Pedro Marcus Pereira Vidigal

Identification of Genes and Pathways Related to Phenol Degradation in Metagenomic Libraries from Petroleum Refinery Wastewater

Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.

Tripping over emerging pathogens around the world: A phylogeographical approach for determining the epidemiology of Porcine circovirus-2 (PCV-2), considering global trading

Porcine circovirus-2 (PCV-2) is an emerging virus associated with a number of different syndromes in pigs known as Porcine Circovirus Associated Diseases (PCVAD). Since its identification and characterization in the early 1990s, PCV-2 has achieved a worldwide distribution, becoming endemic in most pig-producing countries, and is currently considered as the main cause of losses on pig farms. In this study, we analyzed the main routes of the spread of PCV-2 between pig-producing countries using phylogenetic and phylogeographical approaches. A search for PCV-2 genome sequences in GenBank was performed, and the 420 PCV-2 sequences obtained were grouped into haplotypes (group of sequences that showed 100% identity), based on the infinite sites model of genome evolution. A phylogenetic hypothesis was inferred by Bayesian Inference for the classification of viral strains and a haplotype network was constructed by Median Joining to predict the geographical distribution of and genealogical relationships between haplotypes. In order to establish an epidemiological and economic context in these analyses, we considered all information about PCV-2 sequences available in GenBank, including papers published on viral isolation, and live pig trading statistics available on the UN Comtrade database (http://comtrade.un.org/). In these analyses, we identified a strong correlation between the means of PCV-2 dispersal predicted by the haplotype network and the statistics on the international trading of live pigs. This correlation provides a new perspective on the epidemiology of PCV-2, highlighting the importance of the movement of animals around the world in the emergence of new pathogens, and showing the need for effective sanitary barriers when trading live animals.

Identification of an Emergent Porcine Circovirus-2 in Vaccinated Pigs from a Brazilian Farm during a Postweaning Multisystemic Wasting Syndrome Outbreak

ABSTRACT Three porcine circovirus-2 strains were isolated from pigs on a Brazilian farm during an outbreak, indicating a vaccine failure. They present identical genomic sequences, with high identities to other isolates that were also related to vaccination failures, supporting the recent theory about an antigen drift being associated with vaccine failures throughout the world.

UFV-P2 as a member of the Luz24likevirus genus: a new overview on comparative functional genome analyses of the LUZ24-like phages.

BackgroundPhages infecting spoilage microorganisms have been considered as alternative biocontrol agents, and the study of their genomes is essential to their safe use in foods. UFV-P2 is a new Pseudomonas fluorescens-specific phage that has been tested for its ability to inhibit milk proteolysis.ResultsThe genome of the phage UFV-P2 is composed of bidirectional modules and presented 75 functionally predict ORFs, forming clusters of early and late transcription. Further genomic comparisons of Pseudomonas-specific phages showed that these viruses could be classified according to conserved segments that appear be free from genome rearrangements, called locally collinear blocks (LCBs). In addition, the genome organization of the phage UFV-P2 was shown to be similar to that of phages PaP3 and LUZ24 which have recently been classified as a Luz24likevirus.ConclusionsWe have presented the functional annotation of UFV-P2, a new Pseudomonas fluorescens phage. Based on structural genomic comparison and phylogenetic clustering, we suggest the classification of UFV-P2 in the Luz24likevirus genus, and present a set of shared locally collinear blocks as the genomic signature for this genus.

Porcine Circovirus-2 Viral Load versus Lesions in Pigs: Perspectives for Post-weaning Multisystemic Wasting Syndrome

Porcine circovirus-2 (PCV-2) is the main agent related to post-weaning multisystemic wasting syndrome (PMWS) and it is also associated with other syndromes affecting pigs. Not all pigs infected with PCV-2 will develop PMWS and the incidence of PMWS is higher when coinfecting viral and bacterial pathogens are present. In this study, PCV-2 viral loads were evaluated in the tissues of animals with and without PMWS in order to investigate the relationship between viral load and microscopical lesions. Lymph nodes had the highest average viral load, but there was no significant difference between lesion severity and the viral load in these structures. There was no significant difference between the average viral load in inguinal lymph nodes of animals with and without PMWS. However, samples from pigs with PMWS had more severe lesions compared with samples from non-PMWS animals. These findings suggest that other infectious and non-infectious cofactors may be important in the pathogenesis of PMWS.

Tracking the molecular epidemiology of Brazilian Infectious bursal disease virus (IBDV) isolates.

