Synthesis of Two-Dimensional (Cu-S)n Metal-Organic Framework Nanosheets Applied as Peroxidase Mimics for Detection of Glutathione.

Facilely synthesized peroxidase-like nanozymes with high catalytic activity and stability may serve as effective biocatalysts. The present study synthesizes peroxidase-like nanozymes with multinuclear active sites using two-dimensional (2D) metal-organic framework (MOF) nanosheets and evaluates them for their practical applications. A simple method involving a one-pot bottom-up reflux reaction is developed for the mass synthesis of (Cu-S)n MOF 2D nanosheets, significantly increasing production quantity and reducing reaction time compared to traditional autoclave methods. The (Cu-S)n MOF 2D nanosheets with the unique coordination of Cu(I) stabilized in Cu-based MOFs demonstrate impressive activity in mimicking natural peroxidase. The active sites of the peroxidase-like activity of (Cu-S)n MOF 2D nanosheets were predominantly verified as Cu(I) rather than Cu(II) of other Cu-based MOFs. The cost-effective and long-term stability of (Cu-S)n MOF 2D nanosheets make them suitable for practical applications. Furthermore, the inhibition of the peroxidase-like activity of (Cu-S)n MOF nanosheets by glutathione (GSH) could provide a simple strategy for colorimetric detection of GSH against other amino acids. This work remarkably extends the utilization of (Cu-S)n MOF 2D nanosheets in biosensing, revealing the potential for 2D (Cu-S)n MOFs.

A Novel Two-Component Bacteriocin, Acidicin P, and Its Key Residues for Inhibiting Listeria monocytogenes by Targeting the Cell Membrane.

Listeria monocytogenes is an important pathogen which easily contaminates food and causes fatal systemic infections in human. Bacteriocins have received much attention regarding their natural methods of controlling health-related pathogens. Here, we investigated and characterized a novel two-component bacteriocin named acidicin P from Pediococcus acidilactici LAC5-17. Acidicin P showed obvious antimicrobial activity to L. monocytogenes. Through a sequence similarity network analysis for two-component bacteriocin precursors mined in the RefSeq database, acidicin P was observed to belong to an unusual group of two-component bacteriocins. Acidicin P contains two peptides designated Adpα and Adpß which are assessed to interact with each other and form a helical dimer structure which can be inserted into the lipid bilayer of target cell membrane. We demonstrate that A5, N7, and G9 in the A5xxxG9 motif of Adpα and S16, R19, and G20 in the S16xxxG20 motif of Adpß played crucial roles in stabilizing the helix-helix interaction of Adpα and Adpß and were essential for the antilisterial activity of acidicin P by site-directed mutagenesis. A positive residue, R14, in Adpα and a negative residue, D12, in Adpß are also important for acidicin P to fight against L. monocytogenes. These key residues are supposed to form hydrogen bonding, which is crucial for the interaction of Adpα and Adpß. Furthermore, acidicin P induces severe permeabilization and depolarization of the cytoplasmic membrane and causes dramatic changes in L. monocytogenes cell morphology and ultrastructure. Acidicin P has the potential to be applied to inhibit L. monocytogenes efficiently both in the food industry and medical treatments. IMPORTANCE L. monocytogenes can cause widespread food contamination and severe human listeriosis, which amount to a large proportion of the public health and economic burdens. Today, L. monocytogenes is usually treated with chemical compounds in the food industry or antibiotics for human listeriosis. Natural and safe antilisterial agents are urgently required. Bacteriocins are natural antimicrobial peptides that have comparable narrow antimicrobial spectra and are attractive potentials for precision therapy for pathogen infection. In this work, we discover a novel two-component bacteriocin designated acidicin P, which shows obvious antilisterial activity. We also identify the key residues in both peptides of acidicin P and demonstrate that acidicin P is inserted into the target cell membrane and disrupts the cell envelop to inhibit the growth of L. monocytogenes. We believe that acidicin P is a promising lead for further development as an antilisterial drug.

Food and gut originated bacteriocins involved in gut microbe-host interactions.

