Bacterial Stress Research Articles

Phytochemistry, Biological, and Toxicity Study on Aqueous and Methanol Extracts of Chromolaena odorata.

The medicinal plant Chromolaena odorata is traditionally used by people living in different communities of Nepal and the globe against diabetes, soft tissue wounds, skin infections, diarrhea, malaria, and several other infectious diseases. The present study focuses on the qualitative and quantitative phytochemical analyses and antioxidant, antidiabetic, antibacterial, and toxicity of the plant for assessing its pharmacological potential. The extracts of flowers, leaves, and stems were prepared using methanol and distilled water as the extracting solvents. Total phenolic content (TPC) and total flavonoid content (TFC) were estimated by using the Folin-Ciocalteu phenol reagent method and the aluminum chloride colorimetric method. Antioxidant and antidiabetic activities were assessed using the DPPH assay and α-glucosidase inhibition assay. A brine shrimp assay was performed to study the toxicity, and the antibacterial activity test was performed by the agar well diffusion method. Phytochemical analysis revealed the presence of phenols, flavonoids, quinones, terpenoids, and coumarins as secondary metabolites. The methanol extract of leaves and flowers displayed the highest phenolic and flavonoid content with 182.26 ± 1.99 mg GAE/g, 128.57 ± 7.62 mg QE/g and 172.65 ± 0.48 mg GAE/g, 121.74 ± 7.06 mg QE/g, respectively. The crude extracts showed the highest DPPH free radical scavenging activity with half maximal inhibitory concentration (IC50) of 32.81 ± 5.26 µg/mL and 41.00 ± 1.10 µg/mL, respectively. The methanol extract of the leaves was found to be effective against bacterial strains such as K. pneumoniae (ZOI = 9.67 ± 0.32 mm), B. subtilis (ZOI = 15.00 ± 0 mm), and E. coli (7.3 ± 0.32 mm). The methanol extract of the flowers showed the most α-glucosidase inhibitory activity (IC50 227.63 ± 11.38 µg/mL), followed by the methanol extract of leaves (IC50 249.50 ± 0.97 µg/mL). The aqueous extract of the flowers showed the toxic effect with LC50 107.31 ± 49.04 µg/mL against the brine shrimp nauplii. In conclusion, C. odorata was found to be a rich source of plant secondary metabolites such as phenolics and flavonoids with potential effects against bacterial infection, diabetes, and oxidative stress in humans. The toxicity study showed that the aqueous extract of flowers possesses pharmacological activities. This study supports the traditional use of the plant against infectious diseases and diabetes and provides some scientific validation.

Transcriptional biomarker discovery toward building a load stress reporting system for engineered Escherichia coli strains.

Foreign proteins are produced by introducing synthetic constructs into host bacteria for biotechnology applications. This process can cause resource competition between synthetic circuits and host cells, placing a metabolic burden on the host cells which may result in load stress and detrimental physiological changes. Consequently, the host bacteria can experience slow growth, and the synthetic system may suffer from suboptimal function. To help in the detection of bacterial load stress, we developed machine-learning strategies to select a minimal number of genes that could serve as biomarkers for the design of load stress reporters. We identified pairs of biomarkers that showed discriminative capacity to detect the load stress states induced in 41 engineered Escherichia coli strains.

A Mxene@TA/Fe dual-nanozyme composited antifouling hydrogel for burn wound repair

Bacterial infection, oxidative stress, and inflammation response severely hinder burn wound repair. To address these issues, a multifunctional quaternary ammonium modified chitosan-sulfonic betaine acrylamide hydrogel loaded with Mxene@TA/Fe nanosheets was developed for burn wound healing. Since zwitterionic sulfonate betaine acrylamide exhibited good antifouling properties, thus the hydrogel system resisted protein adsorption and bacterial adhesion and avoided wound infection. Moreover, Mxene@TA/Fe nanosheets simultaneously showed catalase (CAT)-like and peroxidase (POD)-like activities because the high conductivity of Mxene augmented the electron transfer rate and promoted the transformation of Fe(Ⅱ) to Fe(Ⅲ) and vice-versa. The POD-like activity combined with photothermal therapy led to a highly effective antibacterial capacity, while the CAT-like activity produced oxygen and alleviated tissue hypoxia. Furthermore, MXene@TA/Fe nanosheets showed a good antioxidant capacity because MXene and TA molecules scavenged toxic reactive oxygen species and reduced oxidative stress. In vivo experiment further proved that the prepared hydrogel could significantly promote burn wound healing by facilitating granulation tissue regeneration, epithelial layer formation, and collagen deposition.

