Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export

Diverse molecules, from small antibacterial drugs to large protein toxins, are exported directly across both cell membranes of gram-negative bacteria. This export is brought about by the reversible interaction of substrate-specific inner-membrane proteins with an outer-membrane protein of the TolC family, thus bypassing the intervening periplasm. Here we report the 2.1-A crystal structure of TolC from Escherichia coli, revealing a distinctive and previously unknown fold. Three TolC protomers assemble to form a continuous, solvent-accessible conduit--a 'channel-tunnel' over 140 A long that spans both the outer membrane and periplasmic space. The periplasmic or proximal end of the tunnel is sealed by sets of coiled helices. We suggest these could be untwisted by an allosteric mechanism, mediated by protein-protein interactions, to open the tunnel. The structure provides an explanation of how the cell cytosol is connected to the external environment during export, and suggests a general mechanism for the action of bacterial efflux pumps.     

Type VI secretion delivers bacteriolytic effectors to target cells

Peptidoglycan is the major structural constituent of the bacterial cell wall, forming a meshwork outside the cytoplasmic membrane that maintains cell shape and prevents lysis. In Gram-negative bacteria, peptidoglycan is located in the periplasm, where it is protected from exogenous lytic enzymes by the outer membrane. Here we show that the type VI secretion system of Pseudomonas aeruginosa breaches this barrier to deliver two effector proteins, Tse1 and Tse3, to the periplasm of recipient cells. In this compartment, the effectors hydrolyse peptidoglycan, thereby providing a fitness advantage for P. aeruginosa cells in competition with other bacteria. To protect itself from lysis by Tse1 and Tse3, P. aeruginosa uses specific periplasmically localized immunity proteins. The requirement for these immunity proteins depends on intercellular self-intoxication through an active type VI secretion system, indicating a mechanism for export whereby effectors do not access donor cell periplasm in transit.     

生物膜和生物膜形成菌的研究

细菌生物膜是一个复杂的微生物群落,生物膜巾除了水和细菌以外,还含有细菌分泌的胞外聚合物、吸附的营养物质、代谢产物及DNA等细菌裂解产物.介绍细菌生物膜的形成过程,并综述了近年来医学领域以几种成膜力强的条件致病菌为研究对象,从基因水平上证实了细菌细胞表面结构鞭毛、纤毛、胞外聚合物和群体感应信号分子等细胞因子对生物膜形成的影响.     

Overexpression and export of Vibrio anguillarum metalloprotease in Escherichia coli

Vibrio anguillarum metalloprotease, an extracellular zinc metalloprotease involved in the virulence mechanism of Vibrio anguillarum, is synthesized from the empA gene as a 611-residue precursor and naturally secreted via Sec secretion pathway in Vibrio anguillarum. In this study, heterologous expression of the empA gene encoding metalloprotease and export of the recombinant metalloprotease in Escherichia coli were examined. The empA gene was subcloned into pBAD24 with arabinose promoter and sequenced. The sequence encoded a polypeptide (611 amino acids) consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. The empA gene inserted in plasmid pBAD24 was overexpressed in TOP10 strain of E. coli after arabinose induction. The 36kDa polypeptide of the recombinant metalloprotease as the mature protease was further confirmed by SDS-PAGE and immunoblotting. It was found that recombinant metalloprotease with the EmpA activity and antigenicity was exported into the periplasm of Escherichia coli cells via Sec translocation pathway, whereas it was secreted into extracellular environments in V. anguillarum. The results imply that the expression, export and processing mechanism of the protein in E. coli are similar to those in V. anguillarum.     

Physiological Study of Stenotrophomonas maltophilia DHHJ in Feather Biodegradation:Structural Remodeling of Cell Surface and Dynamic Change in Kerastinase Activity

Poultry industry produces a vast amount of feather waste annually, which forms a burden for environment protection.However, feathers are valuable bio-resources with high keratinaceous protein content and can be converted into more valuable materials through some approaches such as biodegradation by microorganism-derived keratinases. The characters of keratinases in microorganisms remain largely undetermined. In this study,it is reported that the morphological change of cell surface and the activities of intracellular and extracellular keratinases in Stenotrophomonas maltophilia( S. maltophilia) DHHJ. S. maltophilia DHHJ was cultured on lysogeny broth( LB) and feather broth(FB) through fermenter technology,and ultrastructure of cell and keratinase activity including extracellular and intracellular enzyme were observed respectively. Ultrastructural change on the cell surface was only observed for the bacteria cultured on FB medium,but not on LB,suggesting that the change could be induced by feather keratin. Therefore, the results showed that extracellular keratinase is a kind of induction enzyme while intracellular keratinase is a kind of constitute enzyme in S. maltophilia DHHJ.     

