- Volume 119, Issue 2, 1980
Volume 119, Issue 2, 1980
- Biochemistry
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The Isolation and Characterization of a 1,2-Propanediol Oxidoreductase from Neisseria gonorrhoeae
An enzyme which oxidizes 1,2-propanediol in the presence of NAD+has been purified from lysates of Neisseria gonorrhoeae. The enzyme was activated by monovalent cations, had a pH optimum between 9 and 10, and showed a substrate specificity unlike any known alcohol or glycerol dehydrogenase. The enzyme had an apparent K m of 17 mm for 1,2-propanediol and 0.37 mM for NAD+. When chromatographed on a Sephadex G-150 column, the enzyme eluted as a single peak in the molecular weight region of a bovine serum albumin marker. An antibody to the purified enzyme was prepared in goats. When antiserum was reacted with the enzyme in immunodiffusion experiments, a single precipitin band was detected. When the enzyme was mixed with an excess of antibody and then reacted with substrate, enzyme activity was completely inhibited.
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The Influence of Extracellular Hydrogen on the Metabolism of Bacteroides ruminicola, Anaerovibrio lipolytica and Selenomonas ruminantium
More LessStrains of three anaerobic rumen bacteria, Bacteroides ruminicola, Anaerovibrio lipolytica and Selenomonas ruminantium, were able to use extracellular H2 to reduce fumarate to succinate. Each bacterium possessed membrane-bound hydrogenase and fumarate reductase activity. Membrane-bound cytochrome b was reducible by H2 and oxidizable by fumarate in each bacterium. The apparent K m values for hydrogen of the hydrogenases were 4.5 10−6 m, 1.4 10−5 m and 4.4 10−5 m for B. ruminicola, A. lipolytica and S. ruminantium, respectively. The apparent K m values for fumarate of the fumarate reductases were approximately 1.0 10−4 m for each bacterium.
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- Development And Structure
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Protoplasts from Yeast and Mycelial Forms of Candida albicans
More LessProtoplasts have been obtained in high yields from the yeast and mycelial forms of a variety of strains of Candida albicans by enzymic digestion of cells with commercially available lytic enzymes. The protoplast formation procedure was equally effective for exponential and stationary phase cells. Pretreatment with dithiothreitol and Pronase in the presence of EDTA and Tris was necessary. Other thiol reagents and conditions did not release protoplasts from all the strains of C. albicans tested. Treatment with digestive juice of the snail Helix pomatia required the addition of chitinase for the release of protoplasts from most strains tested. Conditions for maximizing the yield of protoplasts and the activities of β-glucuronidase and chitinase were determined. Electron microscopy of C. albicans showed that the pretreatment conditions removed the outer layers and the treatment itself completely removed the inner layers of the cell wall. More than 90 % of the protoplasts produced by this method were viable as assessed by vital staining with Janus Green B.
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Effects of Streptomycin on the Growth and Sporulation of Saprolegnia spp.
More LessStreptomycin inhibited growth and sporulation of Saprolegnia species, although variations in sensitivity were recorded. In sporulating colonies, streptomycin increased both the length of the post-induction lag phase and the proportion of gemmae. Antibiotic concentrations (> 100 μg ml−1) which resulted in the leakage of potassium ions from sporulating colonies also correlated with the condensation of hyphal cytoplasm into lysed segments. In vegetative colonies grown in > 500 μg streptomycin ml−1, changes were seen in the organization of endoplasmic reticulum, mitochondria and nuclei. These inhibitory effects could be counteracted by the addition of calcium ions. Some observed effects were consistent with an impairment of normal mitochondrial function, but others, particularly in sporulating colonies, were possibly the result of interference with cellular functions regulated by calcium or other divalent cations.
