Myricetin Acts as an Inhibitor of Type II NADH Dehydrogenase from Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a common pathogenic microorganism in humans and animals. Type II NADH oxidoreductase (NDH-2) is the only NADH:quinone oxidoreductase present in this organism and represents a promising target for the development of anti-staphylococcal drugs. Recently, myricetin, a natural flavonoid from vegetables and fruits, was found to be a potential inhibitor of NDH-2 of S. aureus. The objective of this study was to evaluate the inhibitory properties of myricetin against NDH-2 and its impact on the growth and expression of virulence factors in S. aureus. RESULTS: A screening method was established to identify effective inhibitors of NDH-2, based on heterologously expressed S. aureus NDH-2. Myricetin was found to be an effective inhibitor of NDH-2 with a half maximal inhibitory concentration (IC50) of 2 µM. In silico predictions and enzyme inhibition kinetics further characterized myricetin as a competitive inhibitor of NDH-2 with respect to the substrate menadione (MK). The minimum inhibitory concentrations (MICs) of myricetin against S. aureus strains ranged from 64 to 128 µg/mL. Time-kill assays showed that myricetin was a bactericidal agent against S. aureus. In line with being a competitive inhibitor of the NDH-2 substrate MK, the anti-staphylococcal activity of myricetin was antagonized by MK-4. In addition, myricetin was found to inhibit the gene expression of enterotoxin SeA and reduce the hemolytic activity induced by S. aureus culture on rabbit erythrocytes in a dose-dependent manner. CONCLUSIONS: Myricetin was newly discovered to be a competitive inhibitor of S. aureus NDH-2 in relation to the substrate MK. This discovery offers a fresh perspective on the anti-staphylococcal activity of myricetin.

Coelonin protects against PM2 .5 -induced macrophage damage via suppressing TLR4/NF-κB/COX-2 signaling pathway and NLRP3 inflammasome activation in vitro.

One of the important monitoring indicators of the air pollution is atmospheric fine particulate matter (PM2.5 ), which can induce lung inflammation after inhalation. Coelonin can alleviate PM2.5 -induced macrophage damage through anti-inflammation. However, its molecular mechanism remains unclear. We hypothesized that macrophage damage may involve the release of inflammatory cytokines, activation of inflammatory pathways, and pyrosis induced by inflammasome. In this study, we evaluated the anti-inflammation activity of coelonin in PM2.5 -induced macrophage and its mechanism of action. Nitric oxide (NO) and reactive oxygen species (ROS) production were measured by NO Assay kit and dichlorofluorescein-diacetate (DCFH-DA), and apoptosis were measured by Flow cytometry and TUNEL staining. The concentration of inflammatory cytokines production was measured with cytometric bead arrays and ELISA kits. The activation of NF-κB signaling pathway and NLRP3 inflammasome were measured by immunofluorescence, quantitative reverse transcription-polymerase chain reaction and western blot. As expected, coelonin pretreatment reduced NO production significantly as well as alleviated cell damage by decreasing ROS and apoptosis. It decreased generation of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in PM2.5 -induced RAW264.7 and J774A.1 cells. Moreover, coelonin markedly inhibited upregulating the expression of toll-like receptor (TLR)4 and cyclo-oxygenase (COX)-2, blocked activation of p-nuclear factor-kappa B (NF-κB) signaling pathway, and suppressed expression of NLRP3 inflammasome, ASC, GSDMD, IL-18 and IL-1ß. In conclusion, the results showed that coelonin could protect against PM2.5 -induced macrophage damage via suppressing TLR4/NF-κB/COX-2 signaling pathway and NLRP3 inflammasome activation in vitro.

The Antibacterial Properties of 4, 8, 4', 8'-Tetramethoxy (1,1'-biphenanthrene) -2,7,2',7'-Tetrol from Fibrous Roots of Bletilla striata.

