A method is reported for the quantification of 3-oxoacyl homoserine lactones (3-oxo AHLs), a major class of quorum-sensing signals found in Gram-negative bacteria. It is based on the conversion of 3-oxo AHLs to their pentafluorobenzyloxime derivatives followed by gas chromatography-mass spectrometry (electron capture-negative ion). The method used [13C16]-N-3-oxo-dodecanoyl homoserine lactone ([13C16]-OdDHL) as the internal standard, and its validity was tested by spiking the supernatant and cell fractions with three levels of 3-oxo AHLs, i.e. 1, 10 and 100 ng per sample. These showed the method to be both sensitive (S/N ratio >10:1 for 1 ng) and accurate. The assay was applied to the biofilm and effluent of a green fluorescent protein (GFP)-expressing strain of Pseudomonas aeruginosa (6294) culture grown in flow cells. Biofilm volume was determined for three replicate flow cells by confocal scanning laser microscopy. OdDHL was detected in the biofilm at 632 +/- 381 microM and the effluent at 14 +/- 3 nM. The biofilm concentration is the highest level so far reported for an AHL in a wild-type bacterial system. The next most abundant 3-oxo AHL in the biofilm and effluent was N-3-oxo-tetradecanoyl homoserine lactone (OtDHL) at 40 +/- 15 microM and 1.5 +/- 0.7 nM respectively. OtDHL is unreported for P. aeruginosa and has an activity equivalent to OdDHL in a lasR bioassay. Two other 3-oxo AHLs were detected at lower concentrations: N3-oxo-decanoyl homoserine lactone (ODHL) in the biofilm (3 +/- 2 microM) and effluent (1 +/- 0.1 nM); and N-3-oxo-octanoyl homoserine lactone (OOHL) in the effluent (0.1 +/- 0.1 nM).

Hypochlorous acid is the major strong oxidant generated by human neutrophils, and it has the potential to cause much of the tissue damage that these inflammatory cells promote. It is produced from hydrogen peroxide and chloride by the heme enzyme myeloperoxidase. To unequivocally establish that hypochlorous acid contributes to inflammation, a stable and unique marker for its reaction with biomolecules needs to be identified. In this investigation we have found that reagent hypochlorous acid reacts with tyrosyl residues in small peptides and converts them to chlorotyrosine. Purified myeloperoxidase in combination with hydrogen peroxide and chloride, as well as stimulated human neutrophils, chlorinated tyrosine in the peptide Gly-Gly-Tyr-Arg. Rather than reacting directly with the aromatic ring of tyrosine, hypochlorous acid initially reacted with an amine group of the peptide to form a chloramine. The chloramine then underwent an intramolecular reaction with the tyrosyl residue to convert it to chlorotyrosine. This indicates that tyrosyl residues in proteins that are close to amine groups will be susceptible to chlorination. Peroxidases are the only enzymes capable of chlorinating an aromatic ring. Furthermore, myeloperoxidase is the only human enzyme that produces hypochlorous acid under physiological conditions. Therefore, chlorotyrosine will be a specific marker for the production of hypochlorous acid in vivo and for the involvement of myeloperoxidase in inflammatory tissue damage.

SummaryThe bulk of the Earth's biosphere is cold (e.g. 90% of the ocean's waters are ≤ 5°C), sustaining a broad diversity of microbial life. The permanently cold environments vary from the deep ocean to alpine reaches and to polar regions. Commensurate with the extent and diversity of the ecosystems that harbour psychrophilic life, the functional capacity of the microorganisms that inhabitat the cold biosphere are equally diverse. As a result, indigenous psychrophilic microorganisms provide an enormous natural resource of enzymes that function effectively in the cold, and these cold‐adapted enzymes have been targeted for their biotechnological potential. In this review we describe the main properties of enzymes from psychrophiles and describe some of their known biotechnological applications and ways to potentially improve their value for biotechnology. The review also covers the use of metagenomics for enzyme screening, the development of psychrophilic gene expression systems and the use of enzymes for cleaning.

