Bacterial two-component regulatory systems (TCS) are common components of complex regulatory networks and cascades. In Sinorhizobium meliloti, the TCS ExoS/ChvI controls exopolysaccharide succinoglycan production and flagellum biosynthesis. Although this system plays a crucial role in establishing the symbiosis between S. meliloti and its host plant, it is not well characterized. Attempts to generate complete loss-of-function mutations in either exoS or chvI in S. meliloti have been unsuccessful; thus, it was previously suggested that exoS or chvI are essential genes for bacterial cell growth. We constructed a chvI mutant by completely deleting the open reading frame encoding this gene. The mutant strain failed to grow on complex medium, exhibited lower tolerance to acidic condition, produced significantly less poly-3-hydroxybutyrate than the wild type, was hypermotile, and exhibited an altered lipopolysaccharide profile. In addition, this mutant was defective in symbiosis with Medicago truncatula and M. sativa (alfalfa), although it induced root hair deformation as efficiently as the wild type. Together, our results demonstrate that ChvI is intimately involved in regulatory networks involving the cell envelope and metabolism; however, its precise role within the regulatory network remains to be determined.

Sinorhizobium meliloti cells store excess carbon as intracellular poly-3-hydroxybutyrate (PHB) granules that assist survival under fluctuating nutritional conditions. PHB granule-associated proteins (phasins) are proposed to regulate PHB synthesis and granule formation. Although the enzymology and genetics of PHB metabolism in S. meliloti have been well characterized, phasins have not yet been described for this organism. Comparison of the protein profiles of the wild type and a PHB synthesis mutant revealed two major proteins absent from the mutant. These were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as being encoded by the SMc00777 (phaP1) and SMc02111 (phaP2) genes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins associated with PHB granules followed by MALDI-TOF confirmed that PhaP1 and PhaP2 were the two major phasins. Double mutants were defective in PHB production, while single mutants still produced PHB, and unlike PHB synthesis mutants that have reduced exopolysaccharide, the double mutants had higher exopolysaccharide levels. Medicago truncatula plants inoculated with the double mutant exhibited reduced shoot dry weight (SDW), although there was no corresponding reduction in nitrogen fixation activity. Whether the phasins are involved in a metabolic regulatory response or whether the reduced SDW is due to a reduction in assimilation of fixed nitrogen rather than a reduction in nitrogen fixation activity remains to be established.The alfalfa root nodule symbiont Sinorhizobium meliloti stores excess carbon as intracellular poly-3-hydroxybutyrate (PHB) granules as its main carbon storage compound. Mutant analysis has demonstrated that PHB metabolism plays a role in rhizobium-legume symbiosis (19,38,39,43), although the metabolic role of the PHB cycle during nitrogen fixation is still not completely understood (28). While the enzymology and genetics of PHB biosynthesis have been studied extensively with various bacteria (35), less is known about the regulation of this process in S. meliloti. So far, the following two major types of PHB accumulation effectors have been investigated in several bacteria: (i) the granule-associated proteins, or phasins, encoded by phaP genes, which bind to PHB granules and promote PHB synthesis; and (ii) a regulator, encoded by phaR (15). PhaR was first designated AniA in rhizobia because of its expression under anaerobic growth conditions (27). Although the function of aniA has still to be clarified, Povolo and Casella provided evidence that AniA, in partitioning carbon flow in cells, affects not only PHB production but also the production of extracellularly polymeric substances and nitrogen fixation in S. meliloti Rm41 (27). In Rhizobium etli, this protein has been proposed to be involved in directing carbon flow (8, 9). Phasins have not yet been described for rhizobia.Phasins are characterized by low molecular masses (mostly between 11 and 25 kDa), have an amphiphilic character and a high affinity for polyhyd...