Effects of cellobiose and glucose on cellulose hydrolysis by both growing and resting cells of Bacteroides cellulosolvens

The cellulase system of Bacteroides cellulosolvens was subjected to both catabolite repression and feedback inhibition by cellobiose. Cellulose-solubilizing activity was 50% inhibited at a cellobiose concentration of 2.6 g/L and completely inhibited by 12 g/L. Glucose at 12 g/L (the highest concentration tested) had no effect on cellulase activity. Supplementation of B. cellulosolvens cellulase with beta-glucosidase resulted in increased conversion of cellobiose to glucose; however, a constant cellobiose pool size of approximately 7 g/L was maintained.     

Sugar Substrates for l-Lysine Fermentation by Ustilago maydis

The extracellular production of l-lysine in media with cane sugar, blackstrap molasses, or clarified sugar-cane juice by a previously obtained mutant of Ustilago maydis was studied. Enzymatically inverted clarified juice (medium J-3) gave 2.9 g of lysine per liter under the following conditions: inoculum, 5%; pH 5.8; temperature, 30 C; K(La) in the fermentors, 0.41 mmoles of O(2) per liter per min; fermentation time, 72 hr. The concentrate, obtained by direct evaporation and drying of the fermentation broth, could be used as a possible feed supplement because of its amino-acid and vitamin content.     

Genetic diversity of Ustilago maydis strains

Thirty wild isolates belonging to five different locations in Mexico plus two laboratory strains of Ustilago maydis were characterized by restriction fragment length polymorphism (RFLP) analysis using 23 different clones as probes derived from a PstI library and two restriction enzymes. All loci analysed presented a high level of polymorphism, including one locus with thirty one different alleles. Geographical grouping of the populations was based on Nei's genetic distance and there was no correlation between genetic and geographic distances among these isolates. Our results suggest that DNA fingerprinting is a useful method for detecting genetic variation in populations of U. maydis. This work demonstrated that considerable genetic variation may be present within field populations of U. maydis.     

Cloning and disruption of Ustilago maydis genes.

We have demonstrated that genes from Ustilago maydis can be cloned by direct complementation of mutants through the use of genomic libraries made in a high-frequency transformation vector. We isolated a gene involved in amino acid biosynthesis as an illustrative example and showed that integrative and one-step disruption methods can be used to create null mutations in the chromosomal copy of the gene by homologous recombination. The results of this investigation make it clear that one-step gene disruption will be of general utility in investigations of U. maydis, since simple, precise replacement of the sequence under study was readily achieved.     

Initiation of Meiotic Recombination in Ustilago maydis

A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar⁺ recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.     

The generic position of Ustilago maydis, Ustilago scitaminea, and Ustilago esculenta (Ustilaginales)

Three species of smut fungi (Ustilaginales, Basidiomycota) of economic importance, Ustilago maydis on corn, U. scitaminea on sugar cane, and U. esculenta on Zizania latifolia, were investigated in order to define their systematic position using morphological characteristics of the sori, ultrastructure of teliospore walls, and molecular data of the LSU rDNA. LSU rDNA suggests that U. maydis and U. scitaminea belong to the genus Sporisorium. This has already been proposed for U. scitaminea, which develops sori with whip-shaped axes corresponding to columellae. U. maydis and U. scitaminea, like typical species of Sporisorium, present peridia and columellae in their sori. Therefore, U. scitaminea is called Sporisorium scitamineum. U. maydis, however, is not placed in the genus Sporisorium here, because ongoing investigation of molecular data from the ITS rDNA region yields contradictory results and because the name Sporisorium maydis is occupied by an imperfect fungus. U. esculenta is recognized as Yenia esculenta. This placement in a separate genus is based on molecular data and on unique teliospore ultrastructure, i.e. apically enlarged, partly confluent warts developing on a strongly folded plasmalemma, and the exosporium and endosporium forming part of the ornamentation. Part 193 in the series „Studies in Heterobasidiomycetes“ from the Botanical Institute, University of Tübingen.     

Inbreeding levels of two Ustilago maydis populations

Little is known about the population mating behavior of the smut fungus Ustilago maydis DC (Corda). To determine the amount of inbreeding that occurs in local U. maydis populations, two cornfields were sampled, one in North America (NA) at Le Sueur, Minnesota, and one in South America (SA) at Tarariras, Uruguay. These fields were chosen because of their geographic isolation and host management differences. Inbreeding coefficients (F(is)) were calculated using data derived from amplified fragment length polymorphism (AFLP) markers. Mean F(is) values estimated for both the NA (-0.08), and the SA (-0.02) populations statistically are not different from zero. The results of this study demonstrate that the U. maydis population structure in both cornfields results predominately from out-crossing and suggests that teliospores infrequently act as single infection units. The genetic differentiation between populations was high (F(st) = 0.25).     

