Co-inoculation with antibiotic-producing bacteria to increase colonization and nodulation by rhizobia

A study was conducted to determine whether colonization of legume roots and nodulation byRhizobium meliloti andBradyrhizobium japonicum could be enhanced by using inocula containing microorganisms that produce antibiotics suppressing soil or rhizosphere inhabitants but not the root-nodule bacteria. An antibiotic-producing strain of Pseudomonas and one of Bacillus were isolated, and mutants ofR. meliloti andB. japonicum sp. resistant to the antibiotics were used. The colonization of the alfalfa rhizosphere and nodulation byR. meliloti were enhanced by inoculation of soil withPseudomonas sp. in soil initially containing 2.7×10⁵ R. meliloti per g. The colonization of soybean roots byB. japonicum was enhanced by inoculating soil with three cell densities ofBacillus sp., and nodulation was stimulated byBacillus sp. added at two cell densities. In some tests, the dry weights of soybeans and seed yield increased as a result of these treatments, and co-inoculation with Bacillus also increased pod formation. Inoculation of seeds withBacillus sp. and the root-nodule bacterium enhanced nodulation of soybeans and alfalfa, but colonization byB. japonicum andR. meliloti was stimulated only during the early period of plant growth. Studies were also conducted withStreptomyces griseus and isolates ofR. meliloti andB. japonicum resistant to products of the actinomycete. Nodulation of alfalfa byR. meliloti was little or not affected by the actinomycete alone; however, both nodulation and colonization were enhanced if the soil was initially amended with chitin andS. griseus was also added. Chitin itself did not affectR. meliloti. Treatments of seeds with chitin orS. griseus alone did not enhance colonization of alfalfa roots byR. meliloti or soybean roots byB. japonicum, but the early colonization of the roots by both bacterial species was promoted if the seeds received both chitin andS. griseus; this treatment also increased nodulation and dry weights of alfalfa and soybeans and the N content of alfalfa. It is suggested that co-inoculation of legumes with antibiotic-producing microorganisms and root-nodule bacteria resistant to those antibiotics is a promising means of promoting nodulation and possibly nitrogen fixation.     

Factors affecting co-inoculation with antibiotic-producing bacteria to enhance rhizobial colonization and nodulation

Co-inoculation with antibiotic-producing bacteria and rhizobia resistant to those antibiotics has been proposed as a means of promoting colonization and nodulation of legumes by root-nodule bacteria. A study was conducted to establish some of the factors affecting co-inoculation with antibiotic-producing strains of Bacillus and Streptomyces griseus. The stimulation of Rhizobium meliloti and yield and N uptake by alfalfa was enhanced with increasing inoculum size of Bacillus sp. S. griseus and chitin added to soil increased nodulation of soybeans by Bradyrhizobium japonicum and increased nodulation, yield, and number of pods on a second crop grown in the same soil. Bacillus sp. persisted in soil in sufficient numbers for at least 51 days to increase colonization of soybean roots by B. japonicum. The populations of S. griseus, Bacillus sp., and antibiotic-resistant isolates of R. meliloti and B. japonicum fell after their addition to seeds. Nevertheless, a benefical effect by the antibiotic-producing bacteria was evident on R. meliloti colonization of the rhizosphere, nodulation, and yield of alfalfa grown from seeds stored 94 days and on B. japonicum colonization, nodule number, yield, and seed weight of soybeans grown from seeds stored 90 days. Because non-antibiotic-producing derivatives of Bacillus sp. and S. griseus did not promote colonization or nodulation of alfalfa roots by R. meliloti, the benefit of this co-inoculation is a result of antibiotic formation.     

