Should Escherichia coli K-12 substrain MG1655 be classified as NaCl resistant?

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Ana Paula Muche Schiavo, Roberta Almeida Vincenzi, Isabella Gaião Silva, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8882295/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 Extreme environments are defined by conditions that challenge cellular machinery, often compromising survival and biological function. Halophiles, a subclass of extremophiles, thrive in high sodium chloride (NaCl) concentrations. However, current knowledge on salt stress resistance is largely derived from studies on extreme halophiles, even though most halophilic microorganisms are classified as slight or moderate halophiles. This fact poses a question regarding the real representation of state-of-the-art information about salt resistance mechanisms and diversity. To bring light to the problem of the lack of information on slight halophiles, we propose the study of Escherichia coli str. K-12 substr. MG1655 to fill the gap. We evaluated the response of E. coli MG1655 to varying NaCl concentrations, including control without NaCl addition. Our results indicate optimal growth at NaCl concentrations up to 0.5 mol·L⁻¹, suggesting that this strain should be classified as a slight halophile, in contrast to its current classification in the literature. Thus, we propose the study of this strain to understand the molecular mechanisms underlying adaptation to low and moderate salt stress. Biological sciences/Biochemistry Biological sciences/Microbiology sodium chloride extremophile growth rate E. coli halophilic Figures Figure 1 Figure 2 INTRODUCTION Extremophiles are organisms capable of growing in extreme environments 1 . According to the Near Impossibility definition, an extreme environment is an environment that harbors the physical and chemical conditions that make it difficult for cellular machinery to function 2 . Halophiles are a subclass of extremophiles that thrive in high sodium chloride (NaCl) concentrations. They are further branched in respect to the NaCl concentration that exhibited optimum growth: slight (0.2–0.5 mol·L − 1 ), moderate (0.5–2.5 mol·L − 1 ) and extreme halophile (> 2.5 mol·L − 1 ) 3 . Although this classification system has little biological or ecological bases, it has nonetheless been historically adopted in the literature as the standard reference for classifying organisms based on their NaCl response. The interest in the research and application of halophiles includes several areas of knowledge, such as food industries (in processes such as fermentation of fish or soy) 4 , 5 , bioproduction of pigments (e.g. carotenoids) and other small organic molecules (e.g. ectoin) 6 , bioremediation of brines associated with petroleum extraction 7 and the study of the habitability of extraterrestrial brines (existing on past or present-day Mars and the icy moons of the Solar System) 8 , 9 .Whereas there are more than 1000 halophilic species described with representatives in all domains of life ( Bacteria, Archaea and Eukarya ), most of the studied species are prokaryotic 1 . The main halophilic model organisms are Halobacterium salinarum and Haloferax volcanii , both archaeas and classified as extreme halophiles. These species have a wide number of molecular information (e.g. genomic and transcriptomic data) 10 – 24 and delineate most of our knowledge of the resistance to NaCl mechanisms, even though the majority of the microorganisms classified as NaCl resistant lie within the slight or moderate halophile category. This fact poses a question regarding the real significance of the use of extreme halophiles to represent state-of-the-art information about salt resistance mechanisms and diversity. On the other hand, Escherichia coli , the most frequently used model organism in microbiological studies, presents a yet poorly explored resistance to NaCl. This microorganism plays an important role in biological engineering and industrial microbiology 25 – 27 , with more than two hundred thousand sequenced genomes, being widely known and studied (“NCBI Genome Escherichia coli str. K-12 substr. MG1655”). Most of the studies concerning resistance of E. coli to NaCl were published before the year 2000, with little to no information about strain or molecular data 28 – 32 . One more recent work approaches transcriptional data to address the NaCl response of several E. coli strains 33 . Still, it is limited to only one concentration of NaCl (1.03 mol·L − 1 ), which makes it insufficient to provide information about strain salt resistance. Therefore, there is a lack of data about the NaCl resistance of E. coli strains. One E. coli strain in particular caught our attention. The E. coli strain K-12 substrain MG1655 is well known, being the first wild-type laboratory strain completely sequenced 34 and having several articles on gene expression and regulation 35 – 37 . Some articles even show molecular data for osmotic stress caused by NaCl (Nagata et al., 2002; Weber et al., 2006) and other salts 40 . To bring light to the problem of the lack of information on slight halophiles, our group is proposing further studies with Escherichia coli MG1655, a potential slight halophilic microorganism, aiming this work to analyze its resistance to different salt concentrations. RESULTS NaCl response assays To determine the response of E. coli MG1655 to NaCl, we tested the strain in different concentrations of the salt, including a control essay without the addition of NaCl. The resulting curves are shown in Fig. 1 (raw data can be found in Supplementary Information). The figure shows that no growth is observed in NaCl concentrations higher than 1.25 mol·L − 1 , instead, we can see death occurring. By fitting the growth curves with the Baranyi Model (images of the fitted curve are available in Supplementary Information), we were able to get numeric values to the parameters that describe the curve. The values of the parameters are shown in Table 1 as well as the statistical values for goodness of fit of the model. The pseudo-R 2 for all the NaCl concentrations are very close to 1 indicating that the model had a good fit in all cases and the p-values for all the parameters are below 0.001 showing that the values have statistical significance. As expected, the lag phase (λ) increases with the concentration of NaCl. All assays were inoculated using E. coli cultures grown without added NaCl, and the lag phase tends to be longer when the medium is changed 41 . The carrying capacity (y max ) is similar up to 0.75 mol·L − 1 NaCl, showing that the TGY medium can support roughly the same cell population in those cases. This indicates that the energy used in the NaCl resistance mechanisms of E. coli up to 0.75 mol·L − 1 is not significant when compared with the energy supplied by a rich medium such as TGY. Nonetheless, when the NaCl concentration is higher (1.00 to 1.25 mol·L − 1 ) the carrying capacity becomes progressively lower. This shows that these concentrations are more challenging for E. coli to cope with 42 – 44 . Table 1 Fitting parameters for Barany' model, where µ stands for growth rate (h − 1 ), y max for carrying capacity, y 0 for the initial number of cells in culture, λ for lag time (h). Values marked with * were fixed manually to guarantee goodness of fitting. All the y 0 values (marked with †) were determined experimentally, therefore were fixed. The uncertainty values are expressed as standard error. [NaCl] (mol·L − 1 ) Parameter Fitted value t-value p-value Pseudo-R 2 0.00 µ 0.94 ± 0.05 17.35 1.04∙10 − 14 0.95 y max 21.03 ± 0.17 126.04 < 2.00∙10 − 16 y 0 13.40 † - - λ 1.50* - - 0.25 µ 0.81 ± 0.06 13.33 2.65∙10 − 12 0.94 y max 20.73 ± 0.17 125.55 < 2.00∙10 − 16 y 0 14.57 † - - λ 1.50* - - 0.50 µ 0.92 ± 0.05 17.31 1.10∙10 − 14 0.96 y max 21.04 ± 0.18 117.76 < 2.00∙10 − 16 y 0 13.29 † - - λ 2.00* - - 0.75 µ 0.72 ± 0.08 9.59 2.59∙10 − 09 0.98 y max 20.44 ± 0.14 151.27 < 2.00∙10 − 16 y 0 14.63 † - - λ 5.16 ± 0.48 10.80 2.94∙10 − 10 1.00 µ 0.59 ± 0.05 11.58 7.82∙10 − 11 0.98 y max 19.74 ± 0.21 92.07 < 2.00∙10 − 16 y 0 13.39 † - - λ 7.54 ± 0.50 15.15 4.02∙10 − 13 1.25 µ 0.41 ± 0.02 19.11 3.46∙10 − 15 0.99 y max 19.76 ± 0.43 45.52 < 2.00∙10 − 16 y 0 13.24 † - - λ 7.82 ± 0.36 21.98 < 2.00∙10 − 16 However, the most relevant parameter is the µ as it is the growth rate that determines the optimum growth (Perni et al . 