Bacillus subtilis

Anal Bioanal Chem . 2005 Jan 18; {Epub ahead of print}
Isolation of selenium organic species from antarctic krill after enzymatic hydrolysis; Siwek M et al.; Total selenium content and its distribution in the soluble and insoluble protein-bound fractions obtained after aqueous extraction of antarctic krill samples were determined . About 26% of the total selenium (2.4 mug g(-1) dry weight) was found in the supernatant; the rest was in the pellet . Isolation of low molecular selenium-containing fractions was also performed by enzymatic digestion of the protein, followed by size-exclusion chromatography in conjunction with atomic absorption spectrometry . From the applied various proteinases (pronase E, subtilisin Carlsberg, trypsin, chymotrypsin, proteinase and proteinase N from Bacillus subtilis and Novo 0.6 MPX enzyme), the treatment with pronase E led to best recovery of selenium . About 96% of the total Se was found in the hydrolysate, mainly in low molecular weight fractions . Eighty percent of the Se species were in fractions with molecular weights in the range of amino acids and short peptides . High-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC-ICP-MS) allowed the identification of selenomethionine and the assumption that selenocystine or its derivatives were the main species in these fractions.

Nature . 2005 Jan 16; {Epub ahead of print}
Structural basis for substrate binding, cleavage and allostery in the tRNA maturase RNase Z; de la Sierra-Gallay IL et al.; Transfer RNAs (tRNAs) are synthesized as part of longer primary transcripts that require processing of both their 3' and 5' extremities in every living organism known . The 5' side is processed (matured) by the ubiquitously conserved endonucleolytic ribozyme, RNase P, whereas removal of the 3' tails can be either exonucleolytic or endonucleolytic . The endonucleolytic pathway is catalysed by an enzyme known as RNase Z, or 3' tRNase . RNase Z cleaves precursor tRNAs immediately after the discriminator base (the unpaired nucleotide 3' to the last base pair of the acceptor stem, used as an identity determinant by many aminoacyl-tRNA synthetases) in most cases, yielding a tRNA primed for addition of the CCA motif by nucleotidyl transferase . Here we report the crystal structure of Bacillus subtilis RNase Z at 2.1 A resolution, and propose a mechanism for tRNA recognition and cleavage . The structure explains the allosteric properties of the enzyme, and also sheds light on the mechanisms of inhibition by the CCA motif and long 5' extensions . Finally, it highlights the extraordinary adaptability of the metallo-hydrolase domain of the beta-lactamase family for the hydrolysis of covalent bonds.

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 191 - 5
Bacillus velezensis sp . nov., a surfactant-producing bacterium isolated from the river Velez in Malaga, southern Spain; Ruiz-Garcia C et al.; Two Gram-positive, endospore-forming bacterial strains, CR-502(T) and CR-14b, which produce surfactant molecules are described . Phenotypic tests and phylogenetic analyses showed these strains to be members of the genus Bacillus and related to the species Bacillus atrophaeus, Bacillus mojavensis, Bacillus subtilis, Bacillus vallismortis and Bacillus amyloliquefaciens, although they differ from these species in a number of phenotypic characteristics . DNA-DNA hybridization confirmed that they show less than 20 % hybridization with the above-mentioned species and therefore represent a novel species of Bacillus . The DNA G+C content is 46.4 mol% in strain CR-502(T) and 46.1 mol% in strain CR-14b . The main fatty acids in strain CR-502(T) are 15 : 0 anteiso (32.70 %), 15 : 0 iso (29.86 %) and 16 : 0 (13.41 %) . The main quinone in strain CR-502(T) is MK-7 (96.6 %) . In the light of the polyphasic evidence gathered in this study, it is proposed that these strains be classified as a novel species of the genus Bacillus, with the name Bacillus velezensis sp . nov . The type strain (CR-502(T)=CECT 5686(T)=LMG 22478(T)) was isolated from a brackish water sample taken from the river Velez at Torredelmar in Malaga, southern Spain.

Biophys J . 2005 Jan 14; {Epub ahead of print}
Biophysical and kinetic characterization of HemAT, an aerotaxis receptor from Bacillus subtilis; Zhang W et al.; HemAT from B . subtilis is a new type of heme-based sensor protein responsible for sensing oxygen . The structural and functional properties of the full length HemAT protein, the sensor domain (1-178), and Tyr70 mutants have been characterized . Kinetic and equilibrium measurements reveal that both full length HemAT and the sensor domain show two distinct O2 binding components . The high affinity component has a Kdissociation approximately 1-2 microM and a normal O2 dissociation rate constant, kO2 = 50-80 s(-1) . The low affinity component has a Kdissociation approximately 50-100 microM and a large O2 dissociation rate constant equal to ~2,000 s(-1) . The low n-value and biphasic character of the equilibrium curve indicate that O2 binding to HemAT involves either independent binding to high and low affinity subunits in the dimer or negative cooperativity . Replacement of Tyr70(B10) with Phe, Leu or Trp in the sensor domain causes dramatic increases in kO2 for both the high and low affinity components . In contrast, the rates and affinity for CO binding are little affected by loss of the Tyr70 hydroxyl group . These results suggest highly dynamic behavior for the Tyr70 side chain and the fraction of the "up" versus "down" conformation is strongly influenced by the nature of the iron-ligand complex . As result of having both high and low affinity components, HemAT can respond to oxygen concentration gradients under both hypoxic (0 to 10 microM) and aerobic (50-250 microM) conditions, a property which could, in principle, be important for a robust sensing system . The unusual ligand-binding properties of HemAT suggest that asymmetry and apparent negative cooperativity play an important role in the signal transduction pathway.

J Chromatogr B Analyt Technol Biomed Life Sci, 2005 Feb 5, 815(1-2), 227 - 36
Effectiveness and limitation of two-dimensional gel electrophoresis in bacterial membrane protein proteomics and perspectives; Bunai K et al.; Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using isoelectric focusing and SDS-PAGE in the first and second dimensions, respectively, is an established means of simultaneously separating over 1000 proteins and two new types have recently been developed . These procedures have significant shortcomings such as low load ability and poor separation of hydrophobic, acidic and alkaline proteins . We therefore modified the protocols to analyze the Bacillus subtilis membrane proteome . The 2D-PAGE techniques effectively separated membrane proteins having one and two transmembrane segments but not those with more than four . Compared with new LC/MS/MS procedures that are independent of electrophoretic separation, 2D-PAGE can globally analyze and quantify proteins at various stages of the cell cycle when labeled with isotopes such as (35)S-methionine or the stable isotope, (15)N.

J Microbiol, 2004 Dec, 42(4), 319 - 27
Expression of the promoter for the maltogenic amylase gene in Bacillus subtilis 168; Kim DY et al.; An additional amylase, besides the typical alpha-amylase, was detected for the first time in the cytoplasm of B . subtilis SUH4-2, an isolate from Korean soil . The corresponding gene (bbmA) encoded a maltogenic amylase (MAase) and its sequence was almost identical to the yvdF gene of B . subtilis 168, whose function was unknown . Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B . subtilis strains . In an effort to understand the physiological function of the bbmA gene in B . subtilis, the expression pattern of the gene was monitored by measuring the beta-galactosidase activity produced from the bbmA promoter fused to the amino terminus of the lacZ structural gene, which was then integrated into the amyE locus on the B . subtilis 168 chromosome . The promoter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing beta-cyclodextrin (beta-CD), maltose, or starch . On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene . Production of the beta-CD hydrolyzing activity was impaired by the spo0A mutation in B . subtilis 168, indicating the involvement of an additional regulatory system exerting control on the promoter . Inactivation of yvdF resulted in a significant decrease of the beta-CD hydrolyzing activity, if not all . This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing beta-CD in B . subtilis 168 . Based on the results, MAase encoded by bbmA is likely to be involved in maltose and beta-CD utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.

Comp Biochem Physiol B Biochem Mol Biol, 2005 Feb, 140(2), 321 - 31
Kinetic and molecular properties of Bacillus subtilis IBTC-3 subtilisin; Glowacka AE et al.; Bacillus subtilis IBTC-3 subtilisin was purified by gel filtration on Sephadex G 75 and affinity chromatography on bacitracin-CNBr-Sepharose 4B and characterized . Its molecular mass of 27 kDa was determined by SDS-PAGE, and isoelectric pH of 8.4 by chromatofocusing . FT-Raman and FT-IR spectroscopy studies revealed fragments with alpha-helix and irregular secondary structures within the polypeptide chain . The beta-sheet conformation was observed only in second-derivatives of FT-RS and FT-IR spectra, in the range of the amide II, III, and I bands . Tyr residues were shown to be hydrogen bonded and CSCH(3) groups adopted two conformations (P(H)-T and P(C)-G conformers) . Kinetic properties of B . subtilis IBTC-3 subtilisin in hydrolysis of ethyl esters of amino acid derivatives were compared with that of alkaline peptidase from Bacillus alcalophilus PB92 . The first enzyme displayed the highest affinity for NAc-Phe-OEt, both in hydrolysis (K(m) of 0.22 mM) and in synthesis (K(m) of 0.85 mM), whereas PB92 peptidase preferred Tyr derivatives (NAc-Tyr-OEt, K(m) of 0.043 and 0.75 mM, respectively) . In contrast to the latter enzyme, B . subtilis IBTC-3 subtilisin catalyzed hydrolysis and synthesis of Bz-Arg-OEt.

Phytochemistry, 2005 Jan, 66(1), 99 - 104
Antimicrobial and radical scavenging flavonoids from the stem wood of Erythrina latissima; Chacha M et al.; From the stem wood of Erythrina latissima, two isoflavones and a flavanone were isolated and characterized as 7,3'-dihydroxy-4'-methoxy-5'-(gamma,gamma-dimethylallyl)isoflavone (erylatissin A), 7,3'-dihydroxy-6'',6''-dimethyl-4'',5''-dehydropyrano {2'',3'': 4',5'}isoflavone (erylatissin B), (-)-7,3'-dihydroxy-4'-methoxy-5'-(gamma,gamma-dimethylallyl)flavanone (erylatissin C), respectively, in addition to 10 known flavonoids . Structures of these compounds were determined on the basis of their spectroscopic data . These compounds showed antimicrobial activity against Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida mycoderma . The isolated compounds also exhibited weak radical scavenging properties towards DPPH radical.

J Environ Qual, 2005 Jan-Feb, 34(1), 237 - 47
A laboratory study of bacteria-facilitated cadmium transport in alluvial gravel aquifer media; Pang L et al.; Colloids, including bacteria, can dramatically accelerate the transport of heavy metals in ground water . Batch and column experiments were conducted to investigate adsorption of cadmium (Cd) onto Bacillus subtilis spores or Escherichia coli vegetative cells and Cd transport in alluvial gravel aquifer media in the presence of these bacteria . Results of the batch experiments showed that adsorption of Cd onto the bacteria was (i) positively related to solution pH, bacterial concentration, and negative surface charge, but inversely related to Cd concentration and (ii) a rate-limited nonlinear process, but adsorption onto E . coli was much less . For column influent Cd concentrations of about 4 mg/L and bacterial concentrations of >/=10(5) colony-forming units (cfu)/mL, there was a significant increase in total Cd effluent concentrations . In comparison with controls that did not have bacteria-facilitated transport, Cd traveled 17 to 20 times faster when it traveled with mobile bacteria . However, Cd traveled mostly 2 to 3 times slower during the desorption phase under the influence of bacteria retained in the column . The difference between total and dissolved Cd concentrations was significant during Cd cotransport with B . subtilis spores, but this concentration difference was very small during Cd cotransport with E . coli, suggesting an adsorption-dominant mechanism during Cd cotransport with the spores and the possibility of Cd chelation by the dissolved membrane vesicles secreted from E . coli cell walls . Bacteria-facilitated transport of heavy metals may pose a threat to ground water quality in sites such as landfills and following land disposal of industrial and domestic effluent and sludge.

Bioprocess Biosyst Eng, 2004 Dec, 27(1), 63 - 9 Epub 2004 Nov 18.
Enhanced amylase production by Bacillus subtilis using a dual exponential feeding strategy; Huang H et al.; A recombinant Bacillus subtilis strain (ATCC 31784) haboring the plasmid pC194 with a thermostable alpha-amylase gene was cultured in a 22-l B . Braun Biostat C fermenter . Traditional batch operations suffer from low cell mass and protein productions because a high initial glucose concentration causes substrate inhibition and also product inhibition due to acetate accumulation . An exponential fed-batch strategy to prevent these inhibitions was developed in this work . The host strain is auxotrophic for phenylalanine, tyrosine and tryptophan . Due to low solubilities of tyrosine and tryptophan in the feed stream, tyrosine and tryptophan were dissolved separately in ammonia water to form a second feed stream . By dual feeding both streams at different exponential feed rates, a high cell density of 17.6 g/l and a final alpha-amylase activity of 41.4 U/ml and the overall biomass yield of 0.39 g cell/g glucose were achieved.

Can J Microbiol, 2004 Sep, 50(9), 737 - 744
Whole cells of Bacillus subtilis AF 1 proved more effective than cell-free and chitinase-based formulations in biological control of citrus fruit rot and groundnut rust; Manjula K et al.; In foliar and postharvest biocontrol systems, the use of active metabolites produced by antagonistic microorganisms is advantageous compared with the use of living microorganisms . Chitinases, a major group of hydrolytic enzymes produced by biocontrol agents, are involved in the lysis of cell walls of pathogenic fungi . In the present study, an attempt was made to test the partially purified β-1,4-N-acetylglucosaminidase (NAGase) of a biocontrol strain Bacillus subtilis AF 1 for control of rust in groundnut (caused by Puccinia arachidis) and soft rot in lemons (caused by Aspergillus niger) . Four proteins of molecular mass 67, 40, 37, and 32 kDa were isolated from the culture filtrates of AF 1 by affinity chromatography, of which the 67-kDa protein has detectable chitinolytic ability . This protein (NAGase) effectively inhibited the in vitro growth of A . niger in microtitre plates . In the presence of NAGase, germination of urediniospores of P . arachidis was reduced by 96% compared with the control . In a detached leaf bioassay, NAGase reduced the rust lesion frequency by >60% . NAGase significantly reduced the incidence of soft rot in harvested lemon fruits . However, fresh cells and (or) alginate formulation of AF 1 were more effective than NAGase in control of both of the test plant – pathogen systems.

Environ Microbiol, 2005 Jan, 7(1), 40 - 6
Thermal destruction of dried vegetative yeast cells and dried bacterial spores in a convective hot air flow: strong influence of initial water activity; Fine F et al.; Summary Thermal treatment of Bacillus subtilis spores and Saccharomyces cerevisiae cells dried on glass beads was performed at various initial water activities (in the range 0.10-0.90) . Experiments were carried out at 150 degrees C, 200 degrees C and 250 degrees C for 5-120 s . Significant destruction of up to 10(7) vegetative cells and up to 10(5) spores g(-1) was achieved, depending upon treatment conditions . This study demonstrated that the initial water activity (a(w)) value of a sample is very important in the destruction or survival of microorganisms treated with hot air stresses . As described previously, the heat resistance of spores and vegetative cells was strongly enhanced by low initial a(w) values until an optimal a(w) value between 0.30 and 0.50, with maximal viability at 0.35 for both S . cerevisiae and B . subtilis . However, our results highlighted for the first time that very low initial a(w) values (close to 0.10) greatly improved the destruction of spores and vegetative cells . Factors and possible mechanisms involved in the death of vegetative cells and spores are discussed.

Protein Expr Purif, 2005 Feb, 39(2), 219 - 28
The propeptide is not required to produce catalytically active neutral protease from Bacillus stearothermophilus; Mansfeld J et al.; The thermolysin-like neutral protease from Bacillus stearothermophilus (TLP-ste) is usually produced extracellularly in Bacillus subtilis, where it is expressed as preproenzyme and subsequently processed in an autocatalytic, intramolecular process . To create the basis for the production of inactive mutants of TLP-ste, which cannot be processed in B . subtilis, we studied the expression of TLP-ste and its propeptide in cis and in trans in Escherichia coli . In contrast to thermolysin, subtilisin and alpha-lytic protease, which could be obtained only in the presence of the corresponding propeptides, TLP-ste could be produced as an active mature enzyme in E . coli in the absence of its prosequence . Surprisingly, however, a much more effective access to active mature protease was found when TLP-ste (devoid of its prosequence) was expressed as protein with an N-terminal His(6) tag which accumulated in the form of inclusion bodies . Completely unexpected, the protein could be renatured from the inclusion bodies after solubilization in guanidine hydrochloride solutions in high yields . Purification to homogeneity was possible by affinity chromatography on Bacitracin silica as well as by immobilized metal ion affinity chromatography . By addition of separately expressed propeptide to the renaturation mixture yields of renaturation could not be increased significantly, confirming that the propeptide is not essential for proper folding of the enzyme or its stabilization during the folding process . Also in vivo, the expression levels of active mature TLP-ste in Escherichia coli did not significantly differ when the mature sequence was expressed alone or coexpressed with the prosequence in cis or in trans.

Appl Environ Microbiol, 2005 Jan, 71(1), 594 - 6
Novel Keratinase from Bacillus subtilis S14 Exhibiting Remarkable Dehairing Capabilities; Macedo AJ et al.; We report the isolation of a keratinolytic-producing Bacillus subtilis strain and the characterization of the exceptional dehairing properties of its subtilisin-like keratinase . This enzyme can be an alternative to sodium sulfide, the major pollutant from tanneries, and may completely replace it . Its unique nonactivity upon collagen enhances its industrial potential.

J Biotechnol, 2005 Feb 9, 115(3), 249 - 60
Functional display of family 11 endoxylanases on the surface of phage M13; Belien T et al.; Two family 11 endoxylanases (EC 3.2.1.8) were functionally displayed on the surface of bacteriophage M13 . The genes encoding endo-1,4-xylanase I from Aspergillus niger (ExlA) and endo-1,4-xylanase A from Bacillus subtilis (XynA) were fused to the gene encoding the minor coat protein g3p in phagemid vector pHOS31 . Phage rescue resulted in functional monovalent display of the enzymes as was demonstrated by three independent tests . Firstly, purified recombinant phage particles showed a clear hydrolytic activity in an activity assay based on insoluble, chromagenic arabinoxylan substrate . Secondly, specific binding of endoxylanase displaying phages to immobilized endoxylanase inhibitors was demonstrated by interaction ELISA . Finally, two rounds of selection and amplification in a biopanning procedure against immobilized endoxylanase inhibitor were performed . Phages displaying endoxylanases were strongly enriched from background phages displaying unrelated proteins . These results open perspectives to use phage display for analysing protein-protein interactions at the interface between endoxylanases and their inhibitors . In addition, this technology should enable engineering of endoxylanases into novel variants with altered binding properties towards endoxylanase inhibitors.

Pharmazie, 2004 Dec, 59(12), 972 - 6
Sesquiterpenes, lignans and other constituents from Saussurea macrota; Yang M et al.; From the methanol extract of the whole plant of Saussurea macrota Franch, 21 compounds were isolated . Their structures were elucidated by spectroscopic methods and X-ray crystallography . Two of them are new: 3alpha-hydroxy-11alphaH-guaia-4(15),10(14)-diene-12,6alpha-olide (1) and 7'-hydroxyiso-lappaol A (11) . Compound 2 is reported as a natural compound for the first time . In addition, the compounds 12 and 13 showed significant antitumor activity against Bel-7402 and HO-8910 cells . Some of the compounds exhibited weak antibacterial activity against Staphylococcus aureus, Bacillus subtilis and Escherichia coli.

Phytomedicine, 2004 Nov, 11(7-8), 697 - 700
Antibacterial constituents from the berries of Piper nigrum; Reddy SV et al.; Piper nigrum finds an extensive application in antibacterial preparations belonging to Ayurvedic system of medicine . A bioguided extraction and fractionation of the petroleum ether extract of the berries of P . nigrum afforded 2E, 4E, 8Z-N-isobutyleicosatrienamide (1), pellitorine (2), trachyone (3), pergumidiene (4) and isopiperolein B (5) . Pergumidiene and trachyone are isolated for the first time from P . nigrum . All the isolated compounds were active against Bacillus subtilis, Bacillus sphaericus, and Staphylococcus aureus amongst Gram + ve bacteria, and Klebsiella aerogenes and Chromobacterium violaceum among Gram -ve bacterial strains.

Protein J, 2004 Oct, 23(7), 483 - 92
A novel neutral protease from Thermoactinomyces species 27a: sequencing of the gene, purification, and characterization of the enzyme; Zabolotskaya MV et al.; The nucleotide sequence of the previously cloned (Zabolotskaya, M . V., Nosovskaya, E . A., Kaplun, M . A., and Akimkina, T . V . (2001) . Mol . Gen . Mikrobiol . Virusol . No 1, 32-34) DNA fragment from Thermoactinomyces sp . 27a (GenBank Accession No . AY280367) containing the metalloproteinase gene was determined . A continuous open reading frame encoding a polypeptide of 673 aa was revealed . Analysis of this sequence demonstrated that the metalloproteinase from Thermoactinomyces sp . 27a is synthesized as a preproprotein and includes a leader peptide (26 aa), N-terminal propeptide (215 aa), mature region (317 aa), and additional C-terminal domain (115 aa) . The recombinant enzyme from Thermoactinomyces sp . 27a was expressed in Bacillus subtilis AJ73 cells and purified by anion exchange chromatography to an electrophoretically homogeneous state . The determined N-terminal amino acid sequence of the mature protein was identical to that deduced from the gene . The obtained data suggest that the mature protein should include 432 aa and have a calculated molecular weight of 46,262 Da . However, the molecular weight of the mature protein determined by mass spectrometry was 34,190+/-70 Da indicating a C-terminal processing . The proteinase was not inhibited by phenylmethyl sulfonyl fluoride but was inhibited by o-phenanthroline and ethylenediaminetetraacetic acid . The enzyme had maximum activity by azocasein hydrolysis at 55 degrees C and pH 6.5-7.5; it was stable at pH 7.5-8.5 and remained stable at 50 degrees C for several hours . The k(cat)/Km for 3-(2-furyl)acryloyl-glycyl-L-leucine amide hydrolysis was (2.8+/-0.1) x 10(3) M(-1) x s(-1).

Protein Sci . 2005 Jan 4; {Epub ahead of print}
Crystal structures of RsbQ, a stress-response regulator in Bacillus subtilis; Kaneko T et al.; Growth-limiting stresses in bacteria induce the general stress response to protect the cells against future stresses . Energy stress caused by starvation conditions in Bacillus subtilis is transmitted to the sigma(B) transcription factor by stress-response regulators . RsbP, a positive regulator, is a phosphatase containing a PAS (Per-ARNT-Sim) domain and requires catalytic function of a putative alpha/beta hydrolase, RsbQ, to be activated . These two proteins have been found to interact with each other . We determined the crystal structures of RsbQ in native and inhibitor-bound forms to investigate why RsbP requires RsbQ . These structures confirm that RsbQ belongs to the alpha/beta hydrolase superfamily . Since the catalytic triad is buried inside the molecule due to the closed conformation, the active site is constructed as a hydrophobic cavity that is nearly isolated from the solvent . This suggests that RsbQ has specificity for a hydrophobic small compound rather than a macromolecule such as RsbP . Moreover, structural comparison with other alpha/beta hydrolases demonstrates that a unique loop region of RsbQ is a likely candidate for the interaction site with RsbP, and the interaction might be responsible for product release by operating the hydrophobic gate equipped between the cavity and the solvent . Our results support the possibility that RsbQ provides a cofactor molecule for the mature functionality of RsbP.

Biofactors, 2004, 22(1-4), 185 - 7
Fibrinolytic and anti-thrombotic effect of NKCP, the protein layer from Bacillus subtilis (natto); Omura K et al.; NKCP is the completely refined protein layer from cultured Bacillus subtilis (natto) and is supplied in tablet form . It lacks the distinctive fermented odor and bacteria associated with fresh natto . Almost all vitamin K is removed during the manufacturing process . The main component of NKCP is the active fragment of Bacillopeptidase F, which is a serine protease secreted by Bacillus subtilis . In in vivo studies, NKCP shows fibrinolytic and amidolytic activity on plasmin specific synthetic substrate . Daily oral administration of NKCP in 28 volunteers for two weeks and then in 23 volunteers for several months, was examined . The fibrinolytic effect was demonstrated by a shortened euglobulin lysis time in both trials . Furthermore, we observed an improvement in shoulder stiffness . These results suggest that the oral administration of NKCP can be expected to have a fibrinolytic effect and cause positive changes in local blood flow . Further investigations may demonstrate NKCP to be useful in the prevention of thrombosis.

J Bacteriol, 2005 Jan, 187(2), 791 - 4
The Purine Efflux Pump PbuE in Bacillus subtilis Modulates Expression of the PurR and G-Box (XptR) Regulons by Adjusting the Purine Base Pool Size; Nygaard P et al.; In Bacillus subtilis, the expression of genes encoding enzymes and other proteins involved in purine de novo synthesis and salvage is affected by purine bases and phosphoribosylpyrophosphate (PRPP) . The transcription of the genes belonging to the PurR regulon is negatively regulated by the PurR protein and PRPP . The expression of the genes belonging to the G-box (XptR) regulon, including the pbuE gene, is negatively regulated by a riboswitch-controlled transcription termination mechanism . The G-box regulon effector molecules are hypoxanthine and guanine . pbuE encodes a purine base efflux pump and is now recognized as belonging to a third purine regulon . The expression of the pbuE gene is positively regulated by a riboswitch that recognizes adenine . Here we show that the expression of pbuE'-lacZ transcriptional fusions are induced by adenine to the highest extent in mutants which do not express a functional PbuE pump . In a mutant defective in the metabolism of adenine, the ade apt mutant, we found a high intracellular level of adenine and constitutive high levels of PbuE . A growth test using a purine auxotroph provided further evidence for the role of PbuE in lowering the intracellular concentration of purine bases, including adenine . Purine analogs also affect the expression of pbuE, which might be of importance for the protection against toxic analogs . In a mutant that overexpresses PbuE, the expression of genes belonging to the PurR regulon was increased . Our findings provide further evidence for important functions of the PbuE protein, such as acting as a pump that lowers the purine base pool and affects the expression of the G-box and PurR regulons, including pbuE itself, and as a pump involved in protection against toxic purine base analogs.

Biochemistry, 2005 Jan 11, 44(1), 193 - 201
The Crystal Structure of a Quercetin 2,3-Dioxygenase from Bacillus subtilis Suggests Modulation of Enzyme Activity by a Change in the Metal Ion at the Active Site(s); Gopal B et al.; Common structural motifs, such as the cupin domains, are found in enzymes performing different biochemical functions while retaining a similar active site configuration and structural scaffold . The soil bacterium Bacillus subtilis has 20 cupin genes (0.5% of the total genome) with up to 14% of its genes in the form of doublets, thus making it an attractive system for studying the effects of gene duplication . There are four bicupins in B . subtilis encoded by the genes yvrK, yoaN, yxaG, and ywfC . The gene products of yvrK and yoaN function as oxalate decarboxylases with a manganese ion at the active site(s), whereas YwfC is a bacitracin synthetase . Here we present the crystal structure of YxaG, a novel iron-containing quercetin 2,3-dioxygenase with one active site in each cupin domain . Yxag is a dimer, both in solution and in the crystal . The crystal structure shows that the coordination geometry of the Fe ion is different in the two active sites of YxaG . Replacement of the iron at the active site with other metal ions suggests modulation of enzymatic activity in accordance with the Irving-Williams observation on the stability of metal ion complexes . This observation, along with a comparison with the crystal structure of YvrK determined recently, has allowed for a detailed structure-function analysis of the active site, providing clues to the diversification of function in the bicupin family of proteins.

Exp Oncol, 2004 Dec, 26(4), 265 - 70
Characteristics of extracellular DNA containing unmethylated CpG motifs isolated from Bacillus subtilis culture medium filtrate; Olishevsky S et al.; The application of CpG DNA is one of the most modern and sufficiently perspective trends in immunotherapy of cancer . THE AIM of the investigation was to characterize the DNA as a part of nucleoprotein fraction isolated from Bacillus subtilis culture medium filtrate (CMF) and to evaluate the dynamic of accumulation of such DNA and level of its unmethylated CpG motifs enrichment in the process of cultivation of bacterium . METHODS: Two bacterial strains - B . subtilis 7025 and B . subtilis GP1-807-03 were used . Polyacrylamide and agarose gel electrophoresis, restriction analysis and transmission electron microscopy were used . THE RESULTS of the investigations showed that DNA was accumulated in culture medium irregularly . DNA-containing fractions isolated on the 7th-9th day of cultivation have the greatest sensitivity to restriction endonuclease HpaII . The presence of phages in CMF concentrates has been shown . CONCLUSION: The present findings suggest that 7th-9th day of cultivation are the most optimal to obtain the bacterial extracellular DNA for the following immunotherapy application.

Anal Chem, 2005 Jan 1, 77(1), 232 - 42
An integrated metal clad leaky waveguide sensor for detection of bacteria; Zourob M et al.; An integrated optical metal clad leaky waveguide (MCLW) sensor device has been developed for the detection of bacteria . This is more sensitive than waveguide sensors currently in use . The MCLW device has been fabricated to extend the evanescent field to provide significant light intensity over the entire volume of the bacteria bound on the chip surface within this field . This in turn increases the interaction of the light with the entire volume of the bacteria . MCLW devices have been used for detecting refractive index changes, scattering, and fluorescence from bacterial spores captured on an immobilized antibody . The detection limit of Bacillus subtilis var . niger bacterial spores using refractive index detection was 8 x10(4) spores/mL . The scattering intensity of the BG spores was found to be three times greater than the scattering intensity generated using surface plasmon resonance . The extended light propagation along the direction of flow for a few millimeters provides an effective interrogation approach to increase the area of detection to detect low concentrations down to 1 x 10(4) spores/mL . The sensor was then optimized by studying the key factors affecting sensor performance including changing the pH of the medium, type of antibody immobilization matrix, sensor surface regeneration approaches, and longevity of the sensor.

FEMS Microbiol Lett, 2005 Jan 1, 242(1), 137 - 45
The transcriptional regulator pool of the marine bacterium Rhodopirellula baltica SH 1(T) as revealed by whole genome comparisons; Lombardot T et al.; Rhodopirellula baltica (strain SH 1(T)) is a free-living marine representative of the phylogenetically independent and environmentally relevant phylum Planctomycetes . Little is known about the regulatory strategies of free-living bacteria with large (7.15 Mb) genomes . Therefore, a consistent, quantitative and qualitative description was produced by comparing R . baltica's transcriptional regulator pool with that of 123 publicly available bacterial genomes . The overall results are congruous with earlier observations that in Bacteria, the proportion of genes encoding transcriptional regulators generally increases with genome size . However, R . baltica distinctly stands out from this trend with only 2.4% (174) of all genes predicted to encode transcriptional regulators . The qualitative investigation of R . baltica's transcriptional regulators revealed a clear shift towards high numbers of two-component systems (66) as well as high numbers of sigma factors (49), with more than 76% (37) belonging to the extra-cytoplasmic function subfamily of sigma-70 . Only one predicted sigma factor showed a relatively close phylogenetic relationship to that of another bacterium, the sigma factor SigZ of Bacillus subtilis . In summary, analysis of the R . baltica genome revealed disparate regulatory mechanisms and a clear bias towards direct environmental sensing . This strategy might provide a selective advantage for organisms living in habitats with frequently changing environmental conditions.

FEMS Microbiol Lett, 2005 Jan 1, 242(1), 51 - 7
The ylbO gene product of Bacillus subtilis is involved in the coat development and lysozyme resistance of spore; Kuwana R et al.; The Bacillus subtilis YlbO protein is a Myb-like DNA binding domain-containing protein that is expressed under the control of SigE . Here, we analyzed gene expression and protein composition in ylbO-negative cells . SDS-PAGE analysis revealed that the protein profile of ylbO- negative spores differed from that of wild-type . Specifically, the expression of coat proteins CgeA, CotG, and CotY, which are controlled by SigK and GerE, was reduced in ylbO -negative cells . Northern blot analysis revealed that YlbO regulated the transcription of cgeA, cotG, and cotY . These results suggest that YlbO regulates the expression of some coat proteins during sporulation in B . subtilis directly or indirectly.

Colloids Surf B Biointerfaces, 2005 Jan 15, 40(1), 67 - 71
Study on interaction of alpha-amylase from Bacillus subtilis with cetyl trimethylammonium bromide; Bordbar AK et al.; The interaction of cetyl trimethylammonium bromide (CTAB) with alpha-amylase from Bacillus subtilis was investigated at 25 degrees C and various experimental conditions, such as pH, ionic strength and urea concentration . The binding data were measured using CTAB-membrane selective electrodes as a simple, fast, cheap and accurate method . The obtained binding isotherms were analyzed using Wyman binding potential concept . The results represent the highest binding affinity at 10(-3)M of NaBr respect to other salt concentrations . The less binding affinity at pH 9.7 with respect to pH 6.5 is related to increasing of protein self aggregation with pH . The binding data analysis at various urea concentrations also shows that the predominate unfolding of alpha-amylase occurred in the urea concentration range of 3-5M.

J Nat Prod, 2004 Dec 28, 67(12), 2124 - 2126
Triterpenes from Maesopsis eminii; Fokou PA et al.; Two pentacyclic triterpenes, 1alpha,3beta-dihydroxybauer-7-en-28-oic acid (1) and 3beta-hydroxybauer-7-en-28-oic acid (2), together with sitosterol-3-beta-O-d-glucopyranoside and stigmasterol have been isolated from the bark of the plant Maesopsis eminii . Their structures have been elucidated by spectroscopic methods . One of the triterpenes (1) is new, and its structure was confirmed by X-ray crystallographic analysis . This new triterpene displayed moderate antibacterial activity against Bacillus subtilis ATCC 6633.

J Fluoresc, 2004 May, 14(3), 269 - 74
Steady-state and frequency-domain lifetime measurements of an activated molecular imprinted polymer imprinted to dipicolinic acid; Anderson J et al.; We recently demonstrated the synthesis and fluorescence activity associated with an optical detector incorporating a molecular imprinted polymer (MIP) . Steady-state and time-resolved (lifetime) fluorescence measurements were used to characterize the binding activity associated with MIP microparticles imprinted to dipicolinic acid (DPA) . DPA is a unique biomarker associated with the sporulation phase of endospore-forming bacteria . Vinylic monomers were polymerized in a dimethylformamide solution containing DPA as a template . The resulting MIP was then pulverized and sorted into small microscale particles . Tests were conducted on replicate samples of biologically active cultures representing both vegetative stationary phase and sporulation phase of Bacillus subtilis in standard media . Samplers were adapted incorporating the MIP particles within a dialyzer cartridge (500 MW) . The permeability of the dialyzer membrane permitted diffusion of lighter molecular weight constituents from microbial media effluents to enter the dialyzer chamber and come in contact with the MIP . Results showed dramatic (10-fold over background) steady-state fluorescence changes (as a function of excitation, emission and intensity) for samples associated with high endospore biomass (DPA), and a frequency-domain lifetime of 5.3 ns for the MIP-DPA complex.

RNA . 2004 Dec 21; {Epub ahead of print}
Efficient fluorescence labeling of a large RNA through oligonucleotide hybridization; Smith GJ et al.; We present an efficient method of introducing fluorophore labels at selected locations in a large RNA . The method is based on specific and highly efficient hybridization between a fluorophore-containing DNA oligonucleotide and a modular hairpin loop replacing a functionally unimportant hairpin loop in the RNA . We demonstrate its feasibility using a 255-nucleotide RNA derived from the catalytic domain of RNase P from Bacillus subtilis . Hybridization of the DNA oligonucleotide to the modular hairpin loop minimally perturbs the structure and function of this RNA . This labeling scheme should be applicable in studies of RNA conformational dynamics by ensemble and single molecule fluorescence methods.

Mol Microbiol, 2005 Jan, 55(1), 78 - 89
The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex; Kruse T et al.; Summary MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface . The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis . In B . subtilis and C . crescentus, the mreB gene is essential . However, in E . coli, mreB was inferred not to be essential . Using a tight, conditional gene depletion system, we systematically investigated whether the E . coli mreBCD-encoded components were essential . We found that cells depleted of mreBCD became spherical, enlarged and finally lysed . Depletion of each mre gene separately conferred similar gross changes in cell morphology and viability . Thus, the three proteins encoded by mreBCD are all essential and function in the same morphogenetic pathway . Interestingly, the presence of a multicopy plasmid carrying the ftsQAZ genes suppressed the lethality of deletions in the mre operon . Using GFP and cell fractionation methods, we showed that the MreC and MreD proteins were associated with the cell membrane . Using a bacterial two-hybrid system, we found that MreC interacted with both MreB and MreD . In contrast, MreB and MreD did not interact in this assay . Thus, we conclude that the E . coli MreBCD form an essential membrane-bound complex . Curiously, MreB did not form cables in cell depleted for MreC, MreD or RodA, indicating a mutual interdependency between MreB filament morphology and cell shape . Based on these and other observations we propose a model in which the membrane-associated MreBCD complex directs longitudinal cell wall synthesis in a process essential to maintain cell morphology.

Arch Microbiol . 2004 Dec 18; {Epub ahead of print}
bac genes for recombinant bacilysin and anticapsin production in Bacillus host strains; Steinborn G et al.; The genes encoding the biosynthesis of the dipeptide bacilysin and its antibiotic constituent anticapsin were isolated from several strains of Bacillus subtilis as well as B . amyloliquefaciens and B . pumilus . The ywfBCDEF genes of B . subtilis 168 were shown to carry the biosynthetic core functions and were renamed bacABCDE . Mutation of the bacD gene or transformation of the bacABC genes into a B . subtilis Delta (ywfA-bacABCDE) deletion mutant led to the accumulation of anticapsin, which was fourfold higher after transformation of the bacABC genes into a bacD mutant . The genes bacD and bacE proved to encode the functions of amino acid ligation and self-protection to bacilysin, respectively . Amplification of the bacABCDE gene cluster in a bacAB gene-deficient host strain of B . amyloliquefaciens resulted in a tenfold bacilysin overproduction . Some host strains required distinct glucosamine and yeast extract supplements in order to prevent suicidal effects of the recombinant antibiotic production . The bac genes from different Bacillus species revealed the same arrangement and 72.6-88.6% of sequence identity.

