Extracts Of Vegetative Cells Research Articles

High stringency evaluation of the inactivation / exclusion efficacy of a MALDI-TOF MS chemical extraction method, with filtration of extract through 0.1 µm filters, on Bacillus anthracis Vollum vegetative cells and spores.

A previous report indicated that a formic acid chemical extraction method for the preparation of protein extracts for matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) identification, with filtration of extracts through 0.2 μm regenerated cellulose (RC) filters, would not reliably inactivate or exclude Bacillus anthracis Vollum cells or spores when tested under high stringency conditions. B. anthracis was recovered from 13/36 extracts (3/18 from vegetative cell extracts and 10/18 from bacterial spore extracts). In this paper we report the repetition of this study but with the substitution of the 0.2 μm, regenerated cellulose, filters with 0.1 μm polyvinylidene fluoride (PVDF) filters. Experiments were conducted under the same high stringency post-treatment viability test methods (100% of resulting protein content; 7 days Luria (L)-broth and a further 7 days L-agar plate incubation; or 7 days L-agar plate only incubation). B. anthracis was not recovered from any of 18 replicates generated from high concentrations of vegetative cells (107 to 108 cfu), but a single B. anthracis colony was recovered from one of 18 replicates generated from high concentrations of bacterial spores (108 cfu), using a post-treatment viability culture method of 7 days on L-agar plate only. We discuss our results in the context of other similar studies and also a requirement to develop standardised post-treatment viability test methods.

Recombinant spore delivered M. tuberculosis antigens elicit immune response in mice

Abstract BCG, the vaccine against tuberculosis (TB), is one of the mandatory vaccines administered to infants in several parts of the world. However, BCG conferred immunity wanes when children reach adulthood. To develop booster vaccines, we engineered recombinant Bacillus subtilis spores to deliver Mycobacterium tuberculosis (Mtb) antigens. MTAG1 recombinant strain was designed to express a fusion protein of CotC-Ag85B-CFP10 on the spore coat, while MTAG2 and MTAG3 strains were designed to express Ag85B-CFP10 fusion protein in vegetative cells. The difference between MTAG2 and MTAG3 strains is that the latter expresses secreted listeriolysin (LLO) in addition to Ag85B-CFP10. Immunoblot analysis revealed that MTAG1 strain showed positive signals for Ag85B and CFP10 in the spore-coat and other strains in the vegetative cell extracts, indicating that antigens are expressed in these strains as expected. Splenocytes of mice immunized with recombinant spores, through intranasal route, displayed significantly higher antigen specific proliferation of IFN-γ producing cells compared to the splenocytes of control mice. Also, the culture fluids of splenocytes from recombinant spores immunized mice exhibited higher antigen specific release of Th1 cytokines than the culture fluids of splenocytes from control mice. These results indicate that antigens delivered via recombinant spores can be processed and presented to the immune cells. Additional in vivo studies may reveal whether these recombinant spores can be used to boost immunity in BCG vaccinated individuals.

Observations on the Inactivation Efficacy of a MALDI-TOF MS Chemical Extraction Method on Bacillus anthracis Vegetative Cells and Spores.

A chemical (ethanol; formic acid; acetonitrile) protein extraction method for the preparation of bacterial samples for matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) identification was evaluated for its ability to inactivate bacterial species. Initial viability tests (with and without double filtration of the extract through 0.2 μM filters), indicated that the method could inactivate Escherichia coli MRE 162 and Klebsiella pneumoniae ATCC 35657, with or without filtration, but that filtration was required to exclude viable, avirulent, Bacillus anthracis UM23CL2 from extracts. Multiple, high stringency, viability experiments were then carried out on entire filtered extracts prepared from virulent B. anthracis Vollum vegetative cells and spores ranging in concentration from 106-108cfu per extract. B. anthracis was recovered in 3/18 vegetative cell extracts and 10/18 spore extracts. From vegetative cell extracts B. anthracis was only recovered from extracts that had undergone prolonged Luria (L)-broth (7 day) and L-agar plate (a further 7 days) incubations. We hypothesise that the recovery of B. anthracis in vegetative cell extracts is due to the escape of individual sub-lethally injured cells. We discuss our results in view of working practises in clinical laboratories and in the context of recent inadvertent releases of viable B. anthracis.

Cell-specific gene expression in Anabaena variabilis grown phototrophically, mixotrophically, and heterotrophically.

