Units of plasticity in bacterial genomes: new insight from the comparative genomics of two bacteria interacting with invertebrates, Photorhabdus and Xenorhabdus
© Ogier et al; licensee BioMed Central Ltd. 2010
Received: 19 March 2010
Accepted: 15 October 2010
Published: 15 October 2010
Flexible genomes facilitate bacterial evolution and are classically organized into polymorphic strain-specific segments called regions of genomic plasticity (RGPs). Using a new web tool, RGPFinder, we investigated plasticity units in bacterial genomes, by exhaustive description of the RGPs in two Photorhabdus and two Xenorhabdus strains, belonging to the Enterobacteriaceae and interacting with invertebrates (insects and nematodes).
RGPs account for about 60% of the genome in each of the four genomes studied. We classified RGPs into genomic islands (GIs), prophages and two new classes of RGP without the features of classical mobile genetic elements (MGEs) but harboring genes encoding enzymes catalyzing DNA recombination (RGPmob), or with no remarkable feature (RGPnone). These new classes accounted for most of the RGPs and are probably hypervariable regions, ancient MGEs with degraded mobilization machinery or non canonical MGEs for which the mobility mechanism has yet to be described. We provide evidence that not only the GIs and the prophages, but also RGPmob and RGPnone, have a mosaic structure consisting of modules. A module is a block of genes, 0.5 to 60 kb in length, displaying a conserved genomic organization among the different Enterobacteriaceae. Modules are functional units involved in host/environment interactions (22-31%), metabolism (22-27%), intracellular or intercellular DNA mobility (13-30%), drug resistance (4-5%) and antibiotic synthesis (3-6%). Finally, in silico comparisons and PCR multiplex analysis indicated that these modules served as plasticity units within the bacterial genome during genome speciation and as deletion units in clonal variants of Photorhabdus.
This led us to consider the modules, rather than the entire RGP, as the true unit of plasticity in bacterial genomes, during both short-term and long-term genome evolution.
The portion of the bacterial genome common to all strains in a defined set of species and required for basic cellular functions is known as the core genome. The genes variably present between individual strains constitute the flexible genome [1–3]. The estimate of the core and the flexible genomes not only depend on the phylogenetic depth of the group considered, the number of genomes available for comparison but also on the methodology used . Some genes of the flexible genome may play a role in adaptation to special growth conditions, such as those involved in the colonization of new ecological niches, symbiosis, host-cell interaction, and pathogenicity [1, 2]. The plasticity of the flexible genome contributes to bacterial genome evolution [2–4].
The flexible genome is organized principally into polymorphic strain-specific DNA segments that are missing in at least one of the genomes analyzed. These segments are named regions of genomic plasticity (RGPs) without any assumption about the evolutionary origin or genetic basis of these variable chromosomal segments . This terminology covers two classes: hypervariable segments that are likely to be the result of deletions of particular DNA regions in one or more strains, and the mobile genetic elements (MGEs).
The plasticity of MGEs depends on three kinds of molecular events, the duplications, inversions and deletions, mediated by transposases and site-specific recombinases whose genes are located either on core genome or on the RGPs themselves [4, 6]. The MGEs may be excised from one location and reintegrated elsewhere in the genome or may undergo replicative transposition before integration of a new copy of the element elsewhere in the genome (intracellular mobility). Finally, some MGEs may undergo horizontal genetic transfer (HGT) by natural transformation, transduction or well developed and efficient conjugation mechanisms (intercellular mobility) .
The MGE class covers some well characterized elements. Plasmids are stable self-replicating MGEs . Some of them may be transferred in other prokaryotic cells by conjugation. Prophages, the integrated form of temperate bacteriophages, are MGEs that undergo intercellular DNA mobility via transduction . Non replicative MGEs are integrated into the host chromosome and encode at least one enzyme involved in their own excision and integration; these MGEs constitute a large, diverse family [7, 8]. They are referred to as (i) transposable elements, if they do not undergo HGT, (ii) genomic islands (GI) if they present features of HGT (phage and/or plasmid-derived sequences, transfer genes, integrases, insertion sequences (IS), G+C content and codon usage bias) but do not encode genes involved in transfer, (iii) integrative mobilizable elements when they require "helper" elements for mobilization and (iv) integrative conjugative elements (ICEs) when they encode their own complete mobility machinery, generally a type 4 secretion system (T4SS) . However, it is often difficult to apply this nomenclature, because MGEs are generally described on the basis of in silico analysis in large-scale prokaryotic genome sequencing programs. Thus, the effective excision, intracellular or intercellular mobility and subsequent reintegration via site-specific recombination of MGEs have been demonstrated in only a few cases [9–14]. For these reasons, in the course of genomic projects, in the absence of experimental data, the term "GI" is generally used for putative mobilizable MGEs without the organization typical of prophages [1, 2, 4, 15].
