Bacillus subtilis

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 as well . The calculations gave coarse-grained pictures that are limited in resolution to the two sheets of CspB or to the elements of secondary structure . They did not identify the key residues with the high Phi values within these structural elements.

J Clin Invest, 2004 May, 113(10), 1473 - 81
Antagonistic antibody prevents toll-like receptor 2-driven lethal shock-like syndromes; Meng G et al.; Hyperactivation of immune cells by bacterial products through toll-like receptors (TLRs) is thought of as a causative mechanism of septic shock pathology . Infections with Gram-negative or Gram-positive bacteria provide TLR2-specific agonists and are the major cause of severe sepsis . In order to intervene in TLR2-driven toxemia, we raised mAb's against the extracellular domain of TLR2 . Surface plasmon resonance analysis showed direct and specific interaction of TLR2 and immunostimulatory lipopeptide, which was blocked by T2.5 in a dose-dependent manner . Application of mAb T2.5 inhibited cell activation in experimental murine models of infection . T2.5 also antagonized TLR2-specific activation of primary human macrophages . TLR2 surface expression by murine macrophages was surprisingly weak, while both intra- and extracellular expression increased upon systemic microbial challenge . Systemic application of T2.5 upon lipopeptide challenge inhibited release of inflammatory mediators such as TNF-alpha and prevented lethal shock-like syndrome in mice . Twenty milligrams per kilogram of T2.5 was sufficient to protect mice, and administration of 40 mg/kg of T2.5 was protective even 3 hours after the start of otherwise lethal challenge with Bacillus subtilis . These results indicate that epitope-specific binding of exogenous ligands precedes specific TLR signaling and suggest therapeutic application of a neutralizing anti-TLR2 antibody in acute infection.

Bioinformatics, 2004 Nov 1, 20(16), 2719 - 25 Epub 2004 May 14.
Transcription/replication collisions cause bacterial transcription units to be longer on the leading strand of replication; Omont N et al.; MOTIVATION: The costs of head-on versus codirectional collisions between the replication complex and the much slower transcription complex on the circular bacterial chromosomes are much debated . Although it is established that the number of genes on the leading strand is higher than on the lagging strand of replication, the consequences of collisions on the length of transcription units are unknown . RESULTS: Here, we show that transcription units are generally longer on the leading strand, in rough proportion to the bias in number of units between the two strands . We propose a statistical physics model, based on the assumption that the proportion of interrupted transcripts for each unit is the major factor contributing to these biases . Its main prediction is that a large replication/transcription speed ratio implies a low leading/lagging bias for transcription unit length and number . This model is validated by an analysis of proven and predicted units in Escherichia coli and Bacillus subtilis . The results are consistent with an equal cost of head-on versus codirectional collisions.

Biosens Bioelectron, 2004 Jul 15, 19(12), 1627 - 33
Detection of serum uric acid using the optical polymeric enzyme biochip system; Huang SH et al.; An optical polymeric biochip system based on the complementary metal oxide semiconductor (CMOS) photo array sensor and polymeric enzyme biochip for rapidly quantitating uric acid in a one-step procedure was developed . The CMOS sensor was designed with N(+)/P-well structure and manufactured using a standard 0.5 microm CMOS process . The polymeric enzyme biochip was immobilized with uricase-peroxidase and used to fill the reacting medium with the sample . This study encompasses the cloning of the Bacillus subtilis uricase gene and expression in Escherichia coli, as well as the purification of uricase and measurement of its activity . The cloned uricase gene included an open reading frame of 1491 nucleotides that encodes a protein of approximately 55 kDa . The expression of the putative MBP-fusion protein involved approximately 98 kDa of the protein . The CMOS sensor response was stronger at a higher temperature range of 20-40 degrees C, with optimal pH at 8.5 . The calibration curve of purified uric acid was linear in the concentration range from 2.5 to 12.5 mg/dL . The results obtained for serum uric acid correlated quite closely with those obtained using the Beckman Synchron method.

Biotechnol Bioeng, 2004 Jun 20, 86(6), 706 - 17
Fed-batch optimization of alpha-amylase and protease-producing Bacillus subtilis using Markov chain methods; Skolpap W et al.; A stoichiometry-based model for the fed-batch culture of the recombinant bacterium Bacillus subtilis ATCC 6051a, producing extracellular alpha-amylase as a desirable product and proteases as undesirable products, was developed and verified . The model was then used for optimizing the feeding schedule in fed-batch culture . To handle higher-order model equations (14 state variables), an optimization methodology for the dual-enzyme system is proposed by integrating Pontryagin's optimum principle with fermentation measurements . Markov chain Monte Carlo (MCMC) procedures were appropriate for model parameter and decision variable estimation by using a priori parameter distributions reflecting the experimental results . Using a simplified Metropolis-Hastings algorithm, the specific productivity of alpha-amylase was maximized and the optimum path was confirmed by experimentation . The optimization process predicted a further 14% improvement of alpha-amylase productivity that could not be realized because of the onset of sporulation . Among the decision variables, the switching time from batch to fed-batch operation (t(s)) was the most sensitive decision variable .

Biotechnol Bioeng, 2004 Jun 20, 86(6), 622 - 7
A new approach to random mutagenesis in vitro; Lai YP et al.; Random mutagenesis is a powerful tool for studying the effects of a large number of permutations of a particular DNA sequence and its encoded products . Here we describe a new strategy of conducting in vitro random mutagenesis using ethyl methane sulfonate (EMS) . The Bacillus aprN18 gene, coding for a serine protease with fibrinolytic activity, was used as a target gene . To study the mutations of the coding region, rather than the whole plasmid, the 1.4 kb gene fragment was cut out from an expression plasmid and treated with 10 mM EMS at 37 degrees C for 1 h . The treated fragment was then ligated back into the original expression vector and a library of random mutants was constructed in a protease-deficient Bacillus subtilis strain . A plate assay-based screening method was used to select for mutant clones with altered enzyme activity, and the change of activity was then confirmed by a semi-quantitative enzyme assay using liquid culture supernatant . The inserts of five clones with altered enzyme activity were randomly chosen for sequencing analysis . Among the point mutations detected, GC --> AT transition accounts for 42.1%, AT --> GC transition 34.2% and GC/CG transversion 23.7%, respectively . To our knowledge this is the first application of EMS for in vitro mutagenesis of a defined DNA sequence .

J Mol Recognit, 2004 May-Jun, 17(3), 236 - 47
Is there a rational method to purify proteins? From expert systems to proteomics; Asenjo JA et al.; The purification of recombinant proteins for therapeutic or analytical applications requires the use of several chromatographic steps in order to achieve a high level of purity . A range of techniques is available such as anion and cation exchange chromatography, which can be carried out at different pHs, and hence used at different steps, hydrophobic interaction chromatography, gel filtration and affinity chromatography . Evidently when confronted with a complex mixture of partially unknown proteins or a clarified cell extract there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity (e.g . 98, 99.5 or 99.9%) . In this review we will show how an initial "proteomic" characterization of the complex initial mixture of target protein and protein contaminants can be used to select the most efficient chromatographic separation steps in order to achieve a maximum level of purity with a minimum number of steps . The chosen methodology was implemented in a computer based expert system . The first algorithm developed was used to select the most efficient purification method to separate a protein from its contaminants based on the physicochemical properties of the protein product and the protein contaminants . The second algorithm developed was used to predict the number and concentration of contaminants after each separation as well as protein product purity . The successful application of the expert system approach, based on an initial proteomic characterization, to the practical cases of protein mixtures and clarified fermentation supernatant is presented and discussed . The purification strategy proposed was experimentally tested and validated with a mixture of four proteins and the experimental validation was also carried out with an "unknown" supernatant of Bacillus subtilis producing a recombinant beta-1,3-glucanase . The system was robust to errors <10% which is the range that can be found in the experimental determination of the properties in the database of product and contaminants . On the other hand, the system was sensitive both to larger variations (>20%) in the properties of the contaminant database and the protein product and to variations in one protein property (e.g . hydrophobicity) .

J Mol Biol, 2004 May 28, 339(2), 265 - 78
Analysis of the open and closed conformations of the GTP-binding protein YsxC from Bacillus subtilis; Ruzheinikov SN et al.; Genetic analysis has suggested that the product of the Bacillus subtilis ysxC gene is essential for survival of the microorganism and hence may represent a target for the development of a novel anti-infective agent . B.subtilis YsxC is a member of the translation factor related class of GTPases and its crystal structure has been determined in an apo form and in complex with GDP and GMPPNP/Mg2+ . Analysis of these structures has allowed us to examine the conformational changes that occur during the process of nucleotide binding and GTP hydrolysis . These structural changes particularly affect parts of the switch I and switch II region of YsxC, which become ordered and disordered, respectively in the "closed" or "on" GTP-bound state and disordered and ordered, respectively, in the "open" or "off" GDP-bound conformation . Finally, the binding of the magnesium cation results in subtle shifts of residues in the G3 region, at the start of switch II, which serve to optimize the interaction with a key aspartic acid residue . The structural flexibility observed in YsxC is likely to contribute to the role of the protein, possibly allowing transduction of an essential intracellular signal, which may be mediated via interactions with a conserved patch of surface-exposed, basic residues that lies adjacent to the GTP-binding site.

Biomacromolecules, 2004 May-Jun, 5(3), 877 - 82
Permanent, nonleaching antibacterial surfaces . 1 . Synthesis by atom transfer radical polymerization; Lee SB et al.; We have grown an antimicrobial polymer directly on the surfaces of glass and paper using atom transfer radical polymerization (ATRP) . The method described here results in potentially permanent nonleaching antibacterial surfaces without the need to chemically graft the antimicrobial material to the substratum . The tertiary amine 2-(dimethylamino)ethyl methacrylate was polymerized directly onto Whatman #1 filter paper or glass slides via atom transfer radical polymerization . Following the polymerization, the tertiary amino groups were quaternized using an alkyl halide to produce a large concentration of quaternary ammonium groups on the polymer-modified surfaces . Incubating the modified materials with either Escherichia coli or Bacillus subtilis demonstrated that the modified surfaces had substantial antimicrobial capacity . The permanence of the antimicrobial activity was demonstrated through repeated use of a modified glass without significant loss of activity . Quaternary amines are believed to cause cell death by disrupting cell membranes allowing release of the intracellular contents . Atomic force microscopic imaging of cells on modified glass surfaces supports this hypothesis.

J Biol Chem, 2004 Jul 16, 279(29), 29974 - 80 Epub 2004 May 06.
Purification and characterization of the bacterial MraY translocase catalyzing the first membrane step of peptidoglycan biosynthesis; Bouhss A et al.; The MraY translocase catalyzes the first membrane step of bacterial cell wall peptidoglycan synthesis (i.e . the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid), a reversible reaction yielding undecaprenylpyrophosphoryl-N-acetylmuramoyl-pentapeptide (lipid intermediate I) . This essential integral membrane protein, which is considered as a very promising target for the search of new antibacterial compounds, has thus far been clearly underexploited due to its intrinsic refractory nature to overexpression and purification . We here report conditions for the high level overproduction and for the first time the purification to homogeneity of milligram quantities of MraY protein . The kinetic parameters and effects of pH, salts, cations, and detergents on enzyme activity are described, taking the Bacillus subtilis MraY translocase as a model.

Mol Microbiol, 2004 May, 52(4), 1201 - 14
Regulation of type III secretion in Bordetella; Mattoo S et al.; The BvgAS virulence control system regulates the expression of type III secretion genes in Bordetella subspecies that infect humans and other mammals . We have identified five open reading frames, btrS, btrU, btrX, btrW and btrV, that are activated by BvgAS and encode regulatory factors that control type III secretion at the levels of transcription, protein expression and secretion . The btrS gene product bears sequence similarity to ECF (extracytoplasmic function) sigma factors and is required for transcription of the bsc locus . btrU, btrW and btrV encode proteins predicted to contain PP2C-like Ser phosphatase, HPK (His protein kinase)-like Ser kinase and STAS anti-sigma factor antagonist domains, respectively, which are characteristic of Gram-positive partner switching proteins in Bacillus subtilis . BtrU and BtrW are required for secretion of proteins that are exported by the bsc type III secretion system, whereas BtrV is specifically required for protein synthesis and/or stability . Bordetella species have thus evolved a unique cascade to differentially regulate type III secretion that combines a canonical phosphorelay system with an ECF sigma factor and a set of proteins with domain signatures that define partner switchers, which were traditionally thought to function only in Gram-positive bacteria . The presence of multiple layers and mechanisms of regulation most likely reflects the need to integrate multiple signals in controlling type III secretion . The bsc and btr loci are nearly identical between broad-host-range and human-specific Bordetella . Comparative analysis of Bordetella subspecies revealed that, whereas bsc and btr loci were transcribed in all subspecies, only broad-host-range strains expressed a functional type III secretion system in vitro . The block in type III secretion is post-transcriptional in human-adapted strains, and signal recognition appears to be a point of divergence between subspecies.

Mol Microbiol, 2004 May, 52(4), 1091 - 105
The Bacillus subtilis sigmaW anti-sigma factor RsiW is degraded by intramembrane proteolysis through YluC; Schobel S et al.; The Bacillus subtilis sigma(W) regulon is induced by different stresses such as alkaline shock, salt shock, phage infection and certain antibiotics that affect cell wall biosynthesis . The activity of the alternative, extracytoplasmic function (ECF) sigma factor sigma(W) is modulated by a specific anti-sigma factor (RsiW or YbbM) encoded by the rsiW (ybbM) gene located immediately downstream of sigW . The RsiW membrane topology was determined, and a specific reporter system for RsiW function was constructed . Experiments using the yeast two-hybrid system suggested a direct interaction of sigma(W) with the cytoplasmic part of RsiW . Analysis of truncated forms of the RsiW protein revealed that sigma(W) induction by alkaline shock is dependent on both the transmembrane and the extracytoplasmic domain of RsiW . Western blot and pulse-chase experiments demonstrated degradation of RsiW after an alkaline shock . A B . subtilis mutant strain deleted for the Escherichia coli yaeL orthologue yluC, encoding a transmembrane protease, was defective in inducing a sigma(W)-controlled promoter after alkaline shock and accumulated a membrane-bound truncated form of RsiW, suggesting that the activity of sigma(W) is controlled by the proteolysis of RsiW by at least two different proteolytic steps.

Appl Environ Microbiol, 2004 May, 70(5), 2786 - 90
Detection of molecular diversity in Bacillus atrophaeus by amplified fragment length polymorphism analysis; Burke SA et al.; Phenotypically, Bacillus atrophaeus is indistinguishable from the type strain of Bacillus subtilis except by virtue of pigment production on certain media . Several pigmented variants of B . subtilis have been reclassified as B . atrophaeus, but several remain ambiguous in regard to their taxonomic placement . In this study, we examined strains within the American Type Culture Collection originally deposited as Bacillus globigii, B . subtilis var . niger, or Bacillus niger using 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis to determine the level of molecular diversity among these strains and their relationship with closely related taxa . The 16S rRNA gene sequences revealed little variation with one base substitution between the B . atrophaeus type strain ATCC 49337 and the other pigmented bacilli . AFLP analysis produced high-quality DNA fingerprints with sufficient polymorphism to reveal strain-level variation . Cluster analysis of Dice similarity coefficients revealed that three strains, ATCC 31028, ATCC 49760, and ATCC 49822, are much more closely related to B . atrophaeus than to B . subtilis and should be reclassified as B . atrophaeus . A very closely related cluster of B . atrophaeus strains was also observed; this cluster was genetically distinct from the type strain . The level of variation between the two groups was approximately the same as the level of variation observed between members of the two B . subtilis subspecies, subtilis and spizizenii . It is proposed that the cluster of strains typified by ATCC 9372 be designated a new subspecies, B . atrophaeus subsp . globigii.

Appl Environ Microbiol, 2004 May, 70(5), 2734 - 40
Lack of protective osmolytes limits final cell density and volumetric productivity of ethanologenic Escherichia coli KO11 during xylose fermentation; Underwood SA et al.; Limited cell growth and the resulting low volumetric productivity of ethanologenic Escherichia coli KO11 in mineral salts medium containing xylose have been attributed to inadequate partitioning of carbon skeletons into the synthesis of glutamate and other products derived from the citrate arm of the anaerobic tricarboxylic acid pathway . The results of nuclear magnetic resonance investigations of intracellular osmolytes under different growth conditions coupled with those of studies using genetically modified strains have confirmed and extended this hypothesis . During anaerobic growth in mineral salts medium containing 9% xylose (600 mM) and 1% corn steep liquor, proline was the only abundant osmolyte (71.9 nmol x ml(-1) optical density at 550 nm {OD(550)} unit(-1)), and growth was limited . Under aerobic conditions in the same medium, twice the cell mass was produced, and cells contained a mixture of osmolytes: glutamate (17.0 nmol x ml(-1) OD(550) unit(-1)), trehalose (9.9 nmol x ml(-1) OD(550) unit(-1)), and betaine (19.8 nmol x ml(-1) OD(550) unit(-1)) . Two independent genetic modifications of E . coli KO11 (functional expression of Bacillus subtilis citZ encoding NADH-insensitive citrate synthase; deletion of ackA encoding acetate kinase) and the addition of a metabolite, such as glutamate (11 mM) or acetate (24 mM), as a supplement each increased the intracellular glutamate pool during fermentation, doubled cell growth, and increased volumetric productivity . This apparent requirement for a larger glutamate pool for increased growth and volumetric productivity was completely eliminated by the addition of a protective osmolyte (2 mM betaine or 0.25 mM dimethylsulfoniopropionate), consistent with adaptation to osmotic stress rather than relief of a specific biosynthetic requirement.

Biotechnol Lett, 2004 Mar, 26(6), 525 - 8
Michael addition of imidazole with acrylates catalyzed by alkaline protease from Bacillus subtilis in organic media; Cai Y et al.; A new activity of alkaline protease from Bacillus subtilis for Michael addition reactions of imidazole, 4-nitro-1H-imidazole and 2-methyl-4-nitro-1H-imidazole with acrylates and acrylic acid was investigated . The reactions were carried out in pyridine at 50 degrees C for 72 h . Five N-substituted imidazole derivatives were obtained using acrylate esters, but not acrylic acid, in yields from 62% to 76%.

Proc Natl Acad Sci U S A, 2004 May 18, 101(20), 7733 - 8 Epub 2004 May 04.
Lipids in the inner membrane of dormant spores of Bacillus species are largely immobile; Cowan AE et al.; Bacterial spores of various Bacillus species are impermeable or exhibit low permeability to many compounds that readily penetrate germinated spores, including methylamine . We now show that a lipid probe in the inner membrane of dormant spores of Bacillus megaterium and Bacillus subtilis is largely immobile, as measured by fluorescence redistribution after photobleaching, but becomes free to diffuse laterally upon spore germination . The lipid immobility in and the slow permeation of methylamine through the inner membrane of dormant spores may be due to a significant (1.3- to 1.6-fold) apparent reduction of the membrane surface area in the dormant spore relative to that in the germinated spore, but is not due to the dormant spore's high levels of dipicolinic acid and divalent cations.

J Bacteriol, 2004 May, 186(10), 3195 - 201
Evaluation of the kinetic properties of the sporulation protein SpoIIE of Bacillus subtilis by inclusion in a model membrane; Searls T et al.; Starvation induces Bacillus subtilis to initiate a developmental process (sporulation) that includes asymmetric cell division to form the prespore and the mother cell . The integral membrane protein SpoIIE is essential for the prespore-specific activation of the transcription factor sigmaF, and it also has a morphogenic activity required for asymmetric division . An increase in the local concentration of SpoIIE at the polar septum of B . subtilis precedes dephosphorylation of the anti-anti-sigma factor SpoIIAA in the prespore . After closure and invagination of the asymmetric septum, phosphatase activity of SpoIIE increases severalfold, but the reason for this dramatic change in activity has not been determined . The central domain of SpoIIE has been seen to self-associate (I . Lucet et al., EMBO J . 19:1467-1475, 2000), suggesting that activation of the C-terminal PP2C-like phosphatase domain might be due to conformational changes brought about by the increased local concentration of SpoIIE in the sporulating septum . Here we report the inclusion of purified SpoIIE protein into a model membrane as a method for studying the effect of local concentration in a lipid bilayer on activity . In vitro assays indicate that the membrane-bound enzyme maintains dephosphorylation rates similar to the highly active micellar state at all molar ratios of protein to lipid . Atomic force microscopy images indicate that increased local concentration does not lead to self-association.

J Bacteriol, 2004 May, 186(10), 2992 - 5
Drastic differences in Crh and HPr synthesis levels reflect their different impacts on catabolite repression in Bacillus subtilis; Gorke B et al.; In Bacillus subtilis, carbon catabolite repression (CCR) of catabolic genes is mediated by ATP-dependent phosphorylation of HPr and Crh . Here we show that the different efficiencies with which these two proteins contribute to CCR may be due to the drastic differences in their synthesis rates under conditions that cause CCR.

J Bacteriol, 2004 May, 186(10), 2984 - 91
Effects of a gerF (lgt) mutation on the germination of spores of Bacillus subtilis; Igarashi T et al.; One of the proteins of the membrane-bound receptors that recognize individual nutrients that trigger germination of spores of Bacillus subtilis contains the recognition sequence for diacylglycerol addition to a cysteine residue near the protein's N terminus . B . subtilis spores lacking the gerF (lgt) gene that codes for prelipoprotein diacylglycerol transferase exhibited significantly slowed germination in response to nutrient germinants as found previously, but germination of gerF spores with a mixture of Ca2+ and dipicolinic acid or with dodecylamine was normal, as was the spontaneous germination of gerF spores lacking all nutrient germinant receptors . The deleterious effects of the gerF mutation on nutrient germination were highest on germination triggered by the GerA nutrient receptor and were less so (but still significant) on germination triggered by the GerB nutrient receptor . However, there was little, if any, effect on GerK nutrient receptor-mediated spore germination . As predicted from the latter results, replacement by alanine of the cysteine residue to which diacylglycerol is thought to be added to these nutrient receptors had a large effect on GerA receptor function, less effect on GerB receptor function, and little, if any, effect on GerK receptor function.

Biochemistry, 2004 May 11, 43(18), 5474 - 87
Expression, purification, and characterization of Bacillus subtilis cytochromes P450 CYP102A2 and CYP102A3: flavocytochrome homologues of P450 BM3 from Bacillus megaterium; Gustafsson MC et al.; The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically . Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding . CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates . Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes . Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate . Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates . P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles . Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding . Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B . subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.

Vaccine, 2004 May 7, 22(15-16), 1873 - 85
Intracellular fate and immunogenicity of B . subtilis spores; Duc le H et al.; To support our work on the development of bacterial spores as oral vaccines we examined the immunogenicity and intracellular fate of Bacillus subtilis endospores in a murine model . Mice dosed orally with spores developed systemic IgG and mucosal sIgA responses . Analysis of IgG subclasses revealed a predominance of the IgG2a subclass during the early stages of immunisation . Analysis of cytokine mRNA in GALT and lymphoid organs showed early induction of IFN-gamma, a Th1 cytokine, as well as the pro-inflammatory cytokine TNF-alpha . Significant levels of IgG antibodies were produced against vegetative bacilli following dosing with spores . This showed that spores could germinate in the GI tract . In vitro studies detailing the intracellular fate and persistence of spores in a macrophage-like cell line (RAW264.7) demonstrated that spores could germinate efficiently in macrophages, initiate gene expression as well as inducing pro-inflammatory cytokines.

Curr Issues Mol Biol, 2004 Jul, 6(2), 125 - 36
Recent developments in bacterial cold-shock response; Phadtare S; In response to temperature downshift, a number of changes occur in cellular physiology such as, (i) decrease in membrane fluidity, (ii) stabilization of secondary structures of nucleic acids leading to reduced efficiency of mRNA translation and transcription, (iii) inefficient folding of some proteins, and (iv) hampered ribosome function . Cold-shock response and adaptation has been quite extensively studied in Escherichia coli and Bacillus subtilis . A number of cold shock proteins are induced to counteract these harmful effects of temperature downshift . General principles of cold-shock response along with recent findings on desaturase system, RNA chaperone and transcription antitermination function of CspA homologues, cold shock induction of chaperones and synthesis of trehalose, CspA homologues from hyperthermophilic bacteria and possible multiple roles of cold shock proteins in other stress responses of bacteria are discussed.

Nucleic Acids Res, 2004 Apr 26, 32(8), 2306 - 14 Print 2004.
Genome wide, supercoiling-dependent in vivo binding of a viral protein involved in DNA replication and transcriptional control; Gonzalez-Huici V et al.; Protein p6 of Bacillus subtilis bacteriophage Phi29 is essential for phage development . In vitro it activates the initiation of DNA replication and is involved in the early to late transcriptional switch . These activities require the formation of a nucleoprotein complex in which the DNA forms a right-handed superhelix wrapping around a multimeric protein core . However, there was no evidence of p6 binding to Phi29 DNA in vivo . By crosslinking, chromatin immunoprecipitation and real-time PCR we show that protein p6 binds to most, if not all, the viral genome in vivo, although with higher affinity for both DNA ends, which contain the replication origins . In contrast, the affinity for plasmid DNA is negligible, but greatly increases when the negative supercoiling decreases, as shown in vivo by treatment of cells with novobiocin and in vitro by fluorescence quenching with plasmids with different topology . In conclusion, binding of protein p6 all along the Phi29 genome strongly suggests that its functions in replication and transcription control could be local outcomes of a more global role as a histone-like protein . The p6 binding dependence on DNA topology could explain its preferential binding to viral with respect to bacterial DNA, whose level of negative supercoiling is presumably higher than that of Phi29 DNA.

