Comprehensive glycoproteomics shines new light on the complexity and extent of glycosylation in archaea.

Glycosylation is one of the most complex posttranslational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility, and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here, we extend the phenotypic characterization of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type (WT) and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project (ArcPP), we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provide new insights into the roles of N-glycosylation in archaeal cell biology.

Halobacterium hubeiense sp. nov., a haloarchaeal species isolated from a bore core drilled in Hubei Province, PR China.

Eight colonies of live microbes were isolated from an extensively surface-sterilized halite sample which had been retrieved from a depth of 2000 m from a salt mine in the Qianjiang Depression, Hubei Province, PR China. The eight colonies, obtained after 4 weeks of incubation, were named JI20-1T-JI20-8 and JI20-1T was selected as the type strain. The strains have been previously described, including a genomic analysis based on the complete genome for strain JI20-1T and draft genomes for the other strains. In that study, the name Halobacterium hubeiense was suggested, based on the location of the drilling site. Previous phylogenomic analysis showed that strain JI20-1T is most closely related to the Permian isolate Halobacterium noricense from Alpine rock salt. The orthologous average nucleotide identity (orthoANI) and digital DNA-DNA hybridization (dDDH) percentages between the eight strains are 100-99.6 % and 99.8-96.4 %, respectively. The orthoANI and dDDH values of these strains with respect to the type strains of species of the genus Halobacterium are 89.9-78.2 % and 37.3-21.6 %, respectively, supporting their placement in a novel extremely halophilic archaeal species. The phylogenomic tree based on the comparison of sequences of 632 core-orthologous proteins confirmed the novel species status for these haloarchaea. The polar lipid profile includes phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, and sulfated galactosyl mannosyl galactosyl glucosyl diether, a profile compatible with that of Halobacterium noricense. Based on genomic, phenotypic, and chemotaxonomic characterization, we propose strain JI20-1T (=DSM 114402T = HAMBI 3616T) as the type strain of a novel species in the genus Halobacterium, with the name Halobacterium hubeiense sp. nov.

Contribution of mechanosensitive channels to osmoadaptation and ectoine excretion in Halomonas elongata.

For osmoadaptation the halophilic bacterium Halomonas elongata synthesizes as its main compatible solute the aspartate derivative ectoine. H. elongata does not rely entirely on synthesis but can accumulate ectoine by uptake from the surrounding environment with the help of the osmoregulated transporter TeaABC. Disruption of the TeaABC-mediated ectoine uptake creates a strain that is constantly losing ectoine to the medium. However, the efflux mechanism of ectoine in H. elongata is not yet understood. H. elongata possesses four genes encoding mechanosensitive channels all of which belong to the small conductance type (MscS). Analysis by qRT-PCR revealed a reduction in transcription of the mscS genes with increasing salinity. The response of H. elongata to hypo- and hyperosmotic shock never resulted in up-regulation but rather in down-regulation of mscS transcription. Deletion of all four mscS genes created a mutant that was unable to cope with hypoosmotic shock. However, the knockout mutant grew significantly faster than the wildtype at high salinity of 2 M NaCl, and most importantly, still exported 80% of the ectoine compared to the wildtype. We thus conclude that a yet unknown system, which is independent of mechanosensitive channels, is the major export route for ectoine in H. elongata.

Identification of RNA 3´ ends and termination sites in Haloferax volcanii.

Archaeal genomes are densely packed; thus, correct transcription termination is an important factor for orchestrated gene expression. A systematic analysis of RNA 3´ termini, to identify transcription termination sites (TTS) using RNAseq data has hitherto only been performed in two archaea, Methanosarcina mazei and Sulfolobus acidocaldarius. In this study, only regions directly downstream of annotated genes were analysed, and thus, only part of the genome had been investigated. Here, we developed a novel algorithm (Internal Enrichment-Peak Calling) that allows an unbiased, genome-wide identification of RNA 3´ termini independent of annotation. In an RNA fraction enriched for primary transcripts by terminator exonuclease (TEX) treatment we identified 1,543 RNA 3´ termini. Approximately half of these were located in intergenic regions, and the remainder were found in coding regions. A strong sequence signature consistent with known termination events at intergenic loci indicates a clear enrichment for native TTS among them. Using these data we determined distinct putative termination motifs for intergenic (a T stretch) and coding regions (AGATC). In vivo reporter gene tests of selected TTS confirmed termination at these sites, which exemplify the different motifs. For several genes, more than one termination site was detected, resulting in transcripts with different lengths of the 3´ untranslated region (3´ UTR).

