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C Georgopoulos

Publications and source records attributed to C Georgopoulos.

At least 91 records · Page 5Linked to original sources
PubMed1991

The htrM gene, whose product is essential for Escherichia coli viability only at elevated temperatures, is identical to the rfaD gene.

We have identified a new E. coli gene, htrM. The htrM gene was identified because its insertional inactivation by the Tn5 transposon results in E. coli's inability to form colonies at temperatures above 43 degrees C. The corresponding htrM+ gene was cloned on the basis of its ability to correct the temperature-sensitive phenotype of the htrM::Tn5 insertion mutations. The htrM gene has been mapped to 81.2 min on the conventional E. coli genetic map. It was sequenced and shown to code for an acidic, 34,893-Da polypeptide. Three transcriptional starts were located 48, 90 and 123 nucleotides upstream of the ATG, initiation codon referred to as the P1, P2 and P3(hs) promoters, respectively. The -10 and -35 regions of the P1 promoter bear a close similarity to the E sigma 70-recognized consensus sequences, while the -12 region of the P2 promoter resembles the consensus promoter sequence transcribed by the rpoN gene product. Transcripts of the htrM gene accumulate with increasing temperature. The -10 and -35 regions of the P3(hs) promoter, represented by nucleotides 160 to 130 upstream of the ATG initation codon, are similar to the E sigma 32-recognized consensus sequences. The sigma 32 transcription factor is essential for maximal htrM gene transcription, since htrM RNA transcripts are made at reduced rates in a rpoH null mutant background. Surprisingly, the htrM gene turns out to be identical to rfaD, whose product is required for the biosynthesis of the ADP-L-glycero-D manoheptose lipopolyaccharide precursor [Pegues et al. (1990) J. Bacteriol. 172, 4652-4660].

Amino Acid Sequence↗
PubMed1991

Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK.

The products of the Escherichia coli dnaK, dnaJ, and grpE heat shock genes have been previously shown to be essential for bacteriophage lambda DNA replication at all temperatures and for bacterial survival under certain conditions. DnaK, the bacterial heat shock protein hsp70 analogue and putative chaperonin, possesses a weak ATPase activity. Previous work has shown that ATP hydrolysis allows the release of various polypeptides complexed with DnaK. Here we demonstrate that the ATPase activity of DnaK can be greatly stimulated, up to 50-fold, in the simultaneous presence of the DnaJ and GrpE heat shock proteins. The presence of either DnaJ or GrpE alone results in a slight stimulation of the ATPase activity of DnaK. The action of the DnaJ and GrpE proteins may be sequential, since the presence of DnaJ alone leads to an acceleration in the rate of hydrolysis of the DnaK-bound ATP. The presence of GrpE alone increases the rate of release of bound ATP or ADP without affecting the rate of hydrolysis. The stimulation of the ATPase activity of DnaK may contribute to its more efficient recycling, and it helps explain why mutations in dnaK, dnaJ, or grpE genes often exhibit similar pleiotropic phenotypes.

Adenosine Triphosphatases↗
PubMed1991

Analysis of an Escherichia coli dnaB temperature-sensitive insertion mutation and its cold-sensitive extragenic suppressor.

An Escherichia coli mutant, ts121, was isolated following random insertional mutagenesis using phage lambda Mu transposition. The mutant phenotype includes inability to form colonies at temperatures above 38 degrees C and inability to propagate phage lambda at all temperatures. A lambda i434 cI- (ts121)+ transducing phage was isolated on the basis of its ability to form plaques on ts121 mutant bacteria. Using this transducing phage, it was shown through complementation and protein analyses, that the ts121 mutation is located in the dnaB gene. The exact insertion event was identified by polymerase chain reaction amplification of the DNA sequences containing the insertion junction. The mutational insertion event in ts121 was mapped precisely between base pairs 1514 and 1515 of the dnaB gene. This result predicts that the mutant dnaB protein has lost its six terminal amino acids. The reading frame shifts into Mu-specific DNA sequences resulting in an additional 20 amino acid residues. The E. coli wild type dnaB protein participates in host replication and interacts with lambda P protein to initiate phage lambda DNA replication. Our results demonstrate that the extreme carboxyl end of the dnaB protein is required for productive interaction with the lambda P replication protein at all temperatures, and is important for dnaB function at temperatures above 38 degrees C. Cold-sensitive extragenic suppressors of the ts121 mutation were isolated on the basis of their ability to restore colony formation at 42 degrees C. One of these extragenic suppressors was mapped at 54 min on the E. coli genetic map and localized to the suhB gene, whose product may affect the expression of a number of genes at the translational level.

