Electron microscopic observations of prokaryotic surface appendages

Prokaryotic microbes possess a variety of appendages on their cell surfaces. The most commonly known surface appendages of bacteria include flagella, pili, curli, and spinae. Although archaea have archaella (archaeal flagella) and various types of pili that resemble those in bacteria, cannulae, and hami are unique to archaea. Typically involved in cell motility, flagella, the thickest appendages, are 20–26 nm and 10–14 nm wide in bacteria and archaea, respectively. Bacterial and archaeal pili are distinguished by their thin, short, hair-like structures. Curli appear as coiled and aggregative thin fibers, whereas spinae are tubular structures 50–70 nm in diameter in bacteria. Cannulae are characterized by ～25 nm-wide tubules that enter periplasmic spaces and connect neighboring archaeal cells. Hami are 1–3 μm in length and similar to barbed grappling hooks for attachment to bacteria. Recent advances in specimen preparation methods and image processing techniques have made cryo-transmission electron microscopy an essential tool for in situ structural analysis of microbes and their extracellular structures.     

Secretion and processing of the Bacillus subtilis endo-β-1,3-1,4-glucanase in Escherichia coli

The endo-beta-1,3-1,4-glucanase enzyme of Bacillus subtilis C120, when synthesized in Escherichia coli, is located mainly in the cytoplasm, but enzyme activity is also detected in the periplasmic space and in the extracellular medium. The proportion recovered in the extracellular medium is not altered by changes in the levels of synthesis of the enzyme. Lysis of E. coli cells is ruled out as the cause of the secretion by the normal localization of beta-galactosidase, an intracellular protein. However, beta-lactamase, which is normally found in the periplasmic space, is detected in the extracellular medium of E. coli transformants containing beta-glucanase plasmids, suggesting that the presence of beta-glucanase in the cell alters the permeability of the outer membrane. The beta-glucanase proteins found in the extracellular medium, the periplasmic space and the cytoplasm have the same electrophoretic mobilities as the secreted enzyme of B. subtilis. Amino-terminal sequencing has shown that the beta-glucanase enzyme in the intracellular fraction of E. coli is processed at a site two amino acids distant from the processing site used in B. subtilis.     

Morphological properties of a human intestinal spirochete first isolated from a patient with diarrhea in Japan

A human intestinal spirochete isolated from a rectal biopsy specimen was morphologically characterized. The isolate was comma-shaped, 3-6 microm in length, 0.2 micro m in diameter and had tapered ends. The surface layer, external to the outer envelope, was amorphous. Four string-like periplasmic flagella with a diameter of 20 nm were presented at each end of the SDS-treated cells. Thin sections of the bacterial cell revealed a high-density cytoplasmic membrane and flagella in the periplasmic space between the cytoplasmic membrane and the outer envelope. Three segments of equal length were observed in some of the cells, while other cells were bi-segmented and more frequently observed.     

Locked on one side only: ground state dynamics of the outer membrane efflux duct TolC

Playing a major role in the expulsion of antibiotics and the secretion of cell toxins in conjunction with inner membrane transporters of three protein superfamilies, the outer membrane channel TolC occurs in at least two states blocking or permitting the passage of substrates. The details of the underlying gating mechanism are not fully understood. Addressing the questions of extracellular access control and periplasmic gating mechanism, we conducted a series of independent, unbiased 150-300 ns molecular dynamics simulations of wild-type TolC in a phospholipid membrane/150 mM NaCl water environment. We find that TolC opens and closes freely on the extracellular side, suggesting the absence of a gating mechanism on this side in the isolated protein. On the periplasmic side, we observe the outer periplasmic bottleneck region adopting in all simulations a conformation more open than the TolC wild-type crystal structures until in one run the successive binding of two sodium ions induces the transition to a conformation more closed than any of the available TolC X-ray structures. Concurrent with a heightened sodium residence probability near Asp374, the inner periplasmic bottleneck region at Asp374 remains closed throughout the simulations unless all NaCl is removed from the system, inducing a reopening of the outer and inner bottleneck. Our findings suggest that TolC is locked only on the periplasmic side in a sodium-dependent manner.     

