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ObjectivesThe purpose of this study was to determine the structural features and transferability of the multidrug-resistance (MDR) plasmid, and resistance phenotypes for the tested antimicrobials in foodborne Vibrio parahaemolyticus.MethodsPlasmids were isolated from a V. parahaemolyticus strain of seafood origin, then sequenced using the Illumina NovaSeq 6000 and PacBio Sequel II sequencing platforms to obtain the complete genome data. Characterization of the MDR plasmid pVP52-1, including determination of antimicrobial resistance genes (ARGs), plasmid incompatibility groups, and transferability, was carried out.ResultsV. parahaemolyticus strain NJIFDCVp52 contained two circular chromosomes and two circular plasmids (pVP52-1 and pVP52-2). Plasmid typing indicated that pVP52-1 belonged to the incompatibility group IncA/C2 and the sequence type pST3. pVP52-1 carried 12 different ARGs, an IS110-composite transposon consisting of aac(6′)-Ib-cr, qnrVC1, aac(6′)-Ib, dfrA14, and the IS26-mphA-IS6100 unit flanked by inverted sequences of IS5075 and IS4321. pVP52-2 carried no ARGs. A plasmid elimination assay showed that only pVP52-1 and its ARGs were lost, the loss of resistance to several antimicrobials, causing a change from the ampicillin-ampicillin/sulbactam-cefazolin-cefoxitin-ceftazidime-cefotaxime-imipenem-trimethoprim/sulfamethoxazole resistance pattern to the ampicillin resistance pattern. In accordance, a conjugation transfer assay showed that only pVP52-1 and its ARGs were horizontally transferred, leading to increased antimicrobial resistance in Escherichia coli strain EC600, causing a change from the ampicillin-nalidixic acid resistance pattern to the ampicillin-ampicillin/sulbactam-cefazolin-cefoxitin-ceftazidime-cefotaxime-imipenem-nalidixic acid-chloramphenicol-tetracycline-trimethoprim/sulfamethoxazole-azithromycin resistance pattern. Further transferability experiments revealed that pVP52-1 could be transferred to other enterobacterial strains of E. coli and Salmonella.DiscussionThis study emphasizes the urgent need for continued surveillance of resistance plasmids and changes in antimicrobial resistance profiles among the V. parahaemolyticus population.

A paper-based surface enhancement of a Raman scattering substrate consisting of silver-nanowires stacked on glass-fiber filter paper was prepared. At the same time, the DNA-embedding molecule Eva Green was introduced as a signaling molecule for surface-enhanced Raman scattering (SERS) detection. Polymerase chain reaction (PCR) was used to amplify target genes and the method was developed into a rapid molecular diagnostic system. The total detection time of the developed detection method was 40 min, including 30 min of PCR amplification and 10 min of SERS measurement. After 30 PCR cycles, bacterial DNA with an initial concentration of 20 fg/μL and a bacterial suspension with an initial concentration of 7.2 × 101 CFUs/mL could be detected. When the enrichment culture time was 4 h, target bacteria with an initial contamination inoculation volume of 1.5 CFUs/mL could be detected in artificially contaminated samples. The method is fast and highly sensitive, and has not been applied to the detection of V. parahaemolyticus.

The ubiquitous presence of antimicrobial-resistant organisms and antimicrobial resistance genes (ARGs) constitutes a major threat to global public safety. Tetracycline (TET) is a common antimicrobial agent that inhibits bacterial growth and is frequently detected in aquatic environments. Although TET may display coselection for resistance, limited knowledge is available on whether and how it might influence plasmid-mediated conjugation. Subinhibitory concentrations (3.9–250 ng/mL) of TET promoted horizontal gene transfer (HGT) via the mobilizable plasmid pVP52-1 from the donor Vibrio parahaemolyticus NJIFDCVp52 to the recipient Escherichia coli EC600 by 1.47- to 3.19-fold. The transcription levels of tetracycline resistance genes [tetA, tetR(A)], conjugation-related genes (traA, traD), outer membrane protein genes (ompA, ompK, ompV), reactive oxygen species (ROS)-related genes (oxyR, rpoS), autoinducer-2 (AI-2) synthesis gene (luxS), and SOS-related genes (lexA, recA) in the donor and recipient were significantly increased. Furthermore, the overproduced intracellular ROS generation and increased cell membrane permeability under TET exposure stimulated the conjugative transfer of ARGs. Overall, this study provides important insights into the contributions of TET to the spread of antimicrobial resistance.

