Flavin-containing monooxygenase (FMO), which can metabolize numerous foreign chemicals, is a monooxygenase that uses the reducing equivalents of NADPH to reduce one atom of molecular oxygen to water, while the other atom is used to oxidize the substrate [21]. An important issue is that M. huakuii fmoA expression is elevated in nitrogen-fixing bacteroids of the A. sinicus root nodules, but no studies have been published regarding the connection between the legumes-root nodule bacteria nitrogen fixing system and the function of FmoA. In this study, we focus on a fmoA mutant strain of M. huakuii that is affected with regard to its symbiotic capacity and oxidative stress response.
Flavin-containing monooxygenases (FMOs) have been reported to be important for the disposition of many therapeutics, environmental toxicants, and nutrients and, thus, mediate interactions between organisms and their chemical environment [22, 23] Mutation of fmoA can only slightly reduce the growth rate of M. huakuii in AMS with D-glucose as a sole carbon source, but displayed decreased growth capacity in the presence of high concentrations of inorganic peroxide H2O2 and organic peroxide CUOOH (Table 1). Intracellular H2O2 content was measured in the mutant, showing a significantly low H2O2 content compared to the wild type when FmoA was not present (Table 2). No research could demonstrate that FMO functioned as the defence against the peroxides, but human FMOs as a source of hydrogen peroxide released 30–50% of O2 consumed as H2O2 upon addition of NADPH [24], and liver flavin-containing monooxygenase has also been shown to exhibit a stable 4a-flavin hydroperoxide intermediate in the absence of oxygenatable substrate [25], suggesting the presence of FMO may increase cellular peroxide concentration, therefor may improve tolerance of exogenous H2O2. In aerobic cells, GSH is the most abundant antioxidant [26]. The effects of H2O2 stress on the activities of antioxidant enzymes such as peroxidase and glutathione reductase, and the content of GSH were further investigated. The antioxidant enzyme activities of mutant HKfmoA were no difference compared to that of wild type strain 7653R, but its GSH content was significantly lower (Table 2), suggesting that M. huakuii FmoA-deficiency-mediated decrease in glutathione increases the sensitivity of mutant cells to peroxides.
Since M. huakuii fmoA gene expression is significantly up-regulated on the whole nodulation process, and it’s the highest expression level occurred at 14 days after inoculation (Fig. 4). The roles of FmoA in symbiotic nitrogen fixation and the colonization of the plant rhizosphere were further studied by plant experiments. Although the expression levels of symbiotic genes such as nif, fix and nod, were not significantly different in bacteroids from the fmoA mutant and the wild-type strain, A. sinicus plants inoculated with the fmoA mutant exhibited a large decrease in the nitrogen-fixing activity of root nodules (reduced by more than 65%) (Table 3). Further investigation revealed that fmoA mutant HKfmoA-induced nodules were spherical rather than elongated, underwent premature senescence, and PHB granules could be detected in the fmoA mutant bacteroids but not in those of the wild-type strain (Fig. 3). Moreover, the M. huakuii fmoA mutant was unable to compete efficiently in the rhizosphere with its wild-type 7653R. Flavin-containing monooxygenases metabolize a vast array of foreign chemicals including antioxidants, phytochemicals and dietary components, and, thus, mediate interactions between bacteria and their chemical environment [19]. The results showed that bacterial FmoA was important for adaptation to the microenvironment of the plant host (Fig. 2). Overall, considering the poor nitrogen-fixing ability of its nodules, the mutant in fmoA gene has a profound influence on the whole nodulation process.
