Genetic characteristics of novel extreme alkaline-inducible promoter located in five prime upstream region of peptidyl-prolyl cis/trans isomerase from Vibrio anguillarum

Genetic characteristics of novel extreme alkaline-inducible promoter located in five prime upstream region of peptidyl-prolyl cis/trans isomerase from Vibrio anguillarum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Genetic characteristics of novel extreme alkaline-inducible promoter located in five prime upstream region of peptidyl-prolyl cis/trans isomerase from Vibrio anguillarum Dong-Gyun Kim, Dong Nyoung Oh, Eun ji Lee, So Young Park, Jong Min Lee This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4441654/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Vibrio has attracted attention as a promising genetic chassis in the field of synthetic biology. FK506 binding protein (FKBP)-type peptidyl-prolyl cis/trans isomerase (PPIase) is involved in protein folding. In this study, we report, for the first time, the promoter regions in Vibrio that drive increased FKBP expression in the extremely alkaline environment. Proteomic analysis of V. anguillarum NB10 showed that VaFKBP was significantly upregulated under extreme alkaline stress (pH 10) condition. Additionally, the putative core promoter-containing regions and a reporter gene coding a β-galactosidase were introduced into the Escherichia coli system, which showed β-galactosidase activity of 61.47 ± 2.91 and 95.83 ± 6.76 Miller unit (MU) at pH 9 and 10, respectively, after 4 h of stress. This outcome was 1.97- and 2.88-fold higher that that observed under normal conditions of 25°C and pH 7 (31.27 ± 1.15 MU). To the best of our knowledge, this is the first report of a promoter showing increased expression under extremely alkaline conditions. We believe that this is a useful chassis in promoter engineering and can be used as a powerful tool for activating transcriptionally silent biosynthetic gene clusters in specific environments. Biological sciences/Genetics Biological sciences/Microbiology Biological sciences/Molecular biology alkaline-inducible promoter Vibrio anguillarum peptidyl-prolyl cis/trans isomerase synthetic biology biofoundry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The evolutionary history of marine organisms extends back to more than 3 billion years, and marine ecosystems comprise more than thrice the number of phyla than do terrestrial ecosystems [ 1 , 2 ]. Additionally, the marine environment accommodates enormous biodiversity from biological ecological, physical, and chemical perspectives owing to the specificity of environments [ 3 ]. Over a long-term evolutionary process, biological interactions have shaped innumerable organisms to develop unique mechanisms amidst the fierce competition for survival 4 . However, details regarding the kinds of organisms that live in marine environments beyond a few kilometers from the surface remain unknown; hence, determining the genetic diversity in these environments is difficult [ 5 , 6 ]. Unique genetic resources resulting from the biodiversity of terrestrial organisms have been well established; however, despite the great variety of organisms that are adapted to specific conditions in marine environments, reports of a wide range of unique genetic resources are relatively rare. This can be attributed to the fact that the marine microbial community, which has the most extensive biodiversity and unique metabolic, functional, and structural characteristics, has not been sufficiently studied. Although more than 1.2 million species have been taxonomically classified thus far, approximately 91% of the currently existing marine species remain unknown [ 7 ]. Over the past few decades, researchers have shown renewed interest in identifying marine microbes and harnessing these useful resources either for biodiversity research or for their economic significance [ 8 , 9 ]. These efforts have resulted in the discovery of several marine chassis microorganisms that contain useful genetic resources that do not exist in terrestrial ecosystems and are adapted to various and sometimes extreme environments [ 10 – 12 ]. Thus, a section of the biotechnology sector is focused on studying the marine environment and marine microorganisms as promising sources for new genetic resources. Vibrios are ubiquitous bacteria that mainly inhabit fresh-, brackish- and seawater. It belongs to the class Gamma-proteobacteria of the phylum Proteobacteria, which contains the most diverse types of Gram-negative bacteria [ 13 ]. As heterotrophic bacteria, they grow as a free-living organism through association, mutual symbiosis, and parasitism in various aquatic environments and aquatic organisms. They possess the ability to adapt and respond surprisingly quickly to environmental changes. As of 2024, approximately 208 species and 13 subspecies of Vibrios have been recognized, and they are rich sources of diverse genetic resources owing to the following characteristics: viable but non-culturable (VBNC) state and resuscitation to survive in adverse circumstances; fast-growing ability with generation time of < 10 min; swarmer cell phenomenon that enhances mobility and improves the viability of the community; quorum sensing, which is a special interaction system for mutual communication and cooperation; and luminescence capability such as expression of green fluorescent protein to control biological phenomena related to interaction with other organisms and quorum sensing [ 14 – 18 ]. To date, we have attempted to expand our understanding of vibrio survival under extreme environmental stress through genetic and proteomic analyses of Vibrio anguillarum [ 19 – 24 ]. This study continues the search for specific genetic resources from marine bacteria. We observed that under extremely alkaline environments certain proteins involved in protein folding and a specific promoter region showed increased expression. To the best of our knowledge, this is the first report of a promoter whose expression increases under extremely alkaline conditions. Results Protein expression under extreme alkaline stress Proteomic analysis of V. anguillarum NB10 (serotype O1) using two-dimensional (2D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) (2-DE) revealed that protein levels were significantly up-regulated under extreme alkaline stress. The comparative analysis based on MALDI-TOF MS/MS and NB10 genome (accession number: PRJEB5701) analyses identified five upregulated proteins, namely, peptidyl-prolyl cis/trans isomerase (VaFKBP; Protein ID; CDQ51236.1), general secretion pathway protein D (CDQ49073.1), cysteine synthase A (CDQ49756.1), deoxyribose-phosphate aldolase (CDQ51041.1), and transcriptional regulator OmpR (CDQ49100.1) (Table 1 ). Particularly, protein spot density-based expression analysis showed that the peptidyl-prolyl cis/trans isomerase (PPIase; VaFKBP) showed a 768.1 ± 43.2% higher up-regulated expression rate under extreme alkaline stress condition of 25°C and pH 10 than under normal condition of 25°C and pH 7 (Fig. 1 ). Table 1 Profiles of up-regulated proteins in V . anguillarum NB10 against pH 10 stress condition. Protein name Locus (Chr. 1) Protein ID MW (kDa) Molecular function Score MASCOT Peptidyl-prolyl cis–trans isomerase 2,695,469_ 2,696,089 CDQ51236.1 29 Peptidyl-prolyl cis–trans isomerase 164 General secretion pathway protein D 185,777_ 187,810 CDQ49073.1 73 Outer membrane secretin component of the type II secretion system involved in transporting exoproteins from the periplasm 141 Cysteine synthase A 990,531_ 991,499 CDQ49756.1 34 Catalyzes the last reaction of L-cysteine synthesis in bacteria 115 Deoxyribose- phosphate aldolase 2,464,018_ 2,464,794 CDQ51041.1 28 The conversion of exogenous deoxyribonucleosides for energy generation 194 Transcriptional regulator OmpR 213,902_ 214,621 CDQ49100.1 28 Member of the two-component regulatory system envZ/ompR involved in the regulation of osmoregulation 76 6-phospho- β-glucosidase – – 21 Hydrolase; 6-phospho- beta-glucosidase 58 Glucose-1- phosphate adenylyl-transferase – – 45 Glucose-1-phosphate adenylyl transferase; transferase; nucleotidyl transferase 68 Fructose-1- phosphate kinase – – 35 1-phosphofructokinase; phosphotransferase 46 Thr dehydratase – – 30 L-threonine ammonia-lyase 62 Hypothetical – – 44 – 65 In silico characterization of alkaline-inducible VaFKBP Figure 2 shows the location, three-dimensional structure, and ligand-binding site of VaFKBP and its adjacent genes on the NB10 chromosome I (chr. 1). According to genome analysis, NB10 is a multi-chromosome bacterium harboring major chromosomes (3.12 Mbp), extra-chromosomes (chr. 2; 1.12 Mbp), and plasmid (67 Kbp). The VaFKBP gene that is up-regulated under extreme alkaline stress was located on chr 1 (gene location; 2,695,469-2,696,089) (Fig. 2 a). VaFKBP encodes a type of FK506 binding protein (FKBP) and is present as a single gene rather than as an operon. vafkbp is located adjacent to oapA (opacity associated protein A), elaA (putative acetyltransferase), and dbpA (putative ATP-dependent RNA helicase) in the 5'-upstream direction and rplQ (50S ribosomal protein L17), rpoA (RNA polymerase alpha-subunit), and rpsD (30S ribosomal protein S4) genes in the 3'-downstream direction. VaFKBP comprises conserved amino acids, namely, Tyr 127 , Gly 129 , Phe 137 , Asp 138 , Val 154 , Ile 155 , Trp 158 , and Tyr 181 in the C-terminal cavity as part of a FK506 binding pocket, which is a 23-membered macrolide lactone that is known to exert immunosuppressive activity. The putative structure comprises four α-helices and five β-strands in the form of a β-sheet with a 1–4–5–2–3 topology wrapped around helix α4 (Fig. 2 b). Molecular docking analysis showed that FK506 was bound to the C-terminal binding pocket, suggesting that VaFKBP is a type of FKBP (Fig. 2 c). Additionally, it is a homodimer coupled with a hydrophobic N-terminus, suggesting the possibility of a periplasm-localized protein in NB10 (Fig. 2 d). Analysis of putative promoter regions expressed under alkaline conditions Nucleotide sequence homology at the 30–40, 54–59, 129–133, 170–177, 185–200, and 220–231 bp regions was found to be significantly consistent between the sequence upstream of vafkbp and the upstream region of the corresponding FKBP-type PPIase in species related to V. anguillarum NB10 (Fig. 3 a). Therefore, these regions were arbitrarily designated as the − 35 region or Pribnow box of the promoter sequence. Then, five putative promoters (P1–P5) were selected such that each sigma factor-binding region was included in an arbitrary promoter sequence (Fig. 3 b). Based on this, each partial sequence with the 5' prime end deleted was identified and named as VApro/del1, VApro/del2, and VApro/del3, respectively, to identify the core promoter region expressed under alkaline stress. Table 2 lists the sequences of each putative promoter region. Table 2 The sequence of each putative promoter region. Promoter Locus in Putative promoter region (5'→3') Chr. I (bp) -35 ~ 16 ~ 18bp -10 P 1 2,695,242–2,695,271 GTCTCC TTTTCTGTCGAGCTACAG TATAAA P 2 2,695,366–2,695,395 CAGTATA AAAACTCAAGAGGGATC AGTATA P 3 2,695,322–2,695,351 GAACAT TTTTCTTTGGGATCTGCG ATTTGT P 4 2,695,350–2,695,379 GTCACA ACCTGAAGGTTCAATATT GGATAT P 5 2,695,392–2,695,419 TTTCAT CCCATGCTTGAAACTG ATATCC P 6 2,695,408–2,695,437 AAACTG ATATCCTTTGGCACTTGT TTATTT Construction of VaFKBP promoter and lacZ fusion recombinant vector Each transformed E. coli harboring the recombinant plasmids was identified: E. coli VApro/wild, E. coli VApro/del1, E. coli VApro/del2, and E. coli VApro/del3 (Fig. 4 a). The four recombinant plasmids were constructed in combination with a 3.1 kb-fragment of lacZ derived from pcDNA™3.1/His/lacZ and the VaKFBP promoter region designed to remove the 5' prime in stages (VApro/wild, VApro/del1, VApro/del2, and VApro/del3). The 3.1-kbp lacZ and 5.5 Kbp pET-28(a)-VA promoter fragments were identified after double digestion of the pVApro vectors with XhoI and BamHI (Fig. 4 b). Additionally, the VA promoters, which underwent step-by-step deletion, were cross-checked using polymerase chain reaction (PCR) with the primers used for each cloning, and they were confirmed to have suitably sized PCR products: 235 bp, 142 bp, 120 bp and 75 bp (Fig. 4 c). Finally, each recombinant plasmid was sequenced to confirm all gene