Orientation of chromatophores and spheroplast-derived membrane vesicles of Rhodopseudomonas sphaeroides: analysis by localization of enzyme activities.

Intact spheroplasts, vesicles obtained from French-press lysates (chromatophores), and spheroplast-derived vesicles were isolated from photosynthetically grown cells of Rhodopseudomonas sphaeroides. Lysed spheroplasts showed specific activities of succinate, NADH, and l-lactate dehydrogenase which were eight-, six-, and seven-fold higher, respectively, than those of intact spheroplasts when ferricyanide was used as electron acceptor. Mg²⁺-ATPase activity of lysed spheroplasts, measured using an assay system coupled to the oxidation of NADH, was seven-fold higher than the activity of intact sheroplasts. Toluene-treated spheroplast-derived vesicles displayed higher succinate dehydrogenase (ferricyanide reduction) and Mg²⁺-ATPase activities than untreated vesicles whereas no differences were measured between untreated and toluene-treated chromatophores. However, NADH dehydrogenase (ferricyanide reduction) activities of both toluene-treated vesicles and chromatophores were higher than the activities of untreated vesicles and chromatophores. When chromatophores and spheroplast-derived vesicles were preincubated with trypsin, the l-lactate and succinate dehydrogenase activities of chromatophores were preferentially inactivated when phenazine methosulfate was used as electron acceptor. The data indicate that chromatophores are oriented in an opposite direction to the spheroplast-derived vesicles. At least 80% of the latter are oriented in a direction equivalent to the cytoplasmic membrane of intact cells and spheroplasts. Spheroplast-derived vesicles from cells grown with higher light intensities seem to be more uniformly oriented than those obtained from cells grown with lower light intensities.     

RESOLUTION OF GRANULES FROM RABBIT HETEROPHIL LEUKOCYTES INTO DISTINCT POPULATIONS BY ZONAL SEDIMENTATION

Postnuclear supernates from homogenates of essentially pure rabbit heterophil leukocytes were fractionated by means of zonal differential centrifugation through a discontinuous sucrose gradient at various speeds. Three distinct groups of granules were characterized biochemically and morphologically. They were, in order of decreasing sedimentation coefficient: (a) Large, relatively dense granules, identified morphologically as the azurophil or primary granules, and containing essentially all of the myeloperoxidase activity of the preparations, about one-third of their lysozyme activity, and between 50 and 80% of their content in five acid hydrolases typically associated with lysosomes in other cells; (b) smaller, less dense granules, with the morphological appearance of the specific or secondary granules, and carrying most of the alkaline phosphatase and the remainder of the lysozyme activity of the preparations; (c) a second group of lysosome-like particles, associated with a morphologically heterogeneous fraction, and containing the remainder of the acid hydrolases, but little or no myeloperoxidase. When p-nitrophenyl phosphate was used instead of β-glycerophosphate for the assay of acid phosphatase, only small proportions of the total activity accompanied the two main lysosomal bands, and considerable activity was found in a zone slightly retarded with respect to the slowly moving band of acid hydrolases.     

Interactions of alkaline phosphatase and the cell wall of Pseudomonas aeruginosa

Spheroplasts prepared by lysozyme treatment of cells of Pseudomonas aeruginosa, suspended in 20% sucrose or 0.2 m MgCl(2), were examined in detail. Preparation of spheroplasts in the presence of 0.2 m Mg(2+) released periplasmic alkaline phosphatase, whereas preparation in the presence of 20% sucrose did not, even though untreated cells released phosphatase when suspended in sucrose in the absence of lysozyme. Biochemical characterizations of the sucrose-lysozyme preparations indicated that lysozyme mediated a reassociation of the released phosphatase with the spheroplasts. In addition, the enzyme released from whole cells suspended in 20% sucrose (which represents 20 to 40% of the cell-bound phosphatase) reassociates with the cells in the presence of lysozyme. Electron microscopic examinations of various preparations revealed that phosphatase released in sucrose reassociated with the external cell wall layers in the presence of lysozyme, that sucrose-lysozyme prepared spheroplasts did not dissociate phosphatase which remained in the periplasm of sucrose-washed cells, and that phosphatase was never observed to be associated with the cytoplasmic membrane. A model to account for the binding of P. aeruginosa alkaline phosphatase to the internal portion of the tripartite layer of the cell wall rather than to the cytoplasmic membrane or peptidoglycan layer is presented.     

