Chemiluminescence of Acanthamoeba castellanii.

We have investigated ferrocytochrome c-induced proton ejection from reconstituted cytochrome c oxidase-containing vesicles using careful control of the number of enzyme turnovers. Ferrocytochrome c caused the appearance of protons at the vesicle exterior, and this could be abolished by using a protonophore. In addition, its decay was dependent on the permeability of the vesicle membranes to protons and the number of turnovers of the oxidase. These observations indicate that the ejection of protons was the result of genuine translocation. The possibility of this translocation occurring via a Mitchellian loop as a result of the presence of a reduced hydrogen carrier contaminating the enzyme was considered and excluded. Proton-translocating activity in this reconstituted system depended critically on the ratio of enzyme to lipid used in the reconstitution process and we propose a rationale to account for this. We conclude that our data provide strong support for the proposal that cytochrome c oxidase acts as a proton pump and that approx. 0.9 H+ is excluded per ferrocytochrome c molecule oxidized.     

The cytochromes of Acanthamoeba castellanii.

1. Low-temperature difference spectra of gradient-purified mitochondria of Acanthamoeba castellanii reveal the presence of cytochromes b-555, b-562 and c-549, with a-type cytochromes having a broad asymmetrical maximum at 602 nm; these components were also observed in specta of whole cells. 2. The a-type cytochromes are unusual in that they have split Soret absorption maxima (at 442 and 449 nm) and an uncharacteristic CO difference spectrum. 3. CO difference spectra of whole cells and 'microsomal' membranes show large amounts of cytochrome P-420 compared with cytochrome P-450. 4. Difference spectra in the presence of cyanide indicate the presence of an a-type cytochrome and two cyanide-reacting components, one of which may be cytochrome a3. 5. Whole-cell respiration in a N2/O2 (19:1) atmosphere was decreased by 50%, suggesting the presence of a low-affinity oxidase. This lowered respiration is inhibited by 50% by CO, and the inhibition is partially light-reversible; photochemical action spectra suggest that cytochrome a3 contributes to this release of inhibition. Other CO-reacting oxidases are also present. 6. The results are discussed with the view that cytochrome a3 is present in A. castellanii, but its identification in CO difference spectra is obscured by other component(s).     

Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor.

We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers. Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region. Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs). The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases. No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases. ORFtnp is located 5' of ORF3. ORF2 is incomplete and is located 3' of ORF3. Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S. lividans 66 TK24. S. lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs. The G+C content of nec1 suggests that it has moved horizontally from another genus. Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S. scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A. These data suggest that nec1 may have been mobilized into S. scabies through a transposition event mediated by ORFtnp.     

Phagocytosis and pinocytosis in Acanthamoeba castellanii.

Endocytotic activity of Acanthamoeba trophozoites attenuates once the cells enter stationary phase in liquid culture. Phagocytosis, monitored by the ingestion of polystyrene latex beads, essentially ceases and the uptake of [3H]inulin, known to be mediated by pinocytosis, is reduced by about half. The reduced pinocytotic activity of stationary-phase cells remains sensitive to respiratory inhibitors. Preincubation of stationary-phase cells in fresh growth medium for 1-5 h before the initiation of endocytosis has no effect on phagocytosis and only marginally increases pinocytosis. This impairment of ingestion, particularly of pinocytosis, may account for the reduced contractile vacuole activity known to characterize stationary-phase cells of this organism. The unequal responses of phagocytosis and pinocytosis to the onset of stationary-phase growth suggest that they are independent processes subject to different controls.     

Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.     

Identification of the gene encoding transcription factor NusG of Thermus thermophilus.

The nusG gene of Thermus thermophilus HB8 was cloned and sequenced. It is located 388 bp downstream from tufB, which is followed by the genes for ribosomal proteins L11 and L1. No equivalent to secE preceding nusG, as in Escherichia coli, could be detected. The nusG gene product was overproduced in E. coli. A rabbit antiserum raised against the purified recombinant NusG reacted exclusively with one protein band of T. thermophilus crude extracts in Western blot (immunoblot) analyses, and no cross-reaction of the antiserum with E. coli NusG was observed. Recombinant NusG and the reacting T. thermophilus wild-type protein had identical sizes on sodium dodecyl sulfate-polyacrylamide gels. T. thermophilus and E. coli NusG have 45% identical and 22.5% similar amino acids, and similarities between the two proteins are most pronounced in carboxy-terminal regions. The T. thermophilus nusG gene could not rescue a nusG-deficient E. coli mutant strain.