insertions. Expression of the VaFKBP promoter in the E. coli system The promoter that was upregulated at high pH in V. anguillarum NB10 was also effectively implemented in the E. coli system (Fig. 5 ). Significant promoter expression was confirmed in the recombinant E. coli pVApro/wild-type after 4 h under pH 9 and 10 stress. Furthermore, promoter expression increased over time under all tested pH conditions with the highest expression rate observed at 4 h (Fig. 5 a). β-galactosidase activity was 61.47 ± 2.91 and 95.83 ± 6.76 MU, respectively, at pH 9 and 10 after 4 h of stress, which was 1.97- and 2.88-fold higher than that observed at normal conditions of 25°C and pH 7 (31.27 ± 1.15 MU). Additionally, the lacZ mRNA expression rate showed a pattern similar to that of the enzyme activity observed earlier in Miller unit. The mRNA expression level was 137.63 ± 7.50 after 4 h at pH 7, and the expression levels increased to 244.23 ± 14.28 and 438.86 ± 20.31 at pH 9 and 10, respectively (Fig. 5 b). In contrast, β-galactosidase activity and mRNA expression at pH 8 were 33.43 ± 2.85 and 146.48 ± 11.24, respectively, which were higher on average than that at pH 7; however, the difference was not significant. Analysis of the alkaline-inducible promoter region The selective expression of the stepwise-deleted promoter was measured at different pH and time points (Fig. 5 c). β-galactosidase activity pertaining to VApro/del1 and VApro/del2 was 75.12 ± 7.3 MU and 68.6 ± 9.2 MU, respectively, at pH 7 and after 4 h, which was 2.5- and 2.3 times higher compared with the 30 ± 2.8 MU detected for VApro/wild. Similarly, the activities were increased 1.5- and 1.6 times, to 153.4 ± 7.4 and 172.6 ± 12.3 MU, respectively, than the 102.1 ± 6.8 MU detected for VApro/wild at pH 10. The promoter strength of VApro/del2 was higher on average than that of VApro/del1 at pH 10; however, the expression levels of VApro/del1 and VApro/del2 did not differ significantly. VApro/del3 showed significantly lower promoter strength of 4.8 ± 1.2 and 6.8 ± 3.2 MU at pH 7 and pH 10, respectively. Meanwhile, Similar to the no significantly increase in VaFKBP expression observed during heat shock in V . anguillarum NB10, it also did not observe a significant increase in the expression of VApro/del2 during heat shock at 37°C and 50°C compared to 25°C in the E . coli system (Fig. 5 d). Discussion In this study, proteomic analysis was used to identify VaFKBP, which is expressed at high levels in strongly alkaline environments. Additionally, a potential alkaline-inducible promoter region was identified for the first time through genome and reporter gene expression analyses. The probability of evolving unique biological systems in response to geographically localized factors such as pH, temperature, salinity, dissolved oxygen, and nutrient requirements is high in marine microorganisms [ 25 ]. Particularly, fish-related pathogenic microorganisms have developed specific defense systems to survive against various environmental changes on penetrating the host bodies. One of these mechanisms is the expression of chaperone genes to ensure the stability of protein function under alkaline conditions such as those created by the production of ammonia and amines during fish decomposition. VaFKBP is a crucial protein refolding-associated protein. Based on the earlier hypothesis, the promoter that increases VaFKBP expression in an alkaline environment is most certainly a part of an evolved system for the survival of Vibrio in response to changes in the host. Several studies have reported that Fkbp type PPIases in marine-related Gram-negative bacteria are involved in protein refolding as molecular chaperones under various environmental stresses. For example, SIB1 FKBP22 from Shewanella sp. binds to a folding intermediate protein SIB1; PaFkbA from Pseudomonas aeruginosa is a periplasmic chaperone for protein folding; and VaFKBP22 and VaFKBP17 from V. anguillarum act as chaperones and co-chaperones to prevent thermal aggregation [ 19 , 20 , 22 , 26 – 28 ]. These previous studies also support the implication that the alkaline-inducible promoter for VaFKBP expression assists protein refolding under the particular stress environment of V. anguillarum NB10. Notably, the promoter form V. anguillarum was activated under extremely alkaline conditions in E. coli as well. However, the exact mechanism underlying the activation of the foreign Vibrio promoter in E. coli remains unknown. Although many bacteria are vulnerable to harsh pH fluctuations, their defense mechanisms against pH stress remain unclear. Moreover, acid resistance mechanisms are well known but the alkaline resistance mechanisms remain largely uncharacterized. Nevertheless, several mechanisms that enable E . coli to sense external pH fluctuations and convert this information into internal signals for regulating the transcription of specific genes have been identified. The periplasmic protein YceI (multidrug exporter), outer membrane porin proteins OmpC (outer membrane porin C) and OmpA (outer membrane protein A), and membrane-bound redox regulator DsbA (periplasmic dithiol oxidoreductase) have been induced in E. coli to respond to osmotic pressure under extremely high alkaline condition [ 29 – 31 ]. As part of a strategy to produce organic acids to maintain cytosolic pH homeostasis, alkaline pH was used to stimulate the amino acid metabolic enzyme tryptophanase (TnaA) to produce NH 3 and acids [ 32 ]. Moreover, AstD (succinylglutamic acid semialdehyde dehydrogenase), GadA and GadB (glutamate decarboxylases), and GabT (γ-aminobutyric acid transaminase), which participate in the arginine and glutamate catabolic pathways, were also expressed at high levels under alkaline stress [ 30 , 32 , 33 ]. In the case of V. cholera , membrane-embedded transcriptional regulators and their respective partner proteins ToxRS and TcpPH activate toxT (cytoplasmic protein) expression, which encodes the master regulator for the transcription of the downstream genes tcp (toxin co-regulated pilus) and ctx (cholera toxin) [ 34 ]. Furthermore, ToxRS coordinates the inverse regulation of the outer membrane porins OmpU and OmpT to build resistance to alkali stress. Additionally, ompR-induction at alkaline pH results in the transcriptional silencing of acid tolerance response (ATR) and virulence genes. Moreover, OmpR contributes to fitness at alkaline pH and activates the expression of chiP, which is a chitin-specific porin [ 35 ]. Furthermore, another survival strategy is to maintain the cytoplasmic pH at a much lower level than that of the highly alkaline external environment through the regulation of multiple resistance and pH adaptation (Mrp) multi-subunit cation/proton antiporters [ 36 , 37 ]. In summary, the two Gram-negative bacteria use two representative strategies to respond to alkaline stress: maintaining pH homeostasis by producing organic acids and metabolites in the cytoplasm and controlling osmotic pressure by regulating cell membrane proteins. Furthermore, the alkaline response strategies of Gram-negative bacteria such as E. coli and Vibrio appear to overlap with those against salt and extracellular envelope stress due to increased sodium cytotoxicity at high pH and sensitivity of certain cell wall synthesis enzymes at fluctuating pH. This suggests that the intermediate signaling mechanisms for transmitting signals to the promoter in recognition of alkaline conditions may be compatible between Gram-negative bacteria. State-of-the-art genetic engineering technologies include various tools and methods for manipulating gene expression. However, these genetic tools must be expanded into practical tools that can be used to directly control the expression of genes that are selected to design optimized metabolic pathways based on metabolic engineering, synthetic biology, and biofoundry. Among the various tools, promoter engineering has emerged as a powerful tool for redesigning the expression of gene clusters found in bacterial genomes. Generally, intracellular metabolic fluxes are regulated by a series of distinct but intertwined regulatory controls that occur at the transcriptional, translational, and protein levels. One of fundamental means to alter this metabolic flux is to control transcript production at the promoter level. Most of these methods rely on the regulation of transcription initiation using various promoters [ 38 ]. Therefore, designing metabolic pathways has long relied on effective promoter discovery and characterization. Furthermore, the field of promoter engineering attempts to modulate promoter transcriptional ability by mutating the promoter DNA sequence, and the identification and characterization of existing promoters represents a way forward [ 39 ]. Thus, the alkaline-inducible promoter discovered in this study may be effectively applied for selectively expressing the necessary genes in specific environments. Moreover, this promoter is highly accessible for direct use in synthetic biology owing to its compatibility with V. anguillarum and E. coli . We believe that these results for optimizing and validating the alkaline-inducible promoter region could be used as a basis or tool for generating synthetic elements with desirable functions. In conclusion, we leveraged the power of proteomics and genome mining as biophysical tools to discover a logical and regulatory alkaline-inducible promoter that controls bacterial decision making. To the best of our knowledge, this is the first study to report a strong alkaline-inducible promoter. The characterization of novel promoters can help generate the dynamic range required to fine-tune gene expression for metabolic engineering applications. Therefore, this result is significant as it presents another promising and powerful gene regulation tool to control synthetic elements with desirable functions in synthetic biology applications. Methods Bacterial strains, culture conditions, and plasmids V. anguillarum strain NB10 (serotype O1) was standard cultured aerobically under conditions of 125 rpm and 25 ℃ in brain heart infusion (BHI) media (Becton Dickinson and Co., Sparks, MD, USA). Two E. coli strains DH5α and BL21 (DE3) for gene cloning and protein expression, respectively, were standard cultured under conditions of 150 rpm and 37°C in Luria-Bertani (LB) broth. All bacterial strains were stocked at -80°C in 25% glycerol and 7% dimethyl sulfoxide (DMSO) until used later. The pET28a(+) vector (Novagen, Madison, WI, USA) containing the T7 promoter and kanamycin resistance gene was used for cloning and expression. The pcDNA™3.1/His/lacZ vector (Invitrogen, Life Technologies, Carlsbad, CA, USA) was used as the template for LacZ gene. The bacterial strains and plasmids used in this study are listed in Table S1 . Differential expression proteome analysis based on environmental stress Stress condition and of total protein preparation V. anguillarum NB10 was cultured in BHI broth at 25°C and pH 7 until the OD600 of 0.6 as 5×10 8 colony forming units per milliliter (CFU mL − 1 ) was reached. The cells were harvested through centrifugation at 12,000 rpm for 5 min and resuspended in normal (25°C and pH 7) or pH stress-conditioned (25°C and pH 10) media. V. anguillarum NB10 was harvested through centrifugation and vortexed thoroughly for 16 h in a lysis solution containing 7 M urea, 4% 3-(3-cholamidopropyl dimethylammonium) propane sulfonate (CHAPS), 2 M thiourea, 100 mM dithiothreitol (DTT), and 2% Pharmalyte. After centrifugation, the supernatants was recovered and treated using a 2-D Clean-up Kit (GE Healthcare, Piscataway, NJ, USA). The purified total proteins were resuspended in the lysis solution. Protein concentrations were determined using a modified Bradford assay; bovine serum albumin was used as the standard. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) One-dimensional isoelectric focusing (IEF) and two-dimensional (2D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) (2-DE) were performed according to the manufacturer’s protocol (GE Healthcare, Piscataway, NJ, USA). The purified protein samples (200 µg) containing a 0.5% immobilized pH gradient (IPG) buffer (pH 4–7) and 5% bromophenol blue were applied to an immobilized IPG Dry Strip (immobilized pH 4–7, 13 cm; GE Healthcare) and rehydrated for 16 h in lysis solution. The focused IPG strips were equilibrated for 20 min in a solution containing 50 mM Tris–HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, and 1% DTT. Then, the DTT in the solution was replaced with 0.5% iodoacetamide, and the process was continued for an additional 20 min. The equilibrated strips were applied to a 12% polyacrylamide gel (14 × 13 cm, 1.5 mm thick). Silver staining was performed to visualize the proteins, and mass spectrometry was performed according to the manufacturer's protocol (Thermo Fisher Scientific, Cleveland, OH, USA). Protein identification For identifying protein, the proteins were identified by after drying the gel, followed by dehydrating in acetonitrile and overnight digestion at 37°C with sequencing-grade trypsin (Promega, Madison, WI, USA). The resulting tryptic peptides were dissolved in 50% acetonitrile containing 0.5% trifluoroacetic acid and desalted using a ZipTip C18 pipette tip (Millipore, Billerica, MA, USA). The peptides were directly eluted onto matrix-assisted laser desorption ionization (MALDI) plates using a-cyano-4-hydroxy-cinnamic acid (CHCA) matrix solution (10 mg mL − 1 CHCA in 0.5% TFA: 50% acetonitrile, 1:1). MALDI-time of flight/mass spectrometry (MALDI-TOF/MS) and MALDI-TOF/MS/MS were performed in reflection mode using a 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA). The proteins were identified by searching the National Center for Biotechnology Information (NCBI) database using the MASCOT program ( http://www.matrixscience.com/ ; RRID:SCR_014322). In silico method for identifying protein and promoter region Genome analysis The NB10 genome was analyzed using the complete genome sequence deposited at NCBI (accession number: PRJEB5701) to identify FK506 binding protein (FKBP) and its promoter region based on the results of the amino acid sequence confirmed using MALDI-TOF/MS. Genome analysis was performed according to a method reported previously with slight modifications [ 40 ]. Briefly, the NB10 genome annotations were performed using Proksee ( https://proksee.ca/ ), Rapid Annotation using Subsystem Technology 2.0 (RAST; https://rast.nmpdr.org/ ), and Bacterial and Viral Bioinformatics Resource Center (BV-BRC; http://www.bv-brc.org/ ) services. A comparative annotation analysis of all results was performed to complete the commentary. The circular map was constructed using the BV-BRC circular viewer ( https://www.bv-brc.org/view/Genome/ ). Three-dimensional homology-modeling and ligand docking Molecular modeling and docking process were performed according to the procedure described in the reported previously with slight modifications [ 41 ]. Briefly, for 3D structural modeling of FKBP, homologous proteins were screened using the AlphaFold v2 protein database ( https://alphafold.ebi.ac.uk/ ) using an AI system that predicts protein 3D structure based on its amino acid sequence (PBD ID; A0A649YL14.1.A, unknown gene, unknown organism). Secondary screening was performed on the protein templates using the NCBI ( https://www.ncbi.nlm.nih.gov/structure/ ) and RCSB ( https://www.rcsb.org/ ) servers. The candidate templates were applied to a protein structure homology modeling server (Swiss-Model; https://swissmodel.expasy.org/ ) to select templates for the structures that were determined using X-ray crystallography. The final predicted tertiary structures were constructed based on the selected template (PDB ID; 7dek.2.A, Pseudomonas aeruginosa FK506-binding protein PaFkbA). The 3D conformers of tacrolimus (PubChem CID: 445643; C 44 H 69 NO 12 ; FK-506) that act as FKBP ligands were visualized using the PubChem Chemical Molecules Database ( https://pubchem.ncbi.nlm.nih.gov/ ). The intermolecular binding sites and affinities of the proteins and ligands were predicted and evaluated using the CB-Dock2 server ( https://cadd.labshare.cn/cb-dock2/ ). Visualization and further analysis of the molecular complexes for parameters such as density maps, trajectories, and structure matching were performed using UCSF ChimeraX ( https://www.cgl.ucsf.edu/chimerax/ ; RRID:SCR_015872) program. Sequence alignment of the promoter region The protein corresponding to FKBP of NB10 was traced in other Vibrio species ( V. parahaemolyticus ATCC 17802, accession number CP014046.2; V. harveyi ATCC 33843, CP009467.2; V. alginolyticus ATCC 17749, CP006718.1; and V. fluvialis 10M-VF, CP118599.1). The sequences of the FKBP promoter region of each Vibrio were secured through genome comparative analysis. The nucleotide sequence identity matrix and amino acid sequence homology of the fatty acid synthesis-related proteins were calculated using the BioEdit 7.2 program ( https://bioedit.software.informer.com/ ; RRID:SCR_007361). Constructions of recombinant vector pVApro (pET-28a(+)/FKBP promoter/lacZ) was constructed by inserting the FKBP promoter and lacZ gene into pET-28a(+) as the backbone vector. The 3.1 Kbp lacZ gene was amplified using PCR (TaKaRa, Kyoto, Japan) with pcDNA™3.1/His/lacZ as the template and complementary primer sets containing BamHI and XhoI restriction enzyme sites for gene cloning (New England Biolabs (NEB), Cambridge, MA, USA). The PCR conditions were 25 amplification cycles of 97°C for 30 s, 58°C for 60 s, and 72°C for 30 s. The amplified lacZ and pET-28a(+) were digested using BamHI and XhoI and inserted into the multiple cloning site (MCS) of pET-28a(+) using T4 DNA ligase (Takara, Kyoto, Japan). Then, pET-28a(+)/lacZ was transformed into E. coli DH5α using heat shock at 42°C for 60 s to confirm the recombinant plasmid. Next, the stepwise deleted FABP promoter regions were amplified using NB10 chromosomal DNA as the template and complementary primer sets containing the BglII and BamHI restriction sites (PCR condition: 25 amplification cycles of 97°C for 30 s, 58°C for 30 s, and 72°C for 30 s). The PCR products and the purified pET-28a(+)/lacZ were digested using BglII and BamHI restriction enzymes, treated with ligase, and transformed again into E. coli DH5α to complete the pVApro/wild – del3. The insertion of each target gene was confirmed via DNA sequencing with the Applied Bio-systems 3730XL using the BigDye(R) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) [ 42 ]. All the primer sets used in this study are listed in Table S2. Measurements of promoter strength β-galactosidase assay The β-galactosidase assay was performed according to the Miller method with minor modifications to quantify the activation level of the promoter in terms of number of Miller units of enzyme activity [ 43 ]. Briefly, 3 mL of overnight cultured recombinant E. coli DH5α harboring pVApro/wild ( E. coli pVApro/wild) was inoculated into LB-Kanamycin (50 µg/mL) broth (300 mL). The cells were cultured for 6 h at 37°C until the late log phase was 2×10 9 CFU/mL. The cells were collected through centrifugation at 6,000 rpm for 10 min and immediately suspended in 50 mL of fresh LB medium, which was adjusted to pH 5–10 with 6N HCl and NaOH. The samples for promoter strength measurement were collected after 1, 2, and 4 h of incubation at 25°C. To measure promoter strength in response to temperature stress, the samples were incubated at 37 and 50°C and pH 7, followed by centrifugation at 12,000 rpm for 10 min to remove the supernatant completely. The collected cells were resuspended in 50 mL of 50 mM Tris–HCl (pH 7.0 ± 0.2) buffer and disrupted using a sonicator (Sonics & Materials, Inc., Newtown, CT, USA) at 4°C (3 s pulses at 150 W for 30 min with 2 s gap between pulses). After centrifugation, the supernatant was discarded, and the cell debris and inclusion bodies were used as crude enzyme for determining β-galactosidase activity. Each sample was assayed for β-galactosidase assay with o-nitrophenyl-β-D-galactoside (ONPG) as the substrate as described with minor modifications [ 44 ]. The assay mixture (100 µL) containing 5 mM ONPG and crude enzyme solution were incubated for 10 min at 40°C. The reaction was stopped by adding one volume of 1 M Na 2 CO 3 . The optical density of the reactants was read using a Microplate Reader (KLAB, Daejeon, Republic of Korea). The Miller formula was used to calculate the Miller units of enzyme per minute per milliliter of the sample. Each sample was processed in triplicate, and the average values were used to calculate the Miller units of the enzyme in each sample. 1 Miller Unit = 1000 * \(\frac{({Abs}_{420}-\left(1.75\text{*} {Abs}_{550}\right))}{\left(t\text{*}v\text{*} {Abs}_{600}\right)}\) where, Abs 420 indicates the absorbance of yellow o-nitrophenol; Abs 550 indicates the scatter from cell debris; t indicates the reaction time in minutes; v indicates the volume of culture assayed in milliliters; and Abs 600 indicates cell density. Total RNA extraction and cDNA synthesis Cultured bacteria were centrifuged at 12,000 rpm for 10 min, and the pellet was re-suspended in 1 ml TRIzol reagent (Invitrogen Lige Technologies, Burlington, Canada) to isolate the total RNA. Subsequent preparation and washing were performed using the Hybrid-RTM RNA Isolation Kit (GeneAll Biotechnology, LTD, Seoul, Korea). DNA was hydrolyzed using RQ DNase I (Promega, Madison, WI, USA). Complementary DNA (cDNA) was synthesized using PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Otsu, Japan) under the following conditions: initial reaction with random hexamer at 30°C for 10 min, followed by extension at 42°C for 60 min. The RNA and cDNA were quantified using NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression analysis To validate the in vivo differential expression of genes at the transcriptional level, quantitative real-time PCR (qRT-PCR) was performed using a Thermal Cycler Disc™ Real Time System Lite (model TP700/760, software version V5.0x) (Takara Bio Inc, Otsu, Japan) instrument and SYBR Premix Ex Taq™ (Tli RNaseH Plus, Takara, Kyoto, Japan) [ 45 ]. The two-step shuttle PCR protocol was optimized to include by 35 cycles of initial denaturation for 30 s at 95°C, followed denaturation at 95°C for 5 s and annealing and extension at 58°C for 15 s. The PCR mixture (25 µL) contained 12.5 µL of 2x SYBR Premix Ex Taq™, 0.5 µL of each primer (15 µM), 9.5 µl of sterile distilled water, and 2.0 µL of cDNA. V. anguillarum 16S rRNA was used as the housekeeping gene for internal control. The relative quantitative value was expressed in accordance with the 2 −△△Ct method [ 46 ]. Statistical analysis All data were subjected to one-way analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS), followed by Duncan's multiple range test. Statistical significance was set at p < 0.05, unless otherwise noted. Declarations CRediT authorship contribution statement D.G.K. : Conceptualization, Funding acquisition, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing. D.N.O. : Data curation, Methodology, Software, Project administration, Resources, Supervision, Writing – review & editing. E.J.L. : Data curation, Methodology, Software, Validation, Visualization, Writing – original draft. S.Y.P. : Project administration, Supervision, Writing – review & editing. J.M.L. : Conceptualization, Data curation, Funding acquisition, Project administration, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Declaration of Competing Interest The authors declare that they have no competing interest. Funding This research was supported by the National Institute of Fisheries Science, Ministry of Oceans and Fisheries, Korea (R2024053). Data Availability Statement The whole genome sequence data supporting the findings of this study have already been deposited in the National Center for Biotechnology Information with the accession code PRJEB5701. The datasets used and/or analyzed during the current study are available from the corresponding author, Jong Min Lee, upon reasonable request. References McCallum, H. I. et al. 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Marine microbiology: ecology & applications . (CRC Press, 2019). Budiman, C. et al. Engineering of monomeric FK506-binding protein 22 with peptidyl prolyl cis‐trans isomerase: Importance of a V‐shaped dimeric structure for binding to protein substrate. FEBS J. 276, 4091–4101 (2009). Budiman, C., Koga, Y., Takano, K. & Kanaya, S. FK506-binding protein 22 from a psychrophilic bacterium, a cold shock-inducible peptidyl prolyl isomerase with the ability to assist in protein folding. Int. J. Mol. Sci. 12, 5261–5284 (2011). Huang, Q. et al. Structural characterization of PaFkbA: A periplasmic chaperone from Pseudomonas aeruginosa. Comp. Struct. Biotechnol. J.. 19, 2460–2467 (2021). Padan, E., Bibi, E., Ito, M. & Krulwich, T. A. Alkaline pH homeostasis in bacteria: new insights. Biochim. biophys. Acta biomembr. 