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Evolution of the Helicobacter pylori Vacuolating Cytotoxin in a Human Stomach. Francisco Aviles-Jimenez, 2004.We describe two subclones of Helicobacter pylori, isolated contemporaneously from a human stomach, which differ markedly in the vacuolating cytotoxin gene, vacA, but whose near identity in sequences outside this locus implies a very recent common origin . The differences are consistent with homologous recombination with DNA from another strain and result in a changed vacA midregion and, importantly, in changed toxicity . Cloning of S-Adenosyl-L-Methionine:C-24-  -Sterol-Methyltransferase (ERG6) from Leishmania donovani and Characterization of mRNAs in Wild-Type and Amphotericin B-Resistant Promastigotes.Mohammad Pourshafie, 2004.The 24-alkylated sterols have been shown previously to be absent in membranes of amphotericin B (AmB)-resistant Leishmania donovani promastigotes, suggesting that the S- adenosyl-L-methionine:C-24- -sterol-methyltransferase (SCMT or ERG6) was not functional or not expressed in AmB-resistant (AmB-R) parasites . From an L . donovani wild-type clone, we cloned two cDNAs with an identical open reading frame encoding a putative SCMT, the enzyme responsible for a first sterol methylation at the C-24 position . The two cDNAs differed by their 3'-untranslated region (3'-UTR) and 5'-UTR sequences . One transcript (A) had a normal structure with a spliced leader and was highly expressed in normal cells but absent in AmB-R cells . The other (B), which did not possess the spliced leader sequence, was weakly expressed in normal cells but strongly expressed in AmB-R cells . As a functional test, ERG6 null mutant Saccharomyces cerevisiae yeasts were transformed using the pYES2.1 TOPO TA expression vector containing the candidate SCMT1/ERG6 coding sequence cloned from L . donovani . The transformed yeasts exhibited C-24 alkylated sterol expression, mainly ergosterol, within their membranes, proving that the isolated cDNA encodes on a SCMT responsible for sterol methylation . In AmB-R L . donovani promastigotes, the absence of the normal transcript (A) and the expression of an abnormal species (B) devoid of a spliced leader could explain the absence of sterol methylation in these cells . Further studies using a homologous system will allow us to draw conclusions about the relationship between SCMT expression and AmB resistance in Leishmania .
Escherichia coli Cells with Increased Levels of DnaA and Deficient in Recombinational Repair Have Decreased Viability. Aline V. Grigorian, 2003.The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, ß clamp of DNA polymerase III holoenzyme, and RecF . When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs . Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression . To test this, we examined the effects of increasing the intracellular concentrations of DnaA, ß clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability . The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression . A shorter C period was not observed with increased expression of the ß clamp; in fact, many chromosomes did not complete replication in runout experiments . Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability . When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 µm) with extremely low viability and, in runout experiments, most chromosomes had not completed replication . The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways . The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional  100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells .
Enumeration and Characterization of Acidophilic Microorganisms Isolated from a Pilot Plant Stirred-Tank Bioleaching Operation. Naoko Okibe, 2003.Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45°C . Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus-like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma-like isolate) . The relative numbers of these prokaryotes changed in the three reactors sampled, and the Ferroplasma isolate became increasingly dominant as mineral oxidation progressed, eventually accounting for >99% of plate isolates in the third of three in-line reactors . The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g., oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined . More detailed studies were carried out with the Leptospirillum and Ferroplasma isolates . The data presented here represent the first quantitative study of the microorganisms in a metal leaching situation and confirm that mixed cultures of iron- and sulfur-oxidizing prokaryotic acidophiles catalyze the accelerated dissolution of sulfidic minerals in industrial tank bioleaching operations . The results show that indigenous acidophilic microbial populations change as mineral dissolution becomes more extensive .
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