- Volume 130, Issue 3, 1984
Volume 130, Issue 3, 1984
- Biochemistry
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Studies on the Effects of Oxygen on Acetylene Reduction (Nitrogen Fixation) in Gloeothece sp. ATCC 27152
More LessCultures of Gloeothece sp. ATCC 27152 did not reduce acetylene when exposed to O2 concentrations greater than 0·7 atm. However, following exposure to 1 atm O2 for up to 12 h or to 0·8 atm O2 for up to 14 d, the ability to reduce acetylene recovered rapidly when cultures were returned to air. Complete recovery required active protein synthesis, probably for de novo synthesis of nitrogenase. Respiratory O2 consumption by cultures of Gloeothece was stimulated under elevated concentrations of O2. This respiration may contribute to the protection of nitrogenase from inactivation by O2 at concentrations up to 0·4 atm. The role of Ca2+ in nitrogen fixation may be related to respiratory protection.
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Serine Hydroxymethyltransferase and Glycine Cleavage Enzyme from the Cyanogenic Bacterium Chromobacterium violaceum
More LessChromobacterium violaceum forms cyanide as a secondary metabolite with glycine as the precursor and methionine as a stimulator. As the metabolism of glycine and methionine are interrelated by the provision of carbon-one units for the tetrahydrofolate pool by the activities of serine hydroxymethyltransferase (SHMT) and glycine cleavage enzyme (GCE), we have investigated the regulation of formation and activity of these enzymes by C. violaceum. The synthesis of SHMT was unaffected by growth under conditions of high cyanogenesis (on a medium containing glutamate, glycine and methionine) or low cyanogenesis (on glutamate alone), nor was its synthesis affected by addition of potential end-products of carbon-one metabolism to the growth medium. The activity of GCE was very low, less than 1% of the activity of SHMT, irrespective of the growth conditions. This suggests that nearly all the carbon-one units for the tetrahydrofolate pool are supplied by the activity of SHMT; this could lead to excess formation of glycine and might explain the role of cyanogenesis. The activity of SHMT was competitively inhibited by glycine and cysteine.
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Hydrogenation in vitro of α-Linolenic Acid to Stearic Acid by Mixed Cultures of Pure Strains of Rumen Bacteria
More LessThe hydrogenation of α-linolenic acid to stearic acid which occurs with the mixed bacteria of the sheep rumen can be demonstrated in vitro by mixing exponential phase cultures of only two species of rumen bacteria and incubating for a further period with α-linolenic acid. One bacterium must be able to hydrogenate α-linolenic acid to predominantly trans-octadec-11-enoic acid which is then used 11 substrate by a second bacterium. Cultures grown from small mixed inocula failed, with the exception of one pair of bacteria, to hydrogenate α-linolenic acid to stearic acid. The products from these cultures showed that one of the pair of bacteria had outgrown the other. For stearate production it was necessary to use inocula with a minimum number of cells rather than cells in a particular phase of growth. Two of the bacteria used, P2/2 and T344, after several years in pure culture show an increased isomerization of the octadecenoic acid products.
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Isolation, Partial Characterization and Utilization of a Polysaccharide from Bacillus megaterium ATCC 19213
More LessA high molecular weight polysaccharide was isolated from Bacillus megaterium ATCC 19213 cultured on a fructose mineral salts medium. This polymer was a heteropolysaccharide composed of d-glucose, d-xylose, d-galactose and l-arabinose in molar concentrations of 7.7:1.1:1.0:0.6, respectively. The polysaccharide neither bound concanavalin A nor formed a coloured complex with iodine, distinguishing it from many of the glucans isolated from other sporeformers. The concentration of the polymer decreased during forespore development, suggesting that it served as a carbon storage compound for use during formation of the spore.
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Genetic and Structural Evidence for the Presence of Propanediol Oxidoreductase Isoenzymes in Escherichia coli
More LessThe synthesis of propanediol oxidoreductase, an enzyme permitting the anaerobic metabolism of fucose and rhamnose, has been described as being controlled by the prd locus closely linked to the fuc locus in wild-type cells of Escherichia coli. However, strain AA-787, deleted in the fuc and prd loci, grew anaerobically on rhamnose, displaying propanediol oxidoreductase activity. From the deleted strain we derived a constitutive producer of propanediol oxidoreductase able to grow on 1,2-propanediol by oxidizing the diol to lactaldehyde which was further metabolized to lactate. Transduction experiments showed that this ability to use propanediol was closely linked to the rha locus. Peptide mapping of fucose- and rhamnose-induced propanediol oxidoreductase of wild-type cells established structural differences between the two enzymes, indicating two structural genes, one for each sugar metabolizing system.
