Purification and properties of the groES morphogenetic protein of Escherichia coli

The morphogenesis of lambda proheads is governed by the products of at least four bacteriophage-coded genes (B, C, E and Nu3) and two host-coded genes (groES (mopB) and groEL (mopA)). Earlier genetic experiments indicated that the phenotypes of some of the groES- mutations could be suppressed by mutations in the groEL gene, suggesting an interaction between the two groE proteins in vivo (Tilly, K., and Georgopoulos, C. P. (1982) J. Bacteriol. 149, 1082-1088). The Mr 15,000 groES protein was overproduced and purified to homogeneity by monitoring its presence after polyacrylamide gel electrophoresis. Both gel filtration on an AcA34 sizing column and glycerol gradient centrifugation indicate that the groES protein possesses an oligomeric structure of Mr 80,000. In agreement, electron microscopic pictures of the purified groES protein show that it possesses a symmetrical ring-like structure. The sequence of the first five amino acids and the overall composition of the purified protein match those predicted by the nucleotide sequence of the groES gene. The following results implicate a physical association between the groES and groEL proteins in vitro. The groES protein inhibits the weak ATPase activity of the groEL protein, with a maximal effect seen at a 1:1 molar ratio; the two proteins cosediment during glycerol gradient centrifugation in the presence of ATP and Mg2+; and the groES protein binds specifically to a groEL-affinity column. These results help explain why mutations in either of the groE genes exhibit similar phenotypes with respect to both lambda and bacterial growth.     

The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures.

The products of the groES and groEL genes of Escherichia coli, constituting the groE operon, are known to be required for growth at high temperature (42 degrees C) and are members of the heat shock regulon. Using a genetic approach, we examined the requirement for these gene products for bacterial growth at low temperature (17 to 30 degrees C). To do this, we constructed various groES groEL heterodiploid derivative strains. By inactivating one of the groE operons by a polar insertion, it was shown by bacteriophage P1 transduction that at least one of the groE genes was essential for growth at low temperature. Further P1 transduction experiments with strains that were heterodiploid for only one of the groE genes demonstrated that both groE gene products were required for growth at low temperature, which suggested a fundamental role for the groE proteins in E. coli growth and physiology.     

The Escherichia coli groE chaperonins

The E.coli groES and groEL genes have been shown to form an operon, to be essential for E. coli viability, and to belong to the so-called heat-shock class of genes whose expression is regulated by the intracellular levels of sigma factor sigma 32. Both groE chaperonin proteins possess a seven-fold axis of symmetry, groES being composed of seven identical subunits of 97 amino acids each, and groEL of fourteen identical subunits of 548 amino acids each. The two groE chaperonins interact intimately as judged by both genetic and biochemical criteria. This interaction has been shown to be required for both bacteriophage morphogenesis and bacterial growth. The groEL chaperonin has been shown to bind to a number of incomplete or unfolded polypeptides in vitro. Such binding may prevent misfolding and promote rapid intra- or intermolecular folding of polypeptides in vivo. The proposed role of the groES chaperonin is to displace the polypeptides bound to groEL, thus effectively promoting the recycling of groEL.     

An ORFan no more: the bacteriophage T4 39.2 gene product, NwgI, modulates GroEL chaperone function

Bacteriophages are the most abundant biological entities in our biosphere, characterized by their hyperplasticity, mosaic composition, and the many unknown functions (ORFans) encoded by their immense genetic repertoire. These genes are potentially maintained by the bacteriophage to allow efficient propagation on hosts encountered in nature. To test this hypothesis, we devised a selection to identify bacteriophage-encoded gene(s) that modulate the host Escherichia coli GroEL/GroES chaperone machine, which is essential for the folding of certain host and bacteriophage proteins. As a result, we identified the bacteriophage RB69 gene 39.2, of previously unknown function and showed that homologs of 39.2 in bacteriophages T4, RB43, and RB49 similarly modulate GroEL/GroES. Production of wild-type bacteriophage T4 Gp39.2, a 58-amino-acid protein, (a) enables diverse bacteriophages to plaque on the otherwise nonpermissive groES or groEL mutant hosts in an allele-specific manner, (b) suppresses the temperature-sensitive phenotype of both groES and groEL mutants, (c) suppresses the defective UV-induced PolV function (UmuCD) of the groEL44 mutant, and (d) is lethal to the host when overproduced. Finally, as proof of principle that Gp39.2 is essential for bacteriophage growth on certain bacterial hosts, we constructed a T4 39.2 deletion strain and showed that, unlike the isogenic wild-type parent, it is incapable of propagating on certain groEL mutant hosts. We propose a model of how Gp39.2 modulates GroES/GroEL function.     

