne pathogen Listeria monocytogenes . The operon has a conserved orientation in the order groES groEL . Upstream of groES and in the opposite orientation is a gene encoding a homologue of the Bacillus subtilis protein YdiL, while downstream of groEL is a gene encoding a putative bile hydrolase . We used both reverse transcriptase-PCR (RT-PCR) and transcriptional fusions to the UV-optimized Aequorea victoria green fluorescent protein (GFP(UV)) to analyze expression of groESL under various environmental stress conditions, including heat shock, ethanol stress, and acid shock, and during infection of J774 mouse macrophage cells . Strains harboring GFP(UV) transcriptional fusions to the promoter region of groESL demonstrated a significant increase in fluorescence following heat shock that was detected by both fluorimetry and fluorescence microscopy . Using both RT-PCR and GFP technology we detected expression of groESL following internalization by J774 cells . Increased intracellular expression of dnaK was also determined using RT-PCR . We have recently described a system which utilizes L . monocytogenes hemolysin as an in vivo reporter of gene expression within the host cell phagosome (C . G . M . Gahan and C . Hill, Mol . Microbiol . 36:498-507, 2000) . In this study a strain was constructed in which hemolysin expression was placed under the control of the groESL promoter . In this strain hemolysin expression during infection also confirms transcription from the groESL promoter during J774 and murine infection, albeit at lower levels than the known virulence factor plcA. Infect Immun, 2001 Jun, 69(6), 3744 - 54 PerR controls oxidative stress resistance and iron storage proteins and is required for virulence in Staphylococcus aureus; Horsburgh MJ et al.; The Staphylococcus aureus genome encodes three ferric uptake regulator (Fur) homologues: Fur, PerR, and Zur . To determine the exact role of PerR, we inactivated the gene by allelic replacement using a kanamycin cassette, creating strain MJH001 (perR) . PerR was found to control transcription of the genes encoding the oxidative stress resistance proteins catalase (KatA), alkyl hydroperoxide reductase (AhpCF), bacterioferritin comigratory protein (Bcp), and thioredoxin reductase (TrxB) . Furthermore, PerR regulates transcription of the genes encoding the iron storage proteins ferritin (Ftn) and the ferritin-like Dps homologue, MrgA . Transcription of perR was autoregulated, and PerR repressed transcription of the iron homeostasis regulator Fur, which is a positive regulator of catalase expression . PerR functions as a manganese-dependent, transcriptional repressor of the identified regulon . Elevated iron concentrations produced induction of the PerR regulon . PerR may act as a peroxide sensor, since addition of external hydrogen peroxide to 8325-4 (wild type) resulted in increased transcription of most of the PerR regulon, except for fur and perR itself . The PerR-regulated katA gene encodes the sole catalase of S . aureus, which is an important starvation survival determinant but is surprisingly not required for pathogenicity in a murine skin abscess model of infection . In contrast, PerR is not necessary for starvation survival but is required for full virulence (P < 0.005) in this model of infection . PerR of S . aureus may act as a redox sentinel protein during infection, analogous to the in vitro activities of OxyR and PerR of Escherichia coli and Bacillus subtilis, respectively . However, it differs in its response to the metal balance within the cell and has the added capability of regulating iron uptake and storage. Folia Microbiol (Praha), 2000, 45(5), 397 - 406 Cloning of a two-component regulatory system probably involved in the regulation of chitinase in Streptomyces coelicolor A3(2); Kormanec J et al.; Using the method for the identification of promoters recognized by the sporulation specific sigma factor (sigma F), we identified a positive 950 bp Sau3AI DNA fragment in Streptomyces coelicolor A3(2) . High-resolution S1-nuclease mapping identified a potential promoter, PF35, in the E . coli two-plasmid system similar to the consensus sequence of Bacillus subtilis promoters recognized by the general stress-response sigma factor (sigma B) . However, the putative sigF-dependent promoter, PF35, was inactive in S . coelicolor in the course of differentiation, and it was located divergently in the promoter region directing expression of the chiC gene encoding chitinase . Sequence analysis of the region potentially governed by PF35 revealed two translationally coupled genes encoding proteins similar to bacterial two-component regulatory systems, and with the highest similarity to the two-component system chiS, chiR, regulating chitinase activity in Streptomyces thermoviolaceus . However, the genes had a divergent orientation with respect to the PF35 promoter . Disruption of the S . coelicolor chiR gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin . Moreover, the chiR disruption did not affect the overall chitinase activity. J Bacteriol, 2001 Jun, 183(11), 3506 - 14 Biosynthesis of the glycolipid anchor in lipoteichoic acid of Staphylococcus aureus RN4220: role of YpfP, the diglucosyldiacylglycerol synthase; Kiriukhin MY et al.; In Staphylococcus aureus RN4220, lipoteichoic acid (LTA) is anchored in the membrane by a diglucosyldiacylglycerol moiety . The gene (ypfP) which encodes diglucosyldiacylglycerol synthase was recently cloned from Bacillus subtilis and expressed in Escherichia coli (P . Jorasch, F . P . Wolter, U . Zahringer, and E . Heinz, Mol . Microbiol . 29:419-430, 1998) . To define the role of ypfP in this strain of S . aureus, a fragment of ypfP truncated from both ends was cloned into the thermosensitive replicon pVE6007 and used to inactivate ypfP . Chloramphenicol-resistant (ypfP::cat) clones did not synthesize the glycolipids monoglucosyldiacylglycerol and diglucosyldiacylglycerol . Thus, YpfP would appear to be the only diglucosyldiacylglycerol synthase in S . aureus providing glycolipid for LTA assembly . In LTA from the mutant, the glycolipid anchor is replaced by diacylglycerol . Although the doubling time of the mutant was identical to that of the wild type in Luria-Bertani (LB) medium, growth of the mutant in LB medium containing 1% glycine was not observed . This inhibition was antagonized by either L- or D-alanine . Moreover, viability of the mutant at 37 degrees C in 0.05 M phosphate (pH 7.2)-saline for 12 h was reduced to <0.1% . Addition of 0.1% D-glucose to the phosphate-saline ensured viability under these conditions . The autolysis of the ypfP::cat mutant in the presence of 0.05% Triton X-100 was 1.8-fold faster than that of the parental strain . Electron microscopy of the mutant revealed not only a small increase in cell size but also the presence of pleomorphic cells . Each of these phenotypes may be correlated with either (or both) a deficiency of free glycolipid in the membrane or the replacement of the usual glycolipid anchor of LTA with diacylglycerol. J Bacteriol, 2001 Jun, 183(11), 3293 - 302 Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator; Schultz AC et al.; The soil bacterium Bacillus subtilis has developed a highly controlled system for the utilization of a diverse array of low-molecular-weight compounds as a nitrogen source when the preferred nitrogen sources, e.g., glutamate plus ammonia, are exhausted . We have identified such a system for the utilization of purines as nitrogen source in B . subtilis . Based on growth studies of strains with knockout mutations in genes, complemented with enzyme analysis, we could ascribe functions to 14 genes encoding enzymes or proteins of the purine degradation pathway . A functional xanthine dehydrogenase requires expression of five genes (pucA, pucB, pucC, pucD, and pucE) . Uricase activity is encoded by the