Glyphosate-based herbicide use affects individual microbial taxa in strawberry endosphere but not the microbial community composition.

AIMS: In a field study, the effects of treatments of glyphosate-based herbicides (GBHs) in soil, alone and in combination with phosphate fertilizer, were examined on the performance and endophytic microbiota of garden strawberry. METHODS AND RESULTS: The root and leaf endophytic microbiota of garden strawberries grown in GBH-treated and untreated soil, with and without phosphate fertilizer, were analyzed. Next, bioinformatics analysis on the type of 5-enolpyruvylshikimate-3-phosphate synthase enzyme was conducted to assess the potential sensitivity of strawberry-associated bacteria and fungi to glyphosate, and to compare the results with field observations. GBH treatments altered the abundance and/or frequency of several operational taxonomic units (OTUs), especially those of root-associated fungi and bacteria. These changes were partly related to their sensitivity to glyphosate. Still, GBH treatments did not shape the overall community structure of strawberry microbiota or affect plant performance. Phosphate fertilizer increased the abundance of both glyphosate-resistant and glyphosate-sensitive bacterial OTUs, regardless of the GBH treatments. CONCLUSIONS: These findings demonstrate that although the overall community structure of strawberry endophytic microbes is not affected by GBH use, some individual taxa are.

The Mutational Landscape of SARS-CoV-2.

Mutation research is crucial for detecting and treating SARS-CoV-2 and developing vaccines. Using over 5,300,000 sequences from SARS-CoV-2 genomes and custom Python programs, we analyzed the mutational landscape of SARS-CoV-2. Although almost every nucleotide in the SARS-CoV-2 genome has mutated at some time, the substantial differences in the frequency and regularity of mutations warrant further examination. C>U mutations are the most common. They are found in the largest number of variants, pangolin lineages, and countries, which indicates that they are a driving force behind the evolution of SARS-CoV-2. Not all SARS-CoV-2 genes have mutated in the same way. Fewer non-synonymous single nucleotide variations are found in genes that encode proteins with a critical role in virus replication than in genes with ancillary roles. Some genes, such as spike (S) and nucleocapsid (N), show more non-synonymous mutations than others. Although the prevalence of mutations in the target regions of COVID-19 diagnostic RT-qPCR tests is generally low, in some cases, such as for some primers that bind to the N gene, it is significant. Therefore, ongoing monitoring of SARS-CoV-2 mutations is crucial. The SARS-CoV-2 Mutation Portal provides access to a database of SARS-CoV-2 mutations.

Prediction of Recurrent Mutations in SARS-CoV-2 Using Artificial Neural Networks.

Predicting SARS-CoV-2 mutations is difficult, but predicting recurrent mutations driven by the host, such as those caused by host deaminases, is feasible. We used machine learning to predict which positions from the SARS-CoV-2 genome will hold a recurrent mutation and which mutations will be the most recurrent. We used data from April 2021 that we separated into three sets: a training set, a validation set, and an independent test set. For the test set, we obtained a specificity value of 0.69, a sensitivity value of 0.79, and an Area Under the Curve (AUC) of 0.8, showing that the prediction of recurrent SARS-CoV-2 mutations is feasible. Subsequently, we compared our predictions with updated data from January 2022, showing that some of the false positives in our prediction model become true positives later on. The most important variables detected by the model's Shapley Additive exPlanation (SHAP) are the nucleotide that mutates and RNA reactivity. This is consistent with the SARS-CoV-2 mutational bias pattern and the preference of some host deaminases for specific sequences and RNA secondary structures. We extend our investigation by analyzing the mutations from the variants of concern Alpha, Beta, Delta, Gamma, and Omicron. Finally, we analyzed amino acid changes by looking at the predicted recurrent mutations in the M-pro and spike proteins.

Alterations in Metabolome and Microbiome Associated with an Early Stress Stage in Male Wistar Rats: A Multi-Omics Approach.

