- Volume 130, Issue 6, 1984
Volume 130, Issue 6, 1984
- Biochemistry
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A Possible Mechanism for the Cellular Coaggregation between Actinomyces viscosus ATCC 19246 and Streptococcus sanguis ATCC 10557
More LessThe cells of Actinomyces viscosus ATCC 19246 (Av 19246) and Streptococcus sanguis ATCC 10557 (Ss10557) coaggregated immediately after mixing in 40 mm-Tris/HCl buffer. Optimal conditions were pH 7·5 in the presence of Ca2+ at 0·1 mm or higher. Na2 EDTA and its analogues, Na2MgEDTA and Na2MnEDTA at 7·5 mm inhibited the coaggregation. Trypsin and heat treatment impaired the reactive site on Avl 9246 cells, but not on Ss10557 cells. The coaggregates, once formed, dissociated gradually during extended incubation at 37 °C; this was prevented by addition of sufficient Ca2+. The disaggregation appears to be a spontaneous denaturation of the proteinaceous reactive site on Av 19246 cell surface. Thus, the coaggregation involves the interaction of a lectin-like substance on the surface of Av 19246 with a carbohydrate site on Ss10557. Native Ss10557 cell walls possessed reactivity with Avl9246 cells but 5% (w/v) TCA-extracted cell wall residues did not. A carbohydrate moiety extracted from Ss 10557 exhibited a high potency in blocking coaggregation, and coaggregates were dissociated upon addition of the carbohydrate. Lactose, galactose and N-acetyl-d-galactosamine (the latter two are major constituents of the antigen extract) also significantly inhibited the coaggregation, but the other antigen components, glucose and rhamnose, did not. Relative inhibitory activity, expressed as molar potency, of carbohydrate antigen, lactose, galactose and N-acetyl-d-galactosamine respectively, was approximately 26 ×103:16:4:1. Ss10557 cells and cell walls reacted only with a Ricinus communis (castor bean) agglutinin-120 but not with Glycine max (soybean) agglutinin, Arachis hypogaea (peanut) agglutinin or Phaseolus vulgaris agglutinin (phytohaemagglutinin). These results suggest that the reaction site on Ss 10557 cells comprises a d-galactose-(1→4)β-d-glucose-sequence and that N-acetyl-d-galactosamine (an immuno-determinant of the streptococcus strain) appears not to be involved in the coaggregation reaction.
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Properties of Chitinase Activities from Mucor mucedo: Evidence for a Membrane-bound Zymogenic Form
More LessChitinase activities were detected in the supernatant and microsomal fractions from homogenized mycelium of surface-grown cultures of Mucor mucedo. Most properties of the two preparations were very similar, e.g. pH optima were 5·65 and 5·55, respectively, and K m values were 16·7 and 19·2 mg chitin ml−1, respectively. Neither activity had a cationic requirement, but both were inhibited by Cu2+Some properties differed. On freezing, the supernatant activity remained constant over 4 d and then gradually decreased, whereas microsomal activity increased, suggesting that endogenous activation was occurring. Prolonged incubations at 30 °C also allowed endogenous activation of microsomal activity, which could be prevented by incubation with phenylmethanesulphonyl fluoride. This enzyme activity could also be activated by treatment of the microsomes with proteases such as trypsin. Inactivation occurred after treatment with phospholipases, suggesting that microsomal chitinase requires phospholipid-enzyme interactions for activity. Thus the microsomal activity behaved as a membrane-bound zymogen.
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The Preliminary Characterization of the β-Glucosidases of Cellulomonas fimi
More Lessβ-Glucosidase activity in Cellulomonas fimi is strictly intracellular. Cell-free extracts contain at least two β-glucosidases. One of the enzymes is constitutive and it hydrolyses p -nitrophenyl-β-d-glucoside (PNPG) but not cellobiose. A second enzyme is induced about fourfold by growth on Avicel, and about sevenfold by growth on cellobiose; it hydrolyses both PNPG and cellobiose.