The Response of Haloferax volcanii to Salt and Temperature Stress: A Proteome Study by Label-Free Mass Spectrometry.

In-depth proteome analysis of the haloarchaeal model organism Haloferax volcanii has been performed under standard, low/high salt, and low/high temperature conditions using label-free mass spectrometry. Qualitative analysis of protein identification data from high-pH/reversed-phase fractionated samples indicates 61.1% proteome coverage (2509 proteins), which is close to the maximum recorded values in archaea. Identified proteins match to the predicted proteome in their physicochemical properties, with only a small bias against low-molecular-weight and membrane-associated proteins. Cells grown under low and high salt stress as well as low and high temperature stress are quantitatively compared to standard cultures by sequential window acquisition of all theoretical mass spectra (SWATH-MS). A total of 2244 proteins, or 54.7% of the predicted proteome, are quantified across all conditions at high reproducibility, which allowed for global analysis of protein expression changes under these stresses. Of these, 2034 are significantly regulated under at least one stress condition. KEGG pathway enrichment analysis shows that several major cellular pathways are part of H. volcanii's universal stress response. In addition, specific pathways (purine, cobalamin, and tryptophan) are affected by temperature stress. The most strongly downregulated proteins under all stress conditions, zinc finger protein HVO_2753 and ribosomal protein S14, are found oppositely regulated to their immediate genetic neighbors from the same operon.

SLALOM, a flexible method for the identification and statistical analysis of overlapping continuous sequence elements in sequence- and time-series data.

BACKGROUND: Protein or nucleic acid sequences contain a multitude of associated annotations representing continuous sequence elements (CSEs). Comparing these CSEs is needed, whenever we want to match identical annotations or integrate distinctive ones. Currently, there is no ready-to-use software available that provides comprehensive statistical readout for comparing two annotations of the same type with each other, which can be adapted to the application logic of the scientific question. RESULTS: We have developed a method, SLALOM (for StatisticaL Analysis of Locus Overlap Method), to perform comparative analysis of sequence annotations in a highly flexible way. SLALOM implements six major operation modes and a number of additional options that can answer a variety of statistical questions about a pair of input annotations of a given sequence collection. We demonstrate the results of SLALOM on three different examples from biology and economics and compare our method to already existing software. We discuss the importance of carefully choosing the application logic to address specific scientific questions. CONCLUSION: SLALOM is a highly versatile, command-line based method for comparing annotations in a collection of sequences, with a statistical read-out for performance evaluation and benchmarking of predictors and gene annotation pipelines. Abstraction from sequence content even allows SLALOM to compare other kinds of positional data including, for example, data coming from time series.

Phenotypic and genomic comparison of Photorhabdus luminescens subsp. laumondii TT01 and a widely used rifampicin-resistant Photorhabdus luminescens laboratory strain.

BACKGROUND: Photorhabdus luminescens is an enteric bacterium, which lives in mutualistic association with soil nematodes and is highly pathogenic for a broad spectrum of insects. A complete genome sequence for the type strain P. luminescens subsp. laumondii TT01, which was originally isolated in Trinidad and Tobago, has been described earlier. Subsequently, a rifampicin resistant P. luminescens strain has been generated with superior possibilities for experimental characterization. This strain, which is widely used in research, was described as a spontaneous rifampicin resistant mutant of TT01 and is known as TT01-RifR. RESULTS: Unexpectedly, upon phenotypic comparison between the rifampicin resistant strain and its presumed parent TT01, major differences were found with respect to bioluminescence, pigmentation, biofilm formation, haemolysis as well as growth. Therefore, we renamed the strain TT01-RifR to DJC. To unravel the genomic basis of the observed differences, we generated a complete genome sequence for strain DJC using the PacBio long read technology. As strain DJC was supposed to be a spontaneous mutant, only few sequence differences were expected. In order to distinguish these from potential sequencing errors in the published TT01 genome, we re-sequenced a derivative of strain TT01 in parallel, also using the PacBio technology. The two TT01 genomes differed at only 30 positions. In contrast, the genome of strain DJC varied extensively from TT01, showing 13,000 point mutations, 330 frameshifts, and 220 strain-specific regions with a total length of more than 300 kb in each of the compared genomes. CONCLUSIONS: According to the major phenotypic and genotypic differences, the rifampicin resistant P. luminescens strain, now named strain DJC, has to be considered as an independent isolate rather than a derivative of strain TT01. Strains TT01 and DJC both belong to P. luminescens subsp. laumondii.

