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Volume 141,
Issue 7,
1995
Volume 141, Issue 7, 1995
- Pathogenicity And Medical Microbiology
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Adherence of Candida albicans to epithelial cells: studies using fluorescently labelled yeasts and flow cytometry
More LessSummary: Candida albicans adherence to epithelial cells is the first step in the infectious process, but in spite of its importance, current methods for the quantitative measurement of adherence of C. albicans to epithelial cells in vitro have some serious limitations. They are based on filtration assays and either microscopic or radiometric analysis. The adherence reaction is usually carried out with a large excess of yeasts (100-fold) over epithelial cells in order to perform the microscopic analysis, which is slow, subjective and limited to 100-200 cells and thus lacks statistical power. The radiometric analysis fails to measure individual cells. A method for measuring yeast adherence that overcomes these problems has been developed. It is based on labelling the yeasts with the fluorogenic marker 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF) prior to the adherence reaction, and analysing 104 epithelial cells by flow cytometry, while nonbound yeasts are excluded by gating. Two subpopulations of buccal epithelial cells (BECs) which differ in their mean fluorescence intensities per cell (MFIs) were observed: one with MFI which did not exceed nonspecific fluorescence, and the other with MFI as high or higher than the MFI of labelled yeasts. The two subpopulations represent yeast-free and yeast-binding epithelial cells, respectively, and the MFI increment of the BECs is a quantitative measure of the extent of yeast adherence. Control experiments confirming previously described basic features of adherence, such as enhanced adherence at increasing yeast excess, diminished adherence of trypsin-treated or heat-inactivated yeasts, and the differential adherence of various Candida species, supported the validity of the assay. The possibility of studying adherence reliably at low yeast: epithelial cell ratios, which better mimic adhesion as it occurs in vivo, is an important advantage of the assay. New findings, using this method, included the observation that exfoliated BECs from diabetic patients exhibited the same capacity for C. albicans adherence as cells from healthy controls, and that epithelial cells from early human ontogenic stages had a significantly lower adherence level than those from later stages.
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A linear B-cell epitope on the class 3 outer-membrane protein of Neisseria meningitidis recognized after vaccination with the Norwegian group B outer-membrane vesicle vaccine
Summary: The class 3 outer-membrane protein (OMP) of Neisseria meningitidis is a potential target for bactericidal and opsonic antibodies in humans. Synthetic peptides spanning the class 3 OMP from the vaccine strain 44/76 (B: 15:P1.7,16:L3,7) were synthesized on pins and screened with serum obtained from Norwegian adolescents immunized with a meningococcal serogroup B outer-membrane vesicle (OMV) vaccine. A strong IgG response to a single peptide (19FHQNGQVTEVTT30) located within loop 1 (VR1) was stimulated after three doses of OMV vaccine in three vaccinees selected on the basis of their antibody response to class 3 OMP. No clear linear B-cell epitopes were recognized by four different murine serotype 15-specific mAbs. A 23mer peptide (D63b2) containing loop 1 of the class 3 OMP was synthesized, and the IgG responses were measured in pre- and post-vaccination serum from 27 vaccinees. Specific IgG rose significantly in 37% of vaccinees 6 weeks after the second dose and in 74% of the vaccinees 6 weeks after the third dose of the OMV vaccine. Most immune sera reacted distinctly on immunoblots with denatured class 3 OMP, and the immunoblotting reactivity correlated strongly with concentration of the IgG antibodies specific for peptide D63b2. When added to a post-vaccination serum from one vaccinee, peptide D63b2 competed efficiently with the class 3 OMP for specific antibody binding on immunoblots and in pin ELISA. The results show that the significant part of the humoral response to the meningococcal class 3 OMP elicited by vaccination with the Norwegian OMV vaccine was directed against a single continuous epitope.
