Abstract:Vibrio parahaemolyticus, the causative agent of seafood-borne acute gastroenteritis worldwide, benefits from a sessile biofilm lifestyle that enhances survival outside the host, contributes to host colonization and infectivity but also leads to cross-contamination between processing equipment and foods. Insight into the regulatory circuit underlying biofilm formation may propose targeted strategies to interfere with a process that renders this bacterium remarkably adaptable to changing environments. Here, we mainly demonstrated the regulatory of two mobility structures (flagella, pili), extracellular matrix polysaccharides, the second messenger c-di-GMP and quorum-sensing system that contribute to biofilm formation.

Abstract:Norovirus is one of the important foodborne pathogens that cause acute gastroenteritis in humans. Due to the lack of in vitro replication systems and infection models, researchers' understanding of their host's protective immunity has always been limited, leading to greater hindrance in research into controlling viral infections. In recent years, the exogenous expression of viral capsid proteins, the use of surrogate viruses, and the development of volunteer experiments, especially breakthroughs in cell culture models, have made great progress in the study of humoral immunity and cellular immunity. Therefore, this article reviews the innate immunity, humoral immunity, and cellular immune response mechanisms of Norovirus-infected hosts, and looks forward to its future applications in the development of Norovirus candidate vaccines.

Abstract:Quorum sensing (QS) is a ubiquitous cell-cell communication mechanism in which microorganisms secrete and respond to signaling molecules by synthases and regulatory proteins to adapt to environmental changes. Electroactive microorganisms (EAMs) are capable of extracellular electron transfer and have broad application prospects in renewable energy utilization and environmental remediation. Recently, an increasing number of studies have focused on the roles of QS in the extracellular electron transfer of EAMs. In this review, we summarized the effect and mechanisms of QS in EAMs. QS influences pure-culture EAMs through an indirect electron transfer mechanism by stimulating or suppressing electron shuttle production. QS improves the electroactivity of pure-culture EAMs through a direct electron transfer mechanism by stimulating biofilm formation and changing the abundance and structure of the outermost surface proteins. QS enhances the electroactivity of mixed-culture EAMs by promoting biofilm formation and increasing the relative abundance of EAMs inside the biofilm. We also review the construction and application potential of QS and EAM-based AND logic gates and propose future research directions from the perspectives of mechanistic research.

Abstract:[Objective] The regulatory effect of arginine regulator ArgR on the biosynthesis of exopolysaccharides (EPS) was studied in Streptococcus thermophilus. [Methods] ArgR from S. thermophilus was heterologously expressed by Escherichia coli, and purified by urea denaturation refolding and Ni²⁺ affinity chromatography. The interaction and kinetic information between ArgR and eps promoter P_epsA were detected by electrophoretic mobility shift assays (EMSA) and biolayer interferometry (BLI). The yield alteration of EPS was determined by phenol-sulfuric acid assay when the gene argR overexpressed or repressed. [Results] Heterologous expression of ArgR was formed inclusion body, and 2.95 mg/mL of soluble protein was achieved by urea denaturation refolding. EMSA and BLI analysis showed that ArgR can specifically bind with the promoter P_epsA and their affinity was high because of the low dissociation. Increased expression of argR gene reduced EPS synthesis and the suppression raised. [Conclusion] It is the first time to report that ArgR can specifically bind the promoter of eps gene cluster and negatively regulate EPS synthesis in S. thermophilus.

