Energy-dependence of nitrate reductase induction in Candida-Utilis

The requirement of a suitable energy source during the induced synthesis of nitrate reductase in Image was investigated. The levels of nitrate reductase induced were shown to be energy-dependent, and to vary in response to the type of carbon source provided. Glycerol, fructose, ethanol, glucose, and sucrose served as efficient energy sources. Growth rate of the yeast and the induced level of nitrate reductase were dependent on the ratio of carbon to nitrogen in the induction medium, and ratio of 2 being optimal. Induction of nitrate reductase was inhibited by uncouplers, 2,4-dinitrophenol (DNP), dicumarol and carbonyl cyanide Candida-Utilis -trifluoromethoxy phenyl hydrazone (CCCP), and by cyanide and azide, indicating an absolute energy-dependency. The facilitation of induction of a high level of nitrate reductase by exogenously added ATP as sole source of energy confirmed the obligate requirement of ATP for the synthesis of nitrate reductase in Candida-Utilis.

Biomineralization of manganese by Bacillus spp isolated from a marine biofilm

Biomineralization of manganese on titanium condenser material exposed to seawater has been illustrated. Biomineralization occurs when the fouling components, namely, the microbes, are able to oxidize minerals present in water and deposit them as insoluble oxides on biofilm surfaces. Extensive biofilm characterization studies Showed that an alarmingly large number of bacteria in these biofilms are capable of oxidizing manganese and are, thereby, capable of causing biomineralization on the condenser material exposed to seawater. This paper addresses studies on understanding the exact role of the microbes in bringing about oxidation of manganese. The kinetics of manganese oxidation by marine Gram-positive manganese oxidizing bacterium Bacillus spp. that was isolated front the titanium surface was studied in detail. Manganese oxidation in the presence of Bacillus cells, by cell free extract (CFE) and heat-treated cell free extract was also studied. The study confirmed that bacteria mediate manganese oxidation and lead to the formation of biogenic oxides of MnO2 eventually leading to biomineralization on titanium surface exposed to seawater.

5-Methyl-2-Thiouridine in the tRNA of Candida tropicalis and Its Localization in Lysine tRNA

35S incorporation studies showed that Candida tropicalis tRNA contained two thionucleosides, one of which was identified as 5-methyl-2-thiouridine. The other thionucleoside was alkali labile, and it appeared to be an ester. Pulse-chase experiments suggested that the two thionucleosides were structurally related. 5-Methyl-2-thiouridine was present in one of the lysine tRNAs. This is the first report of the presence of this nucleoside in a yeast tRNA.

Studies on the Mechanism of Action of Miconazole: Effect of Miconazole on Respiration and Cell Permeability of Candida albicans

The antifungal drug, miconazole nitrate, inhibits the growth of several species of Candida. Candida albicans, one of the pathogenic species, was totally inhibited at a concentration of approximately 10 μg/ml. Endogenous respiration was unaffected by the drug at a concentration as high as 100 μg/ml, whereas exogenous respiration was markedly sensitive and inhibited to an extent of 85%. The permeability of the cell membrane was changed as evidenced by the leakage of 260-nm absorbing materials, amino acids, proteins, and inorganic cations. The results we present clearly show that the drug alters the cellular permeability, and thus the exogenous respiration becomes sensitive to the drug.

Studies on the Mechanism of Action of Miconazole: Effect of Miconazole on Respiration and Cell Permeability of Candida albicans

The antifungal drug, miconazole nitrate, inhibits the growth of several species of Candida. Candida albicans, one of the pathogenic species, was totally inhibited at a concentration of approximately 10 µg/ml. Endogenous respiration was unaffected by the drug at a concentration as high as 100 µg/ml, whereas exogenous respiration was markedly sensitive and inhibited to an extent of 85%. The permeability of the cell membrane was changed as evidenced by the leakage of 260-nm absorbing materials, amino acids, proteins, and inorganic cations. The results we present clearly show that the drug alters the cellular permeability, and thus the exogenous respiration becomes sensitive to the drug.

