Colicins are not transiently accumulated in the periplasmic space before release from colicinogenic cells

Colicins are released into the spent medium from colicinogenic cells. The pathway of release has been investigated in this study. The localization in producing cells of colicins A, E3 and of cloacin DF13 has been determined at various times after mitomycin C addition: no transient accumulation in the periplasmic space of colicinogenic E. coli K12 strains was detected by electron microscopy for any of the bacteriocins tested. Furthermore, asynchronous induction in individual cells was detected for each bacteriocin tested. These results strongly suggest that colicins, as well as cloacin DF13, do not transit through the periplasmic space before release from colicinogenic cells.     

Lipoteichoic acid is a major component of the Bacillus subtilis periplasm

Cryo-electron microscopy (cryo-EM) of frozen-hydrated specimens allows high-resolution observation of structures in optimally preserved samples. In gram-positive bacteria, this method reveals the presence of a periplasmic space between the plasma membrane and an often differentiated cell wall matrix. Since virtually nothing is known about the composition of its constituent matter (i.e., the periplasm), it is still unclear what structures (or mechanism) sustain a gram-positive periplasmic space. Here we have used cryo-EM of frozen-hydrated sections in combination with various labels to probe the model gram-positive organism Bacillus subtilis for major periplasmic components. Incubation of cells with positively charged gold nanoparticles showed almost similar levels of gold binding to the periplasm and the cell wall. On cells whose cell walls were enzymatically hydrolyzed (i.e., on protoplasts), a surface diffuse layer extending ~30 nm from the membrane was revealed. The thickness and density of this layer were not significantly altered after treatment with a nonspecific protease, whereas it was labeled with anti-lipoteichoic acid (LTA) antibodies conjugated to nanogold. Further, the LTA layer spans most of the thickness of the periplasmic space, which strongly suggests that LTA is a major component of the B. subtilis periplasm.     

Multilayered distribution of peptidoglycan in the periplasmic space of Escherichia coli

When a staining technique using phosphotungstic acid (PTA) in 10% (w/v) chromic acid was applied to cells of Escherichia coli, the periplasmic space was seen as a dark 15-nm-thick layer of uniform appearance and constant width. Our observations are consistent with peptidoglycan being the main material stained. Isolated sacculi as well as purified peptidoglycan (protein free) were also stained by the same procedure, the thickness of the peptidoglycan being 8.8 +/- 1.8 and 6.6 +/- 1.5 nm, respectively. The increased thickness of the PTA-stained layer in stationary phase cells correlated well with the increased thickness of isolated sacculi or purified peptidoglycan and with the increased amount of peptidoglycan in such cells. Thickness measurements on isolated peptidoglycan were compatible with a two to three layer structure for material from exponential phase cells and with a four to five layer structure for that from stationary phase cells. Furthermore, the results indicated an uneven distribution of peptidoglycan material in the periplasmic space, the peptidoglycan spanning the space from the inner to the outer membrane.     

The effect of brefeldin A on the redistribution of osmiophilic particles and the gravitropic response of rye coleoptiles

Summary The occurrence of IAA-inducible osmiophilic particles (OPs) in the periplasmic space of epidermal cells in the upper and lower flank (UF, LF) of gravistimulated rye coleoptile segments was analyzed employing brefeldin A (BFA) as an inhibitor of secretion at the plasma membrane. A 2 h horizontal gravistimulation of untreated samples caused a duplication of OPs in the periplasmic space of epidermal cells at the growth-inhibited UF as compared to the LF of upward bending coleoptile segments. In contrast to this, the number of OPs within the cytoplasm close to the plasma membrane of epidermal cells was similar at both flanks. BFA caused an inhibition of graviresponsive growth and prevented the occurrence of OPs in the periplasmic space of the epidermal cells of the UF and the LF. Likewise, growth of vertically oriented coleoptile segments was inhibited by BFA. Growth inhibition of both gravistimulated and control segments was accompanied by a twofold increase of the occurrence of cytoplasmic OPs. The results illustrate that the occurrence of OPs within the periplasmic space of the epidermal cells depends on secretion processes. Furthermore they provide evidence that their increased occurrence in the growth-inhibited UF during gravistimulation is due to their inhibited infiltration into the cell walls. We suggest that thereby wall loosening is temporarily prevented.Abbreviations BFA brefeldin A - LF lower flank - OP osmiophilic particle - UF upper flank Dedicated to Professor Dr. Dieter Klämbt on the occasion of his 65th birthday     

Extracytoplasmic storage as the nickel resistance mechanism in a natural isolate of Pseudomonas putida S4

