Paenibacillus sabinae T27 performs nitrogen fixation in presence of high concentration of ammonium
The Diazotrophs generally performed nitrogen fixation in low ammonia environments. To determine effect of ammonium on nitrogenase synthesis, the nitrogenase activity was measured by acetylene reduction when P. sabinae T27 were grown in flask containing different ammonium concentrations (0-400 mM) in absence of oxygen. We found that P. sabinae T27 exhibited high nitrogenase activities at low concentration of ammonium (0-3 mM) and high concentration of ammonium (30-300 mM), while very little activities were observed in the presence of 4-20 mM ammonium (Fig. 1). In contrast, nitrogenase activity was totally inhibited in P. polymyxa WLY78 when 2 mM NH4Cl was used [21,25]. Also, nitrogenase activity was totally inhibited in Azospirillum lipoferum and Azospirillum brasilense when 1 mM NH4Cl was used .
Dynamic analysis of nitrogenase activities of P. sabinae T27 in presence of the different concentrations of NH4+ during fermentation process
To investigate whether P. sabinae T27 on scale cultivation exhibited high nitrogenase activities at both low (0-3 mM) and high (30-300 mM) concentrations of NH4+ and little activities in the presence of 4-20 mM NH4+, the bacterial cells were anaerobically cultivated in 7.5 L fermentor containing 20% (w/v) glucose and 2 mM glutamate supplemented with 0 mM, 10 mM and 100 mM NH4CI as the initial nitrogen source, respectively. During each fermentation, the growth rates (OD value) and nitrogenase activities of bacterial cells, and the concentration of NH4+ and glucose in medium were measured at 2 h intervals.
As shown in Fig. 2a, when P. sabinae T27 were grown in the medium containing 0 mM NH4Cl, the cell densities increased and glucose concentration decreased gradually during the fermentation process. Notably, nitrogenase activity was rapidly detected at 2 h and then it increased as bacterial cells grew. Nitrogenase activity reached maximum at 14 h and then decreased.
When P. sabinae T27 cells were grown in the medium containing 10 mM NH4Cl, the cell densities of P. sabinae T27 were much higher than those cultivated in medium containing 0 mM NH4Cl. After 8 h of cultivation, the cell concentrations of the P. sabinae T27 increased gradually from OD600 0.5 to 3, meanwhile the concentration of glucose was decreased. Then glucose was added in order to keep growth of bacterial cells at 10 h and 16 h, respectively. Meanwhile, NH4+ concentration in the medium decreased as culture titer increased and was almost completely depleted after 12 h of fermentation. However, no nitrogenase activity could be detected until after 12 h of cultivation at that time NH4+ in the medium was nearly depleted, consistent with the nitrogenase activities observed in the cultivation on flask scale (Fig. 2b).
When P. sabinae T27 cells were grown in the medium containing 100 mM NH4Cl, the nitrogenase activities increased as the bacterial cells grew. Importantly, nitrogenase activity was rapidly detected after 2 h of cultivation, just as it was observed when P. sabinae T27 cells were grown in the medium containing 0 mM NH4Cl. Then, P. sabinae T27 maintained nitrogenase activities throughout fermentation and the activities of reached a peak of 1169 nmol C2H4 OD600-1 mL-1 h-1 at 16 h. Although NH4+ concentration is also decreased, it was constantly higher than 60 mM during the fermentation process (Figure 2C).
Taken together, fermentation experiments demonstrated that P. sabinae T27 on scale cultivation exhibited a little activity in the presence of 10 mM NH4+, but high nitrogenase activities in the absence of NH4+ and presence of 50-100 mM NH4+. The results were in agreement with the data obtained by cultivating P. sabinae T27 in Erlenmeyer flask containing 0 mM, 10 mM and 100 mM NH4+, respectively.
Transcriptional analysis of the nif and nif-like genes in P. sabinae T27 during the fermentation processes
In order to investigate the effect of NH4+ concentration on the transcription, the transcript levels of the nif and nif-like genes of P. sabinae T27 under fermentation conditions were analyzed by qRT-PCR with the cell samples taken when nitrogenase activity was rapidly increased shown in Fig. 2b, c with arrow. qRT-PCR analysis demonstrated that the transcript levels of the nifBHDKENXorf1hesAnifV genes within the main nif cluster was highly induced under 0 and 100 mM NH4+ conditions in comparison to those under 10 mM NH4+ condition (Fig. 3), suggesting that nifBHDKENXorf1hesAnifV genes were involved in nitrogen fixation in presence of high NH4+. In contrast to nifBHDKENXorf1hesAnifV, nifHDK-like and multiple nifHBNE genes, with the exception of nifH2B2, were not significantly differently expressed under 0, 10 and 100 mM NH4+ conditions, suggesting that these genes did not function in nitrogen fixation under nitrogen-excess condition.
