Telomere-to-Telomere genome assemblies of human-infecting Encephalitozoon species.

BACKGROUND: Microsporidia are diverse spore forming, fungal-related obligate intracellular pathogens infecting a wide range of hosts. This diversity is reflected at the genome level with sizes varying by an order of magnitude, ranging from less than 3 Mb in Encephalitozoon species (the smallest known in eukaryotes) to more than 50 Mb in Edhazardia spp. As a paradigm of genome reduction in eukaryotes, the small Encephalitozoon genomes have attracted much attention with investigations revealing gene dense, repeat- and intron-poor genomes characterized by a thorough pruning of molecular functions no longer relevant to their obligate intracellular lifestyle. However, because no Encephalitozoon genome has been sequenced from telomere-to-telomere and since no methylation data is available for these species, our understanding of their overall genetic and epigenetic architectures is incomplete. METHODS: In this study, we sequenced the complete genomes from telomere-to-telomere of three human-infecting Encephalitozoon spp. -E. intestinalis ATCC 50506, E. hellem ATCC 50604 and E. cuniculi ATCC 50602- using short and long read platforms and leveraged the data generated as part of the sequencing process to investigate the presence of epigenetic markers in these genomes. We also used a mixture of sequence- and structure-based computational approaches, including protein structure prediction, to help identify which Encephalitozoon proteins are involved in telomere maintenance, epigenetic regulation, and heterochromatin formation. RESULTS: The Encephalitozoon chromosomes were found capped by TTAGG 5-mer telomeric repeats followed by telomere associated repeat elements (TAREs) flanking hypermethylated ribosomal RNA (rRNA) gene loci featuring 5-methylcytosines (5mC) and 5-hemimethylcytosines (5hmC), themselves followed by lesser methylated subtelomeres and hypomethylated chromosome cores. Strong nucleotide biases were identified between the telomeres/subtelomeres and chromosome cores with significant changes in GC/AT, GT/AC and GA/CT contents. The presence of several genes coding for proteins essential to telomere maintenance, epigenetic regulation, and heterochromatin formation was further confirmed in the Encephalitozoon genomes. CONCLUSION: Altogether, our results strongly support the subtelomeres as sites of heterochromatin formation in Encephalitozoon genomes and further suggest that these species might shutdown their energy-consuming ribosomal machinery while dormant as spores by silencing of the rRNA genes using both 5mC/5hmC methylation and facultative heterochromatin formation at these loci.

A new microsporidian parasite, Ordospora pajunii sp. nov (Ordosporidae), of Daphnia longispina highlights the value of genomic data for delineating species boundaries.

Speciation is a complex and continuous process that makes the delineation of species boundaries a challenging task in particular in species with little morphological differentiation, such as parasites. In this case, the use of genomic data is often necessary, such as for the intracellular Microsporidian parasites. Here, we characterize the genome of a gut parasite of the cladoceran Daphnia longispina (isolate FI-F-10), which we propose as a new species within the genus Ordospora: Ordospora pajunii sp. nov (Ordosporidae). FI-F-10 closest relative, Ordospora colligata is only found in D. magna. Both microsporidian species share several morphological features. Although it is not possible to estimate divergence times for Microsporidia due to the lack of fossil records and accelerated evolutionary rates, we base our proposal on the phylogenomic and genomic distances between both microsporidian lineages. The phylogenomic reconstruction shows that FI-F-10 forms an early diverging branch basal to the cluster that contains all known O. colligata strains. Whole-genome comparisons show that FI-F-10 presents a greater divergence at the sequence level than observed among O. colligata strains, and its genomic average nucleotide identity (ANI) values against O. colligata are beyond the intraspecific range previously established for yeast and prokaryotes. Our data confirm that the ANI metrics are useful for fine genetic divergence calibration across Microsporidia taxa. In combination with phylogenetic and ecological data, genome-based metrics provide a powerful approach to delimitate species boundaries.

A lack of parasitic reduction in the obligate parasitic green alga Helicosporidium.

