Global effect of the lack of inorganic polyphosphate in the extremophilic archaeon Sulfolobus solfataricus: A proteomic approach.

Inorganic polyphosphates (polyP) are present in all living cells and several important functions have been described for them. They are involved in the response to stress conditions, such as nutrient depletion, oxidative stress and toxic metals amongst others. A recombinant strain of Sulfolobus solfataricus unable to accumulate polyP was designed by the overexpression of its endogenous ppx gene. The overall impact of the lack of polyP on this S. solfataricus polyP (-) strain was analyzed by using quantitative proteomics (isotope-coded protein label, ICPL). Stress-related proteins, such as peroxiredoxins and heat shock proteins, proteins involved in metabolism and several others were produced at higher levels in the ppx expression strain. The polyP deficient strain showed an increased copper sensitivity and an earlier transcriptional up-regulation of copA gene coding for the P-type copper-exporting ATPase. This implies a complementary function of both copper resistance systems. These results strongly suggests that the lack of polyP makes this hyperthermophilic archaeon more sensitive to toxic conditions, such as an exposure to metals or other harmful stimuli, emphasizing the importance of this inorganic phosphate polymers in the adaptations to live in the environmental conditions in which thermoacidophilic archaea thrive. SIGNIFICANCE: Inorganic polyphosphate (polyP) are ubiquitous molecules with many functions in living organisms. Few studies related to these polymers have been made in archaea. The construction of a polyP deficient recombinant strain of Sulfolobus solfataricus allowed the study of the global changes in the proteome of this thermoacidophilic archaeon in the absence of polyP compared with the wild type strain. The results obtained using quantitative proteomics suggest an important participation of polyP in the oxidative stress response of the cells and as having a possible metabolic role in the cell, as previously described in bacteria. The polyP deficient strain also showed an increased copper sensitivity and an earlier transcriptional up-regulation of copA, implying a complementary role of both copper resistance systems.

Expanding the Limits of Thermoacidophily in the Archaeon Sulfolobus solfataricus by Adaptive Evolution.

Extremely thermoacidophilic Crenarchaeota belonging to the order Sulfolobales flourish in hot acidic habitats that are strongly oxidizing. The pH extremes of these habitats, however, often exceed the acid tolerance of type species and strains. Here, adaptive laboratory evolution was used over a 3-year period to test whether such organisms harbor additional thermoacidophilic capacity. Three distinct cell lines derived from a single type species were subjected to high-temperature serial passage while culture acidity was gradually increased. A 178-fold increase in thermoacidophily was achieved after 29 increments of shifted culture pH resulting in growth at pH 0.8 and 80°C. These strains were named super-acid-resistant Crenarchaeota (SARC). Mathematical modeling using growth parameters predicted the limits of acid resistance, while genome resequencing and transcriptome resequencing were conducted for insight into mechanisms responsible for the evolved trait. Among the mutations that were detected, a set of eight nonsynonymous changes may explain the heritability of increased acid resistance despite an unexpected lack of transposition. Four multigene components of the SARC transcriptome implicated oxidative stress as a primary challenge accompanying growth at acid extremes. These components included accelerated membrane biogenesis, induction of the mer operon, and an increased capacity for the generation of energy and reductant.

Insight into the cellular involvement of the two reverse gyrases from the hyperthermophilic archaeon Sulfolobus solfataricus.

