RESUMEN
The effects of mutations in the promoter of the histidine operon of Salmonella typhimurium were examined in vivo. The wild-type chromosomal copy of the his promoter was replaced with mutations in the -10 hexamer sequence and in the region between the -10 hexamer and the transcriptional start point-termed the discriminator sequence. The substitutions were performed with a phage M13 allele replacement system. Expression of the his operon is known to correlate with levels of guanosine 5',3'-bispyrophosphate (ppGpp) in vivo. Strains containing either the wild-type his promoter or his promoter mutations were grown in both nutrient-rich and minimal media under steady-state conditions known to alter intracellular levels of ppGpp in a predictable way. The effect of the presence or absence of the his attenuator was assessed under these conditions as well. Expression of the his operon was studied by measuring the differential rate of beta-galactosidase synthesis with a his-lac transcriptional fusion. Regulation of the his operon in the promoter mutants was also studied under conditions of a transient amino acid downshift induced by the addition of serine hydroxamate to cultures growing in nutrient-rich medium. These growth conditions cause elevated levels of ppGpp. The results provide physiological confirmation of previous evidence obtained with a coupled transcription-translation system in vitro which indicated that ppGpp regulates interaction of RNA polymerase at the his promoter. More specifically, the in vivo evidence shows that the region of the his promoter that includes the -10 hexamer and discriminator sequences is the target at which ppGpp stimulates transcription.
Asunto(s)
Aminoácidos/metabolismo , Histidina/genética , Operón , Regiones Promotoras Genéticas , Salmonella typhimurium/genética , Medios de Cultivo , Regulación Bacteriana de la Expresión Génica , Guanosina Tetrafosfato/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Salmonella typhimurium/metabolismo , Serina/análogos & derivados , Serina/farmacologíaRESUMEN
We studied the expression of the cya promoter(s) in cya-lac fusion strains of Salmonella typhimurium and demonstrated cAMP receptor protein (CRP)-dependent repression by cAMP. Expression of cya was reduced about fourfold in cultures grown in acetate minimal medium as compared to cultures grown in glucose-6-phosphate minimal medium. Expression of cya was also reduced about fourfold by addition of 5 mM cAMP to cultures grown in glucose minimal medium. We constructed in vitro deletion and insertion mutations altering a major cya promoter (P2) and a putative CRP binding site overlapping P2. These mutations were recombined into the chromosome by allele replacement with M13mp::cya recombinant phages and the regulation of the mutant promoters was analyzed. A 4-bp deletion of the CRP binding site and a 4-bp insertion in this site nearly eliminated repression by cAMP. A mutant with the P2 promoter and the CRP binding site both deleted exhibited an 80% reduction in cya expression; the 20% residual expression was insensitive to cAMP repression. This mutant retained a Cya+ phenotype. Taken together, the results establish that the cya gene is transcribed from multiple promoters one of which, P2, is negatively regulated by the cAMP-CRP complex. Correction for the contribution to transcription by the cAMP-CRP nonregulated cya promoters indicates that the P2 promoter is repressed at least eightfold by cAMP-CRP.
Asunto(s)
Adenilil Ciclasas/genética , Regulación Bacteriana de la Expresión Génica , Mutación , Receptores de AMP Cíclico/metabolismo , Salmonella typhimurium/genética , Transcripción Genética , Acetatos/metabolismo , Adenilil Ciclasas/metabolismo , Alelos , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Medios de Cultivo , AMP Cíclico/metabolismo , Glucofosfatos/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Salmonella typhimurium/enzimología , Salmonella typhimurium/crecimiento & desarrolloRESUMEN
We determined the nucleotide sequence of the regulatory region of the cya gene of Salmonella typhimurium. A set of nested BAL-31 deletions originating upstream of the promoter/regulatory region and extending into the cya structural gene was constructed in M13mp::cya phages and was tested for complementation of a chromosomal cya deletion mutation. BAL-31 deletion mutants unable to complement cya localized the major cya promoter. The synthetic tac promoter was inserted upstream of the BAL-31 deletions so that expression of cya was dependent on transcription from tac. Those tac derivative phages unable to complement cya localized the translation initiation region. The cya DNA sequence revealed at least three potential promoters capable of transcribing cya, with a CRP binding site straddling the-10 hexamer of the promoter proximal to the structural gene. The leader RNA sequence initiated at the latter promoter is approximately 140 bases long and includes a region that may form a stable secondary structure (delta G = -23.8 kcal). There exist two possible in-frame translation start points, one of which is TTG and the other of which is ATG. The sequence of the S. typhimurium regulatory region was compared with that reported for Escherichia coli.