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- Genetics And Molecular Biology
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Transformation in Micrococcus radiodurans: Measurement of Various Parameters and Evidence for Multiple, Independently Segregating Genomes per Cell
More LessTransformation frequencies greater than 1% for some single markers were obtained in Micrococcus radiodurans when bacteria in the exponential phase of growth were resuspended in fresh growth medium containing 0·03 m-Ca2+ before being incubated with transforming DNA. Mg2+, Sr2+ or Zn2+ could not replace Ca2+ in giving high frequencies of transformation. The time required for the maximum expression of transformed markers was 2 to 3 h for resistance to rifampicin and acriflavin and 6 to 8 h for resistance to erythromycin, kanamycin and streptomycin. The comparative frequency of transformants at maximum expression for each resistance marker was: kanamycin, 1; streptomycin, 1; acriflavin, 4; erythromycin, 25; rifampicin, 64.
Cultures were competent during all stages of exponential growth, the frequency of transformants only falling during stationary phase. The minimum time for DNA to be taken up by M. radiodurans into a DNAase-resistant form was between 3 and 6 s. From 6 s to 10 min exposure to DNA, the number of transformants increased non-linearly with time as though the process was inducible. The transformation frequency was directly proportional to the DNA concentration up to 1 g ml−1, although even at 88 g ml−1 the bacteria were not saturated. Attempts to measure the fraction of cells which were competent, using the unlinked marker technique, gave values well in excess of one. These were interpreted in terms of multiple genome copies and approximate values of between 0·25 and 0·72 were derived for the competent fractions.
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Nuclear and Mitochondrial Suppression of a Mitochondrially Inherited Cold-sensitive Mutation in Aspergillus nidulans
More LessPartial suppressors of a mitochondrially inherited mutation, [cs-67], conferring cold-sensitivity at 20 0C were identified. These mapped at one mitochondrial and four unlinked nuclear loci. Most suppressors partially restored the cytochrome aa 3 deficiency of the cold-sensitive strain at 20 0C. Strains carrying two or more suppressors and [cs-67] showed considerably impaired growth. This effect was temperature-dependent, being more severe at 37 0C, and was not expressed in the presence of the [cs-67 +] allele. The cytochrome oxidase activity of one of these strains was no more heat-sensitive than that of the wild-type implying that these mutations did not directly modify cytochrome oxidase. The wild-type strain grown in the presence of chloramphenicol and the cold-sensitive strain grown at 20 0C had similar cytochrome spectra and mitochondrial membrane protein profiles on sodium dodecyl sulphate gradient acrylamide gels. [cs-67] conferred pleiotropically a low level of resistance to paromomycin at 37 0C. It is suggested that [cs-67] and the suppressors act at the level of the mitochondrial ribosome.
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Effect of tif Expression, Irradiation of Recipient and Presence of Plasmid pKM101 on Recovery of a Marker from a Donor Exposed to Ultraviolet Light Prior to Conjugation
More LessTo detect the effect of the postulated inducible error-prone repair system (‘SOS repair’) on the bacterial chromosome, an Hfr Escherichia coli strain JC5088 recA was u.v.-irradiated immediately before mating it with recipients in which SOS repair was supposed to be functioning either through tif expression, u.v. irradiation or the presence of plasmid pKM101. The recombinant yields of these crosses were compared with those obtained in corresponding crosses with recipients in which SOS repair either was not induced or was totally eliminated by the lexA mutation. No difference in marker recovery efficiency could be detected between these two sets of recipients and thus no induced repair process acting on donor DNA could be demonstrated. The possible reasons for this finding are discussed.
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A Pigmented Mycelial Antibiotic in Streptomyces coelicolor: Control by a Chromosomal Gene Cluster
More LessStreptomyces coelicolor was found to produce a third secondary metabolite, in addition to the antibiotics methylenomycin A and actinorhodin previously described. This is a red pigmented, highly non-polar compound with antibiotic activity against certain Grampositive bacteria. Mutants lacking the red compound fell into five cosynthetic classes. Representatives of each of the five classes were mapped to the chromosome of the producing organism, in a closely linked cluster. Genetic studies provided evidence that this new metabolite is distinct from actinorhodin and indicated that the two pigments do not share parts of the same biosynthetic pathway.
