Naked Amoebas and Bacteria in an Oil-Impacted Salt Marsh Community.

Populations of soil amoebas were monitored in two salt marshes in Staten Island, NY for 2 years. One site, Gulfport Reach on the Arthur Kill, has been highly impacted by numerous oil spills. In particular, in 1990 a massive no. 2 fuel oil spill from a ruptured pipe flooded the area; its sediments had total petroleum hydrocarbon (TPH) concentrations in the range 800-46,000 ppm. A reference site 11 km away, on the Atlantic coast, had low TPH levels. Amoeba population densities were in general higher in the impacted sediments. In laboratory microcosm experiments, sediment samples from unimpacted sites were treated with added fresh (unweathered) hydrocarbons (no. 2 fuel oil) and cultured; these also yielded higher amoeba numbers than untreated controls. Four distinct amoeba morphotypes were monitored. Changes in population levels of total amoebas were correlated in the two sites, particularly for morphotype 2 (r = 0.83). The ratios of total amoebas to total bacterial numbers were also correlated (r = 0.85) between the sites. This suggests the amoebas may function as generalists, and that their trophic relation to bacterial prey is not much affected by the presence of petroleum hydrocarbons, but rather may reflect regional parameters such as ambient temperature or other physical factors.

Metabolic effects of a methylthioadenosine phosphorylase substrate analog on African trypanosomes.

The effects of 5'-deoxy-5'-(hydroxyethylthio)adenosine (HETA), a trypanocidal analog of 5'-deoxy-5'-(methylthio)adenosine (MTA), on polyamine synthesis and S-adenosylmethionine (AdoMet) metabolism were examined in bloodstream forms of Trypanosoma brucei brucei. HETA was cleaved by trypanosome MTA phosphorylase at the same rate as the natural substrate, MTA, in a phosphate-dependent reaction. Fluorine substitution at the 2-position of the purine ring increased activity by approximately 50%, whereas substitution with an amino group reduced activity to about one-third of the control. HETA was accumulated by trypanosomes with internal concentrations of 100-250 microM and >800 microM after a 15-min incubation with 1 and 10 microM, respectively. Trypanosomes preincubated with HETA metabolized it at a rate of 21.9 nmol/hr/mg protein. Preincubation of cells with HETA at 1 or 10 microM inhibited spermidine synthesis from [3H]ornithine by 22-37%, and increased the cytosolic levels of AdoMet by 2- to 5-fold and that of MTA by up to 8-fold. S-Adenosylhomocysteine (AdoHcy) levels also increased 1.5- to 7-fold in treated cells, whereas decarboxylated AdoMet decreased 65%. Preincubation of trypanosomes with HETA for 4 hr also reduced the incorporation of [35S]methionine in trichloroacetic acid-precipitable material by 50-60%, and reduced the methyl group incorporation into protein from [U-14C]methionine by 65-70%. Thus, HETA interferes with a series of biochemical events involving the participation of AdoMet and methionine in polyamine synthesis, protein synthesis, and transmethylation reactions.

Hydrogenosomal succinate thiokinase in Tritrichomonas foetus and Trichomonas vaginalis.

Succinate thiokinase displays a diversity of nucleotide specificity and molecular size throughout Nature. Eukaryotes and Gram-positive bacteria possess distinct 'small' (dimeric) thiokinase enzymes which are specific for adenine (ADP) or guanine (GDP) nucleotides, whereas Gram-negative bacteria contain a single 'large' (tetrameric) enzyme which utilizes both nucleotides. Succinate thiokinase activities, both ADP- and GDP-dependent, were shown to be hydrogenosomal in Tritrichomonas foetus and Trichomonas vaginalis. Surprisingly, the 'small' enzyme was found in T. foetus whereas T. vaginalis contained a 'large' enzyme.

Molecular analysis of the hydrogenosomal ferredoxin of the anaerobic protist Trichomonas vaginalis.

