This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Rhizobium leguminosarum genome

The genome sequence of the α-proteobacterial N2-fixing symbiont of legumes, Rhizobium leguminosarum, is described, revealing a 'core' and an 'accessory' component.

Until recently, all clostridia producing neurotoxins able to cause paralysis symptomatic of botulism were deemed to be Clostridium botulinum. Defining Cl. botulinum on the basis of this single phenotypic trait has resulted in the species encompassing metabolically very diverse organisms, and four distinct phenotypic groups are recognized within this taxon (designated groups I-IV). Nucleic acid hybridization and 16S ribosomal RNA sequencing studies have revealed the presence of four phylogenetically distinct lineages within the species, which correlate with these phenotypic divisions. In addition to marked phenotypic and genotypic heterogeneity between groups, the taxonomy of the species is further complicated by the existence of strains which are closely related, if not genetically identifiable, to members of each Cl. botulinum group, but are non-toxigenic. Furthermore, strains of species other than Cl. botulinum (viz. Cl. baratii, Cl. butyricum) have been found which express botulinum neurotoxin (BoNT). Great advances have been made in recent years in elucidating the nucleotide sequences of genes encoding the various BoNT antigenic types (A through to G). Genealogical trees derived from BoNTs show marked discordance with those depicting 'natural' relationships inferred from 16S rRNA and phenotypic clusters, and strong evidence exists for BoNT gene transfer between some groups of Cl. botulinum (e.g. groups I and II), and with non-botulinum species. Botulinum neurotoxin is produced by Cl. botulinum as a non-covalently bound progenitor toxin complex of two or more protein components. Information on the evolutionary histories of the various non-toxic progenitor proteins is currently limited, although there is evidence of gene recombination. In particular, chimera-like or mosaic non-toxic-non-haemagglutinins (NTNH) genes in group I Cl. botulinum have been described, and it is now apparent that the phylogeny of the NTNHs is not going to 'mirror' that of botulinal neurotoxins, although their genes are physically contiguous. In this article, the current state of knowledge of the phylogenetics of the species Cl. botulinum and its neurotoxins is reviewed, and a view is presented that a nomenclature based rigidly on BoNT production is no longer tenable.

BackgroundThe rhizosphere is the microbe-rich zone around plant roots and is a key determinant of the biosphere's productivity. Comparative transcriptomics was used to investigate general and plant-specific adaptations during rhizosphere colonization. Rhizobium leguminosarum biovar viciae was grown in the rhizospheres of pea (its legume nodulation host), alfalfa (a non-host legume) and sugar beet (non-legume). Gene expression data were compared to metabolic and transportome maps to understand adaptation to the rhizosphere.ResultsCarbon metabolism was dominated by organic acids, with a strong bias towards aromatic amino acids, C1 and C2 compounds. This was confirmed by induction of the glyoxylate cycle required for C2 metabolism and gluconeogenesis in all rhizospheres. Gluconeogenesis is repressed in R. leguminosarum by sugars, suggesting that although numerous sugar and putative complex carbohydrate transport systems are induced in the rhizosphere, they are less important carbon sources than organic acids. A common core of rhizosphere-induced genes was identified, of which 66% are of unknown function. Many genes were induced in the rhizosphere of the legumes, but not sugar beet, and several were plant specific. The plasmid pRL8 can be considered pea rhizosphere specific, enabling adaptation of R. leguminosarum to its host. Mutation of many of the up-regulated genes reduced competitiveness for pea rhizosphere colonization, while two genes specifically up-regulated in the pea rhizosphere reduced colonization of the pea but not alfalfa rhizosphere.ConclusionsComparative transcriptome analysis has enabled differentiation between factors conserved across plants for rhizosphere colonization as well as identification of exquisite specific adaptation to host plants.

The number of solute-binding protein-dependent transporters in rhizobia is dramatically increased compared with the majority of other bacteria so far sequenced. This increase may be due to the high affinity of solute-binding proteins for solutes, permitting the acquisition of a broad range of growth-limiting nutrients from soil and the rhizosphere. The transcriptional induction of these transporters was studied by creating a suite of plasmid and integrated fusions to nearly all ATP-binding cassette (ABC) and tripartite ATP-independent periplasmic (TRAP) transporters of Sinorhizobium meliloti. In total, specific inducers were identified for 76 transport systems, amounting to Ϸ47% of the ABC uptake systems and 53% of the TRAP transporters in S. meliloti. Of these transport systems, 64 are previously uncharacterized in Rhizobia and 24 were induced by solutes not known to be transported by ABC-or TRAP-uptake systems in any organism. This study provides a global expression map of one of the largest transporter families (transportome) and an invaluable tool to both understand their solute specificity and the relationships between members of large paralogous families.ATP-binding cassette ͉ expression ͉ high throughput ͉ transport ͉ tripartite ATP-independent periplasmic

