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D C Stein

Publications and source records attributed to D C Stein.

At least 19 recordsLinked to original sources
PubMed1992

Plasmids with easily excisable xylE cassettes.

Two new vectors containing the xylE gene (encoding catechol-2,3-dioxygenase) of Pseudomonas putida were constructed that serve as the source of the xylE cassette. These vectors are based on the kanamycin-resistance-encoding plasmid, pKAN18. The promoter-less xylE gene is flanked by several restriction enzyme sites that allow for easy excision of this gene in the form of a cassette containing a ribosome-binding site, 7 bp upstream from the start codon. These cassettes lack any transcriptional termination signals downstream from the stop codon.

Base Sequence↗
PubMed1992

Construction of a Neisseria gonorrhoeae MS11 derivative deficient in NgoMI restriction and modification.

We have cloned from Neisseria gonorrhoeae MS11 the gene encoding a methylase that modifies the sequence GCCGGC. The corresponding restriction enzyme was also encoded by this clone. Sequence analysis demonstrated that the methylase shares sequence similarities with other cytosine methylases, but the sequence organization of M.NgoMI is different from that seen for other cytosine methylases. A deletion was introduced into the chromosome of N. gonorrhoeae MS11 to produce strain MUG701, a strain that is inactivated in both the methylase and the restriction genes. Although this strain no longer methylated its DNA at the NgoMI recognition sequence, cells were viable and had no other significant phenotypic changes. Transformation data indicated that MS11 does not produce enough restriction activity to block plasmid transformation in the gonococcus, even though restriction activity could be demonstrated in E. coli containing the cloned gene.

Amino Acid Sequence↗
PubMed1992

Cloning and linkage analysis of Neisseria gonorrhoeae DNA methyltransferases.

We have cloned DNA methyltransferases (MTases) from various strains of Neisseria gonorrhoeae. Each of these clones represents a single specificity, indicating that the multiple gonococcal MTase specificities are encoded by monospecific MTases. The DNAs of five strains (FA5100, F62, MS11, Pgh3-2, and WR302) were digested with NheI, SpeI, or NheI plus SpeI and subjected to pulsed-field gel electrophoresis. The DNA MTase clones were used to probe Southern blots of these pulsed-field gels to determine whether the MTase genes are linked and whether there are strain-to-strain differences. The results indicate that none of these genes are closely linked, but variable hybridization patterns indicate that there exist restriction fragment length polymorphisms between the strains tested. Most of the chromosomal regions containing these restriction fragment length polymorphisms are clustered in regions containing gonococcal genes known or suspected to antigenically vary via genetic recombination.

Chromosome Mapping↗
PubMed1991

Characterization of melA: a gene encoding melanin biosynthesis from the marine bacterium Shewanella colwelliana.

A recombinant plasmid with the ability to impart melanin synthesis to an Escherichia coli host was isolated from a Shewanella colwelliana genomic library. The genetic determinant of the Mel+ phenotype is carried on a 1.3-kb DNA fragment and sequence analysis of this revealed a single intact open reading frame that was sufficient for melanin synthesis (mel). This gene is expressed as a monocistronic transcript and a putative transcription start point is located 115 nucleotides upstream from the translational start codon. The mel gene encoded a protein of 39.5 kDa [346 amino acids (aa)] that showed no aa sequence homology with other proteins known to mediate melanin synthesis (e.g., tyrosinases).

Amino Acid Sequence↗
PubMed1991

Structural heterogeneity of the lipopolysaccharides of the Neisseriaceae.

Lipopolysaccharides from 5 different genera of the Neisseriaceae were analyzed on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and visualized by silver staining. Significant heterogeneity in the banding patterns was observed with some of the strains producing only low molecular mass molecules and others producing O-repeating units. All genera examined except Branhamella contained strains that were able to produce an O-repeating side chain on their lipopolysaccharides. The ability to produce the repeating subunit did not correlate with the presence of plasmids.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

A new method for the rapid identification of genes encoding restriction and modification enzymes.

