Isolation and characterization of microsatellite markers from Pacific white shrimp (Litopenaeus vannamei)

We report the development of 11 polymorphic microsatellite loci in pacific white shrimp (Litopenaeus vannamei) using an unenriched genomic library. The number of the alleles ranged from two to 18 and observed hererozygosity ranged from 0.0286 to 0.9429, indicating that these markers will be useful for population studies and mapping in pacific white shrimp. Seven loci were detected deviated from Hardy–Weinberg, caused by deficiency of heterozygote, suggesting population genetic structure across the sampled population. No evidence for linkage disequilibrium was found.     

Role of invariant tyrosines in a crustacean mu-class glutathione S-transferase from shrimp Litopenaeus vannamei: site-directed mutagenesis of Y7 and Y116

Y6 and Y115 are key amino acids involved in enzyme-substrate interactions in mu-class glutathione S-transferase (GST). They provide electrophilic assistance and stabilize substrates through their hydroxyl groups. Two site-directed mutants (Y7F and Y116F) and the wild-type shrimp GSTs were expressed in Escherichia coli, and the steady-state kinetic parameters were determined using CDNB as the second substrate. The mutants were modeled based on a crystal structure of a mu-class GST to obtain further insights about the changes at the active site. The Y116F mutant had an increase in kcat contrary to Y7F compared to the wild type. Molecular modeling showed that the shrimp GST has a H108 residue that may contribute to compensate and lead to a less deleterious change when conserved tyrosine residues are mutated. This work indicates that shrimp GST is a useful model to understand the catalysis mechanisms in this critical enzyme.     

Improvement in the growth performance of white shrimp, Litopenaeus vannamei, by a protease-producing probiotic, Bacillus subtilis E20, from natto

Aims:  To isolate and identify a benefic bacterium, Bacillus subtilis E20, from natto (fermented soybeans), and incorporate it into shrimp feed to promote shrimp growth performance.
Methods and Results:  A protease-producing bacterium, E20, isolated from natto was identified as B. subtilis by an API 50 CHB kit and the 16S rDNA sequence. B. subtilis E20 was able to grow at a broad range of temperatures (10–50°C), pH values (5–10), and NaCl levels (0–9%). The best culture conditions for B. subtilis E20 to produce the protease were 40°C, a pH of 6–8 and 0% NaCl. No shrimp died after being injected with B. subtilis E20 [up to 10⁹ colony-forming units (CFU) per shrimp]. Bacillus subtilis E20 was incorporated in diets at the levels of 0 (control), 10⁶, 10⁷, and 10⁸ CFU kg⁻¹ for shrimp grow-out culture, and results showed that after feeding on B. subtilis E20-containing diets (10⁸ CFU kg⁻¹ of diet), shrimp had excellent growth performance and production compared to the control because protease activities in the digestive tract were improved by B. subtilis E20.
Conclusions:  Bacillus subtilis E20 isolated from natto is a great protease producer and is able to improve shrimp growth performance through increasing the digestibility of food.
Significance and Impact of the Study:  Results suggest that B. subtilis E20 is a potential candidate for use as a probiotic to improve shrimp growth performance, and consequently reduce feed costs.     

Immune responses of Litopenaeus vannamei to thermal stress: a comparative study of shrimp in freshwater and seawater conditions

The effects of hypothermal (22–16?°C) and hyperthermal (22–28?°C) stress on the immune system responses of the shrimp Litopenaeus vannamei cultured in either freshwater or seawater were measured and compared. The following immune system indicators were measured for comparison: total hemocyte count (THC), activity of phenoloxidase (PO), nitric oxide synthase (NOS), superoxidase (SOD), and malondialdehyde (MDA) content. Thermal stress significantly decreased THC in both freshwater and seawater shrimp within 6–12?h (P?0.05). After hypothermal stress, all shrimp had a significantly lower THC level than their prechallenge levels (P?0.05). Under both types of thermal stress, the activity of PO, NOS, and SOD first increased and then decreased. After 48?h of thermal stress, shrimp PO and NOS activity decreased in both freshwater and seawater. After 48?h of thermal stress, the reduction in the SOD activity in the hemolymph of freshwater shrimp was greater than that in seawater shrimp. During exposure to stress, the MDA content in freshwater shrimp was significantly higher than in seawater shrimp, which demonstrated that lipids in freshwater shrimp were more susceptible to peroxidation than those in seawater shrimp, particularly at low temperatures. Large temperature fluctuations, particularly sudden cooling, should be avoided when rearing L. vannamei because of high rates of lipid peroxidation and decreased immunity. These effects are more marked in freshwater than in seawater.     