Rhizobium leguminosarum bv. viciae forms nitrogen-fixing nodules on several legumes, including pea (Pisum sativum) and vetch (Vicia cracca), and has been widely used as a model to study nodule biochemistry. To understand the complex biochemical and developmental changes undergone by R. leguminosarum bv. viciae during bacteroid development, microarray experiments were first performed with cultured bacteria grown on a variety of carbon substrates (glucose, pyruvate, succinate, inositol, acetate, and acetoacetate) and then compared to bacteroids. Bacteroid metabolism is essentially that of dicarboxylate-grown cells (i.e., induction of dicarboxylate transport, gluconeogenesis and alanine synthesis, and repression of sugar utilization). The decarboxylating arm of the tricarboxylic acid cycle is highly induced, as is ␥-aminobutyrate metabolism, particularly in bacteroids from early (7-day) nodules. To investigate bacteroid development, gene expression in bacteroids was analyzed at 7, 15, and 21 days postinoculation of peas. This revealed that bacterial rRNA isolated from pea, but not vetch, is extensively processed in mature bacteroids. In early development (7 days), there were large changes in the expression of regulators, exported and cell surface molecules, multidrug exporters, and heat and cold shock proteins. fix genes were induced early but continued to increase in mature bacteroids, while nif genes were induced strongly in older bacteroids. Mutation of 37 genes that were strongly upregulated in mature bacteroids revealed that none were essential for nitrogen fixation. However, screening of 3,072 mini-Tn5 mutants on peas revealed previously uncharacterized genes essential for nitrogen fixation. These encoded a potential magnesium transporter, an AAA domain protein, and proteins involved in cytochrome synthesis.

Plants engineer the rhizosphere to their advantage by secreting various nutrients and secondary metabolites. Coupling transcriptomic and metabolomic analyses of the pea (Pisum sativum) rhizosphere, a suite of bioreporters has been developed in Rhizobium leguminosarum bv viciae strain 3841, and these detect metabolites secreted by roots in space and time. Fourteen bacterial lux fusion bioreporters, specific for sugars, polyols, amino acids, organic acids, or flavonoids, have been validated in vitro and in vivo. Using different bacterial mutants (nodC and nifH), the process of colonization and symbiosis has been analyzed, revealing compounds important in the different steps of the rhizobium-legume association. Dicarboxylates and sucrose are the main carbon sources within the nodules; in ineffective (nifH) nodules, particularly low levels of sucrose were observed, suggesting that plant sanctions affect carbon supply to nodules. In contrast, high myo-inositol levels were observed prior to nodule formation and also in nifH senescent nodules. Amino acid biosensors showed different patterns: a g-aminobutyrate biosensor was active only inside nodules, whereas the phenylalanine bioreporter showed a high signal also in the rhizosphere. The bioreporters were further validated in vetch (Vicia hirsuta), producing similar results. In addition, vetch exhibited a local increase of nod gene-inducing flavonoids at sites where nodules developed subsequently. These bioreporters will be particularly helpful in understanding the dynamics of root exudation and the role of different molecules secreted into the rhizosphere.

By analyzing successive lifestyle stages of a model Rhizobium–legume symbiosis using mariner-based transposon insertion sequencing (INSeq), we have defined the genes required for rhizosphere growth, root colonization, bacterial infection, N2-fixing bacteroids, and release from legume (pea) nodules. While only 27 genes are annotated as nif and fix in Rhizobium leguminosarum, we show 603 genetic regions (593 genes, 5 transfer RNAs, and 5 RNA features) are required for the competitive ability to nodulate pea and fix N2. Of these, 146 are common to rhizosphere growth through to bacteroids. This large number of genes, defined as rhizosphere-progressive, highlights how critical successful competition in the rhizosphere is to subsequent infection and nodulation. As expected, there is also a large group (211) specific for nodule bacteria and bacteroid function. Nodule infection and bacteroid formation require genes for motility, cell envelope restructuring, nodulation signaling, N2 fixation, and metabolic adaptation. Metabolic adaptation includes urea, erythritol and aldehyde metabolism, glycogen synthesis, dicarboxylate metabolism, and glutamine synthesis (GlnII). There are 17 separate lifestyle adaptations specific to rhizosphere growth and 23 to root colonization, distinct from infection and nodule formation. These results dramatically highlight the importance of competition at multiple stages of a Rhizobium–legume symbiosis.

BacA is an integral membrane protein, the mutation of which leads to increased resistance to the antimicrobial peptides bleomycin and Bac7 1-35 and a greater sensitivity to SDS and vancomycin in Rhizobium leguminosarum bv. viciae, R. leguminosarum bv. phaseoli, and Rhizobium etli. The growth of Rhizobium strains on dicarboxylates as a sole carbon source was impaired in bacA mutants but was overcome by elevating the calcium level. While bacA mutants elicited indeterminate nodule formation on peas, which belong to the galegoid tribe of legumes, bacteria lysed after release from infection threads and mature bacteroids were not formed. Microarray analysis revealed almost no change in a bacA mutant of R. leguminosarum bv. viciae in free-living culture. In contrast, 45 genes were more-than 3-fold upregulated in a bacA mutant isolated from pea nodules. Almost half of these genes code for cell membrane components, suggesting that BacA is crucial to alterations that occur in the cell envelope during bacteroid development. In stark contrast, bacA mutants of R. leguminosarum bv. phaseoli and R. etli elicited the formation of normal determinate nodules on their bean host, which belongs to the phaseoloid tribe of legumes. Bacteroids from these nodules were indistinguishable from the wild type in morphology and nitrogen fixation. Thus, while bacA mutants of bacteria that infect galegoid or phaseoloid legumes have similar phenotypes in free-living culture, BacA is essential only for bacteroid development in indeterminate galegoid nodules.