Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli

Cellier, Mathieu; Teyssier, Jacques; Nicolas, Michel; Liautard, Jean‐Pierre; Marti, Jacques; Widada, Joannes Sri

doi:10.1128/jb.174.24.8036-8042.1992

Cited by 58 publications

(42 citation statements)

References 38 publications

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“…Escherichia coli XL-1 Blue competent cells and plating cultures were prepared as described by Sambrook et al (1989) for use as a host for plasmid pBSIIKS + purchased from Stratagene (pBluescriptI1 Exoj mung Kit). They were processed as described previously (Cellier et al, 1992).…”

Section: Methodsmentioning

confidence: 99%

Identification and sequence analysis of IS6501, an insertion sequence in Brucella spp.: relationship between genomic structure and the number of IS6501 copies

Ouahrani

Michaux

Widada

et al. 1993

Journal of General Microbiology

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An insertion sequence (IS) element of Bruceffa ovis, named IS6501, was isolated and its complete nucleotide sequence determined. IS6501 is 836 bp in length and occurs 20-35 times in the B. ovis genome and 5-15 times in other Brucella species. Analysis of the junctions at the sites of insertion revealed a small target site duplication of four bases and inverted repeats of 17 bp with one mismatch. IS6501 presents significant similarity (53.4%) with IS427 identified in Agrobacterium tumefaciens, suggesting a common ancestral sequence. A long ORF of 708 bp was identified encoding a protein with a predicted molecular mass of 26 kDa and sharing sequence identity with the hypothetical protein 1 of A. tumefaciens and with the transposase of Mycobactevium tuberculosis. IS6501 is present in all Bruceffa strains we have tested. Restriction fragment length polymorphism of reference and field strains of two species (B. mefitensis and B. ovis) was studied using either pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA or hybridization of EcoRI-digested DNA using IS6501 as a probe. The genome of B. mefitensis biovar 3 contains about 10 IS copies per genome and field strains of the same species could not be distinguished either by IS hybridization or by XbaI (PFGE) restriction patterns. In contrast, the number of IS copies in the B. ovis genome is around 30 and the different field strains can be differentiated by both methods. As ISs have been shown to be implicated in chromosomal rearrangement, we propose that the chromosomal polymorphism revealed by PFGE and high copy number of IS6501 observed in B. ovis may be related to the presence of an active IS in this species.

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Section: Methodsmentioning

confidence: 99%

Identification and sequence analysis of IS6501, an insertion sequence in Brucella spp.: relationship between genomic structure and the number of IS6501 copies

Ouahrani

Michaux

Widada

et al. 1993

Journal of General Microbiology

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“…A lac fusion of the dnaK operon from Z. mobilis was thermoregulated in E. coli (24), and the dnaK operon of Brucella ovis was expressed in a heat shock-dependent manner in E. coli (8). These two dnaK operons do not contain the IR found in the groESL operons of alpha purple proteobacteria.…”

Section: Vol 178 1996 Regulation Of the A Tumefaciens Groesl Operomentioning

confidence: 99%

Heat shock activation of the groESL operon of Agrobacterium tumefaciens and the regulatory roles of the inverted repeat

Segal

Ron

1996

J Bacteriol

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Deletions were constructed in the conserved inverted repeat (IR) found in the groESL operon of Agrobacterium tumefaciens and in many other groE and dnaK operons and genes in eubacteria. These deletions affected the level of expression of the operon and the magnitude of its heat shock activation. The IR seems to operate at the DNA level, probably as an operator site that binds a repressor under non-heat shock conditions. The IR was also found to function at the mRNA level, since under non-heat shock conditions transcripts containing deletions of one side of the IR had longer half-lives than did transcripts containing the wild-type IR. Under heat shock conditions, the half-life of the mRNA was unaffected by this deletion because of heat shockdependent cleavage. However, the groESL operon was found to be heat shock activated even after most of the IR was deleted. This observation, together with the fact that the groESL operon of A. tumefaciens was heat shock activated in Escherichia coli and vice versa, suggests that a heat shock promoter regulates the heat shock activation of this operon. The primary role of the IR appears to be in reducing the MRNA levels from this promoter under non-heat shock conditions.The heat shock response is a widespread phenomenon found in all living cells examined (10). It is characterized by the induction of several proteins, some of which are highly conserved in evolution, especially those encoded by the groEL (hsp60) and the dnaK (hsp70) genes (4,16,17,48).One of the recent findings concerning the groE and dnaK operons of many eubacteria is the existence of an inverted repeat (IR) (TTAGCACTC-N 9 -GAGTGCTAA) located in the upstream regulatory region. This IR has been found in a large number of phylogenetically distant bacteria (1,2,5,9,13,20,21,25,27,28,30,33,34,39,41,42) and is highly conserved. So far, this IR has been found only in the upstream regions of groE and dnaK operons or genes, except for two cases in which it is located upstream of the dnaJ gene (14, 39). In these two cases, the dnaJ gene is separated from the dnaK gene, while generally they are organized in the same operon.The role of the IR in heat shock induction was characterized in two low-GC gram-positive bacteria, Lactococcus lactis (39) and Bacillus subtilis (46,49). In the dnaJ gene of L. lactis, deletion of the IR resulted in the loss of heat shock activation of this gene. In addition, the transcription level at low temperatures was higher without the IR, in comparison to that of the wild-type gene. In the dnaK operon of B. subtilis, site-directed mutagenesis of the IR resulted in a high level of transcription at low temperatures and a reduction in heat shock activation. In B. subtilis, the protein encoded by orf39 (the first gene in the dnaK operon of B. subtilis) was found to serve as the repressor and to bind the IR at the DNA level (45). In the groE operon of B. subtilis, the IR was also shown to function as an operator site and to be involved in determining the half-life of the transcript to which it is conn...

