Feeding on soybean crops changed gut bacteria diversity of the southern green stinkbug (Nezara viridula) and reduced negative effects of some associated bacteria.

BACKGROUND: The southern green stinkbug (Nezara viridula) is a mayor pest of soybean. However, the mechanism underlying stinkbug resistance to soybean defenses is yet ignored. Although gut bacteria could play an essential role in tolerating plant defenses, most studies testing questions related to insect-plant-bacteria interactions have been performed in laboratory condition. Here we performed experiments in laboratory and field conditions with N. viridula and its gut bacteria, studying gut lipid peroxidaxion levels and cysteine activity in infected and unifected nymphs, testing the hypothesis that feeding on field-grown soybean decreases bacterial abundance in stinkbugs. RESULTS: Gut bacterial abundance and infection ratio were higher in N. viridula adults reared in laboratory than in those collected from soybean crops, suggesting that stinkbugs in field conditions may modulate gut bacterial colonization. Manipulating gut microbiota by infecting stinkbugs with Yokenella sp. showed that these bacteria abundance decreased in field conditions, and negatively affected stinkbugs performance and were more aggressive in laboratory rearing than in field conditions. Infected nymphs that fed on soybean pods had lower mortality, higher mass and shorter development period than those reared in the laboratory, and suggested that field conditions helped nymphs to recover from Yokenella sp. infection, despite of increased lipid peroxidation and decreased cysteine proteases activity in nymphs' guts. CONCLUSIONS: Our results demonstrated that feeding on field-grown soybean reduced bacterial abundance and infection in guts of N. viridula and highlighted the importance to test functional activities or pathogenicity of microbes under realistic field conditions prior to establish conclusions on three trophic interactions. © 2022 Society of Chemical Industry.

Characterization of a novel GH10 alkali-thermostable xylanase from a termite microbiome.

The aim of the present study was to assess the biochemical and molecular structural characteristics of a novel alkali-thermostable GH10 xylanase (Xyl10B) identified in a termite gut microbiome by a shotgun metagenomic approach. This endoxylanase candidate was amplified, cloned, heterologously expressed in Escherichia coli and purified. The recombinant enzyme was active at a broad range of temperatures (37-60 ºC) and pH values (4-10), with optimal activity at 50 ºC and pH 9. Moreover, its activity remained at more than 80% of its maximum at 50 °C for 8 h. In addition, Xyl10B was found to be stable in the presence of salt and several ions and chemical reagents frequently used in the industry. These characteristics make this enzyme an interesting candidate for pulp and paper bleaching industries, since this process requires enzymes without cellulase activity and resistant to high temperatures and alkaline pH (thermo-alkaliphilic enzymes). The products of xylan hydrolysis by Xyl10B (short xylooligosaccharides, xylose and xylobiose) could be suitable for application as prebiotics and in the production of bioethanol.

Characterization of two GH5 endoglucanases from termite microbiome using synthetic metagenomics.

Here, we characterize two novel GH5 endoglucanases (GH5CelA and GH5CelB) from an uncultured bacterium identified in termite gut microbiomes. Both genes were codon-optimized, synthetized, cloned, and expressed as recombinant proteins in Escherichia coli for subsequent purification. Both enzymes showed activity on the pNPC and barley ß-glucan substrates, whereas GH5CelB also showed low activity on carboxymethyl cellulose. The optimum conditions for both enzymes were an acid pH (5) and moderate temperature (35 to 50 °C). The enzymes differed in the kinetic profiles and patterns of the generated hydrolysis products. A structural-based modeling analysis indicated that both enzymes possess a typical (ß/α)8-barrel fold characteristic of GH5 family, with some differential features in the active site cleft. Also, GH5CelB presents a putative secondary binding site. Furthermore, adjacent to the active site of GH5CelA and GH5CelB, a whole subdomain rarely found in GH5 family may participate in substrate binding and thermal stability.Therefore, GH5CelA may be a good candidate for the production of cello-oligosaccharides of different degrees of polymerization applicable for feed and food industries, including prebiotics. On the other hand, GH5CelB could be useful in an enzymatic cocktail for the production of lignocellulosic bioethanol, because of the production of glucose as a hydrolysis product. Key Points • Synthetic metagenomics is a powerful approach for discovering novel enzymes. • Two novel GH5 endoglucanases from nonculturable microorganisms were characterized. • Structural differences between them and other GH5 endoglucanases were observed. • The enzymes may be good candidates for feed, food, and/or bioethanol industries.

