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Showing papers in "Journal of Bacteriology in 1971"


Journal ArticleDOI
TL;DR: Tabulation of adenylate concentrations previously reported for various organisms and tissues supports the prediction, based on enzyme kinetic observations in vitro, that the energy charge is stabilized near 0.85 in intact metabolizing cells of a wide variety of types.
Abstract: The value of the adenylate energy charge, [(adenosine triphosphate) + ½ (adenosine diphosphate)]/[(adenosine triphosphate) + (adenosine diphosphate) + (adenosine monophosphate)], in Escherichia coli cells during growth is about 0.8. During the stationary phase after cessation of growth, or during starvation in carbon-limited cultures, the energy charge declines slowly to a value of about 0.5, and then falls more rapidly. During the slow decline in energy charge, all the cells are capable of forming colonies, but a rapid fall in viability coincides with the steep drop in energy charge. These results suggest that growth can occur only at energy charge values above about 0.8, that viability is maintained at values between 0.8 and 0.5, and that cells die at values below 0.5. Tabulation of adenylate concentrations previously reported for various organisms and tissues supports the prediction, based on enzyme kinetic observations in vitro, that the energy charge is stabilized near 0.85 in intact metabolizing cells of a wide variety of types.

775 citations


Journal ArticleDOI
TL;DR: The R factor was transferable from E. coli to bacterial genera outside the Enterobacteriaceae (Pseudomonas and members of the Rhizobiaceae) to which transfer of F-like and I-like plasmids could not be demonstrated.
Abstract: An R factor from Pseudomonas aeruginosa, which confers resistance to penicillins, kanamycin, and tetracycline, was studied in Escherichia coli K-12. The R factor could coexist with F-like or I-like plasmids and therefore constituted a novel compatibility group. The R factor was transferable from E. coli to bacterial genera outside the Enterobacteriaceae (Pseudomonas and members of the Rhizobiaceae) to which transfer of F-like and I-like plasmids could not be demonstrated.

571 citations


Journal ArticleDOI
TL;DR: Within experimental errors, DNA content was dependent only on growth rate and independent of the type of culture, the carbon source, or the addition of growth factors.
Abstract: The average amount of deoxyribonucleic acid (DNA) per cell was measured in steady-state cultures of Escherichia coli B/r grown at 37 C in glucose-limited chemostats or in batch cultures in the exponential growth phase as maintained with one of several carbon sources. Within experimental errors, DNA content was dependent only on growth rate and independent of the type of culture, the carbon source, or the addition of growth factors. The amount of DNA per cell increased continuously with growth rate over the range of 0.02 to 3 divisions per hour. The data over the entire range of growth rates are in agreement with a constant time for a single replication point to traverse the entire genome, 47 min, and with cell division following 25 min after termination of replication. The measured amount of DNA per genome was 4.2 x 10(-15) g (or 2.5 x 10(9) daltons).

533 citations


Journal ArticleDOI
TL;DR: Comparison of the Triton-soluble and TritOn-insoluble proteins from the cell wall and cytoplasmic membrane fractions by polyacrylamide gel electrophoresis and gel filtration in acidified dimethyl formamide indicated that the detergent specifically solubilized proteins of the cy toplasmi membrane.
Abstract: Treatment of a partially purified preparation of cell walls of Escherichia coli with Triton X-100 at 23 C resulted in a solubilization of 15 to 25% of the protein. Examination of the Triton-insoluble material by electron microscopy indicated that the characteristic morphology of the cell wall was not affected by the Triton extraction. Contaminating fragments of the cytoplasmic membrane were removed by Triton X-100, including the fragments of the cytoplasmic membrane which were normally observed attached to the cell wall. Treatment of a partially purified cytoplasmic membrane fraction with Triton X-100 resulted in the solubilization of 60 to 80% of the protein of this fraction. Comparison of the Triton-soluble and Triton-insoluble proteins from the cell wall and cytoplasmic membrane fractions by polyacrylamide gel electrophoresis after removal of the Triton by gel filtration in acidified dimethyl formamide indicated that the detergent specifically solubilized proteins of the cytoplasmic membrane. The proteins solubilized from the cell wall fraction were qualitatively identical to those solubilized from the cytoplasmic membrane fraction, but were present in different proportions, suggesting that the fragments of cytoplasmic membrane which are attached to the cell wall are different in composition from the remainder of the cytoplasmic membrane of the cell. Treatment of unfractionated envelope preparations with Triton X-100 resulted in the solubilization of 40% of the protein, and only proteins of the cytoplasmic membrane were solubilized. Extraction with Triton thus provides a rapid and specific means of separating the proteins of the cell wall and cytoplasmic membrane of E. coli.

