- Volume 143, Issue 2, 1997
Volume 143, Issue 2, 1997
- Review Article
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- Microbiology Comment
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- Candida Albicans
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WO-2, a stable aneuploid derivative of Candida albicans strain WO-1, can switch from white to opaque and form hyphae
More LessCandida albicans strain WO-2 was isolated as a spontaneous derivative of the white-opaque switching strain WO-1. The electrophoretic karyotype of WO-2 lacks two bands which are found in the parent. These bands correspond to one homologue of chromosome 7 and to a translocation product containing parts of chromosomes 6 and 5. Probing a blot of the karyotype demonstrated that the genetic material in these bands had been lost, yielding an aneuploid strain. UV-irradiation experiments showed that auxotrophs due to mutation in genes located in this region predominated, supporting the conclusion that WO-2 is partially haploid. WO-2 contained about 10% of its genome in the haploid state, and it grew with a doubling time of about twice that of its parent. However, it was able to undergo both the yeast-to-hyphal transition and the white-opaque transition. Hence, these processes do not require perfect diploidy.
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Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans
More LessThe ARG5,6 gene from the dimorphic fungus Candida albicans was cloned by functional complementation of the arginine auxotrophy present in strain EL2 (Arg-) using a gene library constructed in the double autonomously replicating sequence vector pRM1. Sequence analysis revealed a putative 857 amino acid polypeptide (95 kDa) which showed high homology (63% protein identity) to the Saccharomyces cerevisiae ARG5,6 gene. Similarly to the S. cerevisiae gene, the C. albicans ARG5,6 gene is responsible for both the acetylglutamate kinase and acetylglutamyl-phosphate reductase activities, the second and third steps of arginine biosynthesis at the mitochondria. The C. albicans ARG5,6 gene complemented the arg6 mutation present in S. cerevisiae (strain D160-4D) on a yeast episomal plasmid using its own regulatory signals. A set of non-integrative high-efficiency plasmid vectors based on this gene marker was constructed and a null C. albicans arg5,6d strain was obtained using the common URA3-blaster strategy. In addition, we generated an arg5,6d null mutant in a single transformation event, thus improving the basic strategy for generating gene deletions in C. albicans.
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Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans
The green fluorescent protein (GFP) of Aequorea victoria has been developed here as a reporter for gene expression and protein localization in Candida albicans. When wild-type (wt) GFP was expressed in C. albicans, it was not possible to detect fluorescence or a translation product for the wt protein. Since this was probably due in part to the presence of the non-canonical CTG serine codon in the Aequorea sequence, this codon was changed to the leucine codon TTG. C. albicans cells expressing this construct contained GFP mRNA but were non-fluorescent and contained no detectable translation product. Hence a codon-optimized GFP gene was constructed in which all of the 239 amino acids are encoded by optimal codons for C albicans. In this gene were also incorporated two previously identified mutations in the chromophore that increase GFP fluorescence. C. albicans cells expressing this yeast-enhanced GFP gene (yEGFP3) are fluorescent and contain GFP protein. yEGFP3 can be used as a versatile reporter of gene expression in C. albicans and Saccharomyces cerevisiae and the optimized GFP described here should have broad applications in these and other fungal species.
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The highly immunogenic enolase and Hsp70p are adventitious Candida albicans cell wall proteins
More LessScreening cDNA libraries with polyclonal antibody preparations against Candida albicans yeast or mycelial cell walls resulted in isolation of several positive clones. Some of them encoded enolase; others encoded a protein of the 70 kDa heat-shock protein family (Hsp70p), etc. The presence of these cytosolic proteins in the cell wall of actively growing C. albicans was discovered by analytical (SDS-PAGE and Western blot) and cytological (indirect immunofluorescence) experiments. Supplementation of cell cultures with papulacandin B, an antibiotic that inhibits formation of the -glucan skeleton, resulted in the release of enolase to the supernatant fluids; this release was prevented when 0.6 M KCI was present as an osmotic stabilizer. The cell wall of C. albicans incorporated exogenously added proteins (enolase and Escherichia coli and C albicans cytosolic proteins). The presence in the C. albicans cell wall of enolase, Hsp70p, and probably other intracellular proteins that are highly immunogenic might help the fungal cells to evade the host defences, and consequently could represent a survival mechanism for C. albicans in vivo.
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3-Phosphoglycerate kinase: a glycolytic enzyme protein present in the cell wall of Candida albicans
More LessWe have used a polyclonal antiserum to cell wall proteins of Candida albicans to isolate several clones from a cDNA λgt11 expression library. Affinity-purified antibody prepared to the fusion protein of one clone identified a 40 kDa moiety present in cell wall extracts from both morphologies of the organism. Indirect immunofluorescence demonstrated expression of this moiety at the C. albicans cell surface. Sequencing of a pBluescript II genomic clone identified with the cDNA clone revealed an open reading frame for a 417 amino acid protein. The nucleotide sequence showed significant homology with 3-phosphoglycerate kinase (PGK) genes, with 88%, 77% and 76% nucleotide homology with the PGK genes from Candida maltosa, Saccharomyces cerevisiae and Kluyveromyces lactis, respectively. The deduced amino acid sequence was consistent with this identification of the sequence as PGK1 of C. albicans. This finding was confirmed by a positive immunological response of a commercially available purified PGK from S. cerevisiae with the affinity-purified antibody against the fusion protein of the cDNA clone. The presence of PGK in the cell wall was confirmed by two additional methods. Cell wall proteins were biotinylated with a derivative that does not permeate the cell membrane to distinguish extracellular from cytosolic proteins. Biotinylated PGK was detected among the biotinylated proteins obtained following streptavidin affinity chromatography. Immunoelectron microscopy revealed that the protein was present at the outer surface of the cell membrane and cell wall as well as expected in the cytoplasm. Northern blot analysis revealed that the gene transcript was present in C albicans cells growing under different conditions, including different media, temperatures and morphologies. Most of the enzyme activity was found in the cytosol. Low enzymic activity was detected in intact cells but not in culture filtrates. These observations confirmed that PGK is a bona fide cell wall protein of C. albicans.
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Cloning and characterization of a gene (LIP1) which encodes a lipase from the pathogenic yeast Candida albicans
More LessExtracellular phospholipases are demonstrated virulence factors for a number of pathogenic microbes. The opportunistic pathogen Candida albicans is known to secrete phospholipases and these have been correlated with strain virulence. In an attempt to clone C. albicans genes encoding secreted phospholipases, Saccharomyces cerevisiae was transformed with a C. albicans genomic library and screened for lipolytic activity on egg-yolk agar plates, a traditional screen for phospholipase activity. Two identical clones were obtained which exhibited lipolytic activity. Nucleotide sequence analysis identified an ORF encoding a protein of 351 amino acid residues. Although no extensive homologies were identified, the sequence contained the Gly-X-Ser-X-Gly motif found in prokaryotic and eukaryotic lipases, suggesting a similar activity for the encoded protein. Indeed, culture supernatants from complemented yeast cells contained abundant hydrolytic activity against a triglyceride substrate and had no phospholipase activity. The data suggest that C. albicans, in addition to phospholipases, also has lipases. Southern blot analyses revealed that C. albicans may contain a lipase gene (LIP) family, and that a lipase gene(s) may be present in Candida parapsilosis, Candida tropicalis and Candida krusei, but not in Candida pseudotropicalis, Candida glabrata or S. cerevisiae. Northern blot analyses showed that expression of the LIP1 transcript, the cloned gene which encodes a lipase, was detected only when C. albicans was grown in media containing Tween 80, other Tweens or triglycerides as the sole carbon source, and not in Sabouraud Dextrose Broth or yeast/peptone/dextrose media. Additionally, carbohydrate supplementation inhibited LIP1 expression. Cloning this gene will allow the construction of LIP1-deficient null mutants which will be critical in determining the role of this gene in candidal virulence.
