Claudia E. Thompson

New insights on the biology of swine respiratory tract mycoplasmas from a comparative genome analysis

BackgroundMycoplasma hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis live in swine respiratory tracts. M. flocculare, a commensal bacterium, is genetically closely related to M. hyopneumoniae, the causative agent of enzootic porcine pneumonia. M. hyorhinis is also pathogenic, causing polyserositis and arthritis. In this work, we present the genome sequences of M. flocculare and M. hyopneumoniae strain 7422, and we compare these genomes with the genomes of other M. hyoponeumoniae strain and to the a M. hyorhinis genome. These analyses were performed to identify possible characteristics that may help to explain the different behaviors of these species in swine respiratory tracts.ResultsThe overall genome organization of three species was analyzed, revealing that the ORF clusters (OCs) differ considerably and that inversions and rearrangements are common. Although M. flocculare and M. hyopneumoniae display a high degree of similarity with respect to the gene content, only some genomic regions display considerable synteny. Genes encoding proteins that may be involved in host-cell adhesion in M. hyopneumoniae and M. flocculare display differences in genomic structure and organization. Some genes encoding adhesins of the P97 family are absent in M. flocculare and some contain sequence differences or lack of domains that are considered to be important for adhesion to host cells. The phylogenetic relationship of the three species was confirmed by a phylogenomic approach. The set of genes involved in metabolism, especially in the uptake of precursors for nucleic acids synthesis and nucleotide metabolism, display some differences in copy number and the presence/absence in the three species.ConclusionsThe comparative analyses of three mycoplasma species that inhabit the swine respiratory tract facilitated the identification of some characteristics that may be related to their different behaviors. M. hyopneumoniae and M. flocculare display many differences that may help to explain why one species is pathogenic and the other is considered to be commensal. However, it was not possible to identify specific virulence determinant factors that could explain the differences in the pathogenicity of the analyzed species. The M. hyorhinis genome contains differences in some components involved in metabolism and evasion of the host’s immune system that may contribute to its growth aggressiveness. Several horizontal gene transfer events were identified. The phylogenomic analysis places M. hyopneumoniae, M. flocculare and M. hyorhinis in the hyopneumoniae clade.

Predicting the proteins of angomonas deanei, strigomonas culicis and their respective endosymbionts reveals new aspects of the trypanosomatidae family

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.

Genome of the avirulent human-infective trypanosome--Trypanosoma rangeli.

Background Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts. Methodology/Principal Findings The T. rangeli haploid genome is ∼24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heat-shock proteins. Conclusions/Significance Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets.

Metagenomic Analysis of the Microbiota from the Crop of an Invasive Snail Reveals a Rich Reservoir of Novel Genes

The shortage of petroleum reserves and the increase in CO2 emissions have raised global concerns and highlighted the importance of adopting sustainable energy sources. Second-generation ethanol made from lignocellulosic materials is considered to be one of the most promising fuels for vehicles. The giant snail Achatina fulica is an agricultural pest whose biotechnological potential has been largely untested. Here, the composition of the microbial population within the crop of this invasive land snail, as well as key genes involved in various biochemical pathways, have been explored for the first time. In a high-throughput approach, 318 Mbp of 454-Titanium shotgun metagenomic sequencing data were obtained. The predominant bacterial phylum found was Proteobacteria, followed by Bacteroidetes and Firmicutes. Viruses, Fungi, and Archaea were present to lesser extents. The functional analysis reveals a variety of microbial genes that could assist the host in the degradation of recalcitrant lignocellulose, detoxification of xenobiotics, and synthesis of essential amino acids and vitamins, contributing to the adaptability and wide-ranging diet of this snail. More than 2,700 genes encoding glycoside hydrolase (GH) domains and carbohydrate-binding modules were detected. When we compared GH profiles, we found an abundance of sequences coding for oligosaccharide-degrading enzymes (36%), very similar to those from wallabies and giant pandas, as well as many novel cellulase and hemicellulase coding sequences, which points to this model as a remarkable potential source of enzymes for the biofuel industry. Furthermore, this work is a major step toward the understanding of the unique genetic profile of the land snail holobiont.

Patterns of molecular evolution in pathogenesis-related proteins

The genes encoding 13 classes of pathogenesis-related (PR) proteins were examined for positive selection using maximum-likelihood (ML) models of codon substitution. The study involved 194 sequences from 54 species belonging to 37 genera. Although the sizes of the sequences examined varied from 237 bp for PR12 to 1,110 bp for PR7, most classes (9 out of 13) contained sequences made up of more than 400 nucleotides. Signs of positive selection were obtained for sites in PR proteins 4, 6, 8, 9 and 15 using an ML-based Bayesian method and likelihood ratio tests. These results confirm the importance of positive selection in proteins related to defense mechanisms already observed in a wide array of organisms.

Molecular modeling of pathogenesis-related proteins of family 5

The family of pathogenesis-related (PR) 5 proteins have diverse functions, and some of them are classified as thaumatins, osmotins, and inhibitors of α-amylase or trypsin. Although the specific function of many PR5 in plants is unknown, they are involved in the acquired systemic resistance and response to biotic stress, causing the inhibition of hyphal growth and reduction of spore germination, probably by a membrane permeabilization mechanism or by interaction with pathogen receptors. We have constructed three-dimensional models of four proteins belonging to the Rosaceae and Fagaceae botanical families by using the technique of comparative molecular modelling by homology. There are four main structural differences between all the PR5, corresponding to regions with replacements of amino acids. Folding and the secondary structures are very similar for all of them. However, the isoelectric point and charge distributions differ for earch protein.