BACKGROUND: Reverse gyrases are DNA topoisomerases characterized by their unique DNA positive-supercoiling activity. Sulfolobus solfataricus, like most Crenarchaeota, contains two genes each encoding a reverse gyrase. We showed previously that the two genes are differently regulated according to temperature and that the corresponding purified recombinant reverse gyrases have different enzymatic characteristics. These observations suggest a specialization of functions of the two reverse gyrases. As no mutants of the TopR genes could be obtained in Sulfolobales, we used immunodetection techniques to study the function(s) of these proteins in S. solfataricus in vivo. In particular, we investigated whether one or both reverse gyrases are required for the hyperthermophilic lifestyle. RESULTS: For the first time the two reverse gyrases of S. solfataricus have been discriminated at the protein level and their respective amounts have been determined in vivo. Actively dividing S. solfataricus cells contain only small amounts of both reverse gyrases, approximately 50 TopR1 and 125 TopR2 molecules per cell at 80°C. S. solfataricus cells are resistant at 45°C for several weeks, but there is neither cell division nor replication initiation; these processes are fully restored upon a return to 80°C. TopR1 is not found after three weeks at 45°C whereas the amount of TopR2 remains constant. Enzymatic assays in vitro indicate that TopR1 is not active at 45°C but that TopR2 exhibits highly positive DNA supercoiling activity at 45°C. CONCLUSIONS: The two reverse gyrases of S. solfataricus are differently regulated, in terms of protein abundance, in vivo at 80°C and 45°C. TopR2 is present both at high and low temperatures and is therefore presumably required whether cells are dividing or not. By contrast, TopR1 is present only at high temperature where the cell division occurs, suggesting that TopR1 is required for controlling DNA topology associated with cell division activity and/or life at high temperature. Our findings in vitro that TopR1 is able to positively supercoil DNA only at high temperature, and TopR2 is active at both temperatures are consistent with them having different functions within the cells.

Functional curation of the Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 complete genome sequences.

The thermoacidophiles Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 are considered key model organisms representing a major phylum of the Crenarchaeota. Because maintaining current, accurate genome information is indispensable for modern biology, we have updated gene function annotation using the arCOGs database, plus other available functional, structural and phylogenetic information. The goal of this initiative is continuous improvement of genome annotation with the support of the Sulfolobus research community.

VapC6, a ribonucleolytic toxin regulates thermophilicity in the crenarchaeote Sulfolobus solfataricus.

The phylum Crenarchaeota includes hyperthermophilic micro-organisms subjected to dynamic thermal conditions. Previous transcriptomic studies of Sulfolobus solfataricus identified vapBC6 as a heat-shock (HS)-inducible member of the Vap toxin-antitoxin gene family. In this study, the inactivation of the vapBC6 operon by targeted gene disruption produced two recessive phenotypes related to fitness, HS sensitivity and a heat-dependent reduction in the rate of growth. In-frame vapBC6 deletion mutants were analyzed to examine the respective roles of each protein. Since vapB6 transcript abundance was elevated in the vapC6 deletion, the VapC6 toxin appears to regulate abundance of its cognate antitoxin. In contrast, vapC6 transcript abundance was reduced in the vapB6 deletion. A putative intergenic terminator may underlie these observations by coordinating vapBC6 expression. As predicted by structural modeling, recombinant VapC6 produced using chaperone cosynthesis exhibited heat-dependent ribonucleolytic activity toward S. solfataricus total RNA. This activity could be blocked by addition of preheated recombinant VapB6. In vivo transcript targets were identified by assessing the relative expression of genes that naturally respond to thermal stress in VapBC6-deficient cells. Preferential increases were observed for dppB-1 and tetR, and preferential decreases were observed for rpoD and eIF2 gamma. Specific VapC6 ribonucleolytic action could also be demonstrated in vitro toward RNAs whose expression increased in the VapBC6-deficient strain during heat shock. These findings provide a biochemical mechanism and identify cellular targets underlying VapBC6-mediated control over microbial growth and survival at temperature extremes.

Crenarchaeal biofilm formation under extreme conditions.

