Showing papers in "Fems Microbiology Letters in 1994"
TL;DR: The cell wall barrier alone cannot produce significant levels of drug resistance, which requires synergistic contribution from a second factor, such as the enzymatic inactivation of drugs.
Abstract: Mycobacteria show a high degree of intrinsic resistance to most antibiotics and chemotherapeutic agents. The low permeability of the mycobacterial cell wall, with its unusual structure, is now known to be a major factor in this resistance. Thus hydrophilic agents cross the cell wall slowly because the mycobacterial porin is inefficient in allowing the permeation of solutes and exists in low concentration. Lipophilic agents are presumably slowed down by the lipid bilayer which is of unusually low fluidity and abnormal thickness. Nevertheless, the cell wall barrier alone cannot produce significant levels of drug resistance, which requires synergistic contribution from a second factor, such as the enzymatic inactivation of drugs.
552 citations
TL;DR: Most of the small ciliate protozoa, including Dasytricha ruminantium and Entodinium spp.
Abstract: Most of the small ciliate protozoa, including Dasytricha ruminantium and Entodinium spp. living in the rumen of sheep, were found to have intracellular bacteria. These bacteria were not present in digestive vacuoles. They showed characteristic coenzyme F420 autofluorescence and they were detected with a rhodamine-labelled Archaea-specific oligonucleotide probe. The measured volume percent of autofluorescing bacteria (1%) was close to the total volume of intracellular bacteria estimated from TEM stereology. Thus it is likely that all of the bacteria living in the cytoplasm of these ciliates were endosymbiotic methanogens, using H2 evolved by the host ciliate to form methane. Intracellular methanogens appear to be much more numerous than those attached to the external cell surface of ciliates.
330 citations
TL;DR: A HEp-2 cell-vacuolation factor was extracted and purified from the culture supernatant of a Bacillus cereus strain which caused emetic-syndrome food poisoning, and the toxin was named as cereulide, a cyclic dodecadepsipeptide, which is closely related to the potassium ionophore, valinomycin.
Abstract: A HEp-2 cell-vacuolation factor was extracted and purified from the culture supernatant of a Bacillus cereus strain which caused emetic-syndrome food poisoning. The final preparation was chemically pure, and the toxin was named as cereulide. Mass spectrometry, NMR studies and chemical degradation revealed that the cereulide is a cyclic dodecadepsipeptide, (D-O-Leu-D-Ala- L-O-Val-L-Val)3, which is closely related to the potassium ionophore, valinomycin.
258 citations
TL;DR: It is proposed that an increase in plasma membrane fluidity may be correlated with a decrease in the sterol:phospholipid and sterol-protein ratios and a increase in unsaturation index.
Abstract: The lipid composition of a strain of each of two yeasts, Saccharomyces csrevisiae and Kloeckera apiculata, with different ethanol tolerances, was determined for cells grown with or without added ethanol. An increase in the proportion of ergosterol, unsaturated fatty acid levels and the maintenance of phospholipid biosynthesis seemed to be responsible for ethanol tolerance. The association of ethanol tolerance of yeast cells with plasma membrane fluidity, measured by fluorescence anisotropy, is discussed. We propose that an increase in plasma membrane fluidity may be correlated with a decrease in the sterol: phospholipid and sterol: protein ratios and an increase in unsaturation index.
233 citations
TL;DR: In vitro data indicate that an increase in the concentration of fructose-based oligosaccharides in the diet may alter the balance of the gut microflora towards bifidobacteria, a purported health-promoting genus.
Abstract: Chemostat cultures of human faecal bacteria were used to determine the bifidogenic effect of oligofructose, a fermentable carbohydrate found in a number of plants. In single stage continuous culture, oligofructose preferentially enriched for bifidobacteria, in comparison to sucrose and inulin. This stimulatory effect was enhanced at a high dilution rate, high substrate concentration and low pH. These parameters are likely to approximate to those that occur in the proximal colon. Studies with a three-stage continuous culture model of the large intestine confirmed the bifidogenic effect of oligofructose. These in vitro data indicate that an increase in the concentration of fructose-based oligosaccharides in the diet may alter the balance of the gut microflora towards bifidobacteria, a purported health-promoting genus.