Nif protein expression in P. sabinae T27 during the fermentation processes
Western blot analysis with the extracts from fermentation also demonstrated that the Fe protein (NifH) and the α subunit (NifD) of the MoFe protein of nitrogenase were detected at 0 and 100 mM NH4+, respectively, consistent with nitrogenase activity and transcript levels under 0, 10 and 100 mM NH4+ conditions (Fig. 4). The data suggest that nitrogenase was synthesized both in the absence of ammonium and in the presence of high concentration of ammonium.
The specific activities of the purified nitrogenase components in presence of high ammonium
The cells of P. sabinae T27 after 16-20 h of fermentation at 0 and 100 mM NH4+ were anaerobically collected, respectively. Then, MoFe and Fe proteins were purified from the cell-free extracts by sequential anion exchange chromatography and preparative polyacrylamide gel electrophoresis (PAGE), respectively. The homogeneities of the purified MoFe and Fe proteins after preparative PAGE were at least 90% purity, as assessed by SDS-PAGE (Fig. 5). The MoFe protein component showed enrichment of two band (corresponding to α and β subunits) with an apparent molecular size near 55 kDa and the Fe protein component showed enrichment of a band with an apparent molecular size of about 35 kDa. The MoFe protein and the Fe protein purified from the bacterial cultures grown at 0 mM NH4+ had the same sizes as those purified from the bacterial cultures grown at 100 mM NH4+ (Fig. 5).
Furthermore, the bands corresponding to MoFe protein and Fe protein under nitrogen-limited and nitrogen-excess conditions were excised, digested, and analyzed in a qualitative fashion by LC-MS to discern the identity of the bands. The composition of MoFe protein was identified as portions of NifD and NifK, and Fe protein components were recognized as portions of NifH and NifH2 under both nitrogen limitation and nitrogen excess condition (Additional file 1: Figure S1). These data suggest that the nitrogenases of P. sabinae T27 grown under both conditions were similar in composition and that the MoFe encoded by the same nifDK and Fe proteins encoded by the same nifHnifH2 are responsible for nitrogenase activities.
The specific activities of the purified MoFe protein and Fe protein were analyzed by using the acetylene reduction assay after mixing MoFe protein and Fe protein. Nitrogenase activity was observed when the purified MoFe protein and Fe protein were mixed. On the contrary, the purified MoFe protein or Fe proteins along did not show activity. The data suggest that both Fe and MoFe proteins were pure for further assay. Like most of other nitrogenase components, both Fe and MoFe proteins were extremely sensitive to oxygen. The Fe protein levels in the extracts, fractions, or purified preparations were normally established by performing the assays in presence of an excess of MoFe protein, and vice versa. The MoFe and Fe protein activities varied among preparations, and maximum activity of MoFe and Fe proteins (805 and 667 nmol C2H4·mg-1·min-1, respectively) was obtained under nitrogen limitation and (792 and 638 nmol C2H4·mg-1·min-1, respectively) under nitrogen excess (Table 1).
In vitro nitrogenase activities of the purified nitrogenase components from the intraspecies or interspecies
In vitro nitrogenase activities in the mixtures of Fe and MoFe protein components from the intraspecies or interspecies were determined. As shown in Table 2, when Fe protein or MoFe protein from 100 mM NH4+ was correspondingly mixed with MoFe protein or Fe protein from 0 mM NH4+, the similar activities (690 nmol C2H4·mg-1·min-1 and 671 nmol C2H4·mg-1·min-1) were found. The data suggest that Fe protein and MoFe protein purified from 100 mM NH4+ had the specific activities as those purified from 0 mM NH4+ did.
Furthermore, in vitro nitrogenase activities were determined by mixing Fe protein or MoFe protein components of P. sabinae T27 with the corresponding MoFe protein or Fe protein of Azotobacter vinelandii and Klebsiella oxytoca cultivated under nitrogen-fixing conditions. No matter Fe protein or MoFe protein of P. sabinae T27 was purified from 0 mM NH4+ or from 100 mM NH4+, they could exhibit nitrogenase activities when mixed with the corresponding MoFe protein or Fe protein from A. vinelandii and K. oxytoca.