Proc Natl Acad Sci U S A, 2004 Jul 27, 101(30), 10943 - 8 Epub 2004 Jul 13. Attenuation control of pyrG expression in Bacillus subtilis is mediated by CTP-sensitive reiterative transcription; Meng Q et al.; In Bacillus subtilis and other Gram-positive bacteria, pyrimidine-mediated regulation of the pyrG gene, which encodes CTP synthetase, occurs through an attenuation mechanism involving an intrinsic transcription terminator in the pyrG leader region . Low intracellular levels of CTP prevent termination at the attenuator by a mechanism that requires the nontemplate strand sequence GGGC at the pyrG transcription initiation site (first G =+1) and the leader transcript sequence GCUCCC located at the 5' end of the terminator RNA hairpin . In this study, we demonstrate that reiterative transcription adds G residues (up to at least 10) to the 5' end of pyrG transcripts when B . subtilis cells are starved for pyrimidines but not when cells are grown with excess cytidine . Regulated repetitive addition of G residues, as well as pyrimidine-mediated pyrG regulation, requires the sequence GGGC or GGGT at the start of pyrG transcription . Mutational insertion of four extra G residues at the 5' end of the pyrG transcript (i.e., 5'-GGGGGGGC) results in constitutive pyrG expression . We propose that the incorporation of extra G residues by reiterative transcription at the wild-type promoter occurs when normal transcription elongation is stalled at position +4 by low levels of the incoming substrate, CTP, during pyrimidine limitation . The poly(G) extensions on the 5' ends of pyrG transcripts act to prevent transcription attenuation by base pairing with the sequence CUCCCUUUC located in the 5' strand of the terminator hairpin . This control mechanism is likely to operate in other Gram-positive bacteria containing similar pyrG leader sequences. Ann N Y Acad Sci, 2004 Jun, 1022, 195 - 201 Transcession of DNA from bacteria to human cells in culture: a possible role in oncogenesis; Anker P et al.; The human organism is continuously in close contact with microorganisms, especially bacteria . In the present work, by means of a real-time polymerase chain reaction (PCR) technique, we looked for the presence of a distinct bacterial gene in human cells . To this end, we cultured a human cell line, HL60, in a supernatant in which bacteria (Bacillus subtilis) had been grown . A transient transcession of bacterial DNA into the human cells was observed. FEBS Lett, 2004 Jul 16, 570(1-3), 13 - 9 Structures of Bacillus subtilis PdaA, a family 4 carbohydrate esterase, and a complex with N-acetyl-glucosamine; Blair DE et al.; Family 4 carbohydrate esterases deacetylate polymeric carbohydrate substrates such as chitin, acetyl xylan and peptidoglycan . Although some of these enzymes have recently been enzymologically characterised, neither their structure nor their reaction mechanism has been defined . Sequence conservation in this family has pointed to a conserved core, termed the NodB homology domain . We describe the cloning, purification and 1.9 A crystal structure of PdaA, a peptidoglycan deacetylase from Bacillus subtilis . The enzyme assumes a fold related to a (beta/alpha)8 barrel, with a long groove on the surface of the protein that harbours all conserved residues . A complex with the substrate analogue N-acetyl-glucosamine was refined to 2.25 A resolution, revealing interactions of an aspartic acid and three histidines, all conserved in the NodB homology domain, with the ligand . The PdaA structure provides a template for interpreting the wealth of sequence data on family 4 carbohydrate esterases in a structural context and represents a first step towards understanding the reaction mechanism of this family of enzymes. Protein Expr Purif, 2004 Aug, 36(2), 280 - 91 Structural and functional impairment of an Old Yellow Enzyme homologue upon affinity tag incorporation; Fitzpatrick TB et al.; Recently, it has been reported that the previously uncharacterized YqjM protein from Bacillus subtilis is a true homologue of the physiologically enigmatic yeast Old Yellow Enzyme (OYE) . In this study, it was also demonstrated that YqjM is involved in the oxidative stress response of B . subtilis, thus highlighting a novel direction to pursue the role of the OYE family of proteins in the cell . As part of an attempt to pin down the exact physiological role of these enzymes, both a N-terminal glutathione S-transferase and a C-terminal histidine-tagged form of the protein were created to enable "pull-down" assays and identify interacting partners which could aid in the functional definition . However, here we report on a comparison of the biochemical properties of the tagged forms with the native/untagged YqjM, revealing critical differences in the catalytic activities and quaternary structure of the protein forms . UV-visible spectrophotometric features as well as steady state and individual half-reaction kinetic parameters show that the affinity tagged forms are severely impaired both in ligand binding and catalysis . Gel filtration and dynamic light scattering studies show that incorporation of a tag also has major effects on the quaternary structure of the protein by disrupting the native tetrameric conformation which may help to explain the observed differences . The study thus highlights important considerations for expression construct design when isolating members of the OYE family of proteins. Appl Microbiol Biotechnol, 2004 Oct, 65(5), 583 - 92 Epub 2004 Jul 10. Hyper-production of an isomalto-dextranase of an Arthrobacter sp . by a proteases-deficient Bacillus subtilis: sequencing, properties, and crystallization of the recombinant enzyme; Hatada Y et al.; Arthrobacter globiformis T6 is unique in that it produces an enzyme yielding only isomaltose from dextran . In the present study, the organism was re-identified and its classification as a new species of the genus Arthrobacter, A . dextranlyticum, was proposed . The high G+C gene (66.8 mol%) for the isomalto-dextranase was sequenced . The deduced amino acid sequence, with a calculated molecular mass of 65,993 Da (603 amino acids), was confirmed by nanoscale capillary liquid chromatography coupled to tandem mass spectrometry, which covered 71.1% of the amino acid residues of the entire sequence . The enzyme was grouped into glycoside hydrolase family 27, and the C-terminal domain has homology to carbohydrate-binding module family 6 . Hyper-exoproduction of the recombinant enzyme was achieved at a level corresponding to approximately 4.6 g l(-1) of culture broth when proteases-deficient Bacillus subtilis cells were used as the host . The purified enzyme (65.5 kDa) had an optimal pH and temperature for activity of 3.5 and 60 degrees C, respectively . It was crystallized using the sitting-drop vapor-diffusion method at 293 K. Nucleic Acids Res, 2004 Jul 01, 32(11), 3493 - 502 Print 2004. Binding of phage Phi29 architectural protein p6 to the viral genome: evidence for topological restriction of the phage linear DNA; Gonzalez-Huici V et al.; Bacillus subtilis phage Phi29 protein p6 is required for DNA replication and promotes the switch from early to late transcription . In vivo it binds all along the viral linear DNA, which suggests a global role as an architectural protein; in contrast, binding to bacterial DNA is negligible . This specificity could be due to the p6 binding preference for less negatively supercoiled DNA, as is presumably the case with viral (with respect to bacterial) DNA . Here we demonstrate that p6 binding to Phi29 DNA is greatly increased when negative supercoiling is decreased by novobiocin; in addition, gyrase is required for DNA replication . This indicates that, although non-covalently closed, the viral genome is topologically constrained in vivo . We also show that the p6 binding to different Phi29 DNA regions is modulated by the structural properties of their nucleotide sequences . The higher affinity for DNA ends is possibly related to the presence of sequences in which their bendability properties favor the formation of the p6-DNA complex, whereas the lower affinity for the transcription control region is most probably due to the presence of a rigid intrinsic DNA curvature. J Biol Chem, 2004 Sep 17, 279(38), 39340 - 7 Epub 2004 Jul 09. Bacillus subtilis DesR functions as a phosphorylation-activated switch to control membrane lipid fluidity; Cybulski LE et al.; The Des pathway of Bacillus subtilis regulates the synthesis of the cold-shock induced membrane-bound enzyme Delta5-fatty acid desaturase (Delta5-Des) . A central component of the Des pathway is the response regulator, DesR, which is activated by a membrane-associated kinase, DesK, in response to a decrease in membrane lipid fluidity . Despite genetic and biochemical studies, specific details of the interaction between DesR and the DNA remain unknown . In this study we show that only the phosphorylated form of protein DesR is able to bind to a regulatory region immediately upstream of the promoter of the Delta5-Des gene (Pdes) . Phosphorylation of the regulatory domain of dimeric DesR promotes, in a cooperative fashion, the hierarchical occupation of two adjacent, non-identical, DesR-P DNA binding sites, so that there is a shift in the equilibrium toward the tetrameric active form of the response regulator . Subsequently, this phosphorylation signal propagation leads to the activation of the des gene through recruitment of RNA polymerase to Pdes . This is the first dissected example of a transcription factor functioning as a phosphorylation-activated switch for a cold-shock gene, allowing the cell to optimize the fluidity of membrane phospholipids. Biomacromolecules, 2004 Jul-Aug, 5(4), 1219 - 30 Spectroscopic study of extracellular polymeric substances from Bacillus subtilis: aqueous chemistry and adsorption effects; Omoike A et al.; Reactions at ionizable functional groups in extracellular polymeric substances (EPS) from Bacillus subtilis are found to affect aqueous phase conformation and adsorption to mineral surfaces . Characterization by HPSEC, XPS, and FTIR indicates a wide range in apparent molecular mass (0.57-128 kDa), with functional group composition depending on cell growth phase (exponential vs stationary) and location in suspension (free vs cell-bound) . ATR-FTIR spectroscopy shows complexation and dissociation of protons on acidic functional groups that result in alpha-helical protein conformation at pH < 2.6 and random coil (unordered) conformation at higher pH (>6) . EPS exhibit higher affinity for adsorption to alpha-FeOOH than amorphous SiO(2) because of surface charge effects . Increased amide II band intensity and an amide I band shift to higher frequency indicate changes in protein structure upon adsorption . Goethite-EPS spectra show emergent vibrations consistent with P-O-Fe bonding, which suggests a role of phosphodiester groups in the adsorption reaction. Structure (Camb), 2004 Jul, 12(7), 1269 - 80 Crystal structure of activated HutP; an RNA binding protein that regulates transcription of the hut operon in Bacillus subtilis; Kumarevel T et al.; HutP is an L-histidine-activated RNA binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on the hut mRNA . The crystal structure of HutP complexed with an L-histidine analog showed a novel fold; there are four antiparallel beta strands in the central region of each monomer, with two alpha helices each on the front and back . Two HutP monomers form a dimer, and three dimers are arranged in crystallographic 3-fold symmetry to form a hexamer . A histidine analog was located in between the two monomers of HutP, with the imidazole group of L-histidine hydrogen bonded to Glu81 . An activation mechanism is proposed based on the identification of key residues of HutP . The HutP binding region in hut mRNA was defined: it consists of three UAG trinucleotide motifs separated by four spacer nucleotides . Residues of HutP potentially important for RNA binding were identified. Z Naturforsch {C}, 2004 Mar-Apr, 59(3-4), 205 - 8 Enhanced hydrocarbon biodegradation by a newly isolated Bacillus subtilis strain; Christova N et al.; The relation between hydrocarbon degradation and biosurfactant (rhamnolipid) production by a new Bacillus subtilis 22BN strain was investigated . The strain was isolated for its capacity to utilize n-hexadecane and naphthalene and at the same time to produce surface-active compound at high concentrations (1.5 - 2.0 g l(-1)) . Biosurfactant production was detected by surface tension lowering and emulsifying activity . The strain is a good degrader of both hydrocarbons used with degradability of 98.3 +/- 1% and 75 +/- 2% for n-hexadecane and naphthalene, respectively . Measurement of cell hydrophobicity showed that the combination of slightly soluble substrate and rhamnolipid developed higher hydrophobicity correlated with increased utilization of both hydrocarbon substrates . To our knowledge, this is the first report of Bacillus subtilis strain that degrades hydrophobic compounds and at the same time produces rhamnolipid biosurfactant. Mol Genet Genomics, 2004 Aug, 272(1), 98 - 107 Epub 2004 Jul 07. Cold induction of the Bacillus subtilis bkd operon is mediated by increased mRNA stability; Nickel M et al.; Recently it has been demonstrated that the ptb - bcd - buk - lpdV - bkdAA - bkdAB - bkdB operon ( bkdoperon) of Bacillus subtilis, which encodes the enzymes that catalyze the degradation of branched-chain amino acids, is inducible by a temperature downshift from 37 to 18 degrees C . Deamination and oxidative decarboxylation of isoleucine generates 2-methyl-butyryl-CoA, which serves as the precursor of anteiso-branched fatty acid species . Most probably, the induction of this operon upon cold shock ensures an increase in