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Fractionation of Membranes from Metschnikowia reukaufii Protoplasts. Evidence for a Plasma-membrane-bound ATPase
More LessCells of the yeast Metschnikowia reukaufii were transformed to true protoplasts by means of snail gut enzyme. The plasma membrane of purified protoplasts was labelled either by [3H]dansyl chloride or by Na125 I. The crude lysate of osmotically ruptured protoplasts was fractionated by two subsequent centrifugations on sucrose density gradients. The two protein peaks obtained at densities of 1·16 and 1·13 g cm−3 were identified according to their characteristic markers (radioactivity, cytochrome c oxidase, oligomycin-sensitive ATPase at pH 8.5, and sterol content) as mitochondrial and plasma-membrane fractions, respectively. The ATPase activity associated with the plasma-membrane fraction exhibited a pH optimum at 6.5, was insensitive to oligomycin and was inhibited by vanadate. It can therefore be used as an intrinsic plasma-membrane marker.
Adherence of silica microbeads to the protoplasts effectively increased the density of the plasma membrane so that it could be obtained in a purified state by a single density gradient centrifugation. A third ATPase, presumably associated with the tonoplast membrane, was also demonstrated.
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The Isolation of a Mercuric Ion-reducing Flavoprotein from Thiobacillus ferrooxidans
More LessA mercuric ion-reducing flavoprotein was purified from Thiobacillus ferrooxidans TFI 29 by using dye-matrix affinity chromatography. The isolated enzyme had a molecular weight of 130000 and was composed of two subunits (54000 and 62000). The visible absorbance spectrum of the oxidized enzyme had a maximum at 455 nm. Upon addition of NADPH a new absorbance at 540 nm appeared, which was attributed to a charge transfer complex. The K m for mercuric chloride was determined to be 8·9μ m and the enzyme was shown to have a turnover number of 746 min−1 per FAD. A comparison of these physical properties, as well as metal ion inhibition and pH profiles, indicate that the enzyme from T. ferrooxidans is very similar in structure and function to mercuric reductases isolated from other bacterial sources.
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- Development And Structure
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Karyokinesis and Septum Formation during the Regeneration of Incomplete Cell Walls in Protoplasts of Schizosaccharomyces japonicus var. versatilis: a Time-lapse Microcinematographic Study
More LessRegeneration in flattened Schizosaccharomyces japonicus var. versatilis protoplasts was studied with regard to nuclear behaviour, septum formation and lateral wall regeneration. In the course of protoplast regeneration all stages of the cell cycle were realized. However, the time sequence was conserved only between nuclear division and the beginning of septum formation. From this it is suggested that the signal initiating septum formation was issued by the dividing nucleus. Its primary target was not the original cell wall. However, the wall, though incomplete, was an essential prerequisite for the signal to be put into operation, i.e. for septation to begin. Incomplete cell wall regeneration allowed cytokinesis but not reversion of protoplasts into cells with their genetically determined cylindrical cell shape.
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Characterization of Polyagglutinating and Surface Antigens in Pseudomonas aeruginosa
More LessMutants of Pseudomonas aeruginosa PAC1R (serotype O:3) which were resistant to bacteriophage D were isolated and shown to react with O:5d, O:9 and O:13 antisera as well as O:3. Antisera to the parent strain and to the three polyagglutinating (PA) mutants also showed cross-reactions. The mutants differed from the parent strain in their lipopolysaccharide (LPS) composition. The LPS from two of the three mutants yielded high molecular weight polysaccharide fractions. Although the high molecular weight fraction from one of the mutants contained the amino sugars and other components characteristic of the O:3 serotype strains, its mobility on Sephadex G75 was different from that of the parent strain. The high molecular weight material from the second mutant lacked the O-antigenic determinants but these were present in a semi-rough LPS fraction. The third mutant appeared rough and completely lacked the O-antigenic components. These three mutants were compared with the parent strain and with a non-agglutinating LPS-defective mutant which lacked both O-antigenic side chains and all neutral sugars in the outer core. Agglutination with absorbed sera and haemagglutination using purified LPS and ELISA detection suggested that wall components other than LPS were responsible for some of the cross-reactions observed. The components responsible were detected after SDS-PAGE of crude outer membrane fractions by a combination of Coomassie blue and silver-staining and antigenic components were detected by immunoelectrophoresis and ELISA-linked immunoblotting of the gels. The main antigenic determinants detected by antiserum to the parent strain were in the high molecular weight O-polysaccharide fractions and in the semi-rough fractions of the LPS, with some activity due to the H protein of the outer membrane. O:5d antisera reacted with unidentified high molecular weight polysaccharide fractions. Cross-reactions with the O:9 antiserum appeared to be due mainly to the F porin and, to a lesser extent, to the G and E proteins of the outer membrane. O:13 antiserum reacted with high molecular weight polysaccharide fractions but also with the rough core and F and H protein. Cross-reactivity of the other three mutant antisera could largely be interpreted in terms of the outer membrane components exposed in each strain. One reacted strongly with the F porin and the rough core, while the others reacted with a number of protein and LPS-derived fractions. It is suggested that, in these PA mutants, changes in LPS composition modify their O-antigenicity and also expose other outer membrane components to varying extents. It is these protein and rough core components which are responsible for the cross-reactivity with heterologous O-antisera.