506 citations


Journal ArticleDOI
TL;DR: Passive permeabilities of the cell wall and protoplast of Bacillus megaterium strain KM were characterized by use of 50 hydrophilic probing molecules (tritiated water, sugars, dextrans, glycols, and polyglycols) which varied widely in size.
Abstract: Passive permeabilities of the cell wall and protoplast of Bacillus megaterium strain KM were characterized by use of 50 hydrophilic probing molecules (tritiated water, sugars, dextrans, glycols, and polyglycols) which varied widely in size. Weight per cent uptake values (R(w)) were measured at diffusional equilibrium under conditions that negated the influences of adsorption or active transport. Plots of R(w) for intact cells as a function of number-average molecular weight ( M(n)) or Einstein-Stokes hydrodynamic radius ( r(ES)) of the solutes showed three phases: a protoplast uptake phase with a polydisperse exclusion threshold of M(n) = 0.6 x 10(3) to 1.1 x 10(3), r(ES) = 0.6 to 1.1 nm; a cell wall uptake phase with a polydisperse exclusion threshold of M(n) = 0.7 x 10(5) to 1.2 x 10(5), r(ES) congruent with 8.3 nm; and a total exclusion phase. Isolated cell walls showed only the latter two phases. However, it became evident that the cell wall selectively passed only the smallest molecules in a heterodisperse polymer sample. When the molecular-weight distributions of polyglycol samples ( M(n) = 1,000, 1,450, and 3,350) were determined by analytical gel chromatography before and after uptake by intact cells or isolated cell walls, a quasi-monodisperse exclusion threshold was obtained corresponding to M(n) = 1,200, r(ES) = 1.1 nm. The permeability of isolated protoplasts was assessed by the relative ability of solutes to effect osmotic stabilization. An indefinite exclusion threshold, evident even with monodisperse sugars, was attributed to lengthwise orientation of the penetrating rod-shaped molecules. Altogether, the best estimate of the limiting equivalent porosity of the protoplast was 0.4 to 0.6 nm in radius and of the cell wall, 1.1 nm.

422 citations


Journal ArticleDOI
TL;DR: Mutants of Escherichia coli K-12 requiring considerably elevated concentrations of potassium for growth are readily obtained as double mutants combining a kdp mutation with a mutation in one or more of five other loci, referred to as trk, for transport of K, because these mutations result in alterations in K transport.
Abstract: Mutants of Escherichia coli K-12 requiring considerably elevated concentrations of potassium for growth are readily obtained as double mutants combining a kdp mutation with a mutation in one or more of five other loci. These loci are referred to as trk, for transport of K, because these mutations result in alterations in K transport. The kdp mutation is essential in the isolation and identification of this type of mutant; in a Kdp(+) strain, the presence of a trk mutation does not prevent growth of the strain in media containing very low concentrations of K. The trk loci are widely scattered over the E. coli chromosome; none of them is very near any other trk locus or near the kdp genes.

377 citations


Journal ArticleDOI
TL;DR: In this paper, the transfer-deficient mutants of Flac have been isolated; 27 of these bear amber mutations and 1 mutant is temperature-sensitive; all the mutants transfer between 10−2 and <10−5% as well as wild-type Flac, all are curable by acridine orange treatment, and all are resistant to the female-specific phage phi(II).
Abstract: Eighty-four transfer-deficient mutants of Flac have been isolated; 27 of these bear amber mutations and 1 mutant is temperature-sensitive. All the mutants transfer between 10(-2) and <10(-5)% as well as wild-type Flac, all are curable by acridine orange treatment, and all are resistant to the female-specific phage phi(II). Some of the mutants are partially sensitive to female-specific phage tau. Sixty-three of the mutants are resistant to the male-specific phages f1, f2, and Qbeta; 15 are resistant only to f2; and 6 are sensitive to all three male-specific phages. Most of the mutants are still poor recipients in conjugation, but four of the mutants resistant to f1, f2, and Qbeta have become good recipients. Those mutants resistant to all three male-specific phages do not seem to make F-pili.

