Halobacterial adenosine triphosphatases and the adenosine triphosphatase from Halobacterium saccharovorum.

Membranes prepared from various members of the genus Halobacterium contained a Triton X-100 activated adenosine triphosphatase. The enzyme from Halobacterium saccharovorum was unstable in solutions of low ionic strength (< 3 M NaCl) and maximally active in the presence of 3.5 M NaCl. A variety of nucleotide triphosphates was hydrolyzed. MgADP, the product of ATP hydrolysis, was not hydrolyzed and was a competitive inhibitor with respect to MgATP. The enzyme from H. saccharovorum was composed of at least 2 and possibly 4 subunits. The 83-kDa and 60-kDa subunits represented about 90% of total protein. The 60-kDa subunit reacted with dicyclohexylcarbodiimide (DCCD) when inhibition was carried out in an acidic medium. The significance of the two minor components (28 kDa and 12 kDa is not established. The enzyme from H. saccharovorum, which differs from previously described halobacterial ATPases, possesses properties of an F1F0 as well as an E1E2 ATPase.

Studies of a halophilic NADH dehydrogenase. II. Kinetic properties of the enzyme in relation to salt activation.

1. An NADH dehydrogenase, obtained from an extremely halophilic bacterium, was activated by various salts when enzyme activity was measured as the observed velocity, whereas the maximum velocity was unaffected by either the salt concentration or the nature of the salt. 2. Two ion effects were observed; a quantitative cation effect, reflected in changes in the apparent Michaelis constant for 2,6-dichlorophenolindophenol, and a qualitative anion effect, reflected in the apparent Michaelis and dissociation constants for NADH. 3. The data suggest that cations act by neutralizing electrostatic charges surrounding the 2,6-dichlorophenolindophenol-binding site, whereas the anions affect the conformation of the enzyme by altering the accessibility of the NADH-binding site to the bulk solvent. 4. Thus, the apparent activation of this enzyme, obtained from an extremely halophilic bacterium, is a reflection of measuring enzyme activity at non-saturating substrate concentrations.

Amikacin pharmacokinetics in the therapy of childhood urinary tract infection.

Amikacin, a new aminoglycoside antibiotic with a spectrum similar to that of gentamicin, has been used mainly in adults. This report summarizes the first use of this drug in children with urinary tract infection. Organisms were eradicated in all cases and recurrent infection occurred in one half after one week. No evidence of ototoxicity or nephrotoxicity was found. Four children developed transient elevation of serum glutamic oxaloacetic transaminase. Serum level (17 mug/ml) of the drug at one hour and its urinary excretion in six hours (60% of the dose) was comparable to those of adults. This antibiotic is potentially valuable and has thus far not shown major toxicity when given for up to 11 days in patients with normal renal and liver functions.

Rapid diagnosis of tuberculosis in various biopsy and body fluid specimens by the AMPLICOR Mycobacterium tuberculosis polymerase chain reaction test.

STUDY OBJECTIVES: This study was undertaken to determine the usefulness of the AMPLICOR Mycobacterium tuberculosis (MTB) polymerase chain reaction (PCR) test (Roche Diagnostic Systems, Inc; Branchburg, NJ) in diagnosing TB in tissue and body fluid specimens other than respiratory secretions. DESIGN AND SETTING: Prospective analysis of clinical and laboratory data in patients with suspected TB at the four divisional hospitals of Catholic Medical Center, located in New York. PATIENTS AND MEASUREMENTS: A total of 1,090 tissue and body fluid specimens from 1,032 patients with suspected TB were subjected to acid-fast bacillus (AFB) smear, culture, and the AMPLICOR MTB PCR test. RESULTS: Of the 1,090 specimens, 32 grew M tuberculosis complex and 8 specimens grew isolates belonging to the Mycobacterium avium complex (MAC). The AMPLICOR MTB PCR test was positive for 24 of the 32 specimens that grew M tuberculosis. It was also positive for four additional specimens that were culture-negative for M tuberculosis or MAC. Two of these specimens were from patients with a previously recorded positive sputum culture for M tuberculosis. The AMPLICOR test was negative for all eight specimens that yielded MAC only. When AMPLICOR MTB PCR test results were compared with the confirmed clinical diagnosis of TB, the sensitivity, specificity, positive predictive value, and negative predictive value for the AMPLICOR MTB PCR test were 76.4%, 99.8%, 92.8%, and 99.2%, respectively. PCR results were available within 6.5 hours, compared with an average of 3 weeks for culture of M tuberculosis. CONCLUSIONS: These data establish the utility of the AMPLICOR MTB PCR test for the rapid detection of M tuberculosis in tissue and body fluid specimens other than respiratory secretions.

In vitro activity of cefoperazone/sulbactam and other antimicrobials against anaerobic bacteria.