XrvB regulates the type III secretion system by directly repressing hrpG transcription in Xanthomonas oryzae pv. oryzicola

Xrv proteins are a group of regulators in Xanthomonas spp., belonging to the histone-like nucleoid-structuring (H-NS) proteins of Gram-negative bacteria. The rice bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc) harbors three Xrv proteins, the XrvA, XrvB, and XrvC. Here, we report that in Xoc, the XrvB but not XrvA and XrvC is involved in negative regulation of the type III secretion system (T3SS) encoded by hrp genes. As with other Xanthomonas spp., the T3SS is an essential virulence determinant of Xoc and the expression of the hrp genes in Xoc is controlled by the HrpG/HrpX regulatory cascade. HrpG positively regulates the expression of HrpX, which in turn activates the transcription of the hrp genes. We provide evidences to demonstrate that the XrvB binds to the promoter region of hrpG and represses its transcription. Furthermore, we found that XrvB production was induced in the Xoc cells cultured in a nutrient-rich medium compared to a hrp-inducing minimal medium. We also found that in Xoc, the hrpG expression level is inversely correlated with the content of XrvB, and XrvB occupancy at hrpG promoter region is positively correlated with XrvB levels. Our data suggest that XrvB is a determinative factor controlling the expression levels of HrpG. In addition, mutation analysis revealed that the Xoc XrvB also plays positive roles in regulating bacterial growth, cell motility, and stress tolerance. Our findings provide important insights into the molecular mechanism of T3SS expression regulation in Xoc.

High antipersister activity of a promising new quinolone drug candidate in eradicating uropathogenic Escherichia coli persisters and persistent infection in mice.

To develop more potent drugs that eradicate persister bacteria and cure persistent urinary tract infections (rUTIs). We synthesized eight novel clinifloxacin analogs and measured minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), the time-kill curves in uropathogenic Escherichia coli (UPEC) UTI89, and applied the candidate drugs and combinations against biofilm bacteria in vitro and in mice. Transcriptomic analysis was performed for UPEC after candidate drug treatment to shed light on potential mechanism of action. We identified Compound 2, named Qingdafloxacin (QDF), which was more potent than clinafloxacin and clinically used levofloxacin and moxifloxacin, with an MIC of<0.04μg ml-1 and an MBC of 0.08∼0.16μg ml-1. In drug combination studies, QDF+gentamicin+nitrofuran combination but not single drugs completely eradicated all stationary phase bacteria containing persisters and biofilm bacteria, and all bacteria in a persistent UTI mouse model. Transcriptome analysis revealed that the unique antipersister activity of QDF was associated with downregulation of genes involved in bacterial stress response, DNA repair, protein misfolding repair, pyrimidine metabolism, glutamate, and glutathione metabolism, and efflux. QDF has high antipersister activity and its drug combinations proved highly effective against biofilm bacteria in vitro and persistent UTIs in mice, which may have implications for treating rUTIs.

Antimicrobial activity of the Lacticaseibacillus rhamnosus CRL 2244 and its impact on the phenotypic and transcriptional responses in carbapenem resistant Acinetobacter baumannii

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a recognized nosocomial pathogen with limited antibiotic treatment options. Lactic acid bacteria (LAB) constitute a promising therapeutic alternative. Here we studied the antibacterial properties of a collection of LAB strains using phenotypic and transcriptomic analysis against A. baumannii clinical strains. One strain, Lacticaseibacillus rhamnosus CRL 2244, demonstrated a potent inhibitory capacity on A. baumannii with a significant killing activity. Scanning electron microscopy images showed changes in the morphology of A. baumannii with an increased formation of outer membrane vesicles. Significant changes in the expression levels of a wide variety of genes were also observed. Interestingly, most of the modified genes were involved in a metabolic pathway known to be associated with the survival of A. baumannii. The paa operon, Hut system, and fatty acid degradation were some of the pathways that were induced. The analysis reveals the impact of Lcb. rhamnosus CRL 2244 on A. baumannii response, resulting in bacterial stress and subsequent cell death. These findings highlight the antibacterial properties of Lcb. rhamnosus CRL 2244 and its potential as an alternative or complementary strategy for treating infections. Further exploration and development of LAB as a treatment option could provide valuable alternatives for combating CRAB infections.

