In Situ Phylogenetic Structure and Diversity of Wild Bradyrhizobium Communities

Bacteria often infect their hosts from environmental sources, but little is known about how environmental and host-infecting populations are related. Here, phylogenetic clustering and diversity were investigated in a natural community of rhizobial bacteria from the genus Bradyrhizobium. These bacteria live in the soil and also form beneficial root nodule symbioses with legumes, including those in the genus Lotus. Two hundred eighty pure cultures of Bradyrhizobium bacteria were isolated and genotyped from wild hosts, including Lotus angustissimus, Lotus heermannii, Lotus micranthus, and Lotus strigosus. Bacteria were cultured directly from symbiotic nodules and from two microenvironments on the soil-root interface: root tips and mature (old) root surfaces. Bayesian phylogenies of Bradyrhizobium isolates were reconstructed using the internal transcribed spacer (ITS), and the structure of phylogenetic relatedness among bacteria was examined by host species and microenvironment. Inoculation assays were performed to confirm the nodulation status of a subset of isolates. Most recovered rhizobial genotypes were unique and found only in root surface communities, where little bacterial population genetic structure was detected among hosts. Conversely, most nodule isolates could be classified into several related, hyper-abundant genotypes that were phylogenetically clustered within host species. This pattern suggests that host infection provides ample rewards to symbiotic bacteria but that host specificity can strongly structure only a small subset of the rhizobial community.Symbiotic bacteria often encounter hosts from environmental sources (32, 48, 60), which leads to multipartite life histories including host-inhabiting and environmental stages. Research on host-associated bacteria, including pathogens and beneficial symbionts, has focused primarily on infection and proliferation in hosts, and key questions about the ecology and evolution of the free-living stages have remained unanswered. For instance, is host association ubiquitous within a bacterial lineage, or if not, do host-infecting genotypes represent a phylogenetically nonrandom subset? Assuming that host infection and free-living existence exert different selective pressures, do bacterial lineages diverge into specialists for these different lifestyles? Another set of questions addresses the degree to which bacteria associate with specific host partners. Do bacterial genotypes invariably associate with specific host lineages, and is such specificity controlled by one or both partners? Alternatively, is specificity simply a by-product of ecological cooccurrence among bacteria and hosts?Rhizobial bacteria comprise several distantly related proteobacterial lineages, most notably the genera Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium (52), that have acquired the ability to form nodules on legumes and symbiotically fix nitrogen. Acquisition of nodulation and nitrogen fixation loci has likely occurred through repeated lateral transfer of symbiotic loci (13, 74). Thus, the term “rhizobia” identifies a suite of symbiotic traits in multiple genomic backgrounds rather than a taxonomic classification. When rhizobia infect legume hosts, they differentiate into specialized endosymbiotic cells called bacteroids, which reduce atmospheric nitrogen in exchange for photosynthates from the plant (35, 60). Rhizobial transmission among legume hosts is infectious. Rhizobia can spread among hosts through the soil (60), and maternal inheritance (through seeds) is unknown (11, 43, 55). Nodule formation on hosts is guided by reciprocal molecular signaling between bacteria and plant (5, 46, 58), and successful infection requires a compatible pairing of legume and rhizobial genotypes. While both host and symbiont genotypes can alter the outcome of rhizobial competition for adsorption (34) and nodulation (33, 39, 65) of legume roots, little is known about how this competition plays out in nature.Rhizobia can achieve reproductive success via multiple lifestyles (12), including living free in the soil (14, 44, 53, 62), on or near root surfaces (12, 18, 19, 51), or in legume nodules (60). Least is known about rhizobia in bulk soil (not penetrated by plant roots). While rhizobia can persist for years in soil without host legumes (12, 30, 61), it appears that growth is often negligible in bulk soil (4, 10, 14, 22, 25). Rhizobia can also proliferate in the rhizosphere (soil near the root zone) of legumes (4, 10, 18, 19, 22, 25, 51). Some rhizobia might specialize in rhizosphere growth and infect hosts only rarely (12, 14, 51), whereas other genotypes are clearly nonsymbiotic because they lack key genes (62) and must therefore persist in the soil. The best-understood rhizobial lifestyle is the root nodule symbiosis with legumes, which is thought to offer fitness rewards that are superior to life in the soil (12). After the initial infection, nodules grow and harbor increasing populations of bacteria until the nodules senesce and the rhizobia are released into the soil (11, 12, 38, 40, 55). However, rhizobial fitness in nodules is not guaranteed. Host species differ in the type of nodules they form, and this can determine the degree to which differentiated bacteroids can repopulate the soil (11, 12, 38, 59). Furthermore, some legumes can hinder the growth of nodules with ineffective rhizobia, thus punishing uncooperative symbionts (11, 27, 28, 56, 71).Here, we investigated the relationships between environmental and host-infecting populations of rhizobia. A main objective was to test the hypothesis that rhizobia exhibit specificity among host species as well as among host microenvironments, specifically symbiotic nodules, root surfaces, and root tips. We predicted that host infection and environmental existence exert different selective pressures on rhizobia, leading to divergent patterns of clustering, diversity, and abundance of rhizobial genotypes.     

Characterization of the Replication,Transfer, and Plasmid/Lytic Phage Cycle of the Streptomyces Plasmid-Phage pZL12

