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.
Key Message
A resistant
E. grandis
genotype showed a constitutive overexpression of genes related to resistance to myrtle rust caused by
A. psidii
.
Myrtle rust caused by
Austropuccinia psidii
is ...considered one of the most important fungal diseases affecting
Eucalyptus
spp. plantations in Brazil. Although the selection and planting of resistant eucalypt genotypes have been the major strategies to manage the disease in Brazil, the molecular mechanisms involved in resistance are still unclear. In this study, we evaluated the gene expression profile of two contrasting
Eucalyptus grandis
genotypes in resistance level to rust by RNA-Seq. The two genotypes showed a very different background gene expression level even without
A. psidii
infection. The resistant genotype had a constitutive overexpression of a large number of protein-coding genes compared to the susceptible genotype. These genes were mainly associated with signal transduction, photosynthesis, regulation and response to salicylic acid (SA), and protein kinase leucine-rich receptors (PK-LRR). PK-LRR and SA mediated disease resistance are well known to be effective against obligate biotroph pathogens, such as
A. psidii
. In addition, at 24 h after infection, the susceptible genotype was able to activate some response, however, several resistance-related proteins had their expression level reduced with
A. psidii
infection. Here, we present the first analysis of
E. grandis
genotypes transcriptomes infected by
A. psidii
and it reveals a constitutive overexpression of several resistance-related genes in the resistant genotype compared to the susceptible one. Our findings have the potential to be used as candidate molecular markers for resistance to myrtle rust.
Bovine mastitis remains the main cause of economic losses for dairy farmers. Mammary pathogenic Escherichia coli (MPEC) is related to an acute mastitis and its treatment is still based on the use of ...antibiotics. In the era of antimicrobial resistance (AMR), bacterial viruses (bacteriophages) present as an efficient treatment or prophylactic option. However, this makes it essential that its genetic structure, stability and interaction with the host immune system be thoroughly characterized. The present study analyzed a novel, broad host-range anti-mastitis agent, the T4virus vB_EcoM-UFV13 in genomic terms, and its activity against a MPEC strain in an experimental E. coli-induced mastitis mouse model. 4,975 Single Nucleotide Polymorphisms (SNPs) were assigned between vB_EcoM-UFV13 and E. coli phage T4 genomes with high impact on coding sequences (CDS) (37.60%) for virion proteins. Phylogenetic trees and genome analysis supported a recent infection mix between vB_EcoM-UFV13 and Shigella phage Shfl2. After a viral stability evaluation (e.g pH and temperature), intramammary administration (MOI 10) resulted in a 10-fold reduction in bacterial load. Furthermore, pro-inflammatory cytokines, such as IL-6 and TNF-α, were observed after viral treatment. This work brings the whole characterization and immune response to vB_EcoM-UFV13, a biocontrol candidate for bovine mastitis.
Asian soybean rust (ASR), caused by the obligate biotrophic fungus
Phakopsora pachyrhizi,
is one of most important diseases in the soybean (
Glycine max
(L.) Merr.) agribusiness. The identification ...and characterization of genes related to plant defense responses to fungal infection are essential to develop ASR-resistant plants. In this work, we describe four soybean genes,
GmbZIP62
,
GmbZIP105
,
GmbZIPE1
, and
GmbZIPE2
, which encode transcription factors containing a basic leucine zipper (bZIP) domain from two divergent classes, and that are responsive to
P. pachyrhizi
infection. Molecular phylogenetic analyses demonstrated that these genes encode proteins similar to bZIP factors responsive to pathogens. Yeast transactivation assays showed that only GmbZIP62 has strong transactivation activity in yeast. In addition, three of the bZIP transcription factors analyzed were also differentially expressed by plant defense hormones, and all were differentially expressed by fungal attack, indicating that these proteins might participate in response to ASR infection. The results suggested that these bZIP proteins are part of the plant defense response to
P. pachyrhizi
infection, by regulating the gene expression related to ASR infection responses. These bZIP genes are potential targets to obtain new soybean genotypes resistant to ASR.
•Trueperella pyogenes and Escherichia coli can cause economic losses, mainly through mastitis and metritis in dairy cows.•The ability of T. pyogenes to form biofilm is implicated in chronic ...infections by avoiding immune system recognition and antibiotic penetration.•vB_EcoM-UFV13 genome sequencing revealed a broad range of virion-associated hydrolases.•Escherichia phage UFV13 is not able to infect T. pyogenes, however it can act in biofilm disruption.•Multiplicity of infection (MOI) of 10 showed a statistically significant reduction in biofilm formation.
Trueperella pyogenes is an opportunistic pathogen of many animal species. It causes economic losses worldwide, through mastitis, metritis and mainly endometritis in dairy cows. The ability of this bacterium to form biofilms is implicated in chronic infections through hampering immune system recognition and antibiotic penetration. Since it is difficult to eradicate T. pyogenes infections with antibiotics, phage therapy presents itself as a non-toxic, effective and economically viable alternative. The present study evaluated the use of the bacteriophage vB_EcoM-UFV13 (UFV13) in the prevention of T. pyogenes biofilm development. Based upon two different approaches (crystal violet and sessile cell counting) we observed that only a multiplicity of infection (MOI) of 10 showed a statistically significant reduction in biofilm formation. Although the exact mechanisms of biofilm disruption and cell-adhesion inhibition have not been determined, genome sequence analysis of the Escherichia phage UFV13 revealed a repertoire of virion-associated peptidoglycan hydrolases (VAPGHs). The present study presents new findings regarding the disruption of biofilm formation of a Gram-positive bacterium. Subsequent transcriptomic and proteomic research will help us to understand the exact interaction mechanisms between UFV13 and T. pyogenes.
