Protein composition of cell and forespore membranes of Bacillus subtilis

A sporulation mutant of Bacillus subtilis 168 was isolated and characterized. The mutant, designated SB-23, releases viable forespores at the end of the developmental period. Forespores were isolated on linear Renografin gradients and used as a source of forespore membranes. The protein composition of forespore membranes was found to differ from the protein composition of vegetative cell membranes by discgel electrophoresis. The results are discussed in relationship to morphological and physiological differentiation during bacterial sporulation.     

Levels of Small Molecules and Enzymes in the Mother Cell Compartment and the Forespore of Sporulating Bacillus megaterium

We have determined the amounts of a number of small molecules and enzymes in the mother cell compartment and the developing forespore during sporulation of Bacillus megaterium. Significant amounts of adenosine 5'-triphosphate and reduced nicotinamide adenine dinucleotide were present in the forespore compartment before accumulation of dipicolinic acid (DPA), but these compounds disappeared as DPA was accumulated. 3-Phosphoglyceric acid (3-PGA) accumulated only within the developing forespore, beginning 1 to 2 h before DPA accumulation. Throughout its development the forespore contained constant levels of enzymes of both 3-PGA synthesis (phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase) and 3-PGA utilization (phosphoglycerate mutase, enolase, and pyruvate kinase) at levels similar to those in the mother cell and the dormant spore. Despite the presence of enzymes for 3-PGA utilization, this compound was stable within isolated forespores. Two acid-soluble proteins (A and B proteins) also accumulated only in the forespore, beginning 1 to 2 h before DPA accumulation. At this time the specific protease involved in degradation of the A and B proteins during germination also appeared, but only in the forespore compartment. Nevertheless, the A and B proteins were stable within isolated forespores. Arginine and glutamic acid accumulated within the forespore in parallel with DPA accumulation. The forespore also contained the enzyme arginase at a level similar to that in the mother cell and a level of glutamic acid decarboxylase 2- to 25-fold higher than that in the mother cell, depending on when in sporulation the forespores were isolated. The specific activities of several other enzymes (protease active on hemoglobin, ornithine transcarbamylase, malate dehydrogenase, aconitase, and isocitrate dehydrogenase) in forespores were about 10% or less of the values in the mother cell. Aminopeptidase was present at similar levels in both compartments; threonine deaminase was not found in either compartment.     

Bacillus megaterium sporal peptidoglycan synthesis studied by high-resolution autoradiography

Cells of a Dap- Lys- mutant strain of Bacillus megaterium were pulse labeled with [3H]diaminopimelic acid at different times of growth and sporulation. They were processed for radioactivity measurements and high-resolution autoradiography either just after the pulse or after a chase in a nonradioactive medium until refractile forespores started to appear at time (t)4,5. In the pulse-labeled cells, autoradiographs and radioactivity measurements showed that the radioactivity incorporated during a pulse decreased abruptly after t0 and stayed at a low level until t5, although the forespore wall and cortex were formed between t4 and t5. In the pulse-chased bacteria, the acid-insoluble radioactivity, as well as the number of silver grains on autoradiographs, increased during the chase in cells labeled at t1 to t2, whereas it decreased in those labeled before t0. Furthermore, analysis of silver grain distribution showed that, in stage IV bacteria, grains were distributed at the outside of the forespore, mostly on the sporangium cell wall, when pulse-labeling occurred before or at t0; they were located along the cortex and in the forespore cytoplasm when labeling was made at t1 or t2. These facts show that [3H]diaminopimelic acid necessary for spore envelope synthesis was incorporated before their morphological appearance. Free or small diaminopimelic acid precursors entered the sporangium between t1 and t2. The appearance of silver grains in the forespore cytoplasm suggests that the forespore is implicated in sporal peptidoglycan synthesis.     