pucL and pucM genes, and a uric acid transport system is encoded by pucJ and pucK . Allantoinase is encoded by the pucH gene, and allantoin permease is encoded by the pucI gene . Allantoate amidohydrolase is encoded by pucF . In a pucR mutant, the level of expression was low for all genes tested, indicating that PucR is a positive regulator of puc gene expression . All 14 genes except pucI are located in a gene cluster at 284 to 285 degrees on the chromosome and are contained in six transcription units, which are expressed when cells are grown with glutamate as the nitrogen source (limiting conditions), but not when grown on glutamate plus ammonia (excess conditions) . Our data suggest that the 14 genes and the gde gene, encoding guanine deaminase, constitute a regulon controlled by the pucR gene product . Allantoic acid, allantoin, and uric acid were all found to function as effector molecules for PucR-dependent regulation of puc gene expression . When cells were grown in the presence of glutamate plus allantoin, a 3- to 10-fold increase in expression was seen for most of the genes . However, expression of the pucABCDE unit was decreased 16-fold, while expression of pucR was decreased 4-fold in the presence of allantoin . We have identified genes of the purine degradation pathway in B . subtilis and showed that their expression is subject to both general nitrogen catabolite control and pathway-specific control. Nucleic Acids Res, 2001 May 1, 29(9), 1892 - 7 Modular construction for function of a ribonucleoprotein enzyme: the catalytic domain of Bacillus subtilis RNase P complexed with B . subtilis RNase P protein; Loria A et al.; The bacterial RNase P holoenzyme catalyzes the formation of the mature 5'-end of tRNAs and is composed of an RNA and a protein subunit . Among the two folding domains of the RNase P RNA, the catalytic domain (C-domain) contains the active site of this ribozyme . We investigated specific binding of the Bacillus subtilis C-domain with the B.subtilis RNase P protein and examined the catalytic activity of this C-domain-P protein complex . The C-domain forms a specific complex with the P protein with a binding constant of approximately 0.1 microM . The C-domain-P protein complex and the holoenzyme are equally efficient in cleaving single-stranded RNA (approximately 0.9 min(-1) at pH 7.8) and substrates with a hairpin-loop 3' to the cleavage site (approximately 40 min(-1)) . The holoenzyme reaction is much more efficient with a pre-tRNA substrate, binding at least 100-fold better and cleaving 10-500 times more efficiently . These results demonstrate that the RNase P holoenzyme is functionally constructed in three parts . The catalytic domain alone contains the active site, but has little specificity and affinity for most substrates . The specificity and affinity for the substrate is generated by either the specificity domain of RNase P RNA binding to a T stem-loop-like hairpin or RNase P protein binding to a single-stranded RNA . This modular construction may be exploited to obtain RNase P-based ribonucleoprotein complexes with altered substrate specificity. Enzyme Microb Technol, 2001 May 7, 28(7-8), 705 - 712 Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases; Nthangeni MB et al.; The gene coding for an extracellular lipase of Bacillus licheniformis was cloned using PCR techniques . The sequence corresponding to the mature lipase was subcloned into the pET 20b(+) expression vector to construct a recombinant lipase protein containing 6 histidine residues at the C-terminal . High-level expression of the lipase by Escherichia coli cells harbouring the lipase gene-containing expression vector was observed upon induction with IPTG at 30 degrees C . A one step purification of the recombinant lipase was achieved with Ni-NTA resin . The specific activity of the purified enzyme was 130 units/mg with p-nitrophenyl-palmitate as substrate . The enzyme showed maximum activity at pH 10-11.5 and was remarkably stable at alkaline pH values up to 12 . The enzyme was active toward p-nitrophenyl esters of short to long chains fatty acids but with a marked preference for esters with C(6) and C(8) acyl groups . The amino acid sequence of the lipase shows striking similarities to lipases from Bacillus subtilis and Bacillus pumilus . Based on the amino acid identity and biochemical characteristics, we propose that Bacillus lipases be classified into two distinct subfamilies of their own. Biochem J, 2001 May 15, 356(Pt 1), 217 - 22 Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants; Franco R et al.; Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring . Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity . Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme . In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX . Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx . 80% of the total porphyrin corresponds to protoporphyrin IX . Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn(2+) results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate . Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product . Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product. Photochem Photobiol, 2001 Apr, 73(4), 447 - 51 The history of the UV radiation climate of the earth--theoretical and space-based observations; Cockell CS et al.; In the Archean era (3.8-2.5 Ga ago) the Earth probably lacked a protective ozone column . Using data obtained in the Earth's orbit on the inactivation of Bacillus subtilis spores we quantitatively estimate the potential biological effects of such an environment . We combine this practical data with theoretical calculations to propose a history of the potential UV stress on the surface of the Earth over time . The data suggest that an effective ozone column was established at a pO2 of approximately 5 x 10(-3) present atmospheric level . The improvement in the UV environment on the early Proterozoic Earth might have been a much more rapid event than has previously been supposed, with DNA damage rates dropping by two orders of magnitude in the space of just a few tens of millions of years . We postulate that a coupling between reduced UV stress and increased pO2 production could have contributed toward a positive feedback in the production of ozone in the early Proterozoic atmosphere . This would contribute to the apparent rapidity of the oxidation event . The data provide an evolutionary perspective on present-day Antarctic ozone depletion. Genes Dev, 2001 May 1, 15(9), 1093 - 103 Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels; Ratnayake-Lecamwasam M et al.; CodY, a highly conserved protein in the low G + C, gram-positive bacteria, regulates the expression of many Bacillus subtilis genes that are induced as cells make the transition from rapid exponential growth to stationary phase and sporulation . This transition has been associated with a transient drop in the intracellular pool of GTP . Many stationary-phase genes are also induced during exponential-growth phase by treatment of cells with decoyinine, a GMP synthetase inhibitor . The effect of decoyinine on an early-stationary-phase gene is shown here to be mediated through CodY and to reflect a reduction in guanine nucleotide accumulation . CodY proved to bind GTP in vitro . Moreover, CodY-mediated repression of target promoters was dependent on a high concentration of GTP, comparable to that found in rapidly growing exponential-phase cells . Because a codY-null mutant was able to sporulate under conditions of nutrient excess, CodY also appears to be a critical factor that normally prevents sporulation under such conditions . Thus, B . subtilis CodY is a novel GTP-binding protein that senses the intracellular GTP concentration as an indicator of nutritional conditions and regulates the transcription of early-stationary-phase and sporulation