Comparative Genomics Reveals Novel Species and Insights into the Biotechnological Potential, Virulence, and Resistance of Alcaligenes.

Alcaligenes is a cosmopolitan bacterial genus that exhibits diverse properties which are beneficial to plants. However, the genomic versatility of Alcaligenes has also been associated with the ability to cause opportunistic infections in humans, raising concerns about the safety of these microorganisms in biotechnological applications. Here, we report an in-depth comparative analysis of Alcaligenes species using all publicly available genomes to investigate genes associated with species, biotechnological potential, virulence, and resistance to multiple antibiotics. Phylogenomic analysis revealed that Alcaligenes consists of at least seven species, including three novel species. Pan-GWAS analysis uncovered 389 species-associated genes, including cold shock proteins (e.g., cspA) and aquaporins (e.g., aqpZ) found exclusively in the water-isolated species, Alcaligenes aquatilis. Functional annotation of plant-growth-promoting traits revealed enrichment of genes for auxin biosynthesis, siderophores, and organic acids. Genes involved in xenobiotic degradation and toxic metal tolerance were also identified. Virulome and resistome profiles provide insights into selective pressures exerted in clinical settings. Taken together, the results presented here provide the grounds for more detailed clinical and ecological studies of the genus Alcaligenes.

The Soybean Expression Atlas v2: A comprehensive database of over 5000 RNA-seq samples.

Soybean is a crucial crop worldwide, used as a source of food, feed, and industrial products due to its high protein and oil content. Previously, the rapid accumulation of soybean RNA-seq data in public databases and the computational challenges of processing raw RNA-seq data motivated us to develop the Soybean Expression Atlas, a gene expression database of over a thousand RNA-seq samples. Over the past few years, our database has allowed researchers to explore the expression profiles of important gene families, discover genes associated with agronomic traits, and understand the transcriptional dynamics of cellular processes. Here, we present the Soybean Expression Atlas v2, an updated version of our database with a fourfold increase in the number of samples, featuring transcript- and gene-level transcript abundance matrices for 5481 publicly available RNA-seq samples. New features in our database include the availability of transcript-level abundance estimates and equivalence classes to explore differential transcript usage, abundance estimates in bias-corrected counts to increase the accuracy of differential gene expression analyses, a new web interface with improved data visualization and user experience, and a reproducible and scalable pipeline available as an R package. The Soybean Expression Atlas v2 is available at https://soyatlas.venanciogroup.uenf.br/, and it will accelerate soybean research, empowering researchers with high-quality and easily accessible gene expression data.

Molecular Characterization of Salmonella Phage Wara Isolated from River Water in Brazil.

Antimicrobial resistance is increasing despite new treatments being employed, so novel strategies are required to ensure that bacterial infections remain treatable. Bacteriophages (phages; bacteria viruses) have the potential to be used as natural antimicrobial methods to control bacterial pathogens such as Salmonella spp. A Salmonella phage, Wara, was isolated from environmental water samples at the Subaé River Basin, Salvador de Bahia, Brazil. The basin has environmental impacts in its main watercourses arising from the dumping of domestic and industrial effluents and agricultural and anthropological activities. The phage genome sequence was determined by Oxford Nanopore Technologies (ONT) MinION and Illumina HiSeq sequencing, and assembly was carried out by Racon (MinION) and Unicycler (Illumina, Illumina + MinION). The genome was annotated and compared to other Salmonella phages using various bioinformatics approaches. MinION DNA sequencing combined with Racon assembly gave the best complete genome sequence. Phylogenetic analysis revealed that Wara is a member of the Tequintavirus genus. A lack of lysogeny genes, antimicrobial resistance, and virulence genes indicated that Wara has therapeutic and biocontrol potential against Salmonella species in healthcare and agriculture.

Hybrid Genomic Analysis of Salmonella enterica Serovar Enteritidis SE3 Isolated from Polluted Soil in Brazil.