J Orthop Res, 2005 Jan, 23(1), 27 - 33
An articulated antibiotic spacer used for infected total knee arthroplasty: a comparative in vitro elution study of Simplex((R)) and Palacos((R)) bone cements; Stevens CM et al.; For the staged management of infected total knee arthroplasty (TKA), antibiotic laden polymethylmethacrylate (PMMA) spacers have been recommended . Antibiotic-impregnated PMMA spacers target drug delivery, achieving high local levels while limiting the potential for host toxicity associated with parenteral antimicrobial therapy . This study examined the elution characteristics of an articulating PMMA TKA spacer that has been useful clinically . Tobramycin and vancomycin are both active against many organisms leading to joint infections . We used various combined antibiotic concentrations (maintaining a relative ratio of 55% tobramycin to 45% vancomycin w/w), and then assayed the elution profile of the TKA spacer in vitro . Additionally, the elution qualities of two brands of bone cement, Simplex((R)) and Palacos((R)), were compared . Briefly, three groups of PMMA spacers, impregnated with different antibiotic loads, were fashioned from a mold replicating a femoral TKA component . The entire spacer surface area was immersed in sterile phosphate buffered saline (PBS) in a 1:6 ratio of grams of cement to milliliters of PBS and incubated at 37 degrees C for 24 h . After 24 h, aliquot eluates were taken, the PBS discarded, and replaced with fresh, sterile PBS . PBS was changed daily and an aliquot was taken at least weekly for nine weeks . Eluate samples were stored at -70 degrees C until assayed . Each spacer eluate sample's antibiotic concentration was determined by disc diffusion bioassay against Bacillus subtilis . Mean zone inhibition diameters were extrapolated from the standard curve to yield micrograms per milliliter of antibiotic in PBS . In all groups the Palacos((R)) spacers demonstrated higher elution levels, above the MIC for the organism used, for a longer period of time than those made with Simplex((R)) . Based on the observed elution profiles, antibiotic-impregnated Palacos((R)) bone cement may offer a more effective vehicle for local drug delivery during staged treatment of infected TKA.

Appl Spectrosc, 2004 Dec, 58(12), 1408 - 12
Intensities of calcium dipicolinate and Bacillus subtilis spore Raman spectra excited with 244 nm light; Nelson WH et al.; Ultraviolet (UV) resonance Raman spectra of Bacillus subtilis endospores have been excited at 244 nm . Spectra can be interpreted in terms of contributions from calcium dipicolinate and nucleic acid components . Differences between spectra of spores and vegetative cells are very large and are due to the dominance of the dipicolinate features in the spore spectra . Because the DNA and RNA composition of B . subtilis spores is known and because the cross-sections of Raman bands belonging to DNA and RNA bases are known, it is possible to calculate resonance Raman spectral cross-sections for the spore Raman peaks associated with the nucleic acids . The cross-sections of peaks associated with calcium dipicolinate have been measured from aqueous solutions . Cross-section values of the dominant 1017 cm(-1) calcium dipicolinate peak measured from the Bacillus spores have been shown to be consistent with a calcium dipicolinate composition of ten percent or less by weight in the spores . It is suggested that spectral cross-sections of endospores excited at 244 nm can be estimated to be the sum of the cross-sections of the calcium dipicolinate, DNA, and RNA components of the spore . It appears that the peaks due to DNA and RNA can be used as an internal standard in the calculation of spore Raman peak cross-sections, and potentially the amount of calcium dipicolinate in spores . It is estimated on the basis of known nucleic acid base cross-sections that the most intense Raman band of the Bacillus subtilis spore spectra has a cross-section of no more than 4 x 10(-18) cm(2)/mol-sr.

Biotechnol Lett, 2004 Sep, 26(17), 1365 - 9
Highly efficient gene expression of a fibrinolytic enzyme (subtilisin DFE) in Bacillus subtilis mediated by the promoter of alpha-amylase gene from Bacillus amyloliquefaciens; Xiao L et al.; Subtilisin DFE is a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 . The promoter and signal peptide-coding sequence of alpha-amylase gene from B . amyloliquefaciens was cloned and fused to the sequence coding for pro-peptide and mature peptide of subtilisin DFE . This hybrid gene was inserted into the Escherichia coli/Bacillus subtilis shuttle plasmid vector, pSUGV4 . Recombinant subtilisin DFE gene was successfully expressed in B . subtilis WB600 with a fibrinolytic activity of 200 urokinase units ml(-1).

J Bacteriol, 2005 Jan, 187(1), 65 - 76
Comparative analysis of the development of swarming communities of Bacillus subtilis 168 and a natural wild type: critical effects of surfactin and the composition of the medium; Julkowska D et al.; The natural wild-type Bacillus subtilis strain 3610 swarms rapidly on the synthetic B medium in symmetrical concentric waves of branched dendritic patterns . In a comparison of the behavior of the laboratory strain 168 (trp) on different media with that of 3610, strain 168 (trp), which does not produce surfactin, displayed less swarming activity, both qualitatively (pattern formation) and in speed of colonization . On E and B media, 168 failed to swarm; however, with the latter, swarming was arrested at an early stage of development, with filamentous cells and rafts of cells (characteristic of dendrites of 3610) associated with bud-like structures surrounding the central inoculum . In contrast, strain 168 apparently swarmed efficiently on Luria-Bertani (LB) agar, colonizing the entire plate in 24 h . However, analysis of the intermediate stages of development of swarms on LB medium demonstrated that, in comparison with strain 3610, initiation of swarming of 168 (trp) was delayed and the greatly reduced rate of expansion of the swarm was uncoordinated, with some regions advancing faster than others . Moreover, while early stages of swarming in 3610 are accompanied by the formation of large numbers of dendrites whose rapid advance involves packs of cells at the tips, strain 168 advanced more slowly as a continuous front . When sfp+ was inserted into the chromosome of 168 (trp) to reestablish surfactin production, many features observed with 3610 on LB medium were now visible with 168 . However, swarming of 168 (sfp+) still showed some reduced speed and a distinctive pattern compared to swarming of 3610 . The results are discussed in terms of the possible role of surfactin in the swarming process and the different modes of swarming on LB medium.

J Biochem (Tokyo), 2004 Sep, 136(3), 387 - 97
Mutational Analysis of the Helix-Turn-Helix Region of Bacillus subtilis Response Regulator DegU, and Identification of cis-Acting Sequences for DegU in the aprE and comK Promoters; Shimane K et al.; The DegS-DegU two-component system in Bacillus subtilis regulates exoprotease production and competence development . Phosphorylated and unphosphorylated forms of DegU are required for activation of aprE and comK, respectively . Alanine-scanning mutagenesis of the helix-turn-helix region of DegU and in vivo examination of 27 DegU variants revealed five common mutants that showed severe reduction of gene expression of both aprE and comK because of reduced DNA-binding activity . This observation suggested that the DegU-recognized cis-sequences might not be considerably changed for either promoter . We identified a DegU-recognized inverted repeat in the comK promoter using various mutant comK-lacZ fusions . Inspection of the aprE promoter sequence revealed a tandem repeat consisting of short AT-rich sequences containing a consensus one, 5'-TAAAT-3', which was found in the downstream half of the inverted repeat involved in comK activation . Oligonucleotide-directed replacement of the short AT-rich sequences located in the center of each motif decreased DegU-dependent aprE expression, implying that the repeat is required for the activation of aprE . Based on these results, it was concluded that DegU would function through the inverted repeat in the comK promoter and the tandem repeat in the aprE promoter.

J Biochem (Tokyo), 2004 Sep, 136(3), 283 - 91
Characterization of a Polysaccharide Deacetylase Gene Homologue (pdaB) on Sporulation of Bacillus subtilis; Fukushima T et al.; The predicted amino acid sequence of Bacillus subtilis ybaN (renamed pdaB) exhibits high similarity to those of several polysaccharide deacetylases . Northern hybridization analysis with sporulation sigma mutants indicated that the pdaB gene is transcribed by EsigmaE RNA polymerase and negatively regulated by SpoIIID . The pdaB mutant was deficient in spore formation . Phase- and electron microscopic observation showed morphological changes of spores in late sporulation periods . The pdaB spores that had lost their viability were empty . Moreover, GFP driven by the promoter of the sspE gene was localized in the forespore compartment for the wild type, but was localized in both the mother cell and forespore compartments for phase-gray/dark forespores of the pdaB mutant . This indicates that GFP expressed in the forespores of the mutant leaks into the mother cells . Therefore, PdaB is necessary to maintain spores after the late stage of sporulation.

J Inorg Biochem, 2005 Jan, 99(1), 23 - 33
Globin-coupled sensors, protoglobins, and the last universal common ancestor; Freitas TA et al.; The strategy for detecting oxygen, carbon monoxide, nitric oxide, and sulfides is predominantly through heme-based sensors utilizing either a globin domain or a PAS domain . Whereas PAS domains bind various cofactors, globins bind only heme . Globin-coupled sensors (GCSs) were first described as regulators of the aerotactic responses in Bacillus subtilis and Halobacterium salinarum . GCSs were also identified in diverse microorganisms that appear to have roles in regulating gene expression . Functional and evolutionary analyses of the GCSs, their protoglobin ancestor, and their relationship to the last universal common ancestor (LUCA) are discussed in the context of globin-based signal transduction.

Protein Expr Purif, 2005 Jan, 39(1), 1 - 7
Confirmation of Vpr as a fibrinolytic enzyme present in extracellular proteins of Bacillus subtilis; Kho CW et al.; We have previously reported a proteomic approach to detect fibrinolytic enzymes from the secreted proteins of Bacillus subtilis 168 and identified two extracellular fibrinolytic enzymes of Bacillus, namely, Vpr and WprA . In this study, to confirm the fibrinolytic activity of Vpr, we cloned the vpr gene and expressed it in Escherichia coli, where it is predominantly localized to inclusion bodies . After affinity purification and desalting steps, the expressed Vpr is auto-processed to an active form . Interestingly, after the desalting step, several additional bands with fibrinolytic activity were detected in zymography gel along with a mature form (68kDa) of Vpr . MALDI-TOF analyses of these bands revealed that Vpr could exist in multiple forms.

Mikrobiologiia, 2004 Sep-Oct, 73(5), 708 - 15
{Enzyme modification by natural chemical chaperons of microorganisms}; The truncated oxygen-avid hemoglobin from Bacillus subtilis . X-ray structure and ligand binding properties; Department of Biochemical Sciences, University of Rome La Sapienza, Roma, Italy 00185The group II truncated hemoglobin from B . subtilis has been cloned, expressed, purified and characterized . B . subtilis trHb is a monomeric protein endowed with an unusually high oxygen affinity (in the nanomolar range) such that the apparent thermodynamic binding constant for O2 exceeds that for CO by one order of magnitude . The kinetic basis of the high oxygen affinity resides mainly in the very slow rate of ligand release . The extremely stable ferrous oxygenated adduct is resistant to oxidation which can be achieved only with oxidant in large excess, e.g . ferricyanide in 50-fold molar excess . The 3D crystal structure of the cyano-met derivative was determined at 2.15 A resolution . Although the overall fold resembles that of other truncated hemoglobins, the distal heme pocket displays a unique array of hydrophilic side chains in the topological positions that dominate the steric interaction with iron-bound ligands . In fact, the Tyr-B10, Thr-E7 and Gln-E11 oxygens on one side of the heme pocket and the Trp-G8 indole NE1 nitrogen on the other form a novel pattern of the "ligand inclusive hydrogen bond network" described for mycobacterial HbO . On the proximal side, the histidine residue is in an unstrained conformation and the iron-His bond is unusually short (1.91 A).

J Mol Biol, 2005 Jan 28, 345(4), 667 - 79
Bacillus subtilis TRAP binds to its RNA target by a 5' to 3' directional mechanism; Barbolina MV et al.; TRAP is an 11 subunit RNA-binding protein that regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translation control mechanisms . Tryptophan-activated TRAP acts by binding to a site in the 5'-untranslated leader region of trp mRNA consisting of 11 (G/U)AG repeats . We used mung bean nuclease footprinting to analyze the interaction of TRAP with several artificial binding sites composed of 11 GAG repeats in nucleic acids that lack secondary structure . Affinities for individual repeats within a binding site did not vary significantly . In contrast, the association rate constants were highest for repeats at the 5' end and lowest for those at the 3' end of all binding sites tested . These results indicate that TRAP binds to its RNA targets by first associating with one or more repeat at the 5' end of its binding site followed by wrapping the remainder of binding site around the protein in a 5' to 3' direction . This directional binding is novel among RNA-binding proteins . We suggest that this mechanism of binding is important for TRAP-mediated transcription attenuation control of the trp operon.

Biochim Biophys Acta, 2004 Dec 1, 1703(1), 43 - 51
Expression and characterization of a heterodimer of Streptomyces chromofuscus phospholipase D; Yang H et al.; Streptomyces chromofuscus phospholipase D (PLD) is secreted by the bacterium and proteolytically cleaved to a more active form (PLD(37/18)) where the two parts of the molecule are still tightly associated . Based on previous sequencing results of authentic PLD(37/18), we have constructed a vector consisting of separate ORFs for the N-terminal and C-terminal portions of S . chromofuscus PLD and overexpressed active heterodimeric PLD . Neither fragment cloned separately folded properly . The identity of each peptide was confirmed by peptide-mass fingerprinting with MALDI-TOF mass spectrometry . The recombinant complex had a specific activity about six times higher than that of the recombinant intact PLD enzyme and was no longer activated by phosphatidic acid (PA) . Phosphotransferase activity, binding affinity to phospholipid vesicles, loss of product activation, pH profile and pH-related Ca(2+) activation and inhibition were comparable to authentic PLD(37/18) purified from S . chromofuscus growth medium . PLD(37) alone could also be isolated; the enzyme was active but not as stable as PLD(37/18) . These experimental results strongly support the hypothesis that the C-terminal peptide is necessary for correct folding and insertion of catalytic metal ions . However, they suggest the ligands involved in Fe(3+) coordination must be altered upon cleavage of the protein . Asp389, in the C-terminal fragment, whose replacement impairs Fe(3+) binding to the protein, must be replaced by another ligand, since the N-terminal fragment, once folded, is active . In the process of cloning the two peptides, the complete signal sequence for this protein was also determined . The signal peptide of S . chromofuscus PLD enzyme contained a twin arginine motif suggesting that S . chromofuscus PLD, like Bacillus subtilis phoD, is most likely secreted by the TAT translocation pathway under the transcriptional control of the pho regulon.

J Anim Physiol Anim Nutr (Berl), 2004 Dec, 88(11-12), 381 - 92
Field evaluation of the efficacy of a probiotic containing Bacillus licheniformis and Bacillus subtilis spores, on the health status and performance of sows and their litters; Alexopoulos C et al.; Summary The aim of this study was to assess the efficacy of BioPlus 2B, a probiotic containing Bacillus licheniformis and Bacillus subtilis spores, on the health status and productivity of sows and their litters . A total of 109 gilts and sows were allocated into two experimental groups, as follows: untreated controls (UC) and BioPlus 2B (same feeding as the UC group plus BioPlus 2B) at a dose of 400 g/ton of feed (equal to 1.28 x 10(6) viable spores/g of feed) . Treatment started from the day of allocation (14 days prior to the expected farrowing) up to the weaning day . Homogeneity of the groups was satisfied with regard to the parity . From the results it was evident that BioPlus 2B supplementation of the feed improved gilt/sow performance as shown by: (i) the increase of sow feed consumption during the first 14 days postpartum and (ii) the decrease of sow weight loss during the suckling period . Certain blood and milk parameters were significantly improved, as shown by higher serum cholesterol and total lipids concentrations and higher milk fat and protein content at mid-suckling period . As a consequence, a positive effect was also noticed as regard litter health and performance characteristics in terms of: (i) decrease in piglet diarrhoea score, (ii) decrease in pre-weaning mortality thus leading to increase in the number of weaned piglets per litter and (iii) increase in piglet body weight at weaning . Moreover, BioPlus 2B tended to improve the health status and fertility of sows demonstrating: (i) tendency to a lower proportion of sows with Mastitis-Metritus-Agalactia (MMA) problems and (ii) lower proportion of sows returning to oestrus.

J Am Chem Soc, 2004 Dec 15, 126(49), 16148 - 59
Differential transition-state stabilization in enzyme catalysis: quantum chemical analysis of interactions in the chorismate mutase reaction and prediction of the optimal catalytic field; Szefczyk B et al.; Chorismate mutase is a key model system in the development of theories of enzyme catalysis . To analyze the physical nature of catalytic interactions within the enzyme active site and to estimate the stabilization of the transition state (TS) relative to the substrate (differential transition state stabilization, DTSS), we have carried out nonempirical variation-perturbation analysis of the electrostatic, exchange, delocalization, and correlation interactions of the enzyme-bound substrate and transition-state structures derived from ab initio QM/MM modeling of Bacillus subtilis chorismate mutase . Significant TS stabilization by approximately -23 kcal/mol {MP2/6-31G(d)} relative to the bound substrate is in agreement with that of previous QM/MM modeling and contrasts with suggestions that catalysis by this enzyme arises purely from conformational selection effects . The most important contributions to DTSS come from the residues, Arg90, Arg7, Glu78, a crystallographic water molecule, Arg116, and Arg63, and are dominated by electrostatic effects . Analysis of the differential electrostatic potential of the TS and substrate allows calculation of the catalytic field, predicting the optimal location of charged groups to achieve maximal DTSS . Comparison with the active site of the enzyme from those of several species shows that the positions of charged active site residues correspond closely to the optimal catalytic field, showing that the enzyme has evolved specifically to stabilize the TS relative to the substrate.

Biotechnol Bioeng, 2005 Jan 20, 89(2), 219 - 32
Transient expression and flux changes during a shift from high to low riboflavin production in continuous cultures of Bacillus subtilis; Zamboni N et al.; At the onset of glucose-limited continuous cultures, riboflavin production in recombinant Bacillus subtilis declines significantly within 3 generations . This phenomenon was specific to riboflavin production and was not correlated with any other physiological parameter . Physiological analyses excluded genetic degeneration or co-metabolism of previously generated overflow metabolites as possible causes for the riboflavin transients . By developing a novel method for (13)C-based metabolic flux analysis under non-steady-state conditions, we showed that the pentose precursors of riboflavin were exclusively synthesized via the non-oxidative pentose-phosphate (PP) pathway as long as riboflavin production was high . The complete redirection of carbon flux to the oxidative branch of the PP pathway was achieved at unaltered PP pathway gene expression and correlated with the declining riboflavin production . With the possible exception of a slight down-regulation of the purine biosynthesis pathway, genome-wide expression analysis indicated that transcriptional regulation was not responsible for the production decline . (c) 2004 Wiley Periodicals, Inc.

Acta Crystallogr D Biol Crystallogr, 2004 Dec, 60(Pt 12 Pt 2), 2403 - 6 Epub 2004 Dec.
SAD at home: solving the structure of oxalate decarboxylase with the anomalous signal from manganese using X-ray data collected on a home source; Stevenson CE et al.; Oxalate decarboxylase (OxdC) from Bacillus subtilis is a hexamer containing two manganese ions per 43.6 kDa subunit . A single highly redundant data set collected at a medium resolution of 2 A on an in-house X-ray source was sufficient to solve the structure by the single-wavelength anomalous diffraction (SAD) method using the anomalous signal from the manganese ions . The experimentally phased electron-density map was of high quality, enabling 96% of the amino-acid sequence to be automatically traced using ARP/wARP . Further analysis showed that only half of the original raw data were required for successful structure solution . Manganese currently occurs in approximately 2% of PDB entries . A brief survey suggests that several of these structures could also have been determined using manganese SAD . Moreover, the ability of manganese to substitute for other more commonly occurring divalent metal ions may indicate that the use of Mn SAD could have much wider application.

Acta Crystallogr D Biol Crystallogr, 2004 Dec, 60(Pt 12 Pt 2), 2371 - 6 Epub 2004 Dec.
Preliminary crystallographic characterization of BSAP, an extracellular aminopeptidase from Bacillus subtilis; Reiland V et al.; The extracellular aminopeptidase from Bacillus subtilis (BSAP) has recently been cloned, overexpressed and purified from Escherichia coli . It is a monomer with a molecular weight of 46 425 Da, consisting of 425 amino-acid residues and a double-zinc catalytic centre . The recombinant enzyme was found to be stable for 20 min at 353 K, to function optimally in the pH range 8-9 and to prefer basic and large hydrophobic N-terminal amino acids in peptide and protein substrates . As such, this enzyme can be used as a representative model for structural, functional and mechanistic studies of monomeric double-zinc aminopeptidases, many of which have been found to be involved in medically important biological activities . In this report, the crystallization and preliminary crystallographic characterization of wild-type BSAP are described . Two different crystal forms are reported, of which the hexagonal form H2 is the more suitable for structural study, with average unit-cell dimensions a = b = 226.5, c = 42.8 A . A full diffraction data set has been collected from such a crystal of the native enzyme (2.2 A resolution, 91.2% completeness, R(merge) = 7.1%) . A multiwavelength anomalous diffraction (MAD) data set was collected on native (zinc-containing) BSAP at three wavelengths around the zinc absorption edge (peak data set at 2.5 A resolution, 98.8% completeness, R(merge) = 5.3%) . These diffraction data were collected at 95-100 K using a synchrotron X-ray source and a CCD area detector . The data are currently being used to obtain crystallographic phasing and to determine the detailed three-dimensional structure of the enzyme.

Microbiology, 2004 Dec, 150(Pt 12), 4125 - 36
Associations between Bacillus subtilis sigmaB regulators in cell extracts; Kuo S et al.; The general stress regulon of Bacillus subtilis is induced by the activation of the sigma(B) transcription factor . Activation of sigma(B) occurs as a consequence of the dephosphorylation of its positive regulator RsbV by one of two phosphatases that respond to either physical or nutritional stress . The physical stress phosphatase (RsbU) requires a second protein (RsbT) for activity . Stress is thought to initiate a process that triggers the release of RsbT from a large inhibitory complex composed of multiple copies of two protein species, RsbR (and/or its paralogues) and RsbS . The stress-derived signal driving RsbT release is unknown, but it fails to develop in B . subtilis lacking either ribosome protein L11 or the ribosome-associated protein Obg . RsbR, RsbS, RsbT, Obg and ribosomes elute in common high-molecular-mass fractions during gel-filtration chromatography of crude B . subtilis extracts . This paper reports the investigation of the basis of this coelution by the examining of associations between these proteins in extracts prepared from wild-type and mutant B . subtilis, and Escherichia coli engineered to express RsbR, RsbS and RsbT . Large RsbR/RsbS complexes, distinct from ribosomes, were detected in extracts of both B . subtilis and E . coli . In E . coli, high-molecular-mass forms of RsbS were less abundant when RsbR was absent, but in B . subtilis, only when both RsbR and its principal paralogues were missing from the extract was this form less abundant . This finding is consistent with the notion that the RsbR paralogues, present in B . subtilis but not E . coli, can substitute for RsbR in such complexes . RsbT was not bound to RsbR/RsbS in any extract that was examined, including one prepared from a B . subtilis strain with an RsbS variant (RsbS59SA) that is believed to continuously associate with RsbT . The high-molecular-mass forms of RsbT were found to be Triton-sensitive and independent of any other B . subtilis protein for their formation . These probably represent RsbT aggregates . The data suggest that the contribution of ribosomes/Obg to sigma(B) activation does not involve formation of a stable association between these proteins and the Rsb complex . In addition, the binding of RsbT to RsbS/RsbR appears to be more labile than the binding between the previously analysed Rsb proteins which form inhibitory complexes . This, and the apparent proclivity of RsbT to aggregate, suggests an inherent instability in RsbT which may play a role in its regulation.

Microbiology, 2004 Dec, 150(Pt 12), 4115 - 23
Characterization of Bacillus subtilis gamma-glutamyltransferase and its involvement in the degradation of capsule poly-gamma-glutamate; Kimura K et al.; During early stationary phase, Bacillus subtilis NAFM5 produces capsular poly(gamma-glutamic acid) (gammaPGA, 2x10(6) Da), which contains D- and L-glutamate, and then degrades it during late stationary phase . The gamma-glutamyltransferase (EC 2.3.2.2; GGT) of this strain successively hydrolysed gammaPGA from the amino-terminal end, to yield both D- and L-glutamate . This enzyme was specifically synthesized during the stationary phase through transcriptional activation of the corresponding ggt gene by the ComQXPA quorum-sensing system . A ggt knockout mutant degraded gammaPGA into 1x10(5) Da fragments, but not any further, indicating that the capsule gammaPGA is first internally degraded by an endo-type of gammaPGA hydrolase into 1x10(5) Da intermediates, then externally into glutamates via GGT . Due to its inability to generate the glutamates from the capsule, the ggt mutant sporulated more frequently than the wild-type strain . The results show that B . subtilis GGT has a powerful exo-gamma-glutamyl hydrolase activity that participates in capsule gammaPGA degradation to supply stationary-phase cells with constituent glutamates.

Microbiology, 2004 Dec, 150(Pt 12), 3959 - 67
Characterization of the Bacillus cereus Nhe enterotoxin; Lindback T et al.; The non-haemolytic enterotoxin (Nhe) is one of two three-component enterotoxins responsible for the diarrhoeal food-poisoning syndrome caused by Bacillus cereus . Nhe is composed of NheA, NheB and NheC . The three genes encoding the Nhe components constitute an operon, and the transcriptional start site is located 61 bp upstream of the nheA translational start site . The nhe genes were cloned separately, and expressed in either Bacillus subtilis or Escherichia coli . Separate expression showed that all three components were required for biological activity . In addition, NheA and NheB were purified from B . cereus culture supernatants . As NheC seems to be expressed in only small amounts by B . cereus, NheC was expressed and purified as a histidine-tagged fusion protein . The maximum cytotoxic activity was obtained when the molar ratio between NheA : NheB : His6-NheC was 10 : 10 : 1, and it was shown that NheB was the binding component of the enterotoxin complex.

Biochemistry, 2004 Dec 14, 43(49), 15472 - 9
YfiT from Bacillus subtilis is a probable metal-dependent hydrolase with an unusual four-helix bundle topology; Rajan SS et al.; YfiT, a 19-kDa polypeptide from Bacillus subtilis, belongs to a small sequence family with members predominantly from Gram positive bacteria . We have determined the crystal structure of YfiT in complex with Ni(2+) to a resolution of 1.7 A . YfiT exists as a dimer and binds Ni(2+) in a 1:1 stoichiometry . The protein has an unusual four-helix bundle topology and coordinates Ni(2+) in an octahedral geometry with three conserved histidines and three waters . Although there is no similarity in their overall structures, the coordination geometry of the metal and the residues that constitute the putative active site in YfiT are similar to those of metalloproteases such as thermolysin . Our structural analyses suggest that YfiT might function as a metal-dependent hydrolase.

Appl Microbiol Biotechnol . 2004 Dec 2; {Epub ahead of print}
Bacillus subtilis M4 decreases plant susceptibility towards fungal pathogens by increasing host resistance associated with differential gene expression; Ongena M et al.; Results presented in this paper describe the ability of Bacillus subtilis strain M4 to reduce disease incidence caused by Colletotrichum lagenarium and Pythium aphanidermatum on cucumber and tomato, respectively . Disease protection in both pathosystems was most probably due to induction of resistance in the host plant since experiments were designed in order to avoid any direct contact between the biocontrol agent and the pathogen . Pre-inoculation with strain M4 thus sensitised both plants to react more efficiently to subsequent pathogen infection . In cucumber, the use of endospores provided a disease control level similar to that obtained with vegetative cells . In contrast, a mixture of lipopeptides from the surfactin, iturin and fengycin families showed no resistance-inducing potential . Interestingly, treatment with strain M4 was also associated with significant changes in gene transcription in the host plant as revealed by cDNA-AFLP analyses . Several AFLP fragments corresponded to genes not expressed in control plants and specifically induced by the Bacillus treatment . In support to the macroscopic protective effect, this differential accumulation of mRNA also illustrates the plant reaction following perception of strain M4, and constitutes one of the very first examples of defence-associated modifications at the transcriptional level elicited by a non-pathogenic bacterium in a host plant.

J Bacteriol, 2004 Dec, 186(24), 8490 - 8
Regulation of sigmaB by an anti- and an anti-anti-sigma factor in Streptomyces coelicolor in response to osmotic stress; Lee EJ et al.; sigmaB, a homolog of stress-responsive sigmaB of Bacillus subtilis, controls both osmoprotection and differentiation in Streptomyces coelicolor A3 (2) . Its gene is preceded by rsbA and rsbB genes encoding homologs of an anti-sigma factor, RsbW, and its antagonist, RsbV, of B . subtilis, respectively . Purified RsbA bound to sigmaB and prevented sigmaB-directed transcription from the sigBp1 promoter in vitro . An rsbA-null mutant exhibited contrasting behavior to the sigB mutant, with elevated sigBp1 transcription, no actinorhodin production, and precocious aerial mycelial formation, reflecting enhanced activity of sigmaB in vivo . Despite sequence similarity to RsbV, RsbB lacks the conserved phosphorylatable serine residue and its gene disruption produced no distinct phenotype . RsbV (SCO7325) from a putative six-gene operon (rsbV-rsbR-rsbS-rsbT-rsbU1-rsbU) was strongly induced by osmotic stress in a sigmaB-dependent manner . It antagonized the inhibitory action of RsbA on sigmaB-directed transcription and was phosphorylated by RsbA in vitro . These results support the hypothesis that the rapid induction of sigmaB target genes by osmotic stress results from modulation of sigmaB activity by the kinase-anti-sigma factor RsbA and its phosphorylatable antagonist RsbV, which function by a partner-switching mechanism . Amplified induction could result from a rapid increase in the synthesis of both sigmaB and its inhibitor antagonist.

J Bacteriol, 2004 Dec, 186(24), 8424 - 32
Terminal oxidases are essential to bypass the requirement for ResD for full Pho induction in Bacillus subtilis; Schau M et al.; The Bacillus subtilis Pho signal transduction network, which regulates the cellular response to phosphate starvation, integrates the activity of three signal transduction systems to regulate the level of the Pho response . This signal transduction network includes a positive feedback loop between the PhoP/PhoR and ResD/ResE two-component systems . Within this network, ResD is responsible for 80% of the Pho response . To date, the role of ResD in the generation of the Pho response has not been understood . Expression of two terminal oxidases requires ResD function, and expression of at least one terminal oxidase is needed for the wild-type Pho response . Previously, our investigators have shown that strains bearing mutations in resD are impaired for growth and acquire secondary mutations which compensate for the loss of the a-type terminal oxidases by allowing production of cytochrome bd . We report here that the expression of cytochrome bd in a DeltaresDE background is sufficient to compensate for the loss of ResD for full Pho induction . A ctaA mutant strain, deficient in the production of heme A, has the same Pho induction phenotype as a DeltaresDE strain . This demonstrates that the production of a-type terminal oxidases is the basis for the role of ResD in Pho induction . Terminal oxidases affect the redox state of the quinone pool . Reduced quinones inhibit PhoR autophosphorylation in vitro, consistent with a requirement for terminal oxidases for full Pho induction in vivo.

J Bacteriol, 2004 Dec, 186(24), 8380 - 4
Gene ytkD of Bacillus subtilis encodes an atypical nucleoside triphosphatase member of the Nudix hydrolase superfamily; Xu W et al.; Gene ytkD of Bacillus subtilis, a member of the Nudix hydrolase superfamily, has been cloned and expressed in Escherichia coli . The purified protein has been characterized as a nucleoside triphosphatase active on all of the canonical ribo- and deoxyribonucleoside triphosphates . Whereas all other nucleoside triphosphatase members of the superfamily release inorganic pyrophosphate and the cognate nucleoside monophosphate, YtkD hydrolyses nucleoside triphosphates in a stepwise fashion through the diphosphate to the monophosphate, releasing two molecules of inorganic orthophosphate . Contrary to a previous report, our enzymological and genetic studies indicate that ytkD is not an orthologue of E . coli mutT.

Genome Biol . 2004;5(12):R99 . Epub 2004.
Model-independent fluxome profiling from 2H and 13C experiments for metabolic variant discrimination; Zamboni N et al.; We introduce a conceptually novel method for intracellular fluxome profiling from unsupervised statistical analysis of stable isotope labeling . Without a priori knowledge on the metabolic system, we identified characteristic flux fingerprints in 10 Bacillus subtilis mutants from 132 2H and 13C tracer experiments . Beyond variant discrimination, independent component analysis automatically mapped several fingerprints to their metabolic determinants . The approach is flexible and paves the way to large-scale fluxome profiling of any biological system and condition.

Appl Environ Microbiol, 2004 Dec, 70(12), 7321 - 8
Pressure inactivation of Bacillus endospores; Margosch D et al.; The inactivation of bacterial endospores by hydrostatic pressure requires the combined application of heat and pressure . We have determined the resistance of spores of 14 food isolates and 5 laboratory strains of Bacillus subtilis, B . amyloliquefaciens, and B . licheniformis to treatments with pressure and temperature (200 to 800 MPa and 60 to 80 degrees C) in mashed carrots . A large variation in the pressure resistance of spores was observed, and their reduction by treatments with 800 MPa and 70 degrees C for 4 min ranged from more than 6 log units to no reduction . The sporulation conditions further influenced their pressure resistance . The loss of dipicolinic acid (DPA) from spores that varied in their pressure resistance was determined, and spore sublethal injury was assessed by determination of the detection times for individual spores . Treatment of spores with pressure and temperature resulted in DPA-free, phase-bright spores . These spores were sensitive to moderate heat and exhibited strongly increased detection times as judged by the time required for single spores to grow to visible turbidity of the growth medium . The role of DPA in heat and pressure resistance was further substantiated by the use of the DPA-deficient mutant strain B . subtilis CIP 76.26 . Taken together, these results indicate that inactivation of spores by combined pressure and temperature processing is achieved by a two-stage mechanism that does not involve germination . At a pressure between 600 and 800 MPa and a temperature greater than 60 degrees C, DPA is released predominantly by a physicochemical rather than a physiological process, and the DPA-free spores are inactivated by moderate heat independent of the pressure level . Relevant target organisms for pressure and temperature treatment of foods are proposed, namely, strains of B . amyloliquefaciens, which form highly pressure-resistant spores.

Appl Environ Microbiol, 2004 Dec, 70(12), 7241 - 50
New integrative method to generate Bacillus subtilis recombinant strains free of selection markers; Brans A et al.; The novel method described in this paper combines the use of blaI, which encodes a repressor involved in Bacillus licheniformis BlaP beta-lactamase regulation, an antibiotic resistance gene, and a B . subtilis strain (BS1541) that is conditionally auxotrophic for lysine . We constructed a BlaI cassette containing blaI and the spectinomycin resistance genes and two short direct repeat DNA sequences, one at each extremity of the cassette . The BS1541 strain was obtained by replacing the B . subtilis P(lysA) promoter with that of the P(blaP) beta-lactamase promoter . In the resulting strain, the cloning of the blaI repressor gene confers lysine auxotrophy to BS1541 . After integration of the BlaI cassette into the chromosome of a conditionally lys-auxotrophic (BS1541) strain by homologous recombination and positive selection for spectinomycin resistance, the eviction of the BlaI cassette was achieved by single crossover between the two short direct repeat sequences . This strategy was successfully used to inactivate a single gene and to introduce a gene of interest in the Bacillus chromosome . In both cases the resulting strains are free of selection marker . This allows the use of the BlaI cassette to repeatedly further modify the Bacillus chromosome.

Genes Dev, 2004 Dec 1, 18(23), 2916 - 28
Zipper-like interaction between proteins in adjacent daughter cells mediates protein localization; Blaylock B et al.; Protein localization is crucial for cellular morphogenesis and intracellular signal transduction cascades . Here we describe an interaction between two membrane proteins expressed in different cells of the Bacillus subtilis sporangium, the mother cell protein SpoIIIAH and the forespore protein SpoIIQ . We used affinity chromatography, coimmunoprecipitation, and the yeast two-hybrid system to demonstrate that the extracellular domains of these proteins interact, tethering SpoIIIAH to the sporulation septum, and directing its assembly with SpoIIQ into helical arcs and foci around the forespore . We also demonstrate that this interaction can direct proteins made in the same cell to active division sites, as when SpoIIQ is made in the mother cell, it localizes to nascent septa in a SpoIIIAH-dependent manner . Both SpoIIIAH and SpoIIQ are necessary for activation of the second forespore-specific transcription factor (sigma(G)) after engulfment, and we propose that the SpoIIIAH-SpoIIQ complex contributes to a morphological checkpoint coupling sigma(G) activation to engulfment . In keeping with this hypothesis, SpoIIIAH localization depends on the first step of engulfment, septal thinning . The SpoIIQ-SpoIIIAH complex reaches from the mother cell cytoplasm to the forespore cytoplasm and is ideally positioned to govern the activity of engulfment-dependent transcription factors.