Immunological Detection of 22K Protein in Sporulating Cells of Bacillus megaterium ATCC 12872

The synthesis and deposition of 22,000-dalton (22K) spore coat protein were examined immunochemically on the sporulating cells of Bacillus megaterium ATCC 12872 using the antibody to purified 22K spore coat protein. This antibody cross-reacted with 44K and 25K proteins in immunoblot analysis of dormant spore coat proteins. Immunoblot analysis on the sporulating cells showed that 22K protein was detected from t₈ in forespore coat protein fractions. Sandwich enzyme immunoassay revealed that 22K protein in the spore coat protein fraction appeared at t₆ and reached a plateau at t₉, and 22K protein in the mother cell cytoplasmic fraction was detected at only t₇ and t₈ at a very low level.     

Location of peptidoglycan lytic enzymes in Bacillus sphaericus.

The level of three peptidoglycan hydrolases was determined in the mother cell compartment and forespores of Bacillus sphaericus. Vegetative and sporulating cells contained in LD-carboxypeptidase active only on the vegetative cell wall peptidoglycan, and we have previously shown that sporulation is accompanied by the production of two new enzymes active only on the spore cortex peptidoglycan. These gamma-D-glutamyl-meso-diaminopimelate endopeptidase and a meso-diaminopimelate-D-alanine dipeptidase. The LD-carboxypeptidase activity appeared to be located in the membranes of both the mother cells and forespores. Endopeptidase activity was located in the integument fraction of the forespores, and the dipeptidase activity was only found in the forespore cytoplasm. These different locations comply with the probable different functions of these enzymes.     

Measurement of protein synthesis in rat lungs perfused in situ

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs–Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6–690μm-[U-¹⁴C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O₂/CO₂ (19:1) or O₂/N₂/CO₂ (4:15:1)]. [U-¹⁴C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t_½, 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [¹⁴C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [¹⁴C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.     

Regulation of phosphoglycerate mutase in developing forespores and dormant spores ofBacillus megaterium by thein vivo levels of phosphoglycerate mutase inhibitor

Bacillus megaterium accumulated 3-phosphoglycerate during sporulation which was utilized during spore germination. During sporulation a protein was synthesized before or at the start of 3-phosphoglycerate accumulation inside the developing spores about 1.5 h before dipicolinic acid accumulation. This protein has an affinity for Mn²⁺ and other divalent metal ions and inhibits phosphoglycerate mutase activity which has been shown to require Mn²⁺ However, the levels of the inhibitor decreased considerably (75–85%) during spore germination. No appreciable amount of the inhibitor was detected in the vegetable cell and mother cell compartment; however, the forespore compartment possesses an activity comparable to that of dormant spores. The partially purified inhibitor has a molecular weight of 11,000 and possesses both high and low affinity binding sites for Mn²⁺ and Ca²⁺ as determined by Scatchard plot analysis.     

Calcium accumulation during sporulation of Bacillus megaterium KM.

Accumulation of Ca2+ in Bacilli occurs during stages IV to VI of sporulation. Ca2+ uptake into the sporangium was investigated in Bacillus megaterium KM in protoplasts prepared in stage III of sporulation and cultured to continue sporulation. These protoplasts and whole cells exhibit essentially identical Ca2+ uptake, which is compared with that of forespores isolated in stage V of sporulation. Ca2+, uptake into both sporangial protoplasts and isolated forespores occurs by Ca2+-specific carrier-mediated processes. However, protoplasts exhibit a Km value of 31 micrometer, and forespores have a Km value of 2.1 mM. Sporangial protoplasts accumulate Ca2+ against a concentration gradient. In contrast, Ca2+ uptake into isolated forespores is consistent with downhill transfer in which both rate and extent of uptake are affected by the external Ca2+ concontration. Dipicolinic acid has no effect on Ca2+ uptake by isolated forespores, apart from decreasing the external Ca2+ concentration by chelation. A model for sporulation-specific Ca2+ accumulation is proposed, in which Ca2+ is transported into the sporangium, resulting in a concentration of 3--9 mM in the mother-cell cytoplasm. This high concentration of Ca2+ enables carrier-mediated transfer down a concentration gradient into the forespore compartment, where a low free Ca2+ concentration is maintained by complexing with dipicolinic acid.     