Abstract:[Objective] To study Cd-adsorbing bacteria for fixing soil Cd. [Methods] Tolerance of microbe against Cd was measured in the beef extract peptone liquid medium containing Cd²⁺ to select cadmium-resistant strains. The cadmium-tolerant bacteria were identified by 16S rRNA gene similarity and phylogenetic analysis, The adsorption efficiency of bacteria for Cd²⁺ was determined by adding bacteria cells into the solution containing CdCl₂. Soil culture simulation experiment was performed to analyze the effects of cadmium adsorption bacteria on cadmium-contaminated soil by determining soil pH, alkali-hydrolyzed nitrogen, available phosphorus, available potassium, organic matter, CEC, available Cd and the number of microorganisms. [Results] Total of 57 bacteria strains isolated from the rhizosphere soil of Houttuynia cordata at Deyang showed different degrees of resistance to Cd²⁺. Three dominant Cd-resistant bacteria strains were Providencia DY8, Bacillus DY3 and Bacillus dy1-4. They showed good adsorption efficiency to Cd²⁺ in liquid. The adsorption efficiency of Cd²⁺ decreased with the Cd²⁺ concentration increasing. DY8, DY3 and DY1-4 reduced the effective Cd content in cadmium-contaminated soil by 72.11%, 68.55% and 62.32%, respectively, and significantly increased the alkali-dissolved nitrogen and available phosphorus in cadmium-contaminated soil. [Conclusion] The Cd-contaminated farmland soil contains rich resources of Cd-resistant microorganisms. Cd-absorbing bacteria can reduce the content of effective Cd in soil and improve soil nutrient conditions.

Abstract:With increasing emphasis on potato production, the planting scale and professional degree of potato grow very fast in recent years in China. However, ever-increasing application intensity of chemical fertilizers and pesticides, together with succession cropping, leads to more and more serious soil-borne diseases representing by potato common scab. In some areas, the incidence of soil-borne diseases even reaches 90% causing huge economic loss to farmers. [Objective] To monitor the changes of soil bacterial population due to different planting patterns and intensities of fertilization, analyze the relationship between occurrence of soil-borne diseases and change of soil-environment, provide theoretical basis for effectively controlling soil-borne diseases of potato. [Methods] Soil rhizosphere samples were collected from three distinct potato fields continuously cropped for one-year in Xiji (Ningxia, northwest), three-year in Guyuan (Hebei, north) and five-year in Hailar (Inner Mongolia, northeast) with low, high and rare incidence, respectively, of soil borne diseases. Then, high-throughput sequencing technology was used to comparably analyze the bacterial community structure and diversity of these samples. [Results] A total of 617558 effective reads and 3077 sortable operating units (OTUs) were obtained from the 3 groups of samples. Among them, the dominant component was Proteobacteria with a proportion over 33%. Compared with the soil samples from the field without occurrence of potato soil-borne diseases, the bacterial abundance and diversity decreased significantly in the soil samples from the field with high incidence of potato soil-borne diseases. Meanwhile, augmentation and reduction of the relative abundance of pathogenic and probiotic bacteria, respectively, also were observed. Specifically, we found the relative abundance of Actinobacteria increased on a large scale, whereas that of Proteobacteria, Chloroflexi and Acidobacteria decreased dramatically. Moreover, we found that composition changes and quantities of some bacteria (especially Actinomycetes) were closely related to the total phosphorus content of soil. [Conclusion] Excessive application of chemical fertilizers and perennial continuous cropping affected the soil bacterial community and damaged soil ecological environment thus resulting in potato soil-borne diseases eventually. In addition, phosphorus content change may be one of the important factors that alter community structure of soil microbes.

