Transcriptome Analysis Reveals the Important Role of Vitamin B12 in the Response of Natronorubrum daqingense to Salt Stress

Natronorubrum daqingense JX313T is an extremely halophilic archaea that can grow in a NaCl-saturated environment. The excellent salt tolerance of N. daqingense makes it a high-potential candidate for researching the salt stress mechanisms of halophilic microorganisms from Natronorubrum. In this study, transcriptome analysis revealed that three genes related to the biosynthesis of vitamin B12 were upregulated in response to salt stress. For the wild-type (WT) strain JX313T, the low-salt adaptive mutant LND5, and the vitamin B12 synthesis-deficient strain ΔcobC, the exogenous addition of 10 mg/L of vitamin B12 could maximize their cell survival and biomass in both optimal and salt stress environments. Knockout of cobC resulted in changes in the growth boundary of the strain, as well as a significant decrease in cell survival and biomass, and the inability to synthesize vitamin B12. According to the HPLC analysis, when the external NaCl concentration (w/v) increased from 17.5% (optimal) to 22.5% (5% salt stress), the intracellular accumulation of vitamin B12 in WT increased significantly from (11.54 ± 0.44) mg/L to (15.23 ± 0.20) mg/L. In summary, N. daqingense is capable of absorbing or synthesizing vitamin B12 in response to salt stress, suggesting that vitamin B12 serves as a specific compatible solute effector for N. daqingense during salt stress.


Introduction
Salt stress, characterized by high-salinity conditions, poses significant challenges to microbial physiology, affecting cellular processes such as protein function and membrane integrity [1,2].Microorganisms have evolved various tolerance mechanisms: (1) Halophilic microorganisms use Na + /H + exchangers (NHEs) [3] to achieve Na + efflux and accumulate H + or K + intracellularly to maintain osmotic balance.The secondary Na + /H + pumps, named Na + /H + antiporters, are the principal Na + efflux system of halophilic microorganisms and serve as one of the main adaptive response mechanisms to Na + stress [4].
(2) Microorganisms modify the cellular membrane permeability by adjusting the composition and ratio of membrane lipid constituents within cell membranes to enhance their adaptability to high-salinity environments [5,6].(3) Accumulation of compatible solutes is one of the main mechanisms by which halophilic microorganisms tolerate osmotic stress, which is mainly achieved by ingesting compatible solutes from a medium or synthesizing them intracellularly.These compatible solutes can maintain the positive turgor pressure environment required for cell division [7].
Most microorganisms have not evolved extensive genetic changes to adapt to highsalinity environments.As their cytoplasm is unable to tolerate salt, the accumulation of organic or inorganic compatible solutes has become a widely used strategy to adapt to osmotic changes, and most microorganisms rely exclusively on this strategy for osmotic adaption [8].Various organic compounds from different classes have been proven to act as osmotic regulators in different microbial communities, including betaine, polyols, ectoine, volcano plot and M versus A (MA) plot, as shown in Figure 1A,B, with gene information (partial) shown in Table 1.The Euclidean method was used to calculate the distance, and complete linkage was used to conduct the biclustering analysis.The 104 DEGs were clustered into nine clusters, as shown in Figure 1C.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 3 of 17 49 downregulated genes.The calculation of fold change was based on Nd_22.5/Nd_17.5,with the conditions for selecting DEGs being |log2 (Fold Change)| > 1 and p < 0.05.DEGs were plotted in a volcano plot and M versus A (MA) plot, as shown in Figure 1A,B, with gene information (partial) shown in Table 1.The Euclidean method was used to calculate the distance, and complete linkage was used to conduct the biclustering analysis.The 104 DEGs were clustered into nine clusters, as shown in Figure 1C.
Figure 1.Analysis of N. daiqngense differentially expressed genes (DEGs) under salt stress; the control group is the optimal environment, while the treatment group is the additional 5% salt stress environment.(A) Volcano plot of DEGs under salt stress; blue, red, and gray dots, respectively, represent downregulated genes, upregulated genes, and genes with non-significant differential expression.(B) M versus A plot of DEGs under salt stress; A and B, respectively represent the gene expression levels in the two samples, and the color correspondence of the dots is the same as described above.(C) Clustering of DEGs; red represents upregulated genes and green represents downregulated genes.The top 20 gene ontology (GO) terms with the smallest false discovery rates (FDRs) are shown in Figure 2A.Among the GO enrichment results of DEGs caused by salt stress, the highly enriched and more significant terms were binding, cellular protein modification process, the establishment of localization, heterocycle compound binding, localization, nucleoside phosphate binding, nucleoside binding, organic cyclic compound binding, small molecular binding, and transport.As shown in Figure 2B, among the 19 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched by the DEGs caused by salt stress, the highly enriched and significant terms were glycolysis/gluconeogenesis, glyocylate and dicarboxylate metabolism, porphyrin and chloroalkene degradation, propanoate metabolism, pyruvate metabolism, carbon fixation pathways in prokaryotes, quorum sensing, the ribosome pathway, etc.In this study, we focused on the changes in genes related to vitamin B 12 synthesis (as shown in   The top 20 gene ontology (GO) terms with the smallest false discovery rates (FDRs) are shown in Figure 2A.Among the GO enrichment results of DEGs caused by salt stress, the highly enriched and more significant terms were binding, cellular protein modification process, the establishment of localization, heterocycle compound binding, localization, nucleoside phosphate binding, nucleoside binding, organic cyclic compound binding, small molecular binding, and transport.As shown in Figure 2B, among the 19 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched by the DEGs caused by salt stress, the highly enriched and significant terms were glycolysis/gluconeogenesis, glyocylate and dicarboxylate metabolism, porphyrin and chloroalkene degradation, propanoate metabolism, pyruvate metabolism, carbon fixation pathways in prokaryotes, quorum sensing, the ribosome pathway, etc.In this study, we focused on the changes in genes related to vitamin B12 synthesis (as shown in Table 1), which may suggest the important role of vitamin B12 in N. daqingense resistance to salt stress.