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Co-synthesis of Penicillin Following Treatment of Mutants of Aspergillus nidulans Impaired in Antibiotic Production with Lytic Enzymes
More LessMycelia from four mutants of Aspergillus nidulans impaired in penicillin production at separate genetic loci were treated with an enzyme complex capable of lysing cell walls, then mixed in all possible paired combinations and grown in osmotically buffered penicillin production media, containing 2-deoxyglucose and an unrefined mixture of polyoxins to prevent cell wall regeneration. The culture filtrates were assayed after 6 d and significant penicillin yields were observed in four of the six possible combinations. None of these pairs produced penicillin when grown together as normal mycelium, suggesting that intermediates of the penicillin biosynthetic pathway unable to diffuse from untreated mycelium could do so from enzyme-treated mycelium when cell wall regeneration was inhibited. A general method is thus available for examining biochemical pathways with mutants accumulating intermediates unable to cross the cell wall barrier.
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Physiological and Morphological Characteristics of Stationary Phase Vibrio Cells Able to Support Phage Growth
More LessSummary: Growth of phage α3a on stationary phase Vibrio cultures requires micro-aerophilic conditions and is inhibited by aeration. Since pre-conditioning of the bacteria by allowing them to stand for 24 h after shaking for 3 d is an important aspect of the stationary phase phage growth system, various physiological and morphological characteristics of the stationary phase cells during the transition from shaking to standing were investigated. Shaken stationary phase cells were less viable and more sensitive to ultraviolet irradiation and heat than standing stationary phase cells. During pre-conditioning the small, non-flagellated cells present in shaken stationary phase cultures underwent morphological changes and became large, flagellated rods which resembled exponential phase cells. The transition of stationary phase cells from shaking to standing was associated with a marked increase in total RNA synthesis but a rapid and large decrease in total protein synthesis. Intracellular concentrations of ATP in shaken stationary phase cells were 53 % lower than those in standing stationary phase cells. Studies on leucine uptake indicated that its transport was inhibited by isoleucine and that the major part (90 %) of the total leucine uptake was due to a shared system for uptake of both amino acids. Shaken stationary phase cells transported less leucine than standing stationary phase cells. Inhibition of phage growth in aerated stationary phase cultures was not due to the prevention of phage adsorption by shaking. It is suggested that the observed differences between shaken and standing stationary phase cells could be due to aeration affecting the template specificity of the Vibrio RNA polymerase.
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Evidence for Two Mechanisms of Plasmid Transfer in Mixed Cultures of Staphylococcus aureus
More LessNon-lysogenic Staphylococcus aureus strain 1030 was lysogenized with 12 different bacteriophages. Lysogeny was associated with acquisition of phage inducibility by mitomycin C treatment or ultraviolet irradiation, with the presence of plaque-forming phage in culture supernatants and with considerable narrowing in susceptibility to the typing bacteriophages and also with increased sensitivity to trimethoprim and sulphadiazine. The presence of prophages in the donor and/or the recipient could either promote or inhibit transfer of plasmids between mixed cultures. Transfer frequencies in mixed culture after 18 h incubation could be as high as 7.0 × 10−1resistant recipients/final donor, and evidence was adduced for a mechanism distinct from transfer by spontaneous transduction. It is suggested that this method of gene transfer be described as “phage-mediated conjugation”. Chromosomal genes were not transferred by this method. Two similar plasmids cp-2 and cp-3 were able to promote their own transfer through clones of 1030; plasmids coding for resistance to either neomycin or tetracycline could be transferred to a recipient by the presence of cp-2 or cp-3 simultaneously in the donor. The presence of plasmids in 1030 was associated with a small increase in sensitivity to trimethoprim or sulphadiazine.
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Rarity of Gene Transfer Between Animal and Human Isolates of Staphylococcus aureus in Vitro
More LessAntibiotic-resistant Staphylococcus aureus strains of animal origin were studied for transfer of resistance to human strains in vitro, with special reference to tylosin, an antibiotic which is used only in animals. Resistance to tylosin in animal and human strains was part of a constitutive resistance to all macrolide antibiotics. Resistance to tylosin could not be transferred from animal to human strains in vitro. Transfer of other resistances occurred between few strains and at low frequency. Resistance determinants from only seven animal cultures (out of 196 tested) could donate resistance to human strains in mixed cultures. The pattern of metal ion resistance was markedly different in animal and human strains: this supports the conclusion that spontaneous transfer between them is rare in nature.