Protein synthesis and breakdown in the mother-cell and forespore compartments during spore morphogenesis in Bacillus megaterium

Recently developed techniques for isolating forespores from bacilli at all stages of spore morphogenesis have been exploited to investigate the contribution of each of the two compartments of the sporulating cell to the overall pattern of protein synthesis and degradation during sporulation in Bacillus megaterium. These studies have shown: (1) that protein synthesis continues in both compartments throughout spore morphogenesis; (2) that the degradation of proteins made at all times during vegetative growth and sporulation is confined to the mother-cell compartment; (3) that proteins synthesized in the mother-cell compartment during sporulation are subsequently degraded more rapidly than proteins synthesized during vegetative growth. This rate of degradation increases the later the proteins are synthesized in the sporulation sequence. Mature spores were disrupted, and the percentage of the total protein in soluble and particulate fractions was determined. Pulse-labelling experiments were performed to investigate the extent to which the proteins of these two fractions are newly synthesized during sporulation. These data were used to calculate the extent of capture of vegetative cell protein at the time of formation of the forespore septum. The value obtained is consistent with evidence from electron micrographs and supports a model for the origin of spore protein in which there is no protein turnover in the developing forespore.     

Spatiotemporally regulated proteolysis to dissect the role of vegetative proteins during Bacillus subtilis sporulation: cell‐specific requirement of σH and σA

Sporulation in Bacillus subtilis is a paradigm of bacterial development, which involves the interaction between a larger mother cell and a smaller forespore. The mother cell and the forespore activate different genetic programs, leading to the production of sporulation‐specific proteins. A critical gap in our understanding of sporulation is how vegetative proteins, made before sporulation initiation, contribute to spore formation. Here we present a system, spatiotemporally regulated proteolysis (STRP), which enables the rapid, developmentally regulated degradation of target proteins, thereby providing a suitable method to dissect the cell‐ and developmental stage‐specific role of vegetative proteins. STRP has been used to dissect the role of two major vegetative sigma factors, σ^H and σ^A, during sporulation. The results suggest that σ^H is only required in predivisional cells, where it is essential for sporulation initiation, but that it is dispensable during subsequent steps of spore formation. However, evidence has been provided that σ^A plays different roles in the mother cell, where it replenishes housekeeping functions, and in the forespore, where it plays an unexpected role in promoting spore germination and outgrowth. Altogether, the results demonstrate that STRP has the potential to provide a comprehensive molecular dissection of every stage of sporulation, germination and outgrowth.     

Cell viability and protein turnover in nongrowingBacillus megaterium at sporulation suppressing temperature

InBacillus megaterium, a temperature that suppresses sporulation (43°C) only slightly exceeds both the optimum growth temperature and the temperature still permitting sporulation (40–41°C). Here we show that, when cells grown at 35°C and transferred to a sporulation medium, were subjected to shifts between 35°C and the sporulation suppressing temperature (SST, 43°C), their development and proteolytic activities were deeply affected. During the reversible sporulation phase that took place at 35°C for 2–3 h (T₂–T₃), the cells developed forespores and their protein turnover was characterized by degradation of short-lived proteins and proteins made accessible to the proteolytic attack because of starvation. During the following irreversible sporulation phase refractile heat-resistant spores appeared at T₄–T₅. Protein turnover rate increased again after T₂ and up to T₈ 60–70% prelabelled proteins were degraded. The SST suppressed sporulation at its beginning; at T₃ no asymmetric septa were observed and the amount of heat-resistant spores at T₈ was by 4–5 orders lower than at 35°C. However, the cells remained viable and were able to sporulate when transferred to a lower temperature. Protein degradation was increased up to T₃ but then its velocity sharply dropped and the amount of degraded protein at T₈ corresponded to slightly more than one-half of that found at 35°C. The cytoplasmic proteolytic activity was enhanced but the activity in the membrane fraction was decreased. When a temperature shift to SST was applied at the beginning of the irreversible sporulation phase (T_2.5), the sporulation process was impaired. A portion of forespores lyzed, the others were able to complete their development but most spores were not heat-resistant and their coats showed defects. Protein degradation increased again because an effective proteolytic system was developed during the reversible sporulation phase but the amount of degraded protein was slightly lower than at 35°C. A later (T₄) shift to SST had no effect on the sporulation process.     