Sulbactam inhibits the hydrolytic activity of several, clinically important beta-lactamases including those produced by anaerobic bacteria. This study was undertaken to determine the effect of sulbactam on the activity of cefoperazone against 250 anaerobic bacteria including 174 isolates belonging to the Bacteroides fragilis group and to compare the activity of cefoperazone/sulbactam with other antimicrobial agents. beta-lactamase activity was detected in 98% of the isolates of the Bacteroides fragilis group but not in the other species evaluated. Antagonistic activity between cefoperazone and sulbactam was not observed with any of the species. Forty-two percent of the isolates belonging to the B. fragilis group were resistant to cefoperazone. Ninety-four percent of these were converted to either the susceptible or moderately susceptible range upon the addition of sulbactam. Sixty-seven percent were susceptible to the combination cefoperazone/sulbactam and 27% were moderately susceptible. Overall, metronidazole and chloramphenicol were the most active antimicrobials. Significant differences in the antimicrobial susceptibility patterns of members of the B. fragilis group were observed. Sulbactam demonstrated some intrinsic activity against all of the species tested.

In vitro activity of ampicillin/sulbactam against enterococci determined by the time-kill method.

Time-kill studies demonstrated that at clinically achievable serum concentrations, ampicillin/sulbactam was equivalent in activity to ampicillin alone against non-beta-lactamase producing isolates of Enterococcus faecalis and Enterococcus faecium. Sulbactam possessed no antibacterial activity against these organisms. It is not yet known if the activity of ampicillin will be increased with the addition of sulbactam when tested against beta-lactamase-producing enterococci.

Application of the Roche Amplicor Mycobacterium tuberculosis (PCR) test to specimens other than respiratory secretions.

The ability of the Roche AMPLICOR Mycobacterium tuberculosis (MTB) test to detect M. tuberculosis in specimens other than respiratory secretions was evaluated. A total of 249 specimens from 219 patients were tested. Of these, 12 specimens grew isolates of the M. tuberculosis complex and four grew isolates of the M. avium complex. The AMPLICOR MTB test was positive for 10 of the 12 specimens which grew M. tuberculosis and for three specimens which were culture negative. Two of the latter specimens were from patients with a clinical diagnosis of tuberculosis and with multiple sputum specimens which grew M. tuberculosis. Four specimens grew M. avium complex isolates, and all yielded negative AMPLICOR MTB test results. The sensitivity, specificity, and positive and negative predictive values for the AMPLICOR MTB test were 85.7%, 99.5%, 92.3%, and 99.1%, respectively. Our data indicate that the AMPLICOR MTB test will permit the rapid detection of M. tuberculosis in specimens other than respiratory secretions.

ATP synthesis in Halobacterium saccharovorum: evidence that synthesis may be catalysed by an F0F1-ATP synthase.

Halobacterium saccharovorum synthesized ATP in response to a pH shift from 8 to 6.2. Synthesis was inhibited by carbonyl cyanide m-chloro-phenylhydrazone, dicyclohexylcarbodiimide, and azide. Nitrate, an inhibitor of the membrane-bound ATPase previously isolated from this organism, did not inhibit ATP synthesis. N-Ethymaleimide, which also inhibited this ATPase, stimulated the production of ATP. These observations suggested that H. saccharovorum synthesized and hydrolysed ATP using different enzymes and that the vacuolar-like ATPase activity previously described in H. saccharovorum was an ATPase whose function is yet to be identified.

Is the Paracoccus halodenitrificans ATPase a chimeric enzyme?

Membranes from Paracoccus halodenitrificans contain an ATPase that is most active in the absence of NaCl. The most unusual characteristic of the enzyme is its pattern of sensitivity to various inhibitors. Azide and rhodamine 6G, inhibitors of F1F0-ATPases, inhibit ATP hydrolysis as do bafilomycin A1, concanamycin A (folimycin), N-ethylmaleimide, and p-chloromercuriphenylsulfonate which are inhibitors of vacuolar ATPases. This indiscriminate sensitivity suggests that this ATPase may be a hybrid and that caution should be exercised when using inhibition as a diagnostic for distinguishing between F1F0-ATPases and vacuolar ATPases.

Trypsin digestion for determining orientation of ATPase in Halobacterium saccharovorum membrane vesicles.

Membranes prepared by low pressure disruption of cells exhibited no ATPase activity in the absence of Triton X-100, although 43% of the total menadione reductase activity was detected. Trypsin digestion reduced menadione reductase activity by 45% whereas ATPase activity was not affected. Disruption of the membrane fraction at higher pressure solubilized about 45% of the ATPase activity. The soluble activity was still enhanced by Triton X-100, suggesting that the detergent, besides disrupting membrane vesicles, also activated the ATPase. The discrepancy in localization of menadione reductase and ATPase activities raised questions regarding the reliability of using a single marker enzyme as an indicator of vesicle orientation.

