Development of a Streptavidin-Conjugated Single-Chain Antibody That Binds Bacillus cereus Spores

Control of microorganisms such as Bacillus cereus spores is critical to ensure the safety and a long shelf life of foods. A bifunctional single chain antibody has been developed for detection and binding of B. cereus T spores. The genes that encode B. cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin. The fusion protein gene was expressed in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system. Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody. The single-chain antibody-streptavidin fusion protein can be used in an immunoassay of B. cereus spores by applying a biotinylated enzyme detection system. The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 × 10⁴ B. cereus spores per ml. Greater than 90% of the B. cereus spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads.     

Cell cycle analysis in asynchronous cultures using the BUdR-Hoechst technique.

During synchronized germination of spores of Dictyostelium discoideum, protein synthesis begins almost concomitantly with syntheses of messenger-like RNA (mlRNA) and 4–5S RNA (presumably tRNA) in the swollen spore stage and the initiation of ribosomal RNA (rRNA) synthesis is somewhat delayed. DNA synthesis occurs in the early stages of the amoeba emergence phase. Cycloheximide (200 μg/ml) blocked spore germination as well as total protein synthesis, whereas actinomycin D (60 μg/ml) did not affect either. This concentration of actinomycin D selectively inhibited formation of rRNA but did not influence the synthesis of mlRNA. Examinations of RNA labeled with [¹⁴C]uracil during germination indicated that polysomes initially detectable in the course of the germination process contain ¹⁴C-labeled mlRNA. It was concluded that at least some of mRNA synthesized during germination of D. discoideum spores is involved in protein synthesis required for the germination.     

Biochemical Alternations in Bacillus megaterium as Produced by Aflatoxin B1

Bacillus megaterium NRRL B-1368 cells and spores were produced on Trypticase Soy Broth (TSB) and Agar (TSA) containing 3.8 μg of aflatoxin B₁ per ml, analyzed for selected chemical constituents, and compared to cells and spores of B. megaterium produced on nontoxic Trypticase Soy Media. There was an initial 30% kill of cells after inoculation into toxic TSB and during the first 3.5 hr of incubation followed by a logarithmic growth phase in which the generation time was 75 min as compared to 20 min for the control culture. Chemical analyses revealed an increase in protein, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) on both a per cell basis and a per cent dry weight basis when B. megaterium was grown in toxic TSB. There was a concurrent decrease in the total amounts of cellular protein, DNA, and RNA synthesized in toxic TSB. Amino acid analyses of control and test cell walls showed little, if any, difference in cell wall composition. About 97% sporulation of B. megaterium occurred after 3 days on nontoxic TSA although 6 days were required to attain 65% sporulation on toxic TSA. Germination of spores was not inhibited by 4.0 μg of aflatoxin per ml but outgrowth was. No significant differences were observed in the heat resistance, protein, DNA, RNA, or dipicolinic acid content of spores formed on toxic TSA and nontoxic TSA.     

Mutual relationship between antibiotics and resting spores of Bacillus subtilis: morphological changes and macromolecular synthesis after germination of spores treated with cyclic polypeptide and aminoglycoside antibiotics

Morphological changes and synthesis of DNA, RNA, protein, and cell wall were investigated during germination of resting spores of Bacillus subtilis exposed transiently to the cyclic polypeptide antibiotics, polymyxin B and gramicidin S, and the aminoglycoside antibiotics, streptomycin, kanamycin, and gentamicin. Normal germinated spores showed breaks of the spore coat, a diminution in size and a fibrillar appearance of the cortex, a swelling core, a cell wall as thick as that of vegetable cells, some mesosomes and DNA fibrils. On the other hand, no breaks of the spore coat, a spore core with a slight swelling and irregular form, a thin cell wall, no demonstration of the nuclear material and no granularity in the cytoplasm were characteristic of the germinated spores derived from polymyxin B- and gramicidin S-treated resting spores. With gramicidin S-treated germinated spores a few vacuoles were formed in the cytoplasm. Both polymyxin B- and gramicidin S-treated germinated spores showed little or no synthesis of DNA, RNA, and protein. The vegetative cells derived from streptomycin-treated resting spores demonstrated several finely granular regions in the cytoplasm and a disorder of the fibrillar nucleoid, and their autolysis occurred early. Their DNA and RNA synthesis was normal, whereas protein synthesis was low. In spite of no occurrence of cell division and very low protein synthesis, the most striking characteristics of the outgrowing cells derived from kanamycin-treated resting spores were a markedly thickened cell wall and a continuous incorporation of labeled D-alanine suggesting cell wall synthesis; RNA synthesis was slightly lower and DNA synthesis was almost normal. The outgrowing cells from gentamicin-treated resting spores also revealed relatively thick cell walls and a very slight incorporation of labeled D-alanine. Their DNA and RNA synthesis was fairly low and protein synthesis was almost completely inhibited. These results coincide with the growth curves of individual antibiotic-treated resting spores.     

