Identification of the nitrogen-fixing Pseudomonas stutzeri major flagellar gene regulator FleQ and its role in biofilm formation and root colonization

Flagellar biosynthesis and motility are subject to a four-tiered transcriptional regulatory circuit in Pseudomonas,and the master regulator FleQ appears to be the highest-level regulator in this hierarchical regulatory cascade.Pseudomonas stutzeri A1501 is motile by a polar flagellum;however,the motility and regulatory mechanisms involved in this process are unknown.Here,we searched the A1501 genome for flagella and motility genes and found that approximately 50 genes,which were distributed in three non-contiguous chromosomal regions,contribute to the formation,regulation and function of the flagella.The non-polar mutation of fleQ impaired flagellar biosynthesis,motility and root colonization but enhanced biofilm formation.FleQ positively regulates the expression of flagellar class Ⅱ-Ⅳ genes,suggesting a regulatory cascade that is coordinated similar to that of the well-known P.aeruginosa.Based on our results,we propose that flagellar genes in P.stutzeri A1501 are regulated in a cascade regulated by FleQ and that flagellum-driven motility properties may be necessary for competitive rhizosphere colonization.

Mechanical analysis of phase transition experiments of the bacterial flagellar filament

Bacterial flagellar filaments can undergo a polymorphic phase transition in both vitro and vivo environments. Each bacterial flagellar filament has 12 different helical forms which are macroscopically represented by different pitch lengths and helix radii. For external mechanical force induced filament phase transitions, there is so far only one experiment performed by Hotani in 1982, who showed a very beautiful cyclic phase transition phenomenon in his experiment on isolated flagellar filaments. In the present paper, we give a detailed mechanical analysis on Hotani＇s experiments. Through theoretical computations, we obtained a phase transition rule based on the phase transition mechanism. The theoretical analysis provides a foundation facilitating the establishment of phase transition theory for bacterial flagellar filaments.

Simulation of bacterial flagellar phase transition by non-convex and non-local continuum modeling

Bacterial flagellar filament can undergo a stress-induced polymorphic phase transition in both vitro and vivo environments.The filament has 12 different helical forms(phases) characterized by different pitch lengths and helix radii.When subjected to the frictional force of flowing fluid,the filament changes between a left-handed normal phase and a right-handed semi-coiled phase via phase nucleation and growth.This paper develops non-local finite element method(FEM) to simulate the phase transition under a displacement-controlled loading condition(controlled helix-twist).The FEM formulation is based on the Ginzburg-Landau theory using a one-dimensional non-convex and non-local continuum model.To describe the processes of the phase nucleation and growth,viscosity-type kinetics is also used.The non-local FEM simulation captures the main features of the phase transition:two-phase coexistence with an interface of finite thickness,phase nucleation and phase growth with interface propagation.The non-local FEM model provides a tool to study the effects of the interfacial energy/thickness and loading conditions on the phase transition.

Structural analysis of flagellar axonemes from inner arm dynein knockdown strains of Trypanosoma brucei

Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness.Flagellar function in this organism is critical for life cycle progression and pathogenesis,however the regulation of flagellar motility is not well understood.The flagellar axoneme produces a complex beat through the pre-cisely coordinated firing of many proteins,including multiple dynein motors.These motors are found in the inner arm and outer arm complexes.We are studying one of the inner arm dynein motors in the T.brucei flagellum:dynein-f.RNAi knockdown of genes for two components of dynein-f:DNAH10,theαheavy chain,and IC138,an intermediate chain,cause severe motility defects including immotility.To determine if motility defects result from structural disruption of the axoneme,we used two different flagellar preparations to carefully examine axoneme structure in these strains using transmission electron microscopy(TEM).Our analysis showed that inner arm dynein size,axoneme structural integrity and fixed central pair orientation are not significantly different in either knockdown culture when compared to control cultures.These results support the idea that immotility in knockdowns affecting DNAH10 or IC138 results from loss of dynein-f function rather than from obvious structural defects in the axoneme.

