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Brian Henderson - One of the best experts on this subject based on the ideXlab platform.
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identification of the monocyte activating motif in mycobacterium tuberculosis Chaperonin 60 1
Tuberculosis, 2013Co-Authors: Anthony R. M. Coates, Peter A. Lund, Alexander Liu, Brian HendersonAbstract:Evidence is emerging that moonlighting proteins, defined as proteins with more than one biological function, play important roles in bacterial virulence. The Mycobacterium tuberculosis chaperone, Chaperonin 60.1, is a potent stimulator of human monocyte cytokine synthesis and modulator of giant cell and osteoclast formation. Previously, we had shown that these moonlighting activities resided in the equatorial domain of this protein. In this study, through the generation of Chaperonin 60.1 amino acid sequence-deletion mutants and synthetic peptides, we have identified the minimal moonlighting site in this molecular chaperone responsible for monocyte activation as peptide sequence DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD, residues 461-491, in the equatorial domain, Modelling of this biologically active sequence in the M. tuberculosis Chaperonin 60.1 protein reveals a surface-exposed motif with significant α-helical structure.
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Chaperonin 60: a paradoxical, evolutionarily conserved protein family with multiple moonlighting functions
Biological Reviews, 2013Co-Authors: Brian Henderson, Mario A. Fares, Peter A. LundAbstract:Chaperonin 60 is the prototypic molecular chaperone, an essential protein in eukaryotes and prokaryotes, whose sequence conservation provides an excellent basis for phylogenetic analysis. Escherichia coli Chaperonin 60 (GroEL), the prototype of this family of proteins, has an established oligomeric-structure-based folding mechanism and a defined population of folding partners. However, there is a growing number of examples of Chaperonin 60 proteins whose crystal structures and oligomeric composition are at variance with GroEL, suggesting that additional complexities in the protein-folding function of this protein should be expected. In addition, many organisms have multiple Chaperonin 60 proteins, some of which have lost their protein-folding ability. It is emerging that this highly conserved protein has evolved a bewildering variety of additional biological functions - known as moonlighting functions - both within the cell and in the extracellular milieu. Indeed, in some organisms, it is these moonlighting functions that have been left after the loss of the protein-folding activity. This highlights the major paradox in the biology of Chaperonin 60. This article reviews the relationship between the folding and non-folding (moonlighting) activities of the Chaperonin 60 family and discusses current knowledge on their molecular evolution focusing on protein domains involved in the non-folding Chaperonin functions in an attempt to understand the emerging biology of this evolutionarily ancient protein family.
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Chaperonin 60: An Unexpected Cell Surface Receptor in Prokaryotes and Eukaryotes
Heat Shock Proteins, 2012Co-Authors: Brian HendersonAbstract:The Chaperonin (Cpn)60 protein is a fascinating molecule which, under different circumstances, exists on the cell surface or is secreted from the cell. In all the various compartments in which the Cpn60 protein is found in organisms ranging from bacteria, to invertebrates to vertebrates it has evolved a wide range of additional, moonlighting, functions. Among the most unusual of these functions is the ability of this protein to exist on cell surfaces and act like a conventional agonist receptor. This chapter reviews the unexpected receptor and adhesive functions of the Cpn60 pantheon.
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human Chaperonin 60 hsp60 stimulates bone resorption structure function relationships
Bone, 2003Co-Authors: Sajeda Meghji, M Lillicrap, Maria Maguire, P. Tabona, J.s.h Gaston, Stephen Poole, Brian HendersonAbstract:Abstract It is established that the molecular chaperone, Chaperonin 60, from various bacteria and from Homo sapiens has cell-cell signalling activity and is able to induce proinflammatory cytokine synthesis. We previously reported that Chaperonin 60 proteins from Gram-negative bacteria, but not mycobacteria, have the capacity to resorb cultured murine calvarial bone. We now report that lipopolysaccharide-low human recombinant Chaperonin 60 (Hsp60) is a relatively weak cytokine-inducing agonist but is a potent stimulator of murine calvarial bone resorption. The osteolytic activity of Hsp60 was significantly inhibited by indomethacin, interleukin-1 receptor antagonist, and osteoprotegerin, but 5-lipoxygenase inhibitors were less effective. Analysis of Hsp60 truncation mutants revealed that N-terminal mutants (Δ1–137, Δ1–358, and Δ1–465) retained bone resorbing activity. In contrast, a C-terminal truncation mutant (Δ1–26 + Δ466–573) was inactive. This suggests that the active domain in this protein is found within residues 466–573. It is now established that Hsp60 is present in the blood of the majority of the population with the normal range encompassing levels able to activate bone cells. The possibility exists that this protein could play a role in bone remodelling.
