RESUMEN
The sanguis streptococci are primary colonizers of the tooth surface and thus form the foundation for the complex multiple species biofilm known as dental plaque. In addition, these bacteria can colonize native and prosthetic heart valves and are a common cause of endocarditis. Little is known about the molecular mechanisms governing multiple or single species biofilm development within this group of organisms. Using an in vitro assay for biofilm formation, we determined that (i) Streptococcus parasanguis FW213 can form biofilms on inert surfaces such as polystyrene and (ii) environmental and nutritional factors, such as glucose, affect S. parasanguis biofilm formation. Several isogenic mutants of FW213 were tested in the biofilm assay. Strains containing mutations in fap1, a gene encoding a protein required for assembly of fimbriae, were deficient in biofilm formation. Mutants defective in recA, PepO endopeptidase activity, or the production of a fimbriae-associated protein, FimA, were still capable of biofilm formation. Phase-contrast microscopy was used to follow biofilm development by wild-type and fap1 mutant strains on plastic coverslips over time. Wild-type FW213 attached to the surface, formed aggregates of cells, and eventually formed a dense layer of cells that included microcolonies. In contrast, few fap1 mutant cells were observed attached to the surface, and no cell aggregates or microcolonies were formed. These results suggest that the long peritrichous fimbriae of FW213 are critical for the formation of biofilms on solid surfaces.
Asunto(s)
Proteínas Bacterianas/fisiología , Biopelículas , Proteínas Fimbrias , Fimbrias Bacterianas/fisiología , Streptococcus/fisiología , Glucosa/farmacología , Concentración de Iones de Hidrógeno , MutaciónRESUMEN
The gene for the green fluorescent protein, gfp, was cloned, under the control of the Actinobacillus actinomycetemcomitans leukotoxin (ltx) promoter, in the A. actinomycetemcomitans shuttle vector, pSU20. A actinomycetemcomitans containing the ltx-gfp construct emitted bright green fluorescence in the standard invasion assay using epifluorescence microscopy. These data demonstrate that the green fluorescent protein will be a useful tool for the live analysis of A. actinomycetemcomitans interactions with host cells, and that the ltx promoter can be used to drive the expression of non-A. actinomycetemcomitans genes.
Asunto(s)
Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/metabolismo , Proteínas Luminiscentes/biosíntesis , Técnicas Bacteriológicas , Clonación Molecular , Células Epiteliales/microbiología , Escherichia coli/genética , Exotoxinas/genética , Proteínas Fluorescentes Verdes , Indicadores y Reactivos , Proteínas Luminiscentes/genética , Regiones Promotoras GenéticasRESUMEN
Studies in our laboratory have identified two fimbria-associated adhesins, FimA and Fap1, of Streptococcus parasanguis FW213. In this study, we isolated and sequenced DNA fragments linked to fimA to determine if they contained additional factors associated with adherence, virulence, or survival in the host. An open reading frame just upstream and divergently transcribed from the fimA operon was identified and named pepO. Northern hybridization indicated that pepO is transcribed as a monocistronic message. pepO encodes a predicted 631-amino-acid protein with a molecular mass of approximately 70.6 kDa. PepO contains the essential motif HEXXH, typical of many zinc-dependent metalloproteases and metallopeptidases. PepO has significant sequence identity to mammalian metallopeptidases, including endothelin-converting enzyme, which converts a potent vasoconstrictor into its active form, and neutral endopeptidase (NEP), which is involved in terminating the activity of opioid peptides. The opioid peptide metenkephalin is a natural substrate of NEP. Cell extracts of FW213 cleaved metenkephalin at the same site as does NEP, while an extract from an insertionally inactivated pepO mutant did not. These results indicate that FW213 pepO encodes an enzyme with activity similar to that of known mammalian endopeptidases. Phylogenetic analysis of PepO and its homologues suggests lateral genetic exchange between bacteria and eukaryotes.