the content of anteiso-branched fatty acids in the membrane lipids at low temperature, thus permitting maintenance of membrane fluidity at lower temperatures . In the present study, we have analyzed the mechanism of cold induction of the bkd operon and of four further cold-inducible transcriptional units in B . subtilis . We demonstrate that cold induction of these genes is mediated by an increase in the stability of the corresponding mRNAs . None of the promoters that control the five transcriptional units analyzed is actually cold-inducible . Furthermore, the results of this study indicate that the 5' leader regions are not involved in the cold-induced stabilization of the mRNAs . The structural elements that enhance mRNA stability must therefore be restricted to the 3'-ends and/or the coding regions. Appl Environ Microbiol, 2004 Jul, 70(7), 4249 - 55 Enzymatic synthesis of high-molecular-mass poly-gamma-glutamate and regulation of its stereochemistry; Ashiuchi M et al.; For the first time, we succeeded in synthesizing in vitro poly-gamma-glutamate (PGA) with high molecular masses (>1,000 kDa) by the use of enzyme-associated cell membranes from Bacillus subtilis subsp . chungkookjang . The activity for PGA synthesis, however, was readily lost in the presence of critical concentrations of detergents tested in micelles . The optimum pH for the reaction was found to be approximately 7.0 . We examined the effects of some divalent cations on PGA synthesis and found that Mg(2+) was essential in catalysis and that Zn(2+) additionally boosted the activity . In contrast, Fe(2+) and Ca(2+) acted as inhibitors . Mn(2+) did not apparently influence the in vitro formation of PGA . DL-Glutamate (D isomer content, 60 to 80%) apparently served as the best substrate; d-Glutamate was preferable to the L isomer as a substrate . When D- and L-glutamate were used for the reaction, the elongated chains of PGAs were composed of the D- and L-isomers, respectively . Our results suggest that the stereochemical properties of enzymatically synthesized PGAs substantially depend on the stereochemistry (DL ratio) of glutamate as the substrate . Furthermore, genetic analysis indicated that all the pgsB, -C, and -A gene products, which are responsible for PGA production by B . subtilis cells, were also indispensable for enzymatic PGA synthesis. J Bacteriol, 2004 Jul, 186(14), 4655 - 64 Response of Bacillus subtilis to nitric oxide and the nitrosating agent sodium nitroprusside; Moore CM et al.; We examined the effects of nitric oxide (NO) and sodium nitroprusside (SNP) on Bacillus subtilis physiology and gene expression . In aerobically growing cultures, cell death was most pronounced when NO gas was added incrementally rather than as a single bolus, suggesting that the length of exposure was important in determining cell survival . DNA microarrays, Northern hybridizations, and RNA slot blot analyses were employed to characterize the global transcriptional response of B . subtilis to NO and SNP . Under both aerobic and anaerobic conditions the gene most highly induced by NO was hmp, a flavohemoglobin known to protect bacteria from NO stress . Anaerobically, NO also induced genes repressed by the Fe(II)-containing metalloregulators, Fur and PerR, consistent with the known ability of NO to nitrosylate the Fe(II) center in Fur . In support of this model, we demonstrate that NO fails to induce PerR-regulated genes under growth conditions that favor the formation of PerR:Mn(II) rather than PerR:Fe(II) . Aerobically, NO gas induced hmp, the sigmaB general stress regulon, and, to a lesser extent, the Fur and PerR regulons . Surprisingly, NO gas induced the sigmaB regulon via the energy branch of the sigmaB regulatory cascade while induction by SNP was mediated by the environmental stress branch . This emphasizes that NO and SNP elicit genetically distinct stress responses . J Bacteriol, 2004 Jul, 186(14), 4585 - 95 Bacillus subtilis YdiH is a direct negative regulator of the cydABCD operon; Schau M et al.; During aerobic respiration, Bacillus subtilis utilizes three terminal oxidases, cytochromes aa3, caa3, and bd . Cytochrome bd is encoded by the cydABCD operon . We report here the first identification of a regulator for the cydABCD operon, YdiH . While working with DeltaresDE mutant strains, we identified colonies which contained suppressor mutations (cmp) which bypassed the requirement for ResD for all phenotypes not associated with cytochrome aa3 or caa3 . Mapping identified a class of Tn10 insertions which were close to the cmp locus (Tn10-2) and a second class (Tn10-1) which was inserted in cydD, a gene which appears to be essential to the cmp phenotype . Sequencing of the cmp loci from four independent DeltaresDE cmp isolates yielded four loss-of-function alleles of ydiH, a gene encoding a protein with homology to AT-rich DNA-binding proteins . Additionally, we determined that cytochrome bd was aberrantly expressed in the DeltaresDE cmp background . Together these data led to the hypothesis that YdiH serves as a negative regulator of cydABCD expression, a hypothesis supported by both gel-shift and DNase I footprinting analyses . YdiH protected the cydA promoter region at three 22-bp repeats located in the long 5' untranslated region (193 bp) . Induction of the cydABCD operon in a DeltaresDE background showed that expression of the terminal oxidase bd was responsible for the bypass phenotype observed in a DeltaresDE cmp strain, indicating that cytochrome bd expression complemented the loss of cytochromes aa3 and caa3 in the DeltaresDE strain . J Bacteriol, 2004 Jul, 186(14), 4528 - 34 The Bacillus subtilis yqjI gene encodes the NADP+-dependent 6-P-gluconate dehydrogenase in the pentose phosphate pathway; Zamboni N et al.; Despite the importance of the oxidative pentose phosphate (PP) pathway as a major source of reducing power and metabolic intermediates for biosynthetic processes, almost no direct genetic or biochemical evidence is available for Bacillus subtilis . Using a combination of knockout mutations in known and putative genes of the oxidative PP pathway and 13C-labeling experiments, we demonstrated that yqjI encodes the NADP+-dependent 6-P-gluconate dehydrogenase, as was hypothesized previously from sequence similarities . Moreover, YqjI was the predominant isoenzyme during glucose and gluconate catabolism, and its role in the oxidative PP pathway could not be played by either of two homologues, GntZ and YqeC . This conclusion is in contrast to the generally held view that GntZ is the relevant isoform; hence, we propose a new designation for yqjI, gndA, the monocistronic gene encoding the principal 6-P-gluconate dehydrogenase . Although we demonstrated the NAD+-dependent 6-P-gluconate dehydrogenase activity of GntZ, gntZ mutants exhibited no detectable phenotype on glucose, and GntZ did not contribute to PP pathway fluxes during growth on glucose . Since gntZ mutants grew normally on gluconate, the functional role of GntZ remains obscure, as does the role of the third homologue, YqeC . Knockout of the glucose-6-P dehydrogenase-encoding zwf gene was primarily compensated for by increased glycolytic fluxes, but about 5% of the catabolic flux was rerouted through the gluconate bypass with glucose dehydrogenase as the key enzyme . J Bacteriol, 2004 Jul, 186(14), 4441 - 8 Dynamic patterns of subcellular protein localization during spore coat morphogenesis in Bacillus subtilis; van Ooij C et al.; Endospores of Bacillus subtilis are encased in a thick, proteinaceous shell known as the coat, which is composed of a large number of different proteins . Here we report the identification of three previously uncharacterized coat-associated proteins, YabP, YheD, and YutH, and their patterns of subcellular localization during the process of sporulation, obtained by using fusions of the proteins to the green fluorescent protein (GFP) . YabP-GFP was found to form both a shell and a ring around the center of the forespore across the short axis of the sporangium . YheD-GFP, in contrast, formed two rings around the forespore that were offset from its midpoint, before it eventually redistributed to form a shell around the developing spore . Finally, YutH-GFP initially localized to a focus at one end of the forespore, which then underwent transformation into a ring that was located adjacent to the forespore . Next, the ring became a cap at the mother cell pole of the forespore that eventually spread around the entire developing spore . Thus, each protein exhibited its own distinct pattern of subcellular localization during the course of coat morphogenesis . We concluded that spore coat assembly is a dynamic process involving diverse patterns of protein assembly and localization . J Antimicrob Chemother, 2004 Aug, 54(2), 364 - 9 Epub 2004 Jul 01. Effect of substrate exposure and other growth condition manipulations on norA expression; Kaatz GW et al.; OBJECTIVES: Multidrug efflux is a resistance mechanism that simultaneously affects susceptibility to many structurally unrelated compounds . The regulation of norA expression, which encodes the Staphylococcus aureus NorA multidrug efflux pump, is not well understood but the MgrA global regulator and the arlRS locus are involved . The expression of genes encoding proteins related to NorA, such as QacA of S . aureus and Bmr of Bacillus subtilis, is affected by pump substrates . In these instances, substrate interacts with regulatory proteins such that pump gene transcription is increased . The goal of this study was to identify if a similar substrate-level effect exists, or an effect of other growth condition manipulations, on the expression of norA . METHODS: A transcriptional fusion between norA and lacZ was created in single copy on the chromosome of S . aureus SH1000 . beta-Galactosidase activity was quantified following exposure of the fusion strain to various NorA substrates, salicylate, a high salt concentration, putative soluble factors elaborated during growth, and different incubation temperatures . RESULTS AND CONCLUSIONS: Exposure to several substrates significantly increased norA expression whereas salicylate and osmotic stress had no effect and no stable soluble factor affecting norA expression was detectable . An inverse relationship between norA expression and incubation temperature was observed and this effect was related, at least in part, to changes in norA mRNA half-life . However, concomitant changes in translational efficiency at different temperatures could not be ruled out . We conclude that there is a substrate-level effect on norA expression and propose that this may be mediated through substrate interaction with a regulatory protein. Mol Microbiol, 2004 Jul, 53(2), 599 - 611 Activation of the Bacillus subtilis global regulator CodY by direct interaction with branched-chain amino acids; Shivers RP et al.; CodY, a GTP-activated global transcriptional regulator of early stationary phase genes, is conserved in many Gram-positive bacterial species . Recently, a number of novel targets regulated by CodY have been identified, including three Bacillus subtilis operons involved in branched-chain amino acid (BCAA) biosynthesis (Molle, V., et al., 2003, J Bacteriol 185: 1911-1922) . The mechanism of involvement of CodY in regulating the ilvB operon was investigated here using in vivo transcriptional fusions, in vitro gel mobility shift assays and DNase I footprinting assays . CodY was found to mediate regulation of the ilvB operon by GTP and BCAAs and to bind to the ilvB promoter region . BCAAs increased the affinity of CodY for the ilvB promoter and for all other CodY targets tested . This effect of BCAAs in vitro was additive with the effect of GTP on CodY DNA-binding activity. Gene, 2004 Jul 7, 336(1), 25 - 35 Vectors for regulated gene expression in the radioresistant bacterium Deinococcus radiodurans; Lecointe F et al.; Deinococcus radiodurans possesses an exceptional capacity to withstand the lethal and mutagenic effects of most form of DNA damage and has received considerable interest for use in both fundamental and applied research . Here we describe vectors that allow regulated expression of Deinococcal genes for functional analysis . The vectors contain the IPTG-regulated Spac system (Pspac promoter and lacI repressor gene), originally designed for Bacillus subtilis, that we have adapted to be functional in D . radiodurans . We show that the Spac system can control the expression of a lacZ reporter gene over two orders of magnitude depending on the inducer concentration and the copy number of the lacI regulatory gene . Furthermore, we demonstrate that the Spac system can be used to regulate the synthesis of a critical repair protein, such as RecA, resulting in a conditional mitomycin-resistant cell phenotype . We have also developed tools for the construction of conditional mutants where the expression of the target gene is regulated by an inducible promoter . The utility of these conditional gene inactivation systems is exemplified by the conditional lethal phenotype of a mutant expressing gyrA from the Pspac promoter. J Mol Biol, 2004 Jul 16, 340(4), 767 - 82 The crystal structure of YloQ, a circularly permuted GTPase essential for Bacillus subtilis viability; Levdikov VM et al.; yloQ is one of 11 essential genes in Bacillus subtilis with unknown roles in the physiology of the cell . It encodes a polypeptide of 298 residues with motifs characteristic of GTPases . As a contribution to elucidating its indispensable cellular function, we have solved the crystal structure of YloQ to 1.6 A spacing, revealing a three-domain organisation . At the heart of the molecule is the putative GTPase domain, which exhibits a classical alpha/beta nucleotide-binding fold with a topology very