Pseudomonas aeruginosa causes severe and persistent infections in immune compromised individuals and cystic fibrosis sufferers. The infection is hard to eradicate as P. aeruginosa has developed strong resistance to most conventional antibiotics. The problem is further compounded by the ability of the pathogen to form biofilm matrix, which provides bacterial cells a protected environment withstanding various stresses including antibiotics. Quorum sensing (QS), a cell density-based intercellular communication system, which plays a key role in regulation of the bacterial virulence and biofilm formation, could be a promising target for developing new strategies against P. aeruginosa infection. The QS network of P. aeruginosa is organized in a multi-layered hierarchy consisting of at least four interconnected signaling mechanisms. Evidence is accumulating that the QS regulatory network not only responds to bacterial population changes but also could react to environmental stress cues. This plasticity should be taken into consideration during exploration and development of anti-QS therapeutics.

Bacterial cells sense their population density through a sophisticated cell-cell communication system and trigger expression of particular genes when the density reaches a threshold. This type of gene regulation, which controls diverse biological functions including virulence, is known as quorum sensing. Quorum-sensing signals, such as acyl-homoserine lactones (AHLs), are the essential components of the communication system. AHLs regulate virulence gene expression in a range of plant and animal (including human) bacterial pathogens. AHL-producing tobacco restored the pathogenicity of an AHL-negative mutant of Erwinia carotovora. Different bacterial species may produce different AHLs, which vary in the length and substitution of the acyl chain but contain the same homoserine lactone moiety. Here we show that the acyl-homoserine lactonase (AHL-lactonase), a new enzyme from Bacillus sp., inactivates AHL activity by hydrolysing the lactone bond of AHLs. Plants expressing AHL-lactonase quenched pathogen quorum-sensing signalling and showed significantly enhanced resistance to E. carotovora infection. Our results highlight a promising potential to use quorum-sensing signals as molecular targets for disease control, thereby broadening current approaches for prevention of bacterial infections.

N-acylhomoserine lactones, known as autoinducers (AIs), are widely conserved signal molecules present in quorum-sensing systems of many Gram-negative bacteria. AIs are involved in the regulation of diverse biological functions, including expression of pathogenic genes in the plant pathogens Pseudomonas solanacearum, several Erwinia species, and the human pathogen Pseudomonas aeruginosa. A bacterial isolate, Bacillus sp. 240B1, is capable of enzymatic inactivation of AIs. The gene (aiiA) for AI inactivation from Bacillus sp. 240B1 has been cloned and shown to encode a protein of 250 amino acids. Sequence alignment indicates that AiiA contains a ''HXHXDH'' zinc-binding motif that is conserved in several groups of metallohydrolases. Site-directed mutagenesis showed that conserved aspartate and most histidine residues are required for AiiA activity. Expression of aiiA in transformed Erwinia carotovora strain SCG1 significantly reduces the release of AI, decreases extracellular pectolytic enzyme activities, and attenuates pathogenicity on potato, eggplant, Chinese cabbage, carrot, celery, cauliflower, and tobacco. Our results indicate that the AI-inactivation approach represents a promising strategy for prevention of diseases in which virulence is regulated by AIs.

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SummaryN -acylhomoserine lactones (AHLs) are used as signal molecules by many quorum-sensing Proteobacteria. Diverse plant and animal pathogens use AHLs to regulate infection and virulence functions. These signals are subject to biological inactivation by AHLlactonases and AHL-acylases. Previously, little was known about the molecular details underlying the latter mechanism. An AHL signal-inactivating bacterium, identified as a Ralstonia sp., was isolated from a mixed-species biofilm. The signal inactivation encoding gene from this organism, which we call aiiD , was cloned and successfully expressed in Escherichia coli and inactivated three AHLs tested. The predicted 794-amino-acid polypeptide was most similar to the aculeacin A acylase (AAC) from Actinoplanes utahensis and also shared significant similarities with cephalosporin acylases and other N-terminal (Ntn) hydrolases. However, the most similar homologues of AiiD are deduced proteins of undemonstrated function from available Ralstonia , Deinococcus and Pseudomonas genomes. LC-MS analyses demonstrated that AiiD hydrolyses the AHL amide, releasing homoserine lactone and the corresponding fatty acid. Expression of AiiD in Pseudomonas aeruginosa PAO1 quenched quorum sensing by this bacterium, decreasing its ability to swarm, produce elastase and pyocyanin and to paralyse nematodes. Thus, AHLacylases have fundamental implications and hold biotechnological promise in quenching quorum sensing.

