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1.
Rodríguez-Quiñones F Maguire M Wallington EJ Gould PS Yerko V Downie JA Lund PA 《Archives of microbiology》2005,183(4):253-265
Although many bacteria contain only a single groE operon encoding the essential chaperones GroES and GroEL, examples of bacteria containing more than one groE operon are common. The root-nodulating bacterium Rhizobium leguminosarum contains at least three operons encoding homologues to Escherichia coli GroEL, referred to as Cpn60.1, Cpn60.2 and Cpn60.3, respectively. We report here a detailed analysis of the requirement for and relative levels of these three proteins. Cpn60.1 is present at higher levels than Cpn60.2, and Cpn60.3 protein could not be detected under any conditions although the cpn60.3 gene is transcribed under anaerobic conditions. Insertion mutations could not be constructed in cpn60.1 unless a complementing copy was present, showing that this gene is essential for growth under the conditions used here. Both cpn60.2 and cpn60.3 could be inactivated with no loss of viability, and a double cpn60.2 cpn60.3 mutant was also constructed which was fully viable. Thus only Cpn60.1 is required for growth of this organism.Dedicated to the memory of Professor V. Javier Benedí, 1957–2002 相似文献
2.
Mycobacterium tuberculosis expresses two proteins (Cpn60.1 and Cpn60.2) that belong to the chaperonin (Cpn) family of heat shock proteins. Studies have shown that the two proteins have different functional roles in the bacterial life cycle and that Cpn60.2 is essential for cell viability and may be involved in M. tuberculosis pathogenicity. Cpn60.2 does not form a tetradecameric double ring, which is typical of other Cpns. We have determined the crystal structure of recombinant Cpn60.2 to 2.8 Å resolution by molecular replacement; the asymmetric unit (AU) contains a dimer, which is consistent with size-exclusion high-performance liquid chromatography and dynamic light-scattering measurements of the soluble recombinant protein. However, we suggest that the actual Cpn60.2 dimer may be different from that identified within the AU on the basis of surface contact stability, solvation free-energy gain, and functional aspects. Unlike the dimer found in the AU, which is formed through apical domain interactions, the dimeric form we propose here provides a free apical domain that is required for normal chaperone activity and may be involved in M. tuberculosis association with macrophages and arthrosclerosis plaque formation. Here we describe in detail the structural aspects that lead to Cpn60.2 dimer formation and prevent the formation of heptameric rings and tetradecameric double rings. 相似文献
3.
Homologous cpn60 genes in Rhizobium leguminosarum are not functionally equivalent 总被引:1,自引:0,他引:1
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Many bacteria possess 2 or more genes for the chaperonin GroEL and the cochaperonin GroES. In particular, rhizobial species often have multiple groEL and groES genes, with a high degree of amino-acid similarity, in their genomes. The Rhizobium leguminosarum strain A34 has 3 complete groE operons, which we have named cpn.1, cpn.2 and cpn.3. Previously we have shown the cpn. 1 operon to be essential for growth, but the two other cpn operons to be dispensable. Here, we have investigated the extent to which loss of the essential GroEL homologue Cpn60.1 can be compensated for by expression of the other two GroEL homologues (Cnp60.2 and Cpn60.3). Cpn60.2 could not be overexpressed to high levels in R. leguminosarum, and was unable to replace Cpn60.1. A strain that overexpressed Cpn60.3 grew in the absence of Cpn60.1, but the complemented strain displayed a temperature-sensitive phenotype. Cpn60.1 and Cpn60.3, when coexpressed in Escherichia coli, preferentially selfassembled rather than forming mixed heteroligomers. We conclude that, despite their high amino acid similarity, the GroEL homologues of R. leguminosarum are not functionally equivalent in vivo. 相似文献
4.
