Promoter-like mutants with increased expression of the Escherichia coli uridine phosphorylase structural gene.

From an Escherichia coli K-12 strain lacking adenylate cyclase (cya) and cyclic AMP receptor protein (crp), two mutants were isolated that synthesize uridine phosphorylase constitutively. The mutations differ from one another and also from a wild type in the maximum rate of uridine phosphorylase synthesis. They have constitutive expression of the uridine phosphorylase gene (udp) in the presence of repressor protein coded by the cytR regulatory gene and decrease the sensitivity of the udp gene simultaneously with catabolite repression. Both mutations cause a high level of udp expression whether they are in a cya crp or in a cya+ crp+ background. Another mutation (udpP1) isolated previously alters the response of udp gene to the ctyR repressor and produces a higher constitutive level of uridine phosphorylase in a cytR+ than in a cytR background when bacteria are grown in glucose. The synthesis of uridine phosphorylase in this mutant is dependent on an intact cyclic AMP-cyclic AMP receptor protein complex. All mutations studied are cis-acting and extremely closely linked to the udp structural gene, and appear to affect the uridine phosphorylase promoter-operator region. The data obtained are in accordance with a suggestion that the cytR repressor protein normally asserts its function by preventing the positive action of cyclic AMP-cyclic AMP receptor protein complex.     

Interaction of negative (CytT) and positive (cAMP-CRP) regulation in the promoter region of the uridine phosphorylase (udp) gene in Escherichia coli K-12

Interaction of negative (CytR) and positive (cAMP-CRP) control in the promoter region of the uridine phosphorylase (udp) gene of Escherichia coli has been studied by using udp-lac operon fusions in which the structural lacZ gene is expressed from the wild type promoter udpP+ or from mutant promoters udpP1 and udpP18. The specific activity of beta-galactosidase was examined in these fusions in cytR+ and cytR- backgrounds after introduction of specific mutations in crp locus, crp* and crp(a) altering interaction of CRP protein with catabolite-sensitive promoters. The data obtained using crp* mutation confirm the proposed model of the udp gene regulation, according to which CytR repressor protein interferes with CRP binding site in the promoter-operator region of the udp gene and thereby prevents the positive action of cAMP-CRP complex on the udp expression. Additional data in favor of this model were obtained using crp(a) mutation which most probably alters the structure of CRP protein in such a way that it exhibits more high affinity to the udp promoter, as compared to the CytR repressor protein. Indeed, taken by itself, the crp(a) mutation did not lead to any increase in the expression of udpP+-lac fusion under the conditions of cAMP limitation (on glucose-grown cells), in spite of whether or not the CytR repressor was present. However, when combined with the ptsG mutation or when cells were grown on succinate medium, complete constitutive expression of udpP+-lac fusion is observed, even in the presence of the cytR gene product. The effect of the crp(a) mutation was virtually the same in strains harboring udpP1-lac fusion. These data are in accordance with suggestion that udpP1 is a mutation in the site of the promoter-operator region that responds to the cytR gene product, while the corresponding binding site for CRP protein is still unaltered in this mutant. On the other hand, the crp(a) mutation causes only slight alteration in the expression of udpP18-lac fusion, providing additional evidence that udpP18 mutation seems to comprise a modification of the promoter-operator region, where binding sites for CRP and CytR proteins overlap.     

Genetic Characterization of Mutations Which Affect Catabolite-Sensitive Operons in Escherichia coli, Including Deletions of the Gene for Adenyl Cyclase

Sixty-two spontaneous mutations have been characterized which reduce the level of expression of catabolite-sensitive operons. These mutations appear to affect either the crp (catabolite gene activator protein) or cya (adenyl cyclase) loci. No new loci have been discovered. Deletions of the cya gene do not remove an essential function. phi80 transducing phage for the cya gene have been used to do recombination and complementation studies on cya mutants.     

Cyclic AMP and its receptor protein are required for expression of transfer genes of conjugative plasmid F in Escherichia coli

A number of cya and crp mutants of Escherichia coli HfrH were analyzed for several Tra functions of the F plasmid. The mutants were observed to be deficient in conjugal donor ability, absorption of phages MS2 and Q beta and surface exclusion. These defects were suppressed in cya mutants grown with cAMP supplementation. A cAMP concentration of 3 X 10(-4) M produced maximal suppression of donor ability defect in a cya strain. cAMP did not suppress the Tra- phenotype of crp mutants. Latent periods of MS2 were shorter in cya and crp bacteria. Phage T7 development appeared similar in wild type, cya, and crp cells. It is concluded that tra genes of F plasmid are expressed only to a small extent in cya and crp mutants and that cAMP and its receptor protein are required for the normal expression of tra genes.     

Properties of adenyl cyclase and cyclic adenosine 3'',5''-monophosphate receptor protein-deficient mutants of Escherichia coli.

Several spontaneous cya and crp mutants of Escherichia coli have been selected as clones simultaneously resistant to phage lambda and nalidixic acid and characterized. Both cya and crp mutants have been found to grow as cocci with increased doubling times. They have increased resistance to some mutagens (methylmethanesulfonate, ultraviolet light, gamma rays), antibiotics (nalidixic acid, ampicillin), phages (lambda, T6), sublethal heat and hypotonic shock, and decreased resistance to neutral detergents (sodium dodecyl sulfate, sodium deoxycholate), a protein synthesis inhibitor (streptomycin), and a respiratory inhibitor (sodium azide). The nature of changes in cell parameters indicate fundamental alterations in the envelope structure of the cya and crp mutant cells. The new cya and crp mutants have been found to be multiply carbohydrate negative and nonmotile in conformity with similar previously isolated mutants. Studies of revertants and phi80 cya+ and phi80 cya transductants indicated that the pleiotropic phenotype is related to a single mutational event at the cya or the crp locus in the mutants.     

