GR13-type plasmids in Acinetobacter potentiate the accumulation and horizontal transfer of diverse accessory genes

Carbapenem resistance and other antibiotic resistance genes (ARGs) can be found in plasmids in Acinetobacter, but many plasmid types in this genus have not been well-characterised. Here we describe the distribution, diversity and evolutionary capacity of rep group 13 (GR13) plasmids that are found in Acinetobacter species from diverse environments. Our investigation was prompted by the discovery of two GR13 plasmids in A. baumannii isolated in an intensive care unit (ICU). The plasmids harbour distinct accessory genes: pDETAB5 contains blaNDM-1 and genes that confer resistance to four further antibiotic classes, while pDETAB13 carries putative alcohol tolerance determinants. Both plasmids contain multiple dif modules, which are flanked by pdif sites recognised by XerC/XerD tyrosine recombinases. The ARG-containing dif modules in pDETAB5 are almost identical to those found in pDETAB2, a GR34 plasmid from an unrelated A. baumannii isolated in the same ICU a month prior. Examination of a further 41 complete, publicly available plasmid sequences revealed that the GR13 pangenome consists of just four core but 1086 accessory genes, 123 in the shell and 1063 in the cloud, reflecting substantial capacity for diversification. The GR13 core genome includes genes for replication and partitioning, and for a putative tyrosine recombinase. Accessory segments encode proteins with diverse putative functions, including for metabolism, antibiotic/heavy metal/alcohol tolerance, restriction-modification, an anti-phage system and multiple toxin-antitoxin systems. The movement of dif modules and actions of insertion sequences play an important role in generating diversity in GR13 plasmids. Discrete GR13 plasmid lineages are internationally disseminated and found in multiple Acinetobacter species, which suggests they are important platforms for the accumulation, horizontal transmission and persistence of accessory genes in this genus.

Giant Starship elements mobilize accessory genes in fungal genomes

Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a novel group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27-393 kb and last shared a common ancestor ca. 400 mya. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize 4 additional Starships whose past and ongoing activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyager activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first known agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum.

Combined Pan-, Population-, and Phylo-Genomic Analysis of Aspergillus fumigatus Reveals Population Structure and Lineage-Specific Diversity

Aspergillus fumigatus is a deadly agent of human fungal disease, where virulence heterogeneity is thought to be at least partially structured by genetic variation between strains. While population genomic analyses based on reference genome alignments offer valuable insights into how gene variants are distributed across populations, these approaches fail to capture intraspecific variation in genes absent from the reference genome. Pan-genomic analyses based on de novo assemblies offer a promising alternative to reference-based genomics, with the potential to address the full genetic repertoire of a species. Here, we use a combination of population genomics, phylogenomics, and pan-genomics to assess population structure and recombination frequency, phylogenetically structured gene presence-absence variation, evidence for metabolic specificity, and the distribution of putative antifungal resistance genes in A. fumigatus. We provide evidence for three distinct populations of A. fumigatus, structured by both gene variation (SNPs and indels) and distinct gene presence-absence variation with unique suites of accessory genes present exclusively in each clade. Accessory genes displayed functional enrichment for nitrogen and carbohydrate metabolism, hinting that populations may be stratified by environmental niche specialization. Similarly, the distribution of antifungal resistance genes and resistance alleles were often structured by phylogeny. Despite low levels of outcrossing, A. fumigatus demonstrated a large pan-genome including many genes unrepresented in the Af293 reference genome. These results highlight the inadequacy of relying on a single-reference genome based approach for evaluating intraspecific variation, and the power of combined genomic approaches to elucidate population structure, genetic diversity, and putative ecological drivers of clinically relevant fungi.

