超高效液相色谱-串联质谱法检测小菜蛾血清中的β-蜕皮激素

【目的】研究建立可以准确检测小菜蛾血清中痕量β-蜕皮激素的超效液相色谱-串联质谱(UPLC-MS/MS)分析法。【方法】小菜蛾血清β-蜕皮激素样品用乙腈提取并进行蛋白沉淀,以C18色谱柱分离,经UPLC-MS/MS检测,以芸苔素内酯为内标物,内标法定量。【结果】结果表明,在0.5-50μg/L浓度范围内β-蜕皮激素线性相关系数R~2=0.9998;在0.5、10和50μg/L3个添加水平下的平均回收率在91.9%-104.9%之间,相对标准偏差(RSDS,n=5)在3.2%-9.2%之间;方法的定量限为0.5μg/L。【结论】该方法具有操作简单、灵敏度高,稳定性好,应用性强等优点,可为昆虫血清中的β-蜕皮激素检测提供科学准确的方法。     

一株红景天内生细菌的筛选及初步研究

【目的】从红景天根部筛选并鉴定一株产酪醇的细菌,初步研究其产酪醇特性,为寻找红景天替代资源提供新途径。【方法】用NA培养基从大花红景天根部中分离内生细菌,通过薄层层析(TLC)、高效液相色谱(HPLC)、气相色谱-质谱联用(GC-MS)筛选出产量最大的菌株,经菌落形态分析、革兰氏染色分析及16S rRNA基因序列分析其分类学地位。单因素实验确定初始pH、培养温度、发酵时间及接种量对菌株产酪醇活力的影响。【结果】从大花红景天根部分离出14株内生细菌,其中8株能产酪醇,筛选出酪醇产量最大的菌株B3,经菌落形态分析、革兰氏染色分析及16S rRNA基因序列分析初步鉴定为水生拉恩氏菌(Rahnella aquatilis)。研究其发酵条件,其最适pH为6.0,最适温度为32 °C,最佳发酵时间为42 h,最佳接种量为15%。在最适发酵条件下,用改良NA培养基发酵,B3菌株酪醇的产量为15.68 mg/L。【结论】B3菌株是一株具有产酪醇能力的细菌,在最适发酵条件下酪醇产量达到15.68 mg/L,具有潜在的开发价值。     

藤椒精油对腐败解淀粉芽孢杆菌DY1a生物被膜的抑制作用

【背景】芽孢杆菌是豆制品的重要腐败菌,在气液界面形成生物膜,对产品生产带来持续污染。【目的】探讨藤椒精油(Zanthoxylum armatum DC.essential oil,ZA-EO)对腐败解淀粉芽孢杆菌DY1a菌体及生物被膜的抑制作用与机制。【方法】采用气相色谱-质谱(gas chromatography-mass spectrometer,GC-MS)分析藤椒精油主要成分与相对含量,通过二倍稀释法测定藤椒精油对菌株的最低抑菌浓度(minimum inhibitory concentration,MIC)和最低杀菌浓度(minimum bactericidal concentration,MBC),并分析精油对腐败菌胞外蛋白酶活性、腐败菌生物被膜形成抑制及成熟生物被膜的清除作用,采用扫描电镜结合三维光学显微镜分析生物被膜形貌结构变化,测定生物被膜胞外聚合物(extracellular polymeric substance,EPS)多糖与蛋白质含量变化;并通过细菌运动能力、细胞黏附及自聚集能力、细胞表面疏水性和Zeta电位来初步探讨藤椒精油对生物被膜的抑制机理。【结果】藤椒精...     

