Transcriptional regulation by mitogen-activated protein (MAP) kinase signaling cascades is a major control mechanism for eukaryotic development. In budding yeast, Fus3 and Kss1 are two MAP kinases that control two distinct developmental programs-mating and invasive growth. We investigated whether signal-specific activation of mating and invasive growth involves regulation of the transcription factor Tec1 by Fus3 and Kss1. We present evidence that, during mating, Fus3 phosphorylates Tec1 to downregulate this invasive growth-specific transcription factor and its target genes. This function of Fus3 is essential for correct execution of the mating program and is not shared by Kss1. We find that Kss1 controls the activity of Tec1 mainly during invasive growth by control of TEC1 gene expression. Our study suggests that signaling specificity can arise from differential regulation of a single transcription factor by two MAP kinases with shared functions in distinct developmental programs.

Diploid strains of the budding yeast Saccharomyces cerevisiae change the pattern of cell division from bipolar to unipolar when switching growth from the unicellular yeast form (YF) to filamentous, pseudohyphal (PH) cells in response to nitrogen starvation. The functions of two transmembrane proteins, Bud8p and Bud9p, in regulating YF and PH cell polarity were investigated. Bud8p is highly concentrated at the distal pole of both YF and PH cells, where it directs initiation of cell division. Asymmetric localization of Bud8p is independent of the Rsr1p/Bud1p GTPase. rsr1/bud1 mutations are epistatic to bud8 mutations, placing Rsr1p/Bud1p downstream of Bud8p. In YF cells, Bud9p is also localized at the distal pole, yet deletion of BUD9 favours distal bud initiation. In PH cells, nutritional starvation for nitrogen efficiently prevents distal localization of Bud9p. Because Bud8p and Bud9p proteins associate in vivo, we propose Bud8p as a landmark for bud initiation at the distal cell pole, where Bud9p acts as inhibitor. In response to nitrogen starvation, asymmetric localization of Bud9p is averted, favouring Bud8p-mediated cell division at the distal pole.

In Saccharomyces cerevisiae, the transcription factors Tec1p and Ste12p are required for haploid invasive and diploid pseudohyphal growth. Tec1p and Ste12p have been postulated to regulate these developmental processes primarily by cooperative binding to filamentous and invasion-responsive elements (FREs), which are combined enhancer elements that consist of a Tec1p-binding site (TCS) and an Ste12p-binding site (PRE). They are present in the promoter regions of target genes, e.g., FLO11. Here, we show that Tec1p efficiently activates target gene expression and cellular development in the absence of Ste12p. We further demonstrate that TCS elements alone are sufficient to mediate Tec1p-driven gene expression by a mechanism termed TCS control that is operative even when Ste12p is absent. Mutational analysis of TEC1 revealed that TCS control, FLO11 expression, and haploid invasive growth require the C terminus of Tec1p. In contrast, the Ste12p-dependent FRE control mechanism is sufficiently executed by the N-terminal portion of Tec1p, which contains the TEA/ATTS DNA-binding domain. Our study suggests that regulation of haploid invasive and diploid pseudohyphal growth by Ste12p and Tec1p is not only executed by combinatorial control but involves additional control mechanisms in which Ste12p activates TEC1 expression via clustered PREs and where Tec1p regulates expression of target genes, e.g., FLO11, by TCS control.

Comamonas testosteroni ⌬Comamonas (formerly Pseudomonas) testosteroni is a soil microorganism able to grow at the expense of testosterone, progesterone, and various bile acids as unique sources of carbon and energy. Complete assimilation of these substrates is achieved through a complex metabolic pathway involving many enzymatic steps of oxidation responsible for the breakdown of the steroid nucleus (9,25,29). The introduction of double bonds into ring A at the early stage of this catabolic process is critical since it initiates the molecule cleavage. Upon induction with testosterone, C. testosteroni synthesizes three different dehydrogenases able to promote the desaturation of ring A of 3-ketosteroids: one (Fig. 1). All three enzymes have properties specific to flavoproteins, but only ⌬ 4 (5ß)DH was purified and characterized as being a flavin adenine nucleotide (FAD)-dependent enzyme (10,30).In a previous work, we isolated and characterized the gene encoding ⌬ 1 DH from C. testosteroni ATCC 17410 (37). A Tn5-insertion mutant (named 06) defective in testosterone utilization was found to express very low levels of both ⌬ 1 -and ⌬ 4 (5␣)DH activities while producing other quantifiable steroid-modifying enzymes at parental levels. These results strongly suggested that the genes coding for ⌬ 1 DH and ⌬ 4 (5␣)DH belong to the same transcription unit. In the present study, we demonstrate that the two genes are adjacent on the chromosome of C. testosteroni. The putative ⌬ 4 (5␣)DH peptide deduced from the gene sequence showed weak homology with the ⌬ 1 DH protein except in the N terminus, where a highly conserved FAD-binding site is present. These findings indicate that both dehydrogenases, though having closely related enzymatic activities, are probably not derived from a common ancestor. MATERIALS AND METHODSBacterial strains, phages, and plasmids. Bacterial strains, phages, and plasmids used in this study are listed in Table 1.Media and growth conditions. Escherichia coli and Pseudomonas putida were grown routinely in LB broth (38) or on Mueller-Hinton agar plates (Pasteur Diagnostics, France). When necessary, the following antibiotics were added to growth media: ampicillin (50 g/ml), chloramphenicol (20 g/ml), and tetracycline (12.5 g/ml) for E. coli, and chloramphenicol (300 g/ml) for P. putida. C. testosteroni was grown in YE broth as described previously (37). E. coli XL-1 Blue, used for single-stranded DNA production, was cultured in nutrient broth composed of (per liter) Bacto Tryptone (35 g), Bacto Yeast Extract (20 g; Oxoid Ltd., Basingtoke, England), and NaCl (5 g) at pH 7.5. All bacterial cultures were incubated at 30ЊC with shaking.Biotransformation of steroids. Recombinant bacteria harboring DNA fragments from C. testosteroni were assayed for the expression of various enzyme activities of the steroid catabolic pathway (see below). Enzyme-catalyzed conversion of steroids into specific metabolites was demonstrated by thin-layer

In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.