Isolation of stable ribosomal subunits from spores of Bacillus cereus.

Analyses of ribosomes extracted from spores of Bacillus cereus T by a dryspore disruption technique indicated that previously reported defects in ribosomes from spores may arise during the ribosome extraction process. The population of ribosomes from spores is shown to cotain a variable quantity of free 50S subunits which are unstable, giving rise to slowly sedimenting particles in low-Mg2+ sucrose gradients and showing extremely low activity in in vitro protein synthesis. The majority of the ribosomal subunits in spores, obtained by dissociation of 70S ribosomes and polysomes, are shown to be as stable as subunits from vegetative cells, though the activity of spore polysomes was lower than that of vegetative ribosomes. In spite of the instability and inactivity of a fraction of the spore's ribosomal subunits, the activity of the total population obtained from spores by the dry disruption technique was 32% of vegetative ribosome activity, fivefold higher than previously obtained with this species. The improvement in activity and the observed variability of subunit destabilization are taken as evidence for partial degradation of spore ribosomes during extraction.     

RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor

Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time-ordered events. RNA and protein synthesis started during the first 5 min and net DNA synthesis at 60-70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4-5-S RNA were synthesized. Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C-labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.     

Ribosomal proteins of Bacillus subtilis vegetative and sporulating cells

Summary The ribosomal subunit proteins (30S and 50S) from vegetative and sporulating cells of Bacillus subtilis 168M were analyzed by two dimensional acrylamide gel electrophoresis. Twenty two proteins were identified in the 30S subunits and 28 proteins are detectable in the 50S subunits. The number of proteins and their electrophoretic mobility seem to remain unaltered during the sporulation process.The ribosomal proteins of a thermosensitive sporulation mutant (ts-4), isolated from stationary phase cultures, under permissive (for sporulation) and non-permissive conditions, did not show any qualitative difference in either of the subunits.The 21S precursor particles derived from log phase cell ribosomes show two different proteins, in addition to those present in the 30 S subunit. It is suggested that these two proteins either disappear or are modified during the maturation process.     

Unequal accumulation of 26S and 17S RNAs in ribosomes during spore germination in Dictyostelium discoideum

Ribosome synthesis was studied in spores at the swelling stage and compared with freshly emerged and logarithmically growing vegetative amoebae. During the swelling stage of spore germination, ribosome synthesis was abnormal. Newly made ribosomes accumulated unequal amounts of 26S and 17S rRNAs. The stoichiometric ratio 26S:17S was 0.5 in swelling spores, compared with 0.9 in amoebae. The relative level of pre-rRNA persisting in the nucleus was apparently 2- to 3-fold higher in swelling spores than in amoebae. All of the known ribosomal proteins, except for a few, were made during the swelling stage and were associated with the newly made ribosomes in expected amounts. Analysis of the 2'-O-methyl ribose content in the newly made rRNAs suggest that methylation was defective in swelling spores. Compared with growing amoebae, the methyl content was 30 and 64% less in 26S and 17S RNAs from the swelling stage, respectively. It is suggested that undermethylation could be partly responsible for the differential accumulation of newly made 26S and 17S RNAs during the early stages of spore germination in Dictyostelium discoideum.     

Presence of proteins derived from the vegetative cell membrane in the dormant spore coat of Bacillus subtilis

To confirm the presence of the outer spore membrane in dormant spore coats of Bacillus subtilis, the proteins from vegetative cell membrane and dormant spore coat fractions were compared by immunoblot assay with antibodies prepared against both preparations. The spore coat fraction contained at least 11 proteins antigenically identical to those in the vegetative cell membranes. Further, the cytochemical localization of the proteins derived from vegetative cell membrane in dormant spores was examined by an immunoelectron microscopy method with a colloidal gold-immunoglobulin G complex. The colloidal gold particles were observed in the coat region and around the core region of dormant spore. These results have provided evidence that some proteins from vegetative cell membrane remain in the dormant spore coat region of B. subtilis, although it is not clear whether the outer membrane persists as an intact functional entity or not.     