Asunto(s)
Proteínas Bacterianas/fisiología , Metaloendopeptidasas/fisiología , Streptococcus/enzimología , Secuencia de Aminoácidos , Animales , Humanos , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Mutación , Neprilisina/fisiología , Sistemas de Lectura Abierta , Filogenia , Streptococcus/clasificación , Streptococcus/genética , Transcripción GenéticaRESUMEN
PCR was used to amplify an internal region of the recA gene from Streptococcus parasanguis FW213. The PCR fragment was used as a probe to recover the entire streptococcal recA gene from an S. parasanguis genomic library, and the sequence of the gene was determined. The deduced product of the S. parasanguis recA gene showed a high degree of amino acid identity with other prokaryotic RecA proteins. The cloned recA sequence was disrupted in vitro by insertional mutagenesis, and the mutated allele was then introduced into the S. parasanguis chromosome by homologous recombination. Results of Southern hybridizations confirmed the replacement of the wild-type recA gene with the mutated allele. The recA mutant strain was considerably more sensitive to UV light than the parental strain, and this phenotype was consistent with a mutation in recA. The S. parasanguis recA mutant showed no reduction in its ability to adhere in the in vitro tooth surface model, saliva-coated hydroxylapatite (SHA), or in its ability to express the fimbria-associated adhesin Fap1. These results demonstrate that in vitro attachment of S. parasanguis FW213 to SHA and expression of Fap1 are recA independent.
Asunto(s)
Adhesión Bacteriana/genética , Mutación , Rec A Recombinasas/genética , Streptococcus sanguis/genética , Streptococcus sanguis/patogenicidad , Diente/microbiología , Adhesinas Bacterianas/genética , Secuencia de Bases , Cartilla de ADN/genética , ADN Bacteriano/genética , Durapatita , Expresión Génica , Genes Bacterianos , Humanos , Técnicas In Vitro , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Insercional , Fenotipo , Recombinación Genética , Mapeo Restrictivo , Saliva/microbiología , Streptococcus sanguis/fisiologíaRESUMEN
NUT1, a gene homologous to the major nitrogen regulatory genes nit-2 of Neurospora crassa and areA of Aspergillus nidulans, was isolated from the rice blast fungus, Magnaporthe grisea. NUT1 encodes a protein of 956 amino acid residues and, like nit-2 and areA, has a single putative zinc finger DNA-binding domain. Functional equivalence of NUT1 to areA was demonstrated by introducing the NUT1 gene by DNA-mediated transformation into an areA loss-of-function mutant of A. nidulans. The introduced NUT1 gene fully complemented the areA null mutation, restoring to the mutant the ability to utilize a variety of nitrogen sources. In addition, the sensitivity of Aspergillus NUT1 transformants to ammonium repression of extracellular protease activity was comparable to that of wild-type A. nidulans. Thus, NUT1 and areA encode functionally equivalent gene products that activate expression of nitrogen-regulated genes. A one-step disruption strategy was used to generate nut1- mutants of M. grisea by transforming a rice-infecting strain with a disruption vector in which a gene for hygromycin B phosphotransferase (Hyg) replaced the zinc-finger DNA-binding motif of NUT1. Of 31 hygromycin B (hyg-B)-resistant transformants shown by Southern hybridization to contain a disrupted NUT1 gene (nut1 : : Hyg), 26 resulted from single-copy replacement events at the NUT1 locus. Although nut1- transformants of M. grisea failed to grown on a variety of nitrogen sources, glutamate, proline and alanine could still be utilized. This contrasts with A. nidulans where disruption of the zinc-finger region of areA prevents utilization of nitrogen sources other than ammonium and glutamine. The role of NUT1 and regulation of nitrogen metabolism in the disease process was evaluated by pathogenicity assays. The infection efficiency of nut1- transformants on susceptible rice plants was similar to that of the parental strain, although lesions were reduced in size. These studies demonstrate that the M. grisea NUT1 gene activates expression of nitrogen-regulated genes but is dispensable for pathogenicity.
