Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, -lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Mutagenesis induced by bacterial UmuDC proteins and their plasmid homologues

The popular image of a world full of pollutants mutating DNA is only partly true since there are relatively few agents which can subtly and directly change base coding; for example, some alkylating agents alter guanine so that it pairs like adenine. Many more mutagens are less subtle and simply destroy coding altogether rather than changing it. Such mutagens include ultraviolet light, X-rays, DNA cross-linkers and other agents which make DNA breaks or large adducts. In Escherichia coli, mutagenesis by these agents occurs during a DNA repair process which increases cell survival but with an inherent possibility of changing the original sequence. Such mutagenic DNA repair is, in part, encoded by the E. coli umuDC operon. This article reviews the structure, function, regulation and evolution of the umuDC operon and similar genes found both in other species and on naturally occurring plasmids.     

In vivo changes in plasma acute phase protein levels in the rat induced by slow release of IL-1, IL-6 and TNF

Administration of large doses of cytokines by injection is required to induce changes in acute phase protein levels. Comparisons were made in the rat of the effects of administering recombinant human cytokines by injection with continuous release from implanted osmotic minipumps. Continuous release of interleukin-1beta (0.2-2.1 ng h(-1)) induced dose-related changes in the plasma levels of albumin, seromucoid proteins, haptoglobin and caeruloplasmin; interleukin-1alpha had similar effects but required higher doses (2-21 ng h(-1)). Tumour necrosis factor alpha (50 ng h(-1)) only significantly increased seromucoid levels, whereas IL-6 (3-30 ng h(-1)) induced haptoglobin and caeruloplassynthesis. This method provides a better technique for studying the in rive effects of cytokines which may be relevant to the release mechanisms in inflammation.     

Direct ATP photolabeling of Escherichia coli recA proteins: identification of regions required for ATP binding

When the Escherichia coli RecA protein is UV irradiated in the presence of [alpha-32P]ATP, a labeled protein--ATP adduct is formed. All the experimental evidence indicates that, in forming such an adduct, the ATP becomes specifically immobilized in the catalytically relevant ATP binding site. The adduct can also be identified after irradiation of E. coli cell lysates in a similar manner. This direct ATP photolabeling of RecA proteins has been used to identify regions of the polypeptide chain involved in the binding of ATP. The photolabeling of a RecA protein that lacks wild-type carboxy-terminal amino acids is not detectable. A RecA protein in which the amino-terminal sequence NH2-Ala-Ile-Asp-Glu-Asn- is replaced by NH2-Thr-Met-Ile-Thr-Asn-Ser-Ser-Ser- is only about 5% as efficiently photolabeled as the wild-type protein. Both of these RecA protein constructions, however, contain all the elements previously implicated, directly or indirectly, in the binding of ATP. ATP-photolabeled RecA protein has also been chemically cleaved at specific amino acids in order to identify regions of the polypeptide chain to which the nucleotide becomes covalently photolinked. The evidence is consistent with a region comprising amino acids 116-170. Thus, this work and that of others suggest that several disparate regions of the unfolded polypeptide chain may combine to form the ATP binding site upon protein folding or may influence binding through long-range effects.     

Alkaline Sucrose Gradient Sedimentation of Chromosomal Deoxyribonucleic Acid from Escherichia coli PolA+ and PolA− Strains During Thymine Starvation

Single-strand breaks were not detected in the deoxyribonucleic acid of Escherichia coli after thymine starvation for up to 180 min, even in a sensitive PolA(-) strain.     

In vitro proteolytic cleavage of the Escherichia coli Ada protein by the ompT gene product.

Down regulation of the adaptive response to alkylation damage in Escherichia coli has been proposed to occur by proteolytic cleavage of the regulatory Ada protein. In this paper, it is shown that proteolysis of the Ada protein as observed in cell extracts is caused by the ompT gene product. This protease, however, was not involved in switching off the adaptive response in vivo.     

Induction of resistance to alkylating agents in E. coli: the ada+ gene product serves both as a regulatory protein and as an enzyme for repair of mutagenic damage.

The expression of several inducible enzymes for repair of alkylated DNA in Escherichia coli is controlled by the ada+ gene. This regulatory gene has been cloned into a multicopy plasmid and shown to code for a 37-kd protein. Antibodies raised against homogeneous O6-methylguanine-DNA methyltransferase (the main repair activity for mutagenic damage in alkylated DNA) were found to cross-react with this 37-kd protein. Cell extracts from several independently derived ada mutants contain variable amounts of an altered 37-kd protein after an inducing alkylation treatment. In addition, an 18-kd protein identical with the previously isolated O6-methyl-guanine-DNA methyltransferase has been identified as a product of the ada+ gene. The smaller polypeptide is derived from the 37-kd protein by proteolytic processing.