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- Ecology
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Growth of Bacteria on Roots of Grasses: Influence of Mineral Nutrient Supply and Interactions between Species
More LessGrass plants were grown axenically in sand and were then inoculated with either one bacterial species or a mixture of two species. Numbers of bacteria on the root surface were subsequently determined by dilution plating. When six bacterial species were inoculated on to separate Lolium perenne plants, the species with the smallest individual cells produced greatest numbers per mg root after 15 d. When Serratia marcescens was inoculated on to three grass species, L. perenne, Holcus lanatus and Deschampsia flexuosa, there was no significant difference between the grasses in the number of bacteria per mg of root which subsequently developed. Serratia marcescens and a Flavobacterium sp. were inoculated, separately or together, on to L. perenne, grown either with a complete nutrient solution or one deficient in either nitrogen or phosphorus. The Flavobacterium sp. increased more slowly than S. marcescens, a difference enhanced when they were on the same plant. Decreased nitrogen supply, although it reduced plant growth, had no significant effect on bacterial numbers, whereas phosphorus deficiency increased the numbers of both bacterial species.
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- Genetics And Molecular Biology
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Resistance to Apramycin in Escherichia coli Isolated from Animals: Detection of a Novel Aminoglycoside-modifying Enzyme
More LessThe mechanisms of resistance to apramycin of five isolates of Escherichia coli from animals were investigated. Three isolates, which were resistant to all the aminoglycosides tested, did not transfer their resistance and did not produce aminoglycoside-modifying enzymes. The fourth isolate, which was resistant to apramycin, tobramycin, gentamicin, kanamycin and neomycin but not to amikacin, owed its resistance to production of the acetyltransferase AAC(3)IV. The gene specifying this enzyme was carried on a transposon, Tn800, on a plasmid designated R1535. The fifth isolate was resistant to apramycin, neomycin and kanamycin but not to gentamicin, tobramycin or amikacin. It produced an acetyltransferase that readily acetylated only apramycin, neomycin and paromomycin, a compound that is closely related to neomycin. Synthesis of this enzyme was specified by a chromosomal gene located near pyrD at about 20 min on the map of the E. coli K12 chromosome.
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The Effect of D2O on the Growth and Transforming Activities of Streptococcus pneumoniae
More LessAfter an initial period of growth in medium made up in D2O, most strains of pneumococcus tested dramatically lost viability, the extent of the loss depending on the strain and on the amount of contaminating H2O in the D2O. This was followed by a recovery period. Once a strain was ‘adapted’, the ability to grow in D2O-medium without cell death was inherited, even after passage through H2O-medium, indicating the selection of mutants. Cultures that had not reached ‘full adaptation’ also exhibited cell death if transferred into either D2O-medium or H2O-medium, supporting the conclusion that the presence of hydrogen and deuterium together caused the toxicity.
‘Adapted’ cells exhibited an increased mutation frequency to a variety of antibiotic resistances, the propensity for this appearing in the death phase of ‘adaptation’. The specific transforming activity of DNA preparations from cultures undergoing ‘adaptation’ decreased before DNA synthesis ceased indicating damage to the DNA. The integration efficiency of a low-efficiency marker also dropped during ‘adaptation’ before returning to the initial value when measured in a Hex- recipient, but remained constant in a Hex+ recipient, suggesting that the Hex system may be involved in repair of the DNA damage. ‘Adapted’ organisms showed evidence of possessing higher Hex activity and were also able to repair lesions caused by UV-irradiation better than the wild-type.
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Evidence for Two Control Genes Regulating Expression of the Quinic Acid Utilization (qut) Gene Cluster in Aspergillus nidulans
More LessThe first three steps in quinic acid degradation in Aspergillus nidulans are catalysed by highly inducible enzymes encoded by a gene cluster regulated by an adjacent control region. Analysis of two non-inducible mutants has been done in diploid strains, where qutA8 is recessive and all three enzyme activities are fully induced in heterozygous qutA8/qutA+ diploids. In contrast, qutA4/qutA+ heterozygous diploids show semi-dominance of the mutant allele, giving markedly diminished growth on quinic acid and 30–40% decrease of enzyme induction. Strikingly, the qutA4/qutA8 heterozygous diploid grows to the same degree on quinic acid as the qutA4/qutA+ heterozygote and shows the same level of enzyme induction, whereas both the homozygous mutant diploids do not grow on quinic acid and show no enzyme induction. Therefore the two mutant genomes complement, identifying two distinct regulatory gene functions. A genetic model is proposed of a negatively acting gene (qutA) repressing expression of a positively acting gene (qutD, previously designated qutA8+ ) whose product is in turn required for expression of the three structural genes. The qutA4 mutation is interpreted to produce an altered repressor insensitive to quinic acid, and the qutD8 mutation the loss of activator protein. Close similarity in the regulation of the quinic acid gene cluster in Neurospora crassa suggests that the two types of control mutation, qals and qalF, described for N. crassa may also reflect two regulatory genes.
