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| Appl Microbiol Biotechnol . 2004 Dec 22; {Epub ahead of print} Cloning, recombinant expression and biochemical characterisation of novel esterases from Bacillus sp . associated with the marine sponge Aplysina aerophoba; Karpushova A et al.; Two novel esterases (EstB1 and EstB2) were isolated from a genomic library of Bacillus sp . associated with the marine sponge Aplysina aerophoba . EstB1 shows low identity (26-44%) with the published hydrolases of the genus Bacillus, whereas EstB2 shows high identity (73-74%) with the carboxylesterases from B . cereus and B . anthracis . Both esterases were efficiently expressed in Escherichia coli under the control of T7 promoter using the vector pET-22b(+) . Recombinant EstB1 was purified in a single step to electrophoretic homogeneity by IMAC . A method for the refolding of inclusion bodies formed by the recombinant EstB2 was established to obtain active enzyme . Substrate specificity of the two enzymes towards p-nitrophenyl and methyl esters and the respective kinetic parameters K(m) and V(max) were determined . The temperature optima of EstB1 and EstB2 were determined to be in the range of 30-50 degrees C and 20-35 degrees C, respectively . The pH optima were found to be in the range of 6.5-7.5 and 6.5-8.0, respectively . Both enzymes showed the highest stability in up to 50% (v/v) DMSO followed by methanol, ethanol and 2-propanol . The influence of high NaCl and KCl concentrations was tested . The inhibition effect of 10-50 mM Zn(2+) and 50 mM Mg(2+) and Ca(2+) ions was observed for both esterases . One to five millimolar PMSF deactivated the enzymes, whereas beta-mercaptoethanol, DTT and EDTA had no effect on the enzymes activity. J Bacteriol, 2004 Nov, 186(22), 7714 - 25 The bcr1 DNA repeat element is specific to the Bacillus cereus group and exhibits mobile element characteristics; Okstad OA et al.; Bacillus cereus strains ATCC 10987 and ATCC 14579 harbor an approximately 155-bp repeated element, bcr1, which is conserved in B . cereus, B . anthracis, B . thuringiensis, and B . mycoides but not in B . subtilis and B . licheniformis . In this study, we show by Southern blot hybridizations that bcr1 is present in all 54 B . cereus group strains tested but absent in 11 Bacillus strains outside the group, suggesting that bcr1 may be specific and ubiquitous to the B . cereus group . By comparative analysis of the complete genome sequences of B . cereus ATCC 10987, B . cereus ATCC 14579, and B . anthracis Ames, we show that bcr1 is exclusively present in the chromosome but absent from large plasmids carried by these strains and that the numbers of full-length bcr1 repeats for these strains are 79, 54, and 12, respectively . Numerous copies of partial bcr1 elements are also present in the three genomes (91, 128, and 53, respectively) . Furthermore, the genomic localization of bcr1 is not conserved between strains with respect to chromosomal position or organization of gene neighbors, as only six full-length bcr1 loci are common to at least two of the three strains . However, the intergenic sequence surrounding a specific bcr1 repeat in one of the three strains is generally strongly conserved in the other two, even in loci where bcr1 is found exclusively in one strain . This finding indicates that bcr1 either has evolved by differential deletion from a very high number of repeats in a common ancestor to the B . cereus group or is moving around the chromosome . The identification of bcr1 repeats interrupting genes in B . cereus ATCC 10987 and ATCC 14579 and the presence of a flanking TTTAT motif in each end show that bcr1 exhibits features characteristic of a mobile element. BMC Microbiol . 2004 Oct 07;4(1):39. Branched-chain amino acid aminotransferase and methionine formation in Mycobacterium tuberculosis; Venos ES et al.; BACKGROUND: Tuberculosis remains a major world-wide health threat which demands the discovery and characterisation of new drug targets in order to develop future antimycobacterials . The regeneration of methionine consumed during polyamine biosynthesis is an important pathway present in many microorganisms . The final step of this pathway, the conversion of ketomethiobutyrate to methionine, can be performed by aspartate, tyrosine, or branched-chain amino acid aminotransferases depending on the particular species examined . RESULTS: The gene encoding for branched-chain amino acid aminotransferase in Mycobacterium tuberculosis H37Rv has been cloned, expressed, and characterised . The enzyme was found to be a member of the aminotransferase IIIa subfamily, and closely related to the corresponding aminotransferase in Bacillus