Abstract Background Bacillomycin D-C16 can induce resistance in cherry tomato against pathogens; however, the underlying molecular mechanism is poorly understood. Here, the effect of Bacillomycin D-C16 on induction of disease resistance in cherry tomato was investigated using a transcriptomic analysis. Results Transcriptomic analysis revealed a series of obvious enrichment pathways. Bacillomycin D-C16 induced phenylpropanoid biosynthesis pathways and activated the synthesis of defense-related metabolites including phenolic acids and lignin. Moreover, Bacillomycin D-C16 triggered a defense response through both hormone signal transduction and plant-pathogen interactions pathways, and increased the transcription of several transcription factors (e.g., AP2/ERF, WRKY and MYB). These transcription factors might contribute to the further activated the expression of defense-related genes (PR1, PR10 and CHI) and stimulated the accumulation of H2O2. Conclusion Bacillomycin D-C16 can induce resistance in cherry tomato by activating the phenylpropanoid biosynthesis pathway, hormone signal transduction pathway and plant-pathogen interactions pathway, thus activating comprehensive defense reaction against pathogen invasion. These results provided a new insight into the bio-preservation of cherry tomato by the Bacillomycin D-C16.

Nonribosomal peptide synthase (NRPS) is a unique molecular assembly mechanism with high hybridity. Its recombination is conducive to the development of novel lipopeptides. However, there are few reports on NRPS subunit recombination of plipastatin at present. In this paper, plipastatin synthase was modified by the forward movement of subunit PPSE and the replacement of the communication-mediating (COM) domain. The results showed that ppsABE, a new assembly line, could synthesize novel lipopeptides such as cycle pentapeptide (C16-18β-OHFA-E-O-cyclo(Y-T-I), and its antimicrobial activity against Rhizopus stolonifer and Staphylococcus aureus was better than that of plipastatin. However, the reactivity of ppsABCE disappeared, but the substitution of COMDppsC/COMAppsD or COMDppsD/COMAppsE for COMDppsC/COMAppsE could restore its activity and conduct the biosynthesis of linear hexapeptide (C16-17β-OHFA-E-O-Y-T-E-A/V) and heptapeptide (C17-18β-OHFA-E-O-Y-T-E-A-I). Collectively, these findings indicated that the COM donor domain at the C-terminus of PPSB could communicate with the COM acceptor domain at the N-terminus of PPSE and that the compatible COM domain is an important tool for communication between nonpartner subunits. Moreover, the integrity and selective compatibility of the COM acceptor domain of subunit PPSE are essential to promote the interaction between PPSE and other subunits. This work further complemented the rules of NRPS subunit recombination and provided a theoretical basis for the development of novel high-efficiency lipopeptides.

Objective: This paper mainly studied the effects and regulation of exogenous addition of pyruvate, overexpression of pyruvate kinase and acetyl-CoA synthase on the synthesis of Bacillomycin D. Methods: In this study, B. amyloliquefaciens fmbJ was used as the initial strain. Firstly, the effect of exogenous pyruvate with different concentrations on the yield of Bacillomycin D was detected by HPLC. The regulation of pyruvate in this process was detected by RT-PCR. Then, pyruvate kinase (pyk) and acetyl-CoA synthase (acs) overexpression strains B. amyloliquefaciens fmbJ-pyk and fmbJ-acs were constructed by electrotransformation, and their Bacillomycin D production induced by different concentrations of IPTG was studied. Results: 0.075% pyruvate could increase the yield on Bacillomycin D to 1.41-fold that of the control group. The upregulation of Bacillomycin D synthesis gene also verified this enhancement. In addition, pyruvate could up-regulate the expression of regulatory factors such as comA, comP, comQ, sigH, sigM, degU, degQ, spo0A and codY, down-regulate the expression of rapC and abrB, promote the production of acetyl CoA synthase, and finally jointly promote the synthesis of Bacillomycin D. Under the induction of 50 mg/L IPTG, the yield of Bacillomycin D of fmbJ-acs and fmbJ-pyk increased to 1.16-fold and 1.34-fold that of fmbJ, respectively. Conclusion: This study found that exogenous addition of pyruvate and overexpression of acs or pyk could promote the synthesis of Bacillomycin D, clarified the regulation of pyruvate in the synthesis of Bacillomycin D, and widened a new research idea for promoting the efficient production of Bacillomycin D.

