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The identification of orthologous genes is relevant for comparative genomics, phylogenetic analysis, and functional annotation. There are many computational tools for the prediction of orthologous groups as well as web-based resources that offer orthology datasets for download and online analysis. This chapter presents a simple and practical guide to the process of orthologous group prediction, using a dataset of 10 prokaryotic proteomes as example. The orthology methods covered are OrthoMCL, COGtriangles, OrthoFinder2, and OMA. The authors compare the number of orthologous groups predicted by these various methods, and present a brief workflow for the functional annotation and reconstruction of phylogenies from inferred single-copy orthologous genes. The chapter also demonstrates how to explore two orthology databases: eggNOG6 and OrthoDB.

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Metagenome-assembled genomes, or MAGs, are genomes retrieved from metagenome datasets. In the vast majority of cases, MAGs are genomes from prokaryotic species that have not been isolated or cultivated in the lab. They, therefore, provide us with information on these species that are impossible to obtain otherwise, at least until new cultivation methods are devised. Thanks to improvements and cost reductions of DNA sequencing technologies and growing interest in microbial ecology, the rise in number of MAGs in genome repositories has been exponential. This chapter covers the basics of MAG retrieval and processing and provides a practical step-by-step guide using a real dataset and state-of-the-art tools for MAG analysis and comparison.

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Thanks to advancements in genome sequencing and bioinformatics, thousands of bacterial genome sequences are available in public databases. This presents an opportunity to study bacterial diversity in unprecedented detail. This chapter describes a complete bioinformatics workflow for comparative genomics of bacterial genomes, including genome annotation, pangenome reconstruction and visualization, phylogenetic analysis, and identification of sequences of interest such as antimicrobial-resistance genes, virulence factors, and phage sequences. The workflow uses state-of-the-art, open-source tools. The workflow is presented by means of a comparative analysis of Salmonella enterica serovar Typhimurium genomes. The workflow is based on Linux commands and scripts, and result visualization relies on the R environment. The chapter provides a step-by-step protocol that researchers with basic expertise in bioinformatics can easily follow to conduct investigations on their own genome datasets.

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Phylogenomics aims at reconstructing the evolutionary histories of organisms taking into account whole genomes or large fractions of genomes. Phylogenomics has significant applications in fields such as evolutionary biology, systematics, comparative genomics, and conservation genetics, providing valuable insights into the origins and relationships of species and contributing to our understanding of biological diversity and evolution. This chapter surveys phylogenetic concepts and methods aimed at both gene tree and species tree reconstruction while also addressing common pitfalls, providing references to relevant computer programs. A practical phylogenomic analysis example including bacterial genomes is presented at the end of the chapter.

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Bacterial viruses (bacteriophages or phages) are the most abundant and diverse biological entities on Earth. There is a renewed worldwide interest in phage-centered research motivated by their enormous potential as antimicrobials to cope with multidrug-resistant pathogens. An ever-growing number of complete phage genomes are becoming available, derived either from newly isolated phages (cultivated phages) or recovered from metagenomic sequencing data (uncultivated phages). Robust comparative analysis is crucial for a comprehensive understanding of genotypic variations of phages and their related evolutionary processes, and to investigate the interaction mechanisms between phages and their hosts. In this chapter, we present a protocol for phage comparative genomics employing tools selected out of the many currently available, focusing on complete genomes of phages classified in the class Caudoviricetes. This protocol provides accurate identification of similarities, differences, and patterns among new and previously known complete phage genomes as well as phage clustering and taxonomic classification.

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Bacteriophages are recognized as the most abundant members of microbiomes and have therefore a profound impact on microbial communities through the interactions with their bacterial hosts. The International Metagenomics and Metadesign of Subways and Urban Biomes Consortium (MetaSUB) has sampled mass-transit systems in 60 cities over 3 years using metagenomics, throwing light into these hitherto largely unexplored urban environments. MetaSUB focused primarily on the bacterial community. In this work, we explored MetaSUB metagenomic data in order to recover and analyze bacteriophage genomes. We recovered and analyzed 1714 phage genomes with size at least 40 kbp, from the class Caudoviricetes, the vast majority of which (80%) are novel. The recovered genomes were predicted to belong to temperate (69%) and lytic (31%) phages. Thirty-three of these genomes have more than 200 kbp, and one of them reaches 572 kbp, placing it among the largest phage genomes ever found. In general, the phages tended to be site-specific or nearly so, but 194 genomes could be identified in every city from which phage genomes were retrieved. We predicted hosts for 48% of the phages and observed general agreement between phage abundance and the respective bacterial host abundance, which include the most common nosocomial multidrug-resistant pathogens. A small fraction of the phage genomes are carriers of antibiotic resistance genes, and such genomes tended to be particularly abundant in the sites where they were found. We also detected CRISPR-Cas systems in five phage genomes. This study expands the previously reported MetaSUB results and is a contribution to the knowledge about phage diversity, global distribution, and phage genome content.