231 citations
TL;DR: It is reported that nitric oxide (NO) reductase, a bc-type cytochrome involved in denitrification, shares important features with these terminal oxidases as well.
Abstract: Among aerobic prokaryotes, many different terminal oxidase complexes have been described. Sequence comparison has revealed that the aa3-type cytochrome c oxidase and the bo3-type quinol oxidase are variations on the same theme: the heme-copper oxidase. A third member of this family has recently been recognized: the cbb3-type cytochrome c oxidase. Here we give an overview, and report that nitric oxide (NO) reductase, a bc-type cytochrome involved in denitrification, shares important features with these terminal oxidases as well. Tentative structural, functional and evolutionary implications are discussed.
210 citations
TL;DR: The strain designated Tp8T 6331 is differentiated from thermophilic cellulolytic clostridia on the basis of physiological characteristics and phylogenetic position within the Bacillus/Clostridium subphylum of the Gram-positive bacteria.
Abstract: A new obligately anaerobic, extremely thermophilic, cellulolytic bacterium is described. The strain designated Tp8T 6331 is differentiated from thermophilic cellulolytic clostridia on the basis of physiological characteristics and phylogenetic position within the Bacillus/Clostridium subphylum of the Gram-positive bacteria. Strain Tp8T 6331 is assigned to a new genus Caldicellulosiruptor, as Caldicellulosiruptor saccharolyticus gen., nov., sp. nov.
198 citations
TL;DR: The approach enabled analysis of ammonia oxidiser enrichments at an early stage and without the requirement for isolation of pure cultures, significantly reducing the time required and facilitating quantitative assessment of relatedness of strains.
Abstract: Marine ammonia oxidising bacteria were enriched by incubation of sea water, amended with ammonium sulphate, and subsequent subculture in liquid inorganic medium. PCR primers were designed to be specific for rDNA sequences from ammonia oxidisers belonging to the β -rsub-group of the proteobacteria. These primers were then used to amplify rRNA genes from ammonia oxidiser enrichment cultures containing heterotrophs. PCR products were recovered from all cultures in which complete ammonia oxidation occurred. Subsequent rDNA sequence analysis indicated the presence of three new lineages within the clade defined by sequences of cultured β -sub-group ammonia oxidisers. Two of the new lineages showed moderate similarity to sequences from pure cultures of ammonia oxidisers previously isolated from marine and brackish environments. The third lineage (AEM-3) was deep branching and occupied an intermediate position between clades defined by Nitrosomonas or Nitrosospira , which were isolated from soil or sewage. The phylogenetic analysis suggests that, in enrichment cultures, the primers are specific for members of the target group, the β -proteobacteria ammonia oxidisers. The results also indicate the presence of previously unknown ammonia oxidisers in marine samples. The approach enabled analysis of ammonia oxidiser enrichments at an early stage and without the requirement for isolation of pure cultures, significantly reducing the time required and facilitating quantitative assessment of relatedness of strains.
181 citations
TL;DR: A class of proteins that are associated with the cell surface of Gram-positive bacteria has been recognised and site-specific mutants are being used to define their roles in pathogenesis in in vitro and in vivo models of adherence and infection.
Abstract: A class of proteins that are associated with the cell surface of Gram-positive bacteria has been recognised. Common structural features which are implicated in the proper secretion and attachment of these proteins to the cell surface occur in the C-termini. N-terminal domains interact with the host by binding to soluble host proteins, to matrix proteins or to host cells. They probably have important roles in pathogenicity by allowing bacteria to avoid host defences and by acting as adhesins. Four such proteins of Staphylococcus aureus have been characterised: protein A (immunoglobulin binding protein), fibronectin binding proteins, collagen binding protein and the fibrinogen binding protein (clumping factor). Site-specific mutants are being used to define their roles in pathogenesis in in vitro and in vivo models of adherence and infection.