The RNA-seq experiments were performed to provide a foundation for assessing the influence of FmoA on the symbiotic nitrogen fixation. In this study, the carbohydrate transport and metabolism, coenzyme transport and metabolism, and flagellar genes were found to be significantly up-expressed in fmoA mutant induced bacteroids (Fig. 5). Flagellar motility is a critical environmental adaptation for the plant-associated bacteria such as Rhizobium that allows bacteria to escape adverse conditions and populate new environments [27]. The up-regulation of flagellar genes in this case might be due to the hostile environment in which the bacteroids with low nitrogenase activity are embedded [28]. In the fmoA mutant-induced nodules, many symbiosomes were aberrant and the bacteroid membrane showed incrassation (Fig. 3). The peribacteroid membrane may be relatively impermeable to sugars and so dictate the carbon source(s) available to the bacteroids [29]. This might also be the reason for the over-represented up-regulation category “coenzyme transport and metabolism”. The up-expression of carbohydrate transport and metabolism category containing 10 ABC transporter genes might be due to a delicate balance control between sufficient acquisition and overload (Table 4). In this study, PHB was occured in the fmoA mutant bacteroids (Fig. 4). During the formation of bacteroids in indeterminate-type nodules such as M. huakuii, the PHB granules are broken down. PHB can be used as a carbon and energy source for bacteroid formation, but most rhizobial species such as M. huakuii do not accumulate it during symbiosis with legumes. Biochemically, PHB synthesis directly competes with N2 fixation for reductant. PHB synthesis is apparently a concomitant reduction in protein synthesis, a process coupled to ATP formation and utilization [30]. PHB granules occurred in undergoing senescence bacteroids infected by fmoA mutant implied that the energy and carbon metabolism was shifted, NAD(P)H was channeled into other biosynthesis reactions, such as PHB synthesis [31].
Among the the down-exprssion genes, the four categories “Transcription”, “Defense mechanisms”, “Signal transduction mechanisms”, and “Posttranslational modification, protein turnover, chaperones” were significantly over-represented (Fig. 5). One of the remarkable findings of the RNA-seq analysis was that nearly all the genes associated with stress response and virulence were significantly differentially down-expressed (Table 5). The symbiotic nodule is prone to high levels of ROS due to the high rate of respiration necessary to supply energy required for nitrogen reduction by nitrogenase [32], but increased level of ROS causes oxidative damage to important cellular macromolecules [33]. Thiol-containing molecules, such as glutathione, glutathione S-transferases, glutaredoxins, and peroxiredoxins play an important role in maintaining redox homeostasis and redox regulation [34]. Nodules induced by fmoA mutant bacteria presented the lower expression of the thiol-containing molecules, which was associated with increased levels of superoxide accumulation. It has been reported that heat shock proteins play important roles in innate immune responses [35], and in Agrobacterium, the virulence genes are essential for attachment to plant cells [36]. The expression of three heat-shock protein and several virulence genes was also decreased in the fmoA mutant nodules (Table 5). Therefor, the results suggested that the stress response function of M. huakuii is influenced by deletion of the fmoA gene. In addition, it has been reported that the sensor histidine kinase mediated the pathogenesis by the bacterium Rhizobium radiobacter [37], and mutation of a R. leguminosarum histidine kinase gene chvG destabilized the outer membrane of R. leguminosarum, resulting in increased sensitivity to membrane stressors, and caused symbiotic defects on peas, lentils, and vetch [38]. In early senescent nodules induced by the fmoA mutant, the expression of two histidine kinases and a sensory box protein was significantly decreased, suggesting that FmoA could influence the symbiotic interaction between M. huakuii and A. sinicus by decreasing the expression of sensor molecules.
Moreover, 42 transcriptional regulator genes were found among the top 500 genes showing reduced expression (Table 5). MCHK_1343, MCHK_4626, and MCHK_5064 coding for the Crp-Fnr family transcriptional regulators, which is an important transcriptional regulator that controls the expression of a large regulon of more than 100 genes in response to changes in oxygen availability [49]; MCHK_4665 and MCHK_2899 coding for the AraC family transcriptional regulators, which play a critical role in regulating bacterial virulence factors in response to environmental stress [40]; MCHK_5909 coding for Lrp/AsnC family transcriptional regulator, which is known as feast/famine regulatory protein (FFRPs) [41]; MCHK_2407 coding for a DeoR/GlpR-type protein, which serves as transcriptional repressor or activator of either sugar or nucleoside metabolism [42, 43]; MCHK_4182 coding for a PadR family transcriptional regulator that functioned as environmental sensor [44]; MCHK_1555 coding for a MarR family transcriptional regulator, which is involved in the regulation of many cellular processes, including pathogenesis [45]; MCHK_5264 coding for a ArsR family transcriptional regulator involved in symbiosis and virulence [46]; MCHK_5463 coding for transcriptional regulator GcvA, which is required for both glycine-mediated activation and purine-mediated repression of the gcvTHP operon [47]. Taken together, nodules induced by the fmoA mutant are different from those induced by the wild-type strain, and there is also a clear difference in bacteroid aspect, revealing that the fmoA mutant is negatively affected in symbiosis. The reduced nitrogen fixation ability exhibited by the fmoA mutant could be a consequence of a defect in nodule development