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Genetics and Molecular Biology |
Department of Plant Pathology, College of Agriculture, PO Box 210036, The University of Arizona, Tucson, AZ 85721, USA1
Author for correspondence: Christina Kennedy. Tel: +1 520 621 9835. Fax: +1 520 621 9290. e-mail: ckennedy{at}u.arizona.edu
GlnD is a pivotal protein in sensing intracellular levels of fixed nitrogen and has been best studied in enteric bacteria, where it reversibly uridylylates two related proteins, PII and GlnK. The uridylylation state of these proteins determines the activities of glutamine synthetase (GS) and NtrC. Results presented here demonstrate that glnD is an essential gene in Azotobacter vinelandii. Null glnD mutations were introduced into the A. vinelandii genome, but none could be stably maintained unless a second mutation was present that resulted in unregulated activity of GS. One mutation, gln-71, occurred spontaneously to give strain MV71, which failed to uridylylate the GlnK protein. The second, created by design, was glnAY407F (MV75), altering the adenylylation site of GS. The gln-71 mutation is probably located in glnE, encoding adenylyltransferase, because introducing the Escherichia coli glnE gene into MV72, a glnD+ derivative of MV71, restored the regulation of GS activity. GlnK-UMP is therefore apparently required for GS to be sufficiently deadenylylated in A. vinelandii for growth to occur. The 
glnD GSc isolates were Nif-, which could be corrected by introducing a nifL mutation, confirming a role for GlnD in mediating nif gene regulation via 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.
Keywords: nitrogen fixation, nitrogen assimilation, GlnD, NtrC, nif gene regulation
Abbreviations: ATase, adenylyltransferase; GS, glutamine synthetase
a Present address: School of Biological Sciences, Sussex University, Falmer, Brighton BN1 9QG, UK.
b Present address: MRC, Cell Mutation Unit, Sussex University, Falmer, Brighton BN1 9RR, UK.
c Present address: Eli Lilly and Co., Indianapolis, IN 46275, USA.
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