Pseudo-T-even bacteriophage RB49 encodes CocO, a cochaperonin for GroEL, which can substitute for Escherichia coli's GroES and bacteriophage T4's Gp31

Bacteriophage T4-encoded Gp31 is a functional ortholog of the Escherichia coli GroES cochaperonin protein. Both of these proteins form transient, productive complexes with the GroEL chaperonin, required for protein folding and other related functions in the cell. However, Gp31 is specifically required, in conjunction with GroEL, for the correct folding of Gp23, the major capsid protein of T4. To better understand the interaction between GroEL and its cochaperonin cognates, we determined whether the so-called "pseudo-T-even bacteriophages" are dependent on host GroEL function and whether they also encode their own cochaperonin. Here, we report the isolation of an allele-specific mutation of bacteriophage RB49, called epsilon22, which permits growth on the E. coli groEL44 mutant but not on the isogenic wild type host. RB49 epsilon22 was used in marker rescue experiments to identify the corresponding wild type gene, which we have named cocO (cochaperonin cognate). CocO has extremely limited identity to GroES but is 34% identical and 55% similar at the protein sequence level to T4 Gp31, sharing all of the structural features of Gp31 that distinguish it from GroES. CocO can substitute for Gp31 in T4 growth and also suppresses the temperature-sensitive phenotype of the E. coli groES42 mutant. CocO's predicted mobile loop is one residue longer than that of Gp31, with the epsilon22 mutation resulting in a Q36R substitution in this extra residue. Both the CocO wild type and epsilon22 proteins have been purified and shown in vitro to assist GroEL in the refolding of denatured citrate synthase.     

Structures of chaperonins from an intracellular symbiont and their functional expression in Escherichia coli groE mutants.

An intracellular symbiont harbored by the aphid bacteriocyte, a specialized fat body cell, synthesizes in vivo substantially only one protein, symbionin, which is a member of the chaperonin-60 family of molecular chaperones. Nucleotide sequence determination of the symbionin region of the endosymbiont genome revealed that it contains the two-cistron operon sym. Just like the Escherichia coli groE operon, the sym operon was dually led by a heat shock and an ordinary promoter sequence. According to the nucleotide sequence, symbionin was 85.5% identical to GroEL of E. coli at the amino acid sequence level. SymS, another protein encoded in the sym operon, which is a member of chaperonin-10, was 79.6% identical to GroES. Complementation experiments with E. coli groE mutants showed that the chaperonin-10 and chaperonin-60 genes from the endosymbiont are expressed in E. coli and that they can function as molecular chaperones together with endogenous GroEL and GroES, respectively.     

Sequence analysis of the Legionella micdadei groELS operon

A 2.7 kb DNA fragment encoding the 60 kDa common antigen (CA) and a 13 kDa protein of Legionella micdadei was sequenced. Two open reading frames of 57,677 and 10,456 Da were identified, corresponding to the heat shock proteins GroEL and GroES, respectively. Typical -35, -10, and Shine-Dalgarno heat shock expression signals were identified upstream of the L. micdadei groEL gene. Further upstream, a poly-T region, also a feature of the sigma 32-regulated Escherichia coli groELS heat shock operon, was found. Despite the high degree of homology of the expression signals in E. coli and L. micdadei, Western blot analysis with an L. micdadei specific anti-groEL antibody did not reveal a significant increase in the amount of the GroEL protein during heat shock in L. micdadei or in the recombinant E. coli expressing L. micdadei GroEL.     

Cloning and characterization of a gene from Rhizobium melilotii 2011 coding for ribosomal protein S1.