Stress disorders have dramatically increased in recent decades becoming the most prevalent psychiatric disorder in the United States and Europe. However, the diagnosis of stress disorders is currently based on symptom checklist and psychological questionnaires, thus making the identification of candidate biomarkers necessary to gain better insights into this pathology and its related metabolic alterations. Regarding the identification of potential biomarkers, omic profiling and metabolic footprint arise as promising approaches to recognize early biochemical changes in such disease and provide opportunities for the development of integrative candidate biomarkers. Here, we studied plasma and urine metabolites together with metagenomics in a 3 days Chronic Unpredictable Mild Stress (3d CUMS) animal approach that aims to focus on the early stress period of a well-established depression model. The multi-omics integration showed a profile composed by a signature of eight plasma metabolites, six urine metabolites and five microbes. Specifically, threonic acid, malic acid, alpha-ketoglutarate, succinic acid and cholesterol were proposed as key metabolites that could serve as key potential biomarkers in plasma metabolome of early stages of stress. Such findings targeted the threonic acid metabolism and the tricarboxylic acid (TCA) cycle as important pathways in early stress. Additionally, an increase in opportunistic microbes as virus of the Herpesvirales was observed in the microbiota as an effect of the primary stress stages. Our results provide an experimental biochemical characterization of the early stage of CUMS accompanied by a subsequent omic profiling and a metabolic footprinting that provide potential candidate biomarkers.

Reconstruction of the evolution of microbial defense systems.

BACKGROUND: Evolution of bacterial and archaeal genomes is a highly dynamic process that involves intensive loss of genes as well as gene gain via horizontal transfer, with a lesser contribution from gene duplication. The rates of these processes can be estimated by comparing genomes that are linked by an evolutionary tree. These estimated rates of genome dynamics events substantially differ for different functional classes of genes. The genes involved in defense against viruses and other invading DNA are among those that are gained and lost at the highest rates. RESULTS: We employed a stochastic birth-and-death model to obtain maximum likelihood estimates of the rates of gain and loss of defense genes in 35 groups of closely related bacterial genomes and one group of archaeal genomes. We find that on average, the defense genes experience 1.4 fold higher flux than the rest of microbial genes. This excessive flux of defense genes over the genomic mean is consistent across diverse microbial groups. The few exceptions include intracellular parasites with small, degraded genomes that possess few defense systems which are more stable than in other microbes. Generally, defense genes follow the previously established pattern of genome dynamics, with gene family loss being about 3 times more common than gain and an order of magnitude more common than expansion or contraction of gene families. Case by case analysis of the evolutionary dynamics of defense genes indicates frequent multiple events in the same locus and widespread involvement of mobile elements in the gain and loss of defense genes. CONCLUSIONS: Evolution of microbial defense systems is highly dynamic but, notwithstanding the host-parasite arms race, generally follows the same trends that have been established for the rest of the genes. Apart from the paucity and the low flux of defense genes in parasitic bacteria with deteriorating genomes, there is no clear connection between the evolutionary regime of defense systems and microbial life style.

Genomes in turmoil: quantification of genome dynamics in prokaryote supergenomes.

BACKGROUND: Genomes of bacteria and archaea (collectively, prokaryotes) appear to exist in incessant flux, expanding via horizontal gene transfer and gene duplication, and contracting via gene loss. However, the actual rates of genome dynamics and relative contributions of different types of event across the diversity of prokaryotes are largely unknown, as are the sizes of microbial supergenomes, i.e. pools of genes that are accessible to the given microbial species. RESULTS: We performed a comprehensive analysis of the genome dynamics in 35 groups (34 bacterial and one archaeal) of closely related microbial genomes using a phylogenetic birth-and-death maximum likelihood model to quantify the rates of gene family gain and loss, as well as expansion and reduction. The results show that loss of gene families dominates the evolution of prokaryotes, occurring at approximately three times the rate of gain. The rates of gene family expansion and reduction are typically seven and twenty times less than the gain and loss rates, respectively. Thus, the prevailing mode of evolution in bacteria and archaea is genome contraction, which is partially compensated by the gain of new gene families via horizontal gene transfer. However, the rates of gene family gain, loss, expansion and reduction vary within wide ranges, with the most stable genomes showing rates about 25 times lower than the most dynamic genomes. For many groups, the supergenome estimated from the fraction of repetitive gene family gains includes about tenfold more gene families than the typical genome in the group although some groups appear to have vast, 'open' supergenomes. CONCLUSIONS: Reconstruction of evolution for groups of closely related bacteria and archaea reveals an extremely rapid and highly variable flux of genes in evolving microbial genomes, demonstrates that extensive gene loss and horizontal gene transfer leading to innovation are the two dominant evolutionary processes, and yields robust estimates of the supergenome size.

