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D A Siegele

Publications and source records attributed to D A Siegele.

14 recordsLinked to original sources
PubMed1993

Microbial competition: Escherichia coli mutants that take over stationary phase cultures.

Many microorganisms, including Escherichia coli, can survive extended periods of starvation. The properties of cells that survived prolonged incubation in stationary phase were studied by mixture of 10-day-old (aged) cultures with 1-day-old (young) cultures of the same strain of Escherichia coli. Mutants from the aged cultures that could grow eventually took over the population, which resulted in the death of the cells from the young cultures. This phenotype was conferred by mutations in rpoS, which encodes a putative stationary phase-specific sigma factor. These rapid population shifts have implications for the studies of microbial evolution and ecology.

Acridine Orange↗
PubMed1992

Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA.

The sigma 70 subunit of E. coli RNA polymerase is required for sequence-specific recognition of promoter DNA. Genetic studies and sequence analysis have indicated that sigma 70 contains two specific DNA-binding domains that recognize the two conserved portions of the prokaryotic promoter. However, intact sigma 70 does not bind to DNA. Using C-terminal and internal polypeptides of sigma 70, carrying one or both putative DNA-binding domains, we demonstrate that sigma 70 does contain two DNA-binding domains, but that N-terminal sequences inhibit the ability of intact sigma 70 to bind to DNA. Thus, we propose that sigma 70 is a sequence-specific DNA-binding protein that normally functions through an allosteric interaction with the core subunits of RNA polymerase.

Bacterial Proteins↗
PubMed1992

Cloning and sequencing of Escherichia coli murZ and purification of its product, a UDP-N-acetylglucosamine enolpyruvyl transferase.

The Escherichia coli gene murZ, encoding the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase, has been cloned and sequenced. Identified by screening an E. coli genomic library for clones that conferred phosphomycin resistance, murZ encoded a 419-amino-acid polypeptide and was mapped to 69.3 min on the E. coli chromosome. MurZ protein was purified to near homogeneity and found to have the expected UDP-N-acetylglucosamine enolpyruvyl transferase activity. Sequence analysis of the predicted product revealed 44% identity to OrfR from Bacillus subtilis (K. Trach, J.W. Chapman, P. Piggot, D. LeCoq, and J.A. Hoch, J. Bacteriol. 170:4194-4208, 1988), suggesting that orfR may also encode a UDP-N-acetylglucosamine enolpyruvyl transferase enzyme. MurZ is also homologous to the aromatic amino acid biosynthetic enzyme enolpyruvyl shikimate phosphate synthase, the other enzyme known to catalyze an enolpyruvyl transfer.

Alkyl and Aryl Transferases↗
PubMed1992

Life after log.

Explore the source record for details and available documents.

Cell Division↗
PubMed1989

Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase.

We have systematically assayed the in vivo promoter recognition properties of 13 mutations in rpoD, the gene that encodes the sigma 70 subunit of Escherichia coli RNA polymerase holoenzyme, using transcriptional fusions to 37 mutant and wild-type promoters. We found three classes of rpoD mutations: (1) mutations that suggest contacts between amino acid side-chains of sigma 70 and specific bases in the promoter; (2) mutations that appear to affect either sequence independent contacts to promoter DNA or isomerization of the polymerase; and (3) mutations that have little or no effect on promoter recognition. Our results lead us to suggest that a sequence near the C terminus of sigma 70, which is similar to the helix-turn-helix DNA binding motif of phage and bacterial DNA binding proteins, is responsible for recognition of the -35 region, and that a sequence internal to sigma 70, in a region which is highly conserved among sigma factors, recognizes the -10 region of the promoter. rpoD mutations that lie in the recognition helix of the proposed helix-turn-helix motif affect interactions with specific bases in the -35 region, while mutations in the upstream helix, which is thought to contact the phosphate backbone, have sequence-independent effect on promoter recognition.

Base Sequence↗
PubMed1988

Mutations in rpoD, the gene encoding the sigma 70 subunit of Escherichia coli RNA polymerase, that increase expression of the lac operon in the absence of CAP-cAMP.

We have isolated a new class of mutations in rpoD, the gene encoding the sigma 70 subunit of Escherichia coli RNA polymerase, that alter the transcription initiation properties of RNA polymerase holoenzyme. The rpoD(Lac) mutations increase expression of the lac operon in the absence of CAP-cAMP, allowing a strain lacking adenyl cyclase to grow on lactose. Four of the six alleles isolated have three- to fivefold increases in the amount of lac mRNA and beta-galactosidase per cell. We show that these four mutations increase transcription initiation from the same promoter used by wild-type RNA polymerase. The mutations were mapped and sequenced. One mutation occurs in the codon for amino acid 389 of the sigma 70 polypeptide. The remaining five mutations are clustered, affecting residues 570, 571 and 575. These five mutations are within or near a proposed helix-turn-helix motif in the C terminus of sigma 70.

Amino Acid Sequence↗
PubMed1985

The terminase of bacteriophage lambda. Functional domains for cosB binding and multimer assembly.