Arriving at a correlation between the flagellar arrangement and multicellularity

Cilia and flagella are organelles of motility that enable cells to swim or move liquid over its surface. An exhaustive literature survey for the presence of the organelle in organisms across phyla showed that most animal cells harbor cilia in contrast to very few fungal cells. While this was not unexpected, it was the position and arrangement of this organelle in each cell that intrigued our attention. Natural selection might have favored motility over chemotaxis;and it would have done so to evolve a stable structure that could have undergone an optimization process requiring a precise geometry in the shape of cells and the structure that would help cells to move. The positioning of such a structure would play a pre-dominant role in optimal motility. It is now known that the flagellar position of a cell is a genetically distinct trait, occasionally used in phylogeny of bacteria, distributed in distinguishing patterns over cellular surface, but basically are of two types, either polar (one flagellum arising from one pole per cell) or peritrichous (lateral flagella distributed over the entire cell surface). Irrespective of the cellular habitat, flagella origin, ultrastructure and proteome, the present investigation surveyed 26 sub-types of flagellar arrangements from as many species as possible. A peculiar pattern ensued-Prokaryotes harbored predominantly polar and peritrichous types;eukaryotes showed a mere change of the peritrichous one. These numbers when used to create a Similarity tree depicted a similarity distance of 14 between the Eubacteria and Archaebacteria forming the first neighborhood;Protozoans, Algae, Fungi, Plantae and Animalia formed a second neighborhood. We offer a working hypothesis for this pattern and the gradual shift in the flagellar arrangement from polar, peritrichous, sub-apical, and apical to lateral throughout evolution.

Sperm flagellar 2(SPEF2)is essential for sperm flagellar assembly in humans

Spermiogenesis is a complex and tightly regulated process,consisting of acrosomal biogenesis,condensation of chromatin,flagellar assembly,and disposal of extra cytoplasm.Previous studies have reported that sperm flagellar 2(SPEF2)deficiency causes severe asthenoteratozoospermia owing to spermiogenesis failure,but the underlying molecular mechanism in humans remains unclear.Here,we performed proteomic analysis on spermatozoa from three SPEF2 mutant patients to study the functional role of SPEF2 during sperm tail development.A total of 1262 differentially expressed proteins were detected,including 486 upregulated and 776 downregulated.The constructed heat map of the differentially expressed proteins showed similar trends.Among these,the expression of proteins related to flagellar assembly,including SPEF2,sperm associated antigen 6(SPAG6),dynein light chain tctex-type 1(DYNLT1),radial spoke head component 1(RSPH1),translocase of outer mitochondrial membrane 20(TOM20),EF-hand domain containing 1(EFHC1),meiosis-specific nuclear structural 1(MNS1)and intraflagellar transport 20(IFT20),was verified by western blot.Functional clustering analysis indicated that these differentially expressed proteins were specifically enriched for terms such as spermatid development and flagellar assembly.Furthermore,we showed that SPEF2 interacts with radial spoke head component 9(RSPH9)and IFT20 in vitro,which are well-studied components of radial spokes or intra-flagellar transport and are essential for flagellar assembly.These results provide a rich resource for further investigation into the molecular mechanism underlying the role that SPEF2 plays in sperm tail development and could provide a theoretical basis for gene therapy in SPEF2 mutant patients in the future.

MECHANICAL MODELING OF THE BISTABLE BACTERIAL FLAGELLAR FILAMENT

We extend the 2D Landau phase transition theory to the bacterial flagellar filament which displays the phase transition between the left handed normal form and the right handed semi-coiled form. The bacterial flagellar filament is treated as an elastic thin rod based on the Kirchhoff’s thin rod theory. Mechanical analysis is performed on the periodical phase transition of the filament between the two helical structures of the opposite charity. The curvature and twist are chosen as the order parameters in constructing the phase transition model of the filament. The established model is applied to study the instability properties of the filament and to investigate the loading and deformation conditions of the phase transition. In addition, the curvature and twist gradient energy are considered to describe the interface properties of the two phases.

