- Volume 132, Issue 4, 1986
Volume 132, Issue 4, 1986
- Biochemistry
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Extracellular Proteases in Erwinia chrysanthemi
More LessExtracellular proteolytic enzyme activity has been detected in cultures of Erwinia chrysanthemi. This activity, which appears when the cells are grown in the presence of peptides, is rather unstable. A hyperproteolytic mutant was isolated which produces two proteases of apparent polypeptide molecular mass of 50 and 55 kDa, respectively. The 50 kDa protease, which is produced in the largest amounts, has been purified to near homogeneity. It has the properties of a neutral serine protease. The 50 and 55 kDa proteases are unrelated antigenically. Preliminary evidence suggests that both proteases are also produced by the wild-type strain, but that they are either produced in much smaller quantities or are much less stable.
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A Study of Derepression of NAD-specific Glutamate Dehydrogenase of Neurospora crassa
More LessTransfer of Neurospora crassa mycelium from a 1% (w/v) sucrose medium to carbon-free or 1% (w/v) glutamate medium results in the onset of derepression of the catabolic NAD-specific glutamate dehydrogenase (NAD-GDH), within 30 min of the shift. Immunoprecipitation of in vivo pulse-labelled NAD-GDH demonstrated that this enzyme was synthesized de novo, correlating with increasing enzyme activity in shifted cells. Derepression was shown to be under transcriptional control by using the RNA synthesis inhibitor, picolinic acid, and by immunoprecipitation of the in vitro translation products of poly(A)-containing mRNA from repressed and derepressed cells. A brief (5 min) shift to derepression medium followed by a return to 1% (w/v) sucrose medium was sufficient to trigger synthesis of abundant NAD-GDH transcripts and low levels of the active enzyme. A secondary level of translational control is proposed to account for the discrepancy between the detectable levels of NAD-GDH transcripts and protein, following transient derepression.
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Energy-dependence of the Assimilatory Nitrate Uptake in Azotobacter chroococcum
More LessNitrate assimilation by suspensions of Azotobacter chroococcum, as determined by the disappearance of the ion from the external medium, displayed saturation kinetics, was inhibited by nitrite, and exhibited an affinity for nitrate higher than that of nitrate reductase. This suggests that the entry of nitrate into the cell is mediated by a specific transporter. Nitrate assimilation required a readily utilizable carbon source and aerobic conditions and was blocked by the uncouplers carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol (DNP) but not by N, N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of ATPases. The inhibition of nitrate assimilation in the absence of an appropriate carbon source was overcome by the non-physiological energy source ascorbate plus N-methylphenazinium methylsulphate (PMS), a substrate combination that allowed respiration. Though an ATP-dependent nitrate uptake mechanism cannot be ruled out, these data suggest that transport of nitrate into the cell is directly dependent on the proton electrochemical gradient across the cytoplasmic membrane.
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Glutamate Excretion Mechanism in Corynebacterium glutamicum: Triggering by Biotin Starvation or by Surfactant Addition
More LessCells of an industrial strain of the l-glutamate producer Corynebacterium glutamicum grown in biotin-starvation conditions lost 30% of their membrane phospholipids. This was accompanied by a progressive decrease in the rate of accumulation of glutamate and of the intracellular glutamate accumulated at the plateau. Addition of an acylated surfactant to growing cultures of the same strain also induced a progressive loss of glutamate uptake, without changing the K T of the process. The surfactant-treated cells almost completely excreted the labelled glutamate loaded during a preincubation. These results, together with those obtained previously, allow us to propose that glutamate excretion is mediated by a glutamate permease, after uncoupling of this uptake system resulting from a marked loss of membrane phospholipids. This loss of phospholipids can be induced either by biotin starvation or by addition of an acylated surfactant to growing cells.
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- Development And Structure
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Monoclonal Antibodies to Cell Surface Antigens Involved in Sex Pheromone Induced Mating in Streptococcus faecalis
More LessHybridoma cell lines producing monoclonal antibodies to Streptococcus faecalis cell surface antigens were constructed. Some of the antibodies isolated were directed against surface components involved in pheromone-induced mating. This paper describes the use of the monoclonal antibodies to identify antigenically related surface components detected by immunoprecipitation and Western blotting, their use in pheromone response assays, and their use as functional inhibitors in mating experiments.