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Interactions of Ribosomal Antibiotics and Informational Suppressors of Aspergillus nidulans
More LessStrains of Aspergillus nidulans containing informational suppressors were grown on medium containing antibiotics known to affect protein synthesis at the ribosomal level. These strains reacted in the anticipated manner: presumed ribosomal suppressors suaA101, suaA105, suaC109 and sua-115 were sensitive or even hypersensitive to aminoglycoside antibiotics, whereas presumed tRNA-like suppressors suaB111, suaD103 and D108 were only slightly sensitive or wild-type in response. Hygromycin and paromomycin were the most useful antibiotics. All the antibiotics reduced the colony radial growth rate, K r, increased the lag phase and produced wrinkled morphology. Hygromycin was the most toxic. Resistant sectors were produced on paromomycin and hygromycin. The selective action of ‘misreading’ antibiotics on suaA and suaC strains is further evidence that these are ribosomal suppressors, whereas suaB and suaD may code for altered tRNA molecules. The results imply that hygromycin or paromomycin could be used for isolating ribosomal suppressors.
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Spontaneous Duplications and Transpositions of a Large Chromosome Segment in Aspergillus nidulans
More LessSpontaneous revertants of the leaky adE20 mutant of Aspergillus nidulans were obtained as vigorous sectors emerging from stunted colonies on adenine-free medium. Among the genetically heterogeneous sectors up to about 20% were recognized unequivocally as having an additional chromosome segment bearing adE20; two doses of this leaky allele permitted growth without added adenine. Eleven spontaneous duplication strains of independent origin were analysed genetically. Eight carried the duplicate segment on chromosome IIR; three of these, phenotypically similar to all eight, were analysed in detail and were shown - within the limits of such genetic analysis - to have a large, terminal segment of IR duplicated and attached terminally and uninverted to IIR. One strain had a duplication, possibly tandem, on IR and two had duplications attached elsewhere in the genome. The results suggested a preferential site for the initiation of duplicate segments in this system, as well as a preferential site for their attachment. Agents known to modify instability of a previously studied Dp(IR → IIR) strain affected the frequency of duplications among selected adE20 revertant sectors and/or the genomic locations of duplicated segments. Trypan blue and coumarin, which enhance Dp(IR → IIR) instability in a specific way, and Co2+, which stabilizes Dp(IR → IIR), gave 14, 50 and 62% duplication sectors respectively, among revertants. Duplications selected in the presence of Co2+ had mainly IIR attachments; of those from trypan blue and coumarin, about one-quarter were attached to IIL and none to IIR.
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Initiation of Sporulation in Saccharomyces cerevisiae. Mutations Causing Derepressed Sporulation and G1 Arrest in the Cell Division Cycle
More LessMutants of Saccharomyces cerevisiae that are derepressed for meiosis and spore formation have been isolated and characterized genetically. All are the result of single, recessive nuclear mutations that fall into four linkage groups. Three of these groups are represented by spd1, spd3 and spd4 mutations, which in homozygous diploids confer poor growth and extensive sporulation on a range of non-fermentable media. Haploids carrying any of these mutations are arrested under these conditions in the G1 phase of the cell division cycle as large unbudded cells. The alleles of the spd2 mutation complemented all other mutations but were very closely linked to the spd1 locus. The fourth linkage group was represented by a mutation conferring temperature-sensitive growth and derepressed sporulation on homozygous diploids grown between 25 °C and 30 °C on media containing galactose or glycerol, but not glucose, as energy source. Above 30 °C this mutant lysed on all media. The mutation it carried failed to complement available cdc25 mutations. These data bring to five the number of loci at which mutation can lead to derepressed sporulation (spd1, spd3, spd4, cdc25 and cdc35). The spd1 locus has been mapped 13·9 cM to the left of the centromere on chromosome XV, adjacent to the SUP3 gene. Diploid strains homozygous for spd mutations are genetically unstable, giving rise to asporogenous mutants at high frequency, usually as the result of a second mutation unlinked to the spd mutation. Diploids homozygous for these mutations, and for spd mutations, show an altered regulation of the formulation of at least three polypeptides normally subject to carbon source repression.
