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C L Turnbough

Publications and source records attributed to C L Turnbough.

33 records · Page 2Linked to original sources
PubMed1988

Role of the ribosome in suppressing transcriptional termination at the pyrBI attenuator of Escherichia coli K-12.

Pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli K-12 occurs primarily by an attenuation control mechanism. Previous studies have suggested a model for attenuation control in which low intracellular levels of UTP cause close coupling of transcription and translation within the pyrBI leader region. This close coupling apparently prevents transcriptional termination at an attenuator (a rho-independent transcriptional terminator) located 23 base pairs before the pyrBI structural genes within an open reading frame for a 44-amino acid leader polypeptide. Presumably, a ribosome involved in the synthesis of the leader polypeptide disrupts or precludes the formation of the attenuator-encoded RNA hairpin, which is required for transcriptional termination. In this study, we examined the role of the ribosome in inhibiting transcriptional termination at the pyrBI attenuator. Using oligonucleotide-directed mutagenesis, we systematically introduced termination codons into the reading frame for the leader polypeptide to determine the distance a ribosome must translate to suppress transcriptional termination. These mutations were incorporated individually into a pyrB::lacZ gene fusion, which was then introduced into the E. coli chromosome. The resulting fusion strains were used to measure the effect of each mutation on pyrB::lacZ expression. The results show that a ribosome must translate to within 14-16 nucleotides of the attenuator-encoded RNA hairpin to inhibit transcriptional termination efficiently, which indicates a direct interaction between the ribosome and the termination hairpin sequence as proposed in the present model. Additional results indicate that factors not included in the present model for attenuation control contribute to the expression and regulation of the pyrBI operon.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Nucleotide sequence and characterization of the pyrF operon of Escherichia coli K12.

The pyrF gene of Escherichia coli K12, which encodes the pyrimidine biosynthetic enzyme orotidine-5'-monophosphate (OMP) decarboxylase, is part of an operon that includes a downstream gene designated orfF. The orfF gene product is a small polypeptide of unknown function. The nucleotide sequence of a 1549-base pair chromosomal fragment containing this operon was determined. An open reading frame capable of encoding the 27-kDa OMP decarboxylase subunit was identified and shown to be the pyrF structural gene by purifying and characterizing OMP decarboxylase. The subunit molecular weight (Mr = 26,350), amino-terminal amino acid sequence, and amino acid composition of the polypeptide predicted from the nucleotide sequence are in excellent agreement with those properties determined for the purified enzyme. The orfF structural gene was tentatively identified and apparently encodes an 11,396-dalton polypeptide. The orfF translational initiation codon overlaps the pyrF termination codon, which may indicate translational coupling in the expression of these genes. The pyrF promoter was mapped by primer extension of in vivo transcripts. The primary transcriptional initiation site is 51 base pairs upstream of the pyrF structural gene. The level of pyrF transcription and OMP decarboxylase synthesis was found to be coordinately derepressed by pyrimidine limitation, indicating that regulation of pyrF gene expression occurs at the transcriptional level. Inspection of the nucleotide sequence indicates that pyrF gene expression is not regulated by an attenuation control mechanism similar to that described for the pyrBI operon or pyrE gene. Finally, we compared the amino acid sequences of the OMP decarboxylases from E. coli, Saccharomyces cerevisiae, Neurospora crassa, and Ehrlich ascites cells to identify conserved regions.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence and expression of the pyrC gene of Escherichia coli K-12.

The pyrC gene of Escherichia coli K-12, which encodes the pyrimidine biosynthetic enzyme dihydroorotase, was cloned as part of a 1.6-kilobase-pair chromosomal fragment. The nucleotide sequence of this fragment was determined. An open reading frame encoding a 348-amino acid polypeptide (Mr = 38,827) was identified as the pyrC structural gene by comparing the amino acid composition predicted from the DNA sequence with that previously determined for the dihydroorotase subunit. The pyrC promoter was mapped by primer extension of in vivo transcripts. Transcriptional initiation was shown to occur within a region located 36 to 39 base pairs upstream of the pyrC structural gene. Pyrimidine availability appears to affect the use of the minor transcriptional initiation sites. The level of pyrC transcription and dihydroorotase synthesis was coordinately derepressed by pyrimidine limitation, indicating that regulation occurs, at least primarily, at the transcriptional level. Inspection of the pyrC nucleotide sequence indicates that gene expression is not regulated by an attenuation control mechanism similar to that described for the pyrBI operon and the pyrE gene. A possible mechanism of transcriptional control involving a common repressor protein is suggested by the identification of a highly conserved, operatorlike sequence in the promoter regions of pyrC and the other pyrimidine genes (i.e., pyrD and carAB) whose expression is negatively regulated by a cytidine nucleotide effector.

