Comparative Transcriptome Analysis Revealing the Enhanced Volatiles of Cofermentation of Yeast and Lactic Acid Bacteria on Whole Wheat Steamed Bread Dough.

To reveal the underlying mechanism of enhanced volatiles of whole wheat steamed bread, the current study screened Saccharomyces cerevisiae Y5 and Lactiplantibacillus plantarum L7 from sourdough and studied the synergetic effect of cofermentation on the volatiles of steamed bread and fermented dough by comparative transcriptome analysis. Cofermentation significantly improved the types and concentration of volatiles in addition to the improved specific volume and texture. Genes involved in galactose, starch, and glucose metabolism and genes encoding pyruvate oxidase and ß-galactosidase were significantly upregulated in S. cerevisiae and L. plantarum, respectively. Expression of the OPT2 encoding oligopeptide transporter in S. cerevisiae was upregulated, which facilitated the transmembrane transport of oligopeptide and amino acid into yeast cells. Genes involved in the synthesis and metabolism of amino acids, lipids, and ester compounds in L. plantarum changed significantly, and gene encoding acetic acid kinase was upregulated. Moreover, the quorum sensing-related genes in S. cerevisiae and L. plantarum were upregulated.

Effects of monolauroyl-galactosylglycerol on membrane fatty acids and properties of Bacillus cereus.

The purpose of this study was to provide new ideas for the antibacterial mechanism of monolauroyl-galactosylglycerol (MLGG) from the perspective of cell membranes. The changes in cell membrane properties of Bacillus cereus (B. cereus) CMCC 66,301 exposed to different concentrations (1 × MIC (minimum inhibitory concentration), 2 × MIC, 1 × MBC (minimum bacterial concentration)) of MLGG were evaluated. It was found that the lag phase of B. cereus cells was prolonged at low concentration MLGG (1 × MIC and 2 × MIC), while about 2 log CFU/mL reduction in B. cereus populations were observed when exposed to high concentration MLGG (1 × MBC). MLGG treated B. cereus displayed obvious membrane depolarization, while membrane permeability had no change using PI (propidium iodide) staining. Significant increase in the membrane fluidity in response to MLGG exposure occurred, which was consistent with the modification of membrane fatty acids compositions, where the relative content of straight-chain fatty acids (SCFAs) and unsaturated fatty acids (UFAs) increased, while branched-chain fatty acids (BCFAs) decreased significantly. The decreased transition Tm value and cell surface hydrophobicity was also observed. Additionally, effect of MLGG on bacterial membrane compositions were explored at the submolecular level by infrared spectroscopy. Resistance tests of B. cereus to MLGG had demonstrated the advantages of MLGG as a bacteriostatic agent. Collectively, these studies indicate that modifying the fatty acid composition and properties of cellular membranes through MLGG exposure is crucial for inhibiting bacteria growth, providing new insights into the antimicrobial mechanisms of MLGG. KEY POINTS: • Monolauroyl-galactosylglycerol inserted into B. cereus lipid bilayer membrane • Monolauroyl-galactosylglycerol treatment caused B. cereus membrane depolarization • Monolauroyl-galactosylglycerol resulted in B. cereus membrane fatty acids alteration.

Separation, characterization and anti-inflammatory activities of galactoglycerolipids from Perilla frutescens (L.) Britton.

The study was to optimize the separation procedures, characterize the galactoglycerolipids and explore their anti-inflammatory activities. Two monogalactosyldiacylglycerols (MGDGs) and three digalactosyldiacylglycerols (DGDGs) from Perilla frutescens (L.) Britton were obtained through one-step silica gel column chromatography and preparative high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD). The presence of additional MGDG (1-O-9Z,12Z,15Z-octadecatrienoyl-2-O-7Z,10Z,13Z-hexadecatrienoyl-3-O-(ß-D-galactopyranosyl)-sn-glycerol) and DGDG (1-O-9Z,12Z-octadecadienoyl-2-O-9Z,12Z,15Z-octadecatrienoyl-3-O-(ß-D-galactopyranosyl-(1'→6'')-α-D-galactopyranosyl)-sn-glycerol) was concluded for the first time in perilla, by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). In lipopolysaccharide (LPS)-induced RAW264.7 cells, five galactoglycerolipids exhibited good inhibitory activities against nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in a dose-dependent manner, suggesting that fatty acid chain length and unsaturation degree affected their anti-inflammatory activities.

