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PubMed1981

Synthesis of teichoic acid by Bacillus subtilis protoplasts.

Protoplasts of Bacillus subtilis W23 readily synthesized ribitol teichoic acid from nucleotide precursors in the surrounding medium. With cytidine diphosphate-ribitol they made poly(ribitol phosphate), presumably attached to lipoteichoic acid carrier; when cytidine diphosphate-glycerol and uridine diphosphate-N-acetylglucosamine were also present a 10-fold increase in the rate of polymer synthesis occurred, and the product contained both the main chain and the linkage unit. Synthesis was inhibited by trypsin or p-chloromercuribenzenesulfonate in the medium, and we concluded that it occurred at the outer surface of the membrane. During synthesis, which was also achieved readily by whole cells after a brief period of wall lysis, the cytidine phosphate portion of the nucleotide precursors did not pass through the membrane. No evidence could be obtained for a transphosphorylation mechanism for the translocation process. It is suggested that reaction with exogenous substrates was due to temporary exposure of a protein component of the enzyme complex at the outer surface of the membrane during the normal biosynthetic cycle.

4-Chloromercuribenzenesulfonate↗
PubMed1982

Reconstitution of succinate dehydrogenase in Bacillus subtilis by protoplast fusion.

Bacillus subtilis succinate dehydrogenase (SDH) is composed of two unequal subunits designated Fp (Mr, 65,000) and Ip (Mr. 28,000). The enzyme is structurally and functionally complexed to cytochrome b 558 (Mr, 19,000) in the membrane. A total of 21 B. subtilis SDH-negative mutants were isolated. The mutants fall into five phenotypic classes with respect to the presence and localization of the subunits of the SDH-cytochrome b558 complex. One class contains mutants with an inactive membrane-bound complex. Membrane-bound enzymatically active SDH could be reconstituted in fused protoplasts of selected pairs of SDH-negative mutants. Most likely reconstitution is due to the assembly of preformed subunits in the fused cells. On the basis of the reconstitution data, the mutants tested could be divided into three complementation groups. The combined data of the present and previous work indicate that the complementation groups correspond to the structural genes for the three subunits of the membrane-bound SDH-cytochrome b558 complex. A total of 31 SDH-negative mutants of B. subtilis have now been characterized. The respective mutations all map in the citF locus at 255 degrees on the B. subtilis chromosomal map. In the present paper, we have revised the nomenclature for the genetics of SDH in B. subtilis. All mutations which give an SDH-negative phenotype will be called sdh followed by an isolation number. The designation citF will be omitted, and the citF locus will be divided into three genes: sdhA, sdhB, and sdhC. Mutations in sdhA affect cytochrome b558, mutations in sdhB affect Fp, and mutations in sdhC affect Ip.

Bacillus subtilis↗
PubMed1985

High-frequency transformation of Brevibacterium lactofermentum protoplasts by plasmid DNA.

An efficient polyethylene glycol-assisted method for transformation of Brevibacterium lactofermentum protoplasts that uses plasmid vectors has been developed. Two small plasmids, pUL330 (5.2 kilobases) and pUL340 (5.8 kilobases), both containing the kanamycin resistance gene from transposon Tn5 and the replication origin of the natural plasmid pBL1 of B. lactofermentum, were selected as vectors. Supercoiled forms of the plasmids yielded a 100-fold higher transformation frequency than did linear forms. The optimal transformation frequency was achieved with 10 ng of DNA in 1 ml of transformation buffer. Higher concentrations of plasmid DNA resulted in a decrease in transformation frequency per microgram of DNA. Optimal transformation was obtained with 25 to 35% polyethylene glycol 6000. Under optimal conditions, 10(6) transformants per microgram of DNA were obtained.

Brevibacterium↗
PubMed1985

Analysis of transfer of tumor-inducing plasmids from Agrobacterium tumefaciens to Petunia protoplasts.

Petunia protoplasts were infected with the virulent Agrobacterium tumefaciens strain A348 or the avirulent strain A136 (lacking a Ti plasmid). The infection process was stopped at various time intervals up to 24 h after inoculation, and the DNA from the plant cells was isolated. Southern blot analysis indicated that the DNA isolated from infected Petunia cells was not detectably contaminated by bacterial DNA from lysed Agrobacterium cells. Analysis of the DNA from the virulent infections suggested that the transferred DNA (T-DNA) may be transferred to the plant cell rapidly (within 2 to 6 h) after the bacteria bind to the cell wall and that the T-DNA may exist in a rearranged state which is stable over the time period investigated. Dot blot analysis indicated that regions far outside the T-DNA may be transferred to the plant cell. Most of the DNA transferred to the plant cell during the initial hours of infection is rapidly degraded.

