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Disruption of the Candida albicans CYB5 Gene Results in Increased Azole Sensitivity. K. M. Rogers, 2004.Sterol synthesis in fungi is an aerobic process requiring molecular oxygen and, for several cytochrome-mediated reactions, aerobically synthesized heme . Cytochrome b5 is required for sterol C5-6 desaturation and the encoding gene, CYB5, is nonessential in Saccharomyces cerevisiae . Cyb5p and Ncp1p (cytochrome P-450 reductase) appear to have overlapping functions in this organism, with disruptions of each alone being viable . The cytochrome P-450 reductase phenotype has also been shown to demonstrate increased sensitivity to azole antifungals . Based on this phenotype, the CYB5 gene in the human pathogen Candida albicans was investigated to determine whether the cyb5 genotype was viable and would also demonstrate azole sensitivity . Sequential disruption of the CYB5 alleles by direct transformation resulted in viability, presumably conferred by the presence of a third copy of the CYB5 gene . Subsequent disruption procedures with a pMAL2-CYB5 rescue cassette and a CYB5-URA3 blaster cassette resulted in viable cyb5 strains with no third copy . The C . albicans CYB5 gene is concluded to be nonessential . Thus, the essentiality of this gene and whether we observed two or three alleles was dependent upon the gene disruption protocol . The C . albicans cyb5 strains produced a sterol profile containing low ergosterol levels and sterol intermediates similar to that reported for the S . cerevisiae cyb5 . The C . albicans cyb5 shows increased sensitivity to azoles and terbinafine, an inhibitor of squalene epoxidase, and, unexpectedly, increased resistance to morpholines, which inhibit the ERG2 and ERG24 gene products . These results indicate that an inhibitor of Cyb5p would not be lethal but would make the cell significantly more sensitive to azole treatment . A New Pathway for Salvaging the Coenzyme B12 Precursor Cobinamide in Archaea Requires Cobinamide-Phosphate Synthase (CbiB) Enzyme Activity. Jesse D. Woodson, 2003.The ability of archaea to salvage cobinamide has been under question because archaeal genomes lack orthologs to the bacterial nucleoside triphosphate:5'-deoxycobinamide kinase enzyme (cobU in Salmonella enterica) . The latter activity is required for cobinamide salvaging in bacteria . This paper reports evidence that archaea salvage cobinamide from the environment by using a pathway different from the one used by bacteria . These studies demanded the functional characterization of two genes whose putative function had been annotated based solely on their homology to the bacterial genes encoding adenosylcobyric acid and adenosylcobinamide-phosphate synthases (cbiP and cbiB, respectively) of S. enterica . A cbiP mutant strain of the archaeon Halobacterium sp . strain NRC-1 was auxotrophic for adenosylcobyric acid, a known intermediate of the de novo cobamide biosynthesis pathway, but efficiently salvaged cobinamide from the environment, suggesting the existence of a salvaging pathway in this archaeon . A cbiB mutant strain of Halobacterium was auxotrophic for adenosylcobinamide-GDP, a known de novo intermediate, and did not salvage cobinamide . The results of the nutritional analyses of the cbiP and cbiB mutants suggested that the entry point for cobinamide salvaging is adenosylcobyric acid . The data are consistent with a salvaging pathway for cobinamide in which an amidohydrolase enzyme cleaves off the aminopropanol moiety of adenosylcobinamide to yield adenosylcobyric acid, which is converted by the adenosylcobinamide-phosphate synthase enzyme to adenosylcobinamide-phosphate, a known intermediate of the de novo biosynthetic pathway . The existence of an adenosylcobinamide amidohydrolase enzyme would explain the lack of an adenosylcobinamide kinase in archaea . Capsid Functions of Inactivated Human Picornaviruses and Feline Calicivirus. Suphachai Nuanualsuwan, 2003.The exceptional stability of enteric viruses probably resides in their capsids . The capsid functions of inactivated human picornaviruses and feline calicivirus (FCV) were determined . Viruses were inactivated by UV, hypochlorite, high temperature (72°C), and physiological temperature (37°C), all of which are pertinent to transmission via food and water . Poliovirus (PV) and hepatitis A virus (HAV) are transmissible via water and food, and FCV is the best available surrogate for the Norwalk-like viruses, which are leading causes of food-borne and waterborne disease in the United States . The capsids of