similar to that of Ras and Era . However, as anticipated from the order in which the conserved G protein motifs appear in the sequence, the GTPase domain fold in YloQ is circularly permuted with respect to the classical GTPases . The nucleotide-binding pocket in YloQ is unoccupied, and analysis of the phosphate-binding (P) loop indicates that conformational changes in this region would be needed to accommodate GTP . The GTPase domain is flanked at its N terminus by a beta-barrel domain with an oligonucleotide/oligosaccharide-binding (OB) fold, and at its C terminus by an alpha-helical domain containing a coordinated zinc ion . This combination of protein modules is unique to YloQ and its orthologues . Sequence comparisons reveal a clustering of conserved basic and aromatic residues on one face of the OB domain, perhaps pointing to a role for YloQ in nucleic acid binding . The zinc ion in the alpha-helical domain is coordinated by three cysteine residues and a histidine residue in a novel ligand organisation . The juxtaposition of the switch I and switch II regions of the G domain and the OB and zinc-binding domains suggests that chemical events at the GTPase active site may be transduced into relative movements of these domains . The pattern of conserved residues and electrostatic surface potential calculations suggest that the OB and/or Zn-binding domains participate in nucleic acid binding consistent with a possible role for YloQ at some stage during mRNA translation. J Mol Biol, 2004 Jul 16, 340(4), 655 - 64 Bacillus subtilis GabR, a protein with DNA-binding and aminotransferase domains, is a PLP-dependent transcriptional regulator; Belitsky BR; Bacillus subtilis GabR is a member of a poorly characterized but widespread family of chimeric bacterial proteins that have apparent DNA binding and aminotransferase domains . GabR positively regulates expression of the gabTD operon responsible for utilization of gamma-aminobutyric acid (GABA) and represses the divergently transcribed gabR gene . Purified GabR bound specifically to the DNA region overlapping the -35 region of the gabT promoter and the -10 and +1 regions of the gabR promoter . Two 6 bp direct repeats located at the ends of this region appeared to be essential for GabR binding . In transcription reactions in vitro, GabR alone repressed expression from the gabR promoter but activated expression from the gabT promoter only in the presence of GABA and pyridoxal 5'-phosphate, an essential cofactor of aminotransferases . A similar requirement for pyridoxal 5'-phosphate and GABA for GabR-mediated transcription activation was shown in vivo . In vitro this requirement could be partially satisfied with pyridoxamine 5'-phosphate and succinic semialdehyde, the products of a GABA-dependent aminotransferase half-reaction . We hypothesize that the GabR-catalyzed aminotransferase-like reaction between GABA and pyridoxal 5'-phosphate is essential for GabR action as a transcriptional activator. Biotechnol Lett, 2004 Jul, 26(13), 1095 - 9 Application of a quartz crystal microbalance to evaluate biodegradability of starch by Bacillus subtilis; Jenkins M et al.; Biodegradation of solution-cast starch films by Bacillus subtilis was monitored using a quartz crystal microbalance (QCM) . A starch film was formed on the crystal by solution casting and exposed to the Bacillus subtilis culture in a bioreactor . The high sensitivity of the QCM could monitor small weight changes of the starch films on the crystal in the initial stages of biodegradation by secreted exo-enzymes of the bacterium . The feasibility of this approach as a means of quantification and characterisation of biodegradability of different polymeric materials by selected organisms is discussed. Adv Biochem Eng Biotechnol, 2004, 89, 47 - 71 Monitoring of stress responses; Schweder T et al.; New developments in the RNA analysis techniques now enable a comprehensive view on the bacterial physiology under bioprocess conditions . The DNA-chip technology allows a genome wide transcriptional profiling of bacterial cells, whose genome sequence is available . Although the analyses of microbial bioprocesses have still been somewhat limited to date, this technique has already been successfully applied in different laboratories for the investigation of stress responses of selected industrially relevant bacterial hosts . Transcriptome analyses in combination with high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry have been extensively applied for the description of general and specific stress and starvation responses of Escherichia coli and Bacillus subtilis . The consideration of bacterial stress and starvation responses is of crucial importance for the successful establishment of an industrial large scale bioprocess . Stress genes can be used as marker genes in order to monitor the fitness of industrial bacterial hosts during fermentation processes . This chapter gives an overview of current RNA analysis techniques . The bacterial stress and starvation responses, which are of potential importance for industrial microbial bioprocesses are summarised. Biosci Biotechnol Biochem, 2004 Jun, 68(6), 1382 - 4 Region dependent efficiency for recombinational transfer of the Bacillus subtilis 168 genome; Tomita S et al.; Submega-sized regions of the Bacillus subtilis genome were cloned to plasmid by the B . subtilis Recombinational Transfer (BReT) method . BReT efficiency depends not only on the genome location but also on the choice of sequences for simultaneous homologous recombination during BReT . In an extreme case, a 91-kb region that was unsuccessful on the first attempt was obtained when the slightly shifted 98-kb region was targeted. Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W154 - 9 Riboswitch finder--a tool for identification of riboswitch RNAs; Bengert P et al.; We describe a dedicated RNA motif search program and web server to identify RNA riboswitches . The Riboswitch finder analyses a given sequence using the web interface, checks specific sequence elements and secondary structure, calculates and displays the energy folding of the RNA structure and runs a number of tests including this information to determine whether high-sensitivity riboswitch motifs (or variants) according to the Bacillus subtilis type are present in the given RNA sequence . Batch-mode determination (all sequences input at once and separated by FASTA format) is also possible . The program has been implemented and is available both as local software for in-house installation and as a web server at http://www.biozentrum.uni-wuerzburg.de/bioinformatik/Riboswitch/. J Biol Chem, 2004 Aug 27, 279(35), 37087 - 94 Epub 2004 Jun 23. Crystal structure of yeast Ypr118w, a methylthioribose-1-phosphate isomerase related to regulatory eIF2B subunits; Bumann M et al.; Ypr118w is a non-essential, low copy number gene product from Saccharomyces cerevisiae . It belongs to the PFAM family PF01008, which contains the alpha-, beta-, and delta-subunits of eukaryotic translation initiation factor eIF2B, as well as proteins of unknown function from all three kingdoms . Recently, one of those latter proteins from Bacillus subtilis has been characterized as a 5-methylthioribose-1-phosphate isomerase, an enzyme of the methionine salvage pathway . We report here the crystal structure of Ypr118w, which reveals a dimeric protein with two domains and a putative active site cleft . The C-terminal domain resembles ribose-5-phosphate isomerase from Escherichia coli with a similar location of the active site . In vivo, Ypr118w protein is required for yeast cells to grow on methylthioadenosine in the absence of methionine, showing that Ypr118w is involved in the methionine salvage pathway . The crystal structure of Ypr118w reveals for the first time the fold of a PF01008 member and allows a deeper discussion of an enzyme of the methionine salvage pathway, which has in the past attracted interest due to tumor suppression and as a target of aniprotozoal drugs. Antimicrob Agents Chemother, 2004 Jul, 48(7), 2588 - 94 Panel of Bacillus subtilis reporter strains indicative of various modes of action; Hutter B et al.; In a recent project, we collected the transcriptional profiles of Bacillus subtilis 168 after treatment with a large set of diverse antibacterial agents . One result of the data analysis was the identification of marker genes that are indicative of certain compounds or compound classes . We cloned these promoter regions in front of a luciferase reporter gene and reintroduced the constructs individually into the B . subtilis chromosome . Strains were analyzed for their responsiveness after treatment with a set of 37 antibacterials . Twelve functional reporter strains were generated that were selectively and significantly upregulated by the compounds . The selectivity of the reporter strains ranged from generic pathways like protein biosynthesis, cell wall biosynthesis, and fatty acid biosynthesis to compound classes (quinolones and glycopeptides) and individual compounds (rifampin, cycloserine, and clindamycin) . Five of the strains are amenable for high-throughput applications, e.g., pathway-specific screening . In summary, we successfully generated B . subtilis reporter strains that are indicative of the mechanisms of action of various classes of antibacterials . The set of reporter strains presented herein can be used for mode-of-action analyses and for whole-cell screening of compound libraries in a mode-of-action-specific manner. Can J Microbiol, 2004 Apr, 50(4), 279 - 89 Bacterial formyl peptides affect the innate cellular antimicrobial responses of larval Galleria mellonella (Insecta: Lepidoptera); Alavo TB et al.; The non-self cellular (hemocytic) responses of Galleria mellonella larvae, including the attachment to slides and the removal of the bacteria Xenorhabdus nematophila and Bacillus subtilis from the hemolymph, were affected by N-formyl peptides . Both N-formyl methionyl-leucyl-phenylalanine (fMLF) and the ester derivative decreased hemocyte adhesion in vitro, and both elevated hemocyte counts and suppressed the removal of both X . nematophila and B . subtilis from the hemolymph in vivo . The amide derivative and the antagonist tertiary-butoxy-carbonyl-methionyl-leucyl-phenylalanine (tBOC) increased hemocyte attachment to glass . The fMLF suppressed protein discharge from monolayers of granular cells with and without bacterial stimulation, while tBOC stimulated protein discharge . The peptide tBOC offset the effects of fMLF in vitro and in vivo . This is the first report implying the existence of formyl peptide receptors on insect hemocytes in which the compounds fMLF and tBOC inhibited and activated hemocyte activity, respectively. Acta Crystallogr D Biol Crystallogr . 2004 Jul;60(Pt 7):1346. Purification, crystallization and preliminary X-ray analysis of an acetylxylan esterase from Bacillus pumilus . Erratum; Benini S et al.; This erratum is to apologise for having reported the crystallization and X-ray characterization of Bacillus pumilus acetylxylan esterase (AXE) while the protein crystallized was instead an inorganic pyrophosphatase, a contaminant of the expression in E . coli . The protein was purified by hydrophobic interaction, ionic exchange and gel filtration, but still contained traces of contaminant proteins . Crystals were obtained in the R32 space group perfectly compatible with the homohexameric structure of AXE . The cell parameters were compatible with a reasonable crystal packing as in the model cephalosporin C deacetylase from Bacillus subtilis kindly provided before publication by Dr Jim Brannigan et al . (PDB code 1ods crystallized in R3 and 1odt crystallized in R32) . Since every attempt to solve the structure by molecular replacement using 1odt as a model failed, a search of the PDB using the cell parameters of the data collected revealed a match with Escherichia coli inorganic pyrophosphatase (1ipw) . A molecular-replacement solution confirmed that the protein crystallized was indeed E . coli inorganic pyrophosphatase present as a contaminant in the protein preparation used for crystallization . This experience should be kept in mind because proteins used for crystallization should be as pure as possible not only to favour the process itself but also to avoid the crystallization of contaminants. Acta Crystallogr D Biol Crystallogr, 2004 Jul, 60(Pt 7), 1311 - 4 Epub 2004 Jun 22. Anti-TRAP protein from Bacillus subtilis: crystallization and internal symmetry; Shevtsov MB et al.; Anti-TRAP protein regulates the expression of tryptophan biosynthetic genes by binding to TRAP and preventing formation of the TRAP-RNA complex . Anti-TRAP from Bacillus subtilis has been crystallized by vapour diffusion . The crystals belong to space group P1, with unit-cell parameters a = 51.6, b = 60.1, c = 60.4 A, alpha = 114.0, beta = 101.4, gamma = 100.5 degrees . X-ray data have been collected to 2.8 A resolution . Peaks in the self-rotation function correspond to four trimers in the unit cell related by twofold and threefold rotational axes . The symmetry and gel-filtration data suggest that the protein exists as a trimer or a dodecamer in solution. FEMS Microbiol Lett, 2004 Jul 1, 236(1), 115 - 22 Metalloregulation in Bacillus subtilis: the copZ chromosomal gene is involved in cadmium resistance; Solovieva IM et al.; The copZ gene of Bacillus subtilis encodes a copper chaperon CopZ that donates copper to the copper transporter CopA . Both genes copZ and copA are clustered to an operon and its promoter is regulated by Cu ions and CueR, a Mer-like transcriptional activator . Here we show that cadmium ions activate copZA expression as strong as copper ions . Northern hybridization analysis showed that copper and cadmium both induce the