J Bacteriol, 2003 Aug, 185(16), 4717 - 26 Rod shape determination by the Bacillus subtilis class B penicillin-binding proteins encoded by pbpA and pbpH; Wei Y et al.; The peptidoglycan cell wall determines the shape and structural integrity of a bacterial cell . Class B penicillin-binding proteins (PBPs) carry a transpeptidase activity that cross-links peptidoglycan strands via their peptide side chains, and some of these proteins are directly involved in cell shape determination . No Bacillus subtilis PBP with a clear role in rod shape maintenance has been identified . However, previous studies showed that during outgrowth of pbpA mutant spores, the cells grew in an ovoid shape for several hours before they recovered and took on a normal rod shape . It was postulated that another PBP, expressed later during outgrowth, was able to compensate for the lack of the pbpA product, PBP2a, and to guide the formation of a rod shape . The B . subtilis pbpH (ykuA) gene product is predicted to be a class B PBP with greatest sequence similarity to PBP2a . We found that a pbpH-lacZ fusion was expressed at very low levels in early log phase and increased in late log phase . A pbpH null mutant was indistinguishable from the wild-type, but a pbpA pbpH double mutant was nonviable . When pbpH was placed under the control of an inducible promoter in a pbpA mutant, viability was dependent on pbpH expression . Growth of this strain in the absence of inducer resulted in conversion of the cells from rods to ovoid/round shapes and lysis . We conclude that PBP2a and PbpH play redundant roles in formation of a rod-shaped peptidoglycan cell wall. FEMS Microbiol Lett, 2003 Jul 29, 224(2), 169 - 73 A mutant Bacillus subtilis gamma-glutamyltranspeptidase specialized in hydrolysis activity; Minami H et al.; gamma-Glutamyltranspeptidase (GGT) catalyzes the hydrolysis of gamma-glutamyl compounds and the transfer of their gamma-glutamyl moieties to amino acids and peptides . The transpeptidation activity of Bacillus subtilis GGT is about 10-fold higher than its hydrolysis activity . In B . subtilis GGT, substitution of Asp-445 with Ala abolished its transpeptidation activity . The specific activity for hydrolysis of D445A GGT was 40.2% of that of the wild-type GGT . The K(m) value for L-glutamine was 15.3 mM . D445A GGT was salt tolerant like the wild-type GGT . These results indicate that D445A GGT will be highly useful as a 'glutaminase' in food industry. Biotechnol Prog, 2003 Jul-Aug, 19(4), 1355 - 64 Potential of on-line CIMS for bioprocess monitoring; Custer TG et al.; Chemical-ionization mass spectrometry (CIMS) using flow reactors is an emerging method for on-line monitoring of trace concentrations of organic compounds in the gas phase . In this study, a flow-reactor CIMS instrument, employing the H(3)O(+) cation as the ionizing reagent, was used to simultaneously monitor several volatile metabolic products as they are released into the headspace during bacterial growth in a bioreactor . Production of acetaldehyde, ethanol, acetone, butanol, acetoin, diacetyl, and isoprene by Bacillus subtilis is reported . Ion signal intensities were related to solution-phase concentrations using empirical calibrations and, in the case of isoprene, were compared with simultaneous gas chromatography measurements . Identification of volatile and semivolatile metabolites is discussed . Flow-reactor CIMS techniques should be useful for bioprocess monitoring applications because of their ability to sensitively and simultaneously monitor many volatile metabolites on-line. Mol Microbiol, 2003 Aug, 49(4), 1135 - 44 The BceRS two-component regulatory system induces expression of the bacitracin transporter, BceAB, in Bacillus subtilis; Ohki R et al.; BceA and bceB encode a nucleotide-binding domain (NBD) and membrane-spanning domain (MSD) subunit, respectively, of an ATP-binding cassette (ABC) transporter in Bacillus subtilis . Disruption of these genes resulted in hypersensitivity to bacitracin, a peptide antibiotic that is non-ribosomally synthesized in some strains of Bacillus . Northern hybridization analyses showed that expression of the bceAB operon is induced by bacitracin present in the growth medium . The bceRS genes encoding a two-component regulatory system are located immediately upstream of bceAB . Deletion analyses of the bceAB promoter together with DNase I footprinting experiments revealed that a sensor kinase, BceS, responds to extracellular bacitracin either directly or indirectly and transmits a signal to a cognate response regulator, BceR . The regulator binds directly to the upstream region of the bceAB promoter and upregulates the expression of bceAB genes . The bcrC gene product is additionally involved in bacitracin resistance . The expression of bcrC is dependent on the ECF sigma factors, sigmaM and sigmaX, but not on the BceRS two-component system . In view of these results, possible roles of BceA, BceB and BcrC in bacitracin resistance of B . subtilis 168 are discussed. Mol Microbiol, 2003 Aug, 49(4), 1067 - 79 Phosphatidylethanolamine and phosphatidylglycerol are segregated into different domains in bacterial membrane . A study with pyrene-labelled phospholipids; Vanounou S et al.; To detect and characterize membrane domains that have been proposed to exist in bacteria, two kinds of pyrene-labelled phospholipids, 2-pyrene-decanoyl-phosphatidylethanolamine (PY-PE) and 2-pyrene-decanoyl-phosphatidylglycerol (PY-PG) were inserted into Escherichia coli or Bacillus subtilis membrane . The excimerization rate coefficient, calculated from the excimer-to-monomer ratio dependencies on the probe concentration, was two times higher for PY-PE than for PY-PG at 37 degrees C . This was ascribed to different local concentrations rather than to differences in mobility . The extent of mixing between the two fluorescent phospholipids, estimated by formation of their heteroexcimer, was found very low both in E . coli and B . subtilis, in contrast to model membranes . In addition, these two pyrene derivatives exhibited different temperature phase transitions and different detergent extractability, indicating that the surroundings of these phospholipids in bacterial membrane differ in organization and order . Inhibition of protein synthesis, leading to condensation of nucleoid and presumably to dissipation of membrane domains, indeed resulted in increased formation of heteroexcimers, broadening of phase transitions and equal detergent extractability of both probes . It is proposed that in bacterial membranes these phospholipids are segregated into distinct domains that differ in composition, proteo-lipid interaction and degree of order; the proteo-lipid domain being enriched by PE. Mol Microbiol, 2003 Aug, 49(4), 895 - 903 A strand-specific model for chromosome segregation in bacteria; Rocha EP et al.; Chromosome separation and segregation must be executed within a bacterial cell in which the membrane and cytoplasm are highly structured . Here, we develop a strand-specific model based on each of the future daughter chromosomes being associated with a different set of structures or hyperstructures in an asymmetric cell . The essence of the segregation mechanism is that the genes on the same strand in the parental cell that are expressed together in a hyperstructure continue to be expressed together and segregate together in the daughter cell . The model therefore requires an asymmetric distribution of classes of genes and of binding sites and other structures on the strands of the parental chromosome . We show that the model is consistent with the asymmetric distribution of highly expressed genes and of stress response genes in Escherichia coli and Bacillus subtilis . The model offers a framework for interpreting data from genomics. Biotechnol Lett, 2003 Jun, 25(12), 969 - 74 Regioselective synthesis of kojic acid esters by Bacillus subtilis protease; Raku T et al.; The lipophilicity of kojic acid {5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one} was improved by esterifying kojic acid with either divinyl adipate, vinyl hexanoate, vinyl octanoate or vinyl decanoate using protease from Bacillus subtilis for 7 d . 1H-NMR and 13C-NMR showed that the primary hydroxyl group at the C-7 position of kojic acid was regioselectively esterified to afford 7-O-vinyl adipoyl kojic acid, 7-O-hexanoyl kojic acid, 7-O-octanoyl kojic acid and 7-O-decanoyl kojic acid (13-27% yield) . The kojic acid esters had radical scavenging activities, inhibited tyrosinase activity and was biodegradable. Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 9774 - 8 Epub 2003 Jul 29. Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli; Watanabe K et al.; Ansamycins such as rifamycin, ansamitocin, and geldanamycin are an important class of polyketide natural products . Their biosynthetic pathways are especially complex because they involve the formation of 3-amino-5-hydroxybenzoic acid (AHBA) followed by backbone assembly by a hybrid nonribosomal peptide synthetase/polyketide synthase . We have reconstituted the ability to synthesize 2,6-dimethyl-3,5,7-trihydroxy-7-(3'-amino-5'-hydroxyphenyl)-2,4-heptadienoic acid (P8/1-OG), an intermediate in rifamycin biosynthesis, in an extensively manipulated strain of Escherichia coli . The parent strain, BAP1, contains the sfp phosphopantetheinyl transferase gene from Bacillus subtilis, which posttranslationally modifies polyketide synthase and nonribosomal peptide synthetase modules . AHBA biosynthesis in this host required introduction of seven genes from Amycolatopsis mediterranei, which produces rifamycin, and Actinosynnema pretiosum, which produces ansamitocin . Because the four-module RifA protein (530 kDa) from the rifamycin synthetase could not be efficiently produced in an intact form in E . coli, it was genetically split into two bimodular proteins separated by matched linker pairs to facilitate efficient inter-polypeptide transfer of a biosynthetic intermediate . A derivative of BAP1 was engineered that harbors the AHBA biosynthetic operon, the bicistronic RifA construct and the pccB and accA1 genes from Streptomyces coelicolor, which enable methylmalonyl-CoA biosynthesis . Fermentation of this strain of E . coli yielded P8/1-OG, an N-acetyl P8/1-OG analog, and AHBA . In addition to providing a fundamentally new route to shikimate and ansamycin-type compounds, this result enables further genetic manipulation of AHBA-derived polyketide natural products with unprecedented power. J Mol Biol, 2003 Aug 8, 331(2), 473 - 84 A core mutation affecting the folding properties of a soluble domain of the ATPase protein CopA from Bacillus subtilis; Banci L et al.; The two N-terminal domains of the P-type copper ATPase, CopAa and CopAb, from Bacillus subtilis differ in their folding capabilities in vitro . Whereas CopAb has the typical betaalphabetabetaalphabeta structure and is a rigid protein, CopAa is found to be largely unfolded . A sequence analysis of the two and of orthologue homologous proteins indicates that Ser46 in CopAa may destabilise the hydrophobic core, as also confirmed through a bioinformatic energy study . CopAb has a Val in the corresponding position . The S46V and S46A mutants are found to be folded, although the latter displays multiple conformations . S46VCopAa, in both apo and copper(I) loaded forms, has very similar structural and dynamic properties with respect to CopAb, besides a different length of strand beta2 and beta4 . It is intriguing that the oxygen of Thr16 is found close, though at longer than bonding distance, to copper in both domains, as it also occurs in a human orthologue domain . This study contributes to understanding the behaviour of proteins that do not properly fold in vitro . A possible biological significance of the peculiar folding behaviour of this domain is discussed. Zh Mikrobiol Epidemiol Immunobiol, 2003 May-Jun, (3), 91 - 3 {Characterization of highly purified metalloprotease produced by Bacillus subtilis}; Khaziev AF et al.; Proteolytic enzyme produced by Bacillus subtilis is characterized as typical metalloprotease with a molecular weight of 27.9 kD; the enzyme shows its highest activity at pH 7.0-9.0, possesses substrate specificity with respect to different proteins, its temperature optimum is 52 degrees C and its specific activity exceeds that of all known commercial analogues . At 37 degrees C the enzyme is half inactivated in 72 hours. Zh Mikrobiol Epidemiol Immunobiol, 2003 Mar-Apr, (2), 102 - 9 {Biological effects of interferon, produced by recombinant bacteria of the probiotic preparation subalin}; Beliavskaia VA et al.; The present review deals with the analysis of biological and functional activities of recombinant bacteria Bacillus subtilis IF-alpha 2335 are producing a human interferon . The interferon-producing bacteria are constructed on a basis commercial probiotic strain B.subtilis 2335, carrying a recombinant plasmid pMBM 105 with the gene of human alpha-2 interferon . The implementation of the recombinant strain in the preparation probiotic, received a designation "Subalin", necessitates to verify a number of immunologic activities and to perform successive protective effects . Interferon, synthesized by recombinant bacteria shows the activity on macroorganism at oral and rectal application of preparation . Subalin was shown antivirus and antitumor activity and preservation by recombinat bacteria of antagonistic properties . The mechanisms of the positive effect of subalin were considered: this effect was shown to be due to the action of interferon excreted by recombinant bacteria into the mucous of different biotopes of host. Biochemistry, 2003 Aug 5, 42(30), 9060 - 6 Engineering the primary substrate specificity of Streptomyces griseus trypsin; Page MJ et al.; Streptomyces griseus trypsin (SGT) was chosen as a