The complete and fully assembled genome sequence of Aeromonas salmonicida subsp. pectinolytica and its comparative analysis with other Aeromonas species: investigation of the mobilome in environmental and pathogenic strains.

BACKGROUND: Due to the predominant usage of short-read sequencing to date, most bacterial genome sequences reported in the last years remain at the draft level. This precludes certain types of analyses, such as the in-depth analysis of genome plasticity. RESULTS: Here we report the finalized genome sequence of the environmental strain Aeromonas salmonicida subsp. pectinolytica 34mel, for which only a draft genome with 253 contigs is currently available. Successful completion of the transposon-rich genome critically depended on the PacBio long read sequencing technology. Using finalized genome sequences of A. salmonicida subsp. pectinolytica and other Aeromonads, we report the detailed analysis of the transposon composition of these bacterial species. Mobilome evolution is exemplified by a complex transposon, which has shifted from pathogenicity-related to environmental-related gene content in A. salmonicida subsp. pectinolytica 34mel. CONCLUSION: Obtaining the complete, circular genome of A. salmonicida subsp. pectinolytica allowed us to perform an in-depth analysis of its mobilome. We demonstrate the mobilome-dependent evolution of this strain's genetic profile from pathogenic to environmental.

ThiN as a Versatile Domain of Transcriptional Repressors and Catalytic Enzymes of Thiamine Biosynthesis.

Thiamine biosynthesis is commonly regulated by a riboswitch mechanism; however, the enzymatic steps and regulation of this pathway in archaea are poorly understood. Haloferax volcanii, one of the representative archaea, uses a eukaryote-like Thi4 (thiamine thiazole synthase) for the production of the thiazole ring and condenses this ring with a pyrimidine moiety synthesized by an apparent bacterium-like ThiC (2-methyl-4-amino-5-hydroxymethylpyrimidine [HMP] phosphate synthase) branch. Here we found that archaeal Thi4 and ThiC were encoded by leaderless transcripts, ruling out a riboswitch mechanism. Instead, a novel ThiR transcription factor that harbored an N-terminal helix-turn-helix (HTH) DNA binding domain and C-terminal ThiN (TMP synthase) domain was identified. In the presence of thiamine, ThiR was found to repress the expression of thi4 and thiC by a DNA operator sequence that was conserved across archaeal phyla. Despite having a ThiN domain, ThiR was found to be catalytically inactive in compensating for the loss of ThiE (TMP synthase) function. In contrast, bifunctional ThiDN, in which the ThiN domain is fused to an N-terminal ThiD (HMP/HMP phosphate [HMP-P] kinase) domain, was found to be interchangeable for ThiE function and, thus, active in thiamine biosynthesis. A conserved Met residue of an extended α-helix near the active-site His of the ThiN domain was found to be important for ThiDN catalytic activity, whereas the corresponding Met residue was absent and the α-helix was shorter in ThiR homologs. Thus, we provide new insight into residues that distinguish catalytic from noncatalytic ThiN domains and reveal that thiamine biosynthesis in archaea is regulated by a transcriptional repressor, ThiR, and not by a riboswitch.IMPORTANCE Thiamine pyrophosphate (TPP) is a cofactor needed for the enzymatic activity of many cellular processes, including central metabolism. In archaea, thiamine biosynthesis is an apparent chimera of eukaryote- and bacterium-type pathways that is not well defined at the level of enzymatic steps or regulatory mechanisms. Here we find that ThiN is a versatile domain of transcriptional repressors and catalytic enzymes of thiamine biosynthesis in archaea. Our study provides new insight into residues that distinguish catalytic from noncatalytic ThiN domains and reveals that archaeal thiamine biosynthesis is regulated by a ThiN domain transcriptional repressor, ThiR, and not by a riboswitch.

