Organization and expression of the dnaJ and dnaK genes of Escherichia coli K12

Saito, Haruo; Uchida, Hidenobu

doi:10.1007/bf00267592

Cited by 110 publications

(78 citation statements)

References 13 publications

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“…The dnaK gene encodes a 70,000-Mr heat shock protein that is required for X DNA replication and bacterial growth at high temperatures (14,28,29). The dnaK756 mutant was previously shown to be defective in the adaptation phase of the heat shock response, wherein the initial rapid rate of heat shock protein synthesis declines to a new rate characteristic of steady-state growth at that temperature (34).…”

Section: Resultsmentioning

confidence: 99%

Modulation of stability of the Escherichia coli heat shock regulatory factor sigma

1989

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The heat shock response of Escherichia coli is under the positive control of the r32 protein (the product of the rpoH gene). We found that overproduction of the if32 protein led to concomitant overproduction of the heat shock proteins, suggesting that the intracellular i32 levels limit heat shock gene expression. In support of this idea, the intracellular half-life of the if32 protein synthesized from a multicopy plasmid was found to be extremely short, e.g., less than 1 min at 37 and 42°C. The half-life increased progressively with a decrease in temperature, reaching 15 min at 22°C. Finally, conditions known previously to increase the rate of synthesis of the heat shock proteins, i.e., a mutation in the dnaK gene or expression of phage A early proteins, were shown to simultaneously result in a three-to fivefold increase in the half-life of if32.All organisms so far tested respond to a sudden upshift in growth temperature by increasing the synthesis of a set of proteins, a phenomenon called the heat shock response. In Escherichia coli, there is a set of about 20 proteins (called the heat shock proteins) whose synthesis is thereby increased (see reference 27 for a review). The amino acid sequences of at least some heat shock proteins in distantly related organisms, including Drosophila melanogaster and Homo sapiens, are remarkably similar to those in E. coli (4,5,21), suggesting that the heat shock response is of ancient origin and fundamental importance to cellular physiology. The function of the heat shock proteins, however, is unclear, although it has been shown that they play roles in the assembly and disassembly of macromolecular complexes (GroE [15, 16, 21, In E. coli, heat shock protein synthesis rates peak at about 5 min after a temperature upshift (e.g., from 30 to 42°C) and then decline rapidly to new steady-state levels that are characteristic of the new ambient temperature. Initiation of the heat shock response is regulated transcriptionally. It has been shown that the RNA polymerase core (E) binds to a new initiation subunit, c32 (30), and the resulting holoenzyme, E-&32, transcribes only heat shock genes (19), which have promoter sequences that differ from those transcribed by E plus U70, the normal vegetative initiation factor (8). The transcription factor U70 iS itself a heat shock protein, so the increase in its concentration after heat shock may contribute to the decline in heat shock protein synthesis. Furthermore, other heat shock proteins, in particular the dnaK gene product, contribute to the shutoff, since mutations in their genes prolong the high-level synthesis of heat shock proteins (34). The heat shock response must be tightly regulated in order to allow rapid changes in heat shock protein synthesis rates. Although the level of mRNA from the rpoH gene (which encodes U32) increases after heat shock (11,12,36 Technology, Cambridge, MA 02139. this increase is insufficient and too slow to be the sole explanation of the rapid effect of heat shock. In this paper, we show that the concen...

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Section: Resultsmentioning

confidence: 99%

Modulation of stability of the Escherichia coli heat shock regulatory factor sigma

1989

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“…First, the grpE gene is part of the dnaK-dnaJ operon. In E. coli, these three genes form two independent transcriptional units (50,52). Second, there is a fourth potential heat shock gene, orf39, of still unknown biological function.…”

Section: Discussionmentioning

confidence: 99%

“…Subsequently, the dnaK gene product was shown to be essential for E. coli viability at high and low temperatures (22,30,46,51,52), and genetic evidence indicates functions for DnaK after heat shock in the synthesis of RNA and DNA and in cell division (30,46,53).…”