Flow Visualization and Performance Measurements of a Flagellar Propeller

A new type of propeller that is optimized for low Reynolds numbers is required to propel a small object in a medium where the flow is dominated by viscous rather than inertial forces. A propeller in the shape of a bacterial flagellum seems an appro- priate choice for driving a small object. Accordingly, in this study, we visualized the velocity field induced by a spring-like propeller inspired by the Escherichia coli flagellum, using a macroscopic model and applying stereoscopic particle image velocimetry. We also experimentally evaluated the effect of pitch and rotational speed on the performance of this flagellar propeller. Silicone oil, which has a kinematic viscosity 100,000 times that of water, was used as the working fluid to generate a low Reynolds number for the macroscopic model. Thrust, torque, and velocity were measured as functions of pitch and rota- tional speed, and the efficiency of the propeller was calculated from the measured results. We found that the flagellar propeller reached a max!mum efficiency when the pitch angle was approximately 53°. Compared to pitch, rotational speed had a relatively small effect on the efficiency, and the pitch altered the flow pattern behind the rotating propeller.

Non-equilibrium effect in the allosteric regulation of the bacterial flagellar switch

Subject Code:A02With funding support from the National Natural Science Foundation of China,the research group led by Prof.Yuan Junhua(袁军华)and Zhang Rongjing(张榕京)from the University of Science and Technology of China(USTC)has discovered non-equilibrium effect in the regulation of the bacterial flagellar switch,

二甲双胍抑制大肠杆菌生长和鞭毛马达转速

文章运用光学显微成像技术和生物学统计模型等多种生物物理手段研究二甲双胍对大肠杆菌的生长、运动和趋化行为改变过程的影响。研究表明:二甲双胍能够抑制大肠杆菌的生长,并且大肠杆菌的形态受到生长的影响;利用倒置显微镜结合乳胶小球标记鞭毛法和微流控技术探究二甲双胍对大肠杆菌鞭毛马达转速的影响,结果发现,二甲双胍对鞭毛马达的转速具有抑制效果,且不同浓度的二甲双胍影响大肠杆菌对天冬氨酸趋化反应的恢复时间。

鼠伤寒沙门氏菌鞭毛蛋白FliC的原核表达、纯化及其多克隆抗体的制备

利用PCR扩增出鼠伤寒沙门氏菌鞭毛蛋白基因fliC,连接到原核表达载体pET28a(+)上,测序鉴定后转化至大肠杆菌BL21(DE3)pLysS感受态细胞中,构建了原核表达系统。经1mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导及Ni-NTA纯化后,得到了带6×His纯化标签的分子质量大小约为54.6kD的表达产物,和预测蛋白大小一致,且大部分表达产物以可溶形式存在利用Western-blotting进一步鉴定,结果表明,该蛋白能与抗His标签的单抗发生特异性反应。用纯化的融合蛋白免疫BALB/c小鼠,获得多克隆抗体,并对抗血清的效价和特异性进行检测,结果表明,多抗的效价较高,特异性较好。

奇异变形杆菌周期性群集运动的研究

目的 研究奇异变形杆菌(proteusmirabilis ,PM )迁徙生长过程中形态、数量、呼吸酶活性等指标周期性变化。方法 在培养基中加入2 ,3 ,5 -三苯基四唑氯化物(2 ,3 ,5 tetraphenyltetrazoliumchloride ,TTC)作为呼吸酶活性的指示剂观察细菌呼吸酶活性的变化。采用透射电子显微镜对细菌形态、鞭毛进行对比观察。比浊法计数PM在时间、空间上的数量的变化。结果 PM在固体培养基迁徙生长过程中出现菌体数量逐渐减少、形态由短小到细长、鞭毛由少变多、呼吸酶从有活性向无活性转变的周期性变化。结论 PM在培养基迁徙生长过程中,微观上出现菌体长度、鞭毛数量、细菌密度等周期性变化,宏观上出现水波样的环状运动,这是环境、鞭毛、细胞间信号传递和细菌密度等多因素联合作用的结果。

