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Showing papers in "Journal of Bacteriology in 1980"


Journal ArticleDOI
TL;DR: It is suggested that carboxyl groups provide the major site of metal deposition in the B. subtilis wall, whereas extraction of teichoic acid caused a stoichiometric reduction in levels.
Abstract: Amine and carboxyl groups of the cell wall of Bacillus subtilis were chemically modified individually to neutralize their electrochemical charge for determination of their contribution to the metal uptake process. Mild alkali treatment removed ca. 94% of the constituent teichoic acid (expressed as inorganic phosphorus) and allowed estimation of metal interaction with phosphodiester bonds. Chemical modifications of amine functions did not reduce the metal uptake values as compared to native walls, whereas extraction of teichoic acid caused a stoichiometric reduction in levels. In contrast, alteration of carboxyl groups severely limited metal deposition of most of the metals tested. X-ray diffraction and electron microscopy suggested, in this case, that the form and structure of the metal deposit could be different from that found in native walls. The observations suggest that carboxyl groups provide the major site of metal deposition in the B. subtilis wall.

984 citations


Journal ArticleDOI
TL;DR: A series of plasmid vectors suitable for the detection and cloning of translational control signals and 5' coding sequences of exogenously derived genes yielded hybrid proteins useful for purifying peptide sequences encoded by exogenous deoxyribonucleic acid fragments and for studies relating the structure and function of specific peptide segments.
Abstract: We report the construction and use of a series of plasmid vectors suitable for the detection and cloning of translational control signals and 5' coding sequences of exogenously derived genes. In these plasmids, the first eight codons of the amino-terminal end of the lactose operon beta-galactosidase gene, lacZ, were removed, and unique BamHI, EcoRI, and SmaI (XmaI) endonuclease cleavage sites were incorporated adjacent to the eighth codon of lacZ. Introduction of deoxyribonucleic acid fragments containing appropriate regulatory signals and 5' coding sequences into such lac fusion plasmids led to the production of hybrid proteins consisting of the carboxyl-terminal segment of a beta-galactosidase remnant plus a peptide fragment that contained the amino-terminal amino acids encoded by the exogenous deoxyribonucleic acid sequence. These hybrid peptides retained beta-galactosidase enzymatic activity and yielded a Lac+ phenotype. Such hybrid proteins are useful for purifying peptide sequences encoded by exogenous deoxyribonucleic acid fragments and for studies relating the structure and function of specific peptide segments.

971 citations


Journal ArticleDOI
TL;DR: The amounts of glycogen and trehalose have been measured in cells of a prototrophic diploid yeast strain subjected to a variety of nutrient limitations, suggesting that reserve carbohydrate accumulation is a general response to nutrient limitation.
Abstract: The amounts of glycogen and trehalose have been measured in cells of a prototrophic diploid yeast strain subjected to a variety of nutrient limitations. Both glycogen and trehalose were accumulated in cells deprived specifically of nirogen, sulfur, or phosphorus, suggesting that reserve carbohydrate accumulation is a general response to nutrient limitation. The patterns of accumulation and utilization of glycogen and trehalose were not identical under these conditions, suggesting that the two carbohydrates may play distinct physiological roles. Glycogen and trehalose were also accumulated by cells undergoing carbon and energy limitation, both during diauxic growth in a relatively poor medium and during the approach to stationary phase in a rich medium. Growth in the rich medium was shown to be carbon or energy limited or both, although the interaction between carbon source limitation and oxygen limitation was complex. In both media, the pattern of glycogen accumulation and utilization was compatible with its serving as a source of energy both during respiratory adaptation and during a subsequent starvation. In contrast, the pattern of trehalose accumulation and utilization seemed compatible only with the latter role. In cultures that were depleting their supplies of exogenous glucose, the accumulation of glycogen began at glucose concentrations well above those sufficient to suppress glycogen accumulation in cultures growing with a constant concentration of exogenous glucose. The mechanism of this effect is not clear, but may involve a response to the rapid rate of change in the glucose concentration.