Surface potential on the periplasmic side of the photosynthetic membrane of Rhodopseudomonas sphaeroides

Membrane surface potential on the periplasmic side of the photosynthetic membrane was estimated in cells, spheroplasts and chromatophores of Rhodopseudomonas sphaeroides. When the membrane potential (potential difference between bulk aqueous phases) was kept constant in the presence of carbonylcyanide m-chlorophenylhydrazone, addition of salt to a suspension of cells or spheroplasts induced a red shift in the carotenoid absorption spectrum which indicated a change in the intramembrane electrical field. The spectral shift is explained by a rise in electrical potential at the outside surface of the photosynthetic membrane due to a decrease in extent of the negative surface potential.The spectral shift occurred in the direction opposite to that in chromatophores, indicating that the sidedness of the membrane of cells or spheroplasts is opposite to that of chromatophores. The dependences of the extent of the potential change on concentration and valence of cations of salts agreed with the Gouy-Chapman relationship on the electrical diffuse double layer. The charge density on the periplasmic surface of the photosynthetic membrane was estimated to be ?2.9 · 10^?3 elementary charge per Ų, while that on the cytoplasmic side surface was calculated as ?1.9 · 10^?3 elementary charge per Ų (Matsuura, K., Masamoto, K., Itoh, S. and Nishimura, M. (1979) Biochim. Biophys. Acta 547, 91–102). Surface potential on the periplasmic side of the photosynthetic membrane was estimated to be about ?50 mV at pH 7.8 in the presence of 0.1 M monovalent salt.     

Ultrastructural organization of spheroplasts induced in Mycobacterium sp. smegmatis by lysozyme or glycine

Summary The conversion of Mycobacterium sp. smegmatis cells into spheroplasts was achieved at a high rate by their treatment with either lysozyme (100 g/ml) or glycine (2%) in a liquid Dubos medium without albumine, stabilized by sucrose (up to 0.34 M). The dynamic of conversion was followed in hanging-drop preparations.Electron microscopic studies of induced spheroplasts were performed. Two types of membranes (80 Å and 130 Å in the width) together with tubular mesosomes localized either intracytoplasmatically or released were revealed.Differences between lysozyme and glycine induction in conversion rate and in time function were noted. Glycine spheroplasts were moreover characterized by numerous cell wall residues adhering to the cell surface and by the incidence of cytoplasmic exfoldings present in a considerably higher amount than in lysozyme induced spheroplasts.Based on these studies suggestions to the mechanism of inductive processes were pointed out.     

Sidedness of membrane structures in Rhodopseudomonas sphaeroides. Electrochemical titration of the spectrum changes of carotenoid in spheroplasts,spheroplast membrane vesicles and chromatophores

The shift of the carotenoid absorption spectrum induced by illumination and valinomycin-K⁺ addition was investigated in membrane structures with different characteristics and opposite sidednesses isolated from Rhodopseudomonas sphaeroides. Right-side-out membrane structures were prepared by isotonic lysozyme-EDTA treatment of the cells (spheroplasts) and by hypotonic treatment of spheroplasts (spheroplast membrane vesicles). Inside-out membrane structures (“chromatophores”) were obtained by treating spheroplast membrane vesicles by French press or sonication.The membrane structures with either sidedness showed the same light-induced change of the “red shift” type. However, the absorbance change by K⁺ addition in the presence of valinomycin in the right-side-out membrane structures were opposite to that in the inverted vesicles, “blue shift” in the former and “red shift” in the latter. The carotenoid absorbance change was linear to membrane potential, calculated from the concentration of KCl added, with a reference on the cytoplasmic side, through positive and negative ranges.     

Pigment biogenesis in freshwater shrimp ventral nerve chord chromatophores

Summary The possible biogenesis of two pigment granule types present in the monochromatic, brown chromatosomes enveloping the ventral nerve chord of the freshwater palaemonid shrimps Macrobrachium acanthurus, M. heterochirus and M. olfersii is examined by transmission electron microscopy in thin section and freeze fracture replicas. Prominent, membrane limited granules are suggested to have their origin in a complex, juxtanuclear, smooth endoplasmic reticulum labyrinth, continuous with the nuclear envelope. Amembranous, lipocarotenoid granules possibly derive from the external surface of the smooth endoplasmic reticulum. Nuclear envelope and SER membranes contain numerous 11 nm diameter intramembranous particles while pigment granule membranes exhibit fewer particles. A dictyosomal origin for the lipocarotenoid granules is discounted. Granulogenesis is suggested to be a continuous process in crustacean chromatophores.     