The gut microbes interact with each other as well as host, influencing human health and some diseases. Many gut commensals and food originated bacteria produce bacteriocins which can inhibit pathogens and modulate gut microbiota. Bacteriocins have comparable narrow antimicrobial spectrum and are attractive potentials for precision therapy of gut disorders. In this review, the bacteriocins from food and gut microbiomes and their involvement in the interaction between producers and gut ecosystem, along with their characteristics, types, biosynthesis, and functions are described and discussed. Bacteriocins are produced by many intestinal commensals and food microbes among which lactic acid bacteria (many are probiotics) has been paid more attention. Bacteriocin production has been generally regarded as a probiotic trait. They give a competitive advantage to bacteria, enabling their colonization in human gut, and mediating the interaction between the producers and host ecosystem. They fight against unwanted bacteria and pathogens without significant impact on the composition of commensal microbiota. Bacteriocins assist the producers to survive and colonize in the gut microbial populations. There is a great need to evaluate and utilize the potential of bacteriocins for improved therapeutic implications for intestinal health.

Exploring the bacterial community succession and metabolic profiles of Lonicera japonica Thunb. residues during anaerobic fermentation.

Discarding Lonicera japonica Thunb. (LJT) residues containing many active metabolites create tremendous waste. This study aimed to effectively use LJT residues by anaerobic fermentation. Fermentation significantly decreased the pH values and reduced the abundance of undesirable bacteria (potential pathogenic and biofilm-forming) while increasing Lactobacillus abundance. Compound additive use further improved fermentation quality (significantly increased the lactic acid (LA) content and decreased the pH values and ammonia nitrogen (a-N) content) and nutrient quality (significantly decreased the acid detergent fiber (ADF) content and increased the water-soluble carbohydrate (WSC) content) and optimized the microbial community (increased the Lactobacillus abundance). Fermentation also altered the flavonoids, alkaloids and phenols contents in the residues with minor effects on the functional metabolites amounts. The LJT residues metabolic profile was mainly attributed to its epiphytic bacteria, with a small contribution from the compound additive. Thus, compound additives may improve anaerobic LJT residue fermentation without functionally impairing the metabolites.

Dynamic Changes in Fermentation Quality and Structure and Function of the Microbiome during Mixed Silage of Sesbania cannabina and Sweet Sorghum Grown on Saline-Alkaline Land.

Protein-rich Sesbania cannabina and sugar-rich sweet sorghum [Sorghum dochna (Forssk.) Snowden] are characterized by their higher tolerance to saline-alkaline stresses and simultaneous harvests. They could be utilized for coensiling because of their nutritional advantages, which are crucial to compensate protein-rich forage in saline-alkaline regions. The current study investigated the fermentation quality, microbial community succession, and predicted microbial functions of Sesbania cannabina and sweet sorghum in mixed silage during the fermentation process. Before ensiling, the mixtures were treated with compound lactic acid bacteria (LAB) inoculants followed by 3, 7, 14, 30, and 60 days of fermentation. The results revealed that the inoculated homofermentative species Lactobacillus plantarum and Lactobacillus farciminis dominated the early phase of fermentation, and these shifted to the heterofermentative species Lactobacillus buchneri and Lactobacillus hilgardii in the later phase of fermentation. As a result, the pH of the mixed silages decreased significantly, accompanied by the growth of acid-producing microorganisms, especially L. buchneri and L. hilgardii, which actively influenced the bacterial community structure and metabolic pathways. Moreover, the contents of lactic acid, acetic acid, 1,2-propanediol, and water-soluble carbohydrates increased, while the contents of ammonia-N and fiber were decreased, with increasing ratios of sweet sorghum in the mixed silage. Overall, coensiling Sesbania cannabina with >30% sweet sorghum is feasible to attain high-quality silage, and the relay action between homofermentative and heterofermentative LAB species could enhance fermentation quality and conserve the nutrients of the mixed silage. IMPORTANCE The coensiling of Sesbania cannabina and sweet sorghum is of great practical importance in order to alleviate the protein-rich forage deficiency in saline-alkaline regions. Furthermore, understanding the microbial community's dynamic changes, interactions, and metabolic pathways during ensiling will provide the theoretical basis to effectively regulate silage fermentation. Here, we established that coensiling Sesbania cannabina with >30% sweet sorghum was effective at ensuring better fermentation quality and preservation of nutrients. Moreover, the different fermentation types of LAB strains played a relay role during the fermentation process. The homofermentative species L. plantarum and L. farciminis dominated in the early phase of fermentation, while the heterofermentative species L. buchneri and L. hilgardii dominated in the later phase of fermentation. Their relay action in Sesbania cannabina-sweet sorghum mixed silage may help to improve fermentation quality and nutrient preservation.

