Nucleotide sequence of an alfalfa calmodulin cDNA.
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Amino Acid Sequence↗
Biomedical subjects
S R Long
Publications and source records attributed to S R Long.
DNA sequence and translational product of a new nodulation-regulatory locus: syrM has sequence similarity to NodD proteins.
Rhizobium meliloti nodulation (nod) genes are expressed when activated by trans-acting proteins in the NodD family. The nodD1 and nodD2 gene products activate nod promoters when cells are exposed to plant-synthesized signal molecules. Alternatively, the same nod promoters are activated by the nodD3 gene when nodD3 is carried in trans along with a closely linked global regulatory locus, syrM (symbiotic regulator) (J. T. Mulligan and S. R. Long, Genetics 122:7-18, 1989). In this article we report the nucleotide sequence of a 2.6-kilobase SphI fragment from R. meliloti SU47 containing syrM. Expression from this locus was confirmed by using in vitro transcription-translation assays. The open reading frame encoded a protein of either 33 or 36 kilodaltons whose sequence shows similarity to NodD regulatory proteins.
Amino Acid Sequence↗
Rhizobium-legume nodulation: life together in the underground.
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Fabaceae↗
A family of activator genes regulates expression of Rhizobium meliloti nodulation genes.
Nodulation (nod) gene expression in Rhizobium meliloti requires plant inducers and the activating protein product of the nodD gene. We have examined three genes in R. meliloti which have nodD activity and sequence homology. These three nodD genes are designated nodD1, nodD2 and nodD3, and have distinctive properties. The nodD1 gene product activates expression of the nodABC operon, as measured by a nodC-lacZ fusion or by transcript analysis, in the presence of crude seed or plant wash or the inducer, luteolin. The nodD3 gene product can cause a high basal (uninduced) level of nodC-lacZ expression and nodABC transcripts which is relatively unaffected by inducers. The effect of nodD3 is dependent on the presence of another gene, syrM (symbiotic regulator). By primer extension analysis we determined that the transcription start site is the same for nodD1 plus luteolin or nodD3-syrM mediated expression of nodA and nodH mRNAs. syrM also enhances the expression of another symbiotically important trait, production of extracellular polysaccharide. This regulatory effect of syrM requires locus syrA, which is linked to nodD3 and syrM. The syrM-syrA mediated increase in polysaccharide production requires at least some of the previously identified exo genes and may be a parallel regulatory event to the syrM-nodD3 control of nod promoters.
Blotting, Western↗
A non-nodulating alfalfa mutant displays neither root hair curling nor early cell division in response to Rhizobium meliloti.
The early events in the alfalfa-Rhizobium meliloti symbiosis include deformation of epidermal root hairs and the approximately concurrent stimulation of cell dedifferentiation and cell division in the root inner cortex. These early steps have been studied previously by analysis of R. meliloti mutants. Bacterial strains mutated in nodABC, for example, fail to stimulate either root hair curling or cell division events in the plant host, whereas exopolysaccharide (exo) mutants of R. meliloti stimulate host cell division but the resulting nodules are uninfected. As a further approach to understanding early symbiotic interactions, we have investigated the phenotype of a non-nodulating alfalfa mutant, MnNC-1008 (NN) (referred to as MN-1008). Nodulating and non-nodulating plants were inoculated with wild-type R. meliloti and scored for root hair curling and cell divisions. MN-1008 was found to be defective in both responses. Mutant plants inoculated with Exo- bacteria also showed no cell division response. Therefore, the genetic function mutated in MN-1008 is required for both root hair curling and cell division, as is true for the R. meliloti nodABC genes. These observations support the model that the distinct cellular processes of root hair curling and cell division are triggered by related mechanisms or components, or are causally linked.
Cell Division↗
DNA footprint analysis of the transcriptional activator proteins NodD1 and NodD3 on inducible nod gene promoters.
The Rhizobium meliloti nodD1 and nodD3 gene products (NodD1 and NodD3) are members of the lysR-nodD gene regulator family. They are functionally distinct in that NodD1 transcriptionally activates other nod genes in the presence of a flavonoid inducer such as luteolin, while NodD3 is capable of activating nod gene expression at high levels in the absence of inducer. NodD1 and NodD3 are DNA-binding proteins which interact with DNA sequences situated upstream of the transcription initiation sites of at least three sets of inducible nod genes. We report the footprinting of NodD1- and NodD3-DNA complexes with both DNase I and the 1,10-phenanthroline-copper ion reagent. NodD1 and NodD3 both interacted with the nodABC, nodFE, and nodH promoters and protected from cleavage an extensive piece of DNA, including the nod box, from approximately -20 to -75 from the transcription start site for each of the three promoters. The constitutively activating protein NodD3 displayed an additional hypersensitive cleavage site in its footprint compared with NodD1.