1717, 67–88 (2005). Saito, H. & Kobayashi, H. Bacterial responses to alkaline stress. Sci. Prog. 86, 271–282 (2003). Stancik, L. M. et al. pH-dependent expression of periplasmic proteins and amino acid catabolism in Escherichia coli . J. Bacteriol. 184, 4246–4258 (2002). Bordi, C., Théraulaz, L., Méjean, V. & Jourlin-Castelli, C. Anticipating an alkaline stress through the Tor phosphorelay system in Escherichia coli . Mol. Microbiol. 48, 211–223 (2003). Cabo, J. The Alkaline Stress Response in Escherichia coli : RpoS-Controlled Loci are Vital to High pH Resistance. https://digital.kenyon.edu/honorstheses/106 (2013). Pennetzdorfer, N. et al. Regulated proteolysis in Vibrio cholerae allowing rapid adaptation to stress conditions. Front. Cell. Infect. Microbiol. 9, 214; https://doi.org/10.3389/fcimb.2019.00214 (2019). Kunkle, D., Bina, X. & Bina, J. Vibrio cholerae OmpR contributes to virulence repression and fitness at alkaline pH. Infect. Immun. 88, 00141–00120 (2020). Dzioba-Winogrodzki, J., Winogrodzki, O., Krulwich, T. A., Boin, M. A. & Dibrov, P. The Vibrio cholerae Mrp system: cation/proton antiport properties and enhancement of bile salt resistance in a heterologous host. J. Mol. Microbiol. Biotechnol. 16, 176–186 (2009). Preiss, L., Hicks, D. B., Suzuki, S., Meier, T. & Krulwich, T. A. Alkaliphilic bacteria with impact on industrial applications, concepts of early life forms, and bioenergetics of ATP synthesis. Front. Bioeng. Biotechnol. 3, 75; https://doi.org/10.3389/fbioe.2015.00075 (2015). Luo, S., Wang, Z., Zhang, Z., Zhou, T. & Zhang, J. Genome-wide inference reveals that feedback regulations constrain promoter-dependent transcriptional burst kinetics. Nucleic Acids Res. 51, 68–83 (2023). Ji, C. H., Kim, J. P. & Kang, H. S. Library of synthetic Streptomyces regulatory sequences for use in promoter engineering of natural product biosynthetic gene clusters. ACS Synth. Biol. 7, 1946–1955 (2018). Jin, C. Z., Lee, J. M., Kim, C. J., Lee, H. G. & Shin, K. S. Genomic Insight into Shimazuella Soli Sp. Nov. Isolated from Soil and Its Putative Novel Class II Lasso Peptide. Bioengineering 9, 812; https://doi.org/10.3390/bioengineering9120812 (2022). Lee, J. M. et al . Improvement of thermostability and halostability of β-1,3 – 1,4-glucanase by substituting hydrophobic residue for Lys48. Int . J Biol . Macromol . 94, 594–602 (2017). Lee, J. M. et al. The groESL ISR sequence-based species-specific identification of GRAS and non-GRAS Lactiplantibacillus as an alternative to 16S rRNA sequencing. LWT 147, 111504 (2021). Miller, J. H. Assay of β-galactosidase. Experiments in molecular genetics (1972). Lee, J. M. et al. Characterization of salt-tolerant β-glucosidase with increased thermostability under high salinity conditions from Bacillus sp. SJ-10 isolated from jeotgal, a traditional Korean fermented seafood. Bioproc. Biosyst. Eng. 38, 1335–1346 (2015). Park, S. Y. et al. Anti-Obesity Potential through Regulation of Carbohydrate Uptake and Gene Expression in Intestinal Epithelial Cells by the Probiotic Lactiplantibacillus plantarum MGEL20154 from Fermented Food. J. Microbiol. Biotechnol. 33, 621 (2023). Kim, S. K. et al. Characterization of Latilactobacillus curvatus MS2 isolated from Korean traditional fermented seafood and cholesterol reduction effect as synbiotics with isomalto-oligosaccharide in BALB/c mice. Biochem. Biophys. Res. Commun. 571, 125–130 (2021). Additional Declarations No competing interests reported. Supplementary Files SciRepVApromoterSupplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4441654","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":308892381,"identity":"d12a53b4-6137-42d7-af4f-cfdd9f84f0c8","order_by":0,"name":"Dong-Gyun Kim","email":"","orcid":"","institution":"National Institute of Fisheries Science","correspondingAuthor":false,"prefix":"","firstName":"Dong-Gyun","middleName":"","lastName":"Kim","suffix":""},{"id":308892384,"identity":"e1c007b5-82d1-4341-853b-21faedf6e7ae","order_by":1,"name":"Dong Nyoung Oh","email":"","orcid":"","institution":"Pukyong National University","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"Nyoung","lastName":"Oh","suffix":""},{"id":308892387,"identity":"f29cf642-29ef-47d8-84b6-956ba9c4fea2","order_by":2,"name":"Eun ji Lee","email":"","orcid":"","institution":"Pukyong National University","correspondingAuthor":false,"prefix":"","firstName":"Eun","middleName":"ji","lastName":"Lee","suffix":""},{"id":308892389,"identity":"8cb5e2f1-0a28-472f-b3c4-3309564a3200","order_by":3,"name":"So Young Park","email":"","orcid":"","institution":"Pukyong National University","correspondingAuthor":false,"prefix":"","firstName":"So","middleName":"Young","lastName":"Park","suffix":""},{"id":308892390,"identity":"faeca422-9030-4aa7-80e7-2439edb642ff","order_by":4,"name":"Jong Min Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIie3OvQrCMBSG4S8UMgXnlEJ7Cy1CdfJaWgp20b2DQ0WIS3UUoTfhJm516RRxdfQSOokg/lV0NR0F8w7hcMhDAuh0PxhJjRRIqA323nA1IU8iW+3mpEYgwg7TxsRYbMfHSvrxmu1KjlEPZl4o3sjDibdM+sPNbN7nKCNYrUBJhMVkOVztmc9BC9jsu6jJ9HIV99h9kVszIgwiaODuMp8TUcBqQCZmJqm3yWS7G84jZmYK4uXRtjon1OmwgXeoTj2bSxVJ34NbHwGg+hbgfAZXeVWn0+n+tgeVrT7Uv3Wm6wAAAABJRU5ErkJggg==","orcid":"","institution":"Pukyong National University","correspondingAuthor":true,"prefix":"","firstName":"Jong","middleName":"Min","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2024-05-18 15:39:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4441654/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4441654/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57596464,"identity":"c7e4422f-6048-4590-8a82-683684955b1f","added_by":"auto","created_at":"2024-06-03 06:57:06","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":637815,"visible":true,"origin":"","legend":"\u003cp\u003eComparative proteomic analysis of NB10 cultured under normal and alkaline stress condition. Spots pertain to expression of several protein including VaFKBP (marked with a red circle) in response to different conditions; a) normal condition, 25 °C and pH 7; b) stress condition, 25 °C and pH 10\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4441654/v1/728d7220a1fbb0d7357b0690.jpeg"},{"id":57596467,"identity":"6b8fb348-76ba-4a2d-a204-c50277c5fdf5","added_by":"auto","created_at":"2024-06-03 06:57:06","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1601143,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic features and predicted protein structure of the \u003cem\u003eV. anguillarum\u003c/em\u003e NB10 and VaFKBP, respectively. a) Circular plot of the genomes of chromosome I and \u003cem\u003evafkbp\u003c/em\u003e locus in the chromosome. b) VaFKBP monomer topology presented as helices and strands. c) Predicted protein structure schematic of the surfaced form of the VaFKP monomer. d) Protein quaternary structure of the VaFKBP homodimer\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4441654/v1/fdc00e304c7bc4e1f8eb936e.jpeg"},{"id":57596903,"identity":"cd241145-72fe-48c0-ac07-1fc7bd689347","added_by":"auto","created_at":"2024-06-03 07:05:06","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1278127,"visible":true,"origin":"","legend":"\u003cp\u003e5' direction untranslated region and predicted promoter regions of \u003cem\u003evafkbp\u003c/em\u003e. a) Design of stepwise deleted recombinant promoter regions to identify the core promoter regions. b) Comparative sequence analysis of the untranslated region from \u003cem\u003eV. anguillarum\u003c/em\u003e NB10 in the 5' direction of \u003cem\u003evafkbp\u003c/em\u003e with the untranslated regions corresponding to \u003cem\u003evafkbp\u003c/em\u003efrom related species of the genus \u003cem\u003eVibrio\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4441654/v1/10a1d2b22daee9e3f37ca91c.jpeg"},{"id":57596466,"identity":"31b89120-ff83-4f60-a627-a833e51d2dee","added_by":"auto","created_at":"2024-06-03 06:57:06","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":940657,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of VaFKBP promoter and \u003cem\u003elacZ\u003c/em\u003efusion recombinant plasmids. a) Schematic of the construction process for pVApro recombinant plasmids. b and c) DNA fragments of the pVApro/wild digested from restriction enzymes and amplified DNA of VApro/wild-del3. Lane M, DNA ladder; Lane 1, 8.4-Kbp fragment of pVApro/wild digested with Xho1; Lane 2, 3.1-Kbp fragment of \u003cem\u003elacZ\u003c/em\u003e gene and 5.5-Kbp fragment of pET-28(a)-VApro/wild fragments; Lane 3, 235-bp fragment of VApro/wild DNA amplified with cloning primer using pVApro/wild as a template; and Lanes 4–6, VApro/del1-3 DNA: 142 bp, 120 bp and 75 bp, respectively, amplified with the each cloning primer set using pVApro/del1-3 as the template\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4441654/v1/1cfd85822f4d56b78bf00884.jpeg"},{"id":57597360,"identity":"e777b15b-236b-494a-b45b-eecd960a5599","added_by":"auto","created_at":"2024-06-03 07:13:06","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":466320,"visible":true,"origin":"","legend":"\u003cp\u003ePromoter strengths of VaFKBP promoters at different pH and temperature in the \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e system. a) Promoter strength of VApro/wild converted to β-galactosidase activity. b) Promoter strength of VApro/wild measured based on lacZ mRNA expression. c) Promoter strength of stepwise deleted promoters VApro/del1, VApro/del2, and VApro/del3 with VApro/wild at different pH and time durations, and b) the expression level of VApro/del2 after heat shock in an \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e system.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4441654/v1/535420c747f6216430c2544a.jpeg"},{"id":64160338,"identity":"834518c9-5338-418b-95ce-0dceef58d34f","added_by":"auto","created_at":"2024-09-09 07:22:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5737461,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4441654/v1/18e81f39-6ba6-4ca4-94da-b8a22f422c38.pdf"},{"id":57596901,"identity":"752b2e99-b5f5-450c-a094-dbc357ddcfc4","added_by":"auto","created_at":"2024-06-03 07:05:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28766,"visible":true,"origin":"","legend":"","description":"","filename":"SciRepVApromoterSupplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4441654/v1/6cbf783a92a1eb270429e281.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic characteristics of novel extreme alkaline-inducible promoter located in five prime upstream region of peptidyl-prolyl cis/trans isomerase from Vibrio anguillarum","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe evolutionary history of marine organisms extends back to more than 3\u0026nbsp;billion years, and marine ecosystems comprise more than thrice the number of phyla than do terrestrial ecosystems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Additionally, the marine environment accommodates enormous biodiversity from biological ecological, physical, and chemical perspectives owing to the specificity of environments [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Over a long-term evolutionary process, biological interactions have shaped innumerable organisms to develop unique mechanisms amidst the fierce competition for survival\u003csup\u003e4\u003c/sup\u003e. However, details regarding the kinds of organisms that live in marine environments beyond a few kilometers from the surface remain unknown; hence, determining the genetic diversity in these environments is difficult [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnique genetic resources resulting from the biodiversity of terrestrial organisms have been well established; however, despite the great variety of organisms that are adapted to specific conditions in marine environments, reports of a wide range of unique genetic resources are relatively rare. This can be attributed to the fact that the marine microbial community, which has the most extensive biodiversity and unique metabolic, functional, and structural characteristics, has not been sufficiently studied. Although more than 1.2\u0026nbsp;million species have been taxonomically classified thus far, approximately 91% of the currently existing marine species remain unknown [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Over the past few decades, researchers have shown renewed interest in identifying marine microbes and harnessing these useful resources either for biodiversity research or for their economic significance [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These efforts have resulted in the discovery of several marine chassis microorganisms that contain useful genetic resources that do not exist in terrestrial ecosystems and are adapted to various and sometimes extreme environments [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Thus, a section of the biotechnology sector is focused on studying the marine environment and marine microorganisms as promising sources for new genetic resources.