- Volume 132, Issue 6, 1986
Volume 132, Issue 6, 1986
- Biochemistry
-
-
-
Phosphate-polymer-dependent Phosphorylation of Glycolytic Substrates by Brevibacterium Species
More LessPhosphate polymer-utilizing activity for the phosphorylation of glucose, mannose, fructose and fructose 6-phosphate was investigated in 12 Brevibacterium species. Almost all the species were able to phosphorylate sugars using metaphosphate as a phosphoryl donor. However, only B. pusillum was able to phosphorylate fructose 6-phosphate to fructose 1,6-bisphosphate using pyrophosphate. The analysis of cell extracts prepared from B. pusillum indicated that at least two enzymes were responsible for the phosphate-polymer-dependent phosphorylation of sugars and fructose 6-phosphate.
-
-
-
-
Haemoprotein b-590 (Escherichia coli), a Reducible Catalase and Peroxidase: Evidence for its Close Relationship to Hydroperoxidase I and a ‘Cytochrome a 1 b’ Preparation
More LessA reducible hydroperoxidase, haemoprotein b-590, has been purified 16-fold from a soluble fraction of Escherichia coli K12, grown anaerobically with glycerol and fumarate. The M r of the native protein, determined by gel filtration, was 331000 although a minor, smaller species with a M r of 188000 was also detected; both had catalase activities. Based on the subunit M r, determined from SDS gel electrophoresis to be 75000, the above species are tentatively identified as tetramers and dimers, respectively. The isoelectric point of both species was 4·4. The absorption spectrum of the isolated haemoprotein is typical of ferric, high-spin haem. The A 405/A 280 ratio never exceeded 0·27, a value half of that obtained for E. coli hydroperoxidase I. On reduction with dithionite, the ?, ?, and ? bands were at 441, 559 and 590 nm respectively, the ?-band being unusually distinct. Treatment of the reduced form with CO gave a sharp prominent ?-band at 426 nm and caused significant shifts of the ? and ? bands to shorter (574 and 545 nm) wavelengths.
The pyridine haemochrome spectra showed the haem to be protohaem IX; the spectra were featureless between 580 and 630 nm, thus excluding the presence of haem a. However, some features of the difference spectra of the haemoprotein were reminiscent of cytochrome a 1, notably the maxima in reduced minus oxidized spectra at 444 and 593 nm and the peaks and troughs in CO difference spectra at 426 and 446 nm respectively. The haemoprotein had high catalase activity: V max was 2·3 × 106 mol H2O2 (mol haem)?1 min?1 and the K m was 11 mM. At 10 mM-H2O2 the first order rate constant was 0·3 × 107 M?1 s?1. The haemoprotein was also a peroxidase with o-dianisidine or 2,3’,6-trichloroindophenol as substrates; for the latter substrate, the K m was 0·18 mM. It is concluded that haemoprotein b-590 strongly resembles the hydroperoxidase I purified by Claiborne & Fridovich (Journal of Biological Chemistry 254, 4245-4252, 1979) and that a similar haemoprotein was mistaken for a cytochrome a 1 b complex by Barrett & Sinclair (Abstracts of the 7th International Congress of Biochemistry, Tokyo, H-107, p. 907, 1967).
-
-
-
Some Properties of a Saccharomyces cerevisiae Mutant Resistant to 2-Amino-4-methyl-5-?-hydroxyethylthiazole
More LessA mutant of Saccharomyces cerevisiae highly resistant to 2-amino-4-methyl-5-?-hydroxyethylthiazole (2-aminohydroxyethylthiazole), an antimetabolite of 4-methyl-5-?-hydroxyethylthiazole (hydroxyethylthiazole), has been isolated. Its resistance to 2-aminohydroxyethylthiazole was about 104 times that of the sensitive parent strain. The amount of thiamin synthesized in the cells of the resistant strain grown in minimal medium was less than half of that of the sensitive strain. The ability to synthesize thiamin from 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) and hydroxyethylthiazole in the resistant strain was low compared with that of the sensitive strain. These results were found to be due to a deficiency of hydroxyethylthiazole kinase in the resistant strain: in sonic extracts of cells the enzyme activity was only 0·67% of that of the sensitive strain. Although the cells of the sensitive strain could accumulate exogenous hydroxyethylthiazole in the form of hydroxyethylthiazole monophosphate, no significant uptake of hydroxyethylthiazole by the cells of the resistant strain was observed. The possibilities that 2-aminohydroxyethylthiazole monophosphate may be the actual inhibitor of the growth of Saccharomyces cerevisiae, and that hydroxyethylthiazole may not be involved in the pathway of de novo synthesis of thiamin via hydroxyethylthiazole monophosphate, are discussed.
-
-
-
Structural Analysis of Mycolic Acids from the Cell Wall Skeleton of Rhodococcus lentifragmentus AN-115
More LessThe structures of the mycolic acids from the cell wall skeleton of Rhodococcus lentifragmentus AN-115 were established. Field desorption mass spectrometry of underivatized mycolic acids showed that the molecular species of the mycolic acids were distributed from C38 to C50 and the major components were C44, C46 and C48 mono- and dienoic ?-hydroxy fatty acids. Pyrolysis gas chromatography-mass spectrometry confirmed that the mycolic acids consisted of a saturated straight C12 and C14 ?-chain and an unsaturated ?-chain, having mainly C29 and C31 carbons with one or two double bonds. The double bonds in the ?-chain were established as the cis isomers by nuclear magnetic resonance. The absolute configurations of the ?- and ?-asymmetric carbons were both established as R using molecular rotation data.
