Physiologic roles of soluble pyridine nucleotide transhydrogenase in Escherichia coli as determined by homologous recombination

The soluble transhydrogenase is an energy-independent flavoprotein and important in cofactor regenerating system. In order to understand its physiologic roles, the recombinant strain with the deletion of soluble transhydrogenase gene (Delta udhA) in Escherichia coli was constructed using homologous recombination. Then the different genetic backgrounds containing either icd(NADP) or icd(NAD), which encodes NADP-dependent isocitrate clehydrogenase (IDH) or engineered NAD-dependent IDH, were transduced into Delta udhA, creating two strains (icd(NADP)/Delta udhA, icd(NAD)/Delta udhA). During growth on acetate, icd(NADP)/Delta udhA grew poorly and its growth rate was remarkably reduced by 75% as compared with the wild type. However, icd(NAD)/Delta udhA showed significantly better growth than icd(NADP)/Delta udhA. Its growth rate was about 3.7 fold of icd(NADP)/Delta udhA, which was equivalent to the wild type. These results indicated that UdhA is an essential NADH resource for acetate-grown E. coli and is a dominant factor for bacteria to adapt to the stress environment. Furthermore, when UdhA was absence, icd(NAD)/Delta udhA displayed about 1.5 fold increase in the IDH activity after switching the carbon source from glucose to acetate. And RT-PCR showed that the expression of NADH dehydrogenase II (NDH-2) in icd(NAD)/Delta udhA was remarkably up-regulated by about 2.8 fold as compared with icd(NADP)/Delta udhA. The increase of IDH activity and NDH-2 expression can be explained by the reducing excess NADPH production and restoring higher levels of NADH generation in cells.