Base Sequence↗
PubMed1993

Evasion of host defense by in vivo-produced protoplast-like cells of the insect mycopathogen Beauveria bassiana.

In vivo cells (hyphal bodies) of the hyphomycetous insect pathogen Beauveria bassiana collected from host Spodoptera exigua larval hemolymph were osmotically sensitive and lacked a well-defined cell wall. In light and electron microscope studies, a galactose-specific lectin purified from S. exigua hemolymph, concanavalin A (specific for alpha-mannose), and a polyclonal antibody to B. bassiana cell walls all bound to surfaces of in vitro-produced B. bassiana blastospores; however, none of these probes labelled the thin layer of extracellular material covering the plasma membranes of hyphal bodies. These cells were observed freely circulating in S. exigua hemolymph at 36 h postinfection, although immunocompetent hemocytes were known to be present. Additionally, association of hyphal bodies with hemocytes in monolayers was significantly less than for opsonized in vitro blastospores or submerged conidia. The absence of antigenically important galactomannan components on in vivo cells may therefore allow these cells to escape recognition and phagocytosis. Lack of structural components (e.g., chitin, as evidenced by the absence of binding of wheat germ agglutinin) may also be important with respect to evasion of host cellular defense mechanisms. Production of wall material resumed 48 to 60 h postinfection and therefore may coincide with loss of phagocytic capabilities of the hemocytes due to immunosuppressive effects of fungal metabolites. The protoplast-like cells may be formed by the action of hydrolytic enzymes in the hemocytes or by inhibition of fungal cell wall synthetases.

Animals↗
PubMed1993

Genetic analysis of the marine manganese-oxidizing Bacillus sp. strain SG-1: protoplast transformation, Tn917 mutagenesis, and identification of chromosomal loci involved in manganese oxidation.

Mature spores of the marine Bacillus sp. strain SG-1 bind and oxidize manganese(II), thereby becoming encrusted with a manganese(IV) oxide. Both the function and mechanism of this oxidation are unknown, although evidence suggests that spore coat proteins are involved. To further study this phenomenon, methods of genetic analysis were developed for SG-1. By a modified protoplast transformation procedure, SG-1 was transformed (approximately 100 transformants per micrograms of DNA) with several different plasmids of gram-positive origin. Transposon Tn917, delivered on the temperature-sensitive plasmid pLTV1, was used to generate mutants of SG-1. Conditions were established that allowed 98% plasmid loss and insertions to be recovered at a frequency of 10(-3). Each mutant was found to be the result of a single insertion event. Restriction analysis of 27 mutants that do not oxidize manganese but still sporulate localized 17 of the insertions within two regions of the chromosome (termed Mnx regions), and a physical map of these regions was generated. Analysis of 18 transposon integrants in which manganese oxidation was unaffected revealed random transposon integration, with none of their insertions mapping within the Mnx regions. The Mnx regions were cloned from wild-type SG-1, and the largest region, carried on the lactococcal plasmid pGK13, was used to complement in trans one of the nonoxidizing mutants. These results demonstrate that the Mnx regions encode factors that are required for the oxidation of manganese, and this represents the first report identifying genes involved in bacterial manganese oxidation.

Bacillus↗
PubMed1968

Phosphomannanase (PR-factor), an enzyme required for the formation of yeast protoplasts.

The PR-factor, an enzyme necessary for the production of protoplasts from yeast, was identified and was named phosphomannanase. The enzyme released mannan and mannan-proteins from yeasts harvested in the logarithmic phase of growth. The size of the molecules released was greater than 200,000 daltons, which indicated that the enzyme splits very few bonds of the yeast wall. The PR-factor also depolymerized phosphomannans produced by the Hansenula species. The degradation of these substances was due to the splitting of mannosidic bonds. However, the phosphodiester bonds present in these phosphomannans were involved in the specificity of the enzyme, and the number of mannosidic bonds cleaved was dependent on the number of phosphodiester bonds present. We studied the products of degradation of Hansenula phosphomannans and were unable to identify the exact bond split by the enzyme. After enzymatic digestion and subsequent splitting of phosphodiester bonds, phosphomannan Y-2448 yielded products too complex to be separated. Phosphomannan Y-1842 was shown to have a structure more complex than that previously proposed. The action of the enzyme on the phosphate-rich walls of Saccharomyces was studied. Mannan, containing intact phosphodiester bonds, was released from the walls. Mild acid hydrolysis of this released material split the diester bonds to yield monosaccharide and polysaccharide terminated in mannose-6-phosphate. From these products, we deduced that the enzyme cleaved a mannosidic bond adjacent to a mannose, which is also phosphodiester linked through carbon 1. The significance of phosphodiester bonds in the attachment of mannan and mannan-protein enzymes to the wall of yeast is discussed.

