In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies.

A technique is described whereby the addition of low concentrations (millimolar to micromolar) of the fluorescent dye 1,8-ANS to the protein solution prior to crystallization results in crystallization experiments in which protein crystals are strongly contrasted above background artifacts when exposed to low-intensity UV radiation. As 1,8-ANS does not covalently modify the protein sample, no further handling or purification steps are necessary. The system has been tested on a wide variety of protein samples and it has been shown that the addition of 1,8-ANS has no discernible effect on the crystallization frequencies or crystallization conditions of these proteins. As 1,8-ANS interacts with a wide variety of proteins, this is proposed to be a general solution for the automated classification of protein crystallization images and the detection of protein crystals. The results also demonstrate the expected discrimination between salt and protein crystals, as well as allowing the straightforward identification of small crystals that grow in precipitate or under a protein skin.

Development of an ontology for the description of crystallization experiments and results is proposed.

Modern X-ray structure analysis and advances in high-throughput robotics have allowed a significant increase in the number of conditions screened for a given sample volume. An efficient evaluation of the increased amount of crystallization trials in order to identify successful experiments is now urgently required. A novel approach is presented for the visualization of crystallization experiments using fluorescence from trace amounts of a nonspecific dye. The fluorescence images obtained strongly contrast protein crystals against other phenomena, such as precipitation and phase separation. Novel software has been developed to quantitatively evaluate the crystallization outcome based on a biophysical metric correlated with voxel protein concentration. In >1500 trials, 85.6% of the successful crystallization experiments were correctly identified, yielding a 50% reduction in the number of 'missed hits' compared with current automated approaches. The use of the method in the crystallization of three previously uncharacterized proteins from the malarial parasite Plasmodium falciparum is further demonstrated.

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