Ⅳ型菌毛可视化方法及其在菌毛功能研究中的应用

Ⅳ型菌毛(type Ⅳ pili, TFP)作为细菌表面的重要蛋白结构，是细菌的感知器官及运动器官，在细菌生理学、细胞黏附、宿主细胞入侵、DNA摄取、蛋白质分泌、生物被膜形成、细胞运动和电子传递等方面发挥着多种作用。近年来，随着研究方法的深入和技术设备的发展，尤其是随着多种菌毛可视化工具的开发，越来越多的研究揭示了它在生命活动中的各种功能，大大加快了微生物单细胞领域的研究步伐。本文重点讨论了TFP可视化方法及在菌毛功能研究中的应用，为更好地研究和利用TFP功能提供更多的思路，为其未来在生物学、医学以及生态学中的应用提供一定的理论基础。     

细菌平板菌落计数的改进方法

细菌平板菌落计数是微生物教学、科研及生产实践中最常用的活细菌总数测定方法。由于它是根据固体平板上一个菌落代表一个细菌的原理而设计的,因此深层菌落的存在、菌落的大小及疏密程度等因素均影响观察者观察计数。为便于观察.提高菌落的检出率,对常规平板菌落计数法做如下改进: 在倒平板之前,向装有150ml牛肉膏蛋白胨固体培养基的三角瓶内加入1/1000浓度的氧化还原染料     

长江下游地区中国荷斯坦奶牛生乳中菌落总数的影响因素分析

【背景】随着人们生活水平和健康意识的提高,作为部分乳制品的原料,生乳的质量安全问题显得尤为重要。【目的】检测长江下游地区某集约化牧场中国荷斯坦奶牛生乳中菌落总数、总大肠菌群数的真实水平,同时对影响其变化的生理因素和环境因素进行探讨。【方法】以中国荷斯坦奶牛生乳为研究对象,用乳房炎快速诊断剂检测乳房炎感染情况,菌落总数、总大肠菌群数和环境细菌数的检测方法参照国家标准。对采集的生乳样本进行细菌常规分离鉴定,同时收集乳成分变化数据。【结果】多因素方差分析结果表明,不同泌乳阶段和不同产奶量对荷斯坦奶牛生乳中菌落总数的影响显著(P0.05)和极显著(P0.01);体况、泌乳阶段、胎次、产奶量及乳区对大肠菌群数均无显著影响(P0.05)。夏季生乳的菌落总数、大肠菌群数均极显著地高于其他季节(P0.01);场区内各牛舍的不同卫生环境及细菌数对菌落总数、大肠菌群数的影响均在显著以上(P0.05)。不同乳房炎的感染程度对菌落总数影响极显著(P0.01)。细菌鉴定结果是葡萄球菌属和杆菌属感染居多,链球菌属感染几乎没有。菌落总数的变化对乳蛋白率影响不显著(P0.05),对乳脂率的影响极显著(P0.01)。【结论】随机采样泌乳牛群的不同生理和病理状况、外界不同饲养条件及卫生环境对生乳中菌落总数和大肠菌群数有不同程度的影响,生乳的乳脂率与细菌总数变化负相关。     

Evidence of functional and structural changes in the microbial community beneath a succulent invasive plant in coastal dunes

海岸沙地肉质入侵植物对微生物群落功能与结构变化的影响 海岸沙地提供了诸多宝贵的生态系统服务，例如耕地保护、水分供应和生物多样性保护，因此海岸沙地代表了重要的栖息地保护区。因为海岸沙地环境恶劣、水资源匮乏、养分贫瘠，所以土壤微生物群落对维持植物多样性至关重要。就海岸沙地的生态系统保护而言，外来入侵植物是主要威胁。本文探讨了沿海地区入侵植物食用日中花(Carpobrotus edulis)对细菌群落功能及结构的各种影响。沙地中土壤有机质含量低，微生物活性有限，所以沙地是一种颇具挑战性的底物。因此，在评估食用日中花对生态系统的影响之前需要对细菌提取进行优化，并开展功能评估。本文首先从群落水平生理特征(CLPP)的角度，比较了采用不同土壤储量、样品量、提取液等12种提取方法对沙地群落功能活性的影响，然后又探讨了食用日中花入侵的沙地菌落功能(利用Biolog Ecoplates)与结构变化(利用PCR-DGGE)。血细胞计数发现盐溶液持续增殖了细菌细胞(P ≤ 0.001)。主成分分析(PCA)结果显示，在有限时间框架(0–24 h)内，碳底物消耗没有显著变化。通过对比天然区与非天然区(种间)及天然区(种内)之间食用日中花入侵后的变化，发现了一种与众不同的碳底物利用模式，食用日中花入侵对群落功能的影响因地而异，而与之相辅相成的是，PCR-DGGE结果表明原沙地的菌落结构不同于食用日中花入侵后沙地的菌落结构。     

