Transcriptional Regulation of Exopolysaccharide- related Genes in Lactiplantibacillus Plantarum VAL6 Under Environmental Stresses

Trung-Son Le Can Tho University Phu-Tho Nguyen An Giang University, Vietnam National University of Ho Chi Minh City Song-Hao Nguyen-Ho Can Tho University Tang-Phu Nguyen Can Tho University Thi-Tho Nguyen Hutech University My-Ngan Thai An Giang University, Vietnam National University of Ho Chi Minh City To-Uyen Nguyen-Thi Can Tho Science and Technology appliaction Center Minh-Chon Nguyen Can Tho University Quoc-Khanh Hoang Institute of Tropical Biology NGUYEN Huu-Thanh (  nhthanh@agu.edu.vn ) An Giang University https://orcid.org/0000-0003-0179-0705


Introduction
Lactic acid bacteria (LAB) are ubiquitous appearance in food by their generally recognized as safe status (Ismail and Nampoothiri 2010). LAB are capable of producing exopolysaccharides (EPSs) which are widely used in various applications (Baruah et al. 2016). EPS production in LAB has been received a lot of attention in recent years due to the unique biological properties of these biopolymers ). This is exempli ed in Lactiplantibacillus plantarum, which is commonly utilized in the health and food areas owning to its ability to produce EPSs with speci c functions ).
In nature as well as in industrial applications, LAB are often exposed to adverse environmental conditions In LAB, EPSs play an important role in protecting the cells from harsh environmental conditions (Nguyen et al. 2020). In addition, EPSs are also involved in the formation of bio lm and adhesion ) as well as in determining cell interaction characteristics (Lee et al. 2016). Nonetheless, the structure and biological function of EPSs may vary depending on environmental conditions (Vu et al. 2009) that related to the transcriptional levels of eps genes (Boels et al. 2003).
EPS synthesis in LAB is a complex process involved in the regulation of expression of EPS-related genes (Zeidan et al. 2017). Furthermore, changing environmental conditions can alter the expression level of these genes (Wu and Shah 2018). Therefore, in this study, we evaluated the impact of various environmental challenges on the expression levels of genes involved in EPS synthesis in L. plantarum VAL6. The overall aim of the study was to clarify the potential relationships between these regulatory systems and environmental stresses.

Materials And Methods
Bacterial strain and culture conditions L. plantarum VAL6 was obtained from Department of Biotechnology, An Giang University, Vietnam National University Ho Chi Minh City, Vietnam. To perform microbial cell culture for this study, L. plantarum VAL6 was grown in Man-Rogosa-Sharpe medium (MRS) (De Man et al. 1960). L. plantarum VAL6 was stored at -80°C; it was rehydrated in MRS broth with 2% (vol/vol) inoculum, followed by incubating at 37°C for 24 h, agitation rate was set up to 250 rpm under aerobic facultative condition.

Bioreactor operating conditions for stress treatments
The examination was carried out in 5-L bioreactors (BIOSTAT. Sartorius Stadium, Germany). Brie y, 5 L of MRS medium was inoculated with 100 mL of overnight bacterial culture (OD 595 = 1.5). The pH was maintained to 6.8 by regularly adding 10 M NaOH, the temperature was kept at 37 o C, and agitation rate was set up to 250 rpm. After 24 h of culture, stress treatments were then performed independently: For thermal stress, the culture was treated with high temperature either 42 or 47°C for 7 h. The time was calculated when the bioreactor reached the required stress temperature. The pH was maintained to 6.8 and the temperature was kept at 37 o C.
For pH stress, the culture was treated with pH conditions either pH 3 or pH 8 for 7 h. The time was calculated when the bioreactor reached the required pH. The temperature was kept at 37 o C.
For NaCl stress, the culture was treated by adding NaCl to reach 4-10% concentrations for 7 h. The pH was maintained to 6.8 and the temperature was kept at 37 o C.
The non-stress control of these treatments was simultaneously carried out in another bioreactor, where the pH was maintained to 6.8, the temperature was maintained at 37 o C for the entire time.
In the case of CO 2 treatment, right after inoculation, CO 2 was continuously supplied at the rate 250 cm 3 /min for 4 h or 8 h. After the set time, the cultures were continued until 24 h without CO 2 supplement.
The 24-hour CO 2 treatment was supplied with CO 2 (250 cm 3 /min) during the culture period. The nonstress control of this treatment was also carried out at the same time without CO 2 supplement. Extraction of total RNA and synthesis of rst-strand cDNA Total RNA was extracted according to the instructions of TRIzol reagent (Invitrogen, UK). RNA was treated with RQ1 RNase-free DNase (Promega, Madison, USA) to remove contamination of chromosomal DNA. Qualitative test of RNA at 260 and 280 nm was found to be more than 1.8 using a NanoDrop DeNovix DS-11 Spectrophotometer (DeNovix, Wilmington, DE USA). The rst-strand cDNA was synthesized according to the instructions of the GoScript™ Reverse Transcription System Kit (Promega, Madison, USA).

