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Cell Envelope (cell + envelope)
Selected AbstractsPhysiological and morphological responses of the soil bacterium Rhodococcus opacus strain PD630 to water stressFEMS MICROBIOLOGY ECOLOGY, Issue 2 2004Héctor M. Alvarez Abstract Rhodococcus opacus PD630 was investigated for physiological and morphological changes under water stress challenge. Gluconate- and hexadecane-grown cells were extremely resistant to these conditions, and survival accounted for up to 300 and 400 days; respectively, when they were subjected to slow air-drying. Results of this study suggest that strain PD630 has specific mechanisms to withstand water stress. Water-stressed cells were sensitive to the application of ethanol, high temperatures and oxidative stress, whereas they exhibited cross-protection solely against osmotic stress during the first hours of application. Results indicate that the resistance programme for water stress in R. opacus PD630 includes the following physiological and morphological changes, among others: (1) energetic adjustments with drastic reduction of the metabolic activity (,39% decrease during the first 24 h and about 90% after 190 days under dehydration), (2) endogenous metabolism using intracellular triacylglycerols for generating energy and precursors, (3) biosynthesis of different osmolytes such as trehalose, ectoine and hydroxyectoine, which may achieve a water balance through osmotic adjustment and may explain the overlap between water and osmotic stress, (4) adjustments of the cell-wall through the turnover of mycolic acid species, as preliminary experiments revealed no evident changes in the thickness of the cell envelope, (5) formation of short fragmenting-cells as probable resistance forms, (6) production of an extracellular slime covering the surface of colonies, which probably regulates internal and external c anges in water potential, and (7) formation of compact masses of cells. This contributes to understanding the water stress resistance processes in the soil bacterium R. opacus PD630. [source] Heparin-binding hemagglutinin (HBHA) of Mycobacterium leprae is expressed during infection and enhances bacterial adherence to epithelial cellsFEMS MICROBIOLOGY LETTERS, Issue 2 2009Cristiana Soares De Lima Abstract A heparin-binding hemagglutinin (HBHA) expressed on the surface of Mycobacterium tuberculosis is an antigenic protein that has been implicated in bacterial adherence to epithelial cells and systemic dissemination. In this study, the potential role of the Mycobacterium leprae HBHA (ML-HBHA) homologue in leprosy was investigated. Initially, the in vivo expression of HBHA and its association with the M. leprae cell envelope was confirmed by immunoblotting and proteomic analysis. Mycobacterium leprae recombinant HBHA (rML-HBHA) bound to a heparin,Sepharose column, and its capacity to act as an adhesin was demonstrated in experiments showing that the exogenous addition of the protein to latex beads or to M. leprae cells promotes a dramatic increase in association with epithelial cells. Finally, serum anti-HBHA immunoglobulin G levels were investigated in individuals infected with M. leprae. Altogether, our data indicate that HBHA is recognized during the course of bacterial infection in humans and may play a role in leprosy pathogenesis. [source] Extracellular biology of Myxococcus xanthusFEMS MICROBIOLOGY REVIEWS, Issue 2 2010Anna Konovalova Abstract Myxococcus xanthus has a lifecycle characterized by several social interactions. In the presence of prey, M. xanthus is a predator forming cooperatively feeding colonies, and in the absence of nutrients, M. xanthus cells interact to form multicellular, spore-filled fruiting bodies. Formation of both cellular patterns depends on extracellular functions including the extracellular matrix and intercellular signals. Interestingly, the formation of these patterns also depends on several activities that involve direct cell,cell contacts between M. xanthus cells or direct contacts between M. xanthus cells and the substratum, suggesting that M. xanthus cells have a marked ability to distinguish self from nonself. Genome-wide analyses of the M. xanthus genome reveal a large potential for protein secretion. Myxococcus xanthus harbours all protein secretion systems required for translocation of unfolded and folded proteins across the cytoplasmic membrane and an intact type II secretion system. Moreover, M. xanthus contains 60 ATP-binding cassette transporters, two degenerate type III secretion systems, both of which lack the parts in the outer membrane and the needle structure, and an intact type VI secretion system for one-step translocation of proteins across the cell envelope. Also, analyses of the M. xanthus proteome reveal a large protein secretion potential including many proteins of unknown function. [source] Exopolyphosphatases