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SummaryThe opportunistic pathogen Pseudomonas aeruginosa has two acyl-homoserine lactone (acyl-HSL) signalling systems, LasR-I and RhlR-I. LasI catalyses the synthesis of N-3-oxododecanoyl homoserine lactone (3OC12) and LasR is a transcription factor that requires 3OC12 as a ligand. RhlI catalyses the synthesis of N -butanoyl homoserine lactone (C4) and RhlR is a transcription factor that responds to C4. LasR and RhlR control the transcription of hundreds of P. aeruginosa genes. There is a third P. aeruginosa LasR-RhlR homologue encoded by qscR for which there is no cognate acyl-HSL synthase gene. To test the hypothesis that QscR functions by direct control of specific promoters in an acyl-HSL-dependent manner we purified QscR and characterized QscR activity in vitro . We also studied QscR activity in recombinant Escherichia coli . QscR binds to promoters that have elements similar in sequence to those found in LasRor RhlR-dependent promoters but QscR does not bind to the LasR-or RhlR-specific promoters we examined. QscR binding to DNA requires 3OC12, but QscR exhibits a relaxed acyl-HSL specificity compared with the 3OC12-cognate signal receptor LasR. Our results support the hypothesis that there is a specific QscRdependent regulon. We show that QscR controls genes in this regulon directly and that regulation is dependent on an acyl-HSL produced by LasI. Because of its relaxed signal specificity QscR may also respond to acyl-HSLs made by other bacteria in mixed bacterial communities.

SummaryIn Escherichia coli, Fe-S clusters are assembled by gene products encoded from the isc and suf operons. Both the iscRSUA and sufABCDSE operons are induced highly by oxidants, reflecting an increased need for providing and maintaining Fe-S clusters under oxidative stress conditions. Three cis-acting oxidant-responsive elements (ORE-I, II, III) in the upstream of the sufA promoter serve as the binding sites for OxyR, IHF and an uncharacterized factor respectively. Using DNA affinity fractionation, we isolated an ORE-III-binding factor that positively regulates the suf operon in response to various oxidants. MALDI-TOF mass analysis identified it with IscR, known to serve as a repressor of the iscRSUA gene expression under anaerobic condition as a [2Fe-2S]-bound form. The iscR null mutation abolished ORE-III-binding activity in cell extracts, and caused a significant decrease in the oxidant induction of sufA in vivo. OxyR and IscR contributed almost equally to activate the sufA operon in response to oxidants. Purified IscR that lacked Fe-S cluster bound to the ORE-III site and activated transcription from the sufA promoter in vitro. Mutations in Fe-S-binding sites of IscR enabled sufA activation in vivo and in vitro. These results support a model that IscR in its demetallated form directly activates sufA transcription, while it de-represses isc operon, under oxidative stress condition.

The soxRS regulon functions in protecting Escherichia coli cells against superoxide and nitric oxide. When SoxR is activated by oxidation of its [2Fe–2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression. How the oxidized SoxR returns to and is maintained in its reduced state has been under question. To identity genes that constitute the SoxR‐reducing system, we screened an E.coli mutant library carrying a chromosomal soxSp::lacZ fusion, for constitutive mutants. Mutations mapped to two loci: the rsxABCDGE operon (named for reducer of SoxR) that is highly homologous to the rnfABCDGE operon in Rhodobacter capsulatus involved in transferring electrons to nitrogenase, and the rseC gene in the rpoE–rseABC operon. In‐frame deletion of each open reading frame in the rsxABCDGE operon produced a similar constitutive phenotype. The double mutation of rsx and rseC suggested that rsxABCDGE and rseC gene products act together in the same pathway in reducing SoxR. Electron paramagnetic resonance analysis of SoxR and measurement of re‐reduction kinetics support the proposal that rsx and rseC gene products constitute a reducing system for SoxR.

