Degradation of N-Acyl Homoserine Lactone Quorum Sensing Signals by Bacillus thuringiensis AHL Lactonase

Bacterial cells rely on signaling molecules to communicate with others from the same species and induce certain genes in a process known as quorum sensing (QS). A common molecule is N-acyl homoserine lactone (AHL) which is responsible for the expression of virulence and other factors that allow the organisms to compete in a given environment. On the other hand, other bacteria produce certain enzymes such as AHL-lactonase that break down AHL molecules and prevent gene expression of these factors. The aim of this work was to examine the level of degradation of AHL molecules by AHL-lactonase in 62 Bacillus thuringiensis (Bt) strains isolated from Middle Tennessee, Mississippi, and Alabama. N-hexanoyl-homoserine lactone (C6-HSL) and N-3-oxo-hexanoyl homoserine lactone (3-oxo-C6-HSL), which cause Chromobacterium violaceum (CV026) to produce a purple pigment were tested at different concentrations to view the Bt lactonase activity. In addition, PCR was used to test for the presence of the lactonase gene. The results showed that among the 62 Bt strains, there were 58 that possessed the AHL-lactonase (aiiA) gene and 48 strains were able to degrade C6-HSL. At high concentrations of AHL, only 13 strains were able to completely degrade C6-HSL. In addition, degradation of 3-oxo-C6-HSL was weak compared to C6-HSL. The results also revealed that AHL lactonase was thermostable, and it was concluded that the level of degradation varies in Bt strains. Only 13 of the strains studied have potent inhibitory activity against C6-HSL, which may be good to be used in field applications to control agricultural pest.

N-Acylhomoserine Lactones (AHLs), QseB/C Gene Detection, Virulence Factors and Antibiotics Resistance of <i>Aeromonas hydrophila</i>

The aim of this research was to detect the N-acyl homoserine lactones (AHLs) production and QseB/C gene of Aeromonas hydrophila. We analyzed the potentials of these isolates of Aeromonas hydrophila in causing biofilm formation, hemolysis, protease, and lipase. The antibiotic susceptibility of the 15 Aeromonas hydrophila isolates was also investigated. The detection of AHLs was carried out using the Chromobacterium violaceum strain CV026 as biosensors. The isolated strains were tested for the reaction of C. violaceum CV026 by cross-streaking on an agar plate. Production of AHLs was determined by the diffusing via the agar plates and the tinge of the biosensor strains. All isolated strains produced AHLs. A polymerase chain reaction (PCR) showed the isolated strains had qseB and qseC genes. Susceptibility tests of A. hydrophila isolates were administered against 25 different antibiotic disks representing 12 classes of antibiotics. The strains were highly resistant to β-Lactam with 96.7% showing resistibility, whereas 97.7% susceptibility was found towards Aminoglycoside class of the antibiotic used. 60% showed intermediate resistant to Polypeptide. 100% of the strains showed no resistant to Aminoglycoside, Polypeptide, Monobactam, and Carbapenems class of antibiotics. Each of the isolates was found to be associated with at least one virulent factor. Our results clearly demonstrated that there is a presence of QseB/C genes in A. hydrophila and also produces AHLs molecule and virulence factors. The investigated isolates showed the pathogenic potential of Aeromonas hydrophila which makes it a serious threat to public health.

A Preliminary Study of Cell Membrane Mediated Immobilization of a Recombinant Acyl-homoserine Lactonase AidH

The aim of this work was to inhibit biofilm formation by taking advantages of bacterial surface display technology in combination with cell membrane chromatography.A recombinant protein INPAidH was constructed by fusing a quorum signal hydrolase AidH to the C-terminus of the ice nucleation protein(INP).Expression of INP-AidH was achieved on E.coli cell surface at an expression level of 30%of total membrane proteins.Activity of INP-AidH on cell membranes was confirmed in degrading the quorum signal C6-HSL as well as inhibiting bacterial biofilm.Immobilization of INP-AidH anchored cell membranes on silica gel particles was facilitated by taking advantages of cell membrane chromatography.The functionalized silica gel particles also exhibit activities in degrading C6-HSL and inhibiting bacterial biofilm.This article presents a new approach to prevent biofilm formation of silica-based materials.

