A soluble 60 kilo Dalton antigen of Chlamydia spp. is a homologue of Escherichia coli GroEL

Two major 60 kD protein species can be separated by differential detergent extraction in Chlamydia spp. A Sarkosyl-soluble 60 kD protein is (i) structurally and antigenically distinct from the previously characterized 60 kD Omp2 outer membrane protein; and (ii) antigenically related to a bacterial common antigen of similar molecular weight which includes a 65 kD mycobacterial antigen and the GroEL heat-shock protein of Escherichia coli. Among GroEL homologues, the chlamydial protein (chl-GroEL) uniquely displays affinity towards immobilized thiol groups. The significance of this property is discussed with respect to the synthesis and assembly of the chlamydial disulphide-rich cell wall late in the growth cycle. Chl-GroEL is identical to the Triton X-100-soluble, ocular delayed-type hypersensitivity agent (Morrison et al., 1989), an essential component in the development of blinding trachoma. An autoimmune mechanism for chronic chlamydial diseases based on chl-GroEL homology to host proteins is hypothesized.     

Enhancement of ribosomal ribonucleic acid synthesis by deoxyribonucleic acid gyrase activity in Escherichia coli.

The effect of the deoxyribonucleic acid (DNA) gyrase inhibitors coumermycin A1, novobiocin, and oxolinic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was studied in vivo and in vitro. Preferential inhibition of ribosomal RNA (rRNA) synthesis was observed. No effect of oxolinic acid and coumermycin on rRNA synthesis was seen in mutants having a DNA gyrase which is resistant to these inhibitors. In a temperature-sensitive DNA gyrase mutant rRNA synthesis was decreased at nonpermissive temperatures. Thus, a functional DNA gyrase is required for rRNA synthesis. Purified DNA gyrase had no effect on rRNA synthesis in a purified system. However, DNA gyrase does show preferential stimulation of rRNA synthesis in a system supplemented with other proteins. Apparently, DNA gyrase stimulation of rRNA synthesis requires another protein.     

Construction and characterization of mutations at codon 751 of the Escherichia coli gyrB gene that confer resistance to the antimicrobial peptide microcin B17 and alter the activity of DNA gyrase

Microcin B17 is a peptide antibiotic that inhibits DNA replication in Escherichia coli by targeting DNA gyrase. Previously, two independently isolated microcin B17-resistant mutants were shown to harbor the same gyrB point mutation that results in the replacement of tryptophan 751 by arginine in the GyrB polypeptide. We used site-directed mutagenesis to construct mutants in which tryptophan 751 was deleted or replaced by other amino acids. These mutants exhibit altered DNA gyrase activity and different levels of resistance to microcin B17.     

巨大芽孢杆菌分子伴侣GroES和GroEL新基因的克隆及同源性分析

巨大芽孢杆菌作为革兰氏阳性细菌的一种,是良好的重组蛋白的表达宿主.本研究利用PCR技术从巨大芽孢杆菌基因组克隆出一条1.9 Kb的基因片段.核酸序列分析结果表明,该片段全长1 984 bp,包含2个ORF,分别与芽孢杆菌来源的GroES和GroEL基因有高度的相似性.氨基酸序列比对发现,GroES蛋白与枯草芽孢杆菌来源的GroES蛋白氨基酸序列同源性为91%,GroEL蛋白氨基酸序列同源性为90%.     

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.     

Gyrase-dependent initiation of bacteriophage T4 DNA replication: interactions of Escherichia coli gyrase with novobiocin, coumermycin and phage DNA-delay gene products.

