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Volume 116,
Issue 2,
1980
Volume 116, Issue 2, 1980
- Biochemistry
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Adenine Nucleotide Pools During Starvation of Beneckea natriegens
More LessThe marine bacterium Beneckea natriegens was grown in batch culture on a glucose/NH4 +/salts medium; growth terminated due to either carbon or nitrogen depletion from the medium. Nitrogen-limited cultures converted part of the excess glucose into glycogen whereas the carbon-limited cultures formed little glycogen. Glycogen-rich cultures survived longer than glycogen-poor cultures during starvation. Little protein was utilized during starvation and RNA was degraded as the primary endogenous source of energy. Glycogen was consumed only when the RNA content had decreased to about a third of the growth value.
The adenine nucleotide content of nitrogen-limited cultures increased at the start of the stationary phase but the energy charge remained at the growth value of 0·9 to 0·95. The maximum size of the adenine nucleotide pool depended on the concentration of glucose remaining in the medium at the start of the stationary phase but a limiting value of about 60 mol ATP (g protein)−1 was attained, compared with 12 to 14 μmol ATP (g protein)−1 in exponentially growing cultures. During extended starvation of both glycogen-rich and glycogen-poor clutures, there was a large decrease in adenine nucleotide content, but the energy charge remained above 0·6 even when viability was very low.
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Effects of Oligomycin on Glucose Utilization and Calcium Transport in African Trypanosomes
More LessOligomycin (3 μg ml−1) inhibited glucose utilization in Trypanosoma brucei S42 as shown by measurements of oxygen uptake and pyruvate production. Carbonyl cyanide 3-chloro-phenylhydrazone, an uncoupler of oxidative phosphorylation, did not relieve this inhibition, although some relief was afforded by the alternative substrate glycerol. Naturally dyskine-toplastic Trypanosoma evansi MIAG 105 was less sensitive to inhibition by oligomycin although glycerol relief was still observed, reflecting the differential sensitivity of the two pathways. With glucose present as the substrate, 45Ca2+ transport was inhibited by oligomycin in T. brucei, but was stimulated in T. evansi. These results are discussed in terms of alternative systems for maintaining cytoplasmic Ca2+ concentrations in normal and dyskine-toplastic strains of trypanosome.
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Lipid Surface Films: Interaction of Bacteria with Free Fatty Acids and Phospholipids at the Air/Water Interface
More LessThe interactions of bacteria, mainly Serratia marinorubra and Pseudomonas halocrenaea, with different lipid surface films were examined employing the surface balance technique and particle electrophoresis. Three free fatty acids as well as octadecylamine, and two diacyl-phosphatidylcholines were used as monolayer films with phosphate and ammonium acetate buffers as subsurface solutions. The following factors were involved in the interaction: the architecture of the surface film, the charge carried by both the bacteria and the film, hydrophobic interaction, and probably enzyme activity. The presence of Ca2+ seemed to be necessary for interfacial phospholipase activity.
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- Development And Structure
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Origin of the Polysaccharide Component of Ooze from Plants Infected with Erwinia amylovora
More LessThe composition of extracellular polysaccharides produced by Erwinia amylovora in defined culture media was compared with that of the polysaccharide present in the ooze produced during fireblight infection. The results strongly suggested that the ooze polysaccharide was of bacterial origin.
Two distinct polysaccharides were produced in vitro. One contained galactose (61 to 69%), glucose (8 to 11%), mannose (2 to 6%) and uronic acid (16 to 23%) and the other contained only fructose. Both the quantity and composition of the polysaccharide produced was determined by the nature and concentration of the sugar or sugar alcohol supplied and by the nature of the nitrogen source.
Evidence is also presented which suggests that production of the ooze-like polysaccharide is a virulence determinant and that this polysaccharide is similar in composition to the capsule. Preliminary studies indicate the presence of capsule and polysaccharide-hydrolysing enzymes in lysates of phage-infected bacteria.