2005). The values of µ for 0.00 mol·L − 1 (0.94 ± 0.05), 0.25 mol·L − 1 (0.81 ± 0.06) and 0.50 mol·L − 1 (0.92 ± 0.05) NaCl are similar and greater than in any other concentration. This shows that the range of NaCl for optimal growth of E. coli MG1655 is 0.00-0.50 mol·L − 1 . Beyond that range, the growth rate decreases as the NaCl concentration in the medium increases, as shown in Table 1 . NaCl related genes annotation The survey of salt resistance genes across the 48 Escherichia coli K-12 MG1655 genomes revealed a highly conserved and comprehensive repertoire of strategies associated with NaCl stress tolerance (Fig. 2 ). Several genes were detected in all 48 genomes, including the Na⁺/H⁺ antiporters nhaA and nhaB , the trehalose biosynthesis genes otsA and otsB , the complete set of gln genes ( glnA, glnB, glnD, glnE, glnG, glnH, glnK, glnL, glnP, glnQ, glnS ), the proline pathway genes proC , proV , proW , proX , and the regulatory sensor phoQ . Importantly, genes encoding K⁺ transporters ( kdpA, kdpB, kdpD, kdpE and trkA , trkH , trkG ) and Cl⁻ channels ( clcA , clcB ) were also universally or nearly universally conserved, confirming that this strain retains the canonical ionic homeostasis systems typically associated with halotolerance. In addition to this conserved core, other osmoprotectant systems were present at high frequency. The glycine betaine operon ( betA , betB , betT ) was detected in 46 out of 48 genomes, while proA and proB were found in 47 out of 48 genomes, indicating that most strains also retain multiple routes for proline and glycine betaine accumulation. These pathways contribute additional flexibility in the osmotic stress response. By contrast, alternative strategies such as ectoine biosynthesis ( ectABCD ) and trehalose degradation ( treS , treY , treZ ) were completely absent from all genomes, suggesting that they are not relevant mechanisms for NaCl resistance in this lineage. Taken together, these results demonstrate that E. coli K-12 MG1655 harbors a robust and multifaceted molecular toolkit to counteract osmotic stress, comprising Na⁺, K⁺ and Cl⁻ transport systems with osmoprotectant accumulation and global regulators. This conserved genomic repertoire provides additional support for the phenotypic resistance to NaCl previously observed in growth curve experiments, confirming that the strain possesses the molecular machinery necessary to sustain its adaptive response under saline conditions. DISCUSSION Our results have shown that the E. coli MG1655 grows optimally at up to 0.5 mol·L − 1 NaCl. According to the current classification, it should be considered a slight halophile (optimal growth at 0.2–0.5 mol·L − 1 ) 3 . Therefore, the present work brings to light that E. coli MG1655, previously considered not NaCl resistant strain, should in fact be classified as an extremophile, when adopting the current predominant classification system. To further support our novel findings, we identified the presence of several genes related to osmotic stress (Fig. 2 ), which shows clearly the salt-resistance metabolic potential of E. coli K-12 MG1655. The presence/absence analysis of NaCl stress–related genes revealed a largely conserved genetic profile across the Escherichia coli strains obtained from NCBI, suggesting that core mechanisms of salt tolerance are broadly distributed within the strain. Among the genes identified, those involved in osmotic homeostasis, ion transport, and the synthesis or uptake of compatible solutes were consistently detected, in agreement with previous studies highlighting their central role in adaptation of E. coli to high-salinity environments 33 . The genes Otsa und Otsb (present in all strains analyzed) are related to trehalose synthesis, an important osmoprotectant that was also previously described on E. coli salt stress response 45 . Earlier reports show that systems such as the betaine/glycine transporters (e.g., BetT/BetP), the ProP/ProU osmoprotectant uptake systems, and other regulators of osmotic pressure are essential for maintaining cellular volume and preventing protein or membrane damage under elevated NaCl conditions 46 . The recurrent detection of these genes across the analyzed strain therefore supports the interpretation that the slight NaCl tolerance observed in this work is underpinned by a conserved set of physiological and regulatory mechanisms. Overall, these findings reinforce the view that salt stress response in E. coli relies on an evolutionarily stable core of genes whose widespread presence aligns with established models of bacterial osmoadaptation. Through a review of the literature, we have identified that E. coli has never been appropriately classified for its NaCl resistance, as no studies providing such classification were found to the best of our knowledge. This lack of information about one of the most studied model microorganisms significantly impacts many research studies across different fields. These findings have particular impact on the field of extremophiles research, since E. coli is widely used as a control organism for experimental setups (as can be seen in Fisher et al., 2025; Madigan et al., 2008; Siela & Smith, 2019). In addition to the above-mentioned impacts, our findings may appear controversial, as the literature suggests that E. coli is a widely common bacteria present in non-saline environments 49 . However, the presence of several genes related to osmotic stress shows clearly the salt-resistance metabolic potential of E. coli K-12 MG1655. One possible explanation for this apparent contradiction is that the strains routinely cultivated in laboratory environments can develop genotypic and phenotypic differences when compared to other wild strains 50 . Although E. coli K-12 substr. MG1655 can be considered close to the wild strain, since it has no direct modification, it still is a long-used laboratory strain. Therefore, the characterization regarding salt tolerance for this strain may not represent the phenotype encountered in the general population of wild E. coli strains. On the other hand, our results could suggest an alternative resolution to this controversy. The data presented in this study could raise the question of the very definition of what we consider extremophile and, therefore, halophile. By the Statistical Rarity definition of extremophile, extreme environments are those that few species can thrive 2 . The ocean, as one of the major biomes on Earth, has an average salinity of 35 g·L − 1 (equivalent to 0.6 mol·L − 1 ) 51 . To our view, it cannot