The Effect of the Lysine Acetylation Modification of ClpP on the Virulence of Vibrio alginolyticus.

Acetylation modification has become one of the most popular topics in protein post-translational modification (PTM) research and plays an important role in bacterial virulence. A previous study indicated that the virulence-associated caseinolytic protease proteolytic subunit (ClpP) is acetylated at the K165 site in Vibrio alginolyticus strain HY9901, but its regulation regarding the virulence of V. alginolyticus is still unknown. We further confirmed that ClpP undergoes lysine acetylation (Kace) modification by immunoprecipitation and Western blot analysis and constructed the complementation strain (C-clpP) and site-directed mutagenesis strains including K165Q and K165R. The K165R strain significantly increased biofilm formation at 36 h of incubation, and K165Q significantly decreased biofilm formation at 24 h of incubation. However, the acetylation modification of ClpP did not affect the extracellular protease (ECPase) activity. In addition, we found that the virulence of K165Q was significantly reduced in zebrafish by in vivo injection. To further study the effect of lysine acetylation on the pathogenicity of V. alginolyticus, GS cells were infected with four strains, namely HY9901, C-clpP, K165Q and K165R. This indicated that the effect of the K165Q strain on cytotoxicity was significantly reduced compared with the wild-type strain, while K165R showed similar levels to the wild-type strain. In summary, the results of this study indicate that the Kace of ClpP is involved in the regulation of the virulence of V. alginolyticus.

DUF1127-containing protein and ProQ had opposite effects on biofilm formation in Vibrio alginolyticus.

The RNA binding protein is crucial for gene regulation at the post transcription level. In this study, functions of the DUF1127-containing protein and ProQ, which are RNA-binding proteins, were revealed in Vibrio alginolyticus. DUF1127 deletion increased the ability of biofilm formation, whereas ProQ deletion reduced the amount of biofilm. Moreover, extracellular proteinase secretion was significantly reduced in the DUF1127 deletion strain. ProQ, not DUF1127-containing protein, can help the cell to defense oxidative stress. Deletion of DUF1127 resulted in a higher ROS level in the cell, however, ProQ deletion showed no difference. RNA-seq unveiled the expression of genes involved in extracellular protease secretion were significantly downregulated and biofilm synthesis-related genes, such as rbsB and alsS, were differentially expressed in the DUF1127 deletion strain. ProQ affected the expression of genes involved in biofilm synthesis (flgC and flgE), virulence (betB and hutG), and oxidative stress. Moreover, the DUF1127-containing and ProQ affected the mRNA levels of various regulators, such as LysR and BetI. Overall, our study revealed that the DUF1127-containing protein and ProQ have crucial functions on biofilm formation in V. alginolyticus.

Identification of the global regulatory roles of RraA via the integrative transcriptome and proteome in Vibrio alginolyticus.

Bacterial ribonuclease E (RNase E) is vital for posttranscriptional regulation by degrading and processing RNA. The RraA protein inhibits RNase E activity through protein-protein interactions, exerting a global regulatory effect on gene expression. However, the specific role of RraA remains unclear. In this study, we investigated rraA expression in Vibrio alginolyticus ZJ-T and identified three promoters responsible for its expression, resulting in transcripts with varying 5'-UTR lengths. During the stationary phase, rraA was significantly posttranscriptionally inhibited. Deletion of rraA had no impact on bacterial growth in rich medium Luria-Bertani broth with salt (LBS) but resulted in decreased biofilm formation and increased resistance to polymyxin B. Transcriptome analysis revealed 350 differentially expressed genes (DEGs) between the wild type and the rraA mutant, while proteome analysis identified 267 differentially expressed proteins (DEPs). Integrative analysis identified 55 genes common to both DEGs and DEPs, suggesting that RraA primarily affects gene expression at the posttranscriptional level. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis demonstrated that RraA facilitates the conversion of fatty acids, propionic acid, and branched-chain amino acids to acetyl-CoA while enhancing amino acid and peptide uptake. Notably, RraA positively regulates the expression of virulence-associated genes, including those involved in biofilm formation and the type VI secretion system. This study expands the understanding of the regulatory network of RraA through transcriptome analysis, emphasizing the importance of proteomic analysis in investigating posttranscriptional regulation.IMPORTANCERraA is an inhibitor protein of ribonuclease E that interacts with and suppresses its endonucleolytic activity, thereby playing a widespread regulatory role in the degradation and maturation of diverse mRNAs and noncoding small RNAs. However, the physiological functions and associated regulon of RraA in Vibrio alginolyticus have not been fully elucidated. Here, we report that RraA impacts virulence-associated physiological processes, namely, antibiotic resistance and biofilm formation, in V. alginolyticus. By conducting an integrative analysis of both the transcriptome and proteome, we revealed the involvement of RraA in carbon metabolism, amino acid catabolism, and transport, as well as in the type VI secretion system. Collectively, these findings elucidate the regulatory influence of RraA on multiple pathways associated with metabolism and pathogenesis in V. alginolyticus.