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Evidence for Two Virulence Determinants in the Fireblight Pathogen Erwinia amylovora
More LessSeven avirulent strains of Erwinia amylovora were mixed in various combinations and tested for virulence. Only one combination, where the capsulated avirulent strain P66 was combined with the non-capsulated avirulent strain 4S2, induced fireblight symptoms in immature pear fruits and apple shoots. Growth studies in vivo strongly suggested that these strains were acting synergistically and that the non-capsulated strain possessed another, as yet unknown, virulence factor.
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Colony Morphology, Ultrastructure and Morphogenesis in Mycoplasma hominis, Acholeplasma laidlawii and Ureaplasma urealyticum
More LessColonies of Mycoplasma hominis, Acholeplasma laidlawii (three strains) and Ureaplasma urealyticum were examined by light and electron microscopy and their characteristic morphology, ultrastructure and morphogenesis are described. Mycoplasma hominis and A. laidlawii, PG8 and oral strains, developed typical fried-egg colonies which were remarkably heterogeneous in size. The colonies of A. laidlawii strain NCTC 10116 were more homogeneous and grew mainly on the surface of the agar showing a fine granular appearance. Ureaplasma urealyticum produced smaller, granular colonies which grew deeply embedded in the agar and generally without much surface growth. The cellular ultrastructure in these colonies was also examined. The results indicate that several aspects of colony morphogenesis and ultrastructure varied for each of the three species examined.
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Ultrastructural Study of the Interaction between Acholeplasma laidlawii and Antibody
More LessThe ultrastructure of agar-grown Acholeplasma laidlawii incubated with specific antiserum or IgM fractions of this antiserum has been investigated by the thin-sectioning technique. Antiserum treatment resulted in the development of giant cells along the colony circumferences and in the coating of normal-size mycoplasmas with a periodically arranged extra-membranous layer, consisting of attached immunoglobulins as shown by indirect immuno-ferritin labelling. The regular structure of the coat was not influenced by changes in temperature or by fixation of the membrane antigens prior to reaction with antibody. Extracellular enveloped viruses were uniformly covered with antibody in these experiments. IgM fractions of antiserum in high concentrations produced a similarly uniform extramembranous layer both on mycoplasmas and viruses. Possible explanations of the difference demonstrated between the regular arrangement of antigenic determinants on A. laidlawii membranes and the previously observed uniform binding of immunoglobulins to Mycoplasma gallisepticum are discussed.
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Hormonal Regulation of Sexual Reproduction in Phytophthora
More LessInduction of oospore formation by the complementary strain occurred in both cases when A1 and A2 isolates of Phytophthora parasitica were separated by polycarbonate membranes and up to 3 mm of agarose. The number of oospores produced increased as separation distance decreased. The homothallic species P. heveae induced oospore formation in both the A1 and A2 isolates of P. parasitica. Phytophthora megasperma var. sojae stimulated oospore formation in the A2 but not the A1 isolate of P. parasitica, while P. cactorum and P. katsurae stimulated the A1 but not the A2 isolate. Sexual reproduction of homothallic species of Phytophthora may thus also be controlled by α hormones. Sixteen types of chemically regulated sexuality among the members of Phytophthora, differing in hormone production and responsiveness to hormones, are postulated.
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- Genetics And Molecular Biology
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Transfer of Symbiotic Genes with Bacteriocinogenic Plasmids in Rhizobium leguminosarum
More LessTransfer of derivatives of the bacteriocinogenic plasmid pRL1JI into eight symbiotically defective strains of R. leguminosarum resulted in suppression of the mutant phenotype in four cases. These included non-infective, ineffective and temperature-sensitive ineffective phenotypes. However, in none of these strains was the defect suppressible after high frequency transfer of derivatives of two other bacteriocinogenic plasmids, pRL3JI or pRL4JI. Nodulation ability was co-transferred at high frequency (> 95 %) with bacteriocin production by the plasmid pRL1JI. The other two plasmids, pRL3JI and pRL4JI, also mediated the transfer of nodulation ability but at much lower frequency (10−3 to 10−4 per plasmid transfer). Sometimes, transconjugants that had acquired nodulation ability after the transfer of derivatives of plasmids pRL3JI or pRL4JI acted subsequently as high frequency donors for nodulation ability in a manner that was apparently similar to strains containing pRL1JI.