Wild-type bacteriophage T4 and DNA-delay am mutants defective in genes 39, 52, 60 and 58–61 were tested for intracellular sensitivity to the antibiotics coumermycin and novobiocin, drugs which inhibit the DNA gyrase of Escherichia coli. Treatment with these antibiotics drastically reduced the characteristic growth of gene 39, 52 and 60 DNA-delay am mutants in E. coli lacking an amber suppressor (su^?). Wild-type phage-infected cells were unaffected by the drugs while the burst size of a gene 58–61 mutant was affected to an intermediate extent. A su^?E. coli strain which is resistant to coumermycin due to an altered gyrase permitted growth of the DNA-delay am mutants in the presence of the drug. Thus, the characteristic growth of the DNA-delay am mutants in an su^? host apparently depends on the host gyrase. An E. coli himB mutant is defective in the coumermycin-sensitive subunit of gyrase (H. I. Miller, personal communication). Growth of the gene 39, 52 and 60 am mutants was inhibited in the himB mutant while the gene 58–61 mutant and wild-type T4 showed small reductions in burst size in this host. Experiments with nalidixic acid-sensitive and resistant strains of E. coli show that wild-type phage T4 requires a functional nalA protein for growth.Novobiocin and coumermycin inhibit phage DNA synthesis in DNA-delay mutant-infected su^?E. coli if added during the early logarithmic phase of phage DNA synthesis. The gene 58–61 mutant showed the smallest inhibition of DNA synthesis in the presence of the drugs. Addition of the drugs during the late linear phase of phage DNA synthesis had no effect on further synthesis in DNA-delay mutant-infected cells. Coumermycin and novobiocin had no effect on DNA synthesis in wild-type-infected cells regardless of the time of addition of the antibiotics. Models are considered in which the DNA-delay gene products either form an autonomous phage gyrase or interact with the host gyrase and adapt it for proper initiation of phage DNA replication.     

巨大芽孢杆菌分子伴侣GroES和GroEL新基因的克隆及同源性分析

巨大芽孢杆菌作为革兰氏阳性细菌的一种,是良好的重组蛋白的表达宿主.本研究利用PCR技术从巨大芽孢杆菌基因组克隆出一条1.9Kb的基因片段.核酸序列分析结果表明,该片段全长1984bp,包含2个ORF,分别与芽孢杆菌来源的GroES和GroEL基因有高度的相似性.氨基酸序列比对发现,GroES蛋白与枯草芽孢杆菌来源的GroES蛋白氨基酸序列同源性为91%,GroEL蛋白氨基酸序列同源性为90%.     

Increased expression of biodegradative threonine dehydratase of Escherichia coli by DNA gyrase inhibitors

The synthesis of inducible biodegradative threonine dehydratase of Escherichia coli increased several-fold in the presence of the DNA gyrase inhibitors, nalidixic acid and coumermycin. Temperature-sensitive gyrB mutants expressed higher levels of dehydratase as compared to an isogenic gyrB+ strain. Immunoblotting experiments showed increased synthesis of the dehydratase protein in the presence of gyrase inhibitors; addition of rifampicin and chloramphenicol to cells actively synthesizing enzyme preventing new enzyme production. Increased expression of dehydratase by gyrase inhibitors was accompanied by relaxation of supercoiled DNA.     

A cluster of genes that affects nucleoid segregation in Salmonella typhimurium.

Thirteen conditional lethal mutations in genes of Salmonella typhimurium map at the clmF locus and affect both viability and the faithful partitioning of daughter nucleoids. These mutations have now been divided into three complementation groups by using cloned fragments of S. typhimurium DNA and renamed parC, parE, and parF. The proteins produced from the cloned fragments predict that ParC is an 85-kD protein, ParE is 75 kD in size, and ParF, 27 kD. The parE gene is about 5 kb upstream of the parC gene, and parC is just upstream of parF. Genes situated between parC and parE produce at least two proteins of unknown function. The DNA sequence of the S. typhimurium parC gene was determined and has 56% homology with the first 1400 base pairs of the Escherichia coli gryA gene, which encodes the A subunit of DNA gyrase, and 85% homology with the E. coli parC gene. Despite the strong homology between gryA and parC, these two genes cannot substitute for one another. The DNA sequence of the S. typhimurium parF gene was determined and predicts a protein with a hydrophobic N terminus. The ParF protein may interact with ParC and ParE to anchor these proteins to the membrane. These results raise questions about the relative roles of gyrase and ParCEF in nucleoid decatenation. In addition, the parC and gyrA genes provide an example of the evolution of essential functions by gene duplication.     

The C-terminal domain of the Escherichia coli DNA gyrase A subunit is a DNA-binding protein.

We have constructed a clone which over-produces a 33 kDa protein representing the C-terminal portion of the Escherichia coli DNA gyrase A subunit. This protein has no enzymic activity of its own, but will form a complex with a 64 kDa protein (representing the N-terminal part of the A subunit) and the gyrase B subunit, that will efficiently catalyse DNA supercoiling. We show that the 33 kDa protein can bind to DNA on its own in a manner which induces positive supercoiling of the DNA. We propose that the 33 kDa protein represents a domain of the gyrase A subunit which is involved in the wrapping of DNA around DNA gyrase.     

