Putative structure and functions of a poly-beta-hydroxybutyrate/calcium polyphosphate channel in bacterial plasma membranes.

A poly-beta-hydroxybutyrate complex extracted from the plasma membranes of genetically competent Escherichia coli contained polyhydroxybutyrate:polyphosphate:calcium in molar ratios approximating 1:1:0.5. The chain length of the polyhydroxybutyrate was estimated as 120-200 subunits, and that of the polyphosphate was estimated as 130-170 subunits. The extracted complex, when incorporated into liposomes, exhibited a lipid phase transition in the same temperature range as that of the membrane complex in whole cells as well as the same properties of irreversibility, lability, and sensitivity to chelating buffers. Space-filling molecular models and molecular energy minimization methods (Charmm) were used to develop and evaluate a plausible structure for the complex. It is proposed that the polyhydroxybutyrate forms an exolipophilic-endopolarophilic helix around an inner framework helix of calcium polyphosphate. The calcium ions link the two polymers by forming ionic bonds with phosphoryl oxygens of the polyphosphate and ion-dipole bonds with the ester carbonyl oxygens of the polyhydroxybutyrate. This symmetrical structure forms a channel through the membrane and may play a role in the transport of calcium, phosphate, and DNA.

Transport of D- and L-tryptophan in Bacillus megaterium by an inducible permease.

Tryptophan-grown cells of Bacillus megaterium ATCC 19213 contain a permease system that transports both D- and L-tryptophan and is inhibited by sodium azide. Arginine-grown cells contain little tryptophan permease activity, suggesting that the system is inducible. Arginine represses the tryptophan permease as well as the transport system for leucine and phenylalanine. Kynurenine was a more effective inducer of the tryptophan transport system than either D- or L-tryptophan.

Tryptophan catabolism in Bacillus megaterium.

Bacillus megaterium grows in a medium containing L-tryptophan as the sole carbon, nitrogen, and energy source. Kynurenine, anthranilic acid, and catechol are metabolic intermediates, suggesting that this organism used the anthranilic acid pathway for tryptophan degradation. Cells that grow on L-tryptophan oxidize kynurenine, alanine, and anthranilic acid and the presence of tryptophan oxygenase (EC 1.13.1.12), kynureninase (EC 3.7.1.3), and catechol oxygenase (EC 1.13.1.1) in cell extracts provide additional evidence for the degradative pathway in B. megaterium. Tryptophan oxygenase is inhibited by sodium azide, potassium cyanide, and hydroxylamine, indicating that the enzyme has a functional heme group. D-Tryptophan is not a substrate for tryptophan oxygenase, and the D-isomer does not inhibit this enzyme. Formamidase (EC 3.5.1.9) and anthranilate hydroxylase are not detectable in extracts. Tryptophan catabolism is inducible in B megaterium and is subject to catabolite repression by glucose and glutamate. Arginine does not cause repression, and kynurenine induces both tryptophan oxygenase and kynureninase.

Physiological studies of a temperature-sensitive sporulation mutant of Bacillus cereus T.

Growth of temperature-sensitive mutant Bacillus cereus T JS22-C occurred normally at the restrictive temperature (37 degrees C), but sporulation was blocked at stage 0. The production of extracellular and intracellular proteases and of alkaline phosphatase occurred at 37 degrees C, but the expression of a functional tricarboxylic acid cycle did not. At the permissive temperature (26 degrees C), the mutant sporulated at a slightly lower frequency (60%) and at a lower rate than the parent strain. The oxidation of organic acids, which accumulate in the growth medium began at T0 in cultures of the parent strain but was delayed until about T3 in cultures of the mutant. Later events in sporulation were also delayed in the mutant by about 3 h. Experiments in which the temperature of growth was shifted from 37 to 26 degrees C or from 26 to 37 degrees C at various times showed that the temperature-sensitive event began approximately 1 h after the end of exponential growth and ended when the cells reached the end of stage II (septum formation). The absence of a functional tricarboxylic acid cycle in cells of the mutant grown at 37 degrees C or shifted from 26 to 37 degrees C before T1 did not appear to be due to a lesion in one of the structural genes of the tricarboxylic acid cycle but was more likely due to the inability of the cells to derepress the synthesis of some of the enzymes of that cycle.