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BACKGROUND: Lung fibrosis is a major concern in severe COVID-19 patients undergoing mechanical ventilation (MV). Lung fibrosis frequency in post-COVID syndrome is highly variable and even if the risk is proportionally small, many patients could be affected. However, there is still no data on lung extracellular matrix (ECM) composition in severe COVID-19 and whether it is different from other aetiologies of ARDS. METHODS: We have quantified different ECM elements and TGF-ß expression in lung tissue of 28 fatal COVID-19 cases and compared to 27 patients that died of other causes of ARDS, divided according to MV duration (up to six days or seven days or more). In COVID-19 cases, ECM elements were correlated with lung transcriptomics and cytokines profile. RESULTS: We observed that COVID-19 cases presented significant increased deposition of collagen, fibronectin, versican, and TGF-ß, and decreased decorin density when compared to non-COVID-19 cases of similar MV duration. TGF-ß was precociously increased in COVID-19 patients with MV duration up to six days. Lung collagen was higher in women with COVID-19, with a transition of upregulated genes related to fibrillogenesis to collagen production and ECM disassembly along the MV course. CONCLUSIONS: Fatal COVID-19 is associated with an early TGF-ß expression lung environment after the MV onset, followed by a disordered ECM assembly. This uncontrolled process resulted in a prominent collagen deposition when compared to other causes of ARDS. Our data provides pathological substrates to better understand the high prevalence of pulmonary abnormalities in patients surviving COVID-19.

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Two Raphidiopsis (=Cylindrospermopsis) raciborskii metagenome-assembled genomes (MAGs) were recovered from two freshwater metagenomic datasets sampled in 2011 and 2012 in Pampulha Lake, a hypereutrophic, artificial, shallow reservoir, located in the city of Belo Horizonte (MG), Brazil. Since the late 1970s, the lake has undergone increasing eutrophication pressure, due to wastewater input, leading to the occurrence of frequent cyanobacterial blooms. The major difference observed between PAMP2011 and PAMP2012 MAGs was the lack of the saxitoxin gene cluster in PAMP2012, which also presented a smaller genome, while PAMP2011 presented the complete sxt cluster and all essential proteins and clusters. The pangenome analysis was performed with all Raphidiopsis/Cylindrospermopsis genomes available at NCBI to date, with the addition of PAMP2011 and PAMP2012 MAGs (All33 subset), but also without the South American strains (noSA subset), and only among the South American strains (SA10 and SA8 subsets). We observed a substantial increase in the core genome size for the 'noSA' subset, in comparison to 'All33' subset, and since the core genome reflects the closeness among the pangenome members, the results strongly suggest that the conservation level of the essential gene repertoire seems to be affected by the geographic origin of the strains being analyzed, supporting the existence of a distinct SA clade. The Raphidiopsis pangenome comprised a total of 7943 orthologous protein clusters, and the two new MAGs increased the pangenome size by 11%. The pangenome based phylogenetic relationships among the 33 analyzed genomes showed that the SA genomes clustered together with 99% bootstrap support, reinforcing the metabolic particularity of the Raphidiopsis South American clade, related to its saxitoxin producing unique ability, while also indicating a different evolutionary history due to its geographic isolation.

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The retina is a notable tissue with high metabolic needs which relies on specialized vascular networks to protect the neural retina while maintaining constant supplies of oxygen, nutrients, and dietary essential fatty acids. Here we analyzed the lipidome of the mouse retina under healthy and pathological angiogenesis using the oxygen-induced retinopathy model. By matching lipid profiles to changes in mRNA transcriptome, we identified a lipid signature showing that pathological angiogenesis leads to intense lipid remodeling favoring pathways for neutral lipid synthesis, cholesterol import/export, and lipid droplet formation. Noteworthy, it also shows profound changes in pathways for long-chain fatty acid production, vital for retina homeostasis. The net result is accumulation of large quantities of mead acid, a marker of essential fatty acid deficiency, and a potential marker for retinopathy severity. Thus, our lipid signature might contribute to better understand diseases of the retina that lead to vision impairment or blindness.