Environ Sci Technol, 2004 Apr 15, 38(8), 2491 - 5
Experimental study of the adsorption of an ionic liquid onto bacterial and mineral surfaces; Gorman-Lewis DJ et al.; Ionic liquids are being developed as a replacement for volatile organic solvents in a range of industrial applications . These liquids have a vanishingly small vapor pressure, making them an attractive alternative to the volatile organic solvents . However, a thorough assessment of the environmental impact of the use of ionic liquids requires a more complete understanding of their fate and transport in environmental systems . Toward this end, we measured the adsorption of the ionic liquid 1-butyl, 3-methylimidazolium chloride (Bmim CI) onto a range of surfaces meant to represent those commonly found in the near-surface environment . We measured adsorption onto the Gram-positive soil bacterial species Bacillus subtilis, onto gibbsite, onto quartz, and onto Na-montmorillonite . We conducted experiments as a function of pH, solid:solute ratio, time, and ionic strength . The experimental results reveal that Bmim CI is unstable in water below pH 6 and above pH 10 and that it exhibits pH independent and ionic strength dependent adsorption onto Na-montmorillonite with 0.4, 0.8, 1.0, 1.2, and 2.0 g/L of clay . We observed no adsorption of the Bmim CI onto B . subtilis (3.95 or 7.91 g (dry weight) bacteria/L) at pH 5.5-8.5 or onto gibbsite (500 or 1285 g/L) or quartz (1000 and 2000 g/L) over the pH range 6-10 . Calculated distribution coefficient (KD) values for Bmim CI onto the Na-montmorillonite change as a function of ionic strength; the 10(-4) M ionic strength KD value is 1735 +/- 269 L/Kg, and the 10(-1) M ionic strength KD is 1133 +/- 291 L/Kg . Our results suggest that the geologic retardation of this class of ionic liquid, if present as a dissolved contaminant in the subsurface, would be significant when a significant fraction of interlayer clays are present . However, adsorption onto other common geologic and biological surfaces is likely to be minimal, and the ionic liquids may travel unimpeded in groundwater systems in which these types of surfaces dominate.

Carbohydr Res, 2004 May 17, 339(7), 1279 - 83
Regioselective monoacylation of cyclomaltoheptaose at the C-2 secondary hydroxyl groups by the alkaline protease from Bacillus subtilis in nonaqueous media; Xiao YM et al.; Transesterification of cyclomaltoheptaose (beta-CD) with divinyl butanedioate, divinyl hexanedioate, and divinyl decanedioate, catalyzed by the alkaline protease from Bacillus subtilis in anhydrous DMF for 5 days, furnished the corresponding vinyl-beta-CD derivatives . The products were characterized by ESI-MS, (1)H NMR, (13)C NMR, IR, and DSC . The results indicated the products to be monosubstituted esters, with monoacylation occurring at the C-2 secondary hydroxyl groups of beta-CD . The regioselectivity of the monoacylation as catalyzed by alkaline protease was not affected by the chain length of the acyl donor.

Plasmid, 2004 May, 51(3), 238 - 45
New shuttle vectors for ectopic insertion of genes into Bacillus subtilis; Middleton R et al.; We have constructed shuttle vectors for integration of genes via double homologous recombination into three ectopic sites on the chromosome of Bacillus subtilis . The sites of integration are the pyrD, gltA, and sacA genes located at 139 degrees, 172 degrees, and 333 degrees, respectively, on the chromosome . Integration of the vectors into the target genes leads to antibiotic resistance as well as different metabolic phenotypes . B . subtilis strains with integrations of the empty vectors were able to sporulate at rates comparable to wild type cells . Similar levels of expression were obtained from constitutive lacZ fusions integrated at the different sites .

Plasmid, 2004 May, 51(3), 192 - 202
Complete sequence and structural organization of pFL5 and pFL7, two cryptic plasmids from Bacillus licheniformis; Parini C et al.; The complete nucleotide sequences of two plasmids, pFL5 and pFL7, isolated from soil bacteria, Bacillus licheniformis FL5 and FL7, have been determined . The plasmids pFL5 and pFL7 were analyzed and found to be 9150 and 7853 bp in size with a G+C content of 41.0 and 43.6 mol%, respectively . Computer assisted analysis of sequence data revealed 11 possible ORFs in pFL5, four of which could be assigned no function from homology searches . Instead, eight putative ORFs were identified in pFL7, two of which appeared to have no biological function . All the ORFs were preceded by a ribosome binding site . The ORFs 9.5 and 6.7, each of 340 amino acids, were postulated to encode a replication protein similar to known replication proteins of rolling circle replicons, particularly those of the pC194 family . The structural organization of the two pFL plasmids is similar to the pTA plasmids family, with only a few putative coding regions that cannot be attributed to these plasmid backbone genes . In contrast to pTA plasmids, the majority of the genes have an orientation of transcription opposite to the direction of replication . The identified probable sso sequences seem to belong to a different group of those found in Bacillus plasmids; in fact, a significant level of homology was found with ssoA group sequences . These plasmids seem to be related to plasmids identified within the Bacillus subtilis group, confirming the low-level diversity among these replicons .

FEMS Microbiol Lett, 2004 May 1, 234(1), 43 - 9
Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D; Moyne AL et al.; Bacillus subtilis AU195 produces bacillomycin D, a cyclic lipopeptide that is an inhibitor of the aflatoxin producing fungus Aspergillus flavus . Sequence analysis of the bacillomycin D operon revealed four ORFs with the structural organization of the peptide synthetases . Disruption of ORF 2, which links the amino acid moiety to the b-amino fatty acid, resulted in the loss of antifungal activity . By comparing the sequence of bacillomycin D, iturin A and mycosubtilin operons, our results showed that intergenic module replacement have occurred between B . subtilis lipopeptide synthetases including the iturin family and the plipastatin and fengycin family.

FEMS Microbiol Lett, 2004 May 1, 234(1), 37 - 42
Analysis of spontaneous base substitutions generated in mutator strains of Bacillus subtilis; Sasaki M et al.; In the current studies, we investigated base substitutions in the Bacillus subtilis mutT, mutM, and mutY DNA error-prevention system . In the wild type strain, spontaneous mutations were mainly transitions, either G:C --> A:T or A:T --> G:C . Although both transitions and transversions were observed in mutY and mutM mutants, mutM/mutY double mutants contain strictly G:C --> T:A transversions . In the mutT strain, A:T --> C:G transversion was not observed, and over-expression of the B . subtilis mutT gene had no effect on the mutation rate in the Escherichia coli mutT strain . Using 8-oxo-dGTP-induced mutagenesis, transitions especially A:T --> G:C were predominant in the wild type and mutY strains . In contrary, transversion was high on mutY and double mutant (mutM mutY) . Finally, the opuBC and yitG genes were identified from the B . subtilis chromosome as mutator genes that prevented the transition base substitutions.

J Biol Chem, 2004 Jul 9, 279(28), 29718 - 27 Epub 2004 Apr 22.
Structure-function correlation in glycine oxidase from Bacillus subtilis; Mortl M et al.; Structure-function relationships of the flavoprotein glycine oxidase (GO), which was recently proposed as the first enzyme in the biosynthesis of thiamine in Bacillus subtilis, has been investigated by a combination of structural and functional studies . The structure of the GO-glycolate complex was determined at 1.8 A, a resolution at which a sketch of the residues involved in FAD binding and in substrate interaction can be depicted . GO can be considered a member of the "amine oxidase" class of flavoproteins, such as d-amino acid oxidase and monomeric sarcosine oxidase . With the obtained model of GO the monomer-monomer interactions can be analyzed in detail, thus explaining the structural basis of the stable tetrameric oligomerization state of GO, which is unique for the GR(2) subfamily of flavooxidases . On the other hand, the three-dimensional structure of GO and the functional experiments do not provide the functional significance of such an oligomerization state; GO does not show an allosteric behavior . The results do not clarify the metabolic role of this enzyme in B . subtilis; the broad substrate specificity of GO cannot be correlated with the inferred function in thiamine biosynthesis, and the structure does not show how GO could interact with ThiS, the following enzyme in thiamine biosynthesis . However, they do let a general catabolic role of this enzyme on primary or secondary amines to be excluded because the expression of GO is not inducible by glycine, sarcosine, or d-alanine as carbon or nitrogen sources.

Antimicrob Agents Chemother, 2004 May, 48(5), 1541 - 7
Identification and characterization of inhibitors of bacterial enoyl-acyl carrier protein reductase; Ling LL et al.; Bacterial enoyl-acyl carrier protein reductase (ENR) catalyzes an essential step in fatty acid biosynthesis . ENR is an attractive target for narrow-spectrum antibacterial drug discovery because of its essential role in metabolism and its sequence conservation across many bacterial species . In addition, the bacterial ENR sequence and structural organization are distinctly different from those of mammalian fatty acid biosynthesis enzymes . High-throughput screening to identify inhibitors of Escherichia coli ENR yielded four structurally distinct classes of hits . Several members of one of these, the 2-(alkylthio)-4,6-diphenylpyridine-3-carbonitriles ("thiopyridines"), inhibited both purified ENR (50% inhibitory concentration {IC(50)} = 3 to 25 micro M) and the growth of Staphylococcus aureus and Bacillus subtilis (MIC = 1 to 64 micro g/ml) . The effect on cell growth is due in part to inhibition of fatty acid biosynthesis as judged by inhibition of incorporation of {(14)C}acetate into fatty acids and by the increased sensitivity of cells that underexpress an ENR-encoding gene (four- to eightfold MIC shift) . Synthesis of a variety of compounds in this chemical series revealed a correlation between IC(50) and MIC, and the results provided initial structure-activity relationships . Preliminary structure-activity relationships, potency on purified ENR, and activity on bacterial cells indicate that members of the thiopyridine chemical series are effective fatty acid biosynthesis inhibitors suitable for further antibacterial development.

Biotechnol Lett, 2004 Mar, 26(5), 403 - 7
Bacillus subtilis transcriptional regulators interaction; Sanchez A et al.; Bacillus subtilis aprE gene codes for the extracellular protease subtilisin . Its expression is controlled by AbrB, DegU, Hpr, SinI, SinR and Spo0A transition state protein regulators . To determine in vivo the protein-protein interactions among these regulators, we used the LexA-based bacterial genetic two-hybrid system . Our results show homo-dimerization to all the analyzed proteins and hetero-dimerization between SinR-SinI and SinR-Hpr.

Mikrobiol Z, 2004 Jan-Feb, 66(1), 68 - 77
{Effect of UV-radiation and drying on bacterium diversity in soil}; Rokitko PV et al.; It has been shown that after DNA-injuring factors (UV irradiation or drying) action on soil one could observe the decrease of the total quantity of bacteria and the number of species, i.e., the decrease of microbe diversity . At the same time not numerous species were found in soils after their action . Thus the drying or UV-irradiation makes it possible to estimate more completely the microbe diversity in soils as well as to find resistant bacteria . It has been established that the strain Methylobacterium extorquens, M . mesophilicum, Bacillus subtilis, B . cereus, which were isolated after UV irradiation or drying of oil samples, were characterized by high resistance to gamma-irradiation (LD99.99--5-10 kGr) . Bacteria (representatives of Pseudomonas genus) sensitive to drying or UV-irradiation were also sensitive to ionizing radiation (LD99.99--0.09 kGr) . Nocardieforms and representatives of Myxococcus occupied intermediate position between representatives of genera Methylobacterium and Pseudomonas as to resistance to the above-mentioned stress agents.

Mikrobiol Z, 2004 Jan-Feb, 66(1), 36 - 41
{Culture media for growth and spore formation of Bacillus subtilis and Bacillus licheniformis}; Khil'ko TV; The nutrient media for deep cultivation of Bacillus subtilis 3 and Bacillus licheniformis 31 have been optimized . It has been shown, that the media caused much higher biomass production from 2.0 x 10(10) CFU/ml media for B . subtilis 3 to 5.1 x 10(10) CFU/ml for B . licheniformis 31 and 50% production of spores by both cultures under such cultivation conditions.

Biochim Biophys Acta, 2004 Apr 12, 1655(1-3), 256 - 62
Intermediate forms of cytochrome oxidase observed in transient kinetic experiments and those visited in the catalytic cycle; Hill BC; The cytochrome oxidase family of heme-copper oxidases has been the subject of intense kinetic and mechanistic enquiry . Much of this work has focussed on transient kinetic studies of the partial reactions of the enzyme with the goal being to build a kinetic model describing the catalytic cycle that the enzyme undergoes to direct the oxidation of substrate, reduction of oxygen and vectorial proton transfer . A key aspect of such a model is to define the structures of each of the intermediate forms the enzyme takes up as it traverses the catalytic cycle . One complication that has been prevalent with mitochondrial cytochrome c oxidase is the existence of structural variants of the enzyme, as isolated, that may not be participants in catalysis . Studies of structurally simpler procaryotic members of the family may offer new insight on the intermediates of catalysis . In this paper transient-state and steady-state kinetic studies of cytochrome aa(3)-600 from Bacillus subtilis are integrated into a model of the catalytic cycle . This model specifies that the P intermediate accumulates in the steady-state and it is proposed that the step following its formation is limited by proton uptake.

J Mol Biol, 2004 May 7, 338(4), 669 - 82
Interaction of the trp RNA-binding attenuation protein (TRAP) with anti-TRAP; Snyder D et al.; The trp RNA-binding attenuation protein (TRAP) negatively regulates expression of the tryptophan biosynthesis genes of Bacillus subtilis . In the presence of tryptophan, TRAP is activated to bind to the 5'-leader region of the trp mRNA resulting in termination prior to the structural genes . In addition, accumulation of uncharged tRNA(Trp) induces synthesis of anti-TRAP (AT), which binds to TRAP and inhibits its function . Both of these proteins consist of oligomers of identical subunits . Here, we characterize the self-association of each of these proteins and the TRAP-AT interaction in free solution using equilibrium and velocity analytical ultracentrifugation . TRAP exists as a stable 11-mer in the absence and in the presence of tryptophan . Tryptophan binding induces a conformational change in TRAP . AT exists in a reversible equilibrium between trimer and dodecamer with an equilibrium constant of approximately 3 x 10(14)M(-3) . About 20% of the trimer is incompetent to form dodecamer . The AT equilibrium is slow on the time-scale of the velocity experiment . Formation of TRAP-AT complexes occurs only in the presence of tryptophan . A complex containing one TRAP 11-mer and one AT 12-mer forms with high affinity . At higher ratios of TRAP:AT complexes containing two TRAP 11-mers and one AT 12-mer are detected . A model for the structure of the complex is proposed.

Wei Sheng Yan Jiu, 2004 Jan, 33(1), 86 - 8
{Analysis of antibacterial-relative proteins and peptides in housefly larvae}; An C et al.; OBJECTIVE: To study the antibacterial-relative proteins and peptides in housefly larvae . METHODS: Third-instar larvae of housefly were treated with different methods including pricking, pricking with bacteria, heating and ultrasonication and were extracted respectively after 6 h, 16 h, 24 h, 48 h, 65 h . After liquid phase assay, the antibacterial-relative proteins and peptides were confirmed with stepwise regression analysis . RESULTS: The four treatments could all induce antibacterial substances . Among these induced proteins, X17 (20 kDa), X16 (22 kDa) had activity against Staphylococcus aureus and Bacillus subtilis, but X22 (13 kDa), X20 (16 kDa), X12 (26 kDa) had activity only against either Staphylococcus aureus or Bacillus subtilis . X7 (37 kDa), X5 (44 kDa) had negative contribution to antibacterial activity . CONCLUSION: Many antibacterial-relative proteins and peptides which cooperate and establish a whole resisting system will begin to be translated or be translated more.

Wei Sheng Yan Jiu, 2004 Jan, 33(1), 42 - 4, 48
{Synergetic germicidal effect of ultrasonic with glutaraldehyde and its application in clinical disinfection}; Wang C et al.; OBJECTIVE: To study the synergetic germicidal effect of ulstrasonic with glutaraldehyde and the feasibility of combining ulstrasonic and glutaraldehyde to disinfect endocopes and medical instruments . METHODS: We determined the respective and the synergetic killing rates of Bacillus subtilis var . niger of ulstrasonic and glutaraldehyde using quantitative suspenion germicidal tests and simulated on-the-spot tests . RESULTS: In suspenion germicidal tests, because of the synergetic germicidal effect, the concentration of glutaraldehyde needed for sterilize spores was decreased from 20 mg/ml to 5 mg/ml for the same killing time(4 h), and when the concentrations were still 20 mg/ml, its killing time was shorten from 4 h to 1 h . Further more, the solutions containing 5 mg/ml glutaraldehyde could sterilize spores of Bacillus subtilis var . niger in 2 h . In the simulated on-the-spot tests, the concentration of glutaraldehyde needed for sterilization was decreased from 20 mg/ml to 5 mg/ml and its killing time was shorten from 4 h to 1 h under the synergetic germicidal effect . CONCLUSION: It indicats the feasibility of combining ulstrasonic and glutaraldehyde to disinfect instruments in clinic.

Proc Natl Acad Sci U S A, 2004 Apr 27, 101(17), 6421 - 6 Epub 2004 Apr 19.
New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control; Barrick JE et al.; The expression of certain genes involved in fundamental metabolism is regulated by metabolite-binding "riboswitch" elements embedded within their corresponding mRNAs . We have identified at least six additional elements within the Bacillus subtilis genome that exhibit characteristics of riboswitch function (glmS, gcvT, ydaO/yuaA, ykkC/yxkD, ykoK, and yybP/ykoY) . These motifs exhibit extensive sequence and secondary-structure conservation among many bacterial species and occur upstream of related genes . The element located upstream of the glmS gene in Gram-positive organisms functions as a metabolite-dependent ribozyme that responds to glucosamine-6-phosphate . Other motifs form complex folded structures when transcribed as RNA molecules and carry intrinsic terminator structures . These findings indicate that riboswitches serve as a major genetic regulatory mechanism for the control of metabolic genes in many microbial species.

J Mol Biol, 2004 Feb 27, 336(4), 929 - 42
Mechanism of thermostabilization in a designed cold shock protein with optimized surface electrostatic interactions; Makhatadze GI et al.; Using computational and sequence analysis of bacterial cold shock proteins, we designed a protein (CspB-TB) that has the core residues of mesophilic protein from Bacillus subtilis(CspB-Bs) and altered distribution of surface charged residues . This designed protein was characterized by circular dichroism spectroscopy, and found to have secondary and tertiary structure similar to that of CspB-Bs . The activity of the CspB-TB protein as measured by the affinity to a single-stranded DNA (ssDNA) template at 25 degrees C is somewhat higher than that of CspB-Bs . Furthermore, the decrease in the apparent binding constant to ssDNA upon increase in temperature is much more pronounced for CspB-Bs than for CspB-TB . Temperature-induced unfolding (as monitored by differential scanning calorimetry and circular dichroism spectroscopy) and urea-induced unfolding experiments were used to compare the stabilities of CspB-Bs and CspB-TB . It was found that CspB-TB is approximately 20 degrees C more thermostable than CspB-Bs . The thermostabilization of CspB-TB relative to CspB-Bs is achieved by decrease in the enthalpy and entropy of unfolding without affecting their temperature dependencies, i.e . these proteins have similar heat capacity changes upon unfolding . These changes in the thermodynamic parameters result in the global stability function, i.e . Gibbs energy, deltaG(T), that is shifted to higher temperatures with only small changes in the maximum stability . Such a mechanism of thermostabilization, although predicted from the basic thermodynamic considerations, has never been identified experimentally.

J Bacteriol, 2004 May, 186(9), 2789 - 97
Mutational analysis of RsbT, an activator of the Bacillus subtilis stress response transcription factor, sigmaB; Woodbury RL et al.; SigmaB, the stress-activated sigma factor of Bacillus subtilis, requires the RsbT protein as an essential positive regulator of its physical stress pathway . Stress triggers RsbT to both inactivate the principal negative regulator of the physical stress pathway (RsbS) by phosphorylation and activate a phosphatase (RsbU) required for sigmaB induction . Neither the regions of RsbT that are involved in responding to stress signaling nor those required for downstream events have been established . We used alanine scanning mutagenesis to examine the contributions of RsbT's charged amino acids to the protein's stability and activities . Eleven of eighteen rsbT mutations blocked sigmaB induction by stress . The carboxy terminus of RsbT proved to be particularly important for accumulation in Bacillus subtilis . Four of the five most carboxy-terminal mutations yielded rsbT alleles whose products were undetectable in B . subtilis extracts . Charged amino acids in the central region of RsbT were less critical, with four of the five substitutions in this region having no measurable effect on RsbT accumulation or activity . Only when the substitutions extended into a region of kinase homology was sigmaB induction affected . Six other RsbT variants, although present at levels adequate for activity, failed to activate sigmaB and displayed significant changes in their ability to interact with RsbT's normal binding partners in a yeast dihybrid assay . These changes either dramatically altered the proteins' tertiary structure without affecting their stability or defined regions of RsbT that are involved in multiple interactions.

J Bacteriol, 2004 May, 186(9), 2655 - 63
The membrane fluidity sensor DesK of Bacillus subtilis controls the signal decay of its cognate response regulator; Albanesi D et al.; The Bacillus subtilis DesK/DesR two-component system regulates the expression of the des gene coding for the Delta5 acyl lipid desaturase . It is believed that a decrease in membrane lipid fluidity activates the DesK/DesR signal transduction cascade, which results in synthesis of the Delta5 acyl lipid desaturase and desaturation of membrane phospholipids . These newly synthesized unsaturated fatty acids then act as negative signals of des transcription, thus generating a regulatory metabolic loop that optimizes membrane fluidity . We previously suggested that DesK is a bifunctional enzyme with both kinase and phosphatase activities that could assume different signaling states in response to changes in the fluidity of membrane lipids . However, no direct experimental evidence supported this proposed model . In this study, we show that the C-terminal fragment of the DesK protein (DesKC) indeed acts as an autokinase . Addition of the response regulator DesR to phosphorylated DesKC resulted in rapid transfer of the phosphoryl group to DesR . Further, phosphorylated DesR can be dephosphorylated in the presence of DesKC, thus demonstrating that the sensor kinase has the ability to covalently modify DesR through both kinase and phosphatase activities . We also present evidence that DesKC might be locked in a kinase-dominant state in vivo and that its activities are not affected either in vivo or in vitro by unsaturated fatty acids . These findings provide the first direct evidence that the transmembrane segments of DesK are essential to sense changes in membrane fluidity and for regulating the ratio of kinase to phosphatase activities of the cytoplasmic C-terminal domain.

Appl Microbiol Biotechnol, 2004 May, 64(4), 505 - 14 Epub 2004 Feb 20.
Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from a novel species of deep-sea Microbulbifer; Ohta Y et al.; An agar-degrading bacterium, strain JAMB-A7, was isolated from the sediment in Sagami Bay, Japan, at a depth of 1,174 m and identified as a novel species of the genus Microbulbifer . The gene for a novel beta-agarase from the isolate was cloned and sequenced . It encodes a protein of 441 amino acids with a calculated molecular mass of 48,989 Da . The deduced amino acid sequence showed similarity to those of known beta-agarases in glycoside hydrolase family 16, with only 34-55% identity . A sequence similar to a carbohydrate-binding module was found in the C-terminal region of the enzyme . The recombinant agarase was hyper-produced extracellularly using Bacillus subtilis as the host, and the enzyme purified to homogeneity had a specific activity of 398 U (mg protein)(-1) at pH 7.0 and 50 degrees C . It was thermostable, with a half-life of 502 min at 50 degrees C . The optimal pH and temperature for activity were around 7 and 50 degrees C, respectively . The pattern of agarose hydrolysis showed that the enzyme was an endo-type beta-agarase, and the final main product was neoagarotetraose . The activity was not inhibited by NaCl, EDTA, and various surfactants at high concentrations . In particular, sodium dodecyl sulfate had no inhibitory effect up to 2% .

Phytochemistry, 2004 Apr, 65(7), 875 - 80
Antimicrobial and antioxidant flavonoids from the root wood of Bolusanthus speciosus; Erasto P et al.; Three new flavonoids-5,7,4'-trihydroxy-6-{1-hydroxy-2-methylbuten-2-yl}isoflavone (isogancaonin C), 7,2'-dihydroxy-4'-methoxyisoflav-3-ene (bolusanthin III), 6,6'-dihydroxy-4'-methoxy-2-arylbenzofuran (bolusanthin IV), in addition to eight known flavonoids; derrone, medicarpan, genistein, wighteone, lupiwighteone, gancaonin C, 7-hydroxy-4'-methoxyisoflavone and 7,3'-dihydroxy-4'-methoxyisoflavone were isolated from the root wood of Bolusanthus speciosus . The compounds showed strong antimicrobial activity against Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Candida mycoderma . The isolated compounds also showed moderate to strong radical scavenging properties against DPPH radical with the highest activities shown by the 2-arylbenzofuran, the isoflav-3-ene and 7,3'-dihydroxy-4'-methoxyisoflavone in decreasing order.

J Appl Microbiol, 2004, 96(5), 1151 - 60
Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple; Toure Y et al.; AIM: Test of Bacillus subtilis strain GA1 for its potential to control grey mould disease of apple caused by Botrytis cinerea . METHODS AND RESULTS: GA1 was first tested for its ability to antagonize in vitro the growth of a wide variety of plant pathogenic fungi responsible for diseases of economical importance . The potential of strain GA1 to reduce post-harvest infection caused by B . cinerea was tested on apples by treating artificially wounded fruits with endospore suspensions . Strain GA1 was very effective at reducing disease incidence during the first 5 days following pathogen inoculation and a 80% protection level was maintained over the next 10 days . Treatment of fruits with an extract of GA1 culture supernatant also exerted a strong preventive effect on the development of grey mould . Further analysis of this extract revealed that strain GA1 produces a wide variety of antifungal lipopeptide isomers from the iturin, fengycin and surfactin families . A strong evidence for the involvement of such compounds in disease reduction arose from the recovery of fengycins from protected fruit sites colonized by bacterial cells . CONCLUSIONS: The results presented here demonstrate that, despite unfavourable pH, B . subtilis endospores inoculated on apple pulp can readily germinate allowing significant cell populations to establish and efficient in vivo synthesis of lipopeptides which could be related to grey mould reduction . SIGNIFICANCE AND IMPACT OF THE STUDY: This work enables for the first time to correlate the strong protective effect of a particular B . subtilis strain against grey mould with in situ production of fengycins in infected sites of apple fruits.