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Solubilization and Analysis of Mannoprotein Molecules from the Cell Wall of Saccharomyces cerevisiae
More LessPurified walls from Saccharomyces cerevisiae were treated chemically to release intrinsic mannoproteins. Boiling in 2% SDS gave the best results, although treatment in 6 M-urea at room temperature also released significant amounts of mannoprotein radioactivity. Triton X-100, sodium deoxycholate and EDTA were poor solubilizers. Electrophoretic patterns of SDS- or urea-released mannoproteins in SDS-acrylamide gels indicated a great heterogeneity of molecular species, with more than 60 bands. Zymolyase, a glucan-digesting complex, released about half of the mannoproteins, but these species showed an altered mobility on SDS-acrylamide gels and had a lowered capacity for precipitation by ethanol. Action of the enzyme on isolated walls was favoured by dithiothreitol, as is the case with whole cells, and repeated treatments with SDS and Zymolyase released all of the mannoproteins from the wall. Solubilizing treatments other than SDS had a differential effect on recently or formerly incorporated mannoproteins in the wall. The results suggest an asymmetrical arrangement of molecules in the envelope and point to dynamic changes inside the wall as it thickens as a result of cell aging.
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Isolation and Characterization of an Apurinic Endodeoxyribonuclease from the Anaerobic Thermophile Desulfotomaculum nigrificans
More LessAn endodeoxyribonuclease specific to apurinic sites in DNA was purified 12000-fold from vegetative cells of the anaerobic thermophilic bacterium, Desulfotomaculum nigrificans strain IFO 13698. The enzyme specifically hydrolyses phosphodiester bonds of apurinic double-stranded DNA, without action on normal, alkylated, or single-stranded depurinated DNA. The endodeoxyribonuclease has a molecular weight of about 18500 and a sedimentation value of 2·2S. The enzyme has an optimum pH of 7·5–8·0 and an optimum temperature of 55 °C. The half-life of purified enzyme is 55 min at 60 °C but it can be heated at 60 °C for at least 60 min without loss of activity in the presence of BSA. The purified enzyme has an absolute requirement for Mg2+ or Mn2+, and is inhibited by EDTA. Enzyme activity is completely inhibited by 0·5 m-NaCl or 1 mm-p-chloromercuribenzoate. All these properties differ from those of apurinic/apyrimidinic endodeoxyribonucleases isolated from Bacillus subtilis, Bacillus stearothermophilus. and Escherichia coli.
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- Biotechnology
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The Cellulase System of Cellulomonas fimi
Supernatants from cultures of Cellulomonas fimi contained up to 10 components with CM-cellulase activity as determined by non-denaturing PAGE. Some of the active components were glycosylated. The activity profiles of the supernatants, as determined by PAGE, varied with the cellulosic substrate used for the growth of a culture, with culture age, and with storage of the supernatants. These variations were a consequence of proteolysis and a reduction in the glycosylation of some of the components. Proteinase activity in the supernatants was induced by growth of C. fimi on cellulosic substrates. Proteolysis and a reduction in glycosylation resulted in the conversion of slow-moving into fast-moving components on non-denaturing PAGE. The fast-moving components had a reduced ability to bind to an insoluble cellulosic substrate such as Avicel. Several of the CM-cellulase activities in culture supernatants were immunologically related. In contrast to the large number of CM-cellulases found in the supernatant, substrate-bound activity comprised only three slow-moving components, at least some of which were glycosylated. It was concluded that the cellulase system of C. fimi was composed of only three enzymes, and that these enzymes had a great affinity for, and were stabilized by binding to, an insoluble cellulosic substrate. Enzymes which accumulated free in the culture medium were subject to limited proteolysis and de-glycosylation which generated a variety of products, some of which retained enzymic activity.
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Isolation and Characterization of Escherichia coli Clones Expressing Cellulase Genes from Cellulomonas fimi
A sensitive immunoassay developed previously was used to isolate a series of Escherichia coli clones expressing cellulase genes from Cellulomonas fimi. The clones fell into three groups. Clones in the first group contained plasmids with a 6·6 kb insert of C. fimi DNA, were strongly antigenic, and contained low levels of CM-cellulase activity. Those in the second group contained plasmids with a 5·0 kb insert, were weakly antigenic, and contained high levels of CM-cellulase activity. Those in the third group contained plasmids with a 5·6 kb insert, were weakly antigenic, and contained low levels of CM-cellulase activity. Restriction analysis showed that the three groups carried different cellulase genes. All of these CM-cellulase activities were exported to the periplasm in E. coli. but with different efficiencies. These activities represented important components of the C. fimi cellulase complex. Their properties indicated that they were different from each other and that they probably had complementary actions.
