Showing papers in "Journal of Bacteriology in 1974"
TL;DR: A new minimal medium for enterobacteria is developed that supports growth of Escherichia coli and Salmonella typhimurium at rates comparable to those of any of the traditional media that have high phosphate concentrations, but each of the macronutrients is present at a sufficiently low level to permit isotopic labeling.
Abstract: A new minimal medium for enterobacteria has been developed. It supports growth of Escherichia coli and Salmonella typhimurium at rates comparable to those of any of the traditional media that have high phosphate concentrations, but each of the macronutrients (phosphate, sulfate, and nitrogen) is present at a sufficiently low level to permit isotopic labeling. Buffering capacity is provided by an organic dipolar ion, morpholinopropane sulfonate, which has a desirable pK (7.2) and no apparent inhibitory effect on growth. The medium has been developed with the objectives of (i) providing reproducibility of chemical composition, (ii) meeting the experimentally determined nutritional needs of the cell, (iii) avoiding an unnecessary excess of the major ionic species, (iv) facilitating the adjustment of the levels of individual ionic species, both for isotopic labeling and for nutritional studies, (v) supplying a complete array of micronutrients, (vi) setting a particular ion as the crop-limiting factor when the carbon and energy source is in excess, and (vii) providing maximal convenience in the manufacture and storage of the medium.
2,930 citations
TL;DR: A modified transformation procedure that is effective for the introduction of plasmid deoxyribonucleic acid at high frequency into Salmonella typhimurium, as well as into Escherichia coli, is described.
Abstract: A modified transformation procedure that is effective for the introduction of plasmid deoxyribonucleic acid at high frequency into Salmonella typhimurium, as well as into Escherichia coli, is described. Transformed bacteria acquire a circular deoxyribonucleic acid species having the genetic and molecular characteristics of the transforming plasmid.
769 citations
TL;DR: Data to be described are interpreted to suggest that the transfer is by a conjugal mechanism and that the multiple antibiotic resistance is borne by a transferable plasmid of 50 x 10(6) molecular weight, and that in clone JH1-5 this plasmids suffered a large deletion leaving only a 6 x 10 (6) remnant which was incompatible with the complete replicon.
Abstract: A strain of Streptococcus faecalis var. zymogenes, designated JH1, had high-level resistance to the antibiotics streptomycin, kanamycin, neomycin, erythromycin, and tetracycline. These resistances were lost en bloc from approximately 0.1% of cells grown in nutrient broth at 45 C. The frequency of resistance loss was not increased by growth in the presence of the "curing" agents acriflavine or acridine orange, but after prolonged storage in nutrient agar 17% of cells became antibiotic sensitive. Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated from the parental strain and from antibiotic-sensitive segregants by using cesium chloride-ethidium bromide gradients. DNA molecular species were identified by using neutral sucrose gradients. Strain JH1 contained two covalently closed circular DNA species of molecular weights 50 x 10(6) and 38 x 10(6). An antibiotic-sensitive segregant, strain JH1-9, had lost the larger molecular species. A second sensitive segregant, strain JH1-5, had also lost the larger molecular species but a new molecular species of approximate molecular weight 6 x 10(6) was present. The antibiotic resistances that were curable from the parental strain were transferred to antibiotic-sensitive strains of S. faecalis and to strain JH1-9, during mixed incubation in nutrient broth at 37 C. Data to be described are interpreted to suggest that the transfer is by a conjugal mechanism. Analysis of the plasmid species in recipient clones showed that all had received the plasmid of molecular weight 50 x 10(6). Strain JH1-5 was not a good recipient. Analysis of one successful recipient clone of JH1-5 revealed that it had gained the 50 x 10(6) molecular weight plasmid but lost the 6 x 10(6) molecular weight species. These data are interpreted to mean that the multiple antibiotic resistance is borne by a transferable plasmid of 50 x 10(6) molecular weight, and that in clone JH1-5 this plasmid suffered a large deletion leaving only a 6 x 10(6) remnant which was incompatible with the complete replicon.
716 citations
TL;DR: Preliminary results from mutant strains of Pseudomonas putida mt-2 and from a mutant defective in the regulation of the plasmid-carried pathway suggest that the wild type contains two benzoate oxidase systems, one on thePlasmid which is nonspecific in both its catalysis and its induction and one in the chromosome which is more specific to benzoates as substrate and is specifically induced by benzosate.