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Initiation of Sporulation in Saccharomyces cerevisiae. Mutations Preventing Initiation
More LessMutants of Saccharomyces cerevisiae that are unable to initiate sporulation, but can continue vegetative growth under conditions in which the wild-type strain sporulates, have been isolated and characterized. The mutations arose spontaneously as suppressors of the spd1 mutations, restoring the ability of spd1 mutants to grow on glycerol, and also spontaneously in cultures of a wild-type diploid strain undergoing sporulation in continuous culture. The mutations all conferred asporogeny, and were recessive in this respect to the wild-type, but dominant in acting as suppressors of the spd1 mutation. They fell into three complementation groups which corresponded to three unlinked loci, designated spo50, spo51 and spo53. None of these mutations was closely linked to the other initiation mutations defined by the spd1, spd3, spd4, cdc25, cdc28 loci, nor to the cell size control mutations whi1 and whi2. Loose linkage was detected between spd1 and spo53, and spo50, spd3 and spo53 were linked to their respective centromeres. The spo50, spo51 and spo53 mutations are not nonsense suppressors. Mutations in all three genes conferred similar highly pleiotropic phenotypes including: asporogeny; dominant suppression of both spd1 and spd3 mutations; aberrant cell morphology and viability loss on starvation; constitutive ability to reduce tetrazolium (which is subject to carbon source repression in the wild-type); and complete repression of the synthesis of several polypeptides that are subject to carbon source repression in the wild-type strain and derepressed in spd1 mutants derepressed for sporulation. A diploid strain homozygous for the spo50 mutation did not undergo either premeiotic DNA replication or meiotic recombination when transferred to sporulation media.
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Location on the Escherichia coli Genome of a Gene Specifying O-Acetylserine (Thiol)-lyase
More LessThe plasmid pAB65, derived from a specialized transducing phage carrying DNA from about 52 min on the Escherichia coli genome, coded for two polypeptides of M r approx. 34000. The expression of one was regulated by cyst(e)ine and the cysB gene product and the other by the cysB gene product only. One of these polypeptides was a subunit of O-acetylserine (thiol)-lyase (EC 4.2.99.8); the other, associated with the E. coli membrane, was the N-terminus of the product of the ? ben gene. The pattern of peptide synthesis directed by plasmids carrying smaller DNA fragments indicated that the gene for O-acetylserine (thiol)-lyase was transcribed clockwise. The spectrum, amino acid composition and subunit number of the enzyme were determined. The enzyme appears homologous with the Salmonella typhimurium cysK gene product. This provides further evidence for the inversion of this region of the genome.
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Proteus mirabilis Chromosome Mobilization by Plasmid D: a Physical Characterization
More LessPlasmid D, a hybrid of plasmids P-lac and R1drd19, mediates polarized chromosome mobilization from one origin in Proteus mirabilis strain PM5006, while the parental plasmids neither individually nor combined mobilize this chromosome. To elucidate its acquired mobilizing ability plasmid D was characterized physically in relation to P-lac and R1drd19. Restriction patterns of these plasmids were compared and it was shown that D consists of P-lac and only the r-determinant (r-det) of R1drd19. A mechanism for the formation of plasmid D, via transduction of the r-det and subsequent transposon-like integration into P-lac, involving insertion sequence IS1, was suggested. Evidence for aberration in plasmid D DNA as a result of r-det integration into P-lac was attributed to IS1 elements which flank the r-det. Recombination regions of parental plasmid DNA were located on HindIII fragments a and of plasmid D and were subsequently inserted in vitro into IncP-1 plasmid RP4 that fails to mobilize the P. mirabilis chromosome. RP4:: HindIII a plasmids did not mobilize the latter chromosome, but rendered the Proteus host lac+. RP4:: HindIII plasmids pMC1 and pMC17, containing the fragment in opposite orientations, mobilized the P. mirabilis chromosome. For pMC17, mobilization was indistinguishable from that of plasmid D, i.e. having the same orientation and the same single origin. However, mobilization promoted by pMC1 was from two distinctly different origins, different from that of pMC17. This apparently deviates from known examples where inversion of homologous DNA inserted into plasmids leads to mobilization from the same origin but in reverse direction.
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Localization and Characterization of a Gene on the ColE3-CA38 Plasmid that Confers Immunity to Colicin E8
More LessEscherichia coli W3110 cells carrying the ColE3-CA38 plasmid are immune to externally added colicin E8, a newly described member of the E group colicins. By molecular cloning and transposon mutagenesis we localized the colicin E8 immunity gene between the EcoRI site (4·0 kb on the restriction map) and the PvuII site (3·68 kb) of the ColE3-CA38 plasmid. This placed the colicin E8 immunity gene between the colicin E3 immunity gene and lys, the region which determined mitomycin C sensitivity. Insertion of a transposon into the colicin E3 structural gene prevented the synthesis of active colicin and completely abolished mitomycin C sensitivity, but had no effect on the two immunity genes. In contrast, insertion of a transposon into the colicin E8 immunity gene had no effect upon colicin E3 production or colicin E3 immunity but did abolish mitomycin C sensitivity. The phenotype conferred by plasmids with a transposon inserted into the lys region of ColE3-CA38 was dependent upon the site of insertion.