BackgroundWhen the filamentous cyanobacterium Anabaena variabilis grows aerobically without combined nitrogen, some vegetative cells differentiate into N2-fixing heterocysts, while the other vegetative cells perform photosynthesis. Microarrays of sequences within protein-encoding genes were probed with RNA purified from extracts of vegetative cells, from isolated heterocysts, and from whole filaments to investigate transcript levels, and carbon and energy metabolism, in vegetative cells and heterocysts in phototrophic, mixotrophic, and heterotrophic cultures.ResultsHeterocysts represent only 5% to 10% of cells in the filaments. Accordingly, levels of specific transcripts in vegetative cells were with few exceptions very close to those in whole filaments and, also with few exceptions (e.g., nif1 transcripts), levels of specific transcripts in heterocysts had little effect on the overall level of those transcripts in filaments. In phototrophic, mixotrophic, and heterotrophic growth conditions, respectively, 845, 649, and 846 genes showed more than 2-fold difference (p < 0.01) in transcript levels between vegetative cells and heterocysts. Principal component analysis showed that the culture conditions tested affected transcript patterns strongly in vegetative cells but much less in heterocysts. Transcript levels of the genes involved in phycobilisome assembly, photosynthesis, and CO2 assimilation were high in vegetative cells in phototrophic conditions, and decreased when fructose was provided. Our results suggest that Gln, Glu, Ser, Gly, Cys, Thr, and Pro can be actively produced in heterocysts. Whether other protein amino acids are synthesized in heterocysts is unclear. Two possible components of a sucrose transporter were identified that were upregulated in heterocysts in two growth conditions. We consider it likely that genes with unknown function represent a larger fraction of total transcripts in heterocysts than in vegetative cells across growth conditions.ConclusionsThis study provides the first comparison of transcript levels in heterocysts and vegetative cells from heterocyst-bearing filaments of Anabaena. Although the data presented do not give a complete picture of metabolism in either type of cell, they provide a metabolic scaffold on which to build future analyses of cell-specific processes and of the interactions of the two types of cells.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-759) contains supplementary material, which is available to authorized users.

Differential Requirement for Phospholipase D/Spo14 and Its Novel Interactor Sma1 for Regulation of Exocytotic Vesicle Fusion in Yeast Meiosis

During sporulation and meiosis of budding yeast a developmental program determines the formation of the new plasma membranes of the spores. This process of prospore membrane (PSM) formation leads to the formation of meiotic daughter cells, the spores, within the lumen of the mother cell. It is initiated at the spindle pole bodies during meiosis II. Spore formation, but not meiotic cell cycle progression, requires the function of phospholipase D (PLD/Spo14). Here we show that PLD/Spo14 forms a complex with Sma1, a meiotically expressed protein essential for spore formation. Detailed analysis revealed that both proteins are required for early steps of prospore membrane assembly but with distinct defects in the respective mutants. In the Deltaspo14 mutant the initiation of PSM formation is blocked and aggregated vesicles of homogenous size are detected at the spindle pole bodies. In contrast, initiation of PSM formation does occur in the Deltasma1 mutant, but the enlargement of the membrane is impaired. During PSM growth both Spo14 and Sma1 localize to the membrane, and localization of Spo14 is independent of Sma1. Biochemical analysis revealed that Sma1 is not necessary for PLD activity per se and that PLD present in a complex with Sma1 is highly active. Together, our results suggest that yeast PLD is involved in two distinct but essential steps during the regulated vesicle fusion necessary for the assembly of the membranous encapsulations of the spores.

Chemoattractant-induced Ras activation during Dictyostelium aggregation.

Ras proteins are highly conserved molecular switches that regulate cellular response to external stimuli. Dictyostelium discoideum contains an extensive family of Ras proteins that function in regulation of mitosis, cytoskeletal function and motility, and the onset of development. Little is known about the events that lead to the activation of Ras proteins in Dictyostelium, primarily owing to a lack of a biochemical assay to measure the levels of activated Ras. We have adapted an assay, used successfully to measure activated Ras in mammalian cells, to monitor activation of two Dictyostelium Ras proteins, RasC and RasG. We have found that the Ras-binding domain (RBD) of mammalian Raf1 was capable of binding to the activated form of RasG, but not to the activated form of RasC; however, the RBD of Schizosaccharomyces pombe Byr2 was capable of binding preferentially to the activated forms of both RasC and RasG. Using this assay, we discovered that RasC and RasG showed a rapid and transient activation when aggregation-competent cells were stimulated with the chemoattractant cAMP, and this activation did not occur in a number of cAMP signalling mutants. These data provide further evidence of a role for both RasC and RasG in the early development of Dictyostelium.

Protein factors in Blastocladiella emersonii cell extracts recognize similar sequence elements in the promoters of the genes encoding cAMP-dependent protein kinase subunits.

