Presence of acyl-homoserine lactones in 57 members of the Vibrionaceae family.

AIMS: The aim of this study was to use a sensitive method to screen and quantify 57 Vibrionaceae strains for the production of acyl-homoserine lactones (AHLs) and map the resulting AHL profiles onto a host phylogeny. METHODS AND RESULTS: We used a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) protocol to measure AHLs in spent media after bacterial growth. First, the presence/absence of AHLs (qualitative analysis) was measured to choose internal standard for subsequent quantitative AHL measurements. We screened 57 strains from three genera (Aliivibrio, Photobacterium and Vibrio) of the same family (i.e. Vibrionaceae). Our results show that about half of the isolates produced multiple AHLs, typically at 25-5000 nmol l(-1) . CONCLUSIONS: This work shows that production of AHL quorum sensing signals is found widespread among Vibrionaceae bacteria and that closely related strains typically produce similar AHL profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: The AHL detection protocol presented in this study can be applied to a broad range of bacterial samples and may contribute to a wider mapping of AHL production in bacteria, for example, in clinically relevant strains.

Supercooled liquid-like solvent in trypsin crystals: implications for crystal annealing and temperature-controlled X-ray radiation damage studies.

The study of temperature-dependent physical changes in flash-cooled macromolecular crystals is pertinent to cryocrystallography and related issues such as crystal annealing, X-ray radiation damage and kinetic crystallography. In this context, the unit-cell volume of flash-cooled trigonal and orthorhombic trypsin crystals has been monitored upon warming from 100 to 200 K and subsequent re-cooling to 100 K. Crystals of both forms were obtained under the same crystallization conditions, yet they differ in solvent content and channel size. An abrupt non-reversible unit-cell volume decrease is observed at 185 K in orthorhombic and at 195 K in trigonal crystals as the temperature is increased; this result is consistent with ultra-viscous solvent leaving the crystals. Concomitant appearance of ice rings in the diffraction patterns suggests that the transported solvent forms crystalline ice. These results demonstrate that solvent in flash-cooled protein crystals is liquid-like near its crystallization temperature, as has been proposed, yet controversially discussed, for the case of pure water. The use of mineral oil prevents the unit-cell volume decrease in trigonal but not in orthorhombic crystals. The observation of liquid-like solvent has implications in the development of annealing protocols and points a way to the rational design of temperature-controlled crystallographic studies that aim either at studying specific radiation damage or at trapping enzymatic intermediate states.

Atomic resolution structures of trypsin provide insight into structural radiation damage.

Radiation damage is an inherent problem in protein X-ray crystallography and the process has recently been shown to be highly specific, exhibiting features such as cleavage of disulfide bonds, decarboxylation of acidic residues, increase in atomic B factors and increase in unit-cell volume. Reported here are two trypsin structures at atomic resolution (1.00 and 0.95 A), the data for which were collected at a third-generation synchrotron (ESRF) at two different beamlines. Both trypsin structures exhibit broken disulfide bonds; in particular, the bond from Cys191 to Cys220 is very sensitive to synchrotron radiation. The data set collected at the most intense beamline (ID14-EH4) shows increased structural radiation damage in terms of lower occupancies for cysteine residues, more breakage in the six disulfide bonds and more alternate conformations. It appears that high intensity and not only the total X-ray dose is most harmful to protein crystals.

Electrostatics of mesophilic and psychrophilic trypsin isoenzymes: qualitative evaluation of electrostatic differences at the substrate binding site.

A qualitative evaluation of electrostatic features of the substrate binding region of seven isoenzymes of trypsin has been performed by using the continuum electrostatic model for the solution of the Poisson-Boltzmann equation. The sources of the electrostatic differences among the trypsins have been sought by comparative calculations on selective charges: all charges, conserved charges, partial charges, unique cold trypsin charges, and a number of charge mutations. As expected, most of the negative potential at the S(1) region of all trypsins is generated from Asp(189), but the potential varies significantly among the seven trypsin isoenzymes. The three cold active enzymes included in this study possess a notably lower potential at and around the S(1)-pocket compared with the warm active counterparts; this finding may be the main contribution to the increased binding affinity. The source of the differences are nonconserved charged residues outside the specificity pocket, producing electric fields at the S(1)-pocket that are different in both sign and magnitude. The surface charges of the mesophilic trypsins generally induce the S(1) pocket positively, whereas surface charges of the cold trypsins produce a negative electric field of this region. Calculations on mutants, where charged amino acids were substituted between the trypsins, showed that mutations in Loop2 (residues 221B and 224) and residue 175, in particular, were responsible for the low potential of the cold enzymes.

