• Volume 58,Issue 7,2018 Table of Contents
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    • Research progress in the mechanism of IL-1α maturation and secretion

      2018, 58(7):1151-1157. DOI: 10.13343/j.cnki.wsxb.20170411

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      Abstract:Interleukin 1 alpha (IL-1α) is a cytokine of the interleukin 1 family which is existed in many kinds of cells. It is a widespread multiple functional signaling molecule. As an important transcription factor inside the cell, IL-1α can regulate cell growth and differentiation; and secreted extracellular IL-1α can be involved in the host inflammatory responses. Furthermore, IL-1α plays distinct roles in the occurrence and development of cancer and various diseases. Although both the proform and the cleaved form of IL-1α are biologically active, the activity of cleaved form is more enhanced. IL-1α is a potential candidate target for the prevention and treatment of various diseases. Therefore, researchers have paid much attention to the mechanism of IL-1α maturation and secretion. Recent studies have shown that the maturation and secretion of IL-1α are modulated by intracellular calcium concentration and calpain activation. Inflammasome and other signaling pathways might also be involved. In this review, the latest research progresses in the structure and function, the mechanism of maturation and secretion of IL-1α and the relevant diseases are discussed.

    • Roles of Salmonella effectors in manipulating host cell function

      2018, 58(7):1158-1166. DOI: 10.13343/j.cnki.wsxb.20170494

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      Abstract:Salmonella is an intracellular bacterial pathogen that infects both humans and animals. After a long time of hard struggle, Salmonella has evolved numerous mechanisms to evade host immune defenses, and finally accomplished the process of invasion and replication. Although some of the host targets manipulated by Salmonella effectors have been identified, the interaction between Salmonella and host cells remains unclear. This review summarizes the functions and mechanisms of Salmonella effectors in regulating the host cell signaling pathways, including cytoskeletal changes, inflammatory responses, membrane modifications and vacuolar trafficking.

    • >RESEARCH ARTICLES
    • Spatial dynamics of yeast community and its relationship with environmental factors in Lhalu Wetland, Tibet

      2018, 58(7):1167-1181. DOI: 10.13343/j.cnki.wsxb.20170311

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      Abstract:[Objective] We studied the yeast diversity in Lhalu Wetland and analyzed the influence of environmental factors on yeast community. [Methods] Yeasts were isolated by in-situ cultivation and analyzed by the D1/D2 domain of large subunit (26S) ribosomal RNA and morphological characterization. [Results] In total 169 yeast isolates were identified to 15 genera and 31 species. Ustilentyloma graminis and Filobasidium magnumgenus were the dominating species in the wetland. Moreover, Naganishia, Ustilentyloma, Filobasidium and Cystofibasidium were the dominating genera. COD was significantly positive related to yeast abundance, and it had significant influence on Ustilentyloma. [Conclusion] Yeasts were abundant and diverse among various sites in Lhalu Wetland.

    • Quantitative proteomics analysis of isoniazid, streptomycin mono-drug resistant and poly-drug resistant clinical isolates of Mycobacterium tuberculosis

      2018, 58(7):1182-1190. DOI: 10.13343/j.cnki.wsxb.20170335

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      Abstract:[Objective] To investigate the differentially expressed proteins between isoniazid, streptomycin mono-and poly-drug resistant clinical isolate strains of Mycobacterium tuberculosis. [Methods] Cellular proteins were extracted from isoniazid, streptomycin mono-and poly-drug resistant clinical isolates M. tuberculosis and H37Rv. Differentially expressed proteins were identified through isobaric tags for relative and absolute quantification (iTRAQ) combined with Nano LC-MS/MS technology. The biological function and interaction among differentially expressed proteins and isoniazid or streptomycin were analyzed by DAVID 6.7 and STITCH 5.0, respectively. [Results] 58 differentially expressed proteins were identified in isoniazid, streptomycin mono-drug resistant strains and isoniazid, streptomycin poly-drug resistance strain compared with the proteomic profiles of H37Rv. The biological function of differentially expressed proteins mainly relates to oxidoreductase and transferase activity. Compared with isoniazid and streptomycin poly-drug resistance strain, two proteins (Rv2986c and Rv1908c) were up-regulated over 1.25 folds and two proteins (Rv3133c and Rv0577) were down-regulated less than 0.7 fold in isoniazid, streptomycin mono-drug resistant strains. Bioinformatics predicted that the four proteins interact with isoniazid and streptomycin directly or indirectly. [Conclusion] The expressed level or the interactions together of Rv2986c, Rv1908c, Rv3133c and Rv0577 is likely to be related to isoniazid and streptomycin resistance in M. tuberculosis.