J Appl Microbiol, 2004, 96(5), 1133 - 42
Mechanisms of Bacillus subtilis spore resistance to and killing by aqueous ozone; Young SB et al.; AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to aqueous ozone . METHODS AND RESULTS: Killing of B . subtilis spores by aqueous ozone was not due to damage to the spore's DNA, as wild-type spores were not mutagenized by ozone and wild-type and recA spores exhibited very similar ozone sensitivity . Spores (termed alpha-beta-) lacking the two major DNA protective alpha/beta-type small, acid-soluble spore proteins exhibited decreased ozone resistance but were also not mutagenized by ozone, and alpha-beta- and alpha-beta-recA spores exhibited identical ozone sensitivity . Killing of spores by ozone was greatly increased if spores were chemically decoated or carried a mutation in a gene encoding a protein essential for assembly of the spore coat . Ozone killing did not cause release of the spore core's large depot of dipicolinic acid (DPA), but these killed spores released all of their DPA after a subsequent normally sublethal heat treatment and also released DPA much more readily when germinated in dodecylamine than did untreated spores . However, ozone-killed spores did not germinate with either nutrients or Ca(2+)-DPA and could not be recovered by lysozyme treatment . CONCLUSIONS: Ozone does not kill spores by DNA damage, and the major factor in spore resistance to this agent appears to be the spore coat . Spore killing by ozone seems to render the spores defective in germination, perhaps because of damage to the spore's inner membrane . SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanisms of spore killing by and resistance to ozone.

Appl Microbiol Biotechnol, 2004 Aug, 65(3), 263 - 7 Epub 2004 Apr 07.
Batch production of deacetyl 7-aminocephalosporanic acid by immobilized cephalosporin-C deacetylase; Takimoto A et al.; Bacillus subtilis SHS0133 cephalosporin-C deacetylase (CAH) overexpressed in Escherichia coli was immobilized on an anion-exchange resin, KA-890, using glutaraldehyde . The activity yield of immobilized enzyme was approximately 55% of the free enzyme . The pH range for stability of the immobilized enzyme (pH 5-10) was broader than that for free enzyme . The K(m)(app) value of immobilized enzyme for 7-aminocephalosporanic acid (7-ACA) was similar to that of the free enzyme . This immobilized enzyme obeyed Michaelis-Menten kinetics similar to those of the free enzyme . A batch-type reactor with a water jacket was employed for deacetylation of 7-ACA using CAH immobilized on KA-890 . Ten kilograms of 7-ACA were completely converted to deacetyl 7-ACA at pH 8.0 within 90 min . The reaction kinetics agreed well with a computer simulation model . Moreover, the immobilized enzyme exhibited only a slight loss of the initial activity even after repeated use (52 times ) over a period of 70 days . This reaction will thus be useful for the production of cephalosporin-type antibiotics.

Appl Environ Microbiol, 2004 Apr, 70(4), 2535 - 9
Killing of Bacillus subtilis spores by a modified Fenton reagent containing CuCl2 and ascorbic acid; Shapiro MP et al.; Bacillus subtilis spores were killed by CuCl(2)-ascorbic acid, chloride ions were essential for killing of spores, and spores with defective coats were killed more rapidly . CuCl(2)-ascorbic acid did not damage spore DNA, and spores killed by this reagent initiated germination . However, spores killed by CuCl(2)-ascorbic acid may have damage to their inner membrane.

Appl Environ Microbiol, 2004 Apr, 70(4), 2514 - 9
Growth and sporulation of Bacillus cereus ATCC 14579 under defined conditions: temporal expression of genes for key sigma factors; de Vries YP et al.; An airlift fermentor system allowing precise regulation of pH and aeration combined with a chemically defined medium was used to study growth and sporulation of Bacillus cereus ATCC 14579 . Sporulation was complete and synchronous . Expression of sigA, sigB, sigF, and sigG was monitored with real-time reverse transcription-PCR, and the pattern qualitatively resembled that of Bacillus subtilis . This method allows reproducible production of stable spores, while the synchronous growth and defined conditions are excellently suitable for further gene expression studies of cellular differentiation of B . cereus.

Appl Environ Microbiol, 2004 Apr, 70(4), 2508 - 13
Targeted isolation of a designated region of the Bacillus subtilis genome by recombinational transfer; Tomita S et al.; A method for positional cloning of the Bacillus subtilis genome was developed . The method requires a set of two small DNA fragments that flank the region to be copied . A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning . The positional cloning vector for ppsABCDE was constructed using a B . subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium . Structure of the copied DNA was confirmed by restriction enzyme analyses . Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.

Appl Environ Microbiol, 2004 Apr, 70(4), 2349 - 53
Subtilosin production by two Bacillus subtilis subspecies and variance of the sbo-alb cluster; Stein T et al.; Eight different Bacillus subtilis strains and Bacillus atrophaeus were found to produce the bacteriocin subtilosin A . On the basis of the subtilosin gene (sbo) sequences two distinct classes of B . subtilis strains were distinguished, and they fell into the two B . subtilis subspecies (B . subtilis subsp . subtilis and B . subtilis subsp . spizizenii) . The entire sequence of the subtilosin gene cluster of a B . subtilis subsp . spizizenii strain, B . subtilis ATCC 6633, was determined . This sequence exhibited a high level of homology to the sequence of the sbo-alb gene locus of B . subtilis 168 . By using primer extension analysis the transcriptional start sites of sbo in B . subtilis strains ATCC 6633 and 168 were found to be 47 and 45 bp upstream of the sbo start codon, respectively . Our results provide insight into the incipient evolutionary divergence of the two B . subtilis subspecies.

Mol Microbiol, 2004 Apr, 52(2), 529 - 40
The push-pull mechanism of bacteriophage Ø29 DNA injection; Gonzalez-Huici V et al.; The mechanism of bacteriophage DNA injection is poorly understood, often considered a simple process, driven merely by the packing pressure inside the capsid . In contrast to the well-established DNA packaging mechanism of Bacillus subtilis phage O29, that involves a molecular motor formed by the connector and a viral ATPase, nothing is known about its DNA injection into the cell . We have studied this process measuring DNA binding of p6, a viral genome organization protein . The linear DNA penetrates with a right-left polarity, in a two-step process . In the first step approximately 65% of the genome is pushed into the cell most probably by the pressure built inside the viral capsid . Thus, synthesis of viral proteins from the right early operon is allowed . This step is controlled, probably by bacterial protein(s) that slow down DNA entry . In the second step at least one of the viral early proteins, p17, participates in the molecular machinery that pulls the remaining DNA inside the cell . Both steps are energy-dependent, as treatment of cells with azide overrides the whole mechanism, leading to a deregulated, passive entry of DNA.

Mol Microbiol, 2004 Apr, 52(2), 357 - 69
Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility; Kearns DB et al.; Undomesticated strains of Bacillus subtilis, but not laboratory strains, exhibit robust swarming motility on solid surfaces . The failure of laboratory strains to swarm is caused by a mutation in a gene (sfp) needed for surfactin synthesis and a mutation(s) in an additional unknown gene(s) . Insertional mutagenesis of the undomesticated 3610 strain with the transposon mini-Tn10 was carried out to discover genes needed for swarming but not swimming motility . Four such newly identified swarming genes are reported, three of which (swrA, swrB, and efp) had not been previously characterized and one of which (swrC) was known to play a role in resistance to the antibacterial effect of surfactin . Laboratory strains were found to harbour a frameshift mutation in the swrA gene . When corrected for the swrA mutation, as well as the mutation in sfp, laboratory strains regained the capacity to swarm and did so as robustly as the wild strain . The swrA mutation was an insertion of an A:T base pair in a homopolymeric stretch of eight A:T base pairs, and readily reverted to the wild type . These findings suggest that the swrA insertion and its reversion take place by slipped-strand mispairing during DNA replication and that swarming motility is subject to phase variation.

Biochemistry, 2004 Apr 13, 43(14), 4128 - 36
Structure-based mutational analysis of the 4'-phosphopantetheinyl transferases Sfp from Bacillus subtilis: carrier protein recognition and reaction mechanism; Mofid MR et al.; The activation of apo-peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases (NRPSs), apo-acyl carrier proteins (ACPs) of polyketide synthases (PKSs), and fatty acid synthases (FASs) to their active holo form is accomplished with dedicated 4'-phosphopantetheinyl transferases (PPTases) . They catalyze the transfer of the essential prosthetic group 4'-phosphopantetheine (4'-Ppant) from coenzyme A (CoA) to a highly conserved serine residue in all PCPs and ACPs . PPTases, based on sequence and substrate specifity, have been classified into three types: bacterial holo-acyl carrier protein synthase (AcpS), fatty acid synthase of eukaryotes (FAS2) and Sfp, a PPTase of secondary metabolism . The recently solved crystal structures of AcpS and Sfp-type PPTases with CoA revealed a common alpha + beta-fold with a beta(1)alpha(3)beta(2) motif and similarities in CoA binding and polymerization mode . However, it was not possible to discern neither the PCP binding region of Sfp nor the priming reaction mechanism from the Sfp-CoA cocrystal . In this work, we provide a model for the reaction mechanism based on mutational analysis of Sfp that suggests a reaction mechanism in which the highly conserved E151 deprotonates the hydroxyl group of the invariant serine of PCP . That, in turn, acts as a nucleophile to attack the beta-phosphate of CoA . The Sfp mutants K112, E117, and K120 further revealed that the loop region between beta4 and alpha5 (residues T111-S124) in Sfp is the PCP binding region . Also, residues T44, K75, S89, H90, D107, E109, E151, and K155 that have been shown in the Sfp-CoA cocrystal structure to coordinate CoA are now all confirmed by mutational and biochemical analysis.

Curr Opin Microbiol, 2004 Apr, 7(2), 132 - 9
Regulation of transcription attenuation and translation initiation by allosteric control of an RNA-binding protein: the Bacillus subtilis TRAP protein; Babitzke P; Tryptophan allosterically controls the 11-subunit trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis . When activated by tryptophan, TRAP binds to multiple trinucleotide repeats in target transcripts . TRAP is responsible for the decision to terminate transcription in the leader region of the trpEDCFBA operon or to allow transcription to proceed into the structural genes . TRAP also regulates translation of trpE by promoting formation of an RNA structure that prevents ribosome binding . In addition, bound TRAP regulates translation initiation of pabA, trpP and ycbK by directly blocking ribosome binding . The anti-TRAP protein inhibits TRAP activity by competing with RNA for the RNA binding surface of TRAP.

FEMS Microbiol Lett, 2004 Apr 15, 233(2), 247 - 56
DNA-binding studies on the Bacillus subtilis transcriptional regulator and AbrB homologue, SpoVT; Dong TC et al.; The process of spore formation in Bacillus subtilis is dependent upon a sophisticated program of gene expression that is regulated both temporally and spatially by a series of alternate sigma factors, in conjunction with a number of transcriptional regulators . One of these, SpoVT, regulates forespore-specific sigmaG-dependent transcription and is related at the amino acid level to the major stationary phase sentinel, AbrB, whose mode of DNA recognition appears to be non-classical . Here, we report that the C-terminal domain of SpoVT is crucial to its correct folding and function, and how the DNA-binding domain from AbrB cannot complement the closely homologous domain of SpoVT in vivo . We also establish the oligomeric state of SpoVT and its component domains . Finally, we demonstrate that the regulation of transcriptional control by SpoVT is unexpectedly more complicated than its counterpart, AbrB, and that the latent non-specific DNA-binding activity of the N-terminal domain of SpoVT is modulated by the C-terminal domain, which perhaps in combination with another unknown factor, confers specificity.

Peptides, 2004 Feb, 25(2), 171 - 6
A napin-like polypeptide from dwarf Chinese white cabbage seeds with translation-inhibitory, trypsin-inhibitory, and antibacterial activities; Ngai PH et al.; Napins are 1:1 disulfide-linked complexes of a smaller (ca . 4kDa) subunit and a larger (ca . 10kDa) subunit . The intent of the present study was to ascertain the production of napin by the seeds of a Brassica species that has not been examined previously, and also to explore new biological activities of the napin . A heterodimeric 11-kDa napin-like polypeptide has been isolated from Chinese white cabbage (Brassica chinensis cv dwarf) seeds with a protocol comprising ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75 . The N-terminal sequence of the 7-kDa subunit manifests striking similarity to napin large chain, albumin and trypsin inhibitor . The N-terminal sequence of the 4-kDa subunit is homologous to napin large chain and an antimicrobial peptide . The napin-like polypeptide inhibited translation in the rabbit reticulocyte system with an IC50 of 18.5nM . This translation-inhibitory activity was stable between pH 4 and 11, and between 10 and 40 degrees C . The polypeptide inhibited trypsin with a higher potency ( IC50 = 8.5 microM) than it inhibited chymotrypsin (IC50 = 220 microM), but was devoid of ribonuclease and antifungal activities . It manifested antibacterial activity against Pseudomonas aeruginosia, Bacillus subtilis, Bacillus cereus, and Bacillus megaterium . The results revealed that the napin-like polypeptide from Chinese white cabbage seeds exhibited some potentially exploitable activities.

J Bacteriol, 2004 Apr, 186(8), 2481 - 6
Novel rpoB mutations conferring rifampin resistance on Bacillus subtilis: global effects on growth, competence, sporulation, and germination; Maughan H et al.; Previously, spontaneous rifampin resistance mutations were isolated in cluster I of the rpoB gene, resulting in amino acid replacements (Q469R, H482R, H482Y, or S487L) in the Bacillus subtilis RNA polymerase beta subunit (W . L . Nicholson and H . Maughan, J . Bacteriol . 184:4936-4940, 2002) . In this study, each amino acid change in the beta subunit was observed to result in its own unique spectrum of effects on growth and various developmental events, including sporulation, germination, and competence for transformation . The results thus establish the important role played by the RNA polymerase beta subunit, not only in the catalytic aspect of transcription, but also in the regulation of major developmental events in B . subtilis.

J Bacteriol, 2004 Apr, 186(8), 2366 - 75
Properties of Bacillus subtilis sigma A factors with region 1.1 and the conserved Arg-103 at the N terminus of region 1.2 deleted; Hsu HH et al.; sigma factors in the sigma(70) family can be classified into the primary and alternative sigma factors according to their physiological functions and amino acid sequence similarities . The primary sigma factors are composed of four conserved regions, with the conserved region 1 being divided into two subregions . Region 1.1, which is absent from the alternative sigma factor, is poor in conservation; however, region 1.2 is well conserved . We investigated the importance of these two subregions to the function of Bacillus subtilis sigma(A), which belongs to a subgroup of the primary sigma factor lacking a 254-amino-acid spacer between regions 1 and 2 . We found that deletion of not more than 100 amino acid residues from the N terminus of sigma(A), which removed part or all region 1.1, did not affect the overall transcription activity of the truncated sigma(A)-RNA polymerase in vitro, indicating that region 1.1 is not required for the functioning of sigma(A) in RNA polymerase holoenzyme . This finding is consistent with the complementation data obtained in vivo . However, region 1.1 is able to negatively modulate the promoter DNA-binding activity of the sigma(A)-RNA polymerase . Further deletion of the conserved Arg-103 at the N terminus of region 1.2 increased the content of stable secondary structures of the truncated sigma(A) and greatly reduced the transcription activity of the truncated sigma(A)-RNA polymerase by lowering the efficiency of transcription initiation after core binding of sigma(A) . More importantly, the conserved Arg-103 was also demonstrated to be critical for the functioning of the full-length sigma(A) in RNA polymerase.

J Bacteriol, 2004 Apr, 186(8), 2240 - 52
Transcriptional organization and posttranscriptional regulation of the Bacillus subtilis branched-chain amino acid biosynthesis genes; Mader U et al.; In Bacillus subtilis, the genes of the branched-chain amino acids biosynthetic pathway are organized in three genetic loci: the ilvBHC-leuABCD (ilv-leu) operon, ilvA, and ilvD . These genes, as well as ybgE, encoding a branched-chain amino acid aminotransferase, were recently demonstrated to represent direct targets of the global transcriptional regulator CodY . In the present study, the transcriptional organization and posttranscriptional regulation of these genes were analyzed . Whereas ybgE and ilvD are transcribed monocistronically, the ilvA gene forms a bicistronic operon with the downstream located ypmP gene, encoding a protein of unknown function . The ypmP gene is also directly preceded by a promoter sharing the regulatory pattern of the ilvA promoter . The ilv-leu operon revealed complex posttranscriptional regulation: three mRNA species of 8.5, 5.8, and 1.2 kb were detected . Among them, the 8.5-kb full-length primary transcript exhibits the shortest half-life (1.2 min) . Endoribonucleolytic cleavage of this transcript generates the 5.8-kb mRNA, which lacks the coding sequences of the first two genes of the operon and is predicted to carry a stem-loop structure at its 5' end . This processing product has a significantly longer half-life (3 min) than the full-length precursor . The most stable transcript (half-life, 7.6 min) is the 1.2-kb mRNA generated by the processing event and exonucleolytic degradation of the large transcripts or partial transcriptional termination . This mRNA, which encompasses exclusively the ilvC coding sequence, is predicted to carry a further stable stem-loop structure at its 3' end . The very different steady-state amounts of mRNA resulting from their different stabilities are also reflected at the protein level: proteome studies revealed that the cellular amount of IlvC protein is 10-fold greater than that of the other proteins encoded by the ilv-leu operon . Therefore, differential segmental stability resulting from mRNA processing ensures the fine-tuning of the expression of the individual genes of the operon.

Res Microbiol, 2004 Apr, 155(3), 167 - 73
Cloning and expression of a fibrinolytic enzyme (subtilisin DFE) gene from Bacillus amyloliquefaciens DC-4 in Bacillus subtilis; Peng Y et al.; A strong fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4, subtilisin DFE, was isolated from douchi, a traditional Chinese soybean-fermented food . Based on the high homology between the N-terminal sequence of subtilisin DFE and that of subtilisin BPN, PCR primers were designed that allowed for the amplification and cloning of the intact subtilisin DFE gene . Sequence analysis indicated the presence of a 1149-bp open reading frame encoding 382 amino acid residues . The enzyme was actively expressed by the Escherichia coli-Bacillus subtilis shuttle expression vector pSUGV4 in the protease-deficient strain B . subtilis WB600, and its biochemical characteristics were the same as those of the original subtilisin DFE isolated from the donor strain, i.e., its molecular weight is approximately 28 kDa and it is a serine protease.

Biotechnol Prog, 2004 Mar-Apr, 20(2), 491 - 9
Bioprocess parameters and oxygen transfer characteristics in beta-lactamase production by Bacillus species; Celik E et al.; After screening potential beta-lactamase producers in a medium containing penicillin G, an inducible (Bacillus subtilis NRS 1125) and a constitutive (Bacillus licheniformis 749/C ATCC 25972) beta-lactamase producer were selected . As the highest enzyme activity was obtained with B . licheniformis 749/C, the effects of the concentration of carbon sources, i.e., glucose, fructose, sucrose, citric acid, and glycerol, and nitrogen sources, i.e., (NH(4))(2)HPO(4), NH(4)Cl, yeast extract, casamino acids and peptone, pH, and temperature on beta-lactamase production were investigated with B . licheniformis 749/C in laboratory scale bioreactors . Among the investigated media, the highest volumetric activity was obtained as 270 U cm(-)(3) in the medium containing 10.0 kg m(-)(3) glucose, 1.18 kg m(-)(3) (NH(4))(2)HPO(4), 8.0 kg m(-)(3) yeast extract, and the salt solution at 32 degrees C and pH(0) = 6.0 . By using the designed medium, fermentation and oxygen transfer characteristics of the bioprocess were investigated at V = 3.0 dm(3) bioreactor systems with a V(R) = 1.65 dm(3) working volume at Q(O)/V(R) = 0.5 vvm and N = 500 min(-1) . At the beginning of the process the Damkohler number was <1, indicating that the process was at biochemical reaction limited condition; at t = 2-5 h both mass-transfer and biochemical reaction resistances were effective; and at t = 6-10 h (Da >>1) the bioprocess was at mass transfer limited condition . Overall oxygen transfer coefficients (K(L)a) varied between 0.01 and 0.03 s(-)(1), enhancement factor (K(L)a/K(L)a(O)) varied between 1.2 and 2.3, and volumetric oxygen uptake rate varied between 0.001 and 0.003 mol m(-)(3) s(-)(1) throughout the bioprocess . The specific oxygen uptake and the specific substrate consumption rates were the highest at t = 2 h and then decreased with the cultivation . The maximum yield of cells on substrate and the maximum yield of cells on oxygen values were obtained, respectively, as Y(X/S) = 0.34 and Y(X/O) = 1.40, at t = 5 h, whereas the highest yield of substrate on oxygen was obtained as Y(S/O) = 6.94 at t = 3.5 h . The rate of oxygen consumption for maintenance and the rate of substrate consumption for maintenance values were found, respectively, as m(O) = 0.13 kg kg(-)(1) h(-)(1) and m(S) = 3.02 kg kg(-)(1) h(-)(1).

Curr Microbiol, 2004 Apr, 48(4), 276 - 9
Cloning and characterization of xylanase A from the strain Bacillus sp . BP-7: comparison with alkaline pI-low molecular weight xylanases of family 11; Gallardo O et al.; The xynA gene encoding a xylanase from the recently isolated Bacillus sp . strain BP-7 has been cloned and expressed in Escherichia coli . Recombinant xylanase A showed high activity on xylans from hardwoods and cereals, and exhibited maximum activity at pH 6 and 60 degrees C . The enzyme remained stable after incubation at 50 degrees C and pH 7 for 3 h, and it was strongly inhibited by Mn(2+), Fe(3+), Pb(2+), and Hg(2+) . Analysis of xylanase A in zymograms showed an apparent molecular size of 24 kDa and a pI of above 9 . The amino acid sequence of xylanase A, as deduced from xynA gene, shows homology to alkaline pI-low molecular weight xylanases of family 11 such as XynA from Bacillus subtilis . Analysis of codon usage in xynA from Bacillus sp . BP-7 shows that the G+C content at the first and second codon positions is notably different from the mean values found for glycosyl hydrolase genes from Bacillus subtilis.

Curr Microbiol, 2004 Apr, 48(4), 262 - 9
AbrB and Spo0E control the proper timing of sporulation in Bacillus subtilis; Shafikhani SH et al.; We have shown previously that Spo0A approximately P-dependent sinIR operon expression was substantially down-regulated in abrB null mutant backgrounds . In this report, we show that loss of function mutations in abrB also cause phosphorelay gene expression to be down regulated . abrB null mutations caused diminished vegetative growth-associated sporulation and resulted in a significant reduction in sporulation frequencies at T(24) . These mutants, however, sporulated at wild-type levels at T(48), indicating that sporulation timing was affected . The rvtA11 mutation in spo0A, a deletion mutation in spo0E, and a null mutation in hpr ( scoC) rescued sporulation and Spo0A approximately P-dependent gene expression in an abrB mutant background . These data indicate that AbrB and Spo0E may comprise a checkpoint system that regulates the progression of sporulation, allowing exploration of alternate cell states prior to the irrevocable commitment to sporulation.

Biosci Biotechnol Biochem, 2004 Mar, 68(3), 739 - 42
Genetic analysis of an incomplete bio operon in a biotin auxotrophic strain of Bacillus subtilis natto OK2; Sasaki M et al.; We describe the genetic analysis of the bio operon of the biotin auxotrophic Bacillus subtilis natto OK2 strain . The OK2 strain would only cross-feed with the Escherichia coli bioB mutant and also grew well in medium containing dethiobiotin . Sequencing analysis revealed two significant genetic alterations in the bioW and bioF genes within the bio operon of the OK2 strain . Complementation analysis with B . subtilis 168 bio mutants demonstrated that only the bioB gene could complement, but other bio operon genes could not . A bio(+) transformant, isolated from an OK2 strain, has biotin autotrophy.

Dermatol Surg, 2004 Apr, 30(4 Pt 1), 521 - 4
From road rash to top allergen in a flash: bacitracin; Jacob SE et al.; BACKGROUND: Bacitracin is an antibiotic that is produced by Bacillus subtilis, which is used in several types of consumer products, including cosmetics and ophthalmic and cutaneous ointments . OBJECTIVE: To call attention to the rising allergic contact dermatitis associated with bacitracin . RESULTS: Mass usage has resulted in an increasing number of clinically relevant allergic contact dermatitis reactions and near fatal anaphylaxis . The North American Contact Dermatitis Group has recorded its emergence as a leading allergen and continues to monitor the ever-growing allergic reaction rates . CONCLUSION: The clinical impact, scientific evidence, and need for medical cost containment all advocate the discontinuation of routine usage of bacitracin in clean surgical wounds.

Appl Biochem Biotechnol, 2004 Spring, 113-116, 899 - 912
Lipopeptide surfactant production by Bacillus subtilis grown on low-cost raw materials; Reis FA et al.; The production of biosurfactant by Bacillus subtilis ATCC 6633 was investigated using commercial sugar, sugarcane juice and cane molasses, sugarcane juice alcohol stillage, glycerol, mannitol, and soybean oil . Commercial sugar generated the minimum values of surface tension, with the best results (28.7 mN/m, (relative critical micelle concentration {CMC-1} of 78.6) being achieved with 10 g of substrate/L in 48 h . At a pH between 7.0 and 8.0, a higher production of surface-active compounds and a greater emulsifier activity was also observed . Enrichment of the culture medium with trace minerals and EDTA showed maximum yields, whereas supplementation with yeast extract stimulated only cell growth . The kinetic studies revealed that biosurfactant production is a cell growth-associated process; surface tension, CMC, and emulsification index values of 29.6 dyn/cm, 82.3, and 57%, respectively, were achieved, thus indicating that it is feasible to produce biosurfactants from a renewable and low-cost carbon source.