微生物信息传递的研究进展

作为人类,我们应该向我们的微生物邻居学习,它们为了共同利益可以通过群体感应(QS)调控机制团结合作.这种调控机制表达一些对微生物群体有益的物质.美国微生物协会( ASM)第四届细菌信号大会于2011年11月6日至9日在美国迈阿密举行.来自世界各地的超过175名科学家出席了会议,分享他们的最新研究成果.此次会议共有44个报告以及99个海报,它的召开进一步推进了微生物领域的研究进程.本届会议共有三个主要议题:第一,进一步强化了“微生物社会学”的概念,GREENBERG在其报告中强调在微生物世界中同样有互利共生和竞争关系;第二,细菌信号及其受体以及转导的机制,同时展示了一些研究细菌信号转导过程的新技术;第三,细菌间以及细菌与动物植物之间的信号联系.     

数控剪切流微流体技术及其在细菌生物膜研究中的应用

<正>细菌生物膜是指微生物(细菌、真菌等)黏附、聚集形成的一个群体,该群体产生并分泌胞外聚合物(extracellular polymeric substance,EPS),形成一定的三维结构,含有营养物质、氧气等生长必需物质交换的通道,微生物细胞在EPS中增殖、生存[1]。生物膜结构可阻止抗生素或抗体等大分子有效杀伤微生物细胞,目前尚无有效的预防措施和治疗方法控制细菌生物膜所带来的危害。对细菌生物膜形成     

一种从感染的培养细胞中分离细菌RNA的方法

病原菌全基因组表达谱研究是阐明其致病机理的必要的基础 ,已经成为当前生命科学领域的热点和重点 ;然而由于难于从感染的组织中快速固定并分离细菌RNA ,从而极大的制约了该研究的进展。介绍一种从感染的细胞中分离细菌RNA的方法———冷酸酚法 ,其主要特点是 :(1)使用可以破碎真核细胞但不影响细菌细胞完整性的SDS浓度 ,实现了从感染的细胞中快速分离完整的细菌 ,减少了宿主细胞RNA的污染 ;(2 )在将细菌从细胞中分离的同时即利用苯酚 乙醇混合液将其总RNA快速固定 ,既减少了RNA的降解 ,又最大限度地保持了细菌在哺乳细胞内原有的表达模式 ;(3)可以从 10 8个菌体中提取到至少 30 μg的总RNA ,足够用于反转录等其他研究。该方法将为利用DNAMicroarray技术进行的病原菌表达谱研究提供有益的借鉴。     

沼泽红假单胞菌作为微生物细胞工厂的应用

沼泽红假单胞菌(Rhodopseudomonas palustris)是一种紫色非硫细菌,具有新陈代谢代谢方式多样、可利用多种碳源、天然产多种化合物等优点,作为微生物细胞工厂具有很大的研究潜力,目前主要应用于废水处理和水产养殖领域。本文中,笔者从三个方面对沼泽红假单胞菌在微生物细胞工厂领域的研究应用进行总结,包括碳源的多样性、基因工程策略以及微生物细胞工厂的应用,如在产燃料燃气和萜烯化合物、微生物燃料电池、微生物电合成和光催化合成等领域。目前沼泽红假单胞菌作为微生物细胞工厂处于发展的初级阶段,笔者对其目前面临的难点进行了总结,并对其进一步的研究方向进行了展望。     

长期施肥对麦田土壤微生物垂直分布的影响

应用微量热法研究了长期轮作下麦田不同土层中的微生物活性, 以及施肥对微生物垂直分布的影响。结果表明, 微生物活性基本随深度的增加而下降; 随着土层加深, 可培养细菌菌落数减少, 且可培养细菌菌落数目与最大时间(Peak time)P_t值(表示从微生物生长开始到达到峰值的时间)呈负相关; 热功率(P)-时间(t)曲线由陡变缓, 由规则变得起伏不定, 侧翼变得更短, 峰高也降低; 不同土层的微生物生长速率常数(Microbial growth rate constant) µ、峰高(Peak height)P_h值变化明显, 基本随深度增加而减小。施肥土样和不施肥土样的曲线形状不同, 特别是上层土样施肥后的曲线明显比不施肥的曲线陡, 侧翼也明显变短; 施肥土样的µ和P_h值都大于不施肥土样, 且施肥土样的P_t都小于对照。这说明, 在不同的土壤深度有不同的微生物群落结构, 长期的施肥处理改变了土壤微生物的垂直分布特征。     

