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Biomedical subjects

S R Long

Publications and source records attributed to S R Long.

At least 19 recordsLinked to original sources
PubMed1992

Rhizobium--plant signal exchange.

Initial stages in the Rhizobium-legume symbiosis can be thought of as a reciprocal molecular conversation: transmission of a gene inducer from legume host to bacterium, with ensuing bacterial synthesis of a morphogen that is transmitted to the plant, switching the developmental fate of the legume root. These signal molecules have a key role in determining bacterium-host specificity and the purified Nod factor compounds provide useful new tools to probe plant cell function.

Bacterial Proteins↗
PubMed1992

Rhizobium meliloti genes involved in sulfate activation: the two copies of nodPQ and a new locus, saa.

The nitrogen-fixing symbiont Rhizobium meliloti establishes nodules on leguminous host plants. Nodulation (nod) genes used for this process are located in a cluster on the pSym-a megaplasmid of R. meliloti. These genes include nodP and nodQ (here termed nodPQ), which encode ATP sulfurylase and APS kinase, enzymes that catalyze the conversion of ATP and SO(4)2- into the activated sulfate form 3'-phosphoadenosine 5'-phosphosulfate (PAPS), an intermediate in cysteine synthesis. In Rhizobium, PAPS is also a precursor for sulfated and N-acylated oligosaccharide Nod-factor signals that cause symbiotic responses on specific host plants such as alfalfa. We previously found a highly conserved second copy of nodPQ in R. meliloti. We report here the mapping and cloning of this second copy, and its location on the second megaplasmid, pSym-b. The function of nodP2Q2 is equivalent to that of nodP1Q1 in complementation tests of R. meliloti and Escherichia coli mutants in ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase. Mutations in nodP2Q2 do not have as severe an effect on symbiosis or plant host range as do those in nodP1Q1, however, possibly reflecting differences in expression and/or channeling of metabolites to specific enzymes involved in sulfate transfer. Strains mutated or deleted for both copies of nodQ are severely defective in symbiotic phenotypes, but remain prototrophic. This suggests the existence in R. meliloti of a third locus for ATP sulfurylase and APS kinase activities. We have found a new locus saa (sulfur amino acid), which may also encode these activities.

Chromosome Mapping↗
PubMed1991

Genetic and physical analysis of the nodD3 region of Rhizobium meliloti.

The nodulation (nod) genes of the symbiont Rhizobium meliloti are transcriptionally controlled by protein activators in the nodD gene family. While NodD1 and NodD2 act in concert with small molecular weight inducers provided by the host legume plant, NodD3 is an inducer-independent activator of the nod promoters. We determined the sequence of the nodD3 gene, confirmed the expression of a 35 kDa protein in vitro, and determined the insertion points of five Tn5 insertions in the region of the nodD3 gene. We found the NodD3 amino acid sequence to be markedly diverged from the sequences of NodD1 and NodD2, which were more similar to the inducer-dependent NodD of another species, Rhizobium leguminosarum biovar viciae. The expression of nodD3 is not well understood, but involves at least SyrM, another positive activator related to the LysR-NodD family. One of the phenotypically mutant Tn5 insertions used in genetic studies of NodD3-dependent nod regulation lacks NodD3 protein as determined by Western blots, but another expresses about 50-60% of the wild type level. The location of these Tn5 insertions substantially upstream of the open reading frame for NodD3 suggests importance of relatively distant regulatory sequences for nodD3 expression. An insertion that did not cause a NodD3- phenotype is located in the extreme C-terminus of the protein coding region.

Amino Acid Sequence↗
PubMed1991

Laparoscopic nephrectomy: initial case report.

A tumor-bearing right kidney was completely excised from an 85-year-old woman using a laparoscopic approach. A newly devised method for intra-abdominal organ entrapment and a recently developed laparoscopic tissue morcellator made it possible to deliver the 190 gm. kidney through an 11 mm. incision.