MGEs are potent agents of bacterial genome evolution [4, 16]. This property results from both the plasticity of MGE and intra-MGE recombination. Indeed, some MGEs are organized into an array of MGE sub-segments, known as modules [7, 17, 18]. This mosaic organization is the product of the combination of a limited number of constitutive modules : intracellular mobility modules (recombination and replication functions), intercellular mobility modules (transformation, phage propagation and conjugative transfer) and stability modules. The stability modules are responsible for the maintenance of the MGE in the host cell and encode functions such as poison/antidote systems  and antibiotic resistance functions [20, 21]. Recombination between MGEs has been studied in a few cases. Deletions and tandem accretions of modules generate hybrid MGEs [22–24]. The bacterial recA gene or the recombination systems of the MGEs themselves may mediate the generation of hybrid MGEs .
We investigated the plasticity of the flexible genome, by addressing three questions: what are the respective roles of MGEs and hypervariable segments within the flexible genome? Are all RGPs, like MGEs, composed of modules? Do all modules undergo accretion? We addressed these questions by studying the flexible genomes of Photorhabdus and Xenorhabdus. Photorhabdus and Xenorhabdus are closely related Enterobacteriaceae , both of which are appropriate for genomic evolution studies because of their particular lifestyle [27, 28]. Photorhabdus and Xenorhabdus live in monoxenic cultures within the gut of the soil nematodes, Heterorhabditis and Steinernema, respectively. These nematodes infect insect larvae, releasing the bacteria into the hemolymph of the insect. The nematode and the bacteria kill the insect and convert the cadaver into a source of food for nematode growth and development. After several rounds of reproduction, the bacteria recolonize the nematodes, which then emerge from the insect cadaver into the soil, to search for a new host [29–31]. This lifestyle, including obligatory, cyclic pathogenic and mutualistic interactions with invertebrate hosts, restricts the ecological niches colonized by Photorhabus and Xenorhabdus. This biological constraint may favor clonality among bacteria and intrachromosomal rearrangements within the genome. Moreover, Photorhabdus asymbiotica has been recovered in clinical isolates from human wounds, in both North America and Australia [32, 33]. The emergence of pathogenicity in humans is also consistent with a potential for genomic exchange with environmental bacteria.
Genomic plasticity has been studied to different extents in the two genera. Whole-genome analysis has just begun for Xenorhabdus, whereas full genome sequences have been published for two Photorhabdus strains, revealing the presence of a large number of phage remnants, IS, transposases, repeat elements and overrepresented families of paralogs, consistent with a high level of potential plasticity in these genomes [35, 36]. These features are indicative of a general process of genome evolution, as repeatedly observed in host-restricted lineages from many phylogenetic groups . One study described genomic deletion and amplification events in Photorhabdus clonal variants obtained in laboratory conditions . These genomic changes are cryptic, but are always found within the Enterobacteriaceae flexible genome. Finally, some studies have characterized a few Photorhabdus and Xenorhabdus RGPs by in silico analysis [35, 36, 39–43] or by microarray hybridization [38, 44].
In this study, we carried out an exhaustive description of RGPs in the genomes of three entomopathogenic strains isolated from nematodes: Xenorhabdus nematophila ATCC19061 , Xenorhabdus bovienii SS-2004  and Photorhabdus luminescens TT01 , and a strain isolated from humans: Photorhabdus asymbiotica ATCC43949 . For the identification of hypervariable regions, recent MGEs, ancient MGEs and non canonical MGEs with unknown mobility mechanisms, we used a new Web tool, RGPFinder, which identifies both synteny ruptures in the core genome and classical intrinsic and extrinsic MGE features (Roche, D., unpublished data). We then described the fine modular structure of RGPs and showed that (i) each module is a functional unit, (ii) modules have diverse functions, (iii) modules shape the flexible genomes of the various strains studied and, (iv) some modules are deletion units. Overall, our data strongly suggest that modules are the functional integrated systems serving as the real unit of plasticity within RGPs.