Ustilago maydis Produces Cytokinins and Abscisic Acid for Potential Regulation of Tumor Formation in Maize

The infection of maize (Zea mays) by the basidiomycete fungus Ustilago maydis leads to common smut of corn characterized by the production of tumors in susceptible aboveground plant tissues. LC-(ES)MS/MS profiles of abscisic acid (ABA) and 12 different cytokinins (CKs) were determined for infected and uninfected maize tissues over a time course following fungal exposure. Samples were taken at points corresponding to the appearance of disease symptoms. Axenic cultures of haploid and dikaryon forms of U. maydis were also profiled. This study confirmed the capability of Ustilago maydis to synthesize CKs, ABA, and auxin (IAA). It also provided evidence for the involvement of CK and ABA in the U. maydis-maize infection process. Significant quantities of CKs and ABA were detected from axenic cultures of U. maydis as was IAA. CKs and ABA levels were elevated in leaves and stems of maize after infection; notable was the high level of cis-zeatin 9-riboside. Variation among hormone profiles of maize tissues was observed at different time points during infection and between infections with nonpathogenic haploid and pathogenic dikaryon strains. This suggested that CKs and ABA accumulate and are likely metabolized in maize tissue infected with U. maydis. Because U. maydis produced these phytohormones at significant levels, it is possible that the fungal pathogen is a source of these compounds in infected tissue. This is the first study to confirm the production of CKs and document the production of ABA by U. maydis. This study also established an involvement of these phytohormones and a possible functional role for ABA in U. maydis infection of maize.     

Identification of a gene cluster for biosynthesis of mannosylerythritol lipids in the basidiomycetous fungus Ustilago maydis

Many microorganisms produce surface-active substances that enhance the availability of water-insoluble substrates. Although many of these biosurfactants have interesting potential applications, very little is known about their biosynthesis. The basidiomycetous fungus Ustilago maydis secretes large amounts of mannosylerythritol lipids (MELs) under conditions of nitrogen starvation. We recently described a putative glycosyltransferase, Emt1, which is essential for MEL biosynthesis and whose expression is strongly induced by nitrogen limitation. We used DNA microarray analysis to identify additional genes involved in MEL biosynthesis. Here we show that emt1 is part of a gene cluster which comprises five open reading frames. Three of the newly identified proteins, Mac1, Mac2, and Mat1, contain short sequence motifs characteristic for acyl- and acetyltransferases. Mutational analysis revealed that Mac1 and Mac2 are essential for MEL production, which suggests that they are involved in the acylation of mannosylerythritol. Deletion of mat1 resulted in the secretion of completely deacetylated MELs, as determined by mass spectrometry. We overexpressed Mat1 in Escherichia coli and demonstrated that this enzyme acts as an acetyl coenzyme A-dependent acetyltransferase. Remarkably, Mat1 displays relaxed regioselectivity and is able to acetylate mannosylerythritol at both the C-4 and C-6 hydroxyl groups. Based on these results, we propose a biosynthesis pathway for the generation of mannosylerythritol lipids in U. maydis.     

Oxygen transfer and scale-up in lysine production by Ustilago maydis mutant

The oxygen transfer coefficient estimated by both sulfite and dynamic methods and some of the rheological properties of fermentation broths derived from the batch cultivation of a mutant of U. maydis in a sugar cane juice substrate, are used in a scaling-up procedure on the basis of the power consumption per volume unit concept. The fluid was of the Binham plastic type; the Np-N_RE correlations showed that the modified Reynolds numbers of the flat-blade turbine impellers were low, near to or in the laminar region; the Na-Pg/P relations were established and then used in the calculation regardless of the geometrical dissimilarities of the vessels. A change of scale from 5 to 50 liters was calculated and operated keeping the power per volume value constant. Reproduction of lysine yields, 2.5–3.2 g/liter, was repeatedly reached in 8 successive runs.     