A protein isolated from Escherichia coli,identified as GroEL,reacts with anti-beta spectrin antibodies

We found that a protein of molecular weight close to 65kDa, present in Escherichia coli cells, reacts with anti-beta spectrin antibodies. A method of purification of this protein was designed. The method consists of the following: nonionic detergent extraction, gel filtration chromatography, ion-exchange chromatography using DEAE-Servacell, and two FPLC ion-exchange chromatography runs: the first without urea, the second in its presence. This method allowed us to obtain a highly purified protein. The results of mass spectrometry analysis suggest that the investigated protein is GroEL (Hsp60 Class). Using computer programs, by sequence analysis of both proteins we tried to explain why GroEL isolated from E. coli reacts with anti-beta spectrin antibodies. Both proteins may share a single epitope for the antibodies on their surfaces. Additionally, such an assumption is supported by the results of experiments in which antibodies interacting with GroEL were obtained from anti-beta spectrin serum and were shown to react with both GroEL and beta spectrins.     

A 4.2 kDa synthetic peptide as a potential probe to evaluate the antibacterial activity of coumarin drugs.

The coumarin antibiotics are potent inhibitors of DNA replication whose target is the enzyme DNA gyrase, an ATP-dependent bacterial type II topoisomerase. The coumarin drugs inhibit gyrase action by competitive binding to the ATP-binding site of DNA gyrase B protein. The production of new biologically active products has stimulated additional studies on coumarin-gyrase interactions. In this regard, a 4.2 kDa peptide mimic of DNA gyrase B protein from Escherichia coli has been designed and synthesized. The peptide sequence includes the natural fragment 131-146 (coumarin resistance-determining region) and a segment containing the gyrase-DNA interaction region (positions 753-770). The peptide mimic binds to novobiocin (Ka = 1.4+/-0.3 x 10(5) M(-1)), plasmid (Ka = 1.6+/-0.5 x 10(6) M(-1)) and ATP (Ka = 1.9+/-50.4 x 10(3) M(-1)), results previously found with the intact B protein. On the other hand, the binding to novobiocin was reduced when a mutation of Arg-136 to Leu-136 was introduced, a change previously found in the DNA gyrase B protein from several coumarin-resistant clinical isolates of Escherichia coli In contrast, the binding to plasmid and to ATP was not altered. These results suggest that synthetic peptides designed in a similar way to that described here could be used as mimics of DNA gyrase in studies which seek a better understanding of the ATP, as well as coumarin, binding to the gyrase and also the mechanism of action of this class of antibacterial drugs.     

Heat shock protein 60 (GroEL) from Porphyromonas gingivalis: Molecular cloning and sequence analysis of its gene and purification of the recombinant protein

Abstract Porphyromonas gingivalis is associated with human periodontal disease. We cloned and sequenced the gene for heat shock protein 60 (GroEL, HSP60) from P. gingivalis FDC381. The identified clone carried a 2.6 kb DNA fragment which contained two open reading frames (ORFs) encoding a 9.6- and a 58.4-kDa protein. The translated amino acid sequence of these ORFs showed a high degree of homology with known sequences for GroES and GroEL from several bacterial species and humans. Escherichia coli carrying this clone expressed a 65-kDa protein which was recognized by anti- Mycobacterium leprae HSP60 monoclonal antibody. We purified the 65-kDa protein by DEAE-sepharose chromatography and hydroxyapatite chromatography. This protein was immunogenic and was recognized by sera from a number of patients with periodontal disease. This immunological reactivity and the existence of molecular mimicry between the P. gingivalis GroEL and other HSP homologs may indicate an important role for this molecule in periodontal lesion.     

A mutation in a new gene of Escherichia coli, psu, requires secondary mutations for survival: psu mutants express a pleiotropic suppressor phenotype.

A mutation in an apparently new gene of Escherichia coli, psu, maps close to ara (1.3 min). psu mutants express a pleiotropic suppressor phenotype in which several auxotrophic requirements and some deletion mutations are suppressed. psu cloned in pBR322 can be maintained by the transformed cell only in the presence of several secondary mutations which accumulate in cultures of psu mutants and have an apparently compensatory role. The accumulation of secondary mutations is not due to mutator activity. The secondary mutations can each act as a suppressor of an auxotrophic requirement in the absence of psu, while suppression of deletions requires the presence of psu. Thus, the suppressor phenotype of psu mutants is due to both psu and the secondary mutations. The functions of psu and the secondary mutations are not known. However, two observations suggest an association with DNA gyrase and with DNA supercoiling. (i) psu mutants are highly resistant to oxolinic acid, the gyrase A inhibitor, while the secondary mutants vary from being very sensitive to more resistant than the wild-type strain. (ii) Novobiocin, which decreases the level of DNA supercoiling, significantly stimulates suppression of auxotrophy in some secondary mutants.     

