A Rickettsia genome overrun by mobile genetic elements provides insight into the acquisition of genes characteristic of an obligate intracellular lifestyle

We present the draft genome for the Rickettsia endosymbiont of Ixodes scapularis (REIS), a symbiont of the deer tick vector of Lyme disease in North America. Among Rickettsia species (Alphaproteobacteria: Rickettsiales), REIS has the largest genome sequenced to date (>2 Mb) and contains 2,309 genes across the chromosome and four plasmids (pREIS1 to pREIS4). The most remarkable finding within the REIS genome is the extraordinary proliferation of mobile genetic elements (MGEs), which contributes to a limited synteny with other Rickettsia genomes. In particular, an integrative conjugative element named RAGE (for Rickettsiales amplified genetic element), previously identified in scrub typhus rickettsiae (Orientia tsutsugamushi) genomes, is present on both the REIS chromosome and plasmids. Unlike the pseudogene-laden RAGEs of O. tsutsugamushi, REIS encodes nine conserved RAGEs that include F-like type IV secretion systems similar to that of the tra genes encoded in the Rickettsia bellii and R. massiliae genomes. An unparalleled abundance of encoded transposases (>650) relative to genome size, together with the RAGEs and other MGEs, comprise ~35% of the total genome, making REIS one of the most plastic and repetitive bacterial genomes sequenced to date. We present evidence that conserved rickettsial genes associated with an intracellular lifestyle were acquired via MGEs, especially the RAGE, through a continuum of genomic invasions. Robust phylogeny estimation suggests REIS is ancestral to the virulent spotted fever group of rickettsiae. As REIS is not known to invade vertebrate cells and has no known pathogenic effects on I. scapularis, its genome sequence provides insight on the origin of mechanisms of rickettsial pathogenicity.     

CRISPR-Cas systems are widespread accessory elements across bacterial and archaeal plasmids

Many prokaryotes encode CRISPR-Cas systems as immune protection against mobile genetic elements (MGEs), yet a number of MGEs also harbor CRISPR-Cas components. With a few exceptions, CRISPR-Cas loci encoded on MGEs are uncharted and a comprehensive analysis of their distribution, prevalence, diversity, and function is lacking. Here, we systematically investigated CRISPR-Cas loci across the largest curated collection of natural bacterial and archaeal plasmids. CRISPR-Cas loci are widely but heterogeneously distributed across plasmids and, in comparison to host chromosomes, their mean prevalence per Mbp is higher and their distribution is distinct. Furthermore, the spacer content of plasmid CRISPRs exhibits a strong targeting bias towards other plasmids, while chromosomal arrays are enriched with virus-targeting spacers. These contrasting targeting preferences highlight the genetic independence of plasmids and suggest a major role for mediating plasmid-plasmid conflicts. Altogether, CRISPR-Cas are frequent accessory components of many plasmids, which is an overlooked phenomenon that possibly facilitates their dissemination across microbiomes.     

The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts

The roles of restriction-modification (R-M) systems in providing immunity against horizontal gene transfer (HGT) and in stabilizing mobile genetic elements (MGEs) have been much debated. However, few studies have precisely addressed the distribution of these systems in light of HGT, its mechanisms and its vectors. We analyzed the distribution of R-M systems in 2261 prokaryote genomes and found their frequency to be strongly dependent on the presence of MGEs, CRISPR-Cas systems, integrons and natural transformation. Yet R-M systems are rare in plasmids, in prophages and nearly absent from other phages. Their abundance depends on genome size for small genomes where it relates with HGT but saturates at two occurrences per genome. Chromosomal R-M systems might evolve under cycles of purifying and relaxed selection, where sequence conservation depends on the biochemical activity and complexity of the system and total gene loss is frequent. Surprisingly, analysis of 43 pan-genomes suggests that solitary R-M genes rarely arise from the degradation of R-M systems. Solitary genes are transferred by large MGEs, whereas complete systems are more frequently transferred autonomously or in small MGEs. Our results suggest means of testing the roles for R-M systems and their associations with MGEs.     

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.     