Bacterial Proteins↗
PubMed1991

Sequencing, mutational analysis, and transcriptional regulation of the Escherichia coli htrB gene.

The Escherichia coli htrB gene was originally discovered because its insertional inactivation led to an exquisitely temperature-sensitive phenotype in rich media, i.e. the ability to form colonies at temperatures below 32 degrees C, but not above 33 degrees C. The htrB gene has been sequenced. It can potentially code for two proteins, with Mr values of 35,407 Da and 8669 Da, that are encoded by overlapping, divergent open reading frames. Our data are consistent with the 35,407 Da protein being HtrB. Northern blot analysis clearly shows that the monocistronic htrB message is not under heat-shock regulation. We have also sequenced the flanking DNA and have discovered a new gene, designated orf39.9, located immediately adjacent to htrB, but divergently transcribed.

Amino Acid Sequence↗
PubMed1991

Identification of the Escherichia coli sohB gene, a multicopy suppressor of the HtrA (DegP) null phenotype.

We cloned and sequenced the sohB gene of Escherichia coli. The temperature-sensitive phenotype of bacteria that carry a Tn10 insertion in the htrA (degP) gene is relieved when the sohB gene is present in the cell on a multicopy plasmid (30 to 50 copies per cell). The htrA gene encodes a periplasmic protease required for bacterial viability only at high temperature, i.e., above 39 degrees C. The sohB gene maps to 28 min on the E. coli chromosome, precisely between the topA and btuR genes. The gene encodes a 39,000-Mr precursor protein which is processed to a 37,000-Mr mature form. Sequencing of a DNA fragment containing the gene revealed an open reading frame which could encode a protein of Mr 39,474 with a predicted signal sequence cleavage site between amino acids 22 and 23. Cleavage at this site would reduce the size of the processed protein to 37,474 Mr. The predicted protein encoded by the open reading frame has homology with the inner membrane enzyme protease IV of E. coli, which digests cleaved signal peptides. Therefore, it is possible that the sohB gene encodes a previously undiscovered periplasmic protease in E. coli that, when overexpressed, can partially compensate for the missing HtrA protein function.

Amino Acid Sequence↗
PubMed1991

The Escherichia coli htrP gene product is essential for bacterial growth at high temperatures: mapping, cloning, sequencing, and transcriptional regulation of htrP.

We identified and characterized a new Escherichia coli gene, htrP. The htrP gene was identified because its insertional inactivation by the Tn10 transposon results in the inability of E. coli to form colonies at temperatures above 37 degrees C and a slow growth phenotype at 30 degrees C. The htrP gene was cloned and mapped to 66.3 min on the E. coli genetic map, 4 kbp clockwise from the tolC gene. The htrP gene was sequenced and shown to code for an acidic, 27,471-Da polypeptide and to be transcribed counterclockwise with respect to the genetic map. The predicted HtrP protein has two potential transmembrane segments and shares an identity of 64.4% over a length of 210 amino acids with the LuxH protein. Despite the fact that the htrP gene is essential for E. coli growth exclusively at high temperatures, the levels of htrP-specific transcripts decrease with increasing temperature.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of the Escherichia coli htrB gene, whose product is essential for bacterial viability above 33 degrees C in rich media.