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- Ecology
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A Comparative Study of the Biodegradation of the Surfactant Sodium Dodecyltriethoxy Sulphate by Four Detergent-degrading Bacteria
More LessSummary: The 35S-labelled metabolites produced during biodegradation of sodium dodecyltriethoxy [35S]sulphate (SDTES) by four bacterial isolates were identified and quantified. All four isolates used ether-cleavage as the predominant primary degradation pathway. In two of the organisms, the etherase system (responsible for approx. 60–70% of primary biodegradation) liberated mono-, di- and triethylene glycol monosulphates in substantial proportions, the last two esters undergoing some further oxidation to acetic acid 2-(ethoxy sulphate) and acetic acid 2-(diethoxy sulphate), respectively. For these isolates, liberation of SO4 2- directly from SDTES was also significant (30–40%) and the organisms were shown to contain alkylsulphatases active towards SDTES. For the remaining two isolates, etherase action was even more important (responsible for <80% of primary biodegradation) and was restricted almost totally to the alkyl–ether bond to generate mainly triethylene glycol sulphate, some of which was further oxidized. Very small amounts of diethylene glycol monosulphate were also produced, but its mono-homologue, and the oxidation products of both these esters, were absent. Small amounts of inorganic sulphate (approx. 10%) were liberated by these isolates and one of them also produced compounds tentatively identified as intermediates of βoM-oxidation.
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Metabolite Production during the Biodegradation of the Surfactant Sodium Dodecyltriethoxy Sulphate under Mixed-culture Die-away Conditions
More LessSodium dodecyltriethoxy sulphate (SDTES), either pure or as a component of commercial surfactant mixtures, underwent rapid primary biodegradation by mixed bacterial cultures in OECD screen and river-water die-away tests. Inoculation of [35S]SDTES-containing solutions with OECD screen test media acclimatized to surfactants or their degradation products led to production of various 35S-labelled glycol sulphates and their oxidation products, all known to occur during degradation of [35S]SDTES by pure bacterial isolates. Triethylene glycol monosulphate was the major catabolite together with smaller amounts of di- and monoethylene glycol monosulphates implying, by analogy with pure cultures, that ether-cleavage was the major primary biodegradation step. The oxidation product (carboxylate derivative) of each glycol sulphate was also detected together with metabolites tentatively identified as Ω-/β-oxidation products of the dodecyl chain. Relatively little SO4 2- was liberated directly from SDTES but mixed cultures derived from sewage could metabolize the sulphated glycols to SO4 2-. The environmental relevance of these degradation routes was established by following metabolite production from [35S]SDTES in full-scale river-water die-away tests. Triethylene glycol sulphate was formed first, then rapidly oxidized to acetic acid 2-(diethoxy sulphate) which persisted as the major metabolite for 2–3 weeks. Small amounts of sulphated derivatives of di- and monoethylene glycols were also detected during the same period. Very little SO4 2- was formed directly from SDTES but large amounts accompanied the eventual disappearance of glycol sulphate derivatives. None of the 35S-labelled organic metabolites was persistent and, whenever [35S]SDTES was a component of a commercial mixture, all ester sulphate was completely mineralized to 35SO4 2- within 28 d.
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- Genetics And Molecular Biology
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Isolation and Characterization of Ca2+-sensitive Mutants of Saccharomyces cerevisiae
More LessThirty Ca2+-sensitive (cls: calcium sensitive) mutants of Saccharomyces cerevisiae were isolated by replica-plating. These mutants, which each had a single recessive chromosomal mutation, were divided into 18 complementation groups. Some cls mutants showed a phenotype of specific sensitivity to Ca2+, while others showed phenotypes of sensitivities to several divalent cations. From measurements of the calcium contents and initial rates of Ca2+ uptake of the cls mutants, 16 of the 18 cls complementation groups were classified into four types: type I mutants (cls5, cls6, cls13, cls14, cls15, cls16, cls17, and cls18) had both elevated calcium contents and increased uptake activities. A type II mutant (cls4) had a normal calcium content and normal uptake activity; type III mutants (cls1, cls2 and cls3) had elevated calcium contents but normal initial rates of Ca2+ uptake; type IV mutants (cls8, cls9, cls10 and cls11) had normal calcium contents but increased initial rates of Ca2+ uptake. Two of the mutants (cls7 and cls12) had intermediate biochemical properties. The primary defects of these four types of cls mutants were considered in terms of the Ca2+ transport system(s). Both type I and type III mutants, which had elevated calcium contents, simultaneously showed a trifluoperazine-sensitive phenotype, suggesting a close correlation of this phenotype with elevated calcium content. In addition, all type IV mutants were unable to utilize nonfermentable sugars. One CLS gene, CLS7, was located on the left arm of chromosome V.