Variation of Microbial Rhizosphere Communities in Response to Crop Species, Soil Origin, and Inoculation with Sinorhizobium meliloti L33

Abstract A greenhouse study with soil–plant microcosms was conducted in order to compare the effect of crop species, soil origin, and a bacterial inoculant on the establishment of microbial communities colonizing plant roots. Two crop species, alfalfa (Medicago sativa) and rye (Secale cereale), were grown separately in two soils collected from agricultural fields at different locations and with differing histories of leguminous crop rotation. A subset of microcosms was inoculated at 10⁶ cfu g^-1 soil with the luciferase marker gene-tagged Sinorhizobium meliloti strain L33, a symbiotic partner of M. sativa. Microbial consortia were collected from the rhizospheres of alfalfa after 10 weeks of incubation and from rye after 11 weeks. S. meliloti L33 populations were one to two orders of magnitude higher in the rhizospheres of alfalfa than of rye. In soil with previous alfalfa cultivation, 80% of the alfalfa nodules were colonized by indigenous bacteria, while in the other soil alfalfa was colonized almost exclusively (>90%) with S. meliloti L33. Three community-level targeting approaches were used to characterize the variation of the extracted microbial rhizosphere consortia: (1) Community level physiological profiles (CLPP), (2) fatty acid methyl ester analysis (FAME), and (3) diversity of PCR amplified 16S rRNA target sequences from directly extracted ribosomes, determined by temperature gradient gel electrophoresis (TGGE). All approaches identified the crop species as the major determinant of microbial community characteristics. Consistently, the influence of soil was of minor importance, while a modification of the alfalfa-associated microbial community structure after inoculation with S. meliloti L33 was only consistently observed by using TGGE. Received: 20 October 1999; Accepted: 15 January 2000; Online Publication: 18 July 2000     

Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meliloti in the alfalfa (Medicago sativa) rhizosphere

The colonization ability of Pseudomonas fluorescens F113rif in alfalfa rhizosphere and its interactions with the alfalfa microsymbiont Sinorhizobium meliloti EFB1 has been analyzed. Both strains efficiently colonize the alfalfa rhizosphere in gnotobiotic systems and soil microcosms. Colonization dynamics of F113rif on alfalfa were similar to other plant systems previously studied but it is displaced by S. meliloti EFB1, lowering its population by one order of magnitude in co-inoculation experiments. GFP tagged strains used to study the colonization patterns by both strains indicated that P. fluorescens F113rif did not colonize root hairs while S. meliloti EFB1 extensively colonized this niche. Inoculation of F113rif had a deleterious effect on plants grown in gnotobiotic systems, possibly because of the production of HCN and the high populations reached in these systems. This effect was reversed by co-inoculation. Pseudomonas fluorescens F113 derivatives with biocontrol and bioremediation abilities have been developed in recent years. The results obtained support the possibility of using this bacterium in conjunction with alfalfa for biocontrol or rhizoremediation technologies.     

Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and “Sarcina lutea” did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and “S. lutea” were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.     

Synthesis,release, and transmission of alfalfa signals to rhizobial symbionts

In addition to the flavonoids exuded by many legumes as signals to their rhizobial symbionts, alfalfa (Medicago sativa L.) releases two betaines, trigonelline and stachydrine, that induce nodulation (nod) genes inRhizobium meliloti. Experiments with¹⁴C-phenylalanine in the presence and absence of phenylalanine ammonia-lyase inhibitors show that exudation of flavonoidnod-gene inducers from alfalfa roots is linked closely to their concurrent synthesis. In contrast, flavonoid and betainenod-gene inducers are already present on mature seeds before they are released during germination. Alfalfa seeds and roots release structurally differentnod-gene-inducing signals in the absence of rhizobia. WhenR. meliloti is added to roots, medicarpin, a classical isoflavonoid phytoalexin normally elicited by pathogens, and anod-gene-inducing compound, formononetin-7-O-(6-O-malonylglycoside), are exuded. Carbon flow through the phenylpropanoid pathway and into the flavonoid pathway via chalcone synthase is controlled by complexcis-acting sequences andtrans-acting factors which are not completely understood. Even less information is available on molecular regulation of the two other biosynthetic pathways that produce trigonelline and stachydrine. Presumably the three separate pathways for producingnod-gene inducers in some way protect the plant against fluctuations in the production or transmission of the two classes of signals. Factors influencing transmission of alfalfanod-gene inducers through soil are poorly defined, but solubility differences between hydrophobic flavonoids and hydrophilic betaines suggest that the diffusional traits of these molecules are not similar. Knowledge derived from studies of how legumes regulate rhizobial symbionts with natural plant products offers a basis for defining new fundamental concepts of rhizosphere ecology.     