Blastocladiella emersonii contains a single cAMP-dependent protein kinase (PKA), which is similar to the mammalian type II isoforms. Its activity is regulated during development by changes in the levels of the catalytic (C) and regulatory (R) subunits, which occur in parallel with changes in levels of the corresponding mRNAs, suggesting coordinate transcriptional control of the expression of both subunits. Both R and C mRNA levels are low in vegetative cells, rise sharply during sporulation and decrease to basal levels again after germination. To investigate sequence elements common to both Blastocladiella R and C gene promoters, which might be involved in the coordinate regulation of these genes, their 5'-flanking regions were analyzed by gel mobility shift and DNase I footprinting assays. We determined that different DNA-protein complexes are generated when fragments of the R and C gene promoters are incubated with extracts from cells expressing (sporulating cells) or not expressing (vegetative cells) both subunits, and competition experiments suggested that similar protein factors bind to both promoters. DNase I footprinting experiments have indicated that a sequence common to both R and C promoters, and similar to mammalian E-boxes, binds factors present in extracts from vegetative and sporulating cells, whereas sequences flanking the E-boxes in both promoters showed a change in the pattern of DNase I digestion only when the vegetative cell extract was used. This result suggests that the composition of the protein complexes binding to these regions changes during sporulation.

Cyclin B and Cdc2 Expression and Cd2 Kinase Activity DuringDictyosteliumDifferentiation

Although Dictyostelium differentiation occurs in the absence of external nutrients, two periods of mitosis occur, one during early development and one during the formation of the migrating pseudoplasmodium. We showed previously that cyclin B mRNA levels vary in a cell cycle dependent manner during vegetative cell growth. In the present study, we report that cyclin B mRNA levels change dramatically during development, reaching a maximum at the tipped aggregate stage. However, amounts of cyclin B protein vary only slightly, peaking during early development and decreasing during late aggregation and pseudoplasmodial formation. Cdc2 protein levels also remain relatively constant during development. Cdc2-histone H1 kinase activity was considerably higher in vegetative cell extracts of transformants that expressed large amounts of truncated cyclin B protein in comparison to extracts of the parental Ax-2 cells. These results suggest that Cdc2 kinase activity is dependent upon the level of cyclin B in vegetative cells. This result is consistent with the idea that variations in the level of cyclin B during growth regulate the cell cycle. When Cdc2 histone H1 kinase activity was determined during development, it was also found that activity correlated reasonably well with the amount of cyclin B protein. Thus, there was an increase in Cdc2 histone H1 kinase activity early in development, and then levels decreased as development progressed. The increase in Cdc2 histone H1 kinase activity that occurs early in development following starvation may be important in accelerating G2-phase cells through into mitosis. There was no increase in Cdc2 histone H1 kinase that accompanied the previously reported late developmental mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)

A 48 kilodalton enterotoxin-related protein from Clostridium perfringens vegetative and sporulating cells

Sporulating and vegetative cell extracts of enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens were examined by Western blotting using enterotoxin antiserum. A 48-kDa enterotoxin-related protein was found in vegetative and sporulating cell extracts of both toxin types. The results question previous reports about the ability of vegetative cells and presumptive enterotoxin-negative strains of this organism to produce enterotoxin, a protein with a molecular weight of 34 kDa.

Kinetics of spore coat protein synthesis byClostridium perfringens type A

Spore coat proteins of several strains ofClostridium perfringens were analyzed by immunoblotting with antisera against two major spore coat proteins, one [34-kilodalton (kDa)] from an enterotoxin-positive and one (19-kDa) from an enterotoxin-negative (ent−) strain of this organism. The results indicated that spore coat proteins from many strains ofC. perfringens were immunologically related regardless of their ability to produce enterotoxin, but were not immunologically related to enterotoxin. The kinetics of synthesis and deposition of two major spore coat proteins differed depending upon the strain. Coat protein synthesis was sporulationspecific, since coat protein was not detected in vegetative cell extracts. There was no similarity between the amino acid composition of either coat protein and enterotoxin. These results suggest that, contrary to previous reports (W.R. Frieben and C.L. Duncan, Eur J Biochem 39:393–410, 1973), enterotoxin synthesis is not closely related to spore coat protein synthesis in this organism.

Identification of proteins phosphorylated by ATP during sporulation of Bacillus subtilis.