Biphenanthrene compound, 4, 8, 4', 8'-tetramethoxy (1, 1'-biphenanthrene)-2, 7, 2', 7'-tetrol (LF05), recently isolated from fibrous roots of Bletilla striata, exhibits antibacterial activity against several Gram-positive bacteria. In this study, we investigated the antibacterial properties, potential mode of action and cytotoxicity. Minimum inhibitory concentrations (MICs) tests showed LF05 was active against all tested Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA) and staphylococcal clinical isolates. Minimum bactericidal concentration (MBC) tests demonstrated LF05 was bactericidal against S. aureus ATCC 29213 and Bacillus subtilis 168 whereas bacteriostatic against S. aureus ATCC 43300, WX 0002, and other strains of S. aureus. Time-kill assays further confirmed these observations. The flow cytometric assay indicated that LF05 damaged the cell membrane of S. aureus ATCC 29213 and B. subtilis 168. Consistent with this finding, 4 × MIC of LF05 caused release of ATP in B. subtilis 168 within 10 min. Checkerboard test demonstrated LF05 exhibited additive effect when combined with vancomycin, erythromycin and berberine. The addition of rat plasma or bovine serum albumin to bacterial cultures caused significantly loss in antibacterial activity of LF05. Interestingly, LF05 was highly toxic to several tumor cells. Results of these studies indicate that LF05 is bactericidal against some Gram-positive bacteria and acts as a membrane structure disruptor. The application of biphenanthrene in the treatment of S. aureus infection, especially local infection, deserves further study.

Antiviral activity of phenanthrenes from the medicinal plant Bletilla striata against influenza A virus.

BACKGROUND: Influenza represents a serious public health concern. The emergence of resistance to anti-influenza drugs underlines the need to develop new drugs. This study aimed to evaluate the anti-influenza viral activity and possible mechanisms of 12 phenanthrenes from the medicinal plant Bletilla striata (Orchidaceae family). METHODS: Twelve phenanthrenes were isolated and identified from B. striata. Influenza virus A/Sydney/5/97 (H3N2) propagated in embryonated chicken eggs was used. Phenanthrenes mixed with the virus were incubated at 37 °C for 1 h and then inoculated into 9-day-old embryonated chicken eggs via the allantoic route to survey the antiviral activity in vivo. A (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS)-based assay was performed to evaluate the reduction of cytopathic effect induced by H3N2 on Madin-Darby canine kidney (MDCK) cells. The hemagglutination inhibition assay was used to study the blockage of virus receptors by the phenanthrenes, and the neuraminidase (NA) inhibition assay to evaluate the effects of the release of virus. The synthesis of influenza viral matrix protein mRNA in response to compound treatment was measured by real-time polymerase chain reaction. RESULTS: This study showed that phenanthrenes 1, 2, 3, 4, 6, 9, 10, 11, and 12 significantly inhibited the viruses in vivo, with inhibition rates of 20.7, 79.3, 17.2, 34.5, 34.5, 34.5, 44.8, 75.9, and 34.5%, respectively. In MDCK models, the phenanthrenes did not show significant antiviral activity when administered as pretreatment, while phenanthrenes 2, 3, 4, 6, 7 10, and 11 exhibited inhibitory activities as simultaneous treatment with 50% inhibition concentration (IC50) ranging from 14.6 ± 2.4 to 43.3 ± 5.3 µM. The IC50 ranged from 18.4 ± 3.1 to 42.3 ± 3.9 µM in the post-treatment assays. Compounds 1, 3, 4, 6, 10, and 11 exhibited an inhibitory effect on NA; and compounds 2, 3, 4 6, 7, 10, and 11 resulted in the reduced transcription of virus matrix protein mRNA. However, no compound could inhibit hemagglutination by the influenza virus. CONCLUSION: Phenanthrenes from B. striata had strong anti-influenza viral activity in both embryonated eggs and MDCK models, and diphenanthrenes seemed to have stronger inhibition activity compared with monophenanthrenes.

The anti-Staphylococcus aureus activity of the phenanthrene fraction from fibrous roots of Bletilla striata.