Ultraviolet Mutagenesis of Low-Salt Adaptive Mutant LND5
As shown in Figure 3A, when the UV irradiation time was 120 s, the lethality rate of the strain was 69.72%.Generally, the mutagenesis effect is optimal when the lethality rate is around 70%, then the subsequent mutagenesis time is set to 120 s.The effect of NaCl concentration (w/v) on the obtained optimal low-salt adaptive mutant LND5 is shown in Figure 3B.LND5 could not grow normally in the medium lacking NaCl; when the NaCl concentration exceeded 10%, the growth of LND5 was significantly inhibited and almost completely halted at 15%.As shown in Figure 3B, compared with the wild-type (WT) strain JX313 T , LND5 could grow when the NaCl concentration was lower than 10% and exhibited optimal growth when the NaCl concentration was 2.5%, which was five times lower than that of the WT strain.The tolerance of LND5 to the pH of the culture environment ranged from 7.5 to 10.5, the tolerance of LND5 to the strong alkaline conditions was reduced compared with WT, and the optimal pH decreased from 10.0 to 9.0, as shown in Figure 3C.The growth curve of LND5 was measured in the medium containing 2.5% NaCl and pH 9.0, as shown in Figure 3D.LND5 entered the logarithmic phase on the third day and reached the stationary phase on the sixth day.The biomass of LND5 peaked on the eighth day and then entered the decline phase.Therefore, related indicators should be measured on the seventh day in subsequent experiments.Compared with the WT in the optimal environment, the biomass of LND5 during the logarithmic phase was higher in its optimal growth environment.When LND5 entered the decline phase, its biomass decreased more rapidly than that of the WT.No significant difference was found in the maximum biomass that can be achieved using both WT and LND5 during the growth process.

Ultraviolet Mutagenesis of Low-Salt Adaptive Mutant LND5
As shown in Figure 3A, when the UV irradiation time was 120 s, the lethality rate of the strain was 69.72%.Generally, the mutagenesis effect is optimal when the lethality rate is around 70%, then the subsequent mutagenesis time is set to 120 s.The effect of NaCl concentration (w/v) on the obtained optimal low-salt adaptive mutant LND5 is shown in Figure 3B.LND5 could not grow normally in the medium lacking NaCl; when the NaCl concentration exceeded 10%, the growth of LND5 was significantly inhibited and almost completely halted at 15%.As shown in Figure 3B, compared with the wild-type (WT) strain JX313 T , LND5 could grow when the NaCl concentration was lower than 10% and exhibited optimal growth when the NaCl concentration was 2.5%, which was five times lower than that of the WT strain.The tolerance of LND5 to the pH of the culture environment ranged from 7.5 to 10.5, the tolerance of LND5 to the strong alkaline conditions was reduced compared with WT, and the optimal pH decreased from 10.0 to 9.0, as shown in Figure 3C.The growth curve of LND5 was measured in the medium containing 2.5% NaCl and pH 9.0, as shown in Figure 3D.LND5 entered the logarithmic phase on the third day and reached the stationary phase on the sixth day.The biomass of LND5 peaked on the eighth day and then entered the decline phase.Therefore, related indicators should be measured on the seventh day in subsequent experiments.Compared with the WT in the optimal environment, the biomass of LND5 during the logarithmic phase was higher in its optimal growth environment.When LND5 entered the decline phase, its biomass decreased more rapidly than that of the WT.No significant difference was found in the maximum biomass that can be achieved using both WT and LND5 during the growth process.