Tyler B. M. Hickey Hermann J. Ziltener David P. Speert Richard W. Stokes 《Cellular microbiology》2010,12(11):1634-1647
CD43 is a large sialylated glycoprotein found on the surface of haematopoietic cells and has been previously shown to be necessary for efficient macrophage binding and immunological responsiveness to Mycobacterium tuberculosis. Using capsular material from M. tuberculosis and recombinant CD43‐Fc, we have employed affinity chromatography to show that Cpn60.2 (Hsp65, GroEL), and to a lesser extent DnaK (Hsp70), bind to CD43. Competitive inhibition using recombinant protein and polyclonal F(ab′)2 antibody‐mediated epitope masking studies were used to evaluate M. tuberculosis binding to CD43+/+ versus CD43?/? macrophages. Results showed that Cpn60.2, but not DnaK, acts as a CD43‐dependent mycobacterial adhesin for macrophage binding. Assessment of the specific binding between Cpn60.2 and CD43 showed it to be saturable, with a comparatively weak affinity in the low micromolar range. We have also shown that the ability of Cpn60.2 to competitively inhibit M. tuberculosis binding to macrophages is shared by the Escherichia coli homologue, GroEL, but not by the mouse and human Hsp60 homologues. These findings add to a growing field of research that implicates molecular chaperones as having extracellular functions, including bacterial adherence to host cells. Thus, CD43 may act as a Pattern Recognition Receptor (PRR) for bacterial homologues of the 60 kDa molecular chaperone. 相似文献
5.
Chaperonin-60s are large double ring oligomeric proteins with a central cavity where unfolded polypeptides undergo productive folding. In conjunction with their co-chaperonin, Chaperonin-60s bind non-native polypeptides and facilitate their refolding in an ATP-dependent manner. The ATPase activity of Chaperonin-60 is tightly regulated by the 10 kDa co-chaperonin. In contrast to most other bacterial species, Mycobacterium tuberculosis genome carries a duplicate set of cpn60 genes, one of which occurs on the groESL operon (cpn60.1), while the other is separately arranged on the chromosome (cpn60.2). Biophysical characterization of the mycobacterial proteins showed that these proteins exist as lower oligomers and not tetradecamers, an unexpected property much different from the other known Chaperonin-60s. Failure of the M.tuberculosis chaperonins to oligomerize can be attributed to amino acid mutations at the oligomeric interface. Rates of ATP hydrolysis of the M.tuberculosis chaperonins showed that these proteins possess a very weak ATPase activity. Both the M.tuberculosis chaperonins were partially active in refolding substrate proteins. Interestingly, their refolding activity was seen to be independent of the co-chaperonin and ATP. We hypothesize that the ATP independent chaperones might offer benefit to the pathogen by promoting its existence in the latent phase of its life cycle. 相似文献
6.
7.
Bacteriophage-encoded cochaperonins can substitute for Escherichia coli's essential GroES protein
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The Escherichia coli chaperonin machine is composed of two members, GroEL and GroES. The GroEL chaperonin can bind 10–15% of E. coli’s unfolded proteins in one of its central cavities and help them fold in cooperation with the GroES cochaperonin. Both proteins are absolutely essential for bacterial growth. Several large, lytic bacteriophages, such as T4 and RB49, use the host-encoded GroEL in conjunction with their own bacteriophage-encoded cochaperonin for the correct assembly of their major capsid protein, suggesting a cochaperonin specificity for the in vivo folding of certain substrates. Here, we demonstrate that, when the cochaperonin of either bacteriophage T4 (Gp31) or RB49 (CocO) is expressed in E. coli, the otherwise essential groES gene can be deleted. Thus, it appears that, despite very little sequence identity with groES, the bacteriophage-encoded Gp31 and CocO proteins are capable of replacing GroES in the folding of E. coli’s essential, housekeeping proteins. 相似文献
8.
