Bacillus velezensis LG37: Transcriptome profiling and functional verification of GlnK and NmtA in ammonia (NH4+) assimilation

In recent years, interest in Bacillus velezensis has increased considerably due to its role in many industrial water bioremediation processes that include - probiotics. In this study, we isolated and assessed the transcriptome of Bacillus velezensis LG37 (a new strain isolated from aquaculture pond) under different nitrogen sources. Since Bacillus species exhibit heterogeneity, the underlying molecular mechanisms in ammonia nitrogen assimilation are poorly understood; and ammonia’s nitrogen is considered toxic to aquatics.Results Here, a total of 812 differentially expressed genes (DEGs) from the transcriptomic sequencing of LG37 grown with ammonia or glutamine were obtained, from which 56 had Fold Change ≥ 2. BLAST NCBI and UniProt databases revealed 27 out of the 56 DEGs were potentially involved in NH4+ assimilation. Among them, the relative expression level of the two-component regulatory system glnK/glnL were significantly upregulated in the transcriptomic profile, and the same was authenticated by RT-qPCR along with six other genes. In addition, the transcriptome and relative expression analysis showed that the transporter (amtB) gene of LG37 was not involved in ammonia transport even in the highest ammonia concentrations. Besides, CRISPR-Cas9 knockout (Δ) and overexpression (OE) LG37 mutants of glnK further evidenced the exclusion of amtB regulation, suggesting an involvement of alternative transporter. Moreover, in the transcriptomic data, a novel ammonium transporter nmtA was expressed significantly in increased ammonia concentrations. Subsequently, OEnmtA and ΔnmtA LG37 strains showed a unique expression pattern compared to that of wild-LG37 strain.Conclusion Based on the transcriptome data, regulation of nitrogen related genes were promptly screened from the newly isolated LG37 strain to analyse the key regulating factors during ammonia assimilation. The novel NmtA transporter of LG37 became apparent in ammonia

The bacterial growth kinetics of wild-type LG37 was analyzed by culturing in LB broth, and the growth was measured by spectrophotometry. From 25 to 37 °C, the LG37 showed a good growth trend. The peak levels OD (≈7) were maintained for almost 30 h in the static cultivation environment (Additional File 1). As shown in Additional File 1, LG37 on 1 -6 % of salt concentrations had a sharp and stable increase in their growth exceeding 5OD at 28 h, then a gradual decline occurred, yet the levels were maintained above 5 up at to 3% salinity; and the decrease in OD was proportional to salt concentration in the extended period. The LG37 growth was normal at pH 6-8.5 while slow growth was recorded at pH 9.5. The dissolve oxygen (DO) concentration available in the medium mainly influences the growth of the bacterial population [45]. The increasing growth values were observed in all the DO reactors from 0 to 40 h, and the LG37 growth was proportional to the level of DO. Peaked OD of 8.2, 6.1 and 4 -were obtained for LG37 with initial DO of 6.0, 4.2, and 3.0 mg/L respectively at 35 h of the culture period. Conversely, there was no substantial growth recorded in the 1.8 mg/L DO (Additional File 1).

Optimization of Glutamine and ammonia nitrogen concentrations for LG37
The growth characteristics of LG37 was determined in various concentrations of NH 4 + and Gln by OD 600 . The results showed, with the increase of Gln concentration, LG37 displayed an inclination of growth respect to the amount of Gln ( Fig. 2A). The growth curve of LG37 in minimal media with NH 4 + (5, 10, 15, 20, 25, and 30 mmol/L) as the sole nitrogen source showed a similar trend to that of Gln, whereas no significance was observed at 20 -30 mmol/L NH 4 + concentrations (Fig. 2B). However, the growth of LG37 was greater in the increasing levels of nitrogen; the other nutrients in the minimal media might be a limiting factor for their growth above 20 mmol/L. Given that and to avoid overloading, we preferred an average volume (10 mmol/L) of N-source for both Gln and NH 4 + in our supplementary studies.

Transcriptome assembly and functional annotation
LG37 cells acquired from the treatment group (NH 4 + -N) and control group (Gln-N), were sequenced by Illumina Hiseq TM 2500 to obtain the overview of the gene expression pattern. After removing low quality (Q > 20) ambiguous reads from the raw data, a total of 19091060, 19270408, and 19079520 clean reads from the LG37-Gln (Gln-N1, Gln-N2, Gln-  (46), and Photosynthesis (7) (Fig. 4, Additional File 6).