We have constructed derivatives of Escherichia coli that can be used for the rapid identification of recombinant plasmids encoding DNA restriction enzymes and methyltransferases. The induction of the DNA-damage inducible SOS response by the Mcr and Mrr systems, in the presence of methylated DNA, is used to select plasmids encoding DNA methyltransferases. The strains of E. coli that we have constructed are temperature-sensitive for the Mcr and Mrr systems and have been further modified to include a lacZ gene fused to the damage-inducible dinD locus of E. coli. The detection of recombinant plasmids encoding DNA methyltransferases and restriction enzymes is a simple, one step procedure that is based on the induction at the restrictive temperature of the lacZ gene. Transformants encoding DNA methyltransferase genes are detected on LB agar plates supplemented with X-gal as blue colonies. Using this method, we have cloned a variety of DNA methyltransferase genes from diverse species such as Neisseria, Haemophilus, Treponema, Pseudomonas, Xanthomonas and Saccharopolyspora.

Cloning, Molecular↗
PubMed1991

Characterization of a gyrB mutation responsible for low-level nalidixic acid resistance in Neisseria gonorrhoeae.

Nalidixic acid-resistant derivatives of Neisseria gonorrhoeae WR302 were identified and categorized into two classes on the basis of their susceptibilities to this antimicrobial agent. The MIC of nalidixic acid for the derivative strain MUG116 was fourfold greater than that for its isogenic parental strain WR302 (2 versus 0.5 micrograms/ml, respectively). MUG324 was significantly more resistant to nalidixic acid (greater than 64 micrograms/ml). The MICs of other antimicrobial agents known to interact with either the gyrA or gyrB gene products were determined. Although the nalidixic acid MIC for MUG116 increased, no significant increases in the MICs of other agents that interact with the gyrA gene product were seen. The MICs of all agents that interact with the gyrA gene product were significantly increased for MUG324. The gene that imparts low-level nalidixic acid resistance was cloned from strain MUG116. The DNA sequence of this gene was determined, and by comparing the deduced amino acid sequence with sequences of proteins in data bases, this protein was found to be approximately 70% homologous with the gyrB gene product of Escherichia coli.

Amino Acid Sequence↗
PubMed1991

Isolation of temperature-sensitive McrA and McrB mutations and complementation analysis of the McrBC region of Escherichia coli K-12.

We isolated temperature-sensitive mcrA and mcrBC mutants of Escherichia coli. At 42 degrees C, they were unable to restrict the T-even bacteriophages T6gt and T4gt or plasmids encoding cloned DNA methylase genes whose specificities confer sensitivity to the McrA and McrBC nucleases. Complementation analysis of the McrBC region (mcrB251) with the complete cloned McrBC system or a derivative with mcrB alone indicated that the mutation shows an absolute defect for the restriction of DNA containing hydroxymethylcytosine and a thermosensitive defect for the restriction of DNA containing methylcytosine. The properties of the McrA temperature-sensitive mutants suggest that some of these mutations can also influence the restriction of DNA containing hydroxymethylcytosine or methylcytosine residues.

Chromosomes, Bacterial↗
PubMed1991

Analysis of the lsi region involved in lipooligosaccharide biosynthesis in Neisseria gonorrhoeae.

The genetic locus (lsi-1) responsible for the transformation of the lipooligosaccharide (LOS)-defective Neisseria gonorrhoeae mutant FA5100 to LOS expression was studied by deletion mutagenesis and sequence analysis. An open reading frame that was preceded by a leader sequence containing regions with the potential to form hairpin loops was identified. A perfect sigma 70 promoter consensus sequence was found upstream from this open reading frame. Promoter function was screened for functionality by using lac fusion cassettes and in vitro transcription-translation analysis. A frameshift mutation in the lsi-1 gene was constructed by site-directed mutagenesis and introduced into the chromosome of FA19, the LOS-expressing isogenic parent strain of FA5100. The mutant was characterized by Southern blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting (immunoblotting) and found to be phenotypically identical to FA5100.

Amino Acid Sequence↗
PubMed1991

Transformation of Neisseria gonorrhoeae: physical requirements of the transforming DNA.