Pangenome evolution in environmentally transmitted symbionts of deep-sea mussels is governed by vertical inheritance

Microbial pangenomes vary across species; their size and structure are determined by genetic diversity within the population and by gene loss and horizontal gene transfer (HGT). Many bacteria are associated with eukaryotic hosts where the host colonization dynamics may impact bacterial genome evolution. Host-associated lifestyle has been recognized as a barrier to HGT in parentally transmitted bacteria. However, pangenome evolution of environmentally acquired symbionts remains understudied, often due to limitations in symbiont cultivation. Using high-resolution metagenomics, here we study pangenome evolution of two co-occurring endosymbiont populations inhabiting individual Bathymodiolus brooksi mussels from a single cold seep. The symbionts, sulfur-oxidizing (SOX) and methane-oxidizing (MOX) gamma-proteobacteria, are environmentally acquired at an early developmental stage and individual mussels may harbor multiple strains of each species. We found differences in the accessory gene content of both symbionts across individual mussels, which are reflected by differences in symbiont strain composition. Compared to core genes, accessory genes are enriched in functions involved in genome integrity maintenance. We found no evidence for recent horizontal gene transfer between both symbionts. A comparison between the symbiont pangenomes revealed that the MOX population is less diverged and contains fewer accessory genes, supporting the view that the MOX association with B. brooksi is more recent than that of SOX. Our results show that the pangenomes of both symbionts evolved mainly by vertical inheritance. We conclude that association with individual hosts over their lifetime leads to genetically isolated symbiont subpopulations, constraining the frequency of HGT in the evolution of environmentally transmitted symbionts.

Molecular Characterisation of Vancomycin-Resistant Enterococcus faecium Isolates Belonging to the Lineage ST117/CT24 Causing Hospital Outbreaks

Background: Vancomycin-resistant Enterococcus faecium (VREfm) is a successful nosocomial pathogen. The current molecular method recommended in the Netherlands for VREfm typing is based on core genome Multilocus sequence typing (cgMLST), however, the rapid emergence of specific VREfm lineages challenges distinguishing outbreak isolates solely based on their core genome. Here, we explored if a detailed molecular characterisation of mobile genetic elements (MGEs) and accessory genes could support and expand the current molecular typing of VREfm isolates sharing the same genetic background, enhancing the discriminatory power of the analysis.Materials/Methods: The genomes of 39 VREfm and three vancomycin-susceptible E. faecium (VSEfm) isolates belonging to ST117/CT24, as assessed by cgMLST, were retrospectively analysed. The isolates were collected from patients and environmental samples from 2011 to 2017, and their genomes were analysed using short-read sequencing. Pangenome analysis was performed on de novo assemblies, which were also screened for known predicted virulence factors, antimicrobial resistance genes, bacteriocins, and prophages. Two representative isolates were also sequenced using long-read sequencing, which allowed a detailed analysis of their plasmid content.Results: The cgMLST analysis showed that the isolates were closely related, with a minimal allelic difference of 10 between each cluster’s closest related isolates. The vanB-carrying transposon Tn1549 was present in all VREfm isolates. However, in our data, we observed independent acquisitions of this transposon. The pangenome analysis revealed differences in the accessory genes related to prophages and bacteriocins content, whilst a similar profile was observed for known predicted virulence and resistance genes.Conclusion: In the case of closely related isolates sharing a similar genetic background, a detailed analysis of MGEs and the integration point of the vanB-carrying transposon allow to increase the discriminatory power compared to the use of cgMLST alone. Thus, enabling the identification of epidemiological links amongst hospitalised patients.

Core genes can have higher recombination rates than accessory genes within global microbial populations

