Examination of Prokaryotic Multipartite Genome Evolution through Experimental Genome Reduction

Many bacteria carry two or more chromosome-like replicons. This occurs in pathogens such as Vibrio cholerea and Brucella abortis as well as in many N₂-fixing plant symbionts including all isolates of the alfalfa root-nodule bacteria Sinorhizobium meliloti. Understanding the evolution and role of this multipartite genome organization will provide significant insight into these important organisms; yet this knowledge remains incomplete, in part, because technical challenges of large-scale genome manipulations have limited experimental analyses. The distinct evolutionary histories and characteristics of the three replicons that constitute the S. meliloti genome (the chromosome (3.65 Mb), pSymA megaplasmid (1.35 Mb), and pSymB chromid (1.68 Mb)) makes this a good model to examine this topic. We transferred essential genes from pSymB into the chromosome, and constructed strains that lack pSymB as well as both pSymA and pSymB. This is the largest reduction (45.4%, 3.04 megabases, 2866 genes) of a prokaryotic genome to date and the first removal of an essential chromid. Strikingly, strains lacking pSymA and pSymB (ΔpSymAB) lost the ability to utilize 55 of 74 carbon sources and various sources of nitrogen, phosphorous and sulfur, yet the ΔpSymAB strain grew well in minimal salts media and in sterile soil. This suggests that the core chromosome is sufficient for growth in a bulk soil environment and that the pSymA and pSymB replicons carry genes with more specialized functions such as growth in the rhizosphere and interaction with the plant. These experimental data support a generalized evolutionary model, in which non-chromosomal replicons primarily carry genes with more specialized functions. These large secondary replicons increase the organism''s niche range, which offsets their metabolic burden on the cell (e.g. pSymA). Subsequent co-evolution with the chromosome then leads to the formation of a chromid through the acquisition of functions core to all niches (e.g. pSymB).     

Dinucleotide compositional analysis of Sinorhizobium meliloti using the genome signature: distinguishing chromosomes and plasmids

The symbiotic N₂-fixing α-proteobacterium Sinorhizobium meliloti has three replicons: a circular chromosome (3.7 Mb) and two smaller replicons, pSymA (1.4 Mb) and pSymB (1.7 Mb). Sequence analysis has revealed that an essential gene is carried on pSymB, which brings into question whether pSymB should be considered a chromosome or a plasmid. Based on the criterion that essential genes define a chromosome, several species have been shown to have multiple chromosomes. Many of these species are part of the α subdivision of the Proteobacteria family. Here, additional justification is presented for designating the pSymB replicon as a chromosome. It is shown that chromosomes within a species share a more similar dinucleotide composition, or genome signature, than plasmids do with the host chromosome(s). Dinucleotide signatures were determined for each of the S. meliloti replicons, and, consistent with the suggestion that pSymB is a chromosome, it is shown that the pSymB signature more closely resembles that of the S. meliloti chromosome, while the pSymA signature is typical of other α-proteobacterial plasmids. Electronic Publication     

The complete genome sequence of the dominant Sinorhizobium meliloti field isolate SM11 extends the S. meliloti pan-genome