Amidohydrolases↗
PubMed1986

Genetic mapping of novel virulence determinants of Salmonella typhimurium to the region between trpD and supD.

Salmonella typhimurium strains are known to vary greatly in their virulence for mice. A few of the genes affecting their virulence have been described. In this report we have localized at least two genes that affect the ability of S. typhimurium to grow in BALB/cByJ to a 6 unit section of the salmonella chromosome which does not contain any previously described virulence determinants. The genetic mapping was done by interrupted matings using Hfr strains made in a virulent LT2 strain. The Hfr strains were constructed by inserted the plasmid F'(TS)114 lac+ Tn::10 into the LT2 chromosome at specific sites through homologous recombination with chromosomal Tn10s. Short interrupted matings to an avirulent LT2 strain in either direction through the portion of the chromosome from trpD at 34 units to supD at 40 units resulted in transconjugants which were fully virulent. Since we also found several transconjugants with intermediate virulence it appears that more than one virulence gene may exist in this area of the chromosome. The mechanisms of action of these genes are not known.

Animals↗
PubMed1986

Salmonella typhimurium virulence genes necessary to exploit the Itys/s genotype of the mouse.

The studies described in this paper provide evidence that the pathogenesis of Salmonella typhimurium in mice is dependent on interactions between particular genotypes of the infected mice and the infecting Salmonella strain. This conclusion was based on data obtained by infecting a panel of BXD recombinant inbred mice with each of three S. typhimurium strains. One of the S. typhimurium strains was a transconjugant (WB500) produced in an interrupted mating between the two other strains, one (SR-11) of high and the other (LT2-Z) of low virulence for BALB/c mice. We found that strain WB500 appeared to have inherited from SR-11 a gene (or genes) which was required to exploit the Itys/s genotype in mice. However, WB500 apparently lacked other SR-11 virulence gene(s), whose effect on the in vivo growth of SR-11 was independent of the Ity genotype of the mouse.

Animals↗
PubMed1985

The ability of Salmonella typhimurium to produce the siderophore enterobactin is not a virulence factor in mouse typhoid.

One of the nonspecific defense mechanisms of higher animals is their ability to limit iron availability to infecting bacteria. Thus it has been argued that all pathogenic bacteria must have special mechanisms to obtain iron in the host environment. Salmonella typhimurium is known to produce a siderophore, enterobactin, with which it can obtain iron from host transferrin. Previous studies have indicated that the production of this molecule is necessary for the ability of intraperitoneally injected. S. typhimurium cells to cause mouse typhoid, a largely intracellular infection. We have reexamined this finding with wild-type S. typhimurium and isogenic strains carrying the nonenterobactin-producing mutation ent-1 or ent-7. Our findings demonstrate that, although enterobactin production is necessary for growth in normal mouse serum, it does not affect the ability of S. typhimurium to cause mouse typhoid. Based on these findings and published results of other investigators on the role of siderophores in intracellular pathogens, a more comprehensive investigation of the importance of siderophores in intracellular infections may be warranted.

Animals↗
PubMed1985

Toxicity of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate in Salmonella typhimurium.

Growth of Salmonella typhimurium pyrC or pyrD auxotrophs was severely inhibited in media that caused derepressed pyr gene expression. No such inhibition was observed with derepressed pyrA and pyrB auxotrophs. Growth inhibition was not due to the depletion of essential pyrimidine biosynthetic pathway intermediates or substrates. This result and the pattern of inhibition indicated that the accumulation of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate was toxic. This intermediate is synthesized by the sequential action of the first two enzymes of the pathway encoded by pyrA and pyrB and is a substrate for the pyrC gene product. It should accumulate to high levels in pyrC or pyrD mutants when expression of the pyrA and pyrB genes is elevated. The introduction of either a pyrA or pyrB mutation into a pyrC strain eliminated the observed growth inhibition. Additionally, a direct correlation was shown between the severity of growth inhibition of a pyrC auxotroph and the levels of the enzymes that synthesize carbamyl aspartate. The mechanism of carbamyl aspartate toxicity was not identified, but many potential sites of growth inhibition were excluded. Carbamyl aspartate toxicity was shown to be useful as a phenotypic trait for classifying pyrimidine auxotrophs and may also be useful for positive selection of pyrA or pyrB mutants. Finally, we discuss ways of overcoming growth inhibition of pyrC and pyrD mutants under derepressing conditions.