We have determined the primary structure of the [2Fe-2S]ferredoxin of the anaerobic protist Trichomonas vaginalis. This protein, situated in the hydrogenosome, is composed of 93 amino acids. A comparison of T. vaginalis ferredoxin with greater than 80 other ferredoxins shows the closest similarity to [2Fe-2S]putidaredoxin of the aerobic bacterium Pseudomonas putida and a lesser one to mitochondrial [2Fe-2S]ferredoxins of vertebrates. This similarity is reflected in the overall primary structure and in the spacing of cysteine residues coordinating the iron-sulfur center. The primary structure, but not the environment of the iron-sulfur center, also shows similarity with [2Fe-2S]ferredoxins of photosynthetic organisms and halobacteria. We have cloned and analyzed the T. vaginalis ferredoxin gene. The gene is present in a single copy and devoid of introns. It gives rise to a transcript with unusually short 5' and 3' untranslated regions of 16 and 18 nucleotides, respectively. DNA sequence analysis of the gene predicts an additional 8 amino acids at the amino terminus which are absent from the purified protein. This amino-terminal region of the protein is characterized by properties typical of mitochondrial presequences.

In vitro susceptibility of Trichomonas vaginalis to metronidazole and treatment outcome in vaginal trichomoniasis.

We have identified Trichomonas vaginalis strains resistant in vitro to metronidazole, especially under aerobic conditions. Since little is known about the relationship of treatment outcome to metronidazole susceptibility of T. vaginalis, we studied the aerobic and anaerobic susceptibility to metronidazole of 310 clinical isolates of T. vaginalis. Of 199 patients with known outcomes after metronidazole treatment for vaginal trichomoniasis, the geometric mean minimal lethal concentration (MLC) under aerobic conditions for trichomonads associated with cases cured by a single 2-g dose was 24.1 micrograms/ml (n = 146), while that of treatment-resistant isolates (n = 53) was 195.5 micrograms/ml. The corresponding mean anaerobic MLC values were 1.6 and 5.05 micrograms/ml, respectively. The average aerobic:anaerobic MLC ratio was about twofold higher for the resistant isolates. Treatment resistance was more frequent at aerobic MLC values of greater than 25 micrograms/ml or anaerobic values of greater than 1.6 micrograms/ml. Although there was overlap of the metronidazole susceptibility distribution of susceptible and resistant isolates, significant resistance to treatment was common when isolates of T. vaginalis had aerobic MLC values of greater than 100 micrograms/ml or anaerobic MLC values of greater than 3.1 micrograms/ml.

In vitro drug susceptibility and doses of metronidazole required for cure in cases of refractory vaginal trichomoniasis.

There are currently no laboratory or clinical guidelines for the identification and treatment of disease caused by metronidazole-resistant strains of Trichomonas vaginalis. Fifty-three isolates of T. vaginalis from cases of refractory vaginitis in the United States (26 states) and Canada were tested for aerobic and anaerobic metronidazole susceptibility, and after various dosages of metronidazole, the therapeutic outcomes were evaluated for 31 of these cases. The mean aerobic metronidazole susceptibility of these isolates was 195.5 micrograms/ml (range, 12.5-greater than 1,000), which was about eightfold higher than that seen in isolates that were not resistant to metronidazole. The mean anaerobic susceptibility was 5 micrograms/ml (range, 1.6-25), which was about threefold higher than that of isolates from nonresistant strains. The average aerobic-to-anaerobic ratio of metronidazole susceptibility in the highly resistant isolates was more than 3.5-fold greater than that seen in the nonresistant isolates. White women accounted for 88% of the resistant infections. Of 31 cases that were re-treated and monitored, the highest average dose that failed to achieve a cure was 2.1 g of metronidazole/day given over an eight-day period; 27 (87%) of 31 cases were ultimately cured with an average dosage of 2.6 g of metronidazole/day given over a mean period of nine days. Resistance to treatment with metronidazole varied from mild to severe, and the resistance was occasionally more severe than the susceptibility values indicate.

Effect of culture medium iron content on the biochemical composition and metabolism of Trichomonas vaginalis.

Trichomonas vaginalis grown in iron-enriched medium contained increased concentrations of iron-sulfur proteins, including ferredoxin and pyruvate-ferredoxin oxidoreductase. The increases in hydrogenosomal constituents correlated with increased in vivo hydrogenosomal metabolism.