Production of 2-phenethyl alcohol and 2-phenyllactic acid in Candida species

2-Phenethyl alcohol (2-PEA) and 2-phenyllactic acid (2-PLA) were isolated from the culture filtrates of Candida species grown in media containing peptone or phenylalanine as nitrogen source. These compounds were characterized by comparing their UV, IR, and NMR spectral properties with authentic samples. Candida species differed markedly in their production of 2-PEA and 2-PLA. Experiments using [14C]-phenylalanine indicated that both 2-PEA and 2-PLA are synthesised from L-phenylalanine. A pathway for the biosynthesis of 2-PEA from L-phenylalanine has been proposed.

Molecular basis of nitrate reductase induction in Candida utilis

Temporal separaton of transcription and translation during nitrate reductase induction oin Candida utilis was achieved by the use of actinomycin D and cycloheximide. The yeast failed to synthesize nitrate reductase when nitrate was not provided during transcription. Nitrate thus appeared to induce during transcription the capacity to synthesize nitrate reductase. Presence of nitrate, on the other hand, was not obligatory during translation except for its essential role in maintaining the stability of nitrate reductase after its formation as well as its mRNA.

Purification and characterization of assimilatory nitrite reductase from Candida utilis

Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.

CsrA interacting small RNAs in Haemophilus spp genomes: a theoretical analysis

The csrA is a carbon storage regulator gene that encodes a protein with multiple RNA interaction sites. Bacterial non-coding small RNAs like csrB, csrC and their counterparts in diverse bacterial genus are identified to control the regulatory activities of CsrA and its orthologs. An attempt has been made in this study to identify 'novel' non-coding small RNAs that are involved in the regulatory activities of csrA gene. All CsrA-interacting small RNAs are computationally fingerprinted to have multiple occurrence of 7-nucleotide CsrA interacting repeats [CAGGA(U/A/C)G] along with a 18-nucleotide upstream binding site. However, in several of the genomes like Haemophilus spp, the upstream binding site is not identified. The current methodology overcomes this difficulty by identifying small RNA-specific orphan transcriptional units within the intergenic regions of the genome. The results could identify all known CsrA-interacting small RNAs in E. coli, Vibrio cholerae and Pseudomonas aeruginosa genomes and additionally has picked six new possible CsrA-interacting small RNA regions in E. coli. Our computational analysis indicates that known rygD and rprA sRNAs in E. coli could possibly interact with CsrA proteins. The study is extended to three of the Haemophilus genomes that could identify seven new possible CsrA interacting small RNAs.

The key enzyme in galactose metabolism, UDP-galactose-4-epimerase, affects cell-wall integrity and morphology in Candida albicans even in the absence of galactose

The enzyme UDP-galactose-4-epimerase (GAL10) catalyzes a key step in galactose metabolism converting UDP-galactose to UDPglucose which then can get metabolized through glycolysis and TCA cycle thus allowing the cell to use galactose as a carbon and energy source. As in many fungi, a functional homolog of GAL10 exists in Candida albicans. The domainal organization of the homologs from Saccharomyces cerevisiae and C albicans show high degree of homology having both mutarotase and an epimerase domain. The former is responsible for the conversion of beta-D-galactose to alpha-D-galactose and the hitter for epimerization of UDP-galactose to UDP-glucose. Absence of C albicans GAL10 (CaGAL10) affects cell-wall organization, oxidative stress response, biofilm formation and filamentation. Cagal10 mutant cells tend to flocculate extensively as compared to the wild-type cells. The excessive filamentation in this mutant is reflected in its irregular and wrinkled colony morphology. Cagal10 strain is more susceptible to oxidative stress when tested in presence of H2O2. While the S. cerevsiae GAL10 (ScGAL10), essential for survival in the presence of galactose, has not been reported to have defects in the absence of galactose, the C albicans homolog shows these phenotypes during growth in the absence of galactose. Thus a functional CaGal10 is required not only for galactose metabolism but also for normal hyphal morphogenesis, colony morphology, maintenance of cell-wall integrity and for resistance to oxidative stress even in the absence of galactose. (c) 2006 Elsevier Inc. All rights reserved.

