- Volume 132, Issue 2, 1986
Volume 132, Issue 2, 1986
- Biochemistry
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Purification of RNA-core Induced Streptolysin S, and Isolation and Haemolytic Characteristics of the Carrier-free Toxin
More LessSummary: RNA-core (RNAase-resistant fraction of yeast RNA) induced Streptolysin S (SLS) was purified (40% recovery) to apparent electrophoretic homogeneity by hydroxylapatite chromatography followed by gel filtration on Sephadex G-100 in the presence of 6 m-guanidine. HCl. The specific activity of the purified toxin was 3 x 106 haemolytic units (mg protein)−1. The M r of the toxin was below 4000 on the basis of SDS-PAGE and 20000 by gel filtration in guanidine. HCl. High-voltage isoelectric focusing of the purified toxin allowed the isolation of the carrier-free SLS peptide for the first time. This peptide was basic (pI 9.2) as compared to native SLS (pI 3.6). The native toxin and the peptide had similar haemolytic properties except for the high lability of the peptide, which was stabilized by RNA-core. The M r of the denatured peptide was about 1800, as estimated by gel filtration.
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Catabolite Inactivation of the Glucose Transport System in Saccharomyces cerevisiae
More LessSummary: The sugar transport systems of Saccharomyces cerevisiae are irreversibly inactivated when protein synthesis is inhibited. This inactivation is responsible for the drastic decrease in fermentation observed in ammonium-starved yeast and is related to the occurrence of the Pasteur effect in these cells. Our study of the inactivation of the glucose transport system indicates that both the high-affinity and the low-affinity components of this system are inactivated. Inactivation of the high-affinity component evidently requires the utilization of a fermentable substrate by the cells, since (i) inactivation did not occur during carbon starvation, (ii) when a fermentable sugar was added to starved cells, inactivation began, (iii) when the fermentation inhibitors iodoacetate or arsenate were added in addition to sugars, the inactivation was prevented, (iv) when a non-fermentable substrate was added instead of sugars, inactivation was also prevented. The inactivation of the low-affinity component appeared to show similar requirements. It is concluded that the glucose transport system in S. cerevisiae is regulated by a catabolite-inactivation process.
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Proteinases of Legionella: Phenylalanineaminopeptidase of L. pneumophila
Summary: Phenylalanineaminopeptidase was isolated and purified from the culture filtrate of Legionella pneumophila by affinity chromatography on O-tert-butyl-l-threonyl-l-phenylalanyl-l-prolyl-glycyl-aminosilochrom and by gel-filtration; a 401-fold purification with a yield of 18% was achieved. The enzyme was a metalloenzyme with a molecular weight of 35000 and a pI of 5.8. It was stable at pH 7–9 and had an activity optimum in the range of pH 8–9.5 with l-phenylalanine p-nitroanilide as substrate. Enzyme activity was highest towards the latter compound, substantially lower towards l-leucine p-nitroanilide and only marginal towards other p-nitroanilides. Besides phenylalanineaminopeptidase, a metalloproteinase and a serine proteinase were also detected in L. pneumophila culture filtrate.
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- Ecology
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Effect of Growth Conditions and Surface Characteristics of Aquatic Bacteria on Their Attachment to Solid Surfaces
More LessSummary: The physico-chemical basis for the effects of nutrient conditions on the attachment of four freshwater isolates, Pseudomonas fluorescens, Enterobacter cloacae, Chromobacterium sp. and Flexibacter sp., to hydrophobic (PD) and relatively hydrophilic (TCD) polystyrene surfaces was investigated. Different nutrient conditions and growth rates resulted in changes in the physico-chemistry of the bacterial surfaces, measured either by liquid contact angles on lawns of cells or by hydrophobic and electrostatic interaction chromatography of cells, and in different levels of attachment to the substrata. The phenotypic changes in cell surfaces and the levels of attachment were different for each species. Levels of bacterial adhesion differed for the two substrata, indicating different adhesion interactions with PD and TCD surfaces. Treatment of attached cells with chloramphenicol did not cause detachment of any of the bacteria from PD or TCD, whereas periodate and protease treatment removed some attached cells, the degree of detachment depending on the species. The presence of complex organic molecules, in the liquid phase and conditioning the solid surface, influenced the extent of bacterial attachment, the effect depending on the substratum, organic concentration and bacterial species. The results suggest that changes in nutrient conditions in natural aquatic habitats will affect the attachment of individual bacterial species differently, thus influencing the population structure of developing biofilms.
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- Genetics And Molecular Biology
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Arginine Auxotrophs of Candida albicans Deficient in Argininosuccinate Lyase
More LessSummary: Auxotrophic mutants of Candida albicans FC18 were induced by a combination of treatments with nitrous acid and UV irradiation. Arginine (Arg−), histidine (His−) and methionine/cysteine (MetA−) auxotrophs were recovered by this means. The Arg− auxotrophs lacked active argininosuccinate lyase (EC 4.3.2.1), the enzyme catalysing the final step in arginine biosynthesis. Thus the locus may be designated arg-4. The mutant strains bearing this mutation did not form germ tubes unless the germination medium contained arginine.