Molecular evidence of genetic modification of Sinorhizobium meliloti: enhanced PCB bioremediation

The genome of the nitrogen-fixing soil bacterium Sinorhizobium meliloti does not possess genes for bioremediation of aromatic pollutants. It has the well-known ability to interact specifically with the leguminous alfalfa plant, Medicago sativa. Our previous work has shown enhanced degradation of the nitroaromatic compound 2,4-dinitrotoluene (DNT) when a plasmid containing degradative genes was introduced in it. In this study we report molecular evidence of the transfer of a polychlorinated biphenyl (PCB)-biodegradative plasmid pE43 to S. meliloti strain USDA 1936. Several standard analytical tests and plant growth chamber studies were conducted to test the ability of S. meliloti to degrade 2′,3,4-PCB congener. Alfalfa plant alone was able to degrade 30% of PCBs compared with control. No enhanced dechlorination was noted when alfalfa plant was grown with wild-type S. meliloti, and when alfalfa plant was grown with the S. meliloti electrotransformants (genetically modified) dechlorination of PCBs was more than twice that when alfalfa plant was grown with wild-type S. meliloti. When alfalfa plant was grown with uncharacterized mixed culture (containing nodule formers), almost equally significant PCB degradation was observed. The significance of this work is that the naturally occurring nitrogen-fixing soil bacterium S. meliloti (genetically modified) has the ability to enhance fertility of soil in association with the leguminous alfalfa plant while simultaneously enhancing bioremediation of PCB-contaminated soils. Enhanced bioremediation of PCB and robust alfalfa plant growth was also noted when uncharacterized mixed cultures containing alfalfa plant nodule formers were used.

     

Efficient rhizosphere colonization by Pseudomonas fluorescens f113 mutants unable to form biofilms on abiotic surfaces

Motility is a key trait for rhizosphere colonization by Pseudomonas fluorescens. Mutants with reduced motility are poor competitors, and hypermotile, more competitive phenotypic variants are selected in the rhizosphere. Flagellar motility is a feature associated to planktonic, free‐living single cells, and although it is necessary for the initial steps of biofilm formation, bacteria in biofilm lack flagella. To test the correlation between biofilm formation and rhizosphere colonization, we have used P. fluorescens F113 hypermotile derivatives and mutants affected in regulatory genes which in other bacteria modulate biofilm development, namely gacS (G), sadB (S) and wspR (W). Mutants affected in these three genes and a hypermotile variant (V35) isolated from the rhizosphere were impaired in biofilm formation on abiotic surfaces, but colonized the alfalfa root apex as efficiently as the wild‐type strain, indicating that biofilm formation on abiotic surfaces and rhizosphere colonization follow different regulatory pathways in P. fluorescens. Furthermore, a triple mutant gacSsadBwspR (GSW) and V35 were more competitive than the wild‐type strain for root‐tip colonization, suggesting that motility is more relevant in this environment than the ability to form biofilms on abiotic surfaces. Microscopy showed the same root colonization pattern for P. fluorescens F113 and all the derivatives: extensive microcolonies, apparently held to the rhizoplane by a mucigel that seems to be plant produced. Therefore, the ability to form biofilms on abiotic surfaces does not necessarily correlates with efficient rhizosphere colonization or competitive colonization.     

Interactions between Pseudomonas syringae pv. tabaci and Two Rhizosphere Hosts, Medicago sativa and Avena sativa

The interactions between Pseudomonas syringae pv. tabaci and either nodulating alfalfa (Medicago sativa) or oat (Avena sativa) seedlings were examined to further our understanding of this rhizosphere association. P. syringae pv. tabaci produces and releases a toxin, tabtoxinine-β-lactam (TβL), that inactivates glutamine synthetase (GS). Sinorhizobium meliloti grew well in the presence of TβL in culture and on alfalfa roots. The alfalfa symbiont, S. meliloti, and its bacteroids contained TβL-sensitive glutamine synthetases and TβL detoxifying-β-lactamase. The GS of alfalfa leaves is also sensitive to TβL, but GS activity was unaffected in infested plants. Toxin production was apparently suppressed in the alfalfa and nitrate-fed oat rhizospheres since these plants survived and retained significant amounts of leaf GS activity. The water-soluble extracts of these rhizospheres inhibited TPL production in culture and the inhibition was correlated with the amount of reduced nitrogen present. Furthermore, representative mixtures of pure ammonium and amino acids inhibited TβL production in culture in a concentration dependent manner. Thus, a bi-directional interaction occurs between the nitrogen metabolism of alfalfa and oat and TβL production by P. syringae pv. tabaci.     