Abstract: Mutant strains of Pseudomonas putida (arvilla) mt-2 which have lost the ability to grow at the expense of m- or p-toluate (methylbenzoate) but retain the ability to grow with benzoate arise spontaneously during growth on benzoate; this genetic loss occurs to a lesser extent during growth on nonaromatic carbon sources in the presence of mitomycin C. The mutants have totally lost the activity of the enzymes of the divergent meta pathway with the possible exception of 2-oxopent-4-enoate hydratase and 4-hydroxy-2-oxovalerate aldolase; unlike the wild type they utilize benzoate by the ortho pathway. Evidence is presented that these mutants have lost a plasmid coding for the enzymes of the meta pathway, which may be transmitted back to them or into other P. putida strains. Preliminary results from these mutants and from a mutant defective in the regulation of the plasmid-carried pathway suggest that the wild type contains two benzoate oxidase systems, one on the plasmid which is nonspecific in both its catalysis and its induction and one on the chromosome which is more specific to benzoate as substrate and is specifically induced by benzoate.
483 citations
TL;DR: The effect of pH on the K(m) values for ammonia was studied in its oxidation by Nitrosomonas cells and cell-free extracts, suggesting (NH(3)) rather than ( NH(4) (+)) as the actual substrate for oxidation.
Abstract: The effect of pH on the Km values for ammonia was studied in its oxidation by Nitrosomonas cells and cell-free extracts The Km values decreased markedly with increasing pH, suggesting (NH3) rather than (NH4+) as the actual substrate for oxidation
427 citations
TL;DR: Although glycogen synthesis took place in both sporulating and non-sporulating strains, only sporulating strains exhibited a period of glycogen degradation, which coincided with the final maturation of ascospores, which determined the carbohydrate composition of spores isolated from mature asci.
Abstract: Carbohydrate metabolism, under sporulation conditions, was compared in sporulating and non-sporulating diploids of Saccharomyces cerevisiae. Total carbohydrate was fractionated into trehalose, glycogen, mannan, and an alkali-insoluble fraction composed of glucan and insoluble glycogen. The behavior of three fractions was essentially the same in both sporulating and non-sporulating strains; trehalose, mannan, and the insoluble fraction were all synthesized to about the same extent regardless of a strain's ability to undergo meiosis or sporulation. In contrast, aspects of soluble glycogen metabolism depended on sporulation. Although glycogen synthesis took place in both sporulating and non-sporulating strains, only sporulating strains exhibited a period of glycogen degradation, which coincided with the final maturation of ascospores. We also determined the carbohydrate composition of spores isolated from mature asci. Spores contained all components present in vegetative cells, but in different proportions. In cells, the most abundant carbohydrate was mannan, followed by glycogen, then trehalose, and finally the alkali-insoluble fraction; in spores, trehalose was most abundant, followed by the alkali-insoluble fraction, glycogen, and mannan in that order.
394 citations
TL;DR: This work has surveyed a number of chemicals for their ability to repel Escherichia coli and suggested that each class corresponds to a distinct chemoreceptor.
Abstract: Several methods for detecting or measuring negative chemotaxis are described. Using these, we have surveyed a number of chemicals for their ability to repel Escherichia coli. Although most of the repellents are harmful compounds, harmfulness is neither necessary nor sufficient to make a compound a repellent. The repellents can be grouped into at least nine classes according to (i) competition experiments, (ii) mutants lacking certain of the negative taxes, and (iii) their chemical structure. The specificity of each class was studied. It is suggested that each class corresponds to a distinct chemoreceptor. Generally, non-chemotactic mutants lack both positive and negative chemotaxis, and l-methionine is required for both kinds of taxis. Repellents at very low concentrations are not attractants, and attractants at very high concentrations are not repellents.
387 citations
TL;DR: The protein composition of the outer membrane of Salmonella typhimurium has been analyzed by electrophoresis on slabs of sodium dodecyl sulfate-acrylamide gel and multiple major protein components of theouter membrane were identified.
Abstract: The protein composition of the outer membrane of Salmonella typhimurium has been analyzed by electrophoresis on slabs of sodium dodecyl sulfate-acrylamide gel. This powerful technique allows very high resolution of protein mixtures and has permitted the identification of multiple major protein components of the outer membrane; no evidence for a single major component of molecular weight 44,000 was obtained. These proteins were shown to be decreased in amount in mutants which have defective lipopolysaccharides. Mutants of an apparently new type were also found which contain decreased amounts of the proteins and the parent-like lipopolysaccharide, yet are resistant to a lipopolysaccharide-specific phage, C21. Several outer membrane proteins are insoluble in sodium dodecyl sulfate unless heated at high temperature (above 70 C). A purification procedure based on this property is tentatively suggested. Images
347 citations
TL;DR: Three plasmids designated alpha, beta, and gamma, distinguishable by their molecular weights, were isolated from Streptococcus faecalis strain DS-5 (ATCC 14508).