Occurrence of <i>N</i>-Acyl Homoserine Lactones in Extracts of Bacterial Strain of <i>Pseudomonas aeruginosa</i>and in Sputum Sample Evaluated by Gas Chromatography–Mass Spectrometry

This study presents a fast, accurate and sensitive technique using gas chromatography-mass spectrometry (GC-MS) for the identification and quantification of N-acyl homoserine lactones (AHLs) in the extracts of bacterial strain of Pseudomonas aeruginosa and sputum sample of a cystic fibrosis patient. This method involves direct separation and determination of AHLs by using GC-MS as simultaneous separation and characterization of AHLs were possible without any prior derivatiza-tion. Electron ionization resulted in a common fragmentation pattern with the most common fragment ion at m/z 143 and other minor peaks at 73, 57 and 43. The limit of detection for N-butanoyl, N-hexanoyl, N-octanoyl, N-decanoyl, N-dodecanoyl and N-tetradecanoyl homoserine lactones was 2.14, 3.59, 2.71, 2.10, 2.45 and 2.34 μg/L, respectively. The presence of AHLs in the culture of P. aeruginosa strain and spu-tum of a cystic fibrosis patient was achieved in selected ion monitoring (SIM) mode by using the prominent fragment at m/z 143.

AHLs介导的群体感应和群体淬灭对植物-根际微生物相互作用的影响

根际是由植物根系和土壤微生物之间相互作用形成的一种特殊环境,根际微生物群落的宏基因组是植物微生物组的重要组成部分。植物与根际微生物之间的相互作用是一个复杂的过程。在根际环境中,微生物群落利用复杂的种内和种间信号传导机制招募特定的微生物,协调并控制混合群落的行为,从而影响植物的生长发育和健康。根际微生物能够自发产生、释放特定的信号分子,并能感知其浓度变化,从而调节微生物的群体行为,这一调控系统称为群体感应(quorum sensing,QS)。QS系统的特征是合成和释放特定的信号分子。根际土壤细菌中存在多种QS信号分子,如N-酰基高丝氨酸内酯(AHLs)、二酮哌嗪、扩散信号因子、次生代谢物、植物激素类分子等。AHLs作为细菌中被广泛研究的QS信号分子,在植物与根际微生物的相互作用中发挥重要作用。本文综述了AHLs介导的群体感应机制,并讨论了AHLs在植物与根际微生物相互作用中的调节作用,包括AHLs对植物的生长发育、逆境耐受性和抗病性等方面的有益影响,以及AHLs介导的QS系统调控导致的根际致病菌对植物的有害影响,同时还探讨了基于AHLs的群体淬灭对植物-根际微生物相互作用的影响,以期为植物健康与农业生产提供新的思路和方法,推动可持续农业的发展。

Performance and role of N-acyl-homoserine lactone(AHL)-based quorum sensing(QS) in aerobic granules

The present study investigated the relationship between N-acyl-homoserine lactone（AHL）-based quorum sensing（QS） and the physico-chemical properties of aerobic granules.Stable mature granules were observed in SBR2 and SBR3 with average diameters of 0.96,and1.49 mm,respectively. The sludge densities of aerobic granules in SBR2 and SBR3 were1.0246,and 1.0201 g/mL,respectively,which were higher than that of flocculent sludge in SBR1（1.0065 g/mL）. The results showed that the activity of AHL-based QS in SBR2 and SBR3 amounted to 2.4- and 2.1-fold induction,however,that in SBR1 with flocculent sludge was1.6-fold induction. In addition,the results also showed that the activity of AHL-based QS in the three reactors rose in the feast condition,and then dropped with the consumption of substrate. However,the activity of AHL-based QS in these three reactors recovered again in prolonged starvation. Furthermore,the results showed that the enhancement of AHL-based QS favored the extracellular polymeric substance production of microorganisms in activated sludge. Thus,it could be concluded that aerobic granules showed higher AHL-based QS than flocculent sludge,which resulted from the higher sludge density of aerobic granules than flocculent sludge. AHL-based QS was related to the metabolism energy in the feast condition; however,in prolonged starvation,microorganisms would emit more AHL-like molecules to protect themselves to resist starvation. Moreover,the enhancement of AHL-based QS favored the EPS component productivity of the microorganisms in activated sludge,which contributed to maintain the aerobic granular structure.