Appl Biochem Biotechnol, 2004 Spring, 113-116, 827 - 36
Characterization of surfactin from Bacillus subtilis for application as an agent for enhanced oil recovery; Schaller KD et al.; Surfactin produced by Bacillus subtilis (ATCC 21332) was used to examine the effect of altering salt concentration, pH, and temperature on surfactin activity (as measured by reductions in surface tension) . These parameters are some of the conditions that define oil reservoir characteristics and can affect the application of surfactants . The Biotechnology for Oilfield Operations research program at the Idaho National Engineering and Environmental Laboratory (INEEL) has successfully produced surfactin from potato process effluents for possible use as an economical alternative to chemical surfactants for improved oil recovery . Surfactants enhance the recovery of oil through a reduction of the interfacial tension between the oil and water interfaces, or by mediating changes in the wettability index of the system . We investigated changes in surfactin activity under a range of conditions by measuring surface tension . Surface tension was determined using video image analysis of inverted pendant drops . Experimental variables included NaCl (0-10%), pH (3.0-10.0), and temperature (21-70 degrees C) . Each of these parameters, as well as selected combinations, resulted in discrete changes in surfactin activity . It is therefore important to consider the exploration of the studied surfactin as an enhanced oil recovery agent.

Anal Chem, 2004 Apr 1, 76(7), 2103 - 11
Hollow-fiber flow field-flow fractionation for whole bacteria analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Reschiglian P et al.; This work proposes for the first time the use of hollow-fiber flow field-flow fractionation (HF FlFFF) for improved matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOFMS) of whole bacteria . HF FlFFF has proved to be able to prepurify or fractionate different species of whole bacteria . Sample preparation by HF FlFFF gives improved spectra quality because noncellular components possibly present in the sample can be separated from the cells . When a mixture of two bacteria (Bacillus subtilis and Escherichia coli) is fractionated through HF FlFFF, MALDI/TOFMS analysis of each separated bacterial species preserves the most characteristic ion signals of the species without the presence of characteristic signals of the other species . The main advantages of HF FlFFF for MALDI/TOFMS analysis of whole bacteria are miniaturization, simplicity, and low cost of the fractionator components . This low cost makes disposable usage of the fractionator possible, thus eliminating the risk of run-to-run contamination of spectra due to sample carryover . The low fractionator volume yields bacterial fractionation on the order of a few minutes, which is comparable to MALDI/TOFMS analysis time . The small fractionation volume makes sample dilution low enough so that additional sample concentration steps are not strictly required to preserve MALDI/TOFMS detection.

Bioresour Technol, 2004 Jun, 93(2), 175 - 81
Medium optimization for the production of thermal stable beta-glucanase by Bacillus subtilis ZJF-1A5 using response surface methodology; Tang XJ et al.; Polysaccharides, such as barley flour, dextrin and soluble starch, were better carbon sources than monosaccharides and disaccharides, such as glucose and maltose, for cell growth of Bacillus subtilis ZJF-1A5 and beta-glucanase production . beta-Glucanase produced by B . subtilis ZJF-1A5 was associated partially with cell growth and increased significantly when cells entered stationary phase; yeast extract was the best nitrogen source, followed by soybean flour . All inorganic nitrogen sources chosen in the experiments were not favorable for cell growth and enzyme production . A fractional factorial design (2(6-2)) was applied to elucidate medium components that significantly affect beta-glucanase production . The concentration of barley flour, corn flour and soybean flour in medium were significant factors . The steepest ascent method was used to locate the optimal domain and a central composite design was used to estimate the quadratic response surface from which the factor levels for maximum production of beta-glucanase were determined . The composition of fermentation medium optimized with response surface methodology was (g/l): barley flour, 63.5; corn flour, 44.8; KH2PO4, 1.0; MgSO4 x 7H2O, 0.1; CaCl2, 0.1 . beta-Glucanase activity was 251 U/ml at 48 h using optimized medium, 1.4 times higher than that in original medium .

BMC Microbiol . 2004 Mar 30;4(1):13.
Expression of a novel gene, gluP, is essential for normal Bacillus subtilis cell division and contributes to glucose export; Mesak LR et al.; BACKGROUND: The Bacillus subtilis glucokinase operon was predicted to be comprised of the genes, yqgP (now named gluP), yqgQ, and glcK . We have previously established a role for glcK in glucose metabolism . In the absence of enzymes that phosphorylate glucose, such as GlcK and/or enzyme IIGlc, accumulated cytoplasmic glucose can be transported out of the cell . Genes within the glucokinase operon were not previously known to play a role in glucose transport . Here we describe the expression of gluP and its function in glucose transport . RESULTS: We found that transcription of the glucokinase operon was regulated, putatively, by two promoters: sigmaA and sigmaH . Putative sigmaA and sigmaH-recognition sites were located upstream of and within gluP, respectively . Transcriptional glucokinase operon--lacZ fusions and Northern blotting were used to analyze the expression of gluP . GluP was predicted to be an integral membrane protein . Moreover, the prediction of GluP structure revealed interesting signatures: a rhomboid domain and two tetracopeptide repeat (TPR) motifs . Microscopic analysis showed that GluP minus cells were unable to divide completely, resulting in a filamentous phenotype . The cells were grown in either rich or minimal medium . We found GluP may be involved in glucose transport . {14C}-glucose uptake by the GluP minus strain was slightly less than in the wild type . On the other hand, trehalose-derived glucose in the growth medium of the GluP minus strain was detected in very low amounts . Experimental controls comprised of single or multiple genes mutations within the glucose transporting phosphotransferase system . CONCLUSIONS: gluP seems to be regulated only by a putative sigmaA-dependent promoter . The glucose uptake and export assays suggest that GluP is important for glucose export and may act as an exporter . This also supports the role of the glucokinase operon in glucose utilization.

Mol Microbiol, 2004 Apr, 52(1), 273 - 83
Zinc is a key factor in controlling alternation of two types of L31 protein in the Bacillus subtilis ribosome; Nanamiya H et al.; We have analysed changes in the composition of ribosomal proteins during cell growth in Bacillus subtilis . Ribosome fractions were prepared from B . subtilis cells at different phases of growth and were separated by radical-free and highly reducing (RFHR) two-dimensional polyacrylamide gel electrophoresis . We identified 50 ribosomal proteins, including two paralogues of L31 protein (RpmE and YtiA) . Although the ribosome fraction extracted from exponentially growing cells contained RpmE protein, this protein disappeared during the stationary phase . In contrast, YtiA was detected in the ribosome fraction extracted after the end of exponential growth . Expression of the ytiA gene encoding YtiA was found to be negatively controlled by Zur, a zinc-specific transcriptional repressor that controls zinc transport operons . Analysis by inductively coupled plasma mass spectrometry (ICP-MS) indicated that RpmE contains one zinc ion per molecule of protein . In addition, mutagenesis of the rpmE gene encoding RpmE revealed that Cys-36 and Cys-39, located within a CxxC motif, are required not only for binding zinc but also for the accumulation of RpmE in the cell . Taken together, these results indicate that zinc plays an essential role in the alternation between two types of L31 protein in the ribosome of B . subtilis.

Mem Inst Oswaldo Cruz, 2003 Dec, 98(8), 1115 - 20 Epub 2004 Mar 09.
Antibacterial activity of extracts and neolignans from Piper regnellii (Miq.) C . DC . var . pallescens (C . DC.) Yunck; Pessini GL et al.; The evaluation of the activity of the aqueous and ethyl acetate extracts of the leaves of Piper regnellii was tested against gram-positive and gram-negative bacteria . The aqueous extract displayed a weak activity against Staphylococcus aureus and Bacillus subtilis with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1000 micrograms/ml . The ethyl acetate extract presented a good activity against S . aureus and B . subtilis with MIC and MBC at 15.62 micrograms/ml . In contrast to the relative low MICs for gram-positive bacteria, gram-negative bacteria were not inhibited by the extracts at concentrations < or = 1000 mg/ml . The ethyl acetate extract was fractionated on silica gel into nine fractions . The hexane and chloroform fractions were active against S . aureus (MIC at 3.9 micrograms/ml) and B . subtilis (MIC at 3.9 and 7.8 micrograms/ml, respectively) . Using bioactivity-directed fractionation, the hexane fraction was rechromatographed to yield the antimicrobial compounds 1, 2, 5, and 6 identified as eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan, respectively . The pure compounds 1 and 2 showed a good activity against S . aureus with MIC of 1.56 micrograms/ml and 3.12 micrograms/ml, respectively . Both compounds presented MIC of 3.12 micrograms/ml against B . subtilis . The pure compound 6 named as conocarpan was quite active against S . aureus and B . subtilis with MIC of 6.25 micrograms/ml . The antibacterial properties of P . regnellii justify its use in traditional medicine for the treatment of wounds, contaminated through bacteria infections.

J Biol Chem, 2004 May 28, 279(22), 23654 - 60 Epub 2004 Mar 26.
Structural basis of Redox-coupled protein substrate selection by the cytochrome c biosynthesis protein ResA; Crow A et al.; Post-translational maturation of cytochromes c involves the covalent attachment of heme to the Cys-Xxx-Xxx-Cys-His motif of the apo-cytochrome . For this process, the two cysteines of the motif must be in the reduced state . In bacteria, this is achieved by dedicated, membrane-bound thiol-disulfide oxidoreductases with a high reducing power, which are essential components of cytochrome c maturation systems and are also linked to cellular disulfide-bond formation machineries . Here we report high-resolution structures of oxidized and reduced states of a soluble, functional domain of one such oxidoreductase, ResA, from Bacillus subtilis . The structures elucidate the structural basis of the protein's high reducing power and reveal the largest redox-coupled conformational changes observed to date in any thioredoxin-like protein . These redox-coupled changes alter the protein surface and illustrate how the redox state of ResA predetermines to which substrate it binds . Furthermore, a polar cavity, present only in the reduced state, may confer specificity to recognize apo-cytochrome c . The described features of ResA are likely to be general for bacterial cytochrome c maturation systems.

Prep Biochem Biotechnol, 2004 Feb, 34(1), 97 - 107
Enzyme catalyzed synthesis of some vinyl drug esters in organic medium; Lv DS et al.; A series of vinyl drug esters was synthesized using acyclovir and chloramphenicol with different carbon chain length acyl donors by alkaline protease from Bacillus subtilis and Lipozyme respectively, in non-aqueous medium . The corresponding vinyl drug derivatives were confirmed by nuclear magnetic resonance and infrared spectrometry . The influences of different organic solvents, reaction time, temperature, and content of water on synthesis of vinyl chloramphenicol esters were studied.

EMBO J, 2004 Apr 7, 23(7), 1636 - 46 Epub 2004 Mar 25.
Septal localization of forespore membrane proteins during engulfment in Bacillus subtilis; Rubio A et al.; In Bacillus subtilis, many membrane proteins localize to the sporulation septum, where they play key roles in spore morphogenesis and cell-specific gene expression, but the mechanism for septal targeting is not well understood . SpoIIQ, a forespore-expressed protein, is involved in engulfment and forespore-specific gene expression . We find that SpoIIQ dynamically localizes to the sporulation septum, tracks the engulfing mother cell membrane, assembles into helical arcs around the forespore and is finally degraded . Retention of SpoIIQ in the septum requires one or more mother cell-expressed proteins . We also observed that any forespore-expressed membrane protein initially localizes to the septum and later spreads throughout the forespore membrane, suggesting that membrane protein insertion occurs at the forespore septal region . This possibility provides an attractive mechanism for how activation of mother cell-specific gene expression is restricted to adjacent sister cells, since direct insertion of the signaling protein SpoIIR into the septum would spatially restrict its activity . In keeping with this hypothesis, we find that SpoIIR localizes to the septum and is transiently expressed.

Biotechnol Appl Biochem, 2004 Dec, 40(Pt 3), 291 - 8
Purification and biochemical characterization of a thermostable, alkaliphilic, extracellular alpha-amylase from Bacillus subtilis DM-03, a strain isolated from the traditional fermented food of India; Das K et al.; Bacillus subtilis strain DM-03, which is isolated from starter culture used for the production of alcohol by local Assam tribes, grows optimally at 52-55 degrees C and secretes a significant amount of alpha-amylase at pH 8.0 into the culture media . This alpha-amylase, purified by ion-exchange, gel-filtration and reverse-phase HPLC, constitutes 2.9% of the total extracellular protein . This purified enzyme, named Bsamy-I, has a subunit with molecular mass of 42.8 kDa as determined by SDS/PAGE, and optimum temperature and pH values at 52-55 degrees C and 9.0 respectively, which makes it ideal for use in the detergent industries . Maximum alpha-amylase production is obtained by using soluble starch and NH(4)Cl as carbon and nitrogen sources respectively . Thermostability of the enzyme is evident from heating the enzyme at 95 degrees C for 10 min, which results in a loss of 60% of the original enzyme activity . 4-Bromophenacyl bromide and PMSF at 4 and 1.5 mM concentration respectively completely abolish the enzymic activity, documenting the essential role of histidine and carboxylic residues in the catalytic process.

Acta Crystallogr D Biol Crystallogr, 2004 Apr, 60(Pt 4), 755 - 7 Epub 2004 Mar 23.
Single-stranded DNA bound to bacterial cold-shock proteins: preliminary crystallographic and Raman analysis; Bienert R et al.; The cold-shock response has been described for several bacterial species . It is characterized by distinct changes in intracellular protein patterns whereby a set of cold-shock-inducible proteins become abundant . The major cold-shock proteins of Bacillus subtilis (Bs-CspB) and Bacillus caldolyticus (Bc-Csp) are small oligonucleotide/oligosaccharide-binding (OB) fold proteins that have been described as binding single-stranded nucleic acids . Bs-CspB (Mr = 7365) and Bc-Csp (Mr = 7333) were crystallized in the presence of the deoxyhexanucleotide (dT)6 . Crystals of (dT)6 with Bs-CspB grew in the orthorhombic space group C222(1), with unit-cell parameters a = 49.0, b = 53.2, c = 77.0 A . Crystals with Bc-Csp grew in the primitive orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 74.3, b = 64.9, c = 31.2 A . These crystals diffract to maximal resolutions of 1.78 and 1.29 A, respectively . The presence of protein and DNA in the crystals was demonstrated by Raman spectroscopy.

Acta Crystallogr D Biol Crystallogr, 2004 Apr, 60(Pt 4), 638 - 45 Epub 2004 Mar 23.
Metals in the sporulation phosphorelay: manganese binding by the response regulator Spo0F; Mukhopadhyay D et al.; As a part of studies on the structural characterization of the components of the sporulation phosphorelay in Bacillus subtilis, the crystal structure of the manganese derivative of an intermediate signal transducer, Spo0F, has been elucidated at 2.25 A resolution . The calcium complex and the apo structures have been analyzed previously . In apo Spo0F, the active-site cation cavity is only partially formed and it only becomes completed upon metal coordination . The carbonyl of Lys56 is coordinated to the metal and interestingly the side chain of Lys56 exists in a variety of conformations in the three crystal structures of Spo0F . The affinity of the magnesium ion for Spo0F is in fact low; however, it binds Spo0F when it is in complex with Spo0B . It is proposed that the existence of a deep pocket which extends from the surface to the metal site could attract and direct the metal, thereby facilitating the metal binding of the complex.

Biochemistry, 2004 Mar 30, 43(12), 3385 - 95
Structure of subtilosin A, a cyclic antimicrobial peptide from Bacillus subtilis with unusual sulfur to alpha-carbon cross-links: formation and reduction of alpha-thio-alpha-amino acid derivatives; Kawulka KE et al.; The complete primary and three-dimensional solution structures of subtilosin A (1), a bacteriocin from Bacillus subtilis, were determined by multidimensional NMR studies on peptide produced using isotopically labeled {(13)C,(15)N}medium derived from Anabaena sp . grown on sodium {(13)C}bicarbonate and {(15)N}nitrate . Additional samples of 1 were also generated by separate incorporations of {U-(13)C,(15)N}-L-phenylalanine and {U-(13)C,(15)N}-L-threonine using otherwise unlabeled media . The results demonstrate that in addition to having a cyclized peptide backbone (amide between N and C termini), three cross-links are formed between the sulfurs of Cys13, Cys7, and Cys4 and the alpha-positions of Phe22, Thr28, and Phe31, respectively . The stereochemistry of all residues in 1 except for the three modified ones was confirmed to be L by complete desulfurization with nickel boride, acid hydrolysis to the constituent amino acids, and conversion of these to the corresponding pentafluoropropanamide isopropyl esters for chiral GC MS analysis . The stereochemistry at the modified residues was determined by subjecting each of the eight possible stereoisomers of 1 to eight rounds of ARIA structure calculations, starting with the same NMR peak files and assignments . The stereoisomer with the l stereochemistry at Phe22 (alpha-R) and d stereochemistry at Thr28 (alpha-S) and Phe31 (alpha-S) (LDD isomer) fit the NMR data, giving the lowest energy family of structures with the best rmsd . Thus, biochemical formation of the unusual thio links proceeds with net retention of configuration at Phe22, and inversion at Thr28 and Phe31 . Model amino acid derivatives bearing a sulfide moiety at the alpha-carbon were synthesized by reaction of the corresponding alpha-alkoxy compounds with benzyl thiol and SnCl(4) . Separation of their pure stereoisomers and desulfurization with nickel boride demonstrated that the reduction of such compounds proceeds with epimerization, in contrast to the previously reported retention of stereochemistry for analogous reaction of steroidal sulfides . However, desulfurization of subtilosin A to cyclic peptide 14, which is inactive as an antimicrobial agent, occurs with inversion of stereochemistry at the alpha-carbons of Phe22 and Thr28 and with 4:1 retention at Phe31 . This indicates that the desulfurization reaction proceeds via an N-acyl imine and that the structure of the surrounding peptide controls the geometry of reduction . Posttranslational linkage of a thiol to the alpha-carbon of an amino acid residue is unprecedented in ribosomally synthesized peptides or proteins, and very rare in secondary metabolites . Subtilosin A (1) represents a new class of bacteriocins.

J Food Prot, 2004 Mar, 67(3), 596 - 600
Inhibition of Bacillus subtilis and Listeria innocua by nisin in combination with some naturally occurring organic compounds; Olasupo NA et al.; The application of combined preservative factors (hurdle technology) is very effective in controlling the growth of food spoilage and foodborne pathogenic bacteria . Antimicrobial activity of nisin alone and in combination with some natural organic compounds (carvacrol, cinnamic acid, eugenol, diacetyl, and thymol) on the growth of gram-positive bacteria Bacillus subtilis and Listeria innocua was-investigated . All the organic compounds tested exhibited antimicrobial activity against the microorganisms used; however, the MICs varied between 0.8 and 15.0 mM depending on the potency of the compound or the sensitivity of the target strain . Investigation of the interaction between the organic compounds and nisin against the test organisms revealed different patterns, varying from synergistic to antagonistic . Combinations of nisin with carvacrol, eugenol, or thymol resulted in synergistic action against both test organisms . Activity of nisin and cinnamic acid together was synergistic against L . innocua, but only additive against B . subtilis . In contrast, the combination of diacetyl and nisin resulted in an antagonistic effect against both test organisms . This study highlights the potential of the combination of these compounds with nisin to inhibit pathogen growth in food.

Nat Rev Microbiol, 2003 Nov, 1(2), 117 - 26
Regulation of endospore formation in Bacillus subtilis; Errington J; Spore formation in bacteria poses a number of biological problems of fundamental significance . Asymmetric cell division at the onset of sporulation is a powerful model for studying basic cell-cycle problems, including chromosome segregation and septum formation . Sporulation is one of the best understood examples of cellular development and differentiation . Fascinating problems posed by sporulation include the temporal and spatial control of gene expression, intercellular communication and various aspects of cell morphogenesis.

Gene, 2004 Mar 31, 329, 125 - 36
Systematic analysis of SigD-regulated genes in Bacillus subtilis by DNA microarray and Northern blotting analyses; Serizawa M et al.; The SigD-regulated genes in Bacillus subtilis were systematically analyzed by comparing the pattern of transcripts derived from wild-type cells with those from sigD mutant cells using DNA microarray technology . One hundred and fifty-eight genes were found to be SigD-dependent candidates, 46 of which being known SigD-regulated genes . Northern blot analysis revealed that 18 of the remaining genes were SigD-dependent . The SigD consensus sequence was newly identified in the upstream regions of nine operons (11 genes): ybdO, yfmT-yfmS, hemAT, yjcP-yjcQ, yjfB, ylqB, yoaH, yscB and yxkC, and the other seven genes were assumed to be indirectly affected by a SigD mutation . Furthermore, yviE-yviF are likely to be SigD-dependent genes, because three independent sets of array data for yviE and yviF indicated they are SigD-dependent, and these genes are neighbors of flgL and hag transcribed by SigD RNA polymerase.

Biochem Biophys Res Commun, 2004 Apr 9, 316(3), 802 - 8
Conversion of feedback regulation in aspartate kinase by domain exchange; Kato C et al.; To elucidate the mechanism for the regulation of aspartate kinase (AK) via feedback inhibition, we constructed several chimeric enzymes between Bacillus subtilis AK II, a lysine-sensitive mesophilic enzyme, and Thermus flavus AK, a threonine-sensitive thermostable enzyme, each having the same alpha2beta2-type tetrameric structure . A chimeric AK, named BTT, composed of the chimeric alpha subunit that comprises of the N-terminal catalytic region from B . subtilis AK II and the C-terminal region from T . flavus, and the beta subunit from T . flavus, was inhibited only by threonine . Another chimeric enzyme, BT, which has a similar structure to that of BTT but lacks the beta subunit, having alpha2-type homo-dimeric structure, was also responsive only to threonine . However, the addition of threonine enhanced the activity of BT . These results indicate the regulatory function of C-terminal region and beta subunit in AK . BTT showed extremely high thermostability comparable to that of T . flavus, suggesting that the beta subunit also contributed to the stability of the AK.

FEMS Microbiol Lett, 2004 Mar 19, 232(2), 173 - 9
Optimization of a two-plasmid system for the identification of promoters recognized by RNA polymerase containing Staphylococcus aureus alternative sigma factor sigmaB; Homerova D et al.; We optimized a previously established two-plasmid system for the identification of Staphylococcus aureus promoters that are recognized by the alternative transcription factor sigma(B) . The method allowed the identification of 18 S . aureus sigma(B)-dependent promoters, 12 of which are reported here for the first time to be sigma(B)-dependent . S1-nuclease mapping of the respective transcriptional start points revealed that all the promoters contained sequences exhibiting high similarity to the consensus sequence of Bacillus subtilis sigma(B)-dependent promoters . The promoters governed expression of genes encoding proteins proposed to be involved in various cellular functions, including the stress response genes and virulence-associated clfA gene for fibrinogen-binding clumping factor . Comparison of the nucleotide sequences upstream of the identified transcription start points identified a sigma(B) consensus promoter (GttTaa-N(12-15)-gGGTAt) that is highly homologous to that of sigma(B) of B . subtilis.

J Bacteriol, 2004 Apr, 186(7), 2221 - 3
Compartmentalization of gene expression during sporulation of Bacillus subtilis is compromised in mutants blocked at stage III of sporulation; Li Z et al.; Mutations in the spoIIIA and spoIIIJ loci disrupt the compartmentalization of gene expression during sporulation of Bacillus subtilis . The breakdown in compartmentalization is not the cause of their being blocked in spore formation . Rather, it appears to be a consequence of the engulfed prespore's being unstable.

J Bacteriol, 2004 Apr, 186(7), 2212 - 4
Stochastic processes influence stationary-phase decisions in Bacillus subtilis; Maughan H et al.; It has recently been proposed that phenotypic variation in clonal populations of bacterial species results from intracellular "noise," i.e., random fluctuations in levels of cellular molecules, which would be predicted to be insensitive to selective pressure . To test this notion, we propagated five populations of Bacillus subtilis for 5,000 generations with selection for one phenotype: the decision to sporulate . In support of the noise hypothesis, we report that none of the populations responded to selection by improving their efficiency of sporulation, indicating that intracellular noise is independent of heritable genotype.

J Bacteriol, 2004 Apr, 186(7), 2028 - 37
Transcriptional activation by Bacillus subtilis ResD: tandem binding to target elements and phosphorylation-dependent and -independent transcriptional activation; Geng H et al.; The expression of genes involved in nitrate respiration in Bacillus subtilis is regulated by the ResD-ResE two-component signal transduction system . The membrane-bound ResE sensor kinase perceives a redox-related signal(s) and phosphorylates the cognate response regulator ResD, which enables interaction of ResD with ResD-dependent promoters to activate transcription . Hydroxyl radical footprinting analysis revealed that ResD tandemly binds to the -41 to -83 region of hmp and the -46 to -92 region of nasD . In vitro runoff transcription experiments showed that ResD is necessary and sufficient to activate transcription of the ResDE regulon . Although phosphorylation of ResD by ResE kinase greatly stimulated transcription, unphosphorylated ResD, as well as ResD with a phosphorylation site (Asp57) mutation, was able to activate transcription at a low level . The D57A mutant was shown to retain the activity in vivo to induce transcription of the ResDE regulon in response to oxygen limitation, suggesting that ResD itself, in addition to its activation through phosphorylation-mediated conformation change, senses oxygen limitation via an unknown mechanism leading to anaerobic gene activation.

