Sinorhizobium meliloti chemotaxis to quaternary ammonium compounds is mediated by the chemoreceptor McpX

The bacterium Sinorhizobium meliloti is attracted to seed exudates of its host plant alfalfa (Medicago sativa). Since quaternary ammonium compounds (QACs) are exuded by germinating seeds, we assayed chemotaxis of S. meliloti towards betonicine, choline, glycine betaine, stachydrine and trigonelline. The wild type displayed a positive response to all QACs. Using LC–MS, we determined that each germinating alfalfa seed exuded QACs in the nanogram range. Compared to the closely related nonhost species, spotted medic (Medicago arabica), unique profiles were released. Further assessments of single chemoreceptor deletion strains revealed that an mcpX deletion strain displayed little to no response to these compounds. Differential scanning fluorimetry showed interaction of the isolated periplasmic region of McpX (McpX^PR and McpX_34‐306) with QACs. Isothermal titration calorimetry experiments revealed tight binding to McpX^PR with dissociation constants (K_d) in the nanomolar range for choline and glycine betaine, micromolar K_d for stachydrine and trigonelline and a K_d in the millimolar range for betonicine. Our discovery of S. meliloti chemotaxis to plant‐derived QACs adds another role to this group of compounds, which are known to serve as nutrient sources, osmoprotectants and cell‐to‐cell signalling molecules. This is the first report of a chemoreceptor that mediates QACs taxis through direct binding.     

Regulation of motility by the ExpR/Sin quorum-sensing system in Sinorhizobium meliloti

A successful symbiotic relationship between Sinorhizobium meliloti and its host Medicago sativa (alfalfa) depends on several signaling mechanisms, such as the biosynthesis of exopolysaccharides (EPS) by S. meliloti. Previous work in our laboratory has shown that a quorum-sensing mechanism controls the production of the symbiotically active EPS II. Recent microarray analysis of the whole-genome expression profile of S. meliloti reveals that the ExpR/Sin quorum-sensing system regulates additional physiological processes that include low-molecular-weight succinoglycan production, nitrogen utilization, metal transport, motility, and chemotaxis. Nearly half of the flagellar genes and their dependence on quorum sensing are prominently displayed in our microarray analyses. We extend those observations in this work and confirm the findings by real-time PCR expression analysis of selected genes, including the flaF, flbT, flaC, cheY1, and flgB genes, involved in motility and chemotaxis. These genes code for regulators of flagellum synthesis, the chemotactic response, or parts of the flagellar apparatus. Gene expression analyses and visualization of flagella by electron microscopy performed at different points in the growth phase support our proposed model in which quorum sensing downregulates motility in S. meliloti. We demonstrate that the ExpR/Sin quorum-sensing system controls motility gene expression through the VisN/VisR/Rem relay. We also show that the ExoS-dependent two-component system suppresses motility gene expression through VisN and Rem in parallel to quorum sensing. This study contributes to our understanding of the mechanisms that govern motility in S. meliloti.     

Release and Modification of nod-Gene-Inducing Flavonoids from Alfalfa Seeds

Traces of luteolin, an important rhizobial nod gene inducer in Rhizobium meliloti, are released by alfalfa (Medicago sativa L.) seeds, but most luteolin in the seed exudate is conjugated as luteolin-7-O-glucoside (L7G). Processes affecting the production of luteolin from L7G in seed exudate are poorly understood. Results from this study establish that (a) seed coats are the primary source of flavonoids, including L7G, in seed exudate; (b) these flavonoids exist in seeds before imbibition; and (c) both the host plant and the symbiotic R. meliloti probably can hydrolyze L7G to luteolin. Glycolytic cleavage of L7G is promoted by glucosidase activity released from sterile seeds during the first 4 hours of imbibition. Thus, L7G from imbibing alfalfa seeds may serve as a source of the nod-gene-inducing luteolin and thereby facilitate root nodulation by R. meliloti.     