Bacterial Proteins↗
Specific binding of proteins from Rhizobium meliloti cell-free extracts containing NodD to DNA sequences upstream of inducible nodulation genes.
Nodulation (nod) genes in Rhizobium meliloti are transcriptionally induced by flavonoid signal molecules, such as luteolin, produced by its symbiotic host plant, alfalfa. This induction depends on expression of nodD. Upstream of three inducible nod gene clusters, nodABC, nodFE, and nodH, is a highly conserved sequence referred to as a 'nod box.' The upstream sequences have no other obvious similarity. We have found that DNA fragments containing the regions upstream of all three inducible transcripts show altered electrophoretic mobility when treated with R. meliloti extracts. The ability of the extracts to interact specifically with these DNAs correlated with the genetic dosage of nodD1 or nodD3 and with the presence and concentration of the nodD1 or nodD3 protein (NodD1 or NodD3) in the extracts. Antiserum specific to NodD was used to construct an immunoaffinity column that permitted a substantial purification of NodD1; this preparation of NodD1 also displayed specific binding to restriction fragments containing DNA sequences found upstream of inducible nod genes. In addition, NodD-specific antiserum removed the specific DNA-binding activity from total Rhizobium cell extracts. The interaction of total extracts and of partially purified NodD protein with nod promoter sequences was competitive with an oligonucleotide representing the 3' 25-bp portion of the nod box. The interaction of R. meliloti extracts and NodD1 protein with nod gene upstream regions occurred independently of exposure of cells or extracts to flavone inducer.
Bacterial Proteins↗
Alfalfa Root Exudates and Compounds which Promote or Inhibit Induction of Rhizobium meliloti Nodulation Genes.
Using a plate induction assay, we demonstrate that alfalfa exudes inducer of Rhizobium meliloti nodulation genes. The inducer is exuded from the infectible zone of the root, accumulates to at least 1 micromolar, and is not affected by 10 millimolar nitrate. No zones of inhibition are observed. A nodulation minus mutant line of alfalfa, MN-1008, exudes normal levels of inducer. R. meliloti grown in rich medium requires ten-fold higher concentrations of luteolin to achieve half-maximal induction as compared to cells grown in a minimal medium. Flavonoids other than luteolin are found to have activity in R. meliloti nodulation gene induction assays. The compounds apigenin and eriodictyol have activities two-fifths and one-seventh that of luteolin, respectively. Several of the flavonoids tested (morin = naringenin > kaempferol = chrysin > quercetin = fisetin = hesperitin) demonstrate antagonistic activity toward induction by luteolin. The most effective antagonist is the coumarin, umbelliferone.
Journal Article↗
Transcription of Rhizobium meliloti nodulation genes. Identification of a nodD transcription initiation site in vitro and in vivo.
Nodulation genes in Rhizobium are required for invasion of the host plant. The nodABC operon is induced by plant activator molecules; this activation requires the gene product of the constitutively expressed nodD locus, which is transcribed divergently from nodABC. We are employing in vitro transcription to elucidate the molecular mechanism of nod gene activation. We used a micropurification technique to obtain RNA polymerase from Rhizobium meliloti, and here demonstrate that it initiated and terminated accurately at the Escherichia coli trp promoter-leader region. E. coli RNA polymerase, however, apparently fails to recognize R. meliloti promoters. We used the R. meliloti RNA polymerase in a minimal transcription system to attempt to localize the divergent start sites for nodD and nodABC. Transcript sizing and fingerprinting, together with synchronized single-round transcription experiments permit us to designate an in vitro transcription initiation site for nodD. Primer extension analysis of in vivo mRNA demonstrates that the initiation site which is utilized in vitro is the same site used in vivo. While nodABC is not transcribed in our minimal in vitro transcription system, this system should prove useful for the study of factors in induced cells which promote expression of this inducible promoter.
Base Sequence↗
Expression of Rhizobium meliloti nod genes in Rhizobium and Agrobacterium backgrounds.