Isolates of the symbiotic nitrogen-fixing species Sinorhizobium meliloti usually contain a chromosome and two large megaplasmids encoding functions that are absolutely required for the specific interaction of the microsymbiont with corresponding host plants leading to an effective symbiosis. The complete genome sequence, including the megaplasmids pSmeSM11c (related to pSymA) and pSmeSM11d (related to pSymB), was established for the dominant, indigenous S. meliloti strain SM11 that had been isolated during a long-term field release experiment with genetically modified S. meliloti strains. The chromosome, the largest replicon of S. meliloti SM11, is 3,908,022 bp in size and codes for 3785 predicted protein coding sequences. The size of megaplasmid pSmeSM11c is 1,633,319 bp and it contains 1760 predicted protein coding sequences whereas megaplasmid pSmeSM11d is 1,632,395 bp in size and comprises 1548 predicted coding sequences. The gene content of the SM11 chromosome is quite similar to that of the reference strain S. meliloti Rm1021. Comparison of pSmeSM11c to pSymA of the reference strain revealed that many gene regions of these replicons are variable, supporting the assessment that pSymA is a major hot-spot for intra-specific differentiation. Plasmids pSymA and pSmeSM11c both encode unique genes. Large gene regions of pSmeSM11c are closely related to corresponding parts of Sinorhizobium medicae WSM419 plasmids. Moreover, pSmeSM11c encodes further novel gene regions, e.g. additional plasmid survival genes (partition, mobilisation and conjugative transfer genes), acdS encoding 1-aminocyclopropane-1-carboxylate deaminase involved in modulation of the phytohormone ethylene level and genes having predicted functions in degradative capabilities, stress response, amino acid metabolism and associated pathways. In contrast to Rm1021 pSymA and pSmeSM11c, megaplasmid pSymB of strain Rm1021 and pSmeSM11d are highly conserved showing extensive synteny with only few rearrangements. Most remarkably, pSmeSM11b contains a new gene cluster predicted to be involved in polysaccharide biosynthesis. Compilation of the S. meliloti SM11 genome sequence contributes to an extension of the S. meliloti pan-genome.     

Spatiotemporal choreography of chromosome and megaplasmids in the Sinorhizobium meliloti cell cycle

A considerable share of bacterial species maintains multipartite genomes. Precise coordination of genome replication and segregation with cell growth and division is vital for proliferation of these bacteria. The α‐proteobacterium Sinorhizobium meliloti possesses a tripartite genome composed of one chromosome and the megaplasmids pSymA and pSymB. Here, we investigated the spatiotemporal pattern of segregation of these S. meliloti replicons at single cell level. Duplication of chromosomal and megaplasmid origins of replication occurred spatially and temporally separated, and only once per cell cycle. Tracking of FROS (fluorescent repressor operator system)‐labelled origins revealed a strict temporal order of segregation events commencing with the chromosome followed by pSymA and then by pSymB. The repA2B2C2 region derived from pSymA was sufficient to confer the spatiotemporal behaviour of this megaplasmid to a small plasmid. Altering activity of the ubiquitous prokaryotic replication initiator DnaA, either positively or negatively, resulted in an increase in replication initiation events or G1 arrest of the chromosome only. This suggests that interference with DnaA activity does not affect replication initiation control of the megaplasmids.     

Draft genome sequence of Sinorhizobium meliloti RU11/001, a model organism for flagellum structure,motility and chemotaxis

Sinorhizobium meliloti of the order Rhizobiales is a symbiotic nitrogen-fixing bacterium nodulating plants of the genera Medicago, Trigonella and Melilotus and hence is of great agricultural importance. In its free-living state it is motile and capable of modulating its movement patterns in response to chemical attractants. Here, the draft genome consisting of a circular chromosome, the megaplasmids pSymA and pSymB and three accessory plasmids of Sinorhizobium meliloti RU11/001, a model organism for flagellum structure, motility and chemotaxis, is reported.     

The expression of a novel antisense gene mediates incompatibility within the large repABC family of alpha-proteobacterial plasmids

Large extrachromosomal replicons in many members of the alpha-proteobacteria encode genes that are required for plant or animal pathogenesis or symbiosis. Most of these replicons encode repABC genes that control their replication and faithful segregation during cell division. In addition to its chromosome, the plant endosymbiont Sinorhizobium meliloti also maintains the 1.4 Mb pSymA and 1.7 Mb pSymB symbiotic megaplasmids both of which are repABC-type replicons. In all repABC loci that have been characterized, an apparently untranslated intergenic region between the repB and repC genes encodes a strong incompatibility determinant (referred to as incalpha). Here we report the isolation of mutations within the incalpha regions of pSymA and pSymB that eliminate incompatibility. These mutations map to and inactivate a promoter in the intergenic region that drives the expression of an approximately 56 nucleotide untranslated RNA molecule that mediates incompatibility. This gene, that we have named incA, is transcribed antisense to the repABC genes. Our analysis suggests that the incA gene is conserved in repABC loci from a diverse spectrum of bacteria.     

