Marcelo A. Vallim

Metagenomic Analysis of a Tropical Composting Operation at the São Paulo Zoo Park Reveals Diversity of Biomass Degradation Functions and Organisms

Composting operations are a rich source for prospection of biomass degradation enzymes. We have analyzed the microbiomes of two composting samples collected in a facility inside the São Paulo Zoo Park, in Brazil. All organic waste produced in the park is processed in this facility, at a rate of four tons/day. Total DNA was extracted and sequenced with Roche/454 technology, generating about 3 million reads per sample. To our knowledge this work is the first report of a composting whole-microbial community using high-throughput sequencing and analysis. The phylogenetic profiles of the two microbiomes analyzed are quite different, with a clear dominance of members of the Lactobacillus genus in one of them. We found a general agreement of the distribution of functional categories in the Zoo compost metagenomes compared with seven selected public metagenomes of biomass deconstruction environments, indicating the potential for different bacterial communities to provide alternative mechanisms for the same functional purposes. Our results indicate that biomass degradation in this composting process, including deconstruction of recalcitrant lignocellulose, is fully performed by bacterial enzymes, most likely by members of the Clostridiales and Actinomycetales orders.

A Rac Homolog Functions Downstream of Ras1 To Control Hyphal Differentiation and High-Temperature Growth in the Pathogenic Fungus Cryptococcus neoformans

ABSTRACT The Cryptococcus neoformans Ras1 protein serves as a central regulator for several signaling pathways. Ras1 controls the induction of the mating pheromone response cascade as well as a distinct signaling pathway that allows this pathogenic fungus to grow at human physiological temperature. To characterize elements of the Ras1-dependent high-temperature growth pathway, we performed a multicopy suppressor screen, identifying genes whose overexpression allows the ras1 mutant to grow at 37°C. Using this genetic technique, we identified a C. neoformans gene encoding a Rac homolog that suppresses multiple ras1 mutant phenotypes. Deletion of the RAC1 gene does not affect high-temperature growth. However, a rac1 mutant strain demonstrates a profound defect in haploid filamentation as well as attenuated mating. In a yeast two-hybrid assay, Rac1 physically interacts with the PAK kinase Ste20, which similarly regulates hyphal formation in this fungus. Similar to Rac1, overexpression of the STE20α gene also restores high-temperature growth to the ras1 mutant. These results support a model in which the small G protein Rac1 acts downstream of Ras proteins and coordinately with Ste20 to control high-temperature growth and cellular differentiation in this human fungal pathogen.

Ras1 controls pheromone expression and response during mating in Cryptococcus neoformans.

The Cryptococcus neoformans Ras1 signal transduction pathway controls mating, hyphal differentiation, and the ability of this opportunistic human fungal pathogen to grow at elevated temperatures. To further elucidate how Ras1 signals in this organism, the RAS1 gene was disrupted in the congenic serotype D strain background. Genetic epistasis experiments indicated that Ras1 regulates the mating response through the MAP kinase/pheromone response pathway. In fact, Ras1 is required for the transcriptional induction of elements of the pheromone response pathway. However, the ability of C. neoformans Ras1 to allow growth at 37 degrees C is mediated by a separate signaling pathway. Therefore a single Ras protein may differentially activate distinct downstream targets in response to different signals within the same organism. This conserved signaling motif has been coopted in C. neoformans to regulate mating and morphogenesis in addition to being required for its pathogenic potential.

Chemical and Biological Evaluation of Essential Oils from Two Species of Myrtaceae — Eugenia uniflora L. and Plinia trunciflora (O. Berg) Kausel

The chemical composition and antimicrobial activity of essential oils obtained from leaves of two Myrtaceae species–Eugenia uniflora L. and Plinia trunciflora (O. Berg) Kausel–were determined. Analysis by GC/MS as well as determination of Kovatz indexes indicated atractylone (26.78%) and curzerene (17.96%) as major constituents of E. uniflora oil and α-cadinol (19.15%), apiole (11.15%) and cubenol (5.43%) as main components in P. trunciflora oil. Both essential oils were tested for antimicrobial activity against yeasts and bacteria. E. uniflora and P. trunciflora essential oils were active towards two Gram-positive bacteria, Streptococcus equi and Staphylococcus epidermis. In addition, biological activity of both essential oils was detected for pathogenic yeasts of the genus Candida and Cryptococcus. E. uniflora was active towards all yeast tested and exhibited interesting minimal inhibitory concentrations (0.11 to 3.75 mg/mL) across a broad spectrum of activity.