The evolution of an obligate parasitic lifestyle is often associated with genomic reduction, in particular with the loss of functions associated with increasing host-dependence. This is evident in many parasites, but perhaps the most extreme transitions are from free-living autotrophic algae to obligate parasites. The best-known examples of this are the apicomplexans such as Plasmodium, which evolved from algae with red secondary plastids. However, an analogous transition also took place independently in the Helicosporidia, where an obligate parasite of animals with an intracellular infection mechanism evolved from algae with green primary plastids. We characterised the nuclear genome of Helicosporidium to compare its transition to parasitism with that of apicomplexans. The Helicosporidium genome is small and compact, even by comparison with the relatively small genomes of the closely related green algae Chlorella and Coccomyxa, but at the functional level we find almost no evidence for reduction. Nearly all ancestral metabolic functions are retained, with the single major exception of photosynthesis, and even here reduction is not complete. The great majority of genes for light-harvesting complexes, photosystems, and pigment biosynthesis have been lost, but those for other photosynthesis-related functions, such as Calvin cycle, are retained. Rather than loss of whole function categories, the predominant reductive force in the Helicosporidium genome is a contraction of gene family complexity, but even here most losses affect families associated with genome maintenance and expression, not functions associated with host-dependence. Other gene families appear to have expanded in response to parasitism, in particular chitinases, including those predicted to digest the chitinous barriers of the insect host or remodel the cell wall of Helicosporidium. Overall, the Helicosporidium genome presents a fascinating picture of the early stages of a transition from free-living autotroph to parasitic heterotroph where host-independence has been unexpectedly preserved.

Evolution of a morphological novelty occurred before genome compaction in a lineage of extreme parasites.

Intracellular parasitism results in extreme adaptations, whose evolutionary history is difficult to understand, because the parasites and their known free-living relatives are so divergent from one another. Microsporidia are intracellular parasites of humans and other animals, which evolved highly specialized morphological structures, but also extreme physiologic and genomic simplification. They are suggested to be an early-diverging branch on the fungal tree, but comparisons to other species are difficult because their rates of molecular evolution are exceptionally high. Mitochondria in microsporidia have degenerated into organelles called mitosomes, which have lost a genome and the ability to produce ATP. Here we describe a gut parasite of the crustacean Daphnia that despite having remarkable morphological similarity to the microsporidia, has retained genomic features of its fungal ancestors. This parasite, which we name Mitosporidium daphniae gen. et sp. nov., possesses a mitochondrial genome including genes for oxidative phosphorylation, yet a spore stage with a highly specialized infection apparatus--the polar tube--uniquely known only from microsporidia. Phylogenomics places M. daphniae at the root of the microsporidia. A comparative genomic analysis suggests that the reduction in energy metabolism, a prominent feature of microsporidian evolution, was preceded by a reduction in the machinery controlling cell cycle, DNA recombination, repair, and gene expression. These data show that the morphological features unique to M. daphniae and other microsporidia were already present before the lineage evolved the extreme host metabolic dependence and loss of mitochondrial respiration for which microsporidia are well known.

Genetic Diversity of Clostridium sporogenes PA 3679 Isolates Obtained from Different Sources as Resolved by Pulsed-Field Gel Electrophoresis and High-Throughput Sequencing.