model scaffold for the development of serine proteases with enhanced substrate specificity . Recombinant SGT has been produced in a Bacillus subtilis expression system in a soluble active form and purified to homogeneity . The recombinant and native proteases have nearly identical enzymatic properties and structures . Four SGT mutants with alterations in the S1 substrate binding pocket (T190A, T190P, T190S, and T190V) were also expressed . The T190P mutant demonstrated the largest shift to a preference for Arg versus Lys in the P1 site . This was shown by a minor reduction in catalytic activity toward an Arg-containing substrate (k(cat) reduction of 25%) . The crystal structures of the recombinant SGT and the T190P mutant in a complex with the inhibitor benzamidine were obtained at high resolution (approximately 1.9 A) . The increase in P1 specificity, achieved with minimal effect on the catalytic efficiency, demonstrates that the T190P mutant is an ideal candidate for the design of additional substrate specificity engineered into the S2 to S4 binding pockets. Mol Genet Genomics, 2003 Aug, 269(5), 706 - 14 Epub 2003 Jul 23. Genome-wide survey of mRNA half-lives in Bacillus subtilis identifies extremely stable mRNAs; Hambraeus G et al.; We have used DNA microarrays to survey rates of mRNA decay on a genomic scale in early stationary-phase cultures of Bacillus subtilis . The decay rates for mRNAs corresponding to about 1500 genes could be estimated . About 80% of these mRNAs had a half-life of less than 7 min . More than 30 mRNAs, including both mono- and polycistronic transcripts, were found to be extremely stable, i.e . to have a half-life of > or =15 min . Only two such transcripts were known previously in B . subtilis . The results provide the first overview of mRNA decay rates in a gram-positive bacterium and help to identify polycistronic operons . We could find no obvious correlation between the stability of an mRNA and the function of the encoded protein . We have also not found any general features in the 5' regions of mRNAs that distinguish stable from unstable transcripts . The identified set of extremely stable mRNAs may be useful in the construction of stable recombinant genes for the overproduction of biomolecules in Bacillus species. Med Sci Monit, 2003 Jul, 9(7), BR283 - 8 Production of high-molecular-weight ribonuclease Bsn from the recombinant strain of Bacillus subtilis; Kharitonova MA et al.; BACKGROUND: Ribonucleases (RNases) can be used in both basic and clinical sciences, e.g . in research on developmental processes or on antiviral and antitumor therapy . RNases have great potential as therapeutic entities . On the basis of new ribonucleases new medications can be created . Bacilli synthesize two types of secretory ribonucleases, the well-studied low-molecular-weight ribonucleases and high-molecular-weight ribonucleases . Only two RNases of the second type have so far been described: RNase Bsn from B . subtilis and binase II from B . intermedius . MATERIAL/METHODS: The activity of ribonucleases was determined from the amount of the acid-soluble products of RNA hydrolysis . The cultivation media were optimized for maximum RNase production in terms of the experimental factorial design B2 using BIOPT software . RESULTS: Our investigation of a novel secretory ribonuclease, the Bacillus subtilis RNase Bsn expressed in the recombinant B . subtilis strain 168, showed that it is synthesized in the growth retardation phase, when inorganic phosphate is exhausted in the medium . The biosynthesis of Bsn was found to be suppressed by inorganic phosphate in the medium and activated by small amounts of the transcriptional inhibitor actinomycin D . CONCLUSIONS: Our results show that the biosynthesis of the novel secretory ribonuclease Bsn in recombinant strain Bacillus subtilis 168 is subject to negative regulation by inorganic phosphate, and is activated by small doses of actinomycin D . The stimulating effect of this antibiotic is well pronounced during the active synthesis of ribonucleases, but insignificant when ribonuclease synthesis is inhibited by Pi. J Nutr Sci Vitaminol (Tokyo), 2003 Feb, 49(1), 73 - 5 Investigation of 1-deoxy-D-xylulose 5-phosphate synthase and transketolase of Bacillus subtilis in relation to vitamin B6 biosynthesis; Sakai A et al.; In Escherichia coli, 4-(phosphohydroxy)-L-threonine and 1-deoxy-D-xylulose 5-phosphate are believed to be direct precursors of vitamin B6 (B6), and 1-deoxy-D-xylulose 5-phosphate synthase (Dxs) and transketolase could catalyze the formation of each precursor . In this report, the possible involvement Dxs and transketolase (Tkt) in B6 biosynthesis in Bacillus subtilis was investigated . The gene disruptant of tkt and conditional mutants of dxs were constructed, and their ability of B6 biosynthesis was examined . It was found that the tkt disruptants retain the ability to synthesize B6 . The conditional mutant of dxs synthesized the same amount of B6 per dry cell weight as the wild-type strain . Therefore, it is very likely that neither Dxs nor transketolase is involved in B6 biosynthesis in B . subtilis. Biotechnol Lett, 2003 Jan, 25(2), 161 - 6 Enzymatic synthesis of hydrophilic undecylenic acid sugar esters and their biodegradability; Raku T et al.; To enhance water solubility of 10-undecylenic acid, which has anti-fungus, anti-bacterial and anti-virus activity, D-glucose, trehalose and sucrose were regioselectively esterified with vinyl 10-undecylenic acid ester in dimethyl formamide by a commercial protease, Bioprase conc., from Bacillus subtilis . 6-O-(10-Undecylenoyl) D-glucose, 6-O-(10-undecylenoyl) trehalose and 1'-O-(10-undecylenoyl) sucrose were obtained . The influence of structural variation by changing the sugar moiety was analyzed the surface tension and biodegradability. Biotechnol Lett, 2003 Mar, 25(6), 465 - 8 Optimization of cell growth and poly(glutamic acid) production in batch fermentation by Bacillus subtilis; Richard A et al.; Poly(glutamic acid) was produced maximally by Bacillus subtilis in batch fermentations at pH 7 and using glycerol at 20 g l(-1) in a glutamic acid/citric acid medium . Poly(glutamic acid) reached 23 g l(-1) after 30 h. Protein Expr Purif, 2003 Aug, 30(2), 203 - 9 Purification and characterization of YihA, an essential GTP-binding protein from Escherichia coli; Lehoux IE et al.; YihA has previously been characterized as an essential gene of unknown function in both Escherichia coli and Bacillus subtilis . It is conserved in bacteria and represents an attractive target for the discovery of new antibiotics . YihA encodes a putative GTP-binding protein . We have cloned and overexpressed the gene encoding E . coli YihA and initiated biochemical studies as a first step towards understanding its biological function . We showed by circular dichroism that the purified protein has a secondary structure typical of most GTP-binding proteins . It binds guanine nucleotides specifically, as demonstrated by fluorescence resonance energy transfer between 2'-(or-3')-O-(N-methylanthraniloyl) nucleotides (mant-nucleotides) and the tryptophans of YihA . The K(d) values for GDP and GTP were determined by competition with 2'-(or-3')-O-(N-methylanthraniloyl) GDP to be 3 and 27 microM, respectively . Using mutants of YihA we show that nucleotide binding occurs at the putative GTP-binding domain predicted from the primary sequence. Arch Pharm Res, 2003 Jun, 26(6), 449 - 52 Antibacterial coumarins from Angelica gigas roots; Lee S et al.; Systematic fractionation of Angelica gigas roots led to the isolation of linear furano(pyrano)coumarins such as bergapten (1), decursinol angelate (2), decursin (3), nodakenetin (4) and nodakenin (5) . The antibacterial activities of those compounds against pathogenic bacteria were investigated . Among the compounds tested, decursinol angelate (2) and decursin (3) exhibited significant antibacterial activity against Bacillus subtilis with the minimum inhibitory concentrations (MICs) of 50 and 12.5 microg/mL, respectively. Protein Sci, 2003 Aug, 12(8), 1694 - 705 The characterization of mutant Bacillus subtilis adenylosuccinate lyases corresponding to severe human adenylosuccinate lyase deficiencies; Palenchar JB et al.; Adenylosuccinate lyase is a homotetramer that catalyzes two discrete reactions in the de novo synthesis of purines: the cleavage of adenylosuccinate and succinylaminoimidazole carboxamide ribotide (SAICAR) . Several point mutations in the gene encoding the enzyme have been implicated in human disease . Bacillus subtilis adenylosuccinate lyase was used as a model system in which mutations were constructed corresponding to those mutations associated with severe human adenylosuccinate lyase deficiency . Site-directed mutagenesis was utilized to construct amino acid substitutions in B . subtilis adenylosuccinate lyase; Met(10), Ile(123), and Thr(367) were replaced by Leu, Trp, and Arg, respectively, and the altered enzymes were expressed in Escherichia coli . These purified enzymes containing amino acid substitutions were found to have substantial catalytic activity and exhibit relatively small changes in their kinetic parameters . The major deviations from the wild-type-like behavior were observed upon biophysical characterization . All of these enzymes with amino acid replacements are associated with marked thermal instability . I123W adenylosuccinate lyase exhibits notable changes in the circular dichroism spectra, and a native gel electrophoresis pattern indicative of some protein aggregation . T367R also exhibits alterations at the quarternary level, as reflected in native gel electrophoresis . Experimental results, combined with homology modeling, suggest that the altered enzymes are primarily structurally impaired . The enzyme instability was found to be lessened by subunit complementation with the wild-type enzyme, under mild conditions; these studies may have implications for the in vivo behavior of adenylosuccinate lyase in heterozygous patients . Residues Met(10), Ile(123), and Thr(367) appear to be located in regions of the enzyme important for maintaining the structural integrity required for a stable, functional enzyme. Genes Cells, 2003 Aug, 8(8), 699 - 712 A new staphylococcal sigma factor in the conserved gene cassette: functional significance and implication for the evolutionary processes; Morikawa K et al.; BACKGROUND: Staphylococcus aureus is a major human pathogen and causes a serious hospital infection due to the acquired multidrug resistance . Unlike the well-studied bacteria such as Escherichia coli and Bacillus subtilis, which have seven and 18 sigma factors, respectively, only two sigma factors have been known for S . aureus . We searched for possible sigma factor genes by examining the S . aureus genome with a special attention to the gene arrangement around the sigma factor genes of a close relative, B . subtilis . RESULTS: A new sigma factor gene was identified in Staphylococcus . The gene constituted a conserved gene cluster with other genes including translation- and transcription-related genes . Phylogenetic analysis and comparison of the gene sequences among species indicated that the staphylococcal sigma factor originated from a common ancestor of B . subtilis SigH . An over-expression of this sigma factor in S . aureus resulted in a drastic induction of the expression of the com operons that encode proteins required for the natural genetic competence . CONCLUSIONS: We demonstrated that the newly identified staphylococcal sigma factor participated in a regulatory network of transcription that controlled the genetic competence genes . In our phylogenetic tree, the factor was classified as a single group with a common function. Infect Immun, 2003 Aug, 71(8), 4304 - 12 The major cold shock gene, cspA, is involved in the susceptibility of Staphylococcus aureus to an antimicrobial peptide of human cathepsin G; Katzif S et al.; A Tn551 insertional library of Staphylococcus aureus strain ISP479 was challenged with an antimicrobial peptide (CG 117-136) derived from human neutrophil cathepsin G (CG) . After repeated selection and screening of surviving colonies, a mutant was identified that expressed increased resistance to CG 117-136 . Southern hybridization analysis revealed that the Tn551 insert in this mutant (SK1) was carried on a 10.6-kb EcoRI chromosomal DNA fragment . Subsequent physical mapping of this Tn551 insert revealed that it was positioned between a putative promoter sequence and the translational start codon of the cspA gene, which encodes a protein (CspA) highly similar to the major cold shock proteins CspA and CspB of Escherichia coli and Bacillus subtilis, respectively . This Tn551 insertion as well as a separate deletion-insertion mutation in cspA decreased the capacity of S . aureus to respond to the stress of cold shock and increased resistance to CG 117-136 . The results indicate for the first time that a physiologic link exists between bacterial susceptibility to an antimicrobial peptide and a stress response system. Biochemistry, 2003 Jul 29, 42(29), 8739 - 47 Surface salt bridges modulate DNA wrapping by the type II DNA-binding protein TF1; Grove A; The histone-like protein HU is involved in compaction of the bacterial genome . Up to 37 bp of DNA may be wrapped about some HU homologues in a process that has been proposed to depend on a linked disruption of surface salt bridges that liberates cationic side chains for interaction with the DNA . Despite significant sequence conservation between HU homologues, binding sites from 9 to 37 bp have been reported . TF1, an HU homologue that is encoded by Bacillus subtilis bacteriophage SPO1, has nM affinity for 37 bp preferred sites in DNA with 5-hydroxymethyluracil (hmU) in place of thymine . On the basis of electrophoretic mobility shift