RESUMEN
In both prokaryotes and eukaryotes, insight into gene function is typically obtained by in silico homology searches and/or phenotypic analyses of strains bearing mutations within open reading frames. However, the studies herein illustrate how mRNA function is not limited to the expression of a cognate protein. We demonstrate that a stress-induced protein-encoding mRNA (irvA) from the dental caries pathogen Streptococcus mutans directly modulates target mRNA (gbpC) stability through seed pairing interactions. The 5' untranslated region of irvA mRNA is a trans riboregulator of gbpC and a critical activator of the DDAG stress response, whereas IrvA functions independently in the regulation of natural competence. The irvA riboregulatory domain controls GbpC production by forming irvA-gbpC hybrid mRNA duplexes that prevent gbpC degradation by an RNase J2-mediated pathway. These studies implicate a potentially ubiquitous role for typical protein-encoding mRNAs as riboregulators, which could alter current concepts in gene regulation.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , ARN Mensajero/genética , Proteínas Represoras/genética , Streptococcus mutans/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Sistemas de Lectura Abierta , Unión Proteica , Estabilidad del ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Streptococcus mutans/metabolismo , Transcripción GenéticaRESUMEN
Streptococcus mutans is a primary pathogen for dental caries in humans. CiaR and CiaH of S. mutans comprise a two-component signal transduction system (TCS) involved in regulating various virulent factors. However, the signal that triggers the CiaRH response remains unknown. In this study, we show that calcium is a signal for regulation of the ciaRH operon, and that a double-glycine-containing small peptide encoded within the ciaRH operon (renamed ciaX) mediates this regulation. CiaX contains a serine + aspartate (SD) domain that is shared by calcium-binding proteins. A markerless in-frame deletion of ciaX reduced ciaRH operon expression and diminished the calcium repression of operon transcription. Point mutations of the SD domain resulted in the same phenotype as the in-frame deletion, indicating that the SD domain is required for CiaX function. Further characterization of ciaX demonstrated that it is involved in calcium-mediated biofilm formation. Furthermore, inactivation of ciaR or ciaH led to the same phenotype as the in-frame deletion of ciaX, suggesting that all three genes are involved in the same regulatory pathway. Sequence analysis and real-time RT-PCR identified a putative CiaR binding site upstream of ciaX. We conclude that the ciaXRH operon is a three-component, self-regulatory system modulating cellular functions in response to calcium.
Asunto(s)
Proteínas Bacterianas/genética , Calcio/metabolismo , Regulación Bacteriana de la Expresión Génica , Operón , Streptococcus mutans/genética , Secuencia de Aminoácidos , Biopelículas , Eliminación de Gen , Genes Bacterianos , Prueba de Complementación Genética , Homeostasis , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fenotipo , Mutación Puntual , ARN Bacteriano/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Transducción de Señal , Streptococcus mutans/metabolismoRESUMEN
Fusobacterium nucleatum is a gram-negative oral bacterial species associated with periodontal disease progression. This species is perhaps best known for its ability to adhere to a vast array of other bacteria and eukaryotic cells. Numerous studies of F. nucleatum have examined various coaggregation partners and inhibitors, but it is largely unknown whether these interactions induce a particular genetic response. We tested coaggregation between F. nucleatum ATCC strain 25586 and various species of Streptococcus in the presence of a semidefined growth medium containing saliva. We found that this condition could support efficient coaggregation but, surprisingly, also stimulated a similar degree of autoaggregation. We further characterized the autoaggregation response, since few reports have examined this in F. nucleatum. After screening several common coaggregation inhibitors, we identified l-lysine as a competitive inhibitor of autoaggregation. We performed a microarray analysis of the planktonic versus autoaggregated cells and found nearly 100 genes that were affected after only about 60 min of aggregation. We tested a subset of these genes via real-time reverse transcription-PCR and confirmed the validity of the microarray results. Some of these genes were also found to be inducible in cell pellets created by centrifugation. Based upon these data, it appears that autoaggregation activates a genetic program that may be utilized for growth in a high cell density environment, such as the oral biofilm.