Vibrio alginolyticus PEPCK Mediates Florfenicol Resistance through Lysine Succinylation Modification.

Protein succinylation modification is a common post-translational modification (PTM) that plays an important role in bacterial metabolic regulation. In this study, quantitative analysis was conducted on the succinylated proteome of wild-type and florfenicol-resistant Vibrio alginolyticus to investigate the mechanism of succinylation regulating antibiotic resistance. Bioinformatic analysis showed that the differentially succinylated proteins were mainly enriched in energy metabolism, and it was found that the succinylation level of phosphoenolpyruvate carboxyl kinase (PEPCK) was highly expressed in the florfenicol-resistant strain. Site-directed mutagenesis was used to mutate the lysine (K) at the succinylation site of PEPCK to glutamic acid (E) and arginine (R), respectively, to investigate the function of lysine succinylation of PEPCK in the florfenicol resistance of V. alginolyticus. The detection of site-directed mutagenesis strain viability under florfenicol revealed that the survival rate of the E mutant was significantly higher than that of the R mutant and wild type, indicating that succinylation modification of PEPCK protein may affect the resistance of V. alginolyticus to florfenicol. This study indicates the important role of PEPCK during V. alginolyticus antibiotic-resistance evolution and provides a theoretical basis for the prevention and control of vibriosis and the development of new antibiotics.

The Ugd, a capsular polysaccharide synthesis protein, regulates the bacterial motility in Vibrio alginolyticus.

Vibrio alginolyticus is one of the most common opportunistic pathogens in marine animals and humans. In this study, A transposon mutation library of the V. alginolyticus E110 was used to identify motility-related genes, and we found three flagellar and one capsular polysaccharide (CPS) synthesis-related genes were linked to swarming motility. Then, gene deletion and complementation further confirmed that CPS synthesis-related gene ugd is involved in the swarming motility of V. alginolyticus. Phenotype assays showed that the Δugd mutant reduced CPS production, decreased biofilm formation, impaired swimming ability, and increased cytotoxicity compared to the wild-type strain. Transcriptome analysis showed that 655 genes (15%) were upregulated and 914 genes (21%) were downregulated in the Δugd strain. KEGG pathway and heatmap analysis revealed that genes involved in two-component systems (TCSs), chemotaxis, and flagella assembly pathways were downregulated in the Δugd mutant. On the other hand, genes involved in pathways of human diseases, biosynthesis ABC transporters, and metabolism were upregulated in the Δugd mutant. The RT-qPCR further validated that ugd-regulated genes are associated with motility, biofilm formation, virulence, and TCSs. These findings imply that ugd may be an important player in the control of some physiological processes in V. alginolyticus, highlighting its potential as a target for future research and potential therapeutic interventions.

LKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by Vibrio alginolyticus infection.