Ankyrin protein kinases: a novel type of plant kinase gene whose expression is induced by osmotic stress in alfalfa

Interaction between Medicago spp. and Sinorhizobium meliloti leads to the development of a novel organ, the root nodule. A gene, Msapk1, encoding a novel type of plant protein kinase containing a N-terminal region with an ankyrin domain, was identified and shown to be expressed both in S. meliloti-infected and spontaneous nodules in alfalfa. This gene is not exclusively associated to nodulation since its expression was detected in other plant organs. Several genes coding for ankyrin protein kinases (APKs) were detected in various plants and animals. Three closest A. thaliana homologues of Msapk1 were identified in databases and two of them were shown to express differentially in various organs using gene-specific RT-PCR. In contrast, Southern analysis suggests that a single-copy gene exists in diploid M. truncatula. By screening a M. truncatula BAC library the Mtapk1 genomic region was isolated and sequenced. Two neighbouring genes showing homologies to previously identified sequences in data banks were detected in the vicinity of the Mtapk1 gene and compared to similar regions of the three Atapk genes. The distribution of exons/introns was the same for all expressed genes of both species although Mtapk1 contained larger introns. Upon osmotic stress Msapk1 expression was induced in roots of alfalfa starting from three hours up to two days of treatment. These data suggest that Msapk1, involved in alfalfa osmotic stress responses, belongs to a novel class of plant protein kinases.     

Contribution of calystegine catabolic plasmid to competitive colonization of the rhizosphere of calystegine-producing plants by Sinorhizobium meliloti Rm41

Calystegines are plant secondary metabolites produced by the roots of a few plant species, and the ability to catabolize calystegines is infrequent in rhizosphere bacteria. In Sinorhizobium meliloti Rm41, the endosymbiont of the legume Medicago sativa, this ability results from the presence of the genes cac (for calystegine catabolism) located on the nonsymbiotic plasmid pRme41a. The effect of the cac catabolic plasmid pRme41a on the ability of Rm41 to colonize the rhizosphere of calystegine-positive plants was studied using derivatives of Rm41 with or without cac catabolic plasmid. When strains were inoculated alone, the presence of a cac catabolic plasmid had no effect on their colonization of the rhizosphere, regardless of whether plants produced calystegines or not. However, a spontaneous rifampicin-resistant mutant of Rm41 containing a cac catabolic plasmid reached population levels in the rhizosphere of calystegine-positive plants that were several orders of magnitude higher than those of the same strain without the plasmid, when each was co-inoculated with a derivative of Rm41 cured of pRme41a. In contrast, the cac catabolic plasmid provided little or no selective advantage in the rhizosphere of calystegine-negative plants. In conclusion, the cac catabolic plasmid pRme41a can contribute to the ability of S. meliloti Rm41 to colonize the rhizosphere of alternative, nonlegume plant hosts producing calystegines.     

Identification of Sinorhizobium meliloti Early Symbiotic Genes by Use of a Positive Functional Screen

The soil bacterium Sinorhizobium meliloti establishes nitrogen-fixing symbiosis with its leguminous host plant, alfalfa, following a series of continuous signal exchanges. The complexity of the changes of alfalfa root structures during symbiosis and the amount of S. meliloti genes with unknown functions raised the possibility that more S. meliloti genes may be required for early stages of the symbiosis. A positive functional screen of the entire S. meliloti genome for symbiotic genes was carried out using a modified in vivo expression technology. A group of genes and putative genes were found to be expressed in early stages of the symbiosis, and 23 of them were alfalfa root exudate inducible. These 23 genes were further separated into two groups based on their responses to apigenin, a known nodulation (nod) gene inducer. The group of six genes not inducible by apigenin included the lsrA gene, which is essential for the symbiosis, and the dgkA gene, which is involved in the synthesis of cyclic β-1,2-glucan required for the S. meliloti-alfalfa symbiosis. In the group of 17 apigenin-inducible genes, most have not been previously characterized in S. meliloti, and none of them belongs to the nod gene family. The identification of this large group of alfalfa root exudate-inducible S. meliloti genes suggests that the interactions in the early stages of the S. meliloti and alfalfa symbiosis could be complex and that further characterization of these genes will lead to a better understanding of the symbiosis.     

