A novel BLUF photoreceptor modulates the Xanthomonas citri subsp. citri-host plant interaction.

Plant-pathogen interaction is influenced by multiple environmental factors, including temperature and light. Recent works have shown that light modulates not only the defense response of plants but also the pathogens virulence. Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker, an important plant disease worldwide. The Xcc genome presents four genes encoding putative photoreceptors: one bacteriophytochrome and three blue light photoreceptors, one LOV and two BLUFs (bluf1: XAC2120 and bluf2: XAC3278). The presence of two BLUFs proteins is an outstanding feature of Xcc. In this work we show that the bluf2 gene is functional. The mutant strain, XccΔbluf2, was constructed demonstrating that BLUF2 regulates swimming-type motility, adhesion to leaves, exopolysaccharide production and biofilm formation, features involved in the Xcc virulence processes. An important aspect during the plant-pathogen interaction is the oxidative response of the host and the consequent reaction of the pathogen. We observed that ROS detoxification is regulated by Xcc bluf2 gene. The phenotypes of disease in orange plants produced by WT and XccΔbluf2 strains were evaluated, observing different phenotypes. Altogether, these results show that BLUF2 negatively regulates virulence during citrus canker. This work constitutes the first report on BLUF-like receptors in plant pathogenic bacteria.

Genome-resolved metagenome and metatranscriptome analyses of thermophilic composting reveal key bacterial players and their metabolic interactions.

BACKGROUND: Composting is an important technique for environment-friendly degradation of organic material, and is a microbe-driven process. Previous metagenomic studies of composting have presented a general description of the taxonomic and functional diversity of its microbial populations, but they have lacked more specific information on the key organisms that are active during the process. RESULTS: Here we present and analyze 60 mostly high-quality metagenome-assembled genomes (MAGs) recovered from time-series samples of two thermophilic composting cells, of which 47 are potentially new bacterial species; 24 of those did not have any hits in two public MAG datasets at the 95% average nucleotide identity level. Analyses of gene content and expressed functions based on metatranscriptome data for one of the cells grouped the MAGs in three clusters along the 99-day composting process. By applying metabolic modeling methods, we were able to predict metabolic dependencies between MAGs. These models indicate the importance of coadjuvant bacteria that do not carry out lignocellulose degradation but may contribute to the management of reactive oxygen species and with enzymes that increase bioenergetic efficiency in composting, such as hydrogenases and N2O reductase. Strong metabolic dependencies predicted between MAGs revealed key interactions relying on exchange of H+, NH3, O2 and CO2, as well as glucose, glutamate, succinate, fumarate and others, highlighting the importance of functional stratification and syntrophic interactions during biomass conversion. Our model includes 22 out of 49 MAGs recovered from one composting cell data. Based on this model we highlight that Rhodothermus marinus, Thermobispora bispora and a novel Gammaproteobacterium are dominant players in chemolithotrophic metabolism and cross-feeding interactions. CONCLUSIONS: The results obtained expand our knowledge of the taxonomic and functional diversity of composting bacteria and provide a model of their dynamic metabolic interactions.

The rate and role of pseudogenes of the Mycobacterium tuberculosis complex.

Whole-genome sequence analyses have significantly contributed to the understanding of virulence and evolution of the Mycobacterium tuberculosis complex (MTBC), the causative pathogens of tuberculosis. Most MTBC evolutionary studies are focused on single nucleotide polymorphisms and deletions, but rare studies have evaluated gene content, whereas none has comprehensively evaluated pseudogenes. Accordingly, we describe an extensive study focused on quantifying and predicting possible functions of MTBC and Mycobacterium canettii pseudogenes. Using NCBI's PGAP-detected pseudogenes, we analysed 25 837 pseudogenes from 158 MTBC and M. canetii strains and combined transcriptomics and proteomics of M. tuberculosis H37Rv to gain insights about pseudogenes' expression. Our results indicate significant variability concerning rate and conservancy of in silico predicted pseudogenes among different ecotypes and lineages of tuberculous mycobacteria and pseudogenization of important virulence factors and genes of the metabolism and antimicrobial resistance/tolerance. We show that in silico predicted pseudogenes contribute considerably to MTBC genetic diversity at the population level. Moreover, the transcription machinery of M. tuberculosis can fully transcribe most pseudogenes, indicating intact promoters and recent pseudogene evolutionary emergence. Proteomics of M. tuberculosis and close evaluation of mutational lesions driving pseudogenization suggest that few in silico predicted pseudogenes are likely capable of neofunctionalization, nonsense mutation reversal, or phase variation, contradicting the classical definition of pseudogenes. Such findings indicate that genome annotation should be accompanied by proteomics and protein function assays to improve its accuracy. While indels and insertion sequences are the main drivers of the observed mutational lesions in these species, population bottlenecks and genetic drift are likely the evolutionary processes acting on pseudogenes' emergence over time. Our findings unveil a new perspective on MTBC's evolution and genetic diversity.