Results and Discussion
Identification of regions of genomic plasticity (RGPs)
The size of the flexible genome depends on the methodology used, the depth of phylogenetic comparison and the number of genomes compared . Methods based on genomic comparison, detection of composition bias and search of mobility genes are the most performing tools for the flexible genome characterization . Some methods such as IslandViewer and MobileHomeFinder are dedicated to predict genomic islands (GIs) with high stringency. Our goal is the identification of the regions of genomic plasticity (RGPs), which covers not only GIs but also rearrangement events without any assumption about the evolutionary origin or genetic basis of these variable chromosomal segments. For this reason, we developed a new Web tool, RGPFinder, which combines comparison and composition based approaches (Roche et al., unpublished data). Furthermore, RGPFinder is specifically designed to identify regions absent from at least one genome inside the comparison genome set. We applied this tool on the genomes of Photorhabdus luminescens TT01 (Pl), Photorhabdus asymbiotica ATCC43949 (Pa), Xenorhabdus nematophila ATCC19061 (Xn) and Xenorhabdus bovienii SS-2004 (Xb) strains. We compared the results of IslandViewer and RGPFinder on our four genomes (data not shown). RGPFinder carries out a larger description of the flexible genome (more and larger predicted regions) than IslandViewer.
Number and size of regions of genomic plasticity (RGPs) in the P. luminescens TT01, P. asymbiotica ATCC43949, X. nematophila ATCC19061 and X. bovienii SS-2004 genomes, according to the set of bacterial genomes used to search for synteny ruptures in the core genome
Number of predicted RGPs (% of the whole genome), when compared with the
"Photo + Xeno" set2*
"Entero" set after cleaning**
P. luminescens TT01
P. asymbiotica ATCC43949
X. nematophila ATCC19061
X. bovienii SS-2004
After manual inspection of the predicted RGPs (see Methods), we obtained a list of 96, 92, 83 and 71 RGPs sensu lato for Pl, Pa, Xn and Xb, respectively (Table 1 and see RGPs listed in Additional File 2; these lists are the references used throughout this work.). The RGPs were between 5 kb and 316 kb in length, and more than 50% were less than 20 kb long (Additional File 3). No integral RGP was found to be conserved in all four genomes. The flexible genome of the Photorhabdus and Xenorhabdus genera accounted for 52.6 to 61.5% of the entire genome (Table 1). In other studies in conditions similar to those used here, the flexible genome has been found to cover: i) 1 to 10% of the genome when serovar or clinical isolates are compared [5, 46]; ii) 10 to 40% of the genome when strains are compared [47, 48], iii) 25 to 60% of the genome when species are compared [49–51] and iv) 50 to 69% of the genome when genera are compared . Thus, the sizes of the flexible genomes of Photorhabdus and Xenorhabdus within Enterobacteriaceae were consistent with the findings of other studies.
The flexible genome was found to be larger in Photorhabdus than in Xenorhabdus genus. This finding is consistent with previous genomic analyses highlighting the importance of genome plasticity in Photorhabdus genomes, at both the species [36, 40, 44] and clonal  levels.
