Horizontal DNA Transfer Mechanisms of Bacteria as Weapons of Intragenomic Conflict

Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell–cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing “arms race.” Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic’s effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell–cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated.     

Herbicide Selection Promotes Antibiotic Resistance in Soil Microbiomes

Herbicides are one of the most widely used chemicals in agriculture. While they are known to be harmful to nontarget organisms, the effects of herbicides on the composition and functioning of soil microbial communities remain unclear. Here we show that application of three widely used herbicides—glyphosate, glufosinate, and dicamba—increase the prevalence of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in soil microbiomes without clear changes in the abundance, diversity and composition of bacterial communities. Mechanistically, these results could be explained by a positive selection for more tolerant genotypes that acquired several mutations in previously well-characterized herbicide and ARGs. Moreover, herbicide exposure increased cell membrane permeability and conjugation frequency of multidrug resistance plasmids, promoting ARG movement between bacteria. A similar pattern was found in agricultural soils across 11 provinces in China, where herbicide application, and the levels of glyphosate residues in soils, were associated with increased ARG and MGE abundances relative to herbicide-free control sites. Together, our results show that herbicide application can enrich ARGs and MGEs by changing the genetic composition of soil microbiomes, potentially contributing to the global antimicrobial resistance problem in agricultural environments.     

Mobile Genetic Element-Encoded Cytolysin Connects Virulence to Methicillin Resistance in MRSA

Bacterial virulence and antibiotic resistance have a significant influence on disease severity and treatment options during bacterial infections. Frequently, the underlying genetic determinants are encoded on mobile genetic elements (MGEs). In the leading human pathogen Staphylococcus aureus, MGEs that contain antibiotic resistance genes commonly do not contain genes for virulence determinants. The phenol-soluble modulins (PSMs) are staphylococcal cytolytic toxins with a crucial role in immune evasion. While all known PSMs are core genome-encoded, we here describe a previously unidentified psm gene, psm-mec, within the staphylococcal methicillin resistance-encoding MGE SCCmec. PSM-mec was strongly expressed in many strains and showed the physico-chemical, pro-inflammatory, and cytolytic characteristics typical of PSMs. Notably, in an S. aureus strain with low production of core genome-encoded PSMs, expression of PSM-mec had a significant impact on immune evasion and disease. In addition to providing high-level resistance to methicillin, acquisition of SCCmec elements encoding PSM-mec by horizontal gene transfer may therefore contribute to staphylococcal virulence by substituting for the lack of expression of core genome-encoded PSMs. Thus, our study reveals a previously unknown role of methicillin resistance clusters in staphylococcal pathogenesis and shows that important virulence and antibiotic resistance determinants may be combined in staphylococcal MGEs.     

Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm

Our ability to predict evolutionary trajectories of pathogens in response to antibiotic pressure is one of the promising leverage to fight against the present antibiotic resistance worldwide crisis. Yet, few studies tackled this question in situ at the outbreak level, due to the difficulty to link a given pathogenic clone evolution with its precise antibiotic exposure over time. In this study, we monitored the real-time evolution of an Aeromonas salmonicida clone in response to successive antibiotic and vaccine therapies in a commercial fish farm. The clone was responsible for a four-year outbreak of furunculosis within a Recirculating Aquaculture System Salmo salar farm in China, and we reconstructed the precise tempo of mobile genetic elements (MGEs) acquisition events during this period. The resistance profile provided by the acquired MGEs closely mirrored the antibiotics used to treat the outbreak, and we evidenced that two subclonal groups developed similar resistances although unrelated MGE acquisitions. Finally, we also demonstrated the efficiency of vaccination in outbreak management and its positive effect on antibiotic resistance prevalence. Our study provides unprecedented knowledge critical to understand evolutionary trajectories of resistant pathogens outside the laboratory.     