高效液相色谱-串联质谱法同时测定细菌群体感应效应的11种AHLs类信号分子

建立了高效液相色谱-串联质谱(HPLC-MS/MS)法同时测定细菌群体感应分泌的11种AHLs类信号分子的分析方法。细菌所分泌AHLs类信号分子经乙酸乙酯萃取后简单处理,可直接进样分析。采用Sunfire C18色谱柱(50mm×2.1mm,3.5μm),甲醇与水(含2mmoI/L乙酸铵,0.1%甲酸)梯度进样,采用电喷雾离子源正离子模式,多反应监测(MRM)模式,外标法进行质谱定量分析。分析时间为12min。本方法在1～250μg/L范围内呈良好的线性关系(r>0.996);检出限为0.1～1.0μg/kg;定量限为0.3～3.0μg/kg;平均添加回收率为54.4%～128.6%;相对标准偏差为3.5%～13.9%。本方法具有操作简便、快速、准确、灵敏度高等优点,适用于多种AHLs类信号分子化合物的同时定性与定量分析,为微生物群体感应的进一步研究提供了有效的且便利的分析方法。

产生物胺菌株群体感应AHLs检测及分析

为探讨发酵肉制品中生物胺的产生、累积机理,采用高效液相法分析了新疆熏马肠样品产生物胺优势菌株克雷伯氏菌(Klebsiella sp.)、阴沟肠杆菌(Enterobacter cloacae strain)和成团泛菌(Pantoea agglomerans strain)产生物胺能力。并选取克雷伯氏菌、阴沟肠杆菌、成团泛菌作为群体感应现象研究对象,利用微生物传感菌Agrobacterium tumefaciens A136及Agrobacterium tumefaciens KYC55平行线法检测AHLs。检测表明,3株产生物胺菌能够发生群体感应现象。通过β-半乳糖苷酶活性检测法测定AHLs活性试验、HPLC法对产生物胺菌株AHLs定性分析,结果表明,β-半乳糖苷酶活性越高,产生物胺菌株分泌的AHLs越多;Klebsiella sp.、Enterobacter cloacae strain均能分泌:N-butanoyl-L-homoserine lactone(C4-HSL)、N-hexanoyl-DL-homoserine homoserine lactone(C6-HSL)、N-octanoyl-DL-homoserine lactone(C8-HSL)类型的信号分子。

信号分子N-乙酰高丝氨酸内酯(AHLs)诱导铜绿微囊藻细胞凋亡的研究

[目的]研究信号分子N-乙酰高丝氨酸内酯(AHLs)与铜绿微囊藻细胞凋亡的关系。[方法]以铜绿微囊藻为试验材料,采用5μmol/L AHLs处理铜绿微囊藻细胞,用DAPI染色后观察细胞凋亡的形态。[结果]AHLs在1μmol/L浓度下能诱导铜绿微囊藻的细胞凋亡,从而抑制铜绿微囊藻的生长和增殖。[结论]该研究结果为治理铜绿微囊藻水华爆发提供了科学依据。

高氮负荷下硫自养反硝化菌群的胞外聚合物和信号分子特征

硫自养反硝化工艺(SADN)受水力停留时间(HRT)和进水NO_(3)^(-)-N浓度的影响.通过逐步缩短HRT,提高进水NO_(3)^(-)-N浓度的方式探究工艺脱氮极限,同时解析在此过程中胞外聚合物(EPS)和信号分子酰基高丝氨酸内酯(AHLs)的变化特征以及微生物群落结构演替过程.在HRT从4.1h缩短至1.0h的过程中,硫自养反硝化菌群可快速适应环境,当HRT=1.0h,进水NO_(3)^(-)-N含量为40mg/L,系统的TN去除率为99.74%,脱氮负荷高达958.53mg/(L·d).在此过程中,EPS含量的增加以TB-PN和TB-PS为主,AHLs含量的增加以C4-HSL为主.较缩短HRT而言,提高进水NO_(3)^(-)-N浓度会促使C4-HSL含量显著增加并导致污泥结构逐渐趋向不稳定状态.C6-HSL是系统内最主要的AHLs,其含量在G4和G7阶段大幅增长是基于系统脱氮能力弱化后微生物所进行的自我调节.微生物群落组成分析表明,系统运行和氮负荷变化会对微生物群落进行驯化选择.在降HRT组中,Proteobacteria的norank_f_Pleomorphomona-daceae作为优势菌属发挥主要作用,为反硝化菌提供底物以维持系统稳定运行.在提高NO_(3)^(-)-N浓度后,UKL 13-1、Simplicispira、Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium、Thermomonas逐渐演变为优势菌属,这些优势菌属均为具备脱氮功能的关键微生物.