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- Development And Structure
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Ultrastructural Aspects of Autolysis of Pseudomonas fluorescens Induced by Osmotic Shock
More LessAutolysis of exponentially growing Pseudomonas fluorescens was induced by a rapid treatment with distilled water before treatment with 0·5 m-sodium acetate, pH 6·5, and incubation for 2 h at 30 °C. Electron microscopy revealed three stages of autolysis. In the first stage most of the cells were plasmolysed. In the second stage (from 5 to 20 min) the number of plasmolysed cells diminished, the profile of the outer membrane changed from asymmetric to symmetric, fractures appeared in the outer membrane and signs of peptidoglycan degradation became apparent. In the third stage (which began after 20 to 60 min) the ultrastructural alterations of the cell wall became more serious, the cytoplasmic membrane exhibited micromorphological lesions and progressive cellular disintegration occurred. The addition of chloramphenicol (100 μg ml−1) or tetracycline (4·5 μg ml−1), to either the culture or the suspensions of autolysing cells, did not significantly reduce the rate of autolysis. The presence of Ca2+ or Mg2+ (10 mM) in the autolytic medium inhibited autolysis and there were no significant ultrastructural alterations. The presence of 2 mm-N -bromosuccinimide (an inhibitor of bacterial autolytic enzymes) in the autolytic medium strongly inhibited autolysis of P. fluorescens;however, the bacteria incubated under these conditions showed significant ultrastructural alterations (extensive splitting of, and occasional fractures in, the outer membrane; cytoplasmic membrane and ribosomes not visible).
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A Method for Isolation of Protoplasts from Dermatophytes
More LessA method has been developed to isolate protoplasts from dermatophytes using Novozym 234. A simple technique of flotation in MgSO4 has been adapted to separate protoplasts from incubation mixture. Electron microscopic studies confirmed the absence of cell wall material on these protoplasts. The recovery of DNA from protoplasts was higher than from mycelia.
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The Effect of Calcium on Zoospore Differentiation in Phytophthora cinnamomi
More LessThe removal of divalent cations from suspensions of zoospores of Phytophthora cinnamomi decreased cell viability. When zoospores were exposed to combinations of cations, the degree of zoospore encystment was reduced to the mean level of that stimulated by the two cations acting separately. When Ca2+ was present in mM concentrations, any damaging effect of the second cation was reduced. In the absence of added K+ and Ca2+, the ionophores valinomycin and A23187 induced zoospore encystment within minutes. The addition of Ca2+ to zoospores treated with A23187 induced germination of the cysts. Strontium at 25 mM was as effective as Ca2+ in stimulating germination. The calmodulin-inhibitory drugs trifluoperazine, imipramine hydrochloride and dibucaine reduced zoospore viability and, with the exception of dibucaine, were ineffective at inducing encystment.
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- Genetics And Molecular Biology
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Use of Homocysteic Acid for Selecting Mutants at the gltS Locus of Escherichia coli K12
More Lessl-Homocysteic acid is toxic to Escherichia coli K12. Sensitivity to this compound is higher in cells which can utilize glutamate as sole carbon source via the Na+-dependent glutamate transport system. Such cells become resistant by mutation at the gltS locus. Sensitivity of both wild-type and glutamate-utilizing strains is greater if cells are growing on acetate as compared with glucose as major carbon source.
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Two Replication Determinants of an Antibiotic-resistance Plasmid, pTB19, from a Thermophilic Bacillus
More LessTwo different replication determinants were found on an antibiotic resistance plasmid, pTB19, from a thermophilic bacillus. One replication determinant (designated RepA) was functional only in Bacillus subtilis. whereas the other (designated RepB) functioned in both B. subtilis and Bacillus stearothermophilus. A deletion plasmid, pTB90, carrying the RepB derived from pTB19 coincidentally contained the specific 1·0 MDal Eco RI fragment of a cryptic plasmid pBSO2 from B. stearothermophilus. The presence of this 1·0 MDal Eco RI fragment in various deletion plasmids from pTB90 increased transformation frequencies for B. stearothermophilus 103 to 104 times and lowered plasmid copy numbers in the host strain to about one-tenth of those found for plasmids lacking this fragment.