assays, we show that TF1-DNA complex formation is associated with a net release of only approximately 0.5 cations . The structure of TF1 suggests that Asp13 can form a dehydrated surface salt bridge with Lys23; substitution of Asp13 with Ala increases the net release of cations to approximately 1 . These data are consistent with complex formation linked to disruption of surface salt bridges . Substitution of Glu90 with Ala, which would expose Lys87 predicted to contact DNA immediately distal to a proline-mediated DNA kink, causes an increase in affinity and an abrogation of the preference for hmU-containing DNA . We propose that hmU preference is due to finely tuned interactions at the sites of kinking that expose a differential flexibility of hmU- and T-containing DNA . Our data further suggest that the difference in binding site size for HU homologues is based on a differential ability to stabilize the DNA kinks. Proteomics, 2003 Jul, 3(7), 1117 - 27 Using standard positions and image fusion to create proteome maps from collections of two-dimensional gel electrophoresis images; Luhn S et al.; Databases for two-dimensional protein gels pose new challenges in extracting meaningful information from large numbers of experiments . In order to create expression profiles, positions of corresponding protein spots across all gel images have to be established . In larger gel sets errors may accumulate rapidly during this spot matching process, effectively limiting the number of samples available for data mining . Here we present a novel approach for organizing spot data based on the concept of a standard position for a protein species . Standard positions are meaningful average positions that are determined using all occurrences of a protein species . They can be extended to spots that are not annotated via interpolation . The standard position of a spot can serve as a unifying index across all gels in a database, thus allowing creation and analysis of expression profiles that span the whole collection . The standard position gives a much more accurate estimation of a spot's position on a gel than can be obtained using theoretical isoelectric point and molecular weight . Positional indexing is a complement to a priori identifications (e.g . by mass spectrometry or Edman degradation) . Moreover it can be used in advance to select spots that are worth identifying because they show relevant expression profiles . Furthermore, we show how to combine all spots that occur on any of the gels into one synthetic but nevertheless realistic-looking image . This composite image is produced such that all spots have their standard positions . It can serve as a proteome reference map for an organism . As an application, we have computed a reference map from 23 gel images of Bacillus subtilis, using an enhanced prerelease version of the gel analysis software Delta2D (DECODON, Greifswald, Germany). J Food Prot, 2003 Jul, 66(7), 1233 - 40 Characterization of UV-peroxide killing of bacterial spores; Reidmiller JS et al.; Advantage is taken in many sterilization processes, especially for food packaging materials, of the synergy between H2O2 and UV irradiation for spore killing . The nature of the synergy is currently not well defined in terms of targets and mechanisms . We found that under some experimental conditions, the synergistic killing of spores of Bacillus megaterium ATCC 19213 appeared to be mainly UV-enhanced peroxide killing, while under other conditions, it appeared to be mainly peroxide-enhanced UV killing . Lethal combinations of H2O2 and UV irradiation for spores resulted in only modest increases in auxotrophic mutations among survivors, indicative of little DNA damage, in contrast to higher mutation levels after dry-heat damage at 115 degrees C . However, the combination of UV light and peroxide did lead to major inactivation of glucose 6-phosphate dehydrogenase, an enzyme that was used to monitor the damage to bacterial protein . Synergistic UV-H2O2 killing was reduced by agents such as pyruvate, thiosulfate, and iron or copper cations, which appeared to act at least in part by reacting chemically with H2O2, and was only slightly affected by the use of UV light at a wavelength of 222 nn rather than 254 nm . Hydrogen peroxide treatment can precede UV irradiation for synergistic killing by some hours with an interim of drying for spores of Bacillus subtilis A, a spore type used commonly for the validation of aseptic processes . Synergistic killing of dried spores or those in suspensions was accelerated at higher temperatures (50 degrees C) rather than at lower temperatures (25 degrees C). Eur J Biochem, 2003 Aug, 270(15), 3196 - 204 UMP kinase from the Gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity; Gagyi C et al.; The gene encoding Bacillus subtilis UMP kinase (pyrH/smbA) is transcribed in vivo into a functional enzyme, which represents approximately 0.1% of total soluble proteins . The specific activity of the purified enzyme under optimal conditions is 25 units.mg-1 of protein . In the absence of GTP, the activity of B . subtilis enzyme is less than 10% of its maximum activity . Only dGTP and 3'-anthraniloyl-2'-deoxyguanosine-5'-triphosphate (Ant-dGTP) can increase catalysis significantly . Binding of Ant-dGTP to B . subtilis UMP kinase increased the quantum yield of the fluorescent analogue by a factor of more than three . UTP and GTP completely displaced Ant-dGTP, whereas GMP and UMP were ineffective . UTP inhibits UMP kinase of B . subtilis with a lower affinity than that shown towards the Escherichia coli enzyme . Among nucleoside monophosphates, 5-fluoro-UMP (5F-UMP) and 6-aza-UMP were actively phosphorylated by B . subtilis UMP kinase, explaining the cytotoxicity of the corresponding nucleosides towards this bacterium . A structural model of UMP kinase, based on the conservation of the fold of carbamate kinase and N-acetylglutamate kinase (whose crystals were recently resolved), was analysed in the light of physicochemical and kinetic differences between B . subtilis and E . coli enzymes. Appl Opt, 2003 Jul 1, 42(19), 4080 - 7 Native fluorescence and excitation spectroscopic changes in Bacillus subtilis and Staphylococcus aureus bacteria subjected to conditions of starvation; Alimova A et al.; Fluorescence emission and excitation spectra were measured over a 7-day period for Bacillus subtilis (Bs), a spore-forming, and Staphylococcus aureus (Sa), a nonspore-forming bacteria subjected to conditions of starvation . Initially, the Bs fluorescence was predominantly due to the amino acid tryptophan . Later, a fluorescence band with an emission peak at 410 nm and excitation peak at 345 m, from dipicolinic acid, appeared . Dipicolinic acid is produced during spore formation and serves as a spectral signature for detection of spores . The intensity of the 410-nm band continued to increase over the next 3 days . The Sa fluorescence was predominantly from tryptophan and did not change over time . In 6 of the 17 Bs specimens studied, an additional band appeared with a weak emission peak at 460 cm and excitation peaks at 250, 270, and 400 nm . The addition of beta-hydroxybutyric acid to the Bs or the Sa cultures resulted in a two-order of magnitude increase in the 460-nm emission . The addition of Fe2+ quenched the 460 emission, indicating that a source of the 460-nm emission was a siderophore produced by the bacteria . We demonstrate that optical spectroscopy-based instrumentation can detect bacterial spores in real time. J Bacteriol, 2003 Aug, 185(15), 4615 - 9 Analysis of the interaction between the transcription factor sigmaG and the anti-sigma factor SpoIIAB of Bacillus subtilis; Evans L et al.; The activation of sigma(G), a transcription factor, in Bacillus subtilis is coupled to the completion of engulfment during sporulation . SpoIIAB, an anti-sigma factor involved in regulation of sigma(F), is also shown to form a complex with sigma(G) in vitro . SpoIIAA, the corresponding anti-anti-sigma factor, can disrupt the SpoIIAB:sigma(G) complex, releasing free sigma(G) . The data suggest the existence of an as-yet-unknown mechanism to keep sigma(G) inactive prior to engulfment. J Bacteriol, 2003 Aug, 185(15), 4490 - 8 Essential nature of the mreC determinant of Bacillus subtilis; Lee JC et al.; The mre genes of Escherichia coli and Bacillus subtilis are cell shape determination genes . Mutants affected in mre function are spheres instead of the normal rods . Although the mre determinants are not required for viability in E . coli, the mreB determinant is an essential gene in B . subtilis . Conflicting results have been reported as to whether the two membrane-associated proteins MreC and MreD are essential proteins . Furthermore, although the MreB protein has been studied in some detail, the roles of the MreC and MreD proteins in cell shape determination are unknown . We constructed a strain of B . subtilis in which expression of the mreC determinant is dependent upon the addition of isopropyl-beta-D-thiogalactopyranoside to the culture medium . Utilizing this conditional strain, it was shown that mreC is an essential gene in B . subtilis . Furthermore, it was shown that cells lacking sufficient quantities of MreC undergo morphological changes, namely, swelling and twisting of the cells, which is followed by cell lysis . Electron microscopy was utilized to demonstrate that a polymeric material accumulated at one side of the division septum of the cells and that the presence of this material correlated with the bending of the cell . The best explanation for the results is that the MreC protein is involved in the control of septal versus long-axis peptidoglycan synthesis, that cells lacking MreC perform aberrant septal peptidoglycan synthesis, and that lysis results from a deficiency in long-axis peptidoglycan synthesis. J Bacteriol, 2003 Aug, 185(15), 4305 - 14 Chill induction of the SigB-dependent general stress response in Bacillus subtilis and its contribution to low-temperature adaptation; Brigulla M et al.; A variety of environmental and metabolic cues trigger the transient activation of the alternative transcription factor SigB of Bacillus subtilis, which subsequently leads to the induction of more than 150 general stress genes . This general stress regulon provides nongrowing and nonsporulated cells with a multiple, nonspecific, and preemptive stress resistance . By a proteome approach we have detected the expression of the SigB regulon during continuous growth at low temperature (15 degrees C) . Using a combination of Western blot analysis and SigB-dependent reporter gene fusions, we provide evidence for high-level and persistent induction of the sigB operon and the SigB regulon, respectively, in cells continuously exposed to low temperatures . In contrast to all SigB-activating stimuli described thus far, induction by low temperatures does not depend on the positive regulatory protein RsbV or its regulatory phosphatases RsbU and RsbP, indicating the presence of an entirely new pathway for the activation of SigB by chill stress in B . subtilis . The physiological importance of the induction of the general stress response for the adaptation of B . subtilis to low temperatures is emphasized by the observation that growth of a sigB mutant is drastically impaired at 15 degrees C . Inclusion of the compatible solute glycine betaine in the growth medium not only improved the growth of the wild-type strain but rescued the growth defect of the sigB mutant, indicating that the induction of the general stress regulon and the accumulation of glycine betaine are independent means by which B . subtilis cells cope with chill stress. Mol Microbiol, 2003 Aug, 49(3), 581 - 90 Swarming motility in undomesticated Bacillus subtilis; Kearns DB et al.; Swarming motility was identified and characterized in an undomesticated strain of Bacillus subtilis . Rapid surface migration was preceded by a cell density-dependent lag period, which could be eliminated if actively swarming cells were used as the inoculum . The leading edge of the swarm was characterized by multicellular rafts of highly flagellated cells . Flagellum biosynthesis and surfactant production were required for swarming . Swarming was not found in any of several standard laboratory strains . Laboratory strains are characteristically unable to produce surfactant, but such a strain remained unable to swarm even when surfactant was provided by extracellular complementation . We conclude that robust swarming is a feature of undomesticated B . subtilis and that this behaviour has been lost or attenuated in laboratory strains through the accumulation of multiple genetic defects. J Appl Microbiol, 2003, 95(2), 218 - 24 Molecular detection and beta-glucuronidase expression of gus-marked Bacillus subtilis L-form bacteria in developing Chinese cabbage seedlings; Tsomlexoglou E et al.; AIM: To detect L-form bacteria in developing Chinese cabbage seedlings . METHODS AND RESULTS: Stable Bacillus subtilis L-forms were genetically modified to express the gus gene (encoding beta-glucuronidase) . Germinated seeds of Chinese cabbage were soaked in mannitol based suspensions of the L-form bacteria or with mannitol alone and after washing were grown in aseptic conditions on plant growth medium . Histochemical staining of beta-glucuronidase activity (X-gluc) and Polymerase Chain Reaction (PCR) detection of the gus gene were achieved in the L-form associated seedlings . beta-Glucuronidase was localized in discrete spots, mainly in the roots with staining, and was also observed in the cotyledons and base of stems . Correlation was observed between PCR detection of the gus gene and histochemical staining with detection in similar tissues . Stable L-form bacteria were non-culturable after their association with plant material . CONCLUSIONS: The gus reporter gene system with its associated histological staining for enzyme activity was used successfully for detecting B . subtilis L-form bacteria in plant material . SIGNIFICANCE AND IMPACT OF THE STUDY: These molecular marked L-forms should provide a specific and sensitive technique for detecting L-form bacteria in planta and offer a method for further understanding the L-form/plant association. Lett Appl Microbiol, 2003, 37(2), 169 - 73 Genetic diversity and involvement in bread spoilage of Bacillus strains isolated from flour and ropy bread; Sorokulova IB et al.; AIMS: To study Bacillus contamination of wheat flour and ropy bread, to analyse genetic diversity of isolated strains and to evaluate the ability of these strains to produce ropy bread . METHODS AND RESULTS: Classical and molecular methods {16S rDNA sequencing and random amplified polymorphic DNA (RAPD)-PCR} were used to identify and type-isolated strains . The predominant species isolated were Bacillus subtilis and B . licheniformis . RAPD analysis demonstrated that the same sample may harbor different strains . Ten of 15 strains of B . subtilis and four of six strains of B . licheniformis were able to cause rope spoilage of the laboratory-baked bread . CONCLUSION: RAPD typing can be useful in the tracking of Bacillus strains during bakery processing and in the understanding of the role of different Bacillus strains in the rope spoilage of bread . SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate the variability of Bacillus strains isolated from flour and responsible for rope spoilage of bread. Lett Appl Microbiol, 2003, 37(2), 162 - 8 Time series analysis of aerobic bacterial flora during Miso fermentation; Onda T et al.; AIMS: This article reports a microbiological study of aerobic mesophilic bacteria that are present during the fermentation process of Miso . METHODS AND RESULTS: Aerobic bacteria were enumerated and isolated from Miso during fermentation and divided into nine groups using traditional phenotypic tests . The strains were identified by biochemical analysis and 16S rRNA sequence analysis . They were identified as Bacillus subtilis, B . amyloliquefaciens, Kocuria kristinae, Staphylococcus gallinarum and S . kloosii . All strains were sensitive to the bacteriocins produced by the lactic acid bacteria isolated from Miso . CONCLUSIONS: The dominant species among the undesirable species throughout the fermentation process were B . subtilis and B . amyloliquefaciens . It is suggested that bacteriocin-producing lactic acid bacteria are effective in the growth prevention of aerobic bacteria in Miso . SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided useful information for controlling of bacterial flora during Miso fermentation. Pharmazie, 2003 Jun, 58(6), 428 - 30 Iridoids from Pedicularis kansuensis forma albiflora; Yuan CS et al.; A new iridoid glycoside kansuenoside (1) and a new iridoid kansuenin (2), along with eight known compounds (3-10) were isolated from the whole plant of Pedicularis kansuensis forma albiflora Li . Their structures were elucidated by spectroscopic methods . Nine of them were assayed against Bacillus subtilis, Escherichia coli, and Staphylococcus aureus. Science, 2003 Jul 11, 301(5630), 211 - 3 Tandem transcription and translation regulatory sensing of uncharged tryptophan tRNA; Chen G et al.; The Bacillus subtilis AT (anti-TRAP) protein inhibits the regulatory protein TRAP (trp RNA-binding attenuation protein), thereby eliminating transcription termination in the leader region of the trp operon . Transcription of the AT operon is activated by uncharged tryptophan transfer RNA (tRNATrp) . Here we show that translation of AT also is regulated by uncharged tRNATrp . A 10-residue coding region containing three consecutive tryptophan codons is located immediately preceding the AT structural gene . Completion of translation of this coding region inhibits AT synthesis, whereas incomplete translation increases AT production . Tandem sensing of uncharged tRNATrp therefore regulates synthesis of AT, which in turn regulates TRAP's ability to inhibit trp operon expression. Microbiology, 2003 Jul, 149(Pt 7), 1687 - 98 Phosphoenolpyruvate phosphotransferase system and N-acetylglucosamine metabolism in Bacillus sphaericus; Alice AF et al.; Bacillus sphaericus, a bacterium of biotechnological interest due to its ability to produce mosquitocidal toxins, is unable to use sugars as carbon source . However, ptsHI genes encoding HPr and EI proteins belonging to a PTS were cloned, sequenced and characterized . Both HPr and EI proteins were fully functional for phosphoenolpyruvate-dependent transphosphorylation in complementation assays using extracts from Staphylococcus aureus mutants for one of these proteins . HPr(His(6)) was purified from wild-type and a Ser46/Gln mutant of B . sphaericus, and used for in vitro phosphorylation experiments using extracts from either B . sphaericus or Bacillus subtilis as kinase source . The results showed that both phosphorylated forms, P-Ser46-HPr and P-His15-HPr, could be obtained . The findings also proved indirectly the existence of an HPr kinase activity in B . sphaericus . The genetic structure of these ptsHI genes has some unusual features, as they are co-transcribed with genes encoding metabolic enzymes related to N-acetylglucosamine (GlcNAc) catabolism (nagA, nagB and an undetermined orf2) . In fact, this bacterium was able to utilize this amino sugar as carbon and energy source, but a ptsH null mutant had lost this characteristic . Investigation of GlcNAc uptake and streptozotocin inhibition in both a wild-type and a ptsH null mutant strain led to the proposal that GlcNAc is transported and phosphorylated by an EII(Nag) element of the PTS, as yet uncharacterized . In addition, GlcNAc-6-phosphate deacetylase and GlcN-6-phosphate deaminase activities were determined; both were induced in the presence of GlcNAc . These results, together with the authors' recent findings of the presence of a phosphofructokinase activity, are strongly indicative of a glycolytic pathway in B . sphaericus . They also open new possibilities for genetic improvements in industrial applications. FEMS Microbiol Lett, 2003 Jul 15, 224(1), 107 - 12 Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei; Braaz R et al.; The recently discovered extracellular poly{(R)-3-hydroxybutyrate} (PHB) depolymerase PhaZ7 of Paucimonas lemoignei represents the first member of a new subgroup (EC 3.1.1.75) of serine hydrolases with no significant amino acid similarities to conventional PHB depolymerases, lipases or other hydrolases except for a potential lipase box-like motif (Ala-His-Ser136-Met-Gly) and potential candidates for catalytic triad and oxyanion pocket amino acids . In order to identify amino acids essential for activity 11 mutants of phaZ7 were generated by site-directed mutagenesis and expressed in recombinant protease-deficient Bacillus subtilis WB800 . The wild-type depolymerase and 10 of the 11 mutant proteins (except for Ser136Cys) were expressed and efficiently secreted by B . subtilis as shown by Western blots of cell-free culture fluid proteins . No PHB depolymerase activity was detected in strains harbouring one of the following substitutions: His47Ala, Ser136Ala, Asp242Ala, Asp242Asn, His306Ala, indicating the importance of these amino acids for activity . Replacement of Ser136 by Thr resulted in a decrease of activity to about 20% of the wild-type level and suggested that the hydroxy group of the serine side chain is important for activity but can be partially replaced by the hydroxy function of threonine . Alterations of Asp256 to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser136-Met-Gly) to a lipase box consensus sequence (Gly134-His-Ser136-Met-Gly) or to the PHB depolymerase box consensus sequence (Gly134-Leu135-Ser136-Met-Gly) had no significant effect on PHB depolymerase activity, indicating that these amino acids or sequence motifs were not essential for activity . In conclusion, the PHB depolymerase PhaZ7 is a serine hydrolase with a catalytic triad and oxyanion pocket consisting of His47, Ser136, Asp242 and His306. J Biol Chem, 2003 Oct 10, 278(41), 39323 - 9 Epub 2003 Jul 07. Autophosphorylation of the Escherichia coli protein kinase Wzc regulates tyrosine phosphorylation of Ugd, a UDP-glucose dehydrogenase; Grangeasse C et al.; Autophosphorylation of protein-tyrosine kinases (PTKs) involved in exopolysaccharide and capsular polysaccharide biosynthesis and transport has been observed in a number of Gram-negative and Gram-positive bacteria . However, besides their own phosphorylation, little is known about other substrates targeted by these protein-modifying enzymes . Here, we present evidence that the protein-tyrosine kinase Wzc of Escherichia coli is able to phosphorylate an endogenous enzyme, UDP-glucose dehydrogenase (Ugd), which participates in the synthesis of the exopolysaccharide colanic acid . The process of phosphorylation of Ugd by Wzc was shown to be stimulated by previous autophosphorylation of Wzc on tyrosine 569 . The phosphorylation of Ugd was demonstrated to actually occur on tyrosine and result in a significant increase of its dehydrogenase activity . In addition, the phosphotyrosine-protein phosphatase Wzb, which is known to effectively dephosphorylate Wzc, exhibited only a low effect, if any, on the dephosphorylation of Ugd . These data were related to the recent observation that two other UDP-glucose dehydrogenases have been also shown to be phosphorylated by a PTK in the Gram-positive bacterium Bacillus subtilis . Comparative analysis of the activities of PTKs from Gram-negative and Gram-positive bacteria showed that they are regulated by different mechanisms that involve, respectively, either the autophosphorylation of kinases or their interaction with a membrane protein activator. Metab Eng, 2003 Apr, 5(2), 133 - 49 Transcriptional profiling of gene expression in response to glucose in Bacillus subtilis: regulation of the central metabolic pathways; Blencke HM et al.; Chemoheterotrophic bacteria use a few central metabolic pathways for carbon catabolism and energy production as well as for the generation of the main precursors for anabolic reactions . All sources of carbon and energy are converted to intermediates of these central pathways and then further metabolized . While the regulation of genes encoding enzymes used to introduce specific substrates into the central metabolism has already been studied to some detail, much less is known about the regulation of the central metabolic pathways . In this study, we investigated the responses of the Bacillus subtilis transcriptome to the presence of glucose and analyzed the role of the pleiotropic transcriptional regulator CcpA in these responses . We found that CcpA directly represses genes involved in the utilization of secondary carbon sources . In contrast, induction by glucose seems to be mediated by a variety of different mechanisms . In the presence of glucose, the genes encoding glycolytic enzymes are induced . Moreover, the genes responsible for the production of acetate from pyruvate with a concomitant substrate-level phosphorylation are induced by glucose . In contrast, the genes required for the complete oxidation of the sugar (Krebs cycle, respiration) are repressed if excess glucose is available for the bacteria . In the absence of glucose, the genes of the Krebs cycle as well as gluconeogenic genes are derepressed . The genes encoding enzymes of the pentose phosphate pathway are expressed both in the presence and the absence of glucose, as suggested by the central role of this pathway in generating anabolic precursors. Nat Genet, 2003 Aug, 34(4), 377 - 8 Essentiality, not expressiveness, drives gene-strand bias in bacteria; Rocha EP et al.; Preferential positioning of bacterial genes in the leading strand was thought to result from selection to avoid high head-on collision rates between DNA and RNA polymerases . Here we show, however, that in Bacillus subtilis and Escherichia coli, essentiality (the transcript product), not expressiveness (the collision rate), selectively drives the biased gene distribution. Nat Struct Biol, 2003 Aug, 10(8), 652 - 7 Structure of the manganese-bound manganese transport regulator of Bacillus subtilis; Glasfeld A et al.; The Bacillus subtilis manganese transport regulator, MntR, binds Mn2+ as an effector and is a repressor of transporters that import manganese . A member of the diphtheria toxin repressor (DtxR) family of metalloregulatory proteins, MntR exhibits selectivity for Mn2+ over Fe2+ . Replacement of a metal-binding residue, Asp8, with methionine (D8M) relaxes this specificity . We report here the X-ray crystal structures of wild-type MntR and the D8M mutant bound to manganese with 1.75 A and 1.61 A resolution, respectively . The 142-residue MntR homodimer has substantial structural similarity to the 226-residue DtxR but lacks the C-terminal SH3-like domain of DtxR . The metal-binding pockets of MntR and DtxR are substantially different . The cation-to-cation distance between the two manganese ions bound by MntR is 3.3 A, whereas that between the metal ions bound by DtxR is 9 A . D8M binds only a single Mn2+ per monomer, owing to alteration of the metal-binding site . The sole retained metal site adopts pseudo-hexacoordinate geometry rather than the pseudo-heptacoordinate geometry of the MntR metal sites. Mol Genet Genomics, 2003 Aug, 269(5), 640 - 8 Epub 2003 Jul 04. Patterns of protein carbonylation following oxidative stress in wild-type and sigB Bacillus subtilis cells; Mostertz J et al.; Oxidative stress causes damage to nucleic acids, membrane lipids and proteins . One striking effect is the metal-catalyzed, site-specific carbonylation of proteins . In the gram-positive soil