Genome-wide identification of transcriptional start sites in the haloarchaeon Haloferax volcanii based on differential RNA-Seq (dRNA-Seq).

BACKGROUND: Differential RNA-Seq (dRNA-Seq) is a recently developed method of performing primary transcriptome analyses that allows for the genome-wide mapping of transcriptional start sites (TSSs) and the identification of novel transcripts. Although the transcriptomes of diverse bacterial species have been characterized by dRNA-Seq, the transcriptome analysis of archaeal species is still rather limited. Therefore, we used dRNA-Seq to characterize the primary transcriptome of the model archaeon Haloferax volcanii. RESULTS: Three independent cultures of Hfx. volcanii grown under optimal conditions to the mid-exponential growth phase were used to determine the primary transcriptome and map the 5'-ends of the transcripts. In total, 4749 potential TSSs were detected. A position weight matrix (PWM) was derived for the promoter predictions, and the results showed that 64 % of the TSSs were preceded by stringent or relaxed basal promoters. Of the identified TSSs, 1851 belonged to protein-coding genes. Thus, fewer than half (46 %) of the 4040 protein-coding genes were expressed under optimal growth conditions. Seventy-two percent of all protein-coding transcripts were leaderless, which emphasized that this pathway is the major pathway for translation initiation in haloarchaea. A total of 2898 of the TSSs belonged to potential non-coding RNAs, which accounted for an unexpectedly high fraction (61 %) of all transcripts. Most of the non-coding TSSs had not been previously described (2792) and represented novel sequences (59 % of all TSSs). A large fraction of the potential novel non-coding transcripts were cis-antisense RNAs (1244 aTSSs). A strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs was found, which suggested that the negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts corresponded to internal transcripts overlapping with mRNAs (1153 iTSSs) and intergenic small RNA (sRNA) candidates (395 TSSs). CONCLUSION: This study provides a comprehensive map of the primary transcriptome of Hfx. volcanii grown under optimal conditions. Fewer than half of all protein-coding genes have been transcribed under these conditions. Unexpectedly, more than half of the detected TSSs belonged to several classes of non-coding RNAs. Thus, RNA-based regulation appears to play a more important role in haloarchaea than previously anticipated.

The complete genome of a viable archaeum isolated from 123-million-year-old rock salt.

Live microbes have been isolated from rock salt up to Permian age. Only obligatory cellular functions can be performed in halite-buried cells. Consequently, their genomic sequences are likely to remain virtually unchanged. However, the available sequence information from these organisms is scarce and consists of mainly ribosomal 16S sequences. Here, live archaea were isolated from early Cretaceous (∼ 123 million years old) halite from the depth of 2000 m in Qianjiang Depression, Hubei Province, China. The sample was radiologically dated and subjected to rigorous surface sterilization before microbe isolation. The isolates represented a single novel species of Halobacterium, for which we suggest the name Halobacterium hubeiense, type strain Hbt. hubeiense JI20-1. The species was closely related to a Permian (225-280 million years old) isolate, Halobacterium noricense, originating from Alpine rock salt. This study is the first one to publish the complete genome of an organism originating from surface-sterilized ancient halite. In the future, genomic data from halite-buried microbes can become a key factor in understanding the mechanisms by which these organisms are able to survive in harsh conditions deep underground or possibly on other celestial bodies.

Haloferax volcanii archaeosortase is required for motility, mating, and C-terminal processing of the S-layer glycoprotein.