ß-Glucan Extracted from Highland Barley Alleviates Dextran Sulfate Sodium-Induced Ulcerative Colitis in C57BL/6J Mice.

Inflammatory bowel disease (IBD), which significantly affects human health, has two primary presentations: Crohn's disease and ulcerative colitis (UC). Highland barley is the most common food crop for Tibetans and contains much more ß-glucan than any other crop. Highland barley ß-glucan (HBBG) can relieve the gastrointestinal dysfunction and promote intestines health. This study aimed to evaluate whether HBBG can relieve UC in mice. A mouse model of UC was established by adding 2% dextran sulfate sodium (DSS) to drinking water for 1 week. UC was alleviated after the introduction of the HBBG diet, as indicated by reductions in the disease activity index (DAI) score, histopathological damage, and the concentration of colonic myeloperoxidase (MPO), along with an improvement in colonic atrophy. Furthermore, we found that HBBG can increase the relative transcriptional levels of genes encoding ZO-1, claudin-1, occludin, and mucin2 (MUC2), thereby reducing intestinal permeability. Additionally, HBBG maintained the balance of proinflammatory and anti-inflammatory cytokines and modulated the structure of the intestinal flora.

Regulatory mechanisms of energy metabolism and inflammation in oleic acid-treated HepG2 cells from Lactobacillus acidophilus NX2-6 extract.

In this study, the cell-free extracts (CFE) of Lactobacillus acidophilus NX2-6 were utilized to treat oleic acid (OA)-induced hepatic steatosis. It was found that CFE treatment improved lipid metabolism in OA-induced hepatic steatosis model by downregulating several lipogenic genes but increasing expression levels of lipolysis-related genes. In addition, gene expression analysis revealed that CFE treatment promoted mitochondrial biogenesis and fission by upregulating the mRNA levels of PGC-1α, PGC-1ß, Sirt1, NRF1, and Fis1. CFE treatment also increased protein expression of p-AMPKα, PGC-1α, ACOX1, and Sirt1 in OA-treated cells, suggesting that CFE possessed ability to improve energy metabolism. Furthermore, CFE treatment also reversed OA-induced oxidative stress by increasing CAT activity and protein level of Nrf-2 as well as reducing protein expression of ATF6, XBP1, GRP78, p50, and p-ERK, indicating that CFE could inhibit endoplasmic reticulum stress and sterile inflammation. Thus, L. acidophilus NX2-6 had potential to fight against NAFLD. PRACTICAL APPLICATIONS: Diet-induced hepatic steatosis is one of major public health concerns all over the world. Hepatic steatosis is accompanied by disregulation of lipid metabolism and energy metabolism, endoplasmic reticulum stress, oxidative stress as well as chronic inflammation. It is reported that probiotics are considered as emerging therapeutic strategy to alleviate hepatic steatosis. This study indicated potential applications of dead probiotics in the prevention of hepatic steatosis and development of functional foods.

Curative Effect of Early Full Nursing Combined with Postdischarge Continuation Nursing on Patients after Craniocerebral Trauma.

Early full nursing helps patients with some dysfunctions speed up the reorganization of central nervous system functions and coordinate muscle and limb activities. Postdischarge continuation nursing for patients who have not fully recovered after being discharged from the hospital can transfer nursing work from the hospital to the family to meet their nursing needs. In this study, early full nursing combined with postdischarge continuation nursing was used for patients with traumatic brain injury to explore its efficacy and its impact on patients' motor function, quality of life, and complications. The results of the study show that the total effective rate of the observation group (95.92%) was higher than that of the control group (85.71%). At discharge and 1 month, 3 months, and 6 months after discharge, the upper limb Fugl-Meyer score, lower limb Fugl-Meyer score, ARAT score, FIM score, 4 dimensions of GQOLI-74 score, and Barthel index scores of the observation group were higher than those of the control group in the same period. The postoperative complication rate (10.20%) of the observation group was lower than that of the control group (26.53%). Early full nursing combined with postdischarge continuation nursing can improve the rehabilitation effect, effectively promote the recovery of motor function in patients with traumatic brain injury, improve the quality of life, and reduce postoperative complications.

