- Volume 150, Issue 7, 2004
Volume 150, Issue 7, 2004
- Pathogens And Pathogenicity
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ALS3 and ALS8 represent a single locus that encodes a Candida albicans adhesin; functional comparisons between Als3p and Als1p
The ALS (agglutinin-like sequence) gene family of Candida albicans encodes eight cell-surface glycoproteins, some of which are involved in adherence to host surfaces. A mutational analysis of each ALS gene is currently being performed to deduce the functions of the encoded proteins and to better understand the role of these proteins in C. albicans biology and pathogenesis. This paper describes construction of an als3/als3 mutant and comparison of its phenotype to an als1/als1 strain. Efforts to disrupt ALS3 indicated that the gene could be deleted in two transformation steps, suggesting that the gene is encoded by a single locus and that the ALS3-like locus, ALS8, does not exist. Strains lacking ALS3 or ALS1 did not exhibit a defect in germ tube formation when grown in RPMI 1640 medium, but the als1/als1 mutant formed significantly fewer germ tubes in Lee medium. Analysis of ALS3 and ALS1 promoter activity using green fluorescent protein (GFP) reporter strains and flow cytometry showed that when cells are placed into medium that promotes germ tube formation, ALS1 is transcribed prior to ALS3. Comparison of the mutant strains in adhesion assays showed that the als3/als3 strain was defective in adhesion to both human umbilical vein endothelial cells (HUVEC) and buccal epithelial cells (BEC), but not to fibronectin-coated plastic plates. In contrast, the als1/als1 strain showed decreased adherence to HUVEC, but adherence to BEC and fibronectin were the same as wild-type controls. Inoculation of the buccal reconstituted human epithelium (RHE) model of oral candidiasis with the mutant strains showed nearly a total lack of adhesion and epithelial destruction by the als3/als3 mutant while the als1/als1 strain showed only a slightly reduced degree of epithelial destruction compared to the wild-type control. Adhesion data presented here suggest that, in the assays performed, loss of Als3p affects C. albicans adhesion more than loss of Als1p. Collectively, these results demonstrate functional similarities and differences between Als1p and Als3p, and suggest the potential for more complex interrelationships between the ALS genes and their encoded proteins.
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- Physiology
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Ffz1, a new transporter specific for fructose from Zygosaccharomyces bailii
More LessThe basis of fructophily in the yeast Zygosaccharomyces bailii has been shown to reside in the performance of transport systems for hexoses. In this study, a gene encoding a fructose-specific transporter was characterized. The strategy involved the functional complementation of a Saccharomyces cerevisiae strain that does not take up hexoses (hxt-null strain). This strain was transformed with a genomic library of Z. bailii. One transformant capable of growing on fructose, but not on glucose, was obtained. This transformant did not transport d-[14C]glucose, and the kinetic parameters for d-[14C]fructose were V max=3·3 mmol h−1 g−1 and K m=80·4 mM. As in the original strain of Z. bailii, fructose uptake was not inhibited by the presence of other hexoses or uranyl. The plasmid responsible for the observed phenotype was found to carry an ORF encoding a 616 amino acid protein with the characteristics of a membrane transporter, which was designated FFZ1 (fructose facilitator Zygosaccharomyces). The impairment in function observed in an S. cerevisiae transformant expressing a truncated Ffz1 protein lacking 67 amino acids at the C-terminus suggests an important role for this terminal part in the proper structure of the transporter.
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Anaerobic growth of the haloalkaliphilic denitrifying sulfur-oxidizing bacterium Thialkalivibrio thiocyanodenitrificans sp. nov. with thiocyanate
More LessTwo strains of obligate chemolithoautotrophic sulfur-oxidizing bacteria were isolated from soda-lake sediments by enrichment culture with thiocyanate and nitrate at pH 9·9. The isolates were capable of growth with thiocyanate or thiosulfate as electron donor, either aerobically or anaerobically, and with nitrate or nitrite as electron acceptor. Cyanate was identified as an intermediate of thiocyanate oxidation, while sulfate, ammonia and dinitrogen gas were the final products. The anaerobic growth on thiocyanate plus nitrate was much slower (μ max=0·006 h−1) than on thiosulfate plus nitrate (μ max=0·02 h−1), while growth yields were similar (4·8 and 5·1 g protein mol−1, respectively). On the basis of their phenotypic and genetic properties, strains ARhD 1T and ARhD 2 are described as a novel species of the genus Thialkalivibrio, with the highest similarity to Thialkalivibrio denitrificans. The name Thialkalivibrio thiocyanodenitrificans sp. nov. is proposed for this novel species.