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Electrophoretic Analysis of Proteins from Mycoplasma capricolum and Related Serotypes Using Extracts from Intact Cells and from Minicells Containing Cloned Mycoplasma DNA
More LessThe acidic proteins of six different mycoplasma serotypes causing bovine or caprine pleuro-pneumonia were compared by two-dimensional gel electrophoresis of extracts of 35S-labelled cells. The organisms investigated were Mycoplasma mycoides subsp. mycoides (PG1), M. mycoides subsp. mycoides (Y-goat), M. mycoides subsp. capri (PG3), M. capricolum (California kid), the unclassified bovine serogroup 7 of Leach (PG50) and the F38-like group (F38). The results suggested a close relationship between M. capricolum and F38 and a similarly close relationship between the different M. mycoides subspecies, whereas the two M. mycoides subspecies appeared to be quite distant from M. capricolum and F38. The representative strain of the bovine serogroup 7 of Leach was equally distant from F38, M. capricolum and the three strains of M. mycoides. Strikingly, all six mycoplasma strains apparently shared six proteins in the two-dimensional gels. In Escherichia coli minicells, DNA from strain PG50 cloned in the vector pBR325 gave rise to incorporation of radioactive label into proteins which were identified as mycoplasma proteins by two-dimensional electrophoresis and immuno-precipitation.
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Characteristics and Function of Thick and Thin Conjugative Pili Determined by Transfer-derepressed Plasmids of Incompatibility Groups I1, I2, I5, B, K and Z
More LessEleven transfer-derepressed plasmids from incompatibility groups I1, I5, B, K and Z were constructed using the dnaG3 mutant Escherichia coli strain BW86. All were found to determine thin flexible and thick rigid pili constitutively. Immune electron microscopy was used to relate thick and thin pilus serotypes with incompatibility grouping. Mutant plasmids that determined only thick pili constitutively transferred efficiently on an agar surface but not in a liquid, whereas plasmids with both kinds of pili transferred equally well in both environments. A mutant of the Incl2 plasmid R721 determined thin pili constitutively, and thick pili at a repressed level, as indicated by electron microscopy. Experiments with this indicated that thin pili were apparently not involved directly in conjugation but were only used to stabilize mating aggregates.
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Genetic Analysis of Fluorescent Pigment Production in Pseudomonas syringae pv. syringae
More LessGenes involved in the biosynthesis of a fluorescent pigment by Pseudomonas syringae pv. syringae (P. s. syringae) JL2000 were investigated. A genomic library of this strain was constructed using the broad host range cosmid vector, pLAFRl. Nonfluorescent (Flu−) mutants of JL2000, defective in the biosynthesis of a fluorescent pigment, were obtained after ethyl methanesulphonate mutagenesis. Individual recombinant plasmids from the genomic library were introduced into Flu− mutants. Of a total of 146 Flu− mutants, 36 were restored to fluorescence following matings with individual recombinant colonies in the genomic library. Four separate fluorescence restoration groups, each comprised of 5 to 11 Flu− mutants restored to fluorescence by one of four structurally distinct recombinant plasmids, were identified. Whereas the 36 Flu− mutant strains differed in their abilities to grow on an iron-deficient medium, wild-type P. s. syringae strain JL2000 and all Flu+ transconjugants from these 36 crosses grew on an iron-deficient medium. These results indicate that at least four genes or gene clusters are involved in the production of a fluorescent pigment of P. s. syringae strain JL2000. These genes, with few exceptions, also control the ability of strain JL 2000 to grow under iron-limiting conditions.
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Tryptophanase Synthesis in Escherichia coli: the Role of Indole Replacement in Supplying Tryptophan and the Nature of the Constitutive Mutation tnaR3
More LessThe properties of the tryptophanase constitutive mutation tnaR3 have been investigated. It has previously been reported that mutants carrying tnaR3 grow poorly on medium that selects for constitutive expression of tryptophanase. Our results now show that this poor growth can be explained by the inability of tryptophanase to catalyse the synthesis of L-tryptophan from indole, pyruvate and ammonia at a rate sufficient to allow normal growth. Improved media for the characterization of tryptophanase constitutive mutants are described. The mutation tnaR3 rendered tryptophanase synthesis constitutive (at a differential rate that at 37 C is 30% of the fully induced wild-type) and not further inducible. Diploid studies showed that tnaR3 is cis-dominant, but no evidence was found for any effect in trans. In addition to rendering tryptophanase synthesis constitutive, tnaR3 affects the response of tryptophanase synthesis to different growth temperatures.