High-throughput discovery of novel lanthipeptides and producers by metagenomic mining of isolates population (MMIP) from Chinese spicy cabbage.

Lanthipeptides are known antimicrobial agents having great potential for application in food preservation. Many lanthipeptide biosynthetic gene clusters (BGCs) were mined in fermented food microbiota, however, it is difficult to obtain the bioactive lanthipeptides and their producing strains. Here, we established a high-throughput strategy designated Metagenomic Mining of Isolates Population (MMIP) to efficiently excavate and obtain novel lanthipeptides, especially their potential producing strains. MMIP procedure involves gathering bacteria isolates using culturable strategy, metagenomic mining for lanthipeptides and screening their producers, and characterization of specific lanthipeptides. 928 biosynthetic gene clusters including 139 ribosomally synthesized and post-translationally modified peptides (RiPPs) gene clusters were discovered in the metagenomic data of the isolates by antiSMASH. Entianin, lactocin S, lichenicidin, and 17 novel lanthipeptides gene clusters corresponding to 29 possible producers were further found from the harvested isolates population. Entianin and a novel two-component lanthipeptide paralicin were purified from Bacillus subtilis C5B1 and Bacillus paralicheniformis BaC1-8, respectively. They showed strong inhibitory activity to food spoilage bacteria Bacillus cereus and Listeria monocytogenes, and have great potential for application in food preservation. A novel lanthipeptide polysacin was also obtained using semi-in vitro biosynthesis. MMIP affords a novel strategy for effectively excavating lanthipeptides, especially their producers from diverse environmental niches.

Nisin Z attenuates lipopolysaccharide-induced mastitis by inhibiting the ERK1/2 and p38 mitogen-activated protein kinase signaling pathways.

Nisin Z is a possible alternative for treating bovine mastitis by inhibiting mastitis-causing pathogens and having anti-inflammatory activity. However, the anti-inflammatory mechanism of nisin Z on mastitis is unknown. Our study aimed to investigate the mechanisms of nisin Z on mastitis. Our results showed that nisin Z inhibited the activation of the ERK1/2 and p38 mitogen-activated protein kinase (MAPK) signaling pathway, decreased the release of pro-inflammatory cytokines (i.e., tumor necrosis factor-α, IL-1ß, and IL-6), and increased the anti-inflammatory cytokine (IL-10) in lipopolysaccharide (LPS)-induced MCF10A cells. After intraperitoneal injection, nisin Z significantly decreased inflammatory cell infiltration in the mammary gland, as well as decreased myeloperoxidase and pro-inflammatory cytokines in serum and mammary gland. Western blot analysis revealed that nisin Z also dramatically suppressed the activation of the ERK1/2 and p38 MAPK signaling pathways in LPS-induced mastitis mice. We also found that nisin Z treatment could enhance the blood-milk barrier. In summary, our study demonstrated that nisin Z exerted an anti-inflammatory effect by inhibiting the ERK1/2 and p38 MAPK signaling pathway and promoting the blood-milk barrier on LPS-induced mastitis.

Characterization of a Novel Fructosyltransferase from Lactobacillus crispatus, InuCA, That Attaches to the Cell Surface by Electrostatic Interaction.

Fructosyltransferases (FTases), a group of carbohydrate-active enzymes, synthesize fructooligosaccharides (FOS) and fructans, which are promising prebiotics for human health. Here, we identified a novel FTase, InuCA, from Lactobacillus crispatus, a dominant species in the vaginal microbiota of human. InuCA was characterized by the shortest C terminus and the highest isoelectric point among the reported Lactobacillus FTases. InuCA was an inulosucrase and produced a series of FOS using sucrose as the substrate at a moderate temperature. Surprisingly, the C-terminal deletion mutant synthesized oligosaccharides with the fructosyl chain longer than that of the wild type, suggesting that the C-terminal part blocked the binding of long-chain receptor. Moreover, InuCA bound to the cell surface by electrostatic interaction, which was dependent on the environmental pH and represented a distinctive binding mode in FTases. The catalytic and structural properties of InuCA will contribute to FTase engineering and the knowledge of the adaptation of L. crispatus in the vaginal environment. IMPORTANCE L. crispatus is one of the most important species in human vaginal microbiotas, and its persistence is strongly negatively correlated with vaginal diseases. Our research reveals that a novel inulosucrase, InuCA, is present in L. crispatus. InuCA keeps the ability to synthesize prebiotic fructo-oligosaccharides, although it lacks a large part of the C-terminal region compared to other FTases. Remarkably, the short C terminus of InuCA blocks the transfructosylation activity for producing oligosaccharides with longer chains, which is meaningful for the directional modification of FTases and the oligosaccharide products. Besides the catalytic activity, InuCA is anchored on the cell surface, depending on the environmental pH, and also may be involved in the adhesion of L. crispatus to the vaginal epithelial cells. Since L. crispatus plays an essential role in the normal vaginal micro-ecosystem, the described work will be helpful to elucidate the functional genes and colonization mechanism of the dominant species.

