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 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.     

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.     

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.     

The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from Escherichia coli cells

The submission of Escherichia coli cells to heat-shock (45 degrees C, 15 min) caused the intracellular aggregation of endogenous proteins. In the wt cells the aggregates (the S fraction) disappeared 10 min after transfer to 37 degrees C. In contrast, the S fraction in the dnaK and dnaJ mutant strains was stable during approximately one generation time (45 min). This demonstrated that neither the renaturation nor the degradation of the denatured proteins was possible in the absence of DnaK and DnaJ. The groEL44 and groES619 mutations stabilised the aggregates to a lesser extent. It was shown by the use of cloned genes, dnaK/dnaJ or groEL/groES, producing the corresponding proteins in about 4-fold excess, that the appearance of the S fraction in the wt strain resulted from a transiently insufficient supply of the heat-shock proteins. Overproduction of the GroEL/GroES proteins in dnaK756 or dnaJ259 background prevented the aggregation, however, overproduction of the DnaK/DnaJ proteins did not prevent the aggregation in the groEL44 or groES619 mutant cells although it accelerated the disappearance of the aggregates. The properties of the aggregated proteins are discussed from the point of view of their competence to renaturation/degradation by the heat-shock system.     

Evidence that the two Escherichia coli groE morphogenetic gene products interact in vivo.

The Escherichia coli groEL and groES gene products are essential for both phage morphogenesis and bacterial growth. Although the gene products have been identified, their exact roles in these processes are not known. We have isolated mutations in the groEL gene that suppress defects in the groES gene. These intergenic suppressors were shown to map in the groEL gene by a variety of genetic and biochemical analyses. These results suggest that the two morphogenetic gene products interact in vivo and help to explain why mutations in either gene exhibit the same phenotype with respect to lambda head assembly and bacterial growth.     

Error-prone DNA polymerase IV is regulated by the heat shock chaperone GroE in Escherichia coli

An insertion in the promoter of the operon that encodes the molecular chaperone GroE was isolated as an antimutator for stationary-phase or adaptive mutation. The groE operon consists of two genes, groES and groEL; point mutations in either gene conferred the same phenotype, reducing Lac+ adaptive mutation 10- to 20-fold. groE mutant strains had 1/10 the amount of error-prone DNA polymerase IV (Pol IV). In recG+ strains, the reduction in Pol IV was sufficient to account for their low rate of adaptive mutation, but in recG mutant strains, a deficiency of GroE had some additional effect on adaptive mutation. Pol IV is induced as part of the SOS response, but the effect of GroE on Pol IV was independent of LexA. We were unable to show that GroE interacts directly with Pol IV, suggesting that GroE may act indirectly. Together with previous results, these findings indicate that Pol IV is a component of several cellular stress responses.     

Cloning, sequencing, and functional expression in Escherichia coli of chaperonin (groESL) genes from Vibrio cholerae.

Using a series of oligonucleotides synthesized on the basis of conserved nucleotide motifs in heat-shock genes, the groESL heat-shock operon from a Vibrio cholerae TSI-4 strain has been cloned and sequenced, revealing that the presence of two open reading frames (ORFs) of 291 nucleotides and 1,632 nucleotides separated by 54 nucleotides. The first ORF encoded a polypeptide of 97 amino acids, GroES homologue, and the second ORF encoded a polypeptide of 544 amino acids, GroEL homologue. A comparison of the deduced amino acid sequences revealed that the primary structures of the V. cholerae GroES and GroEL proteins showed significant homology with those of the GroES and GroEL proteins of other bacteria. Complementation experiments were performed using Escherichia coli groE mutants which have the temperature-sensitive growth phenotype. The results showed that the groES and groEL from V. cholerae were expressed in E. coli, and groE mutants harboring V. cholerae groESL genes regained growth ability at high temperature. The evolutionary analysis indicates a closer relationship between V. cholerae chaperonins and those of the Haemophilus and Yersinia species.     

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.     

Genetic suppression of a temperature-sensitive groES mutation by an altered subunit of RNA polymerase of Escherichia coli K-12.

Temperature-resistant suppressor mutants were isolated from Escherichia coli mutant strain groES131(Ts). Phage P1-mediated transduction and a two-dimensional gel electrophoretic analysis of cellular proteins indicated that these suppressor mutants carry an additional mutation in either the groEL gene or the rpoA gene.     

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.     

Coexpression of UmuD'' with UmuC suppresses the UV mutagenesis deficiency of groE mutants.

The GroE proteins of Escherichia coli are heat shock proteins which have also been shown to be molecular chaperone proteins. Our previous work has shown that the GroE proteins of E. coli are required for UV mutagenesis. This process requires the umuDC genes which are regulated by the SOS regulon. As part of the UV mutagenesis pathway, the product of the umuD gene, UmuD, is posttranslationally cleaved to yield the active form, UmuD'. In order to investigate what role the groE gene products play in UV mutagenesis, we measured UV mutagenesis in groE+ and groE strains which were expressing either the umuDC or umuD'C genes. We found that expression of umuD' instead of umuD will suppress the nonmutability conferred by the groE mutations. However, cleavage of UmuD to UmuD' is unaffected by mutations at the groE locus. Instead we found that the presence of UmuD' increased the stability of UmuC in groE strains. In addition, we obtained evidence which indicates that GroEL interacts directly with UmuC.     

Cloning, sequence, and expression of the temperature-dependent phage T4 capsid assembly gene 31

The products of the bacteriophage T4 capsid assembly gene 31, the T4 major capsid protein gene 23, and the Escherichia coli heat-shock groE genes participate in an interdependent mechanism in capsid protein oligomerization early in prohead assembly. Gene 31 was cloned, sequenced and expressed, and its regulation during infection was characterized. Gene 31 is more stringently required at high than at low temperature, and this requirement is reduced by temperature adaptation of the bacteria prior to infection. However, T4 gene 31 expression does not appear to be temperature regulated, nor does gene 31 apparently display sequence homology with the E. coli groE and other heat-shock genes.