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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.

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BACKGROUND: There have been significant improvements in Chagas disease therapy and it is now widely accepted that most patients with chronic disease might benefit from therapy. However, there are challenges to monitor drug efficacy and cure for these patients, which are important impediments for current and future therapies. Trypanosoma cruzi-PCR is highly variable while IgG seroconversion takes decades yielding variable results depending on the antigen(s) used for the assay. METHODS AND RESULTS: We used the genomic phage display (gPhage) platform to perform a pairwise comparison of antigens and epitopes recognized by twenty individual patients with chronic Chagas disease before and after treatment with benznidazole. In total, we mapped 54,473 T. cruzi epitopes recognized by IgG from individual patients (N = 20) before benznidazole treatment. After treatment, the number of epitopes recognized by all patients was significantly smaller (21,254), a reduction consistent with a decrease in anti-T. cruzi antibodies. Most of these epitopes represent distinct fragments from the same protein and could, therefore, be grouped into 80 clusters of antigens. After three years of treatment with benznidazole, we observed a 64% reduction in the number of clusters of antigens recognized by patients (59 clusters before versus 21 clusters after treatment). The most abundant antigenic clusters recognized by patients correspond to the surface antigen CA-2 (B13) followed by the microtubule associated antigen, which highlights the value of these epitopes in Chagas disease diagnosis. Most importantly, quantitative pairwise comparison of gPhage data allowed for the prediction of patient response to treatment based on PCR status. PRINCIPAL FINDING: Here, we compiled a list of antigens and epitopes preferentially recognized by Chagas disease patients before and after benznidazole treatment. Next, we observed that gPhage data correlated with patient PCR-status and could, therefore, predict patient response to treatment. Moreover, gPhage results suggest that overall, independent of PCR status, treatment led to a reduction in the presence of T. cruzi-specific antibody levels and the number of antigens and epitopes recognized by these patients. CONCLUSION: The gPhage platform use of unbiased library of antigens, which is different from conventional serological assays that rely on predetermined antigens, is a contribution for the development of novel diagnostic tools for Chagas disease.

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Photosynthetic microorganisms are important components of most ecosystems and have important roles regarding biogeochemical cycles and the basis of the trophic chain. However, they sometimes are present in low abundance compared to other heterotrophic organisms. The Chapada das Mesas National Park (PNCM) is a Conservation Unit in Brazilian Cerrado biome, which is considered a hotspot for biodiversity conservation and possesses important rivers, waterfalls, and springs with economical and touristic importance. The aim of this study was to perform a comparative analysis of enriched and total microbiome of sediments to understand the impact of pre-cultivation in discovery of underrepresented groups like photosynthesizers. All sediment samples were cultivated in BG-11 medium under illumination to enrich for photosynthetic microorganisms and both the raw samples and the enriched ones were submitted to DNA extraction and sequencing of the V3-V4 hypervariable region of the 16S rRNA gene on the Ion Torrent platform. The reads were analyzed using QIIME2 software and the Phyloseq package. The enrichment allowed detection and identification of many genera of cyanobacteria in the Chapada das Mesas National Park (PNCM), which would probably not be possible without the combination of approaches. A total of 58 groups of photosynthetic microorganisms were classified in the samples from the enrichments and their relative abundance based on amplified 16S rRNA sequences were estimated, highlighting the genus Synechocystis which represented 10.10% of the abundance of the phylum Cyanobacteria and the genus Dunaliella, which represented 45.66% of the abundance of algae as the most abundant groups at the PNCM. In the enrichments, microorganisms from the phyla Proteobacteria (45.2%), Bacteroidetes (18%), and Planctomycetes (3.3%) were also identified, since there are ecological associations between the photosynthetic community and other groups of heterotrophic microorganisms. As for the functional analysis, metabolic functions associated with methanotrophy and methylotrophy, hydrocarbon degradation, phototrophy, and nitrogen fixation were predicted. The results highlight a great diversity of photosynthetic microorganisms in Cerrado and the importance of using a combination of approaches when analyzing target groups which are usually underrepresented such as cyanobacteria and microalgae.

