Translocation of proteins across the cell envelope of Gram-positive bacteria

In contrast to Gram-negative bacteria, secretory proteins of Gram-positive bacteria only need to traverse a single membrane to enter the extracellular environment. For this reason, Gram-positive bacteria (e.g. various Bacillus species) are often used in industry for the commercial production of extracellular proteins that can be produced in yields of several grams per liter culture medium. The central components of the main protein translocation system (Sec system) of Gram-negative and Gram-positive bacteria show a high degree of conservation, suggesting similar functions and working mechanisms. Despite this fact, several differences can be identified such as the absence of a clear homolog of the secretion-specific chaperone SecB in Gram-positive bacteria. The now available detailed insight into the organization of the Gram-positive protein secretion system and how it differs from the well-characterized system of Escherichia coli may in the future facilitate the exploitation of these organisms in the high level production of heterologous proteins which, so far, is sometimes very inefficient due to one or more bottlenecks in the secretion pathway. In this review, we summarize the current knowledge on the various steps of the protein secretion pathway of Gram-positive bacteria with emphasis on Bacillus subtilis, which during the last decade, has arisen as a model system for the study of protein secretion in this industrially important class of microorganisms.     

Comparison of the envelope protein compositions of several gram-negative bacteria

Five gram-negative species contained one or two major protein species with electrophoretic mobilities similar to the major protein of the Escherichia coli cell wall.     

A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria.

Seventeen fully sequenced and two partially sequenced extracytoplasmic proteins of purple, gram-negative bacteria constitute a homologous family termed the putative membrane fusion protein (MFP) family. Each such protein apparently functions in conjunction with a cytoplasmic membrane transporter of the ATP-binding cassette family, major facilitator superfamily, or heavy metal resistance/nodulation/cell division family to facilitate transport of proteins, peptides, drugs, or carbohydrates across the two membranes of the gram-negative bacterial cell envelope. Evidence suggests that at least some of these transport systems also function in conjunction with a distinct outer membrane protein. We report here that the phylogenies of these proteins correlate with the types of transport systems with which they function as well as with the natures of the substrates transported. Characterization of the MFPs with respect to secondary structure, average hydropathy, and average similarity provides circumstantial evidence as to how they may allow localized fusion of the two gram-negative bacterial cell membranes. The membrane fusion protein of simian virus 5 is shown to exhibit significant sequence similarity to representative bacterial MFPs.     

Mechanism of protein excretion by gram-negative bacteria: Pseudomonas aeruginosa exotoxin A

Excretion of proteins by a cell with a double membrane may involve mechanisms different from secretion across a single membrane. We studied this problem with Pseudomonas aeruginosa exotoxin A. This 68,000-dalton protein was released as rapidly as it was completed; even after short pulse-labeling the cells contained neither the toxin nor a larger precursor. Excretion is evidently cotranslational, since in fractionated lysates the toxin was formed (almost entirely in the mature form) by the membrane-polysome complexes but not by the free polysomes. When the membrane was perturbed by 10% ethanol, the cells stopped excreting the toxin and they accumulated an immunoprecipitable, enzymatically active precursor of 71,000 daltons. The precursor was located entirely in the outer membrane on its outer surface. On removal of the ethanol, the cells again excreted mature toxin, but they did not process or release the previously accumulated precursor. Based on these data, a model for the excretion of exotoxin A is presented.     

Molecular interaction between bacteriophage and the gram-negative cell envelope

Conclusions Accumulation of molecular data on rceptor recognition by phages over the last years has provided some insight into the underlying molecular mechanisms. Some common motifs emerge. The receptor-recognizing areas of phage proteins seem to be confined to single regions on the polypeptide chains. The repeat structure of these regions, as found in T-even-type phages, seems to be specific for this group since it is not found in other phages such as lambda, T5, and BF23. The maximal number of surface exposed loops of receptor proteins recognized by the phages appears to be limited to four loops. The knowledge of X-ray structures and multiple alignment calculations of related receptor proteins will soon provide exact topological models of these proteins. These models will make possible the positioning of the receptor-binding proteins with respect to the surface-exposed loops involved in phage reception. The knowledge of the exact nature of mutations with new host-ranges will be of great value for such positioning.The TonB-dependent receptor proteins are a group of transport proteins which appear able to undergo considerable conformational changes not only during transport but also during phage adsorption. The isolation of receptor missense mutations impaired solely in phage reception should be very rewarding for TonB-dependent phages, since this could possibly lead to the identification of transiently surface-exposed loops of the receptor proteins.As for DNA uptake, the biochemical and biophysical characterization of pore-forming phage proteins should help elucidate the molecular mechanism of membrane penetration. Studies of this mechanism will be very much facilitated by the accessibilities of the corresponding genes to modern molecular biology techniques.Abbreviation aa amino acid(s)     

On the translocation of proteins across the chloroplast envelope

Most of the chloroplast proteins are coded for in the nucleus and are synthesized in the cytosol from where they are subsequently transported into the different chloroplast compartments. The structural properties of the N-terminal extensions (transit peptides) of these nuclear-coded precursor proteins are discussed as well as the energy requirements for their translocation and the involvement of receptor proteins and that of other (ATP-dependent) factors.     