RT-qPCR verification of selected genes
The Illumina sequence of LG37 expression profile data was biased through randomly selected 8 DEGs that including, 5 up-regulated and 3 down-regulated genes (Additional File 7) using RT-qPCR. The results exhibited a similar expression tendency to expect in a slight variation in the levels with the transcriptome data, which confirmed the reliability of DEGs from the transcriptome sequencing results (Fig. 5).

Glnk are critical for NH 4 + assimilation
To understand the functions of the specific genes associated to NH 4 + assimilation pathway that short-listed by transcriptome data, especially the significantly upregulated glnK (4.26) and glnL (4.12) were primarily analyzed by RT-qPCR following cultivation of wild- LG37 at different concentrations of NH 4 + . The glnK and glnL showed a significant increase in their expression with the increase of NH 4 + concentration, and the glnK showed a greater tendency than the glnL (Fig. 6A). These results demonstrate that the twocomponent regulatory systems glnK/glnL play a synergistic regulation role in the LG37 NH 4 + metabolism.

Functional analysis of related genes using OEglnK and ΔglnK in NH 4 + metabolism
To demonstrate the functionality of the GlnK in NH 4 + assimilation pathway, glnK was knocked out (LG37-ΔglnK) by applying CRISPR/cas9 technique (Additional File 8) and developed overexpression mutant (LG37-OEglnK) strain using pHT1K plasmid (Additional File 9). The mutants were analyzed for the regulation of ammonia assimilation related gene (glnL, amtB, and glnA) by RT-qPCR assays along with wild LG37 strain, following cultivation in 10 mmol/L NH 4 + containing minimal medium. The LG37-OEglnK strain increased the expression of about 2.5 folds and 1.8 folds, while, the LG37-ΔglnK strain lead to decrease in the expression to 0.25 and 0.7 folds for glnL and glnA, respectively. No notable changes were recorded in the amtB gene expression compared to that of control ( Fig. 6B). The growth curve of both the mutated strains represented the differences in growth with increased growth in LG37-OEglnK and decreased growth for LG37-ΔglnK strain when compared to the wild-LG37 control strain (Fig. 6C). These results indicated that the GlnK plays a significant role in the NH 4 + assimilation of LG37.
Based on the above results, we noted that the GlnK sense the NH 4 + concentration and regulate glnL expression thru signal transduction, and further the GlnL promotes glnA and AmtB in favor of NH 4 + assimilation. AmtB, as an ammonium transporter, there was no significant expression among the 3 groups (Fig. 6B), which is similar to that of the transcriptome results of amtB between NH 4 + -N and Gln-N nitrogen groups. Hence, we analyzed the relative expression of AmtB in wild-LG37 at an increased NH 4 + concentration, and the results exhibited no difference in their expression pattern even at the highest NH 4 + -N concentrations (Fig. 6D). The above data suggest that the GlnK plays a vital role in regulating GlnL and GlnA, and still the AmtB were not as a specific positive factor for NH 4 + transporter of LG37 in assimilation. Therefore, it has been ruled out that there might be other NH 4 + transporters in the case of LG37.

Determination of the NH 4 + transporter -NmtA in LG37
Considering the expression of amtB as shown in Fig. 6B and D, we found that the AmtB is not specific in the NH 4 + transport in LG37. Thus, we further analyzed our transcriptome data for another transporter where we noticed nmtA with 2.93 fold change in expression. f (Additional File 4). To verify whether NmtA plays a role in NH 4 + assimilation, we detected the expression of nmtA of LG37 after culturing with different NH 4 + -N concentrations by using RT-qPCR. The expression of nmtA increased with increase in NH 4 + -N concentrations ( Fig. 7A). in the LG37-ΔglnK and LG37-OEnmtA mutant strains, the nmtA expression were increased by a factor of 1.67 and decreased to 0.35, respectively, compared to that of wild-LG37 strain (Fig. 7B). Similarly, we determined the growth of both strains, showed increased growth pattern in LG37-OEnmtA whereas LG37-ΔnmtA strain showed a decreased growth compared to that of wild-LG37 strain (Fig. 7C). These results demonstrated that NmtA was essential for the NH 4 + assimilation process in LG37.
Further, to extend our studies on NmtA, we analyzed the expression of downstream functional genes glnA and glnB in the NH 4 + assimilation pathway for both LG37-OEnmtA and LG37-ΔnmtA strains. The relative-expression levels of glnA (1.7 folds) and glnB (1.6 folds) were increased in the OEnmtA strains, whereas, the ΔnmtA strain the expression of glnA and glnB were only 0.3 and 0.5 folds, respectively (Fig. 7D). These findings evidenced that the NmtA was served as the transporter and play a significant role in the process of NH 4 + assimilation in LG37.