氧化压力下沙门氏菌可培养性检测及其活的非可培养状态形成情况

【背景】氧化压力会导致细菌进入活的非可培养(viable but non-culturable,VBNC)状态,菌落形成能力可能受到亚致死损伤的影响。目前对于VBNC态细菌的定量检测是基于活菌数与可培养数的差值,因此可培养数的检测对于VBNC态定量研究很关键,培养基组成不合适可能会造成漏检。【目的】分析培养基组成对氧化压力下亚致死损伤细菌检测的重要影响;探究常见食源性致病菌肠炎沙门氏菌在氧化压力下形成VBNC态的情况。【方法】分别采用Luria-Bertani (LB)、beef peptone yeast (BPY)和Salmonella Shigella (SS)培养基检测并比较肠炎沙门氏菌的可培养数;采用RT-qPCR、荧光染色-激光共聚焦显微镜观测氧化压力下肠炎沙门氏菌形成VBNC态的情况。【结果】非选择性培养基LB和BPY能检出亚致死细菌,SS培养基中牛胆盐导致可培养数减少;肠炎沙门氏菌经53°C过氧化氢处理1.5 h后进入VBNC态的比例显著高于53°C过氧化氢+亚铁离子和过氧化氢+柠檬酸处理(P<0.05)。【结论】在对VBNC态的检测中应选择合适的固体培养基检测可...     

海洋芽孢杆菌（Bacillus marinus）B-9987菌株抑制病原真菌机理的研究

摘要：【目的】：探讨海洋芽孢杆菌（Bacillus marinus）B-9987菌株的代谢产物BMME-1,对植物病原真菌茄链格孢菌的抑菌作用机理。【方法】分别使用分光光法、气相色谱-质谱GC-MS联用技术、红外光谱法等,检测了BMME-1处理病原真菌后,菌体渗透性、细胞壁及细胞膜成份的变化。【结果】BMME-1对茄链格孢菌的抑菌中浓度（MIC50）为6.2 mg/L,最小杀菌浓度（MFC）为50 mg/L,在MIC50浓度或高于此浓度处理靶标菌,将导致菌体蛋白质、核酸等大分子物质的外流;处理菌株葡聚糖结     

嗜热菌群降解偶氮染料的特性及机制

【背景】印染废水的出水温度高,抑制了微生物对偶氮染料的降解,而关于嗜热菌在高温下降解偶氮染料的报道较少。【目的】富集能在高温下降解偶氮染料的嗜热微生物菌群,并研究其降解潜力和基因组特征。【方法】通过富集的方法获得嗜热微生物菌群,利用分光光度法测定其降解特性;采用全波长扫描、傅里叶变换红外吸收光谱(Fourier transform infrared spectroscopy,FTIR)和气相质谱(gas chromatography-mass spectrometer,GC-MS)分析其降解机理;采用植物毒性的方法比较偶氮染料降解前后的毒性;采用高通量测序技术分析其功能基因和群落结构。【结果】该菌群(SD1)可以在65℃降解偶氮染料,Caldibacillus、unclassified＿f＿＿Bacillaceae、Geobacillus等为优势属,在降解过程中起关键作用;菌群SD1能在较宽泛的p H (5.0-9.0)、温度(50-75℃)、染料浓度(100-500 mg/L)和盐度(1%-5%)降解酸性大红GR;偶氮还原酶和NADH-DCIP是主要的降解酶,GC-MS和FTIR结果...     

