RESUMEN
Clinical isolates that are difficult to identify by conventional means form a valuable source of novel human pathogens. We report on a 5-year study based on systematic 16S rRNA gene sequence analysis. We found 60 previously unknown 16S rRNA sequences corresponding to potentially novel bacterial taxa. For 30 of 60 isolates, clinical relevance was evaluated; 18 of the 30 isolates analyzed were considered to be associated with human disease.
Asunto(s)
Bacterias/aislamiento & purificación , Bacterias/patogenicidad , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/microbiología , Adulto , Bacterias/clasificación , Bacterias/genética , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Femenino , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Spontaneous hydrolytic deamination of DNA bases represents a considerable mutagenic threat to all organisms, particularly those living in extreme habitats. Cytosine is readily deaminated to uracil, which base pairs with adenine during replication, and most organisms encode at least one uracil DNA glycosylase (UDG) that removes this aberrant base from DNA with high efficiency. Adenine deaminates to hypoxanthine approximately 10-fold less efficiently, and its removal from DNA in vivo has to date been reported to be mediated solely by alkyladenine DNA glycosylase. We previously showed that UdgB from Pyrobaculum aerophilum, a hyperthermophilic crenarchaeon, can excise hypoxanthine from oligonucleotide substrates, but as this organism is not amenable to genetic manipulation, we were unable to ascertain that the enzyme also has this role in vivo. In the present study, we show that UdgB from Mycobacterium smegmatis protects this organism against mutagenesis associated with deamination of both cytosine and adenine. Together with Ung-type uracil glycosylase, M. smegmatis UdgB also helps attenuate the cytotoxicity of the antimicrobial agent 5-fluorouracil.
Asunto(s)
Citosina/metabolismo , Mycobacterium smegmatis/enzimología , Uracil-ADN Glicosidasa/metabolismo , Actinomycetales/enzimología , Actinomycetales/genética , Adenina , Secuencia de Aminoácidos , Muerte Celular , Desaminación , Eliminación de Gen , Datos de Secuencia Molecular , Mutagénesis , Pyrobaculum/enzimología , Pyrobaculum/genética , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismo , Uracil-ADN Glicosidasa/genéticaRESUMEN
In this study, we investigated the role of the nucleotide excision repair (NER) pathway in mycobacterial DNA repair. Mycobacterium smegmatis lacking the NER excinuclease component uvrB or the helicase uvrD1 gene and a double knockout lacking both genes were constructed, and their sensitivities to a series of DNA-damaging agents were analyzed. As anticipated, the mycobacterial NER system was shown to be involved in the processing of bulky DNA adducts and interstrand cross-links. In addition, it could be shown to exert a protective effect against oxidizing and nitrosating agents. Interestingly, inactivation of uvrB and uvrD1 significantly increased marker integration frequencies in gene conversion assays. This implies that in mycobacteria (which lack the postreplicative mismatch repair system) NER, and particularly the UvrD1 helicase, is involved in the processing of a subset of recombination-associated mismatches.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas/metabolismo , Reparación del ADN , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Proteínas Bacterianas/genética , Disparidad de Par Base/efectos de la radiación , ADN Helicasas/genética , Reparación del ADN/efectos de la radiación , Conversión Génica/efectos de la radiación , Mutación/efectos de la radiación , Mycobacterium smegmatis/efectos de la radiación , Rayos UltravioletaRESUMEN
SMC (structural maintenance of chromosomes) proteins play fundamental roles in various aspects of chromosome organization and dynamics, including repair of DNA damage. Mutant strains of Mycobacterium smegmatis and Mycobacterium tuberculosis defective in SMC were constructed. Surprisingly, inactivation of smc did not result in recognizable phenotypes in hallmark assays characteristic for the function of these genes. This is in contrast to data for smc null mutants in other species.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Ciclo Celular/genética , Reparación del ADN/genética , Eliminación de Gen , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Mycobacterium/genética , Southern Blotting , Supervivencia Celular , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/ultraestructura , Secuencia Conservada , Daño del ADN , MutaciónRESUMEN
BACKGROUND: The rate at which a stretch of DNA mutates is determined by the cellular systems for DNA replication and repair, and by the nucleotide sequence of the stretch itself. One sequence feature with a particularly strong influence on the mutation rate are nucleotide repeats. Some microbial pathogens use nucleotide repeats in their genome to stochastically vary phenotypic traits and thereby evade host defense. However, such unstable sequences also come at a cost, as mutations are often deleterious. Here, we analyzed how these opposing forces shaped genome stability in the human pathogen Mycobacterium tuberculosis. M. tuberculosis lacks a mismatch repair system, and this renders nucleotide repeats particularly unstable. RESULTS: We found that proteins of M. tuberculosis are encoded by using codons in a context-dependent manner that prevents the emergence of nucleotide repeats. This context-dependent codon choice leads to a strong decrease in the estimated frame-shift mutation rate and thus to an increase in genome stability. CONCLUSION: These results indicate that a context-specific codon choice can partially compensate for the lack of a mismatch repair system, and helps to maintain genome integrity in this pathogen.