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Effect of Transfer of Symbiotic Plasmids and of Hydrogenase Genes (hup) on Symbiotic Efficiency of Rhizobium leguminosarum Strains
More LessSummary: Plasmid-encoded symbiotic determinants from the Rhizobium leguminosarum strain MA1 (817) with uptake hydrogenase activity (Hup+) and from the Hup− strain MC1 (18a) were mobilized by recombination with the self-transmissible plasmid pVW5JI. The symbiotic determinants were transferred by conjugation from strain MA1 to strain MC1 and to a derivative of MC1 without the symbiotic plasmid, and vice versa, thus constructing four types of transconjugants. The determinants for a total recycling hydrogenase in strain MA1 were found to be encoded on the symbiotic plasmid.
Strain MC1 fixed 60% more N2 in pea root nodules, determined as mg nitrogen per plant, than strain MA1. This difference was not increased in the MC1 derivative that obtained hydrogenase activity. Plants inoculated with a derivative of strain MA1, however, where the symbiotic plasmid was replaced by that of strain MC1 had a high percentage nitrogen content. It was concluded that the symbiotic plasmid and the genetic background were more important for plant nitrogen accumulation than uptake hydrogenase.
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Analysis of Inverted Repeat DNA in the Genome of Rhodomicrobium vannielii
More LessSummary: The DNA of Rhodomicrobium vannielii was analysed for the presence of inverted repeat sequences (IR DNA) by S1 nuclease digestion. Approximately 7% of chromosomal DNA was found to be IR DNA which comprised two size classes. The large IR DNA was heterogeneous and contained species in the size range 100–700 bp. The smaller size class contained species of 17 and 27 bp. Both size classes of IR DNA hybridized to many chromosomal restriction fragments, suggesting that these IR DNA sequences are dispersed throughout the genome. Hybridization studies also indicated sequence homology between the two classes of IR DNA and suggested that the 17 and 27 bp IR DNA sequences may exist in clusters.
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Cloning, Expression and Location of the Streptococcus lactis Gene for Phospho-β-d-galactosidase
More LessSummary: Genes for lactose catabolism and proteinase production in Streptococcus lactis 712 are encoded by a 56.5 kb metabolic plasmid, pLP712. A lactose mini-plasmid of only 23.7 kb, pMG820, was constructed by introducing two deletions into pLP712, and was cloned as two segments of DNA into the Escherichia coli vector pAT153 using restriction endonuclease PstI. The lactose genetic region of pLP712, which has been defined by deletion and restriction mapping, was cut into two parts by this process. When the smaller 10.8 kb segment of pMG820 DNA was present, the key lactic streptococcal lactose splitting enzyme, phospho-β-d-galactosidase, was expressed in E. coli. The gene for phospho-β-d-galactosidase was more precisly located by introducing a series of deletions into cloned DNA by in vitro manipulations and then assaying for enzyme activity. The presence of this phospho-β-d-galactosidase activity was correlated with the production of a 58kDa 35S-labelled protein both by E. coli minicells and after coupled transcription and translation of cloned DNA. The product of a second gene, a 37 kDa protein (“protein X”), and a possible truncated phospho-β-d-galactosidase protein of 16 kDa were also detected in minicells.
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spoIID Operon of Bacillus subtilis: Cloning and Sequence
More LessSummary: The locus spoIID, involved in the sporulation of Bacillus subtilis, was cloned into derivatives of the temperate phage ϕ105. Two recombinant phages were obtained which contain chromosomal DNA covering 1∙6 kbp. They are both able to complement mutations spo-68 and spo-298. These mutations, which were believed to be in different loci, spoIID and spoIIC respectively, were shown to be closely linked, and both map at the position assigned to spoIID on the genetic map of B. subtilis. The sequence of 1656 bp carrying the spoIID locus was determined. Only one open reading frame was found; this codes for a protein of 343 amino acids. It is preceded by a ribosome binding site and possible recognition sequences for σ32- and σ29-RNA polymerases. Studies of the locus by means of integrational plasmid vectors defined the outer limits of the transcriptional unit. These results are completely compatible with the sequence data. The combination of sequence and mapping and the information obtained by the use of integrational plasmids confirm that the spoIID locus functions as a monocistronic operon.
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Microcin B17 Blocks DNA Replication and Induces the SOS System in Escherichia coli
More LessSummary: Microcin B17 is a novel peptide antibiotic of low M r (about 4000) produced by Escherichia coli strains carrying plasmid pMccB17. The action of this microcin in sensitive cells is essentially irreversible, follows single-hit kinetics, and leads to an abrupt arrest of DNA replication and, consequently, to the induction of the SOS response. RecA− and RecBC− strains are hypersensitive to microcin B17. Strains producing a non-cleavable SOS repressor (lexA1 mutant) are also more sensitive than wild-type, whereas strains carrying a mutation which causes constitutive expression of the SOS response (spr-55) are less sensitive to microcin. Microcin B17 does not induce the SOS response in cells which do not have an active replication fork. The results suggest that the mode of action of this microcin is different from all other well-characterized microcins and colicins, and from other antibiotics which inhibit DNA replication.
