The NifA-RpoN regulon of Mesorhizobium loti strain R7A and its symbiotic activation by a novel LacI/GalR-family regulator

Abstract

Mesorhizobium loti is the microsymbiont of Lotus species, including the model legume L. japonicus. M. loti differs from other rhizobia in that it contains two copies of the key nitrogen fixation regulatory gene nifA, nifA1 and nifA2, both of which are located on the symbiosis island ICEMlSym(R7A). M. loti R7A also contains two rpoN genes, rpoN1 located on the chromosome outside of ICEMlSym(R7A) and rpoN2 that is located on ICEMlSym(R7A). The aims of the current work were to establish how nifA expression was activated in M. loti and to characterise the NifA-RpoN regulon. The nifA2 and rpoN2 genes were essential for nitrogen fixation whereas nifA1 and rpoN1 were dispensable. Expression of nifA2 was activated, possibly in response to an inositol derivative, by a novel regulator of the LacI/GalR family encoded by the fixV gene located upstream of nifA2. Other than the well-characterized nif/fix genes, most NifA2-regulated genes were not required for nitrogen fixation although they were strongly expressed in nodules. The NifA-regulated nifZ and fixU genes, along with nifQ which was not NifA-regulated, were required in M. loti for a fully effective symbiosis although they are not present in some other rhizobia. The NifA-regulated gene msi158 that encodes a porin was also required for a fully effective symbiosis. Several metabolic genes that lacked NifA-regulated promoters were strongly expressed in nodules in a NifA2-dependent manner but again mutants did not have an overt symbiotic phenotype. In summary, many genes encoded on ICEMlSym(R7A) were strongly expressed in nodules but not free-living rhizobia, but were not essential for symbiotic nitrogen fixation. It seems likely that some of these genes have functional homologues elsewhere in the genome and that bacteroid metabolism may be sufficiently plastic to adapt to loss of certain enzymatic functions.

Figure 1. The fixV-nifA2 region.

A. Map of the fixV-nifA2 region. The location of gene fragments in the intergenic region with homology at the protein level to Msi109 is shown. The inserts in the plasmids pJS104 and pJS100 used for complementation of fixV and nifA2 mutants respectively are indicated above the map. Below the map are the intergenic fragments used to locate the nifA2 promoter. The sizes of the intergenic fragments are shown on the left and the Fix phenotype of the resultant strains on the right. B. BlastN output showing nucleotide identity between the fixV-nifA2 intergenic region and the msi109 region. The ATG corresponding to the start codon of msi109 is bolded and underlined. C. BlastX output showing amino-acid similarity between the two fragments in the fixV-nifA2 intergenic region and Msi109.

Figure 2. Comparison of the genetic organization of gene clusters associated with fixV homologs in a range of rhizobial species.

Genes are shown as arrow symbols and are to scale; colours specific for each gene are used to indicate genes that encode similar proteins in other clusters. Black indicates genes lacking homology to any other genes within the clusters. Fr notes gene fragment, IS denotes insertion sequence.

Figure 3. Model for the regulatory network governing symbiotic nitrogen fixation in M. loti.

In response to an inducer molecule (possibly an inositol derivative), FixV activates expression of nifA2. NifA2, in conjunction with RpoN1 or basal levels of RpoN2 produced from an unknown promoter, activates expression of the prxS-rpoN2 and fixABCX-nifA1 operons. NifA2 and NifA1, in conjunction with the increased levels of RpoN2, then activate expression of operons required for the production of functional nitrogenase along with other NifA-regulated operons that encode auxiliary metabolic functions.