Cell surfaces are decorated by a variety of proteins that facilitate interactions with their environments and support cell stability. These secreted proteins are anchored to the cell by mechanisms that are diverse, and, in archaea, poorly understood. Recently published in silico data suggest that in some species a subset of secreted euryarchaeal proteins, which includes the S-layer glycoprotein, is processed and covalently linked to the cell membrane by enzymes referred to as archaeosortases. In silico work led to the proposal that an independent, sortase-like system for proteolysis-coupled, carboxy-terminal lipid modification exists in bacteria (exosortase) and archaea (archaeosortase). Here, we provide the first in vivo characterization of an archaeosortase in the haloarchaeal model organism Haloferax volcanii. Deletion of the artA gene (HVO_0915) resulted in multiple biological phenotypes: (a) poor growth, especially under low-salt conditions, (b) alterations in cell shape and the S-layer, (c) impaired motility, suppressors of which still exhibit poor growth, and (d) impaired conjugation. We studied one of the ArtA substrates, the S-layer glycoprotein, using detailed proteomic analysis. While the carboxy-terminal region of S-layer glycoproteins, consisting of a putative threonine-rich O-glycosylated region followed by a hydrophobic transmembrane helix, has been notoriously resistant to any proteomic peptide identification, we were able to identify two overlapping peptides from the transmembrane domain present in the ΔartA strain but not in the wild-type strain. This clearly shows that ArtA is involved in carboxy-terminal post-translational processing of the S-layer glycoprotein. As it is known from previous studies that a lipid is covalently attached to the carboxy-terminal region of the S-layer glycoprotein, our data strongly support the conclusion that archaeosortase functions analogously to sortase, mediating proteolysis-coupled, covalent cell surface attachment.

Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii.

BACKGROUND: Thiamine (vitamin B1) is synthesized de novo by certain yeast, fungi, plants, protozoans, bacteria and archaea. The pathway of thiamine biosynthesis by archaea is poorly understood, particularly the route of sulfur relay to form the thiazole ring. Archaea harbor structural homologs of both the bacterial (ThiS-ThiF) and eukaryotic (THI4) proteins that mobilize sulfur to thiazole ring precursors by distinct mechanisms. RESULTS: Based on comparative genome analysis, halophilic archaea are predicted to synthesize the pyrimidine moiety of thiamine by the bacterial pathway, initially suggesting that also a bacterial ThiS-ThiF type mechanism for synthesis of the thiazole ring is used in which the sulfur carrier ThiS is first activated by ThiF-catalyzed adenylation. The only ThiF homolog of Haloferax volcanii (UbaA) was deleted but this had no effect on growth in the absence of thiamine. Usage of the eukaryotic THI4-type sulfur relay was initially considered less likely for thiamine biosynthesis in archaea, since the active-site cysteine residue of yeast THI4p that donates the sulfur to the thiazole ring by a suicide mechanism is replaced by a histidine residue in many archaeal THI4 homologs and these are described as D-ribose-1,5-bisphosphate isomerases. The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205). Deletion of HVO_0665 generated a thiamine auxotroph that was trans-complemented by a wild-type copy of HVO_0665, but not the modified gene encoding an HvThi4 C165A variant. CONCLUSIONS: Based on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine. We extend this finding to a relatively large group of archaea, including haloarchaea, ammonium oxidizing archaea, and some methanogen and Pyrococcus species, by observing that these organisms code for THI4 homologs that have a conserved active site cysteine residue which is likely used in thiamine biosynthesis. Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.

Identification of structural and regulatory cell-shape determinants in Haloferax volcanii.

Archaea play indispensable roles in global biogeochemical cycles, yet many crucial cellular processes, including cell-shape determination, are poorly understood. Haloferax volcanii, a model haloarchaeon, forms rods and disks, depending on growth conditions. Here, we used a combination of iterative proteomics, genetics, and live-cell imaging to identify mutants that only form rods or disks. We compared the proteomes of the mutants with wild-type cells across growth phases, thereby distinguishing between protein abundance changes specific to cell shape and those related to growth phases. The results identified a diverse set of proteins, including predicted transporters, transducers, signaling components, and transcriptional regulators, as important for cell-shape determination. Through phenotypic characterization of deletion strains, we established that rod-determining factor A (RdfA) and disk-determining factor A (DdfA) are required for the formation of rods and disks, respectively. We also identified structural proteins, including an actin homolog that plays a role in disk-shape morphogenesis, which we named volactin. Using live-cell imaging, we determined volactin's cellular localization and showed its dynamic polymerization and depolymerization. Our results provide insights into archaeal cell-shape determination, with possible implications for understanding the evolution of cell morphology regulation across domains.

An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.

Chloride and organic osmolytes: a hybrid strategy to cope with elevated salinities by the moderately halophilic, chloride-dependent bacterium Halobacillus halophilus.