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Identification of salivary basic proline-rich proteins as receptors for Candida albicans adhesion
More LessThe adherence of Candida albicans cells to oral surfaces is believed to be an important step in the development of oral candidosis. Electrophoretically separated parotid salivary proteins were transferred to nitrocellulose membranes and incubated with [35S]methionine-radiolabelled C. albicans cells in a cell overlay adherence assay. A subset of four proteins with apparent molecular masses of 17, 20, 24 and 27 kDa (designated bands A-D) acted as receptors for cells of C. albicans ATCC 10261 and four clinical C. albicans isolates, in overlay assays. The N-terminal amino acid sequence of bands A-D indicated that these proteins were members of the basic proline-rich protein (bPRP) family. Digestion of protein A with endoproteinase Glu-C resulted in a single band (designated Ap) detected by Coomassie blue staining after SDS-PAGE. This band was not bound by C. albicans cells in overlay assays and comprised two fragments, designated ApN and ApC. These fragments had N-terminal sequences corresponding to the N-terminal and post endoproteinase Glu-C cleavage site sequences of bPRP IB-6 and had molecular masses of 618S and 4261 Da as determined by mass spectrometry. Thus intact bPRP IB-6, and other bPRPs, may act as receptors for C. albicans adhesion.
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Analysis of secreted aspartic proteinases from Candida albicans: purification and characterization of individual Sap1, Sap2 and Sap3 isoenzymes
More LessThe recently discovered secreted aspartic proteinase multi-gene (SAP) family in Candida albicans has complicated assessment of proteolytic activity as a factor in the onset and development of Candida infections. Differential expression of the SAP genes under various conditions, as well as possible variation in the properties of the individual isoenzymes, have consequences for immunological detection, for targeted drug design and possibly for pathogenicity. It is therefore important to be able to monitor Sap isoenzyme profiles in different strains of C. albicans cultures, and to know the biochemical properties of each isoenzyme. We have employed a simple purification protocol based on strong anion exchange chromatography for the direct analysis of C. albicans Sap isoenzymes from culture filtrates, as well as recovery of individual Sap1, Sap2 and Sap3 products. In the case of Sap1, this involved development of an overexpression system using the pEMBLyex4 vector transformed into Saccharomyces cerevisiae. The C albicans strains ATCC 10231 and 10261 were shown to produce different ratios of Sap2 and Sap3 under the same conditions. Analysis of all three purified proteins by gel electrophoresis, immunoblotting and proteinase assays which were designed to evaluate pH dependence, thermal stability and substrate specificity revealed similar but distinct properties for each isoenzyme. Although Sap3 was shown to be antigenically more similar to Sap2 than was Sap1, it was less similar in terms of thermal stability and activity at low pH, being more stable and more active.
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N-Myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA:protein N-myristoyltransferase
Myristoyl-CoA:protein N-myristoyltransferase (Nmt) catalyses the covalent attachment of myristate to the N-terminal glycine of a small subset of cellular proteins produced during vegetative growth of Candida albicans. nmt447D is a mutant NMT allele encoding an enzyme with a Gly447 ? Asp substitution and reduced affinity for myristoyl-CoA. Among isogenic NMT/NMT, NMT/dnmt and nmtd/nmt447D strains, only nmtd/nmt447D cells require myristate for growth on yeast/peptone/dextrose media (YPD) at 24 or 37 . When switched from YPD/myristate to YPD alone, 60% of the organisms die within 4 h. Antibodies raised against the C-terminal eight residues of Saccharomyces cerevisiae Arf1p were used to probe Western blots of total cellular proteins prepared from these isogenic Candida strains. N-Myristoylation of C. albicans ADP-ribosylation factor (Arf) produced a change in its electrophoretic mobility during SDS-PAGE: the myristoylated species migrated more rapidly than the nonmyristoylated species. In an NMT/nmtd, strain, 100% of the Arf is N-myristoylated based on this mobility shift assay. When exponentially growing nmtd/nmt447D cells were incubated at 24 in YPD/myristate, < 25% cellular Arf was nonmyristoylated. In contrast, 2 or 4 h after withdrawal of myristate, = 50% of total cellular Arf was nonmyristoylated. This finding suggests that = 50% reduction in Arf N-myristoylation is a biochemical marker of a growth-arrested cell. A similar conclusion was made after assaying isogenic S. cerevisiae strains containing various combinations of NMT1, nmt1-451D, ARF1, arf1d, ARF2 and arf2d alleles and grown at 24-37 on YPD or YPD/myristate. Peptidomimetic inhibitors of C. albicans Nmt were synthesized based on the N-terminal sequence of an S. cerevisiae Arf. SC-59383 has an IC50 of 1.45 + 0.08 M for purified C. albicans Nmt and is 560-fold selective for the fungal compared to human N-myristoyltransf erase. It had an EC50 of 51 + 17 and 67 + 6 M, 24 and 48 h after a single administration of the drug to cultures of C. albicans. The Arf gel mobility shift assay indicated that a single dose of 200 M produced a < 50% reduction in Arf N-myristoylation after 4 h, which is consistent with the fungistatic, but not fungicidal, activity. The effect on Nmt was specific: an enantiomer, SC-59840, had no inhibitory effect on purified C. albicans Nmt (IC50 > 1000 M), and 200 M of the compound produced no detectable reduction in Arf N-myristoylation in vivo. SC-58272, which is related to SC-59383, was a more potent inhibitor in vitro (IC50 0.056 + 001 M), but had no growth inhibitory activity and did not produce any detectable reduction in Arf N-myristoylation. These findings highlight the utility of the Arf protein gel mobility shift assay for demonstrating the mechanism-based antifungal activity of SC-59383, a selective inhibitor of C. albicans Nmt.
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Phenotype in Candida albicans of a disruption of the BGL2 gene encoding a 1,3-β-glucosyltransferase
The BGL2 gene encodes a unique 1,3-β-glucosyltransferase (BgI2p) present in the cell wall of Candida albicans and other fungi. Although believed to be involved in cell wall assembly, disruption of the gene in Saccharomyces cerevisiae showed no apparent phenotype. We performed sequential disruptions of the BGL2 loci in a homozygous ura3 clinical isolate of C. albicans using the URA3 blaster method, in order to investigate the role of BgI2p in this dimorphic, pathogenic fungus. Strain CACW-1 contained disruptions of both homologues of the BGL2 gene and lacked BgI2p, as assessed by protein extraction, SDS-PAGE and Western blot analysis, and enzyme assay; however, residual non-BgI2p transferase activity was detected. CACW-1 was attenuated in virulence for mice when compared to an isogenic parent strain, and fewer organisms were recovered from the kidneys of infected animals. Additional phenotypic changes included: (1) a dramatic increase in the sensitivity to the chitin synthesis inhibitor nikkomycin Z when CACW-1 cells were incubated at 37 or 42 °; (2) an 8.7+1.6% slower growth rate at 37 ° for CACW-1 when compared to its isogenic parent; and (3) aggregation of CACW-1 cells during stationary phase and/or incubation of stationary phase cells in phosphate buffer. Characterization of SDS-extracted cell walls did not reveal any significant differences in the levels of 1,3-β- or 1,6-β-glucan. These data reveal that loss of BgI2p does have a phenotype in C. albicans, and indicate that (1) loss of BgI2p function renders cells more dependent on chitin for wall integrity, and attenuates virulence (probably due to subtle changes in wall structure), and (2) that additional 1,3-β-glucosyltransferases are present in the C. albicans BGL2 disruptant.