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Understanding the biochemistry and metabolic pathways of cyanide degradation is necessary to improve the efficacy of cyanide bioremediation processes and industrial requirements. We have isolated and sequenced the genome of a cyanide-degrading Bacillus strain from water in contact with mine tailings from Lima, Peru. This strain was classified as Bacillus safensis based on 16S rRNA gene sequencing and core genome analyses and named B. safensis PER-URP-08. We searched for possible cyanide-degradation enzymes in the genome of this strain and identified a putative cyanide dihydratase (CynD) gene similar to a previously characterized CynD from Bacillus pumilus C1. Sequence analysis of CynD from B. safensis and B. pumilus allow us to identify C-terminal residues that differentiate both CynDs. We then cloned, expressed in Escherichia coli, and purified recombinant CynD from B. safensis PER-URP-08 (CynDPER-URP-08) and showed that in contrast to CynD from B. pumilus C1, this recombinant CynD remains active at up to pH 9. We also showed that oligomerization of CynDPER-URP-08 decreases as a function of increased pH. Finally, we demonstrated that transcripts of CynDPER-URP-08 in B. safensis PER-URP-08 are strongly induced in the presence of cyanide. Our results suggest that the use of B. safensis PER-URP-08 and CynDPER-URP-08 as potential tool for cyanide bioremediation warrants further investigation. IMPORTANCE Despite being of environmental concern around the world due to its toxicity, cyanide continues to be used in many important industrial processes. Thus, searching for cyanide bioremediation methods is a matter of societal concern and must be present on the political agenda of all governments. Here, we report the isolation, genome sequencing and characterization of cyanide degradation capacity of a bacterial strain isolated from an industrial mining site in Peru. We characterize a cyanide dehydratase (CynD) homolog from one of these bacteria, Bacillus safensis PER-URP-08.

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PURPOSE: Transcriptome analysis of pancreatic ductal adenocarcinoma (PDAC) has been useful to identify gene expression changes that sustain malignant phenotypes. Yet, most studies examined only tumor tissues and focused on protein-coding genes, leaving long non-coding RNAs (lncRNAs) largely underexplored. METHODS: We generated total RNA-Seq data from patient-matched tumor and nonmalignant pancreatic tissues and implemented a computational pipeline to survey known and novel lncRNAs. siRNA-mediated knockdown in tumor cell lines was performed to assess the contribution of PDAC-associated lncRNAs to malignant phenotypes. Gene co-expression network and functional enrichment analyses were used to assign deregulated lncRNAs to biological processes and molecular pathways. RESULTS: We detected 9,032 GENCODE lncRNAs as well as 523 unannotated lncRNAs, including transcripts significantly associated with patient outcome. Aberrant expression of a subset of novel and known lncRNAs was confirmed in patient samples and cell lines. siRNA-mediated knockdown of a subset of these lncRNAs (LINC01559, LINC01133, CCAT1, LINC00920 and UCA1) reduced cell proliferation, migration and invasion. Gene co-expression network analysis associated PDAC-deregulated lncRNAs with diverse biological processes, such as cell adhesion, protein glycosylation and DNA repair. Furthermore, UCA1 knockdown was shown to specifically deregulate co-expressed genes involved in DNA repair and to negatively impact DNA repair following damage induced by ionizing radiation. CONCLUSIONS: Our study expands the repertoire of lncRNAs deregulated in PDAC, thereby revealing novel candidate biomarkers for patient risk stratification. It also provides a roadmap for functional assays aimed to characterize novel mechanisms of action of lncRNAs in pancreatic cancer, which could be explored for therapeutic development.

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Citrus canker is one of the main bacterial diseases that affect citrus crops and is caused by Xanthomonas citri which affects all citrus species worldwide. New strategies to control citrus canker are necessary and the use of bacteriophages as biocontrol agent could be an alternative. Phages that infect Xanthomonas species have been studied, such as XacN1, a myovirus that infects X. citri. Here we report the isolation and characterization of a new jumbo phage, vb_XciM_LucasX, which infects X. citri and X. fuscans. Transmission electron microscopy allowed classification of LucasX in the Myoviridae family, which was corroborated by its genomic sequencing, annotation, and proteome clustering. LucasX has a 305,651 bp-long dsDNA genome. ORF prediction and annotation revealed 157 genes encoding putative structural proteins such as capsid and tail related proteins and phage assembly associated proteins, however, for most of the structural proteins it was not possible assign specific functions. Its genome encodes several proteins related to DNA replication and nucleotide metabolism, five putative RNA polymerases, at least one homing endonuclease mobile element, a terminase large subunit (TerL), an endolysin and many proteins classified as beneficial to the host. Proteome clustering and phylogeny analyses showed that LucasX is a new jumbo phage having as its closest neighbor the Xanthomonas jumbo phage Xoo-sp14. LucasX presented a burst size of 40 PFU/infected cell of X. citri 306, was completely inactivated at temperatures above 50°C, presented survival lower than 25% after 80 s of exposition to artificial UV light and had practically no tolerance to concentrations above 2.5 g/L NaCl or 40% ethanol. LucasX presented optimum pH at 7 and a broad range of Xanthomonas hosts, infecting twenty-one of the twenty-three strains tested. Finally, the LucasX yield was dependent on the host strain utilized, resulting one order of magnitude higher in X. fuscans C 752 than in X. citri 306, which points out to the possibility of phage yield improvement, an usual challenge for biocontrol purposes.