Abstract: Three plasmids designated α, β, and γ, distinguishable by their molecular weights (6, 17, and 34 million, respectively) were isolated from Streptococcus faecalis strain DS-5 (ATCC 14508). Derivatives of this strain “cured” for erythromycin resistance lacked the β-plasmid. In the parent strain the β-plasmid was estimated to be present to the extent of one to two copies per chromosomal genome equivalent whereas the α- and γ-plasmids were about nine and five copies, respectively. Images
340 citations
TL;DR: The composition of the cell envelope of a heptose-deficient lipopolysaccharide mutant of Escherichia coli, GR467, was studied after fractionation into its outer and cytoplasmic membrane components by means of sucrose density gradient centrifugation and it was found that major envelope proteins which were localized in the outer membrane were greatly diminished.
Abstract: The composition of the cell envelope of a heptose-deficient lipopolysaccharide mutant of Escherichia coli, GR467, was studied after fractionation into its outer and cytoplasmic membrane components by means of sucrose density gradient centrifugation. The outer membrane of GR467 had a lower density than that of its parent strain, CR34. Analysis of the fractionated membranes of GR467 indicated that the phospholipid-to-protein ratio had increased 2.4-fold in the outer membrane. The ratio in the mutant cytoplasmic membrane was also increased, although to a lesser extent. By employing a third parameter, the lipid A content of the outer membrane, it was found that the observed phospholipid-to-protein change in the outer membrane was due predominantly to a decrease in the relative amount of protein. This decrease in protein was particularly significant, since it was concomitant with a 68% decrease in the lipid A recovered in the outer membrane of GR467 relative to the lipid A recovered in the outer membrane of CR34. Similar findings were observed in a second heptose-deficient mutant of E. coli, RC-59. The apparent protein deficiency in GR467 was further studied by subjecting solubilized envelope proteins to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that major envelope proteins which were localized in the outer membrane were greatly diminished in GR467. Two revertants of GR467 with the wild-type amounts of heptose had wild-type relative levels of protein in their outer membranes. A partial heptose revertant had a relative level of protein in its outer membrane between those of the mutant and wild type. Images
252 citations
TL;DR: Although the mobilization phenomenon was not found to be associated with a detectable level of direct stable recombinational union between N-SuSm or N-Tc with a transfer factor, it was able to demonstrate a low level of recombination between these replicons and a transfer factors by P1-mediated transduction.
Abstract: Two nonconjugative R-plasmids, N-SuSm and N-Tc, have been characterized. Both were of relatively small size (5 × 106 to 6 × 106 daltons) and present in multiple copies within their respective bacterial hosts. N-SuSm possessed a guanine plus cytosine content of 55%, whereas N-Tc was 49% guanine plus cytosine. Although these plasmids were inherently nontransmissible they could be mobilized by a large variety of transfer agents including Ent, Hly, and K88. The fi− transfer factors tested were far more likely (about 200×) to mobilize these nonconjugative plasmids than were the fi+ transfer factors tested. Although the mobilization phenomenon was not found to be associated with a detectable level of direct stable recombinational union between N-SuSm or N-Tc with a transfer factor, we were able to demonstrate a low level of recombination between these replicons and a transfer factor by P1-mediated transduction. The isolation of recombinants between transfer factors and nonconjugative plasmids presumably represents one means by which unitary molecular types of R-plasmids arise and by which existing R-plasmids may acquire new resistance determinants.
TL;DR: The data indicate that only two pathways are involved in the fermentation of glutamate by the bacteria analyzed, and the methylaspartate pathway appears to be used only by species of Clostridium, whereas the hydroxyglutarate pathway is used by representatives of several genera.
Abstract: Two pathways are involved in the fermentation of glutamate to acetate, butyrate, carbon dioxide, and ammonia-the methylaspartate and the hydroxyglutarate pathways which are used by Clostridium tetanomorphum and Peptococcus aerogenes, respectively. Although these pathways give rise to the same products, they are easily distinguished by different labeling patterns of the butyrate when [4-(14)C]glutamate is used as substrate. Schmidt degradation of the radioactive butyrate from C. tetanomorphum yielded equally labeled propionate and carbon dioxide, whereas nearly all the radioactivity of the butyrate from P. aerogenes was recovered in the corresponding propionate. This procedure was used as a test for the pathway of glutamate fermentation by 15 strains (9 species) of anaerobic bacteria. The labeling patterns of the butyrate indicate that glutamate is fermented via the methylaspartate pathway by C. tetani, C. cochlearium, and C. saccarobutyricum, and via the hydroxyglutarate pathway by Acidaminococcus fermentans, C. microsporum, Fusobacterium nucleatum, and F. fusiformis. Enzymes specific for each pathway were assayed in crude extracts of the above organisms. 3-Methylaspartase was found only in clostridia which use the methylaspartate pathway, including Clostridium SB4 and C. sticklandii, which probably degrade glutamate to acetate and carbon dioxide by using a second amino acid as hydrogen acceptor. High levels of 2-hydroxyglutarate dehydrogenase were found exclusively in organisms that use the hydroxyglutarate pathway. The data indicate that only two pathways are involved in the fermentation of glutamate by the bacteria analyzed. The methylaspartate pathway appears to be used only by species of Clostridium, whereas the hydroxyglutarate pathway is used by representatives of several genera.
