RESUMEN
Ramlibacter tataouinensis TTB310(T) (strain TTB310), a betaproteobacterium isolated from a semi-arid region of South Tunisia (Tataouine), is characterized by the presence of both spherical and rod-shaped cells in pure culture. Cell division of strain TTB310 occurs by the binary fission of spherical "cyst-like" cells ("cyst-cyst" division). The rod-shaped cells formed at the periphery of a colony (consisting mainly of cysts) are highly motile and colonize a new environment, where they form a new colony by reversion to cyst-like cells. This unique cell cycle of strain TTB310, with desiccation tolerant cyst-like cells capable of division and desiccation sensitive motile rods capable of dissemination, appears to be a novel adaptation for life in a hot and dry desert environment. In order to gain insights into strain TTB310's underlying genetic repertoire and possible mechanisms responsible for its unusual lifestyle, the genome of strain TTB310 was completely sequenced and subsequently annotated. The complete genome consists of a single circular chromosome of 4,070,194 bp with an average G+C content of 70.0%, the highest among the Betaproteobacteria sequenced to date, with total of 3,899 predicted coding sequences covering 92% of the genome. We found that strain TTB310 has developed a highly complex network of two-component systems, which may utilize responses to light and perhaps a rudimentary circadian hourglass to anticipate water availability at the dew time in the middle/end of the desert winter nights and thus direct the growth window to cyclic water availability times. Other interesting features of the strain TTB310 genome that appear to be important for desiccation tolerance, including intermediary metabolism compounds such as trehalose or polyhydroxyalkanoate, and signal transduction pathways, are presented and discussed.
Asunto(s)
Adaptación Fisiológica/genética , División Celular/genética , Comamonadaceae/citología , Comamonadaceae/fisiología , Clima Desértico , Genoma Bacteriano , Genómica , Adaptación Fisiológica/efectos de la radiación , Metabolismo de los Hidratos de Carbono/genética , Metabolismo de los Hidratos de Carbono/efectos de la radiación , División Celular/efectos de la radiación , Membrana Celular/metabolismo , Membrana Celular/efectos de la radiación , Movimiento Celular/genética , Movimiento Celular/efectos de la radiación , Forma de la Célula/genética , Forma de la Célula/efectos de la radiación , Ritmo Circadiano/genética , Ritmo Circadiano/efectos de la radiación , Comamonadaceae/enzimología , Comamonadaceae/genética , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , ADN Bacteriano/genética , Espacio Extracelular/genética , Espacio Extracelular/metabolismo , Espacio Extracelular/efectos de la radiación , Ácidos Grasos/metabolismo , Hidrólisis/efectos de la radiación , Luz , Fluidez de la Membrana/genética , Fluidez de la Membrana/efectos de la radiación , Lípidos de la Membrana/metabolismo , Presión Osmótica/efectos de la radiación , Estrés Oxidativo/genética , Estrés Oxidativo/efectos de la radiación , Polisacáridos Bacterianos/biosíntesis , Polisacáridos Bacterianos/metabolismo , Transporte de Proteínas/genética , Transporte de Proteínas/efectos de la radiación , Análisis de Secuencia de ADN , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación , Trehalosa/biosíntesis , Trehalosa/metabolismoRESUMEN
Several enzymes have evolved as sensors in signal transduction pathways to control gene expression, thereby allowing bacteria to adapt efficiently to environmental changes. We recently identified the master regulator of cysteine metabolism in Bacillus subtilis, CymR, which belongs to the poorly characterized Rrf2 family of regulators. We now report that the signal transduction mechanism controlling CymR activity in response to cysteine availability involves the formation of a stable complex with CysK, a key enzyme for cysteine biosynthesis. We carried out a comprehensive quantitative characterization of this regulator-enzyme interaction by surface plasmon resonance and analytical ultracentrifugation. We also showed that O-acetylserine plays a dual role as a substrate of CysK and as an effector modulating the CymR-CysK complex formation. The ability of B. subtilis CysK to bind to CymR appears to be correlated to the loss of its capacity to form a cysteine synthase complex with CysE. We propose an original model, supported by the determination of the intracellular concentrations of the different partners, by which CysK positively regulates CymR in sensing the bacterial cysteine pool.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Cisteína Sintasa/metabolismo , Cisteína/biosíntesis , Modelos Biológicos , Complejos Multienzimáticos/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Cisteína/genética , Cisteína Sintasa/genética , Complejos Multienzimáticos/genética , Resonancia por Plasmón de Superficie/métodosRESUMEN
Translesional DNA polymerases form a large family of structurally related proteins, known as the Y-polymerases. Bacillus subtilis encodes two Y-polymerases, referred herewith as Pol Y1 and Pol Y2. Pol Y1 was expressed constitutively and did not mediate UV mutagenesis. Pol Y1 overexpression increased spontaneous mutagenesis. This effect depended on Pol Y1 polymerase activity, Pol Y1 interaction with the beta-clamp, and did not require the presence of the RecA protein. In addition, Pol Y1 overexpression delayed cell growth at low temperature. The growth delay was mediated by Pol Y1 interaction with the beta-clamp but not by its polymerase activity, suggesting that an excess of Pol Y1 in the cell could sequester the beta-clamp. In contrast, Pol Y2 was expressed during the SOS response, and, in its absence, UV-induced mutagenesis was abolished. Upon Pol Y2 overproduction, both UV-induced and spontaneous mutagenesis were stimulated, and both depended on the Pol Y2 polymerase activity. However, UV mutagenesis did not appear to require the interaction of Pol Y2 with the beta-clamp whereas spontaneous mutagenesis did. In addition, Pol Y2-mediated spontaneous mutagenesis required the presence of RecA. Together, these results show that the regulation and the genetic requirements of the two B. subtilis Y-polymerases are different, indicating that they fulfil distinct biological roles. Remarkably, Pol Y1 appears to exhibit a mutator activity similar to that of Escherichia coli Pol IV, as well as an E. coli UmuD-related function in growth delay. Pol Y2 exhibits an E. coli Pol V-like mutator activity, but probably acts as a single polypeptide to bypass UV lesions. Thus, B. subtilis Pol Y1 and Pol Y2 exhibit distinctive features from the E. coli Y-polymerases, indicating that different bacteria have adapted different solutions to deal with the lesions in their genetic material.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/genética , ADN Polimerasa Dirigida por ADN/genética , Secuencias de Aminoácidos , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Daño del ADN , ADN Bacteriano/efectos de la radiación , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genotipo , Mutación , Conformación Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Temperatura , Rayos UltravioletaRESUMEN
The delivery of a ring-shaped hexameric helicase onto DNA is a fundamental step of DNA replication, conserved in all cellular organisms. We report the biochemical characterization of the bacterial hexameric replicative helicase DnaC of Bacillus subtilis with that of the two replication initiation proteins DnaI and DnaB. We show that DnaI and DnaB interact physically and functionally with the DnaC helicase and mediate its functional delivery onto DNA. Thus, DnaB and DnaI form a pair of helicase loaders, revealing a two-protein strategy for the loading of a replicative helicase. We also present evidence that the DnaC helicase loading mechanism appears to be of the ring-assembly type, proceeding through the recruitment of DnaC monomers and their hexamerization around single-stranded DNA by the coordinated action of DnaI and DnaB.