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...

We describe a query-based web-accessible system (www.neurogadgets.com/bws.php) for facilitating comparative microbial genomics. A variety of query pages are available, each with numerous options, that allow a biologist to pose relevant questions of genomic data. We illustrate with a characterization of species-specific protein-coding genes (so-called 'ORFans'), finding that they are on average smaller, faster evolving, and less G+C-rich, and that they encode proteins more basic in their predicted isoelectric point, compared with nonspecies-specific genes. Using a dual-threshold approach, we conclude that these are characteristics of true species-specific genes, rather than artifacts of mis-annotation. ß

Through hybridization of available probes, we have added nine genes to the macrorestriction map of the Haloferax mediterranei chromosome and five genes to the contig map of Haloferax volcanii. Additionally, we hybridized 17 of the mapped cosmid clones from H. volcanii to the H. mediterranei genome. The resulting 35-point chromosomal comparison revealed only two inversions and a few translocations. Forces known to promote rearrangement, common in the haloarchaea, have been ineffective in changing global gene order throughout the nearly 10 7 years of these species' divergent evolution.One of the most notable characteristics of extremely halophilic archaea is their genetic instability (4,11,12,27). The haloarchaea are rich in insertion sequences which can disrupt genes at frequencies as high as 10 Ϫ2 in the case of Halobacterium salinarium (32,43). Most of the activity of these insertion sequences is confined to plasmid DNA or to FII DNA (which has a lower moles percent GϩC content) (13,28,31), but chromosomal genes are not spared from disruption. The bop gene, for instance, is inactivated by at least eight different types of insertion sequences (11, 27), resulting in a combined risk of about 10 Ϫ4 per generation (32). H. salinarium is known to possess hundreds of insertion sequences, in dozens of families (35). The resulting transpositional cost to the cell is compounded by the potential recombinational chaos mediated by interaction between members of each insertion sequence family. It has been suggested that a genomically pure clone of H. salinarium cannot be grown because of continual rearrangements which occur in plasmid DNA (27,30,36).The genus Haloferax is not as severely infested with insertion sequences as is H. salinarium. Nevertheless, Haloferax volcanii possesses at least 49 copies of the ISH51 family distributed throughout the genome (8), and there is good evidence for the existence of several other types of repeated sequences as well (35,37). Though H. volcanii is not as prone to genetic disruption as is H. salinarium, neither is it immune. Less is known about the number or distribution of insertion sequences in Haloferax mediterranei. Interestingly, the populous ISH51/27 family shared by H. volcanii and H. salinarium (29) is absent from H. mediterranei (37). Regardless, repeated sequences which can potentially facilitate genomic rearrangement are present (1).With physical and genetic maps available, we can now begin to address genomic stability in the haloarchaea. The first researchers to assess the degree of rearrangement in the haloarchaeal chromosome by using a comprehensive comparative mapping approach (15) found that much of the H. salinarium chromosome is conserved in structure among several strains, despite different complements of repetitive sequences in their genomes. Differences in the maps were observed to be confined to a few discrete, hypervariable blocks. H. salinarium NRC-1 and H. salinarium S9 maintain hundreds of insertion sequences within their genomes, and yet gene order is preserve...

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