BACKGROUND: Bletillae Rhizoma, the tuber of Bletilla striata, has been used in Chinese traditional medicine to treat infectious diseases. Chemical studies indicated that phenanthrene was one of the most important components of the herb, with a broad spectrum of antibiotic activity against Gram-positive bacteria. The objective of this study was to further characterize the antibacterial activity of the phenanthrene fraction from the fibrous root of the pseudobulb of B. striata. METHODS: The phenanthrene fraction (EF60) from the ethanol extract of fibrous roots of Bletilla striata pseudobulbs was isolated using polyamide column chromatography. The antibacterial activity of the fraction was evaluated in vitro using a 96-well microtiter plate and microbroth dilution method. The cytotoxicity of EF60 against mammalian cells was tested by hemolysis and MTT assays. RESULTS: EF60 was obtained using alcohol extraction and polyamide column chromatography, with a yield of 14.9 g per 1 kg of the fibrous roots of B. striata. In vitro tests indicated that EF60 was active against all tested strains of Staphylococcus aureus, including clinical isolates and methicillin-resistant S. aureus (MRSA). The minimum inhibitory concentration (MIC) values of EF60 against these pathogens ranged from 8 to 64 µg/mL. Minimum bactericidal concentration tests demonstrated that EF60 was bactericidal against S. aureus 3304 and ATCC 29213 and was bacteriostatic against S. aureus 3211, ATCC 25923, and ATCC 43300. Consistently, the time-kill assay indicated that EF60 could completely kill S. aureus ATCC 29213 at 2× the MIC within 3 h but could kill less than two logarithmic units of ATCC 43300, even at 4× the MIC within 24 h. The postantibiotic effects (PAE) of EF60 (4× MIC) against strains 29213 and 43300 were 2.0 and 0.38 h, respectively. Further studies indicated that EF60 (160 µg/mL) showed no cytotoxicity against human erythrocytes, and was minimally toxic to Human Umbilical Vein Endothelial Cells with an IC50 of 75 µg/mL. CONCLUSIONS: Our studies indicated that EF60 is worthy of further investigation as a potential phytotherapeutic agent for treating infections caused by S. aureus and MRSA.

[Antibacterial Activity of Chemical Constituents Isolated from Fibrous Roots of Bletilla striata].

Objective: To investigate the chemical constituents isolated from the fibrous roots of Bletilla striata, and to research their antibacterial activities. Methods: The native products were isolated and purified by silica gel, Sephadex LH-20 column chromatography and preparative HPLC. Their structures were elucidated on the basis of various spectroscopic analysis, and their antibacterial activities were tested by microbroth dilution method in a 96-well microtiter plate. Results: Seven compounds were isolated from the ethanol extract of the fibrous roots of Bletilla striata, and identified as p-hydroxybenzaldehyde( 1),2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene( 2),4,5-dihydroxy-2-methoxy-9,10-dihydrohenanthrpene( 3),2-dihydroxy-4,7-dimethoxyphenan-threne( 4), militarine( 5), dactylorhin A( 6) and gastrodin( 7). Among them, compounds 2 ~ 4 showed moderate antibacterial activities against several Gram-positive bacterial strains( MIC 8 ~ 128 µg / m L),such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Bacillus subtilis. Conclusion: The fibrous roots and tubers of Bletilla striata contain similar compounds, including glucosyloxybenzyl 2-isobutylmalates,and phenanthrene compounds, which showed antimicrobial activities against Gram-positive bacterial strains. And compounds 3,4 are isolated from Bletilla genus for the first time.

Antibacterial Biphenanthrenes from the Fibrous Roots of Bletilla striata.

