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Volume 135,
Issue 6,
1989
Volume 135, Issue 6, 1989
- Genetics And Molecular Biology
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In situ Localization of Specific RNAs in Whole Fruiting Colonies of Schizophyllum commune
More LesscDNA clones representing eight specific mRNAs abundantly expressed in a dikaryon of Schizophyllum commune but not in the progenitor monokaryons, were used to localize these mRNAs in whole colonies by in situ hybridization. A genomic clone for 18S rRNA was used to probe for rRNA. The colonies were grown on hybridization membranes, fixed, and treated with RNAase-depleted wall-lytic enzymes from Trichoderma harzianum to facilitate permeation of the probes. RNAs in sectors of the colonies were then localized with [32P]DNA probes. Hybridization at different developmental stages showed that the specific mRNAs were only formed in parts of the colony where fruit-body initials arose. At later stages these mRNAs disappeared from abortive fruit bodies but remained high in fruit bodies continuing development.
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Cloning and Expression of the Clostridium thermohydrosulfuricum α-Amylase-pullulanase Gene in Escherichia coli
More LessAn α-amylase-pullulanase gene from Clostridium thermohydrosulfuricum DSM 3783 was cloned in Escherichia coli on a 7·0 kb EcoRI fragment using a λ vector. The gene produced, from an indigenous promoter, active thermostable α-amylase-pullulanase, seemingly mostly a soluble intracellular enzyme in E. coli. Gel filtration separated the active enzyme produced into three peaks, each having both a-amylase and pullulanase activities. Immunoblotting after SDS-PAGE revealed more than ten a-amylase-pullulanase specific polypeptides; the biggest of these had an M r of about 165000, whereas the smallest enzymically active polypeptide had an M r of about 100000. Despite the marked degeneration of its constituent polypeptides, the apparent temperature optimum of the enzyme (80–85C) was only some 5C lower and the heat stability the same as that of the extracellular α-amylase-pullulanase produced by the native host. Oligonucleotide probes prepared according to the NH2-terminal amino acid sequences of the enzyme and its satellite polypeptide (a polypeptide associated with the extracellular enzyme of the native host) hybridized to different regions of the 7·0 kb DNA insert.
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Characterization of the RepFVII Replicon of the Haemolytic Plasmid pSU233: Nucleotide Sequence of an incFVII Determinant
More LessWe have isolated a replication region (designated RepFVII) from the IncFVII plasmid, pSU233. Hybridization experiments showed that RepFVII has high homology to the RepFIII replicon of the plasmid pSU316 and, therefore, that pSU233 is another member of the RepFIIA family. We have also located the incompatibility gene (incFVII) from RepFVII. The analysis of its sequence revealed the same organization described for several other replicons belonging to the same family.
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Regulation of Gene Expression and Cellular Localization of Cloned Klebsiella aerogenes (K. pneumoniae) Urease
More LessThe genes for Klebsiella aerogenes (K. pneumoniae) urease were cloned and the protein was overexpressed (up to 18% of total protein consisted of this enzyme) in several hosts. The small size of the DNA encoding urease (3·5 kb), the restriction map, and the regulation of enzyme expression directed by the recombinant plasmid are distinct from other cloned ureases. Nickel concentration did not affect urease gene expression, as demonstrated by the high levels of apoenzyme measured in cells grown in nickel-free media. However, nickel was required for urease activity. The overproducing recombinant strain was used for immunogold electron microscopic localization studies to demonstrate that urease is a cytoplasmic enzyme.
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- Pathogenicity And Medical Microbiology
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Antibiotic Susceptibility of Salmonella spp. at Different pH Values
More LessWe have examined the effects of acidic pH, in the range of those prevailing within phagosomes and lysosomes, on the growth and the susceptibility to different antibiotics of several strains of Salmonella spp. The minimal inhibitory concentration and the minimal bactericidal concentration of several β-lactams were increased considerably during culture at pH 5·2. The extent of the increase was a function of: (1) the β-lactam structure and, more particularly, the hydrophobicity of the side-chain of the molecule;and (2) the bacterial serotype. This phenotypic resistance at acid pH was not due to β-lactamase activity or to a lower growth rate. In contrast, rifamycin SV was more active at acidic pH than at neutral pH and chloramphenicol, another highly hydrophobic drug, was equally efficacious at both pH values. Membrane lipopolysac-charide mutants, but not porin mutants, cultivated at an acidic pH were inhibited by lower concentrations of the β-lactams. This suggests that the increased resistance to β-lactams, and the increased susceptibility to rifamycin SV, at acidic pH, could have resulted from modified permeability of the outer membrane to antibiotics.