Amylolytic microorganism from são paulo zoo composting: isolation, identification, and amylase production.

Composting is a way of transforming the organic waste into fertilizer, minimizing the use of inorganic compounds that may contaminate the environment. This transformation is the result of the microorganism action, converting complex carbon sources into energy. Enzymes that are exported by the microorganisms to the surrounding environment mediate this process. The aiming of the present work is to prospect the compost produced by the organic composting unit (OCU) of the Fundação Parque Zoológico de São Paulo (FPZSP) to find novel starch hydrolyzing organisms (SHO) that secrete large amounts of amylases under harsh conditions, such as high temperature. We found five bacterial isolates that have amylolytic activity induced by soluble starch and 39°C temperature of growth. These bacterial strains were identified by MALDI-TOF (Matrix-assisted laser desorption/ionization-Time of Flight) analysis, a rapid and efficient methodology for microbe identification in large scale. Our results present amylolytic strains that belong to diverse taxonomic groups (Solibacillus silvestris, Arthrobacter arilaitensis, Isoptericola variabilis, and Acinetobacter calcoaceticus); some of them have never been associated with this kind of hydrolytic activity before. The information regarding enzyme induction will be important to optimize the production by the bacterial isolates, which may be a great value for biotechnological applications.

Internally quenched fluorescent peptide libraries with randomized sequences designed to detect endopeptidases

Identification of synthetic peptide substrates for novel peptidases is an essential step for their study. With this purpose we synthesized fluorescence resonance energy transfer (FRET) peptide libraries Abz (or MCA)-GXXXXXQ-EDDnp and Abz (or MCA)-GXXZXXQ-EDDnp, where X consists of an equimolar mixture of all amino acids, the Z position is fixed with one of the proteinogenic amino acids (cysteine was excluded), Abz (ortho-aminobenzoic acid) or MCA ([7-amino-4-methyl]coumarin) is the fluorescence donor and Q-EDDnp (glutamine-[N-(2,4-dinitrophenyl)-ethylenediamine]) is the fluorescence acceptor. The peptide libraries MCA-GXXX↓XXQ-EDDnp and MCA-GXXZ↓XXQ-EDDnp were cleaved as indicated (↓) by trypsin, chymotrypsin, cathepsin L, pepsin A, and Eqolisin as confirmed by Edman degradation of the products derived from the digestion of these libraries. The best hydrolyzed Abz-GXXZXXQ-EDDnp sublibraries by these proteases, including Dengue 2 virus NS2B-NS3 protease, contained amino acids at the Z position that are reported to be well accepted by their S(1) subsite. The pH profiles of the hydrolytic activities of these canonical proteases on the libraries were similar to those reported for typical substrates. The FRET peptide libraries provide an efficient and simple approach for detecting nanomolar concentrations of endopeptidases and are useful for initial specificity characterization as performed for two proteases secreted by a Bacillus subtilis.

Assessing the Chemical Composition and Antimicrobial Activity of Essential Oils from Brazilian Plants—Eremanthus erythropappus (Asteraceae), Plectrantuns barbatus, and P. amboinicus (Lamiaceae)

The chemical composition and antimicrobial activity of essential oils obtained from three Brazilian plant species—leaves and branches of Eremanthus erythropappus (Asteraceae), leaves of Plectranthus barbatus, and leaves of P. amboinicus (Lamiaceae)—were determined. Analysis by GC/MS and determination of Kovats indexes both indicated δ-elemene (leaves—42.61% and branches—23.41%) as well as (−)-α-bisabolol (leaves—24.80% and stem bark—66.16%) as major constituents of E. erythropappus essential oils. The main components of leaves of P. barbatus were identified as (Z)-caryophyllene (17.98%), germacrene D (17.35%), and viridiflorol (14.13%); whereas those of leaves of P. amboinicus were characterized as p-cymene (12.01%), γ-terpinene (14.74%), carvacrol (37.70%), and (Z)-caryophyllene (14.07%). The antimicrobial activity against yeasts and bacteria was assessed in broth microdilution assays to determine the minimum inhibitory concentration (MIC) necessary to inhibit microbial growth. In addition, the crude oil of branches of E. erythropappus was subjected to chromatographic separation procedures to afford purified (−)-α-bisabolol. This compound displayed biological activity against pathogenic yeasts, thus suggesting that the antimicrobial effect observed with crude oils of E. erythropappus leaves and branches may be related to the occurrence of (−)-α-bisabolol as their main component. Our results showed that crude oils of Brazilian plants, specifically E. erythropappus, P. barbatus, and P. amboinicus and its components, could be used as a tool for the developing novel and more efficacious antimicrobial agents.