bacterium Bacillus subtilis, the PerR-dependent specific stress response and the sigmaB-dependent general stress response act together to make cells more resistant to oxidative stress . In this study, we analyzed the carbonylation of cytoplasmic proteins in response to hydrogen peroxide stress in B . subtilis . Furthermore, we asked whether the sigmaB-dependent response to oxidative stress also confers protection against protein carbonylation . To monitor the amount and specificity of protein damage, carbonyls were derivatized with 2,4-dinitrophenylhydrazine, and the resulting stable hydrazones were detected by immunoanalysis of proteins separated by one- or two-dimensional gel electrophoresis . The overall level of protein carbonylation increased strongly in cells treated with hydrogen peroxide . Several proteins, including the elongation factors EF-G, TufA and EF-Ts, were found to be highly carbonylated . Induction of the peroxide specific stress response by treatment with sub-lethal peroxide concentrations, prior to exposure to otherwise lethal levels of peroxide, markedly reduced the degree of protein carbonylation . Cells starved for glucose also showed only minor amounts of peroxide-mediated protein carbonylation compared to exponentially growing cells . We could not detect any differences between wild-type and deltasigB cells starved for glucose or preadapted by heat treatment with respect to the amount or specificity of protein damage incurred upon subsequent exposure to peroxide stress . However, artificial preloading with proteins that are normally induced by sigmaB-dependent mechanisms resulted in a lower level of protein carbonylation when cells were later subjected to oxidative stress. J Mol Biol, 2003 Jul 11, 330(3), 593 - 606 Multifunctional xylooligosaccharide/cephalosporin C deacetylase revealed by the hexameric structure of the Bacillus subtilis enzyme at 1.9A resolution; Vincent F et al.; Esterases and deacetylases active on carbohydrate ligands have been classified into 14 families based upon amino acid sequence similarities . Enzymes from carbohydrate esterase family seven (CE-7) are unusual in that they display activity towards both acetylated xylooligosaccharides and the antibiotic, cephalosporin C . The 1.9A structure of the multifunctional CE-7 esterase (hereinafter CAH) from Bacillus subtilis 168 reveals a classical alpha/beta hydrolase fold encased within a 32 hexamer . This is the first example of a hexameric alpha/beta hydrolase and is further evidence of the versatility of this particular fold, which is used in a wide variety of biological contexts . A narrow entrance tunnel leads to the centre of the molecule, where the six active-centre catalytic triads point towards the tunnel interior and thus are sequestered away from cytoplasmic contents . By analogy to self-compartmentalising proteases, the tunnel entrance may function to hinder access of large substrates to the poly-specific active centre . This would explain the observation that the enzyme is active on a variety of small, acetylated molecules . The structure of an active site mutant in complex with the reaction product, acetate, reveals details of the putative oxyanion binding site, and suggests that substrates bind predominantly through non-specific contacts with protein hydrophobic residues . Protein residues involved in catalysis are tethered by interactions with protein excursions from the canonical alpha/beta hydrolase fold . These excursions also mediate quaternary structure maintenance, so it would appear that catalytic competence is only achieved on protein multimerisation . We suggest that the acetyl xylan esterase (EC 3.1.1.72) and cephalosporin C deacetylase (EC 3.1.1.41) enzymes of the CE-7 family represent a single class of proteins with a multifunctional deacetylase activity against a range of small substrates. J Mol Biol, 2003 Jul 11, 330(3), 459 - 72 Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis; Madec E et al.; We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases . In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing the kinase and a short juxtamembrane region . This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli . PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction . PrkC also displays kinase activity with myelin basic protein . Using high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc . All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant proteins were tested for in vitro kinase activity . Single and multiple replacement of four threonine residues, clustered between residues 162 and 167 in a putative activation loop, substantially reduced kinase activity and the effect was clearly additive . Replacement of the other three threonine residues, clustered between residues 290 and 320, had relatively little effect on activity . In contrast, substitution of Ser214, which is conserved in closely related receptor kinase-like bacterial proteins, independently affected activity and may represent a novel regulatory mechanism . When projected onto a 3D structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located in the juxtamembrane region . These results indicate that regulation of PrkC kinase activity (and presumably autophosphorylation) includes a conserved activation loop mechanism . The juxtamembrane phospho-threonine residues may be essential, for example for the recruitment of other proteins necessary for a PrkC signalling cascade or for coupling to other signalling pathways . This is the first structure-function analysis of a bacterial receptor-like kinase of the Hanks family. Anal Biochem, 2003 Aug 1, 319(1), 73 - 7 A screening method for endo-beta-1,4-xylanase substrate selectivity; Moers K et al.; Endoxylanase (EC 3.2.1.8) substrate selectivity, i.e., its relative activity toward water-unextractable arabinoxylan (WU-AX) and water-extractable arabinoxylan (WE-AX) substrates, is important for its functionality in biotechnological processes such as bread-making and gluten starch separation . A screening method for rapidly determining said substrate selectivity was developed . Endoxylanase activity toward WU-AX was estimated by incubation of insoluble chromogenic substrate with a range of enzyme concentrations in microtiter plates, followed by colorimetric measurement of the dye released in the supernatant . A similar approach using soluble substrate and ethanol precipitation of unhydrolysed AX fragments was used to estimate enzyme activity toward WE-AX . A substrate selectivity factor was defined as the ratio of enzyme activity toward insoluble substrate over enzyme activity toward soluble substrate . A Bacillus subtilis and an Aspergillus aculeatus endoxylanase, known to have widely varying relative rates of hydrolysis of WU-AX and WE-AX, varied most in their substrate selectivity, while the endoxylanases of Aspergillus niger, Trichoderma longibrachiatum, and Trichoderma viride displayed intermediate such relative activities. Appl Environ Microbiol, 2003 Jul, 69(7), 4144 - 50 A modified Saccharomyces cerevisiae strain that consumes L-Arabinose and produces ethanol; Becker J et al.; Metabolic engineering is a powerful method to improve, redirect, or generate new metabolic reactions or whole pathways in microorganisms . Here we describe the engineering of a Saccharomyces cerevisiae strain able to utilize the pentose sugar L-arabinose for growth and to ferment it to ethanol . Expanding the substrate fermentation range of S . cerevisiae to include pentoses is important for the utilization of this yeast in economically feasible biomass-to-ethanol fermentation processes . After overexpression of a bacterial L-arabinose utilization pathway consisting of Bacillus subtilis AraA and Escherichia coli AraB and AraD and simultaneous overexpression of the L-arabinose-transporting yeast galactose permease, we were able to select an L-arabinose-utilizing yeast strain by sequential transfer in L-arabinose media . Molecular analysis of this strain, including DNA microarrays, revealed that the crucial prerequisite for efficient utilization of L-arabinose is a lowered activity of L-ribulokinase . Moreover, high L-arabinose uptake rates and enhanced transaldolase activities favor utilization of L-arabinose . With a doubling time of about 7.9 h in a medium with L-arabinose as the sole carbon source, an ethanol production rate of 0.06 to 0.08 g of ethanol per g (dry weight) . h(-1) under oxygen-limiting conditions, and high ethanol yields, this yeast strain should be useful for efficient fermentation of hexoses and pentoses in cellulosic biomass hydrolysates. J Bacteriol, 2003 Jul, 185(14), 4099 - 109 Functional dissection of the Bacillus subtilis pur operator site; Bera AK et al.; Bacillus subtilis PurR represses transcription of several genes involved in purine synthesis, metabolism, and transport and cofactor synthesis . PurR binds specifically to DNAs containing an inverted repeat of a 14-nucleotide "PurBox" located in the upstream control regions of genes in the PurR regulon . Further biochemical investigation of the interaction of PurR with a series of shortened upstream DNA fragments of the pur operon determined the minimum length and specificity elements of the operator . The relative affinities of the two PurBoxes differ significantly, such that upstream PurBox1 (-81 to -68 relative to the transcription start site) is designated "strong" and downstream PurBox2 (-49 to -36) is designated "weak." Two PurBoxes are required for high-affinity PurR binding, and one of these must be strong . The shortest DNA construct with high affinity for PurR is a 74-bp perfect palindrome in which weak PurBox2 and its flanking sequences are replaced by strong PurBox1 and flanking sequences . Two PurR dimers bind to this symmetric construct . Phosphoribosylpyrophosphate (PRPP), the effector molecule that reduces affinity of PurR for DNA, requires one weak PurBox in the DNA construct to inhibit PurR binding . PRPP binds, as expected, to a PRPP-motif in PurR . A tracks outside the central conserved CGAA sequence of the PurBox may facilitate DNA bending, leading to a proposal for strong and weak designations of PurBoxes in the control regions of other genes regulated by PurR. J Bacteriol, 2003 Jul, 185(14), 4087 - 98 The purine repressor of Bacillus subtilis: a novel combination of domains adapted for transcription regulation; Sinha SC et al.; The purine repressor from Bacillus subtilis, PurR, represses transcription from a number of genes with functions in the synthesis, transport, and metabolism of purines . The 2.2-A crystal structure of PurR reveals a two-domain protein organized as a dimer . The larger C-terminal domain belongs to the PRT structural family, in accord with a sequence motif for binding the inducer phosphoribosylpyrophosphate (PRPP) . The PRT domain is fused to a smaller N-terminal domain that belongs to the winged-helix family of DNA binding proteins . A positively charged surface on the winged-helix domain likely binds specific DNA sequences in the recognition site . A second positively charged surface surrounds the PRPP site at the opposite end of the PurR dimer . Conserved amino acids in the sequences of PurR homologs in 21 gram-positive bacteria cluster on the proposed recognition surface of the winged-helix domain and around the PRPP binding site at the opposite end of the molecule, supporting a common function of DNA and PRPP binding for all of the proteins . The structure supports a binding mechanism in which extended regions of DNA interact with extensive protein surface . Unlike most PRT proteins, which are phosphoribosyltransferases (PRTases), PurR lacks catalytic activity . This is explained by a tyrosine side chain that blocks the site for a nucleophile cosubstrate in PRTases . Thus, B . subtilis has adapted an enzyme fold to serve as an effector-binding domain and has used it in a novel combination with the DNA-binding winged-helix domain as a repressor of purine genes. Nucleic Acids Res Suppl, 2001, (1), 209 - 10 In vitro hyperprocessing of tRNAs by Bacillus subtilis ribonuclease P RNA; Hori Y et al.; We have previously reported that the catalytic RNA subunit of ribonuclease P (RNase P) of Escherichia coli (M1 RNA) cleaves Drosophila initiator methionine tRNA (tRNA(i)Met), alanine tRNA (tRNA(Ala)) and histidine tRNA (tRNA(His)) within the mature tRNA sequences to produce specific fragments . We call this further cleavage hyperprocessing . These cleavages were dependent on the occurrence of altered conformations of the tRNAs . Here, we found that the RNase P RNA of Bacillus subtilis can hyperprocess these three tRNAs at the same sites as does M1 RNA . The hyperprocessing activity may probably be common feature for Bacterial RNase P RNAs. Acta Crystallogr D Biol Crystallogr, 2003 Jul, 59(Pt 7), 1299 - 301 Epub 2003 Jun 27. Purification, crystallization and preliminary X-ray analysis of a Sco1-like protein from Bacillus subtilis, a copper-binding protein involved in the assembly of cytochrome c oxidase; Imriskova-Sosova I et al.; The putative copper-delivery protein BsSco from Bacillus subtilis is a member of the Sco family of cytochrome c oxidase assembly proteins . BsSco is a membrane protein and the soluble domain has been cloned and expressed in Escherichia coli as a fusion with glutathione-S-transferase . The fusion protein was isolated from the cell lysate using a glutathione-affinity column and the soluble domain of BsSco was released by treatment with thrombin . Sufficient amounts of the soluble domain have been obtained for crystallization . Crystals obtained by hanging-drop vapour diffusion diffract to a resolution of 2.3 A at a synchrotron source . The space group is P6 and the unit-cell parameters are a = 67.74, b = 67.74, c = 189.58 A. Environ Sci Technol, 2003 Jun 1, 37(11), 2376 - 82 