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Human Chaperonin 60 (Hsp60) stimulates bone resorption: structure/function relationships.
Bone, 2003Co-Authors: Sajeda Meghji, M Lillicrap, Maria Maguire, P. Tabona, J.s.h Gaston, Stephen Poole, Brian HendersonAbstract:Abstract It is established that the molecular chaperone, Chaperonin 60, from various bacteria and from Homo sapiens has cell-cell signalling activity and is able to induce proinflammatory cytokine synthesis. We previously reported that Chaperonin 60 proteins from Gram-negative bacteria, but not mycobacteria, have the capacity to resorb cultured murine calvarial bone. We now report that lipopolysaccharide-low human recombinant Chaperonin 60 (Hsp60) is a relatively weak cytokine-inducing agonist but is a potent stimulator of murine calvarial bone resorption. The osteolytic activity of Hsp60 was significantly inhibited by indomethacin, interleukin-1 receptor antagonist, and osteoprotegerin, but 5-lipoxygenase inhibitors were less effective. Analysis of Hsp60 truncation mutants revealed that N-terminal mutants (Δ1–137, Δ1–358, and Δ1–465) retained bone resorbing activity. In contrast, a C-terminal truncation mutant (Δ1–26 + Δ466–573) was inactive. This suggests that the active domain in this protein is found within residues 466–573. It is now established that Hsp60 is present in the blood of the majority of the population with the normal range encompassing levels able to activate bone cells. The possibility exists that this protein could play a role in bone remodelling.
Anthony R. M. Coates - One of the best experts on this subject based on the ideXlab platform.
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identification of the monocyte activating motif in mycobacterium tuberculosis Chaperonin 60 1
Tuberculosis, 2013Co-Authors: Anthony R. M. Coates, Peter A. Lund, Alexander Liu, Brian HendersonAbstract:Evidence is emerging that moonlighting proteins, defined as proteins with more than one biological function, play important roles in bacterial virulence. The Mycobacterium tuberculosis chaperone, Chaperonin 60.1, is a potent stimulator of human monocyte cytokine synthesis and modulator of giant cell and osteoclast formation. Previously, we had shown that these moonlighting activities resided in the equatorial domain of this protein. In this study, through the generation of Chaperonin 60.1 amino acid sequence-deletion mutants and synthetic peptides, we have identified the minimal moonlighting site in this molecular chaperone responsible for monocyte activation as peptide sequence DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD, residues 461-491, in the equatorial domain, Modelling of this biologically active sequence in the M. tuberculosis Chaperonin 60.1 protein reveals a surface-exposed motif with significant α-helical structure.
