Cell Envelope Of Gram-negative Bacteria Research Articles

DNA-Loaded Bacterial Ghosts Efficiently Mediate Reporter Gene Transfer and Expression in Macrophages

There is a demand for efficient and safe DNA delivery vehicles mediating gene transfer and expression. We present bacterial ghosts as a novel platform technology for DNA delivery and targeting of macrophages. Bacterial ghosts are cell envelopes of gram-negative bacteria that are devoid of the cytoplasmic content. Escherichia coli ghosts were loaded with plasmid DNA and linear double-stranded DNA. Confocal laser scanning microscopy and flow cytometry confirmed that the DNA localized to the inner lumen of bacterial ghosts and was not associated with the outer surface of the bacteria. Up to approximately 6000 plasmids could be loaded per single ghost and the amount of loaded DNA correlated with the DNA concentration used for loading. E. coli ghosts loaded with plasmids encoding the enhanced green fluorescent protein (EGFP) targeted efficiently murine macrophages (RAW264.7) and mediated effective gene transfer. The EGFP was expressed by more than 60% of the macrophages as measured by flow cytometry detecting the green fluorescence and immunocytochemical staining with antibodies specific for EGFP.

Bacterial ghost technology for pesticide delivery.

Bacterial ghosts are nondenaturated empty cell envelopes of Gram-negative bacteria produced by E-mediated lysis. Such envelopes from the plant-adhering bacterium Pectobacterium cypripedii were tested for their ability to adhere to plant material and to be used as carriers for pesticide delivery. We show, using fluorescence-labeled P. cypripedii ghosts, that depending on the target plants 55 or 10% (rice or soya, respectively) of the applied bacterial ghosts was retained on the leaves after heavy simulated rain (84 mm). Furthermore, the bacterial ghosts could be loaded with the lipophilic triazole fungicide tebuconazole. In subsequent plant experiments in the glass house, the efficacy of the loaded bacterial ghost for resistance to rainfall and the protective and curative effects against the pathogens Erysiphe graminis, Leptosphaeria nodorum, and Pyrenophora teres on barley and wheat and against Sphaerotheca fuliginea on cucumber were tested. The bacterial ghosts were compared primarily with a commercial tebuconazole formulation, a wettable powder, as it has similar physical characteristics. The comparison revealed similar effects and showed consistently higher or comparable efficacy against the pathogens. The standard operational comparison with the most protective, cereal specific emulsion of oil in water displayed that the bacterial ghosts had equal to or lower efficacy than the emulsion. This study confirmed the potential of bacterial ghost platform technology as a new alternative carrier system for pesticides.

Structure of the translocator domain of a bacterial autotransporter.

Autotransporters are virulence-related proteins of Gram-negative bacteria that are secreted via an outer-membrane-based C-terminal extension, the translocator domain. This domain supposedly is sufficient for the transport of the N-terminal passenger domain across the outer membrane. We present here the crystal structure of the in vitro-folded translocator domain of the autotransporter NalP from Neisseria meningitidis, which reveals a 12-stranded beta-barrel with a hydrophilic pore of 10 x 12.5 A that is filled by an N-terminal alpha-helix. The domain has pore activity in vivo and in vitro. Our data are consistent with the model of passenger-domain transport through the hydrophilic channel within the beta-barrel, and inconsistent with a model for transport through a central channel formed by an oligomer of translocator domains. However, the dimensions of the pore imply translocation of the secreted domain in an unfolded form. An alternative model, possibly covering the transport of folded domains, is that passenger-domain transport involves the Omp85 complex, the machinery required for membrane insertion of outer-membrane proteins, on which autotransporters are dependent.

Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria.

