Molecular Microbiology (1992) 6(11), 1447-1456 Molecular genetic analysis of the Rhizobium meliloti f/x/C promoter: identification of sequences involved in positive and negative regulation F. Waelkens,'*' A. Foglia, J.-B. Morel, J. Fourment, J. Batut and P. Boistard* Laboratoire de Biologie Moleculaire des Interactions Plantes-Microorganismes. INRA/CNRS. BP27. 31326 Castanet-Tolosan Cedex, France. Summary Transcription of the Rhizobium meliloti fixK gene is induced in symbiotic and microaerobic growth condi- tions by the FixL/FixJ modulator/eftector pair. Tran- scription of fixK is also negatively autoregulated. By 5' deletion analysis, the involvement in negative regu- lation of a DNA region between -514 and -450 with respect to the transcription start was demonstrated. Site-directed mutagenesis allowed us to show that a sequence homologous to the binding site of the Escherichia coli Fnr protein, centred at position -487, participates in this effect. However, deletion or muta- genesis of this Fnr-like sequence does not com- pletely eliminate FixK-dependent repression, which suggests that either an additional DNA region is involved in negative regulation or that it is mediated at the level of fixLJ transcription. Deletion analysis also allowed the definition of a DNA region involved in FixJ-mediated activation of the fixK promoter, between -79 and -42. Different point mutations in the -60, -45 and -35 regions were shown to affect pro- moter activity. In some cases, the activity of mutant promoters could be partly or fully restored by increasing the expression of the fixLJ regulatory genes. In an £. coli strain harbouring a plasmid with fixLJ under the control of an inducible (p-tac) pro- moter. Introduction Under conditions of nitrogen fixation, more than 10% of total bacterial protein can be constituted by the products of nitrogen fixation genes. As may be expected, this Received 17 January. 1992; revised and accepled 2 March, 1992. fPre- sent address: F. A, Janssens Laboratory of Genetics, K. U. Leuven, Willem de Croylaan 42, 30001 Heverlee, Belgium. *For corresportdence. Tel, 61 28 50 46; Fax 61 28 50 61. quantitatively important gene expression is regulated to occur only when conditions for nitrogen fixation are favourable. For example, in the free-living diazotrophic bacterium Klebsiella pneumoniae, nitrogen fixation occurs under nifrogen limitation in anaerobic growth con- ditions. Accordingly, expression of the regulatory gene nifA is regulated positively in response to nitrogen starva- tion, and activation of other m/genes by the NifA protein is inhibited in the presence of oxygen or low nitrogen lev- els (Gussin etat.. 1986). In Rhizobium meliloti. nitrogen fixation occurs inside alfalfa nodules. Ammcnia produced by the nitrogenase reaction does not enter the bacterial nitrogen metabolism, but rather is exported to and assimi- lated by the plant (Miflin and Cullimore, 1984). Further- more, as they infect roots and establish inside the devel- oping nodule, the bacteria switch from an aerobic to a micro-aerobic environment. Accordingly, in R. meliloti no relationship has been observed between nifA regulation and the ntrBC control system of nitrogen metabolism (Szeto et al.. 1987), whereas nif and fix genes are induced in micro-aerobic cultures of R. meliloti (Ditta et al.. 1987; David e/a/.. 1988). From recent work on regulation of R. meWof/nitrogen fixation {nif and fix) genes, a model has emerged which involves a two-step regulatory cascade (David et al., 1988; Batut et al.. 1989). At the top of the cascade, the FixUFixJ modulator/effector pair is responsible for the pri- mary oxygen sensitivity of the regulatory system (de Philip et al.. 1990). FixL is a haemoprotein with auto- kinase activity, able to transfer its phosphoryl group to FixJ {Gilles-Gonzales et al.. 1991). This is consistent with a model in which, under low oxygen tension, the trans- membrane FixL protein activates the cytoplasmic FixJ regulator protein by phosphorylation. FixJ consists of two modules: a carboxy-terminal module responsible for tran- scriptional activation, and an amino-terminal phosphoryl acceptor module that regulates the activity of the carbcxy- terminal module (Kahn and Ditta, 1991). Activated FixJ functions as a transcriptional activator of the nifA and fixK genes, both encoding regulator proteins that switch on other nif and fix genes. Since the regulatory protein FixJ contains a putative helix-turn-helix motif in its carboxy-terminal domain, it has been proposed to be a DNA-binding protein (David etai. 1988; Kahn and Ditta, 1991). Deletion analysis of the nifA