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Effects of Microcin B17 on Microcin B17-immune Cells
More LessSummary: When microcin B17-immune cells are treated with microcin B17 they show many of the physiological effects displayed by microcin B17-sensitive cells treated in the same way. DNA replication stops immediately and several SOS functions are subsequently induced. In sensitive cells these effects are irreversible and lead to cell death, whereas in immune cells they are reversible and there is no loss of viability. This is an unusual mechanism of immunity because it does not prevent the primary action of the microcin. The implications of this mechanism concerning the mode of action of microcin B17 and the induction of the SOS system are discussed.
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Resistance of a Bacillus subtilis Mutant to a Group of Temperate Bacteriophages
More LessSummary: A mutant of Bacillus subtilis 168 was isolated which resists infection by all the group III temperate bacteriophages except SPR, while allowing full infection by phages of the other groups (I, II and IV). The mutation conferring this phenotype, pha-3, shows 52–54% PBS1-mediated cotransduction with the hisA1 marker, mapping therefore in the gtaA and gtaB region of the B. subtilis chromosome. Nevertheless, it does not affect the infection by phages sensitive to gta mutations.
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Mapping and Properties of bgl (β-Glucanase) Mutants of Bacillus subtilis
More LessSummary: Among secreted proteins of Bacillus subtilis 168 four polypeptides, separated by electrophoresis in polyacrylamide gradient gels under denaturing conditions, were shown to have lichenanhydrolysing enzyme activity; one of them, a 22 kDa protein, represents the main β-glucanase activity. A number of N-methyl-N-nitro-N-nitrosoguanidine-induced mutants defective in β-glucanase activity at an elevated temperature (44°C) were isolated and characterized. All mutants analysed by PAGE had no active 22 kDa β-glucanase in their supernatants and showed no cross-reactivity with antibodies raised against purified 22 kDa β-glucanase. However, formation of other exoenzymes, including the other minor β-glucanases, was not affected in these mutant strains. The mutations (bgl-1, bgl-12, bgl-35, bgl-101) were mapped by phage PBS1-mediated transduction and found to be located between purA and sacA, close to the hutH marker of the B. subtilis chromosome. A 3.8 kb EcoRI fragment of the B. subtilis chromosome which directs β-glucanase synthesis in Escherichia coli as well as complementing a β-glucanase deficient mutant, bgl-35, of B. subtilis to synthesize active 22 kDa β-glucanase was mapped after homologous recombination by means of an integratable plasmid. The cointegrated chloramphenicol-resistance marker of the plasmid was found at the same map position as the mutations bgl-1, bgl-12, bgl-35 and bgl-101, suggesting that the gene affected encodes for the 22 kDa β-glucanase and lies within the order sacA–sacA–bgl–purA.
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Occurrence of Ploidy Shift in a Strain of the Imperfect Yeast Candida albicans
More LessSummary: A clinical isolate of Candida albicans, a member of the Fungi Imperfecti, was polyploid as shown by the fact that it contained two kinds of nuclei, one of diploid and one of tetraploid DNA content. These determinations were made by fluorescence microscopy-photometry. The nucleus-associated organelles (NAOs), or spindle pole bodies, of yeast cells in this isolate were classified into two groups, one diploid and the other tetraploid, according to their dimensions as determined by serial thin-sectioning electron microscopy. A ploidy shift from diploid to tetraploid was found in individual cells of a culture of this isolate undergoing diphasic growth in minimal salts medium. A process of shift-down or reduction of ploidy from tetraploid to diploid was also observed by electron microscopy during these growth conditions: this appeared to occur in large cells which showed multiple spindle formation during nuclear division, a phenomenon apparently similar to the process of meiosis II during sporogenesis of Saccharomyces cerevisiae, but differing in that it produces diploid daughter nuclei by the vegetative process.
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Genetic Heterogeneity among Isolates of Coxiella burnetii
More LessSummary: Chromosomal and plasmid DNA have been extracted from six isolates of Coxiella burnetii, the aetiological agent of Q fever. Restriction fragment length polymorphisms detected after HaeIII digestions of chromosomal DNA revealed four different patterns that distinguished the American from the European isolates, and the Nine Mile phase I prototype strain from a spontaneously derived, isogenic phase II nonrevertant variant. At least one of the HaeIII fragments visible in the pattern from Nine Mile phase I and not in that from Nine Mile phase II could not be detected by DNA–DNA hybridization, and thus may have been deleted during the phase transition. Comparison of Nine Mile phase II, which does not survive animal passage, with Grita M44 phase II, which does, indicated that the HaeIII fragment was present in the Grita strain. These results suggest that this HaeIII fragment may be concerned with functions necessary to survive the cellular immune response in vivo. Isolates from two human endocarditis cases showed the greatest divergence from all the other isolates, having at least five fragments of unique mobility in the HaeIII digestion pattern of their chromosomal DNA. Also, a plasmid obtained from these two isolates was 2 to 3 kb larger than the plasmid present in the other five isolates, and its restriction pattern could be distinguished from that of the other plasmids by several endonucleases. Detection of chromosomal and plasmid restriction fragment length polymorphisms among strains of phase I or phase II C. burnetii from various geographical locations and environmental sources will facilitate Q fever diagnosis and strain identification.
