Global gene expression mediated by Thermus thermophilus SdrP, a CRP/FNR family transcriptional regulator

Abstract

SummaryThermus thermophilus SdrP is one of four cyclic AMP receptor protein (CRP)/fumarate and nitrate reduction regulator (FNR) family proteins from the extremely thermophilic bacterium T. thermophilus HB8. Expression of sdrP mRNA increased in the stationary phase during cultivation at 70°C. Although the sdrP gene was non-essential, an sdrP-deficient strain showed growth defects, particularly when grown in a synthetic medium, and increased sensitivity to disulphide stress. The expression of several genes was altered in the sdrP disruptant. Among them, we found eight SdrP-dependent promoters using in vitro transcription assays. A predicted SdrP binding site similar to that recognized by Escherichia coli CRP was found upstream of each SdrP-dependent promoter. In the wild-type strain, expression of these eight genes tended to increase upon entry into the stationary phase. Transcriptional activation in vitro was independent of any added effector molecule. The hypothesis that apo-SdrP is the active form of the protein was supported by the observation that the three-dimensional structure of apo-SdrP is similar to that of the DNA-binding form of E. coli CRP. Based on the properties of the SdrP-regulated genes found in this study, it is speculated that SdrP is involved in nutrient and energy supply, redox control, and polyadenylation of mRNA.

TetR-family transcriptional repressor Thermus thermophilus FadR controls fatty acid degradation

“…The T. thermophilus HB8 strain was cultured at 70 uC for 680 min in 1 l TT medium, as described previously (Agari et al, 2008). The crude RNA was extracted from each cell, and after cDNA had been synthesized, it was fragmented and labelled with biotin-dideoxyUTP, as described previously (Agari et al, 2008). The 39-terminally labelled cDNA was hybridized to a TTHB8401a520105F GeneChip (Affymetrix), and then the array was washed, stained and scanned as described previously (Agari et al, 2008).…”

“…The T. thermophilus HB8 strain was cultured at 70 uC for 680 min in 1 l TT medium, as described previously (Agari et al, 2008). The crude RNA was extracted from each cell, and after cDNA had been synthesized, it was fragmented and labelled with biotin-dideoxyUTP, as described previously (Agari et al, 2008).…”

“…The crude RNA was extracted from each cell, and after cDNA had been synthesized, it was fragmented and labelled with biotin-dideoxyUTP, as described previously (Agari et al, 2008). The 39-terminally labelled cDNA was hybridized to a TTHB8401a520105F GeneChip (Affymetrix), and then the array was washed, stained and scanned as described previously (Agari et al, 2008). The raw intensities for the independently cultured four wild-type and three DfadR strains were each summarized to 2266 ORFs, using the GeneChip Operating Software, version 1.2 (Affymetrix).…”

“…The T. thermophilus wild-type or DTTHA0890 strain was pre-cultured at 70 uC for 16 h in 3 ml TT medium (Agari et al, 2008), and then 0.1 ml culture broth was inoculated into 20 ml synthetic medium containing 4 %, w/v, sucrose as the sole carbon source (Agari et al, 2008). When the OD 600 reached~0.5, the cells were collected by centrifugation, washed with 20 ml of the synthetic medium without sucrose, and resuspended in the same medium.…”

LdrP, a cAMP receptor protein/FNR family transcriptional regulator, serves as a positive regulator for the light-inducible gene cluster in the megaplasmid of Thermus thermophilus

“…The T. thermophilus HB8 strain and the litR mutant was cultured at 60 uC for 8 h in TM broth under dark conditions. The crude RNA was extracted from each cell and after the cDNA was synthesized, it was fragmented and labelled with biotin-dideoxy UTP, as described previously (Agari et al, 2008). The 39-terminally labelled cDNA was hybridized to a TTHB8401a520105F GeneChip (Affymetrix), and the array was then washed, stained and scanned as described previously (Agari et al, 2008).…”