Luiz Artur Mendes Bataus

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) widespread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications.

Expression analysis of the exo-β-1,3-glucanase from the mycoparasitic fungus Trichoderma asperellum.

The regulation of the gene encoding the extracellular exo-beta-1,3-glucanase (tag83) produced by the mycoparasite Trichoderma asperellum was studied. Enzyme activity was detected in all carbon sources, but the highest levels were found when starch and purified cell walls from Rhizoctonia solani were used. These results are supported by the appearance of one strong band with enzyme activity in non-denaturing PAGE. Experiments using RT-PCR showed that exo-beta-1,3-glucanase induction in T. asperellum occurred at the transcriptional level. We used RT-PCR and real-time PCR analysis to examine the expression of tag83 gene during in vivo assay of T. asperellum against R. solani. We showed that the expression of tag83 is significantly increased by the presence of R. solani.

Hypothesis testing of genetic similarity based on RAPD data using Mantel tests and model matrices

Clustering and ordination procedures in multivariate analyses have been widely used to describe patterns of genetic distances. However, in some cases, such as when dealing with Jaccard coefficients based on RAPD data, these techniques may fail to represent genetic distances because of the high dimensionality of the genetic distances caused by stochastic variation in DNA fragments among the units analyzed (species or populations). In this note, we show how Mantel tests can be used to test hypotheses about genetic distances and avoid problems with multivariate data representation. The procedure is illustrated with RAPD data comparing 20 anuran species from the cerrado, in which Jaccard genetic distances were compared to three model matrices linking the species at distinct taxonomic levels.

Acetone extract from Streptoverticillium sp., a bacterium isolated from Brazilian Cerrado soil, induces anti-inflammatory activity in mice

The Streptoverticillium sp. Z1 is an actinomycete isolated from the soil under Cerrado vegetation, the extract of this strain was investigated in nociceptive and inflammatory models. The Streptoverticillium extract (ExS) 50 and 100 mg/kg (s.c.) produced a significant inhibition of acetic acid-induced abdominal writhings thereby demonstrating an anti-nociceptive effect. In the tail flick test the ExS (s.c.) was inactive. This result implited that ExS does not contain opioid-like compounds with central analgesic properties. In the inflammatory models, ExS 100 and 200 mg/kg (s.c.) were able to inhibit the croton oil-induced ear edema and, ExS 200 and 500 mg/kg (s.c.) inhibited the leukocyte migration on the carrageenan-induced peritonitis. The phospholipase A2 enzymatic assay showed that the anti-inflammatory activity of ExS was not due to direct effect on phospholipase A2 activity. These data suggest that Streptoverticillium sp. produces metabolites with anti-inflammatory effect and that these metabolites are unable to directly inhibit phospholipase A2 enzyme.

Tetracarboxy-phthalocyanines: From excited state dynamics to photodynamic inactivation against Bovine herpesvirus type 1

Herein we present the excited state dynamic of zinc and aluminum tetracarboxy-phthalocyanines (ZnPc and AlPc) and its application in the photodynamic inactivation (PDI) of Bovine herpesvirus type 1 (BoHV-1) in vitro. The excited state dynamic provides valuable data to describe the excited state properties of potential optical limiters and/or photosensitizers (PSs), such as: the excited state cross-sections, fluorescence lifetime and triplet state quantum yield. The excited state characterization was performed using three different Z-scan techniques: Single Pulse, White Light Continuum and Pulse Train. Considering the photodynamic inactivation of BoHV-1, an initial viral suspension containing 105.75TCID50/mL was incubated with the PSs for 1h at 37°C under agitation and protected from light. The samples were placed in microtiter plates and irradiated (180mW/cm2). During irradiation, a sample was taken every 15min and the viability of the virus was evaluated. The results show that both phthalocyanines were efficient against viruses. However, a higher photodynamic efficiency was observed by ZnPc, which can be attributed to its higher triplet and singlet quantum yields. The results presented here are important for animal health (treatment of BoHV-1) and also open up a field of studies to use AlPc and ZnPc as potential agents against a wide range of microorganisms of veterinary interest.