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Transcriptional Regulation of the Mercury-resistance Genes of Transposon Tn501
More LessSummary: Expression of the mercury-resistance (mer) genes of the transposon Tn501 is positively and negatively controlled by the product of the merR gene. DNA sequence analysis has identified three open reading frames as potential candidates for this gene, one of which is oriented divergently with respect to the mercury-resistance genes. We have demonstrated that although RNA polymerase will bind to fragments containing the potential control regions for all three reading frames, only the control region for this divergent reading frame shows detectable promoter activity in vivo. Transcription of this reading frame is required for repression and induction of mer transcription. We have also shown that the Tn501 merR gene product negatively regulates its own synthesis, and have identified the start point of the transcript for this reading frame and for the mercury-inducible transcript of the mercury-resistance genes.
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Tolerance to Bromodeoxyuridine in a Thymidine-requiring Strain of Bacillus subtilis
J. G. Coote and C. BinnieSummary: Mutations which allow tolerance to 5-bromo-2′-deoxyuridine (BUdR) in a thymidine (TdR)-requiring strain of Bacillus subtilis have been examined. Differences in sensitivity to BUdR existed between isogenic strains harbouring the mutations. Those mutations originally isolated as BUdR-tolerant also bestowed tolerance to 5-bromouracil and vice versa. The strain exhibiting the greatest tolerance to BUdR maintained a normal rate of replication in the presence of BUdR whereas the parent strain did not, but the tolerant strain incorporated less analogue into DNA than the parent strain. The basis of the tolerance mutation appeared to lie at the point of uptake of the analogue into the cell as the tolerant mutant preferentially took up TdR over BUdR into whole cells. DNA polymerase activity measured in vitro did not distinguish between TdR and BUdR in either the parent or the mutant strain and although TdR kinase activity showed a preference for TdR over BUdR as a substrate, the extent of discrimination was similar in both strains.
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Inhibition of Sporulation in Bacillus subtilis by Bromodeoxyuridine and the Effect on DNA Replication
C. Binnie and J. G. CooteSummary: A 5-bromo-2ʹ-deoxyuridine (BUdR)-tolerant derivative of a thymidine (TdR)-requiring strain of Bacillus subtilis was used to examine the effect of BUdR, an analogue of TdR, on sporulation. At a TdR: BUdR ratio which had little effect on growth, sporulation was inhibited if cells were exposed to BUdR during the period of DNA synthesis at the onset of the process. Cells recovered from BUdR inhibition of sporulation if the analogue was removed and DNA replication allowed to continue with TdR alone. BUdR prolonged the period of DNA synthesis during sporulation and experiments with chloramphenicol suggested that this was due in part to unscheduled initiation of new rounds of replication.
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- Immunology
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Immunochemical Characterization of the Cell Surface Carbohydrate Antigens of Peptostreptococcus anaerobius
More LessSummary: Two carbohydrate antigens were isolated from the cell surface of Peptostreptococcus anaerobius. One, extracted from purified cell walls with NaOH, contained glucose and phosphorus, with traces of galactosamine and glucosamine. Serological activity was detected by a ‘dot blot’ procedure. The second antigen, extracted from cell membranes with phenol and purified by chromatography on Sepharose 6B and an immunoadsorbent column, contained glucose, glycerol phosphate, phosphorus and fatty acids. Antigenicity of this extract could also be demonstrated by an ELISA technique.
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- Pathogenicity And Medical Microbiology
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Biochemical and Structural Characterization of an Unusual Group of Gram-negative, Anaerobic Rods from Human Periapical Osteitis
More LessSummary: The biochemical and chemotaxonomic properties of three previously undescribed strains from human dental root canal infections are presented. The strains were obligately anaerobic Gram-negative rods with fimbriae and a thick capsule-like structure. Carbohydrates were not fermented and agglutination tests were negative. The presence of α-galactosidase, α- and β-glucosidase, β-N-acetylglucosaminidase and β-galactosidase was confirmed. The strains produced acetic and succinic acids as metabolic end products. They contained a peptidoglycan structure based upon meso-diaminopimelic acid (A1γ) and lacked respiratory quinones. The cellular fatty acids were mainly straight-chain saturated and methyl-branched molecules. High interstrain DNA homology was observed and the DNA base compositions were between 56 and 59 mol % G + C. These three strains appear to comprise the nucleus of a new genus of anaerobic, Gram-negative rods from odontogenic infections.