763 citations


Journal ArticleDOI
TL;DR: The finding that tetracycline-resistant cells are hypersensitive to lipophilic chelating agents, such as fusaric acid supports the contention that certain metal ions critically facilitate tetr Tracycline uptake and leads to suggest possible molecular mechanisms for tetrACYcline resistance.
Abstract: A simple technique has been devised that allows direct plate selection of tetracycline-sensitive clones from a predominantly tetracycline-resistant population The technique is especially useful in genetic methodologies based on the use of tetracycline resistance transposons, such as Tn10 Potential uses of the method include selection of deletion mutants, fine-structure mapping, generalized mapping, construction of multiply marked strains, elimination of tetracycline resistance transposons and plasmids and cloning The technique is based on our finding that tetracycline-resistant cells are hypersensitive to lipophilic chelating agents, such as fusaric acid This finding supports the contention that certain metal ions critically facilitate tetracycline uptake and leads us to suggest possible molecular mechanisms for tetracycline resistance

713 citations


Journal ArticleDOI
TL;DR: Mutants of Agrobacterium tumefaciens which affect virulence or the ability to catabolize octopine were isolated after Tn5-induced mutagenesis, and three mutants contained insertions in the T-deoxyribonucleic acid.
Abstract: Mutants of Agrobacterium tumefaciens which affect virulence or the ability to catabolize octopine were isolated after Tn5-induced mutagenesis. Of 8,900 colonies tested, 7 mutants with Tn5 insertions in a specific region of other Ti plasmid unable to catabolize octopine were isolated. Thirty-seven mutants affected in tumorigenesis resulted from insertions in the Ti plasmid and the Agrobacterium chromosome. Of these mutations, 12 were chromosomal and 25 mapped on the plasmid. Twenty-three mapped within a 20-megadalton region, which is distinct from the Ti plasmid sequences found stably integrated into the plant cell genome T-deoxyribonucleic acid). Included in these were mutants that were either a virulent or produced tumors with unusual morphologies. Three mutants contained insertions in the T-deoxyribonucleic acid. These three mutants incited tumors which synthesized octopine but had an altered morphology due to either extensive proliferation of shoots or roots from the tumor callus. Three additional mutants not caused by Tn5 contained mutations in the Ti plasmid.

429 citations


Journal ArticleDOI
TL;DR: It is concluded that pyocyanine diverts electron flow and thus increases the production of O2- and H2O2 and that the antibiotic action of this pigment is largely a reflection of the toxicity of these products of oxygen reduction.
Abstract: Exposure of Escherichia coli growing in a rich medium to pyocyanine resulted in increased intracellular levels of superoxide dismutase and of catalase. When these adaptive enzyme syntheses were prevented by nutritional paucity, the toxic action of pyocyanine was augmented. The antibiotic action of pyocyanine was dependent upon oxygen and was diminished by superoxide dismutase and by catalase, added to the suspending medium. Pyocyanine slightly augmented the respiration of E. coli suspended in a rich medium, but greatly increased the cyanide-resistant respiration. Pyocyanine was able to cause the oxidation of reduced nicotinamide adenine dinucleotide, with O2- production, in the absence of enzymatic catalysis. It is concluded that pyocyanine diverts electron flow and thus increases the production of O2- and H2O2 and that the antibiotic action of this pigment is largely a reflection of the toxicity of these products of oxygen reduction.

359 citations


Journal ArticleDOI
TL;DR: The ultrastructure of a magnetotactic bacterium (strain MS-1) was examined by transmission, scanning, and scanning-transmission electron microscopy, and the term "magnetosome" is proposed for the electron-dense particles with their enveloping layer(s) as found in this and other magnetosome bacteria.
Abstract: The ultrastructure of a magnetotactic bacterium (strain MS-1) was examined by transmission, scanning, and scanning-transmission electron microscopy. The organism resembled other spirilla in general cell morphology, although some differences were detected at the ultrastructural level. Electron-dense particles within magnetotactic cells were shown by energy-dispersive X-ray analysis to be localizations containing iron. A non-magnetotactic variant of strain MS-1 lacked these novel bacterial inclusion bodies. A chain of these particles traversed each magnetotactic cell in a specific arrangement that was consistent from cell to cell, seemingly associated with the inner surface of the cytoplasmic membrane. Each particle was surrounded by an electron-dense layer separated from the particle surface by an electron-transparent region. The term "magnetosome" is proposed for the electron-dense particles with their enveloping layer(s) as found in this and other magnetotactic bacteria.

337 citations


Journal ArticleDOI
TL;DR: The observation and the insensitivity of the xylitol transport to low temperature suggest that the facilitator behaves as a membrane channel.
Abstract: The specificity of the glycerol facilitator (glpF) of Escherichia coli was studied with an osmotic method. This transport system allowed the entry of polyols (glycerol and erythritol), pentitols, and hexitols. The analogous sugars were not transported. However, urea, glycine, and DL-glyceraldehyde could use this pathway to enter the cell. The glpF protein allowed the rapid efflux of preequilibrated xylitol. Glycerol surprisingly did not inhibit the uptake of xylitol, and xylitol only slightly reduced the uptake of glycerol. The observation and the insensitivity of the xylitol transport to low temperature suggest that the facilitator behaves as a membrane channel.