Four new 9',10'-dihydro-biphenanthrenes, including an unprecedented 1,2'-linked biphenanthrene, 4,7,3',5'-tetramethoxy-9',10'-dihydro(1,2'-biphenanthrene)-2,7'-diol (1), a new 1,3'-linked biphenanthrene, 4,7,7'-trimethoxy-9',10'-dihydro(1,3'-biphenanthrene)-2,2',5'-triol (2), and two new 1,1'-linked biphenanthrenes, 4,7,4'-trimethoxy-9',10'-dihydro(1,1'-biphenanthrene)-2,2',7'-triol (3) and 4,7,3',5'-tetramethoxy-9',10'-dihydro(1,1'-biphenanthrene)-2,2',7'-triol (4), as well as two known biphenanthrenes (5, 6), were isolated from a 95% ethanol extract of the fibrous roots of Bletilla striata. Their structures were determined by spectroscopic and spectrometric methods. Atropisomerism of these compounds was considered based on their chiral optical properties and potential energy surface scans at the ab initio HF/3-21G level, which revealed their racemic mixture form. Compounds 2-6 showed potent antibacterial activities against six Gram-positive bacterial strains.

Two UDP-glucuronic acid decarboxylases involved in the biosynthesis of a bacterial exopolysaccharide in Paenibacillus elgii.

Xylose is described as a component of bacterial exopolysaccharides in only a limited number of bacterial strains. A bacterial strain, Paenibacillus elgii, B69 was shown to be efficient in producing a xylose-containing exopolysaccharide. Sequence analysis was performed to identify the genes encoding the uridine diphosphate (UDP)-glucuronic acid decarboxylase required for the synthesis of UDP-xylose, the precursor of the exopolysaccharide. Two sequences, designated as Peuxs1 and Peuxs2, were found as the candidate genes for such enzymes. The activities of the UDP-glucuronic acid decarboxylases were proven by heterologous expression and real-time nuclear magnetic resonance analysis. The intracellular activity and effect of these genes on the synthesis of exopolysaccharide were further investigated by developing a thymidylate synthase based knockout system. This system was used to substitute the conventional antibiotic resistance gene system in P. elgii, a natural multi-antibiotic resistant strain. Results of intracellular nucleotide sugar analysis showed that the intracellular UDP-xylose and UDP-glucuronic acid levels were affected in Peuxs1 or Peuxs2 knockout strains. The knockout of either Peuxs1 or Peuxs2 reduced the polysaccharide production and changed the monosaccharide ratio. No polysaccharide was found in the Peuxs1/Peuxs2 double knockout strain. Our results show that P. elgii can be efficient in forming UDP-xylose, which is then used for the synthesis of xylose-containing exopolysaccharide.

Lasiodiplodia sp. ME4-2, an endophytic fungus from the floral parts of Viscum coloratum, produces indole-3-carboxylic acid and other aromatic metabolites.

BACKGROUND: Studies on endophytes, a relatively under-explored group of microorganisms, are currently popular amongst biologists and natural product researchers. A fungal strain (ME4-2) was isolated from flower samples of mistletoe (Viscum coloratum) during a screening program for endophytes. As limited information on floral endophytes is available, the aim of the present study is to characterise fungal endophytes using their secondary metabolites. RESULTS: ME4-2 grew well in both natural and basic synthetic media but produced no conidia. Sequence analysis of its internal transcribed spacer rDNA demonstrated that ME4-2 forms a distinct branch within the genus Lasiodiplodia and is closely related to L. pseudotheobromae. This floral endophyte was thus identified as Lasiodiplodia sp. based on its molecular biological characteristics. Five aromatic compounds, including cyclo-(Trp-Ala), indole-3-carboxylic acid (ICA), indole-3-carbaldehyde, mellein and 2-phenylethanol, were found in the culture. The structures of these compounds were determined using spectroscopic methods combined with gas chromatography. To the best of our knowledge, our work is the first to report isolation of these aromatic metabolites from a floral endophyte. Interestingly, ICA, a major secondary metabolite produced by ME4-2, seemed to be biosynthesized via an unusual pathway. Furthermore, our results indicate that the fungus ME4-2 is a potent producer of 2-phenylethanol, which is a common component of floral essential oils. CONCLUSIONS: This study introduces a fungal strain producing several important aromatic metabolites with pharmaceutical or food applications and suggests that endophytic fungi isolated from plant flowers are promising natural sources of aromatic compounds.