of the yeast Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 3 2003Lidia P Lichko Abstract Separate compartments of the yeast cell possess their own exopolyphosphatases differing from each other in their properties and dependence on culture conditions. The low-molecular-mass exopolyphosphatases of the cytosol, cell envelope, and mitochondrial matrix are encoded by the PPX1 gene, while the high-molecular-mass exopolyphosphatase of the cytosol and those of the vacuoles, mitochondrial membranes, and nuclei are presumably encoded by their own genes. Based on recent works, a preliminary classification of the yeast exopolyphosphatases is proposed. [source] Transglutaminase-1 gene mutations in autosomal recessive congenital ichthyosis: Summary of mutations (including 23 novel) and modeling of TGase-1,HUMAN MUTATION, Issue 4 2009Matthew L. Herman Abstract Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of rare cornification diseases. Germline mutations in TGM1 are the most common cause of ARCI in the United States. TGM1 encodes for the TGase-1 enzyme that functions in the formation of the cornified cell envelope. Structurally defective or attenuated cornified cell envelop have been shown in epidermal scales and appendages of ARCI patients with TGM1 mutations. We review the clinical manifestations as well as the molecular genetics of ARCI. In addition, we characterized 115 TGM1 mutations reported in 234 patients from diverse racial and ethnic backgrounds (Caucasion Americans, Norwegians, Swedish, Finnish, German, Swiss, French, Italian, Dutch, Portuguese, Hispanics, Iranian, Tunisian, Moroccan, Egyptian, Afghani, Hungarian, African Americans, Korean, Japanese and South African). We report 23 novel mutations: 71 (62%) missense; 20 (17%) nonsense; 9 (8%) deletion; 8 (7%) splice-site, and 7 (6%) insertion. The c.877-2A>G was the most commonly reported TGM1 mutation accounting for 34% (147 of 435) of all TGM1 mutant alleles reported to date. It had been shown that this mutation is common among North American and Norwegian patients due to a founder effect. Thirty-one percent (36 of 115) of all mutations and 41% (29 of 71) of missense mutations occurred in arginine residues in TGase-1. Forty-nine percent (35 of 71) of missense mutations were within CpG dinucleotides, and 74% (26/35) of these mutations were C>T or G>A transitions. We constructed a model of human TGase-1 and showed that all mutated arginines that reside in the two beta-barrel domains and two (R142 and R143) in the beta-sandwich are located at domain interfaces. In conclusion, this study expands the TGM1 mutation spectrum and summarizes the current knowledge of TGM1 mutations. The high frequency of mutated arginine codons in TGM1 may be due to the deamination of 5, methylated CpG dinucleotides. Hum Mutat 0, 1,12, 2009. © 2009 Wiley-Liss, Inc. [source] Electron microscopic examination of uncultured soil-dwelling bacteriaMICROBIOLOGY AND IMMUNOLOGY, Issue 5 2008Kazunobu Amako ABSTRACT Bacteria living in soil collected from a rice paddy in Fukuoka, Japan, were examined by electron microscopy using a freeze-substitution fixation method. Most of the observed bacteria could be categorized, based on the structure of the cell envelope and overall morphology, into one of five groups: (i) bacterial spore; (ii) Gram-positive type; (iii) Gram-negative type; (iv) Mycobacterium like; and (v) Archaea like. However, a few of the bacteria could not be readily categorized into one of these groups because they had unique cell wall structures, basically resembling those of Gram-negative bacteria, but with the layer corresponding to the peptidoglycan layer in Gram-negative bacteria being extremely thick, like that of the cortex of a bacterial spore. The characteristic morphological features found in many of these uncultured, soil-dwelling cells were the nucleoid being in a condensed state and the cytoplasm being shrunken. We were able to produce similar morphologies in vitro using a Salmonella sp. by culturing under low-temperature, low-nutrient conditions, similar to those found in some natural environments. These unusual morphologies are therefore hypothesized to be characteristic of bacteria in resting or dormant stages. [source] Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c -type cytochromesMOLECULAR MICROBIOLOGY, Issue 1 2007Liang Shi Summary Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens have developed electron transfer (ET) strategies that require multihaem c -type cytochromes (c -Cyts). In S. oneidensis MR-1, multihaem c -Cyts CymA and MtrA are believed to transfer electrons from the inner membrane quinone/quinol pool through the periplasm to the outer membrane. The type II secretion system of S. oneidensis MR-1 has been implicated in the reduction of metal (hydr)oxides, most likely by translocating decahaem c -Cyts MtrC and OmcA across outer membrane