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Genes Controlling Pigmentation in Nectria haematococca
More LessThe S1 homothallic wild-type strain of Nectria haematococca produces grey-purplish colonies on PDA medium, due to synthesis of fusarubin and related naphthazarins. Seventeen yellow, beige or white mutants were isolated. On the basis of recombination tests they were divided into six distinct chromosomal regions. Four of them, designated befB. befC. belB and belC. may be regarded as regulatory genes which uncouple the processes of ageing and pigmentation that are normally associated in the wild-type. The two others, alyA and yelJ-beiW. control the biosynthesis of naphthazarins. Mutations in alyA were found epistatic to yelJ and beiW mutations, indicating that the alyA locus acts on an earlier biosynthetic step than the yelJ -beiW region. The latter was shown to comprise two closely linked genes, yielding yellow or beige colours when mutated. Analysis of polar mutations suggests that they form a single unit of transcription. One of these two genes probably controls a late oxidation step in naphthazarin biosynthesis.
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Distribution of (l→3)-β- and (l→4)-β-Glucan Synthases Along the Hyphae of Saprolegnia monoica
More LessProtoplasts from hyphae of Saprolegnia monoica. collected from lytic medium after different times of incubation, represented a sequential fractionation of mycelium from apical to distal zones. This method gives the opportunity to study the intrahyphal localization of cell wall glucan synthases in relation to apical growth. (l→4)-β-Glucan synthases were mainly found in early protoplasts (i.e. from apical zones) while the highest (l→3)-β-glucan synthase activities were recovered in late protoplasts (i.e. from the subapical zones). Early protoplasts also exhibited the highest rate of cell wall synthesis during regeneration. The distribution of the enzymes along the fungal hyphae might provide a clue to the mechanisms of cell wall polysaccharide deposition during apical growth.
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The Isolation of λ Phage Carrying DNA from the Histidine and Isoleucine-Valine Regions of the Bacillus subtilis Chromosome
More LessFrom a λgtWES library of the chromosome of Bacillus subtilis. phages carrying DNA from the hisA and ilv-leu regions were isolated. They were identified by their ability to form complementing plaques on hisB. iluC or leuB mutants of Escherichia coli K12 under selective conditions and in the presence of a helper phage. The his phages complemented E. coli his A, B or D mutations and could transform seven mutations in the hisA region of the B. subtilis chromosome; each carried a single Eco RI insert of about 8·2 kb. Phages complementing E. coli ilvC or leuB mutations and carrying the equivalent B. subtilis genes ilvC and leuC transformed a range of mutations in the B. subtilis ilv-leu region. The distribution of genetic markers carried by the phages suggests that the entire ilv -leu cluster from azlA through leuD is covered in the collection of phages obtained and is carried in three Eco RI restriction fragments of approximately 6·7, 4·7 and 2·85 kb.
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Mobilization of Non-conjugative Plasmids into Rhodopseudomonas sphaeroides
More LessThe non-conjugative plasmids pAS9, RSF1010, pBR325 and pML21 were mobilized from Escherichia coli into Rhodopseudomonas sphaeroides by conjugative plasmids of the P-1 incompatibility group or their derivatives. The transfer of plasmids pAS9 and RSF1010 was rec, A -independent and both plasmids could be maintained in the autonomous state in R. sphaeroides cells. In contrast, the transfer of the plasmids pML21 and pBR325 based on the ColEl replicon was dependent on the recA status of donor strains. Replication and maintenance of these plasmids occurred as a result of the production of cointegrates formed by recombination between the mobilized and mobilizing plasmids.
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- Immunology
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Isolation of an Antigenic Oligosaccharide Fraction from Leptospira interrogans serovar canicola with a Monoclonal Antibody
E. Ono, M. Naiki and R. YanagawaAn oligosaccharide fraction containing the antigenic determinant of lipopolysaccharide antigen (TM antigen) from Leptospira interrogans serovar canicola. recognized by a monoclonal antibody (CT3) which agglutinates serovars canicola and broomi. was isolated by formic acid and successive sulphuric acid hydrolyses. Separation of the antigenic compounds was done by Bio-Gel P-2 and Sephadex G-25 gel filtration, and high-performance liquid chromatography with two different columns. The fraction finally obtained was a mixture of two oligosaccharides, both of which migrated as a single spot having a slightly higher mobility than an authentic tetrasaccharide (stachyose) on thin layer chromatography. The fraction contained rhamnose, arabinose and two major and two minor unknown sugars which were shown to be N- or O-acetylated and/or O-methylated sugars by nuclear magnetic resonance. The fraction inhibited the binding of CT3 antibody with TM antigen in enzyme-linked immunosorbent assay and microscopic agglutination of serovar canicola with the antibody. The inhibitory activity was destroyed by periodate oxidation or mild alkaline treatment, but was resistant to sodium borohydride reduction.
