Inhibition mechanisms of secretome proteins from Paenibacillus polymyxa Kp10 and Lactococcus lactis Gh1 against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus

Objective:To determine the inhibition mechanisms of secretome protein extracted from Paenibacillus polymyxa Kp10(Kp10)and Lactococcus lactis Gh1(Gh1)against methicillin-resistant Staphylococcus aureus(MRSA)and vancomycin-resistant Enterococcus(VRE).Methods:The sensitivity and viability of MRSA and VRE treated with secretome proteins of Kp10 and Gh1 were determined using minimal inhibitory concentration,minimum bactericidal concentration,and time-to-kill assays.The morphological changes were observed using scanning electron microscopy and transmission electron microscopy.To elucidate the antimicrobial mechanism of secretome protein of Kp10 and Gh1 against MRSA and VRE,2D gel proteomic analysis using liquid chromatography-mass spectrometry was run by comparing upregulated and downregulated proteins,and the proton motive force study including the efflux of ATP,pH gradient,and the membrane potential study were conducted.Results:MRSA and VRE were sensitive to Kp10 and Gh1 secretome protein extracts and displayed apparent morphological and internal composition changes.Several proteins associated with cellular component functions were either downregulated or upregulated in treated MRSA and VRE by changing the membrane potential gradient.Conclusions:Kp10 and Gh1 secretome proteins reduce the growth of VRE and MRSA by damaging the cell membrane.Cell division,cell wall biosynthesis,and protein synthesis are involved in the inhibition mechanism.

马铃薯淀粉废水资源化制备Paenibacillus polymyxa农用菌剂

马铃薯淀粉废水中含有高浓度的淀粉、蛋白质等有机物。利用马铃薯淀粉废水培养植物促生菌,是实现其资源化利用的方法。采用单因素方法结合中心复合设计(CCD)的方法,对废水体积分数(浓度)、培养温度、初始pH这3个因素进行研究,优化马铃薯淀粉废水培养Paenibacillus polymyxa EBL06菌株的适宜生长条件。实验得到马铃薯淀粉废水培养P.polymyxa的最佳条件:废水COD为13.7 g·L^−1,初始pH为7.17,培养温度为31.4℃。该条件下,培养21 h后,微生物活菌数为6.2×10^9 cfu·mL^−1,与模型预测结果基本一致,可以达到《农用微生物菌剂国家标准》(GB 20287-2006)。为了验证该菌剂的应用效果,进行了蔬菜种植实验。结果表明:P.polymyxa菌剂能有效提高小白菜的产量与品质,作物鲜重、干重、株高,以及维生素C含量别提高了68.6%、13.7%、5.6%、41.3%;相比于只施用化肥的组,菌剂同尿素的混施能提高氮肥的利用效率,小白菜植株中维生素含量提高了25.3%、硝酸盐含量减少了15.3%。以上研究结果可为马铃薯淀粉废水的资源化利用,以及P.polymyxa菌剂的应用推广提供参考。

Production of a newly isolated Paenibacillus polymyxa biocontrol agent using monosodium glutamate wastewater and potato wastewater

A phyllosphere bacterial strain EBL-06 was isolated from wheat leaves. The morphology, cultural characteristics, phospholipid fatty acids, physiological and antagonistic fungus activities of this strain were investigated. A phylogenetic tree was constructed by comparing with the published 16S rDNA sequences of the relevant bacteria. The results showed that the isolate EBL-06 was a strain of Paenibacillus polymyxa; this strain performed a high level of antagonistic fungus activity toward a broad spectrum of phytopathogens, such as Botrytis cinerea, Cladosporium cucumerinum, Fusarium spp. The isolate EBL-06 can grow well using monosodium glutamate wastewater （MGW） and potato wastewater （PW） as culture medium. The maximum yield of 6.5 × 10^9 CFU/mL of the isolate EBL-06 anti-fungus biocontrol agent was reached in 15 hr cultivation at 28℃, pH 6.0-7.5 using the mixture of MGW and PW （1：9）.