For translocation across the cell envelope of Gram-negative bacteria, substances have to overcome two permeability barriers, the inner and outer membrane. Channel-tunnels are outer membrane proteins, which are central to two distinct export systems: the type I secretion system exporting proteins such as toxins or proteases, and efflux pumps discharging antibiotics, dyes, or heavy metals and thus mediating drug resistance. Protein secretion is driven by an inner membrane ATP-binding cassette (ABC) transporter while drug efflux occurs via an inner membrane proton antiporter. Both inner membrane transporters are associated with a periplasmic accessory protein that recruits an outer membrane channel-tunnel to form a functional export complex. Prototypes of these export systems are the hemolysin secretion system and the AcrAB/TolC drug efflux pump of Escherichia coli, which both employ TolC as an outer membrane component. Its remarkable conduit-like structure, protruding 100 A into the periplasmic space, reveals how both systems are capable of transporting substrates across both membranes directly from the cytosol into the external environment. Proteins of the channel-tunnel family are widespread within Gram-negative bacteria. Their involvement in drug resistance and in secretion of pathogenic factors makes them an interesting system for further studies. Understanding the mechanism of the different export apparatus could help to develop new drugs, which block the efflux pumps or the secretion system.

Colicin A hybrids: a genetic tool for selection of type II secretion-proficient Pseudomonas strains.

The gram-negative bacterium Pseudomonas aeruginosa secretes the majority of its extracellular proteins by the type II secretion mechanism, a two-step process initiated by translocation of signal peptide-bearing exoproteins across the inner membrane. The periplasmic forms are transferred across the outer membrane by a machinery consisting of 12 xcp gene products. Although the type II secretion machinery is conserved among gram-negative bacteria, interactions between the secreted proteins and the machinery are specific. The lack of a selectable phenotype has hampered the development of genetic strategies for studying type II secretion. We report a novel strategy to identify rare events, such as those that allow heterologous secretion or identification of extragenic suppressors correcting xcp defects. This is based on creating a host-vector system where the non-secretory phenotype is lethal. The original tool we designed is a hybrid protein containing elastase and the pore-forming domain of colicin A.

Translocases: a bacterial tunnel for drugs and proteins.

Unrelated translocases extrude proteins or antimicrobial agents across both membranes of the cell envelope in Gram-negative bacteria. The TolC protein links the translocases to the external environment. The recently determined crystal structure of TolC shows how this universal tunnel operates.

Autodisplay: functional display of active beta-lactamase on the surface of Escherichia coli by the AIDA-I autotransporter.

Members of the protein family of immunoglobulin A1 protease-like autotransporters comprise multidomain precursors consisting of a C-terminal autotransporter domain that promotes the translocation of N-terminally attached passenger domains across the cell envelopes of gram-negative bacteria. Several autotransporter domains have recently been shown to efficiently promote the export of heterologous passenger domains, opening up an effective tool for surface display of heterologous proteins. Here we report on the autotransporter domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I), which was genetically fused to the C terminus of the periplasmic enzyme beta-lactamase, leading to efficient expression of the fusion protein in E. coli. The beta-lactamase moiety of the fusion protein was presented on the bacterial surface in a stable manner, and the surface-located beta-lactamase was shown to be enzymatically active. Enzymatic activity was completely removed by protease treatment, indicating that surface display of beta-lactamase was almost quantitative. The periplasmic domain of the outer membrane protein OmpA was not affected by externally added proteases, demonstrating that the outer membranes of E. coli cells expressing the beta-lactamase AIDA-I fusion protein remained physiologically intact.

Structure-Bioactivity Relationships of Bacterial Endotoxins

AbstractEndotoxins, chemically lipopolysaccharides (LPS), are major components of the cell envelope of Gram-negative bacteria which are an important contributing factor to septic shock, in general, and Gram-negative septic shock, in particular. The structure of the lipid moiety of LPS, lipid A, has been intensively investigated in Escherichia coli and Salmonella spp. and based on the structural data, synthetic compounds prepared. In general, these compounds exhibit identical endotoxic activities compared with bacterial lipid A and LPS, thus confirming that lipid A is the endotoxic center of LPS. Analysis of lipid A from various Gram-negative bacterial species showed that lipid A is a family of (phospho)glycolipid molecules that are closely related in general architecture, but whose fine structure varies. The various structures of selected lipid As of different bacterial species are reviewed in this article and the influence of their structure on endotoxicity is discussed. The resulting Deductions on struc...