细菌中基因组岛的转移与调控机制研究进展

基因水平转移可导致细菌不同种属间个体DNA的交换,从而使细菌对环境的适应性增强,是细菌进化的重要途径之一。基因组岛是基因水平转移的重要载体,可移动的基因组岛能够整合到宿主的染色体上,并在特定的条件下切除,进而通过转化、接合或转导等方式转移到新的宿主中。基因组岛具有多种生物学功能,如抗生素抗性、致病性、异源物质降解、重金属抗性等。基因组岛的转移造成可变基因在不同种属细菌间的广泛传播,例如毒力和耐药基因的传播导致了多重耐药细菌的产生,威胁人类健康。基因组岛由整合酶介导转移,同时在转移的过程受到多种不同转录因子的调控。本文对细菌中基因组岛的结构特点、转移和调控机制以及预测等方面进行了综述,并最终阐明基因组岛的转移及其调控机制是遏制基因组岛传播的重要策略。     

Influence of a Non-Hospital Medical Care Facility on Antimicrobial Resistance in Wastewater

The global widespread use of antimicrobials and accompanying increase in resistant bacterial strains is of major public health concern. Wastewater systems and wastewater treatment plants are considered a niche for antibiotic resistance genes (ARGs), with diverse microbial communities facilitating ARG transfer via mobile genetic element (MGE). In contrast to hospital sewage, wastewater from other health care facilities is still poorly investigated. At the instance of a nursing home located in south-west Germany, in the present study, shotgun metagenomics was used to investigate the impact on wastewater of samples collected up- and down-stream in different seasons. Microbial composition, ARGs and MGEs were analyzed using different annotation approaches with various databases, including Antibiotic Resistance Ontologies (ARO), integrons and plasmids. Our analysis identified seasonal differences in microbial communities and abundance of ARG and MGE between samples from different seasons. However, no obvious differences were detected between up- and downstream samples. The results suggest that, in contrast to hospitals, sewage from the nursing home does not have a major impact on ARG or MGE in wastewater, presumably due to much less intense antimicrobial usage. Possible limitations of metagenomic studies using high-throughput sequencing for detection of genes that seemingly confer antibiotic resistance are discussed.     

Truncation family selection and nonsystematic inbreeding leads to a rapid fixation of a pattern of mobile genetic elements in a computer model

A computer simulation model of the population dynamics of a polygenic system and a pattern of mobile genetic elements (MGEs) under directional truncation selection for a quantitative trait was developed. Modifier MGEs were shown to be rapidly and adaptively fixed (or lost) together with the modified polygenes. Marker MGEs and independent MGE copies were fixed and lost just as rapidly but in a random manner. Using specific marking of initial haploid genomes and direct computing of the mean proportion of identical encounters at each locus in each generation, it was shown that the mean nonselective inbreeding coefficient F(n) dramatically increases in the course of selection, reaching values 0.7-0.9 in 15-20 generations. As a result, adaptive homozygotization of polygenes and modifier MGEs and random homozygotization of marker MGEs, independent MGE copies, and all other genes of the genome occurs. These results confirm the hypothesis on the "champion" polygene pattern advanced earlier to explain the data of selection experiments.     

Genome-wide analysis of mobile genetic element insertion sites

Mobile genetic elements (MGEs) account for a significant fraction of eukaryotic genomes and are implicated in altered gene expression and disease. We present an efficient computational protocol for MGE insertion site analysis. ELAN, the suite of tools described here uses standard techniques to identify different MGEs and their distribution on the genome. One component, DNASCANNER analyses known insertion sites of MGEs for the presence of signals that are based on a combination of local physical and chemical properties. ISF (insertion site finder) is a machine-learning tool that incorporates information derived from DNASCANNER. ISF permits classification of a given DNA sequence as a potential insertion site or not, using a support vector machine. We have studied the genomes of Homo sapiens, Mus musculus, Drosophila melanogaster and Entamoeba histolytica via a protocol whereby DNASCANNER is used to identify a common set of statistically important signals flanking the insertion sites in the various genomes. These are used in ISF for insertion site prediction, and the current accuracy of the tool is over 65%. We find similar signals at gene boundaries and splice sites. Together, these data are suggestive of a common insertion mechanism that operates in a variety of eukaryotes.     

