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Biomedical subjects

W B Dempsey

Publications and source records attributed to W B Dempsey.

12 recordsLinked to original sources
PubMed1992

Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations.

Complementation analyses using minimal recombinant clones showed that all known pdx point mutations, which cause pyridoxine (vitamin B6) or pyridoxal auxotrophy, are located in the pdxA, pdxB, serC, pdxJ, and pdxH genes. Antibiotic enrichments for chromosomal transposon mutants that require pyridoxine (vitamin B6) or pyridoxal led to the isolation of insertions in pdxA, pdxB, and pdxH but not in pdxJ. This observation suggested that pdxJ, like pdxA, pdxB, and serC, might be in a complex operon. To test this hypothesis, we constructed stable insertion mutations in and around pdxJ in plasmids and forced them into the bacterial chromosome. Physiological properties of the resulting insertion mutants were characterized, and the DNA sequence of pdxJ and adjacent regions was determined. These combined approaches led to the following conclusions: (i) pdxJ is the first gene in a two-gene operon that contains a gene, temporarily designated dpj, essential for Escherichia coli growth; (ii) expression of the rnc-era-recO and pdxJ-dpj operons can occur independently, although the pdxJ-dpj promoter may lie within recO; (iii) pdxJ encodes a 26,384-Da polypeptide whose coding region is preceded by a PDX box, and dpj probably encodes a basic, 14,052-Da polypeptide; (iv) mini-Mud insertions in dpj and pdxJ, which are polar on dpj, severely limit E. coli growth; and (v) three classes of suppressors, including mutations in lon and suppressors of lon, that allow faster growth of pdxJ::mini-Mud mutants can be isolated. A model to account for the action of dpj suppressors is presented, and aspects of this genetic analysis are related to the pyridoxal 5'-phosphate biosynthetic pathway.

ATP-Dependent Proteases↗
PubMed1990

Integration host factor affects expression of two genes at the conjugal transfer origin of plasmid R100.

Integration host factor (IHF) binds to two sites near the origin of transfer of the conjugative antibiotic resistance plasmid, R100. DNase I footprinting shows that one site is immediately adjacent to oriT and the gene X promoter, and another is adjacent to the traM promoter. A third site, known only from retardation gels, is near the traJ promoter. The relative promoter activities of genes X, traJ and traM are reduced in himA mutants (IHF-), as measured by chloramphenicol-resistance assays. Transcript analyses by Northern blots showed a reduction in size of the principal gene X and traJ transcripts in the absence of IHF.

Bacterial Proteins↗
PubMed1989

Sense and antisense transcripts of traM, a conjugal transfer gene of the antibiotic resistance plasmid R100.

The region of the antibiotic resistance plasmid R100 that encodes the plasmid-specific transfer gene traM has two tandemly aligned promoters separated by 145 nucleotides. The principal transcripts are 705 and 562 nucleotides long. Minor transcripts are 1550 and 1700 nucleotides long. The 705-base transcript appears to encode an 11 kD traM protein. The 562-base transcript does not encode a detectable protein. When subcloned on short fragments, the promoter for the 562-base transcript initiates efficiently but that for the 705 site does not. The 3' ends of the 705 and 562 base transcripts end inside the traJ ORF. Thus they provide additional sense RNA to compete with traJ for finP, the antisense translational regulator of traJ. A model is proposed for the participation of these sense and antisense transcripts in the control of expression of the traJ gene.

Bacterial Proteins↗
PubMed1979

Lambda transducing phages derived from a FinO- R100::lambda cointegrate plasmid: proteins encoded by the R100 replication/incompatibility region and the antibiotic resistance determinant.

Three lambda transducing phages have been isolated from pEDR20, an R100::lambda cointegrate plasmid in which the lambda insertion inactivated the R100 finO gene. Physical analysis of the three phages showed that the lambda is inserted at kilobase coordinate 81.3 of R100. All three phages carry different amounts of R100 DNA in the left arm of lambda. Each pahge contains ISlb, the mer genes and the region between coordinate 81.3 and 88.6; thus, all contain the genes necessary for R100 replication. One phage, VA lambda 73, contains the entire r-determination of R100 in addition to the above DNA. Five proteins coded by the region between 81.3 and 88.6 were detected. These had subunit molecular weights of 10,400; 12,200; 16,200; 19,600; and 38,300. The first was made constitutively and the other four only from a lambda promoter. Other constitutive proteins were one from the cml fus region with a molecular weight of 22,400 (cml) and two from the str sul region with molecular weights of 31,500 (str?) and 30,100 (sul?). Mercuric ion induced synthesis of at least 10 proteins. Six of these were known from earlier work. The total size of the proteins which appear to derive from the mer genes exceeds by a factor of 1.5, the coding capacity of this region without overlapping genes. Some, or all of these extra proteins may be chromosomal in origin, possibly derepressed in response to mercury gene products.

