A model industrial workhorse: Bacillus subtilis strain 168 and its genome after a quarter of a century.

The vast majority of genomic sequences are automatically annotated using various software programs. The accuracy of these annotations depends heavily on the very few manual annotation efforts that combine verified experimental data with genomic sequences from model organisms. Here, we summarize the updated functional annotation of Bacillus subtilis strain 168, a quarter century after its genome sequence was first made public. Since the last such effort 5 years ago, 1168 genetic functions have been updated, allowing the construction of a new metabolic model of this organism of environmental and industrial interest. The emphasis in this review is on new metabolic insights, the role of metals in metabolism and macromolecule biosynthesis, functions involved in biofilm formation, features controlling cell growth, and finally, protein agents that allow class discrimination, thus allowing maintenance management, and accuracy of all cell processes. New 'genomic objects' and an extensive updated literature review have been included for the sequence, now available at the International Nucleotide Sequence Database Collaboration (INSDC: AccNum AL009126.4).

Correction: PPanGGOLiN: Depicting microbial diversity via a partitioned pangenome graph.

[This corrects the article DOI: 10.1371/journal.pcbi.1007732.].

ProMetIS, deep phenotyping of mouse models by combined proteomics and metabolomics analysis.

Genes are pleiotropic and getting a better knowledge of their function requires a comprehensive characterization of their mutants. Here, we generated multi-level data combining phenomic, proteomic and metabolomic acquisitions from plasma and liver tissues of two C57BL/6 N mouse models lacking the Lat (linker for activation of T cells) and the Mx2 (MX dynamin-like GTPase 2) genes, respectively. Our dataset consists of 9 assays (1 preclinical, 2 proteomics and 6 metabolomics) generated with a fully non-targeted and standardized approach. The data and processing code are publicly available in the ProMetIS R package to ensure accessibility, interoperability, and reusability. The dataset thus provides unique molecular information about the physiological role of the Lat and Mx2 genes. Furthermore, the protocols described herein can be easily extended to a larger number of individuals and tissues. Finally, this resource will be of great interest to develop new bioinformatic and biostatistic methods for multi-omics data integration.

panRGP: a pangenome-based method to predict genomic islands and explore their diversity.

MOTIVATION: Horizontal gene transfer (HGT) is a major source of variability in prokaryotic genomes. Regions of genome plasticity (RGPs) are clusters of genes located in highly variable genomic regions. Most of them arise from HGT and correspond to genomic islands (GIs). The study of those regions at the species level has become increasingly difficult with the data deluge of genomes. To date, no methods are available to identify GIs using hundreds of genomes to explore their diversity. RESULTS: We present here the panRGP method that predicts RGPs using pangenome graphs made of all available genomes for a given species. It allows the study of thousands of genomes in order to access the diversity of RGPs and to predict spots of insertions. It gave the best predictions when benchmarked along other GI detection tools against a reference dataset. In addition, we illustrated its use on metagenome assembled genomes by redefining the borders of the leuX tRNA hotspot, a well-studied spot of insertion in Escherichia coli. panRPG is a scalable and reliable tool to predict GIs and spots making it an ideal approach for large comparative studies. AVAILABILITY AND IMPLEMENTATION: The methods presented in the current work are available through the following software: https://github.com/labgem/PPanGGOLiN. Detailed results and scripts to compute the benchmark metrics are available at https://github.com/axbazin/panrgp_supdata.

Ancestral state reconstruction of metabolic pathways across pangenome ensembles.

As genome sequencing efforts are unveiling the genetic diversity of the biosphere with an unprecedented speed, there is a need to accurately describe the structural and functional properties of groups of extant species whose genomes have been sequenced, as well as their inferred ancestors, at any given taxonomic level of their phylogeny. Elaborate approaches for the reconstruction of ancestral states at the sequence level have been developed, subsequently augmented by methods based on gene content. While these approaches of sequence or gene-content reconstruction have been successfully deployed, there has been less progress on the explicit inference of functional properties of ancestral genomes, in terms of metabolic pathways and other cellular processes. Herein, we describe PathTrace, an efficient algorithm for parsimony-based reconstructions of the evolutionary history of individual metabolic pathways, pivotal representations of key functional modules of cellular function. The algorithm is implemented as a five-step process through which pathways are represented as fuzzy vectors, where each enzyme is associated with a taxonomic conservation value derived from the phylogenetic profile of its protein sequence. The method is evaluated with a selected benchmark set of pathways against collections of genome sequences from key data resources. By deploying a pangenome-driven approach for pathway sets, we demonstrate that the inferred patterns are largely insensitive to noise, as opposed to gene-content reconstruction methods. In addition, the resulting reconstructions are closely correlated with the evolutionary distance of the taxa under study, suggesting that a diligent selection of target pangenomes is essential for maintaining cohesiveness of the method and consistency of the inference, serving as an internal control for an arbitrary selection of queries. The PathTrace method is a first step towards the large-scale analysis of metabolic pathway evolution and our deeper understanding of functional relationships reflected in emerging pangenome collections.