We have identified and studied the htrB gene of Escherichia coli. Insertional inactivation of the htrB gene leads to bacterial death at temperatures above 33 degrees C. The mutant bacterial phenotype at nonpermissive temperatures includes an arrest of cell division followed by the formation of bulges or filaments. The htrB+ gene has been cloned by complementation and shown to reside at 23.4 min on the E. coli genetic map, the relative order of the neighboring loci being mboA-htrB-pyrC. The htrB gene is transcribed in a counterclockwise fashion, relative to the E. coli genetic map, and its product has been identified as a membrane-associated protein of 35,000 Da. Growth experiments in minimal media indicate that the HtrB function becomes dispensable at low growth rates.

Cloning, Molecular↗
PubMed1990

The E. coli dnaK gene product, the hsp70 homolog, can reactivate heat-inactivated RNA polymerase in an ATP hydrolysis-dependent manner.

Pelham previously proposed that the hsp70 family of heat shock proteins could prevent the formation and/or allow the dissolution of protein aggregates created during stress conditions. We confirmed this hypothesis by showing that the E. coli hsp70 homolog, the dnaK gene product, protects the host RNA polymerase enzyme from heat inactivation in an ATP-independent reaction. In addition, we show that heat-inactivated and aggregated RNA polymerase is both disaggregated and reactivated following simultaneous incubation with DnaK protein and hydrolyzable ATP. The DnaK756 mutant protein has lost the ability to disaggregate the inactivated RNA polymerase enzyme. Our results demonstrate that the DnaK protein contributes to E. coli's growth not only by protecting some enzymes from denaturation but also by reactivating some once they are misfolded or aggregated.

Adenosine Triphosphate↗
PubMed1990

Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication.

The process of initiation of lambda DNA replication requires the assembly of the proper nucleoprotein complex at the origin of replication, ori lambda. The complex is composed of both phage and host-coded proteins. The lambda O initiator protein binds specifically to ori lambda. The lambda P initiator protein binds to both lambda O and the host-coded dnaB helicase, giving rise to an ori lambda DNA.lambda O.lambda P.dnaB structure. The dnaK and dnaJ heat shock proteins have been shown capable of dissociating this complex. The thus freed dnaB helicase unwinds the duplex DNA template at the replication fork. In this report, through cross-linking, size chromatography, and protein affinity chromatography, we document some of the protein-protein interactions occurring at ori lambda. Our results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that the dnaJ protein specifically interacts with the dnaB helicase.

Bacterial Proteins↗
PubMed1990

Mutational analysis of the phage T4 morphogenetic 31 gene, whose product interacts with the Escherichia coli GroEL protein.

The phage T4 morphogenetic gene 31 has been sequenced. Its deduced gene product is a polypeptide of 111 aa, with a predicted Mr of 12064 and a pI of 4.88. The proof that the assigned open reading frame (ORF) encodes Gp31 rests on the sequencing of two known gene 31 amber mutations, amN54 and NG71, demonstrating that these mutations result in translational termination within the assigned ORF. Furthermore, the sequencing of four different T4 epsilon mutations, isolated on the basis of allowing the phage to propagate on Escherichia coli groEL- hosts, showed that they are either missense mutations or 3-bp deletions in the gene 31 reading frame. The sequencing of neighboring DNA revealed the presence of five other ORFs, one of which overlaps gene 31 substantially, but in the opposite orientation.

Amino Acid Sequence↗
PubMed1990

The Escherichia coli heat shock proteins GroEL and GroES modulate the folding of the beta-lactamase precursor.