Taxonomic and functional profiles of soil samples from Atlantic forest and Caatinga biomes in northeastern Brazil

Although microorganisms play crucial roles in ecosystems, metagenomic analyses of soil samples are quite scarce, especially in the Southern Hemisphere. In this work, the microbial diversity of soil samples from an Atlantic Forest and Caatinga was analyzed using a metagenomic approach. Proteobacteria and Actinobacteria were the dominant phyla in both samples. Among which, a significant proportion of stress‐resistant bacteria associated to organic matter degradation was found. Sequences related to metabolism of amino acids, nitrogen, and DNA and stress resistance were more frequent in Caatinga soil, while the forest sample showed the highest occurrence of hits annotated in phosphorous metabolism, defense mechanisms, and aromatic compound degradation subsystems. The principal component analysis (PCA) showed that our samples are close to the desert metagenomes in relation to taxonomy, but are more similar to rhizosphere microbiota in relation to the functional profiles. The data indicate that soil characteristics affect the taxonomic and functional distribution; these characteristics include low nutrient content, high drainage (both are sandy soils), vegetation, and exposure to stress. In both samples, a rapid turnover of organic matter with low greenhouse gas emission was suggested by the functional profiles obtained, reinforcing the importance of preserving natural areas.

A potential source for cellulolytic enzyme discovery and environmental aspects revealed through metagenomics of Brazilian mangroves.

The mangroves are among the most productive and biologically important environments. The possible presence of cellulolytic enzymes and microorganisms useful for biomass degradation as well as taxonomic and functional aspects of two Brazilian mangroves were evaluated using cultivation and metagenomic approaches. From a total of 296 microorganisms with visual differences in colony morphology and growth (including bacteria, yeast and filamentous fungus), 179 (60.5%) and 117 (39.5%) were isolated from the Rio de Janeiro (RJ) and Bahia (BA) samples, respectively. RJ metagenome showed the higher number of microbial isolates, which is consistent with its most conserved state and higher diversity. The metagenomic sequencing data showed similar predominant bacterial phyla in the BA and RJ mangroves with an abundance of Proteobacteria (57.8% and 44.6%), Firmicutes (11% and 12.3%) and Actinobacteria (8.4% and 7.5%). A higher number of enzymes involved in the degradation of polycyclic aromatic compounds were found in the BA mangrove. Specific sequences involved in the cellulolytic degradation, belonging to cellulases, hemicellulases, carbohydrate binding domains, dockerins and cohesins were identified, and it was possible to isolate cultivable fungi and bacteria related to biomass decomposition and with potential applications for the production of biofuels. These results showed that the mangroves possess all fundamental molecular tools required for building the cellulosome, which is required for the efficient degradation of cellulose material and sugar release.

Evaluation of the impact of functional diversification on Poaceae, Brassicaceae, Fabaceae, and Pinaceae alcohol dehydrogenase enzymes

The plant alcohol dehydrogenases (ADHs) have been intensively studied in the last years in terms of phylogeny and they have been widely used as a molecular marker. However, almost no information about their three-dimensional structure is available. Several studies point to functional diversification of the ADH, with evidence of its importance, in different organisms, in the ethanol, norepinephrine, dopamine, serotonin, and bile acid metabolism. Computational results demonstrated that in plants these enzymes are submitted to a functional diversification process, which is reinforced by experimental studies indicating distinct enzymatic functions as well as recruitment of specific genes in different tissues. The main objective of this article is to establish a correlation between the functional diversification occurring in the plant alcohol dehydrogenase family and the three-dimensional structures predicted for 17 ADH belonging to Poaceae, Brassicaceae, Fabaceae, and Pinaceae botanical families. Volume, molecular weight and surface areas are not markedly different among them. Important electrostatic and pI differences were observed with the residues responsible for some of them identified, corroborating the function diversification hypothesis. These data furnish important background information for future specific structure-function and evolutionary investigations.

Genomic analyses and transcriptional profiles of the glycoside hydrolase family 18 genes of the entomopathogenic fungus Metarhizium anisopliae.

Fungal chitin metabolism involves diverse processes such as metabolically active cell wall maintenance, basic nutrition, and different aspects of virulence. Chitinases are enzymes belonging to the glycoside hydrolase family 18 (GH18) and 19 (GH19) and are responsible for the hydrolysis of β-1,4-linkages in chitin. This linear homopolymer of N-acetyl-β-D-glucosamine is an essential constituent of fungal cell walls and arthropod exoskeletons. Several chitinases have been directly implicated in structural, morphogenetic, autolytic and nutritional activities of fungal cells. In the entomopathogen Metarhizium anisopliae, chitinases are also involved in virulence. Filamentous fungi genomes exhibit a higher number of chitinase-coding genes than bacteria or yeasts. The survey performed in the M. anisopliae genome has successfully identified 24 genes belonging to glycoside hydrolase family 18, including three previously experimentally determined chitinase-coding genes named chit1, chi2 and chi3. These putative chitinases were classified based on domain organization and phylogenetic analysis into the previously described A, B and C chitinase subgroups, and into a new subgroup D. Moreover, three GH18 proteins could be classified as putative endo-N-acetyl-β-D-glucosaminidases, enzymes that are associated with deglycosylation and were therefore assigned to a new subgroup E. The transcriptional profile of the GH18 genes was evaluated by qPCR with RNA extracted from eight culture conditions, representing different stages of development or different nutritional states. The transcripts from the GH18 genes were detected in at least one of the different M. anisopliae developmental stages, thus validating the proposed genes. Moreover, not all members from the same chitinase subgroup presented equal patterns of transcript expression under the eight distinct conditions studied. The determination of M. anisopliae chitinases and ENGases and a more detailed study concerning the enzymes’ roles in morphological or nutritional functions will allow comprehensive insights into the chitinolytic potential of this highly infective entomopathogenic fungus.