Specific-pathogen-free pigs were inoculated with one of two hepatitis E viruses (HEV) (one recovered from a pig and the other from a human) to study the relative pathogenesis of the two viruses in swine. Fifty-four pigs were randomly assigned to three groups. Seventeen pigs in group 1 served as uninoculated controls, 18 pigs in group 2 were intravenously inoculated with the swine HEV recovered from a pig in the United States, and 19 pigs in group 3 were intravenously inoculated with the US-2 strain of human HEV recovered from a hepatitis patient in the United States. Two to four pigs from each group were necropsied at 3, 7, 14, 20, 27, or 55 days postinoculation (DPI). Evidence of clinical disease or elevation of liver enzymes or bilirubin was not found in pigs from any of the three groups. Enlarged hepatic and mesenteric lymph nodes were observed in both HEV-inoculated groups. Multifocal lymphoplasmacytic hepatitis was observed in 9 of 17, 15 of 18, and 16 of 19 pigs in groups 1 to 3, respectively. Focal hepatocellular necrosis was observed in 5 of 17, 10 of 18, and 13 of 19 pigs in groups 1 to 3, respectively. Hepatitis lesions were very mild in group 1 pigs, mild to moderate in group 2 pigs, and moderate to severe in group 3 pigs. Hepatic inflammation and hepatocellular necrosis peaked in severity at 20 DPI and were still moderately severe at 55 DPI in the group inoculated with human HEV. Hepatitis lesions were absent or nearly resolved by 55 DPI in the swine-HEV-inoculated pigs. All HEVinoculated pigs seroconverted to anti-HEV immunoglobulin G. HEV RNA was detected by reverse transcriptase PCR in feces, liver tissue, and bile of pigs in both HEV-inoculated groups from 3 to 27 DPI. Based on evaluation of microscopic lesions, the US-2 strain of human HEV induced more severe and persistent hepatic lesions in pigs than did swine HEV. Pig livers or cells from the livers of HEV-infected pigs may represent a risk for transmission of HEV from pigs to human xenograft recipients. Since HEV was shed in the feces of infected pigs, exposure to feces from infected pigs represents a risk for transmission of HEV, and pigs should be considered a reservoir for HEV.Hepatitis E virus (HEV) is the leading cause of enterically transmitted non-A, non-B hepatitis in people in many developing countries (21,28,30). Transmission is thought to be primarily by the fecal-oral route, and waterborne epidemics are characteristic of hepatitis E (1, 28, 30). Clinical disease due to HEV infection is rarely diagnosed in industrialized countries, and most cases of HEV infection in industrialized countries occur in people who have traveled to regions where the disease is endemic (10,21,28,30). Clinical cases occur predominantly in developing countries in Asia, Africa, and Mexico (1, 2, 28, 30). However, sporadic cases of acute hepatitis E in people in the United States and other industrialized countries have recently been reported (7,8,11,18,20,22,31,32,42). Hepatitis E generally affects young adults and usually is not fatal, although mortality r...

Rhodaneses catalyze the transfer of the sulfane sulfur from thiosulfate or thiosulfonates to thiophilic acceptors such as cyanide and dithiols. In this work, we define for the first time the gene, and hence the amino acid sequence, of a 12-kDa rhodanese from Escherichia coli. Well-characterized rhodaneses are comprised of two structurally similar ca. 15-kDa domains. Hence, it is thought that duplication of an ancestral rhodanese gene gave rise to the genes that encode the two-domain rhodaneses. The glpE gene, a member of the sn-glycerol 3-phosphate (glp) regulon of E. coli, encodes the 12-kDa rhodanese. As for other characterized rhodaneses, kinetic analysis revealed that catalysis by purified GlpE occurs by way of an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. The K m s for SSO 3 2؊ and CN ؊ were 78 and 17 mM, respectively. The apparent molecular mass of GlpE under nondenaturing conditions was 22.5 kDa, indicating that GlpE functions as a dimer. GlpE exhibited a k cat of 230 s ؊1 . Thioredoxin 1 from E. coli, a small multifunctional dithiol protein, served as a sulfur acceptor substrate for GlpE with an apparent K m of 34 M when thiosulfate was near its K m , suggesting that thioredoxin 1 or related dithiol proteins could be physiological substrates for sulfurtransferases. The overall degree of amino acid sequence identity between GlpE and the active-site domain of mammalian rhodaneses is limited (ϳ17%). This work is significant because it begins to reveal the variation in amino acid sequences present in the sulfurtransferases. GlpE is the first among the 41 proteins in COG0607 (rhodanese-related sulfurtransferases) of the database Clusters of Orthologous Groups of proteins (http://www.ncbi.nlm.nih.gov/COG/) for which sulfurtransferase activity has been confirmed.Genes of known function belonging to the glp regulon of Escherichia coli encode proteins that are responsible for the metabolism of sn-glycerol 3-phosphate (glycerol-P) and its precursors, glycerol and glycerophosphodiesters (38). The genes comprising this regulon belong to five operons. Transcription of all but the glpEGR operon is negatively regulated by the glp repressor GlpR, a member of the DeoR family of transcriptional regulators (34,65,(69)(70)(71)(72). Operon glpACB, encoding the subunits of the anaerobic glycerol-P dehydrogenase, is located near min 51 of the E. coli genome (38). Divergently transcribed from glpACB is glpTQ. The genes glpT and glpQ encode glycerol-P permease and periplasmic glycerophosphodiesterase, respectively (38). The glpFKX operon, at min 89, encodes glycerol diffusion facilitator, glycerol kinase, and a fructose 1,6-bisphophatase (38; J. L. Donahue, J. L. Bownas, W. G. Niehaus, Jr., and T. J. Larson, unpublished data). The genes glpE and glpG, together with the gene encoding the transcriptional repressor, glpR, form a complex operon at min 77 that is divergently transcribed from glpD (71). The gene glpD encodes the aerobic glycerol-P dehydrogenase (38).Prior to...

