Lineage‐specific plasmid acquisition and the evolution of specialized pathogens in Bacillus thuringiensis and the Bacillus cereus group

Bacterial plasmids can vary from small selfish genetic elements to large autonomous replicons that constitute a significant proportion of total cellular DNA. By conferring novel function to the cell, plasmids may facilitate evolution but their mobility may be opposed by co‐evolutionary relationships with chromosomes or encouraged via the infectious sharing of genes encoding public goods. Here, we explore these hypotheses through large‐scale examination of the association between plasmids and chromosomal DNA in the phenotypically diverse Bacillus cereus group. This complex group is rich in plasmids, many of which encode essential virulence factors (Cry toxins) that are known public goods. We characterized population genomic structure, gene content and plasmid distribution to investigate the role of mobile elements in diversification. We analysed coding sequence within the core and accessory genome of 190 B. cereus group isolates, including 23 novel sequences and genes from 410 reference plasmid genomes. While cry genes were widely distributed, those with invertebrate toxicity were predominantly associated with one sequence cluster (clade 2) and phenotypically defined Bacillus thuringiensis. Cry toxin plasmids in clade 2 showed evidence of recent horizontal transfer and variable gene content, a pattern of plasmid segregation consistent with transfer during infectious cooperation. Nevertheless, comparison between clades suggests that co‐evolutionary interactions may drive association between plasmids and chromosomes and limit wider transfer of key virulence traits. Proliferation of successful plasmid and chromosome combinations is a feature of specialized pathogens with characteristic niches (Bacillus anthracis, B. thuringiensis) and has occurred multiple times in the B. cereus group.     

Characterization of a mobile and multiple resistance plasmid isolated from swine manure and its detection in soil after manure application

Aims: To isolate and characterize multiple antibiotic resistance plasmids found in swine manure and test for plasmid‐associated genetic markers in soil following manure application to an agricultural field. Methods and Results: Plasmids were isolated from an erythromycin enrichment culture that used liquid swine manure as an inoculant. Plasmids were transformed into Escherichia coli DH10β for subsequent characterization. We isolated and DNA sequenced a 22 102‐bp plasmid (pMC2) that confers macrolide, and tetracycline resistances, and carries genes predicted to code for mercury and chromium resistance. Conjugation experiments using an pRP4 derivative as a helper plasmid confirm that pMC2 has a functional mobilization unit. PCR was used to detect genetic elements found on pMC2 in DNA extracted from manure amended soil. Conclusions: The pMC2 plasmid has a tetracycline‐resistant core and has acquired additional resistance genes by insertion of an accessory region (12 762 bp) containing macrolide, mercury and chromium resistance genes, which was inserted between the truncated DDE motifs within the Tn903/IS102 mobile element. Significance and Impact of the Study: Liquid swine manure used for manure spreading contains multiple antibiotic resistance plasmids that can be detected in soil following manure application.     

The genetic organization and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa

Employing the biparental exogenous plasmid isolation method, conjugative plasmids conferring mercury resistance were isolated from the microbial community of the rhizosphere of field grown alfalfa plants. Five different plasmids were identified, designated pSB101–pSB105. One of the plasmids, pSB102, displayed broad host range (bhr) properties for plasmid replication and transfer unrelated to the known incompatibility (Inc) groups of bhr plasmids IncP-1, IncW, IncN and IncA/C. Nucleotide sequence analysis of plasmid pSB102 revealed a size of 55 578 bp. The transfer region of pSB102 was predicted on the basis of sequence similarity to those of other plasmids and included a putative mating pair formation apparatus most closely related to the type IV secretion system encoded on the chromosome of the mammalian pathogen Brucella sp. The region encoding replication and maintenance functions comprised genes exhibiting different degrees of similarity to RepA, KorA, IncC and KorB of bhr plasmids pSa (IncW), pM3 (IncP-9), R751 (IncP-1β) and RK2 (IncP-1α), respectively. The mercury resistance determinants were located on a transposable element of the Tn5053 family designated Tn5718. No putative functions could be assigned to a quarter of the coding capacity of pSB102 on the basis of comparisons with database entries. The genetic organization of the pSB102 transfer region revealed striking similarities to plasmid pXF51 of the plant pathogen Xylella fastidiosa.     

