Identification and characterization of pentose phosphate and Embden Meyerhof Parnas pathway mutants in Sinorhizobium meliloti
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The interaction of Sinorhizobium meliloti¬ and alfalfa is a well-studied model system for symbiotic establishment between rhizobia and legumes. The S. meliloti Rm1021 genome consists of a variety of genes involved in carbon metabolism and transport amongst others. Insertional inactivation of tktA, a gene encoding the major transketolase enzyme for the pentose phosphate pathway, resulted in a strain that was unable to establish an effective symbiosis with alfalfa, or produce symbiotically active exopolysaccharides (EPS) and showed conditional auxotrophy. Strains carrying secondary mutations were isolated that could suppress the effect of a mutation in tktA. The strains carrying the second site mutations were capable of partially restoring symbiosis, as well as reversing the conditional carbon phenotypes that were associated with tktA. Single nucleotide polymorphisms (SNPs) were identified in a Gnt-type negative regulator SMc02340. The suppression was attributed to upregulation of tktB due to mutations in SMc02340. SMc02340 gene product was shown as a negative regulator of tktB, SMc02341 and SMc02339. A unique symbiotic phenotype for tktA mutant strain was also identified by microscopic analysis. The tktA mutant strain is unable to make curled colonized root hairs (CCRH) on alfalfa roots. In S. meliloti, the catabolism of hexoses occurs using the Entner Doudoroff pathway, and the upper part of the Embden Meyerhof Parnas pathway is used in a gluconeogenic manner because of the lack of a gene encoding an ATP dependent phosphofructokinase. We characterized SMc01852 (pfp), which encodes a putative pyrophosphate dependent phosphofructokinase. Overexpression and purification of Pfp showed that the protein had pyrophosphate dependent phosphofructokinase activity. We have also shown that the growth and symbiotic ability of the pfp mutant is dependent on tal by generating ΔpfpΔtal strain. Overall, we have identified the role of tktA and tktB in the PP pathway and symbiosis as well as characterization of the missing phosphofructokinase.