LKB1 (liver kinase B1) is a key upstream kinase of AMPK and plays an important role in various cellular activities. While the function and mechanism of LKB1 have been widely reported in the study of tumor, there are few reports on its role in bacterial infectious diseases, especially in shrimp. In the present study, molecular characterization revealed that LvLKB1 has an open reading frame (ORF) of 1266 bp encoding 421 amino acids with a molecular weight of about 48 KDa, including the kinase region, N-terminal regulatory domain and C-terminal regulatory domain. LvLKB1 in hepatopancreas and hemocytes was significantly upregulated after infection with Vibrio alginolyticus (V. alginolyticus). After silencing LvLKB1 gene in Litopenaeus vannamei (L. vannamei) and artificially infecting V. alginolyticus, the survival rate of L. vannamei was significantly decreased. Subsequently, it was found that the expression of inflammatory factors in hepatopancreas and hemocytes of shrimp was up-regulated, and the expression of lipid oxidation factors was decreased after silencing LKB1, leading to the phenomenon of lipid accumulation in hepatopancreas. In order to explore the mechanism, autophagy levels of shrimp were detected after silencing LKB1, which showed that autophagy levels in hepatopancreas and hemocytes were significantly reduced. Further studies conclusively showed that silencing LvLKB1 inhibited AMPK phosphorylation induced by V. alginolyticus infection, thereby activating TOR pathway and inhibiting autophagy in shrimp. These results indicate that LvLKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by V. alginolyticus infection.

Roles of linker region flanked by transmembrane and peptidoglycan binding region of PomB in energy conversion of the Vibrio flagellar motor.

The flagellar components of Vibrio spp., PomA and PomB, form a complex that transduces sodium ion and contributes to rotate flagella. The transmembrane protein PomB is attached to the basal body T-ring by its periplasmic region and has a plug segment following the transmembrane helix to prevent ion flux. Previously we showed that PomB deleted from E41 to R120 (Δ41-120) was functionally comparable to the full-length PomB. In this study, three deletions after the plug region, PomB (Δ61-120), PomB (Δ61-140), and PomB (Δ71-150), were generated. PomB (Δ61-120) conferred motility, whereas the other two mutants showed almost no motility in soft agar plate; however, we observed some swimming cells with speed comparable for the wild-type cells. When the two PomB mutants were introduced into a wild-type strain, the swimming ability was not affected by the mutant PomBs. Then, we purified the mutant PomAB complexes to confirm the stator formation. When plug mutations were introduced into the PomB mutants, the reduced motility by the deletion was rescued, suggesting that the stator was activated. Our results indicate that the deletions prevent the stator activation and the linker and plug regions, from E41 to S150, are not essential for the motor function of PomB but are important for its regulation.

Exogenous pyruvate promotes gentamicin uptake to kill antibiotic-resistant Vibrio alginolyticus.

OBJECTIVES: Elucidating antibiotic resistance mechanisms is necessary for developing novel therapeutic strategies. The increasing incidence of antibiotic-resistant Vibrio alginolyticus infection threatens both human health and aquaculture, but the mechanism has not been fully elucidated. METHODS: Here, an isobaric tags for relative and absolute quantification (iTRAQ) functional proteomics analysis was performed on gentamicin-resistant V. alginolyticus (VA-RGEN) and a gentamicin-sensitive strain in order to characterize the global protein expression changes upon gentamicin resistance. Then, the bacterial killing assay and bacterial gentamicin pharmacokinetics were performed. RESULTS: Proteomics analysis demonstrated a global metabolic downshift in VA-RGEN, where the pyruvate cycle (the P cycle) was severely compromised. Exogenous pyruvate restored the P cycle activity, disrupting the redox state and increasing the membrane potential. It thereby potentiated gentamicin-mediated killing by approximately 3000- and 150-fold in vitro and in vivo, respectively. More importantly, bacterial gentamicin pharmacokinetics indicated that pyruvate enhanced gentamicin influx to a degree that exceeded the gentamicin expelled by the bacteria, increasing the intracellular gentamicin. CONCLUSION: Thus, our study suggests a metabolism-based approach to combating gentamicin-resistant V. algonolyticus, which paves the way for combating other types of antibiotic-resistant bacterial pathogens.