to the surface of bacterial cells where they form a protein complex. The extracellular MtrC and OmcA can directly reduce solid metal (hydr)oxides. Likewise, outer membrane multihaem c -Cyts OmcE and OmcS of G. sulfurreducens are suggested to transfer electrons from outer membrane to type IV pili that are hypothesized to relay the electrons to solid metal (hydr)oxides. Thus, multihaem c -Cyts play critical roles in S. oneidensis MR-1- and G. sulfurreducens -mediated dissimilatory reduction of solid metal (hydr)oxides by facilitating ET across the bacterial cell envelope. [source] Taking shape: control of bacterial cell wall biosynthesisMOLECULAR MICROBIOLOGY, Issue 5 2005George C. Stewart Summary The characteristic shape of a bacterial cell is a function of the three dimensional architectures of the cell envelope and is determined by the balance between lateral wall extension and synthesis of peptidoglycan at the division septum. The three dimensional patterns of cell wall synthesis in the bacterium Bacillus subtilis is influenced by actin-like proteins that form helical coils in the cell and by the MreCD membrane proteins that link the cytoskeletal elements with the penicillin-binding proteins that carry out peptidoglycan synthesis. Recent genetic studies have provided important clues as to how these proteins are arranged in the cell and how they function to regulate cell shape. [source] VirE2, a Type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciensMOLECULAR MICROBIOLOGY, Issue 6 2003Krishnamohan Atmakuri Summary Agrobacterium tumefaciens transfers oncogenic DNA and effector proteins to plant cells during the course of infection. Substrate translocation across the bacterial cell envelope is mediated by a type IV secretion (TFS) system composed of the VirB proteins, as well as VirD4, a member of a large family of inner membrane proteins implicated in the coupling of DNA transfer intermediates to the secretion machine. In this study, we demonstrate with novel cytological screens , a two-hybrid (C2H) assay and bimolecular fluorescence complementation (BiFC) , and by immunoprecipitation of chemically cross-linked protein complexes that the VirE2 effector protein interacts directly with the VirD4 coupling protein at cell poles of A. tumefaciens. Analyses of truncation derivatives showed that VirE2 interacts via its C terminus with VirD4, and, further, an NH2 -terminal membrane-spanning domain of VirD4 is dispensable for complex formation. VirE2 interacts with VirD4 independently of the virB -encoded transfer machine and T pilus, the putative periplasmic chaperones AcvB and VirJ, and the T-DNA transfer intermediate. Finally, VirE2 is recruited to polar-localized VirD4 as a complex with its stabilizing secretion chaperone VirE1, yet the effector,coupling protein interaction is not dependent on chaperone binding. Together, our findings establish for the first time that a protein substrate of a type IV secretion system is recruited to a member of the coupling protein superfamily. [source] The rpf gene of Micrococcus luteus encodes an essential secreted growth factorMOLECULAR MICROBIOLOGY, Issue 3 2002Galina V. Mukamolova Summary Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signalling functions and is required for the resuscitation of dormant cells. Originally isolated from the supernatant of actively growing cultures, Rpf was also detected on the surface of actively growing bacteria. Most molecules may be sequestered non-productively at the cell surface, as a truncated form of the protein, encompassing only the ,Rpf domain' is fully active. The C-terminal LysM module, which probably mediates binding to the cell envelope, is not required for biological activity. Rpf was essential for growth of M. luteus. Washed cells, inoculated at low density into a minimal medium, could not grow in its absence. Moreover, the incorporation of anti-Rpf antibodies into the culture medium at the time of inoculation also prevented bacterial growth. We were unable to inactivate rpf using a disrupted form of the gene, in which most of the coding sequence was replaced with a selectable thiostrepton resistance marker. Gene disruption was possible in the presence of a second, functional, plasmid-located copy of rpf, but not in the presence of a rpf derivative whose protein product lacked the secretory signal sequence. As far as we are aware, Rpf is the first example of a truly secreted protein that is essential for bacterial growth. If the Rpf-like proteins elaborated by Mycobacterium tuberculosis and other mycobacteria prove similarly essential, interference with their proper functioning may offer novel opportunities for protecting against, and treating, tuberculosis and other mycobacterial disease. [source] How do membrane proteins sense water stress?MOLECULAR MICROBIOLOGY, Issue 4 2002Bert Poolman Summary Maintenance of cell turgor is a