Phage Displayed Short Peptides against Cells of Candida albicans Demonstrate Presence of Species, Morphology and Region Specific Carbohydrate Epitopes

Candida albicans is a commensal opportunistic pathogen, which can cause superficial infections as well as systemic infections in immuocompromised hosts. Among nosocomial fungal infections, infections by C. albicans are associated with highest mortality rates even though incidence of infections by other related species is on the rise world over. Since C. albicans and other Candida species differ in their susceptibility to antifungal drug treatment, it is crucial to accurately identify the species for effective drug treatment. Most diagnostic tests that differentiate between C. albicans and other Candida species are time consuming, as they necessarily involve laboratory culturing. Others, which employ highly sensitive PCR based technologies often, yield false positives which is equally dangerous since that leads to unnecessary antifungal treatment. This is the first report of phage display technology based identification of short peptide sequences that can distinguish C. albicans from other closely related species. The peptides also show high degree of specificity towards its different morphological forms. Using fluorescence microscopy, we show that the peptides bind on the surface of these cells and obtained clones that could even specifically bind to only specific regions of cells indicating restricted distribution of the epitopes. What was peculiar and interesting was that the epitopes were carbohydrate in nature. This gives insight into the complexity of the carbohydrate composition of fungal cell walls. In an ELISA format these peptides allow specific detection of relatively small numbers of C. albicans cells. Hence, if used in combination, such a test could help accurate diagnosis and allow physicians to initiate appropriate drug therapy on time.

Accurate transcription initiation by RNA polymerase II from Candida utilis

An in vitro transcription system from Candida utilis is described. The template used is a hybrid plasmid containing Saccharomyces cerevisiae CYC1 promoter linked to a synthetic 377-bp G-minus casette (1). In vitro transcriptions are carried out in the presence of RNase. T1. Under these conditions only the transcripts that are resistant to RNase T1 accumulate. Using this protocol, it has been shown that in the absence of cytosolic factors RNA polymerase II (pol II) from C. utilis initiated RNA synthesis randomly. But both C. utilis and S. cerevisiae cell-free extracts could direct pol II from C. utilis to initiate transcription accurately. Results also indicated that the general transcription factors are functionally interchangeable between S. cerevisiae and C. utilis

Purification and characterization of DNA-dependent RNA polymerase II from Candida utilis

DNA-dependent RNA polymerase II from Candida utilis has been purified to near homogeneity. The purified enzyme resolved into three subforms, viz. IIO, IIA and IIB. On SDS-PAGE the enzyme showed ten polypeptides with molecular weights in the range of 205 kDa to 14 kDa. By two dimensional electrophoresis (IEF followed by SDS-PAGE) the presence of basic and acidic polypeptides has been demonstrated. The enzyme showed Km values of 5, 5.6 and 8 mu M for GTP, CTP and ATP, respectively, and the activity was inhibited by low levels of oc-amanitin and antibodies raised against bovine RNA polymerase II. By Western blot analysis the enzyme was found to cross-react with antibodies to bovine RNA polymerase II. RNA polymerase II from G. utilis is a phosphoprotein, the subunits RPB1 and RPB10 were found to be phosphorylated. Analysis of carboxy-terminal domain indicated that it was functionally redundant at least in case of nonspecific transcription, implicating its role in other nuclear processes, such as promoter specific initiation or transcription activation or RNA processing.

In vitro and in vivo regulation of assimilatory nitrite reductase from Candida utilis

The nitrate assimilation pathway in Candida utilis, as in other assimilatory organisms, is mediated by two enzymes: nitrate reductase and nitrite reductase. Purified nitrite reductase has been shown to be a heterodimer consisting of 58- and 66-kDa subunits. In the present study, nitrite reductase was found to be capable of utilising both NADH and NADPH as electron donors. FAD, which is an essential coenzyme, stabilised the enzyme during the purification process. The enzyme was modified by cysteine modifiers, and the inactivation could be reversed by thiol reagents. One cysteine was demonstrated to be essential for the enzymatic activity. In vitro, the enzyme was inactivated by ammonium salts, the end product of the path way, proving that the enzyme is assimilatory in function. In vivo, the enzyme was induced by nitrate and repressed by ammonium ions. During induction and repression, the levels of nitrite reductase mRNA, protein, and enzyme activity were modulated together, which indicated that the primary level of regulation of this enzyme was at the transcriptional level. When the enzyme was incubated with ammonium salts in vitro or when the enzyme was assayed in cells grown with the same salts as the source of nitrogen, the residual enzymatic activities were similar. Thus, a study of the in vitro inactivation can give a clue to understanding the mechanism of in vivo regulation of nitrite reductase in Candida utilis.