Eur J Med Chem, 2004 Dec, 39(12), 1059 - 65
Synthesis, antitumour and antimicrobial activities of new peptidyl derivatives containing the 1,3-benzodioxole system; Leite AC et al.; Two series of 5 and 6-substituted 1,3-benzodioxole peptidyl derivatives were synthesized and evaluated as antitumour and antimicrobial agents . The compounds that could be conveniently prepared in a few steps processes from natural safrole have been characterised by IR and 1H-NMR spectroscopy . In vivo antitumor activity tests showed that some of the compounds were able to inhibit carcinoma S-180 tumour growth in mice . The in vitro antimicrobial activity of all compounds revealed that they are able to promote the growth of some organisms, including Bacillus subtilis.

Anal Chem, 2004 Dec 1, 76(23), 6848 - 52
Use of performic acid oxidation to expand the mass distribution of tryptic peptides; Matthiesen R et al.; Significant identification of proteins by mass fingerprinting and partial sequencing of tryptic peptides is central to proteomics . However, peptide masses cluster with distances of approximately 1 Da . Expanding these clusters will give more peptides of unique masses, thereby identifying proteins with a higher significance . The mass clusters can be expanded downward by including more oxygen atoms in the peptides . Classic performic acid oxidation modifies three residues, Cys to CysO(3), Met to MetO(2), and Trp to TrpO(2) . In this study, we compare the mass distributions of tryptic peptides computed from the predicted proteomes of Bacillus subtilis, Drosophila melanogaster, Arabidopsis thaliana, and Homo sapiens modified by oxidation, reduction, and reduction followed by carboxymethylation, carboxamidomethylation, or pyridylethylation . Forty to 46% of the eukaryotic tryptic peptides contain Cys, Met, or Trp . Additionally, the importance of mass accuracy of differentially modified tryptic peptides for significant protein identification by database searches was analyzed . The results show that performic acid oxidation gives markedly extended mass distributions at mass accuracies from +/-0.002 to +/-0.25 Da for the eukaryotes . The effect of the expanded mass distribution on significant protein identification was illustrated by searching simulated mass peak lists against the databases containing oxidized and reduced tryptic peptides . The specificity of formic acid oxidation was tested experimentally, and no general adverse effects were detected . Tryptic peptides provided a 100% sequence coverage of oxidized barley grain peroxidase by LC-MS, and the sequence coverages of oxidized and carboxymethylated bovine serum albumin were similar by MALDI-TOF MS analyses.

Biosci Biotechnol Biochem, 2004 Nov, 68(11), 2374 - 87
Towards structural determination of the ComX pheromone: synthetic studies on peptides containing geranyltryptophan; Okada M et al.; Bacteria produce and respond to signal molecules depending on their cell density . This process is called "quorum sensing" . The ComX pheromone, controlled by quorum sensing, activates natural genetic competence in Bacillus subtilis . ComX is an oligopeptide with a posttranslational modification . It has been suggested that ComX pheromone is modified with an isoprenoid at its tryptophan residue, but the complete chemical structure is unknown . We first determined the molecular formula of ComX(RO-E-2), a competence factor for B . subtilis strain RO-E-2 . Then we synthesized putative pheromones with 1-, 2-, 4-, 5-, 6-, or 7-geranyl substituted tryptophan residues . The regio- and stereo-selective synthesis of the geranyl tryptophans was successful, and we prepared the six peptides with modified tryptophan residues . These peptides had the same molecular formula and showed similar hydrophobicity to the natural ComX(RO-E-2) in LC-MS analysis . But, none of them showed the same retention time as the natural pheromone and none exhibited its biological activity . These results suggest that the isoprenoid modification pattern of the tryptophan residue is more complex than postulated.

Biosci Biotechnol Biochem, 2004 Nov, 68(11), 2319 - 25
Contribution of the second OB fold of ribosomal protein S1 from Escherichia coli to the recognition of TmRNA; Okada T et al.; Escherichia coli ribosomal protein S1 is composed of six repeating homologous oligonucleotide/oligosaccharide-binding fold (OB folds) . In trans-translation, S1 plays a role in delivering transfer-messenger RNA (tmRNA) to stalled ribosomes . The second OB fold of S1 was found to be protected from tryptic digestion in the presence of tmRNA . Truncated S1 mutant Delta2, in which the first and second OB folds were deleted, showed significantly decreased tmRNA-binding activity . Furthermore, the E . coli S1 homolog (BS1) from Bacillus subtilis, which corresponds to the four C-terminal OB folds of E . coli S1, showed no interaction with E . coli tmRNA, as judged by the results of a gel shift assay . Surface plasmon resonance analysis revealed that mutant Delta2 and BS1 had decreased association rate constants (ka, 0.59 x 10(3) M(-1).S(-1); and ka, 1.89 x 10(3) M(-1).S(-1)), while they retained the respective dissociation rate constants (kd, 0.67 x 10(-3) S(-1); and kd, 0.53 x 10(-3) S(-1)), in comparison with wild-type protein S1 (ka, 3.32 x 10(3) M(-1).S(-1); and kd, 0.56 x 10(-3) S(-1)) . These results suggest that the second OB fold in protein S1 is essential for the recognition of tmRNA, while the four C-terminal OB folds play a role in stabilizing the S1-tmRNA complex.

J Basic Microbiol, 2004, 44(6), 451 - 8
The Bacillus megaterium comE locus encodes a functional DNA uptake protein; Lammers M et al.; From Bacillus megaterium, a genomic region was isolated and structurally characterized which strongly resembles the Bacillus subtilis competence locus comE encoding proteins involved in DNA uptake . Functionality of the B . megaterium comEA gene was proven by complementing a DNA-receptor mutant of B . subtilis . This finding provides first evidence for a latent ability of B . megaterium to develop natural competence, although such physiological state has not as yet been identified in this organism . ((c) 2004 WILEY-VCH Verlag GmbH & Co . KGaA, Weinheim).

FEMS Microbiol Lett, 2004 Dec 1, 241(1), 41 - 8
Purification, characterization and functional analysis of an endo-arabinanase (AbnA) from Bacillus subtilis; Leal TF et al.; Bacillus subtilis synthesizes at least one arabinanase encoded by the abnA gene that is able to degrade the polysaccharide arabinan . Here, we report the expression in Escherichia coli of the full-length abnA coding region with a His6-tag fused to the C-terminus . The recombinant protein was secreted to the periplasmic space and correctly processed by the E . coli signal peptidase . The substrate specificity of purified AbnA, the physico-chemical properties and kinetic parameters were determined . Functional analysis studies revealed Glu 215 as a key residue for AbnA hydrolytic activity and indicated that in addition to AbnA B . subtilis secretes other enzyme(s) able to degrade linear 1,5-alpha-l-arabinan.

FEBS Lett, 2004 Nov 19, 577(3), 469 - 72
Identification of enzymes acting on alpha-glycated amino acids in Bacillus subtilis; Wiame E et al.; We have characterized the Bacillus subtilis homologs of fructoselysine 6-kinase and fructoselysine-6-phosphate deglycase, two enzymes that specifically metabolize the Amadori compound fructose-epsilon-lysine in Escherichia coli . The B . subtilis enzymes also catalyzed the phosphorylation of fructosamines to fructosamine 6-phosphates (YurL) and the conversion of the latter to glucose 6-phosphate and a free amino acid (YurP) . However, their specificity was totally different from that of the E . coli enzymes, since they acted on fructoseglycine, fructosevaline (YurL) or their 6-phosphoderivatives (YurP) with more than 30-fold higher catalytic efficiencies than on fructose-alpha-lysine (6-phosphate) . These enzymes are therefore involved in the metabolism of alpha-glycated amino acids.

FEBS Lett, 2004 Nov 19, 577(3), 460 - 4
The Bacillus subtilis DnaD protein: a putative link between DNA remodeling and initiation of DNA replication; Turner IJ et al.; The Bacillus subtilis DnaD protein is an essential protein and a component of the oriC and PriA primosomal cascades, which are responsible for loading the main replicative ring helicase DnaC onto DNA . We present evidence that DnaD also has a global DNA architectural activity, assembling into large nucleoprotein complexes on a plasmid and counteracting plasmid compaction in a manner analogous to that recently seen for the histone-like Escherichia coli HU proteins . This DNA-remodeling role may be an essential function for initiation of DNA replication in the Gram +ve B . subtilis, thus highlighting DnaD as the link between bacterial nucleoid reorganization and initiation of DNA replication.

Curr Opin Microbiol, 2004 Dec, 7(6), 579 - 86
Sporulation of Bacillus subtilis; Piggot PJ et al.; Differentiation of vegetative Bacillus subtilis into heat resistant spores is initiated by the activation of the key transcription regulator Spo0A through the phosphorelay . Subsequent events depend on the cell compartment-specific action of a series of RNA polymerase sigma factors . Analysis of genes in the Spo0A regulon has helped delineate the mechanisms of axial chromatin formation and asymmetric division . There have been considerable advances in our understanding of critical controls that act to regulate the phosphorelay and to activate the sigma factors.

Mol Microbiol, 2004 Dec, 54(5), 1319 - 25
Two minimal Tat translocases in Bacillus; Jongbloed JD et al.; Activity of the Tat machinery for protein transport across the inner membrane of Escherichia coli and the chloroplast thylakoidal membrane requires the presence of three membrane proteins: TatA, TatB and TatC . Here, we show that the Tat machinery of the Gram-positive bacterium Bacillus subtilis is very different because it contains at least two minimal Tat translocases, each composed of one specific TatA and one specific TatC component . A third, TatB-like component is apparently not required . This implies that TatA proteins of B . subtilis perform the functions of both TatA and TatB of E . coli and thylakoids . Notably, the two B . subtilis translocases named TatAdCd and TatAyCy both function as individual, substrate-specific translocases for the twin-arginine preproteins PhoD and YwbN, respectively . Importantly, these minimal TatAC translocases of B . subtilis are representative for the Tat machinery of the vast majority of Gram-positive bacteria, Streptomycetes being the only known exception with TatABC translocases.

Mol Microbiol, 2004 Dec, 54(5), 1237 - 49
Identification of a polar targeting determinant for Bacillus subtilis DivIVA; Perry SE et al.; The Bacillus subtilis protein DivIVA controls both the positioning of the vegetative cell division site and the polar attachment of the chromosome during sporulation . In vegetative growth DivIVA attracts the bipartite cell division inhibitor MinCD away from the cell centre and towards the cell pole . This process ensures the inactivation of old polar division sites and leaves the cell centre free for the assembly of a new cell division complex . During sporulation MinCD and DivIVA levels fall, but DivIVA remains at the cell poles and becomes involved in the migration of the chromosomes to the pole . In order to investigate polar targeting of DivIVA, we undertook a mutational analysis of the 164-amino-acid protein . These studies identified one mutant (divIVA(R18C)) that could not localize to the cell pole but which retained the ability to support both vegetative growth and 50% sporulation efficiency . Further analysis revealed that, in the absence of polar targeting, DivIVA(R18C) localized to the nucleoid during vegetative growth in a Spo0J/Soj-dependent manner and required Spo0J/Soj and MinD to orientate the chromosomes correctly during sporulation . We demonstrate that polar targeting of DivIVA(R18C) is not essential during vegetative growth because the mutant can recognize the cell division site and influences the localization of MinD . Similarly we show that DivIVA(R18C) can function during sporulation because it can support the Spo0J/Soj orientation of the chromosome . In addition, we establish that both residues 18 and 19 constitute a DivIVA polar targeting determinant.

Prikl Biokhim Mikrobiol, 2004 Sep-Oct, 40(5), 551 - 7
{Biological properties of the phosphate-mobilizing Bacillus subtilis strain IMV V-7023}; Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine; Department of Chemistry and Biochemistry, 215 UCB, University of Colorado, Boulder, Colorado 80309, USA . robert.batey@colorado.edu

Riboswitches are genetic regulatory elements found in the 5' untranslated region of messenger RNA that act in the absence of protein cofactors . They are broadly distributed across bacteria and account for the regulation of more than 2% of all genes in Bacillus subtilis, underscoring their importance in the control of cellular metabolism . The 5' untranslated region of many mRNAs of genes involved in purine metabolism and transport contain a guanine-responsive riboswitch that directly binds guanine, hypoxanthine or xanthine to terminate transcription . Here we report the crystal structure at 1.95 A resolution of the purine-binding domain of the guanine riboswitch from the xpt-pbuX operon of B . subtilis bound to hypoxanthine, a prevalent metabolite in the bacterial purine salvage pathway . This structure reveals a complex RNA fold involving several phylogenetically conserved nucleotides that create a binding pocket that almost completely envelops the ligand . Hypoxanthine functions to stabilize this structure and to promote the formation of a downstream transcriptional terminator element, thereby providing a mechanism for directly repressing gene expression in response to an increase in intracellular concentrations of metabolite.

J Bacteriol, 2004 Dec, 186(23), 8089 - 95
Unmasking novel sporulation genes in Bacillus subtilis; Silvaggi JM et al.; The Bacillus subtilis transcription factor sigma(E) directs the expression of a regulon of 262 genes, but null mutations in only a small fraction of these genes severely impair sporulation . We have previously reported that mutations in seven sigma(E)-controlled genes cause a mild (2- to 10-fold) defect in sporulation . In this study, we found that pairwise combinations of some of these seven mutations led to strong synthetic sporulation phenotypes, especially those involving the ytrHI operon and ybaN . Double mutants of ybaN and ytrH and of ybaN and ytrI had >10,000-fold lower sporulation efficiencies than the wild type . Thin-section electron microscopy revealed a block in cortex formation for the ybaN ytrH double mutant and coat defects for the ybaN single and ybaN ytrI double mutants . Sporulating cells of a ybaN ytrI double mutant and of a ybaN ytrHI triple mutant exhibited a pronounced loss of dipicolinic acid (DPA) between hours 8 and 24 of sporulation, in contrast to the constant levels seen for the wild type . An analysis of the spore cortex peptidoglycans of the ybaN ytrI and ybaN ytrHI mutants showed striking decreases in the levels of total muramic acid by hour 24 of sporulation . These data, along with the loss of DPA in the mutants, suggest that the developing spores were unstable and that the cortex underwent degradation late in sporulation . The existence of otherwise hidden sporulation pathways indicates that functional redundancy may mask the role of hitherto unrecognized sporulation genes.

J Bacteriol, 2004 Dec, 186(23), 7971 - 9
Negative transcriptional regulation of the ilv-leu operon for biosynthesis of branched-chain amino acids through the Bacillus subtilis global regulator TnrA; Tojo S et al.; The Bacillus subtilis ilv-leu operon is involved in the synthesis of branched-chain amino acids (valine, isoleucine, and leucine) . The two- to threefold repression of expression of the ilv-leu operon during logarithmic-phase growth under nitrogen-limited conditions, which was originally detected by a DNA microarray analysis to compare the transcriptomes from the wild-type and tnrA mutant strains, was confirmed by lacZ fusion and Northern experiments . A genome-wide TnrA box search revealed a candidate box approximately 200 bp upstream of the transcription initiation base of the ilv-leu operon, the TnrA binding to which was verified by gel retardation and DNase I footprinting analyses . Deletion and base substitution of the TnrA box sequence affected the ilv-leu promoter activity in vivo, implying that TnrA bound to the box might be able to inhibit the promoter activity, possibly through DNA bending . The negative control of the expression of the ilv-leu operon by TnrA, which is considered to represent rather fine-tuning (two- to threefold), is a novel regulatory link between nitrogen and amino acid metabolism.

J Bacteriol, 2004 Dec, 186(23), 7865 - 73
Teichoic acid is an essential polymer in Bacillus subtilis that is functionally distinct from teichuronic acid; Bhavsar AP et al.; Wall teichoic acids are anionic, phosphate-rich polymers linked to the peptidoglycan of gram-positive bacteria . In Bacillus subtilis, the predominant wall teichoic acid types are poly(glycerol phosphate) in strain 168 and poly(ribitol phosphate) in strain W23, and they are synthesized by the tag and tar gene products, respectively . Growing evidence suggests that wall teichoic acids are essential in B . subtilis; however, it is widely believed that teichoic acids are dispensable under phosphate-limiting conditions . In the work reported here, we carefully studied the dispensability of teichoic acid under phosphate-limiting conditions by constructing three new mutants . These strains, having precise deletions in tagB, tagF, and tarD, were dependent on xylose-inducible complementation from a distal locus (amyE) for growth . The tarD deletion interrupted poly(ribitol phosphate) synthesis in B . subtilis and represents a unique deletion of a tar gene . When teichoic acid biosynthetic proteins were depleted, the mutants showed a coccoid morphology and cell wall thickening . The new wall teichoic acid biogenesis mutants generated in this work and a previously reported tagD mutant were not viable under phosphate-limiting conditions in the absence of complementation . Cell wall analysis of B . subtilis grown under phosphate-limited conditions showed that teichoic acid contributed approximately one-third of the wall anionic content . These data suggest that wall teichoic acid has an essential function in B . subtilis that cannot be replaced by teichuronic acid.

Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 311 - 27
Post-translocational folding of secretory proteins in Gram-positive bacteria; Sarvas M et al.; The transport of proteins from their site of synthesis in the cytoplasm to their functional location is an essential characteristic of all living cells . In Gram-positive bacteria the majority of proteins that are translocated across the cytoplasmic membrane are delivered to the membrane-cell wall interface in an essentially unfolded form . They must then be folded into their native configuration in an environment that is dominated by a high density of immobilised negative charge-in essence an ion exchange resin . It is essential to the viability of the cell that these proteins do not block the translocation machinery in the membrane, form illegitimate interactions with the cell wall or, through intermolecular interactions, form insoluble aggregates . Native Gram-positive proteins therefore have intrinsic folding characteristics that facilitate their rapid folding, and this is assisted by a variety of folding factors, including enzymes, peptides and metal ions . Despite these intrinsic and extrinsic factors, secretory proteins do misfold, particularly if the cell is subjected to certain types of stress . Consequently, Gram-positive bacteria such as Bacillus subtilis encode membrane- and cell wall-associated proteases that act as a quality control machine, clearing misfolded or otherwise aberrant proteins from the translocase and the cell wall.

Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 299 - 310
Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism; Westers L et al.; Bacillus subtilis is a rod-shaped, Gram-positive soil bacterium that secretes numerous enzymes to degrade a variety of substrates, enabling the bacterium to survive in a continuously changing environment . These enzymes are produced commercially and this production represents about 60% of the industrial-enzyme market . Unfortunately, the secretion of heterologous proteins, originating from Gram-negative bacteria or from eukaryotes, is often severely hampered . Several bottlenecks in the B . subtilis secretion pathway, such as poor targeting to the translocase, degradation of the secretory protein, and incorrect folding, have been revealed . Nevertheless, research into the mechanisms and control of the secretion pathways will lead to improved Bacillus protein secretion systems and broaden the applications as industrial production host . This review focuses on studies that aimed at optimizing B . subtilis as cell factory for commercially interesting heterologous proteins.

J Appl Microbiol, 2004, 97(6), 1247 - 56
Genetic and functional characterization of a Bacillus sp . strain excreting surfactin and antifungal metabolites partially identified as iturin-like compounds; Souto GI et al.; AIMS: A bacterial strain producing antifungal compounds active against the plant pathogenic fungi Fusarium, Rhizoctonia and Sclerotinia has been characterized and shown to control Rhizoctonia root rot of soya bean . METHODS AND RESULTS: The metabolites excreted by Bacillus BNM 122 remained active after autoclaving, were resistant over a wide pH range and to hydrolytic enzymes . By (1)H-NMR and thin-layer chromatography analyses surfactin and iturin-like compounds were partially identified . Moreover, soya bean seeds bacterization with BNM 122 in a compost-based formulation was as effective controlling Rhizoctonia solani as pentachloronitrobenzene . According to its 16S rDNA sequence BNM 122 was closely related to Bacillus amyloliquefaciens and Bacillus subtilis . PCR analysis of the 16S-23S rRNA intergenic spacer region and repetitive sequence-based PCR (rep-PCR) genomic fingerprinting revealed a close genetic relationship to B . amyloliquefaciens . However, by physiological characterization using API tests, this strain resembled more B . subtilis . CONCLUSIONS: This is the first report describing the co-production of surfactin and iturin-like compounds by a putative strain of B . amyloliquefaciens . The synergistic effect of both lipopetides is a remarkable trait for a candidate biocontrol agent . SIGNIFICANCE AND IMPACT OF THE STUDY: This kind of research has relevance in order to minimize the use of synthetic fungicides and surfactants, contributing to the preservation of the environment.

J Appl Microbiol, 2004, 97(6), 1220 - 7
Effect of microwave radiation on Bacillus subtilis spores; Celandroni F et al.; AIMS: To compare the killing efficacy and the effects exerted by microwaves and conventional heating on structural and molecular components of Bacillus subtilis spores . METHODS AND RESULTS: A microwave waveguide applicator was developed to generate a uniform and measurable distribution of the microwave electric-field amplitude . The applicator enabled the killing efficacy exerted by microwaves on B . subtilis spores to be evaluated in comparison with conventional heating at the same temperature value . The two treatments produced a similar kinetics of spore survival, while remarkably different effects on spore structures were seen . The cortex layer of the spores subjected to conductive heating was 10 times wider than that of the untreated spores; in contrast, the cortex of irradiated spores did not change . In addition, the heated spores were found to release appreciable amounts of dipicolinic acid (DPA) upon treatment, while extracellular DPA was completely undetectable in supernatants of the irradiated spores . These observations suggest that microwave radiation may promote the formation of stable complexes between DPA and other spore components (i.e . calcium ions); thus, making any release of DPA from irradiated spores undetectable . Indeed, while a decrease in measurable DPA concentrations was not produced by microwave radiation on pure DPA solutions, a significant lowering in DPA concentration was detected when this molecule was exposed to microwaves in the presence of either calcium ions or spore suspensions . CONCLUSIONS: Microwaves are as effective as conductive heating in killing B . subtilis spores, but the microwave E-field induces changes in the structural and/or molecular components of spores that differ from those attributable only to heat . SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the effect of microwaves on B . subtilis spore components.

J Mol Biol, 2004 Dec 3, 344(4), 919 - 28
Ligand-induced conformational changes in the Bacillus subtilis chemoreceptor McpB determined by disulfide crosslinking in vivo; Szurmant H et al.; Previously, we characterized the organization of the transmembrane (TM) domain of the Bacillus subtilis chemoreceptor McpB using disulfide crosslinking . Cysteine residues were engineered into serial positions along the two helices through the membrane, TM1 and TM2, as well as double mutants in TM1 and TM2, and the extent of crosslinking determined to characterize the organization of the TM domain . In this study, the organization of the TM domain was studied in the presence and absence of ligand to address what ligand-induced structural changes occur . We found that asparagine caused changes in crosslinking rate on all residues along the TM1-TM1' helical interface, whereas the crosslinking rate for almost all residues along the TM2-TM2' interface did not change . These results indicated that helix TM1 rotated counterclockwise and that TM2 did not move in respect to TM2' in the dimer on binding asparagine . Interestingly, intramolecular crosslinking of paired substitutions in 34/280 and 38/273 were unaffected by asparagine, demonstrating that attractant binding to McpB did not induce a "piston-like" vertical displacement of TM2 as seen for Trg and Tar in Escherichia coli . However, these paired substitutions produced oligomeric forms of receptor in response to ligand . This must be due to a shift of the interface between different receptor dimers, within previously suggested trimers of dimers, or even higher order complexes . Furthermore, the extent of disulfide bond formation in the presence of asparagine was unaffected by the presence of the methyl-modification enzymes, CheB and CheR, or the coupling proteins, CheW and CheV, demonstrating that these proteins must have local structural effects on the cytoplasmic domain that is not translated to the entire receptor . Finally, disulfide bond formation was also unaffected by binding proline to McpC . We conclude that ligand-binding induced a conformational change in the TM domain of McpB dimers as an excitation signal that is likely propagated within the cytoplasmic region of receptors and that subsequent adaptational events do not affect this new TM domain conformation.

Z Naturforsch {C}, 2004 Sep-Oct, 59(9-10), 653 - 6
Antimicrobial activity and chemical composition of the essential oil of Nepeta crispa Willd . from Iran; Sonboli A et al.; The composition and antimicrobial activity of the essential oil of Nepeta crispa Willd., an endemic species from Iran, was studied . The oil was obtained from the aerial parts of the plant and analyzed by GC and GC/MS . Twenty-three compounds, accounting for 99.8% of the total oil, were identified . The main constituents were 1,8-cineol (47.9%) and 4aalpha,7alpha,7abetanepetalactone (20.3%) . The antimicrobial activity of essential oil of N . crispa was tested against seven gram-negative or gram-positive bacteria and four fungi . The results of the bioassays showed the interesting antimicrobial activity, in which the gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, were the most sensitive to the oil . Also, the oil exhibited a remarkable antifungal activity against all the tested fungi.

Proteomics, 2004 Dec, 4(12), 3727 - 50
Towards a comprehensive understanding of Bacillus subtilis cell physiology by physiological proteomics; Hecker M et al.; Using Bacillus subtilis as a model system for functional genomics, this review will provide insights how proteomics can be used to bring the virtual life of genes to the real life of proteins . Physiological proteomics will generate a new and broad understanding of cellular physiology because the majority of proteins synthesized in the cell can be visualized . From a physiological point of view two major proteome fractions can be distinguished: proteomes of growing cells and proteomes of nongrowing cells . In the main analytical window almost 50% of the vegetative proteome expressed in growing cells of B . subtilis were identified . This proteomic view of growing cells can be employed for analyzing the regulation of entire metabolic pathways and thus opens the chance for a comprehensive understanding of metabolism and growth processes of bacteria . Proteomics, on the other hand, is also a useful tool for analyzing the adaptational network of nongrowing cells that consists of several partially overlapping regulation groups induced by stress/starvation stimuli . Furthermore, proteomic signatures for environmental stimuli can not only be applied to predict the physiological state of cells, but also offer various industrial applications from fermentation monitoring up to the analysis of the mode of action of drugs . Even if DNA array technologies currently provide a better overview of the gene expression profile than proteome approaches, the latter address biological problems in which they can not be replaced by mRNA profiling procedures . This proteomics of the second generation is a powerful tool for analyzing global control of protein stability, the protein interaction network, protein secretion or post-translational modifications of proteins on the way towards the elucidation of the mystery of life.

J Agric Food Chem, 2004 Nov 17, 52(23), 6977 - 82
Modified mutation method for screening low citrinin-producing strains of Monascus purpureus on rice culture; Wang JJ et al.; Monascus purpureus NTU 601 is a strain that produces monacolin K, gamma-aminobutyric acid (GABA), and citrinin under solid culture conditions . Because citrinin is a mycotoxin and possesses nephrotoxic and hepatoxic effects, it has a negative impact on the acceptance of red mold rice by people . In this research, a simple and quick selection method for mutant strains with low citrinin production was designed based on the fact that citrinin possesses antibacterial activity for Bacillus subtilis and will form an inhibition zone around the colony of the Monascus strain . The mutant strain M . purpureus N 301 only produced 0.23 +/- 0.01 ppm citrinin, which was 50% less than that of the parent strain, and the monacolin K production was 481.29 +/- 7.98 ppm and maintained 91% productivity . M . purpureus N 310, the other mutant strain, produced 0.27 +/- 0.01 ppm citrinin, which was 41% less than that of the parent strain, and the monacolin K production was 526.29 +/- 5.54 ppm, which showed no significant changes when compared with the parent strain . The GABA content of the two strains was 5000 ppm, which is similar to that of the parent strain . The results showed that the method could be used to select red mold rice with low citrinin production.

Biomacromolecules, 2004 Nov-Dec, 5(6), 2195 - 200
Immobilization and stabilization of recombinant multimeric uridine and purine nucleoside phosphorylases from Bacillus subtilis; Rocchietti S et al.; We selected the PnpI/PupG (PNP) with specificity for ribo- and deoxyriboguanosine and ribo- and deoxyriboinosine and the Up/Pdp (UP) with specificity for uridine, thymidine, and deoxyuridine from the purine and pyrimidine salvage pathway of the Gram-positive bacterium Bacillus subtilis . Then, an extensive study of the UP (uridine phosphorylase) and PNP (purine nucleoside phosphorylase) immobilization and stabilization was carried out: optimal UP preparation was achieved by immobilization onto Sepabeads coated with poly(ethyleneimine) and finally cross-linked with aldehyde dextran (UP-Sep-PEI-Dx); optimal immobilized PNP was prepared onto glyoxyl-agarose . Both derivatives were highly stable and active even under drastic experimental conditions (pH 10, 45 degrees C) unlike the free enzymes which were promptly inactivated . The derivatives prepared were successfully used in the synthesis of 2'-deoxyguanosine by enzymatic transglycosylation in aqueous solution between 2'-deoxyuridine and guanine.

Proteomics, 2004 Nov, 4(11), 3437 - 45
Identification of the degradome of Isp-1, a major intracellular serine protease of Bacillus subtilis, by two-dimensional gel electrophoresis and matrix- assisted laser desorption/ionization-time of flight analysis; Lee AY et al.; Intracellular serine protease-1 (Isp-1) is a major intracellular serine protease of Bacillus subtilis, whose functions still remain largely unknown . Furthermore, physiological substrates are yet to be determined . To identify Isp-1 substrates, we digested extract obtained from an Isp-1 deficient Bacillus mutant with purified Isp-1 and examined eliminated or decreased spots by two-dimensional gel and matrix-assisted laser desorption/ionization-time of flight analyses . Proteins degraded by Isp-1, termed the Isp-1 degradome, are involved in a variety of cellular functions such as DNA packing, genetic competence, and protein secretion . From the degradome we selected ClpC and EF-Tu as putative Isp-1 substrates and studied their in vitro degradation . ClpC and EF-Tu contain putative cleavage sites for Isp-1 . N-terminal sequencing of in vitro proteolytic fragments of ClpC and EF-Tu revealed that these sites are indeed recognized and cleaved by Isp-1 . Moreover, the cellular levels of ClpC and EF-Tu were dramatically reduced at the late stationary phase, where the expression level of Isp-1 was greatly increased . These results suggest that the regulated proteolysis of ClpC by Isp-1 plays an important role in the stationary phase adaptive response . This degradomic approach could provide a powerful tool for finding physiological substrates of many proteolytic enzymes whose functions remain to be determined.

Microbiology, 2004 Nov, 150(Pt 11), 3669 - 79
Autogenous modulation of the Bacillus subtilis sacB-levB-yveA levansucrase operon by the levB transcript; Daguer JP et al.; Silencing of levB, the second structural gene of the tricistronic levansucrase operon encoding the endolevanase LevB, decreases the level of levansucrase expression in Bacillus subtilis . Conversely, independent expression of levB greatly stimulates operon expression . This autogenous effect is mediated by the levB transcript, which carries an internal sequence (5'-AAAGCAGGCAA-3') involved in the enhancing effect . In vitro, the levB transcript displays an affinity for the N-terminal fragment of SacY (K(D) 0.2 microM), the regulatory protein that prevents transcription termination of the levansucrase operon . This positive-feedback loop leads to an increase in the operon expression when B . subtilis is growing in the presence of high sucrose concentrations . Under these conditions, extracellular levan synthesized by the fructosyl polymerase activity of levansucrase can be degraded mainly into levanbiose by the action of LevB . Levanbiose is neither taken up nor metabolized by the bacteria . This work modifies the present view of the status of levansucrase in B . subtilis physiology.

Appl Environ Microbiol, 2004 Nov, 70(11), 6809 - 15
Visualization of differential gene expression by improved cyan fluorescent protein and yellow fluorescent protein production in Bacillus subtilis; Veening JW et al.; The distinguishable cyan and yellow fluorescent proteins (CFP and YFP) enable the simultaneous in vivo visualization of different promoter activities . Here, we report new cloning vectors for the construction of cfp and yfp fusions in Bacillus subtilis . By extending the N-terminal portions of previously described CFP and YFP variants, 20- to 70-fold-improved fluorescent-protein production was achieved . Probably, the addition of sequences encoding the first eight amino acids of the N-terminal part of ComGA of B . subtilis overcomes the slow translation initiation that is provoked by the eukaryotic codon bias present in the original cfp and yfp genes . Using these new vectors, we demonstrate that, within an isogenic population of sporulating B . subtilis cells, expression of the abrB and spoIIA genes is distinct in individual cells.

Biodegradation, 2004 Oct, 15(5), 281 - 7
Effects of Bacillus subtilis O9 biosurfactant on the bioremediation of crude oil-polluted soils; Cubitto MA et al.; The application of a surfactant from Bacillus subtilis O9 (Bs) on the bioremediation of soils polluted with crude oil was assayed in soil microcosms under laboratory conditions . Three concentrations of biosurfactant were assayed (1.9, 19.5, and 39 mg kg(-1) soil) . Microcosms without biosurfactant were prepared as controls . During the experiment, the crude oil-degrading bacterial population, the aliphatic and aromatic hydrocarbons were monitored in each microcosm . The results indicated that applying Bs did not negatively affect the hydrocarbon-degrading microbial population Concentrations of 19 and 19.5mg (Bs) per kilogram of soil stimulated the growth of the population involved in the crude oil degradation, and accelerated the biodegradation of the aliphatic hydrocarbons . However, none of the assayed Bs concentrations stimulated aromatic hydrocarbon degradation.

J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Nov 25, 811(2), 243 - 51
Preparative high-performance liquid chromatographic separation and analysis of the Maltacine complex - a family of cyclic peptide antibiotics from Bacillus subtilis; Hagelin G et al.; Purification of secondary metabolites from fermentation broths can be a challenging task both due to the complexity of the medium, inherently unstable molecular structures or by the action of enzymes present in the fermentation broth leading to poor isolation yield and loss of antibiotic activity . A combination of different purification techniques has usually been used to arrive at acceptable purities for characterisation of the target molecules . Due to rapid decay of antimicrobial activity a rapid preparative high-performance liquid chromatography (HPLC) method was developed that provided separation and resolution of a family of 18 closely related cyclic peptides within 110 min with minimal loss of activity . Characterisation of the peptides with LC-MS, UV/IR spectroscopy and amino acid analysis disclosed 20 different peptides with cyclic structures (lactones) with molecular weights between 1447.7 and 1519.8 Da . No peptide antibiotics with identical molecular weights have previously been reported in the literature, which lead us to conclude that this peptide complex has not been discovered before . We have named them Maltacines.

J Pharm Biomed Anal, 2004 Nov 15, 36(3), 643 - 7
Interference of isonicotinyl hydrazone in the microbiological analysis of rifampicin from anti-tuberculosis FDC products containing isoniazid; Mariappan TT et al.; Microbiological assay is a sensitive method for the estimation of rifampicin (R) . In the present study, interference due to isonicotinyl hydrazone (HYD), an interaction product of R and isoniazid (H), was checked during microbiological analysis of R, employing Bacillus subtilis and Sarcina lutea . The assays were done by disc diffusion method . Both R and HYD showed linear log response curves in the range of 0.01-10microg . In the presence of HYD, R was overestimated when tested against S . lutea and underestimated in case of B . subtilis . The same extent and type of interference was observed on assay of a marketed anti-tuberculosis fixed-dose combination product, subjected to accelerated stability testing (40 degrees C/75% RH) for 1 month . This means that response of organisms used in microbiological assay of R might vary in the presence of HYD, with possibility of incorrect conclusions . Therefore, the study suggests that before a microbiological method involving a particular organism is extended to the determination of R in FDC formulations containing H, it should be tested for the influence of HYD and used only if non-interfering.

FEMS Microbiol Lett, 2004 Nov 15, 240(2), 171 - 9
Characterization of DegU, a response regulator in Listeria monocytogenes, involved in regulation of motility and contributes to virulence; Knudsen GM et al.; The degU (lmo2515) gene encodes a putative response regulator in the food-borne pathogen Listeria monocytogenes . It has 63% amino acid identity to the DegU response regulator of Bacillus subtilis . We have characterized the degU gene product in L . monocytogenes EGD by generation of a deletion mutant . The DeltadegU mutant was found to be non-motile in motility plate assay and no flagellin was detected . The mutant was attenuated in challenge of mice . Northern blot analysis suggested that the degU gene product is a transcriptional activator of the flagellin gene, flaA, at 25 degrees C . However, the degU gene product had no influence on the transcription of prfA encoding the major virulence regulator, PrfA . The results indicate that the putative DegU response regulator is a pleiotropic regulator involved in expression of both motility at low temperature and in vivo virulence in mice.

FEMS Microbiol Lett, 2004 Nov 15, 240(2), 145 - 9
A divIVA null mutant of Staphylococcus aureus undergoes normal cell division; Pinho MG et al.; DivIVA is involved in placement of the division septum and chromosome segregation in Bacillus subtilis and it plays important roles in cell division or morphogenesis in diverse Gram-positive bacteria . In Staphylococcus aureus, DivIVA is localized at the division septum, but it does not colocalize with the chromosomal origin of replication, as labeled with SpoOJ protein . Unexpectedly, a divIVA null mutant is not impaired in growth, nor is it affected in chromosome segregation or cell morphology.

Plasmid, 2004 Nov, 52(3), 212 - 7
Plasmid transfer in bacilli by a self-transmissible plasmid p19 from a Bacillus subtilis soil strain; Poluektova EU et al.; The cryptic 95-kb plasmid p19 of the Bacillus subtilis 19 soil strain promotes the transfer of a small kanamycin resistance plasmid pUB110 . To facilitate direct selection for p19 transfer, a plasmid derivative carrying the chloramphenicol resistance gene was constructed . The frequency of transfer of the large plasmid between cells of B . subtilis 19 approached 100% but was more than two orders of magnitude lower when the strain B . subtilis 168 was a recipient . However, when the restriction-deficient strain B . subtilis 168 was a recipient, the transfer efficiency was almost completely recovered . The effectiveness of pUB110 mobilization was virtually not altered in all these cases . pC194 was not mobilized by p19 . The kinetics of p19 conjugative transfer is also presented.