\u003c/p\u003e \u003cp\u003e \u003cem\u003eVibrios\u003c/em\u003e are ubiquitous bacteria that mainly inhabit fresh-, brackish- and seawater. It belongs to the class Gamma-proteobacteria of the phylum Proteobacteria, which contains the most diverse types of Gram-negative bacteria [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. As heterotrophic bacteria, they grow as a free-living organism through association, mutual symbiosis, and parasitism in various aquatic environments and aquatic organisms. They possess the ability to adapt and respond surprisingly quickly to environmental changes. As of 2024, approximately 208 species and 13 subspecies of \u003cem\u003eVibrios\u003c/em\u003e have been recognized, and they are rich sources of diverse genetic resources owing to the following characteristics: viable but non-culturable (VBNC) state and resuscitation to survive in adverse circumstances; fast-growing ability with generation time of \u0026lt;\u0026thinsp;10 min; swarmer cell phenomenon that enhances mobility and improves the viability of the community; quorum sensing, which is a special interaction system for mutual communication and cooperation; and luminescence capability such as expression of green fluorescent protein to control biological phenomena related to interaction with other organisms and quorum sensing [\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo date, we have attempted to expand our understanding of \u003cem\u003evibrio\u003c/em\u003e survival under extreme environmental stress through genetic and proteomic analyses of \u003cem\u003eVibrio anguillarum\u003c/em\u003e [\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This study continues the search for specific genetic resources from marine bacteria. We observed that under extremely alkaline environments certain proteins involved in protein folding and a specific promoter region showed increased expression. To the best of our knowledge, this is the first report of a promoter whose expression increases under extremely alkaline conditions.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression under extreme alkaline stress\u003c/h2\u003e \u003cp\u003eProteomic analysis of \u003cem\u003eV. anguillarum\u003c/em\u003e NB10 (serotype O1) using two-dimensional (2D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS\u0026ndash;PAGE) (2-DE) revealed that protein levels were significantly up-regulated under extreme alkaline stress. The comparative analysis based on MALDI-TOF MS/MS and NB10 genome (accession number: PRJEB5701) analyses identified five upregulated proteins, namely, peptidyl-prolyl cis/trans isomerase (VaFKBP; Protein ID; CDQ51236.1), general secretion pathway protein D (CDQ49073.1), cysteine synthase A (CDQ49756.1), deoxyribose-phosphate aldolase (CDQ51041.1), and transcriptional regulator OmpR (CDQ49100.1) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Particularly, protein spot density-based expression analysis showed that the peptidyl-prolyl cis/trans isomerase (PPIase; VaFKBP) showed a 768.1\u0026thinsp;\u0026plusmn;\u0026thinsp;43.2% higher up-regulated expression rate under extreme alkaline stress condition of 25\u0026deg;C and pH 10 than under normal condition of 25\u0026deg;C and pH 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProfiles of up-regulated proteins in \u003cem\u003eV\u003c/em\u003e. \u003cem\u003eanguillarum\u003c/em\u003e NB10 against pH 10 stress condition.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocus\u003c/p\u003e \u003cp\u003e(Chr. 1)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW\u003c/p\u003e \u003cp\u003e(kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMolecular function\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eScore\u003c/p\u003e \u003cp\u003eMASCOT\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeptidyl-prolyl\u003c/p\u003e \u003cp\u003ecis\u0026ndash;trans isomerase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,695,469_\u003c/p\u003e \u003cp\u003e2,696,089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDQ51236.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePeptidyl-prolyl cis\u0026ndash;trans isomerase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e164\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGeneral secretion\u003c/p\u003e \u003cp\u003epathway protein D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e185,777_\u003c/p\u003e \u003cp\u003e187,810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDQ49073.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOuter membrane secretin component of the type II secretion system involved in transporting exoproteins from the periplasm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e141\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCysteine\u003c/p\u003e \u003cp\u003esynthase A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e990,531_\u003c/p\u003e \u003cp\u003e991,499\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDQ49756.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCatalyzes the last reaction of L-cysteine synthesis in bacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e115\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDeoxyribose-\u003c/p\u003e \u003cp\u003ephosphate aldolase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,464,018_\u003c/p\u003e \u003cp\u003e2,464,794\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDQ51041.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThe conversion of exogenous deoxyribonucleosides for energy generation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e194\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTranscriptional \u003c/p\u003e \u003cp\u003eregulator\u003c/p\u003e \u003cp\u003eOmpR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e213,902_\u003c/p\u003e \u003cp\u003e214,621\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDQ49100.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMember of the two-component regulatory system envZ/ompR involved in the regulation of osmoregulation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6-phospho-\u003c/p\u003e \u003cp\u003eβ-glucosidase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHydrolase; 6-phospho- beta-glucosidase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose-1-\u003c/p\u003e \u003cp\u003ephosphate\u003c/p\u003e \u003cp\u003eadenylyl-transferase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGlucose-1-phosphate adenylyl transferase; transferase; nucleotidyl transferase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFructose-1-\u003c/p\u003e \u003cp\u003ephosphate kinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1-phosphofructokinase; phosphotransferase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThr dehydratase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eL-threonine ammonia-lyase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypothetical\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn silico\u003c/b\u003e \u003cb\u003echaracterization of alkaline-inducible VaFKBP\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the location, three-dimensional structure, and ligand-binding site of VaFKBP and its adjacent genes on the NB10 chromosome I (chr. 1). According to genome analysis, NB10 is a multi-chromosome bacterium harboring major chromosomes (3.12 Mbp), extra-chromosomes (chr. 2; 1.12 Mbp), and plasmid (67 Kbp). The \u003cem\u003eVaFKBP\u003c/em\u003e gene that is up-regulated under extreme alkaline stress was located on chr 1 (gene location; 2,695,469-2,696,089) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). \u003cem\u003eVaFKBP\u003c/em\u003e encodes a type of FK506 binding protein (FKBP) and is present as a single gene rather than as an operon. \u003cem\u003evafkbp\u003c/em\u003e is located adjacent to \u003cem\u003eoapA\u003c/em\u003e (opacity associated protein A), \u003cem\u003eelaA\u003c/em\u003e (putative acetyltransferase), and \u003cem\u003edbpA\u003c/em\u003e (putative ATP-dependent RNA helicase) in the 5'-upstream direction and \u003cem\u003erplQ\u003c/em\u003e (50S ribosomal protein L17), \u003cem\u003erpoA\u003c/em\u003e (RNA polymerase alpha-subunit), and \u003cem\u003erpsD\u003c/em\u003e (30S ribosomal protein S4) genes in the 3'-downstream direction. VaFKBP comprises conserved amino acids, namely, Tyr\u003csup\u003e127\u003c/sup\u003e, Gly\u003csup\u003e129\u003c/sup\u003e, Phe\u003csup\u003e137\u003c/sup\u003e, Asp\u003csup\u003e138\u003c/sup\u003e, Val\u003csup\u003e154\u003c/sup\u003e, Ile\u003csup\u003e155\u003c/sup\u003e, Trp\u003csup\u003e158\u003c/sup\u003e, and Tyr\u003csup\u003e181\u003c/sup\u003e in the C-terminal cavity as part of a FK506 binding pocket, which is a 23-membered macrolide lactone that is known to exert immunosuppressive activity. The putative structure comprises four α-helices and five β-strands in the form of a β-sheet with a 1\u0026ndash;4\u0026ndash;5\u0026ndash;2\u0026ndash;3 topology wrapped around helix α4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Molecular docking analysis showed that FK506 was bound to the C-terminal binding pocket, suggesting that VaFKBP is a type of FKBP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Additionally, it is a homodimer coupled with a hydrophobic N-terminus, suggesting the possibility of a periplasm-localized protein in NB10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of putative promoter regions expressed under alkaline conditions\u003c/h2\u003e \u003cp\u003eNucleotide sequence homology at the 30\u0026ndash;40, 54\u0026ndash;59, 129\u0026ndash;133, 170\u0026ndash;177, 185\u0026ndash;200, and 220\u0026ndash;231 bp regions was found to be significantly consistent between the sequence upstream of \u003cem\u003evafkbp\u003c/em\u003e and the upstream region of the corresponding FKBP-type PPIase in species related to \u003cem\u003eV. anguillarum\u003c/em\u003e NB10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Therefore, these regions were arbitrarily designated as the \u0026minus;\u0026thinsp;35 region or Pribnow box of the promoter sequence. Then, five putative promoters (P1\u0026ndash;P5) were selected such that each sigma factor-binding region was included in an arbitrary promoter sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Based on this, each partial sequence with the 5' prime end deleted was identified and named as VApro/del1, VApro/del2, and VApro/del3, respectively, to identify the core promoter region expressed under alkaline stress. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e lists the sequences of each putative promoter region.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe sequence of each putative promoter region.