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PubMed1997

Pesticin displays muramidase activity.

Pesticin of Yersinia pestis is the only bacteriocin that converts sensitive cells to stable spheroplasts. The amino acid sequence of pesticin as derived from the nucleotide sequence shows no similarity to those of any of the bacteriocins. The unique properties of pesticin prompted an investigation of its mode of action. Since the pesticin plasmid does not encode a lysis protein for release of pesticin into the culture medium, pesticin was isolated from cells and purified to electrophoretic homogeneity. Highly purified pesticin degraded murein and murein glycan strands lacking the peptide side chains to products that were similar to those obtained by lysozyme, as revealed by high-resolution high-pressure liquid chromatography. After reduction of the murein degradation products with tritium-labeled sodium borohydride, acid hydrolysis, and separation of the products by thin-layer chromatography, radiolabeled muraminitol was identified. This indicates that pesticin is a muramidase, and not an N-acetyl-glucosaminidase, that converts cells into stable spheroplasts by slowly degrading murein.

Bacteriocins↗
PubMed2002

Mutant analysis and cellular localization of the AlgI, AlgJ, and AlgF proteins required for O acetylation of alginate in Pseudomonas aeruginosa.

Alginate is an extracellular polysaccharide produced by mucoid strains of Pseudomonas aeruginosa that are typically isolated from the pulmonary tracts of chronically infected cystic fibrosis patients. Alginate is a linear polymer of D-mannuronate and L-guluronate with O-acetyl ester linkages on the O-2 and/or O-3 position of the mannuronate residues. The presence of O-acetyl groups plays an important role in the ability of the polymer to act as a virulence factor, and the algF, algJ, and algI genes are known to be essential for the addition of O-acetyl groups to alginate. To better understand the mechanism of O acetylation of alginate, the cellular locations of the AlgI, AlgJ, and AlgF proteins were determined. For these studies, defined nonpolar algI, algJ, and algF deletion mutants of P. aeruginosa strain FRD1 were constructed, and each mutant produced alginate lacking O-acetyl groups. Expression of algI, algJ, or algF in trans in the corresponding mutant complemented each O acetylation defect. Random phoA (alkaline phosphatase [AP] gene) fusions to algF, algJ, and algI were constructed. All in-frame fusions to algF and algJ had AP activity, indicating that both AlgF and AlgJ were exported to the periplasm. Immunoblot analysis of spheroplasts and periplasmic fractions showed that AlgF was released with the periplasmic contents but that AlgJ remained with the spheroplast fraction. An N-terminal sequence analysis of AlgJ showed that its putative AlgJ signal peptide was not cleaved, suggesting that AlgJ is anchored to the cytoplasmic membrane by its uncleaved signal peptide. AP gene fusions were also used to map the membrane topology of AlgI, and the results suggest that it is an integral membrane protein with seven transmembrane domains. These results suggest that AlgI-AlgJ-AlgF may form a complex in the membrane that is the reaction center for O acetylation of alginate.

Acetylation↗
PubMed1962

Glycine resistance in Agrobacterium tumefaciens.

Beardsley, Robert E. (Manhattan College, New York, N. Y.). Glycine resistance in Agrobacterium tumefaciens. J. Bacteriol. 83:6-13. 1962.-The application of the fluctuation test of Luria and Delbrück to the distribution of glycine-resistant bacteria among cultures of Agrobacterium tumefaciens strain B6 indicates that resistance arises by mutation in the absence of glycine. On glycine-supplemented medium, additional resistant colonies arise during prolonged periods of incubation. Their appearance is proceded by L-form growth. In general, the number of generations over which glycine resistance is inherited in the absence of glycine is increased by serial transfers on the selection medium. In liquid medium containing glycine, sensitive bacteria form spheroplasts. Resistant bacteria continue to grow as rod forms. In the medium employed, spheroplasts are unstable.

Agrobacterium↗
PubMed1964

ENZYMES OF THE PYRIMIDINE PATHWAY IN ESCHERICHIA COLI. II. INTRACELLULAR LOCALIZATION AND PROPERTIES OF DIHYDROOROTIC DEHYDROGENASE.

