Biology subjects

Gonzalez-Tortuero, E.

Publications and source records attributed to Gonzalez-Tortuero, E..

9 recordsLinked to original sources

bioRxiv2023

Mechanisms of low susceptibility to the disinfectant benzalkonium chloride in a multidrug-resistant environmental isolate of Aeromonas hydrophila

Excessive discharge of quaternary ammonium disinfectants such as benzalkonium chloride (BAC) into aquatic systems can trigger several physiological responses in environmental microorganisms. In this study, we isolated a low susceptible strain of Aeromonas hydrophila, namely INISA09, to from a wastewater treatment plant in Costa Rica. We characterized its phenotypic response upon exposure to three different concentrations of BAC and determined the primary mechanisms related to its resistance using genomic and proteomic tools. The genome of the strain is 4.6-Mb with 4,273 genes, and we found a propound genome rearrangement and thousands of missense mutations when compared to the reference strain A. hydrophila ATCC 7966. We identified 15,762 missense mutations mainly associated with transport, antimicrobial resistance, and outer membrane proteins. In addition, quantitative proteomic analysis revealed the significant upregulation of several efflux pumps and the downregulation of porins when the strain was exposed to three BAC concentrations. Other genes related to membrane fatty acid metabolism and redox metabolic reactions also showed an altered expression. Our findings suggest that the response of A. hydrophila INISA09 to BAC primarily occurs at the membrane level, which is BACs primary target. This study sheds light on the mechanisms of antimicrobial susceptibility in tropical aquatic environments against a widely-used disinfectant, using an environmental strain that could serve as a new study model.

microbiology↗

bioRxiv2022

The impact of PrsA over-expression on the Bacillus subtilis transcriptome during fed-batch fermentation of alpha-amylase production

The production of the alpha-amylase (AMY) enzyme in Bacillus subtilis at a high rate leads to the accumulation of unfolded AMY, which causes secretion stress. The over-expression of the PrsA chaperone aids the enzyme folding and reduces stress. To identify affected pathways and potential mechanisms involved in the reduced growth, we analyzed the transcriptomic differences during fed-batch fermentation between a PrsA over-expressing strain and a control in a time-series RNA-seq experiment. We observe transcription in 542 previously un-annotated regions, of which 234 had significant changes in expression levels between the samples. Moreover, 1,791 protein-coding sequences, 80 non-coding genes, and 20 riboswitches overlapping UTR regions of coding genes had significant changes in expression. Via gene-set over-representation analysis of the differentially expressed genes, we identified putatively regulated biological processes; overall the analysis suggests that the PrsA over-expression affects ATP biosynthesis activity, amino acid metabolism, and cell wall stability. The investigation of the protein interaction network points to a potential impact on cell motility signaling. We discuss the impact of these highlighted mechanisms for reducing secretion stress or detrimental aspects of PrsA over-expression during AMY production.

microbiology↗

bioRxiv2022

CRISPRi screen for enhancing heterologous α-amylase yield in Bacillus subtilis

Enhancing yield during bacterial enzyme production could have positive economic and environmental impacts. For cell factories, such improvements in yields could potentially be obtained by fine-tuning the metabolic processes and their regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two -amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Complex operons that are regulated by a wide variety of transcription factors, non-coding and structured RNAs add to the challenge of finding yield-affecting candidates. Surprisingly, we found that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation (75% of novel RNA predictions had absolute logFC > 2). Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expressed annotations, including both novel candidate and prior annotated ncRNAs, we selected 53 non-coding candidates. The targeting with CRISPRi knockdowns transcription in a genomic region on both the sense and the antisense strand. Thus, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3 UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.