be rightly affirmed that the oceans have low biological diversity, so it should not be considered an extreme environment; by consequence, nor should 0.5 mol·L − 1 (upper limit of optimum NaCl concentration to classify a microorganism as slight halophile) be considered an extreme physicochemical condition. This brings to light the discussion about the classification of extremophiles, especially of halophiles. Due to the availability of extensive molecular data and its ease of use in laboratory settings, E. coli has proven its potential and can be a powerful tool for elucidating the molecular mechanisms underlying the stress response to NaCl. Thus, we propose the study of this strain in the response to low and moderate (up to 1.25 mol·L − 1 ) NaCl concentration even though we argued that the classification of E. coli as a slight halophile is questionable. This information would help us further understand organisms currently classified as slight and moderate halophiles and their molecular adaptation to osmotic stress. Considering the question proposed in the title of the manuscript, our group believes that, regardless of the current halophile classification inconsistencies discussed above, E. coli str. K-12 substr. MG1655 exhibited an unexpected response to NaCl and should be considered a slight halophile by the classification used in literature 52 . As mentioned before, the main current halophilic models are classified as extreme halophiles, consequently most of the known resistance mechanisms to NaCl regard these organisms. Information regarding the mechanisms activated under lower NaCl concentrations is scarce, and it is precisely in this gap that the study of E. coli K-12 substr. MG1655 can make its most significant contribution. This strain shows great potential to further understand the NaCl resistance mechanisms given the amount of molecular data available and its easy handling in the laboratory. The knowledge that can emerge from this approach would be useful to biotechnology, the food industry, Astrobiology among other areas. METHODS Cell culture and media Escherichia coli str. K-12 substr. MG1655 was cultured in TGY medium. TGY liquid medium is composed of tryptone 5 g·L − 1 , glucose 1 g·L − 1 and yeast extract 3 g·L − 1 , sterilized by autoclave for 15 minutes at 121°C. Agar was added at 15 g·L − 1 when solid media was necessary. We first cultivated bacterial cells in a petri dish containing TGY-agar media at 30°C. Then we picked a single colony, inoculated for growth at liquid TGY media at 30°C in a shaking incubator at 150 rpm overnight. Sodium chloride exposure assay Modified TGY liquid media were also prepared by the addition of NaCl in several concentrations (0.25 mol·L − 1 , 0.5 mol·L − 1 , 0.75 mol·L − 1 , 1.0 mol·L − 1 , 1.25 mol·L − 1 , 1.5 mol·L − 1 , 2.0 mol·L − 1 , 3.0 mol·L − 1 and 4.0 mol·L − 1 ). For the assay, we added 10 µL of E. coli str. K-12 substr. MG1655 overnight culture to 10 mL of TGY medium and all the supplemented TGY media. This culture was incubated in a shaking incubator for 24 h. Every 1 h, we collected aliquots of 50 µL for serial dilution (1:10) and Colony Forming Units (CFU) counting. The CFU counting was made by the Drop Plate Method 53 , 54 . Three independent replicas were used for each NaCl concentration. Growth curve model fitting Modeling of the growth curves was done using original scripts in R v.4.3.3. For that, only curves of NaCl concentration where growth is observed were used (y 24h > y 0 ). The model used was the Baranyi 55 , 56 . The Baranyi model has the parameters of natural logarithm of initial CFU counting (y 0 ), carrying capacity (y max , maximum number of individuals of a species that the environment can sustain), growth rate ( µ ) and the length of the lag phase (𝜆). The function of the Baranyi model is given by equations 1 and 2 : \(\:y={y}_{0}+\mu\:\bullet\:A\left(t\right)-ln\left(1+\frac{{e}^{\mu\:\bullet\:A\left(t\right)}-1}{{e}^{{y}_{max}-{y}_{0}}}\right)\) , where(1) $$\:A\left(t\right)=t+\frac{1}{\mu\:}\bullet\:lnln\:\left({e}^{-\mu\:\bullet\:t}+{e}^{-\mu\:\bullet\:\lambda\:}-{e}^{\left[-\mu\:\bullet\:\left(t+\lambda\:\right)\right]}\right)\:$$ 2 This model was fitted using native Non-linear Least Squares from R ( nls function). All y 0 were determined experimentally, so the values were not given by the fitted model. Some of the 𝜆 were fixed with values visually determined to guarantee the best fit. Visual analysis confirmed the general quality of fitting and the pseudo-R 2 calculated to further assess the goodness of the Baranyi Model fitting. Even though the pseudo-R 2 cannot be interpreted as the proportion of data variance explained by the model, pseudo-R 2 (as its linear version, R 2 ) has a maximum value of 1; the closest to 1, the better the model fits. Furthermore, the statistical significance of the fitted parameters was accessed by the p-value given by the nls function. Functional Annotation Analysis For the functional analysis, we used all genomes of the Escherichia coli K-12 MG1655 strain deposited in NCBI that passed our quality control (RefSeq assignment on NCBI, contamination ≤ 3%, and completeness ≥ 95%). In total, 41 genomes were included in the analysis. These genomes were subsequently annotated with Prokka (Seemann, 2014), and their corresponding .faa files were used as input for eggNOG-mapper (v2.1.6) 58 . EggNOG-mapper enables the mapping of protein sequences to evolutionarily conserved orthologs, assigning them to functional categories based on databases such as COG (Clusters of Orthologous Groups), KEGG (Kyoto Encyclopedia of Genes and Genomes), and GO (Gene Ontology). Based on the eggNOG-mapper outputs, we performed a curated search for genes related to NaCl resistance, based on relevant literature. The list of genes and the salt-resistance strategy categories assigned to them are provided in the Supplementary Information. From this filtering, we generated a presence–absence matrix of the genes of interest using a custom Python script, also available in the Supplementary Information. Declarations Acknowledgements Funding for the study was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 16/06160-8 and 2025/11833-0), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 140086/2018-8 and 170387/2018-6) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES 888887.570298/2020-00). The author would like to thank Brazilian Research Unity in Astrobiology (NAP/Astrobio). Author Contributions Ana Paula Muche Schiavo: conceptualization (lead), formal analysis (lead), investigation (equal), project administration (lead), visualization (lead), writing – original draft (lead), writing – review & editing (equal). Roberta Almeida Vincenzi : formal analysis (supporting), investigation (equal), project administration (supporting), visualization (supporting), writing – original draft (supporting), writing – review & editing (equal). Isabella Gaião da Silva : investigation (equal), project administration (supporting), visualization (supporting), writing – original draft (supporting), writing – review & editing (equal). Fabio Rodrigues : funding acquisition (lead), resources (lead), supervision (lead) and writing – review & editing (supporting). Data Availability Statement The datasets and code generated during the current study are available in Supplementary Information. Additional Information No competing interests are declared. References Harrison, J. P., Gheeraert, N., Tsigelnitskiy, D. & Cockell, C. S. The limits for life under multiple extremes. Trends Microbiol. 