Deletion of ArmPT, a LamB-like protein, increases cell membrane permeability and antibiotic sensitivity in Vibrio alginolyticus.

Vibrio bacterial species are dominant pathogens in mariculture animals. However, the extensive use of antibiotics and other chemicals has increased drug resistance in Vibrio bacteria. Despite rigorous investigative studies, the mechanism of drug resistance in Vibrio remains a mystery. In this study, we found that a gene encoding LamB-like outer membrane protein, named ArmPT, was upregulated in Va under antibiotic stress by RT-qPCR. We speculated that ArmPT might play a role in Va's drug resistance. Subsequently, using ArmPT gene knockout and gene complementation experiments, we confirmed its role in resistance against a variety of antibiotics, particularly kanamycin (KA). Transcriptomic and proteomic analyses identified 188 and 83 differentially expressed genes in the mutant strain compared with the wild-type (WT) before and after KA stress, respectively. Bioinformatic analysis predicted that ArmPT might control cell membrane permeability by changing cadaverine biosynthesis, thereby influencing the cell entry of antibiotics in Va. The higher levels of intracellular reactive oxygen species and the infused content of KA showed that antibiotics are more likely to enter the Va mutant strain. These results uncover the drug resistance mechanism of Va that can also exist in other similar pathogenic bacteria.

Rapid visual nucleic acid detection of Vibrio alginolyticus by recombinase polymerase amplification combined with CRISPR/Cas13a.

Vibrio alginolyticus (V. alginolyticus) is a common pathogen in the ocean. In addition to causing serious economic losses in aquaculture, it can also infect humans. The rapid detection of nucleic acids of V. alginolyticus with high sensitivity and specificity in the field is very important for the diagnosis and treatment of infection caused by V. alginolyticus. Here, we established a simple, fast and effective molecular method for the identification of V. alginolyticus that does not rely on expensive instruments and professionals. The method integrates recombinase polymerase amplification (RPA) technology with CRISPR system in a single PCR tube. Using this method, the results can be visualized by lateral flow dipstick (LFD) in less than 50 min, we named this method RPA-CRISPR/Cas13a-LFD. The method was confirmed to achieve high specificity for the detection of V. alginolyticus with no cross-reactivity with similar Vibrio and common clinical pathogens. This diagnostic method shows high sensitivity; the detection limit of the RPA-CRISPR/Cas13a-LFD is 10 copies/µL. We successfully identified 35 V. alginolyticus strains from a total of 55 different bacterial isolates and confirmed their identity by (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS). We also applied this method on infected mice blood, and the results were both easily and rapidly obtained. In conclusion, RPA-CRISPR/Cas13a-LFD offers great potential as a useful tool for reliable and rapid diagnosis of V. alginolyticus infection, especially in limited conditions.

Genome Sequencing-Based Mining and Characterization of a Novel Alginate Lyase from Vibrio alginolyticus S10 for Specific Production of Disaccharides.

Alginate oligosaccharides prepared by alginate lyases attracted great attention because of their desirable biological activities. However, the hydrolysis products are always a mixture of oligosaccharides with different degrees of polymerization, which increases the production cost because of the following purification procedures. In this study, an alginate lyase, Alg4755, with high product specificity was identified, heterologously expressed, and characterized from Vibrio alginolyticus S10, which was isolated from the intestine of sea cucumber. Alg4755 belonged to the PL7 family with two catalytic domains, which was composed of 583 amino acids. Enzymatic characterization results show that the optimal reaction temperature and pH of Alg4755 were 35 °C and 8.0, respectively. Furthermore, Alg4755 was identified to have high thermal and pH stability. Moreover, the final hydrolysis products of sodium alginate catalyzed by Alg4755 were mainly alginate disaccharides with a small amount of alginate trisaccharides. The results demonstrate that alginate lyase Alg4755 could have a broad application prospect because of its high product specificity and desirable catalytic properties.

