Women in biomedicine: encouragement.
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Female↗
Biomedical subjects
S R Long
Publications and source records attributed to S R Long.
Rhizobium meliloti NodP and NodQ form a multifunctional sulfate-activating complex requiring GTP for activity.
The nodulation genes nodP and nodQ are required for production of Rhizobium meliloti nodulation (Nod) factors. These sulfated oligosaccharides act as morphogenic signals to alfalfa, the symbiotic host of R. meliloti. In previous work, we have shown that nodP and nodQ encode ATP sulfurylase, which catalyzes the formation of APS (adenosine 5'-phosphosulfate) and PPi. In the subsequent metabolic reaction, APS is converted to PAPS (3'-phosphoadenosine 5'-phosphosulfate) by APS kinase. In Escherichia coli, cysD and cysN encode ATP sulfurylase; cysC encodes APS kinase. Here, we present genetic, enzymatic, and sequence similarity data demonstrating that nodP and nodQ encode both ATP sulfurylase and APS kinase activities and that these enzymes associate into a multifunctional protein complex which we designate the sulfate activation complex. We have previously described the presence of a putative GTP-binding site in the nodQ sequence. The present report also demonstrates that GTP enhances the rate of PAPS synthesis from ATP and sulfate (SO4(2-)) by NodP and NodQ expressed in E. coli. Thus, GTP is implicated as a metabolic requirement for synthesis of the R. meliloti Nod factors.
Amino Acid Sequence↗
Rhizobium meliloti contains a novel second homolog of the cell division gene ftsZ.
We have identified a second homolog of the cell division gene, ftsZ, in the endosymbiont Rhizobium meliloti. The ftsZ2 gene was cloned by screening a genomic lambda library with a probe derived from PCR amplification of a highly conserved domain. It encodes a 36-kDa protein which shares a high level of sequence similarity with the FtsZ proteins of Escherichia coli and Bacillus subtilis and FtsZ1 (Z1) of R. meliloti but lacks the carboxy-terminal region conserved in other FtsZ proteins. The identity of the ftsZ2 gene product was confirmed both by in vitro transcription-translation in an R. meliloti S-30 extract and by overproduction in R. meliloti cells. As with Z1, the overproduction of FtsZ2 in E. coli inhibited cell division and induced filamentation, although to a lesser extent than with Z1. However, the expression of ftsZ2 in E. coli under certain conditions caused some cells to coil dramatically, a phenotype not observed during Z1 overproduction. Although several Tn3-GUS (glucuronidase) insertions in a plasmid-borne ftsZ2 gene failed to cross into the chromosome, one interruption in the chromosomal ftsZ2 gene was isolated, suggesting that ftsZ2 is nonessential for viability. The two ftsZ genes were genetically mapped to the R. meliloti main chromosome, approximately 100 kb apart.
Amino Acid Sequence↗
Interactions of NodD at the nod Box: NodD binds to two distinct sites on the same face of the helix and induces a bend in the DNA.
The Rhizobium meliloti nodD gene products are positive transcriptional activators of genes required for early stages of nodule morphogenesis in the R. meliloti-alfalfa symbiosis (nod genes). The regulatory activity of NodD, a member of the LysR family of activator proteins, is mediated in part through its binding to conserved DNA sequences termed nod boxes which lie upstream of the inducible nod genes. Here we use interference footprinting to identify two NodD binding sites in the nodA, nodF and nodH nod boxes. These two binding sites are located on the same face of the DNA helix and can be separated by an additional 10 bp with retention of activity. By systematic alteration of the phasing of the two binding sites on the DNA helix, we showed that only constructs which contain both sites on the same side of the helix are recognized by NodD as determined by migration retardation assay and by in vivo activation of nod box-lacZ fusions. Moreover, NodD apparently induces a bend in the DNA upon binding at the nod box as shown by migration retardation behavior of circularly permuted nod box fragments.
Acyltransferases↗
Prokaryotic plant parasites.
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Bacterial Proteins↗
Classics in cytology. VI: The early cytologic discoveries of Lionel S. Beale.
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Cell Biology↗
Regulation of syrM and nodD3 in Rhizobium meliloti.
The early steps of symbiotic nodule formation by Rhizobium on plants require coordinate expression of several nod gene operons, which is accomplished by the activating protein NodD. Three different NodD proteins are encoded by Sym plasmid genes in Rhizobium meliloti, the alfalfa symbiont. NodD1 and NodD2 activate nod operons when Rhizobium is exposed to host plant inducers. The third, NodD3, is an inducer-independent activator of nod operons. We previously observed that nodD3 carried on a multicopy plasmid required another closely linked gene, syrM, for constitutive nod operon expression. Here, we show that syrM activates expression of the nodD3 gene, and that nodD3 activates expression of syrM. The two genes constitute a self-amplifying positive regulatory circuit in both cultured Rhizobium and cells within the symbiotic nodule. We find little effect of plant inducers on the circuit or on expression of nodD3 carried on pSyma. This regulatory circuit may be important for regulation of nod genes within the developing nodule.
Bacterial Proteins↗
Isolation and characterization of a DNA replication origin from the 1,700-kilobase-pair symbiotic megaplasmid pSym-b of Rhizobium meliloti.
A 4-kb fragment active as an autonomously replicating sequence (ARS) from the Rhizobium meliloti symbiotic megaplasmid pSym-b was isolated by selecting for sequences that allowed a normally nonreplicative pBR322 derivative to replicate in R. meliloti. The resulting Escherichia coli-R. meliloti shuttle plasmid (mini-pSym-b) containing the ARS also replicated in the closely related Agrobacterium tumefaciens, but only in strains carrying pSym-b, suggesting that a megaplasmid-encoded trans-acting factor is required. The copy number of mini-pSym-b was approximately the same as that of the resident megaplasmid, and mini-pSym-b was unstable in the absence of antibiotic selection. An 0.8-kb DNA subfragment was sufficient for replication in both R. meliloti and A. tumefaciens. The minimal ARS exhibited several sequence motifs common to other replication origins, such as an AT-rich region, three potential DnA binding sites, a potential 13-mer sequence, and several groups of short direct repeats. Hybridization experiments indicated that there may be a related ARS on the other megaplasmid, pSym-a. The pSym-b ARS was mapped near exoA, within a region nonessential for pSym-b replication. These results suggest that the R. meliloti megaplasmids share conserved replication origins and that pSym-b contains multiple replication origins. Since the mini-pSym-b shuttle vector can coexist with IncP-1 broad-host-range plasmids, it is also now possible to use two compatible plasmids for cloning and genetic manipulation in R. meliloti.
Agrobacterium tumefaciens↗
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↗
Depolarization of alfalfa root hair membrane potential by Rhizobium meliloti Nod factors.
Although much is known about the bacterial genetics of early nodulation, little is known about the plant cell response. Alfalfa root hair cells were impaled with intracellular microelectrodes to measure a membrane potential depolarizing activity in Rhizobium meliloti cell-free filtrates, a plant response dependent on the bacterial nodulation genes. The depolarization was desensitized by repeated exposure to factors and was not observed in a representative nonlegume. A purified extracellular Nod factor, NodRm-IV(S), caused membrane potential depolarization at nanomolar concentrations. This rapid single-cell assay provides a tool for dissecting the mechanisms of host cell response in early nodulation.
Lipopolysaccharides↗
Classics in cytology. V.: William Sanders and early urinary tract cytology.
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Cell Biology↗
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↗
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↗
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↗
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↗
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↗
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↗