Metal resistances in microbes are important to study not only to understand metal homeostasis but also to use such organisms further in environmental bioremediation. Nickel (Ni2+) is an important micronutrient, which at higher concentration becomes toxic. Many Ni2+-resistant organisms are known, which resist metal by active efflux. Pseudomonas putida S4, a natural isolate from India, is reported to show a multi-metal resistance profile. In the present study, the Ni2+-resistance mechanism in strain S4 was examined. Wild-type cells gradually accumulated Ni2+ but kept it preferentially in the periplasmic space in a bound form. In Ni2+-sensitive mutants, periplasmic storage was disturbed and more metal accumulated cytoplasmically, producing toxicity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of periplasmic proteins revealed a band of approximately 18 kDa, which appeared only in Ni2+-exposed wild-type cells, and which was absent from cells not exposed to Ni2+ as well as from Ni2+-sensitive mutants. On the basis of these observations, we propose a Ni2+-resistance mechanism in P. putida S4 based on sequestration of metal in the periplasmic space. This is the first study of sequestration-based Ni2+ resistance.     

Localization of alkaline phosphatase in three gram-negative rumen bacteria

Of the three species (Bacteroides ruminicola, B. succinogenes, and Megasphaera elsdenii) of anaerobic gram-negative rumen bacteria studied, only B. ruminicola produced significant amounts of alkaline phosphatase. This enzyme, which is constitutive, showed a greater affinity for p-nitrophenylphosphate than for sodium-beta-glycerophosphate and was shown to be located exclusively in the periplasmic space of log-phase cells. Small amounts of this enzyme were released from these cells in stationary-phase cultures, but washing in 0.01 M MgCl(2) and the production of spheroplasts by using lysozyme in 0.01 M MgCl(2) did not release significant amounts of the enzyme. Exposure to 0.2 M MgCl(2) did not release significant amounts of the periplasmic alkaline phosphatase of the cell, and when these cells were spheroplasted with lysozyme in 0.2 M MgCl(2) only 25% of the enzyme was released. Spheroplasts were formed spontaneously in aging cultures of B. ruminicola, but even these cells retained most of their periplasmic alkaline phosphatase. It was concluded that the alkaline phosphatase of B. ruminicola is firmly bound to a structural component within the periplasmic area of the cell wall and that the enzyme is released in large amounts only when the cells break down. The behavior of alkaline phosphatase in this bacterium contrasts with that of conventional periplasmic enzymes of aerobic bacteria, which are released upon conversion into spheroplasts by lysozyme and ethylenediaminetetraacetic acid and by other types of cell wall damage. All three species of bacteria studied here, as well as bacteria found in mixed populations in the rumen, have thick, complex layers external to the double-track layer of their cell walls. In addition, B. ruminicola produces a loose extracellular material.     

Occurrence of osmiophilic particles is correlated to elongation growth of higher plants

Summary The occurrence of elongation growth-related osmiophilic particles (OPs) was investigated in hypocotyls of sunflower, bean, and spruce as well as in pea epicotyls and in cress roots of intact seedlings. In all analyzed species, OPs were found to occur specifically within the periplasmic space between plasma membrane and the outer epidermal cell walls of elongating parts of hypocotyls, epicotyls, and roots, whereas cells of nonelongating parts were devoid of OPs. Auxin (IAA) markedly increased the number of OPs in epicotyl and hypocotyl segments. Treatment of pea epicotyl segments with the lectin concanavalin A inhibited their elongation growth in the presence of IAA. At a subcellular level this effect was characterized by the occurrence of a pronounced osmiophilic layer in the periplasmic space of the outer periclinal and the outer part of the anticlinal epidermal cell walls. Treatment of IAA-incubated segments with the secretion inhibitor brefeldin A inhibited both elongation growth and periplasmic occurrence of OPs. This effect was accompanied by complementary accumulation of OPs in the peripheral cytoplasm of epidermal cells. Together the results indicate that IAA-induced epidermis-specific secretion of OPs is closely related to cell elongation growth not only in organs of monocotyledonous species, but also in dicotyledonous angiosperms as well as in gymnosperms.Abbreviations OPs osmiophilic particles - ConA concanavalin A - BFA brefeldin A - IAA -indolyl acetic acid     

The Flat-Ribbon Configuration of the Periplasmic Flagella of Borrelia burgdorferi and Its Relationship to Motility and Morphology

