RESUMEN
The small RNA AbcR1 regulates the expression of ABC transporters in the plant pathogen Agrobacterium tumefaciens, the plant symbiont Sinorhizobium meliloti, and the human pathogen Brucella abortus. A combination of proteomic and bioinformatic approaches suggested dozens of AbcR1 targets in A. tumefaciens. Several of these newly discovered targets are involved in the uptake of amino acids, their derivatives, and sugars. Among the latter is the periplasmic sugar-binding protein ChvE, a component of the virulence signal transduction system. We examined 16 targets and their interaction with AbcR1 in close detail. In addition to the previously described mRNA interaction site of AbcR1 (M1), the CopraRNA program predicted a second functional module (M2) as target-binding site. Both M1 and M2 contain single-stranded anti-SD motifs. Using mutated AbcR1 variants, we systematically tested by band shift experiments, which sRNA region is responsible for mRNA binding and gene regulation. On the target site, we find that AbcR1 interacts with some mRNAs in the translation initiation region and with others far into their coding sequence. Our data show that AbcR1 is a versatile master regulator of nutrient uptake systems in A. tumefaciens and related bacteria.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Alphaproteobacteria/genética , Regulación Bacteriana de la Expresión Génica , ARN Mensajero/genética , ARN Pequeño no Traducido/genética , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Alphaproteobacteria/metabolismo , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Biología Computacional , Proteína de Factor 1 del Huésped/genética , Proteína de Factor 1 del Huésped/metabolismo , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Proteínas de Unión Periplasmáticas/genética , Proteínas de Unión Periplasmáticas/metabolismo , Proteómica , Estabilidad del ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/metabolismo , Reproducibilidad de los Resultados , Alineación de SecuenciaRESUMEN
The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of an hfq mutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineer Agrobacterium tumefaciens binds to the previously described sRNA AbcR1 and its target mRNA atu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and γ-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in an hfq mutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility of A. tumefaciens. The hfq mutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation by A. tumefaciens.
