XPD helicase structures and activities: insights into the cancer and aging phenotypes from XPD mutations.

Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

Modes of action of the archaeal Mre11/Rad50 DNA-repair complex revealed by fast-scan atomic force microscopy.

Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1-2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.

The archaeal triphosphate tunnel metalloenzyme SaTTM defines structural determinants for the diverse activities in the CYTH protein family.

CYTH proteins make up a large superfamily that is conserved in all three domains of life. These enzymes have a triphosphate tunnel metalloenzyme (TTM) fold, which typically results in phosphatase functions, e.g., RNA triphosphatase, inorganic polyphosphatase, or thiamine triphosphatase. Some CYTH orthologs cyclize nucleotide triphosphates to 3',5'-cyclic nucleotides. So far, archaeal CYTH proteins have been annotated as adenylyl cyclases, although experimental evidence to support these annotations is lacking. To address this gap, we characterized a CYTH ortholog, SaTTM, from the crenarchaeote Sulfolobus acidocaldarius. Our in silico studies derived ten major subclasses within the CYTH family implying a close relationship between these archaeal CYTH enzymes and class IV adenylyl cyclases. However, initial biochemical characterization reveals inability of SaTTM to produce any cyclic nucleotides. Instead, our structural and functional analyses show a classical TTM behavior, i.e., triphosphatase activity, where pyrophosphate causes product inhibition. The Ca2+-inhibited Michaelis complex indicates a two-metal-ion reaction mechanism analogous to other TTMs. Cocrystal structures of SaTTM further reveal conformational dynamics in SaTTM that suggest feedback inhibition in TTMs due to tunnel closure in the product state. These structural insights combined with further sequence similarity network-based in silico analyses provide a firm molecular basis for distinguishing CYTH orthologs with phosphatase activities from class IV adenylyl cyclases.

Genetic Study of Four Candidate Holliday Junction Processing Proteins in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius.

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due to the lack of genetic evidence, it is still unclear whether these proteins are actually involved in HR in vivo and how their functional relation is associated with the process. To address the above questions, we constructed hjc-, hje-, hjm-, and pina single-knockout strains and double-knockout strains of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. Notably, we succeeded in isolating the hjm- and/or pina-deleted strains, suggesting that the functions of Hjm and PINA are not essential for cellular growth in this archaeon, as they were previously thought to be essential. Growth retardation in Δpina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δpina Δhjc and Δpina Δhje exhibited severe cold sensitivity. Δhjm exhibited severe sensitivity to interstrand crosslinkers, suggesting that Hjm is involved in repairing stalled replication forks, as previously demonstrated in euryarchaea. Our findings suggest that the function of PINA and HJ resolvases is functionally related at lower temperatures to support robust cellular growth, and Hjm is important for the repair of stalled replication forks in vivo.

Salt Stress Response of Sulfolobus acidocaldarius Involves Complex Trehalose Metabolism Utilizing a Novel Trehalose-6-Phosphate Synthase (TPS)/Trehalose-6-Phosphate Phosphatase (TPP) Pathway.

The crenarchaeon Sulfolobus acidocaldarius has been described to synthesize trehalose via the maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ) pathway, and the trehalose glycosyltransferring synthase (TreT) pathway has been predicted. Deletion mutant analysis of strains with single and double deletions of ΔtreY and ΔtreT in S. acidocaldarius revealed that in addition to these two pathways, a third, novel trehalose biosynthesis pathway is operative in vivo: the trehalose-6-phosphate (T6P) synthase/T6P phosphatase (TPS/TPP) pathway. In contrast to known TPS proteins, which belong to the GT20 family, the S. acidocaldarius TPS belongs to the GT4 family, establishing a new function within this group of enzymes. This novel GT4-like TPS was found to be present mainly in the Sulfolobales The ΔtreY ΔtreT Δtps triple mutant of S. acidocaldarius, which lacks the ability to synthesize trehalose, showed no altered phenotype under standard conditions or heat stress but was unable to grow under salt stress. Accordingly, in the wild-type strain, a significant increase of intracellular trehalose formation was observed under salt stress. Quantitative real-time PCR showed a salt stress-mediated induction of all three trehalose-synthesizing pathways. This demonstrates that in Archaea, trehalose plays an essential role for growth under high-salt conditions.IMPORTANCE The metabolism and function of trehalose as a compatible solute in Archaea was not well understood. This combined genetic and enzymatic approach at the interface of microbiology, physiology, and microbial ecology gives important insights into survival under stress, adaptation to extreme environments, and the role of compatible solutes in Archaea Here, we unraveled the complexity of trehalose metabolism, and we present a comprehensive study on trehalose function in stress response in S. acidocaldarius This sheds light on the general microbiology and the fascinating metabolic repertoire of Archaea, involving many novel biocatalysts, such as glycosyltransferases, with great potential in biotechnology.