    • Effect of ccpA gene on the production of aminopeptidase in Bacillus cereus

      2018, 58(7):1191-1201. DOI: 10.13343/j.cnki.wsxb.20170352

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      Abstract:[Objective] We constructed Bacillus cereus CZ ccpA deletion strains to explore its effect on carbon metabolism and aminopeptidase production. [Methods] Through homologous recombination mediated by the temperature-sensitive plasmid pKSV7, we acquired the mutant strain CZΔccpA successfully. We also constructed ccpA gene revertant strain CZ1 for phenotypic validation of knockout strains. We compared the metabolic differences of three strains cultured with different carbon source and optimized aminopeptidase fermentation conditions. [Results] The ccpA deletion strain CZΔccpA and the revertant strain CZ1 were successfully constructed. There was no difference in the growth of the three strains in LB medium. When fermented with sodium citrate or mannan oligosaccharides as a single carbon source, the CZΔccpA showed significant metabolic differences from the CZ and CZ1. Besides, the activity of aminopeptidase increased by 48.25 percent when D-xylose was used as the sole carbon source. [Conclusion] Bacillus cereus CZ ccpA gene affects the production of aminopeptidase by regulating the carbon metabolism process and we can raise aminopeptidase production by ccpA gene knockout.

    • Effect of carbon sources on production of 2,4-diacetylphoroglucinol in Pseudomonas fluorescens 2P24

      2018, 58(7):1202-1222. DOI: 10.13343/j.cnki.wsxb.20170356

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      Abstract:[Objective] Many environmental factors, such as carbon sources, regulate the biosynthesis of antimicrobial compounds and influence the biocontrol capacity of Pseudomonas fluorescens. P. fluorescens 2P24 protects various crop plants against root diseases caused by plant pathogens. Among a range of antimicrobial compounds secreted by 2P24, 2,4-diacetylphloroglucinol (2,4-DAPG) is the major determinant of its biocontrol potential. This study investigated the impact of exposing strain 2P24 to selected carbon sources on the production of 2,4-DAPG. [Methods] Antifungal activity of strain 2P24 was tested on potato infusion agar with different carbon sources against Rhizoctonia solani. The reporter strain 2P24 (p970Gm-phlAp) was subjected to a random mini-Tn5 insertion mutagenesis. The collection of Tn5 insertion mutants was then screened for improved phlA expression on potato infusion agar with fructose. [Results] Strain 2P24 cultured on potato infusion with glucose strongly inhibited the growth of R. solani, whereas no inhibition was observed on potato infusion agar with or without fructose. Five mutants with significantly increased phlA expression were identified and the interrupted locus in one of them was identified as the cheB gene. Genetic analysis showed that the expression of phlA and the production of phloroglucinol (PG) were strongly increased in the cheB mutant as compared with the parental strain.[Conclusion] Carbon resources influenced the expression of phlA and 2,4-DAPG production and some genetic factors involved in carbon sources to regulate the production of 2,4-DAPG.

    • Characterizations of three diazotrophic Paenibacillus spp. and their effect on Chinese pakchoi yield and soil enzyme activities