Construction of ∆cobC and +cobC
As shown in Figure 4A,B, recombinant plasmids pUC-cobC-ko and pUC-cobC-c, containing linear DNA fragments for cobC knockout and complementation, were constructed by using homologous recombination, and corresponding high-purity linear fragments were prepared.To validate the knockout or complementation of cobC, primers VY-FP/VY-RP were designed for sequencing validation at 750 bp upstream and 740 bp downstream of the gene, respectively.The length of cobC is 717 bp, which was close to the erythromycin resistance sequence (735 bp).Therefore, the gDNA from the strain (Erm + ) obtained after protoplast transformation was PCR amplified with three pairs of primers, VY-FP/VY-RP, VY-FP/VE-RP, and VE-FP/VY-RP, as shown in Figure 4C, respectively, and sequencing data confirmed the knockout of cobC.As shown in Figure 4D, the gDNA from the strain (Hyg + ) obtained after protoplast transformation was PCR amplified with primers VY-FP/VY-RP, and combined with sequencing data, cobC was complimented.

Construction of ΔcobC and +cobC
As shown in Figure 4A,B, recombinant plasmids pUC-cobC-ko and pUC-cobC-c, containing linear DNA fragments for cobC knockout and complementation, were constructed by using homologous recombination, and corresponding high-purity linear fragments were prepared.To validate the knockout or complementation of cobC, primers VY-FP/VY-RP were designed for sequencing validation at 750 bp upstream and 740 bp downstream of the gene, respectively.The length of cobC is 717 bp, which was close to the erythromycin resistance sequence (735 bp).Therefore, the gDNA from the strain (Erm + ) obtained after protoplast transformation was PCR amplified with three pairs of primers, VY-FP/VY-RP, VY-FP/VE-RP, and VE-FP/VY-RP, as shown in Figure 4C, respectively, and sequencing data confirmed the knockout of cobC.As shown in Figure 4D, the gDNA from the strain (Hyg + ) obtained after protoplast transformation was PCR amplified with primers VY-FP/VY-RP, and combined with sequencing data, cobC was complimented.As shown in Figure 5A, both ΔcobC and WT were unable to grow when the NaCl concentration (w/v) was below 10%, and both reached the maximum biomass when the As shown in Figure 5A, both ∆cobC and WT were unable to grow when the NaCl concentration (w/v) was below 10%, and both reached the maximum biomass when the NaCl concentration was 17.5%.However, the biomass of ∆cobC reduced significantly compared with WT. ∆cobC was unable to grow when the NaCl concentration exceeded 30%, whereas the WT could still grow when the NaCl saturation was reached.As shown in Figure 5B, both ∆cobC and WT could tolerate a pH range of 8.0-11.0,and both reached the maximum biomass when the pH was 10.0.∆cobC entered the logarithmic phase on the second day and reached the stationary phase on the seventh day, as shown in Figure 5C.Therefore, the seventh day should be chosen for the measurement of related indicators in the subsequent experiments.The biomass of ∆cobC was mostly lower than that of WT during the logarithmic phase, and the biomass significantly decreased compared with WT after entering the stationary phase.Furthermore, the growth curves of the WT and +cobC were essentially consistent, with no significant differences.Combining the data from Figure 5A,B, the effects of NaCl concentration and pH on the biomass of +cobC were generally consistent with those of WT, indicating that the phenotypic changes in ∆cobC are caused by the knockout of cobC, rather than by non-specific factors.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 7 of 17 NaCl concentration was 17.5%.However, the biomass of ΔcobC reduced significantly compared with WT.ΔcobCwas unable to grow when the NaCl concentration exceeded 30%, whereas the WT could still grow when the NaCl saturation was reached.As shown in Figure 5B, both ΔcobC and WT could tolerate a pH range of 8.0-11.0,and both reached the maximum biomass when the pH was 10.0.ΔcobC entered the logarithmic phase on the second day and reached the stationary phase on the seventh day, as shown in Figure 5C.Therefore, the seventh day should be chosen for the measurement of related indicators in the subsequent experiments.The biomass of ΔcobC was mostly lower than that of WT during the logarithmic phase, and the biomass significantly decreased compared with WT after entering the stationary phase.Furthermore, the growth curves of the WT and +cobC were essentially consistent, with no significant differences.Combining the data from