[Protoplasts isolation, purification and plant regeneration of Pinellia cordata].

The main factors which affected the isolation, purification and cultivation of Pinellia cordata protoplasts from leaves were studied. The results indicated that the optimum enzyme solution for P. cordata leaves was 13% CPW + 1.0% Cellulose +0.1% Pectolase, at pH 6.0, temperature (25-28 degrees C ) for 4 h. The sucrose density gradient centrifugation was adopted to purificate the protoplasts collected, when 25% sucrose was used as mediator, centrifugating at 500 rpm for 10 min. When the protoplasts were shallow liquid and liquid-solid double layer cultured on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA + 13% mannitol at the density of 2.5 x 104 protoplasts/mL, or fed and nursed cultured at the density of 100-500 protoplasts/mL, cell division could be observed for 3 days; granular calli appeared for 30 days. Calli was proliferated on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA solidified by 0.55% agar, and differentiated and regenerated after 5-6 months. Plant generation of P. cordata is successfully established.

The induced and intrinsic resistance of Escherichia coli to sanguinarine is mediated by AcrB efflux pump.

IMPORTANCE: The use of plant extracts is increasing as an alternative to synthetic compounds, especially antibiotics. However, there is no sufficient knowledge on the mechanisms and potential risks of antibiotic resistance induced by these phytochemicals. In the present study, we found that stable drug resistant mutants of E. coli emerged after repetitive exposure to sanguinarine and demonstrated that the AcrB efflux pump contributed to the emerging of induced and intrinsic resistance of E. coli to this phytochemical. Our results offered some insights into comprehending and preventing the onset of drug-resistant strains when utilizing products containing sanguinarine.

Ameliorating role of Tetrastigma hemsleyanum polysaccharides in antibiotic-induced intestinal mucosal barrier dysfunction in mice based on microbiome and metabolome analyses.

The intestinal mucosal barrier is one of the important barriers to prevent harmful substances and pathogens from entering the body environment and to maintain intestinal homeostasis. This study investigated the reparative effect and possible mechanism of Tetrastigma hemsleyanum polysaccharides (THP) on ceftriaxone-induced intestinal mucosal damage. Our results suggested that THP repaired the mechanical barrier damage of intestinal mucosa by enhancing the expression of intestinal tight junction proteins, reducing intestinal mucosal permeability and improving the pathological state of intestinal epithelial cells. Intestinal immune and chemical barrier was further restored by THP via the increment of the body's cytokine levels, intestinal SIgA levels, intestinal goblet cell number, intestinal mucin-2 levels, and short-chain fatty acid levels. In addition, THP increased the abundance of probiotic bacteria (such as Lactobacillus), reduced the abundance of harmful bacteria (such as Enterococcus) to repair the intestinal biological barrier, restored intestinal mucosal barrier function, and maintains intestinal homeostasis. The possible mechanisms were related to sphingolipid metabolism, linoleic acid metabolism, and D-glutamine and D-glutamate metabolism. Our results demonstrated the potential therapeutic effect of THP against intestinal flora disorders and intestinal barrier function impairment caused by antibiotics.

Hinokitiol Selectively Enhances the Antibacterial Activity of Tetracyclines against Staphylococcus aureus.