The O-Mannosyltransferase PMT4 Is Essential for Normal Appressorium Formation and Penetration in Ustilago maydis

In Saccharomyces cerevisiae, the PMT, KRE2/MNT1, and MNN1 mannosyltransferase protein families catalyze the steps of the O-mannosylation pathway, sequentially adding mannoses to target proteins. We have identified members of all three families and analyzed their roles in pathogenesis of the maize smut fungus Ustilago maydis. Furthermore, we have shown that PMT4, one of the three PMT family members in U. maydis, is essential for tumor formation in Zea mays. Significantly, PMT4 seems to be required only for pathogenesis and is dispensable for other aspects of the U. maydis life cycle. We subsequently show that the deletion of pmt4 results in a strong reduction in the frequency of appressorium formation, with the few appressoria that do form lacking the capacity to penetrate the plant cuticle. Our findings suggest that the O-mannosylation pathway plays a key role in the posttranslational modification of proteins involved in the pathogenic development of U. maydis. The fact that PMT homologs are not found in plants may open new avenues for the development of fungal control strategies. Moreover, the discovery of a highly specific requirement for a single O-mannosyltransferase should aid in the identification of the proteins directly involved in fungal plant penetration, thus leading to a better understanding of plant–fungi interactions.     

Induction of phenylalanine ammonia-lyase activity by tryptophan in Ustilago maydis.

To understand the regulation of phenylalanine ammonia-lyase (PAL) activity in the corn smut fungus, Ustilago maydis, we examined the effects of different media, metabolic effectors (including aromatic amino acids), and environmental factors on induction and repression of PAL activity. PAL was detected only in cell extracts and not in the culture medium. U. maydis PAL is constitutively produced at a low level in all media tested but its regulation can be influenced by aromatic amino acids. L-Tryptophan (0.3 mM) induces PAL activity 3- to 5-fold but tryptophan analogs and tryptophan-related metabolites do not. The enzyme is most readily induced during the early stationary phase of growth and the induced activity remains relatively constant during stationary stage. No induction or inhibition of PAL activity was observed as a function of culture temperature, pH or light. PAL induction was repressed by glucose but not by its reaction product, t-cinnamic acid. Induction did not require de novo protein synthesis, suggesting that some form of post-translational protein modification or a metabolic effect may be involved. This study shows that the regulation of U. maydis PAL is very different from the patterns known for plants and other fungi.     

Molecular analysis of the karyotype of Ustilago maydis

A molecular karyotype of the corn smut-inducing fungus Ustilago maydis was prepared using orthogonalfield-alternation gel electrophoresis (OFAGE). At least 20 chromosome-sized DNAs ranging from approximately 300 kb to the maximum limit of resolution of this system were identified in haploid cells of a widely used strain. Although general features of the banding pattern of chromosome-sized DNAs were conserved between strains, no two strains had identical karyotypes, indicating that considerable chromosome length polymorphism exists in this species. This polymorphism was seen in both laboratory strains as well as more recent isolates from nature. Length variation in apparently identical chromosomes was usually small, but was occasionally significant. In one strain Southern DNA hybridization analysis suggested the occurrence of a stable large-scale, inter-chromosomal exchange which had given rise to two novel chromosomes.     

Metamorphosis of the Basidiomycota Ustilago maydis: Transformation of yeast-like cells into basidiocarps

Ustilago maydis (DC) Cda., a phytopathogenic Basidiomycota, is the causal agent of corn smut. During its life cycle U. maydis alternates between a yeast-like, haploid nonpathogenic stage, and a filamentous, dikaryotic pathogenic form that invades the plant and induces tumor formation. As all the members of the Subphylum Ustilaginomycotina, U. maydis is unable to form basidiocarps, instead it produces teliospores within the tumors that germinate forming a septate basidium (phragmobasidium). We have now established conditions allowing a completely different developmental program of U. maydis when grown on solid medium containing auxins in dual cultures with maize embryogenic calli. Under these conditions U. maydis forms large hemi-spheroidal structures with all the morphological and structural characteristics of gastroid-type basidiocarps. These basidiocarps are made of three distinct hyphal layers, the most internal of which (hymenium) contains non-septate basidia (holobasidia) from which four basidiospores develop. In basidiocarps meiosis and genetic recombination occur, and meiotic products (basidiospores) segregate in a Mendelian fashion. These results are evidence of sexual cycle completion of an Ustilaginomycotina in vitro, and the demonstration that, besides its quasi-obligate biotrophic pathogenic mode of life, U. maydis possesses the genetic program to form basidiocarps as occurs in saprophytic Basidiomycota species.