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Inhibition of Growth of Chlamydia trachomatis by the Calcium Antagonist Verapamil
More LessTreatment of BGM (African Green Monkey kidney) cells with the calcium antagonist Verapamil resulted in a reduced yield of chlamydial infectious particles. The inhibitory effect was concentration-dependent, the maximal effect being achieved at 200 μm-Verapamil, which produced a 99·99% reduction of infectious particle yield. Electron microscopy showed that control Chlamydia trachomatis-infected BGM cells contained typical large inclusions in which most of the particles were elementary bodies, whereas Verapamil-treated infected cells contained small inclusions consisting predominantly of reticulate bodies. The findings indicate a possible therapeutic use of this calcium antagonist as an anti-chlamydial drug.
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Analysis of the Axial Filaments of Treponema hyodysenteriae by SDS-PAGE and Immunoblotting
More LessPurified axial filaments from eight serotypes of Treponema hyodysenteriae and two non-pathogenic intestinal spirochaetes were characterized by SDS-PAGE and Western blotting. Axial filaments of all ten strains had similar SDS-PAGE profiles; five major axial filament polypeptides were identified, with molecular masses of 43·8, 38, 34·8, 32·8 and 29·4 kDa. Hyperimmune gnotobiotic pig serum raised against purified axial filaments of strain P18A (serotype 4) cross-reacted with all other serotypes and with the non-pathogens, and convalescent serum taken from a pig with persistent swine dysentery also showed a strong response to the axial filament polypeptides. Hyperimmune gnotobiotic pig serum raised against axial filaments failed to agglutinate viable organisms and did not inhibit growth in vitro. Hence the axial filaments of T. hyodysenteriae have been identified as major immunodominant antigens, although the role that antibodies to these antigens play in protection has yet to be established.
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Staphylococcus aureus Bacteriophages Mediating the Simultaneous Lysogenic Conversion of β-Lysin, Staphylokinase and Enterotoxin A: Molecular Mechanism of Triple Conversion
A new group of serotype F bacteriophages of Staphylococcus aureus has been found which mediates the simultaneous triple-lysogenic conversion of enterotoxin A, staphylokinase and β-lysin. The phages were recovered from methicillin-resistant strains of S. aureus isolated in Irish hospitals between 1971 and 1988 and from strain PS42-D, which has been used as the propagating strain for the S. aureus typing phage 42D since before 1965. The molecular mechanism of triple conversion mediated by three of these phages was determined by molecular cloning, restriction endonuclease site mapping and hybridization analysis, and compared with the mechanism of β-lysin and staphylokinase conversion mediated by the serotype F, double-converting phage 𝜙 13. The genetic determinants mediating expression of enterotoxin A (entA) and staphylokinase (sak) were cloned from the DNA of the triple-converting phage and expression of the cloned determinants detected in Escherchia coli and S. aureus. The entA and sak determinants were closely linked in the phage DNA adjacent to the phage attachment site (attP) in each case and furthermore, the sak determinant of phage 𝜙 13 was also located near its attP. The restriction maps of the entA-, sak- and attP-containing DNA regions of the three triple-converting phages were very similar to each other and to the corresponding sak- and attP-containing DNA region of phage 𝜙 13. Hybridization analysis using a cloned β-lysin determinant (hlb) and cloned attP-containing DNA fragments as probes demonstrated that β-lysin conversion mediated by the triple-converting phages and phage 𝜙 13 was caused by insertional inactivation of the chromosomally encoded hlb determinant by orientation-specific integration of phage DNA following lysogenization.
