Plant seedlings of peas, tomatoes, and cucumbers exude compounds that are needed for growth and chemoattraction of Rhizobium leguminosarum bv. viciae 3841 and Azospirillum brasilense Sp7.

This study characterizes seedling exudates of peas, tomatoes, and cucumbers at the level of chemical composition and functionality. A plant experiment confirmed that Rhizobium leguminosarum bv. viciae 3841 enhanced growth of pea shoots, while Azospirillum brasilense Sp7 supported growth of pea, tomato, and cucumber roots. Chemical analysis of exudates after 1 day of seedling incubation in water yielded differences between the exudates of the three plants. Most remarkably, cucumber seedling exudate did not contain detectable sugars. All exudates contained amino acids, nucleobases/nucleosides, and organic acids, among other compounds. Cucumber seedling exudate contained reduced glutathione. Migration on semi solid agar plates containing individual exudate compounds as putative chemoattractants revealed that R. leguminosarum bv. viciae was more selective than A. brasilense, which migrated towards any of the compounds tested. Migration on semi solid agar plates containing 1:1 dilutions of seedling exudate was observed for each of the combinations of bacteria and exudates tested. Likewise, R. leguminosarum bv. viciae and A. brasilense grew on each of the three seedling exudates, though at varying growth rates. We conclude that the seedling exudates of peas, tomatoes, and cucumbers contain everything that is needed for their symbiotic bacteria to migrate and grow on.

Rhizopine biosensors for plant-dependent control of bacterial gene expression.

Engineering signalling between plants and microbes could be exploited to establish host-specificity between plant-growth-promoting bacteria and target crops in the environment. We previously engineered rhizopine-signalling circuitry facilitating exclusive signalling between rhizopine-producing (RhiP) plants and model bacterial strains. Here, we conduct an in-depth analysis of rhizopine-inducible expression in bacteria. We characterize two rhizopine-inducible promoters and explore the bacterial host-range of rhizopine biosensor plasmids. By tuning the expression of rhizopine uptake genes, we also construct a new biosensor plasmid pSIR05 that has minimal impact on host cell growth in vitro and exhibits markedly improved stability of expression in situ on RhiP barley roots compared to the previously described biosensor plasmid pSIR02. We demonstrate that a sub-population of Azorhizobium caulinodans cells carrying pSIR05 can sense rhizopine and activate gene expression when colonizing RhiP barley roots. However, these bacteria were mildly defective for colonization of RhiP barley roots compared to the wild-type parent strain. This work provides advancement towards establishing more robust plant-dependent control of bacterial gene expression and highlights the key challenges remaining to achieve this goal.

Engineered plant control of associative nitrogen fixation.

Engineering N2-fixing symbioses between cereals and diazotrophic bacteria represents a promising strategy to sustainably deliver biologically fixed nitrogen (N) in agriculture. We previously developed novel transkingdom signaling between plants and bacteria, through plant production of the bacterial signal rhizopine, allowing control of bacterial gene expression in association with the plant. Here, we have developed both a homozygous rhizopine producing (RhiP) barley line and a hybrid rhizopine uptake system that conveys upon our model bacterium Azorhizobium caulinodans ORS571 (Ac) 103-fold improved sensitivity for rhizopine perception. Using this improved genetic circuitry, we established tight rhizopine-dependent transcriptional control of the nitrogenase master regulator nifA and the N metabolism σ-factor rpoN, which drove nitrogenase expression and activity in vitro and in situ by bacteria colonizing RhiP barley roots. Although in situ nitrogenase activity was suboptimally effective relative to the wild-type strain, activation was specific to RhiP barley and was not observed on the roots of wild-type plants. This work represents a key milestone toward the development of a synthetic plant-controlled symbiosis in which the bacteria fix N2 only when in contact with the desired host plant and are prevented from interaction with nontarget plant species.

Minimal gene set from Sinorhizobium (Ensifer) meliloti pSymA required for efficient symbiosis with Medicago.