Aspartic Acid↗
PubMed1985

Role of translation and attenuation in the control of pyrBI operon expression in Escherichia coli K-12.

Expression of the pyrBI operon of Escherichia coli K-12, which encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is negatively regulated by the intracellular levels of UTP. Previous experiments suggested a unique model for regulation of operon expression in which low UTP levels cause close coupling of transcription and translation of the pyrBI leader region. This close coupling suppresses transcriptional termination at an attenuator preceding the structural genes. In this study, we examined the regulatory role of translation and attenuation in operon expression. To determine whether the leader region is translated, we constructed a plasmid, designated pBHM17, in which the pyrBI promoter(s) and the first 11 codons for a putative 44-amino acid leader polypeptide are fused to codon 9 of lacZ. A transformant carrying this plasmid synthesized a beta-galactosidase fusion protein with the amino-terminal sequence of the leader polypeptide, demonstrating that the signals required for leader polypeptide synthesis function in vivo. Synthesis of the fusion protein was nearly insensitive to pyrimidine availability. In uracil-grown cells, the level of fusion protein synthesis encoded by plasmid pBHM17 was much greater than that encoded by a similar plasmid containing a pyrB::lacZ gene fusion, in which the pyrBI promoter-regulatory region is intact. These results indicate that the downstream leader sequence which includes the attenuator is required for regulation and functions as a transcriptional barrier. Oligonucleotide-directed mutagenesis was used to change the ATG leader polypeptide initiation codon of the intact pyrBI operon to ACG, which was shown to strongly inhibit translational initiation. This mutation greatly reduced operon expression and regulation as predicted by the attenuation control model.

Amino Acid Sequence↗
PubMed1983

Attenuation control of pyrBI operon expression in Escherichia coli K-12.

The pyrBI operon of Escherichia coli K-12 encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2). Expression of this operon apparently is negatively regulated by the intracellular levels of UTP. To elucidate the regulatory mechanism in which UTP functions, the nucleotide sequence of the promoter-regulatory region of the pyrBI operon was determined and DNA fragments containing this region were transcribed in vitro. These experiments revealed a rho-independent transcriptional terminator (attenuator) located only 23 base pairs before the promoter-proximal end of the structural genes. Transcription initiated upstream at either of two potential pyrBI promoters was efficiently (approximately equal to 98%) terminated at this site, indicating that the regulation of pyrBI expression involves attenuation control. Additional features identified suggest a model for regulation in which the relative rates of UTP-dependent transcription within the pyrBI leader region and coupled translation of the leader transcript control transcriptional termination at the attenuator.

Amino Acid Sequence↗
PubMed1983

Regulation of Escherichia coli aspartate transcarbamylase synthesis by guanosine tetraphosphate and pyrimidine ribonucleoside triphosphates.

The effects of guanosine tetraphosphate (ppGpp) and pyrimidine ribonucleoside triphosphates on Escherichia coli aspartate transcarbamylase (ATCase) synthesis were examined. To determine the effect of ppGpp, a stringent (relA+) and relaxed (relA) isogenic pair of E. coli K-12 strains was starved for isoleucine, and the residual rate of synthesis of this enzyme was measured. It was necessary to starve the strains for uracil before the isoleucine limitation to maintain similar, low levels of UTP, the putative pyrimidine effector of ATCase synthesis. The isoleucine starvation of the stringent strain caused an immediate 10-fold increase in the intracellular concentration of ppGpp, which was coincident with the cessation of the synthesis of the enzyme. The elevated level of ppGpp then decayed until it reached an intracellular concentration similar to that found in unstarved cells. Enzyme synthesis resumed at this time. In the relaxed strain, the intracellular concentration of ppGpp did not increase upon isoleucine starvation and synthesis of the enzyme was not repressed. These experiments strongly indicated that ppGpp acts as a negative effector of ATCase synthesis. The repression of ATCase synthesis by ppGpp was demonstrated directly by using a Salmonella typhimurium (relA) in vitro coupled transcription-translation system with a lambda specialized transducing phage carrying the E. coli K-12 operon encoding the subunits of this enzyme (pyrBI) as a source of DNA. This in vitro system was also used to measure the effects of UTP and CTP on ATCase synthesis. Increasing the concentration of UTP in the in vitro reaction mixture resulted in strong repression of this synthesis, whereas increasing the CTP concentration did not affect synthesis significantly. Possible mechanisms for the regulation of pyr gene expression, including attenuation control, are discussed.