Intracellular localization of N-formyl chemotactic receptor and Mg2+ dependent ATPase in human granulocytes

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83 000 × g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40–60% were achieved with a 20–70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g·cm^?3. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimanted with specific granules (g9 = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg²⁺ -ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg²⁺ -ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.     

Assessment of Rhodopseudomonas sphaeroides chromatophore membrane asymmetry through bilateral antiserum adsorption studies.

The asymmetric structure of the Rhodopseudomonas sphaeroides chromatophore membrane was examined in detail by crossed immunoelectrophoresis techniques. Because these methods are quantitative and allow increased resolution and sensitivity, it was possible to analyze simultaneously the relative transmembrane distribution of a number of previously identified antigenic components. This was demonstrated by analysis of immunoglobulin samples that were adsorbed by preincubation with either isolated chromatophores or osmotically protected spheroplasts. The photochemical reaction center, the light-harvesting bacteriochlorophyll a-protein complex, the L-lactate dehydrogenase, and reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3) were found to be exposed on the chromatophore surface (cytoplasmic aspect of the membrane within the cell). Other antigenic components were found to be exposed on the surface of spheroplasts (periplasmic aspect of the in vivo chromatophore membrane). Antigens with determinants expressed on both sides of the chromatophore membrane were also identified. Charge shift crossed immunoelectrophoresis confirmed the suggested amphiphilic character of the pigment-protein complexes and identified several additional amphiphilic membrane components.     

Action of chlorophyllase purified from rye seedlings on light-harvesting bacteriochlorophyll of chromatophores and spheroplasts from Rhodospirillum rubrum

1. The chlorophyllase [EC 3.1.1.14] purified from greened rye seedlings hydrolyzed the bacteriochlorophyll isolated from Rhodospirillum rubrum, but not the pigment bound to the membrane of chromatophores or spheroplasts from the bacterium. 2. Acetone, if added at such concentrations that the bound bacteriochlorophyll would not be solubilized, enabled the enzyme to hydrolyze the bound pigment. The acetone concentrations required for half the maximum hydrolysis rates were 16% with chromatophores and 7% with spheroplasts. 3. The enzymic hydrolysis of the bound bacteriochlorophyll in the presence of acetone removed bacteriochlorophyllide from the membrane, leaving its esterifying alcohol, possibly all-trans-geranylgeraniol, in situ. 4. Washing of chromatophores with 30% acetone removed about 10% of the bound bacteriochlorophyll. The bound pigment remaining after washing was not hydrolyzed by the enzyme unless acetone was added. 5. It seems possible that light-harvesting bacteriochlorophyll was mostly, if not all, bound to the inner surface of chromatophores (the outer surface of spheroplasts), having its esterifying alcohol residue buried in the membrane and its porphyrin residue emerging from the membrane into the inside solution; thus, chlorophyllase could not make contact with the ester linkage between the esterifying alcohol and porphyrin moieties of the pigment unless the esterifying alcohol residue was partly exposed.     

OBSERVATIONS ON THE STRUCTURE OF RHODOSPIRILLUM RUBRUM

Sections of Rhodospirillum rubrum cells from cultures of different ages have been examined to obtain information on the development of chromatophores in this organism. Cells from the 12-hour cultures studied contain neither distinct invaginations of the cytoplasmic membrane nor distinct chromatophores. The first structures that can be related to chromatophore development occur peripherally in the cells, are relatively few in number, relatively high in density, and have an indistinct membrane. In cells from 26-hour cultures numerous distinct invaginations of the cytoplasmic membrane are present, and all layers of the cytoplasmic membrane are involved in the formation of each invagination. As the invaginations become more numerous, the ends of the invaginations become constricted to form one or more structures similar to the chromatophores previously described in this organism. Cells of R. rubrum, therefore, develop a structural continuum which initially consists of invaginations of the cytoplasmic membrane, and later of the chromatophores produced by and attached to these invaginations. The presence of this continuum, however, does not necessarily exclude the existence of discrete chromatophores within these cells. Several other structures previously reported in this organism are described in greater detail.     