TL;DR: Theoretically derived thermodynamic equations suggest that the conformation of the flagellar helix affects efficiency of propulsion, and such a relationship between helix waveform and velocity was experimentally observed with Thiospirillum jenese.
Abstract: The behavior of a number of motile flagellated bacteria toward viscosity characteristics of their fluid environments was observed. All showed an increase in velocity (micrometers per second) in more viscous solutions. Velocity reached a maximum at a characteristic value, however, and thereafter decreased with higher viscosities. Peritrichously flagellated bacteria had maximum velocities at higher viscosities than polarly flagellated bacteria. Effects of temperature, and possible utilization of chemical constituents in the viscous solutions, were studied and found to be negligible factors under the experimental conditions used. Different agents produced the same phenomenon, thus indicating that there probably were no chemically induced metabolic effects. Loss of available water and the possibility of a variable energy supply to the flagellar propulsive system were considered but are believed minimal. Theoretically derived thermodynamic equations were utilized and suggest that the conformation of the flagellar helix affects efficiency of propulsion. Such a relationship between helix waveform and velocity was experimentally observed with Thiospirillum jenese.
TL;DR: The SmSu plasmids are closely related and probably have a common evolutionary origin and may be monomer, dimer, and trimer, respectively.
Abstract: Bacterial strains showing linked resistance to streptomycin (Sm) and sulfonamides (Su) were chosen representing a wide taxonomic and geographical range. Their SmSu resistances were transferred to Escherichia coli K-12 and then plasmid deoxyribonucleic acid (DNA) was isolated by ethidium bromide CsCl centrifugation. The plasmid DNA was examined by electron microscopy and analyzed by sedimentation through 5 to 20% neutral sucrose gradients. Plasmid DNA from strains having transmissible SmSu resistance consisted of two or three molecular species, one of which had a molecular mass of about 5.7 Mdal (106 daltons), the others varying between 20 to 60 Mdal. By using transformation or F′ mobilization, we isolated the SmSu-resistance determinant from any fellow resident plasmids in each strain and again isolated the plasmid DNA. Cosedimentation of each of these with a differently labeled reference plasmid DNA (R300B) showed 9 out of 12 of the plasmids to have a molecular mass not significantly different from the reference (5.7 Mdal); two others were 6.3 and 9.2 Mdal, but PB165 consisted of three plasmids of 7.4, 14.7, and 21.4 Mdal. Three separate isolations of the SmSu determinant from PB165 gave the same three plasmids, which we conclude may be monomer, dimer, and trimer, respectively. DNA-DNA hybridizations at 75 C demonstrated 80 to 93% homology between reference R300B DNA and each isolated SmSu plasmid DNA, except for the 9.2-Mdal plasmid which had 45% homology and PB165 which had 35%. All the SmSu plasmids were present as multiple copies (about 10) per chromosome. The conjugative plasmid of R300 (present as 1.3 copies per chromosome) has been shown to have negligible effect on the number of copies of its accompanying SmSu plasmid R300B. We conclude that the SmSu plasmids are closely related and probably have a common evolutionary origin.
TL;DR: There are at least four distinct polypeptide species with apparent molecular masses of about 42,000 daltons in the outer membrane of E. coli O111, and these proteins may contain a small amount of carbohydrate.
Abstract: Previous studies have shown that the outer membrane of Escherichia coli O111 gives a single, major, 42,000-dalton protein peak when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis at neutral pH. Further studies have shown that this peak consists of more than a single polypeptide species, and on alkaline SDS-gel electrophoresis this single peak is resolved into three subcomponents designated as proteins 1, 2, and 3. By chromatography of solubilized, outer membrane protein on diethylaminoethyl-cellulose followed by chromatography on Sephadex G-200 in the presence of SDS, it was possible to separate the 42,000-dalton major protein into four distinct protein fractions. Comparison of cyanogen bromide peptides derived from these fractions indicated that they represented at least four distinct polypeptide species. Two of these proteins migrated as proteins 1 and 2 on alkaline gels. The other two proteins migrated as protein 3 on alkaline gels and cannot be separated by SDS-polyacrylamide gel electrophoresis. In purified form, these major proteins do not contain bound lipopolysaccharide, phospholipid, or phosphate. These proteins may contain a small amount of carbohydrate, as evidenced by the labeling of these proteins by glucosamine, and to a lesser extent by glucose, under conditions where the metabolism of these sugars to amino acids and lipids is blocked. All of the proteins were labeled to the same extent by these sugars. Thus, it was concluded that there are at least four distinct polypeptide species with apparent molecular masses of about 42,000 daltons in the outer membrane of E. coli O111.