Appl Microbiol Biotechnol . 2004 Oct 23; {Epub ahead of print}
Biodegradation of an endocrine-disrupting chemical, di-2-ethylhexyl phthalate, by Bacillus subtilis No . 66; Quan CS et al.; A bacterial strain capable of rapidly degrading di-2-ethylhexyl phthalate (DEHP) was isolated from soil and identified as Bacillus subtilis . The organism also utilized di-butyl phthalate, di-ethyl phthalate, di-pentyl phthalate, di-propyl phthalate, and phthalic acid as sole carbon sources; and their biodegradation ratio was over 99%, when the incubation was performed for 5 days at 30show $132# degrees show $132#C . The microorganism degraded di-2-ethylhexyl phthalate and di-butyl phthalate through the intermediate formation of mono-2-ethylhexyl phthalate and mono-butyl phthalate, which were then metabolized to phthalic acid and further by a protocatechuate pathway, as evidenced by oxygen uptake studies and GC-MS analysis . The decontamination of soil polluted with di-2-ethylhexyl phthalate by B . subtilis was investigated . Experimental results showed that the strain could degrade about 80% of 5 mM DEHP simply by adding 8% culture medium to soil, indicating that the degradation can occur even when other organisms are present.

J Biol Chem, 2005 Jan 14, 280(2), 1669 - 77 Epub 2004 Oct 29.
Determination of selectivity and efficacy of Fatty Acid synthesis inhibitors; Kodali S et al.; Type II fatty acid synthesis (FASII) is essential to bacterial cell viability and is a promising target for the development of novel antibiotics . In the past decade, a few inhibitors have been identified for this pathway, but none of them lend themselves to drug development . To find better inhibitors that are potential drug candidates, we developed a high throughput assay that identifies inhibitors simultaneously against multiple targets within the FASII pathway of most bacterial pathogens . We demonstrated that the inverse t((1/2)) value of the FASII enzyme-catalyzed reaction gives a measure of FASII activity . The K(m) values of octanoyl-CoA and lauroyl-CoA were determined to be 1.1 +/- 0.3 and 10 +/- 2.7 mum in Staphylococcus aureus and Bacillus subtilis, respectively . The effects of free metals and reducing agents on enzyme activity showed an inhibition hierarchy of Zn(2+) > Ca(2+) > Mn(2+) > Mg(2+); no inhibition was found with beta-mercaptoethanol or dithiothreitol . We used this assay to screen the natural product libraries and isolated an inhibitor, bischloroanthrabenzoxocinone (BABX) with a new structure . BABX showed IC(50) values of 11.4 and 35.3 mug/ml in the S . aureus and Escherichia coli FASII assays, respectively, and good antibacterial activities against S . aureus and permeable E . coli strains with minimum inhibitory concentrations ranging from 0.2 to 0.4 mug/ml . Furthermore, the effectiveness, selectivity, and the in vitro and in vivo correlations of BABX as well as other fatty acid inhibitors were elucidated, which will aid in future drug discovery.

Vet Res Commun, 2004 Aug, 28(6), 515 - 24
Pharmacokinetics and bioavailability of florfenicol following intravenous, intramuscular and oral administrations in rabbits; El-Aty AM et al.; This study examined the disposition kinetics and bioavailability of florfenicol after intravenous (i.v.), intramuscular (i.m.) and oral administration to rabbits at a dose of 30 mg/kg BW . Serial blood samples were collected through an indwelling catheter intermittently for 24 h for various routes . Plasma antibacterial concentrations were determined using a microbiological assay method with Bacillus subtilis ATCC 6633 as a reference organism . Plasma concentration-time data generated in the present study were analysed by non-compartmental methods based on statistical moment theory . Following i.v . administration, the overall elimination half-life (t1/2beta) was 1.54 h, mean residence time (MRT) was 1.69 h, mean volume of distribution at steady-state (Vdss) was 0.57 L/kg, and total body clearance (Cltot) was 0.34 L/kg/h . After i.m . and oral dosing, the terminal part of the curve should correspond to the absorption phase, instead of to the elimination phase, with terminal half-lives of 3.01 and 2.57 h, respectively . The mean absorption time (MAT) was 2.65 h for i.m . and 2.01 h for oral administration . Elimination rate constants differed with i.v., i.m . and oral administrations, suggesting a flip-flop situation . The observed mean peak plasma concentrations (Cmax obs) were 21.65 and 15.14 microg/ml achieved at a post-injection time (Tmax obs) of 0.5 h following i.m . and oral dosing, respectively . The absolute systemic availabilities were 88.25% and 50.79%, respectively, and the extent of plasma protein binding percent was 11.65%.

Biosci Biotechnol Biochem, 2004 Oct, 68(10), 2007 - 23
Protein traffic for secretion and related machinery of Bacillus subtilis; Yamane K et al.; Gram-positive sporulating Bacillus subtilis secretes high levels of protein . Its complete genome sequence, published in 1997, encodes 4,106 proteins . Bioinformatic searches have predicted that about half of all B . subtilis proteins are related to the cell membrane through export to the extracellular medium, insertion, and attachment . Key features of the B . subtilis protein secretion machinery are the absence of an Escherichia coli SecB homolog and the presence of an SRP (signal recognition particle) that is structurally rather similar to human SRP . In addition, B . subtilis contains five type I signal peptidases (SipS, T, U, V, and W) . Our in vitro assay system indicated that co-operation between the SRP-protein targeting system to the cell membrane and the Sec protein translocation machinery across the cytoplasmic membrane constitutes the major protein secretion pathway in B . subtilis . Furthermore, the function of the SRP-Sec pathway in protein localization to the cell membrane and spore was analyzed.

Acta Crystallogr D Biol Crystallogr, 2004 Nov, 60(Pt 11), 2031 - 4 Epub 2004 Oct 20.
Expression, purification and preliminary X-ray analysis of crystals of Bacillus subtilis glutamate racemase; Taal MA et al.; Glutamate racemase (MurI, RacE; E.C.5.1.1.3) catalyses the cofactor-independent conversion of L-glutamate to D-glutamate, an essential step in the synthesis of components of the bacterial cell wall . The gene for RacE from Bacillus subtilis has been cloned and the protein expressed in Escherichia coli, purified and crystallized in the presence of L-glutamate using the hanging-drop method of vapour diffusion with diammonium tartrate as the precipitant . The crystals belong to the monoclinic space group C2, with approximate unit-cell parameters a = 133.6, b = 60.1, c = 126.2 A, beta = 117.6 degrees . Consideration of the possible values of V(M) suggests that the asymmetric unit contains either two (V(M) = 3.75 A(3) Da(-1)) or three (V(M) = 2.5 A(3) Da(-1)) subunits . The crystals diffract X-rays to at least 2.1 A resolution on a synchrotron-radiation source and are suitable for structural studies . Determination of the structure may provide insight into the molecular basis of substrate recognition and catalysis by this enzyme.

Bioorg Med Chem Lett, 2004 Dec 6, 14(23), 5943 - 6
Unprecedented olefin-dependent histidine-kinase inhibitory of zerumbone ring-opening material; Kitayama T et al.; Zerumbone ring-opening derivative, 4 (10E/10Z=3/2), inhibited autophosphorylation of the essential histidine-kinase YycG existing in Bacillus subtilis constituting a two-component system (TCS) . Generation of 4E-form could be regulated chemically using the difference from the ring-opening reactivity of the precursor forming of 4 and pure 4E was isolated . The stereoisomer, 4E, showed main inhibition activity of autophosphorylation of YycG (IC(50)=63.5 microM).

Indoor Air, 2004 Dec, 14(6), 385 - 93
Evaluation of a high-volume portable bioaerosol sampler in laboratory and field environments; An HR et al.; This study investigated the physical and biological performances of a portable centrifugal sampler for viable bioaerosols, RCS High Flow . The performance of the test sampler in the laboratory and field environments was compared with that of a reference sampler, BioSampler . The laboratory experiments with non-biological particles of KCl, oleic acid, and polystyrene latex showed that the test sampler's collection efficiency is about 22% for 0.5-microm particles, 48% for 1.0-microm particles, and approximately 100% for particles of 2.5 microm and larger . These tests indicated that the sampler's cut-off size (d50) was 1.1 microm . The test sampler's physical performance when collecting the spores and vegetative cells of Bacillus subtilis var . niger (BG) was similar to that when collecting non-biological particles of the same size . In the laboratory tests, the RCS High Flow sampler was found to enumerate approximately 40% of BG spores and cells relative to the reference sampler, BioSampler . A similar ratio was found during testing in an indoor environment . This ratio decreased to below 10% when testing was performed in an outdoor environment . We hypothesize that the test sampler's underperformance compared with the BioSampler could be caused by the damage to sensitive microorganisms during the collection process, test sampler's sensitivity to wind direction and speed as well as break-up of particle aggregates during the impingement process in BioSampler, which resulted in more colony-forming units (CFUs) being counted by the reference sampler than by the test sampler . Overall, when the RCS High Plus is used to sample culturable airborne microorganisms, the results obtained may have to be adjusted to avoid potential underestimation of microorganism concentration in the air . PRACTICAL IMPLICATIONS: The laboratory testing of the RCS High Flow bioaerosol sampler showed that the sampler collects 1 microm particles and larger with an efficiency of 50% and higher; the efficiency reaches approximately 100% for particles of 2.5 microm and larger . When considering this result, most of the airborne fungal spores would be collected with an efficiency between 50 and 100% . The field testing, however, indicated that the RCS High Flow sampler recovered from 41 to 71% of microorganisms collected relative to the reference sampler, Biosampler, and this ratio dropped to below 5% during outdoor testing . Thus, while the RCS High Flow sampler offers certain advantages over other samplers for viable bioaerosols--it is lightweight, battery operated, and collects viable microorganisms at a high flow rate directly on agar media, the results obtained may have to be adjusted to avoid potential underestimation of microorganism concentration in the air, especially if sampling is performed outdoors.

EMBO J, 2004 Nov 10, 23(22), 4473 - 83 Epub 2004 Oct 21.
An alternative strategy for bacterial ribosome synthesis: Bacillus subtilis rRNA transcription regulation; Krasny L et al.; As an approach to the study of rRNA synthesis in Gram-positive bacteria, we characterized the regulation of the Bacillus subtilis rrnB and rrnO rRNA promoters . We conclude that B . subtilis and Escherichia coli use different strategies to control rRNA synthesis . In contrast to E . coli, it appears that the initiating NTP for transcription from B . subtilis rRNA promoters is GTP, promoter strength is determined primarily by the core promoter (-10/-35 region), and changes in promoter activity always correlate with changes in the intracellular GTP concentration . rRNA promoters in B . subtilis appear to be regulated by changes in the initiating NTP pools, but in some growth transitions, changes in rRNA promoter activity are also dependent on relA, which codes for ppGpp synthetase . In contrast to the situation for E . coli where ppGpp decreases rRNA promoter activity by directly inhibiting RNA polymerase, it appears that ppGpp may not inhibit B . subtilis RNA polymerase directly . Rather, increases in the ppGpp concentration might reduce the available GTP pools, thereby modulating rRNA promoter activity indirectly.

Metab Eng, 2004 Oct, 6(4), 277 - 84
The phosphoenolpyruvate carboxykinase also catalyzes C3 carboxylation at the interface of glycolysis and the TCA cycle of Bacillus subtilis; Zamboni N et al.; Quantitative physiological characterization and isotopic tracer experiments revealed that pyruvate kinase mutants of Bacillus subtilis produced significantly more CO(2) from glucose in the tricarboxylic acid cycle than is explained by the remaining conversion of phosphoenolpyruvate (PEP) to pyruvate catalyzed by the phosphotransferase system . We show here that this additional catabolic flux into the tricarboxylic acid cycle was catalyzed by the PEP carboxykinase . In contrast to its normal role in gluconeogenesis, PEP carboxykinase can operate in the reverse direction from PEP to oxaloacetate upon knockout of pyruvate kinase in a riboflavin-producing B . subtilis strain and in wild-type 168 . At least in the industrial strain, we demonstrate the additional capacity of PEP carboxykinase to function as a substitute anaplerotic reaction when the normal pyruvate carboxylase is inactivated . Presumably as a consequence of the unfavorable kinetics of an ATP-synthesizing anaplerotic PEP carboxykinase reaction, such pyruvate carboxylase mutants grow slowly or, as in the case of wild-type 168, not at all.

Biochemistry, 2004 Oct 26, 43(42), 13541 - 8
Heme O synthase and heme A synthase from Bacillus subtilis and Rhodobacter sphaeroides interact in Escherichia coli; Brown BM et al.; Cytochrome c oxidase requires multiple heme and copper cofactors to catalyze the reduction of molecular oxygen to water . Although significant progress has been made in understanding the transport and incorporation of the copper ions, considerably less is known about the trafficking and insertion of the heme cofactors . Heme O synthase (HOS) and heme A synthase (HAS) from Rhodobacter sphaeroides (Cox10 and Cox15, respectively) and Bacillus subtilis (CtaB and CtaA, respectively) have been cloned and expressed in Escherichia coli . Our results demonstrate that HOS copurifies with HAS and that HAS copurifies with HOS, indicating that HOS and HAS interact and may form a physiologically relevant complex in vivo . Consistent with this hypothesis, the presence of HAS alters the total level of farnesylated hemes, providing further evidence that HOS and HAS interact . Our current working model is that HOS and HAS form a complex and that heme O is transferred directly from HOS to HAS . Because of the strong sequence similarity and evolutionary relationship between R . sphaeroides and mitochondria, our data suggest that this complex may form in eukaryotes as well.

Appl Microbiol Biotechnol . 2004 Oct 15; {Epub ahead of print}
Cloning, expression, and characterization of a glycoside hydrolase family 86 beta-agarase from a deep-sea Microbulbifer-like isolate; Ohta Y et al.; The gene for a novel beta-agarase from a deep-sea Microbulbifer-like isolate was cloned and sequenced . It encoded a mature protein of 126,921 Da (1,146 amino acids), which was a modular protein including two tandem carbohydrate-binding module (CBM)-like sequences and a catalytic module . The catalytic module resembled a glycoside hydrolase family 86 beta-agarase, AgrA, from Pseudoalteromonas atlantica T6c with 31% amino acid identity . Its recombinant agarase was hyper-produced extracellularly using Bacillus subtilis as the host and purified to homogeneity . The activity and stability were strongly enhanced by CaCl(2) . The maximal enzyme activity was observed at 45show $132# degrees show $132#C and pH 7.5 in the presence of 10 mM CaCl(2) . The enzyme was an endo-type beta-agarase and degraded agarose and agarose oligosaccharides more polymerized than hexamers to yield neoagarohexaose as the main product . This is the first glycoside hydrolase family 86 enzyme to be homogeneously purified and characterized.

J Bacteriol, 2004 Nov, 186(21), 7450 - 5
Increased stability of bmr3 mRNA results in a multidrug-resistant phenotype in Bacillus subtilis; Ohki R et al.; A spontaneous mutant isolated in the presence of a high concentration of puromycin acquired a multidrug-resistant phenotype . Expression of the bmr3 gene was dramatically increased . A base substitution, T to A at the +4 position, detected in the mutant resulted in the stabilization of bmr3 mRNA.

J Bacteriol, 2004 Nov, 186(21), 7084 - 90
Multicopy plasmids affect replisome positioning in Bacillus subtilis; Wang JD et al.; The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells . We visualized the location of a multicopy plasmid, pHP13, in living cells of Bacillus subtilis using an array of lac operators and LacI-green fluorescent protein (GFP) . In the majority of cells, plasmids appeared to be highly mobile and randomly distributed . In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole . We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP . Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell . Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid . In contrast, replisome foci in plasmid-free cells were relatively stationary . Our results indicate that in B . subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid . Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location of oriC at the time of initiation of replication.

J Vet Med A Physiol Pathol Clin Med, 2004 Aug, 51(6), 306 - 12
Field evaluation of the effect of a probiotic-containing Bacillus licheniformis and Bacillus subtilis spores on the health status, performance, and carcass quality of grower and finisher pigs; Alexopoulos C et al.; The aim of the study was to assess the efficacy of BioPlus 2B, a probiotic containing Bacillus licheniformis and B . subtilis spores, on the health status and productivity of pigs, during weaning, growing and finishing stages of growth . On a commercial farrow-to-finish farm, five experimental groups were formed, each of 54 weaned piglets . The pigs of the first group (double controls) received normal feed with no probiotic and the pigs of the second group (untreated controls) received BioPlus 2B only during the weaning stage . The pigs of the third, the fourth and the fifth group received the same as the second group feed but, at the growing and at a part of the finishing stages, supplemented with three different doses of Bioplus 2B, a low, medium and high dose, respectively . The results have shown that, compared with the double controls, BioPlus 2B-treated pigs had a lower morbidity and mortality during the whole trial period, compared with the double controls (range from 9.26 to 14.81% versus 25.93% and from 0.00 to 3.70% versus 11.1%, respectively), as a result of the lower incidence of post-weaning diarrhoea due mainly to Escherichia coli . Weight gain, feed conversion ratio and carcass quality of the BioPlus 2B-treated pigs were significantly improved compared with the double controls, whilst the beneficial effects of the probiotic were more pronounced when the medium and high doses were used.

Biochem J . 2004 Oct 14; {Epub ahead of print}
An examination of mitochondrial protein targeting of heme synthetic enzymes . In vivo identification of three functional heme-responsive motifs in 5-aminolevulinate synthase; Dailey TA et al.; The initial and the terminal three enzymes of the mammalian heme biosynthetic pathway are nuclear encoded, cytoplasmically synthesized and post-translationally translocated into the mitochondrion . The first enzyme, 5-aminolevulinate synthase (ALAS), occurs as isoenzymes encoded on different chromosomes and is synthesized either as a housekeeping protein (ALAS1) in all non-erythroid cell types, or only in differentiating erythroid precursor cells (ALAS2) . Both ALAS proteins possess mitochondrial targeting sequences that have putative heme-binding motifs . In the current study evidence is presented demonstrating that two heme-binding motifs in the leader sequence as well as one present in the amino terminus of the mature ALAS1 function in vivo in the heme-regulated translocation of ALAS1 . Coproporphyrinogen oxidase (CPO), the antepenultimate pathway enzyme, possesses a leader sequence that is approximately 120 residues long . In contrast to an earlier report suggesting that only 30 residues were required for translocation of CPO, we report that the complete leader is necessary for translocation and that this process is not heme sensitive in vivo . Protoporphyrinogen oxidase (PPO) lacks a typical mitochondrial targeting leader sequence and was found to be effectively targeted by just seventeen amino-terminal residues . Bacillus subtilis PPO, which is very similar to human PPO at its amino-terminal end, is not targeted to the mitochondrion when expressed in mammalian cells, demonstrating that translocation is highly specific with regard to both the length and spacing of charged residues in this targeting region . Ferrochelatase, the terminal enzyme, possesses a typical amino-terminal leader sequence and no evidence was found for a role for the carboxyl-terminus in mitochondrial targeting.

J Appl Microbiol, 2004, 97(5), 992 - 1000
Cereulide, the emetic toxin of Bacillus cereus, is putatively a product of nonribosomal peptide synthesis; Toh M et al.; AIMS: To determine if cereulide, the emetic toxin produced by Bacillus cereus, is produced by a nonribosomal peptide synthetase (NRPS) . METHODS AND RESULTS: NC Y, an emetic strain of Bacillus cereus, was examined for a NRPS gene using PCR with primers recognizing a fragment of a NRPS gene from the cyanobacterium Microcystis . The amplicon was sequenced and compared with other gene sequences using BLAST analysis, which showed that the amplicon from strain NC Y was similar in sequence to peptide synthetase genes in other micro-organisms, including Bacillus subtilis and B . brevis, while no such sequence was found in the complete genome sequence of a nonemetic strain of B . cereus . Specific PCR primers were then designed and used to screen 40 B . cereus isolates previously implicated in outbreaks of foodborne illness . The isolates were also screened for toxin production using the MTT cell cytotoxicity assay . PCR and MTT assay screening of the B . cereus isolates revealed a high correlation between the presence of the NRPS gene and cereulide production . CONCLUSIONS: The results indicate that cereulide is produced by a NRPS complex . SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide evidence identifying the mechanism of production of cereulide, the emetic toxin of B . cereus . The PCR primers developed in the study allow determination of the potential for cereulide production among isolates of B . cereus.

Nucleic Acids Res, 2004 Oct 11, 32(18), 5392 - 7 Print 2004.
Conservation of adjacency as evidence of paralogous operons; Janga SC et al.; Most of the analyses on the conservation of gene order are limited to orthologous genes . However, the organization of genes into operons might also result in the conservation of gene order of paralogous genes . Thus, we sought computational evidence that conservation of gene order of paralogous genes represents another level of conservation of genes in operons . We found that pairs of genes within experimentally characterized operons of Escherichia coli K12 and Bacillus subtilis tend to have more adjacently conserved paralogs than pairs of genes at transcription unit boundaries . The fraction of same strand gene pairs corresponding to conserved paralogs averages 0.07 with a maximum of 0.22 in Borrelia burgdorferi . The use of evidence from the conservation of adjacency of paralogous genes can improve the prediction of operons in E.coli K12 by approximately 0.27 over predictions using conservation of adjacency of orthologous genes alone.

J Biol Chem, 2004 Dec 17, 279(51), 52840 - 9 Epub 2004 Oct 07.
EPR spectroscopic characterization of the manganese center and a free radical in the oxalate decarboxylase reaction: identification of a tyrosyl radical during turnover; Chang CH et al.; Several molecular mechanisms for cleavage of the oxalate carbon-carbon bond by manganese-dependent oxalate decarboxylase have recently been proposed involving high oxidation states of manganese . We have examined the oxalate decarboxylase from Bacillus subtilis by electron paramagnetic resonance in perpendicular and parallel polarization configurations to test for the presence of such species in the resting state and during enzymatic turnover . Simulation and the position of the half-field Mn(II) line suggest a nearly octahedral metal geometry in the resting state . No spectroscopic signature for Mn(III) or Mn(IV) is seen in parallel mode EPR for samples frozen during turnover, consistent either with a large zero-field splitting in the oxidized metal center or undetectable levels of these putative high-valent intermediates in the steady state . A narrow, featureless g = 2.0 species was also observed in perpendicular mode in the presence of substrate, enzyme, and dioxygen . Additional splittings in the signal envelope became apparent when spectra were taken at higher temperatures . Isotopic editing resulted in an altered line shape only when tyrosine residues of the enzyme were specifically deuterated . Spectral processing confirmed multiple splittings with isotopically neutral enzyme that collapsed to a single prominent splitting in the deuterated enzyme . These results are consistent with formation of an enzyme-based tyrosyl radical upon oxalate exposure . Modestly enhanced relaxation relative to abiological tyrosyl radicals was observed, but site-directed mutagenesis indicated that conserved tyrosine residues in the active site do not host the unpaired spin . Potential roles for manganese and a peripheral tyrosyl radical during steady-state turnover are discussed.

Vaccine, 2004 Oct 22, 22(31-32), 4139 - 43
Oral priming of mice by recombinant spores of Bacillus subtilis; Ciabattini A et al.; Recombinant Bacillus subtilis spores were employed as a vaccine delivery system in a heterologous mucosal priming-parenteral boosting vaccination strategy in the mouse model . BALB/c and C57BL/6 mice were orally immunised with recombinant spores expressing tetanus toxin fragment C (TTFC) fused to the spore outer coat protein CotB, and then subcutaneously boosted with soluble TTFC (without adjuvant) . Two weeks after boosting, a significantly higher serum TTFC-specific IgG response was stimulated in mice primed with recombinant spores (antibody concentration of 2600 +/- 915 in C57BL/6 and 1200 +/- 370 ng/ml in BALB/c) compared to mice inoculated with wild type spores (650 +/- 250 and 250 +/- 130 ng/ml, respectively) . IgG subclass analysis showed a prevalence of IgG1 and IgG2b, indicative of a Th2 type of immune response . Oral administration of recombinant spores stimulated also a significant local TTFC-specific IgA response . These data show that recombinant spores of B . subtilis are able to prime the immune system by the oral route, and that a combined mucosal/parenteral strategy can stimulate both local and systemic antigen-specific immune responses.

Science, 2004 Oct 8, 306(5694), 275 - 9
A glycine-dependent riboswitch that uses cooperative binding to control gene expression; Mandal M et al.; We identified a previously unknown riboswitch class in bacteria that is selectively triggered by glycine . A representative of these glycine-sensing RNAs from Bacillus subtilis operates as a rare genetic on switch for the gcvT operon, which codes for proteins that form the glycine cleavage system . Most glycine riboswitches integrate two ligand-binding domains that function cooperatively to more closely approximate a two-state genetic switch . This advanced form of riboswitch may have evolved to ensure that excess glycine is efficiently used to provide carbon flux through the citric acid cycle and maintain adequate amounts of the amino acid for protein synthesis . Thus, riboswitches perform key regulatory roles and exhibit complex performance characteristics that previously had been observed only with protein factors.

J Biol Chem, 2004 Dec 17, 279(51), 53789 - 97 Epub 2004 Oct 07.
Two novel mutant human adenylosuccinate lyases (ASLs) associated with autism and characterization of the equivalent mutant Bacillus subtilis ASL; Sivendran S et al.; An Australian patient with autism was found to be heterozygous for two mutations in the gene encoding adenylosuccinate lyase (ASL), resulting in the protein mutations E80D and D87E . The patient's mother carried only the E80D mutation . The equivalent positions are 62 and 69 in Bacillus subtilis ASL . Although both human and B . subtilis enzymes normally have Asp at position 87 (or 69), the B . subtilis ASL has Ile and Asp at 62 and 65, respectively, whereas human ASL has Glu and Arg at the equivalent positions . We have constructed, expressed, and purified the double mutant I62E/D65R as a "humanized" normal B . subtilis enzyme to compare with enzymes with a single mutation at position 62 (I62D/D65R), at position 69 (I62E/D65R/D69E), or at both positions (I62D/D65R/D69E) . V(max) for conversion of adenylosuccinate to AMP and fumarate is 0.57 micromol/min/mg for I62E/D65R, 0.064 micromol/min/mg for I62D/D65R, 0.27 micromol/min/mg for I62E/D65R/D69E, and 0.069 micromol/min/mg for I62D/D65R/D69E . The K(m) for adenylosuccinate is elevated in the X62D mutants, and I62D/D65R is the least stable of these ASLs at 37 degrees C . The CD spectra of mutant and wild type enzymes are similar; thus, there are no appreciable structural changes . Clearly the Asp(62) causes the most drastic effect on ASL function, whereas the Glu(69) mutation produces only modest change . These results emphasize the importance of expanding tests for ASL deficiency to individuals with developmental delay of any severity, including individuals with autistic spectrum disorder . This study further demonstrates the usefulness of the B . subtilis ASL as a model to mimic the defective enzyme in ASL deficiency.

Microbiology, 2004 Oct, 150(Pt 10), 3441 - 9
GerE-independent expression of cotH leads to CotC accumulation in the mother cell compartment during Bacillus subtilis sporulation; Baccigalupi L et al.; Evidence is presented that expression of the cotH gene, whose product is required for the correct assembly of the Bacillus subtilis spore coat, is negatively controlled by the transcriptional regulator GerE . Mutations in the GerE-box, present in the cotH promoter region, increased expression of this gene, which also remained elevated during late stages of sporulation, when in wild-type cells cotH is normally turned off . Such alterations of cotH expression did not significantly affect spore coat structure or function but caused the accumulation of CotC molecules in the mother cell compartment, most likely as a consequence of CotH-mediated protection of CotC.

J Biochem Mol Biol, 2004 May 31, 37(3), 298 - 303
Nano-scale proteomics approach using two-dimensional fibrin zymography combined with fluorescent SYPRO ruby dye; Choi NS et al.; In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE) . Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp . proteases . Namely, complex protein mixtures from Bacillus sp . DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel . The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis . Five intracellular fibrinolytic enzymes of Bacillus sp . DJ-4 were detected through 2-D FZ . The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID-TOF MS . Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis . Also, the extracellular proteases of Bacillus sp . DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established . This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).

Mol Microbiol, 2004 Oct, 54(2), 452 - 63
The midcell replication factory in Bacillus subtilis is highly mobile: implications for coordinating chromosome replication with other cell cycle events; Migocki MD et al.; During vegetative growth, rod-shaped bacterial cells such as Escherichia coli and Bacillus subtilis divide precisely at midcell . It is the Z ring that defines the position of the division site . We previously demonstrated that the early stages of chromosome replication are linked to midcell Z ring assembly in B . subtilis and proposed a direct role for the centrally located replication factory in masking and subsequently unmasking the midcell site for Z ring assembly . We now show that the replication factory is significantly more scattered about the cell centre than the Z ring in both vegetative cells and outgrown spores of B . subtilis . This finding is inconsistent with the midcell replication factory acting as a direct physical block to Z ring assembly . Time-lapse experiments demonstrated that the lower precision of replication factory positioning results from its high mobility around the cell centre . Various aspects of this mobility are presented and the results are discussed in the light of current views on the determinants of positional information required for accurate chromosome segregation and cell division.

Mol Microbiol, 2004 Oct, 54(2), 439 - 51
Distinctive genetic features exhibited by the Y-family DNA polymerases in Bacillus subtilis; Duigou S et al.; Translesional DNA polymerases form a large family of structurally related proteins, known as the Y-polymerases . Bacillus subtilis encodes two Y-polymerases, referred herewith as Pol Y1 and Pol Y2 . Pol Y1 was expressed constitutively and did not mediate UV mutagenesis . Pol Y1 overexpression increased spontaneous mutagenesis . This effect depended on Pol Y1 polymerase activity, Pol Y1 interaction with the beta-clamp, and did not require the presence of the RecA protein . In addition, Pol Y1 overexpression delayed cell growth at low temperature . The growth delay was mediated by Pol Y1 interaction with the beta-clamp but not by its polymerase activity, suggesting that an excess of Pol Y1 in the cell could sequester the beta-clamp . In contrast, Pol Y2 was expressed during the SOS response, and, in its absence, UV-induced mutagenesis was abolished . Upon Pol Y2 overproduction, both UV-induced and spontaneous mutagenesis were stimulated, and both depended on the Pol Y2 polymerase activity . However, UV mutagenesis did not appear to require the interaction of Pol Y2 with the beta-clamp whereas spontaneous mutagenesis did . In addition, Pol Y2-mediated spontaneous mutagenesis required the presence of RecA . Together, these results show that the regulation and the genetic requirements of the two B . subtilis Y-polymerases are different, indicating that they fulfil distinct biological roles . Remarkably, Pol Y1 appears to exhibit a mutator activity similar to that of Escherichia coli Pol IV, as well as an E . coli UmuD-related function in growth delay . Pol Y2 exhibits an E . coli Pol V-like mutator activity, but probably acts as a single polypeptide to bypass UV lesions . Thus, B . subtilis Pol Y1 and Pol Y2 exhibit distinctive features from the E . coli Y-polymerases, indicating that different bacteria have adapted different solutions to deal with the lesions in their genetic material.

Proteins . 2004 Oct 1;57(4):850-853 {Epub ahead of print}
Crystal structure of Bacillus subtilis YdaF protein: A putative ribosomal N-acetyltransferase; Brunzelle JS et al.; No abstract.

Macromol Biosci, 2004 Mar 15, 4(3), 324 - 9
Biodegradable water absorbent synthesized from bacterial poly(amino acid)s; Kunioka M; Biodegradable hydrogels prepared by gamma-irradiation from microbial poly(amino acid)s have been studied . pH-Sensitive hydrogels were prepared by the gamma-irradiation of poly(gamma-glutamic acid) (PGA) produced by Bacillus subtilis and poly(epsilon-lysine) (PL) produced by Streptomyces albulus in aqueous solutions . When the gamma-irradiation dose was 19 kGy or more, and the concentration of PGA in water was 2 wt.-% or more, transparent hydrogels could be produced . For the 19 kGy dose, the produced hydrogel was very weak, however, the specific water content (wt . of absorbed water/wt . of dry hydrogel) of this PGA hydrogel was approximately 3,500 . The specific water content decreased to 200, increasing when the gamma-irradiation dose was over 100 kGy . Under acid conditions or upon the addition of electrolytes, the PGA hydrogels shrunk . The PGA hydrogel was pH-sensitive and the change in the volume of the hydrogel depended on the pH value outside the hydrogel in the swelling medium . This PGA hydrogel was hydrodegradable and biodegradable . A new novel purifier reagent (coagulant), made from the PGA hydrogels, for contaminated turbid water has been found and developed by Japanese companies . A very small amount of this coagulant (only 2 ppm in turbid water) with poly(aluminum chloride) can be used for the purification of turbid water . A PL aqueous solution also can change into a hydrogel by gamma-irradiation . The specific water content of the PL hydrogel ranged from 20 to 160 depending on the preparation conditions . Under acid conditions, the PL hydrogel swelled because of the ionic repulsion of the protonated amino groups in the PL molecules . The rate of enzymatic degradation of the respective PL hydrogels by a neutral protease was much faster than the rate of simple hydrolytic degradation.

Biol Pharm Bull, 2004 Oct, 27(10), 1576 - 9
Antimicrobial activity of some pentacyclic triterpenes and their synthesized 3-O-lipophilic chains; Mallavadhani UV et al.; The major metabolites of Diopsyros melanoxylon viz . amyrins and ursolic acid and their lipophilic 3-O-fatty acid ester chains (C12-C18), which are synthesized now under mild esterification conditions in excellent yields (80-95%), were evaluated for their antimicrobial activity against a series of Gram positive and Gram negative bacteria . Significantly these compounds were found to exhibit potent activity against Gram negative bacteria Pseudomonas syringae (ATCC #13457) and fairly good activity against Gram positive bacteria, Bacillus sphaericus (ATCC #14577) and Bacillus subtilis (ATCC #6051).

Appl Environ Microbiol, 2004 Oct, 70(10), 6247 - 56
Comparative analysis of physical maps of four Bacillus subtilis (natto) genomes; Qiu D et al.; The complete SfiI and I-CeuI physical maps of four Bacillus subtilis (natto) strains, which were previously isolated as natto (fermented soybean) starters, were constructed to elucidate the genome structure . Not only the similarity in genome size and organization but also the microheterogeneity of the gene context was revealed . No large-scale genome rearrangements among the four strains were indicated by mapping of the genes, including 10 rRNA operons (rrn) and relevant genes required for natto production, to the loci corresponding to those of the B . subtilis strain Marburg 168 . However, restriction fragment length polymorphism and the presence or absence of strain-specific DNA sequences, such as the prophages SP beta, skin element, and PBSX, as well as the insertion element IS4Bsu1, could be used to identify one of these strains as a Marburg type and the other three strains as natto types . The genome structure and gene heterogeneity were also consistent with the type of indigenous plasmids harbored by the strains.

J Bacteriol, 2004 Oct, 186(20), 6830 - 6
Phosphorylation and RsbX-dependent dephosphorylation of RsbR in the RsbR-RsbS complex of Bacillus subtilis; Chen CC et al.; In the pathway that controls sigmaB activity, the RsbR-RsbS complex plays an important role by trapping RsbT, a positive regulator of sigmaB of Bacillus subtilis . We have proposed that at the onset of stress, RsbR becomes phosphorylated, resulting in an enhanced activity of RsbT towards RsbS . RsbT is then free to interact with and activate RsbU, which in turn ultimately activates sigmaB . In this study with purified proteins, we used mutant RsbR proteins to analyze the role of its phosphorylatable threonine residues . The results show that the phosphorylation of either of the two RsbT-phosphorylatable threonine residues (T171 and T205) in RsbR enhanced the kinase activity of RsbT towards RsbS . However, it appeared that RsbT preferentially phosphorylates T171 . We also present in vitro evidence that identifies RsbX as a potential phosphatase for RsbR T205.

J Biotechnol, 2004 Oct 19, 114(1-2), 209 - 17
Synthesis of levan in water-miscible organic solvents; Castillo E et al.; The synthesis of levan using a levansucrase from a strain of Bacillus subtilis was studied in the presence of the water-miscible solvents: acetone, acetonitrile and 2-methyl-2-propanol (2M2P) . It was found that while the enzyme activity is only slightly affected by acetone and acetonitrile, 2M2P has an activating effect increasing the total activity 35% in 40-50% (v/v) 2M2P solutions at 30 degrees C . The enzyme is highly stable in water at 30 degrees C; however, incubation in the presence of 15 and 50% (v/v) 2M2P reduced the half-life time to 23.6 and 1.8 days, respectively . This effect is reversed in 83% 2M2P, where a half-life time of 11.8 days is observed . The presence of 2M2P in the system increases the transfer/hydrolysis ratio of levansucrase . As the reaction proceeds with 10% (w/v) sucrose in 50/50 water/2M2P sucrose is converted to levan and an aqueous two-phase system (2M2P/Levan) is formed and more sucrose can be added in a fed batch mode . It is shown that high molecular weight levan is obtained as an hydrogel and may be easily recovered from the reaction medium . However, when high initial sucrose concentrations (40% (w/v) in 50/50 water/2M2P) are used, an aqueous two-phase system (2M2P/sucrose) is induce, where the synthesized levan has a similar molecular weight distribution as in water and remains in solution.

Microbiol Res, 2004, 159(3), 257 - 62
FT-IR spectroscopic characteristics of differently cultivated Bacillus subtilis; Filip Z et al.; Bacillus subtilis is an aerobic endospore forming bacterium widely spread in different environments . Because it represents a biological agent of some health relevance, its rapid detection and identification is highly desirable . By using FT-IR spectroscopy for this purpose slightly different characteristics were obtained from cell mass grown in differently composed cultural media, and harvested in different phases of bacterial growth . If cultivated uniformly, i.e., 24h at 30 degrees C in a minimum-strength nutrient broth, cell mass of B . subtilis delivered a well differentiated spectrum with major absorption bands of nucleic acid structures at 3300cm(-1), cell wall constituents at 3000-2800cm(-1), proteinaceous structures at 1660, 1544 and 1235cm(-1), and some aliphatic structural units at 1080cm(-1) . Attenuated total reflectance, and absorption/transmission scanning techniques, delivered structurally identical spectra but those obtained by the former technique were more expressed.