Computational model of the fluid dynamics of a cannula inserted in a vessel: incidence of the presence of side holes in blood flow

Vascular access methods, performed by the insertion of cannulae into vessels, may disturb the physiological flow of blood, giving rise to non-physiological pressure variations and shear stresses. To date, the hydrodynamic behaviour of the cannulae has been evaluated comparing their pressure loss–flow rate relationships, as obtained from in vitro experiments using a monodimensional approach; this methodology neither furnish information about the local fluid dynamics nor the established flow field in specific clinical work conditions. Since the shear stress is a critical factor in the design of artificial circulatory devices, more knowledge should be necessary about the local values assumed by the haemodynamic parameters during cannulation. An alternative way to investigate the fluid dynamic as accurately as possible is given by numeric studies. A 3D model of cannula concentrically placed in a rigid wall vessel is presented, with the finite element methodology used to numerically simulate the steady-state flow field in two different venous cannulation case studies, with two cannulae having a central hole and two or four side holes, respectively, with the same boundary conditions. Lower velocity and shear stress peak values have been computed for the model with four side holes upstream of the central hole, in the region of the cannula where the inlet flows meet and towards cannula's outlet, due to the increased flow symmetry and inlet area with respect to the model with two side holes. Starting from the investigation of different cannula designs, numerically assessing the local fluid dynamics, indications can be drawn to support both the design phase and the device optimal clinical use, in order to limit risks of biomechanical origin. Thus the presence of four side holes implied, as a consequence of the greater inlet area and of the increased symmetry, a less disturbed blood flow, together with reduced shear stress values. Furthermore, results show that the numerical simulations furnished useful informations on the interaction between vessel and cannula, e.g. on the fluid dynamics establishing in the free luminal space left, in the vessel, by the inserted cannula.     

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.     

Biochemical evidence that starch breakdown by Bacteroides thetaiotaomicron involves outer membrane starch-binding sites and periplasmic starch-degrading enzymes.

Bacteroides thetaiotaomicron can utilize amylose, amylopectin, and pullulan as sole sources of carbon and energy. The enzymes that degrade these polysaccharides were found to be primarily cell associated rather than extracellular. Although some activity was detected in extracellular fluid, this appeared to be the result of cell lysis. The cell-associated amylase, amylopectinase, and pullulanase activities partitioned similarly to the periplasmic marker, acid phosphatase, when cells were exposed to a cold-shock treatment. Two other enzymes associated with starch breakdown, alpha-glucosidase and maltase, appeared to be located in the cytoplasm. Intact cells of B. thetaiotaomicron were found to bind 14C-starch. Binding was probably mediated by a protein because it was saturable and was decreased by treatment of cells with proteinase K. Results of competition experiments showed that the starch-binding proteins had a preference for maltodextrins larger than maltohexaose and a low affinity for maltose and maltotriose. Both the degradative enzymes and starch binding were induced by maltose. These findings indicate that starch utilization by B. thetaiotaomicron apparently does not involve secretion of extracellular enzymes. Rather, binding of the starch molecule to the cell surface appears to be a first step to passing the molecule through the outer membrane and into the periplasmic space.     

Immunocytochemical identification and localization of lipase in cells of the mycelium of Penicillium cyclopium variety

The localization of lipase in cells of the fungus Penicillium cyclopium was investigated. It was shown by differential centrifugation of a homogenate of mycelial cells that the activity of the enzyme is associated with the cell wall. A study of ultrathin sections of mycelium fixed using the method of Zvyagintseva in an electron microscope showed that the final products of lipolytic activity of the enzyme is localized on the cell wall. Antibodies were raised against the purified A and B lipases from P. cyclopium and their specificity was assessed by enzyme-linked immunosorbent assay. The antibody preparation was used in cytochemical investigation by immunogold labelling. This study permits the localization of cell-bound lipase mainly in the cell wall and in the periplasmic space. The identity of the cell-bound lipase with one of the two extracellular lipases is also demonstrated.     

Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis

The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp'-'rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured.     