Structures of yeast glutathione‐S‐transferase Gtt2 reveal a new catalytic type of GST family

Glutathione‐S‐transferases (GSTs) are ubiquitous detoxification enzymes that catalyse the conjugation of electrophilic substrates to glutathione. Here, we present the crystal structures of Gtt2, a GST of Saccharomyces cerevisiae, in apo and two ligand‐bound forms, at 2.23 Å, 2.20 Å and 2.10 Å, respectively. Although Gtt2 has the overall structure of a GST, the absence of the classic catalytic essential residues—tyrosine, serine and cysteine—distinguishes it from all other cytosolic GSTs of known structure. Site‐directed mutagenesis in combination with activity assays showed that instead of the classic catalytic residues, a water molecule stabilized by Ser129 and His123 acts as the deprotonator of the glutathione sulphur atom. Furthermore, only glycine and alanine are allowed at the amino‐terminus of helix‐α1 because of stereo‐hindrance. Taken together, these results show that yeast Gtt2 is a novel atypical type of cytosolic GST.     

Effect of Lactobacillus plantarum isolated from digestive tract of wild shrimp on growth and survival of white shrimp (Litopenaeus vannamei) challenged with Vibrio harveyi

Two hundred and two strains of lactic acid bacteria (LAB) isolated from digestive tracts of cultivated and wild adult shrimp, including Litopenaeus vannamei, Metapenaeus brevicornis and Penaeus merguiensis were selected based on their antibacterial activity against Vibrio harveyi. LAB strain of MRO3.12 exhibiting highest reduction of V. harveyi was identified as Lactobacillus plantarum MRO3.12 based on the nucleotide sequence of its 16S rDNA, which showed 99% (780/786 bp) homology to L. plantarum strain L5 (GenBank accession number DQ 239698.1). Co-cultivation of V. harveyi and L. plantarum MRO3.12 showed complete reduction of V. harveyi at 24 h under aerobic and anaerobic conditions, whereas L. plantarum increased from 5.29 to 9.47 log CFU ml⁻¹. After 6-week feeding trial with L. plantarum supplemented diet, white shrimp (L. vannamei) exhibited significant differences (p < 0.05) in relative growth rate (% RGR), feed conversion ratio (FCR) and survival compared to the control group fed with non-supplemented diet. LAB-fed group showed 98.89% survival, whereas only 68.89% survival was observed in the control group. LAB from the digestive tract of probiotic-fed shrimp showed higher level of 5.0 ± 0.14 log CFU/g than the non-supplemented ones (3.34 ± 0.21 log CFU/g). However, total bacterial and non-fermenting vibrios counts decreased in shrimps fed on L. plantarum. Ten days after infection with V. harveyi (5.3-5.5 log CFU ml⁻¹), significant survival (p < 0.05) of 77% was observed in LAB supplemented shrimp, while only 67% survival was observed in the control.     

Thermodynamics of Cd2+ and Zn2+ binding by the phytochelatin (gamma-Glu-Cys)4-Gly and its precursor glutathione

Isothermal titration calorimetry (ITC) was used to study the binding of Cd(2+) and Zn(2+) by glutathione (GSH) and phytochelatins (PC(n)), the metal sequestering compounds in plants and algae. The results are compared with those obtained by differential pulse polarography (DPP) assisted by multivariate curve resolution with alternating least squares (MCR-ALS) and by electrospray ionization mass spectrometry (ESI-MS). ITC allows one to determine (i) the stoichiometries of the different complexes (and confirms those found by DPP/MCR-ALS and ESI-MS) and (ii) their binding and thermodynamic parameters. For Cd-PC(4), the sequential binding sites model with two identical sites yields the best fitting of ITC curves and confirms the presence of CdPC(4) and Cd(2)PC(4) complexes. For Zn-PC(4), exothermic formation of ZnPC(4) is reported. Conditional stability and formation constants for Cd-GSH and Zn-GSH are determined from the fitting of the proper model to experimental ITC curves. The effect of different buffers in the complexation processes shows the key role of the choice of the buffer in calorimetric study.     

Biosorption of cadmium by a Cd2+-hyperresistant Bacillus cereus strain HQ-1 newly isolated from a lead and zinc mine

A Cd²⁺-hyperresistant bacterial strain HQ-1 was isolated from a lead–zinc mine. The strain was characterized and identified as Bacillus cereus based on morphology, physiological tests and 16S rRNA gene analysis. The minimal inhibitory concentration of Cd²⁺ for the bacterium was 0.012 mol/l. Isotherms for cadmium (Cd) biosorption by cells of B. cereus strain HQ-1 were investigated. The equilibrium data could be fitted by a Langmuir isotherm equation. The possible functional sites that might be influenced by the sorption were determined. The results indicate that this B. cereus strain has excellent potential for biosorption of Cd. Physiological characterization of the isolate also indicates possible application of this strain for bioremediation of sites with Cd contamination.     