Electron cryotomography was used to analyze the structure of the Lyme disease spirochete, Borrelia burgdorferi. This methodology offers a new means for studying the native architecture of bacteria by eliminating the chemical fixing, dehydration, and staining steps of conventional electron microscopy. Using electron cryotomography, we noted that membrane blebs formed at the ends of the cells. These blebs may be precursors to vesicles that are released from cells grown in vivo and in vitro. We found that the periplasmic space of B. burgdorferi was quite narrow (16.0 nm) compared to those of Escherichia coli and Pseudomonas aeruginosa. However, in the vicinity of the periplasmic flagella, this space was considerably wider (42.3 nm). In contrast to previous results, the periplasmic flagella did not form a bundle but rather formed a tight-fitting ribbon that wraps around the protoplasmic cell cylinder in a right-handed sense. We show how the ribbon configuration of the assembled periplasmic flagella is more advantageous than a bundle for both swimming and forming the flat-wave morphology. Previous results indicate that B. burgdorferi motility is dependent on the rotation of the periplasmic flagella in generating backward-moving waves along the length of the cell. This swimming requires that the rotation of the flagella exerts force on the cell cylinder. Accordingly, a ribbon is more beneficial than a bundle, as this configuration allows each periplasmic flagellum to have direct contact with the cell cylinder in order to exert that force, and it minimizes interference between the rotating filaments.     

Killing of E coli cells by E group nuclease colicins

The process by which the endonuclease domain of colicin E9 is translocated across the outer membrane, the periplasmic space and the cytoplasmic membrane to reach the cytoplasm of E. coli cells, resulting in DNA degradation and cell death, is a unique event in prokaryotic biology. Although considerable information is known about the role of the BtuB outer membrane receptor, as well as the mostly periplasmic Tol proteins that are essential for the translocation process, the precise nature of the interactions between colicin E9 and these proteins remains to be elucidated. In this review, we consider our current understanding of the key events in this process, concentrating on recent findings concerning receptor-binding, translocation and the mechanism of cytotoxicity.     

A morphologic study of filamentous phage infection of Escherichia coli using biotinylated phages

Using biotinylated phage (BIO-phages), we observed the infection of filamentous phages into Escherichia coli JM109 morphologically. BIO-phages and BIO-phage-derived proteins, mainly pVIII, were detected in E. coli by using the avidin-biotin-peroxidase complex method with electron microscopy. Infected cells revealed positive staining on the outer and inner membranes and in the periplasmic space. Some cells showed specific or predominant staining of the outer membrane, whereas others showed predominant staining of the inner membrane or equivalent staining of the outer and inner membranes. The periplasmic spaces in some infected cells were expanded and filled with reaction products. Some cells showed wavy lines of positive staining in the periplasmic space. BIO-phages were detected as thick filaments or clusters covered with reaction products. The ends of the infecting phages were located on the surface of cells, in the periplasmic space, or on the inner membrane. These findings suggest that phage major coat proteins are integrated into the outer membrane and that phages cause periplasmic expansion during infection.     

Cellular Architecture of Treponema pallidum: Novel Flagellum, Periplasmic Cone, and Cell Envelope as Revealed by Cryo Electron Tomography

High-resolution cryo electron tomography (cryo-ET) was utilized to visualize Treponema pallidum, the causative agent of syphilis, at the molecular level. Three-dimensional (3D) reconstructions from 304 infectious organisms revealed unprecedented cellular structures of this unusual member of the spirochetal family. High-resolution cryo-ET reconstructions provided detailed structures of the cell envelope, which is significantly different from that of Gram-negative bacteria. The 4-nm lipid bilayer of both outer membrane and cytoplasmic membrane resolved in 3D reconstructions, providing an important marker for interpreting membrane-associated structures. Abundant lipoproteins cover the outer leaflet of the cytoplasmic membrane, in contrast to the rare outer membrane proteins visible by scanning probe microscopy. High-resolution cryo-ET images also provided the first observation of T. pallidum chemoreceptor arrays, as well as structural details of the periplasmically located cone-shaped structure at both ends of the bacterium. Furthermore, 3D subvolume averages of periplasmic flagellar motors and flagellar filaments from living organisms revealed the novel flagellar architectures that may facilitate their rotation within the confining periplasmic space. Our findings provide the most detailed structural understanding of periplasmic flagella and the surrounding cell envelope, which enable this enigmatic bacterium to efficiently penetrate tissue and to escape host immune responses.     