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- Physiology And Growth
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Dissimilar Effects of Na+ and K+ on the Promotion of Glucosyltransferase Secretion in Streptococcus salivarius
More LessA defined growth medium (designated AP11), in which the concentrations of Na+ and K+ could be altered independently of one another, was developed for Streptococcus salivarius ATCC 25975. The addition of 100 mm-Na+ to AP11-medium containing 25 mm-K+ initially reduced the rate of expression of extracellular glucosyltransferase (GTFe). However, once S. salivarius had adaptated to grow in the presence of 100 mm-Na+, the rate of GTFe expression was stimulated. In fact once adapted to the presence of Na+ in the environment the same increase in the rate of enzyme expression was observed in all batch cultures irrespective of the K+ concentration (2–50 mm). At 2 mm-K+ there was no change in the level of saturation of the membrane lipids when the Na+ concentration was increased from 0 mm to 100 mm. Na+-stimulation of GTFe expression was confirmed in non-proliferating cell suspensions at different K+ concentrations. In non-proliferating cell suspensions, GTFe expression outlined a rectangular hyperbola with respect to K+ concentration when the K+ concentration was stepped up from 2 mm. The increase in GTFe synthesis and secretion was transient and was similar to that previously reported by us in Na+-rich medium, though it did not reach the same high levels. The reduced transient stimulation of GTFe expression correlated both with an enrichment in the saturated fatty acids of the membrane lipids of S. salivarius, and with the fact that the degree of saturation was only slightly reduced when the K+ concentration was stepped up from 2 mm to 50 mm. Needless to say, the final octadecenoic to octadecanoic (C18:1/C18:0) fatty acid ratio retained its direct correlation with the transient increase in GTFe production following the step up in K+ concentration, giving rise to an apparent biphasic plot when combined with that previously reported.
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Characterization of Methylammonium/Ammonium Transport in Mutant Strains of Anabaena variabilis Resistant to Ammonium Analogues
More LessA methylamine-resistant mutant strain (4m3) of Anabaena variabilis ATCC 29413 was capable of growth at pH 7·0, but not at pH 9·0, in the presence of concentrations of methylamine that totally inhibited growth of the parent strain. Strain 4m3 showed a marked reduction, at pH 7·0 but not at pH 9·0, in the initial rapid and slower second phases of uptake of [14C]methylamine and ammonia, when compared with the parent strain, The rate of inhibition of nitrogenase activity, on adding ammonia at pH 7·0, was lower in strain 4m3 than in the parent strain and was attributable to a defect in the CH3NH+ 3/NH+ 4 transport system. Evidence that the second slower phase of [14C]methylamine uptake is associated with metabolism via glutamine synthetase (GS) was obtained using mutant strains partially deficient in GS. Unlike these GS-deficient strains, strain 4m3 did not liberate ammonia extracellularly unless treated with l-methionine-DL-sulphoximine. Growth of strain 4m3 in medium containing 3 mm-methylamine resulted in a restoration of the first phase of [14C]methylamine uptake, at pH 7·0, but not of ammonia uptake. However, growth in the presence of 3 mm-ammonia did not enhance the uptake of either methylamine or ammonia. Strain 4m3 is apparently deficient in the active CH3NH+ 4/NH+ 4 transport system, but has a CH3NH+ 4 transport system which can be induced by growth in the presence of methylamine.
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The Effect of Growth Temperature on Wax Ester Content and Composition of Euglena gracilis
More LessA streptomycin-bleached mutant of Euglena gracilis strain Z, a non-photosynthetic mutant, was grown aerobically in the dark at 33, 26 and 15 °C respectively. The content of wax esters was greater at higher growth temperatures, whereas the content of paramylon was greater at lower temperatures. The highest temperature (33C) caused the greatest accumulation of wax esters. Wax esters synthesized at the highest temperature had carbon chain lengths of C24-32 and the predominant chain lengths were C27 and C28. The constituent fatty acids and alcohols ranged from C11 to C18 with myristic acid and myristyl alcohol being the main components. At the lowest temperature (15 °C) the mean chain length of wax esters decreased. The most abundant wax ester was C26, and the most abundant fatty acids and fatty alcohols of wax esters were C13. A considerable amount (44–48% of the total) of odd-carbon-numbered wax esters were present. The effect of temperature on odd-carbon-numbered wax formation is discussed in relation to possible sources of propionate or methylmalonate units.
