RESUMEN
CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacterium Streptococcus mutans UA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of other S. mutans. The deletion of the cas genes of CRISPR1 (ΔC1S), CRISPR2 (ΔC2E), or both CRISPR1+2 (ΔC1SC2E) or the removal of spacers 2 and 3 (ΔCR1SP13E) in S. mutans UA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation of S. mutans by the plasmids matching the spacers 2 and 3. Functional analysis of the cas deletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology in S. mutans. Compared to wild-type cells, the ΔC1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ΔC2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression of cas genes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we provide in vivo evidence that the type II-A CRISPR-Cas system of S. mutans may be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.
Asunto(s)
Proteínas Bacterianas/inmunología , Proteínas Asociadas a CRISPR/inmunología , Sistemas CRISPR-Cas , Streptococcus mutans/inmunología , Proteínas Bacterianas/genética , Bacteriófagos/fisiología , Proteínas Asociadas a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Viabilidad Microbiana , Streptococcus mutans/genética , Streptococcus mutans/crecimiento & desarrollo , Streptococcus mutans/virologíaRESUMEN
The regulation of acid production in and the tolerance to low pH of the cariogenic bacterium Streptococcus mutans have garnered considerable attention since both of these properties contribute substantially to the virulence of this organism. Frequent or prolonged exposure to acid end products, mainly lactic acid, that are present following the consumption of dietary sugars erodes the dental enamel, thereby initiating dental caries. Here we report the involvement of the S. mutans VicK sensor kinase in both the acidogenicity and the aciduricity of this bacterium. When cultures were supplemented with glucose, the glycolytic rate of a VicK null mutant was significantly decreased compared to the glycolytic rate of the wild type (P < 0.05), suggesting that there was impaired acid production. Not surprisingly, the VicK deletion mutant produced less lactic acid, while an acid tolerance response assay revealed that loss of VicK significantly enhanced the survival of S. mutans (P < 0.05). Compared to the survival rates of the wild type, the survival rates of the VicK-deficient mutant were drastically increased when cultures were grown at pH 3.5 with or without preexposure to a signal pH (pH 5.5). Global transcriptional analysis using DNA microarrays and S. mutans wild-type UA159 and VicK deletion mutant strains grown at neutral and low pH values revealed that loss of VicK significantly affected expression of 89 transcripts more than twofold at pH 5.5 (P < 0.001). The affected transcripts included genes with putative functions in transport and maintenance of cell membrane integrity. While our results provide insight into the acid-inducible regulon of S. mutans, here we imply a novel role for VicK in regulating intracellular pH homeostasis in S. mutans.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Streptococcus mutans/metabolismo , Adaptación Fisiológica , Proteínas Bacterianas/genética , Medios de Cultivo/química , Eliminación de Gen , Perfilación de la Expresión Génica , Concentración de Iones de Hidrógeno , Streptococcus mutans/genética , Transcripción GenéticaRESUMEN
A toluene-degrading methanogenic consortium enriched from creosote-contaminated aquifer material was maintained on toluene as the sole carbon and energy source for 10 years. The species in the consortium were characterized by using a molecular approach. Total genomic DNA was isolated, and 16S rRNA genes were amplified by using PCR performed with kingdom-specific primers that were specific for 16S rRNA genes from either members of the kingdom Bacteria or members of the kingdom Archaea. A total of 90 eubacterial clones and 75 archaeal clones were grouped by performing a restriction fragment length polymorphism (RFLP) analysis. Six eubacterial sequences and two archaeal sequences were found in the greatest abundance (in six or more clones) based on the RFLP analysis. The relative abundance of each putative species was estimated by using fluorescent in situ hybridization (FISH), and the presence of putative species was determined qualitatively by performing slot blot hybridization with consortium DNA. Both archaeal species and two of the six eubacterial species were detected in the DNA and FISH hybridization experiments. A phylogenetic analysis of these four dominant organisms suggested that the two archaeal species are related to the genera Methanosaeta and Methanospirillum. One of the eubacterial species is related to the genus Desulfotomaculum, while the other is not related to any previously described genus. By elimination, we propose that the last organism probably initiates the attack on toluene.
Asunto(s)
ADN Ribosómico/genética , Euryarchaeota/genética , Euryarchaeota/metabolismo , Filogenia , Tolueno/metabolismo , Archaea/clasificación , Archaea/genética , Archaea/metabolismo , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Biodegradación Ambiental , ADN Bacteriano/genética , Euryarchaeota/clasificación , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , ARN Bacteriano/genética , ARN Ribosómico 16S/genéticaRESUMEN
Gene targeting has indicated that the bHLH transcription factors Myf-5 and MyoD are required for myogenic determination because skeletal myoblasts and myofibers are entirely ablated in mouse embryos lacking both Myf-5 and MyoD. Entrance into the skeletal myogenic program during development occurs following the independent transcriptional induction of either Myf-5 or MyoD. To identify sequences required for the de novo induction of MyoD transcription during development, we investigated the expression patterns of MyoD-lacZ transgenes in embryos deficient in both Myf-5 and MyoD. We observed that a 258-bp fragment containing the core of the -20-kb MyoD enhancer activated expression in newly formed somites and limb buds in compound mutant embryos lacking both Myf-5 and MyoD. Importantly, Myf-5- and MyoD-deficient presumptive muscle precursor cells expressing beta-galactosidase were observed to assume nonmuscle fates primarily as precartilage primordia in the trunk and the limbs, suggesting that these cells were multipotential. Therefore, cells are recruited into the MyoD-dependent myogenic lineage through activation of the -20-kb MyoD enhancer and this occurs independently in somites and limb buds.