21 , 204–212 (2013). Mariscal, C. & Brunet, T. D. P. What are extremophiles? in Social and Conceptual Issues in Astrobiology (eds Smith, K. C. & Mariscal, C.) 157–175 (Oxford University Press, New York, doi: 10.1093/oso/9780190915650.003.0010 . (2020). Anton, J. & Halophile Encyclopedia Astrobiology 1287–1289 doi: 10.1007/978-3-662-65093-6_694 . 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Geosciences (Switzerland) vol. 9 Preprint at (2019). https://doi.org/10.3390/geosciences9090361 Jang, J. et al. Environmental Escherichia coli: ecology and public health implications—a review. J. Appl. Microbiol. 123 , 570–581 (2017). Tenaillon, O. et al. Tempo and mode of genome evolution in a 50,000-generation experiment. Nature 536 , 165–170 (2016). National Oceanic and Atmosferic Administration. Sea Water. (2023). Kushner, D. J. & Kamekura, M. Physiology of halophilic eubacteria. in Halophilic Bacteria (ed Rodriguez-Valera, F.) vol. 1 109–140 (CRC, Boca Raton, (1988). Herigstad, B., Hamilton, M. & Heersink, J. How to optimize the drop plate method for enumerating bacteria. J. Microbiol. Methods . 44 , 121–129 (2001). Naghili, H. et al. Validation of drop plate technique for bacterial enumeration by parametric and nonparametric tests. Vet. Res. Forum . 4 , 179–183 (2013). Chatzidimitriou, K. Fitting modified Gombertz and Baranyi equations for bacterial growth in R. (2019). https://kyrcha.info/2019/10/25/fitting-modified-gompertz-baranyi-equations-bacterial-growth-r https://kyrcha.info/2019/10/25/fitting-modified-gompertz-baranyi-equations-bacterial-growth-r McKellar, R. C. & Lu, X. Modeling Microbial Responses in Food . (2004). Seemann, T. & Prokka Rapid prokaryotic genome annotation. Bioinformatics 30 , (2014). Cantalapiedra, C. P., Hern̗andez-Plaza, A., Letunic, I., Bork, P. & Huerta-Cepas, J. eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale. Mol Biol. Evol 38 , (2021). Additional Declarations No competing interests reported. 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-8882295","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":603418685,"identity":"8b27e3f9-fad1-49bc-97a5-2303bcf10236","order_by":0,"name":"Ana Paula Muche Schiavo","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Paula Muche","lastName":"Schiavo","suffix":""},{"id":603418686,"identity":"ee5530f1-a08f-40be-bc1d-a20a475ea2c8","order_by":1,"name":"Roberta Almeida Vincenzi","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Roberta","middleName":"Almeida","lastName":"Vincenzi","suffix":""},{"id":603418687,"identity":"b73e3dfe-ca35-40e2-89c7-1f6feccefef7","order_by":2,"name":"Isabella Gaião Silva","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Isabella","middleName":"Gaião","lastName":"Silva","suffix":""},{"id":603418688,"identity":"3eb37355-6fdd-4215-94e7-a290169dcb20","order_by":3,"name":"Fabio Rodrigues","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYBAC9gYgwVMhAeExNhChhecAiDhDshbeNgZStLCfffjg7TwL2QaJ9GcPGHfcI0ILT7qx4dxtEsYNEjnmBoxniglrsWdIY5Pm3SaRCNTCJsHYlkCELfzPgFrmgLSkPyNSiwTIlgaQlgQzYrU8Yzacc0zCuI3njblB4hmiHJbG+OBNTZ1sPzswxD7uIEILDDACo4aNgQQNkEhkI0XDKBgFo2AUjCAAADivMN7z7sgbAAAAAElFTkSuQmCC","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":true,"prefix":"","firstName":"Fabio","middleName":"","lastName":"Rodrigues","suffix":""}],"badges":[],"createdAt":"2026-02-14 19:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8882295/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8882295/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104524384,"identity":"7882334f-63a0-48d8-a0b6-f593b16c34ff","added_by":"auto","created_at":"2026-03-12 21:30:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":653573,"visible":true,"origin":"","legend":"\u003cp\u003eNaCl tolerance of \u003cem\u003eE. coli\u003c/em\u003e MG1655. The growth in usual TGY media and TGY media supplemented with different concentrations of NaCl (0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 3 and 4 mol·L-1). Error bars as standard error\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8882295/v1/ac8f25beb5d0dc8789500050.png"},{"id":104780804,"identity":"44d396c2-7a2d-448d-af22-8928abf095c1","added_by":"auto","created_at":"2026-03-17 07:54:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1249056,"visible":true,"origin":"","legend":"\u003cp\u003ePresence/absence of genes related to salt stress response in Escherichia coli K-12 substr. MG1655.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8882295/v1/b80cf487f4b00feecfa73b46.png"},{"id":105923459,"identity":"6858ce0f-ec81-4a5d-addc-342b67f9caaf","added_by":"auto","created_at":"2026-04-01 12:58:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2632917,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8882295/v1/bac9db9c-ff90-4476-85cd-24db56ec1bc8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Should Escherichia coli K-12 substrain MG1655 be classified as NaCl resistant?","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eExtremophiles are organisms capable of growing in extreme environments \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. According to the Near Impossibility definition, an extreme environment is an environment that harbors the physical and chemical conditions that make it difficult for cellular machinery to function \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Halophiles are a subclass of extremophiles that thrive in high sodium chloride (NaCl) concentrations. They are further branched in respect to the NaCl concentration that exhibited optimum growth: slight (0.2\u0026ndash;0.5 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), moderate (0.5\u0026ndash;2.5 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and extreme halophile (\u0026gt;\u0026thinsp;2.5 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) \u003csup\u003e3\u003c/sup\u003e. Although this classification system has little biological or ecological bases, it has nonetheless been historically adopted in the literature as the standard reference for classifying organisms based on their NaCl response.\u003c/p\u003e \u003cp\u003eThe interest in the research and application of halophiles includes several areas of knowledge, such as food industries (in processes such as fermentation of fish or soy) \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, bioproduction of pigments (e.g. carotenoids) and other small organic molecules (e.g. ectoin) \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, bioremediation of brines associated with petroleum extraction \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and the study of the habitability of extraterrestrial brines (existing on past or present-day Mars and the icy moons of the Solar System)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.Whereas there are more than 1000 halophilic species described with representatives in all domains of life (\u003cem\u003eBacteria, Archaea\u003c/em\u003e and \u003cem\u003eEukarya\u003c/em\u003e), most of the studied species are prokaryotic\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe main halophilic model organisms are \u003cem\u003eHalobacterium salinarum\u003c/em\u003e and \u003cem\u003eHaloferax volcanii\u003c/em\u003e, both archaeas and classified as extreme halophiles. These species have a wide number of molecular information (e.g. genomic and transcriptomic data)\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 CR23\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and delineate most of our knowledge of the resistance to NaCl mechanisms, even though the majority of the microorganisms classified as NaCl resistant lie within the slight or moderate halophile category. This fact poses a question regarding the real significance of the use of extreme halophiles to represent state-of-the-art information about salt resistance mechanisms and diversity.