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An Inverted Repeat Sequence of the IncFI Plasmid ColV2-K94 Increases Multimerization-mediated Plasmid Instability
More LessA detailed physical map of the region of the IncFI plasmid ColV2-K94 containing the Rep1 replicon, a Tn903 transposon, and an inverted repeat structure (X1) with unknown properties was prepared by cloning restriction fragments into pBR325. Inserts carrying the 1·2 kb repeated sequence of X1, but not the IS903 sequence of Tn903, had a destabilizing effect on pBR325 and pBR322 plasmid maintenance. One of these derivatives, pWS139, was studied further and was shown to have elevated levels of multimeric DNA forms; this resulted in decreased copy number and plasmid instability, as multimerization reduces the effective number of randomly segregating plasmids per cell. A ColV2-K94 miniplasmid, which has a copy number much lower than that of ColE1-derived vectors, was also less stably inherited if it contained the X1 structure. This destabilizing effect of the X1 repeat sequence was dependent on the RecA function, but not the RecB or the RecC functions of the host. These results suggest that the inverted repeat sequence of the X1 structure serves as a ‘hot-spot’ for generalized recombination. Thus, when present in cis, this sequence can generate plasmid instability because plasmid molecules are readily converted into multimeric forms through enhanced recombination at this site.
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No Correlation Exists between the Conjugative Transfer of the Autotrophic Character and That of Plasmids in Nocardia opaca Strains
More LessA new isolate of Nocardia opaca was obtained by enrichment culture for aerobic lithoautotrophic growth on CO2 and H2. This strain, MR22, is very similar to N. opaca MR11 (formerly 1b) in functioning as a donor for genetic information determining the ability to grow lithoautotrophically (Aut character) in matings with Aut- strains of N. opaca or closely related heterotrophic species. The strain contains a plasmid, pHG33 of about 110 kb. A mutant was isolated from strain MR22 which was plasmid-free, and had lost the Aut character, resistance to 50 μm-thallium salt and susceptibility to the nocardia-specific bacteriophage ϕBl. As a recipient of the Aut character, this plasmid-free mutant was as well suited as plasmid-bearing Aut- strains of N. opaca. In matings with the mutant as recipient the frequency of Aut+ transconjugants per donor was 3 x 10-4 with N. opaca MR11 (pHG31-a, Aut+, Tlr, Strs, ϕBls) and 2 x 10-3 with N. opaca MR22 (pHG33, Aut+, Tlr, Strs, ϕBlr) as donor. Phenotypic characterization of the transconjugants, which had been selected for the Aut marker, revealed that in many cases the Aut marker had been transferred without plasmid transfer. Furthermore, plasmid-free, Aut+ transconjugants functioned as donors for the Aut marker. Both plasmid-free and plasmid-bearing transconjugants transferred the Aut marker to the Aut- strains of N. opaca with a frequency which was one or two orders of magnitude higher than that of the wild-type strains. The plasmids pHG31-a and pHG33 code for thallium resistance (50 μm-thallium acetate). The frequency of thalliuM-resistant transconjugants was 10-1 to 10-2 per donor; all thalliuM-resistant transconjugants contained the donor plasmid.
We conclude that the plasmids pHG31-a of strain MR11 and pHG33 of strain MR22 of N. opaca carry the genetic information for thallium resistance but not the Aut character. As plasmid-free Aut+ strains can function as donors the Aut character is assumed to reside on the chromosome and to function as an independent self-transmissible genetic element.