Discussion
Present-day intensive aquaculture practices are often accompanied by a large volume of excretes and other organic residue accumulations, resulting in the water quality deteriorations. In particular, the building of nitrogenous compounds, namely nitrite and ammonia, are harmful to the aquatic animals although in their least concentrations [46,47]. Proper aquatic animal health management exercises begins with the maintenance of water quality. It can be achieved by assisting with various biological forms of lives that including various Sp., of Bacillus bacterium. Indeed, demonstration of genetic heterogeneity nature of various Bacillus Sp., not only in genetically distinct in subgroups but also within a clonal and synchronized bacterial population [6].  [48], and B. subtilis [49] isolated strains demonstrated substantial ammonia nitrate removal. All of those previous findings suggesting an encouraging signs lead us to isolate the potentially efficient bacillus strain for NH 4 + assimilation and to determine the mechanism of a complex metabolic network like signal inductors, transcriptional regulators, transporters, assimilation enzyme and so on. The increased heterogenetic gene-expression of the bacterial population could not only utilize the existing nutrients for their growth but also get a benefit for the survival of the bacterial population in extreme conditions. One needs to study the interacting molecules and their networks, contribute to understanding the biological system function [50].
In the present study, a Bacillus velezensis LG37 was isolated from the aquaculture pond that exhibited a high nitrogenous compound removal between the three isolated strains.
The LG37 strain illustrated good growth at a wide range of temperature, pH, and salinity.
For the transcriptomic approach of nitrogenous substances, we determined the growth conditions using the inorganic minimal medium that lack rich nutrients incorporated with organic (Glutamine-N) and inorganic nitrogen (NH 4 + -N) for ammonia nitrogen metabolism at the molecular level in LG37. Undoubtedly, the LG37 could able to grow well in both Nsubstrates but to bypass overstressing and to offer other nutrients from the minimal media; the substrates were volume-averaged to 10 mmol/L. Mining the potential genes from the transcriptome data may shed light on the mechanism of N-metabolism of LG37.
To dissect the role of differential gene expression (DEGs) profiles, a global transcriptomic profiling method were employed for LG37 genome that cultivated under two different N- Considering the greater challenges related to the data derived from the transcriptomic study that have to be further verified by other approaches, including RT-qPCR [53,54]. In our study, to eliminate effects of possible amplification bias, we validated the expression patterns of the 8 randomly selected genes using RT-qPCR method. The primary results displayed that the pattern distribution and gene-expression levels were highly correlated with Illumina sequencing data. Additionally, these comparative data presented that not all the functional genes contributed in the same manner but also proved the significance of the genes in the ammonia assimilation process. Although the preliminary results provide a theoretical basis, such information still required an in-depth analysis of potential functional genes in NH 4 + assimilation metabolic network of LG37.
Following our transcriptome data, it was shown that there is no positive correlation between glnK and amtB. We speculate that the AmtB is not specific for NH 4 + transport, as well as there was not a direct interaction also between GlnK and AmtB. To test the role of LG37 specific genes in NH 4 + assimilation mutant strains were constructed using the all-inone CRISPR-Cas9 genome editing system to develop knockout (pJOE8999) [55] and overexpression (pHT1K) LG37 strains. It was again exposed that GlnK does not have a correlated mechanism with the transporter AmtB between the wild-LG37 and mutants (OEglnK and ΔglnK) strains. Whereas, the glnL and glnA expressions were significantly upregulated.
LG37 mutants (OEglnK and ΔglnK) and wild LG37 strain, while the glnL and glnA expressions were significantly upregulated. Similarly, growth curves of the wild-type and mutant strains demonstrated a consistent reduction in the growth of ΔglnK mutant, however, not much difference was observed between OEglnK and the wild-type strain (Fig.   6C). The reduced growth of the knockout cells might be due to the inability to utilize the N-compounds where the minimal media contained only basic nutrients. These results indicated that although glnL and glnA was induced, the amtB gene was found to be nonessential for the transport of NH 4 + in LG37 strain. Thus, LG37 might likely adopted different transporter mechanism similar to that of AmtB in other Bacillus strains for NH 4 + transport.
Conversely, based on the LG37 transcriptomic data, we found a distinct transporter (nmtA) was upregulated to 2.92 folds, and we speculate that NmtA might be an alternate NH 4 + transporter in LG37. The NmtA is a single-polypeptide secondary carrier transmembrane transporter protein belong to the Major Facilitator Superfamily (MFS) that promote small solutes in response to chemiosmotic ion gradients [56,57]. The relative expression levels of nmtA showed remarkable expression levels when LG37 grown in the increasing NH 4 + concentrations as well as the same reflected in the OEglnK mutant strain. The ΔglnK mutant resulted in a reduction of nmtA expression compared to that of wild type.
Furthermore, to confirm this theoretical assumption, we constructed the ΔnmtA and OEnmtA LG37 strains, as mentioned earlier, and analysed for their growth and the expression of downstream genes (glnA and glnB) by RT-qPCR. Our analysis revealed that growth characteristics of the LG37 nmtA mutant strains were almost similar to the growth of glnK mutants and the contrasting expressions of glnA and glnB genes between the strains, compared to that of wild-LG37.