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePromoter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocus in\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003ePutative promoter region (5'\u0026rarr;3')\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr. I (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e~\u0026thinsp;16\u0026thinsp;~\u0026thinsp;18bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,695,242\u0026ndash;2,695,271\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGTCTCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTTTTCTGTCGAGCTACAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTATAAA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,695,366\u0026ndash;2,695,395\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAGTATA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAAAACTCAAGAGGGATC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAGTATA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,695,322\u0026ndash;2,695,351\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAACAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTTTTCTTTGGGATCTGCG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eATTTGT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,695,350\u0026ndash;2,695,379\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGTCACA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eACCTGAAGGTTCAATATT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGGATAT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,695,392\u0026ndash;2,695,419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTTCAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCCCATGCTTGAAACTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eATATCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,695,408\u0026ndash;2,695,437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAACTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eATATCCTTTGGCACTTGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTTATTT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eConstruction of VaFKBP promoter and\u003c/b\u003e \u003cb\u003elacZ\u003c/b\u003e \u003cb\u003efusion recombinant vector\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEach transformed \u003cem\u003eE. coli\u003c/em\u003e harboring the recombinant plasmids was identified: \u003cem\u003eE. coli\u003c/em\u003e VApro/wild, \u003cem\u003eE. coli\u003c/em\u003e VApro/del1, \u003cem\u003eE. coli\u003c/em\u003e VApro/del2, and \u003cem\u003eE. coli\u003c/em\u003e VApro/del3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The four recombinant plasmids were constructed in combination with a 3.1 kb-fragment of \u003cem\u003elacZ\u003c/em\u003e derived from pcDNA\u0026trade;3.1/His/lacZ and the VaKFBP promoter region designed to remove the 5' prime in stages (VApro/wild, VApro/del1, VApro/del2, and VApro/del3). The 3.1-kbp \u003cem\u003elacZ\u003c/em\u003e and 5.5 Kbp pET-28(a)-VA promoter fragments were identified after double digestion of the pVApro vectors with XhoI and BamHI (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Additionally, the VA promoters, which underwent step-by-step deletion, were cross-checked using polymerase chain reaction (PCR) with the primers used for each cloning, and they were confirmed to have suitably sized PCR products: 235 bp, 142 bp, 120 bp and 75 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Finally, each recombinant plasmid was sequenced to confirm all gene insertions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of the VaFKBP promoter in the\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003esystem\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe promoter that was upregulated at high pH in \u003cem\u003eV. anguillarum\u003c/em\u003e NB10 was also effectively implemented in the \u003cem\u003eE. coli\u003c/em\u003e system (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Significant promoter expression was confirmed in the recombinant \u003cem\u003eE. coli\u003c/em\u003e pVApro/wild-type after 4 h under pH 9 and 10 stress. Furthermore, promoter expression increased over time under all tested pH conditions with the highest expression rate observed at 4 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). β-galactosidase activity was 61.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91 and 95.83\u0026thinsp;\u0026plusmn;\u0026thinsp;6.76 MU, respectively, at pH 9 and 10 after 4 h of stress, which was 1.97- and 2.88-fold higher than that observed at normal conditions of 25\u0026deg;C and pH 7 (31.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 MU). Additionally, the \u003cem\u003elacZ\u003c/em\u003e mRNA expression rate showed a pattern similar to that of the enzyme activity observed earlier in Miller unit. The mRNA expression level was 137.63\u0026thinsp;\u0026plusmn;\u0026thinsp;7.50 after 4 h at pH 7, and the expression levels increased to 244.23\u0026thinsp;\u0026plusmn;\u0026thinsp;14.28 and 438.86\u0026thinsp;\u0026plusmn;\u0026thinsp;20.31 at pH 9 and 10, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). In contrast, β-galactosidase activity and mRNA expression at pH 8 were 33.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.85 and 146.48\u0026thinsp;\u0026plusmn;\u0026thinsp;11.24, respectively, which were higher on average than that at pH 7; however, the difference was not significant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the alkaline-inducible promoter region\u003c/h2\u003e \u003cp\u003eThe selective expression of the stepwise-deleted promoter was measured at different pH and time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). β-galactosidase activity pertaining to VApro/del1 and VApro/del2 was 75.12\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3 MU and 68.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2 MU, respectively, at pH 7 and after 4 h, which was 2.5- and 2.3 times higher compared with the 30\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8 MU detected for VApro/wild. Similarly, the activities were increased 1.5- and 1.6 times, to 153.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4 and 172.6\u0026thinsp;\u0026plusmn;\u0026thinsp;12.3 MU, respectively, than the 102.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8 MU detected for VApro/wild at pH 10. The promoter strength of VApro/del2 was higher on average than that of VApro/del1 at pH 10; however, the expression levels of VApro/del1 and VApro/del2 did not differ significantly. VApro/del3 showed significantly lower promoter strength of 4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 and 6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 MU at pH 7 and pH 10, respectively. Meanwhile, Similar to the no significantly increase in VaFKBP expression observed during heat shock in \u003cem\u003eV\u003c/em\u003e. \u003cem\u003eanguillarum\u003c/em\u003e NB10, it also did not observe a significant increase in the expression of VApro/del2 during heat shock at 37\u0026deg;C and 50\u0026deg;C compared to 25\u0026deg;C in the \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e system (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, proteomic analysis was used to identify VaFKBP, which is expressed at high levels in strongly alkaline environments. Additionally, a potential alkaline-inducible promoter region was identified for the first time through genome and reporter gene expression analyses. The probability of evolving unique biological systems in response to geographically localized factors such as pH, temperature, salinity, dissolved oxygen, and nutrient requirements is high in marine microorganisms [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Particularly, fish-related pathogenic microorganisms have developed specific defense systems to survive against various environmental changes on penetrating the host bodies. One of these mechanisms is the expression of chaperone genes to ensure the stability of protein function under alkaline conditions such as those created by the production of ammonia and amines during fish decomposition. VaFKBP is a crucial protein refolding-associated protein. Based on the earlier hypothesis, the promoter that increases VaFKBP expression in an alkaline environment is most certainly a part of an evolved system for the survival of \u003cem\u003eVibrio\u003c/em\u003e in response to changes in the host. Several studies have reported that Fkbp type PPIases in marine-related Gram-negative bacteria are involved in protein refolding as molecular chaperones under various environmental stresses. For example, SIB1 FKBP22 from \u003cem\u003eShewanella\u003c/em\u003e sp. binds to a folding intermediate protein SIB1; PaFkbA from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e is a periplasmic chaperone for protein folding; and VaFKBP22 and VaFKBP17 from \u003cem\u003eV. anguillarum\u003c/em\u003e act as chaperones and co-chaperones to prevent thermal aggregation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These previous studies also support the implication that the alkaline-inducible promoter for VaFKBP expression assists protein refolding under the particular stress environment of \u003cem\u003eV. anguillarum\u003c/em\u003e NB10.\u003c/p\u003e \u003cp\u003eNotably, the promoter form \u003cem\u003eV. anguillarum\u003c/em\u003e was activated under extremely alkaline conditions in \u003cem\u003eE. coli\u003c/em\u003e as well. However, the exact mechanism underlying the activation of the foreign \u003cem\u003eVibrio\u003c/em\u003e promoter in \u003cem\u003eE. coli\u003c/em\u003e remains unknown. Although many bacteria are vulnerable to harsh pH fluctuations, their defense mechanisms against pH stress remain unclear. Moreover, acid resistance mechanisms are well known but the alkaline resistance mechanisms remain largely uncharacterized. Nevertheless, several mechanisms that enable \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e to sense external pH fluctuations and convert this information into internal signals for regulating the transcription of specific genes have been identified. The periplasmic protein YceI (multidrug exporter), outer membrane porin proteins OmpC (outer membrane porin C) and OmpA (outer membrane protein A), and membrane-bound redox regulator DsbA (periplasmic dithiol oxidoreductase) have been induced in \u003cem\u003eE. coli\u003c/em\u003e to respond to osmotic pressure under extremely high alkaline condition [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. As part of a strategy to produce organic acids to maintain cytosolic pH homeostasis, alkaline pH was used to stimulate the amino acid metabolic enzyme tryptophanase (TnaA) to produce NH\u003csub\u003e3\u003c/sub\u003e and acids [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Moreover, AstD (succinylglutamic acid semialdehyde dehydrogenase), GadA and GadB (glutamate decarboxylases), and GabT (γ-aminobutyric acid transaminase), which participate in the arginine and glutamate catabolic pathways, were also expressed at high levels under alkaline stress [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In the case of \u003cem\u003eV. cholera\u003c/em\u003e, membrane-embedded transcriptional regulators and their respective partner proteins ToxRS and TcpPH activate toxT (cytoplasmic protein) expression, which encodes the master regulator for the transcription of the downstream genes \u003cem\u003etcp\u003c/em\u003e (toxin co-regulated pilus) and \u003cem\u003ectx\u003c/em\u003e (cholera toxin) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, ToxRS coordinates the inverse regulation of the outer membrane porins OmpU and OmpT to build resistance to alkali stress. Additionally, ompR-induction at alkaline pH results in the transcriptional silencing of acid tolerance response (ATR) and virulence genes. Moreover, OmpR contributes to fitness at alkaline pH and activates the expression of chiP, which is a chitin-specific porin [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, another survival strategy is to maintain the cytoplasmic pH at a much lower level than that of the highly alkaline external environment through the regulation of multiple resistance and pH adaptation (Mrp) multi-subunit cation/proton antiporters [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In summary, the two Gram-negative bacteria use two representative strategies to respond to alkaline stress: maintaining pH homeostasis by producing organic acids and metabolites in the cytoplasm and controlling osmotic pressure by regulating cell membrane proteins. Furthermore, the alkaline response strategies of Gram-negative bacteria such as \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eVibrio\u003c/em\u003e appear to overlap with those against salt and extracellular envelope stress due to increased sodium cytotoxicity at high pH and sensitivity of certain cell wall synthesis enzymes at fluctuating pH. This suggests that the intermediate signaling mechanisms for transmitting signals to the promoter in recognition of alkaline conditions may be compatible between Gram-negative bacteria.