Genome Biol . 2004;5(10):R77 . Epub 2004 Sep 13.
Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species; Rey MW et al.; BACKGROUND: Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products . This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature . RESULTS: We determined the complete nucleotide sequence of the B . licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes . The B . licheniformis chromosome contains large regions that are colinear with the genomes of B . subtilis and Bacillus halodurans, and approximately 80% of the predicted B . licheniformis coding sequences have B . subtilis orthologs . CONCLUSIONS: Despite the unmistakable organizational similarities between the B . licheniformis and B . subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species . Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B . licheniformis genome . The availability of a completed genome sequence for B . licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.

J Forensic Sci, 2004 Sep, 49(5), 954 - 60
Stable isotope ratios as a tool in microbial forensics--Part 1 . Microbial isotopic composition as a function of growth medium; Kreuzer-Martin HW et al.; The stable isotope ratios of a seized pathogen culture could potentially reveal information about the environment in which the agent was produced . In this paper we describe general relationships between stable isotopes of carbon, nitrogen, and hydrogen in bacteriological culture media and spores of Bacillus subtilis, an endospore-forming soil bacterium . In numerous media that varied both in nutrient composition and water stable isotope ratios, medium to spore enrichment in carbon isotopes was 0.3 +/- 2.0% per thousand (parts per thousand), and in nitrogen, 4.5 +/- 0.7% per thousand . We achieved mass balance for the contribution of hydrogen isotopes from nutrients (70%) and water (30%) to spores in independent experiments by varying the isotope ratios of nutrients or water . A model was derived for predicting the isotope ratio values of spores from those in nutrients and water.

Photochem Photobiol . 2004 Aug 1; {Epub ahead of print}
Effects of the binding of alpha/beta-type small, acid-soluble spore proteins on the photochemistry of DNA in spores of Bacillus subtilis and in vitro; Douki T et al.; The main lesion produced in DNA by UVC irradiation of spores of Bacillus subtilis is 5 thyminyl-5,6-dihydrothymine (spore photoproduct (SP)) . In contrast, cyclobutane dimers (CPDs) and (6-4) photoproducts (6-4PPs) are the main photolesions in other cell types . The novel photochemistry of spore DNA is accounted for in part by its reduced hydration, but largely by the saturation of spore DNA with alpha/beta-type small, acid soluble proteins (SASP) . Using HPLC-mass spectrometry analysis of the photoproducts, we showed in wild-type B . subtilis spores that: 1) UVC irradiation generates almost exclusively SP with little if any CPDs and 6-4PPs; 2) the SP generated is ~99% the intra-strand derivative but ~1% is in the inter-strand form and 3) there is no detectable formation of the SP analog between adjacent C and T residues . UVC irradiation of spores lacking the majority of their alpha/beta-type SASP gave less SP than with wild-type spores and significant levels of CPDs and 6-4PPs . The binding of an alpha/beta-type SASP to isolated DNA either in dry films or in aqueous solution led to a large decrease in the yield of CPDs and 6-4PPs, and a concomitant increase in the yield of SP, although levels of inter-strand photoproducts were extremely low.

Biotechnol Prog, 2004 Sep-Oct, 20(5), 1605 - 7
Secret signatures inside genomic DNA; Arita M et al.; A simple, practical method to watermark short trademarks or signatures into genomic DNA is introduced . Since the marking method is biologically innocuous, it can be applied to all commercialized bacteria to help establish brand names for the engineered strains and to resolve legal disputes regarding gene-related patents . The first such strain of Bacillus subtilis is engineered and is ready to be distributed.

Anal Chem, 2004 Oct 1, 76(19), 5748 - 55
Glucose-sensitive holographic sensors for monitoring bacterial growth; Lee MC et al.; A glucose sensor comprising a reflection hologram incorporated into a thin, acrylamide hydrogel film bearing the cis-diol binding ligand, 3-acrylamidophenylboronic acid (3-APB), is described . The diffraction wavelength (color) of the hologram changes as the polymer swells upon binding cis-diols . The effect of various concentrations of glucose, a variety of mono- and disaccharides, and the alpha-hydroxy acid, lactate, on the holographic response was investigated . The sensor displayed reversible changes in diffraction wavelength as a function of cis-diol concentration, with the sensitivity of the system being dependent on the cis-diol tested . The effect of varying 3-APB concentration in the hydrogel on the holographic response to glucose was investigated, and maximum sensitivity was observed at a functional monomer concentration of 20 mol % . The potential for using this holographic sensor to detect real-time changes in bacterial cell metabolism was demonstrated by monitoring the germination and subsequent vegetative growth of Bacillus subtilis spores.

J Mol Biol, 2004 Oct 15, 343(2), 395 - 406
The structure and ligand binding properties of the B . subtilis YkoF gene product, a member of a novel family of thiamin/HMP-binding proteins; Devedjiev Y et al.; The crystal structure of the Bacillus subtilis YkoF gene product, a protein involved in the hydroxymethyl pyrimidine (HMP) salvage pathway, was solved by the multiwavelength anomalous dispersion (MAD) method and refined with data extending to 1.65 A resolution . The atomic model of the protein shows a homodimeric association of two polypeptide chains, each containing an internal repeat of a ferredoxin-like betaalphabetabetaalphabeta fold, as seen in the ACT and RAM-domains . Each repeat shows a remarkable similarity to two members of the COG0011 domain family, the MTH1187 and YBL001c proteins, the crystal structures of which were recently solved by the Northeast Structural Genomics Consortium . Two YkoF monomers form a tightly associated dimer, in which the amino acid residues forming the interface are conserved among family members . A putative small-ligand binding site was located within each repeat in a position analogous to the serine-binding site of the ACT-domain of the Escherichia coli phosphoglycerate dehydrogenase . Genetic data suggested that this could be a thiamin or HMP-binding site . Calorimetric data confirmed that YkoF binds two thiamin molecules with varying affinities and a thiamine-YkoF complex was obtained by co-crystallization . The atomic model of the complex was refined using data to 2.3 A resolution and revealed a unique H-bonding pattern that constitutes the molecular basis of specificity for the HMP moiety of thiamin.

FEMS Microbiol Lett, 2004 Oct 1, 239(1), 71 - 7
Analysis of the germination of spores of Bacillus subtilis with temperature sensitive spo mutations in the spoVA operon; Vepachedu VR et al.; A Bacillus subtilis strain with a base substitution in the ribosome-binding site of spoVAC was temperature sensitive (ts) in sporulation and spores prepared at the permissive temperature were ts in L-alanine-triggered germination, but not in germination with Ca2+-dipicolinic acid (DPA) or dodecylamine . Spores of a ts spo mutant with a missense mutation in the spoVAC coding region were not ts for germination with l-alanine, dodecylamine or Ca2+-DPA . These findings are discussed in light of the proposal that SpoVA proteins are involved not only in DPA uptake during sporulation, but also in DPA release during nutrient-mediated spore germination.

Biochim Biophys Acta, 2004 Oct 1, 1702(1), 125 - 8
Crystallization and preliminary X-ray diffraction studies of the metal-ion-mediated ternary complex of the HutP protein with L-histidine and its cognate RNA; Kumarevel TS et al.; HutP is an RNA-binding protein that regulates the expression of the Bacillus subtilis hut operon by binding to cis-acting regulatory sequences within hut mRNA, exclusively in the presence of L-histidine . We recently solved the crystal structure of a binary complex (HutP with an L-histidine analog) that revealed a novel RNA-binding fold, and identified the important residues that interact with the L-histidine analog . In addition, we have defined the minimal RNA binding segment that is required for HutP recognition . Interestingly, we showed that ternary complex formation depends on the availability of not only L-histidine but also divalent metal ions . Here we report the crystallization and preliminary X-ray diffraction analysis of the HutP ternary complex . The ternary complex was crystallized in the presence of Mg2+ along with L-histidine and hut mRNA, using the hanging drop vapor diffusion method . The crystal belongs to the R3 space group, with unit cell parameters a=b=75.30 A, c=133.8 A . A complete data set at 1.60 A was collected.

Biochemistry, 2004 Oct 5, 43(39), 12410 - 26
Thermodynamic and biophysical characterization of cytochrome P450 BioI from Bacillus subtilis; Lawson RJ et al.; Cytochrome P450 BioI (CYP107H1) from Bacillus subtilis is involved in the early stages of biotin synthesis . Previous studies have indicated that BioI can hydroxylate fatty acids and may also perform an acyl bond cleavage reaction {Green, A . J., Rivers, S . L., Cheesman, M., Reid, G . A., Quaroni, L . G., Macdonald, I . D . G., Chapman, S . K., and Munro, A . W . (2001) J . Biol . Inorg . Chem . 6, 523-533 . Stok, J . E., and De Voss, J . J . (2000) Arch . Biochem . Biophys . 384, 351-360} . Here we show novel binding features of P450 BioI--specifically that it binds steroids (including testosterone and progesterone) and polycyclic azole drugs with similar affinity to that for fatty acids (K(d) values in the range 0.1-160 microM) . Sigmoidal binding curves for titration of BioI with azole drugs suggests a cooperative process in this case . BioI as isolated from Escherichia coli is in a mixed heme iron spin state . Alteration of the pH of the buffer system affects the heme iron spin-state equilibrium (higher pH increasing the low-spin content) . Steroids containing a carbonyl group at the C(3) position induce a shift in heme iron spin-state equilibrium toward the low-spin form, whereas fatty acids produce a shift toward the high-spin form . Electron paramagnetic resonance (EPR) studies confirm the heme iron spin-state perturbation inferred from optical titrations with steroids and fatty acids . Potentiometric studies demonstrate that the heme iron reduction potential becomes progressively more positive as the proportion of high-spin heme iron increases (potential for low-spin BioI = -330 +/- 1 mV; for BioI as purified from E . coli (mixed-spin) = 228 +/- 2 mV; for palmitoleic acid-bound BioI = -199 +/- 2 mV) . Extraction of bound substrate-like molecule from purified BioI indicates palmitic acid to be bound . Differential scanning calorimetry studies indicate that the BioI protein structure is stabilized by binding of steroids and bulky azole drugs, a result confirmed by resonance Raman studies and by analysis of disruption of BioI secondary and tertiary structure by the chaotrope guanidinium chloride . Molecular modeling of the BioI structure indicates that a disulfide bond is present between Cys250 and Cys275 . Calorimetry shows that structural stability of the protein was altered by addition of the reductant dithiothreitol, suggesting that the disulfide is important to integrity of BioI structure.

Biochemistry, 2004 Oct 5, 43(39), 12390 - 409
Expression and characterization of the two flavodoxin proteins of Bacillus subtilis, YkuN and YkuP: biophysical properties and interactions with cytochrome P450 BioI; Lawson RJ et al.; The two flavodoxins (YkuN and YkuP) from Bacillus subtilis have been cloned, overexpressed in Escherichia coli and purified . DNA sequencing, mass spectrometry, and flavin-binding properties showed that both YkuN and YkuP were typical short-chain flavodoxins (158 and 151 amino acids, respectively) and that an error in the published B . subtilis genome sequence had resulted in an altered reading frame and misassignment of YkuP as a long-chain flavodoxin . YkuN and YkuP were expressed in their blue (neutral semiquinone) forms and reoxidized to the quinone form during purification . Potentiometry confirmed the strong stabilization of the semiquinone form by both YkuN and YkuP (midpoint reduction potential for oxidized/semiquinone couple = -105 mV/-105 mV) with respect to the hydroquinone (midpoint reduction potential for semiquinone/hydroquinone couple = -382 mV/-377 mV) . Apoflavodoxin forms were generated by trichloroacetic acid treatment . Circular dichroism studies indicated that flavin mononucleotide (FMN) binding led to considerable structural rearrangement for YkuP but not for YkuN . Both apoflavodoxins bound FMN but not riboflavin avidly, as expected for short-chain flavodoxins . Structural stability studies with the chaotrope guanidinium chloride revealed that there is moderate destabilization of secondary and tertiary structure on FMN removal from YkuN, but that YkuP apoflavodoxin has similar (or slightly higher) stability compared to the holoprotein . Differential scanning calorimetry reveals further differences in structural stability . YkuP has a lower melting temperature than YkuN, and its endotherm is composed of a single transition, while that for YkuN is biphasic . Optical and fluorimetric titrations with oxidized flavodoxins revealed strong affinity (K(d) values consistently <5 microM) for their potential redox partner P450 BioI, YkuN showing tighter binding . Stopped-flow reduction studies indicated that the maximal electron-transfer rate (k(red)) to fatty acid-bound P450 BioI occurs from YkuN and YkuP at approximately 2.5 s(-1), considerably faster than from E . coli flavodoxin . Steady-state turnover with YkuN or YkuP, fatty acid-bound P450 BioI, and E . coli NADPH-flavodoxin reductase indicated that both flavodoxins supported lipid hydroxylation by P450 BioI with turnover rates of up to approximately 100 min(-1) with lauric acid as substrate . Interprotein electron transfer is a likely rate-limiting step . YkuN and YkuP supported monohydroxylation of lauric acid and myristic acid, but secondary oxygenation of the primary product was observed with both palmitic acid and palmitoleic acid as substrates.

Nucleic Acids Res, 2004 Sep 23, 32(17), 4979 - 91 Print 2004.
Bipartite pattern discovery by entropy minimization-based multiple local alignment; Bi C et al.; Many multimeric transcription factors recognize DNA sequence patterns by cooperatively binding to bipartite elements composed of half sites separated by a flexible spacer . We developed a novel bipartite algorithm, bipartite pattern discovery (Bipad), which produces a mathematical model based on information maximization or Shannon's entropy minimization principle, for discovery of bipartite sequence patterns . Bipad is a C++ program that applies greedy methods to search the bipartite alignment space and examines the upstream or downstream regions of co-regulated genes, looking for cis-regulatory bipartite patterns . An input sequence file with zero or one site per locus is required, and the left and right motif widths and a range of possible gap lengths must be specified . Bipad can run in either single-block or bipartite pattern search modes, and it is capable of comprehensively searching all four orientations of half-site patterns . Simulation studies showed that the accuracy of this motif discovery algorithm depends on sample size and motif conservation level, but results were independent of background composition . Bipad performed equivalent with or better than other pattern search algorithms in correctly identifying Escherichia coli cyclic AMP receptor protein and Bacillus subtilis sigma factor binding site sequences based on experimentally defined benchmarks . Finally, a new bipartite information weight matrix for vitamin D3 receptor/retinoid X receptor alpha (VDR/RXRalpha) binding sites was derived that comprehensively models the natural variability inherent in these sequence elements.

Curr Microbiol, 2004 Sep, 49(3), 186 - 91
Rapid detection and characterization of surfactin-producing Bacillus subtilis and closely related species based on PCR; Hsieh FC et al.; The surfactin production genetic locus ( sfp) is responsible for the ability of Bacillus subtilis to produce the lipopeptide biosurfactant, surfactin . This report demonstrates the utility of using PCR of the sfp gene as a means of identifying Bacillus species that produce surfactin . We carried out a hemolysis zone assay, quantitative HPLC and NMR in parallel to ensure that the PCR provided correct results . PCR analyses were performed for the sfp gene on 15 standard strains and 20 field-collected Bacillus spp . isolates native to Taiwan . Among the 15 standard strains, surfactin was produced by seven strains of B . subtilis and two closely related species, B . amyloliquefaciens B128 and B . circulans ATCC 4513 . Of the 20 field-collected Bacillus spp . isolates; 16 strains yielded surfactin- positive results with PCR and HPLC . A good correlation was observed . Within the 16 field isolates, B . amyloliquefaciens S13 (452.5 mg/L) and B . subtilis S15 (125.6 mg/L) had high productivity of surfactin . The technique is valuable for finding out potential good yields of surfactin-producing strains . The PCR method we used could also be used to find different species or genera containing homologous genes . This is the first report of the detection of surfactin production by B . amyloliquefaciens and B . circulans based on PCR screening.

PLoS Biol . 2004 Oct;2(10):e328 . Epub 2004 Sep 21.
The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis; Eichenberger P et al.; Asymmetric division during sporulation by Bacillus subtilis generates a mother cell that undergoes a 5-h program of differentiation . The program is governed by a hierarchical cascade consisting of the transcription factors: sigma(E), sigma(K), GerE, GerR, and SpoIIID . The program consists of the activation and repression of 383 genes . The sigma(E) factor turns on 262 genes, including those for GerR and SpoIIID . These DNA-binding proteins downregulate almost half of the genes in the sigma(E) regulon . In addition, SpoIIID turns on ten genes, including genes involved in the appearance of sigma(K) . Next, sigma(K) activates 75 additional genes, including that for GerE . This DNA-binding protein, in turn, represses half of the genes that had been activated by sigma(K) while switching on a final set of 36 genes . Evidence is presented that repression and activation contribute to proper morphogenesis . The program of gene expression is driven forward by its hierarchical organization and by the repressive effects of the DNA-binding proteins . The logic of the program is that of a linked series of feed-forward loops, which generate successive pulses of gene transcription . Similar regulatory circuits could be a common feature of other systems of cellular differentiation.

J Mol Microbiol Biotechnol, 2004, 7(4), 204 - 11
The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential; Veith B et al.; The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs . The average G+C ratio is 46.2% . 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified . The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level . B . licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin . From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B . licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose . Many new genes of potential interest for biotechnological applications were found in B . licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes .

Astrobiology, 2004 Fall, 4(3), 377 - 90
Evaluation of various cleaning methods to remove bacillus spores from spacecraft hardware materials; Venkateswaran K et al.; A detailed study was made of the biological cleaning effectiveness, defined in terms of the ability to remove bacterial spores, of a number of methods used to clean hardware surfaces . Aluminum (Al 6061) and titanium (Ti 6Al-4V) were chosen for the study as they were deemed the two materials most likely to be used in spacecraft extraterrestrial sampler construction . Metal coupons (1 cm x 2.5 cm) were precleaned and inoculated with 5.8 x 10(3) cultivable Bacillus subtilis spores, which are commonly found on spacecraft surfaces and in the assembly environments . The inoculated coupons were subsequently cleaned using: (1) 70% isopropyl alcohol wipe; (2) water wipe; (3) multiple-solvent flight-hardware cleaning procedures used at the Jet Propulsion Laboratory (JPL); (4) Johnson Space Center-developed ultrapure water rinse; and (5) a commercial, semi-aqueous, multiple-solvent (SAMS) cleaning process . The biological cleaning effectiveness was measured by agar plate assay, sterility test (growing in liquid media), and epifluorescent microscopy . None of the cleaning protocols tested completely removed viable spores from the surface of the aluminum . In contrast, titanium was capable of being cleaned to sterility by two methods, the JPL standard and the commercial SAMS cleaning process . Further investigation showed that the passivation step employed in the JPL standard method is an effective surface sterilant on both metals but not compatible with aluminum . It is recommended that titanium (Ti 6Al-4V) be considered superior to aluminum (Al 6061) for use in spacecraft sampling hardware, both for its potential to be cleaned to sterilization and for its ability to withstand the most effective cleaning protocols.

Phytochemistry, 2004 Aug, 65(16), 2333 - 6
Composition and antimicrobial activities of volatile components of Lippia javanica; Manenzhe NJ et al.; The volatile oil of Lippia javanica was prepared by hydrodistillation of leaves, flowers and stems, and characterized by GC-MS . The major component was 3-methyl-6-(1-methylethylidene)-cyclohex-2-en-1-one . The oil was tested for antimicrobial activity on cultures of Escherichia coli, Bacillus subtilis and Staphylococcus aureus, and found to inhibit E . coli and S . aureus at 1% dilution . The oil was also active against Plasmodium falciparum in micromolar concentrations.

Res Microbiol, 2004 Oct, 155(8), 605 - 10
Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis; Li W et al.; Bacillus subtilis is an alternative host for expression and secretion of heterologous proteins . However, low yields of protein production limit its use on a wide scale . The secretory pathway of proteins can be divided into three functional stages: the early stage, involving the synthesis of secretory pre-proteins, their interaction with chaperones and binding to the secretory translocase; the second stage, translocation across the cytoplasmic membrane; and the last stage, including removal of the signal peptide, protein refolding and passage through the cell wall . Five bottlenecks for expression and secretion of heterologous proteins are described in this review: transcription, protein folding, translocation, signal peptide processing and proteolysis.

Proteomics, 2004 Oct, 4(10), 2849 - 76
A comprehensive proteome map of growing Bacillus subtilis cells; Eymann C et al.; The proteome of growing cells of Bacillus subtilis was analyzed in order to provide the basis for its application in microbial physiology . DNA arrays were used to calculate the number of genes transcribed in growing cells . From the 4100 B . subtilis genes, 2515 were actively transcribed in cells grown under standard conditions . From these genes 1544 proteins should be covered by our standard gel system pI 4-7 . Using this standard gel system and supplementary zoom gels (pI 5.5-6.7, 5-6, 4.5-5.5, and 4-5) 693 proteins which are expressed in growing cells were detected that cover more than 40% of the vegetative proteome predicted for this region . Particularly broad coverage and thus comprehensive monitoring will be possible for central carbohydrate metabolism (glycolysis, pentose phosphate shunt, and citric acid cycle), amino acid synthesis pathways, purine and pyrimidine metabolism, fatty acid metabolism, and main cellular functions like replication, transcription, translation, and cell wall synthesis . Comparing the theoretical pI and Mr values with those experimentally determined a reasonable correlation was found for the majority of protein spots . By a color code outliers with dramatic deviations in charge or mass were visualized that may indicate post-translational modifications . In addition to the cytosolic neutral and alkaline proteins, 130 membrane proteins were found relying on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation in combination with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) techniques . The vegetative proteome containing 876 proteins in total is now ready for physiological applications . Two main proteome fractions (pI 4-7 and zoom gel pI 4.5-5.5) should be sufficient for such high-throughput physiological proteomics.

J Basic Microbiol, 2004, 44(5), 351 - 9
Tat dependent export of E . coli phytase AppA by using the PhoD-specific transport system of Bacillus subtilis; Gerlach R et al.; It has been shown recently that the twin-arginine signal peptide of Bacillus subtilis phosphodiesterase PhoD (SPPhoD) can mediate Tat dependent transport of proteins via its specific Tat-transport components . In order to test the use of Tat dependent transport signals for heterologous product synthesis, Escherichia coli phytase AppA was expressed under control of PhoD-specific export signals in B . subtilis . Induction of Tat components TatAd/TatCd was mediated by using a functionally altered PhoR/PhoP signal transduction system which regulates the expression of these components . AppA was highly susceptible to host specific extracellular proteases . Expression of appA in B . subtilis wprA strain resulted in the stable production of AppA . A fusion protein consisting of SPPhoD and mature AppA remained unprocessed, while introduction of the AppA signal peptidase cleavage site resulted in efficient processing of the fusion protein .

Appl Opt, 2004 Aug 10, 43(23), 4564 - 70
Measured infrared optical cross sections for a variety of chemical and biological aerosol simulants; Gurton KP et al.; We conducted a series of spectral extinction measurements on a variety of aerosolized chemical and biological simulants over the spectral range 3-13 microm using conventional Fourier-transform IR (FTIR) aerosol spectroscopy . Samples consist of both aerosolized particulates and atomized liquids . Materials considered include Bacillus subtilis endospores, lyophilized ovalbumin, polyethylene glycol, dimethicone (SF-96), and three common background materials: kaolin clay (hydrated aluminum silicate), Arizona road dust (primarily SiO2), and diesel soot . Aerosol size distributions and mass density were measured simultaneously with the FTIR spectra . As a result, all optical parameters presented here are mass normalized, i.e., in square meters per gram . In an effort to establish the utility of using Mie theory to predict such parameters, we conducted a series of calculations . For materials in which the complex indices of refraction are known, e.g., silicone oil (SF-96) and kaolin, measured size distributions were convolved with Mie theory and the resultant spectral extinction calculated . Where there was good agreement between measured and calculated extinction spectra, absorption, total scattering, and backscatter were also calculated.

Appl Microbiol Biotechnol, 2004 Dec, 66(2), 180 - 6 Epub 2004 Dec.
Cloning, expression and characterisation of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtilis; Budde M et al.; The gene encoding CYP102A2, a novel P450 monooxygenase from Bacillus subtilis, was cloned and expressed in Escherichia coli . The recombinant enzyme formed was purified by immobilised metal chelate affinity chromatography (IMAC) and characterised . CYP102A2 is a 119-kDa self-sufficient monooxygenase, consisting of an FMN/FAD-containing reductase domain and a heme domain . The deduced amino acid sequence of CYP102A2 exhibits a high level of identity with the amino acid sequences of CYP102A1 from B . megaterium (59%) and CYP102A3 from B . subtilis (60%) . In reduced, CO-bound form, the enzyme shows a typical Soret band at 449 nm . It catalyses the oxidation of even- and odd-chain saturated and unsaturated fatty acids . In all reactions investigated, the products were the respective omega-3, omega-2 and omega-1 hydroxylated fatty acids . Activity was highest towards oleic acid (K(M)=17.36+/-1.4 microM, k(cat)=2,244+/-72 min(-1)) and linoleic acid (K(M)=12.25+/-1.8 microM, k(cat)=1,950+/-84 min(-1)) . Comparison of a CYP102A2 homology model with the CYP102A1 crystal structure revealed significant differences in the substrate access channels, which might explain the differences in the catalytic properties of these two enzymes.

Appl Microbiol Biotechnol . 2004 Sep 16; {Epub ahead of print}
Fed-batch production of d-ribose from sugar mixtures by transketolase-deficient Bacillus subtilis SPK1; Park YC et al.; d-Ribose, a five-carbon sugar, is used as a key intermediate for the production of various biomaterials, such as riboflavin and inosine monophosphate . A high d-ribose-producing Bacillus subtilis SPK1 strain was constructed by the chemical mutation of the transketolase-deficient strain, B . subtilis JY1 . Batch fermentation of B . subtilis SPK1 with 20 g l(-1) xylose and 20 g l(-1) glucose resulted in 4.78 g l(-1) dry cell mass, 23.0 g l(-1) d-ribose concentration, and 0.72 g l(-1) h(-1) productivity, corresponding to a 1.5- to 1.7-fold increase when compared with values for the parental strain . A late-exponential phase was chosen as the best point for switching to a fed-batch process . Optimized fed-batch fermentation of B . subtilis SPK1, feeding a mixture of 200 g l(-1) xylose and 50 g l(-1) glucose after the late-exponential phase reduced the residual xylose and glucose concentrations to less than 7.0 g l(-1) and gave the best results of 46.6 g l(-1) d-ribose concentration and 0.88 g l(-1) h(-1) productivity which were 2.0- and 1.2-fold higher than the corresponding values in a simple batch fermentation.

J Bacteriol, 2004 Oct, 186(19), 6485 - 91
Contribution of the mismatch DNA repair system to the generation of stationary-phase-induced mutants of Bacillus subtilis; Pedraza-Reyes M et al.; A reversion assay system previously implemented to demonstrate the existence of adaptive or stationary-phase-induced mutagenesis in Bacillus subtilis was utilized in this report to study the influence of the mismatch DNA repair (MMR) system on this type of mutagenesis . Results revealed that a strain deficient in MutSL showed a significant propensity to generate increased numbers of stationary-phase-induced revertants . These results suggest that absence or depression of MMR is an important factor in the mutagenesis of nongrowing B . subtilis cells because of the role of MMR in repairing DNA damage . In agreement with this suggestion, a significant decrease in the number of adaptive revertant colonies, for the three markers tested, occurred in B . subtilis cells which overexpressed a component of the MMR system . Interestingly, the single overexpression of mutS, but not of mutL, was sufficient to decrease the level of adaptive mutants in the reversion assay system of B . subtilis . The results presented in this work, as well as in our previous studies, appear to suggest that an MMR deficiency, putatively attributable to inactivation or saturation with DNA damage of MutS, may occur in a subset of B . subtilis cells that differentiate into the hypermutable state.

J Bacteriol, 2004 Oct, 186(19), 6477 - 84
Bacillus subtilis ResD induces expression of the potential regulatory genes yclJK upon oxygen limitation; Hartig E et al.; Transcription of the yclJK operon, which encodes a potential two-component regulatory system, is activated in response to oxygen limitation in Bacillus subtilis . Northern blot analysis and assays of yclJ-lacZ reporter gene fusion activity revealed that the anaerobic induction is dependent on another two-component signal transduction system encoded by resDE . ResDE was previously shown to be required for the induction of anaerobic energy metabolism . Electrophoretic mobility shift assays and DNase I footprinting experiments showed that the response regulator ResD binds specifically to the yclJK regulatory region upstream of the transcriptional start site . In vitro transcription experiments demonstrated that ResD is sufficient to activate yclJ transcription . The phosphorylation of ResD by its sensor kinase, ResE, highly stimulates its activity as a transcriptional activator . Multiple nucleotide substitutions in the ResD binding regions of the yclJ promoter abolished ResD binding in vitro and prevented the anaerobic induction of yclJK in vivo . A weight matrix for the ResD binding site was defined by a bioinformatic approach . The results obtained suggest the existence of a new branch of the complex regulatory system employed for the adaptation of B . subtilis to anaerobic growth conditions.

J Bacteriol, 2004 Oct, 186(19), 6457 - 64
Hetero- and autoprocessing of the extracellular metalloprotease (Mpr) in Bacillus subtilis; Park CH et al.; Most proteases are synthesized as inactive precursors which are processed by proteolytic cleavage into a mature active form, allowing regulation of their proteolytic activity . The activation of the glutamic-acid-specific extracellular metalloprotease (Mpr) of Bacillus subtilis has been examined . Analysis of Mpr processing in defined protease-deficient mutants by activity assay and Western blotting revealed that the extracellular protease Bpr is required for Mpr processing . pro-Mpr remained a precursor form in bpr-deficient strains, and glutamic-acid-specific proteolytic activity conferred by Mpr was not activated in bpr-deficient strains . Further, purified pro-Mpr was processed to an active form by purified Bpr protease in vitro . We conclude that Mpr is activated by Bpr in vivo, and that heteroprocessing, rather than autoprocessing, is the major mechanism of Mpr processing in vivo . Exchange of glutamic acid for serine in the cleavage site of Mpr (S93E) allowed processing of Mpr into its mature form, regardless of the presence of other extracellular proteases, including Bpr . Thus, a single amino acid change is sufficient to convert the Mpr processing mechanism from heteroprocessing to autoprocessing.

J Bacteriol, 2004 Oct, 186(19), 6443 - 56
The fur homologue in Borrelia burgdorferi; Katona LI et al.; Borrelia burgdorferi contains a gene that codes for a Fur homologue . The function of this Fur protein is unknown; however, spirochetes grown at 23 or 35 degrees C expressed fur as determined by reverse transcriptase PCR . The fur gene (BB0647) was cloned and overexpressed as a His-Fur fusion protein in Escherichia coli . The fusion protein was purified by zinc-chelate chromatography, and the N-terminal His tag was removed to generate recombinant Fur for use in mobility shift studies . Fur bound DNA containing the E . coli Fur box sequence (GATAATGATAATCATTATC) or Bacillus subtilis Per box sequence (TTATAAT-ATTATAA) with an apparent Kd of approximately 20 nM . Fur also bound the upstream sequences of three Borrelia genes: BB0646 (gene encoding a hydrolase of the alpha/beta-fold family), BB0647 (fur), and BB0690 (napA) . Addition of metal ions was not required . Binding activity was greatly decreased by either exposure to oxidizing agents (H2O2, t-butyl hydroperoxide, cumene hydroperoxide, or diamide) or by addition of Zn2+ . B . burgdorferi NapA is a homologue of Dps . Dps functions in E . coli to protect DNA against damage during periods of redox stress . Fur may function in B . burgdorferi as a repressor and regulate oxidative stress genes . Additional genes (10 chromosomal and 15 plasmid) that may be Fur regulated were identified by in silico analysis.

Peptides, 2004 Sep, 25(9), 1415 - 24
Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of the spa-box in subtilin-responsive promoters; Kleerebezem M et al.; The production of the type I antimicrobial peptide (AMP) subtilin by Bacillus subtilis is regulated in a cell-density-dependent manner {Kleerebezem M, de Vos WM, Kuipers OP . The lantibiotics nisin and subtilin act as extracellular regulators of their own biosynthesis . In: Dunny GM, Winans SC, editors . Cell-cell signaling in bacteria . Washington, D.C., USA: ASM Press; 1999 . p . 159-74; Stein T, Borchert S, Kiesau P, Heinzmann S, Kloss S, Klein C, Helfrich M, Entian KD . Dual control of subtilin biosynthesis and immunity in Bacillus subtilis . Mol Microbiol 2002;44:403-16; Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD . The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis . Mol Microbiol 2003;47:1627-36} . Three subtilin-responsive promoter elements within the spaBTCSIFEGRK are controlled by the specific cis-acting sequence element called the spa-box, which represents the binding site of the subtilin regulator SpaR {Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD . The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis . Mol Microbiol 2003;47:1627-36} . Here, we describe the functional characterization of the spaB, spaS and spaI promoters by transcriptional fusion with a promoterless beta-glucuronidase encoding gusA gene . Within these gusA fusion constructs, transcription initiation start sites of the spaS and spaI promoters were mapped to be located downstream of the spa-box, which is in contrast to previous reports {Banerjee S, Hansen JN . Structure and expression of a gene encoding the precursor of subtilin, a small protein antibiotic . J Biol Chem 1988;263:9508-14; Stein T, Heinzmann S, Kiesau P, Himmel B, Entian KD . The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis . Mol Microbiol 2003;47:1627-36} . Nevertheless, all spa-promoters displayed typical cell-density-dependent activity in a subtilin-producing strain B . subtilis ATCC6633 . Moreover, analysis of beta-glucuronidase activities in a spaB mutant of B . subtilis ATCC6633 and a derivative of strain 168 that harbors the spaRK genes integrated in the chromosomal amyE locus, confirmed that these promoters are activated by subtilin-triggered, SpaRK-mediated, quorum-sensing control . Quantitative analysis showed that the spaS promoter strength at a given subtilin concentration appeared to be approximately five-fold higher than the spaB promoter, which in turn is approximately two-fold higher than the spaI promoter . Finally, it is shown that the elementary components involved in subtilin-mediated regulation are the two-component system, SpaRK, and a spa-box containing promoter.

Biochemistry, 2004 Sep 7, 43(35), 11331 - 43
Initiation of surfactin biosynthesis and the role of the SrfD-thioesterase protein; Steller S et al.; In this paper, the initiation reactions in surfactin biosynthesis by Bacillus subtilis OKB 105 were investigated . Evidence for a specific role of the SrfD protein, the external thioesterase enzyme in surfactin biosynthesis, was obtained for the first time . The action of SrfD was investigated both with the native, but only partially purified, enzyme and the highly purified, His-tagged protein overexpressed in Escherichia coli . Surfactin can be formed by the interaction of the three amino acid activating components of surfactin synthetase SrfA, B and C alone . This process is stimulated by SrfD . In the initiation reactions, the beta-hydroxy fatty acid substrate is transferred from beta-hydroxymyristoyl-coenzyme A to the start enzyme SrfA followed by formation of beta-hydroxymyristoyl-glutamate . The same reactions were also observed with the recombinant L-Glu-activating module of surfactin synthetase . Lipopeptide formation can be initiated by these function units alone, but SrfD efficiently supports and stimulates the formation of initiation products . From these results, we infer that SrfD functions as the thioesterase/acyltransferase enzyme in the initiation process previously postulated by Menkhaus et al . {Menkhaus et al . (1993) J . Biol . Chem . 268, 7678-7684}, thus enhancing surfactin formation.

J Mol Biol, 2004 Oct 1, 342(5), 1359 - 65
Reduced contact order and RNA folding rates; Sosnick TR et al.; We investigated the relationship between RNA structure and folding rates accounting for hierarchical structural formation . Folding rates of two-state folding proteins correlate well with relative contact order, a quantitative measure of the number and sequence distance between tertiary contacts . These proteins do not form stable structures prior to the rate-limiting step . In contrast, most secondary structures are stably formed prior to the rate-limiting step in RNA folding . Accordingly, we introduce "reduced contact order", a metric that reflects only the number of residues available to participate in the conformational search after the formation of secondary structure . Plotting the folding rates and the reduced contact order from ten different RNAs suggests that RNA folding can be divided into two classes . To examine this division, folding rates of circularly permutated isomers are compared for two RNAs, one from each class . Folding rates vary by tenfold for circularly permuted Bacillus subtilis RNase P RNA isomers, whereas folding rates vary by only 1.2-fold for circularly permuted catalytic domains . This difference is likely related to the dissimilar natures of their rate-limiting steps.

J Struct Biol, 2004 Oct, 148(1), 98 - 109
Crystal structure of Bacillus subtilis YckF: structural and functional evolution; Sanishvili R et al.; The crystal structure of the YckF protein from Bacillus subtilis was determined with MAD phasing and refined at 1.95A resolution . YckF forms a tight tetramer both in crystals and in solution . Conservation of such oligomerization in other phosphate sugar isomerases indicates that the crystallographically observed tetramer is physiologically relevant . The structure of YckF was compared to with its ortholog from Methanococcus jannaschii, MJ1247 . Both of these proteins have phosphate hexulose isomerase activity, although neither of the organisms can utilize methane or methanol as source of energy and/or carbon . Extensive sequence and structural similarities with MJ1247 and with the isomerase domain of glucosamine-6-phosphate synthase from Escherichia coli allowed us to group residues contributing to substrate binding or catalysis . Few notable differences among these structures suggest possible cooperativity of the four active sites of the tetramer . Phylogenetic relationships between obligatory and facultative methylotrophs along with B . subtilis and E . coli provide clues about the possible evolution of genes as they loose their physiological importance.