J Bacteriol, 2004 Apr, 186(7), 1999 - 2005
Sporulation phenotype of a Bacillus subtilis mutant expressing an unprocessable but active sigmaE transcription factor; McBride S et al.; SigmaE, a sporulation-specific sigma factor of Bacillus subtilis, is formed from an inactive precursor (pro-sigmaE) by a developmentally regulated processing reaction that removes 27 amino acids from the proprotein's amino terminus . A sigE variant (sigE335) lacking 15 amino acids of the prosequence is not processed into mature sigmaE but is active without processing . In the present work, we investigated the sporulation defect in sigE335-expressing B . subtilis, asking whether it is the bypass of proprotein processing or a residual inhibition of sigmaE activity that is responsible . Fluorescence microscopy demonstrated that sigE335-expressing B . subtilis progresses further into sporulation (stage III) than do strains lacking sigmaE activity (stage II) . Consistent with its stage III phenotype, and a defect in sigmaE activity rather than its timing, the sigE335 allele did not disturb early sporulation gene expression but did inhibit the expression of late sporulation genes (gerE and sspE) . The Spo- phenotype of sigE335 was found to be recessive to wild-type sigE . In vivo assays of sigmaE activity in sigE, sigE335, and merodiploid strains indicate that the residual prosequence on sigmaE335, still impairs its activity to function as a transcription factor . The data suggest that the 11-amino-acid extension on sigmaE335 allows it to bind RNA polymerase and direct the resulting holoenzyme to sigmaE-dependent promoters but reduces the enzyme's ability to initiate transcription initiation and/or exit from the promoter.

J Bacteriol, 2004 Apr, 186(7), 1983 - 90
Contrasting effects of sigmaE on compartmentalization of sigmaF activity during sporulation of Bacillus subtilis; Hilbert DW et al.; Spore formation by Bacillus subtilis is a primitive form of development . In response to nutrient starvation and high cell density, B . subtilis divides asymmetrically, resulting in two cells with different sizes and cell fates . Immediately after division, the transcription factor sigmaF becomes active in the smaller prespore, which is followed by the activation of sigmaE in the larger mother cell . In this report, we examine the role of the mother cell-specific transcription factor sigmaE in maintaining the compartmentalization of gene expression during development . We have studied a strain with a deletion of the spoIIIE gene, encoding a DNA translocase, that exhibits uncompartmentalized sigmaF activity . We have determined that the deletion of spoIIIE alone does not substantially impact compartmentalization, but in the spoIIIE mutant, the expression of putative peptidoglycan hydrolases under the control of sigmaE in the mother cell destroys the integrity of the septum . As a consequence, small proteins can cross the septum, thereby abolishing compartmentalization . In addition, we have found that in a mutant with partially impaired control of sigmaF, the activation of sigmaE in the mother cell is important to prevent the activation of sigmaF in this compartment . Therefore, the activity of sigmaE can either maintain or abolish the compartmentalization of sigmaF, depending upon the genetic makeup of the strain . We conclude that sigmaE activity must be carefully regulated in order to maintain compartmentalization of gene expression during development.

Mol Cell, 2004 Mar 12, 13(5), 703 - 11
Features of a leader peptide coding region that regulate translation initiation for the anti-TRAP protein of B . subtilis; Chen G et al.; The rtpA gene of Bacillus subtilis encodes the Anti-TRAP protein, AT . AT can bind and inhibit the TRAP regulatory protein, preventing TRAP from promoting transcription termination in the trpEDCFBA operon leader region . AT synthesis is upregulated transcriptionally and translationally in response to the accumulation of uncharged tRNA(Trp) . Here we analyze AT's translational regulation by rtpLP, a 10 residue leader peptide coding region located immediately preceding the rtpA Shine-Dalgarno sequence . Our findings suggest that, whenever the charged tRNA(Trp) level is sufficient to allow the ribosome translating rtpLP to reach its stop codon, it blocks the adjacent rtpA Shine-Dalgarno sequence, inhibiting AT synthesis . However, when there is a charged tRNA(Trp) deficiency, the translating ribosome presumably stalls at one of three adjacent rtpLP Trp codons . This stalling exposes the rtpA Shine-Dalgarno sequence, permitting AT synthesis . RNA-RNA pairing may also influence AT synthesis . Production of AT would inactivate TRAP, thereby increasing trp operon expression.

Biochemistry, 2004 Mar 23, 43(11), 3120 - 8
Fluorescence and kinetic analysis of the SpoIIAB phosphorylation reaction, a key regulator of sporulation in Bacillus subtilis; Clarkson J et al.; Sporulation in Bacillus subtilis provides a valuable model system for studying differential gene expression . The anti-sigma factor SpoIIAB is a bifunctional protein, responsible for regulating the activity of the first sporulation-specific sigma factor, sigma(F) . SpoIIAB can either bind to (and thus inhibit) sigma(F) or phosphorylate the anti-anti-sigma factor SpoIIAA . The phosphorylation reaction follows an unusual time course in which a pre-steady-state phase is succeeded by a slower steady-state phase . Previous experiments have shown that in the steady-state phase SpoIIAB is unable to inhibit sigma(F) . A fluorescent derivative of SpoIIAB (AB-F97W) was made that was indistinguishable from the wild type in its interactions with SpoIIAA and sigma(F) . AB-F97W exhibited distinctive changes in its fluorescence intensity when bound to ATP, ADP, or SpoIIAA . By following changes in the fluorescence properties of AB-F97W during the phosphorylation reaction, we confirmed a previous hypothesis that during the steady-state phase the predominant species are SpoIIAA.SpoIIAB.ADP complexes . The formation of these complexes is responsible for the slowing of the reaction, an important feature during sporulation since it reduces the loss of ATP in the nutrient-deprived cell . We also show that, to form a complex with SpoIIAA and ADP during the reaction, SpoIIAB must undergo a change in state which increases its affinity for ADP, and that this change in state is stimulated by its interaction with SpoIIAA . We derive a model of the reaction using previously determined kinetic and binding constants, and relate these findings to the known structure of SpoIIAB.

Comput Biol Chem, 2004 Feb, 28(1), 3 - 10
The operons, a criterion to compare the reliability of transcriptome analysis tools: ICA is more reliable than ANOVA, PLS and PCA; Carpentier AS et al.; The number of statistical tools used to analyze transcriptome data is continuously increasing and no one, definitive method has so far emerged . There is a need for comparison and a number of different approaches has been taken to evaluate the effectiveness of the different statistical tools available for microarray analyses . In this paper, we describe a simple and efficient protocol to compare the reliability of different statistical tools available for microarray analyses . It exploits the fact that genes within an operon exhibit the same expression patterns . In order to compare the tools, the genes are ranked according to the most relevant criterion for each tool; for each tool we look at the number of different operons represented within the first twenty genes detected . We then look at the size of the interval within which we find the most significant genes belonging to each operon in question . This allows us to define and estimate the sensitivity and accuracy of each statistical tool . We have compared four statistical tools using Bacillus subtilis expression data: the analysis of variance (ANOVA), the principal component analysis (PCA), the independent component analysis (ICA) and the partial least square regression (PLS) . Our results show ICA to be the most sensitive and accurate of the tools tested . In this article, we have used the protocol to compare statistical tools applied to the analysis of differential gene expression . However, it can also be applied without modification to compare the statistical tools developed for other types of transcriptome analyses, like the study of gene co-expression.

Arch Microbiol, 2004 Apr, 181(4), 255 - 68 Epub 2004 Mar 12.
Incidence and function of sigma factors in Ralstonia metallidurans and other bacteria; Nies DH; Bacterial sigma factors are essential for directing the bacterial RNA polymerase to promoter regions during transcription initiation . Genomic sequencing of the highly heavy-metal-resistant beta-proteobacterium Ralstonia metallidurans strain CH34 revealed 17 candidate genes for sigma factors . This review compares the sigma factor machinery of R . metallidurans to that of other bacteria . The sigma factors of 105 bacterial genomes were assigned to sigma factor clusters and families formed around the factors from Escherichia coli, Bacillus subtilis, and R . metallidurans . Genes for between 1 and 65 sigma-factor-related proteins were found in these genomes . Although prediction of sigma factor function from sequence comparisons can be misleading, organization of the R . metallidurans sigma factors into clusters and protein families, together with a discussion of the physiological function of members of these clusters, might yield insight into the cellular roles of bacterial sigma factors and the genes that depend on them for their expression.

J Mol Biol, 2004 Mar 26, 337(3), 661 - 73
Structural and catalytic diversity in the two family 11 aldo-keto reductases; Ehrensberger AH et al.; Aldo-keto reductases (AKRs) are a large superfamily of NAD(P)H-dependent enzymes that function in a wide range of biological processes . The structures of two enzymes from the previously uncharacterized family 11 (AKR11A and AKR11B), the products of the iolS and yhdN genes of Bacillus subtilis have been determined . AKR11B appears to be a relatively conventional member of the superfamily with respect to structural and biochemical properties . It is an efficient enzyme, specific for NADPH and possesses a catalytic triad typical for AKRs . AKR11A exhibits catalytic divergence from the other members of the superfamily and, surprisingly, AKR11B, the most closely related aldo-keto reductase in sequence . Although both have conserved catalytic residues consisting of an acidic tyrosine, a lysine and an aspartate, a water molecule interrupts this triad in cofactor-bound AKR11A by inserting between the lysine and tyrosine side-chains . This results in a unique architecture for an AKR active site with scant catalytic power . In addition, the absence of a bulky tryptophan side-chain in AKR11A allows an unconventional conformation of the bound NADP+ cosubstrate, raising the possibility that it donates the 4-pro-S hydride rather than the 4-pro-R hydride seen in most other AKRs . Based upon the architecture of the active site and the resulting reaction velocities, it therefore appears that functioning as an efficient oxido-reductase is probably not the primary role of AKR11A . A comparison of the apo and holo forms of AKR11A demonstrates that the cosubstrate does not play the dramatic role in active site assembly seen in other superfamily members.

J Mol Biol, 2004 Mar 26, 337(3), 585 - 96
Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo; Beale RC et al.; To investigate the extent to which in vivo mutation spectra might reflect the intrinsic specificities of active mutators, genetic and biochemical assays were used to analyse the DNA target specificities of cytidine deaminases of the APOBEC family . The results reveal the critical importance of nucleotides immediately 5' of the targeted C for the specificity of all three enzymes studied (AID, APOBEC1 and APOBEC3G) . At position -1, APOBEC1 showed a marked preference for dT, AID for dA/dG and APOBEC3G a strong preference for dC . Furthermore, AID and APOBEC3G showed distinct dependence on the nucleotide at position -2 with dA/dT being favoured by AID and dC by APOBEC3G . Most if not all activity of the recombinant deaminases on free dC could be attributed to low-level contamination by host enzymes . The target preference of APOBEC3G supports it being a major but possibly not sole contributor to HIV hypermutation without making it a dominant contribution to general HIV sequence variation . The specificity of AID as deduced from the genetic assay (which relies on inactivation of sacB of Bacillus subtilis) agrees well with that deduced by Pham et al . using an in vitro assay although we postulate that major intrinsic mutational hotspots in immunoglobulin V genes in vivo might reflect favoured sites of AID action being generated by proximal DNA targets located on opposite DNA strands . The target specificity of AID also accords with the spectrum of mutations observed in B lymphoma-associated oncogenes . The possibility of deaminase involvement in non-lymphoid human tumours is hinted at by tissue-specific differences in the spectra of dC transitions in tumour-suppressor genes . Thus, the patterns of hypermutation in antibodies and retroviruses owe much to the intrinsic sequence preferences of the AID/APOBEC family of DNA deaminases: analogous biases might also contribute to the spectra of cancer-associated mutation.

FEMS Microbiol Lett, 2004 Mar 12, 232(1), 93 - 9
Altered gene expression in the transition phase by disruption of a Na+/H+ antiporter gene (shaA) in Bacillus subtilis; Kosono S et al.; The shaA gene (sodium-hydrogen antiporter gene A, identical to mrpA) is largely responsible for Na+ extrusion in Bacillus subtilis . The disruption of shaA combined with a low concentration of NaCl completely abolishes sporulation but allows normal growth . To investigate the role of shaA and shaA-mediated sodium ion homeostasis in sporulation, we performed a comprehensive study of expression profiles of eight alternative sigma factors, sigmaB and the seven extracytoplasmic function sigma factors (sigmaM, sigmaV, sigmaW, sigmaX, sigmaY, sigmaZ, and sigmaYlaC) in an attempt to determine the global change of gene expression that results from a disturbance of Na+ homeostasis caused by shaA disruption . Induction of sigmaB activity in the transition phase was impaired in the shaA mutant, and this effect was enhanced in the presence of 30 mM NaCl . Salt stress activation of sigmaB occurred normally in the shaA mutant . sigmaM-, sigmaW-, sigmaX-dependent transcription and sigZ transcription was also induced in the transition phase of the wild-type, which was modulated by shaA disruption . The induction of sigmaM-dependent transcription was enhanced in the shaA mutant, while that of sigmaX-dependent transcription and sigZ transcription was decreased . sigmaW-dependent transcription was increased throughout the growth phase of the shaA mutant, which was consistent with the result of proteome analysis . We conclude that shaA disruption resulted in the modulated induction of alternative sigma factor activities, which would be problematic for the cell upon entering the sporulation stage.

Biosens Bioelectron, 2004 Apr 15, 19(9), 1021 - 8
Ultrasonic deposition of cells on a surface; Hawkes JJ et al.; Bacteria in water have been driven to a glass surface by an ultrasonic standing wave . On an antibody coated surface capture of Bacillus subtilis var niger (BG) spores (6.6 x 10(6) ml(-1)) was increased more than 200-fold over above the efficiency in the absence of ultrasound . In microfluidic (non-turbulent) systems detection of particles by sensors operating at a surface is diffusion limited . This results in very low detection abilities particularly for particles with diameters greater than 1 microm . Ultrasound is used here to drive bacterial spores to a wall and overcome this limitation . The results confirm: (1) pressure nodes can be formed close to the water-glass interface when the glass thickness is near half the ultrasonic wavelength; (2) the antibody used was able to capture spores in the presence of an ultrasonic standing wave.

BMC Microbiol . 2004 Feb 03;4(1):6.
Bacillus subtilis GlcK activity requires cysteines within a motif that discriminates microbial glucokinases into two lineages; Mesak LR et al.; BACKGROUND: Bacillus subtilis glucokinase (GlcK) (GenBank NP_390365) is an ATP-dependent kinase that phosphorylates glucose to glucose 6-phosphate . The GlcK protein has very low sequence identity (13.7%) to the Escherichia coli glucokinase (Glk) (GenBank P46880) and some other glucokinases (EC 2.7.1.2), yet glucose is merely its substrate . Our lab has previously isolated and characterized the glcK gene . RESULTS: Microbial glucokinases can be grouped into two different lineages . One of the lineages contains three conserved cysteine (C) residues in a CXCGX(2)GCXE motif . This motif is also present in the B . subtilis GlcK . The GlcK protein occurs in both monomer and homodimer . Each GlcK monomer has six cysteines . All cysteine residues have been mutated, one-by-one, into alanine (A) . The in vivo GlcK enzymatic activity was assayed by functional complementation in E . coli UE26 (ptsG ptsM glk) . Mutation of the three motif-specific residues led to an inactive enzyme . The other mutated forms retained, or in one case (GlcKC321A) even gained, activity . The fluorescence spectra of the GlcKC321A showed a red shift and enhanced fluorescence intensity compare to the wild type's . CONCLUSIONS: Our results emphasize the necessity of cysteines within the CXCGX(2)GCXE motif for GlcK activity . On the other hand, the C321A mutation led to higher GlcKC321A enzymatic activity with respect to the wild type's, suggesting more adequate glucose phosphorylation.

Curr Microbiol, 2004 Jan, 48(1), 62 - 7
Identification and immunochemical location of UMP kinase from Bacillus subtilis; Gagyi C et al.; Phosphorylation of CMP and UMP is accomplished in Bacillus subtilis, as in Escherichia coli, by two different enzymes exhibiting characteristic structural and catalytic properties . UMP kinase from B . subtilis is an oligomer whose activity is strictly dependent on GTP . The B . subtilis enzyme is unstable in the absence of UTP, which acts as an allosteric inhibitor . Antibodies raised against recombinant B . subtilis UMP kinase recognized the protein both in soluble extract and in immunoelectron microscopy . UMP kinase from B . subtilis has a peripheral distribution which is related most probably to its role in the synthesis of membrane sugar components and its putative role in cell division.

Curr Microbiol, 2004 Jan, 48(1), 57 - 61
Evaluation of OxoPlate for real-time assessment of antibacterial activities; Hutter B et al.; We have evaluated the OxoPlate OP96F for its usefulness in the characterization of antibacterial compounds . Each well of the plate carries two different fluorescent dyes, an oxygen-sensitive indicator dye and a reference dye . Fluorescence intensity is proportional to the amount of dissolved oxygen in the medium . Growth of bacterial cells is measured indirectly via the gradual depletion of oxygen . In this study, Bacillus subtilis was treated with 14 different antibacterials of various modes of action . The OxoPlate was not only useful to deduce MIC values directly from the oxygen-depletion curves, but was also able to discriminate bactericidal from bacteriostatic compounds . Oxygen levels of cultures treated with bactericidal compounds dropped initially and then increased after the cells had died . In contrast, oxygen levels remained low when cultures were treated with bacteriostatic compounds . The system described herein gives valuable insight into the kinetics of growth inhibition and killing, and we conclude that the OxoPlate is highly useful for the initial characterization of antibacterials.

J Antibiot (Tokyo), 2003 Dec, 56(12), 1045 - 52
Isolation and characterization of inhibitors of the essential histidine kinase, YycG in Bacillus subtilis and Staphylococcus aureus; Watanabe T et al.; The set of sensor kinase YycG and response regulator YycF is the only essential two-component system (TCS) in Bacillus subtilis and Staphylococcus aureus . We have developed a screening method for antibacterial agents that inhibit YycG, the essential histidine kinase (HK) . To increase screening sensitivity, a temperature-sensitive yycF mutant (CNM2000) of B . subtilis with super-sensitivity to HK inhibitors was constructed, which was used for the screening of acetone extracts from 4000 microbes . A total of 11 samples showed higher sensitivity to CNM2000 than to wild-type parent 168, and seven of those were characterized to be potent inhibitors against autophosphorylation of YycG . One sample compound was purified and identified as aranorosinol B, a known antibacterial agent against Gram-positive bacteria including B . subtilis and S . aureus . Aranorosinol B inhibited YycG from both B . subtilis and S . aureus with a half-maximum inhibitory concentration (IC50) of 223 and 211 microM, respectively.

J Am Chem Soc, 2004 Mar 17, 126(10), 3091 - 6
The biosynthesis of the thiazole phosphate moiety of thiamin (vitamin B1): the early steps catalyzed by thiazole synthase; Dorrestein PC et al.; Thiazole synthase (ThiG) catalyzes an Amadori-type rearrangement of 1-deoxy-d-xylulose-5-phosphate (DXP) via an imine intermediate . In support of this, we have demonstrated enzyme-catalyzed exchange of the C2 carbonyl of DXP . Borohydride reduction of the enzyme DXP imine followed by top-down mass spectrometric analysis localized the imine to lysine 96 . On the basis of these observations, a new mechanism for the biosynthesis of the thiazole phosphate moiety of thiamin pyrophosphate in Bacillus subtilis is proposed . This mechanism involves the generation of a ketone at C3 of DXP by an Amadori-type rearrangement of the imine followed by nucleophillic addition of the sulfur carrier protein (ThiS-thiocarboxylate) to this carbonyl group.

Mol Microbiol, 2004 Mar, 51(6), 1629 - 40
The bacterial condensin/cohesin-like protein complex acts in DNA repair and regulation of gene expression; Dervyn E et al.; Structural maintenance of chromosome (SMC) and the SMC-interacting kleisin protein families have key functions in the chromosome organization of most organisms . Here, we report that the Bacillus subtilis kleisin, ScpA, can form a ternary complex with the SMC and ScpB proteins in a yeast tri-hybrid assay, supporting the notion of a bacterial cohesin/condensin-like complex . Furthermore, ScpA interacts in two-hybrid assays with the AddAB complex, essential for recombinational repair, with DegS, a two-component sensor kinase, as well as with other potential transcription regulators . Point mutations in scpA allowing growth under conditions not permissive for the spcA null and not affecting chromosome condensation were isolated . Among these mutations, some affected DNA repair and gene regulation, thus separating ScpA functions in these two pathways from its functions in chromosome condensation and segregation . Some separation-of-function mutations in scpA caused a deficiency in the repair of mitomycin C DNA lesions that was suppressed by increasing the intracellular dosage of the interacting AddAB complex . Another mutation in scpA deregulated the expression of genes encoding degradative enzymes that are known to be controlled by the interacting DegS kinase . We propose that the SMC-ScpA-ScpB complex could: (i) recruit the AddAB helicase/nuclease to act in post-replicative repair; and (ii) form a complex with the DegS sensor kinase that inhibits its kinase activity . Moreover, our results indicate that the role of cohesin and condensin complexes in DNA repair and gene regulation is evolutionary conserved.

Appl Biochem Biotechnol, 2004 Mar, 112(3), 163 - 72
Biosurfactant production by Bacillus subtilis using cassava-processing effluent; Nitschke M et al.; A cassava flour-processing effluent (manipueira) was evaluated as a substrate for surfactant production by two Bacillus subtilis strains . B . subtilis ATCC 21332 reduced the surface tension of the medium to 25.9 mN/m, producing a crude biosurfactant concentration of 2.2 g/L . The wild-type strain, B . subtilis LB5a, reduced the surface tension of the medium to 26.6 mN/m, giving a crude biosurfactant concentration of 3.0 g/L . A decrease in surfactant concentration observed for B . subtilis ATCC 21332 seemed to be related to an increase in protease activity . The biosurfactant produced on cassava effluent medium by B . subtilis LB5a was similar to surfactin.

Chemosphere, 2004 Apr, 55(4), 631 - 9
Identification and molecular characterization of a Bacillus subtilis IS13 strain involved in the biodegradation of 4,5,6-trichloroguaiacol; Andretta CW et al.; 4,5,6-Trichloroguaiacol (4,5,6-TCG) is a recalcitrant organochlorine compound produced during pulp bleaching and a potential environmental hazard in paper mill effluents . We report here the identification by biochemical tests and molecular biological analysis, using 16S ribotyping, of a 4,5,6-TCG-degrading bacterium, identified as a strain of Bacillus subtilis that is most closely related according to the phylogenetic analysis to B . subtilis strain Lactipan (alignment score 99%) . Biodegradation of 4,5,6-TCG by this organism in a mineral salts medium was shown to occur only when the inoculum was composed of cells in the stationary phase of growth and to be accelerated by an additional carbon source, such as glucose, sucrose, glycerol or molasses . An additional nitrogen source (as ammonium sulfate) did not affect the rate of 4,5,6-TGC removal . No plasmids were detected in the bacterial cells . This is the first strain of B . subtilis which degrades chlorophenols and shows that 4,5,6-TCG is not degraded by cometabolism and that the gene encoding this characteristic is probably located on the chromosome . The lack of requirement for additional nitrogen source, the ability to enhance biodegradation by adding cheap carbon sources such as molasses, and the fact the trait is likely to be stable since it is encoded on the cell chromosome, are all characteristics that make the organism an attractive possibility for treatment of wastes and environments polluted with organochlorine compounds.

J Biotechnol, 2004 Mar 18, 108(3), 207 - 17
An efficient heat-inducible Bacillus subtilis bacteriophage 105 expression and secretion system for the production of the Streptomyces clavuligerus beta-lactamase inhibitory protein (BLIP); Liu HB et al.; The Streptomyces clavuligerus beta-lactamase inhibitory protein (BLIP) has been shown to be a potent inhibitor of class A beta-lactamases including the Escherichia coli TEM-1 beta-lactamase (Ki = 0.6 nM) . A heat-inducible BLIP expression system was constructed based on a derivative of Bacillus subtilis phage phi105 . The recombinant BLIP produced by this system was secreted to the culture medium, purified to homogeneity, and fully active . We have shown that the signal peptide of BLIP functions well in B . subtilis to secrete BLIP out of the cells, which facilitates purification . The absence of a His-tag also avoids the activity and structure of BLIP being altered . An unprecedented high yield of recoverable protein in culture supernatant (3.6mg of >95% pure BLIP/l culture) was achieved by a simple purification protocol . We have developed an efficient production process in which the culture time before heat-induction was 3-4h and the culture supernatant could be collected 5h after induction . This total time of 8-9h is considered to be very short compared to that of the native S . clavuligerus culturing (60-70h) . We achieved a very efficient BLIP production rate of 0.8-0.9mg/l/h . Heterologous gene expression was tightly controlled and no production of BLIP was observed before heat-induction, suggesting that cell density can be further increased to improve enzyme yield.

Plasmid, 2004 Mar, 51(2), 108 - 15
Construction of a shuttle vector for the overexpression of recombinant proteins in Actinobacillus succinogenes; Kim P et al.; To express foreign proteins in Actinobacillus succinogenes, a shuttle vector was constructed based on the Actinobacillus pleuropneumoniae-Escherichia coli shuttle vector, pGZRS-19 . We demonstrated that A . succinogenes is transformed by electroporation at reasonably high efficiency, that pGZRS-19 is stable in A . succinogenes, and that the ampicillin resistance gene carried by pGZRS-19 is expressed in A . succinogenes . Three steps were then required to develop our A . succinogenes-E . coli shuttle vector . (i) The constitutively expressed A . succinogenes phosphoenolpyruvate carboxykinase gene, pckA, was cloned and sequenced . (ii) Its promoter region and ribosome-binding site were subcloned into pGZRS-19 . (iii) Finally, the ColE1 origin of replication was added to the vector to increase its stability in E . coli . High levels of A . succinogenes phosphoenolpyruvate carboxykinase, E . coli NADP-dependent malic enzyme, and Bacillus subtilis NAD-dependent malic enzyme activities detected in recombinant A . succinogenes strains confirmed that A . succinogenes and foreign proteins could be expressed in A . succinogenes under control of the A . succinogenes pckA promoter carried by pLGZ920 . A . succinogenes is sensitive to chloramphenicol and tetracycline . Although not expressed from their own promoters, the Tn9 chloramphenicol and the Tn10 tetracycline resistance genes are expressed under control of the pckA promoter, and they can be used as additional selection markers in A . succinogenes.