Asunto(s)
Adenilil Ciclasas/genética , Regulación Bacteriana de la Expresión Génica , Secuencias Reguladoras de Ácidos Nucleicos , Salmonella typhimurium/genética , Adenilil Ciclasas/metabolismo , Secuencia de Bases , Deleción Cromosómica , Cromosomas Bacterianos , Genes Bacterianos , Prueba de Complementación Genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Mapeo Restrictivo , Salmonella typhimurium/enzimología , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
We isolated a collection of 67 independent, spontaneous Salmonella typhimurium his operon promoter mutants with decreased his expression. The mutants were isolated by selecting for resistance to the toxic lactose analog o-nitrophenyl-beta-D-thiogalactoside in a his-lac fusion strain. The collection included base pair substitutions. small insertions, a deletion, and one large insertion identified as IS30 (IS121), which is resident on the Mu d1 cts(Apr lac) phage used to construct the his-lac fusion. Of the 37 mutations that were sequenced, 14 were unique. Six of the 14 were isolated more than once, with the IS30 insertion occurring 16 times. The mutations were located throughout the his promoter region, with two in the conserved - 35 hexamer sequence, four in the conserved - 10 hexamer sequence (Pribnow box), seven in the spacer between the - 10 and -35 hexamer sequences, and the IS30 insertions just upstream of the -35 hexamer sequence. Four of the five substitution mutations changed a consensus base pair recognized by E sigma 70 RNA polymerase in the -10 or -35 hexamer. Decreased his expression caused by the 14 different his promoter mutations was measured in vivo. Relative to the wild-type promoter, the mutations resulted in as little as a 4-fold decrease to as much as a 357-fold decrease in his expression, with the largest decreases resulting from changes in the most highly conserved features of E sigma 70 promoters.
Asunto(s)
Histidina/biosíntesis , Mutación , Operón , Regiones Promotoras Genéticas , Salmonella typhimurium/genética , Secuencia de Bases , Deleción Cromosómica , Elementos Transponibles de ADN , Farmacorresistencia Microbiana/genética , Genes Bacterianos , Genotipo , Datos de Secuencia Molecular , Nitrofenilgalactósidos/farmacología , Salmonella typhimurium/efectos de los fármacos , beta-Galactosidasa/genéticaRESUMEN
We have analyzed the correlation of attenuator-independent expression of the Salmonella typhimurium histidine operon in vivo with levels of the "alarmone" guanosine 5'-diphosphate 3'-diphosphate. Amino acid downshift caused by serine hydroxamate addition increased his expression in a relA+ strain and decreased his expression in a relA mutant, whereas levels of guanosine 5'-diphosphate-3'-diphosphate varied in parallel with the changes in his expression in the two strains. In several experiments, overall variations in his expression ranged from 20- to 60-fold after downshift. The mild downshift allowed growth of the cultures to continue at near-preshift rates. Serine hydroxamate addition was also used to analyze the effect of amino acid downshift on induced expression of wild-type and mutant lac promoters. There was a 12-fold difference in lac expression when a relA+-relA1 pair was subjected to mild starvation but only a 3-fold difference when the strains carried the lacZpL8UV5 promoter mutation. These results suggest that guanosine 5'-diphosphate-3'-diphosphate stimulates gene expression in vivo at the level of transcription initiation.