Functional analysis of nine putative chemoreceptor proteins in Sinorhizobium meliloti

The genome of the symbiotic soil bacterium Sinorhizobium meliloti contains eight genes coding for methyl-accepting chemotaxis proteins (MCPs) McpS to McpZ and one gene coding for a transducer-like protein, IcpA. Seven of the MCPs are localized in the cytoplasmic membrane via two membrane-spanning regions, whereas McpY and IcpA lack such hydrophobic regions. The periplasmic regions of McpU, McpV, and McpX contain the small-ligand-binding domain Cache. In addition, McpU possesses the ligand-binding domain TarH. By probing gene expression with lacZ fusions, we have identified mcpU and mcpX as being highly expressed. Deletion of any one of the receptor genes caused impairments in the chemotactic response toward most organic acids, amino acids, and sugars in a swarm plate assay. The data imply that chemoreceptor proteins in S. meliloti can sense more than one class of carbon source and suggest that many or all receptors work as an ensemble. Tactic responses were virtually eliminated for a strain lacking all nine receptor genes. Capillary assays revealed three important sensors for the strong attractant proline: McpU, McpX, and McpY. Receptor deletions variously affected free-swimming speed and attractant-induced chemokinesis. Noticeably, cells lacking mcpU were swimming 9% slower than the wild-type control. We infer that McpU inhibits the kinase activity of CheA in the absence of an attractant. Cells lacking one of the two soluble receptors were impaired in chemokinetic proficiency by more than 50%. We propose that the internal sensors, IcpA and the PAS domain containing McpY, monitor the metabolic state of S. meliloti.     

Chemotaxis of Rhizobium meliloti towards Nodulation Gene-Inducing Compounds from Alfalfa Roots

Luteolin, a flavone present in seed exudates of alfalfa, induces nodulation genes (nod) in Rhizobium meliloti and also serves as a biochemically specific chemoattractant for the bacterium. The present work shows that R. meliloti RCR2011 is capable of very similar chemotactic responses towards 4′,7-dihydroxyflavone, 4′,7-Dihydroxyflavanone, and 4,4′-dihydroxy-2-methoxychalcone, the three principal nod gene inducers secreted by alfalfa roots. Chemotactic responses to the root-secreted nod inducers in capillary assays were usually two- to four-fold above background and, for the flavone and flavonone, occurred at concentrations lower than those required for half-maximal induction of the nodABC genes. Complementation experiments indicated that the lack of chemotactic responsiveness to luteolin seen in nodD1 and nodA mutants of R. meliloti was not due to mutations in the nod genes, as previously thought. Thus, while nod gene induction and flavonoid chemotaxis have the same biochemical specificity, these two functions appear to have independent receptors or transduction pathways. The wild-type strain was found to suffer selective, spontaneous loss of chemotaxis towards flavonoids during laboratory subculture.     

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.     

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.     

Effect of Canavanine from Alfalfa Seeds on the Population Biology of Bacillus cereus

Bacillus cereus UW85 suppresses diseases of alfalfa seedlings, although alfalfa seed exudate inhibits the growth of UW85 in culture (J. L. Milner, S. J. Raffel, B. J. Lethbridge, and J. Handelsman, Appl. Microbiol. Biotechnol. 43:685–691, 1995). In this study, we determined the chemical basis for and biological role of the inhibitory activity. All of the alfalfa germ plasm tested included seeds that released inhibitory material. We purified the inhibitory material from one alfalfa cultivar and identified it as canavanine, which was present in the cultivar Iroquois seed exudate at a concentration of 2 mg/g of seeds. Multiple lines of evidence suggested that canavanine activity accounted for all of the inhibitory activity. Both canavanine and seed exudate inhibited the growth of UW85 on minimal medium; growth inhibition by either canavanine or seed exudate was prevented by arginine, histidine, or lysine; and canavanine and crude seed exudate had the same spectrum of activity against B. cereus, Bacillus thuringiensis, and Vibrio cholerae. The B. cereus UW85 populations surrounding canavanine-exuding seeds were up to 100-fold smaller than the populations surrounding non-canavanine-exuding seeds, but canavanine did not affect the growth of UW85 on seed surfaces. The spermosphere populations of canavanine-resistant mutants of UW85 were larger than the spermosphere populations of UW85, but the mutants and UW85 were similar in spermoplane colonization. These results indicate that canavanine exuded from alfalfa seeds affects the population biology of B. cereus.     