Salt acclimation in moderately halophilic bacteria is the result of action of a grand interplay orchestrated by signals perceived from the environment. To elucidate the cellular players involved in sensing and responding to changing salinities we have determined the genome sequence of Halobacillus halophilus, a Gram-positive moderate halophilic bacterium that has a strict requirement for the anion chloride. Halobacillus halophilus synthesizes a multitude of different compatible solutes and switches its osmolyte strategy with the external salinity and growth phase. Based on the emerging genome sequence, the compatible solutes glutamate, glutamine, proline and ectoine have already been experimentally studied. The biosynthetic routes for acetyl ornithine and acetyl lysine are also delineated from the genome sequence. Halobacillus halophilus is nutritionally very versatile and most compatible solutes cannot only be produced but also used as carbon and energy sources. The genome sequence unravelled isogenes for many pathways indicating a fine regulation of metabolism. Halobacillus halophilus is unique in integrating the concept of compatible solutes with the second fundamental principle to cope with salt stress, the accumulation of molar concentrations of salt (Cl(-)) in the cytoplasm. Extremely halophilic bacteria/archaea, which exclusively rely on the salt-in strategy, have a high percentage of acidic proteins compared with non-halophiles with a low percentage. Halobacillus halophilus has an intermediate position which is consistent with its ability to integrate both principles.

The ring of confidence: a haloarchaeal CRISPR/Cas system.

To survive the constant invasions by foreign genetic elements, prokaryotes have evolved various defensive systems. Almost all sequenced archaea, and half of the analysed bacteria use the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system, a recently identified prokaryotic immune system that can fend off invading elements in a sequence-specific manner. Few archaeal CRISPR/Cas systems have been analysed so far, and the molecular details of many of the steps involved in adaptation and defence are yet to be understood. In the present paper, we summarize our current knowledge about the CRISPR/Cas system in Haloferax volcanii, an extremely halophilic archaeon that was isolated from the Dead Sea. H. volcanii encodes a type I-B CRISPR/Cas system, and carries three CRISPR loci and eight Cas proteins. Although in laboratory culture for more than three decades, this defence system was shown to be still active. All three CRISPR loci are transcribed and processed into mature crRNAs (CRISPR RNAs). Cells challenged with engineered plasmids can recognize and eliminate these invading elements if they contain the correct PAM (protospacer adjacent motif) and a sequence that can be recognized by one of the CRISPR spacers.

Genome comparison reveals that Halobacterium salinarum 63-R2 is the origin of the twin laboratory strains NRC-1 and R1.

The genome of Halobacterium strain 63-R2 was recently reported and provides the opportunity to resolve long-standing issues regarding the source of two widely used model strains of Halobacterium salinarum, NRC-1 and R1. Strain 63-R2 was isolated in 1934 from a salted buffalo hide (epithet "cutirubra"), along with another strain from a salted cow hide (91-R6T , epithet "salinaria," the type strain of Hbt. salinarum). Both strains belong to the same species according to genome-based taxonomy analysis (TYGS), with chromosome sequences showing 99.64% identity over 1.85 Mb. The chromosome of strain 63-R2 is 99.99% identical to the two laboratory strains NRC-1 and R1, with only five indels, excluding the mobilome. The two reported plasmids of strain 63-R2 share their architecture with plasmids of strain R1 (pHcu43/pHS4, 99.89% identity; pHcu235/pHS3, 100.0% identity). We detected and assembled additional plasmids using PacBio reads deposited at the SRA database, further corroborating that strain differences are minimal. One plasmid, pHcu190 (190,816 bp) corresponds to pHS1 (strain R1) but is even more similar in architecture to pNRC100 (strain NRC-1). Another plasmid, pHcu229, assembled partially and completed in silico (229,124 bp), shares most of its architecture with pHS2 (strain R1). In deviating regions, it corresponds to pNRC200 (strain NRC-1). Further architectural differences between the laboratory strain plasmids are not unique, but are present in strain 63-R2, which contains characteristics from both of them. Based on these observations, it is proposed that the early twentieth-century isolate 63-R2 is the immediate ancestor of the twin laboratory strains NRC-1 and R1.

Unidirectional gene pairs in archaea and bacteria require overlaps or very short intergenic distances for translational coupling via termination-reinitiation and often encode subunits of heteromeric complexes.