A Phenotypic Characterization of Two Isolates of a Multidrug-Resistant Outbreak Strain of Mycobacterium tuberculosis with Opposite Epidemiological Fitness.

Tuberculosis (TB) is an infectious disease, caused by Mycobacterium tuberculosis, primarily affecting the lungs. The M. tuberculosis strain of the Haarlem family named M was responsible for a large multidrug-resistant TB (MDR-TB) outbreak in Buenos Aires. This outbreak started in the early 1990s and in the mid 2000s still accounted for 29% of all MDR-TB cases in Argentina. By contrast, a clonal variant of strain M, named 410, has caused a single tuberculosis case since the onset of the outbreak. The molecular bases of the high epidemiological fitness of the M strain remain unclear. To assess its unique molecular properties, herein, we performed a comparative protein and lipid analysis of a representative clone of the M strain (Mp) and the nonprosperous M variant 410. We also evaluated their growth in low pH. The variant 410 had higher levels of latency proteins under standard conditions and delayed growth at low pH, suggesting that it is more sensitive to stress stimuli than Mp. Moreover, Mp showed higher levels of mycolic acids covalently attached to the cell wall and lower accumulation of free mycolic acids in the outer layer than the 410 strain. The low expression of latency proteins together with the reduced content of surface mycolic acids may facilitate Mp to evade the host immune responses.

Neotropical termite microbiomes as sources of novel plant cell wall degrading enzymes.

In this study, we used shotgun metagenomic sequencing to characterise the microbial metabolic potential for lignocellulose transformation in the gut of two colonies of Argentine higher termite species with different feeding habits, Cortaritermes fulviceps and Nasutitermes aquilinus. Our goal was to assess the microbial community compositions and metabolic capacity, and to identify genes involved in lignocellulose degradation. Individuals from both termite species contained the same five dominant bacterial phyla (Spirochaetes, Firmicutes, Proteobacteria, Fibrobacteres and Bacteroidetes) although with different relative abundances. However, detected functional capacity varied, with C. fulviceps (a grass-wood-feeder) gut microbiome samples containing more genes related to amino acid metabolism, whereas N. aquilinus (a wood-feeder) gut microbiome samples were enriched in genes involved in carbohydrate metabolism and cellulose degradation. The C. fulviceps gut microbiome was enriched specifically in genes coding for debranching- and oligosaccharide-degrading enzymes. These findings suggest an association between the primary food source and the predicted categories of the enzymes present in the gut microbiomes of each species. To further investigate the termite microbiomes as sources of biotechnologically relevant glycosyl hydrolases, a putative GH10 endo-ß-1,4-xylanase, Xyl10E, was cloned and expressed in Escherichia coli. Functional analysis of the recombinant metagenome-derived enzyme showed high specificity towards beechwood xylan (288.1 IU/mg), with the optimum activity at 50 °C and a pH-activity range from 5 to 10. These characteristics suggest that Xy110E may be a promising candidate for further development in lignocellulose deconstruction applications.

In silico identification of immunotherapeutic and diagnostic targets in the glycosylphosphatidylinositol metabolism of the coccidian Sarcocystis aucheniae.

Meat of the South American camelids (SACs) llama and alpaca is an important source of animal protein and income for rural families in the Andes, and a product with significant growth potential for local and international markets. However, infestation with macroscopic cysts of the coccidian protozoon Sarcocystis aucheniae, a parasitosis known as SAC sarcocystosis, significantly hampers its commercialization. There are no validated methods to diagnose the presence of S. aucheniae cysts other than carcass examination. Moreover, there are no available drugs or vaccines to cure or prevent SAC sarcocystosis. Identification of relevant molecules that act at the host-pathogen interface can significantly contribute to the control of this disease. It has been shown for other pathogenic protozoa that glycosylphosphatidylinositol (GPI) is a critical molecule implicated in parasite survival and pathogenicity. This study focused on the identification of the enzymes that participate in the S. aucheniae GPI biosynthetic pathway and the repertoire of the parasite GPI-anchored proteins (GPI-APs). To this aim, RNA was extracted from parasite cysts and the transcriptome was sequenced and translated into amino acid sequences. The generated database was mined using sequences of well-characterized GPI biosynthetic enzymes of Saccharomyces cerevisiae and Toxoplasma gondii. Eleven enzymes predicted to participate in the S. aucheniae GPI biosynthetic pathway were identified. On the other hand, the database was searched for proteins carrying an N-terminal signal peptide and a single C-terminal transmembrane region containing a GPI anchor signal. Twenty-four GPI-anchored peptides were identified, of which nine are likely S. aucheniae-specific, and 15 are homologous to membrane proteins of other coccidians. Among the latter, 13 belong to the SRS domain superfamily, an extensive group of coccidian GPI-anchored proteins that mediate parasite interaction with their host. Phylogenetic analysis showed a great degree of intra- and inter-specific divergence among SRS family proteins. In vitro and in vivo experiments are needed to validate S. aucheniae GPI biosynthetic enzymes and GPI-APs as drug targets and/or as vaccine or diagnostic antigens.

