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PubMed1986

Activation of Bordetella pertussis adenylate cyclase by the carboxyl-terminal tryptic fragment of calmodulin.

Highly purified tryptic fragments of calmodulin were tested for their ability to stimulate adenylate cyclase activity of Bordetella pertussis spheroplast membranes and were compared to their activities on brain Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase. The C-terminal fragment, consisting of residues 78-148, was a full agonist for the cyclase with 0.1-0.15 the potency of calmodulin but did not stimulate phosphodiesterase. Fragments 1-77, 1-90, and 107-148 stimulated adenylate cyclase (and not phosphodiesterase) at low potency; this was not due to calmodulin contamination, but contamination by fragment 78-148 could not be excluded with certainty. An adduct of norchlorpromazine isothiocyanate and calmodulin showed full agonist activity for adenylate cyclase at 0.01-0.02 the potency of calmodulin. Stimulation of adenylate cyclase by a number of the fragments occurred in the absence of Ca2+, but stimulator potency was enhanced 20-60-fold in its presence. The similarity of Ca2+ requirements of fragment 78-148 and calmodulin suggests that occupancy of the two C-terminal Ca2+ binding sites of calmodulin accounts for most of the Ca2+ enhancement of calmodulin stimulation of adenylate cyclase.

Adenylyl Cyclases↗

PubMed1977

Inhibition of Escherichia coli growth and respiration by polymyxin B covalently attached to agarose beads.

Polymyxin B was attached to agarose beads by stable covalent bonds and the antimicrobial activity of the immobilized peptide was examined. Polymyxin-agarose inhibited the growth of Escherichia coli and Pseudomonas aeruginosa, but not Bacillus subtilis. In addition, the respiration of E. coli, E. coli spheroplasts, and B. subtilis protoplasts was inhibited by immobilized polymyxin, whereas the respiration of B. subtilis was unaffected by polymyxin-agarose. The activity of polymyxin-agarose was not due to the release of free peptide from the derivative. These data indicate that polymyxin can inhibit the growth and respiration of gram-negative bacteria by interacting with the outer surface of these cells. It is proposed that perturbation of outer membrane structure by polymyxin-agarose indirectly affected the selective permeability of the inner membrane and inhibited respiration. The results of this study emphasize the importance of outer membrane structural integrity for the normal functions of gram-negative bacteria.

Bacillus subtilis↗

PubMed1976

Control of ribonucleic acid synthesis in eukaryotes. 2. The effect of protein synthesis on the activities of nuclear and total DNA-dependent RNA polymerase in yeast.

A thermosensitive conditional yeast mutant (ts-187) which suppresses protein synthesis at the nonpermissive temperature (36 degrees C) also suppresses RNA synthesis. The effect of temperature on the mutant is similar to the addition of cycloheximide--it inhibits the incorporation of labeled precursors into RNA in both whole cells and isolated nuclei. The effect of temperature is selective for the RNA polymerases bound to the nuclear template but not for the total RNA polymerases. Thus, the specific activities and total amounts of RNA polymerase species extracted and assayed with exogenous DNA template are similar in the ts-187 cultured at 23 degrees C and at 36 degrees C. On the contrary, the nuclear polymerases, i.e., RNA synthesis in isolated nuclei, are dramatically inhibited in cells cultured at 36 degrees C. When amino acid starved ts-187 cells are transferred to 36 degrees C, release from the inhibtion of RNA synthesis is observed. As with the addition of cycloheximide, this relaxation is observed in cells but not in isolated nuclei. The parental strain, A364A, which responds by stimulating instead of inhibiting protein synthesis when the temperature is increased to 36 degrees C, also exhibits an inhibition in the incorporation of labeled precursor into RNA as well as reducing RNA synthesis in isolated nuclei. However, these are transitory inhibitions and afterward there is reinitiation of both processes. Reinitiation of RNA synthesis in isolated nuclei is similar to the relaxed phenomenon and it is called "nuclear relaxation". This relaxation can only be obtained if protein synthesis is not inhibited; however, cellular relaxation occurs in the absence of protein synthesis. The repression of the nuclear RNA polymerase activities which starvation and inhibition of protein synthesis produce appears to be due to a restriction in the nuclear DNA template. This notion is supported by the fact that a net diminution of these nuclear enzyme activities is observed in spheroplasts cultured under starving conditions. Studies of the four main ribonucleotide pools indicate that stringency and inhibition of protein synthesis (ts-187 cultured at 36 degrees C) produce an increase in UTP and CTP pools. This is consistent with the concept that stringency and inhibition of protein synthesis affect the rate of utilization rather than the synthesis of these ribonucleotide residues. In the A364A and ts-187 yeast strains, the conversion of uracil but not of uridine into the UTP and CTP is inhibited when there is inhibition of the nuclear RNA polymerases. This indicates that the uracil phosphoribosyltransferase but not the uridine-cytidine kinase is allosterically inhibited by UTP and CTP in yeast. The feedback inhibition in the metabolic pathway of the base explains why relaxation cannot be detected when uracil instead of uridine is used as the labeled RNA precursor.

