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Membrane Association and Kinase-Like Motifs of the RamC Protein of Streptomyces coelicolor. Michael E. Hudson, 2002.The protein RamC is required for the production of the spore-forming cells called aerial hyphae by the filamentous bacterium Streptomyces coelicolor. We showed that RamC, which contains several weakly predicted membrane-spanning sequences, is located exclusively in the S . coelicolor membrane . By constructing site-directed mutants in the cloned ramC gene and complementing a ramC null mutant, we showed that protein kinase-like sequence motifs in the amino-terminal half of the protein are required for function in vivo. These data suggest that RamC is a membrane-associated receptor kinase . Novel spoIIE Mutation That Causes Uncompartmentalized  F Activation in Bacillus subtilis.David W. Hilbert, 2003.During sporulation, Bacillus subtilis undergoes an asymmetric division that results in two cells with different fates, the larger mother cell and the smaller forespore . The protein phosphatase SpoIIE, which is required for activation of the forespore-specific transcription factor F, is also required for optimal efficiency and timing of asymmetric division . We performed a genetic screen for spoIIE mutants that were impaired in sporulation but not
F activity and isolated a strain with the mutation spoIIEV697A . The mutant exhibited a 10- to 40-fold reduction in sporulation and a sixfold reduction in asymmetric division compared to the parent . Transcription of the
F-dependent spoIIQ promoter was increased more than 10-fold and was no longer confined to the forespore . The excessive
F activity persisted even when asymmetric division was prevented . Disruption of spoIIGB did not restore asymmetric division to the spoIIEV697A mutant, indicating that the deficiency is not a consequence of predivisional activation of the mother cell-specific transcription factor
E . Deletion of the gene encoding
F (spoIIAC) restored asymmetric division; however, a mutation that dramatically reduced the number of promoters responsive to
F, spoIIAC561 (spoIIACV233 M), failed to do so . This result suggests that the block is due to expression of one of the small subset of
F-dependent genes expressed in this background or to unregulated interaction of
F with some other factor . Our results indicate that regulation of SpoIIE plays a critical role in coupling asymmetric division to
F activation in order to ensure proper spatial and temporal expression of forespore-specific genes .
Adaptation of Saccharomyces cerevisiae to the Herbicide 2,4-Dichlorophenoxyacetic Acid, Mediated by Msn2p- and Msn4p-Regulated Genes: Important Role of SPI1. T. Simões, 2003.The possible roles of 13 Msn2p- and Msn4p-regulated genes in the adaptation of Saccharomyces cerevisiae to the herbicide 2,4-D-dichlorophenoxyacetic acid (2,4-D) were examined . Single deletion of genes involved in defense against oxidizing agents (CTT1, GRX1, and GRX2/TTR1) or encoding chaperones of the HSP70 family (SSA1, SSA4, and SSE2) showed a slight effect . A more significant role was observed for the heat shock genes HSP78, HSP26, HSP104, HSP12, and HSP42, most of which encode molecular chaperones . However, the SPI1 gene, encoding a member of the glycosylphosphatidylinositol-anchored cell wall protein family, emerged as the major determinant of 2,4-D resistance . SPI1 expression reduced the loss of viability of an unadapted yeast population suddenly exposed to the herbicide, allowing earlier growth resumption . Significantly, yeast adaptation to 2,4-D involves the rapid and transient Msn2p- and Msn4p-mediated activation (fivefold) of SPI1 transcription . SPI1 mRNA levels were reduced to values slightly above those in unstressed cells when the adapted population started duplication in the presence of 2,4-D . Since SPI1 deletion leads to the higher ß-1,3-glucanase sensitivity of 2,4-D-stressed cells, it was hypothesized that adaptation may involve an Spi1p-mediated increase in the diffusional restriction of the liposoluble acid form of the herbicide across the cell envelope . Such a cell response would avoid a futile cycle due to acid reentry into the cell counteracting the active export of the anionic form, presumably through an inducible plasma membrane transporter(s) . Consistent with this concept, the concentration of 14C-labeled 2,4-D in 2,4-D-energized adapted  spi1 mutant cells and the consequent intracellular acidification are higher than in wild-type cells .
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