      2018, 58(7):1223-1223. DOI: 10.13343/j.cnki.wsxb.20170357

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      Abstract:[Objective] Nitrogen-fixing (diazotrophic) bacteria are considered possible alternative to nitrogen fertilizers for promoting plant growth and yield. This study was to run a phylogenetic analysis and determine the PGPR traits of the isolated diazotrophic bacteria and characterize the effect of diazotrophic bacteria on Chinese pakchoi yield and soil enzyme activities.[Methods] We isolated 30 diazotrophic bacteria from rhizosphere of wheat (11 isolates), Chinese pakchoi (16 isolates), and lotus (3 isolates) on N-free medium plates. Based on 16S rRNA sequence analysis, the dominant diazotrophic bacteria of wheat, Chinese pakchoi, and lotus belonged to the genus Paenibacillus. [Results] Three multi-function diazotrophic bacteria designated as Paenibacillus spp. P-4, W-7 and L-3 were screened from these 30 diazotrophic. The nitrogenase activities of P-4, W-7 and L-3 were higher than the that of Azotobacter chroococcum used as the control. Strains P-4, W-7 and L-3 can inhibit the growth of two or three plant pathogens of Sclerotinia sclerotiorum, Gibberella zeae and Verticillium dahliae. Strain W-7 has also the phosphate solubilizing ability. Inoculation with Paenibacillus spp. W-7 or L-3 significantly increased the shoot fresh weight of Chinese pakchoi and changed the activity of soil sucrase, phosphatase, and catalase under field conditions, whereas inoculation with Paenibacillus sp. P-4 had no significant effect on plant growth or enzyme activity. [Conclusion] Paenibacillus sp. W-7 and L-3 have good potential to promote plant yield and improve soil quality.

    • Effect of oral feeding maternal fecal microbiota on intestinal microbiota development of newborn piglets

      2018, 58(7):1224-1232. DOI: 10.13343/j.cnki.wsxb.20170363

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      Abstract:[Objective] Fecal microbiota transplantation (FMT) has been used in human intestinal disease, however, information on the use of FMT in newborn piglets is still limited. In this work, we investigated the effects of oral feeding maternal fecal intervention on the development of intestinal microbiota in newborn piglets. [Methods] Twelve Duroc×Landrace×Large piglets from the same litter were randomly divided into the feces treatment and control groups. Each piglet in the feces treatment group was fed with 3 mL fecal inoculation solution at the ages of 1 to 5 days. Piglets in the control group were treated with equivalent saline. Piglets' fecal sample was collected at the ages of 1, 3, 5, 7, 10, 14, 18 and 22 days for the microbiota analysis by Miseq sequencing. [Results] Oral administration of maternal fecal fluid increased the diversity of gut microbiota in piglets. Principal coordinate analysis showed that the bacterial clusterings from two groups were not separated completely, and the two clusterings from 18-and 22-d piglets closed to the samples of sows. With the growth of piglets, the relative abundance of Proteobacteria was decreased significantly (P<0.05), while Bacteroidetes gradually became one of the predominant phyla. FMT significantly increased the relative abundance of genus Escherichia-Shigella in piglets' fecal samples on day 10, but decreased the abundance of this genus on day 18. The relative abundance of Enterococcus and Prevotella in the feces treatment group was significantly higher than those in the control group on day 18. [Conclusion] During the first 3 days after birth, oral administration of maternal fecal microbes had low effect on the intestinal microbial structure in piglets. The impact of oral maternal fecal solution on intestinal microbial colonization of piglets lasted for a maximum of 10 to 14 days. And the microbial community of piglets was close to the sows' at the age of 22 days.

    • Acetylation reduced the DNA binding activity of Ku protein

      2018, 58(7):1233-1244. DOI: 10.13343/j.cnki.wsxb.20170394

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      Abstract:[Objective] To understand the role of acetylation modification on the activity of Ku protein.[Methods] Acetylated Ku protein and its non-acetylated mutant were expressed and purified using M smegmatis expression system, and then their biochemical activities were compared. The effect of oxidative stress and acidic environment on Ku protein acetylation level were analyzed in M. smegmatis. [Results] Ku protein over-expression M. smegmatis strain grew slower than the control strain transformed with the empty plasmid. Acetylated Ku protein had lower DNA repair activity and DNA binding activity than the non-acetylated Ku. Quantity of Ku protein in M. smegmatis cells under oxidative and acidic stress decreased, whereas there was subtle change of acetylated Ku protein. [Conclusion] Acetylation modification can regulate the DNA binding activity of Ku protein, thus regulate the activity of Non-homologous end joining system. The increase of acetylation level of Ku protein is the response of mycobacteria against the adverse growth environment.