Cell Nucleus↗

PubMed1976

Properties of a major protein released from Escherichia coli by osmotic shock.

A large fraction of a constitutively synthesized polypeptide, comprising 5% of the total Escherichia coli protein, is released when plasmolysed cells are subjected to osmotic shock into ice-cold water. Since the protein is not liberated by the conversion of cells to spheroplasts, it is not a typical periplasmic protein. A complex pattern of association with the cell envelope indicates that it is bound to this structure in vivo. Its susceptibility to trypsin and its interaction with specific antibodies vary with the type of preparations used. Based on these observations, we postulate a peripheral location at the inner surface of the plasma membrane. The protein has been purified to homogeneity from osmotic shock fluid. It has a mass of 44 000 daltons. Some of its physical and chemical properties have been investigated. Most remarkable are its strongly aggregating and adhesive characteristics and its precipitation by vinblastine and calcium ions. These unusual properties, its presumed location, and the observation that it is present in large amounts (approximately 70 000 molecules per cell) suggest a structural role for this protein.

Amino Acids↗

PubMed2003

Bestowing antifungal and antibacterial activities by lipophilic acid conjugation to D,L-amino acid-containing antimicrobial peptides: a plausible mode of action.

The dramatically increased frequency of opportunistic fungal infections has prompted research to diversify the arsenal of antifungal agents. Antimicrobial peptides constitute a promising family for future antibiotics with a new mode of action. However, only a few are effective against fungal pathogens because of their ability to self-assemble. Recently, we showed that the conjugation of fatty acids to the potent antibacterial peptide magainin endowed it with antifungal activity concomitant with an increase in its oligomeric state in solution. To investigate whether a high potency of the parental peptide is prerequisite for antifungal activity, we conjugated undecanoic acid (UA) and palmitic acid (PA) to inactive diastereomers of magainin containing four d-amino acids ([D]-4-magainin), as well as to a weakly active diastereomeric lytic peptide containing Lys and Leu ([D]-K(5)L(7)). All lipopeptides gained potent activity toward Cryptococcus neoformans. Most importantly, [D]-K(5)L(7)-UA was highly potent against all microorganisms tested, including bacteria, yeast, and opportunistic fungi. All lipopeptides increased the permeability of Escherichia coli spheroplasts and intact C. neoformans, as well as their corresponding membranes, phosphatidylethanol (PE)/phosphatidylglycerol (PG) and phosphatidylcholine (PC)/PE/phosphatidylinositol (PI)/ergosterol, respectively. The extent of membrane-permeating activity correlated with their biological function, suggesting that the plasma membrane was one of their major targets. Circular dichroism (CD) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy revealed that their mode of oligomerization in solution, structure, and organization in membranes have important roles regarding their antibacterial and antifungal activities. Together with the advantage of using diastereomers versus all l-amino acid peptides, this study paves the way to the design of a new group of potent antifungal peptides urgently needed to combat opportunistic fungal infection.