A 7 kb chromosomal DNA fragment from R. melilotii was cloned, which complemented temperature-sensitivity of an E. coli amber mutant in rpsA, the gene for ribosomal protein S1 (ES1). From complementation and maxicell analysis a 58 kd protein was identified as the homolog of protein S1 (RS1). DNA sequence analysis of the R. melilotii rpsA gene identified a protein of 568 amino acids, which showed 47% identical amino acid homology to protein S1 from E. coli. The RS1 protein lacked the two Cys residues which had been reported to play an important role for the function of ES1. Two repeats containing Shine-Dalgarno sequences were identified upstream of the structural gene. Binding studies with RNA polymerase from E. coli and Pseudomonas putida located one RNA-polymerase binding site close to the RS1 gene and another one several hundred basepairs upstream. One possible promoter was also identified by DNA sequence comparison with the corresponding E. coli promoter.     

Expression and control of an operon from an intracellular symbiont which is homologous to the groE operon.

Members of the genus Buchnera are intracellular symbionts harbored by the aphid bacteriocyte which selectively synthesize symbionin, a homolog of the Escherichia coli GroEL protein, in vivo. Symbionin and SymS, a GroES homolog, are encoded in the symSL operon. Northern blotting and primer extension analyses revealed that the symSL operon invariably gives rise to a bicistronic mRNA under the control of a heat shock promoter, though the amount of the symSL mRNA in the isolated symbiont did not increase in response to heat shock. The sigma32 protein that recognizes the heat shock promoter in E. coli was scarcely detected in Buchnera cells even after heat shock. Although the functionally essential regions of the Buchnera sigma32 protein were well conserved, the Buchnera rpoH gene did not complement an E. coli delta rpoH mutant. On the one hand, the A-T evolutionary pressure imposed on the Buchnera genome may have not only decreased the activity of its sigma32 but also ruined the nucleotide sequences necessary for the expression of rpoH; on the other hand, it may have facilitated expression of the symSL operon without activation by sigma32.     

Asymmetric segregation of heat-shock proteins upon cell division in Caulobacter crescentus

Three Caulobacter crescentus heat-shock proteins were shown to be immunologically related to the Escherichia coli heat-shock proteins GroEL, Lon and DnaK. A fourth heat-shock protein was detected with antibody to the C. crescentus RNA polymerase. This 37,000 Mr heat-shock protein might be related to the E. coli 32,000 Mr heat-shock sigma subunit. The synthesis of the major C. crescentus RNA polymerase sigma factor was not induced by heat shock. The E. coli GroEL protein and the related protein from C. crescentus were also induced by treatment with hydrogen peroxide. Like some of the proteins in the heat-shock protein families of Drosophila and yeast, the four heat-shock proteins in C. crescentus were found to be regulated developmentally under normal conditions. All four proteins were synthesized in the predivisional cell, but the progeny showed cell type-specific bias in the level of enhanced synthesis after heat shock. The 92,000 Mr Lon homolog and the 37,000 Mr RNA polymerase subunit were preferentially synthesized in the stalked cell, whereas the synthesis of the 62,000 Mr GroEL homolog was enhanced in the progeny swarmer cell. Furthermore, the four heat-shock proteins synthesized in the predivisional cell were partitioned in a specific manner upon cell division. The stalked cell, which initiates chromosome replication immediately upon division, received the Lon homolog, the DnaK homolog and the 37,000 Mr RNA polymerase subunit. The GroEL homolog, however, was distributed equally to both the stalked cell and the swarmer cell. These results provide access to the functions of C. crescentus heat-shock proteins under both normal and stress conditions. They also allow an investigation of the regulatory signals that modulate the asymmetric distribution of proteins and their subsequent cell type-specific expression in the initial stages of a developmental program.     

Escherichia coli cells resistant to the DNA gyrase inhibitor, ciprofloxacin, overproduce a 60 kD protein homologous to GroEL

Using a variety of mutagenic methods, we have generated a series of ciprofloxacin-resistant mutants derived from Escherichia coli strains which overproduce the DNA gyrase A protein. Many of these mutants are found to overexpress a 60 kD protein which is shown to be highly homologous in terms of N-terminal amino acid sequence to the E. coli heat-shock protein, GroEL. Other evidence confirms that the 60 kD protein is unrelated to DNA gyrase and is similar, but not identical, to GroEL.     

