Abstract Filamentous fungi are the organisms of choice for most industrial biotechnology. Some species can produce a variety of secondary metabolites and enzymes of commercial interest, and the production of valuable molecules has been enhanced through different molecular tools. Methods for genetic manipulation and transformation have been essential for the optimization of these organisms. The genus Simplicillium has attracted increased attention given several potential biotechnological applications. The Simplicillium genus harbors several entomopathogenic species and some isolates have been explored for bioremediation of heavy metal contaminants. Furthermore, the myriad of secondary metabolites isolated from Simplicillium spp. render these organisms as ideal targets for deep exploration and further biotechnological mining possibilities. However, the lack of molecular tools hampered the exploration of this genus. Thus, an Agrobacterium tumefaciens-mediated transformation method was established for Simplicillium subtropicum, employing the far-red fluorescent protein TURBOFP635/Katushka, as a visual marker, and the selection marker SUR gene, that confers resistance to chlorimuron ethyl. Notably, one round of transformation using the established method yielded almost 400 chlorimuron resistant isolates. Furthermore, these transformants displayed mitotic stability for, at least, five generations. We anticipate that this method can be useful for deep molecular exploration and improvement of strains in the Simplicillium genus.

Abstract Carbonic anhydrases (CA) belong to the family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the present work, we characterized the cDNAs of four Paracoccidioides CAs (CA1, CA2, CA3, and CA4). In the presence of CO2, there was not a significant increase in fungal ca1, ca2 and ca4 gene expression. The ca1 transcript was induced during the mycelium-to-yeast transition, while ca2 and ca4 gene expression was much higher in yeast cells, when compared to mycelium and mycelium-to-yeast transition. The ca1 transcript was induced in yeast cells recovered directly from liver and spleen of infected mice, while transcripts for ca2 and ca4 were down-regulated. Recombinant CA1 (rCA1) and CA4 (rCA4), with 33 kDa and 32 kDa respectively, were obtained from bacteria. The enzymes rCA1 (β-class) and rCA4 (α-class) were characterized regarding pH, temperature, ions and amino acids addition influence. Both enzymes were stable at pHs 7.5-8.5 and temperatures of 30-35 °C. The enzymes were dramatically inhibited by Hg+2 and activated by Zn+2, while only rCA4 was stimulated by Fe2+. Among the amino acids tested (all in L configuration), arginine, lysine, tryptophan and histidine enhanced residual activity of rCA1 and rCA4.

Paracoccidioides brasiliensis causes infection through inhalation by the host of airborne propagules from the mycelium phase of the fungus. This fungus reaches the lungs, differentiates into the yeast form and is then disseminated to virtually all parts of the body. Here we review the identification of differentially-expressed genes in host-interaction conditions. These genes were identified by analyzing expressed sequence tags (ESTs) from P. brasiliensis cDNA libraries. The P. brasiliensis was recovered from infected mouse liver as well as from fungal yeast cells incubated in human blood and plasma, mimicking fungal dissemination to organs and tissues and sites of infection with inflammation, respectively. In addition, ESTs from a cDNA library of P. brasiliensis mycelium undergoing the transition to yeast were previously analyzed. Together, these studies reveal significant changes in the expression of a number of genes of potential importance in the host-fungus interaction. In addition, the unique and divergent representation of transcripts when the cDNA libraries are compared suggests differential gene expression in response to specific niches in the host. This analysis of gene expression patterns provides details about host-pathogen interactions and peculiarities of sites within the host.

Mycoplasma synoviae and Mycoplasma hyopneumoniae are wall-less eubacteria belonging to the class of Mollicutes. These prokaryotes have a reduced genome size and reduced biosynthetic machinery. They cause great losses in animal production. M. synoviae is responsible for an upper respiratory tract disease of chickens and turkeys. M. hyopneumoniae is the causative agent of enzootic pneumonia in pigs. The complete genomes of these organisms showed 17 ORFs encoding kinases in M. synoviae and 15 in each of the M. hyopneumoniae strain. Four kinase genes were restricted to the avian pathogen while three were specific to the pig pathogen when compared to each other. All deduced kinases found in the non pathogenic strain (J[ATCC25934]) were also found in the pathogenic M. hyopneumoniae strain. The enzymes were classified in nine families composing five fold groups.