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confidence: 99%

Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis

et al. 1992

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By using an internal part of the dnaK gene from BaciUlus megaterium as a probe, a 5.2-kb Hindlll fragment of chromosomal DNA of BaciUlus subtilis was cloned. Downstream sequences were isolated by in vivo chromosome walking. Sequencing of 5,085 bp revealed four open reading frames in the order orf9-grpEdnaK-dnaJ. orJ39 encodes a 39-kDa polypeptide of unknown biological function with no noticeable homology to any other protein within the data bases. Alignment of the GrpE protein with those of three other bacterial species revealed a low overall homology, but a higher homology restricted to two regions which might be involved in interactions with other proteins. Alignment of the DnaK protein with six bacterial DnaK polypeptides revealed that a contiguous region of 24 amino acids is absent from the DnaK proteins of all known gram-positive species. Primer extension studies revealed three potential transcription start sites, two preceding orJ39 (Si and S2) and a third one in front of grpE (S3). S2 and S3 were activated at a high temperature. Northern (RNA) analysis led to the detection of three mRNA species of 4.9, 2.6, and 1.5 kb. RNA dot blot experiments revealed an at-least-fivefold increase in the amount of specific mRNA from 0 to 5 min postinduction and then a rapid decrease. A transcriptional fusion between dnaK and the amyL reporter gene exhibited a slight increase in a-amylase activity after heat induction. A 9-bp inverted repeat was detected in front of the coding region of orJ39. This inverted repeat is present in a number of other heat shock operons in other microorganisms ranging from cyanobacteria to mycobacteria. The biological property of this inverted repeat as a putative key element in the induction of heat shock genes is discussed. The dnaK locus was mapped at about 2230 on the B. subtilis genetic map.The heat shock response is a homeostatic mechanism that enables cells to survive a variety of environmental stresses. It is characterized by the increased synthesis of a group of evolutionarily conserved proteins, heat shock proteins (HSPs), and is a universal feature of both prokaryotic and eukaryotic cells (35). When Eschertichia coli cells are shifted to a high temperature, the synthesis of a set of about 20 HSPs transiently increases and then declines rapidly to steady-state levels characteristic of the new ambient temperature (44).The highly conserved HSPs perform similar functions in all organisms. One of these functions is the well-established regulation of protein-protein interactions by the chaperonins (69). One of the most abundant HSPs, HSP70, is highly conserved in evolution. It is found in such diverse organisms as E. coli, Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens (14, 35).The dnaK gene of E. coli was originally discovered because mutations in it blocked bacteriophage lambda DNA replication at all temperatures (22,23). Subsequently, the dnaK gene product was shown to be essential for E. coli viability at high and low temperatures (22,30,46,51,52), and genetic evid...

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“…In most genomes, dnaK is a single copy gene, often found in the same operon as dnaJ. In Escherichia coli (Gammaproteobacteria), the dnaK operon is bicistronic, comprising the dnaK (1917 bp) and dnaJ (1131 bp) genes (Saito & Uchida, 1978). The dnaJ gene is sometimes found in more than one copy, as in the high-G+C-content Gram-positive actinobacteria.…”

Section: Introductionmentioning

confidence: 99%

dnaJ is a useful phylogenetic marker for alphaproteobacteria

Alexandre

Laranjo

Young

et al. 2008

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLO

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In the past, bacterial phylogeny relied almost exclusively on 16S rRNA gene sequence analysis. More recently, multilocus sequence analysis has been used to infer organismal phylogenies. In this study, the dnaJ chaperone gene was investigated as a marker for phylogeny studies in alphaproteobacteria. Preliminary analysis of G+C contents and G+C3s contents (the G+C content of the synonymous third codon position) showed no clear evidence of horizontal transfer of this gene in proteobacteria. dnaJ-based phylogenies were then analysed at three taxonomic levels: the Proteobacteria, the Alphaproteobacteria and the genus Mesorhizobium. Dendrograms based on DnaJ and 16S rRNA gene sequences revealed the same topology described previously for the Proteobacteria. These results indicate that the DnaJ phylogenetic signal is able to reproduce the accepted relationships among the five classes of the Proteobacteria. At a lower taxonomic level, using 20 alphaproteobacteria, the 16S rRNA gene-based phylogeny is distinct from the one based on DnaJ sequence analysis. Although the same clusters are generated, only the topology of the DnaJ tree is consistent with broader phylogenies from recent studies based on concatenated alignments of multiple core genes. For example, the DnaJ tree shows the two clusters within the Rhizobiales as closely related, as expected, while the 16S rRNA gene-based phylogeny shows them as distantly related. In order to evaluate the phylogenetic performance of dnaJ at the genus level, a multilocus analysis based on five housekeeping genes (atpD, gapA, gyrB, recA and rplB) was performed for ten Mesorhizobium species. In contrast to the 16S rRNA gene, the DnaJ sequence analysis generated a tree similar to the multilocus dendrogram. For identification of chickpea mesorhizobium isolates, a dnaJ nucleotide sequence-based tree was used. Despite different topologies, 16S rRNA gene-and dnaJ-based trees led to the same species identification. This study suggests that the dnaJ gene is a good phylogenetic marker, particularly for the class Alphaproteobacteria, since its phylogeny is consistent with phylogenies based on multilocus approaches. INTRODUCTIONThe phylum Proteobacteria is composed of five classes, the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria and Epsilonproteobacteria (Garrity et al., 2005;Stackebrandt et al., 1988). By the end of 2008, more than 380 complete genomes of proteobacteria were available in public databases (http://www. ncbi.nlm.nih.gov/genomes/lproks.cgi). Alphaproteobacteria exhibit an enormous diversity in their morphological and metabolic characteristics and the class is presently recognized solely as a clade in the 16S rRNA gene-based phylogeny (Stackebrandt et al., 1988). Based on 16S rRNA gene trees, the Alphaproteobacteria has been divided into seven orders: Caulobacterales, Rhizobiales, Rhodobacterales, Rhodospirillales, Rickettsiales, Sphingomonadales and Parvularculales (Kersters et al., 2006). The Alphaproteobacteria includes important bacteria tha...

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Organization and expression of the dnaJ and dnaK genes of Escherichia coli K12

Cited by 110 publications

References 13 publications

Modulation of stability of the Escherichia coli heat shock regulatory factor sigma

Modulation of stability of the Escherichia coli heat shock regulatory factor sigma

Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis

dnaJ is a useful phylogenetic marker for alphaproteobacteria

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