杜氏盐藻S腺苷高半胱氨酸水解酶基因的克隆及功能分析

目的:探讨杜氏盐藻S腺苷高半胱氨酸水解酶(SAHase)基因在鞭毛再生中的作用。方法:通过设计简并引物及RACE法克隆了杜氏盐藻SAHase基因全长,使用pH休克法对杜氏盐藻进行鞭毛去除及再生,通过实时荧光定量PCR研究在转录水平上SAHase基因的变化。结果:所克隆的杜氏盐藻SAHase基因cDNA全长1926bp,包含ORF1458bp、3’UTR61bp和5’UTR407bp。实时荧光定量PCR结果显示,在鞭毛再生的过程中,SAHasemRNA转录量升高,其水平在3h时达到了未处理组的3倍左右。结论:杜氏盐藻的SAHase基因与鞭毛再生有关。

水稻白叶枯病菌核苷酸信号受体蛋白Clpxoo的分子鉴定及其功能

【目的】本文的目的为阐明水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,简称Xoo)核苷酸信号途径及其作用机理。【方法】本研究对推导的信号受体蛋白Clpxoo进行了基因克隆、序列分析、缺失突变和互补及其相关表型的鉴定。【结果】克隆的clpxoo基因序列与大肠杆菌crp和铜绿假单胞菌vfr的同源性较高,与其它几种病原黄单胞菌clp高度保守。Clpxoo序列N端具有环化核苷酸cNMP结合结构域(CAP-ED),C端具有保守的DNA结合结构域(HTH-CRP)。用双交换法构建了基因缺失突变体(△clpxoo)。与野生型菌株PXO99A相比,△clpxoo的运动性、胞外多糖产生能力和对H2O2的抗性均显著降低,基因互补可使之部分恢复;△clpxoo胞外酶产生和对烟草致敏性无显著改变。【结论】Clpxoo可能作为全局性的保守调控因子之一,调控了Xoo的鞭毛运动性、胞外多糖产生和对H2O2的抗性。

不同温度条件下鲁氏耶尔森氏菌的链特异性转录组分析

为鉴定鱼源鲁氏耶尔森氏菌(Yersinia ruckeri)SC09菌株水生环境中不同温度的转录组水平上的差异,研究采用链特异性转录组测序(Strand-specific RNA-seq)技术对菌体生理温度(28℃)和实验培养温度(37℃)下进行链特异性测序,原始数据质控后,筛选得到差异表达基因,通过KEGG(Kyoto Encyclopedia of Genes and Genomes)数据库对差异表达基因进行富集分析,并利用Rockhopper软件筛选出的重要原核生物基因簇进行验证。结果显示,共筛选获得173个显著差异表达基因(P<0.05),其中包括58个上调基因,主要富集到一些特殊的碳水化合物代谢相关的通路中;以及115个下调基因,主要富集到双组份信号系统中与三羧酸循环相关的代谢通路上,同时部分基因富集到编码鞭毛素相关的基因簇中。结果表明,相对于37℃的实验室培养温度,在水生环境的生理温度条件下(28℃)SC09菌株拥有较高的运动性和较强的葡萄糖代谢,但相对的SC09菌株代谢一些特殊糖类的能力减弱。

一种lacZ报告基因T载体的构建及其在沙门氏菌鞭毛主调控基因flhDC表达活性测定中的应用

为研究5'-非翻译区(UTR)对沙门氏菌鞭毛主调控基因flhDC表达的影响,本研究以鼠伤寒沙门氏菌542(STM542)基因组DNA为模板,扩增含不同长度5'-UTR的flhDC全长基因(共5种),并将其克隆至含阿拉伯糖启动子的质粒PBAD33中。通过测定含阿拉伯糖的半固体平板上包含不同长度flhDC基因的重组大肠杆菌菌落的直径,初步评估不同5'-UTR调控序列对flhDC基因表达的影响。为精确测定不同调控序列的活性差异,本实验室进一步构建了以lacZ为报告基因的T载体,并将扩增的对应于前4种flhDC基因调控序列片段克隆于构建的T载体,通过测定其β-半乳糖苷酶活性获得相应调控序列的活性参数。结果显示,在阿拉伯糖诱导下,对应于1572bp的flhDC基因片段的调控序列活性最低,对应于1201bp的flhDC基因片段的调控序列活性最高,本实验为进一步研究flhDC基因的调控功能奠定基础。

