Binding Specificity of a Novel Cyclo/Maltodextrin-Binding Protein and Its Role in the Cyclodextrin ABC Importer System from Thermoanaerobacterales.

Extracellular synthesis of functional cyclodextrins (CDs) as intermediates of starch assimilation is a convenient microbial adaptation to sequester substrates, increase the half-life of the carbon source, carry bioactive compounds, and alleviate chemical toxicity through the formation of CD-guest complexes. Bacteria encoding the four steps of the carbohydrate metabolism pathway via cyclodextrins (CM-CD) actively internalize CDs across the microbial membrane via a putative type I ATP-dependent ABC sugar importer system, MdxEFG-(X/MsmX). While the first step of the CM-CD pathway encompasses extracellular starch-active cyclomaltodextrin glucanotransferases (CGTases) to synthesize linear dextrins and CDs, it is the ABC importer system in the second step that is the critical factor in determining which molecules from the CGTase activity will be internalized by the cell. Here, structure-function relationship studies of the cyclo/maltodextrin-binding protein MdxE of the MdxEFG-MsmX importer system from Thermoanaerobacter mathranii subsp. mathranii A3 are presented. Calorimetric and fluorescence studies of recombinant MdxE using linear dextrins and CDs showed that although MdxE binds linear dextrins and CDs with high affinity, the open-to-closed conformational change is solely observed after α- and ß-CD binding, suggesting that the CM-CD pathway from Thermoanaerobacterales is exclusive for cellular internalization of these molecules. Structural analysis of MdxE coupled with docking simulations showed an overall architecture typically found in sugar-binding proteins (SBPs) that comprised two N- and C-domains linked by three small hinge regions, including the conserved aromatic triad Tyr193/Trp269/Trp378 in the C-domain and Phe87 in the N-domain involved in CD recognition and stabilization. Structural bioinformatic analysis of the entire MdxFG-MsmX importer system provided further insights into the binding, internalization, and delivery mechanisms of CDs. Hence, while the MdxE-CD complex couples to the permease subunits MdxFG to deliver the CD into the transmembrane channel, the dimerization of the cytoplasmatic promiscuous ATPase MsmX triggers active transport into the cytoplasm. This research provides the first results on a novel thermofunctional SBP and its role in the internalization of CDs in extremely thermophilic bacteria.

Comparison of Single and Combined Use of Catechin, Protocatechuic, and Vanillic Acids as Antioxidant and Antibacterial Agents against Uropathogenic Escherichia Coli at Planktonic and Biofilm Levels.

The objective of this study was to evaluate the effect of combining catechin, protocatechuic, and vanillic acids against planktonic growing, adhesion, and biofilm eradication of uropathogenic Escherichia coli (UPEC), as well as antioxidant agents. The minimum inhibitory concentrations (MIC) of protocatechuic, vanillic acids and catechin against the growth of planktonic bacteria were 12.98, 11.80, and 13.78 mM, respectively. Mixing 1.62 mM protocatechuic acid + 0.74 mM vanillic acid + 0.05 mM catechin resulted in a synergistic effect acting as an MIC. Similarly, the minimum concentrations of phenolic compounds to prevent UPEC adhesion and biofilm formation (MBIC) were 11.03 and 7.13 mM of protocatechuic and vanillic acids, respectively, whereas no MBIC of catechin was found. However, combinations of 1.62 mM protocatechuic acid + 0.74 mM vanillic acid + 0.05 mM catechin showed a synergistic effect acting as MBIC. On the other hand, the minimum concentrations to eradicate biofilms (MBEC) were 25.95 and 23.78 mM, respectively. The combination of 3.20 mM protocatechuic acid, 2.97 mM vanillic acid, and 1.72 mM catechin eradicated pre-formed biofilms. The antioxidant capacity of the combination of phenolics was higher than the expected theoretical values, indicating synergism by the DPPH•, ABTS, and FRAP assays. Effective concentrations of catechin, protocatechuic, and vanillic acids were reduced from 8 to 1378 times when combined. In contrast, the antibiotic nitrofurantoin was not effective in eradicating biofilms from silicone surfaces. In conclusion, the mixture of phenolic compounds was more effective in preventing cell adhesion and eradicating pre-formed biofilms of uropathogenic E. coli than single compounds and nitrofurantoin, and showed antioxidant synergy.

Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei.

Triosephosphate isomerase (TIM; EC 5.3.1.1) is a key enzyme involved in glycolysis and gluconeogenesis. Glycolysis is one of the most regulated metabolic pathways, however little is known about the structural mechanisms for its regulation in non-model organisms, like crustaceans. To understand the structure and function of this enzyme in invertebrates, we obtained the crystal structure of triosephosphate isomerase from the marine Pacific whiteleg shrimp (Litopenaeus vannamei, LvTIM) in complex with its inhibitor 2-phosphogyceric acid (2-PG) at 1.7Å resolution. LvTIM assembles as a homodimer with residues 166-176 covering the active site and residue Glu166 interacting with the inhibitor. We found that LvTIM is the least stable TIM characterized to date, with the lowest range of melting temperatures, and with the lowest activation enthalpy associated with the thermal unfolding process reported. In TIMs dimer stabilization is maintained by an interaction of loop 3 by a set of hydrophobic contacts between subunits. Within these contacts, the side chain of a hydrophobic residue of one subunit fits into a cavity created by a set of hydrophobic residues in the neighboring subunit, via a "ball and socket" interaction. LvTIM presents a Cys47 at the "ball" inter-subunit contact indicating that the character of this residue is responsible for the decrease in dimer stability. Mutational studies show that this residue plays a role in dimer stability but is not a solely determinant for dimer formation.

Arginine kinase shows nucleoside diphosphate kinase-like activity toward deoxythymidine diphosphate.

Arginine kinase (AK) (ATP: L-arginine phosphotransferase, E.C. 2.7.3.3) catalyzes the reversible transfer of ATP γ-phosphate group to L-arginine to synthetize phospho-arginine as a high-energy storage. Previous studies suggest additional roles for AK in cellular processes. Since AK is found only in invertebrates and it is homologous to creatine kinase from vertebrates, the objective of this work was to demonstrate nucleoside diphosphate kinase-like activity for shrimp AK. For this, AK from marine shrimp Litopenaeus vannamei (LvAK) was purified and its activity was assayed for phosphorylation of TDP using ATP as phosphate donor. Moreover, by using high-pressure liquid chromatography (HPLC) the phosphate transfer reaction was followed. Also, LvAK tryptophan fluorescence emission changes were detected by dTDP titration, suggesting that the hydrophobic environment of Trp 221, which is located in the top of the active site, is perturbed upon dTDP binding. The kinetic constants for both substrates Arg and dTDP were calculated by isothermal titration calorimetry (ITC). Besides, docking calculations suggested that dTDP could bind LvAK in the same cavity where ATP bind, and LvAK basic residues (Arg124, 126 and 309) stabilize the dTDP phosphate groups and the pyrimidine base interact with His284 and Ser122. These results suggest that LvAK bind and phosphorylate dTDP being ATP the phosphate donor, thus describing a novel alternate nucleoside diphosphate kinase-like activity for this enzyme.

Exploring structural and computational contrasts in Myoglobins: Implications for thermal treatment-induced Sulfmyoglobin formation.

Greening of tuna metmyoglobin (MetMb) by thermal treatment (TT) and free cysteine is associated with sulfmyoglobin (SulfMb) production. This greening reaction (GR) was once thought to occur only in tuna species. However, recent research has revealed that not all tuna species exhibit this behavior, and it can also occur in horse MetMb. Thus, the present study aimed to compare the GR-reactive (Katsuwonus pelamis and Equus caballus) and GR-unreactive (Sarda chiliensis and Euthynnus lineatus) MetMb using UV-vis spectrometry during TT (60 °C/30 min and free cysteine) to monitor the GR. We used molecular dynamics (MD) simulation to assess the stability of the heme group during TT. We discovered that using GR-unreactive MetMb resulted in an incomplete GR without producing SulfMb. Additionally, our MD simulations indicated that Met85 presence in the heme cavity from GR-unreactive is responsible for the heme group instability and displacement of distal His during TT.

Binding of green tea epigallocatechin gallate to the arginine kinase active site from the brown recluse spider (Loxosceles laeta): A potential synergist to chemical pesticides.