\u003c/p\u003e \u003cp\u003eOn the other hand, \u003cem\u003eEscherichia coli\u003c/em\u003e, the most frequently used model organism in microbiological studies, presents a yet poorly explored resistance to NaCl. This microorganism plays an important role in biological engineering and industrial microbiology\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, with more than two hundred thousand sequenced genomes, being widely known and studied (\u0026ldquo;NCBI Genome Escherichia coli str. K-12 substr. MG1655\u0026rdquo;). Most of the studies concerning resistance of \u003cem\u003eE. coli\u003c/em\u003e to NaCl were published before the year 2000, with little to no information about strain or molecular data \u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. One more recent work approaches transcriptional data to address the NaCl response of several \u003cem\u003eE. coli\u003c/em\u003e strains \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Still, it is limited to only one concentration of NaCl (1.03 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), which makes it insufficient to provide information about strain salt resistance. Therefore, there is a lack of data about the NaCl resistance of \u003cem\u003eE. coli\u003c/em\u003e strains.\u003c/p\u003e \u003cp\u003eOne \u003cem\u003eE. coli\u003c/em\u003e strain in particular caught our attention. The \u003cem\u003eE. coli\u003c/em\u003e strain K-12 substrain MG1655 is well known, being the first wild-type laboratory strain completely sequenced \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and having several articles on gene expression and regulation \u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Some articles even show molecular data for osmotic stress caused by NaCl (Nagata et al., 2002; Weber et al., 2006) and other salts \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. To bring light to the problem of the lack of information on slight halophiles, our group is proposing further studies with \u003cem\u003eEscherichia coli\u003c/em\u003e MG1655, a potential slight halophilic microorganism, aiming this work to analyze its resistance to different salt concentrations.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eNaCl response assays\u003c/h2\u003e \u003cp\u003eTo determine the response of \u003cem\u003eE. coli\u003c/em\u003e MG1655 to NaCl, we tested the strain in different concentrations of the salt, including a control essay without the addition of NaCl. The resulting curves are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (raw data can be found in Supplementary Information). The figure shows that no growth is observed in NaCl concentrations higher than 1.25 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, instead, we can see death occurring. By fitting the growth curves with the Baranyi Model (images of the fitted curve are available in Supplementary Information), we were able to get numeric values to the parameters that describe the curve.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe values of the parameters are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e as well as the statistical values for goodness of fit of the model. The pseudo-R\u003csup\u003e2\u003c/sup\u003e for all the NaCl concentrations are very close to 1 indicating that the model had a good fit in all cases and the p-values for all the parameters are below 0.001 showing that the values have statistical significance. As expected, the lag phase (λ) increases with the concentration of NaCl. All assays were inoculated using \u003cem\u003eE. coli\u003c/em\u003e cultures grown without added NaCl, and the lag phase tends to be longer when the medium is changed \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The carrying capacity (y\u003csub\u003emax\u003c/sub\u003e) is similar up to 0.75 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl, showing that the TGY medium can support roughly the same cell population in those cases. This indicates that the energy used in the NaCl resistance mechanisms of \u003cem\u003eE. coli\u003c/em\u003e up to 0.75 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is not significant when compared with the energy supplied by a rich medium such as TGY. Nonetheless, when the NaCl concentration is higher (1.00 to 1.25 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) the carrying capacity becomes progressively lower. This shows that these concentrations are more challenging for \u003cem\u003eE. coli\u003c/em\u003e to cope with \u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\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\u003eFitting parameters for Barany' model, where \u0026micro; stands for growth rate (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), y\u003csub\u003emax\u003c/sub\u003e for carrying capacity, y\u003csub\u003e0\u003c/sub\u003e for the initial number of cells in culture, λ for lag time (h). Values marked with * were fixed manually to guarantee goodness of fitting. All the y\u003csub\u003e0\u003c/sub\u003e values (marked with \u0026dagger;) were determined experimentally, therefore were fixed. The uncertainty values are expressed as standard error.\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=\"left\" 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\u003e[NaCl] (mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFitted value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003et-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePseudo-R\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026micro;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.04∙10\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e126.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2.00∙10\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.40\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eλ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.50*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026micro;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.65∙10\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e125.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2.00∙10\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.57\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eλ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.50*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026micro;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.10∙10\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e117.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2.00∙10\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.29\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eλ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.00*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026micro;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.59∙10\u003csup\u003e\u0026minus;\u0026thinsp;09\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e151.