Ion selectivity and rotor coupling of the Vibrio flagellar sodium-driven stator unit.

Bacteria swim using a flagellar motor that is powered by stator units. Vibrio spp. are highly motile bacteria responsible for various human diseases, the polar flagella of which are exclusively driven by sodium-dependent stator units (PomAB). However, how ion selectivity is attained, how ion transport triggers the directional rotation of the stator unit, and how the stator unit is incorporated into the flagellar rotor remained largely unclear. Here, we have determined by cryo-electron microscopy the structure of Vibrio PomAB. The electrostatic potential map uncovers sodium binding sites, which together with functional experiments and molecular dynamics simulations, reveal a mechanism for ion translocation and selectivity. Bulky hydrophobic residues from PomA prime PomA for clockwise rotation. We propose that a dynamic helical motif in PomA regulates the distance between PomA subunit cytoplasmic domains, stator unit activation, and torque transmission. Together, our study provides mechanistic insights for understanding ion selectivity and rotor incorporation of the stator unit of the bacterial flagellum.

Interaction of FlhF, SRP-like GTPase with FliF, MS ring component assembling the initial structure of flagella in marine Vibrio.

Vibrio alginolyticus forms a single flagellum at its cell pole. FlhF and FlhG are known to be the main proteins responsible for the polar formation of single flagellum. MS-ring formation in the flagellar basal body appears to be an initiation step for flagellar assembly. The MS-ring is formed by a single protein, FliF, which has two transmembrane (TM) segments and a large periplasmic region. We had shown that FlhF was required for the polar localization of Vibrio FliF, and FlhF facilitated MS-ring formation when FliF was overexpressed in Escherichia coli cells. These results suggest that FlhF interacts with FliF to facilitate MS-ring formation. Here, we attempted to detect this interaction using Vibrio FliF fragments fused to a tag of Glutathione S-transferase in E. coli. We found that the N-terminal 108 residues of FliF, including the first TM segment and the periplasmic region, could pull FlhF down. In the first step, signal recognition particle (SRP) and its receptor are involved in the transport of membrane proteins to target them, which delivers them to the translocon. FlhF may have a similar or enhanced function as SRP, which binds to a region rich in hydrophobic residues.

Modeling and optimization of antibacterial effect of lichen-associated bacteria, Bacillus subtilis KSRLAB3 strain against marine fouling bacteria, Vibrio alginolyticus.

One of the most commonly occurring bacteria, Bacillus subtilis, can produce a wide variety of secondary metabolites. In this study, the antimicrobial effect of B. subtilis KSRLAB3 against Vibrio alginolyticus was optimized using the Plackett-Burman design (PBD) method, response surface methodology (RSM), and genetic algorithm (GA). Initially, the effects of carbon source, nitrogen source, NaCl concentration, pH, temperature, and incubation time on antimicrobial effects were studied. Among the carbon and nitrogen sources investigated, mannose and peptone elicited maximum antimicrobial effect. Using PBD, the most significant variables that influence the antimicrobial effect were identified, including incubation time, peptone concentration, and temperature. The optimum conditions required for attaining maximum antimicrobial effect was identified using the RSM-GA hybrid method, and the optimum condition includes 49.999 h of incubation time, 4.39 g/L of peptone concentration, and 27.629°C of incubation temperature. The confirmatory experiments performed around the optimum condition showed a zone of inhibition of 35 ± 0.52 mm. Methanolic extract also proved the presence of antibacterial lipopeptide surfactin. Therefore, the RSM-GA hybrid method was successfully used in this study to model the antimicrobial effect of B. subtilis KSRLAB3 against V. alginolyticus. The effective inhibition of V. alginolyticus can be investigated further for the development of antifouling coatings.

Measurements of the Ion Channel Activity of the Transmembrane Stator Complex in the Bacterial Flagellar Motor.