Influence of Bacillus subtilis cell walls and EDTA on calcite dissolution rates and crystal surface features; Friis AK et al.; This study investigates the influence of EDTA and the Gram-positive cell walls of Bacillus subtilis on the dissolution rates and development of morphological features on the calcite {1014} surface . The calcite dissolution rates are compared at equivalent saturation indicies (SI) and relative to its dissolution behavior in distilled water (DW) . Results indicate that the presence of metabolically inactive B . subtilis does not affect the dissolution rates significantly . Apparent increases in dissolution rates in the presence of the dead bacterial cells can be accounted for by a decrease of the saturation state of the solution with respect to calcite resulting from bonding of dissolved Ca2+ by functional groups on the cell walls . In contrast, the addition of EDTA to the experimental solutions results in a distinct increase in dissolution rates relative to those measured in DW and the bacterial cell suspensions . These results are partly explained by the 6.5-8 orders of magnitude greater stability of the Ca-EDTA complex relative to the Ca-B . subtilis complexes as well as its free diffusion to and direct attack of the calcite surface . Atomic force microscopy images of the {1014} surface of calcite crystals exposed to our experimental solutions reveal the development of dissolution pits with different morphologies according to the nature and concentration of the ligand . Highly anisotropic dissolution pits develop in the early stages of the dissolution reaction at low B . subtilis concentrations (0.004 mM functional group sites) and in DW . In contrast, at high functional group concentrations (4.0 mM EDTA or equivalent B . subtilis functional group sites), dissolution pits are more isotropic . These results suggest that the mechanism of calcite dissolution is modified by the presence of high concentrations of organic ligands . Since all the pits that developed on the calcite surfaces display some degree of anisotropy and dissolution rates are strongly SI dependent, the rate-limiting step is most likely a surface reaction for all systems investigated in this study . Results of this study emphasize the importance of solution chemistry and speciation in determining calcite reaction rates and give a more accurate and thermodynamically sound representation of dead bacterial cell wall-mineral interactions . In studies of natural aquatic systems, the presence of organic ligands is most often ignored in speciation calculations . This study clearly demonstrates that this oversight may lead to an overestimation of the saturation state of the solutions with respect to calcite and thermodynamic inconsistencies. FEMS Microbiol Lett, 2003 Jun 27, 223(2), 221 - 5 Differences in effects on DNA gyrase activity between two glutamate racemases of Bacillus subtilis, the poly-gamma-glutamate synthesis-linking Glr enzyme and the YrpC (MurI) isozyme; Ashiuchi M et al.; Bacillus subtilis possesses two isogenes encoding glutamate racemases, the poly-gamma-glutamate synthesis-linking Glr enzyme and the YrpC isozyme, and produces abundant amounts of the Glr enzyme . The YrpC isozyme, but not the Glr enzyme, was found to influence the activity of DNA gyrase, as did the MurI-type glutamate racemase of Escherichia coli, which is involved in peptidoglycan synthesis during cell division. FEMS Microbiol Lett, 2003 Jun 27, 223(2), 153 - 7 The response regulator Spo0A from Bacillus subtilis is efficiently phosphorylated in Escherichia coli; Ladds JC et al.; The response regulator proteins of two-component systems mediate many adaptations of bacteria to their ever-changing environment . Most response regulators are transcription factors that alter the level of transcription of specific sets of genes . Activation of response regulators requires their phosphorylation on a conserved aspartate residue by a cognate sensor kinase . For this reason, expression of a recombinant response regulator in the absence of the requisite sensor kinase is expected to yield an unphosphorylated product in the inactive state . For Spo0A, the response regulator controlling sporulation in Bacillus subtilis however, we have found that a significant fraction of the purified recombinant protein is phosphorylated . This phosphorylated component is dimeric and binds to Spo0A recognition sequences in DNA . Treatment with the Spo0A-specific phosphatase, Spo0E, leads to dissociation of the dimers and loss of DNA binding . It is therefore necessary to pre-treat recombinant Spo0A preparations with the cognate phosphatase, to generate the fully inactive state of the molecule. Mycol Res, 2003 Apr, 107(Pt 4), 421 - 7 Efficacy of microorganisms antagonistic to Rhizoctonia cerealis and their cell wall degrading enzymatic activities; Innocenti G et al.; The effect of Trichoderma atroviride, T . harzianum, T . longibrachiatum, Clonostachys rosea and Bacillus subtilis isolates applied to wheat seeds against Rhizoctonia cerealis disease of seedlings was investigated under controlled greenhouse conditions . Most Trichoderma isolates significantly reduced the incidence of disease compared with the infected control . Bacillus subtilis was also effective against sharp eyespot, although less active than Trichoderma spp . Interactions between the antagonistic microorganisms and the cereal pathogenic fungus in dual culture experiments on agar growth medium were also studied . Almost all tested antagonists showed competitive activity against R . cerealis: inhibition of its mycelial growth and hyphal interaction . The production of extracellular beta-N-acetylhexosaminidase, chitin 1,4-beta-chitobiosidase, glucan 1,3-beta-glucosidase and protease activity by the tested microorganisms in the presence of cell walls of R . cerealis was then determined . All isolates showed glucosaminidase and chitobiosidase activity . They also produced glucosidase activity, except B . subtilis, whereas only C . rosea, B . subtilis and one isolate of T . harzianum showed detectable levels of protease activity. Mol Microbiol, 2003 Jul, 49(1), 157 - 65 Identification of additional TnrA-regulated genes of Bacillus subtilis associated with a TnrA box; Yoshida K et al.; Bacillus subtilis TnrA is a global regulator that responds to the availability of nitrogen sources and both activates and represses many genes during nitrogen-limited growth . In order to obtain a holistic view of the gene regulation depending on TnrA, we performed a genome-wide screening for TnrA-regulated genes associated with a TnrA box . A combination of DNA microarray hybridization and a genome-wide search for TnrA boxes allowed us to find 36 TnrA-regulated transcription units associated with a putative TnrA box . Gel retardation assaying, using probes carrying at least one putative TnrA box and the deletion derivatives of each box, indicated that 17 out of 36 transcription units were likely TnrA targets associated with the TnrA boxes, two of which (nasA and nasBCDEF) possessed a common TnrA box . The sequences of these TnrA boxes contained a consensus one, TGTNANAWWWTMTNACA . The TnrA targets detected in this study were nrgAB, pucJKLM, glnQHMP, nasDEF, oppABCDF, nasA, nasBCDEF and ywrD for positive regulation, and gltAB, pel, ywdIJK, yycCB, yttA, yxkC, ywlFG, yodF and alsT for negative regulation, nrgAB and gltAB being well-studied TnrA targets . It was unexpected that the negatively regulated TnrA targets were as many as the positively regulated targets . The physiological role of the TnrA regulon is discussed. Science, 2003 Jul 25, 301(5632), 510 - 3 Epub 2003 Jun 19. Cannibalism by sporulating bacteria; Gonzalez-Pastor JE et al.; Spore formation by the bacterium Bacillus subtilis is an elaborate developmental process that is triggered by nutrient limitation . Here we report that cells that have entered the pathway to sporulate produce and export a killing factor and a signaling protein that act cooperatively to block sister cells from sporulating and to cause them to lyse . The sporulating cells feed on the nutrients thereby released, which allows them to keep growing rather than to complete morphogenesis . We propose that sporulation is a stress-response pathway of last resort and that B . subtilis delays a commitment to spore formation by cannibalizing its siblings. J Bacteriol, 2003 Jul, 185(13), 3905 - 17 Expression of spoIIIJ in the prespore is sufficient for activation of sigma G and for sporulation in Bacillus subtilis; Serrano M et al.; During sporulation in Bacillus subtilis, the prespore-specific developmental program is initiated soon after asymmetric division of the sporangium by the compartment-specific activation of RNA polymerase sigma factor sigma(F) . sigma(F) directs transcription of spoIIIG, encoding the late forespore-specific regulator sigma(G) . Following synthesis, sigma(G) is initially kept in an inactive form, presumably because it is bound to the SpoIIAB anti-sigma factor . Activation of sigma(G) occurs only after the complete engulfment of the prespore by the mother cell . Mutations in spoIIIJ arrest sporulation soon after conclusion of the engulfment process and prevent activation of sigma(G) . Here we show that sigma(G) accumulates but is mostly inactive in a spoIIIJ mutant . We also show that expression of the spoIIIGE155K allele, encoding a form of sigma(G) that is not efficiently bound by SpoIIAB in vitro, restores sigma(G)-directed gene expression to a spoIIIJ mutant . Expression of spoIIIJ occurs during vegetative growth . However, we show that expression of spoIIIJ in the prespore is sufficient for sigma(G) activation and for sporulation . Mutations in the mother cell-specific spoIIIA locus are known to arrest sporulation just after completion of the engulfment process . Previous work has also shown that sigma(G) accumulates in an inactive form in spoIIIA mutants and that the need for spoIIIA expression for sigma(G) activation can be circumvented by the spoIIIGE155K allele . However, in contrast to the case for spoIIIJ, we show that expression of spoIIIA in the prespore does not support efficient sporulation . The results suggest that the activation of sigma(G) at the end of the engulfment process involves the action of spoIIIA from the mother cell and of spoIIIJ from the prespore. J Bacteriol, 2003 Jul, 185(13), 3813 - 20 Characterization of LytH, a differentiation-associated peptidoglycan hydrolase of Bacillus subtilis involved in endospore cortex maturation; Horsburgh GJ et al.; The cortex peptidoglycan from endospores of Bacillus subtilis is responsible for the maintenance of dormancy . LytH (YunA) has been identified as a novel sporulation-specific component with a role in cortex structure determination . The lytH gene was expressed only during sporulation, under the control of the mother cell-specific sigma factor sigma(K) . Spores of a lytH mutant have slightly reduced heat resistance and altered staining when viewed by electron microscopy . Analysis of the peptidoglycan structure of lytH mutant spores shows the loss of muramic acid residues substituted with L-alanine and a corresponding increase in muramic acid residues substituted with tetrapeptide compared to those in the parent strain . In a lytH cwlD mutant, the lack of muramic acid residues substituted with L-alanine and delta-lactam leaves 97% of residues substituted with tetrapeptide . These results suggest that lytH encodes an L-Ala-D-Glu peptidase involved in production of single L-alanine side chains from tetrapeptides in the spore cortex . The lack of di- or tripeptides in a lytH mutant reveals the enzyme is an endopeptidase. J Bacteriol, 2003 Jul, 185(13), 3711 - 7 Electrophoretic mobility of Bacillus subtilis knockout mutants with and without flagella; Okuda S et al.; Mutants of Bacillus subtilis 168 strain were obtained by inactivation of a specific gene by homologous recombination with the plasmid pMutinT3 . The cell surface properties of these strains were characterized by measuring the electrophoretic mobility of the cells as a function of pH and ionic strength . The surface properties were different for the strains possessing flagella on their cells and strain FlgB, having no flagellum, due to knockout of the corresponding gene . The cell surface properties of the strains possessing flagella become similar to those of strain FlgB after acid treatment . It was confirmed that the acid treatment degraded the flagella without causing any apparent structural change on the cell surface via observations made using atomic force microscopy, transmission electron microscopy, and scanning electron microscopy . These results indicate that the flagella are a key factor influencing cell surface properties. J Appl Microbiol, 2003, 95(1), 167 - 79 Studies on the mechanism of the osmoresistance of spores of Bacillus subtilis; Tovar-Rojo F et al.; AIMS: To determine the reason that spores of Bacillus species, in particular Bacillus subtilis, are able to form colonies with high efficiency on media with very high salt concentrations . METHODS AND RESULTS: Spores of various Bacillus species have a significantly higher plating efficiency on media with high salt concentration (termed osmoresistance) than do log or stationary phase cells . This spore osmoresistance is higher on richer media . Bacillus subtilis spores lacking various small, acid-soluble spore proteins (SASP) were generally significantly less osmoresistant than were wild-type spores, as shown previously (Ruzal et al . 