细菌显微追踪技术在生物被膜中的应用

细菌生物被膜是粘附于物体表面的由细菌细胞及其胞外物质组成的复杂膜样物聚集体,具有很强的耐药性和免疫逃逸能力。生物被膜内细菌的代谢活性、运动状态等与浮游细菌有明显区别。近年来,先进的显微成像技术结合新型图像处理方法,在研究细菌的运动、生理等方面发挥了重要作用。本文围绕生物被膜,概述了细菌显微追踪技术在其研究中的应用。主要从细菌的运动方式和生物被膜形成过程的调控两方面出发,介绍了在单细胞水平上利用该技术研究生物被膜的进展,包括细菌的游泳、蹭行、群集运动和多种信号通路调控下生物被膜的形成过程等,并展望了该技术在生物被膜其他相关研究领域的应用前景。     

Dynamics of Snake-like Swarming Behavior of Vibrio alginolyticus

Swarming represents a special case of bacterial behavior where motile bacteria migrate rapidly and collectively on surfaces. Swarming and swimming motility of bacteria has been studied well for rigid, self-propelled rods. In this study we report a strain of Vibrio alginolyticus, a species that exhibits similar collective motility but a fundamentally different cell morphology with highly flexible snake-like swarming cells. Investigating swarming dynamics requires high-resolution imaging of single cells with coverage over a large area: thousands of square microns. Researchers previously have employed various methods of motion analysis but largely for rod-like bacteria. We employ temporal variance analysis of a short time-lapse microscopic image series to capture the motion dynamics of swarming Vibrio alginolyticus at cellular resolution over hundreds of microns. Temporal variance is a simple and broadly applicable method for analyzing bacterial swarming behavior in two and three dimensions with both high-resolution and wide-spatial coverage. This study provides detailed insights into the swarming architecture and dynamics of Vibrio alginolyticus isolate B522 on carrageenan agar that may lay the foundation for swarming studies of snake-like, nonrod-shaped motile cell types.     

Lateral Flagella and Swarming Motility in Aeromonas Species

Swarming motility, a flagellum-dependent behavior that allows bacteria to move over solid surfaces, has been implicated in biofilm formation and bacterial virulence. In this study, light and electron microscopic analyses and genetic and functional investigations have shown that at least 50% of Aeromonas isolates from the species most commonly associated with diarrheal illness produce lateral flagella which mediate swarming motility. Aeromonas lateral flagella were optimally produced when bacteria were grown on solid medium for approximately 8 h. Transmission and thin-section electron microscopy confirmed that these flagella do not possess a sheath structure. Southern analysis of Aeromonas reference strains and strains of mesophilic species (n = 84, varied sources and geographic regions) with a probe designed to detect lateral flagellin genes (lafA1 and lafA2) showed there was no marked species association of laf distribution. Approximately 50% of these strains hybridized strongly with the probe, in good agreement with the expression studies. We established a reproducible swarming assay (0.5% Eiken agar in Difco broth, 30 degrees C) for Aeromonas spp. The laf-positive strains exhibited vigorous swarming motility, whereas laf-negative strains grew but showed no movement from the inoculation site. Light and scanning electron microscopic investigations revealed that lateral flagella formed bacterium-bacterium linkages on the agar surface. Strains of an Aeromonas caviae isolate in which lateral flagellum expression was abrogated by specific mutations in flagellar genes did not swarm, proving conclusively that lateral flagella are required for the surface movement. Whether lateral flagella and swarming motility contribute to Aeromonas intestinal colonization and virulence remains to be determined.     

Swarming populations of<Emphasis Type="Italic">Salmonella</Emphasis> represent a unique physiological state coupled to multiple mechanisms of antibiotic resistance

Salmonella enterica serovar Typhimurium is capable of swarming over semi-solid surfaces. Although its swarming behavior shares many readily observable similarities with other swarming bacteria, the phenomenon remains somewhat of an enigma in this bacterium since some attributes skew away from the better characterized systems. Swarming is quite distinct from the classic swimming motility, as there is a prerequisite for cells to first undergo a morphological transformation into swarmer cells. In some organisms, swarming is controlled by quorum sensing, and in others, swarming has been shown to be coupled to increased expression of important virulence factors. Swarming in serovar Typhimurium is coupled to elevated resistance to a wide variety of structurally and functionally distinct classes of antimicrobial compounds. As serovar Typhimurium differentiates into swarm cells, thepmrHFIJKLM operon is up-regulated, resulting in a more positively charged LPS core. Furthermore, as swarm cells begin to de-differentiate, thepmr operon expression is down-regulated, rapidly reaching the levels observed in swim cells. This is one potential mechanism which confers swarm cells increased resistance to antibiotics such as the cationic antimicrobial peptides. However, additional mechanisms are likely associated with the cells in the swarm state that confer elevated resistance to such a broad spectrum of antimicrobial agents. Published: September 26, 2003     