MGEScan-non-LTR: computational identification and classification of autonomous non-LTR retrotransposons in eukaryotic genomes

Computational methods for genome-wide identification of mobile genetic elements (MGEs) have become increasingly necessary for both genome annotation and evolutionary studies. Non-long terminal repeat (non-LTR) retrotransposons are a class of MGEs that have been found in most eukaryotic genomes, sometimes in extremely high numbers. In this article, we present a computational tool, MGEScan-non-LTR, for the identification of non-LTR retrotransposons in genomic sequences, following a computational approach inspired by a generalized hidden Markov model (GHMM). Three different states represent two different protein domains and inter-domain linker regions encoded in the non-LTR retrotransposons, and their scores are evaluated by using profile hidden Markov models (for protein domains) and Gaussian Bayes classifiers (for linker regions), respectively. In order to classify the non-LTR retrotransposons into one of the 12 previously characterized clades using the same model, we defined separate states for different clades. MGEScan-non-LTR was tested on the genome sequences of four eukaryotic organisms, Drosophila melanogaster, Daphnia pulex, Ciona intestinalis and Strongylocentrotus purpuratus. For the D. melanogaster genome, MGEScan-non-LTR found all known ‘full-length’ elements and simultaneously classified them into the clades CR1, I, Jockey, LOA and R1. Notably, for the D. pulex genome, in which no non-LTR retrotransposon has been annotated, MGEScan-non-LTR found a significantly larger number of elements than did RepeatMasker, using the current version of the RepBase Update library. We also identified novel elements in the other two genomes, which have only been partially studied for non-LTR retrotransposons.     

Airborne and indigenous microbiomes co-drive the rebound of antibiotic resistome during compost storage

Composting is widely used to reduce the abundance of antibiotic resistance genes (ARGs) in solid waste. While ARG dynamics have been extensively investigated during composting, the fate and abundance of residual ARGs during the storage remain unexplored. Here, we tested experimentally how ARG and mobile genetic element (MGE) abundances change during compost storage using metagenomics, quantitative PCR and direct culturing. We found that 43.8% of ARGs and 39.9% of MGEs quickly recovered already during the first week of storage. This rebound effect was mainly driven by the regrowth of indigenous, antibiotic-resistant bacteria that survived the composting. Bacterial transmission from the surrounding air had a much smaller effect, being most evident as MGE rebound during the later stages of storage. While hyperthermophilic composting was more efficient at reducing the relative abundance of ARGs and MGEs, relatively greater ARG rebound was observed during the storage of hyperthermophilic compost, exceeding the initial levels of untreated sewage sludge. Our study reveals that residual ARGs and MGEs left in the treated compost can quickly rebound during the storage via airborne introduction and regrowth of surviving bacteria, highlighting the need to develop better storage strategies to prevent the rebound of ARGs and MGEs after composting.     

Interplay between the cell envelope and mobile genetic elements shapes gene flow in populations of the nosocomial pathogen Klebsiella pneumoniae