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The topoisomerase I gene from Candida albicans
More LessWe report here the cloning of the Candida albicans genomic topoisomerase I gene (TOP1) by use of PCR and subsequent hybridization. The predicted protein sequence shared 58.8% identity with the Saccharomyces cerevisiae topoisomerase I and 30-50% identity with other eukaryotic topoisomerase I proteins. A conditional gene disruption strain (CWJ477) was constructed so that one copy of TOP1 was deleted and the other copy of TOP1 was placed under a regulatable promoter. Under repressed conditions, cells grew slowly and cell morphology was abnormal. The virulence of CWJ477 was markedly reduced in a mouse model system, and that of the single gene knockout strain was slightly attenuated, indicating that TOP1 might play a role in the infection of C. albicans in mice in a dose-dependent manner. Despite the reduced virulence of both the single and double knockout strains, viable cells of the pathogen were recovered from the kidneys as late as 22 d post-infection.
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An oligopeptide transport gene from Candida albicans
More LessA Candida albicans oligopeptide transport gene, OPT1, was cloned from a C. albicans genomic library through heterologous expression in the Saccharomyces cerevisiae di-/tripeptide transport mutant PB1X-9B. When transformed with a plasmid harbouring OPT1, S. cerevisiae PB1X-9B, which did not express tetra-/pentapeptide transport activity under the conditions used, was conferred with an oligopeptide transport phenotype, as indicated by growth on the tetrapeptide Lys-Leu-Leu-Gly, sensitivity to toxic tetra- and pentapeptides, and an increase in the initial uptake rate of the radiolabeled tetrapeptide Lys-Leu-Gly-[3H]Leu. The level of oligopeptide transport was found to be influenced in the heterologous host by the source of nitrogen used for growth. The entire 3.8 kb fragment containing the oligopeptide transport activity was sequenced and an ORF of 2349 nucleotides containing a 58 nucleotide intron was identified. The deduced protein product of 783 amino acid residues contained 12 hydrophobic regions suggestive of a membrane transport protein. Sequence comparisons revealed that similar proteins are encoded by genes from S. cerevisiae and Schizosaccharomyces pombe and that OPT1 is not a member of the ABC or PTR membrane transport families.
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Functional reconstitution of a purified proline permease from Candida albicans: interaction with the antifungal cispentacin
More LessWe have purified proline permease to homogeneity from Candida albicans using an L-proline-linked agarose matrix as an affinity column. The eluted protein produced two bands of 64 and 67 kDa by SDS-PAGE, whereas it produced a single band of 67 kDa by native PAGE and Western blotting. The apparent K m for L-proline binding to the purified protein was 153 �M. The purified permease was reconstituted into proteoliposomes and its functionality was tested by imposing a valinomycin-induced membrane potential. The main features of L-proline transport in reconstituted systems, viz. specificity and sensitivity to N-ethylmaIeimide, were very similar to those of intact cells. The antifungal cispentacin, which enters C. albicans cells via an inducible proline permease, competitively inhibited the L-proline binding and translocation in reconstituted proteoliposomes. However, the uptake of L-proline in proteoliposomes reconstituted with the purified protein displayed monophasic kinetics with an apparent K m of 40 �M.
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Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene
More LessResistance to azole antifungal agents in Candida albicans can be mediated by multidrug efflux transporters. In a previous study, we identified at least two such transporters, Cdr1p and Benp, which belong to the class of ATP-binding cassette (ABC) transporters and of major facilitators, respectively. To isolate additional factors potentially responsible for resistance to azole antifungal agents in C. albicans, the hypersusceptibility of a Saccharomyces cerevisiae multidrug transporter mutant, δpdr5, to these agents was complemented with a C. albicans genomic library. Several new genes were isolated, one of which was a new ABC transporter gene called CDR2 ( Candida drug resistance). The protein Cdr2p encoded by this gene exhibited 84% identity with Cdr1p and could confer resistance to azole antifungal agents, to other antifungals (terbinafine, amorolfine) and to a variety of metabolic inhibitors. The disruption of CDR2 in the C. albicans strain CAF4-2 did not render cells more susceptible to these substances. When the disruption of CDR2 was performed in the background of a mutant in which CDR1 was deleted, the resulting double δcdr1 δcdr2 mutant was more susceptible to these agents than the single δcdr1 mutant. The absence of hypersusceptibility of the single δcdr2 mutant could be explained by the absence of CDR2 mRNA in azole-susceptible C albicans strains. CDR2 was overexpressed, however, in clinical C. albicans isolates resistant to azole antifungal agents as described previously for CDR1, but to levels exceeding or equal to those reached by CDR1. Interestingly, CDR2 expression was restored in δcdr1 mutants reverting spontaneously to wild-type levels of susceptibility to azole antifungal agents. These data demonstrate that CDR2 plays an important role in mediating the resistance of C. albicans to azole antifungal agents.
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A DNA-binding protein from Candida albicans that binds to the RPG box of Saccharomyces cerevisiae and the telomeric repeat sequence of C. albicans
Electromobility shift assays with a DNA probe containing the Saccharomyces cerevisiae ENO1 RPG box identified a specific DNA-binding protein in total protein extracts of Candida albicans. The protein, named Rbf1p (RPG-box-binding protein 1), bound to other S. cerevisiae RPG boxes, although the nucleotide recognition profile was not completely the same as that of S. cerevisiae Rap1p (repressor-activator protein 1), an RPG-box-binding protein. The repetitive sequence of the C. albicans chromosomal telomere also competed with RPG-box binding to Rbf1p. For further analysis, we purified Rbf1p 57600-fold from C. albicans total protein extracts, raised mAbs against the purified protein and immunologically cloned the gene, whose ORF specified a protein of 527 aa. The bacterially expressed protein showed RPG-box-binding activity with the same profile as that of the purified one. The Rbf1p, containing two glutamine-rich regions that are found in many transcription factors, showed transcriptional activation capability in S. cerevisiae and was predominantly observed in nuclei. These results suggest that Rbf1p is a transcription factor with telomere-binding activity in C. albicans.
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Biochemical and genetic characterization of Rbf1p, a putative transcription factor of Candida albicans
More LessA Candida albicans gene encoding a novel DNA-binding protein that bound to the RPG box of Saccharomyces cerevisiae and the telomeric repeat sequence o C albicans was previously cloned and designated RBF1 (RPG-box-binding factor). In this report, determination of the functional domains of the protein is described. The DNA-binding domain was 140 aa in length, was centrally located between two glutamine-rich regions, and correlated with transcriptional activation in S. cerevisiae. The results, together with the previous finding that showed its predominant localization in the nucleus, suggest that this DNA-binding protein could be a transcription factor. Disruption of the functional RBF1 gene of C. albicans strains caused an alteration in cell morphology to the filamentous form on all solid and liquid media tested. Thus, we speculate that Rbf1p may be involved in the regulation of the transition between yeast and filamentous forms at the level of transcription.
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Metabolism of inositol 1,4,5-trisphosphate in Candida albicans: significance as a precursor of inositol polyphosphates and in signal transduction during the dimorphic transition from yeast cells to germ tubes
More LessThe metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was examined in yeast cells and germ tubes of Candida albicans. Methods have been developed for analysis of the two key metabolic enzymes, Ins(1,4,5)P3 kinase and phosphatase. ATP-dependent Ins(1,4,5)P3 kinase activity was detected predominantly in the soluble fraction of cell extracts and exhibited a K m of approximately 9 μM. The apparent K m of Ins(1,4,5)P3 phosphatase for Ins(1,4,5)P3 was approximately 480 μM. The slow rate of dephosphorylation of Ins(1,4,5)P3 to inositol bisphosphate suggests a lower importance of the phosphatase within cells compared to the kinase. Since both yeast cells and germ tubes of C. albicans rapidly phosphorylated Ins(1,4,5)P3 to inositol tetrakisphosphate and inositol penta/hexakisphosphate, it is suggested that Ins(1,4,5)P3 has an important role as a precursor for production of these compounds. A sustained increase in cellular Ins(1,4,5)P3 levels was observed during germ tube formation and, prior to the onset of germination between 1 and 2 h incubation, the Ins(1,4,5)P3 content increased up to eightfold. Transien increases in the level of Ins(1,4,5)P3 were also observed during yeast-like growth of C. albicans. The possible role and relative importance of Ins(1,4,5)P3 as a precursor for inositol polyphosphates and in signal transduction involving Ca2+ release from internal stores is discussed.