    • Inhibition of antifungal substances from Bacillus amyloquefaciens B15 against Botrytis Cinerea—the agent of “gray mold” of grape

      2018, 58(7):1245-1254. DOI: 10.13343/j.cnki.wsxb.20170395

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      Abstract:[Objective] The present study aimed to investigate the inhibition of cyclic lipopeptides (iturin A and fengycin) produced by Bacillus amyloquefaciens B15 against Botrytis cinerea by using laser confocal scanning microscopy and fluorescence microscopy. [Methods] The agar diffusion method was applied to investigate the antifungal activity of B15 strain against Botrytis cinerea. A 7-mm agar plug of Botrytis cinerea was placed on the PDA plate containing B15 culture supernatant at the concentration of 1:20. The hyphal viability was determined by staining the hyphae of Botrytis cinerea with trypan blue. Then the classical cell apoptotic assay was applied by staining cell nucleus, reactive oxygen species, calcium ions, and phosphatidylserine of hyphae with DAPI, Dihydrorhodamine 123, fluo-3/am and Annexin V-PI probe. [Results] The antagonistic assay showed that the B15 strain displayed evident antifungal activity against Botrytis cinerea. From the trypan blue assay, it was evident to observe the distorted hyphal structure of Botrytis cinerea in treated group, including conglobation of branch tips, swollen hyphae, hyphal vacuolation, and shrinkage under fluorescence microscopy. The laser confocal scanning microscopy observation of Botrytis cinerea showed that the typical cell apoptosis pattern appeared in the treated group, including the nuclear fragmentation and condensation, accumulation in reactive oxygen species and Ca2+, and the flip-flop of phosphatidylserine. [Conclusion] The combined results of the trypan blue assay and cell apoptotic assay indicated that B15 culture supernatant inhibited the growth of Botrytis cinerea in the way of inducing apoptotic-like cell death in Botrytis cinerea, instead of damaging the cell membrane and directly contributing to the death of mycelium.

    • Physiological and transcriptional responses of Actinobacillus succinogenes to acid stress

      2018, 58(7):1255-1265. DOI: 10.13343/j.cnki.wsxb.20170400

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      Abstract:[Objective] We explored by physiological and transcriptional responses of Actinobacillus succinogenes CGMCC1593 to acid stress. [Methods] We studied the effects of different initial pH on cell growth, activity of H+-ATPase and intracellular pH before and after acid stress as well as the protection mechanism of glutamate on CGMCC1593. The transcriptomics technique was used to explore the differentially expressed genes under acid stress. [Results] Cell growth was inhibited and the activity of H+-ATPase decreased with the decrease of initial pH. After acid stress at pH 4.7, the cell membrane was severely damaged. Glutamate had a protective effect on the cells under acid stress. In total 39 genes were differently expressed altogether under acid stress, of which 49% were stress and transport proteins, and a small part was related to metabolism. [Conclusion] Understanding the physiological and transcriptional responses of Actinobacillus succinogenes to acid stress provides references for improving acid resistance of Actinobacillus succinogenes.

    • Analysis of a CRISPR-Cas system and a genomic island, MGIVvuTF3, in complete genome of Vibrio vulnificus TF3

      2018, 58(7):1266-1273. DOI: 10.13343/j.cnki.wsxb.20170402

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      Abstract:[Objective] In order to dig interesting genetic regions and explore whether CRISPR-Cas systems exist, the draft genome of Vibrio vulnificus TF3 was analyzed. [Methods] We isolated V. vulnificus strain TF3 from the diseased shrimp Litopenaeus vannamei, and then obtained its genome sequence by Illumina Miseq sequencing. The genomic annotation showed that there was a CRISPR-Cas system in V. vulnificus TF3, named CRISPR-CasTF3. After further analysis, we found that the CRISPR-CasTF3 located in a genomic island, named MGIVvuTF3. The characteristics and sources of CRISPR-CasTF3 and MGIVvuTF3 were analyzed using bioinformatic methods. [Results] We found the CRISPR-CasTF3 belonged to type I-E CRISPR-Cas system similar to that of Escherichia coli. The CRISPR-CasTF3 contained eight cas genes with the order, cas3-cas8e-cse2-cas6-cas7-cas5-cas1-cas2. CRISPR-CasTF3 had 50 repeats sequences separated by 49 spacer sequences. MGIVvuTF3 featured attL/attR sequences, and key genes participating in site-specific integration, excision, and transfer, and thus it manifested that MGIVvuTF3 represents a genomic island. MGIVvuTF3 had highly similarity to a genomic island, MGIVch0395, found in V. cholerae O395. However, the two genomic islands also had two distinct differences, namely, they had completely different spacer sequences and each of them had several particular predicted genes. [Conclusion] MGIVvuTF3 harboring CRISPR-CasTF3is most likely obtained through horizontal gene transfer, and therefore the CRISPR-CasTF3 system may transfer horizontally by means of MGIVvuTF3.