Analysing nonsynonymous mutations between two Mycobacterium bovis strains with contrasting pathogenic profiles.

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis, a chronic infectious disease that can affect cattle, other domesticated species, wild animals and humans. This disease produces important economic losses worldwide. Two M. bovis strains (04-303 and 534) have been isolated in Argentina. Whereas the 04-303 strain was isolated from a wild boar, the 534 strain was obtained from cattle. In a previous study, six weeks after infection, the 04-303 strain induced 100% mortality in mice. By contrast, mice infected with the 534 strain survived, with limited tissue damage, after four months. In this study we compared all predictive proteins encoded in both M. bovis genomes. The comparative analysis revealed 141 polymorphic proteins between both strains. From these proteins, nine virulence proteins showed polymorphisms in 04-303, whereas five did it in the 534 strain. Remarkably, both strains contained a high level of polymorphism in proteins related to phthiocerol dimycocerosate (PDIM) synthesis or transport. Further experimental evidence indicated that only mutations in the 534 strain have an impact on PDIM synthesis. The observed reduction in PDIM content in the 534 strain, together with its low capacity to induce phagosome arrest, may be associated with the reported deficiency of this strain to replicate and survive inside bovine macrophages. The findings of this study could contribute to a better understanding of pathogenicity and virulence aspects of M. bovis, which is essential for further studies aiming at developing new vaccines and diagnostic techniques for bovines.

Modelling growth/no growth interface of Zygosaccharomyces bailii in simulated acid sauces as a function of natamycin, xanthan gum and sodium chloride concentrations.

Probabilistic microbial modelling using logistic regression was used to predict the growth/no growth (G/NG) interfaces of Zygosaccharomyces bailii in simulated acid sauces as a function of natamycin, xanthan gum (XG) and sodium chloride concentrations. The growth was assessed colorimetrically by using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride and 2-methoxy-1,4-naphthoquinone as detection reagents. The logistic regression model successfully predicted G/NG probability. The detection reagents used allowed the evaluation of G/NG interfaces in opaque systems with an excellent agreement with the plate count method. Natamycin concentration of 12 mg/L was needed to inhibit Z. bailii growth independently of the presence of XG and/or NaCl. Addition of 3.00 and 6.00% of NaCl exerted an antagonistic effect on natamycin action. Furthermore, addition of 0.25 and 0.50% XG decreased natamycin and/or NaCl action. However, an increased in XG concentration to 1.00% decreased yeast growth. Mentioned results highlighted the importance of the correct selection of stress factors applied to inhibit Z. bailii growth.

Data supporting the growth/no growth interface of Zygosaccharomyces bailii in simulated acid sauces.

This article contains experimental data, images and methods for the growth/no growth interface of Zygosaccharomyces bailii in simulated acid sauces. Mentioned data are related to the research article "Modeling growth/no growth interface of Zygosaccharomyces bailii in simulated acid sauces as a function of natamycin, xanthan gum and sodium chloride concentrations" (Zalazar et al., 2018) [1]. The growth was assessed colorimetrically by using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride and 2-methoxy-1,4-naphthoquinone as detection reagents. Furthermore, yeast growth was confirmed by plate count.

Characterization of the two component regulatory system PhoPR in Mycobacterium bovis.

Mycobacterium bovis is the causative agent of bovine tuberculosis and is a member of Mycobacterium tuberculosis complex, which causes tuberculosis in a number of mammals including humans. Previous studies have shown that the genes encoding the two-component system PhoPR, which regulates several genes involved in the virulence of M. tuberculosis, are polymorphic in M. bovis, when compared to M. tuberculosis, which results in a dysfunctional two-component system. In this study we investigated the role of PhoPR in two M. bovis strains with differing degrees of virulence. We found that the deletion of phoP in an M. bovis isolate reduced its capacity of inducing phagosomal arrest in bovine macrophages. By gene expression analysis, we demonstrated that, in both M. bovis strains, PhoP regulates the expression of a putative lipid desaturase Mb1404-Mb1405, a protein involved in redox stress AhpC, the sulfolipid transporter Mmpl8 and the secreted antigen ESAT-6. Furthermore, the lack of PhoP increased the sensitivity to acidic stress and alteration of the biofilm/pellicle formation of M. bovis. Both these phenotypes are connected to bacterial redox homeostasis. Therefore, the results of this study suggest a role of PhoPR in M. bovis to be linked to the mechanisms that mycobacteria display to maintain their redox balance.