Bacillus↗
PubMed1969

Inhibitory effect of 2-deoxy-d-glucose on the formation of the cell wall in yeast protoplasts.

2-Deoxy-d-glucose (2DG) acted as a competitive inhibitor of the synthesis of cell wall components in Saccharomyces cerevisiae protoplasts. The synthesis of fibrillar glucan cell wall component was inhibited at a glucose to 2DG ratio of 4:1 in the cultivating medium. The completion of the formation of cell wall by the synthesis of the amorphous mannan-protein cell wall component was inhibited at a glucose to 2DG ratio of about 20:1. The inhibition could be reversed by increasing the glucose to 2DG ratio in the nutrient medium. No incorporation of 2DG into fibrillar glucan cell wall component was observed.

Cell Wall↗
PubMed1969

Permeability properties of Aspergillus nidulans protoplasts.

The regeneration of mycelial forms from protoplasts of mutant and wild-type strains of Aspergillus nidulans was followed microscopically under conditions with which the rate of regeneration depends upon a specific transport function. This method has shown that at least four uptake systems-one for divalent anions, two for purines, and one for sugars-remain functional after removal of the cell wall.

Aspergillus↗
PubMed2003

cis-acting elements required for efficient packaging of brome mosaic virus RNA3 in barley protoplasts.

Brome mosaic virus (BMV) is a positive-sense RNA plant virus, the tripartite genomic RNAs of which are separately packaged into virions. RNA3 is copackaged with subgenomic RNA4. In barley protoplasts coinoculated with RNA1 and RNA2, an RNA3 mutant with a 69-nucleotide (nt) deletion in the 3'-proximal region of the 3a open reading frame (ORF) was very poorly packaged compared with other RNA3 mutants and wild-type RNA3, despite their comparable accumulation in the absence of coat protein. Computer analysis of RNA secondary structure predicted two stem-loop (SL) structures (i.e., SL-I and SL-II) in the 69-nt region. Disruption of SL-II, but not of SL-I, significantly reduced RNA3 packaging. A chimeric BMV RNA3 (B3Cmp), with the BMV 3a ORF replacing that of cucumber mosaic virus (CMV), was packaged negligibly, whereas RNA4 was packaged efficiently. Replacement of the 3'-proximal region of the CMV 3a ORF in B3Cmp with the 3'-proximal region of the BMV 3a ORF significantly improved packaging efficiency, and the disruption of SL-II in the substituted BMV 3a ORF region greatly reduced packaging efficiency. These results suggest that the 3'-proximal region of the BMV 3a ORF, especially SL-II predicted between nt 904 and 933, plays an important role in the packaging of BMV RNA3 in vivo. Furthermore, the efficient packaging of RNA4 without RNA3 in B3Cmp-infected cells implies the presence of an element in the 3a ORF of BMV RNA3 that regulates the copackaging of RNA3 and RNA4.

Bromovirus↗
PubMed1993

Activity of chimeric U small nuclear RNA (snRNA)/mRNA genes in transfected protoplasts of Nicotiana plumbaginifolia: U snRNA 3'-end formation and transcription initiation can occur independently in plants.

Formation of the 3' ends of RNA polymerase II (Pol II)-specific U small nuclear RNAs (U snRNAs) in vertebrate cells is dependent upon transcription initiation from the U snRNA gene promoter. Moreover, U snRNA promoters are unable to direct the synthesis of functional polyadenylated mRNAs. In this work, we have investigated whether U snRNA 3'-end formation and transcription initiation are also coupled in plants. We have first characterized the requirements for 3'-end formation of an Arabidopsis U2 snRNA expressed in transfected protoplasts of Nicotiana plumbaginifolia. We found that the 3'-end-adjacent sequence CA (N)3-10AGTNNAA, conserved in plant Pol II-specific U snRNA genes, is essential for the 3'-end formation of U2 transcripts and, similar to the vertebrate 3' box, is highly tolerant to mutation. The 3'-flanking regions of an Arabidopsis U5 and a maize U2 snRNA gene can effectively substitute for the Arabidopsis U2 3'-end formation signal, indicating that these signals are functionally equivalent among different Pol II-transcribed snRNA genes. The plant U snRNA 3'-end formation signal can be recognized irrespective of whether transcription initiation occurs at U snRNA or mRNA gene promoters, although efficiency of 3' box utilization is higher when transcription initiation occurs at the U snRNA promoter. Moreover, transcripts initiated from the U2 gene promoter can be spliced and polyadenylated. Transcription from a Pol III-specific plant U snRNA gene promoter is not compatible with polyadenylation. Finally, we reveal that initiation at a Pol II-specific plant U snRNA gene promoter can occur in the absence of the snRNA coding region and a functional snRNA 3'-end formation signal, demonstrating that these sequences play no role in determining the RNA polymerase specificity of plant U snRNA genes.