Plasmid carriage can limit bacteria–phage coevolution

Coevolution with bacteriophages is a major selective force shaping bacterial populations and communities. A variety of both environmental and genetic factors has been shown to influence the mode and tempo of bacteria–phage coevolution. Here, we test the effects that carriage of a large conjugative plasmid, pQBR103, had on antagonistic coevolution between the bacterium Pseudomonas fluorescens and its phage, SBW25ϕ2. Plasmid carriage limited bacteria–phage coevolution; bacteria evolved lower phage-resistance and phages evolved lower infectivity in plasmid-carrying compared with plasmid-free populations. These differences were not explained by effects of plasmid carriage on the costs of phage resistance mutations. Surprisingly, in the presence of phages, plasmid carriage resulted in the evolution of high frequencies of mucoid bacterial colonies. Mucoidy can provide weak partial resistance against SBW25ϕ2, which may have limited selection for qualitative resistance mutations in our experiments. Taken together, our results suggest that plasmids can have evolutionary consequences for bacteria that go beyond the direct phenotypic effects of their accessory gene cargo.     

Increased Abundance of IncP-1β Plasmids and Mercury Resistance Genes in Mercury-Polluted River Sediments: First Discovery of IncP-1β Plasmids with a Complex mer Transposon as the Sole Accessory Element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1β replicon-specific sequences detected in total community DNA. The isolation of IncP-1β plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1β plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1β plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTΔP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1β plasmids R751 and pB8. However, this is the first example of an IncP-1β plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.     

High‐resolution mapping of the recombination landscape of the phytopathogen Fusarium graminearum suggests two‐speed genome evolution

Recombination is a major evolutionary force, increasing genetic diversity and permitting efficient coevolution of fungal pathogen(s) with their host(s). The ascomycete Fusarium graminearum is a devastating pathogen of cereal crops, and can contaminate food and feed with harmful mycotoxins. Previous studies have suggested a high adaptive potential of this pathogen, illustrated by an increase in pathogenicity and resistance to fungicides. In this study, we provide the first detailed picture of the crossover events occurring during meiosis and discuss the role of recombination in pathogen evolution. An experimental recombinant population (n = 88) was created and genotyped using 1306 polymorphic markers obtained from restriction site‐associated DNA sequencing (RAD‐seq) and aligned to the reference genome. The construction of a high‐density linkage map, anchoring 99% of the total length of the reference genome, allowed the identification of 1451 putative crossovers, positioned at a median resolution of 24 kb. The majority of crossovers (87.2%) occurred in a relatively small portion of the genome (30%). All chromosomes demonstrated recombination‐active sections, which had a near 15‐fold higher crossover rate than non‐active recombinant sections. The recombination rate showed a strong positive correlation with nucleotide diversity, and recombination‐active regions were enriched for genes with a putative role in host–pathogen interaction, as well as putative diversifying genes. Our results confirm the preliminary analysis observed in other F. graminearum strains and suggest a conserved ‘two‐speed’ recombination landscape. The consequences with regard to the evolutionary potential of this major fungal pathogen are also discussed.     

Independent and interactive effects of two facilitators on their habitat‐providing host plant,Spartina alterniflora

The role of habitat‐providing species in facilitating associated species abundance and diversity is recognized as a key structuring force in many ecosystems. Reciprocal facilitation by associates, often involving multiple species, can be important for the maintenance of the host species. As with other multi‐species interactions (e.g. multiple predator effects), non‐additive relationships may be common among these associates, yet relatively few studies have examined potential interactions among multiple facilitator species. We combined field surveys and a mesocosm experiment to examine the independent and interactive effects of two co‐occurring facilitator species, ribbed mussels Geukensia demissa and fiddler crabs Uca pugilator, on their host salt marsh plant species, cordgrass Spartina alterniflora. We also experimentally examined how these relationships varied across different host plant genotypes. Overall, facilitator effects increased with increasing facilitator density. There was a significant interaction between mussel and fiddler crab presence, indicating that the effects of each species on cordgrass were dependent on the presence of the other facilitator species. In addition, there were strong interactions among mussels, fiddler crabs, and plant genotype, with greater variation in the performance of individual genotypes when fiddler crabs were absent. Our work reinforces the importance of considering multiple responses when assessing the functional redundancy of co‐occurring facilitators, as species are seldom completely redundant across the range of services they provide. It also highlights that the strength and direction of species interactions can vary due to genetic variation within the interacting species.     