321 citations


Journal ArticleDOI
TL;DR: Nine groups which had deoxyribonucleic acid (DNA) containing 45 to 48 moles per cent guanine plus cytosine (GC) were assigned to a redefined genus Beneckea, and all of the strains in this genus, when grown in liquid medium, had a single, polar flagellum.
Abstract: One-hundred-and-forty-five isolates of marine origin were submitted to an extensive physiological, nutritional, and morphological characterization. All strains were gram-negative, facultatively anaerobic, straight or curved rods which were motile by means of flagella. Glucose was fermented with the production of acid but no gas. Sodium but no organic growth factors were required. None of the strains were able to denitrify or fix molecular nitrogen. The results of nutritional and physiological tests were submitted to a numerical analysis. On the basis of phenotypic similarity, nine groups were established. These groups could be distinguished from one another by multiple, unrelated, phenotypic traits. Six groups which had deoxyribonucleic acid (DNA) containing 45 to 48 moles per cent guanine plus cytosine (GC) were assigned to a redefined genus Beneckea . All of the strains in this genus, when grown in liquid medium, had a single, polar flagellum. When grown on a solid medium, many strains had peritrichous flagella. Two groups were similar to previously described species and were designated B. alginolytica and B. natriegens . The remaining four groups were designated B. campbellii, B. neptuna, B. nereida , and B. pelagia . An additional group of phenotypically similar strains having the properties of the genus Beneckea was not included in the numerical analysis. These strains were readily separable from species of this genus and were designated B. parahaemolytica . Of the remaining groups, one was identified as Photobacterium fischeri . The other group (B-2) which had about 41 moles% GC content in its DNA could not be placed into existing genera. Images

312 citations


Journal ArticleDOI
TL;DR: Mutants of Saccharomyces cerevisiae capable of growth on a minimal medium supplemented with ureidosuccinic and glutamic acids have been isolated from a pyrimidineless strain and it is suggested that the mutation is nonchromosomal and could be mitochondrial.
Abstract: Mutants of Saccharomyces cerevisiae capable of growth on a minimal medium supplemented with ureidosuccinic and glutamic acids have been isolated from a pyrimidineless strain. One of these mutants consistently yielded a non-Mendelian meiotic segregation. Moreover, the mitotic transmission of the mutation was very high. It is suggested that the mutation is nonchromosomal and could be mitochondrial. However, this mutation behaves very differently from other mitochondrial mutations.

310 citations


Journal ArticleDOI
TL;DR: The results are interpreted in terms of the types of noncovalent bonds involved in maintaining the organized structure of the cell wall and suggest that the main forces involved are hydrophobic protein-protein interactions between thecell wall proteins and to a lesser degree a stabilization of protein- protein and protein-lipopolysaccharide interactions by divalent cations.
Abstract: Extraction of a partially purified preparation of cell walls from Escherichia coli with the nonionic detergent Triton X-100 removed all cytoplasmic membrane contamination but did not affect the normal morphology of the cell wall. This Triton-treated preparation, termed the "Triton-insoluble cell wall," contained all of the protein of the cell wall but only about half of the lipopolysaccharide and one-third of the phospholipid of the cell wall. This Triton-insoluble cell wall preparation was used as a starting material in an investigation of several further treatments. Reextraction of the Triton-insoluble cell wall with either Triton X-100 or ethylenediaminetetraacetic acid (EDTA) caused no further solubilization of protein. However, when the Triton-insoluble cell wall was extracted with a combination of Triton X-100 and EDTA, about half of the protein and all of the remaining lipopolysaccharide and phospholipid were solubilized. The material which remained insoluble after this combined Triton and EDTA extraction still retained some of the morphological features of the intact cell wall. Treatment of the Triton-insoluble cell wall with lysozyme resulted in a destruction of the peptidoglycan layer as seen in the electron microscope and in a release of diaminopimelic acid from the cell wall but did not solubilize any cell wall protein. Extraction of this lysozyme-treated preparation with a combination of Triton X-100 and EDTA again solubilized about half of the cell wall protein but resulted in a drastic change in the morphology of the Triton-EDTA-insoluble material. After this treatment, the insoluble material formed lamellar structures. These results are interpreted in terms of the types of noncovalent bonds involved in maintaining the organized structure of the cell wall and suggest that the main forces involved are hydrophobic protein-protein interactions between the cell wall proteins and to a lesser degree a stabilization of protein-protein and protein-lipopolysaccharide interactions by divalent cations. A model for the structure of the E. coli cell wall is presented.