Arabidopsis↗
PubMed1984

Efficient transfer of cloned DNA into human diploid cells: protoplast fusion in suspension.

A method for fusion of protoplasts bearing amplified plasmids and human diploid fibroblasts or other cell types in suspension is described. Transient expression of plasmid-encoded proteins occurs in up to 50% of the human cells, as demonstrated for simian virus 40 T antigen by immunofluorescence and the Escherichia coli xanthine-guanine phosphoribosyl transferase by autoradiography. In contrast, frequencies of stable transformants were similar to those obtained by the CaPO4 coprecipitation technique. However, experiments with both methods involving the recombinant pRSVneo (in which the Rous sarcoma virus long terminal repeat regulates expression of the antibiotic-inactivating aminoglycoside phosphotransferase) revealed a much higher frequency of colonies in G418 selective medium with constructions in which the early region of simian virus 40 DNA was present as well. We propose a role for the simian virus 40 T antigen in enhancing stable transformation in this system.

Antigens, Viral, Tumor↗
PubMed1984

Transfer of herpes simplex virus thymidine kinase synthesized in bacteria by a high-expression plasmid to tissue culture cells by protoplast fusion.

The introduction of a protein into living tissue culture cells may permit the in vivo study of functions of the protein. We have previously described a high-efficiency-expression plasmid, pHETK2, containing the herpes simplex virus type 1 thymidine kinase (TK) gene which, upon temperature induction, causes TK to be synthesized as greater than 4% of the bacterial protein. In this report we show that enzymatically active TK was transferred to mouse Ltk- cells by polyethylene glycol-mediated fusion with protoplasts prepared from bacteria containing induced levels of TK. The presence of TK in the Ltk- cells was detected by the incorporation of [3H]thymidine into cell nuclei as measured by autoradiography.

Animals↗
PubMed1987

ESR study of copper(II) retention by entire cell, cells walls, and protoplasts of Saccharomyces cerevisiae.

Copper retention by whole cells, protoplasts, and isolated cell walls of Saccharomyces cerevisiae was investigated in the absence of any energy source in the medium. The cell walls accounted only for a small fraction of the cation retention by whole cells. ESR results showed that copper was not bound only at the outer face of the plasma membrane, but it was also distributed in the plasma membrane and (or) in the cytoplasm. ESR studies also showed that, in all three systems, copper was chelated by peptides or proteins. The binding sites were formed by an amide and a strongly complexing ligand such as an amine. Their configuration depended upon pH: in slightly acidic conditions, copper was bound by the oxygen of the amide; at basic pH, NHCO became deprotonated and the negatively charged nitrogen bound to the metal.

Cell Wall↗
PubMed2000

Amylase hyper-producing haploid recombinant strains of Thermomyces lanuginosus obtained by intraspecific protoplast fusion.

Amylase hyper-producing, catabolite-repression-resistant, recombinant strains were produced by intraspecific protoplast fusion of thermophilic fungus Thermomyces lanuginosus strains, using well-characterized, morphological, and 2-deoxy-D-glucose resistant markers. The fusant heterokaryons exhibited enhanced amylase activities as compared to the amylase hyper-producing parental strain (T2). Diploids derived from heterokaryons segregated to stable haploid recombinant strains. In the haploid strain (Tlh 4q), approximately 5-fold higher specific activities of alpha-amylase and glucoamylase in the culture filtrate were observed as compared to the wild-type strain (W0).

Amylases↗
PubMed2000

Ligninolytic characteristics of Pleurotus ostreatus strain F6 and its monokaryotic protoplast derivative P19.

A stable isolate of Pleurotus ostreatus P19 differing in some morphological and physiological characteristics from its parental wild-type strain F6 was obtained via protoplast isolation during the preparation of strains with altered ligninolytic abilities. The isolate is monokaryotic, does not form clamp-connections, and produces much higher activities of enzymes involved in lignin modification (laccase, manganese peroxidase). Cellulase activity was comparable to that of wild-type strain F6, but the xylanase activity was slightly higher in isolate P19. However, this monokaryotic derivative degrades lignin at a slightly lower rate than its parental strain F6. Electron microscopy observations of wood degradation as a function of mycelium growth were performed on three zones of birch wafers delimited according to the distance from the point of inoculation. The different stages of fungal mycelium growth showed differences in the ultrastructural patterns of the decay not only between the strains P19 and F6, but also depending on the distance from the point of inoculation. This suggests a spatio-temporally controlled secretion of enzymes along the hyphae. The enhanced ability of P19 to degrade the condensed forms of lignin in middle lamellae is correlated to its higher laccase activity.

Biodegradation, Environmental↗