Rhizobium meliloti nod genes are required for the infection of alfalfa. Induction of the nodC gene depends on a chemical signal from alfalfa and on nodD gene expression. By using a nodC-lacZ fusion, we have shown that the induction of the R. meliloti nodC gene and the expression of nodD occur at almost normal levels in other Rhizobium backgrounds and in Agrobacterium tumefaciens, but not in Escherichia coli. Xanthomonas campestris, or Pseudomonas savastanoi. Our results suggest that bacterial genes in addition to nodDABC are required for nod gene response to plant cells. We have found that inducing activity is present in other plant species besides alfalfa. Acetosyringone, the A. tumefaciens vir gene inducer, does not induce nodC.
Acetophenones↗
A plant flavone, luteolin, induces expression of Rhizobium meliloti nodulation genes.
The symbiotic interaction of Rhizobium meliloti and alfalfa results in the formation of nitrogen-fixing root nodules. Rhizobium meliloti nodABC genes are required for the early host responses of cortical cell divisions and root hair curling. The induction of nodABC expression by alfalfa exudates demonstrates host-symbiont signaling at an early stage in nodule development. The inducer molecule for nodABC expression was isolated from plant exudate by constructing a nodABC-lacZ fusion to monitor the inducing activity. From ultraviolet-visible absorption spectra, proton nuclear magnetic resonance, and mass spectrometry, the inducer was determined to be 3',4', 5,7-tetrahydroxyflavone (luteolin). Luteolin is a normal secondary plant metabolite found throughout the plant kingdom that may serve to control nodABC expression during nodule development. This regulatory role for a flavone contrasts with the function of some flavonoids as defense compounds.
Flavonoids↗
Induction of Rhizobium meliloti nodC expression by plant exudate requires nodD.
The soil bacterium Rhizobium meliloti invades and establishes a symbiosis with host plants such as alfalfa. Bacterial nodulation (nod) genes are required for this invasion, but their mechanism of action and the timing of their expression are not known. We have used translational lacZ fusions to monitor expression of nodD and nodC, which are located in the cluster of four nod genes on the R. meliloti megaplasmid (pSym). nodD is expressed at comparable levels by broth-grown bacterial cells and by cells exposed to exudates from aseptically grown plants. Activity of the nodC-lacZ protein fusion in broth-grown bacterial cells is very low. nodC-lacZ activity is increased approximately equal to 30-fold by plant exudate when nodD is expressed at a high level but not when nodD expression is low. Both fusions show differences in expression when borne on inc-P vectors as compared to when located on the pSym megaplasmid. nodD expression from vector-borne copies of the nod segment and response of nodC to plant exudate appear to require additional loci on the megaplasmid. Our results suggest that regulation of bacterial nod gene expression is an important control mechanism early in the symbiosis, and that the biochemical nature of some nod gene products may be cryptic except in cells grown in the presence of plant exudate.
Bacterial Proteins↗
Nucleotide sequence of Rhizobium meliloti 1021 nodulation genes: nodD is read divergently from nodABC.
Nodulation (nod) genes are required for Rhizobium meliloti to invade and stimulate nodule formation in its host, alfalfa. We have established the DNA sequence of nodD, nodA, and nodB, which are part of a gene cluster located 20 kb downstream of nifHDK on the R. meliloti pSym megaplasmid. The nodD open reading frame (308 amino acids) reads from proximal to nifHDK toward distal to nifHDK, divergently from nodA (196 aa) and nodB (217 aa). These two genes read from distal to nifHDK toward proximal, and are just upstream from the previously defined open reading frame for nodC. Fourteen Tn5 insertion sites have been sequenced in nodD, nodA, and nodB, revealing no major hotspots for insertion, but an overall preference for G/C bases at positions 1 and 9 of the 9-bp repeat.
Amino Acid Sequence↗
Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii.
Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into NodR. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod mutants. All seven mutants were restored to Nod on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.
Journal Article↗
Nodules are induced on alfalfa roots by Agrobacterium tumefaciens and Rhizobium trifolii containing small segments of the Rhizobium meliloti nodulation region.
