Replicon-specific regulation of small heat shock genes in Agrobacterium tumefaciens

Abstract

Four genes coding for small heat shock proteins (sHsps) were identified in the genome sequence of Agrobacterium tumefaciens, one on the circular chromosome (hspC), one on the linear chromosome (hspL), and two on the pAT plasmid (hspAT1 and hspAT2). Induction of sHsps at elevated temperatures was revealed by immunoblot analyses. Primer extension experiments and translational lacZ fusions demonstrated that expression of the pAT-derived genes and hspL is controlled by temperature in a regulon-specific manner. While the sHsp gene on the linear chromosome turned out to be regulated by RpoH (sigma32), both copies on pAT were under the control of highly conserved ROSE (named for repression of heat shock gene expression) sequences in their 5' untranslated region. Secondary structure predictions of the corresponding mRNA strongly suggest that it represses translation at low temperatures by masking the Shine-Dalgarno sequence. The hspC gene was barely expressed (if at all) and not temperature responsive.

FIG. 1.

Overview of the four small heat shock genes encoded by A. tumefaciens. The genomic locations (pAT plasmid, linear chromosome, and circular chromosome) are indicated in the gene designations. Potential transcription start sites are represented by arrows. Presumed regulatory mechanisms are listed, and ROSE-type sequences are shown as grey boxes.

FIG. 2.

Heat-induced production of sHsps in A. tumefaciens. Cells were grown in YEB medium at the temperatures indicated. For heat treatment, aliquots of the cells were shifted to higher temperatures for 30 min. Harvested cells were subjected to Western blot analysis as described in Materials and Methods using an anti-HspL serum. The detected band migrates at 18 kDa.

FIG. 3.

Transcription start site mapping of pAT-encoded small heat shock genes. Primer extension experiments were performed with total RNA from A. tumefaciens grown in YEB medium at 30°C or heat shocked from 30 to 42°C for 30 min. (A) Reverse transcriptase reaction with primer Sig212, and (B) reverse transcriptase reaction with primer Sig214. The corresponding sequencing reactions (AGCT) with pUC18-derived plasmids carrying the hspAT1 and hspAT2 regions, respectively, are shown. The positions of reverse transcripts corresponding to +1 in panel C are indicated by black arrowheads to the left of the gels. (C) Alignment of the deduced promoter regions and ROSE sequences of hspAT1 (top strand) and hspAT2 (bottom strand). Transcription start sites (+1), −10 and −35 regions, Shine-Dalgarno sequences (SD), and translational start sites are indicated. (D) Secondary structure prediction of the hspAT2 ROSE region. The mfold program version 3.1 (http://www.bioinfo.rpi.edu/applications/mfold/old/rna/) was used (47). Nucleotides are numbered starting from the transcription start site (+1). The Shine-Dalgarno sequence and start codon are labeled. A conserved bulged G residue present in all known ROSE elements is indicated by a black arrow.

FIG. 4.

Transcription start site mapping of the hspL gene. Primer extension experiments were performed with total RNA from A. tumefaciens grown in YEB medium at 30°C or heat shocked from 30 to 42°C for 30 min. Primers Sig225 (A) and Sig218 (not shown) producing identical results were used. The corresponding sequencing reactions (AGCT) with a pUC18-derived plasmid carrying the hspL region, respectively, are shown. The position of the reverse transcript from which the transcription start site was deduced is indicated by an arrowhead to the right of the gel. (B) The deduced promoter region of hspL is listed and compared with consensus sequences of RpoH-dependent promoters from A. tumefaciens (Atu) and E. coli (Eco) (24).

FIG. 5.

Temperature-dependent expression of A. tumefaciens small heat shock genes. Cultures were grown to exponential phase in AB medium with glucose at the temperatures indicated before β-galactosidase activity (in Miller units [MU]) was measured.

FIG. 6.

Expression of lacZ fusions to hspAT2 and hspL after a temperature upshift in A. tumefaciens and E. coli. A. tumefaciens cells grown in AB medium with glucose and E. coli grown in LB medium were shifted from 20 to 37°C before β-galactosidase activity was measured at the time points indicated. The activities at timepoint zero were set to 1.