Coupling efficiency in light-driven hybrid P450BM3 and CYP119 enzymes.

The light-driven hybrid P450 enzyme approach utilizing the photochemical properties of a covalently attached Ru(II)-diimine photosensitizer was extended to the archaeal Sulfolobus acidocaldarius CYP119 enzyme leading to high photocatalytic activity in the hydroxylation of the chromogenic substrate, 11-nitrophenoxyundecanoic acid. The determined kcat was greater than those reported with various natural redox partners. In addition, the sacrificial electron donor, diethyldithiocarbamate, used in the photocatalytic reaction is shown to play a dual role. It acts as an efficient quencher of the Ru(II) excited state leading to a highly reducing species necessary to inject electrons into the heme. It is also known for its antioxidant properties and is shown herein to be a useful probe to determine coupling efficiency in the light-driven hybrid enzymes.

Endonucleases responsible for DNA repair of helix-distorting DNA lesions in the thermophilic crenarchaeon Sulfolobus acidocaldarius in vivo.

The DNA repair mechanisms of hyperthermophiles can provide important insights for understanding how genetic information is maintained under extreme environments. Recent biochemical studies have identified a novel endonuclease in hyperthermophilic archaea, NucS/EndoMS, that acts on branched DNA substrates and mismatched bases. NucS/EndoMS is thought to participate in the DNA repair of helix-distorting DNA lesions, including UV-induced DNA damage and DNA adducts, and mismatched bases; however, the specific in vivo role of NucS/EndoMS in hyperthermophilic archaeal DNA repair has not been reported. To explore the role of this protein, we knocked out the nucS/endoMS gene of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. While the nucS/endoMS-deleted strain exhibited sensitivity to DNA adducts, it did not have high mutation rates or any sensitivity to UV irradiation. It has been proposed that the XPF endonuclease is involved in homologous recombination-mediated stalled-fork DNA repair. The xpf-deficient strain exhibited sensitivity to helix-distorting DNA lesions, but the sensitivity of the nucS/endoMS and xpf double knockout strain did not increase compared to that of the single knockout strains. We conclude that the endonuclease NucS/EndoMS works with XPF in homologous recombination-mediated stalled-fork DNA repair for the removal of helix-distorting DNA lesions in S. acidocaldarius.

High-efficiency expression of Sulfolobus acidocaldarius maltooligosyl trehalose trehalohydrolase in Escherichia coli through host strain and induction strategy optimization.