Terminase is a protein complex involved in lambda DNA packaging. The subunits of terminase, gpNul and gpA, are the products of genes Nul and A. The actions of terminase include DNA binding, prohead binding and DNA nicking. Phage 21 is a lambdoid phage that also makes a terminase, encoded by genes 1 and 2. The terminases of 21 and lambda are not interchangeable. This specificity involves two actions of terminase; DNA binding and prohead binding. In addition, the subunits of lambda terminase will not form functional multimers with the subunits of 21 terminase. lambda-21 hybrid phages can be produced as a result of recombination. We describe here lambda-21 hybrid phages that have hybrid terminase genes. The packaging specificities of the hybrids and the structure of their genes were compared in order to identify functional domains of terminase. The packaging specificities were determined in vivo by complementation tests and helper packaging experiments. Restriction enzyme site mapping and sequencing located the sites at which recombination occurred to produce the hybrid phages. lambda-21 hybrid 51 carries the lambda A gene, and a hybrid 1/Nul gene. The crossover that produced this phage occurred near the middle of the 1 and Nul genes. The amino-terminal portion of the hybrid protein is homologous to gp1 and the carboxy-terminal portion is homologous to gpNul. It binds to 21 DNA and forms functional multimers with gpA, providing evidence that the amino-terminal portion of gpNul is involved in DNA binding and the carboxy-terminal portion of gpNul is involved in the interaction with gpA. lambda-21 hybrid 54 has a hybrid 2/A gene. The amino terminus of the hybrid protein of lambda-21 hybrid 54 is homologous with gp2. This protein forms functional multimers only with gp1, providing evidence that the amino terminus of gpA is involved in the interaction with gpNul. These studies identify three functional domains of terminase.

Bacteriophage lambda↗
PubMed1984

A functional domain of bacteriophage lambda terminase for prohead binding.

Terminase is a multifunctional protein complex involved in DNA packaging during bacteriophage lambda assembly. Terminase is made of gpNul and gpA, the products of the phage lambda Nu1 and A genes. Early during DNA packaging terminase binds to lambda DNA to form a complex called complex I. Terminase is required for the binding of proheads by complex I to form a DNA: terminase: prohead complex known as complex II. Terminase remains associated with the DNA during encapsidation. The other known role for terminase in packaging is the production of staggered nicks in the DNA thereby generating the cohesive ends. Lambdoid phage 21 has cohesive ends identical to those of lambda. The head genes of lambda and 21 show partial sequence homology and are analogous in structure, function and position. The terminases of lambda and 21 are not interchangeable. At least two actions of terminase are involved in this specificity: (1) DNA binding; (2) prohead binding. The 1 and 2 genes at the left end of the 21 chromosome were identified as coding for the 21 terminase. gp1 and gp2 are analogous to gpNu1 and gpA, respectively. We have isolated a phage, lambda-21 hybrid 33, which is the product of a crossover between lambda and 21 within the terminase genes. Lambda-21 hybrid 33 DNA and terminase have phage 21 packaging specificity, as determined by complementation and helper packaging studies. The terminase of lambda-21 hybrid 33 requires lambda proheads for packaging. We have determined the position at which the crossover between lambda DNA and 21 DNA occurred to produce the hybrid phage. Lambda-21 hybrid 33 carries the phage 21 1 gene and a hybrid phage 2/A gene. Sequencing of lambda-21 hybrid 33 DNA shows that it encodes a protein that is homologous at the carboxy terminus with the 38 amino acids of the carboxy terminus of lambda gpA; the remainder of the protein is homologous to gp2. The results of these studies define a specificity domain for prohead binding at the carboxy terminus of gpA.

Bacteriophage lambda↗
PubMed1983

The head genes of bacteriophage 21.

Physical and genetic maps of the head genes of lambdoid phage 21 have been made and compared with the head gene map of lambda. Because 21 and lambda have partial sequence homology throughout the head genes it was expected that the head genes of 21 would be analogous to those of lambda. Eight head genes of 21 have been identified and it was found that each of the genes is analogous in position, structure, and/or function to a lambda head gene. Phage 21 genes analogous to the lambda D and FI genes were not identified by mutation. Complementation studies between phage 21 and lambda mutants indicate that only gpFII (the protein product of a gene is referred to as gp (gene product] is fully interchangeable, gpW and gpD are partially interchangeable, and the rest of the head morphogenetic proteins are phage specific. In analogy with phage lambda, it is found that the gpNu3 analog (gp6) of phage 21 is synthesized from the same reading frame as the gpC analog (gp5), resulting in a protein identical to the carboxy terminus of gp5.

Bacteriophage lambda↗
PubMed1982

Cosmid DNA packaging in vivo.

The packaging of cosmid DNA into phage particles during phage lambda growth is described. Evidence is presented supporting the work of others that cosmid transducing phages contain linear multimers of cosmid DNA in which the number of cosmid copies is that required to make a packagable DNA length (greater than 0.77 of the lambda DNA length). The yield of cosmid transducing phages declines sharply as the number of cosmid copies required to make a packagable DNA length increases. The cosmid DNA replication that produces the packaging substrate shares with lambda rolling-circle replication a dependence on the lambda gam gene product.

Bacteriophage lambda↗