光氧环境对紫色硫细菌YL28去除无机三态氮的影响

【背景】光和氧是制约光合细菌生长代谢进而影响其除氮效果的重要因素。不产氧光合细菌紫色硫细菌——海洋着色菌(Marichromatium gracile) YL28能以亚硝氮为唯一氮源进行光合生长,对高浓度无机三态氮具有良好去除能力。【目的】阐明YL28菌株除氮效率与光氧环境的交互联系,获得其生物除氮的最适光氧条件。【方法】以高浓度无机三态氮共存海水水体为研究体系,在有光/无光条件下考查装样量(表征体系溶氧状态)对YL28菌株生物除氮活性的影响,并通过响应面分析法对装样量、光照强度和光周期3个主要因素进行优化。【结果】光照且氧浓度较低时(80%装样量),YL28具有最佳生长和无机三态氮去除能力;装样量在10%-100%时,菌体生物量(OD_(660))在0.938-2.719之间,当氨氮、亚硝氮和硝氮分别为7.16、5.67和4.83mmol/L时,其去除率分别在71.44%-89.09%、99.22%-99.83%和91.60%-97.33%。黑暗条件下,装样量在20%-100%时,氨氮、亚硝氮和硝氮去除率分别在48.07%-64.27%、73.51%-86.42%和42.57%-46.34%,但菌体生物量(OD_(660)为0.615-0.903)明显降低。通过响应面优化,当装样量、光照强度和光周期分别为80.0%(溶氧量约为0.32 mg/L)、2 800 lx和24L:0D时,细胞生长和氨氮去除活性达到最佳状态,分别比优化前提高了21.28%和14.11%。在实际应用中,选取72%-89%装样量(溶氧量约为0.26-0.63mg/L)、2240-3460lx光照强度和21L:3D-24L:0D光周期,细胞活性可达95%以上。【结论】80%装样量有助于促进菌体光照生长和除氮;在黑暗有氧和无氧环境下,YL28菌株也具有较好除氮活性,这为不产氧光合细菌在生物反应器中高效去除无机三态氮的应用提供了有价值的参考数据。     

镍胁迫下产铁载体细菌对花生的促生性

【目的】挖掘镍耐受性强、产铁载体活性高的植物根际促生细菌,研究镍胁迫下产铁载体细菌对花生的促生作用及其对花生吸收镍的影响。【方法】利用CAS(Chrome azurol S)培养基对花生根际产铁载体细菌定性筛选及定量测试获得产铁载体能力强的菌株,16S r RNA基因相似性及系统进化分析鉴定产铁载体细菌,并用含Ni~(2+)牛肉膏蛋白胨培养基测试细菌对Ni的耐受性;通过花生盆栽实验,测试花生的株高、根长、生物量、氮磷钾含量及镍含量来分析镍胁迫下产铁载体细菌对花生的影响。【结果】从花生根际分离筛选产铁载体芽孢杆菌5株,其中HSGJ1产铁载体能力最强,培养2 d后产156.56 mg/L的铁载体。HSGJ1对Ni~(2+)具有较强的耐受性,最小致死浓度为150 mg/L。在50、100 mg/kg的Ni~(2+)盆栽基质中,HSGJ1能够有效地促进花生的生长、增加花生的生物量及氮磷钾含量,并使花生根部和地上部分的镍含量降低。【结论】产铁载体芽孢杆菌HSGJ1是一株优良的植物根际促生细菌,可应用于镍污染农耕土壤的作物种植中,以提高作物在镍胁迫下的抗逆性,降低作物对镍的富集量,具有较好的应用价值。     

粪臭素高效降解菌YKSW-6的分离、鉴定及降解特性

【背景】粪臭素是畜牧堆肥中有机污染物的主要成分,造成养殖场及周边环境恶化,粪臭素污染问题亟待解决,利用微生物降解粪臭素是一种环保节能的有效方法。【目的】分离鉴定粪臭素高效降解菌株,研究其降解特性,为粪臭素降解提供高效的菌种资源,为该菌株应用于臭味污染环境的净化提供基础。【方法】以粪臭素为唯一碳源的无机盐培养基作为培养基质,从猪粪堆肥样品中分离筛选粪臭素高效降解菌株,通过形态特征和16S rRNA基因序列分析进行分离菌株的初步鉴定,分析其生长规律及粪臭素降解特性,并利用气相色谱质谱联用(GC-MS)对菌株代谢粪臭素的产物进行分析。【结果】从样品中分离获得一株能以粪臭素为唯一碳源的细菌YKSW-6菌株,形态学和16S rRNA基因序列分析初步鉴定该菌株为戈登氏红球菌(Rhodococcus gordoniae)。接种量为10%时,该菌培养14 h对100 mg/L的粪臭素降解率达到100%。其能够利用D-山梨醇、溴-丁二酸等18种碳源,对亚碲酸钾、溴酸钾等13种化学敏感物具有抗性。菌株YKSW-6在5%接种量、温度30-42℃和pH值为6.0-9.0时对100 mg/L的粪臭素降解效率均能达到100%,菌株生长和降解粪臭素的最佳条件为：pH 7.2,温度37℃,转速180 r/min。GC-MS结果表明,粪臭素在菌株的作用下C2先被氧化,转变为3-甲基羟基吲哚,随后进一步被氧化为N-(2-乙酰基苯基)甲酰胺。同时中间产物还有苯乙醛和苯乙酸。【结论】红球菌YKSW-6为目前已报道的降解粪臭素能力较强的菌株,丰富了粪臭素降解菌种的资源库,为实际环境微生物修复应用提供了理论参考。     

