Priestia megaterium NCT-2 was inoculated into an inorganic salt medium with K¹⁵NO₃ as a nitrogen source at 2.0% inoculum and incubated (200 r min⁻¹ and 35 °C for 78 h). The sterile medium was used as a control group. At 0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 60 h, and 72 h, the culture medium was centrifuged at 4 °C and 5,000 rpm for 10 min, and the supernatant was collected. Cells were collected after repeated suspension, centrifugation, and washing in phosphate buffer (30 mmol Na₂HPO₄ + 20 mmol K₂HPO₄). The contents of NO₃⁻, NO₂⁻, and NH₄⁺ in the supernatant were determined by CleverChem ONE spectrophotometer (Alliance company, France). The kjeldahl nitrogen analyzer was used to determine the total nitrogen content in supernatants and cells. The dry cell weight was measured by weighing. The ¹⁵N atomic percent (atom%) of NO₃⁻, NO₂⁻, and NH₄⁺ in supernatants and the ¹⁵N atom% in cells were determined by a stable isotope mass spectrometer (ThermoFisher, Germany), respectively.

Priestia megaterium NCT-2 was inoculated into a 250 mL fermentation flask containing 80 mL of inorganic salt medium with K¹⁵NO₃ as a nitrogen source, and cultured in an anaerobic incubator at 35 °C. At 0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 60 h, and 72 h, gas samples were collected using a 25 mL closed syringe (with stopper) and injected into a vacuum bag. The N₂O concentration was measured by a greenhouse gas analyzer, and the N₂O-¹⁵N atom% was measured by a stable isotope mass spectrometer. Similarly, the seed liquid was inoculated into a medium with ¹⁵NH₄NO₃ as a nitrogen source. The content and ¹⁵N atom % of NO₃⁻, NO₂⁻, NH₄⁺ were analyzed on a CleverChem ONE spectrophotometer (Alliance company, France) by extraction with KCl solution - automated method with segmented flow analysis (Li et al., 2012; You et al., 2021). Total nitrogen was determined by an Element analyzer (ThermoFisher, Germany).

The assimilatory nitrate reductase gene (nasC) and nitrite reductase gene (nasD) were selected for functional validation because they encode the key enzymes in the dissimilatory nitrate reduction pathway, which is central to the proposed mechanism of nitrate removal by P. megaterium NCT-2 from saline soil. This selection was based on our laboratory’s prior genomic sequencing of the NCT-2 strain and preliminary pathway analysis.

The knockout vectors were constructed using an allele replacement strategy via homologous recombination. The upstream and downstream homology arms for nasC (300 bp for nasC-L and 297 bp for nasC-R) and for nasD (312 bp for nasD-L and 309 bp for nasD-R) were amplified from the genomic DNA of wild-type P. megaterium NCT-2. A chloramphenicol resistance gene (Cm^r) was amplified from plasmid pBR325. The fragments [Homology Arm L - Cm^r - Homology Arm R] for both nasC and nasD were assembled by overlap PCR, resulting in the nasCL-Cm-nasCR and nasDL-Cm-nasDR cassettes, respectively. These cassettes were then cloned into the BamHI/BglII site of the E. coli-P. megaterium shuttle vector pHIS1525. The resulting plasmids, designated pHIS1525-nasC and pHIS1525-nasD, were verified by sequencing in E. coli JM109. The primer sequences and PCR conditions are listed in Supplementary Table S1.

The recombinant plasmids pHIS1525-nasC and pHIS1525-nasD were independently transformed into wild-type P. megaterium NCT-2 protoplasts (Biedendieck et al., 2011). Transformants were initially selected on LB agar plates containing chloramphenicol.

To ensure the selection of true double-crossover mutants and to cure the replicative pHIS1525 plasmid, a critical screening step was implemented. Positive transformants were subcultured for more than 10 generations in antibiotic-free LB medium to allow for plasmid loss. These cultures were then plated onto LB agar without antibiotics. Individual colonies were replica-plated onto LB agar with and without chloramphenicol. Colonies that grew on the non-selective medium but failed to grow on the chloramphenicol-containing medium (i.e., chloramphenicol-sensitive) were selected as potential double-crossover mutants.

