Heat shock response of Archaeoglobus fulgidus

Abstract

The heat shock response of the hyperthermophilic archaeon Archaeoglobus fulgidus strain VC-16 was studied using whole-genome microarrays. On the basis of the resulting expression profiles, approximately 350 of the 2,410 open reading frames (ORFs) (ca. 14%) exhibited increased or decreased transcript abundance. These span a range of cell functions, including energy production, amino acid metabolism, and signal transduction, where the majority are uncharacterized. One ORF called AF1298 was identified that contains a putative helix-turn-helix DNA binding motif. The gene product, HSR1, was expressed and purified from Escherichia coli and was used to characterize specific DNA recognition regions upstream of two A. fulgidus genes, AF1298 and AF1971. The results indicate that AF1298 is autoregulated and is part of an operon with two downstream genes that encode a small heat shock protein, Hsp20, and cdc48, an AAA+ ATPase. The DNase I footprints using HSR1 suggest the presence of a cis-binding motif upstream of AF1298 consisting of CTAAC-N5-GTTAG. Since AF1298 is negatively regulated in response to heat shock and encodes a protein only distantly related to the N-terminal DNA binding domain of Phr of Pyrococcus furiosus, these results suggest that HSR1 and Phr may belong to an evolutionarily diverse protein family involved in heat shock regulation in hyperthermophilic and mesophilic Archaea organisms.

FIG. 1.

Schematic plan of heat shock experiments. An initial A. fulgidus 500-ml culture was divided into six smaller flasks and incubated at 78°C. At an OD₆₀₀ of 0.3, 30 ml of medium was removed from each flask and used as the reference and control. Each flask was then transferred from 78 to 89°C, and samples were removed at 5, 10, 15, 30, and 60 min after the temperature shift. Samples were taken so that each time point shared a common flask with the previous time point. RNA was purified from each sample and used for hybridization with two microarray slides. The reference samples were pooled together prior to slide hybridization.

FIG. 2.

Temporal pattern of gene expression in response to heat shock. Data for each gene were plotted if a threefold or greater abundance of mRNA was observed at 5 min post-temperature shift. The x axis is expressed in minutes, while the y axis is the expression change in fold mRNA abundance. The error bars represent one standard deviation.

FIG. 3.

Comparison of real-time RT-PCR data versus the corresponding DNA microarray data. The mRNA change in response to heat shock was measured as described in Materials and Methods using microarray hybridization and real-time RT-PCR. Data for the 5- and the 10-min changes relative to the 0-min time are indicated, where the correlation coefficient was 0.944.

FIG. 4.

Multiple amino acid sequence alignments of putative heat shock regulatory proteins. The indicated sequences were aligned using ClustalW 1.83 with the default settings. The sequences were the following: AF1298, Archaeoglobus fulgidus; cmi2, Haloferax volcanii; VNG1843C, Halobacterium sp. (strain NRC-1); MA4576, Methanosarcina acetivorans; MM1257, Methanosarcina mazei; METH0903, Methanosarcina barkeri; PF1790, Pyrococcus furiosus; PAB0208, Pyrococcus abyssi; PH1744, Pyrococcus horikoshii; MTH1288, Methanothermobacter thermautotrophicus strain Delta H.

FIG. 5.

Electrophoretic mobility shift assays for the AF1298, AF1971, and AF1813, DNA fragments. The concentration of HSR1 protein from left to right was 0, 125, 250, 500, 1,000, and 2000 nM. EMSA for the promoter regions from position bp −175 to +50 relative to the start of translation.

FIG. 6.

DNase I footprint of AF1971 DNA on the coding and noncoding strands. Coding strand (lanes 1 to 7): lane 1, G reaction; lanes 2 to 7, increasing levels of HSR1 protein (from left to right) of 0, 125, 250, 500, 1,000, and 2,000 nM. Noncoding strand (lanes 8 to 14): lane 8 to 13, increasing levels of HSR1 protein (from left to right) of 0, 125, 250, 500, 1,000, and 2,000 nM; lane 14, G reaction. The numbering of the DNA is relative to the start of translation.

FIG. 7.

DNase I footprinting of AF1298 DNA on the coding and noncoding strands. Coding strand (lanes 1 to 7): lane 1, G reaction: lanes 2 to 7, increasing concentrations of the HSR1 protein (from left to right) of 0, 125, 250, 500, 1,000, and 2,000 nM. Noncoding strand (lanes 8 to 14): lane 8, G reaction as ladder; lanes 9 to 14, increasing HSR1 protein (from left to right) of 0, 125, 250, 500, 1,000, and 2,000 nM. The numbering of the DNA is relative to the start of translation.

FIG. 8.

Alignment of promoter DNA sequences for AF1298, AF1971, and AF1813. The sequences were aligned using GAP alignment from the GCG Wisconsin package 10.3 (Accelrys Inc., San Diego, CA). Three conserved regions are marked with gray boxes: the TATA box, the BRE interaction site, and a downstream potential binding site for HSR1. A conserved palindromic motif, CTAAC-N5-GTTAG, is indicated by the opposing arrows. The horizontal lines above (AF1298) and below (AF1971) the DNA sequences indicate the regions protected in the DNase I footprint experiments.