J Mol Biol, 2004 Mar 12, 337(1), 31 - 47
Exploring the regulation of tRNA distribution on the genomic scale; Dittmar KA et al.; Though up to 20% of the total RNA in bacterial cells is tRNA, the regulation of tRNA distribution on the genomic level remains unclear . tRNA distribution is governed by four processes: transcription, processing of precursor tRNA, degradation of precursor tRNA and degradation of mature tRNA . To elucidate the relationship between these processes in the regulation of tRNA production, the relative tRNA distribution was measured using a microarray specifically designed for tRNA . We developed a procedure that selectively labels 3'-CCA-containing RNAs with the fluorophores Cy3 or Cy5 . The labeled tRNAs were then hybridized to microarrays printed with complementary DNA probes . The regulation of tRNA distribution in Bacillus subtilis was explored for a wild-type strain and a mutant strain with significantly decreased levels of RNase P, the enzyme required for the 5' maturation of all tRNA . The strains were either grown under a variety of conditions at doubling times ranging from 0.1 to 2.2 doublings per hour to investigate growth-related changes in the tRNA abundance or treated with the transcriptional inhibitor rifampicin to analyze mature tRNA degradation . Our results confirm that transcription and processing contribute significantly to the distribution of the 35 tRNA species in B.subtilis, and suggest a role for the degradation of precursor tRNA . Mature tRNA degradation occurs with little specificity for individual tRNA species and on the hour time-scale, indicating that degradation of mature tRNA plays only a minor role in the regulation of tRNA distribution . Aside from transcription, the final tRNA distribution appears to be derived from a balance between processing and precursor degradation activities.

Cell Mol Life Sci, 2004 Feb, 61(4), 387 - 92
Protein structure to function: insights from computation; Ringe D et al.; Computation plays an important role in functional genomics . THEMATICS is a computational method that predicts chemical and electrostatic properties of residues in enzymes and utilizes information contained in those predictions to identify active sites . The only input required is the three-dimensional structure of the query protein . The identification of residues involved in catalysis and in recognition is discussed . The two serine proteases Kex2 from Saccharomyces cerevisiae and subtilisin from Bacillus subtilis are used as examples to illustrate how the method finds the catalytic residues for both enzymes . In addition, Kex2 is specific for dibasic sites and THEMATICS finds the recognition residues for both the S1 and S2 sites of Kex2 . In contrast, no such recognition sites are found for the non-specific enzyme subtilisin . The ability to identify sites that govern recognition opens the door to better understanding of specificity and to the design of highly specific inhibitors.

J Bacteriol, 2004 Mar, 186(6), 1785 - 92
Roles of genes 44, 50, and 51 in regulating gene expression and host takeover during infection of Bacillus subtilis by bacteriophage SPO1; Sampath A et al.; We show that the products of SPO1 genes 44, 50, and 51 are required for the normal transition from early to middle gene expression during infection of Bacillus subtilis by bacteriophage SPO1; that they are also required for control of the shutoff of host DNA, RNA, and protein synthesis; and that their effects on host shutoff could be accounted for by their effects on the regulation of gene expression . These three gene products had four distinguishable effects in regulating SPO1 gene expression: (i) gp44-50-51 acted to restrain expression of all SPO1 genes tested, (ii) gp44 and/or gp50-51 caused additional specific repression of immediate-early genes, (iii) gp44 and/or gp50-51 stimulated expression of middle genes, and (iv) gp44 and/or gp50-51 stimulated expression of some delayed-early genes . Shutoff of immediate-early gene expression also required the activity of gp28, the middle-gene-specific sigma factor . Shutoff of host RNA and protein synthesis was accelerated by either the 44- single mutant or the 50(-)51(-) double mutant and more so by the 44(-)50(-)51(-) triple mutant . Shutoff of host DNA synthesis was accelerated by the mutants early in infection but delayed by the 44(-)50(-)51(-) triple mutant at later times . Although gp50 is a very small protein, consisting almost entirely of an apparent membrane-spanning domain, it contributed significantly to each activity tested . We identify SPO1 genes 41 to 51 and 53 to 60 as immediate-early genes; genes 27, 28, and 37 to 40 as delayed-early genes; and gene 52 as a middle gene.

J Bacteriol, 2004 Mar, 186(6), 1694 - 704
Mutational analysis of the signal-sensing domain of ResE histidine kinase from Bacillus subtilis; Baruah A et al.; The Bacillus subtilis ResD-ResE two-component regulatory system activates genes involved in nitrate respiration in response to oxygen limitation or nitric oxide (NO) . The sensor kinase ResE activates the response regulator ResD through phosphorylation, which then binds to the regulatory region of genes involved in anaerobiosis to activate their transcription . ResE is composed of an N-terminal signal input domain and a C-terminal catalytic domain . The N-terminal domain contains two transmembrane subdomains and a large extracytoplasmic loop . It also has a cytoplasmic PAS subdomain between the HAMP linker and C-terminal kinase domain . In an attempt to identify the signal-sensing subdomain of ResE, a series of deletions and amino acid substitutions were generated in the N-terminal domain . The results indicated that cytoplasmic ResE lacking the transmembrane segments and the extracytoplasmic loop retains the ability to sense oxygen limitation and NO, which leads to transcriptional activation of ResDE-dependent genes . This activity was eliminated by the deletion of the PAS subdomain, demonstrating that the PAS subdomain participates in signal reception . The study also raised the possibility that the extracytoplasmic region may serve as a second signal-sensing subdomain . This suggests that the extracytoplasmic region could contribute to amplification of ResE activity leading to the robust activation of genes required for anaerobic metabolism in B . subtilis.

J Bacteriol, 2004 Mar, 186(6), 1683 - 93
Thermoprotection of Bacillus subtilis by exogenously provided glycine betaine and structurally related compatible solutes: involvement of Opu transporters; Holtmann G et al.; Bacillus subtilis possesses five osmotically regulated transporters (Opu) for the uptake of various compatible solutes for osmoprotective purposes . We have now found that compatible solutes also function as thermoprotectants for B . subtilis . Low concentrations of glycine betaine enhanced the growth of the B . subtilis wild-type strain JH642 at its maximal growth temperature (52 degrees C) but did not allow an extension of the upper growth limit . A similar enhancement in the growth of B . subtilis was also observed by the addition of several other compatible solutes that are structurally related to glycine betaine or by the addition of proline . Each of these compatible solutes was taken up under heat stress by the cell through the same Opu transporters that are used for their acquisition under osmostress conditions . Northern blot analysis revealed a moderate increase in transcription of the structural genes for each of the Opu transport systems in cells that were propagated at 52 degrees C . In contrast, the uptake level of radiolabeled glycine betaine was very low under high-temperature growth conditions but nevertheless allowed the buildup of an intracellular glycine betaine pool comparable to that found in cells grown at 37 degrees C in the absence of salt stress . Although exogenously added glutamate has only a limited osmoprotective potential for B . subtilis, it was found to be a very effective thermoprotectant . Collectively, our data demonstrate thermoprotection by a variety of compatible solutes in B . subtilis, thus ascribing a new physiological function for this class of compounds in this microorganism and broadening the physiological role of the known osmoprotectant uptake systems (Opu).

J Agric Food Chem, 2004 Mar 10, 52(5), 1072 - 6
Antibacterial activity of akyl gallates against Bacillus subtilis; Kubo I et al.; The antibacterial activity of a series of alkyl gallates (3,4,5-trihydroxybenzoates) against Gram-positive bacteria was tested using a broth dilution method . All of the Gram-positive bacteria tested were susceptible to alkyl gallates, and this activity was found to correlate with the alkyl chain length . The antibacterial activity of alkyl gallates against Bacillus subtilis was a parabolic function of their lipophilicity and maximized with alkyl chain length between C(8) and C(11) . Notably, alkyl gallates were found to be bactericidal against B . subtilis ATCC 9372, but this activity was significantly affected by the endospore formation in the culture . The antibacterial activity of alkyl gallates likely comes at least in part from their ability to inhibit the membrane respiratory chain but is not due to the prooxidant action.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 555 - 7 Epub 2004 Feb 25.
Crystallization and preliminary X-ray diffraction study of two complexes of a TAXI-type xylanase inhibitor with glycoside hydrolase family 11 xylanases from Aspergillus niger and Bacillus subtilis; Sansen S et al.; Endo-beta-1,4-xylanases hydrolyze arabinoxylan, a major constituent of cereal cell walls, and are nowadays widely used in biotechnological processes . Purified complexes of family 11 xylanases from Aspergillus niger and Bacillus subtilis with TAXI I, a TAXI-type xylanase inhibitor from Triticum aestivum L., were prepared . In both cases the complex was crystallized using the hanging-drop vapour-diffusion method . The needle-like crystals of TAXI I in complex with A . niger xylanase belong to the trigonal space group P3(1) or P3(2), with unit-cell parameters a = b = 88.43, c = 128.99 A, and diffract to 1.8 A resolution . TAXI I in complex with B . subtilis xylanase crystallizes in the monoclinic space group C2, with a = 107.89, b = 95.33, c = 66.31 A, beta = 122.24 degrees . Complete data sets were collected for both crystal types using synchrotron radiation.

Microbiology, 2004 Mar, 150(Pt 3), 591 - 9
Interaction of Bacillus subtilis extracytoplasmic function (ECF) sigma factors with the N-terminal regions of their potential anti-sigma factors; Yoshimura M et al.; Extracytoplasmic function (ECF) sigma factors constitute a diverse family of proteins, within the class of the sigma 70 subunit of RNA polymerase . Most members of the family studied to date are known to regulate gene expression in response to stress conditions . The Bacillus subtilis genome encodes at least 17 distinct sigma factors, seven of which are members of the ECF subfamily . Among these, five sigma factors, namely SigV, SigW, SigX, SigY and SigM, are encoded by the first genes of the cognate sigma operons . Disruption or repressed expression of the downstream gene(s) resulted in transcriptional activation of the cognate sigma operon . Moreover, in vivo protein-protein interaction analyses by yeast two-hybrid experiments indicated that these immediate downstream gene products bind the cognate ECF sigma factor, suggesting that they function as anti-sigma factors by capturing sigma factor on the inner surface of the cytoplasmic membrane . Interaction with other sigma factors was not observed . The results presented here also show that these anti-sigma factors interact with ECF sigma factors through their N-terminal region, implying that the N-terminal domain resides inside the cytoplasmic membrane.

Microbiology, 2004 Mar, 150(Pt 3), 581 - 9
Effect of loss of CheC and other adaptational proteins on chemotactic behaviour in Bacillus subtilis; Saulmon MM et al.; Bacillus subtilis has a more complex mechanism of chemotaxis than does the paradigm organism, Escherichia coli . In order to understand better the role of the novel chemotaxis proteins--CheC, CheD and CheV--mutants in which increasing numbers of the corresponding genes had been deleted were studied as tethered cells and their biases and sometimes durations of counterclockwise (CCW) and clockwise (CW) flagellar rotations in response to addition and removal of the attractant asparagine were observed . The cheC mutant was found to have considerably reduced switching frequency (that is, prolonged CCW and CW rotations) without a significantly different prestimulus CCW bias, compared with wild-type . This result may indicate that in absence of CheC the switch might be in a conformation less resembling the transition state than in presence of CheC . Conversely, the cheB (methylesterase) mutant showed considerably increased switching frequency without affecting CCW bias, compared with wild-type . Removal of all known adaptation systems--the methylation, CheC and CheV systems--resulted in a mutant (cheRBCDV) that still retained some adaptation following the addition of attractant.

Microbiology, 2004 Mar, 150(Pt 3), 571 - 80
The fifth gene of the iol operon of Bacillus subtilis, iolE, encodes 2-keto-myo-inositol dehydratase; Yoshida K et al.; The myo-inositol catabolism pathway of Bacillus subtilis has not been fully characterized but was proposed to involve step-wise multiple reactions that finally yielded acetyl-CoA and dihydroxyacetone phosphate . It is known that the iolABCDEFGHIJ operon is responsible for the catabolism of inositol . IolG catalyses the first step of myo-inositol catabolism, the dehydrogenation of myo-inositol, producing 2-keto-myo-inositol (inosose) . The second step was thought to be the dehydration of inosose . Genetic and biochemical analyses of the iol genes led to the identification of iolE, encoding the enzyme for the second step of inositol catabolism, inosose dehydratase . The reaction product of inosose dehydratase was identified as D-2,3-diketo-4-deoxy-epi-inositol.

Pac Symp Biocomput . 2004;:276-87.
Predicting the operon structure of Bacillus subtilis using operon length, intergene distance, and gene expression information; De Hoon MJ et al.; We predict the operon structure of the Bacillus subtilis genome using the average operon length, the distance between genes in base pairs, and the similarity in gene expression measured in time course and gene disruptant experiments . By expressing the operon prediction for each method as a Bayesian probability, we are able to combine the four prediction methods into a Bayesian classifier in a statistically rigorous manner . The discriminant value for the Bayesian classifier can be chosen by considering the associated cost of misclassifying an operon or a non-operon gene pair . For equal costs, an overall accuracy of 88.7% was found in a leave-one-out analysis for the joint Bayesian classifier, whereas the individual information sources yielded accuracies of 58.1%, 83.1%, 77.3%, and 71.8% respectively . The predicted operon structure based on the joint Bayesian classifier is available from the DBTBS database .

Res Microbiol, 2004 Mar, 155(2), 113 - 27
A new form of bacterial movement, dragging of multicellular aggregate structures over solid surfaces, is powered by macrofiber supercoiling; Mendelson NH et al.; Growing Bacillus subtilis macrofibers use twist and supercoiling to: power their own self-assembly, join fibers together into multiclonal aggregates, move themselves over solid surfaces, and to drag other structures (cargo) over solid surfaces . The dragging of multiclonal aggregates attached to the ends of growing macrofibers is analyzed here . The linkage between fibers and cargo arose naturally in macrofiber cultures . Dragging was triggered when growing macrofibers became linked to cargo at both of their ends . Such macrofibers supercoiled, reduced their length, and dragged the cargo toward one another . In parallel experiments immobile wire was used in place of cargo at one end of macrofibers that were linked to cargo at the other . The cargo was dragged toward the wire when these fibers supercoiled . To estimate the force required for dragging we determined the dimensions of the cargo, the buoyant density of macrofibers in the growth medium where dragging occurred, the rate and distance over which the aggregate structures were dragged, and the viscosity of the growth medium . Friction resulting from contact with the solid surface over which the structures were dragged was estimated using the measured parameters . The results indicate that the supercoiling tension required to overcome limiting friction must have been approximately 10 nN, while that needed to overcome fluid drag was of the order of 1 nN . These values suggest that only a small fraction of the total power available from macrofiber supercoiling was needed to drive this new form of multicellular bacterial movement.

Res Microbiol, 2004 Mar, 155(2), 80 - 6
The metNPQ operon of Bacillus subtilis encodes an ABC permease transporting methionine sulfoxide, D- and L-methionine; Hullo MF et al.; The Bacillus subtilis yusCBA operon, which encodes an ABC-type transporter, contains an S-box motif in its promoter region . We showed that the expression of these genes is repressed via the S-box system when methionine is available . The YusCB proteins are involved in the transport of both d- and l-methionine but also methionine sulfoxide . A yusCB mutant is unable to grow in the presence of 5 microM l-methionine or 100 microM methionine sulfoxide, while it grows similarly to the wild type with 100 microM l-methionine and 1 mM methionine sulfoxide . Other uptake systems are therefore present for these two compounds . In contrast, the Yus ABC transporter corresponds to the sole d-methionine uptake system . We propose to rename yusC, yusB and yusA as metN, metP and metQ, respectively.

FEMS Microbiol Lett, 2004 Feb 16, 231(2), 211 - 7
Modulation of the K+ efflux activity of Bacillus subtilis YhaU by YhaT and the C-terminal region of YhaS; Fujisawa M et al.; The cation/proton antiporter 2 (CPA2) family is a large family of cation transporters and putative channel proteins that are found in bacteria, archaea as well as eukaryotes . Consistent with a K+ efflux capacity that is found in several other CPA2 proteins, it is shown here that the YhaU protein of Bacillus subtilis greatly increased the concentration of K+ required for growth of a K+ uptake-defective mutant of Escherichia coli . No YhaU-dependent K+(Na+)/H+ antiport activity was found in membrane vesicles . Two genes, yhaS and yhaT, are located upstream of yhaU and form an apparent operon with it . The YhaS protein has no reported homologues while the YhaT protein has sequence similarity to a sub-domain of KTN proteins that are associated with potassium-translocating channels and transporters . YhaT and the C-terminal region of YhaS were shown to modulate the K+ transport capacities of YhaU in complementation experiments . Expression studies, conducted by monitoring the beta-galactosidase levels in pMutin-disrupted mutants of the yhaU locus, indicated that yhaU is strongly induced by alkaline pH- plus salt-induced stress and that there are additional sodium-specific responses of yhaS and yhaT.

Astrobiology, 2003 Winter, 3(4), 709 - 17
Spore UV and acceleration resistance of endolithic Bacillus pumilus and Bacillus subtilis isolates obtained from Sonoran desert basalt: implications for lithopanspermia; Benardini JN et al.; Bacterial spores have been used as model systems for studying the theory of interplanetary transport of life by natural processes such as asteroidal or cometary impacts (i.e., lithopanspermia) . Because current spallation theory predicts that near-surface rocks are ideal candidates for planetary ejection and surface basalts are widely distributed throughout the rocky planets, we isolated spore-forming bacteria from the interior of near-subsurface basalt rocks collected in the Sonoran desert near Tucson, Arizona . Spores were found to inhabit basalt at very low concentrations (</=28 colony-forming units/g) in these samples . Six isolates identified as being most closely related to Bacillus pumilus and one Bacillus subtilis isolate were recovered from near-subsurface basalt samples . Populations of purified spores prepared from the isolated strains were subjected to 254-nm UV and ballistics tests in order to assess their resistance to UV radiation and to extreme acceleration shock, two proposed lethal factors for spores during interplanetary transfer . Specific natural isolates of B . pumilus were found to be substantially more resistant to UV and extreme acceleration than were reference laboratory strains of B . subtilis, the benchmark organism, suggesting that spores of environmental B . pumilus isolates may be more likely to survive the rigors of interplanetary transfer.

Anal Chem, 2004 Mar 1, 76(5), 1419 - 28
Two-dimensional separation method for analysis of Bacillus subtilis metabolites via hyphenation of micro-liquid chromatography and capillary electrophoresis; Jia L et al.; A novel two-dimensional separation method, which hyphenated chromatography and electrophoresis, was developed for analysis of Bacillus subtilis metabolites . Micro-liquid chromatography (LC) with a monolithic silica-ODS column was used as the first dimension, from which the effluent fractions were further analyzed by capillary electrophoresis (CE) acting as the second dimension . Concentration strategies, namely, dynamic pH junction and sweeping, were selectively employed to interface the two dimensions, which proved to be beneficial for the detection of metabolites . For system evaluation, an artificial sample containing 54 standard metabolites was separated according to their hydrophobicity by micro-LC with gradient mode . The early-eluting fractions were separated by capillary zone electrophoresis in combination with dynamic pH junction, while the late-eluting fractions were separated by sweeping micellar electrokinetic chromatography . The middle fractions were analyzed by both modes of CE . Under the optimum conditions, all the components in the artificial sample could be well resolved . The method was applied to profile B . subtilis metabolites . Some crucial metabolites were identified . This method provided great potential for resolving complex biological samples containing compounds having different characteristics.

J Org Chem, 2004 Mar 5, 69(5), 1513 - 23
Application of the synthetic aminosugars for glycodiversification: synthesis and antimicrobial studies of pyranmycin; Elchert B et al.; A divergent approach was employed for the synthesis of aminosugars, from which a novel library of aminoglycoside antibiotics (pyranmycins) was synthesized . Pyranmycins have comparable antibacterial activity as neomycin, a clinically used aminoglycoside antibiotic, against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Mycobacterium smegmatis . In addition, pyranmycins, like streptomycin, are bacteriocidal while isoniazid (INH) is bacteriostatic . Therefore, pyranmycins may provide new therapeutic options in the treatment against tuberculosis . Several members of pyranmycins also manifest modest anti-Tat and anti-Rev activities, which may aid in the development of new anti-HIV agents . Although the antibacterial activity of pyranmycins against aminoglycoside resistant bacteria is less than expected, the synthetic methodologies of utilizing a library of aminosugars can be a model for future studies of glycodiversification or glycorandomization.

J Biol Chem, 2004 May 14, 279(20), 21121 - 7 Epub 2004 Feb 25.
Differential activation of the NF-kappaB-like factors Relish and Dif in Drosophila melanogaster by fungi and Gram-positive bacteria; Hedengren-Olcott M et al.; The current model of immune activation in Drosophila melanogaster suggests that fungi and Gram-positive (G(+)) bacteria activate the Toll/Dif pathway and that Gram-negative (G(-)) bacteria activate the Imd/Relish pathway . To test this model, we examined the response of Relish and Dif (Dorsal-related immunity factor) mutants to challenge by various fungi and G(+) and G(-) bacteria . In Relish mutants, the Cecropin A gene was induced by the G(+) bacteria Micrococcus luteus and Staphylococcus aureus, but not by other G(+) or G(-) bacteria . This Relish-independent Cecropin A induction was blocked in Dif/Relish double mutant flies . Induction of the Cecropin A1 gene by M . luteus required Relish, whereas induction of the Cecropin A2 gene required Dif . Intact peptidoglycan (PG) was necessary for this differential induction of Cecropin A . PG extracted from M . luteus induced Cecropin A in Relish mutants, whereas PGs from the G(+) bacteria Bacillus megaterium and Bacillus subtilis did not, suggesting that the Drosophila immune system can distinguish PGs from various G(+) bacteria . Various fungi stimulated antimicrobial peptides through at least two different pathways requiring Relish and/or Dif . Induction of Attacin A by Geotrichum candidum required Relish, whereas activation by Beauvaria bassiana required Dif, suggesting that the Drosophila immune system can distinguish between at least these two fungi . We conclude that the Drosophila immune system is more complex than the current model . We propose a new model to account for this immune system complexity, incorporating distinct pattern recognition receptors of the Drosophila immune system, which can distinguish between various fungi and G(+) bacteria, thereby leading to selective induction of antimicrobial peptides via differential activation of Relish and Dif.

Comp Biochem Physiol C Toxicol Pharmacol, 2004 Jan, 137(1), 65 - 74
Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02; Ko JH et al.; Two fibrinolytic enzymes (QK-1 and QK-2) purified from the supernatant of Bacillus subtilis QK02 culture broth had molecular masses of 42,000 Da and 28,000 Da, respectively . The first 20 amino acids of the N-terminal sequence are AQSVPYGISQ IKAPALHSQG . The deduced protein sequence and its restriction enzyme map of the enzyme QK-2 are different from those of other proteases . The enzyme QK-2 digested not only fibrin but also a subtilisin substrate, and PMSF inhibited its fibrinolytic and amidolytic activities completely; while QK-1 hydrolyzed fibrin and a plasmin substrate, and PMSF as well as aprotinin inhibited its fibrinolytic activity . These results indicated QK-1 was a plasmin-like serine protease and QK-2 a subtilisin family serine protease . Therefore, these enzymes were designated subtilisin QK . The sequence of a DNA fragment encoding subtilisin QK contained an open reading frame of 1149 base pairs encoding 106 amino acids for signal peptide and 257 amino acids for subtilisin QK, which is highly similar with that of a fibrinolytic enzyme, subtilisin NAT (identities 96.8%) . Asp32, His64 and Ser221 in the amino acid sequence deduced from the QK gene are identical to the active site of nattokinase (NK) produced by B . subtilis natto.

Protein Eng, 2003 Dec, 16(12), 1055 - 61
Functional properties of subunit interactions in human cytidine deaminase; Vincenzetti S et al.; An intersubunit interactions study related to the active site has been performed on the wild-type cytidine deaminase (CDA) and on the mutant enzyme F137W/W113F . F137 is the homologous to the Bacillus subtilis CDA F125 involved in the subunit interactions . In the presence of SDS, wild-type human CDA dissociates into enzymatically inactive monomers without intermediate forms via a non-cooperative transition . Extensive dialysis or dilution of the inactivated monomers restores completely the activity . Circular dichroism measurements show that the secondary/tertiary structure organization of each subunit is unaffected by the SDS concentration, while the mutation Phe/Trp causes weakening in quaternary structure . The presence of the strong human CDA competitive inhibitor 5-fluorozebularine disfavours dissociation of the tetramer into subunits in the wild-type CDA, but not in mutant enzyme F137W/W113F . The absence of tyrosine fluorescence and the much higher quantum yield of the double mutant protein spectrum suggest the occurrence of an energy transfer effect between the protein subunits . This assumption is confirmed by the crystallographic studies on B.subtilis in which it is shown that three different subunits concur with the formation of each of the four active sites and that F125, homologous to the human CDA F137, is located at the interface between two different subunits contributing to the formation of active site.

