Fine Structure of Thermus aquaticus, an Extreme Thermophile

Electron microscopic studies using thin sections revealed that Thermus aquaticus has a structure similar to that of most other gram-negative bacteria. The cell envelope is tripartite: plasma membrane, thin middle layer, and a thicker and irregular outer layer. The outer layer appears to be joined to the plasma membrane by a series of connections and, when seen in tangential section, the outer layer appears as a series of parallel bands. The cell division mechanism resembles that of typical gram-negative bacteria. Large spherical bodies designated “rotund bodies” are formed as a result of the association of a number of separate cells. In this association the outer envelope layers of the cells fuse and pull away from the middle layer. The rotund body thus appears as a series of rods, usually lying in parallel around the periphery of the sphere, completely connected by means of the fused outer layer.     

Characteristics of major outer membrane proteins of Haemophilus influenzae.

Several properties of Haemophilus influenzae outer membrane proteins were analyzed to define related proteins in various isolates. H. influenzae type b 760705 had six major outer membrane proteins with the following characteristics. Protein a (Mr, 47,000) demonstrated heat modifiability in sodium dodecyl sulfate; its apparent molecular weight was 34,000 at temperatures below 60 degrees C. This protein was extracted from cell envelopes by using Triton X-100-10 mM MgCl2; in cell envelope preparations, the protein was degraded by trypsin. Proteins b (Mr, 41,000) and c (Mr, 40,000) were insensitive to trypsin degradation, were not heat modifiable in sodium dodecyl sulfate, and were peptidoglycan associated in 0.5% Triton X-100-0.2% sodium dodecyl sulfate. The amount of protein b was reduced in ultrasonically obtained cell envelopes. Protein d (Mr, 37,000) was heat modifiable in sodium dodecyl sulfate with an Mr of 28,000 at temperatures below 100 degrees C and was degraded by trypsin, leaving a membrane-bound fragment of Mr, 27,000. Both the intact and degraded proteins were immunologically cross-reactive with the heat-modifiable OmpA protein of Escherichia coli K-12. Protein d was absent in LiCl-EDTA extracts of cells. Protein e (Mr, 30,000), invariably present in all H. influenzae strains tested, was insensitive to trypsin and absent in LiCl-EDTA extracts of cells. Protein k (Mr, 58,000) was extracted from cell envelopes with 2% Triton X-100-10 mM MgCl2 and, in cell envelopes, appeared to be sensitive to trypsin degradation. Proteins with similar properties to those of proteins a to k were found in 10 other H. influenzae b strains, reference strains with serotype a, c, d, e, and f capsules, and 18 of 20 nonencapsulated strains. Their relative molecular weights, however, varied.     

Chemical alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

Cell envelopes from Pseudomonas aeruginosa strains resistant to polymyxin were compared with cell envelopes from polymyxin-sensitive strains as to their content of total protein, carbohydrate, and 2-keto-3-deoxyoctonate and as to their protein composition as determined by slab polyacrylamide gel electrophoresis. The cell envelopes of the polymyxin-resistant strains had reduced amounts of lipopolysaccharide, as indicated a reduction in both carbohydrate and 2-keto-3-deoxyoctonate concentrations, and a greatly altered protein composition as shown by polyacrylamide gel electrophoresis. There was a quantitative increase in total cell envelop protein in these strains. However, those protein bands identified as being major outer membrane proteins upon polyacrylamide gel electrophoresis of separated outer and cytoplasmic membranes were reduced greatly in concentration in the polymyxin-resistant cell envelopes. Thus, it appears that polymyxin resistance in these strains is associated with the alteration of the outer membrane through a loss of lipopolysaccharide and outer membrane proteins.     