The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation of alfalfa plants

Background  

Soil bacteria collectively known as Rhizobium, characterized by their ability to establish beneficial symbiosis with legumes, share several common characteristics with pathogenic bacteria when infecting the host plant. Recently, it was demonstrated that a fadD mutant of Sinorhizobium meliloti is altered in the control of swarming, a type of co-ordinated movement previously associated with pathogeniCity, and is also impaired in nodulation efficiency on alfalfa roots. In the phytopathogen Xanthomonas campestris, a fadD homolog (rpfB) forms part of a cluster of genes involved in the regulation of pathogeniCity factors. In this work, we have investigated the role in swarming and symbiosis of SMc02161, a S. meliloti fadD-linked gene.     

A nodule endophytic Bacillus megaterium strain isolated from Medicago polymorpha enhances growth,promotes nodulation by Ensifer medicae and alleviates salt stress in alfalfa plants

A Gram‐positive, fast‐growing, endophytic bacterium was isolated from root nodules of Medicago polymorpha and identified as Bacillus megaterium. The isolate, named NMp082, co‐inhabited nodules with the symbiotic rhizobium Ensifer medicae. B. megaterium NMp082 contained nifH and nodD genes that were 100% identical to those of Ensifer meliloti, an unusual event that suggested previous lateral gene transfer from a different rhizobial species. Despite the presence of nodulation and nitrogen fixation genes, the endophyte was not able to form effective nodules; however, it induced nodule‐like unorganised structures in alfalfa roots. Axenic inoculation promoted plant growth in M. polymorpha, Medicago lupulina, Medicago truncatula and Medicago sativa, and co‐inoculation with E. medicae enhanced growth and nodulation of Medicago spp. plants compared with inoculation with either bacterium alone. B. megaterium NMp082 also induced tolerance to salt stress in alfalfa and Arabidopsis plants. The ability to produce indole acetic acid (IAA) and the 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase activity displayed by the endophyte in vitro might explain the observed plant growth promotion and salt stress alleviation. The isolate was also highly tolerant to salt stress, water deficit and to the presence of different heavy metals. The newly characterised endophytic bacterium possessed specific characteristics that point at potential applications to sustain plant growth and nodulation under abiotic stress.     

Indirect effects of polycyclic aromatic hydrocarbon contamination on microbial communities in legume and grass rhizospheres

Background and aims

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) is accelerated in the presence of plants, due to the stimulation of rhizosphere microbes by plant exudates (nonspecific enhancement). However, plants may also recruit specific microbial groups in response to PAH stress (specific enhancement). In this study, plant effects on the development of rhizosphere microbial communities in heterogeneously contaminated soils were assessed for three grasses (ryegrass, red fescue and Yorkshire fog) and four legumes (white clover, chickpea, subterranean clover and red lentil).

Methods

Plants were cultivated using a split-root model with their roots divided between two independent pots containing either uncontaminated soil or PAH-contaminated soil (pyrene or phenanthrene). Microbial community development in the two halves of the rhizosphere was assessed by T-RFLP (bacterial and fungal community) or DGGE (bacterial community), and by 16S rRNA gene tag-pyrosequencing.

Results

In legume rhizospheres, the microbial community structure in the uncontaminated part of the split-root model was significantly influenced by the presence of PAH-contamination in the other part of the root system (indirect effect), but this effect was not seen for grasses. In the contaminated rhizospheres, Verrucomicrobia and Actinobacteria showed increased populations, and there was a dramatic increase in Denitratisoma numbers, suggesting that this genus may be important in rhizoremediation processes.

Conclusion

Our results show that Trifolium and other legumes respond to PAH-contamination stress in a systemic manner, to influence the microbial diversity in their rhizospheres.     

