Search

We identified a temperature-sensitive mutant of the plant pathogenic fungus Ustilago maydis that is defective in the polar distribution of cell wall components and shows abnormal morphology. The affected gene, yup1, was cloned by complementation. It encodes a putative target soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (t-SNARE), suggesting a function in membrane fusion. A Yup1-GFP fusion protein localized to vesicles that showed rapid saltatory motion along microtubules. These vesicles are part of the endocytic pathway and accumulate at sites of active growth, thereby supporting the expansion of the hyphal tip. In yup1(ts) cells, endocytosis is impaired and accumulation of Yup1-carrying endosomes at cell poles is abolished, resulting in apolar distribution of wall components and morphological alterations. This suggests that a membrane recycling process via early endosomes supports polar growth of U. maydis.

FIS belongs to the group of small abundant DNA-binding proteins of Escherichia coli. We recently demonstrated that, in vivo, FIS regulates the expression of several genes needed for catabolism of sugars and nucleic acids, a majority of which are also transcriptionally regulated by cAMP-cAMP-receptor protein (CRP) complex. Here we provide evidence that FIS represses transcription of the crp gene both in vivo and in vitro. Employing crp promoter-lacZ fusions, we demonstrate that both FIS and cAMP-CRP are required to keep the crp promoter in a repressed state. We have identified in the crp promoter other transcription initiation sites which are located 73, 79 and 80 bp downstream from the previously mapped start site. Two CRP- and several FIS-binding sites with different affinities are located in the crp promoter region, one of them overlapping the downstream transcription initiation sites. We show that initiation of transcription at the crp promoter is affected by the composition of nucleoprotein complexes resulting from the outcome of competition between proteins for overlapping binding sites. Our results suggest that the control of crp transcription is achieved by oscillation in the composition of these regulatory nucleoprotein complexes in response to the physiological state of the cell.

Ustilago maydis, a fungal pathogen of corn, can alternate between yeast-like and filamentous growth. This dimorphic switch is governed by the mating-type loci. We have identified an abundant class of small SDS-insoluble cell wall proteins, designated repellents, specifically present in the filamentous form. Genetic analysis revealed that these peptides are processed from a single precursor protein, Rep1. Rep1 comprises 652 amino acids with a leader sequence for secretion. A characteristic feature of Rep1 is 12 repeats of a 37 amino acid consensus sequence; 10 of these repeats are separated by Kex2 protease cleavage sites. In (delta)rep1 mutants formation of aerial hyphae and surface hydrophobicity were reduced dramatically. This and the fact that expression of rep1 is regulated by the mating-type loci indicates that repellents play a structural role in the formation of aerial hyphae.

The Escherichia coli DNA binding protein FIS activates stable RNA promoters during outgrowth of cells from stationary phase. The upstream activating sequences (UASs) of these promoters contain three highly conserved FIS binding sites positioned in helical register. Neither the apparent requirement for three sites nor the mechanism of FIS-mediated activation has been established. We demonstrate here that on saturation of its three binding sites in the UAS, FIS forms a specific nucleoprotein complex which 'traps' RNA polymerase (RNAP) at the promoter of the tyrT operon. This effect is abolished by a change in helical phasing between FIS sites II and III, which impaires cooperative interactions between DNA-bound FIS dimers. The sigma 70 subunit of RNAP stimulates the formation of higher order FIS complexes, a property that is indicative of protein-protein interactions. We propose that after initiation of transcription, the released sigma 70 subunit may be recaptured by the FIS nucleoprotein 'trap' and recycled in successive rounds of holoenzyme assembly. Such a mechanism could overcome transient limitations on the availability of sigma 70 or core polymerase after a prolonged stationary phase.

Cell recognition and mating in the smut fungus Ustilago maydis have been proposed to involve specific pheromones and pheromone receptors. The respective structural genes are located in the a mating type locus that exists in the alleles a1 and a2. We demonstrate that binding of pheromone to the receptor can induce a morphological switch from yeast-like to filamentous growth in certain strains. Using this as biological assay we were able to purify both the a1 and a2 pheromone. The structure of the secreted pheromones was determined to be 13 amino acids for a1 and nine amino acids for a2. Both pheromones are post-translationally modified by farnesylation and carboxyl methyl esterification of the C-terminal cysteine. An unmodified a1 peptide exhibits dramatically reduced activity. The pheromone alone is able to induce characteristic conjugation tubes in cells of opposite mating type and confers mating competence; even cells of the same mating type undergo fusion. We discuss the role of pheromones in initiating filamentous growth and pathogenic development.

The heavy chain of dynein forms a globular motor domain that tightly couples the ATP-cleavage region and the microtubule-binding site to transform chemical energy into motion along the cytoskeleton. Here we show that, in the fungus Ustilago maydis, two genes, dyn1 and dyn2, encode the dynein heavy chain. The putative ATPase region is provided by dyn1, while dyn2 includes the predicted microtubule-binding site. Both genes are located on different chromosomes, are transcribed into independent mRNAs and are translated into separate polypeptides. Both Dyn1 and Dyn2 co-immunoprecipitated and co-localized within growing cells, and Dyn1–Dyn2 fusion proteins partially rescued mutant phenotypes, suggesting that both polypeptides interact to form a complex. In cell extracts the Dyn1–Dyn2 complex dissociated, and microtubule affinity purification indicated that Dyn1 or associated polypeptides bind microtubules independently of Dyn2. Both Dyn1 and Dyn2 were essential for cell survival, and conditional mutants revealed a common role in nuclear migration, cell morphogenesis and microtubule organization, indicating that the Dyn1–Dyn2 complex serves multiple cellular functions.