Germination proteins in the inner membrane of dormant Bacillus subtilis spores colocalize in a discrete cluster

Dormant bacterial spores are extraordinarily resistant to environmental insults and are vectors of various illnesses. However, spores cannot cause disease unless they germinate and become vegetative cells. The molecular details of initiation of germination are not understood, but proteins essential in early stages of germination, such as nutrient germinant receptors (GRs) and GerD, are located in the spore inner membrane. In this study, we examine how these germination proteins are organized in dormant Bacillus subtilis spores by expressing fluorescent protein fusions that were at least partially functional and observing spores by fluorescence microscopy. We show that GRs and GerD colocalize primarily to a single cluster in dormant spores, reminiscent of the organization of chemoreceptor signalling complexes in Escherichia coli. GRs require all their subunits as well as GerD for clustering, and also require diacylglycerol addition to GerD and GRs' C protein subunits. However, different GRs cluster independently of each other, and GerD forms clusters in the absence of all the GRs. We predict that the clusters represent a functional germination unit or 'germinosome' in the spore inner membrane that is necessary for rapid and cooperative response to nutrients, as conditions known to block nutrient germination also disrupt the protein clusters.     

Permeability of dormant spores of Bacillus subtilis to gramicidin S

Abstract Gramicidin S, dissolved in ethanol, penetrated into the inside of the dormant spores of Bacillus subtilis , had a partial inhibitory effect on l-alanine-initiated germination and completely inhibited their outgrowth and vegetative growth. The activity of particulate NADH oxidase of the antibiotic-treated dormant spores was also influenced significantly. Abnormal morphological changes were observed in germinated spores from gramicidin S-treated dormant spores. An immunoelectron microscopy method with colloidal gold-IgG complex showed that the penetration site of gramicidin S inside dormant spores was mainly the core region. These facts suggest that gramicidin S induces the damage of not only the outer membrane-spore coat complex but also the inner membrane surrounding the spore protoplast, and is able to penetrate into the core region of B. subtilis dormant spores.     

Content, distribution and stability of protein-synthesis elongation factor Tu in subcellular fractions of vegetative cells and spores of Streptomyces aureofaciens

The protein synthesis elongation factor Tu (EF-Tu) was identified in dormant spores of Streptomyces aureofaciens and its content and distribution in vegetative cells and dormant spores were determined. Cell-free homogenates from spores were found to contain a EF-Tu cleaving membrane bound protease. The protease cleaved aggregated EF-Tu much less efficiently than non-aggregated factor in cell homogenates. The relative content of EF-Tu and ribosomes in dormant spores was very similar to that found in exponentially growing vegetative cells.     

Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis

Bacillus subtilis spores that germinated poorly with saturating levels of nutrient germinants, termed superdormant spores, were separated from the great majority of dormant spore populations that germinated more rapidly. These purified superdormant spores (1.5 to 3% of spore populations) germinated extremely poorly with the germinants used to isolate them but better with germinants targeting germinant receptors not activated in superdormant spore isolation although not as well as the initial dormant spores. The level of β-galactosidase from a gerA-lacZ fusion in superdormant spores isolated by germination via the GerA germinant receptor was identical to that in the initial dormant spores. Levels of the germination proteins GerD and SpoVAD were also identical in dormant and superdormant spores. However, levels of subunits of a germinant receptor or germinant receptors activated in superdormant spore isolation were 6- to 10-fold lower than those in dormant spores, while levels of subunits of germinant receptors not activated in superdormant spore isolation were only ≤ 2-fold lower. These results indicate that (i) levels of β-galactosidase from lacZ fusions to operons encoding germinant receptors may not be an accurate reflection of actual germinant receptor levels in spores and (ii) a low level of a specific germinant receptor or germinant receptors is a major cause of spore superdormancy.     

Identification of proteins involved in the peptidyl transferase activity of ribosomes by chemical modification.

Photochemical oxidation of Escherichia coli 50 S ribosomal subunits in the presence of methylene blue or Rose Bengal causes rapid loss of peptidyl transferase activity. Reconstitution experiments using mixtures of components from modified and unmodified ribosomes reveal that both RNA and proteins are affected, and that among the proteins responsible for inactivation there are both LiCl-split and core proteins. The proteins L2 and L16 from the split fraction and L4 from the core fraction of unmodified ribosomes were together nearly as effective as total unmodified proteins in restoring peptidyl transferase activity to reconstituted ribosomes when added with proteins from modified ribosomes. These three proteins are therefore the most important targets identified as responsible for loss of peptidyl transferase activity on photo-oxidation of 50 S ribosomal subunits.     

Surface topography of the Bacillus stearothermophilus ribosome.

Summary The surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more ¹²⁵I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of ¹²⁵I. Iodinated 70S ribosomes and subunits retained 62–78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were S4, S7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins S2, S3, S7, S13, Sa, L5/9, L10, L11 and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including S5, S9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared.     

Altered chloroplast ribosomal proteins in a yellow mutant of Chlamydomonas reinhardii.