Amino Acid Sequence↗

PubMed2004

Neosynthesis of cardiolipin in Rhodobacter sphaeroides under osmotic stress.

The phospholipid composition of Rhodobacter sphaeroides cells resuspended in various hypertonic solutions has been examined by thin-layer chromatography and ESI mass spectrometry. R. sphaeroides responds to hyperosmotic stress by increasing the amount of cardiolipin in the membranes; this phenomenon occurs in spheroplasts also. Cardiolipin increases quickly and continuously during the time when the cells are resuspended in hypertonic medium. The optimum of stimulation of the neosynthesis of cardiolipin during osmotic stress was found to be at external 1 osm. ESI-MS analyses allowed the identification of two different cardiolipins in R. sphaeroides: the tetravaccenylcardiolipin ([M - H](-), m/z 1456.9) and the trivaccenylmonopalmitoylcardiolipin ([M - H](-), m/z 1430.0).

Cardiolipins↗

PubMed1996

Identification of retained N-formylmethionine in bacterial recombinant mammalian cytochrome P450 proteins with the N-terminal sequence MALLLAVFL...: roles of residues 3-5 in retention and membrane topology.

An N-terminal block to Edman degradation was observed when any of five different mammalian cytochrome P450 (P450) proteins was expressed in Escherichia coli using the N-terminal sequence MALLLAVFL... This block was also seen in Salmonella typhimurium. With all proteins examined, the block could be removed by mild acid hydrolysis (0.6--6 N HCl, 23 degrees C) to expose Met as the N-terminus, suggesting N-formylMet retention. The N-terminal peptide of a modified P450 1A2 ("mutant 1", containing a thrombin-sensitive site inserted at residue 25) was released with thrombin and analyzed by electrospray mass spectrometry and found to yield the M(r) expected for the N-formyl derivative (+/- 0.8 amu). The region of positions 3--5 was altered by random mutagenesis, and three P450 1A2-expressing clones were analyzed for nucleotide and amino acid sequences. The changes from LLL were to RER (P450 1A2a), VDS (P450 1A2b), and WRH (P450 1A2c); these all show slightly dissimilar hydropathy plots compared to the MALLLAVFL... sequence. Mutant P450 1A2a had the N-terminal Met removed to yield N-terminal Ala; P450 1A2b contained an unmodified Met at the N-terminus; P450 1A2c had an approximately 80% block of the N-terminal Met. Experiments with bacterial membranes containing expressed P450 1A2 mutant 1 and P450 1A2 mutant 2 (thrombin-sensitive site inserted at residue 46) suggest that thrombin site 2, but not 1, is sequestered in the membrane. Spheroplasts of bacteria expressing P450 1A2 and the mutants at positions 3--5 were treated with proteinase K; amino acid analysis indicated that no cleavage occurred. These results are interpreted in a model in which most of the mammalian P450 expressed in the bacterium is located in the cytosol, the region near residue 46 is in the inner membrane, the region near residue 25 is in the cytosol, and the N-terminus is either imbedded in the membrane or free in the cytosolic space, depending upon the sequence. However, the possibility that the differences in N-terminal processing are the result of direct changes in interactions with the deformylase and Met aminopeptidase cannot be excluded.

Amino Acid Sequence↗

PubMed2002

NADH is specifically channeled through the mitochondrial porin channel in Saccharomyces cerevisiae.