菌群感应AHLs分子对小鼠肠道菌群多样性影响的分析

目的探讨菌群感应信号分子N-酰基高丝氨酸内酯(N-acyl homoserine lactones,AHLs)对小鼠肠道微生物菌群构成的影响,为感染状态下肠道微生态的紊乱补充理论依据。方法 3-oxo-C_(10)-HSL分子经腹腔注射小鼠后,于24 h分别在小鼠十二指肠及空回肠部位用拭子涂抹法取样,并通过Illumina Hiseq 2500高通量测序平台对样品16S rDNA测序,并进行微生物菌群分类构成及多样性分析。结果在实验小鼠肠道内共检出22个细菌门,109个细菌属;对照组和3-oxo-C_(10)-HSL处理组小鼠肠道不同部位菌群均以厚壁菌门(>50%)和变形菌门(>20%)为主,但两组在属水平菌群构成有明显差异,对照组优势菌属依次为葡萄球菌、埃希氏-志贺菌、乳球菌、假单胞菌、肠球菌,3-oxo-C_(10)-HSL处理组优势菌属主要为肠球菌、埃希氏-志贺菌、葡萄球菌;多样性和丰度分析提示3-oxo-C_(10)-HSL处理组肠道菌群多样性均有提高,大肠埃希菌属等主要优势菌丰度显著上升,而且空回肠部位的菌落丰度最高。结论 AHLs对小鼠肠道菌群构成影响较小,而对菌群内部某些特定类群结构变化影响较明显。

酰基-高丝氨酸内酯(AHL)对鼠伤寒沙门氏菌生物菌膜形成的影响

[目的]研究铜绿假单胞菌所产生的群体感应信号分子酰基-高丝氨酸内酯(AHL)对鼠伤寒沙门氏菌生物菌膜形成的影响,为不同菌体间群体感应的研究提供参考,为沙门氏菌的控制提供帮助。[方法]利用薄层层析法分析铜绿假单胞菌所产生AHL的成分,并通过添加0%、1.0%、1.5%、2.0%(体积分数)的AHL来研究其对鼠伤寒沙门氏菌生物菌膜形成、浮游菌体生长以及菌体泳动能力的影响。[结果]铜绿假单胞菌产生2种相同类型的AHL分子(C12-HSL和3-oxo-C8-HSL),100℃加热5 min并不会改变AHL的成分。当AHL添加量低于1.5%时,虽然浮游菌体指数生长时期受到一定抑制,但随着时间的延长,稳定期浮游菌体的生长并无显著差异,反观生物菌膜,其形成明显受到抑制。当AHL添加量达到2%时,浮游菌体生长和生物菌膜形成均受到显著抑制,说明AHL对鼠伤寒沙门氏菌生物菌膜的形成有明显抑制作用。AHL对单一菌体泳动性有显著抑制作用,而对群体泳动性有不同程度的促进作用。[结论]群体感应信号分子AHL对鼠伤寒沙门氏菌浮游菌体生长的影响受添加量调控,当AHL添加量增加到一定值时浮游菌体生长才受到抑制,但对生物菌膜的形成有明显抑制作用,且随着AHL添加量提高,抑制作用增强。

污水生物处理中信号分子AHL介导的群体感应

介绍了信号分子酰基高丝氨酸内酯(AHL)的结构及性质、合成感应机制,叙述了对AHL介导的群体感应(QS)在2种污水生物处理过程(生物膜法及颗粒污泥法)中的研究进展。污水生物处理需要微生物的协同作用以实现污染物的去除,这种协作主要依赖于AHL介导的QS作用,生物膜及颗粒污泥中的AHL种类及含量均表现出明显的时空差异,且容易受到环境因子的影响;AHL介导的QS可以通过改善生物膜、颗粒污泥的相关性能,以及调节微生物群落结构来提高它们的污水处理效果。未来应进一步探索更简便、有效的AHL定性及定量方法,深入研究AHL对生物膜及颗粒污泥性能的影响机制,以实现AHL介导的QS在实际废水处理中的应用及调控。