We report here the isolation and recombinational cloning of a large plasmid, pZL12, from endophytic Streptomyces sp. 9R-2. pZL12 comprises 90,435 bp, encoding 112 genes, 30 of which are organized in a large operon resembling bacteriophage genes. A replication locus (repA) and a conjugal transfer locus (traA-traC) were identified in pZL12. Surprisingly, the supernatant of a 9R-2 liquid culture containing partially purified phage particles infected 9R-2 cured of pZL12 (9R-2X) to form plaques, and a phage particle (φZL12) was observed by transmission electron microscopy. Major structural proteins (capsid, portal, and tail) of φZL12 virions were encoded by pZL12 genes. Like bacteriophage P1, linear φZL12 DNA contained ends from a largely random pZL12 sequence. There was also a hot end sequence in linear φZL12. φZL12 virions efficiently infected only one host, 9R-2X, but failed to infect and form plaques in 18 other Streptomyces strains. Some 9R-2X spores rescued from lysis by infection of φZL12 virions contained a circular pZL12 plasmid, completing a cycle comprising autonomous plasmid pZL12 and lytic phage φZL12. These results confirm pZL12 as the first example of a plasmid-phage in Streptomyces.Streptomyces species, a major source of antibiotics and pharmacologically active metabolites, are Gram-positive, mycelial bacteria with high G+C content in their DNA (15). They usually harbor conjugative circular and/or linear plasmids, propagating in autonomous and/or chromosomally integrated forms (14). Most Streptomyces circular plasmids reported are small (8 to 14 kb), including rolling-circle-replication (RCR) plasmids (pIJ101, pJV1, pSG5, pSN22, pSVH1, pSB24.2, pSY10, pSNA1, pSLG33, pEN2701, etc.) (12, 14) and chromosomally integrating/autonomous plasmids (SLP1 and pSAM2) (4, 27, 28). Some theta replication plasmids are of intermediate size (31 to 39 kb), such as SCP2, pFP1, and pFP11 (13, 40). These theta replication loci comprise a rep gene and an adjacent noncoding or iteron sequence, to which Rep protein binds specifically in vitro (10, 40). The occurrence of an ∼163-kb large plasmid, pSV1, in Streptomyces violaceoruber SANK95570 was confirmed (1, 37), but this plasmid could not be physically isolated by standard procedures for plasmid preparation (17). In contrast to more than 30 genes for conjugal transfer on the Escherichia coli F plasmid (20), Streptomyces plasmids usually need a single tra gene (encoding a DNA translocase containing a cell division FtsK/SpoIIIE domain) (15, 29). The transfer of Streptomyces circular plasmids involves binding of the nonnicked double-stranded DNA (dsDNA) by multimers of Tra proteins at a noncoding sequence and ATP hydrolysis-dependent translocation of this DNA through the hyphal tips of the Streptomyces mycelium (15, 32).Numerous Streptomyces phages have been described, including φC31 (22), SAt1 (26), TG1 (11), FP43 (24), φSPK1 (19), φSC623 (34), DAH2/DAH4/DAH5/DAH6 (6), and mu1/6 (9). They range in size from 36 kb (19) to 121 kb (6), with 50 to 71.2% GC content (9, 23, 35). Streptomyces phages often have a wide host range; for example, 16 of 27 Streptomyces strains are susceptible to infection by φSPK1 (19), and phage FP43 transduces species of Streptoverticillium, Chainia, and Sacchropolyspora (24). φC31 is the most-studied Streptomyces phage and cloning vector (8). The sequences of the φC31 head proteins (e.g., portal, capsid, and head protease) resemble those of other bacterial dsDNA phages, suggesting evolutionary relationships to other viruses (35).We report here the isolation and recombinational cloning of a 90,435-bp plasmid, pZL12, from endophytic Streptomyces sp. 9R-2 and the characterization of its replication and transfer. Surprisingly, the supernatant of 9R-2 liquid culture infected 9R-2 cured of pZL12 to form plaques. A cycle comprising autonomous plasmid pZL12 and lytic phage φZL12 is described.     

Novel Antibiotic-Free Plasmid Selection System Based on Complementation of Host Auxotrophy in the NAD De Novo Synthesis Pathway

The use of antibiotic resistance genes in plasmids causes potential biosafety and clinical hazards, such as the possibility of horizontal spread of resistance genes or the rapid emergence of multidrug-resistant pathogens. This paper introduces a novel auxotrophy complementation system that allowed plasmids and host cells to be effectively selected and maintained without the use of antibiotics. An Escherichia coli strain carrying a defect in NAD de novo biosynthesis was constructed by knocking out the chromosomal quinolinic acid phosphoribosyltransferase (QAPRTase) gene. The resistance gene in the plasmids was replaced by the QAPRTase gene of E. coli or the mouse. As a result, only expression of the QAPRTase gene from plasmids can complement and rescue E. coli host cells in minimal medium. This is the first time that a vertebrate gene has been used to construct a nonantibiotic selection system, and it can be widely applied in DNA vaccine and gene therapy. As the QAPRTase gene is ubiquitous in species ranging from bacteria to mammals, the potential environmental biosafety problems caused by horizontal gene transfer can be eliminated.Antibiotic resistance genes are the most commonly used markers for selecting and maintaining recombinant plasmids in hosts, such as Escherichia coli. However, the use of these genes has several drawbacks. For example, horizontal transfer of the antibiotic resistance gene can potentially contribute to the rapid emergence of multidrug-resistant organisms (e.g., superbacteria) (11, 29). Another significant concern is that the antibiotic resistance genes in DNA vaccines may become integrated into human chromosomes (23). The possibility arises, although the probability is low, that once the antibiotic resistance gene is translated into a functional protein, the vaccinee might be resistant to the corresponding antibiotic. This would add to the difficulty of curing diseases caused by infectious pathogens. Accordingly, the use of antibiotic resistance genes is undesirable in many areas of biotechnology, especially in gene therapy products and genetically engineered microorganisms (17, 23, 28). Furthermore, the addition of antibiotics is costly in large-scale cultivation, and there are risks of contamination of the final product with antibiotics (2, 3). Finally, the constitutively expressed antibiotic resistance genes impose a metabolic burden on the host cells, resulting in reduced growth rate and cell density (4, 27). An alternative strategy is to utilize antibiotic-free host-plasmid balanced lethal systems to select and maintain the recombinant plasmids.To date, several such systems have been developed to replace traditional antibiotic selection systems. They include auxotrophy complementation (AC), postsegregational killing (PSK), and operator-repressor titration (ORT) (8). The AC system is based on a strain auxotrophic for an essential metabolite, obtained by mutating or knocking out the corresponding chromosomal gene, which can be complemented with the plasmid-borne selection gene. The choice of the essential gene used for complementation of host auxotrophy is critical, and it is mainly involved in DNA precursor, amino acid, or cell wall biosynthetic pathways. Various essential genes, such as asd, thyA, and glnA, have been utilized to construct AC systems (5, 9, 21, 22, 24, 26, 28). However, all of these systems require extra nutrients or expensive reagents. The PSK system relies on the balance between toxin and antitoxin, expressed from genome and plasmid, respectively. If a cell loses the plasmid, the corresponding antitoxin is degraded and the toxin then kills the cell. Unfortunately, this system has proven ineffective for plasmid maintenance during prolonged culture (6, 14). The ORT system utilizes plasmids with the lac operator to derepress a modified essential chromosomal gene. Loss of these types of plasmids no longer titrates the repressor and leads to the death of the bacterium. This system requires short, nonexpressed lac operator functions as the vector-borne selection marker and enables the selection and maintenance of plasmids free from expressed selectable marker genes (7, 8, 15, 30). Additionally, several other nonantibiotic selection systems (e.g., the fabI-triclosan system) have recently been developed (12, 17, 18).Among the antibiotic-free selection systems that have been developed, the AC system has drawn much attention and has now been applied in numerous bacterial species, such as Lactococcus lactis, Salmonella spp., Vibrio cholerae, Mycobacterium bovis, and E. coli (5, 16, 21, 22, 24). However, all of the AC systems utilize plasmid-borne bacterial-origin genes to complement the auxotrophy. These systems may suffer from a potential risk that the bacterial-origin genes may be integrated into human chromosome when they are used in transgenic products, such as DNA vaccines. Therefore, a better strategy would be to use the genes of the vaccinees themselves to construct an AC system. Not only would this type of approach select and maintain plasmids in bacteria, but it could also be widely applied in the production of safer DNA vaccines.In the present study, we successfully developed a novel antibiotic-free plasmid selection system based on complementation of host auxotrophy in the NAD synthesis pathway. The NAD synthesis pathway, including de novo and salvage pathways, differs among species. However, by comparison of NAD metabolism in different species, quinolinic acid phosphoribosyltransferase (QAPRTase) appears to be a common enzyme for de novo NAD biosynthesis in both prokaryotes and eukaryotes (13). Therefore, the QAPRTase gene was viewed as a favorable candidate that could potentially be utilized to construct a new AC system.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Identification of Diverse Antimicrobial Resistance Determinants Carried on Bacterial,Plasmid, or Viral Metagenomes from an Activated Sludge Microbial Assemblage