Asunto(s)
Aminoácidos/metabolismo , Nucleótidos de Guanina/metabolismo , Guanosina Tetrafosfato/metabolismo , Histidina/biosíntesis , Operón , Salmonella typhimurium/genética , Genotipo , Cinética , Mutación , Regiones Promotoras Genéticas , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/crecimiento & desarrollo , Serina/análogos & derivados , Serina/farmacología , Especificidad de la EspecieRESUMEN
We have developed an allele exchange system for shuttling sequences of DNA to and from their original chromosomal loci. Cloned segments of the histidine operon of Salmonella typhimurium and the lactose operon of Escherichia coli served as target sequences and were used to develop the system. Replacement and retrieval of target sequences used the phage M13mp vectors and proceeded through an M13 lysogen intermediate. The intermediates and products of allele exchange were characterized by genetic and hybridization analyses. Several unique properties of M13 lysogens were exploited to devise positive selections to detect integration and excision. These positive selections were used to manipulate phenotypically silent alleles.
Asunto(s)
Colifagos/genética , Escherichia coli/genética , Genes Bacterianos , Operón , Salmonella typhimurium/genética , Deleción Cromosómica , Técnicas Genéticas , Histidina/biosíntesis , Cinética , Lactosa/biosíntesis , Lisogenia , FenotipoRESUMEN
We have analyzed the effects of the "alarmone" guanosine 5'-diphosphate 3'-diphosphate (ppGpp) on regulation of the Salmonella typhimurium histidine operon in vitro. Expression of the wild-type promoter, measured in a DNA-dependent transcription-translation system, was strongly dependent on ppGpp; addition of ppGpp stimulated his expression 22-fold with plasmid DNA templates. Oligonucleotide-directed, site-specific mutations that increase the homology of the -10 hexamer to the consensus sequence of the E sigma 70 promoters dramatically increased his expression in the absence of ppGpp and reduced the stimulation to less than a factor of 2. A deletion mutation that alters the sequence between the -10 hexamer and the start point of transcription, generated by BAL-31 nuclease, affected ppGpp regulation in a similar manner. We propose that the -10 hexamer sequence and the adjacent downstream region are both important in regulating transcription by ppGpp. Mechanisms to account for activation and repression of transcription by ppGpp are discussed.
Asunto(s)
Nucleótidos de Guanina/farmacología , Guanosina Tetrafosfato/farmacología , Histidina/genética , Operón , Regiones Promotoras Genéticas , Salmonella typhimurium/genética , ADN Bacteriano/genética , ADN Superhelicoidal/genética , Regulación de la Expresión Génica , Transcripción Genética/efectos de los fármacosRESUMEN
Phage Mud1 cts (Apr lac), or Mud1, insertion mutations may be accompanied by adjacent deletion formation which can complicate use of lac fusions generated with this phage for gene regulatory studies. As for phage Mu insertion mutations, phage Mud1 insertions fail to revert at significant frequency (whether or not accompanied by an adjacent deletion). We describe isolation of revertible (X mutant) derivatives of phage Mud1 in Salmonella typhimurium. The X mutant derivatives allow use of reversion as a simple test to determine whether a Mud1 insertion has occurred precisely without an adjacent deletion that may have fused the lac genes to a promoter outside of the gene of interest. In addition, a simple method for stabilizing Mud1 generated lac fusions against subsequent transposition is described.