Clostridium sporogenes PA 3679 is a nonpathogenic, nontoxic model organism for proteolytic Clostridium botulinum used in the validation of conventional thermal food processes due to its ability to produce highly heat-resistant endospores. Because of its public safety importance, the uncertain taxonomic classification and genetic diversity of PA 3679 are concerns. Therefore, isolates of C. sporogenes PA 3679 were obtained from various sources and characterized using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. The phylogenetic relatedness and genetic variability were assessed based on 16S rRNA gene sequencing and whole-genome single nucleotide polymorphism (SNP) analysis. All C. sporogenes PA 3679 isolates were categorized into two clades (clade I containing ATCC 7955 NCA3679 isolates 1961-2, 1990, and 2007 and clade II containing PA 3679 isolates NFL, UW, FDA, and Campbell and ATCC 7955 NCA3679 isolate 1961-4). The 16S maximum likelihood (ML) tree clustered both clades within proteolytic C. botulinum strains, with clade I forming a distinct cluster with other C. sporogenes non-PA 3679 strains. SNP analysis revealed that clade I isolates were more similar to the genomic reference PA 3679 (NCTC8594) genome (GenBank accession number AGAH00000000.1) than clade II isolates were. The genomic reference C. sporogenes PA 3679 (NCTC8594) genome and clade I C. sporogenes isolates were genetically distinct from those obtained from other sources (University of Wisconsin, National Food Laboratory, U.S. Food and Drug Administration, and Campbell's Soup Company). Thermal destruction studies revealed that clade I isolates were more sensitive to high temperature than clade II isolates were. Considering the widespread use of C. sporogenes PA 3679 and its genetic information in numerous studies, the accurate identification and genetic characterization of C. sporogenes PA 3679 are of critical importance.

Isolation and characterization of an interactive culture of two Paenibacillus species with moderately thermophilic desulfurization ability.

OBJECTIVE: To isolate and characterize novel thermophilic bacteria capable of biodesulfurization of petroleum. RESULTS: A culture containing two Paenibacillus spp. (denoted "32O-W" and "32O-Y") was isolated by repeated passage of a soil sample at up to 55 °C in medium containing dibenzothiophene (DBT) as sulfur source. Only 32O-Y metabolized DBT, apparently via the 4S pathway; maximum activity occurred from 40 to 45 °C, with some activity up to at least 50 °C. 32O-W enhanced DBT metabolism by 32O-Y (by 22-74 % at 40-50 °C). With sulfate as sulfur source, 32O-Y and 32O-W grew well up to 58 and 63 °C, respectively. Selection of a mixed culture of 32O-Y and 32O-W at 54 °C increased DBT metabolism 36-42 % from 40 to 45 °C. Genome sequencing identified desulfurization gene homologs in the strains consistent with their desulfurization properties. CONCLUSION: The 32O-Y/32O-W culture may be a useful starting point for development of an improved thermophilic petroleum biodesulfurization process.

Gain and loss of multiple functionally related, horizontally transferred genes in the reduced genomes of two microsporidian parasites.

Microsporidia of the genus Encephalitozoon are widespread pathogens of animals that harbor the smallest known nuclear genomes. Complete sequences from Encephalitozoon intestinalis (2.3 Mbp) and Encephalitozoon cuniculi (2.9 Mbp) revealed massive gene losses and reduction of intergenic regions as factors leading to their drastically reduced genome size. However, microsporidian genomes also have gained genes through horizontal gene transfers (HGT), a process that could allow the parasites to exploit their hosts more fully. Here, we describe the complete sequences of two intermediate-sized genomes (2.5 Mbp), from Encephalitozoon hellem and Encephalitozoon romaleae. Overall, the E. hellem and E. romaleae genomes are strikingly similar to those of Encephalitozoon cuniculi and Encephalitozoon intestinalis in both form and content. However, in addition to the expected expansions and contractions of known gene families in subtelomeric regions, both species also were found to harbor a number of protein-coding genes that are not found in any other microsporidian. All these genes are functionally related to the metabolism of folate and purines but appear to have originated by several independent HGT events from different eukaryotic and prokaryotic donors. Surprisingly, the genes are all intact in E. hellem, but in E. romaleae those involved in de novo synthesis of folate are all pseudogenes. Overall, these data suggest that a recent common ancestor of E. hellem and E. romaleae assembled a complete metabolic pathway from multiple independent HGT events and that one descendent already is dispensing with much of this new functionality, highlighting the transient nature of transferred genes.

Complete genome sequences from three genetically distinct strains reveal high intraspecies genetic diversity in the microsporidian Encephalitozoon cuniculi.