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Location of the Site-specific Recombination System of R46: a Function Necessary for Plasmid Maintenance
More LessThe R46 site-specific recombination system comprises a per (plasmid-encoded recombinase) gene and a site at which the gene product acts, the per site. The two functions have been cloned into pACYC184. They are encoded by sequences within a region of approximately 2 kb on the R46 genome. These R46 sequences are closely related to the site-specific recombination systems of the ampicillin resistance transposons collectively designated TnA. The R46 per function is interchangeable with the tnpR gene product of TnA. Both enzymes can mediate recombination between the related res and per sites in R46::TnA recombinant plasmids to generate site-specific deletions and inversions. Similar DNA rearrangements occur when TnA inserts into pACYC184 derivatives carrying the cloned R46 per functions. Carriage of this site-specific recombination system contributes to the stable maintenance of R46. By converting plasmid dimers to monomers the R46 per functions help to ensure equal partitioning at cell division.
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Characterization and Incompatibility Properties of ROM- Derivatives of pBR322-based Plasmids
More LessWe have characterized a copy number mutant of the pBR322-based plasmid pWT111. A single nucleotide transversion in loop II’ of RNAI results in an eightfold increase in plasmid copy number. Removal of the rom coding region from pWT111cop results in a further sixfold increase in copy number. We present evidence that ROM is involved in the strong incompatibility effect seen between pMB1 and ColE1 type plasmids.
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Novel Transmissible Factors in a Non-O1 Vibrio cholerae and a Vibrio sp.
More LessTransmissible factors encoding production of lacunae (L factors) were demonstrated in a non-O1 Vibrio cholerae and a Vibrio sp. of recent environmental origin. Lacunae were produced in lawns of non-O1 V. cholerae indicator strains under the same assay conditions as those where lacunae were produced by the well characterized P fertility plasmid of V. cholerae O1 and the V fertility factor found in a non-cholera vibrio strain. The origin of the lacunae produced by strains habouring the V and L factors was examined. No vibriocin or phage activity was found in culture supernates or in lacunae produced by the strains, suggesting that, as in the case of the P plasmid, the lacunae probably represent sites of active mating. Unlike the P plasmid, neither the Vn or L factor could be detected or isolated by conventional plasmid techniques.
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Characterization of SE1, a New General Transducing Phage of Salmonella typhimurium
More LessA transducing phage, SE1, which is able to infect Salmonella typhimurium was isolated from a Salmonella enteritidis strain. SE1 is a temperate phage which is heteroimmune with respect to phages P22, L, KB1 and ES18. It is similar in morphology and size to phages P22, L and KB1 and is serologically related to phages P22 and L but not to KB1. Efficiencies of generalized transduction effected by phage SE1 are similar to those for P22HT (int7), a mutant which mediates a high frequency of chromosomal gene transduction. The lengths of chromosomal DNA transduced by SE1 and P22HT (int7) are similar. Furthermore, the SE1 prophage does not exclude the transducing particles from cells it has lysogenized; consequently it is possible to use both SE1 lysogens and non-lysogenic strains as recipients in SE1-mediated transduction experiments, and obtain similar transduction efficiencies. However, the SE1 prophage gives rise to a lysogenic conversion that decreases the rate of adsorption of SE1 and L phages by about 50%, but does not affect adsorption of P22. Altogether these results suggest that phage SE1 may be a useful tool in the genetic manipulation of S. typhimurium.
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Isolation of a cDNA Encoding a Portion of the Cyclic Nucleotide Phosphodiesterase of Dictyostelium discoideum
More LessThe cyclic nucleotide phosphodiesterase (phosphodiesterase) of Dictyostelium discoideum is one of a group of developmentally regulated proteins which enable cells to aggregate by chemotaxis during the early stages of development. We report the identification and DNA sequence of a cDNA clone encoding the amino-terminal region of the phosphodiesterase. The clone, pPD-3, was selected from a cDNA library created by priming first strand synthesis using a set of oligonucleotides with sequences predicted from the amino-terminal amino acid sequence of purified phosphodiesterase. The DNA sequence of pPD-3 encodes perfectly the available phosphodiesterase amino acid sequence, and pPD-3 selects an mRNA which can be translated into material recognized by phosphodiesterase antisera. The nucleotide sequence of pPD-3 indicates there are 49 amino acids, which contain a segment possessing the characteristics of a signal peptide, that separate the amino-terminal residue identified in the purified protein from the methionine codon at which translation originates. DNA blot analysis demonstrates that the phosphodiesterase gene exists as a single copy in the nuclear genome. Analysis of RNA indicates that the phosphodiesterase transcript is 2·1 kb long, which is approximately 0·8 kb more than the minimum required to encode this protein.