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- Antigens And Immunity
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O-antigenic determinants in Salmonella species of serogroup C1 are expressed in distinct immunochemical populations of chains
More LessThe O-antigenic specificities found among salmonellae of serogroup C1 are O:61,7, O:62,7, O:61,62,7 and O:6,7,14, as defined by classical serology. Factor O:7 is the group-wide determinant while factors O:61, O:62 and O:14 are found in some strains but not others. Strains of the O:62,7 specificity are subject to lysogenic conversion by phages 61 and 14 to the O:61,7 and O:6,7,14 specificities, respectively. To further delineate antigenic complexity and serological relationships among strains of this serogroup monoclonal antibodies (mAbs) were generated against the O:61,62,7 polysaccharide of Salmonella thompson. Five mAbs of either the O:61, or the O:62 specificities did not bind O:6,7,14 strains or LPS, showing that the O:6 determinant in these strains is neither O:61, nor O:62. Thus antigenic conversion of O:62,7 strains by phage 14 is accompanied by addition of O:14 as well as loss of O:62 Three mAbs which demonstrated group-wide reactivity, and were thus specific for O:7, recognized clearly separable epitopes hereby defined as sub-specificities, O:71 O:72 and O:73 Immunoblotting of mAbs against electrophoretically resolved LPS showed that factors O:61 and O:62 are expressed only in LPS molecules of high molecular mass whereas O:72 and O:73 are expressed only in relatively low-molecular-mass chains. These results are consistent with the expression of different antigenic determinants in structurally distinct subpopulations of O chains. The implication of the existence of distinct subpopulation of chains is that the published structure of the O:6,7 repeat unit is not fully representative of the O-antigenic structure of this group.
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Identification of novel species-specific antigens of Mycoplasma hyopneumoniae by preparative SDS-PAGE ELISA profiling
More LessMycoplasma hyopneumoniae, M. hyorhinis and M. flocculare are commonly isolated from the respiratory tract of pigs and are phylogenetically related. The identification and characterization of antigens specific for M. hyopneumoniae is crucial for the development of serological reagents and for understanding the mechanisms of pathogenicity of this pathogen. Protein and antigen profiles of six strains of M. hyopneumoniae, four strains of M. hyorhinis and a type strain of M. flocculare were compared using SDS-PAGE and immunoblotting. Five strains of M. hyopneumoniae originally isolated from diverse geographical regions produced similar protein and antigen profiles. One strain, C1735/2, produced a unique protein profile and was poorly immunoreactive, suggesting that some strains of M. hyopneumoniae may possess a structurally modified repertoire of antigens. Major M. hyopneumoniae antigens with molecular masses of approximately 36, 43, 48, 52, 76, 78, 80, 82, 94, 106, 114 and 200 kDa were identified by immunoblotting using hyperimmune pig sera raised against both high and low passage strains of M. hyopneumoniae. Porcine hyperimmune sera raised against the GDL type strain of M. hyorhinis reacted strongly with all M. hyorhinis strains although the profiles displayed considerable variation. Major antigens of molecular mass 42, 49, 52, 78, 80 and 82 kDa were identified in type strains GDL and BTS-7 and field strain 2; however, field strain 1 produced a unique profile. A preparative SDS-PAGE profiling (PPP) technique was developed which enabled quantification of the immiunoreactivity of denatured antigens with porcine serum by ELISA. PPP facilitated the rapid identification of species-specific and cross-reactive antigens among the three mycoplasma species. PPP studies revealed several strongly immunoreactive M. hyopneumoniae-specific antigens of 43, 76, 94, 114 and 200 kDa as well as antigens of molecular mass between 52 and 62 kDa which were not apparent in immunoblotting studies. Rabbit monospecific anti-43 kDa serum reacted specifically with a 43 kDa antigen in whole cell lysates of geographically diverse strains of M. hyopneumoniae and failed to cross-react with M. flocculare or M. hyorhinis whole cell lysates. This study has identified a number of M. hyopneumoniae-specific antigens which warrant further investigation to determine their potential as diagnostic reagents and the role they play, if any, in pathogenicity.
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The Mycoplasma hominis P120 membrane protein contains a 216 amino acid hypervariable domain that is recognized by the human humoral immune response
More LessIn the antigenically heterogeneous species Mycoplasma hominis a monoclonal antibody, mAb 26.7D, was previously found to recognize a 120 kDa polypeptide from M. hominis 7488. This antibody did not react with the type strain PG21. The homologous gene from M. hominis PG21 was cloned and sequenced and found to have a sequence identity of 91% with the gene of strain 7488. One hypervariable and two semivariable regions were detected. The epitope for mAb 26.7D was mapped to the hypervariable domain by expression of various parts of this domain in Escherichia coli using expression vector systems. A polyclonal antiserum (pAb 121) generated against the hypervariable region of P120 from PG21 identified the P120 homologue in M. hominis PG21. Fusion proteins of the hypervariable and constant parts of the proteins were constructed and tested for reactivity with 21 human sera. Twelve sera reacted with the 7488 hypervariable fusion protein, but only four reacted with the PG21 hypervariable fusion protein. No reactivity was seen with a fusion protein containing part of the constant region of P120. Gene fragments amplified from 18 M. hominis isolates by PCR confirmed the heterogeneity of the hypervariable domain. Based on restriction endonuclease cleavage patterns of the hypervariable domain the 18 isolates could be divided into four classes. Reactivity with both mAb 26.7D and pAb 121 confirmed these classes. The hypervariable, but not the constant, part of P120 was recognized by the human humoral immune response. Such a variable domain may be important in evasion of the host's immune response, and thus aid survival of the micro-organism.
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- Biochemistry
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Properties of NAD+- and NADP+-dependent malic enzymes of Rhizobium (Sinorhizobium) meliloti and differential expression of their genes in nitrogen-fixing bacteroids
More LessThe wild-type NAD+-dependent malic enzyme (dme) gene of Rhizobium (now Sinorhizobium) meliloti was cloned and localized to a 3.1 kb region isolated on the cosmid pTH69. This cosmid complemented the symbiotic nitrogen fixation (Fix-) phenotype of R. meliloti dme mutants. The dme gene was mapped by conjugation to between the cys-11 and leu-53 markers on the R. meliloti chromosome. β-Galactosidase activities measured in bacterial strains carrying either dme-lacZ or tme-lacZ gene fusions (the tme gene encodes NADP+-dependent malic enzyme) indicated that the dme gene was expressed constitutively in free-living cells and in N2-fixing bacteroids whereas expression of the tme gene was repressed in bacteroids. The R. meliloti dme gene product (DME) was overexpressed in and partially purified from Escherichia coli. The properties of this enzyme, together with those of the NADP+-dependent malic enzyme (TME) partially purified from R. meliloti dme mutants, were determined. Acetyl-CoA inhibited DME but not TME activity. This result supports the hypothesis that DME, together with pyruvate dehydrogenase, forms a pathway in which malate is converted to acetyl-CoA.