Infectious bursal disease is a highly contagious disease of young chickens caused by Infectious bursal disease virus (IBDV). Genome segment A encodes the capsid protein (VP2), while segment B encodes the RNA-dependent RNA polymerase (VP1). In the present study, we trace the molecular epidemiology of IBDV in Brazil by analyzing 29 isolates collected in the major regions of poultry production. To genetically characterize the isolates, phylogenetic and population dynamic analyses were conducted using 68 VP1 (2634 nt) and 102 VP2 (1356 nt) coding sequences from IBDV isolates from different regions of the world. Furthermore, the evolution of IBDV was analyzed by characterizing the selective forces that operated during the diversification of viral isolates. We show that IBDV isolates were introduced into Brazil mainly from the Netherlands and the USA. These introductions were associated with all Brazilian poultry production regions analyzed in this work. In addition, we show that the evolution of IBDV has been shaped by a combination of very low recombination rates and relatively high rates of nucleotide substitution (2.988×10(-4) for VP1 and 3.2937×10(-4) for VP2), which themselves are a function of purifying selection operating on VP1 and VP2. Furthermore, our extended Bayesian skyline plot suggests that the increase in the effective population size of isolates of IBDV is consistent with its epidemiological history, with a large increase during the emergence of acute outbreaks of IBD in the 1980s.

Genomic Sequence of the Yeast Kluyveromyces marxianus CCT 7735 (UFV-3), a Highly Lactose-Fermenting Yeast Isolated from the Brazilian Dairy Industry

ABSTRACT Here, we present the draft genome sequence of Kluyveromyces marxianus CCT 7735 (UFV-3), including the eight chromosomes and the mitochondrial genomic sequences.

Genetic evaluation of IS900 partial sequence of Mycobacterium avium subsp. paratuberculosis Brazilian isolates from bovine milk

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis. Insertion sequence IS900 is used for the identification of MAP. The objective of this study was to verify the genetic conservation of IS900 sequences in raw milk samples. To evaluate genetic conservation, 206 quarter milk samples and 16 bulk-tank milk samples were collected. DNA extraction and IS900 PCR were performed in all samples. Six samples amplified the expected fragment. To confirm the identity of the amplified fragments, PCR products were cloned and sequenced. The resulting sequences were compared with other MAP sequences from GenBank, and it was possible to identify eight polymorphic regions and to form five distinct haplotypes. The number of mutations in each haplotype was verified. IS900 sequence is a very well-conserved sequence that could be used as tool for the molecular detection of this agent and epidemiological purposes. The results showed the first genetic analysis on Brazilian isolates of MAP.

A deep insight into the whole transcriptome of midguts, ovaries and salivary glands of the Amblyomma sculptum tick

Ticks stand out as the second most important vector of human pathogens and their control is a key issue for public health strategies. The success of these arthropods to parasitize a large range of vertebrate hosts and their importance as a vector of rickettsiosis are intimately related with the physiology of its internal organs. The midguts, ovaries and salivary glands all participate as key sites for rickettsial entry into tick hemolymph, ovarian amplification and transmission to vertebrate hosts, respectively. Although transcriptomic efforts have documented several tick sialomes aspects, very little is known about the repertoire of genes that orchestrate the physiology of midguts and ovaries. The aim of this study was to construct a deep catalogue from the repertoire of genes and putative proteins expressed in the selected internal organs of Amblyomma scultpum, a key vector of spotted fever rickettsiosis, through the employment of a RNAseq strategy. A total of 200 million reads derived from midguts, ovaries and salivary glands and sequenced by Illumina HiSeq, Ion Torrent PGM and 454 pyrosequencing, were assembled into contigs. The mapping of the contigs allowed for the identification of 25,569 CDS expressed in midguts, 21,230 in the ovaries and 10,697 in salivary glands. In depth analysis of the transcriptomes revealed several organ specific metabolic processes that expand the knowledge base of tick biology and its role in infectious diseases. The annotation approach used in this work facilitated the discovery of a higher number of novel genes and processes and gives insight into the roles that they play in the A.scultpum transcriptome.

Transcriptome analysis of the thermotolerant yeast Kluyveromyces marxianus CCT 7735 under ethanol stress

The thermotolerant yeast Kluyveromyces marxianus displays a potential to be used for ethanol production from both whey and lignocellulosic biomass at elevated temperatures, which is highly alluring to reduce the cost of the bioprocess. Nevertheless, contrary to Saccharomyces cerevisiae, K. marxianus cannot tolerate high ethanol concentrations. We report the transcriptional profile alterations in K. marxianus under ethanol stress in order to gain insights about mechanisms involved with ethanol response. Time-dependent changes have been characterized under the exposure of 6% ethanol and compared with the unstressed cells prior to the ethanol addition. Our results reveal that the metabolic flow through the central metabolic pathways is impaired under the applied ethanol stress. Consistent with these results, we also observe that genes involved with ribosome biogenesis are downregulated and gene-encoding heat shock proteins are upregulated. Remarkably, the expression of some gene-encoding enzymes related to unsaturated fatty acid and ergosterol biosynthesis decreases upon ethanol exposure, and free fatty acid and ergosterol measurements demonstrate that their content in K. marxianus does not change under this stress. These results are in contrast to the increase previously reported with S. cerevisiae subjected to ethanol stress and suggest that the restructuration of K. marxianus membrane composition differs in the two yeasts which gives important clues to understand the low ethanol tolerance of K. marxianus compared to S. cerevisiae.