Sequence Analysis of the 144-Kilobase Accessory Plasmid pSmeSM11a, Isolated from a Dominant Sinorhizobium meliloti Strain Identified during a Long-Term Field Release Experiment

The genome of Sinorhizobium meliloti type strain Rm1021 consists of three replicons: the chromosome and two megaplasmids, pSymA and pSymB. Additionally, many indigenous S. meliloti strains possess one or more smaller plasmids, which represent the accessory genome of this species. Here we describe the complete nucleotide sequence of an accessory plasmid, designated pSmeSM11a, that was isolated from a dominant indigenous S. meliloti subpopulation in the context of a long-term field release experiment with genetically modified S. meliloti strains. Sequence analysis of plasmid pSmeSM11a revealed that it is 144,170 bp long and has a mean G+C content of 59.5 mol%. Annotation of the sequence resulted in a total of 160 coding sequences. Functional predictions could be made for 43% of the genes, whereas 57% of the genes encode hypothetical or unknown gene products. Two plasmid replication modules, one belonging to the repABC replicon family and the other belonging to the plasmid type A replicator region family, were identified. Plasmid pSmeSM11a contains a mobilization (mob) module composed of the type IV secretion system-related genes traG and traA and a putative mobC gene. A large continuous region that is about 42 kb long is very similar to a corresponding region located on S. meliloti Rm1021 megaplasmid pSymA. Single-base-pair deletions in the homologous regions are responsible for frameshifts that result in nonparalogous coding sequences. Plasmid pSmeSM11a carries additional copies of the nodulation genes nodP and nodQ that are responsible for Nod factor sulfation. Furthermore, a tauD gene encoding a putative taurine dioxygenase was identified on pSmeSM11a. An acdS gene located on pSmeSM11a is the first example of such a gene in S. meliloti. The deduced acdS gene product is able to deaminate 1-aminocyclopropane-1-carboxylate and is proposed to be involved in reducing the phytohormone ethylene, thus influencing nodulation events. The presence of numerous insertion sequences suggests that these elements mediated acquisition of accessory plasmid modules.     

Formate-dependent autotrophic growth in Sinorhizobium meliloti

It was found that S. meliloti strain SmA818, which is cured of pSymA, could not grow on defined medium containing only formate and bicarbonate as carbon sources. Growth experiments showed that Rm1021 was capable of formate/bicarbonate-dependent growth, suggesting that it was capable of autotrophic-type growth. The annotated genome of S. meliloti Rm1021 contains three formate dehydrogenase genes. A systematic disruption of each of the three formate dehydrogenase genes, as well as the genes encoding determinants of the Calvin-Benson-Bassham, cycle was carried out to determine which of these determinants played a role in growth on this defined medium. The results showed that S. meliloti is capable of formate-dependent autotrophic growth. Formate-dependent autotrophic growth is dependent on the presence of the chromosomally located fdsABCDG operon, as well as the cbb operon carried by pSymB. Growth was also dependent on the presence of either of the two triose-phosphate isomerase genes (tpiA or tpiB) that are found in the genome. In addition, it was found that fdoGHI carried by pSymA encodes a formate dehydrogenase that allows Rm1021 to carry out formate-dependent respiration. Taken together, the data allow us to present a model of how S. meliloti can grow on defined medium containing only formate and bicarbonate as carbon sources.     

Replicon-Dependent Differentiation of Symbiosis-Related Genes in Sinorhizobium Strains Nodulating Glycine max