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Polythionate degradation by tetrathionate hydrolase of Thiobacillus ferrooxidans
More LessCell-free extracts of Thiobacillus ferrooxidans grown with thiosulfate as energy source and prepared at high ammonium sulfate concentrations and at low pH are capable of polythionate hydrolysis. The enzyme responsible for the hydrolysis of tetrathionate (S4O2- 6) and pentathionate (S4O2- 6) was purified to homogeneity. Enzyme activity during the purification procedure was based on a continuous spectrophotometric method that detects soluble intermediates that absorb in the UV region. The end products of hydrolysis of both polythionates by the pure enzyme were thiosulfate, sulfur and sulfate. The purified enzyme has a pH optimum of around 4 and a temperature optimum of 65 �. The activity is strongly influenced by the presence of sulfate ions. The purified enzyme is a dimer with two identical subunits of molecular mass 52 kDa. During purification of tetrathionate hydrolase, fractions able to hydrolyse trithionate and tetrathionate were separated, indicating that the two substrates are hydrolysed by different enzymes.
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Distribution of amine oxidases and amine dehydrogenases in bacteria grown on primary amines and characterization of the amine oxidase from Klebsiella oxytoca
More LessThe bacteria Klebsiella oxytoca LMD 72.65 (ATCC 8724), Arthrobacter P1 LMD 81.60 (NCIB 11625), Paracoccus versutus LMD 80.62 (ATCC 25364), Escherichia coli W LMD 50.28 (ATCC 9637), E. coli K12 LMD 93.68, Pseudomonas aeruginosa PAO1 LMD 89.1 (ATCC 17933) and Pseudomonas putida LMD 68.20 (ATCC 12633) utilized primary amines as a carbon and energy source, although the range of amines accepted varied from organism to organism. The Gram-negative bacteria K. oxytoca and E. coli as well as the Gram-positive methylotroph Arthrobacter P1 used an oxidase whereas the pseudomonads and the Gram-negative methylotroph Paracoccus versutus used a dehydrogenase for amine oxidation. K. oxytoca utilized several primary amines but showed a preference for those containing a phenyl group moiety. Only a single oxidase was used for oxidation of the amines. After purification, the following characteristics of the enzyme indicated that it belonged to the group of copper-quinoprotein amine oxidases (EC 1.4.3.6): the molecular mass (172000 Da) of the homodimeric protein; the UV/visible and EPR spectra of isolated and p-nitrophenylhydrazine-inhibited enzyme; the presence and the content of copper and topaquinone (TPQ). The amine oxidase appeared to be soluble and localized in the periplasm, but catalase and NAD-dependent aromatic aldehyde dehydrogenase, enzymes catalysing the conversion of its reaction products, were found in the cytoplasm. From the amino acid sequence of the N-terminal part as well as that of a purified peptide, it appears that K. oxytoca produces a copper-quinoprotein oxidase which is very similar to that found in other Enterobacteriaceae.
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- Genetics And Molecular Biology
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2-Phenylethylamine catabolism by Escherichia coli K-12: gene organization and expression
More LessA gene encoding phenylacetaldehyde dehydrogenase (PAD), the enzyme involved together with a copper-topaquinone-containing amine oxidase in the initial steps of 2-phenylethylamine catabolism, was located at 31.1 min on the Escherichia coli K-12 genetic map. It was immediately adjacent to the gene encoding the amine oxidase but transcribed in the opposite direction. The purified PAD acted almost equally well on phenylacetaldehyde, 4-hydroxyphenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde. It had a subunit size of 54 kDa and its deduced amino acid sequence was approximately 40% identical to various eukaryotic and prokaryotic aldehyde dehydrogenases. A third gene encoding a positive regulatory protein required for expression of the amine oxidase and PAD genes was located next to the PAD gene. A gene previously located in this position was reported to encode a second amine oxidase but this was not confirmed. The nucleotide sequence from 1447 to 1450 kb on the E. coli K-12 physical map has been determined.
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Oligonucleoticle-tnediated genetic transformation of Borrelia burgdorferi
More LessWe have used short oligonucleotides to genetically transform the Lyme disease spirochaete Borrelia burgdorferi. The oligonucleotides are derived from the sequence of an Arg-133 to He mutant gyrB (chromosomal) gene that confers resistance to the antibiotic coumermycin A1. Oligonucleotides were about 10000-fold less efficient at transformation, on a molar basis, than longer PCR-generated substrates. All of the transformants tested contained the predicted site-directed silent mutation in their gyrB genes. Antisense oligonucleotides were more efficient at transformation than either sense or double-stranded oligonucleotides. This is the first demonstration of oligonucleotides used to introduce site-directed mutations directly into the genome of a bacterium.
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Electrotransformation of Streptococcus pneumoniae: evidence for restriction of DNA on entry
More LessElectrotransformation is a method generally used in biotechnology to introduce recombinant DNA into a wide range of bacteria. However, the mechanism of DNA entry is poorly understood. We report that in Streptococcus pneumoniae, a naturally transformable species, electrotransformation efficiently introduces a plasmid replicon. DNA is strongly restricted by the restriction-modification systems Dpnl and Dpnll which degrade methylated and non-methylated DNA, respectively, at GATC sequences. This suggests that in electrotransformation double-stranded DNA penetrates into these bacteria without a single-stranded DNA step in contrast to natural transformation. Single-stranded DNA by Itself is able to electrotransform very weakly and linearized double-stranded plasmid DNA yields barely detectable levels of transformants.
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Lactobacillus delbrueckii subsp. lactis DSM7290 pepG gene encodes a novel cysteine aminopeptidase
More LessA number of Escherichia coli clones were isolated from a Lactobacillus delbrueckii subsp. lactis gene library capable of hydrolysing the chromogenic substrate Gly-Ala-β-naphthylamide (Gly-Ala-βNA). Some of the recombinant plasmids carried by these clones have been shown to encode the cysteine aminopeptidase gene pepC. Nucleotide sequence analyses of the plasmid inserts of the remaining clones resulted in the identification of two adjacent ORFs encoding proteins exhibiting a high degree of similarity between themselves (72.6%) and with PepC. One gene, designated pepG, was overexpressed in E. coli and the crude extracts obtained were shown to be peptidolytically active both against chromogenic substrates and peptides, and in a Salmonella typhimurium growth test. PepC and PepG activities were compared using chromogenic βNA and p-nitroanilide substrates and leucine or proline-containing peptides were applied in growth experiments of recombinant Sal. typhimurium. The results indicate that the enzymes, although structurally related, have different substrate preferences. No enzyme activity could be ascribed to the second ORF (orfW), despite the production of a visible protein using a T7 RNA polymerase system. Primer extension analysis, using mRNA isolated from Lb. delbrueckii subsp. lactis DSM7290 did establish that orfW was transcribed.
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A new insertion sequence IS1452 from Acetobacter pasteurianus
More LessA new insertion sequence element, IS1452, was found to be associated with inactivation of the alcohol dehydrogenase by insertion in the adhS gene encoding subunit III of the three-component membrane-bound alcohol dehydrogenase complex in Acetobacter pasteurianus. Cloning and sequencing analyses of the mutated subunit III gene locus revealed that IS 1452 was inserted at or near the ribosome-binding sequence of adhS. Analysis of transcription using the chloramphenicol acetyltransferase gene as the reporter indicated that IS1452 abolished transcription of adhS by separating its promoter from the subunit III structural gene. IS1452 was 1411 bp in length and had a terminal inverted repeat of 21 bp. IS1452 contained one long ORF of 416 amino acids rich in basic amino acids. This protein showed homology with a putative transposase, Tra1, of IS701 isolated from the cyanobacterium Calothrix species PCC 7601. Like IS701, IS7452 was found to generate a 4 bp direct repeat at the site of insertion upon transposition. The target site specificity was rather strict, and a CTA(A or G) sequence appeared to be preferentially recognized. Transposition of IS1452 was replicative, since it was accompanied by an increase in the copy number of IS1452. Several strains belonging to the genus Acetobacter also contained IS1452 at varying copy numbers from one to more than ten. These observations suggest that IS1452 is one of the insertion sequences that are responsible for genetic instability leading to deficiencies in various physiological properties in acetic acid bacteria.