Conclusions
The newly isolated bacterium Bacillus velezensis was named as B. velezensis LG37, showed its ability of well growing in various basic parameters and its heterotrophic capacity to utilize both NH 4 + -N and Glutamine-N as a sole nitrogen source to grow. The transcriptome sequence analysis under different nitrogenous source proposed that LG37 up-regulated (18) or down-regulated (9) genes predominantly related to nitrogen metabolism. Moreover, 18 up-regulated DEGs associated with a few hypothetical proteins, transcriptional regulator, transporter, transporter permease, and GlnK/GlnL regulatory system for assimilating the N-compounds. Further, we applied genome editing technology to the genes whose expression was impacted in nitrogen assimilation; the glnK mutants led to uncover that amtB was not associated with ammonium transport (Fig. 6). However, knock out of glnK in LG37 showed comparable growth to wild-type/OEglnK on ammonia as a sole nitrogen source, which demanded us to expose the unidentified ammonia transport system. In particular, the upregulation of nmtA transcriptional expression was shown to specifically correlate with the ammonia transporter and exhibited the regulation of related downstream genes (glnA and glnB). Altogether, the NmtA was proved as a novel ammonium transporter in Bacillus velezensis LG37 and our results provide a theoretical basis and new clues to NH 4 + assimilation mechanism. Furthermore, we hypothesize that more than one ammonia transporter might have involved from the transcriptome of LG37 differential expression (Additional File 4), and this can be evaluated through subsequent experiments.

Bacterial Isolation
The Chloroform (200 µL) was addedd and solution further shaken thoroughly. It was then kept on ice for 5 minutes. Centrifugation (10,000×g, 15 minutes) was carried out before 1:1 (v/v) of supernatant and isoporpyl alcohol were uniformly and gently mixed with a pipette.
The mixture was returned to ice for 20, then centrifuged (10,000×g, 30 minutes). The supernatant was gently discarded, 1 ml of 70% ethanol pipetted into the tubes to wash the residual pellet by gently allowing the ethanol to flow over the pellets, while partially rotating the tubes. The ethanol was then discarded carefully. The wash-discard process repeated twice; then the tubes were air-dried for 5 minutes. The RNA pellets were then dissoled in 80 µL of RNase-free water. The quality and quantity of the RNA were assessed

Construction of glnK and nmtA overexpression LG37 strains
To investigate the exact role of glnK and nmtA in ammonia assimilation, the P xyl promoter DNA region from LG37 genomic DNA was amplified and the PCR products were inserted into the pHT1K expression vector on NcoI and BamHI restriction endonuclease recognition site. Subsequently, PCR amplified products of LG37 genomic glnK and nmtA were cloned into the integrating plasmid pHT1K-P xyl at BamHI and KpnI restriction endonuclease recognition site to obtain pHT1K-P xyl -glnk and pHT1K-P xyl -nmtA recombinant plasmids, respectively. The DNA sequences were amplified using primers listed in (Additional File 11). The acquired plasmids were transformed into competent Escherichia coli DH5 α cells for overnight cultivation in LBampicillin plates at 37°C. Positive clones containing inserts of the expected size for P xyl , glnK and nmtA fragments were verified by sequencing and by using specific restriction enzyme digestion analysis. Both recombinants were transformed separately into LG37 by electroporation, and successful transformants were screened with 25 μg/mL erythromycin in LB solid medium. Diagrammatic representation of a common OE plasmid construction was shown in Additional File 9.

Statistical analysis
Statistical analyses were performed using a statistical package for social sciences (SPSS,        These results are means ± SD. *P < 0.05, **P < 0.01 versus control.

Supplementary Files
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