Genotypic diversity among rhizospheric bacteria of three legumes assessed by cultivation-dependent and cultivation-independent techniques

The genotypic diversity of rhizospheric bacteria of 3 legumes including Vigna radiata, Arachis hypogaea and Acacia mangium was compared by using cultivation-dependent and cultivation-independent methods. For cultivation-dependent method, Random amplified polymorphic DNA (RAPD) profiles revealed that the bacterial genetic diversity of V. radiata and A. mangium rhizospheres was higher than that of A. hypogaea rhizosphere. For cultivation-independent method, Denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA genes revealed the difference in bacterial community and diversity of rhizospheres collected from 3 legumes. The ribotype richness which indicates species diversity, was highest in V. radiata rhizosphere, followed by A. hypogaea and A. mangium rhizospheres, respectively. Three kinds of media were used to cultivate different target groups of bacteria. The result indicates that the communities of cultivable bacteria in 3 rhizospheres recovered from nutrient agar (NA) medium were mostly different from each other, while Bradyrhizobium selective medium (BJSM) and nitrogen-free medium shaped the communities of cultivable bacteria. Nine isolates grown on BJSM were identified by 16S rRNA gene sequence analysis. These isolates were very closely related (with 96% to 99% identities) to either one of the three groups including Cupriavidus-Ralstonia group, Bacillus group and Bradyrhizobium-Bosea-Afipia group. The rhizospheres were also examined for their enzymatic patterns. Of 19 enzymes tested, 3 rhizospheres were distinguishable by the presence or the absence of leucine acrylamidase and acid phosphatase. The selected cultivable bacteria recovered from NA varied in their abilities to produce indole-acetic acid and ammnonia. The resistance to 10 antibiotics was indistinguishable among bacteria isolated from different rhizospheres.     

Quantitation of Adsorption of Rhizobia in Low Numbers to Small Legume Roots

Bacteria adsorbed in low numbers to alfalfa or clover root surfaces were counted after incubation of seedlings in mineral solution with very dilute inocula (less than 10⁵ bacteria per ml) of an antibiotic-resistant strain under defined conditions. After specified washing, bacteria which remained adsorbed to roots were selectively quantitated by culturing the roots embedded in yeast extract-mannitol-antibiotic agar and counting the microcolonies along the root surface; the range was from about 1 bacterium per root (estimated as the most probable number) to 50 bacteria per cm of root length (by direct counting). This simple procedure can be used with any pair of small-rooted plant and antibiotic-resistant bacterium, requires bacterial concentrations comparable to those frequently found in soils, and yields macroscopic localization and distribution data for adsorbed bacteria over the root surface. The number of adsorbed bacteria was proportional to the size of the inoculum. One of every four Rhizobium meliloti cells adsorbed in very low numbers to alfalfa roots resulted in the formation of a nodule. Overall adsorption of various symbiotic and nonsymbiotic bacterial strains to alfalfa and clover roots did not reflect the specificities of these legumes for their respective microsymbionts, R. meliloti and R. trifolii.     

A New Genetic Locus in Sinorhizobium meliloti Is Involved in Stachydrine Utilization

Stachydrine, a betaine released by germinating alfalfa seeds, functions as an inducer of nodulation genes, a catabolite, and an osmoprotectant in Sinorhizobium meliloti. Two stachydrine-inducible genes were found in S. meliloti 1021 by mutation with a Tn5-luxAB promoter probe. Both mutant strains (S10 and S11) formed effective alfalfa root nodules, but neither grew on stachydrine as the sole carbon and nitrogen source. When grown in the absence or presence of salt stress, S10 and S11 took up [¹⁴C]stachydrine as well as wild-type cells did, but neither used stachydrine effectively as an osmoprotectant. In the absence of salt stress, both S10 and S11 took up less [¹⁴C]proline than wild-type cells did. S10 and S11 appeared to colonize alfalfa roots normally in single-strain tests, but when mixed with the wild-type strain, their rhizosphere counts were reduced more than 50% (P ≤ 0.01) relative to the wild type. These results suggest that stachydrine catabolism contributes to root colonization. DNA sequence analysis identified the mutated locus in S11 as putA, and the luxAB fusion in that gene was induced by proline as well as stachydrine. DNA that restored the capacity of mutant S10 to catabolize stachydrine contained a new open reading frame, stcD. All data are consistent with the concept that stcD codes for an enzyme that produces proline by demethylation of N-methylproline, a degradation product of stachydrine.