181 citations
TL;DR: It is shown by complementation that the gacA gene is also essential for the expression of two extracellular enzymes in P. fluorescens CHA0: phospholipase C and a 47-kDa metalloprotease.
Abstract: Pseudomonas fluorescens strain CHA0 protects plants from various root diseases. Antibiotic metabolites synthesized by this strain play an important role in disease suppression; their production is mediated by the global activator gene gacA. Here we show by complementation that the gacA gene is also essential for the expression of two extracellular enzymes in P. fluorescens CHA0: phospholipase C and a 47-kDa metalloprotease. In contrast, the production of another exoenzyme, lipase, is not regulated by the gacA gene. Protease, phospholipase and antibiotics of P. fluorescens are all known to be optimally produced at the end of exponential growth; thus, the gacA gene appears to be a general stationary-phase regulator.
174 citations
TL;DR: Results indicate that hydrophobicity of the substratum is a primary determinant and is sufficient to induce appressorium formation in M. grisea.
Abstract: Infection by Magnaporthe grisea, the causal agent of rice blast, requires the formation of a melanized, dome-shaped infection cell, called an appressorium. Little is known about the signals and mechanisms regulating this important developmental process. We have previously observed a correlation between hydrophobicity of the contact surface and appressorium formation. To evaluate this thigmotropic response more precisely, we measured appressorium formation on the surfaces of silicon wafers modified to create various degrees of hydrophobicity. We also examined the effects of artificial ridges created on polystyrene surfaces. Hydrophobic surfaces induced a high level of appressorium formation, whereas hydrophilic surfaces did not. Tips of germ-tubes did not respond to ridges of any particular height, but formed appressoria in a random manner. These results indicate that hydrophobicity of the substratum is a primary determinant and is sufficient to induce appressorium formation in M. grisea.
TL;DR: Phylogenetic relationships between isolates defined by 16S rRNA sequence divergence represent a selection clearly different from the multi-enzyme activities responsible for the PLFA patterns.
Abstract: Twenty-five isolates of dissimilatory sulfate-reducing bacteria were clustered based on similarity analysis of their phospholipid ester-linked fatty acids (PLFA). Of these, 22 showed that phylogenetic relationships based on the sequence similarity of their 16S rRNA directly paralleled the PLFA relationships. Desulfobacter latus and Desulfobacter curvatus grouped with the other Desulfobacter spp. by 16S rRNA comparison but not with the PLFA analysis as they contained significantly more monoenoic PLFA than the others. Similarly, Desulfovibrio africanus clustered with the Desulfovibrio spp. by 16S rRNA but not with them when analyzed by PLFA patterns because of higher monoenoic PLFA content. Otherwise, clustering obtained with either analysis was essentially congruent. The relationships defined by PLFA patterns appeared robust to shifts in nutrients and terminal electron acceptors. Additional analyses utilizing the lipopolysaccharide-lipid A hydroxy fatty acid patterns appeared not to shift the relationships based on PLFA significantly except when completely absent, as in Gram-positive bacteria. Phylogenetic relationships between isolates defined by 16S rRNA sequence divergence represent a selection clearly different from the multi-enzyme activities responsible for the PLFA patterns. Determination of bacterial relationships based on different selective pressures for various cellular components provides more clues to evolutionary history leading to a more rational nomenclature.
TL;DR: Nitrilase, which catalyses the hydrolysis of nitriles to the corresponding acids and ammonia, was originally discovered as a plant hormone indoleacetic acid-synthesising enzyme as mentioned in this paper.
Abstract: Nitrilase, which catalyses the hydrolysis of nitriles to the corresponding acids and ammonia, was originally discovered as a plant hormone indoleacetic acid-synthesising enzyme It is expected to be useful as a catalyst in organic chemical processing, and to have versatile functions in academic and applied fields
TL;DR: It is shown that in sucrose medium, A. niger produced high amounts of gluconic and oxalic acids, whereas in lactose permeate medium only oxalal acid was produced.
