Biochemical,Transcriptional and Translational Evidences of the Phenol-meta-Degradation Pathway by the Hyperthermophilic Sulfolobus solfataricus 98/2

Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D) and meta involving a catechol 2,3 dioxygenase (C23D). Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively) and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg.L⁻¹) suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.     

An insertion element of the extremely thermophilic archaeon Sulfolobus solfataricus transposes into the endogenous β-galactosidase gene

Three phenotypically stable mutants of the extremely thermophilic archaeon Sulfolobus solfataricus have been isolated by screening for β-galactosidase negative colonies on plates with X-Gal (5-bromo-4-chloro-3-indolyl-(3-d-galactopyranoside). From one of these mutants an insertion element, designated ISC1217, was isolated and characterized. Sequence analysis of ISC1217 and of the regions adjacent to the insertion site in the β-galactosidase gene revealed features typical of a transposable element: ISC1217 contained terminal inverted repeats and was flanked by a direct repeat of 6 bp. The 1147 by sequence contained an open reading frame encoding a putative protein of 354 amino acid residues and, overlapping this, two smaller open reading frames on the opposite strand. There were approximately 8 copies of the insertion element in the S. solfataricus genome. ISC1217 did not cross-hybridize with DNA of other Sulfolobus species. All three independently isolated β-galactosidase mutants of S. solfataricus arose by transposition of ISC1217 or a related element.     

A spreadable, non-integrative and high copy number shuttle vector for Sulfolobus solfataricus based on the genetic element pSSVx from Sulfolobus islandicus

The pSSVx genetic element from Sulfolobus islandicus REY15/4 is a hybrid between a plasmid and a fusellovirus, able to be maintained in non-integrative form and to spread when the helper SSV2 virus is present in the cells. In this work, the satellite virus was engineered to obtain an Escherichia coli–Sulfolobus solfataricus shuttle vector for gene transfer and expression in S.solfataricus by fusing site-specifically the pSSVx chromosome with an E.coli plasmid replicon and the ampicillin resistance gene. The pSSVx-based vector was proven functional like the parental virus, namely it was able to spread efficiently through infected S.solfataricus cells. Moreover, the hybrid plasmid stably transformed S.solfataricus and propagated with no rearrangement, recombination or integration into the host chromosome. The high copy number of the artificial genetic element was found comparable with that calculated for the wild-type pSSVx in the new host cells, with no need of genetic markers for vector maintenance in the cells and for transfomant enrichment.

The newly constructed vector was also shown to be an efficient cloning vehicle for the expression of passenger genes in S.solfataricus. In fact, a derivative plasmid carrying an expression cassette of the lacS gene encoding the β-glycosidase from S.solfataricus under the control of the Sulfolobus chaperonine (thermosome tf55) heat shock promoter was also able to drive the expression of a functional enzyme. Complementation of the β-galactosidase deficiency in a deletion mutant strain of S.solfataricus demonstrated that lacS gene was an efficient marker for selection of single transformants on solid minimal lactose medium.

     

The identification and molecular characterization of the first archaeal bifunctional exo-β-glucosidase/N-acetyl-β-glucosaminidase demonstrate that family GH116 is made of three functionally distinct subfamilies

Background

β-N-acetylhexosaminidases, which are involved in a variety of biological processes including energy metabolism, cell proliferation, signal transduction and in pathogen-related inflammation and autoimmune diseases, are widely distributed in Bacteria and Eukaryotes, but only few examples have been found in Archaea so far. However, N-acetylgluco- and galactosamine are commonly found in the extracellular storage polymers and in the glycans decorating abundantly expressed glycoproteins from different Crenarchaeota Sulfolobus sp., suggesting that β-N-acetylglucosaminidase activities could be involved in the modification/recycling of these cellular components.

Methods

A thermophilic β-N-acetylglucosaminidase was purified from cellular extracts of S. solfataricus, strain P2, identified by mass spectrometry, and cloned and expressed in E. coli. Glycosidase assays on different strains of S. solfataricus, steady state kinetic constants, substrate specificity analysis, and the sensitivity to two inhibitors of the recombinant enzyme were also reported.

Results

A new β-N-acetylglucosaminidase from S. solfataricus was unequivocally identified as the product of gene sso3039. The detailed enzymatic characterization demonstrates that this enzyme is a bifunctional β-glucosidase/β-N-acetylglucosaminidase belonging to family GH116 of the carbohydrate active enzyme (CAZy) classification.