A range of gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorumsensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene, aiiA, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species Bacillus thuringiensis. Enzymatic analysis with known Bacillus strains confirmed that all of the strains of B. thuringiensis and the closely related species B. cereus and B. mycoides tested produced AHL-inactivating enzymes but B. fusiformis and B. sphaericus strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.N-Acyl-homoserine lactones (AHLs), also known as autoinducers, are widely conserved signal molecules that are present in the quorum-sensing systems of many gram-negative bacteria. The bacteria release, detect, and respond to accumulation of these signal molecules for synchronizing expression of particular sets of genes and for coordinating cellular activities. It has been found that AHLs are involved in regulation of a range of biological functions, including bioluminescence in Vibrio species (4, 13), Ti plasmid conjugal transfer in Agrobacterium tumefaciens (32), induction of virulence genes in Burkholderia cepacia, Erwinia carotovora, Erwinia chrysanthemi, Erwinia stewartii, Pseudomonas aeruginosa, and Xenorhabdus nematophilus (3,6,12,17,[20][21][22][23]25), regulation of antibiotic production in Pseudomonas aureofaciens and E. carotovora (6, 25), swarming motility in Serratia liquifaciens (14), and biofilm formation in Pseudomonas fluorescens and P. aeruginosa (1,8). More bacterial species are known to produce AHLs, but the relevant biological functions have not been investigated (2, 5, 11).AHL quorum-sensing signals are a fascinating group of molecular targets for genetic and chemical manipulation. These molecules are highly conserved; they have the same homoserine lactone moiety but differ in the length and structure of the acyl side chain. Although different target genes are regulated by AHLs, the basic mechanisms of AHL biosynthesis and gene regulation seem to be conserved in different bacterial species. The general feature of AHL-mediated gene regulation is cell population-dependent regulation, which is known as quorum sensing. The concentration of an AHL increases along with the growth of bacterial cells. When the AHL concentr...

Conjugal opines secreted by crown gall tumours induce strains of Agrobacterium tumefaciens that are donors of Ti plasmids to produce a diffusible conjugation factor. This enhances the conjugal transfer efficiency of the Ti plasmid in other strains of A. tumefaciens. This factor behaves as a secondary messenger, transmitting the environmental information to tra genes. Here we report the use of spectrometry to show that this factor is identical to synthetic N-(beta-oxo-octan-1-oyl)-L-homoserine lactone and confirm that the synthetic compound is biologically active. N-(Hexan-1-oyl)-L-homoserine lactone has also been detected. A closely related molecule, N-(beta-oxo-hexan-1-oyl)-L-homoserine lactone, autoinduces bioluminescence in the distantly related bacterium, Vibrio fischeri. N-Acyl-homoserine lactones thus seem to be conserved molecules in which the length and nature of the lipophilic acyl chain determines the biological function to be regulated. Mutants that do not produce the factor fail to conjugate unless supplied with it in the induction medium (our unpublished data). These data indicate that the conjugation factor is an autoinducer and a key signal molecule in the conjugation system of A. tumefaciens. It is, to our knowledge, the first example of a second messenger molecule in a bacterial conjugation system.

Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42°C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquidchromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other ␥-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.Many bacterial species control and modulate their physiology in response to increases in their population densities in a process known as quorum sensing (reviewed in references 11 and 25). Several dozen species of Proteobacteria use acyl-homoserine lactones (AHLs) as dedicated signal molecules in this process. A diversity of AHL structures and the enzymes and proteins involved in their synthesis and recognition have been elucidated (12,27,31,35). One of the best studied quorum-sensing species is the opportunistic pathogen Pseudomonas aeruginosa, which makes and responds to two distinct AHLs: 3-oxododecanoyl-homoserine lactone (3OC12HSL, also known as PAI, the signal component of the Pseudomonas las quorum-sensing system), and butanoyl-HSL (C4HSL, also known as PAI-2, the autoinducer of the rhl quorum-sensing system). The two quorum circuits are known to control several physiologies and virulence factors associated with the infection of immunocompromised individuals and those with cystic fibrosis (40). Recently, the influence...