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S R Long

Publications and source records attributed to S R Long.

83 records · Page 5Linked to original sources
PubMed1984

Generalized transduction in Rhizobium meliloti.

Generalized transduction of Rhizobium meliloti 1021 was carried out by bacteriophage N3. Genetic markers on the chromosome and the pSym megaplasmid were transduced, along with markers on several IncP plasmids. Cotransduction between transposon Tn5 insertions and integrated recombinant plasmid markers permitted correlation of cotransductional frequencies and known physical distances. Bacteriophage N3 was capable of infecting several commonly used strains of R. meliloti.

Bacteriophages↗
PubMed1983

Physical and genetic characterization of Rhizobium meliloti symbiotic mutants.

A set of 19 symbiotic mutants of Rhizobium meliloti obtained by a Tn5 "suicide plasmid" mutagenesis procedure was characterized genetically and physically. As part of this characterization, we showed that R. meliloti strain 1021, like other R. meliloti strains, contains a very large indigenous plasmid (greater than 300 Md) that carries the structural genes for nitrogenase (nifHDK genes). Among the 19 symbiotic mutations studied, at least six were shown to reside on the megaplasmid. By a "walking procedure" we obtained from a cosmid clone bank a set of overlapping cosmids that contained megaplasmid sequences contiguous to nifHDK. A 90 kb region of contiguous DNA from these cosmids was used to probe the mutant strains for rearrangements within this region. The same six mutations that were located on the megaplasmid mapped within the 90 kb region examined, which included the structural genes for nitrogenase (nifHDK). A majority of the mutations characterized in this study could not be correlated with a bona fide Tn5 insertion into a symbiotic gene.

DNA, Bacterial↗
PubMed1982

Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants.

We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK290 (Ditta et al., 1980) by inserting a 1.6-kb Bg/II fragment containing lambda cos into the unique Bg/II site in pRK290. The new vector, pLAFR1, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFR1 using a partial EcoRI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tcr) transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007. pLAFR1 cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tcr transconjugants were prototrophic.

Bacteriophage lambda↗
PubMed1982

Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis.

We have physically and genetically characterized 20 symbiotic and 20 auxotrophic mutants of Rhizobium meliloti, the nitrogen-fixing symbiont of alfalfa (Medicago sativa), isolated by transposon Tn5 mutagenesis. A "suicide plasmid" mutagenesis procedure was used to generate TN-5-induced mutants, and both auxotrophic and symbiotic mutants were found at a frequency of 0.3% among strains containing random TN5 insertions. Two classes of symbiotic mutants were isolated: 4 of the 20 formed no nodules at all (Nod-), and 16 formed nodules which failed to fix nitrogen (Fix-). We used a combination of physical and genetic criteria to determine that in most cases the auxotrophic and symbiotic phenotypes could be correlated with the insertion of a single Tn5 elements. Once the Tn5 element was inserted into the R. meliloti genome, the frequency of its transposition to a new site was approximately 10-8 and the frequency of precise excision was less than 10-9. In approximately 25% of the mutant strains, phage Mu DNA sequences, which originated from the suicide plasmid used to generate the Tn5 transpositions, were also found in the R. meliloti genome contiguous with Tn5. These later strains exhibited anomalous conjugation properties, and therefore we could not correlate the symbiotic phenotype with a Tn5 insertion. In general, we found that both physical and genetic tests were required to fully characterize transposon-induced mutations.

DNA Transposable Elements↗
PubMed1982

Structural studies of alfalfa roots infected with nodulation mutants of Rhizobium meliloti.

Alfalfa roots infected with four nodulation defective (Nod-) mutants of Rhizobium meliloti which were generated by transposon Tn5 mutagenesis were examined by light and electron microscopy. In one class of Nod- mutants, which we can nonreactive, the bacteria did not induce root hair curling or penetrate host cells. In a second class of Nod- mutants, which we call reactive, the bacteria induced some root hair curling and entered root epidermal cells, although no infection threads were formed. In addition, reactive Nod- mutants induced extensive root hair proliferation and hypertrophied roots. This study presents the details of the phenotype of the association between each mutant strain and alfalfa roots.

Medicago sativa↗
PubMed1982

ISRm1: A Rhizobium meliloti insertion sequence that transposes preferentially into nitrogen fixation genes.