genes, allowing the cell to adapt to nutrient limitation. Biosci Biotechnol Biochem, 2001 Mar, 65(3), 579 - 83 Green marker for colonies of Bacillus subtilis; Itaya M et al.; A Bacillus subtilis plasmid encoding a green fluorescence protein gene (gfp) was constructed . The fluorescence of B . subtilis colonies having this plasmid on agar plates was so high that they could be readily discerned visually under UV light . The fluorescence could be effectively expressed in three ways (i) through use of a strong bsr promoter (blasticidin S resistance gene), (ii) by efficient translation with the bsr translation system, and (iii) through increase in the copy number per cell . The high stability of the GFP plasmid was demonstrated by using more complicated growth conditions without any antibiotic for selection. Sheng Wu Gong Cheng Xue Bao, 2001 Jan, 17(1), 11 - 5 {Cloning of neutral phytase gene nphy from Bacillus subtilis and its expression in Escherichia coli}; Yao B et al.; The gene encoding the neutral phytase nphy was cloned from Bacillus subtilis by polymerase chain reaction (PCR) . Nucleotide sequence analysis of nphy revealed the presence of an open reading frame of 1152 bp coding for 383 aa . The start codon was followed by a sequence coding for a putative signal peptide of 26 aa in length . The nphy without original signal peptide encoding sequence was cloned into E . coli expression plasmid pTYB40 . The result of SDS-PAGE of the phytase expressed in E . coli showed that the nphy had been overexpressed . The expressed phytase was over 40% of the total soluble protein of E . coli, and has normal bioactivity. Biochemistry, 2001 Feb 27, 40(8), 2340 - 50 A structural variation for MurB: X-ray crystal structure of Staphylococcus aureus UDP-N-acetylenolpyruvylglucosamine reductase (MurB); Benson TE et al.; The X-ray crystal structure of the substrate free form of Staphylococcus aureus UDP-N-acetylenolpyruvylglucosamine reductase (MurB) has been solved to 2.3 A resolution with an R-factor of 20.3% and a free R-factor of 22.3% . While the overall fold of the S . aureus enzyme is similar to that of the homologous Escherichia coli MurB X-ray crystal structure, notable distinctions between the S . aureus and E . coli MurB protein structures occur in residues involved in substrate binding . Analysis of available MurB sequences from other bacteria suggest that the S . aureus MurB structure is representative of a distinct structural class of UDP-N-acetylenolpyruvylglucosamine reductases including Bacillus subtilis and Helicobacter pylori that are characterized by a modified mechanism for substrate binding. J Bacteriol, 2001 May, 183(10), 3237 - 46 Two ResD-controlled promoters regulate ctaA expression in Bacillus subtilis; Paul S et al.; The Bacillus subtilis ResDE two-component system plays a positive role in global regulation of genes involved in aerobic and anaerobic respiration . ctaA is one of the several genes involved in aerobic respiration that requires ResD for in vivo expression . The ctaAB-divergent promoter regulatory region has three ResD binding sites; A1, A2, and A3 . The A2 site is essential for in vivo promoter activity, while binding sites A2 and A3 are required for full ctaA promoter activity . In this study, we demonstrate the role of ResD~P in the activation of the ctaA promoter using an in vitro transcription system . The results indicate that the ctaA promoter (binding sites A2 and A3) has two transcriptional start sites . Binding site A2 was sufficient for weak transcription of the upstream promoter (Pv) by Esigma(A), transcription which was enhanced approximately 1.5-fold by ResD and 5-fold by ResD~P . The downstream promoter (Ps) required both binding sites A2 and A3 and was not transcribed by Esigma(A) with or without ResD~P . RNA polymerase (RNAP) isolated from B . subtilis when cells were at the end of exponential growth (T(0)) or 3, 4, or 5 h into the stationary phase (T(3), T(4), or T( 5), respectively) was used in in vitro transcription assays . Maximal transcription from Ps required T(4) RNAP plus ResD~P . RNAP isolated from a spo0A or a sigE mutant strain was not capable of Ps transcription . Comparison of the Ps promoter sequence with the SigE binding consensus suggests that the ctaA Ps promoter may be a SigE promoter . The collective data from ResD footprinting, in vivo promoter deletion analysis, and in vitro transcription assays suggest that ctaA is transcribed during late exponential to early stationary phases of growth from the Pv promoter, which requires ResD binding site A2, Esigma(A), and ResD~P, and during later stationary phase from Ps, which requires binding sites A2 and A3, ResD~P, and Esigma(E) or a sigma factor whose transcription is dependent on SigE. J Bacteriol, 2001 May, 183(10), 3041 - 9 SpoVID guides SafA to the spore coat in Bacillus subtilis; Ozin AJ et al.; Bacteria assemble complex structures by targeting proteins to specific subcellular locations . The protein coat that encases Bacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides . The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID . In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface . SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures . However, it is not known which spore coat proteins interact directly with SpoVID . In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro . We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself . Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID . Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore . We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID. J Bacteriol, 2001 May, 183(10), 3032 - 40 Response of Bacillus subtilis to cerulenin and acquisition of resistance; Schujman GE et al.; Cerulenin is a fungal mycotoxin that potently inhibits fatty acid synthesis by covalent modification of the active site thiol of the chain-elongation subtypes of beta-ketoacyl-acyl carrier protein (ACP) synthases . The Bacillus subtilis fabF (yjaY) gene (fabF(b)) encodes an enzyme that catalyzes the condensation of malonyl-ACP with acyl-ACP to extend the growing acyl chain by two carbons . There were two mechanisms by which B . subtilis adapted to exposure to this antibiotic . First, reporter gene analysis demonstrated that transcription of the operon containing the fabF gene increased eightfold in response to a cerulenin challenge . This response was selective for the inhibition of fatty acid synthesis, since triclosan, an inhibitor of enoyl-ACP reductase, triggered an increase in fabF reporter gene expression while nalidixic acid did not . Second, spontaneous mutants arose that exhibited a 10-fold increase in the MIC of cerulenin . The mutation mapped at the B . subtilis fabF locus, and sequence analysis of the mutant fabF allele showed that a single base change resulted in the synthesis of FabF(b){I108F} . The purified FabF(b) and FabF(b){I108F} proteins had similar specific activities with myristoyl-ACP as the substrate . FabF(b) exhibited a 50% inhibitory concentration (IC(50)) of cerulenin of 0.1 microM, whereas the IC(50) for FabF(b){I108} was 50-fold higher (5 microM) . These biochemical data explain the absence of an overt growth defect coupled with the cerulenin resistance phenotype of the mutant strain. Biochim Biophys Acta, 2001 May 3, 1526(2), 131 - 40 Isolation and characterization of four bactericidal domains in the bovine beta-lactoglobulin; Pellegrini A et al.; Proteolytic digestion of bovine beta-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity . The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15-20), AASDISLLDAQSAPLR (residues 25-40), IPAVFK (residues 78-83) and VLVLDTDYK (residues 92-100) . The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only . In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified . The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis . By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55-64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision . A peptide with this sequence was synthesized and assayed for bactericidal activity . VLVATLRYKK was strongly active against all the bacterial strains tested . Our results suggest a possible antimicrobial function of beta-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of beta-lactoglobulin could be useful to increase its antimicrobial function. J Nat Prod, 2001 Apr, 64(4), 527 - 30 New macrolides and furan carboxylic acid derivative from the sponge-derived fungus Cladosporium herbarum; Jadulco R et al.; Bioassay-guided fractionation of organic extracts of Cladosporium herbarum, isolated from the marine sponge Callyspongia aerizusa, yielded two new macrolide metabolites: pandangolide 3 and 4 (1 and 2) and the known fungal metabolites pandangolide 2 (3), cladospolide B (4), and iso-cladospolide B (5) . Also isolated were the antimicrobially active (against Bacillus subtilis and Staphylococcus aureus) furan carboxylic acids: Sumiki's acid (6) and its new derivative, acetyl Sumiki's acid (7) . All structures were elucidated by spectroscopic methods. J Mol Microbiol Biotechnol, 2001 Apr, 3(2), 237 - 46 Functions of tetracycline efflux proteins that do not involve tetracycline; Krulwich TA et al.; Tet(L) and Tet(K) are specific antibiotic-resistance determinants . They catalyze efflux of a tetracycline(Tc)-divalent metal complex in exchange for protons, as do other Tet efflux proteins . These Tet proteins also catalyze Na+ and K+ exchange for protons . Each of the "cytoplasmic substrates", Na+, K+ and the Tc-metal ion complex, can also be exchanged for K+, a catalytic mode that accounts for the long-recognized K+ uptake capacity conferred by some Tet proteins . The multiple catalytic modes of Tet(L) and Tet(K) provide potential new avenues for development of inhibitors of these efflux systems as well as avenues for exploration of structure-function relationships . The multiple catalytic modes of Tet(L), which is chromosomally encoded in Bacillus subtilis, also correspond to diverse physiological roles, including roles in antibiotic-, Na+-, and alkali-resistance as well as K+ acquisition . The use of K+ as an external coupling ion may contribute not only to the organism's K+ uptake capacity but also to its ability to exclude Na+ and Tc at elevated pH values . Regulation of the chromosomal tetL gene by Tc has been proposed to involve a translational re-initiation mechanism that is novel for an antibiotic-resistance gene and increases Tet expression seven-fold . Other elements of tetL expression and its regulation are already evident, including gene amplification and use of multiple promoters . However, further studies are required to clarify the full panoply of regulatory mechanisms, and their integration to ensure different levels of tetL expression that are optimal for its different functions . It will also be of interest to investigate the implications of Tet(L) and Tet(K) multifunctionality on the emergence and persistence of these antibiotic-resistance genes. J Mol Microbiol Biotechnol, 2001 Apr, 3(2), 151 - 4 The ostensible paradox of multidrug recognition; Neyfakh AA; The ability of multidrug-efflux transporters to recognize scores of dissimilar organic compounds has always been considered paradoxical because of its apparent contradiction to some of the basic dogmas of biochemistry . In order to understand, at least in principle, how a protein can recognize multiple compounds, we analysed the transcriptional regulator of the Bacillus subtilis multidrug transporter Bmr . This regulator, BmrR, binds multiple dissimilar hydrophobic cations and, by activating expression of the Bmr transporter, causes their expulsion from the cell . Crystallographic analysis of the complexes of the inducer-binding domain of BmrR with some of its inducers revealed that ligands penetrate the hydrophobic core of the protein, where they form multiple van der Waals and stacking interactions with hydrophobic amino acids and an electrostatic bond with the buried glutamate . Mutational analysis of the binding site suggests that each ligand forms a unique set of atomic contacts with the protein: each tested mutation exerted disparate effects on the binding of different ligands . The example of BmrR demonstrates that a protein can bind multiple hydrophobic compounds with micromolar affinities by using only electrostatic and hydrophobic interactions . Its ligand specificity can be further broadened by the flexibility of the binding site . It appears, therefore, that the commonly expressed fascination with the relaxed substrate specificity of multidrug transporters is misdirected and originates from an almost exclusive familiarity with the more sophisticated processes of specific molecular recognition that predominate among proteins analyzed to date. Biotechnol Bioeng, 2001 Jun 5, 73(5), 390 - 9 Scale-down model to simulate spatial pH variations in large-scale bioreactors; Amanullah A et al.; For the first time a laboratory-scale two-compartment system was used to investigate the effects of pH fluctuations consequent to large scales of operation on microorganisms . pH fluctuations can develop in production-scale fermenters as a consequence of the combined effects of poor mixing and adding concentrated reagents at the liquid surface for control of the bulk pH . Bacillus subtilis was used as a model culture since in addition to its sensitivity to dissolved oxygen levels, the production of the metabolites, acetoin and 2,3-butanediol, is sensitive to pH values between 6.5 and 7.2 . The scale-down model consisted of a stirred tank reactor (STR) and a recycle loop containing a plug flow reactor (PFR), with the pH in the stirred tank being maintained at 6.5 by addition of alkali in the loop . Different residence times in the loop simulated the exposure time of fluid elements to high values of pH in the vicinity of the addition point in large bioreactors and tracer experiments were performed to characterise the residence time distribution in it . Since the culture was sensitive to dissolved oxygen, for each experiment with pH control by adding base into the PFR, equivalent experiments were conducted with pH control by addition of base into the STR, thus ensuring that any dissolved oxygen effects were common to both types of experiments . The present study indicates that although biomass concentration remained unaffected by pH variations, product formation was influenced by residence times in the PFR of 60 sec or longer . These changes in metabolism are thought to be linked to both the sensitivity of the acetoin and 2,3-butanediol-forming enzymes to pH and to the inducing effects of dissociated acetate on the acetolactate synthase enzyme . Microbiology, 2001 May, 147(Pt 5), 1331 - 41 Transcripts of the genes sacB, amyE, sacC and csn expressed in Bacillus subtilis under the control of the 5' untranslated sacR region display different stabilities that can be modulated; Pereira Y et al.; When Bacillus subtilis levanase (SacC), alpha-amylase (AmyE) and chitosanase (Csn) structural genes were expressed under the regulated control of sacR, the inducible levansucrase (SacB) leader region in a degU32(Hy) mutant, it was observed that the production yields of the various extracellular proteins were quite different . This is mainly due to differences in the stabilities of their corresponding mRNAs which lead to discrepancies between the steady-state level of mRNA of sacB and csn on the one hand and amyE and sacC on the other . In contrast to levansucrase mRNA, the decay curves of alpha-amylase and levanase mRNAs obtained by Northern blotting analysis did not match the decay curves of their functional mRNA . This suggested that only a part of the population of the amyE and sacC