Abstract Xenograft bone scaffolds have certain advantages such as mechanical strength, osteoinductive properties, sufficient source and safety. This study aimed to compare osteogenesis of the two main bovine bone xenografts namely true bone ceramics (TBC) and decalcified bone matrix (DBM), and TBC or DBM combined with bone morphogenetic protein (BMP)-2 (TBC&BMP-2 and DBM&BMP-2). The characteristics of TBC and DBM were investigated by observing the appearance and scanning electron microscopic images, examining mechanical strength, evaluating cytotoxicity and detecting BMP-2 release after being combined with BMP-2 in vitro. The femoral condyle defect and radial defect models were successively established to evaluate the performance of the proposed scaffolds in repairing cortical and cancellous bone defects. General observation, hematoxylin and eosin (HE) staining, mirco-CT scanning, calcein double labeling, X-ray film observation, three-point bending test in vivo were then performed. It indicated that the repair with xenograft bone scaffolds of 8 weeks were needed and the repair results were better than those of 4 weeks whatever the type of defects. To femoral condyle defect, TBC and TBC&BMP-2 were better than DBM and DBM&BMP-2, and TBC&BMP-2 was better than TBC alone; to radial defect, DBM and DBM&BMP-2 were better than TBC and TBC&BMP-2, and DBM&BMP-2 was better than DBM alone. This study has shown that TBC and DBM xenograft scaffolds can be more suitable for the repair of cancellous bone and cortical bone defects for 8 weeks in rats, respectively. We also have exhibited the use of BMP-2 in combination with DBM or TBC provides the possibility to treat bone defects more effectively. We thus believe that we probably need to select the more suitable scaffold according to bone defect types, and both TBC and DBM are promising xenograft materials for bone tissue engineering and regenerative medicine. Graphical abstract

Bacillomycin D is a cyclic lipopeptide produced by Bacillus amyloliquefaciens fmbJ. At present, no relevant report has described the combinatorial biosynthesis of bacillomycin D. Due to the strong biosynthetic potential of the communication-mediating (COM) domains, its crosstalk between NRPS subunits has been studied to some extent, but the interaction of COM domain between modules is rarely reported. Therefore, in this study, we conducted the combinatorial biosynthesis of bacillomycin D through the deletion of the COM donor and acceptor domains between the modules and elucidated the interaction between the NRPS modules. The results showed that the deletion of the donor domain between modules 2 and 3 did not affect catalysis by upstream modules, but prevented downstream modules from catalysing the extension of the lipopeptide product, ultimately resulting in mutant complexes that could form linear dipeptides with the sequence β-NH2FA-Asn-Tyr. However, the engineered hybrid bacillomycin D NRPSs lacking the donor domains between modules 3 and 4 and modules 6 and 7 could form multiple assembly lines that produced bacillomycin D and its analogs (linear tripeptides, cyclic hexapeptides and linear hexapeptides). In addition, all the acceptor domain deletion strains failed to produce bacillomycin D, only truncated peptides produced by module interruption (except for the acceptor domain deletion strains between modules 3 and 4, which also produced cyclic hexapeptides). In conclusion, deletion of the inter-module donor domains led to a more flexible hybrid biosynthetic system for the production of diverse peptide products; compared with the inter-subunit donor domain deletion strains that could only produce truncated peptides, the former had a greater biosynthetic capacity. Meanwhile, the acceptor domains between modules were an important part of module-module interactions and efficient communication within bacillomycin D synthetase.