In many kinds of permeabilized cells, the restriction of metabolite diffusion by a mitochondrial porin "closed state" has been shown to control the respiration rate. However, since in isolated mitochondria the porin appears to be always "open," the physiological relevance of a putative regulation via this channel status is now a subject of discussion. In Saccharomyces cerevisiae, in which some of the NADH dehydrogenase active sites are facing the intermembrane space, this regulatory mechanism might play an important role for the regulation of the cytosolic redox status. Using permeabilized spheroplasts from wild-type and porin-deficient mutant, we show that the NADH produced in the cytosol is channeled to the mitochondrial NADH dehydrogenases through a metabolic network involving the porin channel. Thus, the control exerted by the porin (i.e., "open" or "closed" state) seems to be determined through its participation or not in organized metabolic networks.

Cytosol↗

PubMed2001

Conversion of Borrelia garinii cystic forms to motile spirochetes in vivo.

Cystic forms (also called spheroplasts or starvation forms) and their ability to reconvert into normal motile spirochetes have already been demonstrated in the Borrelia burgdorferi sensu lato complex. The aim of this study was to determine whether motile B. garinii could develop from cystic forms, not only in vitro but also in vivo, in cyst-inoculated mice. The cysts prepared in distilled water were able to reconvert into normal motile spirochetes at any time during in vitro experiments, lasting one month, even after freeze-thawing of the cysts. Motile spirochetes were successfully isolated from 2 out of 15 mice inoculated intraperitoneally with cystic forms, showing the infectivity of the cysts. The demonstrated capacity of the cysts to reconvert into motile spirochetes in vivo and their surprising resistance to adverse environmental conditions should lead to further studies on the role and function of these forms in Lyme disease.

Animals↗

PubMed1993

Germ-line transmission and expression of a human-derived yeast artificial chromosome.

Introduction of DNA fragments, hundreds of kilobases in size, into mouse embryonic stem (ES) cells would greatly advance the ability to manipulate the mouse genome. Mice generated from such modified cells would permit investigation of the function and expression of very large or crudely mapped genes. Large DNA molecules cloned into yeast artificial chromosomes (YACs) are stable and genetically manipulable within yeast, suggesting yeast-cell fusion as an ideal method for transferring large DNA segments into mammalian cells. Introduction of YACs into different cell types by this technique has been reported; however, the incorporation of yeast DNA along with the YAC has raised doubts as to whether ES cells, modified in this way, would be able to recolonize the mouse germ line. Here we provide, to our knowledge, the first demonstration of germ-line transmission and expression of a large human DNA fragment, introduced into ES cells by fusion with yeast spheroplasts. Proper development was not impaired by the cointegration of a large portion of the yeast genome with the YAC.

Animals↗

PubMed1976

Competition between trichodermin and several other sesquiterpene antibiotics for binding to their receptor site(s) on eukaryotic ribosomes.

1. Of the five sesquiterpene antibiotics tested and found to inhibit protein synthesis in yeast spheroplasts, trichothecin, trichodermol or trichodermin stabilized polyribosomes whereas, in contrast, verrucarin A or T-2 toxin induced 'run off' of polyribosomes with a corresponding increase in 80S monoribosomes. The effect of fusarenon X on the system could not be determined as the drug failed to enter the cells. 2. [acetyl-14C]Trichodermin bound to yeast polyribosomes with a dissociation constant of 2.10 muM and to yeast 'run off' ribosomes with a dissociation constant of 0.72 muM. 3. Trichothecin, trichodermol, fusarenon X, T-2 toxin and verrucarin A competed with [acetyl-14C]trichodermin for binding to its receptor site on 'run off' ribosomes. The observed competition was quantitatively similar for all drugs tested. In contrast, the five drugs competed to different extents with trichodermin for binding to its receptor site on polyribosomes. Thus trichothecin competed with relative efficiency, whereas verrucarin A competed poorly, and the other drugs occupied intermediate positions between these two extremes. 4. Studies were also carried out with yeast 'run off' ribosomes prepared from both a wild-type strain and a strain resistant to trichodermin. Competition experiments between verrucarin A and [3H]anisomycin indicated that verrucarin A bound to 'run off' ribosomes from the mutant strain less efficiently than to those from the wild-type.

