Lethality of glnD null mutations in Azotobacter vinelandii is suppressibleby prevention of glutamine synthetase adenylylation

GlnD is a pivotal protein in sensing intracellular levels of fixed nitrogen and has been best studied in enteric bacteria, where it reversibly uridyly lates two related proteins, PII and GlnK, The uridylylation state of these proteins determines the activities of glutamine synthetase (GS) and NtrC, R esults presented here demonstrate that glnD is an essential gene in Azotoba cter vinelandii, Null glnD mutations were introduced into the A. vinelandii genome, but none could be stably maintained unless a second mutation was p resent that resulted in unregulated activity of GS, One mutation, gln-71, o ccurred spontaneously to give strain MV71, which failed to uridylylate the GlnK protein. The second, created by design, was glnA Y407F (MV75), alterin g the adenylylation site of GS, The gln-71 mutation is probably located in glnE, encoding adenylyltransferase, because introducing the Escherichia col i glnE gene into MV72, a glnD(+) derivative of MV71, restored the regulatio n of GS activity. GlnK-UMP is therefore apparently required for GS to be su fficiently deadenylylated in A. vinelandii for growth to occur. The Delta g lnD GS(c) isolates were Nif(-), which could be corrected by introducing a n ifL mutation, confirming a role for GlnD in mediating nif gene regulation v ia some aspect of the NifL/NifA interaction. MV71 was unexpectedly NtrC(+), suggesting that A. vinelandii NtrC activity might be regulated differently than in enteric organisms.