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the effect of m tuberculosis Chaperonin 60 1 on leukocyte migration
European Respiratory Journal, 2011Co-Authors: Yanira Riffovasquez, Anthony R. M. Coates, Clive P Page, Domenico SpinaAbstract:We have shown that M. tuberculosis Cpn60.1 inhibits allergic lung inflammation and bronchial hyperresponsiveness in mice (Riffo-Vasquez Y et al. Clin Exp All. 2004). Here we have evaluated the effect of lower concentrations of Cpn60.1 on lung eosinophilia and leukocyte/endothelial cell interaction in vivo. Balbl/c mice were immunized twice with ovalbumin (ova, 10mg/mouse i.p. in alum). From day 14 all mice were exposed to ova (3%) once daily for 3 days and Cpn 60.1 was given i.n.15 min earlier. Lung lavages were performed 24 h after the last exposure. To examine the recruitment of cells in the microvasculature of the cremaster muscle, Cpn60.1 at 1μg/mouse was given s.c. into the scrotal sac of ova-sensitized mice followed by 100ng of eotaxin 10 minutes later. Four hours later the animals were prepared for intravital microscopy. Cpn60.1 at 0.001-1 μg/mouse inhibited the migration of eosinophils into the airways (ova 44.3±8.6 vs ova/60.1: 12.7±1.42; 13.3±3.4; 13.4±4.3 and 9.4±2.2×104/ml, n=10 for 0.001-1 μg of Cpn60.1 respectively, p≤ 0.05). Four hours after eotaxin injection, saline treated mice showed a significant accumulation of cells in the extra vascular tissue (saline: 40±5 vs Cpn60.1: 2.3±1 cells/50μm2, n=6) and higher number of cells rolling along the vessel wall in 30 sec (saline: 12±1.12 vs Cpn60.1: 2.8±1.02) compared to Cpn60.1 treated mice. In contrast, the adherence of cells to the endothelial layer was higher in Cpn60.1 compared to saline treated mice (saline adhesion: 15±1.7 vs Cpn60.1: 22.2±4.8 cells/50μm, n=6). Cn60.1 inhibits leukocyte migration to the lung in response to ova and prevented leukocyte rolling along and transmigration across the vessel wall in vivo.
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Mycobacterial Heat Shock Protein 60s in the Induction and Regulation of Infectious Disease
Heat Shock Proteins, 2009Co-Authors: Anthony R. M. Coates, Ana CehovinAbstract:Mycobacterium tuberculosis expresses several heat shock protein 60s, also called Chaperonin 60 proteins. As well as being protein folding molecules, the Chaperonin 60 proteins have implications in bacterial diseases. Here we discuss the effect of the mycobacterial Chaperonins in the regulation of tuberculosis. The disease is characterized by the formation of granulomas, which are necessary for completing the life cycle of bacteria. The mycobacterial Chaperonins are released into the cellular environment and are potent pro-inflammatory inducers. An M. tuberculosis mutant lacking the Chaperonin 60.1 is not capable of inducing the granulomas in vivo. In vitro data also suggest an inhibitory role of mycobacterial Chaperonins. Taken together, the mycobacterial Chaperonin 60 proteins may control the immune responses during the tuberculosis infection.
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Chaperonin 60 and macrophage activation
Novartis Foundation Symposium, 2008Co-Authors: Anthony R. M. Coates, Ana CehovinAbstract:Eukaryotic and prokaryotic Chaperonin 60s (Cpn60s) activate macrophages to produce pro-inflammatory cytokines. CD14 and TLR4 have been proposed as potential Cpn receptors. In addition, Cpn60s can block LPS-induced activation. This is a dose-related effect, low concentrations block, and high concentrations activate. This may relate to the ability of Cpn60s to block inflammatory disease. Cpns are multiplex or moon-lighting proteins, with functions as molecular chaperones, in stress survival and as inflammatory modulators. A cpn60.1 knockout mutant does not induce a granulomatous response and cytokine levels, such as tumour necrosis factor are reduced in the tissues. These data suggest that Cpn60.1 may also function as a virulence factor.
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Cloning, expression and purification of three Chaperonin 60 homologues.
Journal of Chromatography B, 2003Co-Authors: Maria Maguire, Anthony R. M. Coates, Brian HendersonAbstract:The Chaperonin 60 (Cpn60) proteins have, in addition to their well-known functions of protein folding and protection, a range of intercellular signalling activities. As part of a study to investigate the biological activity of the Cpn60 proteins, particularly from pathogenic organisms, we have cloned and expressed three Cpn60 proteins from Homo sapiens, Helicobacter pylori and Chlamydia pneumoniae. The Cpn60 proteins were purified to apparent homogeneity using a combination of nickel column affinity chromatography and Reactive Red dye affinity columns. Insoluble protein was solubilised using 8 M urea and then re-folded on the nickel column by stepwise removal of the urea. The immunostimulant LPS was removed by addition of the antibiotic polymyxin B as part of the purification process.