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.     

Construction and Environmental Release of a Sinorhizobium meliloti Strain Genetically Modified To Be More Competitive for Alfalfa Nodulation

Highly efficient nitrogen-fixing strains selected in the laboratory often fail to increase legume production in agricultural soils containing indigenous rhizobial populations because they cannot compete against these populations for nodule formation. We have previously demonstrated, with a Sinorhizobium meliloti PutA⁻ mutant strain, that proline dehydrogenase activity is required for colonization and therefore for the nodulation efficiency and competitiveness of S. meliloti on alfalfa roots (J. I. Jiménez-Zurdo, P. van Dillewijn, M. J. Soto, M. R. de Felipe, J. Olivares, and N. Toro, Mol. Plant-Microbe Interact. 8:492–498, 1995). In this work, we investigated whether the putA gene could be used as a means of increasing the competitiveness of S. meliloti strains. We produced a construct in which a constitutive promoter was placed 190 nucleotides upstream from the start codon of the putA gene. This resulted in an increase in the basal expression of this gene, with this increase being even greater in the presence of the substrate proline. We found that the presence of multicopy plasmids containing this putA gene construct increased the competitiveness of S. meliloti in microcosm experiments in nonsterile soil planted with alfalfa plants subjected to drought stress only during the first month. We investigated whether this construct also increased the competitiveness of S. meliloti strains under agricultural conditions by using it as the inoculum in a contained field experiment at León, Spain. We found that the frequency of nodule occupancy was higher with inoculum containing the modified putA gene for samples that were analyzed after 34 days but not for samples that were analyzed later.     

Flavonoids Released Naturally from Alfalfa Seeds Enhance Growth Rate of Rhizobium meliloti

Alfalfa (Medicago sativa L.) releases different flavonoids from seeds and roots. Imbibing seeds discharge 3′,4′,5,7-substituted flavonoids; roots exude 5-deoxy molecules. Many, but not all, of these flavonoids induce nodulation (nod) genes in Rhizobium meliloti. The dominant flavonoid released from alfalfa seeds is identified here as quercetin-3-O-galactoside, a molecule that does not induce nod genes. Low concentrations (1-10 micromolar) of this compound, as well as luteolin-7-O-glucoside, another major flavonoid released from germinating seeds, and the aglycones, quercetin and luteolin, increase growth rate of R. meliloti in a defined minimal medium. Tests show that the 5,7-dihydroxyl substitution pattern on those molecules was primarily responsible for the growth effect, thus explaining how 5-deoxy flavonoids in root exudates fail to enhance growth of R. meliloti. Luteolin increases growth by a mechanism separate from its capacity to induce rhizobial nod genes, because it still enhanced growth rate of R. meliloti lacking functional copies of the three known nodD genes. Quercetin and luteolin also increased growth rate of Pseudomonas putida. They had no effect on growth rate of Bacillus subtilis or Agrobacterium tumefaciens, but they slowed growth of two fungal pathogens of alfalfa. These results suggest that alfalfa can create ecochemical zones for controlling soil microbes by releasing structurally different flavonoids from seeds and roots.     

Growth and Movement of Spot Inoculated Rhizobium meliloti on the Root Surface of Alfalfa