Novel lipid components of the Azotobacter vinelandii cyst membrane.

Phospholipids are ubiquitous components of biological membranes. In the vegetative cells of Azotobacter vinelandii, a Gram-negative free-living aerobic soil bacterium, the membrane lipids are phospholipids with polar head group and fatty acyl compositions similar to those of Escherichia coli. We report here that when A. vinelandii differentiates to form metabolically dormant cysts, the phospholipids in the membranes are replaced by a family of 5-n-alkylresorcinols and 6-n-alkylpyrones. These novel amphiphilic lipids form a unique membrane matrix which may contribute to the physiology and desiccation resistance of the cyst.

Unique lipids in Azotobacter vinelandii cysts: synthesis, distribution, and fate during germination.

Unique lipids found in Azotobacter vinelandii cysts are derived primarily from beta-hydroxybutyrate used to induce encystment. Tracer studies with beta-[14C]hydroxybutyrate showed that the biosynthesis of these compounds during encystment began at 8 to 12 h after induction and reached maximal levels after 2 days, Seventy percent of these unique lipids were found in the central body of the cysts, and 23% were found in the exine. Pyronic compounds, which are located mostly in the central body, were degraded during germination of the cysts, but little change occurred in the phenolic compounds, which are more uniformly distributed in the cysts.

Lipid metabolism during encystment of Azotobacter vinelandii.

The formation of cysts by Azotobacter vinelandii involves the synthesis of lipids as major metabolic products. Cells which encyst at low levels in aging glucose cultures undergo the same pattern of lipid synthesis as cells which undergo reasonably synchronous encystment in beta-hydroxybutyrate or n-butanol. The accumulation of poly-beta-hydroxybutyrate (PHB) precedes the synthesis of 5-n-heneicosylresorcinol and 5-n-tricosylresorcinol (AR1), which is then followed in about 6 h by the synthesis of the 5-n-alkylresorcinol galactosides (AR2). In the mature cyst, PHB, AR1, and AR2 account for 8, 5.6, and 4.5%, respectively, of the dry weight. Phospholipid formation levels off 4 h postinduction, which coincides with the final cell division, but fatty acids synthesis continues at a very low level throughout encystment, suggesting some turnover of fatty acid. Distribution studies show that AR1 and AR2 are found in roughly equal amounts in the exine and central body of the cysts, with only trace amounts recovered from the intine. Studies of cysts labeled during encystment with [14C]beta-hydroxybutyrate or during vegetative growth with [14C]glucose suggest that the exine structure is synthesized during encystment, but that the intine is composed largely of vegetative cell components.

D-(-)-poly-beta-hydroxybutyrate in membranes of genetically competent bacteria.

D-(-)-Poly-beta-hydroxybutyrate is a constituent of the membranes and the cytoplasms of genetically competent Azotobacter vinelandii, Bacillus subtilis, and Haemophilus influenzae cells. Within each species the concentration of D-(-)-poly-beta-hydroxybutyrate in the membranes and cytoplasm correlates with transformability. Fluorescence analysis of the thermotropic lipid phase transitions in A. vinelandii and B. subtilis cells indicates that D-(-)-poly-beta-hydroxybutyrate forms an organized gel structure in the membranes which is very labile. The concentration of organized D-(-)-poly-beta-hydroxybutyrate in the membranes, which can be estimated from the intensity of its phase transition, can be used to assess the competence of a culture.

Production of bacteriophage by temperature-sensitive sporulation mutants of Bacillus cereus T.