Reduction of N2 gas to ammonia in legume root nodules is a key component of sustainable agricultural systems. Root nodules are the result of a symbiosis between leguminous plants and bacteria called rhizobia. Both symbiotic partners play active roles in establishing successful symbiosis and nitrogen fixation: while root nodule development is mostly controlled by the plant, the rhizobia induce nodule formation, invade, and perform N2 fixation once inside the plant cells. Many bacterial genes involved in the rhizobia-legume symbiosis are known, and there is much interest in engineering the symbiosis to include major nonlegume crops such as corn, wheat, and rice. We sought to identify and combine a minimal bacterial gene complement necessary and sufficient for symbiosis. We analyzed a model rhizobium, Sinorhizobium (Ensifer) meliloti, using a background strain in which the 1.35-Mb symbiotic megaplasmid pSymA was removed. Three regions representing 162 kb of pSymA were sufficient to recover a complete N2-fixing symbiosis with alfalfa, and a targeted assembly of this gene complement achieved high levels of symbiotic N2 fixation. The resulting gene set contained just 58 of 1,290 pSymA protein-coding genes. To generate a platform for future synthetic manipulation, the minimal symbiotic genes were reorganized into three discrete nod, nif, and fix modules. These constructs will facilitate directed studies toward expanding the symbiosis to other plant partners. They also enable forward-type approaches to identifying genetic components that may not be essential for symbiosis, but which modulate the rhizobium's competitiveness for nodulation and the effectiveness of particular rhizobia-plant symbioses.

qPCR assay targeting Bradyrhizobium japonicum shows that row spacing and soybean density affects Bradyrhizobium population.

The ability for a soybean plant to be efficiently nodulated when grown as a crop is dependent on the number of effective Bradyrhizobium japonicum that can be found in close proximity to the developing seedling shortly after planting. In Manitoba, the growing of soybean as a crop has increased from less than 500 000 acres in 2008 to over 2.3 million acres in 2017. Since the large increase in soybean production is relatively recent, populations of B. japonicum have not yet developed. In response to this, we developed a primer pair that can identify B. japonicum, and be used to determine the titre found in field soil. Their utility was demonstrated by being used to determine whether row spacing of soybean affects B. japonicum populations, as well as to follow B. japonicum populations in a soybean field over the course of a field season. The data show that plant density can affect B. japonicum populations. Moreover, evidence is presented that suggests plant development affects overall B. japonicum populations.

Optimizing Rhizobium-legume symbioses by simultaneous measurement of rhizobial competitiveness and N2 fixation in nodules.

Legumes tend to be nodulated by competitive rhizobia that do not maximize nitrogen (N2) fixation, resulting in suboptimal yields. Rhizobial nodulation competitiveness and effectiveness at N2 fixation are independent traits, making their measurement extremely time-consuming with low experimental throughput. To transform the experimental assessment of rhizobial competitiveness and effectiveness, we have used synthetic biology to develop reporter plasmids that allow simultaneous high-throughput measurement of N2 fixation in individual nodules using green fluorescent protein (GFP) and barcode strain identification (Plasmid ID) through next generation sequencing (NGS). In a proof-of-concept experiment using this technology in an agricultural soil, we simultaneously monitored 84 different Rhizobium leguminosarum strains, identifying a supercompetitive and highly effective rhizobial symbiont for peas. We also observed a remarkable frequency of nodule coinfection by rhizobia, with mixed occupancy identified in ∼20% of nodules, containing up to six different strains. Critically, this process can be adapted to multiple Rhizobium-legume symbioses, soil types, and environmental conditions to permit easy identification of optimal rhizobial inoculants for field testing to maximize agricultural yield.

Control of nitrogen fixation in bacteria that associate with cereals.

Legumes obtain nitrogen from air through rhizobia residing in root nodules. Some species of rhizobia can colonize cereals but do not fix nitrogen on them. Disabling native regulation can turn on nitrogenase expression, even in the presence of nitrogenous fertilizer and low oxygen, but continuous nitrogenase production confers an energy burden. Here, we engineer inducible nitrogenase activity in two cereal endophytes (Azorhizobium caulinodans ORS571 and Rhizobium sp. IRBG74) and the well-characterized plant epiphyte Pseudomonas protegens Pf-5, a maize seed inoculant. For each organism, different strategies were taken to eliminate ammonium repression and place nitrogenase expression under the control of agriculturally relevant signals, including root exudates, biocontrol agents and phytohormones. We demonstrate that R. sp. IRBG74 can be engineered to result in nitrogenase activity under free-living conditions by transferring a nif cluster from either Rhodobacter sphaeroides or Klebsiella oxytoca. For P. protegens Pf-5, the transfer of an inducible cluster from Pseudomonas stutzeri and Azotobacter vinelandii yields ammonium tolerance and higher oxygen tolerance of nitrogenase activity than that from K. oxytoca. Collectively, the data from the transfer of 12 nif gene clusters between 15 diverse species (including Escherichia coli and 12 rhizobia) help identify the barriers that must be overcome to engineer a bacterium to deliver a high nitrogen flux to a cereal crop.