Abstract:[Objective] To use xylan as co-substrate to enhance cofactor recycling in chiral catalytic reaction, we constructed a fusion expression system containing (S)-carbonyl reductase (SCRII) from Candida parapsilosis CCTCC M203011, glucose dehydrogenase mutant Ala258Phe (Ala258Phe/GDH) from Bacillus sp. YX-1, and xylanase 2 from Trichoderma reesei Rut C-30 in Escherichia coli BL21. The recombinant E. coli strains efficiently catalyzed 2-hydroxyacetophenone to (S)-phenyl-1,2-ethanediol. [Methods] By adjusting the 3 encoding genes' locations in the pET-28a, 2 recombinant plasmids pET-SCRII-A258F-XYN2 and pET-A258F-SCRII-XYN2 were constructed by using the overlap extension PCR technology. The optimal temperature, pH value and the best ratio of 2-hydroxyacetophenone and co-substrate xylan for catalyzing (S)-phenyl-1,2-ethanediol by the recombinant E. coli/pET-SCRII-A258F-XYN2 and E. coli/pET-A258F-SCRII-XYN2 were determined. [Results] Through the optimization of pH, temperature and the ratios between substrate and co-substrate, the recombinant E. coli/pET-SCRII-A258F-XYN2 produced (S)-phenyl-1,2-ethanediol with a yield of 98.8% (W/W) under the optimal conditions:35℃, pH 7.0 and a 1:1 substrate-co-substrate ratio, while the recombinant E. coli/pET-A258F-SCRII-XYN2 produced (S)-phenyl-1,2-ethanediol with a yield of 95.6% (W/W) under the optimal conditions:35℃, pH 7.0 and a 2:1 substrate-co-substrate ratio. The two recombinant strains catalyzed (S)-phenyl-1,2-ethanediol with an optical purity >99%. [Conclusion] In the fusion expression system containing three enzymes, xylanase and glucose dehydrogenase mutant mediated cofactor regeneration was introduced into asymmetric biosynthesis reactions, which efficiency improved chiral biotransformation. This work supplied a more solid foundation by using the naturally abundant xylan for chiral catalysis.

Abstract:[Objective] The aim of this study was to isolate venturicidins from Streptomyces sp. NO1W98 and identify their biosynthetic gene cluster.[Methods] The secondary metabolites were extracted from enlarged-scale fermentation broth of Streptomyces sp. NO1W98 with organic solvent, purified with normal and reverse phase silica gel chromatography and structure elucidated with spectroscopic approaches. The genomic DNA of Streptomyces sp. NO1W98 was extracted and sequenced by Illumina Hiseq technology. Gene cluster of venturicidins (vtd) was preliminarily located by bioinformatic analysis and verified by gene disruption and trans complementation. Different in fermentation extracts between Streptomyces sp. NO1W98 wild type and related mutants were analyzed with HPLC. [Results] Two macrolides, venturicidins A and B were isolated and their structures were identified from Streptomyces sp. NO1W98. Draft genome of Streptomyces sp. NO1W98 harbors 49 proposed secondary metabolite biosynthetic gene clusters, including the vtd located in Region 3.3 of scaffold 3 that is responsible for the biosynthesis of venturicidins. The vtd was preliminarily characterized by gene disruption of vtdA1, orf(-1), orf(+1) and then verified by gene complementation of vtdA1 to ΔvtdA1. The vtd was found to include 6 PKS skeleton genes, 5 transporter genes, 2 regulator genes and 9 post-PKS tailoring genes. [Conclusion] The isolation and identification of venturicidins and their biosynthetic gene cluster from Streptomyces sp. NO1W98 provide the basis for future genetic engineering and strain improvement.

Abstract:[Objective] To enhance the catalytic activity of alanine racemase from Salmonella typhimurium by error-prone PCR. [Methods] A mutant library of alanine racemase from S. typhimurium was constructed by error-prone PCR using plasmid pTrc99A-StAlr or pTrc99A-Y343H as template, and DNA recombination with improved catalytic activity were screened by serine auxotroph strain UT5028. Racemase activities for converting both L-alanine to D-alanine and L-serine to D-serine were calculated based on the absorbance at 550 nm using Epoch Microplate Spectrophotometer. The cell lysate was separated by gel filtration chromatography, and each fraction was detected for the alanine racemase activity to analyze the oligomerization states. Kinetic parameters of StAlr and mutants were determined by measuring the total amount of L/D-alanine or L/D-serine by high performance liquid chromatography with a spectrofluorometer. [Results] Three mutants Y343H, A3VY343H and A3V with improved catalytic activities were obtained by two rounds of error-prone PCR and site-directed mutagenesis, separately. Based on the kinetic parameters, the mutant Y343H only displayed a 2.01 and 3.68-fold improvement in catalytic efficiency (k_cat/K_m) towards L/D-serine compared to the wild type StAlr, while the mutant A3V showed a distinct reduction in K_m value and dramatic increase in k_cat and k_cat/K_m values towards L/D-alanine and L/D-serine. For substrate L-alanine and D-alanine, the k_cat/K_m values of A3V were 105.51 and 97.36-fold of that of wild type StAlr, whereas for L-serine and D-serine, the k_cat/K_m values of A3V were 4.63 and 10.73-fold of that of wild type StAlr. Gel filtration chromatography revealed that only the mutant A3V eluted as two distinct peaks at a very low amount of protein, which may correspond to the dimeric and monomeric form, respectively. As the amount of protein increased, the oligomerization states of all proteins were gradually shifted from monomeric to dimeric form. These indicated that the monomer-dimer transition of mutant A3V might be much faster than that of protein StAlr and A3VY343H. [Conclusion] The residue A3 located at N-terminus of StAlr might be a key residue for its catalytic activity and oligomerization state.