Genomic DNA from these potential mutants was subjected to a rigorous PCR-based verification. Using primers that anneal to regions flanking the upstream and downstream homology arms, a PCR product of the expected size (nasCL-Cm-nasCR: 1257 bp; nasDL-Cm-nasDR: 1281 bp) was obtained. The identity of these PCR products was conclusively confirmed by Sanger sequencing, verifying the precise replacement of the wild-type allele with the knockout cassette. To definitively rule out single-crossover events (plasmid integration) or the persistence of the free plasmid, PCR was performed using primers specific to the pHIS1525 vector backbone sequence outside the cloned region. Clones that yielded a negative result in this PCR were considered clean, unambiguous chromosomal knockouts. The two verified mutant strains were designated P. megaterium NCT-2-ΔnasC and NCT-2-ΔnasD.

Complementation strains were constructed to verify that the observed phenotypes were due to the specific gene knockouts. The full-length nasC or nasD gene, including its native promoter, was amplified and fused to a kanamycin resistance gene (Km) via overlap PCR. The fusion fragments (nasC-Km and nasD-Km) were cloned into the P. megaterium expression vector pWH1520. The resulting plasmids, pWH1520-nasC-Km and pWH1520-nasD-Km, were transformed into the corresponding mutant strains to generate the complementation strains NCT-2-nasC-Km and NCT-2-nasD-Km.

The wild-type, knockout mutants, and complementation strains were inoculated into an inorganic salt medium with nitrate as the sole nitrogen source. Bacterial growth (OD₆₀₀) was monitored, and the concentrations of NO₃⁻, NO₂⁻, and NH₄⁺ in the supernatant were determined to assess the functional impact of gene knockout and complementation (Li et al., 2012; You et al., 2021a).

Total RNA of P. megaterium NCT-2 was extracted using TRIzol® reagent according to the manufacturer’s instructions (Invitrogen, USA), and genomic DNA was removed using DNase I (Takara, China). RNA quality was determined with an Agilent 2,100 Bioanalyzer, and RNA was quantified using an ND-2000 (NanoDrop Technologies). High-quality RNA samples (OD260/280 = 1.8 ~ 2.0, OD260/230 ≥ 2.0, RNA Integrity Number (RIN) ≥ 6.5, 23S:16S ≥ 1.0, total amount≥100 ng μL⁻¹, concentration≥2 μg) were used for library construction and Real-time PCR.

RNA libraries were constructed using the TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA). The rRNA was removed using the Ribo-Zero Magnetic kit (epicenter), and the mRNA was randomly fragmented into small fragments of about 200 bp. Double-stranded cDNA was synthesized by reverse transcription using mRNA template, random primers, and SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA). The second strand of cDNA was synthesized by dUTP instead of dTTP. Double-stranded cDNA was blunt-ended by adding End Repair Mix. Then the 5′ end was phosphorylated, an ‘A’ base is added to the 3′ end, and it is ligated into a Y-shaped sequencing adapter. The second strand of cDNA containing dUTP was eliminated with UNG enzyme, so that only the first strand of cDNA was included in the library.

The enriched library was extracted by PCR amplification with Phusion DNA polymerase (NEB) for 15 cycles. Quantification was performed with TBS380 (Picogreen), and RNA-seq paired-end sequencing was performed using Illumina HiSeq X Ten (2 × 150 bp). Subsequently, the sequencing results were compared, annotated, and analyzed.

The data generated from the Illumina platform were used for bioinformatics analysis. All of the analyses were performed using the free online platform of Majorbio Cloud Platform¹ from Shanghai Majorbio Bio-pharm Technology Co., Ltd. The major software and parameters are as follows. High-quality reads in each sample were mapped to the reference genome of Priestia megaterium NCT-2 (assembly ASM33487v3, obtained from NCBI RefSeq) using Bowtie2.² Analysis tool: Bowtie2 (see footnote 2).

In this step, randomly selected 10,000 raw reads in each sample are aligned to the Rfam database⁴ with the blast method. Based on the annotation results, the percentage of rRNA in each sample is calculated, which is estimated as rRNA contamination. The rRNA contamination was less than 5% in all samples in Supplementary Table S9. Analysis tool: Blast.

Gene and isoform abundances were quantified using RSEM (v1.3.0) (Li and Dewey, 2011). RSEM employs an Expectation–Maximization (EM) algorithm to compute maximum likelihood abundance estimates, accounting for paired-end reads, fragment length distributions, and sequencing quality scores. Expression levels were reported in both FPKM (Fragments Per Kilobase of transcript per Million mapped reads) and TPM (Transcripts Per Million) units. These normalized metrics eliminate the confounding effects of gene length and sequencing depth variations, thereby enabling direct comparison of gene expression levels across different samples (Wagner et al., 2012). For downstream differential expression analysis, we utilized the TPM values due to their superior cross-sample comparability.

