Isolation and identification of NFAPSMs
Seven PSMs were selectively obtained from the mangrove sediments based on the obvious phosphate-solubilizing halos around colonies on SRSM medium (Fig. 1). The bromocresol purple indicator on the SRSM medium changed from purple to yellow (Fig. 1), which was a sign of pH drop. Thus, the seven PSMs might dissolve mineral phosphate by secreting organic acids or expelling protons.
We performed the PCR amplifications using specific primers (Additional file 1: Table S1) for 16S rDNA. 16S rDNA amplicons were subjected to agarose gel electrophoresis, and bright bands were detected at 1, 500 bp (Fig. 2a). The results showed that the seven PSMs were pure strains. Then they were sequenced and submitted to NCBI for homology comparison (Table S2). The deduced amino acid sequences were aligned with the 16S rDNA sequences from other species and the alignments were used to generate a neighbor-joining molecular phylogenetic tree (Fig. 2b). The result suggested that NM1-A2 and HM3 were identified as B. aryabhattai. HM2 was Metabacillus halosaccharovorans. HM4 was B. haynesii. HM5 and HM6 were B. licheniformis. HM7 was B. velezensis.
Evaluation Of The Ps Ability Of Seven Psms
The PS activity of seven PSMs after 48 h varied from 22.65 mg/L to 139.46 mg/L. Except for HM2 and HM7, the content of soluble phosphate decreased as time progressed. The amount of soluble phosphate of B. aryabhattai NM1-A2 was much higher than that of other strains, and it reached 139.46 mg/L at 48 h (Table 1).
Table 1
Evaluation of the PS ability of seven PSMs screened in this study.
Strain
|
PS ability (mg/L)
|
48 h
|
72 h
|
96 h
|
120 h
|
NM1-A2
|
139.46 ± 0.42 a
|
95.00 ± 1.35 a
|
54.28 ± 0.30 b
|
32.12 ± 1.04 bc
|
HM2
|
37.18 ± 3.96 c
|
40.29 ± 2.06 d
|
34.65 ± 0.93 c
|
30.76 ± 3.13 bc
|
HM3
|
116.19 ± 2.99 b
|
97.19 ± 1.53 a
|
74.69 ± 4.99 a
|
35.33 ± 2.49 b
|
HM4
|
85.14 ± 5.28 c
|
35.48 ± 3.16 d
|
25.52 ± 1.96 c
|
18.62 ± 4.31 d
|
HM5
|
85.04 ± 2.62 c
|
65.36 ± 4.07 bc
|
25.03 ± 0.95 c
|
26.44 ± 1.30 bcd
|
HM6
|
62.88 ± 9.08 d
|
57.59 ± 5.93 c
|
29.35 ± 15.77 c
|
22.65 ± 3.07 cd
|
HM7
|
64.39 ± 8.07 d
|
72.60 ± 5.74 b
|
62.06 ± 1.60 ab
|
57.68 ± 5.47 a
|
The PS process of B. aryabhattai NM1-A2 on SRSM medium containing different components was examined to better understand its PS characteristics. We found that the solubilization of tricalcium phosphate (TCP) in the SRSM liquid medium was accompanied with the decrease of pH after 12 h (Fig. 3). The correlation analysis showed a significant negative correlation between the soluble phosphate concentration and pH (Table 2), which indicated that the P-solubilizing mechanism was related to acid production [10]. The highest amount of soluble phosphate reached 124.24 mg/L when B. aryabhattai NM1-A2 was cultivated with glucose as the sole C source, followed by sucrose, starch, fructose, and maltose (117, 73.07, 45.38 and 40.68 mg/L, p < 0.05) (Fig. 3a). As shown in Fig. 3b, the optimum N source for PS was (NH4)2SO4 (136.29 mg/L), followed by NH4Cl, CO(NH2)2, KNO3 (124.99, 87.51, and 56.08 mg/L, p < 0.05). In the present study, the PS ability decreased rapidly with the increase in C/N ratio in the SRSM medium (Fig. 3c). C/N could affect the process of PS. The change of pH value directly affected the survival and metabolism of microorganisms. As shown in Fig. 3d, B. aryabhattai NM1-A2 grew normally in the range of pH 5.0–10.0. The peak value of soluble phosphate reached 142.79 mg/L when the initial pH value was 6.0, followed by 7.0, 5.0, 8.0, 9.0 and 10.0 (137.65, 132.87, 126.504, 114.74, and 65.37 mg/L). In addition, the content of soluble phosphate reached the highest when the concentration of NaCl was 2% (Fig. 3e). Subsequently, the amount of soluble P decreased continuously with the increase of NaCl concentration. The process of PS was inhibited under salt stress. The content of soluble phosphate increased from 9.9 mg/L to 186.66 mg/L with (NH4)2SO4 concentration ranging from 1 mM to 300 mM. The maximum PS efficiency (196.96 mg/L) was observed at (NH4)2SO4 concentration of 250 mM (Fig. 3f).