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Regulation of Proline Transport in Aspergillus nidulans
More LessAspergillus nidulans has at least two permeases for l-proline. The prnB gene of the prn gene cluster specifies the major proline permease, which is inducible by proline. Synthesis of the prnB permease is subject to repression by ammonium at 37 °C but not at 25 °C. A genetically unidentified minor proline permease(s) does not respond to proline induction or ammonium repression but is inhibited by ammonium. areAr mutants are unable to utilize nitrogen sources other than ammonium because they lack a positive-acting regulatory product required for expression of ammonium-repressible activities. However, there are very few cases in which the lack of growth of areAr mutants on a particular nitrogen source can be attributed to a reduction in the level of a particular enzyme activity or permease. Reduced expression of the prnB permease can account for the inability of areAr mutants to utilize proline. This is demonstrated by the ability of cis-acting regulatory mutations designated prnd , which derepress synthesis of the prnB permease, to suppress areAr mutations for proline utilization. The apparent ability of prnd mutations to derepress synthesis of proline oxidase and l-pyrroline-5-carboxylate dehydrogenase is probably an indirect consequence of their ability to derepress synthesis of the prnB permease, preventing inducer exclusion. There is presently no evidence that prnd mutations directly affect expression of the prnA, prnC or prnD genes, but this possibility has not been definitively eliminated.
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Streptomyces albus G Mutants Defective in the SalGI Restriction-Modification System
More LessStreptomyces albus G mutants (at least 12 of which were independent) defective in SalGI-mediated restriction (R−) were isolated after mutagenesis. Some of them lacked detectable SalGI activity in cell-free extracts. Some were also partially or completely defective in SalGI-associated modification (M−). Loss of restriction rendered S. albus G sensitive to many phages to which it was normally totally resistant. DNA from one such phage had many SalGI target sites (mean, one site per 1·35 kilobases). A mutant was isolated which was heat-sensitive for growth, apparently because it was restriction-proficient but temperature-sensitive for modification. At a rather high frequency, this mutant generated spontaneous heat-tolerant derivatives which were nearly all R−. Such R− mutants were always M− rather than being temperature-sensitive for modification. In a limited genetic analysis, the determinants of restriction and modification did not recombine with each other, and since there was no reassortment of these phenotypes among the parental output of crosses it appeared that the determinants were located close together on the chromosome.
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Genes and Enzymes of Lysine Catabolism in Pseudomonas aeruginosa
More LessPseudomonas aeruginosa strain PAO1 cannot utilize l-lysine effectively as a carbon source for growth but grows on cadaverine and glutarate. Strains PAO1 and PAO1632 (Hut−Ami−) have low activities for l-lysine uptake and for l-lysine decarboxylase but both strains gave rise to mutants that grew well on l-lysine. Strain PAO2087, isolated from PAO1, had an active l-lysine uptake system and an inducible l-lysine decarboxylase. Strain PAO2070, isolated from strain PAO1632, had an active l-lysine uptake system and a constitutive l-lysine decarboxylase. We suggest that the genetic defect of strain PAO1 (and PAO1632) that prevents growth on l-lysine is in a regulatory gene controlling the expression of linked genes for l-lysine permease and l-lysine decarboxylase.
Mutants unable to utilize l-lysine as a carbon source, isolated from strain PAO2070, exhibited four distinct growth phenotypes. Transductional analysis showed that the genetic defects of these mutants could be distinguished from each other and from that of strain PAO1. Group I mutants, unable to utilize glutarate, formed a single transduction linkage group and were mapped at about 20 min on the chromosome. The mutations of groups II, III and IV appeared to be in separate but linked genes. The group II mutants had no detectable l-lysine decarboxylase activity and the gene locus was mapped by interrupted mating in the 50 to 60 min region of the chromosome. The group III mutants possessed all the early enzymes of the l-lysine decarboxylase pathway and lacked only an active l-lysine uptake system.