One of the fundamental problems in biochemistry is the role of accessory proteins in the process of protein folding. The Escherichia coli heat shock protein complex GroEL/ES has been suggested to be a 'chaperonin' and be involved in both oligomer assembly as well as protein transport through the membrane. We show here that the folding of the purified precursor of beta-lactamase is inhibited by purified GroEL or the GroEL/ES complex with a stoichiometry of one particle per molecule of pre-beta-lactamase. Purified GroES alone has no effect on folding. After Mg2+ ATP addition folding resumes and the yield of active enzyme is higher than in the absence of GroEL or GroEL/ES. Unexpectedly, GroEL or GroEL/ES, when added to folded pre-beta-lactamase, lead to an apparent net 'unfolding', probably to a collapsed state of the protein, which can be reversed by the addition of Mg2+ ATP. The reversible and Mg2+ ATP-dependent association of GroEL/ES with non-native proteins might explain its postulated role in both protein transport and oligomer assembly.

Enzyme Precursors↗
PubMed1990

Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress.

We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis.

Animals↗
PubMed1990

Role of Escherichia coli heat shock proteins DnaK and HtpG (C62.5) in response to nutritional deprivation.

Because of the highly conserved pattern of expression of the eucaryotic heat shock genes hsp70 and hsp84 or their cognates during sporulation in Saccharomyces cerevisiae and development in higher organisms, the role of the Escherichia coli homologs dnaK and htpG was examined during the response to starvation. The htpG deletion mutant was found to be similar to its wild-type parent in its ability to survive starvation for essential nutrients and to induce proteins specific to starvation conditions. The dnaK103 mutant, however, was highly susceptible to killing by starvation for carbon and, to a lesser extent, for nitrogen and phosphate. Analysis of proteins induced under starvation conditions on two-dimensional gels showed that the dnaK103 mutant was defective for the synthesis of some proteins induced in wild-type cells by carbon starvation and of some proteins induced under all starvation conditions, including the stationary phase in wild-type cells. In addition, unique proteins were synthesized in the dnaK103 mutant in response to starvation. Although the synthesis of some proteins under glucose starvation control was drastically affected by the dnaK103 mutation, the synthesis of proteins specifically induced by nitrogen starvation was essentially unaffected. Similarly, the dnaK103 mutant was able to grow, utilizing glutamine or arginine as a source of nitrogen, at a rate approximate to that of the wild-type parent, but it inefficiently utilized glycerol or maltose as carbon sources. Several differences between the protein synthetic pattern of the dnaK103 mutant and the wild type were observed after phosphate starvation, but these did not result in a decreased ability to survive phosphate starvation, compared with nitrogen starvation.

Bacterial Proteins↗
PubMed1990

Identification, cloning, and characterization of the Escherichia coli sohA gene, a suppressor of the htrA (degP) null phenotype.

Two extragenic suppressors which allow temperature-sensitive htrA mutant Escherichia coli bacteria to grow at 42 degrees C and simultaneously acquire a cold-sensitive phenotype at 30 degrees C were isolated. The cold-sensitive phenotype exhibited by one of the mutants was used to clone the corresponding wild-type copy of the suppressor gene. This was done through complementation with a mini-mu plasmid E. coli DNA library, by selection for colonies which were no longer cold sensitive, at 30 degrees C. The cloned suppressor gene was shown to complement the cold-sensitive phenotype of both suppressor mutations. It was mapped to 68 min on the E. coli chromosome through hybridization to the Kohara library of overlapping lambda transducing bacteriophages, which covers the entire E. coli chromosome. The complementing gene was further subcloned on an 830-base-pair (bp) DNA fragment. DNA sequencing revealed the presence of an open reading frame (ORF) of 333 bp which could encode a protein of 12,359 Mr. Subcloning of various DNA fragments from within this 830-bp DNA fragment suggests that this ORF is most likely responsible for suppression of the cold-sensitive phenotype of the htrA suppressor bacteria. By using a T7 polymerase system to overproduce plasmid-encoded proteins, a protein of approximately 12,000 Mr was produced by this cloned DNA fragment. This ORF defines a previously undiscovered gene in E. coli, called sohA (suppressor of htrA).

Amino Acid Sequence↗
PubMed1990

The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase.