prerequisite for almost any form of life as it provides a mechanical force for the expansion of the cell envelope. As changes in extracellular osmolality will have similar physicochemical effects on cells from all biological kingdoms, the responses to osmotic stress may be alike in all organisms. The primary response of bacteria to osmotic upshifts involves the activation of transporters, to effect the rapid accumulation of osmo-protectants, and sensor kinases, to increase the transport and/or biosynthetic capacity for these solutes. Upon osmotic downshift, the excess of cytoplasmic solutes is released via mechanosensitive channel proteins. A number of breakthroughs in the last one or two years have led to tremendous advances in our understanding of the molecular mechanisms of osmosensing in bacteria. The possible mechanisms of osmosensing, and the actual evidence for a particular mechanism, are presented for well studied, osmoregulated transport systems, sensor kinases and mechanosensitive channel proteins. The emerging picture is that intracellular ionic solutes (or ionic strength) serve as a signal for the activation of the upshift-activated transporters and sensor kinases. For at least one system, there is strong evidence that the signal is transduced to the protein complex via alterations in the protein,lipid interactions rather than direct sensing of ion concentration or ionic strength by the proteins. The osmotic downshift-activated mechanosensitive channels, on the other hand, sense tension in the membrane but other factors such as hydration state of the protein may affect the equilibrium between open and closed states of the proteins. [source] FtsQ, FtsL and FtsI require FtsK, but not FtsN, for co-localization with FtsZ during Escherichia coli cell divisionMOLECULAR MICROBIOLOGY, Issue 2 2001Joseph C. Chen During cell division in Gram-negative bacteria, the cell envelope invaginates and constricts at the septum, eventually severing the cell into two compartments, and separating the replicated genetic materials. In Escherichia coli, at least nine essential gene products participate directly in septum formation: FtsA, FtsI, FtsL, FtsK, FtsN, FtsQ, FtsW, FtsZ and ZipA. All nine proteins have been localized to the septal ring, an equatorial ring structure at the division site. We used translational fusions to green fluorescent protein (GFP) to demonstrate that FtsQ, FtsL and FtsI localize to potential division sites in filamentous cells depleted of FtsN, but not in those depleted of FtsK. We also constructed translational fusions of FtsZ, FtsA, FtsQ, FtsL and FtsI to enhanced cyan or yellow fluorescent protein (ECFP or EYFP respectively), GFP variants with different fluorescence spectra. Examination of cells expressing different combinations of the fusions indicated that FtsA, FtsQ, FtsL and FtsI co-localize with FtsZ in filaments depleted of FtsN. These localization results support the model that E. coli cell division proteins assemble sequentially as a multimeric complex at the division site: first FtsZ, then FtsA and ZipA independently of each other, followed successively by FtsK, FtsQ, FtsL, FtsW, FtsI and FtsN. [source] Analysis of the periplasmic proteome of Pseudomonas aeruginosa, a metabolically versatile opportunistic pathogenPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2009Francesco Imperi Abstract The Gram-negative bacterium Pseudomonas aeruginosa is a main cause of infection in hospitalized, burned, immunocompromised, and cystic fibrosis patients. Many processes essential for P. aeruginosa pathogenesis, e.g., nutrient uptake, antibiotic resistance, and virulence, take place in the cell envelope and depend on components residing in the periplasmic space. Recent high-throughput studies focused on P. aeruginosa membrane compartments. However, the composition and dynamics of its periplasm remain largely uncharacterized. Here, we report a detailed description of the periplasmic proteome of the wild-type P. aeruginosa strain PAO1 by 2-DE and MALDI-TOF/TOF analysis. Three extraction methods were compared at proteome level in order to achieve the most reliable and comprehensive periplasmic protein map. A total of 495 spots representing 395 different proteins were identified. Most of the high intensity spots corresponded to periplasmic proteins, while cytoplasmic contaminants were mainly detected among faint spots. The majority of the identified periplasmic proteins is involved in transport, cell-envelope integrity, and protein folding control. Notably, more than 30% still has an unpredicted function. This work provides the first overview of the P. aeruginosa periplasm and offers the basis for future studies on periplasmic proteome changes occurring during P. aeruginosa adaptation to different environments and/or antibiotic treatments. [source] A dual infection by infectious cuticular epithelial necrosis virus