321 citations


Journal ArticleDOI
TL;DR: Pseudomonas aeruginosa PAO mutants defective in elastase were isolated by plate assays of nitrosoguanidine-mutagenized clones and one mutant (PAO-E64) was apparently identical to the parental strain except for its deficiency inElastase activity.
Abstract: Pseudomonas aeruginosa PAO mutants defective in elastase were isolated by plate assays of nitrosoguanidine-mutagenized clones. A total of 75 elastase mutants were isolated from 43,000 mutagenized clones. One mutant (PAO-E64) was apparently identical to the parental strain except for its deficiency in elastase activity. This mutant produced an enzyme which was antigenically indistinguishable from parental elastase. Furthermore, equal levels of elastase antigen were produced by this mutant and its parental strain. The mutant elastase, however, had greatly reduced enzymatic activity. Mutant PAO-E64 is presumed to have a mutation in the structural gene for elastase. We have designated the genotype of the mutation in PAO-E64 as lasA1.

317 citations


Journal ArticleDOI
TL;DR: It is suggested that aerobic growth of E. coli in batch cultures is limited by the rate of respiration and the concomitant rate of ATP generation through oxidative phosphorylation.
Abstract: Batch cultures of Escherichia coli were grown in minimal media supplemented with various carbon sources which supported growth at specific growth rates from 0.2 to 1.3/h. The respiration rates of the cultures were measured continuously. With few exceptions, the specific rate of oxygen consumption was about 20 mmol of O2/h per g (dry weight), suggesting that the respiratory capacity was limited at this value. The adenosine triphosphate (ATP) required for the production of cell material from the different carbon sources was calculated on the basis of known ATP requirements in the biochemical pathways and routes of macromolecular synthesis. The calculated ATP requirements, together with the measured growth rates and growth yields on the different carbon sources, were used to calculate the rate of ATP synthesis by oxidative phosphorylation. This rate was closely related to the respiration rate. We suggest that aerobic growth of E. coli in batch cultures is limited by the rate of respiration and the concomitant rate of ATP generation through oxidative phosphorylation.

297 citations


Journal ArticleDOI
TL;DR: A partially purified fraction of the enzymes, easily obtained with a single purification step, had a high lytic specific activity and was superior to commercial preparations in regard to nuclease, protease, and chitinase contamination.
Abstract: Yeast lytic activity was purified from the culture supernatant of Oerskovia xanthineolytica grown on minimal medium with insoluble yeast glucan as the carbon source. The lytic activity was found to consist of two synergistic enzyme activities which copurified on carboxymethyl cellulose and Sephadex G-150, but were resolved on Bio-Gel P-150. The first component was a beta-1,3-glucanase with a molecular weight of 55,000. The K(m) for yeast glucan was 0.4 mg/ml; that for laminarin was 5.9 mg/ml. Hydrolysis of beta-1,3-glucans was endolytic, yielding a mixture of products ranging from glucose to oligomers of 10 or more. The size distribution of products was pH dependent, smaller oligomers predominating at the lower pH. The glucanase was unable to lyse yeast cells without 2-mercaptoethanol or the second lytic component, an alkaline protease. Neither of these agents had any effect on the glucanase activity on polysaccharide substrates. The protease had a molecular weight of 30,000 and hydrolyzed Azocoll and a variety of denatured proteins. The enzyme was unusual in that it had an affinity for Sephadex. Although the activity was insensitive to most protease inhibitors, it was affected by polysaccharides; yeast mannan was a potent inhibitor. The enzyme did not have any mannanase activity, however. Neither pronase nor trypsin could substitute for this protease in promoting yeast cell lysis. A partially purified fraction of the enzymes, easily obtained with a single purification step, had a high lytic specific activity and was superior to commercial preparations in regard to nuclease, protease, and chitinase contamination. Lyticase has been applied in spheroplast, membrane, and nucleic acid isolation, and has proved useful in yeast transformation procedures.

