Integration of Complete Plasmids Containing Bont Genes into Chromosomes of Clostridium parabotulinum, Clostridium sporogenes, and Clostridium argentinense

At least 40 toxin subtypes of botulinum neurotoxins (BoNTs), a heterogenous group of bacterial proteins, are produced by seven different clostridial species. A key factor that drives the diversity of neurotoxigenic clostridia is the association of bont gene clusters with various genomic locations including plasmids, phages and the chromosome. Analysis of Clostridium sporogenes BoNT/B1 strain CDC 1632, C. argentinense BoNT/G strain CDC 2741, and Clostridium parabotulinum BoNT/B1 strain DFPST0006 genomes revealed bont gene clusters within plasmid-like sequences within the chromosome or nested in large contigs, with no evidence of extrachromosomal elements. A nucleotide sequence (255,474 bp) identified in CDC 1632 shared 99.5% identity (88% coverage) with bont/B1-containing plasmid pNPD7 of C. sporogenes CDC 67071; CDC 2741 contig AYSO01000020 (1.1 MB) contained a ~140 kb region which shared 99.99% identity (100% coverage) with plasmid pRSJ17_1 of C. argentinense BoNT/G strain 89G; and DFPST0006 contig JACBDK0100002 (573 kb) contained a region that shared 100% identity (99%) coverage with the bont/B1-containing plasmid pCLD of C. parabotulinum Okra. This is the first report of full-length plasmid DNA-carrying complete neurotoxin gene clusters integrated in three distinct neurotoxigenic species: C. parabotulinum, C. sporogenes and C. argentinense.

Ecogenomics of Groundwater Phages Suggests Niche Differentiation Linked to Specific Environmental Tolerance

To our knowledge, this is the first study to identify the bacteriophage distribution in a groundwater ecosystem shedding light on their prevalence and distribution across metal-contaminated and background sites. Our study is uniquely based on selective sequencing of solely the extrachromosomal elements of a microbiome followed by analysis for viral signatures, thus establishing a more focused approach for phage identifications.

Exploring short k-mer profiles in cells and mobile elements from Archaea highlights the major influence of both the ecological niche and evolutionary history

Background K-mer-based methods have greatly advanced in recent years, largely driven by the realization of their biological significance and by the advent of next-generation sequencing. Their speed and their independence from the annotation process are major advantages. Their utility in the study of the mobilome has recently emerged and they seem a priori adapted to the patchy gene distribution and the lack of universal marker genes of viruses and plasmids. To provide a framework for the interpretation of results from k-mer based methods applied to archaea or their mobilome, we analyzed the 5-mer DNA profiles of close to 600 archaeal cells, viruses and plasmids. Archaea is one of the three domains of life. Archaea seem enriched in extremophiles and are associated with a high diversity of viral and plasmid families, many of which are specific to this domain. We explored the dataset structure by multivariate and statistical analyses, seeking to identify the underlying factors. Results For cells, the 5-mer profiles were inconsistent with the phylogeny of archaea. At a finer taxonomic level, the influence of the taxonomy and the environmental constraints on 5-mer profiles was very strong. These two factors were interdependent to a significant extent, and the respective weights of their contributions varied according to the clade. A convergent adaptation was observed for the class Halobacteria, for which a strong 5-mer signature was identified. For mobile elements, coevolution with the host had a clear influence on their 5-mer profile. This enabled us to identify one previously known and one new case of recent host transfer based on the atypical composition of the mobile elements involved. Beyond the effect of coevolution, extrachromosomal elements strikingly retain the specific imprint of their own viral or plasmid taxonomic family in their 5-mer profile. Conclusion This specific imprint confirms that the evolution of extrachromosomal elements is driven by multiple parameters and is not restricted to host adaptation. In addition, we detected only recent host transfer events, suggesting the fast evolution of short k-mer profiles. This calls for caution when using k-mers for host prediction, metagenomic binning or phylogenetic reconstruction.

The Distinctive Evolution of orfX Clostridium parabotulinum Strains and Their Botulinum Neurotoxin Type A and F Gene Clusters Is Influenced by Environmental Factors and Gene Interactions via Mobile Genetic Elements

Of the seven currently known botulinum neurotoxin-producing species of Clostridium, C. parabotulinum, or C. botulinum Group I, is the species associated with the majority of human botulism cases worldwide. Phylogenetic analysis of these bacteria reveals a diverse species with multiple genomic clades. The neurotoxins they produce are also diverse, with over 20 subtypes currently represented. The existence of different bont genes within very similar genomes and of the same bont genes/gene clusters within different bacterial variants/species indicates that they have evolved independently. The neurotoxin genes are associated with one of two toxin gene cluster types containing either hemagglutinin (ha) genes or orfX genes. These genes may be located within the chromosome or extrachromosomal elements such as large plasmids. Although BoNT-producing C parabotulinum bacteria are distributed globally, they are more ubiquitous in certain specific geographic regions. Notably, northern hemisphere strains primarily contain ha gene clusters while southern hemisphere strains have a preponderance of orfX gene clusters. OrfX C. parabotulinum strains constitute a subset of this species that contain highly conserved bont gene clusters having a diverse range of bont genes. While much has been written about strains with ha gene clusters, less attention has been devoted to those with orfX gene clusters. The recent sequencing of 28 orfX C. parabotulinum strains and the availability of an additional 91 strains for analysis provides an opportunity to compare genomic relationships and identify unique toxin gene cluster characteristics and locations within this species subset in depth. The mechanisms behind the independent processes of bacteria evolution and generation of toxin diversity are explored through the examination of bacterial relationships relating to source locations and evidence of horizontal transfer of genetic material among different bacterial variants, particularly concerning bont gene clusters. Analysis of the content and locations of the bont gene clusters offers insights into common mechanisms of genetic transfer, chromosomal integration, and development of diversity among these genes.