Maltooligosyl trehalose trehalohydrolase (MTHase, EC 3.2.1.141) catalyzes the release of trehalose, a novel food ingredient, by splitting the α-1,4-glucosidic linkage adjacent to the α-1,1-glucosidic linkage of maltooligosyl trehalose. However, the high-yield preparation of recombinant MTHase has not yet been reported. In this study, a codon-optimized synthetic gene encoding Sulfolobus acidocaldarius MTHase was expressed in Escherichia coli. In initial expression experiments conducted using pET-24a (+) and E. coli BL21 (DE3), the MTHase activity was 10.4 U/mL and a large amount of the expression product formed inclusion bodies. The familiar strategies, including addition of additives, co-expression with molecular chaperones, and expression with a fusion partner, failed to enhance soluble MTHase expression. Considering the intermolecular disulfide bond of MTHase, expression was investigated using a system comprising plasmid pET-32a (+) and host E. coli Origami (DE3), which is conducive to cytoplasmic disulfide bond formation. The MTHase activity increased to 55.0 U/mL, a 5.3-fold increase. Optimization of the induction conditions in a 3-L fermentor showed that when the lactose was fed at 0.2 g/L/h beginning at an OD600 of 40 and the induction temperature was maintained at 30 °C, the MTHase activity reached a maximum of 204.6 U/mL. This is the first report describing a systematic effort to obtain high-efficiency MTHase production. The high yield obtained using this process provides the basis for the industrial-scale production of trehalose. This report is also expected to be valuable in the production of other enzymes containing disulfide bonds.

High yield production and purification of two recombinant thermostable phosphotriesterase-like lactonases from Sulfolobus acidocaldarius and Sulfolobus solfataricus useful as bioremediation tools and bioscavengers.

BACKGROUND: Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. But nowadays their manufacturing in high yields is still an issue that limits their industrial applications. In this work we aimed to set up a high yield production and purification biotechnological process of two recombinant PLLs expressed in E. coli, the wild type SacPox from Sulfolobus acidocaldarius and a triple mutated SsoPox C258L/I261F/W263A, originally from Sulfolobus solfataricus. To follow this aim new induction approaches were investigated to boost the enzyme production, high cell density fermentation strategies were set-up to reach higher and higher enzyme yields up to 22-L scale, a downstream train was studied to meet the requirements of an efficient industrial purification process. RESULTS: Physiological studies in shake flasks demonstrated that the use of galactose as inducer increased the enzyme concentrations up to 4.5 folds, compared to the production obtained by induction with IPTG. Optimising high cell density fed-batch strategies the production and the productivity of both enzymes were further enhanced of 26 folds, up to 2300 U·L- 1 and 47.1 U·L- 1·h- 1 for SacPox and to 8700 U·L- 1 and 180.6 U·L- 1·h- 1 for SsoPox C258L/I261F/W263A, and the fermentation processes resulted scalable from 2.5 to 22.0 L. After being produced and extracted from the cells, the enzymes were first purified by a thermo-precipitation step, whose conditions were optimised by response surface methodology. A following ultra-filtration process on 100 and 5 KDa cut-off membranes drove to a final pureness and a total recovery of both enzymes of 70.0 ± 2.0%, suitable for industrial applications. CONCLUSIONS: In this paper, for the first time, a high yield biotechnological manufacturing process of the recombinant enzymes SacPox and SsoPox C258L/I261F/W263A was set-up. The enzyme production was boosted by combining a new galactose induction approach with high cell density fed-batch fermentation strategies. An efficient enzyme purification protocol was designed coupling a thermo-precipitation step with a following membrane-based ultra-filtration process.

Two trehalose-hydrolyzing enzymes from Crenarchaeon Sulfolobus acidocaldarius exhibit distinct activities and affinities toward trehalose.

Two archaeal trehalase-like genes, Saci1250 and Saci1816, belonging to glycoside hydrolase family 15 (GH15) from the acidophilic Crenarchaeon Sulfolobus acidocaldarius were expressed in Escherichia coli. The gene products showed trehalose-hydrolyzing activities, and the names SaTreH1 and SaTreH2 were assigned to Saci1816 and Saci1250 gene products, respectively. These newly identified enzymes functioned within a narrow range of acidic pH values at elevated temperatures, which is similar to the behavior of Euryarchaeota Thermoplasma trehalases. SaTreH1 displayed high KM and kcat values, whereas SaTreH2 had lower KM and kcat values despite a high degree of identity in their primary structures. A mutation analysis indicated that two glutamic acid residues in SaTreH1, E374 and E574, may be involved in trehalase catalysis because SaTreH1 E374Q and E574Q showed greatly reduced trehalose-hydrolyzing activities. Additional mutations substituting G573 and H575 residues with serine and glutamic acid residues, respectively, to mimic the TVN1315 sequence resulted in a decrease in trehalase activity and thermal stability. Taken together, the results indicated that Crenarchaea trehalases adopt active site structures that are similar to Euryarchaeota enzymes but have distinct molecular features. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 trehalases as well as other family enzymes and will provide insights into archaeal trehalose metabolism.