The Cell Envelope of Gram-Negative Bacteria -- Structure and Function: Summary of Session

The section on the structure and function of the cell envelope of gram-negative bacteria begins with a paper by Dr. Nikaido, who summarizes the important structures present on the surface of bacteria, in particular those integral to or associated with the outer membrane. A good understanding of how bacteria exclude deleterious molecules or form barriers to prevent these molecules from entering the organism has emerged from recent studies, but additional information is needed to define control mechanisms that enable bacteria to accommodate to changing conditions in the host. The presence of sites on organisms recognized by host and vice versa is reconciled from an evolutionary point of view as a means whereby the organism can avoid potentially damaging host factors, e.g., immunologic factors. As an example, capsular structures or long O side chains present on lipopolysaccharides of smooth gramnegative bacteria may create a barrier between host and the integral portion of the outer membrane. This may protect the organism by redirecting potentially lethal host responses to distal sites away from the membrane and may permit the organism to survive. The Ki polysaccharide, which is a poor immunogen because of its biochemical structural similarity to human host tissue, is-because of immunologic tolerance particularly effective in thwarting effective host responses. Dr. Silver and colleagues have initiated studies to map the K1 genes and to identify the structure and function of the coded proteins. With the identification of specific enzyme proteins required for K1 assembly and of proteins associated with transport of this molecule, a clearer picture has emerged of how this carbohydrate is produced by Escherichia coli. These studies illustrate the complex interaction of mechanisms of production of carbohydrate on capsular or other polysaccharides or

Structure and Functions of the Cell Envelope of Gram-Negative Bacteria

The surface of gram-negative bacteria contains structures that prevent the entry of noxious compounds into the cell and that help the cell evade recognition by host elements such as antibodies and complement while allowing the bacteria to obtain nutrients from the environment and to attach to structures of the host cell. This review discusses the role of outer-membrane components such as lipopolysaccharides and porin, as well as appendages such as capsules and fimbriae in such functions.

Interaction of polymyxin B nonapeptide with anionic phospholipids.

The interaction of polymyxin B nonapeptide (PMBN) and polymyxin B (PMB) with the anionic phospholipids phosphatidylserine (PS), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidic acid (DPPA), and 1:1 mixtures (w/w) of DPPA and distearoylphosphatidylcholine (DSPC) was studied by calorimetry, electron spin resonance, and fluorescence spectrometry, electron microscopy, and fusion and leakage assays. The phase transition temperatures of DPPA and DPPG were very similar when bound to PMB or PMBN, indicating that the lipids are in a similar state when bound to the cationic peptides. Both PMB and PMBN caused the interdigitation of DPPG bilayers, suggesting that the penetration of hydrophobic side chains from a peptide bound electrostatically on the surface is sufficient to induce this phenomenon. Stopped-flow experiments revealed that PMBN and PMB induced the fusion of small unilamellar PS and large unilamellar DPPA-DSPC vesicles. The aggregation of vesicles was found to be diffusion-controlled process; the subsequent fusion took place with a frequency of 10(2)-(5 X 10(2] s-1 for small vesicles and 1-100 s-1 for large vesicles. The freeze-fracture replicas of the PMB-treated vesicles displayed 12-50-nm depressions on several superimposed bilayers, indicating the formation of stable lipid-PMB domains. Since the incubation with PMBN produced similar depressions only if the specimens were fixed, PMBN-induced domain formation seems to be a reversible rapid process. The differences in the phospholipid-peptide interactions are correlated with the differences in the physiological action of the antibiotic PMB and the nonbactericidal PMBN on the cell envelope of Gram-negative bacteria.

The cell envelope of gram-negative bacteria: new aspects of its function in transport and chemotaxis.