Abstract:[Objective] The effects of walnut shell extracts (WSE) on the growth and lipid accumulation of Monoraphidium sp. QLZ-3 were explored. [Methods] Different volumes of WSE were added to BG-11 medium (all nutrients in BG-11 were retained in the medium). [Results] The biomass and lipid productivity reached (534.7±4.07) mg/(L·d) and (296.4±15.36) mg/(L·d) with addition of 40% WSE in the culture medium, increasing 14.82% and 33.50%, respectively, with upregulation of protein and downregulation of carbohydrate. [Conclusion] Exogenous WSE could enhance the biomass and lipid productivities in microalgae.

Abstract:[Objective] The purpose of this test is to elucidate the pathogenicity and molecular epidemic characteristics of chicken-derived Escherichia coli, and to provide new ideas for exploring reasonable ways to prevent and control E. coli. [Methods] Livers of dead chicken samples were collected in Hebei Province from 2018 to 2019, and the isolated strains were systematically identified through selection of media selection, biochemical identification, and serum agglutination test. Detection of virulence genes in isolates was conducted by PCR. Cluster analysis of E. coli was performed with reference to phylogenetic classification. Multi-sequence typing analysis of housekeeping gene sequences with reference to 7 databases provided on the McMLST website database. [Results] The results show that 56 isolates conformed to the biochemical characteristics of E. coli were divided into 8 biochemical phenotypes. Among them, B4 (30.36%), B5 (25%), and B2 (23.21%) were the main biochemical phenotypes. 56 isolates of E. coli were positive for serum agglutination test, divided into 11 serotypes. O₇₈ (26.79%), O₂ (23.21%), O₁₅₇ (17.86%), and O₁ (14.29%) were the main epidemic serotypes. A total of 15 E. coli virulence genes were detected in 56 strains of E. coli, but no papC, ibeA, and ibeB genes were detected. The gene carrying rate of Adhesion-related gene fimC and antiserum survival factor-related gene ompA in all isolates is 100%. The detection rates of the genes of aatA, yijP, irp2, mat, and iss were 98.21%, 98.21%, 98.21%, 96.43% and 92.86%. The detection rates of iroN, fyuA, iucD and irp2 of E. coli and iron transport-related genes were all above 80%. Of the 56 strains of E. coli, 20 are Enterohemorrhagic E. coli (EHEC), followed by Enteroaggregative E. coli (EAEC) (n=4) and enterotoxigenic E. coli (ETEC) (n=2). These strains have more D group isolates, followed by B2 group. According to MLST typing analysis, there are 22 ST types in total, of which ST88, ST85 and ST243 are the main epidemic types. [Conclusion] The serotypes of E. coli were diverse and the virulence factors were various. The pathogenic E. coli also carried multiple virulence genes, indicating that animal-derived E. coli has a strong virulence basis.