Membrane Lipid Composition Influences the Hydration of Proton Half-Channels in FoF1-ATP Synthase.

The membrane lipid composition plays an important role in the regulation of membrane protein activity. To probe its influence on proton half-channels' structure in FoF1-ATP synthase, we performed molecular dynamics simulations with the bacterial protein complex (PDB ID: 6VWK) embedded in three types of membranes: a model POPC, a lipid bilayer containing 25% (in vivo), and 75% (bacterial stress) of cardiolipin (CL). The structure proved to be stable regardless of the lipid composition. The presence of CL increased the hydration of half-channels. The merging of two water cavities at the inlet half-channel entrance and a long continuous chain of water molecules directly to cAsp61 from the periplasm were observed. Minor conformational changes in half-channels with the addition of CL caused extremely rare direct transitions between aGlu219-aAsp119, aGlu219-aHis245, and aGln252-cAsp61. Deeper penetration of water molecules (W1-W3) also increased the proton transport continuity. Stable spatial positions of significant amino acid (AA) residue aAsn214 were found under all simulation conditions indicate a prevailing influence of AA-AA or AA-W interactions on the side-chain dynamics. These results allowed us to put forward a model of the proton movement in ATP synthases under conditions close to in vivo and to evaluate the importance of membrane composition in simulations.

Antibacterial activity and mechanism of cinnamon essential oil nanoemulsion against Pseudomonas deceptionensis CM2

This work aimed to evaluate the antibacterial activity and mechanism of cinnamon essential oil nanoemulsion (CON) against Pseudomonas deceptionensis CM2. The results revealed that CON could effectively inhibit the proliferation of P. deceptionensis CM2 cells in a time- and concentration-dependent manner. After 4 h of incubation with CON at the minimum inhibitory concentration (0.125 mg/mL), the relative fluorescence intensity of propidium iodide and 1-N-phenylnapthylamine (NPN) was increased by 32.0% and 351.4%, respectively. The membrane permeability of P. deceptionensis CM2 cells was significantly disrupted after CON treatment, resulting in the leakage of intracellular substances (such as proteins and electrolytes). CON also caused significant increases in the DiBAC4(3) fluorescence intensity of P. deceptionensis CM2 cells. These results demonstrate that CON induced inactivation of P. deceptionensis CM2 by destroying the integrity and function of bacterial membrane. A higher level of intracellular reactive oxygen species (ROS) was observed in CON-treated cells (p < 0.05), compared with control cells. Moreover, the addition of glutathione to the growth medium remarkably decreased the antimicrobial activity of CON against P. deceptionensis CM2, further confirming that oxidative stress played an important role in the antimicrobial activity of CON. Overall, CON may exhibit antibacterial effects by causing damage to the bacterial membranes and oxidative stress.

Biosynthesis of antioxidant xanthan gum by Xanthomonas campestris using substrates added with moist olive pomace

Moist olive pomace (MOP) is a high moisture content by-product of the olive oil industry. Managing this recalcitrant residue (transport, storage, and drying) is a priority demanding investment in finding alternative valorisation routes. In this context, the biosynthesis of xanthan gum (XG) incorporating MOP in the substrate (0.0 %, 5.0 %, 10.0 %, 15.0 %, 20.0 %, 25.0 %, 30.0 % and 50.0 %) to induce bacterial stress was attempted. XG biosynthesis yield was quantified, and the product was characterised by structural analysis (FTIR), thermal behaviour (TG), rheology and antioxidant capacity. Relative to the control (sample with no added MOP), a significant increase in XG biosynthesis was found for concentrations up to 30.0 % MOP. In particular, for XG produced with 15 % MOP, a 50.91 % (p < 0.0001) increase was achieved, together with 395.78 % for viscosity. In general, XG produced with MOP presence showed antioxidant activity, a value-added property, especially for applications in the food, pharmaceutical and cosmetic areas. The results indicated that the stress imposed by the MOP induced a microbial response leading to XG production increase, structural and viscosity modifications, and antioxidant properties incorporation. Overall, this work points out a new MOP application contributing to the sustainability of the olive oil productive chain from a biobased circular economy perspective.