Extracellular Production of Yeast Protease

The groEL gene of the alkaliphilic Bacillus sp. strain C-125 was cloned in Escherichia coli and sequenced. The groEL gene encoded a polypeptide of 544 amino acids and was preceded by the incomplete groES gene, lacking its 5′-end. The sequence of the derived amino acids was 87.5% identical to that of B. subtilis, 85.4% identical to that of B. stearothemophilus, and 60.9% identical to that of E. coli. The GroEL protein was expressed in E. coli. Purified GroEL protected yeast a-glucosidase from irreversible aggregation at a high temperature and the addition of Mg-ATP was essential for reactivation of the a-glucosidase. The addition of E. coli GroES increased recovery of the enzyme activity, indicating that C-125 GroEL could function in coordination with E. coli GroES.     

Expression of a highly toxic protein, Bax, in Escherichia coli by attachment of a leader peptide derived from the GroES cochaperone

Expression of the human apoptosis modulator protein Bax in Escherichia coli is highly toxic, resulting in cell lysis at very low concentrations (Asoh, S., et al., J. Biol. Chem. 273, 11384-11391, 1998). Attempts to express a truncated form of murine Bax in the periplasm by using an expression vector that attached the OmpA signal sequence to the protein failed to alleviate this toxicity. In contrast, attachment of a peptide based on a portion of the E. coli cochaperone GroES reduced Bax's toxicity significantly and allowed good expression. The peptide, which was attached to the N-terminus, included the amino acid sequence of the mobile loop of GroES that has been demonstrated to interact with the chaperonin, GroEL. Under normal growth conditions, expression of this construct was still toxic, but generated a small amount of detectable recombinant Bax. However, when cells were grown in the presence of 2% ethanol, which stimulated overproduction of the molecular chaperones GroEL and DnaK, toxicity was reduced and good overexpression occurred. Two-dimensional gel electrophoresis analysis showed that approximately 15-fold more GroES-loop-Bax was produced under these conditions than under standard conditions and that GroEL and DnaK were elevated approximately 3-fold.     

Cloning, Expression, and Homology Modeling of GroEL Protein from Leptospira interrogans Serovar Autumnalis Strain N2

Leptospirosis is an infectious bacterial disease caused by Leptospira species. In this study, we cloned and se- quenced the gene encoding the immunodominant protein GroEL from L. interrogans serovar Autumnalis strain N2, which was isolated from the urine of a patient during an outbreak of leptospirosis in Chennai, India. This groEL gene encodes a protein of 60 kDa with a high degree of homology (99% similarity) to those of other leptospiral serovars. Recombinant GroEL was overexpressed in Escherichia coli. Immunoblot analysis indi- cated that the sera from confirmed leptospirosis patients showed strong reactivity with the recombinant GroEL while no reactivity was observed with the sera from seronegative control patient. In addition, the 3D structure of GroEL was constructed using chaperonin complex cpn60 from Thermus thermophilus as template and vali- dated. The results indicated a Z-score of -8.35, which is in good agreement with the expected value for a pro- tein. The superposition of the Cα traces of cpn60 structure and predicted structure of leptospiral GroEL indi- cates good agreement of secondary structure elements with an RMSD value of 1.5 . Further study is necessary to evaluate GroEL for serological diagnosis of leptospirosis and for its potential as a vaccine component.     

Phosphocarrier proteins in an intracellular symbiotic bacterium of aphids.