Journal ArticleDOI
TL;DR: Significant quantitative differences in end-product yields by two strains of Clostridium thermocellum and one strain of Thermoanaerobium brockii were observed during cellobiose fermentation, mainly related to ethanol/H(2) and lactate/acetate ratios.
Abstract: Significant quantitative differences in end-product yields by two strains of Clostridium thermocellum and one strain of Thermoanaerobium brockii were observed during cellobiose fermentation. Most notably, the ethanol/H2 and lactate/acetate ratios were drastically higher for T. brockii as compared with C. thermocellum strains LQRI and AS39. Exogenous H2 addition (0.4 to 1.0 atm) during culture growth increased the ethanol/acetate ratio of both T. brockii and AS39 but had no effect on LQRI. All strains had an operative Embden-Meyerhof glycolytic pathway and displayed catabolic activities of fructose-1,6-diphosphate–activated lactate dehydrogenase, coenzyme A acetylating pyruvate and acetaldehyde dehydrogenase, hydrogenase, ethanol dehydrogenase, and acetate kinase. Enzyme kinetic properties (apparent Km, Vmax, and Q10 values) and the specificity of electron donors/acceptors for different oxidoreductases involved in pyruvate conversion to fermentation products were compared in the three strains. Both species contained ferredoxin-linked pyruvate dehydrogenase and pyridine nucleotide oxidoreductases. Ferredoxin-nicotinamide adenine dinucleotide (NAD) reductase activity was significantly higher in T. brockii than in AS39 and was not detectable in LQRI. H2 production and hydrogenase activity were inversely related to ferredoxin-NAD reductase activity in the three strains. Ferredoxin-NAD phosphate reductase activity was present in cell extracts of both species. Alcohol dehydrogenase activity in C. thermocellum was NAD dependent, unidirectional, and inhibited by low concentrations of NAD and ethanol. Ethanol dehydrogenase activity of T. brockii was both NAD and NADP linked, reversible, and not inhibited by low levels of reaction products. The high lactate yield of T. brockii correlated with increased fructose-1,6-diphosphate. The relation of catabolic enzyme activity and quantitative differences in intracellular electron flow and fermentation product yields of these thermophilic bacteria is discussed.

Journal ArticleDOI
TL;DR: The pathology induced by the virulent transconjugants containing only pAr15834b was identical to that produced by the wild-type strain of A. rhizogenes, and restriction endonuclease fragment analysis of plasmids from the trans Conjugants and the donor revealed that p Ar15834c is a cointegrate of pAr 15834a and pAr15934b.
Abstract: Agrobacterium rhizogenes strain 15834, which incites hairy root disease in plants, harbors three large plasmids: pAr15834a (107 x 10(6) daltons), pAr15834b (154 x 10(6) daltons), and pAr15834c (258 x 10(6) daltons). Kanamycin-resistant transconjugants were selected in a cross of kanamycin-resistant derivate of strain 15834 and an avirulent recipient. The transconjugants belonging to one class were virulent and contained all three donor plasmids. These transconjugants also acquired sensitivity to the bacteriocin agrocin 84. The loss of plasmids from virulent transconjugants during growth at 37 degrees C indicated that virulence genes reside on pAr15834b, whereas agrocin 84 sensitivity genes reside on pAr15834a. The pathology induced by the virulent transconjugants containing only pAr15834b was identical to that produced by the wild-type strain of A. rhizogenes. Restriction endonuclease fragment analysis of plasmids from the transconjugants and the donor revealed that pAr15834c is a cointegrate of pAr15834a and pAr15834b. Kanamycin-resistant transconjugants belonging to a second class were avirulent and contained an altered form of pAr15834b. Strain 15834 can utilize octopine. However, this trait was not detected in any of the transconjugants. Octopine is not synthesized by infected plant tissue.

Journal ArticleDOI
TL;DR: Initially, cointegrate molecules, whose molecular weights were approximately the sum of pAD1 and pAD2, accounted for these transconjugants; however, as the induction time increased, p AD1::Tn917 became increasingly prominent.
Abstract: Streptococcus faecalis strain DS16 harbors two plasmids, a conjugative plasmid, pAD1, which encodes hemolysin and bacteriocin activities, and a nonconjugative plasmid, pAD2, encoding resistance to streptomycin, kanamycin, and erythromycin, the latter of which is inducible. The erythromycin resistance determinant is located on a 3.3-megadalton transposable element designated Tn917, which could be transposed to pAD1 as well as to two other plasmids, pAm gamma 1 and pAM alpha 1. When strain DS16 was exposed to low (inducing) concentrations of erythromycin for a few hours, the frequency of Tn917 transposition from pAD2 to pAD1 increased by an order of magnitude. This induction paralleled induction of erythromycin resistance and was prevented by exposing the cells to inhibitors of deoxyribonucleic acid, ribonucleic acid or protein synthesis. The exposure of strain DS16 to inducing concentrations of erythromycin also enhanced the frequency of erythromycin-resistant transconjugants appearing during mating. Initially, cointegrate molecules, whose molecular weights were approximately the sum of pAD1 and pAD2, accounted for these transconjugants; however, as the induction time increased, pAD1::Tn917 became increasingly prominent.