ClpP protease modulates bacterial growth, stress response, and bacterial virulence in Brucella abortus

The process of intracellular proteolysis through ATP-dependent proteases is a biologically conserved phenomenon. The stress responses and bacterial virulence of various pathogenic bacteria are associated with the ATP-dependent Clp protease. In this study, a Brucella abortus 2308 strain, ΔclpP, was constructed to characterize the function of ClpP peptidase. The growth of the ΔclpP mutant strain was significantly impaired in the TSB medium. The results showed that the ΔclpP mutant was sensitive to acidic pH stress, oxidative stress, high temperature, detergents, high osmotic environment, and iron deficient environment. Additionally, the deletion of clpP significantly affected Brucella virulence in macrophage and mouse infection models. Integrated transcriptomic and proteomic analyses of the ΔclpP strain showed that 1965 genes were significantly affected at the mRNA and/or protein levels. The RNA-seq analysis indicated that the ΔclpP strain exhibited distinct gene expression patterns related to energy production and conversion, cell wall/membrane/envelope biogenesis, carbohydrate transport, and metabolism. The iTRAQ analysis revealed that the differentially expressed proteins primarily participated in amino acid transport and metabolism, energy production and conversion, and secondary metabolites biosynthesis, transport and catabolism. This study provided insights into the preliminary molecular mechanism between Clp protease to bacterial growth, stress response, and bacterial virulence in Brucella strains.

The tRNA methyltransferase TrmB is critical for Acinetobacter baumannii stress responses and pulmonary infection.

As deficiencies in tRNA modifications have been linked to human diseases such as cancer and diabetes, much research has focused on the modifications' impacts on translational regulation in eukaryotes. However, the significance of tRNA modifications in bacterial physiology remains largely unexplored. In this paper, we demonstrate that the m7G tRNA methyltransferase TrmB is crucial for a top-priority pathogen, Acinetobacter baumannii, to respond to stressors encountered during infection, including oxidative stress, low pH, and iron deprivation. We show that loss of TrmB dramatically attenuates a murine pulmonary infection. Given the current efforts to use another tRNA methyltransferase, TrmD, as an antimicrobial therapeutic target, we propose that TrmB, and other tRNA methyltransferases, may also be viable options for drug development to combat multidrug-resistant A. baumannii.

Toxin-Antitoxin System HigBA in Xylella fastidiosa Is Expressed at Low Temperature but Does Not Impact Virulence or Grapevine Recovery in Greenhouse Trials

Xylella fastidiosa has multiple chromosomally encoded type II toxin-antitoxin (TA) systems, several of which have unknown functions in the bacterial lifecycle. In many bacterial species, type II TA systems are associated with bacterial stress survival under various detrimental environmental conditions, and with influencing disease-associated phenotypes such as biofilm development and expression of virulence factors. Chromosomal TA systems are often found in genomic regions with evidence of phage insertion, pointing to potential acquisition of these elements by horizontal gene transfer. One of the TA systems found in X. fastidiosa, higBA, is transcriptionally expressed at higher levels at 15°C than at 28°C. However, a higBA deletion mutant (Δ higBA) in X. fastidiosa subsp. fastidiosa Stag's Leap is not deficient in growth or viability under in vitro cold stress and is able to infect and cause disease in grapevines comparable to the wild-type strain. Grapevines infected with X. fastidiosa Δ higBA recover from infection following cold treatment at 4°C at a similar rate to wild-type infected plants, suggesting that this TA system does not contribute significantly to X. fastidiosa survival at a low temperature in planta. Although a definitive function for HigBA in X. fastidiosa has yet to be determined, this study adds to a growing body of work describing the functions and expression dynamics of TA systems in plant pathogenic bacteria. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

S-Adenosylmethionine Decreases Bacterial Translocation, Proinflammatory Cytokines, Oxidative Stress and Apoptosis Markers in Hepatic Ischemia-Reperfusion Injury in Wistar Rats.