synthesis of a 2.7 kb copZA transcript and a 250 bp copZ transcript . A copA deletion mutant was sensitive to copper, whereas a copZ deletion resulted in an increased sensitivity to cadmium and copper . Transcription of the cadmium resistance gene cadA, which is adjacent to the copZA cluster, is extremely reduced in a copZ deletion strain . Transformation of copZ in trans restores wild type resistance to cadmium and copper in a copZ deletion strain . This excludes any polar effect and proves that the copZ encoded protein is important for copper and cadmium resistance. J Agric Food Chem, 2004 Jun 30, 52(13), 4296 - 302 Impact of inhibition sensitivity on endoxylanase functionality in wheat flour breadmaking; Trogh I et al.; A Bacillus subtilis endoxylanase (XBS(i)) sensitive to inhibition by Triticum aestivum L . endoxylanase inhibitor (TAXI) and a mutant thereof (XBS(ni)), uninhibited by TAXI, were used in straight-dough breadmaking to assess the importance of endoxylanase inhibition sensitivity on endoxylanase functionality in the process . With two European wheat flours, the loaf volume improving effect of XBS(ni) at much lower enzyme dosages was substantially larger than that brought about by XBS(i) . This coincided with differences in arabinoxylan (AX) hydrolysis . Although XBS(ni) had a lower substrate selectivity for water-unextractable arabinoxylan (WU-AX) than XBS(i), the former solubilized significantly more WU-AX than XBS(i) . Because of inhibition, XBS(i) solubilized most of the WU-AX during mixing, whereas, with XBS(ni), the rate of solubilization decreased less with increasing processing time than that with XBS(i) . During fermentation and baking and at the highest dosage (600 U/kg of flour of XBS(i) and 60 U/kg of flour of XBS(ni)), XBS(ni) induced a stronger degradation of enzymically solubilized and water-extractable AX than XBS(i) . Taken together, the data clearly demonstrate that endoxylanases, which in vitro are inhibited by endoxylanase inhibitors and still are active in the breadmaking process, as demonstrated by their functional (bread volume) enhancing effect, gradually lose their activity in the process. J Agric Food Chem, 2004 Jun 30, 52(13), 4240 - 9 Enzymatic solubilization of arabinoxylans from native, extruded, and high-shear-treated rye bran by different endo-xylanases and other hydrolyzing enzymes; Figueroa-Espinoza MC et al.; The overall objective of this research was to find a new way to valorize rye bran, by producing a gellifier from the enzymatic solubilization of arabinoxylans (AX) . The effects of three pure endo-xylanases from Aspergillus niger (Xyl-1), Talaromyces emersonii (Xyl-2), and Bacillus subtilis (Xyl-3) and of Grindamyl S100 (GS100), a commercial enzyme preparation containing a Xyl-1 type endo-xylanase, were tested on rye bran to study the solubilization of water-unextractable arabinoxylans (WUAX) . Eight different extrusion-treated rye brans were also used as substrates to find the best physical treatment to facilitate enzymatic arabinoxylan (AX) solubilization . Arabinoxylans were better solubilized from the bran extruded at high temperature using Xyl-3 . This enzyme was then tested in combination with pure (1,4)-beta-d-arabinoxylan arabinofuranohydrolase (AXH) and endo-beta-d-glucanase or ferulic acid esterase (FAE), from A . niger . Only beta-glucanase in combination with Xyl-3 improved the AX extraction, but it did not have a marked effect on the viscosity of the extracts . Xyl-3 was then tested on a high-shear-treated rye bran, and results were compared to those obtained with the high-temperature-extruded rye bran . The high-shear treatment did not improve the bran AX enzymatic solubilization . The combination of FAE with Xyl-1 or Xyl-3 did not improve the AX extraction from untreated and high-shear-treated rye bran . Finally, to study the gelation capacity of the enzymatically solubilized AX, the effect of the hydrogen peroxide/horseradish peroxidase (H(2)O(2)/POD) was tested on the Xyl-3 high-temperature-extruded bran extracts . Solubilized AX did not gel in the presence of the oxidizing system. Biotechnol Bioeng, 2004 Jul 5, 87(1), 81 - 9 Intracellular reactive oxygen species mediate suppression of sporulation in Bacillus subtilis under shear stress; Sahoo S et al.; Sporulation is an important cellular response to stress that is also significant from a bioreactor operation viewpoint . While sporulating organisms are known to show an enhanced sporulation response under several stress situations, the sporulation response to shear stress has not been investigated thus far . Such a study could be of interest since shear stress, to a greater or lesser degree, is always present in bioreactor operation . In this article, we investigate the sporulation extents of the Gram-positive bacteria Bacillus subtilis at various defined shear levels . We show that, contrary to expectations, shear inhibits sporulation . We found an inverse correlation between the shear rate-dependent specific intracellular reactive oxygen species level (siROS), and the sporulation extent . A 10-fold increase in siROS resulted in about 17-fold decrease in sporulation extent . The involvement of reactive oxygen species (ROS) in sporulation was unknown thus far . Further, through experiments that specifically increased and reduced intracellular ROS (iROS), we established that siROS is responsible for the inhibition of sporulation under shear stress . In addition, we found that shear induced siROS regulated the expression levels of the general stress proteins Ctc and sigma(B) . Based on the above, we hypothesize that siROS may regulate suppression of sporulation under high shear by altering sigma(B) and Ctc expression levels, and a model for the same is presented . Cell, 2004 Jun 25, 117(7), 915 - 25 Coordination of cell division and chromosome segregation by a nucleoid occlusion protein in Bacillus subtilis; Wu LJ et al.; A range of genetical and physiological experiments have established that diverse bacterial cells possess a function called nucleoid occlusion, which acts to prevent cell division in the vicinity of the nucleoid . We have identified a specific effector of nucleoid occlusion in Bacillus subtilis, Noc (YyaA), as an inhibitor of division that is also a nonspecific DNA binding protein . Under various conditions in which the cell cycle is perturbed, Noc prevents the division machinery from assembling in the vicinity of the nucleoid . Unexpectedly, cells lacking both Noc and the Min system (which prevents division close to the cell poles) are blocked for division, apparently because they establish multiple nonproductive accumulations of division proteins . The results help to explain how B . subtilis specifies the division site under a range of conditions and how it avoids catastrophic breakage of the chromosome by division through the nucleoid. Food Chem Toxicol, 2004 Aug, 42(8), 1323 - 37 Chemical analysis and genotoxicological safety assessment of paper and paperboard used for food packaging; Ozaki A et al.; This study presents the research on the chemical analysis and genotoxicity of 28 virgin/recycled paper products in food-contact use . In the chemical analysis, paper products were extracted by reflux with ethanol, and analyzed by gas chromatography/mass spectrometry . 4,4'-bis(dimethylamino)benzophenone (Michler's ketone: MK), 4,4'-bis(diethylamino)benzophenone (DEAB), 4-(dimethylamino)benzophenone (DMAB) and bisphenol A (BPA) were found characteristically in recycled products . Seventy-five percent of the recycled paper products contained MK (1.7-12 microg/g), 67% contained DEAB (0.64-10 micro g/g), 33% contained DMAB (0.68-0.9 microg/g) and 67% contained BPA (0.19-26 microg/g) . Although, BPA was also detected in virgin paper products, the detection levels in the recycled products were ten or more times higher than those in the virgin products . The genotoxicity of paper and paperboard extracts and compounds found in them were investigated by Rec-assay and comet assay . Of the 28 products tested by Rec-assay using Bacillus subtilis, 13 possessed DNA-damaging activity . More recycled than virgin products (75% against 25%) exhibited such activity, which, of the compounds, was observed in BPA, 1,2-benzisothiazoline-3-one (BIT), 2-(thiocyanomethylthio)benzothiazole, 2,4,5,6-tetrachloro-isophthalonitrile, 2,4,6-trichlorophenol (TCP), and pentachlorophenol . The critical toxicant in one virgin paper product was concluded to be BIT . Eight samples with DNA-damaging activity were also tested by comet assay using HL-60 cells; six induced comet cells significantly (five times or higher than the control) without a decrease of viable cells . TCP, BZ, DEAB, and BIT also caused a slight increase in comet cells . In conclusion, we showed that most recycled paper products contain chemicals such as MK, DEAB, DMAB, and BPA, and possess genotoxicity . However, the levels of the chemicals in the recycled products could not explain their genotoxic effects. J Bacteriol, 2004 Jul, 186(13), 4390 - 4 Insulation of the sigmaF regulatory system in Bacillus subtilis; Carniol K et al.; The transcription factors sigmaF and sigmaB are related RNA polymerase sigma factors that govern dissimilar networks of adaptation to stress conditions in Bacillus subtilis . The two factors are controlled by closely related regulatory pathways, involving protein kinases and phosphatases . We report that insulation of the sigmaF pathway from the sigmaB pathway involves the integrated action of both the cognate kinase and the cognate phosphatase. J Bacteriol, 2004 Jul, 186(13), 4307 - 14 Group I intron homing in Bacillus phages SPO1 and SP82: a gene conversion event initiated by a nicking homing endonuclease; Landthaler M et al.; Many group I introns encode endonucleases that promote intron homing by initiating a double-stranded break-mediated homologous recombination event . In this work we describe intron homing in Bacillus subtilis phages SPO1 and SP82 . The introns encode the DNA endonucleases I-HmuI and I-HmuII, respectively, which belong to the H-N-H endonuclease family and possess nicking activity in vitro . Coinfections of B . subtilis with intron-minus and intron-plus phages indicate that I-HmuI and I-HmuII are required for homing of the SPO1 and SP82 introns, respectively . The homing process is a gene conversion event that does not require the major B . subtilis recombination pathways, suggesting that the necessary functions are provided by phage-encoded factors . Our results provide the first examples of H-N-H endonuclease-mediated intron homing and the first demonstration of intron homing initiated by a nicking endonuclease. J Bacteriol, 2004 Jul, 186(13), 4262 - 75 Autoinduction of Bacillus subtilis phoPR operon transcription results from enhanced transcription from EsigmaA- and EsigmaE-responsive promoters by phosphorylated PhoP; Paul S et al.; The phoPR operon encodes a response regulator, PhoP, and a histidine kinase, PhoR, which activate or repress genes of the Bacillus subtilis Pho regulon in response to an extracellular phosphate deficiency . Induction of phoPR upon phosphate starvation required activity of both PhoP and PhoR, suggesting autoregulation of the operon, a suggestion that is supported here by PhoP footprinting on the phoPR promoter . Primer extension analyses, using RNA from JH642 or isogenic sigE or sigB mutants isolated at different stages of growth and/or under different growth conditions, suggested that expression of the phoPR operon represents the sum of five promoters, each responding to a specific growth phase and environmental controls . The temporal expression of the phoPR promoters was investigated using in vitro transcription assays with RNA polymerase holoenzyme isolated at different stages of Pho induction, from JH642 or isogenic sigE or sigB mutants . In vitro transcription studies using reconstituted EsigmaA, EsigmaB, and EsigmaE holoenzymes identified PA4 and PA3 as EsigmaA promoters and PE2 as an EsigmaE promoter . Phosphorylated PhoP (PhoP approximately P) enhanced transcription from each of these promoters . EsigmaB was sufficient for in vitro transcription of the PB1 promoter . P5 was active only in a sigB mutant strain . These studies are the first to report a role for PhoP approximately P in activation of promoters that also have activity in the absence of Pho regulon induction and an activation role for PhoP approximately P at an EsigmaE promoter . Information concerning PB1 and P5 creates a basis for further exploration of the regulatory coordination or overlap of the PhoPR and SigB regulons during phosphate starvation. J Bacteriol, 2004 Jul, 186(13), 4085 - 99 Microarray-based analysis of the Staphylococcus aureus sigmaB regulon; Bischoff M et al.; Microarray-based analysis of the transcriptional profiles of the genetically distinct Staphylococcus aureus strains COL, GP268, and Newman indicate that a total of 251 open reading frames (ORFs) are influenced by sigmaB activity . While sigmaB was found to positively control 198 genes by a factor of > or =2 in at least two of the three genetic lineages analyzed, 53 ORFs were repressed in the presence of sigmaB . Gene products that were found to be influenced by sigmaB are putatively involved in all manner of cellular processes, including cell envelope biosynthesis and turnover, intermediary metabolism, and signaling pathways . Most of the genes and/or operons identified as upregulated by sigmaB were preceded by a nucleotide sequence that resembled the sigmaB consensus promoter sequence of Bacillus subtilis . A conspicuous number of virulence-associated genes were identified as regulated by sigmaB activity, with many adhesins upregulated and prominently represented in this group, while transcription of various exoproteins and toxins were repressed . The data presented here suggest that the sigmaB of S . aureus controls a large regulon and is an important modulator of virulence gene expression that is likely to act conversely to RNAIII, the effector molecule of the agr locus . We propose that this alternative transcription factor may be of importance for the invading pathogen to fine-tune its virulence factor production in response to changing host environments. J Mol Biol, 2004 Jul 2, 340(2), 203 - 9 Physical evidence for the induced release of the Bacillus subtilis transcription factor, sigma(F), from its inhibitory complex; Clarkson J et al.; The release of the transcription factor sigma(F) from its inhibitory complex with SpoIIAB is a key regulatory step in the control of sporulation in Bacillus subtilis as it initiates a pattern of differential gene expression in the mother cell and prespore compartments . The sigma(F).SpoIIAB complex is dissociated by the unphosphorylated form of the protein SpoIIAA, the alternative binding partner of SpoIIAB . Here, we employ fluorescence spectroscopy to examine the mechanism by which SpoIIAA acts on the sigma(F).SpoIIAB complex . We constructed a mutant of sigma(F), sigma(F)-W46L, which displayed a reproducible fluorescence response on binding to SpoIIAB . Using this mutant we were able to quantify the amount of sigma(F) bound to SpoIIAB in real time . The results provide physical evidence for the "induced release" mechanism of sigma(F) activation . We demonstrate that SpoIIAA interacts directly with the sigma(F).SpoIIAB complex, greatly decreasing the affinity of SpoIIAB for sigma(F) and thus causing the release of the latter . We also demonstrate that sigma(F) is released before SpoIIAA is phosphorylated and that release occurs on a similar time scale to the binding of SpoIIAA to SpoIIAB. AAPS PharmSciTech . 