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2.00∙10\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.63\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eλ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.94∙10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026micro;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.82∙10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2.00∙10\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.39\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eλ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.02∙10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026micro;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.46∙10\u003csup\u003e\u0026minus;\u0026thinsp;15\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2.00∙10\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.24\u003csup\u003e\u0026dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eλ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2.00∙10\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e\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\u003eHowever, the most relevant parameter is the \u0026micro; as it is the growth rate that determines the optimum growth (Perni \u003cem\u003eet al\u003c/em\u003e. 2005). The values of \u0026micro; for 0.00 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05), 0.25 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06) and 0.50 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05) NaCl are similar and greater than in any other concentration. This shows that the range of NaCl for optimal growth of \u003cem\u003eE. coli\u003c/em\u003e MG1655 is 0.00-0.50 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Beyond that range, the growth rate decreases as the NaCl concentration in the medium increases, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNaCl related genes annotation\u003c/h3\u003e\n\u003cp\u003eThe survey of salt resistance genes across the 48 \u003cem\u003eEscherichia coli\u003c/em\u003e K-12 MG1655 genomes revealed a highly conserved and comprehensive repertoire of strategies associated with NaCl stress tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Several genes were detected in all 48 genomes, including the Na⁺/H⁺ antiporters \u003cem\u003enhaA\u003c/em\u003e and \u003cem\u003enhaB\u003c/em\u003e, the trehalose biosynthesis genes \u003cem\u003eotsA\u003c/em\u003e and \u003cem\u003eotsB\u003c/em\u003e, the complete set of \u003cem\u003egln\u003c/em\u003e genes (\u003cem\u003eglnA, glnB, glnD, glnE, glnG, glnH, glnK, glnL, glnP, glnQ, glnS\u003c/em\u003e), the proline pathway genes \u003cem\u003eproC\u003c/em\u003e, \u003cem\u003eproV\u003c/em\u003e, \u003cem\u003eproW\u003c/em\u003e, \u003cem\u003eproX\u003c/em\u003e, and the regulatory sensor \u003cem\u003ephoQ\u003c/em\u003e. Importantly, genes encoding K⁺ transporters (\u003cem\u003ekdpA, kdpB, kdpD, kdpE\u003c/em\u003e and \u003cem\u003etrkA\u003c/em\u003e, \u003cem\u003etrkH\u003c/em\u003e, \u003cem\u003etrkG\u003c/em\u003e) and Cl⁻ channels (\u003cem\u003eclcA\u003c/em\u003e, \u003cem\u003eclcB\u003c/em\u003e) were also universally or nearly universally conserved, confirming that this strain retains the canonical ionic homeostasis systems typically associated with halotolerance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to this conserved core, other osmoprotectant systems were present at high frequency. The glycine betaine operon (\u003cem\u003ebetA\u003c/em\u003e, \u003cem\u003ebetB\u003c/em\u003e, \u003cem\u003ebetT\u003c/em\u003e) was detected in 46 out of 48 genomes, while \u003cem\u003eproA\u003c/em\u003e and \u003cem\u003eproB\u003c/em\u003e were found in 47 out of 48 genomes, indicating that most strains also retain multiple routes for proline and glycine betaine accumulation. These pathways contribute additional flexibility in the osmotic stress response. By contrast, alternative strategies such as ectoine biosynthesis (\u003cem\u003eectABCD\u003c/em\u003e) and trehalose degradation (\u003cem\u003etreS\u003c/em\u003e, \u003cem\u003etreY\u003c/em\u003e, \u003cem\u003etreZ\u003c/em\u003e) were completely absent from all genomes, suggesting that they are not relevant mechanisms for NaCl resistance in this lineage.\u003c/p\u003e \u003cp\u003eTaken together, these results demonstrate that \u003cem\u003eE. coli\u003c/em\u003e K-12 MG1655 harbors a robust and multifaceted molecular toolkit to counteract osmotic stress, comprising Na⁺, K⁺ and Cl⁻ transport systems with osmoprotectant accumulation and global regulators. This conserved genomic repertoire provides additional support for the phenotypic resistance to NaCl previously observed in growth curve experiments, confirming that the strain possesses the molecular machinery necessary to sustain its adaptive response under saline conditions.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur results have shown that the \u003cem\u003eE. coli\u003c/em\u003e MG1655 grows optimally at up to 0.5 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl. According to the current classification, it should be considered a slight halophile (optimal growth at 0.2\u0026ndash;0.5 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) \u003csup\u003e3\u003c/sup\u003e. Therefore, the present work brings to light that \u003cem\u003eE. coli\u003c/em\u003e MG1655, previously considered not NaCl resistant strain, should in fact be classified as an extremophile, when adopting the current predominant classification system. To further support our novel findings, we identified the presence of several genes related to osmotic stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which shows clearly the salt-resistance metabolic potential of \u003cem\u003eE. coli\u003c/em\u003e K-12 MG1655.\u003c/p\u003e \u003cp\u003eThe presence/absence analysis of NaCl stress\u0026ndash;related genes revealed a largely conserved genetic profile across the \u003cem\u003eEscherichia coli\u003c/em\u003e strains obtained from NCBI, suggesting that core mechanisms of salt tolerance are broadly distributed within the strain. Among the genes identified, those involved in osmotic homeostasis, ion transport, and the synthesis or uptake of compatible solutes were consistently detected, in agreement with previous studies highlighting their central role in adaptation of \u003cem\u003eE. coli\u003c/em\u003e to high-salinity environments \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The genes Otsa und Otsb (present in all strains analyzed) are related to trehalose synthesis, an important osmoprotectant that was also previously described on \u003cem\u003eE. coli\u003c/em\u003e salt stress response \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Earlier reports show that systems such as the betaine/glycine transporters (e.g., BetT/BetP), the ProP/ProU osmoprotectant uptake systems, and other regulators of osmotic pressure are essential for maintaining cellular volume and preventing protein or membrane damage under elevated NaCl conditions \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The recurrent detection of these genes across the analyzed strain therefore supports the interpretation that the slight NaCl tolerance observed in this work is underpinned by a conserved set of physiological and regulatory mechanisms. Overall, these findings reinforce the view that salt stress response in \u003cem\u003eE. coli\u003c/em\u003e relies on an evolutionarily stable core of genes whose widespread presence aligns with established models of bacterial osmoadaptation.