In Silico Prediction and Design of Uropathogenic Escherichia coli Alpha-Hemolysin Generate a Soluble and Hemolytic Recombinant Toxin.

The secretion of α-hemolysin by uropathogenic Escherichia coli (UPEC) is commonly associated with the severity of urinary tract infections, which makes it a predictor of poor prognosis among patients. Accordingly, this toxin has become a target for diagnostic tests and therapeutic interventions. However, there are several obstacles associated with the process of α-hemolysin purification, therefore limiting its utilization in scientific investigations. In order to overcome the problems associated with α-hemolysin expression, after in silico prediction, a 20.48 kDa soluble α-hemolysin recombinant denoted rHlyA was constructed. This recombinant is composed by a 182 amino acid sequence localized in the aa542-723 region of the toxin molecule. The antigenic determinants of the rHlyA were estimated by bioinformatics analysis taking into consideration the tertiary form of the toxin, epitope analysis tools, and solubility inference. The results indicated that rHlyA has three antigenic domains localized in the aa555-565, aa600-610, and aa674-717 regions. Functional investigation of rHlyA demonstrated that it has hemolytic activity against sheep red cells, but no cytotoxic effect against epithelial bladder cells. In summary, the results obtained in this study indicate that rHlyA is a soluble recombinant protein that can be used as a tool in studies that aim to understand the mechanisms involved in the hemolytic and cytotoxic activities of α-hemolysin produced by UPEC. In addition, rHlyA can be applied to generate monoclonal and/or polyclonal antibodies that can be utilized in the development of diagnostic tests and therapeutic interventions.

Genomic epidemiology of the SARS-CoV-2 epidemic in Brazil.

The high numbers of COVID-19 cases and deaths in Brazil have made Latin America an epicentre of the pandemic. SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, but important gaps remain in our understanding of virus transmission dynamics at a national scale. We use 17,135 near-complete genomes sampled from 27 Brazilian states and bordering country Paraguay. From March to November 2020, we detected co-circulation of multiple viral lineages that were linked to multiple importations (predominantly from Europe). After November 2020, we detected large, local transmission clusters within the country. In the absence of effective restriction measures, the epidemic progressed, and in January 2021 there was emergence and onward spread, both within and abroad, of variants of concern and variants under monitoring, including Gamma (P.1) and Zeta (P.2). We also characterized a genomic overview of the epidemic in Paraguay and detected evidence of importation of SARS-CoV-2 ancestor lineages and variants of concern from Brazil. Our findings show that genomic surveillance in Brazil enabled assessment of the real-time spread of emerging SARS-CoV-2 variants.

Correction: Lesbon et al. Nucleocapsid (N) Gene Mutations of SARS-CoV-2 Can Affect Real-Time RT-PCR Diagnostic and Impact False-Negative Results. Viruses 2021, 13, 2474.

The authors hereby request the inclusion of two authors (Olivia Teixeira and Maria Cristina Nonato) in the recently published article in Viruses entitled "Nucleocapsid (N) gene mutations of SARS-CoV-2 can affect real-time RT-PCR diagnostic and impact false-negative results" [...].

Genomic epidemiology reveals the impact of national and international restrictions measures on the SARS-CoV-2 epidemic in Brazil.

