Characterization of a (pckA) mutant of the non-nodulating bacterium Rhizobium pusense NRCPB10 induced by transposon Tn5 mutagenesis

Subrata K Das,Pran K Chakrabartty

doi:10.1007/s13205-012-0057-5

Abstract

Phosphoenolpyruvate carboxykinase (PCK) catalyzes the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate in the gluconeogenic pathway in most organisms. A pckA gene encoding PCK was cloned and sequenced from strain Rhizobium pusense NRCPB100, a spontaneous rifampicin resistant mutant of R. pusense NRCPB10T (JCM16209T) of a recently described new species of a non-nodulating and non-tumorigenic bacterium. The mapping of the pck gene region following Tn5 mutagenesis located the gene downstream of a transcriptional regulatory protein gene (chvI) and upstream of a conserved hypothetical protein gene. The pck of 1,611 bp was deduced to encode 536 amino acids and showed high homology to the genes of known ATP-dependent PCK enzymes. Phylogenetic analysis of the gene placed it in a cluster with pck of other known members of Rhizobiales. Amino acid sequences of the putative functional regions of the deduced enzyme were found to be conserved.

Highlights

Gluconeogenesis refers to the central metabolic pathway in which tricarboxylic acid (TCA) cycle intermediatesP
The mutant NRC43 failed to grow in rhizobium minimal medium (RMM) containing the glucogenic substrate succinate as was shown for pckA mutants of E. meliloti, R. leguminosarum bv. viciae and Rhizobium sp
The data indicated that the mutation in NRC43 did not allow it to grow in glucogenic substrates as the sole carbon and energy source, presumably the strain lacked the key enzyme of gluconeogenesis

Summary

Introduction

Gluconeogenesis refers to the central metabolic pathway in which tricarboxylic acid (TCA) cycle intermediates. The first step of the gluconeogenic pathway is catalyzed by the enzyme phosphoenolpyruvate carboxykinase (PCK) (EC 4.1.1.49) which decarboxylates and simultaneously phosphorylates oxaloacetate to phosphoenolpyruvate (PEP) (Utter and Kolenbrander 1972). Fructose-1,6-bisphosphatase, which converts fructose-1,6bisphosphate to fructose-6-phosphate, is required for gluconeogenesis (Østeras et al 1997; Voet et al 2008). PCK has been demonstrated to play a crucial role in several metabolic processes associated with cataplerosis (Yang et al 2009). PCKs have been classified according to their specificities for nucleotides, GTP and ATP. The enzymes from mammals and a number of other eukaryotes have specificity for GTP, while the enzymes from bacteria, yeast and plants have specificity for ATP (Utter and Kolenbrander 1972). PCK appears to have an absolute requirement for divalent metal ions, such as Mn2?, but other divalent ions, such as Mg2? or Co2?, can act as substitutes but with reduced activity (Utter and Kolenbrander 1972)

Methods

Results

Conclusion