The effect of the locus pstB on phosphate binding in the phosphate specific transport (PST) system of Escherichia coli

Summary The periplasmic phosphate binding protein is a product of the phoS gene and is an essential component of the phosphate specific transport (PST) system, which mediates Pi uptake in Escherichia coli. The binding of Pi to periplasmic protein(s) and the kinetic parameters of Pi uptake were studied in phoT and pstB mutants of E. coli. These mutants are impaired in Pi uptake but have a periplasmic Pi-binding protein whose Pi-binding acpacity was estimated by the retention kinetics. The Pi-binding activity in two pstB mutants was found to be weaker as compared to phoT9 and the wild type. The K _D values for Pi binding to periplasmic protein were determined by equilibrium dialysis. In the pstB mutants the K _D value was found to be 9–31 times higher than the values obtained for the wild type and the phoT mutant. The apparent K _m values for Pi uptake in one pstB mutant is 14.3 times higher than in the wild type. V _max of the mutant is 8.3 times lower that of the wild type. The data indicate that pstB, an essential gene of the PST transport system, is promoting the binding capacity of the Pi-binding protein.Abbreviations AP alkaline phosphatase - Pi inorganic orthophosphate - Km kanamycin     

Mutants of Escherichia coli "cryptic" for certain periplasmic enzymes: evidence for an alteration of the outer membrane.

Mutants in which the expression of periplasmic enzymes by whole cells is reduced (termed "cryptic") are also found to show greatly reduced uptake of labeled adenosine 5'-monophosphate (5'-AMP), providing a rapid assay for crypticity. The crypticity of 3'- and 5'-nucleotidase has been examined as a function of substrate concentration. The Km for 3'- or 5'-AMP increases in the cryptic mutants when whole cells are used as the enzyme source. The Vmax is not altered. Electrophoretic analysis of protein prepared from cell envelopes showed that three cryptic mutants have a polypeptide absent from the outer membrane and a relatively high proportion of a polypeptide in the inner membrane. Analysis of the molar ratios of constituent sugars of the lipopolysaccharides showed no differences between three cryptic mutants and the parent strain. One cryptic mutant (3--41), however, has altered sensitivity to phage T4. By selection for phage resistance, derivatives of the cryptic mutants that are deoxycholate sensitive have been obtained. These mutants are no longer cryptic. We suggest that cryptic mutants have an altered outer membrane, with decreased permeability to 3'- and 5'-AMP, as a result of an altered polypeptide.     

Lambda integrase cleaves DNA in cis.

In the Int family of site-specific recombinases, DNA cleavage is accomplished by nucleophilic attack on the activated scissile phosphodiester bond by a specific tyrosine residue. It has been proposed that this tyrosine is contributed by a protomer bound to a site other than the one being cleaved ('trans' cleavage). To test this hypothesis, the difference in DNA binding specificity between closely related integrases (Ints) from phages lambda and HK022 was exploited to direct wild type Ints and cleavage- or activation-defective mutants to particular sites on bispecific substrates. Analysis of Int cleavage at individual sites strongly indicates that DNA cleavage is catalyzed by the Int bound to the cleaved site ('cis' cleavage). This conclusion contrasts with those from previous experiments with two members of the Int family, FLP and lambda Int, that supported the hypothesis of trans cleavage. We suggest explanations for this difference and discuss the implications of the surprising finding that Int-family recombinases appear capable of both cis and trans mechanisms of DNA cleavage.     

Decay of Normal and 5-Fluorouracil-Substituted Messenger Ribonucleic Acid of Alkaline Phosphatase in Escherichia coli

The decay of alkaline phosphatase messenger ribonucleic acid (mRNA) in Escherichia coli was studied in cells sensitized to actinomycin D by ethylenediaminetetra-acetate treatment. It was found that in the wild-type strain (K-10) as well as in a nonsense mutant (S26C200), which is phenotypically reversible by treatment with 5-fluorouracil, mRNA decays with a half-life of 2.0 to 2.5 min. Similarly, in a 3-min pulse of labeled 5-fluorouracil, 70% of the incorporated analogue is contained in the labile RNA fraction decomposing with a half-life of 1.8 min.     

The use of anti-immunoglobulin for the immunoprecipitation of labeled proteins from tissue homogenates

An improved immunochemical procedure for the quantitative isolation of labeled minor proteins from tissue homogenates is worked out and is applied to the isolation of glucose-6-phosphate dehydrogenase from mouse liver. Goat anti-enzyme serum is used as primary reagent, followed by rabbit anti-goat IgG, and not by carrier enzyme as in currently used methods. The resulting immunoprecipitates are analyzed by acrylamide gel electrophoresis, so that only counts in enzyme bands are registered. An equivalent precipitate formed with serum from nonimmunized goat serves as an efficient control for coprecipitation.