Detection of relationships among microsporidan isolates by electrophoretic analysis: hydrophobic extracts

Hydrophobic spore proteins were extracted from 11 microsporidan isolates obtained from 9 species of insects for which these microorganisms are pathogenic. Hydrophobic protein spectra were found to be stable when (1) two different genera of hosts were used for spore propagation, (2) hosts were reared at a variety of temperatures, or (3) protein was extracted from spores harvested at different stages of sporogenesis. Five consistent and distinct electrophoretic spectra were observed. Spectrum I was represented by 6 isolates including Nosema necatrix, Thelohania diazoma, Nosema plodiae, and Nosema sphingidis; spectrum II by Pleistophora sp; Spectrum III by Nosema whitei; spectrum IV by Thelohania legeri; and spectrum V by Nosema trichoplusia. The highly reproducible nature of these analyses indicated polyacrylamide gel disc electrophoresis of hydrophobic extracts can be used for the identification of Microsporida. Moreover, these analyses do not support the present classification, based mainly on the number of spores in a pansporoblast, inasfar as (1) some species of Nosema have the same pattern (I) as a species of Thelohania and (2) two species of Nosema have different patterns (III and V) in contrast to the Nosema species showing pattern I.     

Isolation and characterization of a mycovirus in Lentinula edodes

A mycovirus was isolated from an edible mushroom, Lentinula edodes, that was suffering from a severe epidemic. Fractionation of the diseased cell extract by isopycnic centrifugation with 50% CsCl revealed that the diseased mushroom was infected by Lentinula edodes spherical virus (LeSV), a new spherical virus with a diameter of 55 nm. The particle of LeSV encapsidated the 12 kb RNA genome by a 120 kDa coat protein. BLAST analysis of the partially sequenced LeSV genome showed 95% sequence identity with a putative RNA-dependent RNA polymerase (RdRp) gene of the mycovirus HKB, which was previously reported as being a double-stranded RNA (dsRNA) element. In contrast to HKB, the RNA genome in LeSV is encapsidated by the 120 kDa coat protein. To confirm that the LeSV coat protein is encoded by the viral genome, the N-terminal amino acid sequence of the coat protein was determined. The resulting N-terminal amino acid sequence, N-SALDVAPVVPELYFXXLEV-C, was found to be located in the middle of the HKB ORF1, suggesting that the LeSV coat protein was indeed encoded by the virus. To detect LeSV in L. edodes, a primer set targeting the RdRp gene was designed based on the partial sequence of the LeSV genome. RT-PCR analysis showed that 56 of the 84 commercially available dikaryotic cultivars carry LeSV. The transmission pattern of the virus was determined by analysing basidiospores from LeSV-infected and LeSV-free fruiting bodies. Nine out of 10 basidiospores from the LeSV-infected cultivars contained the virus while the spores from the LeSV-free parent were free of LeSV, suggesting that vertical transmission is the primary mode of LeSV propagation.     

Photocontrol of the germination of onoclea spores: I. Action spectrum

Light stimulates the germination of spores of the fern Onoclea sensibilis L. At high dosages, broad band red, far red, and blue light promote maximal germination. Maximal sensitivity to these spectral regions is attained from 6 to 48 hours of dark presoaking, and all induced rapid germination after a lag of 30 to 36 hours. Maximal germination is attained approximately 70 hours after irradiation. Dose response curves suggest log linearity. The action spectrum to cause 50% germination shows that spores are most sensitive to irradiation in the red region (620-680 nm) with an incident energy less than 1000 ergs cm⁻²; sensitivity decreases towards both shorter and longer wavelengths. Although the action spectrum is suggestive of phytochrome involvement, photoreversibility of germination between red and far red light has not been demonstrated with Onoclea spores. An absorption spectrum of the intact spores reveals the presence of chlorophylls and carotenoids. Since the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea does not inhibit germination, it is concluded that photosynthesis does not play a role in the germination process.     