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“…The dnaKJ operons are abbreviated as follows: CcK, C. crescentus (19); BoK, B. ovis (9); AtK, A. tumefaciens (this work). The groESL operons are abbreviated as follows: AtE, A. tumefaciens (40); BaE, Brucella abortus (22); BjE, Bradyrhizobium japonicum (16); CcE, C. crescentus (accession no.…”

Section: Fig 6 (A)mentioning

confidence: 99%

“…A lacZ fusion of the dnaK operon from Zymomonas mobilis was thermoregulated in E. coli (26), and the dnaK operon of B. ovis was expressed in a heat shock-dependent manner in E. coli (9). In both cases, the transcription start site of the operon was not determined, and there is no evidence that transcription in E. coli occurred from the same promoter as that in the original bacteria.…”

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confidence: 99%

The dnaKJ operon of Agrobacterium tumefaciens: transcriptional analysis and evidence for a new heat shock promoter

Segal

Ron

1995

J Bacteriol

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The dnaKJ operon of Agrobacterium tumefaciens was cloned and sequenced and was found to be highly homologous to previously analyzed dnaKJ operons. Transcription of this operon in A. tumefaciens was stimulated by heat shock as well as by exposure to ethanol and hydrogen peroxide. There were two transcripts representing the dnaKJ operon: one containing the dnaK and dnaJ genes and the second containing only the dnaK gene. Primer extension analysis indicated that transcription started from the same site in heat-shocked cells and in untreated cells. The upstream regulatory region of the dnaKJ operon of A. tumefaciens does not contain the highly conserved inverted repeat sequence previously found in the groESL operon of this bacterium, as well as in many other groE and dnaK operons. Sequence analysis of the promoter region of several groESL and dnaK operons from ␣-purple proteobacteria indicates the existence of a putative promoter sequence different from the known consensus promoter sequences recognized by the Escherichia coli vegetative or heat shock sigma factor. This promoter may constitute the heat shock promoter of these ␣-purple proteobacteria.The heat shock response is a widespread phenomenon that was found in all living cells examined (11). It is characterized by the induction of several proteins, some of which are highly conserved in evolution, especially those encoded by the groEL (hsp60) and dnaK (hsp70) genes (5, 14, 53). Several of the heat shock proteins are also induced by other stress conditions such as exposure to ethanol, heavy metals, and hydrogen peroxide (30,34,45).In Escherichia coli the heat shock response is mediated by the positive regulator protein sigma-32. This sigma factor recognizes a promoter sequence different from that of the vegetative sigma factor (sigma-70) and in this way specifically transcribes heat shock genes (7, 52).Recently, it was observed that gram-positive bacteria and several gram-negative bacteria contain a highly conserved inverted repeat sequence in the upstream regulatory region of groESL and dnaK operons (1, 6, 10, 15, 21, 27-29, 31, 32, 38, 40, 44, 47, 48). In Bacillus subtilis (54) and Lactococcus lactis (44), it was shown that this inverted repeat is involved in the heat shock response and that these heat shock operons contain a promoter sequence recognized by the vegetative sigma factor. In B. subtilis, other heat shock-activated genes contain a promoter sequence recognized by the SigB sigma factor, which is known as a general stress factor (49).In a previous paper (40), we showed that the groESL operon of Agrobacterium tumefaciens contains the highly conserved inverted repeat sequence and that the operon is heat shock activated. Here, we present results from the cloning and sequencing of the dnaKJ operon of A. tumefaciens. The operon was shown to be heat shock activated, although it does not contain the inverted repeat sequence found in the groESL operon of this bacterium. The results presented in this paper and sequence analysis of heat shock promoters from o...

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Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli

Abstract: The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe

Cited by 58 publications

References 38 publications

Identification and sequence analysis of IS6501, an insertion sequence in Brucella spp.: relationship between genomic structure and the number of IS6501 copies

Identification and sequence analysis of IS6501, an insertion sequence in Brucella spp.: relationship between genomic structure and the number of IS6501 copies

Heat shock activation of the groESL operon of Agrobacterium tumefaciens and the regulatory roles of the inverted repeat

The dnaKJ operon of Agrobacterium tumefaciens: transcriptional analysis and evidence for a new heat shock promoter

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