The Geminiviridae family encompasses a group of single-stranded DNA viruses with twinned and quasi-isometric virions, which infect a wide range of dicotyledonous and monocotyledonous plants and are ...responsible for significant economic losses worldwide. Geminiviruses are divided into nine genera, according to their insect vector, host range, genome organization, and phylogeny reconstruction. Using rolling-circle amplification approaches along with high-throughput sequencing technologies, thousands of full-length geminivirus and satellite genome sequences were amplified and have become available in public databases. As a consequence, many important challenges have emerged, namely, how to classify, store, and analyze massive datasets as well as how to extract information or new knowledge. Data mining approaches, mainly supported by machine learning (ML) techniques, are a natural means for high-throughput data analysis in the context of genomics, transcriptomics, proteomics, and metabolomics.
Here, we describe the development of a data warehouse enriched with ML approaches, designated geminivirus.org. We implemented search modules, bioinformatics tools, and ML methods to retrieve high precision information, demarcate species, and create classifiers for genera and open reading frames (ORFs) of geminivirus genomes.
The use of data mining techniques such as ETL (Extract, Transform, Load) to feed our database, as well as algorithms based on machine learning for knowledge extraction, allowed us to obtain a database with quality data and suitable tools for bioinformatics analysis. The Geminivirus Data Warehouse (geminivirus.org) offers a simple and user-friendly environment for information retrieval and knowledge discovery related to geminiviruses.
•Ceratocystis fimbriata LPF1912 has a compact genome with 43% as coding sequencing.•A set of 4919 orthologous gene clusters represent the minimum quorum for the three pathogens.•Two Ceratocystis ...pathogens showed a different pathogenicity-related genes compared to Calonectria pseudoreteaudii.•The most of DEGs of C. fimbriata LPF1912 is related to its pathogenicity in Eucalyptus spp.
Ceratocystis fimbriata is an important plant pathogen known to cause Ceratocystis Wilt (CW), a prevalent fungal disease known to affect Eucalyptus spp. plantations in Brazil. To better understand the molecular mechanisms related to pathogenicity in eucalyptus, we generated a high-quality assembly and annotation of the Ce. fimbriata LPF1912 isolate (LPF1912) genome, as well as the first transcriptome of LPF1912 from 16 eucalyptus clones at three infection incubation periods (12, 18, and 24 h). The LPF1912 genome assembly contains 805 scaffolds, totaling 31.8 Mb, with 43% of the genome estimated to be coding sequence comprised of 7,390 protein-coding genes of which 626 (8.5%) were classified as secreted proteins, 120 ribosomal RNAs, and 532 transfer RNAs. Comparative genomic analysis among three eucalyptus fungal pathogens (Ce. fimbriata, Ce. eucalypticola, and Calonectria pseudoreteaudii), showed high similarity in the proteome (21.81%) and secretome (52.01%) of LPF1912 and Ce. eucalypticola. GO annotation of pathogenicity-related genes of LPF1912 and Ce. eucalypticola, revealed enrichment in cell wall degrading enzymes (CWDEs), and lipid/cutin metabolism for Ca. pseudoreteaudii. Additionally, a transcriptome analysis between resistant and susceptible eucalyptus clones to CW infection indicated that a majority (11) of LPF1912 differentially expressed genes had GO terms associated with enzymatic functions, such as the polygalacturonase gene family, confirming the crucial role of CWDEs for Ce. fimbriata pathogenicity. Finally, our genomic and transcriptomic analysis approach provides a better understanding of the mechanisms involved in Ce. fimbriata pathogenesis, as well as a framework for further studies.
► We analyzed the main routes of the spread of PCV-2 between pig-producing countries. ► PCV-2 is widely distributed throughout pig-producing countries. ► Can., Den., Fr., Neth., and the USA are the ...main origins of the predicted routes. ► The dispersal routes of PCV-2 have a strong correlation with the trading of live pigs.
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.
Erwinia psidii is a gram‐negative bacterium that threatens both guava and eucalypt plantations in several countries. Despite the economic importance of both crops, nothing is currently known about ...the molecular mechanisms underlying E. psidii pathogenicity and, consequently, how it evolved to infect Eucalyptus species besides its presumed native host Psidium guajava. In this study, we predicted putative type III secretion system effectors that may play important roles during plant–E. psidii interactions and conducted effector structure and phylogenetic analyses to gain important insights into their function and evolution. For that, the whole genomes of four E. psidii strains that exhibit differential aggressiveness towards eucalypt clones were sequenced and their effector repertoires predicted based on sequence identity with known effectors of the model phytopathogen Erwinia amylovora. Only proteins sharing significant sequence identity with the DspE and Eop1 effectors were found. Here, it is shown that these two E. psidii effectors retain all structural characteristics of their corresponding protein superfamilies, but exhibit allelic variations that are consistent with the observed aggressiveness differences between strains. Phylogenetic analyses revealed that whereas E. psidii housekeeping gene sequences are more closely related to those from Erwinia tracheiphila, the effector (either nucleotide or amino acid) sequences are more closely related to their Pantoea agglomerans counterparts, suggesting that dspE and eop1 were both acquired through horizontal gene transfer from the latter bacterial species. The results of this study provide important insights on E. psidii pathogenicity and set the stage for future effector functional studies.
Erwinia psidii type III effectors DspE and Eop1 retain structural characteristics of their protein superfamily members and their encoding genes seem to have been acquired by horizontal transfer.
► The molecular epidemiology of Brazilian IBDV isolates is assessed. ► Very low recombination rates and high rates of nucleotide substitution were detected. ► IBDV isolates were introduced into ...Brazil mainly from the Netherlands and the USA. ► IBDV population had a high increase during the emergence of acute outbreaks in the 1980s.
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 (2634nt) and 102 VP2 (1356nt) 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.
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