Brazil has experienced some of the highest numbers of COVID-19 cases and deaths globally and from May 2021 made Latin America a pandemic epicenter. Although SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, important gaps remain in our understanding of virus transmission dynamics at the national scale. Here, we describe the genomic epidemiology of SARS-CoV-2 using near-full genomes sampled from 27 Brazilian states and a bordering country - Paraguay. We show that the early stage of the pandemic in Brazil was characterised by the co-circulation of multiple viral lineages, linked to multiple importations predominantly from Europe, and subsequently characterized by large local transmission clusters. As the epidemic progressed under an absence of effective restriction measures, there was a local emergence and onward international spread of Variants of Concern (VOC) and Variants Under Monitoring (VUM), including Gamma (P.1) and Zeta (P.2). In addition, we provide a preliminary genomic overview of the epidemic in Paraguay, showing evidence of importation from Brazil. These data reinforce the usefulness and need for the implementation of widespread genomic surveillance in South America as a toolkit for pandemic monitoring that provides a means to follow the real-time spread of emerging SARS-CoV-2 variants with possible implications for public health and immunization strategies.

Genomic monitoring of the SARS-CoV-2 B1.1.7 (WHO VOC Alpha) in the Sao Paulo state, Brazil.

The SARS-CoV-2 alpha VOC (also known as lineage B.1.1.7) initially described in the autumn, 2020 in UK, rapidly became the dominant lineage across much of Europe. Despite multiple studies reporting molecular evidence suggestive of its circulation in Brazil, much is still unknown about its genomic diversity in the state of São Paulo, the main Brazilian economic and transportation hub. To get more insight regarding its transmission dynamics into the State we performed phylogenetic analysis on all alpha VOC strains obtained between February and August 2021 from the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants. The performed phylogenetic analysis showed that most of the alpha VOC genomes were interspersed with viral strains sampled from different Brazilian states and other countries suggesting that multiple independent Alpha VOC introductions from Brazil and overseas have occurred in the São Paulo State over time. Nevertheless, large monophyletic clusters were also observed especially from the Central-West part of the São Paulo State (the city of Bauru) and the metropolitan region of the São Paulo city. Our results highlight the Alpha VOC molecular epidemiology in the São Paulo state and reinforce the need for continued genomic surveillance strategies for the real-time monitoring of potential emerging SARS-CoV-2 variants during the ever-growing vaccination process.

Nucleocapsid (N) Gene Mutations of SARS-CoV-2 Can Affect Real-Time RT-PCR Diagnostic and Impact False-Negative Results.

The current COVID-19 pandemic demands massive testing by Real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction), which is considered the gold standard diagnostic test for the detection of the SARS-CoV-2 virus. However, the virus continues to evolve with mutations that lead to phenotypic alterations as higher transmissibility, pathogenicity or vaccine evasion. Another big issue are mutations in the annealing sites of primers and probes of RT-PCR diagnostic kits leading to false-negative results. Therefore, here we identify mutations in the N (Nucleocapsid) gene that affects the use of the GeneFinder COVID-19 Plus RealAmp Kit. We sequenced SARS-CoV-2 genomes from 17 positive samples with no N gene detection but with RDRP (RNA-dependent RNA polymerase) and E (Envelope) genes detection, and observed a set of three different mutations affecting the N detection: a deletion of 18 nucleotides (Del28877-28894), a substitution of GGG to AAC (28881-28883) and a frameshift mutation caused by deletion (Del28877-28878). The last one cause a deletion of six AAs (amino acids) located in the central intrinsic disorder region at protein level. We also found this mutation in 99 of the 14,346 sequenced samples by the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants, demonstrating the circulation of the mutation in Sao Paulo, Brazil. Continuous monitoring and characterization of mutations affecting the annealing sites of primers and probes by genomic surveillance programs are necessary to maintain the effectiveness of the diagnosis of COVID-19.

Identification of Novel Interspersed DNA Repetitive Elements in the Trypanosoma cruzi Genome Associated with the 3'UTRs of Surface Multigenic Families.

