JOURNAL OF BACTERIOLOGY, Dec. 2007, p. 9050–9056 Vol. 189, No. 24 0021-9193/07/$08.00+0 doi:10.1128/JB.01190-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved. Influence of the Poly-3-Hydroxybutyrate (PHB) Granule-Associated Proteins (PhaP1 and PhaP2) on PHB Accumulation and Symbiotic Nitrogen Fixation in Sinorhizobium meliloti Rm1021  Chunxia Wang, 1 * Xiaoyan Sheng, 1 Raymie C. Equi, 1 Maria A. Trainer, 2 Trevor C. Charles, 2 and Bruno W. S. Sobral 1 Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia 24061, 1 and Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada 2 Received 26 July 2007/Accepted 25 September 2007 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 pro- posed 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 inoc- ulated 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 met- abolic role of the PHB cycle during nitrogen fixation is still not completely understood (28). While the enzymology and genet- ics 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, en- coded by phaP genes, which bind to PHB granules and pro- mote 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 polyhydroxyalkanoate (PHA) inclusions, and can comprise a significant fraction of total cell proteins (13, 44). Phasins and their structural genes, phaP, have been found in various bacteria. These proteins have been shown to play a major role in the synthesis and degradation of PHB and in the formation of PHB granules (26). For example, Ralstonia eu- tropha H16 has four phasin genes, namely, phaP1, phaP2, phaP3, and phaP4. These genes are all expressed (24, 33), but only PhaP1, the major phasin, appears to influence PHB ac- cumulation (25). Moreover, York et al. demonstrated that PhaP promotes PHB synthesis by regulating the surface/vol- ume ratio of PHB granules or by interacting with PHB syn- thase, and the levels of PhaP generally parallel levels of PHB in cells (45). Methylobacterium extorquens AM1 has two major phasins, and mutations in their genes result in defective PHB production and also in inhibited growth on C 2 compounds, while not affecting growth on C 1 or multicarbon compounds (15). Phasins appear to be present in all PHA-synthesizing bacteria, and even though they generally are not conserved in sequence, they are believed to fulfill the same functions, bind- ing to PHA granules and promoting PHA granule formation in a manner that is still poorly understood (14). In this study, we identified two major proteins associated with PHB granules, namely, PhaP1, encoded by SMc00777 (phaP1), and PhaP2, encoded by SMc02111 (phaP2), in S. meliloti Rm1021. To understand the functions of phaP1 and phaP2, mutations in these genes were generated. The effects of the phaP mutations on PHB formation and accumulation were investigated. Furthermore, we also investigated the effects of mutation of these genes on nodulation and nitrogen fixation. * Corresponding author. Mailing address: Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24061. Phone: (540) 231- 1958. Fax: (540) 231-2606. E-mail: cwang@vbi.vt.edu.  Published ahead of print on 5 October 2007. 9050