Filter-feeding oyster is an important food transmission vector for the foodborne norovirus. To investigate the contamination level and genetic diversity of norovirus in commercial oysters in Guangzhou and better assess the risk of consumption, oyster samples were collected from local aquatic market from June 2020 to May 2021 in this study. The contamination content and genotype distribution of virus in shellfish were detected by protease K treatment coupled with polyethylene glycol precipitation small system previously established in the laboratory，including the real-time reverse transcription polymerase chain reaction and the nested RT-PCR. 110 oyster samples were detected, the positive rate of norovirus genogroup II(GII) was 52.7% (58/110), and the contents of virus was 1.56×103~1.09×106 copies/g (digestive gland). The positive rate of norovirus in oysters collected in spring and summer (March to August) was 35.7% (20/56), which was lower than the positive rate of 70.4% (38/54) in autumn and winter (September to February). However, there was no significant difference in content of virus detected in all seasons, in spring, content of virus was (2.69±1.46)×105 copies/g (digestive gland), in summer, content of virus was (1.97±2.16)×105 copies/g (digestive gland), in autumn, content of virus was (6.91±6.16)×104 copies/g (digestive gland), and winter was (4.83±2.99)×104 copies/g (digestive gland). Positive samples were sequenced and analyzed, the result showed that except for one was GII.17 genotype, the rest were all GII.4 genotypes (n=13), which was consistent with the local clinical epidemic genotypes. The results of this study showed that the level of norovirus contamination in commercial oysters of Guangzhou was severe, it is necessary to strengthen control work of prevention for virus, especially to remind consumers to fully process when eating oysters.

Salmonela enterica serovar Typhimurium (S. Typhimurium) is a food-borne pathogen that can form biofilms to increase its resistance to the external environment. Through the detection of biofilm of several S. Typhimurium strains in this study, strain CDC3 with strong biofilm forming capacity and strain CVCC3384 with weak biofilm forming capacity were identified. The genes expressed in planktonic and biofilm cells of two S. Typhimurium strains were analysed by transcriptome sequencing. Results showed that the genes related to the signal transduction pathway were upregulated and genes related to motility were downregulated in strain CDC3. By comparing biofilms and planktonic cells of the two strains, we found that CDC3 regulates biofilm formation mainly through the two-component system kdpABC, while strain CVCC3384 does so mainly through motility and quorum sensing. This study revealed regulation mechanism of biofilms formation between different biofilm forming capacity strains, and provided a theoretical basis for subsequent research.

To extend the application range of L-asparaginase in food pre-processing, the thermostability improvement of the enzyme is essential. Herein, two non-conserved cysteine residues with easily oxidized free sulfhydryl groups, Cys8 and Cys283, of Acinetobacter soli L-asparaginase (AsA) were screened out via consensus design. After saturation mutagenesis and combinatorial mutation, the mutant C8Y/C283Q with highly improved thermostability was obtained with a half-life of 361.6 min at 40 °C, an over 34-fold increase compared with that of the wild-type. Its melting temperature (Tm) value reaches 62.3 °C, which is 7.1 °C higher than that of the wild-type. Molecular dynamics simulation and structure analysis revealed the formation of new hydrogen bonds of Gln283 and the aromatic interaction of Tyr8 formed with adjacent residues, resulting in enhanced thermostability. The improvement in the thermostability of L-asparaginase could efficiently enhance its effect on acrylamide inhibition; the contents of acrylamide in potato chips were efficiently reduced by 86.50% after a mutant C8Y/C283Q treatment, which was significantly higher than the 59.05% reduction after the AsA wild-type treatment. In addition, the investigation of the mechanism behind the enhanced thermostability of AsA could further direct the modification of L-asparaginases for expanding their clinical and industrial applications.

Abstract Background Lipoxygenase (LOX) from Anabaena sp. PCC 7120 (Ana-rLOX) offers important applications in the food industry, especially for improving aroma and dough rheological properties. However, industrial applications of LOXs have been limited by their poor thermostability. Herein, we report a bioinformatics-based consensus concept approach for the engineering of thermostable Ana-rLOX. Results A series of mutations (N130D, G260A, S437T, N130D/G260Q, N130D/S437Y) showed higher thermostability and activity than the wild-type enzyme. Thus, N130D/G260Q exhibited a 6.6-fold increase in half-life and 2.45 °C increase in unfolding temperature; N130D/S437Y showed a 10 °C increase in optimal temperature. The secondary structure did not change much that contributed to improved thermostability were investigated in detail using circular dichroism. Homology modeling suggested that enhanced thermostability and specific activity may result from favorable hydrophobic interactions. Conclusions A series of mutations were achieved, showing higher thermostability and activity than the wild-type enzyme by semi-rational mutagenesis with limited structure information. Our findings provide important new insights into molecular modifications aimed at improving Ana-rLOX thermostability and activity.

