Preferential Utilization of Sugars and Their Effect on Phosphate Solubilization in Rhizobium sp. RM

Abstract

Rhizobia are a group of bacteria existing symbiotically in and around roots of leguminous plants. Members of Rhizobium species have received extensive importance owing to their ability to form nodules on roots where they carry out nitrogen fixation leading to increment in soil fertility. They have hence, been considered as potent candidates for biofertilizers. Although benefitting the plants by their nitrogen-fixing trait at lab-scale, they are often found futile in field trials. The complex signalling between the plants and rhizobia in the rhizosphere to establish the symbiotic relation has been a major area of research over decades. However, metabolism and physiology of these bacteria have received less attention. The limited understanding of biochemical pathways and responses to various energy conditions is a barrier in harnessing them as successful biofertilizers. Another major blind spot with respect to understanding the bacterial physiology is the phenomenon of carbon catabolite repression that helps bacteria to survive in the competitive environment. It is known that rhizobia, unlike E. coli and B. subtilis, and like pseudomonads, prefer organic acids over sugars, however, the preference among sugars and the molecular basis underlying it has not been explored. There are many sugars present in the rhizosphere as a result of plant root exudates. Majority of the plants have glucose (G), fructose (F), arabinose (A) and xylose (X) present in their root exudations. The present study aims to explore the effect of utilization of these sugars on the mineral phosphate solubilization (MPS) trait of Rhizobium sp. RM. Further, sequential uptake of these rhizospheric sugars and involvement of global regulators is investigated. We hypothesize that utilization of different sugars by Rhizobium sp. RM releases various organic acids mediating the MPS phenotype. Additionally, preferential utilization of sugars might be governed at the transporter level, initial steps of sugar metabolism and /or via the regulation of few regulatory proteins. Growth, organic acid profile, real-time transcriptional profile of genes corresponding to transport, sugar specific metabolism and regulation in the presence of the four common rhizospheric sugars and their combinations was carried out in this work. In the present work, we have demonstrated that the Vigna radiata nodulating Rhizobium sp. RM solubilizes complex phosphates like tricalcium phosphate (TCP) and rock phosphate (RP) in higher amount than many other reported rhizobacteria like Bacillus sp. and Pseudomonas fluorescens. Isolate RM solubilized complex phosphate(P) via organic acid production leading to a drop in media pH, upon utilization of multiplesugars. Gluconate was found to be the prominent organic acid in HPLC analysesresponsible for P solubilization followed by oxalate and malate. Oxalate and malate weresecreted upon utilization of arabinose and xylose, respectively. Fructose utilizationsupported organic acid (malate) production along with lowering of pH to solubilize TCPalone, however, no acid and hence no pH change was detected when buffered RP wasused as complex phosphate source. In contrast to this observation, the dual combinationof aldose- G/A/X and fructose yielded maximum TCP solubilization of 644, 575, 446 µgml-1respectively and RP solubilization of 75, 68, 63 µg ml-1respectively, as compared toindividual sugars.The second part of the study comprises of fructose being considered as asecondary carbon source by RM. Concentration independent hierarchy among aldoses(G/A/X) and fructose was evident from the diauxic growth curves with distinct 30 minmid-lag phase. The aldoses, however, when present in dual combinations in the media,might be taken up simultaneously as the growth curves of RM were monoauxic in nature.To elucidate the basis of this peculiar hierarchy, quantitative real time PCR analysis ofthe selected genes of fructose transport, sugar metabolism and regulatory proteins wascarried out. We found that the prevention of fructose uptake by aldoses was through theregulation of genes of fructose transporter (fructose specific ABC transporter componentB (frcB) and C (frcC)) as well as fructokinase (frk). The 1.8-3.7-fold downregulation offrcB and frcC genes, and 4.2-8.8-fold downregulation of frk gene in presence of aldosesand 3.5-6.9-fold upregulation only during fructose utilization phases of growth was a cuefor fructose being utilized later to aldoses by isolate RM. The regulatory genes hfq andhprK were 1.4-1.9-fold upregulated only in cells from aldose utilization phase however,1.1-4.3-fold downregulated in cells from fructose utilization phase of fructose+aldosecombinations (FG/FA/FX). This observation indicated that the regulatory proteins, Hfqand HPrK, might govern the prevention of fructose uptake by regulating fructose specificgenes when aldoses are supplemented with fructose in dual combination as carbonsources for Rhizobium sp. RM.An attempt was made to check the binding energies of sugars to the fructosetransporter by in silico docking technique using Autodoc Vina. However, due to the crudestructure of fructose transporter obtained from the sequencing of Rhizobium sp. RM, theresults need further investigation.