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).

Analyzing Prokaryotic Transcriptomics in the Light of Genome Data with the MicroScope Platform.

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 occurring in our environment. However, despite the development of powerful bioinformatics approaches, full interpretation of the content of these genomes remains a difficult task. To address this challenge, the MicroScope platform has been developed. It is an integrated Web platform for management, annotation, comparative analysis, and visualization of microbial genomes ( https://mage.genoscope.cns.fr/microscope ). Launched in 2005, the platform has been under continuous development and provides analyzes for complete and ongoing genome projects together with metabolic network reconstruction and transcriptomic experiments allowing users to improve the understanding of gene functions. MicroScope platform is widely used by microbiologists from academia and industry all around the world for collaborative studies and expert annotation. It enables collaborative work in a rich comparative genomic context and improves community-based curation efforts. Here, we describe the protocol to follow for the integration and analysis of transcriptomics data in the Microscope platform. The chapter reviews each key step from the experimental design to the analysis and interpretation of the experiment data and results. The integration of transcriptomics data gives a dynamic view of the genome by allowing the users to improve the understanding of gene functions by interpreting them in the light of regulatory cell processes. Moreover, they can also contribute to the refinement of genome annotation through the discovery of new genes and help to fill metabolic gaps.

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

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

From Strain Characterization to Field Authorization: Highlights on Bacillus velezensis Strain B25 Beneficial Properties for Plants and Its Activities on Phytopathogenic Fungi.

Agriculture is in need of alternative products to conventional phytopharmaceutical treatments from chemical industry. One solution is the use of natural microorganisms with beneficial properties to ensure crop yields and plant health. In the present study, we focused our analyses on a bacterium referred as strain B25 and belonging to the species Bacillus velezensis (synonym B. amyloliquefaciens subsp. plantarum or B. methylotrophicus), a promising plant growth promoting rhizobacterium (PGPR) and an inhibitor of pathogenic fungi inducing crops diseases. B25 strain activities were investigated. Its genes are well preserved, with their majority being common with other Bacillus spp. strains and responsible for the biosynthesis of secondary metabolites known to be involved in biocontrol and plant growth-promoting activities. No antibiotic resistance genes were found in the B25 strain plasmid. In vitro and in planta tests were conducted to confirm these PGPR and biocontrol properties, showing its efficiency against 13 different pathogenic fungi through antibiosis mechanism. B25 strain also showed good capacities to quickly colonize its environment, to solubilize phosphorus and to produce siderophores and little amounts of auxin-type phytohormones (around 13,051 µg/mL after 32 h). All these findings combined to the fact that B25 demonstrated good properties for industrialization of the production and an environmental-friendly profile, led to its commercialization under market authorization since 2018 in several biostimulant preparations and opened its potential use as a biocontrol agent.

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.

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.

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.

Physiological and evolutionary implications of tetrameric photosystem I in cyanobacteria.

Photosystem I (PSI) is present as trimeric complexes in most characterized cyanobacteria and as monomers in plants and algae. Recent reports of tetrameric PSI have raised questions regarding its structural basis, physiological role, phylogenetic distribution and evolutionary significance. Here, we examined PSI in 61 cyanobacteria, showing that tetrameric PSI, which correlates with the psaL gene and a distinct genomic structure, is widespread among heterocyst-forming cyanobacteria and their close relatives. Physiological studies revealed that expression of tetrameric PSI is favoured under high light, with an increased content of novel PSI-bound carotenoids (myxoxanthophyll, canthaxanthan and echinenone). In sum, this work suggests that tetrameric PSI is an adaptation to high light intensity, and that change in PsaL leads to monomerization of trimeric PSI, supporting the hypothesis of tetrameric PSI being the evolutionary intermediate in the transition from cyanobacterial trimeric PSI to monomeric PSI in plants and algae.

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.

A novel species of the marine cyanobacterium Acaryochloris with a unique pigment content and lifestyle.

All characterized members of the ubiquitous genus Acaryochloris share the unique property of containing large amounts of chlorophyll (Chl) d, a pigment exhibiting a red absorption maximum strongly shifted towards infrared compared to Chl a. Chl d is the major pigment in these organisms and is notably bound to antenna proteins structurally similar to those of Prochloron, Prochlorothrix and Prochlorococcus, the only three cyanobacteria known so far to contain mono- or divinyl-Chl a and b as major pigments and to lack phycobilisomes. Here, we describe RCC1774, a strain isolated from the foreshore near Roscoff (France). It is phylogenetically related to members of the Acaryochloris genus but completely lacks Chl d. Instead, it possesses monovinyl-Chl a and b at a b/a molar ratio of 0.16, similar to that in Prochloron and Prochlorothrix. It differs from the latter by the presence of phycocyanin and a vestigial allophycocyanin energetically coupled to photosystems. Genome sequencing confirmed the presence of phycobiliprotein and Chl b synthesis genes. Based on its phylogeny, ultrastructural characteristics and unique pigment suite, we describe RCC1774 as a novel species that we name Acaryochloris thomasi. Its very unusual pigment content compared to other Acaryochloris spp. is likely related to its specific lifestyle.