all 37°C-inactivated viruses still protected the viral RNA against RNase, even in the presence of proteinase K, which contrasted with findings with viruses inactivated at 72°C . The loss of ability of the virus to attach to homologous cell receptors was universal, regardless of virus type and inactivation method, except for UV-inactivated HAV, and so virus inactivation was almost always accompanied by the loss of virus attachment . Inactivated HAV and FCV were captured by homologous antibodies . However, inactivated PV type 1 (PV-1) was not captured by homologous antibody and 37°C-inactivated PV-1 was only partially captured . The epitopes on the capsids of HAV and FCV are evidently discrete from the receptor attachment sites, unlike those of PV-1 . These findings indicate that the primary target of UV, hypochlorite, and 72°C inactivation is the capsid and that the target of thermal inactivation (37°C versus 72°C) is temperature dependent . Growth of Hyperthermophilic Archaeon Pyrococcus furiosus on Chitin Involves Two Family 18 Chitinases. Jun Gao, 2003.Pyrococcus furiosus was found to grow on chitin, adding this polysacharide to the inventory of carbohydrates utilized by this hyperthermophilic archaeon . Accordingly, two open reading frames (chiA [Pf1234] and chiB [Pf1233]) were identified in the genome of P . furiosus, which encodes chitinases with sequence similarity to proteins from the glycosyl hydrolase family 18 in less-thermophilic organisms . Both enzymes contain multiple domains that consist of at least one binding domain and one catalytic domain . ChiA (ca . 39 kDa) contains a putative signal peptide, as well as a binding domain (ChiABD), that is related to binding domains associated with several previously studied bacterial chitinases . chiB, separated by 37 nucleotides from chiA and in the same orientation, encodes a polypeptide with two different proline-threonine-rich linker regions (6 and 3 kDa) flanking a chitin-binding domain (ChiBBD [11 kDa]), followed by a catalytic domain (ChiBcat [35 kDa]) . No apparent signal peptide is encoded within chiB . The two chitinases share little sequence homology to each other, except in the catalytic region, where both have the catalytic glutamic acid residue that is conserved in all family 18 bacterial chitinases . The genes encoding ChiA, without its signal peptide, and ChiB were cloned and expressed in Escherichia coli. ChiA exhibited no detectable activity toward chitooligomers smaller than chitotetraose, indicating that the enzyme is an endochitinase . Kinetic studies showed that ChiB followed Michaelis-Menten kinetics toward chitotriose, although substrate inhibition was observed for larger chitooligomers . Hydrolysis patterns on chitooligosaccharides indicated that ChiB is a chitobiosidase, processively cleaving off chitobiose from the nonreducing end of chitin or other chitooligomers . Synergistic activity was noted for the two chitinases on colloidal chitin, indicating that these two enzymes work together to recruit chitin-based substrates for P . furiosus growth . This was supported by the observed growth on chitin as the sole carbohydrate source in sulfur-free media . Bacterial Chemotaxis to Naphthalene Desorbing from a Nonaqueous Liquid. Aaron M. J. Law, 2003.Bacterial chemotaxis has the potential to increase the rate of degradation of chemoattractants, but its influence on degradation of hydrophobic attractants initially dissolved in a non-aqueous-phase liquid (NAPL) has not been examined . We studied the effect of chemotaxis by Pseudomonas putida G7 on naphthalene mass transfer and degradation in a system in which the naphthalene was dissolved in a model NAPL . Chemotaxis by wild-type P . putida G7 increased the rates of naphthalene desorption and degradation relative to rates observed with nonchemotactic and nonmotile mutant strains . While biodegradation alone influenced the rate of substrate desorption by increasing the concentration gradient against which desorption occurred, chemotaxis created an even steeper gradient as the cells accumulated near the NAPL source . The extent to which chemotaxis affected naphthalene desorption and degradation depended on the initial bacterial and naphthalene concentrations, reflecting the influences of these variables on concentration gradients and on the relative rates of mass transfer and biodegradation . The results of this study suggest that chemotaxis can substantially increase the rates of mass transfer and degradation of NAPL-associated hydrophobic pollutants .
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