As a preliminary step in the understanding of the function of the Escherichia coli HtrA (DegP) protein, which is indispensable for bacterial survival only at elevated temperatures, the protein was purified and partially characterized. The HtrA protein was purified from cells carrying the htrA gene cloned into a multicopy plasmid, resulting in its overproduction. The sequence of the 13 N-terminal amino acids of the purified HtrA protein was determined and was identical to the one predicted for the mature HtrA protein by the DNA sequence of the cloned gene. Moreover, the N-terminal sequence showed that the 48-kilodalton HtrA protein is derived by cleavage of the first 26 amino acids of the pre-HtrA precursor polypeptide and that the point of cleavage follows a typical target sequence recognized by the leader peptidase enzyme. The HtrA protein was shown to be a specific endopeptidase which was inhibited by diisopropylfluorophosphate, suggesting that HtrA is a serine protease.

Amino Acid Sequence↗
PubMed1990

A new Escherichia coli heat shock gene, htrC, whose product is essential for viability only at high temperatures.

We identified and characterized a new Escherichia coli gene, htrC. Inactivation of the htrC gene results in the inability to form colonies at 42 degrees C. An identical bacterial phenotype is found whether the htrC gene is inactivated either by Tn5 insertions or by a deletion spanning the entire gene. The htrC gene has been localized at 90 min, immediately downstream of the rpoC gene, and has been previously sequenced. It codes for a basic polypeptide with an Mr of 21,130. The htrC gene is under heat shock regulation, since it is transcribed actively only in bacteria possessing functional sigma 32. Inactivation of htrC results in (i) bacterial filamentation at intermediate temperatures, (ii) cell lysis at temperatures above 42 degrees C, (iii) overproduction of sigma 32-dependent heat shock proteins at all temperatures, (iv) overproduction of a few additional polypeptides, (v) underproduction of many polypeptides, and (vi) an overall defect in cellular proteolysis as judged by the reduced rate of puromycyl polypeptide degradation. In addition, the presence of an htrC mutation eliminates the UV sensitivity normally exhibited by lon mutant bacteria.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of dnaJ null mutants of Escherichia coli.

Bacteriophage lambda requires the lambda O and P proteins for its DNA replication. The rest of the replication proteins are provided by the Escherichia coli host. Some of these host proteins, such as DnaK, DnaJ, and GrpE, are heat shock proteins. Certain mutations in the dnaK, dnaJ, or grpE gene block lambda growth at all temperatures and E. coli growth above 43 degrees C. We have isolated bacterial mutants that were shown by Southern analysis to contain a defective, mini-Tn10 transposon inserted into either of two locations and in both orientations within the dnaJ gene. We have shown that these dnaJ-insertion mutants did not grow as well as the wild type at temperatures above 30 degrees C, although they blocked lambda DNA replication at all temperatures. The dnaJ-insertion mutants formed progressively smaller colonies at higher temperatures, up to 42 degrees C, and did not form colonies at 43 degrees C. The accumulation of frequent, uncharacterized suppressor mutations allowed these insertion mutants to grow better at all temperatures and to form colonies at 43 degrees C. None of these suppressor mutations restored the ability of the host to propagate phage lambda. Radioactive labeling of proteins synthesized in vivo followed by immunoprecipitation or immunoblotting with anti-DnaJ antibodies demonstrated that no DnaJ protein could be detected in these mutants. Labeling studies at different temperatures demonstrated that these dnaJ-insertion mutations resulted in altered kinetics of heat shock protein synthesis. An additional eight dnaJ mutant isolates, selected spontaneously on the basis of blocking phage lambda growth at 42 degrees C, were shown not to synthesize DnaJ protein as well. Three of these eight spontaneous mutants had gross DNA alterations in the dnaJ gene. Our data provide evidence that the DnaJ protein is not absolutely essential for E. coli growth at temperatures up to 42 degrees C under standard laboratory conditions but is essential for growth at 43 degrees C. However, the accumulation of extragenic suppressors is necessary for rapid bacterial growth at higher temperatures.

Bacterial Proteins↗