2003 Nov 05;4(4):E56. Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11; Adinarayana K et al.; The purpose of the research was to study the purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11 . The enzyme was purified in a 2-step procedure involving ammonium sulfate precipitation and Sephadex G-200 gel permeation chromatography . The enzyme was shown to have a relative low molecular weight of 15 kd by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and was purified 21-fold with a yield of 7.5% . It was most active at 60 degrees C, pH 10, with casein as substrate . It was stable between pH 8 and 10 . This enzyme was almost 100% stable at 60 degrees C even after 350 minutes of incubation . It was strongly activated by metal ions such as Ca+2, Mg+2, and Mn+2 . Enzyme activity was inhibited strongly by phenylmethyl sulphonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP) but was not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with iodoacetate, p-chloromercuric benzoate (pCMB), and beta-mercaptoethanol (beta-ME) . The compatibility of the enzyme was studied with commercial and local detergents in the presence of 10mM CaCl2 and 1M glycine . The addition of 10mM CaCl2 and 1M glycine, individually and in combination, was found to be very effective in improving the enzyme stability where it retained 52% activity even after 3 hours . This enzyme improved the cleansing power of various detergents . It removed blood stains completely when used with detergents in the presence of 10mM CaCl2 and 1M glycine. J Ind Microbiol Biotechnol, 2004 Jun, 31(5), 199 - 203 Epub 2004 Jun 12. Antifungal activity of Bacillus subtilis 355 against wood-surface contaminant fungi; Feio SS et al.; A strain of Bacillus subtilis was examined for antifungal activity against phytopathogenic and wood-surface contaminant fungi . The bacterium was grown in five culture media with different incubation times in order to study cell development, sporulation, and the production of metabolites with antifungal activity . The anti-sapstain and anti-mould activity of the bacterium grown in yeast extract glucose broth (YGB) medium in wood was also evaluated . In YGB, the bacterium inhibited the growth of several fungi and displayed a broader spectrum of activity than in the other media tested . A relationship between bacterial spore production and the formation of metabolites with antifungal activity was detected . YGB medium displayed effective control in wood block tests . YGB medium was extracted with solvents of increasing polarity and the dry residues were applied to silicagel plates, resolved with the appropriate solvent and sprayed with different solutions, detecting the presence, of amines, and higher alcohols . The bioautographic method revealed the presence of at least two active compounds against the blue-stain fungus Cladosporium cucumerinum. Lett Appl Microbiol, 2004, 39(1), 98 - 102 Application of electrospray ionization mass spectrometry in rapid typing of fengycin homologues produced by Bacillus subtilis; Wang J et al.; AIMS: To rapidly type the fengycin homologues produced by Bacillus subtilis strains with electrospray ionization/collision-induced dissociation (ESI/CID) mass spectrometry . METHODS AND RESULTS: Fengycin homologues produced by Bacillus subtilis JA were analysed . When each homologue was subjected to ESI/CID analysis, ions representing characteristic fragmentations were detected . These ions can help to identify the homologues; even homologues of the same nominal mass can be discriminated by their ESI/CID spectra . Based on the CID results, fengycin homologues can be correctly assigned . CONCLUSIONS AND SIGNIFICANCE OF THIS STUDY: ESI/CID leads to rapid detection and structural characterization of fengycin homologues or lipopeptides with similar properties . It will be very useful in studying the regulatory expression of these peptides. Lett Appl Microbiol, 2004, 39(1), 65 - 73 The Bacillus secretion stress response is an indicator for alpha-amylase production levels; Westers H et al.; AIMS: Overproduced alpha-amylases in Bacillus subtilis provoke a specific stress response involving the CssRS two-component system, which controls expression of the HtrA and HtrB proteases . Previously, the B . subtilis TepA protein was implicated in high-level alpha-amylase secretion . Our present studies were aimed at investigating a possible role of TepA in secretion stress management, and characterizing the intensity of the secretion stress response in relation to alpha-amylase production . METHODS AND RESULTS: The expression of a transcriptional htrB-lacZ gene fusion, and the levels of alpha-amylase production were monitored simultaneously using tepA mutant B . subtilis strains . TepA was shown to be dispensable for secretion stress management . Importantly, however, the levels of htrB-lacZ expression can be correlated with the levels of alpha-amylase production . CONCLUSION: Our observations show that the secretion stress response can serve as an indicator for alpha-amylase production levels . SIGNIFICANCE AND IMPACT OF STUDY: Conceivably, this stress response can be employed to monitor the biotechnological production of various secretory proteins by the Bacillus cell factory. Zh Mikrobiol Epidemiol Immunobiol, 2004 Mar-Apr, (2), 91 - 4 {Properties of the isolated Bacillus subtilis strains and their influence on the intestinal microflora of experimental mice}; Gataullin AG et al.; The cultural, physiologo-biochemical adhesive and antagonistic properties of B . subtilis strains with good prospects for use as biotherapeutic preparations were studied . For further studies B . subtilis strain No . 1719 was chosen . In experiments on non-inbred white mice the animals were treated by the preparation Cifran used for their selective decontamination from opportunistic microflora and for the creation of the state of dysbiosis . The influence of the spore-forming microbe on the parietal microflora of the large intestine of the animals was shown . Reliable data on the changes in the number of microorganisms (CFU/ml) per 1 cm of the surface of the large intestine were established . As markers making it possible to evaluate the action of biotherapeutic and other medicinal remedies, easily determinable ratios of lac+/lac- of bacteria and Staphylococcus aureus/Staphylococcus spp . was proposed. Electrophoresis, 2004 Jun, 25(10-11), 1695 - 704 Insulator-based dielectrophoresis for the selective concentration and separation of live bacteria in water; Lapizco-Encinas BH et al.; Insulator-based dielectrophoresis (iDEP) was utilized to separate and concentrate selectively mixtures of two species of live bacteria simultaneously . Four species of bacteria were studied: the Gram-negative Escherichia coli and the Gram-positive Bacillus subtilis, B . cereus, and B . megaterium . Under an applied direct current (DC) electric field all the bacterial species exhibited negative dielectrophoretic behavior . The dielectrophoretic separations were carried out in a glass microchannel containing an array of insulating posts . The insulating posts in the microchannel produced nonuniformities in the electric field applied along the channel . Mixtures of two species of bacteria were introduced into the microchannel and the electric field was applied . The bacterial species exhibited different dielectrophoretic mobilities under the influence of the nonuniform field . From these experiments a trapping order was established with E . coli trapping at the weakest applied electric field, while the Bacillus species were trapped at different characteristic threshold fields . At stronger applied electric fields, the two different species of bacteria in the microchannel were dielectrophoretically trapped into two spatially distinct bands . The results showed that iDEP has the potential to selectively concentrate and separate different species of bacteria. Proc Natl Acad Sci U S A, 2004 Jun 15, 101(24), 8882 - 7 Epub 2004 Jun 08. Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells; Zhang Z et al.; An orthogonal tryptophanyl-transfer RNA (tRNA) synthetase (TrpRS)-mutant opal suppressor tRNA(Trp) (mutRNA(UCA)(Trp)) pair was generated for use in mammalian cells . The anticodon loop of the Bacillus subtilis tRNA(Trp) was mutated to UCA, three positions in the D arm were mutated to generate an internal promoter sequence, and the mutRNA(UCA)(Trp) gene was inserted between the 5' and 3' flanking sequences of the tRNA(Trp-1) gene from Arabidopsis to enhance its expression in mammalian cells . In vitro aminoacylation assays and in vivo opal suppression assays showed that B . subtilis TrpRS (BsTrpRS) charges only the cognate mutRNA(UCA)(Trp) and no endogenous mammalian tRNAs . Similarly, the mutRNA(UCA)(Trp) is specifically charged by B . subtilis TrpRS and not by endogenous synthetases in mammalian cells . Site-directed mutagenesis was then used to alter the specificity of BsTrpRS to uniquely charge 5-hydoxy-l-tryptophan . The resulting mutant BsTrpRS-mutRNA(UCA)(Trp) pair allows the efficient and selective incorporation of 5-hydroxy-l-tryptophan into mammalian proteins in response to the codon, TGA . This amino acid can be used as a fluorescence probe and also undergoes electrochemical oxidation in situ to generate an efficient protein crosslinking. Microbiol Mol Biol Rev, 2004 Jun, 68(2), 234 - 62 Compartmentalization of gene expression during Bacillus subtilis spore formation; Hilbert DW et al.; Gene expression in members of the family Bacillaceae becomes compartmentalized after the distinctive, asymmetrically located sporulation division . It involves complete compartmentalization of the activities of sporulation-specific sigma factors, sigma(F) in the prespore and then sigma(E) in the mother cell, and then later, following engulfment, sigma(G) in the prespore and then sigma(K) in the mother cell . The coupling of the activation of sigma(F) to septation and sigma(G) to engulfment is clear; the mechanisms are not . The sigma factors provide the bare framework of compartment-specific gene expression . Within each sigma regulon are several temporal classes of genes, and for key regulators, timing is critical . There are also complex intercompartmental regulatory signals . The determinants for sigma(F) regulation are assembled before septation, but activation follows septation . Reversal of the anti-sigma(F) activity of SpoIIAB is critical . Only the origin-proximal 30% of a chromosome is present in the prespore when first formed; it takes approximately 15 min for the rest to be transferred . This transient genetic asymmetry is important for prespore-specific sigma(F) activation . Activation of sigma(E) requires sigma(F) activity and occurs by cleavage of a prosequence . It must occur rapidly to prevent the formation of a second septum . sigma(G) is formed only in the prespore . SpoIIAB can block sigma(G) activity, but SpoIIAB control does not explain why sigma(G) is activated only after engulfment . There is mother cell-specific excision of an insertion element in sigK and sigma(E)-directed transcription of sigK, which encodes pro-sigma(K) . Activation requires removal of the prosequence following a sigma(G)-directed signal from the prespore. Microbiol Mol Biol Rev, 2004 Jun, 68(2), 207 - 33 Proteomics of protein secretion by Bacillus subtilis: separating the "secrets" of the secretome; Tjalsma H et al.; Secretory proteins perform a variety of important "remote-control" functions for bacterial survival in the environment . The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes . Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes . Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which approximately 90 extracellular proteins were identified . Analysis of these proteins disclosed various "secrets of the secretome," such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome . This showed that genome-based predictions reflect only approximately 50% of the actual composition of the extracellular proteome of B . subtilis . Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment . In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B . subtilis and other gram-positive bacteria . Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance. Mol Microbiol, 2004 Jun, 52(6), 1757 - 67 Control of DNA replication initiation by recruitment of an essential initiation protein to the membrane of Bacillus subtilis; Rokop ME et al.; The Bacillus subtilis proteins DnaD and DnaB are essential for replication initiation and are conserved in low G+C content Gram-positive bacteria . Previous work indicated that DnaD and DnaB are involved in helicase loading during the process of restarting stalled replication forks . We have investigated the roles of DnaD and DnaB in replication initiation at oriC in vivo . Using chromatin immunoprecipitation (ChIP), we found that DnaD and DnaB functions are needed to load the replicative helicase at oriC . To investigate further the functions of DnaD and DnaB in replication initiation, we isolated and characterized suppressors of the temperature sensitivity of dnaD and dnaB mutant cells . In both cases, we isolated the identical missense mutation in dnaB, dnaBS371P . Using yeast two-hybrid analysis, we found that dnaBS371P uncovers a previously undetected physical interaction between DnaD and DnaB . We also found that DnaBS371P constitutively recruits DnaD to the membrane fraction of cells, where DnaB and oriC are enriched . Phenotypes of cells expressing DnaBS371P are consistent with aberrant replication control . We hypothesize that B . subtilis regulates replication initiation by regulating a physical interaction between two proteins essential for helicase loading at chromosomal origins. Nat Struct Mol Biol, 2004 Jul, 11(7), 643 - 9 Epub 2004 Jun 06. DNA transport into Bacillus subtilis requires proton motive force to generate large molecular forces; Maier B et al.; Bacteria can acquire genetic diversity, including antibiotic resistance and virulence traits, by horizontal gene transfer . In particular, many bacteria are naturally competent for uptake of naked DNA from the environment in a process called transformation . Here, we used optical tweezers to demonstrate that the DNA transport machinery in Bacillus subtilis is a force-generating motor . Single DNA molecules were processively transported in a linear fashion without observable pausing events . Uncouplers inhibited DNA uptake immediately, suggesting that the transmembrane proton motive force is needed for DNA translocation . We found an uptake rate of 80 +/- 10 bp s(-1) that was force-independent at external forces <40 pN, indicating that a powerful molecular machine supports DNA transport. Microbiology, 2004 Jun, 150(Pt 6), 1839 - 49 Branched swarming patterns on a synthetic medium formed by wild-type Bacillus subtilis strain 3610: detection of different cellular morphologies and constellations of cells as the complex architecture develops; Julkowska D et al.; After optimizing the conditions, including nutrients and temperature, swarming of Bacillus subtilis 3610 was obtained on a synthetic, fully defined medium . The swarms formed highly branched (dendritic) patterns, generated by successive waves of moving cells . A detailed microscopic in situ analysis of swarms 1 and 2 revealed varied cell morphologies and a remarkable series of events, with cells assembling into different 'structures', as the architecture of the swarm developed . Long filamentous cells begin to form before the onset of the first swarming (11 h) and are again observed at later stages in the interior of individual mature dendrites . Swarm 2, detected at 18-22 h, is accompanied by the rapid movement of a wave of dispersed (non-filamentous) cells . Subsequently at the forward edge of this swarm, individual cells begin to cluster together, gradually forming de novo the shape of a dendrite tip with progressive lengthening of this new structure 'backwards' towards the swarm centre . In both swarms 1 and 2, after the initial clustering of cells, there is the progressive appearance of a spreading monolayer of rafts (4-5 non-filamented cells, neatly aligned) . The alternative possible roles of the rafts in the development of the swarm are discussed. FEMS Microbiol Lett, 2004 Jun 15, 235(2), 393 - 9 Cloning and characterization of the Halobacillus trueperi betH gene, encoding the transport system for the compatible solute glycine betaine; Lu W et al.; Halobacillus trueperi accumulates glycine betaine under condition of high osmolarity . A fragment of the glycine betaine transporter betH gene was obtained from the genome of H . trueperi with degenerate primers . Through Southern blot hybridization and inverse PCR, a 5.1 kb EcoRI fragment containing the complete betH gene was identified and subsequently sequenced . The betH gene was predicted to encode a 55.2 kDa protein (504 amino acid residues) with 12 transmembrane regions . BetH showed 56% identity to the OpuD of Bacillus subtilis which belongs to the betaine/carnitine/choline transporter (BCCT) family . Its putative promoter region was highly homologous to sigmaB-dependent promoter of B . subtilis . A 2.6 kb fragment containing the betH gene was cloned into pUC18 and transformed into the Escherichia coli MKH13 . The accumulation of glycine betaine in transformed E . coli MKH13 bacteria was confirmed using 13C nuclear magnetic resonance spectroscopy. Biochim Biophys Acta, 2004 Jun 11, 1672(3), 184 - 91 Activation and inhibition of Candida rugosa and Bacillus-related lipases by saturated fatty acids, evaluated by a new colorimetric microassay; Ruiz C et al.; Research on lipase inhibitors could help in the therapy of diseases caused by lipase-producing microorganisms and in the design of novel lipase substrate specificities for biotechnology . Here we report a fast and sensitive colorimetric microassay that is low-cost and suitable for high-throughput experiments for the evaluation of lipase activity and inhibition . Comparison of Candida rugosa activity and inhibition with previous HPLC results validated the method, and revealed the importance of the reaction mixture composition . The assay was used to evaluate the effect of saturated fatty acids on Bacillus-related lipases . Cell-bound esterases were strongly inhibited by fatty acids, suggesting a negative feedback regulation by product, and a role of these enzymes in cell membrane turnover . Bacillus subtilis LipA was moderately activated by low concentrations of fatty acids and was inhibited at greater concentrations . LipB-like esterases were highly activated by myristic and lauric acids and were only slightly inhibited by high capric acid concentrations . Such an activation, reported here for the first time in bacterial lipases, seems to be part of a regulatory system evolved to ensure a high use of carbon sources, and could be related to the successful adaptation of Bacillus strains to nutrient-rich environments with strong microbial competition. Biochemistry, 2004 Jun 15, 43(23), 7391 - 402 Gln212, Asn270, and Arg301 are critical for catalysis by adenylosuccinate lyase from Bacillus subtilis; Segall ML et al.; In adenylosuccinate lyase from Bacillus subtilis, Gln(212), Asn(270), and Arg(301) are conserved and located close to the succinyl moiety of docked adenylosuccinate . We constructed mutant enzymes with Gln(212) replaced by Glu and Met, Asn(270) by Asp and Leu, and Arg(301) by Gln or Lys . The wild-type and mutant enzymes were expressed in Escherichia coli and purified to homogeneity . The specific activities of the Q212M and the 270 and 301 mutant enzymes were decreased more than 3000-fold as compared to the wild type . Only Q212E retained sufficient activity for determination of its kinetic parameters: V(max) was decreased approximately 1000-fold, and K(m) was increased 6-fold, as compared to the wild-type enzyme . Adenylosuccinate binding studies of the other mutants revealed greatly weakened affinities that contributed to, but did not account entirely for, the loss of activity . These mutant enzymes did not differ greatly from the wild-type enzyme in secondary structure or subunit association state, as shown by circular dichroism spectroscopy and light-scattering photometry . Incubation of pairs of inactive mutant enzymes led to reconstitution of some functional sites by subunit complementation, with recovery of up to 25% of the specific activity of the wild-type enzyme . Subunit complementation occurs only if the two mutations are contributed to the active site by different subunits . Thus, mixing Q212E with N270L enzyme yielded a specific activity of approximately 20% of the wild-type enzyme, while mixing Q212M with R301K enzyme did not restore activity . As supported by computer modeling, the studies presented here indicate that Gln(212), Asn(270), and Arg(301) are indispensable to catalysis by adenylosuccinate lyase and probably interact noncovalently with the carboxylate anions of the substrates 5-aminoimidazole-4(N-succinylocarboxamide)ribonucleotide and adenylosuccinate, optimizing their bound orientations. J Biol Chem, 2004 Aug 20, 279(34), 35479 - 85 Epub 2004 Jun 04. Crystal structure of Bacillus subtilis guanine deaminase: the first domain-swapped structure in the cytidine deaminase superfamily; Liaw SH et al.; Guanine deaminase, a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine . The crystal structure of the 156-residue guanine deaminase from Bacillus subtilis has been solved at 1.17-A resolution . Unexpectedly, the C-terminal segment is swapped to form an intersubunit active site and an intertwined dimer with an extensive interface of 3900 A(2) per monomer . The essential zinc ion is ligated by a water molecule together with His(53), Cys(83), and Cys(86) . A transition state analog was modeled into the active site cavity based on the tightly bound imidazole and water molecules, allowing identification of the conserved deamination mechanism and specific substrate recognition by Asp(114) and Tyr(156') . The closed conformation also reveals that substrate binding seals the active site entrance, which is controlled by the C-terminal tail . Therefore, the domain swapping has not only facilitated the dimerization but has also ensured specific substrate recognition . Finally, a detailed structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites found in the guanine, cytosine, and cytidine deaminases and suggests putative specific substrate-interacting residues for other members such as dCMP deaminases. Protein Expr Purif, 2004 Jul, 36(1), 124 - 30 Overexpression, purification, and characterization of ATP-NAD kinase of Sphingomonas sp . A1; Ochiai A et al.; The NAD kinase gene (nadK) of Sphingomonas sp . A1 was cloned and then overexpressed in Escherichia coli, and the gene product (NadK) was purified from the E . coli cells through five steps with a 25% yield of activity . NadK was a homodimer of 32 kDa subunits, utilized ATP or other nucleoside triphosphates, but not inorganic polyphosphates, as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 8.0 and 50-55 degrees C, and was designated as ATP-NAD kinase (NadK) . NadK showed no NADH kinase activity and was slightly inhibited by NADP(H) . Precursors for NAD biosynthesis such as quinolinic acid, nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, and nicotinic acid had no effect on the NadK activity, as observed in the cases of the NAD kinases of Micrococcus flavus, Mycobacterium tuberculosis, and E . coli . Taken together with the report that the NAD kinase of Bacillus subtilis is activated by quinolinic acid {J . Bacteriol . 185 (2003) 4844}, it is indicated that the regulatory patterns of NAD kinases differ even among bacterial NAD kinases. Biotechnol Prog, 2004 May-Jun, 20(3), 979 - 83 Optimizing iron supplement strategies for enhanced surfactin production with Bacillus subtilis; Wei YH et al.; Supplement of Fe(2+) into fermentation medium was utilized as a tool to optimize the iron-mediated enhancement of surfactin production from Bacillus subtilis ATCC 21332 . Up to 3000 mg L(-)(1) of surfactin was produced using an iron-enriched minimal salt (MS) medium amended with an optimal Fe(2+) dosage of 4.0 mM, leading to 8-fold and 10-fold increase in cell concentration and surfactin yield, respectively, as compared to those without Fe(2+) supplement . In addition to resulting in an optimal production yield of surfactin, a supplement of 4.0 mM of Fe(2+) also propelled maximum overall surfactin production rate to a highest value of 24 mg L(-)(1) h(-)(1) . Our results also show that production of surfactin followed a growth-associated kinetic model . The best yield coefficient estimated from the model was ca . 