A Megaplasmid-Borne Anaerobic Ribonucleotide Reductase in Alcaligenes eutrophus H16

Abstract

ABSTRACTThe conjugative 450-kb megaplasmid pHG1 is essential for the anaerobic growth ofAlcaligenes eutrophusH16 in the presence of nitrate as the terminal electron acceptor. We identified two megaplasmid-borne genes (nrdDandnrdG) which are indispensable under these conditions. Sequence alignment identified significant similarity of the 76.2-kDa gene product NrdD and the 30.9-kDa gene product NrdG with anaerobic class III ribonucleotide reductases and their corresponding activases. Deletion ofnrdDandnrdGinA. eutrophusabolished anaerobic growth and led to the formation of nondividing filamentous cells, a typical feature of bacteria whose DNA synthesis is blocked. Enzyme activity of NrdD-like ribonucleotide reductases is dependent on a stable radical at a glycine residue in a conserved C-terminal motif. A mutant ofA. eutrophuswith a G650A exchange in NrdD showed the DNA-deficient phenotype as the deletion strain, suggesting that G650 forms the glycyl radical. Analysis of transcriptional and translational fusions indicate thatnrdDandnrdGare cotranscribed and that the translation efficiency ofnrdDis 40-fold higher than that ofnrdG. Thus, the two proteins NrdD and NrdG are not synthesized at a stoichiometric level.