We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodiumproton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.

SummaryHalophilic archaea thrive in environments with salt concentrations approaching saturation. However, little is known about the way in which these organisms stabilize their secreted proteins in such 'hostile' conditions. Here, we present data suggesting that the utilization of protein translocation pathways for protein secretion by the Halobacteriaceae differs significantly from that of non-haloarchaea, and most probably represents an adaptation to the high-salt environment. Although most proteins are secreted via the general secretion (Sec) machinery, the twinarginine translocation (Tat) pathway is mainly used for the secretion of redox proteins and is distinct from the Sec pathway, in that it allows cytoplasmic folding of secreted proteins. TATFIND (developed in this study) was used for systematic whole-genome analysis of Halobacterium sp. NRC-1 and several other prokaryotes to identify putative Tat substrates. Our analyses revealed that the vast majority of haloarchaeal secreted proteins were predicted substrates of the Tat pathway. Strikingly, most of these putative Tat substrates were non-redox proteins, the homologues of which in non-haloarchaea were identified as putative Sec substrates. We confirmed experimentally that the secretion of one such putative Tat substrate depended on the twin-arginine motif in its signal sequence. This extensive utilization of the Tat pathway in haloarchaea suggests an evolutionary adaptation to high-salt conditions by allowing cytoplasmic folding of secreted proteins before their secretion.

Most secreted archaeal proteins are targeted to the membrane via a tripartite signal composed of a charged N terminus and a hydrophobic domain, followed by a signal peptidase-processing site. Signal peptides of archaeal flagellins, similar to class III signal peptides of bacterial type IV pilins, are distinct in that their processing sites precede the hydrophobic domain, which is crucial for assembly of these extracytoplasmic structures. To identify the complement of archaeal proteins with class III signal sequences, a PERL program (FlaFind) was written. A diverse set of proteins was identified, and many of these FlaFind positives were encoded by genes that were cotranscribed with homologs of pilus assembly genes. Moreover, structural conservation of primary sequences between many FlaFind positives and subunits of bacterial pilus-like structures, which have been shown to be critical for pilin assembly, have been observed. A subset of pilin-like FlaFind positives contained a conserved domain of unknown function (DUF361) within the signal peptide. Many of the genes encoding these proteins were in operons that contained a gene encoding a novel euryarchaeal prepilin-peptidase, EppA, homolog. Heterologous analysis revealed that Methanococcus maripaludis DUF361-containing proteins were specifically processed by the EppA homolog of this archaeon. Conversely, M. maripaludis preflagellins were cleaved only by the archaeal preflagellin peptidase FlaK. Together, the results reveal a diverse set of archaeal proteins with class III signal peptides that might be subunits of as-yet-undescribed cell surface structures, such as archaeal pili.

The twin-arginine translocation (Tat) pathway, which has been identified in plant chloroplasts and prokaryotes, allows for the secretion of folded proteins. However, the extent to which this pathway is used among the prokaryotes is not known. By using a genomic approach, a comprehensive list of putative Tat substrates for 84 diverse prokaryotes was established. Strikingly, the results indicate that the Tat pathway is utilized to highly varying extents. Furthermore, while many prokaryotes use this pathway predominantly for the secretion of redox proteins, analyses of the predicted substrates suggest that certain bacteria and archaea secrete mainly nonredox proteins via the Tat pathway. While no correlation was observed between the number of Tat machinery components encoded by an organism and the number of predicted Tat substrates, it was noted that the composition of this machinery was specific to phylogenetic taxa.Prokaryotes have a number of distinct pathways dedicated to the process of protein secretion. In general, these organisms translocate the majority of their secretory proteins in an unfolded conformation via the universally conserved and essential Sec pathway (15,16,20). Proteins secreted by this pathway are directed to the membrane-embedded proteinaceous Sec pore by an N-terminal signal peptide (9). While Sec signal peptides are similar structurally, they do not show sequence conservation (33). Once targeted to the membrane, Sec substrates can be translocated through the pore by the energetics of translation and/or ATP hydrolysis (reference 15 and references therein).An alternate secretion mechanism, the twin-arginine translocation (Tat) pathway, was originally identified in chloroplasts and has recently been found in bacteria and archaea (24,27,32,37). It is distinct from the Sec pathway in that (i) Tat substrates are secreted in a folded conformation (11,22,31), (ii) Tat signal peptides contain a highly conserved twin-arginine motif (3, 6, 18), (iii) the energy driving translocation is provided solely by the proton motive force (7, 24), and (iv) the Tat pathway is not a universally conserved secretion mechanism (36, 37).Previous analyses of Escherichia coli Tat mutants and substrates suggested that the major role of this pathway in prokaryotes is to translocate redox proteins that integrate their cofactors in the cytoplasm and therefore possess some degree of tertiary structure prior to secretion (3,22,35). However, the recent identification of nonredox Tat substrates (such as virulence factors from Pseudomonas aeruginosa) indicates a broader role for the pathway than merely the secretion of redox proteins (19,34). Furthermore, genomic data suggest that one group of organisms, the halophilic archaea, have routed nearly all of their secretome to the Tat pathway (23).While Tat components have been identified in many prokaryotes (36, 37), the extent to which this secretory pathway is utilized in bacteria and archaea is not well characterized. We have identified putative Tat substrates from 84 diverse pr...

