DUF2285 is a novel helix-turn-helix domain variant that orchestrates both activation and antiactivation of conjugative element transfer in proteobacteria.

Horizontal gene transfer is tightly regulated in bacteria. Often only a fraction of cells become donors even when regulation of horizontal transfer is coordinated at the cell population level by quorum sensing. Here, we reveal the widespread 'domain of unknown function' DUF2285 represents an 'extended-turn' variant of the helix-turn-helix domain that participates in both transcriptional activation and antiactivation to initiate or inhibit horizontal gene transfer. Transfer of the integrative and conjugative element ICEMlSymR7A is controlled by the DUF2285-containing transcriptional activator FseA. One side of the DUF2285 domain of FseA has a positively charged surface which is required for DNA binding, while the opposite side makes critical interdomain contacts with the N-terminal FseA DUF6499 domain. The QseM protein is an antiactivator of FseA and is composed of a DUF2285 domain with a negative surface charge. While QseM lacks the DUF6499 domain, it can bind the FseA DUF6499 domain and prevent transcriptional activation by FseA. DUF2285-domain proteins are encoded on mobile elements throughout the proteobacteria, suggesting regulation of gene transfer by DUF2285 domains is a widespread phenomenon. These findings provide a striking example of how antagonistic domain paralogues have evolved to provide robust molecular control over the initiation of horizontal gene transfer

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

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

Comparison of the genetic organization of gene clusters associated with <i>fixV</i> homologs in a range of rhizobial species.

<p>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.</p

Symbiotic expression of various genes in wild-type, Δ<i>nifA1</i> and Δ<i>nifA2</i> backgrounds.

a<p>ß-galactosidase assays were performed on bacteroid suspensions from nodules harvested 14 days post-inoculation. All activity values are the average of at least two assays ± Standard Deviation. Significant differences in expression observed between R7A and R7AΔ<i>nifA1</i>:: Ωkan **  = <i>P</i><0.005, between R7A and R7AΔ<i>nifA2</i>:: Ωkan ⁺⁺ =  <i>P</i><0.005, between R7AΔ<i>nifA2</i>:: Ωkan and R7AΔ<i>nifA1</i>:: Ωkan^• = <i>P</i><0.05 (as determined by unpaired t test).</p

Symbiotic properties of partially effective and ineffective mutants.

a<p>Mean wet foliage weight of 30 plants ± Standard Deviation. Data were analysed using the Students T-test. ^b Percentage of acetylene reduction relative to R7A. Nitrogen fixation activity was measured as the amount of C₂H₂ reduced (µmol h⁻¹) per plant root for 10 plants ± standard deviation. Data were analysed using the Students T-test. A single asterisk represents <i>P</i><0.05 and two asterisks <i>P</i><0.005 when compared to R7A.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p