Fitness costs limit influenza A virus hemagglutinin glycosylation as an immune evasion strategy.

Here, we address the question of why the influenza A virus hemagglutinin (HA) does not escape immunity by hyperglycosylation. Uniquely among dozens of monoclonal antibodies specific for A/Puerto Rico/8/34, escape from H28-A2 neutralization requires substitutions introducing N-linked glycosylation at residue 131 or 144 in the globular domain. This escape decreases viral binding to cellular receptors, which must be compensated for by additional substitutions in HA or neuraminidase that enable viral replication. Sequence analysis of circulating H1 influenza viruses confirms the in vivo relevance of our findings: natural occurrence of glycosylation at residue 131 is always accompanied by a compensatory mutation known to increase HA receptor avidity. In vaccinated mice challenged with WT vs. H28-A2 escape mutants, the selective advantage conferred by glycan-mediated global reduction in antigenicity is trumped by the costs of diminished receptor avidity. These findings show that, although N-linked glycosylation can broadly diminish HA antigenicity, fitness costs restrict its deployment in immune evasion.

Use of the Phylobone database for the annotation of bone extracellular matrix proteins in reindeer (Rangifer tarandus).

Bone extracellular matrix (ECM) proteins play a key role in bone formation and regeneration, including structural and regulatory functions. The Phylobone database consists of 255 ECM protein groups from 39 species and can be used to support bone research. Here, we gathered bone ECM proteins from reindeer (Rangifer tarandus), a member of the Cervidae family. The importance of reindeer lies in their ability to regenerate their antlers, in both male and female individuals. Protein sequences were extracted from the National Center for Biotechnology Information's repository and selected by homology searches. We identified 215 proteins and their corresponding functional domains, which are putatively present in the bone ECM of reindeer. Protein sequence alignments have shown a high degree of conservation between R. tarandus and other members of the Cervidae family. This update expands the Phylobone database and shows that it is a useful resource for the preliminary annotation of bone ECM proteins in novel proteomes.

Pollution-related changes in nest microbiota: Implications for growth and fledging in three passerine birds.

Non-ferrous smelters emit toxic metals into the environment, posing a threat to wildlife health. Despite the acknowledged role of microbes in host health, the impact of such emissions on host-associated microbiota, especially in wild birds, remains largely unexplored. This study investigates the associations of metal pollution, fitness, and nest microbiota (serving as a proxy for early-life microbial environment) which may influence the nestling health and development. Our study focuses on three passerine birds, the great tit (Parus major), blue tit (Cyanistes caeruleus), and pied flycatcher (Ficedula hypoleuca), within control and metal-polluted sites around a Finnish copper-nickel smelter. The polluted sites had been contaminated with arsenic (As), cadmium (Cd), copper (Cu), nickel (Ni), and zinc (Zn). We performed bacterial 16S rRNA sequencing and metal analyses on 90 nests and monitored nestling body mass, fledging success, and various biotic and abiotic factors. Our findings revealed species-specific responses to metal exposure in terms of both fitness and nest microbiota. P. major and C. caeruleus showed sensitivity to pollution, with decreased nestling growth and fledging in the polluted zone. This was accompanied by a shift in the bacterial community composition, which was characterized by an increase in some pathogenic bacteria (in P. major and C. caeruleus nests) and by a decrease in plant-associated bacteria (within C. caeruleus nests). Conversely, F. hypoleuca and their nest microbiota showed limited responses to pollution, indicating greater tolerance to pollution-induced environmental changes. Although pollution did not correlate with nest alpha diversity or the most abundant bacterial taxa across all species, certain potential pathogens within the nests were enriched in polluted environments and negatively correlated with nestling fitness parameters. Our results suggest that metal pollution may alter the nest bacterial composition in some bird species, either directly or indirectly through environmental changes, promoting pathogenic bacteria and potentially impacting bird survival.