The cell envelope of the hyperthermophilic archaebacterium Pyrobaculum organotrophum consists of two regularly arrayed protein layers: three-dimensional structure of the outer layer

The cell envelope of the hyperthermophilic sulphur-reducing archaebacterium Pyrobaculum organotrophum H10 was found to be composed of two distinct hexagonally arranged crystalline protein arrays. Electron microscopic analysis of freeze-etched cells and isolated envelopes in conjunction with image processing showed that the inner layer (lattice centre-to-centre spacing 27.9 nm) is essentially identical to the protein array of Pyrobaculum islandicum GEO3, a complex, rigid structure implicated in the maintenance of cell shape. The outer layer has clear p6 symmetry and a lattice spacing of 20.6 nm. Its three-dimensional structure was reconstructed from a negative stain tilt series of an intact double-layered envelope using Fourier filtration to separate the desired information from the other lattices present. The outer layer is a unique, porous network of blocklike dimers disposed around six-fold axes, and exhibits minimal asymmetry between its inner and outer faces. It appears to be rather loosely associated with the outer surface of the inner layer. In most H10 envelopes, the inner layer is orientated with one base vector exactly perpendicular to the long axis of the cell, so that the cylindrical portion is composed of a series of parallel cell-girdling hoops of hexameric morphological units. All the other known Pyrobaculum strains were found to have a GEO3-type envelope structure, consisting of a single rigid protein array and a fibrous capsule. Although H10 does not possess a capsule, fibrils appear to be sandwiched between the two protein layers.     

Electron Microscopic Observations on the Structure of the Envelopes of Mature Elementary Bodies and Developmental Reticulate Forms of Chlamydia psittaci

Purified suspensions of Chlamydia psittaci were prepared from L cells. Thin sections of intact elementary bodies and intact developmental reticulate bodies and of their purified envelopes were observed by electron microscopy. In both intact organisms and partially purified envelopes, two membranous structures, each appearing in electron micrographs as two darkly stained layers, were observed. In the elementary body sections, the outer membrane was round, apparently rigid, and was not soluble in 0.5% sodium dodecyl sulfate. The inner layer was irregular in shape and was completely removed by detergent treatment. We interpret these results to indicate that the outer rigid layer of the envelope is the cell wall and the inner layer is the cytoplasmic membrane. When the fragile reticulate body envelopes were similarly studied, the outer cell wall was clearly visible, and some evidence of an inner membrane was seen. After treatment with nucleases and detergent, all evidence of inner or cytoplasmic membrane was removed, but the outer cell wall remained. Thus, it appears that the cell wall of this organism is continuous throughout the growth cycle and that the fragility and lack of rigidity of the reticulate body cell is due to changes in chemical composition or structure of the cell wall.     

Toward the assembly of a minimal divisome

The construction of an irreducible minimal cell having all essential attributes of a living system is one of the biggest challenges facing synthetic biology. One ubiquitous task accomplished by any living systems is the division of the cell envelope. Hence, the assembly of an elementary, albeit sufficient, molecular machinery that supports compartment division, is a crucial step towards the realization of self-reproducing artificial cells. Looking backward to the molecular nature of possible ancestral, supposedly more rudimentary, cell division systems may help to identify a minimal divisome. In light of a possible evolutionary pathway of division mechanisms from simple lipid vesicles toward modern life, we define two approaches for recapitulating division in primitive cells: the membrane deforming protein route and the lipid biosynthesis route. Having identified possible proteins and working mechanisms participating in membrane shape alteration, we then discuss how they could be integrated into the construction framework of a programmable minimal cell relying on gene expression inside liposomes. The protein synthesis using recombinant elements (PURE) system, a reconstituted minimal gene expression system, is conceivably the most versatile synthesis platform. As a first step towards the de novo synthesis of a divisome, we showed that the N-BAR domain protein produced from its gene could assemble onto the outer surface of liposomes and sculpt the membrane into tubular structures. We finally discuss the remaining challenges for building up a self-reproducing minimal cell, in particular the coupling of the division machinery with volume expansion and genome replication.     