Trypanosoma cruzi is the etiological agent of Chagas disease, which affects millions of people in Latin America. No transcriptional control of gene expression has been demonstrated in this organism, and 50% of its genome consists of repetitive elements and members of multigenic families. In this study, we applied a novel bioinformatics approach to predict new repetitive elements in the genome sequence of T. cruzi. A new repetitive sequence measuring 241 nt was identified and found to be interspersed along the genome sequence from strains of different DTUs. This new repeat was mostly on intergenic regions, and upstream and downstream regions of the 241 nt repeat were enriched in surface protein genes. RNAseq analysis revealed that the repeat was part of processed mRNAs and was predominantly found in the 3' untranslated regions (UTRs) of genes of multigenic families encoding surface proteins. Moreover, we detected a correlation between the presence of the repeat in the 3'UTR of multigenic family genes and the level of differential expression of these genes when comparing epimastigote and trypomastigote transcriptomes. These data suggest that this sequence plays a role in the posttranscriptional regulation of the expression of multigenic families.

Global Distribution and Evolution of Mycobacterium bovis Lineages.

Mycobacterium bovis is the main causative agent of zoonotic tuberculosis in humans and frequently devastates livestock and wildlife worldwide. Previous studies suggested the existence of genetic groups of M. bovis strains based on limited DNA markers (a.k.a. clonal complexes), and the evolution and ecology of this pathogen has been only marginally explored at the global level. We have screened over 2,600 publicly available M. bovis genomes and newly sequenced four wildlife M. bovis strains, gathering 1,969 genomes from 23 countries and at least 24 host species, including humans, to complete a phylogenomic analyses. We propose the existence of four distinct global lineages of M. bovis (Lb1, Lb2, Lb3, and Lb4) underlying the current disease distribution. These lineages are not fully represented by clonal complexes and are dispersed based on geographic location rather than host species. Our data divergence analysis agreed with previous studies reporting independent archeological data of ancient M. bovis (South Siberian infected skeletons at ∼2,000 years before present) and indicates that extant M. bovis originated between 715 and 3,556 years BP, with later emergence in the New World and Oceania, likely influenced by trades among countries.

Complete genome sequence and analysis of Alcaligenes faecalis strain Mc250, a new potential plant bioinoculant.

Here we present and analyze the complete genome of Alcaligenes faecalis strain Mc250 (Mc250), a bacterium isolated from the roots of Mimosa calodendron, an endemic plant growing in ferruginous rupestrian grasslands in Minas Gerais State, Brazil. The genome has 4,159,911 bp and 3,719 predicted protein-coding genes, in a single chromosome. Comparison of the Mc250 genome with 36 other Alcaligenes faecalis genomes revealed that there is considerable gene content variation among these strains, with the core genome representing only 39% of the protein-coding gene repertoire of Mc250. Mc250 encodes a complete denitrification pathway, a network of pathways associated with phenolic compounds degradation, and genes associated with HCN and siderophores synthesis; we also found a repertoire of genes associated with metal internalization and metabolism, sulfate/sulfonate and cysteine metabolism, oxidative stress and DNA repair. These findings reveal the genomic basis for the adaptation of this bacterium to the harsh environmental conditions from where it was isolated. Gene clusters associated with ectoine, terpene, resorcinol, and emulsan biosynthesis that can confer some competitive advantage were also found. Experimental results showed that Mc250 was able to reduce (~60%) the virulence phenotype of the plant pathogen Xanthomonas citri subsp. citri when co-inoculated in Citrus sinensis, and was able to eradicate 98% of juveniles and stabilize the hatching rate of eggs to 4% in two species of agricultural nematodes. These results reveal biotechnological potential for the Mc250 strain and warrant its further investigation as a biocontrol and plant growth-promoting bacterium.

A novel Sporothrix brasiliensis genomic variant in Midwestern Brazil: evidence for an older and wider sporotrichosis epidemic.