Classification of RGPs
Classification of RGPs in the P. luminescens TT01, P. asymbiotica ATCC43949, X. nematophila ATCC19061 and X. bovienii SS-2004 genomes as a function of their genetic composition (the proportion of the modules belonging to a given class is indicated in parentheses)
RGP sensu stricto
P. luminescens TT01
P. asymbiotica ATCC 43949
X. nematophila ATCC19061
X. bovienii SS-2004
RGPs showing at least one of the typical features of MGEs acquired by HGT (insertion near a tRNA gene, an integrase-coding gene or a G+C content different from that of the core genome) and that are not prophages were named GIs. No ICE class was created since no T4SS loci was identified in the four studied genomes. GI85_PL and GI25_PA, in the Pl and Pa genomes, respectively, were found to harbor type three secretion system (T3SS) loci similar to those of Yersinia pestis and Pseudomonas aeruginosa[35, 36, 40, 59]. Many pathogenic Gram-negative bacteria encode T3SSs of the Ysc type . In Pl, this T3SS is involved in bacterial adaptation to the insect host, as it prevents the uptake of bacteria by the immunity organs of Locusta migratoria. As previously described [30, 59], no such loci are found in Xenorhabdus genomes. The GI27_PL of Pl is another cluster potentially involved in interactions between bacteria and host, as it harbors a homolog of the Yersinia adhesion pathogenicity island (YAPI) . The YAPI encodes a type IV pilus, which contributes to pathogenicity in Yersinia pseudotuberculosis serotype O:1, but also includes genes encoding proteins involved in general metabolism, a gene cluster for a restriction-modification system and a large number of mobile genetic elements . This YAPI cluster was detected only on the Pl chromosome. Finally, GI63_XN from Xn is a gene cluster potentially involved in nematode interaction. It harbors the nil locus, enabling X. nematophila strains to colonize their nematode host, S. carpocapsae, specifically . It also encodes putative peptide synthetases, which may be involved in antibiotic production, thereby facilitating the eviction of competing bacteria during the association of nematodes and bacteria before the nematodes leave the insect cadaver. The GI63_XN is specific to Xn.
The other predicted regions were named RGPs sensu stricto. Several of these RGPs contained ISs and genes encoding enzymes catalyzing DNA recombination, such as resolvase, invertase and excisionase. We named these RGPs RGPmob. RGPs with no remarkable features were named RGPnone. This last group of RGPs probably consists of hypervariable regions with intracellular mobility mediated by chromosomal rearrangements, such as deletion, duplication or inversion. RGPmob and RGPnone may also be ancient mobilizable MGEs with degraded mobility machinery. The membership to multiple RGP classes of Photorhabdus virulence cassettes (PVCs) and the toxin complex (Tc) loci is consistent with this hypothesis. PVCs are phage-like elements flanked by variable putative or identified toxin genes [63–65]). They are found in Photorhabdus genomes, but not in Xenorhabdus genomes. Yang and coworkers speculated that PVCs might encode phage-like structures, acting as syringes to deliver toxins to the interior of eukaryotic cells . Six of the eight previously described PVC families , belong to the prophage and GI classes (PVCcif: GI52_PA, GI60_PL, PVClopT of Pl: GI56_PL, PVCpnf of Pa: GI81_PA, PVClmt of Pa: GI57_PA, PVCphx of Pl and its tandem repeat: P41_PL). The remaining three families were classified as RGPmob, despite their putative ancestral origin from phages (PVClopT of Pa: RGP54_PA, PVCphx of PA: RGP66_PA). The Tc loci of Photorhabdus and Xenorhabdus encoded families of insecticidal toxins active by ingestion [35, 36, 39, 41]. These loci are conserved in several other entomopathogenic bacteria (Serratia entomophila, Pseudomonas entomophila) and bacteria associated with insects (Yersinia spp., Pseudomonas syringae) [43, 63, 66, 67], strongly suggesting that HGT of Tc loci has occurred between soil bacteria from different genera known to interact with insects. Some Tc loci are embedded in GIs (GI94_PL, GI27_PL, GI22_PL, GI23_PA, GI91_PA, GI50_XN, GI56_XN, GI23_XB), whereas others are found in RGPnone (RGP14_PL, RGP58_PL, RGP47_XN, RGP14_XB, RGP30_XB). In these two examples, the genomic erosion of HGT features may lead to a loss of information in some RGPs. This process has classically been reported for nucleotide usage: with increasing time since the HGT event, codon usage in the inserted DNA is gradually modified to match the background of the recipient genome . RGPmob and RGPnone may also be MGE that can be mobilizable in trans by other MGEs or non canonical MGEs with mobility mechanisms that have yet to be described. Our classification thus opens up promising new lines of research that may lead to the identification of new classes of MGE.