Abstract:[Objective] The foodborne pathogen Listeria monocytogenes is well-adapted outside of the environmental conditions and inside of the host niche by delicately controlling flagellar production. We here aimed to explore the regulatory roles of L. monocytogenes glutathione synthetase (encoded by gshF) in bacterial motility and flagella production.[Methods] The gene knock-out mutant and complemented strains (ΔgshF and CΔgshF) were constructed and then subjected to in vitro bacterial growth and motility assays compared with the wild-type strain EGD-e. In addition, transcriptional changes of the flagella-associated genes were determined for the wild-type and gshF mutant strains using the Real-time Quantitative Reverse Transcription PCR (qRT-PCR) method.[Results] The data showed that in vitro growth ability was not significantly affected by deletion of gshF while the swarming ability of ΔgshF was markedly impaired. More importantly, the transcriptional levels of the flagellar-associated factors GmaR, a protein thermometer that controls temperature-dependent transcription of flagellar genes, and FlaA, encoding flagellin, were most significantly down-regulated at 30℃ in the absence of gshF.[Conclusion] We in this study showed that the glutathione synthetase of L. monocytogenes plays a vital role in controlling transcription of flagellar genes and contributes to bacterial swarming motility and flagellar production. These data would be helpful to better understand the regulatory mechanisms employed by this intracellular pathogen to favor environmental adaption and host infection.

Abstract:[Objective] We investigated the acid tolerance mechanisms of Lactobacillus delbrueckii subsp. bulgaricus by heterologous expression of genes related to acid adaptation regulated by two-component system TCS1 (JN675228/JN675229) of Lb. bulgaricus in Lactococcus lactis subsp. cremoris NZ9000. [Methods] We used reverse transcription polymerase chain reaction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to verify the expression of acid adaptation related genes, such as adenine phosphoribosyltransferase (aprt), D-alanine-D-alanine ligase (ddl), oligopeptide ABC transporter (oppDII) and elongation factor Ts (tsf), regulated by TCS1 of Lb. bulgaricus, in Lc. lactis NZ9000. We validated acid treatment experiment to verify the effect of gene expression on acid stress tolerance of host bacteria. We confirmed the interactions between TCS1 and the expressed genes related to acid adaptation by yeast two hybrid. [Results] Our results show that aprt, ddl, oppDII and tsf were successfully expressed in Lc. lactis NZ9000. The expression of aprt and ddl genes increased the resistance of recombinant strains to acid stress by 75 and 114 times. The expression of oppDII and tsf genes had no significant effect on the acid resistance of the recombinant strains. Yeast two hybrid system showed that there was interaction between histidine protein kinase (HPK1) of TCS1 and Ddl, and HPK1-C domain was the key region of the interaction. [Conclusion] The acid tolerance of strains expressing Aprt and Ddl was significantly higher than that of the control strain. The results of this study can provide references for the strategies of obtaining acid resistance of Lb. bulgaricus and other similar strains.

Abstract:[Objective] In this study, we aimed to characterize the community structure of arsenite-oxidizing bacteria in Xikuangshan of Hunan Province, and the whole genome of an arsenite-antimonite-oxidizing strain Bosea sp. AS-1 (Abbreviation:AS-1). [Methods] Arsenite-oxidizing bacterial strains were isolated from the samples collected in Xikuangshan, and 16S rRNA genes were sequenced for phylogenic analysis. Whole genome of strain AS-1 was sequenced and analyzed using relevant software and databases for genome assembly, gene prediction and functional annotation. [Results] The arsenite-oxidizing bacteria in Xikuangshan were mainly distributed in α-, β-, γ-Proteobacteria and Firmicutes. AS-1's genome contained one circular chromosome with a size of 5.536 Mb and two plasmids of 189.9 kb and 112.1 kb, respectively. Further analysis on AS-1's genomic data reveal many genes related to arsenic and antimony metabolism, as well as flagella formation, flagellar movement, and biofilm formation. These genes may be involved in AS-1's resistance to high level of arsenic and antimony in environment. Besides, several carbon-fixation genes and sulfur-oxidizing genes were also found in the genome of AS-1, suggesting that AS-1 may grow autotrophically and oxidize sulfur. [Conclusion] Subsequent experiments confirmed autotrophic growth of AS-1.