Sean M. Hemmingsen - One of the best experts on this subject based on the ideXlab platform.
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Pyrosequencing of Chaperonin-60 (cpn60) amplicons as a means of determining microbial community composition.
Methods in Molecular Biology, 2011Co-Authors: John J. Schellenberg, Sean M. Hemmingsen, Janet E. Hill, Matthew G. Links, Geoffrey A. Peters, Tim J DumonceauxAbstract:The Chaperonin-60 universal target (cpn60 UT) is generated from a set of PCR primers and provides a universally conserved, phylogenetically informative sequence signature for determining the composition of microbial communities by DNA sequencing. Pyrosequencing of cpn60 UT amplicons is emerging as a next-generation tool for providing unprecedented sequencing depth and resolution of microbial communities in individual samples. Owing to the increase in sequencing depth, the dynamic range across which the presence and abundance of individual species can be sampled experimentally also increases, significantly improving our ability to investigate microbial community richness and diversity. The flexible format of the pyrosequencing reaction setup combined with the ability to pool samples through the use of multiplexing IDs makes the generation of microbial profiles based on the cpn60 UT both feasible and cost-effective. We describe here the methods we have developed for determining microbial community profiles by pyrosequencing of cpn60 UT amplicons, from generating amplicons to sequencing and data analysis.
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Enumeration of specific bacterial populations in complex intestinal communities using quantitative PCR based on the Chaperonin-60 target
Journal of Microbiological Methods, 2006Co-Authors: Tim J Dumonceaux, Sean M. Hemmingsen, Janet E. Hill, Seth A. Briggs, Kingsley Kwaku Amoako, Andrew G Van KesselAbstract:We used qPCR and the target gene Chaperonin-60 (cpn60) to enumerate Clostridium perfringens genomes in DNA extracts from contents of the chicken gastrointestinal tract with the aim of optimizing this methodology to enumerate any bacterium of interest. To determine the most accurate protocols for determining target species abundance, we compared various DNA extraction methods in combination with four methods for producing standard curves. Factors affecting accuracy included the co-purification of PCR inhibitors and/or fluorescence quenchers and the yield of target DNA in the extract. Anion exchange chromatography of the spiked test samples enabled accurate enumeration of C. perfringens using a standard curve comprised of a plasmid containing a fragment of C. perfringens cpn60. We used qPCR to enumerate C. perfringens and other intestinal bacteria in ileum and cecum samples from chickens that had been challenged with C. perfringens and compared the results with viable counts on corresponding selective agars. We conclude that qPCR-based molecular enumeration of target species in the gastrointestinal tract is feasible, but care must be taken in order to mitigate the effects of confounding factors that can affect the apparent cell count.
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Identification of pathogenic Helicobacter species by Chaperonin-60 differentiation on plastic DNA arrays.
Genomics, 2006Co-Authors: Luke Masson, Sean M. Hemmingsen, Janet E. Hill, Swee-han Goh, Christine Maynard, Roland Brousseau, Ana Paccagnella, Ryuichi Oda, Naoki KimuraAbstract:A microarray method for bacterial species identification based on cpn60 and 16S rDNA hybridization was developed. Specific cpn60 or 16S rDNA oligonucleotides from various Helicobacter or Campylobacter species were printed and immobilized onto a proprietary plastic solid support. Using universal primers, fragments derived from either cpn60 or 16S rDNA genes from single isolates or from a complex human waste sludge DNA sample spiked with Helicobacter pylori were biotinylated and hybridized to the plastic slide. Subsequent querying with a streptavidin-horseradish peroxidase conjugate followed by color development using tetramethylbenzidine resulted in accurate Helicobacter species identification with no cross-hybridization to either the 16S rDNA or the cpn60 sequence of a closely related strain of Campylobacter jejuni. The combination of a nonfluorescence visual detection system with a polymer-based DNA microarray slide has resulted in a molecular tool that should prove useful in numerous applications requiring rapid, low-cost bacterial species identification.