From the viewpoint of whole-genome evolution, our classification highlights the unusual position of the Pa genome. The proportions of RGPmob and RGPnone are fairly similar in the Pl, Xb and Xn genomes, whereas the Pa genome contains a much higher proportion of RGPnone (Table 2). This difference probably results from differences in genome evolution and/or plasticity between Pa, which was recovered from a human patient in North America , and the other three strains, which were isolated from nematodes. The evolutionary implications of the higher proportion of RGPnone in Pa remain unclear, but a systematic functional analysis of RGPnone specific to Pa would complement virulence mapping techniques , thereby contributing to the identification of new MGEs involved in the emergence of human pathogens.
RGPs in the core genome architecture
We mapped the different classes of RGPs on schematic circular maps, to visualize their chromosomal distribution (Additional File 4). GIs, prophages and RGPs sensu stricto were found to be evenly distributed throughout the four genomes. Replication, by its inherent asymmetry, shapes the global structure of the prokaryotic chromosome, and some regions of the chromosome are much less accessible for internal recombination than others . However, GI, prophages and RGPs sensu stricto were equally likely to be located in the region of the origin of replication, the replication termination region or other regions. This permissiveness probably results from compensatory lateral transfers and/or recombination events, preventing dramatic effects on gene order or the large-scale organization of the genome .
RGP sites (genes flanking RGPs on the right or left) conserved in the P. luminescens TT01 (Pl), P. asymbiotica ATCC43949 (Pa), X. nematophila ATCC19061 (Xn) and X. bovienii strain SS-2004 (Xb) genomes.
location sites *
yjdC _t RNA-phe
rpo D _tRNA-met
tRNA gene integration site
pgsA _ tRNAleu_ tRNAcys
yjeM _tRNA - pro
mltC _ tRNAphe
gltX _ tRNA-val _ 2tRNAlys _ 3tRNA - val
vacJ _ tRNA - arg
rsmC _ tRNA-leu
Protein encoding gene integration site
In addition to the trmE CDS-site, a number of other integration hotspots are conserved in other bacteria. The CS54 genomic island of Salmonella enterica serotype Typhimurium  and the 14 kb genomic island of E. coli CFT073  are both flanked by the xseA integration hotspot. The rpoS gene is considered to be a recombination hotspot within the Enterobacteriaceae . Integration hotspots are also conserved in more distantly related bacteria. flgL and fbaA are the integration sites of the flagellin glycosylation island of Pseudomonas aeruginosa and the ICESt1 and ICESt3 of Streptococcus thermophilus, respectively. The tropism of MGEs for particular integration hotspots in phylogenetically unrelated taxa highlights the probable ancestral role of such sites in integration and recombination.
Modules are the functional units within RGPs
We have shown that no single RGP is conserved between the four genomes. Indeed, the RGPs identified were either unique or shared a limited number of subregions. An exhaustive in silico analysis provided evidence for the structuring of each RGP (P, GI, RGPmob and RCPnone) into several subregions or modules. These modules are blocks of genes 0.5 to 60 kb in length, with a conserved gene order (synteny) in at least two genomes of the Entero set or specific to the strain (see Materials and Methods).
For each genome, a list of modules is given in Additional File 5 and the module to which each gene belongs is identified in the gene file accessible on PhotoScope and XenorhabduScope (see Materials and Methods). We distinguished eight functional classes of modules: 1) metabolic modules, consisting of genes involved in primary metabolism, metabolite transport and cell component biosynthesis; 2) drug resistance modules; 3) antibiotic synthesis modules encoding bacteriocins, non ribosomal peptide synthetase, polyketide synthases; 4) phage modules, 5) recombination modules encoding enzymes involved in DNA recombination, such as transposase, invertase, excisionase; 6) environment interaction modules encoding proteins or proteinaceous structures involved in interactions with the environment (iron uptake, adhesion to surfaces etc.), 7) host-interaction modules encoding virulence or symbiosis factors essential for interaction with the insect or nematode host; 8) modules of unknown function.
Antibiotic modules (3 to 6%) and drug resistance modules (4 to 5%) constitute another canonical module class [17, 20]. The Photorhabdus and Xenorhabdus flexible genomes contain numerous genes and operons encoding antibiotic molecules and secondary metabolites, such as non-ribosomal peptides, polyketides and bacterocins. These products are prime candidates for HGT, because they provide a gain-of-function phenotype and are not essential to the microbial cell . Furthermore, one of the major challenges for entomopathogenic nematode symbiosis is the maintenance of a monoxenic infection in an insect cadaver in the soil. Flexibility, resulting in the renewal of antimicrobial factors, is therefore likely to be determinant for Photorhabdus and Xenorhabdus.