Aspartate Carbamoyltransferase↗
PubMed1981

Purification and properties of glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis.

A procedure for the rapid and efficient purification of glutamine phosphoribosylpyrophosphate amidotransferase to better than 98% homogeneity from depressed Bacillus subtilis cells is described. The molecular weight of the subunit was estimated to be about 50 000. The purified enzyme exhibits microheterogeneity on electrophoresis on highly resolving polyacrylamide gels; it is suggested that this heterogeneity results from limited proteolytic modification of the native subunit. The native enzyme exists in equilibrium among tetrameric, dimeric, and monomeric forms. The influence of enzyme concentration and the presence of substrates and allosteric inhibitors on this equilibrium are described. There is no simple correlation between allosteric inhibition and stabilization of dimeric or tetrameric states. The amino acid composition of the amidotransferase is reported; presence of a 4Fe-4S center in the enzyme was described previously. Preparation of inactive apoprotein by treatment with 1,10-phenanthroline and general characteristics of the apoprotein are presented.

Amidophosphoribosyltransferase↗
PubMed1981

Infection of Salmonella typhimurium with coliphage Mu d1 (Apr lac): construction of pyr::lac gene fusions.

A procedure was developed for introducing the coliphage Mu d1 (Apr lac) into Salmonella typhimurium in order to construct gene fusions that place the structural genes of the lac operon under the control of the promoter-regulatory region of other genes. To introduce Mu d1 from Escherichia coli K-12 into S. typhimurium, which is normally not a host for Mu, we first constructed an E. coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid helper phage (MuhP1) that confers the P1 host range. A lysate prepared from this strain was used to infect a P1-sensitive (i.e., galE), restriction-deficient, modification-proficient strain of S. typhimurium, and a double lysogen carrying Mu d1 and MuhP1 was isolated. Induction of the latter strain produced lysates capable of infecting and generating gene fusions in P1-sensitive strains of S. typhimurium. In this paper we describe the construction of pyr::lac fusions by this technique.

Bacteriophage mu↗
PubMed1975

Oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase in stationary-phase cultures of Bacillus subtilis.

Glutamine phosphoribosylpyrophosphate amidotransferase (ATase) activity is rapidly inactivated in stationary-phase cells of Bacillus subtilis. The inactivation of APase requires both the cessation of rapid cell growth and the presence of oxygen. ATase is inactivated in two protease-deficient mutant strains at a rate similar to that seen in the wild type, and is stable in anaerobic cell-free extracts of the parent strain. These results suggest that the inactivation of ATase is not the result of general proteolysis. The inactivation of ATase in stationary-phase cultures can be inhibited by oxygen starvation. This oxygen requirement does not reflect a dependence on the generation of metabolic energy, but appears to be a direct requirement for molecular oxygen. ATase synthesis is repressed by the addition of adenosine, and is inactivated only after the cessation of exponential growth. Addition of chloramphenicol or rifampin to exponential- and stationary-phase cells does not inhibit ATase inactivation, suggesting that protein or ribonucleic acid synthesis is not required for inactivation. ATase is inactivated at the end of exponential growth in cells that have exhausted a required amino acid.

Adenosine↗
PubMed1975

Oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase in vitro inactivation.

The oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase (ATase) is demonstrated in cell extracts of Bacillus subtilis. The rate of inactivation of ATase in vitro is apparently first order with respect to oxygen concentration and ATase activity. ATase inactivation in vitro (or in vivo) cannot be reactivated by a variety of reductants. ATase is significantly stabilized to oxygen-dependent inactivation in vitro in the presence of tetrasodium phosphoribosylpyrophosphate and glutamine together. The effects of the end product inhibitors, adenosine 5-monophosphate (AMP) and guanosine 5-monophosphate (GMP), on the stability of ATase are antagonistic. AMP stabilizes ATase, whereas GMP destabilizes the enzyme. The stability of ATase can be manipulated over wide ranges by variations in the AMP/GM ratio. The effects of AMP and GMP on the inactivation of ATase in vitro are very specific. ATase is partially inhibited by 1,10-phenanthroline, suggesting that the enzyme contains iron (or some other chelatable metal ion). The inactivation of ATase in vitro is proposed to present a model for the reconstruction of the inactivation of ATase in stationary-phase cells of B. subtilis.

Adenosine Monophosphate↗