Spirochete periplasmic flagella and motility

Spirochetes have a unique structure, and as a result their motility is different from that of other bacteria. They also have a special attribute: spirochetes can swim in a highly viscous, gel-like medium, such as that found in connective tissue, that inhibits the motility of most other bacteria. In spirochetes, the organelles for motility, the periplasmic flagella, reside inside the cell within the periplasmic space. A given periplasmic flagellum is attached only at one end of the cell, and depending on the species, may or may not overlap in the center of the cell with those attached at the other end. The number of periplasmic flagella varies from species to species. These structures have been shown to be directly involved in spirochete motility, and they function by rotating within the periplasmic space. The mechanics of motility also vary among the spirochetes. In Leptospira, a motility model developed several years ago has been extensively tested, and the evidence supporting this model is convincing. Borrelia burgdorferi swims differently, and a model of its motility has been recently put forward. This model is based on analyzing the motion of swimming cells, high voltage electron microscopy of fixed cells, and mutant analysis. To better understand spirochete motility on a more molecular level, the proteins and genes involved in motility are being analyzed. Spirochete periplasmic flagellar filaments are among the most complex of bacterial flagella. They are composed of the FlaA sheath proteins, and in many species, multiple FlaB core proteins. Allelic exchange mutagenesis of the genes which encode these proteins is beginning to yield important information with respect to periplasmic flagellar structure and function. Although we are at an early stage with respect to analyzing the function, organization, and regulation of many of the genes involved in spirochete motility, unique aspects have already become evident. Future studies on spirochete motility should be exciting, as only recently have complete genome sequences and tools for allelic exchange mutagenesis become available.     

Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant

E. coli is one of the most commonly used host strains for recombinant protein production. However, recombinant proteins are usually found intracellularly, in either cytoplasm or periplasmic space. Inadequate secretion to the extracellular environment is one of its limitations. This study addresses the outer membrane barrier for the translocation of recombinant protein directed to the periplasmic space. Specifically, using recombinant maltose binding protein (MalE), xylanase, and cellulase as model proteins, we investigated whether the lpp deletion could render the outer membrane permeable enough to allow extracellular protein production. In each case, significantly higher excretion of recombinant protein was observed with the lpp deletion mutant. Up to 90% of the recombinant xylanase activity and 70% of recombinant cellulase activity were found in the culture medium with the deletion mutant, whereas only 40-50% of the xylanase and cellulase activities were extracellular for the control strain. Despite the weakened outer membrane in the mutant strain, cell lysis did not occur, and increased excretion of periplasmic protein was not due to cell lysis. The lpp deletion is a simple method to generate an E. coli strain to effect significant extracellular protein production. The phenotype of extracellular protein production without cell lysis is useful in many biotechnological applications, such as bioremediation and plant biomass conversion.     

Purification and Comparison of the Periplasmic and Extracellular Forms of the Cellodextrinase from Bacteroides succinogenes

Both the periplasmic and the extracellular cellodextrinases from Bacteroides succinogenes S85 grown on Avicel microcrystalline cellulose were purified to homogeneity by column chromatography and characterized. Over 70% of the total cellobiosidase activity displayed by cells was accounted for by these enzymes. The periplasmic and extracellular cellodextrinases had identical molecular weights (50,000), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identical isoelectric points (4.9). In addition, the two enzymes were similar in catalytic properties, with K_m and V_max values of approximately 0.24 mM and 21 μmol/min per mg of protein, respectively. Examination of the two enzymes by using peptide mapping and immunoblotting techniques provided additional evidence indicating their identical nature. Immunoblotting of the extracellular culture fluid with affinity-purified antibody to the periplasmic cellodextrinase revealed one band with a molecular weight corresponding to that of the periplasmic cellodextrinase. The stability of the purified periplasmic cellodextrinase in aqueous solution was markedly enhanced by increased protein content. This enzyme showed a low affinity for crystalline cellulose.     

Three Years' Experience with Bypass Cannulae for Chronic Hemodialysis

In 14 patients over the past three years, “bypass” cannulae have been inserted to relieve obstruction in cannulae used for chronic hemodialyses. Experience with these patients has prompted changes resulting in improved technique.Clotting was the major cause of cannulae failure and infection was the second most important cause. Other causes of failure included: venous atheroma, cannulae extrusion, aneurysms of the vessel at the cannulae tips, obstructive vegetation on the vessel wall, calcium deposits on the vessel wall, and thrombi on the vein walls.The mean survival time was improved from 1.9 months in 1963-1964 to over 9.0 months in 1965-1966. The longest surviving cannula set was two years and the shortest one week.It is considered that the most important factors contributing to increased cannula survival are improved cannula care by patients and staff, the use of angiography for accurate diagnosis, and prompt anticoagulation if atheromatous stenosis of the venous component is encountered.     

