RESUMEN
The chemosensory system in Sinorhizobium meliloti has several important deviations from the widely studied enterobacterial paradigm. To better understand the differences between the two systems and how they are optimally tuned, we determined the cellular stoichiometry of the methyl-accepting chemotaxis proteins (MCPs) and the histidine kinase CheA in S. meliloti Quantitative immunoblotting was used to determine the total amount of MCPs and CheA per cell in S. meliloti The MCPs are present in the cell in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY and McpZ), whereas McpT was below the detection limit. The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 23.5:1, highly similar to that seen in Bacillus subtilis (23:1) and about 10 times higher than that in Escherichia coli (3.4:1). Different from E. coli, the high-abundance receptors in S. meliloti are lacking the carboxy-terminal NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. Using transcriptional lacZ fusions, we showed that chemoreceptors are positively controlled by the master regulators of motility, VisNR and Rem. In addition, FlbT, a class IIA transcriptional regulator of flagellins, also positively regulates the expression of most chemoreceptors except for McpT and McpY, identifying chemoreceptors as class III genes. Taken together, these results demonstrate that the chemosensory complex and the adaptation system in S. meliloti deviates significantly from the established enterobacterial paradigm but shares some similarities with B. subtilisIMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti is of great agricultural importance because of its nitrogen-fixing properties, which enhances growth of its plant symbiont, alfalfa. Chemotaxis provides a competitive advantage for bacteria to sense their environment and interact with their eukaryotic hosts. For a better understanding of the role of chemotaxis in these processes, detailed knowledge on the regulation and composition of the chemosensory machinery is essential. Here, we show that chemoreceptor gene expression in S. meliloti is controlled through the main transcriptional regulators of motility. Chemoreceptor abundance is much lower in S. meliloti than in Escherichia coli and Bacillus subtilis Moreover, the chemoreceptor-to-kinase CheA ratio is different from that of E. coli but similar to that of B. subtilis.
Asunto(s)
Quimiotaxis/fisiología , Histidina Quinasa/genética , Proteínas Quimiotácticas Aceptoras de Metilo/genética , Sinorhizobium meliloti/química , Sinorhizobium meliloti/genética , Transcripción Genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Factores Quimiotácticos , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Eliminación de Gen , Histidina Quinasa/análisis , Medicago sativa/microbiología , Proteínas de la Membrana/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo/análisis , Proteínas Quimiotácticas Aceptoras de Metilo/química , Movimiento , Sinorhizobium meliloti/fisiología , SimbiosisRESUMEN
Type IVb pili in enteropathogenic bacteria function as a host colonization factor by mediating tight adherence to host cells, but their role in bacterium-plant symbiosis is currently unknown. The genome of the symbiotic soil bacterium Sinorhizobium meliloti contains two clusters encoding proteins for type IVb pili of the Flp (fimbrial low-molecular-weight protein) subfamily. To establish the role of Flp pili in the symbiotic interaction of S. meliloti and its host, Medicago sativa, we deleted pilA1, which encodes the putative pilin subunit in the chromosomal flp-1 cluster and conducted competitive nodulation assays. The pilA1 deletion strain formed 27% fewer nodules than the wild type. Transmission electron microscopy revealed the presence of bundle-forming pili protruding from the polar and lateral region of S. meliloti wild-type cells. The putative pilus assembly ATPase CpaE1 fused to mCherry showed a predominantly unilateral localization. Transcriptional reporter gene assays demonstrated that expression of pilA1 peaks in early stationary phase and is repressed by the quorum-sensing regulator ExpR, which also controls production of exopolysaccharides and motility. Binding of acyl homoserine lactone-activated ExpR to the pilA1 promoter was confirmed with electrophoretic mobility shift assays. A 17-bp consensus sequence for ExpR binding was identified within the 28-bp protected region by DNase I footprinting analyses. Our results show that Flp pili are important for efficient symbiosis of S. meliloti with its plant host. The temporal inverse regulation of exopolysaccharides and pili by ExpR enables S. meliloti to achieve a coordinated expression of cellular processes during early stages of host interaction.