Using both sequence- and function-based metagenomic approaches, multiple antibiotic resistance determinants were identified within metagenomic libraries constructed from DNA extracted from bacterial chromosomes, plasmids, or viruses within an activated sludge microbial assemblage. Metagenomic clones and a plasmid that in Escherichia coli expressed resistance to chloramphenicol, ampicillin, or kanamycin were isolated, with many cloned DNA sequences lacking any significant homology to known antibiotic resistance determinants.Activated sludge in wastewater treatment plants is an open system with a dynamic and phylogenetically diverse microbial community (2, 3, 6, 7, 10, 11). Since the activated sludge process promotes cellular interactions among diverse microorganisms, there is great potential for the lateral transfer of antibiotic resistance genes between microbes in activated sludge and in downstream environments. Several studies have previously identified antibiotic resistance determinants from wastewater communities that are carried on bacterial chromosomes (1, 4, 14) and plasmids (9, 12, 13), but to our knowledge, a simultaneous metagenomic survey of antibiotic resistance determinants from all three genetic reservoirs (i.e., chromosomes, plasmids, and viruses) has never been performed within the same environment. To achieve a more comprehensive assessment of antibiotic resistance genes in the activated sludge microbial community, this study used both function- and sequence-based metagenomic approaches to identify antibiotic resistance determinants carried on bacterial chromosomes, plasmids, or viruses within an activated sludge microbial assemblage.     

A Bacillus anthracis S-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC

The sequestration of iron by mammalian hosts represents a significant obstacle to the establishment of a bacterial infection. In response, pathogenic bacteria have evolved mechanisms to acquire iron from host heme. Bacillus anthracis, the causative agent of anthrax, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, including B. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Here, we present evidence that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation of B. anthracis to changing iron reservoirs during an infection. Understanding the mechanism of heme uptake in pathogenic bacteria is important for the development of novel therapeutics to prevent and treat bacterial infections.Pathogenic bacteria need to acquire iron to survive in mammalian hosts (12). However, the host sequesters most iron in the porphyrin heme, and heme itself is often bound to proteins such as hemoglobin (14, 28, 85). Circulating hemoglobin can serve as a source of heme-iron for replicating bacteria in infected hosts, but the precise mechanisms of heme extraction, transport, and assimilation remain unclear (25, 46, 79, 86). An understanding of how bacterial pathogens import heme will lead to the development of new anti-infectives that inhibit heme uptake, thereby preventing or treating infections caused by these bacteria (47, 68).The mechanisms of transport of biological molecules into a bacterial cell are influenced by the compositional, structural, and topological makeup of the cell envelope. Gram-negative bacteria utilize specific proteins to transport heme through the outer membrane, periplasm, and inner membrane (83, 84). Instead of an outer membrane and periplasm, Gram-positive bacteria contain a thick cell wall (59, 60). Proteins covalently anchored to the cell wall provide a functional link between extracellular heme reservoirs and intracellular iron utilization pathways (46). In addition, several Gram-positive and Gram-negative bacterial genera also contain an outermost structure termed the S (surface)-layer (75). The S-layer is a crystalline array of protein that surrounds the bacterial cell and may serve a multitude of functions, including maintenance of cell architecture and protection from host immune components (6, 7, 18, 19, 56). In bacterial pathogens that manifest an S-layer, the “force field” function of this structure raises questions concerning how small molecules such as heme can be successfully passed from the extracellular milieu to cell wall proteins for delivery into the cell cytoplasm.Bacillus anthracis is a Gram-positive, spore-forming bacterium that is the etiological agent of anthrax disease (30, 33). The life cycle of B. anthracis begins after a phagocytosed spore germinates into a vegetative cell inside a mammalian host (2, 40, 69, 78). Virulence determinants produced by the vegetative cells facilitate bacterial growth, dissemination to major organ systems, and eventually host death (76-78). The release of aerosolized spores into areas with large concentrations of people is a serious public health concern (30).Heme acquisition in B. anthracis is mediated by the action of IsdX1 and IsdX2, two extracellular hemophores that extract heme from host hemoglobin and deliver the iron-porphyrin to cell wall-localized IsdC (21, 45). Both IsdX1 and IsdX2 harbor near iron transporter domains (NEATs), a conserved protein module found in Gram-positive bacteria that mediates heme uptake from hemoglobin and contributes to bacterial pathogenesis upon infection (3, 8, 21, 31, 44, 46, 49, 50, 67, 81, 86). Hypothesizing that B. anthracis may contain additional mechanisms for heme transport, we provide evidence that B. anthracis S-layer protein K (BslK), an S-layer homology (SLH) and NEAT protein (32, 43), is surface localized and binds and transfers heme to IsdC in a rapid, contact-dependent manner. These results suggest that the Isd system is not a self-contained conduit for heme trafficking and imply that there is functional cross talk between differentially localized NEAT proteins to promote heme uptake during infection.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Wide Dispersal and Possible Multiple Origins of Low-Copy-Number Plasmids in Rickettsia Species Associated with Blood-Feeding Arthropods