Raw read counts for each gene were obtained from the alignment files. Differential expression analysis was performed using the DESeq2.⁵ Genes with an adjusted p-value (Benjamini-Hochberg procedure) of less than 0.05 and an absolute fold change greater than 2 (|log2FoldChange| > 1) were considered statistically significant and differentially expressed.

The Gene Ontology⁶ project provides an ontology of defined terms representing gene properties, which covers three domains: Cellular Component, Molecular Function, and Biological Process. GO enrichment analysis will find gene ontology (GO) terms in which differentially expressed genes (DEGs) are enriched. It also helps to illustrate the difference between two particular samples on functional levels.

Goatools⁷ is used to identify statistically significantly enriched GO terms using Fisher’s exact test. The purpose of performing false discovery rate (FDR) Bonferroni correction is to reduce the Type-1 error by bonferroni, holm-bonferroni method (Holm), Benjamini-Yekutieli procedure (BY), Benjamini-Hochberg (BH) (multiple hypothesis test method). After multiple testing corrections, GO terms with adjusted p-value ≤ 0.05 are significantly enriched in DEGs.

Different expressed genes (DEGs) usually interact with each other in vivo to play roles in certain biological functions. Compared with the whole genome background, Kyoto Encyclopedia of Genes and Genomes enrichment analysis could identify the most important biological metabolic pathways and signal transduction pathways of DEGs.

KOBAS 2.0⁸ is used to identify statistically significantly enriched pathways using Fisher’s exact test. The purpose of performing FDR correction is to reduce the Type-1 error by bonferroni, Holm, BY, BH (multiple hypothesis test method). The calculating formula of the p-value and corrected p-value is similar to that in GO analysis. After multiple testing corrections, we chose pathways with a p-value ≤ 0.05, which are significantly enriched in DEGs.

The previous experimental results found that the most salt ions removed by NCT-2 inoculant were NO₃⁻, followed by Cl⁻ and Na⁺. Therefore, we further analyzed the tolerance of this strain to NO₃⁻, Cl⁻, and Na⁺ under pure culture conditions. The strain was inoculated into the medium (CK) with ammonium as a nitrogen source, the medium with nitrate as a nitrogen source (NCTa), and the medium with nitrate and salt stress (NCTb). The results demonstrated that the growth of P. megaterium NCT-2 was similar in ammonium (CK) and nitrate (NCTa) as nitrogen sources (Figure 2). These findings confirmed that the strain can transform and utilize nitrate. In addition, the strain growth was slower than NCTa from 0–48 h in salt stress (NCTb) (Figure 2). However, the final growth was the same for the three treatments (Figure 2). Therefore, the results of this analysis show that P. megaterium NCT-2 is capable of efficiently using nitrate in salt stress conditions.

Even though it has been demonstrated that P. megaterium NCT-2 can transform and utilize nitrate, the nitrate metabolic pathway of P. megaterium NCT-2 remains unknown. Therefore, this study explored the reduction dynamics of NO₃⁻ by P. megaterium NCT-2. The growth curve, nitrogenous compounds, and ¹⁵N atom% were determined in NO₃⁻ as a nitrogen source. These results showed that the logarithmic growth phase of the strain was comprised of 6–18 h (Figures 3a,b). After 18 h of culture, NO₃⁻ had been entirely metabolized, and approximately 0.23 mg L⁻¹ NO₂⁻ and 2.2 mg L⁻¹ NH₄⁺ had been accumulated in the medium, respectively. Subsequently, the NO₂⁻ and NH₄⁺ were gradually decreased and fully utilized (Figures 3b–d). The NO₃⁻ content of supernatant was 0 mg L⁻¹ at 18 ~ 72 h, indicating that no NO₃⁻ was generated. Furthermore, NO₂⁻-¹⁵N and NH₄⁺-¹⁵N atom% were identical with NO₃⁻-¹⁵N atom% of the marker during the culture period, which further proved that the production of NO₂⁻ and NH₄⁺ was derived from NO₃⁻. Therefore, the reduction process of NO₃⁻ was NO₃⁻—NO₂⁻—NH₄⁺.