Asunto(s)
Proteínas de Unión al ADN , Músculo Esquelético/embriología , Transactivadores , Animales , Movimiento Celular , Elementos de Facilitación Genéticos , Extremidades/embriología , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Operón Lac , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Proteína MioD/genética , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico , Somitos/citología , Somitos/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
Mounting evidence supports the notion that Myf-5 and MyoD play unique roles in the development of epaxial (originating in the dorso-medial half of the somite, e.g. back muscles) and hypaxial (originating in the ventro-lateral half of the somite, e.g. limb and body wall muscles) musculature. To further understand how Myf-5 and MyoD genes cooperate during skeletal muscle specification, we examined and compared the expression pattern of MyoD-lacZ (258/2.5lacZ and MD6.0-lacZ) transgenes in wild-type, Myf-5, and MyoD mutant embryos. We found that the delayed onset of muscle differentiation in the branchial arches, tongue, limbs, and diaphragm of MyoD-/- embryos was a consequence of a reduced ability of myogenic precursor cells to progress through their normal developmental program and not because of a defect in migration of muscle progenitor cells into these regions. We also found that myogenic precursor cells for back, intercostal, and abdominal wall musculature in Myf-54-/- embryos failed to undergo normal translocation or differentiation. By contrast, the myogenic precursors of intercostal and abdominal wall musculature in MyoD-/- embryos underwent normal translocation but failed to undergo timely differentiation. In conclusion, these observations strongly support the hypothesis that Myf-5 plays a unique role in the development of muscles arising after translocation of epithelial dermamyotome cells along the medial edge of the somite to the subjacent myotome (e.g., back or epaxial muscle) and that MyoD plays a unique role in the development of muscles arising from migratory precursor cells (e.g., limb and branchial arch muscles, tongue, and diaphragm). In addition, the expression pattern of MyoD-lacZ transgenes in the intercostal and abdominal wall muscles of Myf-5-/- and MyoD-/- embryos suggests that appropriate development of these muscles is dependent on both genes and, therefore, these muscles have a dual embryonic origin (epaxial and hypaxial).
Asunto(s)
Proteínas de Unión al ADN , Proteínas Musculares/fisiología , Músculos/embriología , Proteína MioD/fisiología , Proteínas del Tejido Nervioso , Transactivadores , Músculos Abdominales/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Cruzamientos Genéticos , Desmina/metabolismo , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genotipo , Inmunohistoquímica , Hibridación in Situ , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Ratones Transgénicos , Proteínas Musculares/metabolismo , Músculos/metabolismo , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico , Cadenas Pesadas de Miosina/metabolismo , Nestina , Factores de Tiempo , Distribución Tisular , beta-Galactosidasa/metabolismoRESUMEN
The myogenic progenitors of epaxial (paraspinal and intercostal) and hypaxial (limb and abdominal wall) musculature are believed to originate in dorsal-medial and ventral-lateral domains, respectively, of the developing somite. To investigate the hypothesis that Myf-5 and MyoD have different roles in the development of epaxial and hypaxial musculature, we further characterized myogenesis in Myf-5- and MyoD-deficient embryos by several approaches. We examined expression of a MyoD-lacZ transgene in Myf-5 and MyoD mutant embryos to characterize the temporal-spatial patterns of myogenesis in mutant embryos. In addition, we performed immunohistochemistry on sectioned Myf-5 and MyoD mutant embryos with antibodies reactive with desmin, nestin, myosin heavy chain, sarcomeric actin, Myf-5, MyoD and myogenin. While MyoD(-/-) embryos displayed normal development of paraspinal and intercostal muscles in the body proper, muscle development in limb buds and brachial arches was delayed by about 2.5 days. By contrast, Myf-5(-/-) embryos displayed normal muscle development in limb buds and brachial arches, and markedly delayed development of paraspinal and intercostal muscles. Although MyoD mutant embryos exhibited delayed development of limb musculature, normal migration of Pax-3-expressing cells into the limb buds and normal subsequent induction of Myf-5 in myogenic precursors was observed. These results suggest that Myf-5 expression in the limb is insufficient for the normal progression of myogenic development. Taken together, these observations strongly support the hypothesis that Myf-5 and MyoD play unique roles in the development of epaxial and hypaxial muscle, respectively.