Phylobone: a comprehensive database of bone extracellular matrix proteins in human and model organisms.

The bone extracellular matrix (ECM) contains minerals deposited on highly crosslinked collagen fibrils and hundreds of non-collagenous proteins. Some of these proteins are key to the regulation of bone formation and regeneration via signaling pathways, and play important regulatory and structural roles. However, the complete list of bone extracellular matrix proteins, their roles, and the extent of individual and cross-species variations have not been fully captured in both humans and model organisms. Here, we introduce the most comprehensive resource of bone extracellular matrix (ECM) proteins that can be used in research fields such as bone regeneration, osteoporosis, and mechanobiology. The Phylobone database (available at https://phylobone.com ) includes 255 proteins potentially expressed in the bone extracellular matrix (ECM) of humans and 30 species of vertebrates. A bioinformatics pipeline was used to identify the evolutionary relationships of bone ECM proteins. The analysis facilitated the identification of potential model organisms to study the molecular mechanisms of bone regeneration. A network analysis showed high connectivity of bone ECM proteins. A total of 214 functional protein domains were identified, including collagen and the domains involved in bone formation and resorption. Information from public drug repositories was used to identify potential repurposing of existing drugs. The Phylobone database provides a platform to study bone regeneration and osteoporosis in light of (biological) evolution, and will substantially contribute to the identification of molecular mechanisms and drug targets.

Ecosystem consequences of herbicides: the role of microbiome.

Non-target organisms are globally exposed to herbicides. While many herbicides - for example, glyphosate - were initially considered safe, increasing evidence demonstrates that they have profound effects on ecosystem functions via altered microbial communities. We provide a comprehensive framework on how herbicide residues may modulate ecosystem-level outcomes via alteration of microbiomes. The changes in soil microbiome are likely to influence key nutrient cycling and plant-soil processes. Herbicide-altered microbiome affects plant and animal performance and can influence trophic interactions such as herbivory and pollination. These changes are expected to lead to ecosystem and even evolutionary consequences for both microbes and hosts. Tackling the threats caused by agrochemicals to ecosystem functions and services requires tools and solutions based on a comprehensive understanding of microbe-mediated risks.

Glyphosate and a glyphosate-based herbicide affect bumblebee gut microbiota.

Pollinator decline is one of the gravest challenges facing the world today, and the overuse of pesticides may be among its causes. Here, we studied whether glyphosate, the world's most widely used pesticide, affects the bumblebee gut microbiota. We exposed the bumblebee diet to glyphosate and a glyphosate-based herbicide and quantified the microbiota community shifts using 16S rRNA gene sequencing. Furthermore, we estimated the potential sensitivity of bee gut microbes to glyphosate based on previously reported presence of target enzyme. Glyphosate increased, whereas the glyphosate-based herbicide decreased gut microbiota diversity, indicating that negative effects are attributable to co-formulants. Both glyphosate and the glyphosate-based herbicide treatments significantly decreased the relative abundance of potentially glyphosate-sensitive bacterial species Snodgrasella alvi. However, the relative abundance of potentially glyphosate-sensitive Candidatus Schmidhempelia genera increased in bumblebees treated with glyphosate. Overall, 50% of the bacterial genera detected in the bee gut microbiota were classified as potentially resistant to glyphosate, while 36% were classified as sensitive. Healthy core microbiota have been shown to protect bees from parasite infections, change metabolism, and decrease mortality. Thus, the heavy use of glyphosate-based herbicides may have implications on bees and ecosystems.

Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-specific genes.