ParI,an orphan ParA family protein from Pseudomonas putida KT2440‐specific genomic island,interferes with the partition system of IncP‐7 plasmids

Pseudomonas putida KT2440 is an ideal soil bacterium for expanding the range of degradable compounds via the recruitment of various catabolic plasmids. In the course of our investigation of the host range of IncP‐7 catabolic plasmids pCAR1, pDK1 and pWW53, we found that the IncP‐7 miniplasmids composed of replication and partition loci were exceptionally unstable in KT2440, which is the authentic host of the archetypal IncP‐9 plasmid pWW0. This study identified ParI, a homologue of ParA family of plasmid partitioning proteins encoded on the KT2440‐specific cryptic genomic island, as a negative host factor for the maintenance of IncP‐7 plasmids. The miniplasmids were destabilized by ectopic expression of ParI, and the loss rate correlated with the copy number of ParB binding sites in the centromeric parS region. Mutations in the conserved ATPase domains of ParI abolished destabilization of miniplasmids. Furthermore, ParI destabilized miniplasmid derivatives carrying the partition‐deficient parA mutations but failed to impact the stability of miniplasmid derivatives with parB mutations in the putative arginine finger. Altogether, these results indicate that ParI interferes with the IncP‐7 plasmid partition system. This study extends canonical partition‐mediated incompatibility of plasmids beyond heterogeneous mobile genetic elements, namely incompatibility between plasmid and genomic island.     

Genetic structure among arable populations of Capsella bursa‐pastoris is linked to functional traits and in‐field conditions

Wild arable plants can be an economic burden but they also support diverse arable food webs and contribute to valuable ecosystem functions. These benefits may have been compromised over recent decades by declining weed diversity. The decline in wild arable plant diversity has been viewed predominantly in terms of species shifts a view that ignores the genetic and functional variation existing within species and the impact on ecological and evolutionary processes which this may have. To examine within‐species diversity, ISSR markers were used in parallel with environmental and phenotypic characterisation, to investigate the population structure and diversity of Capsella bursa‐pastoris (shepherd's purse) from arable fields in the UK. Analysis of 338 ISSR products for 109 individuals from 51 accessions obtained from the seed banks of 33 arable fields showed that in‐field populations of shepherd's purse were genetically differentiated between individuals, and among accessions and fields. In addition, cluster analysis identified three genetically distinct regional‐scale populations. Phenotypic variation was present at all scales of genetic differentiation, including the regional scale where populations differed in their key life‐history traits: flowering time, fecundity and dormancy. Genetic drift is proposed as a contributor to differentiation among genetically isolated but locally co‐occurring accessions. In addition, the genetic and phenotypic variation in shepherd's purse exhibited large scale, spatial trends and showed statistically significant associations with cropping intensity and soil‐pH. These results suggest that adaptation as a result of selection by cropping practise and soil‐pH has played a role in the ability of shepherd's purse to colonise and persist in arable fields.     