PPanGGOLiN: Depicting microbial diversity via a partitioned pangenome graph.

The use of comparative genomics for functional, evolutionary, and epidemiological studies requires methods to classify gene families in terms of occurrence in a given species. These methods usually lack multivariate statistical models to infer the partitions and the optimal number of classes and don't account for genome organization. We introduce a graph structure to model pangenomes in which nodes represent gene families and edges represent genomic neighborhood. Our method, named PPanGGOLiN, partitions nodes using an Expectation-Maximization algorithm based on multivariate Bernoulli Mixture Model coupled with a Markov Random Field. This approach takes into account the topology of the graph and the presence/absence of genes in pangenomes to classify gene families into persistent, cloud, and one or several shell partitions. By analyzing the partitioned pangenome graphs of isolate genomes from 439 species and metagenome-assembled genomes from 78 species, we demonstrate that our method is effective in estimating the persistent genome. Interestingly, it shows that the shell genome is a key element to understand genome dynamics, presumably because it reflects how genes present at intermediate frequencies drive adaptation of species, and its proportion in genomes is independent of genome size. The graph-based approach proposed by PPanGGOLiN is useful to depict the overall genomic diversity of thousands of strains in a compact structure and provides an effective basis for very large scale comparative genomics. The software is freely available at https://github.com/labgem/PPanGGOLiN.

TbD1 deletion as a driver of the evolutionary success of modern epidemic Mycobacterium tuberculosis lineages.

Mycobacterium tuberculosis (Mtb) strains are classified into different phylogenetic lineages (L), three of which (L2/L3/L4) emerged from a common progenitor after the loss of the MmpS6/MmpL6-encoding Mtb-specific deletion 1 region (TbD1). These TbD1-deleted "modern" lineages are responsible for globally-spread tuberculosis epidemics, whereas TbD1-intact "ancestral" lineages tend to be restricted to specific geographical areas, such as South India and South East Asia (L1) or East Africa (L7). By constructing and characterizing a panel of recombinant TbD1-knock-in and knock-out strains and comparison with clinical isolates, here we show that deletion of TbD1 confers to Mtb a significant increase in resistance to oxidative stress and hypoxia, which correlates with enhanced virulence in selected cellular, guinea pig and C3HeB/FeJ mouse infection models, the latter two mirroring in part the development of hypoxic granulomas in human disease progression. Our results suggest that loss of TbD1 at the origin of the L2/L3/L4 Mtb lineages was a key driver for their global epidemic spread and outstanding evolutionary success.

MicroScope: an integrated platform for the annotation and exploration of microbial gene functions through genomic, pangenomic and metabolic comparative analysis.

Large-scale genome sequencing and the increasingly massive use of high-throughput approaches produce a vast amount of new information that completely transforms our understanding of thousands of microbial species. However, despite the development of powerful bioinformatics approaches, full interpretation of the content of these genomes remains a difficult task. Launched in 2005, the MicroScope platform (https://www.genoscope.cns.fr/agc/microscope) has been under continuous development and provides analysis for prokaryotic genome projects together with metabolic network reconstruction and post-genomic experiments allowing users to improve the understanding of gene functions. Here we present new improvements of the MicroScope user interface for genome selection, navigation and expert gene annotation. Automatic functional annotation procedures of the platform have also been updated and we added several new tools for the functional annotation of genes and genomic regions. We finally focus on new tools and pipeline developed to perform comparative analyses on hundreds of genomes based on pangenome graphs. To date, MicroScope contains data for >11 800 microbial genomes, part of which are manually curated and maintained by microbiologists (>4500 personal accounts in September 2019). The platform enables collaborative work in a rich comparative genomic context and improves community-based curation efforts.

Specialization of small non-conjugative plasmids in Escherichia coli according to their family types.