In addition to the most common building blocks found in MGEs, we identified modules more specifically involved in metabolism, environment/host interaction and unknown functions. Metabolic modules constituted the most frequently represented class, regardless of the strain considered (22-27%). This finding highlights the contribution of the acquisition of additional metabolic traits to adaptability and competitiveness under certain circumstances, such as during the colonization of a new niche or rapidly changing growth conditions . Interactions between host and environment also account for a large proportion of modules (22-31%). This may reflect the complex life style of these bacteria, which interact with two invertebrate hosts, an insect and a nematode. No known function could be attributed to 13 to 18% of the modules, and the hypothetical proteins encoded by the genes of these modules are good candidates for identifying new genes playing a role in the particular lifestyle of these bacteria.
The modular structure of some MGEs has already been reported in previous studies. This structure consists principally of intracellular mobility modules, intercellular mobility modules and antibiotic resistance modules [17, 20]. Furthermore, it has been described principally for phages, transposons, ICEs and GIs [24, 69, 83–89]. By measuring features describing the rate of change of DNA in the locus of enterocyte and effacement of Escherichia coli, Castillo and coworkers also concluded that the GI had a mosaic structure and identified subregions, rather than the whole GI, as the true units of selection .
In this study, we show that (i) the modular structure relates not only to prophages and GIs but also to RGPmob and RGPnone, (ii) module functions cover a broad range of functions involved in adaptation to the bacterial environment. RGPs sensu lato are therefore polyfunctional, and the functional unit of the RGPs is the module. This RGP organization is observed in the Enterobacteriaceae family, but probably also throughout the prokaryotic kingdom.
Modules are the plasticity units of RGPs during long-term genome evolution
As discussed above, no entire RGP is conserved between the four genomes analyzed in this study and RGPs are composed of functional units, the modules. We investigated whether the mobility of these modules within and between cells could have contributed to shaping the RGPs in the different strains or species of a taxon. We compared the distribution of modules throughout the genome and their organization, between the four genomes studied.
Our data show that the RGPs of the flexible genome are shaped by the acquisition and loss of modules, and that RGP diversity probably results from intrachromosomal or interchromosomal rearrangements between module units.
Modules are the units of deletion of RGPs involved in short-term genome rearrangements
If modules do indeed shape the RGPs during genome evolution, some rearrangements are likely to be detectable in clonal populations during growth in the laboratory. We therefore searched for intrachromosomal rearrangements of modules within the genome of Pl clonal variants available in our laboratory. TT01α is a Pl variant collected from a laboratory-maintained symbiotic nematode . TT01α differs from the Pl reference genome by nine large-scale deletion events in the flexible genome, but these genomic changes have cryptic phenotypic consequences. The boundaries of the deleted regions in TT01α were located in the Pl genome by a combination of macrorestriction and DNA microarray experiments .
In conclusion, whatever the molecular mechanism involved in these deletion scenarios, in the case of clonal genomic plasticity, the modules may be deleted over a timescale corresponding to growth in the laboratory and may be considered units of deletion within RGPs.
The data presented here participate to a better vision of the bacterial flexible genome organization. The characterization of RGPs by the RGPFinder method showed the flexible genome to be much broader than the sum of GIs and prophage elements. Additional elements -- RGPmob and RGPnone elements -- lacking classical mobility features may be hypervariable regions that undergo deletions, ancient mobile elements with a degraded mobilization machinery, MGE that can be mobilizable in trans by other MGEs or non canonical MGE for which the mobility mechanism has yet to be described. Furthermore, we provide evidence that not only GIs and prophages, but all RGPs sensu lato have a mosaic structure composed of modules that are both functional and plasticity units.