Schlüter A Wiesgigl M Hoyer C Fleischer S Klaholz L Schmetz C Clos J 《Biochimica et biophysica acta》2000,1491(1-3):65-74
We have identified two diverged members of the cpn60 gene family in Leishmania donovani, causative agent of Indian Kala Azar. One of the genes, cpn60.1, although actively transcribed, is not expressed to detectable levels of protein in cultured L. donovani. The other gene, cpn60.2, which, compared with cpn60.1, shows a higher sequence conservation with the hsp60 genes from Trypanosoma brucei and Trypanosoma cruzi is expressed constitutively in cultured promastigotes. The abundance of the gene product, Cpn60.2, increases by 2.5-fold under heat stress and in axenic amastigotes of L. donovani. Cpn60.2 is also found enriched in mitochondrial cell fractions and localizes to the mitochondrial matrix. We conclude that Cpn60.2 is the major mitochondrial chaperonin in Leishmania. 相似文献
9.
Rhizobium leguminosarum chaperonin 60.3, but not chaperonin 60.1, induces cytokine production by human monocytes: activity is dependent on interaction with cell surface CD14
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Lewthwaite J George R Lund PA Poole S Tormay P Sharp L Coates AR Henderson B 《Cell stress & chaperones》2002,7(2):130-136
As part of a program of work to understand the interaction of bacterial chaperonins with human leukocytes, we have examined 2 of the 3 chaperonin 60 (Cpn 60) gene products of the nonpathogenic plant symbiotic bacterium, Rhizobium leguminosarum, for their capacity to induce the production of pro- and antiinflammatory cytokines by human cells. Recombinant R. leguminosarum Cpn 60.1 and 60.3 proteins were added to human monocytes at a range of concentrations, and cytokine production was measured by sandwich enzyme-linked immunosorbent assay. In spite of the fact that the 2 R. leguminosarum Cpn 60 proteins share 74.5% amino acid sequence identity, it was found that Cpn 60.3 induced the production of interleukin (IL)-1beta, tumor necrosis factor alpha, IL-6, IL-8, IL-10, and IL-12, but not IL-4, interferon gamma, or GM-CSF (granulocyte-macrophage colony-stimulating factor), whereas the Cpn 60.1 protein failed to demonstrate any cytokine-inducing activity. The use of neutralizing monoclonal antibodies showed that the cytokine-inducing activity of Cpn 60.3 was dependent on its interaction with CD14. This demonstrates that CD14 mediates not only lipopolysaccharide but also R. leguminosarum Cpn 60.3 cell signaling in human monocytes. 相似文献
10.
Kathryn M. Schultz Jimmy B. Feix Candice S. Klug 《Protein science : a publication of the Protein Society》2013,22(11):1639-1645
The lipopolysaccharide (LPS)‐rich outer membrane (OM) is a unique feature of Gram‐negative bacteria, and LPS transport across the inner membrane (IM) and through the periplasm is essential to the biogenesis and maintenance of the OM. LPS is transported across the periplasm to the outer leaflet of the OM by the LPS transport (Lpt) system, which in Escherichia coli is comprised of seven recently identified proteins, including LptA, LptC, LptDE, and LptFGB2. Structures of the periplasmic protein LptA and the soluble portion of the membrane‐associated protein LptC have been solved and show these two proteins to be highly structurally homologous with unique folds. LptA has been shown to form concentration dependent oligomers that stack end‐to‐end. LptA and LptC have been shown to associate in vivo and are expected to form a similar protein–protein interface to that found in the LptA dimer. In these studies, we disrupted LptA oligomerization by introducing two point mutations that removed a lysine and glutamine side chain from the C‐terminal β‐strand of LptA. This loss of oligomerization was characterized using EPR spectroscopy techniques and the affinity of the interaction between the mutant LptA protein and WT LptC was determined using EPR spectroscopy (Kd = 15 µM) and isothermal titration calorimetry (Kd = 14 µM). Kd values were also measured by EPR spectroscopy for the interaction between LptC and WT LptA (4 µM) and for WT LptA oligomerization (29 µM). These data suggest that the affinity between LptA and LptC is stronger than the affinity for LptA oligomerization. 相似文献
11.
Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies
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Ghader Bashiri Edward N. Baker 《Protein science : a publication of the Protein Society》2015,24(1):1-10
Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over‐express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non‐pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics. 相似文献
12.
Chaperonin (Cpn)60 proteins have the ability to activate human and murine myeloid cells. There is contradictory evidence that the receptor for this protein is either similar to that of lipopolysaccharide--CD14 and one or other toll-like receptor (e.g. TLR4) or is some other, undidentified, receptor. In an attempt to directly identify the receptor for Mycobacterium tuberculosis Cpn60.1 we have used two approaches. The first is to use Cpn60.1 as an affinity ligand to pull out the receptor from lysates of the murine monocyte cell line RAW 264.7. The second is to crosslink Cpn60.1 to its receptor on RAW cells and isolate the complex by immunoprecipitation. These methods have worked for other receptors. Using affinity chromatography, 2D SDS-PAGE and peptide mass fingerprinting with MALDI-TOF MS it was found that a number of proteins had the ability to bind to Cpn60.1 on an affinity matrix. We identified five proteins, three of which were likely to be on the cell surface. One of these proteins, the endoplasmic reticulum molecular chaperone, BiP did bind to Cpn60.1 with low affinity. Protein crosslinking studies proved inadequate as insufficient protein could be isolated for mass spectrometric identification. Thus, it appears that Cpn60.1, like Hsp70, may bind to a number of cell surface proteins. BiP appears to be one of these receptor proteins but more work is needed to identify those responsible for signalling. Of interest, CD14 and TLR4 were not identified in this study as a receptor for Cpn60.1. 相似文献
13.
Group II chaperonins (Cpns) are essential mediators of cellular protein folding in eukaryotes and archaea. They consist of two back-to-back rings forming symmetrical cavities in which non-native substrates undergo appropriate folding, but the primary structural basis for the double ring formation remains unclear. To address this, we carried out systematic mutagenesis on the Cpn from the hyperthermophilic archaeon Pyrococcus furiosus, which is assembled from identical subunits. In our study, 21GRDAQRMNIL30 was found to be a critical domain for double ring formation. Deletion of this section stepwise beyond residue 20 resulted in failure to assemble double-ring oligomers and the progressive loss of chaperone function. A key domain spanning the residues 21–50 that is essential for the formation of tetramers that appear to be the intermediates for double ring assembly. Mutation of either Arg22 to Ala22 or Glu37 to Ala37 resulted in similar defects in double-ring assembly and functional deficits. A mutant with Arg22 and Glu37 switched assembled double rings efficiently and exhibited chaperone functions similar to the wild-type. Therefore, Arg22 and Glu37 could form inter-ring salt bridges critical for double ring formation. In addition, Asn28 and Ile29 were found to contribute significantly to ring formation. Sequence alignment revealed that these four residues are highly conserved among group II Cpns. This is the first report of a comprehensive N-terminal mutational analysis for elucidating the oligomerization of group II Cpns. 相似文献
14.
The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. In Escherichia coli three membrane proteins, TatA, TatB and TatC, are essential components of the machinery. TatA from Providencia stuartii is homologous to E. coli TatA but is synthesized as an inactive pre‐protein with an N‐terminal extension of eight amino acids. Removal of this extension by the rhomboid protease AarA is required to activate P. stuartii TatA. Here we show that P. stuartii TatA can functionally substitute for E. coli TatA provided that the E. coli homologue of AarA, GlpG, is present. The oligomerization state of the P. stuartii TatA pro‐protein was compared with that of the proteolytically activated protein and with E. coli TatA. The pro‐protein still formed small homo‐oligomers but cannot form large TatBC‐dependent assemblies. In the absence of TatB, E. coli TatA or the processed form of P. stuartii TatA form a complex with TatC. However, this complex is not observed with the pro‐form of P. stuartii TatA. Taken together our results suggest that the P. stuartii TatA pro‐protein is inactive because it is unable to interact with TatC and cannot form the large TatA complexes required for transport. 相似文献
15.