subtilis, but not to that found in B . anthracis or B . cereus . The amino donor preference for the formation of methionine from ketomethiobutyrate was for isoleucine, leucine, valine, glutamate, and phenylalanine . The enzyme catalysed branched-chain amino acid and ketomethiobutyrate transamination with a Km of 1.77 - 7.44 mM and a Vmax of 2.17 - 5.70 micromol/min/mg protein, and transamination of ketoglutarate with a Km of 5.79 - 6.95 mM and a Vmax of 11.82 - 14.35 micromol/min/mg protein . Aminooxy compounds were examined as potential enzyme inhibitors, with O-benzylhydroxylamine, O-t-butylhydroxylamine, carboxymethoxylamine, and O-allylhydroxylamine yielding mixed-type inhibition with Ki values of 8.20 - 21.61 microM . These same compounds were examined as antimycobacterial agents against M . tuberculosis and a lower biohazard M . marinum model system, and were found to completely prevent cell growth . O-Allylhydroxylamine was the most effective growth inhibitor with an MIC of 78 microM against M . marinum and one of 156 microM against M . tuberculosis . CONCLUSION: Methionine formation from ketomethiobutyrate is catalysed by a branched-chain amino acid aminotransferase in M . tuberculosis . This enzyme can be inhibited by selected aminooxy compounds, which also have effectiveness in preventing cell growth in culture . These compounds represent a starting point for the synthesis of branched-chain aminotransferase inhibitors with higher activity and lower toxicity. Org Lett, 2004 Oct 14, 6(21), 3801 - 4 Preparation of peptide p-nitroanilides using an aryl hydrazine resin; Kwon Y et al.; {reaction: see text} Peptide p-nitroanilides are useful compounds for studying protease activity; however, the poor nucleophilicity of p-nitroaniline makes their preparation difficult . We describe a new efficient approach for the Fmoc-based synthesis of peptide p-nitroanilides using an aryl hydrazine resin . Mild oxidation of the peptide hydrazide resin yields a highly reactive acyl diazene that efficiently reacts with weak nucleophiles . We have prepared several peptide p-nitroanilides, including substrates for the Lethal Factor protease from B . anthracis. J Mass Spectrom, 2004 Oct, 39(10), 1113 - 21 Complete sequences of small acid-soluble proteins from Bacillus globigii; Whiteaker JR et al.; Three abundant small acid-soluble proteins (SASPs) from spores of Bacillus globigii were sequenced using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with post-source decay and nanoelectrospray collision-induced dissociation tandem mass spectrometry . The proteins were extracted from spores with 1 M HCl . Scanning electron micrographs of spores before and after acid extraction show that the spores retain their overall structure but have a shriveled texture following the acid treatment . Extracted SASPs were purified by high-performance liquid chromatography and molecular masses of the SASPs were identified at 7068 (SASP-1), 7332 (SASP-2), and 8889 (gamma-SASP) . De novo peptide sequencing was used to determine the protein sequences . The correct ordering of peptide sequences was aided by mapping overlapping enzymatic digests and by comparison with homologous SASPs from Bacillus stearothermophilus . B . globigii is used in many field tests as a surrogate for B . anthracis . Thus complete SASP sequences from B . globigii will facilitate the development of methods for rapid identification of bacteria based on mass spectrometry and the examination of taxonomic relationships between Bacillus species . 2004 John Wiley & Sons, Ltd. Proteomics, 2004 Oct, 4(10), 2877 - 92 A targeted proteomics approach to the rapid identification of bacterial cell mixtures by matrix-assisted laser desorption/ionization mass spectrometry; Warscheid B et al.; A proteomic approach to the rapid identification of bacteria is presented, which relies on the solubilization of a limited number of proteins from intact cells combined with on-probe tryptic digestion . Within 20 min, complete cleavage products of a limited set of bacterial proteins with molecular masses of about 4-125 kDa were obtained by on-probe digestion with immobilized trypsin . Bacterial peptides suitable for unimolecular decomposition analysis were generated within 5 min, and the sequence information obtained allowed identification of abundant proteins, and accordingly, their bacterial sources via searches in the NCBI database . Analysis of fragmentation products was also shown to allow for identification of bacterial peptides identical in mass but differing slightly in amino acid sequence by manual data analysis . In this work, Bacillus subtilis 168, B . globigii, B . sphaericus 14577, B . cereus T, and B . anthracis Sterne