and a Chlamydia -like organism in cultured Litopenaeus vannamei (Boone) in EcuadorAQUACULTURE RESEARCH, Issue 11 2001R Jimenez During 1996, microscopic examinations of post larvae and juveniles of moribund Litopenaeus vannamei showed multifocal necrosis in the cuticular epithelial tissues. In addition to these severe degenerative alterations in the epithelial cells typical of infectious cuticular epithelial necrosis virus (ICENV), columnar cells of the epithelium displayed small round intracytoplasmic inclusions in the necrotic tissue. Examination by electron microscopy of affected tissues demonstrated prokaryotic organisms in the cytoplasm of epithelial cells delineated by a distinct cytoplasmic vesicle; the prokaryotic organisms were morphologically similar to the genus Chlamydia. The necrotic tissue also showed the presence of particles of ICENV; the double infection by two different organisms in cuticular epithelial cells has not been reported previously. Two distinct stages in the intracellular development of a Chlamydia -like organism were recognized: (1) pleomorphic elementary bodies (EBs) that were spherical to oval were often observed in the process of division or in forming a common chain of three cells, the cells were surrounded by a rigid cell envelope and the presence of a cap or plaque hexagonally arrayed; (2) the reticular bodies (RBs) were forms often in the process of division. These cells had an electron-dense cytoplasm and contained a loose network of nuclear fibrils and a more fragile cell envelope. Regardless of the development stages of the Chlamydia -like organism within the cytoplasmic vesicles, ICENV particles were observed, either dispersed or in clusters, surrounded or inside the vesicles. The potential adverse impact of this dual infection on shrimp culture should be considered, especially in high-density operations. [source] Formation of palladium(0) nanoparticles at microbial surfacesBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Michael Bunge Abstract The increasing demand and limited natural resources for industrially important platinum-group metal (PGM) catalysts render the recovery from secondary sources such as industrial waste economically interesting. In the process of palladium (Pd) recovery, microorganisms have revealed a strong potential. Hitherto, bacteria with the property of dissimilatory metal reduction have been in focus, although the biochemical reactions linking enzymatic Pd(II) reduction and Pd(0) deposition have not yet been identified. In this study we investigated Pd(II) reduction with formate as the electron donor in the presence of Gram-negative bacteria with no documented capacity for reducing metals for energy production: Cupriavidus necator, Pseudomonas putida, and Paracoccus denitrificans. Only large and close-packed Pd(0) aggregates were formed in cell-free buffer solutions. Pd(II) reduction in the presence of bacteria resulted in smaller, well-suspended Pd(0) particles that were associated with the cells (called "bioPd(0)" in the following). Nanosize Pd(0) particles (3,30,nm) were only observed in the presence of bacteria, and particles in this size range were located in the periplasmic space. Pd(0) nanoparticles were still deposited on autoclaved cells of C. necator that had no hydrogenase activity, suggesting a hydrogenase-independent formation mechanism. The catalytic properties of Pd(0) and bioPd(0) were determined by the amount of hydrogen released in a reaction with hypophosphite. Generally, bioPd(0) demonstrated a lower level of activity than the Pd(0) control, possibly due to the inaccessibility of the Pd(0) fraction embedded in the cell envelope. Our results demonstrate the suitability of bacterial cells for the recovery of Pd(0), and formation and immobilization of Pd(0) nanoparticles inside the cell envelope. However, procedures to make periplasmic Pd(0) catalytically accessible need to be developed for future nanobiotechnological applications. Biotechnol. Bioeng. 2010;107: 206,215. © 2010 Wiley Periodicals, Inc. [source] Localization of sphingomyelin during the development of dorsal and tail epidermis of miceBRITISH JOURNAL OF DERMATOLOGY, Issue 5 2001Y. Yoshida Background The water permeability barrier of the stratum corneum seems to be regulated primarily by lamellar bodies situated between the corneocytes; the lamellar bodies originate largely from polar lipid precursors, mainly sphingomyelin (SM), provided by the cells of the stratum granulosum via exocytosis of their lamellar body content. Objectives The aim of our study was to evaluate the cellular distribution of SM during development of the epidermis. Methods In this study, we investigated the expression and localization of SM in both adult and fetal mouse skin by a cytochemical detection method, immunofluorescence microscopy and immunoelectron microscopy, using anti-SM antibody, a specific