We investigated the regulation of the two of the three groE operons (cpn.1 and cpn.2) of the root-nodulating bacterium R. leguminosarum strain A34. Both are heat inducible, and both have a CIRCE sequence in their upstream regions, suggesting regulation by an
HrcA repressor. Mutagenesis of the CIRCE sequence upstream of cpn.1 led to an increase in the levels of cpn.1 mRNA, and knock-out of the hrcA gene increased the level of Cpn60.1 protein (the GroEL homologue encoded by the cpn.1 operon). Inactivation of the hrcA gene also caused increased expression of a 29 kDa protein that was identified as RhiA, a component of a quorum-sensing system.
However, neither loss of the upstream CIRCE sequence, nor loss of HrcA function, had any effect on expression from the cpn.2 promoter. Further analysis of the cpn.2 upstream region suggested regulation could be mediated by an RpoH system, and this was confirmed by deleting the rpoH gene from the chromosome, which led to a decreased level of Cpn60.2 expression. Inactivation of RpoH led to a reduction in
growth rate which could be partly compensated for by inactivation of HrcA, indicating an overlap in the in vivo function of
the proteins regulated by these two systems.
Accession numbers: DQ173160 (hrcA operon); DQ173161 (rpoH gene). 相似文献
16.
17.
18.
Peter Lund 《BioEssays : news and reviews in molecular, cellular and developmental biology》1994,16(4):229-231
The process of protein folding in the cell is now known to depend on the action of other proteins. These proteins include molecular chaperones, Which interact non-covalently with proteins as they fold and improve the final yields of active protein in the cell. The precise mechanism by which molecular chaperones act is obscure. Experiments reported recently(1) show that for one molecular chaperone (Cpn60, typified by the E. coli protein GroEL), the folding reaction is driven by cycles of binding and release of the co-chaperone Cpn10 (known as GroES in E. coli). These alternate with binding and release of the unfolded protein substrate. These cycles come about because of the opposite effects of Cpn10 and unfolded protein on the Cpn60 complex: the former stabilises the ADP-bound state of Cpn60, whereas the latter stimulates ADP-ATP exchange. This model proposes that the substrate protein goes through multiple cycles of binding and release, and is released into the cavity of the Cpn60 complex where it can undergo folding without interacting with other nearby folding intermediates. This is consistent with the ability of Cpn60 proteins to enhance folding by blocking pathways to aggregation. 相似文献
19.
Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor 总被引:8,自引:3,他引:5
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Ehrt S Guo XV Hickey CM Ryou M Monteleone M Riley LW Schnappinger D 《Nucleic acids research》2005,33(2):e21
Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome. 相似文献
20.
H‐NS is an abundant DNA‐binding protein that has been implicated in the silencing of foreign DNA in several different bacteria. The ability of H‐NS dimers to form higher‐order oligomers is thought to aid the polymerization of the protein across AT‐rich stretches of DNA and facilitate gene silencing. Although the oligomerization of H‐NS from enteric bacteria has been the subject of intense investigation, little is known regarding the oligomerization of H‐NS family members from bacteria outside of the enterobacteriaceae, many of which share little sequence similarity with their enteric counterparts. Here we show that MvaT, a member of the H‐NS family of proteins from Pseudomonas aeruginosa, can form both dimers and higher‐order oligomers, and we identify a region within MvaT that mediates higher‐order oligomer formation. Using genetic assays we identify mutants of MvaT that are defective for higher‐order oligomer formation. We present evidence that these mutants are functionally impaired and exhibit DNA‐binding defects because of their inability to form higher‐order oligomers. Our findings support a model in which the ability of MvaT to bind efficiently to the DNA depends upon protein–protein interactions between MvaT dimers and suggest that the ability to form higher‐order oligomers is a conserved and essential feature of H‐NS family members. 相似文献