Asunto(s)
Elementos Transponibles de ADN , Mutación , Fagos de Salmonella/genética , Salmonella typhimurium/genética , Genes Bacterianos , Genes Reguladores , Genes Virales , Genotipo , Especificidad de la EspecieRESUMEN
We have sequenced the hisD-hisC gene border of the Salmonella typhimurium histidine operon. The translation termination codon of the hisD gene overlaps with the translation initiation codon of the hisC gene in the manner AUGA. The Shine-Dalgarno sequence of the hisC gene is contained entirely within hisD and there is no intercistronic space since all of the bases are utilized in coding. Two mutations that alter the hisD-hisC gene border are analyzed. Both mutations simultaneously abolish the termination codon of hisD and modify the initiation codon of hisC. One of the mutations changes the hisC initiation codon from AUG to AUU. The AUU codon is 10 to 20% as efficient as AUG for initiation of translation of the hisC gene. The mutant hisC ribosome binding site is compared to the ribosome binding site of the Escherichia coli infC gene which has been reported to contain an AUU initiation codon. The role of overlapping termination/initiation codons in regulating translation of polycistronic mRNAs in bacterial operons is discussed.
Asunto(s)
Genes Bacterianos , Genes , Histidina/biosíntesis , Operón , Salmonella typhimurium/genética , Secuencia de Aminoácidos , Secuencia de Bases , Biosíntesis de Proteínas , ARN Mensajero/genética , Fagos de Salmonella/genética , Moldes GenéticosRESUMEN
A restriction map was determined for a phi 80 lambda dhis transducing phage DNA carrying the Salmonella typhimurium histidine operon. DNA fragments containing the promoter/regulatory region and the first two structural genes of the histidine operon (hisOGD) were identified by their ability to direct regulated synthesis of histidinol dehydrogenase (product of hisD) in a coupled in vitro protein synthesizing system. A 3.1-kb SalI-EcoRI restriction fragment containing the hisOGD region, was subcloned into phage M13mp8 and M13mp9 RF DNAs. Methods are described for shuttling mutant and wild-type bacterial DNA sequences between the M13mp::his phage and host bacterial genomes. Of novel importance is the use of the phage M13 gene II amber mutation to obtain integration of the M13mp::his phage genome into the homologous his region of the bacterial chromosome following transduction of recipients lacking an amber suppressor. This method can be used to facilitate allele replacement with genes carried on M13 transducing phages.
Asunto(s)
Genes Bacterianos , Histidina/genética , Operón , Salmonella typhimurium/genética , Bacteriófagos/genética , Mapeo Cromosómico , Clonación Molecular , Enzimas de Restricción del ADN , ADN Bacteriano/genética , ADN Viral/genética , Regulación de la Expresión Génica , Transducción GenéticaRESUMEN
The hisT mutation in Salmonella typhimurium which results in loss of pseudouridine base modifications in the anticodon regions of many tRNAs was shown to reduce the rate of protein synthesis in vivo by about 20 to 25% as compared with that measured in hisT strains. Reduced protein synthesis rate occurred predominantly at the level of translation rather than transcription. Increased sensitivity of hisT mutants to growth inhibition by antibiotics that inhibit translation elongation, but not by those that inhibit translation initiation, transcription initiation, or transcription elongation, indicates that the hisT mutation leads to a defect in one or more of the steps in the polypeptide chain elongation mechanism. These results can account for effects of the hisT mutation on regulation of certain amino acid biosynthetic operons, including the his, leu, and ilv operons.
Asunto(s)
Regulación de la Expresión Génica , Genes Bacterianos , Transferasas Intramoleculares , Extensión de la Cadena Peptídica de Translación , Seudouridina/metabolismo , Salmonella typhimurium/genética , Uridina/análogos & derivados , Proteínas Bacterianas/biosíntesis , Inducción Enzimática , Cinética , Mutación , Seudouridina/genética , Salmonella typhimurium/metabolismo , Transcripción Genética , beta-Galactosidasa/biosíntesisRESUMEN
Equilibrium constants, defined on the basis of the moles of all reactants and products including water, were found to be 4.6, 1.0 and 0.8 for the esterification of the glycinc cation at 20 degrees in a dilute aqueous sytem, and systems having 0.5 : 1 and 1 : 1 molar ratios of ethanol to water, respectively. When corrections were made for deviations from ideality a value of 5 +/- 1 was obtained for all three systems. Enthalpy changes were determined calorimetrically for the dilute aqueous and equimolar ethanol-water systems, and in each case was close to 1 kcal mole(-). The entropy change was calculated as 6 cal mole(-1) deg(-1).DeltaG(0') for the hydrolysis of the glycine ethyl ester cation at 20 degrees and pH 7 was calculated as -7.5 kcal mole(-1).