Biochemistry, 2004 Sep 21, 43(37), 11647 - 57
Thiamin biosynthesis in Bacillus subtilis: structure of the thiazole synthase/sulfur carrier protein complex; Settembre EC et al.; Thiazole synthase is the key enzyme involved in the formation of the thiazole moiety of thiamin pyrophosphate . We have determined the structure of this enzyme in complex with ThiS, the sulfur carrier protein, at 3.15 A resolution . Thiazole synthase is a tetramer with 222 symmetry . The monomer is a (betaalpha)(8) barrel with similarities to the aldolase class 1 and flavin mononucleotide dependent oxidoreductase and phosphate binding superfamilies . The sulfur carrier protein (ThiS) is a compact protein with a fold similar to that of ubiquitin . The structure allowed us to model the substrate, deoxy-D-xylulose 5-phosphate (DXP), in the active site . This model identified Glu98 and Asp182 as new active site residues likely to be involved in the catalysis of thiazole formation . The function of these residues was probed by mutagenesis experiments, which confirmed that both residues are essential for thiazole formation and identified Asp182 as the base involved in the deprotonation at C3 of the thiazole synthase DXP imine . Comparison of the ThiS binding surface to the surface of ubiquitin identified a conserved hydrophobic patch of unknown function on ubiquitin that may be involved in complex formation between ubiquitin and one of its binding partners.

Carbohydr Res, 2004 Aug 23, 339(12), 2059 - 67
Regiospecific alkaline protease-catalyzed divinyl acyl transesterifications of primary hydroxyl groups of mono- and di-saccharides in pyridine; Wu Q et al.; This paper describes highly selective transesterification reactions, catalyzed by an alkaline protease from Bacillus subtilis in pyridine, of several mono- and di-saccharides with divinyl dicarboxylates ranging from 4 to 10 carbon atoms . A series of polymerizable vinyl fatty acid sugar esters were obtained with good selectivity and high yields . Most products had high proportions of the alpha anomer . The influences of the enzymes, solvents, temperature, and acyl donor chain length on the reaction were studied . Vinyl sugar esters offer a new family of functional water-soluble monomers for preparation of sugar-containing polymers.

J Appl Microbiol, 2004, 97(4), 838 - 52
Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress; Cortezzo DE et al.; AIMS: To determine if treatment of Bacillus subtilis spores with a variety of oxidizing agents causes damage to the spore's inner membrane . METHODS AND RESULTS: Spores of B . subtilis were killed 80-99% with wet heat or a variety of oxidizing agents, including betadine, chlorine dioxide, cumene hydroperoxide, hydrogen peroxide, Oxone, ozone, sodium hypochlorite and t-butylhydroperoxide, and the agents neutralized and/or removed . Survivors of spores pretreated with oxidizing agents exhibited increased sensitivity to killing by a normally minimal lethal heat treatment, while spores pretreated with wet heat did not . In addition, spores treated with wet heat or the oxidizing agents, except sodium hypochlorite, were more sensitive to high NaCl in plating media than were untreated spores . The core region of spores treated with at least two oxidizing agents was also penetrated much more readily by methylamine than was the core of untreated spores, and spores treated with oxidizing agents but not wet heat germinated faster with dodecylamine than did untreated spores . Spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents . CONCLUSIONS: Treatment of spores with oxidizing agents has been suggested to cause damage to the spore's inner membrane, a membrane whose integrity is essential for spore viability . The sensitization of spores to killing by heat and to high salt after pretreatment with oxidizing agents is consistent with and supports this suggestion . Presumably mild pretreatment with oxidizing agents causes some damage to the spore's inner membrane . While this damage may not be lethal under normal conditions, the damaged inner membrane may be less able to maintain its integrity, when dormant spores are exposed to high temperature or when germinated spores are faced with osmotic stress . Triggering of spore germination by dodecylamine likely involves action by this agent on the spore's inner membrane allowing release of the spore core's depot of dipicolinic acid . Presumably dodecylamine more readily alters the permeability of a damaged inner membrane and thus more readily triggers germination of spores pretreated with oxidizing agents . Damage to the inner spore membrane by oxidizing agents is also consistent with the more rapid penetration of methylamine into the core of treated spores, as the inner membrane is likely the crucial permeability barrier to methylamine entry into the spore core . As spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents, it is not through oxidation of unsaturated fatty acids that oxidizing agents kill and/or damage spores . Perhaps these agents work by causing oxidative damage to key proteins in the spore's inner membrane . SIGNIFICANCE AND IMPACT OF THE STUDY: The more rapid heat killing and germination with dodecylamine, the greater permeability of the spore core and the osmotic stress sensitivity in outgrowth of spores pretreated with oxidizing agents is consistent with such agents causing damage to the spore's inner membrane, even if this damage is not lethal under normal conditions . It may be possible to take advantage of this phenomenon to devise improved, less costly regimens for spore inactivation.

J Pept Res, 2004 Oct, 64(4), 159 - 69
Antimicrobial activity of arginine- and tryptophan-rich hexapeptides: the effects of aromatic clusters, D-amino acid substitution and cyclization; Wessolowski A et al.; Many antimicrobial peptides bear arginine (R)- and tryptophan (W)-rich sequence motifs . Based on the sequence Ac-RRWWRF-NH2, sets of linear and cyclic peptides were generated by changes in the amino acid sequence, L-D-amino acid exchange and naphthylalanine substituted for tryptophan . Linear RW-peptides displayed moderate activity towards Gram-positive Bacillus subtilis (15 < MIC < 31 microm) and were inactive against Gram-negative Escherichia coli at peptide concentrations < 100 microm . Cyclization induced high antimicrobial activity . The effect of cyclization was most pronounced for peptides with three adjacent aromatic residues . Incorporation of d-amino acid residues had minor influence on the biological activity . The haemolytic activity of all RW-peptides at 100 microm concentration was low (< 7% lysis for linear R/W-rich peptides and < 28% for the cyclic analogues) . Introduction of naphthylalanine enhanced the biological activities of both the linear and cyclic peptides . All peptides induced permeabilization of large unilamellar vesicles (LUVs) composed of lipids of the membrane of B . subtilis and erythrocytes, but surprisingly had no effect on LUVs composed of lipids of the E . coli inner membrane . The profiles of peptide activity against B . subtilis and red blood cells correlated with the permeabilizing effects on the corresponding model membranes and were related to hydrophobicity parameters as derived from reversed phase high-performance liquid chromatography (HPLC) . The results underlined the importance of amphipathicity as a driving force for cell lytic activity and suggest that conformational constraints and an appropriate position of aromatic residues allowing the formation of hydrophobic clusters are highly favourable for antimicrobial activity and selectivity.

J Microbiol, 2004 Jun, 42(2), 111 - 6
Growth inhibition of Escherichia coli during heterologous expression of Bacillus subtilis glutamyl-tRNA synthetase that catalyzes the formation of mischarged glutamyl-tRNA1 Gln; Baick JW et al.; It is known that Bacillus subtilis glutamyl-tRNA synthetase (GluRS) mischarges E . coli tRNA1 Gln with glutamate in vitro . It has also been established that the expression of B . subtilis GluRS in Escherichia coli results in the death of the host cell . To ascertain whether E . coli growth inhibition caused by B . subtilis GluRS synthesis is a consequence of Glu-tRNA1 Ghn formation, we constructed an in vivo test system, in which B . subtilis GluRS gene expression is controlled by IPTG . Such a system permits the investigation of factors affecting E . coli growth . Expression of E . coli glutaminyl-tRNA synthetase (GlnRS) also ameliorated growth inhibition, presumably by competitively preventing tRNA1 Gln misacylation . However, when amounts of up to 10 mM L-glutamine, the cognate amino acid for acylation of tRNA1 Gln, were added to the growth medium, cell growth was unaffected . Overexpression of the B . subtilis gatCAB gene encoding Glu-tRNAGln amidotransferase (Glu-AdT) rescued cells from toxic effects caused by the formation of the mischarging GluRS . This result indicates that B . subtilis Glu-AdT recognizes the mischarged E . coli GlutRNA1 Gln, and converts it to the cognate Gln-tRNA1 Gln species . B . subtilis GluRS-dependent Glu-tRNA1 Gln formation may cause growth inhibition in the transformed E . coli strain, possibly due to abnormal protein synthesis.

DNA Seq, 2004 Feb, 15(1), 51 - 7
cDNA cloning and characterization of enolase from Chinese cabbage, Brassica campestris ssp . Pekinensis; Zhao J et al.; An enolase-encoding cDNA clone from Chinese cabbage, Brassica campestris ssp . Pekinensis, was isolated . This gene (Accession number: AY307448) had a total length of 1580bp with an open reading frame of 1335bp, and encoded a predicted polypeptide of 444 amino acids with a molecular weight of 47.38 kDa . The deduced amino acid (aa) sequence shared identity with a number of enolases ranging from Bacillus subtilis to human beings and had much higher identity with other plant enolases than with enolases from Bacillus, yeast and human beings . Comparison of its primary structure with those of other enolases revealed the presence of an insertion of 5 amino acids in enolase of Chinese cabbage . Expression of the cloned enolase gene decreased under salt stress, but increased in response to low temperature . Southern blot analysis of genomic DNA indicated that low-copies of enolase gene were present in the genome of Chinese cabbage.

J Mol Biol, 2004 Sep 24, 342(4), 1187 - 95
Efficient regulation of sigmaF, the first sporulation-specific sigma factor in B.subtilis; Clarkson J et al.; Differential gene expression is established in the prespore and mother-cell compartments of Bacillus subtilis through the successive activation of a series of cell-type-specific sigma factors . Crucial to the success of this process is the control of the first prespore-specific sigma factor, sigmaF . sigmaF is regulated by the proteins SpoIIAB, SpoIIAA and SpoIIE . SpoIIAB forms an inhibitory complex with sigmaF, which can be dissociated by interaction with SpoIIAA . During this interaction SpoIIAA is phosphorylated . SpoIIE is a membrane-bound phosphatase that dephosphorylates SpoIIAA, thereby re-activating it . It is not understood how sigmaF is activated specifically in the prespore but not in the mother cell . Here, we use a recently developed fluorescence spectroscopy technique to follow in real time the formation of sigmaF.SpoIIAB complexes and their dissociation by SpoIIAA . We show that complete activation of sigmaF is induced by a tenfold increase in SpoIIE activity . This result demonstrates that relatively small changes in SpoIIE activity, which could arise from asymmetric septation, can achieve the all-or-nothing response in sigmaF activity required by the cell . For long-term sigmaF activation, we find that sustained SpoIIE activity is required to counteract the activity of SpoIIAB . Even though the continual phosphorylation and dephosphorylation of SpoIIAA by these two enzymes will expend some ATP, the formation of SpoIIAA.SpoIIAB.ADP complexes greatly diminishes the rate of the phosphorylation reaction, and thus minimizes the wastage of energy . These features provide a very efficient system for regulating sigmaF.

Peptides, 2004 Aug, 25(8), 1223 - 33
SPAG11/isoform HE2C, an atypical anionic beta-defensin-like peptide; von Horsten HH et al.; A human caput epididymidal cDNA, HE2C, was cloned based on its homology to the known chimpanzee counterpart, suggesting that the encoded beta-defensin-like peptide represented a conserved component of the innate epididymidal epithelial defense system in primates . An approximately 6kDa HE2- related peptide was co-purified together with other HE2 isoforms from human seminal plasma by affinity chromatography . By its antibody reactivity as shown by Western blot analysis, this peptide was distinct from the more abundant HE2 isoforms and was concluded to correspond to HE2C . Similar to other HE2-encoded isoforms, the endogenous HE2C was proteolytically processed from a larger precursor by a furin-like prohormone convertase . This was confirmed by N-terminal sequencing . In order to study the structural and functional properties of HE2C it was recombinantly expressed in insect cells . Post-translational processing also occurred within these cells, yielding the mature processed HE2C peptide . Correct disulfide bonding of the recHE2C peptide was shown by p-aminophenylarsineoxide(PAPAO)-agarose binding assay . Purified recHE2C strongly bound to Escherichia coli DH5alpha and Bacillus subtilis; however, it did not exhibit microbicidal activity when tested in a radial diffusion assay against these bacteria . Different from the previously described beta-defensins, the mature HE2C peptide has an anionic pI and an algebraic net charge of -1 . Also, it lacks the amphipathic transitions, which, according to the Shai-Matzusaki-Huang model, are prerequisite for the membranolytic activity of antimicrobial peptides.

Microbiology, 2004 Sep, 150(Pt 9), 2911 - 20
Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis; Kimura K et al.; Many bacteria, including Escherichia coli, have a unique gene that encodes glutamate racemase . This enzyme catalyses the formation of d-glutamate, which is necessary for cell wall peptidoglycan synthesis . However, Bacillus subtilis has two glutamate racemase genes, named racE and yrpC . Since racE appears to be indispensable for growth in rich medium, the role of yrpC in d-amino acid synthesis is vague . Experiments with racE- and yrpC-knockout mutants confirmed that racE is essential for growth in rich medium but showed that this gene was dispensable for growth in minimal medium, where yrpC executes the anaplerotic role of racE . LacZ fusion assays demonstrated that racE was expressed in both types of media but yrpC was expressed only in minimal medium, which accounted for the absence of yrpC function in rich medium . Neither racE nor yrpC was required for B . subtilis cells to synthesize poly-gamma-dl-glutamate (gamma-PGA), a capsule polypeptide of d- and l-glutamate linked through a gamma-carboxylamide bond . Wild-type cells degraded the capsule during the late stationary phase without accumulating the degradation products, d-glutamate and l-glutamate, in the medium . In contrast, racE or yrpC mutant cells accumulated significant amounts of d- but not l-glutamate . Exogenous d-glutamate utilization was somewhat defective in the mutants and the double mutation of race and yrpc severely impaired d-amino acid utilization . Thus, both racemase genes appear necessary to complete the catabolism of exogenous d-glutamate generated from gamma-PGA.

Microbiology, 2004 Sep, 150(Pt 9), 2815 - 24
Dynamic localization of membrane proteins in Bacillus subtilis; Johnson AS et al.; The subcellular localization of membrane proteins in Bacillus subtilis was examined by using fluorescent protein fusions . ATP synthase and succinate dehydrogenase were found to localize within discrete domains on the membrane rather than being homogeneously distributed around the cell periphery as expected . Dual labelling of cells indicated partial colocalization of ATP synthase and succinate dehydrogenase . Further analysis using an ectopically expressed phage protein gave the same localization patterns as ATP synthase and succinate dehydrogenase, implying that membrane proteins are restricted to domains within the membrane . 3D reconstruction of images of the localization of ATP synthase showed that domains were not regular and there was no bias for localization to cell poles or any other positions . Further analysis revealed that this localization was highly dynamic, but random, implying that integral membrane proteins are free to diffuse two-dimensionally around the cytoplasmic membrane.

Appl Environ Microbiol, 2004 Sep, 70(9), 5349 - 56
RsbT and RsbV contribute to sigmaB-dependent survival under environmental, energy, and intracellular stress conditions in Listeria monocytogenes; Chaturongakul S et al.; Sigma B (sigma(B)) is a stress-responsive alternative sigma factor that has been identified in various gram-positive bacteria . Seven different regulators of sigma B (Rsbs) are located in the sigB operons of both Bacillus subtilis and Listeria monocytogenes . In B . subtilis, these proteins contribute to regulation of sigma(B) activity by conveying environmental and energy stress signals through two well-established branches of a signal transduction pathway . RsbT contributes to regulation of sigma(B) activity in response to environmental stresses, while RsbV contributes to sigma(B) activation under both environmental and energy stresses in B . subtilis . To probe L . monocytogenes Rsb roles in sigma(B)-mediated responses to various stresses, in-frame deletions were created in rsbT and rsbV . Phenotypic characterization of the L . monocytogenes rsbT and rsbV null mutants revealed that both mutants were similar to the DeltasigB strain in their abilities to survive under environmental stress conditions (exposure to synthetic gastric fluid, pH 2.5, acidified brain heart infusion broth {BHI}, or oxidative stress {13 mM cumene hydroperoxide}) . Under energy stress conditions (carbon starvation in defined media, entry into stationary phase, or reduced intracellular ATP), both DeltarsbT and DeltarsbV showed survival reductions similar to that of the DeltasigB strain . These observations suggest that the pathways for Rsb-dependent regulation of sigma(B) activity differ between L . monocytogenes and B . subtilis . As sigma(B) also activates transcription of the L . monocytogenes prfAP2 promoter, we evaluated virulence-associated characteristics of DeltaprfAP1rsbT and DeltaprfAP1rsbV double mutants in hemolysis and tissue culture assays . Both double mutants showed identical phenotypes to DeltaprfAP1P2 and DeltaprfAP1sigB double mutants, i.e., reduced hemolysis activity and reduced plaque size in mouse fibroblast cells . These findings indicate that RsbT and RsbV both contribute to sigma(B) activation in L . monocytogenes during exposure to environmental and energy stresses as well as during tissue culture infection.

Appl Environ Microbiol, 2004 Sep, 70(9), 5252 - 7
Anaerobic growth of Bacillus mojavensis and Bacillus subtilis requires deoxyribonucleosides or DNA; Folmsbee MJ et al.; Bacillus mojavensis strains JF-2 (ATCC 39307), ROB2, and ABO21191(T) and Bacillus subtilis strains 168 (ATCC 23857) and ATCC 12332 required four deoxyribonucleosides or DNA for growth under strict anaerobic conditions . Bacillus licheniformis strains L89-11 and L87-11, Bacillus sonorensis strain TG8-8, and Bacillus cereus (ATCC 14579) did not require DNA for anaerobic growth . The requirement for the deoxyribonucleosides or DNA did not occur under aerobic growth conditions . The addition of a mixture of five nucleic acid bases, four ribonucleotides, or four ribonucleosides to the basal medium did not replace the requirement of B . mojavensis JF-2 for the four deoxyribonucleosides . However, the addition of salmon sperm DNA, herring sperm DNA, Escherichia coli DNA, or synthetic DNA (single or double stranded) to the basal medium supported anaerobic growth . The addition of four deoxyribonucleosides to the basal medium allowed aerobic growth of B . mojavensis JF-2 in the presence of hydroxyurea . B . mojavensis did not grow in DNA-supplemented basal medium that lacked sucrose as the energy source . These data provide strong evidence that externally supplied deoxyribonucleosides can be used to maintain a balanced deoxyribonucleotide pool for DNA synthesis and suggest that ribonucleotide reductases may not be essential to the bacterial cell cycle nor are they necessarily part of a minimal bacterial genome.

Appl Environ Microbiol, 2004 Sep, 70(9), 5168 - 76
Genotyping and toxigenic potential of Bacillus subtilis and Bacillus pumilus strains occurring in industrial and artisanal cured sausages; Matarante A et al.; Artisanal and industrial sausages were analyzed for their aerobic, heat-resistant microflora to assess whether new emerging pathogens could be present among Bacillus strains naturally contaminating cured meat products . Sixty-four isolates were characterized by randomly amplified polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (fAFLP) . The biotypes, identified by partial 16S rRNA gene sequence analysis, belonged to Bacillus subtilis, Bacillus pumilus, and Bacillus amyloliquefaciens species . Both RAPD-PCR and fAFLP analyses demonstrated that a high genetic heterogeneity is present in the B . subtilis group even in strains harvested from the same source, making it possible to isolate 56 different biotypes . Moreover, fAFLP analysis made it possible to distinguish B . subtilis from B . pumilus strains . The strains were characterized for their toxigenic potential by molecular, physiological, and immunological techniques . Specific PCR analyses revealed the absence of DNA sequences related to HBL, BcET, NHE, and entFM Bacillus cereus enterotoxins and the enzymes sphingomyelinase Sph and phospholipase PI-PLC in all strains; also, the immunological analyses showed that Bacillus strains did not react with NHE- and HBL-specific antibodies . However, some isolates were found to be positive for hemolytic and lecithinase activity . The absence of toxigenic potential in Bacillus strains from the sausages analyzed indicates that these products can be considered safe under the processing conditions they were produced; however, great care should be taken when the ripening time is shortened, particularly in the case of traditional sausages, which could contain high amounts of Bacillus strains and possibly some B . cereus cells.

Pneumologie, 2004 Sep, 58(9), 666 - 9
{Misting-fountain-alveolitis}; Koschel D et al.; A 22-year-old woman developed recurrent episodes of fever, cough and dyspnea after repeated exposure to a misting fountain at home . A diagnosis of extrinsic allergic alveolitis (EAA) was made by detection of serum antibodies against the fountain water, by culture of Bacillus subtilis, Mucor racemosus, Mucor mucedo, and Saccharomyces cerevisiae from the water, and by detection of specific IgG antibodies against Bacillus subtilis and the Mucores . The diagnosis was confirmed by a restrictive lung function pattern, and a highly increased total cell count with a lymphocytosis of 39 % in the bronchoalveolar lavage . An inhalation challenge with the misting fountain resulted in a positive reaction . Because this humidifier system has recently become widespread at home, clinicians should be aware of this specific type of EAA which may be called "misting fountain alveolitis".

J Bacteriol, 2004 Sep, 186(18), 6230 - 8
Bacillus subtilis StoA Is a thiol-disulfide oxidoreductase important for spore cortex synthesis; Erlendsson LS et al.; Bacillus subtilis is an endospore-forming bacterium . There are indications that protein disulfide linkages occur in spores, but the role of thiol-disulfide chemistry in spore synthesis is not understood . Thiol-disulfide oxidoreductases catalyze formation or breakage of disulfide bonds in proteins . CcdA is the only B . subtilis thiol-disulfide oxidoreductase that has previously been shown to play some role in endospore biogenesis . In this work we show that lack of the StoA (YkvV) protein results in spores sensitive to heat, lysozyme, and chloroform . Compared to CcdA deficiency, StoA deficiency results in a 100-fold-stronger negative effect on sporulation efficiency . StoA is a membrane-bound protein with a predicted thioredoxin-like domain probably localized in the intermembrane space of the forespore . Electron microscopy of spores of CcdA- and StoA-deficient strains showed that the spore cortex is absent in both cases . The BdbD protein catalyzes formation of disulfide bonds in proteins on the outer side of the cytoplasmic membrane but is not required for sporulation . Inactivation of bdbD was found to suppress the sporulation defect of a strain deficient in StoA . Our results indicate that StoA is a thiol-disulfide oxidoreductase that is involved in breaking disulfide bonds in cortex components or in proteins important for cortex synthesis.

J Bacteriol, 2004 Sep, 186(18), 6150 - 8
RsbV-independent induction of the SigB-dependent general stress regulon of Bacillus subtilis during growth at high temperature; Holtmann G et al.; General stress proteins protect Bacillus subtilis cells against a variety of environmental insults . This adaptive response is particularly important for nongrowing cells, to which it confers a multiple, nonspecific, and preemptive stress resistance . Induction of the general stress response relies on the alternative transcription factor, SigB, whose activity is controlled by a partner switching mechanism that also involves the anti-sigma factor, RsbW, and the antagonist protein, RsbV . Recently, the SigB regulon has been shown to be continuously induced and functionally important in cells actively growing at low temperature . With the exception of this chill induction, all SigB-activating stimuli identified so far trigger a transient expression of the SigB regulon that depends on RsbV . Through a proteome analysis and Northern blot and gene fusion experiments, we now show that the SigB regulon is continuously induced in cells growing actively at 51 degrees C, close to the upper growth limit of B . subtilis . This heat induction of SigB-dependent genes requires the environmental stress-responsive phosphatase RsbU, but not the metabolic stress-responsive phosphatase RsbP . RsbU dependence of SigB activation by heat is overcome in mutants that lack RsbV . In addition, loss of RsbV alone or in combination with RsbU triggers a hyperactivation of the general stress regulon exclusively at high temperatures detrimental for cell growth . These new facets of heat induction of the SigB regulon indicate that the current view of the complex genetic and biochemical regulation of SigB activity is still incomplete and that SigB perceives signals independent of the RsbV-mediated signal transduction pathways under heat stress conditions.

J Bacteriol, 2004 Sep, 186(18), 6124 - 32
In vivo phosphorylation of partner switching regulators correlates with stress transmission in the environmental signaling pathway of Bacillus subtilis; Kim TJ et al.; Exposure of bacteria to diverse growth-limiting stresses induces the synthesis of a common set of proteins which provide broad protection against future, potentially lethal stresses . Among Bacillus subtilis and its relatives, this general stress response is controlled by the sigmaB transcription factor . Signals of environmental and energy stress activate sigmaB through a multicomponent network that functions via a partner switching mechanism, in which protein-protein interactions are governed by serine and threonine phosphorylation . Here, we tested a central prediction of the current model for the environmental signaling branch of this network . We used isoelectric focusing and immunoblotting experiments to determine the in vivo phosphorylation states of the RsbRA and RsbS regulators, which act in concert to negatively control the RsbU environmental signaling phosphatase . As predicted by the model, the ratio of the phosphorylated to unphosphorylated forms of both RsbRA and RsbS increased in response to salt or ethanol stress . However, these two regulators differed substantially with regard to the extent of their phosphorylation under both steady-state and stress conditions, with RsbRA always the more highly modified . Mutant analysis showed that the RsbT kinase, which is required for environmental signaling, was also required for the in vivo phosphorylation of RsbRA and RsbS . Moreover, the T171A alteration of RsbRA, which blocks environmental signaling, also blocked in vivo phosphorylation of RsbRA and impeded phosphorylation of RsbS . These in vivo results corroborate previous genetic analyses and link the phosphorylated forms of RsbRA and RsbS to the active transmission of environmental stress signals.

J Bacteriol, 2004 Sep, 186(18), 6003 - 14
DegU-P represses expression of the motility fla-che operon in Bacillus subtilis; Amati G et al.; Bacillus subtilis implements several adaptive strategies to cope with nutrient limitation experienced at the end of exponential growth . The DegS-DegU two-component system is part of the network involved in the regulation of postexponential responses, such as competence development, the production of exoenzymes, and motility . The degU32(Hy) mutation extends the half-life of the phosphorylated form of DegU (DegU-P); this in turn increases the production of alkaline protease, levan-sucrase, and other exoenzymes and inhibits motility and the production of flagella . The expression of the flagellum-specific sigma factor SigD, of the flagellin gene hag, and of the fla-che operon is strongly reduced in a degU32(Hy) genetic background . To investigate the mechanism of action of DegU-P on motility, we isolated mutants of degU32(Hy) that completely suppressed the motility deficiency . The mutations were genetically mapped and characterized by PCR and sequencing . Most of the mutations were found to delete a transcriptional termination signal upstream of the main flagellar operon, fla-che, thus allowing transcriptional readthrough from the cod operon . Two additional mutations improved the sigmaA-dependent promoter sequence of the fla-che operon . Using an electrophoretic mobility shift assay, we have demonstrated that purified DegU binds specifically to the PA promoter region of the fla-che operon . The data suggest that DegU represses transcription of the fla-che operon, and they indicate a central role of the operon in regulating the synthesis and assembly of flagella.

Structure (Camb), 2004 Sep, 12(9), 1619 - 29
Novel catalytic mechanism of glycoside hydrolysis based on the structure of an NAD+/Mn2+ -dependent phospho-alpha-glucosidase from Bacillus subtilis; Rajan SS et al.; GlvA, a 6-phospho-alpha-glucosidase from Bacillus subtilis, catalyzes the hydrolysis of maltose-6'-phosphate and belongs to glycoside hydrolase family GH4 . GH4 enzymes are unique in their requirement for NAD(H) and a divalent metal for activity . We have determined the crystal structure of GlvA in complex with its ligands to 2.05 A resolution . Analyses of the active site architecture, in conjunction with mechanistic studies and precedent from the nucleotide diphosphate hexose dehydratases and other systems, suggest a novel mechanism of glycoside hydrolysis by GlvA that involves both the NAD(H) and the metal.

Arch Microbiol, 2004 Oct, 182(2-3), 182 - 92 Epub 2004 Aug 31.
Sporulation genes in members of the low G+C Gram-type-positive phylogenetic branch ( Firmicutes); Onyenwoke RU et al.; Endospore formation is a specific property found within bacteria belonging to the Gram-type-positive low G+C mol% branch ( Firmicutes) of a phylogenetic tree based on 16S rRNA genes . Within the Gram-type-positive bacteria, endospore-formers and species without observed spore formation are widely intermingled . In the present study, a previously reported experimental method (PCR and Southern hybridization assays) and analysis of genome sequences from 52 bacteria and archaea representing sporulating, non-spore-forming, and asporogenic species were used to distinguish non-spore-forming (void of the majority of sporulation-specific genes) from asporogenic (contain the majority of sporulation-specific genes) bacteria . Several sporulating species lacked sequences similar to those of Bacillus subtilis sporulation genes . For some of the genes thought to be sporulation specific, sequences with weak similarity were identified in non-spore-forming bacteria outside of the Gram-type-positive phylogenetic branch and in archaea, rendering these genes unsuitable for the intended classification into sporulating, asporogenic, and non-spore-forming species . The obtained results raise questions regarding the evolution of sporulation among the Firmicutes.

Chem Commun (Camb), 2004 Sep 7, (17), 2006 - 7 Epub 2004 Jul 29.
A single-enzyme, two-step, one-pot synthesis of N-substituted imidazole derivatives containing a glucose branch via combined acylation/Michael addition reaction; Yao SP et al.; Combined regioselective acylation/Michael addition reaction catalyzed by alkaline protease from Bacillus subtilis in anhydrous pyridine for synthesis of N-substituted imidazole derivatives containing a glucose branch via a novel single-enzyme, two-step, one-pot procedure is reported.

Chem Pharm Bull (Tokyo), 2004 Sep, 52(9), 1117 - 22
Study on the stevioside analogues of steviolbioside, steviol, and isosteviol 19-alkyl amide dimers: synthesis and cytotoxic and antibacterial activity; Lin LH et al.; A new group of steviolbioside amide dimers 2a-g, derivatives 2h-i and their related steviol and isosteviol amide dimers 3a and 4a were prepared by reacting aliphatic alkylamine and alkyldiamines with PyBOP and DIEA . The synthesized compounds had cytotoxic effects on cancer and human embryonic lung cells . Compounds 3a, 4a, 2b and 2h were cytotoxic to cancer cells and to a lesser extent to human embryo lung cells . Compounds 2f, 2g and 4 of this series had favorable antibacterial effects, and were superior to penicillin G at inhibiting growth of Bacillus subtilis (BCRC 10029) . The cytotoxicity and antibacterial effects may depend on the dimerization and derivative moieties in relation to the respective aglycons.

Photochem Photobiol, 2004 Jul-Aug, 80, 150 - 3
Tryptophan fluorescence in the Bacillus subtilis phototropin-related protein YtvA as a marker of interdomain interaction; Losi A et al.; The Bacillus subtilis protein YtvA, related to plant phototropins (phot), binds flavin mononucleotide (FMN) within the N-terminal light, oxygen and voltage (LOV) domain . The blue light-triggered photocycle of YtvA and phot involves the reversible formation of a covalent photoadduct between FMN and a cysteine (cys) residue . YtvA contains a single tryptophan, W103, localized on the LOV domain and conserved in all phot-LOV domains . In this study, we show that the fluorescence parameters of W103 in YtvA-LOV are markedly different from those observed in the full-length YtvA . The fluorescence quantum yields are ca 0.03 and 0.08, respectively . In YtvA-LOV, the maximum is redshifted (ca 345 vs 335 nm) and the average fluorescence lifetime shorter (2.7 vs 4.7 ns) . These data indicate that W103 is located in a site of tight contact between the two domains of YtvA . In the FMN-cys adduct, selective excitation of W103 at 295 nm results in minimal changes of the fluorescence parameters with respect to the dark state . On 280 nm excitation, however, there is a detectable decrease in the fluorescence emitted from tyrosines, with concomitant increase in W103 fluorescence . This effect is reversible in the dark and might arise from a light-regulated energy transfer process from a yet unidentified tyrosine to W103.

RNA, 2004 Oct, 10(10), 1595 - 608 Epub 2004 Aug 30.
Ionic interactions between PRNA and P protein in Bacillus subtilis RNase P characterized using a magnetocapture-based assay; Day-Storms JJ et al.; Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the cleavage of the 5' end of precursor tRNA . To characterize the interface between the Bacillus subtilis RNA (PRNA) and protein (P protein) components, the intraholoenzyme KD is determined as a function of ionic strength using a magnetocapture-based assay . Three distinct phases are evident . At low ionic strength, the affinity of PRNA for P protein is enhanced as the ionic strength increases mainly due to stabilization of the PRNA structure by cations . Lithium substitution in lieu of potassium enhances the affinity at low ionic strength, whereas the addition of ATP, known to stabilize the structure of P protein, does not affect the affinity . At high ionic strength, the observed affinity decreases as the ionic strength increases, consistent with disruption of ionic interactions . These data indicate that three to four ions are released on formation of holoenzyme, reflecting the number of ion pairs that occur between the P protein and PRNA . At moderate ionic strength, the two effects balance so that the apparent KD is not dependent on the ionic strength . The KD between the catalytic domain (C domain) and P protein has a similar triphasic dependence on ionic strength . Furthermore, the intraholoenzyme KD is identical to or tighter than that of full-length PRNA, demonstrating that the P protein binds solely to the C domain . Finally, pre-tRNAasp (but not tRNAasp) stabilizes the PRNA*P protein complex, as predicted by the direct interaction between the P protein and pre-tRNA leader .

Biochem Biophys Res Commun, 2004 Sep 17, 322(2), 565 - 9
Guanine nucleotides stabilize the binding of Bacillus subtilis Obg to ribosomes; Zhang S et al.; Obg is a GTP-binding protein of Bacillus subtilis with essential, but undefined roles in the bacterium's growth, sporulation, and stress responses . Obg orthologs are widely conserved among both bacteria and eukaryotes . Gel filtration and affinity blot assays have suggested that Obg may be ribosome-associated . In the current work, we continue an examination of the putative Obg:ribosome interaction . Velocity centrifugation analyses of crude B . subtilis extracts or purified Obg:ribosome mixtures suggest that Obg is initially ribosome-bound, but can separate from ribosomes during sedimentation in the absence of added nucleotides . Addition of either GTP, GDP or ATP to the gradient prolonged the Obg:ribosome association, while inclusion of a nonhydrolyzable GTP analog (5-guanylyl-imidodiphosphate) preserved it . The data strengthen the notion that Obg is a ribosome-associated protein, demonstrate that Obg's association with ribosomes is stabilized by GTP, and indicate that the ribosome-bound Obg can likely hydrolyze GTP and be released as a consequence.

J Antibiot (Tokyo), 2004 Jun, 57(6), 367 - 72
Mutactimycin PR, a new anthracycline antibiotic from Saccharothrix sp . SA 103 . I . Taxonomy, fermentation, isolation and biological activities; Zitouni A et al.; In the course of screening for new antibacterial agents, a new isolate collected from a soil sample of an arid area in south Algeria, produced a red pigment which was shown an antagonistic action against a gram-positive bacterium Bacillus subtilis . The isolate was identified as Saccharothrix sp . and named SA 103 . The red pigment, eluted by HPLC on reverse phase C18 column, contained two compounds of an anthracycline antibiotics group . The structure of the major product (2) was characterized as mutactimycin C, and PR (1) was a new member of this group, designated as mutactimycin PR . These compounds showed an antibiotic activity against certain gram-positive bacteria in vitro . This is the first report of mutactimycins production by the genus Saccharothrix.

Biosci Biotechnol Biochem, 2004 Aug, 68(8), 1672 - 80
Spontaneous transformation and its use for genetic mapping in Bacillus subtilis; Murayama R et al.; Using a simple semi-synthetic competence and sporulation medium (CSM), we found evidence that Bacillus subtilis cells transformed in the competence phase can sporulate, indicating that genetic information acquired during the competence phase is inherited by the next generation after germination of the transformed spores . Moreover, the results from mixed cell culture experiments suggest that spontaneous genetic transformation can occur between competent cells and DNA released from lysed cells in the natural environment . We also found evidence that the spontaneous transformation system can be used for genetic mapping in B . subtilis.

J Mol Biol, 2004 Aug 27, 341(5), 1271 - 81
Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase; Acharya P et al.; Variation in gene sequences generated by directed evolution approaches often does not assure a minimalist design for obtaining a desired property in proteins . While screening for enhanced thermostability, structural information was utilized in selecting mutations that are generated by error-prone PCR . By this approach we have increased the half-life of denaturation by 300-fold compared to the wild-type Bacillus subtilis lipase through three point mutations generated by only two cycles of error-prone PCR . At lower temperatures the activity parameters of the thermostable mutants are unaltered . High-resolution crystal structures of the mutants show subtle changes, which include stacking of tyrosine residues, peptide plane flipping and a better anchoring of the terminus, that challenge rational design and explain the structural basis for enhanced thermostability . The approach may offer an efficient and minimalist solution for the enhancement of a desired property of a protein.

FEMS Microbiol Lett, 2004 Aug 15, 237(2), 377 - 84
Lactoferricin B inhibits bacterial macromolecular synthesis in Escherichia coli and Bacillus subtilis; Ulvatne H et al.; Most antimicrobial peptides have an amphipathic, cationic structure, and an effect on the cytoplasmic membrane of susceptible bacteria has been postulated as the main mode of action . Other mechanisms have been reported, including inhibition of cellular functions by binding to DNA, RNA and proteins, and the inhibition of DNA and/or protein synthesis . Lactoferricin B (Lfcin B), a cationic peptide derived from bovine lactoferrin, exerts slow inhibitory and bactericidal activity and does not lyse susceptible bacteria, indicating a possible intracellular target . In the present study incorporation of radioactive precursors into DNA, RNA and proteins was used to demonstrate effects of Lfcin B on macromolecular synthesis in bacteria . In Escherichia coli UC 6782, Lfcin B induces an initial increase in protein and RNA synthesis and a decrease in DNA synthesis . After 10 min, the DNA-synthesis increases while protein and RNA-synthesis decreases significantly . In Bacillus subtilis, however, all synthesis of macromolecules is inhibited for at least 20 min . After 20 min RNA-synthesis increases . The results presented here show that Lfcin B at concentrations not sufficient to kill bacterial cells inhibits incorporation of radioactive precursors into macromolecules in both Gram-positive and Gram-negative bacteria.