Structural and functional insights into tRNA binding and adenosine N1-methylation by an archaeal Trm10 homologue.

Purine nucleosides on position 9 of eukaryal and archaeal tRNAs are frequently modified in vivo by the post-transcriptional addition of a methyl group on their N1 atom. The methyltransferase Trm10 is responsible for this modification in both these domains of life. While certain Trm10 orthologues specifically methylate either guanosine or adenosine at position 9 of tRNA, others have a dual specificity. Until now structural information about this enzyme family was only available for the catalytic SPOUT domain of Trm10 proteins that show specificity toward guanosine. Here, we present the first crystal structure of a full length Trm10 orthologue specific for adenosine, revealing next to the catalytic SPOUT domain also N- and C-terminal domains. This structure hence provides crucial insights in the tRNA binding mechanism of this unique monomeric family of SPOUT methyltransferases. Moreover, structural comparison of this adenosine-specific Trm10 orthologue with guanosine-specific Trm10 orthologues suggests that the N1 methylation of adenosine relies on additional catalytic residues.

Hole Hopping through Tryptophan in Cytochrome P450.

Electron-transfer kinetics have been measured in four conjugates of cytochrome P450 with surface-bound Ru-photosensitizers. The conjugates are constructed with enzymes from Bacillus megaterium (CYP102A1) and Sulfolobus acidocaldarius (CYP119). A W96 residue lies in the path between Ru and the heme in CYP102A1, whereas H76 is present at the analogous location in CYP119. Two additional conjugates have been prepared with (CYP102A1)W96H and (CYP119)H76W mutant enzymes. Heme oxidation by photochemically generated Ru3+ leads to P450 compound II formation when a tryptophan residue is in the path between Ru and the heme; no heme oxidation is observed when histidine occupies this position. The data indicate that heme oxidation proceeds via two-step tunneling through a tryptophan radical intermediate. In contrast, heme reduction by photochemically generated Ru+ proceeds in a single electron tunneling step with closely similar rate constants for all four conjugates.

Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius.

Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/µg DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr-) and arginine decarboxylase- (argD-) deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

AglH, a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius, is capable of complementing the eukaryal Alg7.

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D100), IV (F220) and V (F264) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

Analysis of cytochrome P450 CYP119 ligand-dependent conformational dynamics by two-dimensional NMR and X-ray crystallography.

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional (1)H,(15)N HSQC chemical shift perturbation mapping of (15)N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.

Disruption of the gene encoding restriction endonuclease SuaI and development of a host-vector system for the thermoacidophilic archaeon Sulfolobus acidocaldarius.

Sulfolobus acidocaldarius is a useful model organism for the genetic study of thermophilic archaea due to its ease of cultivation. Here we describe the development of a host-vector system for S. acidocaldarius consisting of SuaI restriction system-deficient strain SK-1 and shuttle vector pSAV2. The new host strain SK-1 was constructed by pop-out recombination based on the pyrE marker gene. Plasmid pSAV2 was constructed from the S. islandicus native plasmid pRN1, in which selectable markers and functional genes were inserted in suitable locations and orientations followed by the deletion of non-essential open reading frames. SK-1 allowed direct transformation without N(4)-methylation at SuaI restriction sites, so unmethylated vector pSAV2 could be introduced directly into SK-1 by electroporation. The transformants were selected by pyrEF complementation on xyrose-tryptone solid medium without prior liquid culturing. The transformation efficiency was approximately 1.0 × 10(3)/µg DNA. After replication in S. acidocaldarius, pSAV2 was successfully recovered from transformant cultures by the standard alkaline lysis method. Plasmid yield was approximately 40-50 ng/ml from late-log through stationary phase cultures. In addition, pSAV2 was maintained stably and at relatively high copy number in S. acidocaldarius.

