Abstract:[Background] Misgurin is an important component of nonspecific immune defense system in Misgurnus anguillicaudatus. It has broad spectrum and strong antimicrobial ability. Therefore, it is necessary to obtain large quantities of antimicrobial peptides. [Objective] To achieve high-efficient expression of misgurin. [Methods] Misgurin gene was cloned into expression vector pPIC9K in order to construct recombinant expression plasmid pPIC9K-misgurin. Using Sal I restriction endonuclease digest linearization scheme, the recombinant expression vector was integrated into the chromosome of Pichia pastoris SMD1168 by electric shock method. The positive single colonies was screened through minimal dextrose (MD) solid medium, which was inoculated MD liquid medium at 30 °C, 200 r/min shake flask for 96 h before transfer to BMMY liquid medium to induce expression, adding 5% methanol every 24 h. By comparing the diameter of bacteriostasis circle of Escherichia coli and Staphylococcus aureus, strain pPIC9K-misgurin-22 with the better antimicrobial activity was screened out and inoculated to a 100 L of fermentor induction for 48 h and then detected of activity. [Results] This active substance of pPIC9K-misgurin-22 strain was identified as misgurin by Tricine-SDS-PAGE protein gel detection and mass spectrometry analysis. In comparison with shaking flask fermentation, the bioavailability of Escherichia coli and Staphylococcus aureus in fermentor fermentation increased by 1.47 fold, 1.43 fold, respectively; misgurin has weak antimicrobial activity to Acinetobacter baumannii and Salmonella. It has no antimicrobial activity to Clostridium perfringens and probiotics (Bacillus subtilis, Enterococcus faecalis, Lactobacillus), and has no obvious hemolytic activity; When the temperature reaches 90 °C, the antimicrobial activity of fermentation broth is obviously weakened, the pH value of fermentation broth at 1.0?12.0, everyone has antimicrobial activity; The antimicrobial activity is weakened after adding trypsin, pepsin and protease K. [Conclusion] A strain producing misgurin is obtained, which has high expression and industrialized production potential in Pichia pastoris.

Abstract:[Objective] We studied the hemolytic activity of Vibrio alginolyticus strains, and the distribution of a hemolysin gene vah in these strains; we analyzed the functions of vah gene and the related promoter, and assessed their contributions to the hemolytic activity. [Methods] Hemolytic test was performed using 47 V. alginolyticus strains, including one type strain 1.1587 and 46 environmental strains isolated from seawater and various marine animals. The difference of hemolytic ability was compared among a wild V. alginolyticus strain ZJ051 that showed hemolytic activity, a recombinant Escherichia coli BL21 aiming at a predicted vah gene, a vah-deletion mutant, and a vah-recovery strain. The distribution of vah gene was further detected in V. alginolyticus followed by the sequence analysis of vah genes and their promoters among the hemolytic and non-hemolytic strains. [Results] Among the V. alginolyticus strains 47.8% showed hemolytic activities, and thus hemolysis was ubiquitous in environmental V. alginolyticus strains. Hemolysis was observed in the recombinant Escherichia coli BL21 and the vah-recovery strain but not in the vah-deletion mutant. The vah gene widely distributed in V. alginolyticus. The vah sequences of V. alginolyticus strains were highly similar, and they shared identical amino acid sequences. Through the vah promoter comparison between the hemolytic and non-hemolytic strains, a difference in 188-190 base sites of the promoters was discovered. [Conclusion] Hemolysis is directly caused by the vah gene, and the discrepancy of hemolytic ability among various V. alginolyticus strains has no relation with the difference of vah sequences, which is likely determined by the diversity in 188-190 base sites of vah promoters.

