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Journal of Bacteriology, October 2003, p. 5854-5861, Vol. 185, No. 19
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.19.5854-5861.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification of Three Genes Encoding P_II-Like Proteins in Gluconacetobacter diazotrophicus: Studies of Their Role(s) in the Control of Nitrogen Fixation

Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden,² Department of Gene Technology and Microbiology, University of Bielefeld, DE-33615 Bielefeld, Germany¹

Received 24 March 2003/ Accepted 8 July 2003

In our studies on the regulation of nitrogen metabolism in Gluconacetobacter diazotrophicus, an endophytic diazotroph of sugarcane, three glnB-like genes were identified and their role(s) in the control of nitrogen fixation was studied. Sequence analysis revealed that one P_II protein-encoding gene, glnB, was adjacent to a glnA gene (encoding glutamine synthetase) and that two other P_II protein-encoding genes, identified as glnK1 and glnK2, were located upstream of amtB1 and amtB2, respectively, genes which in other organisms encode ammonium (or methylammonium) transporters. Single and double mutants and a triple mutant with respect to the three P_II protein-encoding genes were constructed, and the effects of the mutations on nitrogenase expression and activity in the presence of either ammonium starvation or ammonium sufficiency were studied. Based on the results presented here, it is suggested that none of the three P_II homologs is required for nif gene expression, that the GlnK2 protein acts primarily as an inhibitor of nif gene expression, and that GlnB and GlnK1 control the expression of nif genes in response to ammonium availability, both directly and by relieving the inhibition by GlnK2. This model includes novel regulatory features of P_II proteins.