Role of Crc in Regulation of PQQ GDH Dependent Mineral Phosphate Solubilization Phenotype and its Repression in Acinetobacter Species

Abstract

lant growth-promoting rhizobacteria (PGPR) are healthful bacteria present in or on the root rhizosphere of the plant and have been explored as bio-inoculants in the agricultural fields for several decades. Many PGPR strains are routinely employed but their positive responses to plants are often limited to laboratory or greenhouse and the results are inconsistent in field conditions. The plant roots secrete organic compounds in root exudates that help microbes for survival in the rhizosphere and they in return promote plant growth. The compositions of root exudates vary according to the plant species and often comprise complex mixtures. Presence of various organic acids and sugars in rhizosphere execute CCR for preferential utilization of carbon sources. This facilitates the bacterium to compete and survive among existing microbial communities. While it is not yet clear how bacteria choose the substrate in a rhizosphere where CCR could be regulating metabolism, various experiments have been conducted in vitro with commonly found organic acids present in the root exudate. Here in this study, we looked for the mechanisms underlying the succinate mediated catabolite repression (SMCR) of the gluconate arbitrated mineral phosphate solubilization (MPS) in Acinetobacter sp. SK2. Acinetobacter sp. SK2, a potential plant growth promoting rhizobacteria isolated from the rhizosphere of Vigna radiata that solubilized 682 μg/ml of tricalcium phosphate (TCP) and 86 μg/ml of rock phosphate (RP) with concomitant decrease in pH up to 4 due to the production of gluconate. The mechanism of MPS in Acinetobacter sp. SK2 was studied in detail and the role of enzymes responsible for periplasmic glucose oxidation (membrane glucose dehydrogenase-mGDH) and sGDH (soluble glucose dehydrogenase) was elucidated. Gluconate mediated MPS phenotype of Acinetobacter sp. SK2 was post-transcriptionally repressed by Crc in presence of succinate+glucose. Gluconate production and expression of mGDH and sGDH was found only in glucose but not in glucose+succinate. Genes for mGDH, sGDH and Crc were inactivated using single strand homologous recombination. The absence of mGDH encoding gene gdhA resulted in loss of MPS phenotype due to loss of mGDH activity which further confirmed its role in periplasmic glucose oxidation and gluconate mediated MPS phenotype. The insertional inactivation in sGDH encoding gene gdhB resulted in loss of sGDH activity and confirmed that it was indispensable for sGDH activity but the lack of gdhB did not alter MPS phenotype as well as the mGDH activity. Thus, it was also concluded that it has no role to play in periplasmic glucose oxidation. Inactivation of Crc resulted in increased activity of the mGDH enzymes in glucose+succinate grown cells. An increase in phosphate solubilization up to 44% in glucose+succinate grown crc － compared to glucose grown cells, was recorded which was totally repressed in wild-type cells grown in glucose+succinate. The data indicated that Hfq also plays a critical role in utilization of preferred and non-preferred carbon source by perhaps modulating Crc-Hfq-sRNA machinery. This study illustrates the role of Crc in regulation of SMCR of MPS phenotype in Acinetobacter sp. SK2. The gene expression data showed inducible nature of PQQ synthesis genes by glucose because the presence of succinate alone or along with glucose repressed expression of pqqBCDE. This implies that the repressed PQQ synthesis put down the GDH activity and therefore limiting MPS phenotype under glucose+succinate conditions. Vigna radiata plants inoculated with wild-type SK2 improved both root and shoot length by 1.3-1.4 fold. However, crc insertional inactivation strains increased root and shoot length by 1.6 folds, compared to uninoculated controls. While mimicking the soil condition (the presence of multiple carbon sources at a time like succinate along with glucose), the crc inactivated phosphate solubilizers better served the purpose of constitutive phosphate solubilization in pot experiments. A strategy of similar kind may be employed when phosphate solubilization or other PGP traits are influenced by the components of root exudates and CCR. By these means, repression relieved strains can be developed which may further improve plant growth under natural soil conditions also.