Journal ArticleDOI
TL;DR: Hill plots of equilibrium dialysis data suggest that metal binding to cell walls involves negative cooperativity, and competition between various metals for binding sites suggested that the cations complex with identical sites on the cell walls.
Abstract: The use of equilibrium dialysis techniques established that isolated cell walls of Bacillus subtilis possess selective affinities for several cations. The binding of these cations to the cell wall was influenced by the presence of various functional groups in the peptidoglycan matrix. Selective chemical modification of the free carboxyl and amino groups showed that when amino groups were replaced by neutral, bulky, or negatively charged groups, the sites available for cation complexing generally increased. Introduction of positive charges into the wall resulted in a marked decrease in the numbers of metal binding sites and usually a decrease in the apparent association constants. Both teichoic acid and peptidoglycan contribute to the sites available for interaction with metals. Hill plots of equilibrium dialysis data suggest that metal binding to cell walls involves negative cooperativity. Competition between various metals for binding sites suggested that the cations complex with identical sites on the cell walls. When the hydrogen ion concentration was increased, the affinity of the walls for metals decreased, but the numbers of metal binding sites remained constant, suggesting that cations and protons also compete for the same sites.

Journal ArticleDOI
TL;DR: Results clearly distinguished a number of surface mating systems where most plasmids were derepressed for transfer and determined conjugative pili constitutively, and led to the reclassification of R831b as IncM.
Abstract: Representative plasmids for most incompatibility groups in Escherichia coli K-12 were transferred to a "bald" strain to compare transfer frequencies for liquid and solid media. Standard broth matings were used for a liquid environment, but for solid surface mating, conjugation was allowed to take place on nutrient plates before washing off the cells for transconjugant selection on plates containing appropriate drugs. Plasmids that determine rigid pili transferred at least 2,000x better on plates than in broth. Some plasmids that determine thick flexible pili transferred 45 to 470x better, whereas others transferred equally well in both environments, as did plasmids of the I complex, which determine thin flexible pili. These results clearly distinguished a number of surface mating systems where most plasmids were derepressed for transfer and determined conjugative pili constitutively. The temperature-independent IncH2 plasmid R831b transferred best on plates, but other IncH plasmids transferred equally well in broth. This inconsistency led to the reclassification of R831b as IncM.

Journal ArticleDOI
TL;DR: Tn5's ability to turn on distal gene expression is not due to a promoter in its central region and therefore is not dependent on the overall orientation of Tn5 in the operon.
Abstract: We assessed the effect of insertions of the kanamycin resistance transposon Tn5 in the lac operon of Escherichia coli on the expression of distal genes lacY and lacA (melibiose fermentation at 41 degrees C and thiogalactoside transacetylase synthesis, respectively). Every insertion mutation tested (41 in lacZ and 23 in lacY) was strongly polar. However, approximately one-third of the insertion mutants expressed distal genes at low levels due to a promoter associated with Tn5. To localize this promoter, we (i) reversed the orientation of Tn5 at several sites and (ii) replaced wild-type Tn5 with several substitution derivatives which lack Tn5's central region. Neither alteration changed the expression of distal genes. Thus, in contrast to transposons IS2 and TnA. Tn5's ability to turn on distal gene expression is not due to a promoter in its central region and therefore is not dependent on the overall orientation of Tn5 in the operon. Our results suggest that the promoter is within 186 base pairs of the ends of Tn5. It is possible that the promoter is detected in only a fraction of insertions because it overlaps Tn5-target sequence boundary.

Journal ArticleDOI
TL;DR: Evidence is presented that Pi is transported as Pi or a very labile intermediate and that accumulated Pi does not exit through the Pst or Pit systems from glucose-grown cells.
Abstract: Inorganic phosphate (Pi) transport by wild-type cells of Escherichia coli grown in excess phosphate-containing media involves two genetically separable transport systems. Cells dependent upon the high affinity-low velocity Pst (phosphate specific transport) system have a Km of 0.43 +/- 0.2 microM Pi and a Vmax of 15.9 +/- 0.3 nmol of Pi (mg [dry weight]-1min-1) and will grow in the presence of arsenate in the medium. However, cells dependent upon the low affinity-high velocity Pit (Pi transport) system have a Km of 38.2 +/- 0.4 microM and a Vmax of 55 +/- 1.9 nmol of Pi (mg [dry weight]-1min-1), and these cells cannot grow in the presence of an arsenate-to-Pi ratio of 10 in the medium. Pi transport by both systems was sensitive to the energy uncoupler 2,4-dinitrophenol and the sulfhydryl reagent N-ethylmaleimide, whereas only the Pst system was very sensitive to sodium cyanide. Evidence is presented that Pi is transported as Pi or a very labile intermediate and that accumulated Pi does not exit through the Pst or Pit systems from glucose-grown cells. Kinetic analysis of Pi transport in the wild-type strain containing both the Pst and Pit transport systems revealed that each system was not operating at full capacity. In addition, Pi transport in the wild-type strain was completely sensitive to sodium cyanide (a characteristic of the Pst system).