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The Physiology of l-Methionine Catabolism to the Secondary Metabolite Ethylene by Escherichia coli
More LessCatabolism of l-methionine by Escherichia coli strain B SPAO led to the formation of ethylene nas a secondary metabolite (ethylenogenesis). Methionine was initially deaminated by a transamination reaction to the 2-oxo acid 2-oxo-4-methylthiobutyric acid (KMBA) which was then converted to ethylene. The utilization of l-methionine as an additional nitrogen source was investigated by examining ethylene synthesis under different nitrogen supply conditions. Ethylene formation in batch culture was unaffected by the concentration of the precursor l-methionine in the medium although increasing concentrations of NH4Cl resulted in progressively less ethylene formation. Cultures grown without l-methionine did not produce ethylene but were able to synthesize ethylene when l-methionine or KMBA was provided. Addition of l-tyrosine to batch cultures reduced the yield of ethylene after 42 h by 54%. Under these conditions the maximum transient level of KMBA was reduced by 32% and occurred later compared to when l-methionine was the only amino acid supplement. Continuous cultures grown under ammonia limitation produced both ethylene and KMBA. In contrast, when glucose was limiting, neither of these metabolites were produced. Cells harvested from continuous cultures grown under glucose or ammonia limitation were able to synthesize ethylene from either l-methionine or KMBA although their capacity for ethylene synthesis (ethylenogenic capacity) was optimal under ammonia limitation (C:N ratio = 20).
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Determination of Murein Precursors during the Cell Cycle of Escherichia coli
More LessA convenient and reliable method has been established that allows a quantitative determination of m-diamino[3H]pimelic acid-labelled murein precursors in 1 ml culture samples of Escherichia coli. Prior to separation by reversed-phase high-pressure liquid chromatography the lipid-linked intermediates were hydrolysed to release the muropeptides. The accuracy for the measurement of UDP-N-acetylmuramylpentapeptide (UDP-MurNAc-pentapeptide) was ± 1·9% (sd), for undecaprenyl-P-P-MurNAc-pentapeptide (lipid I) ± 10% (sd) and for undecaprenyl-P-P-(GlcNAc-β1→4)MurNAc-pentapeptide (lipid II) ± 5% (sd). The ratio of UDP-MurNAc-pentapeptide:lipid I:lipid II was about 300:1:3 for E. coli MC4100. The relative cellular concentrations of all three precursor molecules were found not to vary throughout the cell cycle. It is concluded that elongation and division of the murein sacculus is not controlled by oscillations in the concentrations of these late murein precursors.
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Inhibition of DNA Synthesis in Saccharomyces cerevisiae by Yeast Killer Toxin KT28
More LessTreatment of sensitive cells of Saccharomyces cerevisiae with killer toxin KT28 affected cell viability after 2 h: the effect was dependent upon the availability of a utilizable energy source. Treatment led to an interruption of cell growth. The mother cells contained nuclear DNA, whereas their daughter buds did not. Using a killer-toxin-sensitive thymidine auxotroph of S. cerevisiae carrying a temperature-sensitive thymidylate uptake mutation, it was shown that the incorporation of dTMP at the permissive temperature was inhibited within 30 min of the addition of KT28. When cells labelled at the permissive temperature were incubated at the restrictive temperature, the level of radioactivity declined in the absence but not in the presence of KT28. No other effects of KT28 were observed within 2 h of its addition, and it is concluded that the inhibition of DNA synthesis is an early effect of the action of KT28.
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Negative Chemotaxis to Ammonia and Other Weak Bases by Migrating Slugs of the Cellular Slime Moulds
More LessAmmonia is known to be a repellent gas for rising sorogens of Dictyostelium discoideum, and it has been suggested that it is also a repellent gas for migrating slugs of the same species. Here we present evidence that migrating slugs of D. discoideum and two related species, D. mucoroides and Polysphondylium violaceum, indeed orient away from high concentrations of NH3. In D. discoideum, brief exposure of a slug to an NH3 gradient of about 1 p.p.m. mm−1 (10−5 atm cm−1 or 0·00076 mmHg mm−1) was sufficient to alter the direction of its migration. The gases of other weak bases, such as methylamine, trimethylamine, ethylamine and pyrrolidine, were also effective.