Taylor, W. Herman (Portland State College, Portland, Ore.), and Mary L. Taylor. Enzymes of the pyrimidine pathway in Escherichia coli. II. Intracellular localization and properties of dihydroorotic dehydrogenase. J. Bacteriol. 88:105-111. 1964.-Intracellular localization of three enzymes of the pyrimidine pathway in Escherichia coli was studied. Dihydroorotic dehydrogenase was found to be associated with the membrane portion of lysed spheroplasts. Centrifugal fractionation of cell-free extracts showed all the dihydroorotic dehydrogenase activity to be associated with large structures, probably cell wall-membrane fragments. In contrast, all orotidylic decarboxylase activity was found in the cytoplasm in both lysed spheroplasts and cell-free extracts. Aspartate transcarbamylase activity appeared to be particulate in repressed cells, but only 25% was particulate in derepressed cells. Dihydroorotic dehydrogenase was shown to be bound to oxidative particles by oxygen uptake and orotate production from dihydroorotate. A ferricyanide reduction assay, suitable for measuring soluble and particulate enzyme, was devised for dihydroorotic dehydrogenase. Soluble dihydroorotic dehydrogenase was prepared by use of deoxycholate. A 20-fold purification of the enzyme compared to whole-cell activity was achieved by ammonium sulfate fractionation of the deoxycholate-soluble enzyme. Although cytochromes were implicated by cyanide inhibition of aerobic orotate production by particles, the purified enzyme appeared to be separated from the cytochromes, as shown by lack of cyanide inhibition in the ferricyanide assay. The purified soluble enzyme did not react in the aerobic assay previously used by others for assay of this enzyme. In contrast to the degradative dihydroorotic dehydrogenases reported by other workers, the biosynthetic dihydroorotic dehydrogenase of E. coli did not link to pyridine nucleotides.

Carboxy-Lyases↗
PubMed1966

Parasitic interaction of Bdellovibrio bacteriovorus with other bacteria.

Starr, Mortimer P. (University of California, Davis), and Nancy L. Baigent. Parasitic interaction of Bdellovibrio bacteriovorus with other bacteria. J. Bacteriol. 91:2006-2017. 1966.-The interactions of the predatory parasite, Bdellovibrio bacteriovorus, with Erwinia amylovora, Pseudomonas tabaci, and P. phaseolicola were examined by means of phase-contrast and electron microscopy. Attachment of the bdellovibrio to the host cell is apparently initially reversible; detachment occurs infrequently in the later stages. Formation of a pore in the host cell wall is followed by disorganization of the host nucleus and of the murein layer of the host cell wall. Short host cells become totally spheroplasted; the longer rods of Pseudomonas usually are partially spheroplasted. The parasite completely invades the host cell, and the cell contents of the host are digested. Bdellovibrios living as parasites inside the host increase considerably in size in comparison with those which have been living away from the host for a time. When the host protoplast is entirely lysed, the parasites leave the disintegrating "ghosted" cell envelope, and are ready to reinitiate the parasitic cycle. The time taken for a mature Bdellovibrio cell to complete the parasitic cycle may vary depending on the length of time the parasite has been away from its hosts.

Erwinia↗
PubMed1969

Thermus aquaticus gen. n. and sp. n., a nonsporulating extreme thermophile.

The isolation of a new thermophilic bacterium, Thermus aquaticus gen. n. and sp. n., is described. Successful enrichment requires incubation at 70 to 75 C, and the use of nutrient media relatively dilute with respect to the organic components. Strains of T. aquaticus have been isolated from a variety of thermal springs in Yellowstone National Park and from a thermal spring in California. The organism has also been isolated from man-made thermal habitats, such as hot tap water, in geographical locations quite distant from thermal springs. Isolates of T. aquaticus are gram-negative nonsporulating nonmotile rods which frequently form long filaments at supraoptimal temperatures or in the stationary phase. All isolates form a yellow cellular pigment, probably a carotenoid. A characteristic structure formed by all isolates is a large sphere, considerably larger than a spheroplast. These large spheres, as well as lysozyme-induced spheroplasts, are resistant to osmotic lysis. Deoxyribonucleic acid base compositions of four strains were determined by CsCl density gradient ultracentrifugation and found to be between 65.4 and 67.4 moles per cent guanine plus cytosine. The growth of all isolates tested is inhibited by fairly low concentrations of cycloserine, streptomycin, penicillin, novobiocin, tetracycline, and chloramphenicol. Nutritional studies on one strain showed that it did not require vitamins or amino acids, although growth was considerably faster in enriched than in synthetic medium. Several sugars and organic acids served as carbon sources, and either NH(4) (+) or glutamate could serve as nitrogen source. The organism is an obligate aerobe and has a pH optimum of 7.5 to 7.8. The optimum temperature for growth is 70 C, the maximum 79 C, and the minimum about 40 C. The generation time at the optimum is about 50 min. The possible relationships of this new genus to the myxobacteria, flexibacteria, and flavobacteria are discussed.