The groESL operon of Agrobacterium tumefaciens: evidence for heat shock-dependent mRNA cleavage.

The heat shock response of the groESL operon of Agrobacterium tumefaciens was studied at the RNA level. The operon was found to be activated under heat shock conditions and transcribed as a polycistronic mRNA that contains the groES and groEL genes. After activation, the polycistronic mRNA appeared to be cleaved between the groES and groEL genes and formed two monocistronic mRNAs. The groES cleavage product appeared to be unstable and subjected to degradation, while the groEL cleavage product appeared to be stable and became the major mRNA representing the groESL operon after long periods of growth at a high temperature. The polycistronic mRNA containing the groES and groEL genes was the major mRNA representing the groESL operon at a low temperature, and it reappeared when the cells were returned to the lower growth temperature after heat shock induction. These findings indicate that the cleavage event is part of the heat shock regulation of the groESL operon in A. tumefaciens.     

Assessment of plant chaperonin-60 gene function in Escherichia coli.

Brassica napus chaperonin-60 alpha and chaperonin-60 beta genes expressed separately and in combination produce three novel Escherichia coli strains: alpha, beta, and alpha beta. In beta and alpha beta cells, the plant gene products assemble efficiently into tetradecameric cpn60(14) species, including novel hybrids containing both bacterial and plant gene products. The levels of authentic groEL14 are reduced in these cells (Cloney, L. P., Wu, H. B., and Hemmingsen, S. M. (1992) J. Biol. Chem. 267, 23327-23332). The assembly of cyanobacterial ribulose-P2 carboxylase (rubisco) in E. coli requires the activities of the endogenous chaperonin proteins. Furthermore, the extent to which assembly occurs is limited by the normal levels of expression of the groE operon (Goloubinoff, P., Gatenby, A. A., and Lorimer, G. H. (1989) Nature 337, 44-47). We have now monitored the accumulation of cyanobacterial rubisco in E. coli alpha, beta, and alpha beta cells to assess the activity of the plant cpn60 gene products and effects on endogenous chaperonin functions. Expression of cpn-60 alpha alone did not enhance rubisco assembly, which is consistent with our previous observation that p60cpn-60 alpha required the presence of p60cpn-60 beta for assembly into cpn60(14) species. In contrast, expression of cpn-60 beta alone resulted in markedly enhanced rubisco assembly in cells that accumulated normal levels of both endogenous chaperonin polypeptides (groEL and groES). This demonstrates that assembled p60cpn-60 beta is functional as a chaperonin in E. coli. Co-expression of cpn-60 alpha and cpn-60 beta in cells with normal levels of expression of groES and groEL suppressed rubisco assembly. Increased expression of groES in cells in which cpn-60 alpha and cpn-60 beta were co-expressed relieved this suppression and resulted in enhanced rubisco assembly. Implications with respect to dependence of chloroplast cpn60 function on cpn10 are discussed.     

Control of cell division by sex factor F in Escherichia coli. III. Participation of the groES (mopB) gene of the host bacteria

Cell division of F+ bacteria is coupled to DNA replication of the F plasmid. Two plasmid coded genes, letA (ccdA) and letD (ccdB) are indispensable for this coupling. To investigate bacterial genes that participate in this coupling, we attempted to identify the target of the division inhibitor (the letD gene product) of the F plasmid. Two temperature-sensitive growth defective mutants were screened from bacterial mutants that escaped the letD product growth inhibition that occurs in hosts carrying an FletA mutant. Phage P1-mediated transduction and complementation analysis indicated that the temperature-sensitive mutations are located in the groES (mopB) gene, which is essential for the morphogenesis of several bacteriophages and also for growth of the bacteria. The nucleotide sequence of the promoter region of the gene in which the temperature-sensitive mutations had occurred was virtually identical with that of the groES gene of Escherichia coli; furthermore the sequence of the first five amino acid residues and the overall amino acid composition predicted from the nucleotide sequence of the gene match those of the purified GroES protein. The temperature-sensitive mutants did not allow the propagation of phage lambda at 28 degrees C and formed long filamentous structures without septa at 41 degrees C, as is observed in the case of groES mutants. Growth of the two groES mutants tested was not inhibited by the F plasmid with the letA mutation. These observations suggest to us that the morphogenesis gene groES plays a key role in coupling between replication of the F plasmid and cell division of the host cells.     