THE CELL-FREE SYNTHESIS OF ALKALOID-BINDING PROTEINS IN IMMATURE RAT BRAIN1

Abstract— cell-free amino acid incorporating system from immature rat brain, consisting of ribosomal and soluble fractions, has been investigated for its capacity to incorporate [¹⁴C]amino acids into specific soluble proteins that interact with vinblastine sulfate and colchicine. The soluble ¹⁴C-labeled proteins formed in the cell-free system during incubation were compared with similar soluble proteins from immature rat brain which had been labeled in vivo by the incorporation of ¹⁴C-labeled amino acids. Criteria for the formation of vinblastine-binding, ¹⁴C-labeled proteins were: (1) aggregation of ¹⁴C-labeled soluble protein by one mm -vinblastine sulfate and (2) immunoprecipitation of ¹⁴C-labeled soluble protein by an antiserum against vinblastine sulfate-precipitable material. Criteria for the formation of [³H]colchicine-binding, ¹⁴C-labeled protein were based upon: (1) co-precipitation of the ³H-and ¹⁴C-labeled materials by vinblastine sulfate and (2) the coincidence of ³H- and ¹⁴C-labeled elution peaks from columns of Sephadex G-200, DEAE-Sephadex A-50 and isoelectric focusing. Both in the in vitro and in the in vivo system, ¹⁴C-labeled amino acids were incorporated into soluble proteins of the post-microsomal supernatant fraction. Proteins labeled with ¹⁴C-labeled amino acids in vitro and in vivo yielded comparable and qualitatively identical results by the criteria tested, including the formation of immunoprecipitates. In the in vitro system, ¹⁴C-labeled amino acids were incorporated into protein with a molecular weight of approx 120,000, an isoelectric point of 5.3 and with a chromatographic mobility on Sephadex G-200 which is identical to [³H]colchicine-binding protein. The above experimental results are presumptive evidence for the synthesis of vinblastine-binding and colchicine-binding proteins in the in vitro cell-free system.     

The DnaA inhibitor SirA acts in the same pathway as Soj (ParA) to facilitate oriC segregation during Bacillus subtilis sporulation

DNA replication and chromosome segregation must be carefully regulated to ensure reproductive success. During Bacillus subtilis sporulation, chromosome copy number is reduced to two, and cells divide asymmetrically to produce the future spore (forespore) compartment. For successful sporulation, oriC must be captured in the forespore. New rounds of DNA replication are prevented in part by SirA, a protein that utilizes residues in its N‐terminus to directly target Domain I of the bacterial initiator, DnaA. Using a quantitative forespore chromosome organization assay, we show that SirA also acts in the same pathway as another DnaA regulator, Soj, to promote oriC capture in the forespore. By analyzing loss‐of‐function variants of both SirA and DnaA, we observe that SirA's ability to inhibit DNA replication can be genetically separated from its role in oriC capture. In addition, we identify substitutions near the C‐terminus of SirA and in DnaA Domain III that enhance interaction between the two proteins. One such variant, SirA_P141T, remained functional in regard to inhibiting replication, but was unable to support oriC capture. Collectively, our results support a model in which SirA targets DnaA Domain I to inhibit DNA replication, and DnaA Domain III to facilitate Soj‐dependent oriC capture in the forespore.     