The 110kDa glutathione transferase of Yarrowia lipolytica is encoded by a homologue of the TEF3 gene from Saccharomyces cerevisiae: cloning, expression, and homology modeling of the recombinant protein

The TEF4 gene of the non-saccharomyces yeast Yarrowia lipolytica encodes an EF1Bgamma protein with structural similarity to the glutathione transferases (GSTs). This 1203bp gene was cloned, over-expressed in Escherichia coli, and the recombinant protein characterized. DNA sequencing of the cloned gene agreed with the recently completed Y. lipolytica genome and showed 100% identity to a previously reported 30-residue N-terminal sequence for a 110kDa Y. lipolytica GST, except that it encoded two additional N-terminal residues (N-Met-Ser-). The recombinant protein (subunit M(r) 52kDa) was found not to possess GST activity with 1-chloro-2,4-dinitrobenzene. Partial tryptic digestion released two fragments of M(r) 22 and 18kDa, which we interpret as N- and C-terminal domains. Homology modeling confirmed that the N-terminal domain of Y. lipolytica TEF4 encodes a GST-like protein.     

Bis(2-methyltetrazole)dichlorocopper(II) is a layered coordination polymer built from dinuclear chloro-bridged Cu units linked by bridging tetrazoles. The first structurally and magnetically characterized complex of 2-monosubstituted tetrazoles

The data on synthesis, crystal structure, magnetic susceptibility and thermal properties of coordination compound of copper(II) chloride with 2-methyltetrazole (2mtet) of Cu(2mtet)₂Cl₂ composition are reported. The Cu atom environment forms an elongated octahedron, with two 2-methyltetrazole ligands (N4 bound) and two Cl atoms in the equatorial positions. Symmetry related 2-methyltetrazole ligand and Cl atom are in the axial positions. One of the two 2-methyltetrazole molecules of the asymmetric unit exhibits bridging properties being linked to two Cu atoms through two N atoms (i.e., N4 and N1) of the tetrazole ring, while the other ligand molecule is coordinated in monodentate fashion via one tetrazole N4 atom. The Cu-octahedra form dinuclear building bricks by sharing edges with equatorial and axial Cl atoms. These dinuclear units are linked together via bridging 2-methyltetrazole ligands to form infinite layers parallel to the plane. Magnetic properties of Cu(2mtet)₂Cl₂ and the data of quantum-chemical calculations of molecular electrostatic potential and energies of hydronation of nitrogen atoms for 2mtet using B3LYP/6-31G^* level of theory are in agreement with the structural data obtained.     

Isolation of a highly copper-tolerant yeast, Cryptococcus sp., from the Japan Trench and the induction of superoxide dismutase activity by Cu2+

Thirteen yeast strains were isolated from deep-sea sediment samples collected at a depth of 4500 m to 6500 m in the Japan Trench. Amongst them, strain N6 possessed high tolerance against Cu²⁺ and could grow on yeast extract/peptone/dextrose/agar containing 50 mM CuSO₄. Analysis of the 18S rDNA sequence indicates strain N6 belongs to the genus Cryptococcus. In contrast, the type strain of C. albidus, a typical marine yeast Rhodotorula ingeniosa and Saccharomyces cerevisiae did not grow at high concentrations of CuSO₄. Superoxide dismutase (SOD) catalyzes the scavenging of superoxide radicals. The activity of SOD in cell extract of strain N6 was very weak (<1 mU g^–1 total protein) when the strain was grown in the absence of CuSO₄. However, the activity was stimulated (25.8 mU g^–1 total protein) when cells were grown with 1 mM CuSO₄ and further enhanced to 110 mU g^–1 total protein with 10 mM CuSO₄. Catalase activity was increased only 1.4 or 1.1-fold with 1 mM or 10 mM CuSO₄ in the growth medium, respectively. These results suggest that SOD may have a role in the defensive mechanisms against high concentrations of CuSO₄ in strain N6.     

Secretory expression of a β‐xylosidase gene from Thermomyces lanuginosus in Escherichia coli and characterization of its recombinant enzyme

Aims: To characterize a β‐xylosidase from the thermophilic fungus Thermomyces lanuginosus and to investigate its potential in saccharification of hemicellulosic xylans. Methods and Results: A gene (designated TlXyl43) encoding β‐xylosidase was cloned from T. lanuginosus CAU44 and expressed in Escherichia coli. The gene consists of a 1017‐bp open reading frame without introns. It encodes a mature protein of 338 residues with no predicted signal peptide, belonging to glycoside hydrolase (GH) family 43. Over 60% of the recombinant β‐xylosidase (TlXyl43) was secreted into the culture medium. TlXyl43 was purified 2·6‐fold to homogeneity with an estimated mass of 51·6 kDa by SDS‐PAGE. The purified enzyme exhibited optimal activity at pH 6·5 and 55°C and was stable at 50°C. It was competitively inhibited by xylose with a K_i value of 63 mmol l^?1. Conclusions: In this study, a GH family 43 β‐xylosidase gene (TlXyl43) from T. lanuginosus CAU44 was cloned and functionally expressed in E. coli, and over 60% of recombinant protein was secreted into the culture. Significance and Impact of the Study: This is the first report of the cloning and functional expression of a β‐xylosidase gene from Thermomyces species. TlXyl43 holds great potential for variety of industries.