Catalytic linkage between caspase activity and proteostasis in Archaea.

The model haloarchaeon, Haloferax volcanii possess an extremely high, and highly specific, basal caspase activity in exponentially growing cells that closely resembles caspase-4. This activity is specifically inhibited by the pan-caspase inhibitor, z-VAD-FMK, and has no cross-reactivity with other known protease families. Although it is one of the dominant cellular proteolytic activities in exponentially growing H. volcanii cells, the interactive cellular roles remain unknown and the protein(s) responsible for this activity remain elusive. Here, biochemical purification and in situ trapping with caspase targeted covalent inhibitors combined with genome-enabled proteomics, structural analysis, targeted gene knockouts and treatment with canavanine demonstrated a catalytic linkage between caspase activity and thermosomes, proteasomes and cdc48b, a cell division protein and proteasomal degradation facilitating ATPase, as part of an 'interactase' of stress-related protein complexes with an established link to the unfolded protein response (UPR). Our findings provide novel cellular and biochemical context for the observed caspase activity in Archaea and add new insight to understanding the role of this activity, implicating their possible role in the establishment of protein stress and ER associated degradation pathways in Eukarya.

Light-dependent expression of four cryptic archaeal circadian gene homologs.

Circadian rhythms are important biological signals that have been found in almost all major groups of life from bacteria to man, yet it remains unclear if any members of the second major prokaryotic domain of life, the Archaea, also possess a biological clock. As an initial investigation of this question, we examined the regulation of four cyanobacterial-like circadian gene homologs present in the genome of the haloarchaeon Haloferax volcanii. These genes, designated cirA, cirB, cirC, and cirD, display similarity to the KaiC-family of cyanobacterial clock proteins, which act to regulate rhythmic gene expression and to control the timing of cell division. Quantitative RT-PCR analysis was used to examine the expression of each of the four cir genes in response to 12 h light/12 h dark cycles (LD 12:12) in H. volcanii during balanced growth. Our data reveal that there is an approximately two to sixteen-fold increase in cir gene expression when cells are shifted from light to constant darkness, and this pattern of gene expression oscillates with the light conditions in a rhythmic manner. Targeted single- and double-gene knockouts in the H. volcanii cir genes result in disruption of light-dependent, rhythmic gene expression, although it does not lead to any significant effect on growth under these conditions. Restoration of light-dependent, rhythmic gene expression was demonstrated by introducing, in trans, a wild-type copy of individual cir genes into knockout strains. These results are noteworthy as this is the first attempt to characterize the transcriptional expression and regulation of the ubiquitous kaiC homologs found among archaeal genomes.

Specificity of archaeal caspase activity in the extreme halophile Haloferax volcanii.

Caspase-like proteases are key initiators and executioners of programmed cell death (PCD), which is initiated by environmental stimuli and manifests in organisms ranging from unicellular microbes to higher eukaryotes. Archaea had been absent from the caspase inheritance discussion due to a lack of gene homologues. We recently demonstrated extremely high, basal caspase-like catalytic activity in the model haloarcheon, Haloferax volcanii, which was linked to the cellular stress response and was widespread among diverse Archaea. Here, we rigorously tested the catalytic specificity of the observed archaeal caspase-like activities using hydrolytic assays with a diverse suite of protease substrates and inhibitors compared with known model serine and cysteine proteases (trypsin, cathepsin, papain, and human caspase-8). Our experiments demonstrate that exponentially growing H. volcanii possesses a highly specific caspase-like activity that most closely resembles caspase-4, is preferentially inhibited by the pancaspase inhibitor, zVAD-FMK, and has no crossreactivity with other known protease families. Our findings firmly root the extremely high levels of caspase-like activity as the dominant proteolytic activity in this extreme haloarcheaon, thereby providing further support for housekeeping functions in Haloarchaea. Given the deep archaeal roots of eukaryotes, we suggest that this activity served as a foundation for stress pathways in higher organisms.

Microbial Diversity of the Hypersaline Sidi Ameur and Himalatt Salt Lakes of the Algerian Sahara.

Microbial populations within hypersaline lakes often exhibit high activities of photosynthesis, dissimilatory sulphate reduction and other processes and, thus, can have profound impacts on biogeochemical cycles of carbon, nitrogen, sulphur and other important elements within arid lands. To further understand these types of ecosystems, the physicochemical and biological properties of Sidi Ameur and Himalatt Salt Lakes in the Algerian Sahara were examined and compared. Both lakes were relatively neutral in pH (7.2 to 7.4) and high in salt, at 12% and 20 % (w/v) salinity for Himalatt and Sidi Ameur Lakes, respectively, with dominant ions of sodium and chloride. The community compositions of microbes from all three domains (Bacteria, Archaea and Eukarya) were surveyed through the use of 16S and 18S ribosomal gene amplification and clone library clustering using amplified ribosomal DNA restriction analysis (ARDRA) in conjunction with DNA sequencing and analysis. A high level of microbial diversity, particularly among the bacteria of the Himalatt Salt Lake and archaea of Sidi Ameur Lake, was found within these environments. Representatives from all known halophilic bacterial phyla as well as 6 different genera of halophilic archaea were identified. Moreover, several apparently novel phylotypes among both archaea and bacteria were revealed.

Tantalizing evidence for caspase-like protein expression and activity in the cellular stress response of Archaea.