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Physiological Characteristics of the Mycobacterium tuberculosis–M. bovis Group of Organisms with Particular Reference to Heterogeneity within M. bovis
More LessSummary: By examining the abilities of mycobacterial strains to remove amino acids from solution, differences were found between Mycobacterium tuberculosis and other closely related taxa. Heterogeneity was observed in most taxa. Strains of M. bovis were examined in greater detail. By using the amino acid typing method in combination with tests routinely used for differentiating mycobacterial species, and SDS-polyacrylamide gel electrophoresis of whole cell soluble proteins, a great degree of heterogeneity was observed in this taxon.
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The Effect of Protein II and Pili on the Interaction of Neisseria gonorrhoeae with Human Polymorphonuclear Leucocytes
More LessSummary: Colonial variants of Neisseria gonorrhoeae strain P9 expressing different pili and/or outer membrane protein II (P.II) were investigated with respect to their interaction with human polymorphonuclear leucocytes (PMN). Two assay systems were used. A phagocytic killing assay measured the intracellular survival of gonococci, and PMN chemiluminescence (CL) was used to determine the initial surface interactions. All variants expressing P.II were killed effectively by PMN and also greatly stimulated PMN CL. The P.II− variants, on the other hand, were resistant to phagocytic killing and stimulated a much lower CL response. The presence of different P.II species was associated with different CL profiles and therefore different modes of interaction with the PMN membrane. A P.II-specific monoclonal IgG was opsonic and greatly increased PMN CL in contrast to F(abʹ)2 prepared from the same antibody, which inhibited it, thus confirming the role of P.II in the PMN interaction. Phagocytic killing assays revealed that with the loss of P.II, gonococcal variants acquired resistance to killing. Comparison of piliated and non-piliated pairs of variants with the same P.II profile showed that PMN–gonococcal interactions are dominated by the nature of the P.II species present whereas pili have little effect.
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- Physiology And Growth
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A Radiorespirometric Study on the Contribution of the Hexose Monophosphate Pathway to Glucose Metabolism in Candida utilis CBS 621 Grown in Glucose-limited Chemostat Cultures
More LessSummary: A radiorespirometric study of glucose metabolism in Candida utilis CBS 621 was done using glucose-limited chemostat cultures growing at a dilution rate of 0.1 h−1 with ammonium or nitrate as the nitrogen source. From a steady-state analysis of 14CO2 yields from [1-14C]- and [6-14C]glucose supplied in the medium feed it appeared that during growth with nitrate the flow of glucose through the hexose monophosphate (HMP) pathway was much higher than during growth with ammonium as the nitrogen source. The same phenomenon was apparent from an analysis of the rate of 14CO2 production after administration of small amounts of labelled glucose to samples withdrawn from steady-state cultures. Additionally, these experiments revealed not only that the initial fraction of glucose 6-phosphate routed into the HMP pathway increases, but also that recycling of hexose phosphates via this pathway increases when nitrate is used as the nitrogen source. From a quantitative analysis of the results it is concluded that the contribution of the HMP pathway to glucose metabolism is close to the theoretical minimum required to cover the NADPH requirement for biosynthesis.
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Stabilization of Glucose-starved Escherichia coli K12 and Salmonella typhimurium LT2 by Peptidase-deficient Mutants
More LessSummary: Escherichia coli K12 and Salmonella typhimurium LT2 cells were stabilized during carbon starvation in the presence of peptidase-deficient mutant strains. The rate of loss of viability of the wild-type S. typhimurium strain was decreased an average of 2-fold, and the rate for the wild-type E. coli strain was decreased about 2.3-fold, when either was starved in the presence of the multiply peptidase-deficient S. typhimurium strain TN852; other peptidase-deficient strains exhibited similar stabilizing effects. Starving wild-type S. typhimurium LT2 cells utilized peptides excreted by the starving peptidase-deficient cells for protein synthesis, and, to a lesser extent, as respiratory substrates. Provision of free amino acids in steady-state levels to starving E. coli K12 cells in a cell recycle apparatus had a stabilizing effect similar to that of mixing with peptidase-deficient cells.
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>Nitrogen Fixation by Pseudomonas saccharophila Doudoroff ATCC 15946
More LessSummary: Pseudomonas saccharophila Doudoroff (ATCC 15946) reduced C2H2 and fixed 15N2 heterotrophically in N-free semi-solid medium supplemented with 100 mg yeast extract l−1. Nitrogenase activity was markedly stimulated by addition of sugars and organic acids and was expressed microaerobically. Autotrophically-grown cells showed nitrogenase activity. This is the first report of N2 fixation in a well-characterized Pseudomonas species capable of chemoautotrophic growth with H2 as the electron donor.
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The Use of Activities of Carbon Catabolic Enzymes as a Probe for the Carbon Nutrition of Snakebean Nodule Bacteroids
More LessSummary: In sugar-grown cells of cowpea Rhizobium strain NGR234 activities for enzymes of the Entner-Doudoroff and pentose phosphate pathways were present while the virtual absence of phospho-fructokinase and fructose-bisphosphate aldolase indicated that the Embden–Meyerhof–Parnas pathway was unlikely to be significant. Invertase, fructokinase, glucose-6-phosphate dehydrogenase and the Entner–Doudoroff enzymes were present at only low activities in succinate grown cells, but were induced in sugar-grown cells. Isolated snakebean bacteroids contained very low activities of these four enzymes. Although C4-dicarboxylic acids exerted some repressive effect on induction of these enzymes, there was substantial enzyme activity induced in cells grown on sucrose plus a C4 dicarboxylic acid. The data suggest that the peribacteroid membrane may be relatively impermeable to sugars and so dictate the carbon source(s) available to the bacteroids.