Ancestral Genome Estimation Reveals the History of Ecological Diversification in Agrobacterium.

Horizontal gene transfer (HGT) is considered as a major source of innovation in bacteria, and as such is expected to drive adaptation to new ecological niches. However, among the many genes acquired through HGT along the diversification history of genomes, only a fraction may have actively contributed to sustained ecological adaptation. We used a phylogenetic approach accounting for the transfer of genes (or groups of genes) to estimate the history of genomes in Agrobacterium biovar 1, a diverse group of soil and plant-dwelling bacterial species. We identified clade-specific blocks of cotransferred genes encoding coherent biochemical pathways that may have contributed to the evolutionary success of key Agrobacterium clades. This pattern of gene coevolution rejects a neutral model of transfer, in which neighboring genes would be transferred independently of their function and rather suggests purifying selection on collectively coded acquired pathways. The acquisition of these synapomorphic blocks of cofunctioning genes probably drove the ecological diversification of Agrobacterium and defined features of ancestral ecological niches, which consistently hint at a strong selective role of host plant rhizospheres.

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.

Genomic Characterization of Flavobacterium psychrophilum Serotypes and Development of a Multiplex PCR-Based Serotyping Scheme.

Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonids reared in freshwater worldwide. So far, serological diversity between isolates has been described but the underlying molecular factors remain unknown. By combining complete genome sequence analysis and the serotyping method proposed by Lorenzen and Olesen (1997) for a set of 34 strains, we identified key molecular determinants of the serotypes. This knowledge allowed us to develop a robust multiplex PCR-based serotyping scheme, which was applied to 244 bacterial isolates. The results revealed a striking association between PCR-serotype and fish host species and illustrate the use of this approach as a simple and cost-effective method for the determination of F. psychrophilum serogroups. PCR-based serotyping could be a useful tool in a range of applications such as disease surveillance, selection of salmonids for bacterial coldwater disease resistance and future vaccine formulation.

Rearranged Biosynthetic Gene Cluster and Synthesis of Hassallidin E in Planktothrix serta PCC 8927.

Cyanobacteria produce a wide range of natural products with antifungal bioactivity. The cyclic glycosylated lipopeptides of the hassallidin family have potent antifungal activity and display a great degree of chemical diversity. Here, we report the discovery of a hassallidin biosynthetic gene cluster from the filamentous cyanobacterium Planktothrix serta PCC 8927. The hassallidin gene cluster showed heavy rearrangement and marks of genomic plasticity. Nucleotide bias, differences in GC content, and phylogenetic incongruence suggested the acquisition of the hassallidin biosynthetic gene cluster in Planktothrix serta PCC 8927 by horizontal gene transfer. Chemical analyses by liquid chromatography and mass spectrometry demonstrated that this strain produced hassallidin E, a new glycosylated hassallidin variant. Hassallidin E was the only structural variant produced by Planktothrix serta PCC 8927 in all tested conditions. Further evaluated on human pathogenic fungi, hassallidin E showed an antifungal bioactivity. Hassallidin production levels correlated with nitrogen availability, in the only nitrogen-fixing Planktothrix described so far. Our results provide insights into the distribution and chemical diversity of cyanobacterial antifungal compounds as well as raise questions on their ecological relevance.

Insights into the Planktothrix genus: Genomic and metabolic comparison of benthic and planktic strains.

Planktothrix is a dominant cyanobacterial genus forming toxic blooms in temperate freshwater ecosystems. We sequenced the genome of planktic and non planktic Planktothrix strains to better represent this genus diversity and life style at the genomic level. Benthic and biphasic strains are rooting the Planktothrix phylogenetic tree and widely expand the pangenome of this genus. We further investigated in silico the genetic potential dedicated to gas vesicles production, nitrogen fixation as well as natural product synthesis and conducted complementary experimental tests by cell culture, microscopy and mass spectrometry. Significant differences for the investigated features could be evidenced between strains of different life styles. The benthic Planktothrix strains showed unexpected characteristics such as buoyancy, nitrogen fixation capacity and unique natural product features. In comparison with Microcystis, another dominant toxic bloom-forming genus in freshwater ecosystem, different evolutionary strategies were highlighted notably as Planktothrix exhibits an overall greater genetic diversity but a smaller genomic plasticity than Microcystis. Our results are shedding light on Planktothrix evolution, phylogeny and physiology in the frame of their diverse life styles.

MicroScope in 2017: an expanding and evolving integrated resource for community expertise of microbial genomes.