Effects of Exogenous Vitamin B12 Addition
As shown in Figure 6A-C, the cell survival of WT, LND5, and ΔcobC under 5% salt stress was significantly reduced compared with optimal conditions.Overall, the exogenous addition of vitamin B12 at a lower concentration resulted in a pronounced positive effect on the cell survival of strains under salt stress, and even the cell survival was higher than that of the strains in the optimal environments at the same concentration.The most significant enhancement was observed when the addition of vitamin B12 reached 10 mg/L.When the concentration of exogenously added vitamin B12 exceeded 10 mg/L, the cell survival of strains was inhibited.As shown in Figure 6D-F, when the concentration of exogenously added vitamin B12 was low, the biomass of strains except LND5 in the optimal

Effects of Exogenous Vitamin B 12 Addition
As shown in Figure 6A-C, the cell survival of WT, LND5, and ∆cobC under 5% salt stress was significantly reduced compared with optimal conditions.Overall, the exogenous addition of vitamin B 12 at a lower concentration resulted in a pronounced positive effect on the cell survival of strains under salt stress, and even the cell survival was higher than that of the strains in the optimal environments at the same concentration.The most significant enhancement was observed when the addition of vitamin B 12 reached 10 mg/L.When the concentration of exogenously added vitamin B 12 exceeded 10 mg/L, the cell survival of strains was inhibited.As shown in Figure 6D-F, when the concentration of exogenously added vitamin B 12 was low, the biomass of strains except LND5 in the optimal environments was significantly promoted, while the enhancement effect was significant for strains under salt stress.When the exogenous addition of vitamin B 12 was 10 mg/L, the biomass of strains reached the maximum, and the biomass of WT under salt stress recovered to the optimal environment closely.When the concentration of exogenously added vitamin B 12 exceeded 10 mg/L, the biomass of the strains was inhibited.As shown in Figure 6, the exogenous addition of appropriate concentrations of vitamin B 12 helps to enhance the cell survival and biomass of N. daqingense under salt stress, indicating that N. daqingense can tolerate salt stress by taking up vitamin B 12 from the culture environment.Taken together, it is speculated that N. daqingense can absorb vitamin B 12 from the environment and accumulate it intracellularly to resist salt stress, and vitamin B 12 is likely to be a specific compatible solute effector in this process.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 8 of 17 environments was significantly promoted, while the enhancement effect was significant for strains under salt stress.When the exogenous addition of vitamin B12 was 10 mg/L, the biomass of strains reached the maximum, and the biomass of WT under salt stress recovered to the optimal environment closely.When the concentration of exogenously added vitamin B12 exceeded 10 mg/L, the biomass of the strains was inhibited.As shown in Figure 6, the exogenous addition of appropriate concentrations of vitamin B12 helps to enhance the cell survival and biomass of N. daqingense under salt stress, indicating that N. daqingense can tolerate salt stress by taking up vitamin B12 from the culture environment.
Taken together, it is speculated that N. daqingense can absorb vitamin B12 from the environment and accumulate it intracellularly to resist salt stress, and vitamin B12 is likely to be a specific compatible solute effector in this process.

Detection of Intracellular Vitamin B 12 Content
To investigate whether N. daqingense can synthesize and accumulate vitamin B 12 intracellularly to resist salt stress, this study measured the intracellular vitamin B 12 content of WT and ∆cobC in both optimal and salt stress environments using HPLC.The standard curve correlating the concentration of vitamin B 12 standards with the peak area is shown in Figure 7A, and the intracellular content of vitamin B 12 per liter of fermentation broth was calculated according to the fitting equation and the dilution factor from the "bacterial milking" described in Section 4.6.According to the HPLC results, vitamin B 12 was not detected in ∆cobC from either the optimal or salt stress environments, which indicated the knockout of cobC made N. daqingense unable to synthesize vitamin B 12 , and the results for WT are shown in Figure 7B.In the optimal environment, the intracellular vitamin B 12 content of WT of fermentation broth was (11.54 ± 0.44) mg/L; when an additional 5% salt stress was applied in the medium, the intracellular vitamin B 12 significantly increased to (15.23 ± 0.20) mg/L.Taken together, N. daqingense can synthesize and accumulate vitamin B 12 intracellularly to resist salt stress, and vitamin B 12 is likely to be a specific compatible solute effector in this process.