Conclusions

This study allowed us to propose that family GH116 is composed of three subfamilies, which show distinct substrate specificities and inhibitor sensitivities.

General significance

The characterization of SSO3039 allows, for the first time in Archaea, the identification of an enzyme involved in the metabolism β-N-acetylhexosaminide, an essential component of glycoproteins in this domain of life, and substantially increases our knowledge on the functional role and phylogenetic relationships amongst the GH116 CAZy family members.     

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.     

Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea

The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolobus–Escherichia coli shuttle vectors based on the small multicopy plasmid pRN1 from Sulfolobus islandicus. Selection in suitable uracil auxotrophs is provided through inclusion of pyrEF genes in the plasmid. The shuttle vectors do not integrate into the genome and do not rearrange. The plasmids allow functional overexpression of genes, as could be demonstrated for the β-glycosidase (lacS) gene of S. solfataricus. In addition, we demonstrate that this β-glycosidase gene could function as selectable marker in S. solfataricus. The shuttle plasmids differ in their interruption sites within pRN1 and allowed us to delineate functionally important regions of pRN1. The orf56/orf904 operon appears to be essential for pRN1 replication, in contrast interruption of the highly conserved orf80/plrA gene is tolerated. The new vector system promises to facilitate genetic studies of Sulfolobus and to have biotechnological uses, such as the overexpression or optimization of thermophilic enzymes that are not readily performed in mesophilic hosts.     

Deficiency of the tRNATyr:Ψ35-synthase aPus7 in Archaea of the Sulfolobales order might be rescued by the H/ACA sRNA-guided machinery

Up to now, Ψ formation in tRNAs was found to be catalysed by stand-alone enzymes. By computational analysis of archaeal genomes we detected putative H/ACA sRNAs, in four Sulfolobales species and in Aeropyrum pernix, that might guide Ψ35 formation in pre-tRNA^Tyr(GUA). This modification is achieved by Pus7p in eukarya. The validity of the computational predictions was verified by in vitro reconstitution of H/ACA sRNPs using the identified Sulfolobus solfataricus H/ACA sRNA. Comparison of Pus7-like enzymes encoded by archaeal genomes revealed amino acid substitutions in motifs IIIa and II in Sulfolobales and A. pernix Pus7-like enzymes. These conserved RNA:Ψ-synthase- motifs are essential for catalysis. As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13. We showed that the presence of an A residue 3′ to the target U residue is required for P. abyssi aPus7 activity, and that this is not the case for the reconstituted S. solfataricus H/ACA sRNP. In agreement with the possible formation of Ψ35 in tRNA^Tyr(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA^Tyr(GUA) abolished Ψ35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA^Tyr did not affect Ψ35 formation in this RNA when using an S. solfataricus extract.     

Biochemical characterization and homology modeling of a purine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus: Insights into mechanisms of protein stabilization

We report the biochemical and structural characterization of the purine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus (SsIAG-NH). SsIAG-NH is a homodimer of 70 kDa specific for adenosine, guanosine and inosine. SsIAG-NH is highly thermophilic and is characterized by extreme thermodynamic stability (T_m, 107 °C), kinetic stability and remarkable resistance to guanidinium chloride-induced unfolding. A disulfide bond that, on the basis of SDS-PAGE is positioned intersubunits, plays an important role in thermal stability. SsIAG-NH shares 43% sequence identity with the homologous pyrimidine-specific nucleoside hydrolase from S. solfataricus (SsCU-NH). The comparative sequence alignment of SsIAG-NH, SsCU-NH, purine non-specific nucleoside hydrolase from Crithidia fasciculata and purine-specific nucleoside hydrolase from Trypanosoma vivax shows that, only few changes in the base pocket are responsible for different substrate specificity of two S. solfataricus enzymes. The structure of SsIAG-NH predicted by homology modeling allows us to infer the role of specific residues in substrate specificity and thermostability.     