were examined, and various cold shock proteins were identified in all species . In addition, DNA-binding, 60 kDa-heat shock, surface-related and other stress-protective proteins were identified in the bacterial cell digests, and species-specific tryptic peptides could be generated from each of the Bacillus species studied . Bacterial peptides could be analyzed with greater sensitivity and mass accuracy than the parent proteins . The applicability of this targeted proteomics approach to the rapid identification of Bacillus species was further established by analyzing binary cell mixtures. Infect Immun, 2004 Sep, 72(9), 5253 - 61 Population structure of the Bacillus cereus group as determined by sequence analysis of six housekeeping genes and the plcR Gene; Ko KS et al.; The population structure of the Bacillus cereus group (52 strains of B . anthracis, B . cereus, and B . thuringiensis) was investigated by sequencing seven gene fragments (rpoB, gyrB, pycA, mdh, mbl, mutS, and plcR) . Most of the strains were classifiable into two large subgroups in six housekeeping gene trees but not in the plcR tree . In addition, several consistent clusters were identified, which were unrelated to species distinction . Moreover, interrelationships among these clusters were incongruent in each gene tree . The incongruence length difference test and split decomposition analyses also showed incongruences between genes, suggesting horizontal gene transfer . The plcR gene was observed to have characteristics that differed from those of the other genes in terms of phylogenetic topology and pattern of sequence diversity . Thus, we suggest that the evolutionary history of the PlcR regulon differs from those of the other chromosomal genes and that recombination of the plcR gene may be frequent . The homogeneity of B . anthracis, which is depicted as an independent lineage in phylogenetic trees, is suggested to be of recent origin or to be due to the narrow taxonomic definition of species. Anal Bioanal Chem, 2004 Mar, 378(6), 1587 - 93 A generic sandwich-type biosensor with nanomolar detection limits; Baeumner AJ et al.; A quantitative and highly sensitive, yet simple and rapid, biosensor system was developed for the detection of nucleic acid sequences that can also be adapted to the detection of antigens . A dipstick-type biosensor with liposome amplification, based on a sandwich assay format with optical detection, was combined with a simple coupling reaction that allows the transformation of the generic biosensor components to target specific ones by a mere incubation step . This biosensor platform system was developed and optimized, and its principle was proven using DNA oligonucleotides that provided a nucleic acid biosensor for the specific detection of RNA and DNA sequences . However, the coupling reaction principle chosen can also be used for the immobilization of antibodies or receptor molecules, and therefore for the development of immunosensors and receptor-based biosensors . The generic biosensor consists of liposomes entrapping sulforhodamine B that are coated with streptavidin on the outside, and polyethersulfone membranes with anti-fluorescein antibodies immobilized in the detection zone . In order to transform the generic biosensor into a specific DNA/RNA biosensor, two oligonucleotides that are able to hybridize to the target sequence were labeled with a biotin and a fluorescein molecule, respectively . By simultaneously incubating the liposomes, both oligonucleotides, and the target sequence in a hybridization buffer for 20-30 min at 42 degrees C, a sandwich complex was formed . The mixture was applied to the polyethersulfone membrane . The complex was captured in the detection zone and quantified using a hand-held reflectometer . The system was tested using RNA sequences from B . anthracis, C . parvum and E . coli . Quantitation of concentrations between 10 fmol and 1000 fmol (10-1000 nM) was possible without altering any biosensor assay conditions . In addition, no changes to hybridization conditions were required when using authentic nucleic acid sequence-based amplified RNA sequences, and the generic biosensor compared favorably with those previously developed specifically for the RNA sequences . Therefore, the universal biosensor described is an excellent tool, for use in laboratories or at test sites, for rapidly investigating and quantifying any nucleic acid sequence of interest, as well as potentially any antigen of interest that can be bound by two antibodies simultaneously. J Clin Microbiol, 2004 Apr, 42(4), 1626 - 30 Reassessment of sequence-based targets for identification of bacillus species; Blackwood KS et al.; The Bacillus genus