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IS900 targets translation initiation signals in Mycobacterium avium subsp. paratuberculosis to facilitate expression of its hed gene
The Mycobacterium avium subsp. paratuberculosis (formerly Mycobacterium paratuberculosis) atypical insertion sequence, IS900, encodes a novel gene on the complementary strand to the putative transposase, p43. This gene requires a promoter, ribosome binding site (RBS) and termination codon to be acquired upon insertion into the M. avium subsp. paratuberculosis genome and hence is designated the hed (host expression-dependent) gene of IS900. Analysis of IS900 insertion sites suggests that this element targets translation initiation signals in M. avium subsp. paratuberculosis, specifically inserting between the RBS and start codon of a putative gene sequence. This aligns the hed initiation codon adjacent to a functional RBS and possibly downstream of an active promoter, driving expression of Hed protein. We have confirmed this unique targeting process by detecting expression of hed in M. avium subsp. paratuberculosis at the level of transcription by reverse transcription-PCR. Further, two Hed-specific antibodies detected Hed translation products in Western blots of protein extracts from M. avium subsp. paratuberculosis. A recombinant form of Hed expressed and purified from Escherichia coli will facilitate studies of IS900 transposition and will also be assessed as a diagnostic antigen for M. avium subsp. paratuberculosis disease. Implications of IS900 insertion in M. avium subsp. paratuberculosis pathogenicity are discussed.
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Analysis of sequences flanking the vap regions of Dichelobacter nodosus: evidence for multiple integration events, a killer system, and a new genetic element
More LessDichelobacter nodosus is the causative agent of ovine footrot. The vap regions of the D. nodosus genome may have arisen by the integration of a genetic element and may have a role in virulence. The virulent D. nodosus strain A198 has multiple copies of the vap regions. In the present study, sequences to the left and right of vap regions 1, 2 and 3 of strain A198 were analysed by Southern blotting and DMA sequencing. The results suggest that vap regions 1 and 2 arose by independent integration events into different tRNA genes. The discovery of a second integrase gene (intB), a gene with similarity to bacteriophage repressor proteins (regA), and a gene similar to an ORF from a conjugative transposon (gepA), suggests that a second genetic element, either a bacteriophage or a conjugative transposon, is integrated next to vap region 3 in the D. nodosus genome. The arrangement of intB and the vap regions in three other virulent strains and one benign strain was determined using using Southern blotting and PCR. One strain, H1215, contained vapE’ and not vapE, and thus resembles vap region 3, suggesting that vap region 3 also may have arisen by an independent integration event. In all strains, a copy of intB was found next to the vap regions. The vap regions contain two genes, vapA and toxA, with similarity to the hig genes of the killer plasmid Rts1. Evidence is presented that vapA and toxA have a similar function in D. nodosus.
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The Paracoccus denitrificans ccmA, B and C genes: cloning and sequencing, and analysis of the potential of their products to form a haem or apo- c-type cytochrome transporter
More LessTwo c-type cytochrome deficient mutants of Paracoccus denitrificans, HN49 and HN53, were isolated by Tn5 mutagenesis and screening for failure to oxidize dimethylphenylenediamine (the Nadi test). Both were completely deficient in c-type cytochromes. Genomic DNA flanking the site of Tn5 insertion in HN53 was cloned by marker rescue and a 3.1 kb region sequenced. Three of the genes, designated ccmA, ccmB and ccmC, present in this region are proposed to encode the components of a membrane transporter of the ABC (ATP-binding cassette) super-family, which is similar to a group of transporters postulated to translocate either haem or apocytochromes c. The Tn5 elements in HN49 and HN53 were shown to be inserted in ccmB and ccmA, respectively. Sequence analysis suggested that both CcmB and CcmC have the potential to interact with CcmA and thus that the three gene products probably associate to form a complex with (CcmA)2-CcmB-CcmC stoichiometry; it also indicated a lack of similarity between CcmB and CcmC and the membrane-integral components of transporters mediating uptake of haem or other iron complexes. Supplementation of growth media with haem did not stimulate c-type cytochrome formation in HN49 or HN53, although it elevated levels of soluble haemoproteins and membrane-bound cytochromes b, suggesting that exogenous haem can traverse both outer and inner membranes of P. denitrificans. HN49 and HN53 accumulated apocytochrome c 550 to much lower levels than other c-type cytochrome deficient mutants of P. denitrificans but expression and translocation of an apocytochrome c 550-alkaline phosphatase fusion protein and apocytochrome cd 1 were unaffected in HN53. The results suggest that the substrate for the putative CcmABC-transporter is probably neither haem nor c-type apocytochromes.
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Gain-of-function mutation of sapB that affects formation of alkaline phosphatase by Bacillus subtilis in sporulation conditions
More LessThe sapB locus was defined by mutations that render sporulation alkaline phosphatase formation independent of sf and sE without affecting the temporal control of formation. The sapB locus has been cloned and sequenced. The deduced polypeptide is 232 amino acids long, with a molecular mass of 26 kDa. It is very similar to four sequences in the database, none of which has a known function. Analysis of the transcription of sapB indicates that it is induced during late exponential phase, and that maximum expression is reached during the first hour of stationary phase, both under sporulation and non-sporulation conditions. The defining mutations of the locus, sapB2 and sapB10, have been sequenced and found to contain the same change, a G → A transition resulting in an Ala111 Thr switch. This mutation apparently results in a gain-of-function, as sapB null mutants are indistinguishable from sap + strains in terms of their APase production during sporulation.
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Missense mutations in the 3' end of the Escherichia coli dnaG gene do not abolish primase activity but do confer the chromosome-segregation-defective (par) phenotype
More LessIsogenic dnaG strains of Escherichia coli with the parB and dnaG2903 alleles in the MG1655 chromosomal background displayed the classic par phenotype at the nonpermissive temperature of 42 �. These strains synthesized DNA at 42 �, but remained chromosome segregation defective as determined by cytology. A strain with the dnaG2903 allele was tested for its ability to support DNA replication of a primase-dependent G4oric-containing M13 phage derivative by quantitative competitive PCR (QC-PCR). The dnaG2903 strain converted the single-stranded DNA into double-stranded replicative form DNA at 42 �. These results indicate that DnaG2903 retains primase activity at the restrictive temperature. Nucleoids remained unsegregated in the central region of cell filaments at 42 �. The observed suppression of cell filamentation in dnaG sfiA or dnaG lexA double mutants suggests that the SOS response is induced at the restrictive temperature in parB and dnaG2903 strains but fails to account entirely for the cell filamentation phenotype. ParB and DnaG2903 presumably can synthesize primer RNA for DNA replication, but may be defective in their interactions with DNA replication proteins, cell cycle regulatory factors, or the chromosome segregation apparatus itself.
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Sequence analysis of pqq genes required for biosynthesis of pyrroloquinoline quinone in Methylobacterium extorquens AM1 and the purification of a biosynthetic intermediate
More LessMethylobacterium extorquens AM1 produces pyrroloquinoline quinone (PQQ), the prosthetic group of methanol dehydrogenase. Two gene clusters have been shown to be required for PQQ biosynthesis in this micro-organism and complementation analysis has identified seven pqq genes, pqqDGCBA and pqqEF. The DNA sequence of pqqDGC’ was reported previously. This paper reports the sequence of the genomic region corresponding to pqqC'BA. For consistency, the nomenclature of pqq genes in Klebsiella pneumoniae will be followed. The new nomenclature for pqq genes of M. extorquens AM1 is pqqABCDE and pqqFG. In the genomic region sequenced in this study, two open reading frames were found. One of these encodes PqqE, which showed high identity to analogous pqq genes in other bacteria. PqqE also showed identity to MoaA and NifB in the N-terminal region, where a conserved CxxxCxYC sequence was identified. The sequence of the second open reading frame covered both the pqqC and pqqD regions, suggesting that both functions were encoded by this gene. It is proposed to designate this gene pqqC/D. The deduced amino acid sequence of the pqqC/D product showed identity to PqqC of K. pneumoniae and Pqql of Acinetobacter calcoaceticus in the N-terminal region, and to PqqD of K. pneumoniae and Pqqll of A. calcoaceticus in the C-terminal region. A fragment of M. extorquens AM1 DNA containing only pqqC/D produced a protein of 42 kDa in Escherichia coli, which corresponds to the size of the deduced amino acid sequence of PqqC/D, confirming the absence of a separate pqqD. This genomic region complemented the growth of pqqC mutants of M. extorquens AM1 and Methylobacterium organophilum DSM 760 on methanol. As previously reported for pqq genes of K. pneumoniae, a pqqC mutant of M. extorquens AM1 produced an intermediate of PQQ biosynthesis, which was converted to PQQ by incubation with a crude extract from E. coli cells expressing PqqC/D. The intermediate was found in both crude extract and culture supernatant, and it was purified from the crude extract. The PqqC/D enzyme reaction appeared to require molecular oxygen and reduced nicotinamide adenine dinucleotides.