\u003c/p\u003e \u003cp\u003eThrough a review of the literature, we have identified that \u003cem\u003eE. coli\u003c/em\u003e has never been appropriately classified for its NaCl resistance, as no studies providing such classification were found to the best of our knowledge. This lack of information about one of the most studied model microorganisms significantly impacts many research studies across different fields. These findings have particular impact on the field of extremophiles research, since \u003cem\u003eE. coli\u003c/em\u003e is widely used as a control organism for experimental setups (as can be seen in Fisher et al., 2025; Madigan et al., 2008; Siela \u0026amp; Smith, 2019).\u003c/p\u003e \u003cp\u003eIn addition to the above-mentioned impacts, our findings may appear controversial, as the literature suggests that \u003cem\u003eE. coli\u003c/em\u003e is a widely common bacteria present in non-saline environments \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. However, the presence of several genes related to osmotic stress shows clearly the salt-resistance metabolic potential of \u003cem\u003eE. coli\u003c/em\u003e K-12 MG1655. One possible explanation for this apparent contradiction is that the strains routinely cultivated in laboratory environments can develop genotypic and phenotypic differences when compared to other wild strains \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Although \u003cem\u003eE. coli\u003c/em\u003e K-12 substr. MG1655 can be considered close to the wild strain, since it has no direct modification, it still is a long-used laboratory strain. Therefore, the characterization regarding salt tolerance for this strain may not represent the phenotype encountered in the general population of wild \u003cem\u003eE. coli\u003c/em\u003e strains.\u003c/p\u003e \u003cp\u003eOn the other hand, our results could suggest an alternative resolution to this controversy. The data presented in this study could raise the question of the very definition of what we consider extremophile and, therefore, halophile. By the Statistical Rarity definition of extremophile, extreme environments are those that few species can thrive \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The ocean, as one of the major biomes on Earth, has an average salinity of 35 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (equivalent to 0.6 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) \u003csup\u003e51\u003c/sup\u003e. To our view, it cannot be rightly affirmed that the oceans have low biological diversity, so it should not be considered an extreme environment; by consequence, nor should 0.5 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (upper limit of optimum NaCl concentration to classify a microorganism as slight halophile) be considered an extreme physicochemical condition. This brings to light the discussion about the classification of extremophiles, especially of halophiles.\u003c/p\u003e \u003cp\u003eDue to the availability of extensive molecular data and its ease of use in laboratory settings, \u003cem\u003eE. coli\u003c/em\u003e has proven its potential and can be a powerful tool for elucidating the molecular mechanisms underlying the stress response to NaCl. Thus, we propose the study of this strain in the response to low and moderate (up to 1.25 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) NaCl concentration even though we argued that the classification of \u003cem\u003eE. coli\u003c/em\u003e as a slight halophile is questionable. This information would help us further understand organisms currently classified as slight and moderate halophiles and their molecular adaptation to osmotic stress.\u003c/p\u003e \u003cp\u003eConsidering the question proposed in the title of the manuscript, our group believes that, regardless of the current halophile classification inconsistencies discussed above, \u003cem\u003eE. coli\u003c/em\u003e str. K-12 substr. MG1655 exhibited an unexpected response to NaCl and should be considered a slight halophile by the classification used in literature \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. As mentioned before, the main current halophilic models are classified as extreme halophiles, consequently most of the known resistance mechanisms to NaCl regard these organisms. Information regarding the mechanisms activated under lower NaCl concentrations is scarce, and it is precisely in this gap that the study of \u003cem\u003eE. coli\u003c/em\u003e K-12 substr. MG1655 can make its most significant contribution. This strain shows great potential to further understand the NaCl resistance mechanisms given the amount of molecular data available and its easy handling in the laboratory. The knowledge that can emerge from this approach would be useful to biotechnology, the food industry, Astrobiology among other areas.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and media\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e str. K-12 substr. MG1655 was cultured in TGY medium. TGY liquid medium is composed of tryptone 5 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, glucose 1 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and yeast extract 3 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, sterilized by autoclave for 15 minutes at 121\u0026deg;C. Agar was added at 15 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e when solid media was necessary. We first cultivated bacterial cells in a petri dish containing TGY-agar media at 30\u0026deg;C. Then we picked a single colony, inoculated for growth at liquid TGY media at 30\u0026deg;C in a shaking incubator at 150 rpm overnight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSodium chloride exposure assay\u003c/h2\u003e \u003cp\u003eModified TGY liquid media were also prepared by the addition of NaCl in several concentrations (0.25 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 0.5 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 0.75 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1.0 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1.25 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1.5 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2.0 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 3.0 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 4.0 mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). For the assay, we added 10 \u0026micro;L of \u003cem\u003eE. coli\u003c/em\u003e str. K-12 substr. MG1655 overnight culture to 10 mL of TGY medium and all the supplemented TGY media. This culture was incubated in a shaking incubator for 24 h. Every 1 h, we collected aliquots of 50 \u0026micro;L for serial dilution (1:10) and Colony Forming Units (CFU) counting. The CFU counting was made by the Drop Plate Method \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Three independent replicas were used for each NaCl concentration.