Anti-Bacterial Agents↗
PubMed1992

Reversion of Q beta RNA phage mutants by homologous RNA recombination.

Q beta phage RNAs with inactivating insertion (8-base) or deletion (17-base) mutations within their replicase genes were prepared from modified Q beta cDNAs and transfected into Escherichia coli spheroplasts containing Q beta replicase provided in trans by a resident plasmid. Replicase-defective (Rep-) Q beta phage produced by these spheroplasts were detected as normal-sized plaques on lawns of cells containing plasmid-derived Q beta replicase, but were unable to form plaques on cells lacking this plasmid. When individual Rep- phage were isolated and grown to high titer in cells containing plasmid-derived Q beta replicase, revertant (Rep+) Q beta phage were obtained at a frequency of ca. 10(-8). To investigate the mechanism of this reversion, a point mutation was placed into the plasmid-derived Q beta replicase gene by site-directed mutagenesis. Q beta mutants amplified on cells containing the resultant plasmid also yielded Rep+ revertants. Genomic RNA was isolated from several of the latter phage revertants and sequenced. Results showed that the original mutation (insertion or deletion) was no longer present in the phage revertants but that the marker mutation placed into the plasmid was now present in the genomic RNAs, indicating that recombination was one mechanism involved in the reversion of the Q beta mutants. Further experiments demonstrated that the 3' noncoding region of the plasmid-derived replicase gene was necessary for the reversion-recombination of the deletion mutant, whereas this region was not required for reversion or recombination of the insertion mutant. Results are discussed in terms of a template-switching model of RNA recombination involving Q beta replicase, the mutant phage genome, and plasmid-derived replicase mRNA.

Amino Acid Sequence↗
PubMed1994

Functional expression of P-glycoprotein in Saccharomyces cerevisiae confers cellular resistance to the immunosuppressive and antifungal agent FK520.

We have recently reported that expression in yeast cells of P-glycoprotein (P-gp) encoded by the mouse multidrug resistance mdr3 gene (Mdr3) can complement a null ste6 mutation (M. Raymond, P. Gros, M. Whiteway, and D. Y. Thomas, Science 256:232-234, 1992). Here we show that Mdr3 behaves as a fully functional drug transporter in this heterologous expression system. Photolabelling experiments indicate that Mdr3 synthesized in yeast cells binds the drug analog [125I]iodoaryl azidoprazosin, this binding being competed for by vinblastine and tetraphenylphosphonium bromide, two known multidrug resistance drugs. Spheroplasts expressing wild-type Mdr3 (Ser-939) exhibit an ATP-dependent and verapamil-sensitive decreased accumulation of [3H]vinblastine as compared with spheroplasts expressing a mutant form of Mdr3 with impaired transport activity (Phe-939). Expression of Mdr3 in yeast cells can confer resistance to growth inhibition by the antifungal and immunosuppressive agent FK520, suggesting that this compound is a substrate for P-gp in yeast cells. Replacement of Ser-939 in Mdr3 by a series of amino acid substitutions is shown to modulate both the level of cellular resistance to FK520 and the mating efficiency of yeast mdr3 transformants. The effects of these mutations on the function of Mdr3 in yeast cells are similar to those observed in mammalian cells with respect to drug resistance and transport, indicating that transport of a-factor and FK520 in yeast cells is mechanistically similar to drug transport in mammalian cells. The ability of P-gp to confer cellular resistance to FK520 in yeast cells establishes a dominant phenotype that can be assayed for the positive selection of intragenic revertants of P-gp inactive mutants, an important tool for the structure-function analysis of mammalian P-gp in yeast cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp silvaticum in long term cultures from Crohn's disease and control tissues.