Loxosceles spp. spiders can cause serious public health issues. Chemical control is commonly used, leading to health and environmental problems. Identifying molecular targets and using them with natural compounds can help develop safer and eco-friendlier biopesticides. We studied the kinetics and predicted structural characteristics of arginine kinase (EC 2.7.3.3) from Loxosceles laeta (LlAK), a key enzyme in the energy metabolism of these organisms. Additionally, we explored (-)-epigallocatechin gallate (EGCG), a green tea flavonoid, as a potential lead compound for the LlAK active site through fluorescence and in silico analysis, such as molecular docking and molecular dynamics (MD) simulation and MM/PBSA analyses. The results indicate that LlAK is a highly efficient enzyme (K m Arg 0.14 mM, K m ATP 0.98 mM, k cat 93 s-1, k cat/K m Arg 630 s-1 mM-1, k cat/K m ATP 94 s-1 mM-1), which correlates with its structure similarity to others AKs (such as Litopenaeus vannamei, Polybetes pythagoricus, and Rhipicephalus sanguineus) and might be related to its important function in the spider's energetic metabolism. Furthermore, the MD and MM/PBSA analysis suggests that EGCG interacted with LlAK, specifically at ATP/ADP binding site (RMSD <1 nm) and its interaction is energetically favored for its binding stability (-40 to -15 kcal/mol). Moreover, these results are supported by fluorescence quenching analysis (K d 58.3 µM and K a 1.71 × 104 M-1). In this context, LlAK is a promising target for the chemical control of L. laeta, and EGCG could be used in combination with conventional pesticides to manage the population of Loxosceles species in urban areas.

Crystal structure of shrimp arginine kinase in binary complex with arginine-a molecular view of the phosphagen precursor binding to the enzyme.

Arginine kinase (AK) is a key enzyme for energetic balance in invertebrates. Although AK is a well-studied system that provides fast energy to invertebrates using the phosphagen phospho-arginine, the structural details on the AK-arginine binary complex interaction remain unclear. Herein, we determined two crystal structures of the Pacific whiteleg shrimp (Litopenaeus vannamei) arginine kinase, one in binary complex with arginine (LvAK-Arg) and a ternary transition state analog complex (TSAC). We found that the arginine guanidinium group makes ionic contacts with Glu225, Cys271 and a network of ordered water molecules. On the zwitterionic side of the amino acid, the backbone amide nitrogens of Gly64 and Val65 coordinate the arginine carboxylate. Glu314, one of proposed acid-base catalytic residues, did not interact with arginine in the binary complex. This residue is located in the flexible loop 310-320 that covers the active site and only stabilizes in the LvAK-TSAC. This is the first binary complex crystal structure of a guanidine kinase in complex with the guanidine substrate and could give insights into the nature of the early steps of phosphagen biosynthesis.

Sulfmyoglobin production by free cysteine during thermal treatment: Involvement of heme iron in the production of free radicals.

The meat greening is an abnormal pigmentation related to microbiological contamination and lipid oxidation during storage. This color change results from sulfmyoglobin (SulfMb) production promoted by the reaction between metmyoglobin (MetMb), H2O2, and thiol compounds. Spectral studies on cooked meat suggested the production of SulfMb, probably due to the increment of free radicals during thermal treatment. Thus, we evaluated the involvement of free radicals and heme iron in the SulfMb production from horse MetMb and free cysteine (Cys) during thermal treatment. The results confirm that the reaction of SulfMb production at meat muscle pH (5.7-7.2) during heat treatment is a product of free radicals formed from Cys oxidation (SH) and reactive oxygen species (O2-, H2O2). This is catalyzed by the release of heme iron, thus promoting a consecutive reaction having MbFe(IV)O as a reaction intermediate.

Review of the Greening Reaction by Thermal Treatment: New Insights Exploring the Structural Implications of Myoglobin.

Myoglobin is the main factor responsible for muscle pigmentation in tuna; muscle color depends upon changes in the oxidative state of myoglobin. The tuna industry has reported muscle greening after thermal treatment involving metmyoglobin (MetMb), trimethylamine oxide (TMAO), and free cysteine (Cys). It has been proposed that this pigmentation change is due to a disulfide bond between a unique cysteine residue (Cys10) found in tuna MetMb and free Cys. However, no evidence has been given to confirm that this reaction occurs. In this review, new findings about the mechanism of this greening reaction are discussed, showing evidence of how free radicals produced from Cys oxidation under thermal treatment participate in the greening of tuna and horse muscle during thermal treatment. In addition, the reaction conditions are compared to other green myoglobins, such as sulfmyoglobin, verdomyoglobin, and cholemyoglobin.