Characterized non-transient microbiota from stinkbug (Nezara viridula) midgut deactivates soybean chemical defenses.

The Southern green stinkbug (N. viridula) feeds on developing soybean seeds in spite of their strong defenses against herbivory, making this pest one of the most harmful to soybean crops. To test the hypothesis that midgut bacterial community allows stinkbugs to tolerate chemical defenses of soybean developing seeds, we identified and characterized midgut microbiota of stinkbugs collected from soybean crops, different secondary plant hosts or insects at diapause on Eucalyptus trees. Our study demonstrated that while more than 54% of N. viridula adults collected in the field had no detectable bacteria in the V1-V3 midgut ventricles, the guts of the rest of stinkbugs were colonized by non-transient microbiota (NTM) and transient microbiota not present in stinkbugs at diapause. While transient microbiota Bacillus sp., Micrococcus sp., Streptomyces sp., Staphylococcus sp. and others had low abundance, NTM microbiota was represented by Yokenella sp., Pantoea sp. and Enterococcus sp. isolates. We found some isolates that showed in vitro ß-glucosidase and raffinase activities plus the ability to degrade isoflavonoids and deactivate soybean protease inhibitors. Our results suggest that the stinkbugs´ NTM microbiota may impact on nutrition, detoxification and deactivation of chemical defenses, and Enterococcus sp., Yokenella sp. and Pantoea sp. strains might help stinkbugs to feed on soybean developing seeds in spite of its chemical defenses.

ERAP1 and PDE8A Are Downregulated in Cattle Protected against Bovine Tuberculosis.

Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium bovis that is responsible for significant economic losses worldwide. In spite of its relevance, the limited knowledge about the host immune responses that provide effective protection against the disease has long hampered the development of an effective vaccine. The identification of host proteins with an expression that correlates with protection against bTB would contribute to the understanding of the cattle defence mechanisms against M. bovis infection. In this study, we found that ERAP1 and PDE8A were downregulated in vaccinated cattle that were protected from experimental M. bovis challenge. Remarkably, both genes encode proteins that have been negatively associated with immune protection against bTB.

Single nucleotide polymorphisms may explain the contrasting phenotypes of two variants of a multidrug-resistant Mycobacterium tuberculosis strain.

Globally, about 4.5% of new tuberculosis (TB) cases are multi-drug-resistant (MDR), i.e. resistant to the two most powerful first-line anti-TB drugs. Indeed, 480,000 people developed MDR-TB in 2015 and 190,000 people died because of MDR-TB. The MDR Mycobacterium tuberculosis M family, which belongs to the Haarlem lineage, is highly prosperous in Argentina and capable of building up further drug resistance without impairing its ability to spread. In this study, we sequenced the whole genomes of a highly prosperous M-family strain (Mp) and its contemporary variant, strain 410, which produced only one recorded tuberculosis case in the last two decades. Previous reports have demonstrated that Mp induced dysfunctional CD8+ cytotoxic T cell activity, suggesting that this strain has the ability to evade the immune response against M. tuberculosis. Comparative analysis of Mp and 410 genomes revealed non-synonymous polymorphisms in eleven genes and five intergenic regions with polymorphisms between both strains. Some of these genes and promoter regions are involved in the metabolism of cell wall components, others in drug resistance and a SNP in Rv1861, a gene encoding a putative transglycosylase that produces a truncated protein in Mp. The mutation in Rv3787c, a putative S-adenosyl-l-methionine-dependent methyltransferase, is conserved in all of the other prosperous M strains here analysed and absent in non-prosperous M strains. Remarkably, three polymorphic promoter regions displayed differential transcriptional activity between Mp and 410. We speculate that the observed mutations/polymorphisms are associated with the reported higher capacity of Mp for modulating the host's immune response.

Polymorphisms of 20 regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis.