The application of comparative genome sequencing to experimental evolution studies provides us with an opportunity to study the link between genome dynamics and adaptive evolution. Nevertheless, such studies are generally carried out on bacterial populations evolving in a synthetic broth culture, and they mostly identify point mutations [95, 96]. Here, by carrying out comparative genomics studies on variants obtained from their host in the laboratory, we showed experimentally that the same modules undergo genomic rearrangements during genome speciation and short-term genomic rearrangements. This work improves our understanding of the process responsible for bacterial genome diversification and evolution.
Obtaining of these data were made possible by the use of the Photorhabdus and Xenorhabdus genera for our comparative genomic study. Indeed, the life cycle of these genera is restricted to two successive ecological niches. We argue that this unusual pattern of selective pressure is responsible for an alternation of genomic shuffling: HGT in the insect cadaver, which constitutes an abundant nutrient resource potentially shared with many other microorganisms and intrachromosomal rearrangements of recently acquired modules in the bacterial monoxenic culture within the nematode gut, as observed in the Pl variant isolated from a laboratory-maintained symbiotic nematode. We therefore suggest that Photorhabdus and Xenorhabdus are suitable new bacterial models for studies of the evolution of bacterial genomes.
Finally, the data obtained in this study contribute to our understanding of the fluid nature of genomes throughout the kingdoms of life. According to J. A. Shapiro, prokaryotic and eukaryotic cells are genetic engineers and mobile elements are "natural genetic engineering systems" that facilitate the evolutionary rewriting of genomic information . Shapiro's hypothesis is that repeated evolutionary challenges have selected systems that (i) reduce the size of the genomic search space and (ii) maximize the chance of success by using combinatorial processes based on basal functional components . We argue that the modules described here are entirely consistent with this vision, as these functional units recombined at a limited number of hotspots shaping and delimiting the flexible genome.
Bacterial strains and genome sequences
Photorhabdus luminescens subspecies laumondii strain TT01 is a symbiont of the nematode Heterorhabditis bacteriophora isolated in Trinidad and Tobago [35, 98]. The genome of strain TT01 consists of a single circular chromosome of 5,688,987 bp (accession number NC_005126). Photorhabdus asymbiotica subspecies asymbiotica strain ATCC43949 is a North American clinical isolate. This strain was isolated in 1977 from a female patient with endocarditis, in Maryland, USA [32, 99]. The genome of strain ATCC43949 consists of a single circular chromosome of 5,064,808 bp and a 29,732 bp plasmid  (accession numbers NC_012962 and NC_012961, respectively). Xenorhabdus nematophila ATCC19061, the type strain of the species, is a symbiont of the nematode Steinernema carpocapsae, isolated from Georgia, USA . The genome of strain ATCC19061 comprises a single circular chromosome of 4,432,590 bp and a 155,327 bp plasmid  (accession numbers FN667742 and FN667743, respectively). Xenorhabdus bovienii SS-2004 is a symbiont of the nematode Steinernema jollieti sp. isolated from a woodland in the Missouri valley, USA, in 1999 . The genome of strain SS-2004 comprises a single circular chromosome of 4,225,498 bp  (accession number FN667741). The four genomes were input into the PhotoScope and XenorhabduScope databases http://www.genoscope.cns.fr/agc/mage.
Regions of genomic plasticity (RGPs) were sought in the P. luminescens TT01, P. asymbiotica ATCC43949, X. nematophila ATCC19061 and X. bovienii SS-2004 genomes, with the RGPfinder web tool implemented in the MaGe annotation platform (http://www.genoscope.cns.fr/agc/mage; Roche et al., unpublished data). Briefly, RGPFinder searches for breaks in synteny between a reference genome and the genomes of a set of related bacteria -- the bacterial genome set (Figure 1.A). A RGP sensu lato is the sum of overlapping subregions missing in at least one of the bacterial genomes of the comparison set. RGPs have a minimal size of 5 kb. This excludes the isolated insertion sequences of the RGPs, but favors regions with several genes of potential functional interest in the bacterial biology. This definition does not involve any underlying assumption about the evolutionary origin or genetic basis of these variable chromosomal segments. RGPFinder also provides information about composition abnormalities (GC% deviation, codon adaptation index) and about the features flanking the RGPs, such as tRNA, IS, integrase (int) and genetic elements involved in DNA mobility (mob), which are common characteristics of foreign DNA acquired by horizontal genetic transfer. The results obtained with this web tool include those for Alien Hunter , a method detecting atypical sequences (i.e., sequences potentially acquired by horizontal genetic transfer) through the analysis of composition bias.