Bacteriocins: Potential for Human Health.

Due to the challenges of antibiotic resistance to global health, bacteriocins as antimicrobial compounds have received more and more attention. Bacteriocins are biosynthesized by various microbes and are predominantly used as food preservatives to control foodborne pathogens. Now, increasing researches have focused on bacteriocins as potential clinical antimicrobials or immune-modulating agents to fight against the global threat to human health. Given the broad- or narrow-spectrum antimicrobial activity, bacteriocins have been reported to inhibit a wide range of clinically pathogenic and multidrug-resistant bacteria, thus preventing the infections caused by these bacteria in the human body. Otherwise, some bacteriocins also show anticancer, anti-inflammatory, and immune-modulatory activities. Because of the safety and being not easy to cause drug resistance, some bacteriocins appear to have better efficacy and application prospects than existing therapeutic agents do. In this review, we highlight the potential therapeutic activities of bacteriocins and suggest opportunities for their application.

Effects of Lactobacillus hilgardii 60TS-2, with or without homofermentative Lactobacillus plantarum B90, on the aerobic stability, fermentation quality and microbial community dynamics in sugarcane top silage.

This study investigated the effects of Lactobacillus hilgardii (LH), alone or in combination with Lactobacillus plantarum (LP), on the aerobic stability, fermentation quality and dynamics of the bacterial and fungal communities of sugarcane top silage. Results demonstrated that LH and LHLP (LH combined with LP) improved the aerobic stability of sugarcane top silages. As the exposure time increased, the pH values and the contents of lactic acid, acetic acid, as well as propionic acid remained stable in silage treated with LH and LHLP. The abundance of L. hilgardii was enriched and the undesirable microorganisms, such as Acetobacter pasteurianus, Paenibacillus amylolyticus and yeasts like Kazachstania humilis, were suppressed in silages treated with LH and LHLP. In conclusion, LH-treated silage, whether with LP or not, positively impacted the fungal and bacterial microbes. This improved the quality of fermentation, the aerobic stability, and reduced aerobic spoilage in sugarcane top silage.

Transcriptional Regulator AcrR Increases Ethanol Tolerance through Regulation of Fatty Acid Synthesis in Lactobacillus plantarum.

Lactobacillus plantarum is a versatile bacterium with significant adaptability to harsh habitats containing excessive ethanol concentrations. It was found that the L. plantarum NF92-TetR/AcrR family regulator, AcrR, significantly enhanced the growth rate of this lactic acid bacterium in the presence of ethanol. Through screening 172 ethanol-resistant related genes by electrophoretic mobility shift and quantitative reverse transcription-PCR (RT-qPCR) assays, six genes were identified to be regulated by AcrR under ethanol stress. Among these was a gene coding for a 3-hydroxyacyl-ACP dehydratase (fabZ1) regulated by AcrR under ethanol stress. AcrR regulated fabZ1 under ethanol stress by binding to its promoter, P fabZ1 DNase I footprinting analysis indicated that there were two specific AcrR binding sites on P fabZ1 RT-PCR results showed fabZ1 could cotranscribe with its downstream 12 genes and conform a fatty acid de novo biosynthesis (fab) gene cluster under the control of P fabZ1 Both RT-qPCR of the fab gene cluster in acrR knockout and overexpression strains and fatty acid methyl ester analysis of the acrR knockout strain showed that AcrR could promote fatty acid synthesis in L. plantarum NF92. Membrane fluorescence anisotropy analysis of acrR knockout and overexpression strains showed that AcrR could increase membrane fluidity under ethanol stress. Thus, AcrR could regulate fatty acid synthesis and membrane fluidity to promote the adaption of L. plantarum NF92 to a high ethanol concentration.IMPORTANCE Ethanol tolerance is essential for L. plantarum strains living in substances with more than 9% ethanol, such as wine and beer. The details regarding how L. plantarum adapts to ethanol are still lacking. This study demonstrates that AcrR regulates the de novo synthesis of fatty acids in L. plantarum adapting to toxic levels of ethanol. We also identified the ability of the TetR/AcrR family regulator to bind to the fatty acid biosynthesis gene promoter, P fabZ1 , in L. plantarum and defined the binding sites. This finding facilitates the induction of the adaptation of L. plantarum strains to ethanol for food fermentation applications.

