Cecillia M. Joseph

Alfalfa (Medicago sativa L.) Root Exudates Contain Isoflavonoids in the Presence of Rhizobium meliloti

Root exudates of alfalfa (Medicago sativa L.) inoculated with symbiotic Rhizobium meliloti bacteria contained three isoflavonoids that were not found in exudates of uninoculated plants. Data from proton nuclear magnetic resonance, mass spectrometry, and ultraviolet-visible absorbance analyses indicated that root exudates of inoculated plants contained aglycone and glycoside forms of the phytoalexin medicarpin and a formononetin-7-O-(6″-O-malonylglycoside), a conjugated form of the medicarpin precursor formononetin. The medicarpin molecules did not induce nod gene transcription in R. meliloti, but the formononetin-7-O-(6″-O-malonylglycoside) induced nod genes regulated by both NodD1 and NodD2 proteins in R. meliloti. Hydrolysis of either the malonyl or the glycosyl linkage from the formononetin conjugate eliminated nod gene-inducing activity. The nod gene-inducing activity of crude root exudates was increased 200 and 65% upon inoculation with R. meliloti or R. leguminosarum bv phaseoli, respectively. When root exudate from uninoculated alfalfa was incubated with R. meliloti, high performance liquid chromatography analyses showed no evidence that bacterial metabolism produced medicarpin. These results indicate that alfalfa responds to symbiotic R. meliloti by exuding a phytoalexin normally elicited by pathogens and that the microsymbiont can use a precursor of the phytoalexin as a signal for inducing symbiotic nod genes.

Organization of the nif genes in cyanobacteria in symbiotic association with Azolla and Anthoceros

The sizes of endonuclease digestion fragments of DNA from cyanobacteria in symbiotic association with Azolla caroliniana or Anthoceros punctatus, or in free-living culture, were compared by Southern hybridization using cloned nitrogenase (nif) genes from Anabaena sp. PCC 7120 as probes. The restriction fragment pattern produced by cyanobacteria isolated from A. caroliniana by culture through symbiotic association with Anthoceros differed from that of the major symbiotic cyanobacterium freshly separated from A. caroliniana. The results indicate that minor cyanobacterial symbionts occur in association with Azolla and that the dominant symbiont was not cultured in the free-living state. Both the absence of hybridization to an xisA gene probe and the mapping of restriction fragments indicated a contiguous nifHDK organization in all cells of the symbiont in association with Azolla. On the other hand, in the cultured isolate from Azolla and in Nostoc sp. 7801, the nifD and nifK genes are nominally separated by an interval of unknown length, compatible with the interruption of the nifHDK operon by a DNA element as observed in Anabaena sp. PCC 7120. In the above cultured strains, restriction fragments consistent with a contiguous nifHDK operon were also present at varying hybridization intensities, especially in Nostoc sp. 7801 grown in association with Anthoceros, presumably due to gene rearrangement in a fraction of the cells.

Metabolites from soil bacteria affect plant water relations

Water-soluble compounds move naturally in soil moisture toward roots of transpiring plants. To test for effects of rhizosphere food-web molecules on plants, low concentrations of common microbial products were supplied to bean (Phaseolus vulgaris L.) roots. Stomatal conductance and transpiration increased significantly (+20 to +30%, P ≤ 0.05) 42 h after 10 nM homoserine lactone (HL) was supplied to roots. Because transpiration helps both a plant and its root-colonizing bacteria obtain diffusion-limited mineral nutrients, such as phosphorus, any increase triggered by a degradation product of N-acyl-homoserine lactone (AHL) regulatory signals commonly used among plant-associated bacteria may represent a mutualistic plant-microbe interaction. Results from these initial physiological tests justify further screening to identify other rhizosphere compounds that control plant functions important for root-colonizing microorganisms.

Fixation of [13N]N2 and transfer of fixed nitrogen in the Anthoceros-Nostoc symbiotic association

The initial product of fixation of [13N]N2 by pure cultures of the reconstituted symbiotic association between Anthoceros punctatus L. and Nostoc sp. strain ac 7801 was ammonium; it accounted for 75% of the total radioactivity recovered in methanolic extracts after 0.5 min and 14% after 10 min of incubation. Glutamine and glutamate were the primary organic products synthesized from [13N]N2 after incubation times of 0.5–10 min. The kinetics of labeling of these two amino acids were characteristic of a precursor (glutamine) and product (glutamate) relationship. Results of inhibition experiments with methionine sulfoximine (MSX) and diazo-oxonorleucine were also consistent with the assimilation of N2-derived NH4+by Anthoceros-Nostoc through the sequential activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1), with little or no assimilation by glutamate dehydrogenase (EC 1.3.1.3). Isolated symbiotic Nostoc assimilated exogenous 13NH4+into glutamine and glutamate and their formation was inhibited by MSX, indicating operation of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway: However, relative to free-living cultures, isolated symbiotic Nostoc assimilated 80% less exogenous ammonium into glutamine and glutamate, implying that symbiotic Nostoc could assimilate only a fraction of N2-derived NH4+. This implication was tested by using Anthoceros associations reconstituted with wild-type or MSX-resistant strains of Nostoc incubated with [13N]N2 in the presence of MSX. The results of these experiments indicated that, in situ, symbiotic Nostoc assimilated about 10% of the N2-derived NH4+and that NH4+was made available to Anthoceros tissue where it was apparently assimilated by the GS-GOGAT pathway. Since less than 1% of the fixed N2 was lost to the suspension medium, it appears that transfer of NH4+from symbiont to host tissue was very efficient in this extracellular symbiotic association.

