AniA Regulates Reserve Polymer Accumulation and Global Protein Expression in Rhizobium etli

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Abstract
Previously, it was reported that the oxidative capacity and ability to grow on carbon sources such as pyruvate and glucose were severely diminished in the Rhizobium etli phaC ::ΩSm r /Sp r mutant CAR1, which is unable to synthesize poly-β-hydroxybutyric acid (PHB) (M. A. Cevallos, S. Encarnación, A. Leija, Y. Mora, and J. Mora, J. Bacteriol. 178:1646-1654, 1996). By random Tn 5 mutagenesis of the phaC strain, we isolated the mutants VEM57 and VEM58, both of which contained single Tn 5 insertions and had recovered the ability to grow on pyruvate or glucose. Nucleotide sequencing of the region surrounding the Tn 5 insertions showed that they had interrupted an open reading frame designated aniA based on its high deduced amino acid sequence identity to the aniA gene product of Sinorhizobium meliloti . R. etli aniA was located adjacent to and divergently transcribed from genes encoding the PHB biosynthetic enzymes β-ketothiolase (PhaA) and acetoacetyl coenzyme A reductase (PhaB). An aniA ::Tn 5 mutant (VEM5854) was constructed and found to synthesize only 40% of the wild type level of PHB. Both VEM58 and VEM5854 produced significantly more extracellular polysaccharide than the wild type. Organic acid excretion and levels of intracellular reduced nucleotides were lowered to wild-type levels in VEM58 and VEM5854, in contrast to those of strain CAR1, which were significantly elevated. Proteome analysis of VEM58 showed a drastic alteration of protein expression, including the absence of a protein identified as PhaB. We propose that the aniA gene product plays an important role in directing carbon flow in R. etli.