TheglnD gene in enteric bacteria encodes a uridylyltransferase/uridylyl-removing enzyme which acts as the primary nitrogen sensor in the nitrogen regulation (Ntr) system. We have investigated the role of this enzyme in transcriptional regulation of nitrogen fixation genes inKlebsiella pneumoniae by cloningglnD from this organism and constructing a null mutant by insertional inactivation of the chromosomal gene using the interposon.K. pneumoniae glnD encodes a 102.3 kDa polypeptide which is highly homologous to the predicted products of bothEscherichia coli glnD andAzotobacter vinelandii nfrX. TheglnD- mutant was unable to uridylylate P_II and was altered in adenylylation/deadenylylation of glutamine synthetase. Uridylyltransferase was required for derepression ofntr-regulated promoters such asglnAp2 and pnifL but was not involved in thenifspecific response to changes in nitrogen status mediated by thenifL product. We conclude that a separate, as yet uncharacterised, nitrogen control system may be responsible for nitrogen sensing by NifL.