Distinct roles of PII-like signal transmitter proteins and amtB in regulation of nif gene expression, nitrogenase activity, and posttranslational modification of NifH in Azoarcus sp strain BH72

P-H-like signal transmitter proteins, found in Bacteria, Archaea, and plants, are known to mediate control of carbon and nitrogen assimilation. They indirectly regulate the activity of key metabolic enzymes and transcription factors by protein-protein interactions with signal transduction proteins. Many Proteobacteria harbor two paralogous Pu-like proteins, GinB and UnK, whereas a novel third P-H paralogue (GInY) was recently identified in Azoarcus sp. strain BH72, a diazotrophic endophyte of grasses. In the present study, evidence was obtained that the Pu-like proteins have distinct roles in mediating nitrogen and oxygen control of nif gene transcription and nitrogenase activity. Full repression of nif gene transcription in the presence of a combined nitrogen source or high oxygen concentrations was observed in wild-type and glnB and g1nK knockout mutants, revealing that GlnB and GlnK can complement each other in mediating the repression. In contrast, in a gInBK double mutant strain in the presence of only GlnY, nif gene transcription was still detectable, albeit at a lower level, on nitrate or 20% oxygen. As another level of control, nitrogenase activity was regulated by at least three types of mechanisms in strain BR72: covalent modification of dinitrogenase reductase (NifH), probably by ADP-ribosylation, and two other, unknown means. Functional inactivation upon ammonium addition (switchoff) required the putative high-affinity ammonium transporter Amtl3 and GlnK, but not Gln13 or GlnY. Functional inactivation in response to anaerobiosis did not depend on AmtB, GlnK, or GlnB. In contrast, covalent modification of NifH required both GlnB and GlnK and AmtB as response to ammonium addition, whereas it required either GlnB or GlnK and not AmtB when cells were shifted to anaerobiosis. In a glnBK double mutant expressing only GlnY, NifH modification was completely abolished, further revealing functional differences between the three P-H paralogues.