Engineering transkingdom signalling in plants to control gene expression in rhizosphere bacteria.

The root microbiota is critical for agricultural yield, with growth-promoting bacteria able to solubilise phosphate, produce plant growth hormones, antagonise pathogens and fix N2. Plants control the microorganisms in their immediate environment and this is at least in part through direct selection, the immune system, and interactions with other microorganisms. Considering the importance of the root microbiota for crop yields it is attractive to artificially regulate this environment to optimise agricultural productivity. Towards this aim we express a synthetic pathway for the production of the rhizopine scyllo-inosamine in plants. We demonstrate the production of this bacterial derived signal in both Medicago truncatula and barley and show its perception by rhizosphere bacteria, containing bioluminescent and fluorescent biosensors. This study lays the groundwork for synthetic signalling networks between plants and bacteria, allowing the targeted regulation of bacterial gene expression in the rhizosphere for delivery of useful functions to plants.

A Bacterial Expression Vector Archive (BEVA) for Flexible Modular Assembly of Golden Gate-Compatible Vectors.

We present a Bacterial Expression Vector Archive (BEVA) for the modular assembly of bacterial vectors compatible with both traditional and Golden Gate cloning, utilizing the Type IIS restriction enzyme Esp3I, and have demonstrated its use for Golden Gate cloning in Escherichia coli. Ideal for synthetic biology and other applications, this modular system allows a rapid, low-cost assembly of new vectors tailored to specific tasks. Using the principles outlined here, new modules (e.g., origin of replication for plasmids in other bacteria) can easily be designed for specific applications. It is hoped that this vector construction system will be expanded by the scientific community over time by creation of novel modules through an open source approach. To demonstrate the potential of the system, three example vectors were constructed and tested. The Golden Gate level 1 vector pOGG024 (pBBR1-based broad-host range and medium copy number) was used for gene expression in laboratory-cultured Rhizobium leguminosarum. The Golden Gate level 1 vector pOGG026 (RK2-based broad-host range, lower copy number and stable in the absence of antibiotic selection) was used to demonstrate bacterial gene expression in nitrogen-fixing nodules on pea plant roots formed by R. leguminosarum. Finally, the level 2 cloning vector pOGG216 (RK2-based broad-host range, medium copy number) was used to construct a dual reporter plasmid expressing green and red fluorescent proteins.

Succinoglycan Production Contributes to Acidic pH Tolerance in Sinorhizobium meliloti Rm1021.

In this work, the hypothesis that exopolysaccharide plays a role in the survival of Sinorhizobium meliloti at low pH levels is addressed. When S. meliloti was grown at pH 5.75, synthesis of succinoglycan increased, whereas synthesis of galactoglucan decreased. Succinoglycan that was isolated from cultures grown at low pH had a lower degree of polymerization relative to that which was isolated from cultures grown at neutral pH, suggesting that low-molecular weight (LMW) succinoglycan might play a role in adaptation to low pH. Mutants unable to produce succinoglycan or only able to produce high-molecular weight polysaccharide were found to be sensitive to low pH. However, strains unable to produce LMW polysaccharide were 10-fold more sensitive. In response to low pH, transcription of genes encoding proteins for succinoglycan, glycogen, and cyclic ß(1-2) glucans biosynthesis increased, while those encoding proteins necessary for the biosynthesis of galactoglucan decreased. While changes in pH did not affect the production of glycogen or cyclic ß(1-2) glucan, it was found that the inability to produce cyclic ß(1-2) glucan did contribute to pH tolerance in the absence of succinoglycan. Finally, in addition to being sensitive to low pH, a strain carrying mutations in exoK and exsH, which encode the glycanases responsible for the cleavage of succinoglycan to LMW succinoglycan, exhibited a delay in nodulation and was uncompetitive for nodule occupancy. Taken together, the data suggest that the role for LMW succinoglycan in nodule development may be to enhance survival in the colonized curled root hair.

Common dyes used to determine bacterial polysaccharides on agar are affected by medium acidification.