Protein phosphorylation in Bacillus subtilis was assayed in vitro by using extracts prepared from cells at various times during growth and sporulation. At least six proteins were labeled in vitro by using [gamma-32P]ATP and extracts of vegetative cells. In extracts prepared at the end of exponential growth and during stationary phase, 12 to 13 proteins were labeled. Seven of the phosphoproteins were purified by fast-performance liquid chromatography and polyacrylamide gel electrophoresis, blotted to Immobilon membranes, and subjected to partial protein sequencing. One of the sequences had sequence homology (greater than 45%) to elongation factor G from several bacterial species, and four sequences matched the predicted amino-terminal sequences of the outB, orfY-tsr, orfU, and ptsH genes.

Influence of target preparation in secondary ion mass spectrometry: molecular mass determination of a pterin isolated from dictyostelium discoideum

The molecular mass of a major pterin derivative isolated by reverse phase high performance liquid chromatography (HPLC) from cellular extracts of vegetative cells of Dictyostelium discoïdeum has been determined using secondary ion mass spectrometry. The importance of the target preparation procedure is discussed. In particular, for samples originating from HPLC eluates, the sensitivity is drastically reduced compared to what is observed with pure commercial compounds. The mass spectral sensitivity can be largely improved by addition of sodium iodide to the target.

Myxococcus xanthus protein C is a major spore surface protein

Fruiting body formation in Myxococcus xanthus involves the aggregation of cells to form mounds and the differentiation of rod-shaped cells into spherical myxospores. The surface of the myxospore is composed of several sodium dodecyl sulfate (SDS)-soluble proteins, the best characterized of which is protein S (Mr, 19,000). We have identified a new major spore surface protein called protein C (Mr, 30,000). Protein C is not present in extracts of vegetative cells but appears in extracts of developing cells by 6 h. Protein C, like protein S, is produced during starvation in liquid medium but is not made during glycerol-induced sporulation. Its synthesis is blocked in certain developmental mutants but not others. When examined by SDS-polyacrylamide gel electrophoresis, two forms of protein C are observed. Protein C is quantitatively released from spores by treatment with 0.1 N NaOH or by boiling in 1% SDS. It is slowly washed from the spore surface in water but is stabilized by the presence of magnesium. Protein C binds to the surface of spores depleted of protein C and protein S. Protein C is a useful new marker for development in M. xanthus because it is developmentally regulated, spore associated, abundant, and easily purified.

A sequence-specific DNA-binding factor (VF1) from Anabaena sp. strain PCC 7120 vegetative cells binds to three adjacent sites in the xisA upstream region

A DNA-binding factor (VF1) partially purified from Anabaena sp. strain PCC 7120 vegetative cell extracts by heparin-Sepharose chromatography was found to have affinity for the xisA upstream region. The xisA gene is required for excision of an 11-kilobase element from the nifD gene during heterocyst differentiation. Previous studies of the xisA upstream sequences demonstrated that deletion of this region is required for the expression of xisA from heterologous promoters in vegetative cells. Mobility shift assays with a labeled 250-base-pair fragment containing the binding sites revealed three distinct DNA-protein complexes. Competition experiments showed that VF1 also bound to the upstream sequences of the rbcL and glnA genes, but the rbcL and glnA fragments showed only single complexes in mobility shift assays. The upstream region of the nifH gene formed a weak complex with VF1. DNase footprinting and deletion analysis of the xisA binding site mapped the binding to a 66-base-pair region containing three repeats of the consensus recognition sequence ACATT.

A segment of Myxococcus xanthus ops DNA functions as an upstream activation site for tps gene transcription

A segment of DNA located between 131 and 311 base pairs (bp) upstream from the transcriptional start of the Myxococcus xanthus ops gene (-131 to -311) was shown to function as an upstream activation site (UAS) for developmentally regulated transcription from the tps gene promoter region. The activation of early developmental transcription by the ops UAS was independent of orientation and could be increased by the addition of a second copy of the UAS. The ops UAS segment continued to function when placed 1.5 kbp upstream from the transcription initiation site. DNA from the tps promoter region was required for transcriptional activation by the ops UAS, and a specific requirement for the sequence of tps DNA between -34 and -66 was demonstrated. Several specific ops UAS DNA-protein complexes were observed after incubation of this DNA segment with an extract of early developmental M. xanthus cells. Extracts of vegetative cells contained much less ops UAS-specific DNA-binding activity. When the distance between the tps and ops genes was increased from 2 to 15 kbp by insertion of a transduced segment of DNA, the amount of developmentally induced tps RNA was found to be about one-third that found in wild-type M. xanthus. Our observations suggest that the regulatory region of the ops gene functions not only to control ops gene expression but also to increase early developmental expression of the tps gene located about 2 kbp downstream on the M. xanthus chromosome.

Effect of light quality on the acetylene reducing activity of the isolated heterocysts of Anabaena sp. strain CA.

