Polyhydroxybutyrate (PHB) reserve amounts correlates with competition for nodulation behavior but not with nitrogen fixation in Bradyrhizobium japonicum-soybean symbiosis

J.I. QUELAS; J. PÉREZ GIMÉNEZ; A.R. LODEIRO

Munich

Conferencia; 10th European Nitrogen Fixation Conference; 2012

Ludwig-Maximilian-University München

Bradyrhizobium japonicum is a soil bacterium that associates with soybean in specializedroot organs (named nodules). Inside them, bacteria (as bacteroids) fix atmospheric N2 andprovide legumes with ammonia, supplying with this element on soils with low N content(common in soybean-producing countries like Argentina). In arable soils, soybean seeds areinoculated year after year with B. japonicum. So, the inoculated rhizobia compete withdifferent rhizobial strains from indigenous bacterial population or introduced by the inoculantsused in previous crop seasons both of them naturally more competitive. In this sense, Creservesare vital not only for survival but in supplying rapid fuel to invade the plants. PHB isan important C-reserve, which may constitute more than 75% of rhizobia total dry weight andaccumulates as hydrophobic cytoplasmic granules.PHB is synthesized in three steps: condensation of two acetyl-CoA molecules to giveacetoacetyl-CoA, reduction of acetoacetyl-CoA to hydroxybutyryl-CoA, and thepolymerization of hydroxybutyryl-CoA into PHB. This last step is catalyzed by PHB-synthase(PhbC) and appears essential for PHB synthesis in all rhizobial species.While most sequenced rhizobia have one or two phbC homologs in their genomes, fiveputative phbC ORFs were detected in B. japonicum USDA 110. However, conclusiveexperimental evidence about the expression of these five ORFs is available for only two ofthem.In our lab, we obtained and characterized B. japonicum deletion mutants in these two ORFs:bll4360 (phbC1) and bll6073 (phbC2). The single mutants were named LP 4360 (ΔphbC1)and LP 6073 (ΔphbC2), and double mutant was named LP 6060 (ΔphbC1/ΔphbC2).Using bioinformatics tools, we found that PhbC1 was grouped with rhizobial Class I PHBsynthases while PhbC2 was associated with sequences that cannot be assigned to any classof PHB-synthases. Indeed, mutation of phbC1 or phbC2 leads to different phenotypes onPHB accumulation: while LP 4360 and LP 6060 were unable to synthesize PHB in free-livingor bacteroids cells, LP 6073 produced approximately three-fold the wild-type PHB amounts inboth states.In defined synthetic liquid medium without C-source, phbC mutant cells survival was similarto the wild-type strain after 60 days incubation.In competition for nodulation experiments against the wild-type in 1:1 proportion, LP 4360and LP 6060 occupied only 13-15 % of the nodules respectively, while LP 6073 occupied 81% of the nodules, suggesting that PHB levels strong correlates with competitiveness.However, its amounts in the bacteroids did not affect nitrogen fixation, as judged by shoot dryweight accumulation in plants inoculated with each strain separately.These results are promising in order to improve inoculant?s competitiveness to obtainsignificantly higher percentages of nodule occupation.