Anisomycin↗

PubMed1978

The location of purine phosphoribosyltransferase activities in Escherichia coli.

During the preparation of spheroplasts, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) were released in parallel with cytidine deaminase (EC 3.5.4.5) and uridine phosphorylase (EC 2.4.2.3), which, on other evidence, are considered to be located intracellularly. The two phosphoribosyltransferases and uridine phosphorylase were not significantly associated with purified membrane fractions as was purine nucleoside phosphorylase (EC 2.4.2.1). The effects of the poorly permeable enzyme-inactivating reagents, 4-diazoniumbenzenesulphonate, 7-diazonium-1,3-naphthalene-disulphonate and 2,4,6-trinitrobenzenesulphonate, on Escherichia coli indicate that all the above-mentioned enzymes and also the xanthine-guanine phosphoribosyltransferase [Miller, Ramsey, Krenitsky & Elion (1972) Biochemistry 11, 4723--4731] are located intracellularly.

Adenine Phosphoribosyltransferase↗

PubMed1980

Phosphatidylethanolamine distribution and fluidity in outer and inner membranes of the gram-negative bacterium Erwinia carotovora.

1. The distribution of phosphatidylethanolamine, the major lipid of Erwinia carotovora, was investigated in intact bacteria, spheroplasts and outer- and inner-membrane preparations, with the amino-group reagent 2,4,6-trinitrobenzenesulphonic acid. Only 4% was found on the external surface of the outer membrane with 30% on the internal surface, whereas the inner membrane had 27 and 38% on its external and internal surfaces respectively. Some comparative studies were made with three other bacteria. 2. The fluidity of the membranes of E. carotovora was studied by using the fluorescent probe 1,6-diphenylhexa-1,3,5-triene. Results were consistent with the hydrocarbon region of the outer membrane bilayer being less fluid than that of the inner one. 3. On the basis of these and other results a model for the outer- and inner-membrane structures of E. carotovora is proposed.

Cell Membrane↗

PubMed1980

The role of the membrane-bound hydrogenase in the energy-conserving oxidation of molecular hydrogen by Escherichia coli.

H2-dependent reduction of fumarate and nitrate by spheroplasts from Escherichia coli is coupled to the translocation of protons across the cytoplasmic membrane. The leads to H+/2e- stoicheiometry (g-ions of H+ translocated divided by mol of H2 added) is approx. 2 with fumarate and approx. 4 with nitrate as electron acceptor. This proton translocation is dependent on H2 and a terminal electron acceptor and is not observed in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide. H2-dependent reduction of menadione and ubiquinone-1 is coupled to a protonophore-sensitive, but 2-n-heptyl-4-hydroxy-quinoline N-oxide-insensitive, proton translocation with leads to H+/2e- stoicheiometry of approx. 2. H2-dependent reduction of Benzyl Viologen (BV++) to its radical (BV+) liberates protons at the periplasmic aspect of the cytoplasmic membrane according to the reaction: H2 + 2BV++ leads to 2H+ + 2BV+. It is concluded that the effective proton translocation observed in the H2-oxidizing segment of the anaerobic respiratory chain of Escherichia coli arises as a direct and inevitable consequence of transmembranous electron transfer between protolytic reactions that are spatially separated by a membrane of low proton-permeability.

Escherichia coli↗

PubMed1990

The cellular location and specificity of bacterial cytochrome c peroxidases.