Crystallization and X-ray diffraction studies of arginine kinase from the white Pacific shrimp Litopenaeus vannamei.

Crystals of an unligated monomeric arginine kinase from the Pacific whiteleg shrimp Litopenaeus vannamei (LvAK) were successfully obtained using the microbatch method. Crystallization conditions and preliminary X-ray diffraction analysis to 1.25 Šresolution are reported. Data were collected at 100 K on NSLS beamline X6A. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.5, b = 70.2, c = 81.7 Å. One monomer per asymmetric unit was found, with a Matthews coefficient (V(M)) of 2.05 Å(3) Da(-1) and 40% solvent content. Initial phases were determined by molecular replacement using a homology model of LvAK as the search model. Refinement was performed with PHENIX, with final R(work) and R(free) values of 0.15 and 0.19, respectively. Biological analysis of the structure is currently in progress.

Crystallization and X-ray diffraction studies of crustacean proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA), a member of the sliding clamp family of proteins, interacts specifically with DNA replication and repair proteins through a small peptide motif called the PCNA-interacting protein or PIP box. PCNA is recognized as one of the key proteins involved in DNA metabolism. In the present study, the recombinant PCNA from Litopenaeus vannamei (LvPCNA) was heterologously overexpressed and purified using metal ion-affinity chromatography. Crystals suitable for diffraction grew overnight using the hanging-drop vapour-diffusion method. LvPCNA crystals belong to space group C2 with unit-cell parameters a=144.6, b=83.4, c=74.3 Å, ß=117.6°. One data set was processed to 3 Šresolution, with an overall Rmeas of 0.09 and a completeness of 93.3%. Initial phases were obtained by molecular replacement using a homology model of LvPCNA as the search model. Refinement and structural analysis are underway. This report is the first successful crystallographic analysis of a marine crustacean decapod shrimp (L. vannamei) proliferating cell nuclear antigen.

The greening reaction of skipjack tuna (Katsuwonus pelamis) metmyoglobin promoted by free cysteine during thermal treatment.

Background: Tuna muscle greening is a problem that occurs after heating. A hypothesis has been postulated to address this problem, involving a conserved Cys residue at position 10 (Cys-10) present on tuna myoglobin (Mb) that is exposed during the thermic treatment, forming a disulfide bond with free cysteine (Cys) in the presence of trimethylamine oxide (TMAO), resulting in the greening of the tuna Mb. Methods: We present a study using skipjack tuna (Katsuwonus pelamis) metmyoglobin (MbFe(III)-H2O) where the effect of free Cys (1-6 mM), TMAO (1.33 mM), and catalase on the greening reaction (GR) was monitored by UV-vis spectrometry during thermal treatment at 60 °C for 30 min. Moreover, the participation of Cys-10 on the GR was evaluated after its blocking with N-ethymaleimide. Results: The GR occurred in tuna MbFe(III)-H2O after heat treatment with free Cys, forming sulfmyoglobin (MbFe(II)-S) as the responsible pigment for the tuna greening. However, the rate constants of MbFe(II)-S production depended on Cys concentration (up to 4 mM) and occurred regardless of the TMAO presence. We postulate that two consecutive reactions involve an intermediate ferrylmyoglobin (promoted by H2O2) species with a subsequent MbFe(II)-S formation since the presence of catalase fosters the reduction of the rate reaction. Moreover, GR occurred even with blocked Cys-10 residues in tuna Mb and horse Mb (without Cys in its sequence). Discussion: We found that GR is not exclusive to tuna Mb´s, and it can be promoted in other muscle systems. Moreover, Cys and thermal treatment are indispensable for promoting this pigmentation anomaly.

Agavin induces beneficial microbes in the shrimp microbiota under farming conditions.