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- Pathogenicity And Medical Microbiology
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Ampicillin Inactivation in the Caecum of Axenic, Gnotoxenic and Conventional Lambs: Interaction with Resistant or Sensitive Escherichia coli
M. Coste, Ph. Gouet and L. EscoulaThe fate of orally administered ampicillin was studied in axenic lambs, in gnotoxenic lambs given a complex microflora and a mixture of ampicillin resistant and/or sensitive strains of Escherichia coli, and in conventional lambs. In axenic lambs or animals with a sensitive micro-flora, antibiotic concentrations of 500-1600 g ml−1 were detected in the intestine, and most of the ampicillin passed through the small intestine and entered the large intestine, within 12-15 h of administration. These antibiotic concentrations were sufficient to decrease the numbers of ampicillin-sensitive E. coli from 108 109 bacteria ml−1 to about 105-106bacteria ml−1 by 8 h after ampicillin administration. Second and third doses of antibiotic had no further effect on the bacterial count. Administration of ampicillin to animals hosting ampicillin-resistant E. coli resulted in a significant inactivation of the antibiotic in the intestine. As might be expected there was little reduction in the numbers of these organisms. These results are similar to those observed in conventional lambs hosting resistant E. coli as the dominant colibacillary flora.
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Adherence of Haemophilus influenzae to Monkey Respiratory Tissue in Organ Culture
More LessWe studied the adherence of Haemophilus influenzae to monkey respiratory mucosa using nasal turbinates maintained in organ culture. Adherence of capsulated and rough strains was not inhibited by monosaccharides, sucrose, human albumin, foetal calf serum or polyribophos-phate. However, antisera directed against surface components decreased bacterial adherence. Although variation in adherence capacity in individual strains was observed there was no correlation with capsulation, anatomical site of strain isolation or biotype. Bacterial surface structures other than capsular material appear important in effecting upper respiratory tract colonization.
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Immunocytochemical Labelling of Bacteroides nodosus Pili Using an Immunogold Technique
More LessAn immunogold labelling technique was used to label the pili of the bacterium Bacteroides nodosus. The labelling was distinct and highly specific, and individual pili could be recognised beneath the gold probe. The labelling of somatic antigens could be distinguished from that of pilus antigens. Furthermore, labelling of fragments of cytoplasm released by cell lysis and trapped in the pili could be distinguished from pilus labelling. An antiserum that had been raised against strain 80200 (serotype N) labelled pili of strain 215 (serotype B). Double labelling experiments with this antiserum and the antiserum against strain 215 (serotype B) showed that both antisera label the same pili bundles. The ease of detection of the immunocytochemical reaction should enable this technique to be used as a routine screen for pilus antigens. It also possesses the potential for much wider applications for immunolabelling other antigens, such as viruses, that can be obtained in suspension.
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- Physiology And Growth
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Temperature-sensitive β-Lactam-tolerant Mutants of Escherichia coli
Seven temperature-sensitive penicillin-tolerant mutants of Escherichia coli strain LD5 (thi lysA dapD) were isolated and characterized. Treatment with β-lactams caused lysis of the mutants at 30 °C. Although growth of the mutants at 42 °C was inhibited by β-lactams, no lysis occurred. The mutants were also slightly tolerant to d-cycloserine at 42 °C but lysed readily when deprived of diaminopimelate or when treated with moenomycin. The minimum inhibitory concentrations of various antibiotics were the same for the mutants and their parent. The mutations conferring penicillin tolerance were phenotypically suppressed in the presence of a variety of compounds which may act as chaotropic or antichaotropic agents. No defects in penicillin-binding proteins and peptidoglycan hydrolases were detected. Temperature-resistant revertants of the mutants were no longer tolerant to penicillin-induced autolysis at 42 °C. The mutations in five isolates were localized to the 56 to 61 min region of the E. coli linkage map and to the 44 to 51 min region in the case of two other isolates.