Improvement of bread making quality by supplementation with a recombinant xylanase produced by Pichia pastoris

Xylanases (EC 3.2.1.8) are hydrolytic enzymes, which randomly cleave the β-1,4-linked xylose residues from xylan. The synthetic gene xynBS27 from Streptomyces sp. S27 was successfully cloned and expressed in Pichia pastoris. The full-length gene consists of 729 bp and encodes 243 amino acids including 51 residues of a putative signal peptide. This enzyme was purified in two steps and was shown to have a molecular weight of 20 kDa. The purified r-XynBS27 was active against beechwood xylan and oat spelt xylan as expected for GH 11 family. The optimum pH and temperature values for the enzyme were 6.0 and 75 °C, respectively. The Km and Vmax were 12.38 mg/mL and 13.68 μmol min/mg, respectively. The r-XynBS27 showed high xylose tolerance and was inhibited by some metal ions and by SDS. r-XynBS27 was employed as an additive in the bread making process. A decrease in firmness, stiffness and consistency, and improvements in specific volume and reducing sugar content were recorded.

Cultivable microbiome and its resistance to antimicrobials isolated from Zaprionus indianus

The objective of this study was to identify Z. indianus in PEJC and PESCAN. Listed are the following methods: to isolate bacteria from the integument of the Z. indianus species collected and to check the resistance of microorganisms to antibiotics. Collections of Z. indianus were performed in four seasons of the two parks. The results obtained suggest that the low amount of Z. indianus collected can be justified by environmental factors such as high average temperature and low average humidity. It is noted that there is a predominance of bacteria of the Enterobacteriaceae family found in both the PEJC and the PESCAN. The antibiogram performed for the isolated PEJC bacteria shows statistical significance when comparing the edge and inside values of the park. Studies with fungi were also carried out, and it was evidenced that Trichophyton spp. was the genus that most inhabited the two environments studied. The ability of fluconazole and ketoconazole to inhibit fungal growth was also investigated, and considering the concentration tested may suggest that they have good action spectra. Plasmid profile data show that 60% of antibiotic-resistant bacteria have plasmids. The values found show that Z. indianus can act as vectors of microorganisms that affect the healthy animals and humans and that these organisms may be influenced by seasons.

Development of a nanogold slot blot inhibition assay for the detection of antibodies against bovine herpesvirus type 1

Bovine herpesvirus type 1 (BoHV-1) is recognized as an important pathogen causing respiratory, reproductive, and neurological disorders in cattle and is associated with economic losses to animal industry. Accurate diagnostic methods are needed for prevention of disease transmission. While the virus neutralization test is considered the gold standard method, it requires maintenance of the virus and cell cultures, which is time consuming and expensive. Serological techniques such as enzyme-linked immunosorbent assay (ELISA) are widely applied, as these are easy to perform and provide quick results. In the present study, a nanogold slot blot inhibition assay was developed for the serological diagnosis of BoHV-1 and compared with standard ELISA and horseradish peroxidase (HRP) slot blot assays. Of 42 serum samples tested by ELISA, 32 (76.2%) were positive and 10 (23.8%), were negative. The sensitivity and specificity of the nanogold slot blot inhibition assay was similar to that observed for ELISA and HRP slot blot assays, and a strong correlation was observed between the tests. Thus, the nanogold slot blot inhibition assay may serve as an efficient and rapid alternative to ELISA in settings, where plate-reading equipment is lacking.

A conformational epitope mapped in the bovine herpesvirus type 1 envelope glycoprotein B by phage display and the HSV-1 3D structure.

The selected dodecapeptide (1)DRALYGPTVIDH(12) from a phage-displayed peptide library and the crystal structure of the envelope glycoprotein B (Env gB) from Herpes Simplex Virus type 1 (HSV-1) led us to the identification of a new discontinuous epitope on the Bovine herpesvirus type 1 (BoHV-1) Env gB. In silico analysis revealed a short BoHV-1 gB motif ((338)YKRD(341)) within a epitope region, with a high similarity to the motifs shared by the dodecapeptide N-terminal region ((5)YxARD(1)) and HSV-1 Env gB ((326)YARD(329)), in which the (328)Arg residue is described to be a neutralizing antibody target. Besides the characterization of an antibody-binding site of the BoHV-1 Env gB, we have demonstrated that the phage-fused peptide has the potential to be used as a reagent for virus diagnosis by phage-ELISA assay, which discriminated BoHV-1 infected serum samples from negative ones.