毕赤酵母中外源表达脂肪酰-CoA还原酶生产脂肪醇

【目的】通过在毕赤酵母Komagataella pastoris GS115中外源表达来源于霍霍巴[Simmondsia chinensis(Jojoba)]的脂肪酰-Co A还原酶Jojoba FAR,利用微生物发酵生产脂肪醇。【方法】以质粒p RL105为模板PCR扩增获得霍霍巴脂肪酰-Co A还原酶的编码基因,以p GAPZαA为载体构建重组表达质粒p GAP-far,并通过电转化法转入K.pastoris GS115,筛选转化子并发酵,气相色谱-质谱联用检测发酵产物。【结果】构建了毕赤酵母重组菌株p GAPZ-far-GS115,通过摇瓶发酵检测到脂肪醇的合成。随后在7 L规模的发酵罐上发酵验证,得到脂肪醇产量为134.74 mg/L,产率为1.22 mg/(L·h)。【结论】实现了脂肪醇在毕赤酵母中的生物合成,为工业上利用毕赤酵母生产脂肪醇奠定了一定基础。     

Production of phleichrome by Cladosporium phlei as stimulated by diketopiperadines of Epichloe typhina

Epichloe typhina is an endophytic fungus, while Cladosporium phlei is a pathogenic fungus of the timothy plant (Phleum pretense L.). We found two activities in the culture filtrate of E. typhina: one stimulated the pathogenic fungus, C. phlei, to produce phleichrome and the other inhibited its growth. The active ingredients that stimulated the production of phleichrome and inhibited the growth of C. phlei were isolated and characterized. The isolated compounds were identified as cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe), which were stimulatory compounds, and p-hydroxybenzaldehyde, which was the growth inhibitory compound, based on an analysis of their spectral data. Of the two stimulatory compounds, cyclo-(L-Pro-L-Phe) showed higher activity. However, when 500 microg of cyclo-(L-Pro-L-Phe) was spotted on the TLC plate for bio-autography, a growth inhibitory zone was identified in the central red region, which contained phleichrome. On the other hand, phleichrome showed antifungal activity against E. typhina in the light, so it is assumed that there might be antagonism between the endophytic fungus, E. typhina, and the pathogenic fungus, C. phlei.     

Diketopiperazines Produced by the Halophilic Archaeon, <Emphasis Type="Italic">Haloterrigena hispanica</Emphasis>, Activate AHL Bioreporters

The generic term “quorum sensing” has been adopted to describe the bacterial cell-to-cell communication mechanism which coordinates gene expression when the population has reached a high cell density. Quorum sensing depends on the synthesis of small molecules that diffuse in and out of bacterial cells. There are few reports about this mechanism in Archaea. We report the isolation and chemical characterization of small molecules belonging to class of diketopiperazines (DKPs) in Haloterrigena hispanica, an extremely halophilic archaeon. One of the DKPs isolated, the compound cyclo-(l-prolyl–l-valine) activated N-acyl homoserine lactone (AHL) bioreporters, indicating that Archaea may have the ability to interact with AHL-producing bacteria within mixed communities.     