Plasmid-borne mercury resistance in aquatic bacteria

Abstract Bacteria isolated from the River Mersey were analysed for their tolerance to mercury (HgCl₂). About 40% of the population was tolerant to mercury and in 13 of 52 mercury-tolerant isolates tested the mercury resistance (Hg^®) was transferred to Escherichia coli in conjugal matings. These 13 isolates represented a range of gram-negative genera and in each case mercury resistance was coded by a conjugative plasmid. These plasmids (75 kb to > 250 kb in size) all expressed mercury resistance of the narrow spectrum variety, volatilised HgCl₂ to elemental Hg° vapour and showed some degree of temperature sensitivity of transfer. None expressed resistance to nine different antibiotics. These 13 Hg^R plasmids were classified by restriction mapping into three distinct groups typified by pMER11, pMER327 and pMER610. The eight pMER610 group plasmids are identical and belong to the IncHI-2 group. Two of the four pMER327 group plasmids are closely related while the other two contain some common restriction fragments. pMER11 is quite distinct from the other groups. These results imply that within this aquatic environment plasmids play an important role in the response of bacteria to contaminating mercury and that there is widespread plasmid transfer and considerable genetic rearrangement.     

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.     

Strengths and potential pitfalls of hay transfer for ecological restoration revealed by RAD‐seq analysis in floodplain Arabis species

Achieving high intraspecific genetic diversity is a critical goal in ecological restoration as it increases the adaptive potential and long‐term resilience of populations. Thus, we investigated genetic diversity within and between pristine sites in a fossil floodplain and compared it to sites restored by hay transfer between 1997 and 2014. RAD‐seq genotyping revealed that the stenoecious floodplain species Arabis nemorensis is co‐occurring with individuals that, based on ploidy, ITS‐sequencing and morphology, probably belong to the close relative Arabis sagittata, which has a documented preference for dry calcareous grasslands but has not been reported in floodplain meadows. We show that hay transfer maintains genetic diversity for both species. Additionally, in A. sagittata, transfer from multiple genetically isolated pristine sites resulted in restored sites with increased diversity and admixed local genotypes. In A. nemorensis, transfer did not create novel admixture dynamics because genetic diversity between pristine sites was less differentiated. Thus, the effects of hay transfer on genetic diversity also depend on the genetic make‐up of the donor communities of each species, especially when local material is mixed. Our results demonstrate the efficiency of hay transfer for habitat restoration and emphasize the importance of prerestoration characterization of microgeographic patterns of intraspecific diversity of the community to guarantee that restoration practices reach their goal, that is maximize the adaptive potential of the entire restored plant community. Overlooking these patterns may alter the balance between species in the community. Additionally, our comparison of summary statistics obtained from de novo‐ and reference‐based RAD‐seq pipelines shows that the genomic impact of restoration can be reliably monitored in species lacking prior genomic knowledge.     

Effects of species co‐occurrence on the trophic‐niche breadth of characids in Amazon forest streams

This study assessed the width of the trophic niche of four characid species (Bryconops giacopinii, Bryconops inpai, Hyphessobrycon aff. melazonatus and Iguanodectes geisleri) found under different co‐occurrence circumstances in Amazonian upland streams. The study was conducted during the rainy season of 2011 at eight sites of two micro‐basins of the Ducke Reserve, Manaus, Amazonas, Brazil. The four species were studied in the following circumstances: only one of the species occurring in the stream; two species co‐occurring; three species co‐occurring. The relative volume of the food items in the fish stomachs was used to calculate Hurlbert's trophic‐niche breadth for the individuals of each species in the different co‐occurrence circumstances. Hyphessobrycon aff. melazonatus changed their diet when occurring in syntopy with other characid species of similar feeding habits, as shown by a significant narrowing of its trophic niche. The opportunistic habits and great feeding flexibility of these characid species make the partitioning of food resources possible and act as an important ecological mechanism that facilitates the coexistence of different species, possibly by attenuating the effects of direct competition for food. In addition, the low carrying capacity of these upland forest streams may be an important environmental factor influencing the results of this study.     