-
-
-
Regulation of Formaldehyde Oxidation by the Methanol Dehydrogenase Modifier Proteins of Methylophilus methylotrophus and Pseudomonas AM1
More LessThe modifier protein (M-protein) for methanol dehydrogenase (MDH) of the obligate methylotroph Methylophilus methylotrophus was purified almost to homogeneity (98%pure), characterized and shown to be similar to that previously described in the facultative methylotroph Pseudomonas AM1. It was a dimer of M r 140000, its isoelectric point was 5·6 and it showed no spectral absorbance above 320 nm. In the dye-linked MDH assay system, the M-proteins facilitated oxidation of alcohols not normally oxidized (1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol and 3-methylbutanol) by increasing the affinity of MDH for these alcohols. The effect of M-protein on the oxidation of formaldehyde was to decrease the affinity of MDH for formaldehyde by more than 97%, thus preventing any significant oxidation of formaldehyde. The M-protein also exerted its effect on the activity of MDH in the cytochrome-linked system of M. methylotrophus. The usual function of M-protein in methylotrophs is apparently to prevent formaldehyde oxidation. A dye-linked aldehyde dehydrogenase of wide specificity was partially purified and characterized; it failed to oxidize formaldehyde at a significant rate but it oxidized lactaldehyde at a rate sufficient to account for the oxidation of propanediol to lactate in whole bacteria.
-
- Development And Structure
-
-
-
Isolation and Characterization of the Tubular Organelles Induced by Fumarate Reductase Overproduction in Escherichia coli
More LessStrains of Escherichia coli amplifying the intrinsic membrane enzyme fumarate reductase accommodate the overproduced enzyme by increasing the amount of membrane material, in the form of intracellular tubular structures. These tubules have been observed in strains harbouring multicopy frd plasmids and in ampicillin hyper-resistant strains. A procedure has been developed for isolation of tubules nearly free of cytoplasmic membrane. Using protein A-gold labelling and optical diffraction of electron micrographs, a model for tubule structure is proposed. The tubules have a lower lipid/protein ratio than the cytoplasmic membrane, with the enzyme accounting for greater than 90% of the protein in the tubules. Both cytoplasmic membranes and tubules from amplified strains are enriched in cardiolipin and have a more fluid fatty acid composition than wild-type strains. Mutants defective in cardiolipin synthesis produce tubules in response to excess fumarate reductase, but these tubules have an altered appearance, indicating that lipid-protein interactions may be important for tubule assembly.
-
-
-
-
Polyoxin D Inhibits Colloidal Gold–Wheat Germ Agglutinin Labelling of Chitin in Dimorphic Forms of Candida albicans
More LessYeasts and mycelia of the pathogen Candida albicans grown in the presence of polyoxin D, a competitive inhibitor of chitin synthase, formed chains of swollen bulbous cells as observed by fluorescence microscopy. Wheat germ agglutinin (WGA) complexed to colloidal gold (Au) was used as a specific label at the ultrastructural level to visualize chitin in walls of control and polyoxin-treated cells. In control cells, Au-WGA labelling was preferentially localized in the innermost wall layers and was predominant at bud scars and septa. After 4·5 h in 4 mM-polyoxin D, budding in yeasts and lateral wall growth in mycelia continued, but primary septa failed to form and no Au-WGA labelling was detected in the walls. These results demonstrated that the morphological alterations caused by polyoxin D were due to the absence of chitin, a wall component important for formation of primary septa and for maintenance of structural integrity during morphogenesis.
-
-
-
Involvement of Ca2+/Calmodulin in Sexual Differentiation Induced by Mating Pheromone Rhodotorucine A in Rhodosporidium toruloides
More LessRhodotorucine A is a mating pheromone produced by mating type A cells of Rhodosporidium toruloides which induces mating tube formation in mating type a cells. Rhodotorucine A induced conspicuous changes in Ca2+ fluxes in the target cells which consisted of a very rapid, transient Ca2+ influx immediately after pheromone addition and a gradual Ca2+ accumulation during mating tube elongation. The magnitude of the pheromone-induced changes of Ca2+ fluxes was correlated with the efficiency with which the pheromone elicits biological responses in mating type a cells. Compared to vegetative growth, the response to the pheromone was highly sensitive to trifluoperazine, an inhibitor of calmodulin. Trifluoperazine inhibited exclusively the emergence and the elongation of mating tubes but not the signalling reaction.
-
-
-
Identification of a Diploid Self-sporulating Cycle in the Basidiomycetous Yeast Rhodosporidium toruloides
More LessSelf-sporulating diploid and aneuploid cells were identified in the life cycle of Rhodosporidium toruloides by microscopic observation of nuclear behaviour, microphotometry of DNA content, and genetic characterization of progeny. From a sexual cross between haploid A- and a-type strains, diploid progeny with both A and a mating-type loci and aneuploid progeny with either A or a mating-type locus were isolated in addition to haploid progeny. The diploid isolates propagated by budding in yeast form and eventually developed monokaryotic hyphae. The diploid hyphae formed uninucleate teliospores and blastospores from which diploid yeast cells were isolated (self-sporulating). Aneuploids carrying the A or a mating-type isolated from a sexual cross and from self-sporulating diploids entered the sexual cycle like haploid cells. The life cycle consisting of sexual and self-sporulating cycles is presented.