Activity of chloramphenicol acetyltransferase overproduced in E. coli with wild-type and mutant GroEL.

The homo-oligomeric protein chloramphenicol acetyltransferase (CAT) has previously been shown to interact with a chaperone GroEL in vitro, suggesting a possible involvement of GroEL in CAT assembly. CAT was overproduced to various levels in the presence and absence of GroEL overproduction, and in groEL mutants. CAT was accumulated to 9-45% of total cellular protein in a fully soluble form, without formation of inclusion bodies. In all cases, even with groEL mutants, CAT specific activity was shown proportional to the amount of protein produced, indicating the formation of active trimer CAT structure does not need GroEL in Escherichia coli.     

Structure and Expression of a GroE-homologous Operon of a Macronucleus-Specific Symbiont Holospora obtusa of the Ciliate Paramecium caudatum

ABSTRACT The reproductive form of a macronucleus-specific symbiont Holospora obtuse , when harbored by the macronucleus of the ciliate Paramecium caudatum , selectively synthesized a 63-kDa protein which is immunologically related to GroEL, or HSP60, of Escherichia coli. Heat shock treatment of isolated cells of the reproductive and infectious form of the bacterium also induced the synthesis of the GroEL homolog. Immunoblotting showed that the amount of this protein per cell, whether the reproductive or infectious form, is roughly constant. Cloning and sequencing of a gene coding for the GroEL homolog suggested that the protein is 55.2% identical to GroEL of E. coli at the amino acid sequence level, and that the gene is preceded by an open reading frame which encodes a protein 39.6% identical to GroES of E. coli. Northern blot hybridization showed that the GroEL homologous gene is highly expressed in the reproductive form, but only in a trace amount in the intermediate and infectious form. Immunoelectron microscopy revealed that the GroEL homolog is localized in the cytoplasm of the reproductive and infectious form.     

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.     

Mechanism of DNA gyrase-mediated illegitimate recombination: characterization of Escherichia coli gyrA mutations that confer hyper-recombination phenotype.

To study the mechanism of DNA gyrase-mediated illegitimate recombination in Escherichia coli, we isolated temperature-sensitive gyrA mutants that confer spontaneous illegitimate recombination and spontaneous induction of lambda prophage at higher frequencies than that in the wild-type. After reconstruction of single mutations by targeted mutagenesis, we confirmed that two single mutations, gyrAL492P and gyrAL488P, and a double mutation, gyrAI203V+gyrAI205V, show the same properties as those described above. With respect to the phenotypes of hyper-recombination and higher induction of lambda prophage, these mutations were dominant over the wild-type. Analysis of recombination junctions of lambdabio transducing phages formed spontaneously in these mutants showed that the parental E. coli bio and lambda recombination sites have a homologous sequence of only 0. 7 base-pair on average, indicating that homology is not required for this illegitimate recombination. Analysis of nucleotide sequences of mutant gyrA genes revealed that the gyrAL492P and gyrAL488P mutations contain amino acid substitutions of Leu492-->Pro and Leu488-->Pro, respectively, which correspond to the alpha18 helix in the breakage-reunion domain of DNA gyrase A subunit. The gyrAI203V and gyrAI205V mutations contain Ile203-->Val and Ile205-->Val, respectively, which correspond to the alpha10' helix, also in the breakage-reunion domain of DNA gyrase A subunit. Biochemical analysis indicated that the GyrA63 protein that contains the L492P mutation has an apparently normal supercoiling activity, but it also produces a small amount of linear DNA in the absence of DNA gyrase inhibitor during the supercoiling reaction, suggesting that the mutant DNA gyrase may have a defect at the step of religation or a defect in the subunit interaction. These results suggest that the recombination is induced by defects of religation and/or dimer formation in the mutant DNA gyrases, implying that two alpha helices, alpha10' and alpha18, of DNA gyrase A subunit have crucial roles in subunit interaction and/or resealing of DNA.