Plasmids are mobile genetic elements of bacteria that can impart important adaptive traits, such as increased virulence or antibiotic resistance. We report the existence of plasmids in Rickettsia (Rickettsiales; Rickettsiaceae) species, including Rickettsia akari, “Candidatus Rickettsia amblyommii,” R. bellii, R. rhipicephali, and REIS, the rickettsial endosymbiont of Ixodes scapularis. All of the rickettsiae were isolated from humans or North and South American ticks. R. parkeri isolates from both continents did not possess plasmids. We have now demonstrated plasmids in nearly all Rickettsia species that we have surveyed from three continents, which represent three of the four major proposed phylogenetic groups associated with blood-feeding arthropods. Gel-based evidence consistent with the existence of multiple plasmids in some species was confirmed by cloning plasmids with very different sequences from each of two “Ca. Rickettsia amblyommii” isolates. Phylogenetic analysis of rickettsial ParA plasmid partitioning proteins indicated multiple parA gene origins and plasmid incompatibility groups, consistent with possible multiple plasmid origins. Phylogenetic analysis of potentially host-adaptive rickettsial small heat shock proteins showed that hsp2 genes were plasmid specific and that hsp1 genes, found only on plasmids of “Ca. Rickettsia amblyommii,” R. felis, R. monacensis, and R. peacockii, were probably acquired independently of the hsp2 genes. Plasmid copy numbers in seven Rickettsia species ranged from 2.4 to 9.2 per chromosomal equivalent, as determined by real-time quantitative PCR. Plasmids may be of significance in rickettsial evolution and epidemiology by conferring genetic plasticity and host-adaptive traits via horizontal gene transfer that counteracts the reductive genome evolution typical of obligate intracellular bacteria.The alphaproteobacteria of the genus Rickettsia (Rickettsiales; Rickettsiaceae) have undergone the reductive genome evolution typical of obligate intracellular bacteria, resulting in A/T-rich genomes (1.1 × 10⁶ to 1.5 × 10⁶ bp) with a high content of pseudogenes undergoing elimination (3, 10, 20, 26). Initial sequencing of rickettsial genomes focused on the important arthropod-borne pathogens Rickettsia prowazekii, Rickettsia conorii, and Rickettsia typhi and appeared to confirm the prevailing belief that plasmids were absent and transposons were rare among Rickettsia spp. (2, 28, 39, 44). As mobile genetic elements in bacteria, plasmids and transposons drive horizontal gene transfer (HGT) and the acquisition of virulence determinants and environmental adaptive traits (30, 43, 60, 70). Subsequent sequencing of the Rickettsia felis genome revealed the surprising presence of abundant transposase paralogs and the 63-kbp pRF plasmid, with 68 open reading frames (ORFs) encoding predicted proteins, as well as a 39-kbp deletion form, pRFδ (45). Although pRF was suggested to be conjugative, it was initially thought to be unique among the rickettsiae, a reasonable inference given that plasmids are uncommon among the reduced genomes of obligate intracellular bacteria and were previously unknown in the Rickettsiales (3, 4, 13). However, a phylogenetic analysis implied an origin for pRF in ancestral rickettsiae and the possible existence of other rickettsial plasmids (28), which was soon confirmed by the cloning of the 23.5-kbp pRM plasmid from Rickettsia monacensis (6). Some of the 23 ORFs on pRM had close pRF homologs, and both plasmids carried transposon genes and the molecular footprints of transposition events associated with HGT from other bacterial taxa.The discoveries of pRF and pRM made obsolete the long-held dogma that plasmids were not present in members of the genus Rickettsia and implied a source of unexpected genetic diversity in the reduced rickettsial genomes, particularly if potentially conjugative plasmids carrying transposon genes proved to be common among members of the genus. That hypothesis gained credence when pulsed-field gel electrophoresis (PFGE) and Southern blot surveys (7) using plasmid gene-specific probes demonstrated plasmids in Rickettsia helvetica, “Candidatus Rickettsia hoogstraalii” (38), and Rickettsia massiliae and possible multiple plasmids in “Candidatus Rickettsia amblyommii” (71) isolates. The same study demonstrated the loss of a plasmid in the nonpathogenic species Rickettsia peacockii during long-term serial passage in cultured cells and the absence of a plasmid in Rickettsia montanensis M5/6, an isolate with a long laboratory passage history. Genome sequencing of R. massiliae and Rickettsia africae revealed the 15.3-kbp pRMA and 12.4-kbp pRAF sequences, with 12 and 11 ORFs, respectively, that were more similar to those of pRF than to those of pRM (11, 24).The absence of plasmids in R. montanensis and important Rickettsia pathogens maintained as laboratory isolates has left unresolved the question of the true extent of plasmid distribution among Rickettsia spp. Until recently, the genus was thought to consist of closely related species, known chiefly as typhus and spotted fever pathogens transmitted by lice, fleas, mites, and ticks (31). It is now apparent that many, and possibly most, Rickettsia spp. inhabit a diverse range of arthropods that do not feed on blood, as well as leeches, helminths, crustaceans, and protozoans, suggesting an ancient and complex evolutionary history (54). A multigene phylogenetic analysis of the Rickettsiales resulted in a “molecular clock” which indicated that the order arose from a presumably free-living ancestor and then adapted to intracellular growth during the appearance of metazoan phyla in the Cambrian explosion (76). A transition to a primary association with arthropods followed during the Ordovician and Silurian periods. The genus Rickettsia arose approximately 150 million years ago and evolved into several clades, including the early-diverging hydra and torix lineages associated with leeches and protozoans. A rapid radiation occurred about 50 million years ago in the arthropod-associated lineages (76).Whole-genome sequencing has led to a revision of phylogenetic relationships among Rickettsia spp. associated with blood-feeding arthropods (10, 26, 28). A newly defined ancestral group (AG) contains the earliest-diverging species, Rickettsia bellii and Rickettsia canadensis, while R. prowazekii and R. typhi, transmitted by lice and fleas, respectively, constitute the typhus group (TG). A proposed transitional group (TRG), consisting of the mite-borne Rickettsia akari, the flea-borne R. felis, and the tick-borne Rickettsia australis, bridges the genotypic and phenotypic differences between the TG and the much larger spotted fever group (SFG), consisting of tick-borne rickettsiae (28). However, some presumptive SFG rickettsiae remain poorly characterized and are of uncertain phylogenetic status, while the accumulation of genomic data from rickettsiae found in a diverse range of invertebrate hosts may have profound impacts on the currently understood phylogeny of rickettsiae associated with blood-feeding arthropods. For example, it appears that the above AG and TRG species have many close relatives in insects (76). Despite the recent phylogenomic advances, the genetic and host-adaptive mechanisms underlying the evolution of arthropod-transmitted pathogens of vertebrates from ancestral Rickettsia spp., including any possible role of plasmids, remain poorly understood.In this report, we have taken advantage of recent isolations of rickettsiae from North and South America to conclusively demonstrate that low-copy-number plasmids are indeed common in low-passage isolates of AG, TRG, and SFG rickettsiae. The only exceptions were multiple isolates of R. parkeri, obtained from ticks and human eschar biopsy specimens and newly recognized as a mildly pathogenic SFG rickettsia (49, 50, 52, 79), and the previously characterized species R. montanensis (7). We confirmed that some Rickettsia isolates harbor more than one plasmid by cloning and sequencing multiple plasmids from “Ca. Rickettsia amblyommii” isolates AaR/SC and Ac/Pa, and we obtained PCR- and gel-based evidence that supported genome sequence evidence for the existence of multiple plasmids in REIS, the rickettsial endosymbiont of Ixodes scapularis. Phylogenetic analysis provided strong evidence for multiple plasmid incompatibility groups and possible multiple origins of plasmid-carried parA genes in the genus Rickettsia. Other than genes encoding plasmid replication initiation and partitioning proteins, the newly sequenced “Ca. Rickettsia amblyommii” plasmids resembled the previously sequenced rickettsial plasmids in sharing limited similarities in coding capacity (6, 7, 22). However, we have previously drawn attention to the presence of hsp genes, encoding α-crystalline small heat shock proteins, as a conserved feature of most rickettsial plasmids that may play a role in host adaptation (7). Phylogenetic analysis indicated that the hsp2 genes were plasmid specific, while the hsp1 genes found on four rickettsial plasmids may have been acquired by a chromosome-to-plasmid transfer event in a TRG-like species.     