RESUMEN
The nitrogen regulatory locus "glnR" of Escherichia coli and Salmonella typhimurium is composed of two cistrons, which we propose to call ntrB and ntrC (nitrogen regulation B and C). Frameshift mutations in ntrB and ntrC were isolated on a lambda phage that carries the E. coli ntrB and ntrC genes and the closely linked glnA gene, the structural gene encoding glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]; mutations were selected as suppressors of glnF (which we propose to rename ntrA), a selection used previously to isolate glnR mutations. Phage DNA from one mutant (ntrB) failed to direct synthesis of a 36-kilodalton (kDal) protein whose synthesis was directed by DNA from the parent phage (ntrB+) in a coupled in vitro transcription/translation system. DNA from three other mutants (ntrC) failed to direct synthesis of a 54-kDal protein; DNA from two of these mutants instead directed synthesis of smaller proteins, 53 and 50 kDal, respectively. In all four cases, DNA from frameshift revertants directed synthesis of both the 36-kDal and 54-kDal proteins. These results suggested that ntrB and ntrC were separate genes which encoded 36-kDal and 54-kDal protein products, respectively. Frameshift mutations in ntrB and ntrC complemented each other with regard to regulation of glnA expression in vivo and growth on arginine as nitrogen source, another nitrogen-controlled phenotype; this confirmed that ntrB and ntrC are separate cistrons that encode diffusible products. The ntrB and ntrC genes were also defined in S. typhimurium. Studies of mutant strains provided information on the roles of the ntrB and ntrC products in activation and repression of glnA expression and raised the possibility that these products function as a protein complex in regulating expression of nitrogen-controlled genes.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Genes Reguladores , Nitrógeno/metabolismo , Salmonella typhimurium/genética , Bacteriófago lambda , ADN Recombinante , Regulación de la Expresión Génica , Genes , Prueba de Complementación Genética , Glutamato-Amoníaco Ligasa/genética , MutaciónRESUMEN
Maximal expression of the Escherichia coli lactose operon in a coupled in vitro transcription-translation system from a Salmonella typhimurium relA mutant was strongly dependent upon addition of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). Without added ppGpp, at saturating 3',5'-cyclic AMP (cAMP) concentrations, synthesis of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was reproducibly only 5-7% of that which can be obtained with 0.5-0.8 mM ppGpp. Experiments in which transcription was uncoupled from translation indicated that this 14- to 20-fold stimulation by ppGpp occurred at the level of transcription. When coupled beta-galactosidase synthesis was primed with a template containing a well-characterized mutant lac promoter (lacP(r)L8UV5), the dependence on ppGpp was greatly reduced. This result provides an important experimental control previously unavailable for verifying the significance of ppGpp effects on gene regulation in vitro; it indicates that activation of lacP(+) expression by ppGpp is specifically an effect of increased transcription initiations. Furthermore, the large ppGpp stimulation of lacP(+) DNA enabled the level of expression of this template to approach that of lacP(r)L8UV5 DNA, an observation expected from results in vivo but not obtained with other transcription-translation systems in vitro. The importance of these results is considered with respect to previous ideas on the physiological role of ppGpp as a supercontrol molecule in bacterial regulation.