In Brazil, Salmonella enterica serovar Enteritidis is a significant health threat. Salmonella enterica serovar Enteritidis SE3 was isolated from soil at the Subaé River in Santo Amaro, Brazil, a region contaminated with heavy metals and organic waste. Illumina HiSeq and Oxford Nanopore Technologies MinION sequencing were used for de novo hybrid assembly of the Salmonella SE3 genome. This approach yielded 10 contigs with 99.98% identity with S. enterica serovar Enteritidis OLF-SE2-98984-6. Twelve Salmonella pathogenic islands, multiple virulence genes, multiple antimicrobial gene resistance genes, seven phage defense systems, seven prophages and a heavy metal resistance gene were encoded in the genome. Pangenome analysis of the S. enterica clade, including Salmonella SE3, revealed an open pangenome, with a core genome of 2137 genes. Our study showed the effectiveness of a hybrid sequence assembly approach for environmental Salmonella genome analysis using HiSeq and MinION data. This approach enabled the identification of key resistance and virulence genes, and these data are important to inform the control of Salmonella and heavy metal pollution in the Santo Amaro region of Brazil.

Integrating omics approaches to discover and prioritize candidate genes involved in oil biosynthesis in soybean.

Soybean is a major source of edible protein and oil. Oil content is a quantitative trait that is significantly determined by genetic and environmental factors. Over the past 30 years, a large volume of soybean genetic, genomic, and transcriptomic data have been accumulated. Nevertheless, integrative analyses of such data remain scarce, in spite of their importance for crop improvement. We hypothesized that the co-occurrence of genomic regions for oil-related traits in different studies may reveal more stable regions encompassing important genetic determinants of oil content and quality in soybean. We integrated publicly available data, obtained with distinct techniques, to discover and prioritize candidate genes involved in oil biosynthesis and regulation in soybean. We detected key fatty acid biosynthesis genes (e.g., BCCP2 and ACCase, FADs, KAS family proteins) and several transcription factors, which are likely regulators of oil biosynthesis. In addition, we identified new candidates for seed oil accumulation and quality, such as Glyma.03G213300 and Glyma.19G160700, which encode a translocator protein homolog and a histone acetyltransferase, respectively. Further, oil and protein genomic hotspots are strongly associated with breeding and not with domestication, suggesting that soybean domestication prioritized other traits. The genes identified here are promising targets for breeding programs and for the development of soybean lines with increased oil content and quality.

Characterization of cellular, biochemical and genomic features of the diazotrophic plant growth-promoting bacterium Azospirillum sp. UENF-412522, a novel member of the Azospirillum genus.

Given their remarkable beneficial effects on plant growth, several Azospirillum isolates currently integrate the formulations of various commercial inoculants. Our research group isolated a new strain, Azospirillum sp. UENF-412522, from passion fruit rhizoplane. This isolate uses carbon sources that are partially distinct from closely-related Azospirillum isolates. Scanning electron microscopy analysis and population counts demonstrate the ability of Azospirillum sp. UENF-412522 to colonize the surface of passion fruit roots. In vitro assays demonstrate the ability of Azospirillum sp. UENF-412522 to fix atmospheric nitrogen, to solubilize phosphate and to produce indole-acetic acid. Passion fruit plantlets inoculated with Azospirillum sp. UENF-41255 showed increased shoot and root fresh matter by 13,8% and 88,6% respectively, as well as root dry matter by 61,4%, further highlighting its biotechnological potential for agriculture. We sequenced the genome of Azospirillum sp. UENF-412522 to investigate the genetic basis of its plant-growth promotion properties. We identified the key nif genes for nitrogen fixation, the complete PQQ operon for phosphate solubilization, the acdS gene that alleviates ethylene effects on plant growth, and the napCAB operon, which produces nitrite under anoxic conditions. We also found several genes conferring resistance to common soil antibiotics, which are critical for Azospirillum sp. UENF-412522 survival in the rhizosphere. Finally, we also assessed the Azospirillum pangenome and highlighted key genes involved in plant growth promotion. A phylogenetic reconstruction of the genus was also conducted. Our results support Azospirillum sp. UENF-412522 as a good candidate for bioinoculant formulations focused on plant growth promotion in sustainable systems.

