Synthesis and degradation of poly(A) in permeable cells of Escherichia coli.

Poly(A) synthesis and degradation have been examined in Escherichia coli cells made permeable to nucleotides by treatment with toluene. Although newly synthesized poly(A) is normally rapidly degraded in this system, extraction of the soluble portion of the cell effectively eliminates this process without affecting poly(A) synthesis. Poly(A) synthesis in this system displays many properties associated with poly(A) synthesis by purified poly(A) polymerase in vitro including a lag in polymerization, stimulation by increased ionic strength, and a low Mg2+ optimum. As with the purified enzyme, this system uses both ADP and ATP as substrates, requires conversion of ATP to ADP, and is strongly inhibited by dADP, orthophosphate, and pyrophosphate. In contrast to the purified poly(A) polymerase, the permeable cell system displays some properties suggestive of in vivo poly(A) metabolism. Thus, the permeable cells require an endogenous RNA primer for activity, the poly(A) product remains with the cells, and the reaction is greatly stimulated by polyamines. This system should prove extremely useful for studies of poly(A) metabolism in E. coli. A surprising feature of these studies was the finding that mutant strains deficient in polynucleotide phosphorylase were unable to synthesize poly(A). The possible roles of polynucleotide phosphorylase and poly(A) in E. coli are discussed.     

Methylation of chemotaxis-specific proteins in Escherichia coli cells permeable to S-adenosylmethionine

Using a modification of the EGTA treatment of Oishi and Smith [Oishi, M., & Smith, C. L. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3569], Escherichia coli cells have been made permeable to S-adenosylmethionine and other related molecules in order to facilitate the study of methylation in chemotaxis. The permeable cells are nonmotile but respond to chemotactic stimuli by reversible methylation of their methyl-accepting chemotactic proteins (MCP I and MCP II) in a manner similar to that of untreated, motile cells. Addition of S-adenosyl-L-[methyl-3H]methionine to the permeable cells specifically labels two proteins, MCP I and MCP II. Methylation of these MCP's is dependent on the presence of wild-type gene products of flaI, flaA, cheB, cheX, tsr, and tar. The extent of methylation of the MCP's is affected by the presence of attractants or repellents: addition of attractant increases the steady-state level of methylation; addition of repellent causes rapid demethylation to a new steady-state level. Methylation is inhibited by the addition of the transmethylase inhibitors A9145C and Sinefungin, which are S-adenosylmethionine analogues, and by S-adenosylhomocysteine.     

Enzymatic synthesis of [beta-32P]ADP using adenylate kinase and [gamma-32P]ATP

An enzymatic method for the synthesis of [beta-32P]ADP from [gamma-32P]ATP is described. This substrate is required for the assay of ADPase and is not commercially available. The method described results in a preparation of [beta-32P]ADP of high purity with a yield of approximately 40% the theoretical obtainable.     

Deoxyribonucleic acid replication in permeable and fully viable Escherichia coli cells.

Escherichia coli cells made permeable with a hypotonic tris(hydroxymethyl)aminomethane buffer utilized exogenous deoxyribonucleoside triphosphates to perform semiconservative replication. The rate of replication was the same as in cells made permeable with toluene or sucrose.     

Preparation of 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for incorporation into transfer RNA. Photoaffinity labeling of Escherichia coli ribosomes

2-Azidoadenosine was synthesized from 2-chloroadenosine by sequential reaction with hydrazine and nitrous acid and then bisphosphorylated with pyrophosphoryl chloride to form 2-azidoadenosine 3',5'-bisphosphate. The bisphosphate was labeled in the 5'-position using the exchange reaction catalyzed by T4 polynucleotide kinase in the presence of [gamma-32P]ATP. Polynucleotide kinase from a T4 mutant which lacks 3'-phosphatase activity (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) was required to facilitate this reaction. 2-Azidoadenosine 3',5'-[5'-32P]bisphosphate can serve as an efficient donor in the T4 RNA ligase reaction and can replace the 3'-terminal adenosine of yeast tRNAPhe with little effect on the amino acid acceptor activity of the tRNA. In addition, we show that the modified tRNAPhe derivative can be photochemically cross-linked to the Escherichia coli ribosome.     

Preparation of Escherichia coli elongation factor Tu-guanosine 5'-triphosphate analogs

A simple procedure for the bulk preparation of 20 mg of Escherichia coli elongation factor (EF)-Tu-GTP analogs is described. The protocol is based upon the preparation and stabilization of nucleotide-free EF-Tu using an EF-Ts affinity chromatographic resin. The procedure is a general one for the preparation of any GTP analog of EF-Tu.     