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comparison of ileum microflora of pigs fed corn wheat or barley based diets by Chaperonin 60 sequencing and quantitative pcr
Applied and Environmental Microbiology, 2005Co-Authors: Janet E. Hill, Sean M. Hemmingsen, Swee-han Goh, Blair G Goldade, Tim J Dumonceaux, Jonathan L Klassen, R T Zijlstra, Andrew G Van KesselAbstract:We have combined the culture-independent methods of high-throughput sequencing of Chaperonin-60 PCR product libraries and quantitative PCR to profile and quantify the small-intestinal microflora of pigs fed diets based on corn, wheat, or barley. A total of 2,751 Chaperonin-60 PCR product clones produced from samples of ileum digesta were examined. The majority (81%) of these clones contained sequences independently recovered from all three libraries; 372 different nucleotide sequences were identified, but only 14% of the 372 different sequences were recovered from all three libraries. Taxonomic assignments of the library sequences were made by comparison to a reference database of Chaperonin-60 sequences combined with phylogenetic analysis. The taxa identified are consistent with previous reports of pig ileum microflora. Frequencies of each sequence in each library were calculated to identify taxa that varied in frequency between the corn, barley, and wheat libraries. The Chaperonin-60 sequence inventory was used as a basis for designing PCR primer sets for taxon-specific quantitative PCR. Results of quantitative PCR analysis of ileum digesta confirmed the relative abundances of targeted taxa identified with the library sequencing approach. The results of this study indicate that Chaperonin-60 clone libraries can be valid profiles of complex microbial communities and can be used as the basis for producing quantitative PCR assays to measure the abundance of taxa of interest during experimentally induced or natural changes in a community.
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Characterization of vaginal microflora of healthy, nonpregnant women by Chaperonin-60 sequence-based methods.
American Journal of Obstetrics and Gynecology, 2005Co-Authors: Janet E. Hill, Swee-han Goh, Deborah Money, Melissa Doyle, William L. Crosby, Matthew G. Links, Amy Leung, Debbie Chan, Sean M. HemmingsenAbstract:Objective The purpose of this study was to use a novel method that was based on the application of Chaperonin-60 sequencing to describe the vaginal microflora of 16 healthy women. Study design Asymptomatic women consented for vaginal swabs to be collected at the time of a clinical pelvic examination. Total genomic DNA was isolated from the vaginal swabs. Degenerate, universal polymerase chain reaction primers were used to amplify an approximately 555 base pair region of the universal Chaperonin-60 gene, which is found in all eubacteria and eukaryotes, from the total genomic DNA and libraries of cloned polymerase chain reaction products were constructed. Library clones were sequenced, and the resulting sequences were assigned to taxonomic groups on the basis of similarity to reference sequence data. Presence of Chlamydophila psittaci sequences in the samples was confirmed by species-specific polymerase chain reaction. Results Sixteen of the 23 women who were enrolled had normal flora by Nugent's score of Lactobacillus spp L crispatus , L iners , L gasseri , L jensenii, and L buchneri . Other sequences that were identified included representatives of Gardnerella spp, sequences with similarity to Porphyromonas spp and Megasphaera spp and sequences identical to C psittaci . Conclusion Culture-independent, Chaperonin-60 sequence-based molecular methods can lead to the identification of greater diversity within defined taxa compared with those that are identified by standard culture-based methods and to the identification of novel organisms that were not previously associated with vaginal flora.
Janet E. Hill - One of the best experts on this subject based on the ideXlab platform.
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Pyrosequencing of Chaperonin-60 (cpn60) amplicons as a means of determining microbial community composition.