Structural comparison of psychrophilic and mesophilic trypsins. Elucidating the molecular basis of cold-adaptation.

Structural rationalizations for differences in catalytic efficiency and stability between mesophilic and cold-adapted trypsins have been suggested from a detailed comparison of eight trypsin structures. Two trypsins, from Antarctic fish and Atlantic cod, have been constructed by homology modeling techniques and compared with six existing X-ray structures of both cold-adapted and mesophilic trypsins. The structural analysis focuses on the cold trypsin residue determinants found in a more extensive comparison of 27 trypsin sequences, and reveals a number of structural features unique to the cold-adapted trypsins. The increased substrate affinity of the psychrophilic trypsins is probably achieved by a lower electrostatic potential of the S1 binding pocket particularly arising from Glu221B, and from the lack of five hydrogen bonds adjacent to the catalytic triad. The reduced stability of the cold trypsins is expected to arise from reduced packing in two distinct core regions, fewer interdomain hydrogen bonds and from a destabilized C-terminal alpha-helix. The helices of the cold trypsins lack four hydrogen bonds and two salt-bridges, and they have poorer van der Waals packing interactions to the body of the molecule, compared to the mesophilic counterparts.

Cold adapted enzymes.

The number of reports on enzymes from cold adapted organisms has increased significantly over the past years, and reveals that adaptive strategies for functioning at low temperature varies among enzymes. However, the high catalytic efficiency at low temperature seems, for the majority of cold active enzymes, to be accompanied by a reduced thermal stability. Increased molecular flexibility to compensate for the low working temperature, is therefore still the most dominating theory for cold adaptation, although there also seem to be other adaptive strategies. The number of experimentally determined 3D structures of enzymes possessing cold adaptation features is still limited, and restricts a structural rationalization for cold activity. The present summary of structural characteristics, based on comparative studies on crystal structures (7), homology models (7), and amino acid sequences (24), reveals that there are no common structural feature that can account for the low stability, increased catalytic efficiency, and proposed molecular flexibility. Analysis of structural features that are thought to be important for stability (e.g. intra-molecular hydrogen bonds and ion-pairs, proline-, methionine-, glycine-, or arginine content, surface hydrophilicity, helix stability, core packing), indicates that each cold adapted enzyme or enzyme system use different small selections of structural adjustments for gaining increased molecular flexibility that in turn give rise to increased catalytic efficiency and reduced stability. Nevertheless, there seem to be a clear correlation between cold adaptation and reduced number of interactions between structural domains or subunits. Cold active enzymes also seem, to a large extent, to increase their catalytic activity by optimizing the electrostatics at and around the active site.

Residue determinants and sequence analysis of cold-adapted trypsins.

The digestive enzyme trypsin is among the most extensively studied proteins, and its structure has been reported from a large number of organisms. This article focuses on the trypsins from vertebrates adapted to life at low temperatures. Cold-adapted organisms seem to have compensated for the reduced reaction rates at low temperatures by evolving more active and less temperature-stable enzymes. We have analyzed 27 trypsin sequences from a variety of organisms to find unique attributes for the cold-adapted trypsins, comparing trypsins from salmon, Antarctic fish, cod, and pufferfish to other vertebrate trypsins. Both the "cold" and the "warm" active trypsins have about 50 amino acids that are unique and conserved within each class. The main unique features of the cold-adapted trypsins attributable to low-temperature adaptation seem to be (1) reduced hydrophobicity and packing density of the core, mainly because of a lower (Ile + Leu)/(Ile + Leu + Val) ratio, (2) reduced stability of the C-terminal, (3) lack of one warm trypsin conserved proline residue and one proline tyrosine stacking, (4) difference in charge and flexibility of loops extending the binding pocket, and (5) different conformation of the "autolysis" loop that is likely to be involved in substrate binding.