J Bacteriol, 2004 Sep, 186(17), 5956 - 60
FlhF, the third signal recognition particle-GTPase of Bacillus subtilis, is dispensable for protein secretion; Zanen G et al.; Bacillus subtilis contains three proteins of the signal recognition particle-GTPase family known as Ffh, FtsY, and FlhF . Here we show that FlhF is dispensable for protein secretion, unlike Ffh and FtsY . Although flhF is located in the fla/che operon, B . subtilis 168 flhF mutant cells assemble flagella and are motile.

J Bacteriol, 2004 Sep, 186(17), 5926 - 32
Transcription regulation of ezrA and its effect on cell division of Bacillus subtilis; Chung KM et al.; The EzrA protein of Bacillus subtilis is a negative regulator for FtsZ (Z)-ring formation . It is able to modulate the frequency and position of Z-ring formation during cell division . The loss of this protein results in cells with multiple Z rings located at polar as well as medial sites; it also lowers the critical concentration of FtsZ required for ring formation (P . A . Levin, I . G . Kurster, and A . D . Grossman, Proc . Natl . Acad . Sci . USA 96:9642-9647, 1999) . We have studied the regulation of ezrA expression during the growth of B . subtilis and its effects on the intracellular level of EzrA as well as the cell length of B . subtilis . With the aid of promoter probing, primer extension, in vitro transcription, and Western blotting analyses, two overlapping sigmaA-type promoters, P1 and P2, located about 100 bp upstream of the initiation codon of ezrA, have been identified . P1, supposed to be an extended -10 promoter, was responsible for most of the ezrA expression during the growth of B . subtilis . Disruption of this promoter reduced the intracellular level of EzrA very significantly compared with disruption of P2 . Moreover, deletion of both promoters completely abolished EzrA in B . subtilis . More importantly, the cell length and percentage of filamentous cells of B . subtilis were significantly increased by disruption of the promoter(s) . Thus, EzrA is required for efficient cell division during the growth of B . subtilis, despite serving as a negative regulator for Z-ring formation.

J Bacteriol, 2004 Sep, 186(17), 5856 - 64
The ClpP peptidase is the major determinant of bulk protein turnover in Bacillus subtilis; Kock H et al.; Measurements of overall protein degradation rates in wild-type and clpP mutant Bacillus subtilis cells revealed that stress- or starvation-induced bulk protein turnover depends virtually exclusively on the ClpP peptidase . ClpP is also essential for intracellular protein quality control, and in its absence newly synthesized proteins were highly prone to aggregation even at 37 degrees C . Proteomic comparisons between the wild type and a DeltaclpP mutant showed that the absence of ClpP leads to severe perturbations of "normal" physiology, complicating the detection of ClpP substrates . A pulse-chase two-dimensional gel approach was therefore used to compare wild-type and clpP mutant cultures that had been radiolabeled in mid-exponential phase, by quantifying changes in relative spot intensities with time . The results showed that overall proteolysis is biased toward proteins with vegetative functions which are no longer required (or are required at lower levels) in the nongrowing state . The identified substrate candidates for ClpP-dependent degradation include metabolic enzymes and aminoacyl-tRNA synthetases . Some substrate candidates catalyze the first committed step of certain biosynthetic pathways . Our data suggest that ClpP-dependent proteolysis spans a broad physiological spectrum, with regulatory processing of key metabolic components and regulatory proteins on the one side and general bulk protein breakdown at the transition from growing to nongrowing phases on the other.

J Bacteriol, 2004 Sep, 186(17), 5775 - 81
Assembly dynamics of FtsZ rings in Bacillus subtilis and Escherichia coli and effects of FtsZ-regulating proteins; Anderson DE et al.; FtsZ is the major cytoskeletal component of the bacterial cell division machinery . It forms a ring-shaped structure (the Z ring) that constricts as the bacterium divides . Previous in vivo experiments with green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching have shown that the Escherichia coli Z ring is extremely dynamic, continually remodeling itself with a half time of 30 s, similar to microtubules in the mitotic spindle . In the present work, under different experimental conditions, we have found that the half time for fluorescence recovery of E . coli Z rings is even shorter (approximately 9 s) . As before, the turnover appears to be coupled to GTP hydrolysis, since the mutant FtsZ84 protein, with reduced GTPase in vitro, showed an approximately 3-fold longer half time . We have also extended the studies to Bacillus subtilis and found that this species exhibits equally rapid dynamics of the Z ring (half time, approximately 8 s) . Interestingly, null mutations of the FtsZ-regulating proteins ZapA, EzrA, and MinCD had only modest effects on the assembly dynamics . This suggests that these proteins do not directly regulate FtsZ subunit exchange in and out of polymers . In B . subtilis, only 30 to 35% of the FtsZ protein was in the Z ring, from which we conclude that a Z ring only 2 or 3 protofilaments thick can function for cell division.

J Bacteriol, 2004 Sep, 186(17), 5640 - 8
Bacillus subtilis LmrA is a repressor of the lmrAB and yxaGH operons: identification of its binding site and functional analysis of lmrB and yxaGH; Yoshida K et al.; The Bacillus subtilis lmrAB operon is involved in multidrug resistance . LmrA is a repressor of its own operon, while LmrB acts as a multidrug efflux transporter . LmrA was produced in Escherichia coli cells and was shown to bind to the lmr promoter region, in which an LmrA-binding site was identified . Genome-wide screening involving DNA microarray analysis allowed us to conclude that LmrA also repressed yxaGH, which was not likely to contribute to the multidrug resistance . LmrA bound to a putative yxaGH promoter region, in which two tandem LmrA-binding sites were identified . The LmrA regulon was thus determined to comprise lmrAB and yxaGH . All three LmrA-binding sites contained an 18-bp consensus sequence, TAGACCRKTCWMTATAWT, which could play an important role in LmrA binding.

J Bacteriol, 2004 Sep, 186(17), 5567 - 75
Transglutaminase-mediated cross-linking of GerQ in the coats of Bacillus subtilis spores; Ragkousi K et al.; The spores of Bacillus subtilis show remarkable resistance to many environmental stresses, due in part to the presence of an outer proteinaceous structure known as the spore coat . GerQ is a spore coat protein essential for the presence of CwlJ, an enzyme involved in the hydrolysis of the cortex during spore germination, in the spore coat . Here we show that GerQ is cross-linked into higher-molecular-mass forms due in large part to a transglutaminase . GerQ is the only substrate for this transglutaminase identified to date . In addition, we show that cross-linking of GerQ into high-molecular-mass forms occurs only very late in sporulation, after mother cell lysis . These findings, as well as studies of GerQ cross-linking in mutant strains where spore coat assembly is perturbed, lead us to suggest that coat proteins must assemble first and that their cross-linking follows as a final step in the spore coat formation pathway.

J Bacteriol, 2004 Sep, 186(17), 5557 - 66
Genetic recombination in Bacillus subtilis 168: contribution of Holliday junction processing functions in chromosome segregation; Carrasco B et al.; Bacillus subtilis mutants classified within the epsilon (ruvA, DeltaruvB, DeltarecU, and recD) and eta (DeltarecG) epistatic groups, in an otherwise rec+ background, render cells impaired in chromosomal segregation . A less-pronounced segregation defect in DeltarecA and Deltasms (DeltaradA) cells was observed . The repair deficiency of addAB, DeltarecO, DeltarecR, recH, DeltarecS, and DeltasubA cells did not correlate with a chromosomal segregation defect . The sensitivity of epsilon epistatic group mutants to DNA-damaging agents correlates with ongoing DNA replication at the time of exposure to the agents . The Deltasms (DeltaradA) and DeltasubA mutations partially suppress the DNA repair defect in ruvA and recD cells and the segregation defect in ruvA and DeltarecG cells . The Deltasms (DeltaradA) and DeltasubA mutations partially suppress the DNA repair defect of DeltarecU cells but do not suppress the segregation defect in these cells . The DeltarecA mutation suppresses the segregation defect but does not suppress the DNA repair defect in DeltarecU cells . These results result suggest that (i) the RuvAB and RecG branch migrating DNA helicases, the RecU Holliday junction (HJ) resolvase, and RecD bias HJ resolution towards noncrossovers and that (ii) Sms (RadA) and SubA proteins might play a role in the stabilization and or processing of HJ intermediates.

Bioprocess Biosyst Eng, 2004 Dec, 27(1), 3 - 7 Epub 2004 Aug 13.
Partial purification of alpha-amylase from culture supernatant of Bacillus subtilis in aqueous two-phase systems; Zhi W et al.; A study was made of the partition and purification of alpha-amylase from a culture supernatant of Bacillus subtilis in the polyethylene glycol (PEG)--citrate aqueous two-phase system (ATPS) . Factors that influenced the partition of the protein in this system, including the molecular weight of the PEG, the tie line length of ATPS, the pH value and the sodium chloride concentration, were investigated . Purification of alpha-amylase was attained with a purification factor (PF) of 1.8 and 90% yield at pH 6.0 in a PEG1000-citrate ATPS with short tie line length . By utilizing the salt-out effect of neutral salt, the purification of alpha-amylase was further improved to 2.0 of PF and 80% yield in a PEG3350-citrate ATPS with 4% sodium chloride.

Mikrobiologiia, 2004 May-Jun, 73(3), 335 - 42
{The regulation of Bacillus intermedius glutamyl endopeptidase biosynthesis in the recombinant Bacillus subtilis strain during sporulation}; Chastukhina IB et al.; The growth of the recombinant Bacillus subtilis strain AJ73 carrying the Bacillus intermedius 3-19 glutamyl endopeptidase gene on a multicopy plasmid and the effect of some nutrients on the efficiency of extracellular glutamyl endopeptidase production in the stationary growth phase were studied . In this phase, the concentration of glutamyl endopeptidase in the culture liquid peaked at the 48th and 78th h of cultivation and depended on the composition of the cultivation medium . Unlike the synthesis of glutamyl endopeptidase in the trophophase (i.e., during vegetative growth), which was suppressed by glucose, the synthesis of this enzyme during sporulation was resistant to glucose present in the cultivation medium . A multifactorial experimental design allowed optimal proportions between the concentrations of major nutrients (peptone and inorganic phosphate) to be determined . Inorganic phosphate and ammonium ions augmented the production of glutamyl endopeptidase by 30-150%, and complex organic substrates, such as casein and gelatin, enhanced the production of glutamyl endopeptidase by 50-100% . During sporulation, the production of glutamyl endopeptidase was stimulated by some bivalent cations (Ca2+, Mg2+, and Co2+) and inhibited by others (Zn2+, Fe2+, and Cu2+) . The inference is drawn that the regulatory mechanisms of glutamyl endopeptidase synthesis during vegetative growth and sporulation are different.

Arch Biochem Biophys, 2004 Sep 15, 429(2), 204 - 14
A gene cluster for biosynthesis of kanamycin from Streptomyces kanamyceticus: comparison with gentamicin biosynthetic gene cluster; Kharel MK et al.; Gene clusters for the biosynthesis of kanamycin (Km) and gentamicin (Gm) were isolated from the genomic libraries of Streptomyces kanamyceticus and Micromonospora echinospora, respectively . The sequencing of the 47 kb-region of S . kanamyceticus genomic DNA revealed 40 putative open reading frames (ORFs) encoding Km biosynthetic proteins, regulatory proteins, and resistance and transport proteins . Similarly, the sequencing of 32.6 kb genomic DNA of M . echinospora revealed a Gm biosynthetic gene cluster flanked by resistant genes . Biosynthetic pathways for the formation of Km were proposed by the comparative study of biosynthetic genes . Out of 12 putative Km biosynthetic genes, kanA was expressed in Escherichia coli and determined its function as a 2-deoxy-scyllo-inosose synthase . Furthermore, the acetylations of aminoglycoside-aminocyclitols (AmAcs) by Km acetyltransferase (KanM) were also demonstrated . The acetylated derivatives completely lost their antibacterial activities against Bacillus subtilis . The comparative genetic studies of Gm, Km, tobramycin (Tm), and butirosin (Bn) reveal their similar biosynthetic routes and provide a framework for the further biosynthetic studies.

J Mol Biol, 2004 Jul 30, 341(1), 135 - 50
A multicomponent protein complex mediates environmental stress signaling in Bacillus subtilis; Kim TJ et al.; Activity of the general stress transcription factor sigma(B) of Bacillus subtilis is regulated directly by a partner-switching mechanism in which key protein interactions are governed by serine phosphorylation . Signals of energy or environmental stress are conveyed to sigma(B) by independent pathways, each terminating with a differentially regulated serine phosphatase, whose activity is required to control the partner-switching regulators . We present genetic and biochemical evidence that activation of the RsbU environmental signaling phosphatase is modulated by a second, atypical partner switch that comprises redundant negative regulatory proteins in a large, multicomponent signaling complex . In the current model, negative regulation of the RsbU phosphatase depends solely on the RsbS antagonist protein . Here, we perform a critical genetic test that invalidates this model and demonstrates that the RsbS antagonist alone is insufficient to prevent environmental signaling . Also required is one of a family of four co-antagonist proteins, here renamed RsbRA, RsbRB, RsbRC, and RsbRD, each with a carboxyl-terminal domain closely resembling the entire RsbS protein . Because any single member of the RsbR family, together with RsbS, was sufficient for environmental signaling, we conclude that the RsbR proteins serve as redundant co-antagonists necessary for RsbS antagonist function . Moreover, purification of RsbRA from cell extracts by nickel affinity and gel-filtration chromatography found a multicomponent complex containing the RsbRA and RsbRB co-antagonists together with the RsbS antagonist . We propose that this complex serves as a machine to transmit stress signals to sigma(B), and that the properties of the complex may contribute to environmental stress sensing.

Biotechnol Appl Biochem . 2004 Aug 13; {Epub ahead of print}
High-level expression of a neoagarobiose-producing beta-agarase gene from Agarivorans sp . strain JAMB-A11 in Bacillus subtilis and enzymatic properties of the recombinant enzyme; Ohta Y et al.; The structural gene for a neoagarobiose-producing beta-agarase of an Agarivorans isolate was expressed in Bacillus subtilis . High-level production of the recombinant enzyme was achieved corresponding to a level of 1.9 x 10 4 U/l of the culture broth . The efficiency of production is thus 30-fold greater than that of the original strain . The recombinant enzyme had a molecular mass of 105 kDa and a specific activity of 371 U/mg . The optimal pH and temperature of the enzyme was 7.5-8.0 and 40 degrees C, respectively . The enzyme is an endo-type beta-agarase hydrolyzing not only agarose but also neoagarotetraose to yield neoagarobiose as the final main product, representing about 90 mol% of total products . RagaA11 would be useful for industrial production of neoagarobiose.

J Org Chem, 2004 Aug 20, 69(17), 5588 - 94
Rapid preparation of isotopolog libraries by in vivo transformation of 1)C-glucose . Studies on 6,7-dimethyl-8-ribityllumazine, a biosynthetic precursor of vitamin B2; Illarionov B et al.; An Escherichia coli strain engineered for expression of the ribABGH genes of Bacillus subtilis was shown to produce 100 mg of the riboflavin precursor 6,7-dimethyl-8-ribityllumazine per liter of minimal medium . Growth of the recombinant strain in medium supplemented with {U-13C6}glucose and/or 15NH4Cl as single sources of carbon and/or nitrogen afforded 6,7-dimethyl-8-ribityllumazine universally labeled with 13C and/or 15N . The yield of {U-13C13}-6,7-dimethyl-8-ribityllumazine based on {U-13C6}glucose was 25 mg/g . Fermentation with {1-13C1}-, {2-13C1}-, or {3-13C1}glucose afforded mixtures of 6,7-dimethyl-8-ribityllumazine isotopologs, predominantly with 13C enrichment of single carbon atoms . The isotope-labeled samples enabled a comprehensive NMR analysis of 6,7-dimethyl-8-ribityllumazine . Isotopolog libraries of a wide variety of microbial metabolites can be produced by the same experimental approach .

Biochemistry, 2004 Aug 17, 43(32), 10490 - 501
4-Oxalocrotonate tautomerase, its homologue YwhB, and active vinylpyruvate hydratase: synthesis and evaluation of 2-fluoro substrate analogues; Johnson WH Jr et al.; A series of 2-fluoro-4-alkene and 2-fluoro-4-alkyne substrate analogues were synthesized and examined as potential inhibitors of three enzymes: 4-oxalocrotonate tautomerase (4-OT) and vinylpyruvate hydratase (VPH) from the catechol meta-fission pathway and a closely related 4-OT homologue found in Bacillus subtilis designated YwhB . All of the compounds were potent competitive inhibitors of 4-OT with the monocarboxylated 2E-fluoro-2,4-pentadienoate and the dicarboxylated 2E-fluoro-2-en-4-ynoate being the most potent . Despite the close mechanistic and structural similarities between 4-OT and YwhB, these compounds were significantly less potent inhibitors of YwhB with K(i) values ranging from 5- to 633-fold lower than those determined for 4-OT . The study of VPH is complicated by the fact that the enzyme is only active as a complex with the metal-dependent 4-oxalocrotonate decarboxylase (4-OD), the enzyme following 4-OT in the catechol meta-fission pathway . A structure-based sequence analysis identified 4-OD as a member of the fumarylacetoacetate hydrolase (FAH) superfamily and implicated Glu-109 and Glu-111 as potential metal-binding ligands . Changing these residues to a glutamine verified their importance for enzymatic activity and enabled the production of soluble E109Q4-OD/VPH or E111Q4-OD/VPH complexes, which retained full hydratase activity but had little decarboxylase activity . Subsequent incubation of the E109Q4-OD/VPH complex with the substrate analogues identified the 2E and 2Z isomers of the monocarboxylated 2-fluoropent-2-en-4-ynoate as competitive inhibitors . The combined results set the stage for crystallographic studies of 4-OT, YwhB, and VPH using these inhibitors as ligands.

Biochemistry, 2004 Aug 17, 43(32), 10370 - 8
Evolution of enzymatic activities in the enolase superfamily: structure of a substrate-liganded complex of the L-Ala-D/L-Glu epimerase from Bacillus subtilis; Klenchin VA et al.; The members of the mechanistically diverse enolase superfamily share a bidomain structure formed from a (beta/alpha)7beta-barrel domain {a modified (beta/alpha)8- or TIM-barrel} and a capping domain formed from N- and C-terminal segments of the polypeptide . The active sites are located at the interface between the C-terminal ends of the beta-strands in the barrel domain and two flexible loops in the capping domain . Within this structure, the acid/base chemistry responsible for formation and stabilization of an enediolate intermediate derived from a carboxylate anion substrate and the processing of it to product is "hard-wired" by functional groups at the C-terminal ends of the beta-strands in the barrel domain; the identity of the substrate is determined in part by the identities of residues located at the end of the eighth beta-strand in the barrel domain and two mobile loops in the capping domain . On the basis of the identities of the acid/base functional groups at the ends of the beta-strands, the currently available structure-function relationships derived from functionally characterized members are often sufficient for "deciphering" the identity of the chemical reaction catalyzed by sequence-divergent members discovered in genome projects . However, insufficient structural information for liganded complexes for specifying the identity of the substrate is available . In this paper, the structure of the complex of L-Ala-L-Glu with the L-Ala-D/L-Glu epimerase from Bacillus subtilis is reported . As expected for the 1,1-proton transfer reaction catalyzed by this enzyme, the alpha-carbon of the substrate is located between Lys 162 and Lys 268 at the ends of the second and sixth beta-strands in the barrel domain . The alpha-ammonium group of the l-Ala moiety is hydrogen bonded to both Asp 321 and Asp 323 at the end of the eighth beta-strand, revealing a novel strategy for substrate recognition in the superfamily . The delta-carboxylate group of the Glu moiety is hydrogen bonded to Arg 24 in one of the flexible loops in the capping domain, thereby providing a structural explanation for the restricted substrate specificity of this epimerase {Schmidt, D . M., Hubbard, B . K., and Gerlt, J . A . (2001) Biochemistry 40, 15707-15715} . These studies provide important new information about the structural bases for substrate specificity in the enolase superfamily.

Biochemistry, 2004 Aug 17, 43(32), 10314 - 27
The formylglycinamide ribonucleotide amidotransferase complex from Bacillus subtilis: metabolite-mediated complex formation; Hoskins AA et al.; Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP- and glutamine-dependent formation of formylglycinamidine ribonucleotide, ADP, P(i), and glutamate in the fourth step of de novo purine biosynthesis . Like all amidotransferases (ATs), FGAR-AT is proposed to channel ammonia between a glutaminase and AT domain . In Gram-negative bacteria and eukaryotes, FGAR-AT is a single approximately 140 kDa protein . In archae and Gram-positive bacteria, the FGAR-AT is formed from three proteins: PurS (10 kDa), PurQ (25 kDa, a glutaminase), and smPurL (80 kDa, an AT) . This is the only known AT to require a third structural component (PurS) for activity . Here we report the first purification and biochemical characterization of a three-component AT from Bacillus subtilis . Efforts to isolate an intact FGAR-AT focused initially on coexpression of PurS, smPurL, and PurQ . However, all attempts to purify the complex resulted in separation of the constituent proteins . PurS, smPurL, and PurQ were therefore separately expressed and purified to homogeneity . PurQ had a glutaminase activity of 0.002 s(-1), and smPurL had an ammonia-dependent AT activity of 0.044 s(-1) . Reconstitution of PurS, smPurL, and PurQ at a ratio of 2:1:1 gave an activity of 2.49 s(-1), similar to that previously reported for the Escherichia coli 140 kDa FGAR-AT (5.00 s(-1)) . PurS was essential for the glutamine-dependent FGAR-AT activity . Surprisingly, activity was found to be absolutely dependent on the presence of Mg2+ and ADP, and a stable FGAR-AT complex of 2PurS/1smPurL/1PurQ was detected only in the presence of Mg2+, ADP, and glutamine . The implications of these observations are discussed with respect to ammonia channeling.

Acta Crystallogr D Biol Crystallogr, 1996 May, 52(Pt 3), 589 - 90
Crystallization and preliminary X-ray analysis of a Y13S mutant of Spo0F from Bacillus subtilis; Madhusudan; Spo0F, a member of a superfamily of bacterial response regulatory proteins, is crucial to the regulation of sporulation in Bacillus subtilis . As there were difficulties in reproducing crystals of wild-type Spo0F, we report here the crystallization and preliminary studies of a mutant, Y13S protein, which gave well diffracting reproducible crystals . The crystals of the mutant obtained by the hanging-drop method belong to the tetragonal space group P4(1)2(1)2 (P4(3)2(1)2) a = b = 105.1, c = 85.9 A . Diffraction data were collected at 2.8 A at the laboratory source and subsequently 2.05 . A data were collected upon flash freezing the crystal at the Stanford Synchrotron Radiation Laboratory . This mutant participates in the phosphorelay in a similar manner to the wild-type protein . The presence of divalent cations are essential for wild-type phosphorylation and the present mutant crystal form is obtained in the presence of calcium.

Acta Crystallogr D Biol Crystallogr, 1996 Nov, 52(Pt 6), 1194 - 5
Crystallization of a novel esterase which inactivates the macrolide toxin brefeldin A; Wei Y; A novel esterase obtained from Bacillus subtilis and capable of hydrolyzing the phytotoxin brefeldin A was crystallized using the hanging-drop technique . The crystals have two forms and both are monoclinic: form I, space group P2(1) with a = 101.7, b = 64.1, c = 55.4 A and beta = 102.5 degrees, and form II, space group C2 with a = 140.7, b = 82.6, c = 81.5 A and beta = 112.5 degrees . There are two molecules related by a pronounced non-crystallographic dyad per asymmetric unit in both crystal forms . The crystals diffract to 2.3 A using a rotating-anode X-ray source.

Appl Environ Microbiol, 2004 Aug, 70(8), 4711 - 9
Identification of an Na(+)-dependent transporter associated with saxitoxin-producing strains of the cyanobacterium Anabaena circinalis; Pomati F et al.; Blooms of the freshwater cyanobacterium Anabaena circinalis are recognized as an important health risk worldwide due to the production of a range of toxins such as saxitoxin (STX) and its derivatives . In this study we used HIP1 octameric-palindrome repeated-sequence PCR to compare the genomic structure of phylogenetically similar Australian isolates of A . circinalis . STX-producing and nontoxic cyanobacterial strains showed different HIP1 (highly iterated octameric palindrome 1) DNA patterns, and characteristic interrepeat amplicons for each group were identified . Suppression subtractive hybridization (SSH) was performed using HIP1 PCR-generated libraries to further identify toxic-strain-specific genes . An STX-producing strain and a nontoxic strain of A . circinalis were chosen as testers in two distinct experiments . The two categories of SSH putative tester-specific sequences were characterized by different families of encoded proteins that may be representative of the differences in metabolism between STX-producing and nontoxic A . circinalis strains . DNA-microarray hybridization and genomic screening revealed a toxic-strain-specific HIP1 fragment coding for a putative Na(+)-dependent transporter . Analysis of this gene demonstrated analogy to the mrpF gene of Bacillus subtilis, whose encoded protein is involved in Na(+)-specific pH homeostasis . The application of this gene as a molecular probe in laboratory and environmental screening for STX-producing A . circinalis strains was demonstrated . The possible role of this putative Na(+)-dependent transporter in the toxic cyanobacterial phenotype is also discussed, in light of recent physiological studies of STX-producing cyanobacteria.

Biochem J, 2004 Nov 15, 384(Pt 1), 169 - 78
Studies of SpoIIAB mutant proteins elucidate the mechanisms that regulate the developmental transcription factor sigmaF in Bacillus subtilis; Shu JC et al.; SigmaF, the first compartment-specific sigma factor of sporulation, is regulated by an anti-sigma factor, SpoIIAB (AB) and its antagonist SpoIIAA (AA) . AB can bind to sigmaF in the presence of ATP or to AA in the presence of ADP; in addition, AB can phosphorylate AA . The ability of AB to switch between its two binding partners regulates sigmaF . Early in sporulation, AA activates sigmaF by releasing it from its complex with AB . We have previously proposed a reaction scheme for the phosphorylation of AA by AB which accounts for AA's regulatory role . A crucial feature of this scheme is a conformational change in AB that accompanies its switch in binding partner . In the present study, we have studied three AB mutants, all of which have amino-acid replacements in the nucleotide-binding region; AB-E104K (Glu104-->Lys) and AB-T49K (Thr49-->Lys) fail to activate sigmaF, and AB-R105A (Arg105-->Ala) activates it prematurely . We used techniques of enzymology, surface plasmon resonance and fluorescence spectroscopy to analyse the defects in each mutant . AB-E104K was deficient in binding to AA, AB-T49K was deficient in binding to ADP and AB-R105A bound ADP exceptionally strongly . Although the release of sigmaF from all three mutant proteins was impaired, and all three failed to undergo the wild-type conformational change when switching binding partners, the phenotypes of the mutant cells were best accounted for by the properties of the respective AB species in forming complexes with AA and ADP . The behaviour of the mutants enables us to propose convincing mechanisms for the regulation of sigmaF in wild-type bacteria.

J Biol Chem, 2004 Oct 15, 279(42), 43555 - 9 Epub 2004 Jul 28.
A genetic screen for the identification of thiamin metabolic genes; Lawhorn BG et al.; A genetic screen was developed for the identification of genes related to thiamin biosynthesis and degradation . Genes conferring resistance to bacimethrin or 4-amino-2-trifluoromethyl-5-hydroxymethylpyrimidine were selected from Escherichia coli and Bacillus subtilis genomic libraries . Hits from the selection included the known thiamin biosynthetic genes thiC, thiE, and dxs as well as five genes of previously unknown function (E . coli yjjX, yajO, ymfB, and cof and B . subtilis yveN) . The gene products YmfB and Cof catalyze the hydrolysis of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate to 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate . YmfB also converts thiamin pyrophosphate into thiamin phosphate.

J Biol Chem, 2004 Oct 15, 279(42), 43468 - 78 Epub 2004 Jul 29.
The PDZ domain of the SpoIVB transmembrane signaling protein enables cis-trans interactions involving multiple partners leading to the activation of the pro-sigmaK processing complex in Bacillus subtilis; Dong TC et al.; In sporulating cells of Bacillus subtilis, the serine peptidase SpoIVB is the essential component of a transmembrane signaling cascade between the two intracellular compartments (forespore and mother cell) that leads to activation of the sigmaK transcription factor in the mother cell chamber . This regulatory process, referred to as the sigmaK checkpoint, is essential for ensuring proper development of the spore and introduces an appropriate level of fidelity to the developmental process . This work unravels the signaling process and establishes how SpoIVB interacts with other protein partners in the sigmaK checkpoint . SpoIVB is synthesized as a zymogen that is autoproteolytically activated and carries a PDZ domain that is responsible for at least three distinct binding reactions, a phenomenon not previously demonstrated for an individual PDZ domain . First, binding to the SpoIVB NH2 terminus to maintain the protein in its zymogen form . Second, following secretion across a spore membrane, binding in trans to the COOH terminus of another SpoIVB molecule . Binding in trans facilitates the first cleavage event of SpoIVB near the NH2 terminus releasing it from the inner forespore membrane . We show that at least two further cis cleavage events occur at specific sites near the NH2 terminus after which the PDZ domain targets SpoIVB to the pro-sigmaK processing complex in the outer forespore membrane . Specifically, SpoIVB binds to the COOH terminus of BofA . In turn, this allows SpoIVB to cleave the COOH terminus of SpoIVFA an event pivotal to activating the SpoIVFB zinc metalloprotease by disruption of the heteroligomeric pro-sigmaK complex.

J Bacteriol, 2004 Aug, 186(16), 5450 - 9
spoIVH (ykvV), a requisite cortex formation gene, is expressed in both sporulating compartments of Bacillus subtilis; Imamura D et al.; It is well known that the ykvU-ykvV operon is under the regulation of the sigma(E)-associated RNA polymerase (Esigma(E)) . In our study, we observed that ykvV is transcribed together with the upstream ykvU gene by Esigma(E) in the mother cell and monocistronically under Esigma(G) control in the forespore . Interestingly, alternatively expressed ykvV in either the forespore or the mother cell increased the sporulation efficiency in the ykvV background . Studies show that the YkvV protein is a member of the thioredoxin superfamily and also contains a putative Sec-type secretion signal at the N terminus . We observed efficient sporulation in a mutant strain obtained by replacing the putative signal peptide of YkvV with the secretion signal sequence of SleB, indicating that the putative signal sequence is essential for spore formation . These results suggest that YkvV is capable of being transported by the putative Sec-type signal sequence into the space between the double membranes surrounding the forespore . The ability of ykvV expression in either compartment to complement is indeed intriguing and further introduces a new dimension to the genetics of B . subtilis spore formation . Furthermore, electron microscopic observation revealed a defective cortex in the ykvV disruptant . In addition, the expression levels of sigma(K)-directed genes significantly decreased despite normal sigma(G) activity in the ykvV mutant . However, immunoblotting with the anti-sigma(K) antibody showed that pro-sigma(K) was normally processed in the ykvV mutant, indicating that YkvV plays an important role in cortex formation, consistent with recent reports . We therefore propose that ykvV should be renamed spoIVH.

J Bacteriol, 2004 Aug, 186(16), 5392 - 9
Kinetic analysis of tRNA-directed transcription antitermination of the Bacillus subtilis glyQS gene in vitro; Grundy FJ et al.; Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription antitermination mechanism . Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA . tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix . tRNA(Gly)-dependent antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system . This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and antitermination . Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on antitermination efficiency was analyzed . We found that addition of tRNA(Gly) can promote antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.

J Bacteriol, 2004 Aug, 186(16), 5376 - 83
Bacillus subtilis YhcR, a high-molecular-weight, nonspecific endonuclease with a unique domain structure; Oussenko IA et al.; In a continuing effort to identify ribonucleases that may be involved in mRNA decay in Bacillus subtilis, fractionation of a protein extract from a triple-mutant strain that was missing three previously characterized 3'-to-5' exoribonucleases (polynucleotide phosphorylase {PNPase}, RNase R, and YhaM) was undertaken . These experiments revealed the presence of a high-molecular-weight nuclease encoded by the yhcR gene that was active in the presence of Ca(2+) and Mn(2+) . YhcR is a sugar-nonspecific nuclease that cleaves endonucleolytically to yield nucleotide 3'-monophosphate products, similar to the well-characterized micrococcal nuclease of Staphylococcus aureus . YhcR appears to be located principally in the cell wall and is likely to be a substrate for a B . subtilis sortase . Zymogram analysis suggests that YhcR is the major Ca(2+)-activated nuclease of B . subtilis . In addition to having a unique overall domain structure, YhcR contains a hitherto unknown structural domain that we have named "NYD," for "new YhcR domain."

J Bacteriol, 2004 Aug, 186(16), 5355 - 65
Differential and cross-transcriptional control of duplicated genes encoding alternative sigma factors in Streptomyces ambofaciens; Roth V et al.; The duplicated hasR and hasL genes of Streptomyces ambofaciens encode alternative sigma factors (named sigma(B(R)) and sigma(B(L))) belonging to the sigma(B) general stress response family in Bacillus subtilis . The duplication appears to be the result of a recent event that occurred specifically in S . ambofaciens . The two genes are 98% identical, and their deduced protein products exhibit 97% identity at the amino acid level . In contrast with the coding sequences, their genetic environments and their transcriptional control are strongly divergent . While hasL is monocistronic, hasR is arranged in a polycistronic unit with two upstream open reading frames, arsR and prsR, that encode putative anti-anti-sigma and anti-sigma factors, respectively . Transcription of each has gene is initiated from two promoters . In each case, one promoter was shown to be developmentally controlled and to be similar to those recognized by the B . subtilis general stress response sigma factor sigma(B) . Expression from this type of promoter for each of the has genes dramatically increases during the course of growth in liquid or on solid media and following oxidative and osmotic stresses . Reverse transcription-PCR measurements indicate that hasR is 100 times more strongly expressed than hasL from the sigma(B)-like promoter . Transcription from the second promoter of each gene (located upstream of arsR in the case of the hasR locus) appears to be constitutive and weak . Quantitative transcriptional analysis in single and double has mutant strains revealed that sigma(B(R)) and sigma(B(L)) direct their own transcription as well as that of their duplicates . Only a slight sensitivity in response to oxidative conditions could be assigned to either single or double mutants, revealing the probable redundancy of the sigma factors implied in stress response in Streptomyces.

J Agric Food Chem, 2004 Aug 11, 52(16), 5052 - 6
Extracellular production of a functional soy cystatin by Bacillus subtilis; Kang IS et al.; A recombinant Bacillus subtilis producing soy cystatin was developed by subcloning with a soy cystatin gene cloned in Escherichia coli . An active form of cystatin against the cysteine protease from Pacific whiting fillets contaminated with Myxosporidia parasite was constitutively expressed and secreted extracelluarly into the medium . Two gene fragments of signal peptides from kerA and sacB were introduced and compared for secretion efficiency of cystatin . The secretion level of active cystatin improved with the signal peptide of kerA when compared to that of sacB . Inhibitor activity was reduced rapidly after peak expression of the target protein at 36 h of fermentation . The addition of 1% glucose, a suppressor of protease, into the medium sustained the increase of the cystatin activity during fermentation . This study introduced a potential new method for fermentation production of cystatin.

Microbiology, 2004 Aug, 150(Pt 8), 2619 - 28
Post-transcriptional regulation of the Bacillus subtilis pst operon encoding a phosphate-specific ABC transporter; Allenby NE et al.; During phosphate starvation, Bacillus subtilis regulates genes in the PhoP regulon to reduce the cell's requirement for this essential substrate and to facilitate the recovery of inorganic phosphate from organic sources such as teichoic and nucleic acids . Among the proteins that are highly induced under these conditions is PstS, the phosphate-binding lipoprotein component of a high-affinity ABC-type phosphate transporter . PstS is encoded by the first gene in the pst operon, the other four members of which encode the integral membrane and cytoplasmic components of the transporter . The transcription of the pst operon was analysed using a combination of methods, including transcriptional reporter gene technology, Northern blotting and DNA arrays . It is shown that the primary transcript of the pst operon is processed differentially to maintain higher concentrations of PstS relative to other components of the transporter . The comparative studies have revealed limitations in the use of reporter gene technology for analysing the transcription of operons in which the messenger RNA transcript is differentially processed.

Microbiology, 2004 Aug, 150(Pt 8), 2609 - 17
Characterization of the Bacillus subtilis YxdJ response regulator as the inducer of expression for the cognate ABC transporter YxdLM; Joseph P et al.; The genome of Bacillus subtilis, like those of some other AT-rich Gram-positive bacteria, has the uncommon feature of containing several copies of arrangements in which the genes encoding two-component and cognate ABC transporter systems are adjacent . As the function of one of these systems, the product of the yxd locus, is still unknown, it was analysed further in order to get some clues on the physiological role of the gene products it encodes . The yxdJ gene was shown to encode a DNA-binding protein that directly controls transcription of the neighbouring operon encoding the ABC transporter YxdLM . Primer extension and DNase protection experiments allowed precise definition of the yxdLM transcription start and controlling region . Two putative direct repeats were identified that are proposed to be the YxdJ response regulator binding sites . Whole-cell transcriptome analyses revealed that the YxdJ regulon is extremely restricted . In addition to the yxdJKLMyxeA operon, only a few genes involved in modifications of the bacterial cell wall were shown to be regulated by YxdJ.