In Vitro Screening of Chicken-Derived Lactobacillus Strains that Effectively Inhibit Salmonella Colonization and Adhesion.

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.

L. johnsonii, L. plantarum, and L. rhamnosus alleviated Enterohaemorrhagic Escherichia coli-induced diarrhoea in mice by regulating gut microbiota.

Enterohaemorrhagic Escherichia coli (EHEC) is a prominent foodborne pathogen that causes infectious intestinal diarrhoea. Lactobacillus is a recognized probiotic that inhibits intestinal pathogens and maintains the balance of the intestinal flora. The purpose of this study was to investigate the regulatory effects of three Lactobacillus strains, L. johnsonii, L. plantarum, and L. rhamnosus, on the intestinal flora of EHEC-infected mice. The initial weight and diarrhoea index of the mice were recorded. After 21 days, the faeces of the mice were subjected to 16S rDNA high-throughput sequencing. The diarrhoea index of mice treated with Lactobacillus improved, their body weight continued to rise, and their liver index gradually decreased. The α diversity analysis showed that the intestinal flora diversity and abundance were lower in mice infected with EHEC than in healthy mice. L. plantarum, L. johnsonii, and L. rhamnosus significantly improved the diversity of the flora species. In terms of flora composition, the three main phyla present were Bacteroidetes, Firmicutes, and Proteobacteria. The abundance of these three phyla was reduced to 93.81% after infection and restored to over 96.30% after treatment. At the genus level, Lactobacillus reduced the abundance of Bacteroides, Helicobacter pylori, and Shigella, while increasing the abundance of butyric acid-producing bacteria and Lactobacillus. Finally, a heat map and non-metric multidimensional scaling analysis showed that the intestinal flora structures in the L. johnsonii, L. plantarum, and L. rhamnosus treatment groups were closest to those of healthy mice. In conclusion, L. johnsonii, L. plantarum, and L. rhamnosus regulated and improved the structure of intestinal flora and relieved diarrhoea caused by EHEC infection.

Influence of different factors on biofilm formation of Listeria monocytogenes and the regulation of cheY gene.

In a food-processing environment, bacterial cells often adhere to surfaces and form biofilms to protect themselves from external adverse influences. Our study aimed to identify the influence of environmental factors and cell properties on Listeria monocytogenes biofilm formation. Biofilm formation was quantified through measuring the optical density at 590 nm (OD590 nm) after crystal violet staining. Neutral pH and 37oC were beneficial for biofilm formation whereas the influence of glucose (0.0-1.0%) and sodium chloride (0.0-1.0%) were strain-dependent. In general, the addition of sodium chloride and glucose increased biofilm formation in most strains compared to that in controls with no sodium chloride or glucose added. Bacteria with strong biofilm-forming capacity always produced large amounts of biofilm in most instances. Biofilm formation positively correlated with the cell surface hydrophobicity and motility but was independent from planktonic cell growth. The expression of flagella-related flaA, motB, and the two-component chemotactic system cheA/Y genes in biofilm cells increased compared to that in planktonic cells. Meanwhile, a cheY knockout mutant was constructed, and decreased biofilm-formation ability along with reduced cell-surface hydrophobicity were found in the non-motile mutant. Furthermore, the cheY knockout mutant showed no change in growth, and pH susceptibility compared to that in the wild-type strain.

Development of a test kit for visual loop-mediated isothermal amplification of Salmonella in spiked ready-to-eat fruits and vegetables.