Five temperature-sensitive sporulation mutants of Bacillus cereus T have been isolated. These mutants are blocked at stage 0 of sporulation at the restrictive temperature (37 C) but are able to sporulate at nearly normal frequencies at the permissive temperature (26 C). A bacteriophage that forms a stable lysogen in the parent strain is induced at increased frequencies in the mutants. This induction is accompanied, in some of the mutants, by a reduction in immunity to the phage. Revertants, selected for their ability to sporulate normally at both temperatures, lose their ability to produce high titers of the phage. In addition to this lytic phage, an apparently defective phage has been found in lysates of the mutants. Strains cured of the plaque-forming phage still carry the defective phage. Comparisons of physical and biological properties of the plaque-forming phage with those of the two Bacillus cereus phages most similar to it have shown that this phage is not identical to either of them. The maximal titer of phage produced in cultures of the parent strain is about 10(3) plaque-forming units (PFU) per ml at both temperatures. The maximal titers of phage produced by the mutant are 4 x 10(9) PFU/ml at 37 C and 7 x 10(8) PFU/ml at 26 C. Both mutant and parent strains release over 90% of the phage they produce after the onset of stationary phase.

Purification of the extracellular protease of Bacillus licheniformis and its inhibition by bacitracin.

Sporulating cells of Bacillus licheniformis excrete three seryl proteases that are of similar size, 28,000 daltons, but of different charge at pH 6. The peptide antibiotic bactracin is released from the cells at the same time and exists, in part, as a bacitracin-protease complex that is stable throughout chromatographic procedures employed in enzyme purification. However, preextraction of crude protease with CHCl3 and subsequent gel filtration effect separation of the antibiotic and the enzyme. Three purified, bacitracin-free proteases, designated CMC I, CMC II, and CMC III and whose ratios of total activity are 1:3.7:10.3, respectively, are obtained by chromatography on carboxymethyl cellulose. The major component, CMC III, is inhibited by commercial bacitracin at near-physiological concentrations of the antibiotic.

In vitro production of bacitracin by proteolysis of vegetative Bacillus licheniformis cell protein.

The action of a sporulation-specific seryl protease on antibiotic-free extracts of Bacillus licheniformis cells yields a peptide that is identified as bacitracin by its biological activity, its spectral properties, and its comigration with genuine bacitracin in both paper and thin-layer chromatography. During proteolysis, a chemical structure is generated with the spectral properties of a delta-2 thiazoline ring. The yield in vitro, 4 microgram of bacitracin per mg of protein, is less than the maximal yield from sporulating cells, 75 microgram of bacitracin per mg of cell protein, but is a linear function of the amount of protein in the reaction system. Approximately 30% of the protein yielding the antibiotic is ribosomal associated, and only 25% of that amount can be removed by washing with 1 M NH4Cl. The substrate protein is a constant fraction of the cell protein throughout exponential growth and very early sporulation stages of culture development.

Preparation and ultrastructure of the outer coats of Azotobacter vinelandii cysts.

Cysts of Azotobacter vinelandii were ruptured with 3.0 mm ethylenediaminetetraacetate in 0.05 m tris(hydroxymethyl)aminomethane buffer (pH 7.8) and fractionated by differential centrifugation in density gradients of sucrose or Ficoll (polysucrose) at 10,000 x g for 2 hr at 4 C. The average densities of the cyst exine, the cyst central body, the vegetative cell, and the intact cyst were found to be 1.13, 1.26, 1.28, and 1.31 g/cm(3) in sucrose, and 1.08, 1.12, 1.13, and 1.17 g/cm(3) in Ficoll, respectively. These data were utilized to devise procedures for the isolation of cyst components. Cyst exine appeared as a multilayered structure in ultrathin sections. This same sheetlike structure was seen when exines were subjected to detergent treatment, metal-shadowed, and examined in an electron microscope. The exine was lysed by trypsin and was resistant to lysozyme, indicating that protein may be the principal structural material of the outer cyst coat.