Detection of Intracellular Vitamin B12 Content
To investigate whether N. daqingense can synthesize and accumulate vitamin B12 intracellularly to resist salt stress, this study measured the intracellular vitamin B12 content of WT and ΔcobC in both optimal and salt stress environments using HPLC.The standard curve correlating the concentration of vitamin B12 standards with the peak area is shown in Figure 7A, and the intracellular content of vitamin B12 per liter of fermentation broth was calculated according to the fitting equation and the dilution factor from the "bacterial milking" described in Section 4.6.According to the HPLC results, vitamin B12 was not detected in ΔcobC from either the optimal or salt stress environments, which indicated the knockout of cobC made N. daqingense unable to synthesize vitamin B12, and the results for WT are shown in Figure 7B.In the optimal environment, the intracellular vitamin B12 content of WT of fermentation broth was (11.54 ± 0.44) mg/L; when an additional 5% salt stress was applied in the medium, the intracellular vitamin B12 significantly increased to (15.23 ± 0.20) mg/L.Taken together, N. daqingense can synthesize and accumulate vitamin B12 intracellularly to resist salt stress, and vitamin B12 is likely to be a specific compatible solute effector in this process.The effect of salt stress on intracellular vitamin B12 content; the significance analysis represents the comparison between the optimal environment and the salt stress environment.*** indicates that the analysis of significant difference between the two groups is p < 0.001.

Discussion
Compatible solutes can act as osmotic protectants, alleviating the inhibitory effects of high osmotic stress on microorganisms when added to a medium.These compatible solutes can provide osmotic protection via uptake from the medium or via de novo synthesis and intracellular accumulation [39].Glucosylglycerol is present at the reducing terminus of the polysaccharides in Bifidobacterium and exists in free form in a few mesophilic bacteria and thermophilic archaea.In recent years, glucosylglycerol has been identified in some microorganisms as an accumulable compatible solute to tolerate salt stress or nitrogen nutrient deficiency [40].In a study of compatible solutes of Spiribacter salinus, León et al. [41] found that the intracellular content of ectoine increased from a basal level of 80 μM to 170 μM under conditions ranging from 0.6 M NaCl to the optimal concentration of 0.8 M NaCl.Although further increasing the NaCl concentration to 1.3 M significantly reduced the biomass, there was no corresponding increase in the intracellular content of ectoine, which only increased when the NaCl concentration exceeded 1.6 M. Betaine can act as a compatible solute in most microorganisms, and its concentration varies with the

Discussion
Compatible solutes can act as osmotic protectants, alleviating the inhibitory effects of high osmotic stress on microorganisms when added to a medium.These compatible solutes can provide osmotic protection via uptake from the medium or via de novo synthesis and intracellular accumulation [39].Glucosylglycerol is present at the reducing terminus of the polysaccharides in Bifidobacterium and exists in free form in a few mesophilic bacteria and thermophilic archaea.In recent years, glucosylglycerol has been identified in some microorganisms as an accumulable compatible solute to tolerate salt stress or nitrogen nutrient deficiency [40].In a study of compatible solutes of Spiribacter salinus, León et al. [41] found that the intracellular content of ectoine increased from a basal level of 80 µM to 170 µM under conditions ranging from 0.6 M NaCl to the optimal concentration of 0.8 M NaCl.Although further increasing the NaCl concentration to 1.3 M significantly reduced the biomass, there was no corresponding increase in the intracellular content of ectoine, which only increased when the NaCl concentration exceeded 1.6 M. Betaine can act as a compatible solute in most microorganisms, and its concentration varies with the external NaCl concentration.Studies have shown that when the external NaCl concentrations were 0.51 M, 1.7 M, and 3.4 M, the intracellular concentrations of betaine were (0.21 ± 0.2) M, (0.65 ± 0.06) M, and (0.97 ± 0.09) M [42].Moreover, trehalose was shown to act as a compatible solute for Chromohalobacter israelensis when the external NaCl concentration was below 0.6 M [43].Trehalose is also one of the major compatible solutes of Desulfovibrio halophilus; when the medium contains 2.5 M NaCl without a betaine source, the strain can accumulate 8 µM/mg of protein and about 2.5 µM/mg of protein intracellularly [44].
In this study, transcriptome sequencing analysis revealed that three genes related to vitamin B 12 biosynthesis were upregulated by salt stress, as shown in Table 1.The gene cbiE, which encodes for cobalt-precorrin-7-[C(5)]-methyltransferase, catalyzes the conversion of cobalt-precorrin 7 to cobalt-precorrin 8.The gene cobH, which encodes for precorrin 8X methylmutase, catalyzes the conversion of precorrin 8X to hydrogenobyrinate.The gene cobC, which encodes for cobalamin biosynthesis protein, catalyzes the conversion of adenosyl-GDP-cobinamide to cobalamin [45,46].In current research on halophilic microorganisms, vitamin B 12 typically acts as a growth factor.Some vitamin B 12 -dependent microorganisms may have growth restriction or complete cessation in the absence of vitamin B 12 .However, the exogenous addition of vitamin B 12 (50 µg/L) to Methylophaga lonarensis, a methanogenic halophile isolated from a saline lake, can promote its growth at higher salinity, although this strain is not a vitamin B 12 -dependent strain [47].In this study, preliminary experiments showed that adding trace amounts of vitamin B 12 (measured in µg/L) to the medium had no significant effect on biomass.As shown in Figure 6, when the exogenous addition of vitamin B 12 was at 10 mg/L, WT, LND5, and ∆cobC all achieved the highest cell survival and biomass under the optimal and salt stress environments.Moreover, the intracellular vitamin B 12 concentration of WT increased, and the ∆cobC growth boundary, cell survival, and biomass changed under salt stress.These results indicated that N. daqingense did not use vitamin B 12 as a regulatory factor to participate in other metabolic pathways, but instead ingested vitamin B 12 from the medium as a compatible solute or an effector to maintain osmotic balance [8].
In this study, we detected that the intracellular vitamin B 12 accumulated by N. daqingense under salt stress can reach (15.23 ± 0.20) mg/L, which shows a certain development potential compared with other vitamin B 12 -producing strains [19].Future studies will focus on the role of vitamin B 12 in the response of closely related species to salt stress and analyze the molecular mechanism of vitamin B 12 acting as a compatible solute effector in the response of N. daqingense to salt stress.Studies of the relationship between the biosynthesis of vitamin B 12 and other salt-stress-related genes in N. daqingense, as well as the transcriptome sequencing of the low-salt adaptive mutant LND5, can further elucidate the mechanisms of N. daqingense in response to salt stress.Combined with the genetic modification and optimization of fermentation conditions, N. daiqngense can be utilized for the high production of vitamin B 12 .