In order to investigate the genetic differentiation of Sinorhizobium strains nodulating Glycine max and related microevolutionary mechanisms, three housekeeping genes ({"type":"entrez-protein","attrs":{"text":"SMc00019","term_id":"1174089704","term_text":"SMC00019"}}SMc00019, truA, and thrA) and 16 symbiosis-related genes on the chromosome (7 genes), pSymA (6 genes), and pSymB (3 genes) were analyzed. Five distinct species were identified among the test strains by calculating the average nucleotide identity (ANI) of {"type":"entrez-protein","attrs":{"text":"SMc00019","term_id":"1174089704","term_text":"SMC00019"}}SMc00019-truA-thrA: Sinorhizobium fredii, Sinorhizobium sojae, Sinorhizobium sp. I, Sinorhizobium sp. II, and Sinorhizobium sp. III. These species assignments were also supported by population genetics and phylogenetic analyses of housekeeping genes and symbiosis-related genes on the chromosome and pSymB. Different levels of genetic differentiation were observed among these species or different replicons. S. sojae was the most divergent from the other test species and was characterized by its low intraspecies diversity and limited geographic distribution. Intergenic recombination dominated the evolution of 19 genes from different replicons. Intraspecies recombination happened frequently in housekeeping genes and symbiosis-related genes on the chromosome and pSymB, whereas pSymA genes showed a clear pattern of lateral-transfer events between different species. Moreover, pSymA genes were characterized by a lower level of polymorphism and recombination than those on the chromosome and pSymB. Taken together, genes from different replicons of rhizobia might be involved in the establishment of symbiosis with legumes, but these symbiosis-related genes might have evolved differently according to their corresponding replicons.     

Application of Multilocus Sequence Typing To Study the Genetic Structure of Megaplasmids in Medicago-Nodulating Rhizobia

A multilocus sequence typing (MLST) analysis was used to examine the genetic structure and diversity within the two large extrachromosomal replicons in Medicago-nodulating rhizobia (Sinorhizobium meliloti and Sinorhizobium medicae). The allelic diversity within these replicons was high compared to the reported diversity within the corresponding chromosomes of the same strains (P. van Berkum et al., J. Bacteriol. 188:5570-5577, 2006). Also, there was strong localized linkage disequilibrium (LD) between certain pSymA loci: e.g., nodC and nifD. Although both of these observations could be explained by positive (or diversifying) selection by plant hosts, results of tests for positive selection did not provide consistent support for this hypothesis. The strong LD observed between the nodC and nifD genes could also be explained by their close proximity on the pSymA replicon. Evidence was obtained that some nodC alleles had a history of intragenic recombination, while other alleles of this locus had a history of intergenic recombination. Both types of recombination were associated with a decline in symbiotic competence with Medicago sativa as the host plant. The combined observations of LD between the nodC and nifD genes and intragenic recombination within one of these loci indicate that the symbiotic gene region on the pSymA plasmid has evolved as a clonal segment, which has been laterally transferred within the natural populations.Plants of the genus Medicago are legumes that often benefit from a mutualistic symbiosis with rhizobia. The most agriculturally significant species of rhizobia that nodulate these plants are Sinorhizobium meliloti (9) and Sinorhizobium medicae (22). Previously reported population genetic analyses of these bacteria have focused on the study of how allelic variants at multiple loci are distributed within and among natural populations (2, 3, 10, 26, 31, 32). This was also the focus of the present study, but it was extended by examining more loci in many more strains of both species of Sinorhizobium coupled with an analysis having a range of symbiotic genotypes. One goal was to determine if there were any obvious correlations between the megaplasmid genotypes observed and their symbiotic competence. A second goal was to determine if selection by their host plants may have influenced the evolution of their symbiotic relationships.The genes for symbiosis reside on the extrachromosomal replicons pSymA (1,354,226 nucleotides [nt]) and pSMED02 (1,245,408 nt) in the genomes of S. meliloti Rm1021 and S. medicae WSM419, respectively (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE006469","term_id":"25168258","term_text":"AE006469"}}AE006469 and {"type":"entrez-nucleotide","attrs":{"text":"CP000740","term_id":"150031715","term_text":"CP000740"}}CP000740, respectively). Besides these two plasmids, these two strains each harbor one other large extrachromosomal replicon, pSymB (1,683,333 nt) and pSMED01 (1,570,951 nt), respectively (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AL591985","term_id":"30407151","term_text":"AL591985"}}AL591985 and {"type":"entrez-nucleotide","attrs":{"text":"CP000739","term_id":"150030273","term_text":"CP000739"}}CP000739, respectively).Multilocus sequence typing (MLST) (16) is a form of genomic indexing that is commonly used to study the population genetic structure and phylogenetic relatedness within diverse groups of bacteria. In this method, nucleotide sequences of a fixed set of common loci are obtained from a collection of strains, and polymorphic sites among these sequences are used to derive an allelic profile or sequence type (ST) for each genome. Comparisons of the resulting data can be used to infer phylogenetic relationships among the organisms in the sample population, and they also can be used to infer how evolutionary processes, such as recombination and selection, have shaped the genetic structure of the population. For example, levels of intergenic recombination among chromosomal genes in natural populations of Neisseria meningitidis reportedly are relatively high, while corresponding levels within populations of Staphylococcus aureus were low (28). Depending on the specific pairs of loci examined, the levels of linkage disequilibrium (LD) (a lack of intergenic recombination) among several chromosomally carried core genes of S. meliloti were reported to be generally moderate to high (26).The MLST approach has been used to confirm that the chromosomes of S. meliloti and S. medicae are sexually isolated (2, 3, 31) and to provide evidence that horizontal gene transfer (HGT) does occur between the symbiotic megaplasmids of these species (3, 32). It has also been used to demonstrate that levels of intergenic recombination, as indicated by linkage disequilibrium, differ between the three replicons of S. meliloti (26). Levels of intergenic recombination within the pSymB replicons of these strains are generally high, unlike the chromosomes and pSymA replicons within the same strains (26). Bailly et al. (3) hypothesized that the region of the pSymA plasmid that contains the nodulation (nod) genes is frequently transferred in natural populations. They also suggested that selective pressures from the host plant may have influenced both nod gene diversity and patterns of polymorphism across the entire nod gene region.In the present study, multilocus allelic variation of the two megaplasmids was examined among 231 Medicago-nodulating rhizobia that originated primarily from southwest Asia (10). Previously, 91 different chromosomal sequence types (STs) were identified among the same strains from sequence variation in 10 loci (31). This collection of strains had earlier been divided into two closely related groups based on results of multilocus enzyme electrophoresis (10), and this result was subsequently cited in support of separating the Medicago-nodulating rhizobia into the two species S. meliloti and S. medicae (22).The objectives of this study were (i) to use MLST to examine the genetic relationships within and among the large extrachromosomal replicons in S. meliloti and S. medicae, (ii) to estimate levels of intergenic and intragenic recombination in these replicons, (iii) to evaluate the nitrogen-fixing competence of representative symbiotic genotypes with Medicago sativa, and (iv) to determine whether positive (or diversifying) selection may have influenced the genetic structure of the megaplasmids.     