1994) . Other results included: (a) spore osmoresistance varied significantly between species; (b) the osmoresistance of spores lacking SASP was not restored well by amino acid osmolytes added to plating media, but was completely restored by glucose; (c) the osmoresistance of spores lacking SASP was restored upon brief germination in the absence of salt in a process that did not require protein synthesis; (d) significant amounts of amino acids generated by SASP degradation were retained within spores upon germination in a medium with high but not low salt; (e) slowing but not abolishing SASP degradation by loss of the SASP-specific germination protease (GPR) did not affect spore osmoresistance; (f) sporulation at higher temperatures produced less osmoresistant spores; and (g) spore osmoresistance was not decreased markedly by the absence of the stress sigma factor for RNA polymerase, sigmaB . CONCLUSIONS: Spore osmoresistance appears as a result of three major factors: (1) specific characteristics of spores and cells of individual species; (2) the precise sporulation conditions that produce the spores; and (3) sufficient energy generation by the germinating and outgrowing spore to allow the spore to adapt to conditions of high osmotic strength; the substrates for this energy generation can come from either the endogenous generation of amino acids by SASP degradation or from the spore's environment, in the form of a readily taken up and metabolized energy source such as glucose . SIGNFICANCE AND IMPACT OF STUDY: These results provide information on the mechanisms of spore osmoresistance, a spore property that can be of major applied significance given the use of high osmotic strength with or without high salt as a means of food preservation. J Appl Microbiol, 2003, 95(1), 135 - 41 Modelling the combined effects of pH, temperature and sodium chloride stresses on the thermal inactivation of Bacillus subtilis spores in a buffer system; Jagannath A et al.; AIMS: The inactivation of Bacillus subtilis 168 spores subjected to the combined stress of pH, temperature and sodium chloride in a buffer system was modelled . METHODS AND RESULTS: Bacillus subtilis 168 spore suspension in 50 mmol l-1 potassium phosphate buffer was heated in an open system using a block heater . A second order polynomial equation was used to describe the relationship between pH, temperature, sodium chloride concentration and the logarithm of the decimal reduction time (D-value) of the spores . Response surface graphs were constructed to predict the inactivation within the experimental domain . The data obtained were also compared with those reported for B . subtilis in different media and foods included in a large reference-based database of thermal inactivation (ThermoKill Database, TKDB R9100), which was constructed in the laboratory . CONCLUSIONS: All the variables studied seemed to have a significant effect on the inactivation of B . subtilis 168 spores in potassium phosphate buffer . The coefficient of determination, r2, and an analysis of the residuals from the model indicated the adequacy of the model to predict the inactivation of B . subtilis spores within the range of the experimental variables studied . SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this study will enable a better understanding of the inactivation of B . subtilis spores under the influence of the studied environmental variables . The model can be used by food industries to assess and monitor the shelf life of food products in the event of a chance contamination by B . subtilis spores. J Appl Microbiol, 2003, 95(1), 54 - 67 Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide; Young SB et al.; AIMS: To determine the mechanisms of Bacillus subtilis spore killing by hypochlorite and chlorine dioxide, and its resistance against them . METHODS AND RESULTS: Spores of B . subtilis treated with hypochlorite or chlorine dioxide did not accumulate damage to their DNA, as spores with or without the two major DNA protective alpha/beta-type small, acid soluble spore proteins exhibited similar sensitivity to these chemicals; these agents also did not cause spore mutagenesis and their efficacy in spore killing was not increased by the absence of a major DNA repair pathway . Spore killing by these two chemicals was greatly increased if spores were first chemically decoated or if spores carried a mutation in a gene encoding a protein essential for assembly of many spore coat proteins . Spores prepared at a higher temperature were also much more resistant to these agents . Neither hypochlorite nor chlorine dioxide treatment caused release of the spore core's large depot of dipicolinic acid (DPA), but hypochlorite- and chlorine dioxide-treated spores much more readily released DPA upon a subsequent normally sub-lethal heat treatment than did untreated spores . Hypochlorite-killed spores could not initiate the germination process with either nutrients or a 1 : 1 chelate of Ca2+-DPA, and these spores could not be recovered by lysozyme treatment . Chlorine dioxide-treated spores also did not germinate with Ca2+-DPA and could not be recovered by lysozyme treatment, but did germinate with nutrients . However, while germinated chlorine dioxide-killed spores released DPA and degraded their peptidoglycan cortex, they did not initiate metabolism and many of these germinated spores were dead as determined by a viability stain that discriminates live cells from dead ones on the basis of their permeability properties . CONCLUSIONS: Hypochlorite and chlorine dioxide do not kill B . subtilis spores by DNA damage, and a major factor in spore resistance to these agents appears to be the spore coat . Spore killing by hypochlorite appears to render spores defective in germination, possibly because of severe damage to the spore's inner membrane . While chlorine dioxide-killed spores can undergo the initial steps in spore germination, these germinated spores can go no further in this process probably because of some type of membrane damage . SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanisms of the killing of bacterial spores by hypochlorite and chlorine dioxide. EMBO J, 2003 Jun 16, 22(12), 2893 - 902 Molecular architecture of the ATP-dependent CodWX protease having an N-terminal serine active site; Kang MS et al.; CodWX in Bacillus subtilis is an ATP-dependent, N-terminal serine protease, consisting of CodW peptidase and CodX ATPase . Here we show that CodWX is an alkaline protease and has a distinct molecular architecture . ATP hydrolysis is required for the formation of the CodWX complex and thus for its proteolytic function . Remarkably, CodX has a 'spool-like' structure that is formed by interaction of the intermediate domains of two hexameric or heptameric rings . In the CodWX complex, CodW consisting of two stacked hexameric rings (WW) binds to either or both ends of a CodX double ring (XX), forming asymmetric (WWXX) or symmetric cylindrical particles (WWXXWW) . CodWX can also form an elongated particle, in which an additional CodX double ring is bound to the symmetric particle (WWXXWWXX) . In addition, CodWX is capable of degrading EzrA, an inhibitor of FtsZ ring formation, implicating it in the regulation of cell division . Thus, CodWX appears to constitute a new type of protease that is distinct from other ATP-dependent proteases in its structure and proteolytic mechanism. DNA Cell Biol, 2003 Mar, 22(3), 209 - 15 First complete nucleotide sequence and heterologous gene organization of the two rRNA operons in the phytoplasma genome; Jung HY et al.; Phytoplasmas are cell-wallless Gram-positive low G + C bacteria belonging to the Mollicutes that inhabit the cytoplasm of plants and insects . Although phytoplasmas possess two ribosomal RNA (rrn) operons, only one has been fully sequenced . Here, we determined the complete nucleotide sequence of both rrn operons (designated rrnA and rrnB) of onion yellows (OY) phytoplasma . Both operons have rRNA genes organized as 5'-16S-23S-5S-3' with very highly conserved sequences; the 16S, 23S, and 5S rRNA genes are 99.9, 99.8, and 99.1% identical between the two operons . However, the organization of tRNA genes in the upstream region from 16S rRNA gene and in the downstream region from 5S rRNA gene differs markedly . Several promoter candidates were detected upstream from both operons, which suggests that both operons are functional . Interestingly, both have a tRNA(Ile) gene in the 16S-23S spacer region, while the reported rrnB operon of loofah witches' broom phytoplasma does not, indicating heterogenous gene organization of rrnB within phytoplasmas . The phytoplasma tRNA gene organization is similar to that of acholeplasmas, a closely related mollicute, and different from that of mycoplasmas, another mollicute . Moreover, the organization suggests that the rrn operons were derived from that of a related nonmollicute bacterium, Bacillus subtilis . This data should shed light on the evolutionary relationships and phylogeny of the mollicutes. J Med Chem, 2003 Jun 19, 46(13), 2731 - 9 Synthesis of substituted 6-anilinouracils and their inhibition of DNA polymerase IIIC and Gram-positive bacterial growth; Zhi C et al.; Certain substituted 6-anilinouracils are potent and selective inhibitors of Gram+ bacterial DNA polymerase IIIC (pol IIIC) . In addition, analogues with 3-substituents in the uracil ring have potent antibacterial activity against Gram+ organisms in culture . In an attempt to find optimal anilino substituents for pol IIIC binding and optimal 3-substituents for antibacterial activity, we have prepared several series of 3-substituted-6-aminouracils and assayed their activity against pol IIIC from Bacillus subtilis and a panel of Gram+ and Gram- bacteria in culture . The 6-(3-ethyl-4-methylanilino) group and closely related substituent patterns maximized pol IIIC inhibition potency . Among a series of 3-(substituted-butyl)-6-(3-ethyl-4-methylanilino)uracils, basic amino substituents increased pol IIIC inhibition, but decreased antibacterial activity . The most potent antibacterials were simple hydroxybutyl and methoxybutyl derivatives, and hydrophobically substituted piperidinylbutyl derivatives. Microbiol Immunol, 2003, 47(4), 279 - 83 An effective sporicidal reagent against Bacillus subtilis spores; Kida N et al.; We developed a reagent which showed significant sporicidal activity against Bacillus subtilis spores . This reagent was composed of ethylenediaminetetraacetic acid, disodium salt (EDTA-2Na), ferric chloride hexahydrate (FeCl3 x 6H2O) and ethanol (tentatively designated as the ethanol reagent) . The ethanol reagent showed pH- and temperature-dependent sporicidal activity . At pH 0.3, its activity was almost the same as that of 0.05% sodium hypochlorite at 20 C and was higher at 37 C than at 20 C . The activity of the ethanol reagent was similar both with and without 10% serum . The ethanol reagent might be applicable for disinfecting Bacillus spores. J Dairy Res, 2003 May, 70(2), 189 - 97 Antibacterial activity of casein-derived peptides isolated from rabbit (Oryctolagus cuniculus) milk; Baranyi M et al.; Acid-precipitated rabbit 'whole casein' was digested by trypsin, chymotrypsin, pepsin, and clostripain to screen for possible peptides with antibacterial properties . The peptide fragments were separated by reversed-phase chromatography . The collected fractions were pooled and their antibacterial properties tested against Escherichia coli, Bacillus subtilis and Staphylococcus lentus . Three antibacterial peptide fragments derived from tryptic digestion of rabbit casein were isolated and identified . Their sequences were found as follows: HVEQLLR (residues 50-56 of beta-casein), ILPFIQSLFPFAER (residues 64-77 of beta-casein), and FHLGHLK (residues 19-25 of alpha(s1)-casein) . The three peptides were synthesized and found to exert antibacterial effect against gram positive bacteria only . Proteolytic digestion of rabbit casein by chymotrypsin, pepsin and clostripain yielded several peptide fragments with antibacterial activity . Since antibiotic peptides can be released from casein during the digestion of milk proteins, our results suggest a possible antibacterial function of rabbit caseins . It is conceivable that antibacterial peptides can be generated by endopeptidases of the mammalian gastrointestinal tract possibly providing protection for new-born rabbits against aggression of micro-organisms. Nucleic Acids Res, 2003 Jun 15, 31(12), 3071 - 7 The nicking homing endonuclease I-BasI is encoded by a group I intron in the DNA polymerase gene of the Bacillus thuringiensis phage Bastille; Landthaler M et al.; Here we describe the discovery of a group I intron in the DNA polymerase gene of Bacillus thuringiensis phage Bastille . Although the intron insertion site is identical to that of the Bacillus subtilis phages SPO1 and SP82 introns, the Bastille intron differs from them substantially in primary and secondary structure . Like the SPO1 and SP82 introns, the Bastille intron encodes a nicking DNA endonuclease of the H-N-H family, I-BasI, with a cleavage site identical to that of the SPO1-encoded enzyme I-HmuI . Unlike I-HmuI, which nicks both intron-minus and intron-plus DNA, I-BasI cleaves only intron-minus alleles, which is a characteristic of typical homing endonucleases . Interestingly, the C-terminal portions of these H-N-H phage endonucleases contain a conserved sequence motif, the intron-encoded endonuclease repeat motif (IENR1) that also has been found in endonucleases of the GIY-YIG family, and which likely comprises a small DNA-binding module with a globular betabetaalphaalphabeta fold, suggestive of module shuffling between different homing endonuclease families. FEMS Microbiol Lett, 2003 Jun 6, 223(1), 101 - 6 Analysis of a DNA-binding motif of the Bacillus subtilis HrcA repressor protein; Wiegert T et al.; The hrcA gene of Bacillus subtilis encodes a transcriptional repressor protein which negatively controls the heat shock operons dnaK and groESL . Alignment of the HrcA protein with repressor proteins from the NCBI database revealed that it exhibits a