Gene activity during germination of spores of the fern, Onoclea sensibilis. Cell-free translation analysis of mRNA of spores and the effect of alpha-amanitin on spore germination

Poly(A)-RNA fractions of dormant, dark-imbibed (non-germinating) and photoinduced (germinating) spores of Onoclea sensibilis were poor templates in the rabbit reticulocyte lysate protein synthesizing system, but the translational efficiency of poly(A)+RNA was considerably higher than that of unfractionated RNA. Poly(A)+RNA isolated from photoinduced spores had a consistently higher translational efficiency than poly(A)+RNA from dark-imbibed spores. Analysis of the translation products by one-dimensional polyacrylamide gel electrophoresis showed no qualitative differences in the mRNA populations of dormant, dark-imbibed, and photoinduced spores. However, poly(A)+RNA from dark-imbibed spores appeared to encode in vitro fewer detectable polypeptides at a reduced intensity than photoinduced spores. A DNA clone encoding the large subunit of maize ribulose bisphosphate carboxylase hybridized at strong to moderate intensity to RNA isolated from dark-imbibed spores, indicating the absence of mRNA degradation. Although alpha-amanitin did not inhibit the germination of spores, the drug prevented the elongation of the rhizoid and protonemal initial with a concomitant effect on the synthesis of poly(A)+RNA. These results are consistent with the view that some form of translational control involving stored mRNA operates during dark-imbibition and photoinduced germination of spores.     

Structure of DNA in Spores of Bacillus cereus

The structure of DNA extracted from dormant and germinating spores of B. cereus T was investigated using circular dichroism and other methods. No significant differences between DNAs extracted from vegetative cells and from spores of various stages could be found by analyses of CD spectra, CsCl density gradient centrifugation, melting profiles and template activity. All the DNA preparations were in B conformation and had the same density (1.695), Tm (83°C) and template activity in the reaction of DNA-dependent RNA polymerase. An abnormal DNA fraction of high density which was formerly found in B. cereus spores or a stable DNA complex with protein and/or RNA was not detected in the present extracts of spores. Preliminary X-ray analyses of intact spores indicate that the structure of DNA in spores is not so different from B form.     

Mitochondrial biogenesis during fungal spore germination. Development of cytochrome c oxidase activity.

Mitochondria from dormant spores of the fungus Botryodiplodia theobromae did not contain extractable cyctochrome c oxidase (EC 1.9.3.1) activity; however, this enzyme activity was elaborated rapidly after 150 min of the 240-min germination sequence. The absence of cytochrome c oxidase activity in the dormant spores apparently is not an artifact caused by spore disruption and fractionation procedures, transient enzyme instability, or insensitivity of the enzyme assay. Mitochondria from dormant spores of three other phylogenetically diverse genera of fungi were observed to contain readily detectable quantities of cytochrome c oxidase, suggesting that the absence of the enzyme in B. theobromae may be relatively novel. The elaboration of cytochrome c oxidase activity in germinating spores was abolished by cycloheximide if the drug was added at or before 95 min of germination, but development of enzyme activity was initially insensitive to inhibitors of the mitochondrial genetic system, chloramphenicol or ethidium bromide. Incubation of spores in both ethionine and S-2-aminoethyl-l-cysteine reduced the amount of extracted cytochrome c oxidase activity. Elaboration of enzyme activity was severely retarded by cerulenin, an inhibitor of fatty acid biosynthesis and of spore germination. This enzyme activity developed in water-incubated or 1% Tween 80-incubated spores in which only the cytoplasmic ribosomes are functional in translation of a stored nuclear messenger RNA. The results of this study show that cytoplasmic (but not mitochondrial) ribosome function is required for development of this enzyme activity during spore germination, and they suggest that a portion of the cytochrome c oxidase enzyme or some other protein required for its activity is synthesized de novo upon germination.     

Differential scanning calorimetric observations concerning the activation mechanism of spores of Phycomyces blakesleeanus.

Spores of Phycomyces were scanned in a Differential Scanning Calorimeter. The spectrum obtained was clearly influenced by previous activation of spores by heat or by acetate.When spores were allowed to return to dormancy the original spectrum of dormant spores was restored. The high temperature at which the difference in the spectrum between activated and dormant spores was found points to a protein denaturation. It is suggested therefore that the activation of spores is obtained through a conformational change of a protein.     