Phytother Res, 2004 Jun, 18(6), 497 - 500
Antibacterial activity of the Chinese traditional medicine, Zi Hua Di Ding; Xie C et al.; The antibacterial activity of extracts from species of plants used in the Chinese medicine, Zi Hua Di Ding (Viola yedoensis, V . prionantha, Corydalis bungeana and Gueldenstaedtia verna), was tested against Bacillus subtilis and Pseudomonas syringae using a bioautographic assay . The petroleum ether and ethyl acetate extracts of all four species of plants showed activity against both species of bacteria, whereas the methanol and aqueous methanol extracts were inactive . Three fractions from the petroleum ether extracts of V . yedoensis, V . prionantha and C . bungeana showed activity against B . subtilis at 6.25 microg/mL . Preliminary analysis of these active fractions indicates that they contain long chain carboxylic acids.

Biotechnol Bioeng, 2004 Aug 20, 87(4), 501 - 15
Empirical modeling of batch fermentation kinetics for poly(glutamic acid) production and other microbial biopolymers; Richard A et al.; An empirical kinetic model is proposed for the batch production of poly(glutamic acid) from Bacillus subtilis IFO 3335 . In addition, the proposed model was used to fit the kinetic data of poly(glutamic acid) production from other bacterial strains using different media, as well as kinetic data from different strains for the production of the exocellular biopolymers dextran, hyaluronic acid, xanthan, alginate, and the endocellular biopolymer polyhydroxybutyrate . The empirical model treats the biopolymer as a component of the biomass and fits the experimental biomass data using a sigmoidal relationship that includes the maximum specific growth rate, mu(max), and the substrate saturation parameter, K(S) . An empirical parameter, the relative coefficient (r), quantifies, in relative terms, the degree of nongrowth-associated biopolymer formation.

Biotechnol Bioeng, 2004 Aug 20, 87(4), 459 - 64
Increased production of Bacillus keratinase by chromosomal integration of multiple copies of the kerA gene; Wang JJ et al.; To increase the production of keratinase, stable strains of Bacillus licheniformis carrying multiple keratinase gene copies in the chromosome were developed . Integrative vectors carrying kerA with or without P43-promoter were constructed and subcloned into B . licheniformis T399D and Bacillus subtilis DB104 . In T399D, multiple copies of kerA integration into the chromosome were identified and determined by Southern blot . The optimal integration of kerA was found in the range of 3-5 copies . Higher integration of gene copies (>5) caused reduced processing and secretion of the extracellular keratinase . In DB104, kerA was cloned in the plasmid, not integrated into the chromosome . The strong constitutive promoter P43 not only increased the keratinase production in plasmid-based expression in DB104 but also improved the enzyme yield of the integrants of T399D . New strains were able to enhance cell growth and enzyme yield at higher concentrations of medium substrate . When they were grown in either soy or feather medium, the keratinase activity was stable and improved by about 4-6 times.

Mol Biol (Mosk), 2004 May-Jun, 38(3), 437 - 41
{The replication system of plasmids from Bacillus subtilis environmental isolates}; Lagodich AV et al.; Restriction enzyme analysis, cloning, and sequencing showed that large (more than 90-kb) plasmids isolated from different Bacillus subtilis strains are identical in structure of the region ensuring stable inheritance of plasmid replicons and are widespread in Belarussian environmental strains of B . subtilis.

Can J Microbiol, 2004 Jun, 50(6), 451 - 4
Enhancement of the antifungal activity of Bacillus subtilis F29-3 by the chitinase encoded by Bacillus circulans chiA gene; Chen CY et al.; Bacillus subtilis F29-3 is an antagonistic bacterium against a wide range of fungal species . In order to determine the effect of chitinase on the antifungal activity of B . subtilis F29-3, a 2.4-kb DNA fragment containing the chiA gene of Bacillus circulans WL-12 was ligated into a shuttle vector pHY300PLK and transformed into B . subtilis F29-3 . A bioassay conducted on the culture supernatant showed that, in comparison to the B . subtilis control strain, B . subtilis F29-3 expressing the chiA gene exhibited a greater inhibition of spore germination of Botrytis elliptica, indicating that chitinase could enhance the antifungal function conferred by B . subtilis F29-3.

Avian Dis, 2004 Apr-Jun, 48(2), 287 - 99
Construction, characterization, and evaluation of the vaccine potential of three genetically defined mutants of avian pathogenic Escherichia coli; Kariyawasam S et al.; The delta galE, delta purA, and delta aroA derivatives of avian septicemic Escherichia coli EC99 strain (O78 serogroup) were constructed with a suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis . The resultant isogenic mutants were stable and lacked approximately 45%, 36%, and 52% of the genes for galE, purA, and aroA, respectively . The delta purA and delta aroA mutants did not grow on minimal medium, whereas the delta galE mutant grew on minimal medium but was sensitive to galactose-induced lysis . The reversion frequencies of all three mutants were <10(-12) . The mutants were highly attenuated for virulence as determined by administration of approximately 10(7) colony-forming units of each mutant to 1-day-old chicks by the subcutaneous route . Chickens were vaccinated with the mutants by spray (droplet size approximately 20 microm) at 1 and 14 days of age to determine safety, immunogenicity, and efficacy . The mutants were found to be safe . Seven days after a second vaccination, immunoglobulin (Ig)Y antibodies to E . coli in serum and air sac washings were detected by enzyme-linked immunosorbent assay . In both serum and air sac washings, IgY antibodies were significantly higher in chickens vaccinated with the mutants as compared with phosphate-buffered saline-treated controls but were significantly lower compared with chickens that were vaccinated with the parent strain . In serum, but not in air sac washings, IgY antibodies were significantly lower in chickens vaccinated with the mutants compared with the parent strain . The vaccinated chickens were given infectious bronchitis virus intranasally at 17 days of age and were challenged with homologous (EC99 strain) or heterologous (O2 serogroup) E . coli 4 days later . Chickens that received wild-type EC99 strain or its mutant derivatives were protected from homologous but not from heterologous challenge . This study indicates that the delta galE, delta purA, and delta aroA mutants are safe and moderately immunogenic but the protection conferred by the mutants is serogroup specific.

Antonie Van Leeuwenhoek, 2004 Aug, 86(2), 149 - 58
Nucleotides downstream of start codons show marked non-randomness in Escherichia coli but not in Bacillus subtilis; Fuglsang A; This study aimed at measuring the nucleotide non-randomness in the region downstream of start codons in bacterial genes and to see if the non-randomness differs between biased and unbiased genes, in terms of the effective number of codons (Nc) and the codon adaptation index (CAI) . In Escherichia coli, there was a marked elevation in nucleotide conservation for the genes having low Nc-values compared to the genes having high Nc-values, i.e the more biased genes showed a higher level of non-randomness . Likewise, the genes displaying high CAI-values showed stronger nucleotide conservation than the genes of low CAI-values . This elevated conservation is visible up to approximately 15-17 nucleotides downstream of the start codon, after which there is little difference . This indicates that there may be distinct selectional mechanisms acting upon the first 5-6 codons within genes in E . coli . In B . subtilis, these effects are less pronounced, if present at all . Furthermore, analyses of codons used in this region were not in support of the hypothesis that the elevation in nucleotide non-randomness is a question of selection for certain optimal codons.

J Microbiol Methods, 2004 Sep, 58(3), 327 - 34
Development of a robust microtiter plate-based assay method for assessment of bioactivity; Casey JT et al.; A microtiter plate-based assay was developed for the quantitative monitoring of bioactive compound production in Streptomyces hygroscopicus fermentation samples . The method reported demonstrates the successful application of the theories of disk diffusion based methods of bioactivity assessment, to a microtiter assay for high throughput analysis . The assay method facilitates the generation of the dose-response curve of test organisms (Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae) to a bioactive compound . Using this dose-response curve, the method facilitates definition of three distinct Minimum Inhibitory Concentration (MIC) values for use in the characterisation of the bioactive attributes of a sample . The assay uses established standard procedures to facilitate adaptation of the assay for use with a wider range of test microorganisms . Errors due to the assumption of a linear relationship between turbidity and biomass concentration are also reduced, due to incorporation of a step to convert turbidity to biomass concentration, for use in the calculation of bioactivity.

Proteomics, 2004 Aug, 4(8), 2408 - 24
Quantitative proteome profiling during the fermentation process of pleiotropic Bacillus subtilis mutants; Antelmann H et al.; Using a combined quantitative proteomic and bioinformatic approach, we monitored the cytoplasmic proteome profile of the Gram-positive bacterium Bacillus subtilis during a fermentation process in complex medium . Proteome signatures were applied to elucidate the physiological changes occurring in the gene expression profile during growth . Furthermore, we determined the significance level of quantitative proteome changes, identified relative to the threshold of scatter in replicated samples and developed a statistically rigorous method that allowed us to determine significant fold-changes at 95% confidence between different proteomes . Different functional groups of proteins were clustered according to their pattern of significant expression changes . The largest group is induced by the exhaustion of glucose and the presence of alternative carbon and nitrogen sources . Furthermore, depletion of glucose caused the induction of the trichloroacetic acid (TCA) cycle enzymes and the downregulation of glycolytic enzymes . The onset of the transition phase may be provoked by amino acid starvation, resulting in the RelA-dependent repression of proteins involved in the translation process and in the induction of amino acid biosynthetic pathways . Comparisons between the parental strain and two subtilisin-hypersecreting strains revealed only small cytoplasmic differences in the main metabolic pathways . Instead, the overproduction of degradative enzymes in both of these mutants was reflected in the extracellular proteome.

Protein Sci, 2004 Aug, 13(8), 2184 - 95
The C-terminal domain of dimeric serine hydroxymethyltransferase plays a key role in stabilization of the quaternary structure and cooperative unfolding of protein: domain swapping studies with enzymes having high sequence identity; Bhatt AN et al.; The serine hydroxymethyltransferase from Bacillus subtilis (bsSHMT) and B . stearothermophilus (bstSHMT) are both homodimers and share approximately 77% sequence identity; however, they show very different thermal stabilities and unfolding pathways . For investigating the role of N- and C-terminal domains in stability and unfolding of dimeric SHMTs, we have swapped the structural domains between bs- and bstSHMT and generated the two novel chimeric proteins bsbstc and bstbsc, respectively . The chimeras had secondary structure, tyrosine, and pyridoxal-5'-phosphate microenvironment similar to that of the wild-type proteins . The chimeras showed enzymatic activity slightly higher than that of the wild-type proteins . Interestingly, the guanidium chloride (GdmCl)-induced unfolding showed that unlike the wild-type bsSHMT, which undergoes dissociation of native dimer into monomers at low guanidium chloride (GdmCl) concentration, resulting in a non-cooperative unfolding of enzyme, its chimera bsbstc, having the C-terminal domain of bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding from native dimer to unfolded monomer . In contrast, the wild-type dimeric bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding, whereas its chimera bstbsc, having the C- terminal domain of bsSHMT, showed dissociation of native dimer into monomer at low GdmCl concentration and a GdmCl-induced non-cooperative unfolding . These results clearly demonstrate that the C-terminal domain of dimeric SHMT plays a vital role in stabilization of the oligomeric structure of the native enzyme hence modulating its unfolding pathway.

Nucleic Acids Res, 2004 Jul 25, 32(13), 3904 - 12 Print 2004.
Identification of important chemical groups of the hut mRNA for HutP interactions that regulate the hut operon in Bacillus subtilis; Kumarevel TS et al.; HutP is an RNA binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus species by binding to cis-acting regulatory sequences on hut mRNA . We recently solved the HutP crystal structure, which revealed a novel fold where three dimers are arranged in a 3-fold axis to form the hexamer . We also identified a minimal RNA binding element sufficient for HutP binding: three UAG trinucleotide motifs, each separated by 4 nt, located just upstream of the terminator . In the present study we have identified important RNA chemical groups essential for HutP interactions, by combining an in vitro selection strategy and analyses by site-specific base substitutions . These analyses suggest that each HutP molecule recognizes one UAG motif, where the first base (U) can be substituted with other bases, while the second and third bases (A and G) are required for the interactions . Further analyses of the chemical groups of the A and G bases in the UAG motif by modified base analogs suggested the importance of the exocyclic NH2 group in these bases . Also, in this motif, only the 2'-OH group of A is important for HutP recognition . Considering the important chemical groups identified here, as well as the electrostatic potential analysis of HutP, we propose that Glu137 is one of the important residues for the HutP-RNA interactions.

Antimicrob Agents Chemother, 2004 Aug, 48(8), 2888 - 96
Antibiotic-inducible promoter regulated by the cell envelope stress-sensing two-component system LiaRS of Bacillus subtilis; Mascher T et al.; Soil bacteria are among the most prodigious producers of antibiotics . The Bacillus subtilis LiaRS (formerly YvqCE) two-component system is one of several antibiotic-sensing systems that coordinate the genetic response to cell wall-active antibiotics . Upon the addition of vancomycin or bacitracin, LiaRS autoregulates the liaIHGFSR operon . We have characterized the promoter of the lia operon and defined the cis-acting sequences necessary for antibiotic-inducible gene expression . A survey for compounds that act as inducers of the lia promoter revealed that it responds strongly to a subset of cell wall-active antibiotics that interfere with the lipid II cycle in the cytoplasmic membrane (bacitracin, nisin, ramoplanin, and vancomycin) . Chemicals that perturb the cytoplasmic membrane, such as organic solvents, are also weak inducers . Thus, the reporter derived from P(liaI) (the liaI promoter) provides a tool for the detection and classification of antimicrobial compounds.

Antimicrob Agents Chemother, 2004 Aug, 48(8), 2838 - 44
Prediction of mechanisms of action of antibacterial compounds by gene expression profiling; Hutter B et al.; We have generated a database of expression profiles carrying the transcriptional responses of the model organism Bacillus subtilis following treatment with 37 well-characterized antibacterial compounds of different classes . The database was used to build a predictor for the assignment of the mechanisms of action (MoAs) of antibacterial compounds by the use of support vector machines . This predictor was able to correctly classify the MoA class for most compounds tested . Furthermore, we provide evidence that the in vivo MoA of hexachlorophene does not match the MoA predicted from in vitro data, a situation frequently faced in drug discovery . A database of this kind may facilitate the prioritization of novel antibacterial entities in drug discovery programs . Potential applications and limitations are discussed.

Genetika, 2004 May, 40(5), 716 - 20
{Study of the mechanism for regulating ribR gene activity in Bacillus subtilis}; Dynamic movement of actin-like proteins within bacterial cells; Biochemie, Fachbereich Chemie, Hans-Meerwein-Strasse, Philipps-Universitat Marburg, 35032 Marburg, GermanyActin proteins are present in pro- and eukaryotes, and have been shown to perform motor-like functions in eukaryotic cells in a variety of processes . Bacterial actin homologues are essential for cell viability and have been implicated in the formation of rod cell shape, as well as in segregation of plasmids and whole chromosomes . We have generated functional green fluorescent protein fusions of all three Bacillus subtilis actin-like proteins (MreB, Mbl and MreBH), and show that all three proteins form helical filaments underneath the cell membrane, the pattern of which is distinct for each protein . Time-lapse microscopy showed that the filaments are highly dynamic structures . A number of separate filaments of MreB and Mbl continuously move through the cell along helical tracks underneath the cell membrane . The speed of extension of the growing end of filaments is within the range of known actin polymerization (0.1 microm/s), generating a potential poleward or centreward pushing velocity at 0.24 microm/min for MreB or Mbl, respectively . During nutritional downshift and a block in topoisomerase IV activity, the filaments rapidly disintegrated, showing that movement occurs only in growing cells . Contrary to Mbl and MreBH filaments, MreB filaments were generally absent in cells lacking DNA, providing a further distinction between the three orthologues.

Acta Crystallogr D Biol Crystallogr, 2004 Aug, 60(Pt 8), 1435 - 7 Epub 2004 Jul 21.
Expression, purification, crystallization and preliminary X-ray diffraction data of methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis; Dubourg H et al.; Methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis was cloned and overexpressed in Escherichia coli . Suitable crystals for X-ray diffraction experiments were obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant . The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 195.2, b = 192.5, c = 83.5 A, and contain one tetramer per asymmetric unit . X-ray diffraction data were collected to 2.5 A resolution using a synchrotron-radiation source . The crystal structure was solved by the molecular-replacement method.

J Nat Prod, 2004 Jul, 67(7), 1178 - 81
Cyclic peptides from a Ruegeria strain of bacteria associated with the sponge Suberites domuncula; Mitova M et al.; Two new cyclic peptides, cyclo-(glycyl-L-seryl-L-prolyl-L-glutamyl) and cyclo-(glycyl-L-prolyl-L-glutamyl), have been isolated from the cell extract of a Ruegeria strain associated with cell cultures of Suberites domuncula . Three other cyclopeptides have been isolated for the first time from a natural source . Additionally, a new diastereoisomer of a known compound is reported . The structures of isolated compounds have been elucidated by means of spectroscopic data (1D-, 2D-NMR, HRESIMS) and chiral HPLC analysis . The new cyclopeptides exhibited moderate antimicrobial activity against Bacillus subtilis.

Biotechnol Lett, 2004 Jun, 26(12), 975 - 9
Cloning and over-expression of an alkaline protease from Bacillus licheniformis; Tang XM et al.; The alkaline protease gene, apr, from Bacillus licheniformis 2709 was cloned into a Bacillus shuttle expression vector, pHL, to yield the recombinant plasmid pHL-apr . The pHL-apr was expressed in Bacillus subtilis WB600, yielding a high expression strain BW-016 . The amount of alkaline protease produced in the recombinant increased by 65% relative to the original strain . SDS-PAGE analysis indicated a Mr of 30.5 kDa . The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 6816.

Proc Natl Acad Sci U S A, 2004 Aug 3, 101(31), 11386 - 91 Epub 2004 Jul 21.
A specific gene expression program triggered by Gram-positive bacteria in the cytosol; McCaffrey RL et al.; Innate and adaptive immunity depends critically on host recognition of pathogen-associated molecules . Toll-like receptors (TLRs) are key mediators of pathogen surveillance at the cell or phagocytic vacuole surface . However, mechanisms underlying recognition of pathogens in other cellular compartments remain unclear, and responses elicited by cytosolic challenge are poorly characterized . We therefore used mouse cDNA microarrays to investigate gene expression triggered by infection of bone marrow-derived macrophages with cytosol- and vacuole-localized Listeria monocytogenes (Lm), a model cytosolic pathogen . The resulting gene expression program included two basic categories of induced genes: an "early/persistent" cluster consistent with NF-kappaB-dependent responses downstream of TLRs, and a subsequent "late response" cluster largely composed of IFN-responsive genes (IRGs) . The early/persistent cluster was observed upon infection with WT, heat-killed, or mutant Lm lacking listeriolysin O, the pore-forming hemolysin that promotes escape from phagocytic vacuoles . However, the IRG cluster depended on entry of WT Lm into the cytosol . Infection with listeriolysin O-expressing, cytosolic Bacillus subtilis (Bs) strikingly recapitulated the expression profile associated with WT Lm, including IRG induction . IRG up-regulation was associated with MyD88-independent induction of IFN-beta transcription and activity . Whereas Staphylococcus aureus (Sa) lipoteichoic acid treatment confirmed that many late-response genes could also be stimulated through TLRs, our study identified a cytosol-specific transcriptional program independent of TLR signaling through MyD88 . Further characterization of cytosolic surveillance pathway(s) and their points of convergence with TLR- and IFN-dependent pathways will enhance our understanding of the means by which mammals detect and respond to pathogens.

Indian J Exp Biol, 2003 Jun, 41(6), 614 - 9
Isolation and purification of an extracellular protease from a new strain of Bacillus subtilis, viz.NCIM 2711; Surti AM et al.; A new extracellular protease having a prospective application in the food industry was isolated from Bacillus sUbtilis NCIM 2711 by (NH4)2SO4 precipitation from the cell broth . It was purified using DEAE-Cellulose and CM-Sephadex C-50 ion-exchange chromatography . With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 7.0 and temperature 55 degrees C with Km 1.06 mg/ml . The enzyme was stable over a pH range 6.5-8.0 at 30 degrees C for 1 hr in presence of CaCl2 x 2H2O . At 55 degrees C, the enzyme retained 60% activity up to 15 min in presence of CaCl2 x 2H2O . EDTA and o-phenanthroline (OP) completely inhibited the enzyme activity while DFP, PMSF and iodoacetamide were ineffective . The enzyme was completely inhibited by Hg2+ and partially by Cd2+, Cu2+, Ni2+, Pb2+ and Fe2+ . The OP inhibited enzyme could be reactivated by Zn2+ and Co2+ up to 75% and 69% respectively . It is a neutral metalloprotease showing a single band of 43 kDa on SDS-PAGE.

Biotechnol Lett, 2004 Jul, 26(14), 1115 - 8
Construction and application of a promoter-trapping vector with methyl parathion hydrolase gene mpd as the reporter; Cui ZL et al.; A facilitative and efficient promoter-trapping vector, pUC-mpd, was constructed with the promoterless methyl parathion hydrolase gene as the reporter . This reporter gene is easily used to clone promoters with different promoting strength on selective plates . Promoter regions of the ytkA and ywoF genes with strong promoting and signal peptide functions were cloned from the Bacillus subtilis 168 genomic promoter library with this vector.

Anal Biochem, 2004 Aug 15, 331(2), 340 - 8
Development of an enzymatic method for site-specific incorporation of desthiobiotin to recombinant proteins in vitro; Wu SC et al.; To extend the (strept)avidin-biotin technology for affinity purification of proteins, development of reusable biochips and immobilized enzyme bioreactors, selective immobilization of a protein of interest from a crude sample to a protein array without protein purification and many other possible applications, the (strept)avidin-biotin interaction is better when reversible . A gentle enzymatic method to introduce a biotin analog, desthiobiotin, in a site-specific manner to recombinant proteins carrying a biotinylation tag has been developed . The optimal condition for efficient in vitro desthiobiotinylation catalyzed by Escherichia coli biotin ligase (BirA) in 1-4h has been established by systematically varying the substrate concentrations, reaction time, and pH . Real desthiobiotinylation in the absence of any significant biotinylation using this enzymatic method was confirmed by mass spectrometric analysis of the desthiobiotinylated tag . This approach was applied to affinity purify desthiobiotinylated staphylokinase secreted by recombinant Bacillus subtilis to high purity and with good recovery using streptavidin-agarose . The matrix can be regenerated for reuse . This study represents the first successful application of E . coli BirA to incorporate biotin analog to recombinant proteins in a site-specific manner.

Eur J Biochem, 2004 Aug, 271(15), 3146 - 54
Mode of action of the microbial metabolite GE23077, a novel potent and selective inhibitor of bacterial RNA polymerase; Sarubbi E et al.; GE23077, a novel microbial metabolite recently isolated from Actinomadura sp . culture media, is a potent and selective inhibitor of bacterial RNA polymerase (RNAP) . It inhibits Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) RNAPs with IC50 values (i.e . the concentration at which the enzyme activity is inhibited by 50%) in the 10(-8) m range, whereas it is not active on E . coli DNA polymerase or on eukaryotic (wheat germ) RNAP II (IC50 values > 10(-4) m in both cases) . In spite of its potent activity on purified bacterial RNAPs, GE23077 shows a narrow spectrum of antimicrobial activity on Gram-positive and Gram-negative bacteria . To investigate the molecular basis of this behaviour, the effects of GE23077 on macromolecular biosynthesis were tested in E . coli cells permeabilized under different conditions . The addition of GE23077 to plasmolyzed cells resulted in an immediate and specific inhibition of intracellular RNA biosynthesis, in a dose-response manner, strongly suggesting that cell penetration is the main obstacle for effective antimicrobial activity of the antibiotic . Biochemical studies were also conducted with purified enzymes to obtain further insights into the mode of action of GE23077 . Interestingly, the compound displays a behaviour similar to that of rifampicin, an antibiotic structurally unrelated to GE23077: both compounds act at the level of transcription initiation, but not on the sigma subunit and not on the formation of the promoter DNA-RNAP complex . Tests on different rifampicin-resistant E . coli RNAPs did not show any cross-resistance between the two compounds, indicating distinct binding sites on the target enzyme . In conclusion, GE23077 is an interesting new molecule for future mechanistic studies on bacterial RNAP and for its potential in anti-infective drug discovery.

J Struct Funct Genomics, 2004, 5(1-2), 103 - 9
Genome-scale expression of proteins from Bacillus subtilis; Moy S et al.; We have applied high throughput methods for cloning and expression of more than 850 genes from the Bacillus subtilis genome . The process uses 96-well plates and is automated from the level of primer design to the detection of soluble protein by a tag detection screen . This process was applied to a set of cytoplasmic targets from Bacillus subtilis to produce clones expressing soluble protein for incorporation into the structure determination pipeline of the Midwest Center for Structural Genomics . We also evaluated the feasibility of these plate-based methods for domain-based cloning and expression of secretory proteins and putative soluble domains of membrane proteins . This approach shows promise for implementation in a high throughput format and could provide additional target resources for structure determination . The continued development of new technologies that can be implemented in an automated format will be essential for continued success in the structural genomic programs.

J Biol Chem, 2004 Sep 24, 279(39), 40927 - 37 Epub 2004 Jul 19.
Functional and structural characterization of RsbU, a stress signaling protein phosphatase 2C; Delumeau O et al.; RsbU is a positive regulator of the activity of sigmaB, the general stress-response sigma factor of Gram+ microorganisms . The N-terminal domain of this protein has no significant sequence homology with proteins of known function, whereas the C-terminal domain is similar to the catalytic domains of PP2C-type phosphatases . The phosphatase activity of RsbU is stimulated greatly during the response to stress by associating with a kinase, RsbT . This association leads to the induction of sigmaB activity . Here we present data on the activation process and demonstrate in vivo that truncations in the N-terminal region of RsbU are deleterious for the activation of RsbU . This conclusion is supported by comparisons of the phosphatase activities of full-length and a truncated form of RsbU in vitro . Our determination of the crystal structure of the N-terminal domain of RsbU from Bacillus subtilis reveals structural similarities to the regulatory domains from ubiquitous protein phosphatases and a conserved domain of sigma-factors, illuminating the activation processes of phosphatases and the evolution of "partner switching." Finally, the molecular basis of kinase recruitment by the RsbU phosphatase is discussed by comparing RsbU sequences from bacteria that either possess or lack RsbT .

J Bacteriol, 2004 Aug, 186(15), 5157 - 9
Cellular levels of trp RNA-binding attenuation protein in Bacillus subtilis; McCabe BC et al.; Expression of the Bacillus subtilis trp genes is negatively regulated by an 11-subunit trp RNA-binding attenuation protein (TRAP), which is activated to bind RNA by binding l-tryptophan . We used Western blotting to estimate that there are 200 to 400 TRAP 11-mer molecules per cell in cells grown in either minimal or rich medium.

J Bacteriol, 2004 Aug, 186(15), 5153 - 6
PBP1 is a component of the Bacillus subtilis cell division machinery; Scheffers DJ et al.; Bacillus subtilis penicillin-binding protein PBP1 has been implicated in cell division . We show here that a PBP1 knockout strain is affected in the formation of the asymmetric sporulation septum and that green fluorescent protein-PBP1 localizes to the sporulation septum . Localization of PBP1 to the vegetative septum is dependent on various cell division proteins . This study proves that PBP1 forms part of the B . subtilis cell division machinery.

J Bacteriol, 2004 Aug, 186(15), 4875 - 84
Three different systems participate in L-cystine uptake in Bacillus subtilis; Burguiere P et al.; The symporter YhcL and two ATP binding cassette transporters, YtmJKLMN and YckKJI, were shown to mediate L-cystine uptake in Bacillus subtilis . A triple DeltayhcL DeltaytmJKLMN DeltayckK mutant was unable to grow in the presence of L-cystine and to take up L-cystine . We propose that yhcL, ytmJKLMN, and yckKJI should be renamed tcyP, tcyJKLMN, and tcyABC, respectively . The L-cystine uptake by YhcL (K(m) = 0.6 microM) was strongly inhibited by seleno-DL-cystine, while the transport due to the YtmJKLMN system (K(m) = 2.5 microM) also drastically decreased in the presence of DL-cystathionine, L-djenkolic acid, or S-methyl-L-cysteine . Accordingly, a DeltaytmJKLMN mutant did not grow in the presence of 100 microM DL-cystathionine, 100 microM L-djenkolic acid, or 100 microM S-methyl-L-cysteine . The expression of the ytmI operon and the yhcL gene was regulated in response to sulfur availability, while the level of expression of the yckK gene remained low under all the conditions tested.

Bioinformatics, 2004 Aug 4, 20 Suppl 1, I101 - I108
Predicting gene regulation by sigma factors in Bacillus subtilis from genome-wide data; De Hoon MJ et al.; MOTIVATION: Sigma factors regulate the expression of genes in Bacillus subtilis at the transcriptional level . We assess the accuracy of a fold-change analysis, Bayesian networks, dynamic models and supervised learning based on coregulation in predicting gene regulation by sigma factors from gene expression data . To improve the prediction accuracy, we combine sequence information with expression data by adding their log-likelihood scores and by using a logistic regression model . We use the resulting score function to discover currently unknown gene regulations by sigma factors . RESULTS: The coregulation-based supervised learning method gave the most accurate prediction of sigma factors from expression data . We found that the logistic regression model effectively combines expression data with sequence information . In a genome-wide search, highly significant logistic regression scores were found for several genes whose transcriptional regulation is currently unknown . We provide the corresponding RNA polymerase binding sites to enable a straightforward experimental verification of these predictions.

Trends Genet, 2004 Aug, 20(8), 367 - 74
The different roles of tryptophan transfer RNA in regulating trp operon expression in E . coli versus B . subtilis; Yanofsky C; Escherichia coli and Bacillus subtilis use different mechanisms of sensing and responding to tryptophan and uncharged tRNA(Trp) as regulatory signals . In E . coli, tryptophan activates a repressor that binds to the trp promoter- operator, inhibiting transcription initiation . In B . subtilis, tryptophan activates an RNA-binding protein, TRAP, which binds to the trp operon leader RNA, causing transcription termination . In E . coli uncharged tRNA(Trp) accumulation stalls the ribosome attempting translation of tandem Trp codons in the leader-peptide coding region of the operon . This stalling permits the formation of an RNA antiterminator structure, preventing transcription termination . In B . subtilis uncharged tRNA(Trp) accumulation activates transcription and translation of the at operon . AT protein inhibits tryptophan-activated TRAP, thereby preventing TRAP-mediated transcription termination . These differences might reflect the unique organizational features of the respective trp operons and their ancestry.

Life Sci, 2004 Aug 13, 75(13), 1635 - 47
Cytotoxic effects of mammea type coumarins from Calophyllum brasiliense; Reyes-Chilpa R et al.; Calophyllum brasiliense (Clusiaceae) is a big tree from the Tropical Rain Forests of the American continent . The organic extracts from the leaves yielded coumarins of the mammea type: mammea A/BA, A/BB, B/BA, B/BB, C/OA, C/OB, B/BA cyclo F, B/BB cyclo F, and isomammeigin . The triterpenoids friedelin and canophyllol, as well as the biflavonoid amentoflavone, protocatechuic and shikimic acids, were also obtained . Most of the isolated compounds were tested in vitro against K562, U251, and PC3 human tumor cell lines . The coumarins were cytotoxic against the three cell lines, the highest activity was shown by mammea A/BA (IC50 = 0.04 to 0.59 microM) . The mixtures of mammea A/BA + A/BB, mammea B/BA + B/BB and mammea C/OA + C/OB were also highly active (IC50 < 4.05 microM) . Friedelin was cytotoxic only against PC3, and U251 lines . Inhibition of HIV-1 reverse transcriptase was also assayed in vitro; however, none of the tested compounds (250 microM) prevented the activity of this enzyme . Most of the isolated compounds were also inactive against fourteen bacterial strains; however mammea A/BA + A/BB, and mammea C/OA + C/OB inhibited the growth of Staphylococcus aureus, S . epidermidis and Bacillus subtilis.

Proc Natl Acad Sci U S A, 2004 Jul 27, 101(30), 10937 - 42 Epub 2004 Jul 15.
A large conformational change of the translocation ATPase SecA; Osborne AR et al.; The ATPase SecA mediates the posttranslational translocation of a wide range of polypeptide substrates through the SecY channel in the cytoplasmic membrane of bacteria . We have determined the crystal structure of a monomeric form of Bacillus subtilis SecA at a 2.2-A resolution . A comparison with the previously determined structures of SecA reveals a nucleotide-independent, large conformational change that opens a deep groove similar to that in other proteins that interact with diverse polypeptides . We propose that the open form of SecA represents an activated state.

Microbiology, 2004 Jul, 150(Pt 7), 2465 - 74
Extended phenotype of an mreB-like mutant in Azospirillum brasilense; Biondi EG et al.; Tn5 mutagenesis was used to generate an Azospirillum brasilense SPF94 mutant . Genetic analysis of this mutant revealed that a homologue of the mreB gene, which controls cell shape in Bacillus subtilis and Escherichia coli, was inactivated . The cell-surface properties of the mutant were different from those of the parental strain . The mutant colonies were highly fluorescent when grown on plates containing Calcofluor White . Light and electron microscopy revealed that the mutant cells were round and had thicker capsules than the spiral parental strain . The mutants contained up to ten times more capsule protein than the parental strain, but lacked a 40 kDa protein that is abundant in the parental strain . The phenotype of the isolated mutant resembled that of the cyst-like differentiated forms of Azospirillum, suggesting that the mreB homologue could be involved in differentiation.

Microbiology, 2004 Jul, 150(Pt 7), 2277 - 87
Genetic analysis of the Bacillus subtilis sigG promoter, which controls the sporulation-specific transcription factor sigma G; Evans L et al.; At the onset of sporulation in Bacillus subtilis, an asymmetric cell division gives rise to two unequal-sized compartments with distinct developmental fates . The smaller compartment, or prespore, becomes the spore, whilst the larger compartment, or mother cell, eventually lyses after contributing to spore maturation . The fate of each compartment is determined by differential gene expression, controlled by the activation of four compartment-specific sigma-factors . The expression and activity of all four sigma-factors are tightly regulated to ensure the correct sequence of morphological events . Prespore-specific genes are transcribed by two sigma-factors, sigma(F) followed by sigma(G) . The gene encoding sigma(G) (sigG) is transcribed by sigma(F), but also requires the activity of one of the mother-cell-specific sigma-factors, sigma(E), for its expression . The minimal promoter required for dependence on sigma(E) was found to stretch to just upstream of the -35 site . Analysis of mutant sigG promoters generated by site-directed mutagenesis and sigG promoters from other species suggests the presence of a binding site for a transcriptional repressor within the sigG promoter region . Replacement of the wild-type promoter with sigma(E)-independent promoters resulted in impairment of sporulation . These data support the idea that sigma(E) activity is required for the transcription of sigG.

J Environ Qual, 2004 Jul-Aug, 33(4), 1343 - 53
Polycyclic aromatic hydrocarbon release from a soil matrix in the in vitro gastrointestinal tract; Van de Wiele TR et al.; Soil ingestion is an important exposure route by which immobile soil contaminants enter the human body . We assessed polycyclic aromatic hydrocarbon (PAH) release from a contaminated soil, containing 49 mg PAH kg(-1), using a SHIME (Simulator of the Human Intestinal Microbial Ecosystem) reactor comprising the stomach, duodenal, and colon compartments . Polycyclic aromatic hydrocarbon release was defined as that fraction remaining in the digest supernatant after centrifugation for 5 min at 1500 x g . The PAH release in the stomach digest was only 0.44% of the total PAH present in soil, resulting in PAH concentrations of 23 micrograms PAH L(-1) chyme . The lower PAH releases in duodenum (0.13%) and colon (0.30%) digests, compared with the stomach digest, were thought to be attributed to combined complexation and precipitation with bile salts, dissolved organic matter, or colon microbiota . We studied these complexation processes in an intestinal suspension more in depth by preparing mixtures of 9-anthracenepropionic acid, a Bacillus subtilis culture, and cholin as model compounds for PAHs, organic matter, and bile salts, respectively . Bile salts or organic matter in the aqueous phase initially enhance PAH desorption from soil . However, desorbed PAHs may form large aggregates with bile and organic matter, lowering the freely dissolved PAH fraction in the supernatant . Using the model compounds, mathematical equations were developed and validated to predict PAH complexation processes in the gastrointestinal tract . Contaminant release and subsequent complexation in the gut is an important prerequisite to intestinal absorption and thus bioavailability of that contaminant . The data from this research may help in understanding the processes to which PAHs are subjected in the gastrointestinal tract, before intestinal absorption.

Arch Microbiol, 2004 Sep, 182(1), 80 - 9 Epub 2004 Jul 14.
Purification and characterization of ferredoxin-NADP+ reductase encoded by Bacillus subtilis yumC; Seo D et al.; From Bacillus subtilis cell extracts, ferredoxin-NADP+ reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing . YumC is a approximately 94-kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit . In a diaphorase assay with 2,6-dichlorophenol-indophenol as electron acceptor, the affinity for NADPH was much higher than that for NADH, with Km values of 0.57 microM vs >200 microM . Kcat values of YumC with NADPH were 22.7 s(-1) and 35.4 s(-1) in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively . The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low . The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR . A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and which have been annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc . These genes are found notably in gram-positive bacteria, except Clostridia, and less frequently in archaea and proteobacteria . We propose that YumC and C . tepidum FNR constitute a new group of FNR that should be added to the already established plant-type, bacteria-type, and mitochondria-type FNR groups.

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