Reduction of nitroimidazole derivatives by hydrogenosomal extracts of Trichomonas vaginalis.

The reduction rate of nitroimidazole derivatives by pyruvate:ferredoxin oxidoreductase activity in ferredoxin depleted hydrogenosomal extracts of Trichomonas vaginalis depended on the one-electron midpoint potential (E7(1)) of 15 compounds out of the 16 tested. The results showed a linear correlation with a positive slope between the logarithm of the rate and the E7(1) in the range from -564 to -260 mV. Addition of T. vaginalis ferredoxin stimulated the reduction. The additional rate (stimulated rate minus basal rate) was proportional to the concentration of ferredoxin and independent of the E7(1) of the compounds. The compound with the most positive E7(1) (-243 mV) was, however, reduced more slowly than expected. These findings indicate that reduction in the presence of ferredoxin is the sum of two processes, i.e. electron transfer directly from pyruvate:ferredoxin oxidoreductase and from reduced ferredoxin generated by pyruvate:ferredoxin oxidoreductase activity. The relative role of ferredoxin in reductive activation of nitroimidazole derivatives is greater for compounds with more negative E7(1) values. This observation correlates with the high selectivity of the more negative 5-nitroimidazoles against anaerobic prokaryotic and eukaryotic microorganisms in which ferredoxin plays an important metabolic role.

Bacillus Calmette-Guérin-activated murine macrophages kill syngeneic melanoma cells under strict anaerobic conditions.

We have studied the spontaneous killing of B5(59) melanoma cells by Bacillus Calmette-Guérin (BCG)-elicited macrophages under strictly anaerobic conditions to investigate the role of oxygen in macrophage-mediated cytotoxicity. The number of melanoma cells capable of forming colonies after aerobic or anaerobic incubation with BCG-macrophages was used as the index of cytotoxicity. The BCG-macrophages killed melanoma cells regardless of the amount of oxygen present. The killing observed was proportional to the ratio of effector cells added; a ratio of 25:1 effector to target cells was required to achieve nearly 90% cytotoxicity both aerobically and anaerobically. This cytotoxicity was not dependent on a diffusible macrophage product nor on alteration of the medium by macrophages, since tumor cells incubated in the same culture medium, but not in contact with a mixed population of tumor cells and macrophages, were not killed. These results also indicated that macrophage-mediated cytotoxicity was dependent on macrophage-tumor cell contact. The mechanism responsible for the oxygen-independent cytotoxicity is unknown at present.

Metabolism and metronidazole uptake in Trichomonas vaginalis isolates with different metronidazole susceptibilities.

Three Trichomonas vaginalis isolates with low in vivo susceptibilities to metronidazole (95% curative dose, greater than 3 X 100 mg kg-1 in subcutaneous infections in mice) were compared with strain ATCC 30001 and with four isolates exhibiting high in vivo susceptibilities (95% curative dose, less than 3 X 15 mg kg-1). Activity of pyruvate:ferredoxin oxidoreductase, anaerobic fermentation, and anaerobic intracellular accumulation of [14C]metronidazole label showed no significant isolate-dependent differences which could be correlated with drug susceptibility. The results suggest that processes providing electrons for metronidazole activation are not defective in the resistant strains. Aerobiosis, known to inhibit the antimicrobial action of metronidazole, inhibited accumulation of label more strongly in resistant isolates than in susceptible ones. No differences were detected, however, between resistant and susceptible isolates in respiration, aerobic fermentation, and the specific activity of NADH and NADPH oxidases, the main terminal oxidases of T. vaginalis. These findings suggest that the production of electrons is not diminished under aerobic conditions. The inhibitory effect of aerobic conditions on metronidazole activation, possibly due to competition for the electrons, is markedly enhanced in the resistant isolates compared to the susceptible ones. The mechanism of this effect, however, remains unknown.

Hydrogenosomal ferredoxin of the anaerobic protozoon, Tritrichomonas foetus.

