Novel Genes and Regulators That Influence Production of Cell Surface Exopolysaccharides in Sinorhizobium meliloti

Abstract

ABSTRACT Sinorhizobium meliloti is a soil-dwelling alphaproteobacterium that engages in a nitrogen-fixing root nodule symbiosis with leguminous plants. Cell surface polysaccharides are important both for adapting to stresses in the soil and for the development of an effective symbiotic interaction. Among the polysaccharides characterized to date, the acidic exopolysaccharides I (EPS-I; succinoglycan) and II (EPS-II; galactoglucan) are particularly important for protection from abiotic stresses, biofilm formation, root colonization, and infection of plant roots. Previous genetic screens discovered mutants with impaired EPS production, allowing the delineation of EPS biosynthetic pathways. Here we report on a genetic screen to isolate mutants with mucoid colonial morphologies that suggest EPS overproduction. Screening with Tn 5 -110, which allows the recovery of both null and upregulation mutants, yielded 47 mucoid mutants, most of which overproduce EPS-I; among the 30 unique genes and intergenic regions identified, 14 have not been associated with EPS production previously. We identified a new protein-coding gene, emmD , which may be involved in the regulation of EPS-I production as part of the EmmABC three-component regulatory circuit. We also identified a mutant defective in EPS-I production, motility, and symbiosis, where Tn 5 -110 was not responsible for the mutant phenotypes; these phenotypes result from a missense mutation in rpoA corresponding to the domain of the RNA polymerase alpha subunit known to interact with transcription regulators. IMPORTANCE The alphaproteobacterium Sinorhizobium meliloti converts dinitrogen to ammonium while inhabiting specialized plant organs termed root nodules. The transformation of S. meliloti from a free-living soil bacterium to a nitrogen-fixing plant symbiont is a complex developmental process requiring close interaction between the two partners. As the interface between the bacterium and its environment, the S. meliloti cell surface plays a critical role in adaptation to varied soil environments and in interaction with plant hosts. We isolated and characterized S. meliloti mutants with increased production of exopolysaccharides, key cell surface components. Our diverse set of mutants suggests roles for exopolysaccharide production in growth, metabolism, cell division, envelope homeostasis, biofilm formation, stress response, motility, and symbiosis.