The Decay of the Chromosomally Encoded ccdO157 Toxin–Antitoxin System in the Escherichia coli Species

The origin and the evolution of toxin–antitoxin (TA) systems remain to be uncovered. TA systems are abundant in bacterial chromosomes and are thought to be part of the flexible genome that originates from horizontal gene transfer. To gain insight into TA system evolution, we analyzed the distribution of the chromosomally encoded ccd_O157 system in 395 natural isolates of Escherichia coli. It was discovered in the E. coli O157:H7 strain in which it constitutes a genomic islet between two core genes (folA and apaH). Our study revealed that the folA–apaH intergenic region is plastic and subject to insertion of foreign DNA. It could be composed (i) of a repetitive extragenic palindromic (REP) sequence, (ii) of the ccd_O157 system or subtle variants of it, (iii) of a large DNA piece that contained a ccdA_O157 antitoxin remnant in association with ORFs of unknown function, or (iv) of a variant of it containing an insertion sequence in the ccdA_O157 remnant. Sequence analysis and functional tests of the ccd_O157 variants revealed that 69% of the variants were composed of an active toxin and antitoxin, 29% were composed of an active antitoxin and an inactive toxin, and in 2% of the cases both ORFs were inactive. Molecular evolution analysis showed that ccdB_O157 is under neutral evolution, suggesting that this system is devoid of any biological role in the E. coli species.     