Characterization of a major envelope protein from the rumen anaerobe Selenomonas ruminantium OB268

Cell envelopes from the Gram-negative staining but phylogenetically Gram-positive rumen anaerobe Selenomonas ruminantium OB268 contained a major 42 kDa heat modifiable protein. A similarly sized protein was present in the envelopes of Selenomonas ruminantium D1 and Selenomonas infelix. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of Triton X-100 extracted cell envelopes from S. ruminantium OB268 showed that they consisted primarily of the 42 kDa protein. Polyclonal antisera produced against these envelopes cross-reacted only with the 42 kDa major envelope proteins in both S. ruminantium D1 and S. infelix, indicating a conservation of antigenic structure among each of the major envelope proteins. The N-terminus of the 42 kDa S. ruminantium OB268 envelope protein shared significant homology with the S-layer (surface) protein from Thermus thermophilus, as well as additional envelope proteins containing the cell surface binding region known as a surface layer-like homologous (SLH) domain. Thin section analysis of Triton X-100 extracted envelopes demonstrated the presence of an outer bilayer over-laying the cell wall, and a regularly ordered array was visible following freeze-fracture etching through this bilayer. These findings suggest that the regularly ordered array may be composed of the 42 kDa major envelope protein. The 42 kDa protein has similarities with regularly ordered outer membrane proteins (rOMP) reported in certain Gram-negative and ancient eubacteria.     

Demonstration of a missing outer membrane protein in tolG mutants of Escherichia cell

A class of Escherichia coli mutants called tolG are specifically tolerant to bacteriocin JF246. Cell envelopes were prepared from three independent spontaneous E. coli. tolG mutants and the parental strain (tolG⁺). Electrophoresis of these preparations in polyacrylamide gels containing sodium dodecyl sulfate showed that the tolG strains lacked a cell envelope protein found in the tolG⁺ strain. It was estimated that this protein accounted for 10% of the total cell envelope proteins by densitometer tracings of gels stained with Fast Green. Membrane fractionation by isopycnic centrifugation in a sucrose density gradient showed that this protein was located in the outer membrane of tolG⁺ cells. Genetic studies using conjugation, transduction and reversion showed that, in the limited number of recombinants or revertants studied, strains exhibiting the tolerant phenotype lacked the outer membrane protein, whereas the protein was present in bacteriocin-sensitive strains.     

The timing and manner of disassembly of the apparatuses for chloroplast and mitochondrial division in the red alga Cyanidioschyzon merolae

The timing and manner of disassembly of the apparatuses for chloroplast division (the plastid-dividing ring; PD ring) and mitochondrial division (the mitochondrion-dividing ring; MD ring) were investigated in the red alga Cyanidioschyzon merolae De Luca, Taddei and Varano. To do this, we synchronized cells both at the final stage of and just after chloroplast and mitochondrial division, and observed the rings in three dimensions by transmission electron microscopy. The inner (beneath the stromal face of the inner envelope) and middle (in the inter-membrane space) PD rings disassembled completely, and disappeared just before completion of chloroplast division. In contrast, the outer PD and MD rings (on the cytoplasmic face of the outer envelope) remained in the cytosol between daughter organelles after chloroplast and mitochondrial division. The outer rings started to disassemble and disappear from their surface just after organelle division, initially clinging to the outer envelopes at both edges before detaching. The results suggest that the two rings inside the chloroplast disappear just before division, and that this does not interfere with completion of division, while the outer PD and MD rings function throughout and complete chloroplast and mitochondrial division. These results, together with previous studies of C. merolae, disclose the entire cycle of change of the PD and MD rings. Received: 19 May 2000 / Accepted: 3 August 2000     

Localization of enterobacterial common antigen: Proteus mirabilis and its various L-forms.

An investigation of Proteus mirabilis wild-type strains and their various derived L-forms shows that the enterobacterial common antigen (ECA) is localized in the outer membrane of the cell envelope of these strains. In strains where the outer membrane is lacking (stable protoplast L-forms) or where its amount is reduced (spheroplast UL19) no ECA or only reduced amounts of it are detected by serological tests or by ferritin-labeling techniques.     

Protein alterations in isolated outer membranes of polymyxin-resistantPseudomonas aeruginosa isolates

Isolated outer membranes fromPseudomonas aeruginosa strains resistant to polymyxin were compared with isolated outer membranes from polymyxin-sensitive strains as to their protein composition as determined by slab polyacrylamide gel electrophoresis. Both the porin protein and the H1 protein were decreased greatly in concentration in the outer membranes from the polymyxin-resistant strains. This confirms previous findings reduced concentrations of these proteins in cell envelopes of these strains. No evidence was found for these decreases being the result of an artifactual loss of these proteins from the outer membrane due to conditions used for growth or for preparation of cell envelopes. Nevertheless, the role of these outer membrane proteins in mediating polymyxin resistance is uncertain.     