Encystment and polymer production by Azotobacter vinelandii in the presence of beta-hydroxybutyrate.

Cells of Azotobacter vinelandii encysted in Burk's nitrogen-free liquid media which had been supplemented with n-butyl alcohol, beta-hydroxybutyrate, or crotonate. Butyraldehyde and butyrate did not influence the extent of encystment. In the absence of glucose, beta-hydroxybutyrate enhanced the rate and extent of encystment. In the presence of glucose, it promoted abortive encystment, which was manifested by the disorganization of the exine and the release of a highly viscous material into the medium. The soluble, viscous polymer was separated from the medium by a series of ethyl alcohol precipitations and identified as a mucopeptide. It was cleaved by treatment with lysozyme and lysostaphin with a concomitant increase in reducing power. It contained 13.9% N; 56% amino acids, as alanine (alanine, lysine, and glutamic acids); and 42% hexosamines. The polymer appeared to be similar to a noncross-linked peptidoglycan.

Chemical composition of Azotobacter vinelandii cysts.

Cysts of Azotobacter vinelandii ATCC 12837 were germinated by exposure to 3.0 mm ethylenediaminetetraacetic acid (EDTA)-tris(hydroxymethyl)aminomethane buffer at pH 7.8, and their outer coats (exines) were purified by differential and isopycnic centrifugation. Electron micrographs of exine showed it to consist of multilayers of a three-membered sheet structure whose thickness was 7.0 to 7.5 nm. The inner, less electron-dense layer (intine) was also prepared from cysts by EDTA treatment, centrifugation, concentration, and dialysis. The exine consisted of 32% carbohydrate, 28% protein, 30% lipid, and 3.2% ash, with the ash comprised of 1.62% calcium, 0.02% magnesium, and 0.34% phosphorus. The amino acid composition of exine was similar to that of gram-negative bacterial cell walls. The intine consisted of 44% carbohydrate, 9.1% protein, 37% lipid, and 4.1% ash, with the ash comprised of 2.45% calcium, 0.02% magnesium, and 0.38% phosphorus. The carbohydrates of both exine and intine contained glucose, mannose, xylose, and rhamnose. Glucosamine and galactosamine were found only in the exines. The fatty acids consisted of normal, iso, and anteiso saturated fatty acids with 10 to 18 carbon atoms and mono-unsaturated C(11), C(16), and C(18) fatty acids. The exines contained mostly bound lipid, but intines contained primarily free lipid.

Morphogenesis of cysts in Azotobacter vinelandii.

Cultures of Azotobacter vinelandii were induced to encystment with beta-hydroxybutyrate. The morphological events in the transition from cell to cyst were observed by electron microscopy of thin sections. Upon induction of encystment, cells became rounded and nonmotile. The exine coat developed by the continuous excretion of membranous components into the capsule surrounding the cell.

5-n-Alkylresorcinols from encysting Azotobacter vinelandii: isolation and characterization.

Azotobacter vinelandii was found to form novel lipid compounds when encystment was initiated by 0.2% beta-hydroxybutyrate. An examination of these compounds led to the isolation and characterization of 5-n-heneicosylresorcinol, 5-n-tricosylresorcinol, and their galactoside derivatives.

Properties of a thermosensitive asporogenous filamentous mutant of Bacillus megaterium.