305 citations


Journal ArticleDOI
TL;DR: Two types of basal body structures are proposed, as exemplified by E. coli and B. subtilis, which directly reflect the structure of the gram-negative and gram-positive cell envelopes.
Abstract: The hook-basal body complex comprising the basal end of purified intact flagella from Escherichia coli and Bacillus subtilis was studied in detail with an electron microscope. The E. coli hook can be described as having five or six concentric helical coils. The basal body from E. coli is 27 nm in length and consists of four rings, 22.5 nm in diameter, arranged in two pairs and mounted on a rod. The top pair of rings is connected near their periphery, resembling a closed cylinder. In B. subtilis the basal body looks like that from E. coli, except that the top pair of rings is missing. Hook-basal body complexes from both organisms could be isolated by dissociating the filaments with either urea or acid. Based on our results, two types of basal body structures are proposed, as exemplified by E. coli and B. subtilis, which directly reflect the structure of the gram-negative and gram-positive cell envelopes.

Journal ArticleDOI
TL;DR: The phosphatidic acid synthesized in vitro shows a striking enrichment of oleate at the beta position analogous to the positional specificity observed in phospholipid biosynthesis synthesizing in vivo.
Abstract: The higher the growth temperature of Escherichia coli cultures the greater is the proportion of saturated fatty acids in the bacterial phospholipids. When fatty acids are exogenously supplied to E. coli, higher growth temperatures will likewise increase the relative incorporation of saturated fatty acids into phospholipids. One of the steps in the utilization of fatty acids for phospholipid biosynthesis is, therefore, temperature-controlled. The temperature effect observed in vivo with mixtures of (3)H-oleate and (14)C-palmitate is demonstrable in vitro by using mixtures of the coenzyme A derivative of these fatty acids for the acylation of alpha-glycerol phosphate to lysophosphatidic and phosphatidic acids. In E. coli extracts, the relative rates of transacylation of palmityl and oleyl coenzyme A vary as a function of incubation temperature in a manner which mimics the temperature control observed in vivo. The phosphatidic acid synthesized in vitro shows a striking enrichment of oleate at the beta position analogous to the positional specificity observed in phospholipids synthesized in vivo.

Journal ArticleDOI
TL;DR: Novobiocin-supersensitive mutants, with incomplete LPS, lacking phosphates in their backbone structure were found to be resistant to phage T4, and those which also lacked heptose wereresistant to phages T4 and T7.
Abstract: Novobiocin-supersensitive (NS) mutants which could not grow on plates containing 40 μg or less of novobiocin per ml were isolated from Escherichia coli strain JE1011 (derived from E. coli K-12). Most of these NS mutants were found to have incomplete lipopolysaccharides (LPS), and they lack phosphate diester bridges in their backbone structure, with or without total loss of heptose, to which the phosphate diester is linked, and consequently lack external outer-core oligosaccharides. The phosphate diester bridges in the LPS backbone are apparently very important in forming a cell surface structure resistant to the penetration of antibiotics such as novobiocin, spiramycin, and actinomycin D. NS mutants, with incomplete LPS, lacking phosphates in their backbone structure were found to be resistant to phage T4, and those which also lacked heptose were resistant to phages T4 and T7. In contrast to the generally accepted idea that resistances to phages T3, T4, and T7 are linked genetically, no NS mutant was found to be resistant to T3. The possible structures of the receptors for T4 and T7 are discussed. The positions of novobiocin-supersensitive genes on the chromosome of several of the NS mutants defective in LPS were mapped. The genes were designated lpcA (between ara and lac) and lpcB (between 55 min and 60 min). The latter seemed to be a group of several related genes.

Journal ArticleDOI
TL;DR: It was demonstrated that DNA prepared from log-phase cells renatures faster than stationary-phase DNA and also departs from theoretical second-order kinetics.
Abstract: The base composition of a deoxyribonucleic acid (DNA) sample affects its intrinsic rate of renaturation. In agreement with the information of Wetmur and Davidson, it was established that high guanosine plus cytosine (GC) DNA renatures faster than expected from analytical measurement of its molecular weight. A calculated correction factor of 1.8% of the observed C0t.5 is required for every mole per cent GC difference from 51% GC. The correction factor is now established in the range of 32 to 65% GC. Renaturation of DNA mixtures prepared from pairs of organisms has been studied. When no similarity existed between the two organisms, the observed C0t.5 of the mixture was the sum of the independently determined C0t.5 values. Lack of additivity was correlated with similarities in polynucleotide sequence of the reassociating DNA molecules. A quantitative relationship was formulated to relate C0t.5 values of renatured DNA mixtures to per cent binding (“homology”). Finally, it was demonstrated that DNA prepared from log-phase cells renatures faster than stationary-phase DNA and also departs from theoretical second-order kinetics.