Novel Toxin-Antitoxin System Composed of Serine Protease and AAA-ATPase Homologues Determines the High Level of Stability and Incompatibility of the Tumor-Inducing Plasmid pTiC58

Stability of plant tumor-inducing (Ti) plasmids differs among strains. A high level of stability prevents basic and applied studies including the development of useful strains. The nopaline type Ti plasmid pTiC58 significantly reduces the transconjugant efficiency for incoming incompatible plasmids relative to the other type, such as octopine-type plasmids. In this study we identified a region that increases the incompatibility and stability of the plasmid. This region was located on a 4.3-kbp segment about 38 kbp downstream of the replication locus, repABC. We named two open reading frames in the segment, ietA and ietS, both of which were essential for the high level of incompatibility and stability. Plasmid stabilization by ietAS was accomplished by a toxin-antitoxin (TA) mechanism, where IetS is the toxin and IetA is the antitoxin. A database search revealed that putative IetA and IetS proteins are highly similar to AAA-ATPases and subtilisin-like serine proteases, respectively. Amino acid substitution experiments in each of the highly conserved characteristic residues, in both putative enzymes, suggested that the protease activity is essential and that ATP binding activity is important for the operation of the TA system. The ietAS-containing repABC plasmids expelled Ti plasmids even in strains which were tolerant to conventional Ti-curing treatments.Agrobacterium tumefaciens strains bearing a tumor-inducing (Ti) plasmid are the etiological agents of crown gall disease. Most genes required for pathogenicity are located on the plasmids (17, 33). Ti plasmids are kept stable at a low copy number equivalent to that of the chromosomal DNA in the bacterial cells (32) due to the repABC locus (16, 30, 34). The stability of Ti plasmids differs among strains (11).Many genes for keeping plasmids stable have been reported in eubacteria, and these are divided into three categories based on their mechanism: multimer resolution systems, active partitioning systems, and toxin-antitoxin (TA) systems (15). Multimer resolution systems increase the number of plasmid molecules by resolving a multimer plasmid into monomers, resulting in a higher probability of plasmid distribution to daughter cells during cell division even when plasmid distribution occurs randomly (29). Active partitioning systems deliver plasmid copies to each progeny cell at cell division (21). In the repABC locus, the RepA and RepB proteins and parS site(s) ensure stable plasmid inheritance by the active partitioning system (2). TA systems contribute to plasmid maintenance in cell populations by initiating growth inhibition or death of plasmid-free cells and are widely distributed among eubacterial and archaeal plasmids as well as their chromosomes (9). Generally, the TA module consists of two genes which encode toxin and antitoxin. The antitoxin neutralizes the action of a cognate toxin by interaction with the toxin or its target molecules. When a plasmid harboring the TA module is lost from a host cell, the antitoxin molecules decrease to an ineffective level because the antitoxin is degraded quickly or diluted by cell division (15). Thereafter, the toxin exerts its toxicity and inhibits the host cell growth. RNA antitoxins can suppress toxin expression by binding to the toxin mRNA as an antisense RNA or repress toxicity effects by an unknown mechanism (6, 4). In pTi-SAKURA, the Ti plasmid in the A. tumefaciens strain MAFF301001, it was shown that the tiorf24 and tiorf25 module increased plasmid stability by the TA mechanism (40; also S. Yamamoto, unpublished data).Differences in Ti plasmid stability are critical for plasmid engineering and evolution (33). However, little is known about the stability factors of Ti plasmids other than the repABC locus. In our previous study (40), tiorf24 and tiorf25 were shown to increase the segregational stability and incompatibility of Ti plasmids and reduce the efficiency of transconjugants by the introduction of incompatible plasmids into host cells. The two genes are located 2.5-kbp downstream of repABC (8). Generally, incompatibility has been defined as a situation where two plasmids contain a related replication and/or partitioning system and are unable to exist in a cell simultaneously without external selection (1). A. tumefaciens strain C58, which contains a Ti plasmid pTiC58, allows entry of an incompatible repABC plasmid into the cell 60-fold less efficiently than a derivative of C58. The derivative of C58 harbors a small repABC vector instead of pTiC58 (40). This suggests the presence of incompatibility-enhancing genes on pTiC58.In this study, we located the responsible genes in pTiC58 and found that the novel genes ietA and ietS enhance the incompatibility and stability of the plasmid by the TA mechanism.     

A Novel Genotype H9N2 Influenza Virus Possessing Human H5N1 Internal Genomes Has Been Circulating in Poultry in Eastern China since 1998

Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.Wild birds, including wild waterfowls, gulls, and shorebirds, are the natural reservoirs for influenza A viruses, in which they are thought to be in evolutionary stasis (2, 33). However, when avian influenza viruses are transmitted to new hosts such as terrestrial poultry or mammals, they evolve rapidly and may cause occasional severe systemic infection with high morbidity (20, 29). Despite the fact that avian influenza virus infection occurs commonly in chickens, it is unable to persist for a long period of time due to control efforts and/or a failure of the virus to adapt to new hosts (29). In the past 20 years, greater numbers of outbreaks in poultry have occurred, suggesting that the avian influenza virus can infect and spread in aberrant hosts for an extended period of time (5, 14-16, 18, 32).During the past 10 years, H9N2 influenza viruses have become panzootic in Eurasia and have been isolated from outbreaks in poultry worldwide (3, 5, 11, 14-16, 18, 24). A great deal of previous studies demonstrated that H9N2 influenza viruses have become established in terrestrial poultry in different Asian countries (5, 11, 13, 14, 18, 21, 24, 35). In 1994, H9N2 viruses were isolated from diseased chickens in Guangdong province, China, for the first time (4), and later in domestic poultry in other provinces in China (11, 16, 18, 35). Two distinct H9N2 virus lineages represented by A/Chicken/Beijing/1/94 (H9N2) and A/Quail/Hong Kong/G1/98 (H9N2), respectively, have been circulating in terrestrial poultry of southern China (9). Occasionally these viruses expand their host range to other mammals, including pigs and humans (6, 17, 22, 34). Increasing epidemiological and laboratory findings suggest that chickens may play an important role in expanding the host range for avian influenza virus. Our systematic surveillance of influenza viruses in chickens in China showed that H9N2 subtype influenza viruses continued to be prevalent in chickens in mainland China from 1994 to 2008 (18, 19, 36).Eastern China contains one metropolitan city (Shanghai) and five provinces (Jiangsu, Zhejiang, Anhui, Shandong, and Jiangxi), where domestic poultry account for approximately 50% of the total poultry population in China. Since 1996, H9N2 influenza viruses have been isolated regularly from both chickens and other minor poultry species in our surveillance program in the eastern China region, but their genetic diversity and the interrelationships between H9N2 influenza viruses and different types of poultry have not been determined. Therefore, it is imperative to explore the evolution and properties of these viruses. The current report provides insight into the genesis and evolution of H9N2 influenza viruses in eastern China and presents new evidence for the potential crossover between H9N2 and H5N1 influenza viruses in this region.     

