Identifying Fishes through DNA Barcodes and Microarrays

Background: International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. Methodology/Principal Findings: This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of ‘‘DNA barcoding’’ and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the ‘‘position of label’’ effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (.90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. Conclusions/Significance: Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products

(A) Effect of pH, (B) NaCl and (C) temperature on the lytic activity of Ts2631 endolysin.

<p>Relative activity against <i>T</i>. <i>thermophilus</i> HB8 cells was calculated by comparing the lytic activity at specific condition with the maximal lytic activity among the dataset. Each experiment was repeated in triplicate; error bars indicate the standard deviation.</p

Examination of thermal stability of the Ts2631 endolysin.

<p>(A) Samples of Ts2631 endolysin (5 μg) and hen egg white lysozyme (HEWL, 5 μg) were incubated in 10 mM potassium phosphate buffer pH 8.0 at 95°C (0–4 h). Then samples were placed on ice before measuring their activity against <i>T</i>. <i>thermophilus</i> HB8 cells at optimal temperature (60°C for Ts2631 endolysin and 37°C for HEWL). The activity of Ts2631 endolysin and HEWL lysozyme is indicated as percentage relative to untreated samples. Each experiment was repeated in triplicate; error bars indicate the standard deviation. (B) Heat-capacity curves of Ts2631 endolysin determined by: DSC (black dotted line) and the fit to the two-state model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137374#pone.0137374.ref031" target="_blank">31</a>] (black solid line) recorded in MES buffer, pH 6.0.</p

Substrate specificity of Ts2631 endolysin.

<p>Relative activities are expressed as the percentage of activity to <i>T</i>. <i>thermophilus</i> HB8 DSM579. Values represent the mean ± standard deviation (n = 3).</p

Thermostability of Ts2631 endolysin substitution variants.

<p>(A) Clear lysates of Ts2631 endolysin substitution variants (1) and supernatants after heat treatment (2) were mixed with Laemmli buffer and loaded on 12.5% SDS-PAGE. Ts2631 endolysin was used as an experiment positive control. Gels were stained with Coomassie Blue R-250 and visualized with use of Gel Doc XR+ Imager (BioRad). (B) Ts2631 endolysin and its substitution variants (20 μl) at concentration 0.5 mg ml^-1 were incubated in thermocycler at indicated temperature gradient for 15 min. Samples were subsequently centrifuged (10,000 × g; 20 min; 4°C) to remove aggregated proteins. Supernatant (6 μl) from each fraction was mixed with Laemmli buffer and loaded on 12.5% SDS-PAGE. Densitometric analysis was performed with use of Image Lab 5.1 BETA software (Bio-Rad). At 75°C 73% of H131N and more than 32% of H30N and C139S aggregate. At 85°C native enzyme and T137K variant were soluble, while at 90.4°C only Ts2631 endolysin remained in the supernatant.</p

Effect of divalent metal cations on lytic activity of Ts2631 endolysin.

<p>^a Lytic activities under standard conditions before EDTA treatment of Ts2631 endolysin.</p><p>^b Lytic activities of EDTA treated endolysins.</p><p>^c Lytic activities of EDTA treated and dialyzed endolysins against chloroform treated <i>T</i>. <i>thermophilus</i> HB8 cells supplemented with different divalent metal ions at 0.1 and 1 mM concentrations.</p><p>Activities are expressed as percentage in relation to the non-treated endolysin control. Values represent the mean ± standard deviation (n = 3).</p

Structural model of Ts2631 endolysin.

<p>His30, Tyr58, His131, and Cys139, represented as blue sticks, are involved in Zn²⁺ binding. The secondary-structure elements alpha-helices, beta-strands, and loops are shown in red, cyan and grey respectively.</p

Biochemical Characterization and Validation of a Catalytic Site of a Highly Thermostable Ts2631 Endolysin from the <i>Thermus scotoductus</i> Phage vB_Tsc2631

<div><p>Phage vB_Tsc2631 infects the extremophilic bacterium <i>Thermus scotoductus</i> MAT2631 and uses the Ts2631 endolysin for the release of its progeny. The Ts2631 endolysin is the first endolysin from thermophilic bacteriophage with an experimentally validated catalytic site. <i>In silico</i> analysis and computational modelling of the Ts2631 endolysin structure revealed a conserved Zn²⁺ binding site (His³⁰, Tyr⁵⁸, His¹³¹ and Cys¹³⁹) similar to Zn²⁺ binding site of eukaryotic peptidoglycan recognition proteins (PGRPs). We have shown that the Ts2631 endolysin lytic activity is dependent on divalent metal ions (Zn²⁺ and Ca²⁺). The Ts2631 endolysin substitution variants H30N, Y58F, H131N and C139S dramatically lost their antimicrobial activity, providing evidence for the role of the aforementioned residues in the lytic activity of the enzyme. The enzyme has proven to be not only thermoresistant, retaining 64.8% of its initial activity after 2 h at 95°C, but also highly thermodynamically stable (T_m = 99.82°C, ΔH_cal = 4.58 × 10⁴ cal mol^-1). Substitutions of histidine residues (H30N and H131N) and a cysteine residue (C139S) resulted in variants aggregating at temperatures ≥75°C, indicating a significant role of these residues in enzyme thermostability. The substrate spectrum of the Ts2631 endolysin included extremophiles of the genus <i>Thermus</i> but also Gram-negative mesophiles, such as <i>Escherichia coli</i>, <i>Salmonella panama</i>, <i>Pseudomonas fluorescens</i> and <i>Serratia marcescens</i>. The broad substrate spectrum and high thermostability of this endolysin makes it a good candidate for use as an antimicrobial agent to combat Gram-negative pathogens.</p></div

Lytic activity of Ts2631 endolysin.

<p>Different concentrations of Ts2631 endolysin were added to chloroform treated <i>T</i>. <i>thermophilus</i> HB8 DSM 579 cell suspension in 200 μl of 10 mM potassium phosphate buffer pH 8.0 and a decrease in OD₆₀₀ was measured for 40 min in 5 min intervals. Results of the control (buffer only) were subtracted from the sample values. The experiment was repeated in triplicate; error bars indicate the standard deviation.</p