is a large heterogeneous group in need of an efficient method for species differentiation . To determine the current validity of a sequence-based method for identification and provide contemporary data, PCR and sequencing of a 500-bp product encompassing the V1 to V3 regions of the 16S rRNA gene were undertaken using 65 of the 83 type strains of this genus . This region proved discriminatory between most species (70.0 to 100% similarity), the exceptions being clinically relevant B . cereus and B . anthracis as well as nonpathogenic B . psychrotolerans and B . psychrodurans . Consequently, 27 type and clinical strains from the B . cereus group were used to test alternate targets (rpoB, vrrA, and the 16S-23S spacer region) for identification . The rpoB gene proved the best alternate target, with a conserved 4-nucleotide difference between B . cereus and B . anthracis . The high 16S rRNA gene sequence similarities between some strains demonstrated the need for a polyphasic approach to the systematics of this genus . This approach is one focus of the Ribosomal Differentiation of Medical Microorganisms mandate . Accordingly, the 16S rRNA gene sequences generated in this study have been submitted for inclusion into its publicly accessible, quality-controlled database at http://www.ridom_rdna.de/. J Public Health Manag Pract, 2004 Jan-Feb, 10(1), 77 - 85 Evaluation of the Washington State National Pharmaceutical Stockpile dispensing exercise, part II--dispensary site worker findings; Beaten RD et al.; On January 24, 2002, the Washington State Department of Health, in collaboration with local and federal agencies, conducted an exercise of the Centers for Disease Control and Prevention's National Pharmaceutical Stockpile dispensing portion of the Washington State plan . This exercise included predrill planning, training, and the orchestration of services of more than 40 dispensary site workers . These workers provided education and post-exposure prophylaxis for over 230 patient volunteers in the aftermath of a simulated exposure to B . anthracis . This article discusses findings of a postdrill questionnaire completed by 90% of these dispensary site workers who provided triage, education, dispensary, security and other services during this exercise . In general, this dispensing drill promoted confidence in the worker participants and provided an opportunity for these participants to coordinate their activities . This mock bioterrorist preparedness exercise allowed worker participants and observers to review and evaluate the Washington State plan for dispensing the National Pharmaceutical Stockpile . This article is apparently the first published account of dispensary site workers' subjective impressions and quantitative analysis of their postdrill opinions following a simulated bioterrorist post-exposure chemoprophylaxis dispensing exercise. BMC Bioinformatics . 2004 Jan 12;5(1):4. Identification of polymorphic tandem repeats by direct comparison of genome sequence from different bacterial strains: a web-based resource; Denoeud F et al.; BACKGROUND: Polymorphic tandem repeat typing is a new generic technology which has been proved to be very efficient for bacterial pathogens such as B . anthracis, M . tuberculosis, P . aeruginosa, L . pneumophila, Y . pestis . The previously developed tandem repeats database takes advantage of the release of genome sequence data for a growing number of bacteria to facilitate the identification of tandem repeats . The development of an assay then requires the evaluation of tandem repeat polymorphism on well-selected sets of isolates . In the case of major human pathogens, such as S . aureus, more than one strain is being sequenced, so that tandem repeats most likely to be polymorphic can now be selected in silico based on genome sequence comparison . RESULTS: In addition to the previously described general Tandem Repeats Database, we have developed a tool to automatically identify tandem repeats of a different length in the genome sequence of two (or more) closely related bacterial strains . Genome comparisons are pre-computed . The results of the comparisons are parsed in a database, which can be conveniently queried over the internet according to criteria of practical value, including repeat unit length, predicted size difference, etc . Comparisons are available for 16 bacterial species, and the orthopox viruses, including the variola virus and three of its close neighbors . CONCLUSIONS: We are presenting an internet-based resource to help develop and perform tandem repeats based bacterial strain typing . The tools accessible at now comprise four parts . The Tandem Repeats Database enables the identification