Microsporidia from the Encephalitozoonidae are obligate intracellular parasites with highly conserved and compacted nuclear genomes: they have few introns, short intergenic regions, and almost identical gene complements and chromosome arrangements. Comparative genomics of Encephalitozoon and microsporidia in general have focused largely on the genomic diversity between different species, and we know very little about the levels of genetic diversity within species. Polymorphism studies with Encephalitozoon are so far restricted to a small number of genes, and a few genetically distinct strains have been identified; most notably, three genotypes (ECI, ECII, and ECIII) of the model species E. cuniculi have been identified based on variable repeats in the rRNA internal transcribed spacer (ITS). To determine if E. cuniculi genotypes are genetically distinct lineages across the entire genome and at the same time to examine the question of intraspecies genetic diversity in microsporidia in general, we sequenced de novo genomes from each of the three genotypes and analyzed patterns of single nucleotide polymorphisms (SNPs) and insertions/deletions across the genomes. Although the strains have almost identical gene contents, they harbor large numbers of SNPs, including numerous nonsynonymous changes, indicating massive intraspecies variation within the Encephalitozoonidae. Based on this diversity, we conclude that the recognized genotypes are genetically distinct and propose new molecular markers for microsporidian genotyping.

SYNY: a pipeline to investigate and visualize collinearity between genomes.

Investigating collinearity between chromosomes is often used in comparative genomics to help identify gene orthologs, pinpoint genes that might have been overlooked as part of annotation processes and/or perform various evolutionary inferences. Collinear segments, also known as syntenic blocks, can be inferred from sequence alignments and/or from the identification of genes arrayed in the same order and relative orientations between investigated genomes. To help perform these analyses and assess their outcomes, we built a simple pipeline called SYNY (for synteny) that implements the two distinct approaches and produces different visualizations. The SYNY pipeline was built with ease of use in mind and runs on modest hardware. The pipeline is written in Perl and Python and is available on GitHub (https://github.com/PombertLab/SYNY) under the permissive MIT license.

Near chromosome-level genome assembly of the microsporidium Hamiltosporidium tvaerminnensis.

Microsporidia are intracellular parasitic fungi whose genomes rank among the smallest of all known eukaryotes. A number of outstanding questions remain concerning the evolution of their large-scale variation in genome architecture, responsible for genome size variation of more than an order of magnitude. This genome report presents the first near-chromosomal assembly of a large-genome microsporidium, Hamiltosporidium tvaerminnensis. Combined Oxford Nanopore, Pacific Biosciences (PacBio), and Illumina sequencing led to a genome assembly of 17 contigs, 11 of which represent complete chromosomes. Our assembly is 21.64 Mb in length, has an N50 of 1.44 Mb, and consists of 39.56% interspersed repeats. We introduce a novel approach in microsporidia, PacBio Iso-Seq, as part of a larger annotation pipeline for obtaining high-quality annotations of 3,573 protein-coding genes. Based on direct evidence from the full-length Iso-Seq transcripts, we present evidence for alternative polyadenylation and variation in splicing efficiency, which are potential regulation mechanisms for gene expression in microsporidia. The generated high-quality genome assembly is a necessary resource for comparative genomics that will help elucidate the evolution of genome architecture in response to intracellular parasitism.

The evolutionary history of haptophytes and cryptophytes: phylogenomic evidence for separate origins.

An important missing piece in the puzzle of how plastids spread across the eukaryotic tree of life is a robust evolutionary framework for the host lineages. Four assemblages are known to harbour plastids derived from red algae and, according to the controversial chromalveolate hypothesis, these all share a common ancestry. Phylogenomic analyses have consistently shown that stramenopiles and alveolates are closely related, but haptophytes and cryptophytes remain contentious; they have been proposed to branch together with several heterotrophic groups in the newly erected Hacrobia. Here, we tested this question by producing a large expressed sequence tag dataset for the katablepharid Roombia truncata, one of the last hacrobian lineages for which genome-level data are unavailable, and combined this dataset with the recently completed genome of the cryptophyte Guillardia theta to build an alignment composed of 258 genes. Our analyses strongly support haptophytes as sister to the SAR group, possibly together with telonemids and centrohelids. We also confirmed the common origin of katablepharids and cryptophytes, but these lineages were not related to other hacrobians; instead, they branch with plants. Our study resolves the evolutionary position of haptophytes, an ecologically critical component of the oceans, and proposes a new hypothesis for the origin of cryptophytes.