Mutant TH14 of Bacillus megaterium ATCC 19213 is thermosensitive and defective in cell-division septation and spore formation at the restrictive temperature (39 C). As a consequence, the mutant forms multinucleate aseptate filaments and is asporogenic. The mutation does not result in any qualitative compositional changes in extractable membrane proteins. At the restrictive temperature, the mutant membrane has a reduced content of a small molecular weight protein(s). A membrane protein(s) with a molecular weight of nearly 80,000 appears to be partially derepressed in the mutant grown at the restrictive temperature. In addition, numerous unidentified spherical inclusions of fairly uniform size (diameter approximately 100 nm) are present in the cytoplasm at the restrictive temperature. They are especially concentrated at only one pole of each filament. Filamentous growth of the mutant is less sensitive to penicillin than growth in the rod form. Growth in either form is equally sensitive to d-cycloserine at the concentrations used for selection of the mutant. Temperature shift-up experiments suggest that one to two rounds of deoxyribonucleic acid (DNA) replication occur before the phenotypic expression of the mutation occurs. The septations after these replication events can be either two-division septations or a single-division septation plus a subsequent sporulation septation. This conclusion, coupled with previously reported work, supports the hypothesis that the early stages of sporulation represent a modified cell division.

Sequential metabolic events during encystment of Azobacter vinelandii.

Cells of Azotobacter vinelandii are specifically induced to encyst by beta-hydroxybutyrate (BHB). The process of differentiation, which occurs over a period of 36 h, was characterized by an ordered sequence of biochemical events. Upon initiation of encystment, nitrogen fixation and glucose-6-phosphate dehydrogenase activities decreased immediately to very low levels. This was followed by an increase in the specific activities of BHB dehydrogenase, isocitrate dehydrogenase, isocitrate lyase, and malate synthase first at 3 h and then again at 21 h. The peak activity of fructose 1,6-diphosphate aldolase occurred at 6 h, and the enzyme activity then decreased gradually. Fructose 1,6-diphosphatase had peak activities at 9 and 27 h. Deoxyribonucleic acid synthesis ceased just prior to the final cell division at 4 to 6 h, but ribonucleic acid synthesis continued until the 12th h. From labeling studies and the appearance of new enzyme activities, it appeared that protein synthesis continued throughout encystment.

Germination of Azotobacter vinelandii cysts: sequence of macromolecular synthesis and nitrogen fixation.

Azotobacter vinelandii cysts undergo conversion to vegetative cells in Burk's nitrogen-free medium utilizing glucose, sucrose, or acetate. In 1% glucose, this overall process was complete in 8 hr and consisted of a germination and an outgrowth phase. Respiration, ribonucleic acid, and protein synthesis began soon after the addition of the germinant, and these processes proceeded at rates characteristic of the germination. The rates of respiration and synthesis increased sharply between 4 and 5 hr, the beginning of the outgrowth, at which time deoxyribonucleic acid synthesis and nitrogen fixation began. Respiration, macromolecular synthesis and nitrogen fixation continued at high rates until the emergence of vegetative cells from the cyst coats.

Physiological factors affecting transformation of Azotobacter vinelandii.

Cells of Azotobacter vinelandii (ATCC 12837) can be transformed by exogenous deoxyribonucleic acid towards the end of exponential growth. Transformation occurs at very low frequencies when the deoxyribonucleic acid is purified or when the transformation is carried out in liquid medium. Optimal transformation occurs on plates of Burk nitrogen-free glucose medium containing either high phosphate (10 mM) or low calcium (0 to 0.29 mM) content. Higher levels of calcium are inhibitory, whereas magnesium ions are essential for transformation and growth. Extracellular polymer and capsule are increasingly inhibitory to transformation and are most abundant when the calcium content of the medium is high. Transformation is optimal at pH 7.0 to 7.1 and at 30 C, conditions which also coincide with minimal extracellular polymer production. Nonencapsulated strains are excellent transformation recipients. Glycine-induced pleomorphism reduces the transformation frequency and the degree of inhibition is dependent on the phosphate concentration of the medium. Rifampin resistance and shifts from adenine, hypoxanthine, uracil, and nitrogenase auxotrophy to prototrophy can be achieved. Although single marker transfer is always greater than double marker transfer, the data suggest that rifampin resistance is linked to hypoxanthine, adenine and uracil protorophy at intervals of increasing distance. Rifampin resistance did not appear to be linked to nitrogenase.