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Arginine Uptake by a Psychrophilic Marine Vibrio sp. During Starvation-induced Morphogenesis
More LessThe effects of starvation on the arginine transport system in ANT-300, a psychrophilic marine Vibrio sp., were investigated. A rapid rate of uptake was detected on the first day of starvation, followed by a much lower but constant rate of uptake until day 35. Transport was found to require maintenance of a proton gradient across the membrane, but not to involve ATP. Respiration of the transported arginine increased during the 35 d of starvation. Thus, ANT-300 is able to maintain a high-affinity, active transport system for arginine during extended periods of starvation even in the absence of an exogenously supplied energy source.
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Growth and Sugar Metabolism of a Thermoaddophilic Iron-oxidizing Mixotrophic Bacterium
More LessMixotrophic growth of a thermoacidophilic iron-oxidizing, facultatively autotrophic bacterium is described. Rapid growth with sucrose, fructose, glucose and ribose required the concurrent oxidation of ferrous iron, which, it was concluded, provided most of the energy required for biosynthesis. Specific iron oxidation rate during growth and biomass production depended on the sugar supplied. About 20% of the cell carbon was obtained from CO2-fixation during growth on glucose. The presence of ribulosebisphosphate carboxylase indicated this to be fixed by the Calvin cycle. The kinetics and inhibition of glucose transport indicated uptake by diffusion and by energy-dependent processes. Sensitivity to fluoride indicated the possible involvement of a phosphoenolpyruvate-phosphotransferase system. Radiorespirometry, using specifically labelled glucose molecules, demonstrated that glucose was oxidized by the oxidative pentose phosphate cycle with further oxidation of glyceraldehyde 3-phosphate by means of the tricarboxylic acid cycle. Dehydrogenases for glucose 6-phosphate and 6-phosphogluconate were present but 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase were not detected in cell-free extracts, showing absence of the Entner-Doudoroff pathway.
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Origin and Fate of the Lysophosphatidylethanolamine in a Chain-forming Mutant (envC) of Escherichia coli
More LessThe role of phospholipid metabolism in the functioning of the bacterial envelope was investigated in the chain-forming Escherichia coli envC. Lysophosphatidylethanolamine (LPE) which accumulated in this strain during growth was indentified as the product of phosphatidylethanolamine (PE) hydrolysis by a phospholipase A1, i.e. 2-acylLPE. Isotopically labelled LPE transferred into intact mutant and parent cells by liposome/bacteria interaction was rapidly reacylated to PE. However, in envC the final PE/LPE ratio was lower than that in the parent, thus showing that the fate of LPE is modified. Crude cell extracts degraded LPE to a lesser extent in envC than in the parent but were unable to promote reacylation activity under our experimental conditions. In both strains, the lysophospholipase activity was neither calcium-dependent nor inhibited by the SH-group inhibitors p HMB or p CMPS, and hydrolysed 1-acylLPE as well as 2-acylLPE. These results indicate the existence of a deacylation-reacylation cycle in E. coli and show that this cycle is perturbed in envC cells, especially at the lysophospholipase step.
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Binding Sites for Bacterial Flagella at the Surface of the Soil Amoeba Acanthamoeba
More LessAcanthamoeba castellanii (CCAP 1534/3) was found to bind avidly the common soil bacterium Pseudomonas fluorescens. This adhesion was mediated not by pili nor by the general bacterial surface but by the polar flagella. Because of the nature of the flagellar rotary motor, the cell bodies of the attached bacteria could be seen rotating clearly. While initially bacterial binding occurred uniformly over the cell membrane of Acanthamoeba. the bacteria were soon swept posteriorly to form a cap and either endocytosed or sloughed off, still agglutinated by their flagella. Such capped amoebae would not bind Pseudomonas if challenged immediately, indicating a depletion of flagella-binding sites. The bacteria could not bind to amoebae pre-treated with concanavalin A (Con A) even after the lectin had been capped to the uroid. However, capping of flagella-binding sites did not co-cap all the Con A-binding sites on the surface of the amoeba. The flagella-binding sites were not affected by pre-treatment with Pronase (1 mg ml−1) or anti-Acanthamoeba surface antibody. Proteus mirabilis also bound avidly by its flagella to Acanthamoeba and, furthermore, competition experiments suggested that Proteus and Pseudomonas adhere to a common surface site on the amoeba. The presence of sites on the cell membrane of A. castellanii that are specific for flagellin would enhance strongly the adsorption of motile bacteria prior to endocytosis. This would represent an excellent feeding strategy for a soil-dwelling phagotroph.