The purpose of this study was to assemble two types of loop-mediated isothermal amplification (LAMP) kit that have the ability to visually detect Salmonella in ready-to-eat fruits and vegetables. The reaction results were obtained within 20-40 min after addition of DNA and can be discerned by the naked eye or an amplification plot. The stability of the LAMP wet kit was evident after multiple freezing and thawing cycles, and the one-step LAMP lyophilized kit was further evolved to allow ambient temperature transport for deployment in resource-limited settings. The cost-effective wet kit had the ability to detect minimum amounts of 1.8 CFU/ml Salmonella DNA without enrichment, while the sensitivity of the one-step LAMP lyophilized kit was only 9.8 × 103 CFU/ml. They both have good anti-interference, as they were both able to detect 2.1 × 102 CFU/ml Salmonella mixed with 106 CFU/ml four non-Salmonella strain mixture. Moreover, cucumber and lettuce that were contaminated with an initial inoculation of 1.7 CFU of Salmonella/10 g showed detection within a reaction time of 30 min after 10 h enrichment. The present research setup is a convenient and practical kit for Salmonella rapid detection that has good application prospects in food safety monitoring.

Antibiofilm activity and modes of action of a novel ß-sheet peptide against multidrug-resistant Salmonella enterica.

S. enterica is an important foodborne pathogen worldwide. As some strains can form biofilms which may offer protection against antimicrobials, it is of interest to explore ways to prevent biofilm formation by S. enterica. In this study, we engineered a short ß-sheet peptide WK2 (WKWKCTKSGCKWKW-NH2) and examined its antimicrobial and anti-biofilm activities against various S. enterica strains, including the multidrug-resistant S. Typhimurium DT104. WK2 displayed bacteriostatic activity with a geometric mean (GM) minimum inhibitory concentration (MIC) of 4.17 µg/mL, and bactericidal activity, with a GM lethal concentration (LC) of 7.51 µg/mL. Crystal violet staining and fluorescence measurements demonstrated that WK2 inhibited S. Typhimurium DT104 biofilm formation at 0.5 µg/mL and killed the sessile cells in biofilms at 8 µg/mL. Real-time polymerase chain reaction (qPCR) and microscopic observation revealed that the anti-biofilm activity of WK2 likely arises through the formation of complexes with bacterial DNA, inhibition of surface organelle biosynthesis and interference with autoinducer-2 (AI-2)-mediated quorum sensing (QS). Therefore, WK2 is a promising antimicrobial agent for the prevention and control of biofilms produced by multidrug-resistant S. enterica.

Propidium monoazide real-time PCR amplification for viable Salmonella species and Salmonella Heidelberg in pork.

Salmonella enterica serovar Heidelberg causes foodborne infections and is a major threat to the food chain and public health. In this study, we aimed to develop a rapid molecular typing approach to identify Salmonella enterica serovar Heidelberg. Using comparative genomics, four serovar-specific gene fragments were identified, and a real-time polymerase chain reaction (PCR) combined with a propidium monoazide (PMA) pretreatment method was developed for simultaneous detection of viable Salmonella sp. (invA) and Salmonella Heidelberg (SeHA_C3258). The assay showed 100% specificity for all strains tested. The assay was able to distinguish effectively viable or dead cells with the PMA. The detection limit was 2.4 CFU/mL following 6 h of incubation in enrichment Luria-Bertani medium, and the assay could detect 1.7 × 102 CFU/mL in the presence of pork background flora. In artificially contaminated pork, real-time PCR detected inoculum levels of 1.15 CFU/25 g of pork after a 6 h enrichment. Thus, our findings indicated that this comparative genomics approach could be used to screen for serovar-specific fragments and that real-time PCR with PMA was a simple and reliable method for detecting viability of Salmonella species and Salmonella Heidelberg.

Development of a real-time nucleic acid sequence-based amplification assay for the rapid detection of Salmonella spp. from food.

Salmonella spp. is one of the most common foodborne infectious pathogen. This study aimed to develop a real-time nucleic acid sequence-based amplification (NASBA) assay for detecting Salmonella in foods. Primers and a molecular beacon targeting the Salmonella-specific xcd gene were designed for mRNA transcription, and 48 Salmonella and 18 non-Salmonella strains were examined. The assay showed a high specificity and low detection limit for Salmonella (7 × 10-1 CFU/mL) after 12 h of pre-enrichment. Importantly, it could detect viable cells. Additionally, the efficacy of the NASBA assay was examined in the presence of pork background microbiota; it could detect Salmonella cells at 9.5 × 103 CFU/mL. Lastly, it was successfully used to detect Salmonella in pork, beef, and milk, and its detection limit was as low as 10 CFU/25 g (mL). The real-time NASBA assay developed in this study may be useful for rapid, specific, and sensitive detection of Salmonella in food of animal origin.

