Late stages of protein secretion in bacilli

This review highlights the later stages of protein secretion in bacilli, which include protein release from the membrane and their translocation through the cell wall. Mechanisms of release of secreted polypeptides into the medium differ in Gram-positive and Gram-negative bacteria due to different structure of their cell envelope. Exogenous factors including molecular chaperons that can influence polypeptide folding may be also involved in later stages of protein secretion in bacilli. In Gram-positive bacteria protein secretion also depends on structural components of the cell wall. Certain evidence exists that maintenance of the secretory function is important for normal development of the sporulation process in these bacteria.     

Are S-layers exoskeletons? The basic function of protein surface layers revisited

Surface protein or glycoprotein layers (S-layers) are common structures of the prokaryotic cell envelope. They are either associated with the peptidoglycan or outer membrane of bacteria, and constitute the only cell wall component of many archaea. Despite their occurrence in most of the phylogenetic branches of microorganisms, the functional significance of S-layers is assumed to be specific for genera or groups of organisms in the same environment rather than common to all prokaryotes. Functional aspects have usually been investigated with isolated S-layer sheets or proteins, which disregards the interactions between S-layers and the underlying cell envelope components. This study discusses the synergistic effects in cell envelope assemblies, the hypothetical role of S-layers for cell shape formation, and the existence of a common function in view of new insights.     

Induction of coordinated movement of Myxococcus xanthus cells.

Rhythmically advancing waves of cells, called ripples, arise spontaneously during the aggregation of Myxococcus xanthus into fruiting bodies. Extracts prepared by washing rippling cells contain a substance that will induce quiescent cells to ripple. Three lines of evidence indicate that murein (peptidoglycan) is the ripple-inducing substance in the extracts. First, ripple-inducing activity is associated with the cell envelope of sonically disrupted M. xanthus cells. Second, whole cells, cell extracts, or purified murein from a variety of different bacteria are capable of inducing ripples. In contrast, extracts prepared from Methanobacterium spp. which contain pseudomurein instead of typical bacterial murein fail to induce ripples. Third, four components of M. xanthus murein, N-acetylglucosamine, N-acetylmuramic acid, diaminopimelate, and D-alanine, are able to induce ripples. Ripples produced by aggregating cells have a wavelength of 45 micrometers and a maximum velocity of 2 micrometers/min. Both of the multigene systems that control gliding motility appear to be required for rippling, and all known mutations at the spoC locus eliminate both rippling and sporulation.     

Cell envelope composition and organisation in the genus Rhodococcus

A knowledge of the organisation of the rhodococcal cell envelope is of fundamental importance if the environmental and biotechnological significance of these bacteria are to be understood and succesfully exploited. The genus Rhodococcus belongs to a distinctive suprageneric taxon, the mycolata, which includes among others the genera Corynebacterium, Mycobacterium and Nocardia. Members of this taxon exhibit an unusual complexity in their cell envelope composition and organisation compared to other Gram-positive bacteria. Models that describe the architecture of the mycobacterial cell envelope are extrapolated here to provide a model of the rhodococcal cell envelope. The rhodococcal cell envelope is dominated by the presence of an arabinogalactan cell wall polysaccharide and large 2-alkyl 3-hydroxy branched-chain fatty acids, the mycolic acids, which are covalently assembled into a peptidoglycan–arabinogalactan–mycolic acid matrix. This review further emphasises that the mycolic acids in this complex form the basis of an outer lipid permeability barrier. The localisation and roles of other cell envelope components, notably complex free lipids, lipoglycans, proteins and lipoproteins are also considered.     

The structure of infection threads,bacteria and bacteroids in pea and clover root nodules

Summary The bacteria and infection threads of the pea root nodule were examined by light and electron microscopy. The bacteria in the infection thread are enclosed in a microcapsule. This capsule disappears when the bacterium is released into the host cytoplasm. The membrane envelope which surrounds the bacteria in the host cell is shown to be derived from the plant cell membrane. The infection of the host cell is by means of a process akin to pinocytosis and the bacteria are confined to vacuoles in the host cytoplasm. As each membrane envelope contains only one bacterium, the envelopes must divide with the bacteria. The bacteria increase 40fold in volume from the infection thread to the stage of the mature bacteroid. The mature infected host cell contains few organelles. The mitochondria become confined to the periphery of the cell. Differences of membrane structure in gram negative bacteria found by other workers have been attributed to fixation artifacts.     