Inactivation of Gram-Negative Bacteria by Lysozyme, Denatured Lysozyme, and Lysozyme-Derived Peptides under High Hydrostatic Pressure

We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 μg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.     

GRANULES ASSOCIATED WITH THE CHLOROPLAST LAMELLAE OF PORPHYRIDIUM CRUENTUM

Small granules with a diameter of approximately 350 A are attached to the chloroplast lamellae of the red alga Porphyridium cruentum. To some extent, their size depends on the culture conditions and the age of the cell. It was possible to preserve the granules only with aldehyde prefixation. It can be seen that fixed or negatively stained granules are comprised of smaller subunits. The granules are arranged regularly on the lamellae in repeating rows with a center-to-center granule distance of 400 to 500 A. Attempts at characterization of these chloroplast granules revealed that they are resistant to hydrolysis by ribonuclease and appear to be structurally unaffected by methanol-acetone extraction. Because of their close association with the chloroplast lamellae, they are considered as possible sites of phycobilin concentration. This possibility is supported by two observations: when the phycobilins are removed, the granules disappear; and, when the chlorophyll and stainable membrane portions are selectively removed, the phycobilins and granules are still present. It was found that all other marine red algae examined had granules which were associated with the chloroplast lamellae.     

CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-¹⁴C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.     

How does lysozyme penetrate through the bacterial outer membrane?

Lysozyme fails to penetrate through the outer membrane of stationary phase cells of Escherichia coli when it is simply added to suspensions of plasmolyzed cells. Lysozyme penetrates the outer membrane only when these cells are exposed to a mild osmotic shock in the presence of EDTA and lysozyme.In the presence of Mg²⁺, the outer membrane is stabilized sufficiently so that there is no lysozyme penetration during osmotic shock. If Mg²⁺ is added after an osmotic shock has been used to cause lysozyme to penetrate a destabilized outer membrane, the outer membrane is stabilized once again. In this case however, cells are converted to spheroplasts by the lysozyme which has gained access to the murein layer prior to the addition of Mg²⁺. Mg²⁺ stabilizes the outer membranes of these spheroplasts sufficiently so that they remain immune to lysis even in the absence of osmotic stabilizers such as sucrose.These results are discussed in terms of current information on the structure of the murein layer and the outer membrane.     

A highly efficient procedure for the quantitative formation of intact and viable lysozyme spheroplasts from escherichia coli

This paper describes a highly efficient procedure for the quantitative conversion of Escherichia coli cells to spheroplasts utilizing 100- to 1000-fold less lysozyme than in the most efficient procedures used to date. The resulting spheroplasts have intact outer and inner membranes and are fully viable on agar plates. The spheroplasting procedure is a refinement of earlier procedures and enables regulation of the translocation of minute amounts of lysozyme into the periplasmic space of E. coli cells, based on a Ca²⁺ pretreatment, an EDTA incubation, and a heat shock. About 1000 lysozyme molecules per cell are sufficient for complete spheroplast formation (>98%). Some of the characteristics of these spheroplasts prior to and after recovery are described. It is anticipated that such viable spheroplasts will be useful in the study of fusion of gram-negative cells and other membrane systems, in the introduction of DNA and proteins into refractory gram-negative cell, and in investigating envelope-related synthesis and assembly processes.     

FINE STRUCTURAL CHANGES IN RESPONSE TO HORMONAL STIMULATION OF THE PERFUSED CANINE PANCREAS

The dog pancreas isolated in situ was perfused with oxygenated dog blood and stimulated with pancreozymin, secretin, or both. There were no significant changes in the fine structure of the acinar, centroacinar, or duct cells attributable to the perfusion. Combined glutaraldehyde and osmium fixation gave good preservation of the secretory products of the acinar cell. Before stimulation, the lumen of the acini is filled with material similar in texture to the content of the zymogen granules, but of somewhat lower density. Release of secretion commonly takes place by coalescence of the limiting membrane of zymogen granules with the plasmalemma, but one granule opening at the surface may frequently be joined by others coalescing with its membrane and forming an interconnected series all with contents having the same texture as the released zymogen. Such a mechanism seems to permit a more rapid release of secretory product than discharge of individual granules. Pancreozymin stimulation caused marked depletion of zymogen granules, but no obvious changes in the Golgi apparatus. It is clear, therefore, that this hormone exerts its effect upon release of granules rather than upon their formation. Secretin stimulation of water and bicarbonate secretion caused a marked washing out of the luminal contents, but had little detectable effect on cellular structure.     