Table 2
Correlation coefficient between soluble phosphate concentration and pH while Bacillus aryabhattai NM1-A2 was cultured under different conditions.
Factor
|
Pearson correlation coefficient (r)
|
p-value
|
Carbon (C) sources
|
-0.958
|
p < 0.01
|
N sources
|
-0.968
|
p < 0.01
|
C/N
|
-0.924
|
P < 0.01
|
NaCl concentrations
|
-0.831
|
P < 0.01
|
pH
|
-0.779
|
P < 0.01
|
(NH4)2SO4 concentrations
|
-0.902
|
P < 0.01
|
Effect Of Nh Stress On The Types And Concentrations Of Organic Acids
High performance liquid chromatography (HPLC) analysis showed the presence of multiple organic acids during PS. Four different organic acids with low molecular weight, namely, formic acid, malic acid, acetic acid and succinic acid, were detected in all treatments, while lactic acid and citric acid were not detected (Fig. S1 and Table S3). Among them, formic acid showed the highest content, followed by malic acid, acetic acid and succinic acid (Fig. 4). The concentrations of formic acid and acetic acid in the high-NH4+ group were 443.7 and 120.5 mg/L at 12 h, respectively. These concentrations were significantly higher than those in low-NH4+ group (160.1 and 57.3 mg/L at 12 h, p < 0.001). The concentrations of succinic acid in the high-NH4+ group was 7.5 mg/L at 12 h, which was lower than that in the low-NH4+ group (20.8 mg/L, p < 0.05). However, there is no variation on the concentrations of malic acid. The results showed that the secretion of organic acids was affected by the concentration of NH4+. The mechanism of PS by B. aryabhattai NM1-A2 occurred mainly through the production of organic acids.
Whole-genome assembly and annotation of B. aryabhattai NM1-A2
NH4+ was a preferred N source for most bacteria [1]. The high-affinity NH4+ transporters AMT (encoded by amt) were used to absorb NH4+ at low NH4+ concentrations, while some low-affinity transporters were responsible for NH4+ at high NH4+ concentrations [20]. The whole-genome of B. aryabhattai NM1-A2 (Table S4) contained genes coding for glutamine synthetase (GS, encoded by glnA), glutamine 2-oxoglutarate amidotransferase (GOGAT, encoded by gltBD) and glutamate dehydrogenase (GDH, encoded by gdhA), which were key enzymes for NH4+ assimilation [21].
In addition, there were many types of PS-related genes, mainly including genes involved in organic acid synthesis and the phosphate regulatory system (Table S4). Inorganic phosphate (Pi) was mainly obtained through two independent uptake systems, Pit and Pst [22]. The Pst system was a typical ABC transporter composed of PstS, PstC, PstA, PstB and PhoU [22, 23]. Pi was transported by the Pst system under P starvation [23, 24]. B. aryabhattai NM1-A2 also had histidine kinase PhoR, response regulator PhoB for induction of PHO regulon under P-limiting conditions [25]. Genes for the main enzymes involved in TCA cycle and organic acid synthesis were found to be: glucose dehydrogenase (GDH) gene gdhB, formate dehydrogenase (FDH) gene fdhD, acetate kinase (AK) gene ackA.