Isolation and characterization of the outer membrane of Neisseria gonorrhoeae

The cell envelope of Neisseria gonorrhoeae strain 2686, colonial type 4, was isolated from spheroplasts formed by the action of ethylenediaminetetraacetic acid and lysozyme. Isopycnic centrifugation of osmotically ruptured spheroplasts resolved the cell envelope into two main membrane fractions. Chemical and enzymatic analyses were used to characterize these isolated membranes. Succinic dehydrogenase, reduced nicotinamide adenine dinucleotide oxidase, and d-lactate dehydrogenase were localized in the membrane fraction of buoyant density, rho degrees = 1.141 g/cm(3). Lipopolysaccharide and over half of the cell envelope protein were associated with the membrane that banded in sucrose at rho degrees = 1.219 g/cm(3). These fractions were consequently designated cytoplasmic and outer or L-membrane, respectively. Sodium dodecyl sulfate-polyacrylamide electrophoresis of isolated membranes demonstrated the relative simplicity of the protein spectrum of the outer membrane. The majority of the protein in this membrane could be accounted for by proteins of molecular weights 34,500, 22,000, and 11,500. The protein of molecular weight 34,500 accounted for 66% of the total protein of the L-membrane. Isoelectric precipitation at pH 4.6 with 10% acetic acid selectively removed this protein from a 150 mM NaCl in 10 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.4, extract of purified outer membrane. At pH 4.0, the other proteins of the L-membrane were precipitated. It was concluded that the membrane components of the cell envelope of N. gonorrhoeae were similar to those of other gram-negative bacteria. The cell envelope fractions described here, in particular the outer membrane, are sufficiently well defined to provide a valuable tool for future biochemical and immunological studies on N. gonorrhoeae.     

Cell wall of Thermoanaerobacterium thermosulfurigenes EM1: isolation of its components and attachment of the xylanase XynA

Thermoanaerobacterium thermosulfurigenes EM1 has a gram-positive type cell wall completely covered by a surface layer (S-layer) with hexagonal lattice symmetry. The components of the cell envelope were isolated, and the S-layer protein was purified and characterized. S-layer monomers assembled in vitro into sheets with the same hexagonal symmetry as in vivo. Monosaccharide analysis revealed that the S-layer is associated with fucose, rhamnose, mannosamine, glucosamine, galactose, and glucose. The N-terminal 31 amino acid residues of the S-layer protein showed significant similarity to SLH (S-layer homology) domains found in S-layer proteins of different bacteria and in the exocellular enzymes pullulanase, polygalacturonate hydrolase, and xylanase of T. thermosulfurigenes EM1. The xylanase from T. thermosulfurigenes EM1 was copurified with the S-layer protein during isolation of cell wall components. Since SLH domains of some structural proteins have been shown to anchor these proteins noncovalently to the cell envelope, we propose a common anchoring mechanism for the S-layer protein and exocellular enzymes via their SLH domains in the peptidoglycan-containing layer of T. thermosulfurigenes EM1. Received: 23 October 1998 / Accepted: 21 December 1998     

Enzymes responsible for the bactericidal effect in extracts of vitelline and fertilisation envelopes of rainbow trout eggs

Extracts from both the vitelline envelope (VE) and fertilisation envelopes (FE) of rainbow trout eggs have the ability to exert a bactericidal effect on Gram-positive and -negative bacteria. The effect may be due to the presence of phospholipase D (PLD), lysozyme, proteinase and DNases, as the extracts contain these enzyme activities. The intensity of chorionic PLD and lysozyme activities in the VE extract was maintained in the FE without any alteration in activity even after transformation in the course of the cortical reaction, as components of a fundamental architecture of the envelope. Both extracts also contain different types of proteinase activities. Treatment with VE or FE extract seriously damaged the outer membrane of Gram-negative bacteria and the plasma membrane of Gram-positive and -negative bacteria at the ultrastructural level. Chorionic DNases probably degrade DNA of bacterial cells killed by virtue of the action of PLD and/or lysozyme and contribute to the transmigration of nucleosides and/or nucleotides produced by degrading bacterial DNA after degradation of bacterial components by the actions of the chorionic PLD, lysozyme and proteinase. These results suggest that the bactericidal process manifested by the VE or FE extract may start with the action of PLD and/or lysozyme against bacteria and be completed by subsequent degradation of constitutive proteins and DNA by the action of proteinases and DNases, respectively. Thus the VE and FE are able to protect the egg itself and the embryo, respectively, from bacterial infection in the internal or external environments.     