The bacterial flagellum is driven by a rotational motor located at the base of the flagellum. The stator unit complex conducts cations such as protons (H+) and sodium ions (Na+) along the electrochemical potential across the cytoplasmic membrane and interacts with the rotor to generate the rotational force. Escherichia coli and Salmonella have the H+-type stator complex, which serves as a transmembrane H+ channel that couples H+ flow through an ion channel to torque generation whereas Vibrio and some Bacillus species have the Na+-type stator complex. In this chapter, we describe how to measure the ion conductivity of the transmembrane stator complex over-expressed in E. coli cells using fluorescent indicators. Intensity measurements of fluorescent indicators using either a fluorescence spectrophotometer or microscope allow quantitative detection of changes in the intracellular ion concentrations due to the ion channel activity of the transmembrane protein complex.

Purification of the Na+-Driven PomAB Stator Complex and Its Analysis Using ATR-FTIR Spectroscopy.

The flagellar motor of marine Vibrio is driven by the sodium-motive force across the inner membrane. The stator complex, consisting of two membrane proteins PomA and PomB, is responsible for energy conversion in the motor. To understand the coupling of the Na+ flux with torque generation, it is essential to clearly identify the Na+-binding sites and the Na+ flux pathway through the stator channel. Although residues essential for Na+ flux have been identified by using mutational analysis, it has been difficult to observe Na+ binding to the PomAB stator complex. Here we describe a method to monitor the binding of Na+ to purified PomAB stator complex using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. This method demonstrates that Na+-binding sites are formed by critical aspartic acid and threonine residues located in the transmembrane segments of PomAB.

Hidden Resistances: How Routine Whole-Genome Sequencing Uncovered an Otherwise Undetected blaNDM-1 Gene in Vibrio alginolyticus from Imported Seafood.

Vibrio alginolyticus causes vibriosis of marine vertebrates, invertebrates, and humans, and while there have been several reports of multidrug resistance in V. alginolyticus, carbapenem resistance is rare. V. alginolyticus strain AUSMDU00064140 was isolated in Melbourne, Australia, from imported prawns. Routine genomic surveillance detected the presence of a full-length blaNDM-1 gene, subsequently shown to be collocated with additional acquired antimicrobial resistance genes on a resistance cassette on the largest chromosome, flanked by mobilization gene annotations. Comparisons to a previously described V. alginolyticus plasmid, pC1349, revealed differing gene content and arrangements between the resistance cassettes. Phylogenetic analysis was performed against a local and global data set (n = 109), demonstrating that AUSMDU00064140 was distinct and did not cluster with any other strains. Despite the presence of the complete blaNDM-1 gene and positive phenotypic assays for carbapenemase production, carbapenem MICs were low (meropenem MIC ≤0.5 mg/liter). However, it is still possible that this gene may be transferred to another species in the environment or a host, causing phenotypic carbapenem resistance and presenting a risk of great public health concern. IMPORTANCE Carbapenems are last-line antimicrobials, vital for use in human medicine. Antimicrobial resistance determinants such as blaNDM (New Delhi metallo-ß-lactamase producing) genes conferring resistance to the carbapenem class of antimicrobials, are typically found in Enterobacterales (first described in 2009 from a Klebsiella pneumoniae isolate). Our study shows that Vibrio alginolyticus isolated from cooked prawn is able to harbor antimicrobial resistance (AMR) genes of public health concern, specifically a chromosomally located blaNDM-1 gene, and there is the potential for transmission of resistance genes. This may be linked with antimicrobial use in low- and middle-income settings, which has typically been high, unregulated, or not reported. Many countries, including Thailand, have implemented national strategic plans to incorporate the World Health Organization (WHO)'s Global Action Plan (2015) recommendations of a global One Health approach, including increased resources for surveillance of antimicrobial usage and AMR; however, efficient antimicrobial surveillance systems incorporating genomic and phenotypic testing of isolates are still lacking in many jurisdictions.