Mycobacteria contain two groEL genes: the second Mycobacterium leprae groEL gene is arranged in an operon with groES

In contrast to other bacterial species, mycobacteria were thus far considered to contain groEL and groES genes that are present on separate loci on their chromosomes, Here, by screening a Mycobacterium leprae lambda gt11 expression library with serum from an Ethiopian lepromatous leprosy patient, two DNA clones were isolated that contain a groEL gene arranged in an operon with a groES gene. The complete DNA sequence of this groESL operon was determined. The predicted amino acid sequences of the GroES and GroEL proteins encoded by this operon are 85-90% and 59-61% homologous to the sequences from previously characterized mycobacterial GroES and GroEL proteins. Southern blotting analyses with M. leprae groES- and groEL-specific probes demonstrate that similar groESL homologous DNA is present in the genomes of other mycobacteria, including Mycobacterium tuberculosis. This strongly suggests that mycobacteria contain a groESL operon in addition to a separately arranged second groEL gene. Using five T-cell clones from two leprosy patients as probes, expression of the M. leprae GroES protein in Escherichia coli after heat shock was demonstrated. Four of these clones recognized the same M. leprae-specific GroES-derived peptide in a DR2-restricted fashion. No expression of the groEL gene from this operon was detected in E. coli after heat shock, as tested with a panel of T-cell clones and monoclonal antibodies reactive to previously described GroEL proteins of mycobacteria.     

Sequence analysis and phenotypic characterization of groEL mutations that block lambda and T4 bacteriophage growth.

The groES and groEL genes of Escherichia coli have been shown previously to belong to a single operon under heat shock regulation. Both proteins have been universally conserved in nature, as judged by the presence of similar proteins throughout evolution. The GroEL protein has been shown to bind promiscuously to many unfolded proteins, thus preventing their aggregation. ATP hydrolysis by GroEL results in the release of the bound polypeptides, a process that often requires the action of GroES. In an effort to understand GroEL and GroES structure and function, we have determined the nucleotide changes of nine mutant alleles of groEL. All of these mutant alleles were isolated because they block bacteriophage lambda growth. Our sequencing results demonstrate that (i) many of these alleles are identical, in spite of the fact that they were independently isolated, and (ii) most of the different alleles are clustered in the same region of the gene. One of the mutant alleles was shown to possess two nucleotide alterations in the groEL coding phase, one of which is located in a putative ATP-binding domain. The two nucleotide changes were separated by genetic engineering, and each individual change was shown to exert an effect on bacteriophage growth. But, using genetic analyses, we demonstrate that the restriction on bacterial growth at elevated temperatures is conferred only by the mutation within the putative ATP-binding domain. We have cloned the mutant alleles on multicopy plasmids and overexpressed their products. By testing for the ability of bacteriophage either to propagate or to form colonies at 43 degrees C, we have been able to divide the mutant proteins into those with no activity and those with residual activity under the various conditions tested.     

Physiological roles of the DnaK and GroE stress proteins: catalysts of protein folding or macromolecular sponges?

When organisms ranging from microbes to man are subjected to certain environmental stresses a characteristic 'heat shock' response is observed. In Escherichia coli this response is characterized by the induction of several proteins, three of which are the 70 kilodalton product of the dnaK gene, the 60 kilodalton product of the groEL (mopA) gene and the 15 kilodalton product of the groES (mopB) gene. In this review, utilizing enteric bacteria as model organisms, we focus on the role of these proteins within the context provided by well-established functions of other heat shock products. These facts serve as a starting point from which to speculate upon the in vivo role of these proteins during steady-state growth.     

Coexpression of molecular chaperones to enhance functional expression of anti-BNP scFv in the cytoplasm of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the detection of B-type natriuretic peptide