Quorum-sensing cross talk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other Gram-negative bacteria

In cell-free Pseudomonas aeruginosa culture supernatants, we identified two compounds capable of activating an N-acylhomoserine lactone (AHL) biosensor. Mass spectrometry and NMR spectroscopy revealed that these compounds were not AHLs but the diketopiperazines (DKPs), cyclo(DeltaAla-L-Val) and cyclo(L-Pro-L-Tyr) respectively. These compounds were also found in cell-free supernatants from Proteus mirabilis, Citrobacter freundii and Enterobacter agglomerans [cyclo(DeltaAla-L-Val) only]. Although both DKPs were absent from Pseudomonas fluorescens and Pseudomonas alcaligenes, we isolated, from both pseudomonads, a third DKP, which was chemically characterized as cyclo(L-Phe-L-Pro). Dose-response curves using a LuxR-based AHL biosensor indicated that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) activate the biosensor in a concentration-dependent manner, albeit at much higher concentrations than the natural activator N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL). Competition studies showed that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) antagonize the 3-oxo-C6-HSL-mediated induction of bioluminescence, suggesting that these DKPs may compete for the same LuxR-binding site. Similarly, DKPs were found to be capable of activating or antagonizing other LuxR-based quorum-sensing systems, such as the N-butanoylhomoserine lactone-dependent swarming motility of Serratia liquefaciens. Although the physiological role of these DKPs has yet to be established, their activity suggests the existence of cross talk among bacterial signalling systems.     

Biocontrol of Aspergillus Species on Peanut Kernels by Antifungal Diketopiperazine Producing Bacillus cereus Associated with Entomopathogenic Nematode

The rhabditid entomopathogenic nematode associated Bacillus cereus and the antifungal compounds produced by this bacterium were evaluated for their activity in reducing postharvest decay of peanut kernels caused by Aspergillus species in in vitro and in vivo tests. The results showed that B. cereus had a significant effect on biocontrol effectiveness in in vitro and in vivo conditions. The antifungal compounds produced by the B. cereus were purified using silica gel column chromatography and their structure was elucidated using extensive spectral analyses. The compounds were identified as diketopiperazines (DKPs) [cyclo-(L-Pro-Gly), cyclo(L-Tyr-L-Tyr), cyclo-(L-Phe-Gly) and cyclo(4-hydroxy-L-Pro-L-Trp)]. The antifungal activities of diketopiperazines were studied against five Aspergillus species and best MIC of 2 µg/ml was recorded against A. flavus by cyclo(4-hydroxy-L-Pro-L-Trp). To investigate the potential application of cyclo(4-hydroxy-L-Pro-L-Trp) to eliminate fungal spoilage in food and feed, peanut kernels was used as a food model system. White mycelia and dark/pale green spores of Aspergillus species were observed in the control peanut kernels after 2 days incubation. However the fungal growth was not observed in peanut kernels treated with cyclo(4-hydroxy-L-Pro-L-Trp). The cyclo(4-hydroxy-L-Pro-L-Trp) was nontoxic to two normal cell lines [fore skin (FS) normal fibroblast and African green monkey kidney (VERO)] up to 200 µg/ml in MTT assay. Thus the cyclo(4-hydroxy-L-Pro-L-Trp) identified in this study may be a promising alternative to chemical preservatives as a potential biopreservative agent which prevent fungal growth in food and feed. To the best of our knowledge, this is the first report demonstrating that the entomopathogenic nematode associated B. cereus and cyclo(4-hydroxy-L-Pro-L-Trp) could be used as a biocontrol agents against postharvest fungal disease caused by Aspergillus species.     