Bacillomycin D-C16 triggers apoptosis of gastric cancer cells through the PI3K/Akt and FoxO3a signaling pathways.

Bacillomycin D can inhibit the growth of Aspergillus ochraceus in food samples. In addition, it can induce apoptosis in and inhibit the proliferation of cancer cells, although the details of this mechanism are unknown. In this study, we separated bacillomycin D-C14, D-C15, D-C16 monomers from the Bacillus subtilis strain fmbJ. The bacillomycin D monomers containing longer fatty acid chains better induced apoptosis in Bgc-823, Sgc-7901, and Hgc-27 gastric cancer cells. The Bgc-823 cell line was the most sensitive. Acridine orange-ethidium bromide staining indicated that bacillomycin D-C16-induced Bgc-823 cell death by triggering apoptosis, characterized by membrane blebbing, cellular shrinkage, and DNA fragmentation. Flow cytometric analysis showed a bacillomycin D-C16 dose-dependent trigger of Bgc-823 apoptosis. Bacillomycin D-C16-induced the mitochondrial pathway, as indicated by a reduced Bcl-2/Bax expression ratio, enhanced cytochrome C release, and higher levels of cleaved caspase-3. Furthermore, bacillomycin D-C16 effectively repressed phosphorylation of the serine-threonine protein kinase Akt at Ser-473 and increased the levels of the FoxO3a protein. The combination of the PI3K/Akt-inhibitor BEZ235 with bacillomycin D-C16 enhanced the apoptosis of Bgc-823 cells. Together, these findings indicated that bacillomycin D-C16 induces apoptosis through the PI3K/Akt and FoxO3a signaling pathways.

Effect of the luxS gene on biofilm formation and antibiotic resistance by Salmonella serovar Dublin.

Biofilms are communities of bacterial cells that serve to protect them from external adverse influences and enhance bacterial resistance to antibiotics and sanitizers. Here, we studied the regulatory effects of glucose and sodium chloride on biofilm formation in Salmonella serovar Dublin (S. Dublin). To analyze expression levels of the quorum sensing gene luxS, we created a luxS knockout mutant. Also, antimicrobial resistance, hydrophobicity and autoinducer-2 (AI-2) activity of both the wild-type (WT) and the mutant strain were investigated. Our results revealed that glucose was not essential for S. Dublin biofilm formation but had an inhibitory effect on biofilm formation when the concentration was over 0.1%. NaCl was found to be indispensable in forming biofilm, and it also exerted an inhibitory effect at high concentrations (>1.0%). Both the WT and the mutant strains displayed significant MIC growth after biofilm formation. An increase of up to 32,768 times in the resistance of S. Dublin in biofilm phonotype against antibiotic (ampicillin) compared to its planktonic phonotype was observed. However, S. Dublin luxS knockout mutant only showed slight differences compared to the WT strain in the antimicrobial tests although it displayed better biofilm-forming capacity than the WT strain. The mutant strain also exhibited higher hydrophobicity than the WT strain, which was a feature related to biofilm formation. The production of the quorum sensing autoinducer-2 (AI-2) was significantly lower in the mutant strain than in the WT strain since the LuxS enzyme, encoded by the luxS gene, plays an essential role in AI-2 synthesis. However, the limited biofilm-forming ability in the WT strain indicated AI-2 was not directly related to S. Dublin biofilm formation. Furthermore, gene expression analysis of the WT and mutant strains revealed upregulation of genes related to biofilm stress response and enhanced resistance in the luxS mutant strain, which may provide evidence for the regulatory role of the luxS gene in biofilm formation.

The antibacterial activity of LI-F type peptide against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and inhibition of infections in murine scalded epidermis.