Control of Gluconate Utilization in Sinorhizobium meliloti

The Sinorhizobium meliloti megaplasmid pSymA has previously been implicated in gluconate utilization. We report a locus on pSymA encoding a putative tripartite ATP-independent periplasmic (TRAP) transporter that is required for gluconate utilization. The expression of this locus is negatively regulated by a GntR family regulator encoded adjacent to the transporter operon.     

New Recombination Methods for Sinorhizobium meliloti Genetics

The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative α-proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system. Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S. meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots.     

The ClpXP Protease Is Responsible for the Degradation of the Epsilon Antidote to the Zeta Toxin of the Streptococcal pSM19035 Plasmid

Most bacterial genomes contain different types of toxin-antitoxin (TA) systems. The ω-ϵ-ζ proteinaceous type II TA cassette from the streptococcal pSM19035 plasmid is a member of the ϵ/ζ family, which is commonly found in multiresistance plasmids and chromosomes of various human pathogens. Regulation of type II TA systems relies on the proteolysis of antitoxin proteins. Under normal conditions, the Epsilon antidote neutralizes the Zeta toxin through the formation of a tight complex. In this study, we show, using both in vivo and in vitro analyses, that the ClpXP protease is responsible for Epsilon antitoxin degradation. Using in vivo studies, we examined the stability of the plasmids with active or inactive ω-ϵ-ζ TA cassettes in B. subtilis mutants that were defective for different proteases. Using in vitro assays, the degradation of purified His₆-Epsilon by the His₆-Lon_Bs, ClpP_Bs, and ClpX_Bs proteases from B. subtilis was analyzed. Additionally, we showed that purified Zeta toxin protects the Epsilon protein from rapid ClpXP-catalyzed degradation.     

Functional and Structural Insights of a Staphylococcus aureus Apoptotic-like Membrane Peptide from a Toxin-Antitoxin Module

We report a functional type I toxin-antitoxin (TA) module expressed by a human pathogen, Staphylococcus aureus. TA systems consist of stable toxins and labile antitoxins encoded within small genetic modules widespread in eubacteria and archaea. TA genes provide stress adaptation and protection against DNA loss or invasion. The genes encoding the SprA1 toxic peptide (PepA1) and the SprA1_AS RNA antitoxin are within a pathogenicity island on opposite strands and possess a 3′ overlap. To prevent peptide toxicity during S. aureus growth, PepA1 expression from stable (half-life > 3 h) SprA1 is repressed by elevated amounts of unstable (half-life = ∼10 mn) SprA1_AS. In vivo, PepA1 localizes at the bacterial membrane and triggers S. aureus death. Based on NMR and CD data, its solution structure was solved and is a long bent, interrupted helix. Molecular dynamics simulations indicate that PepA1 compaction and helical content fluctuate in accordance with its cytoplasm or membrane location. When inserted into the S. aureus membrane, the PepA1 conformation switches to a ∼7-nm-long continuous helix, presumably forming pores to alter membrane integrity. PepA1 expression is induced upon acidic and oxidative stresses by reducing SprA1_AS levels. As an altruistic behavior during infection, some cells may induce the expression of that toxin that would facilitate departure from the host immune cells for spreading.     

Mycobacterium tuberculosis Type II Toxin-Antitoxin Systems: Genetic Polymorphisms and Functional Properties and the Possibility of Their Use for Genotyping

Various genetic markers such as IS-elements, DR-elements, variable number tandem repeats (VNTR), single nucleotide polymorphisms (SNPs) in housekeeping genes and other groups of genes are being used for genotyping. We propose a different approach. We suggest the type II toxin-antitoxin (TA) systems, which play a significant role in the formation of pathogenicity, tolerance and persistence phenotypes, and thus in the survival of Mycobacterium tuberculosis in the host organism at various developmental stages (colonization, infection of macrophages, etc.), as the marker genes. Most genes of TA systems function together, forming a single network: an antitoxin from one pair may interact with toxins from other pairs and even from other families. In this work a bioinformatics analysis of genes of the type II TA systems from 173 sequenced genomes of M. tuberculosis was performed. A number of genes of type II TA systems were found to carry SNPs that correlate with specific genotypes. We propose a minimally sufficient set of genes of TA systems for separation of M. tuberculosis strains at nine basic genotype and for further division into subtypes. Using this set of genes, we genotyped a collection consisting of 62 clinical isolates of M. tuberculosis. The possibility of using our set of genes for genotyping using PCR is also demonstrated.     