The Role of Amino Acid Permeases and Tryptophan Biosynthesis in Cryptococcus neoformans Survival.

Metabolic diversity is an important factor during microbial adaptation to different environments. Among metabolic processes, amino acid biosynthesis has been demonstrated to be relevant for survival for many microbial pathogens, whereas the association between pathogenesis and amino acid uptake and recycling are less well-established. Cryptococcus neoformans is an opportunistic fungal pathogen with many habitats. As a result, it faces frequent metabolic shifts and challenges during its life cycle. Here we studied the C. neoformans tryptophan biosynthetic pathway and found that the pathway is essential. RNAi indicated that interruptions in the biosynthetic pathway render strains inviable. However, auxotroph complementation can be partially achieved by tryptophan uptake when a non preferred nitrogen source and lower growth temperature are applied, suggesting that amino acid permeases may be the target of nitrogen catabolism repression (NCR). We used bioinformatics to search for amino acid permeases in the C. neoformans and found eight potential global permeases (AAP1 to AAP8). The transcriptional profile of them revealed that they are subjected to regulatory mechanisms which are known to respond to nutritional status in other fungi, such as (i) quality of nitrogen (Nitrogen Catabolism Repression, NCR) and carbon sources (Carbon Catabolism Repression, CCR), (ii) amino acid availability in the extracellular environment (SPS-sensing) and (iii) nutritional deprivation (Global Amino Acid Control, GAAC). This study shows that C. neoformans has fewer amino acid permeases than other model yeasts, and that these proteins may be subjected to complex regulatory mechanisms. Our data suggest that the C. neoformans tryptophan biosynthetic pathway is an excellent pharmacological target. Furthermore, inhibitors of this pathway cause Cryptococcus growth arrest in vitro.

The role of the de novo pyrimidine biosynthetic pathway in Cryptococcus neoformans high temperature growth and virulence.

Fungal infections are often difficult to treat due to the inherent similarities between fungal and animal cells and the resulting host toxicity from many antifungal compounds. Cryptococcus neoformans is an opportunistic fungal pathogen of humans that causes life-threatening disease, primarily in immunocompromised patients. Since antifungal therapy for this microorganism is limited, many investigators have explored novel drug targets aim at virulence factors, such as the ability to grow at mammalian physiological temperature (37°C). To address this issue, we used the Agrobacterium tumefaciens gene delivery system to create a random insertion mutagenesis library that was screened for altered growth at elevated temperatures. Among several mutants unable to grow at 37°C, we explored one bearing an interruption in the URA4 gene. This gene encodes dihydroorotase (DHOase) that is involved in the de novo synthesis of pyrimidine ribonucleotides. Loss of the C. neoformans Ura4 protein, by targeted gene interruption, resulted in an expected uracil/uridine auxotrophy and an unexpected high temperature growth defect. In addition, the ura4 mutant displayed phenotypic defects in other prominent virulence factors (melanin, capsule and phospholipase) and reduced stress response compared to wild type and reconstituted strains. Accordingly, this mutant had a decreased survival rate in macrophages and attenuated virulence in a murine model of cryptococcal infection. Quantitative PCR analysis suggests that this biosynthetic pathway is induced during the transition from 30°C to 37°C, and that transcriptional regulation of de novo and salvage pyrimidine pathway are under the control of the Ura4 protein.

A simple and effective method to synthesize fluorescent nanoparticles using tryptophan and light and their lethal effect against bacteria.

A simple, environmentally friendly and cost-effective method was used to synthesize silver nanoparticles using tryptophan and light. To prepare the nanoparticles, the following components were used: deionized water, silver nitrate, light and tryptophan. The effects of the tryptophan concentration and light exposure time on the formation of tryptophan silver nanoparticles (Tnnps) were studied. The synthesized Tnnps were characterized using transmission electron microscopy (TEM), absorption and fluorescence spectroscopy and zeta potential measurements. The synthesized Tnnps were nearly spherical, with sizes of approximately 17 nm. In addition, the antibacterial activity of Tnnps was determined by monitoring the growth curves of strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Serratia marcescens, and Enterococcus faecalis using the microdilution test. The Minimum Inhibitory Concentration (MIC) for 4 of 5 tested bacteria was determined to be between 20.0 and 17.5 μg/mL for 48 h and between 22.5 and 20.0 μg/mL for 72 h.