Sporotrichosis is a subcutaneous infection caused by fungi from the genus Sporothrix. It is transmitted by inoculation of infective particles found in plant-contaminated material or diseased animals, characterizing the classic sapronotic and emerging zoonotic transmission, respectively. Since 1998, southeastern Brazil has experienced a zoonotic sporotrichosis epidemic caused by S. brasiliensis, centred in the state of Rio de Janeiro. Our observation of feline sporotrichosis cases in Brasília (Midwestern Brazil), around 900 km away from Rio de Janeiro, led us to question whether the epidemic caused by S. brasiliensis has spread from the epicentre in Rio de Janeiro, emerged independently in the two locations, or if the disease has been present and unrecognized in Midwestern Brazil. A retrospective analysis of 91 human and 4 animal cases from Brasília, ranging from 1993 to 2018, suggests the occurrence of both sapronotic and zoonotic transmission. Molecular typing of the calmodulin locus identified S. schenckii as the agent in two animals and all seven human patients from which we were able to recover clinical isolates. In two other animals, the disease was caused by S. brasiliensis. Whole-genome sequence typing of seven Sporothrix spp. strains from Brasília and Rio de Janeiro suggests that S. brasiliensis isolates from Brasília are genetically distinct from those obtained at the epicentre of the outbreak in Rio de Janeiro, both in phylogenomic and population genomic analyses. The two S. brasiliensis populations seem to have separated between 2.2 and 3.1 million years ago, indicating independent outbreaks or that the zoonotic S. brasiliensis outbreak might have started earlier and be more widespread in South America than previously recognized.

Gut transcriptome analysis on females of Ornithodoros mimon (Acari: Argasidae) and phylogenetic inference of ticks.

Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also harmful to humans. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to know and ascertain the main molecules expressed in the gut of argasid after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon using 454-based RNA sequencing. The gut transcriptome analysis reveals several transcripts associated with hemoglobin digestion, such as serine, cysteine, aspartic proteases and metalloenzymes. The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. We recorded the presence a cathepsin O peptidase-coding transcript in ticks. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on mitochondrial genome and nuclear rRNA sequences. We deposited 2,213 sequence of O. mimon to the public databases. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity.

Gut transcriptome analysis on females of Ornithodoros mimon (Acari: Argasidae) and phylogenetic inference of ticks / Transcriptoma do intestino de fêmeas de Ornithodoros mimon (Acari: Argasidae) e inferência filogenética de carrapatos

Abstract Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also harmful to humans. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to know and ascertain the main molecules expressed in the gut of argasid after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon using 454-based RNA sequencing. The gut transcriptome analysis reveals several transcripts associated with hemoglobin digestion, such as serine, cysteine, aspartic proteases and metalloenzymes. The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. We recorded the presence a cathepsin O peptidase-coding transcript in ticks. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on mitochondrial genome and nuclear rRNA sequences. We deposited 2,213 sequence of O. mimon to the public databases. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity.

Resumo Ornithodoros mimon é um carrapato argasídeo parasita de morcegos, aves e marsupiais, além de ser bastante agressivo aos humanos. O conhecimento dos transcritos presentes no intestino dos carrapatos auxilia no entendimento do papel de moléculas vitais no processo de digestão e na relação parasito-hospedeiro, além de fornecer também informações sobre a evolução dos artrópodes hematófagos. Desta maneira, o presente estudo teve como objetivo conhecer e identificar as principais moléculas expressas no intestino de uma espécie de carrapato argasídeo após o repasto sanguíneo, através de uma análise transcritômica descritiva do intestino de fêmeas ingurgitadas de O. mimon, utilizando um sequenciamento de RNA de nova geração da plataforma 454. Além de inferir a relação filogenética de carrapatos através de um conjunto de dados transcritômicos. O transcriptoma do intestino revelou diversos transcritos associados com a digestão da hemoglobina, como proteinases das classes serino, cisteína, aspártica e metalo. Registramos a presença de um transcrito de uma cisteína peptidase do tipo catepsina O em carrapatos. A inferência filogenética baseada em conjunto de dados transcritos homólogos tem uma resolução topológica similar a de outros conjuntos de dados moleculares. Foram depositados no banco de dados gênico público 2213 transcritos de O. mimon. Os achados obtidos no presente estudo podem contribuir para compreensão dos importantes processos, como digestão, nutrição e imunidade dos carrapatos da família Argasidae, além de fornecer informações sobre a filogenia da ordem Ixodida.