Genome sequence of Halobacterium species NRC-1.

We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodium-proton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.

Purification and characterization of a membrane-associated ATPase from Natronococcus occultus, a haloalkaliphilic archaeon.

Isolated membranes of the extreme haloalkaliphilic archaeon Natronococcus occultus were able to hydrolyze ATP via an ATPase, which required the presence of Mg(2+), high concentrations of NaCl, and a pH value of 9. The native molecular mass of the purified ATPase was 130 kDa and was composed of 74- and 61-kDa subunits. Enzyme activity was specific for the hydrolysis of ATP with slight activity towards GTP, CTP, and ITP. The enzyme required NaCl for maximal activity but Na(2)SO(4) and (NH(4))(2)SO(4) could substitute. The enzyme showed no activity if Na(2)SO(3) or sodium citrate was substituted for NaCl. The ATPase from N. occultus was inhibited by NBD-Cl, NaN(3), and ouabain, and was sensitive to nitrate, vanadate, DCCD, and bafilomycin A(1). It was not inhibited by NEM in contrast to other previously characterized halophile ATPases. The ATPase had a K(M) of 0.5 mM and appeared to be non-competitively inhibited by NaN(3) with a K(I) of 3.1 mM.

2-Sulfotrehalose, a novel osmolyte in haloalkaliphilic archaea.

A novel 1-->1 alpha-linked glucose disaccharide with sulfate at C-2 of one of the glucose moieties, 1-(2-O-sulfo-alpha-D-glucopyranosyl)-alpha-D-glycopyranose, was found to be the major organic solute accumulated by a Natronococcus sp. and several Natronobacterium species. The concentration of this novel disaccharide, termed sulfotrehalose, increased with increasing concentrations of external NaCl, behavior consistent with its identity as an osmolyte. A variety of noncharged disaccharides (trehalose, sucrose, cellobiose, and maltose) were added to the growth medium to see if they could suppress synthesis and accumulation of sulfotrehalose. Sucrose was the most effective in suppressing biosynthesis and accumulation of sulfotrehalose, with levels as low as 0.1 mM being able to significantly replace the novel charged osmolyte. Other common osmolytes (glycine betaine, glutamate, and proline) were not accumulated or used for osmotic balance in place of the sulfotrehalose by the halophilic archaeons.

A minimal medium for growth of Pasteurella multocida.

We developed a minimal medium supporting the growth of both toxigenic and nontoxigenic strains of Pasteurella multocida to optical densities of > 0.5 (600 nm). P. multocida P1059 (ATCC 15742), one of a number of strains which can cause fowl cholera, was used as the model strain in this study. The medium was composed of 17 ingredients including cysteine, glutamic acid, leucine, methionine, inorganic salts, nicotinamide, pantothenate, thiamine, and an energy source. Leucine was not required for growth but was stimulatory, and thiamine could be replaced by adenine. An additional 46 strains of P. multocida were tested, and 40 out of 46 (87%) strains grew as well as strain P1059 through a minimum of 10 serial transfers. P. multocida toxin (PMT) was produced when cells of a known toxigenic strain (P4261) were cultivated in the minimal medium. No growth of Pasteurella Haemolytica or Pasteurella trehalosi strains was observed in this minimal medium.

Purification of recombinant Shiga-like toxin type I A1 fragment from Escherichia coli.

Shiga-like toxin I A1 (Slt-IA1) is a RNA N-glycosidase which depurinates a specific adenosine of 28 S eukaryotic rRNA thus inhibiting protein synthesis and ultimately leading to cell death. We have overexpressed this protein in Escherichia coli using a high copy number plasmid and purified the enzyme to homogeneity using a three-step process. Slt-IA1 is released from the periplasm of cells using polymyxin B sulfate, precipitated with ammonium sulfate, and adsorbed to a Matrex Gel Green A dye-ligand agarose column. The enzyme is eluted from the Green A agarose as a single peak with 0.32 M NaCl. Slt-IA1 was purified approximately 1979-fold and routinely gave yields greater than 100%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular weight of 28,000. An isoelectric point of 5.1 was determined using analytical isoelectric focusing gels. In an in vitro protein synthesis inhibition assay, 0.02 pM of purified Slt-IA1 inhibited protein synthesis by 50%.

Evidence that the A2 fragment of Shiga-like toxin type I is required for holotoxin integrity.