Methods in Molecular Biology, 2011Co-Authors: John J. Schellenberg, Sean M. Hemmingsen, Janet E. Hill, Matthew G. Links, Geoffrey A. Peters, Tim J DumonceauxAbstract:The Chaperonin-60 universal target (cpn60 UT) is generated from a set of PCR primers and provides a universally conserved, phylogenetically informative sequence signature for determining the composition of microbial communities by DNA sequencing. Pyrosequencing of cpn60 UT amplicons is emerging as a next-generation tool for providing unprecedented sequencing depth and resolution of microbial communities in individual samples. Owing to the increase in sequencing depth, the dynamic range across which the presence and abundance of individual species can be sampled experimentally also increases, significantly improving our ability to investigate microbial community richness and diversity. The flexible format of the pyrosequencing reaction setup combined with the ability to pool samples through the use of multiplexing IDs makes the generation of microbial profiles based on the cpn60 UT both feasible and cost-effective. We describe here the methods we have developed for determining microbial community profiles by pyrosequencing of cpn60 UT amplicons, from generating amplicons to sequencing and data analysis.
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Predicting relatedness of bacterial genomes using the Chaperonin-60 universal target (cpn60 UT): Application to Thermoanaerobacter species
Systematic and Applied Microbiology, 2011Co-Authors: Tobin J. Verbeke, Janet E. Hill, Matthew G. Links, Richard Sparling, David B. Levin, Tim J DumonceauxAbstract:D.R. Zeigler determined that the sequence identity of bacterial genomes can be predicted accurately using the sequence identities of a corresponding set of genes that meet certain criteria [32]. This three-gene model for comparing bacterial genome pairs requires the determination of the sequence identities for recN, thdF, and rpoA. This involves the generation of approximately 4.2 kb of genomic DNA sequence from each organism to be compared, and also normally requires that oligonucleotide primers be designed for amplification and sequencing based on the sequences of closely related organisms. However, we have developed an analogous mathematical model for predicting the sequence identity of whole genomes based on the sequence identity of the 542–567 base pair Chaperonin-60 universal target (cpn60 UT). The cpn60 UT is accessible in nearly all bacterial genomes with a single set of universal primers, and its length is such that it can be completely sequenced in one pair of overlapping sequencing reads via di-deoxy sequencing. These mathematical models were applied to a set of Thermoanaerobacter isolates from a wood chip compost pile and it was shown that both the one-gene cpn60 UT-based model and the three-gene model based on recN, rpoA, and thdF predicted that these isolates could be classified as Thermoanaerobacter thermohydrosulfuricus. Furthermore, it was found that the genomic prediction model using cpn60 UT gave similar results to whole-genome sequence alignments over a broad range of taxa, suggesting that this method may have general utility for screening isolates and predicting their taxonomic affiliations.
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pyrosequencing of the Chaperonin 60 universal target as a tool for determining microbial community composition
Applied and Environmental Microbiology, 2009Co-Authors: John J. Schellenberg, Janet E. Hill, Tim J Dumonceaux, Matthew G. Links, Geoffrey A. Peters, Shaun Tyler, Blake T Ball, Alberto Severini, Francis A PlummerAbstract:We compared dideoxy sequencing of cloned Chaperonin-60 universal target (cpn60 UT) amplicons to pyrosequencing of amplicons derived from vaginal microbial communities. In samples pooled from a number of individuals, the pyrosequencing method produced a data set that included virtually all of the sequences that were found within the clone library and revealed an additional level of taxonomic richness. However, the relative abundances of the sequences were different in the two datasets. These observations were expanded and confirmed by the analysis of paired clone library and pyrosequencing datasets from vaginal swabs taken from four individuals. Both for individuals with a normal vaginal microbiota and for those with bacterial vaginosis, the pyrosequencing method revealed a large number of low-abundance taxa that were missed by the clone library approach. In addition, we showed that the pyrosequencing method generates a reproducible profile of microbial community structure in replicate amplifications from the same community. We also compared the taxonomic composition of a vaginal microbial community determined by pyrosequencing of 16S rRNA amplicons to that obtained using cpn60 universal primers. We found that the profiles generated by the two molecular targets were highly similar, with slight differences in the proportional representation of the taxa detected. However, the number of operational taxonomic units was significantly higher in the cpn60 data set, suggesting that the protein-encoding gene provides improved species resolution over the 16S rRNA target. These observations demonstrate that pyrosequencing of cpn60 UT amplicons provides a robust, reliable method for deep sequencing of microbial communities.