Sequence and domain structure of talin

Talin is a high-molecular-weight cytoskeletal protein concentrated at regions of cell-substratum contact and, in lymphocytes, at cell-cell contacts. Integrin receptors are involved in the attachment of adherent cells to extracellular matrices and of lymphocytes to other cells. In these situations, talin codistributes with concentrations of integrins in the cell surface membrane. Furthermore, in vitro binding studies suggest that integrins bind to talin, although with low affinity. Talin also binds with high affinity to vinculin, another cytoskeletal protein concentrated at points of cell adhesion. Finally, talin is a substrate for the Ca2(+)-activated protease, calpain II, which is also concentrated at points of cell-substratum contact. To learn more about the structure of talin and its involvement in transmembrane connections between extracellular adhesions and the cytoskeleton, we have cloned and sequenced murine talin. We describe a model for the structure of talin based on this sequence and other data. Homologies between talin and other proteins define a novel family of submembranous cytoskeleton-associated proteins all apparently involved in connections to the plasma membrane.     

Biomineralization process occurring in iron mud of coastal seepage area of Zhoushan Island, Zhejiang province

The biomineralization process of iron oxidizing bacteria and its influence on accumulation of metals were investigated by modern biological observation techniques （i.e., SEM and TEM） and geochemical methods, in coastal area of Zhoushan Island, Zhejiang province where a thick ancient wood layers were buried, Results show that the iron mud samples mainly contain Leptothrix-like sheaths and Gallionella-like stalks, which are known as neutrophilic iron-oxidizing bacteria. These two bacteria are present as obviously different abundance in two sampling sites, which may be regulated by the geochemistry of seepage water. The biomineralization product of iron oxidizing bacteria is ferrihydrite, a poorly ordered iron oxide, and formation of amorphous mineral is affected by the factors of bacteria, minor Si and temperature preventing any further transformation into more crystalline phases. Organic functional groups, extracellular polymers and surface charges can provide favorable nucleation sites or template for formation of iron precipitates on the bacterial surface. The mineralization process of the iron oxidizing bacteria is divided into different stages, i.e., extracellular mineralization, intracellular mineralization and the whole cell mineralization. Furthermore, due to BIOS containing the bacterial organic matter, the accumulation capacity of metals specially Fe and Co is highly increased, suggesting that BIOS exert a degree of controlling in the cycling of metal elements in seepage area.     

Crystal structure of bacterial multidrug efflux transporter AcrB

AcrB is a major multidrug exporter in Escherichia coli. It cooperates with a membrane fusion protein, AcrA, and an outer membrane channel, TolC. We have determined the crystal structure of AcrB at 3.5 A resolution. Three AcrB protomers are organized as a homotrimer in the shape of a jellyfish. Each protomer is composed of a transmembrane region 50 A thick and a 70 A protruding headpiece. The top of the headpiece opens like a funnel, where TolC might directly dock into AcrB. A pore formed by three alpha-helices connects the funnel with a central cavity located at the bottom of the headpiece. The cavity has three vestibules at the side of the headpiece which lead into the periplasm. In the transmembrane region, each protomer has twelve transmembrane alpha-helices. The structure implies that substrates translocated from the cell interior through the transmembrane region and from the periplasm through the vestibules are collected in the central cavity and then actively transported through the pore into the TolC tunnel.     

Pilus chaperones represent a new type of protein-folding catalyst

Adhesive type 1 pili from uropathogenic Escherichia coli strains have a crucial role during infection by mediating the attachment to and potentially the invasion of host tissue. These filamentous, highly oligomeric protein complexes are assembled by the 'chaperone-usher' pathway, in which the individual pilus subunits fold in the bacterial periplasm and form stoichiometric complexes with a periplasmic chaperone molecule that is essential for pilus assembly. The chaperone subsequently delivers the subunits to an assembly platform (usher) in the outer membrane, which mediates subunit assembly and translocation to the cell surface. Here we show that the periplasmic type 1 pilus chaperone FimC binds non-native pilus subunits and accelerates folding of the subunit FimG by 100-fold. Moreover, we find that the FimC-FimG complex is formed quantitatively and very rapidly when folding of FimG is initiated in the presence of both FimC and the assembly-competent subunit FimF, even though the FimC-FimG complex is thermodynamically less stable than the FimF-FimG complex. FimC thus represents a previously unknown type of protein-folding catalyst, and simultaneously acts as a kinetic trap preventing spontaneous subunit assembly in the periplasm.     