Regions of the Rhizobium meliloti nodulation genes from the symbiotic plasmid were transferred to Agrobacterium tumefaciens and Rhizobium trifolii by conjugation. The A. tumefaciens and R. trifolii transconjugants were unable to elicit curling of alfalfa root hairs, but were able to induce nodule development at a low frequency. These were judged to be genuine nodules on the basis of cytological and developmental criteria. Like genuine alfalfa nodules, the nodules were initiated from divisions of the inner root cortical cells. They developed a distally positioned meristem and several peripheral vascular bundles. An endodermis separated the inner tissues of the nodule from the surrounding cortex. No infection threads were found to penetrate either root hairs or the nodule cells. Bacteria were found only in intercellular spaces. Thus, alfalfa nodules induced by A. tumefaciens and R. trifolii transconjugants carrying small nodulation clones of R. meliloti were completely devoid of intracellular bacteria. When these strains were inoculated onto white clover roots, small nodule-like protrusions developed that, when examined cytologically, were found to more closely resemble roots than nodules. Although the meristem was broadened and lacked a root cap, the protrusions had a central vascular bundle and other rootlike features. Our results suggest that morphogenesis of alfalfa root nodules can be uncoupled from infection thread formation. The genes encoded in the 8.7-kilobase nodulation fragment are sufficient in A. tumefaciens or R. trifolii backgrounds for nodule morphogenesis.
Conjugation, Genetic↗
Physical and genetic map of a Rhizobium meliloti nodulation gene region and nucleotide sequence of nodC.
Infection of alfalfa by the soil bacterium Rhizobium meliloti proceeds by deformation of root hairs and bacterial invasion of host tissue by way of an infection thread. We studied an 8.7-kilobase (kb) segment of the R. meliloti megaplasmid, which contains genes required for infection. Site-directed Tn5 mutagenesis was used to examine this fragment for nodulation genes. A total of 81 R. meliloti strains with mapped Tn5 insertions in the 8.7-kb fragment were evaluated for nodulation phenotype on alfalfa plants; 39 of the insertions defined a 3.5-kb segment containing nodulation functions. Of these 39 mutants, 37 were completely nodulation deficient (Nod-), and 2 at the extreme nif-distal end were leaky Nod-. Complementation analysis was performed by inoculating plants with strains carrying a genomic Tn5 at one location and a plasmid-borne Tn5 at another location in the 3.5-kb nodulation segment. Mutations near the right border of the fragment behaved as two distinct complementation groups. The segment in which these mutations are located was analyzed by DNA sequencing. Several open reading frames were found in this region, but the one most likely to function is 1,206 bases long, reading from left to right (nif distal to proximal) and spanning both mutation groups. The genetic behavior of this segment may be due either to the gene product having two functional domains or to a recombinational hot spot between the apparent complementation groups.
Amino Acid Sequence↗
Rhizobium meliloti nodulation genes: identification of nodDABC gene products, purification of nodA protein, and expression of nodA in Rhizobium meliloti.
A set of conserved, or common, bacterial nodulation (nod) loci is required for host plant infection by Rhizobium meliloti and other Rhizobium species. Four such genes, nodDABC, have been indicated in R. meliloti 1021 by genetic analysis and DNA sequencing. An essential step toward understanding the function of these genes is to characterize their protein products. We used in vitro and maxicell Escherichia coli expression systems, together with gel electrophoresis and autoradiography, to detect proteins encoded by nodDABC. We facilitated expression of genes on these DNA fragments by inserting them downstream of the Salmonella typhimurium trp promoter, both in colE1 and incP plasmid-based vectors. Use of the incP trp promoter plasmid allowed overexpression of a nodABC gene fragment in R. meliloti. We found that nodA encodes a protein of 21 kilodaltons (kDa), and nodB encodes one of 28 kDa; the nodC product appears as two polypeptide bands at 44 and 45 kDa. Expression of the divergently read nodD yields a single polypeptide of 33 kDa. Whether these represent true Rhizobium gene products must be demonstrated by correlating these proteins with genetically defined Rhizobium loci. We purified the 21-kDa putative nodA protein product by gel electrophoresis, selective precipitation, and ion-exchange chromatography and generated antiserum to the purified gene product. This permitted the immunological demonstration that the 21-kDa protein is present in wild-type cells and in nodB- or nodC-defective strains, but is absent from nodA::Tn5 mutants, which confirms that the product expressed in E. coli is identical to that produced by R. meliloti nodA. Using antisera detection, we found that the level of nodA protein is increased by exposure of R. meliloti cells to plant exudate, indicating regulation of the bacterial nod genes by the plant host.
Bacterial Proteins↗