Genome sequencing of the vermicompost strain Stenotrophomonas maltophilia UENF-4GII and population structure analysis of the S. maltophilia Sm3 genogroup.

The Stenotrophomonas maltophilia complex (Smc) is a cosmopolitan bacterial group that has been proposed an emergent multidrug-resistant pathogen. Taxonomic studies support the genomic heterogeneity of Smc, which comprises genogroups exhibiting a range of phenotypically distinct strains from different sources. Here, we report the genome sequencing and in-depth analysis of S. maltophilia UENF-4GII, isolated from vermicompost. This genome harbors a unique region encoding a penicillin-binding protein (pbpX) that was carried by a transposon, as well as horizontally-transferred genomic islands involved in anti-phage defense via DNA modification, and pili glycosylation. We also analyzed all available Smc genomes to investigate genes associated with resistance and virulence, niche occupation, and population structure. S. maltophilia UENF-4GII belongs to genogroup 3 (Sm3), which comprises three phylogenetic clusters (PC). Pan-GWAS analysis uncovered 471 environment-associated and 791 PC-associated genes, including antimicrobial resistance (e.g. blaL1 and blaR1) and virulence determinants (e.g. treS and katG) that provide insights on the resistance and virulence potential of Sm3 strains. Together, the results presented here provide the grounds for more detailed clinical and ecological investigations of S. maltophilia.

The global population structure and beta-lactamase repertoire of the opportunistic pathogen Serratia marcescens.

Serratia marcescens is a global spread nosocomial pathogen. This rod-shaped bacterium displays a broad host range and worldwide geographical distribution. Here we analyze an international collection of this multidrug-resistant, opportunistic pathogen from 35 countries to infer its population structure. We show that S. marcescens comprises 12 lineages; Sm1, Sm4, and Sm10 harbor 78.3% of the known environmental strains. Sm5, Sm6, and Sm7 comprise only human-associated strains which harbor smallest pangenomes, genomic fluidity and lowest levels of core recombination, indicating niche specialization. Sm7 and Sm9 lineages exhibit the most concerning resistome; blaKPC-2 plasmid is widespread in Sm7, whereas Sm9, also an anthropogenic-exclusive lineage, presents highest plasmid/lineage size ratio and plasmid-diversity encoding metallo-beta-lactamases comprising blaNDM-1. The heterogeneity of resistance patterns of S. marcescens lineages elucidated herein highlights the relevance of surveillance programs, using whole-genome sequencing, to provide insights into the molecular epidemiology of carbapenemase producing strains of this species.

Systematic analysis of 1298 RNA-Seq samples and construction of a comprehensive soybean (Glycine max) expression atlas.

Soybean (Glycine max [L.] Merr.) is a major crop in animal feed and human nutrition, mainly for its rich protein and oil contents. The remarkable rise in soybean transcriptome studies over the past 5 years generated an enormous amount of RNA-seq data, encompassing various tissues, developmental conditions and genotypes. In this study, we have collected data from 1298 publicly available soybean transcriptome samples, processed the raw sequencing reads and mapped them to the soybean reference genome in a systematic fashion. We found that 94% of the annotated genes (52 737/56 044) had detectable expression in at least one sample. Unsupervised clustering revealed three major groups, comprising samples from aerial, underground and seed/seed-related parts. We found 452 genes with uniform and constant expression levels, supporting their roles as housekeeping genes. On the other hand, 1349 genes showed heavily biased expression patterns towards particular tissues. A transcript-level analysis revealed that 95% (70 963 of 74 490) of the assembled transcripts have intron chains exactly matching those from known transcripts, whereas 3256 assembled transcripts represent potentially novel splicing isoforms. The dataset compiled here constitute a new resource for the community, which can be downloaded or accessed through a user-friendly web interface at http://venanciogroup.uenf.br/resources/. This comprehensive transcriptome atlas will likely accelerate research on soybean genetics and genomics.