Thirty one cultures were established in MG3 medium from the intestinal tissues of 29 patients, including 18 with Crohn's disease, five with ulcerative colitis, and six non-inflammatory bowel disease controls. All cultures grew either acid fast bacilli or uncharacterized spheroplasts. Pellets from these cultures were coded and assayed blind for M paratuberculosis and M avium subsp silvaticum using IS900- and IS902-PCR (polymerase chain reaction) assays, respectively. IS900 and IS902 are multicopy DNA insertion elements specific for these two organisms. Six Crohn's disease cultures and a single non-inflammatory bowel disease control were positive for M paratuberculosis. A further six cultures were positive for M avium subsp silvaticum, of which two each were from Crohn's disease, ulcerative colitis, and non-inflammatory bowel disease controls. The intensity of the IS900-PCR signals indicated very low numbers of M paratuberculosis organisms and bore no relation to visible spheroplastic or bacillary mycobacterial growth. The results suggest that M paratuberculosis isolated from man exists in a form which hardly replicates if at all when cultured in MG3 medium in vitro, and are consistent with the involvement of this known animal enteric pathogen in a proportion of chronic enteritis in man.

Base Sequence↗
PubMed1976

Stable L-forms of Clostridium perfringens and their growth on glass surfaces.

L-forms of Clostridium perfringens were induced in brain heart infusion broth containing 10% sucrose and 2 units of penicillin. After a few hours of growth, spheroplasts, granules, and elongated bacilli were apparent. At 24-h intervals, serial subcultures were made in the above medium which resulted in a culture composed entirely of spheroplasts (or protoplasts) and granules. Upon the withdrawal of penicillin these L-form cultures grew well and, after 100 passages, there was no reversion to the bacillary form. Sucrose could also be withdrawn from the medium. The effects of centrifugation, osmotic stabilizer, ultraviolet light, temperature, pH, and lyophilization upon stable L-forms were examined. L-forms were found to attach to the walls of culture tubes during trowth and sheets of L-form growth were obtained on cover slips in Leighton tubes and on the sides of medicine bottles.

Agar↗
PubMed1994

Interaction of mutants of Xenorhabdus nematophilus (Enterobacteriaceae) with antibacterial systems of Galleria mellonella larvae (Insecta: Pyralidae).

Xenorhabdus nematophilus mutants that took longer to kill insects than did the wild type were used to determine the relationship of the physicochemical properties and outer membrane composition to bacterial interaction with the antibacterial systems of Galleria mellonella larvae and to bacterial virulence. Insect serum slowed the growth of the wild-type and mutant bacteria. This was attributed to increased spheroplast formation for the mutants. Spheroplast formation was associated with an increased sensitivity to insect lysozyme and a reduction in overall bacterial cationic charge. Increasing bacterial hydrophobicity was correlated with both increased bacterial attachment to the insect's haemocytes and the accelerated removal of the bacteria from the haemolymph. Attachment of the mutants to the insect haemocytes also increased as the bacterial lipopolysaccharide content increased, the level of prophenoloxidase activation increased, and cationic charge declined. Bacterial emergence into the haemolymph occurred in parallel with haemocyte damage but neither the total lipopolysaccharide levels in the bacteria nor the rate of bacterial emergence were associated with virulence. The rate of lipopolysaccharide release into the haemolymph influenced the rate of haemocyte damage. The contribution of outer membrane proteins to lipopolysaccharide release, bacterial adhesion to haemocytes, and virulence is discussed. Virulence reflects bacterial tolerance to the host's antibacterial defences, favouring an increase in bacteria and toxic lipopolysaccharides.

Animals↗
PubMed1980

Yeast cell wall, membrane, and soluble marker polypeptides identified by comparative two-dimensional electrophoresis.