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Enumeration of specific bacterial populations in complex intestinal communities using quantitative PCR based on the Chaperonin-60 target
Journal of Microbiological Methods, 2006Co-Authors: Tim J Dumonceaux, Sean M. Hemmingsen, Janet E. Hill, Seth A. Briggs, Kingsley Kwaku Amoako, Andrew G Van KesselAbstract:We used qPCR and the target gene Chaperonin-60 (cpn60) to enumerate Clostridium perfringens genomes in DNA extracts from contents of the chicken gastrointestinal tract with the aim of optimizing this methodology to enumerate any bacterium of interest. To determine the most accurate protocols for determining target species abundance, we compared various DNA extraction methods in combination with four methods for producing standard curves. Factors affecting accuracy included the co-purification of PCR inhibitors and/or fluorescence quenchers and the yield of target DNA in the extract. Anion exchange chromatography of the spiked test samples enabled accurate enumeration of C. perfringens using a standard curve comprised of a plasmid containing a fragment of C. perfringens cpn60. We used qPCR to enumerate C. perfringens and other intestinal bacteria in ileum and cecum samples from chickens that had been challenged with C. perfringens and compared the results with viable counts on corresponding selective agars. We conclude that qPCR-based molecular enumeration of target species in the gastrointestinal tract is feasible, but care must be taken in order to mitigate the effects of confounding factors that can affect the apparent cell count.
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Identification of pathogenic Helicobacter species by Chaperonin-60 differentiation on plastic DNA arrays.
Genomics, 2006Co-Authors: Luke Masson, Sean M. Hemmingsen, Janet E. Hill, Swee-han Goh, Christine Maynard, Roland Brousseau, Ana Paccagnella, Ryuichi Oda, Naoki KimuraAbstract:A microarray method for bacterial species identification based on cpn60 and 16S rDNA hybridization was developed. Specific cpn60 or 16S rDNA oligonucleotides from various Helicobacter or Campylobacter species were printed and immobilized onto a proprietary plastic solid support. Using universal primers, fragments derived from either cpn60 or 16S rDNA genes from single isolates or from a complex human waste sludge DNA sample spiked with Helicobacter pylori were biotinylated and hybridized to the plastic slide. Subsequent querying with a streptavidin-horseradish peroxidase conjugate followed by color development using tetramethylbenzidine resulted in accurate Helicobacter species identification with no cross-hybridization to either the 16S rDNA or the cpn60 sequence of a closely related strain of Campylobacter jejuni. The combination of a nonfluorescence visual detection system with a polymer-based DNA microarray slide has resulted in a molecular tool that should prove useful in numerous applications requiring rapid, low-cost bacterial species identification.
F R Tabita - One of the best experts on this subject based on the ideXlab platform.
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interaction of inactivated and active ribulose 1 5 bisphosphate carboxylase oxygenase of rhodobacter sphaeroides with nucleotides and the Chaperonin 60 groel protein
Journal of Bacteriology, 1992Co-Authors: Xing Wang, F R TabitaAbstract:Purified inactivated form I ribulose 1,5-bisphosphate carboxylase/oxygenase (form I RubisCO) of Rhodobacter sphaeroides was activated by ATP and, to some extent, by other adenylates and nucleotides. Reactivation in the presence of ATP occurred by a time-dependent and concentration-dependent process which appeared to be irreversible. The carbamylated form of inactivated form I RubisCO was less susceptible to ATP-mediated reactivation than the uncarbamylated inactivated enzyme. In some cases, ATP analogs could mimic the reactivation process; one analog, adenylyl(beta, gamma-methylene)-diphosphonate, was found to partially block ATP-mediated reactivation but could not block reactivation induced by Mg(II). Concomitant with the recovery of enzymatic activity, the migration of the inactivated form I RubisCO on nondenaturing and sodium dodecyl sulfate gels changed from a pattern that was characteristic of inactivated enzyme to a pattern that was identical to that of the active protein. It was further found that discrete proportions of active enzyme and the Chaperonin 60 protein of R. sphaeroides aggregated in the presence of ATP. The form I RubisCO is thus proposed to contain a specific ATP-binding site that may contribute to both the regulation of activity and the assembly of active enzyme.