INCORPORATION OF AXONALLY TRANSPORTED SUBSTANCES INTO MYELIN LIPIDS

Abstract— The possibility that axonally transported lipids and/or proteins might undergo transaxonal migration and become incorporated into surrounding myelin lamellae was studied by isolating myelin from optic tracts of myelinating rabbits at various times following intraocular injection of [3-¹⁴C]-serine and [2-³H]glycerol. Myelin isolated by a procedure employing ethylene glycol-bis(β-aminoethyl ether)-.N,N'-tetraacetic acid had relatively constant specific radioactivity with respect to both isotopes over a 21 day period. Myelin lipids showed a gradual increase in ¹⁴C specific radioactivity, attributed to reutilization of [¹⁴C]serine from the axon by a compartment of the oligodendrocyte. Free serine is postulated to arise in the axon from catabolism of axonally transported proteins (and possibly lipids) and to migrate transaxonally into the neighboring oligodendroglia. This reutilization mechanism resulted in synthesis of myelin cerebrosides, sphingomyelin, ethanolamine phosphoglycerides and possibly sulfatides, but not gangliosides or serine phosphoglycerides. The data for choline- and inositol-phosphoglycerides are inconclusive. [³H]Glycerol-labeled myelin lipids decreased slowly in ³H specific radioactivity with time, indicating either that [2-³H]glycerol does not participate in the reutilization pathway or that the label is lost in the process. Evidence is presented that ³H- and ¹⁴C-labeled lipids are true myelin constituents. Lipids from the myelin, axolemma- and axon-enriched fractions tended to converge in specific radioactivity over the 21 days, especially the former two fractions. These results together with isotope ratio changes point to an equilibration process whereby lipids are able to transfer. (or exchange) between the 3 compartments. Protein radioactivity in isolated myelin was suggested to arise from residual axon/axolemma contamination, and no evidence was found for transaxonal migration of protein into myelin. The 2 mechanisms elucidated here are believed to account for a quantitatively small portion of myelin lipid and are considered to represent a form of axon-glia interaction.     

Studies on Sporopollenin Biosynthesis in Tulipa Anthers

Investigations were carried out to clarify sporopollenin biosynthesis. Tracer experiments were focussed on the incorporation of specifically labeled ¹⁴C-phenylalanine into sporopollenin. In addition, the incorporation of further ¹⁴C-labeled substances, such as glucose, acetate, malonic acid, mevalonate and tyrosine, was investigated. The sporopollenin fraction was isolated and purified by a gentle method including extractions by different solvents, incubations with hydrolyzing enzymes and fractionated saponifications. During the purification procedure the whereabouts of the initially applied radioactivity was followed. After each step the remaining as well as the released radioactivity was determined. Saponification of samples labeled after application of phenylalanine yielded p-coumaric acid and p-coumaric acid methyl ester as labeled products. In comparison with the other substances applied, the highest incorporation rates were obtained with phenylalanine, regardless of the position of labeling. After degradation of the sporopollenin sample labeled with ring-¹⁴C-phenylalanine, p-hydroxybenzoic acid was detected as the main labeled product. These results unequivocally show that an integral incorporation of the aromatic ring system occurred. Tracer experiments were carried out at different stages of development. Their results show that, although the incorporation rates of ¹⁴C-phenylalanine into sporopollenin differ, the substantial incorporation of this substance is not bound to defined stages of development.     

Phytochemical Studies on the Tobacco Alkaloids

Duplicate feeding experiments of dl-ornithine-2-¹⁴C to the excised tobacco root culture were made, and the radioactive nornicotine was isolated. Approximately two thirds of the radioactivity was located in the 2-position of the pyrrolidine of the nornicotine in these experiments. This fact indicates that there are two modes in nornicotine biosynthesis: exclusive incorporation to the C-2 and equal incorporation to C-2 and C-5 from C-2 of ornithine.