Mutation and reversion frequencies of different Sulfolobus species and strains

We have determined the apparent and actual spontaneous mutation frequencies and rates for different species and strains of the thermoacidophilic crenarchaeote Sulfolobus. The proportion of mutations caused by insertion sequences has also been analyzed. Mutation frequencies for S. islandicus (0.08–0.6 mutations per cell division and 10⁷ cells) were below those determined for S. solfataricus and comparable to or lower than those for S. acidocaldarius. The proportion of insertion sequence mutations for the S. islandicus strains REN1H1 (9 out of 230) and HVE10/4 (0 out of 24) was found to be considerably lower than in S. solfataricus P1 and P2 and also low in comparison to other S. islandicus strains. Mutants defective in either the pyrEF genes or the lacS gene have been isolated. Their growth phenotype on selective and non-selective medium was examined and the inactivating mutations in either of the genes were determined. In addition the reversion frequencies for these mutants were measured and found to be in the range of <0.6–1.5 mutations per cell division and 10⁸ cells. However, when being subjected to electroporation as a transformation procedure, increased reversion was observed.     

Insights into a Viral Lytic Pathway from an Archaeal Virus-Host System

Archaeal host cells infected by Sulfolobus turreted icosahedral virus (STIV) and Sulfolobus islandicus rod-shaped virus 2 (SIRV2) produce unusual pyramid-like structures on the cell surface prior to virus-induced cell lysis. This viral lysis process is distinct from known viral lysis processes associated with bacterial or eukaryal viruses. The STIV protein C92 and the SIRV2 protein 98 are the only viral proteins required for the formation of the pyramid lysis structures of STIV and SIRV2, respectively. Since SIRV2 and STIV have fundamentally different morphotypes and genome sequences, it is surprising that they share this lysis system. In this study, we have constructed a collection of C92/P98 chimeric proteins and tested their abilities, both in the context of virus replication and alone, to form pyramid lysis structures in S. solfataricus. The results of this study illustrate that these proteins are functionally homologous when expressed as individual chimeric proteins but not when expressed in the context of complete STIV infection.     

The Exopolyphosphatase Gene from Sulfolobus solfataricus: Characterization of the First Gene Found To Be Involved in Polyphosphate Metabolism in Archaea

Inorganic polyphosphate (polyP) polymers are widely distributed in all kinds of organisms. Although the presence of polyP in members of the domain Archaea has been described, at present nothing is known about the enzymology of polyP metabolism or the genes involved in this domain. We have cloned, sequenced, and overexpressed an exopolyphosphatase (PPX) gene (ppx) from thermophilic Sulfolobus solfataricus. The gene codes for a functional PPX and possesses an open reading frame for 417 amino acids (calculated mass, 47.9 kDa). The purified recombinant PPX was highly active, degrading long-chain polyP (700 to 800 residues) in vitro at 50 to 60°C. The putative PPXs present in known archaeal genomes showed the highest similarity to yeast PPXs. In contrast, informatic analysis revealed that the deduced amino acid sequence of S. solfataricus PPX showed the highest similarity (25 to 45%) to sequences of members of the bacterial PPXs, possessing all of their conserved motifs. To our knowledge, this is the first report of an enzyme characterized to be involved in polyP metabolism in members of the Archaea.     

A thermostable phosphotriesterase from the archaeon Sulfolobus solfataricus: cloning, overexpression and properties

A new gene from the hyperthermophilic archaeon Sulfolobus solfataricus MT4, coding for a putative protein reported to show sequence identity with the phosphotriesterase-related protein family (PHP), was cloned by means of the polymerase chain reaction from the S. solfataricus genomic DNA. In order to analyse the biochemical properties of the protein an overexpression system in Escherichia coli was established. The recombinant protein, expressed in soluble form at 5 mg/l of E. coli culture, was purified to homogeneity and characterized. In contrast with its mesophilic E. coli counterpart that was devoid of any tested activity, the S. solfataricus enzyme was demonstrated to have a low paraoxonase activity. This activity was dependent from metal cations with Co²⁺, Mg²⁺ and Ni²⁺ being the most effective and was thermophilic and thermostable. The enzyme was inactivated with EDTA and o-phenantroline. A reported inhibitor for Pseudomonas putida phosphotriesterase (PTE) had no effect on the S. solfataricus paraoxonase. The importance of a stable paraoxonase for detoxification of chemical warfare agents and agricultural pesticides will be discussed.     