A GroEL homolog produced by Buchnera, an intracellular symbiotic bacterium of aphids, is not only a molecular chaperone but also a novel phosphocarrier protein, suggesting that this protein plays a role in a signal transducing system specific to bacteria living in an intracellular environment. This prompted us to look into phosphocarrier proteins of Buchnera that may be shared in common with other bacteria. As a result, no evidence was obtained for the presence of sensor kinases of the two-component system in Buchnera, which are found in many bacteria. It is possible that the lack of sensor kinases is compensated for by the mulitifunctional GroEL homolog in this symbiotic bacteria. In contrast, we successfully identified three phosphotransferase system genes, ptsH, ptsI, and crr in Buchnera, and provide evidence for their active expression. While the deduced amino acid sequences of these gene products, histidine-containing phosphocarrier protein, Enzyme I, and Enzyme III were similar to their counterparts in Escherichia coli, the predicted isoelectric points of the Buchnera proteins were strikingly higher. It was also suggested that Buchnera Enzyme I, when produced in E. coli, is able to accept the phosphoryl group from phosphoenolpyruvate, but not from ATP.     

Molecular cloning and functional analysis of a human cDNA encoding an Escherichia coli AlkB homolog, a protein involved in DNA alkylation damage repair.

The Escherichia coli AlkB protein is involved in protecting cells against mutation and cell death induced specifically by SN2-type alkylating agents such as methyl methanesulfonate (MMS). A human cDNA encoding a polypeptide homologous to E.coli AlkB was discovered by searching a database of expressed sequence tags (ESTs) derived from high throughput cDNA sequencing. The full-length human AlkB homolog (hABH) cDNA clone contains a 924 bp open reading frame encoding a 34 kDa protein which is 52% similar and 23% identical to E.coli AlkB. The hABH gene, which maps to chromosome 14q24, was ubiquitously expressed in 16 human tissues examined. When hABH was expressed in E.coli alkB mutant cells partial rescue of the cells from MMS-induced cell death occurred. Under the conditions used expression of hABH in skin fibroblasts was not regulated by treatment with MMS. Our findings show that the AlkB protein is structurally and functionally conserved from bacteria to human, but its regulation may have diverged during evolution.     

Cloning and nucleotide sequence of the Myxococcus xanthus lon gene: indispensability of lon for vegetative growth.

The lon gene of Escherichia coli is known to encode protease La, an ATP-dependent protease associated with cellular protein degradation. A lon gene homolog from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon nutrient starvation, was cloned and characterized by use of the lon gene of E. coli as a probe. The nucleotide sequence of the M. xanthus lon gene was determined. It contains an open reading frame that encodes a 92-kDa protein consisting of 817 amino acid residues. The deduced amino acid sequence of the M. xanthus lon gene product showed 60 and 56% identity with those of the E. coli and Bacillus brevis lon gene products, respectively. Analysis of an M. xanthus strain carrying a lon-lacZ operon fusion suggested that the lon gene is similarly expressed during vegetative growth and development in M. xanthus. In contrast to that of E. coli, the M. xanthus lon gene was shown to be essential for cell growth, since a null mutant could not be isolated.     

Surface-associated GroEL facilitates the adhesion of Escherichia coli to macrophages through lectin-like oxidized low-density lipoprotein receptor-1

The Escherichia coli homolog of GroEL, a 60 kDa heat shock protein (HSP), is a dominant protein produced not only in response to heat stress but also under in vitro growth condition. Beside its traditional cytoplasmic location, the surface exposures of GroEL have been observed in many pathogenic bacteria. To investigate the role of the surface-associated GroEL in the binding of E. coli to macrophages, we constructed a new strain of E. coli displaying GroEL on the outer membrane. We found that surface-associated GroEL increases the clearance ratio of E. coli by macrophages. It has been previously demonstrated that lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is the receptor for Hsp60 from different species. Our present results showed that GroEL on E. coli was recognized by LOX-1 on macrophages, leading to the phagocytosis of pathogen by macrophages. In addition, surface-associated GroEL made mice more susceptible to E. coli-induced peritonitis. These findings add to the research that clarifies the factors mediating bacterial adherence to host cells. Our results suggest that GroEL is a novel therapeutic target for modulating the immune response in infectious and inflammatory conditions.     