Spores of microorganisms

Spores ofBacillus cereus were germinated in a germination limited medium (GL-medium) which facilitates only germination but not the postgerminative development of spores. Under these conditions a limited protein synthesis occurs. However, this protein synthesis is stopped after a short time interval. The rate of synthesis of new proteins, as well as their total amount, is influenced by the length of the activation heat shock. Synthesis of the wall material continues for several hours and thick-walled cells with a changed ultrastructure are formed. Synthesis of the diaminopimelic acid (dap) containing material of the cell wall is sensitive to actinomycin D and relatively resistant to chloramphenicol. Similarly, protein synthesis is relatively chloramphenicol-resistant but is fully inhibited by azauracil or spiramycin. Whereas RNA formed in the control culture is partially decomposed after 30 min of incubation, chloramphenicol accelerates its synthesis and prevents its decay. Exudate components apparently stimulate synthesis of ribonucleic acid, proteins and the wall material. The¹⁴C-dap containing material released by prelabelled spores in the form of the exudate during the germination is not re-utilized by the spores germinated in the GL-medium. The results are discussed with respect to the atypical primary synthetic activities of spores under conditions when the postgerminative development is prevented and from the point of view of participation of the germination exudate during these syntheses.     

Gene Activity during Germination of Spores of the Fern, Onoclea sensibilis: RNA and Protein Synthesis and the Role of Stored mRNA

Pattern of ³H-uridine incorporation into RNA of spores of Onocleasensibilis imbibed in complete darkness (non-germinating conditions)and induced to germinate in red light was followed by oligo-dTcellulose chromatography, gel electrophoresis coupled with fluorographyand autoradiography. In dark-imbibed spores, RNA synthesis wasinitiated about 24 h after sowing, with most of the label accumulatingin the high mol. wt. poly(A)^–RNA fraction. There was noincorporation of the label into poly(A) ⁺ RNA until 48 h aftersowing. In contrast, photo-induced spores began to synthesizeall fractions of RNA within 12 h after sowing and by 24 h, incorporationof ³H-uridine into RNA of irradiated spores was nearly 70-foldhigher than that into dark-imbibed spores. Protein synthesis,as monitored by ³H-arginine incorporation into the acid-insolublefraction and by autoradiography, was initiated in spores within1–2 h after sowing under both conditions. Autoradiographicexperiments also showed that the onset of protein synthesisin the cytoplasm of the germinating spore is independent ofthe transport of newly synthesized nuclear RNA. One-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis of³⁵S-methionine-labelled proteins revealed a good correspondencebetween proteins synthesized in a cell-free translation systemdirected by poly(A) ⁺RNA of dormant spores and those synthesizedin vivo by dark-imbibed and photo-induced spores. These resultsindicate that stored mRNAs of O. sensibilis spores are functionallycompetent and provide templates for the synthesis of proteinsduring dark-imbibition and germination. Key words: Onoclea sensibilis, fern spore germination, gene expression, protein synthesis, sensitive fern, stored mRNA     

A Method for Extracting RNA from Dormant and Germinating Bacillus subtilis Strain 168 Endospores

RNA was extracted from dormant and germinating Bacillus subtilis 168 spores (intact spores and chemically decoated spores) by using rapid rupture followed by acid–phenol extraction. Spore germination progress was monitored by assaying colony forming ability before and after heat shock and by reading the optical density at 600 nm. The purity, yield, and composition of the extracted RNA were determined spectrophotometrically from the ratio of absorption at 260 nm to that at 280 nm; in a 2100 BioAnalyzer, giving the RNA yield/10⁸ spores or cells and the distribution pattern of rRNA components. The method reported here for the extraction of RNA from dormant spores, as well as during different phases of germination and outgrowth, has proven to be fast, efficient and simple to handle. RNA of a high purity was obtained from dormant spores and during all phases of germination and growth. There was a significant increase in RNA yield during the transition from dormant spores to germination and subsequent outgrowth. Chemically decoated spores were retarded in germination and outgrowth compared with intact spores, and less RNA was extracted; however, the differences were not significant. This method for RNA isolation of dormant, germinating, and outgrowing bacterial endospores is a valuable prerequisite for gene expression studies, especially in studies on the responses of spores to hostile environmental conditions.     