transcripts was fully translated, while the others were possibly poorly bound to ribosomes and thus were only partially translated or not at all and consequently submitted to rapid endonuclease degradation . This hypothesis was substantiated by the finding that the introduction of a Shine-Dalgarno sequence upstream from the ribosome-binding site in the sacC transcript resulted in a fourfold increase in both the half-life of this transcript and the production of levanase . An additional cause of low-level levanase production is the premature release of mRNA by the polymerase . It was attempted to correlate this event with internal secondary structures of sacC mRNA. Appl Biochem Biotechnol, 2001 Mar, 90(3), 211 - 20 Purification of alpha-amylases using magnetic alginate beads; Teotia S et al.; Magnetic alginate beads were used to purify alpha-amylases from porcine pancreas, starchzyme, BAN 240L (a commercial purification from Bacillus subtilis), and wheat germ . The beads bound a significant level of alpha-amylase activity from porcine pancreas, BAN 240L, and wheat germ . In each case, the enzyme activity could be eluted by using 1.0 M maltose, a known competitive inhibitor of alpha-amylase . In the case of BAN 240L, 3.6-fold purification with 72% recovery of activity was observed . In the case of wheat germ enzyme, starting from the crude extract, 48-fold purification with 70% activity recovery was observed . Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis also indicated considerable purification in the latter case. Appl Biochem Biotechnol, 2001 Mar, 90(3), 199 - 210 Lichenysin: a more efficient cation chelator than surfactin; Grangemard I et al.; The lipopeptide lichenysin (cyclo-{L-Gln1-->D-Leu2-->L-Leu3-->L-Val4--> L-Asp5-->D-Leu6-->L-Ile7-beta-OH fatty acid}) produced by Bacillus licheniformis structurally resembles surfactin from Bacillus subtilis . The main difference is the presence of a glutaminyl residue in position 1 of the peptide sequence in place of glutamic acid in surfactin . This local variation causes significant changes in the properties of the molecule compared to surfactin . Lichenysin has a higher surfactant power, the critical micellar concentration (c.m.c.) being strongly reduced from 220 to 22 microM and a much higher hemolytic activity because 100% hemolysis was observed with only 15 microM instead of 200 microM . Lichenysin is also a better chelating agent because its association constants with Ca2+ and Mg2+ are increased by a factor of 4 and 16, respectively . This effect is assigned to an increase in the accessibility of the carboxyl group to cations owing to a change in the side chain topology induced by the Glu/Gln exchange . Additionally, the propensity of the lipopeptide for extensive hydrophobic interactions, as illustrated by its low c.m.c., contributes to further stabilization of the cation and an increase in the partitioning of lichenysin into the erythrocyte membrane . Our data support the formation of a lichensyin-Ca2+ complex in a molar ratio of 2:1 instead of 1:1 with surfactin, suggesting an intermolecular salt bridge between two lichenysin molecules . Therefore, when Ca2+ ions are present in the solution, micellization occurs via a dimer assembly, with a possible long-range effect on the spatial arrangement of the micelles or other supramolecular structures . Finally, among all the surfactin peptidic variants so far known, lichenysin is the one for which the three tested activities are the most substantially improved. Mikrobiologiia, 2000 Sep-Oct, 69(5), 681 - 5 {Genetic transformation of Bacillus subtilis cells in the presence of clay minerals montmorillonite and kaolinite}; Lotareva OV et al.; The effect of the clay minerals montmorillonite and kaolinite on the transformation of competent Bacillus subtilis cells with chromosomal DNA was studied . Clay particles were found to substantially increase the transformation frequency of competent cells, as well as the rate of their spontaneous chromosomal and plasmid transformation . The effect was ascribed to the adsorption of bacterial cells on the surface of mineral particles. Mikrobiologiia, 2000 Sep-Oct, 69(5), 653 - 9 {Medium for the production of Bacillus intermedius glutamyl endopeptidase by the recombinant Bacillus subtilis}; Gabdrakhmanova LA et al.; A nutrient medium was elaborated for the efficient production of glutamyl endopeptidase by the recombinant Bacillus subtilis strain AJ73 bearing the Bacillus intermedius 3-19 glutamyl endopeptidase gene within a multicopy plasmid . Optimal concentrations of the main nutrients, peptone and inorganic phosphate, were found using a multifactor approach . To provide for active growth and efficient glutamyl endopeptidase production, the cultivation medium of the recombinant strain should be enriched in phosphorus, organic and inorganic nitrogen sources, and yeast extract . Complex protein substrates, such as casein and gelatin, enhanced the biosynthesis of glutamyl endopeptidase . At the same time, easily metabolizable carbon sources suppressed it . The production of glutamyl endopeptidase was stimulated by the bivalent cations Ca2+, Mg2+, and Co2+. J Basic Microbiol, 2001, 41(1), 17 - 24 Amplification of the Escherichia coli lacZ gene in Bacillus subtilis and its expression on a by-product growth medium; el-Helow ER et al.; A Bacillus subtilis wild type strain and a kinA (spoIIJ) isogenic mutant were compared as hosts for the expression of the Escherichia coli beta-galactosidase gene, lacZ, driven by the B . subtilis aprE promoter in a chromosomal system . The 2 x SG sporulation formula, with some modifications, was used as a basal medium . The specific activity values recorded by the mutant strain at the stationary phase were markedly higher than those achieved by the wild type host . Exposure of the cells to increasing levels of chloramphenicol resulted in significant amplifications of the lacZ region . Gene copy numbers of 19 and 11 were estimated in the amplified wild type and kinA strains, respectively, with high segregational stability records . The magnitude of beta-galactosidase over-expression was dependent on, and roughly proportional to antibiotic resistance levels . Among five examined by-products, a 2.3-times diluted concentration of neutralized cheese whey was successfully used as a sole medium component for over-expression of the recombinant beta-galactosidase gene in B . subtilis. J Biol Chem, 2001 Jun 29, 276(26), 23471 - 9 Epub 2001 Apr 19. A single domain of the replication termination protein of Bacillus subtilis is involved in arresting both DnaB helicase and RNA polymerase; Gautam A et al.; The current models that have been proposed to explain the mechanism of replication termination are (i) passive arrest of a replication fork by the terminus (Ter) DNA-terminator protein complex that impedes the replication fork and the replicative helicase in a polar fashion and (ii) an active barrier model in which the Ter-terminator protein complex arrests a fork not only by DNA-protein interaction but also by mechanistically significant terminator protein-helicase interaction . Despite the existence of some evidence supporting in vitro interaction between the replication terminator protein (RTP) and DnaB helicase, there has been continuing debate in the literature questioning the validity of the protein-protein interaction model . The objective of the present work was two-fold: (i) to reexamine the question of RTP-DnaB interaction by additional techniques and different mutant forms of RTP, and (ii) to investigate if a common domain of RTP is involved in the arrest of both helicase and RNA polymerase . The results validate and confirm the RTP-DnaB interaction in vitro and suggest a critical role for this interaction in replication fork arrest . The results also show that the Tyr(33) residue of RTP plays a critical