Lactobacillus plantarum PFM 105 Promotes Intestinal Development Through Modulation of Gut Microbiota in Weaning Piglets.

Lactobacillus plantarum is a widespread bacterial species and is commonly used as a probiotic. L. plantarum PFM105 was isolated from the rectum of a healthy sow. Here we found that L. plantarum PFM105 showed probiotic effect on weaning piglets in which intestinal inflammation and unbalanced gut microbiota happened frequently. L. plantarum PFM105 was identified to improve the growth of weaning piglet and promote the development of small intestinal villi. Antibiotics are often used in weaning piglet to prevent intestinal infection and promote the growth of animal. We found that weaning piglets feeding with L. plantarum PFM105 showed similar growth promotion but decreased diarrhea incidence compared with those feeding with antibiotics. High-throughput sequencing was used to analyze the gut microbiota in weaning piglets treated with L. plantarum PFM105 or antibiotics. The relative abundance of beneficial microbes Prevotellaceae and Bifidobacteriaceae were increased in colon of weaning piglet feeding L. plantarum PFM105, while antibiotics increased the relative abundance of bacteria associated with pathogenicity, such as Spirochaeta and Campylobacteraceae. L. plantarum PFM 105 increased indicators of intestinal health including serum levels of IgM, IL-10, and TGF-ß, and colonic levels of SCFAs. We found strong correlations between the alterations in gut microbiota composition caused by feeding antibiotics and probiotics and the measured growth and health parameters in weaning piglets. The addition of L. plantarum PFM105 could significantly increase the relative abundance of metabolic genes which may important to intestinal microbiota maturation. Altogether, we demonstrated here that L. plantarum PFM 105 could promote intestinal development through modulation of gut microbiota in weaning piglets.

AcrR and Rex Control Mannitol and Sorbitol Utilization through Their Cross-Regulation of Aldehyde-Alcohol Dehydrogenase (AdhE) in Lactobacillus plantarum.

Lactobacillus plantarum is a versatile bacterium that occupies a wide range of environmental niches. In this study, we found that a bifunctional aldehyde-alcohol dehydrogenase-encoding gene, adhE, was responsible for L. plantarum being able to utilize mannitol and sorbitol through cross-regulation by two DNA-binding regulators. In L. plantarum NF92, adhE was greatly induced, and the growth of an adhE-disrupted (ΔadhE) strain was repressed when sorbitol or mannitol instead of glucose was used as a carbon source. The results of enzyme activity and metabolite assays demonstrated that AdhE could catalyze the synthesis of ethanol in L. plantarum NF92 when sorbitol or mannitol was used as the carbon source. AcrR and Rex were two transcriptional factors screened by an affinity isolation method and verified to regulate the expression of adhE DNase I footprinting assay results showed that they shared a binding site (GTTCATTAATGAAC) in the adhE promoter. Overexpression and knockout of AcrR showed that AcrR was a novel regulator to promote the transcription of adhE The activator AcrR and repressor Rex may cross-regulate adhE when L. plantarum NF92 utilizes sorbitol or mannitol. Thus, a model of the control of adhE by AcrR and Rex during L. plantarum NF92 utilization of mannitol or sorbitol was proposed.IMPORTANCE The function and regulation of AdhE in the important probiotic genus Lactobacillus are rarely reported. Here we demonstrated that AdhE is responsible for sorbitol and mannitol utilization and is cross-regulated by two transcriptional regulators in L. plantarum NF92, which had not been reported previously. This is important for L. plantarum to compete and survive in some harsh environments in which sorbitol or mannitol could be used as carbon source. A novel transcriptional regulator AcrR was identified to be important to promote the expression of adhE, which was unknown before. The cross-regulation of adhE by AcrR and Rex is important to balance the level of NADH in the cell during sorbitol or mannitol utilization.