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The nucleoside-specific Tsx channel from the outer membrane of Salmonella typhimurium, Klebsiella pneumoniae and Enterobacter aerogenes: functional characterization and DNA sequence analysis of the tsx genes
More LessThe Escherichia coli tsx gene encodes an integral outer-membrane protein (Tsx) that functions as a substrate-specific channel for deoxynucleosides and the antibiotic albicidin, and also serves as a receptor for bacteriophages and colicins. We cloned the structural genes of the Tsx proteins from Salmonella typhimurium, Klebsiella pneumoniae and Enterobacter aerogenes and expressed them in an E. coli tsx mutant. The heterologous Tsx proteins fully substituted the E. coli Tsx protein with respect to its function in deoxynucleoside and albicidin uptake, and as receptor for colicin K. The Tsx proteins from K. pneumoniae and Ent. aerogenes were also proficient as receptors for several Tsx-specific bacteriophages, whereas the corresponding protein from S. typhimurium did not confer sensitivity against these phages. The nucleotide sequence of the tsx genes from S. typhimurium, K. pneumoniae and Ent. aerogenes was established. Each of the Tsx proteins is initially synthesized with typical bacterial signal sequence peptides and the predicted mature forms of the Tsx proteins have a calculated Mr of 30567 (265 residues), 31412 (272 residues) and 31477 (272 residues), respectively. Multiple sequence alignments between the Tsx proteins showed a high degree of sequence identity and revealed the presence of four hypervariable regions, which are thought to constitute segments of the polypeptide chain exposed at the cell surface. Most notable was a deletion of 8 amino acids in one of these hypervariable domains in the S. typhimurium Tsx protein. When this deletion was introduced by site-directed mutagenesis into the corresponding region of the E. coli tsx gene, the mutant Tsx-515 protein lost its phage receptor function but still served as a colicin K receptor and as a substrate-specific channel, indicating that the region between residues 198 and 207 might be part of the bacteriophage receptor area. Multiple sequence alignments, structural predictions and the properties of previously characterized Tsx missense mutants were taken into account to develop a two-dimensional model for the topological organization of the Tsx protein within the outer membrane.
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Natural kirromycin resistance of elongation factor Tu from the kirrothricin producer Streptomyces cinnamoneus
More LessThe antibiotic kirromycin (Kr) inhibits bacterial protein synthesis by binding to elongation factor Tu (EF-Tu). Streptomyces cinnamoneus and Nocardia lactamdurans, producers of antibiotics of the Kr class, are known to possess an EF-Tu resistant to Kr. Both micro-organisms appear to possess a single tuf gene and we have characterized the one from S. cinnamoneus, which belongs to the tuf 1 family. To assess the molecular determinants of Kr resistance, the S. cinnamoneus tuf gene was expressed in Escherichia coli as a translational fusion to malE, which enabled the recovery by affinity chromatography of the recombinant protein uncontaminated by the host factor. The recombinant EF-Tu was able to catalyse polyU-directed polyPhe synthesis in two heterologous cell-free systems, even as an uncleaved fusion. When tested for antibiotic sensitivity it behaved like the natural S. cinnamoneus protein, showing equivalent resistance to Kr but sensitivity to the antibiotic GE2270, indicating that all determinants for Kr resistance are intrinsic to the EF-Tu sequence. Multiple sequence analysis of EF-Tu proteins, together with knowledge of mutations conferring Kr resistance, allowed the identification of key residues as likely candidates for the natural Kr resistance of the S. cinnamoneus EF-Tu. One of these, Thr378, was mutated to the consensus Ala and the resulting mutant protein was sensitive to Kr. Interestingly, it retained some activity (30% of the control) even at. high Kr concentrations.
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- Pathogenicity And Medical Microbiology
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Arginine-, hypoxanthine-, uracil-requiring isolates of Neisseria gonorrhoeae are a clonal lineage within a non-clonal population
More LessMultilocus enzyme electrophoresis has shown that a collection of 101 arginine-, hypoxanthine-, uracil-requiring (AHU-) isolates of Neisseria gonorrhoeae, recovered over a 39 year period from the UK and Denmark, were of a single electrophoretic type (91% of strains), or differed from the predominant electrophoretic type at only a single locus. The striking uniformity of the AHU- isolates, and the correlation between auxotype, serovar and overall genetic background, contrasts with previous studies of gonococcal populations (that included very few AHU- strains), and a small sample of non-AHU- isolates studied here, which demonstrated a non-clonal population structure and a lack of association between auxotype, serovar and genetic background. There was no marked difference in the ability of AHIT isolates to be transformed with their own DNA, or with DNA from gonococci of other auxotypes, and the relative genetic stability of AHU- isolates does not appear to be due to a defect in their ability to be transformed. An alternative possibility is that AHU- gonococci recombine with other lineages, but that the resulting recombinants are not maintained in the population. This would occur, for example, if AHU- gonococci competed poorly in mixed infections, within which effective recombination between lineages occurs, and are usually only transmitted from individuals who are singly infected with an AHU- strain. The association between AHU- gonococci and asymptomatic infections may lead to an increased rate of transmission of these strains which under this scenario would be needed to prevent them from being lost from the population.
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A novel gene, algK, from the alginate biosynthetic cluster of Pseudomonas aeruginosa
More LessColonization of the cystic fibrosis lung by Pseudomonas aeruginosa is greatly facilitated by the production of an exopolysaccharide called alginate. Many of the enzymes involved in alginate biosynthesis are clustered in an operon at 34 min on the P. aeruginosa chromosome. This paper reports the nucleotide sequence of a previously uncharacterized gene, algK, which lies between the alg44 and algE genes of the operon. DNA sequencing data for algK predicted a protein product of approximately 52.5 kDa which contains a putative 27 amino acid N-terminal signal sequence and a consensus cleavage and lipid attachment site for signal peptidase II. Expression of algK using either T7 or tac promoter expression systems, and in vivo labelling studies with [35S]methionine, indicated that algK encodes a polypeptide of approximately 53 kDa which is processed to a mature protein of approximately 50 kDa when expressed in Escherichia coli or P. aeruginosa, in agreement with the nucleotide sequence analysis. Results from an AlgK-β-lactamase fusion survey support this interpretation and also provide evidence that mature AlgK is entirely periplasmic and is probably membrane-anchored.
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- Physiology And Growth
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Investigation of space flight effects on Escherichia coli and a proposed model of underlying physical mechanisms
More LessPrevious investigations have reported that space flight may produce a stimulating effect on microbial metabolism; however, the specific underlying mechanisms associated with the observed changes have not yet been identified. In an effort to systematically evaluate the effect of space flight on each phase of microbial growth (lag, exponential and stationary), a series of experiments was carried out using in vitro suspension cultures of Escherichia coli aboard seven US Space Shuttle missions. The results indicated that, as a result of space flight, the lag phase was shortened, the duration of exponential growth was increased, and the final cell population density was approximately doubled. A model was derived from these cumulative data in an attempt to associate gravity-dependent, extracellular transport phenomena with unique changes observed in each specific phase of growth. It is suggested that a cumulative effect of gravity may have a significant impact on suspended cells via their fluid environment, where an immediate, direct influence of gravity might otherwise be deemed negligible.