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Regeneration of Protoplasts of Bacillus subtilis 168 and Closely Related Strains
More LessSummary: Regeneration of protoplasts to bacilli was attempted in several strains of Bacillus closely related to Bacillus subtilis 168. On DM3 and similar media using succinate as osmotic support, only B. subtilis 168 and Bacillus natto ATCC 15245 were able to regenerate. Media containing mannitol as osmotic support, and agar as gelling agent gave rise to L-form colonies with Bacillus licheniformis NCTC 6346. Many of the L-form colonies were able to regenerate to the bacillary form when plated on the mannitol medium solidified with gelatin. All of the Bacillus species tested were able to regenerate on the latter medium at rates sufficient to allow protoplast transformation and fusion experiments.
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Ammonia Transport in Free-living and Symbiotic Rhizobium sp. ANU289
More LessSummary: [14C]Methylamine uptake by free-living Rhizobium sp. ANU289 had Michaelis–Menten kinetics (apparent K m 6.6 μm). Uptake was competitively inhibited by ammonia (K i 0.4 μm) and was dependent on an energized membrane. Uptake by bacteria had an optimum at pH 7.0. Methylamine uptake by bacteroids from siratro root nodules was much slower than that by free-living bacteria at pH 7.0 but increased exponentially with the pH of the medium. Uptake by bacteroids did not show saturation kinetics and was insensitive to the presence of ammonia or uncouplers. These results suggest that free-living bacteria (grown under conditions where ammonia is limiting) have an active transport mechanism for the uptake of ammonium ions; this carrier is not operative in the symbiotic state, where passive diffusion of ammonia occurs. In the free-living state, the ammonium carrier is under genetic control, being repressed by growth on high concentrations of ammonia. Derepression occurs under conditions of nitrogen starvation.
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Effects of Water Activity and Culture Age on the Glycerol Accumulation Patterns of Five Fungi
More LessSummary: One non-xerophilic fungus, Penicillium digitatum, and four xerophilic fungi, Penicillium janczewskii, Eurotium chevalieri, Wallemia sebi and Polypaecilum pisce, were grown at six different water activities (a w) on media containing various concentrations of sodium chloride. Each species was sampled as soon as visible growth appeared and up to six times thereafter during various stages of the growth cycle. The fungal mycelium was extracted and assayed for glycerol using a specific enzymic method. At the highest a w, 0.997, only small amounts of glycerol were present in the fungi. At lower a w values, glycerol concentrations rose rapidly at first, then declined as the cultures aged. There appeared to be a correlation between the amount of glycerol accumulated, and the complexity of the spore-bearing structures. Glycerol depletion appeared to be related to the formation of spores and their maturation.
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Decreased Particulate NADH Oxidase Activity in Bacillus subtilis Spores After Polymyxin B Treatment
More LessSummary: The activities of several enzymes of polymyxin B-treated dormant and germinated spores of Bacillus subtilis were examined. The particulate NADH oxidase of the antibiotic-treated spores showed considerably lower specific and total activities compared with those of untreated ones. The specific and total NADH oxidase activities of untreated spores increased 12- and 15-fold respectively during germination, whereas increases during germination of polymyxin B-treated spores were inhibited. The specific and total activities of particulate NADH cytochrome c reductase of dormant spores were decreased by polymyxin B treatment in almost the same proportion as those of the particulate NADH oxidase. The specific activity of NADH dehydrogenase of dormant spores remained unchanged after antibiotic treatment but the total activity fell considerably. The activities of other enzymes examined were similar for untreated dormant and germinated spores and antibiotic-treated spores. The respiration of polymyxin B-treated dormant spores was inhibited at the same time as the start of germination. Morphologically, polymyxin B-treated dormant spores lost a laminar structure of the cortex and details of the spore protoplast. The inhibitory mechanism of particulate NADH oxidase activity of polymyxin B-treated dormant spores is discussed.