162 mg surfactin (g dry cell)(-)(1) . The supernatant of the iron-enriched culture of B . subtilis ATCC 21332 exhibited the ability to emulsify kerosene and achieved a maximum emulsion index (E(24)) of 80% for culture supplemented with 4.0 mM of Fe(2+) . Comparison of emulsion index and the corresponding surfactin production indicates that the emulsification activity was essentially contributed by surfactin. EMBO J, 2004 Jul 7, 23(13), 2664 - 73 Epub 2004 Jun 03. Positive and negative regulation of SMC-DNA interactions by ATP and accessory proteins; Hirano M et al.; Structural maintenance of chromosomes (SMC) proteins are central regulators of higher-order chromosome dynamics from bacteria to humans . The Bacillus subtilis SMC (BsSMC) homodimer adopts a V-shaped structure with an ATP-binding catalytic domain at each end . We report here that two small proteins, ScpA and ScpB, associate with the catalytic domains of BsSMC in an ordered fashion and suppress its ATPase activity . When combined with a 'transition state' mutant of BsSMC that poorly hydrolyzes ATP, ScpA promotes stable engagement of two catalytic domains in an ATP-dependent manner . In solution, this occurs intramolecularly and closes the DNA-entry gate of an SMC dimer . ScpB further stabilizes this conformation and prevents BsSMC from binding to double-stranded DNA (dsDNA) . In contrast, when the mutant BsSMC is first allowed to interact with dsDNA, subsequent addition of ScpA leads to assembly of large nucleoprotein complexes, possibly by stabilizing intermolecular engagement of the catalytic domains from different SMC dimers . We propose that the ATP-modulated engagement/disengagement cycle of SMC proteins plays both positive and negative roles in their dynamic interactions with dsDNA. J Bacteriol, 2004 Jun, 186(12), 4025 - 9 The last gene of the fla/che operon in Bacillus subtilis, ylxL, is required for maximal sigmaD function; Werhane H et al.; ylxL was found to be the last gene of the fla/che operon in Bacillus subtilis and is cotranscribed with the gene for the flagellum-specific alternate sigma factor, sigma(D) . The ylxL gene was disrupted by insertional mutagenesis, and the resultant mutant strain was found to be compromised for sigma(D)-dependent functions. J Bacteriol, 2004 Jun, 186(12), 4000 - 13 Role of the anti-sigma factor SpoIIAB in regulation of sigmaG during Bacillus subtilis sporulation; Serrano M et al.; RNA polymerase sigma factor sigma(F) initiates the prespore-specific program of gene expression during Bacillus subtilis sporulation . sigma(F) governs transcription of spoIIIG, encoding the late prespore-specific regulator sigma(G) . However, transcription of spoIIIG is delayed relative to other genes under the control of sigma(F), and after synthesis, sigma(G) is initially kept in an inactive form . Activation of sigma(G) requires the complete engulfment of the prespore by the mother cell and expression of the spoIIIA and spoIIIJ loci . We screened for random mutations in spoIIIG that bypassed the requirement for spoIIIA for the activation of sigma(G) . We found a mutation (spoIIIGE156K) that resulted in an amino acid substitution at position 156, which is adjacent to the position of a mutation (E155K) previously shown to prevent interaction of SpoIIAB with sigma(G) . Comparative modelling techniques and in vivo studies suggested that the spoIIIGE156K mutation interferes with the interaction of SpoIIAB with sigma(G) . The sigma(GE156K) isoform restored sigma(G)-directed gene expression to spoIIIA mutant cells . However, expression of sspE-lacZ in the spoIIIA spoIIIGE156K double mutant was delayed relative to completion of the engulfment process and was not confined to the prespore . Rather, beta-galactosidase accumulated throughout the entire cell at late times in development . This suggests that the activity of sigma(GE156K) is still regulated in the prespore of a spoIIIA mutant, but not by SpoIIAB . In agreement with this suggestion, we also found that expression of spoIIIGE156K from the promoter for the early prespore-specific gene spoIIQ still resulted in sspE-lacZ induction at the normal time during sporulation, coincidently with completion of the engulfment process . In contrast, transcription of spoIIIGE156K, but not of the wild-type spoIIIG gene, from the mother cell-specific spoIID promoter permitted the rapid induction of sspE-lacZ expression . Together, the results suggest that SpoIIAB is either redundant or has no role in the regulation of sigma(G) in the prespore. Photochem Photobiol Sci, 2004 Jun, 3(6), 575 - 9 Epub 2004 Apr 15. Functional variations among LOV domains as revealed by FT-IR difference spectroscopy; Bednarz T et al.; The two LOV domains, LOV1 and LOV2, from Chlamydomonas reinhardtii were investigated by light-induced FT-IR difference spectroscopy and compared to the LOV domain of Bacillus subtilis (YtvA-LOV) . It is shown that the two S-H conformations of the reactive LOV1 cysteine C57(1) are exposed to environments of different hydrogen bonding strength . Thus, the two rotamer configurations of C57 might be related to the fact that the triplet state decays bi-exponentially into the LOV1-390 photoproduct . Exchange of the two other cysteines of LOV1 (C32S and C83S) does not alter the S-H stretching band providing evidence that this band feature arises solely from C57 . The reactive cysteine of LOV2 from Chlamydomonas reinhardtii (C250) and of YtvA-LOV (C62) exhibit both a homogenous S-H stretching vibrational band which suggests a single conformer of the amino acid side chain . Finally, the FT-IR difference spectrum of YtvA from Bacillus subtilis comprising the light absorbing LOV domain and the putative signaling STAS (sulfate transporter/antisigma-factor antagonist) domain, reveals conformational changes in the latter after blue-light excitation. Photochem Photobiol Sci, 2004 Jun, 3(6), 566 - 74 Epub 2004 Mar 10. The bacterial counterparts of plant phototropins; Losi A; We review and analyze the growing family of bacterial proteins carrying the LOV (light oxygen voltage) motif, a flavin-binding photoactive domain first characterized in plant blue-light receptors, the phototropins . A total of 29 sequences encoding LOV-proteins can be detected in the genomes of 24 bacterial species . In the bacterial LOV domains, the majority of the amino acids known to interact with the flavin mononucleotide (FMN) chromophore in phototropin LOVs are conserved, supporting the suggestion of their possible role as blue-light sensors . The Bacillus subtilis protein YtvA has been the first bacterial LOV-protein shown to bind FMN and to undergo the same light-induced reactions as plant phototropins . The photocycle involves the reversible formation of a covalent adduct between FMN and a conserved cysteine . In this work we report preliminary results on a Caulobacter crescentus LOV-kinase, that undergoes the same photochemistry as YtvA . The bacterial LOV-proteins exhibit a variety of effector domains associated to the light-responsive LOV-domain, e.g . histidine kinase, transcriptional regulators, putative phosphodiesterases and regulators of stress factors, pointing to their physiological role as sensing and signalling proteins. Curr Microbiol, 2004 Jun, 48(6), 401 - 4 The delta subunit of RNA polymerase functions in sporulation; Gao H et al.; Purified RNA polymerase from Bacillus subtilis and other Gram-positive organisms contains a novel subunit designated delta encoded by the rpoE gene . There is no distinctive phenotype of strains with a disruption of this gene, so the function of delta is very subtle or redundant . We have found, however, that suppression of a block in sporulation of B . subtilis at early stage III owing to disruption of the pdhC gene encoding the E2 subunit of pyruvate dehydrogenase (PDH) was attributable to a Tn10 insertion in the rpoE gene . An independent disruption of this gene also caused suppression . An earlier sporulation block due to absence of the E1beta subunit of PDH was not suppressed . This specific suppression indicates that the delta subunit does have some direct or indirect role in sporulation, probably in the transcription of selected genes at stage II-III of sporulation, which is critical but only when there is functional E2. Biosci Biotechnol Biochem, 2004 May, 68(5), 1073 - 81 Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from the novel marine isolate, JAMB-A94; Ohta Y et al.; A gene, agaA, for a novel beta-agarase from the marine bacterium JAMB-A94 was cloned and sequenced . The 16S rDNA of the isolate had the closest match, of only 94.8% homology, with that from Microbulbifer salipaludis JCM11542(T) . The agaA gene encoded a protein with a calculated molecular mass of 48,203 Da . The deduced amino acid sequence showed 37-66% identity to those of known agarases in glycoside hydrolase family 16 . A carbohydrate-binding module-like amino acid sequence was found in the C-terminal region . The recombinant enzyme was hyper-produced extracellularly when Bacillus subtilis was used as a host . The purified enzyme was an endo-type beta-agarase, yielding neoagarotetraose as the main final product . It was very thermostable up to 60 degrees C . The optimal pH and temperature for activity were around 7.0 and 55 degrees C respectively . The activity was not inhibited by EDTA (up to 100 mM) and sodium dodecyl sulfate (up to 30 mM). Mol Microbiol, 2004 Jun, 52(5), 1281 - 90 Oligomeric structure of the Bacillus subtilis cell division protein DivIVA determined by transmission electron microscopy; Stahlberg H et al.; DivIVA from Bacillus subtilis is a bifunctional protein with distinct roles in cell division and sporulation . During vegetative growth, DivIVA regulates the activity of the MinCD complex, thus helping to direct cell division to the correct mid-cell position . DivIVA fulfils a quite different role during sporulation in B . subtilis when it directs the oriC region of the chromosome to the cell pole before asymmetric cell division . DivIVA is a 19.5 kDa protein with a large part of its structure predicted to form a tropomyosin-like alpha-helical coiled-coil . Here, we present a model for the quaternary structure of DivIVA, based on cryonegative stain transmission electron microscopy images . The purified protein appears as an elongated particle with lateral expansions at both ends producing a form that resembles a 'doggy-bone' . The particle mass estimated from these images agrees with the value of 145 kDa measured by analytical ultracentrifugation suggesting 6- to 8-mers . These DivIVA oligomers serve as building blocks in the formation of higher order assemblies giving rise to strings, wires and, finally, two-dimensional lattices in a time-dependent manner. J AOAC Int, 2004 Mar-Apr, 87(2), 429 - 34 Studies of polyester fiber as carrier for microbes in a quantitative test method for disinfectants; Miner N et al.; Tests were conducted by a Task Force on Disinfectant Test Methods that was appointed to investigate controversies regarding the accuracy of AOAC test methods for disinfectants as presented in AOAC's Official Methods of Analysis, Chapter 6 . The general principles for new and improved AOAC tests are discussed, and a disinfectant test using microbes labeled onto a polyester fiber surface is described . The quantitative test measures the survival of test microbes as a function of exposure time as well as the exposure conditions required to kill 6 log10 of the test microbes . The time required was similar to that for the kinetics of the kill of Bacillus subtilis-labeled cylinders as tested by methods of the AOAC Sporicidal Test 966.04. Acta Crystallogr D Biol Crystallogr, 2004 Jun, 60(Pt 6), 1152 - 4 Epub 2004 May 21. Crystallization and preliminary crystallographic analysis of Bacillus subtilis guanine deaminase; Chang YJ et al.; Guanine deaminase, a key enzyme in nucleotide metabolism, catalyzes the hydrolytic deamination of guanine to xanthine . The first guanine deaminase crystal from Bacillus subtilis was grown in the absence or presence of the inhibitor hypoxanthine in 30% polyethylene glycol 4000, 0.2 M ammonium acetate and 0.1 M sodium citrate pH 6.5 . The crystals belong to space group C222(1), with unit-cell parameters a = 84.91, b = 90.90, c = 80.19 angstroms, with one dimer per asymmetric unit . The crystals diffract X-rays to beyond 1.2 angstroms resolution and an initial atomic model has been built based on selenomethionyl multiwavelength anomalous data at 2 angstroms resolution . Unexpectedly, this is the first domain-swapped structure in the cytidine deaminase superfamily . Acta Crystallogr D Biol Crystallogr, 2004 Jun, 60(Pt 6), 1101 - 7 Epub 2004 May 21. Harvesting the high-hanging fruit: the structure of the YdeN gene product from Bacillus subtilis at 1.8 angstroms resolution; Janda I et al.; High-throughput (HT) protein crystallography is severely impeded by the relatively low success rate of protein crystallization . Proteins whose structures are not solved in the HT pipeline owing to attrition in any phase of the project are referred to as the high-hanging fruit, in contrast to those proteins that yielded good-quality crystals and crystal structures, which are referred to as low-hanging fruit . It has previously been shown that proteins that do not crystallize in the wild-type form can have their surfaces engineered by site-directed mutagenesis in order to create patches of low conformational entropy that are conducive to forming intermolecular interactions . The application of this method to selected proteins from the Bacillus subtilis genome which failed to crystallize in the HT mode is now reported . In this paper, the crystal structure of the product of the YdeN gene is reported . Of three prepared double mutants, i.e . E124A/K127A, E167A/E169A and K88A/Q89A, the latter gave high-quality crystals and the crystal structure was solved by SAD at 1.8 angstroms resolution . The protein is a canonical alpha/beta hydrolase, with an active site that is accessible to solvent . Acta Crystallogr D Biol Crystallogr, 2004 Jun, 60(Pt 6), 1094 - 100 Epub 2004 May 21. Structure analysis of PH1161 protein, a transcriptional activator TenA homologue from the hyperthermophilic archaeon Pyrococcus horikoshii; Itou H et al.; The crystal structure of the Bacillus subtilis TenA-homologue protein PH1161 from the hyperthermophilic archaebacterium Pyrococcus horikoshii was determined . TenA is known to belong to a new family of activators that stimulate the production of extracellular proteases in B . subtilis . A sequence-similarity search revealed that TenA-homologue proteins are widespread in bacteria and archaea, suggesting that this family of proteins plays an essential role in these organisms . In the present study, the first