Abstract:[Background] Vibrio parahemolyticus is an important food-borne pathogen causing severe threats to public health. Toxin-antitoxin (TA) systems are widely distributed in the genomes of bacteria and archaea, and possess important biological functions. [Objective] To identify novel TA systems in V. parahemolyticus, and lay the foundation for exploring the underlying mechanism of bacterial pathogenicity and drug resistance from the aspect of TA systems. [Methods] The putative chromosomal type II TA systems in V. parahemolyticus were predicted by a web-based tool. The toxic effects of the putative toxins on Escherichia coli and the antitoxic effects of the corresponding antitoxins were assessed by growth curves analyses and spot dilution assays. Reverse transcription PCR was used to determine whether the genes encoding the toxin and antitoxin were co-transcribed. The homologous proteins of the newly identified TA system were determined by bioinformatics analysis. The regulation of their own promoters by the antitoxin and antitoxin-toxin complex was detected by LacZ reporter assay. [Results] Six putative chromosomal TA systems were identified in V. parahemolyticus. The product of the vp1820 gene (VP1820) had bactericidal activity against E. coli, which could be counteracted by the product of the vp1821 gene (VP1821). The genes vp1821 and vp1820 are co-transcribed. The vp1821-vp1820 locus encodes the YefM-YoeB TA system. The YefM antitoxin positively regulates the promoter, while the YefM-YoeB complex negatively regulates the promoter. [Conclusion] This study identifies a novel type II TA system in V. parahemolyticus, namely YefM-YoeB, and lays the foundation for further research on the role of this system in the pathogenicity and drug resistance of V. parahemolyticus.

Abstract:[Background] Diseases caused by aquatic pathogens continue to break out, and finding safe and effective alternatives to antibiotics is an urgent need. Probiotics are often influenced by interests and profit-making, whereas not enough attention has been paid to their safety evaluation. [Objective] To find green and safe probiotics with multiple bacteriostatic activities based on the phenotypic and genetic characteristics of Bacillus subtilis in China’s mariculture system. [Methods] Taking 37 B. subtilis isolated from China’s mariculture system from 2009 to 2021 as the research object, this study used K-B method to detect the resistance of B. subtilis to different antibiotics. The amylase, protease, and hemolytic capacity of B. subtilis were determined by medium plate method. Polymerase chain reaction (PCR) was used to detect the risk of carrying genes related to hemolysis in B. subtilis. The Oxford cup method was used to determine its bacteriostatic effects on six pathogens, including Vibrio parahaemolyticus, Vibrio algaelyticus, Edwardsiella tarda, Vibrio harveyi, Pseudoalteromonas sp., and Photobacterium damselae. The safety of candidate probiotic B. subtilis was evaluated. [Results] The results of drug susceptibility test showed that 37 isolates of B. subtilis showed strong resistance to trimethoprim, pipemidic acid, and streptomycin, moderate resistance to sulfadiazine, low resistance to cefotaxime, ciprofloxacin, and sulbactam, and complete sensitivity to clarithromycin, norfloxacin, florfenicol, flumequine, cotrimoxazole, and tetracycline. The test results of protease and amylase activity showed that 37 isolates of B. subtilis hydrolyzed casein and starch to varying degrees. The hemolytic test results showed that 4/37 isolates of B. subtilis had hemolytic phenomenon, while 8 hemolysis-related genes were detected in 37 isolates of B. subtilis. The analysis of the correlation between hemolytic phenotype and detection gene showed that there was no direct correlation between the strain that produced hemolysis and its hemolytic gene carrier. The analysis of bacteriostatic experiments showed that all isolates of B. subtilis had inhibitory effect on 2 or more pathogenic bacteria, and 2 isolates (strains Bs4 and Bs7) had good bacteriostatic effects on 6 pathogenic bacteria. Safety experiments on Litopenarus vanmamei showed that strain Bs4 had high safety against L. vanmamei, and the survival rate of 7 d was 100%. [Conclusion] Through the comparative analysis of the physiological metabolic phenotype, genetic characteristics, and pathogen antagonism characteristics of B. subtilis isolates, it is revealed that B. subtilis has diversified phenotypes and genetic characteristics in China’s mariculture system. An ecologically safe and probiotic B. subtilis with multiple bacteriostatic activities is screened out, which provides a theoretical basis and technical support for the prevention and control of aquaculture diseases, the development of bacteriostatic microecological preparations, and the healthy and green development of aquaculture industry.

Abstract:Vibrio parahemolyticus is a saltophilic and important food-borne pathogen which widely exists in all kinds of seafood. The general method costs more time and has low specificity in isolating and identificating of V. parahemolyticus. In order to enhance the efficiency and shorten the detection periods, a new chromogenic medium (HKMVPM) was designed in this study. The sensitivity, specificity and detection efficiency of HKMVPM were studied and compared with that of KHVPM and TCBS by detecting the reference strains and natural samples. The results showed that the selectivity and specificity of HKMVPM were same as that of KHVPM. The detection efficiency of HKMVPM was better than that of KHVPM. The detection efficiency of KHVPM was better than that of TCBS. V. parahemolyticus chromogenic medium shows a high degree of higher sensitivity and specificity and has a good prospect in applying.