基质冲击对ANAMMOX颗粒释放AHLs及颗粒特性的影响

从群体感应角度考察基质冲击对ANAMMOX颗粒特性的影响,以期为提高ANAMMOX颗粒抗基质冲击能力提供理论指导与借鉴.结果表明,24h 1500mg/L总氮冲击刺激颗粒AHLs释放量由4.3大幅增加至10.0,同时颗粒结合性胞外聚合物(B-EPS)过量释放(增加了107.3mg/g VSS),导致颗粒沉降性能下降(密度和沉速分别降低了53%和33%).但基质浓度恢复至稳定期水平后,AHLs释放量逐渐恢复至稳定期水平,同时B-EPS产量和颗粒性状也逐渐恢复至稳定期水平,推测基质冲击导致AHLs释放量的变化进而引起B-EPS产量的变化.批次试验结果进一步证实了基质浓度显著影响AHLs释放量,B-EPS产量与AHLs释放量密切相关.总氮浓度为1000mg/L时,AHLs释放量高达11.7,导致B-EPS中的松散结合性EPS(LB-EPS)相比总氮浓度为200mg/L时增加了69mg/g VSS,颗粒沉降性能下降.然而紧密结合性EPS(TB-EPS)含量不受基质浓度影响,认为LB-EPS是决定颗粒沉降能力的关键因素.低浓度抑制剂可有效抑制高基质浓度引起的AHLs过量释放,使LB-EPS产量降低36%,颗粒沉降性能和活性得以提高.

Effects of exogenous short-chain N-acyl homoserine lactone on denitrifying process of Paracoccus denitrificans

N-acyl-homoserine lactones（AHLs） serve as quorum-sensing signals, which control a number of bacterial processes in many proteobacteria. Here we report the effects of exogenous short-chain AHL on the denitrifying process of Paracoccus denitrificans, which are capable of aerobic and anaerobic growth by utilizing nitrate. The denitrification activity of these cells was monitored by measuring denitrification products（including nitrate, nitrite,and nitrous oxide）, and the individual messenger ribonucleic acid（m RNA） levels of nitrate,nitrite, nitric oxide and nitrous oxide reductases. The results indicated that 2 μmol/L C6-homoserine lactone（HSL） has little effect on cell density under either anaerobic or aerobic culture conditions, and the nitrate reduction activity appeared slightly affected by N-hexanoyl-DL-homoserine lactone（C6-HSL）. However, exogenous C6-HSL significantly affected the transcription of nitrite reductase and nitric oxide reductase genes in P.denitrificans regardless of the presence of oxygen, and N2O accumulation activity in P. denitrificans was suppressed by C6-HSL under aerobic condition. In contrast, exogenous C6-HSL stimulated the production of N2O under anaerobic condition, suggesting that the regulation of denitrification by quorum sensing may be important in N2O release.

Responses of bacterial strains isolated from drinking water environments to N-acyI-L-homoserine lactones and their analogs during biofilm formation

Often as a result ofbiofilm formation, drinking water distribution systems （DWDS） are regularly faced with the problem of microbial contamination. Quorum sensing （QS） systems play a marked role in the regulation of microbial biofilm formation; thus, inhibition of QS systems may provide a promising approach to biofilm formation control in DWDS. In the present study, 22 bacterial strains were isolated from drinking water-related environments. The following properties of the strains were investigated： bacterial biofilm formation capacity, QS signal molecule N-acyl-L-homoserine lactones （AHLs） production ability, and responses to AHLs and AHL analogs, 3-chloro-4-（dichloromethyl）-5-hydroxy-2（5H）- furanone （MX） and 2（5H）-furanone. Four AHLs were added to developed biofilms at dosages ranging from 0.1 nmol.L J to 100nmol.L1. As a result, the biofilm growth of more than 1/4 of the isolates, which included AHL producers and non-producers, were significantly promoted. Further, the biofilm biomasses were closely associated with respective AHLs concentrations. These results provided evidence to support the idea that AHLs play a definitive role in biofilm formation in many of the studied bacteria. Meanwhile, two AHLs analogs demon- strated unexpectedly minimal negative effects on biofilm formation. This suggested that, in order to find an applicable QS inhibition approach for biofilm control in DWDS, the testing and analysis of more analogs is needed.