Three classes of low-G+C Gram-positive bacteria (Firmicutes), Bacilli, Clostridia and Negativicutes, include numerous members that are capable of producing heat-resistant endospores. Spore-forming firmicutes include many environmentally important organisms, such as insect pathogens and cellulose-degrading industrial strains, as well as human pathogens responsible for such diseases as anthrax, botulism, gas gangrene and tetanus. In the best-studied model organism Bacillus subtilis, sporulation involves over 500 genes, many of which are conserved among other bacilli and clostridia. This work aimed to define the genomic requirements for sporulation through an analysis of the presence of sporulation genes in various firmicutes, including those with smaller genomes than B. subtilis. Cultivable spore-formers were found to have genomes larger than 2300 kb and encompass over 2150 protein-coding genes of which 60 are orthologues of genes that are apparently essential for sporulation in B. subtilis. Clostridial spore-formers lack, among others, spoIIB, sda, spoVID and safA genes and have non-orthologous displacements of spoIIQ and spoIVFA, suggesting substantial differences between bacilli and clostridia in the engulfment and spore coat formation steps. Many B. subtilis sporulation genes, particularly those encoding small acid-soluble spore proteins and spore coat proteins, were found only in the family Bacillaceae, or even in a subset of Bacillus spp. Phylogenetic profiles of sporulation genes, compiled in this work, confirm the presence of a common sporulation gene core, but also illuminate the diversity of the sporulation processes within various lineages. These profiles should help further experimental studies of uncharacterized widespread sporulation genes, which would ultimately allow delineation of the minimal set(s) of sporulation-specific genes in Bacilli and Clostridia.

Glycosylation focuses sequence variation in the influenza A virus H1 hemagglutinin globular domain.

Antigenic drift in the influenza A virus hemagglutinin (HA) is responsible for seasonal reformulation of influenza vaccines. Here, we address an important and largely overlooked issue in antigenic drift: how does the number and location of glycosylation sites affect HA evolution in man? We analyzed the glycosylation status of all full-length H1 subtype HA sequences available in the NCBI influenza database. We devised the "flow index" (FI), a simple algorithm that calculates the tendency for viruses to gain or lose consensus glycosylation sites. The FI predicts the predominance of glycosylation states among existing strains. Our analyses show that while the number of glycosylation sites in the HA globular domain does not influence the overall magnitude of variation in defined antigenic regions, variation focuses on those regions unshielded by glycosylation. This supports the conclusion that glycosylation generally shields HA from antibody-mediated neutralization, and implies that fitness costs in accommodating oligosaccharides limit virus escape via HA hyperglycosylation.

Rethinking the Intrinsic Sensitivity of Fungi to Glyphosate.

The 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) is the central enzyme of the shikimate pathway to synthesize the three aromatic amino acids in fungi, plants, and prokaryotes. This enzyme is the target of the herbicide glyphosate. In most plants and prokaryotes, the EPSPS protein is constituted by a single domain family, the EPSP synthase (PF00275) domain, whereas in fungi, the protein is formed by a multi-domain structure from combinations of 22 EPSPS-associated domains. The most common multi-domain EPSPS structure in fungi involves five EPSPS-associated domains of the shikimate pathway. In this article, we analyze 390 EPSPS proteins of fungi to determine the extent of the EPSPS-associated domains. Based on the current classification of the EPSPS protein, most fungal species are intrinsically sensitive to glyphosate. However, complex domain architectures may have multiple responses to the herbicide. Further empirical studies are needed to determine the effect of glyphosate on fungi, taking into account the diversity of multi-domain architectures of the EPSPS. This research opens the door to novel biotechnological applications for microbial degradation of glyphosate.

Why Is the UAG (Amber) Stop Codon Almost Absent in Highly Expressed Bacterial Genes?

The genome hypothesis postulates that genes in a genome tend to conform to their species' usage of the codon catalog and the GC content of the DNA. Thus, codon frequencies differ across organisms, including the three termination codons in the standard genetic code. Here, we analyze the frequencies of stop codons in a group of highly expressed genes from 196 prokaryotes under strong translational selection. The occurrence of the three translation termination codons is highly biased, with UAA (ochre) being the most prevalent in almost all bacteria. In contrast, UAG (amber) is the least frequent termination codon, e.g., only 321 occurrences (7.4%) in E. coli K-12 substr. W3110. Of the 253 highly expressed genes, only two end with an UAG codon. The strength of the selective bias against UAG in highly expressed genes varies among bacterial genomes, but it is not affected by the GC content of these genomes. In contrast, increased GC content results in a decrease in UAA abundance with a concomitant increase in UGA abundance. We propose that readthrough efficiency and context effects could explain the prevalence of UAA over UAG, particularly in highly expressed genes. Findings from this communication can be utilized for the optimization of gene expression.