    • Study of the structure of intestinal microflora in patients with mild hepatic encephalopathy based on probability topic model

      2018, 58(7):1274-1286. DOI: 10.13343/j.cnki.wsxb.20170409

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      Abstract:[Objective] We used Latent Dirichlet Allocation (LDA) probabilistic topic model to analyze the effect of rifaximin combined with probiotics orally taken by patients with mild hepatic encephalopathy (MHE) on structural heterogeneity and clinical efficacy of intestinal microflora. [Methods] We adopted Monte Carlo algorithm for folding Gibbs sampling of LDA probabilistic topic model included in R language to analyze the OTUs (operational taxonomic unit) data set with temporal heterogeneity of MHE patients' intestinal microflora structure. [Results] Using LDA model we divided MHE patients' 42 fecal samples into 3 topics and determined OTUs genus with largest impact on MHE patients' heterostructure of intestinal microflora. They were Escherichia, Bacteroides and Streptococcus. Compared conditions before and after treatment, the variation patterns of 3 species in the group were the genus of the same type. Their variation times and frequency were higher than those of different types of bacteria. Both MHE patients in Rifaximin combined with probiotics treatment group and rifaximin alone treatment group changed the intestinal microflora structure after treatment (P<0.05). In addition, according to the clinical curative effect index:comparing serum IL-2, IL-4, IL-6, IL-10, TNF-α, TBIL, ALT, CRP, NCT-A, γ-GGT and blood ammonia level after treatment, the observation group was obviously superior to the control group with significant differences and the difference has statistical significance (P<0.05). Total effective rate for treatment group was 88.8%, with 22.2% total incidence of adverse reactions; Total effective rate for control group was 75%, with 38.5% total incidence of adverse reactions (P<0.05). [Conclusion] LDA model can effectively quantifying the heterogeneity of flora structure and identify the OTUs with the greatest impact on heterogeneity. Rifaximin combined with probiotics treatment can significantly improve the level of blood ammonia and serum inflammatory factors in patients with MHE. It can also change intestinal microflora structure in MHE patients, which is mainly represented by the reduction of pathogens and the increase of probiotics. As a result, it has good clinical application value.

    • Effects of different probiotics on mice obesity induced by high-fat diet

      2018, 58(7):1287-1297. DOI: 10.13343/j.cnki.wsxb.20170416

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      Abstract:[Objective] To study the effects of different probiotics on mice obesity based on the interaction between microbiota and host metabolism. [Methods] In total 50 C57BL/6J male mice were equally divided into 10 groups and fed with normal feed, high-fat feed and high-fat feed with eight different probiotics compositions (5 billion CFU/mice). All mice were continuously fed for nine weeks and weighted weekly. Fasting blood glucose, GTT, blood fat indexes and organ weight were measured at last week. Microbiota total DNA was extracted from cecum contents and sequenced by 16S rDNA sequencing. [Results] Some probiotic compositions accelerated mice body weight accumulation, whereas some probiotic compositions reduced obesity, decreased visceral fat and relieved hyperlipemia. Compared to the normal feed group, Firmicutes/Bacteroidetes (F/B) ratio was 22.8-fold in accelerated body weight group fed with Danisco probiotic, and none Akkermansia muciniphila (Akkermansia) was detected, while F/B ratio was similar and the Akkermansia content was 0.5% (half of normal feed group) in reduced obesity group fed with Junlading probiotic. [Conclusion] Some probiotics influence mice body weight and metabolism, but different compositions may lead to opposite effects. Specific probiotic composition improves obesity and hyperlipemia possibly by its selected strain characteristics and relative ratio through decreasing F/B ratio and increasing Akkermansia level in gut microbiota. This study contributes to developing metabolism-improving probiotic product in the future.