Transcriptome sequencing of B. aryabhattai NM1-A2 and annotation analysis of DEGs
Transcriptome data results showed 2422 significantly differentially expressed genes (DEGs) between the low- and high-NH4+ groups. A total of 739 significantly upregulated genes and 1683 significantly downregulated genes were detected (Fig. 5a and Fig. 5b). KEGG enrichment results showed that the DEGs were mainly enriched in carbohydrate metabolism, amino acid metabolism, metabolism of cofactors and vitamins, and energy metabolism (Fig. 5c). C and N metabolism played an important role in response to NH4+ stress [20]. Moreover, up or down expression of DEGs in the 20 most enriched KEGG pathways was selected for display (Fig. 5d). The genes related to oxidative phosphorylation, C fixation pathways in prokaryotes, glycolysis, and TCA cycle were mostly upregulated. The GO enrichment analysis of DEGs between the low- and high-NH4+ groups (Fig. 5e and Fig. 5f) showed that the main enriched biological processes were cellular process, single organization and metabolic processes. The main enriched cellular components were cell and cell part. The main enriched functions were binding, catalytic activity, and transporter activity. These results indicated that catalytic and transport activities in B. aryabhattai NM1-A2 were significantly affected by high NH4+ stress.
The transcriptome data showed that the expression level of amt was downregulated by 3.52 fold, which indicated that the NH4+ transported to the bacteria was reduced (Table 3 and Fig. 6). NH4+ assimilation is regulated by two PII proteins (GlnB and GlnK) [26]. GlnB and GlnK can be reversibly uridylylated by uridilyl-transferase/uridylyl-removase (UT/UR), controlling the bifunctional enzyme adenylyl-transferase/adenylyl-removase (AT/AR), which can reversibly adenylylate (inactivate) or deadenylylate (activate) GS [26, 27]. GlnK is commonly cotranscribed with the NH4+ transporter AMT and reversibly inhibits AMT’s transport activity [27]. The expression level of glnB and glnK was downregulated by 2.779 and 0.9080 folds, respectively, under NH4+ stress (Table 3). AR enzymatic activity was promoted through the deuridylylation of GlnK-U and GlnB-U to rapidly inhibited the activity of GS. Then GlnK binded to AMT to form a complex to inhibit NH4+ uptake.
Table 3
DEGs related to NH4+ assimilation, phosphate transport and metabolism in B. aryabhattai NM1-A2.
Locus
|
Genes symbol
|
Gene description
|
Log2(B/A)
|
K8Z47_RS03380
|
amt
|
Ammonium transporter
|
-3.5183
|
K8Z47_RS21460
|
gudB, gdhA
|
Glutamate dehydrogenase
|
0.4648
|
K8Z47_RS20175
|
glnA
|
Glutamine synthetase, type I
|
-0.765
|
K8Z47_RS21810
|
gltD
|
Glutamate synthase [NADPH] small
chain
|
-3.377
|
K8Z47_RS10265
|
gltB
|
Glutamate synthase (NADPH) large chain
|
-0.9738
|
K8Z47_RS25960
|
phoR
|
Phosphate regulon sensor histidine kinase PhoR
|
-5.0795
|
K8Z47_RS04795
|
phoB
|
Phosphate regulon response regulator PhoB
|
0.4406
|
K8Z47_RS22250
|
phoU
|
Phosphate transport system regulatory protein PhoU
|
-4.0229
|
K8Z47_RS22810
|
pstS
|
Phosphate-binding protein PstS 1 precursor
|
-5.2930
|
K8Z47_RS22805
|
pstC
|
Phosphate ABC transporter, permease protein PstC
|
-2.3054
|
K8Z47_RS22800
|
pstA
|
Phosphate transport system permease protein PstA
|
-2.3193
|
K8Z47_RS22255
|
pstB
|
Phosphate ABC transporter, ATP-binding protein PstB
|
-5.7066
|
K8Z47_RS25095
|
ppaX
|
Pyrophosphatase PpaX
|
2.062
|
K8Z47_RS23660
|
ackA
|
Acetate kinase
|
1.3045
|
K8Z47_RS24905
|
fdhD
|
Formate dehydrogenase accessory protein
|
-1.5832
|
K8Z47_RS23540
|
icd
|
Isocitrate dehydrogenase [NADP]
|
1.2557
|
K8Z47_RS03260
|
aceA
|
Isocitrate lyase
|
-2.9359
|
K8Z47_RS11450
|
fumC, FH
|
Fumarate hydratase, class II
|
0.9909
|
K8Z47_RS23535
|
mdh
|
Malate dehydrogenase
|
1.2396
|
K8Z47_RS05365
|
glnK
|
PII family nitrogen regulator
|
-0.908
|
K8Z47_RS03375