-
- Ecology
-
-
-
Chemotaxis of Actinoplanes missouriensis Zoospores to Fungal Conidia, Chlamydospores and Sclerotia
More LessThe chemotactic response of Actinoplanes missouriensis zoospores to three different types of fungal spores (conidia of Curvularia lunata, chlamydospores of Fusarium solani and sclerotia of Macrophomina phaseolina) and their exudates was examined in vitro and in soil. Zoospores were attracted in vitro to substances exuded by fungal spores and to a number of organic compounds but not to sodium phosphate buffer solution. Numbers of zoospores attracted to spores of C. lunata, F. solani or M. phaseolina and their exudates, mixed in a sterilized soil, were significantly (P = 0·05) greater than to soil only supplemented with buffer. Movement of zoospores in an unsterilized soil to conidia, chlamydospores or sclerotia was significantly greater (P = 0·05) than background populations occurring in soil held at 0, -10 and -50 mbar matric potential. Attraction of zoospores to fungal spores and (or) their exudates was generally in the order of conidia > chlamydospores > sclerotia both in vitro and in soil. The results suggest that compounds exuded by living fungal spores may act as attractant for motile Actinoplanes zoospores.
-
-
-
-
The Effect of Temperature on Recovery of Buoyancy by Microcystis
More LessColonies of Microcystis in Abbots Pool, Avon, UK, were found to regulate their buoyancy according to light (photon flux density). The autumnal decline of the population was associated with an increase in the proportion of colonies that were non-buoyant, and with declining temperatures in the pond. Non-buoyant colonies taken from the pond regained buoyancy in the dark rapidly at 20°C but only slowly at 12°C and below. A laboratory strain of Microcystis behaved in a similar manner. Comparisons of the behaviour of this organism placed at 8°C and 20°C were made; in high photon flux density buoyancy was lost at both temperatures due to accumulation of dense carbohydrate. When transferred to the dark cells at 20°C became buoyant again as carbohydrate was utilized and more gas vesicles were made; at 8°C much less carbohydrate was used and no increase in gas-vacuolation occurred. The failure to regain buoyancy in the dark at low temperatures accounts for the loss of buoyancy and sedimentation of the Microcystis in Abbots Pool.
-
- Genetics And Molecular Biology
-
-
-
Identification, Mapping, Cloning and Characterization of a Gene (sbmA) Required for Microcin B17 Action on Escherichia coli K12
More LessWe have identified mutations in three different chromosomal genes of Escherichia coli K12 which reduce sensitivity to microcin B17. Mutations in ompF and ompR genes affected production of an outer membrane porin protein, OmpF, and resulted in reduced sensitivity to a number of other agents (colicins, bacteriophages) besides microcin B17. The third class of mutants were specifically and highly resistant to microcin B17. The mutations in these strains were mapped to a gene (sbmA), located at 8·7 min on the E. coli K12 chromosome, which is closely linked to phoA. The wild-type sbmA allele was cloned into multiple copy number plasmids, and its location within the cloned DNA fragment was further defined by mutagenesis with MiniMudII1681. These insertion mutations resulted in in-frame fusions between the sbmA and lacZ genes, thereby allowing us to determine the direction of sbmA gene transcription. Plasmids carrying these gene fusions produced low levels of β-galactosidase, indicating that the sbmA gene is poorly expressed. We have been unable to identify the sbmA gene product, but indirect evidence indicates that it might be an envelope protein involved in microcin uptake.
-
-
-
-
Characteristics of the Developmental Cycle of Actinophage ϕC31
More LessSummary: Some characteristics of the lytic development of the temperate phage ϕC31 in Streptomyces coelicolor A3(2) were studied using a thermoinducible lysogen. The physiological state of the host and the culture medium influenced the production of progeny virus after induction. The latent period lasted 45 min and the rise period 20-30 min. RNA synthesis in induced cultures was reduced with respect to controls. This reduction was restricted to cellular transcription as evidenced by: (i) no stable RNA being synthesized in induced cultures, and (ii) the proportion of phage specific RNA increasing from 0·5% before induction to more than 30% in induced cultures. Host RNA synthesis proceeded throughout the lytic cycle. Protein synthesis was also reduced in induced cultures, although to a lesser extent than RNA synthesis. Phage DNA synthesis started at around 10 min postinduction, marking the division between the early and late periods of phage development. Host DNA synthesis occurred during the first 20 min after induction, and gradually decreased later.
-
-
-
RecE-dependent Lysogenic Induction in the Absence of Repressor in Bacillus subtilis Non-complementing Diploids
More LessThe RecE protein of Bacillus subtilis, known to be required for induction of the SOS response and of ϕ105 prophage, was shown to be involved in mitomycin C induction of B. subtilis diploid lysogens carrying a silent ϕ105 prophage in their unexpressed chromosome. These stable non-complementing diploid lysogens, formed by protoplast fusion and regeneration, did not synthesize repressor, so that the induction observed must have resulted from RecE-dependent activation of the prophage rather than from RecE-dependent inactivation of repressor. Mitomycin C treatment does not induce permanent expression of the silent chromosome, so the activation seems to be temporary, perhaps reflecting the action of an SOS function under RecE control.
-
-
-
The Isolation and Characterization of Bacteriophages Infecting Obligately Thermophilic Strains of Bacillus
More LessTwenty-four thermophilic bacteriophages have been isolated from diverse sources such as compost, soil, silage and rotting straw. Although considerable individual host specificity was observed, the phages were able to infect most of the major taxonomic groups of Bacillus thermophiles. The phages varied considerably in morphology and size; the phage heads were either cylindrical or polyhedral with tails varying in length between 15 and 500 nm. Most of the phages were stable at 50°C for 4-5 h but at 70°C the plaque-forming units decreased by between 102- and 107-fold in 2 h. The DNA of morphologically similar phages was examined by restriction enzyme analysis, and some differences in the DNA fragment patterns were found. Efficiency of plating data indicated that ‘B. caldotenax’ has a restriction and modification system. These phages may be valuable for the study of the genetics of thermophilic bacilli: transduction of ‘B. caldotenax’ and ‘B. caldovelox’ by phage JS017 has been observed.