Selection-Driven Extinction Dynamics for Group II Introns in Enterobacteriales

Transposable elements (TEs) are one of the major driving forces of genome evolution, raising the question of the long-term dynamics underlying their evolutionary success. Some TEs were proposed to evolve under a pattern of periodic extinctions-recolonizations, in which elements recurrently invade and quickly proliferate within their host genomes, then start to disappear until total extinction. Depending on the model, TE extinction is assumed to be driven by purifying selection against colonized host genomes (Sel-DE model) or by saturation of host genomes (Sat-DE model). Bacterial group II introns are suspected to follow an extinction-recolonization model of evolution, but whether they follow Sel-DE or Sat-DE dynamics is not known. Our analysis of almost 200 group II intron copies from 90 sequenced Enterobacteriales genomes confirms their extinction-recolonization dynamics: patchy element distributions among genera and even among strains within genera, acquisition of new group II introns through plasmids or other mobile genetic elements, and evidence for recent proliferations in some genomes. Distributions of recent and past proliferations and of their respective homing sites further provide strong support for the Sel-DE model, suggesting that group II introns are deleterious to their hosts. Overall, our observations emphasize the critical impact of host properties on TE dynamics.     

Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae

IncX plasmids are narrow host range plasmids of Enterobactericeae that have been isolated for over 50years. They are known to encode type IV fimbriae enabling their own conjugative transfer, and to provide accessory functions to their host bacteria such as resistance towards antimicrobial agents and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid diversity and prevalence is underappreciated. To address these possible shortcomings, we generated additional plasmid sequences of IncX plasmids of interest and compared them to the genomes of all sequenced IncX-like plasmids. These comparisons revealed that IncX plasmids possess a highly syntenic plasmid backbone, but that they are quite divergent with respect to nucleotide and amino acid similarity. Based on phylogenetic comparisons of the sequenced IncX plasmids, the IncX plasmid group has been expanded to include at least four subtypes, IncX1-IncX4. A revised IncX plasmid replicon typing procedure, based upon these sequences and subtypes, was then developed. Use of this revised typing procedure revealed that IncX plasmid occurrence among bacterial populations is much more common than had previously been acknowledged. Thus, this revised procedure can be used to better discern the occurrence of IncX type plasmids among enterobacterial populations.     

Genome sequence analyses of Pseudomonas savastanoi pv. glycinea and subtractive hybridization-based comparative genomics with nine pseudomonads

Bacterial blight, caused by Pseudomonas savastanoi pv. glycinea (Psg), is a common disease of soybean. In an effort to compare a current field isolate with one isolated in the early 1960s, the genomes of two Psg strains, race 4 and B076, were sequenced using 454 pyrosequencing. The genomes of both Psg strains share more than 4,900 highly conserved genes, indicating very low genetic diversity between Psg genomes. Though conserved, genome rearrangements and recombination events occur commonly within the two Psg genomes. When compared to each other, 437 and 163 specific genes were identified in B076 and race 4, respectively. Most specific genes are plasmid-borne, indicating that acquisition and maintenance of plasmids may represent a major mechanism to change the genetic composition of the genome and even acquire new virulence factors. Type three secretion gene clusters of Psg strains are near identical with that of P. savastanoi pv. phaseolicola (Pph) strain 1448A and they shared 20 common effector genes. Furthermore, the coronatine biosynthetic cluster is present on a large plasmid in strain B076, but not in race 4. In silico subtractive hybridization-based comparative genomic analyses with nine sequenced phytopathogenic pseudomonads identified dozens of specific islands (SIs), and revealed that the genomes of Psg strains are more similar to those belonging to the same genomospecies such as Pph 1448A than to other phytopathogenic pseudomonads. The number of highly conserved genes (core genome) among them decreased dramatically when more genomes were included in the subtraction, suggesting the diversification of pseudomonads, and further indicating the genome heterogeneity among pseudomonads. However, the number of specific genes did not change significantly, suggesting these genes are indeed specific in Psg genomes. These results reinforce the idea of a species complex of P. syringae and support the reclassification of P. syringae into different species.     

New insights into the diversity and evolution of the archaeal mobilome from three complete genomes of Saccharolobus shibatae

Saccharolobus (formerly Sulfolobus) shibatae B12, isolated from a hot spring in Beppu, Japan in 1982, is one of the first hyperthermophilic and acidophilic archaeal species to be discovered. It serves as a natural host to the extensively studied spindle-shaped virus SSV1, a prototype of the Fuselloviridae family. Two additional Sa. shibatae strains, BEU9 and S38A, sensitive to viruses of the families Lipothrixviridae and Portogloboviridae, respectively, have been isolated more recently. However, none of the strains has been fully sequenced, limiting their utility for studies on archaeal biology and virus–host interactions. Here, we present the complete genome sequences of all three Sa. shibatae strains and explore the rich diversity of their integrated mobile genetic elements (MGE), including transposable insertion sequences, integrative and conjugative elements, plasmids, and viruses, some of which were also detected in the extrachromosomal form. Analysis of related MGEs in other Sulfolobales species and patterns of CRISPR spacer targeting revealed a complex network of MGE distributions, involving horizontal spread and relatively frequent host switching by MGEs over large phylogenetic distances, involving species of the genera Saccharolobus, Sulfurisphaera and Acidianus. Furthermore, we characterize a remarkable case of a virus-to-plasmid transition, whereby a fusellovirus has lost the genes encoding for the capsid proteins, while retaining the replication module, effectively becoming a plasmid.     