Abstract: The complex-forming compound oxalic acid can effectively solubilise metals such as aluminium, iron, lithium, and manganese. In order to produce high amounts of oxalic acid for biohydrometallurgical processes, it was the aim of this work to optimise oxalic acid production by Aspergillus niger, a fungus well known for its ability to produce oxalic acid. A. niger excreted 427 mmol oxalic acid 1−1 if it was cultivated in a pH-controlled (pH 6.0) fed-batch run in a 2-1 stirred tank reactor. Sucrose and lactose permeate were suitable carbon sources for oxalic acid production. In sucrose medium, A. niger produced high amounts of gluconic and oxalic acids, whereas in lactose permeate medium only oxalic acid was produced. Cultivation in green syrup and molasses media lead to high yields of biomass, but low oxalic acid production (<20 mmol 1−1).
TL;DR: Results revealed that the human ileocecal adenocarcinoma (HCT-8) cell line supported nearly twice the number of parasite developmental stages than MDBK cells or any of the other host cell types.
Abstract: Using standardized media, incubation, and parasite inoculating procedures, we compared development of Crytosporidium parvum between Madin-Darby bovine kidney (MDBK) cells and 10 additional host cell lines available through the American Type Culture Collection. Parasite development was assessed by counting parasite numbers atop monlayers in 25 random oil fields 68 h post-infection using Nomarski interference-contrast optics. Results revealed that the human ileocecal adenocarcinoma (HCT-8) cell line supported nearly twice the number of parasite developmental stages than MDBK cells or any of the other host cell types.
TL;DR: In the oral streptococci, two families of surface protein receptors with highly conserved amino acid sequences have been identified and ligand-binding specificities of these receptor polypeptides may account for species-specific adherence and site-directed colonization of streptitis within the human oral cavity.
Abstract: Streptococci have a vast repertoire of adherence properties which include binding to human tissue components, epithelial cells and to other bacterial cells These interactions are determined by the expression of cell-surface receptors some of which are species-specific In the oral streptococci, two families of surface protein receptors with highly conserved amino acid sequences have been identified The antigen I/II family of polypeptides are wall-associated high molecular mass proteins (158-166 kDa) with several binding functions that may be attributed to different domains of the receptor molecules The LraI family of polypeptides are surface-associated lipoproteins (32-33 kDa) involved in adherence of streptococci to salivary glycoprotein pellicle and to oral Actinomyces A region of amino acid sequence similarity is evident amongst members of the two protein families in Streptococcus gordonii Ligand-binding specificities of these receptor polypeptides may account for species-specific adherence and site-directed colonization of streptococci within the human oral cavity
TL;DR: The majority of thermophilic strains of the genus Bacillus were found to cluster in two groups represented by B. stearothermophilus and B. pallidus, and the as yet undescribed taxon 'B. flavothermus' warrants species status.
Abstract: Sixteen thermophilic strains of the genus Bacillus, representing eight validly described and six invalidly described species, as well as one unassigned strain, were investigated by comparative 16S rDNA analyses and the sequences compared to the existing database for the genera Bacillus and Alicyclobacillus. The majority of strains were found to cluster in two groups represented by B. stearothermophilus and B. pallidus. Bacillus smithii, B. thermocloacae, and B. thermoruber are phylogenetically well separated and cluster within the radiation of mesophilic bacilli. The as yet undescribed taxon ‘B. flavothermus’ warrants species status. B. schlegelii and B. tusciae group peripherally with members of Alicyclobacillus and may be reclassified when more phenotypic data support their phylogenetic position.
TL;DR: The presence of dolichyl phosphate and a family of saturated isoprenoid lipids in Archaebacteria suggests a possible evolutionary link between the bacterial and eukaryotic systems.