In iron-replete environments, the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein represses expression of two small regulatory RNAs encoded by prrF1 and prrF2. Here we describe the effects of iron and PrrF regulation on P. aeruginosa physiology. We show that PrrF represses genes encoding enzymes for the degradation of anthranilate (i.e. antABC), a precursor of the Pseudomonas quinolone signal (PQS). Under ironlimiting conditions, PQS production was greatly decreased in a ⌬prrF1,2 mutant as compared with wild type. The addition of anthranilate to the growth medium restored PQS production to the ⌬prrF1,2 mutant, indicating that its defect in PQS production is a consequence of anthranilate degradation. PA2511 was shown to encode an anthranilate-dependent activator of the ant genes and was subsequently renamed antR. AntR was not required for regulation of antA by PrrF but was required for optimal iron activation of antA. Furthermore, iron was capable of activating both antA and antR in a ⌬prrF1,2 mutant, indicating the presence of two distinct yet overlapping pathways for iron activation of antA (AntR-dependent and PrrF-dependent). Additionally, several quorum-sensing regulators, including PqsR, influenced antA expression, demonstrating that regulation of anthranilate metabolism is intimately woven into the quorum-sensing network of P. aeruginosa. Overall, our data illustrate the extensive control that both iron regulation and quorum sensing exercise in basic cellular physiology, underlining how intermediary metabolism can affect the regulation of virulence factors in P. aeruginosa.Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes serious infections in immuno-compromised individuals, such as burn victims, and in cystic fibrosis (CF) 2 patients. To cause disease, P. aeruginosa expresses several virulence factors that allow it to colonize and survive within its host, as well as a variety of systems that allow for the acquisition of nutrients required for metabolism and growth. P. aeruginosa must be able to coordinate the expression of each of these factors to successfully establish and maintain infection. For example, a shortage of iron availability leads to the increased expression of iron acquisition systems and decreased expression of pathways that rely on relatively large amounts of iron. Conversely, the potential for iron toxicity necessitates the tight regulation of iron acquisition in response to iron availability, a function mediated through the action of the ferric uptake regulator (Fur) protein. Under iron-replete conditions, the Fur protein becomes ferrated and binds to a 19-bp consensus sequence, called the Fur box, in the promoters of genes required for iron uptake, thereby preventing their transcription (1, 2). In P. aeruginosa, Fur directly or indirectly controls the expression of a large number of genes and operons involved in iron uptake, as well an assortment of virulence genes (3-6). Fur can also contribute to the increased expression of genes via the repressi...

Ellagic acid, ellagic acid glycosides, and ellagitannins found in various fruits and nuts, including muscadine grape, are reported to have potential health-promoting benefits and antioxidant properties. This study isolated and identified several ellagic acid derivatives present in muscadine grapes and determined their relative antioxidant properties (AOX). Compounds were extracted from grape skins and pulp using methanol, and the solvent was evaporated. Isolates were dissolved in citric acid buffer (pH 3.5) and absorbed onto C18 cartridges. Nonretained polyphenolics were collected separately and again partitioned from Sephadex LH-20, whereas retained polyphenolics were first eluted with ethyl acetate followed by methanol. Ellagic acid derivatives were identified on the basis of UV and mass spectra, and the presence of ellagitannins was confirmed by a significant increase in free ellagic acid with HPLC followed by acid hydrolysis. Muscadine grapes contained phenolic acids, flavonols, anthocyanins, ellagic acid, and numerous ellagic acid derivatives. AOX varied in the order ethyl acetate > methanol > C18 nonretained fractions; each correlated to both total phenolics (r = 0.90) and total ellagic acid (r = 0.99) contents. Results of this study revealed previously unidentified ellagic acid derivatives in muscadine grapes.

The opportunistic pathogen Pseudomonas aeruginosa possesses two complete acyl-homoserine lactone (acyl-HSL) signaling systems. One system consists of LasI and LasR, which generate a 3-oxododecanoyl-homoserine lactone signal and respond to that signal, respectively. The other system is RhlI and RhlR, which generate butanoyl-homoserine lactone and respond to butanoyl-homoserine lactone, respectively. These quorum-sensing systems control hundreds of genes. There is also an orphan LasR-RhlR homolog, QscR, for which there is no cognate acyl-HSL synthetic enzyme. We previously reported that a qscR mutant is hypervirulent and showed that QscR transiently represses a few quorum-sensing-controlled genes. To better understand the role of QscR in P. aeruginosa gene regulation and to better understand the relationship between QscR, LasR, and RhlR control of gene expression, we used transcription profiling to identify a QscR-dependent regulon. Our analysis revealed that QscR activates some genes and represses others. Some of the repressed genes are not regulated by the LasR-I or RhlR-I systems, while others are. The LasI-generated 3-oxododecanoyl-homoserine lactone serves as a signal molecule for QscR. Thus, QscR appears to be an integral component of the P. aeruginosa quorum-sensing circuitry. QscR uses the LasI-generated acyl-homoserine lactone signal and controls a specific regulon that overlaps with the already overlapping LasR-and RhlR-dependent regulons.