Spontaneous heteromerization of gammaretrovirus envelope proteins: a possible novel mechanism of retrovirus restriction

The env gene of gammaretroviruses encodes a glycoprotein conserved among diverse retroviruses, except for the domains involved in receptor binding. Here we show that pairs of gammaretrovirus envelope proteins (from Friend virus and GALV or xenotropic viruses) assemble into heteromers when coexpressed. This assembly results in a strong inhibition of infectivity. An unrelated envelope protein does not assemble in heteromers with the gammaretrovirus glycoproteins tested and does not affect their infectivity, demonstrating the specificity of the mechanism we describe. We propose that the numerous copies of endogenous retroviral env genes conserved within mammalian genomes act as restriction factors against infectious retroviruses.     

Detection of alternative nitrogenases in aerobic gram-negative nitrogen-fixing bacteria.

Strains of aerobic, microaerobic, nonsymbiotic, and symbiotic dinitrogen-fixing bacteria were screened for the presence of alternative nitrogenase (N2ase) genes by DNA hybridization between genomic DNA and DNA encoding structural genes for components 1 of three different enzymes. A nifDK gene probe was used as a control to test for the presence of the commonly occurring Mo-Fe N2ase, a vnfDGK gene probe was used to show the presence of V-Fe N2ase, and an anfDGK probe was used to detect Fe N2ase. Hitherto, all three enzymes have been identified in Azotobacter vinelandii OP, and all but the Fe N2ase are present in Azotobacter chroococcum ATCC 4412 (MCD1). Mo-Fe N2ase and V-Fe N2ase structural genes only were confirmed in this strain and in two other strains of A. chroococcum (ATCC 480 and ATCC 9043). A similar pattern was observed with Azotobacter beijerinckii ATCC 19360 and Azotobacter nigricans ATCC 35009. Genes for all three systems are apparently present in two strains of Azotobacter paspali (ATCC 23367 and ATCC 23833) and also in Azomonas agilis ATCC 7494. There was no good evidence for the existence of any genes other than Mo-Fe N2ase structural genes in several Rhizobium meliloti strains, cowpea Rhizobium strain 32H1, or Bradyrhizobium japonicum. Nitrogenase and nitrogenase genes in Azorhizobium caulinodans behaved in an intermediate fashion, showing (i) the formation of ethane from acetylene under Mo starvation, a characteristic of alternative nitrogenases, and (ii) a surprising degree of cross-hybridization to the vnfDGK, but not the anfDGK, probe. vnfDGK- and anfDGK-like sequences were not detected in two saccharolytic Pseudomonas species or Azospirillum brasilense Sp7. The occurrence of alternative N2ases seems restricted to members of the family Azotobacteraceae among the aerobic and microaerobic diazotrophs tested, suggesting that an ability to cope with O2 when fixing N2 may be an important factor influencing the distribution of alternative nitrogenases.     

Transfer of phospholipid and protein into the envelope of gram-negative bacteria by liposome fusion

A liposome-bacterial fusion system was developed in order to introduce preformed terminal complement complexes, C5b-9, into the outer membrane of Gram-negative bacteria. Liposomes were prepared from a total phospholipid extract of Salmonella minnesota Re595. Fusion between liposomes and Salmonella sp. or Escherichia coli 17 was dependent on time, temperature, pH, and Ca2+ and PO4- concentration. Only Salmonella sp. with attenuated LPS core regions were able to fuse efficiently with liposomes. It was demonstrated that fusion of liposomes with S. minnesota Re595 or E. coli 17 under optimum conditions resulted in (i) quantitative transfer of the self-quenching fluorescent membrane probe octadecyl rhodamine B chloride from the liposomal bilayer to the bacterial envelope, (ii) transfer of radiolabeled liposomal phospholipid to the bacterial outer membrane and its subsequent translocation to the cytoplasmic membrane, demonstrated by isolation of the bacterial membranes following fusion, and (iii) delivery of liposome-entrapped horseradish peroxidase (HRP) to the periplasmic space, confirmed by a chemiluminescent assay. Following fusion of liposomes incorporating C5b-9 complexes with S. minnesota Re595 or E. coli 17, immunological analysis of the isolated membranes revealed C5b-9 complexes located exclusively in the outer membrane.     

Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope.

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins.     