Prebiotics and probiotics have shown a number of beneficial impacts preventing diseases in cultured shrimps. Complex soluble carbohydrates are considered ideal for fostering microbiota biodiversity by fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPS). Here we evaluated the growth performance and microbiota composition of the white shrimp Litopenaeus vannamei after dietary intervention using agavin as a FODMAP prebiotic under farming conditions. Adult L. vannamei were raised at a shrimp farm and the effect of agavin supplemented at 2% (AG2) or 10% (AG10) levels were compared to an agavin-free basal diet (BD). After 28 days-trial, the feed conversion ratio, total feed ingested, and protein efficiency ratio was significantly improved on animals fed with AG2. At the same time, no effect on growth performance was observed in AG10. Surprisingly, after sequencing the V3-V4 regions of the 16S rRNA gene a higher microbial richness and diversity in the hepatopancreas and intestine was found only in those animals receiving the AG10 diet, while those receiving the AG2 diet had a decreased richness and diversity, both diets compared to the BD. The beta diversity analysis showed a clear significant microbiota clustering by agavin diets only in the hepatopancreas, suggesting that agavin supplementation had a more substantial deterministic effect on the microbiota of hepatopancreas than on the intestine. We analyzed the literature to search beneficial microbes for shrimp's health and found sequences for 42 species in our 16S data, being significantly increased Lactobacillus pentosus, Pseudomonas putida and Pseudomonas synxantha in the hepatopancreas of the AG10 and Rodopseudomonas palustris and Streptococcus thermophiles th1435 in the hepatopancreas of the AG2, both compared to BD. Interestingly, when we analyzed the abundance of 42 beneficial microbes as a single microbial community "meta-community," found an increase in their abundance as agavin concentration increases in the hepatopancreas. In addition, we also sequenced the DNA of agavin and found 9 of the 42 beneficial microbes. From those, Lactobacillus lactis and Lactobacillus delbrueckii were found in shrimps fed with agavin (both AG2 and AG10), and Lysinibacillus fusiformis in AG10 and they were absent the BD diet, suggesting these three species could be introduced with the agavin to the diet. Our work provides evidence that agavin supplementation is associated with an increase of beneficial microbes for the shrimp microbiota at farming conditions. Our study provides the first evidence that a shrimp prebiotic may selectively modify the microbiota in an organ-dependent effect.

OTUs and ASVs Produce Comparable Taxonomic and Diversity from Shrimp Microbiota 16S Profiles Using Tailored Abundance Filters.

The interplay between shrimp immune system, its environment, and microbiota contributes to the organism's homeostasis and optimal production. The metagenomic composition is typically studied using 16S rDNA profiling by clustering amplicon sequences into operational taxonomic units (OTUs) and, more recently, amplicon sequence variants (ASVs). Establish the compatibility of the taxonomy, α, and ß diversity described by both methods is necessary to compare past and future shrimp microbiota studies. Here, we used identical sequences to survey the V3 16S hypervariable-region using 97% and 99% OTUs and ASVs to assess the hepatopancreas and intestine microbiota of L. vannamei from two ponds under standardized rearing conditions. We found that applying filters to retain clusters >0.1% of the total abundance per sample enabled a consistent taxonomy comparison while preserving >94% of the total reads. The three sets turned comparable at the family level, whereas the 97% identity OTU set produced divergent genus and species profiles. Interestingly, the detection of organ and pond variations was robust to the clustering method's choice, producing comparable α and ß-diversity profiles. For comparisons on shrimp microbiota between past and future studies, we strongly recommend that ASVs be compared at the family level to 97% identity OTUs or use 99% identity OTUs, both using tailored frequency filters.

Biochemical characterization and inhibition of thermolabile hemolysin from Vibrio parahaemolyticus by phenolic compounds.

Vibrio parahaemolyticus (Vp), a typical microorganism inhabiting marine ecosystems, uses pathogenic virulence molecules such as hemolysins to cause bacterial infections of both human and marine animals. The thermolabile hemolysin VpTLH lyses human erythrocytes by a phospholipase B/A2 enzymatic activity in egg-yolk lecithin. However, few studies have been characterized the biochemical properties and the use of VpTLH as a molecular target for natural compounds as an alternative to control Vp infection. Here, we evaluated the biochemical and inhibition parameters of the recombinant VpTLH using enzymatic and hemolytic assays and determined the molecular interactions by in silico docking analysis. The highest enzymatic activity was at pH 8 and 50 °C, and it was inactivated by 20 min at 60 °C with Tm = 50.9 °C. Additionally, the flavonoids quercetin, epigallocatechin gallate, and morin inhibited the VpTLH activity with IC50 values of 4.5 µM, 6.3 µM, and 9.9 µM, respectively; while phenolics acids were not effective inhibitors for this enzyme. Boltzmann and Arrhenius equation analysis indicate that VpTLH is a thermolabile enzyme. The inhibition of both enzymatic and hemolytic activities by flavonoids agrees with molecular docking, suggesting that flavonoids could interact with the active site's amino acids. Future research is necessary to evaluate the antibacterial activity of flavonoids against Vp in vivo.