Inoculum droplets of approximately 10 nanoliter volume and containing about 10 Rhizobium meliloti cells were placed onto the root surface of alfalfa seedlings in plastic growth pouches at either the root tip, the position of the smallest emergent root hairs, or at a site midway between these points. The droplets were initially confined to an area of about 0.2 square millimeter at the point of application. By 48 and 96 hours after inoculation, the inoculum bacteria and their progeny were distributed over several centimeters of the root between the initial site of deposition and the growing root tip, reaching densities of 10³ to 10⁴ bacteria per centimeter near the site of initial deposition and decreasing exponentially from that point toward the root tip. Graphite particles deposited on the root surface close to the growing tip were similarly distributed along the root length by 48 and 96 hours, suggesting that passive displacement by root cell elongation was primarily responsible for the spread of bacteria. A nonmotile mutant of R. meliloti colonized alfalfa roots to the same extent as the wild type and was usually distributed in the same manner, indicating that bacterial motility contributed little under these conditions to long distance spread of the bacteria. However, when applied in low numbers, R. meliloti mutants defective in motility or chemotaxis were considerably less efficient in initiating nodules near the point of inoculation than the wild type. This implies that motility and/or chemotaxis contribute significantly to local exploration for suitable infection sites. Almost all nodules on the primary root formed within a few millimeters of the spot-inoculation site, indicating that, under our experimental conditions, movement and multiplication of R. meliloti on the root surface were not sufficient to maintain an adequate population in the infectible region of the root during root growth.     

Hydrogen Evolution from Alfalfa and Clover Nodules and Hydrogen Uptake by Free-Living Rhizobium meliloti

A series of Rhizobium meliloti and Rhizobium trifolii strains were used as inocula for alfalfa and clover, respectively, grown under bacteriologically controlled conditions. Replicate samples of nodules formed by each strain were assayed for rates of H₂ evolution in air, rates of H₂ evolution under Ar and O₂, and rates of C₂H₂ reduction. Nodules formed by all strains of R. meliloti and R. trifolii on their respective hosts lost at least 17% of the electron flow through nitrogenase as evolved H₂. The mean loss from alfalfa nodules formed by 19 R. meliloti strains was 25%, and the mean loss from clover nodules formed by seven R. trifolii strains was 35%. R. meliloti and R. trifolii strains also were cultured under conditions that were previously established for derepression of hydrogenase synthesis. Only strains 102F65 and 102F51 of R. meliloti showed measurable activity under free-living conditions. Bacteroids from nodules formed by the two strains showing hydrogenase activity under free-living conditions also oxidized H₂ at low rates. The specific activity of hydrogenase in bacteroids formed by either strain 102F65 or strain 102F51 of R. meliloti was less than 0.1% of the specific activity of the hydrogenase system in bacteroids formed by H₂ uptake-positive Rhizobium japonicum USDA 110, which has been investigated previously. R. meliloti and R. trifolii strains tested possessed insufficient hydrogenase to recycle a substantial proportion of the H₂ evolved from the nitrogenase reaction in nodules of their hosts. Additional research is needed, therefore, to develop strains of R. meliloti and R. trifolii that possess an adequate H₂-recycling system.     

The ligand‐binding domain of a chemoreceptor from Comamonas testosteroni has a previously unknown homotrimeric structure

Transmembrane chemoreceptors are widely present in Bacteria and Archaea. They play a critical role in sensing various signals outside and transmitting to the cell interior. Here, we report the structure of the periplasmic ligand‐binding domain (LBD) of the transmembrane chemoreceptor MCP2201, which governs chemotaxis to citrate and other organic compounds in Comamonas testosteroni. The apo‐form LBD crystal revealed a typical four‐helix bundle homodimer, similar to previously well‐studied chemoreceptors such as Tar and Tsr of Escherichia coli. However, the citrate‐bound LBD revealed a four‐helix bundle homotrimer that had not been observed in bacterial chemoreceptor LBDs. This homotrimer was further confirmed with size‐exclusion chromatography, analytical ultracentrifugation and cross‐linking experiments. The physiological importance of the homotrimer for chemotaxis was demonstrated with site‐directed mutations of key amino acid residues in C. testosteroni mutants.     

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.     