Regional specialization of retinal glial cell membrane

Neural activity generates increases in extracellular K+ concentration, [K+]0, which must be regulated in order to maintain normal brain function. Glial cells are thought to play an important part in this regulation through the process of K+ spatial buffering: K+-mediated current flow through glial cells redistributes extracellular K+ following localized [K+]0 increases. As is the case in other glia, the retinal Müller cell is permeable almost exclusively to K+ . Recent experiments have suggested that this K+ conductance may not be distributed uniformly over the cell surface. In the present study, two novel techniques have been used to assess the Müller cell K+ conductance distribution. The results demonstrate that 94% of all membrane conductance lies in the endfoot process of the cell. This strikingly asymmetric distribution has important consequences for theories concerning K+ buffering and should help to explain the generation of the electroretinogram.     

Ecto-protein kinase activity on the external surface of neural cells

ATP is secreted in association with neurotransmitters at certain synapses and neuromuscular junctions. Extracellular ATP is known to exert potent effects on the activity of cells in the nervous system, where it can act as a neurotransmitter or as a modulator regulating the activity of other neurohormones. We have suggested that such modulation may involve the activity of extracellular protein phosphorylation systems. It is well known that intracellular protein kinases are important in the regulation of various neuronal functions, but protein kinases which use extracellular ATP to phosphorylate proteins localized at the external surface of the plasma membrane (ecto-protein kinases) have not been demonstrated in neuronal cells. Here we present direct evidence for the existence of an ecto-protein kinase and demonstrate endogenous substrates for its activity at the surface of intact neural cells. The phosphorylation of one of these surface proteins is selectively stimulated during cell depolarization. In addition, neuronal cell adhesion molecules (N-CAMs) appear to be among the substrates of ecto-protein kinase activity. These results suggest a role for surface protein phosphorylation in regulating specific functions of developing and mature neurones.     

开封发展出口贸易存在的问题及解决途径

近年来，开封出口创汇继续保持较高增长，主要出口市场相对稳定，出口贸易规模不断扩大，对拉动开封经济的增长发挥了积极作用。但是，在我国入世的新环境下，受自身所处区位、经济发展水平、产业结构等多方面因素的影响，开封市与沿海开放城市相比，还存在着出口市场相对集中、出口产品结构层次较低、出口企业经营规模小、经济外向度低等突出问题。对此，应加快产业和产品结构调整，继续实施市场多元化战略，建立外贸出口发展基金和出口奖励制度，加强适应市场竞争需要的高素质外贸人才队伍建设。     

Localization of VP4 neutralization sites in rotavirus by three-dimensional cryo-electron microscopy

Three-dimensional structures of several spherical viruses have been determined by electron microscopy and X-ray crystallography. We report here the first three-dimensional structure of the complex between an intact virus and Fab fragments of a neutralizing monoclonal antibody. The antibody is against VP4, one of the two outer capsid proteins of rotaviruses. These large icosahedral viruses cause gastroenteritis in children and young animals and account for over a million human deaths annually. VP4 in these viruses has been implicated in several important functions such as cell penetration, haemagglutination, neutralization and virulence. Here we demonstrate that the surface spikes on rotavirus particles are made up of VP4. Antigenic sites are located near the distal ends of the spikes and two Fab fragments bind to each of the sixty spikes. The mass of the spike indicates that it is a dimer of VP4. The bilobed structure at the distal end of the spike may be involved in both the attachment to the cell and in viral penetration. A novel feature in the virus-Fab complex is the structural difference between the two chemically equivalent Fab fragments on each spike, which could be indicative of variations in the Fab elbow angles.     

Transforming binding affinities from three dimensions to two with application to cadherin clustering

Membrane-bound receptors often form large assemblies resulting from binding to soluble ligands, cell-surface molecules on other cells and extracellular matrix proteins. For example, the association of membrane proteins with proteins on different cells (trans-interactions) can drive the oligomerization of proteins on the same cell (cis-interactions). A central problem in understanding the molecular basis of such phenomena is that equilibrium constants are generally measured in three-dimensional solution and are thus difficult to relate to the two-dimensional environment of a membrane surface. Here we present a theoretical treatment that converts three-dimensional affinities to two dimensions, accounting directly for the structure and dynamics of the membrane-bound molecules. Using a multiscale simulation approach, we apply the theory to explain the formation of ordered, junction-like clusters by classical cadherin adhesion proteins. The approach features atomic-scale molecular dynamics simulations to determine interdomain flexibility, Monte Carlo simulations of multidomain motion and lattice simulations of junction formation. A finding of general relevance is that changes in interdomain motion on trans-binding have a crucial role in driving the lateral, cis-, clustering of adhesion receptors.     

