To analyze if the three fnr genes in H. seropedicae influence either the expression level or the activity of NifA, we monitored expression of a nifB::lacZ fusion, as a reporter of NifA activity. We compared nifB expression in the wild-type strain (SmR1) with a double deletion strain, which lacks both fnr1 and fnr3 (MB13) and a strain carrying deletions in all three fnr genes (MB231). Multiple fnr deletion strains were not analysed in these experiments since single gene deletions did not influence NifA activity. As nifB gene expression is tightly regulated by nitrogen and oxygen levels in the cell [6], the activity of the nifB::lacZ reporter fusion is only observed when the cultures exhaust the supply of fixed nitrogen and oxygen becomes limited, as the culture reaches a high cell density. Although the fnr deletions impaired growth under these conditions as observed previously [18], the activity of the nifB::lacZ fusion was significantly higher after 12–16 hours incubation in the strain lacking both fnr1 and fnr3 (MB13) and also in the triple fnr deletion strain (MB231) when compared with the wild-type (Figure 1A). This suggests that either NifA expression or its activity is more highly induced in cultures of these multiple fnr deletion strains.
We considered the possibility that the multiple fnr deletion strains exhaust dissolved oxygen in the media faster than the wild type strain, thus leading to higher activity or stability of NifA in cultures of the fnr deletion mutants. To examine this further, we assayed nifB::lacZ activity in cultures grown in the absence of fixed nitrogen under defined initial oxygen concentrations of oxygen in the gas phase (Figure 1B). As anticipated, nifB expression was not detected under either 8% or 20.8% oxygen in both wild-type and the fnr triple deletion mutant, presumably because H. seropedicae NifA is inactivated at high oxygen concentrations [8,19]. However, the activity of the nifB::lacZ promoter fusion was markedly higher in the triple fnr deletion strain (MB231) compared with the parental strain, when cultures were incubated under an initial oxygen concentration of 4% or 6% in the gas phase (Figure 1B). To ensure that the increase of nifB expression observed in the mutant strains was NifA-dependent, we prepared single nifA
− and multiple deletion strains carrying a nifA deletion in addition to the fnr mutations (Additional file 1) and confirmed that the influence of Fnr proteins on nifB promoter activation requires NifA protein (Additional file 2).
Since expression of the nifA gene itself is subject to autoactivation in H. seropedicae [20], we tested the influence of fnr deletions on nifA expression using various nifA::lacZ promoter constructs (Figure 2). Transcriptional regulation of nifA is complex, since this σ54-dependent promoter is subject to nitrogen regulation by the enhancer binding protein NtrC in addition to autogenous activation by NifA under oxygen-limiting conditions (see Figure 2A). Notably, single deletions in each of the three fnr genes had no apparent influence on nifA expression. However, as in the case of nifB, an increase in promoter activation was apparent in the double fnr1, fnr3 deletion mutant (MB13) and the triple fnr deletion strain (MB231) (Figure 2B). In all cases, promoter activation significantly decreased when cultures were grown in the presence of a high concentration of fixed nitrogen (Figure 2C), or when the −24 to −12 region of the promoter was disrupted (plasmid pRW22, Figure 2B), indicating that activation is rpoN-dependent and subject to nitrogen regulation by NtrC as expected [20]. In all cases, irrespective of the presence of fnr mutations, nifA expression decreased when promoter constructs (plasmids pRWC and pRW3) carried mutations in the upstream activation sequence (UAS2) of the promoter (Figure 2B), presumably as a consequence of decreased autoactivation by NifA [20]. Overall, these results demonstrate that in the absence of both fnr1 and fnr3, activation of the nifA promoter is increased. Since higher expression of the nifA::lacZ fusion is not observed when the NifA binding site (UAS2) is deleted, it is likely that the increased expression results from autoactivation of the nifA promoter due to increased activity or stability of NifA protein.