Genome analysis of bovine-mastitis-associated Escherichia coli O32:H37 strain P4

Escherichia coli is a major pathogen of bovine intramammary infections. Here we report the first draft of the genome sequence of the E. coli O32:H37 P4 strain, which is widely used in experimental bovine mastitis studies.     

Preparation of [32P]phosphatidylcholine and [32P]lysophosphatidylcholine by using soya beans.

This method describes a procedure that can be carried out easily to obtain large amounts of [32P]phosphatidylcholine and [32P]lysophosphatidylcholine. The method involves germinating soya beans in the presence of [32P]Pi. The yield was 0.58% for [P]phosphatidylcholine and 0.52% for [32P]lysophosphatidylcholine, and the specific radioactivity of both was 10(7) d.p.m./mumol.     

Hydrolysis of bis(5''-nucleosidyl) polyphosphates by Escherichia coli 5''-nucleotidase.

Two enzymatic activities that split diadenosine triphosphate have been reported in Escherichia coli: a specific Mg-dependent bis(5'-adenosyl) triphosphatase (EC 3.6.1.29) and the bis(5'-adenosyl) tetraphosphatase (EC 3.6.1.41). In addition to the activities of these two enzymes, a different enzyme activity that hydrolyzes dinucleoside polyphosphates is described. After purification and study of its molecular and kinetic properties, we concluded that it corresponded to the 5'-nucleotidase (EC 3.1.3.5) that has been described in E. coli. The enzyme was purified from sonic extracts and osmotic shock fluid. From sonic extracts, two isoforms were isolated by chromatography on ion-exchange Mono Q columns; they had a molecular mass of about 100 kilodaltons (kDa). From the osmotic shock fluid, a unique form of 52 kDa was recovered. Mild heating transformed the 100-kDa isoform to a 52-kDa form, with an increase in activity of about threefold. The existence of a 5'-nucleotidase inhibitor described previously, which associates with the enzyme and is not liberated in the osmotic shock fluid, may have been responsible for these results. The kinetic properties and substrate specificities of both forms (52 and 100 kDa) were almost identical. The enzyme, which is known to hydrolyze AMP and uridine-(5')-diphospho-(1)-alpha-D-glucose, but not adenosine-(5')-diphospho-(1)-alpha-D-glucose, was also able to split adenosine-(5')-diphospho-(5)-beta-D-ribose, ribose-5-phosphate, and dinucleoside polyphosphates [diadenosine 5',5'-P1,P2-diphosphate,diadenosine 5',5'-P1,P3-triphosphate, diadenosine 5',5'-P1,P4-tetraphosphate, and bis(5'-guanosyl) triphosphate]. The effects of divalent cations and pH on the rate of the reaction with different substrates were studied.     

NAD(P)H oxidation by hydrogen peroxide in Escherichia coli

A protein fraction from Escherichia Coli soluble extracts contain a NAD(P)H:hydrogen peroxide oxidoreductase activity. This activity is compared to and found to be distinct from well-known E. Coli enzymes involved in the protection from peroxides: hydroperoxidase I (HPI) and its o-dianisidine peroxidase component and the alkyl hydroperoxide reductase.     

Mutants of Escherichia coli K12 permeable to haemin.

Mutants of Escherichia coli which require 5-aminolaevulinic acid (5-ALA), the first intermediate of haem biosynthesis, do not respond to haemin and porphyrins. The probable explanation of the lack of response is that E. coli may be impermeable to haemin and porphyrins. Mutants are described which responded to haemin and porphyrins as well as to 5-ALA. Indirect evidence is presented that the mutants were permeable to haemin. The mutants showed other phenotypic changes, and resembled some mutants which are known to have changes in the cell envelope.     

Puromycin interacts with the donor (P) site of Escherichia coli ribosomes

Puromycin inhibits the interaction of peptidyl-tRNA analogs AcPhe-tRNA Phe ox-red, AcPhe-tRNA Phe and FMet-tRNA f Met with the donor (P) site of Escherichia coli ribosomes. It affects both template-free and poly(U)-dependent systems. The inhibition is apparently due to direct competition for the P-site. On isolated 30S ribosomal subunits it was shown that the puromycin binding site is situated far from the peptidyl transferase center. Quantitative measurements of the inhibition revealed that the affinity constant of puromycin for the P-site is not less than its affinity for the A-moiety of the peptidyl transferase center [1.1 divided by 3.8) X 10(3) M-1).