Complete sequence of the IncP-9 TOL plasmid pWW0 from Pseudomonas putida

The TOL plasmid pWW0 (117 kb) is the best studied catabolic plasmid and the archetype of the IncP-9 plasmid incompatibility group from Pseudomonas. It carries the degradative (xyl) genes for toluenes and xylenes within catabolic transposons Tn4651 and Tn4653. Analysis of the complete pWW0 nucleotide sequence revealed 148 putative open reading frames. Of these, 77 showed similarity to published sequences in the available databases predicting functions for: plasmid replication, stable maintenance and transfer; phenotypic determinants; gene regulation and expression; and transposition. All identifiable transposition functions lay within the boundaries of the 70 kb transposon Tn4653, leaving a 46 kb sector containing all the IncP-9 core functions. The replicon and stable inheritance region was very similar to the mini-replicon from IncP-9 antibiotic resistance plasmid pM3, with their Rep proteins forming a novel group of initiation proteins. pWW0 transfer functions exist as two blocks encoding putative DNA processing and mating pair formation genes, with organizational and sequence similarity to IncW plasmids. In addition to the known Tn4651 and IS1246 elements, two additional transposable elements were identified as well as several putative transposition functions, which are probably genetic remnants from previous transposition events. Genes likely to be responsible for known resistance to ultraviolet light and free radicals were identified. Other putative phenotypic functions identified included resistance to mercury and other metal ions, as well as to quaternary ammonium compounds. The complexity and size of pWW0 is largely the result of the mosaic organization of the transposable elements that it carries, rather than the backbone functions of IncP-9 plasmids.     

ParA‐mediated plasmid partition driven by protein pattern self‐organization

DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low‐copy plasmids, such as the plasmids P1 and F, employ a three‐component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker‐type ATPase, typically called ParA, which also binds non‐specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP‐driven patterning is involved in partition is unknown. We reconstituted and visualized ParA‐mediated plasmid partition inside a DNA‐carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB‐stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion‐ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid.     

Increased abundance of IncP-1beta plasmids and mercury resistance genes in mercury-polluted river sediments: first discovery of IncP-1beta plasmids with a complex mer transposon as the sole accessory element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1beta replicon-specific sequences detected in total community DNA. The isolation of IncP-1beta plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1beta plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1beta plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTDeltaP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1beta plasmids R751 and pB8. However, this is the first example of an IncP-1beta plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.     

Genetic Rearrangement of Plasmids: In Vivo Recombination between a Dehalogenation Plasmid and Multiple-Resistance Plasmid RP4 in Pseudomonas sp

When Moraxella plasmid pUO1 encoding haloacetate dehalogenase and mercury resistance coexisted with IncP-1 plasmid RP4 in Pseudomonas sp., genetic exchange between the plasmids often occurred, probably by site-specific recombination. The recombinant plasmids obtained were classified into four groups on the basis of phenotype. Representative plasmids for each group were analyzed for DNA composition and function, and the mechanism for the formation of these plasmids was sought. They were inherited stably in Escherichia coli and a Pseudomonas sp.     

The indigenous Pseudomonas plasmid pQBR103 encodes plant-inducible genes, including three putative helicases

Plasmid pQBR103 ( approximately 400 kb) is representative of many self-transmissible, mercury resistant plasmids observed in the Pseudomonas community colonising the phytosphere of sugar beet. A promoter trapping strategy (IVET) was employed to identify pQBR103 genes showing elevated levels of expression on plant surfaces. Thirty-seven different plant-inducible gene fusions were isolated that were silent in laboratory media, but active in the plant environment. Three of the fusions were to DNA sequences whose protein products show significant homology to DNA-unwinding helicases. The three helicase-like genes, designated helA, helB and helC, are restricted to a defined group of related Pseudomonas plasmids. They are induced in both the root and shoot environments of sugar beet seedlings. Sequence analysis of the three plasmid-encoded helicase-like genes shows that they are phylogenetically distinct and likely to have independent evolutionary histories. The helA gene is predicted to encode a protein of 1121 amino acids, containing conserved domains found in the ultraviolet (UV) resistance helicase, UvrD. A helA knockout mutant was constructed and no phenotypic changes were found with plasmid-conferred UV resistance or plasmid conjugation. The other 34 fusions are unique with no homologues in the public gene databases, including the Pseudomonas genomes. These data demonstrate the presence of plant responsive genes in plasmid DNA comprising a component of the genomes of plant-associated bacteria.