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans and animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and other members of the MTC evolved. The genome of M. bovis is over >99.95% identical to that of M. tuberculosis but with seven deletions ranging in size from 1 to 12.7 kb. In addition, 1200 single nucleotide mutations in coding regions distinguish M. bovis from M. tuberculosis. In the present study, we assessed 75 M. tuberculosis genomes and 23 M. bovis genomes to identify non-synonymous mutations in 202 coding sequences of regulatory genes between both species. We identified species-specific variants in 20 regulatory proteins and confirmed differential expression of hypoxia-related genes between M. bovis and M. tuberculosis.

Effect of Different Lignocellulosic Diets on Bacterial Microbiota and Hydrolytic Enzyme Activities in the Gut of the Cotton Boll Weevil (Anthonomus grandis).

Cotton boll weevils, Anthonomus grandis, are omnivorous coleopteran that can feed on diets with different compositions, including recalcitrant lignocellulosic materials. We characterized the changes in the prokaryotic community structure and the hydrolytic activities of A. grandis larvae fed on different lignocellulosic diets. A. grandis larvae were fed on three different artificial diets: cottonseed meal (CM), Napier grass (NG) and corn stover (CS). Total DNA was extracted from the gut samples for amplification and sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. Proteobacteria and Firmicutes dominated the gut microbiota followed by Actinobacteria, Spirochaetes and a small number of unclassified phyla in CM and NG microbiomes. In the CS feeding group, members of Spirochaetes were the most prevalent, followed by Proteobacteria and Firmicutes. Bray-Curtis distances showed that the samples from the CS community were clearly separated from those samples of the CM and NG diets. Gut extracts from all three diets exhibited endoglucanase, xylanase, ß-glucosidase and pectinase activities. These activities were significantly affected by pH and temperature across different diets. We observed that the larvae reared on a CM showed significantly higher activities than larvae reared on NG and CS. We demonstrated that the intestinal bacterial community structure varies depending on diet composition. Diets with more variable and complex compositions, such as CS, showed higher bacterial diversity and richness than the two other diets. In spite of the detected changes in composition and diversity, we identified a core microbiome shared between the three different lignocellulosic diets. These results suggest that feeding with diets of different lignocellulosic composition could be a viable strategy to discover variants of hemicellulose and cellulose breakdown systems.

Protease and hemicellulase assisted extraction of dietary fiber from wastes of Cynara cardunculus.

The action of protease and hemicellulase for the extraction of fractions enriched in soluble fiber from bracts and stems of Cynara cardunculus was evaluated. Using a two-factor simplex design comprising protease amounts of 0-200 µL and hemicellulase amounts of 0-200 mg for 5 g of material, we explored the effect of a 5 h enzymatic treatment at 40 °C on the chemical composition and yield of the fractions isolated. The fractions contained inulin and pectin. In general, the protein, inulin, and polyphenol contents and also the yields were higher for fractions obtained from stems. The most marked effects were observed when enzymes were used at higher concentrations, especially for hemicellulase. The inclusion of a pre-heating step increased the yield and the inulin content for fractions isolated from bracts and stems and decreased the protein and polyphenol contents, and the galacturonic acid for bracts. These fractions, in general, contained the polyphenolic compounds monocaffeoylquinic acid, apigenin, and pinoresinol.

Pyrosequencing reveals changes in soil bacterial communities after conversion of Yungas forests to agriculture.

The Southern Andean Yungas in Northwest Argentina constitute one of the main biodiversity hotspots in the world. Considerable changes in land use have taken place in this ecoregion, predominantly related to forest conversion to croplands, inducing losses in above-ground biodiversity and with potential impact on soil microbial communities. In this study, we used high-throughput pyrosequencing of the 16S ribosomal RNA gene to assess whether land-use change and time under agriculture affect the composition and diversity of soil bacterial communities. We selected two areas dedicated to sugarcane and soybean production, comprising both short- and long-term agricultural sites, and used the adjacent native forest soils as a reference. Land-use change altered the composition of bacterial communities, with differences between productive areas despite the similarities between both forests. At the phylum level, only Verrucomicrobia and Firmicutes changed in abundance after deforestation for sugarcane and soybean cropping, respectively. In cultivated soils, Verrucomicrobia decreased sharply (~80%), while Firmicutes were more abundant. Despite the fact that local diversity was increased in sugarcane systems and was not altered by soybean cropping, phylogenetic beta diversity declined along both chronosequences, evidencing a homogenization of soil bacterial communities over time. In spite of the detected alteration in composition and diversity, we found a core microbiome resistant to the disturbances caused by the conversion of forests to cultivated lands and few or none exclusive OTUs for each land-use type. The overall changes in the relative abundance of copiotrophic and oligotrophic taxa may have an impact in soil ecosystem functionality. However, communities with many taxa in common may also share many functional attributes, allowing to maintain at least some soil ecosystem services after forest conversion to croplands.