Chaperone mediated solubilization of 69-kDa recombinant maltodextrin glucosidase in Escherichia coli

Aims:  To investigate the factors affecting expression and solubilization of Escherichia coli maltodextrin glucosidase in E. coli .
Methods and Results:  Expression level and solubilization of the recombinant E. coli maltodextrin glucosidase was studied in E. coli at different temperatures, in presence of overexpressed GroEL, GroES and externally supplemented glycerol. Aggregation of maltodextrin glucosidase in the cytoplasm was partially prevented by the co-expression of GroEL and GroES, and using externally supplemented glycerol or lowering the culture temperature. Co-expression of GroEL and GroES or simultaneous presence of overexpressed GroEL, GroES and externally supplemented glycerol together resulted significant increase of the activity of maltodextrin glucosidase. The growth rate of E. coli was inhibited by the formation of inclusion bodies whereas the presence of overexpressed GroEL, GroES alone or together with glycerol enhanced the growth rate of E. coli substantially.
Conclusions:  The results indicated that lowering the temperature, use of GroEL, GroES and glycerol could be few controlling factors for the solubilization of recombinant aggregation-prone maltodextrin glucosidase in E. coli.
Significance and Impact of the Study:  Our study could help in developing the strategy for enhancing the production of soluble industrial enzymes and finding the therapeutic agents against protein misfolding diseases.     

Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli production of soluble apoenzyme and in vitro formation of active holoenzyme.

Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli BL21(DE3) was accomplished by introducing a derivative of pET-23a(+) containing a copy of the coding gene into the multicloning site. E. coli BL21(DE3)/pETMNCAT produced abundant quantities of manganese catalase as insoluble inclusion bodies. Regeneration of active catalase was achieved by denaturation in guanidine hydrochloride and subsequent dialysis in the presence of manganese ion. When the E. coli chaperone genes GroEL, GroES, DnaK, DnaJ and GrpE were coexpressed with manganese catalase, a significant fraction of the overproduced protein was partitioned into the soluble fraction. However, almost all of the soluble enzyme was isolated in a manganese-deficient apo form which could subsequently be converted into active holoenzyme by incubation with manganese ion at high temperatures. Further experiments on this apo catalase suggested that the structure of this protein was virtually identical to the active holoenzyme.     

Crystal structure of the native chaperonin complex from Thermus thermophilus revealed unexpected asymmetry at the cis-cavity

The chaperonins GroEL and GroES are essential mediators of protein folding. GroEL binds nonnative protein, ATP, and GroES, generating a ternary complex in which protein folding occurs within the cavity capped by GroES (cis-cavity). We determined the crystal structure of the native GroEL-GroES-ADP homolog from Thermus thermophilus, with substrate proteins in the cis-cavity, at 2.8 A resolution. Twenty-four in vivo substrate proteins within the cis-cavity were identified from the crystals. The structure around the cis-cavity, which encapsulates substrate proteins, shows significant differences from that observed for the substrate-free Escherichia coli GroEL-GroES complex. The apical domain around the cis-cavity of the Thermus GroEL-GroES complex exhibits a large deviation from the 7-fold symmetry. As a result, the GroEL-GroES interface differs considerably from the previously reported E. coli GroEL-GroES complex, including a previously unknown contact between GroEL and GroES.     

Expression of human 60-kD heat shock protein (HSP60 or P1) in Escherichia coli and the development and characterization of corresponding monoclonal antibodies.

Human P1 protein, which is the homolog of the 60- to 65-kD heat shock "common" antigenic protein of numerous pathogenic organisms (synonyms: HSP60, GroEL homolog, or chaperonin), has been expressed to high level in Escherichia coli cells. A large number of well-characterized deletions of this protein spanning the entire sequence have been constructed and expressed. Methods to purify recombinant human HSP60 protein and its deletions from E. coli have been worked out. In addition, monoclonal antibodies to the human HSP60 protein have been raised and partially characterized. The availability of these materials should greatly aid in understanding the role of this highly conserved and immunologically important protein in autoimmune diseases and in cell structure and function.