Abstract Background Plipastatin, an antifungal lipopeptide, is synthesized by a non-ribosomal peptide synthetase (NRPS) in Bacillus subtilis. However, little information is available on the combinatorial biosynthesis strategies applied in plipastatin biosynthetic pathway. In this study, we applied module or individual domain deletion strategies to engineer the plipastatin biosynthetic pathway, and investigated the effect of deletions on the plipastatin assembly line, as well as revealed the synthetic patterns of novel lipopeptides. Results Module deletion inactivated the entire enzyme complex, whereas individual domain (A/T domain) deletion within module 7 truncated the assembly line, resulting in truncated linear hexapeptides (C16~17β-OHFA-Glu-Orn-Tyr-Thr-Glu-Ala/Val). Interestingly, within the module 6 catalytic unit, the effect of thiolation domain deletion differed from that of adenylation deletion. Absence of the T6-domain resulted in a nonproductive strain, whereas deletion of the A6-domain resulted in multiple assembly lines via module-skipping mechanism, generating three novel types of plipastatin derivatives, pentapeptides (C16~17β-OHFA-Glu-Orn-Tyr-Thr-Glu), hexapeptides (C16~17β-OHFA-Glu-Orn-Tyr-Thr-Glu-Ile), and octapeptides (C16~17β-OHFA-Glu-Orn-Tyr-Thr-Glu-Gln-Tyr-Ile). Conclusions Notably, a unique module-skipping process occurred following deletion of the A6-domain, which has not been previously reported for engineered NRPS systems. This finding provides new insight into the lipopeptides engineering. It is of significant importance for combinatorial approaches and should be taken into consideration in engineering non-ribosomal peptide biosynthetic pathways for generating novel lipopeptides.

L-asparaginase (E.C.3.5.1.1) is a well-known agent that prevents the formation of acrylamide both in the food industry and against childhood acute lymphoblastic leukemia in clinical settings. The disadvantages of L-asparaginase, which restrict its industrial application, include its narrow range of pH stability and low thermostability. In this study, a novel L-asparaginase from Mycobacterium gordonae (GmASNase) was cloned and expressed in Escherichia coli BL21 (DE3). GmASNase was found to be a tetramer with a monomeric size of 32 kDa, sharing only 32% structural identity with Helicobacter pylori L-asparaginases in the Protein Data Bank database. The purified GmASNase had the highest specific activity of 486.65 IU mg−1 at pH 9.0 and 50 °C. In addition, GmASNase possessed superior properties in terms of stability at a wide pH range of 5.0–11.0 and activity at temperatures below 40 °C. Moreover, GmASNase displayed high substrate specificity towards L-asparagine with Km, kcat, and kcat/Km values of 6.025 mM, 11,864.71 min−1 and 1969.25 mM−1min−1, respectively. To evaluate its ability to mitigate acrylamide, GmASNase was used to treat potato chips prior to frying, where the acrylamide content decreased by 65.09% compared with the untreated control. These results suggest that GmASNase is a potential candidate for applications in the food industry.

The soft rot disease caused by Rhizopus stolonifer is an important disease in cherry tomato fruit. In this study, the effect of iturin A on soft rot of cherry tomato and its influence on the storage quality of cherry tomato fruit were investigated. The results showed that 512 μg/mL of iturin A could effectively inhibit the incidence of soft rot of cherry tomato fruit. It was found that iturin A could induce the activity of resistance-related enzymes including phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), glucanase (GLU), and chitinase (CHI), and active oxygen-related enzymes including ascorbate peroxidases (APX), superoxide dismutases (SOD), catalases (CAT), and glutathione reductase (GR) of cherry tomato fruit. In addition, iturin A treatment could slow down the weight loss of cherry tomato and soften the fruit. These results indicated that iturin A could retard the decay and improve the quality of cherry tomato fruit by both the inhibition growth of R. stolonifera and the inducing the resistance.