AHLs投加方式对好氧颗粒污泥稳定性的影响

为探究N-乙酰基高丝氨酸内酯类化合物投加方式对好氧颗粒污泥稳定性的影响,利用SBAR反应器培养4组好氧颗粒污泥,在不同时期先后投加C12-HSL、C14-HSL两种化合物,监测运行过程中污泥的颗粒粒径、质量浓度、EPS质量分数等参数的变化。结果表明:采用混合投加方式可以使好氧颗粒污泥更稳定,但颗粒粒径明显减小,平均粒径约为0.1 mm;两种化合物共同调控的好氧颗粒污泥更利于维持EPS中酪氨酸类蛋白质、色氨酸类蛋白质、蛋白质二级结构β-折叠的相对质量分数。

Profile of the culturable microbiome capable of producing acyl-homoserine lactone in the tobacco phyllosphere

Bacterial populations coexisting in the phyllosphere niche have important effects on plant health. Quorum sensing （QS） allows bacteria to communicate via diffusible signal molecules, but QS-dependent behaviors in phyllosphere bacterial populations are poorly understood. We investigate the dense and diverse N-acyl-homoserine lactone （AHL）-producing phyllosphere bacteria living on tobacco leaf surfaces via a culture-dependent method and 16S rRNA gene sequencing. Our results indicated that approximately 7.9%-11.7% of the culturable leaf-associated bacteria have the ability to produce AHL based on the assays using whole-cell biosensors. Sequencing of the 16S rRNA gene assigned the AHL-producing strains to two phylogenetic groups, with Gammaproteobacteria （93%） as the predominant group, followed by Alphaproteobacteria. All of the AHL-producing Alphaproteobacteria were affiliated with the genus Rhizobium, whereas the AHL-producing bacteria belonging to the Gammaproteobacteria mainly fell within the genera Pseudomonas, Acinetobacter, Citrobacter, Enterobacter, Pantoea and Serratia. The bioassays of supernatant extracts revealed that a portion of the strains have a remarkable AHL profile for AHL induction activity using the two different biosensors, and one compound in the active extract of a representative isolate, NTL223, corresponded to 3-oxo-hexanoyl-homoserine lactone. A large population size and diversity of bacteria capable of AHL-driven QS were found to cohabit on leaves, implying that cross-communication based AHL-type QS may be common in the phyllosphere. Furthermore, this study provides a general snapshot of a potential valuable application of AHL-producing bacteria inhabiting leaves for their presumable ecological roles in the phyllosphere.

厌氧生物处理中AHLs群体感应调控研究进展

厌氧生物处理技术具有能耗低、能源化特点,在可持续发展污水处理工艺应用中备受关注。基于群体感应厌氧生物处理工艺具有良好应用前景,本文介绍厌氧生物处理技术功能菌群、菌群代谢途径和AHLs群感理论,并归纳AHLs对厌氧颗粒污泥EPS分泌、厌氧颗粒污泥钙化及菌群结构影响方面的研究进展,以期为推动AHLs群感促进厌氧生物处理提高运行效能提供新的理论思路和技术支持。

不同载体材料固定Sphingobium sp.PHE3对溶液中菲的降解研究

为探究不同载体材料对微生物降解菲的影响,以蒙脱石、针铁矿、玉米秸秆炭为载体,采用吸附挂膜法对菲降解菌株(革兰氏阴性菌鞘脂菌PHE3)进行固定化,并应用固定化微生物降解溶液中的菲。根据扫描电镜结果,该菌株在玉米秸秆炭载体表面附着生长数量最多,形态最优。添加蒙脱石和玉米秸秆炭显著(P<0.05)提高了微生物对菲的降解率,加快了菲的微生物降解速率,同时促进了细菌胞外聚合物(EPS)中多糖和蛋白质产量的增加,但添加针铁矿对菲的微生物降解和EPS的产生促进作用较弱。此外,鞘脂菌PHE3在形成生物膜的过程中可能有群体感应现象发生,能够分泌信号分子C_(8)-HSL和C_(12)-HSL,可能在一定程度上调控EPS的生成和污染物的降解。