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Localization of the Alkaline Phosphatase and 5′-Nucleotidase Activities of the Diatom Phaeodactylum tricornutum
More LessSummary: Intact cells of phosphate-depleted Phaeodactylum tricornutum possessed non-specific alkaline phosphatase (EC 3.1.3.1; PMEase) and 5′-nucleotidase (EC 3.1.3.5; 5′-NDase) activities; there was also an extracellular PMEase. The optimum pH for the cell-bound PMEase was > 10.3, and for the 5′-NDase was 9–9.5. The extracellular PMEase had an optimum pH > 10.3 and accounted for > 30% of the total PMEase activity at this pH; there was no extracellular 5′-NDase activity. The activities of these enzymes increased during phosphate-deprivation, but the rate of AMP hydrolysis (by the action of both PMEase and 5′-NDase) always exceeded that of p-nitrophenylphosphate at the physiological pH (for a marine organism) of 8–8.5. By the use of differential centrifugation after cell disruption in a French pressure cell, a highly purified fraction of cell walls was prepared. This fraction was virtually devoid of membranous material as viewed by electron microscopy, and exhibited PMEase, but no 5′-NDase activity. By using a different centrifugation procedure after disruption by shaking with glass beads, a microsomal fraction (pelleted by forces of 14000–156000 g) was prepared. This fraction was free of cell wall fragments as viewed by electron microscopy, and exhibited 5′-NDase activity but no PMEase activity. It is concluded that the PMEase was associated with cell walls, whilst the membrane-bound 5′-NDase which sedimented as vesicles in the microsomal fraction was associated with plasma membranes.
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A Decrease in GTP Content is Associated with Aerial Mycelium Formation in Streptomyces MA406-A-1
More LessSummary: Aerial mycelium formation by Streptomyces sp. MA406-A-1, a formycin-producing strain, was suppressed by the presence of excess nutrient. In such suppressed cultures, decoyinine, which specifically inhibits GMP synthetase, initiated the formation of aerial mycelium at concentrations which only partially inhibited growth. The intracellular GTP pool of organisms growing in liquid culture markedly decreased on the addition of decoyinine. Decoyinine was also effective in initiating aerial mycelium formation of three other Streptomyces spp. examined. Regardless of the successful initiation of aerial mycelium formation, the ability of the cells to produce antibiotics (formycin or actinomycin D) did not increase, but decreased, on the addition of decoyinine. It is concluded that aerial mycelium formation by Streptomyces results from a decrease in the pool of GTP (or GDP), whereas antibiotic synthesis results from a different signal(s).
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Phage Adsorption and Cell Adherence Are Motility-dependent Characteristics of the Gliding Bacterium Cytophaga johnsonae
More LessSummary: Mutants of the gliding bacterium Cytophaga johnsonae that are incapable of movement are called truly nonmotile (TNM) to distinguish them from other mutants that are motile but produce nonspreading colonies. All TNM mutants are pleiotropic, being nonmotile, unable to digest chitin, resistant to all phages that infect wild-type cells, nonadherent and having less hydrophobic surfaces than do wild-type cells. In this study, we tested the idea that the TNM pleiotropy is the result of blocking cell surface movement, rather than of loss or alteration of a specific cell surface component. Motility of wild-type cells was blocked by addition of chemicals, and treated cells were compared with untreated cells for their ability to adhere to spheroidal hydroxyapatite (SHA) beads and to adsorb phages, two characteristics used as an index of the TNM pleiotropy. All the chemicals tested that blocked motility also reduced phage adsorption and adherence to SHA to approximately the same extent seen with TNM mutants. The chemicals tested (carbonyl cyanide m-chlorophenylhydrazone, cyanide, azide and photoactivated eosin Y and rose bengal) were sufficiently different from each other to reduce the possibility that each chemical inhibited phage adsorption and cell adherence by similar secondary effects, independent of their effects on motility. It was also shown that the pleiotropy of TNM mutants is not caused by their inability to maintain a membrane potential. The results are compatible with the conclusion that the TNM pleiotropy is manifested whenever cell movement is stopped, whether by mutation or by use of chemical inhibitors, and they are inconsistent with the idea that all TNM mutants are pleiotropic because they all carry a lesion in the same gene that codes for expression of surface components required for all characteristics affected. The reason that stopping motility influences several seemingly unrelated properties is not known but is probably related to adaptations required for the organism to interact with its environment through a cell surface covered with a slime that is normally kept in motion by components of the machinery of gliding motility.
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Ethanol Dissipates the Proton-motive Force across the Plasma Membrane of Saccharomyces cerevisiae
Summary: Populations of Saccharomyces cerevisiae NCYC 431, harvested after 16 h incubation from self-induced anaerobic cultures, were more tolerant to the inhibitory effect of ethanol on fermentation rate and viability than organisms harvested from 8 h cultures. Ethanol increased the rate of passive influx of protons into de-energized organisms at a rate which was greater with organisms from 8 h compared with 16 h cultures. Rates of passive influx of protons into spheroplasts were significantly greater than into intact organisms, although culture age did not affect rates of ethanol-induced influx of protons into spheroplasts. Ethanol retarded both the initial net rate of proton efflux and the final extent of acidification produced by suspensions of energized organisms, both effects being more pronounced with organisms from 8 h as compared with 16 h cultures. The magnitude of the proton-motive force (Δp) was decreased by ethanol in both energized and de-energized organisms. Although culture age did not affect the extent of ethanol-induced decrease in Δp in de-energized organisms, in energized organisms harvested from 8 h cultures ethanol produced a significantly greater decrease in Δp as compared with organisms from 16 h cultures. If the ability of ethanol to decrease the Δp value is important in its inhibitory effect on growth, it is suggested that some phenomenon other than proton uncoupling is involved.