Alterations in Sindbis Viral Envelope Proteins by Treating BHK cells with Glucosamine

Addition of d-glucosamine to BHK cells infected with Sindbis virus inhibited the formation of the E-2 viral envelope from its precursor PE-2. Release of virus was blocked, and two new viral protein bands replaced the normal envelope protein bands detected in SDS-gel electropherograms of infected cell extracts.     

Microreview: Type IV secretion systems: versatility and diversity in function

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.     

Structural basis for the coordination of cell division with the synthesis of the bacterial cell envelope

Bacteria are surrounded by a complex cell envelope made up of one or two membranes supplemented with a layer of peptidoglycan (PG). The envelope is responsible for the protection of bacteria against lysis in their oft‐unpredictable environments and it contributes to cell integrity, morphology, signaling, nutrient/small‐molecule transport, and, in the case of pathogenic bacteria, host–pathogen interactions and virulence. The cell envelope requires considerable remodeling during cell division in order to produce genetically identical progeny. Several proteinaceous machines are responsible for the homeostasis of the cell envelope and their activities must be kept coordinated in order to ensure the remodeling of the envelope is temporally and spatially regulated correctly during multiple cycles of cell division and growth. This review aims to highlight the complexity of the components of the cell envelope, but focusses specifically on the molecular apparatuses involved in the synthesis of the PG wall, and the degree of cross talk necessary between the cell division and the cell wall remodeling machineries to coordinate PG remodeling during division. The current understanding of many of the proteins discussed here has relied on structural studies, and this review concentrates particularly on this structural work.     

Structural analysis of precursor and product forms of type-common envelope glycoprotein D (CP-1 antigen) of herpes simplex virus type 1.

The type-common CP-1 antigen of herpes simplex virus type 1 (HSV-1) is associated in the infected cell with two components, a 52,000-molecular-weight glycoprotein (gp52 or pD) and a 59,000-molecular-weight glycoprotein (gp59 or D). The larger form (D) is also found in the virion envelope. It was postulated that pD is a precursor of D. We found that pD shared methionine and arginine tryptic peptides with D isolated from infected cell extracts. D isolated from infected extracts had the same trypric methionine peptide profile as D isolated from the virion envelope. Thus, processing of pD to D does not involve any major alterations in polypeptide structure. Furthermore, D did not share tryptic methionine peptides with the other major glycoproteins of HSV-1. Using [2-3H]mannose as a specific glycoprotein label, we found that pD, which is a basic protein (isoelectric point = 8.0) contained a 1,800-molecular-weight oligomannosyl core moiety and was processed by further glycosylation and sialyation to a more acidic and heterogeneous molecule D, which as a molecular weight of at least 59,000.     

Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure

In the course of evolution, Gram-positive bacteria, defined here as prokaryotes from the domain Bacteria with a cell envelope composed of one biological membrane (monodermita) and a cell wall composed at least of peptidoglycan and covalently linked teichoic acids, have developed several mechanisms permitting to a cytoplasmic synthesized protein to be present on the bacterial cell surface. Four major types of cell surface displayed proteins are currently recognized: (i) transmembrane proteins, (ii) lipoproteins, (iii) LPXTG-like proteins and (iv) cell wall binding proteins. The subset of proteins exposed on the bacterial cell surface, and thus interacting with extracellular milieu, constitutes the surfaceome. Here, we review exhaustively the current molecular mechanisms involved in protein attachment within the cell envelope of Gram-positive bacteria, from single protein to macromolecular protein structure.     