Abstract:[Objective] We studied the diversity and traced the source of microbial community in Pixian broad bean paste and its two semi-fermented products, including broad bean meju and chili moromi. In addition, we also characterized the flavor compounds in Pixian broad bean paste. [Methods] High throughput sequencing was used to explore the microbial community structure and succession. Meanwhile, high throughput liquid chromatography and gas chromatography-mass spectrometry were used to determine the concentrations of nonvolatile and volatile flavor compounds. Multiple bioinformatic methods were used to trace the microbes and flavor compounds in Pixian broad bean paste. [Results] Chili moromi contributes 44% to 59% bacterial communities and 42% to 77% fungal communities to Pixian broad bean paste fermentation. Broad bean meju contributes 5% to 22% bacterial communities and 2% to 18% fungal communities to Pixian broad bean paste fermentation. In addition, 16 bacterial genera were only contributed by chili moromi. Two bacterial genus and two fungal genera are only contributed by broad bean meju. In addition, 1-octen-3-ol, phenylacetaldehyde, isobutyraldehyde, malic acid and furfural were only traced from broad bean meju. Capsaicin, 3-methyl-1-butanol, hexanol and isobutanol were only traced from chili moromi. [Conclusion] Chili moromi contributes the main microbial communities to Pixian broad bean paste, while broad bean meju contributes the main substrates (including amino acids and glucose). Two semi-fermented products, including chili moromi and broad bean meju, contributed their unique microbes and flavor compounds to Pixian broad bean paste, which might be the reason why Pixian broad bean paste has the characteristic aroma and taste.

Abstract:[Objective] Plant pathogenic bacteria can directly deliver the secreted type III effectors (T3SEs) into different sites of host cells through the type III secretion system (T3SS), thereby exercising various pathogenic functions. The purpose of this study was to determine the subcellular localization of the largest T3SE protein XopXccR1 of Xcc 8004 in plants. [Methods] The bioinformatics methods were used to analyze the putative transmembrane region of XopXccR1. To obtain the transient expression Agrobacterium strains, the full-length XopXccR1 without stop codon, the N-terminus (1-1220 aa), and the C-terminus (1221-2030 aa) were ligated to the plant expression vector pCAMBIA-2300-35S::EGFP respectively by homologous recombination. Utilizing Agrobacterium-mediated transient expression to infect Nicotiana benthamiana, the results of subcellular localization were then observed with a laser confocal microscope. [Results] The full-length and the N-terminus of XopXccR1 are mainly localized in the cell membrane of N. benthamiana, while the C-terminus of XopXccR1 in the cytoplasm. [Conclusion] XopXccR1 N-and C-terminus might both have the targeting signals. The targeting signals in the N-terminus of XopXccR1 mainly contributes the final sub-localization of XopXccR1.

Abstract:[Objective] To study the effect of genistein on methicillin-resistant Staphylococcus aureus (MRSA) efflux proteins. [Methods] Combined drug sensitivity test was used to detect the effect of genistein on the sensitivity of MRSA to ciprofloxacin. The isobaric tags for relative and absolute quantitation (iTRAQ) technology were used to detect the change of bacterial cell protein expression after genistein was applied to MRSA41577. The bioinformatics method was used to analyze the significantly different proteins. qPCR and Nile Red efflux assay were used to explore the mechanism of drug resistance-related proteins mediating bacterial resistance. [Results] Combined drug sensitivity test showed that genistein enhanced the sensitivity of MRSA to ciprofloxacin. A total of 129 significantly different proteins were detected by iTRAQ technology, including 60 proteins with up-regulated expression and 69 proteins with down-regulated expression. Bioinformatics analysis showed that there were about 14 proteins related to bacterial resistance, of which PstB, PstS and other proteins mainly mediated bacterial resistance through the active efflux system. qPCR results showed that compared with the control group, PstB and PstS gene expression decreased by 51.6% and 78.6%, respectively. Nile Red efflux assay showed there was a competitive relationship between genistein and Nile Red, so that genistein was a competitive inhibitor of MRSA41577. [Conclusion] Genistein reverses bacterial resistance by reducing mRNA expression of the efflux genes pstB and pstS, and affected the expression of efflux proteins PstB and PstS of MRSA41577. Besides, genistein is also a competitive efflux inhibitor of MRSA41577 and plays antibacterial effect by competing with substrates for efflux, which makes antibacterial drugs stay in the bacteria.

