AniA, a regulatory protein involved in polymer accumulation in Ensifer meliloti

Bacterial biosynthesis of different polymers depends upon the genetic traits of the strain in use and by the culture conditions adopted. Although the separate production of exopolysaccharides (EPS) or poly-3-hydroxybutyrate [P(3HB)] is well documented, much less attention has been paid to the relationships between the synthesis of these two major compounds produced by bacteria. The biosynthetic pathways of these polymers are not directly linked, but they can be produced at the same time in some microorganisms. In rhizobia the general conditions governing the biosynthetic pathways (the nature and availability of carbon and nitrogen sources, the oxygenation, the energetic and redox states of cells, the environmental constraints, etc.) seem to be the same. Since the extent of EPS and P(3HB) production implies a significant energetic cost, a sensitive regulatory mechanism is required. A control of the synthesis and degradation of P(3HB) in bacteroids is important to maintain an effective symbiosis. Therefore, a well-regulated P(3HB) cycle results as a key factor for an optimal use of the available energy and for a balanced distribution of the carbon resources. Regulation of carbon flux into P(3HB) production occurs at multiple levels, and the comprehension of this regulation is essential for understanding the physiological functions of P(3HB) and possibly for applying this knowledge to industrial production of polyesters. AniA, a putative regulatory protein previously described (Povolo and Casella, 2000), and identified in the polyhydroxyalkanoates locus in Ensifer meliloti (Tombolini et al. , 1995), was found to be involved in carbon/energy regulation under normal growth conditions. The occurrence of AniA orthologs (described in some cases as PhaR) and organization of the respective genes were described in detail in many different bacteria (Pötter et al., 2005). The present work gives a better inside of the role of the carbon flux regulator (aniA) in E. meliloti 41. Previous studies on other bacterial species indicated that the impaired synthesis of one polymer causes other reserve materials to be turned over (Breuer and Babel, 1999). A strain carrying a lacZ transcriptional fusion inside the aniA gene was constructed from E. meliloti 41 and from the mutant strain E. meliloti 41003 unable to accumulate polyhydroxyalkanoates (Povolo and Casella, 2008). E. meliloti 41003 accumulates also less exopolysaccharides as compared to the wild-type strain 41 (Povolo and Casella, 2008). The transcription of aniA-lacZ fusions was studied in the wild-type and in the phaC-mutant backgrounds under different conditions. We also showed that an EPS negative mutant of E. meliloti 2011 (strain H3a) could accumulate more P(3HB) than the wild-type strain 2011. All together these results indicate a clear correlation between P(3HB) and EPS biosynthesis. On the other hand, an aniA-Km mutation was transferred to different E. meliloti strains carrying exp-lacZ and exo-lacZ fusions. Phenotypic analysis of these double mutants showed a change from rough colonies of the single mutants to mucoid colonies in the double mutants (strains exoY-lacZ/aniA-, exoL-lacZ/aniA- and exoP-lacZ/aniA-) indicating an effect of aniA on EPS production.