role both in the arrest of helicase and RNA polymerase. Biotechnol Prog, 2001 Mar-Apr, 17(2), 273 - 7 Production of extracellular protease from crude substrates with dregs in an external-loop airlift bioreactor with lower ratio of height to diameter; Yuguo Z et al.; Bacillus subtilis AS1.398 was cultivated in a 11.5-L total volume external-loop airlift bioreactor with a low height-to-diameter ratio of 2.9 and a riser-to-downcomer diameter ratio of 6.6 for the production of protease from crude substrates with dregs . The influence of aeration rate, liquid volume, and sparger hole diameter on protease production was investigated . An average of 8197 u/mL protease activity was obtained after a total fermentation time of 32 h in the external-loop airlift bioreactor with a liquid volume of 8.5 L, air flow rate of 1.5 vvm, and sparger hole diameter of 1.5 mm . The addition of one stainless steel sieve plate in the riser of the airlift bioreactor increased productivity of protease . After 32 h of fermentation, an average of 8718 u/mL protease activity was achieved in the external-loop airlift bioreactor with one sieve plate and an air flow rate of 1.2 vvm, liquid volume of 8.5 L, and gas sparger hole diameter of 1.5 mm . This was 9.0% higher than the typical averages of about 8000 u/mL protease activity in the mechanically stirred tank bioreactors of the enzyme factory using the same microorganism . It is possible to make a scale-up of the external-loop airlift bioreactor and feasible to operate it for production of protease from crude substrate with dregs. J Microbiol Methods, 2001 Jun, 45(2), 119 - 26 Influence of electrical properties on the evaluation of the surface hydrophobicity of Bacillus subtilis; Ahimou F et al.; The surface hydrophobicity of nine Bacillus subtilis strains in different states (spores, vegetative cells, and dead cells) was assessed by water contact angle measurements, hydrophobic interaction chromatography (HIC) and bacterial adhesion to hydrocarbon (BATH) . Electrokinetic properties of B . subtilis strains were characterized by zeta potential measurements and found to differ appreciably according to the strain . Correlations between HIC data, BATH data and zeta potential showed that HIC and BATH are influenced by electrostatic interactions . Water contact angle measurements thus provide a better estimate of cell surface hydrophobicity . The water contact angle of B . subtilis varied according to the strain and the state, the spores tending to be more hydrophobic than vegetative cells. J Biol Chem, 2001 Jul 6, 276(27), 25230 - 5 Epub 2001 Apr 17. Distinction between major and minor Bacillus signal peptidases based on phylogenetic and structural criteria; van Roosmalen ML et al.; The processing of secretory preproteins by signal peptidases (SPases) is essential for cell viability . As previously shown for Bacillus subtilis, only certain SPases of organisms containing multiple paralogous SPases are essential . This allows a distinction between SPases that are of major and minor importance for cell viability . Notably, the functional difference between major and minor SPases is not reflected clearly in sequence alignments . Here, we have successfully used molecular phylogeny to predict major and minor SPases . The results were verified with SPases from various bacilli . As predicted, the latter enzymes behaved as major or minor SPases when expressed in B . subtilis . Strikingly, molecular modeling indicated that the active site geometry is not a critical parameter for the classification of major and minor Bacillus SPases . Even though the substrate binding site of the minor SPase SipV is smaller than that of other known SPases, SipV could be converted into a major SPase without changing this site . Instead, replacement of amino-terminal residues of SipV with corresponding residues of the major SPase SipS was sufficient for conversion of SipV into a major SPase . This suggests that differences between major and minor SPases are based on activities other than substrate cleavage site selection. J Appl Microbiol, 2001 Apr, 90(4), 622 - 9 Bacillomycin D: an iturin with antifungal activity against Aspergillus flavus; Moyne AL et al.; AIMS: In a search for an antifungal peptide with a high activity against Aspergillus flavus, Bacillus subtilis AU195 was selected from a collection of isolates with antagonistic activity against A . flavus . METHODS AND RESULTS: To identify the antifungal peptides, a protein purification scheme was developed based on the detection of the antifungal activity in purified fractions against A . flavus . Two lipopeptides were purified with anion exchange and gel filtration chromatography . Their masses were determined to be 1045 and 1059 m/z with mass spectrometry, and their peptide moiety was identical to bacillomycin D . CONCLUSION: AU195 synthesized a mixture of two antifungal bacillomycin D analogues with masses of 1045 and 1059, the 14 mass unit difference representing the difference between a C15 and a C16 lipid chain . SIGNIFICANCE AND IMPACT OF THE STUDY: Both bacillomycin D analogues were active at the same concentration against A . flavus, but the different lipid chain length apparently affected the activity of the lipopeptide against other fungi. Phys Rev E Stat Nonlin Soft Matter Phys . 2001 Apr;63(4 Pt 1):041103 . Epub 2001 Mar 19. Non-Hermitian delocalization from Hermitian Hamiltonians; Moiseyev N et al.; Here we show that using Galilean transformations the non-Hermitian delocalization phenomenon, which is relevant in different fields, such as bacteria population (e.g., Bacillus subtilis), vortex pinning in superconductors, and stability solutions of hydrodynamical problems discovered by Hatano and Nelson {Phys . Rev . Lett . 77, 5706 (1996)}, can be obtained from solutions of the time-dependent Schrodinger equation with a Hermitian Hamiltonian . Using our approach, one avoids the numerical complications and instabilities which result form the calculations of left and right eigenfunctions of the non-Hermitian Hamiltonian which are associated with the non-Hermitian delocalization phenomenon . One also avoids the need to replace the non-Hermitian Hamiltonian H by a supermatrix with twice the dimension of H, where the complex frequencies serve as variational parameters rather than eigenvalues of H. Biosci Biotechnol Biochem, 2001 Feb, 65(2), 446 - 8 A simple and rapid method for intra- and interspecific transformation of Bacillus subtilis on solid media by DNA in protoplast lysates; Akamatsu T et al.; A simple method for intra- and interspecific transformation of Bacillus subtilis on solid media has been devised with DNA in protoplast lysates, 0.8% agar, glutamate, and yeast extract . The transformation frequency is 2.3 x 10(3) transformants per microg DNA, 10-20 times higher than that for conventional transformation on solid media . The method can be applicable to transformation in microtiter plates. Mikrobiol Z, 2000 Jan-Feb, 62(1), 20 - 9 {Effect of microelements on accumulation of biomass and exopolysaccharides in Bacillus subtilis strains}; Osadchaia AI et al.; Different trace elements in the composition of nine synthesized complex compounds of N-oxides of pyridine derivatives have been studied . It has been shown that the effect of trace elements included in the composition of nutrient media on accumulation of cells and exopolysaccharides (EPS) by the strains of B . subtilis is sufficiently expressed; it is also diverse and depends on the element nature, concentration as well as on peculiarities of the bacillus cultures . The studied trace elements are separated into the conditional groups: the elements intensifying the above processes (manganese and lithium); elements repressing the processes (copper and cadmium); those retarding the accumulation of biomass but activating the secretion of EPS (nickel, boron, cobalt, zinc, iron) . Concentrations of trace elements optimal for the studied productivity indices of strains have been established . Peculiarities of the trace element effect in paired and ternary combinations on the growth and biosynthesis of EPS during cultivation of strains in liquid media have been revealed. Mol Microbiol, 2001 Apr, 40(1), 52 - 64 A ComGA-dependent checkpoint limits growth during the escape from competence; Haijema BJ et al.; In Bacillus subtilis, competence for transformation develops in 5-10% of the cells in a stationary phase culture . These cells exhibit a prolonged lag in the resumption of growth and cell division during the escape from competence . To better understand the basis of this lag, we have characterized competent cultures microscopically . To distinguish the minority of competent cells, a translational fusion between ComK, the competence transcription factor, and the green fluorescent protein (GFP) was used as a marker . Only 5-10% of the cells in a competent culture were fluorescent, indicating that ComK synthesis is an all or nothing event . To validate the identification of competent cells, we demonstrated the coincident expression of comEA, a late competence gene, and comK-gfp . Competent cells resemble stationary phase cells; the majority are single (not in chains), contain single nucleoids, and rarely contain FtsZ rings . Upon dilution into fresh medium, competent cells maintain this appearance for about 2 h . In contrast, the majority of non-competent cells rapidly resume growth, exhibiting chaining, nuclear division and FtsZ-ring formation . The late competence protein ComGA is required for the competence-related block in chromosome replication and cell division . In the competent cells of a comGA mutant culture, chromosomal replication and FtsZ-ring formation were no longer blocked, although competent comGA mutant cells were abnormal in appearance . It is likely that one role for ComGA is to prevent growth, chromosome replication and cell division until ComK can be eliminated by degradation . A mutation in the ATP-binding site of comGA inactivated the protein for transformation but did not prevent it from inhibiting DNA replication and cell division . The buoyant density difference between competent and non-competent cells depends on the competence-specific growth arrest. Mol Microbiol, 2001 Apr, 40(1), 9 - 19 Bacillus subtilis mutations that alter the pathway of phosphorylation of the anti-anti-sigmaF factor SpoIIAA lead to a Spo- phenotype; Lee CS et al.; Sigma-F, the first sporulation-specific transcription factor of Bacillus subtilis, is regulated by an anti-sigma factor SpoIIAB, which can also act as a protein kinase that phosphorylates the anti-anti-sigma factor SpoIIAA . The time course of phosphorylation reaction is biphasic, a fact that has been interpreted in terms of a mechanism for sequestering SpoIIAB away from sigmaF and thus allowing activation of sigmaF when needed . Site-directed mutagenesis of SpoIIAA has allowed us to isolate two mutants that cannot activate sigmaF and which are therefore Spo- . The two mutant SpoIIAA proteins, SpoIIAAL61A and SpoIIAAL90A, are phosphorylated with linear kinetics; in addition they are less able to form the stable non-covalent complex that wild-type SpoIIAA makes with SpoIIAB in the presence of ADP . The phosphorylated form of SpoIIAAL90A was hydrolysed by the specific phosphatase SpoIIE at the same rate as wild-type SpoIIAA-P, but the rate of hydrolysis of SpoIIAAL61A-P was much slower . The secondary structure and the global fold of the mutant proteins were unchanged from the wild type . The results are interpreted in terms of a model for the wild type in which SpoIIAB, after phosphorylating SpoIIAA, is released in a form that is tightly bound to ADP and which then makes a ternary complex with an unreacted SpoIIAA . We propose that it is the inability to make this ternary complex that deprives the mutant cells of a means of keeping SpoIIAB from inhibiting sigmaF. Int J Food Microbiol, 2001 Mar 20, 64(3), 333 - 41 A study on the effects of high pressure and heat on Bacillus subtilis spores at low pH; Wuytack EY et al.; Bacillus subtilis spore suspensions were subjected to pressure treatments at 100 and 600 MPa at 40 degrees C and over a pH range from 3 to 8 . Inactivation of spores under these conditions was maximally 80% and was not increased at low pH . However, higher levels of inactivation were obtained when spores were first pressure treated at neutral pH and then exposed for 1 h to low pH . This large difference in inactivation could be explained by the finding that pressure-induced spore germination, which is known to occur at neutral pH, was inhibited at low pH (< 5) . Pressure treatment at low pH made spores more sensitive to heat inactivation, suggesting that demineralized H-spores had been formed . Changes in spore core hydration and pH upon exposure of spores at low pH were studied in a more direct way using green fluorescent protein expressed in recombinant B . subtilis as a reporter protein, and it was confirmed that pressure and heat increase spore permeability for protons . Based on these results, the potential of low temperature, high pressure processes for spore inactivation in acid products is discussed. Acta Microbiol Pol, 2000, 49(3-4), 225 - 35 Growth, zearalenone production and some metabolic activities of Fusarium moniliforme under salt stress; Abou-Zeid AM; With a increasing salinity, a decrease in the growth rate of Fusarium moniliforme was observed . The percentage of germinated conidia decreased with increasing salinity (% germination ranged from 80.3% at 0.0% NaCl to 0.0% at 15% NaCl) . Concentration of 12.5% NaCl produced the highest chlamydospore-like structures . Concentration of 5% NaCl increased zearalenone production which decreased with increasing salt stress . Escherichia coli was more tolerant to the toxin than Bacillus subtilis . The total amount of lipids produced by F . moniliforme changed with increasing the concentration of NaCl in the growth medium . The genomic DNA of the control and treated samples showed a common band of more than 20 kilobases . Similar RAPD-PCR patterns were also produced. J Bacteriol, 2001 May, 183(9), 2963 - 8 Growth phase variation in cell and nucleoid morphology in a Bacillus subtilis recA mutant; Sciochetti SA et al.; The major role of RecA is thought to be in helping repair and restart stalled replication forks . During exponential growth, Bacillus subtilis recA cells exhibited few microscopically observable nucleoid defects . However, the efficiency of plating was about 12% of that of the parent strain . A substantial and additive defect in viability was also seen for addB and recF mutants, suggesting a role for the corresponding recombination paths during normal growth . Upon entry into stationary phase, a subpopulation (approximately 15%) of abnormally long cells and nucleoids developed in B . subtilis recA mutants . In addition, recA mutants showed a delay in, and a diminished capacity for, effecting prespore nucleoid condensation. J Bacteriol, 2001 May, 183(9), 2795 - 802 Dissection of the functional and structural domains of phosphorelay histidine kinase A of Bacillus subtilis; Wang L et al.; The initiation of sporulation in Bacillus subtilis results primarily from phosphoryl group input into the phosphorelay by histidine kinases, the major kinase being kinase A . Kinase A is active as a homodimer, the protomer of which consists of an approximately 400-amino-acid N-terminal putative signal-sensing region and a 200-amino-acid C-terminal autokinase . On the basis of sequence similarity, the N-terminal region may be subdivided into three PAS domains: A, B, and C, located from the N- to the C-terminal end . Proteolysis experiments and two-hybrid analyses indicated that dimerization of