Predicted RGPs were then manually inspected, to eliminate false-positive results. Indeed, point mutations may lead RGPFinder to identify a region as an RGP when it actually belongs to the core genome. Finally, the boundaries of the RGP were homogenized between the compared genomes and potential insertion sites were defined. The genomes used in the bacterial genome set were those of P. luminescens TT01, P. asymbiotica ATCC43949, X. nematophila ATCC19061, X. bovienii SS-2004, Yersinia pestis CO92 (accession number 003143); Salmonella enterica subsp. enterica Typhi CT18 (accession number NC_003198), Erwinia carotovora subsp. atroseptica SCRI1043 (accession number NC_004547) and E. coli K12 (accession number NC_000913). Finally, Prophinder was used to detect prophages among the RGP sensu lato, http://aclame.ulb.ac.be/Tools/Prophinder/.
Definition and distribution of modules
The MaGe web interface  was used to divide RGPs manually into subregions corresponding to blocks of genes specific to the strain or blocks of syntenic genes (i.e. genes with a conserved genomic organization in at least two genomes of the Entero set). These subregions, which often contain genes of similar biological function, were named "modules". The distribution of modules among the "Enterobacteriaceae" genome set was analyzed manually: a module was considered present (or partially present) in a genome if it had more than 80% (25%) of syntenic orthologous genes (orthologous genes shared at least 30% of identity on 80% of the shortest sequence by BlastP) with the module of the reference genome. The module was otherwise considered to be absent. Descriptions of the modules and their distributions are available from PhotoScope https://www.genoscope.cns.fr/agc/mage/wwwpkgdb/Login/log.php?pid=13 and XenorhabduScope https://www.genoscope.cns.fr/agc/mage/wwwpkgdb/Login/log.php?pid=24, by opening the Genomic Object Editor of a gene and consulting the "Module" results.
Multiplex PCR procedure and sequencing
Genomic DNA was extracted as previously described  and stored at 4°C. Primers flanking the right (primers P1/P2) and left (primers P3/P4) module borders were designed with Primer 3 http://frodo.wi.mit.edu/primer3/. Primer sequences are listed in Additional File 6. Multiplex PCR with the four primers (P1, P2, P3, P4) was performed with a Bio-Rad thermocycler (Bio-Rad, Marne La Vallée, France). Fragments with a predicted size smaller than 3 kb were amplified with Invitrogen Taq polymerase (Invitrogen, France), according to the manufacturer's protocol. Fragments with a predicted size greater than 3 kb were amplified with the Herculase Enhanced DNA polymerase (Stratagene, Amsterdam Zuidoost, Pays Bas) in accordance with the manufacturer's recommendations. Samples of reaction mixtures were analyzed by electrophoresis in an agarose gel. The fragments amplified by PCR [P3-P4] were purified from the gel with the high purity purification kit from Roche (Roche Diagnostic, France) and sequenced with the PCR primers described in Additional File 6, via a chromosome walking process, by Macrogen (South Korea).
List of abbreviations
Escherichia coli K12
Erwinia carotovora subsp. atroseptica SCRI1043
horizontal genetic transfer
integrative conjugative element
mobile genetic element
, Photorhabdus asymbiotica ATCC43949
Photorhabdus luminescens TT01
Photorhabdus virulence cassette
region of genomic plasticity
Salmonella enterica subsp. enterica Typhi CT18
type three secretion system
type six secretion system
Xenorhabdus nematophila ATCC19061
Xenorhabdus bovienii SS-2004
Yersinia adhesion pathogenicity island
Yersinia pestis CO92
We thank Christine Laroui for technical assistance. We thank the Xenorhabdus genome consortium for access to the Xenorhabdus genomes, R. Ffrench-constant and N. Waterfield for access to the Photorhabdus asymbiotica genomes before public access. We thank Eric Duchaud for critical reading of parts of the manuscript. This study received financial support from the Institut National de la Recherche Agronomique (grant SPE 2007_1133_03), the Agence Nationale de la Recherche (ANR PFTV MicroScope) and the GIS IBiSA.
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