TL;DR: A new class of mutants of Escherichia coli K-12 defective in conjugation (Con(-) mutants), which lack a major protein of the outer membrane and are defective as recipients with Hfr and F' donors and as recipients for several F-like R factors and Col factors are described.
Abstract: We describe a new class of mutants of Escherichia coli K-12 defective in conjugation (Con− mutants). They lack a major protein of the outer membrane and are defective as recipients with Hfr and F′ donors and as recipients for several F-like R factors and Col factors. In the case of crosses with an Hfr donor, we have shown that the Con− recipient is defective in pair formation. The mutants are resistant to certain phages due to loss of receptor activity.
TL;DR: A role for glutamine synthetase as a positive control element for nitrogen fixation in K. pneumoniae is indicated and the synthesis of nitrogenase is allowed to continue under conditions that completely repress nitrogenase synthesis in the wild-type strain.
Abstract: Mutations causing constitutive synthesis of glutamine synthetase (GlnC(-) phenotype) were transferred from Klebsiella aerogenes into Klebsiella pneumoniae by P1-mediated transduction. Such GlnC(-) strains of K. pneumoniae have constitutive levels of glutamine synthetase. Two of three GlnC(-) strains of K. pneumoniae studied, each containing independently isolated mutations that confer the GlnC(-) phenotype, continue to synthesize nitrogenase in the presence of NH(4) (+). One strain, KP5069, produces 30% as much nitrogenase when grown in the presence of 15 mM NH(4) (+) as in its absence. The GlnC(-) phenotype allows the synthesis of nitrogenase to continue under conditions that completely repress nitrogenase synthesis in the wild-type strain. Glutamine auxotrophs of K. pneumoniae, that do not produce catalytically active glutamine synthetase, are unable to synthesize nitrogenase during nitrogen limited growth. Complementation of K. pneumoniae Gln(-) strains by an Escherichia coli episome (F'133) simultaneously restores glutamine synthetase activity and the ability to synthesize nitrogenase. These results indicate a role for glutamine synthetase as a positive control element for nitrogen fixation in K. pneumoniae.
TL;DR: The results indicate that the ratios RNA/DNA, RNA/protein, and protein/DNA give linear relationships with the growth rate, whereas RNA/cell andprotein/cell show a more complex growth rate dependency.
Abstract: By using Escherichia coli B/r, the cellular amounts of ribonucleic acid (RNA) and protein were determined as a function of the steady-state growth rate (0.67 to 2.40 doublings per h) by a method which combines measurements of the RNA to deoxyribonucleic acid (DNA) ratio and the differential rate of ribosomal protein synthesis with the Cooper and Helmstetter theory of DNA replication. The results indicate that the ratios RNA/DNA, RNA/protein, and protein/DNA give linear relationships with the growth rate (above 1.2 doublings per h), whereas RNA/cell and protein/cell show a more complex growth rate dependency. The significance of these relationships is discussed. Finally, a detailed description of the growth parameters and composition of E. coli B/r is presented.
TL;DR: A mutator gene, mutD5, whose phenotype is conditional, has been identified in Escherichia coli and by P1 transduction it has been shown to lie at about 5.7 min on the chromosome, being co-transduced with proA and argF.
Abstract: A mutator gene, mutD5, whose phenotype is conditional, has been identified in Escherichia coli By P1 transduction it has been shown to lie at about 57 min on the chromosome, being co-transduced with proA and argF In rich medium, streptomycin- and nalidixic acid-resistant mutation frequencies are 50 to 100 times higher than those in minimal medium In minimal medium, the mutD5-induced mutation frequencies are still 50 to 100 times above co-isogenic wild-type (mut+) levels Similar results were obtained with all markers tested Mutant frequencies can be raised by thymidine in the medium at concentrations as low as 004 μM, or by the endogenous generation of thymidine from thymine plus a deoxyribosyl donor Deoxyadenosine, various ribonucleosides, thymine, and 2-deoxyribose do not stimulate mutation None of these effects are related to growth rate, since growth rate and mutation rate can be decoupled completely
TL;DR: A method has been developed to isolate mutants sensitive to coliphage P1 from bacterial genera normally not sensitive to this phage, providing a well-known genetic system for the study of manyacterial genera that previously had either no such system or only a marginally useful means of genetic manipulation.
Abstract: A method has been developed to isolate mutants sensitive to coliphage P1 from bacterial genera normally not sensitive to this phage. P1clr100KM was used. This phage is heat inducible and confers kanamycin resistance when present as a prophage (in lysogens). P1-sensitive mutants of Klebsiella, Enterobacter, Citrobacter, and Erwinia have been found. This technique provides a well-known genetic system for the study of many bacterial genera that previously had either no such system or only a marginally useful means of genetic manipulation. It also extends the range of possible intergeneric hybrids that may be constructed and studied.