A Novel Glutathione S-Transferase Gtt2 Class (VpGSTT2) Is Found in the Genome of the AHPND/EMS Vibrio parahaemolyticus Shrimp Pathogen.

Glutathione S-transferases are a family of detoxifying enzymes that catalyze the conjugation of reduced glutathione (GSH) with different xenobiotic compounds using either Ser, Tyr, or Cys as a primary catalytic residue. We identified a novel GST in the genome of the shrimp pathogen V. parahaemolyticus FIM- S1708+, a bacterial strain associated with Acute Hepatopancreatic Necrosis Disease (AHPND)/Early Mortality Syndrome (EMS) in cultured shrimp. This new GST class was named Gtt2. It has an atypical catalytic mechanism in which a water molecule instead of Ser, Tyr, or Cys activates the sulfhydryl group of GSH. The biochemical properties of Gtt2 from Vibrio parahaemolyticus (VpGSTT2) were characterized using kinetic and crystallographic methods. Recombinant VpGSTT2 was enzymatically active using GSH and CDNB as substrates, with a specific activity of 5.7 units/mg. Low affinity for substrates was demonstrated using both Michaelis-Menten kinetics and isothermal titration calorimetry. The crystal structure showed a canonical two-domain structure comprising a glutathione binding G-domain and a hydrophobic ligand H domain. A water molecule was hydrogen-bonded to residues Thr9 and Ser 11, as reported for the yeast Gtt2, suggesting a primary role in the reaction. Molecular docking showed that GSH could bind at the G-site in the vicinity of Ser11. G-site mutationsT9A and S11A were analyzed. S11A retained 30% activity, while T9A/S11A showed no detectable activity. VpGSTT2 was the first bacterial Gtt2 characterized, in which residues Ser11 and Thr9 coordinated a water molecule as part of a catalytic mechanism that was characteristic of yeast GTT2. The GTT2 family has been shown to provide protection against metal toxicity; in some cases, excess heavy metals appear in shrimp ponds presenting AHPND/EMS. Further studies may address whether GTT2 in V. parahaemolyticus pathogenic strains may provide a competitive advantage as a novel detoxification mechanism.

Doing More with Less: A Comparison of 16S Hypervariable Regions in Search of Defining the Shrimp Microbiota.

The shrimp has become the most valuable traded marine product in the world, and its microbiota plays an essential role in its development and overall health status. Massive high-throughput sequencing techniques using several hypervariable regions of the 16S rRNA gene are broadly applied in shrimp microbiota studies. However, it is essential to consider that the use of different hypervariable regions can influence the obtained data and the interpretation of the results. The present study compares the shrimp microbiota structure and composition obtained by three types of amplicons: one spanning both the V3 and V4 hypervariable regions (V3V4), one for the V3 region only (V3), and one for the V4 region only (V4) using the same experimental and bioinformatics protocols. Twenty-four samples from hepatopancreas and intestine were sequenced and evaluated using the GreenGenes and silva reference databases for clustering and taxonomic classification. In general, the V3V4 regions resulted in higher richness and diversity, followed by V3 and V4. All three regions establish an apparent clustering effect that discriminates between the two analyzed organs and describe a higher richness for the intestine and a higher diversity for the hepatopancreas samples. Proteobacteria was the most abundant phyla overall, and Cyanobacteria was more common in the intestine, whereas Firmicutes and Actinobacteria were more prevalent in hepatopancreas samples. Also, the genus Vibrio was significantly abundant in the intestine, as well as Acinetobacter and Pseudomonas in the hepatopancreas suggesting these taxa as markers for their respective organs independently of the sequenced region. The use of a single hypervariable region such as V3 may be a low-cost alternative that enables an adequate description of the shrimp microbiota, allowing for the development of strategies to continually monitor the microbial communities and detect changes that could indicate susceptibility to pathogens under real aquaculture conditions while the use of the full V3V4 regions can contribute to a more in-depth characterization of the microbial composition.