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- Pathogenicity And Medical Microbiology
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Purification and Characterization of a Bacteriocin from Klebsiella pneumoniae 158
More LessKlebocin, a bacteriocin produced by Klebsiella pneumoniae 158, was purified to homogeneity by ammonium sulphate fractionation and sequential DEAE-Sephacel and Sephadex G-150 column chromatography. The purified preparation had an M r of approximately 40000 on SDS-PAGE. Chemical analysis of the purified preparation showed it to be a protein, and it was sensitive to digestion by various proteolytic enzymes.
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Interaction of Chlamydia trachomatis with Human Genital Epithelium in Culture
More LessPrimary cultures of human endometrial and ectocervical epithelial cells were examined as a new model system to study genital infection by Chlamydia trachomatis. Initial studies demonstrated that these cells were indeed susceptible to chlamydial infection. Inocula, adjusted to produce inclusions in 50 to 80% of equivalent numbers of standard McCoy cells, resulted in infection rates of approximately 15 to 30% for the columnar cells of the endometrium and 5 to 10% for the squamous cells of the ectocervix. Exposure of cultures to DEAE-dextran and centrifugation-assisted inoculation, manipulations reported to enhance infection of HeLa and McCoy cells, did not alter the number of inclusion-positive genital cells. Addition of cycloheximide to the post-inoculation culture medium slightly increased numbers of inclusion-bearing cells while growth of genital cells in hormone-supplemented medium resulted in a variable effect on inclusion development and a significant reduction in the association of radiolabelled organisms with these cells. The basis for the different levels of infection in McCoy versus genital cell cultures was revealed by immunofluorescence analysis of chlamydial association with host cells immediately after inoculation. Chlamydiae failed to adhere to many cells in the genital cell cultures while adherence to McCoy cells was uniform. In addition, the association of radiolabelled C. trachomatis was significantly lower with genital cells than with McCoy cells. Finally, culture conditions were defined which markedly inhibited inclusion development without an immediate loss of chlamydial growth potential. This investigation indicates that primary genital cell cultures are susceptible to chlamydial infection and will be valuable for studies on the nature of C. trachomatis interactions with natural human target cells.
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Outer Membrane Proteins of Fusobacterium nucleatum Fev1
More LessOuter membrane enriched material from six strains of Fusobacterium nucleatum was analysed by SDS-PAGE. The protein profiles of all the strains were dominated by proteins with molecular masses of about 40 kDa, and a very high degree of homology in relation to apparent molecular masses was observed. In all strains except Fev1, one of the most dominant proteins exhibited heat modifiable properties, having an apparent molecular mass of about 38 kDa and 42 kDa when heated in SDS at 50 and 100° respectively. None of the proteins of the outer membrane of F. nucleatum Fev1 demonstrated such heat modifiable properties. The 40 kDa protein, and several other proteins, appear to be both exposed on the cell surface and peptidoglycan associated.
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Purification, Characterization and Immunological Properties of the Serotype-specific Capsular Polysaccharide of Pasteurella haemolytica Serotype A7 Organisms
More LessThe serotype-specific capsular polysaccharide from two strains of Pasteurella haemolytica serotype A7 organisms was purified and characterized by chemical analysis and by 1H and 13C NMR spectroscopy using one- and two-dimensional methods. The polymer has the repeating unit → 3)-β-2-acetamido-2-deoxygalactopyranose-(1 → 3)-α-2-acetamido-2-deoxy-6-O-acetylglucopyranose-(1-phosphate →. It was immunogenic (capable of eliciting antibodies) for sheep. Chemical removal of O-acetyl groups destroyed both the ability of the polymer to adhere to sheep erythrocytes at neutral pH and the ability to form immune precipitates with specific antisera. Studies using the protein A-gold technique in the electron microscope showed the polysaccharide to be peripherally localized on the bacterial surface.
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Partial Purification of an Osteolytic Toxin from Pasteurella multocida
More LessA protein toxin apparently composed of one polypeptide with an estimated M r of 155000 was purified from sonicated cells of a type D strain of Pasteurella multocida (LFB3) by preparative polyacrylamide gel electrophoresis (PAGE) and DEAE-Sephadex A50 chromatography. Its specific activity was 150-fold greater than that of the crude extract. The partially purified protein was cytotoxic for embryonic bovine lung cells, lethal for mice and caused turbinate atrophy in gnotobiotic pigs; a single intraperitoneal injection of approximately 360 ng kg-1 caused 50% turbinate atrophy. Reversal of the two-step purification procedure using DEAE-Sephacel chromatography followed by preparative PAGE increased the yield of toxin 30-fold; the specific activity of the partially purified toxin was 1970-fold greater than that of the crude extract.