Does Glyphosate Affect the Human Microbiota?

Glyphosate is the world's most widely used agrochemical. Its use in agriculture and gardening has been proclaimed safe because humans and other animals do not have the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). However, increasing numbers of studies have demonstrated risks to humans and animals because the shikimate metabolic pathway is present in many microbes. Here, we assess the potential effect of glyphosate on healthy human microbiota. Our results demonstrate that more than one-half of human microbiome are intrinsically sensitive to glyphosate. However, further empirical studies are needed to determine the effect of glyphosate on healthy human microbiota.

Quantification of the Potential Impact of Glyphosate-Based Products on Microbiomes.

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. The target of glyphosate is the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway, which is virtually universal in plants. The inhibition of the enzyme stops the production of three essential amino acids: phenylalanine, tyrosine, and tryptophan. EPSPS is also present in fungi and prokaryotes, such as archaea and bacteria; thus, the use of GBP may have an impact on the microbiome composition of soils, plants, herbivores, and secondary consumers. This article aims to present general guidelines to assess the effect of GBP on microbiomes from field experiments to bioinformatics analyses and provide a few testable hypotheses. Two field experiments are presented to test the GBP on non-target organisms. First, plant-associated microbes from 10 replicated control and GBP treatment plots simulating no-till cropping are sampled and analyzed. In the second experiment, samples from experimental plots fertilized by either poultry manure containing glyphosate residues or non-treated control manure were obtained. Bioinformatics analysis of EPSPS protein sequences is utilized to determine the potential sensitivity of microbes to glyphosate. The first step in estimating the effect of GBP on microbiomes is to determine their potential sensitivity to the target enzyme (EPSPS). Microbial sequences can be obtained either from public repositories or by means of PCR amplification. However, in the majority of field studies, microbiome composition has been determined based on universal DNA markers such as the 16S rRNA and the internal transcribed spacer (ITS). In these cases, sensitivity to glyphosate can only be estimated through a probabilistic analysis of EPSPS sequences using closely related species. The quantification of the potential sensitivity of organisms to glyphosate, based on the EPSPS enzyme, provides a robust approach for further experiments to study target and non-target resistant mechanisms.

Gaining and losing the thermophilic adaptation in prokaryotes.

We studied the evolution of thermophily in prokaryotes using the phylogenetic relationships between 279 bacteria and archaea and their thermophilic amino acid composition signature. Our findings suggest several examples in which the capacity of thermophilic adaptation has been gained or lost over relatively short evolutionary periods throughout the evolution of prokaryotes.

Genomics of the proteorhodopsin-containing marine flavobacterium Dokdonia sp. strain MED134.

Proteorhodopsin phototrophy is expected to have considerable impact on the ecology and biogeochemical roles of marine bacteria. However, the genetic features contributing to the success of proteorhodopsin-containing bacteria remain largely unknown. We investigated the genome of Dokdonia sp. strain MED134 (Bacteroidetes) for features potentially explaining its ability to grow better in light than darkness. MED134 has a relatively high number of peptidases, suggesting that amino acids are the main carbon and nitrogen sources. In addition, MED134 shares with other environmental genomes a reduction in gene copies at the expense of important ones, like membrane transporters, which might be compensated by the presence of the proteorhodopsin gene. The genome analyses suggest Dokdonia sp. MED134 is able to respond to light at least partly due to the presence of a strong flavobacterial consensus promoter sequence for the proteorhodopsin gene. Moreover, Dokdonia sp. MED134 has a complete set of anaplerotic enzymes likely to play a role in the adaptation of the carbon anabolism to the different sources of energy it can use, including light or various organic matter compounds. In addition to promoting growth, proteorhodopsin phototrophy could provide energy for the degradation of complex or recalcitrant organic matter, survival during periods of low nutrients, or uptake of amino acids and peptides at low concentrations. Our analysis suggests that the ability to harness light potentially makes MED134 less dependent on the amount and quality of organic matter or other nutrients. The genomic features reported here may well be among the keys to a successful photoheterotrophic lifestyle.