Hepatic ischemia/reperfusion injury (IRI) can seriously impair liver function. It is initiated by oxidative stress, resulting in inflammation and apoptosis-induced cellular damage. Glutathione (GSH) prevents oxidative stress. S-Adenosylmethionine (SAMet) is a GSH synthesis precursor that avoids the deficit in SAMet-synthetase activity and contributes to intracellular ATP repletion. It also acts as a methyl group donor, stabilizing hepatocyte membranes, among other functions. This study investigated the effect of SAMet on bacterial translocation and levels of proinflammatory cytokines, oxidative stress and apoptosis markers in male Wistar rats subjected to hepatic IRI. Animals were randomly divided into six groups: (1) sham operation, (3) animals undergoing 60 min of ischemia of the right lateral lobe for temporary occlusion of the portal vein and hepatic artery plus 10 min of reperfusion, and (5) the same as (3) but with a reperfusion period of 120 min. Groups 2, 4 and 6, respectively, are the same as (1), (3) and (5), except that animals received SAMet (20 mg/kg) 15 min before ischemia. GSH, ATP, lipid peroxidation (LPO), TNF-α, IL-1β, IL-6, total caspase-1 and caspase-9, total and cleaved caspase-3, and phosphatidylcholine were determined in the liver. Endotoxin, TNF-α, IL-1β, IL-6, IL-10 and LPO in vena cava and portal vein blood samples were also measured. Endotoxin and LPO levels as well as proinflammatory cytokines and apoptotic markers increased significantly in animals undergoing IRI, both after 10 and 120 min of reperfusion. IRI produced a significant decrease in GSH, ATP, portal IL-10 and phosphatidylcholine. SAMet treatment prevented these effects significantly and increased survival rate. The study suggests that SAMet exerts protective effects in hepatic IRI.

Tannic acid-Fe3+ dual catalysis induced rapid polymerization of injectable poly(lysine) hydrogel for infected wound healing

Infected wounds are difficult to heal because they are vulnerable to bacterial attacks, inflammatory responses, and oxidative stress. To promote the healing of infected wounds, we developed an injectable dual-network hydrogel TFAEP (TA-Fe, APS, EPL-GMA, PVA) based on ε-poly-l-lysine-graft-glycidyl methacrylate (EPL-GMA), polyvinyl alcohol (PVA), and tannic acid-iron (TA-Fe). TA-Fe formed a stable redox pair, which acted as a dual-autocatalytic system to activate ammonium persulfate, generate free radicals, and subsequently induce EPL-GMA polymerization. Then PVA formed hydrogen bonds with TA molecules. Here, TA-Fe not only simulated peroxidase to convert H2O2 into hydroxyl radicals (OH), but also exhibited good near-infrared photothermal conversion efficiency, which all endowed the hydrogel with excellent antibacterial ability. In addition, the hydrogel could remove excessive reactive oxygen species and reactive nitrogen species, alleviating oxidative stress and reducing inflammation response due to the presence of TA molecules. Moreover, the hydrogel showed good injectability and tissue adhesion, ensuring the close adhesion of the hydrogel to the wound and achieving the maximum function. In vivo experiments demonstrated that the hydrogel promoted infected wound healing by accelerating epidermal regeneration, promoting angiogenesis and collagen deposition, and facilitating the expression of anti-inflammatory factors.

Bacterial outer-membrane vesicles promote Vγ9Vδ2 T cell oncolytic activity.

Increasing evidence suggests the immune activation elicited by bacterial outer-membrane vesicles (OMVs) can initiate a potent anti-tumor immunity, facilitating the recognition and destruction of malignant cells. At present the pathways underlying this response remain poorly understood, though a role for innate-like cells such as γδ T cells has been suggested. Peripheral blood mononuclear cells (PBMCs) from healthy donors were co-cultured with E. coli MG1655 Δpal ΔlpxM OMVs and corresponding immune activation studied by cell marker expression and cytokine production. OMV-activated γδ T cells were co-cultured with cancer cell lines to determine cytotoxicity. The vesicles induced a broad inflammatory response with γδ T cells observed as the predominant cell type to proliferate post-OMV challenge. Notably, the majority of γδ T cells were of the Vγ9Vδ2 type, known to respond to both bacterial metabolites and stress markers present on tumor cells. We observed robust cytolytic activity of Vγ9Vδ2 T cells against both breast and leukaemia cell lines (SkBr3 and Nalm6 respectively) after OMV-mediated expansion. Our findings identify for the first time, that OMV-challenge stimulates the expansion of Vγ9Vδ2 T cells which subsequently present anti-tumor capabilities. We propose that OMV-mediated immune activation leverages the anti-microbial/anti-tumor capacity of Vγ9Vδ2 T cells, an axis amenable for improved future therapeutics.