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- Physiology And Growth
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Basis of cerulenin resistance of two strains of Candida albicans
More LessSummary: The basis of cerulenin resistance of Candida albicans strains 4918-2 and 4918-10 has been investigated. Parasexual genetic analyses established that cerulenin resistance to concentrations of at least 5 μg ml-1 is dominant in both strains. The results also showed that strain 4918-2 is heterozygous for resistance, while the change from resistance to sensitivity of strain 4918-10 is reversible. Experiments to define the mechanism(s) responsible for resistance focused on cerulenin uptake and fatty-acid synthase activity. Cerulenin uptake by strains 4918-2 and 4918-10 was 24% of that of the wild-type (strain 4918). Uptake was restored in UV-induced cerulenin-sensitive segregants of strains 4918-2 and 4918-10, and varied from 63% to 200% of parental values. Fatty-acid synthase from strains 4918-2 and 4918-10 was resistant to cerulenin as judged by differences in the inactivation of the enzyme by the agent. However, inactivation kinetics of fatty-acid synthase of cerulenin-sensitive segregants did not revert to the parental inactivation profile. Further investigation showed that nine out of ten segregants were resistant to cerulenin at concentrations between 1 and 4 μg ml-1 while strain 4918 was sensitive to cerulenin at all concentrations tested. Thus, the results suggest that alteration of fatty-acid synthase and changes in permeability contribute to total cerulenin resistance of each strain.
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Constitutive glucose-induced activation of the Ras-cAMP pathway and aberrant stationary-phase entry on a glucose-containing medium in the Saccharomyces cerevisiae glucose-repression mutant hex2
More LessSummary: Addition of glucose to cells of the yeast Saccharomyces cerevisiae growing on a nonfermentable carbon source triggers a rapid, transient increase in the cAMP level. The occurrence of this cAMP spike appears to be correlated inversely with the glucose-repression state of the cells. This was also observed for the hex2 mutant, which is deficient in glucose repression and which displayed the cAMP signal constitutively. When cells of the hex2 mutant were starved for nitrogen on a glucose-containing medium, they rapidly lost viability, similarly to mutants with overactivation of the Ras-adenylate cyclase pathway. Flow cytometry measurements showed that G1 arrest of the hex2 mutant under such conditions was incomplete. Trehalose accumulation, a typical feature of cells entering the stationary phase G0, was very short-lived in the hex2 mutant under the same conditions. These results are in agreement with the presence of continuous glucose-triggered activation of cAMP synthesis in hex2 cells on a glucose-containing nitrogen-starvation medium. In the course of these experiments a spontaneous suppressor mutant, shx (for suppressor of hex2), was isolated which survived nitrogen starvation on a glucose-containing medium much better than the hex2 strain. It also showed normal G1 arrest and much longer accumulation of trehalose. The suppressor mutation also caused inability to grow on nonfermentable carbon sources and absence of invertase derepression, and it was epistatic to hex2 for these characteristics also. The isolation of this epistatic derepression mutation supports the idea that the defect in glucose repression of the hex2 mutant is the cause of its rapid loss of viability during nitrogen starvation on a glucose-containing medium. Substitution of glucose for glycerol partially abolished the rapid loss of viability in the hex2 mutant. These results suggest that the glucose-repressible character of the pathway involved in glucose-triggered activation of cAMP synthesis might have a physiological role in preventing overstimulation of cAMP synthesis and allowing proper entrance into the stationary-phase G0 in a medium containing ample glucose but lacking another essential nutrient for growth. Such a situation might be quite common in the glucose-rich natural environment of S. cerevisiae.