-
-
-
Detection of an Integrated Tetracycline Resistance Plasmid in the Chromosome of Methicillin-resistant Staphylococcus aureus
More LessThe majority of multiresistant Staphylococcus aureus strains isolated in Australian hospitals since 1970 carry a chromosomally-encoded minocycline and tetracycline resistance determinant. By using DNA-DNA hybridization, some of these multiresistant strains were shown to possess also a tetracycline resistance plasmid, equivalent to pT181, integrated into a unique site in the chromosome. By relating the hybridization data to the map of pT181, the site of integration on this plasmid was established to be between the genes for replication and tetracycline resistance.
-
-
-
Expression of the Aspergillus nidulans argB Gene in Escherichia coli
More LessThe Aspergillus nidulans argB gene coding for ornithine carbamoyltransferase (OTCase) is not expressed in Escherichia coli. However, E. coli OTCase-deficient strains transformed with plasmids carrying the argB gene from A. nidulans reverted to prototrophy at a high frequency. In these derivatives the argB gene became functional due to DNA rearrangements upstream of the coding sequence. Two types of rearrangement were characterized. One was identified as an insertion of IS2. The second was a deletion that resulted in transcription of the argB gene from the TcR gene promoter and translation from a newly created ribosome-binding site formed at the junction between the A. nidulans and vector DNA sequences.
-
-
-
The Expression of the Escherichia coli lacZ Gene in Streptomyces
More LessThe Escherichia coli lacZ gene was stably introduced into ϕC31-based phage cloning vectors in Streptomyces lividans. However, lacZ could not be stably introduced into S. lividans on the plasmid vectors pIJ702 or pIJ41. Studies of the expression of lacZ in S. lividans and S. coelicolor were facilitated by the use of mutants and/or growth conditions in which endogenous β-galactosidase activity was low or absent. Plaques and lysogens of °C31::lacZ constructs involving transcriptional fusions to the pBR322 tet gene contained β-galactosidase activity. Activities were markedly higher in S. lividans than in S. coelicolor. Insertion of the major transcriptional terminator of coliphage fd between the tet promoter and lacZ reduced lacZ expression more in lysogens than in lytically infected cultures, suggesting the existence of a phage-specified anti-termination function. Several ϕC31 derivatives suitable for the construction of different kinds of transcriptional and translational fusions in Streptomyces were derived. The expression of lacZ in one transcriptional fusion vector (KC659) was increased both in plaques and in lysogens by the appropriately positioned insertion of a previously characterized promoter from the aph gene of S. fradiae. When ϕC31 DNA fragments were inserted into KC659, at least one recombinant phage gave high β-galactosidase activity in lytic infections, but not in lysogens. The potential usefulness of lacZ fusions in Streptomyces is discussed.
-
-
-
Cloning and Expression of the Succinyl-CoA Synthetase Genes of Escherichia coli K12
More LessThe genes encoding both subunits of the succinyl-CoA synthetase of Escherichia coli have been identified as distal genes of the suc operon, which also encodes the dehydrogenase (Elo; sucA) and succinyltransferase (E20; sucB) components of the 2-oxoglutarate dehydrogenase complex. The newly defined genes express polypeptides of 41 kDa (sucC) and 31 kDa (sucD), corresponding to the β and α subunits of succinyl-CoA synthetase, respectively. The genes are thus located at 16·8 min in the E. coli linkage map, together with the citrate synthase (gltA) and succinate dehydrogenase (sdh) genes, in a cluster of nine citric acid cycle genes: gltA-sdhCDABE-sucABCb. Four deletion strains lacking all of these citric acid cycle enzymes were characterized. The succinyl-CoA synthetase activities of strains harbouring plasmids containing the sucC and sucD genes were amplified some fourfold. Further emzymological studies indicated that expression of succinyl-CoA synthetase is coordinately regulated with 2-oxoglutarate dehydrogenase.
-
- Immunology
-
-
-
Immunochemical Properties of the Lipopolysaccharide O-Antigen of Vibrio cholerae O1 in Relation to Its Chemical Structure
More LessD-Glucuronic acid and D-glucosamine have an immunodominant role in the lipopolysaccharide (LPS) O-antigen of both the Ogawa and the Inaba subtypes of Vibrio cholerae O1. This was evident from the pronounced inhibitory effect on the LPS precipitin reaction demonstrated by these monosaccharides and by oligosaccharides containing either of them which were isolated from LPS hydrolysate. There was a considerable decrease in the antibody-combining capacity of chemically modified LPS in which the carboxyl group of the glucuronic acid had been reduced. Similarly, on deamination, the O-specific polysaccharide fraction of the LPS molecule from both subtypes completely lost the ability to precipitate the LPS antibody.
-
-
- Pathogenicity And Medical Microbiology
-
-
-
Separation of Legionella pneumophila Proteases and Purification of a Protease Which Produces Lesions Like Those of Legionnaires’ Disease in Guinea Pig Lung
More LessSix discrete protease activities were recovered from the supernatant broth of Legionella pneumophila cultures by ion-exchange chromatography. One of these demonstrated in vitro activity against collagen, casein and gelatin. When administered into the lungs of guinea-pigs this protease elicited lesions which were pathologically similar to those seen in clinical and experimentally induced Legionnaires’ disease.
-
-
-
-
Cell-surface Proteins of Streptococcus sanguis Associated with Cell Hydrophobicity and Coaggregation Properties
More LessIncubating cells of Streptococcus sanguis with sodium lauroyl sarcosinate, under conditions that did not cause lysis, solubilized material comprising 5-8% of the cell dry weight. The treatment reduced cell hydrophobicity, and reduced the ability of the cells to coaggregate with Actinomyces spp. The extract contained about 20 polypeptides and these were identified as being cell-surface components on the basis of one or more of the following criteria: (a) being degraded when cells were incubated with protease; (b) being labelled when cells were iodinated using a lactoperoxidase-catalysed reaction; (c) reacting with antibodies raised to fixed whole cells. Eight of the polypeptides accounted for more than 70% of the total protein extracted, and one component (molecular mass 16 kDa) was hydrophobic. The cell-surface proteins described are implicated in cell hydrophobicity and coaggregation.