We undertook a comprehensive comparative analysis of a collection of 30 small (<25 kb) non-conjugative Escherichia coli plasmids previously classified by the gene sharing approach into 10 families, as well as plasmids found in the National Center for Biotechnology Information (NCBI) nucleotide database sharing similar genomic sequences. In total, 302 mobilizable (belonging to 2 MOBrep and 5 MOBRNA families) and 106 non-transferable/relaxase-negative (belonging to three ReLRNA families) plasmids were explored. The most striking feature was the specialization of the plasmid family types that was not related to their transmission mode and replication system. We observed a range of host strain specificity, from narrow E. coli host specificity to broad host range specificity, including a wide spectrum of Enterobacteriaceae. We found a wide variety of toxin/antitoxin systems and colicin operons in the plasmids, whose numbers and types varied according to the plasmid family type. The plasmids carried genes conferring resistance spanning almost all of the antibiotic classes, from those to which resistance developed early, such as sulphonamides, to those for which resistance has only developed recently, such as colistin. However, the prevalence of the resistance genes varied greatly according to the family type, ranging from 0 to 100 %. The evolutionary history of the plasmids based on the family type core genes showed variability within family nucleotide divergences in the range of E. coli chromosomal housekeeping genes, indicating long-term co-evolution between plasmids and host strains. In rare cases, a low evolutionary divergence suggested the massive spread of an epidemic plasmid. Overall, the importance of these small non-conjugative plasmids in bacterial adaptation varied greatly according to the type of family they belonged to, with each plasmid family having specific hosts and genetic traits.

The Arginine Deiminase Operon Is Responsible for a Fitness Trade-Off in Extended-Spectrum-ß-Lactamase-Producing Strains of Escherichia coli.

We previously identified an operon involved in an arginine deiminase (ADI) pathway (arc operon) on a CTX-M-producing plasmid from an O102-ST405 strain of Escherichia coli As the ADI pathway was shown to be involved in the virulence of various Gram-positive bacteria, we tested whether the ADI pathway could be involved in the epidemiological success of extended-spectrum-ß-lactamase (ESBL)-producing E. coli strains. We studied two collections of human E. coli isolated in France (n = 493) and England (n = 1,509) and show that the prevalence of the arc operon (i) is higher in ESBL-producing strains (12.1%) than in nonproducers (2.5%), (ii) is higher in CTX-M-producing strains (16%) than in other ESBL producers (3.5%), and (iii) increased over time in ESBL-producing strains from 0% before 2000 to 43.3% in 2011 to 2012. The arc operon, found in strains from various phylogenetic backgrounds, is carried by IncF plasmids (85%) or chromosomes (15%) in regions framed by numerous insertion sequences, indicating multiple arrivals. Competition experiments showed that the arc operon enhances fitness of the strain in vitro in lysogeny broth with arginine. In vivo competition experiments showed that the arc operon is advantageous for the strain in a mouse model of urinary tract infection (UTI), whereas it is a burden in a mouse model of intestinal colonization. In summary, we have identified a trait linked to CTX-M-producing strains that is responsible for a trade-off between two main E. coli lifestyles, UTI and gut commensalism. This trait alone cannot explain the wide spread of ESBLs in E. coli but merits epidemiological surveillance.

MicroScope-an integrated resource for community expertise of gene functions and comparative analysis of microbial genomic and metabolic data.

The overwhelming list of new bacterial genomes becoming available on a daily basis makes accurate genome annotation an essential step that ultimately determines the relevance of thousands of genomes stored in public databanks. The MicroScope platform (http://www.genoscope.cns.fr/agc/microscope) is an integrative resource that supports systematic and efficient revision of microbial genome annotation, data management and comparative analysis. Starting from the results of our syntactic, functional and relational annotation pipelines, MicroScope provides an integrated environment for the expert annotation and comparative analysis of prokaryotic genomes. It combines tools and graphical interfaces to analyze genomes and to perform the manual curation of gene function in a comparative genomics and metabolic context. In this article, we describe the free-of-charge MicroScope services for the annotation and analysis of microbial (meta)genomes, transcriptomic and re-sequencing data. Then, the functionalities of the platform are presented in a way providing practical guidance and help to the nonspecialists in bioinformatics. Newly integrated analysis tools (i.e. prediction of virulence and resistance genes in bacterial genomes) and original method recently developed (the pan-genome graph representation) are also described. Integrated environments such as MicroScope clearly contribute, through the user community, to help maintaining accurate resources.

Extended-spectrum ß-lactamase-encoding genes are spreading on a wide range of Escherichia coli plasmids existing prior to the use of third-generation cephalosporins.