three-dimensional structure of a member of the TenA family of proteins was determined, unexpectedly revealing that the protein has a fold identical to that of haem oxygenase-1 . Analysis has also shown that the protein has a unique ligand-binding pocket . Electron density of a bound ligand molecule was observed in this pocket . These results provide a valuable insight into the functional understanding of the TenA family of proteins . Appl Biochem Biotechnol, 2004 May, 117(2), 123 - 32 Interaction of an intermediate structure of Bacillus subtilis alpha-amylase with alkyl-substituted sepharose 4B: a model of membrane translocation; Karbalaei-Heidari HR et al.; An intermediate form of alpha-amylase from Bacillus subtilis was prepared following a previously reported procedure . Stabilization of this protein structure by various additives and its interaction with alkyl-substituted Sepharose 4B (Sepharose-lipid), used to mimic the role of the alkyl chains of the phospholipid bilayer, were investigated . Exposure of hydrophobic clusters in the protein structure on denaturation was indicated by a greater affinity of the intermediate form for interaction with the alkyl chains on the matrix, as compared with the original native structure . Near- and far-ultraviolet circular dichroism studies supported loss of tertiary structure with retention of secondary structure, as expected from molten globular intermediate forms . Based on the results presented, we suggest that interaction of a protein in its native and nonnative forms with an alkyl-substituted matrix may provide useful information regarding its capacity for insertion into and translocation across the biologic membrane. Biochim Biophys Acta, 2004 Jun 1, 1699(1-2), 65 - 75 Two extracellular proteins with alkaline peroxidase activity, a novel cytochrome c and a catalase-peroxidase, from Bacillus sp . No.13; Ogawa J et al.; A novel cytochrome c and a catalase-peroxidase with alkaline peroxidase activity were purified from the culture supernatant of Bacillus sp . No.13 and characterized . The cytochrome c exhibited absorption maxima at 408 nm (Soret band) in its oxidized state, and 550 (alpha-band), 521 (beta-band), and 415 (Soret band) nm in its reduced state . The native cytochrome c with a relative molecular mass of 15,000 was composed of two identical subunits . The cytochrome c showed over 50 times higher peroxidase activity than those of known c-type cytochromes from various sources . The optimum pH and temperature of the peroxidase activity were about 10.0 and 70 degrees C, respectively . The peroxidase activity is stable in the pH range of 6.0 to 10.8 (30 degrees C, 1-h treatment), and at temperatures up to 80 degrees C (pH 8.5, 20-min treatment) . The heme content was determined to be 1 heme per subunit . The amino acid sequence of the cytochrome c showed high homology with those of the c-type cytochromes from Bacillus subtilis and Bacillus sp . PS3 . The catalase-peroxidase showed high catalase activity and considerable peroxidase activity, the specific activities being 55,000 and 0.94 micromol/min/mg, respectively . The optimum pH and temperature of the peroxidase activity were in the range of 6.4 to 10.1 and 60 degrees C, respectively . The catalase-peroxidase showed a lower K(m) value (0.67 mM) as to H(2)O(2) than known catalase-peroxidases. Nucleic Acids Res, 2004 May 20, 32(9), 2853 - 64 Print 2004. A protein-dependent riboswitch controlling ptsGHI operon expression in Bacillus subtilis: RNA structure rather than sequence provides interaction specificity; Schilling O et al.; The Gram-positive soil bacterium Bacillus subtilis transports glucose by the phosphotransferase system . The genes for this system are encoded in the ptsGHI operon . The expression of this operon is controlled at the level of transcript elongation by a protein-dependent riboswitch . In the absence of glucose a transcriptional terminator prevents elongation into the structural genes . In the presence of glucose, the GlcT protein is activated and binds and stabilizes an alternative RNA structure that overlaps the terminator and prevents termination . In this work, we have studied the structural and sequence requirements for the two mutually exclusive RNA structures, the terminator and the RNA antiterminator (the RAT sequence) . In both cases, the structure seems to be more important than the actual sequence . The number of paired and unpaired bases in the RAT sequence is essential for recognition by the antiterminator protein GlcT . In contrast, mutations of individual bases are well tolerated as long as the general structure of the RAT is not impaired . The introduction of one additional base in the RAT changed its structure and resulted in complete loss of interaction with GlcT . In contrast, this mutant RAT was efficiently recognized by a different B.subtilis antitermination protein, LicT. J Antibiot (Tokyo), 2004 Mar, 57(3), 205 - 9 New aminocoumarin antibiotics from a cloQ-defective mutant of the clorobiocin producer Streptomyces roseochromogenes DS12.976; Freitag A et al.; Three new antibiotics, vanillobiocin, isovanillobiocin and declovanillobiocin, were isolated from the culture broth of a cloQ-defective mutant of the clorobiocin producer Streptomyces roseochromogenes, which is blocked in the biosynthesis of the prenylated 4-hydroxybenzoic acid moiety of clorobiocin . Spectroscopic analysis showed that the isolated compounds were similar to clorobiocin, but contained vanillic acid as the acyl component instead of the prenylated 4-hydroxybenzoic acid present in clorobiocin . Isovanillobiocin differs from vanillobiocin by the position of the pyrrole unit attached to the sugar moiety of the antibiotic . Declovanillobiocin lacks the chlorine atom at the aminocoumarin ring . All three compounds had lower antibiotic activity against Bacillus subtilis than clorobiocin. J Bacteriol, 2004 Jun, 186(11), 3660 - 2 Identification of the two missing bacterial genes involved in thiamine salvage: thiamine pyrophosphokinase and thiamine kinase; Melnick J et al.; The genes encoding thiamine kinase in Escherichia coli (ycfN) and thiamine pyrophosphokinase in Bacillus subtilis (yloS) have been identified . This study completes the identification of the thiamine salvage enzymes in bacteria. J Bacteriol, 2004 Jun, 186(11), 3399 - 407 Modulation of activity of Bacillus subtilis regulatory proteins GltC and TnrA by glutamate dehydrogenase; Belitsky BR et al.; The Bacillus subtilis gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression and is repressed by the global regulatory protein TnrA . The factor that controls TnrA activity, a complex of glutamine synthetase and a feedback inhibitor, such as glutamine, is known, but the signal for modulation of GltC activity has remained elusive . GltC-dependent gltAB expression was drastically reduced when cells were grown in media containing arginine or ornithine or proline, all of which are inducers and substrates of the Roc catabolic pathway . Analysis of gltAB expression in mutants with various defects in the Roc pathway indicated that rocG-encoded glutamate dehydrogenase was required for such repression, suggesting that the substrates or products of this enzyme are the real effectors of GltC . Given that RocG is an enzyme of glutamate catabolism, the main regulatory role of GltC may be prevention of a futile cycle of glutamate synthesis and degradation in the presence of arginine-related amino acids or proline . In addition, high activity of glutamate dehydrogenase was incompatible with activity of TnrA. J Bacteriol, 2004 Jun, 186(11), 3392 - 8 CcpA-dependent regulation of Bacillus subtilis glutamate dehydrogenase gene expression; Belitsky BR et al.; The Bacillus subtilis rocG gene, encoding catabolic glutamate dehydrogenase, was found to be subject to direct CcpA-dependent glucose repression . The effect of CcpA required the presence of both the HPr and Crh proteins . The primary CcpA binding site was identified by mutational analysis and DNase I footprinting . In the absence of inducers of the Roc pathway, rocG was still expressed at a low level due to readthrough transcription . CcpA-dependent repression of rocG readthrough transcription proved to contribute to the slow growth rate of B . subtilis cells in glucose-glutamate medium . Increased readthrough expression of rocG was shown to be partially responsible for the growth defect of ccpA strains in glucose-ammonium medium. J Bacteriol, 2004 Jun, 186(11), 3331 - 45 Differential gene expression in response to hydrogen peroxide and the putative PerR regulon of Synechocystis sp . strain PCC 6803; Li H et al.; We utilized a full genome cDNA microarray to identify the genes that comprise the peroxide stimulon in the cyanobacterium Synechocystis sp . strain PCC 6803 . We determined that a gene (slr1738) encoding a protein similar to PerR in Bacillus subtilis was induced by peroxide . We constructed a PerR knockout strain and used it to help identify components of the PerR regulon, and we found that the regulatory properties were consistent with the hypothesis that PerR functions as a repressor . This effort was guided by finding putative PerR boxes in positions upstream of specific genes and by careful statistical analysis . PerR and sll1621 (ahpC), which codes for a peroxiredoxin, share a divergent promoter that is regulated by PerR . We found that isiA, encoding a Chl protein that is induced under low-iron conditions, was strongly induced by a short-term peroxide stress . Other genes that were strongly induced by peroxide included sigD, sigB, and genes encoding peroxiredoxins and Dsb-like proteins that have not been studied yet in this strain . A gene (slr1894) that encoded a protein similar to MrgA in B . subtilis was upregulated by peroxide, and a strain containing an mrgA knockout mutation was highly sensitive to peroxide . A number of genes were downregulated, including key genes in the chlorophyll biosynthesis pathway and numerous regulatory genes, including those encoding histidine kinases . We used PerR mutants and a thioredoxin mutant (TrxA1) to study differential expression in response to peroxide and determined that neither PerR nor TrxA1 is essential for the peroxide protective response. J Biol Chem, 2004 Jul 23, 279(30), 31804 - 12 Epub 2004 May 17. Crystal structure of unsaturated glucuronyl hydrolase, responsible for the degradation of glycosaminoglycan, from Bacillus sp . GL1 at 1.8 A resolution; Itoh T et al.; Unsaturated glucuronyl hydrolase (UGL) is a novel glycosaminoglycan hydrolase that releases unsaturated d-glucuronic acid from oligosaccharides produced by polysaccharide lyases . The x-ray crystallographic structure of UGL from Bacillus sp . GL1 was first determined by multiple isomorphous replacement (mir) and refined at 1.8 A resolution with a final R-factor of 16.8% for 25 to 1.8 A resolution data . The refined UGL structure consists of 377 amino acid residues and 478 water molecules, four glycine molecules, two dithiothreitol (DTT) molecules, and one 2-methyl-2,4-pentanediol (MPD) molecule . UGL includes an alpha(6)/alpha(6)-barrel, whose structure is found in the six-hairpin enzyme superfamily of an alpha/alpha-toroidal fold . One side of the UGL alpha(6)/alpha(6)-barrel structure consists of long loops containing three short beta-sheets and contributes to the formation of a deep pocket . One glycine molecule and two DTT molecules surrounded by highly conserved amino acid residues in UGLs were found in the pocket, suggesting that catalytic and substrate-binding sites are located in this pocket . The overall UGL structure, with the exception of some loops, very much resembled that of the Bacillus subtilis hypothetical protein Yter, whose function is unknown and which exhibits little amino acid sequence identity with UGL . In the active pocket, residues possibly involved in substrate recognition and catalysis by UGL are conserved in UGLs and Yter . The most likely candidate catalytic residues for glycosyl hydrolysis are Asp(88) and Asp(149) . This was supported by site-directed mutagenesis studies in Asp(88) and Asp(149). J Mol Biol, 2004 Jun 4, 339(3), 555 - 69 The folding transition state of the cold shock protein is strongly polarized; Garcia-Mira MM et al.; The cold shock protein CspB from Bacillus subtilis consists of a three-stranded (beta1-beta3) and a two stranded (beta4-beta5) sheet, which form a closed beta barrel structure . CspB folds and unfolds rapidly in a two-state reaction, and the unfolded and the folded molecules interconvert with a time constant of 30 ms at the midpoint of the urea-induced transition (at 25 degrees C) . The transition state of folding is native-like, as judged by the Tanford betaT value of > or =0.9 . By using a mutational approach and Phi value analysis, we find that the folding transition state of CspB is energetically polarized . Despite the high betaT value, most Phi values are low . Values close to 1 were found for only a few residues, particularly in strand beta1 (Lys5, Val6, Lys7, Asn10) . The interactions of the Asn10 side-chain with the backbone at positions 12 and 13 define the turn that connects the strands beta1 and beta2 . Lys5 and Val6 in beta1 interact with residues in beta4, and their high Phi values indicate that an energetic linkage between beta1 and beta4 and thus between the two sheets exists already in the transition state . We compared our experimental Phi values with theoretical predictions of the folding pathway of cold shock proteins . Several of them suggest that the entire first sheet is formed in the transition state, and some identify the beta1-beta4 pairing as a crucial step in folding . Alternative paths that involve formation of the second sheet and beta3-beta5 pairing reactions were, however, suggested a