Staphylococcus aureus is a common food pathogen and has a strong tolerance to environmental stress. Here, the biofilm formation of S. aureus strains after cold stress for 24 weeks were investigated. It was found that the biofilm formation of S. aureus CICC 21600, CICC 22942, W1, W3, and C1 cells was enhanced after cold stress for 20 weeks. What is more, the mRNA levels of the clfA, icaA, icaB, icaC or icaD genes in these strains were increased for >2-fold. The increased gene transcription levels were consistent with the increase in the polysaccharide content in the biofilm matrix of these S. aureus strains after cold stress. Meanwhile, hydrophobicity and the adhesion proteins also played a role in the formation of biofilms. The biofilm of S. aureus cells can be effectively degraded by snailase and proteinase K (125 µg/mL + 20 µg/mL) mixture. In summary, S. aureus frozen at −20 °C for 12 to 20 weeks is still a potential hazard. Food factory equipment should be cleaned in a timely manner to avoid outbreaks of foodborne pathogenic bacteria due to contamination.

Deoxynivalenol (DON) is a widely distributed mycotoxin that frequently occurs in various agricultural raw materials and feeds. DON acts as a virulence factor that accelerates the spread of plant diseases; moreover, its accumulation in grains causes yield loss and serious health problems to humans and livestock. Biodegradation of DON into less- or non-toxic substances using naturally existing microorganisms is considered the best approach for DON detoxification. Although various single isolates and mixed cultures capable of detoxifying DON have been reported, details of the metabolic pathways and the degrading enzymes/coding genes involved are scarce. In this study, we aimed to isolate DON-degrading bacteria from soil samples and explore the mechanisms. Toward this end, 85 soil samples collected from different provinces in China were enriched under aerobic conditions with mineral media containing 50 μg/ml of DON as the sole carbon source. The bacterial consortium LZ-N1 exhibited highly efficient and steady DON-transforming activity. High-throughput sequencing was used to characterize the composition of the involved microflora, and analysis of 16S rRNA sequences indicated that LZ-N1 was composed of at least 11 bacterial genera, with Pseudomonas accounting for nearly half the relative abundance. Coincubation of a mixed culture of two novel strains from the LZ-N1 consortium, namely Pseudomonas sp. Y1 and Lysobacter sp. S1, showed sustained transformation of DON into the metabolite 3-epi-deoxynivalenol, with no degradation products detected after 72 h. The cell-free supernatant, lysate, and cell debris of the mixed culture possessed DON-degrading ability, with the supernatant reaching a DON degradation rate of 100% within 48 h with 50 μg/ml of DON. This is the first report of two-step enzymatic epimerization of DON by a mixed culture, which may provide a new insight into this pathway for future applications in detoxification of DON-contaminated cereals and feed.

Inhibition of Salmonella by Lactobacillus has been a popular research topic for decades; however, the inhibition potential of chicken-derived Salmonella by chicken-derived Lactobacillus has not yet been studied. In this study, 89 strains of Lactobacillus from chicken intestines were isolated by national standard method, Gram staining, physiological, and biochemical experiments and molecular sequencing; The inhibition characteristics of 89 strains of chicken derived Lactobacillus against 10 strains Salmonella (S. Enteritidis SE05, SC31, SC21, SC72 SC74, SC79, SC83, SC87; S. bongori SE47; S. Typhimurium, SC85) were detected by agar inhibition zone, The results showed that the inhibition zone of 24 strains of chicken derived Lactobacillus was more than 10 mm, which indicated that the isolated chicken derived Lactobacillus could effectively inhibit the growth of Salmonella; The drug resistance and bile salt tolerance of these 24 strains were analyzed, The results showed that the standard strains LG and L76 were not resistant, and the other 22 Lactobacillus strains showed different degrees of resistance. The strains LAB24, LAB26, LAB53, LAB69, and L76 showed good tolerance at the concentration of 3 g/L bile salt; Caco-2 cell experiment and flow cytometry were used to analyze the inhibitory effect of chicken derived Lactobacillus on the adhesion of Salmonella to Caco-2 cells, The results showed that 16 probiotics could effectively inhibit the adhesion of Salmonella to Caco-2 cells. Twelve probiotics were identified by molecular biology. The results showed that L76 was Enterococcus faecalis, and the other 11 strains were Lactobacillus.