binding protein to SM (lysenin), and Nile red stain. In addition, we measured transepidermal water loss to estimate the barrier function of the fetal skin. Results We observed that SM was widely distributed from the basal layer to the granular layer in the adult mouse epidermis. An intense cytochemical reaction for SM was observed on embryonic day E14·5 of gestation just before the differentiation of the granular and squamous cells from the intermediate cells. The immunofluorescence indicating SM was detected in two regions, i.e. the most superficial zone of the granular layer and the upper spinous layer after the cell differentiation at the late gestational age. This distribution was not detected by conventional lipid staining, such as with Nile red stain. Immunoelectron microscopy revealed that SM was mainly localized in the intercellular spaces of the adult mouse epidermis and in the intracellular vesicles without a complete lamellar structure in the cytoplasm of epidermal cells of E14·5 fetuses. It is well known that the formation of the structurally mature cornified cell envelope occurs at E15·5 of development. The skin of fetuses at E16·5 showed a definite barrier function. Conclusions These findings suggest that SM dynamics is related to the formation of the lipid envelope, cell differentiation, and epidermal barrier function during development. [source] Microreview: Type IV secretion systems: versatility and diversity in functionCELLULAR MICROBIOLOGY, Issue 9 2010Karin Wallden Summary Type IV secretion systems (T4SSs) are large protein complexes which traverse the cell envelope of many bacteria. They contain a channel through which proteins or protein,DNA complexes can be translocated. This translocation is driven by a number of cytoplasmic ATPases which might energize large conformational changes in the translocation complex. The family of T4SSs is very versatile, shown by the great variety of functions among family members. Some T4SSs are used by pathogenic Gram-negative bacteria to translocate a wide variety of virulence factors into the host cell. Other T4SSs are utilized to mediate horizontal gene transfer, an event that greatly facilitates the adaptation to environmental changes and is the basis for the spread of antibiotic resistance among bacteria. Here we review the recent advances in the characterization of the architecture and mechanism of substrate transfer in a few representative T4SSs with a particular focus on their diversity of structure and function. [source] Mycobacterial survival strategies in the phagosome: defence against host stressesCELLULAR MICROBIOLOGY, Issue 8 2009Sabine Ehrt Summary Infections with Mycobacterium tuberculosis remain a major cause of disease and death in humans. Among the factors that contribute to M. tuberculosis's success as a pathogen is its ability to withstand potentially bactericidal host defences and to resist elimination by an activated immune system. This resistance to killing by the host is in part due to the low permeability of the mycobacterial cell envelope for many toxic molecules. In addition, it depends upon the detoxification of reactive oxygen and reactive nitrogen molecules produced by the host, the repair of the damage these molecules cause and maintenance of a neutral intrabacterial pH within acidic environments. The latter three mechanisms are the focus of this review. [source] Identification of phospholipids as new components that assist in the in vitro trimerization of a bacterial pore proteinFEBS JOURNAL, Issue 3 2001Hans De Cock The in vitro trimerization of folded monomers of the bacterial pore protein PhoE, into its native-like, heat- and SDS-stable form requires incubations with isolated cell envelopes and Triton X-100. The possibility that membranes could be isolated that are enriched in assembly factors required for assembly of the pore protein was now investigated. Fractionation of total cell envelopes of Escherichia coli via various techniques indeed revealed the existence of membrane fractions with different capacities to support assembly in vitro. Fractions containing mainly inner membrane vesicles supported the formation of trimers that were associated with these membrane vesicles. However, only a proportion of these trimers were heat- and SDS-stable and these were formed with slow kinetics. In contrast, fractions containing mainly outer membrane vesicles supported formation of high amounts of heat-stable trimers with fast kinetics. We identified phospholipids as active assembly components in these membranes that support trimerization of folded monomers in a process with similar characteristics as observed with inner membrane vesicles. Furthermore, phospholipids strongly stimulate the kinetics of trimerization and increase the final yield of heat-stable trimers in the context of outer membranes. We propose that lipopolysaccharides stabilize