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Studies on Protein Synthesis in Kappa Particles
More LessFurther evidence that kappa endosymbionts of stock 7 Paramecium biaurelia[Caedibacter varicaedens] are bacteria is given by the discovery of a prokaryotic translational system in these particles. Kappa proteins incorporate 35S in in vivo experiments. However, there is no evidence that the translational systems of the host paramecia and the symbionts are shared. On the contrary, experiments with symbiont-bearing and symbiont-free strains strongly indicate that all major proteins found in kappa are synthesized in kappa. In this respect, kappa clearly differs from cell organelles. Protein synthesis in kappa is not inhibited by cycloheximide and the aminoglycoside G418, which do inhibit, although not completely, protein synthesis of the host cells. On the other hand, chloramphenicol quantitatively blocks translation in kappa without any obvious effects on the translational system of the paramecium cytoplasm.
Stock 7 kappa was isolated and purified using a filter paper column. The purity of kappa preparations thus obtained was significantly improved by adding a preliminary deciliation step.
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Carbon Source Regulation of Nicotinamide Adenine Dinucleotide (Phosphate) Glycohydrolase in Neurospora crassa: Induction and Repression of Enzyme Synthesis
More LessSynthesis and release of NAD(P)ase by Neurospora crassa wild type was studied in experiments in which mycelia grown in Vogel minimal medium were transferred to media containing protein as the only carbon source. Several results are presented suggesting that the NAD(P)ase may be induced by the presence of protein in the culture medium. Low concentrations of sucrose or glucose (0·1%), Casamino acids or some amino acids such as methionine, cysteine, phenylalanine and tryptophan strongly repressed the enzyme synthesis. Under induction conditions NAD(P)ase and alkaline protease appeared together in the culture medium. It would appear that NAD(P)ase and alkaline protease are coordinately regulated by a common control mechanism related to carbon catabolism.
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An Investigation into the Recycling of Gas Vesicle Protein Derived from Collapsed Gas Vesicles
More LessGas vesicle protein may account for more than 6% of the cell dry weight and over 10% of the total cell protein in the planktonic cyanobacterium Anabaena Jlos-aquae. The gas vesicles collapsed by rising cell turgor pressure during buoyancy regulation can therefore represent a significant protein pool within this organism. We have looked for evidence of gas vesicle protein recycling. Gas vesicles were isolated from cells of A.80s-aquae that had been pressurized and then cultured in a radioactively labelled medium. The new gas vesicles that formed were less highly labelled than the new vesicles formedin cells from a second culture that had not been pressurized. The results suggest that the collapsed gas vesicles are disaggregated and that the constituent gas vesicle protein is reassembled into new gas vesicles. Proof of this will require experiments using in vitro assembly systems.
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- Systematics
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Distinctive Electrophoretic and Isoelectric Focusing Patterns of Esterases from Yersinia enterocolitica and Yersinia pseudotuberculosis
Ph Goullet and B. PicardEsterases of 53 strains of Yersinia enterocolitica sensu stricto. including five previously defined biotypes, and 30 strains of Yersinia pseudotuberculosis were analysed by horizontal poly-acrylamide-agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. Esterase bands were defined by their range of activity towards several synthetic substrates, their resistance to heat and to di-isopropyl fluorophosphate. The two species were characterized by distinct electrophoretic patterns of their esterases. The apparent molecular weights of the heat-resistant esterase of Y. enterocolitica and of the major heat-resistant esterase of Y. pseudotuberculosis. as determined by polyacrylamide gradient gel electrophoresis, were estimated to be 52000 and 250000, respectively. On the basis of electrophoretic mobilities and isoelectric points of esterases produced by strains of Y. enterocolitica. five principal zymotypes were observed: two for strains of biotype 1, two for strains of biotypes 2 and 3, respectively, and only one for strains of both biotypes 4 and 5. The zymotypes of strains of biotypes 2, 3, 4 and 5 appeared to be more closely related to one another than to zymotypes of strains of biotype 1. Variations in number or mobility of bands observed within each biotype of Y. enterocolitica and within some serotypes of Y. pseudotuberculosis could represent an additional marker for epidemiological analysis.
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