The mitochondrial genome of the arbuscular mycorrhizal fungus Gigaspora margarita reveals two unsuspected trans-splicing events of group I introns.

• Arbuscular mycorrhizal fungi (AMF) are ubiquitous organisms that benefit ecosystems through the establishment of an association with the roots of most plants: the mycorrhizal symbiosis. Despite their ecological importance, however, these fungi have been poorly studied at the genome level. • In this study, total DNA from the AMF Gigaspora margarita was subjected to a combination of 454 and Illumina sequencing, and the resulting reads were used to assemble its mitochondrial genome de novo. This genome was annotated and compared with those of other relatives to better comprehend the evolution of the AMF lineage. • The mitochondrial genome of G. margarita is unique in many ways, exhibiting a large size (97 kbp) and elevated GC content (45%). This genome also harbors molecular events that were previously unknown to occur in fungal mitochondrial genomes, including trans-splicing of group I introns from two different genes coding for the first subunit of the cytochrome oxidase and for the small subunit of the rRNA. • This study reports the second published genome from an AMF organelle, resulting in relevant DNA sequence information from this poorly studied fungal group, and providing new insights into the frequency, origin and evolution of trans-spliced group I introns found across the mitochondrial genomes of distantly related organisms.

The Rad9-Rad1-Hus1 DNA Repair Clamp is Found in Microsporidia.

DNA repair is an important component of genome integrity and organisms with reduced repair capabilities tend to accumulate mutations at elevated rates. Microsporidia are intracellular parasites exhibiting high levels of genetic divergence postulated to originate from the lack of several proteins, including the heterotrimeric Rad9-Rad1-Hus1 DNA repair clamp. Microsporidian species from the Encephalitozoonidae have undergone severe streamlining with small genomes coding for about 2,000 proteins. The highly divergent sequences found in Microsporidia render functional inferences difficult such that roughly half of these 2,000 proteins have no known function. Using a structural homology-based annotation approach combining protein structure prediction and tridimensional similarity searches, we found that the Rad9-Rad1-Hus1 DNA clamp is present in Microsporidia, together with many other components of the DNA repair machinery previously thought to be missing from these organisms. Altogether, our results indicate that the DNA repair machinery is present and likely functional in Microsporidia.

Branching network of proteinaceous filaments within the parasitophorous vacuole of Encephalitozoon cuniculi and Encephalitozoon hellem.

The microsporidia are a diverse phylum of obligate intracellular parasites that infect all major animal groups and have been recognized as emerging human pathogens for which few chemotherapeutic options currently exist. These organisms infect every tissue and organ system, causing significant pathology, especially in immune-compromised populations. The microsporidian spore employs a unique infection strategy in which its contents are delivered into a host cell via the polar tube, an organelle that lies coiled within the resting spore but erupts with a force sufficient to pierce the plasma membrane of its host cell. Using biochemical and molecular approaches, we have previously identified components of the polar tube and spore wall of the Encephalitozoonidae. In this study, we employed a shotgun proteomic strategy to identify novel structural components of these organelles in Encephalitozoon cuniculi. As a result, a new component of the E. cuniculi developing spore wall was identified. Surprisingly, using the same approach, a heretofore undescribed filamentous network within the lumen of the parasitophorous vacuole was discovered. This network was also present in the parasitophorous vacuole of Encephalitozoon hellem. Thus, in addition to further elucidating the molecular composition of seminal organelles and revealing novel diagnostic and therapeutic targets, proteomic analysis-driven approaches exploring the spore may also uncover unknown facets of microsporidian biology.

3DFI: a pipeline to infer protein function using structural homology.