Strains, Plasmids and Growth Conditions
The strains and plasmids used in the current study are detailed in Table 2. High-salt Luria Bertani (HLB) medium was used to culture N.daqingense JX313 T with an optimal NaCl concentration (17.5%, w/v) [34].When an additional 5% salt stress was applied, the NaCl concentration was adjusted to 22.5% (w/v) and the medium was named HLB+.The culture conditions were maintained at both 35 • C and pH 10.0.The culture conditions for N. daqingense LND5 differed from the aforementioned ones in that the NaCl concentration was 3% (w/v) and the pH was adjusted to 9.0.E. coli DH5α was cultured in LB medium (10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of NaCl) at 37 • C and pH 7.0.The antibiotic concentrations used for selection in this study were ampicillin 100 µg•mL −1 , hygromycin B 50 µg•mL −1 , and erythromycin 200 µg•mL −1 .

Transcriptome Sequencing and Analysis
The transcriptome sequencing of N. daqingense JX313 T was based on two different NaCl concentrations.The NaCl concentration of the control group was 17.5% (w/v), while that of the salt-stressed group was 22.5% (w/v); each group had three replicates.N. daqingense was cultured under the above conditions until it reached the logarithmic growth phase, respectively.Then, 1 L of culture was collected for the extraction of total RNA.After removing tRNA, RNA was fragmented and a specific cDNA library was synthesized.The library fragments were enriched via PCR amplification and sequenced using the Illumina platform.The filtered data were aligned to the reference genome and the expression levels of genes were calculated.Based on this, the samples were further subjected to differential expression analysis, enrichment analysis, and cluster analysis.

Ultraviolet Mutagenesis of Low-Salt Adaptive Mutants
To obtain low-salt adaptive mutants, the bacterial solution was exposed, respectively, to UV light for 50 s, 60 s, 70 s, 80 s, 90 s, 100 s, 110 s, and 120 s under aseptic conditions.Each bacterial solution was then coated on an LB solid medium containing 10% NaCl (WT cannot grow in environments with a NaCl concentration less than 10%), with inoculated plates not exposed to UV light serving as the control group.Each experiment was performed in triplicate.After wrapping the plates with aluminum foil, they were incubated upside down at 35 • C for 7 days.Based on the number of single colonies on the plates, the lethality rates at different UV exposure times were calculated to determine the lethality curve.The exposure time with a lethality rate of about 70% was chosen for mutagenesis treatment.Mutants capable of maintaining their characteristics stably after 30 generations were screened.Their 16S rDNA was amplified using the 22F/1540R primers to eliminate bacterial interference.The optimal NaCl concentration and pH were determined for each mutant.The strain with the lowest optimal NaCl concentration was selected, and its growth curve was measured under optimal conditions. of 17