Population structure and pangenome analysis of Enterobacter bugandensis uncover the presence of blaCTX-M-55, blaNDM-5 and blaIMI-1, along with sophisticated iron acquisition strategies.

Enterobacter bugandensis is a recently described species that has been largely associated with nosocomial infections. We report the genome of a non-clinical E. bugandensis strain, which was integrated with publicly available genomes to study the pangenome and general population structure of E. bugandensis. Core- and whole-genome multilocus sequence typing allowed the detection of five E. bugandensis phylogroups (PG-A to E), which contain important antimicrobial resistance and virulence determinants. We uncovered several extended-spectrum ß-lactamases, including blaCTX-M-55 and blaNDM-5, present in an IncX replicon type plasmid, described here for the first time in E. bugandensis. Genetic context analysis of blaNDM-5 revealed the resemblance of this plasmid with other IncX plasmids from other bacteria from the same country. Three distinctive siderophore producing operons were found in E. bugandensis: enterobactin (ent), aerobactin (iuc/iut), and salmochelin (iro). Our findings provide novel insights on the lifestyle, physiology, antimicrobial, and virulence profiles of E. bugandensis.

Genomic analysis unveils important aspects of population structure, virulence, and antimicrobial resistance in Klebsiella aerogenes.

Klebsiella aerogenes is an important pathogen in healthcare-associated infections. Nevertheless, in comparison to other clinically important pathogens, K. aerogenes population structure, genetic diversity, and pathogenicity remain poorly understood. Here, we elucidate K. aerogenes clonal complexes (CCs) and genomic features associated with resistance and virulence. We present a detailed description of the population structure of K. aerogenes based on 97 publicly available genomes by using both multilocus sequence typing and single-nucleotide polymorphisms extracted from the core genome. We also assessed virulence and resistance profiles using Virulence Finder Database and Comprehensive Antibiotic Resistance Database, respectively. We show that K. aerogenes has an open pangenome and a large effective population size, which account for its high genomic diversity and support that negative selection prevents fixation of most deleterious alleles. The population is structured in at least 10 CCs, including two novel ones identified here, CC9 and CC10. The repertoires of resistance genes comprise a high number of antibiotic efflux proteins as well as narrow- and extended-spectrum ß-lactamases. Regarding the population structure, we identified two clusters based on virulence profiles because of the presence of the toxin-encoding clb operon and the siderophore production genes, irp and ybt. Notably, CC3 comprises the majority of K. aerogenes isolates associated with hospital outbreaks, emphasizing the importance of constant monitoring of this pathogen. Collectively, our results may provide a foundation for the development of new therapeutic and surveillance strategies worldwide.

Molecular epidemiology of 16S rRNA methyltransferase in Brazil: RmtG in Klebsiella aerogenes ST93 (CC4).

Aminoglycosides are a class of antibiotics that play a key role in antimicrobial treatment of Multidrug resistant (MDR) Gram-negative bacilli, typically in combination with ß-lactams. Ribosomal 16S RNA modification by methyltransferases (e.g. RmtG) is an aminoglycoside resistance mechanism that, along with the occurrence carbapenem-resistant Enterobacteriaceae (CRE), has become a clinical concern. In Brazil, rmtG genes were initially reported in Klebsiella pneumoniae, and monitoring isolates from other species carrying this gene is critical for epidemiological studies and to prevent dissemination. Here we report the presence of rmtG in Klebisella aerogenes D3 and characterize its genetic context in comparison to isolates from other species. Further, we performed a phylogenetic reconstruction of 900 16S rRNA methyltransferases (16S-RMTases) and methyltransferase-related proteins. We show that, in K. aerogenes D3, rmtG co-occurs with sul2, near a transposon with an IS91-like insertion sequence. Resistome analysis revealed the co-production of RmtG and CTX-M-59. Ongoing surveillance of 16S-RMTases is crucial to delay the dissemination of such multiresistant isolates. Our results also highlight the reduction in treatment options for CRE infections, as well as the need of expanding prevention measures of these pathogens worldwide.