Probiotic Paenibacillus polymyxa 10 and Lactobacillus plantarum 16 enhance growth performance of broilers by improving the intestinal health

With the ever-growing strict prohibitions on antibiotic growth promoters(AGP)in animal production,in-feed probiotics are becoming attractive alternatives to antibiotics in the poultry industry.To investigate the effects of Paenibacillus polymyxa 10 and Lactobacillus plantarum 16 on the growth performance and intestinal health of broilers,540 male Cobb 500 broilers of 1 d old were randomly divided into 3 groups with 6 replicates per group and 30 chicks per replicate.Broilers were fed with either a basal diet or basal diets supplemented with 1×108 colony-forming units(CFU)/kg P.polymyxa 10(BSC10)or L.plantarum 16(Lac16)for 42 d.Results showed that Lac16 treatment improved(P<0.05)the growth performance(body weight and feed conversion)of broilers at the starter phase,while BSC10 treatment slightly improved(P>0.05)the growth performance of the starter phase broilers.The increased villus height(P<0.05)at d 14,21 and 42 and villus height to crypt depth ratio(P<0.05)at d 14 and 21 were observed in the ileum of the 2 probiotic groups.Besides,transmission electron microscopy results showed that the 2 probiotics enhanced the intestinal epithelial barrier.Both probiotic treatments up-regulated(P<0.05)the mRNA expression of fatty acid binding protein 1(FABP1)and sodium-dependent glucose transporters-1(SGLT-1)in the ileal mucosa of broilers at d 21.In addition,BSC10 and Lac16 treatments significantly(P<0.05)increased the relative abundance of short-chain fatty acids-producing bacteria,such as Butyricicoccus pullicaecorum,Faecalibacterium prausnitzii,Lachnospira and Cop-rococcu,and significantly(P<0.05)decreased the relative abundance of enteric pathogens(Escherichia coli,Bacteroides fragilis and Shigella sonnei).Furthermore,the 2 probiotic treatments also increased the positive connection among the intestinal microbes and the carbohydrate metabolism-related pathways of the intes-tinal bacteria(P<0.05),with decreasing(P<0.05)nucleotides biosynthesis-related pathways of the intestinal bacteria.Overall,these results suggest that the 2 probiotics,especially Lac16,have a potential beneficial effect on the growth performance and intestinal health of starter phase broilers.

Fusaricidins in Paenibacillus polymyxa A21 and their antagonistic activity against Botrytis cinerea on tomato

Four kinds of antifungal compounds from an extract of Paenibacillus polymyxa A21 with molecular masses of 883.56, 897.59, 947.55, and 961.58 Da were characterized as the members of fusaricidin-type of antibiotics according to LC-MS analysis. Fusaricidins isolated from culture filtrate displayed high antagonistic activity against several plant fungal pathogens, especially Botrytis cinerea, the causal agent of gray mold. The fusaricidins biosynthetic gene cluster(BGC) from A21 was cloned by PCR and comparative cluster analysis revealed that gene fus TE, the 3′ boundary of the fusaricidin BGC in strain PKB1, was not present in fusaricidin BGC of A21, indicating that fus TE is not necessary for fusaricidin synthesis. Fusaricidin extract from A21 significantly reduced gray mold disease incidence and severity on tomato. The m RNA levels for three pathogenesis-related proteins(PRs) revealed that treatment of tomato leaves with fusaricidin extract induced the expression of PR genes to different levels, suggesting that one reason for the reduction of gray mold infection by fusaricidin is induction of PR proteins, which lead to increased resistance to pathogens. This is the first report of the application of fusaricidins to control tomato gray mold and the comparative cluster analysis provides important molecular basis for research on fusaricidin biosynthesis.

Coinoculation of bioinoculants improve Acacia auriculiformis seedling growth and quality in a tropical Alfisol soil

We conducted a study to find out if arbuscular mycorrhizal(AM) fungi(Acaulospora scrobiculata,Scutellospora calospora) and phosphate solubilizing bacteria(PSB, Paenibacillus polymyxa) inoculation either individually or in combinations can improve Acacia auriculiformis seedling growth, uptake of nutrients and quality in a phosphorus deficient tropical Alfisol. The seedlings were assessed for various growth and nutrient uptake parameters after 60 days of treatment. Inoculation with P.polymyxa stimulated mycorrhizal formation. Seedling height, stem girth, taproot length, number of leaves and leaf area, plant dry matter production, nodulation, and nodular dry weight were significantly higher for seedlings that were either dual inoculated or triple inoculated compared to individual inoculation of AM fungi or PSB, and uninoculated seedlings. Dual and triple application of AM fungi and PSB also significantly improved the nutrient contents of shoots and roots and nutrient uptake efficiencies. The calculated seedling quality indexes of the AM fungi and PSB inoculated seedling were 25–208% higher than uninoculated seedlings. These findings show that A.auriculiformis seedlings when dual inoculated or triple inoculated performed better than seedlings inoculated with the microbes individually and compared with uninoculated control seedlings. We conclude that bioinoculation is important for the production of high-quality A.auriculiformis seedlings in tree nurseries for planting in nutrient deficient soils.