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Enzymological and physiological consequences of restructuring the lipoyl domain content of the pyruvate dehydrogenase complex of Escherichia coli
The core-forming lipoate acetyltransferase (E2p) subunits of the pyruvate dehydrogenase (PDH) complex of Escherichia coli contain three tandemly repeated lipoyl domains although one lipoyl domain is apparently sufficient for full catalytic activity in vitro. Plasmids containing IPTG-inducible aceEF-IpdA operons which express multilip-PDH complexes bearing one N-terminal lipoyl domain and up to seven unlipoylated (mutant) domains per E2p chain, were constructed. Each plasmid restored the nutritional lesion of a strain lacking the PDH complex and expressed a sedimentable PDH complex, although the catalytic activities declined significantly as the number of unlipoylated domains increased above four per E2p chain. It was concluded that the extra domains protrude from the 24-meric E2p core without affecting assembly of the E1p and E3 subunits, and that the lipoyl cofactor bound to the outermost domain can participate successfully at each of the three types of active site in the assembled complex. Physiological studies with two series of isogenic strains expressing multilip-PDH complexes from modified chromosomal pdh operons (pdhR-aceEF-IpdA) showed that three lipoyl domains per E2p chain is optimal and that only the outermost domain need be lipoylated for optimal activity. It is concluded that the reason for retaining three lipoyl domains is to extend the reach of the outermost lipoyl cofactor rather than to provide extra cofactors for catalysis.
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Growth competition between Halobacterium salinarium strain PHH1 and mutants affected in gas vesicle synthesis
More LessTo investigate the role of the buoyancy provided by gas vesicles in the facultative anaerobe Halobacterium salinarium PHH1, the growth of a gas-vacuolate (Gv+) strain in competition with two gas-vesicle-defective (Gvdef) mutants was examined. The Gv+ strain synthesized gas vesicles throughout its growth cycle, and floated up to form a thick surface scum during the exponential growth phase in static culture. Mutant Gvdef1 produced significantly fewer gas vesicles than the Gv+ strain in corresponding stages of growth, although in late stationary phase a small proportion of cells floated up to the surface of static cultures. Mutant Gvdef2 had a much lower gas vesicle content in shaken culture and produced negligible amounts of gas vesicles in static culture. The Gv+ and the two Gvdef strains grew equally well in shaken cultures, but in static cultures, where steep vertical gradients of oxygen concentration were established, Gvdef1 was outgrown by the Gv+ strain. Gvdef2 outcompeted the Gv+ strain in shallow static cultures, perhaps because Gvdef2 carried a smaller protein burden, which offset the benefits of buoyancy. This selection for Gvdef2 was lost in deeper static cultures, although it could be restored by aerating static cultures from below. The results support the hypothesis that the role of buoyancy in halobacteria is to maintain cells at the more aerated surface of brine pools.
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A physiological model for the control of erythromycin production in batch and cyclic fed batch culture
More LessReported differences in antibiotic production dynamics resulting from altering the growth-limiting nutrient (growth-dissociated production in carbon-limited culture and apparent growth-associated production in nitrogen-limited culture) are due to the different effects on growth kinetics. The substrate affinity for nitrate is significantly lower than that for glucose, resulting in nitrogen limitation effectively occurring throughout the culture. Glucose limitation occurs later in the culture, coinciding with the induction of antibiotic production. Induction occurs at the start of nitrogen-limited culture so that production appears to be growth-associated. Evidence that this hypothesis is consistent with production kinetics in cyclic fed batch culture was also obtained.
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Effects of growth-inhibitory concentrations of copper on alginate biosynthesis in highly mucoid Pseudomonas aeruginosa
More LessAlginate production and degree of polymerization were affected when the highly mucoid Pseudomonas aeruginosa 8821M was grown with growth-inhibitory concentrations of Cu2+ (supplied as CuCl2; 1-5 mM). The inhibition of alginate biosynthesis was consistent with the decreased activity in Cu2+-stressed cells of phosphomannose isomerase/GDP-mannose pyrophosphorylase (encoded by algA), phosphomannomutase (encoded by algC) and GDP-mannose dehydrogenase (encoded by algD). However, in cells grown with concentrations of CuCl2 below 2 mM, the steady-state mRNA levels from algA, algC, algD and from the regulatory gene algR1 increased moderately. This observation is consistent with the suggested linkage between the control of alginate gene expression and the global regulation involved in the oxidative stress response. At highly inhibitory concentrations the levels of the four alginate gene transcripts decreased from maximal values. The bell-shaped curves, representing the effect of Cu2+ concentration on mRNA levels from the four alginate genes, exhibited similar patterns but did not concur. The decrease of the specific activity of enzymes necessary for GDP-mannuronic acid synthesis in Cu2+-grown cells was correlated with changes in gene expression, with the inhibitory effect of Cu2+ on enzyme activities and with Cu2+-induced oxidative inactivation of enzymes, especially the particularly sensitive phosphomannose isomerase activity.
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- Plant-Microbe Interactions
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Fire blight protection with avirulent mutants of Erwinia amylovora
More LessFire blight is a necrotic disease caused by the bacterium Erwinia amyiovora, which affects pears, apples and ornamentals including Crataegus, Pyracantha, and Cotoneaster. The disease can be only partially controlled, through the use of resistant genotypes, cultural measures and antibacterial compounds, thus other methods must be investigated. It has long been established that avirulent isolates of the pathogen can control the disease, under experimental conditions. However, field use of avirulent isolates is not acceptable because of their unknown genetic stability. The protective ability under controlled conditions of genetically characterized avirulent insertion mutants of E. amylovora was examined. A bioassay on apple seedlings was used for the determination of the protective ability of 34 insertion mutants (hrp, dsp, ams). Some protective effect could be observed with most of the mutants tested and was dependent on the avirulent/virulent inoculum ratio as well as on the level of virulence of the pathogen; a minimal concentration of the avirulent mutant was necessary to give a significant level of protection. An early competition between avirulent and virulent strains for putative infection sites might be involved. For six of the mutants tested, the protective ability was particularly high and might be related to the alteration of regulatory functions of hrp genes. Results obtained with Ams- and Ams- Hrp- mutants suggested that the bacterial exopolysaccharide might play a role in the protection.
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Volume 87 (1975)
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Volume 86 (1975)
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Volume 85 (1974)
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Volume 84 (1974)
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Volume 83 (1974)
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Volume 82 (1974)
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Volume 81 (1974)
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Volume 80 (1974)
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Volume 79 (1973)
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Volume 78 (1973)
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Volume 77 (1973)
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Volume 76 (1973)
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Volume 75 (1973)
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Volume 74 (1973)
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Volume 73 (1972)
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Volume 72 (1972)
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Volume 71 (1972)
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Volume 70 (1972)
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Volume 69 (1971)
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Volume 68 (1971)
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Volume 67 (1971)
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Volume 66 (1971)
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Volume 65 (1971)
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Volume 64 (1970)
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Volume 63 (1970)
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Volume 62 (1970)
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Volume 61 (1970)
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Volume 60 (1970)
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Volume 59 (1969)
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Volume 58 (1969)
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Volume 57 (1969)
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 52 (1968)
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Volume 51 (1968)
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Volume 50 (1968)
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Volume 49 (1967)
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Volume 48 (1967)
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Volume 47 (1967)
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Volume 46 (1967)
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Volume 45 (1966)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 38 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)