An enigmatic feature of microbial evolution is the emergence of programmed cell death (PCD), a genetically controlled form of cell suicide triggered by environmental stimuli. Archaea, the second major prokaryotic domain of life, have been notably absent from the PCD inheritance discussion, due to a lack of genetic homologues. Using the model haloarchaeon Haloferax volcanii, we document extremely high caspase-specific activity and expression of immunoreactive proteins to human caspase 8 antisera, both of which were induced by salt stress and death and were abolished by in vivo addition of a broad-spectrum caspase inhibitor. Caspase inhibition severely impaired cell growth under low and high salt stress, demonstrating a critical role in the cellular stress response. In silico analysis of the H. volcanii proteome identified a subset of 18 potential target proteins containing a signature tetrapeptide caspase cleavage motif (IETD), some with putative roles in allosteric regulation, signal transduction, osmotic stress and cell communication. Detection of similarly high activity and expression in other haloarchaea (Halorubrum and Haloarcula) and in diverse members of Euryarchaeota (the methanogen Methanosarcina acetivorans and the hyperthermophile Pyrococcus furiosus) and Crenarchaeota (the acidophile Sulfolobus solfataricus) argue for a broad representation within the archaeal domain. By playing a role in normal cell function, caspase-like proteases in Archaea appear to have co-evolved with other metabolic pathways, broadening their biological roles beyond apoptosis and cell death.

Proteomic analysis of Haloferax volcanii reveals salinity-mediated regulation of the stress response protein PspA.

A proteomic survey of the halophilic archaeon Haloferax volcanii was performed by comparative two-dimensional gel electrophoresis in order to determine the molecular effects of salt stress on the organism. Cells were grown under optimal (2.1 M) and high (3.5 M) NaCl conditions. From this analysis, over 44 protein spots responsive to these conditions were detected. These spots were excised, digested in-gel with trypsin, subjected to QSTAR tandem mass spectrometry (LC/MS/MS) analysis, and identified by comparing the MS/MS-derived peptide sequence to that deduced from the H. volcanii genome. Approximately 40 % of the proteins detected (18 in total) displayed differential abundance based on the detection of at least two peptide fragments per protein and overall MOWSE scores of >or=75 per protein. All of these identified proteins were either uniquely present or 2.3- to 26-fold higher in abundance under one condition compared to the other. The majority of proteins identified in this study were preferentially displayed under optimal salinity and primarily involved in translation, transport and metabolism. However, one protein of interest whose transcript levels were confirmed in these studies to be upregulated under high salt conditions was identified as a homologue of the phage shock protein PspA. The pspA gene belongs to the psp stress-responsive regulon commonly found among Gram-negative bacteria where its transcription is stimulated by a wide variety of stressors, including heat shock, osmotic shock and prolonged stationary-phase incubation. Homologues of PspA are also found among the genomes of cyanobacteria, higher plants and other Archaea, suggesting that this protein may retain some aspects of functional conservation across the three domains of life. Given its integral role in sensing a variety of membrane stressors in bacteria, these results suggest that PspA may play an important role in hypersaline adaptation in H. volcanii.

HMG-CoA reductase is regulated by salinity at the level of transcription in Haloferax volcanii.

The moderately halophilic archaeon Haloferax volcanii was surveyed for protein profile changes correlated with growth at high and low salinity. A single polypeptide with an approximate mass of 46 kDa was conspicuously more abundant during growth at high salinity. This protein was identified as HMG-CoA reductase (HMGR), encoded by the hmgR gene. HMGR is a key enzyme in the mevalonate pathway of isoprenoid biosynthesis, the sole route in haloarchaea for lipid and carotenoid production. Enzymatic assays confirmed that HMGR activity is more abundant in cells grown at high salinity. Low salt cultures of H. volcanii contained lower amounts of hmgR transcript compared to cells grown in high salt suggesting that the observed regulation occurs at the level of transcription. Paradoxically, both lipid and carotenoid content decreased in H. volcanii grown at high salinity despite the increased levels of HMGR specific activity. To our knowledge, this is the first report demonstrating that the expression of HMGR is regulated in response to non-optimal salinity in a halophilic archaeon.

Differential expression of genes influenced by changing salinity using RNA arbitrarily primed PCR in the archaeal halophile Haloferax volcanii.

Extreme halophiles belonging to the domain Archaea require a minimum of approximately 10% NaCl for growth. Many of these obligate halophiles will continue to grow even as NaCl concentrations approach saturation. The haloarchaeon Haloferax volcanii is a model organism in which to study the effects of changes in medium salinity on gene expression, as this organism grows over a wide range of NaCl concentrations, between 12% and 23%, with little effect on growth rate. An RNA arbitrarily primed PCR (RAP-PCR) approach has been applied to identify those genes that are differentially expressed in response to changing salinity. Differences in gene expression can be detected using this methodology, as each sample generates its own unique RNA fingerprint for each growth condition examined. RNA was prepared from H. volcanii cultures grown with two different NaCl concentrations in the medium, RAP-PCR was performed, and seven differentially expressed transcripts were identified. These fragments were cloned, sequenced, and subjected to transcript analysis to confirm their regulation. One of the sequences identified in this study displays homology to the eukaryotic Ser/Thr protein kinase Ire1p, a sensor of protein unfolding in yeast and mammalian cells. Evidence for serine phosphorylation in H. volcanii is also presented.