The cell envelope structure of the lipopolysaccharide-lacking gram-negative bacterium Sphingomonas paucimobilis.

From the cell envelope preparation of Sphingomonas paucimobilis two membrane fractions with different densities were separated by sucrose density gradient ultracentrifugation. The high-density fraction contained several major proteins, phospholipids, and glycosphingolipids, which are the only glycolipids of this lipopolysaccharide-lacking gram-negative bacterium. The low-density fraction showed many minor bands of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NADH oxidase activity was localized in this fraction. Combined with morphological data of vesicles formed by these membrane fractions, the high-density and low-density fractions were proposed to be an outer membrane and a cytoplasmic membrane, respectively. The localization of the glycosphingolipid was investigated also by means of immunoelectron microscopic analysis using a glycosphingolipid-specific antibody. The glycosphingolipid was shown to localize at the cell envelope, and the antigenic sugar portion was exposed to the bacterial cell surface. From these results the glycosphingolipid was assumed to have a function similar to that of the lipopolysaccharide of other gram-negative bacteria.     

The secretion pathway of IgA protease-type proteins in gram-negative bacteria

The pathogenic, Gram-negative bacteria, Neisseria gon-orrhoeae, Neisseria meningitidis and Haemophilus influenzae, secrete immunoglobulin A1 proteases into their extracellular surroundings. An extraordinary feature in the secretory pathway of these putative virulence factors is a self-directed outer membrane transport step allowing the proteins to be secreted autonomously, even from foreign Gram-negative host cells like Escherichia coli. Here we summarize recent achievements in the understanding of IgA protease outer membrane translocation.     

A possible mechanism for self-coordination of bidirectional traffic across nuclear pores

Nuclear pore complexes are constantly confronted by large fluxes of macromolecules and macromolecular complexes that need to get into and out of the nucleus. Such bidirectional traffic occurring in a narrow channel can easily lead to jamming. How then is passage between the nucleus and cytoplasm maintained under the varying conditions that arise during the lifetime of the cell? Here, we address this question using computer simulations in which the behaviour of the ensemble of transporting cargoes is analysed under different conditions. We suggest that traffic can exist in two distinct modes, depending on the concentration of cargoes and dissociation rates of the transport receptor-cargo complexes from the pores. In one mode, which prevails when dissociation is quick and cargo concentration is low, transport in either direction proceeds uninterrupted by transport in the other direction. The result is that the overall traffic direction fluctuates rapidly and unsystematically between import and export. Remarkably, when cargo concentrations are high and disassociation is slow, another mode takes over in which traffic proceeds in one direction for a certain extent of time, after which it flips direction for another period. The switch between this, more regulated, mode of transport and the other, quickly fluctuating state, does not require an active gating mechanism but rather occurs spontaneously through the dynamics of the transported particles themselves. The determining factor for the behaviour of traffic is found to be the exit rate from the pore channel, which is directly related to the activity of the Ran system that controls the loading and release of cargo in the appropriate cellular compartment.     

Barrier properties of the gram-negative cell envelope towards high molecular weight polyhexamethylene biguanides

The antimicrobial activities of four discrete molecular weight fractions of polyhexamethylene biguanides towards a number of Escherichia coli strains have been investigated. Whilst activity of the polymers was observed to increase in proportion to polymerization number, the dependence of activity upon molecular weight was five times greater towards sphaeroplasts than towards whole cells. This suggested that the cell envelope, whilst not conferring complete resistance to the agents, did provide a significant exclusion barrier. Comparison of the activities towards rough and deep-rough lipopolysaccharide strains showed growth inhibitory activity, but not bactericidal activity nor respiratory inhibition, to be enhanced in the rough strains. Uptake studies showed mixed H- and C-type adsorption with significantly greater numbers of high-affinity binding sites being associated with rough than deep-rough lipopolysaccharide. The binding affinity of polyhexamethylene biguanides towards cells was also enhanced in the rough strains. Binding affinity was, in all cases, significantly reduced in the presence of magnesium and suggested a mechanism of self-promoted uptake for these biocides, facilitated through core lipopolysaccharide.     

State of the rigid-layer in cell walls of some gram-negative bacteria

Summary Rigid-layers in cell walls of Enterobacteriaceae and Pseudomonadales contain murein sacculi of identical chemotype. However, lipoprotein in covalent linkage to the murein participates to a different extent in rigid layer construction in the two taxonomic groups. In Proteus mirabilis and Escherichia coli lipoprotein particles of similar size and spatial arrangement are visible in the electron microscope as major rigid layer components. In the cell walls of Pseudomonadales covalent (lipo-)protein plays a far less prominent role. It is present as a minor constituent in rigid layers of Pseudomonas aeruginosa. Rigid layers of Spirillum serpens are naked murein sacculi devoid of any covalently attached protein.