Metabolically active heterocysts isolated from Anabaena sp, strain CA showed high rates of light-dependent nitrogenase activity. When these active heterocysts were incubated under light of various wavelengths, they were most active under blue light, followed by yellow and red light respectively. Involvement of phycobiliproteins in transferring light energy to photosystem I in isolated heterocysts is discussed. Aldolase, phosphofructokinase and catalase activities were also studied in extracts of vegetative cells as were heterocysts from Anabaena sp. strain CA.

Examination of 20 Bacillus species by crossed immunoelectrophoresis under taxonomic aspects

In order to examine the efficiency of crossed immunoelectrophoresis (CIE) for the differentiation of species and for the determination of taxonomic relationships within the genus Bacillus, an investigation including strains of 20 species was performed. Ultrasonic extracts (USE) of sporefree-grown vegetative cells were used. With rabbit antisera against USE of type strains, the CIE was accomplished in homologous and heterologous combinations. USE's of additional strains were included into this investigation. In order to evaluate the immunoelectropherograms, the number of precipitates was counted. A mean of 103 precipitates was found in homologous reactions. With few exceptions, heterologous combinations showed less similarity in the number of antigens. Because of their high cross-reactions, the following species could not be differentiated: B. subtilis from B. amyloliquefaciens and strains of B. coagulans, furthermore, B. cereus from B. thuringiensis. The different species revealed lower numbers of precipitates with decreasing taxonomic relationships in heterologous combinations. This observation was used to classify the investigated species by a 'position-frequency analysis' (PFA). After sorting the matrix of precipitate numbers in the ascertained optimal sequence of species, a cluster analysis was carried out. The phenogram showed 6 (respectively 8) group clusters. The members of the morphologic group I (Smith et al., 1952) was found only in group cluster 2. The phenogram was partly in agreement with phenograms based on other characteristics, e.g. DNA hybridization.

Stage-specific in vitro expression of the Bacillus subtilis spoVE gene.

Synthesis of the spo VE gene product (a 32 kilodalton protein) of Bacillus subtilis was studied using a DNA-directed protein-synthesizing system from B. subtilis. The spo VE gene product was synthesized specifically by T2 (2 hours after the end of logarithmic growth) cell extracts but not by vegetative cell extracts. A dot blot hybridization experiment with a spoVE-specific probe showed that the spoVE mRNA was synthesized by vegetative as well as by T2-cell extracts, and was present in most spo0 mutants. These results suggest that the transcription of the spoVE gene is not dependent on the spo0 gene function and the synthesis of the SpoVE protein is somehow controlled at the level of translation. Heterologous combination experiments using supernatant fractions from the vegetative and T2 cells, and ribosomes from the vegetative and T2 cells showed that T2-specific synthesis of the SpoVE protein is dependent on the T2-cell supernatant. The addition of the T2-cell supernatant to the vegetative cell extract i...

Enzymes of the intermediary carbohydrate metabolism of Polyangium cellulosum

Crude extracts of vegetative cells of the cellulolytic myxobacter Polyangium cellulosum contained significant levels of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle. Key enzymes of glycolysis and the pentose phosphate shunt were also detected. Specific activities of hexokinase and fructose- 1,6-diphosphate aldolase exhibited a 10-fold increase when the cells were grown in complex medium containing glucose. Cytochromes of a, b, and c type were demonstrated. By the use of a dispersly growing strain of P. cellulosum, its generation time was determined to be 22–24 h. This study suggests that the organism probably uses glycolysis and citric acid cycle for complete oxidation of glucose. The exact role of the glyoxylate cycle and pentose phosphate shunt cannot be deduced from this study. This is the first report on the study of intermediary carbohydrate metabolism in any member of the family Polyangiaceae.

Endospores of Sporosarcina halophila: characteristics and ultrastructure

Sporosarcina halophila forms endospores. Electron micrographs revealed ultrastructural similarity to spores of S. ureae. Spore germination indicated by loss of refractility, darkening, swelling and formation of new vegetative cells was followed by phase contrast light microscopy. To induce spore germination, the endospores needed to be heat avtivated. After activation, they were inoculated into nutrient broth medium supplemented with sea-water. Double concentrated sea-water was found to be optimal for germination. Similar to other bacterial endospores, the spores were found to be resistant to heat and ethanol. An ultraviolet absorbing substance was isolated from suspensions of free spores; it was identified to be pyridine-2,6-dicarboxylic acid (DPA) usually present in bacterial spores. DPA was detected in amounts ranging from 5–7% of the spore dry weight; it was not detected in extracts of vegetative cells.