Escherichia coli Shiga-like toxin type I (SLT-I) is a potent cytotoxin consisting of an enzymatically active A subunit and a pentameric B subunit that mediates toxin binding to susceptible eukaryotic cells. Evidence that the carboxy-terminal 38 amino acids of the A subunit are involved in holotoxin 1A:5B association is presented. We compared the ability of purified recombinant SLT-I B subunit (Slt-IB) to combine in vitro with purified recombinant SLT-I A subunit (Slt-IA; full-length subunit A includes amino acids 1 to 293) and its ability to combine with purified recombinant SLT-I A1 subunit (Slt-IA1; truncated subunit A includes amino acids 1 to 255). Each mixture was analyzed for biological and physical evidence of toxin assembly. Although Slt-IA successfully combined with Slt-IB to form a molecular species similar to holotoxin that was detectable by nondenaturing polyacrylamide gel electrophoresis and immunoblotting and yielded a molecule which was cytotoxic to cultured Vero cells, Slt-IA1 did not have this ability. Slt-IA1 was 36-fold more active than Slt-IA in an in vitro protein synthesis inhibition assay. These findings suggest that the Slt-IA2 fragment is crucial for formation of SLT holotoxin and stabilizes the interaction between the A and B subunits.

Characterization of the metal centers of the Ni/Fe-S component of the carbon-monoxide dehydrogenase enzyme complex from Methanosarcina thermophila.

Methanosarcina thermophila contains a multienzyme complex called the carbon-monoxide dehydrogenase complex, which has been resolved into a nickel/iron-sulfur and a corrinoid/iron-sulfur component. This complex plays a central role in acetoclastic methanogenesis. The Ni/Fe-S component catalyzes CO oxidation and has been proposed to be involved in cleavage of acetyl-CoA into its methyl, carbonyl, and CoA moieties. In the work reported here, three metal centers in the Ni/Fe-S component were characterized by electron paramagnetic resonance (EPR) spectroscopy and spectroelectrochemistry and pre-steady state kinetics. Center A contains nickel and iron and forms an EPR active adduct with CO, which is called the NiFeC species. The EPR spectrum of the NiFeC species has g values of 2.059, 2.051, and 2.029 and is observable at temperatures as high as 150 K. This signal had previously been observed only in the carbon-monoxide dehydrogenase complex of M. thermophila and the acetyl-CoA synthase from acetate-producing bacteria. Incubation of the CO-reduced Ni/Fe-S component with acetyl-CoA resulted in an increase in intensity of the NiFeC signal, which supports a role for the component in the cleavage of acetyl-CoA. Generation of the NiFeC EPR signal occurs with a rate constant of 0.4 s-1, a result that demonstrates the kinetic competence of this species in the acetyl-CoA cleavage reaction but rules it out as the site of oxidation of CO to CO2. Center B is likely to be a [4Fe-4S]2+/1+ center with g values of 2.04, 1.93, and 1.89 (gav = 1.95) and a standard reduction potential (E'0) of -444 mV. At potentials less than -500 mV, another EPR signal develops that appears to originate from another state of Center B. Center C is a fast relaxing center with g values of 2.02, 1.88, and 1.71 (gav = 1.87) and an E'0 of -154 mV.

Characterization of the metal centers of the corrinoid/iron-sulfur component of the CO dehydrogenase enzyme complex from Methanosarcina thermophila by EPR spectroscopy and spectroelectrochemistry.

The multienzyme carbon monoxide dehydrogenase complex from Methanosarcina thermophila contains at least two protein components: a CO-oxidizing nickel/iron-sulfur (Ni/Fe-S) component and a cobalt-containing corrinoid/iron-sulfur component (Co/Fe-S). The CO dehydrogenase complex has been shown to synthesize acetyl-CoA from CoA, CH3I, and CO as well as to cleave acetyl-CoA into its methyl, carbonyl, and CoA components as the first step in the catabolism of acetyl-CoA to methane and CO2. Presumed to serve as an acceptor of the methyl group of acetyl-CoA en route to methane, the Co/Fe-S component contains iron, acid-labile sulfur, and a corrinoid cofactor (factor III) that is the site of methylation. Using EPR spectroscopy and spectroelectrochemistry, we characterized the cobalt and Fe-S centers of the Co/Fe-S component. The redox and EPR properties of the metal centers in the isolated Co/Fe-S component are similar to those of the Co/Fe-S component in the CO dehydrogenase enzyme complex, a result that indicates that any protein-protein interaction between components in the complex has little influence on the properties of the metal centers. The corrinoid is maintained in the base-off state with a formal equilibrium reduction potential (E'o) at pH 7.8 of -486 mV for the Co2+/1+ couple that facilitates reduction of the Co2+ state by approximately 12 kcal/mol relative to base-on cobamides. The Co/Fe-S component also contains a [4Fe-4S]2+/1+ cluster with an E'o at pH 7.8 of -502 mV, which is nearly isopotential with the Co2+/1+ couple of the cobamide.

Reductive dechlorination of trichloroethylene by the CO-reduced CO dehydrogenase enzyme complex from Methanosarcina thermophila.