LI-F type peptides are a family of cyclic lipodepsipeptide antibiotics isolated from Paenibacillus polymyxa and display potent activities against positive bacteria including methicillin-resistant S. aureus (MRSA). In this study, we investigated the mechanism of action of LI-F type peptide AMP-jsa9 against a MRSA (S. aureus CICC10790), which is resistant to ciprofloxacin, gentamicin, kanamycin, chloramphenicol, methicillin, and tetracycline. It was found that AMP-jsa9 mainly targets the cell membrane of MRSA and is able to inhibit biofilm formation through killing planktonic bacteria cells. Moreover, AMP-jsa9 can bind to DNA in vitro, which represents another pathway for the action on MRSA. Furthermore, in vivo treatment of scalded mice with AMP-jsa9 resulted in inhibiting MRSA infections and healing of the scalded wound. In addition, it was demonstrated that AMP-jsa9 can effectively inhibit MRSA infections in scalded murine epidermis and that inflammatory cytokines including IL-8, IL-6, tumor necrosis factor alpha (TNF-α), and monocyte chemotactic factor-1 (MCP-1) were reduced; moreover, both protein and gene expression levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (e-NOS) were enhanced, which promote neovascularization and proliferation of new granulation tissue.

Mechanism of action of AMP-jsa9, a LI-F-type antimicrobial peptide produced by Paenibacillus polymyxa JSa-9, against Fusarium moniliforme.

LI-F type peptides (AMP-jsa9) are a group of cyclic lipodepsipeptides that exhibit broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi. We sought to assess the toxicity of AMP-jsa9 and the mechanism of AMP-jsa9 action against Fusarium moniliforme. AMP-jsa9 exhibited weak hemolytic activity and weak cytotoxicity at antimicrobial concentrations (32µg/ml). Confocal laser microscopy, SEM, and TEM indicated that AMP-jsa9 primarily targets the cell wall, plasma membrane, and cytoskeleton, increases membranepermeability, and enhances cytoplasm leakage (e.g., K+, protein). Quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) detected a total of 162 differentially expressed proteins (59 up-regulated and 103 down-regulated) following treatment of F. moniliforme with AMP-jsa9. AMP-jsa9 treatment also led to reductions in chitin, ergosterol, NADH, NADPH, and ATP levels. Moreover, fumonisin B1 expression and biosynthesis was suppressed in AMP-jsa9-treated F. moniliforme. Our results provide a theoretical basis for the application of AMP-jsa9 as a natural and effective antifungal agent in the agricultural, food, and animal feed industries.

iTRAQ-based proteomic analysis of LI-F type peptides produced by Paenibacillus polymyxa JSa-9 mode of action against Bacillus cereus.

LI-F type peptides (AMP-jsa9) produced by Paenibacillus polymyxa JSa-9 are a group of cyclic lipodepsipeptide antibiotics that exhibit a broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi, especially Bacillus cereus and Fusarium moniliforme. In this study, to better understand the antibacterial mechanism of AMP-jsa9 against B. cereus, the ultrastructure of AMP-jsa9-treated B. cereus cells was observed by both atomic force microscopy and transmission electron microscopy, and quantitative proteomic analysis was performed on proteins extracted from treated and untreated bacterial cells by using isobaric tag for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to access differentially expressed proteins. Furthermore, multiple experiments were conducted to validate the results of the proteomic analysis, including determinations of ATP, NAD(+)H, NADP(+)H, reactive oxygen species (ROS), the activities of catalase (CAT) and superoxide dismutase (SOD), and the relative expression of target genes by quantitative real-time PCR. Bacterial cells exposed to AMP-jsa9 showed irregular surfaces with bleb projections and concaves; we hypothesize that AMP-jsa9 penetrated the cell wall and was anchored on the cytoplasmic membrane and that ROS accumulated in the cell membrane after treatment with AMP-jsa9, modulating the bacterial membrane properties and increasing membrane permeability. Consequently, the blebs were formed on the cell wall by the impulsive force of the leakage of intercellular contents. iTRAQ-based proteomic analysis detected a total of 1317 proteins, including 176 differentially expressed proteins (75 upregulated (fold >2) and 101 downregulated (fold <0.5)). Based on proteome analysis, the putative pathways of AMP-jsa9 action against B. cereus can be summarized as: (i) inhibition of bacterial sporulation, thiamine biosynthesis, energy metabolism, DNA transcription and translation, and cell wall biosynthesis, through direct regulation of protein levels; and (ii) indirect effects on the same pathways through the accumulation of ROS and the consequent impairment of cellular functions, resulting from downregulation of antioxidant proteins, especially CAT and SOD. BIOLOGICAL SIGNIFICANCE: The mode of action of LI-F type antimicrobial peptides (AMP-jsa9) against B. cereus was elucidated at the proteomic level. Two pathways of AMP-jsa9 action upon B. cereus cells were identified and the mechanism of bleb formation on the surfaces of bacterial cells was predicted based on the results of ultrastructural observation and proteomic analysis. These results are helpful in understanding the mechanism of LI-F type peptides and in providing the theoretical base for applying AMP-jsa9 or its analogs to combat Gram-positive pathogenic bacteria in the food and feed industries.

