Revealing a New Family of D-2-Hydroxyglutarate Dehydrogenases in Escherichia coli and Pantoea ananatis Encoded by ydiJ.

In E. coli and P. ananatis, L-serine biosynthesis is initiated by the action of D-3-phosphoglycerate dehydrogenase (SerA), which converts D-3-phosphoglycerate into 3-phosphohydroxypyruvate. SerA can concomitantly catalyze the production of D-2-hydroxyglutarate (D-2-HGA) from 2-ketoglutarate by oxidizing NADH to NAD+. Several bacterial D-2-hydroxyglutarate dehydrogenases (D2HGDHs) have recently been identified, which convert D-2-HGA back to 2-ketoglutarate. However, knowledge about the enzymes that can metabolize D-2-HGA is lacking in bacteria belonging to the Enterobacteriaceae family. We found that ydiJ encodes novel D2HGDHs in P. ananatis and E. coli, which were assigned as D2HGDHPa and D2HGDHEc, respectively. Inactivation of ydiJ in P. ananatis and E. coli led to the significant accumulation of D-2-HGA. Recombinant D2HGDHEc and D2HGDHPa were purified to homogeneity and characterized. D2HGDHEc and D2HGDHPa are homotetrameric with a subunit molecular mass of 110 kDa. The pH optimum was 7.5 for D2HGDHPa and 8.0 for D2HGDHEc. The Km for D-2-HGA was 208 µM for D2HGDHPa and 83 µM for D2HGDHEc. The enzymes have strict substrate specificity towards D-2-HGA and displayed maximal activity at 45 °C. Their activity was completely inhibited by 0.5 mM Mn2+, Ni2+ or Co2+. The discovery of a novel family of D2HGDHs may provide fundamental information for the metabolic engineering of microbial chassis with desired properties.

DcrA and dcrB Escherichia coli genes can control DNA injection by phages specific for BtuB and FhuA receptors.

We had previously shown that the Escherichia coli proteins DcrA (SdaC) and DcrB, located, respectively, in the inner membrane and periplasm, are involved in the early development of virulent bacteriophage C1, which recognises BtuB as an outer membrane receptor. In the present work it is demonstrated that the DcrA and DcrB proteins, coordinately with another outer membrane receptor protein, FhuA, are also involved in an early stage of development of a newly isolated virulent phage, C6. In both cases, DcrA and DcrB probably are required in the second stage of phage adsorption-the DNA injection process. This means that DcrA and DcrB proteins can participate in phage DNA transport pathways in cooperation with different outer membrane receptors, including FhuA and BtuB. The increased sensitivity of bacterial cells to SDS following C1 and C6 adsorption suggests that adsorption by these phages triggers the opening of diffusion channels through the outer membrane. Our results also indirectly demonstrate that the DcrA and DcrB proteins participate in the opening or formation of these channels.