Molecular characterization and expression analysis of the chicken-type and goose-type lysozymes from totoaba (Totoaba macdonaldi).

Lysozymes play a key role in innate immune response to bacterial pathogens, catalyzing the hydrolysis of the peptidoglycan layer of bacterial cell walls. In this study, the genes encoding the c-type (TmLyzc) and g-type (TmLyzg) lysozymes from Totoaba macdonaldi were cloned and characterized. The cDNA sequences of TmLyzg and TmLyzc were 582 and 432 bp, encoding polypeptides of 193 and 143 amino acids, respectively. Amino acid sequences of these lysozymes shared high identity (60-90%) with their counterparts of other teleosts and showed conserved functional-structural signatures of the lysozyme superfamily. Phylogenetic analysis indicated a close relationship with their vertebrate homologues but distinct evolutionary paths for each lysozyme. Expression analysis by qRT-PCR revealed that TmLyzc was expressed in stomach and pyloric caeca, while TmLyzg was highly expressed in stomach and heart. These results suggest that both lysozymes play important roles in defense of totoaba against bacterial infections or as digestive enzyme.

Refolding and Activation from Bacterial Inclusion Bodies of Trypsin I from Sardine (Sardinops sagax caerulea).

BACKGROUND: Trypsin from fish species is considered as a cold-adapted enzyme that may find potential biotechnological applications. In this work, the recombinant expression, refolding and activation of Trypsin I (TryI) from Monterey sardine (Sardinops sagax caerulea) are reported. METHODS: TryI was overexpressed in Escherichia coli BL21 as a fusion protein of trypsinogen with thioredoxin. Refolding of trypsinogen I was achieved by dialysis of bacterial inclusion bodies with a recovery of 16.32 mg per liter of Luria broth medium. RESULTS: Before activation, the trypsinogen fusion protein did not show trypsin activity. Trypsinogen I was activated by adding 0.002 U of native TryI purified from the sardine pyloric caeca (nonrecombinant). The activated recombinant trypsin showed three times more activity than the nonrecombinant trypsin alone. CONCLUSION: The described protocol allowed obtaining sufficient amounts of recombinant TryI from Monterey sardine fish for further biochemical and biophysical characterization of its coldadaptation parameters.

A novel thymidylate synthase from the Vibrionales, Alteromonadales, Aeromonadales, and Pasteurellales (VAAP) clade with altered nucleotide and folate binding sites.

Thymidylate synthase (TS, E.C. 2.1.1.45) is a crucial enzyme for de novo deoxythymidine monophosphate (dTMP) biosynthesis. The gene for this enzyme is thyA, which encodes the folate-dependent TS that converts deoxyuridine monophosphate group (dUMP) into (dTMP) using the cofactor 5,10-methylenetetrahydrofolate (mTHF) as a carbon donor. We identified the thyA gene in the genome of the Vibrio parahaemolyticus strain FIM-S1708+ that is innocuous to humans but pathogenic to crustaceans. Surprisingly, we found changes in the residues that bind the substrate dUMP and mTHF, previously postulated as invariant among all TSs known (Finer-Moore, Santi & Stroud, 2003). Interestingly, those amino acid changes were also found in a clade of microorganisms that contains Vibrionales, Alteromonadales, Aeromonadales, and Pasteurellales (VAAP) from the Gammaproteobacteria class. In this work, we studied the biochemical properties of recombinant TS from V. parahemolyticus FIM-S1708+ (VpTS) to address the natural changes in the TS amino acid sequence of the VAAP clade. Interestingly, the Km for dUMP was 27.3 ± 4.3 µM, about one-fold larger compared to other TSs. The Km for mTHF was 96.3 ± 18 µM, about three- to five-fold larger compared to other species, suggesting also loss of affinity. Thus, the catalytic efficiency was between one or two orders of magnitude smaller for both substrates. We used trimethoprim, a common antibiotic that targets both TS and DHFR for inhibition studies. The IC50 values obtained were high compared to other results in the literature. Nonetheless, this molecule could be a lead for the design antibiotics towards pathogens from the VAAP clade. Overall, the experimental results also suggest that in the VAAP clade the nucleotide salvage pathway is important and should be investigated, since the de novo dTMP synthesis appears to be compromised by a less efficient thymidylate synthase.