Summary Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analysed and compared by two-dimensional gel electrophoresis.Mixothrophycally grown yellow-27 mutant differs from wild-type cells in lowered chlorophyll content and grana fromation of the chloroplast.Analytical ultracentrifuge analyses of cell extracts show a reduced amount of free 70S ribosomes and increased level of 50S subunits in the mutant cells. Similar results were obtained by electronmicroscopical method.Two-dimensional gel electrophoresis shows alterations in protein composition of 70S ribosomes of the mutant. Two proteins of 70S ribosomes have been altered. One of them with high molecular weight is practically absent while there is an additional, intensively stained spot in the mutant.Since the mutation is inherited in a non-Mendelian manner it is possible that the protein alterations in 70S ribosome are localized in the chloroplast DNA.     

Proteins, associated with 50S-ribosomal subunits of Escherichia coli MRE600]

The proteins associated with the ribosomal subunits having the molecular masses from 158 to 47 kDa were isolated from hyaloplasmic, nucleoid and membrane fractions of Escherichia coli MRE600 cells. The proteins are eliminated from 50S subunits of ribosomes by thrice washing with the 1 M ammonium chloride buffer. 50S subunit proteins were found to be immunologically related to the inner membrane proteins. The native 50S subunits of ribosomes possess the expressed ATP-ase activity, while the washed off subunits lose it completely.     

In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.

From the high salt wash of the ribosomes of the yeast Saccharomyces cerevisiae, three protein kinases have been isolated and separated by DEAE-cellulose chromatography. The three kinases differ in their abilities to phosphorylate substrates such as histones (calf thymus), casein, and S. cerevisiae ribosomes; two of the kinases showed increased activity in the presence of cyclic adenosine 3',5'-monophosphate when histones and 40S ribosomal subunits were used as substrates. The protein kinases catalyzed phosphorylation of certain proteins of the 40S and 60S ribosomal subunits, and 80S ribosomes in vitro. Nine proteins of the 80S ribosome, seven proteins of the 40S subunit, and eleven of the 60S subunit were phosphorylated; different proteins were modified to various extents when different kinases were used. We have identified several proteins of 40S and 60S ribosomal subunits which are not available to the kinases in the 80S particles. Ribosomes isolated from S. cerevisiae cells growing in logarithmic phase of growth were found to contain a number of phosphorylated proteins. Studies by two-dimensional polyacrylamide gel electrophoresis indicated that the ribosomal proteins phosphorylated in vivo correspond with those phosphorylated in vitro. The relationship of in vivo phsophorylation of ribosomes to the growth and physiology of S. cerevisiae is not known.     

Expression of proteins and protein kinase activity during germination of aerial spores of Streptomyces granaticolor

Dormant aerial spores of Streptomyces granaticolor contain pre-existing pool of mRNA and active ribosomes for rapid translation of proteins required for earlier steps of germination. Activated spores were labeled for 30 min with [35S]methionine/cysteine in the presence or absence of rifamycin (400 microg/ml) and resolved by two-dimensional electrophoresis. About 320 proteins were synthesized during the first 30 min of cultivation at the beginning of swelling, before the first DNA replication. Results from nine different experiments performed in the presence of rifamycin revealed 15 protein spots. Transition from dormant spores to swollen spores is not affected by the presence of rifamycin but further development of spores is stopped. To support existence of pre-existing pool of mRNA in spores, cell-free extract of spores (S30 fraction) was used for in vitro protein synthesis. These results indicate that RNA of spores possesses mRNA functionally competent and provides templates for protein synthesis. Cell-free extracts isolated from spores, activated spores, and during spore germination were further examined for in vitro protein phosphorylation. The analyses show that preparation from dormant spores catalyzes phosphorylation of only seven proteins. In the absence of phosphatase inhibitors, several proteins were partially dephosphorylated. The activation of spores leads to a reduction in phosphorylation activity. Results from in vitro phosphorylation reaction indicate that during germination phosphorylation/dephosphorylation of proteins is a complex function of developmental changes.     