microbiology↗

bioRxiv2021

Comparative analysis of gene prediction tools for viral genome annotation

The number of newly available viral genomes and metagenomes has increased exponentially since the development of high throughput sequencing platforms and genome analysis tools. Bioinformatic annotation pipelines are largely based on open reading frame (ORF) calling software, which identifies genes independently of the sequence taxonomical background. Although ORF-calling programs provide a rapid genome annotation, they can misidentify ORFs and start codons; errors that might be perpetuated and propagated over time. This study evaluated the performance of multiple ORF-calling programs for viral genome annotation against the complete RefSeq viral database. Programs outputs varied when considering the viral nucleic acid type versus the viral host. According to the number of ORFs, Prodigal and Metaprodigal were the most accurate programs for DNA viruses, while FragGeneScan and Prodigal generated the most accurate outputs for RNA viruses. Similarly, Prodigal outperformed the benchmark for viruses infecting prokaryotes, and GLIMMER and GeneMarkS produced the most accurate annotations for viruses infecting eukaryotes. When the coordinates of the ORFs were considered, Prodigal scored high for all scenarios except for RNA viruses, where GeneMarkS generated the most reliable results. Overall, the quality of the coordinates predicted for RNA viruses was poorer than for DNA viruses, suggesting the need for improved ORF-calling programs to deal with RNA viruses. Moreover, none of the ORF-calling programs reached 90% accuracy for annotation of DNA viruses. Any automatic annotation can still be improved by manual curation, especially when the presence of ORFs is validated with wet-lab experiments. However, our evaluation of the current ORF-calling programs is expected to be useful for the improvement of viral genome annotation pipelines and highlights the need for more expression data to improve the rigor of reference genomes.

bioinformatics↗

bioRxiv2020

Amino acid oxidation constrained protein sequences during evolution: an analysis of a model bacterium.

Within the cell, there are compartments and local conditions that may constrain amino acid sequence. Life emerged in an anoxic world but releasing a photosynthesis by-product, the molecular oxygen, forced adaptive changes to counteract its toxicity. Several mechanisms evolved to balance the intracellular redox state and maintain a reductive environment more compatible with many essential biological functions. Here, we statistically interrogate the aminoacidic composition of E. coli proteins and investigate how the proneness or susceptibility to oxidation of amino acid biased their sequences. By sorting the proteins in five different compartments: cytoplasm, internal membrane, periplasm, outer membrane and extracellular; we found that various oxidative lesions constrain protein composition, that there is a dependency of the cellular compartments and there is an impact on the evenness distribution or frequency. This analysis is necessary from an evolutionary point of view to reflect how the oxidative atmosphere could restrict protein composition and probably imposed a trend in the codon bias.

evolutionary biology↗

bioRxiv2019

The BSGatlas: An enhanced annotation of genes and transcripts for the Bacillus subtilis genome with improved information access

The genome of Bacillus subtilis continues to provide exiting genomic insights. However, the growing collective genomic knowledge about this micro-organism is spread across multiple annotation resources. Thus, the full annotation is not directly accessible neither for specific genes nor for large-scale high-throughput analyses. Furthermore, access to annotation of non-coding RNA genes (ncRNAs) and polycistronic mRNAs is difficult. To address these challenges we introduce the Bacillus subtilis genome atlas, BSGatlas, in which we integrate and unify multiple existing annotation resources. Our integration provides twice as many ncRNAs than the individual resources, improves the positional annotation for 70% of the combined ncRNAs, and makes it possible to infer specific ncRNA types. Moreover, we unify known transcription start sites, termination, and transcriptional units (TUs) as a comprehensive transcript map. This transcript map implies 815 new TUs and 6, 164 untranslated regions (UTRs), which is a five-fold increase over existing resources. We furthermore, find 2, 309 operons covering the transcriptional annotation for 93% of all genes, corresponding to an improvement by 11%. The BSGatlas is available in multiple formats. A user can either download the entire annotation in the standardized GFF3 format, which is compatible with most bioinformatics tools for omics and high-throughput studies, or view the annotation in an online browser at http://rth.dk/resources/bsgatlas.\n\nImportanceThe Bacillus subtilis genome has been studied in numerous context and consequently multiple efforts have been made in providing a complete annotation. Unfortunately, a number of resources are no longer maintained, and (i) the collective annotation knowledge is dispersed over multiple resources, of which each has a different focus of what type of annotation information they provide. (ii) Thus, it is difficult to easily and at a large scale obtain information for a genomic region or genes of interest. (iii) Furthermore, all resources are essentially incomplete when it comes to annotating non-coding and structured RNA, and transcripts in general. Here, we address all three problems by first collecting existing annotations of genes and transcripts start and termination sites; afterwards resolving discrepancies in annotations and combining them, which doubled the number of ncRNAs; inferring full transcripts and 2,309 operons from the combined knowledge of known transcript boundaries and meta-information; and critically providing it all in a standardized UCSC browser. That interface and its powerful set of functionalities allow users to access all the information in a single resource as well as enables them to include own data on top the full annotation.

genomics↗

bioRxiv2019

Seawater salt-trapped Pseudomonas aeruginosa survives for years and gets primed for salinity tolerance

