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Molecular and Phenotypic Analysis of CaVRG4, Encoding an Essential Golgi Apparatus GDP-Mannose Transporter. Akiko Nishikawa, 2002.Cell surface mannan is implicated in almost every aspect of pathogenicity of Candida albicans . In Saccharomyces cerevisiae, the Vrg4 protein acts as a master regulator of mannan synthesis through its role in substrate provision . The substrate for mannosylation of proteins and lipids in the Golgi apparatus is GDP-mannose, whose lumenal transport is catalyzed by Vrg4p . This nucleotide sugar is synthesized in the cytoplasm by pathways that are highly conserved in all eukaryotes, but its lumenal transport (and hence Golgi apparatus-specific mannosylation) is a fungus-specific process . To begin to study the role of Golgi mannosylation in C . albicans, we isolated the CaVRG4 gene and analyzed the effects of loss of its function . CaVRG4 encodes a functional homologue of the S . cerevisiae GDP-mannose transporter . CaVrg4p localized to punctate spots within the cytoplasm of C . albicans in a pattern reminiscent of localization of Vrg4p in the Golgi apparatus in S . cerevisiae . Like partial loss of ScVRG4 function, partial loss of CaVRG4 function resulted in mannosylation defects, which in turn led to a number of cell wall-associated phenotypes . While heterozygotes displayed no growth phenotypes, a hemizygous strain, containing a single copy of CaVRG4 under control of the methionine-repressible MET3 promoter, did not grow in the presence of methionine and cysteine, demonstrating that CaVRG4 is essential for viability . Mutant Candida vrg4 strains were defective in hyphal formation but exhibited a constitutive polarized mode of pseudohyphal growth . Because the VRG4 gene is essential for yeast viability but does not have a mammalian homologue, it is a particularly attractive target for development of antifungal therapies . Control of Anthrax Toxin Gene Expression by the Transition State Regulator abrB. Elke Saile, 2002.Bacillus anthracis produces the anthrax toxin proteins protective antigen (PA), lethal factor (LF), and edema factor (EF) in a growth phase-dependent manner when cultured in liquid medium . Expression of the toxin genes pagA, lef, and cya peaks in late log phase, and steady-state levels of the toxin proteins are highest during the transition into stationary phase . Here we show that an apparent transition state regulator negatively regulates toxin gene expression . We identified two orthologues of the B . subtilis transition state regulator abrB in the B . anthracis genome: one on the chromosome and one on the 182-kb virulence plasmid pXO1 . The orthologue located on the chromosome is predicted to encode a 94-amino-acid protein that is 85% identical to B . subtilis AbrB . The hypothetical protein encoded on pXO1 is 41% identical to B . subtilis AbrB but missing 27 amino acid residues from the amino terminus compared to the B . subtilis protein . Deletion of the pXO1-encoded abrB orthologue did not affect toxin gene expression under the conditions tested . However, a B . anthracis mutant in which the chromosomal abrB gene was deleted expressed pagA earlier and at a higher level than the parent strain . Expression of a transcriptional pagA-lacZ fusion in the abrB mutant was increased up to 20-fold during early exponential growth compared to the parent strain and peaked in mid-exponential rather than late exponential phase . In contrast to the strong effect of abrB on pagA expression, lef-lacZ and cya-lacZ expression during early-log-phase growth was increased only two- to threefold in the abrB null mutant . Western hybridization analysis showed increased PA, LF, and EF synthesis by the mutant . As is true in B . subtilis, the B . anthracis abrB gene is negatively regulated by spo0A . Our findings tie anthrax toxin gene expression to the complex network of postexponential phase adaptive responses that have been well studied in B . subtilis . Bioenergetic Properties of the Thermoalkaliphilic Bacillus sp . Strain TA2.A1. Karen Olsson, 2003.The thermoalkaliphilic Bacillus sp . strain TA2.A1 was able to grow in pH-controlled batch culture containing a nonfermentable growth substrate from pH 7.5 to 10.0 with no significant change in its specific growth rate, demonstrating that this bacterium is a facultative alkaliphile . Growth at pH 10.0 was sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that a proton motive force (  p) generated via aerobic respiration was an obligate requirement for growth of strain TA2.A1 . Strain TA2.A1 exhibited intracellular pH homeostasis as the external pH increased from 7.5 to 10.0; however, the maximum
pH generated over this pH range was only 1.1 units at an external pH of 9.5 . The membrane potential ( ) was maintained between -114 mV and -150 mV, and little significant change was observed over the pH range for growth . In contrast, the
p declined from -164 mV at pH 7.5 to approximately -78 mV at pH 10.0 . An inwardly directed sodium motive force (pNa+) of -100 mV at pH 10.0 indicated that cellular processes (i.e., solute transport) dependent on a sodium gradient would not be affected by the adverse
p . The phosphorylation potential of strain TA2.A1 was maintained between -300 mV and -418 mV, and the calculated H+/ATP stoichiometry of the ATP synthase increased from 2.0 at pH 7.5 to 5.7 at pH 10.0 . Based on these data, vigorous growth of strain TA2.A1 correlated well with the
pNa+, phosphorylation potential, and the ATP/ADP ratio, but not with
p . This communication represents the first report on the bioenergetics of an extremely thermoalkaliphilic aerobic bacterium .
In Situ Accessibility of Small-Subunit rRNA of Members of the Domains Bacteria, Archaea, and Eucarya to Cy3-Labeled Oligonucleotide Probes. Sebastian Behrens, 2003.Low accessibility of the rRNA is together with cell wall impermeability and low cellular ribosome content a frequent reason for failure of whole-cell fluorescence hybridization with fluorescently labeled oligonucleotide probes . In this study we compare accessibility data for the 16S rRNA of Escherichia coli (gamma Proteobacteria, Bacteria) with the phylogenetically distantly related organisms Pirellula sp . strain 1 (Planctomycetes, Bacteria) and Metallosphaera sedula (Crenarchaeota, Archaea) and the 18S rRNA accessibility of Saccharomyces cerevisiae (Eucarya) . For a total of 537 Cy3-labeled probes, the signal intensities of hybridized cells were quantified under standardized conditions by flow cytometry . The relative probe-conferred fluorescence intensities are shown on color-coded small-subunit rRNA secondary-structure models . For Pirellula sp., most of the probes belong to class II and III (72% of the whole data set), whereas most of the probes targeting sites on M . sedula were grouped into class V and VI (46% of the whole data set) . For E . coli, 45% of all probes of the data set belong to class III and IV . A consensus model for the accessibility of the small-subunit rRNA to oligonucleotide probes is proposed which uses 60 homolog target sites of the three prokaryotic 16S rRNA molecules . In general, open regions were localized around helices 13 and 14 including target positions 285 to 338, whereas helix 22 (positions 585 to 656) and the 3' half of helix 47 (positions 1320 to 1345) were generally inaccessible . Finally, the 16S rRNA consensus model was compared to data on the in situ accessibility of the 18S rRNA of S . cerevisiae .
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