Chemoreceptors from the commensal gut Roseburia rectibacter bind to mucin and trigger chemotaxis

Chemotaxis is crucial for bacterial adherence and colonization of the host gastrointestinal tract. Previous studies have demonstrated that chemotaxis affects the virulence of causative pathogens and the infection in the host. However, the chemotactic abilities of non-pathogenic and commensal gut bacteria have rarely been explored. We observed that Roseburia rectibacter NSJ-69 exhibited flagella-dependent motility and chemotaxis to a variety of molecules, including mucin and propionate. A genome-wide analysis revealed that NSJ-69 has 28 putative chemoreceptors, 15 of which have periplasmic ligand-binding domains (LBDs). These LBD-coding genes were chemically synthesized and expressed heterologously in Escherichia coli. Intensive screening of ligands revealed four chemoreceptors bound to mucin and two bound to propionate. When expressed in Comamonas testosteroni or E. coli, these chemoreceptors elicited chemotaxis toward mucin and propionate. Hybrid chemoreceptors were constructed, and results showed that the chemotactic responses to mucin and propionate were dependent on the LBDs of R. rectibacter chemoreceptors. Our study identified and characterized R. rectibacter chemoreceptors. These results will facilitate further investigations on the involvement of microbial chemotaxis in host colonization.     

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO₂ evolution or as O₂ uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 10⁷ bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO₂ production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO₂, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.     

Growth Dynamics of Salmonella enterica Strains on Alfalfa Sprouts and in Waste Seed Irrigation Water

Alfalfa sprouts and other seed sprouts have been implicated in numerous outbreaks of salmonellosis. The source of these epidemics appears to have been low-level contamination of seeds by Salmonella bacteria that developed into clinically significant populations during the seed germination process. To test the possibility that Salmonella enterica strains carry host range determinants that allow them to grow on alfalfa, strains isolated from alfalfa or other sources were surveyed for their ability to grow on germinating alfalfa seeds. An S. enterica serovar Cubana strain originally isolated from contaminated alfalfa sprouts multiplied most rapidly during the initial 24 h of the seed germination process. Germinating alfalfa seeds supported the multiplication of S. enterica cells prior to the emergence of the root radicle at 72 h. Thereafter, much lower rates of multiplication were apparent. The ability of S. enterica to grow on germinating alfalfa seeds was independent of the serovar, isolation source, or virulence of the strain. Isolates obtained from alfalfa attained population levels similar to those observed for strains isolated from contaminated meat products or stools. Each of the strains could be detected in the waste irrigation water, with populations being strongly correlated with those detected on the germinating alfalfa seeds. The S. enterica strains were capable of utilizing the waste irrigation water as a sole carbon and nitrogen source. S. enterica strains thus appear to grow saprophytically on soluble organics released from seeds during early phases of germination. The ability to detect S. enterica in the waste irrigation water early in the germination process indicates that this method may be used as a simple way to monitor the contamination of sprouts during commercial operations.     

Structural Basis for Polyamine Binding at the dCACHE Domain of the McpU Chemoreceptor from Pseudomonas putida

Many bacteria can move chemotactically to a variety of compounds and the recognition of chemoeffectors by the chemoreceptor ligand binding domain (LBD) defines the specificity of response. Many chemoreceptors were found to recognize different amino and organic acids, but the McpU chemoreceptor from Pseudomonas putida was identified as the first chemoreceptor that bound specifically polyamines. We report here the three-dimensional structure of McpU-LBD in complex with putrescine at a resolution of 2.4 Å, which fitted well a solution structure generated by small-angle X-ray scattering. Putrescine bound to a negatively charged pocket in the membrane distal module of McpU-LBD. Similarities exist in the binding of putrescine to McpU-LBD and taurine to the LBD of the Mlp37 chemoreceptor of Vibrio cholerae. In both structures, the primary amino group of the respective ligand is recognized by hydrogen bonds established by two aspartate and a tyrosine side chain. This feature may be used to predict the ligands of chemoreceptors with unknown function. Analytical ultracentrifugation revealed that McpU-LBD is monomeric in solution and that ligand binding does not alter this oligomeric state. This sensing mode thus differs from that of the well-characterised four-helix bundle domains where ligands bind to two sites at the LBD dimer interface. Although there appear to be different sensing modes, results are discussed in the context of data, indicating that chemoreceptors employ the same mechanism of transmembrane signaling. This work enhances our understanding of CACHE domains, which are the most abundant sensor domains in bacterial chemoreceptors and sensor kinases.