Comparative genomic hybridisation and ultrafast pyrosequencing revealed remarkable differences between the Sinorhizobium meliloti genomes of the model strain Rm1021 and the field isolate SM11

Genomic variation between the Sinorhizobium meliloti model strain Rm1021 and the field isolate SM11 was assessed by using the genome-wide S. meliloti Rm1021 Sm6k-oligonucleotide microarray in a comparative genomic hybridisation experiment. Several gene clusters present in the Rm1021 genome are missing in the SM11 genome. In detail, three missing gene clusters were identified for the chromosome, five for megaplasmid pSymA and two for megaplasmid pSymB. To confirm these hybridisation results, the draft genome sequence of the S. meliloti field isolate SM11 was established by 454-pyrosequencing. Three sequencing runs on the ultrafast Genome Sequencer 20 System yielded 112.5 million bases. These could be assembled into 905 larger contigs resulting in a nearly 15-fold coverage of the 7.1Mb SM11 genome. The missing gene regions identified by comparative genomic hybridisation could be confirmed by the results of the 454-sequencing project. An in-depth analysis of these gene regions resulted in the following findings: (i) a complete type I restriction/modification system encoded by a composite transposon is absent in the chromosome of strain SM11. (ii) Most of the Rm1021 denitrification genes and the complete siderophore biosynthesis operon were found to be missing on SM11 megaplasmid pSymA. (iii) S. meliloti SM11 megaplasmid pSymB lacks a complete cell surface carbohydrate synthesis gene cluster. (iv) Several genes that are absent in the SM11 genome could be assigned to insertion sequences and transposons.     

Chromosomal toxin-antitoxin loci can diminish large-scale genome reductions in the absence of selection

Superintegrons (SIs) are chromosomal genetic elements containing assemblies of genes, each flanked by a recombination sequence (attC site) targeted by the integron integrase. SIs may contain hundreds of attC sites and intrinsic instability is anticipated; yet SIs are remarkably stable. This implies that either selective pressure maintains the genes or mechanisms exist which favour their persistence in the absence of selection. Toxin/antitoxin (TA) systems encode a stable toxin and a specific, unstable antitoxin. Once activated, the continued synthesis of the unstable antitoxin is necessary for cell survival. A bioinformatic search of accessible microbial genomes for SIs and TA systems revealed that large SIs harboured TA gene cassettes while smaller SIs did not. We demonstrated the function of TA loci in different genomic contexts where large-scale deletions can occur; in SIs and in a 165 kb dispensable region of the Escherichia coli genome. When devoid of TA loci, large-scale genome loss was evident in both environments. The inclusion of two TA loci, relBE1 and parDE1, which we identified in the Vibrio vulnificus SI rendered these environments refractory to gene loss. Thus, chromosomal TA loci can stabilize massive SI arrays and limit the extensive gene loss that is a hallmark of reductive evolution.     

Directed Construction and Analysis of a Sinorhizobium meliloti pSymA Deletion Mutant Library

Resources from the Sinorhizobium meliloti Rm1021 open reading frame (ORF) plasmid libraries were used in a medium-throughput method to construct a set of 50 overlapping deletion mutants covering all of the Rm1021 pSymA megaplasmid except the replicon region. Each resulting pSymA derivative carried a defined deletion of approximately 25 ORFs. Various phenotypes, including cytochrome c respiration activity, the ability of the mutants to grow on various carbon and nitrogen sources, and the symbiotic effectiveness of the mutants with alfalfa, were analyzed. This approach allowed us to systematically evaluate the potential impact of regions of Rm1021 pSymA for their free-living and symbiotic phenotypes.