Conjugative Plasmid Transfer and Adhesion Dynamics in an Escherichia coli Biofilm

A conjugative plasmid from the catheter-associated urinary tract infection strain Escherichia coli MS2027 was sequenced and annotated. This 42,644-bp plasmid, designated pMAS2027, contains 58 putative genes and is most closely related to plasmids belonging to incompatibility group X (IncX1). Plasmid pMAS2027 encodes two important virulence factors: type 3 fimbriae and a type IV secretion (T4S) system. Type 3 fimbriae, recently found to be functionally expressed in E. coli, played an important role in biofilm formation. Biofilm formation by E. coli MS2027 was specifically due to expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027 and enabled a non-biofilm-forming strain to grow as part of a mixed biofilm following acquisition of this plasmid. Thus, the importance of conjugation as a mechanism to spread biofilm determinants was demonstrated. Conjugation may represent an important mechanism by which type 3 fimbria genes are transferred among the Enterobacteriaceae that cause device-related infections in nosocomial settings.Bacterial biofilms are complex communities of bacterial cells living in close association with a surface (17). Bacterial cells in these protected environments are often resistant to multiple factors, including antimicrobials, changes in the pH, oxygen radicals, and host immune defenses (19, 38). Biofilm formation is a property of many bacterial species, and a range of molecular mechanisms that facilitate this process have been described (2, 3, 11, 14, 16, 29, 33, 34). Often, the ability to form a biofilm is dependent on the production of adhesins on the bacterial cell surface. In Escherichia coli, biofilm formation is enhanced by the production of certain types of fimbriae (e.g., type 1 fimbriae, type 3 fimbriae, F1C, F9, curli, and conjugative pili) (14, 23, 25, 29, 33, 39, 46), cell surface adhesins (e.g., autotransporter proteins such as antigen 43, AidA, TibA, EhaA, and UpaG) (21, 34, 35, 40, 43), and flagella (22, 45).The close proximity of bacterial cells in biofilms creates an environment conducive for the exchange of genetic material. Indeed, plasmid-mediated conjugation in monospecific and mixed E. coli biofilms has been demonstrated (6, 18, 24, 31). The F plasmid represents the best-characterized conjugative system for biofilm formation by E. coli. The F pilus mediates adhesion to abiotic surfaces and stabilizes the biofilm structure through cell-cell interactions (16, 30). Many other conjugative plasmids also contribute directly to biofilm formation upon derepression of the conjugative function (16).One example of a conjugative system employed by gram-negative Enterobacteriaceae is the type 4 secretion (T4S) system. The T4S system is a multisubunit structure that spans the cell envelope and contains a secretion channel often linked to a pilus or other surface filament or protein (8). The Agrobacterium tumefaciens VirB-VirD4 system is the archetypical T4S system and is encoded by 11 genes in the virB operon and one gene (virD4) in the virD operon (7, 8). Genes with strong homology to genes in the virB operon have also been identified on other conjugative plasmids. For example, the pilX1 to pilX11 genes on the E. coli R6K IncX plasmid and the virB1 to virB11 genes are highly conserved at the nucleotide level (28).We recently described identification and characterization of the mrk genes encoding type 3 fimbriae in a uropathogenic strain of E. coli isolated from a patient with a nosocomial catheter-associated urinary tract infection (CAUTI) (29). The mrk genes were located on a conjugative plasmid (pMAS2027) and were strongly associated with biofilm formation. In this study we determined the entire sequence of plasmid pMAS2027 and revealed the presence of conjugative transfer genes homologous to the pilX1 to pilX11 genes of E. coli R6K (in addition to the mrk genes). We show here that biofilm formation is driven primarily by type 3 fimbriae and that the T4S apparatus is unable to mediate biofilm growth in the absence of the mrk genes. Finally, we demonstrate that conjugative transfer of pMAS2027 within a mixed biofilm confers biofilm formation properties on recipient cells due to acquisition of the type 3 fimbria-encoding mrk genes.     

Evolutionary Dynamics of Insertion Sequences in Relation to the Evolutionary Histories of the Chromosome and Symbiotic Plasmid Genes of Rhizobium etli Populations