Mol Microbiol, 2004 Mar, 51(5), 1321 - 32
MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus; Figge RM et al.; The bacterial actin homologue, MreB, is required for the maintenance of a rod-shaped cell and has been shown to form spirals that traverse along the longitudinal axis of Bacillus subtilis and Escherichia coli cells . The depletion of MreB in Caulobacter crescentus resulted in lemon-shaped cells that possessed defects in the integrity of the cell wall . MreB localization appeared as bands or spirals that encircled the cell along its entire length and switched to a mid-cell location at a time that coincided with the initiation of cell division . The formation of smaller MreB spirals or bands at the mid-cell was dependent on the presence on the cytokinetic protein, FtsZ . Penicillin-binding protein 2 (PBP2) also formed band-like structures perpendicular to the cell periphery that resembled, and depended upon, MreB localization . PBP2 co-immunoprecipitated with several other penicillin-binding proteins, suggesting that these proteins are in association in Caulobacter cells . We hypothesize that MreB filaments function as a cytoskeleton that serves as an organizer or tracking device for the PBP2-peptidoglycan biosynthesis complex.

Mol Microbiol, 2004 Mar, 51(5), 1251 - 66
Differential roles of multiple signal peptidases in the virulence of Listeria monocytogenes; Bonnemain C et al.; Most bacteria contain one type I signal peptidase (Spase I) for cleavage of signal peptides from exported and secreted proteins . Here, we identified a locus encoding three contiguous Spase I genes in the genome of Listeria monocytogenes . The deduced Sip proteins (denoted SipX, SipY and SipZ) are significantly similar to SipS and SipT, the major SPase I proteins of Bacillus subtilis (38% to 44% peptidic identity) . We studied the role of these multiple signal peptidases in bacterial pathogenicity by constructing a series of single- and double-chromosomal knock-out mutants . Inactivation of sipX did not affect intracellular multiplication of L . monocytogenes but significantly reduced bacterial virulence (approximately 100-fold) . Inactivation of sipZ impaired the secretion of phospholipase C (PC-PLC) and listeriolysin O (LLO), restricted intracellular multiplication and almost abolished virulence (LD(50) of 10(8.3)), inactivation of sipY had no detectable effects . Most importantly, a mutant expressing only SipX was impaired in intracellular survival and strongly attenuated in the mouse (LD(50) of 10(7.2)), whereas, a mutant expressing only SipZ behaved like wild-type EGD in all the assays performed . The data establish that SipX and SipZ perform distinct functions in bacterial pathogenicity and that SipZ is the major Spase I of L . monocytogenes . This work constitutes the first report on the differential role of multiple Spases I in a pathogenic bacterium and suggests a possible post-translational control mechanism of virulence factors expression.

Proc Natl Acad Sci U S A, 2004 Mar 23, 101(12), 4142 - 7 Epub 2004 Feb 23.
FamClash: a method for ranking the activity of engineered enzymes; Saraf MC et al.; This article introduces the computational procedure FamClash for analyzing incompatibilities in engineered protein hybrids by using protein family sequence data . All pairs of residue positions in the sequence alignment that conserve the property triplet of charge, volume, and hydrophobicity are first identified, and significant deviations are denoted as residue-residue clashes . This approach moves beyond earlier efforts aimed at solely classifying hybrids as functional or nonfunctional by correlating the rank ordering of these hybrids based on their activity levels . Experimental testing of this approach was performed in parallel to assess the predictive ability of FamClash . As a model system, single-crossover ITCHY (incremental truncation for the creation of hybrid enzymes) libraries were prepared from the Escherichia coli and Bacillus subtilis dihydrofolate reductases, and the activities of functional hybrids were determined . Comparisons of the predicted clash map as a function of crossover position revealed good agreement with activity data, reproducing the observed V shape and matching the location of a local peak in activity.

Proc Natl Acad Sci U S A, 2004 Mar 2, 101(9), 2747 - 51 Epub 2004 Feb 19.
Recycling of a regulatory protein by degradation of the RNA to which it binds; Deikus G et al.; When Bacillus subtilis is grown in the presence of excess tryptophan, transcription of the trp operon is regulated by binding of tryptophan-activated TRAP to trp leader RNA, which promotes transcription termination in the trp leader region . Transcriptome analysis of a B . subtilis strain lacking polynucleotide phosphorylase (PNPase; a 3'-to-5' exoribonuclease) revealed a striking overexpression of trp operon structural genes when the strain was grown in the presence of abundant tryptophan . Analysis of trp leader RNA in the PNPase(-) strain showed accumulation of a stable, TRAP-protected fragment of trp leader RNA . Loss of trp operon transcriptional regulation in the PNPase(-) strain was due to the inability of ribonucleases other than PNPase to degrade TRAP-bound leader RNA, resulting in the sequestration of limiting TRAP . Thus, in the case of the B . subtilis trp operon, specific ribonuclease degradation of RNA in an RNA-protein complex is required for recycling of an RNA-binding protein . Such a mechanism may be relevant to other systems in which limiting concentrations of an RNA-binding protein must keep pace with ongoing transcription.

J Biol Chem, 2004 Apr 30, 279(18), 19018 - 25 Epub 2004 Feb 19.
A conserved Val to Ile switch near the heme pocket of animal and bacterial nitric-oxide synthases helps determine their distinct catalytic profiles; Wang ZQ et al.; Nitric oxide (NO) release from nitric oxide synthases (NOSs) is largely dependent on the dissociation of an enzyme ferric heme-NO product complex (Fe(III)NO) . Although the NOS-like protein from Bacillus subtilis (bsNOS) generates Fe(III)NO from the reaction intermediate N-hydroxy-l-arginine (NOHA), its NO dissociation is about 20-fold slower than in mammalian NOSs . Crystal structures suggest that a conserved Val to Ile switch near the heme pocket of bsNOS might determine its kinetic profile . To test this we generated complementary mutations in the mouse inducible NOS oxygenase domain (iNOSoxy, V346I) and in bsNOS (I224V) and characterized the kinetics and extent of their NO synthesis from NOHA and their NO-binding kinetics . The mutations did not greatly alter binding of Arg, (6R)-tetrahydrobiopterin, or alter the electronic properties of the heme or various heme-ligand complexes . Stopped-flow spectroscopy was used to study heme transitions during single turnover NOHA reactions . I224V bsNOS displayed three heme transitions involving four species as typically occurs in wild-type NOS, the beginning ferrous enzyme, a ferrous-dioxy (Fe(II)O(2)) intermediate, Fe(III)NO, and an ending ferric enzyme . The rate of each transition was increased relative to wild-type bsNOS, with Fe(III)NO dissociation being 3.6 times faster . In V346I iNOSoxy we consecutively observed the beginning ferrous, Fe(II)O(2), a mixture of Fe(III)NO and ferric heme species, and ending ferric enzyme . The rate of each transition was decreased relative to wild-type iNOSoxy, with the Fe(III)NO dissociation being 3 times slower . An independent measure of NO binding kinetics confirmed that V346I iNOSoxy has slower NO binding and dissociation than wild-type . Citrulline production by both mutants was only slightly lower than wild-type enzymes, indicating good coupling . Our data suggest that a greater shielding of the heme pocket caused by the Val/Ile switch slows down NO synthesis and NO release in NOS, and thus identifies a structural basis for regulating these kinetic variables.

J Biol Chem, 2004 Apr 23, 279(17), 17397 - 403 Epub 2004 Feb 18.
The Bacillus subtilis response regulator Spo0A stimulates sigmaA-dependent transcription prior to the major energetic barrier; Seredick SD et al.; At the spoIIG promoter phosphorylated Spo0A (Spo0A approximately P) binds 0A boxes overlapping the -35 element, interacting with RNA polymerase to facilitate open complex formation . We have compared in vitro transcription from a series of heteroduplex templates containing denatured regions within the promoters . Transcription from heteroduplex templates with 12, 8, or 6 base pairs denatured was independent of Spo0A approximately P, but heteroduplexes with 4 or 2 base pairs denatured required Spo0A approximately P for maximal levels of transcription . Investigation of the thermal dependence of transcription suggested that strand separation was the primary thermodynamic barrier to transcription initiation but indicated that Spo0A approximately P does not reduce this energetic barrier . Kinetic assays revealed that Spo0A approximately P stimulated both the rate of formation of initiated complexes as well as increasing the number of complexes capable of initiating transcription . These results imply that Spo0A approximately P stimulates transcription at least in part by stabilizing the RNA polymerase-spoIIG complex until contacts between RNA polymerase and the -10 element induce strand separation.

J Biol Chem, 2004 Apr 30, 279(18), 19302 - 14 Epub 2004 Feb 19.
Structure-function analysis of PrsA reveals roles for the parvulin-like and flanking N- and C-terminal domains in protein folding and secretion in Bacillus subtilis; Vitikainen M et al.; The PrsA protein of Bacillus subtilis is an essential membrane-bound lipoprotein that is assumed to assist post-translocational folding of exported proteins and stabilize them in the compartment between the cytoplasmic membrane and cell wall . This folding activity is consistent with the homology of a segment of PrsA with parvulin-type peptidyl-prolyl cis/trans isomerases (PPIase) . In this study, molecular modeling showed that the parvulin-like region can adopt a parvulin-type fold with structurally conserved active site residues . PrsA exhibits PPIase activity in a manner dependent on the parvulin-like domain . We constructed deletion, peptide insertion, and amino acid substitution mutations and demonstrated that the parvulin-like domain as well as flanking N- and C-terminal domains are essential for in vivo PrsA function in protein secretion and growth . Surprisingly, none of the predicted active site residues of the parvulin-like domain was essential for growth and protein secretion, although several active site mutations reduced or abolished the PPIase activity or the ability of PrsA to catalyze proline-limited protein folding in vitro . Our results indicate that PrsA is a PPIase, but the essential role in vivo seems to depend on some non-PPIase activity of both the parvulin-like and flanking domains.

Nucleic Acids Res, 2004 Feb 18, 32(3), 1166 - 76 Print 2004.
Relevance of UP elements for three strong Bacillus subtilis phage phi29 promoters; Meijer WJ et al.; Various Escherichia coli promoters contain, in addition to the classical -35 and -10 hexamers, a third recognition element, named the UP element . Located upstream of the -35 box, UP elements stimulate promoter activity by forming a docking site for the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) . Accumulating genetic, biochemical and structural information has provided a detailed picture on the molecular mechanism underlying UP element-dependent promoter stimulation in E.coli . However, far less is known about functional UP elements of Bacillus subtilis promoters . Here we analyse the strong early sigma(A)-RNA polymerase-dependent promoters C2, A2c and A2b of the lytic B.subtilis phage phi29 . We demonstrate that the phage promoters contain functional UP elements although their contribution to promoter strength is very different . Moreover, we show that the UP element of the A2b promoter, being critical for its activity, is located further upstream of the -35 box than most E.coli UP elements . The importance of the UP elements for the phage promoters and how they relate to other UP elements are discussed.

J Bacteriol, 2004 Mar, 186(5), 1493 - 502
Residues required for Bacillus subtilis PhoP DNA binding or RNA polymerase interaction: alanine scanning of PhoP effector domain transactivation loop and alpha helix 3; Chen Y et al.; Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency . Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase . We describe alanine scanning mutagenesis of the PhoP alpha loop and alpha helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins . Eight residues important for DNA binding were clustered between Val202 and Arg210 . Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins . Class I proteins (PhoP(I206A), PhoP(R210A), PhoP(L209A), and PhoP(H208A)) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro . Class II proteins (PhoP(H205A) and PhoP(V204A)) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation . Members of this class had higher transcription activation in vitro than in vivo . The class III mutants, PhoP(V202A) and PhoP(D203A), had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro . Seven alanine substitutions in PhoP (PhoP(V190A), PhoP(W191A), PhoP(Y193A), PhoP(F195A), PhoP(G197A,) PhoP(T199A), and PhoP(R200A)) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200 . PhoP(W191A) and PhoP(R200A) could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both . The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short alpha helix 3, more similar to OmpR than to PhoB of Escherichia coli.

J Bacteriol, 2004 Mar, 186(5), 1381 - 7
Studies of the interaction of Escherichia coli YjeQ with the ribosome in vitro; Daigle DM et al.; Escherichia coli YjeQ represents a conserved group of bacteria-specific nucleotide-binding proteins of unknown physiological function that have been shown to be essential to the growth of E . coli and Bacillus subtilis . The protein has previously been characterized as possessing a slow steady-state GTP hydrolysis activity (8 h(-1)) (D . M . Daigle, L . Rossi, A . M . Berghuis, L . Aravind, E . V . Koonin, and E . D . Brown, Biochemistry 41: 11109-11117, 2002) . In the work reported here, YjeQ from E . coli was found to copurify with ribosomes from cell extracts . The copy number of the protein per cell was nevertheless low relative to the number of ribosomes (ratio of YjeQ copies to ribosomes, 1:200) . In vitro, recombinant YjeQ protein interacted strongly with the 30S ribosomal subunit, and the stringency of that interaction, revealed with salt washes, was highest in the presence of the nonhydrolyzable GTP analog 5'-guanylylimidodiphosphate (GMP-PNP) . Likewise, association with the 30S subunit resulted in a 160-fold stimulation of YjeQ GTPase activity, which reached a maximum with stoichiometric amounts of ribosomes . N-terminal truncation variants of YjeQ revealed that the predicted OB-fold region was essential for ribosome binding and GTPase stimulation, and they showed that an N-terminal peptide (amino acids 1 to 20 in YjeQ) was necessary for the GMP-PNP-dependent interaction of YjeQ with the 30S subunit . Taken together, these data indicate that the YjeQ protein participates in a guanine nucleotide-dependent interaction with the ribosome and implicate this conserved, essential GTPase as a novel factor in ribosome function.

J Bacteriol, 2004 Mar, 186(5), 1287 - 96
Transcriptional regulation of genes encoding arabinan-degrading enzymes in Bacillus subtilis; Raposo MP et al.; Bacillus subtilis produces hemicellulases capable of releasing arabinosyl oligomers and arabinose from plant cell walls . In this work, we characterize the transcriptional regulation of three genes encoding arabinan-degrading enzymes that are clustered with genes encoding enzymes that further catabolize arabinose . The abfA gene comprised in the metabolic operon araABDLMNPQ-abfA and the xsa gene located 23 kb downstream most probably encode alpha-L-arabinofuranosidases (EC 3.2.1.55) . Here, we show that the abnA gene, positioned immediately upstream from the metabolic operon, encodes an endo-alpha-1,5-arabinanase (EC 3.2.1.99) . Furthermore, by in vivo RNA studies, we inferred that abnA and xsa are monocistronic and are transcribed from sigma(A)-like promoters . Transcriptional fusion analysis revealed that the expression of the three arabinases is induced by arabinose and arabinan and is repressed by glucose . The levels of induction by arabinose and arabinan are higher during early postexponential growth, suggesting a temporal regulation . Moreover, the induction mechanism of these genes is mediated through negative control by the key regulator of arabinose metabolism, AraR . Thus, we analyzed AraR-DNA interactions by in vitro quantitative DNase I footprinting and in vivo analysis of single-base-pair substitutions within the promoter regions of xsa and abnA . The results indicate that transcriptional repression of the abfA and xsa genes is achieved by a tightly controlled mechanism but that the regulation of abnA is more flexible . We suggest that the expression of genes encoding extracellular degrading enzymes of arabinose-containing polysaccharides, transport systems, and intracellular enzymes involved in further catabolism is regulated by a coordinate mechanism triggered by arabinose via AraR.

J Bacteriol, 2004 Mar, 186(5), 1462 - 74
Assembly of an oxalate decarboxylase produced under sigmaK control into the Bacillus subtilis spore coat; Costa T et al.; Over 30 polypeptides are synthesized at various times during sporulation in Bacillus subtilis, and they are assembled at the surface of the developing spore to form a multilayer protein structure called the coat . The coat consists of three main layers, an amorphous undercoat close to the underlying spore cortex peptidoglycan, a lamellar inner layer, and an electron-dense striated outer layer . The product of the B . subtilis oxdD gene was previously shown to have oxalate decarboxylase activity when it was produced in Escherichia coli and to be a spore constituent . In this study, we found that OxdD specifically associates with the spore coat structure, and in this paper we describe regulation of its synthesis and assembly . We found that transcription of oxdD is induced during sporulation as a monocistronic unit under the control of sigma(K) and is negatively regulated by GerE . We also found that localization of a functional OxdD-green fluorescent protein (GFP) at the surface of the developing spore depends on the SafA morphogenetic protein, which localizes at the interface between the spore cortex and coat layers . OxdD-GFP localizes around the developing spore in a cotE mutant, which does not assemble the spore outer coat layer, but it does not persist in spores produced by the mutant . Together, the data suggest that OxdD-GFP is targeted to the interior layers of the coat . Additionally, we found that expression of a multicopy allele of oxdD resulted in production of spores with increased levels of OxdD that were able to degrade oxalate but were sensitive to lysozyme.

J Bacteriol, 2004 Mar, 186(5), 1475 - 83
Cardiolipin domains in Bacillus subtilis marburg membranes; Kawai F et al.; Recently, use of the cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) revealed CL-rich domains in the Escherichia coli membrane (E . Mileykovskaya and W . Dowhan, J . Bacteriol . 182: 1172-1175, 2000) . Staining of Bacillus subtilis cells with NAO showed that there were green fluorescence domains in the septal regions and at the poles . These fluorescence domains were scarcely detectable in exponentially growing cells of the clsA-disrupted mutant lacking detectable CL . In sporulating cells with a wild-type lipid composition, fluorescence domains were observed in the polar septa and on the engulfment and forespore membranes . Both in the clsA-disrupted mutant and in a mutant with disruptions in all three of the paralogous genes (clsA, ywjE, and ywiE) for CL synthase, these domains did not vanish but appeared later, after sporulation initiation . A red shift in the fluorescence due to stacking of two dye molecules and the lipid composition suggested that a small amount of CL was present in sporulating cells of the mutants . Mass spectrometry analyses revealed the presence of CL in these mutant cells . At a later stage during sporulation of the mutants the frequency of heat-resistant cells that could form colonies after heat treatment was lower . The frequency of sporulation of these cells at 24 h after sporulation initiation was 30 to 50% of the frequency of the wild type . These results indicate that CL-rich domains are present in the polar septal membrane and in the engulfment and forespore membranes during the sporulation phase even in a B . subtilis mutant with disruptions in all three paralogous genes, as well as in the membranes of the medial septa and at the poles during the exponential growth phase of wild-type cells . The results further suggest that the CL-rich domains in the polar septal membrane and engulfment and forespore membranes are involved in sporulation.

J Bacteriol, 2004 Mar, 186(5), 1571 - 3
Characterization of two kinases involved in thiamine pyrophosphate and pyridoxal phosphate biosynthesis in Bacillus subtilis: 4-amino-5-hydroxymethyl-2methylpyrimidine kinase and pyridoxal kinase; Park JH et al.; Two Bacillus subtilis genes encoding two proteins (currently annotated ThiD and YjbV) were overexpressed and characterized . YjbV has 4-amino-5-hydroxymethyl-2-methylpyrimidine and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate kinase activity and should be reannotated ThiD, and B . subtilis ThiD has pyridoxine, pyridoxal, and pyridoxamine kinase activity and should be reannotated PdxK.

RNA, 2004 Mar, 10(3), 482 - 92
Interaction of the Bacillus subtilis RNase P with the 30S ribosomal subunit; Barrera A et al.; Ribonuclease P (RNase P) is a ribozyme required for the 5' maturation of all tRNA . RNase P and the ribosome are the only known ribozymes conserved in all organisms . We set out to determine whether this ribonucleoprotein enzyme interacts with other cellular components, which may imply other functions for this conserved ribozyme . Incubation of the Bacillus subtilis RNase P holoenzyme with fractionated B . subtilis cellular extracts and purified ribosomal subunits results in the formation of a gel-shifted complex with the 30S ribosomal subunit at a binding affinity of approximately 40 nM in 0.1 M NH(4)Cl and 10 mM MgCl(2) . The complex does not form with the RNase P RNA alone and is disrupted by a mRNA mimic polyuridine, but is stable in the presence of high concentrations of mature tRNA . Endogenous RNase P can also be detected in the 30S ribosomal fraction . Cleavage of a pre-tRNA substrate by the RNase P holoenzyme remains the same in the presence of the 30S ribosome, but the cleavage of an artificial non-tRNA substrate is inhibited eightfold . Hydroxyl radical protection and chemical modification identify several protected residues located in a highly conserved region in the RNase P RNA . A single mutation within this region significantly reduces binding, providing strong support on the specificity of the RNase P-30S ribosome complex . Our results also suggest that the dimeric form of the RNase P is primarily involved in 30S ribosome binding . We discuss several models on a potential function of the RNase P-30S ribosome complex.

Biochemistry, 2004 Feb 24, 43(7), 1874 - 82
Molecular modeling of CPT-11 metabolism by carboxylesterases (CEs): use of pnb CE as a model; Wierdl M et al.; CPT-11 is a prodrug that is converted in vivo to the topoisomerase I poison SN-38 by carboxylesterases (CEs) . Among the CEs studied thus far, a rabbit liver CE (rCE) converts CPT-11 to SN-38 most efficiently . Despite extensive sequence homology, however, the human homologues of this protein, hCE1 and hiCE, metabolize CPT-11 with significantly lower efficiencies . To understand these differences in drug metabolism, we wanted to generate mutations at individual amino acid residues to assess the effects of these mutations on CPT-11 conversion . We identified a Bacillus subtilis protein (pnb CE) that could be used as a model for the mammalian CEs . We demonstrated that pnb CE, when expressed in Escherichia coli, metabolizes both the small esterase substrate o-NPA and the bulky prodrug CPT-11 . Furthermore, we found that the pnb CE and rCE crystal structures show an only 2.4 A rmsd variation over 400 residues of the alpha-carbon trace . Using the pnb CE model, we demonstrated that the "side-door" residues, S218 and L362, and the corresponding residues in rCE, L252 and L424, were important in CPT-11 metabolism . Furthermore, we found that at position 218 or 252 the size of the residue, and at position 362 or 424 the hydrophobicity and charge of the residue, were the predominant factors in influencing drug activation . The most significant change in CPT-11 metabolism was observed with the L424R variant rCE that converted 10-fold less CPT-11 than the wild-type protein . As a result, COS-7 cells expressing this mutant were 3-fold less sensitive to CPT-11 than COS-7 cells expressing the wild-type protein.

PLoS Biol . 2004 Feb;2(2):E49 . Epub 2004 Feb 17.
Design and diversity in bacterial chemotaxis: a comparative study in Escherichia coli and Bacillus subtilis; Rao CV et al.; Comparable processes in different species often involve homologous genes . One question is whether the network structure, in particular the feedback control structure, is also conserved . The bacterial chemotaxis pathways in E . coli and B . subtilis both regulate the same task, namely, excitation and adaptation to environmental signals . Both pathways employ many orthologous genes . Yet how these orthologs contribute to network function in each organism is different . To investigate this problem, we propose what is to our knowledge the first computational model for B . subtilis chemotaxis and compare it to previously published models for chemotaxis in E . coli . The models reveal that the core control strategy for signal processing is the same in both organisms, though in B . subtilis there are two additional feedback loops that provide an additional layer of regulation and robustness . Furthermore, the network structures are different despite the similarity of the proteins in each organism . These results demonstrate the limitations of pathway inferences based solely on homology and suggest that the control strategy is an evolutionarily conserved property.

Protein Expr Purif, 2003 Dec, 32(2), 293 - 301
Co-expression of a prophage system and a plasmid system in Bacillus subtilis; Ho KM et al.; A dual expression system for overexpressing two proteins by a single cell strain has been developed in Bacillus subtilis . This dual expression system combines the phi105MU331 prophage system and a plasmid system within a single cell . Protein expression by the prophage system is heat inducible, while that of the plasmid system is constitutive . Three candidate genes, BPN, BT, and amyE, all of Bacillus origin, were used as test models . Seven strains (BPN, BT, AMY, BS168K, MU331K, BPNK, and BTK) were constructed to investigate the influences of the prophage system and the plasmid system on each other, and to compare the efficiency of the individual expression systems with that of the dual expression system . Individually, the yield of the plasmid system is higher than that of the prophage system, which could be attributed to the constitutive nature of the expression of the plasmid system . Nonetheless, for the dual expression strains, the expression of two enzymes in a single fermentation run can reduce costs in facilities, manpower, and utilities . Fed-batch fermentation of BPNK strains confirmed the feasibility of applying this dual expression system in industrial-scale production.

Pharmazie, 2004 Jan, 59(1), 65 - 70
Proteomic signatures for daunomycin and adriamycin in Bacillus subtilis; Sender U et al.; The influence of the two anthracyclin antibiotics daunomycin and adriamycin on the proteome of Bacillus subtilis was investigated . They intercalate in the double helix causing strand breaks . Both compounds induce proteins related to DNA damage and oxidative stress as indicated by the induction of some members of the PerR and the DinR-regulon . The mild induction of some members of the HrcA- and the CtsR-regulon may indicate protein oxidation as well . Furthermore, an influence on the sigmaB-dependent general stress response was shown . These data show that the proteomic signature is a valuable experimental tool for a comprehensive evaluation of the action of different drugs.

J Appl Microbiol, 2004, 96(3), 569 - 78
Engineering of quorum-sensing systems for improved production of alkaline protease by Bacillus subtilis; Tjalsma H et al.; AIM: Engineering of Rap-Phr quorum-sensing systems of Bacillus subtilis and subsequent evaluation of the transcription of the aprE gene, encoding a major extracellular alkaline protease . METHODS AND RESULTS: Addition of synthetic Phr pentapeptides to the growth medium, or overproduction of pre-Phr peptides, slightly improved the transcription of the aprE gene in B . subtilis . Disruption of certain rap genes similarly improved the transcription of the aprE gene . The production of extracellular proteolytic enzymes was increased when the rapA mutation was combined with a degU32 (Hy) mutation for hyper-secretion . CONCLUSIONS: Certain Rap-Phr systems of B . subtilis seem to suppress extracellular AprE production . Although this may be an important feature under natural conditions, repression of AprE production by these systems is not desirable under fermentation conditions . SIGNIFICANCE AND IMPACT OF THE STUDY: Although the levels of aprE transcriptional increase in this study are moderate, engineering of Rap-Phr systems may be used to improve the yield of Bacillus strains that are used for the production of the extracellular protease AprE, or Bacillus strains that use of the aprE promoter for the production of a heterologous protein.