The changes in cell dry weight and cell total nitrogen were similar to the growth curve. The cell dry weight and cell total nitrogen gradually increased in the logarithmic growth phase, and then stabilized in the stationary phase (Figures 3e,f). On the contrary, the total nitrogen content first gradually decreased and then stabilized in the supernatant (Figures 3e,f). These indicated that P. megaterium NCT-2 could transport NO₃⁻ into cells. Thus, P. megaterium NCT-2 primarily utilizes the aerobic assimilation pathway to metabolize nitrate.

When ¹⁵NH₄⁺ and NO₃⁻ are present in a medium, the N dilution occurs through DNRA or cell N mineralization. In order to investigate the DNRA process, P. megaterium NCT-2 (NCT-2) was cultured in medium containing ¹⁵NH₄NO₃ as a nitrogen source, and sterile medium (CK) serving as a control, in anaerobic conditions. The NH₄⁺ in the medium first gradually increased and then decreased (Figure 4a). Over the course of the culture period, the NO₃⁻ level decreased, whereas the metabolized NO₃⁻ quantity was only 16 mg L⁻¹ (Figure 4b). These results suggested that the strain consumed NO₃⁻ and produced NH₄⁺. The initial NH₄⁺-¹⁵N atom% in the medium was 9.98%, which was decreased to 9.69% after 12 h and 9.46% after 24 h (Figure 4c). This demonstrated that NH₄⁺-¹⁵N was continuously diluted by unlabeled NH₄⁺. NO₃⁻ was the only unlabeled nitrogen source, so the unlabeled NH₄⁺ can only come from NO₃⁻. These results indicated that the strain could carry out the DNRA pathway, even though its NO₃⁻ metabolism was significantly lower than that of the assimilation pathway.

Some microorganisms can use NO₃⁻ to produce N₂O and N₂ in anaerobic conditions. However, the finding showed that the N₂O content was consistent with that in the air, and ¹⁵N atom% did not change during the culture period (Figure 4d). Thus, proving that the NCT-2 strain could not produce N₂O and could not perform denitrification.

The sequence alignment of transcriptomics is presented in Supplementary Table S4. The DEGs of P. megaterium NCT-2 in nitrate and salt stress were further screened according to the standard of difference significance. The FC > 4 and FDR < 0.05 are significantly up-regulated genes. The FC < 0.25 and FDR < 0.05 are significantly down-regulated genes. The result showed that there were 1,315 DEGs in the CK vs. NCTa group, including 944 significantly up-regulated genes and 371 significantly down-regulated genes (p < 0.05) (Figure 6a). There were 767 DEGs in NCTa vs. NCTb group, of which 448 were significantly up-regulated and 319 were significantly down-regulated (p < 0.05) (Figure 6b).

In order to further understand the biological functions of these DEGs, GO and KEGG annotations were performed. Both groups were enriched to 26 GO secondary classification functions (Figure 7). Moreover, DEGs were mainly annotated in amino acid metabolism, carbohydrate metabolism, energy metabolism, metabolism of cofactors and vitamins, membrane transport, and signal transduction (Figure 8).

We explored the functions of DEGs in more depth on the basis of GO and KEGG annotations. The results showed that DEGs were mainly related to stress regulation functions, energy metabolism, and transport processes. Specifically, in the CK vs. NCTa group, the number of up-regulated DEGs in spore formation and germination was the largest (122) (p < 0.05) (Figure 9a). Fifty-three DEGs were involved in amino acid metabolism (p < 0.05) (Figure 9a). Forty-three up-regulated DEGs were attributed to the ABC transport (p < 0.05) (Figure 9a). There were 41 DEGs related to energy metabolism, including oxidative phosphorylation, glycolysis, pentose phosphate pathway, and tricarboxylic acid (TCA) cycle (p < 0.05) (Figure 9a). The key enzymes and rate-limiting enzymes of these pathways were significantly up-regulated, including cytochrome panthenol oxidase, cytochrome C oxidase, phosphofructokinase-1, phosphoenolpyruvate carboxykinase, 6-phosphoglucose dehydrogenase, and citrate synthase. There were 36 up-regulated DEGs related to stress and antioxidant function, including universal stress protein, catalase (CAT), superoxide dismutase (SOD), Hsp20/alpha crystallin family protein, and poly-γ-glutamate synthase genes (p < 0.05) (Figure 9a). There were 10 up-regulated DEGs related to flagellar assembly, including flagellar matrix rod protein, flagellar matrix M-loop protein, and flagellar hook matrix complex protein (p < 0.05) (Figure 9a). Six up-regulated DEGs were functional genes in vesicle formation, including GvpA, GvpL, and GvpF genes (p < 0.05) (Figure 9a). However, the functions of down-regulated DEGs were mainly attributed two-component system (54) (p < 0.05) (Figure 9b).