Insertion sequences (IS) are mobile genetic elements that are distributed in many prokaryotes. In particular, in the genomes of the symbiotic nitrogen-fixing bacteria collectively known as rhizobia, IS are fairly abundant in plasmids or chromosomal islands that carry the genes needed for symbiosis. Here, we report an analysis of the distribution and genetic conservation of the IS found in the genome of Rhizobium etli CFN42 in a collection of 87 Rhizobium strains belonging to populations with different geographical origins. We used PCR to generate presence/absence profiles of the 39 IS found in R. etli CFN42 and evaluated whether the IS were located in consistent genomic contexts. We found that the IS from the symbiotic plasmid were frequently present in the analyzed strains, whereas the chromosomal IS were observed less frequently. We then examined the evolutionary dynamics of these strains based on a population genetic analysis of two chromosomal housekeeping genes (glyA and dnaB) and three symbiotic sequences (nodC and the two IS elements). Our results indicate that the IS contained within the symbiotic plasmid have a higher degree of genomic context conservation, lower nucleotide diversity and genetic differentiation, and fewer recombination events than the chromosomal housekeeping genes. These results suggest that the R. etli populations diverged recently in Mexico, that the symbiotic plasmid also had a recent origin, and that the IS elements have undergone a process of cyclic infection and expansion.Insertion sequences (IS) are the smallest transposable elements found in prokaryotes (usually less than 3 kb in size). They encode a transposase and may also encode small hypothetical proteins (4, 9). IS are distinguished by their ability to move within prokaryotic replicons, including both the chromosome and plasmids, and to copy themselves into various genomic sites. In this manner, IS elements can inactivate or alter the expression of adjacent genes (4). When IS occur in two or more identical copies within a genome, they can participate in various types of genetic rearrangements (e.g., duplications, inversions, and deletions), suggesting that IS may play an important role in the evolution of their hosts by promoting genomic plasticity (34). Due to these evolutionary dynamics, the diversity and distribution of IS elements differ greatly between taxa and even within strains of the same species (27).Various theories have been proposed to explain the evolution of IS elements in laboratory model strains and environmental bacterial populations (8, 18, 25, 29). Two main hypotheses seek to explain how these elements are maintained over the long term in their host genomes. The first proposes that they occasionally generate beneficial mutations and therefore may represent a selective advantage to their hosts (34). The second suggests that IS elements are genomic parasites that are maintained by their high rate of transposition and might be disseminated among different bacterial lineages by horizontal gene transfer (HGT). Data supporting the second hypothesis have shown that some IS elements may transpose at high rates upon entering a new host (42). After the initial infection, however, purifying selection may continuously remove these elements from the genome. Thus, IS may undergo an infection-expansion-extinction cycle that allows them to remain in different bacterial populations within the gene pool (42). These two hypotheses are not contradictory, and the evolutionary dynamics and distribution of IS may differ greatly depending on several factors, including (most notably) the rate of transposition and HGT, as well as selective pressures, population size, and the host''s habitat (6, 18, 21, 25, 27, 29).In the nitrogen-fixing symbiotic bacteria of the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium (of the alphaproteobacteria), Cupriavidus, and Burkholderia (of the betaproteobacteria), IS are particularly abundant in symbiotic plasmids (pSym) and symbiotic chromosomal islands (SI) (2, 12, 14, 19, 20, 43). SI and pSym include most of the genes needed to establish symbiosis in the roots of leguminous plants through nodule formation and nitrogen fixation (11). It is generally believed that these elements entered the rhizobial genomes through HGT (39, 40). Comparative genomic analyses have shown that both pSym and SI are highly variable, with the exception of a common set of genes encoding factors critical to nitrogen fixation (nif) and nodulation (nod) (5, 14). SI and pSym have been found to have lower GC contents and different codon usages than the corresponding chromosomal and nonsymbiotic plasmid sequences, suggesting that they were recently acquired by HGT.Some of these symbiotic elements, such as in pSym of Rhizobium etli CFN42 and the SI of Mesorhizobium loti, are conjugative and mobile (30, 32). Genomic analysis of R. etli CFN42 revealed the presence of 39 IS belonging to different families (14); they were found in the chromosome (11 IS); the 371-kb symbiotic plasmid (13 IS); the smaller 192-kb conjugative plasmid, p42a (13 IS); and two other plasmids, p42b and p42c (2 IS). Interestingly, this particular strain shows no evidence of IS disrupting open reading frames (ORFs) or having transpositional activity. However, another 42 incomplete IS may be found in the chromosome, pSym, and the conjugative plasmid; these incomplete sequences are truncated or contain stop codons in their coding sequences.Here, we focused on the dynamics and distribution of IS in different populations of the nitrogen-fixing symbiont R. etli. Since the maintenance of IS in bacterial species might depend on their transpositional activities and horizontal transfer rates, the identification of IS in the same genomic contexts across different strains of the same species could provide new insights into their persistence and divergence over short evolutionary periods. To examine the evolutionary dynamics of IS in natural populations of R. etli, we characterized the distributions, genomic contexts, and sequence variations of IS in isolates of R. etli from three populations with different origins, as well as in some other Rhizobium species. More specifically, we used PCR to generate presence/absence profiles of the 39 IS found in R. etli CFN42 in a collection of 87 strains representing different geographical sites and a gradient of domestication of the bacterial host, the common bean (Phaseolus vulgaris). We also evaluated whether the IS were conserved in the same genomic context relative to their position in R. etli CFN42 and determined the nucleotide sequences of two IS found in most of the isolates. Several population genetic tests applied to these IS, another pSym gene (nodC), and two chromosomal housekeeping genes (dnaB and glyA) suggest that these two IS elements have been inherited vertically and represent recent components of the R. etli gene pool. Finally, the present study strongly suggests that symbiotic plasmids have a recent origin within the R. etli populations.     

Rickettsia felis Infection in a Common Household Insect Pest,Liposcelis bostrychophila (Psocoptera: Liposcelidae)

Many species of Rickettsia are well-known mammalian pathogens transmitted by blood-feeding arthropods. However, molecular surveys are continually uncovering novel Rickettsia species, often in unexpected hosts, including many arthropods that do not feed on blood. This study reports a systematic molecular characterization of a Rickettsia infecting the psocid Liposcelis bostrychophila (Psocoptera: Liposcelidae), a common and cosmopolitan household pest. Surprisingly, the psocid Rickettsia is shown to be Rickettsia felis, a human pathogen transmitted by fleas that causes serious morbidity and occasional mortality. The plasmid from the psocid R. felis was sequenced and was found to be virtually identical to the one in R. felis from fleas. As Liposcelis insects are often intimately associated with humans and other vertebrates, it is speculated that they acquired R. felis from fleas. Whether the R. felis in psocids causes disease in vertebrates is not known and warrants further study.Many species of Rickettsia are well-known mammalian pathogens that are transmitted by blood-feeding arthropods via bites or feces and can cause mild to fatal diseases in humans (33). Some species are also considered potential bioterrorism agents (4). Most Rickettsia research has focused on pathogens that are found in two closely related species groups, the typhus and spotted fever groups, such as Rickettsia prowazekii, Rickettsia rickettsii, and Rickettsia typhi, the causal agents of epidemic typhus, Rocky Mountain spotted fever, and murine typhus, respectively (3, 4, 33). However, recent surveys suggest that Rickettsia bacteria are much more widespread than previously suspected and that they are being detected in novel hosts, the vast majority of which are arthropods, including many that do not feed on blood (29, 45).The number of new rickettsial species that cause diseases in humans is rapidly increasing (33). One such species that has been generating much interest in recent years is Rickettsia felis, the causative agent of a murine typhus-like disease (1, 2, 13, 16, 17, 28, 44). The disease is often unrecognized, and even though it is considered clinically mild, it can cause severe illness and death in older patients and in cases of delayed diagnosis (2). R. felis was identified only in 1990 (1) and has since been found worldwide in fleas, where it is maintained transovarially and can reach high infection rates (e.g., 86% to 94% in cat fleas) (2, 3, 44), as well as in ticks and mites (34). While experimental infections have confirmed that R. felis is transmitted to vertebrate hosts via blood feeding and that R. felis occurs in an infectious extracellular state (39), it is not known whether transmission can also occur through contamination of broken skin by infected vector feces, as in R. typhi (3, 34).A number of features distinguish R. felis from species in both the typhus and spotted fever groups. Lately, it has been proposed that R. felis be in its own group, allied with Rickettsia akari and Rickettsia australis, the causal agents of rickettsial pox and Queensland tick typhus, respectively, and a number of recently discovered strains infecting insects that do not feed on blood (16, 17, 29, 45). Moreover, R. felis was the first Rickettsia species shown to have a plasmid (28). While plasmids now appear to be quite widespread in the genus, the R. felis plasmid stands out with respect to its relatively large size and distinctive gene content (5, 6, 9, 14, 17).This study reports that a common and cosmopolitan insect, the psocid Liposcelis bostrychophila (Psocoptera: Liposcelidae) harbors R. felis. Liposcelids are the closest free-living relatives of parasitic lice (19) and are well-known for their close proximity to humans, particularly as pests in houses and grain storage facilities (8, 41). Through 16S rRNA gene sequencing, L. bostrychophila was recently shown to harbor a strain of Rickettsia (29, 30, 42). A systematic molecular characterization of this Rickettsia was conducted, demonstrating that it is authentic R. felis. Furthermore, the psocid symbiont plasmid was sequenced and was shown to be virtually identical to the plasmid from R. felis that infects cat fleas.     