To understand the evolutionary dynamics of extended-spectrum ß-lactamase (ESBL)-encoding genes in Escherichia coli, we undertook a comparative genomic analysis of 116 whole plasmid sequences of human or animal origin isolated over a period spanning before and after the use of third-generation cephalosporins (3GCs) using a gene-sharing network approach. The plasmids included 82 conjugative, 22 mobilizable and 9 non-transferable plasmids and 3 P-like bacteriophages. ESBL-encoding genes were found on 64 conjugative, 6 mobilizable, 2 non-transferable plasmids and 2 P1-like bacteriophages, indicating that these last three types of mobile elements also play a role, albeit modest, in the diffusion of the ESBLs. The network analysis showed that the plasmids clustered according to their genome backbone type, but not by origin or period of isolation or by antibiotic-resistance type, including type of ESBL-encoding gene. There was no association between the type of plasmid and the phylogenetic history of the parental strains. Finer scale analysis of the more abundant clusters IncF and IncI1 showed that ESBL-encoding plasmids and plasmids isolated before the use of 3GCs had the same diversity and phylogenetic history, and that acquisition of ESBL-encoding genes had occurred during multiple independent events. Moreover, the blaCTX-M-15 gene, unlike other CTX-M genes, was inserted at a hot spot in a blaTEM-1-Tn2 transposon. These findings showed that ESBL-encoding genes have arrived on wide range of pre-existing plasmids and that the successful spread of blaCTX-M-15 seems to be favoured by the presence of well-adapted IncF plasmids that carry a Tn2-blaTEM-1 transposon.

Expansion of the SOS regulon of Vibrio cholerae through extensive transcriptome analysis and experimental validation.

BACKGROUND: The SOS response is an almost ubiquitous response of cells to genotoxic stresses. The full complement of genes in the SOS regulon for Vibrio species has only been addressed through bioinformatic analyses predicting LexA binding box consensus and in vitro validation. Here, we perform whole transcriptome sequencing from Vibrio cholerae treated with mitomycin C as an SOS inducer to characterize the SOS regulon and other pathways affected by this treatment. RESULTS: Comprehensive transcriptional profiling allowed us to define the full landscape of promoters and transcripts active in V. cholerae. We performed extensive transcription start site (TSS) mapping as well as detection/quantification of the coding and non-coding RNA (ncRNA) repertoire in strain N16961. To improve TSS detection, we developed a new technique to treat RNA extracted from cells grown in various conditions. This allowed for identification of 3078 TSSs with an average 5'UTR of 116 nucleotides, and peak distribution between 16 and 64 nucleotides; as well as 629 ncRNAs. Mitomycin C treatment induced transcription of 737 genes and 28 ncRNAs at least 2 fold, while it repressed 231 genes and 17 ncRNAs. Data analysis revealed that in addition to the core genes known to integrate the SOS regulon, several metabolic pathways were induced. This study allowed for expansion of the Vibrio SOS regulon, as twelve genes (ubiEJB, tatABC, smpA, cep, VC0091, VC1190, VC1369-1370) were found to be co-induced with their adjacent canonical SOS regulon gene(s), through transcriptional read-through. Characterization of UV and mitomycin C susceptibility for mutants of these newly identified SOS regulon genes and other highly induced genes and ncRNAs confirmed their role in DNA damage rescue and protection. CONCLUSIONS: We show that genotoxic stress induces a pervasive transcriptional response, affecting almost 20% of the V. cholerae genes. We also demonstrate that the SOS regulon is larger than previously known, and its syntenic organization is conserved among Vibrio species. Furthermore, this specific co-localization is found in other γ-proteobacteria for genes recN-smpA and rmuC-tatABC, suggesting SOS regulon conservation in this phylum. Finally, we comment on the limitations of widespread NGS approaches for identification of all RNA species in bacteria.

GROOLS: reactive graph reasoning for genome annotation through biological processes.

BACKGROUND: High quality functional annotation is essential for understanding the phenotypic consequences encoded in a genome. Despite improvements in bioinformatics methods, millions of sequences in databanks are not assigned reliable functions. The curation of protein functions in the context of biological processes is a way to evaluate and improve their annotation. RESULTS: We developed an expert system using paraconsistent logic, named GROOLS (Genomic Rule Object-Oriented Logic System), that evaluates the completeness and the consistency of predicted functions through biological processes like metabolic pathways. Using a generic and hierarchical representation of knowledge, biological processes are modeled in a graph from which observations (i.e. predictions and expectations) are propagated by rules. At the end of the reasoning, conclusions are assigned to biological process components and highlight uncertainties and inconsistencies. Results on 14 microbial organisms are presented. CONCLUSIONS: GROOLS software is designed to evaluate the overall accuracy of functional unit and pathway predictions according to organism experimental data like growth phenotypes. It assists biocurators in the functional annotation of proteins by focusing on missing or contradictory observations.