Journal ArticleDOI
TL;DR: In this article, it was shown that Pseudomonas aeruginosa cells grown in Mg2+-deficient medium acquire nonmutational resistance to the chelator ethylenediaminetetraacetate and to the cationic antibiotic polymyxin B; this type of resistance can be reversed by transferring the cells to Mg 2+-sufficient medium for a few generations.
Abstract: It is well established that Pseudomonas aeruginosa cells grown in Mg2+-deficient medium acquire nonmutational resistance to the chelator ethylenediaminetetraacetate and to the cationic antibiotic polymyxin B; this type of resistance can be reversed by transferring the cells to Mg2+-sufficient medium for a few generations. Stable mutants resistant to polymyxin B were isolated and shown to have also gained ethylenediaminetetraacetate resistance. Both the mutants and strains grown on Mg2+-deficient medium had greatly enhanced levels of outer membrane protein H1 when compared with the wild-type strain or with revertants grown in Mg2+-sufficient medium. It was determined that in all strains and at all medium Mg2+ concentrations, the cell envelope Mg2+ concentration varied inversely with the amount of protein H1. In addition, the increase in protein H1 in the mutants was associated with an increase in resistance to another group of cationic antibiotics, the aminoglycosides, e.g., gentamicin. We propose that protein H1 acts by replacing Mg2+ at a site on the lipopolysaccharide which can otherwise be attacked by the cationic antibiotics or ethylenediaminetetraacetate.

Journal ArticleDOI
TL;DR: Cell diameter decreases with increasing cell length but increases again at the end of the cell cycle as indicated by an increase of average diameter in the three consecutive classes of constriction.
Abstract: Extensive measurements of steady-state populations of several Escherichia coli strains have consistently indicated that cell diameter decreases with increasing cell length. This was observed both after electron microscopy of air-dried cells and after phase-contrast microscopy of living cells. The analysis was made by considering separately the unconstricted cells and three classes (slight, medium, and deep) of constricted cells in the population. During slow growth, cells with the average newborn length were up to 8% thicker than unconstricted cells twice as long. This decrease in diameter is less at higher growth rates. Despite the small changes and the large variation of the diameter in any particular length class, significant negative correlations between diameter and length were obtained. Cell diameter increases again at the end of the cell cycle as indicated by an increase of average diameter in the three consecutive classes of constriction.

Journal ArticleDOI
TL;DR: A positive selection for the presence of tryptophan 2-monooxygenase was devised based on its capacity to use as a substrate the toxic tryPTophan analogue 5-methyltryptophan, and this plasmid, here called pIAA1, when reintroduced into Iaa- mutants by transformation, restored tryptophilease and indoleacetamide hydrolase activities and production of IAA.
Abstract: Olive (or oleander) knot is a plant disease incited by Pseudomonas savastanoi. Disease symptoms consist of tumorous outgrowths induced in the plant by bacterial production of indole-3-acetic acid (IAA). Synthesis of IAA occurs by the following reactions: L-tryptophan leads to indoleacetamide leads to indoleacetic acid, catalyzed by tryptophan 2-monooxygenase and indoleacetamide hydrolase, respectively. Whereas the enzymology of IAA synthesis is well characterized, nothing is known about the genetics of the system. We devised a positive selection for the presence of tryptophan 2-monooxygenase based on its capacity to use as a substrate the toxic tryptophan analogue 5-methyltryptophan. Efficient curing of the bacterium of tryptophan 2-monoxygenase, indoleacetamide hydrolase, and IAA production was obtained by acridine orange treatment. Further, loss of capacity to produce IAA by curing was correlated with loss of a plasmid of 34 X 10(6) molecular weight. This plasmid, here called pIAA1, when reintroduced into Iaa- mutants by transformation, restored tryptophan 2-monooxygenase and indoleacetamide hydrolase activities and production of IAA.

Journal ArticleDOI
TL;DR: In bacterial promoters subject to stringent control the heptanucleotide sequence spanning nucleotide positions -5 to +2 (defining the -10 region [D. Pribnow, J. Biol. 99:419-443, 1975] as positions -12 to -6) is highly conserved.
Abstract: In bacterial promoters subject to stringent control the heptanucleotide sequence spanning nucleotide positions −5 to +2 (defining the −10 region [D. Pribnow, J. Mol. Biol. 99:419–443, 1975] as positions −12 to −6) is highly conserved. This conserved region, for which the best consensus sequence is [Formula: see text] is absent from promoters which are not under stringent control.