Mobile genetic elements (MGEs) drive genetic transfers between bacteria using mechanisms that require a physical interaction with the cellular envelope. In the high-priority multidrug-resistant nosocomial pathogens (ESKAPE), the first point of contact between the cell and virions or conjugative pili is the capsule. While the capsule can be a barrier to MGEs, it also evolves rapidly by horizontal gene transfer (HGT). Here, we aim at understanding this apparent contradiction by studying the covariation between the repertoire of capsule genes and MGEs in approximately 4,000 genomes of Klebsiella pneumoniae (Kpn). We show that capsules drive phage-mediated gene flow between closely related serotypes. Such serotype-specific phage predation also explains the frequent inactivation of capsule genes, observed in more than 3% of the genomes. Inactivation is strongly epistatic, recapitulating the capsule biosynthetic pathway. We show that conjugative plasmids are acquired at higher rates in natural isolates lacking a functional capsular locus and confirmed experimentally this result in capsule mutants. This suggests that capsule inactivation by phage pressure facilitates its subsequent reacquisition by conjugation. Accordingly, capsule reacquisition leaves long recombination tracts around the capsular locus. The loss and regain process rewires gene flow toward other lineages whenever it leads to serotype swaps. Such changes happen preferentially between chemically related serotypes, hinting that the fitness of serotype-swapped strains depends on the host genetic background. These results enlighten the bases of trade-offs between the evolution of virulence and multidrug resistance and caution that some alternatives to antibiotics by selecting for capsule inactivation may facilitate the acquisition of antibiotic resistance genes (ARGs).

A study of how the complex interaction between capsules and mobile genetic elements shapes gene flow in populations of Klebsiella pneumoniae reveals that capsule inactivation by phage pressure facilitates its subsequent re-acquisition by conjugation, and this loss and re-gain process influences the gene flow towards other lineages whenever it leads to serotype changes.     

Network‐based visualisation reveals new insights into transposable element diversity

Transposable elements (TEs) are widespread across eukaryotic genomes, yet their content varies widely between different species. Factors shaping the diversity of TEs are poorly understood. Understanding the evolution of TEs is difficult because their sequences diversify rapidly and TEs are often transferred through non‐conventional means such as horizontal gene transfer. We developed a method to track TE evolution using network analysis to visualise TE sequence and TE content across different genomes. We illustrate our method by first using a monopartite network to study the sequence evolution of Tc1/mariner elements across focal species. We identify a connection between two subfamilies associated with convergent acquisition of a domain from a protein‐coding gene. Second, we use a bipartite network to study how TE content across species is shaped by epigenetic silencing mechanisms. We show that the presence of Piwi‐interacting RNAs is associated with differences in network topology after controlling for phylogenetic effects. Together, our method demonstrates how a network‐based approach can identify hitherto unknown properties of TE evolution across species.     

Xylella fastidiosa comparative genomic database is an information resource to explore the annotation, genomic features, and biology of different strains

The Xylella fastidiosa comparative genomic database is a scientific resource with the aim to provide a user-friendly interface for accessing high-quality manually curated genomic annotation and comparative sequence analysis, as well as for identifying and mapping prophage-like elements, a marked feature of Xylella genomes. Here we describe a database and tools for exploring the biology of this important plant pathogen. The hallmarks of this database are the high quality genomic annotation, the functional and comparative genomic analysis and the identification and mapping of prophage-like elements. It is available from web site http://www.xylella.lncc.br.     

Complex interactions between diverse mobile genetic elements drive the evolution of metal-resistant bacterial genomes

In this study, we compared the genomes of three metal-resistant bacteria isolated from mercury-contaminated soil. We identified diverse and novel MGEs with evidence of multiple LGT events shaping their genomic structure and heavy metal resistance. Among the three metal-resistant strains, Sphingobium sp SA2 and Sphingopyxis sp SE2 were resistant to multiple metals including mercury, cadmium, copper, zinc and lead. Pseudoxanthomonas sp SE1 showed resistance to mercury only. Whole genome sequencing by Illumina and Oxford Nanopore technologies was undertaken to obtain comprehensive genomic data. The Sphingobium and Sphingopyxis strains contained multiple chromosomes and plasmids, whereas the Pseudoxanthomonas strain contained one circular chromosome. Consistent with their metal resistance profiles, the strains of Sphingobium and Sphingopyxis contained a higher quantity of diverse metal resistance genes across their chromosomes and plasmids compared to the single-metal resistant Pseudoxanthomonas SE1. In all three strains, metal resistance genes were principally associated with various novel MGEs including genomic islands (GIs), integrative conjugative elements (ICEs), transposons, insertion sequences (IS), recombinase in trio (RIT) elements and group II introns, indicating their importance in facilitating metal resistance adaptation in a contaminated environment. In the Pseudoxanthomonas strain, metal resistance regions were largely situated on a GI. The chromosomes of the strains of Sphingobium and Sphingopyxis contained multiple metal resistance regions, which were likely acquired by several GIs, ICEs, numerous IS elements, several Tn3 family transposons and RIT elements. Two of the plasmids of Sphingobium were impacted by Tn3 family transposons and ISs likely integrating metal resistance genes. The two plasmids of Sphingopyxis harboured transposons, IS elements, an RIT element and a group II intron. This study provides a comprehensive annotation of complex genomic regions of metal resistance associated with novel MGEs. It highlights the critical importance of LGT in the evolution of metal resistance of bacteria in contaminated environments.     

