Characterization of two heat shock genes from Haloferax volcanii: a model system for transcription regulation in the Archaea

Abstract

The expression of two heat-responsive cct (chaperonin-containing Tcp-1) genes from the archaeon Haloferax volcanii was investigated at the transcription level. The cct1 and cct2 genes, which encode proteins of 560 and 557 amino acids, respectively, were identified on cosmid clones of an H. volcanii genomic library and subsequently sequenced. The deduced amino acid sequences of these genes exhibited a high degree of similarity to other archaeal and eucaryal cct family members. Expression of the cct genes was characterized in detail for the purpose of developing a model for studying transcription regulation in the domain Archaea. Northern (RNA) analysis demonstrated that the cct mRNAs were maximally induced after heat shock from 37 to 55°C and showed significant heat inducibility after 30 min at 60°C. Transcription of cct mRNAs was also stimulated in response to dilute salt concentrations. Transcriptional analysis of cct promoter regions coupled to a yeast tRNA reporter gene demonstrated that 5 flanking sequences up to position ؊233 (cct1) and position ؊170 (cct2) were sufficient for promoting heat-induced transcription. Transcript analysis indicated that both basal transcription and stress-induced transcription of the H. volcanii cct genes were directed by a conserved archaeal consensus TATA motif (5-TTTATA-3) centered at ؊25 relative to the mapped initiation site. Comparison of the cct promoter regions also revealed a striking degree of sequence conservation immediately 5 and 3 of the TATA element.All living organisms adapt to adverse environmental conditions by evolving specific molecular responses. In particular, the universally conserved heat shock response occurs when cells are exposed to elevated temperatures, resulting in the rapid and transient overproduction of a limited class of proteins called the heat shock proteins (HSPs). The HSPs produced in the domains Bacteria and Eucarya are highly conserved both in structure and function, and their induction is generally regulated at the transcription initiation level (recently reviewed in reference 31).Among the HSPs, the Cct family is a recently identified group of molecular chaperonins that are distinct from members of the well-studied Hsp60/GroEL family. So far, members of the Cct family have been identified only in the domains Archaea and Eucarya, where they are known variously as Tcomplex polypeptide, or TCP (15); chaperonin-containing Tcp-1, or Cct (25, 62); TCP-1 ring complex, or TriC (13); thermosome complex (59); and thermophilic factor-55, or TF55 (54). Protein members of the Cct and Hsp60/GroEL families display similar double-ringed toroidal structures as their active forms. Differences between the two families include the absence of an HSP10/GroES-like accessory protein necessary for Cct function (25) and the hetero-oligomeric structure of Cct complexes, which contain either 2 (58) or 8 to 10 different subunits (61). Among the members of the Archaea, members of the Cct protein family have been documented in many hyperthermophiles, including Sulfolo...

Natural chaperonin of the hyperthermophilic archaeum, Thermococcus strain KS-1: a hetero-oligomeric chaperonin with variable subunit composition

“…It has been reported that both the a-and the b-subunits of chaperonin accumulate in response to a high growth temperature and/or heat shock in archaea such as S. shibatae (Trent et al, 1991;Kagawa et al, 1995), P. occultum (Phipps et al, 1991), Haloferax volcanii (Kuo et al, 1997) and Archaeoglobus fulgidus (Emmerhoff et al, 1998). The ratio of the a-to b-subunit is constant at different growth temperatures, and their expression is thought to be co-regulated (Phipps et al, 1991;Trent et al, 1991;Kagawa et al, 1995).…”

“…This is consistent with the hypothesis that the bsubunit is adapted to a higher temperature than the asubunit. The a-subunit may be the prototype from which the b-subunit has evolved, because it has the GGM repeat sequence that is conserved in group I chaperonin (McLennan et al, 1993;Brocchieri and Karlin, 2000) and in some chaperonins of archaea belonging to Euryarchaeota (Kuo et al, 1997;Yoshida et al, 1997;Furutani et al, 1998). The a-subunit might have been lost after the divergence of the Pyrococcus species from the Thermococcus species.…”

Purification and Characterization of Sa-Lrp, a DNA-Binding Protein from the Extreme Thermoacidophilic Archaeon Sulfolobus acidocaldarius Homologous to the Bacterial Global Transcriptional Regulator Lrp

“…Indeed, at the present time, regulation of archaeal transcription initiation and mRNA stability have been addressed mostly in methanogens and halophiles, representatives of the Euryarchaeota, but very little information is available on extreme-and hyperthermophilic archaea. Moreover, though mobility shift experiments performed with archaeal protein extracts and analyses of cisacting regulatory elements (11,13,21,22,24,31,39,46,48,52,53) have indicated the existence of sequence-specific DNAbinding proteins and of target sites located close to the transcription initiation sites of specific genes, these potential regulatory elements have not been characterized thoroughly (for a recent review, see reference 33). Only one detailed study has been performed (2) (see Discussion).…”

New insights into microbial adaptation to extreme saline environments

“…This suggests that under moderate salt deprivation, a first level of response consists in minimizing the loss of ions. Interestingly, in halophilic strains, many of the genes encoding for thermal stress response proteins are also induced under low salt conditions, thus confirming that low salt represents a physico-chemical stressor (68). We studied the accumulation of specific proteins in H. marismortui and H. salinarum cells exposed to prolonged low salt stress conditions, similar to the one we used for neutrons experiments.…”

“…We specifically searched for large complexes in the hydrophobic fractions of the total cytosolic extracts of H. salinarum and H. marismortui. These studies revealed that the type II chaperonin complex, called "thermosome" in archaea, is strongly accumulated within cells exposed to low salt conditions (68)(69)(70). Gene expression studies indicated that the heat shock promoter of the thermosome is also strongly salt regulated, suggesting that the low salt and the heat and salt stressors use similar, if not identical, transcriptional regulators.…”