the assembly competent state of folded monomers as a lipochaperone. Phospholipids are involved in converting the folded monomer into new assembly competent intermediate with a short half-life that will form heat-stable trimers most efficiently in the context of outer membrane vesicles. These results provide biochemical evidence for the involvement of different lipidic components at distinct stages of the porin assembly process. [source] Cellular effects of monohydrochloride of l -arginine, N, -lauroyl ethylester (LAE) on exposure to Salmonella typhimurium and Staphylococcus aureusJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2004E. Rodríguez Abstract Aims:, Here we study the effect of monohydrochloride of l -arginine, N, -lauroyl ethylester (LAE), a cationic preservative derived from lauric acid and arginine, on the cell envelopes of Salmonella typhimurium and Staphylococcus aureus at sub-lethal concentration such as their respective minimal inhibitory concentrations, 32 and 8 ,g ml,1, respectively. Methods and Results:, Bacterial populations were studied by using transmission electron and fluorescence microscopy (TEM and FM), flow cytometry (FC) and ion-flux across the cellular membrane. Cell integrity was altered mainly in the outer membrane of S. typhimurium, but there was no significant change in the cytoplasm. However, in Staph. aureus, clear zones, abnormal septation and mesosome-like structures were observed in the cytoplasm. Bacterial populations were double-stained with propidium iodide (PI) and SYTO-13 for FC analysis. In S. typhimurium the proportion of damaged cells after 24 h was 97% and in Staph. aureus 56·3%. LAE induced transmembrane ion flux, the increase of potassium leakage after 30 min of contact was 7·7 and 3·34 ,g ml,1 for Staph. aureus and S. typhimurium, respectively. Membrane disruption was detected by measuring the proton flow across the membrane. Conclusions:, Disturbance in membrane potential and structural changes was caused by LAE, although cells were not disrupted. Significance and Impact of the Study:, This is the first time the cellular effects of LAE on bacterial cells were studied. [source] Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c -type cytochromesMOLECULAR MICROBIOLOGY, Issue 1 2007Liang Shi Summary Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens have developed electron transfer (ET) strategies that require multihaem c -type cytochromes (c -Cyts). In S. oneidensis MR-1, multihaem c -Cyts CymA and MtrA are believed to transfer electrons from the inner membrane quinone/quinol pool through the periplasm to the outer membrane. The type II secretion system of S. oneidensis MR-1 has been implicated in the reduction of metal (hydr)oxides, most likely by translocating decahaem c -Cyts MtrC and OmcA across outer membrane to the surface of bacterial cells where they form a protein complex. The extracellular MtrC and OmcA can directly reduce solid metal (hydr)oxides. Likewise, outer membrane multihaem c -Cyts OmcE and OmcS of G. sulfurreducens are suggested to transfer electrons from outer membrane to type IV pili that are hypothesized to relay the electrons to solid metal (hydr)oxides. Thus, multihaem c -Cyts play critical roles in S. oneidensis MR-1- and G. sulfurreducens -mediated dissimilatory reduction of solid metal (hydr)oxides by facilitating ET across the bacterial cell envelope. [source] Lipoprotein Mutation Accelerates Substrate Permeability-Limited Toluene Dioxygenase-Catalyzed ReactionBIOTECHNOLOGY PROGRESS, Issue 3 2005Ye Ni One of the major problems in whole-cell biocatalysis is its low reaction rate. The underlying cause is the substrate permeation barrier presented by cell envelopes. The present research investigates mutation effects of the Braunapos;s lipoprotein, the most abundant outer membrane structural protein in Escherichia coli, on toluene dioxyengase (TDO)-catalyzed reaction. Dramatic enhancement of the reaction rate, an increase of up to 6-fold, was observed with the mutant for all three small, hydrophobic substrates tested (toluene, ethylbenzene, and 2-indanone). The increase was observed over a wide range of substrate concentrations (0.1,5 mM). The mutant exhibited a normal growth rate and expressed the recombinant multicomponent enzyme as well as the isogenic parent strain. Taken together, the lipoprotein mutant expressing TDO is a much better whole-cell catalyst for the oxidation reaction. The beneficial effect of the lipoprotein mutation may be general for a broad range of substrates and enzyme systems as the mutation affects the global integrity of the cell membrane. A comparison of the mutation effect with a common permeabilizing procedure, the EDTA treatment, further illustrates the clear advantages of using genetic modification in cellular membrane engineering for improved whole-cell catalysts. [source] | ||||||||||||||||||||||||||||