TL;DR: Investigations on the growth and macromolecular synthesis in a G3P acyltransferase K(m) mutant revealed that upon glycerol deprivation, net phospholipid synthesis stopped immediately and growth continued for about one doubling, and that the rates of these processes are not coupled during this time period.
Abstract: sn-Glycerol 3-phosphate (G3P) auxotrophs of Escherichia coli have been selected from a strain which cannot aerobically catabolize G3P. The auxotrophy resulted from loss of the biosynthetic G3P dehydrogenase (EC 1.1.1.8) or from a defective membranous G3P acyltransferase. The apparent K(m) of the acyltransferase for G3P was 11- to 14-fold higher (from about 90 mum to 1,000 to 1,250 mum) in membrane preparations from the mutants than those of the parent. All extracts prepared from revertants of the G3P dehydrogenase mutants showed G3P dehydrogenase activity, but most contained less than 10% of the wild-type level. Membrane preparations from revertants of the acyltransferase mutants had apparent K(m)'s for G3P similar to that of the parent. Strains have been derived in which the G3P requirement can be satisfied with glycerol in the presence of glucose, presumably because the glycerol kinase was desensitized to inhibition by fructose 1,6-diphosphate. Investigations on the growth and macromolecular synthesis in a G3P acyltransferase K(m) mutant revealed that upon glycerol deprivation, net phospholipid synthesis stopped immediately; growth continued for about one doubling; net ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein nearly doubled paralleling the growth curve; the rate of phospholipid synthesis assessed by labeling cells with (32)P-phosphate, (14)C-acetate, or (3)H-serine was reduced greater than 90%; the rates of RNA and DNA synthesis increased as the cells grew and then decreased as the cells stopped growing; the rate of protein synthesis showed no increase and declined more slowly than the rates of RNA and DNA synthesis when the cells stopped growing. The cells retained and gained in the capacity to synthesize phospholipids upon glycerol deprivation. These data indicate that net phospholipid synthesis is not required for continued macromolecular synthesis for about one doubling, and that the rates of these processes are not coupled during this time period.
TL;DR: Data indicate that, in both prokaryotes and in eukaryotes, superoxide dismutase is an important component of the defenses against oxygen toxicity.
Abstract: Saccharomyces cerevisiae var. ellipsoideus contained 6.5 times more superoxide dismutase and 2.3 times more catalase when grown under 100% O2 than when grown anaerobically. Growth under oxygen caused equal increases in both the cyanide-sensitive and the cyanide-insensitive superoxide dismutases of this organism. Experience with other eukaryotes has shown that cyanide sensitivity is a property of the cupro-zinc superoxide dismutase of the cytosol, whereas cyanide insensitivity is a property of the corresponding mangani-enzyme found in mitochondria. Cu2+, which has been shown to increase the radioresistance of yeast, also caused an increase of both of the superoxide dismutases of S. cerevisiae. Yeast which had been grown under 1 atm of O2 were more resistant toward the lethal effects of 20 atm of O2 than were yeast which had been grown in the absence of O2. Escherichia coli K-12 his− responded to growth under 1 atm of O2 by increasing its content of catalase and of peroxidase, but not of superoxide dismutase. This contrasts with E. coli B, which was previously shown to respond to O2 by a striking increase in superoxide dismutase. E. coli K-12 his− did not gain resistance toward 20 atm of O2 because of having been grown under 1 atm of O2. Once again, this contrasts with the behavior of E. coli B. These data indicate that, in both prokaryotes and in eukaryotes, superoxide dismutase is an important component of the defenses against oxygen toxicity.
TL;DR: The limiting sizes of molecules that can permeate the intact cell wall and protoplast membrane of Saccharomyces cerevisiae were determined from the inflection points in a triphasic pattern of passive equilibrium uptake values obtained with a series of inert probing molecules varying in molecular size.
Abstract: The limiting sizes of molecules that can permeate the intact cell wall and protoplast membrane of Saccharomyces cerevisiae were determined from the inflection points in a triphasic pattern of passive equilibrium uptake values obtained with a series of inert probing molecules varying in molecular size. In the phase identified with the yeast protoplast, the uptake-exclusion threshold corresponded to a monodisperse ethylene glycol of molecular weight = 110 and Einstein-Stokes hydrodynamic radius (rES) = 0.42 nm. In the cell wall phase, the threshold corresponded to a polydisperse polyethylene glycol of number-average molecular weight (¯Mn) = 620 and average radius (rES) = 0.81 nm. The third phase corresponded to complete exclusion of larger molecules. The assessment of cell wall porosity was confirmed by use of a second method involving analytical gel chromatographic analyses of the molecular weight distribution for a single polydisperse polyglycol before and after uptake by the cells, which indicated a quasi-monodisperse threshold for the cell wall of Mn = 760 and rES = 0.89 nm. The results were reconciled with two situations in which much larger protein molecules previously have been reported able to penetrate the yeast cell wall.