Abstract: The peptidoglycan layer of bacterial cell walls is biosynthesised using a lipid carrier undecaprenyl phosphate to assemble and transport the MurNAc(GlcNAc)-pentapeptide precursor. Similar lipid-linked cycles are involved in the biosynthesis of other bacterial exopolysaccharides and eukaryotic asparagine-linked glycoproteins, the latter involving the structurally related dolichyl phosphate as a lipid carrier. Recent protein sequence data and common inhibitors of the bacterial and eukaryotic systems have revealed functional similarities between the two systems. Biological and physical studies on the lipid carriers themselves have provided clues to their role in oligosaccharide translocation, but have not revealed significant differences in function between undecaprenyl phosphate and dolichyl phosphate. The presence of dolichyl phosphate and a family of saturated isoprenoid lipids in Archaebacteria suggests a possible evolutionary link between the two systems.
TL;DR: Comparative sequence analysis of the 16S rDNA of 14 alkaliphilic or alkalitolerant, Gram-positive, aerobic, endo-spore forming bacterial strains was performed and showed the majority of isolates clustered with B. alcalophilus DSM 485T forming a distinct phylogenetic group within the radiation of the genus Bacillus and related taxa.
Abstract: Comparative sequence analysis of the 16S rDNA of 14 alkaliphilic or alkalitolerant, Gram-positive, aerobic, endo-spore forming bacterial strains was performed. Bacillus alcalophilus DSM 485T and Bacillus cohnii DSM 6307T were included to represent the two validly described alkaliphiles assigned to the genus Bacillus. The majority of isolates (8 strains) clustered with B. alcalophilus DSM 485T forming a distinct phylogenetic group (rRNA group 6) within the radiation of the genus Bacillus and related taxa. Bacillus cohnii DSM 6307T and two of the isolates, DSM 8719 and DSM 8723, grouped with B. fastidiosus and B. megaterium and are allocated to rRNA group 1. The remaining two strains DSM 8720 and DSM 8721 show an equidistant relationship to both groups.
TL;DR: An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector and methylation of the plasmid with PstI methylase increased the methanococcal transformation frequency at least four-fold.
Abstract: An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector. The frequency of transformation with 0.8 μg of plasmid and 3×109 cells was 4.8×10−5 transformants cfu−1, or 1.8×105 transformants μg−1, which was four orders of magnitude greater than with the natural transformation method. A PstI restriction activity in M. maripaludis was also identified. Methylation of the plasmid with PstI methylase increased the methanococcal transformation frequency at least four-fold. Also, chromosomal DNA from M. maripaludis was resistant to digestion by the PstI endonuclease.
TL;DR: The nature of amino acid 241 is critical in conferring resistance or susceptibility to beta-lactamase inhibitors, and these basic to neutral amino acid replacements explain the more acidic pI of these IRT enzymes compared to that of TEM-1.
Abstract: Two blaTEM-like genes were characterized that encoded IRT β-lactamases (previously called TRI) in clinical isolates of Escherichia coli resistant to amoxycillin alone and to combinations of amoxycillin with β-lactamase inhibitors. Plasmids carrying this resistance were isolated from E. coli K 12 transconjugants and the genes were sequenced after amplification of defined fragments, using TEM-1-specific primers. The gene for IRT-1 β-lactamase resembled the blaTEM-1B gene, and that for IRT-2 resembled blaTEM-2. However, both IRT enzymes have a glutamine residue at position 37, which is characteristic of TEM-1. The unique nucleotide difference with parental genes corresponding to amino acid variation was observed at nucleotide position 929. The consequence of C to T transition in the blaIRT-1 gene and C to A transversion in the blaIRT-2 gene was the substitution of arginine 241 in the native protein by cysteine and serine, respectively, in the mutants. Thus, the nature of amino acid 241 is critical in conferring resistance or susceptibility to β-lactamase inhibitors. Furthermore, these basic to neutral amino acid replacements explain the more acidic pI(pI=5.2) of these IRT enzymes compared to that of TEM-1 (pI=5.4). The presence of cysteine-241 in IRT-1 also explains the selective sensitivity of this β-lactamase to inhibition by p-chloromercuribenzoate.