BACKGROUND: Biofilm formation has been studied in much detail for a variety of bacterial species, as it plays a major role in the pathogenicity of bacteria. However, only limited information is available for the development of archaeal communities that are frequently found in many natural environments. METHODOLOGY: We have analyzed biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii. We established a microtitre plate assay adapted to high temperatures to determine how pH and temperature influence biofilm formation in these organisms. Biofilm analysis by confocal laser scanning microscopy demonstrated that the three strains form very different communities ranging from simple carpet-like structures in S. solfataricus to high density tower-like structures in S. acidocaldarius in static systems. Lectin staining indicated that all three strains produced extracellular polysaccharides containing glucose, galactose, mannose and N-acetylglucosamine once biofilm formation was initiated. While flagella mutants had no phenotype in two days old static biofilms of S. solfataricus, a UV-induced pili deletion mutant showed decreased attachment of cells. CONCLUSION: The study gives first insights into formation and development of crenarchaeal biofilms in extreme environments.

Structural analysis of BldR from Sulfolobus solfataricus provides insights into the molecular basis of transcriptional activation in Archaea by MarR family proteins.

The multiple antibiotic resistance regulator (MarR) family constitutes a significant class of transcriptional regulators whose members control a variety of important biological functions such as regulation of response to environmental stress, control of virulence factor production, resistance to antimicrobial agents, and regulation of aromatic catabolic pathways. Although the majority of MarR family members have been characterized as transcriptional repressors, a few examples of transcriptional activators have also been reported. BldR is a newly identified member of this family that has been demonstrated to act as a transcriptional activator in stress response to aromatic compounds in the crenarchaeon Sulfolobus solfataricus. In this work, we report findings on the BldR X-ray crystal structure and present a molecular modeling study on the complex that this protein forms with its cognate DNA sequence, thus providing the first detailed description of the DNA-binding mechanism of an archaeal activator belonging to the MarR family. Two residues responsible for the high binding specificity of this transcriptional regulator were also identified. Our studies demonstrated that, in Archaea, the capability of MarR family members to act as activators or repressors is not related to a particular DNA-binding mechanism but rather could be due to the position of the binding site on the target DNA. Moreover, since genes encoding MarR proteins often control transcription of operons that encode for multisubstrate efflux pumps, our results also provided important insights for the identification of new tools to overcome the microorganism's multidrug resistance.

UV-inducible cellular aggregation of the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by pili formation.

The hyperthermophilic archaeon Sulfolobus solfataricus has been shown to exhibit a complex transcriptional response to UV irradiation involving 55 genes. Among the strongest UV-induced genes was a putative pili biogenesis operon encoding a potential secretion ATPase, two pre-pilins, a putative transmembrane protein and a protein of unknown function. Electron microscopy and image reconstruction of UV-treated cells showed straight pili with 10 nm in diameter, variable in length, not bundled or polarized and composed of three evenly spaced helices, thereby clearly being distinguishable from archaeal flagella. A deletion mutant of SSO0120, the central type II/IV secretion ATPase, did not produce pili. It could be complemented by reintroducing the gene on a plasmid vector. We have named the operon ups operon for UV-inducible pili operon of Sulfolobus. Overexpression of the pre-pilins, Ups-A/B (SSO0117/0118) in Sulfolobus resulted in production of extremely long filaments. Pronounced cellular aggregation was observed and quantified upon UV treatment. This aggregation was a UV-dose-dependent, dynamic process, not inducible by other physical stressors (such as pH or temperature shift) but stimulated by chemically induced double-strand breaks in DNA. We hypothesize that pili formation and subsequent cellular aggregation enhance DNA transfer among Sulfolobus cells to provide increased repair of damaged DNA via homologous recombination.

Proteome and transcriptional analysis of ethanol-grown Sulfolobus solfataricus P2 reveals ADH2, a potential alcohol dehydrogenase.