Journal ArticleDOI
TL;DR: The relationship between specific growth rate of Escherichia coli and the concentration of limiting nutrient (glucose or phosphate or tryptophan) has been determined for populations in a steady state and the results indicate that mean cell volume depends not only on thespecific growth rate but also on the nature of the limiting nutrient.
Abstract: The relationship between specific growth rate of Escherichia coli and the concentration of limiting nutrient (glucose or phosphate or tryptophan) has been determined for populations in a steady state. At high concentrations the specific growth rate is independent of the concentration of nutrient, but at low concentrations the specific growth rate is a strong function of the nutrient concentration. Such a relationship was predicted by Monod; however, Monod's equation does not predict the relationship over the entire range of nutrient concentration. If parameters of the equation are estimated from the results obtained at low concentrations, then at high concentrations of nutrient, the specific growth rate is significantly higher than that predicted by Monod's equation. These results were interpreted on the basis that the rate of growth is controlled by at least two parallel reactions and that the affinities of the enzymes catalyzing these reactions are different. The relationship between specific growth rate and mean cell volume was also measured, and the results indicate that mean cell volume depends not only on the specific growth rate but also on the nature of the limiting nutrient. There are different mean cell volumes at the same specific growth rate established by different limiting nutrients. Therefore, the mean cell volume is not uniquely determined by the specific growth rate.

Journal ArticleDOI
TL;DR: A model for flagellar attachment is deduced from a purification of the Escherichia coli outer membrane with flagella still attached and a knowledge of the structure and dimensions of the E. coliFlagella were bound to the purified lipopolysaccharide membrane specifically at the basal body ring closest to the hook (the L ring).
Abstract: A procedure is described for the purification of the Escherichia coli outer membrane (lipopolysaccharide or L membrane) with flagella still attached. The resulting lipopolysaccharide membrane was in the form of vesicles that had a trilaminar structure in thin section and contained about 55% lipopolysaccharide and 45% protein. T2 or T4 phage preadsorbed to E. coli were found attached to the purified lipopolysaccharide membrane. Flagella were bound to the purified lipopolysaccharide membrane specifically at the basal body ring closest to the hook (the L ring). The cytoplasmic membrane in preparations from osmotically lysed E. coli spheroplasts or Bacillus subtilis protoplasts was specifically attached to flagella at the basal body ring farthest from the hook (the M ring). In the E. coli preparation, lipopolysaccharide membrane was also present and was attached to the L ring. From these data and a knowledge of the structure and dimensions of the E. coli flagellar basal body and cell envelope, a model for flagellar attachment is deduced.

Journal ArticleDOI
TL;DR: Cell respiration in wild type and poky was studied as part of a long-term investigation of cyanide-resistant respiration on the basis of its sensitivity to antimycin A or cyanide.
Abstract: Cell respiration in wild type and poky was studied as part of a long-term investigation of cyanide-resistant respiration in Neurospora. Respiration in wild type proceeds via a cytochrome chain which is similar to that of higher organisms; it is sensitive to antimycin A or cyanide. Poky, on the other hand, respires by means of two alternative oxidase systems. One of these is analogous to the wild-type cytochrome chain in that it can be inhibited by antimycin A or cyanide; this system accounts for as much as 15% of the respiration of poky f− and 34% of the respiration of poky f+. The second oxidase system is unaffected by antimycin A or cyanide at concentrations which inhibit the cytochrome chain maximally. It can, however, be specifically inhibited by salicyl hydroxamic acid. The cyanide-resistant oxidase is not exclusive to poky, but is also present in small quantities in wild type grown under ordinary circumstances. These quantities may be greatly increased (as much as 20-fold) by growing wild type in the presence of antimycin A, cyanide, or chloramphenicol.