The twin-arginine translocation (Tat) pathway is a protein transport system for the export of folded proteins. Substrate proteins are targeted to the Tat translocase by N-terminal signal peptides harboring a distinctive R-R-x-⌽-⌽ ''twin-arginine'' amino acid motif. Using a combination of proteomic techniques, the protein contents from the cell wall of the model Gram-positive bacterium Streptomyces coelicolor were identified and compared with that of mutant strains defective in Tat transport. The proteomic experiments pointed to 43 potentially Tat-dependent extracellular proteins. Of these, 25 were verified as bearing bona fide Tat-targeting signal peptides after independent screening with a facile, rapid, and sensitive reporter assay. The identified Tat substrates, among others, include polymerdegrading enzymes, phosphatases, and binding proteins as well as enzymes involved in secondary metabolism. Moreover, in addition to predicted extracellular substrates, putative lipoproteins were shown to be Tat-dependent. This work provides strong experimental evidence that the Tat system is used as a major general export pathway in Streptomyces.Protein transport ͉ secondary metabolism ͉ Tat pathway ͉ twin arginine signal peptide ͉ proteome

Although the genome of Haloferax volcanii contains genes (flgA1-flgA2) that encode flagellins and others that encode proteins involved in flagellar assembly, previous reports have concluded that H. volcanii is nonmotile. Contrary to these reports, we have now identified conditions under which H. volcanii is motile. Moreover, we have determined that an H. volcanii deletion mutant lacking flagellin genes is not motile. However, unlike flagella characterized in other prokaryotes, including other archaea, the H. volcanii flagella do not appear to play a significant role in surface adhesion. While flagella often play similar functional roles in bacteria and archaea, the processes involved in the biosynthesis of archaeal flagella do not resemble those involved in assembling bacterial flagella but, instead, are similar to those involved in producing bacterial type IV pili. Consistent with this observation, we have determined that, in addition to disrupting preflagellin processing, deleting pibD, which encodes the preflagellin peptidase, prevents the maturation of other H. volcanii type IV pilin-like proteins. Moreover, in addition to abolishing swimming motility, and unlike the flgA1-flgA2 deletion, deleting pibD eliminates the ability of H. volcanii to adhere to a glass surface, indicating that a nonflagellar type IV pilus-like structure plays a critical role in H. volcanii surface adhesion.To escape toxic conditions or to acquire new sources of nutrients, prokaryotes often depend on some form of motility. Swimming motility, a common means by which many bacteria move from one place to another, usually depends on flagellar rotation to propel cells through liquid medium (24,26,34). These motility structures are also critical for the effective attachment of bacteria to surfaces.As in bacteria, rotating flagella are responsible for swimming motility in archaea, and recent studies suggest that archaea, like bacteria, also require flagella for efficient surface attachment (37, 58). However, in contrast to bacterial flagellar subunits, which are translocated via a specialized type III secretion apparatus, archaeal flagellin secretion and flagellum assembly resemble the processes used to translocate and assemble the subunits of bacterial type IV pili (34,38,54).

membrane proteins, known as the SecYEG Boston, Massachusetts 02115 complex in bacteria (Brundage et al., 1990), and the ‡ Max Delbru ¨ck Center for Molecular Medicine Sec61 complex in eukaryotes (Go ¨rlich and Rapoport, Robert-Ro ¨ssle-Stra␤e 10 1993). These complexes probably form a pore, through D-13122 Berlin Buch which proteins pass on their way out of the cytoplasm Germany (Hanein et al., 1996). Two of the subunits of these complexes are conserved in the domains-the SecY/Sec61␣ and the SecE/Sec61␥ proteins (Hartmann et al., 1994) in bacteria and eukaryotes, respectively. These proteins In all cells, extracytoplasmic proteins must be transloare essential for protein translocation in vivo and in vitro. cated across lipid bilayers to reach their final destina-Without exception, archaeal homologs of SecY/ tion. Most protein translocation across hydrophobic Sec61␣ and SecE/Sec61␥ are more closely related to membranes occurs through an evolutionarily conserved the eukaryotic than to the bacterial members of these proteinaceous complex. In addition to this complex, the families (Figure 2). protein translocation machinery in bacteria and eukary-Analyses of SecY/Sec61␣ and SecE/Sec61␥ homootes employs a number of other proteins that do not logs from a number of organisms have led to the identifiappear to be shared between these two domains of cation of regions that are conserved in all cells and living organisms (Murphy and Beckwith, 1996; Rapoport regions of conservation that are limited to only one or et al., 1996; Schatz and Dobberstein, 1996). The functwo domains. Such information could provide clues tions of many of these proteins and the mechanism of about which portions of SecY/Sec61␣ and SecE/Sec61␥ protein translocation remain largely unknown.interact with each other or with additional components Genome sequencing projects are providing the basis of the protein translocation apparatus. For example, as for a novel approach to learning more about protein will be discussed below, homologs of SecD and SecF, translocation in all cells. Protein translocation has been two components that are thought to interact with the studied only in bacteria and eukaryotes, two of the three bacterial heterotrimeric complex, have been identified domains of life. The completion of the genome sein archaea but not in yeast. Regions of bacterial SecY

Diversity-generating retroelements (DGRs) are novel genetic elements that use reverse transcription to generate vast numbers of sequence variants in specific target genes. Here, we present a detailed comparative bioinformatic analysis that depicts the landscape of DGR sequences in nature as represented by data in GenBank. Over 350 unique DGRs are identified, which together form a curated reference set of putatively functional DGRs. We classify target genes, variable repeats and DGR cassette architectures, and identify two new accessory genes. The great variability of target genes implies roles of DGRs in many undiscovered biological processes. There is much evidence for horizontal transfers of DGRs, and we identify lineages of DGRs that appear to have specialized properties. Because GenBank contains data from only 10% of described species, the compilation may not be wholly representative of DGRs present in nature. Indeed, many DGR subtypes are present only once in the set and DGRs of the candidate phylum radiation bacteria, and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea archaea, are exceptionally diverse in sequence, with little information available about functions of their target genes. Nonetheless, this study provides a detailed framework for classifying and studying DGRs as they are uncovered and studied in the future.