In this work, we highlight effects of pH on bacterial phenotypes when using the bacteriological dyes Aniline blue, Congo red, and Calcofluor white to analyze polysaccharide production. A study of galactose catabolism in Sinorhizobium meliloti led to the isolation of a mutation in dgoK1, which was observed to overproduce exopolysaccharides when grown in the presence of galactose. When this mutant strain was spotted onto plates containing Aniline blue, Congo red, or Calcofluor white, the intensity of the associated staining was strikingly different from that of the wild type. Additionally, a Calcofluor dull phenotype was observed, suggesting production of a polysaccharide other than succinoglycan. Further investigation of this phenotype revealed that these results were dependent on medium acidification, as buffering at pH 6 had no effect on these phenotypes, while medium buffered at pH 6.5 resulted in a reversal of the phenotypes. Screening for mutants of the dgoK1 strain that were negative for the Aniline blue phenotype yielded a strain carrying a mutation in tkt2, which is annotated as a putative transketolase. Consistent with the plate phenotypes, when this mutant was grown in broth cultures, it did not acidify its growth medium. Overall, this work shows that caution should be exercised in evaluating polysaccharide phenotypes based strictly on the use of dyes.

Role of O2 in the Growth of Rhizobium leguminosarum bv. viciae 3841 on Glucose and Succinate.

Insertion sequencing (INSeq) analysis of Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O2 was used to understand how O2 concentration alters metabolism. Two transcriptional regulators were required for growth on glucose (pRL120207 [eryD] and RL0547 [phoB]), five were required on succinate (pRL100388, RL1641, RL1642, RL3427, and RL4524 [ecfL]), and three were required on 1% O2 (pRL110072, RL0545 [phoU], and RL4042). A novel toxin-antitoxin system was identified that could be important for generation of new plasmidless rhizobial strains. Rlv3841 appears to use the methylglyoxal pathway alongside the Entner-Doudoroff (ED) pathway and tricarboxylic acid (TCA) cycle for optimal growth on glucose. Surprisingly, the ED pathway was required for growth on succinate, suggesting that sugars made by gluconeogenesis must undergo recycling. Altered amino acid metabolism was specifically needed for growth on glucose, including RL2082 (gatB) and pRL120419 (opaA, encoding omega-amino acid:pyruvate transaminase). Growth on succinate specifically required enzymes of nucleobase synthesis, including ribose-phosphate pyrophosphokinase (RL3468 [prs]) and a cytosine deaminase (pRL90208 [codA]). Succinate growth was particularly dependent on cell surface factors, including the PrsD-PrsE type I secretion system and UDP-galactose production. Only RL2393 (glnB, encoding nitrogen regulatory protein PII) was specifically essential for growth on succinate at 1% O2, conditions similar to those experienced by N2-fixing bacteroids. Glutamate synthesis is constitutively activated in glnB mutants, suggesting that consumption of 2-ketoglutarate may increase flux through the TCA cycle, leading to excess reductant that cannot be reoxidized at 1% O2 and cell death. IMPORTANCE: Rhizobium leguminosarum, a soil bacterium that forms N2-fixing symbioses with several agriculturally important leguminous plants (including pea, vetch, and lentil), has been widely utilized as a model to study Rhizobium-legume symbioses. Insertion sequencing (INSeq) has been used to identify factors needed for its growth on different carbon sources and O2 levels. Identification of these factors is fundamental to a better understanding of the cell physiology and core metabolism of this bacterium, which adapts to a variety of different carbon sources and O2 tensions during growth in soil and N2 fixation in symbiosis with legumes.

Symbiotic Nitrogen Fixation and the Challenges to Its Extension to Nonlegumes.

Access to fixed or available forms of nitrogen limits the productivity of crop plants and thus food production. Nitrogenous fertilizer production currently represents a significant expense for the efficient growth of various crops in the developed world. There are significant potential gains to be had from reducing dependence on nitrogenous fertilizers in agriculture in the developed world and in developing countries, and there is significant interest in research on biological nitrogen fixation and prospects for increasing its importance in an agricultural setting. Biological nitrogen fixation is the conversion of atmospheric N2 to NH3, a form that can be used by plants. However, the process is restricted to bacteria and archaea and does not occur in eukaryotes. Symbiotic nitrogen fixation is part of a mutualistic relationship in which plants provide a niche and fixed carbon to bacteria in exchange for fixed nitrogen. This process is restricted mainly to legumes in agricultural systems, and there is considerable interest in exploring whether similar symbioses can be developed in nonlegumes, which produce the bulk of human food. We are at a juncture at which the fundamental understanding of biological nitrogen fixation has matured to a level that we can think about engineering symbiotic relationships using synthetic biology approaches. This minireview highlights the fundamental advances in our understanding of biological nitrogen fixation in the context of a blueprint for expanding symbiotic nitrogen fixation to a greater diversity of crop plants through synthetic biology.

