We present a practical description of polyethylene glycol mediated spheroplast transformation of Halobacterium halobium and Halobacterium volcanii. This method has been applied to phage DNA transfection, plasmid DNA transformation, and transformation with linear fragments of high molecular weight genomic DNA. Efficient spheroplast regeneration allows uncomplicated recovery of transformed progeny. Transformations can be performed equally well using fresh or frozen cell preparations. These methods should find application in molecular cloning, genetic fine mapping, and strain construction.

We determined the complete nucleotide sequence of the 6354-base-pair plasmid pHV2 of the archaebacterium Halobacterium vokcanii. This plasmid is present in approximately six copies per chromosome. We have generated a strain, H. volcanii WFD11, cured of pHV2 by treatment of liquid cultures with ethidium bromide. We describe PEGmediated transformation of H. volcanii WFD11 with intact pHV2 and with a form of pHV2 marked by a 93-base-pair deletion generated in vitro.Since the recognition that the archaebacteria are a form of life distinct from both eubacteria and eukaryotes (1, 2), a considerable amount of research has focused on elucidating the biological and biochemical characteristics of this ancient lineage (for reviews see refs. 3 and 4). Although there has been some progress in understanding the molecular genetics ofarchaebacteria, this work has been hampered until recently by the inability to introduce exogenous DNA into cells. We have demonstrated transfection of halobacteria (5) with naked DNA from OH, a natural bacteriophage of Halobacterium halobium (6). In addition, Bertani and Baresi (7) have observed low-frequency transformation of Methanococcus voltae histidine and purine auxotrophs to prototrophy, using total wild-type DNA. However, there remained a need to obtain transformation with a plasmid as a first step toward the construction of vectors to shuttle between an archaebacterium and Escherichia coli.The halobacteria have, for purposes of research in molecular biology, an advantage over other groups of archaebacteria in that they can be maintained and manipulated in the laboratory easily, by methods similar to those for E. coli. Mevarech and Werczberger (8) (5). E. coli, strain JM101, was grown and maintained according to standard procedures (13).Preparation of DNA. Total DNA was isolated from halobacteria by quickly lysing resuspended cell pellets in 50 mM Tris'HCI, pH 8/50 mM EDTA/0.2% N-lauroylsarcosine, followed by digestion with proteinase K at 0.1 mg/ml for several hours with gentle shaking at 37°C, before phenol extraction and ethanol precipitation. The 6.4-kilobase-pair (kbp) plasmid pHV2 (14) was prepared from H. volcanii DS2 using the following methods: (i) CsCl/ethidium bromide buoyant density ultracentrifugation (14), (ii) alkaline extraction (method B without column purification of ref. 15), or (iii) rate zonal centrifugation on a sucrose density gradient (16). Phage 4OH was prepared as described (6). Plasmids and M13 RF forms from E. coli JM101 were prepared by alkaline extraction. The single-stranded M13 DNA was isolated and purified for sequencing as described by Messing (13).Cloning and Sequencing. The 6.4-kbp plasmid pHV2 of H. volcanii DS2 cloned at its unique Pst I site into a pUC plasmid vector was kindly provided by C. J. Daniels (Ohio State University). Ordered series of deletions for each strand of three restriction fragments subcloned into various pUC or M13mp series vectors (17) were prepared using BAL-31 (18) or exonuclease III followed by nuclease S1 (19). Deleted...

BackgroundSince Darwin's Origin of Species, reconstructing the Tree of Life has been a goal of evolutionists, and tree-thinking has become a major concept of evolutionary biology. Practically, building the Tree of Life has proven to be tedious. Too few morphological characters are useful for conducting conclusive phylogenetic analyses at the highest taxonomic level. Consequently, molecular sequences (genes, proteins, and genomes) likely constitute the only useful characters for constructing a phylogeny of all life. For this reason, tree-makers expect a lot from gene comparisons. The simultaneous study of the largest number of molecular markers possible is sometimes considered to be one of the best solutions in reconstructing the genealogy of organisms. This conclusion is a direct consequence of tree-thinking: if gene inheritance conforms to a tree-like model of evolution, sampling more of these molecules will provide enough phylogenetic signal to build the Tree of Life. The selection of congruent markers is thus a fundamental step in simultaneous analysis of many genes.ResultsHeat map analyses were used to investigate the congruence of orthologues in four datasets (archaeal, bacterial, eukaryotic and alpha-proteobacterial). We conclude that we simply cannot determine if a large portion of the genes have a common history. In addition, none of these datasets can be considered free of lateral gene transfer.ConclusionOur phylogenetic analyses do not support tree-thinking. These results have important conceptual and practical implications. We argue that representations other than a tree should be investigated in this case because a non-critical concatenation of markers could be highly misleading.

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...