Sulfolobus solfataricus P2 was shown to survive on ethanol at various concentrations (0.08-3.97% w/v) as the sole carbon source. The highest ethanol consumption rate was 15.1 mg/L/hr (via GC-MS analysis) in cultures grown on 0.79% w/v ethanol. In vivo metabolic labeling, using 13C universally labeled ethanol, provided evidence for both ethanol uptake and metabolic utilization. Results obtained from isobaric mass tag-facilitated shotgun proteomics (iTRAQ) indicate that on average, 21 and 31% of the 284 proteins identified (with > or = 2 MS/MS) are increased and decreased expression in ethanol cultures compared to glucose control cultures. Preliminary analysis shows >2-fold increase of the zinc-dependent alcohol dehydrogenase, ADH-10 (SSO2536), and the putative ADH-2 (SSO0764) in both translational and transcriptional data (using quantitative RT-PCR), suggesting both proteins are integral to ethanol metabolism. Evidence that ethanol was catabolised into central metabolism via acetyl-CoA intermediates was further indicated by another >2-fold increase in protein expression levels of various acetyl-CoA synthetases. The decreased expression (>2-fold) of isocitrate dehydrogenase at the protein level suggests that the ethanol grown cultures shifted toward the glyoxylate cycle. Subsequently, the activity of ADH-2 was confirmed by overexpression in Escherichia coli, with the resultant purified in vitro enzyme exhibiting an activity that increased with temperature up to 95 degrees C, and giving a specific activity of 1.05 U/mg.

Three eukaryote-like Orc1/Cdc6 proteins functionally interact and mutually regulate their activities of binding to the replication origin in the hyperthermophilic archaeon Sulfolobus solfataricus P2.

The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins. However, it is not known whether these SsoCdc6 proteins can functionally interact and collectively contribute to DNA replication initiation. In the current work, we found that SsoCdc6-1 stimulates DNA-binding activities of SsoCdc6-3. In contrast, SsoCdc6-3 inhibits those of both SsoCdc6-1 and SsoCdc6-2. These regulatory functions are differentially affected by the C-terminal domains of these SsoCdc6 proteins. These data, in conjunction with studies on physical interactions between these replication initiators by bacterial two-hybrid and pull-down/Western blot assays, lead us to propose the possibility that multiple SsoCdc6 proteins might coordinately regulate DNA replication in the archaeon species. This is the first report on the functional interaction among the archaeal multiple Cdc6 proteins to regulate DNA replication.

Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus.

Flagellation in archaea is widespread and is involved in swimming motility. Here, we demonstrate that the structural flagellin gene from the crenarchaeaon Sulfolobus solfataricus is highly expressed in stationary-phase-grown cells and under unfavorable nutritional conditions. A mutant in a flagellar auxiliary gene, flaJ, was found to be nonmotile. Electron microscopic imaging of the flagellum indicates that the filaments are composed of right-handed helices.

Equal rates of repair of DNA photoproducts in transcribed and non-transcribed strands in Sulfolobus solfataricus.

The nucleotide excision repair (NER) pathway removes bulky lesions such as photoproducts from DNA. In both bacteria and eukarya, lesions located in transcribed strands are repaired significantly faster than those located in non-transcribed strands due to damage signalling by stalled RNA polymerase molecules: a phenomenon known as transcription-coupled repair (TCR). TCR requires a mechanism for coupling the detection of stalled RNA polymerase molecules to the NER pathway, provided in bacteria by the Mfd protein. In the third domain of life, archaea, the pathway of NER is not well defined, there are no Mfd homologues and the existence of TCR has not been investigated. In this report we looked at rates of removal of photoproducts in three different operons of the crenarchaeon Sulfolobus solfataricus following UV irradiation. We found no evidence for significantly faster repair in the transcribed strands of these three operons. The rate of global genome repair in S. solfataricus is relatively rapid, and this may obviate the requirement for a specialized TCR pathway. Significantly faster repair kinetics were observed in the presence of visible light, consistent with the presence of a gene for photolyase in the genome of S. solfataricus.

Dynamic metabolic adjustments and genome plasticity are implicated in the heat shock response of the extremely thermoacidophilic archaeon Sulfolobus solfataricus.

Approximately one-third of the open reading frames encoded in the Sulfolobus solfataricus genome were differentially expressed within 5 min following an 80 to 90 degrees C temperature shift at pH 4.0. This included many toxin-antitoxin loci and insertion elements, implicating a connection between genome plasticity and metabolic regulation in the early stages of stress response.