Abstract:[Objective] Antibiotic resistance is a severe public health problem. Recently, the relationship between bacterial metabolic regulation and antibiotic efficacy has been proposed in several pathogens. In the present study, we explored the effect of adding additional chemicals on drug susceptibility of Vibrio parahaemolyticus. [Methods] We studied the phenotypic change and compared the survival of V. parahemolyticus after treatment with antibiotics and different chemicals. The experimental results were conformed with carbonylcyanide-p-chlorophenyl hydrazine (CCCP). [Results] These results indicated that specific chemicals effectively potentiate kanamycin to eliminate multidrug resistant V. parahemolyticus. Among them, the addition of exogenous glucose could significantly enhance the sterilization ability with subinhibitory concentrations of kanamycin, but without effect on other antibiotics. The enhancement of antimicrobial susceptibility of Vibrio parahaemolyticus caused by exogenous chemicals could be eliminated after treatment with CCCP. [Conclusion] The sensitivity of V. parahemolyticus to aminoglycosides can be improved by regulating the metabolism of bacterial cells. Therefore, we present a novel approach in fighting against multidrug resistant V. parahemolyticus with practical application value.

Abstract:[Objective] We established the mutation library of pathogenic Vibrio parahaemolyticus (Vp), analyzed the phenotype characteristic of hemolytic ability changed mutations, to provide the research basis for deeper exploration and understanding of the regulation system and mechanism of tdh. [Methods] In this study, the Mini-Tn5-Km2 was inserted randomly into the genome of pathogenic Vp (ATCC 33846) by biparental mating. The conjugants (KmRAmpS) were investigated using thiosulfate citrate bile salts sucrose agar medium with kanamycin (Km) and ampicillin (Amp) respectively. After detection of the Km gene by polymerase chain reaction, library of mutations was established, which the Tn5 was inserted in different gene locus randomly. The hemolytic activity was evaluated with human blood agar. Different characters of these conjugants were evaluated, including growth rate, biofilm formation and motility ability. [Results] Library of mutations was successfully established including 490 mutants. After evaluating hemolytic activity, the phenotype of 5 strains was changed, including 2 up-regulated strains and 3 down-regulated strains. There was significant difference between the 5 conjugants and parent strain on growth rate, biofilm formation and motility ability. Among 3 down-regulated strains, the biofilm formation ability of 2 conjugants was increased (P<0.05). The residual 3 strains demonstrated the significant difference (P<0.05) on growth rate, biofilm formation ability and motility, comparing with their parent strain. [Conclusion] Tn5 transposon can be used to establish the library of Vp mutations; Hemolytic ability of Vp is related to the phenotype; The five conjugants will be contributed on investigation of regulation system and mechanism of tdh in further study.

Abstract:[Objective] To study the transcriptional regulation of vopT by AphA in Vibrio parahemolyticus. [Methods] Total RNAs were extracted from the wide-type (WT) strain and the aphA mutant (ΔaphA). Primer extension assay was employed to detect the transcription start site and the promoter activity of vopT in WT, and that in ΔaphA. Quantitative RT-PCR was also applied to calculate the transcriptional variation of target genes between WT and ΔaphA. The entire promoter region of vopT was cloned into the pHRP309 containing a promoterless lacZ gene. The recombinant lacZ reporter plasmid was transformed into WT and ΔaphA, respectively, to measure the promoter activity (the β-Galactosidase activity) of vopT in WT and ΔaphA by using the β-Galactosidase Enzyme Assay System. The over-expressed His-AphA was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham), and the entire promoter region of the target genes was amplified by PCR. Then, the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-AphA to target promoter regions in vitro. [Results] Primer extension assay detected only one transcriptional start site located at 86 bp upstream of vopT, whose transcript was negative regulated by AphA in an indirectly manner. Moreover, RT-PCR and EMSA results showed that the transcription of vtrA was also indirectly controlled by AphA. [Conclusion] AphA repressed the transcription of vopT indirectly, and this indirect inhibition was not dependent on VtrA.