Genetic Determinants of Swarming in Rhizobium etli

Swarming motility is considered to be a social phenomenon that enables groups of bacteria to move coordinately atop solid surfaces. The differentiated swarmer cell population is embedded in an extracellular slime layer, and the phenomenon has previously been linked with biofilm formation and virulence. The gram-negative nitrogen-fixing soil bacterium Rhizobium etli CNPAF512 was previously shown to display swarming behavior on soft agar plates. In a search for novel genetic determinants of swarming, a detailed analysis of the swarming behavior of 700 miniTn5 mutants of R. etli was performed. Twenty-four mutants defective in swarming or displaying abnormal swarming patterns were identified and could be divided into three groups based on their swarming pattern. Fourteen mutants were completely swarming deficient, five mutants showed an atypical swarming pattern with no completely smooth edge and local extrusions, and five mutants displayed an intermediate swarming phenotype. Sequence analysis of the targeted genes indicated that the mutants were likely affected in quorum-sensing, polysaccharide composition or export, motility, and amino acid and polyamines metabolism. Several of the identified mutants displayed a reduced symbiotic nitrogen fixation activity.     

Salmonella enterica serovar typhimurium swarming mutants with altered biofilm-forming abilities: surfactin inhibits biofilm formation

Swarming motility plays an important role in surface colonization by several flagellated bacteria. Swarmer cells are specially adapted to rapidly translocate over agar surfaces by virtue of their more numerous flagella, longer cell length, and encasement of slime. The external slime provides the milieu for motility and likely harbors swarming signals. We recently reported the isolation of swarming-defective transposon mutants of Salmonella enterica serovar Typhimurium, a large majority of which were defective in lipopolysaccharide (LPS) synthesis. Here, we have examined the biofilm-forming abilities of the swarming mutants using a microtiter plate assay. A whole spectrum of efficiencies were observed, with LPS mutants being generally more proficient than wild-type organisms in biofilm formation. Since we have postulated that O-antigen may serve a surfactant function during swarming, we tested the effect of the biosurfactant surfactin on biofilm formation. We report that surfactin inhibits biofilm formation of wild-type S. enterica grown either in polyvinyl chloride microtiter wells or in urethral catheters. Other bio- and chemical surfactants tested had similar effects.     

Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes

Swarming is a specialized form of surface motility displayed by several flagellated bacterial genera, which shares features with other surface phenomenon such as biofilm formation and host invasion. Swarmer cells are generally more flagellated and longer than vegetative cells of the same species propagated in liquid media, and move within an encasement of polysaccharide 'slime'. Signals and signalling pathways controlling swarm cell differentiation are largely unknown. In order to test whether there is a genetic programme specific to swarming, we have determined global gene expression profiles of Salmonella typhimurium over an 8 h time course during swarming, and compared the microarray data with a similar time course of growth in liquid media as well as on harder agar where the bacteria do not swarm. Our data show that bacteria growing on the surface of agar have a markedly different physiology from those in broth, as judged by differential regulation of nearly one-third of the functional genome. The large number of genes showing surface-specific upregulation included those for lipopolysaccharide synthesis, iron metabolism and type III secretion. Although swarming-specific induction of flagellar gene expression was not generally apparent, genes for iron metabolism were strongly induced specifically on swarm agar. Surface-dependent regulation of many virulence genes suggests that growth on an agar surface could serve as a model for gene expression during the initial stages of host infection. Based on cluster analysis of distinctive expression patterns, we report here the identification of putative new genes involved in motility and virulence.     

Social interactions in myxobacterial swarming

Swarming, a collective motion of many thousands of cells, produces colonies that rapidly spread over surfaces. In this paper, we introduce a cell-based model to study how interactions between neighboring cells facilitate swarming. We chose to study Myxococcus xanthus, a species of myxobacteria, because it swarms rapidly and has well-defined cell–cell interactions mediated by type IV pili and by slime trails. The aim of this paper is to test whether the cell contact interactions, which are inherent in pili-based S motility and slime-based A motility, are sufficient to explain the observed expansion of wild-type swarms. The simulations yield a constant rate of swarm expansion, which has been observed experimentally. Also, the model is able to quantify the contributions of S motility and A motility to swarming. Some pathogenic bacteria spread over infected tissue by swarming. The model described here may shed some light on their colonization process.     

Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili

We describe swarming in Pseudomonas aeruginosa as a third mode of surface translocation in addition to the previously described swimming and twitching motilities. Swarming in P. aeruginosa is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids. Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming. Cells from the edge of the swarm were about twice as long as cells from the swarm center. In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy. While a fliC mutant of P. aeruginosa displayed slightly diminished swarming, a pilR and a pilA mutant, both deficient in type IV pili, were unable to swarm. Furthermore, cells with mutations in the las cell-to-cell signaling system showed diminished swarming behavior, while rhl mutants were completely unable to swarm. Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of P. aeruginosa in this type of surface motility.