The appearance of protein synthesis controlled by nucleic acids was one of the crucial events in prebiotic evolution. Such systems, able to perform metabolism and replication of macromolecules, existed long before the first cells. We sometimes overlook the evolutionary significance of the inception of membranes. The evolution of nucleic acids coding for the primitive enzymes was extremely slow in prebiotic times before development of membranes. If a variant of nucleic acid (RNA) arose that made a superior type of enzyme, the new enzyme could not selectively contribute to the replication of the new RNA in its competition with the neighboring old RNA copies. Sequestering of macro-molecules within a cell envelope greatly accelerated evolution because it linked selective propagation of this new, advantageous RNA much more tightly to the function of the superior gene product (Eigen et al. 1981).KeywordsOuter MembraneOuter Membrane ProteinCell EnvelopeMaltose Binding ProteinMajor Outer Membrane ProteinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Problems and changing patterns of resistance with gram-negative bacteria.

Throughout the antibiotic era, the emergence of drug-resistant bacteria has paralleled the development of new antimicrobial agents. As a result of selection pressures and invasive techniques that prolong the lives of seriously ill hospital patients, gram-negative bacilli have become the dominant causes of nosocomial infection. These microorganisms produce a diversity of antibiotic-inactivating enzymes. Moreover, the cell envelope of gram-negative bacteria provides a series of barriers that keep antibiotics from reaching their targets. Resistance factors can be transmitted among bacteria of different genera and species, thus conferring multidrug resistance. These problems continue to challenge scientists to better understand resistance mechanisms and to develop new compounds to circumvent them.

Effects of bacterial endotoxins on neutrophil function.

The neutrophil is a key element in host resistance to bacterial infection. Bacterial products capable of subverting the antimicrobial properties of neutrophils can have a potentially deleterious effect on the host. Current knowledge of the effects of endotoxins derived from the outer cell envelope of gram-negative bacteria on neutrophil function is summarized. Available evidence indicates that endotoxins bind to neutrophils, both in vitro and in vivo. The lipid A region of the endotoxin macromolecule appears to be important in promoting the association of endotoxin with the neutrophil cell membrane. Endotoxin-neutrophil interactions can result in altered neutrophil adhesive and locomotory properties. Moreover, endotoxins have been demonstrated to induce selective degranulation of specific (secondary) granule constituents and to alter the oxidative and microbicidal properties of the neutrophil. Further studies are needed to define on a molecular level the nature of the endotoxin receptor, the precise structural components of endotoxin responsible for altering neutrophil behavior, and the transductional event(s) leading to neutrophil activation as a result of endotoxin exposure.

A possible alternative mechanism of excretion of proteins across the cell envelope of gram-negative bacteria

A possible alternative mechanism of excretion of proteins across the cell envelope of gram-negative bacteria

Control of N-acetylglucosaminidase specific activity in myxamoebae of Dictyostelium discoideum

Myxamoebae of Dictyostelium discoideum were grown with Aerobacter aerogenes as substrate, on nutrient agar plates or in shaken culture, with mean doubling times that varied between 3 and 6 hr. Growth in axenic culture was with mean doubling times of 5–6 or 8 hr. The specific activity of N-acetylglucosaminidase was three to four times higher in myxamoebae grown axenically than in myxamoebae grown with A. aerogenes and there was no correlation between enzyme specific activity and myxamoebal growth rate. High specific activities of N-acetylglucosaminidase were also found in myxamoebae grown with gram-positive bacteria ( Bacillus megaterium, Staphylococcus lactis) as substrate while low specific activities were found in myxamoebae grown with the gram-negative bacteria Acinetobacter lwoffi and Pseudomonas aeruginosa as well as with A. aerogenes. A preparation of the cell envelope of A. aerogenes was nearly as effective as the intact bacteria at depressing myxamoebal N-acetylglucosaminidase specific activity. Lipopolysaccharide extracted from the cell envelope of gram-negative bacteria, or the lipid A component of the lipopolysaccharide, also depressed N-acetylglucosaminidase specific activity when added to axenic cultures of myxamoebae.

Biosynthesis and assembly of envelope lipoprotein in a glycerol-requiring mutant of Salmonella typhimurium.