of tandem repeats across entire genomes . The Strain Comparison Page identifies tandem repeats differing between different genome sequences from the same species . The "Blast in the Tandem Repeats Database" facilitates the search for a known tandem repeat and the prediction of amplification product sizes . The "Bacterial Genotyping Page" is a service for strain identification at the subspecies level. Appl Environ Microbiol, 2004 Jan, 70(1), 191 - 201 Multilocus sequence typing scheme for bacteria of the Bacillus cereus group; Helgason E et al.; In this study we developed a multilocus sequence typing (MLST) scheme for bacteria of the Bacillus cereus group . This group, which includes the species B . cereus, B . thuringiensis, B . weihenstephanensis, and B . anthracis, is known to be genetically very diverse . It is also very important because it comprises pathogenic organisms as well as bacteria with industrial applications . The MLST system was established by using 77 strains having various origins, including humans, animals, food, and soil . A total of 67 of these strains had been analyzed previously by multilocus enzyme electrophoresis, and they were selected to represent the genetic diversity of this group of bacteria . Primers were designed for conserved regions of housekeeping genes, and 330- to 504-bp internal fragments of seven such genes, adk, ccpA, ftsA, glpT, pyrE, recF, and sucC, were sequenced for all strains . The number of alleles at individual loci ranged from 25 to 40, and a total of 53 allelic profiles or sequence types (STs) were distinguished . Analysis of the sequence data showed that the population structure of the B . cereus group is weakly clonal . In particular, all five B . anthracis isolates analyzed had the same ST . The MLST scheme which we developed has a high level of resolution and should be an excellent tool for studying the population structure and epidemiology of the B . cereus group. Anal Chem, 2003 Oct 15, 75(20), 5618 - 27 Characterization of Bacillus spore species and their mixtures using postsource decay with a curved-field reflectron; Warscheid B et al.; A strategy is proposed for the rapid identification of Bacillus spores, which relies on the selective release of a family of proteins, referred to as small, acid-soluble spore proteins (SASPs) . In this work, SASPs were selectively solubilized from Bacillus spores on the MALDI sample plate by using 10% TFA . Proteolytic digests of SASPs generated in situ from spores of B . subtilis 168, B . globigii, B . thuringiensis subs . Kurstaki HD-1, B . cereus T, and the nonpathogenic strain B . anthracis Sterne were prepared in 5-25 min by using trypsin immobilized on Agarose beads and subsequently analyzed by MALDI-TOFMS using a curved-field reflectron . Protein identification was obtained by partial sequencing of distinctive tryptic peptides from Bacillus spores via post-source decay analysis combined with genome-based database searches by Mascot Sequence Query . Various unique SASPs were identified, allowing the characterization of Bacillus species by obtaining sequence-specific information on single peptides . The applicability of this approach for the rapid identification of Bacillus species was further established by analyzing spore mixtures. Med Dosw Mikrobiol, 2003, 55(1), 41 - 6 {Survival of Bacillus antracis spores in baths using modern technologic tannery processes}; Mendrycka M et al.; The influence of skin tannery baths, according as wet-blue, wet-white and plant technology, on B . anthracis spores survival was investigated . As a result of this study there was explained that lime bath do not inactivated of all spores that are present in infected bath . As a result of these experiments the spores were inactivated completely not before pickle bath. Public Health Rep, 2003 Mar-Apr, 118(2), 92 - 8 Epidemiologic clues to bioterrorism; Treadwell TA et al.; Public health investigators have successfully carried out epidemiologic investigations of outbreaks of disease for many years . By far the majority of these outbreaks have occurred naturally . With the recent illnesses resulting from deliberate dissemination of B . anthracis on an unsuspecting population, public health investigation of diseases must now include consideration of bioterrorism as a potential cause of outbreaks of disease . The features of naturally occurring outbreaks have a certain amount of predictability in terms of consistency with previous occurrences, or at least biological plausibility . However, with a deliberately introduced outbreak or infection among a population, this predictability is minimized . In this paper, the authors propose some epidemiologic clues that highlight