J Biol Chem, 2004 May 7, 279(19), 19867 - 74 Epub 2004 Feb 10.
A closed conformation of Bacillus subtilis oxalate decarboxylase OxdC provides evidence for the true identity of the active site; Just VJ et al.; Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalate to formate and carbon dioxide and utilizes dioxygen as a cofactor . By contrast, the evolutionarily related oxalate oxidase (EC 1.2.3.4) converts oxalate and dioxygen to carbon dioxide and hydrogen peroxide . Divergent free radical catalytic mechanisms have been proposed for these enzymes that involve the requirement of an active site proton donor in the decarboxylase but not the oxidase reaction . The oxidase possesses only one domain and manganese binding site per subunit, while the decarboxylase has two domains and two manganese sites per subunit . A structure of the decarboxylase together with a limited mutagenesis study has recently been interpreted as evidence that the C-terminal domain manganese binding site (site 2) is the catalytic site and that Glu-333 is the crucial proton donor (Anand, R., Dorrestein, P . C., Kinsland, C., Begley, T . P., and Ealick, S . E . (2002) Biochemistry 41, 7659-7669) . The N-terminal binding site (site 1) of this structure is solvent-exposed (open) and lacks a suitable proton donor for the decarboxylase reaction . We report a new structure of the decarboxylase that shows a loop containing a 3(10) helix near site 1 in an alternative conformation . This loop adopts a "closed" conformation forming a lid covering the entrance to site 1 . This conformational change brings Glu-162 close to the manganese ion, making it a new candidate for the crucial proton donor . Site-directed mutagenesis of equivalent residues in each domain provides evidence that Glu-162 performs this vital role and that the N-terminal domain is either the sole or the dominant catalytically active domain.

J Biochem (Tokyo), 2003 Dec, 134(6), 935 - 46
Organization and expression of the Bacillus subtilis sigY operon; Tojo S et al.; We investigated the organization and expression of the Bacillus subtilis sigY operon, the first gene of which codes for sigmaY, a member of the extracytoplasmic function (ECF) family of sigma factors . The sigY operon, comprising six genes (sigY, yxlC, D, E, F, and G), was induced upon nitrogen starvation; it was continuously transcribed from the 31st base upstream of sigY to a neighboring convergent gene, yxlH, resulting in a 4.2-kb mRNA . The expression of the sigY operon was also positively autoregulated through sigmaY, suggesting that its transcription is likely to be directed by sigmaY . Deletion analysis of the sigY promoter, which was localized by primer extension, revealed the promoter region of sigY with the "-10" and "-35" sequences of CGTC and TGAACG, respectively . The latter sequence was distinct from those recognized by sigmaW, sigmaX, and sigmaM . The sigmaY-directed transcription of sigY was under negative regulation involving YxlD . sigY disruption affected sporulation induced by nitrogen starvation, but sigY induction upon nitrogen starvation was not associated with the sporulation process . The organization and function of the sigY operon are significantly conserved in several microorganisms living in adverse living environments.

FEMS Microbiol Lett, 2004 Feb 9, 231(1), 99 - 110
Hpr (ScoC) and the phosphorelay couple cell cycle and sporulation in Bacillus subtilis; Shafikhani SH et al.; Bacillus subtilis sporulation is a developmental process that culminates in the formation of a highly resistant and persistent endospore . Inhibiting DNA synthesis prior to the completion of the final round of DNA replication blocks sporulation at an early stage . Conditions that prevent compartmentalization of gene expression, i.e . inhibition of asymmetric septum formation or chromosome partitioning, also block sporulation at an early stage . Multiple mechanisms including a RecA-dependent, a RecA-independent, and the soj-spo0J operon have been implicated in signal transduction, connecting DNA replication and chromosome partitioning to the onset of sporulation in B . subtilis . We suggest that a single mechanism involving Hpr (ScoC) and Sda couple cell cycle signaling to sporulation initiation . We show that transcription of phosphorelay sensory chain genes is adversely affected by post-exponential perturbation of the cell cycle . DNA replication arrest by chemical treatments, such as hydroxyphenylazouracil, hydroxyurea, nalidixic acid, and through genetic means using dnaA1ts and dnaB19ts temperature-sensitive mutants caused substantial down-regulation of spo0F and kinA expression and elevated the expression of spo0A and spo0H (sigH) . Despite the elevation in spo0A expression, Spo0A approximately P-dependent sinI expression was substantially down-regulated indicating that in vivo Spo0A approximately P levels may be diminished . Similar alterations in gene expression patterns were observed in an ftsA279ts mutant background, indicating that cytokinesis and sporulation may also be coupled by a similar mechanism . Loss of function mutation in hpr (scoC) restored sporulation in a dnaA1ts mutant, blocked the DNA replication arrest induction of spo0A expression and restored expression of spo0F, kinA and sinI . Moreover, hpr expression was up-regulated in response to DNA replication arrest . The increase in hpr expression required Sda . These results suggest a role for Hpr (ScoC) in mediating the coupling of cell cycle events to the onset of sporulation.

Acta Pharm, 2003 Dec, 53(4), 275 - 85
Analysis of propolis from the continental and Adriatic regions of Croatia; Kosalec I et al.; Thin-layer chromatography of ethanolic extract of propolis (EEP) from the continental and Adriatic regions of Croatia showed that 72.2% of propolis samples contain galangin, 88.8% of samples contain kaempferol, naringenin and apigenin and 66.6% of samples contain caffeic acid . Caffeic acid, pinocembrin, galangin, chrysin and naringenin were analyzed by HPLC . In all samples, pinocembrin was the dominant flavonoid . In samples from the Adriatic region, concentration of pinocembrin ranged from 0.03 to 6.14% (x = 2.87%) and in the continental region samples from 0 to 4.74% (x = 2.84%) . Chrysin was found in all propolis samples in a concentration ranging from 0.22 to 5.32% (x = 1.86%) in the continental region samples and from 0.03 to 3.64% (x = 1.96%) in samples from the Adriatic region . Chrysin was followed by naringenin, ranging from 0 to 1.14% (x = 0.42%) in samples from the Adriatic region and from 0.22 to 2.41% (x = 0.60%) in the continental region samples . Concentration of caffeic acid ranged from 0 to 10.11% (x = 2.69%) in the Adriatic region samples and from 0.27 to 2.67% (x = 1.37%) in samples from the continental region of Croatia . Results of HPLC analyses suggest that propolis samples collected from various parts of Croatia do not differ markedly in contents of chrysin, pinocembrin, naringenin and galangin but differ in the concentration of caffeic acid . All EEPs significantly inhibited the growth of Bacillus subtilis in comparison with the control (80% ethanol) (p < 0.05), showing inhibition zones of 16 +/- 2 mm for samples from the continental region, and of 18 +/- 3 mm for samples from the Adriatic region . There was no significant difference in antimicrobial activity of EEPs from the continental and Adriatic regions of Croatia, suggesting that bactericidal activity depends on synergism of all phenolic compounds.

Mikrobiologiia, 2003 Nov-Dec, 72(6), 780 - 4
{Conjugal plasmid transfer in Bacillus subtilis in soil microcosms}; Lotareva OV et al.; Conjugal transfer of the small plasmid pUB110 between Bacillus subtilis strains was studied under conditions of microcosms with sterile and nonsterile soil . Plasmid transfer proved to be possible after soil inoculation with vegetative partner cells or with their spores . Plasmid transfer occurred at temperatures of 30 degrees C and 22-23 degrees C.

J Basic Microbiol, 2004, 44(1), 3 - 9
Evaluation of some fungi and bacteria for biocontrol of anthracnose disease of cowpea; Adebanjo A et al.; The efficacy of some fungal and bacterial isolates obtained from cowpea phylloplane in inhibiting the in vitro and in vivo growth of Colletotrichum lindemuthianum, causal agent of anthracnose of cowpea was investigated . Inhibition of growth of the pathogen with production of zones of inhibition was observed for Aspergillus flavus, A . ochraceus, Penicillium aurantiogriseum, Bacillus subtilis-BS21, B . subtilis-BS22 and B . subtilis-BS23 . Inhibition of growth on contact was recorded for A . niger while Trichoderma viride-TH14 and T . viride-TH31hyperparasitized the pathogen . The two isolates of T . viride and all tested bacteria significantly reduced seedling infection from anthracnose infested seeds in pot experiments . Spray application of T . viride-TH31 on inoculated cowpea plants in the field effectively suppressed the incidence and severity of anthracnose disease, and significantly increased yield over the control . The antagonist was more effective when applied twice weekly than once in a week.

Chem Soc Rev, 2004 Feb 20, 33(2), 98 - 107 Epub 2003 Dec 09.
Theoretical insights in enzyme catalysis; Marti S et al.; In this tutorial review we show how the methods and techniques of computational chemistry have been applied to the understanding of the physical basis of the rate enhancement of chemical reactions by enzymes . This is to answer the question: Why is the activation free energy in enzyme catalysed reactions smaller than the activation free energy observed in solution? Two important points of view are presented: Transition State (TS) theories and Michaelis Complex (MC) theories . After reviewing some of the most popular computational methods employed, we analyse two particular enzymatic reactions: the conversion of chorismate to prephenate catalysed by Bacillus subtilis chorismate mutase, and a methyl transfer from S-adenosylmethionine to catecholate catalysed by catechol O-methyltransferase . The results and conclusions obtained by different authors on these two systems, supporting either TS stabilisation or substrate preorganization, are presented and compared . Finally we try to give a unified view, where a preorganized enzyme active site, prepared to stabilise the TS, also favours those reactive conformations geometrically closer to the TS.

Protein Sci, 2004 Mar, 13(3), 668 - 77 Epub 2004 Feb 06.
Crystal structure of the phosphorolytic exoribonuclease RNase PH from Bacillus subtilis and implications for its quaternary structure and tRNA binding; Harlow LS et al.; RNase PH is a member of the family of phosphorolytic 3' --> 5' exoribonucleases that also includes polynucleotide phosphorylase (PNPase) . RNase PH is involved in the maturation of tRNA precursors and especially important for removal of nucleotide residues near the CCA acceptor end of the mature tRNAs . Wild-type and triple mutant R68Q-R73Q-R76Q RNase PH from Bacillus subtilis have been crystallized and the structures determined by X-ray diffraction to medium resolution . Wild-type and triple mutant RNase PH crystallize as a hexamer and dimer, respectively . The structures contain a rare left-handed beta alpha beta-motif in the N-terminal portion of the protein . This motif has also been identified in other enzymes involved in RNA metabolism . The RNase PH structure and active site can, despite low sequence similarity, be overlayed with the N-terminal core of the structure and active site of Streptomyces antibioticus PNPase . The surface of the RNase PH dimer fit the shape of a tRNA molecule.

Microbiology, 2004 Feb, 150(Pt 2), 497 - 512
Transcriptome and proteome analysis of Bacillus subtilis gene expression in response to superoxide and peroxide stress; Mostertz J et al.; The Gram-positive soil bacterium Bacillus subtilis responds to oxidative stress by the activation of different cellular defence mechanisms . These are composed of scavenging enzymes as well as protection and repair systems organized in highly sophisticated networks . In this study, the peroxide and the superoxide stress stimulons of B . subtilis were characterized by means of transcriptomics and proteomics . The results demonstrate that oxidative-stress-responsive genes can be classified into two groups . One group encompasses genes which show similar expression patterns in the presence of both reactive oxygen species . Examples are members of the PerR and the Fur regulon which were induced by peroxide and superoxide stress . Similarly, both kinds of stress stimulated the activation of the stringent response . The second group is composed of genes primarily responding to one stimulus, like the members of the SOS regulon which were particularly upregulated in the presence of peroxide, and many genes involved in sulfate assimilation and methionine biosynthesis which were only induced by superoxide . Several genes encoding proteins of unknown function could be assigned to one of these groups.

Microbiology, 2004 Feb, 150(Pt 2), 427 - 36
The fate of extracellular proteins tagged by the SsrA system of Bacillus subtilis; Kolkman MA et al.; In bacteria, SsrA, a highly conserved RNA molecule, functions in a mechanism meant to rescue stalled ribosomes . In this process, a peptide tag encoded by SsrA is cotranslationally added to truncated polypeptides, thereby targeting these molecules for proteolytic degradation, at least when they stay inside the cell . This study examined the fate of two extracellular proteins that were tagged by the SsrA system of Bacillus subtilis . Gene constructs encoding human interleukin-3 (hIL-3) fused to a signal peptide and B . subtilis alpha-amylase, both lacking an in-frame stop codon, were used as models to achieve ribosome stalling and activation of the SsrA system . Introduction of these gene constructs into B . subtilis led to tagging of the gene products by SsrA RNA . The tagged protein products bound to antibodies that were raised against the proteolysis tag encoded by B . subtilis SsrA {(A)GKTNSFNQNVALAA} . The apolar C-terminal SsrA-tag does not function as a specific signal for proteolytic degradation of SsrA-tagged amylase directly after trans-translation or during the secretion process . Also, SsrA-tagged amylase appeared to be very stable once outside the cell . In contrast, hIL-3 molecules tagged with the native, apolar SsrA-tag were considerably less stable than hIL-3 molecules that received a negatively charged control tag . Not one specific protease, but several non-specific proteases seem to play a role in the rapid degradation of SsrA-tagged hIL-3 . The polarity of the C-terminus of heterologous hIL-3 protein proved to be an important determinant for protein stability when produced by B . subtilis . As observed previously in Escherichia coli and B . subtilis, SsrA tagging also occurs frequently in normally growing Gram-positive bacilli and it appears that intracellular proteins are the predominant natural substrates of SsrA.

Microbiology, 2004 Feb, 150(Pt 2), 415 - 25
CtaG is required for formation of active cytochrome c oxidase in Bacillus subtilis; Bengtsson J et al.; The Gram-positive bacterium Bacillus subtilis contains two respiratory oxidases of the haem-copper superfamily: cytochrome aa(3), which is a quinol oxidase, and cytochrome caa(3), which is a cytochrome c oxidase . Cytochrome c oxidase uniquely contains a di-copper centre, Cu(A) . B . subtilis CtaG is a membrane protein encoded by the same gene cluster as that which encodes the subunits of cytochrome c oxidase . The role of B . subtilis CtaG and orthologous proteins present in many other Gram-positive bacteria has remained unexplored . The sequence of CtaG is unrelated to that of CtaG/Cox11p of proteobacteria and eukaryotic cells . This study shows that B . subtilis CtaG is essential for the formation of active cytochrome caa(3) but is not required for assembly of the core subunits I and II with haem in the membrane and it has no role in the synthesis of active cytochrome aa(3) . B . subtilis YpmQ, a homologue to Sco1p of eukaryotic cells, is also a membrane-bound cytochrome c oxidase-specific assembly factor . Properties of CtaG- and YpmQ-deficient mutants were compared . Cells lacking YpmQ showed a low cytochrome c oxidase activity and this defect was suppressed by the supplementation of the growth medium with copper ions . It has previously been proposed that YpmQ/Sco1p is involved in synthesis of the Cu(A) centre . The results of this study are consistent with this proposal but the exact role of YpmQ in assembly of cytochrome c oxidase remains to be elucidated.

Chem Commun (Camb), 2004 Feb 21, (4), 430 - 1 Epub 2004 Jan 19.
Interaction of modified liposomes with Bacillus spores; Kazakov S et al.; The interaction between liposomes modified with a particular peptide sequence and Bacillus subtilis spores was experimentally observed as (1) an increase in the average diameter of spore-related particles, and (2) the formation of dense and structured shells around the spores at higher concentrations of liposomes.

J Biol Chem, 2004 May 28, 279(22), 23472 - 6 Epub 2004 Feb 04.
Substrate and dioxygen binding to the endospore coat laccase from Bacillus subtilis; Enguita FJ et al.; The CotA laccase from the endospore coat of Bacillus subtilis has been crystallized in the presence of the non-catalytic co-oxidant 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and the structure was determined using synchrotron radiation . The binding site for this adduct is well defined and indicates how ABTS, in conjunction with laccases, could act as an oxidative mediator toward non-phenolic moieties . In addition, a dioxygen moiety is clearly defined within the solvent channel oriented toward one of the T3 copper atoms in the trinuclear center.

Mol Microbiol, 2004 Feb, 51(4), 1169 - 83
Peptidoglycan hydrolytic activities associated with bacteriophage virions; Moak M et al.; Murein hydrolases appear to be widespread in the virions of bacteriophages infecting Gram-positive or Gram-negative bacteria . Muralytic activity has been found in virions of the majority of a diverse collection of phages . Where known, the enzyme is either part of a large protein or found associated with other structural components of the virion that limit enzyme activity . In most cases, the lack of genetic and structural characterization of the phage precludes making a definitive identification of the enzymatic protein species . However, three proteins with muralytic activity have been unequivocally identified . T7gp16 is a 144 kDa internal head protein that is ejected into the cell at the initiation of infection; its enzyme activity is required only when the cell wall is more highly cross-linked . P22gp4 is part of the neck of the particle and is essential for infectivity . The activity associated with virions of Bacillus subtilis phage o29 and its relatives lies in the terminal protein gp3 . These studies lead to a general mechanism describing how phage genomes are transported across the bacterial cell wall.

Mol Microbiol, 2004 Feb, 51(4), 1155 - 68
Analysis of HutP-dependent transcription antitermination in the Bacillus subtilis hut operon: identification of HutP binding sites on hut antiterminator RNA and the involvement of the N-terminus of HutP in binding of HutP to the antiterminator RNA; Oda M et al.; We investigated HutP-dependent transcription antitermination of the Bacillus subtilis hut operon . In vitro transcription assays with the B . subtilissigmaA-containing RNA polymerase indicated that HutP inhibits transcription termination at the internal terminator by binding to the antiterminator on hut mRNA in the presence of histidine . Ethylnitrosourea modification interference assays and mutational analyses of the interference sites showed that interaction of HutP with a region containing three UAG trinucleotide sequences, which is located on top of the antiterminator structure, is critical for hut antitermination in vivo . Results from kinetic analysis of binding of HutP to RNA containing various portions of the antiterminator sequences indicated that secondary structure is required for binding of HutP to the region containing three UAG triplets in the antiterminator . The in vivo HutP antiterminator activity was reduced by the mutations in the N-terminal region of HutP . The HutP variants with H4A, R7A, I9A and Q26A mutations exhibited reduced binding affinities to the antiterminator RNA in vitro . A 25-mer peptide consisting of amino acid residues 2-26 of HutP bound to the antiterminator RNA . These results indicated that the N-terminus of HutP is involved in binding of HutP to the antiterminator RNA.

Mol Microbiol, 2004 Feb, 51(4), 1087 - 102
MurAA, catalysing the first committed step in peptidoglycan biosynthesis, is a target of Clp-dependent proteolysis in Bacillus subtilis; Kock H et al.; The carboxyvinyl transfer from phosphoenolpyruvate to UDP-N-acetylglucosamine is the first committed step in the pathway of peptidoglycan formation . This crucial reaction for bacterial cell growth is catalysed by the MurA enzymes . Gram-negative bacteria carry one murA gene, whereas in a subgroup of Gram-positive bacteria two separate paralogues, MurAA and MurAB, exist . This study provides evidence that in the Gram-positive bacterium Bacillus subtilis, the MurAA protein is specifically degraded by the ClpCP protease . This Clp-dependent degradation is especially enhanced upon entry into stationary phase, thus ensuring an immediate growth arrest due to stalled murein biosynthesis . The MurAA protein can therefore be addressed as a target of Clp-dependent regulatory proteolysis such as the transcriptional regulators CtsR, ComK, Spx in B . subtilis, CtrA in Caulobacter crescentus or RpoS in Escherichia coli . Taking into account all other known regulatory targets of ATP-dependent proteases, MurAA of B . subtilis represents the first example of a metabolic enzyme which is a unique regulatory substrate of Clp-dependent proteolysis . Its function as a regulatory metabolic checkpoint resembles that of homoserine trans-succinylase (MetA) in E . coli which is similarly ATP-dependently degraded.

J Bacteriol, 2004 Feb, 186(4), 1213 - 4
Gamma-glutamyltranspeptidase, but not YwrD, is important in utilization of extracellular blutathione as a sulfur source in Bacillus subtilis; Minami H et al.; gamma-Glutamyltranspeptidase (EC 2.3.2.2) of Bacillus subtilis, which is an extracellular enzyme, hydrolyzes the gamma-glutamyl linkage of glutathione . YwrD, which is homologous to gamma-glutamyltranspeptidase, was speculated to have a similar physiological role . It was shown that gamma-glutamyltranspeptidase, but not YwrD, is important in utilizing glutathione as the sole sulfur source in Bacillus subtilis.

J Bacteriol, 2004 Feb, 186(4), 1191 - 6
Physical and enzymological interaction of Bacillus subtilis proteins required for de novo pyridoxal 5'-phosphate biosynthesis; Belitsky BR; Bacillus subtilis synthesizes pyridoxal 5'-phosphate, the active form of vitamin B(6), by a poorly characterized pathway involving the yaaD and yaaE genes . The pdxS (yaaD) mutant was confirmed to be a strict B(6) auxotroph, but the pdxT (yaaE) mutant turned out to be a conditional auxotroph depending on the availability of ammonium in the growth medium . The PdxS and PdxT proteins copurified during affinity chromatography and apparently form a complex that has glutaminase activity . PdxS and PdxT appear to encode the synthase and glutaminase subunits, respectively, of a glutamine amidotransferase of as-yet-unknown specificity essential for B(6) biosynthesis.

J Bacteriol, 2004 Feb, 186(4), 1182 - 90
Transcriptional regulation of the phoPR operon in Bacillus subtilis; Pragai Z et al.; When Bacillus subtilis is subjected to phosphate starvation, the Pho regulon is activated by the PhoP-PhoR two-component signal transduction system to elicit specific responses to this nutrient limitation . The response regulator, PhoP, and its cognate histidine sensor kinase, PhoR, are encoded by the phoPR operon that is transcribed as a 2.7-kb bicistronic mRNA . The phoPR operon is transcribed from two sigma(A)-dependent promoters, P(1) and P(2) . Under conditions where the Pho regulon was not induced (i.e., phosphate-replete conditions or phoR-null mutant), a low level of phoPR transcription was detected only from promoter P(1) . During phosphate starvation-induced transition from exponential to stationary phase, the expression of the phoPR operon was up-regulated in a phosphorylated PhoP (PhoP approximately P)-dependent manner; in addition to P(1), the P(2) promoter becomes active . In vitro gel shift assays and DNase I footprinting experiments showed that both PhoP and PhoP approximately P could bind to the control region of the phoPR operon . The data indicate that while low-level constitutive expression of phoPR is required under phosphate-replete conditions for signal perception and transduction, autoinduction is required to provide sufficient PhoP approximately P to induce other members of the Pho regulon . The extent to which promoters P(1) and P(2) are activated appears to be influenced by the presence of other sigma factors, possibly the result of sigma factor competition . For example, phoPR is hyperinduced in a sigB mutant and, later in stationary phase, in sigH, sigF, and sigE mutants . The data point to a complex regulatory network in which other stress responses and post-exponential-phase processes influence the expression of phoPR and, thereby, the magnitude of the Pho regulon response.

J Bacteriol, 2004 Feb, 186(4), 1175 - 81
Identification of genes controlled by the essential YycG/YycF two-component system of Staphylococcus aureus; Dubrac S et al.; The YycG/YycF essential two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low-G+C gram-positive bacteria, including several pathogens such as Staphylococcus aureus . By studying growth of S . aureus cells where the yyc operon is controlled by an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter, we have shown that this system is essential in S . aureus during growth at 37 degrees C and that starvation for the YycG/YycF regulatory system leads to cell death . During a previous study of the YycG/YycF TCS of B . subtilis, we defined a potential YycF consensus recognition sequence, consisting of two hexanucleotide direct repeats, separated by five nucleotides {5'-TGT(A/T)A(A/T/C)-N(5)-TGT(A/T)A(A/T/C)-3'} . A detailed DNA motif analysis of the S . aureus genome indicates that there are potentially 12 genes preceded by this sequence, 5 of which are involved in virulence . An in vitro approach was undertaken to determine which of these genes are controlled by YycF . The YycG and YycF proteins of S . aureus were overproduced in Escherichia coli and purified . Autophosphorylation of the YycG kinase and phosphotransfer to YycF were shown in vitro . Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified YycF to the promoter region of the ssaA gene, encoding a major antigen and previously suggested to be controlled by YycF . YycF was also shown to bind specifically to the promoter regions of two genes, encoding the IsaA antigen and the LytM peptidoglycan hydrolase, in agreement with the proposed role of this system in controlling virulence and cell wall metabolism.

J Bacteriol, 2004 Feb, 186(4), 1136 - 46
The Bacillus subtilis extracytoplasmic-function sigmaX factor regulates modification of the cell envelope and resistance to cationic antimicrobial peptides; Cao M et al.; Bacillus subtilis contains seven extracytoplasmic-function sigma factors that activate partially overlapping regulons . We here identify four additional members of the sigma(X) regulon, pbpX (penicillin-binding protein), ywnJ, the dlt operon (D-alanylation of teichoic acids), and the pss ybfM psd operon (phosphatidylethanolamine biosynthesis) . Modification of teichoic acids by esterification with D-alanine and incorporation of phosphatidylethanolamine into the cell membrane have a common consequence: in both cases positively charged amino groups are introduced into the cell envelope . The resulting reduction in the net negative charge of the cell envelope has been previously implicated as a resistance mechanism specific for cationic antimicrobial peptides . Consistent with this notion, we find that both sigX and dltA mutants are more sensitive to nisin than wild-type cells . We conclude that activation of the sigma(X) regulon serves to alter cell surface properties to provide protection against antimicrobial peptides.

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