\u003c/p\u003e \u003cp\u003eState-of-the-art genetic engineering technologies include various tools and methods for manipulating gene expression. However, these genetic tools must be expanded into practical tools that can be used to directly control the expression of genes that are selected to design optimized metabolic pathways based on metabolic engineering, synthetic biology, and biofoundry. Among the various tools, promoter engineering has emerged as a powerful tool for redesigning the expression of gene clusters found in bacterial genomes. Generally, intracellular metabolic fluxes are regulated by a series of distinct but intertwined regulatory controls that occur at the transcriptional, translational, and protein levels. One of fundamental means to alter this metabolic flux is to control transcript production at the promoter level. Most of these methods rely on the regulation of transcription initiation using various promoters [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, designing metabolic pathways has long relied on effective promoter discovery and characterization. Furthermore, the field of promoter engineering attempts to modulate promoter transcriptional ability by mutating the promoter DNA sequence, and the identification and characterization of existing promoters represents a way forward [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Thus, the alkaline-inducible promoter discovered in this study may be effectively applied for selectively expressing the necessary genes in specific environments. Moreover, this promoter is highly accessible for direct use in synthetic biology owing to its compatibility with \u003cem\u003eV. anguillarum\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. We believe that these results for optimizing and validating the alkaline-inducible promoter region could be used as a basis or tool for generating synthetic elements with desirable functions.\u003c/p\u003e \u003cp\u003eIn conclusion, we leveraged the power of proteomics and genome mining as biophysical tools to discover a logical and regulatory alkaline-inducible promoter that controls bacterial decision making. To the best of our knowledge, this is the first study to report a strong alkaline-inducible promoter. The characterization of novel promoters can help generate the dynamic range required to fine-tune gene expression for metabolic engineering applications. Therefore, this result is significant as it presents another promising and powerful gene regulation tool to control synthetic elements with desirable functions in synthetic biology applications.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains, culture conditions, and plasmids\u003c/h2\u003e \u003cp\u003e \u003cem\u003eV. anguillarum\u003c/em\u003e strain NB10 (serotype O1) was standard cultured aerobically under conditions of 125 rpm and 25 ℃ in brain heart infusion (BHI) media (Becton Dickinson and Co., Sparks, MD, USA). Two \u003cem\u003eE. coli\u003c/em\u003e strains DH5α and BL21 (DE3) for gene cloning and protein expression, respectively, were standard cultured under conditions of 150 rpm and 37\u0026deg;C in Luria-Bertani (LB) broth. All bacterial strains were stocked at -80\u0026deg;C in 25% glycerol and 7% dimethyl sulfoxide (DMSO) until used later. The pET28a(+) vector (Novagen, Madison, WI, USA) containing the T7 promoter and kanamycin resistance gene was used for cloning and expression. The pcDNA\u0026trade;3.1/His/lacZ vector (Invitrogen, Life Technologies, Carlsbad, CA, USA) was used as the template for \u003cem\u003eLacZ\u003c/em\u003e gene. The bacterial strains and plasmids used in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDifferential expression proteome analysis based on environmental stress\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eStress condition and of total protein preparation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eV. anguillarum\u003c/em\u003e NB10 was cultured in BHI broth at 25\u0026deg;C and pH 7 until the OD600 of 0.6 as 5\u0026times;10\u003csup\u003e8\u003c/sup\u003e colony forming units per milliliter (CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was reached. The cells were harvested through centrifugation at 12,000 rpm for 5 min and resuspended in normal (25\u0026deg;C and pH 7) or pH stress-conditioned (25\u0026deg;C and pH 10) media. \u003cem\u003eV. anguillarum\u003c/em\u003e NB10 was harvested through centrifugation and vortexed thoroughly for 16 h in a lysis solution containing 7 M urea, 4% 3-(3-cholamidopropyl dimethylammonium) propane sulfonate (CHAPS), 2 M thiourea, 100 mM dithiothreitol (DTT), and 2% Pharmalyte. After centrifugation, the supernatants was recovered and treated using a 2-D Clean-up Kit (GE Healthcare, Piscataway, NJ, USA). The purified total proteins were resuspended in the lysis solution. Protein concentrations were determined using a modified Bradford assay; bovine serum albumin was used as the standard.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTwo-dimensional polyacrylamide gel electrophoresis (2D-PAGE)\u003c/h2\u003e \u003cp\u003eOne-dimensional isoelectric focusing (IEF) and two-dimensional (2D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS\u0026ndash;PAGE) (2-DE) were performed according to the manufacturer\u0026rsquo;s protocol (GE Healthcare, Piscataway, NJ, USA). The purified protein samples (200 \u0026micro;g) containing a 0.5% immobilized pH gradient (IPG) buffer (pH 4\u0026ndash;7) and 5% bromophenol blue were applied to an immobilized IPG Dry Strip (immobilized pH 4\u0026ndash;7, 13 cm; GE Healthcare) and rehydrated for 16 h in lysis solution. The focused IPG strips were equilibrated for 20 min in a solution containing 50 mM Tris\u0026ndash;HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, and 1% DTT. Then, the DTT in the solution was replaced with 0.5% iodoacetamide, and the process was continued for an additional 20 min. The equilibrated strips were applied to a 12% polyacrylamide gel (14 \u0026times; 13 cm, 1.5 mm thick). Silver staining was performed to visualize the proteins, and mass spectrometry was performed according to the manufacturer's protocol (Thermo Fisher Scientific, Cleveland, OH, USA).\u003c/p\u003e \u003cp\u003eProtein identification\u003c/p\u003e \u003cp\u003eFor identifying protein, the proteins were identified by after drying the gel, followed by dehydrating in acetonitrile and overnight digestion at 37\u0026deg;C with sequencing-grade trypsin (Promega, Madison, WI, USA). The resulting tryptic peptides were dissolved in 50% acetonitrile containing 0.5% trifluoroacetic acid and desalted using a ZipTip C18 pipette tip (Millipore, Billerica, MA, USA). The peptides were directly eluted onto matrix-assisted laser desorption ionization (MALDI) plates using a-cyano-4-hydroxy-cinnamic acid (CHCA) matrix solution (10 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e CHCA in 0.5% TFA: 50% acetonitrile, 1:1). MALDI-time of flight/mass spectrometry (MALDI-TOF/MS) and MALDI-TOF/MS/MS were performed in reflection mode using a 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA). The proteins were identified by searching the National Center for Biotechnology Information (NCBI) database using the MASCOT program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.matrixscience.com/\u003c/span\u003e\u003cspan address=\"http://www.matrixscience.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; RRID:SCR_014322).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn silico\u003c/b\u003e \u003cb\u003emethod for identifying protein and promoter region\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGenome analysis\u003c/h2\u003e \u003cp\u003eThe NB10 genome was analyzed using the complete genome sequence deposited at NCBI (accession number: PRJEB5701) to identify FK506 binding protein (FKBP) and its promoter region based on the results of the amino acid sequence confirmed using MALDI-TOF/MS. Genome analysis was performed according to a method reported previously with slight modifications [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Briefly, the NB10 genome annotations were performed using Proksee (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://proksee.ca/\u003c/span\u003e\u003cspan address=\"https://proksee.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Rapid Annotation using Subsystem Technology 2.0 (RAST; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rast.nmpdr.org/\u003c/span\u003e\u003cspan address=\"https://rast.nmpdr.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and Bacterial and Viral Bioinformatics Resource Center (BV-BRC; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bv-brc.org/\u003c/span\u003e\u003cspan address=\"http://www.bv-brc.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) services. A comparative annotation analysis of all results was performed to complete the commentary. The circular map was constructed using the BV-BRC circular viewer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bv-brc.org/view/Genome/\u003c/span\u003e\u003cspan address=\"https://www.bv-brc.org/view/Genome/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eThree-dimensional homology-modeling and ligand docking\u003c/h2\u003e \u003cp\u003eMolecular modeling and docking process were performed according to the procedure described in the reported previously with slight modifications [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Briefly, for 3D structural modeling of FKBP, homologous proteins were screened using the AlphaFold v2 protein database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/\u003c/span\u003e\u003cspan address=\"https://alphafold.ebi.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using an AI system that predicts protein 3D structure based on its amino acid sequence (PBD ID; A0A649YL14.1.A, unknown gene, unknown organism). Secondary screening was performed on the protein templates using the NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/structure/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/structure/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and RCSB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) servers. The candidate templates were applied to a protein structure homology modeling server (Swiss-Model; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to select templates for the structures that were determined using X-ray crystallography. The final predicted tertiary structures were constructed based on the selected template (PDB ID; 7dek.2.A, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e FK506-binding protein PaFkbA). The 3D conformers of tacrolimus (PubChem CID: 445643; C\u003csub\u003e44\u003c/sub\u003eH\u003csub\u003e69\u003c/sub\u003eNO\u003csub\u003e12\u003c/sub\u003e; FK-506) that act as FKBP ligands were visualized using the PubChem Chemical Molecules Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The intermolecular binding sites and affinities of the proteins and ligands were predicted and evaluated using the CB-Dock2 server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cadd.labshare.cn/cb-dock2/\u003c/span\u003e\u003cspan address=\"https://cadd.labshare.cn/cb-dock2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Visualization and further analysis of the molecular complexes for parameters such as density maps, trajectories, and structure matching were performed using UCSF ChimeraX (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cgl.ucsf.edu/chimerax/\u003c/span\u003e\u003cspan address=\"https://www.cgl.ucsf.edu/chimerax/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; RRID:SCR_015872) program.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSequence alignment of the promoter region\u003c/h2\u003e \u003cp\u003eThe protein corresponding to FKBP of NB10 was traced in other \u003cem\u003eVibrio\u003c/em\u003e species (\u003cem\u003eV. parahaemolyticus\u003c/em\u003e ATCC 17802, accession number CP014046.2; \u003cem\u003eV. harveyi\u003c/em\u003e ATCC 33843, CP009467.2; \u003cem\u003eV. alginolyticus\u003c/em\u003e ATCC 17749, CP006718.1; and \u003cem\u003eV. fluvialis\u003c/em\u003e 10M-VF, CP118599.1). The sequences of the FKBP promoter region of each \u003cem\u003eVibrio\u003c/em\u003e were secured through genome comparative analysis. The nucleotide sequence identity matrix and amino acid sequence homology of the fatty acid synthesis-related proteins were calculated using the BioEdit 7.2 program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioedit.software.informer.com/\u003c/span\u003e\u003cspan address=\"https://bioedit.software.informer.