Journal ArticleDOI
TL;DR: It was possible to locate the ftsZ gene between ftsA and envA and it was concluded that these cell division genes are expressed independently of the neighboring murein genes.
Abstract: Complementation tests have revealed that the mutation in the filamenting mutant PAT84 is distinct from ftsA and has been designated ftsZ. By isolating transducing phages carrying various amounts of the bacterial deoxyribonucleic acid in this region, it was possible to locate the ftsZ gene between ftsA and envA. It is concluded that these cell division genes are expressed independently of the neighboring murein genes.

Journal ArticleDOI
TL;DR: Microbial methanogenesis was examined in thermal waters, muds, and decomposing algal-bacterial mats associated with volcanic activity in Yellowstone National Park and revealed that the microbial population was predominantly rod shaped and asporogenous.
Abstract: Microbial methanogenesis was examined in thermal waters, muds, and decomposing algal-bacterial mats associated with volcanic activity in Yellowstone National Park. Radioactive tracer studies with [(14)C]glucose, acetate, or carbonate and enrichment culture techniques demonstrated that methanogenesis occurred at temperatures near 70 degrees C but below 80 degrees C and correlated with hydrogen production from either geothermal processes or microbial fermentation. Three Methanobacterium thermoautotrophicum strains (YT1, YTA, and YTC) isolated from diverse volcanic habitats differed from the neotype sewage strain DeltaH in deoxyribonucleic acid guanosine-plus-cytosine content and immunological properties. Microbial methanogenesis was characterized in more detail at a 65 degrees C site in the Octopus Spring algal-bacterial mat ecosystem. Here methanogenesis was active, was associated with anaerobic microbial decomposition of biomass, occurred concomitantly with detectable microbial hydrogen formation, and displayed a temperature activity optimum near 65 degrees C. Enumeration studies estimated more than 10(9) chemoorganotrophic hydrolytic bacteria and 10(6) chemolithotrophic methanogenic bacteria per g (dry weight) of algal-bacterial mat. Enumeration, enrichment, and isolation studies revealed that the microbial population was predominantly rod shaped and asporogenous. A prevalent chemoorganotrophic organism in the mat that was isolated from an end dilution tube was a taxonomically undescribed gram-negative obligate anaerobe (strain HTB2), whereas a prevalent chemolithotrophic methanogen isolated from an end dilution tube was identified as M. thermoautotrophicum (strain YTB). Taxonomically recognizable obligate anaerobes that were isolated from glucose and xylose enrichment cultures included Thermoanaerobium brockii strain HTB and Clostridium thermohydrosulfuricum strain 39E. The nutritional properties, growth temperature optima, growth rates, and fermentation products of thermophilic bacterial strains 39E, HTB2, and YTB were determined.

Journal ArticleDOI
TL;DR: Three mutants of the yeast Saccharomyces cerevisiae which require exogenous ethanolamine or choline were isolated and the lipid composition suggests that cho1 mutants do not synthesize phosphatidylserine under any growth conditions.
Abstract: Three mutants of the yeast Saccharomyces cerevisiae which require exogenous ethanolamine or choline were isolated. The mutants map to a single locus (cho1) on chromosome V. The lipid composition suggests that cho1 mutants do not synthesize phosphatidylserine under any growth conditions. If phosphatidylethanolamine or phosphatidylcholine, which are usually derived from phosphatidylserine, were synthesized from exogenous ethanolamine or choline, the mutants grew and divided relatively normally. However, mitochondrial abnormalities were evident even when ethanolamine and choline were supplied. Diploids homozygous for the cho1 mutation were defective in sporulation. Growth on nonfermentable carbon sources was slow, and a high proportion of respiratory-deficient (petite) cells were generated in cho1 cultures.

Journal ArticleDOI
TL;DR: Strain JC10240 (Hfr PO45 srlC300::Tn10 recA56 thr-300 ilv-318 rpsE300) was constructed and can be moved to other Escherichia coli strains in transductional or conjugational crosses selecting resistance to tetracycline.
Abstract: Strain JC10240 (Hfr PO45 srlC300::Tn10 recA56 thr-300 ilv-318 rpsE300) was constructed. On account of the close linkage of Tn10 to recA56, the latter can be moved to other Escherichia coli (and closely related) strains in transductional or conjugational crosses selecting resistance to tetracycline.