Plasmid metagenome reveals high levels of antibiotic resistance genes and mobile genetic elements in activated sludge

The overuse or misuse of antibiotics has accelerated antibiotic resistance, creating a major challenge for the public health in the world. Sewage treatment plants (STPs) are considered as important reservoirs for antibiotic resistance genes (ARGs) and activated sludge characterized with high microbial density and diversity facilitates ARG horizontal gene transfer (HGT) via mobile genetic elements (MGEs). However, little is known regarding the pool of ARGs and MGEs in sludge microbiome. In this study, the transposon aided capture (TRACA) system was employed to isolate novel plasmids from activated sludge of one STP in Hong Kong, China. We also used Illumina Hiseq 2000 high-throughput sequencing and metagenomics analysis to investigate the plasmid metagenome. Two novel plasmids were acquired from the sludge microbiome by using TRACA system and one novel plasmid was identified through metagenomics analysis. Our results revealed high levels of various ARGs as well as MGEs for HGT, including integrons, transposons and plasmids. The application of the TRACA system to isolate novel plasmids from the environmental metagenome, coupled with subsequent high-throughput sequencing and metagenomic analysis, highlighted the prevalence of ARGs and MGEs in microbial community of STPs.     

Genome analyses of Icelandic strains of Sulfolobus islandicus, model organisms for genetic and virus-host interaction studies

The genomes of two Sulfolobus islandicus strains obtained from Icelandic solfataras were sequenced and analyzed. Strain REY15A is a host for a versatile genetic toolbox. It exhibits a genome of minimal size, is stable genetically, and is easy to grow and manipulate. Strain HVE10/4 shows a broad host range for exceptional crenarchaeal viruses and conjugative plasmids and was selected for studying their life cycles and host interactions. The genomes of strains REY15A and HVE10/4 are 2.5 and 2.7 Mb, respectively, and each genome carries a variable region of 0.5 to 0.7 Mb where major differences in gene content and gene order occur. These include gene clusters involved in specific metabolic pathways, multiple copies of VapBC antitoxin-toxin gene pairs, and in strain HVE10/4, a 50-kb region rich in glycosyl transferase genes. The variable region also contains most of the insertion sequence (IS) elements and high proportions of the orphan orfB elements and SMN1 miniature inverted-repeat transposable elements (MITEs), as well as the clustered regular interspaced short palindromic repeat (CRISPR)-based immune systems, which are complex and diverse in both strains, consistent with them having been mobilized both intra- and intercellularly. In contrast, the remainder of the genomes are highly conserved in their protein and RNA gene syntenies, closely resembling those of other S. islandicus and Sulfolobus solfataricus strains, and they exhibit only minor remnants of a few genetic elements, mainly conjugative plasmids, which have integrated at a few tRNA genes lacking introns. This provides a possible rationale for the presence of the introns.     

The minimal genome paradox

The concept of a 'minimal genome' has appeared as an attempt to answer the question what the minimum number of genes or minimum amount of DNA to support life is. Since bacteria are cells bearing the smallest genomes, it has been generally accepted that the minimal genome must belong to a bacterial species. Currently the most popular chromosome in studies on a minimal genome belongs to Mycoplasma genitalium, a parasite bacterium whose total genetic material is as small as approximately 580 kb. However, the problem is how we define life, and thus also a minimal genome. M. genitalium is a parasite and requires substances provided by its host. Therefore, if a genome of a parasite can be considered as a minimal genome, why not to consider genomes of bacteriophages? Going further, bacterial plasmids could be considered as minimal genomes. The smallest known DNA region playing the function of the origin of replication, which is sufficient for plasmid survival in natural habitats, is as short as 32 base pairs. However, such a small DNA molecule could not form a circular form and be replicated by cellular enzymes. These facts may lead to an ostensibly paradoxical conclusion that the size of a minimal genome is restricted by the physical size of a DNA molecule able to replicate rather, than by the amount of genetic information.     

Mobile genetic elements colonizing the genomes of metazoan parasites

A substantial fraction of the genome of most eukaryotes, including those of metazoan parasites, is predicted to comprise repetitive sequences. Mobile genetic elements (MGEs) will make up much of these repetitive sequences, particularly the interspersed sequences. This article reviews information on MGEs that have colonized the genomes of metazoan parasites (i.e. parasites of parasites). Helminth and mosquito genomes, in particular, are compared with those of better-understood model organisms. MGEs from the genomes of metazoan parasites can be expected to have practical uses in transgenesis and epidemiological studies.     