The fully resolved genome of Bacillus thuringiensis HER1410 reveals a cry-containing chromosome, two megaplasmids & an integrative plasmidial prophage

Bacillus thuringiensis is the most used biopesticide in agriculture. Its entomopathogenic capacity stems from the possession of plasmid-borne insecticidal crystal genes (cry), traditionally used as discriminant taxonomic feature for that species. As such, crystal and plasmid identification are key to the characterization of this species. To date, about 600 B. thuringiensis genomes have been reported, but less than 5% have been resolved, while the other draft genomes are incomplete, precluding plasmid delineation. Here we present the complete genome of Bacillus thuringiensis HER1410, a strain closely related to B. thuringiensis entomocidus and a known host for a variety of Bacillus phages. The combination of short and long-reads techniques allowed fully resolving the genome and delineation of three plasmids. This enabled the accurate detection of an unusual location of a unique cry gene, cry1Ba4, located in a genomic island near the chromosome replication origin. Two megaplasmids, pLUSID1 and pLUSID2 could be delineated: pLUSID1 (368kb), a likely conjugative plasmid involved in virulence, and pLUSID2 potentially related to the sporulation process. A smaller plasmidial prophage pLUSID3, with a dual lifestyle whose integration within the chromosome, causes the disruption of a flagellar key component. Finally, phylogenetic analysis located this strain within a clade comprising members from the B. thuringiensis serovar thuringiensis and other serovars and with B.cereus s. s. This highlights the intermingled taxonomy of B. cereus sensu lato group, where genomics alone does not support the present taxonomy between B. cereus s. s. and B. thuringiensis as species designation currently relies solely on the presence of entomocidal genes.ImportanceBacillus cereus group species have been extensively studied due to their economical and clinical relevance. This importance originally set the basis for B. cereus group members classification which are commonly based on phenotypical criteria. Sequencing era has shed light about genomic characterization of these species, showing their chromosomal genomic similarity and highlighting the role of mobile genetic elements, especially megaplasmids, in the classification and characterization of this group. However, only the 5% of the sequenced B. thuringiensis genomes have been fully resolved. Thus, here we addressed efficiently the study B. thuringiensis HER1410 genomic features by the use of a combination of short and long-reads sequencing. This methodology resulted in the high-quality assembly, which led to the identification of an uncommon location of a cry gene close to the chromosomal origin, as well as three fully resolved extrachromosomal elements, two megaplasmids and an integrative plasmidial prophage.

Complete Genome Sequence of Collinsella aerofaciens JCM 10188T

We report a complete genome sequence of Collinsella aerofaciens JCM 10188T (=VPI 1003T). The genome consists of a circular chromosome (2,428,218 bp with 60.6% G+C content) and two extrachromosomal elements. The genome was predicted to contain 5 sets of rRNA genes, 58 tRNA genes, and 2,079 protein-encoding sequences.

The Hypercomplex Genome of an Insect Reproductive Parasite Highlights the Importance of Lateral Gene Transfer in Symbiont Biology

ABSTRACT Mobile elements—plasmids and phages—are important components of microbial function and evolution via traits that they encode and their capacity to shuttle genetic material between species. We here report the unusually rich array of mobile elements within the genome of Arsenophonus nasoniae, the son-killer symbiont of the parasitic wasp Nasonia vitripennis. This microbe’s genome has the highest prophage complement reported to date, with over 50 genomic regions that represent either intact or degraded phage material. Moreover, the genome is predicted to include 17 extrachromosomal genetic elements, which carry many genes predicted to be important at the microbe-host interface, derived from a diverse assemblage of insect-associated gammaproteobacteria. In our system, this diversity was previously masked by repetitive mobile elements that broke the assembly derived from short reads. These findings suggest that other complex bacterial genomes will be revealed in the era of long-read sequencing. IMPORTANCE The biology of many bacteria is critically dependent on genes carried on plasmid and phage mobile elements. These elements shuttle between microbial species, thus providing an important source of biological innovation across taxa. It has recently been recognized that mobile elements are also important in symbiotic bacteria, which form long-lasting interactions with their host. In this study, we report a bacterial symbiont genome that carries a highly complex array of these elements. Arsenophonus nasoniae is the son-killer microbe of the parasitic wasp Nasonia vitripennis and exists with the wasp throughout its life cycle. We completed its genome with the aid of recently developed long-read technology. This assembly contained over 50 chromosomal regions of phage origin and 17 extrachromosomal elements within the genome, encoding many important traits at the host-microbe interface. Thus, the biology of this symbiont is enabled by a complex array of mobile elements.