After transposon Tn5 mutagenesis, a high proportion of Rhizobium meliloti symbiotic mutants do not contain Tn5 insertions in symbiotic genes. Instead, the mutations in these strains are correlated with the presence of an endogenous insertion sequence (ISRm1) in nitrogen fixation (nif) or symbiotic genes which are adjacent to the nif genes. ISRm1 is 1.4 kb and transposes to at least three restriction fragments in the nif region at a frequency between 10(-2) and 10(-3). A nif region restriction fragment containing ISRm1 was cloned from one of the mutant strains unable to fix nitrogen symbiotically (Fix-) and the resulting plasmid was used as a hybridization probe. ISRm1 is present at least ten times in the R. meliloti genome but is not present in any other R. meliloti strains, E. coli strains, or Rhizobium species tested. We demonstrated that the Fix- phenotype correlated with ISRm1 transposition is indeed caused by ISRm1 insertion by conjugating a cloned fragment containing ISRm1 into a wild type Fix+ R. meliloti host and replacing the normal genomic nif fragment with the nif::ISRm1 fragment. The resulting strain was Fix-.

DNA Transposable Elements↗
PubMedDate not supplied

Mass spectrometric investigations on Conus peptides.

Molecular masses and primary structure determination of Conus peptides, such as alpha-, mu- and omega-conotoxins, conantokins and conopressins, were accurately measured by state-of-the-art mass spectrometric techniques using only 1-2 pmole quantities. Soft ionization of Conus peptides under electrospray, matrix-assisted laser desorption and continuous flow frit-FAB conditions produced their corresponding singly and multiply charged molecular ions which can be detected by mass spectrometric analysis. The molecular masses of Conus peptides were obtained by the deconvolution of the multiply charged pseudo-molecular ions. Mixture analysis without chromatographic separation can be accomplished by this approach. The ions formed during collision-induced dissociation of either singly or multiply charged ions of any reduced and derivatized peptide provided the corresponding sequences of the amino acids. Preliminary investigations indicate that the developed techniques and procedures could be applied in order to characterize the peptides present in unknown Conus venoms from the Bay of Bengal region.

Animals↗
PubMedDate not supplied

Nucleotide sequence and protein products of two new nodulation genes of Rhizobium meliloti, nodP and nodQ.

Previous studies had suggested the existence of nodulation (nod) genes downstream of nodG in Rhizobium meliloti strain 1021. We have established the DNA sequence and analyzed the translation products of the genes located in this position. Computer analysis of the DNA sequence revealed a number of overlapping putative open-reading frames (ORFs), so we constructed several clones that contained either full-length or truncated ORFs. The protein products of these clones were expressed in both R. meliloti and Escherichia coli in vitro transcription-translation systems. These assays unambiguously defined the expressed ORFs, which we named nodP and nodQ. In addition, we found homology to these genes, via Southern hybridizations, elsewhere in the genome of R. meliloti strain 1021, and in other species of Rhizobium. The nodP gene also displayed homology to E. coli. A computer search revealed significant homology between NodQ and the GDP binding domain of elongation factor Tu (EF-Tu).

Amino Acid Sequence↗
PubMedDate not supplied

Homology of Rhizobium meliloti NodC to polysaccharide polymerizing enzymes.

Rhizobium bacteria form nitrogen-fixing nodules on legume roots. As part of the nodulation process, they secrete Nod factors that are beta-1,4-linked oligomers of N-acetylglucosamine. These factors depend on nodulation (nod) genes, but most aspects of factor synthesis are not yet known. We show here that one gene, nodC, shows striking similarity to genes encoding proteins known to be involved in polysaccharide synthesis in yeast and bacteria, specifically chitin and cellulose synthases, as well as a protein with unknown function in Xenopus embryos, DG42. This similarity is consistent with a role for the NodC protein in the formation of the beta-1,4-linkage in Nod factors.

Amino Acid Sequence↗
PubMedDate not supplied

An open reading frame downstream of Rhizobium meliloti nodQ1 shows nucleotide sequence similarity to an Agrobacterium tumefaciens insertion sequence.

We sequenced a small uncharacterized region in the Rhizobium meliloti nod gene cluster downstream of nodQ1. We found the beginning of a large open reading frame (260 amino acids) in this fragment. The sequence reported here has striking similarity to IS66 (Y. Machida, M. Sakurai, S. Kiyokawa, A. Ubasawa, and S. Yasushiro, 1984, Proc. Natl. Acad. Sci. USA 81:7495-7499), an insertion element found in an Agrobacterium tumefaciens mutant.

Agrobacterium tumefaciens↗