Genomes of bacteria and archaea contain a much larger fraction of unidirectional (serial) gene pairs than convergent or divergent gene pairs. Many of the unidirectional gene pairs have short overlaps of -4 nt and -1 nt. As shown previously, translation of the genes in overlapping unidirectional gene pairs is tightly coupled. Two alternative models for the fate of the post-termination ribosome predict either that overlaps or very short intergenic distances are essential for translational coupling or that the undissociated post-termination ribosome can scan through long intergenic regions, up to hundreds of nucleotides. We aimed to experimentally resolve the contradiction between the two models by analyzing three native gene pairs from the model archaeon Haloferax volcanii and three native pairs from Escherichia coli. A two reporter gene system was used to quantify the reinitiation frequency, and several stop codons in the upstream gene were introduced to increase the intergenic distances. For all six gene pairs from two species, an extremely strong dependence of the reinitiation efficiency on the intergenic distance was unequivocally demonstrated, such that even short intergenic distances of about 20 nt almost completely abolished translational coupling. Bioinformatic analysis of the intergenic distances in all unidirectional gene pairs in the genomes of H. volcanii and E. coli and in 1,695 prokaryotic species representative of 49 phyla showed that intergenic distances of -4 nt or -1 nt (= short gene overlaps of 4 nt or 1 nt) were by far most common in all these groups of archaea and bacteria. A small set of genes in E. coli, but not in H. volcanii, had intergenic distances of around +10 nt. Our experimental and bioinformatic analyses clearly show that translational coupling requires short gene overlaps, whereas scanning of intergenic regions by the post-termination ribosome occurs rarely, if at all. Short overlaps are enriched among genes that encode subunits of heteromeric complexes, and co-translational complex formation requiring precise subunit stoichiometry likely confers an evolutionary advantage that drove the formation and conservation of overlapping gene pairs during evolution.

A comparative genomics perspective on the genetic content of the alkaliphilic haloarchaeon Natrialba magadii ATCC 43099T.

BACKGROUND: Natrialba magadii is an aerobic chemoorganotrophic member of the Euryarchaeota and is a dual extremophile requiring alkaline conditions and hypersalinity for optimal growth. The genome sequence of Nab. magadii type strain ATCC 43099 was deciphered to obtain a comprehensive insight into the genetic content of this haloarchaeon and to understand the basis of some of the cellular functions necessary for its survival. RESULTS: The genome of Nab. magadii consists of four replicons with a total sequence of 4,443,643 bp and encodes 4,212 putative proteins, some of which contain peptide repeats of various lengths. Comparative genome analyses facilitated the identification of genes encoding putative proteins involved in adaptation to hypersalinity, stress response, glycosylation, and polysaccharide biosynthesis. A proton-driven ATP synthase and a variety of putative cytochromes and other proteins supporting aerobic respiration and electron transfer were encoded by one or more of Nab. magadii replicons. The genome encodes a number of putative proteases/peptidases as well as protein secretion functions. Genes encoding putative transcriptional regulators, basal transcription factors, signal perception/transduction proteins, and chemotaxis/phototaxis proteins were abundant in the genome. Pathways for the biosynthesis of thiamine, riboflavin, heme, cobalamin, coenzyme F420 and other essential co-factors were deduced by in depth sequence analyses. However, approximately 36% of Nab. magadii protein coding genes could not be assigned a function based on Blast analysis and have been annotated as encoding hypothetical or conserved hypothetical proteins. Furthermore, despite extensive comparative genomic analyses, genes necessary for survival in alkaline conditions could not be identified in Nab. magadii. CONCLUSIONS: Based on genomic analyses, Nab. magadii is predicted to be metabolically versatile and it could use different carbon and energy sources to sustain growth. Nab. magadii has the genetic potential to adapt to its milieu by intracellular accumulation of inorganic cations and/or neutral organic compounds. The identification of Nab. magadii genes involved in coenzyme biosynthesis is a necessary step toward further reconstruction of the metabolic pathways in halophilic archaea and other extremophiles. The knowledge gained from the genome sequence of this haloalkaliphilic archaeon is highly valuable in advancing the applications of extremophiles and their enzymes.