Lipoprotein PssN of Rhizobium leguminosarum bv. trifolii: subcellular localization and possible involvement in exopolysaccharide export

Surface expression of exopolysaccharides (EPS) in gram-negative bacteria depends on the activity of proteins found in the cytoplasmic membrane, the periplasmic space, and the outer membrane. pssTNOP genes identified in Rhizobium leguminosarum bv. trifolii strain TA1 encode proteins that might be components of the EPS polymerization and secretion system. In this study, we have characterized PssN protein. Employing pssN-phoA and pssN-lacZ gene fusions and in vivo acylation with [3H]palmitate, we demonstrated that PssN is a 43-kDa lipoprotein directed to the periplasm by an N-terminal signal sequence. Membrane detergent fractionation followed by sucrose gradient centrifugation showed that PssN is an outer membrane-associated protein. Indirect immunofluorescence with anti-PssN and fluorescein isothiocyanate-conjugated antibodies and protease digestion of spheroplasts and intact cells of TA1 provided evidence that PssN is oriented towards the periplasmic space. Chemical cross-linking of TA1 and E. coli cells overproducing PssN-His6 protein showed that PssN might exist as a homo-oligomer of at least two monomers. Investigation of the secondary structure of purified PssN-His6 protein by Fourier transform infrared spectroscopy revealed the predominant presence of beta-structure; however, alpha-helices were also detected. Influence of an increased amount of PssN protein on the TA1 phenotype was assessed and correlated with a moderate enhancement of EPS production.     

Outer membrane protein e of Escherichia coli K-12 is co-regulated with alkaline phosphatase.

Outer membrane protein e is induced in wild-type cells, just like alkaline phosphatase and some other periplasmic proteins, by growth under phosphatase limitation. nmpA and nmpB mutants, which synthesize protein e constitutively, are shown also to produce the periplasmic enzyme alkaline phosphatase constitutively. Alternatively, individual phoS, phoT, and phoR mutants as well as pit pst double mutants, all of which are known to produce alkaline phosphatase constitutively, were found to be constitutive for protein e. Also, the periplasmic space of most nmpA mutants and of all nmpB mutants grown in excess phosphate was found to contain, in addition to alkaline phosphatase, at least two new proteins, a phenomenon known for individual phoT and phoR mutants as well as for pit pst double mutants. The other nmpA mutants as well as phoS mutants lacked one of these extra periplasmic proteins, namely the phosphate-binding protein. From these data and from the known positions of the mentioned genes on the chromosomal map, it is concluded that nmpB mutants are identical to phoR mutants. Moreover, some nmpA mutants were shown to be identical to phoS mutants, whereas other nmpA mutants are likely to contain mutations in one of the genes phoS, phoT, or pst.     

Periplasmic chaperones--new structural and functional insights

Although chaperones exist in the periplasmic compartment of Gram-negative bacterial cells, how they function is not well understood. New intriguing functional insights are provided by the solved crystal structure of the periplasmic chaperone SurA.     

Secretion of human superoxide dismutase in Escherichia coli using the condensed single-protein-production system

A secretion vector, pColdV for the Single-Protein-Production (SPP) system was constructed using the E. coli OmpA signal peptide. Using this vector, human superoxide dismutase (hSOD) was co-expressed with MazF, an ACA-specific mRNA interferase, allowing E. coli cells to produce only hSOD, which was secreted into the periplasmic space with a yield of ～20% of total cellular proteins. The signal peptide was properly cleaved. Using cells overproducing DsbA protein, two S-S bridges were also properly formed to yield enzymatically active SOD. A well resolved heteronuclear single quantum coherence (HSQC) spectrum of hSOD isotope-labeled in the condensed SPP (cSPP) system was obtained by simply isolating the periplasmic fraction. These results indicate that human secretory proteins can be expressed well in the cSPP system using pColdV.     

Ultrastructural localization of alkaline phosphatase in the blue-green bacterium Plectonema boryanum.

Histochemical techniques applied at the ultrastructural level have clearly established the periplasmic space as the site of alkaline phosphatase activity in Plectonema boryanum. Considerable enzyme activity is found after the organism is placed in a phosphate-free medium for 5 days. This activity is found only in the cellular fraction of the culture with no activity present in the culture medium. Localization of the site of enzyme activity in cells was investigated by a modification of the method of Costerton. Unfixed cells were reacted with calcium nitrate, which acts as the initial capture reagent. After this deposition, the cells were suspended in 2% lead nitrate to convert the calcium phosphate to more electron-dense lead phosphate. The majority of activity appears associated with layer 3 (periplasmic space) of the cell wall.     