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Capsulation of in vitro and in vivo Grown Bacteroides Species
More LessBy centrifugation on a four step Percoll density gradient cells of Bacteroides species could be separated according to the size of extracellular structure. The difference in size was visible by both light and electron microscopy. Two structures were observed on Bacteroides fragilis by electron microscopy, namely a fibrous network and an electron dense layer. An electron dense layer was visible on Bacteroides ovatus only when stained with ruthenium red. B. fragilis cells grown in the mouse peritoneal cavity did not produce a large fibrous network. An electron dense layer was observed on some cells in the presence of ruthenium red stain and cells possessing this layer were phagocytosed in vivo.
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Cell-surface Hydrophobicity of Candida Species as Determined by the Contact-angle and Hydrocarbon-adherence Methods
More LessCell-surface hydrophobicities of six Candida species were studied by two methods: measurement of the contact angle, and partitioning with aqueous-hydrocarbon (n-octane, n-hexadecane and p-xylene) mixtures. C. tropicalis, C. glabrata and C. krusei adhered better to the hydrocarbons than did C. albicans, C. stellatoidea and C. parapsilosis. Contact angles for the less adherent species were smaller than those for the more adherent species. Thus the two methods gave results that were similar overall and indicated that C. tropicalis, C. glabrata and C. krusei have greater cell-surface hydrophobicities than C. albicans, C. stellatoidea and C. parapsilosis.
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Oxidative and Phagocytic Functions of Macrophages during Infections Induced in Mice by Mycobacterium intracellulare and Listeria monocytogenes
More LessThe oxidative metabolism (chemiluminescence and H2O2 release) and phagocytic activity of mouse peritoneal macrophages during chronic infections induced by Mycobacterium intracellulare and more acute infections due to Listeria monocytogenes were studied. In M. intracellulare infections, macrophage chemiluminescence in response to phorbol myristate acetate (PMA) was greatest at around 2 weeks, with a 1 week lag phase after infection, while the PMA-triggered H2O2 release was markedly enhanced even 1 d after challenge, and remained high thereafter for up to 10 weeks. The pattern of changes in the phagocytic activity of host macrophages in response to latex beads during this infection resembled the pattern seen with macrophage H2O2 release. In the L. monocytogenes infections, the PMA-triggered chemiluminescence of the host macrophages increased 4 d (in a sublethal infection) and 2 d (in a lethal infection) after bacterial challenge, whereas the PMA-triggered H2O2 release was markedly enhanced as early as 1 d after infection and the elevated level persisted until either the bacteria were eliminated or the animals died. The patterns of changes in phagocytic activity of the host macrophages during L. monocytogenes infection at sublethal and lethal doses differed. In the former, phagocytosis was most active in the early phase of infection, with a peak around day 2, followed by a rapid decrease; in the latter, the phagocytic ability increased more slowly, and remained elevated until the animals died. The results suggest that the macrophages induced by M. intracellulare are in a more activated state than are those induced by L. monocytogenes.
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Preliminary Characterization of an Organism Isolated from a Case of Viluy Encephalomyelitis Indicates a Protozoal, Rather Than Viral, Aetiology
More LessA microbial agent was isolated previously from a case of Viluy encephalomyelitis and named the ‘KPN agent’ after the initials of the patient. Here a detailed characterization of nucleic acids extracted from the purified KPN agent is presented. The agent contains both DNA and RNA, and has its own tRNAs and some other low-M r RNAs, including 5S RNA. These findings, and the isolation of eukaryotic-type ribosomes, suggest that the KPN agent is not a virus, as believed before, but a more complex micro-organism, with protein-synthesizing capacity. The nucleotide sequence of the 5S RNA in the ribosomes of the KPN agent is identical with the sequence of 5S RNA of Acanthamoeba castellanii. The novel protozoan nature of the KPN agent is discussed in relation to other unusual properties of this micro-organism. Some implications of these results for the aetiology of Viluy encephalomyelitis are also discussed.
