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