Decreased activity of Blastocladiella emersonii zoospore ribosomes: correlation with developmental changes in ribosome-associated proteins

Ribosomal proteins isolated from dormant zoospores were compared to the ribosomal proteins found in the active growth phase by two-dimensional polyacrylamide gel electrophoresis. Zoospore ribosomes were found to contain a set of five proteins, designated Z1 to Z5, which were not present in growth phase ribosomes. The Z1-Z5 proteins were not removed by high-salt washes using either 1 M KCl or 1 M NH4 Cl. The Z1 protein is found associated with zoospore 60 S subunits while Z2-Z5 are bound to 40 S subunits. Zoospore monoribosomes and polyribosomes contain comparable levels of each of the five proteins. Approximately 60 min. after sporulation is induced, the Z1-Z5 proteins begin to accumulate on the ribosomes with the highest levels of these proteins found associated with ribosomes at the zoospore stage. During germination, the proteins gradually disappear and are not detectable on the ribosomes after 4 hr of germination. The presence of the Z1-Z5 proteins correlates with a decrease in in vitro protein synthetic activity of the fungal ribosomes. The data are consistent with the hypothesis that the proteins regulate translation by completely blocking protein synthesis on a subset of ribosomes while the remainder of the ribosomes function at normal rates.     

Induction of cell-specific ribosomal proteins in aggregation-competent nonmorphogenetic Dictyostelium discoideum

Vegetatively growing amoebae, if shaken in a starvation (nonnutrient) buffer, acquired aggregation competence, but do not embark on a morphogenetic program. The quantitative variation of ribosomal proteins in vegetative and aggregation-competent cells was compared by labeling the different cell types with [35S]methionine. Vegetative cells were examined at various phases of the growth cycle. No changes could be detected in the content of ribosomes or the apparent stoichiometry of ribosomal proteins in growing cells. In stationary phase cells, the net ribosome content declined to 15% of that observed in logarithmic phase, but the relative amounts of individual ribosomal proteins were not altered. Although aggregation-competent cells contained 30% less ribosomes compared with logarithmic phase cells, the total fraction of newly made ribosomal proteins was the same in both. In contrast to vegetative cells, distinct changes were induced in the ribosomal proteins of aggregation-competent cells. The composition of ribosomes in aggregation-competent phase resembled in every respect that observed in spore cells. As reported earlier, changes were found in all 12 of the developmentally regulated ribosomal proteins. For the majority of newly made ribosomal proteins during aggregation competence, the stoichiometry was similar to that in logarithmically growing cells. However, the relative synthesis of some was particularly higher (13- to 46-fold for A and L; 3- to 8-fold for D, E, S24, L3, S6, and L4) compared with logarithmic phase cells. About 18 proteins, which included the cell-specific ribosomal proteins L18, S10, S14, S16, and L11, were synthesized in lesser amounts than in logarithmic phase cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

High molecular weight ribosomal ribonucleic acid from vegetative hyphae and spores of Streptomyces griseus.

High molecular weight ribosomal ribonucleic acids (rRNAs) were isolated from young vegetative cells and spores of a streptomycin non-producing Streptomyces griseus, and their electrophoretic mobility was compared to each other and to that of rRNAs of Escherichia coli K-12. The electrophoretic mobility of 23 and 16S rRNAs from vegetative cells and spores of S. griseus was identical, but the 23S rRNAs of streptomyces ribosomes migrated more slowly on polyacrylamide gel than those of E. coli ribosomes. Intact, electrophoretically homogenous rRNAs could be isolated from S. griseus (No. 45-H) only in the presence of diethyl 1 pyrocarbonate (DEP), and intact rRNAs could be obtained from spores only if DEP had been added before breaking the spores. Otherwise instead of two distinct bands, three were obtained on polyacrylamide gel.     

Immunochemical analysis of the structure of eukaryotic ribosomes

Summary Antibodies were prepared in rabbits and sheep to rat liver ribosomes, ribosomal subunits, and to mixtures of proteins from the particles. The antisera were characterized by quantitative immunoprecipitation, by passive hemagglutination, by immunodiffusion on Ouchterlony plates, and by immunoelectrophoresis. While all the antisera contained antibodies specific for ribosomal proteins, none had precipitating antibodies against ribosomal RNA. Rat liver ribosomal proteins were more immunogenic in sheep than rabbits, and the large ribosomal subunit and its proteins were more immunogenic than those of the 40S subparticle. Antisera specific for one or the other ribosomal subunit could be prepared; thus it is unlikely that there are antigenic determinants common to the proteins of the two subunits. When ribosomes, ribosomal subunits, or mixtures of proteins were used as antigens the sera contained antibodies directed against a large number of the ribosomal proteins.Abbreviations TP total proteins—used to designate mixtures of proteins from ribosomal particles, hence TP80 is a mixtures of all the proteins from 80S ribosomes - TP60 the proteins from 60S subunits - TP40 the proteins from 40S particles