Protein sorting to the cell wall envelope of Gram-positive bacteria

The covalent anchoring of surface proteins to the cell wall envelope of Gram-positive bacteria occurs by a universal mechanism requiring sortases, extracellular transpeptidases that are positioned in the plasma membrane. Surface protein precursors are first initiated into the secretory pathway of Gram-positive bacteria via N-terminal signal peptides. C-terminal sorting signals of surface proteins, bearing an LPXTG motif or other recognition sequences, provide for sortase-mediated cleavage and acyl enzyme formation, a thioester linkage between the active site cysteine residue of sortase and the C-terminal carboxyl group of cleaved surface proteins. During cell wall anchoring, sortase acyl enzymes are resolved by the nucleophilic attack of peptidoglycan substrates, resulting in amide bond formation between the C-terminal end of surface proteins and peptidoglycan cross-bridges within the bacterial cell wall envelope. The genomes of Gram-positive bacteria encode multiple sortase genes. Recent evidence suggests that sortase enzymes catalyze protein anchoring reactions of multiple different substrate classes with different sorting signal motif sequences, protein linkage to unique cell wall anchor structures as well as protein polymerization leading to the formation of pili on the surface of Gram-positive bacteria.     

Green fluorescent chimeras indicate nonpolar localization of pullulanase secreton components PulL and PulM

The Klebsiella oxytoca pullulanase secreton (type II secretion system) components PulM and PulL were tagged at their N termini with green fluorescent protein (GFP), and their subcellular location was examined by fluorescence microscopy and fractionation. When produced at moderate levels without other secreton components in Escherichia coli, both chimeras were envelope associated, as are the native proteins. Fluorescent GFP-PulM was evenly distributed over the cell envelope, with occasional brighter foci. Under the same conditions, GFP-PulL was barely detectable in the envelope by fluorescence microscopy. When produced together with all other secreton components, GFP-PulL exhibited circumferential fluorescence, with numerous brighter patches. The envelope-associated fluorescence of GFP-PulL was almost completely abolished when native PulL was also produced, suggesting that the chimera cannot compete with PulL for association with other secreton components. The patches of GFP-PulL might represent functional secretons, since GFP-PulM also appeared in similar patches. GFP-PulM and GFP-PulL both appeared in spherical polar foci when made at high levels. In K. oxytoca, GFP-PulM was evenly distributed over the cell envelope, with few patches, whereas GFP-PulL showed only weak envelope-associated fluorescence. These data suggest that, in contrast to their Vibrio cholerae Eps secreton counterparts (M. Scott, Z. Dossani, and M. Sandkvist, Proc. Natl. Acad. Sci. USA 98:13978-13983, 2001), PulM and PulL do not localize specifically to the cell poles and that the Pul secreton is distributed over the cell surface.     

Outer membrane vesicle production by Escherichia coli is independent of membrane instability

It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we conducted a screen in Escherichia coli to identify gene disruptions that caused vesicle over- or underproduction. Only a few low-vesiculation mutants and no null mutants were recovered, suggesting that vesiculation may be a fundamental characteristic of gram-negative bacterial growth. Gene disruptions were identified that caused differences in vesicle production ranging from a 5-fold decrease to a 200-fold increase relative to wild-type levels. These disruptions included loci governing outer membrane components and peptidoglycan synthesis as well as the sigma(E) cell envelope stress response. Mutations causing vesicle overproduction did not result in upregulation of the ompC gene encoding a major outer membrane protein. Detergent sensitivity, leakiness, and growth characteristics of the novel vesiculation mutant strains did not correlate with vesiculation levels, demonstrating that vesicle production is not predictive of envelope instability.     

Chloroplast division: squeezing the photosynthetic captive

Chloroplasts have evolved from a cyanobacterial endosymbiont and have been retained in eukaryotic cells for more than one billion years via chloroplast division and inheritance by daughter cells during cell division. Recent studies revealed that chloroplast division is performed by a large protein complex at the division site, encompassing both the inside and the outside of the two envelope membranes. The division complex has retained a few components of the cyanobacterial division complex to go along with other components supplied by the host cell. On the basis of the information about the division complex, we are beginning to understand how the division complex evolved, and how eukaryotic host cells regulate chloroplast division during proliferation and differentiation.