The S2 Gene of Equine Infectious Anemia Virus Is Dispensable for Viral Replication In Vitro

Equine infectious anemia virus (EIAV) contains the simplest genome among lentiviruses in that it encodes only three putative regulatory genes (S1, S2, S3) in addition to the canonical gag, pol, and env genes, presumably reflecting its limited tropism to cells of monocyte/macrophage lineage. Tat and Rev functions have been assigned to S1 and S3, respectively, but the specific function for the S2 gene has yet to be determined. Thus, the function of S2 in virus replication in vitro was investigated by using an infectious molecular viral clone, EIAV_UK. Various EIAV_UK mutants lacking S2 were constructed, and their replication kinetics were examined in several equine cell culture systems, including the natural in vivo target equine macrophage cells. The EIAV S2 mutants showed replication kinetics similar to those of the parental virus in all of the tested primary and transformed equine cell cultures, without any detectable reversion of mutant genomes. The EIAV_UK mutants also showed replication kinetics similar to those of the parental virus in an equine blood monocyte differentiation-maturation system. These results demonstrate for the first time that the EIAV S2 gene is not essential and does not appear to affect virus infection and replication properties in target cells in vitro.     

Sequence of the 16S rRNA gene from the thermoacidophilic archaebacteriumSulfolobus solfataricus and its evolutionary implications

Summary The sequence of the small-subunit rRNA from the thermoacidophilic archaebacteriumSulfolobus solfataricus has been determined and compared with its counterparts from halophilic and methanogenic archaebacteria, eukaryotes, and eubacteria. TheS. solfataricus sequence is specifically related to those of the other archaebacteria, to the exclusion of the eukaryotic and eubacterial sequences, when examined either by evolutionary distance matrix analyses or by the criterion of minimum change (maximum parsimony). The archaebacterial 16S rRNA sequences all conform to a common secondary structure, with theS. solfataricus structure containing a higher proportion of canonical base pairs and fewer helical irregularities than the rRNAs from the mesophilic archaebacteria.S. solfataricus is unusual in that its 16S rRNA-23S rRNA intergenic spacer lacks a tRNA gene.     

Peptidyl-tRNA hydrolase from Sulfolobus solfataricus

An enzyme capable of liberating functional tRNA^Lys from Escherichia coli diacetyl-lysyl-tRNA^Lys was purified from the archae Sulfolobus solfataricus. Contrasting with the specificity of peptidyl- tRNA hydrolase (PTH) from E.coli, the S.solfataricus enzyme readily accepts E.coli formyl-methionyl-tRNA^fMet as a substrate. N-terminal sequencing of this enzyme identifies a gene that has homologs in the whole archaeal kingdom. Involvement of this gene (SS00175) in the recycling of peptidyl-tRNA is supported by its capacity to complement an E.coli strain lacking PTH activity. The archaeal gene, the product of which appears markedly different from bacterial PTHs, also has homologs in all the available eukaryal genomes. Since most of the eukaryotes already display a bacterial-like PTH gene, this observation suggests the occurrence in many eukaryotes of two distinct PTH activities, either of a bacterial or of an archaeal type. Indeed, the bacterial- and archaeal-like genes encoding the two full-length PTHs of Saccharomyces cerevisiae, YHR189w and YBL057c, respectively, can each rescue the growth of an E.coli strain lacking endogeneous PTH. In vitro assays confirm that the two enzymes ensure the recycling of tRNA^Lys from diacetyl-lysyl-tRNA^Lys. Finally, the growth of yeast cells in which either YHR189w or YBL057c has been disrupted was compared under various culture conditions. Evidence is presented that YHR189w, the gene encoding a bacterial-like PTH, should be involved in mitochondrial function.     

Specificities and pH profiles of adenine and hypoxanthine–guanine–xanthine phosphoribosyltransferases (nucleotide synthases) of the thermoacidophile archaeon Sulfolobus solfataricus

Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine phosphoribosyltransferase (PRTase), the protein product turned out to be a PRTase highly specific for adenine and we suggest that the reading frame should be renamed apT. The other reading frame SSO2424 (gpT-2) proved to be a true 6-oxopurine PRTase active with hypoxanthine, xanthine and guanine as substrates, and we suggest that the gene should be renamed gpT. Both enzymes exhibited unusual profiles of activity versus pH. The adenine PRTase showed the highest activity at pH 7.5–8.5, but had a distinct peak of activity also at pH 4.5. The 6-oxo PRTase showed maximal activity with hypoxanthine and guanine around pH 4.5, while maximal activity with xanthine was observed at pH 7.5. We discuss likely reasons why SSO2341 in S. solfataricus and similar open reading frames in other Crenarchaeota could not be identified as genes encoding APRTase.