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Coordination of sucrose uptake and respiration in the yeast Debaryomyces yamadae
More LessSummary: Screening in batch cultures identified Debaryomyces yamadae as a yeast that exhibits the Kluyver effect for sucrose: this disaccharide can be respired but, even under oxygen-limited conditions, alcoholic fermentation of sucrose does not occur. Ethanol, glycerol and arabitol were the main fermentation products during oxygen-limited growth on glucose in chemostat cultures. None of these fermentation products were produced in oxygen-limited chemostat cultures grown on sucrose and the fraction of the sucrose that could not be respired remained unused in the culture medium. This absence of alcoholic fermentation was not due to repression of the key fermentative enzymes pyruvate decarboxylase and alcohol dehydrogenase. In contrast to some other yeasts that exhibit a Kluyver effect, D. yamadae did not exhibit a preference for ethanol in batch cultures grown on mixtures of ethanol and sucrose. Sucrose metabolism in D. yamadae involves intracellular hydrolysis by an α-glucosidase. Incubation of weakly buffered cell suspensions with sucrose led to a rapid transient alkalinization, indicating the presence of a sucrose-proton symport system. The apparent substrate saturation constant of the sucrose-uptake system was 0.2 mmol l-1. Sucrose-dependent alkalinization rates were much lower in samples from oxygen-limited cultures than in samples from aerobic cultures. Transient responses of D. yamadae to oxygen limitation were investigated by applying a sudden decrease in the oxygen feed to aerobic sugar-limited chemostat cultures. In glucose-grown cultures, this led to alcoholic fermentation and no significant accumulation of sugar occurred after the switch. In sucrose-limited cultures, sugar accumulation occurred instantaneously after the switch, and ethanol formation was virtually absent. The results indicate that the Kluyver effect for sucrose in D. yamadae, i.e. the adjustment of the glycolytic flux to the cells' respiratory capacity, is effected by rapid down-regulation of the capacity of the sucrose carrier under oxygen-limited conditions.
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Production of Escherichia coli STb enterotoxin is subject to catabolite repression
More LessSummary: Enterotoxigenic Escherichia coli are known to secrete several types of toxins including STb, a heat-stable enterotoxin. STb enterotoxin production was studied in wild-type E. coli strains. Using a quantitative STb-specific inhibition ELISA, the amount of toxin present in the culture supernatant fractions of various E. coli strains was determined. Variation in the production of STb toxin was observed for the wild-type strains. For E. coli strain 82-4247 grown in trypticase soy broth, the toxin was produced after 4 h of growth and was maximal after about 57 h of growth. The amount of toxin in the culture supernatant fraction increased concomitantly with bacterial growth. Using the rat loop assay, the biological activity of STb was retained even after the logarithmic phase of growth when STb production levelled off (i.e. from 24 to 74 h). STb production by E. coli strain 82-4247 varied with the culture medium used. In particular, addition of 1-0% (w/v) compared to 0-1 % glucose to Davis minimal medium decreased STb production, whereas addition of 1-0% (w/v) glycerol did not affect STb production. Addition of exogenous cAMP reversed the repressive effect of glucose. Using mutant strains, STb production was shown to be subject to catabolite repression.
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Mutations showing specificity for normal growth or Mn(II)-dependent post-exponential-phase cell division in Deinococcus radiodurans
More LessSummary: We have previously reported that in the presence of small amounts of Mn(II) ions, stationary-phase cultures of the radioresistant bacterium Deinococcus radiodurans IR are capable of undergoing about three new rounds of cell division which are non-exponential and reductive. This novel Mn(II)-induced cell division (Mn-CD) phenomenon was studied further. Two mutants were isolated from D. radiodurans IR and were found to harbour mutations affecting the continuation of cell division and DNA replication, one of which affected vegetative growth but not Mn-CD, while the other affected Mn-CD but not vegetative growth. Moreover, a DNA synthesis transition was observed when growth was switched from the normal to the Mn-CD process. These results suggest the presence in D. radiodurans of an alternative type of cell division induction, which is characterized by being Mn(II)-dependent and stationary-phase specific.
