Among the prokaryotae, the nucleotide ppGpp is a second messenger of physiological stress and starvation. The target of ppGpp is RNA polymerase, where it putatively binds and alters the enzyme's activity. Previous data had implicated the ␤-subunit of Escherichia coli RNA polymerase as containing a single ppGpp binding site. In this study, a photocross-linkable derivative of ppGpp, 6-thioguanosine-3,5-(bis)pyrophosphate (6-thio-ppGpp), was used to localize the ppGpp binding site. In in vitro transcription assays, 6-thio-ppGpp inhibited transcription from the argT promoter identically to bona fide ppGpp. The thio group of 6-thio-ppGpp is directly photoactivatable and is thus a zero-length crosslinker. Cross-linking of RNA polymerase was directed primarily to the ␤-subunit and could be competed efficiently by native ppGpp but not by GTP or GDP. Cyanogen bromide digestion analysis of the cross-linked ␤-subunit was consistent with an extreme N-terminal cross-link. To assess allosteric consequences of ppGpp binding to RNA polymerase, high level trypsin resistance in the presence and absence of ppGpp was monitored. Trypsin digestion of RNA polymerase bound to ppGpp leads to protection of an N-terminal fragment of the ␤-subunit and a C-terminal fragment of the ␤-subunit. We propose that the N terminus of ␤ together with the C terminus of ␤ constitute a modular ppGpp binding site.Within the bacterial domain of the kingdom prokaryotae exists a general and ubiquitous response to nutritional and environmental stress, the stringent response (1). This general stress response is mediated by high level accumulation of the transcription effector guanosine-3Ј,5Ј-(bis)pyrophosphate (ppGpp). A major effect of elevated ppGpp levels is an immediate and severe reduction of stable rRNA and tRNA gene transcription (2). The cessation of stable RNA syntheses halts the major energy consuming activities of the cell, transcription and translation. This period of metabolic inactivity allows the cell to utilize its remaining energy reserves to adapt to stressful growth conditions through induction of specific "stress genes" (3). Once adaptation is near completion, ppGpp levels decrease and growth resumes. Failure to reduce ppGpp levels results in a severe reduction of cell viability (4).Several lines of evidence suggest that ppGpp exerts its effects by directly binding to RNA polymerase (RNAP).1 Certain rifampicin-resistant mutants of the ␤-subunit of RNAP display increased intracellular sensitivity to ppGpp (5, 6). Spontaneously occurring mutants that confer survival under artificial and prolonged exposure to toxic levels of ppGpp were mapped to the rpoB gene, encoding the ␤-subunit of RNAP (7). Mutant strains devoid of ppGpp, ppGpp 0 strains, are incapable of surviving nutritional deprivation; however, specific mutants of the 70 -, ␤-, or ␤Ј-subunits of RNAP restores normal survivability to ppGpp 0 strains (8). Fluorescence quenching studies of RNAP in the presence of increasing concentrations of a fluorescently labeled ppGpp analogue (1-ami...