Antifouling diketopiperazines produced by a deep-sea bacterium, Streptomyces fungicidicus

Modern antifouling coatings use heavy metals and toxic organic molecules to prevent biofouling, the undesirable growth of marine organisms on man-made substrata. In an ongoing survey of deep-sea microorganisms aimed at finding low toxic antifouling metabolites, an actinomycete bacterium was isolated from the Pacific sediment at the depth of about 5000 m. The bacterium was closely related to Streptomyces fungicidicus (99% similarity) according to 16S ribosomal RNA sequence information. The spent culture medium of this bacterium inhibited barnacle larval attachment. Bioassay-guided fractionation was employed to isolate antifouling compounds. The ethyl acetate extract was fractionated by using an open silica gel column. Active fractions were further purified on a HPLC C18 column. Five diketopiperazines, cyclo-(L-Leu-L-Pro), cyclo-(L-Phe-L-Pro), cyclo-(L-Val-L-Pro), cyclo-(L-Trp-L-Pro), and cyclo-(L-Leu-L-Val) were isolated for the first time from a deep sea bacterium, and the structures of the compounds were elucidated by nuclear magnetic resonance spectroscopy and mass spectrometry. The pure diketopiperazines were tested for antilarval activity using the barnacle Balanus amphitrite. Effective concentrations that inhibited 50% larval attachment (EC50) after 24 h ranged from 0.10- 0.27 mM. The data suggest that diketopiperazines and other compounds from deep-sea bacteria may be used as novel antifoulants.     

In vitro synergistic activity of diketopiperazines alone and in combination with amphotericin B or clotrimazole against Candida albicans

The synergistic anticandidal activity of three diketopiperazines [cyclo-(l-Pro-l-Leu) (1), cyclo-(d-Pro-l-Leu) (2), and cyclo-(d-Pro-l-Tyr) (3)] purified from a Bacillus sp. N strain associated with entomopathogenic nematode Rhabditis (Oscheius) in combination with amphotericin B and clotrimazole was investigated using the macrodilution method. The minimum inhibitory concentration and minimum fungicidal concentration of the diketopiperazines was compared with that of the standard antibiotics. The synergistic anticandidal activities of diketopiperazines with amphotericin B or clotrimazole were assessed using the checkerboard and time-kill methods. The results of the present study showed that the combined effects of diketopiperazines with amphotericin B or clotrimazole predominantly recorded synergistic (<0.5). Time-kill study showed that the growth of the Candida was completely attenuated after 12–24 h of treatment with 50:50 ratios of diketopiperazines and antibiotics. These results suggest that diketopiperazines combined with antibiotics may be microbiologically beneficial and not antagonistic. These findings have potential implications in delaying the development of resistance as the anticandidal effect is achieved with lower concentrations of both drugs (diketopiperazines and antibiotics). The cytotoxicity of diketopiperazines was also tested against two normal human cell lines (L231 lung epithelial and FS normal fibroblast) and no cytotoxicity was recorded for diketopiperazines up to 200 μg/mL. The in vitro synergistic activity of diketopiperazines with antibiotics against Candida albicans is reported here for the first time.     

Antifouling diketopiperazines produced by a deep-sea bacterium,Streptomyces fungicidicus

Abstract

Modern antifouling coatings use heavy metals and toxic organic molecules to prevent biofouling, the undesirable growth of marine organisms on man-made substrata. In an ongoing survey of deep-sea microorganisms aimed at finding low toxic antifouling metabolites, an actinomycete bacterium was isolated from the Pacific sediment at the depth of about 5000 m. The bacterium was closely related to Streptomyces fungicidicus (99% similarity) according to 16S ribosomal RNA sequence information. The spent culture medium of this bacterium inhibited barnacle larval attachment. Bioassay-guided fractionation was employed to isolate antifouling compounds. The ethyl acetate extract was fractionated by using an open silica gel column. Active fractions were further purified on a HPLC C₁₈ column. Five diketopiperazines, cyclo-(L-Leu-L-Pro), cyclo-(L-Phe-L-Pro), cyclo-(L-Val-L-Pro), cyclo-(L-Trp-L-Pro), and cyclo-(L-Leu-L-Val) were isolated for the first time from a deep sea bacterium, and the structures of the compounds were elucidated by nuclear magnetic resonance spectroscopy and mass spectrometry. The pure diketopiperazines were tested for antilarval activity using the barnacle Balanus amphitrite. Effective concentrations that inhibited 50% larval attachment (EC₅₀) after 24 h ranged from 0.10 – 0.27 mM. The data suggest that diketopiperazines and other compounds from deep-sea bacteria may be used as novel antifoulants.     