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- Immunology
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Crossed Immunoelectrophoresis and Enzyme-linked Immunosorbent Assay of the Cell-surface Antigens of Bacteroides fragilis
More LessAntisera were raised to whole, live cells of a reference strain (NCTC 9344) and two clinical isolates (GNAB 92 and GNAB 4) of Bacteroides fragilis. Each antiserum was reacted in crossed line immunoelectrophoresis (CLIE) with EDTA-heat-sonication-prepared outer membrane (OM) complex from 10 B. fragilis strains. In addition, the antisera were reacted with these antigens in an enzyme-linked immunosorbent assay (ELISA). In CLIE, the antisera raised to the reference strain and one of the clinical isolates (GNAB 92) demonstrated a heat labile antigen which was common to all 10 of the test strains. Lipopolysaccharide (LPS) prepared from both the clinical isolates produced three major precipitin lines when reacted with their homologous antisera in crossed immunoelectrophoresis (CIE). In both cases, these three antigens were present as major components of the OM complex. Each antiserum reacted significantly in ELISA with all test OM complex preparations. Inhibition of ELISA showed that carbohydrates were the predominant cross-reactive antigens in ELISA and that in the case of the clinical isolate GNAB 4, most of the cross-reactive antigenic activity was present in the homologous LPS preparation.
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- Pathogenicity And Medical Microbiology
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Interactions of Candida albicans Yeast Cells, Germ Tubes and Hyphae with Human Polymorphonuclear Leucocytes in vitro
More LessSuspensions of Candida albicans yeast cells, germ tubes and hyphae with biomass standardized by ATP measurement were compared for their relative susceptibilities to phagocytosis and intracellular killing by human polymorphonuclear leucocytes. All three forms were ingested to a similar extent, but significantly fewer yeast cells were killed intracellularly after ingestion than were filamentous forms of the fungus. Ketoconazole pretreatment significantly enhanced the susceptibility of hyphae, but not of germ tubes, to phagocytosis and intracellular killing. The opsonic requirements of the yeasts and filamentous forms for efficient phagocytosis and killing differed.
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Gas Chromatography-Mass Spectrometry of Mycolic Acids as a Tool in the Identification of Medically Important Coryneform Bacteria
More LessThe mycolic acid derivatives of 11 unidentified pathogenic coryneform bacteria were examined by TLC, GLC and GLC-mass spectrometry. The resulting mycolic acid profiles of the unidentified isolates were compared with those of type or reference strains of possibly related coryneform species, namely Corynebacterium bovis, C. diphtheriae, C. xerosis and Rhodococcus equi. It was apparent that most of the unidentified strains showed a distinctive mycolic acid profile, with predominant amounts of relatively high molecular weight mycolic acids (C32-C36) and a high degree of unsaturation, and could thus be distinguished from both C. bovis, which had exceptionally low molecular weight mycolic acids (C24-C30), and C. diphtheriae (C28-C34), which had large amounts of saturated mycolic acids. The mycolates of C. xerosis and R. equi (C28-C36) were generally similar to those of the unidentified coryneforms but their overall mycolic acid patterns were different from one another as well as the unidentified strains. The mycolic acid profiles exhibited by the pathogenic coryneforms examined here were very similar to one another but unlike that of any of the type or reference strains included in the study.
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Comparative Studies on Surface Hydrophobicity of Streptococcal Strains of Groups A, B, C, D and G
More LessCell surface hydrophobicity of group A, B, C, D and G streptococcal strains has been studied and compared in a new test based on the fact that the degree of bacterial aggregation in ammonium sulphate depends on amphiphilic surface antigens. M-positive group A strains showing good growth in normal human blood aggregated in the standard salt aggregation test at very low concentrations of ammonium sulphate, while M-negative strains, which were killed in normal human blood, usually aggregated at high salt concentrations. Agents such as 2 M-KSCN, 2 M-guanidine. HCl or 2 M-urea decreased the aggregation of the M-positive strains in the salt aggregation test while non ionic detergents such as Tween 20 (1%, w/v) and ethylene glycol (2 m) did not affect cell aggregation. Binding of fibrinogen and albumin resulted in a decrease of surface hydrophobicity of the group A M-positive strains. Group B strains possess a hydrophilic surface character and did not aggregate, while group C and G strains behaved in the salt aggregation test like M-negative strains of group A streptococci. Group D strains did not aggregate even at high ammonium salt concentrations. The results are discussed in relation to the influence of lipoteichoic acid and other surface antigens on strains of the various groups, and it is suggested that M protein and possibly also other surface proteins contribute to the high surface hydrophobicity of group A strains.