|
glnB
|
P family nitrogen regulator
|
-2.7789
|
The GS-GOGAT cycle was the most crucial pathway for primary N assimilation [28, 29]. NH4+ and glutamate (GLU) to form glutamine (GLN) under the catalysis of GS (GlnA) [27]. The large subunit (GltB) and small subunit (GltD) of GOGAT complete the metabolic pathway of GLU regeneration from GLN and 2-oxoglutarate (2-OG) [27]. The expression levels of glnA, gltB, and gltD were downregulated by 0.7650, 0.9738, and 3.377 folds, respectively (Table 3), which revealed that the GS − GOGAT pathway was not the main approach for NH4+ assimilation in B. aryabhattai NM1-A2. GDH has been reported to incorporate excess NH4+ into glutamate when exposed to NH4+ stress [21]. The expression level of gudB was upregulated in the high-NH4+ group compared with that in the low-NH4+ group (Table 3). Therefore, the GDH pathway played a critical role in responding to NH4+ stress (Fig. 6). The assimilation of NH4+ was accompanied by the release of protons, which was one of the mechanisms of PS (Fig. 6) [8].
The production of organic acids is the main mechanism for solubilizing insoluble Pi [9]. Transcriptome analysis showed that ackA was upregulated while fdhD was downregulated in high-NH4+ group, which led to the increasing concentration of acetic acid and formic acid under NH4+ stress (Fig. 4). Thus, the synthesis and secretion of formic acid and acetic acid played an important role in the process of PS of B. aryabhattai NM1-A2 under NH4+ stress (Fig. 6) [30]. In addition, idh-encoding isocitrate dehydrogenase (IDH) was upregulated. Therefore, more 2-OG was produced under NH4+ stress, which provided sufficient organic acid skeletons for the subsequent amino acid synthesis [31–33]. The expression level of aceA was downregulated (Table 3), which might be the reason behind the decrease in succinic acid in the high-NH4+ group (Fig. 4). fumC and mdh were upregulated in the high-NH4+ group compared with those in the low-NH4+ group (Table 3). The catabolism of malic acid was greater than the synthesis, which led to a decrease in malic acid content under NH4+ stress.
As shown in the Fig. 3f, the content of soluble phosphate in the high-NH4+ group (196.96 mg/L) was significantly higher than that in the low-NH4+ group (62.5 mg/L, p < 0.001). The Pst system played an important role in transporting Pi under conditions of Pi limitation [25]. PstS and PstB were the key proteins in Pst system [22]. Genes related to the Pst system and PhoR were downregulated under NH4+ stress, especially pstS and pstB (downregulated by 5.29 and 5.71 folds). As a result, the Pst transporter was inhibited. The signal was transmitted from the Pst transporter to PhoR through PhoU, which inhibited the kinase function of PhoR, and thus dephosphorylating PhoB [25]. When Pi was abundant, excess Pi was stored as polyphosphate (polyP) [34]. PolyP could be separated into two groups, pyrophosphates (two phosphate residues) and high molecular weight polyP [35]. Pyrophosphates (PPi) is a metabolic product of biosynthetic reactions, and pyrophosphatase (PPaX) catalyzed the hydrolysis of PPi [35]. ppaX was upregulated in high-NH4+ group, which can maintain cellular PPi homeostasis and provide energy for the PPi-generating bio-synthetic reactions (Table 3 and Fig. 6).
qRT-PCR of genes involved in PS of B. aryabhattai NM1-A2 under NH 4 + stress
Quantitative real-time polymerase chain reaction (qRT-PCR). According to the abovemetioned results, eight genes (gltD, ackA, pstB, ppa, pstB, amtB, phoR, and glnA) were selected for qRT-PCR analysis. The expression levels of ackA and ppa were upregulated in the high-NH4+ group, whereas gltD, pstB, pstB, amtB, phoR, and glnA were downregulated (Fig. S2). The expression trends of eight genes were consistent with the RNA-seq data, which confirmed the reliability of the transcriptomic data.