We report for the first time the production of acyl homoserine lactones (AHLs) by bacteria associated with marine sponges. Given the involvement of AHLs in bacterial colonization of many higher organisms, we speculate that such quorum sensing signals could play a part in interactions between sponges and the dense bacterial communities living within them.Many gram-negative bacteria utilize acyl homoserine lactone (AHL)-mediated signaling systems to communicate with one another (9,27,33). These systems involve the production of low-molecular-weight molecules that accumulate with increasing bacterial numbers and thus provide an index of population density. When a threshold bacterial density (and corresponding AHL concentration) is reached, AHLs interact with transcriptional activators to trigger the expression of target genes. Many terrestrial bacteria produce AHLs, yet, beyond the well-characterized Vibrio fischeri-squid symbioses (3, 22, 24), relatively little is known about the occurrence of AHLs in marine environments. The recent finding that bacteria in marine snow are capable of producing AHLs (12) suggests that such signaling molecules could be widespread. In other studies, the addition of AHLs and cyclic AMP (4) or siderophores (14) to marine growth media increased bacterial culturability and growth, respectively.Many AHL-regulated phenotypes are involved in bacterial colonization of higher organisms, e.g., bioluminescence in V. fischeri (22,24) and conjugal plasmid transfer in Agrobacterium tumefaciens (23,36). Given the dense bacterial communities associated with marine eukaryotes such as sponges (8, 34, 35), macroalgae (18, 20), or ascidians (29, 30) and the densitydependent nature of AHL signaling, we targeted benthic eukaryotes as likely sources of AHL-producing bacteria. Marine sponges in particular are notable for their diverse microbial biota (15,16,32), with bacterial densities in some sponges exceeding those in surrounding seawater by 2 to 3 orders of magnitude (7). Here, we demonstrate for the first time the production of AHLs by bacteria isolated from sponges and the apparent in situ production of AHLs within sponge tissue.Marine organisms, or portions thereof, were collected by scuba diving at depths of 1 to 10 m from several coastal locations near Sydney, in southeastern Australia. Duplicate individuals were sampled for species from the following taxa: sponges (31 species); macroalgae (30 species); and sea grasses, bryozoans, ascidians, and corals (2 species each). Direct screening of small tissue samples by the Chromobacterium violaceum CV026 bioassay (21) revealed the presence of AHLproducing bacteria in all host phyla examined (Table 1). While AHLs appeared to be widespread in marine eukaryote-bacterium associations (especially those involving sponges), there was also substantial variability (both over time and within species) in their occurrence. For example, in a separate short-term sampling program, AHLs were detected in only two out of eight sponge species tested on day 1 but were foun...

Quorum-sensing systems provide Pseudomonas aeruginosa with a sensitive regulatory mechanism that allows for the induction of several phenotypic genes in a cell density fashion. In this work, a mathematical model of the acylated homoserine lactones regulatory network system in P. aeruginosa has been developed. It is the first integrated model to consider both quorum-sensing systems. The model has allowed us to disentangle the complex behavior exhibited by the system as the concentration of extracellular OdDHL is increased. At either low or high levels of extracellular OdDHL, the bacterium remains in an uninduced or induced state, respectively. At moderate levels, the behavior is characterized by several states. Here, the bacteria can switch suddenly from an uninduced to an induced phenotype in response to small changes in the concentration of extracellular OdDHL. Additionally, we have been able to address the roles of RsaL and Vfr as regulators of the quorum-sensing system. An important result from this analysis suggests that RsaL will increase the concentration of extracellular OdDHL required to induce the system, and it is a key regulator of the inhibition of the quorum-sensing system under low cell densities. Most importantly, our results suggest that Vfr has strong regulatory effects on the system as an increased affinity between the LasR/OdDHL complex, and the lasR promoter leads to significant qualitative changes in induction patterns. We also show experimental data that demonstrate that Vfr is required for signal production in the early phase of growth, but that in the latter stages of growth, the vfr mutant is able to synthesize wild-type levels of signal.