The increasing prevalence of antibiotic resistance causes an urgent need for alternative agents to combat drug-resistant bacterial pathogens. Plant-derived compounds are promising candidates for the treatment of infections caused by antibiotic-resistant bacteria. Hinokitiol (ß-thujaplicin), a natural tropolone derivative found in the heartwood of cupressaceous plants, has been widely used in oral and skin care products as an antimicrobial agent. The aim of this work was to study the synergy potential of hinokitiol with antibiotics against Staphylococcus aureus, which is an extremely successful opportunistic pathogen capable of causing nosocomial and community-acquired infections worldwide. The MIC was determined by the broth microdilution method, and the effect of combinations was evaluated through fractional inhibitory concentration indices (FICI). The mechanism behind this synergy was also investigated by using fluorescence spectroscopy and high-performance liquid chromatography (HPLC). The MICs of hinokitiol alone against most S. aureus strains were 32 µg/mL. Selectively synergistic activities (FICIs of ≤0.5) were observed for combinations of this phytochemical with tetracyclines against all tested strains of S. aureus. Importantly, hinokitiol at 1 µg/mL completely or partially reversed tetracycline resistance in staphylococcal isolates. The increased accumulation of tetracycline inside S. aureus in the presence of hinokitiol was observed. In addition, hinokitiol promoted the uptake of ethidium bromide (EB) in bacterial cells without membrane depolarization, suggesting that it may be an efflux pump inhibitor. IMPORTANCE The disease caused by S. aureus is a public health issue due to the continuing emergence of drug-resistant strains, particularly methicillin-resistant S. aureus (MRSA). Tetracyclines, one of the old classes of antimicrobials, have been used for the treatment of infections caused by S. aureus. However, the increased resistance to tetracyclines together with their toxicity have limited their use in the clinic. Here, we demonstrated that the combination of hinokitiol and tetracyclines displayed synergistic antibacterial activity against S. aureus, including tetracycline-resistant strains and MRSA, offering a potential alternative approach for the treatment of infections caused by this bacterium.

Battacin (Octapeptin B5), a new cyclic lipopeptide antibiotic from Paenibacillus tianmuensis active against multidrug-resistant Gram-negative bacteria.

Hospital-acquired infections caused by drug-resistant bacteria are a significant challenge to patient safety. Numerous clinical isolates resistant to almost all commercially available antibiotics have emerged. Thus, novel antimicrobial agents, specifically those for multidrug-resistant Gram-negative bacteria, are urgently needed. In the current study, we report the isolation, structure elucidation, and preliminary biological characterization of a new cationic lipopeptide antibiotic, battacin or octapeptin B5, produced from a Paenibacillus tianmuensis soil isolate. Battacin kills bacteria in vitro and has potent activity against Gram-negative bacteria, including multidrug-resistant and extremely drug-resistant clinical isolates. Hospital strains of Escherichia coli and Pseudomonas aeruginosa are the pathogens most sensitive to battacin, with MICs of 2 to 4 µg/ml. The ability of battacin to disrupt the outer membrane of Gram-negative bacteria is comparable to that of polymyxin B, the last-line therapy for infections caused by antibiotic-resistant Gram-negative bacteria. However, the capacity of battacin to permeate bacterial plasma membranes is less extensive than that of polymyxin B. The bactericidal kinetics of battacin correlate with the depolarization of the cell membrane, suggesting that battacin kills bacteria by disrupting the cytoplasmic membrane. Other studies indicate that battacin is less acutely toxic than polymyxin B and has potent in vivo biological activity against E. coli. Based on the findings of the current study, battacin may be considered a potential therapeutic agent for the treatment of infections caused by antibiotic-resistant Gram-negative bacteria.

Gene cluster analysis for the biosynthesis of elgicins, novel lantibiotics produced by Paenibacillus elgii B69.

BACKGROUND: The recent increase in bacterial resistance to antibiotics has promoted the exploration of novel antibacterial materials. As a result, many researchers are undertaking work to identify new lantibiotics because of their potent antimicrobial activities. The objective of this study was to provide details of a lantibiotic-like gene cluster in Paenibacillus elgii B69 and to produce the antibacterial substances coded by this gene cluster based on culture screening. RESULTS: Analysis of the P. elgii B69 genome sequence revealed the presence of a lantibiotic-like gene cluster composed of five open reading frames (elgT1, elgC, elgT2, elgB, and elgA). Screening of culture extracts for active substances possessing the predicted properties of the encoded product led to the isolation of four novel peptides (elgicins AI, AII, B, and C) with a broad inhibitory spectrum. The molecular weights of these peptides were 4536, 4593, 4706, and 4820 Da, respectively. The N-terminal sequence of elgicin B was Leu-Gly-Asn-Tyr, [corrected] which corresponded to the partial sequence of the peptide ElgA encoded by elgA. Edman degradation suggested that the product elgicin B is derived from ElgA. By correlating the results of electrospray ionization-mass spectrometry analyses of elgicins AI, AII, and C, these peptides are deduced to have originated from the same precursor, ElgA. CONCLUSIONS: A novel lantibiotic-like gene cluster was shown to be present in P. elgii B69. Four new lantibiotics with a broad inhibitory spectrum were isolated, and these appear to be promising antibacterial agents.