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; RRID:SCR_007361).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eConstructions of recombinant vector\u003c/h2\u003e \u003cp\u003epVApro (pET-28a(+)/FKBP promoter/lacZ) was constructed by inserting the FKBP promoter and \u003cem\u003elacZ\u003c/em\u003e gene into pET-28a(+) as the backbone vector. The 3.1 Kbp \u003cem\u003elacZ\u003c/em\u003e gene was amplified using PCR (TaKaRa, Kyoto, Japan) with pcDNA\u0026trade;3.1/His/lacZ as the template and complementary primer sets containing BamHI and XhoI restriction enzyme sites for gene cloning (New England Biolabs (NEB), Cambridge, MA, USA). The PCR conditions were 25 amplification cycles of 97\u0026deg;C for 30 s, 58\u0026deg;C for 60 s, and 72\u0026deg;C for 30 s. The amplified \u003cem\u003elacZ\u003c/em\u003e and pET-28a(+) were digested using BamHI and XhoI and inserted into the multiple cloning site (MCS) of pET-28a(+) using T4 DNA ligase (Takara, Kyoto, Japan). Then, pET-28a(+)/lacZ was transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α using heat shock at 42\u0026deg;C for 60 s to confirm the recombinant plasmid. Next, the stepwise deleted FABP promoter regions were amplified using NB10 chromosomal DNA as the template and complementary primer sets containing the BglII and BamHI restriction sites (PCR condition: 25 amplification cycles of 97\u0026deg;C for 30 s, 58\u0026deg;C for 30 s, and 72\u0026deg;C for 30 s). The PCR products and the purified pET-28a(+)/lacZ were digested using BglII and BamHI restriction enzymes, treated with ligase, and transformed again into \u003cem\u003eE. coli\u003c/em\u003e DH5α to complete the pVApro/wild \u0026ndash; del3. The insertion of each target gene was confirmed via DNA sequencing with the Applied Bio-systems 3730XL using the BigDye(R) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. All the primer sets used in this study are listed in Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMeasurements of promoter strength\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eβ-galactosidase assay\u003c/h2\u003e \u003cp\u003eThe β-galactosidase assay was performed according to the Miller method with minor modifications to quantify the activation level of the promoter in terms of number of Miller units of enzyme activity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Briefly, 3 mL of overnight cultured recombinant \u003cem\u003eE. coli\u003c/em\u003e DH5α harboring pVApro/wild (\u003cem\u003eE. coli\u003c/em\u003e pVApro/wild) was inoculated into LB-Kanamycin (50 \u0026micro;g/mL) broth (300 mL). The cells were cultured for 6 h at 37\u0026deg;C until the late log phase was 2\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU/mL. The cells were collected through centrifugation at 6,000 rpm for 10 min and immediately suspended in 50 mL of fresh LB medium, which was adjusted to pH 5\u0026ndash;10 with 6N HCl and NaOH. The samples for promoter strength measurement were collected after 1, 2, and 4 h of incubation at 25\u0026deg;C. To measure promoter strength in response to temperature stress, the samples were incubated at 37 and 50\u0026deg;C and pH 7, followed by centrifugation at 12,000 rpm for 10 min to remove the supernatant completely. The collected cells were resuspended in 50 mL of 50 mM Tris\u0026ndash;HCl (pH 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2) buffer and disrupted using a sonicator (Sonics \u0026amp; Materials, Inc., Newtown, CT, USA) at 4\u0026deg;C (3 s pulses at 150 W for 30 min with 2 s gap between pulses). After centrifugation, the supernatant was discarded, and the cell debris and inclusion bodies were used as crude enzyme for determining β-galactosidase activity. Each sample was assayed for β-galactosidase assay with o-nitrophenyl-β-D-galactoside (ONPG) as the substrate as described with minor modifications [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The assay mixture (100 \u0026micro;L) containing 5 mM ONPG and crude enzyme solution were incubated for 10 min at 40\u0026deg;C. The reaction was stopped by adding one volume of 1 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e. The optical density of the reactants was read using a Microplate Reader (KLAB, Daejeon, Republic of Korea). The Miller formula was used to calculate the Miller units of enzyme per minute per milliliter of the sample. Each sample was processed in triplicate, and the average values were used to calculate the Miller units of the enzyme in each sample.\u003c/p\u003e \u003cp\u003e1 Miller Unit\u0026thinsp;=\u0026thinsp;1000 * \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{({Abs}_{420}-\\left(1.75\\text{*} {Abs}_{550}\\right))}{\\left(t\\text{*}v\\text{*} {Abs}_{600}\\right)}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003ewhere, Abs\u003csub\u003e420\u003c/sub\u003e indicates the absorbance of yellow o-nitrophenol; Abs\u003csub\u003e550\u003c/sub\u003e indicates the scatter from cell debris; \u003cem\u003et\u003c/em\u003e indicates the reaction time in minutes; \u003cem\u003ev\u003c/em\u003e indicates the volume of culture assayed in milliliters; and Abs\u003csub\u003e600\u003c/sub\u003e indicates cell density.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTotal RNA extraction and cDNA synthesis\u003c/h2\u003e \u003cp\u003eCultured bacteria were centrifuged at 12,000 rpm for 10 min, and the pellet was re-suspended in 1 ml TRIzol reagent (Invitrogen Lige Technologies, Burlington, Canada) to isolate the total RNA. Subsequent preparation and washing were performed using the Hybrid-RTM RNA Isolation Kit (GeneAll Biotechnology, LTD, Seoul, Korea). DNA was hydrolyzed using RQ DNase I (Promega, Madison, WI, USA). Complementary DNA (cDNA) was synthesized using PrimeScript\u0026trade; 1st Strand cDNA Synthesis Kit (Takara, Otsu, Japan) under the following conditions: initial reaction with random hexamer at 30\u0026deg;C for 10 min, followed by extension at 42\u0026deg;C for 60 min. The RNA and cDNA were quantified using NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGene expression analysis\u003c/h2\u003e \u003cp\u003eTo validate the \u003cem\u003ein vivo\u003c/em\u003e differential expression of genes at the transcriptional level, quantitative real-time PCR (qRT-PCR) was performed using a Thermal Cycler Disc\u0026trade; Real Time System Lite (model TP700/760, software version V5.0x) (Takara Bio Inc, Otsu, Japan) instrument and SYBR Premix Ex Taq\u0026trade; (Tli RNaseH Plus, Takara, Kyoto, Japan) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The two-step shuttle PCR protocol was optimized to include by 35 cycles of initial denaturation for 30 s at 95\u0026deg;C, followed denaturation at 95\u0026deg;C for 5 s and annealing and extension at 58\u0026deg;C for 15 s. The PCR mixture (25 \u0026micro;L) contained 12.5 \u0026micro;L of 2x SYBR Premix Ex Taq\u0026trade;, 0.5 \u0026micro;L of each primer (15 \u0026micro;M), 9.5 \u0026micro;l of sterile distilled water, and 2.0 \u0026micro;L of cDNA. \u003cem\u003eV. anguillarum\u003c/em\u003e 16S rRNA was used as the housekeeping gene for internal control. The relative quantitative value was expressed in accordance with the 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e method [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were subjected to one-way analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS), followed by Duncan's multiple range test. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, unless otherwise noted.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.G.K.\u003c/strong\u003e: Conceptualization, Funding acquisition, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eD.N.O.\u003c/strong\u003e: Data curation, Methodology, Software, Project administration, Resources, Supervision, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eE.J.L.\u003c/strong\u003e: Data curation, Methodology, Software, Validation, Visualization, Writing \u0026ndash; original draft. \u003cstrong\u003eS.Y.P.\u003c/strong\u003e: Project administration, Supervision, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eJ.M.L.\u003c/strong\u003e: Conceptualization, Data curation, Funding acquisition, Project administration, Software, Supervision, Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Institute of Fisheries Science, Ministry of Oceans and Fisheries, Korea (R2024053).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe whole genome sequence data supporting the findings of this study have already been deposited in the National Center for Biotechnology Information with the accession code PRJEB5701. The datasets used and/or analyzed during the current study are available from the corresponding author, Jong Min Lee, upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcCallum, H. I. \u003cem\u003eet al.\u003c/em\u003e Marine ecosystems as \u0026lsquo;open\u0026rsquo;systems. Trends Ecol. Evol. 11, 585\u0026ndash;591 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStincone, P. \u0026amp; Brandelli, A. Marine bacteria as source of antimicrobial compounds. Crit. Rev. Biotechnol. 40, 306\u0026ndash;319 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSlijepcevic, P. \u003cem\u003eBiocivilisations: a new look at the science of life\u003c/em\u003e. (Chelsea Green Publishing, 2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoger-Reischer, R. Z. \u003cem\u003eet al.\u003c/em\u003e Evolution of a minimal cell. 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Commun. 571, 125\u0026ndash;130 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"alkaline-inducible promoter, Vibrio anguillarum, peptidyl-prolyl cis/trans isomerase, synthetic biology, biofoundry","lastPublishedDoi":"10.21203/rs.3.rs-4441654/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4441654/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eVibrio\u003c/em\u003e has attracted attention as a promising genetic chassis in the field of synthetic biology. FK506 binding protein (FKBP)-type peptidyl-prolyl cis/trans isomerase (PPIase) is involved in protein folding. In this study, we report, for the first time, the promoter regions in \u003cem\u003eVibrio\u003c/em\u003e that drive increased FKBP expression in the extremely alkaline environment. Proteomic analysis of \u003cem\u003eV. anguillarum\u003c/em\u003e NB10 showed that VaFKBP was significantly upregulated under extreme alkaline stress (pH 10) condition. Additionally, the putative core promoter-containing regions and a reporter gene coding a β-galactosidase were introduced into the \u003cem\u003eEscherichia coli\u003c/em\u003e system, which showed β-galactosidase activity of 61.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91 and 95.83\u0026thinsp;\u0026plusmn;\u0026thinsp;6.76 Miller unit (MU) at pH 9 and 10, respectively, after 4 h of stress. This outcome was 1.97- and 2.88-fold higher that that observed under normal conditions of 25\u0026deg;C and pH 7 (31.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 MU). To the best of our knowledge, this is the first report of a promoter showing increased expression under extremely alkaline conditions. We believe that this is a useful chassis in promoter engineering and can be used as a powerful tool for activating transcriptionally silent biosynthetic gene clusters in specific environments.\u003c/p\u003e","manuscriptTitle":"Genetic characteristics of novel extreme alkaline-inducible promoter located in five prime upstream region of peptidyl-prolyl cis/trans isomerase from Vibrio anguillarum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-03 06:57:02","doi":"10.21203/rs.3.rs-4441654/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a4bb05b7-2334-4c0c-8a60-138385244a97","owner":[],"postedDate":"June 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":32635927,"name":"Biological sciences/Genetics"},{"id":32635928,"name":"Biological sciences/Microbiology"},{"id":32635929,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2024-12-11T04:53:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-03 06:57:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4441654","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4441654","identity":"rs-4441654","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}