Conflicting selection alters the trajectory of molecular evolution in a tripartite bacteria–plasmid–phage interaction

Bacteria engage in a complex network of ecological interactions, which includes mobile genetic elements (MGEs) such as phages and plasmids. These elements play a key role in microbial communities as vectors of horizontal gene transfer but can also be important sources of selection for their bacterial hosts. In natural communities, bacteria are likely to encounter multiple MGEs simultaneously and conflicting selection among MGEs could alter the bacterial evolutionary response to each MGE. Here, we test the effect of interactions with multiple MGEs on bacterial molecular evolution in the tripartite interaction between the bacterium, Pseudomonas fluorescens, the lytic bacteriophage, SBW25φ2, and conjugative plasmid, pQBR103, using genome sequencing of experimentally evolved bacteria. We show that individually, both plasmids and phages impose selection leading to bacterial evolutionary responses that are distinct from bacterial populations evolving without MGEs, but that together, plasmids and phages impose conflicting selection on bacteria, constraining the evolutionary responses observed in pairwise interactions. Our findings highlight the likely difficulties of predicting evolutionary responses to multiple selective pressures from the observed evolutionary responses to each selective pressure alone. Understanding evolution in complex microbial communities comprising many species and MGEs will require that we go beyond studies of pairwise interactions.     

Identification and annotation of noncoding RNAs in Saccharomycotina

The importance of ncRNAs in biological processes makes their annotation an essential component of any genome-sequencing project. The identification of ncRNAs in genomes requires specific expertise and tools that are distinct from the traditional protein gene annotation tools. Here, we describe the assembly of two automatic annotation pipelines, integrating publicly available tools, for homology and de novo ncRNA search in genomes. We applied both pipelines to 10 Saccharomycotina genomes and were able to find and annotate 693 ncRNA genes, corresponding to 81% of the ncRNAs expected for those genomes assuming the number of ncRNAs in Saccharomyces cerevisiae (86) as a reference. Several new ncRNAs, not yet known in the Saccharomycotina clade, were also detected. The results show the feasibility of automatic search for ncRNAs in full genomes and the utility of such approaches in large multi-genome sequencing and annotation projects.     

Detecting motifs and patterns at mobile genetic element insertion site

Mobile genetic elements (MGEs) occupy major proportion of eukaryotic genomes and are present in significant numbers in prokaryote genomes also. Here we report a new method which extracts a motif at the site of insertion of MGE using tools such as DNA SCANNER. The flanking region of the insertion site is extracted and is analyzed in DNA Scanner for physiochemical properties like protein-interaction measures, energy profiles as well as structural parameters. In case significant signals are observed, the most frequently occurring n-mer (5E. histolytica, signals for EhSine1 are found at around 5 bps upstream of insertion and most frequently occurring motif is found to be AAGGT and TCGAA. Signals for Ty3 element in S. cerevisiae are found at 0-3 bps upstream of tRNA, and most frequent motif is GTTCGA (6 bps), GGTTCGA (7 bps) and GGTTCGAT (8 bps). P-element of Drosophila showed remarkable dyad peaks suggesting palindromic site of insertion.