Assimilation of exogenous and dinitrogen-derived13NH 4 + byAnabaena azollae separated fromAzolla caroliniana Willd

Anabaena azollae was isolated fromAzolla caroliniana by the “gentle roller” method and differential centrifugation. Incubation of suchAnabaena preparations for 10 min with [13N]N2 resulted in the formation of four radioactive compounds; ammonium, glutamine, glutamate and alanine. Ammonium accounted for 66% of the total radioactivity recovered and 58% of the ammonium was in an extracellular fraction. Since essentially no extracellular13N-labeled organic compounds were found, it appears that ammonium is the compound most probably made available toAzolla during dinitrogen-dependent growth of the association.The kinetics of incorporation of exogenous13NH4+ into glutamine and glutamate were characteristic of a precursor (glutamine)-product (glutamate) relationship and consistent with assimilation by the glutamine synthetase-glutamate synthase pathway. The results of experiments using the glutamine synthetase inhibitor, methionine sulfoximine, the glutamate synthase inhibitor, diazo-oxonorleucine, and increasing the ammonium concentration to greater than 1 mM, provided evidence for assimilation primarily by the glutamine synthetase-glutamate synthase pathway with little or no contribution from biosynthetic glutamate dehydrogenase.While showing that N2 fixation and NH4+ assimilation were not tightly coupled metabolic processes in symbioticAnabaena, these results reflect a composite picture and do not indicate the extent to which ammonium assimilatory enzymes might be regulated in filaments associated with specific stages in theAzolla-Anabaena developmental profile.

Roles for Riboflavin in the Sinorhizobium-Alfalfa Association

Genes contributing to riboflavin production in Sinorhizobium meliloti were identified, and bacterial strains that overproduce this vitamin were constructed to characterize how additional riboflavin affects interactions between alfalfa (Medicago sativa) and S. meliloti. Riboflavin-synthesis genes in S. meliloti were found in three separate linkage groups and designated as ribBA, ribDribC, and ribH for their similarities to Escherichia coli genes. The ribBA and ribC loci complemented corresponding E. coli rib mutants. S. meliloti cells containing extra copies of ribBA released 10 to 20% more riboflavin than a control strain but grew at similar rates in a defined medium lacking riboflavin. Cells carrying extra copies of ribBA colonized roots to densities that were 55% higher than that of a control strain. No effect of extra rib genes was detected on alfalfa grown in the absence or presence of combined N. These results support the importance of extracellular riboflavin for alfalfa root colonization by S. meliloti and are consistent with the hypothesis that this molecule benefits bacteria indirectly through an effect on the plant.

Assimilation of 13NH 4 + by Anthoceros grown with and without symbiotic Nostoc

The pathways of assimilation of ammonium by pure cultures of symbiont-free Anthoceros punctatus L. and the reconstituted Anthoceros-Nostoc symbiotic association were determined from time-course (5–300 s) and inhibitor experiments using 13NH4+. The major product of assimilation after all incubation times was glutamine, whether the tissues were cultured with excess ammonium or no combined nitrogen. The 13N in glutamine was predominantly in the amide-nitrogen position. Formation of glutamine and glutamate by Anthoceros-Nostoc was strongly inhibited by either 1mM methionine sulfoximine (MSX) or 1 mM exogenous ammonium. These data are consistent with the assimilation of 13NH4+and formation of glutamate by the glutamine synthetase (EC 6.3.1.2)-glutamate synthase (EC 1.4.7.1) pathway in dinitrogen-grown Anthoceros-Nostoc. However, in symbiont-free Anthoceros, grown with 2.5 mM ammonium, formation of glutamine, but not glutamate, was decreased by either MSX or exogenous ammonium. These results indicate that during short incubation times ammonium is assimilated in nitrogenreplete Anthoceros by the activities of both glutamine synthetase and glutamate dehydrogenase (EC 1.4.1.2). In-vitro activities of glutamine synthetase were similar in nitrogen-replete Anthoceros and Anthoceros-Nostoc, indicating that the differences in the routes of glutamate formation were not based upon regulation of synthesis of the initial enzyme of the glutamine synthetase-glutamate synthase pathway. When symbiont-free Anthoceros was cultured for 2 d in the absence of combined nitrogen, total 13NH4+assimilation, and glutamine and glutamate formation in the presence of inhibitors, were similar to dinitrogen-grown Anthoceros-Nostoc. The routes of immediate (within 2 min) glutamate formation and ammonium assimilation in Anthoceros were apparently determined by the intracellular levels of ammonium; at low levels the glutamine synthetase-glutamate synthase pathway was predominant, while at high levels independent activities of both glutamine synthetase and glutamate dehydrogenase were expressed.

Cloning and expression of a Nostoc sp. leucine biosynthetic gene in Escherichia coli

Genomic DNA extracted from the symbiotically-competent, heterocyst-forming cyanobacterium Nostoc sp. strain 7801 was resistant to cleavage by a number of restriction endonucleases. A cosmid library of Nostoc DNA was prepared and maintained in the modification-limited Escherichia coli strain HB101. Analysis of cloned Nostoc DNA fragments indicated infrequent occurrence of restriction endonuclease recognition sites in the Nostoc genome.The Nostoc genomic library was screened for sequences complementing mutations in the E. coli leucine and proline biosynthetic operons. Two cosmids complementing leuB were isolated but none for leuA, leuC, leuD, or proA were detected in 1000 cosmids. A 3.0 kb fragment subcloned from one of the cosmids complemented mutations in leuB when inserted into the HindIII site of pBR322 in either orientation, demonstrating that transcription of leuB originated within the cloned fragment. The cloned fragment also carries a second site capable of initiating transcription of fused antibiotic resistance genes. While transcription of Nostoc DNA sequences did occur in E. coli, unknown barriers must also exist that prevented additional biological complementation of specific E. coli mutations.