the N-terminal region is accomplished through the PAS-B/PAS-C region of the molecule, whereas the most amino-proximal PAS-A domain is not dimerized . N-terminal deletions generated with maltose binding fusion proteins showed that an intact PAS-A domain is very important for enzymatic activity . Amino acid substitution mutations in PAS-A as well as PAS-C affected the in vivo activity of kinase A, suggesting that both PAS domains are required for signal sensing . The C-terminal autokinase, when produced without the N-terminal region, was a dimer, probably because of the dimerization required for formation of the four-helix-bundle phosphotransferase domain . The truncated autokinase was virtually inactive in autophosphorylation with ATP, whereas phosphorylation of the histidine of the phosphotransfer domain by back reactions from Spo0F~P appeared normal . The phosphorylated autokinase lost the ability to transfer its phosphoryl group to ADP, however . The N-terminal region appears to be essential both for signal sensing and for maintaining the correct conformation of the autokinase component domains. J Biol Chem, 2001 Jul 13, 276(28), 26154 - 63 Epub 2001 Apr 04. Crystal structure of Bacillus subtilis isocitrate dehydrogenase at 1.55 A . Insights into the nature of substrate specificity exhibited by Escherichia coli isocitrate dehydrogenase kinase/phosphatase; Singh SK et al.; Isocitrate dehydrogenase from Bacillus subtilis (BsIDH) is a member of a family of metal-dependent decarboxylating dehydrogenases . Its crystal structure was solved to 1.55 A and detailed comparisons with the homologue from Escherichia coli (EcIDH), the founding member of this family, were made . Although the two IDHs are structurally similar, there are three notable differences between them . First, a mostly nonpolar beta-strand and two connecting loops in the small domain of EcIDH are replaced by two polar alpha-helices in BsIDH . Because of a 13-residue insert in this region of BsIDH, these helices protrude over the active site cleft of the opposing monomer . Second, a coil leading into this cleft, the so-called "phosphorylation" loop, is bent inward in the B . subtilis enzyme, narrowing the entrance to the active site from about 12 to 4 A . Third, although BsIDH is a homodimer, the two unique crystallographic subunits of BsIDH are not structurally identical . The two monomers appear to differ by a domain shift of the large domain relative to the small domain/clasp region, reminiscent of what has been observed in the open/closed conformations of EcIDH . In Escherichia coli, IDH is regulated by reversible phosphorylation by the bifunctional enzyme IDH kinase/phosphatase (IDH-K/P) . The site of phosphorylation is Ser(113), which lies deep within the active site crevice . Structural differences between EcIDH and BsIDH may explain disparities in their abilities to act as substrates for IDH-K/P. Cell, 2001 Mar 23, 104(6), 913 - 22 Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis; Jones LJ et al.; In the absence of an overt cytoskeleton, the external cell wall of bacteria has traditionally been assumed to be the primary determinant of cell shape . In the Gram-positive bacterium Bacillus subtilis, two related genes, mreB and mbl, were shown to be required for different aspects of cell morphogenesis . Subcellular localization of the MreB and Mbl proteins revealed that each forms a distinct kind of filamentous helical structure lying close to the cell surface . The distribution of the proteins in different species of bacteria, and the similarity of their sequence to eukaryotic actins, suggest that the MreB-like proteins have a cytoskeletal, actin-like role in bacterial cell morphogenesis. J Mass Spectrom, 2001 Feb, 36(2), 124 - 39 Negative and positive ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and positive ion nano-electrospray ionization quadrupole ion trap mass spectrometry of peptidoglycan fragments isolated from various Bacillus species; Bacher G et al.; A general approach for the detailed characterization of sodium borohydride-reduced peptidoglycan fragments (syn . muropeptides), produced by muramidase digestion of the purified sacculus isolated from Bacillus subtilis (vegetative cell form of the wild type and a dacA mutant) and Bacillus megaterium (endospore form), is outlined based on UV matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) quadrupole ion trap (QIT) mass spectrometry (MS) . After enzymatic digestion and reduction of the resulting muropeptides, the complex glycopeptide mixture was separated and fractionated by reversed-phase high-performance liquid chromatography . Prior to mass spectrometric analysis, the muropeptide samples were subjected to a desalting step and an aliquot was taken for amino acid analysis . Initial molecular mass determination of these peptidoglycan fragments (ranging from monomeric to tetrameric muropeptides) was performed by positive and negative ion MALDI-MS using the thin-layer technique with the matrix alpha-cyano-4-hydroxycinnamic acid . The results demonstrated that for the fast molecular mass determination of large sample numbers in the 0.8-10 pmol range and with a mass accuracy of +/-0.07%, negative ion MALDI-MS in the linear TOF mode is the method of choice . After this kind of muropeptide screening often a detailed primary structural analysis is required owing to ambiguous data . Structural data could be obtained from peptidoglycan monomers by post-source decay (PSD) fragment ion analysis, but not from dimers or higher oligomers and not with the necessary sensitivity . Multistage collision-induced dissociation (CID) experiments performed on an nESI-QIT instrument were found to be the superior method for structural characterization of not only monomeric but also of dimeric and trimeric muropeptides . Up to MS4 experiments were sometimes necessary to obtain unambiguous structural information . Three examples are presented: (a) CID MSn (n = 2-4) of a peptidoglycan monomer (disaccharide-tripeptide) isolated from B . subtilis (wild type, vegetative cell form), (b) CID MSn (n = 2-4) of a peptidoglycan dimer (bis-disaccharide-tetrapentapeptide) obtained from a B . subtilis mutant (vegetative cell form) and (c) CID MS2 of a peptidoglycan trimer (a linear hexasaccharide with two peptide side chains) isolated from the spore cortex of B . megaterium . All MS(n) experiments were performed on singly charged precursor ions and the MS2 spectra were dominated by fragments derived from interglycosidic bond cleavages . MS3 and MS4 spectra exhibited mainly peptide moiety fragment ions . In case of the bis-disaccharide-tetrapentapeptide, the peptide branching point could be determined based on MS3 and MS4 spectra . The results demonstrate the utility of nESI-QIT-MS towards the facile determination of the glycan sequence, the peptide linkage and the peptide sequence and branching of purified muropeptides (monomeric up to trimeric forms) . The wealth of structural information generated by nESI-QIT-MSn is unsurpassed by any other individual technique. Proc Natl Acad Sci U S A, 2001 Apr 10, 98(8), 4397 - 402 Epub 2001 Apr 03. A ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Chlorobium tepidum that is involved with sulfur metabolism and the response to oxidative stress; Hanson TE et al.; A gene encoding a product with substantial similarity to ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) was identified in the preliminary genome sequence of the green sulfur bacterium Chlorobium tepidum . A highly similar gene was subsequently isolated and sequenced from Chlorobium limicola f.sp . thiosulfatophilum strain Tassajara . Analysis of these amino acid sequences indicated that they lacked several conserved RubisCO active site residues . The Chlorobium RubisCO-like proteins are most closely related to deduced sequences in Bacillus subtilis