In the NCTa vs. NCTb group, the function of up-regulated DEGs was similar to CK vs. NCTa group. There were 73 DEGs involved in spore formation and germination (Figure 9c). There were 29 up-regulated DEGs associated with stress and antioxidant functions, including universal stress protein, CAT, SOD, TetR/AcrR family transcriptional regulator (TetRs), and SOS response-associated peptidase (p < 0.05) (Figure 9c). There are 17 DEGs belonging to amino acid metabolism (p < 0.05) (Figure 9c). There were 4 up-regulated DEGs belonging to flagellum assembly (p < 0.05) (Figure 9c). Four up-regulated DEGs were functional genes in vesicle formation (p < 0.05) (Figure 9c). Down-regulated DEGs were mainly involved in ABC transport (27) and oxidative phosphorylation (11) (p < 0.05) (Figure 9d). Partial resistance mechanisms of P. megaterium NCT-2 to nitrate and salt stress are depicted in Figure 9e. In addition, all genes and expression levels are detailed in Supplementary Tables S5–S8.

In the CK vs. NCTa group, the most up-regulated genes were mainly focused on spore formation and germination, including three YjcZ family sporulation proteins (982, 846, and 787 times, respectively), outer spore coat protein CotE (475 times), small acid-soluble spore protein SspI (407 times), alpha/beta-type small acid-soluble spore protein (384 times), and spore germination protein GerE (382 times) (Supplementary Table S5). In NCTa compared to NCTb, the largest up-regulations were observed in amino acid metabolism, including two carbamoyl phosphate synthases (21 and 17 times), pyrroline-5-carboxylate reductase (20 times), and carbamoyl phosphate synthase small subunit (18 times). There are also two flagellins (17 and 16 times) and a sporulation protein (17 times) (Supplementary Table S7).

The utilization of nitrogen sources is inseparable from the synergistic effect of cellular metabolism. It is well known that amino acid metabolism is the basic metabolism of microorganisms. Sugar catabolism and energy metabolism are the main ways for organisms to obtain energy. ATP transporter is a protein family with transport functions (Canback et al., 2002). The rate-limiting enzyme genes or key genes of these processes in P. megaterium were significantly up-regulated by NO₃⁻, which directly affects the metabolic capacity of cells (Sosa-Saavedra et al., 2001). P. megaterium NCT-2 accelerated the extracellular transport of nitrate and other nutrients for amino acid metabolism through the ATP transporter (Beis, 2015). Subsequently, substances (such as acetyl-CoA) were produced and entered into energy metabolism, including the TCA cycle, glycolysis, and oxidative phosphorylation (Kadenbach, 2018; Steffens et al., 2021). These processes produced large amounts of nucleotide and amino acid precursors for the expression of metabolic enzymes. Therefore, P. megaterium regulated metabolism and transport processes synergistically to promote transformation and absorption of nutrients in NO₃⁻ as a nitrogen source. This is consistent with previous studies, which found that bacteria maintained growth by promoting basal and energy metabolism in stress conditions (Qiao et al., 2019).

In addition, bacteria use two-component systems as a means of adapting to their environment (Nguyen and Hong, 2008). Two-component system-related genes of P. megaterium were significantly down-regulated, especially histidine kinase. Histidine kinase is the core protein in the two-component system, which has kinase, phosphotransferase, and phosphatase activities (Nguyen and Hong, 2008). This implied that the NO₃⁻ did not threaten the survival of P. megaterium, which could adapt to NO₃⁻.

The key enzyme genes of ATP transport and oxidative phosphorylation pathways were significantly down-regulated in salt stress, which could reduce bacterial utilization of nutrients. This was consistent with the results of bacterial growth, where salt stress was found to interfere with the conversion and utilization of NO₃⁻ by P. megaterium. However, four genes related to amino acid metabolism were found to have the largest up-regulation in salt stress. The importance of amino acid metabolism in adaptation to salt stress has been demonstrated in microorganisms and plants. Amino acids can be used as osmotic protectants to restore osmotic homeostasis in salt stress (Zhang et al., 2017). Therefore, these genes may play a key role in the response of P. megaterium to salt stress.

Salt stress has been reported to induce cell damage and oxidative damage in microorganisms. Microorganisms must control damage and repair themselves by regulating functional processes and activating interlocking defense functions (Bi et al., 2018). This study suggested that P. megaterium responded to salt stress through multiple strategies.