Lactobacillus reuteri HCM2 protects mice against Enterotoxigenic Escherichia coli through modulation of gut microbiota.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of infectious diarrhea in children and postweaning piglets. ETEC infection results in induced pro-inflammatory responses in intestinal epithelial cells and dysbiosis of intestinal microbiota. Here, a Lactobacillus reuteri strain, HCM2, isolated from a healthy piglet showed a high survival rate in the harsh gastrointestinal tract environment and inhibited the growth of ETEC and its adherence to intestinal epithelial cells. Pre-supplementation with L. reuteri HCM2 for 14 days reduced the ETEC load in the jejunum of ETEC-infected mice and prevented the disruption of intestinal morphology by ETEC. The colonic microbiota of mice with or without HCM2 pre-supplementation were analyzed, and this analysis revealed that HCM2 could prevent dysbiosis caused by ETEC infection by stabilizing the relative abundance of dominant bacteria. These results indicate that L. reuteri HCM2 has the potential to attenuate the effect of ETEC on the colonic microbiota in infected mice.

CerR, a Single-Domain Regulatory Protein of the LuxR Family, Promotes Cerecidin Production and Immunity in Bacillus cereus.

Cerecidins are small lantibiotics from Bacillus cereus that were obtained using a semi-in vitro biosynthesis strategy and showed prominent antimicrobial activities against certain Gram-positive bacteria. However, the parental strain B. cereus As 1.1846 is incapable of producing cerecidins, most probably due to the transcriptional repression of the cerecidin gene cluster. Located in the cerecidin gene cluster, cerR encodes a putative response regulator protein that belongs to the LuxR family transcriptional regulators. CerR (84 amino acids) contains only a conserved DNA binding domain and lacks a conventional phosphorylation domain, which is rarely found in lantibiotic gene clusters. To investigate its function in cerecidin biosynthesis, cerR was constitutively expressed in B. cereus As 1.1846. Surprisingly, Constitutive expression of cerR enabled the production of cerecidins and enhanced self-immunity of B. cereus toward cerecidins. Reverse transcription-PCR analysis and electrophoresis mobility shift assays indicated, respectively, that the cer cluster was transcribed in two transcripts (cerAM and cerRTPFE) and that CerR regulated the cerecidin gene cluster directly by binding to the two predicted promoter regions of cerA and cerR DNase I footprinting experiments further confirmed that CerR specifically bound to the two promoter regions at a conserved inverted repeat sequence that was designated a CerR binding motif (cerR box). The present study demonstrated that CerR, as the first single-domain LuxR family transcriptional regulator, serves as a transcriptional activator in cerecidin biosynthesis and activates the cerecidin gene cluster, which was otherwise cryptic in B. cereusIMPORTANCE Lantibiotics with intriguing and prominent bioactivities are potential peptide antibiotics that could be applied in many areas, including food and pharmaceutical industries. The biosynthesis of lantibiotics is generally controlled by two-component regulatory systems consisting of histidine kinases and response regulators, while some unique and interesting regulatory systems are also revealed with the ever-increasing discovery of lantibiotic gene clusters among diverse microorganisms. Dissection of diverse lantibiotic regulation machineries would permit deep understanding of the biological functions of lantibiotics in different niches and even enable genetic activation of lantibiotic gene clusters that are otherwise cryptic. The significance of our study is to illuminate the regulatory mechanism of a special single-domain protein, CerR, in regulating cerecidin biosynthesis in Bacillus cereus, providing a possible novel approach to activate cryptic lantibiotic clusters.

Transcriptome responses of Lactobacillus acetotolerans F28 to a short and long term ethanol stress.

Lactobacillus acetotolerans is a major microbe contributing to the Chinese liquor fermentation with unknown function. It can be grown well in a high concentration of ethanol. RNA sequencing (RNA-seq) was performed on L. acetotolerans F28 growing in 12% ethanol to determine important genetic mechanisms for both a short and long term adaption to this environment. A genome-wide transcriptional analysis revealed that the most important genetic elements for L. acetotolerans F28 grown in ethanol are related to high levels of stress response and fatty acid biosynthesis, and a reduction of amino acid transport and metabolism after both a short and long time stress. The fatty acid methyl ester analyses showed that most fatty acids were increased in L. acetotolerans F28 after exposure to ethanol while the unsaturated fatty acid octadecenoic acid (C18:1) was significantly increased. The increasing unsaturated fatty acid biosynthesis in L. acetotolerans F28 might enhance cell membrane fluidity and protect the cells against high concentration of ethanol. Overall, the transcriptome and functional analysis indicated that the elevated stress response and fatty acid biosynthesis, and the decrease of amino acid transport and metabolism might play important roles for L. acetotolerans F28 to adapt to environmental ethanol.