Characterization of two ß-decarboxylating dehydrogenases from Sulfolobus acidocaldarius.

Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two ß-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg2+, thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.

Structural Adaptability Facilitates Histidine Heme Ligation in a Cytochrome P450.

Almost all known members of the cytochrome P450 (CYP) superfamily conserve a key cysteine residue that coordinates the heme iron. Although mutation of this residue abolishes monooxygenase activity, recent work has shown that mutation to either serine or histidine unlocks non-natural carbene- and nitrene-transfer activities. Here we present the first crystal structure of a histidine-ligated P450. The T213A/C317H variant of the thermostable CYP119 from Sulfolobus acidocaldarius maintains heme iron coordination through the introduced ligand, an interaction that is accompanied by large changes in the overall protein structure. We also find that the axial cysteine C317 may be substituted with any other amino acid without abrogating folding and heme cofactor incorporation. Several of the axial mutants display unusual spectral features, suggesting that they have active sites with unique steric and electronic properties. These novel, highly stable enzyme active sites will be fruitful starting points for investigations of non-natural P450 catalysis and mechanisms.

Membrane-bound amylopullulanase is essential for starch metabolism of Sulfolobus acidocaldarius DSM639.

Sulfolobus acidocaldarius DSM639 produced an acid-resistant membrane-bound amylopullulanase (Apu) during growth on starch as a sole carbon and energy source. The physiological role of Apu in starch metabolism was investigated by the growth and starch degradation pattern of apu disruption mutant as well as biochemical properties of recombinant Apu. The Δapu mutant lost the ability to grow in minimal medium in the presence of starch, and the amylolytic activity observed in the membrane fraction of the wild-type strain was not detected in the Δapu mutant when the cells were grown in YT medium. The purified membrane-bound Apu initially hydrolyzed starch, amylopectin, and pullulan into various sizes of maltooligosaccharides, and then produced glucose, maltose, and maltotriose in the end, indicating Apu is a typical endo-acting glycoside hydrolase family 57 (GH57) amylopullulanase. The maltose and maltotriose observed in the culture medium during the exponential and stationary phase growth indicates that Apu is the essential enzyme to initially hydrolyze the starch into small maltooligosaccharides to be transported into the cell.

Archaeal signal transduction: impact of protein phosphatase deletions on cell size, motility, and energy metabolism in Sulfolobus acidocaldarius.

In this study, the in vitro and in vivo functions of the only two identified protein phosphatases, Saci-PTP and Saci-PP2A, in the crenarchaeal model organism Sulfolobus acidocaldarius were investigated. Biochemical characterization revealed that Saci-PTP is a dual-specific phosphatase (against pSer/pThr and pTyr), whereas Saci-PP2A exhibited specific pSer/pThr activity and inhibition by okadaic acid. Deletion of saci_pp2a resulted in pronounced alterations in growth, cell shape and cell size, which could be partially complemented. Transcriptome analysis of the three strains (Δsaci_ptp, Δsaci_pp2a and the MW001 parental strain) revealed 155 genes that were differentially expressed in the deletion mutants, and showed significant changes in expression of genes encoding the archaella (archaeal motility structure), components of the respiratory chain and transcriptional regulators. Phosphoproteome studies revealed 801 unique phosphoproteins in total, with an increase in identified phosphopeptides in the deletion mutants. Proteins from most functional categories were affected by phosphorylation, including components of the motility system, the respiratory chain, and regulatory proteins. In the saci_pp2a deletion mutant the up-regulation at the transcript level, as well as the observed phosphorylation pattern, resembled starvation stress responses. Hypermotility was also observed in the saci_pp2a deletion mutant. The results highlight the importance of protein phosphorylation in regulating essential cellular processes in the crenarchaeon S. acidocaldarius.