Molecular chaperones are a ubiquitous family of cellular proteins that mediate the correct folding of other target polypeptides. In our previous study, the recombinant anti-BNP scFv, which has promising applications for diagnostic, prognostic, and therapeutic monitoring of heart failure, was expressed in the cytoplasm of Escherichia coli. However, when the anti-BNP scFv was expressed, 73.4% of expressed antibodies formed insoluble inclusion bodies. In this study, molecular chaperones were coexpressed with anti-BNP scFv with the goal of improving the production of functional anti-BNP in the cytoplasm of E. coli. Five sets of molecular chaperones were assessed for their effects on the production of active anti-BNP scFv. These sets included the following: trigger factor (TF); groES/groEL; groES/groEL/TF; dnaK/dnaJ/grpE; groES/groEL/dnaK/dnaJ/grpE. Of these chaperones, the coexpression of anti-BNP scFv with the groES/groEL chaperones encoded in plasmid pGro7 exhibited the most efficient functional expression of anti-BNP scFv as an active form. Coexpressed with the groES/groEL chaperones, 64.9% of the total anti-BNP scFv was produced in soluble form, which is 2.4 times higher scFv than that of anti-BNP scFv expressed without molecular chaperones, and the relative binding activity was 1.5-fold higher. The optimal concentration of l-arabinose required for induction of the groES/groEL chaperone set was determined to be 1.0 mM and relative binding activity was 3.5 times higher compared with that of no induction with l-arabinose. In addition, soluble anti-BNP scFv was increased from 11.5 to 31.4 μg/ml with optimized inducer concentration (1.0 mM l-arabinose) for the coexpression of the groES/groEL chaperones. These results demonstrate that the functional expression of anti-BNP scFv can be improved by coexpression of molecular chaperones, as molecular chaperones can identify and help to refold improperly folded anti-BNP scFv.     

Cloning and nucleotide sequence of the Brucella abortus groE operon.

The cloning and sequencing of the Brucella abortus groES and groEL genes are reported. The genes are adjacent on the Brucella chromosome, and presumably comprise a functional operon. Putative promoter and terminator sequences are also identified. The groES gene exhibits 60%, and the groEl gene 69%, sequence identity with the corresponding Escherichia coli genes.     

Only one of five groEL genes is required for viability and successful symbiosis in Sinorhizobium meliloti

Many bacterial species contain multiple copies of the genes that encode the chaperone GroEL and its cochaperone, GroES, including all of the fully sequenced root-nodulating bacteria that interact symbiotically with legumes to generate fixed nitrogen. In particular, in Sinorhizobium meliloti there are four groESL operons and one groEL gene. To uncover functional redundancies of these genes during growth and symbiosis, we attempted to construct strains containing all combinations of groEL mutations. Although a double groEL1 groEL2 mutant cannot be constructed, we demonstrate that the quadruple groEL1 groESL3 groEL4 groESL5 and groEL2 groESL3 groEL4 groESL5 mutants are viable. Therefore, like E. coli and other species, S. meliloti requires only one groEL gene for viability, and either groEL1 or groEL2 will suffice. The groEL1 groESL5 double mutant is more severely affected for growth at both 30 degrees C and 40 degrees C than the single mutants, suggesting overlapping functions in stress response. During symbiosis the quadruple groEL2 groESL3 groEL4 groESL5 mutant acts like the wild type, but the quadruple groEL1 groESL3 groEL4 groESL5 mutant acts like the groEL1 single mutant, which cannot fully induce nod gene expression and forms ineffective nodules. Therefore, the only groEL gene required for symbiosis is groEL1. However, we show that the other groE genes are expressed in the nodule at lower levels, suggesting minor roles during symbiosis. Combining our data with other data, we conclude that groESL1 encodes the housekeeping GroEL/GroES chaperone and that groESL5 is specialized for stress response.     

Lysis of Escherichia coli by the bacteriophage phi X174 E protein: inhibition of lysis by heat shock proteins.

Lysis of Escherichia coli by the cloned E protein of bacteriophage phi X174 was more rapid than expected when bacteria were shifted from 30 to 42 degrees C at the time of E induction. Since such treatment also induces the heat shock response, we investigated the effect of heat shock proteins on lysis. An rpoH mutant was more sensitive to lysis by E, but a secondary suppressor mutation restored lysis resistance to parental levels, which suggests that the sigma 32 subunit itself did not directly increase lysis resistance. At 30 degrees C, mutants in five heat shock genes (dnaK, dnaJ, groEL, groES, and grpE) were more sensitive to lysis than were their wild-type parents. The magnitude of lysis sensitivity varied with mutation and strain background, with dnaK, dnaJ, and groES mutants consistently exhibiting the greatest sensitivities. Extended protection against lysis occurred when overproduction of heat shock proteins was induced artificially in cells that contained a plasmid with the rpoH+ gene under control of the tac promoter. This protective effect was completely abolished by mutations in dnaK, dnaJ, or groES but not by grpE or groEL mutations. Altered membrane behavior probably explains the contradiction whereby an actual temperature shift sensitized cells to lysis, but production of heat shock proteins exhibited protective effects. The results demonstrate that E-induced lysis can be divided into two distinct operations which may now be studied separately. They also emphasize a role for heat shock proteins under non-heat-shock conditions and suggest cautious interpretation of lysis phenomena in systems where E protein production is under control of a temperature-sensitive repressor.