Adenoma↗
PubMed1991

Analysis of Rhizobium meliloti nodulation mutant WL131: novel insertion sequence ISRm3 in nodG and altered nodH protein product.

Nodulation (nod) genes are required for invasion of legumes by Rhizobium bacteria. Mutant WL131 is a derivative of 102F51 that has a severe Nod- phenotype on alfalfa. Upon examination of the extended DNA region containing host-specific nodulation genes nodFEG and nodH, we found that the nodG gene of WL131 bears a novel insertion sequence, ISRm3. Complementation studies implied, however, that the phenotype on alfalfa correlated with the nodH locus. We found that nodH in WL131 encodes an altered gene product. Correlation of the WL131 defect with nodH was also supported by phenotypic behavior. Each mutation affected nodulation more severely on alfalfa (Medicago sativa) than on sweet clover (Melilotus albus). However, we found that the degree of requirement for nodH in nodulation varied with the conditions under which the plant was grown.

Blotting, Southern↗
PubMed1991

Cloning and characterization of a Rhizobium meliloti homolog of the Escherichia coli cell division gene ftsZ.

The ftsZ gene is essential for initiation of cell division in Escherichia coli and Bacillus subtilis. To begin our studies of division arrest during differentiation of Rhizobium meliloti bacteroids, we isolated a R. meliloti ftsZ homolog, ftsZRm. Degenerate primers directed towards a conserved region of ftsZ were used to amplify a segment of R. meliloti DNA by polymerase chain reaction, and the product of this reaction was then used to isolate positive clones from a bacteriophage library. The DNA sequence of an open reading frame containing the region of homology indicated that the R. meliloti FtsZ protein (FtsZRm) is 50% homologous to the known E. coli and B. subtilis FtsZ proteins, but at 590 amino acids (63 kDa), it is predicted to be nearly 50% larger. Strong expression of an approximately 70-kDa labeled protein in a coupled in vitro transcription-translation system supports this prediction. The additional 200 amino acids appear to fall in a single internal domain highly enriched for proline and glutamine residues. When we regulated R. meliloti ftsZ (ftsZRm) expression on a high-copy-number plasmid in E. coli with Plac and laclq, cells were smaller than normal in the presence of low FtsZRm levels (with no isopropyl-beta-D-thiogalactopyranoside [IPTG]) and filamentous when FtsZRm was overproduced (with IPTG). These results suggest that low levels of FtsZRm stimulate E. coli cell division, while high levels may be inhibitory.

Amino Acid Sequence↗
PubMed1990

ATP sulphurylase activity of the nodP and nodQ gene products of Rhizobium meliloti.

The symbiotic bacterium Rhizobium meliloti stimulates alfalfa (Medicago sativa L.) roots to undergo morphogenesis and form nitrogen-fixing nodules. It has been proposed that the bacterial genes nodABC, common to all Rhizobium, are required for synthesis of an oligosaccharide factor, which is converted to a sulphated form (NodRm-1) by the products of the R. meliloti-specific genes nodH and nodQ1-5; NodRm-1 elicits host-specific plant responses. Previously we have shown that the nodP gene is homologous to a segment of the Escherichia coli genome; when we cloned this E. coli fragment we found that it mapped near 59 minutes, corresponding to the cysDNC locus. The genes cysD and cysN encode proteins that catalyse the synthesis of adenosine 5'-phosphosulphate, the first step in the activation of inorganic sulphate. Here we demonstrate that nodP and nodQ correspond to cysD and cysN, and that their proteins have ATP sulphurylase activity both in vivo and in vitro. We propose that nodP and nodQ synthesize an activated sulphate that is an intermediate in the formation of the alfalfa-specific sulphated nodRm-1 factor.

Cloning, Molecular↗
PubMed1990

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1989

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↗
PubMed1989

Rhizobium genetics.

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Genes, Bacterial↗
PubMed1988

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↗
PubMed1987

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↗