The phytopathogenic fungus Ustilago maydis exists in two stages, the yeast-like haploid form and the filamentous dikaryon. Both pathogenicity and dimorphism are genetically controlled by two mating-type loci, with only the filamentous stage being pathogenic on corn. We have identified two genes (kin1 and kin2) encoding motor proteins of the kinesin family. Kin1 is most similar to the human CENP-E gene product, while Kin2 is most closely related to the conventional kinesin Nkin of Neurospora crassa. Deletion mutants of kin1 had no discernible phenotype; delta kin2 mutants, however, were severely affected in hyphal extension and pathogenicity. The wild-type dikaryon showed rapid tip growth, with all the cytoplasm being moved to the tip compartment. Left behind are septate cell wall tubes devoid of cytoplasm. In delta kin2 mutants, dikaryotic cells were formed after cell fusion, but these hyphal structures remained short and filled with cytoplasm. A functional green fluorescent protein (GFP)-Kin2 fusion was generated and used to determine the localization of the motor protein by fluorescence microscopy. Inspection of the hyphal tips by electron microscopy revealed a characteristic accumulation of darkly stained vesicles which was absent in mutant cells. We suggest that the motor protein Kin2 is involved in organizing this specialized growth zone at the hyphal tip, probably by affecting the vectorial transport of vesicles.

In the phytopathogenic fungus Ustilago maydis, cell fusion is governed by a pheromone signalling system. The pheromone receptors belong to the seven transmembrane class that are coupled to heterotrimeric G proteins. We have isolated four genes (gpa1 to gpa4) encoding alpha subunits of G proteins. Gpa1, Gpa2 and Gpa3 have homologues in other fungal species, while Gpa4 is novel. Null mutants in individual genes were viable and only disruption of gpa3 caused a discernible phenotype. gpa3 mutant strains were unable to respond to pheromone and thus were mating-deficient. A constitutively active allele of gpa3 (gpa3(Q206L)) was generated by site-directed mutagenesis. Haploid strains harbouring gpa3(Q206L) were able to mate without pheromone stimulation, indicating that Gpa3 plays an active role in transmission of the pheromone signal. Surprisingly, Gpa3 is also required for pathogenic development, although pheromone signalling is not essential for this process.

The DNA binding protein FIS is involved in processes like site specific DNA inversion, lambda excision and stimulation of stable RNA synthesis in Escherichia coli. The amount of FIS protein is subject to dramatic changes during growth. We demonstrate that fis is part of an operon with one ORF of unknown function preceding the fis gene. Regulation of fis synthesis occurs at the transcriptional level. Within 15 min after nutritional upshift a large burst of fis mRNA is produced which levels off when cells begin to grow. By mutational analysis using promoter-lacZ fusions we demonstrate that the fis promoter is autoregulated by FIS. Growth phase regulation of the fis promoter depends on the presence of a GC motif downstream of the -10 region. We show that the fis promoter is subject to stringent control and discuss this unusual feature with respect to the known and putative functions FIS serves in E. coli.

The DNA invertase Gin encoded by bacteriophage Mu catalyses efficient site-specific recombination between inverted repeat sequences (IR) in vivo and in vitro in the presence of the host factor FIS and the recombinational enhancer. We demonstrate that Gin alone is able to introduce single strand breaks into duplex DNA fragments which contain the IR sequence. Strand cleavage is site-specific and can occur on either strand within the IR. Cleaved molecules contain Gin covalently attached to DNA. The covalent complex is formed through linkage of Gin to the 5' DNA phosphate at the site of the break via a phosphoserine. Extensive site-directed mutational analysis showed that all mutants altered at serine position 9 were completely recombination deficient in vivo and in vitro. The mutant proteins bind to DNA but lack topoisomerase activity and are unable to introduce nicks. This holds true even for a conservative amino acid substitution at position 9. We conclude that serine at position 9 is part of the catalytic domain of Gin. The intriguing finding that the DNA invertase Gin has the same catalytic center as the DNA resolvases that promote deletions without recombinational enhancer and host factor FIS is discussed.

Site-specific DNA inversion in phage Mu is catalysed by the phage-encoded DNA invertase Gin and a host factor FIS. We demonstrate that purified Gin protein binds specifically to 34-bp sequences that flank the G segment as inverted repeats. Each inverted repeat (IR) contains two binding sites for Gin which have to be arranged in a specific configuration to constitute a recombinogenic site. While one of these sites is bound when present alone, the other site is bound only in conjunction with the first one, suggesting cooperative binding. In addition to the sites within the IR, Gin binds with lower affinity to AT-rich sequences adjacent to the IR. We demonstrate that these sites do not participate in the inversion reaction. The IR itself can be shortened to 25 bp without effect on inversion frequency. Using gel mobility shift experiments on circular permuted fragments containing the IR we show that Gin bends DNA upon binding. We discuss the possibility that DNA bending is related to the formation of a productive synaptic complex.

Inversion of the G segment in bacteriophage Mu DNA occurs by a site-specific recombination event and determines the host specificity of Mu phage particles produced. Inversion is mediated by a Mu function (Gin). The gin gene has been placed under control of the inducible lambda pL promoter and a synthetic Shine-Dalgarno linker upstream of the initiation codon. The Gin protein content in induced cells is boosted to 10% of total protein. Partially purified extracts from overproducing strains promote efficient inversion of the G DNA segment in vitro which is visualized by agarose gel electrophoresis of the substrate DNA after cutting with appropriate restriction endonucleases. The in vitro reaction requires Mg, a super-coiled DNA substrate and occurs in the absence of exogenous ATP. Inversion from the G(+) to the G(-) orientation is as efficient as the switch from G(-) to G(+).