Candida albicans and Saccharomyces cerevisiae switch from a yeast to a filamentous form. In Saccharomyces, this switch is controlled by two regulatory proteins, Ste12p and Phd1p. Single-mutant strains, ste12/ste12 or phd1/phd1, are partially defective, whereas the ste12/ste12 phd1/phd1 double mutant is completely defective in filamentous growth and is noninvasive. The equivalent cph1/cph1 efg1/efg1 double mutant in Candida (Cph1p is the Ste12p homolog and Efg1p is the Phd1p homolog) is also defective in filamentous growth, unable to form hyphae or pseudohyphae in response to many stimuli, including serum or macrophages. This Candida cph1/cph1 efg1/efg1 double mutant, locked in the yeast form, is avirulent in a mouse model.

The contribution of seven known and nine predicted genes or operons associated with multidrug resistance to the susceptibility of Escherichia coli W3110 was assessed for 20 different classes of antimicrobial compounds that include antibiotics, antiseptics, detergents, and dyes. Strains were constructed with deletions for genes in the major facilitator superfamily, the resistance nodulation-cell division family, the small multidrug resistance family, the ATP-binding cassette family, and outer membrane factors. The agar dilution MICs of 35 compounds were determined for strains with deletions for multidrug resistance (MDR) pumps. Deletions in acrAB or tolC resulted in increased susceptibilities to the majority of compounds tested. The remaining MDR pump gene deletions resulted in increased susceptibilities to far fewer compounds. The results identify which MDR pumps contribute to intrinsic resistance under the conditions tested and supply practical information useful for designing sensitive assay strains for cell-based screening of antibacterial compounds.

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To better understand the molecular basis of posaconazole (POS) resistance in Aspergillus fumigatus, resistant laboratory isolates were selected. Spontaneous mutants arose at a frequency of 1 in 10 8 and fell into two susceptibility groups, moderately resistant and highly resistant. Azole resistance in A. fumigatus was previously associated with decreased drug accumulation. We therefore analyzed the mutants for changes in levels of transcripts of genes encoding efflux pumps (mdr1 and mdr2) and/or alterations in accumulation of [ 14 C]POS. No changes in either pump expression or drug accumulation were detected. Similarly, there was no change in expression of cyp51A or cyp51B, which encode the presumed target site for POS, cytochrome P450 14␣-demethylase. DNA sequencing revealed that each resistant isolate carried a single point mutation in residue 54 of cyp51A. Mutations at the same locus were identified in three clinical A. fumigatus isolates exhibiting reduced POS susceptibility but not in susceptible clinical strains. To verify that these mutations were responsible for the resistance phenotype, we introduced them into the chromosome of a POS-susceptible A. fumigatus strain under the control of the glyceraldehyde phosphate dehydrogenase promoter. The transformants exhibited reductions in susceptibility to POS comparable to those exhibited by the original mutants, confirming that point mutations in the cyp51A gene in A. fumigatus can confer reduced susceptibility to POS.

When Drosophila cells are shifted from 25°C to 37C, protein synthesis is rapidly redirected from the complex pattern characteristic of normal growth to the simple pattern of heat shock proteins (HSPs). On return to 250C, synthesis ofnormal proteins is gradually reactivated and that of HSPs is repressed.In quantifying many different recovery experiments, we found that preexisting mRNAs always behaved as a cohort, with messages for different proteins returning to translation at the same rate. Heat shock mRNAs (HS mRNAs), on the other hand, never behaved as a cohort. Their repression was asynchronous, with translation of hsp7O always the first and translation of hsp82 always the last to be repressed. Although recovery times varied enormously (depending on the severity of the heat treatment), repression of hsp7O was always correlated with restoration of normal synthesis, suggesting a link between the two events. hsp7O repression was not simply due to competition with reactivated 250C mRNAs. A general decline in the translation efficiency of hsp7O mRNA was not observed. Instead, an increasing number of messages were translationally inactivated, while those remaining in the translational pool retained full ribosome loading. Unlike inactive 25°C mRNAs, which are stable during heat shock, inactive HSP mRNAs are degraded during recovery.When eukaryotic cells are exposed to temperatures 5-15°C above their optimum for growth, they respond by inducing a small number of evolutionarily conserved proteins, the socalled heat shock proteins (HSPs). The response of Drosophila cells is particularly rapid and dramatic [see Ashburner and Bonner (1) for review]. Within minutes ofa shift from 25°C to 37°C, transcription is blocked at most previously active sites and is vigorously initiated at HS gene loci (2-4). At the same time, a mechanism oftranslational control is activated that clears preexisting messages from polysomes and allows heat shock messages to be translated with maximum efficiency as soon as they appear (5,(8)(9)(10)(11)(12)(13)(14). This response provides the most dramatic example of selective translational regulation known in any eukaryotic cell.The commitment to heat shock synthesis is not a terminal event; when cells are returned to 250C, normal patterns oftranscription and translation are gradually restored (6-8). Thus, an equally profound change in the specificity of translation occurs during recovery. We examined several parameters of recovery after subjecting cells to a variety of different heat treatments. The data indicate that the shift away from heat shock-synthesis is accomplished by a different mechanism than the shift into 'heat shock synthesis.MATERIALS cells are exposed to elevated temperatures, they shift entirely to heat shock synthesis within 20 min. This contrasts with a much more gradual restoration of normal protein synthesis when cells are returned to 25°C. Fig. 1 displays a time course of recovery following a 30-min treatment at 36.5°C, the optimum temperature for HS induction with our ...