Pullulanase plays an important role as a starch hydrolysis enzyme in the production of bio-fuels and animal feed, and in the food industry. Compared to the methods currently used for pullulanase production, synthesis by Bacillus subtilis would be safer and easier. However, the current yield of pullulanase from B. subtilis is low to meet industrial requirements. Therefore, it is necessary to improve the yield of pullulanase by B. subtilis. In this study, we mined 10 highly active promoters from B. subtilis based on transcriptome and bioinformatic data. Individual promoters and combinations of promoters were used to improve the yield of pullulanase in B. subtilis BS001. Four recombinant strains with new promoters (Phag, PtufA, PsodA, and PfusA) had higher enzyme activity than the control (PamyE). The strain containing PsodA+fusA (163 U/mL) and the strain containing PsodA+fusA+amyE (336 U/mL) had the highest activity among the analyzed dual- and triple-promoter construct stains in shake flask, which were 2.29 and 4.73 times higher than that of the strain with PamyE, respectively. Moreover, the activity of the strain containing PsodA+fusA+amyE showed a maximum activity of 1,555 U/mL, which was 21.9 times higher than that of the flask-grown PamyE strain in a 50-liter fermenter. Our work showed that these four strong promoters mined from transcriptome data and their combinations could reliably increase the yield of pullulanase in quantities suitable for industrial applications.

To improve the antibacterial and antioxidant properties of chitosan (CS), CS grafted with gallic acid (GA) using recombinant bacterial laccase from Bacillus vallismortis fmb-103 (fmb-rL103) as a catalyst. The structures of grafted chitosans were identified using Fourier transform infrared spectroscopy (FT-IR) and UV visible spectrum (UV–Vis spectroscopy). After gallic acid grafting, the antibacterial properties of chitosans against Pseudomonas, Acinetobacter, Brochothrix thermosphacta, Escherichia coli, Staphylococcus aureus, Salmonella, and Listeria monocytogenes were significantly improved. Meanwhile, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging results showed that the antioxidant properties of grafted CS increased as well. The preservative effects of the grafted chitosan on chilled meat were then investigated. For this purpose, the quality indexes of the chilled meat during the storage were monitored, including total bacterial count, total basic volatile nitrogen (TVB-N) content, pH value, color and thiobarbituric acid reactive substances (TBARS) and so on. The results showed that coating with the grafted chitosan retarded the growth of spoilage bacteria, and decreased TVB-N and TBARS values of meat. The shelf life of chilled meat coated by CS grafted with GA (GA-g-CS) also extended from 6 days to 18 days at 4°C. These results provided a theoretical basis for the future application of the GA-g-CS in the preservation of chilled meat.Highlights:(1)The temperature and pH-stable bacterial laccase was used to synthesize gallic acid grafted chitosan.(2)Antioxidant and antibacterial properties of chitosan were improved through grafting gallic acid.(3)Storage properties of chilled meat were improved by coating with gallic acid grafted chitosan.

This paper is the first public report that Streptomyces flavogriseus can produce both actinomycin D and holomycin. The actinomycete strain NJ-4 isolated from the soil of Nanjing Agricultural University was identified as S. flavogriseus. This S. flavogriseus strain was found for the first time to produce two antimicrobial compounds that were identified as actinomycin D and holomycin. GS medium, CS medium and GSS medium were used for the production experiments. All three media supported the production of actinomycin D, while holomycin was detected only in GS medium and was undetectable by HPLC in the CS and GSS media. The antimicrobial activity against B. pumilus, S. aureus, Escherichia coli, F. moniliforme, F. graminearum and A. niger was tested using the agar well diffusion method. Actinomycin D exhibited strong antagonistic activities against all the indicator strains. Holomycin exhibited strong antagonistic activities against B. pumilus, S. aureus and E. coli and had antifungal activity against F. moniliforme and F. graminearum but had no antifungal activity against A. niger. The cell viability was determined using an MTT assay. Holomycin exhibited cytotoxic activity against A549 lung cancer cells, BGC823 gastric cancer cells and HepG2 hepatocellular carcinoma cells. The yield of actinomycin D from S. flavogriseus NJ-4 was 960 mg/l. S. flavogriseus NJ-4 exhibits a distinct capability and has the industrial potential to produce considerable yields of actinomycin D under unoptimized conditions.