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Human Serum Bactericidal Activity against Haemophilus influenzae Type b
We examined bactericidal and opsonizing activity of pooled adult ‘immune’ serum against Haemophilus influenzae type b with and without the addition of phagocytes. Four type b strains from cerebrospinal fluid (CSF) and three such strains from the nasopharynx (NP) of healthy children were examined. Duplicate reaction mixtures contained organisms in exponential (E) or stationary phase (S) of growth, serum, a complement source (human agammaglobulinaemic serum), and culture medium (bactericidal assay); separate assays contained the above components and polymorphonuclear leucocytes (opsonization system). A decrease in bacterial density of ⩾ 1 log10 unit was considered significant. All four S-CSF strains, three of four E-CSF strains and one of three S-NP strains were sensitive to the bactericidal activity of pooled serum. The other E-CSF strain, two S-NP strains and all three E-NP strains were resistant to the bactericidal activity of pooled serum. Two of three E-NP strains were opsonized by pooled serum; the other strains resistant to the bactericidal activity of pooled serum were also resistant to opsonization. Bactericidal and opsonizing activity of serum from an immunized adult was greater than or equal to that of pooled serum against each strain. Assuming normal adults are immune to invasive H. influenzae type b infection, an experimental test reflecting this immunity is the bactericidal activity against CSF isolates tested in stationary phase. We conclude that protection against invasive disease due to H. influenzae type b appears more complex than the presence of bactericidal and opsonizing activity in serum.
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- Physiology And Growth
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Regulation of Autotrophic and Heterotrophic Metabolism in Pseudomonas oxalaticus OX1. Growth on Fructose and on Mixtures of Fructose and Formate in Batch and Continuous Cultures
More LessIn Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic CO2 fixation via the Calvin cycle is regulated by repression/derepression. During growth of the organism on fructose alone, the synthesis of ribulosebisphosphate carboxylase (RuBPCase) remained fully repressed, both in batch culture and in fructose-limited continuous cultures at various dilution rates. Growth in batch culture on a mixture of fructose and formate resulted in the simultaneous utilization of both substrates. Under these conditions we observed synthesis of RuBPCase up to high levels, indicating that formate did not merely function as an ancillary energy source in the metabolism of fructose, but stimulated autotrophic CO2 fixation via the Calvin cycle. In subsequent experiments growth of P. oxalaticus on mixtures of fructose and formate was studied in carbon source-limited continuous cultures. In these experiments further evidence was obtained that fructose is a poor source of (co-)repressor molecules for the synthesis of RuBPCase in the presence of formate. Thus, addition of formate to the medium reservoir of a fructose-limited continuous culture resulted in derepression of RuBPCase synthesis at (relatively) high ratios of fructose over formate. In the reverse experiment the specific activity of RuBPCase decreased with increasing concentrations of fructose in the medium reservoir. However, it can be calculated that the total capacity of RuBPCase in the culture to fix CO2 remained constant. In these experiments the dry weight produced on the various mixtures equalled the sum of the dry weight values obtained during growth on the same amounts of the two substrates separately. This indicated that, once RuBPCase was present, autotrophic and heterotrophic carbon assimilation pathways functioned simultaneously and independently of each other. Possible explanations for the low repressing effect of fructose on autotrophic CO2 fixation in P. oxalaticus are discussed.
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Methylamine Uptake by the Facultative Methylotroph Hyphomicrobium X
More LessHyphomicrobium X grows on methylamine as sole source of carbon, energy and nitrogen. The uptake of the compound occurred via a single inducible transport system irrespective of whether methylamine was used as a carbon and energy source or a nitrogen source only. The transport system had the following properties: temperature optimum 30 °C, pH optimum 7·0, K m approx. 70–100 nm, V max 12·5 nmol min−1 (mg dry weight)−1. Methylamine uptake was inhibited by azide, cyanide, N-ethylmaleimide and carbonyl cyanide-m-chlorophenyl-hydrazone. The uptake was not inhibited by ammonium ions, was slightly inhibited by amino acids and amides but was competitively inhibited by short-chain alkylamines (K i ethylamine 200 nm). Cells grown on formate, methanol or ethanol as C-source and ammonium sulphate as N-source did not possess the transport system but this could be induced by the presence of methylamine for 45–50 min. The addition of formate, methanol or ethanol to methylamine-grown cells did not affect the rate of methylamine uptake. Bacteria pre-grown on dimethylamine or trimethylamine transported methylamine into the cells at rates of 99% and 52%, respectively, when compared to rates with cells pre-grown on methylamine.