Journal ArticleDOI
TL;DR: It is demonstrated that the synthesis observed in minicell preparations is a property of the plasmid-containing minICElls and not a result of the few cells contaminating the preparations, suggesting that the observed synthesis is of biological importance.
Abstract: Unlike the deoxyribonucleic acid (DNA)-deficient minicells produced by F(-) parents, minicells produced by plasmid-containing strains contain significant amounts of plasmid DNA. We examined the ability of plasmid-containing minicells to synthesize ribonucleic acid (RNA) and protein. In vivo, minicells produced by F(-) parents are unable to incorporate radioactive precursors into acid-insoluble RNA or protein, whereas minicells produced by F', R(+), or Col(+) parents are capable of such synthesis. Using a variety of approaches, including polyacrylamide gel analysis of the RNA species produced and electron microscope autoradiography, we demonstrated that the synthesis observed in minicell preparations is a property of the plasmid-containing minicells and not a result of the few cells (approximately 1 per 10(6) minicells) contaminating the preparations. That the observed synthesis is of biological importance is suggested by the ability of plasmid-containing minicells to yield viable phage upon infection with T4.

Journal ArticleDOI
TL;DR: Screening of different genera of yeasts has shown that the inactivation of fructose diphosphatase is a relatively widespread phenomenon and that reappearance of enzyme activity implies de novo synthesis.
Abstract: Fructose-1,6-diphosphatase was derepressed in Saccharomyces cerevisiae by incubation in media containing non-sugar carbon sources. Addition of glucose to a derepressed culture led to a rapid loss of the measurable activity of the enzyme. Fructose and mannose also produced inactivation, but 2-deoxyglucose was ineffective. Experiments with cycloheximide indicated that the inactivation does not require protein synthesis. It was also shown that the process is not energy-dependent. The reappearance of the enzyme was dependent on an energy source and was prevented by cycloheximide. These results suggest that fructose diphosphatase inactivation is irreversible and that reappearance of enzyme activity implies de novo synthesis. Screening of different genera of yeasts has shown that the inactivation of fructose diphosphatase is a relatively widespread phenomenon.

Journal ArticleDOI
TL;DR: Only one group (B-2) of marine bacteria included in this study was found to have polar, unsheathed flagella.
Abstract: Species of marine bacteria belonging to the genus Beneckea and strains of Photobacterium fischeri were negatively stained and examined by means of the electron microscope to determine the structure and arrangement of their flagella. All of the species of the genus Beneckea had single, polar, sheathed flagella when grown in liquid medium. When grown on solid medium, most strains of B. campbellii and B. neptuna and all strains of B. alginolytica and B. parahaemolytica had unsheathed, peritrichous flagella in addition to the single, sheathed, polar flagellum. The remaining species, B. nereida, B. pelagia, and B. natriegens, had a single, polar, sheathed flagellum when grown on solid medium. Strains of P. fischeri had sheathed flagella arranged in polar tufts. Only one group (B-2) of marine bacteria included in this study was found to have polar, unsheathed flagella.

Journal ArticleDOI
TL;DR: The same procedure developed for Escherichia coli was also successful for purifying intact flagella from Bacillus subtilis for the purification of intact flagescens free from detectable cell wall, membrane, or cytoplasmic material.
Abstract: A procedure is described for the purification of bacterial flagella in the form of a filament-hook-basal body complex (intact flagella) free from detectable cell wall, membrane, or cytoplasmic material. Spheroplasts produced with lysozyme and ethylenediaminetetraacetic acid were lysed with Triton X-100, and the flagella were purified by (NH4)2SO4 precipitation, differential centrifugation, and CsCl gradient centrifugation. As much as 40% of the flagella were recovered, and they contained about one basal body per 4 to 6 μm of flagella. The same procedure developed for Escherichia coli was also successful for purifying intact flagella from Bacillus subtilis.

Journal ArticleDOI
Ronald S. Cole1
TL;DR: Since the biological inactivation by psoralen correlates well with cross-link formation, it is suggested that the sensitizing action of this drug primarily derives from its ability to form DNA cross-links.
Abstract: We have investigated some biological consequences of light-induced psoralen-deoxyribonucleic acid (DNA) adducts and find that for several Escherichia coli functions (killing of strain AB2480 recA13 uvrA6, inactivation of phage lambda plaque-forming ability in wild type and uvrA6 hosts, loss of ability to transmit intact Flac+ episomes), a light exposure sufficient for production of a single cross-link per DNA molecule correlates well with the biological consequence. Although one cross-link per genome is apparently lethal to recA13 uvr− strains, mutants carrying the recA13 or uvrA6 markers survive light exposures producing 6.7 and 16 cross-links per genome, respectively, and wild-type cells recover from 65 psoralen cross-links. Evidently, the excision and recombinational repair systems complement one another in reconstructing an intact genome from cellular DNA containing psoralen photoproducts. The above bacterial and phage strains, in which DNA repair processes are minimized, are also extremely sensitive to pyrimidine dimer-forming 254-nm UV light (without psoralen), and were expected to respond similarly to formation of psoralen-pyrimidine base monoadducts in their DNA. Since the biological inactivation by psoralen correlates well with cross-link formation, we suggest that the sensitizing action of this drug primarily derives from its ability to form DNA cross-links.