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- Physiology And Growth
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Extracellular Vesicle Formation and Biosurfactant Production by Serratia marcescens
More LessPigmented and non-pigmented strains of Serratia marcescens produced extracellular vesicles and had wetting activity when grown at 30°C but not at 37°C. Light microscopy showed that the red pigment was present in vesicles and intracellular granules. Electron microscopy revealed the presence of vesicles surrounded by the bacterial membrane. Three lipids having the wetting activity, W1, W2 and W3, were isolated by thin-layer chromatography of lipids from different strains of S. marcescens. Dispersions of the isolated wetting agents had small contact angles on a polystyrene surface and the ability to lower surface tension. Wetting agent W1 was the aminolipid serratamolide. Wetting agents W2 and W3 were also aminolipids but were shown to be different from serratamolide by chemical analyses. Wetting agent and prodigiosin (in a pigmented strain) were the main lipids of isolated vesicles.
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Determination of the Spiral Conformation of Aquaspirillum spp. by Scanning Electron Microscopy of Elongated Cells Induced by Cephalexin Treatment
More LessThe effect of the β-lactam antibiotic cephalexin on the spiral conformation of cells of Aquaspirillum spp. was examined by scanning electron microscopy. A. itersonii and A. peregrinum, which are known to have a left-handed spiral shape, elongated and still showed left-handed spirals in medium containing cephalexin. The spiral conformation of the elongated cells is therefore considered to represent the natural condition. The spiral conformations of A. metamorphum and A. psychrophilum grown in ordinary cultures were difficult to determine because they have short cells without a complete spiral. After cephalexin treatment, the cells of these species elongated and displayed spiral forms, right-handed in A. metamorphum and left-handed in A. psychrophilum. This elongation method may be useful for checking and determination of the spiral handedness of short spiral or curved bacteria such as vibrios.
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Asymmetric Location of the Septum in Morphologically Altered Cells of the Fission Yeast Schizosaccharomyces pombe
More LessCells of the fission yeast Schizosaccharomyces pombe, normally sausage-shaped, changed to a round-bottomed flask (RBF)-like morphology during growth in the presence of aculeacin A (Acu), an antifungal antibiotic. The volume of RBF-like cells was comparable to that of the control cells. After being transferred to normal conditions (without Acu at 25°C), the RBF-like cells continued to grow at the cylindrical and/or spherical end(s) and then the septum at the subsequent division of the cells was formed without exception at the boundary plane between the spheroidal and the cylindrical region; it is at this boundary that the nucleus was located before mitosis. Hence the RBF-like cell divided into a spheroidal and a cylindrical sib at the first cell division. At the end of the second cell cycle, the spheroidal and the cylindrical progeny divided into two spheroidal and two cylindrical sibs respectively. The values of the mean length (long/short) and volume (big/small) ratios of paired sibs were larger in order of (a) cylindrical normal, with both mean ratios 1·06; (b) cylindrical control; (c) cylindrical progeny of RBF-like cell; (d) spheroidal progeny of RBF-like cell; and (e) RBF-like cell, whose mean length ratio was 1·25 but whose mean volume ratio was 1·94. That is, the more the morphology deviated from the cylindrical form, the greater was the degree of asymmetry. There was no rule relating the biases to the growth pole in these asymmetries.
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Isolation and Characterization of a Vibrio alginolyticus Mutant That Overproduces Extracellular Proteases
More LessThe Vibrio alginolyticus prot-T1 mutant was able to produce haloes of clearing on skim milk/peptone agar plates at 42°C whereas proteolysis by the wild-type strain was inhibited at 37°C. The prot-T1 mutant overproduced the three major alkaline proteases with apparent molecular masses of approximately 28000, 22500 and 19500 (proteases 1a, 2 and 3, respectively). Their synthesis was not markedly repressed by incubation at 37°C or by non-aeration. Both treatments inhibited protease synthesis in the wild-type strain, which only produced proteases during the stationary growth phase. The prot-T1 mutant synthesized proteases throughout the exponential and stationary growth phases in peptone medium. High protease activities were induced by glucose or glutamine in stationary phase prot-T1 that were pre-grown in peptone medium. Glucose or glutamine had the opposite effect on protease activities in stationary phase prot-T1 cultures that were pre-grown in minimal medium. Collagenase synthesis was not altered in the prot-T1 mutant and was repressed by growth at 37° or without aeration. The independent control of collagenase synthesis supports the conclusion that there are no regulatory proteins responsible for the overall control of extracellular protease synthesis by temperature, aeration and growth phase in V. alginolyticus.