-
-
-
Analysis of EDTA-soluble Cell Surface Components of Gram-positive Anaerobic Cocci
More LessThe protein content of EDTA extracts from 76 strains of Gram-positive anaerobic cocci was examined using SDS-PAGE. Strains of Peptostreptococcus anaerobius produced almost identical profiles; greater heterogeneity was observed within the species Peptococcus magnus, Peptococcus prevotii and Peptococcus asaccharolyticus, but several strains within each biotype produced similar patterns. Serological investigation of these extracts by ELISA revealed numerous cross-reactions among the different biotypes. Immunoblot transfers from polyacrylamide gels demonstrated two common antigens within strains of the species Ps. anaerobius, but these were not species-specific.
-
-
-
Antigenic Specificity of Human Antibody to Chlamydia in Trachoma and Lymphogranuloma Venereum
More LessAn understanding of the molecular basis of the humoral immune response to chlamydial infections in man requires the identification of target antigens to which antibodies are directed. The antigenic specificity of antibody from patients with lymphogranuloma venereum (LGV) or trachoma was therefore assessed by Western blotting. Surface polypeptides were first identified using purified chlamydial outer membrane complex as antigen. Antibodies in sera from patients with LGV but not from control negative sera reacted with a wide range of chlamydial surface polypeptides with molecular masses of 19, 29, 41, 58, 63 and 65 kDa. The major component of the antibody response detected by both immunoblotting and immunoprecipitation assay was directed against the major outer membrane protein (MOMP). Antibody to MOMP was species-specific on Western blotting, whereas antibody to several other polypeptides recognized common immunodeterminants on polypeptides of C. psittaci Cal-10 of equivalent molecular mass. Immunologically C. psittaci Cal-10 was more closely related to LGV strains of C. trachomatis than a guinea pig inclusion conjunctivitis strain of C. psittaci.
Trachoma sera collected from a village in southern Iran showed predominantly type-specific antibody on micro-immunofluorescence to serotype A or B trachoma agents. These sera showed a weak immune response to MOMP, a pronounced response to a polypeptide of 36 kDa and much less widespread reactivity with other chlamydial polypeptides. The lack of an immune response to SDS-stable immunodeterminants on MOMP might contribute to the susceptibility of trachoma patients to repeated cycles of ocular infection with chlamydiae.
-
-
-
Monoclonal Antibodies to Gonococcal Outer Membrane Protein I: Location of a Conserved Epitope on Protein IB
More LessHybrid cell lines have been derived which produce monoclonal antibodies reacting with outer membrane protein I from Neisseria gonorrhoeae strain P9. The antibodies obtained showed variable reactivity with other strains but one antibody recognized an epitope present on all of the strains tested which expressed the protease sensitive protein IB. Purified IgG labelled with 125I was used in competitive radioimmunoassays with unlabelled antibody to investigate the spacial distribution of the epitopes recognized. Each pair of antibodies showed some degree of inhibition. The relative magnitude of inhibition suggested that the conserved epitope lies within a variable region containing other epitopes which determine the antigenic specificity of the protein. Western blotting of peptides derived by proteolytic digestion of protein IB revealed that the conserved epitope is located close to the chymotrypsin cleavage site within a 7000 M r surface exposed region of the molecule.
-
-
-
Monoclonal Antibodies to Gonococcal Outer Membrane Protein IB: Use in Investigation of the Potential Protective Effect of Antibodies Directed against Conserved and Type-specific Epitopes
M. Virji, K. Zak and J. E. HeckelsSeveral monoclonal antibodies directed against gonococcal outer membrane protein IB have been used in in vitro assays to investigate their potential efficacy in protection against gonococcal infection. In a cytotoxicity assay, virulence of the variant P9-17 for epithelial cells in tissue culture was reduced in the presence of three of the four antibodies which recognized type-specific epitopes. Similarly, virulence of P9-17 as well as a recent isolate was reduced in the presence of the one antibody, SM24, which reacted with a conserved epitope. This antibody was also bactericidal in the presence of complement, and in addition was opsonic for several protein IB-expressing strains as determined by polymorphonuclear leucocyte chemiluminescence measurements. Similarly, all the type-specific antibodies were opsonic for P9 variants. However, only two of these antibodies mediated complement-dependent killing although those which were ineffective were nevertheless complement-fixing antibodies. These results indicate that antibodies to closely positioned epitopes on protein I vary in their biological activities and that the conserved epitope recognized by the antibody SM24 is potentially an effective target on the gonococcal surface for immunoprophylaxis.
-
-
-
Host Modification of the Adherence Properties of Chlamydia trachomatis
More LessThe adherence of Chlamydia trachomatis LGV440(L1) to human HeLa 229 and mouse McCoy cells was stimulated by the lectin wheat germ agglutinin (WGA) and inhibited by the sugars N-acetyl-D-glucosamine, N-acetyl-D-galactosamine and chitobiose, but only when the chlamydiae had been passaged several times in HeLa cells. After passage in McCoy cells, the lectin and the sugars elicited little response. The non-LGV serovar UW-31(K), however, differed from LGV440(L1) in that, regardless of passage, the lectin and sugar effects were observed only in HeLa cells. Affinity chromatography on WGA-agarose confirmed that HeLa-grown LGV-440(L1) bound to a significantly greater extent relative to McCoy-grown chlamydiae. In addition, participation of heterogeneous chlamydial ligands was suggested by the observation that the adherence of heated (60°C, 5 min) UW-31(K) to HeLa cells at 37°C was not inhibited at all, but at 5°C, the adherence rate was greatly reduced, indicating the participation of heat-stable as well as heat-labile ligands. These data are interpreted to indicate that the passage history of C. trachomatis results in the acquisition of altered surface components that participate in the initial interaction of the bacterium with the host.