Host alternation of chikungunya virus increases fitness while restricting population diversity and adaptability to novel selective pressures

The mechanisms by which RNA arboviruses, including chikungunya virus (CHIKV), evolve and maintain the ability to infect vertebrate and invertebrate hosts are poorly understood. To understand how host specificity shapes arbovirus populations, we studied CHIKV populations passaged alternately between invertebrate and vertebrate cells (invertebrate ↔ vertebrate) to simulate natural alternation and contrasted the results with those for populations that were artificially released from cycling by passage in single cell types. These CHIKV populations were characterized by measuring genetic diversity, changes in fitness, and adaptability to novel selective pressures. The greatest fitness increases were observed in alternately passaged CHIKV, without drastic changes in population diversity. The greatest increases in genetic diversity were observed after serial passage and correlated with greater adaptability. These results suggest an evolutionary trade-off between maintaining fitness for invertebrate ↔ vertebrate cell cycling, where maximum adaptability is possible only via enhanced population diversity and extensive exploration of sequence space.Emergence of pathogenic RNA viruses is associated with their genomic variability and environmental changes that lead to novel host contacts. Many new and reemerging RNA viruses have been introduced into humans (10). Arthropod-borne viruses (arboviruses), including dengue virus (DENV), have caused recent epidemics by changing their host ranges to increase infection of humans (54). Adaptation to the urban mosquito Aedes albopictus may have expanded a 2005-2006 outbreak of Chikungunya virus (CHIKV) in Reunion Island, France (46), that subsequently circulated among humans in the absence of other amplifying hosts.Despite these emergence events, the evolutionary processes that mediate arbovirus host range changes are poorly understood, partly since arbovirus evolution is understudied. Arboviruses are transmitted horizontally between arthropod vectors and vertebrate reservoir hosts. They replicate rapidly and achieve large population sizes. Polymerases of RNA viruses lack proofreading to repair errors, leading to one substitution per ∼10⁻⁴ nucleotides (nt) copied (11, 36), corresponding to one error per 10-kb genome. This polymerase infidelity leads to diversification that produces closely related but nonidentical RNAs that together form a spectrum of mutants. Although arbovirus mutant spectra have been observed in nature (1, 8, 20, 55, 57), the diversity and divergence within the spectrum are not well described, and the phenotypic roles of minority RNAs are unknown. Understanding the mutation distribution in a heterogeneous arbovirus population is important, given that any variant can be favored by selection and ultimately affect fitness (12, 13). However, the relationships between fitness and RNA virus population diversity are poorly understood.Studies with other RNA viruses, including human immunodeficiency virus (HIV) (3, 58, 60), hepatitis C virus (14, 15, 25), and poliovirus (30, 52), indicate that intrahost population diversity is important for virus evolution, fitness, and pathogenesis. Unlike these vertebrate-only RNA viruses, arboviruses obligately cycle between vertebrates and arthropods, a process that imposes additional selective constraints on evolution and adaptation. Sequence comparisons of RNA arbovirus isolates show that they are relatively stable (18, 19), and genetic studies reveal that evolution is dominated by purifying selection (20-22, 57). This constancy of consensus sequence may derive from the need to infect disparate hosts that present conflicting demands for adaptation where sequence changes that improve fitness in one host may not be maintained in the alternate organism.Experimental evolution studies have been performed to study fitness trade-offs and the unique ability of arboviruses to simultaneously evolve to vertebrate and invertebrate hosts. In vitro evolution studies reveal three general patterns of arbovirus evolution: (i) fitness gains after serial passage in vertebrate or invertebrate cells (except in certain cases [7]) and losses in bypassed host cell types (DENV, Eastern equine encephalitis virus [EEEV], Sindbis virus [SINV], and vesicular stomatitis virus [VSV]) (17, 28, 51, 56); (ii) reduced fitness in new cells (VSV) (28), and (iii) fitness increases after alternating (invertebrate ↔ vertebrate) passage (DENV, EEEV, SINV, VSV) (17, 28, 51, 56). Together, these studies suggest that constraints on fitness differ in insect and vertebrate cells and can be virus specific but that arbovirus fitness is not limited by alternating between vertebrate and invertebrate hosts.An in vivo study revealed a similar pattern of arbovirus evolution: vertebrate-passaged Venezuelan equine encephalitis virus (VEEV) was five times more fit than its unpassaged parent, and mosquito-passaged VEEV was more infectious for vectors (6). Consensus sequences revealed that, despite becoming more fit, mutations in passaged populations were slight or absent. This suggests that fitness increases were mediated by minority genomes in the mutant spectrum. However, a major limitation of the in vivo experiments and other arbovirus evolution studies is that sequencing of individual RNAs from the mutant spectrum in passaged intrahost populations was not performed (although notable exceptions for flaviviruses exist [5, 22]). The identity of minority variants within intrahost arbovirus populations and their influences on phenotype have not been extensively examined.The goal of this study, therefore, is to understand how obligate host cycling shapes an intrahost arbovirus population. RNA viruses can tolerate increased niche breadth when they evolve in heterogeneous environments (48), a trait which may promote easier emergence and therefore help explain why arboviruses frequently emerge or reemerge to cause human and veterinary disease. One limitation of increasing breadth may be restricted genetic diversity, where only genomes accepted in both hosts can be maintained. To explore this possibility, we described arbovirus populations after invertebrate ↔ vertebrate cell cycling and compared them to populations that were artificially released from alternating passage via serial vertebrate or invertebrate cell transfer. Since previous studies indicate that intrahost RNA virus population diversity can determine phenotype (3, 37, 52) and given that increases in arbovirus fitness are not always associated with consensus genome changes (6, 27), we determined how arbovirus population diversity (mutation frequency) and distance (number of mutations by which each RNA differs from the consensus) relate to fitness. We predicted that genetic diversity and distance between RNAs in the mutant spectrum and the corresponding consensus are correlated with fitness and that diverse populations better escape varied selective pressures than genetically homogeneous populations since, by chance, they are more likely to contain an advantageous mutation(s) when the pressure is applied.Chikungunya virus (family Togaviridae, genus Alphavirus) was selected primarily because of its medical relevance. CHIKV has caused outbreaks of human disease characterized by arthralgia and myalgia since the 18th century and since 2004 in Africa, Indian Ocean islands, Southeast Asia, and Italy (32). Furthermore, no adaptation studies have been performed for CHIKV, and no intrahost population studies have been conducted for an alphavirus. An infectious clone was generated from the strain used for passages and was genetically marked to differentiate its RNAs from passaged CHIKV, so that after direct competition, the fitnesses of passaged populations could be compared to those for progenitors.