Identification and functional analysis of gene cluster involvement in biosynthesis of the cyclic lipopeptide antibiotic pelgipeptin produced by Paenibacillus elgii.

BACKGROUND: Pelgipeptin, a potent antibacterial and antifungal agent, is a non-ribosomally synthesised lipopeptide antibiotic. This compound consists of a ß-hydroxy fatty acid and nine amino acids. To date, there is no information about its biosynthetic pathway. RESULTS: A potential pelgipeptin synthetase gene cluster (plp) was identified from Paenibacillus elgii B69 through genome analysis. The gene cluster spans 40.8 kb with eight open reading frames. Among the genes in this cluster, three large genes, plpD, plpE, and plpF, were shown to encode non-ribosomal peptide synthetases (NRPSs), with one, seven, and one module(s), respectively. Bioinformatic analysis of the substrate specificity of all nine adenylation domains indicated that the sequence of the NRPS modules is well collinear with the order of amino acids in pelgipeptin. Additional biochemical analysis of four recombinant adenylation domains (PlpD A1, PlpE A1, PlpE A3, and PlpF A1) provided further evidence that the plp gene cluster involved in pelgipeptin biosynthesis. CONCLUSIONS: In this study, a gene cluster (plp) responsible for the biosynthesis of pelgipeptin was identified from the genome sequence of Paenibacillus elgii B69. The identification of the plp gene cluster provides an opportunity to develop novel lipopeptide antibiotics by genetic engineering.

Polysaccharides From the Aerial Parts of Tetrastigma Hemsleyanum Diels et Gilg Induce Bidirectional Immunity and Ameliorate LPS-Induced Acute Respiratory Distress Syndrome in Mice.

Tetrastigma hemsleyanum Diels et Gilg (Sanyeqing, SYQ) has traditionally been used to treat inflammation, high fever and improve immune function of patients. Polysaccharides have been proved to be one of the important components of SYQ. Previous studies have confirmed the antipyretic and antitumor effects of polysaccharides from SYQ (SYQP), and clarified that SYQP could enhance immunity through TLR4 signalling pathway. However, there were more possibilities for the mechanism by which SYQP exerted immunomodulatory effects and the role of SYQP in acute respiratory distress syndrome (ARDS) is elusive. The purpose of this study was further to explain the bidirectional modulation of immunity mechanism of SYQP in vitro and its effect in LPS-induced ARDS in vivo. Experimental results showed that SYQP significantly stimulated gene expressions of TLR1, TLR2 and TLR6 and secretion of cytokines in RAW264.7 cells. Individual or combined application of TLR2 antagonist C29 and TLR4 antagonist TAK-242 could reduce SYQP-mediated stimulation of cytokine secretion in RAW264.7 cells and mouse peritoneal macrophages (MPMs) to varying degrees. On the other hand, SYQP markedly inhibited the expression levels of inflammatory cytokines, NO, iNOS and COX-2 in LPS-treatment RAW264.7 cells. Moreover, in vivo results indicated that SYQP significantly reduced LPS-induced damage in ARDS mice through alleviating LPS-induced pulmonary morphological damage, inhibiting myeloperoxidase (MPO) expression levels, ameliorating the inflammatory cells in bronchoalveolar lavage fluid (BALF) and improving hematological status. Meanwhile, SYQP evidently reduced IL-6, TNF-α and IFN-γ secretion, the overexpression levels of TLR2 and TLR4, as well as the phosphorylation of NF-κB p65. In addition, SYQP reduced the phosphorylation of JAK2 and STAT1 and the overexpression of NLRP3, caspase-1, caspase-3 and caspase-8 in lung tissues of ARDS mice. In summary, our study confirmed that SYQP induced bidirectional immunity and ameliorated LPS-induced acute respiratory distress syndrome in mice through TLR2/TLR4-NF-κB, NLRP3/caspase and JAK/STAT signaling pathways, which provided a theoretical basis for further use of SYQP.