Journal ArticleDOI
TL;DR: All of the lactic streptococci examined except Streptococcus lactis ML8 fermented galactose to lactate, formate, acetate, and ethanol, and reduced intracellular levels of both the lactate dehydrogenase activator and pyruvate-formate lyase inhibitors appeared to be the main factors involved in the diversion of lactate to the other products.
Abstract: All of the lactic streptococci examined except Streptococcus lactis ML8 fermented galactose to lactate, formate, acetate, and ethanol. The levels of pyruvate-formate lyase and lactate dehydrogenase were elevated and reduced, respectively, in galactose-grown cells compared with glucose- or lactose-grown cells. Reduced intracellular levels of both the lactate dehydrogenase activator (fructose, 1,6-diphosphate) and pyruvate-formate lyase inhibitors (triose phosphates) appeared to be the main factors involved in the diversion of lactate to the other products. S. lactis ML8 produced only lactate from galactose, apparently due to the maintenance of high intracellular levels of fructose 1,6-diphosphate and triose phosphates. The growth rates of all 10 Streptococcus cremoris strains examined decreased markedly with galactose concentrations below about 30 mM. This effect appeared to be correlated with uptake predominantly by the low-affinity galactose phosphotransferase system and initial metabolism via the D-tagatose 6-phosphate pathway. In contrast, with four of the five S. lactis strains examined, galactose uptake and initial metabolism involved more extensive use of the high-affinity galactose permease and Leloir pathway. With these strains the relative flux of galactose through the alternate pathways would depend on the exogenous galactose concentration.

Journal ArticleDOI
TL;DR: It is concluded that nmpB mutants are identical to phoR mutants, whereas other nmpA mutants are likely to contain mutations in one of the genes phoS, phoT, or pst.
Abstract: Outer membrane protein e is induced in wild-type cells, just like alkaline phosphatase and some other periplasmic proteins, by growth under phosphatase limitation. nmpA and nmpB mutants, which synthesize protein e constitutively, are shown also to produce the periplasmic enzyme alkaline phosphatase constitutively. Alternatively, individual phoS, phoT, and phoR mutants as well as pit pst double mutants, all of which are known to produce alkaline phosphatase constitutively, were found to be constitutive for protein e. Also, the periplasmic space of most nmpA mutants and of all nmpB mutants grown in excess phosphate was found to contain, in addition to alkaline phosphatase, at least two new proteins, a phenomenon known for individual phoT and phoR mutants as well as for pit pst double mutants. The other nmpA mutants as well as phoS mutants lacked one of these extra periplasmic proteins, namely the phosphate-binding protein. From these data and from the known positions of the mentioned genes on the chromosomal map, it is concluded that nmpB mutants are identical to phoR mutants. Moreover, some nmpA mutants were shown to be identical to phoS mutants, whereas other nmpA mutants are likely to contain mutations in one of the genes phoS, phoT, or pst.

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TL;DR: The results indicate that at least two plasmid-encoded genes, capable of acting in trans, and a replication origin are required for RK2 replication and maintenance.
Abstract: The sites of cleavage on the map of the broad-host-range plasmid RK2 (56 kilobases) were determined for the BglII, PstI, and SmaI restriction enzymes, and the determinants for tetracycline and ampicillin resistance were localized. The cleavage sites were clustered at or near the drug resistance genes. To localize regions required for plasmid replication and maintenance in Escherichia coli, we deleted nonessential regions of RK2 by partial digestion with the restriction endonuclease HaeII to produce small derivatives. The smallest stable replicon obtained contained five HaeII fragments of RK2 which total 5.4 kilobases. These fragments were derived from three regions of RK2 that are separated from each other by antibiotic resistance genes. One of these HaeII fragments (0.75 kilobases) has the properties expected of the origin of replication. The outer four fragments, located in two separate regions of RK2, were found to provide, in trans, functions that permit the replication of the HaeII fragment carrying the origin of the replication. These results indicate that at least two plasmid-encoded genes, capable of acting in trans, and a replication origin are required for RK2 replication and maintenance.

Journal ArticleDOI
TL;DR: Energy-independent association of iron with bacteria and energy-dependent accumulation of iron in the presence of ferripyochelin varied concomitantly in cells grown under various conditions, but pyochelin synthesis appeared to be controlled separately.
Abstract: Pyochelin is an iron-binding compound produced by Pseudomonas aeruginosa and demonstrates siderophore activity by its involvement in iron transport. During the transport process, an energy-independent association of [55Fe]ferripyochelin with bacteria occurred within the initial 30 s of reaction, followed by an energy-dependent accumulation of iron. The energy-independent association with iron appeared to be at the surface of the bacteria because the iron could be washed from the cells with thioglycolate, whereas accumulated iron was not washed from the bacteria. Energy-independent association of iron with bacteria and energy-dependent accumulation of iron in the presence of ferripyochelin varied concomitantly in cells grown under various conditions, but pyochelin synthesis appeared to be controlled separately. 55Fe complexed with citrate was also taken up by P. aeruginosa with a lower level of initial cell association. Bacterial mechanisms for iron uptake from ferric citrate were present in cells grown in a variety of media and were in lowest levels in cells grown in citrate. The synthesis of bacterial components for iron uptake from ferric citrate and from ferripyochelin was inhibited by high concentrations of iron supplied in growth media.