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Cyclic AMP, Trehalase and Germination of Phycomyces blakesleeanus Spores
More LessWhen heat activated Phycomyces blakesleeanus spores were incubated at room temperature a rapid and transient increase in cyclic AMP content was observed. A similar increase was observed during the heat treatment itself but the cyclic AMP content had returned to the value of dormant spores at the end of the 5 min treatment. The cyclic AMP content increased only after treatments at temperatures which also induced spore germination. A more gradual rise in cyclic AMP was also found during activation at room temperature with acetate and after activation of the spores with pyrosulphite. The increase in cyclic AMP content is the earliest event known to occur after activation of the spores. It clearly preceded the increase in trehalase activity observed during and/or after all the activating treatments, suggesting that, as shown for in vitro activation, the in vivo activation of trehalase is due to a cyclic AMP-dependent phosphorylation. An increase in cyclic AMP was a common feature of all activation methods investigated and appears to be the factor triggering spore germination.
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Variation of Intracellular Cyclic AMP and Cyclic GMP Following Chemical Stimulation in Relation to Contractility in Physarum polycephalum
More LessThe plasmodium of Physarum polycephalum reacts to external stimuli tactically. Positive and negative taxes correspond to relaxation and contraction, respectively. Variations of intracellular cAMP and cGMP concentrations in response to chemical stimuli were examined in relation to the regulation of contractility. Concentrations of the two cyclic nucleotides oscillated, with a gradual shift, for some time after stimulation. The period of oscillation was 4-5 min, with phases being the same in response to repellents, but different when attractants were tested. Therefore, changes in the accumulation of the nucleotides summed over 15 min were taken as a quantitative measure of the external stimuli. Attractants (glucose, 2-deoxyglucose, galactose, maltose) induced decreases both in cAMP and in cGMP concentration, the latter being larger than the former. Repellents (KCl, CaCl2, MgCl2, sucrose) induced decreases both in cAMP and in cGMP concentration, the former being larger than the latter. Variations of the intracellular cAMP concentration for repellents and those of cGMP concentration for attractants, took place at similar concentrations of stimulants as variations of contraction and relaxation, respectively. Microinjection of cAMP and cGMP into the plasmodial strands induced contraction, cGMP being about 10 times as effective as cAMP. The results indicate that both cAMP and cGMP regulate the ability to contract, not antagonistically, but cooperatively, in the sensory transduction of the Physarum plasmodium.
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Induction of Cellular Efflux by a Galactosamine Polymer from Neurospora crassa
More LessA cationic polymer of d-galactosamine was isolated from culture filtrates of a colonial temperature-sensitive strain of Neurospora crassa. Adsorption of the polymer to the cell surface initiated immediate efflux of low molecular weight metabolites and subsequent loss of viability. The polymer appeared to bind to those sites on the cell surface that normally bind calcium ions. Chemical analysis of the polymer showed it to be partially N-acetylated. The polymer had an isoelectric point of 8·4. Thirty percent of the d-galactosamine residues contained free amino groups. A rapid assay that has potential application for monitoring the effect of a variety of other membrane-active factors on membrane permeability has been developed.
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- Systematics
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Mycoplasma glycophilum, a New Species of Avian Origin
More LessA mycoplasma, designated strain 486, was isolated from the oviduct of an adult chicken. On the basis of its morphological, physical and cultural characteristics the organism was assigned to the class Mollicutes, order Mycoplasmatales. The guanine plus cytosine content of its DNA was estimated to be 27·5 mol%. The organism was inhibited by digitonin and it showed a growth response to sterol, although its minimal requirement was low. It neither showed helical forms nor hydrolysed urea and was hence assigned to the family Mycoplasmataceae, genus Mycoplasma. Strain 486 fermented glucose and reduced tetrazolium under anaerobic conditions. It did not hydrolyse arginine, aesculin or arbutin, nor did it produce films and spots or digest serum. Phosphatase activity was negative or very weak, and the organism adsorbed turkey but not chicken red blood cells. Serological tests (growth inhibition, indirect fluorescent antibody, double immunodiffusion and metabolism inhibition) with 75 currently accepted Mycoplasma species or serovars failed to identify the isolate. Thus strain 486 appears to represent a new species, for which the name Mycoplasma glycophilum is proposed. (NCTC 10194, ATCC 35277).
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- Short Communication
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A New Solidifying Agent for Culture Media Which Liquefies on Cooling
More LessPluronic polyol F127, a block copolymer of polypropylene oxide and ethylene oxide, may be used to solidify culture media for the enrichment, isolation and growth of micro-organisms. A stable gel is formed at temperatures above 10 °C, the exact temperature depending on the concentration of polyol used. The most important feature of the polyol gel is that it liquefies as the temperature drops below the critical value for the concentration used. The gel may be auto-claved, and will alternate repeatedly between liquid and gel form. It does not appear to be toxic to aquatic bacteria, is more transparent than agar and may be used to isolate heat-sensitive organisms.
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