Combined metabolomics and transcriptomics analysis reveals the mechanism of antibiotic resistance of Salmonella enterica serovar Typhimurium after acidic stress

Drug-resistant Salmonella is widely distributed in the meat production chain, endangering food safety and public health. Acidification of meat products during processing can induce acid stress, which may alter antibiotic resistance. Our study investigated the effects of acid stress on the antibiotic resistance and metabolic profile of Salmonella Typhimurium, and explored the underlying mechanisms using metabolomic and transcriptomic analysis. We found that acid-stressed 14028s was more sensitive to small molecule hydrophobic antibiotics (SMHA) while more resistant to meropenem (MERO). Metabolomic analysis revealed that enhanced sensitivity to SMHA was correlated with increased purine metabolism and tricarboxylic acid cycle. Transcriptomic analysis revealed the downregulation of chemotaxis-related genes, which are also associated with SMHA sensitivity. We also found a significant downregulation of the ompF gene, which encodes a major outer membrane protein OmpF of Salmonella. The decreased expression of OmpF porin hindered the influx of MERO, leading to enhanced resistance of the bacteria to the drug. Our findings contribute to greatly improve the understanding of the relationship between Salmonella metabolism, gene expression, and changes in drug resistance after acid stress, while providing a structural framework for exploring the relationship between bacterial stress responses and antibiotic resistance.

CpxA/R-Controlled Nitroreductase Expression as Target for Combinatorial Therapy against Uropathogens by Promoting Reactive Oxygen Species Generation.

The antibiotic resistances emerged in uropathogens lead to accumulative treatment failure and recurrent episodes of urinary tract infection (UTI), necessitating more innovative therapeutics to curb UTI before systematic infection. In the current study, the combination of amikacin and nitrofurantoin is found to synergistically eradicate Gram-negative uropathogens in vitro and in vivo. The mechanistic analysis demonstrates that the amikacin, as an aminoglycoside, induced bacterial envelope stress by introducing mistranslated proteins, thereby constitutively activating the cpxA/R two-component system (Cpx signaling). The activation of Cpx signaling stimulates the expression of bacterial major nitroreductases (nfsA/nfsB) through soxS/marA regulons. As a result, the CpxA/R-dependent nitroreductases overexpression generates considerable quantity of lethal reactive intermediates via nitroreduction and promotes the prodrug activation of nitrofurantoin. As such, these actions together disrupt the bacterial cellular redox balance with excessively-produced reactive oxygen species (ROS) as "Domino effect", accelerating the clearance of uropathogens. Although aminoglycosides are used as proof-of-principle to elucidate the mechanism, the synergy between nitrofurantoin is generally applicable to other Cpx stimuli. To summarize, this study highlights the potential of aminoglycoside-nitrofurantoin combination to replenish the arsenal against recurrent Gram-negative uropathogens and shed light on the Cpx signaling-controlled nitroreductase as a potential target to manipulate the antibiotic susceptibility.

Molecular cloning and expression profiles of MnSOD and CAT genes from the turbot Scophthalmus maximus

Manganese superoxide dismutase (MnSOD) and catalase (CAT) could eliminate reactive oxygen species (ROS) and maintain the reduction-oxidation balance in cells. This study aimed to investigate their functions in turbot (Scophthalmus maximus) response to the Vibro anguillarum challenge. SmMnSOD, the full-length liver cDNA of MnSOD from S. maximus, was cloned by fast amplification of cDNA ends (RACE). Sequencing of nucleotides indicated that the SmMnSOD cDNA was 1267 base pairs with a 684-base-pair open reading frame, encoding a 228 amino acid protein with 28 amino acid residues. The SmMnSOD sequence contains MnSOD signatures (DVWEHAYY) and probable N-glycosylation sites (NVT, NHT, and NLS). The deduced sequence of SmMnSOD revealed sequence homology between 85.3% and 92.9% with those of other species. A phylogenetic study found that SmMnSOD clustered with other fish MnSOD, indicating that SmMnSOD was a member of the MnSOD family. The SmMnSOD transcript was discovered by qRT-PCR in the gill, stomach, head-kidney, muscle, liver, intestine, and heart of S. maximus, with the highest expression in the liver. Upon intervention by V. anguillarum, the liver and head kidney transcript levels of SmMnSOD were up-regulated at 12 and 48 h, whereas the temporal expression profiles of the CAT transcript increased at 6 and 24 h. As the pathogenic bacterial stress processing was prolonged to 72 h, the liver and head kidney transcript levels of SmMnSOD and CAT decreased gradually. Thus, SmMnSOD was triggered and may be related to S. maximus’ immunological responses against V. anguillarum.