Spores are dormant bodies produced by bacteria in a certain environment and can remain viable for several years to decades. It is extremely resistant to high temperatures, ultraviolet light, and many toxic chemicals. The sporulation is catalyzed by a series of spore-forming proteins (Tan and Ramamurthi, 2014). The NO₃⁻ and salt stress significantly up-regulated the main genes of the sporulation (Kim et al., 2006). Moreover, YjcZ family sporulation protein, CotE, and small acid-soluble spore protein genes were the most up-regulated genes in NO₃⁻ as a nitrogen source. CotE and small acid-soluble spore protein genes are key genes for sporulation and protect DNA (Tan and Ramamurthi, 2014; Wetzel and Fischer, 2015). The function of the YjcZ family sporulation proteins is unclear. However, the results of this study suggested that they could have a key role in sporulation. Therefore, the spore formation may be a possible strategy of P. megaterium in response to salt stress. This could be caused by the nutrient deficiency of the strain and the stress of NO₂⁻ in NO₃⁻ as a nitrogen source. In most cases, spores can form in adverse conditions. However, some bacteria can only produce spores in rich nutrition and suitable conditions. For example, Bacillus thuringiensis must be cultured in adequate nutrition and suitable temperature to form spores in large numbers (Lv et al., 2019). Therefore, spores cannot be simply understood as a product of adverse conditions. However, regardless of growth conditions, bacteria are highly resistant to adverse conditions after forming spores.

Reactive oxygen species (ROS) are produced naturally during mitochondrial aerobic metabolism, which maintains a dynamic equilibrium in normal conditions (Yang and Lee, 2015b). However, ROS homeostasis is disrupted in salt stress (Yang and Lee, 2015a). Therefore, the first antioxidant mechanisms of P. megaterium NCT-2 will be activated, such as SOD, CAT, and universal stress protein (Kvint et al., 2003). Moreover, heat shock proteins can form the first line of defense against protein aggregation in stress responses (Carra et al., 2017). Poly-γ-glutamate synthase can help bacteria survive in salt stress and participate in detoxification (Candela and Fouet, 2006). This is consistent with previous studies, which found that microorganisms can resist salt stress by up-regulating stress proteins (Jiang et al., 2019). Therefore, the significant up-regulation of these genes may be the key strategy of P. megaterium in response to NO₃⁻ and salt stress. In addition, TetRs monitor cellular dynamics and regulate genes, including osmotic stress and metabolic regulation (Deng et al., 2013). The SOS response can stop DNA replication and cell division to protect bacteria (Janion, 2008). The up-regulation of TetRs and SOS response-associated peptidase genes highlighted the important role of TetRs and SOS response in P. megaterium response to salt stress.

Furthermore, key genes associated with both vesicle formation and flagellar assembly were significantly upregulated in P. megaterium NCT-2 under nitrate and salt stress. First, the formation of bacterial vesicles is considered an adaptive mechanism. Some studies suggest that vesicles can alter cell buoyancy, encouraging cells to float to the liquid surface, which might represent a microenvironment with lower salinity or more favorable nutrients (Winter et al., 2018; Tan et al., 2021). Therefore, the upregulation of vesicle-related genes in P. megaterium NCT-2 under salt stress may aid in relocating to a more suitable living space. More critically, the upregulation of flagellar assembly provides a direct motile advantage for coping with salt stress. The primary function of flagella is to drive bacterial motility, enabling cells to actively seek nutrients and escape harmful conditions via chemotaxis (Winter et al., 2018; Tan et al., 2021). Under high-salt stress, this motility is crucial. Flagella-driven locomotion allows bacteria to actively escape local microenvironments with critically high salt concentrations and migrate toward zones more conducive to growth. Furthermore, salt stress causes drastic osmotic changes, and the activation of the flagellar system is likely linked to the perception of such environmental stress signals. Research indicates that flagellar gene expression is often regulated by complex networks that integrate various environmental cues, including osmotic pressure (Lertsethtakarn et al., 2011). Thus, the upregulation of flagellar assembly genes represents not merely an enhancement of motility but is likely an active adaptive response initiated upon sensing salt stress, aimed at mitigating it by altering the bacterium’s spatial position.

Therefore, we conclude that by increasing vesicle and flagella formation, P. megaterium NCT-2 enhances its ability to spatially locate and migrate to more favorable environments and promote nutrient uptake, collectively alleviating the damage caused by salt stress.