A Method for Collection of Bile in the Conscious Sheep

A method to collect bile directly from the hepatic duct is described for use in the sheep. The technique is a combination of the intestinal re-entrant cannulae and a catheter from the duodenal lumen to the hepatic duct. The cystic duct is ligated near its junction with the common bile duct. The catheter is fixed in the proximal visible end of the hepatic duct. One plastic cannula is fixed to the duodenum opposite to the opening of the common bile duct and the other is fixed in the same way about 15 cm posterior to the first one. The two plastic cannulae fixed together with a plastic tube serve as an extra-abdominal anastomosis. During the collection periods the bile duct catheter is passed through an opening in the wall of the connection tube into a collection bag that is fixed to the plastic cannulae. Between the collection periods the catheter ends in the lumen of the anastomosis.     

Rhizobium extracellular structures in the symbiosis

The extracellular and surface polysaccharides produced by Rhizobium species constitute a composite macromolecular interface between the bacterial cell and its environment. Several of these polysaccharides are involved in the complex series of interactions leading to the establishment of an effective Rhizobium-legume symbiosis. Extracellular heteropolysaccharides (EPSs) are found in culture supernatants, while capsular polysaccharides adhere to the cell surface. Cyclic (1–2)--d glucan is a periplasmic oligosaccharide that has also been found in the culture supernatants of some strains. The lipopolysaccharides (LPSs), which form part of the outer membrane and contain the O-somatic antigens, comprise the other major group of extracellular polysaccharides. In this review we will describe the major Rhizobium extracellular structures and their role in symbiosis with leguminous plants.The authors are with the Departamento de Microbiologia y Parasitologia, Facultad de Farmacia. Universidad de Sevilla, 41012 Sevilla, Spain     

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.     

Native cell wall organization shown by cryo-electron microscopy confirms the existence of a periplasmic space in Staphylococcus aureus

The current perception of the ultrastructure of gram-positive cell envelopes relies mainly on electron microscopy of thin sections and on sample preparation. Freezing of cells into a matrix of amorphous ice (i.e., vitrification) results in optimal specimen preservation and allows the observation of cell envelope boundary layers in their (frozen) hydrated state. In this report, cryo-transmission electron microscopy of frozen-hydrated sections of Staphylococcus aureus D2C was used to examine cell envelope organization. A bipartite wall was positioned above the plasma membrane and consisted of a 16-nm low-density inner wall zone (IWZ), followed by a 19-nm high-density outer wall zone (OWZ). Observation of plasmolyzed cells, which were used to artificially separate the membrane from the wall, showed membrane vesicles within the space associated with the IWZ in native cells and a large gap between the membrane and OWZ, suggesting that the IWZ was devoid of a cross-linked polymeric cell wall network. Isolated wall fragments possessed only one zone of high density, with a constant level of density throughout their thickness, as was previously seen with the OWZs of intact cells. These results strongly indicate that the IWZ represents a periplasmic space, composed mostly of soluble low-density constituents confined between the plasma membrane and OWZ, and that the OWZ represents the peptidoglycan-teichoic acid cell wall network with its associated proteins. Cell wall differentiation was also seen at the septum of dividing cells. Here, two high-density zones were sandwiched between three low-density zones. It appeared that the septum consisted of an extension of the IWZ and OWZ from the outside peripheral wall, plus a low-density middle zone that separated adjacent septal cross walls, which could contribute to cell separation during division.     

Hypoglycosylation in the alg12delta yeast mutant destabilizes protease A and causes proteolytic loss of external invertase

The Saccharomyces cerevisiae alg12delta mutant accumulates oligosaccharide lipid with a Man(7)GlcNAc(2) oligosaccharide. To determine the N-glycan structures present on S. cerevisiae glycoproteins in the alg12delta strain, we made attempts to purify external invertase, a highly glycosylated secreted protein. These efforts revealed that, in the alg12delta background, external invertase was mildly hypoglycosylated and rapidly destroyed proteolytically. Although secreted alg9delta invertase was more severely hypoglycosylated than the alg12delta form, it was paradoxically stable during purification. The loss of periplasmic invertase was prevented by addition of pepstatin A to the cell cultures, suggesting that aspartyl proteases were active. We found that during overexpression of invertase in alg12delta yeast, sufficient protease A was mistargeted to the periplasmic space, where it hydrolyzed the invertase. Even though alg9delta invertase is underglycosylated in comparison to the alg12delta form, it is more stable because in this genetic background much less protease A is secreted compared to alg12delta cells. These observations may be relevant to studies using other extracellular proteins (e.g., mating factors, alpha-glucosidase) as probes when characterizing glycosylation defects in yeast.     

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