A glycerol-requiring mutant of Salmonella typhimurium was used in a study of the biosynthesis and assembly of a structural lipoprotein in the cell envelope of gram-negative bacteria. Upon removal of glycerol from the growth medium, the biosynthesis of lipoprotein, as measured by radioactive arginine incorporation, was reduced by the same extent as that of other envelope proteins, the cumulative incorporation of arginine being 20% of that of the unstarved control cells. However, the incorporation of radioactive palmitate into lipoprotein was more severely curtailed after glycerol starvation, the cumulative rate of which was 8% of that observed in the unstarved cells. It was further observed that the lipoprotein synthesized in the glycerol-starved cells was more enriched in unmodified cysteine, which is known to be the N-terminal amino acid of lipoprotein, than that synthesized in the unstarved cells. We conclude that the synthesis of the apoprotein portion of Braun's lipoprotein proceeds independently of the attachment of diglyceride to the sulfhydryl group of the N-terminal cysteine and may, in fact, precede the incorporation of the diglyceride moiety.

Action of Polymyxin B on Bacterial Membranes: Morphological Changes in the Cytoplasm and in the Outer Membrane of Salmonella typhimurium and Escherichia coli B

Though the primary action of the cationic antibiotic polymyxin B is against the membrane of susceptible bacteria, severe morphological changes are detected in the cytoplasm. Using fluorescence microscopy and a mono-N-dansyl-polymyxin B derivative, we could demonstrate aggregations of the antibiotic with cellular material, possibly nucleic acids and/or ribosomes. These aggregations were only produced by minimum inhibitory or higher concentrations of the antibiotic as shown with Salmonella and Escherichia strains differing in their polymyxin susceptibility. The outer membrane of Salmonella typhimurium revealed characteristic blebs when treated with polymyxin B. This was investigated by the gentle methods of spray-freezing and freeze-etching. The obtained electron micrographs suggest that the polymyxin-induced blebs are projections of the outer monolayer of the outer membrane. A possible mechanism of penetration of polymyxin B through the cell envelope of gram-negative bacteria is presented.

Structure and biogenesis of the cell envelope of gram-negative bacteria.

Annals of the New York Academy of SciencesVolume 235, Issue 1 p. 52-65 STRUCTURE AND BIOGENESIS OF THE CELL ENVELOPE OF GRAM-NEGATIVE BACTERIA* M. J. Osborn, M. J. Osborn Microbiology Department University of Connecticut Health Center Farmington, Connecticut 06032Search for more papers by this authorP. D. Rick, P. D. Rick Microbiology Department University of Connecticut Health Center Farmington, Connecticut 06032Search for more papers by this authorV. Lehmann, V. Lehmann Microbiology Department University of Connecticut Health Center Farmington, Connecticut 06032Search for more papers by this authorE. Rupprecht, E. Rupprecht Microbiology Department University of Connecticut Health Center Farmington, Connecticut 06032Search for more papers by this authorM. Singh, M. Singh Microbiology Department University of Connecticut Health Center Farmington, Connecticut 06032Search for more papers by this author M. J. Osborn, M. J. Osborn Microbiology Department University of Connecticut Health Center Farmington, Connecticut 06032Search for more papers by this authorP. D. Rick, P. D. Rick Microbiology Department University of Connecticut Health Center Farmington, Connecticut 06032Search for more papers by this authorV. Lehmann, V. Lehmann Microbiology Department University of Connecticut Health Center Farmington, Connecticut 06032Search for more papers by this authorE. Rupprecht, E. Rupprecht Microbiology Department University of Connecticut Health Center Farmington, Connecticut 06032Search for more papers by this authorM. Singh, M. Singh Microbiology Department University of Connecticut Health Center Farmington, Connecticut 06032Search for more papers by this author First published: May 1974 https://doi.org/10.1111/j.1749-6632.1974.tb43256.xCitations: 50 * This investigation was supported by United States Public Health Service Grant AI-08650. P. D. Rick is a Fellow of the Helen Hay Whitney Foundation, and V. Lehmann and E. Rupprecht hold Postdoctoral Fellowships from the Deutsche Forschungsgemeinshaft. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume235, Issue1Mode of Action of Antibiotics on Microbial Walls and MembranesMay 1974Pages 52-65 RelatedInformation

Structure and function of the cell envelope of gram-negative bacteria.

Structure and function of the cell envelope of gram-negative bacteria.