Draft genome sequence of Paenibacillus elgii B69, a strain with broad antimicrobial activity.

Here, we report the draft genome sequence of Paenibacillus elgii B69, which was isolated from soil and has broad-spectrum antimicrobial activity. As far as we know, the P. elgii genome is the largest of the Paenibacillus genus for which genome sequences are available. Multiple sets of genes related to antibiotic biosynthetic pathways have been found in the genome.

Identification and analysis of the gene cluster involved in biosynthesis of paenibactin, a catecholate siderophore produced by Paenibacillus elgii B69.

Bacteria belonging to the genus Paenibacillus are recognized as rich sources of bioactive natural products. To date, there are few characterized siderophores from this genus. Here, through genome analysis, we identified a non-ribosomal peptide biosynthetic gene cluster (pae) responsible for siderophore assembly in Paenibacillus elgii B69. The 12.8 kb gene cluster comprises six open reading frames encoding proteins similar to the components of the bacillibactin biosynthetic machinery and bacillibactin esterase. To examine the product of the pae gene cluster, we cultured P. elgii B69 in iron-deficient medium for siderophore expression. A novel siderophore structurally similar to bacillibactin, designated paenibactin, was purified and characterized. Its structure was determined as a cyclic trimeric lactone of 2,3-dihydroxybenzoyl-alanine-threonine. The involvement of the pae gene cluster in paenibactin biosynthesis was confirmed by the biochemical assay of adenylation domain specificity. Furthermore, we demonstrated that the pae gene cluster evolves from an ancestral bacillibactin biosynthetic gene cluster via sequence and phylogenetic analyses. The structural difference between paenibactin and bacillibactin may stem from a mutation-induced change in the adenylation domain specificity. Based on these findings and published models for bacillibactin, we proposed models for paenibactin biosynthesis, ferric-paenibactin uptake and paenibactin-bounded iron release.

Paenibacillus tianmuensis sp. nov., isolated from soil.

Two closely related, Gram-stain-negative, rod-shaped, spore-forming strains, B27(T) and F6-B70, were isolated from soil samples of Tianmu Mountain National Natural Reserve in Zhejiang, China. Phylogenetic analysis based on 16S rRNA gene and rpoB sequences indicated that the isolates were members of the genus Paenibacillus. Both isolates were closely related to Paenibacillus ehimensis IFO 15659(T), Paenibacillus elgii SD17(T) and Paenibacillus koreensis YC300(T) (≥ 95.2 % 16S rRNA gene sequence similarity). DNA-DNA relatedness between strain B27(T) and P. ehimensis DSM 11029(T), P. elgii NBRC 100335(T) and P. koreensis KCTC 2393(T) was 21.2, 28.6 and 16.8 %, respectively. The major cellular fatty acids of strains B27(T) and F6-B70 were anteiso-C(15 : 0) and iso-C(15 : 0). The cell wall contained meso-diaminopimelic acid. The two isolates differed from their closest neighbours in terms of phenotypic characteristics and cellular fatty acid profiles (such as variable for oxidase, negative for methyl red test, unable to produce acid from d-fructose and glycogen and relatively higher amounts of iso-C(15 : 0) and lower amounts of C(16 : 0) and iso-C(16 : 0)). Strains B27(T) and F6-B70 represent a novel species of the genus Paenibacillus, for which the name Paenibacillus tianmuensis sp. nov. is proposed. The type strain is B27(T) ( = DSM 22342(T)  = CGMCC 1.8946(T)).