Trichloroethylene (TCE) was reductively dechlorinated to cis-dichloroethylene, trans-dichloroethylene, 1,1-dichloroethylene, vinyl chloride, and ethylene by the CO-reduced CO dehydrogenase enzyme complex from Methanosarcina thermophila; the apparent Km and Vmax values were 1.7 +/- 0.3 mM TCE and 26.2 +/- 1.7 mol TCE dechlorinated/min/mmol factor III. Factor III also catalysed the dechlorination of TCE when in the presence of titanium(III) citrate; the apparent Km and Vmax values were 1.2 +/- 0.3 mM TCE and 34.9 +/- 3.6 mol TCE dechlorinated/min/mmol factor III. The enzyme complex was resolved into the two-subunit nickel/iron-sulfur (Ni/Fe-S) component and the two-subunit factor III-containing corrinoid/iron-sulfur (Co/Fe-S) component. The Ni/Fe-S component was unable to dechlorinate TCE in the presence of CO; however, reconstitution with the Co/Fe-S component yielded the same dechlorinated products as with the CO dehydrogenase enzyme complex.

Purification and properties of methyl coenzyme M methylreductase from acetate-grown Methanosarcina thermophila.

Methyl coenzyme M methylreductase from acetate-grown Methanosarcina thermophila TM-1 was purified 16-fold from a cell extract to apparent homogeneity as determined by native polyacrylamide gel electrophoresis. Ninety-four percent of the methylreductase activity was recovered in the soluble fraction of cell extracts. The estimated native molecular weight of the enzyme was between 132,000 (standard deviation [SD], 1,200) and 141,000 (SD, 1,200). Denaturing polyacrylamide gel electrophoresis revealed three protein bands corresponding to molecular weights of 69,000 (SD, 1,200), 42,000 (SD, 1,200), and 33,000 (SD, 1,200) and indicated a subunit configuration of alpha 1 beta 1 gamma 1. As isolated, the enzyme was inactive but could be reductively reactivated with titanium (III) citrate or reduced ferredoxin. ATP stimulated enzyme reactivation and was postulated to be involved in a conformational change of the inactive enzyme from an unready state to a ready state that could be reductively reactivated. The temperature and pH optima for enzyme activity were 60 degrees C and between 6.5 and 7.0, respectively. The active enzyme contained 1 mol of coenzyme F430 per mol of enzyme (Mr, 144,000). The Kms for 2-(methylthio)ethane-sulfonate and 7-mercaptoheptanoylthreonine phosphate were 3.3 mM and 59 microM, respectively.

Protein content and enzyme activities in methanol- and acetate-grown Methanosarcina thermophila.

The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila TM-1 was examined by two-dimensional polyacrylamide gel electrophoresis. More than 100 mutually exclusive spots were present in acetate- and methanol-grown cells. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the carbon monoxide dehydrogenase complex were identified in acetate-grown cells. Activities of formylmethanofuran dehydrogenase, formylmethanofuran:tetrahydromethanopterin formyltransferase, 5,10-methenyltetrahydromethanopterin cyclohydrolase, methylene tetrahydromethanopterin:coenzyme F420 oxidoreductase, formate dehydrogenase, and carbonic anhydrase were examined in acetate- and methanol-grown Methanosarcina thermophila. Levels of formyltransferase in either acetate- or methanol-grown Methanosarcina thermophila were approximately half the levels detected in H2-CO2-grown Methanobacterium thermoautotrophicum. All other enzyme activities were significantly lower in acetate- and methanol-grown Methanosarcina thermophila.

Oxacillin-induced inhibition of protein and RNA synthesis in a tolerant Staphylococcus aureus isolate.

A clinical isolate of Staphylococcus aureus was found to be tolerant (MBC much greater than MIC) to a number of beta-lactam antibiotics, including oxacillin. Biophotometric analysis showed that a number of concentrations of oxacillin were capable of stimulating rapid cellular lysis in this organism, but the extent of lysis was antibiotic concentration dependent and limited. Cell cultures treated with an antibiotic concentration yielding the maximum rate and extent of lysis were analyzed for protein and RNA synthesis by pulse-labeling techniques. RNA synthesis was initially stimulated and then severely inhibited. Protein synthesis was not inhibited initially; however, the increase in the rate of synthesis expected as the result of logarithmic growth was not observed. Instead, the antibiotic-treated culture maintained for approximately 50 min the rate of protein synthesis ongoing at the time of antibiotic addition. The rate of protein synthesis declined thereafter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein samples taken 1 and 3 h after antibiotic addition showed that the shutdown of protein synthesis was not coordinate but rather was suggestive of the operation of a stress regulon perhaps similar to those responsible for heat shock, SOS, and oxidation stress.