Characterization of Sinorhizobium meliloti Triose Phosphate Isomerase Genes

Abstract

A Tn5 mutant strain of Sinorhizobium meliloti with an insertion in tpiA (systematic identifier SMc01023), a putative triose phosphate isomerase (TPI)-encoding gene, was isolated. The tpiA mutant grew more slowly than the wild type on rhamnose and did not grow with glycerol as a sole carbon source. The genome of S. meliloti wild-type Rm1021 contains a second predicted TPI-encoding gene, tpiB (SMc01614). We have constructed mutations and confirmed that both genes encode functional TPI enzymes. tpiA appears to be constitutively expressed and provides the primary TPI activity for central metabolism. tpiB has been shown to be required for growth with erythritol. TpiB activity is induced by growth with erythritol; however, basal levels of TpiB activity present in tpiA mutants allow for growth with gluconeogenic carbon sources. Although tpiA mutants can be complemented by tpiB, tpiA cannot substitute for mutations in tpiB with respect to erythritol catabolism. Mutations in tpiA or tpiB alone do not cause symbiotic defects; however, mutations in both tpiA and tpiB caused reduced symbiotic nitrogen fixation.Triose phosphate isomerase (TPI) catalyzes the reversible interconversion of glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). This activity makes TPI a key enzyme of central carbon metabolism, allowing it to play a role in the glycolysis (Embden-Meyerhof-Parnas [EMP]), gluconeogenesis, pentose phosphate (PP), and Entner-Doudoroff (ED) pathways. Past research on the symbiotic soil bacterium Sinorhizobium meliloti and other rhizobia has shown that hexose catabolism proceeds through the ED and PP pathways, while the EMP pathway functions at very low levels, if at all (49). Gluconeogenesis, however, is functional, and mutations in gluconeogenic enzymes have been shown to cause complex symbiotic phenotypes, including reduced or abolished nitrogen-fixing activity (6,15,16,19,24).Recent work has confirmed the roles of the ED and PP pathways as catabolic pathways in S. meliloti. Analyses of carbon flux carried out using labeled carbon compounds and gas chromatography-mass spectrometry have confirmed the absence of the EMP pathway during growth with glucose (17). Another set of experiments done using labeled carbon and nuclear magnetic resonance is in agreement, confirming that glucose is degraded primarily through the ED pathway (22, 40). The gas chromatography-mass spectrometry and nuclear magnetic resonance experiments also confirm that some of the G3P produced by the catabolism of hexoses through the ED pathway is converted back to higher-molecular-weight compounds through TPI and fructose bisphosphate aldolase (FBA) in a cyclic pathway. Because FBA requires G3P and DHAP as substrates, the interconversion of G3P and DHAP is necessary for the cyclic metabolism of hexoses as well as gluconeogenesis. An S. meliloti strain missing TPI activity would therefore be compromised metabolically.Based on gene homology, S. meliloti has two putative chromosomal genes predicted to encode TPI enzymes (18). The gene ...

Formate-Dependent Autotrophic Growth in Sinorhizobium meliloti

“…Glyceraldehyde 3-phosphate is subsequently isomerized by triose phosphate isomerase to dihydroxyacetone phosphate, and a hexose is synthesized via an aldolase reaction, yielding fructose 1,6-bisphosphate. It has recently been shown that S. meliloti contains two triose phosphate isomerases (48). It has been shown that tpiA plays a role in central metabolic metabolism (48), whereas tpiB appears to be necessary for erythritol catabolism in S. meliloti and Rhizobium leguminosarum (48,61).…”

Proteomics-Based Analysis of <i>Phalaenopsis amabilis</i> in Response toward <i>Cymbidium</i> Mosaic Virus and/or <i>Odontoglossum</i> Ringspot Virus Infection

“…In this study, CymMV infection also dramatically induces TRXh expression in P. amabilis leaves, in consistence with the previous result. PGK is involved in glycolysis and tricarboxylic acid (TCA) cycle and plays important roles in energy production [21]. PGK expression was significantly decreased only in CymMV-infected P. amabilis leaves, indicating that the energy metabolism of the plant host was damaged severely after CymMV infection.…”

“…Similarly, our results showed that ORSV infection enhanced Rubisco expression in leaves, implying that the modified Rubisco struc-ture is associated with different viral pathogens. TPI is involved in glycolysis and TCA cycle and plays important roles in energy production [21]. IDH is a ratelimiting enzyme in TCA cycle and plays important roles in energy metabolism, amino acid synthesis and vitamin synthesis [23,24].…”

Characterization of a Two-Component Regulatory System That Regulates Succinate-Mediated Catabolite Repression in Sinorhizobium meliloti

“…The in-frame deletion mutations of sma0113 and sma0114 were complemented by inserting wild-type copies of the genes into the rhamnose-binding protein gene (smc02324) using pCAP87. pCAP87 is a suicide plasmid containing a constitutive trp promoter upstream of the multiple cloning site and an internal fragment of smc02324 which targets the plasmid to an operon that encodes a rhamnose transport system (1,35,45). Wild-type copies of sma0113 and sma0114 were cloned into plasmid pCAP87.…”