    • High-throughput screening method for high-yield nisin strain

      2018, 58(7):1298-1308. DOI: 10.13343/j.cnki.wsxb.20170559

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      Abstract:[Objective] Nisin, a bioactive antimicrobial peptide, inhibits many Gram-positive bacteria causing food spoilage and infection. In this study, cost-effective screening method for high-yield nisin strains was developed using high-throughput screening technology to provide a practical method for screening during the industrial production process. [Methods] Mutants (2511) were obtained by applying ultraviolet mutagenesis treatments to Lactococcus lactis ATCC 11454, followed by development of a 96-well plate high-throughput screening method using a Biomek FXP automatic workstation to select mutant strains for culture dilution. Diluted bacterial solutions were transferred to another 96-well plate with Micrococcus luteus grown to logarithmic phase, and an improved turbidimetric method was used to rapidly detect the biological activity of nisin and screen the mutants associated with the high-yield strains. High-yield strains were fermented by shake-flask fermentation to evaluate the screening method. [Results] Turbidimetric detection revealed nisin activity in the range between 10 and 25 IU/mL, with M. luteus biomass detected after a 2 h reaction with nisin. After two rounds of high-throughput screening, about 50 high-yield strains were obtained from the 2511 mutants, with 8 accurately detected by shake-flask fermentation. Results indicated that the nisin yield of the tested strains all increased, with one strain showing a 30% increase in production, indicating the efficacy of the high-throughput screening method. [Conclusion] Our high-throughput screening method was capable of screening high nisin-producing bacteria.

    • Regulation of AI-2/LuxS gene on biofilm formation and spoilage of Shewanella baltica

      2018, 58(7):1309-1321. DOI: 10.13343/j.cnki.wsxb.20170565

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      Abstract:[Objective] Shewanella baltica is a specific spoilage bacterium in marine fish during refrigerated storage. In the present study regulation of quorum sensing AI-2/LuxS on the biofilm formation and spoilage of S. baltica was elucidated. [Methods] LuxS gene in SB11 strain was amplified and knocked out by suicide plasmid. Biofilm formation, adhesion, swimming and amine metabolites between wild strain and △luxS mutant at 4℃ and 28℃ were comparatively measured by using crystal violet staining, bead vortexing, microscopy and HPLC, respectively. [Results] S. baltica SB11 luxS gene was amplified, and bioinformatics analysis revealed that deduced LuxS protein with 169 aa contained conserve His-Thr-Leu-Glu-His (HTLEH) motif and key amino acid functional sites. Three-dimensional structure of LuxS protein was similar to that from various bacteria. Compared with wild strain, supernatant of △luxS mutant lost bioluminance activity, although their growth was the same. △luxS exhibited lower biofilm development and maturation than the wild strain, and decreased by 20.1% and 27.9% at 4℃ for 96 h and at 28℃ for 24 h, respectively. Compared with the wild strain, △luxS reduced adherent cell by 6.48% at 4℃ for 72 h, and 6.57% at 28℃ for 24 h, indicating that the adherence of mutant on steel slide was significantly weak. The observation by fluorescence microscopy showed that the wild strain rapidly adhered to the coverslip and formed a large number of biofilm, whereas, △luxS mutant only seemed to form a flat sparse biofilm and failed to aggregate into clusters. Confocal Laser Scanning Microscope (CLSM) revealed that the thickness of maturing biofilm in the wild and mutant strains was 68.95 μm and 36.44 μm, respectively. Furthermore, the absence of luxS gene significantly promoted the bacterial swimming at 4 and 28℃. However, the production of trimethylamine and putrescine between the wild and mutant strain was similar. [Conclusion] LuxS protein in S. baltica SB11 was conserve, and AI-2/LuxS was involved in biofilm formation, adhesion, swimming, but not a functional signal in the regulation of spoilage potential.

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