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The putative permease PhlE of Pseudomonas fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance
More Less2,4-Diacetylphloroglucinol (PHL) is the primary determinant of the biological control activity of Pseudomonas fluorescens F113. The operon phlACBD encodes enzymes responsible for PHL biosynthesis from intermediate metabolites. The phlE gene, which is located downstream of the phlACBD operon, encodes a putative permease suggested to be a member of the major facilitator superfamily with 12 transmembrane segments. PhlE has been suggested to function in PHL export. Here the sequencing of the phlE gene from P. fluorescens F113 and the construction of a phlE null mutant, F113-D3, is reported. It is shown that F113-D3 produced less PHL than F113. The ratio of cell-associated to free PHL was not significantly different between the strains, suggesting the existence of alternative transporters for PHL. The phlE mutant was, however, significantly more sensitive to high concentrations of added PHL, implicating PhlE in PHL resistance. Furthermore, the phlE mutant was more susceptible to osmotic, oxidative and heat-shock stresses. Osmotic stress induced rapid degradation of free PHL by the bacteria. Based on these results, we propose that the role of phlE in general stress tolerance is to export toxic intermediates of PHL degradation from the cells.
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A multisubunit membrane-bound [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase in the fermenting bacterium Thermoanaerobacter tengcongensis
More LessThermoanaerobacter tengcongensis is a thermophilic Gram-positive bacterium able to dispose of the reducing equivalents generated during the fermentation of glucose to acetate and CO2 by reducing H+ to H2. A unique combination of hydrogenases, a ferredoxin-dependent [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase, were found to be responsible for H2 formation in this organism. Both enzymes were purified and characterized. The tightly membrane-bound [NiFe] hydrogenase belongs to a small group of complex-I-related [NiFe] hydrogenases and has highest sequence similarity to energy-converting [NiFe] hydrogenase (Ech) from Methanosarcina barkeri. A ferredoxin isolated from Ta. tengcongensis was identified as the physiological substrate of this enzyme. The heterotetrameric Fe-only hydrogenase was isolated from the soluble fraction. It contained FMN and multiple iron–sulfur clusters, and exhibited a typical H-cluster EPR signal after autooxidation. Sequence analysis predicted and kinetic studies confirmed that the enzyme is an NAD(H)-dependent Fe-only hydrogenase. When H2 was allowed to accumulate in the culture, the fermentation was partially shifted to ethanol production. In cells grown at high hydrogen partial pressure [p(H2)] the NADH-dependent hydrogenase activity was fourfold lower than in cells grown at low p(H2), whereas aldehyde dehydrogenase and alcohol dehydrogenase activities were higher in cells grown at elevated p(H2). These results indicate a regulation in response to the p(H2).
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- Plant-Microbe Interactions
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Extended phenotype of an mreB-like mutant in Azospirillum brasilense
Tn5 mutagenesis was used to generate an Azospirillum brasilense SPF94 mutant. Genetic analysis of this mutant revealed that a homologue of the mreB gene, which controls cell shape in Bacillus subtilis and Escherichia coli, was inactivated. The cell-surface properties of the mutant were different from those of the parental strain. The mutant colonies were highly fluorescent when grown on plates containing Calcofluor White. Light and electron microscopy revealed that the mutant cells were round and had thicker capsules than the spiral parental strain. The mutants contained up to ten times more capsule protein than the parental strain, but lacked a 40 kDa protein that is abundant in the parental strain. The phenotype of the isolated mutant resembled that of the cyst-like differentiated forms of Azospirillum, suggesting that the mreB homologue could be involved in differentiation.
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An aspartic proteinase gene family in the filamentous fungus Botrytis cinerea contains members with novel features
More LessBotrytis cinerea, an important fungal plant pathogen, secretes aspartic proteinase (AP) activity in axenic cultures. No cysteine, serine or metalloproteinase activity could be detected. Proteinase activity was higher in culture medium containing BSA or wheat germ extract, as compared to minimal medium. A proportion of the enzyme activity remained in the extracellular glucan sheath. AP was also the only type of proteinase activity in fluid obtained from B. cinerea-infected tissue of apple, pepper, tomato and zucchini. Five B. cinerea genes encoding an AP were cloned and denoted Bcap1–5. Features of the encoded proteins are discussed. BcAP1, especially, has novel characteristics. A phylogenetic analysis was performed comprising sequences originating from different kingdoms. BcAP1 and BcAP5 did not cluster in a bootstrap-supported clade. BcAP2 clusters with vacuolar APs. BcAP3 and BcAP4 cluster with secreted APs in a clade that also contains glycosylphosphatidylinositol-anchored proteinases from Saccharomyces cerevisiae and Candida albicans. All five Bcap genes are expressed in liquid cultures. Transcript levels of Bcap1, Bcap2, Bcap3 and Bcap4 are subject to glucose and peptone repression. Transcripts from all five Bcap genes were detected in infected plant tissue, indicating that at least part of the AP activity in planta originates from the pathogen.
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