Use of plant colonizing bacteria as chassis for transfer of N2-fixation to cereals.

Engineering cereal crops that are self-supported by nitrogen fixation has been a dream since the 1970s when nitrogenase was transferred from Klebsiella pneumoniae to Escherichia coli. A renewed interest in this area has generated several new approaches with the common aim of transferring nitrogen fixation to cereal crops. Advances in synthetic biology have afforded the tools to rationally engineer microorganisms with traits of interest. Nitrogenase biosynthesis has been a recent target for the application of new synthetic engineering tools. Early successes in this area suggest that the transfer of nitrogenase and other supporting traits to microorganisms that already closely associate with cereal crops is a logical approach to deliver nitrogen to cereal crops.

Exopolysaccharide production in response to medium acidification is correlated with an increase in competition for nodule occupancy.

Sinorhizobium meliloti strains unable to utilize galactose as a sole carbon source, due to mutations in the De-Ley Doudoroff pathway (dgoK), were previously shown to be more competitive for nodule occupancy. In this work, we show that strains carrying this mutation have galactose-dependent exopolysaccharide (EPS) phenotypes that were manifested as aberrant Calcofluor staining as well as decreased mucoidy when in an expR(+) genetic background. The aberrant Calcofluor staining was correlated with changes in the pH of the growth medium. Strains carrying dgoK mutations were subsequently demonstrated to show earlier acidification of their growth medium that was correlated with an increase expression of genes associated with succinoglycan biosynthesis as well as increased accumulation of high and low molecular weight EPS in the medium. In addition, it was shown that the acidification of the medium was dependent on the inability of S. meliloti strains to initiate the catabolism of galactose. To more fully understand why strains carrying the dgoK allele were more competitive for nodule occupancy, early nodulation phenotypes were investigated. It was found that strains carrying the dgoK allele had a faster rate of nodulation. In addition, nodule competition experiments using genetic backgrounds unable to synthesize either succinoglycan or EPSII were consistent with the hypothesis that the increased competition phenotype was dependent upon the synthesis of succinoglycan. Fluorescent microscopy experiments on infected root-hair cells, using the acidotropic dye Lysotracker Red DND-99, provide evidence that the colonized curled root hair is an acidic compartment.

Physiology, genetics, and biochemistry of carbon metabolism in the alphaproteobacterium Sinorhizobium meliloti.

A large proportion of genes within a genome encode proteins that play a role in metabolism. The Alphaproteobacteria are a ubiquitous group of bacteria that play a major role in a number of environments. For well over 50 years, carbon metabolism in Rhizobium has been studied at biochemical and genetic levels. Here, we review the pre- and post-genomics literature of the metabolism of the alphaproteobacterium Sinorhizobium meliloti. This review provides an overview of carbon metabolism that is useful to readers interested in this organism and to those working on other organisms that do not follow other model system paradigms.

Phylogenetic analysis of erythritol catabolic loci within the Rhizobiales and proteobacteria.

BACKGROUND: The ability to use erythritol as a sole carbon source is not universal among the Rhizobiaceae. Based on the relatedness to the catabolic genes in Brucella it has been suggested that the eryABCD operon may have been horizontally transferred into Rhizobium. During work characterizing a locus necessary for the transport and catabolism of erythritol, adonitol and L-arabitol in Sinorhizobium meliloti, we became interested in the differences between the erythritol loci of S. meliloti and R. leguminosarum. Utilizing the Ortholog Neighborhood Viewer from the DOE Joint Genome Institute database it appeared that loci for erythritol and polyol utilization had distinct arrangements that suggested these loci may have undergone genetic rearrangements. RESULTS: A data set was established of genetic loci containing erythritol/polyol orthologs for 19 different proteobacterial species. These loci were analyzed for genetic content and arrangement of genes associated with erythritol, adonitol and L-arabitol catabolism. Phylogenetic trees were constructed for core erythritol catabolic genes and contrasted with the species phylogeny. Additionally, phylogenetic trees were constructed for genes that showed differences in arrangement among the putative erythritol loci in these species. CONCLUSIONS: Three distinct erythritol/polyol loci arrangements have been identified that reflect metabolic need or specialization. Comparison of the phylogenetic trees of core erythritol catabolic genes with species phylogeny provides evidence that is consistent with these loci having been horizontally transferred from the alpha-proteobacteria into both the beta and gamma-proteobacteria. ABC transporters within these loci adopt 2 unique genetic arrangements, and although biological data suggests they are functional erythritol transporters, phylogenetic analysis suggests they may not be orthologs and probably should be considered analogs. Finally, evidence for the presence of paralogs, and xenologs of erythritol catabolic genes in some of the genomes included in the analysis is provided.