Motivation: Inferring protein function is an integral part of genome annotation and analysis. This process is usually performed in silico, and most in silico inferences are based on sequence homology approaches, which can fail when in presence of divergent sequences. However, because protein structures and their biological roles are intertwined, protein function can also be inferred by searching for structural homology. Many excellent tools have been released in recent years with regards to protein structure prediction, structural homology searches and protein visualization. Unfortunately, these tools are disconnected from each other and often use a web server-based approach that is ill-suited to high-throughput genome-wide analyses. To help assist genome annotation, we built a structural homology-based pipeline called 3DFI (for tridimensional functional inference) leveraging some of the best structural homology tools. This pipeline was built with simplicity of use in mind and enables genome-wide structural homology inferences. Availability and implementation: 3DFI is available on GitHub https://github.com/PombertLab/3DFI under the permissive MIT license. The pipeline is written in Perl and Python.

Microsporidia with Vertical Transmission Were Likely Shaped by Nonadaptive Processes.

Microsporidia have the leanest genomes among eukaryotes, and their physiological and genomic simplicity has been attributed to their intracellular, obligate parasitic life-style. However, not all microsporidia genomes are small or lean, with the largest dwarfing the smallest ones by at least an order of magnitude. To better understand the evolutionary mechanisms behind this genomic diversification, we explore here two clades of microsporidia with distinct life histories, Ordospora and Hamiltosporidium, parasitizing the same host species, Daphnia magna. Based on seven newly assembled genomes, we show that mixed-mode transmission (the combination of horizontal and vertical transmission), which occurs in Hamiltosporidium, is found to be associated with larger and AT-biased genomes, more genes, and longer intergenic regions, as compared with the exclusively horizontally transmitted Ordospora. Furthermore, the Hamiltosporidium genome assemblies contain a variety of repetitive elements and long segmental duplications. We show that there is an excess of nonsynonymous substitutions in the microsporidia with mixed-mode transmission, which cannot be solely attributed to the lack of recombination, suggesting that bursts of genome size in these microsporidia result primarily from genetic drift. Overall, these findings suggest that the switch from a horizontal-only to a mixed mode of transmission likely produces population bottlenecks in Hamiltosporidium species, therefore reducing the effectiveness of natural selection, and allowing their genomic features to be largely shaped by nonadaptive processes.

Evolutionary relationships among salivarius streptococci as inferred from multilocus phylogenies based on 16S rRNA-encoding, recA, secA, and secY gene sequences.

BACKGROUND: Streptococci are divided into six phylogenetic groups, i.e, anginosus, bovis, mitis, mutans, pyogenic, and salivarius, with the salivarius group consisting of only three distinct species. Two of these species, Streptococcus salivarius and Streptococcus vestibularis, are members of the normal human oral microflora whereas the third, Streptococcus thermophilus, is found in bovine milk. Given that S. salivarius and S. vestibularis share several physiological characteristics, in addition to inhabiting the same ecosystem, one would assume that they would be more closely related to each other than to S. thermophilus. However, the few phylogenetic trees published so far suggest that S. vestibularis is more closely related to S. thermophilus. To determine whether this phylogenetic relationship is genuine, we performed phylogenetic inferences derived from secA and secY, the general secretion housekeeping genes, recA, a gene from a separate genetic locus that encodes a major component of the homologous recombinational apparatus, and 16S rRNA-encoding gene sequences using other streptococcal species as outgroups. RESULTS: The maximum likelihood (ML) and maximum parsimony (MP) phylogenetic inferences derived from the secA and recA gene sequences provided strong support for the S. vestibularis/S. thermophilus sister-relationship, whereas 16S rRNA-encoding and secY-based analyses could not discriminate between alternate topologies. Phylogenetic analyses derived from the concatenation of these sequences unambiguously supported the close affiliation of S. vestibularis and S. thermophilus. CONCLUSION: Our results corroborated the sister-relationship between S. vestibularis and S. thermophilus and the concomitant early divergence of S. salivarius at the base of the salivarius lineage.

