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PubMed1974

Localization of polyribosomes containing alkaline phosphatase nascent polypeptides on membranes of Escherichia coli.

A procedure has been developed for extracting membranes from bacterial cells under conditions that keep a large fraction of bacterial polyribosomes intact. Freeze-thawing spheroplasts in the presence of deoxyribonuclease, followed by differential centrifugation, permits a separation of free and membrane-associated polyribosomes. The latter fraction contains as much as 40% of cell ribosomal ribonucleic acid (RNA) and 55% of cell messenger RNA (mRNA). Nascent polypeptides were divided almost equally between the two fractions, but 70 to 80% of alkaline phosphatase nascent chains, detected both chemically and immunologically, were derived from polyribosomes associated with the bacterial membrane. Analysis of the fractions for mRNA specific for the lac and trp operons by RNA-deoxyribonucleic acid hydridization showed somewhat larger amounts on membrane than on free polyribosomes, but enrichment for nascent alkaline phosphatase (a secreted protein) on membranes was consistently greater, suggesting that polyribosomes making secreted proteins are more tightly bound to membranes. Electron micrographs of the membrane preparations show relatively intact membranes with clusters of polyribosomes on their inner surfaces.

Alkaline Phosphatase↗
PubMed1974

Localization of proteinase(s) near the cell surface of Streptococcus lactis.

Two criteria suggest that most of the proteinase of Streptococcus lactis is localized in the cell wall. (i) Intact cells possess proteinase activity when incubated with a high-molecular-weight substrate. (ii) Most of the cell-bound proteinase activity is released during spheroplast formation under conditions which result in the release of only 1% of the intracellular enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase. The solubilized cell wall, plasma membrane, and cytoplasm fractions contained 84, 0, and 16%, respectively, of the total proteinase activity with casein as substrate. The physiological role of a surface-bound proteinase in this organism is discussed.

Caseins↗
PubMed1974

Size of the chromosome of Pseudomonas aeruginosa PAO.

Electron microscope examination and velocity sedimentation analysis of the deoxyribonucleic acid released from Pseudomonas aeruginosa spheroplasts indicate that this organism carries the bulk of its genetic determinants in a single duplex deoxyribonucleic acid molecule having a molecular mass of 2.1 x 10(9) daltons.

Carbon Radioisotopes↗
PubMed1974

Subcellular localization of isoleucine-valine biosynthetic enzymes in yeast.

By using the method of stepwise homogenization of yeast spheroplast lysates employed previously with the leucine biosynthetic enzymes, it is shown that threonine deaminase, acetohydroxy acid synthase, Mg(2+)-dependent isomero-reductase, and dihydroxy acid dehydratase are particulate. Density gradient centrifugation and the behavior of marker enzymes suggest that all of the above enzymes of the isoleucine-valine biosynthetic pathway are associated with the mitochondria.

Cell-Free System↗
PubMed1974

Expression of cryptic beta-fructofuranosidase in Saccharomyces rouxii.

Raffinose hydrolysis was studied in Saccharomyces rouxii. The responsible enzyme was identified as a beta-fructofuranosidase (EC 3.2.1.26), which has a pH optimum of 5.5 and a K(m) of 83 mM for raffinose. This enzyme was cryptic in cells from a 3-day culture. A 2-min treatment with 0.1 volume of ethyl acetate in sodium acetate buffer (pH 6) gave complete expression of the enzyme, which was still retained by the cell. Ghosts were prepared by modifying membrane structure with small basic proteins in distilled water, and after washing they showed the full complement of enzymatic activity. The enzyme remained cryptic in osmotically protected spheroplasts; however, after lysis (by dilution) release, as well as expression, was effected. Mechanical disruption of fresh cells revealed and released all of the enzyme. Cells in early stationary phase had all of their beta-fructofuranosidase in a cryptic state. Aging yielded fractional expression of activity; initially this was proportional to cell death, but later the degree of expression exceeded the death rate. Media from aged cultures or cell-free extracts of aged cells were not effective in revealing the cryptic enzyme of younger cells. S. rouxii beta-fructofuranosidase has a different autolytic-release pattern from its counterpart in S. cerevisiae. Also, high concentrations of glucose do not repress the S. rouxii enzyme. The beta-fructofuranosidase in young cells of S. rouxii must be enclosed by the protoplasmic membrane or a special vesicular structure. This system was compared with other Saccharomyces species in connection with the translocation of enzymes across the protoplasmic membrane.