The locations of cytochrome c peroxidase and catalase activities in the two Gram-negative bacteria Pseudomonas stutzeri (N.C.I.B. 9721) and Paracoccus denitrificans (N.C.I.B. 8944) were investigated by the production of spheroplasts. In both species the cytochrome c peroxidase was predominantly periplasmic: 92% of total activity in Ps. stutzeri and 98% of nonmembrane-bound activity in Pa. denitrificans were found in this cellular compartment. In contrast, the catalase was mostly in the cytoplasmic fraction. Purification of the Pa. denitrificans cytochrome c peroxidase showed it to be the haem c-containing polypeptide of Mr 42,000 that has already been described by Bosma, Braster, Stouthamer & Van Versefeld [(1987) Eur. J. Biochem. 165, 665-670] but was not identified by them as a peroxidase. The visible-absorption spectra of the enzyme closely resemble those of cytochrome c peroxidase from Pseudomonas aeruginosa but the donor specificity is different, with the Pa. denitrificans enzyme preferring the basic mitochondrial cytochromes c to the acidic cytochromes c-551 and reacting well with the Pa. denitrificans cytochrome c-550.

Bacterial Proteins↗

PubMed1995

Topological organization of the Rieske iron-sulphur protein and subunit IV in the cytochrome bc1 complex of Rhodobacter sphaeroides.

The ubiquinol-cytochrome c2 oxidoreductases (cytochrome bc1 complex) of Rhodobacter sphaeroides contains highly conserved cytochrome b, cytochrome c1 and Rieske FeS subunits, as well as a unique 14 kDa polypeptide, designated as subunit IV, thought to function as a ubiquinol-binding protein [Yu and Yu (1991) Biochemistry 30, 4934-4939]. As the topology of subunit IV is unknown and that of the FeS subunit remains a matter of debate, both the inner (cytoplasmic) and outer (periplasmic) surfaces of the intracytoplasmic membrane (ICM) were digested with proteinase K, and cleavage products were identified by immunoblotting. In uniformly oriented chromatophore vesicles (inner ICM surface exposed), fragments of approx. 4 and 1 kDa were removed from subunit IV and the FeS protein respectively. Neither subunit IV nor the FeS protein was cleaved from the outer ICM surface as exposed in osmotically protected spheroplasts or as presented to proteinase K after microencapsulation of the protease in unilamellar liposomes and fusion of these structures to chromatophore vesicles. Studies with the isolated bc1 complex, however, suggested that the C-terminal domain of the Rieske FeS, thought to reside on the periplasmic side of the ICM, was resistant to proteinase K. Overall, these results suggest a single N-terminal transmembrane helix for the FeS protein, with exposure of the N-terminus to the cytoplasm and an orientation in which a major, N-terminal portion of subunit IV is located in the cytoplasm with the predicted C-terminal transmembrane domain anchoring this polypeptide to the membrane.

Bacterial Chromatophores↗

PubMed1972

Precursor and product in bacteriophage lambda recombination.

Analyses of the time of recombination in bacteria infected with phage lambda and in spheroplasts infected with purified phage lambda DNA indicate a preponderance of recombination occurring before replication ("early recombination") after infection with tandem dimers formed in vitro compared to linear monomer DNA and phage infections. Among the early recombinants is a large fraction of cells that produces one recombinant type exclusively. Two suggestions are discussed-that a dimer-like molecule is the precursor of the recombinant progeny molecule and that one recombinant chromosome is the frequent or exclusive product of a recombinational event.

Centrifugation, Density Gradient↗

PubMed1974

Correlation between thermal death and membrane fluidity in Bacillus stearothermophilus.

Paramagnetic resonance spectra of spin labels partitioned into spheroplast membranes of Bacillus stearothermophilus indicate lateral lipid phase separations. Cells adjust their lipid composition in response to temperature changes so that the same change of state in membrane phospholipids is achieved at the respective growth temperature. A temperature-sensitive mutant that fails to change its lipid composition above a certain temperature can survive only up to the higher temperature boundary for lateral phase separation. These data are interpreted to indicate that the maximal and minimal growth temperatures of thermophiles are regulated by the onset and conclusion of phase separations of the particular lipid composition they synthesize. It is suggested that isolated lipid domains are required for functional membrane assembly.

Bacillus↗