A streamlined and predominantly diploid genome in the tiny marine green alga Chloropicon primus.

Tiny marine green algae issued from two deep branches of the Chlorophyta, the Mamiellophyceae and Chloropicophyceae, dominate different regions of the oceans and play key roles in planktonic communities. Considering that the Mamiellophyceae is the sole lineage of prasinophyte algae that has been intensively investigated, the extent to which these two algal groups differ in their metabolic capacities and cellular processes is currently unknown. To address this gap of knowledge, we investigate here the nuclear genome sequence of a member of the Chloropicophyceae, Chloropicon primus. Among the main biological insights that emerge from this 17.4 Mb genome, we find an unexpected diploid structure for most chromosomes and a propionate detoxification pathway in green algae. Our results support the notion that separate events of genome minimization, which entailed differential losses of genes/pathways, have occurred in the Chloropicophyceae and Mamiellophyceae, suggesting different strategies of adaptation to oceanic environments.

Characterization of Listeria monocytogenes enhanced cold-tolerance variants isolated during prolonged cold storage.

In this study, we show that growth and prolonged storage of Listeria monocytogenes at 4 °C can promote the selection of variants with enhanced cold and heat tolerance. Enhanced cold-tolerance (ECT) variants (n = 12) were successfully isolated from a strain with impaired cold growth abilities following 84 days of storage at 4 °C in brain heart infusion broth (BHIB). Whole genome sequencing, membrane fatty acid analysis, and stress tolerance profiling were performed on the parent strain and two ECT variants: one displaying regular-sized colonies and the other displaying small colonies when grown at 37 °C on BHI agar. Under cold stress conditions, the parent strain exhibited an impaired ability to produce branched-chain fatty acids which are known to be important for cold adaptation in L.monocytogenes. The ECT variants were able to overcome this limitation, a finding which is hypothesized to be associated with the identification of two independent single-nucleotide polymorphisms in genes encoding subunits of acetyl-coA carboxylase, an enzyme critical for fatty acid biosynthesis. While the ECT phenotype was not found to be associated with improved salt (BHIB + 6% NaCl, 25 °C), acid (BHIB pH 5, 25 °C) or desiccation (33% RH, 20 °C) tolerance, the small-colony variant exhibited significantly (p < 0.05) enhanced heat tolerance at 52 °C in buffered peptone water compared to the parent strain and the other variant. The results from this study demonstrate that the continuous use of refrigeration along the food-supply chain has the potential to select for L.monocytogenes variants with enhanced cold and heat tolerance, highlighting the impact that microbial intervention strategies can have on the evolution of bacterial strains and likewise, food safety.

The bacteriocin from the prophylactic candidate Streptococcus suis 90-1330 is widely distributed across S. suis isolates and appears encoded in an integrative and conjugative element.

The Gram-positive α-hemolytic Streptococcus suis is a major pathogen in the swine industry and an emerging zoonotic agent that can cause several systemic issues in both pigs and humans. A total of 35 S. suis serotypes (SS) have been identified and genotyped into > 700 sequence types (ST) by multilocus sequence typing (MLST). Eurasian ST1 isolates are the most virulent of all S. suis SS2 strains while North American ST25 and ST28 strains display moderate to low/no virulence phenotypes, respectively. Notably, S. suis 90-1330 is an avirulent Canadian SS2-ST28 isolate producing a lantibiotic bacteriocin with potential prophylactic applications. To investigate the suitability of this strain for such purposes, we sequenced its complete genome using the Illumina and PacBio platforms. The S. suis 90-1330 bacteriocin was found encoded in a locus cargoed in what appears to be an integrative and conjugative element (ICE). This bacteriocin locus was also found to be widely distributed across several streptococcal species and in a few Staphylococcus aureus strains. Because the locus also confers protection from the bacteriocin, the potential prophylactic benefits of using this strain may prove limited due to the spread of the resistance to its effects. Furthermore, the S. suis 90-1330 genome was found to code for genes involved in blood survival, suggesting that strain may not be a benign as previously thought.