Identification of potential biomarkers of disease progression in bovine tuberculosis.

Bovine tuberculosis (bTB) remains an important animal and zoonotic disease in many countries. The diagnosis of bTB is based on tuberculin skin test and IFN-γ release assays (IGRA). Positive animals are separated from the herd and sacrificed. The cost of this procedure is difficult to afford for developing countries with high prevalence of bTB; therefore, the improvement of diagnostic methods and the identification of animals in different stages of the disease will be helpful to control the infection. To identify biomarkers that can discriminate between tuberculin positive cattle with and without tuberculosis lesions (ML+ and ML-, respectively), we assessed a group of immunological parameters with three different classification methods: lineal discriminant analysis (LDA), quadratic discriminant analysis (QDA) and K nearest neighbors (k-nn). For this purpose, we used data from 30 experimentally infected cattle. All the classifiers (LDA, QDA and k-nn) selected IL-2 and IL-17 as the most discriminatory variables. The best classification method was LDA using IL-17 and IL-2 as predictors. The addition of IL-10 to LDA improves the performance of the classifier to discriminate ML-individuals (93.3% vs. 86.7%). Thus, the expression of IL-17, IL-2 and, in some cases, IL-10 would serve as an additional tool to study disease progression in herds with a history of bTB.

Study of the in vivo role of Mce2R, the transcriptional regulator of mce2 operon in Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the agent of human tuberculosis, has developed strategies involving proteins and other compounds called virulence factors to subvert human host defences and damage and invade the human host. Among these virulence-related proteins are the Mce proteins, which are encoded in the mce1, mce2, mce3 and mce4 operons of M. tuberculosis. The expression of the mce2 operon is negatively regulated by the Mce2R transcriptional repressor. Here we evaluated the role of Mce2R during the infection of M. tuberculosis in mice and macrophages and defined the genes whose expression is in vitro regulated by this transcriptional repressor. RESULTS: We used a specialized transduction method for generating a mce2R mutant of M. tuberculosis H37Rv. Although we found equivalent replication of the MtΔmce2R mutant and the wild type strains in mouse lungs, overexpression of Mce2R in the complemented strain (MtΔmce2RComp) significantly impaired its replication. During in vitro infection of macrophages, we observed a significantly increased association of the late endosomal marker LAMP-2 to MtΔmce2RComp-containing phagosomes as compared to MtΔmce2R and the wild type strains. Whole transcriptional analysis showed that Mce2R regulates mainly the expression of the mce2 operon, in the in vitro conditions studied. CONCLUSIONS: The findings of the current study indicate that Mce2R weakly represses the in vivo expression of the mce2 operon in the studied conditions and argue for a role of the proteins encoded in Mce2R regulon in the arrest of phagosome maturation induced by M. tuberculosis.

Separating homeologs by phasing in the tetraploid wheat transcriptome.

BACKGROUND: The high level of identity among duplicated homoeologous genomes in tetraploid pasta wheat presents substantial challenges for de novo transcriptome assembly. To solve this problem, we develop a specialized bioinformatics workflow that optimizes transcriptome assembly and separation of merged homoeologs. To evaluate our strategy, we sequence and assemble the transcriptome of one of the diploid ancestors of pasta wheat, and compare both assemblies with a benchmark set of 13,472 full-length, non-redundant bread wheat cDNAs. RESULTS: A total of 489 million 100 bp paired-end reads from tetraploid wheat assemble in 140,118 contigs, including 96% of the benchmark cDNAs. We used a comparative genomics approach to annotate 66,633 open reading frames. The multiple k-mer assembly strategy increases the proportion of cDNAs assembled full-length in a single contig by 22% relative to the best single k-mer size. Homoeologs are separated using a post-assembly pipeline that includes polymorphism identification, phasing of SNPs, read sorting, and re-assembly of phased reads. Using a reference set of genes, we determine that 98.7% of SNPs analyzed are correctly separated by phasing. CONCLUSIONS: Our study shows that de novo transcriptome assembly of tetraploid wheat benefit from multiple k-mer assembly strategies more than diploid wheat. Our results also demonstrate that phasing approaches originally designed for heterozygous diploid organisms can be used to separate the close homoeologous genomes of tetraploid wheat. The predicted tetraploid wheat proteome and gene models provide a valuable tool for the wheat research community and for those interested in comparative genomic studies.