The occurrence of denitrification in extremely halophilic bacteria.

The ability of Halobacterium vallismortis, Halobacterium mediterranei and Halobacterium marismortui (Ginzburg strain) to grow anaerobically and denitrify was determined. Each organism grew anaerobically only in the presence of nitrate. H. marismortui produced nitrite and dinitrogen from nitrate during exponential growth. However, as the culture entered stationary phase, dinitrogen production ceased and nitrous oxide was detected. H. vallismortis produced nitrous oxide and dinitrogen during exponential growth, with dinitrogen production ceasing at the onset of stationary phase. H. mediterranei produced dinitrogen during exponential growth and did not produce nitrous oxide. These results confirm the occurrence of denitrification in the halobacteria.

Denitrification by extremely halophilic bacteria.

Extremely halophilic bacteria were isolated from widely separated sites by anaerobic enrichment in the presence of nitrate. The anaerobic growth of several of these isolates was accompanied by the production of nitrate, nitrous oxide, and dinitrogen. These results are a direct confirmation of the existence of extremely halophilic denitrifying bacteria, and suggest that such bacteria may be common inhabitants of hypersaline environments.

Polar lipid composition of a new halobacterium.

Investigations of the polar lipid composition of a new aerobic, extremely halophilic aracheabacterium capable of nitrate reduction have shown that this organism contains two previously unknown phospholycolipids derived from diphytanyl glycerol diethers. Comparison of the lipid pattern from this new isolate with other known strains indicate that this organism is novel. On the basis of the unique polar lipid pattern it can be concluded that this organism represents a new taxon, at least at the species level.

The enzymes associated with denitrification.

NASA: The enzymes involved in the reduction of nitrogenous oxides are thought to be intermediates in denitrification processes. This review examines the roles of nitrate reductase, nitrite reductases, nitric oxide reductase, mechanisms of N-N bond formation, and nitrous oxide reductases.^ieng

A comparison of an ATPase from the archaebacterium Halobacterium saccharovorum with the F1 moiety from the Escherichia coli ATP synthase.

A purified ATPase associated with membranes from Halobacterium saccharovorum was compared with the F1 moiety from the Escherichia coli ATP synthase. The halobacterial enzyme was composed of two major (I and II) and two minor subunits (III and IV), whose molecular masses were 87 kDa, 60 kDa, 29 kDa and 20 kDa, respectively. The isoelectric points of these subunits ranged from 4.1 to 4.8, which in the case of the subunits I and II was consistent with the presence of an excess of acidic amino acids (20-22 mol/100 mol). Peptide mapping of subunits I and II denatured with sodium dodecyl sulfate showed no relationship between the primary structures of the individual halobacterial subunits or similarities to the subunits of the F1 ATPase from E. coli. Trypsin inactivation of the halobacterial ATPase was accompanied by the partial degradation of the major subunits. This observation, taken in conjunction with molecular masses of the subunits and the native enzyme, was consistent with the previously proposed stoichiometry of 2:2:1:1. These results suggest that H. saccharovorum, and possibly, halobacteria in general, possess an ATPase which is unlike the ubiquitous F0F1 ATP synthase.

Salt specificity of a reduced nicotinamide adenine dinucleotide oxidase prepared from a halophilic bacterium.

Extracts prepared from a halophilic bacterium contained a reduced nicotinamide adenine dinucleotide (NADH(2)) oxidase active at high solute concentrations. The cation requirement was nonspecific, since KCl, RbCl, and CsCl replaced NaCl with little or no loss of activity, and NH(4)Cl was only partially effective. Only LiCl failed to replace NaCl. No specific chloride requirement was observed although not all anions replaced chloride. Bromide, nitrate, and iodide were essentially ineffective, whereas acetate, formate, citrate, and sulfate proved suitable. The presence of sulfate affected the ability of a cation to satisfy the solute requirement. Sulfate enhanced the rate of NADH(2) oxidation when compared with the rate observed in the presence of chloride. Cations which were inactive as chlorides (LiCl and MgCl(2) at high concentrations) satisfied the cation requirement when added as sulfate salts. Although magnesium satisfied the cation requirement, a concentration effect, as well as an anion effect, was observed. In the presence of MgCl(2), little NADH(2) oxidation was observed at concentrations greater than 1 m. At lower concentrations, the rate of oxidation increased, reaching a maximal value at 0.1 m and remaining constant up to a concentration of 0.05 m MgCl(2). Magnesium acetate and MgSO(4) also replaced NaCl, and the maximal rate of oxidation occurred at 0.05 m with respect to magnesium. There was no change in the rate of oxidation at high magnesium acetate concentrations, whereas the rate of NADH(2) oxidation increased at higher concentrations of MgSO(4).