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Alginate Synthesis in Mucoid Pseudomonas aeruginosa: a Chromosomal Locus Involved in Control
More LessMucoid variants of Pseudomonas aeruginosa isolated in vitro or in vivo could be classified into two phenotypic groups based on whether alginate was produced on a chemically defined medium. Mucoid strains yielded lower recombination frequencies than the non-mucoid parent when used as donors in FP2-mediated plate matings. The mucoid characteristic (muc) was co-inherited by a proportion of recombinants selected for the inheritance of chromosomal markers his-5075 +or cys-5605 +. The results of further experiments using either a mucoid recipient or a mucoid donor carrying plasmid R68.45 suggested that the control of alginate production in P. aeruginosa involves at least one chromosomal locus.
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Naturally Occurring R. ColBM Plasmids Belonging to the IncFIII Incompatibility Group
More LessTwo Escherichia coli strains isolated from urinary tract infections were resistant to streptomycin, kanamycin, neomycin, tetracycline and sulphonamides. The strains also produced colicins B and M. The resistance to streptomycin, kanamycin and neomycin and the ability to produce colicins B and M could be transferred to an E. coli K12 recipient. Resistance and colicinogeny markers were transferred together by conjugation, and did not segregate even after interrupted mating or phage P1-mediated transduction. Hence, the drug-resistance and colicinogeny markers were carried by the same plasmid, designated as R. ColBM plasmid. The two R. ColBM plasmids were Fi+and produced an F-like pilus. They belonged to the IncFIII incompatibility group, being thus the first R plasmids identified in this group. The two plasmids were isolated and their molecular sizes were determined by electrophoresis in agarose gels and by contour length measurements. Both methods showed that both plasmids were about 52 megadaltons.
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The Sodium Effect of Bacillus subtilis Growth on Aspartate
More LessaspH mutants of Bacillus subtilis have a constitutive aspartase activity and grow well on as partate as sole carbon source. aspH aspT mutants, which are deficient in high affinity aspartate transport as a result of the aspT mutation, grow as well as aspH mutants in medium containing high concentrations of aspartate and Na+. This Na+ effect is not due to an enhancement of aspartate transport but is the result of increased cellular metabolism. The ability to grow rapidly in sodium aspartate is induced by prior growth in the presence of Na+. In potassium aspartate, the addition of arginine, citrulline, ornithine, Δ1-pyrroline-5-carboxylate or proline instead of Na+also allows rapid growth; but in a mutant deficient in ornithine-oxo-acid aminotransferase, only pyrroline-carboxylate or proline can replace Na+. The amino acid pool of cells growing slowly in potassium aspartate contains proline at a low concentration which increases upon addition of proline (but not Na+) to the medium. Thus, Na+addition does not increase the synthesis of proline, but proline or pyrroline-carboxylate acts similarly to Na+either in preventing some inhibitory effect (by aspartate or the accumulating NH4+) or in overcoming some deficiency (e.g. in further proline metabolism).
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Life-span and Senescence in Podospora anserina: Effect of Mitochondrial Genes and Functions
More LessThe life-span of a mitochondrial mutant of Podospora anserina resistant to chloramphenicol was at least five times that of isonuclear chloramphenicol-sensitive strains. This property was maternally inherited. A study of the segregation of heteroplasmic mycelia showed that, in addition to the mitochondrial alleles cap r I/cap s I, another cytoplasmic factor, whose nature is discussed, controlled the life-span. Cycloheximide decreased the life-spans of all the strains studied, whereas they were greatly increased by chloramphenicol and ethidium bromide. Chloramphenicol seemed to act mainly by lowering the probability of commitment to senescence, while ethidium bromide seemed to affect both the commitment probability and the incubation period. Furthermore, chloramphenicol and ethidium bromide were able to rejuvenate senescent mycelia. These results are discussed in connection with previous results on the mitochondrial origin of senescence in Podospora anserina.