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- Systematics
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A Numerical Classification of Some Thermus Isolates
More LessSummary: A numerical classification was performed on a collection of 45 Thermus isolates recovered from New Zealand hot pools and on six type strains including T. aquaticus and ‘T. thermophilus’. Unweighted average linking (UPGMA) and single linkage clustering methods were applied to similarity matrices derived from simple matching (SSM ) and Jaccard similarity (SJ ) coefficients. Differences were observed between phenograms derived from SSM and SJ coefficients, indicating that some of the clusters formed were derived from a significant component of negative matches. Test error was estimated at 2∙9%. In the UPGMA/SSM phenogram, seven clusters were formed. A majority of the New Zealand isolates did not cluster with non-New Zealand isolates. Analysis of variance showed that there was a relationship between the composition of the clusters and the temperature and pH of the source of the isolate. Chi-squared testing showed that, within New Zealand, the geographical source of the isolate had no bearing on the clusters formed.
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Isolation and Restriction Endonuclease Analysis of Mycobacterial DNA
More LessSummary: A method for the isolation of DNA from mycobacteria propagated in vitro is described that utilizes organic solvents to extract lipoidal components from the outer membrane, and digestion with a protease (nagarse) and lysozyme to penetrate the cell wall. The mycobacterial cells were lysed by the addition of detergent and the DNA was purified by digestion with pronase, sequential phenol and chloroform extractions, and digestion with RNAase A. The isolated DNA, which was obtained in good yields, was of a relatively high M r and could be readily digested by restriction endonucleases. By this method, the genomes of Mycobacterium avium, M. intracellulare, M. lepraemurium, ‘M. lufu’, M. marinum, M. phlei, M. scrofulaceum, M. smegmatis and M. tuberculosis were isolated and the restriction endonuclease digestion patterns analysed. Each species could be distinguished by the digestion patterns, indicating that this approach can be used for identifying mycobacterial species. This approach is also sufficiently sensitive to differentiate strains since we were able to distinguish two independently isolated strains of M. tuberculosis, H37 and H4. In addition, no evidence was obtained for the presence of methylcytosine residues in the sequences 5ʹ.CCGG.3ʹ, 5ʹ.CCCGGG.3ʹ, 5ʹ.CC(A/T)GG.3ʹ or for methyladenine at 5ʹ.GATC.3ʹ in the DNA of the nine mycobacterial species examined using pairs of restriction enzymes that recognize and cleave at the same nucleotide sequence but differ in their sensitivity to 5-methylcytosine or 6 N-methyladenine.
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- Short Communication
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Antigenic Heterogeneity in Subunit S1 of Pertussis Toxin
More LessSummary: Culture supernates containing pertussis toxin (PT) from four strains of Bordetella pertussis were examined for both immunological reactivity and biological activity. PT from all four strains sensitized mice to histamine and toxin was detectable in supernates of all strains when examined by Western blotting with polyclonal antiserum to PT. In supernates of three of the four strains, PT was detectable by an enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody to subunit S1 of PT as the third antibody layer. However, supernates from one strain, 18323, failed to react in ELISA. Electroblots probed with the monoclonal antibody labelled subunit S1 of PT from all strains except that of strain 18323. PT of strain 18323, whilst retaining histamine-sensitizing activity, differed antigenically from that of other strains.
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Virulence and Immunogenicity in Experimental Animals of Bacillus anthracis Strains Harbouring or Lacking 110 MDa and 60 MDa Plasmids
More LessSummary: A comparative study was made of the virulence and immunogenicity in mice or guinea pigs of Bacillus anthracis strains harbouring 110 MDa and/or 60 MDa plasmids. Strains cured of the 110 MDa or the 60 MDa plasmid were more than 100-fold less virulent to mice than were the parental strains harbouring these plasmids. Guinea-pigs immunized with plasmid-free derivatives of the non-encapsulated vaccine strain 34F2 showed no resistance to challenge with strain 17JB, which harbours both 110 MDa and 60 MDa plasmids, suggesting that the derivative strains had lost their immunizing ability against anthrax.
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Adenylate Kinase Activity in Mycobacterium leprae
More LessSummary: Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) was detected in partially purified preparations of cell-free extracts of Mycobacterium leprae. The apparent K m values of M. leprae adenylate kinase for ADP and Mg2+ were 1 × 10−4 m and 4 × 10−4 m, respectively. The enzyme was heat-labile: loss of activity by 80% at 45°C and over 90% at 60°C occurred within 5 min. M. leprae adenylate kinase was distinct from armadillo adenylate kinase in respect of affinity for substrate and heat-sensitivity.
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Activity of Two Strong Promoters Cloned into Bacillus subtilis
More LessSummary: Two DNA fragments, one encoding the Escherichia coli trc promoter and the other encoding a sequence from the early region of Bacillus subtilis phage SPO1, were cloned into the B. subtilis promoter-probe vector pPL603. Both fragments effected strong in vivo promoter activity in vegetative B. subtilis cells.
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- Corrigendum
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