features of outbreaks that may be suggestive of bioterrorism . They also describe briefly the general process of involvement of agencies at various levels of government, public health and non-public health, depending on the extent of an outbreak or level of suspicion. Wei Sheng Wu Xue Bao, 2001 Apr, 41(2), 141 - 7 {Cloning of parasporal body protein gene resembling to S-layer protein genes from Bacillus thuringiensis CTC strain}; Sun M et al.; Bacillus CTC strain was identified as Bacillus thuringiensis subsp . finitimus (serotype H2) and Pasteur Institute confirmed this identification . The parasporal body formed by CTC strain is oval shaped, and consists of 100 kD protein . The determination of the N-terminal amino acid sequence showed this protein shares 93% similarity to that of B . anthracis S-layer proteins . The restriction map covering the related protein gene (ctc) was deduced according to southern hybridization . The DNA fragments containing the 5' and 3' end of ctc gene with 0.6 kb overlap were isolated, respectively . Full length ctc gene was then constructed . B . thuringiensis is a species similar to B . cereus and B . anthracis, and only can be distinguished by forming parasporal bodies . The growth of E . coli maintaining ctc gene is similar to that of E . coli expressing S-layer protein gene . The above preliminary results showed that the parasporal body protein in CTC strain is a cell surface-like protein . So identifying CTC strain as a species of B . thuringiensis and the unique criterion for the identification of B . thuringiensis by forming parasporal body should be reconsidered. Curr Top Microbiol Immunol, 2002, 271, 115 - 41 Macrophage interactions; Guidi-Rontani C et al.; B . anthracis virulence is the sum of the contributions of factors involved in toxicity, growth and persistence in the host . Recent data has revealed that the interactions between B . anthracis and macrophage is central to the B . anthracis pathogenesis . This review presents and describes tactics by which B . anthracis not only overcomes and avoids macrophages but also perverts the host defense immune system and defense-related products to its advantage . The understanding of the complex network of such interactions is likely to allow new therapeutic and preventative strategies to be developed. Mol Cell Probes, 2002 Apr, 16(2), 119 - 27 Sequence-specific identification of 18 pathogenic microorganisms using microarray technology; Wilson WJ et al.; We have developed a Multi-Pathogen Identification (MPID) microarray for high confidence identification of eighteen pathogenic prokaryotes, eukaryotes and viruses . Analysis of amplified products from pathogen genomic DNA using microarray hybridization allows for highly specific and sensitive detection, and allows the discrimination between true amplification products and false positive amplification products that might be derived from primers annealing to non-target sequences . Species-specific primer sets were used to amplify multiple diagnostic regions unique to each individual pathogen . Amplified products were washed over the surface of the microarray, and labelled with phycoerythrin-streptavidin for fluorescence detection . A series of overlapping 20-mer oligonucleotide probes hybridize to the entire diagnostic region, while parallel hybridizations on the same surface allow simultaneous screening for all organisms . Comparison to probes that differ by a single mismatch at the central position reduced the contribution of non-specific hybridization . Samples containing individual pathogens were analyzed in separate experiments and the corresponding species-specific diagnostic regions were identified by fluorescence among their highly redundant probe sets . On average, 91% of the 53 660 pathogen probes on the MPID microarray performed as predicted . The limit of detection was found to be as little as 10 fg of B . anthracis DNA in samples that were amplified with six diagnostic primer-pairs . In contrast, PCR products were not observed at this concentration when identical samples were prepared and visualized by agarose gel electrophoresis . Eur J Biochem, 2002 Jan, 269(2), 458 - 69 Identification and properties of type I-signal peptidases of Bacillus amyloliquefaciens; Chu HH et al.; The use of Bacillus amyloliquefaciens for enzyme production and its exceptional high protein export capacity initiated this study where the presence and function of multiple type I signal peptidase isoforms was investigated . In addition to type I signal peptidases SipS(ba) {Meijer, W.J.J., de Jong, A., Bea, G., Wisman, A., Tjalsma, H., Venema, G., Bron, S . & van Dijl, J.M . (1995) Mol . Microbiol . 