Acetates↗
PubMed1975

Effect of glucose and its analogues on the accumulation and release of cyclic adenosine 3',5'-monophosphate in a membrane fraction of Escherichia coli: relation to beta-galactosidase synthesis.

Correlation between beta-galactosidase synthesis and cyclic adenosine 3',5'-monophosphate (cAMP) levels in a membrane fraction obtained from disrupted spheroplasts of Escherichia coli was investigated. Repression of beta-galactosidase synthesis in the membrane fraction by glucose-6-phosphate and by 2-deoxyglucose differed in sensitivity to reversal by cAMP. The difference between the two repressions could be due to the fact that glucose-6-phosphate inhibited severely the accumulation of exogenous [3-H]cAMP by the membrane fraction, whereas 2-deoxyglucose had little effect on the accumulation of the nucleotide. On the other hand, a quick decrease in the level of [3-H]cAMP preaccumulated in the membrane fraction resulted from addition of either glucose-6-phosphate or 2-deoxyglucose. Results reported here suggest that repression of beta-galactosidase synthesis is associated with anabrupt decrease in cAMP levels at the intramembranal sites where beta-galactosidase is synthesized, and the major, if not sole, mechanism which leads to instantaneous drop of cAMP level is via the release of cAMP, but not by degradation of the nucleotide since the membrane fraction retained less than 10 percent of cellular cyclic phosphodiesterase and the activity of the enzyme was not affected by repressing sugars.

Adenylyl Cyclases↗
PubMed1975

Attachment of lipoprotein to murein (peptidoglycan) of Escherichia coli in the presence and absence of penicillin FL 1060.

In vivo studies on the attachment of lipoprotein to the murein (peptidoglycan) of Escherichia coli showed that it takes several generations of growth until the amount of lipoprotein on newly made murein is equilibrated. The technique used involves degradation of the sodium dodecyl sulfate-insoluble murein-lipoprotein complex (sacculus, rigid layer) with lysozyme and separation of the labeled products on paper. No lipoprotein was found on murein subunits incorporated during a pulse of [3H]diaminopimelate for 1 min in logarithmically growing cells at 37 C. Even after one doubling of the cell mass, only 4 to 8% of the labeled murein was isolated as bound to lipoprotein. With uniformly labeled murein, 30% remains bound to lipoprotein after lysozyme treatment, corresponding to three murein subunits. Therefore it can be concluded that during pulse labeling either no lipoprotein is incorporated into the newly synthesized murein or no murein subunits are inserted into existing murein around lipoprotein attachment sites. Longer pulse and pulse-chase experiments argue for the latter interpretation. It is therefore concluded that incorporation of murein subunits into the growing murein polymer is not at all a random process. Instead, quite large areas of murein, on which lipoprotein is situated, seem to be preserved. Under the influence of penicillin FL 1060 murein synthesis is 50% inhibited. The rate of lipoprotein attachment is less affected so that increasing amounts of lipoprotein become attached during spheroplast formation. By the time the stationary growth phase has been reached, the lipoprotein content of the murein has doubled. Diaminopimelate auxotrophic mutants require, in the presence of penicillin FL 1060, more diaminopimelate for full growth than in the absence of penicillin FL 1060. This finding and the fact that murein synthesis is always inhibited by 50% over a wide range of penicillin concentration (1 to 1,000 mug/ml) point to the inhibition of an enzymatic step of murein synthesis which can be partially bypassed by a second enzyme, less efficient but resistant to penicillin FL 1060.

Binding Sites↗
PubMed1975

Methionine transport in Yersinia pestis.

Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the methionine transport system because of this requirement. The methionine system showed saturation kinetics with a Km for transport of approximately 9 times 10(-7) M. After 8 s of methionine transport, essentially all of the methionine label appeared in S-adenosyl-L-methionine (SAM) as detected in ethanol extracts. Small amounts of free methionine was detected intracellularly after 1 min of transport. Addition of glucose increased significantly the amount of intracellular methionine at 1 min. A series of SAM metabolic products was detected after 90 s to 5 min of transport including: 5'-thiomethyladenosine, homoserine lactone, S-adenosyl homoserine, and a fluorescent methyl receptor compound. Results from assays for SAM synthetase in spheroplast fractions showed a small (16%) but significant portion of synthetase associated with the membrane. However, most of the enzyme activity was associated with the cytoplasmic fraction. Methionine transport was characterized by a high degree of stereospecificity. No competition occurred from structurally unrelated amino acids. Although uptake was inhibited by uncoupling and sulfhydryl reagents, no efflux was observed. Results using energy inhibitors on unstarved and starved cells showed that respiratory inhibitors such as potassium cyanide (KCN) and amytal were most effective, and that arsenate was least effective. KCN plus arsenate completely blocked utilization of energy derived from glucose, and KCN completely blocked utilization of energy deived from D-lactate. The data indicate that methionine transport in Y. pestis is linked to the trapping of methionine in SAM. The results further suggest that this transport system can be classified as a permease-bound system where transport is coupled to an energized membrane state and to respiration.

Amobarbital↗
PubMed1975

Localization of D-lactate dehydrogenase in membrane vesicles prepared by using a french press or ethylenediaminetetraacetate-lysozyme from Escherichia coli.

The localization of D-lactate dehydrogenase in membrane vesicles prepared from Escherichia coli was studied using antibody against the purified enzyme. The activity of D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by using a French press were completely inhibited by this antibody, suggesting that the enzyme is localized on the outside of these vesicles. This and previous results (Futai, 1974) strongly indicate the inversion of these vesicles. The D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by treatment with ethylenediaminetetraacetate-lysozyme were inhibited about 15% by the antibody, whereas proline transport of the vesicles was insensitive to antibody. These results suggest that most of the membrane vesicles have D-lactate dehydrogenase on the inside of the membrane and that such vesicles transport amino acids. This essentially confirms the results of Short, Kaback, and Kohn (1975). However, unlike them we observed that a small but significant portion of activity was sensitive to the antibody as shown above. This portion may represent the completely inverted vesicles in the preparation. Ferricyanide reductase activity cannot be detected in spheroplasts, but about 30 to 50% of the total was detected in membrane vesicles prepared by treatment with ethylenediaminetetraacetate. This confirms our previous findings with membrane prepared by a slightly different procedure. It is concluded that in these vesicles about half the reactive sites for ferricyanide are moved from inside to outside the membrane, whereas 85% of the D-lactate dehydrogenase remains inside the membrane.

Antibodies, Bacterial↗
PubMed1976

Turnover of polyadenylate-containing ribonucleic acid in Saccharomyces cerevisiae.

We examined the kinetics of incorporation of [3H]adenine into polyadenylate-containing ribonucleic acid [poly(A)-containing RNA] in yeast. The total poly(A)-containing RNA from spheroplasts and intact cells and the polysomal poly(A)-containing RNA exhibited similar incorporation kinetics. At 30 C half-saturation of the pool of poly(A)-containing RNA with label occurred in approximately 22 min. Since precursor pools appeared to require 5 min to saturate with label, we conclude that at 30 C messenger RNA molecules in yeast decay with an average half-life of 17 min.

Adenine↗
PubMed1976

Cell division in Pseudomonas aeruginosa: participation of alkaline phosphatase.

Pseudomonas aeruginosa grows at an apparent reduced rate at 46 C as compared with the rate at 37 C, when growth is measured as an increase in absorbance. Cells at 46 C are long, plasmolyzed, nonmotile filaments. The filaments contain phase-dark material that may be chromosomal in nature. When the 46 C culture is shifted to 37 C, the filaments fragment at polar ends after flagella form, and the final number of cells is equal to the number of chromosomal "packets" observed within the filament. The outer envelope of the filament appears to be structurally complete as determined by biochemical, thin section, and freeze-etch examination. When filaments are treated with lysozyme, they form large spheroplasts, suggesting that the outer wall and the cytoplasmic membrane are continuous within the filament. Filaments produce little or no periplasm-located alkaline phosphatase (APase), but activity appears immediately after a shift to 37 C. Cells grown at 37 C and shifted to 46 C remain as single, nonmotile, rods or doublets, and the APase formed at 37 C remains stable at 46 C. The addition of APase or inorganic phosphate is partially or completely effective as an inducer of filament fragmentation at 46 C. The results suggest that periplasm-located APase is an important enzyme in the final stages of cell division when P. aeruginosa is cultured on inorganic phosphate-limiting media.