Gene Knockout and Complementation
The gene knockout and complementation methods used in this study were improved according to the method of Wang et al. [48]; two common antibiotics were selected as selection markers.The gene knockout or complementation was achieved through spontaneous homologous recombination between the extremely halophilic archaea genome and prepared linear DNA fragments, traditionally known as the "gene replacement" method in archaeal gene manipulation [49].The linear DNA fragment used for gene knockout consists of the following (from 5 ′ to 3 ′ ): the upstream homologous arm sequence (500 bp), erythromycin resistance sequence (735 bp), and downstream homologous arm sequence (500 bp).The linear DNA fragment used for gene complementation consisted of the following (from 5 ′ to 3 ′ ): the upstream homologous arm sequence (500 bp), hygromycin resistance sequence (1026 bp), target gene, and downstream homologous arm sequence (500 bp).The upstream and downstream homologous arms and target gene sequences were amplified via PCR using the gDNA as a template, and the antibiotic resistance sequences were amplified via PCR using the corresponding plasmids as a template.After purification, the PCR products and the pUC18 clone vector (linearized with BamHI) were mixed gently (the addition amount of each component is 0.03 × length, ng; e.g., if the sequence length is 1000 bp, the required mass is 30 ng) and incubated at 50 • C for 30 min.After ligation, the samples were immediately cooled on ice for 3 min and then transformed into competent E. coli DH5α via the heat shock process.Through PCR identification, restriction enzyme digestion, and sequencing verification, the correct transformants with the correct connection sequence were selected.Recombinant plasmids were then extracted and used as templates for PCR amplification with the 5 ′ primer of the upstream homologous arm and the 3 ′ primer of the downstream homologous arm.Subsequently, the linear DNA fragments that can be used for gene knockout or complementation were acquired using gel extraction.The prepared linear DNA fragments were introduced into the recipient archaea via protoplast transformation.The obtained transformants were inoculated into 5 mL of HLB liquid medium and cultured at 35 • C and 180 rpm, for 7 days.The extracted gDNA of transformants were used as the template, and the pair of primers VY-FP/VY-RP was used to verify whether the knockout or complementation of the target gene was successful via PCR amplification.Then, the correct transformants were selected for sequencing verification.

Archaeal Protoplast Transformation
A total of 4.5 mL of archaeal broth (O.D. 600nm 0.8) was enriched in a 2 mL tube via centrifuging at 5600× g.Then, the broth was gently resuspended in 1 mL of protoplast formation buffer (Solution I) and centrifuged at 5600× g for 2 min.The supernatant was removed, and the process was repeated twice.Then, the cells were gently suspended in 150 µL of protoplast formation solution (Solution II), combined with 15 µL of 0.5 M EDTA solution (pH 8.0), and left at room temperature (RT) for 10 min.An amount of 2 µg of the purified linear homologous fragment was added, gently mixed, and then left at RT for 15 min.A total of 175 µL of 60% PEG600 solution (preheated at 37 • C) was added, gently mixed, and then left at RT for 30 min.An amount of 1 mL of protoplast dilution (Solution III) was added to the tube along the wall to rinse the cells without mixing, then centrifuged at 3500× g for 2 min after standing at RT for 5 min; the supernatant was removed, and this process was repeated once.The cells were gently resuspended using 1 mL of protoplast regeneration solution (Solution IV), left at 37 • C for 2 h, and then revived at 37 • C and 40 rpm, for 8-12 h.An amount of 1 mL of protoplast transformation diluent (Solution V) was added along the tube wall to rinse the cells, and then mixed gently and centrifuged at 3500× g for 2 min.The supernatant was removed and this process was repeated once.Finally, the cells were resuspended with 200 µL Solution V, coated on HLB solid medium containing the corresponding antibiotics, and cultured at 37 • C for 7-10 days.The solution formulation used for protoplast transformation is shown in Table 3. of 17 analysis was performed using the Pheatmap software (1.0.12) package of Rstudio 2022.Functional enrichment analysis of differentially expressed genes was conducted using the GO database http://geneontology.org/ (accessed on 1 July 2022) and the KEGG database https://www.kegg.jp/(accessed on 1 July 2022).

