RESUMEN
The genome of Streptococcus sanguinis is a circular DNA molecule consisting of 2,388,435 bp and is 177 to 590 kb larger than the other 21 streptococcal genomes that have been sequenced. The G+C content of the S. sanguinis genome is 43.4%, which is considerably higher than the G+C contents of other streptococci. The genome encodes 2,274 predicted proteins, 61 tRNAs, and four rRNA operons. A 70-kb region encoding pathways for vitamin B(12) biosynthesis and degradation of ethanolamine and propanediol was apparently acquired by horizontal gene transfer. The gene complement suggests new hypotheses for the pathogenesis and virulence of S. sanguinis and differs from the gene complements of other pathogenic and nonpathogenic streptococci. In particular, S. sanguinis possesses a remarkable abundance of putative surface proteins, which may permit it to be a primary colonizer of the oral cavity and agent of streptococcal endocarditis and infection in neutropenic patients.
Asunto(s)
Genoma Bacteriano , Streptococcus/genética , Proteínas Bacterianas/análisis , Proteínas Bacterianas/metabolismo , Composición de Base , Placa Dental/microbiología , Transferencia de Gen Horizontal , Humanos , Datos de Secuencia Molecular , Infecciones Oportunistas/microbiología , ARN Bacteriano/metabolismo , ARN de Transferencia/metabolismo , Infecciones Estreptocócicas/microbiología , Streptococcus/patogenicidad , Operón de ARNrRESUMEN
Modifications were made to published arbitrary primed polymerase chain reaction (AP-PCR) procedures that resulted in increased specificity and sensitivity. Several arbitrary primer sequences were also evaluated, resulting in recommendations for primer design.
Asunto(s)
Elementos Transponibles de ADN/genética , Reacción en Cadena de la Polimerasa/métodos , Cartilla de ADN , Streptococcus sanguis/genéticaRESUMEN
Streptococcus sanguinis is a gram-positive, facultative anaerobe and a normal inhabitant of the human oral cavity. It is also one of the most common agents of infective endocarditis, a serious endovascular infection. To identify virulence factors for infective endocarditis, signature-tagged mutagenesis (STM) was applied to the SK36 strain of S. sanguinis, whose genome is being sequenced. STM allows the large-scale creation, in vivo screening, and recovery of a series of mutants with altered virulence. Screening of 800 mutants by STM identified 38 putative avirulent and 5 putative hypervirulent mutants. Subsequent molecular analysis of a subset of these mutants identified genes encoding undecaprenol kinase, homoserine kinase, anaerobic ribonucleotide reductase, adenylosuccinate lyase, and a hypothetical protein. Virulence reductions ranging from 2-to 150-fold were confirmed by competitive index assays. One putatively hypervirulent strain with a transposon insertion in an intergenic region was identified, though increased virulence was not confirmed in competitive index assays. All mutants grew comparably to SK36 in aerobic broth culture except for the homoserine kinase mutant. Growth of this mutant was restored by the addition of threonine to the medium. Mutants containing an insertion or in-frame deletion in the anaerobic ribonucleotide reductase gene failed to grow under strictly anaerobic conditions. The results suggest that housekeeping functions such as cell wall synthesis, amino acid and nucleic acid synthesis, and the ability to survive under anaerobic conditions are important virulence factors in S. sanguinis endocarditis.
Asunto(s)
Proteínas Bacterianas/genética , Endocarditis Bacteriana/microbiología , Mutagénesis , Infecciones Estreptocócicas/microbiología , Streptococcus sanguis/patogenicidad , Animales , Proteínas Bacterianas/fisiología , Modelos Animales de Enfermedad , Conejos , Ratas , Streptococcus sanguis/genética , Streptococcus sanguis/fisiología , Virulencia/genéticaRESUMEN
Streptococcus mutans belongs to the viridans group of oral streptococci, which is the leading cause of endocarditis in humans. The LraI family of lipoproteins in viridans group streptococci and other bacteria have been shown to function as virulence factors, adhesins, or ABC-type metal transporters. We previously reported the identification of the S. mutans LraI operon, sloABCR, which encodes components of a putative metal uptake system composed of SloA, an ATP-binding protein, SloB, an integral membrane protein, and SloC, a solute-binding lipoprotein, as well as a metal-dependent regulator, SloR. We report here the functional analysis of this operon. By Western blotting, addition of Mn to the growth medium repressed SloC expression in a wild-type strain but not in a sloR mutant. Other metals tested had little effect. Cells were also tested for aerobic growth in media stripped of metals then reconstituted with Mg and either Mn or Fe. Fe at 10 micro M supported growth of the wild-type strain but not of a sloA or sloC mutant. Mn at 0.1 micro M supported growth of the wild-type strain and sloR mutant but not of sloA or sloC mutants. The combined results suggest that the SloABC proteins transport both metals, although the SloR protein represses this system only in response to Mn. These conclusions are supported by (55)Fe uptake studies with Mn as a competitor. Finally, a sloA mutant demonstrated loss of virulence in a rat model of endocarditis, suggesting that metal transport is required for endocarditis pathogenesis.