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.

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.

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.

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.

Global Distribution and Diversity of Haloarchaeal pL6-Family Plasmids.

Australian isolates of Haloquadratum walsbyi, a square-shaped haloarchaeon, often harbor small cryptic plasmids of the pL6-family, approximately 6 kb in size, and five examples have been previously described. These plasmids exhibit a highly conserved gene arrangement and encode replicases similar to those of betapleolipoviruses. To assess their global distribution and recover more examples for analysis, fifteen additional plasmids were reconstructed from the metagenomes of seven hypersaline sites across four countries: Argentina, Australia, Puerto Rico, and Spain. Including the five previously described plasmids, the average plasmid size is 6002 bp, with an average G+C content of 52.5%. The tetramers GGCC and CTAG are either absent or significantly under-represented, except in the two plasmids with the highest %G+C. All plasmids share a similar arrangement of genes organized as outwardly facing replication and ATPase modules, but variations were observed in some core genes, such as F2, and some plasmids had acquired accessory genes. Two plasmids, pCOLO-c1 and pISLA-c6, shared 92.7% nt identity despite originating from Argentina and Spain, respectively. Numerous metagenomic CRISPR spacers matched sequences in the fifteen reconstructed plasmids, indicating frequent invasion of haloarchaea. Spacers could be assigned to haloarchaeal genera by mapping their associated direct repeats (DR), with half of these matching Haloquadratum. Finally, strand-specific metatranscriptome (RNA-seq) data could be used to demonstrate the active transcription of two pL6-family plasmids, including antisense transcripts.

Beyond bacterial paradigms: uncovering the functional significance and first biogenesis machinery of archaeal lipoproteins.

Lipoproteins are major constituents of prokaryotic cell surfaces. In bacteria, lipoprotein attachment to membrane lipids is catalyzed by prolipoprotein diacylglyceryl transferase (Lgt). However, no Lgt homologs have been identified in archaea, suggesting the unique archaeal membrane lipids require distinct enzymes for lipoprotein lipidation. Here, we performed in silico predictions for all major archaeal lineages and revealed a high prevalence of lipoproteins across the domain Archaea. Using comparative genomics, we identified the first set of candidates for archaeal lipoprotein biogenesis components (Ali). Genetic and biochemical characterization confirmed two paralogous genes, aliA and aliB , are important for lipoprotein lipidation in the archaeon Haloferax volcanii . Disruption of AliA- and AliB-mediated lipoprotein lipidation results in severe growth defects, decreased motility, and cell-shape alterations, underscoring the importance of lipoproteins in archaeal cell physiology. AliA and AliB also exhibit different enzymatic activities, including potential substrate selectivity, uncovering a new layer of regulation for prokaryotic lipoprotein lipidation.

Genome sequence of Pseudomonas silesiensis strain G3, a member of a bacterial consortium capable of polyethylene degradation.

A consortium of landfill bacteria including strain G3 can break down polyethylene, a long-lasting plastic that accumulates in the environment. The complete genome sequence of strain G3 was determined by PacBio and Nanopore sequencing and consists of three circular replicons. Genome-based classification assigned strain G3 to the species Pseudomonas silesiensis.

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.

Iron starvation results in up-regulation of a probable Haloferax volcanii siderophore transporter.

The response of the haloarchaeal model organism Haloferax volcanii to iron starvation was analyzed at the proteome level by data-independent acquisition mass spectrometry. Cells grown in minimal medium with normal iron levels were compared to those grown under low iron conditions, with samples being separated into membrane and cytoplasmic fractions in order to focus on import/export processes which are frequently associated with metal homeostasis. Iron starvation not only caused a severe retardation of growth but also altered the levels of many proteins. Using a comprehensive annotated spectral library and data-independent acquisition mass spectrometry (DIA-MS), we found that iron starvation resulted in significant changes to both the membrane and the soluble proteomes of Hfx. volcanii. The most affected protein is the RND family permease HVO_A0467, which is 44-fold enriched in cells grown under iron starvation. The gene HVO_A0467 can be deleted suggesting that it is not essential under standard conditions. Compared to wild type cells the deletion strain shows only slight changes in growth and cell morphologies show no differences. Molecular docking predictions indicated that HVO_A0467 may be an exporter of the siderophore schizokinen for which a potential biosynthesis cluster is encoded in the Hfx. volcanii genome. Together, these findings confirm the importance of iron for archaeal cells and suggest HVO_0467 as a siderophore exporter.

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.