Yeast cell wall, plasma membrane, total spheroplast, and total soluble protein fractions were isolated from exponentially growing Saccharomyces cerevisiae batch cultures. The cell wall, plasma membrane, and soluble protein fractions were obtained by mechanical disruption of intact yeast cells under identical osmotic conditions. Electron micrographs of purified wall fractions appeared free of vesicular membrane contamination and micrographs of plasma membrane vesicles were free of cell wall contamination. Various stages of cell wall purification were monitored by electron microscopy and comparative two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This resulted in the identification of a glycopeptide designated 16w in the cell wall fraction, with an apparent isoelectric point of 5.0 and an apparent molecular weight of 25 000. Protein analyses of soluble and plasma membrane protein fractions failed to detect component 16w. Two-dimensional protein analyses of total cellular homogenates were capable of resolving the cell wall glycopeptide 16w. However, protein separations of spheroplasts formed by glusulase degradation of the cell wall complex did not detect 16w. These observations suggest that component 16w is unique to the cell wall fraction. In addition, comparison of two-dimensional gels of soluble and plasma membrane proteins, with a total cellular homogenate, tentatively identified several polypeptides unique to each of the soluble and plasma membrane fractions.

Cell Fractionation↗
PubMed1987

Human preproparathyroid hormone synthesized in Escherichia coli is transported to the surface of the bacterial inner membrane but not processed to the mature hormone.

cDNA encoding human preproPTH (hpreproPTH) was expressed in Escherichia coli to study the processing of the precursor to hPTH and its secretion by the bacterial secretory apparatus. We first constructed hybrid genes that differed randomly in the distance between the E. coli lac promoter's ribosomal binding site and DNA encoding a fusion protein with beta-galactosidase activity and the prepro sequence of hpreproPTH on the aminoterminus. Starting with clones identified as efficient producers of beta-galactosidase on indicator agar plates, the coding sequence for hpreproPTH was reconstituted intact. In a different construction we placed the hpreproPTH coding sequence downstream from the lac promoter at a distance of 12 base pairs from the ribosomal binding site. PTH immunoreactive proteins from multiple clones were identified by protein gel electrophoresis and by protein microsequencing. PTH-related proteins encoded by different plasmids were shown to be hpreproPTH with amino-terminal extensions of either two or four amino acids and as authentic hpreproPTH. Two hPTH fragments, hPTH(3-84) and hPTH(8-84), were also observed. The trypsin accessibility of hpreproPTH and of the two hPTH fragments in pulse-chase, cell-fractionation experiments using intact and lysed spheroplasts lets us conclude that the mammalian signal sequence directs hpreproPTH to the surface of the spheroplast membrane but is not appropriately cleaved by the signal peptidase.

Animals↗
PubMed1987

Isolation of morphologically intact mitochondrial nucleoids from the yeast, Saccharomyces cerevisiae.

Mitochondrial nucleoids (mt-nucleoids) of the yeast, Saccharomyces cerevisiae, were isolated from spheroplasts of stationary phase cells and their structure and organization were investigated by fluorescence microscopy, electron microscopy, and biochemical techniques. Isolated mt-nucleoids were spherical or ovoid and 0.3-0.6 micron in diameter, and were about the same size and shape as those observed in the cell by the DAPI staining technique. Measurement of DNA content of mt-nucleoids, using a video-intensified microscope system, after DAPI staining revealed that a mt-nucleoid in spheroplasts from stationary phase cells contains, on average, 3.9 mtDNA molecules and an isolated mt-nucleoid contains, on average, 3.1. Negatively stained electron micrographs showed that mt-nucleoids consist of particles 20-50 nm in diameter. SDS-polyacrylamide gel electrophoresis of mt-nucleoids detected 20 species of polypeptides in the molecular weight range from 10 X 10(3) to 70 X 10(3). Acid-urea/SDS two-dimensional electrophoresis of acid extract from mt-nucleoids indicated that a polypeptide of 20 X 10(3) is the only major polypeptide with basic property like histones.

DNA, Mitochondrial↗
PubMedDate not supplied

Sensitivity of Escherichia coli to viral nucleic acid, X. Ba2+-induced competence for transfecting DNA.