Translocation of the thioesterase domain for the redesign of plipastatin synthetase.

Non-ribosomal peptide synthetases (NRPSs) are large enzymatic complexes that catalyse the synthesis of biologically active peptides in microorganisms. Genetic engineering has recently been applied to reprogram NRPSs to produce lipopeptides with a new sequence. The carboxyl-terminal thioesterase (TE) domains from NRPSs catalyse cleavage products by hydrolysis or complex macrocyclization. In this study, we modified plipastatin synthetase by moving the intrinsic TE region to the end of the internal thiolation (T) domains, thus generating Bacillus subtilis strains that could produce new truncated cyclic or linear peptides of the predicted sequence, which further provided an important insight into the regioselectivity of plipastatin TE. The TE was capable of recognizing and catalysing the lactone formation between L-Try3 with the last few residues L-Pro7 and L-Gln8 at the C-terminus. Additionally, the unmatched linkers connecting the TE region and T domain resulted in nonproduction strains, suggesting that the native T-TE linker is necessary and sufficient for the TE domain to release the products from the hybrid enzymes. This is the first report to demonstrate truncated cyclic lipopeptides production and module skipping by simply moving the TE domain forward in an NRPS system.

Purification and Characterization of Plantaricin JLA-9: A Novel Bacteriocin against Bacillus spp. Produced by Lactobacillus plantarum JLA-9 from Suan-Tsai, a Traditional Chinese Fermented Cabbage.

Bacteriocins are ribosomally synthesized peptides with antimicrobial activity produced by numerous bacteria. A novel bacteriocin-producing strain, Lactobacillus plantarum JLA-9, isolated from Suan-Tsai, a traditional Chinese fermented cabbage, was screened and identified by its physiobiochemical characteristics and 16S rDNA sequence analysis. A new bacteriocin, designated plantaricin JLA-9, was purified using butanol extraction, gel filtration, and reverse-phase high-performance liquid chromatography. The molecular mass of plantaricin JLA-9 was shown to be 1044 Da by MALDI-TOF-MS analyses. The amino acid sequence of plantaricin JLA-9 was predicted to be FWQKMSFA by MALDI-TOF-MS/MS, which was confirmed by Edman degradation. This bacteriocin exhibited broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, especially Bacillus spp., high thermal stability (20 min, 121 °C), and narrow pH stability (pH 2.0-7.0). It was sensitive to α-chymotrypsin, pepsin, alkaline protease, and papain. The mode of action of this bacteriocin responsible for outgrowth inhibition of Bacillus cereus spores was studied. Plantaricin JLA-9 had no detectable effects on germination initiation over 1 h on monitoring the hydration, heat resistance, and 2,6-pyridinedicarboxylic acid (DPA) release of spores. Rather, germination initiation is a prerequisite for the action of plantaricin JLA-9. Plantaricin JLA-9 inhibited growth by preventing the establishment of oxidative metabolism and disrupting membrane integrity in germinating spores within 2 h. The results suggest that plantaricin JLA-9 has potential applications in the control of Bacillus spp. in the food industry.