Inability to catabolize galactose leads to increased ability to compete for nodule occupancy in Sinorhizobium meliloti.

A mutant unable to utilize galactose was isolated in Sinorhizobium meliloti strain Rm1021. The mutation was found to be in a gene annotated dgoK1, a putative 2-keto-3-deoxygalactonokinase. The genetic region was isolated on a complementing cosmid and subsequently characterized. Based on genetic and bioinformatic evidence, the locus encodes all five enzymes (galD, dgoK, dgoA, SMc00883, and ilvD1) involved in the De Ley-Doudoroff pathway for galactose catabolism. Although all five genes are present, genetic analysis suggests that the galactonase (SMc00883) and the dehydratase (ilvD1) are dispensable with respect to the ability to catabolize galactose. In addition, we show that the transport of galactose is partially facilitated by the arabinose transporter (AraABC) and that both glucose and galactose compete with arabinose for transport. Quantitative reverse transcription-PCR (qRT-PCR) data show that in a dgoK background, the galactose locus is constitutively expressed, and the induction of the ara locus seems to be enhanced. Assays of competition for nodule occupancy show that the inability to catabolize galactose is correlated with an increased ability to compete for nodule occupancy.

Genetic characterization of a complex locus necessary for the transport and catabolism of erythritol, adonitol and L-arabitol in Sinorhizobium meliloti.

The Sinorhizobium meliloti locus necessary for the utilization of erythritol as a sole carbon source, contains 17 genes, including genes that encode an ABC transporter necessary for the transport of erythritol, as well as the genes encoding EryA, EryB, EryC, TpiB and the regulators EryD and EryR (SMc01615). Construction of defined deletions and complementation experiments show that the other genes at this locus encode products that are necessary for the catabolism of adonitol (ribitol) and l-arabitol, but not d-arabitol. These analyses show that aside from one gene that is specific for the catabolism of l-arabitol (SMc01619, lalA), the rest of the catabolic genes are necessary for both polyols (SMc01617, rbtC; SMc01618, rbtB; SMc01622, rbtA). Genetic and biochemical data show that in addition to utilizing erythritol as a substrate, EryA is also capable of utilizing adonitol and l-arabitol. Similarly, transport experiments using labelled erythritol show that adonitol, l-arabitol and erythritol share a common transporter (MptABCDE). Quantitative RT-PCR experiments show that transcripts containing genes necessary for adonitol and l-arabitol utilization are induced by these sugars in an eryA-dependent manner.

Glycerol utilization by Rhizobium leguminosarum requires an ABC transporter and affects competition for nodulation.

Plasmid curing has shown that the ability to use glycerol as a carbon source is plasmid-encoded in Rhizobium leguminosarum. We isolated the locus responsible for glycerol utilization from plasmid pRleVF39c in R. leguminosarum bv. viciae VF39. This region was analyzed by DNA sequencing and mutagenesis. The locus encompasses a gene encoding GlpR (a DeoR regulator), genes encoding an ABC transporter, and genes glpK and glpD, encoding a kinase and dehydrogenase, respectively. All the genes except the regulatory gene glpR were organized into a single operon, and were required for growth on glycerol. The glp operon was strongly induced by both glycerol and glycerol 3-phosphate, as well as by pea seed exudate. GlpR repressed the operon in the absence of inducer. Mutation of genes encoding the ABC transporter abolished all transport of glycerol in transport assays using radiolabelled glycerol. This confirms that, unlike in other organisms such as Escherichia coli and Pseudomonas aeruginosa, which use facilitated diffusion, glycerol uptake occurs by an active process in R. leguminosarum. Since the glp locus is highly conserved in all sequenced R. leguminosarum and Rhizobium etli strains, as well as in Sinorhizobium spp. and Agrobacterium spp. and other alphaproteobacteria, this process for glycerol uptake is probably widespread. Mutants unable to use glycerol were deficient in competitiveness for nodulation of peas compared with the wild-type, suggesting that glycerol catabolism confers an advantage upon the bacterium in the rhizosphere or in the infection thread.