TL;DR: Purified MalE-AmpD fusion protein was found to have murein amidase activity with a pronounced specificity for 1,6-anhydromuropeptides, the characteristic muresin turnover products in Escherichia coli, and it is suggested that the negative regulatory effect of AmpD is due to the hydrolysis of anhydro-muropePTides which may function as signals for beta-lactamase induction.
Abstract: Construction of a malE-ampD gene fusion allowed purification of biologically active fusion protein by affinity chromatography. The cloned malE-ampD gene fusion complemented a chromosomal ampD mutation. Purified MalE-AmpD fusion protein was found to have murein amidase activity with a pronounced specificity for 1,6-anhydromuropeptides, the characteristic murein turnover products in Escherichia coli. Being a N-acetyl-anhydromuramyl-L-alanine amidase AmpD is likely to be involved in recycling of the turnover products. It is suggested that the negative regulatory effect of AmpD is due to the hydrolysis of anhydro-muropeptides which may function as signals for β-lactamase induction.
TL;DR: Methanogens appeared in the rumen of 30-h-old lambs, and as they developed there was a proportional decrease in the numbers of acetogens, indicating a competition for hydrogen between these two groups.
Abstract: The development of hydrogenotrophic bacteria in the rumen of lambs was investigated by culture and labeling experiments. 14CO2 and 13CO2 incorporation by the rumen microflora of a 24-h-old lamb showed that while there was no labeled methane, double-labeled acetate was formed indicating the presence of hydrogen-dependent acetogenesis. In vitro counts from rumen fluid of 20-h-old lambs confirmed an extensive colonization of acetogenic bacteria while methanogens were absent. Methanogens appeared in the rumen of 30-h-old lambs, and as they developed there was a proportional decrease in the numbers of acetogens, indicating a competition for hydrogen between these two groups. Hydrogen-utilizing sulfate-reducing bacteria, which were established by the 3rd day after birth, did not seem to be affected by this competition.
TL;DR: In this article, the influence of culture conditions on FTIR spectra and the discrimination of Lactobacillus species found in breweries was investigated. But the results were limited.
Abstract: Fourier Transform infrared (FTIR) spectroscopy can be used to identify microorganisms. This study describes the influence of culture conditions on FTIR spectra and the discrimination of Lactobacillus species found in breweries. Fifty three Lactobacillus strains were analysed by FTIR spectroscopy and identification at the species level was correct for 94% of the strains, and at the strain level for 91% of the strains.
TL;DR: Analysis of SfiI-digested genomic DNA by zero integrated-field electrophoresis followed by Southern hybridization revealed vanB-containing chromosomal insertions of approximately 90-250 kb in the transconjugants, indicating transfer of VanB-type resistance is associated with the movement of large genetic elements from chromosome to chromosome.
Abstract: Resistance to various levels of vancomycin and susceptibility to teicoplanin in enterococci (VanB phenotype) is mediated by the vanB gene cluster. VanB-type resistance was transferred by intra- and inter-specific conjugation between different strains of Enterococcus . Analysis of S⨍i I-digested genomic DNA by zero integrated-field electrophoresis followed by Southern hybridization revealed vanB -containing chromosomal insertions of approximately 90–250 kb in the transconjugants. Thus, transfer of VanB-type resistance is associated with the movement of large genetic elements from chromosome to chromosome.
TL;DR: Both approaches show, under conditions in which the growth of a limited number of viable cells during resuscitation is excluded, that a significant portion of the apparently non-viable cell population in an extended stationary phase is dormant, and not dead.
Abstract: Micrococcus luteus starved for 2–7 months in spent medium following growth to stationary phase in batch culture exhibited a culturability (as estimated by direct plating on nutrient agar plates) of < 0.001%. However, following a lag, some 70% of the cells could be lysed upon inoculation into and cultivation in fresh lactate minimal medium containing penicillin, showing the capability of a significant portion of the cells at least to enlarge (and thus potentially to resuscitate). When the viable cell count was estimated using the most probable number method, by incubation of high dilutions of starved cells in liquid growth media, the number of culturable or resuscitable cells was very low, and little different from the viable cell count as assessed by plating on solid media. However, the apparent viability of these populations evidenced with the most probable number method was 1000–100 000-fold greater when samples were diluted into liquid media containing supernatants taken from the stationary phase of batch cultures of the organism, suggesting that viable cells can produce a factor which stimulates the resuscitation of dormant cells. Both approaches show, under conditions in which the growth of a limited number of viable cells during resuscitation is excluded, that a significant portion of the apparently non-viable cell population in an extended stationary phase is dormant, and not dead.