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Effect of Thymine Deprivation on the Restoration of DNA Synthesis in Ultraviolet-irradiated Escherichia coli B/r Hcr+
More LessThe influence of a prior period of thymine and amino acid deprivation on the restoration of DNA synthesis and on the fraction of cells surviving after u.v. irradiation and subsequent incubation with chloramphenicol in Escherichia coli B/r Hcr+has been studied. Thymine and amino acid deprivation stimulated post-irradiation DNA synthesis and increased the fraction of surviving cells if, in the period between deprivation and u.v. irradiation, protein synthesis occurred. It is concluded that proteins induced by thymine starvation participated in the repair of u.v.-irradiated cells.
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- Medical Microbiology
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Inapparent Chlamydial Infection in the Urogenital Tract of Guinea-pigs
More LessMicrobiologically inapparent urogenital infection appeared to be induced in male guinea-pigs inoculated intra-urethrally with low doses of the guinea-pig inclusion conjunctivitis strain (GP-IC) of Chlamydia psittaci. This state was indicated by the ability of inoculated animals to donate eye infection to normal animals caged with them. Donors failed to develop overt urogenital infection throughout the period of transmission judged by both absence of infected cells in urethral scrapings and failure to isolate GP-IC in cell culture; however, inoculation of donors with 5-iododeoxyuridine led to transient appearance of infectivity in scrapings. In distinction from overtly infected animals, donors failed to develop serum antibody and remained susceptible to urethral challenge with larger doses of GP-IC. Animals that had recovered from overt urethral infection were resistant to challenge and appeared unable to transmit eye infection.
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The Occurrence of 1,2-Propanediol Oxidoreductase in Micro-organisms and its Use as a Possible Diagnostic Marker for Neisseria gonorrhoeae
The cervical microbial flora of 25 females and stock cultures of various micro-organisms which may be present in the human female cervix were examined using a fluorimetric assay for 1,2-propanediol oxidoreductase. Results indicated that only members of the genera Neisseria and Acinetobacter possess appreciable activities of the enzyme, whose physiological function is not yet known. The activity of this enzyme in N. gonorrhoeae appeared to be significantly higher than the activities observed in most of the other Neisseria species and in the Acinetobacter species. These results indicated that it may be possible to utilize this enzyme as a presumptive diagnostic marker for N. gonorrhoeae in cervical secretions. 1,2-Propanediol oxidoreductase may also be of taxonomic significance for the classification of various bacterial species.
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Immunological Properties of the Cell Envelope Components of Vibrio cholerae
More LessSeveral immunobiological properties of cell envelope components of Vibrio cholerae such as mitogenicity, antigenicity, adjuvanticity and toxicity were tested in mice. Killed whole bacteria, spheroplasts, lipopolysaccharide and outer membrane proteins possessed mitogenic activity as determined by [3H]thymidine uptake in spleen cell cultures. All these components predominantly stimulated murine bone-marrow derived (B) lymphocytes. The mitogenicity induced by V. cholerae lipopolysaccharide was similar in magnitude to that observed with Salmonella typhimurium lipopolysaccharide. Vibrio cholerae lipopolysaccharide was mitogenic for gut-associated lymphocytes such as those obtained from Peyer's patches and small intestine. Antibody formation at the cellular level was detected by the haemolytic plaque assay. Plaque-forming cells to V. cholerae lipopolysaccharide were only detected when mice were immunized intraperitoneally with intact cells or with spheroplasts. Among the various cell envelope components, lipopolysaccharide alone possessed adjuvant properties as it increased the number of plaque-forming cells to sheep erythrocytes fourfold in mouse spleens. Also, lipopolysaccharide was the only component found to be toxic for the mouse (LD50 0.5 mg). Neither spheroplasts nor outer membrane of V. cholerae showed adjuvanticity or toxicity in mice.
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