TL;DR: The cell envelope of Neisseria gonorrhoeae strain 2686, colonial type 4, was isolated from spheroplasts formed by the action of ethylenediaminetetraacetic acid and lysozyme and the membrane components were similar to those of other gram-negative bacteria.
Abstract: The cell envelope of Neisseria gonorrhoeae strain 2686, colonial type 4, was isolated from spheroplasts formed by the action of ethylenediaminetetraacetic acid and lysozyme. Isopycnic centrifugation of osmotically ruptured spheroplasts resolved the cell envelope into two main membrane fractions. Chemical and enzymatic analyses were used to characterize these isolated membranes. Succinic dehydrogenase, reduced nicotinamide adenine dinucleotide oxidase, and d-lactate dehydrogenase were localized in the membrane fraction of buoyant density, rho degrees = 1.141 g/cm(3). Lipopolysaccharide and over half of the cell envelope protein were associated with the membrane that banded in sucrose at rho degrees = 1.219 g/cm(3). These fractions were consequently designated cytoplasmic and outer or L-membrane, respectively. Sodium dodecyl sulfate-polyacrylamide electrophoresis of isolated membranes demonstrated the relative simplicity of the protein spectrum of the outer membrane. The majority of the protein in this membrane could be accounted for by proteins of molecular weights 34,500, 22,000, and 11,500. The protein of molecular weight 34,500 accounted for 66% of the total protein of the L-membrane. Isoelectric precipitation at pH 4.6 with 10% acetic acid selectively removed this protein from a 150 mM NaCl in 10 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.4, extract of purified outer membrane. At pH 4.0, the other proteins of the L-membrane were precipitated. It was concluded that the membrane components of the cell envelope of N. gonorrhoeae were similar to those of other gram-negative bacteria. The cell envelope fractions described here, in particular the outer membrane, are sufficiently well defined to provide a valuable tool for future biochemical and immunological studies on N. gonorrhoeae.
TL;DR: The growth response of these recombinants to leucine peptides shows that all of these peptidases can function in the catabolism of peptides and that they display overlapping substrate specificities in vivo.
Abstract: Six peptidase activities have been distinguished electrophoretically in cell extracts of Salmonella typhimurium with the aid of a histochemical stain. The activities can also be partially separated by chromatography on diethylaminoethyl-cellulose. These peptidases show overlapping substrate specificities. Mutants ( pepN ) of the parent strain leu-485 lacking one of these enzymes ( peptidase N ) were obtained by screening for colonies that do not hydrolyze the chromogenic substrate l-alanyl-β-naphthylamide. The absence of this broad-specificity peptidase in leu-485 pepN − mutants allowed the selection of mutants unable to use l-leucyl-l-alaninamide as a leucine source. These mutants ( leu-485 pepN − pepA − ) lack a broad-specificity peptidase (peptidase A) similar to aminopeptidase I previously described in Escherichia coli . Mutants ( pepD ) lacking a dipeptidase (peptidase D) have been isolated from a leu-485 pepN − pepA − parent by penicillin selection for mutants unable to use l-leucyl-l-glycine as a leucine source. Mutants ( pepB ) lacking a fourth peptidase (peptidase B) have been isolated from a leu-485 pepN − pepA − pepD − strain by penicillin selection for failure to utilize l-leucyl-l-leucine as a source of leucine. Single recombinants were obtained by transduction for each of the peptidases missing in a leu-485 pepN − pepA − pepD − pepB − strain. The growth response of these recombinants to leucine peptides shows that all of these peptidases can function in the catabolism of peptides and that they display overlapping substrate specificities in vivo. Images
TL;DR: This work isolated and characterized a series of mutants (cfs) that restored flagella-forming ability in a Crp strain of E. coli and proposed a regulatory function for the product of the flaI gene, which is suggested to be the step in flagellar synthesis that is catabolite sensitive and requires cAMP.
Abstract: In Escherichia coli, the synthesis of the flagellar organelle is sensitive to catabolite repression. Synthesis requires the presence of the cyclic adenosine monophosphate receptor protein (Crp) and 3′,5′-cyclic adenosine monophosphate (cAMP); i.e., mutants that lack Crp or adenylcyclase (Cya) synthesize no flagella. We isolated and characterized a series of mutants (cfs) that restored flagella-forming ability in a Crp strain of E. coli. The mutations in these strains were transferred onto episomes and they were then introduced into a variety of other strains. The presence of the mutation resulted in flagella synthesis in Cya and Crp strains as well as in the wild type grown under conditions of catabolite repression. Deletion analysis and other genetic studies indicated that: (i) the cfs mutations had a dominant effect when they were in the transconfiguration in merodiploids: (ii) they occurred in or very close to the flaI gene: and (iii) their expression required the presence of an intact flaI gene adjacent to the cfs mutation. Biochemical studies showed that the synthesis of at least two flagellar polypeptides, the hook subunit and an amber fragment of flagellin, were absent in strains that carried a cya mutation. Their synthesis was depressed in strains grown under conditions of catabolite repression. The presence of the cfs mutation restored the specific synthesis of these two polypeptides. We suggest that the formation of the flaI gene product is the step in flagellar synthesis that is catabolite sensitive and requires cAMP. We propose a regulatory function for the product of the flaI gene.