Asunto(s)
Escherichia coli/metabolismo , Galactosidasas/biosíntesis , Nucleótidos de Guanina/farmacología , Guanosina Tetrafosfato/farmacología , Operón Lac , Salmonella typhimurium/metabolismo , Transcripción Genética/efectos de los fármacos , beta-Galactosidasa/biosíntesis , Oxidorreductasas de Alcohol/biosíntesis , Histidinol , CinéticaRESUMEN
Massive gynecomastia can produce severe psychological problems for the patient. The surgical techniques employed for simple gynecomastia are not effective in correcting the displaced nipple and excess skin. Utilizing a dermal predicle technique, the excess fat and breast tissue have been removed, along with relocation of the nipple. Satisfactory results and acceptable scars have been obtained in four patients.
Asunto(s)
Mama/cirugía , Ginecomastia/cirugía , Adulto , Procedimientos Quirúrgicos Dermatologicos , Estudios de Seguimiento , Ginecomastia/etiología , Humanos , Síndrome de Klinefelter/complicaciones , Masculino , Pezones/cirugía , Cirugía Plástica/métodosRESUMEN
Mutations are described which delete all or part of the first structural gene (hisG) of the histidine operon of Salmonella typhimurium. Physiological regulation of histidine enzymes occurs normally in strains carrying any deletion that has both endpoints within the hisG gene. Constitutive high operon expression is observed in strains carrying any hisG deletion and an unlinked regulatory mutation, hisT1504. These results strongly indicate that the hisG protein is not an essential component of the mechanism for regulating expression of the histidine operon.
Asunto(s)
Genes , Histidina/biosíntesis , Operón , Salmonella typhimurium/metabolismo , División Celular , Mapeo Cromosómico , Genotipo , Mutación , Transducción GenéticaRESUMEN
Maximal expression of the histidine operon of Salmonella typhimurium in a coupled in vitro transcription-translation system is strongly dependent upon addition of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). This requirement for ppGpp is exerted at the level of transcription through a mechanism distinct from the his-operon-specific regulatory mechanism. In vivo derepression of the his operon is markedly defective when histidine starvation is imposed on a relA mutant--unable to rapidly increase synthesis of ppGpp--growing in amino-acid-rich medium. Increased sensitivity of relA mutants to growth inhibition by a number of amino-acid analogs suggests that ppGpp is generally important in adjusting expression of amino-acid-producing systems. Analysis of these findings leads us to propose that ppGpp is a positive effector in a system that enables the cell to balance endogenous amino-acid production with environmental conditions of amino-acid availability, and to compensate efficiently for transient changes in these conditions. We propose a unifying theory of the role of ppGpp as the general signal molecule (alarmone) in a "super-control" which senses an amino-acid deficiency and redirects the cell's economy in response.
Asunto(s)
Aminoácidos/biosíntesis , Nucleótidos de Guanina/farmacología , Histidina/biosíntesis , Operón/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Aminoácidos/farmacología , Genes Reguladores/efectos de los fármacos , Nucleótidos de Guanina/metabolismo , Mutación , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/crecimiento & desarrolloRESUMEN
An activator-attenuator model of positive control, a s opposed to the classic repressor-operator model of negative control, is proposed for the major operon-specific mechanism governing expression of the histidine gene cluster of Salmonella typhimurium. Evidence for this mechanism is derived from experiments performed with a coupled in vitro transcription-translation system, as well as with a minimal in vitro transcription system [Kasai, T. (1974) Nature 249, 523--527]. The product (G enzyme, or N-1-[5'-phosphoribosyl]adenosine triphosphate:pyrophosphate phosphoribosyltransferase; EC 2.4.2.17) of the first structural gene (hisG) of the histidine operon is not involved in the positive control mechanism. However, a possible role for G enzyme as an accessory negative control element interacting at the attenuator can be accommodated in our model. The operon-specific mechanism works in conjunction with an independent mechanism involving guanosine 5'-diphosphate 3'-diphosphate (ppGpp) which appears to be a positive effector involved in regulating amino-acid-producing systems, in general [Stephens, J.C., Artz, S.W. & Ames, B.N. (1975) Proc. Nat. Acad. Sci. USA, in press].