Enteropathogenic Escherichia coli (EPEC) infection triggers the release of ATP from host intestinal cells, and the ATP is broken down to ADP, AMP, and adenosine in the lumen of the intestine. Ecto-5-nucleotidase (CD73) is the main enzyme responsible for the conversion of 5-AMP to adenosine, which triggers fluid secretion from host intestinal cells and also has growth-promoting effects on EPEC bacteria. In a recent study, we examined the role of the host enzyme CD73 in EPEC infection by testing the effect of ecto-5-nucleotidase inhibitors. Zinc was a less potent inhibitor of ecto-5-nucleotidase in vitro than the nucleotide analog ␣,␤-methylene-ADP, but in vivo, zinc was much more efficacious in preventing EPEC-induced fluid secretion in rabbit ileal loops than ␣,␤-methylene-ADP. This discrepancy between the in vitro and in vivo potencies of the two inhibitors prompted us to search for potential targets of zinc other than ecto-5-nucleotidase. Zinc, at concentrations that produced little or no inhibition of EPEC growth, caused a decrease in the expression of EPEC protein virulence factors, such as bundle-forming pilus (BFP), EPEC secreted protein A, and other EPEC secreted proteins, and reduced EPEC adherence to cells in tissue culture. The effects of zinc were not mimicked by other transition metals, such as manganese, iron, copper, or nickel, and the effects were not reversed by an excess of iron. Quantitative real-time PCR showed that zinc reduced the abundance of the RNAs encoded by the bfp gene, by the plasmid-encoded regulator (per) gene, by the locus for the enterocyte effacement (LEE)-encoded regulator (ler) gene, and by several of the esp genes. In vivo, zinc reduced EPEC-induced fluid secretion into ligated rabbit ileal loops, decreased the adherence of EPEC to rabbit ileum, and reduced histopathological damage such as villus blunting. Some of the beneficial effects of zinc on EPEC infection appear to be due to the action of the metal on EPEC bacteria as well as on the host.Enteropathogenic Escherichia coli (EPEC) infection is a common cause of watery diarrhea in children in developing countries. The mechanism by which EPEC triggers watery diarrhea is obscure, since unlike several other types of diarrheaproducing E. coli strains, EPEC produces no toxins. We recently theorized that the release of adenine nucleotides from the host intestinal cells, followed by the breakdown to adenosine, could trigger watery diarrhea by the activation of adenosine receptors in the intestine (8).Our interest in zinc was prompted by studies of ecto-5Ј-nucleotidase, a key extracellular enzyme which catalyzes the hydrolysis of extracellular 5Ј-AMP to adenosine. Zinc and the nucleotide analog ␣,␤-methylene-ADP are the classical inhibitors of this enzyme that were tested in vitro and in vivo in a recent study by one of our laboratories (9). Although ␣,␤-methylene-ADP is about eightfold more potent than zinc acetate in the inhibition of ecto-5Ј-nucleotidase, in vivo, zinc acetate was superior to ␣,␤-methylene-ADP in its ability to...

Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5′-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5′-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5′-AMP. EPEC infection triggered a release of ecto-5′-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5′-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5′-nucleotidase was susceptible to inhibition by zinc acetate and by α,β-methylene-adenosine diphosphate (α,β-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5′-AMP. In addition, α,β-methylene-ADP and zinc blocked the ability of 5′-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5′-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5′-nucleotidase, such as α,β-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection.

Guanosine tetraphosphate (ppGpp) is a signal of nutritional stress that regulates transcription. An RNA polymerase rudder mutant rpoC (Delta 312-315) is found to suppress ppGpp deficiency phenotypes by restoring both negative and positive activities of promoter fusions in vivo, as if ppGpp were present. Measurements of defects in transcription of the PargT tRNA promoter with mutant RNA polymerase reveal that the mutant enzyme quantitatively mimics the presence of added ppGpp. DNaseI footprints and mobility shifts under RNA polymerization conditions reveal that the promoter-specific transcription defect of the mutant enzyme can be ascribed to the presence of inactive dead-end promoter complexes with features similar to those of a stable closed complex. We propose that formation of such inactive complexes represents an alternative explanation of "stringent RNA polymerase" mutant behavior to those currently published, and it represents a newly discovered mode of action of ppGpp.

Enteropathogenic E. coli (EPEC) is a common cause of diarrhea in children in developing countries. After adhering to intestinal cells, EPEC secretes effector proteins into host cells, causing cell damage and eventually death. We previously showed that EPEC infection triggers the release of ATP from host cells and that ATP is broken down to ADP, AMP, and adenosine. Adenosine produced from the breakdown of extracellular ATP triggers fluid secretion in intestinal monolayers and may be an important mediator of EPEC-induced diarrhea. Here we examined whether adenosine has any effects on EPEC bacteria. Adenosine stimulated EPEC growth in several types of media in vitro. Adenosine also altered the pattern of EPEC adherence to cultured cells from a localized adherence pattern to a more diffuse pattern. Adenosine changed the expression of virulence factors in EPEC, inhibiting the expression of the bundle-forming pilus (BFP) and enhancing expression of the EPEC secreted proteins (Esps). In vivo, experimental manipulations of adenosine levels had strong effects on the outcome of EPEC infection in rabbit intestinal loops. In addition to its previously reported effects on host tissues, adenosine has strong effects on EPEC bacteria, stimulating EPEC growth, altering its adherence pattern, and changing the expression of several important virulence genes. Adenosine, like noradrenaline, is a small, host-derived molecule that is utilized as a signal by EPEC.