Structural identification of a bacterial quorum-sensing signal containing boron

Cell-cell communication in bacteria is accomplished through the exchange of extracellular signalling molecules called autoinducers. This process, termed quorum sensing, allows bacterial populations to coordinate gene expression. Community cooperation probably enhances the effectiveness of processes such as bioluminescence, virulence factor expression, antibiotic production and biofilm development. Unlike other autoinducers, which are specific to a particular species of bacteria, a recently discovered autoinducer (AI-2) is produced by a large number of bacterial species. AI-2 has been proposed to serve as a 'universal' signal for inter-species communication. The chemical identity of AI-2 has, however, proved elusive. Here we present the crystal structure of an AI-2 sensor protein, LuxP, in a complex with autoinducer. The bound ligand is a furanosyl borate diester that bears no resemblance to previously characterized autoinducers. Our findings suggest that addition of naturally occurring borate to an AI-2 precursor generates active AI-2. Furthermore, they indicate a potential biological role for boron, an element required by a number of organisms but for unknown reasons.     

12C6+离子辐射下海迪茨氏菌及其表面活性物质研究

 生物表面活性剂具备无毒、生物降解等优点,这些特性尤其适合于石油的生物降黏、提高原油采收率、重油污染土壤的生物修复等。本文以一株在唯一碳源培养基中能产生胞外生物表面活性剂的海地茨开菌(Dietzia maris)为对象,利用不同剂量¹²C⁶⁺离子束辐射该菌,从大量突变株中筛选出一株正突变菌株YR9。研究表明,经25Gy剂量辐照至该菌突变率最佳,生物表面活性剂产量达到6336.45U/mL,比未经诱变处理菌株提高了2.25倍,能够完全降解n-C₅、n-C₆、n-C₁₆、甲苯、喹啉、咔唑、邻二甲苯、对二甲苯、汽油、萘、邻苯二酚,但对n-C₅、萘降解力降低。分析了该菌产生物表面活性剂的理化性质,对其产物进行了鉴定,对产生生物表面活性剂的条件进行了优化研究。     

分光光度法测定芦丁颗粒剂中总黄酮含量

建立芦丁颗粒剂中总黄酮分光光度测定方法.在样品溶液中分别加入亚硝酸钠试液、碱溶液及硝酸铝试液,放置一段时间后,于506 nm波长处测定总黄酮含量.总黄酮浓度在8.8~52.8μg/mL范围内,吸光度与浓度呈良好线性关系(r=0.9992);低、中、高浓度的平均空白加样回收率分别为100.2%、100.3%及99.6%;同一批样品6次测定值的RSD为1.2%.本方法简便、准确、精密,适于测定芦丁颗粒剂中总黄酮含量.     

微生物燃料电池技术发展及其应用前景

微生物燃料电池可以借助微生物的催化作用直接将燃料(如有机酸,糖类等)的化学能转化为电能.某些类型的细菌具有将电子传递到细胞外并与外界电子受体接合的能力,可以用于构建微生物燃料电池.微生物燃料电池的研究集中于产电细菌、电极材料和电池反应器构型等方面,同时,微生物燃料电池在废水处理、生物修复等方面具有广阔的应用前景.     

A new class of synthetic antibacterials acting on lipopolysaccharide biosynthesis

Although there is a need for antibacterial agents that act only on Gram-negative bacteria, there are at present few such compounds. The 2-deoxy analogue of beta-KDO (3-deoxy-beta-D-manno-2-octulopyranosonic acid) is a potent inhibitor of a key enzyme (CMP-KDO synthetase) in lipopolysaccharide biosynthesis of Gram-negative bacteria, but it fails to penetrate intact bacteria. Coupling an L-L-dipeptide to the 8-amino-2,8-dideoxy analogue of beta-KDO enabled it to be recognized and actively accumulated by certain peptide permeases of the cytoplasmic membrane. The dipeptide was hydrolysed in the cell and the inhibitor released. Subsequent inhibition of CMP-KDO synthetase led to the accumulation of large amounts of lipid A precursor and bacterial death. These compounds represent a new class of synthetic antimicrobials with a novel mechanism of action and considerable potential as chemotherapeutic agents.     

Cell-surface regulation of beta 1-integrin activity on developing retinal neurons.

Integrins are a family of alpha beta heterodimeric receptors that mediate cell-cell and cell-substratum interactions. Integrin binding to extracellular ligands regulates cell adhesion, shape, motility, intracellular signalling and gene expression. Mechanisms that regulate integrin function are, therefore, central to the participation of integrins in a diverse set of cellular events. Here we report the identification of TASC, a monoclonal antibody to a novel epitope on the integrin beta 1 subunit, which inhibits cell adhesion to vitronectin but promotes adhesion to laminin and collagen types I and IV. We show that developing retinal neurons that have lost responsiveness to laminin regain the ability to bind laminin in the presence of TASC. Thus, beta 1-class integrins are likely to occupy multiple affinity states that can be modulated at the cell surface.