The 1600-bp (base pair) fragment encoding a portion of the nalidixic acid resistant DNA gyrase, subunit B, was characterized to determine what parameters effect transformation in the gonococcus. When this DNA (pSY2) was isolated from Escherichia coli, it was able to transform a variety of gonococcal strains to resistance to nalidixic acid via DNA-mediated transformation, irrespective of their restriction-modification phenotype. Nalidixic acid resistant transformants contained no plasmid DNA sequences that corresponded to the vector, as measured by plasmid screening procedures and colony hybridization techniques. Supercoiled and linear DNA transformed the gonococcus at the same efficiency. DNA fragments as small as 615 bp were able to transform the gonococcus. The presence of a 10-bp uptake sequence enhanced a DNA fragment's ability to transform the gonococcus by four orders of magnitude. When the fragment encoding the nalidixic acid resistant DNA gyrase was subcloned into M13mp18, both the replicative form and the single-stranded form of the phage were able to transform the gonococcus to nalidixic acid resistance.

Cloning, Molecular↗
PubMed1990

Expression of cloned restriction and modification genes, hjaIRM from Hyphomonas jannaschiana in Escherichia coli.

A type-II RM system, HjaI, was identified in the marine bacterium, Hyphomonas jannaschiana. The ENase recognizes GATATC, and DNA fragments generated after cleavage with this enzyme contain blunt ends. A DNA fragment encoding these enzymes was cloned and expressed in Escherichia coli, although the level of expression of the cloned genes was low. DNA methylated by M.HjaI was not restricted by the Mcr or Mrr restriction systems of E. coli. Although H. jannaschiana is a marine bacterium isolated near the thermal vents on the floor of the Pacific Ocean, the biochemical properties of the ENase were similar to those of EcoRV, an isoschizomer isolated from E. coli.

Bacteria↗
PubMed1989

Molecular analysis of lipooligosaccharide biosynthesis in Neisseria gonorrhoeae.

A HindIII gene bank of Neisseria gonorrhoeae MUG116 was constructed in the cosmid vector pHC79. A cosmid (pSY81) was isolated that was able to convert N. gonorrhoeae FA5100 to reactivity with monoclonal antibody (MAb) 2-1-L8. Several MAb-reactive transformants were isolated and characterized with respect to lipooligosaccharide (LOS) production as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their ability to react with two other LOS-specific MAbs (3F11 and O6B4), and Southern blot analysis. Escherichia coli containing the clone had altered lipopolysaccharide expression as determined by electrophoretic analysis; however, no reactivity was seen with gonococcus-specific MAbs. The introduction of pSY81 into FA5100 had a pleiomorphic effect, giving rise to transformants having the full parental phenotype or transformants lacking reactivity to a combination of LOS-specific MAbs. Southern blot analysis indicated that the LOS biosynthetic mutation in FA5100 was not due to chromosomal rearrangement or large deletions.

Blotting, Southern↗
PubMed1988

Neisseria gonorrhoeae M.Ngo AI DNA methyltransferase: physical and catalytic properties of the homogeneous enzyme.

A DNA methyltransferase, M.NgoAI, was purified to homogeneity from Neisseria gonorrhoeae strain WR220 by successive column chromatography. Its Mr is 25,000, as determined by both gel filtration and denaturing polyacrylamide gel electrophoresis. Maximal enzymatic activity was obtained in 50 mM Tris.HCl (pH 7.4), 10 mM EDTA, with incubation at 37 degrees C. An apparent Km value for S-adenosylmethionine and 5' -GGCC sites was determined to be 1.25 microM and 89.6 nM, respectively.

Bacterial Proteins↗
PubMed1988

Purification and characterization of DNA methyltransferases from Neisseria gonorrhoeae.

Three DNA methyltransferases, M.NgoAI, and M.NgoBI and M.NgoBII, free of any nuclease activities were isolated from Neisseria gonorrhoeae strains WR220 and MUG116 respectively. M.NgoAI recognizes the sequence 5' GGCC 3' and methylates the first 5' cytosine on both strands. M.NgoBI and M.NgoBII recognize 5' TCACC 3' and 5' GTAN5CTC 3' respectively. M.NgoBII methylates cytosine on only one strand to produce 5' GTAN5mCTC 3'.

Cytosine↗