Bacteriophage lambda↗
PubMed1978

Pyridoxine-requiring mutants of Escherichia coli: glycolaldehyde dehydrogenase is not coded for by the pdxB gene.

Twenty-seven independent pyridoxineless mutants belonging to genetic linkage group I were assayed for glycolaldehyde dehydrogenase. Some mutants lacked enzyme activity entirely, and others showed activity ranging from very low to wild-type levels. Reversion to pyridoxine independence usually had no effect upon this activity. Transfer of the pyridoxine genes to a common host that had wild-type levels of enzyme activity made the recipient pyridoxineless without affecting the activity. These results negate the idea of an obligatory role for glycolaldehyde dehydrogenase in pyridoxine biosynthesis.

Acetaldehyde↗
PubMed1978

3-hydroxypyruvate substitutes for pyridoxine in serC mutants of Escherichia coli K-12.

Escherichia coli K-12 mutants with serC genotype required pyridoxine and serine for normal growth, as do E. coli B mutants of this type. Mutants of the K-12 strain, however, reverted easily to pyridoxine independence without regaining activity in the 3-phosphoserine oxoglutarate transaminase coded for by the serC gene. Both these revertants and the parental type synthesized pyridoxine in normal amounts when 3-hydroxypyruvate was used as a supplement, although neither of these mutants could use this compound to satisfy their serine requirement. Since serine alone was inadequate to provide the nutritional requirement of serC mutants, these mutants must have been unable to synthesize 3-hydroxypyruvate from serine. We suggest that 3-phosphoserine oxoglutarate transaminase in normal E. coli serves as a catalyst for transaminating small amounts of serine to 3-hydroxypyruvate, which is then used in pyridoxine biosynthesis. In serC mutants, this activity is blocked, and these mutants then show a double requirement for serine and pyridoxine.

Escherichia coli↗
PubMed1978

Properties of lambda transducing bacteriophages carrying R100 plasmid DNA: mercury resistance genes.

Three lambdamer (resistance to Hg2+ and mercurials) transducing phages were prepared from three independent cointegrate isolates of bacteriophage lambda and plasmid R100. DNA heteroduplex and restriction nuclease analyses of the lambdamer DNA showed that all three phages had resulted from lambda insertion at kilobase coordinate 8.6 of plasmid R100, followed by loss of different lengths of lambda DNA and replacement with different lengths of R100 DNA. Two of the lambdamer phages were defective, containing deletions from lambdaatt through the lambdaN gene and into the lambdarex gene; the third, VAlambda14, was an N+ Spi- plaque-forming phage. With VAlambda14, N-dependent transcription of R100 mer from the lambdapL promoter suggested that transcription of mer proceeded in the direction from IS1b toward the sulfonamide resistance determinant (i.e., from a plasmid promoter in restriction nuclease fragment EcoRI-H toward fragment EcoRI-I). Phage-directed protein synthesis in a UV-irradiated lambdaind- lysogen showed the Hg2+-inducible synthesis of three major polypeptides of molecular weights 68,000, 11,500, and 8,500 and three minor ones of molecular weights 54,000, 33,000, and 13,500. The largest of the major polypeptides is identified as the subunit of the mercuric reductase enzyme. The functions of the smaller polypeptides are not known. Hg2+ reductase enzyme assays confirmed the regulation of mer synthesis during phage infection.

Bacterial Proteins↗
PubMed1976

Plasmid co-integrates of prophage lambda and R factor R100.

Single and tandem insertions of prophage lambda into R100 have been isolated. Insertions into the transfer genes, insertions into the transfer control gene finO, and insertions into regions that result in no detectable phenotypic change were found. From the last type, deletion mutants were isolated which established the sequence of antibiotic resistance genes as tet-cml-fus-str-sul-mer in R100. High frequency transducing phage preparations lambdamer, lambdasul str, and lambdasul str cml were also isolated from this type.

Anti-Bacterial Agents↗
PubMed1975

Incorporation of pantothenate into citrate lyase by a pantothenateless mutant of Klebsiella pneumoniae.

A pantothenate-requiring mutant of Klebsiella pneumoniae was isolated. The mutant showed an absolute dependence on pantothenate for growth. When grown in the presence of [14C]pantothenate, the mutant incorporated [14C]pantothenate into citrate lyase (3.4 mol/mol of enzyme). Analysis of a double-labeled enzyme ([14C]pantothenate and [3H]acetate) by gel electrophoresis in sodium dodecyl sulfate showed that both 3H and 14C were associated solely with the smallest subunit, the acyl carrier protein of citrate lyase.

Acetates↗