A low molecular weight iron-sulfur protein has been purified from Tritrichomonas foetus by deoxycholate extraction of whole cells, ion exchange chromatography, and gel filtration. The purified protein was essentially homogeneous as judged by isoelectric focusing, polyacrylamide gel electrophoresis, and gel filtration. A pI of 4.3 was observed. The molecular weight of the protein was estimated to be 12,000. Chemical and spectral analysis showed the protein to have a [2Fe-2S] cluster. The absorbance spectrum of the oxidized protein showed maxima at 280, 340, 458 and shoulders at 410 and 550 nm. The maximum observed A458/A280 ratio was 0.82 and the absorbance of the oxidized protein at 458 nm was 8,000 M-1 X cm-1. The low temperature EPR spectrum of the protein reduced with dithionite revealed axial symmetry with features at g values of g = 1.94 and g = 2.02. The oxidized protein gave no EPR signal in the g = 1.8 to 2.2 range. Cell fractionation studies indicated the localization of this protein in the hydrogenosome. The protein was able to function as an electron transport component in the reduction of metronidazole (a 5-nitroimidazole derivative) by pyruvate:ferredoxin oxidoreductase and hydrogenase from T. foetus and also from Trichomonas vaginalis and Clostridium pasteurianum as well as in the reduction of cytochrome c by plant NADPH:ferredoxin oxidoreductase. This protein has the characteristics of a ferredoxin and is likely to be a physiological electron carrier in hydrogenosomal pyruvate oxidation.

Novel microbial and chemical components of a specific black-band region in the cockroach hindgut.

An area of the hindgut of the cockroach, Eublaberus posticus, is characterized by its black color. This area is the site of accumulation of metal sulfides in the lumen next to the gut wall. In addition to the normal hindgut flora, two unusual procaryotic organisms are seen by both scanning and transmission electron microscopy only in this area of the hindgut. They are (i) a large rod (1.2 by 6 micrometers) with a tuft of polar flagella, many inclusion bodies, and a distinctive complex wall and (ii) an apparently flexible rod with a helically ridged wall. In addition, phagelike particles are described which are apparently infecting gram-positive bacteria attached to the gut wall in the black band area.

Reduction of nicotinamide adenine dinucleotide by pyruvate:lipoate oxidoreductase in anaerobic, dark-grown Rhodospirillum rubrum mutant C.

Cell extracts from fermentatively grown Rhodospirillum rubrum reduced about 80 nmol of nicotinamide adenine dinucleotide (NAD) per mg of protein per min under anaerobic conditions with sodium pyruvate. The reaction was specific for pyruvate and NAD; NAD phosphate was not reduced. Results indicated that pyruvate-linked NAD reduction occurred via pyruvate:lipoate oxidoreductase. The reaction required catalytic amounts of both coenzyme A and thiamine pyrophosphate. Addition of sodium arsenite inhibited enzyme activity by 90%. Pyruvate:lipoate oxidoreductase was the only system detected in anaerobic, dark-grown R. rubrum cell extracts which operated to produce reduced NAD. The low activity of the enzyme system suggested that it was not quantitatively important in ATP formation.

Fermentative metabolism of pyruvate by Rhodospirillum rubrum after anaerobic growth in darkness.

Rhodospirillum rubrum grew anaerobically in darkness and fermented sodium pyruvate by a pyruvate formate-lyase reaction. During 30 min of anaerobic dark or light incubation with sodium pyrivate, crude extracts from fermentatively grown cells produced about 6 micronmol of acetylphosphate and formate per mg of protein in reactions performed at pH 8.3. Cell extracts also catalyzed the exchange of sodium [14C]formate into sodium pyruvate at an apparent pH optimum of 7.3 to 7.5, but only about 2.5 micronmol of acetylphosphate was produced at this lower pH value. R. rubrum may also form pyruvate:ferredoxin oxidoreductase activity, as evidenced by low bicarbonate exchange activity. However, its participation in pyruvate metabolism in anaerobic dark-grown cells was not understood. During anaerobic, dark growth with pyruvate, formate was an intermediate in H2 and CO2 gas evolution. In contrast with H2 production by a light-dependent H2-nitrogenase system in photosynthetically grown cells, H2 formation in fermenting R. rubrum occurred through a carbon monoxide-sensitive formic hydrogenlyase reaction not influenced by light.