Identification and characterization of RNA polymerase II (RNAP) C-Terminal domain phosphatase-like 3 (SlCPL3) in tomato under biotic stress.

Bacterial diseases are a huge threat to the production of tomatoes. During infection intervals, pathogens affect biochemical, oxidant and molecular properties of tomato. Therefore, it is necessary to study the antioxidant enzymes, oxidation state and genes involved during bacterial infection in tomato. Different bioinformatic analyses were performed to conduct homology, gene promoter analysis and determined protein structure. Antioxidant, MDA and H2O2 response was measured in Falcon, Rio grande and Sazlica tomato cultivars. In this study, RNA Polymerase II (RNAP) C-Terminal Domain Phosphatase-like 3 (SlCPL-3) gene was identified and characterized. It contained 11 exons, and encoded for two protein domains i.e., CPDCs and BRCT. SOPMA and Phyre2, online bioinformatic tools were used to predict secondary structure. For the identification of protein pockets CASTp web-based tool was used. Netphos and Pondr was used for prediction of phosphorylation sites and protein disordered regions. Promoter analysis revealed that the SlCPL-3 is involved in defense-related mechanisms. We further amplified two different regions of SlCPL-3 and sequenced them. It showed homology respective to the reference tomato genome. Our results showed that SlCPL-3 gene was triggered during bacterial stress. SlCPL-3 expression was upregulated in response to bacterial stress during different time intervals. Rio grande showed a high level of SICPL-3 gene expression after 72 hpi. Biochemical and gene expression analysis showed that under biotic stress Rio grande cultivar is more sensitive to Pst DC 3000 bacteria. This study lays a solid foundation for the functional characterization of SlCPL-3 gene in tomato cultivars. All these findings would be beneficial for further analysis of SlCPL-3 gene and may be helpful for the development of resilient tomato cultivars.

Species-specific CgCaspase-Cg-5 in the pacific oyster induces haemocyte apoptosis by regulating the mRNA expression of apoptosis-related genes in the early stage of immune response.

Caspases are cysteinyl aspartate-specific proteinases, playing critical roles in apoptotic pathway to induce apoptosis and inflammatory response. In this study, the expanded repertoire of Caspases was revealed in the Pacific oyster Crassostrea gigas, and a total of 30 Caspases were identified from the genomic and stress-induced transcriptomic databases of the Pacific oyster. They were clustered into CgCaspase-2/9, CgCaspase-8/10, CgCaspase-3/6/7, CgCaspase-Cg, and CgCaspase-L. CgCaspase-Cg subgroup was found to be specifically expanded after a positive selection in oyster with average Ka/Ks of 0.50. The mRNA expression of CgCaspase-Cg-5 was found to be obviously induced against various bacterial and viral stimulations or environmental stresses. The relative expression level of CgCaspase-Cg-5 in haemocytes increased and reached the peak at 6h after Vibrio splendidus stimulation, which was 5.57-fold of that in the control group (p<0.01). In the oysters whose CgCaspase-Cg-5 expression was knocked down, the mRNA expression of apoptosis-related genes including CgBcl2, CgBax, CgCaspase3 and CgCaspase9 changed significantly at 12h after V. splendidus stimulation. The expression of CgBax, CgCaspase3 and CgCaspase9 decreased, which was 0.64-fold (p<0.05), 0.53-fold (p<0.05) and 0.62-fold (p<0.01), while the expression of CgBcl2 increased, which was 2.81-fold (p<0.01) of that in the EGFP-dsRNA group, respectively. Meanwhile, the apoptotic rate of haemocytes (1.90±0.71%) significantly decreased compared to that in the EGFP-dsRNA group (5.40±0.72%) (p<0.05), and the histological damages of widened cell spacing, gill filament swelling and loose cytoplasm were observed in the CgCaspase-Cg-5-knockdown oysters after V. splendidus stimulation. Collectively, CgCaspase-Cg subgroup was specifically expanded in oyster and some bivalve species, and species-specific CgCaspase-Cg-5 regulated the mRNA expression of the apoptosis-related genes to induce haemocyte apoptosis in the early stage of immune response. This provided insight into the evolutionary and functional characteristics of Caspase repertoire in the Pacific oyster and highlighted the important role of CgCaspase-Cg-5 in the response to pathogen infection and environmental stresses.