A novel mutation FruS, altering synthesis of components of the phosphoenolpyruvate: fructose phosphotransferase system in Escherichia coli K12.

A novel mutation, FruS localised in the fru operon was obtained. It uncouples expression of the genes determining synthesis of the fructose-specific transport proteins and fructose-1-phosphate kinase. In FruS bacteria the fruA and fruF genes (coding for Enzyme IIfru and FPr, respectively) are constitutive by expressed while fruK (encoding fructose-1-phosphate kinase) remains inducible. In contrast to other mutations, which render expression of the whole fru operon constitutive, the FruS mutation: (1) does not lead to D-xylitol sensitivity; (2) does not inhibit growth on D-lactate, pyruvate and L-alanine; (3) does not decrease phosphoenolpyruvate (PEP) synthase activity.

Effect of ptsI and ptsH mutations on initiation of transcription of the Escherichia coli lactose operon.

Mutations in the pts genes (which code for the enzyme I and HPr protein - the general components of the phosphoenolypyruvate-dependent phosphotransferase system) lead to decreases in enzyme-inducible synthesis at the level of transcription. The intracellular content of cyclic AMP in the ptsIH mutant was severely diminished, while the ptsH bacteria contain the same amounts of this nucleotide as the wild-type cells. Nevertheless expression of the lac operon was diminished in the ptsH as well as in the ptsIH mutant. The exogenous cyclic AMP did not prevent repression of beta-galactosidase synthesis in a delta cya ptsI mutant in a wide range of concentrations in the growth medium (from 0.05 mM to 5 mM). The combination of ptsI or ptsH mutations with rpoC1 (synthesis of thermosensitive beta' subunit of RNA polymerase) leads to greater disturbance of beta-galactosidase production at the nonpermissive temperature than demonstrated in the pts+ rpoC1 strain. The stimulatory effect of exogenous cyclic AMP was more pronounced in pts rpoC1 than in pts+ rpoC1 bacteria. The data presented confirm the hypothesis that pts mutations alter the function of CRP in initiation of transcription.

Involvement of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system in regulation of transcription of catabolic genes.

Synthesis of catabolite-sensitive enzymes is repressed in mutants defective in the general proteins (enzyme I and HPr) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system (ptsI and ptsH mutations). To elucidate the mechanism of this phenomenon we constructed isogenic strains carrying pts mutations as well as different lesions of regulation of the lac operon or mutations affecting adenylate cyclase activity (cya mutation) and synthesis of cyclic AMP-receptor protein (crp mutation) Measurements of the differential rate of beta-galactosidase synthesis in these strains showed that the repressive effect of pts mutations was revealed in lac+, lacI, lacOc and cya bacteria, but it was lost in lacP and crp strains. It was concluded that mutational damage to the general components of the phosphoenolpyruvate-dependent phosphotransferase system diminishes activity of the lac promoter. The results obtained led to the conclusion that pts gene products (apparently phospho approximately HPr) are necessary for the initiation of transcription of catabolite-sensitive operons in E. coli.

Repression of inducible enzyme synthesis in a mutant of Escherichia coli K 12 deleted for the ptsH gene.

The genome of lambda phage with thermosensitive repressor was inserted into the pts region of the Escherichia coli chromosome. This lysogenic culture possessed the PTS1 phenotype at 30 degrees C. A mutant strain with a deletion covering the ptsH gene was isolated after a prophage curing procedure. The deletion nature of the pts mutation was confirmed in genetical and biochemical experiments. The deletion covered a small fragment of the bacterial genome not extending in the ptsI and lig genes. The isolated deltaptsH mutant possessed all characteristics of known pts mutants: pleiotropical disturbances of transport and utilization of a number of carbohydrates, repression of the enzyme inducible synthesis, and resistance to glucose catabolite repression. From these and other data we can conclude that the phosphorylated form of the heat-stable protein HPr is involved (directly or indirectly) in activation of the DNA transcription process.

Glucose effect in tgl mutant of Escherichia col K12 defective in methyl-alpha-D-glucoside transport.

1. The dependence of the rate of accumulation of methyl-alpha-D-glucoside on its extracellular concentration was studied in the tgl mutant of Escherichia coli K12, isolated earlier. It has been shown that the kinetics of methyl-alpha-D-glucoside transport differ sharply from those in wild-type bacteria. 2. The beta-galactosidase synthesis in tgl strain is much less sensitive both to permanent and transient glucose catabolite repression. The level of cyclic AMP in mutant cells under the conditions of glucose catabolite repression is several times higher than in the parent strain. 3. The tgl mutation does not affect the manifestation of catabolite inhibition and inducer exclusion with glucose. 4. The data obtained are discussed in the light of a hypothesis concerning the existence of two sites, binding and pecific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system. The tgl mutation alters the first site, and the second one is damaged by the pgt mutation. 5. It is suggested that the products of the tgl and gpt genes are necessary for the manifestation of the phenomena of glucose permanent and transient repression. The effects of catabolite inhibition and inducer exclusion are realized irrespective of the existence or absence of the tgl product.

Catabolite repression in Escherichia coli K12 mutants defective in glucose transport.

The phenomenon of glucose catabolite repression was studied in Escherichia coli mutants unable to transport this carbohydrate. The pts I,H mutant P34 was much less sensitive to permanent and transient repressive effect of glucose on beta-galactosidase synthesis than parental type. The 1103 mutant with lack of enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (ptsI) behaves as well as P34 mutant after addition of glucose to casamino acids mineral medium. But in minimal medium with succinate as the sole source of carbon cells of the 1103 mutant (in accordance with the data of Perlman and Pastan, 1969) show hightened sensibility to transient glucose repression. The effect of hypersensibility disappears when the lacI mutation rendering the beta-galactosidase synthesis to costitutivity is introduced in 1103 mutant. It is shown that the hightened sensibility of beta-galactosidase synthesis to glucose transient repression in 1103 mutant is not an effect of the pts mutation and most probably is due to "inducer exclusion" of the lac operon. It is also shown that if one introduces the P34 mutation in strain devoided of one of the enzymes II for glucose (gptA) (and due to this resistant to glucose catabolite repression) then the level of resistance in double mutant does not increase in spite of considerable supression of 14C glucose accumulation. It is discussed the role of separate components of Escherichia coli K12 glucose transport system in realization of the phenomenon of catabolite repression.

Glucose catabolite repression in Escherichia coli K12 mutants defective in methyl-alpha-d-glucoside transport.

1. Two spontaneous Escherichia coli K12 mutants resistant to glucose catabolite repression were isolated using minimal agar plates with methyl alpha-D-glucoside. Mutants grow well on glucose and mannitol. 2. Glucose does not inhibit the inducible enzyme synthesis in isolated mutants: mutant cell (in contrast to parent cells) produce high levels of beta-galactosidase and L-tryptophanase under the conditions of glucose catabolite repression. 3. The isolated mutants are negative in methyl-alpha-D-glucoside transport; glucose uptake is not severely damaged. But the mutants (named tgl, transport of glucose) retained the ability to phosphorylate methyl alpha-D-glucoside in vitro at the expense of phosphoenolpyruvate. 4. The tgl mutation is cotransduced with purB and pyrC markers, i.e. locates near 24 min of the E. coli chromosome map. 5. It is thought that E. coli cells possess two glucose transport systems. The first one is represented by the glucose-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system. The second glucose transport system (coded for tgl gene) functions as permease and possesses high affinity to methyl alpha-D-glucoside. The integrity of glucose permease determine the sensitivity of the cell to glucose catabolite repression.