The dynamic structure of the Escherichia coli cell envelope as probed by 15N nuclear magnetic resonance spectroscopy.

Proton decoupled 15N NMR spectroscopy is shown to be a useful tool for probin the dynamic structure of the bacterial cell envelope. The proton decoupled 15N NMR spectra of Escherichia coli whole cells, cell envelopes and outer membranes were obtained and displayed resonances originating from protein side-chain groups, phosphatidylethanolamine, and peptidoglycan. Removal of phospholipids from the cell envelope resulted in a decrease in the motional freedom of peptidoglycan and cell envelope proteins. The mobility of the protein Arg side-chain groups is increased in the absence of peptidoglycan. These data provide insights into the effect of supramolecular organization on the dynamic structure of the E. coli cell envelope.     

FINE STRUCTURE AND ORGANELLE ASSOCIATIONS IN BROWN ALGAE

The structural interrelationships among several membrane systems in the cells of brown algae have been examined by electron microscopy. In the brown algae the chloroplasts are surrounded by two envelopes, the outer of which in some cases is continuous with the nuclear envelope. The pyrenoid, when present, protrudes from the chloroplast, is also surrounded by the two chloroplast envelopes, and, in addition, is capped by a third dilated envelope or "pyrenoid sac." The regular apposition of the membranes around the pyrenoid contrasts with their looser appearance over the remainder of the chloroplast. The Golgi apparatus is closely associated with the nuclear envelope in all brown algae examined, but in the Fucales this association may extend to portions of the cytoplasmic endoplasmic reticulum as well. Evidence is presented for the derivation of vesicles, characteristic of those found in the formative region of the Golgi apparatus, from portions of the underlying nuclear envelope. The possibility that a structural channeling system for carbohydrate reserves and secretory precursors may be present in brown algae is considered. Other features of the brown algal cell, such as crystal-containing bodies, the variety of darkly staining vacuoles, centrioles, and mitochondria, are examined briefly, and compared with similar structures in other plant cells.     

Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium.

Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water.     

Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium

Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water.     

The periplasmic space in Thermus thermophilus: evidence from a regulation-defective S-layer mutant overexpressing an alkaline phosphatase

The presence of a periplasmic space within the cell envelope of Thermus thermophilus was analyzed in a mutant (HB8(Delta)UTR1) defective in the regulation of its S-layer (surface crystalline layer). This mutant forms round multicellular bodies (MBs) surrounded by a common envelope as the culture approaches the stationary phase. Confocal microscopy revealed that the origin of the MBs is the progressive detachment of the external layers coupled with the accumulation of NH(2)-containing material between the external envelopes and the peptidoglycan. A specific pattern of proteins was found as soluble components of the intercellular space of the MBs by a single fractionation procedure, suggesting that they are periplasmic-like components. To demonstrate this, we cloned a gene ( phoA) from T. thermophilus HB8 encoding a signal peptide-wearing alkaline phosphatase (AP), and engineered it to be overexpressed in the mutant from a shuttle vector. Most of the AP activity (>80%) was found as a soluble component of the MBs' intercellular fraction. All these data indicate that Thermus thermophilus actually has a periplasmic space which is functionally similar to that of Proteobacteria. The potential application of the HB8(Delta)UTR1 mutant for the overexpression of periplasmic thermophilic proteins is discussed.     

A common-immunogenic Vibrio outer membrane protein

Abstract The presence of antibodies in rabbit antisera to cell envelope proteins of Vibrio cholerae has been examined using a two-dimensional system, in which the cell envelopes are electrophoresed in sodium dodecyl sulfate (SDS) in polyacrylamide gels in the first dimension, and in agarose containing antibodies in the second. The results show that a 25-kDa protein is markedly immunogenic and appears to be common to Vibrio strains; it is not present in a number of other organisms. This 25-kDa protein is an outer membrane protein as judged by sucrose gradient centrifugation and it is accessible to iodination by lactoperoxidase, suggesting that it is exposed on the cell surface.     

Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.

Late in the terminal differentiation of epidermis and cultured epidermal cells, a protein envelope located beneath the plasma membrane becomes cross-linked by cellular transglutaminase. The process of cross-linking can be initiated in cultured epidermal cells by agents affecting cell membrane permeability--nonionic detergents, high salt concentrations and ionophores. These agents initiate the cross-linking process by making calcium ions available to the transglutaminase. A soluble precursor of the cross-linked envelope has been identified in crude extracts of cultured epidermal cells by its ability to incorporate labeled amines through the action of transglutaminase. The protein has been purified to homogeneity by gel filtration and chromatography on columns of DEAE-cellulose and hydroxyapatite. Comprising an estimated 5--10% of the soluble cell proteins, it has a molecular weight of about 92,000, is isoelectric at pH 4.5 +/- 0.3 and has an unusual amino acid composition (46% Glx residues). It is chemically and immunochemically unrelated to keratins. The following evidence confirms that the protein becomes incorporated into cross-linked envelopes: first, washed cross-linked envelopes bind antibody to the purified protein, as shown by indirect immunofluorescence; second, absorption of the antiserum with washed envelopes removes all detectable antibodies to the purified protein; and third, the protein cannot be extracted from keratinocytes after their envelopes have become cross-linked. Examination of sections of epidermis by immunofluorescence, using antiserum to the purified protein, reveals that in addition to the stratum corneum, the living cells of the outer half of the spinous layer react strongly. The envelope precursor is present in the cytoplasm, but becomes concentrated at the cell periphery, where it will be cross-linked later, when the cells have passed through the granular layer. The protein is also concentrated in a peripheral location in cultured epidermal cells.     

Constitution of the cell envelope of Haemophilus influenzae in relation to competence for genetic transformation.

Cell envelopes of Haemophilus influenzae have been prepared by breakage in a French pressure cell followed by differential centrifugation. The envelope fraction may be resolved into an inner-membrane (light) and an outer-membrane (heavy) fraction on density gradients. Envelopes from competent cells possess elevated levels of lipopolysaccharide with a composition different from that of log-phase cell envelopes. Three apparently new polypeptides have been observed in envelopes from competent cells by gel electrophoresis in sodium dodecyl sulfate; additional quantitative alterations in the profiles of membrane polypeptides also company the development of the capacity to transport deoxyribonucleic acid. Most of the polypeptide changes are confined to the outer membrane; one new polypeptide is associated with the inner cytoplasmic membrane of competent cells. Protein synthesis during competence developement is rquired for the change in lipopolysaccharides and in the envelope polypeptides to occur.     

Localization of a 64-kDa phosphoprotein in the lumen between the outer and inner envelopes of pea chloroplasts

The identification and localization of a marker protein for the intermembrane space between the outer and inner chloroplast envelopes is described. This 64-kDa protein is very rapidly labeled by [gamma-32P]ATP at very low (30 nM) ATP concentrations and the phosphoryl group exhibits a high turnover rate. It was possible to establish the presence of the 64-kDa protein in this plastid compartment by using different chloroplast envelope separation and isolation techniques. In addition comparison of labeling kinetics by intact and hypotonically lysed pea chloroplasts support the localization of the 64-kDa protein in the intermembrane space. The 64-kDa protein was present and could be labeled in mixed envelope membranes isolated from hypotonically lysed plastids. Mixed envelope membranes incorporated high amounts of 32P from [gamma-32P]ATP into the 64-kDa protein, whereas separated outer and inner envelope membranes did not show significant phosphorylation of this protein. Water/Triton X-114 phase partitioning demonstrated that the 64-kDa protein is a hydrophilic polypeptide. These findings suggest that the 64-kDa protein is a soluble protein trapped in the space between the inner and outer envelope membranes. After sonication of mixed envelope membranes, the 64-kDa protein was no longer present in the membrane fraction, but could be found in the supernatant after a 110,000 x g centrifugation.