Sulfolobus solfataricus contains a membrane-associated protein kinase activity that displays a strong preference for threonine as the phospho-acceptor amino acid residue. When a partially purified detergent extract of the membrane fraction from the archaeon S. solfataricus that had been enriched for this activity was incubated with [␥-32 P]ATP, radiolabeled phosphate was incorporated into roughly a dozen polypeptides, several of which contained phosphothreonine. One of the phosphothreonine-containing proteins was identified by mass peptide profiling as the product of open reading frame [ORF] sso0469. Inspection of the DNA-derived amino acid sequence of the predicted protein product of ORF sso0469 revealed the presence of sequence characteristics faintly reminiscent of the "eukaryotic" protein kinase superfamily. ORF sso0469 therefore was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein formed insoluble aggregates that could be dispersed using urea or detergents. The solubilized polypeptide phosphorylated several exogenous proteins in vitro, including casein, myelin basic protein, and bovine serum albumin. Mutagenic alteration of amino acids predicted to be essential for catalytic activity abolished or severely reduced catalytic activity. Phosphorylation of exogenous substrates took place on serine and, occasionally, threonine. This new archaeal protein kinase displayed no catalytic activity when GTP was substituted for ATP as the phospho-donor substrate, while Mn 2؉ was the preferred cofactor.The versatility of covalent phosphorylation-dephosphorylation as a mechanism for regulating protein function and transducing extracellular signals has been compellingly demonstrated in numerous studies encompassing a broad spectrum of eucaryal and bacterial organisms (reviewed in references 5, 15, 19, 22, 23, and 28). However, while protein phosphorylation has been detected in several members of the third phylogenetic domain, the Archaea (24,41,50,51,53,54,56,57), we know relatively little concerning the chemical nature, enzymatic catalysts, and physiological roles of archaeal protein phosphorylation-dephosphorylation events (reviewed in reference 27). Only a few archaeal proteins have been implicated as the targets of protein phosphorylation to date. They include a CheY homolog in Halobacterium salinarium (45), a methyltransferase-activating protein from Methanosarcina barkeri (11), an aminopeptidase from Sulfolobus solfataricus (9), and a glycogen synthase from Sulfolobus acidocaldarius (7). In addition, the N-terminal sequences of three phosphotyrosine-containing proteins from Thermococcus kodakaraensis KOD1 have been determined, although the full sequences of these phosphoproteins have yet to be identified (24 (26,27,32,42,49). However, in only a few instances have the inferences of these in silico analyses been translated into the direct, empirical identification and characterization of defined gene products displaying the predicted functional capabilities. Included among ...

When soluble extracts of the extreme acidothermophilic archaeon Sulfolobus solfataricus were incubated with [␥-32 P]ATP, several proteins were radiolabeled. One of the more prominent of these, which migrated with a mass of ϳ46 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was purified by column chromatography and SDS-PAGE and subjected to amino acid sequence analysis via both the Edman technique and mass spectroscopy. The best match to the partial sequence obtained was the potential polypeptide product of open reading frame sso0417, whose DNA-derived amino acid sequence displayed many features reminiscent of the 2,3-diphosphoglycerate-independent phosphoglycerate (PGA) mutases [iPGMs]. Open reading frame sso0417 was therefore cloned, and its protein product was expressed in Escherichia coli. Assays of its catalytic capabilities revealed that the protein was a moderately effective PGA mutase that also exhibited low levels of phosphohydrolase activity. PGA mutase activity was dependent upon the presence of divalent metal ions such as Co 2؉ Interest in the members of the Archaea has grown steadily over the last two decades, stimulated to a large degree by their extremophilic lifestyles and distinct phylogenetic status. Within archaeal proteomes resides a wealth of information concerning unique metabolic pathways for extracting energy from sulfur, producing methane from CO 2 , etc., as well as biochemical and biophysical mechanisms for sustaining life processes under conditions of temperature, pH, and salinity hostile to "conventional" organisms (reviewed in references 1, 12, 23, and 37). Archaeal genomes combine unique, bacterial, and eucaryal features in a complex puzzle whose deconvolution will provide important insights into the nature and order of the evolutionary process (reviewed in references 13, 14, and 17). However, our current library of information concerning fundamental metabolic, sensory, regulatory, and other processes in the Archaea remains lacking in both breadth and depth.In a previous study, we observed that when extracts of the hyperthermophilic archaeon Sulfolobus solfataricus were incubated with [␥-32 P]ATP, numerous polypeptides incorporated radiolabeled phosphate (41). One of the more visually prominent of these was a phosphoserine-containing polypeptide with a mass of ϳ46 kDa. Using the recently released genome sequence of S. solfataricus P2 (38), we have been able to link this phosphoprotein to the open reading frame (ORF) that encodes it. We describe here the identification of the ϳ46-kDa phosphoprotein as an enzyme with phosphoglycerate (PGA) mutase activity that is encoded by ORF sso0417. MATERIALS AND METHODS Materials. [␥-32 P]ATP, [␣-32 P]ATP, and [␥-32 P]GTP were purchased from NEN Research Products (Boston, Mass.). Matrix Gel Blue A and Matrix Gel Blue B were purchased from Amicon Corp. (Danvers, Mass.). Hydroxyapatite was from Bio-Rad (Richmond, Calif.). Enolase was obtained from Sigma (St. Louis, Mo.). Oligonucleotide primers were from Life Technol...