Crosstalk between Tryptophan Metabolism and Cardiovascular Disease, Mechanisms, and Therapeutic Implications.

The cardiovascular diseases (CVD) associated with the highest rates of morbidity are coronary heart disease and stroke, and the primary etiological factor leading to these conditions is atherosclerosis. This long-lasting inflammatory disease, characterized by how it affects the artery wall, results from maladaptive immune responses linked to the vessel wall. Tryptophan (Trp) is oxidized in a constitutive manner by tryptophan 2,3-dioxygenase in liver cells, and for alternative cell types, it is catalyzed in the presence of a differently inducible indoleamine 2,3-dioxygenase (IDO1) in the context of a specific pathophysiological environment. Resultantly, this leads to a rise in the production of kynurenine (Kyn) metabolites. Inflammation in the preliminary stages of atherosclerosis has a significant impact on IDO1, and IDO1 and the IDO1-associated pathway constitute critical mediating agents associated with the immunoinflammatory responses that characterize advanced atherosclerosis. The purpose of this review is to survey the recent literature addressing the kynurenine pathway of tryptophan degradation in CVD, and the author will direct attention to the function performed by IDO1-mediated tryptophan metabolism.

Identification of Key Residues in the NisK Sensor Region for Nisin Biosynthesis Regulation.

Histidine kinase (HK) NisK is well known to sense lantibiotic nisin for regulating the biosynthesis of nisin. NisK possesses two trans-membrane segments and a large extracellular region and nisin contains 34 amino acids with five lanthionine rings. Unlike most peptide sensing HK with multi trans-membrane segments, NisK is a representative of a group of rarely reported HK that sense peptide as ligand. To reveal how NisK senses nisin molecule to regulate nisin biosynthesis, we constructed a reporter Lactococcus lactis strain with nisRK constitutively expressed and a reporter gene lacZ expressed under the control of promoter P nisA . We showed that the extracellular region of NisK was involved in recognizing nisin. Conserved residues in this group of HK were found in the extracellular region of NisK and mutagenesis of these residues in the reporter strain revealed that several hydrophobic residues including two aromatic residues are crucial for NisK sensing nisin and regulating nisin biosynthesis. Substitutions of hydrophobic regions in NisK extracellular domain showed that the first strand that was rich of hydrophobic amino acids was involved in regulating nisin biosynthesis. A negatively charged residue in the first ßstrand also contributed to nisin biosynthesis. Protein binding analyses demonstrated that nisin could not interact with key NisK mutants, indicating these site in the extracellular region of NisK was involved in recognizing nisin.

One-pot synthesis of class II lanthipeptide bovicin HJ50 via an engineered lanthipeptide synthetase.

Lanthipeptides are a large class of bacteria-produced, ribosomally-synthesized and post-translationally modified peptides. They are recognized as peptide antibiotics because most of them exhibit potent antimicrobial activities against Gram-positive bacteria especially those that are phylogenetically related to producers. Maturation of class II lanthipeptide like bovicin HJ50 undergoes precursor modification by LanM and a subsequent leader peptide cleavage by LanT. Herein, via co-expression of precursor gene bovA, modification gene bovM and transporter gene bovT in Escherichia coli C43 (DE3), bioactive bovicin HJ50 was successfully produced and secreted. To further achieve in vitro one-pot synthesis of bovicin HJ50, an engineered bovicin HJ50 synthetase BovT150M was obtained by fusing the peptidase domain of BovT (BovT150) to the N-terminus of BovM. BovT150M exhibited dual functions of precursor modification and leader peptide cleavage to release mature bovicin HJ50. Under the guidance of BovA leader peptide, BovT150M exhibited substrate tolerance to modify non-native substrates including suicin and lacticin 481. This work exemplifies the feasibility of enzyme chimera of peptidase domain (LanT150) and modification enzyme (LanM) as a one-pot lanthipeptide synthetase.