Germination of Heat- and Alkali-Altered Spores of Clostridium perfringens Type A by Lysozyme and an Initiation Protein

The normal system functioning in the utilization of metabolizable germinants by both heat-sensitive and heat-resistant spores of Clostridium perfringens was inactivated by heat or by treatment of the spores with alkali to remove a soluble coat protein layer. Altered spores were incapable of germination (less than 1%) and outgrowth (less than 0.0005%) in complex media without the addition of either lysozyme or an initiation protein produced by C. perfringens. The addition of either of these agents permitted, in the case of alkali-treated spores, both 90 to 95% germination and outgrowth, as measured by colony formation. In the case of heat-damaged spores, only 50% germination and 2% outgrowth resulted from addition of the initiation protein, whereas lysozyme permitted 85% germination and 8% outgrowth. Alteration of the spores by heat or alkali apparently inactivated the normal lytic system responsible for cortical degradation during germination. Kinetics of production of the initiation protein and conditions affecting both its activity and that of lysozyme on altered spores are described.     

Crystal Structure of the GerBC Component of a Bacillus subtilis Spore Germinant Receptor

The nutrient germinant receptors (nGRs) of spores of Bacillus species are clusters of three proteins that play a critical role in triggering the germination of dormant spores in response to specific nutrient molecules. Here, we report the crystal structure of the C protein of the GerB germinant receptor, so-called GerBC, of Bacillus subtilis spores at 2.3 Å resolution. The GerBC protein adopts a previously uncharacterized type of protein fold consisting of three distinct domains, each of which is centered by a β sheet surrounded by multiple α helices. Secondary-structure prediction and structure-based sequence alignment suggest that the GerBC structure represents the prototype for C subunits of nGRs from spores of all Bacillales and Clostridiales species and defines two highly conserved structural regions in this family of proteins. GerBC forms an interlocked dimer in the crystalline state but is predominantly monomeric in solution, pointing to the possibility that GerBC oligomerizes as a result of either high local protein concentrations or interaction with other nGR proteins in spores. Our findings provide the first structural view of the nGR subunits and a molecular framework for understanding the architecture, conservation, and function of nGRs.     

Developmental gene regulation in Aspergillus nidulans

The Ascomycete fungus Aspergillus nidulans reproduces asexually by differentiating conidiophores and conidia. Gene regulation during asexual reproduction was investigated by comparing poly(A) RNA populations derived from somatic hyphae, conidiating cultures and purified conidia. Single-copy and complementary DNA hybridization experiments showed that vegetative cells contained 5600–6000 diverse, average-sized poly(A) RNA sequences distributed into three prevalence classes. cDNA hybridization experiments indicated that a significant proportion of the poly(A) RNA derived from either conidiating cultures or spores consisted of sequences absent from somatic hyphae. To assess accurately the degree to which the poly(A) RNA populations differed, cDNA preparations were isolated which were complementary to sequences present only in conidia or in conidiating cultures. Hybridization of these cDNAs with poly(A) RNA from conidiating cultures showed that approximately 18.5% of the poly(A) RNA mass comprised 1300 diverse sequences not present in somatic cells. Of these, about 300 were present only in conidia. The remainder were accumulated specifically during sporulation, but were absent from spores. Analogous experiments showed that the great majority of the poly(A) RNA sequences accumulated by vegetative hyphae were also present in conidiating cultures. Thus, cell differentiation during A. nidulans asexual reproduction involves the accumulation of many new poly(A) RNA sequences, but not the loss of preexisting ones.     

Characteristics of developing mitochondrial genetic and respiratory functions in germinating fungal spores

Spores of the fungus Botryodiplodia theobromae began a cyanide-sensitive oxygen consumption immediately upon exposure to a liquid medium, and spore germination and respiration were not affected by ethidium bromide, d-threochloramphenicol, and acriflavin until later during germ tube emergence. These inhibitors of the mitochondrial genetic system all inhibited total cell protein synthesis to the same intermediate degree from the outset of incubation. When spores were incubated in water under non-germinating conditions, protein synthesis and oxygen uptake proceeded at initial rates almost identical to those seen in spores germinating in the presence of the three mitochondrial system inhibitors. Although the spores respired at rapid rates from the onset of incubation, no cytochrome absorption peaks could be observed in mitochondrial fractions prepared from ungerminated spores; they were readily observed in germinated spores, however. When the spores were germinated in the presence of inhibitors of the mitochondrial system, an excess of cytochrome c was observed in the near absence of cytochromes a and b. The results indicate that the ungerminated spores of this organism contain a preserved, potentially functional aerobic respiratory system which requires cycloheximide-sensitive ribosome activity to become functional when the spores are inoculated into a liquid medium.