On the basis of this finding, biosynthetic route was discussed.

dl-Ornithine-2-¹⁴C, dl-methionine-¹⁴CH₃ and partially racemized l-nornicotine-2,5-¹⁴C were administered to aseptically grown excised roots (N. rustica var. Brasilia). Incorporation of their radioactivity to nicotine was compared. The extent of their radioactive incorporation to nicotine was high in the order of ornithine, methionine and nornicotine; incorporation of radioactivity of nornicotine to nicotine was extraordinarily low. ¹⁵N-Labeled nornicotine was also fed to the same materials and ¹⁵N distribution was examined. Most of ¹⁵N still remained in the nornicotine reisolated. Marked amounts of ¹⁵N were located in the ethanol-insoluble fraction, the amino acid fraction and the substances having chromatographic R_F value close to that of nicotine. Only small amount of ¹⁵N was incorporated to the isolated nicotine.

Nornicotine is generally accepted to be a direct precursor of nicotine in tobacco plants. From these findings, however, it can be said that the biosynthesis of nicotine can occur through other routes without going through nornicotine.     

Biosynthesis of Phenylalanine from Phenylacetate by Chromatium and Rhodospirillum rubrum

Cultures of Chromatium strain D and Rhodospirillum rubrum incorporated ¹⁴C from phenylacetate-1-¹⁴C during anaerobic growth. The radioactivity in the protein fraction of cells was mainly in phenylalanine. Phenylalanine from Chromatium cells grown in phenylacetate-1-¹⁴C was labeled at carbon 2. Incorporation of phenylacetate by Chromatium was decreased in the presence of exogenous phenylalanine, and de novo synthesis of phenylalanine from bicarbonate was less in medium containing either phenylalanine or phenylacetate. These organisms, and also certain anaerobic rumen bacteria, apparently carboxylate phenylacetate to synthesize the phenylalanine carbon skeleton. The mechanism of the carboxylation is unknown; however, it appears to be dependent upon anaerobic conditions, since R. rubrum did not synthesize phenylalanine from phenylacetate during aerobic growth in the dark.     

Polypeptide Synthesis with Microsomes from Pressure-Treated Tetrahymena1

SYNOPSIS High hydrostatic pressure is known to interfere with mitosis, cytokinesis and synthesis of DNA, RNA and protein. In Tetrahymena, incorporation of phenylalanine and formation of polysomes are known to be pressure-sensitive. Microsomal preparations from Tetrahymena pyriformis GL can incorporate [¹⁴C]-phenylalanine into polypeptides. Incorporation was enhanced by addition of supernatant fraction and 14.5 mM Mg⁺⁺ An energy-generating system and exogenous messenger (poly U) were essential for polypeptide biosynthesis, Microsomes from pressurized cells (14,000 psi for 5 min) incorporated [¹⁴C]phenylalanine as efficiently as control microsomes. Microsomal function was not grossly damaged by pressure in a test system containing exogenous messenger, crude microsomal preparation, exogenous energy-generating system and supernatant fraction containing activating enzymes.     

Studies on the biosynthesis of coenzyme F420 in methanogenic bacteria

Coenzyme F₄₂₀ is a 8-hydroxy-5-deazaflavin present in methanogenic bacteria. We have investigated whether the pyrimidine ring of the deazaflavin originates from guanine as in flavin biosynthesis, in which the pyrimidine ring of guanine is conserved. For this purpose the incorporation of [2-¹⁴C]guanine and of [8-¹⁴C]guanine into F₄₂₀ by growing cultures of Methanobacterium thermoautotrophicum was studied. Only in the case of [2-¹⁴C]guanine did F₄₂₀ become labeled. The specific radioactivity of the deazaflavin and of guanine isolated from nucleic acids of [2-¹⁴C]guanine grown cells were identical. This finding suggests that the pyrimidine ring of the deazaflavin and of flavins are synthesized by the same pathway.F₄₂₀ did not become labeled when M. thermoautotrophicum was grown in the presence of methyl-[¹⁴C] methionine, [U-¹⁴C]phenylalanine or [U-¹⁴C]tyrosine. This excludes that C-5 of the deazaflavin is derived from the methyl group of methionine and that the benzene ring comes from phenylalanine or tyrosine.