-
- Physiology And Growth
-
-
-
Arginine Deiminase of Mycoplasma hominis: Cytoplasmic and Membrane-associated Forms
More LessMembrane and cytoplasmic fractions of Mycoplasma hominis inhibited the multiplication of this mycoplasma. Arginine deiminase (EC 3.5.3.6), isolated from both fractions, reproduced the inhibition. The purified cytoplasmic deiminase had a subunit M r of 49000, a specific activity of 53 units (mg protein)−1 and an A 280/A 260 ratio of 1·76. The membrane-associated enzyme had an identical M r but lower values for specific activity [39 units (mg protein)−1] and the A 280/A 260 ratio (1·46). In experiments in vitro, recent clinical isolates of M. hominis produced less arginine deiminase, but grew faster than the laboratory reference strain PG 21. In addition, other growth inhibitory components associated with membrane preparations were detected in recent clinical isolates but were absent from strain PG 21.
-
-
-
-
Effect of Cadmium on the Morphology, Membrane Integrity and Permeability of Pseudomonas putida
More LessCadmium-adapted Pseudomonas putida exhibited a long lag phase (6 h) on incubation in a defined medium containing 3 mM-Cd2+. During this time extensive blebbing of the outer membrane was observed by electron microscopy and polyphosphate granules containing Cd2+ were present in the cells. Cells from exponential-phase cultures of cadmium-adapted P. putida were found in clusters. They were much smaller than control cells grown without cadmium, and contained electron-dense aggregates. Cadmium-adapted cells released more lipopolysaccharide and protein into the external medium than did control cells; the addition of Ca2+, but not Mg2+, to the medium prevented this increased release. Cadmium-adapted cells also showed greatly increased sensitivity towards certain antibiotics, including the aminoglycosides, cyclic polypeptides and doxycycline. It is suggested that this is related to changes in membrane structure.
-
-
-
Effect of Growth Conditions on the Production, Composition and Viscosity of Xanthomonas campestris Exopolysaccharide
More LessA streptomycin-resistant variant of Xanthomonas campestris was grown in defined nutrient-deficient media in both batch and continuous culture. The production, composition and viscosity of the extracellular polysaccharide (xanthan) synthesized by this strain were influenced by the fermentation time and nutrient exhaustion in batch culture and by the dilution rate in continuous culture. The specific rate of exopolysaccharide synthesis was maximal during exponential growth in all of the nutrient-deficient media studied although some xanthan was formed during stationary phase. The concentration of exopolysaccharide decreased at later stages of stationary phase in some cultures. Both the extent of acylation and the consistency index of xanthan isolates were low or minimal during exponential growth, maximal in polysaccharide isolated as the growth rate fell and usually lower after this time. Between dilution rates of 0·03 and 0·06 h−1 in chemostat culture, the cell and exopolysaccharide dry weights were independent of dilution rate, the specific rate of xanthan synthesis decreasing at lower growth rates. Although the variation in the acyl content and consistency index was less than that observed in batch culture, exopolysaccharide isolated at higher dilution rates tended to have a higher acetyl content, lower pyruvyl content and lower consistency index.
-
-
-
Utilization of Aliphatic Amides and Nitriles by Nocardia rhodochrous LL100-21
More LessA wide range of nitriles and amides act as carbon and/or nitrogen sources for Nocardia rhodochrous LL100-21. Growth on a number of aliphatic nitriles or their corresponding amides resulted in the coinduction of both acetonitrile hydratase and acetamidase activities. In each case the specific activity of acetonitrile hydratase was higher than that of acetamidase. The substrate and inducer specificities were determined for the two enzymes and found to be quite distinct. Propionitrile, although an excellent inducer of both enzymes, was only slowly hydrolysed by acetonitrile hydratase. Conversely acrylonitrile was a good enzyme substrate but had a complete lack of effect as an inducer. Butyramide and methacrylamide acted as poor substrates for acetamidase but induced high levels of both acetonitrile hydratase and acetamidase. Dinitriles of long chain length, lactonitrile and iodoacetamide were inhibitory to growth and enzyme activity.
-
-
-
Production of N-Coronafacoyl-L-Amino Acid Analogues of Coronatine by Pseudomonas syringae pv. atropurpurea in Liquid Cultures Supplemented with L-Amino Acids
More LessNeutral L-amino acids were added to liquid cultures of Pseudomonas syringae pv. atropurpurea that were entering the exponential phase of growth. For each amino acid addition experiment the production of the usual coronafacoyl compounds, coronatine and N-coronafacoyl-L-valine, was diminished and a new product was detected. This was isolated and purified, and established by mass spectrometry and GC analysis of the amino acid released by acid hydrolysis to be the N-coronafacoyl amide of each L-amino acid added to the culture. Coupling was established to occur between coronafacic acid and the L-amino acids alanine, ?-aminobutyric acid, norvaline, isoleucine, alloisoleucine, leucine and norleucine. These results suggest that an intracellular enzyme system is operating which lacks an overall rigid specificity in the coupling (amide bond formation) between coronafacic acid and aliphatic neutral amino acids.