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Role of Protein Synthesis in the Cell Division and Starvation Induced Resistance to Autolysis of a Marine Vibrio during the Initial Phase of Starvation
More LessStarvation of a marine Vibrio sp. S14 for carbon, nitrogen and phosphorus resulted in a fourfold increase in cell number during the first 6 h in the starvation regime. This initial cell division of non-growing cells was dependent on both DNA and peptidoglycan synthesis as deduced from inhibition experiments using nalidixic acid and ampicillin. Inhibition of protein synthesis by the addition of chloramphenicol led to the cessation of both cell division and DNA synthesis after 40–60 min in the starvation regime. Starvation also induced resistance against autolytic cell wall degradation. Resistance to ampicillin-induced murein degradation was most extensive in the portion of the cell wall that was synthesized after the onset of starvation and was dependent on de novo protein synthesis. The amount of d-alanine per unit dry weight increased twofold during 24 h of starvation and an increased resistance to lysis induced by sonication was observed during this period. It is suggested that the fourfold increase in cell number during the first six hours of starvation requires proteins synthesized de novo and that new rounds of DNA replication may be initiated during non-growth subsequent to 40–60 min of starvation. While the rate of DNA synthesis during the initial 40–60 min was unaffected by the blockage of protein synthesis, the mechanism conferring autolysis resistance was effectively inhibited.
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Influence of Oestradiol on Protein Expression and Methionine Utilization during Morphogenesis of Paracoccidioides brasiliensis
More LessThe temporal sequence of cytosolic protein expression during phase transition of Paracoccidioides brasiliensis was examined. Electrophoretic analysis of cytosol proteins by one-dimensional SDS-PAGE revealed numerous differences between the mycelial and yeast forms as well as alterations induced by 17β-oestradiol. Using either protein staining or fluorography of [35S]methionine-labelled proteins 30 phase-specific bands were detected, 12 mycelial-associated bands (range 30 to 140 kDa) and 18 yeast-associated bands (range 22 to 127 kDa). In cells undergoing mycelial to yeast transition after a shift from 25 °C to 37 °C, the protein patterns showed a temporal progression toward the yeast profile with the accumulation of yeast bands prior to observable morphogenesis. Five novel protein bands (range 23 to 50 kDa) were detected by silver staining during transition. Treatment of temperature-shifted mycelial cultures with 26 × 10−7 m-oestradiol altered observed profiles; 4 of 12 mycelial-associated bands were maintained whereas the appearance of the 5 novel transition bands and 9 of 18 yeast-associated bands was blocked or delayed. Analysis of [35S]methionine-labelled proteins revealed that oestradiol induced label uptake by mycelial cells, blocked the synthesis of a 92 kDa yeast-specific band 72 h into transition, and diminished label incorporation 120 h into transition. In conjunction with these steroid-induced alterations of protein expression, little or no morphological transformation occurred. These results support our hypothesis that, analogous to mammalian steroid receptor action, the functional responses of P. brasiliensis to oestradiol are related to regulation of protein expression, presumably mediated via a specific binding proteinligand complex.
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- Systematics
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Nocardia pinensis sp. nov., an Actinomycete Found in Activated Sludge Foams in Australia
A new nocardioform actinomycete was isolated by filament micromanipulation during the course of a study into foaming in activated sludge plants in Australia. It constitutes the second most prevalent foaming organism in Australia after Nocardia amarae. These two foaming organisms can be differentiated morphologically, biochemically and chemotaxonomically. The microscopic appearance of the filaments of the new taxon resembles a pine tree. The filaments are Gram-positive, non-acid-fast, non-motile, non-sheathed and about 0·5–1·0 μm in diameter. On a complex medium, the colonies are orange, opaque, macroscopically dry and friable, microscopically moist and shiny, with a pasty texture and an entire edge. The strains are positive for catalase, oxidase and urease and are oxidative in their metabolism of glucose. No strain could degrade hypoxanthine, xanthine, tyrosine, casein, gelatin or aesculin and none could grow with lysozyme. The strains contain peptidoglycan type Alγ, cell wall type IV, whole cell sugar pattern type A, phospholipid type PII, menaquinones ω-cyclo-MK-8 (H4), fatty acids comprising straight chain saturated and unsaturated acids and tuberculostearic acid, and mycolic acids with 58–64 carbons containing substantial amounts of unsaturated chains in the 2-position. The name Nocardia pinensis has been chosen for the new taxon because of the pine tree like appearance of the organism on microscopy. The type strain, UQM3063, is deposited at the University of Queensland, Department of Microbiology Culture Collection.
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