Genetic Characterization of ExPEC-Like Virulence Plasmids among a Subset of NMEC

Neonatal Meningitis Escherichia coli (NMEC) is one of the most common causes of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or neurologic deficits in survivors. Large plasmids have been shown experimentally to increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9 ExPEC-like plasmids were isolated from NMEC and sequenced to identify the core and accessory plasmid genes of ExPEC-like virulence plasmids in NMEC and create an expanded plasmid phylogeny. Results showed sequenced virulence plasmids carry a strongly conserved core of genes with predicted functions in five distinct categories including: virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The major functions of virulence-associated and plasmid core genes serve to increase in vivo fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate NMEC’s survival in the host’s low iron environment, and systems to enhance bacterial resistance to host innate immunity. Phylogenetic analysis based on these core plasmid genes showed that at least two lineages of ExPEC-like plasmids could be discerned. Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could not be differentiated based solely on the genes of the core plasmid genome.     

Long-term episomal maintenance of bovine papillomavirus type 1 plasmids is determined by attachment to host chromosomes, which Is mediated by the viral E2 protein and its binding sites

Papillomavirus genomes are stably maintained as extrachromosomal nuclear plasmids in dividing host cells. To address the mechanisms responsible for stable maintenance of virus, we examined nuclear compartmentalization of plasmids containing the full-length upstream regulatory region (URR) from the bovine papillomavirus type 1 (BPV1) genome. We found that these plasmids are tightly associated with the nuclear chromatin both in the stable cell lines that maintain episomal copies of the plasmids and in transiently transfected cells expressing the viral E1 and E2 proteins. Further analysis of viral factors revealed that the E2 protein in trans and its multiple binding sites in cis are both necessary and sufficient for the chromatin attachment of the plasmids. On the other hand, the BPV1 URR-dependent plasmid replication and chromatin attachment processes are clearly independent of each other. The ability of the plasmids to stably maintain episomes correlates clearly with their chromatin association function. These data suggest that viral E2 protein-mediated attachment of BPV1 genomes to the host cell chromatin could provide a mechanism for the coupling of viral genome multiplication and partitioning to the host cell cycle during viral latent infection.     

Rhizobial plasmids — replication,structure and biological role

Soil bacteria, collectively named rhizobia, can establish mutualistic relationships with legume plants. Rhizobia often have multipartite genome architecture with a chromosome and several extrachromosomal replicons making these bacteria a perfect candidate for plasmid biology studies. Rhizobial plasmids are maintained in the cells using a tightly controlled and uniquely organized replication system. Completion of several rhizobial genome-sequencing projects has changed the view that their genomes are simply composed of the chromosome and cryptic plasmids. The genetic content of plasmids and the presence of some important (or even essential) genes contribute to the capability of environmental adaptation and competitiveness with other bacteria. On the other hand, their mosaic structure results in the plasticity of the genome and demonstrates a complex evolutionary history of plasmids. In this review, a genomic perspective was employed for discussion of several aspects regarding rhizobial plasmids comprising structure, replication, genetic content, and biological role. A special emphasis was placed on current post-genomic knowledge concerning plasmids, which has enriched the view of the entire bacterial genome organization by the discovery of plasmids with a potential chromosome-like role.     

Adaptive plasmid evolution results in host-range expansion of a broad-host-range plasmid

Little is known about the range of hosts in which broad-host-range (BHR) plasmids can persist in the absence of selection for plasmid-encoded traits, and whether this "long-term host range" can evolve over time. Previously, the BHR multidrug resistance plasmid pB10 was shown to be highly unstable in Stenotrophomonas maltophilia P21 and Pseudomonas putida H2. To investigate whether this plasmid can adapt to such unfavorable hosts, we performed evolution experiments wherein pB10 was maintained in strain P21, strain H2, and alternatingly in P21 and H2. Plasmids that evolved in P21 and in both hosts showed increased stability and decreased cost in ancestral host P21. However, the latter group showed higher variability in stability patterns, suggesting that regular switching between distinct hosts hampered adaptive plasmid evolution. The plasmids evolved in P21 were also equally or more stable in other hosts compared to pB10, which suggested true host-range expansion. The complete genome sequences of four evolved plasmids with improved stability showed only one or two genetic changes. The stability of plasmids evolved in H2 improved only in their coevolved hosts, not in the ancestral host. Thus a BHR plasmid can adapt to an unfavorable host and thereby expand its long-term host range.