Vibrio tapetis Displays an Original Type IV Secretion System in Strains Pathogenic for Bivalve Molluscs.

The Brown Ring Disease (BRD) caused high mortality rates since 1986 in the Manila clam Venerupis philippinarum introduced and cultured in Western Europe from the 1970s. The causative agent of BRD is a Gram-Negative bacterium, Vibrio tapetis, which is also pathogenic to fish. Here we report the first assembly of the complete genome of V. tapetis CECT4600T, together with the genome sequences of 16 additional strains isolated across a broad host and geographic range. Our extensive genome dataset allowed us to describe the pathogen pan- and core genomes and to identify putative virulence factors. The V. tapetis core genome consists of 3,352 genes, including multiple potential virulence factors represented by haemolysins, transcriptional regulators, Type I restriction modification system, GGDEF domain proteins, several conjugative plasmids, and a Type IV secretion system. Future research on the coevolutionary arms race between V. tapetis virulence factors and host resistance mechanisms will improve our understanding of how pathogenicity develops in this emerging pathogen.

Bacillus subtilis, the model Gram-positive bacterium: 20 years of annotation refinement.

Genome annotation is, nowadays, performed via automatic pipelines that cannot discriminate between right and wrong annotations. Given their importance in increasing the accuracy of the genome annotations of other organisms, it is critical that the annotations of model organisms reflect the current annotation gold standard. The genome of Bacillus subtilis strain 168 was sequenced twenty years ago. Using a combination of inductive, deductive and abductive reasoning, we present a unique, manually curated annotation, essentially based on experimental data. This reveals how this bacterium lives in a plant niche, while carrying a paleome operating system common to Firmicutes and Tenericutes. Dozens of new genomic objects and an extensive literature survey have been included for the sequence available at the INSDC (AccNum AL009126.3). We also propose an extension to Demerec's nomenclature rules that will help investigators connect to this type of curated annotation via the use of common gene names.

Carrageenan catabolism is encoded by a complex regulon in marine heterotrophic bacteria.

Macroalgae contribute substantially to primary production in coastal ecosystems. Their biomass, mainly consisting of polysaccharides, is cycled into the environment by marine heterotrophic bacteria using largely uncharacterized mechanisms. Here we describe the complete catabolic pathway for carrageenans, major cell wall polysaccharides of red macroalgae, in the marine heterotrophic bacterium Zobellia galactanivorans. Carrageenan catabolism relies on a multifaceted carrageenan-induced regulon, including a non-canonical polysaccharide utilization locus (PUL) and genes distal to the PUL, including a susCD-like pair. The carrageenan utilization system is well conserved in marine Bacteroidetes but modified in other phyla of marine heterotrophic bacteria. The core system is completed by additional functions that might be assumed by non-orthologous genes in different species. This complex genetic structure may be the result of multiple evolutionary events including gene duplications and horizontal gene transfers. These results allow for an extension on the definition of bacterial PUL-mediated polysaccharide digestion.

Genome Sequence of the Dichloromethane-Degrading Bacterium Hyphomicrobium sp. Strain GJ21.

The genome sequence of Hyphomicrobium sp. strain GJ21, isolated in the Netherlands from samples of environments contaminated with halogenated pollutants and capable of using dichloromethane as its sole carbon and energy source, was determined.

Parallel evolution of non-homologous isofunctional enzymes in methionine biosynthesis.

Experimental validation of enzyme function is crucial for genome interpretation, but it remains challenging because it cannot be scaled up to accommodate the constant accumulation of genome sequences. We tackled this issue for the MetA and MetX enzyme families, phylogenetically unrelated families of acyl-L-homoserine transferases involved in L-methionine biosynthesis. Members of these families are prone to incorrect annotation because MetX and MetA enzymes are assumed to always use acetyl-CoA and succinyl-CoA, respectively. We determined the enzymatic activities of 100 enzymes from diverse species, and interpreted the results by structural classification of active sites based on protein structure modeling. We predict that >60% of the 10,000 sequences from these families currently present in databases are incorrectly annotated, and suggest that acetyl-CoA was originally the sole substrate of these isofunctional enzymes, which evolved to use exclusively succinyl-CoA in the most recent bacteria. We also uncovered a divergent subgroup of MetX enzymes in fungi that participate only in L-cysteine biosynthesis as O-succinyl-L-serine transferases.