Study of the regulation of crp gene expression in Escherichia coli K12

The regulation of crp gene expression by CRP-cAMP complex was studied in E. coli strain by the crp-lac operon fusion. F'141 crp+ episome decreased 5-7 fold the high level of crp-lac expression in crp strains while F'141 crp episome had no effect. The hybrid plasmid pCAP2 crp+ with the intact crp gene did not affect the crp gene expression level in crp mutants, though they had acquired the Crp+ phenotype just as they did in F'141 crp+ presence. The F'141 crp+ and pCAP2 crp+ combination in crp mutants also resulted in decrease of the crp gene expression comparable to the registered in the presence of the F'141 crp+ plasmid. Similar repression occurred only in cya+ strains but not in cya strains. The crp gene is supposed to possess negative regulation by CRP-cAMP complex with a complementary factor also necessary. The latter is evidently located in an E. coli chromosome site overlapped by F'141 episome.     

A possible involvement of cya gene in the synthesis of cyclic guanosine 3':5'-monophosphate in E. coli.

Based on the following genetical experiments, the cya gene in E. coli was shown to be involved in the synthesis of both cyclic AMP and cyclic GMP. First, all five independent cya-deficient mutants accumulated exceedingly low amounts of cyclic GMP. Second, the ability to form both cyclic AMP and cyclic GMP was simultaneously restored by transduction of an intact cya locus to one of the above cya-deficient mutants. Third, a spontaneous revertant from one of the above mutants regained the synthetic activity for cyclic GMP as well as for cyclic AMP. Fourth, the characteristic of a strain overproducing cyclic GMP was co-transduced with the cya locus. These results suggest that the synthesis of both cyclic GMP and cyclic AMP is mediated by the same enzyme, adenylate cyclase, Interestingly, a reciprocal effect of glucose starvation was observed on the accumulation of both cyclic nucleotides. The formation of cyclic AMP was greatly enhanced on glucose starvation, whereas that of cyclic GMP proceeded at a slower rate than in the presence of glucose. This effect was observed only in cells carrying normal cya and crp genes, but not in a cya-altered or a crp-deficient strain.     

Different functions assigned to NAD(H) and NADP(H) in light-dependent nitrogen fixation by heterocysts of Anabaena variabilis

Abstract We have directly selected thermosensitive alleles of cya and crp genes from the wild-type, and present preliminary characterization of these mutants. The selection procedure is based on prior growth of a mutagenized wild-type culture in a medium that counterselects, at low temperature, non-conditional relative to thermosensitive mutants, followed by routine selection of mutants at high temperature. This method should be applicable to various genetic systems.     

Identification of the Escherichia coli deoR and cytR gene products.

The protein products encoded by the Escherichia coli deoR and cytR structural genes have been identified based on results obtained from E. coli maxicells harboring (i) recombinant plasmids carrying wild-type deoR and cytR genes, (ii) deletion derivatives of the deoR+ and cytR+ plasmids, (iii) plasmids containing site-specific mutations in the deoR and cytR structural genes, and (iv) plasmids which have transposon Tn1000 inserted into the deoR and cytR structural genes. Analysis of the protein profiles obtained from all the maxicell experiments demonstrated that the deoR gene encodes a protein with a subunit molecular weight of 30,500 and that the product of the cytR gene is a protein with a subunit molecular weight of 37,000.     

Glucose repression of anaerobic genes of Escherichia coli is independent of cyclic AMP

Abstract Several anaerobically regulated gene fusions were examined for the effects of catabolite repression. Glucose repressed the expression of most of the genes represented in our collection of anaerobically induced fusions. However, addition of cyclic AMP did not reverse the effects of glucose. Furthermore, introduction of cya and crp mutations into selected anaerobically induced fusion strains did not reduce anaerobic gene expression as expected from the known mechanism of aerobic catabolite repression. In fact, in different fusion strains, cya or crp mutations caused from 2 to 20-fold increases in gene expression. Although glucose repression occurs anaerobically its mechanism would appear to be quite different from that under aerobic conditions.     

Effects of the Escherichia coli sfsA gene on mal genes expression and a DNA binding activity of SfsA

The sfsA gene was identified as one of the sfs genes the over-expression of which stimulates maltose fermentation of the Mal- Escherichia coli strain MK2001 (crp*1, cya:Km(r)). Expression from the malPQ promoter, which was measured using a chromosomally integrated malPp-lacZ fusion, was induced by over-expressing the sfsA gene in the crp*1, cya:Km(r) strain. The level of the MalE protein was increased in crp*1, cya:Km(r) cells over-producing SfsA. The SfsA protein was purified to homogeneity and tested for DNA binding activity. The purified SfsA protein binds to DNA non-specifically. All these results may suggest that SfsA functions as a DNA binding protein to induce the mal genes in coordination with CRP*1.     

Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli

Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in delta cya or delta crp mutants during starvation. Most of the cyclic AMP-dependent proteins were synthesized in the delta cya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, beta-galactosidase induction of several carbon starvation response gene fusions occurred only in a cya+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coli require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since delta cya or delta crp mutants of either E. coli or Salmonella typhimurium survived starvation as well as their wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya- and crp-independent proteins were also induced during nitrogen starvation and, as shown previously, during phosphate starvation. Proteins whose synthesis is independent of cya- and crp control are referred to as Pex (postexponential).