BackgroundIn nature, microorganisms have to adapt to long-term stressful conditions often with growth limitations. However, little is known about the evolution of the adaptability of new bacteria to such environments. Pseudomonas aeruginosa, an opportunistic pathogen, after natural evaporation of seawater, was shown to be trapped in laboratory-grown halite crystals and to remain viable after entrapment for years. However, how this bacterium persists and survives in such hypersaline conditions is not understood.\n\nResultsIn this study, we aimed to understand the basis of survival, and to characterise the physiological changes required to develop salt tolerance using P. aeruginosa as a model. Several clones of P. aeruginosa were rescued after fourteen years in naturally evaporated marine salt crystals. Incubation of samples in nutrient-rich broth allowed re-growth and subsequent plating yielded observable colonies. Whole genome sequencing of the P. aeruginosa isolates confirmed the recovery of the original strain. The re-grown strains, however, showed a new phenotype consisting of an enhanced growth in growing salt concentration compared to the ancestor strain. The intracellular accumulation of K+ was elicited by high concentration of Na+ in the external medium to maintain the homeostasis. Whole transcriptomic analysis by microarray indicated that seventy-eight genes had differential expression between the parental strain and derivative clones. Sixty-one transcripts were up-regulated, while seventeen were down-regulated. Based on a collection of single-gene knockout mutants and gene ontology analysis, we suggest that the adaptive response in P. aeruginosa to hyper-salinity relies on multiple gene product interactions.\n\nConclusionsThe individual gene contributions build up the observed phenotype, but do not ease the identification of salinity-related metabolic pathways. The long-term inclusion of P. aeruginosa in salt crystals primes the bacteria, mediating a readjustment of the bacterial physiology to growth in higher salt concentrations. Our findings provide a starting point to understand how P. aeruginosa, a relevant environmental and pathogenic bacterium, survives to long-term salt stress.

microbiology↗

bioRxiv2018

Biology and taxonomy of crAss-like bacteriophages, the most abundant virus in the human gut

CrAssphage is yet to be cultured even though it represents the most abundant virus in the gut microbiota of humans. Recently, sequence based classification was performed on distantly related crAss-like phages from multiple environments, leading to the proposal of a familial level taxonomic group [Yutin N, et al. (2018) Discovery of an expansive bacteriophage family that includes the most abundant viruses from the human gut. Nat Microbiol 3(1):38-46]. Here, we assembled the metagenomic sequencing reads from 702 human faecal virome/phageome samples and obtained 98 complete circular crAss-like phage genomes and 145 contigs [≥]70kb. In silico comparative genomics and taxonomic analysis was performed, resulting in a classification scheme of crAss-like phages from human faecal microbiomes into 4 candidate subfamilies composed of 10 candidate genera. Moreover, laboratory analysis was performed on faecal samples from an individual harbouring 7 distinct crAss-like phages. We achieved propagation of crAss-like phages in ex vivo human faecal fermentations and visualised Podoviridae virions by electron microscopy. Furthermore, detection of a crAss-like phage capsid protein could be linked to metagenomic sequencing data confirming crAss-like phage structural annotations.\n\nSignificanceCrAssphage is the most abundant biological entity in the human gut, but it remains uncultured in the laboratory and its host(s) is unknown. CrAssphage was not identified in metagenomic studies for many years as its sequence is so different from anything present in databases. To this day, it can only be detected from sequences assembled from metagenomics or viromic datasets (crAss - cross Assembly). In this study, we identified 243 new crAss-like phages from human faecal metagenomic studies. Taxonomic analysis of these crAss-like phages highlighted their extensive diversity within the human microbiome. We also present the first propagation of crAssphage in faecal fermentations and provide the first electron micrographs of this extraordinary bacteriophage.

microbiology↗

bioRxiv2018

VIGA: a sensitive, precise and automatic de novo VIral Genome Annotator.

Viral (meta)genomics is a rapidly growing field of study that is hampered by an inability to annotate the majority of viral sequences; therefore, the development of new bioinformatic approaches is very important. Here, we present a new automatic de novo genome annotation pipeline, called VIGA, to annotate prokaryotic and eukaryotic viral sequences from (meta)genomic studies. VIGA was benchmarked on a database of known viral genomes and a viral metagenomics case study. VIGA generated the most accurate outputs according to the number of coding sequences and their coordinates, outputs also had a lower number of non-informative annotations compared to other programs.

bioinformatics↗