Down-Regulation of the Rhizobium Ntr Regulatory System: A Mechanism to Uncouple Nitrogen Fixation and Assimilation in Bacteroids

Abstract

The soil bacterium Rhizobium etli induces nitrogen-fixing nodules on roots of common beans (Phaseolus vulgaris). During the symbiotic interaction, the regulation of ammonium metabolism is of particular interest because marked changes in nitrogen metabolism occur when bacteria differentiate into nitrogen-fixing bacteroids. Free-living Rhizobium assimilates nitrogen by direct uptake of NH₄+ or by nitrate reduction, but nothing is presently known about genes involved in the NH₄+ transport in the family Rhizobiaceae. We cloned, sequenced and characterized the R. etli glnK-amtB operon coding for a protein (GlnK) belonging to the P_II family of signal transduction proteins and a protein (AmtB) with 11 or 12 putative membrane-spanning regions, which is very similar to previously identified proteins required for high-affinity ammonium transport (Ninnemann et al, 1994; Marini et al, 1994). The glnK-amtB operon is transcribed from a σ54(σN)-dependent promoter, located upstream of glnK and requires the nitrogen regulatory protein NtrC for expression. Transcription of this operon is enhanced under nitrogen-limiting conditions in the wild-type strain. It is undetectable in a ntrC- strain of R. etli., which takes up [14C]-methylammonium (14C-MA) only when harbouring a plasmid carrying the R. etli amtB gene under the control of the inducible dctA promoter from R. meliloti. We conclude that the expression of AmtB is sufficient to restore the ntrC- mutant ability to transport (methyl)ammonium.