striking homology near its N-terminal part with proteins of the DeoR family . This region contains a helix-turn-helix motif and has been shown to be involved in DNA binding . To investigate whether this is also true for the HrcA protein, three critical amino acid residues were changed within or adjacent to the recognition helix . While single amino acid replacements barely influenced the binding activity, alteration of two consecutive amino acid residues within the recognition helix completely abolished the binding activity . When this mutant hrcA allele was expressed together with the wild-type allele within the same cell, it conferred a dominant-negative phenotype to the cells underlining that these amino acid residues are crucial for specific DNA binding and that HrcA binds to DNA in an oligomeric form. FEMS Microbiol Lett, 2003 Jun 6, 223(1), 47 - 51 Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03; Cho SJ et al.; The Bacillus subtilis strain KS03 was isolated, and identified as a biological control agent that inhibits the anthracnose disease fungus Gloeosporium gloeosporioides . The antifungal compound was purified from its culture broth through butanol extraction, diethylaminoethyl (DEAE) Sepharose CL-6B chromatography, and preparative thin layer chromatography . Tandem mass spectrometric analyses (MS/MS), with matrix-assisted laser desorption ionization (MALDI) time-of-fight/time-of-flight (TOF/TOF) mass spectrometry, showed that the antifungal compound was iturin A, a cyclic lipopeptide antibiotic . The major compound, with a molecular mass of 1042 Da, was identified as iturin A(2). J Mol Biol, 2003 Jun 20, 329(5), 973 - 82 Crystal structure of the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase from Bacillus subtilis; Steinbacher S et al.; Two types of isopentenyl diphosphate:dimethylallyl diphosphate isomerases (IDI) have been characterized at present . The long known IDI-1 is only dependent on divalent metals for activity, whereas IDI-2 requires a metal, FMN and NADPH . Here, we report the first structure of an IDI-2 from Bacillus subtilis at 1.9A resolution in the ligand-free form and of the FMN-bound form at 2.8A resolution . The enzyme is an octamer that forms a D4 symmetrical open, cage-like structure . The monomers of 45 kDa display a classical TIM barrel fold . FMN is bound only with very moderate affinity and is therefore completely lost during purification . However, the enzyme can be reconstituted in the crystals by soaking with FMN . Three glycine-rich sequence stretches that are characteristic for IDI-2 participate in FMN binding within the interior of the cage . Regions harboring strictly conserved residues that are implicated in substrate binding or catalysis remain largely disordered even in the presence of FMN. J Agric Food Chem, 2003 Jun 18, 51(13), 3770 - 5 TAXI type endoxylanase inhibitors in different cereals; Goesaert H et al.; An affinity-based purification procedure with the immobilized family 11 Bacillus subtilis endoxylanase XynA allowed us to obtain high yields of highly pure endoxylanase inhibitor fractions from rye, barley, and durum wheat . In contrast, no inhibitors interacting with the B . subtilis endoxylanase affinity column are present in corn, buckwheat, rice, and oats . Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and inhibitor specificity showed that the isolated inhibitors belonged to the TAXI endoxylanase inhibitor family, thus providing a view on the diversity of this cereal inhibitor family . The isolated inhibitors are basic proteins of ca . 40 kDa, occurring in two molecular forms, with pI values of ca . 8.5 (durum wheat) and ca . 9.0 (rye, barley) . They are, in general, strong inhibitors of family 11 endoxylanases but not of family 10 endoxylanases . Because cereal endogenous endoxylanases belong to the latter family, this probably indicates that they do not influence cereal metabolic processes . For the first time, endoxylanase inhibitors, similar to TAXI I and TAXI II from wheat, were isolated from durum wheat and characterized . For each cereal, high-resolution cation exchange chromatography revealed the presence of multiple isoinhibitors, each of which occurs in two molecular forms . However, in durum wheat and barley, a single isoform is predominantly present. Chembiochem, 2003 Jun 6, 4(6), 485 - 93 A molecular mechanism of enantiorecognition of tertiary alcohols by carboxylesterases; Henke E et al.; Carboxylesterases containing the sequence motif GGGX catalyze the hydrolysis of esters of chiral tertiary alcohols, albeit with only low to moderate enantioselectivity, for three model substrates (linalyl acetate, methyl-1-pentin-1-yl acetate, 2-phenyl-3-butin-2-yl acetate) . In order to understand the molecular mechanism of enantiorecognition and to improve enantioselectivity for this interesting substrate class, the interaction of both enantiomers with the substrate binding sites of acetylcholinesterases and p-nitrobenzyl esterase from Bacillus subtilis was modeled and correlated to experimental enantioselectivity . For all substrate-enzyme pairs, enantiopreference and ranking by enantioselectivity could be predicted by the model . In p-nitrobenzyl esterase, one of the key residues in determining enantioselectivity was G105: exchange of this amino acid for an alanine residue led to a sixfold increase of enantioselectivity (E = 19) towards 2-phenyl-3-butin-2-yl acetate . However, the effect of this mutation is specific: the same mutant had the opposite enantiopreference towards the substrate linalyl acetate . Thus, depending on the substrate structure, the same mutant has either increased enantioselectivity or opposite enantiopreference compared to the wild-type enzyme. Microbiol Mol Biol Rev, 2003 Jun, 67(2), 157 - 74, table of contents RNA processing and degradation in Bacillus subtilis; Condon C; This review focuses on the enzymes and pathways of RNA processing and degradation in Bacillus subtilis, and compares them to those of its gram-negative counterpart, Escherichia coli . A comparison of the genomes from the two organisms reveals that B . subtilis has a very different selection of RNases available for RNA maturation . Of 17 characterized ribonuclease activities thus far identified in E . coli and B . subtilis, only 6 are shared, 3 exoribonucleases and 3 endoribonucleases . Some enzymes essential for cell viability in E . coli, such as RNase E and oligoribonuclease, do not have homologs in B . subtilis, and of those enzymes in common, some combinations are essential in one organism but not in the other . The degradation pathways and transcript half-lives have been examined to various degrees for a dozen or so B . subtilis mRNAs . The determinants of mRNA stability have been characterized for a number of these and point to a fundamentally different process in the initiation of mRNA decay . While RNase E binds to the 5' end and catalyzes the rate-limiting cleavage of the majority of E . coli RNAs by looping to internal sites, the equivalent nuclease in B . subtilis, although not yet identified, is predicted to scan or track from the 5' end . RNase E can also access cleavage sites directly, albeit less efficiently, while the enzyme responsible for initiating the decay of B . subtilis mRNAs appears incapable of direct entry . Thus, unlike E . coli, RNAs possessing stable secondary structures or sites for protein or ribosome binding near the 5' end can have very long half-lives even if the RNA is not protected by translation. J Biol Chem, 2003 Aug 22, 278(34), 32219 - 26 Epub 2003 Jun 06. The functional glycosyltransferase signature sequence of the human beta 1,3-glucuronosyltransferase is a XDD motif; Gulberti S et al.; The human beta 1,3-glucuronosyltransferase I (GlcAT-I) is the key enzyme responsible for the completion of glycosaminoglycan-protein linkage tetrasaccharide of proteoglycans (GlcA beta 1,3Gal beta 1,3Gal beta 1,4Xyl beta 1-O-serine) . We have investigated the role of aspartate residues Asp194-Asp195-Asp196 corresponding to the glycosyltransferase DXD signature motif, in GlcAT-I function by UDP binding experiments, kinetic analyses, and site-directed mutagenesis . We presented the first evidence that Mn2+ is not only essential for GlcAT-I activity but is also required for cosubstrate binding . In agreement, kinetic studies were consistent with a metal-activated enzyme model whereby activation probably occurs via binding of a Mn2+.UDP-GlcA complex to the enzyme . Mutational analysis showed that the Asp194-Asp195-Asp196 motif is a major element of the UDP/Mn2+ binding site . Furthermore, determination of the individual role of each aspartate showed that substitution of Asp195 as well as Asp196 to alanine strongly impaired GlcAT-I activity, whereas Asp194 replacement produced only a moderate alteration of the enzyme activity . These findings along with molecular modeling and three-dimensional structure comparison of the GlcAT-I catalytic center with that of the Bacillus subtilis glycosyltransferase SpsA provided evidence that the interactions of Asp195 with the ribose moiety of UDP and of Asp196 with the metal cation Mn2+ were crucial for GlcAT-I function . Altogether, these results indicated that, similarly to the SpsA enzyme, the nucleotide binding site of GlcAT-I contains a XDD motif rather than a DXD motif. Mol Microbiol, 2003 Jun, 48(6), 1491 - 500 Consequences of reductive evolution for gene expression in an obligate endosymbiont; Wilcox JL et al.; The smallest cellular genomes are found in obligate symbiotic and pathogenic bacteria living within eukaryotic hosts . In comparison with large genomes of free-living relatives, these reduced genomes are rearranged and have lost most regulatory elements . To test whether reduced bacterial genomes incur reduced regulatory capacities, we used full-genome microarrays to evaluate transcriptional response to environmental stress in Buchnera aphidicola, the obligate endosymbiont of aphids . The 580 genes of the B . aphidicola genome represent a subset of the 4500 genes known from the related organism, Escherichia coli . Although over 20 orthologues of E . coli heat stress (HS) genes are retained by B . aphidicola, only five were differentially expressed after near-lethal heat stress treatments, and only modest shifts were observed . Analyses of upstream regulatory regions revealed loss or degradation of most HS (sigma32) promoters . Genomic rearrangements downstream of an intact HS promoter yielded upregulation of a functionally unrelated and an inactivated gene . Reanalyses of comparable experimental array data for E . coli and Bacillus subtilis revealed that genome-wide differential expression was significantly lower in B . aphidicola . Our demonstration of a diminished stress response validates reports of temperature sensitivity in B . aphidicola and suggests that this reduced bacterial genome exhibits transcriptional inflexibility. Cell, 2003 May 30, 113(5), 577 - 86 Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria; Mandal M et al.; Riboswitches are metabolite binding domains within certain messenger RNAs that serve as precision sensors for their corresponding targets . Allosteric rearrangement of mRNA structure is mediated by ligand binding, and this results in modulation of gene expression . We have identified a class of riboswitches that selectively recognizes guanine and becomes saturated at concentrations as low as 5 nM . In Bacillus subtilis, this mRNA motif is located on at least five separate transcriptional units that together encode 17 genes that are mostly involved in purine transport and purine nucleotide biosynthesis . Our findings provide further examples of mRNAs that sense metabolites and that control gene expression without the need for protein factors . Furthermore, it is now apparent that riboswitches contribute to the regulation of numerous fundamental metabolic pathways in certain bacteria. Appl Microbiol Biotechnol, 2003 Nov, 63(1), 35 - 41 Epub 2003 May 29. Development, validation, and applications of a new laboratory-scale indirect impedancemeter for rapid microbial control; Ribeiro T et al.; We introduce a new laboratory-scale impedance-meter which is specially intended for indirect technique . It consists of a software system enabling data acquisition via a connected bus which is wired to the measuring cells . These measuring cells are individual impedance-meters that can be activated independently of one another . In the current configuration, the device is slightly affected by temperature, but it can register as little as 10.9 micromol of CO(2) . With Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae cultures, the conductance responses were highly replicable and repeatable for inocula concentrations of 1-10(8) colony-forming units (CFU) ml(-1) . The main use for such devices could be the detection of contamination in foodstuffs . Several of these foodstuffs, when incubated at 37 degrees C, spontaneously release quite large amounts of CO(2) . Our impedance meter, however, was able to detect an E . coli presence in canned French beans at 2.35 x 10(-2) CFU ml(-1) and a S . cerevisiae contamination of apple puree in glass jars at 6.1 x 10(-3) CFU ml(-1) . The conductance response and the detection time (the time needed for a significant change in conductance) were correlated to the concentration of ampicillin (an antibiotic added to E . coli cultures) . The device is thus able to detect the presence of inhibitory compounds in milk or other foodstuffs . Some industrial assays are in process to complement these laboratory tests . Compared with other available techniques for CO(2) measurement (manometry, infrared, radioactive labeling), the technique put forward here appears to be the best compromise between sensitivity, technical constraints, and cost . A commercial version of the