Given that the nifA promoter is subjected to complex regulation, we designed experiments to confirm that NifA activity is enhanced in fnr mutant strains. Firstly, using the combined fnr
− and nifA deletion strains described above (Additional files 1 and 2) we complemented the nifA mutation with nifA expressed ectopically from the lac promoter (plasmid pRAMM1), which is constitutive in H. seropedicae. In this complementation assay we observed that the levels of nifH mRNA were higher in the fnr deletion strains complemented with constitutively expressed NifA in comparison with the complemented strain containing wild-type fnr alleles (Figure 3). This implies that an increase in NifA activity, rather than its expression, is responsible for increased activation of nif promoters in the fnr deletion mutants. Secondly, we constructed strains expressing NifA fused to a 3XFlag peptide to allow detection of the protein in both wild type and fnr mutant backgrounds (Additional file 3). Western blots of strains carrying the nifA-3Xflag allele revealed higher levels of NifA expression in the double fnr1, fnr3 deletion (MBN5) and also in the triple fnr deletion (MBN6) backgrounds compared with the strain carrying wild-type fnr alleles (MBN4) (Figure 4). This confirms the additional level of autoactivation of the nifA promoter conferred by the multiple fnr deletions (Figure 2B), again indicating that NifA activity is higher in the fnr mutant strains.
As the H. seropedicae NifA protein is sensitive to oxygen, being inactivated and degraded upon exposure to O2 [8,19], we hypothesized that NifA might be protected in its active form in fnr deletion strains if these strains exhibit a higher oxygen consumption rate. To further test this hypothesis we measured oxygen depletion during the growth of bacterial cultures in the same growth conditions as described for the assay of the nifA::lacZ fusions. We first analyzed the decrease in oxygen concentration in the gas phase of Suba-seal stoppered flasks (Figure 5A) and additionally compared the profiles of dissolved oxygen consumption using a Clark type electrode (Figure 5B). These assays revealed that the consumption of oxygen was higher in multiple fnr deletion strains, implying that these strains have a higher respiratory rate when compared to the wild type (Figure 5). Notably, the oxygen consumption data in Figure 5A directly correlates with the increased activity of NifA observed in strains lacking both Fnr1 and Fnr3 (Figure 2B) implying that the absence of both these transcription factors results in higher respiration rates.
In a previous study, we showed that the triple fnr mutant is deficient in the expression of the cytochrome c-type branch of the electron transport chain [18]. An alternative route of electron transport from the quinol pool to oxygen via the terminal quinol oxidases is likely to occur in the triple fnr mutant. As the quinol branch of the respiratory chain results in a lower number of proton-translocation events it is conceivable that the activity of this branch, rather than the expression of the bo
3
and bd-type oxidases, is enhanced in the fnr mutant strains to compensate for the lower level of energy production. This may result in increased electron flux through the respiratory chain and hence enhanced oxygen consumption as observed in our experiments.
We demonstrated previously that nitrogenase activity is severely impaired when the triple fnr deletion strain is cultured in ammonium-limiting liquid medium, potentially as a consequence of energy depletion [18]. We also showed that diazotrophic growth is impaired in the fnr ablated strain, after subjecting cultures to severe nitrogen starvation [18]. Under these conditions, cultures divide at extremely low growth rates, requiring 24 days post-inoculation to achieve an OD600 of ~ 1.6 (Additional file 4). However, it is notable that the triple fnr mutant grew faster than the wild-type for the first 12 days of incubation under these conditions. Potentially, the enhanced rate of O2 consumption by the triple fnr deletion allows higher levels of NifA activity and consequently higher nitrogenase activity during the ‘early’ stages of growth. However, it is possible that as the bacterial population increases and the oxygen levels in the culture drop further, the triple fnr mutant strain can no longer maintain the necessary electron flux to support nitrogenase activity and as a consequence, diazotrophic growth is impaired.
In summary, these studies have not identified a direct role for the H. seropedicae Fnr proteins in regulating NifA activity and nitrogen fixation, but rather suggest that they may influence both, by means of altering the composition of the electron transport chain and the oxygen consumption rate. Since we only observe such effects in strains deleted for both fnr1 and fnr3, there is apparently some redundancy in the physiological functions of the three fnr paralogs in H. seropedicae. It is feasible that H. seropedicae can take advantage of the three fnr genes to differentially modulate respiratory chain composition. This is likely to influence nitrogen fixation during different phases of growth and enable efficient adaptation during plant-bacterial colonization.