RESUMO
An expression vector (CIp10-MAL2p) for use in Candida albicans has been constructed in which a gene of interest can be placed under the control of the CaMAL2 maltase promoter and stably integrated at the CaRP10 locus. Using this vector to express the Candida URA3 gene from the CaMAL2 promoter, we have demonstrated tight regulation of CaURA3 expression by carbon source. Thus under conditions when the CaMAL2 promoter is not induced, expression of Candida URA3 was unable either to complement a C. albicans ura3 mutation or to confer sensitivity to 5-fluoroorotic acid, a compound which is highly toxic to URA3 strains. Since Candida albicans is an obligate diploid organism, analysis of gene function requires manipulation of both copies of any gene of interest. Our expression vector provides a strategy by which the remaining copy of a gene of interest can be placed under CaMAL2 promoter control in a strain where the first copy has been deleted, permitting analysis of gene function by manipulation of carbon source. CIp10-MAL2p should therefore provide a useful means for functional analysis of genes in C. albicans. We have used this strategy with C. albicans DPB2 to demonstrate that the gene is essential and that loss of function leads cells to adopt a hypha-like morphology as they cease proliferation.
Assuntos
Antígenos de Fungos , Candida albicans/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Regiões Promotoras Genéticas , alfa-Glucosidases/genética , Candida albicans/crescimento & desenvolvimento , Meios de Cultura , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Vetores Genéticos , Glucose/metabolismo , Maltose/metabolismo , Proteínas Ribossômicas/genéticaRESUMO
The Staphylococcus aureus repeat (STAR) element is a sequence identified in two intergenic regions in S. aureus. The element is found in 13 to 21 copies in individual S. aureus strains, and elements in the homologous intergenic location are variable in length. The element sequence consists of several small and unusually GC-rich direct repeats with recurring intervening sequences. In addition, STAR-like elements may be present in related staphylococcal species.
Assuntos
Repetições de Dinucleotídeos/genética , Proteínas de Escherichia coli , Staphylococcus aureus/genética , Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Ligação a DNA/genética , Bases de Dados Factuais , Genótipo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/genética , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The Staphylococcus xylosus gene hprK, encoding HPr kinase (HPrK), has been isolated from a genomic library. The HPrK enzyme, purified as a His(6) fusion protein, phosphorylated HPr, the phosphocarrier protein of the bacterial phosphotransferase system, at a serine residue in an ATP-dependent manner, and it also catalyzed the reverse reaction. Therefore, the enzyme constitutes a bifunctional HPr kinase/phosphatase. Insertional inactivation of the gene in the genome of S. xylosus resulted in the concomitant loss of both HPr kinase and His serine-phosphorylated-HPr phosphatase activities in cell extracts, strongly indicating that the HPrK enzyme is also responsible for both reactions in vivo. HPrK deficiency had a profound pleiotropic effect on the physiology of S. xylosus. The hprK mutant strain showed a severe growth defect in complex medium upon addition of glucose. Glucose uptake in glucose-grown cells was strongly enhanced compared with the wild type. Carbon catabolite repression of three tested enzyme activities by glucose, sucrose, and fructose was abolished. These results clearly demonstrate the prominent role of HPr kinase in global control to adjust catabolic capacities of S. xylosus according to the availability of preferred carbon sources.
Assuntos
Proteínas de Bactérias , Mutação/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Staphylococcus/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Metabolismo dos Carboidratos , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Glicosídeo Hidrolases/metabolismo , Dados de Sequência Molecular , Mutagênese Insercional/genética , Fenótipo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/isolamento & purificação , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/isolamento & purificação , Aldeído Pirúvico/metabolismo , RNA Bacteriano/análise , RNA Bacteriano/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Staphylococcus/genética , Staphylococcus/crescimento & desenvolvimento , Staphylococcus/metabolismo , Transcrição Gênica/genéticaRESUMO
Nosocomial infections that result in the formation of biofilms on the surfaces of biomedical implants are a leading cause of sepsis and are often associated with colonization of the implants by Staphylococcus epidermidis. Biofilm formation is thought to require two sequential steps: adhesion of cells to a solid substrate followed by cell-cell adhesion, creating multiple layers of cells. Intercellular adhesion requires the polysaccharide intercellular adhesin (PIA), which is composed of linear beta-1,6-linked glucosaminylglycans and can be synthesized in vitro from UDP-N-acetylglucosamine by products of the intercellular adhesion (ica) locus. We have investigated a variety of Staphylococcus aureus strains and find that all strains tested contain the ica locus and that several can form biofilms in vitro. Sequence comparison with the S. epidermidis ica genes revealed 59 to 78% amino acid identity. Deletion of the ica locus results in a loss of the ability to form biofilms, produce PIA, or mediate N-acetylglucosaminyltransferase activity in vitro. Cross-species hybridization experiments revealed the presence of icaA in several other Staphylococcus species, suggesting that cell-cell adhesion and the potential to form biofilms is conserved within this genus.