Journal ArticleDOI
TL;DR: The amino acid analogue l-serine hydroxamate, which is bacteriostatic for Escherichia coli, has been shown to inhibit protein synthesis, and mutants resistant to l-Serine Hydroxamate have been selected, and three were shown to have seryl-tRNA synthetases with increased K(i) values.
Abstract: The amino acid analogue l-serine hydroxamate, which is bacteriostatic for Escherichia coli, has been shown to inhibit protein synthesis. The antimetabolite is a competitive inhibitor of seryl-transfer ribonucleic acid (tRNA) synthetase with a K(i) value of 30 mum. Mutants resistant to l-serine hydroxamate have been selected, and three were shown to have seryl-tRNA synthetases with increased K(i) values. One mutant contains a 3-phosphoglycerate dehydrogenase which is insensitive to inhibition by l-serine.

Journal ArticleDOI
TL;DR: It is proposed that the same formate dehydrogenase is involved in nitrate reduction, hydrogen production, and in aerobic formate oxidation.
Abstract: The effects of adding molybdate and selenite to a glucose-minimal salts medium on the formation of enzymes involved in the anaerobic metabolism of formate and nitrate in Escherichia coli have been studied. When cells were grown anaerobically in the presence of nitrate, molybdate stimulated the formation of nitrate reductase and a b-type cytochrome, resulting in cells that had the capacity for active nitrate reduction in the absence of formate dehydrogenase. Under the same conditions, selenite in addition to molybdate was required for forming the enzyme system which permits formate to serve as an effective electron donor for nitrate reduction. When cells were grown anaerobically on a glucose-minimal salts medium without nitrate, active hydrogen production from formate as well as formate dehydrogenase activity depended on the presence of both selenite and molybdate. The effects of these metals on the formation of formate dehydrogenase was blocked by chloramphenicol, suggesting that protein synthesis is required for the increases observed. It is proposed that the same formate dehydrogenase is involved in nitrate reduction, hydrogen production, and in aerobic formate oxidation.

Journal ArticleDOI
TL;DR: The purification and properties of an ultraviolet (UV) repair endonuclease are described and the specific substrate in UV-irradiated DNA appears to be pyrimidine dimer sites.
Abstract: The purification and properties of an ultraviolet (UV) repair endonuclease are described. The enzyme is induced by infection of cells of Escherichia coli with phage T4 and is missing from extracts of cells infected with the UV-sensitive and excision-defective mutant T4V(1). The enzyme attacks UV-irradiated deoxyribonucleic acid (DNA) containing either hydroxymethylcytosine or cytosine, but does not affect native DNA. The specific substrate in UV-irradiated DNA appears to be pyrimidine dimer sites. The purified enzyme alone does not excise pyrimidine dimers from UV-irradiated DNA. However, dimer excision does occur in the presence of the purified endonuclease plus crude extract of cells infected with the mutant T4V(1).

Journal ArticleDOI
TL;DR: Several diaminopimelic acid (DAP)- and lysine-requiring mutants of Escherichia coli were isolated and studied by genetic, physiological, and biochemical means.
Abstract: Several diaminopimelic acid (DAP)- and lysine-requiring mutants of Escherichia coli were isolated and studied by genetic, physiological, and biochemical means. The genes concerned with DAP-lysine synthesis map at several different sites on the E. coli chromosome and, therefore, do not constitute a single operon. Three separate loci affecting DAP synthesis are located in the 0 to 2.5 min region of the genetic map. The order of the loci in this region is thr-dapB-pyrA-ara-leu-pan-dapC-tonA-dapD. Two additional DAP genes map in the region between min 47 and 48, with the gene order being gua-dapA-dapE-ctr. The lys locus at min 55 determines the synthesis of the enzyme DAP decarboxylase, which catalyzes the conversion of DAP into lysine. The order of the genes in this region is serA-lysA-thyA.