-
-
-
Cuticle-degrading Enzymes of Entomopathogenic Fungi: Regulation of Production of Chitinolytic Enzymes
More LessSynthesis of chitinase and chitosanase by the entomopathogenic fungus Metarhizium anisopliae is regulated by products of chitin and chitosan degradation through an inducer-repressor mechanism. Slow-feeding with sugars or alanine (about 20 μg ml−1 h−1) in a carbon deficient medium to prevent catabolite repression (restricted cultures) demonstrated that the most effective inducers of chitinase and chitosanase were the principal monomeric constituents of chitin (N-acetylglucosamine) and chitosan (glucosamine) respectively. Increasing the rate of release of N-acetylglucosamine decreased chitinase synthesis by about 87% while causing a sevenfold increase in growth. In batch cultures high chitinase activities were present only in chitin-containing medium. There was a negative correlation between accessibility and amount of chitin substrates, levels of free N-acetylglucosamine in culture fluids and chitinase production. Addition of carbohydrates, lipid or proteins to chitin-grown cultures repressed chitinase production. Basal levels of chitinase were produced in non-inducing media. Production of chitobiase (N-acetylglucosaminidase) was enhanced from high basal levels by amino sugars, but was less inducible and less susceptible to catabolite repression than chitinase.
-
-
-
Protein Degradation by Human Intestinal Bacteria
More LessAnalysis of human gut contents showed that substantial quantities of soluble protein, ammonia and branched chain volatile fatty acids occurred throughout the large intestine [0·1-24·4 g (kg contents)−1, 7·7-66·0 mmol (kg contents)−1 and 1·5-11·1 mmol (kg contents)−1 respectively]. The presence of these metabolites suggested that substantial proteolysis was occurring. In vitro studies showed that casein and bovine serum albumin were partly degraded in slurries of human faeces over a 96 h incubation period, to produce TCA-soluble peptides, ammonia and volatile fatty acids. Proteolytic activity detected in the stools of five individuals ranged from 3·5 to 19·8 mg azocasein hydrolysed h−1 (g faecal material)−1. Washed cell and washed particulate faecal fractions accounted for 24-67% of total activity. The predominant proteolytic bacteria in the faecal samples examined were identified as Bacteroides spp. [1·0 × 1011-1·3 × 1012 (g dry wt faeces)−1] and Propionibacterium spp. [1·2 × 108-1·0 × 1010 (g dry wt faeces)−1]. Other proteolytic bacteria which occurred in lesser numbers were identified as belonging to the genera Streptococcus, Clostridium, Bacillus and Staphylococcus. These results demonstrate that the gut microflora could potentially play a major role in proteolysis in the human colon.
-
- Systematics
-
-
-
On the Aetiology of Bovine Farcy in the Sudan
More LessFifteen strains of the agent of bovine farcy were isolated from lymph nodes of affected cattle. Quantitative analyses of mycolic acids revealed values that allowed the assignment of these strains to the genus Mycobacterium. The organisms bore a greater resemblance to Mycobacterium farcinogenes than to Mycobacterium senegalense.
-
-
-
-
Isolation and Preliminary Taxonomic Studies of Thermus Strains Isolated from Yellowstone National Park, USA
More LessForty-eight strains of Thermus isolated from hot springs in Yellowstone National Park, Wyomming, USA, and eight reference strains were subjected to a numerical taxonomic analysis using Gower's coefficient (SG ) with single and average linkage clustering. Two major groups were distringuished, which could be differentiated by colony morphology, ability to reduce nitrate and proteolytic activity. Cluster 1 contained Thermus aquaticus YT-1, the type strain of the species, and cluster 2 contained authentic strains of ‘T. flavus’ and ‘T. thermophilus’. T. ruber was recovered as a single member cluster. The mol% G + C of DNA from representative strains from each cluster was 64·4 to 66·8 for cluster 1, 62·2 to 67·1 for cluster 2 and 62·5 for T. ruber.
-
- Short Communication
-
-
-
Cloning of the Aconitase Gene (acn) of Escherichia coli K12
More LessLambda phages containing the aconitase gene (acn) of Escherichia coli K12 have been isolated by hybridization with an M 13 probe containing part of the aconitase gene (citB) of Bacillus subtilis. Aconitase specific activities are amplified 5- to 18-fold in thermally induced λacn lysogens and threefold in a strain transformed with a plasmid derivative (pGS181).
-
-
-
-
An Efficient Method for the Introduction of Viral DNA into Brevibacterium lactofermenturn Protoplasts
More LessA method for the introduction of a bacteriophage DNA into Brevibacterium lactofermenturn protoplasts is described. Frequencies of 105 infective centres per μg DNA were easily achieved, the relationship between the number of infective centres and the amount of DNA being linear up to 5 μg DNA per assay. This method can be used to introduce foreign DNA into these bacteria.
-
-
-
Aluminium Uptake by Anabaena cylindrica
More LessAtomic absorption spectrophotometry was used to characterize aluminium uptake by the cyanobacterium Anabaena cylindrica. An EDTA-washing procedure was used to distinguish between adsorbed and intracellular aluminium. The intracellular aluminium content increased with increasing external concentration and time. The phosphorus concentration in the growth medium did not affect the rate of aluminium uptake nor did dark treatment or addition of CCCP, an uncoupler of phosphorylation. We therefore conclude that aluminium toxicity is due to intracellular aluminium rather than to interactions with nutrients in the growth medium and that aluminium uptake is independent of phosphorus uptake. The accumulation of aluminium in polyphosphate granules and cell walls of phosphorus-rich cells noted earlier is rather due to an increased binding capacity in these cellular compartments. Also, the rapid uptake of aluminium by A. cylindrica mainly occurs via passive diffusion.
-
-
-
Phenotypic Expression of the Major 47 kDa Surface Immunogen of Treponerna pallidurn in Virulent, Tissue-cultured Treponemes
More LessSpecific monoclonal antibody and Western blot analysis were used to examine the phenotypic expression of the major 47 kDa surface immunogen of Treponerna pallidurn among organisms cultivated in oitro. Tissue-cultured treponemes synthesized the 47 kDa immunogen as well as, or better than, organisms cultivated in vivo (rabbit testicles).