Asunto(s)
Ascomicetos/genética , Proteínas de Unión al ADN/fisiología , Genes Fúngicos/fisiología , Genes Reguladores/fisiología , Nitrógeno/metabolismo , Secuencia de Aminoácidos , Ascomicetos/crecimiento & desarrollo , Ascomicetos/patogenicidad , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Genes Fúngicos/genética , Genes Reguladores/genética , Datos de Secuencia Molecular , Mutagénesis , Oryza/microbiología , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Dedos de ZincRESUMEN
Two unlinked incompatibility loci, a and b, control mating, pathogenicity, and sexual development in Ustilago maydis, a fungal pathogen of corn. Fusion of nonpathogenic haploid cells occurs when alleles of the a locus differ; fusion products that differ at the b locus will be pathogenic and complete sexual development. The two alleles of the a locus have been cloned. The a2 allele was isolated by exploiting the close linkage of the a locus to the genetic marker pan1. Several cosmids from a clonal a2 mating-type library allowed, via DNA-mediated transformation, an a1 mating type, pan1-1 auxotrophic recipient to grow prototrophically on medium lacking pantothenic acid. Cosmids containing functional a2 mating-type regions were identified by their ability to confer a2 mating behavior to the a1 recipient strain. The a1 allele was isolated from a plasmid library using DNA from the a2 region as a hybridization probe. The isolated clones contain a1 or a2 mating-type specificity as demonstrated by a plate mating assay, by pathogenicity tests on corn, and by gene replacement experiments. The a1 and a2 mating-type clones contain nonhomologous DNA segments that are flanked by similar nucleotide (nt) sequences. Restriction fragments containing a mating-type function are embedded within the nonhomologous DNA segments. U. maydis contains a single copy of either the a1 or a2 mating-type sequence within each haploid genome. This structural organization demonstrates that the a mating-type alleles of U. maydis are idiomorphs sensu Metzenberg and Glass (1990), nt sequences mapping to the same genetic locus that share no obvious evolutionary relationship.
Asunto(s)
Ustilago/genética , Alelos , Clonación Molecular , Cruzamientos Genéticos , Diploidia , Prueba de Complementación Genética , Genotipo , Fusión de Membrana , Hibridación de Ácido Nucleico , Mapeo Restrictivo , Transformación Genética , Ustilago/fisiologíaRESUMEN
The URA1 gene (encoding orotidine-5'-monophosphate decarboxylase) of the basidiomycete fungus Schizophyllum commune was mapped to a 1.4-kb BglI-BamHI fragment of two independent phage lambda clones previously isolated from a Schizophyllum genomic library. The fragment was identified by its ability to complement Schizophyllum ura1 mutants via transformation. The complete nucleotide sequence of the fragment containing the URA1 gene was determined. Sequence analysis revealed that the coding region of the URA1 gene encompasses a polypeptide of 279 amino acids (aa) interrupted by two small introns. The deduced aa sequence corresponds to 30.3 kDa and is substantially similar to the sequences of analogous polypeptides from other organisms. No canonical 5'-TATA sequence nor 3'-AATAAA polyadenylation signal are evident in the flanking regions of the URA1 gene.
Asunto(s)
Basidiomycota/genética , Carboxiliasas/genética , Genes Fúngicos , Orotidina-5'-Fosfato Descarboxilasa/genética , Schizophyllum/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Intrones , Datos de Secuencia Molecular , Mapeo Restrictivo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Schizophyllum/enzimología , Homología de Secuencia de Ácido NucleicoRESUMEN
We have developed a routine way to isolate genes directly from the basidiomycete fungus, Schizophyllum commune. Plasmid DNA from a genomic gene library was used to isolate five specific genes by complementation of Schizophyllum mutations via transformation. The mutant strains were deficient in the ability to synthesize either adenine (ade2 and ade5), uracil (ura1, encoding orotidine-5'-phosphate decarboxylase; OMPdecase), tryptophan (trp1, encoding indole-3-glycerol phosphate synthetase; IGPS) or para aminobenzoic acid (pab1). In each case, Southern analysis revealed that transformation to prototrophy was concomitant with the integration of vector sequence into the genome of the S. commune mutant. Total DNA from transformants was restricted, religated, and used to transform E. coli. Ampicillin resistant plasmids were recovered from E. coli and tested for their ability to transform the corresponding mutant of S. commune. Plasmids complementing the ade2, ade5, pab1, trp1, and ura1 mutations were recovered.