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

Abstract

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...

Replicon-Specific Regulation of Small Heat Shock Genes in Agrobacterium tumefaciens

“…The dnaKJ operon, clpB, and several other heat shock genes are under control of RpoH (19,24,38). The groESL operon was found to be under dual control of RpoH and the HrcA repressor (24,39). Analysis of the heat shock proteome by two-dimensional protein gel electrophoresis confirmed these results but also raised additional questions, since 32 of 56 heat shock proteins were induced independently of RpoH and HrcA (34).…”

Transcriptional Analysis of Major Heat Shock Genes of Helicobacter pylori

“…If CIRCE is part of the mRNA and localized close to the ribosome binding site of the following gene, it decreases the stability of mRNA by impairing ribosome binding and consequently deprotecting the 5Ј end of mRNA against 5Ј-binding RNases (20,32,46). This destabilizing mechanism is not realized in the case of the H. pylori dnaK operon.…”

Heat Shock Proteome of Agrobacterium tumefaciens : Evidence for New Control Systems

“…A second class of heat shock proteins is regulated by B (7)(8)(9), and the others are HrcA and B independent (7,9). In Agrobacterium tumefaciens and other ␣-proteobacteria, two heat shock control elements were identified, the RpoH homologue and the CIRCE-HrcA regulatory system (16,24,29,30,32). In contrast to the gram-positive bacteria the CIRCE-HrcA system is found only in the groESL operon and heat shock transcription of the major chaperone genes is controlled by RpoH (16,18,(27)(28)(29)(30)(31)(32).…”