-
Volumes and issues
-
Volume 170 (2024)
-
Volume 169 (2023)
-
Volume 168 (2022)
-
Volume 167 (2021)
-
Volume 166 (2020)
-
Volume 165 (2019)
-
Volume 164 (2018)
-
Volume 163 (2017)
-
Volume 162 (2016)
-
Volume 161 (2015)
-
Volume 160 (2014)
-
Volume 159 (2013)
-
Volume 158 (2012)
-
Volume 157 (2011)
-
Volume 156 (2010)
-
Volume 155 (2009)
-
Volume 154 (2008)
-
Volume 153 (2007)
-
Volume 152 (2006)
-
Volume 151 (2005)
-
Volume 150 (2004)
-
Volume 149 (2003)
-
Volume 148 (2002)
-
Volume 147 (2001)
-
Volume 146 (2000)
-
Volume 145 (1999)
-
Volume 144 (1998)
-
Volume 143 (1997)
-
Volume 142 (1996)
-
Volume 141 (1995)
-
Volume 140 (1994)
-
Volume 139 (1993)
-
Volume 138 (1992)
-
Volume 137 (1991)
-
Volume 136 (1990)
-
Volume 135 (1989)
-
Volume 134 (1988)
-
Volume 133 (1987)
-
Volume 132 (1986)
-
Volume 131 (1985)
-
Volume 130 (1984)
-
Volume 129 (1983)
-
Volume 128 (1982)
-
Volume 127 (1981)
-
Volume 126 (1981)
-
Volume 125 (1981)
-
Volume 124 (1981)
-
Volume 123 (1981)
-
Volume 122 (1981)
-
Volume 121 (1980)
-
Volume 120 (1980)
-
Volume 119 (1980)
-
Volume 118 (1980)
-
Volume 117 (1980)
-
Volume 116 (1980)
-
Volume 115 (1979)
-
Volume 114 (1979)
-
Volume 113 (1979)
-
Volume 112 (1979)
-
Volume 111 (1979)
-
Volume 110 (1979)
-
Volume 109 (1978)
-
Volume 108 (1978)
-
Volume 107 (1978)
-
Volume 106 (1978)
-
Volume 105 (1978)
-
Volume 104 (1978)
-
Volume 103 (1977)
-
Volume 102 (1977)
-
Volume 101 (1977)
-
Volume 100 (1977)
-
Volume 99 (1977)
-
Volume 98 (1977)
-
Volume 97 (1976)
-
Volume 96 (1976)
-
Volume 95 (1976)
-
Volume 94 (1976)
-
Volume 93 (1976)
-
Volume 92 (1976)
-
Volume 91 (1975)
-
Volume 90 (1975)
-
Volume 89 (1975)
-
Volume 88 (1975)
-
Volume 87 (1975)
-
Volume 86 (1975)
-
Volume 85 (1974)
-
Volume 84 (1974)
-
Volume 83 (1974)
-
Volume 82 (1974)
-
Volume 81 (1974)
-
Volume 80 (1974)
-
Volume 79 (1973)
-
Volume 78 (1973)
-
Volume 77 (1973)
-
Volume 76 (1973)
-
Volume 75 (1973)
-
Volume 74 (1973)
-
Volume 73 (1972)
-
Volume 72 (1972)
-
Volume 71 (1972)
-
Volume 70 (1972)
-
Volume 69 (1971)
-
Volume 68 (1971)
-
Volume 67 (1971)
-
Volume 66 (1971)
-
Volume 65 (1971)
-
Volume 64 (1970)
-
Volume 63 (1970)
-
Volume 62 (1970)
-
Volume 61 (1970)
-
Volume 60 (1970)
-
Volume 59 (1969)
-
Volume 58 (1969)
-
Volume 57 (1969)
-
Volume 56 (1969)
-
Volume 55 (1969)
-
Volume 54 (1968)
-
Volume 53 (1968)
-
Volume 52 (1968)
-
Volume 51 (1968)
-
Volume 50 (1968)
-
Volume 49 (1967)
-
Volume 48 (1967)
-
Volume 47 (1967)
-
Volume 46 (1967)
-
Volume 45 (1966)
-
Volume 44 (1966)
-
Volume 43 (1966)
-
Volume 42 (1966)
-
Volume 41 (1965)
-
Volume 40 (1965)
-
Volume 39 (1965)
-
Volume 38 (1965)
-
Volume 37 (1964)
-
Volume 36 (1964)
-
Volume 35 (1964)
-
Volume 34 (1964)
-
Volume 33 (1963)
-
Volume 32 (1963)
-
Volume 31 (1963)
-
Volume 30 (1963)
-
Volume 29 (1962)
-
Volume 28 (1962)
-
Volume 27 (1962)
-
Volume 26 (1961)
-
Volume 25 (1961)
-
Volume 24 (1961)
-
Volume 23 (1960)
-
Volume 22 (1960)
-
Volume 21 (1959)
-
Volume 20 (1959)
-
Volume 19 (1958)
-
Volume 18 (1958)
-
Volume 17 (1957)
-
Volume 16 (1957)
-
Volume 15 (1956)
-
Volume 14 (1956)
-
Volume 13 (1955)
-
Volume 12 (1955)
-
Volume 11 (1954)
-
Volume 10 (1954)
-
Volume 9 (1953)
-
Volume 8 (1953)
-
Volume 7 (1952)
-
Volume 6 (1952)
-
Volume 5 (1951)
-
Volume 4 (1950)
-
Volume 3 (1949)
-
Volume 2 (1948)
-
Volume 1 (1947)