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- Plant-Microbe Interactions
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Rhizobium meliloti lacking mosA synthesizes the rhizopine scyllo-inosamine in place of 3-O-methyl-scyllo-inosamine
More LessSummary: The Rhizobium meliloti Rm220-3 mos locus producing the rhizopine scyllo-inosamine (SI) in nodules is shown to consist of three ORFs (ORF1, mosB and mosC) arranged in an operon structure. This differs from the R. meliloti L5-30 mos locus, which produces 3-O-methyl-scyllo-inosamine (3-O-MSI), by the complete absence of mosA. The deletion covers a region of 1235 nt and includes the entire mosA gene as well as the sequence both upstream and downstream. As a result, Rm220-3 mos ORF1 shares a 5′ region common with L5-30 ORF1 but includes an additional 10 bp insertion and a section in the L5-30 mosA and mosB intercistronic region. Antibodies against L5-30 Mos proteins detected MosB and MosC proteins in Rm220-3-induced nodules but no translation product for either ORF1 or mosA was detected. A construct prepared from the L5-30 mos locus which has a truncated mosA gene produces SI rather than 3-O-MSI, confirming this gene is involved in a methylation step in the production of 3-O-MSI. Further, changes made to this ORF result in reduced levels of the rhizopine.
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-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other Gram-negative bacteria
Summary: A series of transposons are described which contain the gusA gene, encoding -glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out.
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- Systematics
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Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals
More LessSummary: Atypical oral Candida isolates were recovered from 60 HIV-infected and three HIV-negative individuals. These organisms were germ-tube-positive and produced abundant chlamydospores which were frequently arranged in triplets or in contiguous pairs. They belonged to C. albicans serotype A and had atypical carbohydrate assimilation profiles. Fingerprinting the genomic DNA of a selection of these organisms with the C. albicans-specific probe 27A and five separate oligonucleotides, homologous to eukaryotic microsatellite repeat sequences, demonstrated that they had a very distinct genomic organization compared to C. albicans and C. stellatoidea. This was further established by random amplified polymorphic DNA (RAPD) and karyotype analysis. Comparison of 500 bp of the V3 variable region of the large ribosomal subunit genes from nine atypical isolates and the corresponding sequences determined from C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. glabrata, C. kefyr and C. krusei showed that the atypical organisms formed a homogeneous cluster (100% similarity) that was significantly different from the other Candida species analysed, but was most closely related to C. albicans and C. stellatoidea. These genetic data combined with the phenotypic characteristics of these atypical organisms strongly suggest that they constitute a novel species within the genus Candida for which the name Candida dubliniensis is proposed.
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Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species
More LessSummary: One hundred and nineteen strains of alkaliphilic and alkalitolerant, aerobic endospore-forming bacteria were examined for 47 physiological and biochemical characters, and DNA base composition. Numerical analysis (S J and S SM/UPGMA clustering) revealed 11 clusters that comprised three or more strains. Most of the phena were further characterized by analysis of carbohydrate utilization profiles using the API 50CH system, but strains of two taxa could not be cultured by this method. DNA reassociation studies showed that nine of the phena were homogeneous, but strains of phenon 4 and phenon 8 were each subdivided into two DNA hydridization groups. The strains could therefore be classified into 13 taxa plus a number of unassigned single-membered clusters. Two taxa were equated with Bacillus cohnii and B. alcalophilus and nine of the remainder are proposed as new species with the following names: B. agaradhaerens sp. nov., B. clarkii sp. nov., B. clausii sp. nov., B. gibsonii sp. nov., B. halmapalus sp. nov., B. halodurans comb, nov., B. horikoshii sp. nov., B. pseudalcalophilus sp. nov. and B. pseudofirmus sp. nov. Two taxa were insufficiently distinct to allow confident identification and these have therefore not been proposed as new species.
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- Genome Analysis
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An operon encoding a novel ABC-type transport system in Bacillus subtilis
More LessSummary: Downstream from the surfactin synthetase operon in Bacillus subtillis a new operon-type structure has been localized which, on the basis of sequence determination, potentially encodes an ABC-type transport system. The 268 amino acid protein, the product of orf1, represents the solute-binding component of the system whereas the orf2 product a 234 amino acid protein, is the transmembrane component. Finally orf3 potentially encodes a typical 241 amino acid ATP-binding protein involved in energy supply. Comparison of the three proteins with the subunits of other ABC-type systems suggests that this new system is involved in amino acid transport.
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- Guidelines For Authors
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