TL;DR: Bulge formation, due to the presumed action of an autolysin(s), may be an initial step in the septation sequence when the mucopeptide is modified to allow construction of the sePTum.
Abstract: Septa can be demonstrated in sections of Escherichia coli strains B and B/r after fixation with acrolein and glutaraldehyde. The septum consists of an ingrowth of the cytoplasmic membrane and the mucopeptide layer; the outer membrane is excluded from the septum until the cells begin to separate. Mesosomes have also been observed. The septum is highly labile and, except in the chain-forming strains, E. coli D22 env A and CRT 97, not easily preserved by standard procedures. The labile nature of the septum may be due to the presence of autolysin(s) located at the presumptive division site. Blocking division by addition of ampicillin (2 to 5 μg/ml) to cells of E. coli B/r produces a bulge at the middle of the cells; bulge formation is stopped by addition of chloramphenicol. Cephalosporins also induce bulge formation but may stop cell elongation as well as division. Bulge formation, due to the presumed action of an autolysin(s), may be an initial step in the septation sequence when the mucopeptide is modified to allow construction of the septum. In a nonseptate filament-forming strain, PAT 84, which ceases to divide at 42 C, bulge formation only occurs in the presence of ampicillin at the time of a shift-down at 30 C or at 42 C in the presence of NaCl (0.25 to 0.34 M). Experiments with chloramphenicol suggest that the filaments are fully compartmentalized but fail to divide owing to the inactivation, rather than loss of synthesis, of an autolysin at 42 C.
TL;DR: Beta (beta)-lactamases determined by 29 ampicillin resistance plasmids could be divided into two types, TEM-type and less common, which showed lower absolute levels of activity, and was heterogeneous as regards substrate specificities.
Abstract: Beta (β)-lactamases determined by 29 ampicillin resistance plasmids could be divided into two types. One, TEM-type, was very uniform with respect to substrate specificity but heterogeneous in absolute levels of β-lactamase activity. The TEM-type β-lactamase was determined by R factors of compatibility groups FII, Iα, Ie, N, C, A, T, W, P, L, and X, and by prophage φ Amp. The other type, characterized by the ability to hydrolyze oxacillin, was less common, showed lower absolute levels of activity, and was heterogeneous as regards substrate specificities. Oxacillin-hydrolyzing β-lactamases were determined by R factors of compatibility groups FI, Iα, N, C, and O.
TL;DR: The time course of synthesis and breakdown of various macromolecules has been compared for sporulating and nonsporulating yeast cells transferred to potassium acetate sporulation medium to find out which types of cells incorporate label into ribonucleic acid and protein.
Abstract: The time course of synthesis and breakdown of various macromolecules has been compared for sporulating (a/alpha) and nonsporulating (a/a and alpha/alpha) yeast cells transferred to potassium acetate sporulation medium. Both types of cells incorporate label into ribonucleic acid and protein. The gel electrophoresis patterns of proteins synthesized in sporulation medium are identical for sporulating and nonsporulating diploids; both are different from electropherograms of vegetative cells. Sporulating and nonsporulating strains differ with respect to deoxyribonucleic acid synthesis; no deoxyribonucleic acid is synthesized in the latter case, whereas the deoxyribonucleic acid complement is doubled in the former. Glycogen breakdown occurs only in sporulating strains. Breakdown of preexisting vegetative ribonucleic acid and protein molecules occurs much more extensively in sporulating than in nonsporulating cells. A timetable of these data is presented.
TL;DR: The location of methane oxidizers in a dimictic lake indicates that these organisms prefer less than saturating levels of dissolved oxygen, and laboratory experiments confirmed the preference of these organisms for atmospheres containing less oxygen than air.
Abstract: Bacteria capable of growth on methane and a variety of complex organic substrates as sole sources of carbon and energy have been isolated Conditions used to rigorously establish the purity of the cultures are described One facultative methylotroph has been studied in detail This organism has peripherally arranged pairs of intracytoplasmic membranes characteristic of obligate methylotrophs This isolate apparently utilizes the serine pathway of formaldehyde fixation The location of methane oxidizers in a dimictic lake indicates that these organisms prefer less than saturating levels of dissolved oxygen Laboratory experiments confirmed the preference of these organisms for atmospheres containing less oxygen than air