Conclusions
this study, transcriptome sequencing revealed the important role of vitamin B 12 in the response of N. daqingense to salt stress.The addition of 10 mg/L of exogenous vitamin B 12 significantly enhanced cell survival and biomass in N. daqingense under both optimal and salt-stressed conditions.The experimental validation showed that N. daqingense can resist salt stress by either the uptake of vitamin B 12 from the environment or synthesizing it internally, which suggests that vitamin B 12 acts as a specific compatible solute effector in the response of N. daqingense to salt stress.

Figure 1 .
Figure1.Analysis of N. daiqngense differentially expressed genes (DEGs) under salt stress; the control group is the optimal environment, while the treatment group is the additional 5% salt stress environment.(A) Volcano plot of DEGs under salt stress; blue, red, and gray dots, respectively, represent downregulated genes, upregulated genes, and genes with non-significant differential expression.(B) M versus A plot of DEGs under salt stress; A and B, respectively represent the gene expression levels in the two samples, and the color correspondence of the dots is the same as described above.(C) Clustering of DEGs; red represents upregulated genes and green represents downregulated genes.

Figure 2 .
Figure 2. Functional enrichment analysis of N. daiqngense DEGs (FDR stands for false discovery rate).(A) GO enrichment analysis bubble plot; (B) KEGG enrichment analysis bubble plot.

Figure 2 .
Figure 2. Functional enrichment analysis of N. daiqngense DEGs (FDR stands for false discovery rate).(A) GO enrichment analysis bubble plot; (B) KEGG enrichment analysis bubble plot.

Figure 3 .
Figure 3. Ultraviolet mutagenesis of N. daiqngense low-salt adaptive mutant LND5 and detection of physiological characteristics.(A) Ultraviolet mutagenesis lethality curve; (B) detection of the optimal NaCl concentration of LND5; (C) detection of the optimal pH of LND5; and (D) growth curves of WT and LND5 in their respective optimal environments.

Figure 3 .
Figure 3. Ultraviolet mutagenesis of N. daiqngense low-salt adaptive mutant LND5 and detection of physiological characteristics.(A) Ultraviolet mutagenesis lethality curve; (B) detection of the optimal NaCl concentration of LND5; (C) detection of the optimal pH of LND5; and (D) growth curves of WT and LND5 in their respective optimal environments.
Figure 5A,B, the effects of NaCl concentration and pH on the biomass of +cobC were generally consistent with those of WT, indicating that the phenotypic changes in ΔcobC are caused by the knockout of cobC, rather than by non-specific factors.

Figure 5 .
Figure 5. Detection of the physiological characteristics of N. daiqngense ΔcobC and +cobC.(A) Detection of the optimal NaCl concentration of ΔcobC and +cobC; (B) detection of the optimal pH of ΔcobC and +cobC; and (C) growth curve of WT, ΔcobC, and +cobC.

Figure 6 .
Figure 6.The effect of exogenous addition of vitamin B12.(A-C) The effect of exogenous addition of vitamin B12 on the cell survival of N. daiqngense JX313 T (WT), LND5, and ΔcobC.(D-F) The effect of the exogenous addition of vitamin B12 on the biomass of N. daiqngense JX313 T (WT), LND5, and ΔcobC; the significance analysis represents the comparison between the optimal environment and the salt stress environment.*** represents p < 0.001, ** represents p < 0.01, * represents p < 0.05, and no mark represents non-significant.

Figure 6 .
Figure 6.The effect of exogenous addition of vitamin B 12 .(A-C) The effect of exogenous addition of vitamin B 12 on the cell survival of N. daiqngense JX313 T (WT), LND5, and ∆cobC.(D-F) The effect of the exogenous addition of vitamin B 12 on the biomass of N. daiqngense JX313 T (WT), LND5, and ∆cobC; the significance analysis represents the comparison between the optimal environment and the salt stress environment.*** represents p < 0.001, ** represents p < 0.01, * represents p < 0.05, and no mark represents non-significant.

Figure 7 .
Figure 7. Detection of intracellular vitamin B12 content.(A) Vitamin B12 standard curve.(B)The effect of salt stress on intracellular vitamin B12 content; the significance analysis represents the comparison between the optimal environment and the salt stress environment.*** indicates that the analysis of significant difference between the two groups is p < 0.001.

Figure 7 .
Figure 7. Detection of intracellular vitamin B 12 content.(A) Vitamin B 12 standard curve.(B) The effect of salt stress on intracellular vitamin B 12 content; the significance analysis represents the comparison between the optimal environment and the salt stress environment.*** indicates that the analysis of significant difference between the two groups is p < 0.001.

Table
), which may suggest the important role of vitamin B 12 in N. daqingense resistance to salt stress.

Table 2 .
Strains and plasmids employed in the current study.

Table 4 .
Primers used in this study.