Abstract:[Objective] A gene encoding the esterase to hydrolyze R, S-metalaxyl high enantioseclectively to R-metalaxyl acid was cloned and expressed in E. coli. The properties of the recombinant esterase were studied. [Methods] Based on known N-terminal 10 amino acid sequence of the target esterase, a candidate esterase gene matching with it was found in the sequenced Albibacters sp. zjut528 genome. It had a length of 969 bp encoding 322 amino acids and was named RMest. The RMest DNA segment was amplified by PCR, ligated with pET-28a(+), transformed into E. coli BL21Gold(DE3) to construct the recombinant strain. The recombinant esterase was expressed by induction with IPTG and purified with Ni²⁺ resin. [Results] The recombinant RMesterase was expressed successfully in RMest-pET-28a(+)-E. coli BL21 Gold (DE3). Its size was about 46 kDa in SDS-PAGE. Hydrolysis of 10 g/L of R,S-metalaxyl for 6 h catalyzed by intracellular crude enzyme from the recombinant strain, the substrate conversion rate reached 49.8% with product (metalaxyl acid) ee_p of 99.3%. The main product was R-metalaxyl acid. The optimal temperature and pH of RMestrase was 40℃ and 9.0 respectively. The activity of RMesterase was inhibited by methanol, the other product of metalaxyl hydrolysis. After aligning the amino acid sequence of RMest with other proteins in Uniprot KB database by Blast+, the neighboring-joining phylogenetic tree was constructed. It shows that the esterase RMest had a long evolution distance with homologous Lysophospholipases, AB hydrolase-1 domain-containing proteins and other esterases. That indicates that the esterase RMest was a new one that evolved relative independently. [Conclusion] The esterase gene RMest was successfully cloned and expressed in RMest-pET-28a(+)-E. coli BL21 Gold (DE3). The recombinant RMesterase could hydrolyze R, S-metalaxyl high R-enantioselectively to produce R-metalaxyl acid.

Abstract:[Objective] The aim of this research is to study the temporal and spatial changes of atypical virulence genes and drug resistance of the marine fish pathogen Photobacterium damselae in South China, and analyze the potential environmental factors driving the change of virulence and drug resistance in P. damselae, and consequently provide suggestions for disease prevention and control caused by P. damselae. [Methods] Based on PCR and Kirby-Bauer diffusion method, we analyzed the presence of 5 atypical virulence genes and the resistance to 15 tested antibiotics of 35 P. damselae strains isolated from diseased marine fishes in South China coastal area. [Results] The results show that:(1) 19 strains contained 1-2 atypical virulence genes, particularly, the detection rate of hlyA and vvh were higher than 20%; (2) 27 resistance types were observed in these 35 strains, the multi-antibiotic resistance index of was 0.00-0.67 with the multi-antibiotic resistance rate (one strain shows resistance to more than 3 antibiotics) up to 60.00%. Therein, more than 50% strains showed resistance to vancomycin, amoxicillin, midecamycin and rifampin, but less than 10% strains resisted to gentamicin, norfloxacin, ciprofloxacin, chloramphenicol and florfenicol; (3) The atypical virulence gene contents and drug resistance were marginally increased with the year grows, and more/stronger in Hainan than in Guangdong. Further, the richness of resistance types, multi-antibiotic resistance rate, the most resistant number in one strain, and multi-antibiotic resistance index were observed higher/more in Hainan (1.00, 69.23%, 10, 0.32, respectively) than in Guangdong (0.82, 54.55%, 9, 0.25, respectively). [Conclusion] These results indicated that atypical virulence genes probably be transferred into P. damselae by horizontal gene transfer and the variation of virulence and drug resistance of P. damselae isolated form Hainan and Guangdong are mainly affected by temperature and antibiotic pollution.