TL;DR: Variations in culture and assay conditions affected both the rate of denitrification and the ratio of end products (N2O:N2).
Abstract: The production of nitrogen-containing gases by denitrification in three organisms was examined using membrane inlet mass spectrometry. The effects of O2 (during both growth and maintenance) and of pH, nitrate concentration and carbon source were tested in non-proliferating cell suspensions. Two strains of Pseudomonas aeruginosa were capable of co-respiration of NO3- and O2 and, under controlled O2 supply, gave oscillatory denitrification. Variations in culture and assay conditions affected both the rate of denitrification and the ratio of end products (N2O:N2). Higher rates were seen following anaerobic growth. Optimum values of pH and nitrate concentration for denitrification are given. Generally, the optimum pH was 7.0-7.5, approximately that of the growth medium. Optimum nitrate concentration was generally 20 mM.
TL;DR: Two Cryptococcus neoformans strains isolated from an AIDS patient were investigated and the sterol composition of CN3 indicated a defect in sterol delta 8-->7 isomerase in this strain and depletion of ergosterol, the major sterol of the CN1.
Abstract: Two Cryptococcus neoformans strains isolated from an AIDS patient were investigated, a pretreatment isolate (CN1) and a second isolate (CN3) following failure of fluconazole and amphotericin B treatment. No difference in fluconazole sensitivity, but relative resistance to amphotericin B was observed for CN3. The sterol composition of CN3 indicated a defect in sterol delta 8-->7 isomerase in this strain and depletion of ergosterol, the major sterol of the CN1.
TL;DR: Two technical modifications of a novel approach for the type-specific identification of emm genes have been developed and the feasibility of these rapid and easy to perform methods was shown for the unequivocal identification of reference strains and clinical isolates belonging to 16 different M serotypes.
Abstract: Because of the allelic variations within the M protein gene (emm gene) of group A streptococci, reliable typing of this important human pathogen can be accomplished by the use of emm gene-specific oligonucleotide probes. Two technical modifications (a reverse dot blot and a reverse line blot hybridization assay) of a novel approach for the type-specific identification of emm genes have been developed. Both procedures involved amplification of an emm gene by polymerase chain reaction. The non-radioactively labeled amplicon was subsequently hybridized to a membrane carrying an array of immobilized emm gene-specific oligonucleotide probes, thus allowing the simultaneous analysis of the gene polymorphism in a single hybridization reaction. The feasibility of these rapid and easy to perform methods was shown for the unequivocal identification of reference strains and clinical isolates belonging to 16 different M serotypes.
TL;DR: Physical mapping with restriction endonucleases and nucleotide sequence determination revealed the existence of a 702 bp long deletion, occurring between two short direct repeats, in the chromosome of B19, which rendered the B19 strain sensitive to erythritol.
Abstract: Brucella abortus B19, an avirulent strain obtained by spontaneous mutation, is used worldwide as a vaccine for the control of bovine brucellosis. B19 differs from other B. abortus strains in its sensitivity to erythritol. We took advantage of a previously obtained erythritol sensitive Tn5 insertion mutant of B. abortus 2308 to clone the chromosomal region containing erythritol catabolic genes from this representative pathogenic strain and from the vaccine strain B19. Physical mapping with restriction endonucleases and nucleotide sequence determination revealed the existence of a 702 bp long deletion, occurring between two short direct repeats, in the chromosome of B19. This deletion rendered the B19 strain sensitive to erythritol. Two oligonucleotides whose sequences flank this deletion provided an easy method to differentiate B19 from all other B. abortus isolates.