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Interaction of inactivated and active ribulose 1,5-bisphosphate carboxylase/oxygenase of Rhodobacter sphaeroides with nucleotides and the Chaperonin 60 (GroEL) protein.
Journal of Bacteriology, 1992Co-Authors: Xing Wang, F R TabitaAbstract:Purified inactivated form I ribulose 1,5-bisphosphate carboxylase/oxygenase (form I RubisCO) of Rhodobacter sphaeroides was activated by ATP and, to some extent, by other adenylates and nucleotides. Reactivation in the presence of ATP occurred by a time-dependent and concentration-dependent process which appeared to be irreversible. The carbamylated form of inactivated form I RubisCO was less susceptible to ATP-mediated reactivation than the uncarbamylated inactivated enzyme. In some cases, ATP analogs could mimic the reactivation process; one analog, adenylyl(beta, gamma-methylene)-diphosphonate, was found to partially block ATP-mediated reactivation but could not block reactivation induced by Mg(II). Concomitant with the recovery of enzymatic activity, the migration of the inactivated form I RubisCO on nondenaturing and sodium dodecyl sulfate gels changed from a pattern that was characteristic of inactivated enzyme to a pattern that was identical to that of the active protein. It was further found that discrete proportions of active enzyme and the Chaperonin 60 protein of R. sphaeroides aggregated in the presence of ATP. The form I RubisCO is thus proposed to contain a specific ATP-binding site that may contribute to both the regulation of activity and the assembly of active enzyme.
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purification and characterization of the Chaperonin 10 and Chaperonin 60 proteins from rhodobacter sphaeroides
Biochemistry, 1991Co-Authors: K C Terlesky, F R TabitaAbstract:Two heat-shock proteins that show high identity with the Escherichia coli Chaperonin 60 (groEL) and Chaperonin 10 (groES) Chaperonin proteins were purified and characterized from photolithoautotrophically grown Rhodobacter sphaeroides. The proteins were purified by using sucrose density gradient centrifugation and Mono-Q anion-exchange chromatography. In the presence of 1 mM ATP, the Chaperonin 10 and Chaperonin 60 proteins bound to each other and comigrated as a large complex during sucrose density gradient centrifugation. The native molecular weights of each protein as determined by gel filtration chromatography were 889,200 for Chaperonin 60 and 60,000 for Chaperonin 10. Chaperonin 60 is comprised of monomers with a molecular weight of 61,000 and Chaperonin 10 is comprised of monomers with a molecular weight of 12,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chaperonin 60 was 9.3% of the total soluble cell protein during photolithoautotrophic growth which increased to 28.5% following heat-shock treatment. When cells were grown photoheterotrophically or chemoheterotrophically, Chaperonin 60 was reduced to 6.7% and 3.5%, respectively, of the total soluble protein. The N-terminal amino acid sequence of each protein was determined; Chaperonin 60 of R. sphaeroides showed 72% identity to E. coli Chaperonin 60 protein, and R. sphaeroides Chaperonin 10 showed 45% identity with E. coli Chaperonin 10. R. sphaeroides Chaperonin 60 catalyzed ATP hydrolysis with a specific activity of 134 nmol min-1 mg-1 (kcat = 0.13 s-1) and was inhibited by R. sphaeroides Chaperonin 10, but not E. coli Chaperonin 10. The E. coli Chaperonin 60 ATPase activity was inhibited by Chaperonin 10 from both R. sphaeroides and E. coli.