Identification of Antifungal Compounds Produced by Lactobacillus casei AST18

Lactobacillus casei AST18 was screened as an antifungal lactic acid bacteria which we have reported before. In this research, the antifungal properties of cell-free culture filtrate (CCF) from L. casei AST18 were detected, and the antifungal compounds of CCF were prepared by ultrafiltration, and semi-preparative HPLC, and then determined by GC-MS. CCF was sensitive to pH and heat treatment but it was not affected by the treatment of trypsin and pepsin. Through the treatment of ultrafiltration and semi-preparative HPLC there were two parts of CCF which showed antifungal activities: part 1 and part 4. Lactic acid was identified as the main antifungal compound in part 1. In part 4, three small molecular substances were detected with GC-MS. The three potential antifungal substances were cyclo-(Leu-Pro), 2,6-diphenyl-piperidine, and 5,10-diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a;1',2'-d]pyrazine. The antifungal activity of L. casei AST18 was a synergistic effect of lactic acid and cyclopeptides.     

Plant growth-promoting Pseudomonas putida WCS358 produces and secretes four cyclic dipeptides: cross-talk with quorum sensing bacterial sensors

The most universal cell-cell signaling mechanism in Gram-negative bacteria occurs via the production and response to a class of small diffusible molecules called N-acylhomoserine lactones (AHLs). This communication is called quorum sensing and is responsible for the regulation of several physiological processes and many virulence factors in pathogenic bacteria. The detection of these molecules has been rendered possible by the utilization of genetically engineered bacterial biosensors which respond to the presence of exogenously supplied AHLs. In this study, using diverse bacterial biosensors, several biosensor activating fractions were purified by organic extraction, HPLC and TLC of cell-free culture supernatants of plant growth-promoting Pseudomonas putida WCS358. Surprisingly, it was observed that the most abundant compounds in these fractions were cyclic dipeptides (diketopiperazines, DKPs), a rather novel finding in Gram-negative bacteria. The purification, characterization, chemical synthesis of four DKPs are reported and their possible role in cell-cell signaling is discussed. Received: 19 October 2001 / Accepted: 8 January 2002     

Inhibition of biofilm in <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> Q-426 by diketopiperazines

Biofilm formation can make significant effects on bacteria habits and biological functions. In this study, diketopiperazines (DKPs) produced by strain of Bacillus amyloliquefaciens Q-426 was found to inhibit biofilm formed in the gas–liquid interface. Four kinds of DKPs were extracted from B. amyloliquefaciens Q-426, and we found that 0.04 mg ml^?1 DKPs could obviously inhibit the biofilm formation of the strain. DKPs produced by B. amyloliquefaciens Q-426 made a reduction on extracellular polymeric substance (EPS) components, polysaccharides, proteins, DNAs, etc. Real-time PCR was performed to determine that whether DKPs could make an obvious effect on the expression level for genes related to biofilm formation in the strain. The relative expression level of genes tasA, epsH, epsG and remB which related to proteins, extracellular matrix, and polysaccharides, were downregulated with 0.04 mg ml^?1 DKPs, while the expression level of nuclease gene nuc was significantly upregulated. The quantitative results of the mRNA expression level for these genes concerted with the quantitative results on EPS levels. All of the experimental results ultimately indicated that DKPs could inhibit the biofilm formation of the strain B. amyloliquefaciens Q-426.