Forming biofilm is a strategy utilized by Shiga toxin-producing Escherichia coli (STEC) to survive and persist in food processing environments. We investigated the biofilm-forming potential of STEC strains from 10 clinically important serogroups on stainless steel at 22 °C or 13 °C after 24, 48, and 72 h of incubation. Results from crystal violet staining, plate counts, and scanning electron microscopy (SEM) identified a single isolate from each of the O113, O145, O91, O157, and O121 serogroups that was capable of forming strong or moderate biofilms on stainless steel at 22 °C. However, the biofilm-forming strength of these five strains was reduced when incubation time progressed. Moreover, we found that these strains formed a dense pellicle at the air-liquid interface on stainless steel, which suggests that oxygen was conducive to biofilm formation. At 13 °C, biofilm formation by these strains decreased (P < 0.05), but gradually increased over time. Overall, STEC biofilm formation was most prominent at 22 °C up to 24 h. The findings in this study identify the environmental conditions that may promote STEC biofilm formation in food processing facilities and suggest that the ability of specific strains to form biofilms contributes to their persistence within these environments.

Both donors and acceptors of communication-mediating (COM) domains are essential for coordinating intermolecular communication within nonribosomal peptides synthetases (NRPSs) complexes. Different sets of COM domains provide selectivity, allowing NRPSs to utilise different natural biosynthetic templates. In this study, novel lipopeptides were synthesised by reprogramming the plipastatin biosynthetic machinery. A Thr-to-Asp point mutation was sufficient to shift the selectivity of the donor COM domain of ppsB toward that of ppsD. Deletion and/or interchangeability established donor and acceptor function. Variations in acceptor COM domain did not result in novel product formation in the presence of its partner donor, whereas plipastatin formation was completely abrogated by altering donor modules. Five novel lipopeptides (cyclic pentapeptide, linear hexapeptide, nonapeptide, heptapeptide and cyclic octapeptide) were identified and verified by high-resolution LC-ESI-MS/MS. In addition, we demonstrated the potential to generate novel strains with the antimicrobial activity by selecting compatible COM domains, and the novel lipopeptides exhibited antimicrobial activity against five of the fungal species at a contention of 31.25-125 μg/ml.

Previous studies have shown the tumor-suppressive role of selenium-binding protein 1 (SBP1), but the underlying mechanisms are unclear. In this study, we found that induction of SBP1 showed significant inhibition of colorectal cancer cell growth and metastasis in mice. We further employed isobaric tags for relative and absolute quantitation (iTRAQ) to identify proteins that were involved in SBP1-mediated anti-cancer effects in tumor tissues. We identified 132 differentially expressed proteins, among them, 53 proteins were upregulated and 79 proteins were downregulated. Importantly, many of the differentially altered proteins were associated with lipid/glucose metabolism, which were also linked to Glycolysis, MAPK, Wnt, NF-kB, NOTCH and epithelial-mesenchymal transition (EMT) signaling pathways. These results have revealed a novel mechanism that SBP1-mediated cancer inhibition is through altering lipid/glucose metabolic signaling pathways.

A loop-mediated isothermal amplification (LAMP) method combined with calcein was established for the detection of Staphylococcus aureus. LAMP primers were designed targeting the ASL18_04625 species-specific gene screened through bioinformatics and PCR analysis. The target gene and detection method demonstrated 100% specificity for S. aureus and no cross-reactions with other pathogens. For LAMP reactions, the results could be directly observed with the naked eye or evaluated by S-shaped amplification curves of fluorescence signals. The assay's detection limit was 3.395 × 10-4 ng/µL genomic DNA or 3.21 CFU/mL pure bacterial culture. Furthermore, the target bacteria of 8.5 × 105-8.5 × 100 CFU/mL could be accurately detected in spiked milk samples, and the overall detection process could be completed within 40 min. This method improved the accuracy and convenience for S. aureus detection and provied a dependable tool for the rapid screening of S. aureus. [ABSTRACT FROM AUTHOR]