等鞭金藻属(等鞭金藻目)1新种——湛江等鞭金藻(Isochrysis zhanjiangensis sp .nov)及其超微结构的观察

描述了从广东湛江南三岛沿岸采集、分离的等鞭金藻1新种——湛江等鞭金藻，应用电镜技术观察了湛江等鞭金藻运动细胞的超微结构．电镜观察证明，其运动细胞表面覆盖2～4层鳞片，鳞片有两种类型，一种较大，椭圆形；另一种较小，圆形．细胞顶端具有2条等长的平滑的鞭毛，两鞭毛间具有一条退化的定鞭（haptonema），很短，仅有1～2条微管．湛江等鞭金藻鞭毛过渡区中央轴丝与周边微管之间有5～6条螺旋条纹．两个金褐色色素体位于细胞两侧．3条类囊体为一组，无带片层．每个质体内侧具1个单层膜包裹的裸露的蛋白核．蛋白核内常具1对横列的类囊体．高尔基器位于鞭毛基体基部．线粒体长管状，具多个不分枝或分枝的脊．细胞中下部具1个至多个金藻昆布糖（Chrysolaminaran）颗粒．细胞核位于两质体之间、细胞基部．

细菌鞭毛马达——一种卓越的分子机器

鞭毛马达 (flagellarmotor)是一种分子旋转马达 ,它在细菌鞭毛的结构与功能中起着中心作用 .鞭毛马达的结构已基本清楚 ,主要由MotA、MotB、FliG、FliM和FliN 5种蛋白组成定子 (stator)和转子 (rotor) ,其驱动力来自于跨膜的H+或Na+流 .目前对鞭毛马达的旋转动力学及旋转力矩产生机制已有初步的了解 .鞭毛马达可作为研究分子旋转马达的理想模型 。

青岛海洋趋磁球菌QH-3的鞭毛特征

趋磁细菌(magnetotactic bacteria,MTB)是一类能够沿着磁力线运动的特殊细菌。MTB在胞内磁小体的导向下借助自身鞭毛进行趋磁趋氧运动,以到达适宜的生境。本研究在青岛潮间带沉积物中发现大量的趋磁球菌,通过光镜与透射电镜对其运动方式与显微结构进行观察。结果显示优势趋磁细菌为卵球形,大小为2.5μm×2.1μm,宽长比为0.85,将其命名为QH-3。QH-3为趋北型(North-seeking)MTB,运动速度88μm/s,细胞内两侧各有一条由棱柱形颗粒组成的磁小体链。QH-3在菌体同一侧有两个由直径62 nm±9 nm的鞘包裹7根鞭毛组成的鞭毛束。这种特殊的鞭毛结构与已报道的致病菌的不同,而与法国地中海趋磁球菌MO-1的相似。

外源钙对盐胁迫下匍枝萎陵菜生长及生理特性的影响

采用盆栽试验法，对匍枝萎陵菜进行盐胁迫处理，同时施用不同浓度氯化钙，通过对新梢生长量，叶片中丙二醛（MDA）、可溶性糖、脯氨酸（Pro）的含量以及超氧化物歧化酶（SOD）活性等指标进行测定分析，来探讨其耐盐能力及氯化钙的作用。结果表明：随盐浓度升高，匍枝萎陵菜新梢生长量下降，叶片中MDA和Pro含量增加，叶片中SOD活性、可溶性糖含量均呈先升后降趋势。各盐浓度处理下，随外源钙浓度的增加，新梢生长量、SOD活性、可溶性糖含量、脯氨酸含量基本呈现先升后降趋势，MDA含量在低盐浓度下（3，4mg·g^-1）呈下降趋势，在高盐浓度下（5，6mg·g^-1）呈现先降后升趋势。综合各测定指标及外部形态的表现认为：匍枝萎陵菜可以在含盐量4．00mg·g^-1及以下的土壤中正常生长；氯化钙对匍枝萎陵菜遭受盐胁迫有一定缓解作用，在较低盐浓度下（3—4mg·g^-1），CaCl2缓解作用较明显，以20mmol·L^-1的浓度较适宜；较高盐浓度下（5-6mg·g^-1）CaCl：的作用不明显。