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Background: The experimental determination of a bacteriophage host is a laborious procedure. Thus, there is a pressing need for reliable computational predictions of bacteriophage hosts. Materials and Methods: We developed the program vHULK for phage host prediction based on 9504 phage genome features, which consider alignment significance scores between predicted proteins and a curated database of viral protein families. The features were fed to a neural network, and two models were trained to predict 77 host genera and 118 host species. Results: In controlled random test sets with 90% redundancy reduction in terms of protein similarity, vHULK obtained on average 83% precision and 79% recall at the genus level, and 71% precision and 67% recall at the species level. The performance of vHULK was compared against three other tools on a test data set with 2153 phage genomes. On this data set, vHULK achieved better performance at both the genus and the species levels than the other tools. Conclusions: Our results suggest that vHULK represents an advance on the state of art in phage host prediction.

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Abstract We present a survey of projects that have been funded by FAPESP under the BIOTA-Microorganisms program. These projects generated a wide variety of results, including the identification of novel antibacterial-producing microorganisms, the characterization of novel microbial enzymes for industrial applications, taxonomic classification of novel microorganisms in several environments, investigation of the soil and mangrove microbial ecosystems and its influence on endangered plant species, and the sequencing of novel metagenome-assembled genomes. The results surveyed demonstrate the importance of microorganisms in environments that play important roles in human activities as well as the potential that many of these microorganisms have in contributing to biotechnological applications crucial for human survival in the 21st century.

Resumo Apresentamos um levantamento comentado de projetos financiados pelo programa BIOTA-Micro-organismos. Estes projetos geraram uma variada gama de resultados, incluindo a identificação de novos micro-organismos produtores de compostos antibacterianos, a caracterização de novas enzimas microbianas para usos industriais, classificação taxonômica de novos micro-organismos presentes em diversos ambientes, investigação de ecossistemas microbianos em solos e mangues e sua influência sobre plantas ameaçadas, e o sequenciamento de vários novos genomas microbianos derivados de metagenomas. Os resultados descritos demonstram o papel-chave de micro-organismos em ecossistemas importantes para atividades humanas, assim como o potencial que vários desses micro-organismos tem de contribuir para aplicações biotecnológicas cruciais para a sobrevivência humana no século 21.

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The malaria parasite Plasmodium falciparum possesses a unique Acetyl-CoA Synthetase (PfACS), which provides acetyl moieties for different metabolic and regulatory cellular pathways. We characterized PfACS and studied its role focusing on epigenetic modifications using the var gene family as reporter genes. For this, mutant lines to modulate plasmodial ACS expression by degron-mediated protein degradation and ribozyme-induced transcript decay were created. Additionally, an inhibitor of the human Acetyl-CoA Synthetase 2 was tested for its effectiveness in interfering with PfACS. The knockdown of PfACS or its inhibition resulted in impaired parasite growth. Decreased levels of PfACS also led to differential histone acetylation patterns, altered variant gene expression, and concomitantly decreased cytoadherence of infected red blood cells containing knocked-down parasites. Further, ChIP analysis revealed the presence of PfACS in many loci in ring stage parasites, underscoring its involvement in the regulation of chromatin. Due to its central function in the plasmodial metabolism and significant differences to human ACS, PfACS is an interesting target for drug development.

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This protocol describes the genomic phage (gPhage) display platform, a large-scale antigen and epitope mapping technique. We constructed a gPhage display peptide library of a eukaryotic organism, Trypanosoma cruzi (causative agent of Chagas disease), to map the antibody response landscape against the parasite. Here, we used an organism with a relatively large but intronless genome, although future applications could include other prevalent or (re)emerging infectious organisms carrying genomes with a limited number of introns. For complete details on the use and execution of this protocol, please refer to Teixeira et al. (2021).

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