Effect of alkaline earth metal ions on induction of the competence for DNA transfection was investigated. Unlike spheroplasts, the bulk of the bacteria treated with these ions retains colony-forming ability. The order of effectiveness for transfection of phiA replicative-form DNA has been found to be Ba2+ greater than Ca2+ greater than Sr2+ greater than Mg2+. The competence of Ba2+-treated cells is 3 to 5 times higher that that of Ca2+-treated bacteria and about 40 times higher than that of lysozyme-EDTA spheroplasts. The Ba2+-dependent transfection is cryophilic and formation of the infective complex occurs very rapidly at 0 degrees C, But not at 37 degrees C.

Barium↗
PubMed2002

Lytic and nonlytic mechanism of inactivation of gram-positive bacteria by lysozyme under atmospheric and high hydrostatic pressure.

A different behavior was observed in three gram-positive bacteria exposed to hen egg white lysozyme by plate counts and phase-contrast microscopy. The inactivation of Lactobacillus johnsonii was accompanied by spheroplast formation, which is an indication of peptidoglycan hydrolysis. Staphylococcus aureus was resistant to lysozyme and showed no signs of peptidoglycan hydrolysis, and Listeria innocua was inactivated and showed indications of cell leakage but not of peptidoglycan hydrolysis. Under high hydrostatic pressure, S. aureus also became sensitive to lysozyme but did not form spheroplasts and was not lysed. These results suggested the existence of a nonlytic mechanism of bactericidal action of lysozyme on the latter two bacteria, and this mechanism was further studied in L. innocua. Elimination of the enzymic activity of lysozyme by heat denaturation or reduction with beta-mercaptoethanol eliminated this bactericidal mechanism. By means of a LIVE/DEAD viability stain based on a membrane-impermeant fluorescent dye, the nonlytic mechanism was shown to involve membrane perturbation. In the absence of lysozyme, high-pressure treatment was shown to induce autolytic activity in S. aureus and L. innocua.

Anti-Bacterial Agents↗
PubMedDate not supplied

[Atypical cell forms overproducing extracellular substances in population of cycad cyanobionts].

The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycas circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. The cyanobiont microcolonies grown in the intercellular space of the cyanobacterial zone of cortical parenchyma in the cycad coralloid roots contained two specific forms of vegetative cells with a reduced cell wall, namely, protoplasts and spheroplasts. The protoplasts and spheroplasts exhibited ultrastructural changes indicating the overproduction of two extracellular substances, one of which resembled the mucilage polysaccharides and the other was proteinous. The substances were likely to be synthesized intracellularly and then be excreted with the aid of surface vesicles or by channels in the cytoplasmic membrane to form, respectively, a slimy extracellular matrix and an additional electron-opaque envelope around the cell. At the late developmental stages, the excretion of these substances was accompanied by degradative changes in the cells, leading eventually to cell death. The physiological role of these specific cell forms and the factors that induce their development and death in the cell populations of cyanobionts are discussed.

Cyanobacteria↗
PubMed1991

Acidic cytochrome c6 of unicellular cyanobacteria is an indispensable and kinetically competent electron donor to cytochrome oxidase in plasma and thylakoid membranes.

Cytochromes c6 from three cyanobacteria were tested as substrates for membranous cyt. c oxidase(aa3) of Anacystis and Synechocystis using intact spheroplasts or isolated plasma(CM) and thylakoid(ICM) membranes. Neither spheroplasts nor CM/ICM gave significant O2 uptake rates with NADH without added cyt. c. Horse cyt. c (at low ionic strength) or cyt. c6 from Anacystis, Synechocystis or Microcystis (at high ionic strength) supported substantial HCN- & CO-sensitive NADH oxidase activity, consistent with in vivo O2 uptake. Cyanobacterial respiratory electron transport involves NADH dehydrogenase(fpN), plastoquinone, cyt. b/c(f), cyt. c6 & cyt. aa3, in both CM & ICM. In ICM, fpN and cyt. aa3 are functionally replaced in the light by PS II and PS I, respectively. In both membranes, cyt. c6 is an obligatory electron donor to cyt. aa3 &/or to P700. Respiratory action of acidic cyt. c6 (in unicellular species) may be unmasked only under conditions of elevated ionic strength.

Animals↗