Localization of reaction center and B800-850 antenna pigment proteins in membranes of Rhodobacter sphaeroides.

The localization of the N- and C-terminal regions of pigment-binding polypeptides of the bacterial photosynthetic apparatus of Rhodobacter sphaeroides was investigated by proteinase K treatment of chromatophore and spheroplast-derived vesicles and amino acid sequence determination. Under conditions of proteinase K treatment of chromatophores, which left the in vivo absorption spectrum and the membrane intact, 15 and 46 amino acyl residues from the N-terminal regions of the L and M subunits, respectively, of the reaction center polypeptides were removed. The N termini are therefore exposed on the cytoplasmic surface of the membrane. The C-terminal domain of the light-harvesting B800-850 alpha and B870 alpha polypeptides was found to be exposed on the periplasmic surface of the membrane. A total of 9 and 13 amino acyl residues were cleaved from the B800-850 alpha and B870 alpha polypeptides, respectively, when spheroplasts were treated with proteinase K. The N-terminal regions of the alpha polypeptides were not digested in either membrane preparation and were apparently protected from proteolytic attack. Seven N-terminal amino acyl residues of the B800-850 beta polypeptide were removed after the digestion of chromatophores. C-terminal residues were not removed after the digestion of chromatophores or spheroplasts. The C termini seem to be protected from protease attack by interaction with the membrane. Therefore, the N-terminal regions of the beta polypeptides are exposed on the cytoplasmic membrane surface. The C termini of the beta polypeptides are believed to point to the periplasmic space.     

Location and chemical composition of anionic sites in Bruch's membrane of the rat

The location and chemical composition of anionic sites in Bruch's membrane (BM) were examined using cationic probe molecules demonstrable in electron microscopic preparations and tissue digestion with specific degradative enzymes. Ruthenium red and native lysozyme revealed densities distributed at regular intervals in two major components of BM: the basal laminae of the retinal pigment epithelium (RPE) and choriocapillary endothelium (EN). Staining was not observed with succinylated lysozyme (anionic). Colloidal iron also failed to stain BM components. Following crude heparinase treatment at 43 degrees C (specific for heparan sulfate) anionic sites in the RPE basal lamina were not demonstrable with either ruthenium red or native lysozyme. Sites in the EN basal lamina were not affected. Chondroitinase treatment removed almost all of the ruthenium red-positive material in the EN basal lamina; lysozyme binding here was markedly reduced. No changes were observed in the RPE basal lamina after chondroitinase digestion. There was no morphological evidence for site removal by either neuraminidase or leech hyaluronidase, although a detachment of the RPE from BM often occurred after incubation of eye tissue in the latter. Pronase E removed all stainable material. These findings indicate that anionic sites in BM consist to a large extent of chondroitin sulfates and heparan sulfate.     

Poly(3-Hydroxybutyrate) Granules at the Early Stages of Formation Are Localized Close to the Cytoplasmic Membrane in Caryophanon latum

Localization of newly synthesized poly(3hydroxybutyrate) (PHB) granules was determined by confocal laser scanning fluorescence microscopy of Nile red-stained cells and by transmission electron microscopy (TEM). PHB granules of Nile red-stained living cells of Caryophanon latum at the early stages of PHB accumulation were frequently found at or close to the cytoplasmic membrane. TEM analysis of the same culture revealed electron-translucent globular structures resembling PHB granules that were nonrandomly distributed in the cell lumen but were frequently found at or close to the cytoplasmic membrane. Immunogold labeling using PHB-specific antiserum confirmed that the electron-translucent structures represented PHB granules. Electron microscopy examination of PHB granules after cell lysis revealed that PHB granules were often associated with membrane vesicles. Nonrandom localization of PHB granules was also found in Beijerinckia indica. Cells of this species harbored one PHB granule at each cell pole. Our results show that newly synthesized PHB granules often are close to or even in physical contact with the cytoplasmic membrane. Possible explanations for this unexpected finding and a hypothetical model of PHB granule formation in C. latum are discussed.