17, 621-631} and SipT(ba) {Hoang, V . & Hofemeister, J . (1995) Biochim . Biophys . Acta 1269, 64-68} which were previously identified, here we present evidence for two other Sip-like genes in B . amyloliquefaciens . Same map positions as well as sequence motifs verified that these genes encode homologues of Bacillus subtilis SipV and SipW . SipU-encoding DNA was not found in B . amyloliquefaciens . SipW-encoding DNA was also found for other Bacillus strains representing different phylogenetic groups, but not for Bacillus stearothermophilus and Thermoactinomyces vulgaris . The absence of these genes, however, could have been overlooked due to sequence diversity . Sequence alignments of 23 known Sip-like proteins from Bacillus origin indicated further branching of the P-group signal peptidases into clusters represented by B . subtilis SipV, SipS-SipT-SipU and B . anthracis Sip3-Sip5 proteins, respectively . Each B . amyloliquefaciens sip(ba) gene was expressed in an Escherichia coli LepBts mutant and tested for genetic complementation of the temperature sensitive (TS) phenotype as well as pre-OmpA processing . Although SipS(ba) as well as SipT(ba) efficiently restored processing of pre-OmpA in E . coli, only SipS(ba) supported growth at TS conditions, indicating functional diversity . Changed properties of the sip(ba) gene disruption mutants, including cell autolysis, motility, sporulation, and nuclease activities, seemed to correlate with specificities and/or localization of B . amyloliquefaciens SipS, SipT and SipV isoforms. PDA J Pharm Sci Technol, 2001 May-Jun, 55(3), 150 - 61 Application of recovery tests in the validation of immunoassays for assessing the immunogenicity of B . anthracis PA vaccine; Halperin G et al.; In the quantitative assessment of polyclonal serum antibodies, the complex composition and characteristics of the analyte population (serum antibodies) restricts the capability of constructing appropriately defined calibration standards . This fact limits the application of the conservative recovery tests to the validation of immunoassays aimed at determining serum antibody levels . The present report describes a modification of recovery tests that overcomes this impediment . The modified approach is based on a dilution analysis system, where a given immune serum is serially diluted in normal serum and the antibody titers in each of the derived diluted samples are then determined . Expected sample titers (calculated on the basis of the relevant dilution factors) are plotted against the respective observed results, and the resulting recovery curve is then examined by means of a regression analysis, according to the standard rules of the conservative recovery analysis . This approach was tried with two immunoassay systems, Enzyme Linked Immunosorbent Assay (ELISA) and Neutralizing Antibodies (NtAb) immunoassays, aimed at assessing the immunogenicity in guinea pigs of B . anthracis protective antigen (PA) vaccine . In a series of feasibility studies using a recovery simulation model (dilutions made in the immunoassay diluent, rather than in normal serum) the average recovery levels in ELISA and NtAb immunoassays were 0.99 +/- 0.011 and 1.02 +/- 0.04 respectively, and the 99% confidence intervals contained the target 100% value . Regression lines were proved to be linear demonstrating R > 0.97 in all cases . The 99% confidence intervals around the observed slopes and intercepts always contained the corresponding target values 1 and 0 . The relative standard deviation (RSD) in the ELISA and NtAb immunoassays was found to be 0.01 and 0.025 respectively . All of the above experimental results were not affected by the serum antibody titer, or by day-to-day variations embodied in these immunoassay systems . When true recovery tests were applied to the above immunoassays, essentially identical results were obtained . In both assays the correlation coefficients were in the range of 0.96-1, recoveries were found to be in the range of 0.90-1.06, and RSD values were in the range of 0.02-0.025 . All the recovery deviations from the target value of 1 were not statistically significant . The hitherto observed experimental findings illustrate the capability of the dilution analysis system to allow the application of recovery tests to the validation of quantitative immunoassays, which are based on the procedure of serum titrations. Lett Appl Microbiol, 2000 Sep, 31(3), 242 - 6 Molecular recognition specificity of Bacillus globigii spore antibodies; Longchamp P et al.; Western blotting methods have been used to assess the specificity of polyclonal antibodies raised against Bacillus globigii spore and vegetative cell preparations . None of the antibodies studied