Journal ArticleDOI
TL;DR: All three proteolytic enzymes isolated from sporulating cultures of Bacillus subtilis were shown to exhibit a rapid increase in specific activity at a time coinciding with the appearance of refractile bodies in cells.
Abstract: Three proteolytic enzymes have been isolated from sporulating cultures of Bacillus subtilis. These activities were, respectively, a protease inhibited by ethylenediaminetetraacetic acid (EDTA) but not phenylmethylsulfonyl fluoride (PMSF), a protease active on both protein and ester substrates, and an ester-active enzyme with low activity on proteins. The latter two enzymes were inhibited by PMSF but not by EDTA. The specific activity of each was determined both intra- and extra-cellularly during growth and sporulation in a single-defined medium. All three enzymes were shown to exhibit a rapid increase in specific activity at a time coinciding with the appearance of refractile bodies in cells.

Journal ArticleDOI
TL;DR: The following hypothesis is suggested to account for the low viability of Rec(-)E.
Abstract: We have studied the growth properties of 17 isogenic strains of Escherichia coli K-12 differing only in the recA, recB, recC, and sbcA alleles. We have observed the following. (i) All recombination deficient strains have decreased growth rates and decreased viabilities compared with recombination proficient strains. The large populations of nonviable cells in Rec(-) cultures may arise by spontaneous lethal sectoring (9). (ii) A recA mutant strain which is entirely recombination deficient and which shows high ultraviolet sensitivity and "reckless" deoxyribonucleic acid (DNA) breakdown has approximately the same growth rate and twice the viability as recB and recC mutant strains which have residual recombination proficiency, moderate ultraviolet sensitivity, and "cautious" DNA breakdown. (iii) Indirectly suppressed (sbcA(-)) recombination proficient (Rec(+)) revertants of recB and recC mutant strains have approximately normal growth rates and are three times as viable as their Rec(-) ancestors (but not as viable as rec(+) cells). We suggest the following hypothesis to account for the low viability of Rec(-)E. coli. Single-strand breaks in the DNA duplex, necessary for normal bacterial growth, may be repaired in a Rec(+) cell. Failure of Rec(-) cells to repair this normal DNA damage may lead to the observed loss of viability.

Journal ArticleDOI
TL;DR: A method is described for calculating k (the rate constant for inactivation of individual sites), N (sites per cell), and X(L) (the number which must be inactivated to cause death under the experimental conditions used) from experimental data, which is consistent with all experimental observations on thermal injury and death of bacteria.
Abstract: Experimental observations on thermal injury and death of bacteria in the stationary phase can be explained by assuming that death results from inactivation of (XL) of N critical sites. It is assumed: (i) that inactivation of individual sites occurs at random and follows first-order kinetics, (ii) that the critical sites are identical and of equal heat resistance, and (iii) the bacterial population is homogeneous in heat resistance. A method is described for calculating k (the rate constant for inactivation of individual sites), N (sites per cell), and XL (the number which must be inactivated to cause death under the experimental conditions used) from experimental data. Theoretical curves calculated by using this model are identical with experimental curves, providing support for the assumptions used. Calculated values of N and XL were 130 and 21.7 for Pseudomonas viscosa and 175 and 2.7 for Salmonella anatum. There is considerable uncertainty in the absolute values of N, but they are probably > 100. It is predicted that XL will vary depending on the recovery medium used after heating. This theory is consistent with all experimental observations on thermal injury and death of bacteria.

Journal ArticleDOI
TL;DR: It is concluded that the chlD gene product cannot be a structural component of the formate-hydrogenlyase pathway or the formates-nitrate reductase pathway, but that it must have an indirect role in processing molybdate to a form necessary for both electron transport systems.
Abstract: ChlD mutants of Escherichia coli are pleiotropic, lacking formate-nitrate reductase activity as well as formate-hydrogenlyase activity. Whole-chain formate-nitrate reductase activity, assayed with formate as the electron donor and measuring the amount of nitrite produced, was restored to wild-type levels in the mutants by addition of 10(-4)m molybdate to the growth medium. Under these conditions, the activity of each of the components of the membrane-bound nitrate reductase chain increased after molybdate supplementation. In the absence of nitrate, the activities of the formate-hydrogenlyase system were also restored by molybdate. Strains deleted for the chlD gene responded in a similar way to molybdate supplementation. The concentration of molybdenum in the chlD mutant cells did not differ significantly from that in the wild-type cells at either low or high concentrations of molybdate in the medium. However, the distribution of molybdenum between the soluble protein and membrane fractions differed significantly from wild type. We conclude that the chlD gene product cannot be a structural component of the formate-hydrogenlyase pathway or the formate-nitrate reductase pathway, but that it must have an indirect role in processing molybdate to a form necessary for both electron transport systems.