Novel applications of acrylamide for cryosectioning of isolated cells, tissues, and arthropods

Cross-linked and uncross-linked acrylamide polymers were used to alleviate technical difficulties in cryosectioning and autoradiographic processing of marine arthropods (Pycnogonum litorale), larval insect tissues (Sarcophaga bullata), and amphibian (Xenopus laevis) oocytes. Rapidly polymerized cross-linked acrylamide was used to prepare sections from an animal with a hard thick cuticle, P. litorale. Fragmentation and compression artifacts caused by tissue density differences were avoided and internal tissues were well preserved. Our results indicate that closely applied external support eliminates difficulties typically associated with arthropod cryosectioning. Pre-polymerized, uncross-linked acrylamide provided similar protection when used for sectioning larval tissue from S. bullata. Soft tissues under the cuticle were undamaged in tracheal and epidermal preparations. This polymer was also an excellent embedding material for soft tissues such as salivary glands, quickly penetrating tissue cavities and eliminating air bubbles. Uncross-linked acrylamide was used as an embedding and culturing medium for oocytes from X. laevis. The polymers were non-toxic and allowed the preparation of thin frozen sections containing as many as 50 large oocytes per section.     

Direct measurement of free Ca(2+) shows different regulation of Ca(2+) between the periplasm and the cytosol of Escherichia coli

As in eukaryotes, bacterial free Ca(2+) can play an important role as an intracellular signal. However, because free Ca(2+) is difficult to measure in live bacteria, most of the evidence for such a role is indirect. Gram-negative bacteria also have an outer membrane separating the external fluid from the periplasm as well as the cytosol where most bacterial metabolism takes place. Here we report, for the first time, direct measurement of free Ca(2+) in the periplasmic space of living Escherichia coli. Periplasmic free Ca(2+) was measured by targeting the Ca(2+)-activated photoprotein aequorin to this compartment using the N-terminal OmpT signal sequence. Cytosolic free Ca(2+) was determined using aequorin alone. We show that, under certain conditions, the periplasm can concentrate free Ca(2+), resulting in the inner membrane being exposed to free Ca(2+) concentrations several fold higher than in the bulk external fluid. Manipulation of periplasmic membrane-derived oligosaccharides (MDOs) altered the free Ca(2+) as predicted by the Donnan potential. With micromolar concentrations of external free Ca(2+), the periplasm concentrated free Ca (2+) some three to sixfold with respect to the external medium. A Ca(2+) gradient also existed between the periplasm and the cytosol under these conditions, the periplasmic free Ca(2+) being some one to threefold higher. At millimolar levels of external free Ca(2+), a similar concentration was detected in the periplasm, but the bacteria still maintained tight control of cytosolic free Ca(2+) in the micromolar range. We propose that the highly anionic MDOs in the periplasmic space generate a Donnan potential, capable of concentrating Ca(2+) in this compartment, where it may constitute a sink for regulation of Ca(2+)-dependent processes in the cytoplasm.     

Induction conditions for maximizing recombinant staphylokinase expression in Escherichia coli

Staphylokinase, a profibrinolytic bacterial protein, was cloned into Escherichia coli, following the amplification of its gene via PCR. The amplificated gene was inserted in a pKK223-3 plasmid vector. The recombinant protein (STAR), expressed from a tac promoter, was obtained in the periplasmic space when IPTG was added to the culture medium. Both the concentration of the inducer as well as the growth phase of recombinant cells at which it was added affected the final yield of periplasmic STAR. The protein was purified by a one-step procedure in an acylated-plasminogen Sepharose coupled column.     

The time course of hydrolysis of a beta-lactam antibiotic by intact gram-negative bacteria possessing a periplasmic beta-lactamase.

An equation is derived from first principles for describing the change in concentration with time of a beta-lactam antibiotic in the presence of intact Gram-negative bacteria possessing a beta-lactamase located in the periplasmic space. The equation predicts a first-order decline in beta-lactam concentration of the form [S] = [Si]e lambda t, where [S] is the exogenous concentration of beta-lactam, [Si] is the value of [S] at time zero, t is the time from mixing of cells and antibiotic and lambda (less than 0) is the decay constant. The value of lambda is exactly described by the theory in terms of experimentally measurable quantities. Quantitative data concerning cephaloridine hydrolysis by intact cells of Haemophilus influenzae agreed well with the theory, as did data concerning the uptake of 2-nitrophenyl galactoside by intact cells of Escherichia coli. Cephalosporin C hydrolysis by intact cells of Pseudomonas aeruginosa did not progress as predicted by the theory. The theory is applicable to any substrate which is acted on by an enzyme that is located solely in the periplasmic space and that obeys the Michaelis-Menten equation of enzyme kinetics.