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The Catabolism of Arginine by Pseudomonas aeruginosa
More LessMutants isolated from Pseudomonas aeruginosa strain PAO1632 (Hut−Ami−) were unable to utilize l-arginine or l-ornithine as the carbon source for growth. Arginine deiminase (AD), catabolic ornithine carbamoyltransferase (cOTC) and N 2-acetylornithine 5-aminotrans-ferase (ACOAT) were present in the mutants but these enzymes were not induced to higher levels by exogenous l-arginine. One group of mutants could utilize l-ornithine but not l-arginine and in these strains l-arginine induced the synthesis of ACOAT but not AD or cOTC. The mutations of the arginine utilization-negative mutants were all in genes of the same transductional linkage group and mapped in the 45 to 50 min region of the chromosome. Revertants isolated on l-arginine or l-ornithine plates were derepressed for the synthesis of ACOAT. It is suggested that l-arginine is normally catabolized by the wild-type strain via the arginine deiminase pathway and requires a threshold level of ACOAT. The regulatory factors controlling the functioning of the divergent arginine deiminase and arginine carboxylase pathways are discussed.
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Mitotic Arrest and Chromosome Doubling Using Thiabendazole, Cambendazole, Nocodazole and Ben Late in the Slime Mould Dictyostelium discoideum
More LessThiabendazole and cambendazole induced mitotic arrest and isogenic diploid formation through chromosome doubling in amoebae of the cellular slime mould Dictyostelium discoideum grown either axenically or in a bacterial suspension. The effect of nocodazole on axenically grown cells was similar to that of thiabendazole and cambendazole, but with cells grown on bacteria nocodazole was a much less effective inducer of mitotic arrest and did not lead to isogenic diploid formation. Benomyl, the active ingredient of ben late, had little or no effect on the induction of mitotic arrest and was a poor inducer of isogenic diploids in cells grown either axenically or on bacteria. All four benzimidazole derivatives were effective in haploidizing pre-existing diploids.
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Examination of the Chromosomes ofPolysphondylium pallidum Following Metaphase Arrest by Benzimidazole Derivatives and Colchicine
More LessTechniques used previously to examine the mitotic chromosomes of Dictyostelium discoideum have been applied to the cellular slime mould Polysphondylium pallidum. Mitosis in P. pallidum is similar to that in D. discoideum except that a metaphase plate configuration is rare and that it has more chromosomes (n = 11 or 12) than D. discoideum (n = 7). Some metaphase arrest (mitotic index 14 %) was achieved in P. pallidum when colchicine was used at a high concentration (10 mg ml−1). Low concentrations of benzimidazole derivatives (thiabendazole, cambendazole, ben late and nocodazole) caused the arrest of cell division, mitotic indices greater than 50%, chromosome doubling and lowered plating efficiency. Growth of P. pallidum in axenic medium was investigated.
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Heterokaryosis in Nectria haematococca: Complementation Between Mutants Affecting the Expression of Two Differentiated States
More LessCertain mycelia of the ascomycete Nectria haematococca show two morphological differentiated states, rings and sectors, which are cytoplasmically transmissible. Two nuclear loci A and S, appear to be involved in the control of these differentiated states. Mutations in the A region either prevent ring formation and the synthesis of the corresponding infectious factor (type a) or modify ring expression (type 58). Using forced heterokaryons, it was demonstrated that locus A corresponds to a single gene which exists in three allelic forms, wild-type, a and 58.