The hematopoietic cytokine Astakine play a vital role in hemocyte proliferation and innate immunity in Scylla paramamosain.

Astakine may induce hematopoietic response in crustaceans, as it is necessary for hemocyte proliferation. In this study, we produced the recombinant Scylla paramamosain Astakine (rspAstakine) and assessed its immunomodulatory function. We analyzed its amino acid sequences and generated a three-dimensional model, then ligand binding sites and enzyme commission of spAstakine were predicted. The rspAstakine was identified at 21.3 kDa by Western blot and liquid chromatography-mass spectrometry. The results showed that rspAstakine induced proliferation of hemocytes in mud crab in vivo and in vitro. The expression of immune-related genes was up-regulated after rspAstakine treatment, similarly to the immunity-related parameters, activities of superoxide dismutase, phenoloxidase, lysozyme, and peroxidase. Additionally, the intracellular content of reactive oxygen species was higher in the rspAstakine treatment group than PBS group. The rspAstakine also enhanced the rate of phagocytosis, while reduced the apoptosis rate of hemocytes after Vibrio alginolyticus infection. The mortalities of the V. alginolyticus only group and rspAstakine + V. alginolyticus group were 83.3 % and 58.3 %, respectively, which illustrated that rspAstakine plays a protective role against V. alginolyticus infection in S. paramamosain. Our results demonstrate the potential of Astakine to enhance the proliferation and immunomodulatory function of hemocytes in crustaceans.

Cloning and expression of two anti-lipopolysaccharide factors in Eriocheir hepuensis under Vibrio alginolyticus-induced stress.

Anti-lipopolysaccharide factors (ALFs) are small basic proteins that exhibit broad-spectrum antiviral properties and antibacterial activity. In this research, we cloned and studied two Eriocheir hepuensis ALFs, EhALF2 and EhALF3. The results showed that the open reading frame lengths of EhALF2 and EhALF3 were 363 and 372 bp, encoding 120 and 123 amino acids, respectively. Their sequences both contained an Lipopolysaccharide-binding (LPS) domain and were highly similarity to other crab ALFs. qRT-PCR showed that EhALF2 and EhALF3 were detected in nine examined tissues and were expressed the highest in the haemocytes. After challenge with Vibrio alginolyticus, in the hepatopancreas, the expression levels of EhALF2 and EhALF3 reached the highest levels at 48 and 3 h, respectively. In the heart, the expression levels of the two genes were highest at 12 h. These results indicate that EhALF2 and EhALF3 could participate in the resistance of E. hepuensis to V. alginolyticus stress within a short time. They have potential applications in the study of environmental stress markers and disease-resistance factors in E. hepuensis.

Transposon insertion sequencing analysis unveils novel genes involved in luxR expression and quorum sensing regulation in Vibrio alginolyticus.

Vibrio alginolyticus is an important conditional pathogen of fish, shrimp, shellfish, and other marine aquaculture animals that causes huge economic losses to the marine aquaculture industries. Temperature has a significant influence on its quorum sensing (QS) system, which is essential for its various physiological functions. Using transposon insertion sequencing (Tn-seq) technology, we identified 218 putative regulatory factors of LuxR, the master regulator of QS in V. alginolyticus. In addition to established regulators, novel regulatory factors involved in LuxR expression are related to multiple processes. OmpH, 00189, TolC, VscY, and NirD are validated upstream regulatory factors of LuxR. Interestingly, OmpH and 00189 repress luxR expression at lower temperatures and activate its expression at higher temperatures. In contrast, TolC, VscY, and NirD enhance luxR expression at lower temperatures but suppress it at higher temperatures. Moreover, the abovementioned regulators are essential for QS-associated phenotypes, including Asp yields, motility, and biofilm formation, in temperature-dependent or temperature-independent manners. Thus, these novel regulators appear to relay various physiological signals in addition to temperature, effecting population phenotype modifications via QS regulation and warranting future investigation into the underlying mechanisms of opportunistic outbreaks of vibriosis.