Recombination is essential to microbial evolution, and is involved in the spread of antibiotic resistance, antigenic variation, and adaptation to the host niche. Yet quantifying the impact of homologous recombination on different gene classes, which is critical to understanding how selection acts on variation to shape species diversity and genome structure, remains challenging. This is largely due to the dynamic nature of bacterial genomes, whose high intraspecies genome content diversity and complex phylogenetic relationships present difficulties for inferring rates of recombination, particularly for rare genes. In this work, we apply a computationally efficient, non-phylogenetic approach to measure homologous recombination rates in the core and accessory genome (genes present in all strains and only a subset of strains, respectively) using >100,000 whole genome sequences from 12 microbial species. Our analysis suggests that even well-resolved sequence clusters sampled from global populations interact with overlapping gene pools, which has implications for the role of population structure in genome evolution. We show that in a majority of species, core genes have shorter coalescence times and higher recombination rates than accessory genes, and that gene frequency is often positively correlated with increased recombination. Our results provide a new line of population genomic evidence supporting the hypothesis that core genes are under strong, purifying selection, and indicate that homologous recombination may play a key role in increasing the efficiency of selection in those parts of the genome most conserved within each species.

Biofilm associated genotypes of multiple antibiotic resistant Pseudomonas aeruginosa

Background Pseudomonas aeruginosa is a ubiquitous environmental microorganism and also a common cause of infection. Its ability to survive in many different environments and persistently colonize humans is linked to its presence in biofilms formed on indwelling device surfaces. Biofilm promotes adhesion to, and survival on surfaces, protects from desiccation and the actions of antibiotics and disinfectants. Results We examined the genetic basis for biofilm production on polystyrene at room (22 °C) and body temperature (37 °C) within 280 P. aeruginosa. 193 isolates (69 %) produced more biofilm at 22 °C than at 37 °C. Using GWAS and pan-GWAS, we found a number of accessory genes significantly associated with greater biofilm production at 22 °C. Many of these are present on a 165 kb region containing genes for heavy metal resistance (arsenic, copper, mercury and cadmium), transcriptional regulators and methytransferases. We also discovered multiple core genome SNPs in the A-type flagellin gene and Type II secretion system gene xpsD. Analysis of biofilm production of isolates of the MDR ST111 and ST235 lineages on stainless-steel revealed several accessory genes associated with enhanced biofilm production. These include a putative translocase with homology to a Helicobacter pylori type IV secretion system protein, a TA system II toxin gene and the alginate biosynthesis gene algA, several transcriptional regulators and methytransferases as well as core SNPs in genes involved in quorum sensing and protein translocation. Conclusions Using genetic association approaches we discovered a number of accessory genes and core-genome SNPs that were associated with enhanced early biofilm formation at 22 °C compared to 37 °C. These included a 165 kb genomic island containing multiple heavy metal resistance genes, transcriptional regulators and methyltransferases. We hypothesize that this genomic island may be associated with overall genotypes that are environmentally adapted to survive at lower temperatures. Further work to examine their importance in, for example gene-knockout studies, are required to confirm their relevance. GWAS and pan-GWAS approaches have great potential as a first step in examining the genetic basis of novel bacterial phenotypes.

Metatranscriptomic comparison of endophytic and pathogenic Fusarium–Arabidopsis interactions reveals plant transcriptional plasticity

Plants are continuously exposed to beneficial and pathogenic microbes, but how plants recognize and respond to friends versus foes remains poorly understood. Here, we compared the molecular response of Arabidopsis thaliana independently challenged with a Fusarium oxysporum endophyte Fo47 versus a pathogen Fo5176. These two Fusarium oxysporum strains share a core genome of about 46 Mb, in addition to unique 1,229 and 5,415 accessory genes. Metatranscriptomic data reveal a shared pattern of expression for most plant genes (~80%) in responding to both fungal inoculums at all time points from 12 to 96 h post inoculation (HPI). However, the distinct responding genes depict transcriptional plasticity, as the pathogenic interaction activates plant stress responses and suppresses plant growth/development related functions, while the endophytic interaction attenuates host immunity but activates plant nitrogen assimilation. The differences in reprogramming of the plant transcriptome are most obvious in 12 HPI, the earliest time point sampled and are linked to accessory genes in both fungal genomes. Collectively, our results indicate that the A. thaliana and F. oxysporum interaction displays both transcriptome conservation and plasticity in the early stages of infection, providing insights into the fine-tuning of gene regulation underlying plant differential responses to fungal endophytes and pathogens.