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Studies of Temperature-sensitive Transfer and Maintenance of H Incompatibility Group Plasmids
More LessThe mechanism of temperature-sensitive transfer was studied for plasmids of the H incompatibility group. Transfer depended on the temperature of the mating mixture but the growth temperature of the donor was also important, and donor cells previously grown at 26 °C could not facilitate transfer at 37°C. Comparison of transfer characteristics of a non-thermosensitive H plasmid R831b and thermosensitive H plasmids from Salmonella from Ontario during a 2 h mating period showed that the thermosensitive phenotype inhibited transfer by about 200-fold at 37 °C and by 10-fold at 42 °C. At temperatures between 15 and 30 °C, the thermotolerant H plasmid transferred at about the same frequency as the temperature-sensitive plasmid. Elimination of some H plasmids after growth of host cells was also observed and physical evidence of this was obtained. The characteristic of high-temperature elimination (Hte) was limited to plasmids from similar bacterial and geographical sources. Plasmids from Salmonella spp. isolated in Ontario did not possess this phenotype, whereas plasmids from Serratia marcescens isolated in the United States did. Although the Tra(ts) and Hte phenotypes may both be characteristic of H plasmids, they were shown to be separate and distinct properties. The H plasmids used in this study were isolated and their molecular weights determined by agarse gel electrophoresis. All were large, with molecular weights often exceeding 140 × 106. In contrast, the thermostable H plasmid R831b had a molecular weight of only 49 × 106.
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Genetic Analysis of a Pleiotropic Mutant of Klebsiella pneumoniae Affected in Nitrogen Metabolism
More LessGenetic and reversion analyses of a thermosensitive pleiotropic mutant strain of Klebsiella pneumoniae with defects in nitrogen fixation and nitrogen metabolism have shown that the pleiotropic behaviour of the mutant is due to a single mutation in a gene designated nim. This gene is cotransducible with trp at a frequency of about 30% (using bacteriophage P1) and with cys at a frequency of about 14%. The gene order is cys, trp, nim. The defect in the nim mutant is complemented by the E. coli F′ element, F′148.
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- Medical Microbiology
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Crossed Immunoelectrophoresis and Crossed-line Immunoelectrophoresis of Salmonella dublin Antigens
More LessCrossed immunoelectrophoresis and crossed-line immunoelectrophoresis were used to detect 62 antigens in extracts of sonicated Salmonella dublin against an homologous antiserum. Comparison with six extracts of closely related bacteria showed that all but two of these antigens cross-reacted with at least one other extract.
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Immunochemical Characterization of Neisseria meningitidis Serotype Antigens by Immunodiffusion and SDS-Polyacrylamide Gel Electrophoresis Immunoperoxidase Techniques and the Distribution of Serotypes Among Cases and Carriers
More LessThe chemical nature of the antigens of the meningococcal serotypes described by Frasch and colleagues was determined by a combination of immunodiffusion and the SDS-poly-acrylamide gel electrophoresis immunoperoxidase technique (SGIP). It was confirmed that the serotype antigens of the outer membrane of serotypes 1, 2, 6, 9, 11 and 12 were proteins, whilst those of serotypes 4, 5 and 8 were lipopolysaccharides. Serotype 2 can now be divided into three related types, provisionally called 2a (originally serotype 2), 2b and 2c with the specific antigens being proteins having molecular weights of 41000, 41500 and 41500, respectively. A total of 195 strains of meningococci isolated from patients and carriers in the Netherlands and 20 serogroup Y strains from patients in the U.S.A. were serotyped by means of immunodiffusion. Serotype 2a could be demonstrated in some strains belonging to the serogroups B (only those from carriers), C, W-135 and Y (only those from the U.S.A.). The W-135 strains isolated from patients in this series more often belonged to serotype 2a than did the W-135 strains from carriers. Serotype 2b was present in about half of the serogroup B and a few serogroup C strains isolated from patients with meningitis, but absent in serogroup B and C strains from carriers. Serotype 2c could only be demonstrated in serogroup Y strains, both from the Netherlands and the U.S.A. The other serotypes were found only sporadically.
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