were completely species-specific in their recognition of spore surface epitopes . One polyclonal serum recognized several spore surface epitopes and demonstrated limited cross-reaction with the spore surface of the near-neighbour species B . subtilis . A second polyclonal serum, raised against aged spore antigens, recognized damaged spore epitopes primarily . Both of these antibodies also cross-reacted with vegetative cell epitopes present in all four Bacillus species (B . globigii, B . subtilis, B . cereus and B . anthracis) studied. Appl Environ Microbiol, 2000 Sep, 66(9), 3828 - 34 Rapid characterization of spores of Bacillus cereus group bacteria by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry; Ryzhov V et al.; Matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry was used to characterize the spores of 14 microorganisms of the Bacillus cereus group . This group includes the four Bacillus species B . anthracis, B . cereus, B . mycoides, and B . thuringiensis . MALDI mass spectra obtained from whole bacterial spores showed many similarities between the species, except for B . mycoides . At the same time, unique mass spectra could be obtained for the different B . cereus and B . thuringiensis strains, allowing for differentiation at the strain level . To increase the number of detectable biomarkers in the usually peak-poor MALDI spectra of spores, the spores were treated by corona plasma discharge (CPD) or sonicated prior to MALDI analysis . Spectra of sonicated or CPD-treated spores displayed an ensemble of biomarkers common for B . cereus group bacteria . Based on the spectra available, these biomarkers differentiate B . cereus group spores from those of Bacillus subtilis and Bacillus globigii . The effect of growth medium on MALDI spectra of spores was also explored. Res Microbiol, 2000 Jun, 151(5), 361 - 8 On the fate of ingested Bacillus spores; Spinosa MR et al.; Spores of various Bacillus species, including B . subtilis, B . cereus and B . clausii, are used as probiotics, although they are generally absent from the normal microflora of man . We used two nonpathogenic Bacillus species, B . subtilis and B . clausii, to follow the fate of spores inoculated intragastrically in mice . We did not find detectable amounts of vegetative cells in intestinal samples, probably because of high toxicity of the conjugated bile salt taurodeoxycholic acid against Bacillus species . Both spores and cells were detected in the lymph nodes and spleen of one mouse . Our results indicate that Bacillus is present in the intestinal tract solely as spores and that nonpathogenic Bacillus spores may germinate in lymphoid organs, a finding reminiscent of B . anthracis germination in macrophages . These results indicate that any claimed probiotic effect of B . subtilis should be due to spores or, alternatively, to vegetative growth outside the intestine. J Appl Microbiol, 1999 Oct, 87(4), 481 - 90 Enterotoxigenic profiles and polymerase chain reaction detection of Bacillus cereus group cells and B . cereus strains from foods and food-borne outbreaks; Hsieh YM et al.; Bacillus cereus is one of the important food pathogens . Since B . cereus group cells, such as B . cereus, B . thuringiensis, B . anthracis and B . mycoides, share many phenotypical properties and a high level of chromosomal sequence similarity, it is interesting to investigate the virulence profiles for B . cereus group cells, including B . cereus strains isolated from foods and samples associated with food-poisoning outbreaks . For this investigation, the presence of enterotoxin genes, such as those of haemolysin BL, B . cereus enterotoxin T and enterotoxin FM, were assayed by polymerase chain reaction (PCR) methods . Meanwhile, their enterotoxin activities were assayed using the BCET-RPLA kit, haemolytic patterns on sheep blood agar and their cytotoxicity to Chinese hamster ovary (CHO) cells . Results showed that there were 12 enterotoxigenic profiles for the 98 B . cereus group strains collected . In addition, if any of the three types of enterotoxins was present in the B . cereus group cells, these cells were shown to be cytotoxic to the CHO cells . Similar enterotoxigenic profiles could be found among strains of B . cereus, B . mycoides and B . thuringiensis . Thus, all B . cereus group strains may be potentially toxigenic and the detection of these cells in foods is important . We thus designed PCR primers, termed Ph1/Ph2, from the sphingomyelinase gene of B . cereus cells . These primers were specific for all B . cereus group strains and could be used for the detection of B . cereus cells contaminated in food samples.
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