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The Tricarboxylic Acid Cycle of Heterogeneous and Swarmer Cell Populations of Rhodomicrobium vannielii Rm5
More LessStudies on the tricarboxylic acid cycle of Rhodomicrobium vannielii Rm5 demonstrated that, unlike other Rhodospirillaceae, this organism has a functionally incomplete cycle, broken at 2-oxoglutarate dehydrogenase, under photoheterotrophic conditions. This enzyme was, however, synthesized when Rm. vannielii was grown under microaerophilic chemoheterotrophic conditions. The citrate synthase exhibited responses to inhibitors characteristic of Gram-negative organisms but, unlike many microbes exhibiting an incomplete cycle, was not inhibited by 2-oxoglutarate. Both NAD- and NADP-linked isocitrate dehydrogenase activity was detectable but the functional roles of these enzymes are unclear. No significant differences in enzyme activities or inhibitor sensitivities of enzymes were detected between heterogeneous cultures and synchronous swarmer cell populations.
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Differential Scanning Calorimetry of Bacteria
More LessThermograms obtained by differential scanning calorimetry of a range of bacteria of different heat resistances were compared. Equations were derived to calculate the rate at which the numbers of viable organisms in a calorimeter decline as the temperature is raised at a constant rate. Vegetative bacteria scanned at 10°C min-1 showed multi-peaked thermograms with four major peaks (denoted m, n, p and q) occurring in the regions 68–73, 77–84, 89–99 and 105–110°C respectively. Exceptions were that peak m (the largest peak) occurred at 79–82°C in Bacillus stearothermophilus and an additional peak, r, was detected in Escherichia coli at 119°C. At temperatures below the main peak m there were major differences in thermograms between species. There was a direct relationship between the onset of thermal denaturation and the thermoresistance of different organisms. Heat-sensitive organisms displayed thermogram features which were absent in the more heat-resistant types. When samples were cooled to 5°C and re-heated, a small endothermic peak, pr , was observed at the same temperature as p. Peaks p and pr were identified as the melting endotherms of DNA. In all vegetative organisms examined, maximum death rates, computed from published D and z values, occurred at temperatures above the onset of thermal denaturation, i.e. cell death and irreversible denaturation of cell components occurred within the same temperature range.
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Effect of l(–)Sorbose on the Release of β-Glucosidase by Trichoderma reesei QM9414
More Lessl(–)Sorbose, a sugar known to cause paramorphogenesis in fungi, was tested for its effect on morphology and release of cell-wall bound β-glucosidase (EC 3.2.1.21) in the cellulolytic fungus Trichoderma reesei QM9414. Sorbose caused an increase in branching and septation in the growing mycelium. Extracellular β-glucosidase activity was enhanced when cellobiose or cellulose growth medium was supplemented with sorbose. In sorbose-supplemented cultures, the β-glucosidase activity associated with the wall fraction was less than half that in unsupplemented cultures. The intracellular activity was also lower in the sorbose-supplemented cultures than in unsupplemented controls. The glucosamine/glucose ratio of wall hydrolysates from sorbose-supplemented cultures was about twice that of control hydrolysates. Since β-glucosidase is closely associated with 1,3-β-glucan in the walls of T. reesei, a decrease in wall glucan content, and the resulting weakened association of the enzyme with the walls, is probably responsible for its increased release into the culture medium in the presence of sorbose.
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Heterogeneity of Lipopolysaccharide Banding Patterns in Leptospira spp.
More LessStrains of Leptospira interrogans and Leptospira biflexa, examined by electrophoresis after whole cell lysis and protein digestion, revealed the presence of 2-keto-3-deoxyoctonate and an heterogeneous lipopolysaccharide electrophoretic banding pattern, which was characteristic of the species.
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Methyl Chloride: Naturally Occurring Toxicant and C-1 Growth Substrate
More LessA strain of Hyphomicrobium that could utilize methyl chloride as the sole carbon and energy source for growth was isolated from industrial sewage. The methylotroph utilized methyl chloride quantitatively with stoichiometric release of chloride ion. A specific growth rate (μ) of 0·09 h-1 was observed with about 1% (v/v) methyl chloride in the gas phase.
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