The annotation of genomes from NGS platforms needs to be automated and fully integrated. However, maintaining consistency and accuracy in genome annotation is a challenging problem because millions of protein database entries are not assigned reliable functions. This shortcoming limits the knowledge that can be extracted from genomes and metabolic models. Launched in 2005, 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. Effective comparative analysis requires a consistent and complete view of biological data, and therefore, support for reviewing the quality of functional annotation is critical. MicroScope allows users to analyze microbial (meta)genomes together with post-genomic experiment results if any (i.e. transcriptomics, re-sequencing of evolved strains, mutant collections, phenotype data). It combines tools and graphical interfaces to analyze genomes and to perform the expert curation of gene functions in a comparative context. Starting with a short overview of the MicroScope system, this paper focuses on some major improvements of the Web interface, mainly for the submission of genomic data and on original tools and pipelines that have been developed and integrated in the platform: computation of pan-genomes and prediction of biosynthetic gene clusters. Today the resource contains data for more than 6000 microbial genomes, and among the 2700 personal accounts (65% of which are now from foreign countries), 14% of the users are performing expert annotations, on at least a weekly basis, contributing to improve the quality of microbial genome annotations.

A single regulatory gene is sufficient to alter Vibrio aestuarianus pathogenicity in oysters.

Oyster diseases caused by pathogenic vibrios pose a major challenge to the sustainability of oyster farming. In France, since 2012 a disease affecting specifically adult oysters has been associated with the presence of Vibrio aestuarianus. Here, by combining genome comparison, phylogenetic analyses and high-throughput infections of strains isolated before or during the recent outbreaks, we show that virulent strains cluster into two V. aestuarianus lineages independently of the sampling dates. The bacterial lethal dose was not different between strains isolated before or after 2012. Hence, the emergence of a new highly virulent clonal strain is unlikely. Each lineage comprises nearly identical strains, the majority of them being virulent, suggesting that within these phylogenetically coherent virulent lineages a few strains have lost their pathogenicity. Comparative genomics allowed the identification of a single frameshift in a non-virulent strain. This mutation affects the varS gene that codes for a signal transduction histidine-protein kinase. Genetic analyses confirmed that varS is necessary for infection of oysters and for a secreted metalloprotease expression. For the first time in a Vibrio species, we show here that VarS is a key factor of pathogenicity.

Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters.

Disease in oysters has been steadily rising over the past decade, threatening the long-term survival of commercial and natural stocks. Our understanding and management of such diseases are of critical importance as aquaculture is an important aspect of dealing with the approaching worldwide food shortage. Although some bacteria of the Vibrio genus isolated from diseased oysters have been demonstrated to be pathogenic by experimental infection, direct causality has not been established. Little is known about the dynamics of how the bacterial population hosted by oysters changes during disease progression. Combining experimental ecology, a high-throughput infection assay and genome sequencing, we show that the onset of disease in oysters is associated with progressive replacement of diverse benign colonizers by members of a phylogenetically coherent virulent population. Although the virulent population is genetically diverse, all members of that population can cause disease. Comparative genomics across virulent and nonvirulent populations identified candidate virulence factors that were clustered in population-specific genomic regions. Genetic analyses revealed that one gene for a candidate virulent factor, a putative outer membrane protein, is necessary for infection of oysters. Finally, analyses of oyster mortality following experimental infection suggest that disease onset can be facilitated by the presence of nonvirulent strains. This is a new form of polymicrobial disease, in which nonpathogenic strains contribute to increase mortality.

Phylum-wide comparative genomics unravel the diversity of secondary metabolism in Cyanobacteria.

BACKGROUND: Cyanobacteria are an ancient lineage of photosynthetic bacteria from which hundreds of natural products have been described, including many notorious toxins but also potent natural products of interest to the pharmaceutical and biotechnological industries. Many of these compounds are the products of non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways. However, current understanding of the diversification of these pathways is largely based on the chemical structure of the bioactive compounds, while the evolutionary forces driving their remarkable chemical diversity are poorly understood. RESULTS: We carried out a phylum-wide investigation of genetic diversification of the cyanobacterial NRPS and PKS pathways for the production of bioactive compounds. 452 NRPS and PKS gene clusters were identified from 89 cyanobacterial genomes, revealing a clear burst in late-branching lineages. Our genomic analysis further grouped the clusters into 286 highly diversified cluster families (CF) of pathways. Some CFs appeared vertically inherited, while others presented a more complex evolutionary history. Only a few horizontal gene transfers were evidenced amongst strongly conserved CFs in the phylum, while several others have undergone drastic gene shuffling events, which could result in the observed diversification of the pathways. CONCLUSIONS: Therefore, in addition to toxin production, several NRPS and PKS gene clusters are devoted to important cellular processes of these bacteria such as nitrogen fixation and iron uptake. The majority of the biosynthetic clusters identified here have unknown end products, highlighting the power of genome mining for the discovery of new natural products.