Alkaline Phosphatase↗
PubMed1976

Comparative studies on membrane-associated, folded chromosomes from Escherichia coli.

Nuclear bodies were isolated from Escherichia coli spheroplasts by two different lysis procedures. Their lipid and protein content and the superhelix density was measured. The preparations differed mainly in respect to the amount of the attached membrane material, which seems to be an essential factor in maintaining the stability of the nuclear bodies. The superhelix density was found to be higher than that of naturally occurring, covalently closed, circular deoxyribonucleic acids. The influence of the preparative method on the superhelicity was comparatively small.

Bacterial Proteins↗
PubMed1976

Magnitude of the protonmotive force in respiring Staphylococcus aureus and Escherichia coli.

The membrane potential and pH gradient developed across the plasma membranes of whole cells of Staphylococcus aureus and spheroplasts of Escherichia coli were estimated. The distributions of potassium ions in the presence of valinomycin and the pH gradient across the membrane were determined from the changes in pK and pH observed in the external medium during transition from the energized respiring state to the de-engerized resting condition. The protonmotive force in respiring cells was estimated at 211 mV for S. aureus and 230 mV for E. coli at external pH values of approximately 6.5. The adequacy of these protonmotive forces as a driving force for substrate accumulation or adenosine 5'-triphosphate synthesis is discussed.

Adenosine Triphosphate↗
PubMed1977

Functional mosaicism of membrane proteins in vesicles of Escherichia coli.

Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source. However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles. Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis. These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles. Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.

Adenosine Triphosphatases↗
PubMed1977

Intracellular localization of the superoxide dismutases of Escherichia coli: a reevaluation.

All of the superoxide dismutase isozymes of Escherichia coli have been shown to occur in the cell matrix, and none have been found in the periplasm. This was the case with both E. coli B and E. coli K-12, whether grown on a low phosphate medium or on a Trypticase soy-yeast extract medium. Alkaline phosphatase was used as a marker of the periplasm; adenosine deaminase and glucose 6-phosphate dehydrogenase were used as matrix markers, and consistent results were obtained by osmotic shock, spheroplast formation, and use of a diazonium salt that penetrates the periplasm but cannot cross the plasma membrane. A previous report that the iron-containing superoxide dismutase of E. coli is a periplasmic enzyme is now seen to have been in error.

Adenosine Deaminase↗
PubMed1977

Lysozyme-promoted association of protein I molecules in the outer membrane of Escherichia coli.

Incubation of whole envelopes prepared from sonically oscillated Escherichia coli K-12 cultures with lysozyme in vitro resulted in the appearance of a protein species with an apparent molecular weight double that of outer membrane protein I. Similar dimers were also detected in purified outer membranes and whole envelopes from lysozyme-induced spheroplasts of E. coli K-12. This was confirmed by two-dimensional electrophoresis in which the dimers were resolved in the second dimension to run as single polypeptides of protein I. Formation of dimers was correlated with peptidoglycan degradation, but the ability of protein I molecules to associate may vary between strains of E. coli, since dimers were found only in outer membranes from E. coli W7. We suggest that extensive degradation of peptidoglycan leads to nonspecific formation of protein I aggregates, but that these aggregates do not occur in vivo.

Bacterial Proteins↗
PubMed1977

Inhibitory action of a non-metabolizable fatty acid on the growth of Escherichia coli: role of metabolism and outer membrane integrity.

The inhibitory action of decanoic acid on both Escherichia coli K-12/154 (normal lipopolysaccharide) and E. coli RC59 (defective lipopolysaccharide) was studied. A correlation was found between the doubling time of E. coli 154 growing in different media and the lethal effect of 0.4% decanoic acid on this bacterium. Decanoic acid (0.4%) exerted a lytic action on glucose-starved and NaN3-inhibited cells of E. coli 154 and RC59. Exponentially growing cultures of both strains were not affected by the addition of 0.4% methyldecanoate, but cells of E. coli RC59 reaching the stationary phase were attacked by that compound. A bactericidal action of 0.4% methyldecanoate on exponential E. coli 154 and RC59 was observed when sodium azide was also present in the media. Concentrations lower than 0.01% methyldecanoate had a lytic effect on spheroplasts from E. coli 154 and RC59. These results indicate that the inhibitory action of a non-metabolizable fatty acid on E. coli depends on the cellular metabolic activity and the outer membrane integrity.

Azides↗