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGrowth curve model fitting\u003c/h3\u003e\n\u003cp\u003eModeling of the growth curves was done using original scripts in R v.4.3.3. For that, only curves of NaCl concentration where growth is observed were used (y\u003csub\u003e24h\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;y\u003csub\u003e0\u003c/sub\u003e). The model used was the Baranyi \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The Baranyi model has the parameters of natural logarithm of initial CFU counting (y\u003csub\u003e0\u003c/sub\u003e), carrying capacity (y\u003csub\u003emax\u003c/sub\u003e, maximum number of individuals of a species that the environment can sustain), growth rate (\u003cem\u003e\u0026micro;\u003c/em\u003e) and the length of the lag phase (\u0026#120582;). The function of the Baranyi model is given by equations 1 and \u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e2\u003c/span\u003e:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:y={y}_{0}+\\mu\\:\\bullet\\:A\\left(t\\right)-ln\\left(1+\\frac{{e}^{\\mu\\:\\bullet\\:A\\left(t\\right)}-1}{{e}^{{y}_{max}-{y}_{0}}}\\right)\\)\u003c/span\u003e \u003c/span\u003e, where(1)\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:A\\left(t\\right)=t+\\frac{1}{\\mu\\:}\\bullet\\:lnln\\:\\left({e}^{-\\mu\\:\\bullet\\:t}+{e}^{-\\mu\\:\\bullet\\:\\lambda\\:}-{e}^{\\left[-\\mu\\:\\bullet\\:\\left(t+\\lambda\\:\\right)\\right]}\\right)\\:$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThis model was fitted using native Non-linear Least Squares from R (\u003cem\u003enls\u003c/em\u003e function). All y\u003csub\u003e0\u003c/sub\u003e were determined experimentally, so the values were not given by the fitted model. Some of the \u0026#120582; were fixed with values visually determined to guarantee the best fit. Visual analysis confirmed the general quality of fitting and the pseudo-R\u003csup\u003e2\u003c/sup\u003e calculated to further assess the goodness of the Baranyi Model fitting. Even though the pseudo-R\u003csup\u003e2\u003c/sup\u003e cannot be interpreted as the proportion of data variance explained by the model, pseudo-R\u003csup\u003e2\u003c/sup\u003e (as its linear version, R\u003csup\u003e2\u003c/sup\u003e) has a maximum value of 1; the closest to 1, the better the model fits. Furthermore, the statistical significance of the fitted parameters was accessed by the p-value given by the \u003cem\u003enls\u003c/em\u003e function.\u003c/p\u003e\n\u003ch3\u003eFunctional Annotation Analysis\u003c/h3\u003e\n\u003cp\u003eFor the functional analysis, we used all genomes of the \u003cem\u003eEscherichia coli\u003c/em\u003e K-12 MG1655 strain deposited in NCBI that passed our quality control (RefSeq assignment on NCBI, contamination\u0026thinsp;\u0026le;\u0026thinsp;3%, and completeness\u0026thinsp;\u0026ge;\u0026thinsp;95%). In total, 41 genomes were included in the analysis. These genomes were subsequently annotated with Prokka (Seemann, 2014), and their corresponding .faa files were used as input for eggNOG-mapper (v2.1.6) \u003csup\u003e58\u003c/sup\u003e. EggNOG-mapper enables the mapping of protein sequences to evolutionarily conserved orthologs, assigning them to functional categories based on databases such as COG (Clusters of Orthologous Groups), KEGG (Kyoto Encyclopedia of Genes and Genomes), and GO (Gene Ontology).\u003c/p\u003e \u003cp\u003eBased on the eggNOG-mapper outputs, we performed a curated search for genes related to NaCl resistance, based on relevant literature. The list of genes and the salt-resistance strategy categories assigned to them are provided in the Supplementary Information. From this filtering, we generated a presence\u0026ndash;absence matrix of the genes of interest using a custom Python script, also available in the Supplementary Information.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eFunding for the study was provided by Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo (FAPESP 16/06160-8 and 2025/11833-0), Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq 140086/2018-8 and 170387/2018-6) and Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES 888887.570298/2020-00). The author would like to thank Brazilian Research Unity in Astrobiology (NAP/Astrobio).\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAna Paula Muche Schiavo:\u0026nbsp;\u003c/strong\u003econceptualization (lead), formal analysis (lead), investigation (equal), project administration (lead), visualization (lead), writing \u0026ndash; original draft (lead), writing \u0026ndash; review \u0026amp; editing (equal). \u003cstrong\u003eRoberta Almeida Vincenzi\u003c/strong\u003e: formal analysis (supporting), investigation (equal), project administration (supporting), visualization (supporting), writing \u0026ndash; original draft (supporting), writing \u0026ndash; review \u0026amp; editing (equal). \u003cstrong\u003eIsabella Gai\u0026atilde;o da Silva\u003c/strong\u003e: investigation (equal), project administration (supporting), visualization (supporting), writing \u0026ndash; original draft (supporting), writing \u0026ndash; review \u0026amp; editing (equal). \u003cstrong\u003eFabio Rodrigues\u003c/strong\u003e: funding acquisition (lead), resources (lead), supervision (lead) and writing \u0026ndash; review \u0026amp; editing (supporting).\u003c/p\u003e\n\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The datasets and code generated during the current study are available in Supplementary Information.\u003c/p\u003e\n\u003cp\u003eAdditional Information\u003c/p\u003e\n\u003cp\u003eNo competing interests are declared.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHarrison, J. 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Evol\u003c/em\u003e \u003cb\u003e38\u003c/b\u003e, (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":"sodium chloride, extremophile, growth rate, E. coli, halophilic","lastPublishedDoi":"10.21203/rs.3.rs-8882295/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8882295/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExtreme environments are defined by conditions that challenge cellular machinery, often compromising survival and biological function. Halophiles, a subclass of extremophiles, thrive in high sodium chloride (NaCl) concentrations. However, current knowledge on salt stress resistance is largely derived from studies on extreme halophiles, even though most halophilic microorganisms are classified as slight or moderate halophiles. This fact poses a question regarding the real representation of state-of-the-art information about salt resistance mechanisms and diversity. To bring light to the problem of the lack of information on slight halophiles, we propose the study of \u003cem\u003eEscherichia coli\u003c/em\u003e str. K-12 substr. MG1655 to fill the gap. We evaluated the response of \u003cem\u003eE. coli\u003c/em\u003e MG1655 to varying NaCl concentrations, including control without NaCl addition. Our results indicate optimal growth at NaCl concentrations up to 0.5 mol\u0026middot;L⁻\u0026sup1;, suggesting that this strain should be classified as a slight halophile, in contrast to its current classification in the literature. Thus, we propose the study of this strain to understand the molecular mechanisms underlying adaptation to low and moderate salt stress.\u003c/p\u003e","manuscriptTitle":"Should Escherichia coli K-12 substrain MG1655 be classified as NaCl resistant?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-12 21:30:45","doi":"10.21203/rs.3.rs-8882295/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":"5e65caa8-0786-4277-9365-5673ccb45893","owner":[],"postedDate":"March 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":64209812,"name":"Biological sciences/Biochemistry"},{"id":64209813,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-01T12:57:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-12 21:30:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8882295","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8882295","identity":"rs-8882295","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}