Design and synthesis of primers
The reference gene was selected from the housekeeping gene, i.e. 16S rRNA. Primers of the six target genes were designed for quantitative real-time polymerase chain reaction (qPCR) analysis based on the genome sequence of L. plantarum WCFS1 (Genbank: AL935263.2) using Primer-BLAST software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primers were synthesized by The Shanghai Brilliance Biotechnology Co. Ltd.. The primers were designed with sequence length from 18-23 nucleotides, coupling temperature (T m ) from 57-60°C and GC rate not more than 70% (Table 1).

Statistical analysis
The experiments were repeated three times. All the data were expressed as means ± standard deviations.
Signi cance of difference was evaluated with one way ANOVA, followed by Fisher's least signi cant difference (LSD) procedure to identify statistically signi cant differences at the 95.0% con dence interval. One-way analysis of variance was performed. LSD multiple-range tests were applied to the individual variables to compare means and to assess if there was a signi cant difference.

Results
The expression of EPS-related genes under thermal stress The effect of high-temperature stresses (42 and 47 o C) on the expression of EPS-related gene in L. plantarum VAL6 was investigated via RNA sequencing. Through analysis and comparison of the gene expression pro le of the stressed L. plantarum VAL6 and the control (37 o C), we found that cps4F, cps4E and cps4J genes were signi cantly (p < 0.05) upregulated in response to thermal stress. Also, the exposure time played an important role in the regulation of these genes (Fig. 1).
The expression level of cps4E (Fig. 1d) (Fig. 1c) was 27.6-fold under 42 o C treatment and 32.8-fold under 47 o C treatment. However, the expression levels of two genes decreased after exposure to thermal stress for 7 h. Meanwhile, the expression level of cps4J was steadily increased over time of stress and reached more than 18-fold after 7 h of treatment (Fig. 1e).

The expression of EPS-related genes under acid or alkaline stress
In order to survive and adapt to acid or alkaline stress, microorganisms have developed complex mechanisms at physiological and molecular levels (Guan and Liu 2020). In this work, the response of L. plantarum VAL6 to acid or alkaline stress via the transcriptional analysis of EPS-related genes was also studied. The results disclosed that cps4H was signi cantly (p < 0.05) upregulated in exposure to acid at pH 3. Meanwhile, cps4F and cps4E were signi cantly (p < 0.05) upregulated in response to alkaline stress condition (Fig. 2).
Under stress at pH 3, there was a great increase in the expression level of cps4H (~ 4-fold) (Fig. 2f), but the expression levels of glmU, pgmB1 and cps4E decreased (Fig. 2a, Fig. 2b and Fig. 2e). In the case of stress at pH 8, the expression level of cps4F increased steadily from 2-fold at 1 h to 3.3-fold at 7 h (Fig. 2c). As a similar pattern with cps4F, the expression level of cps4E was also rose gradually from 2.8 to 3.9-fold during the time of stress (Fig. 2d).
The expression of EPS-related genes under NaCl stress The alteration in gene expression under osmotic stress is also to adjust cellular metabolisms (Le Marrec 2011). Therefore, it is important to consider transcriptional regulation in exposure to osmotic stress. In this study, we also investigated the effect of the addition of NaCl at different concentrations on the expression of genes involved in EPS synthesis in L. plantarum VAL6. The overall results indicated that the expression of glmU, pgmB1, cps4J and cps4H genes were signi cantly (p < 0.05) downregulated in exposure to NaCl at 7-10% concentrations (Fig. 3).

The expression of EPS-related genes under CO 2 intensi cation culture
Unlike other stress treatments, CO 2 intensi cation culture increased the expression of all tested genes between 1.2 and 3-fold. Furthermore, with the exception of cps4E, the other genes were upregulated with increasing time of CO 2 supplementation (Fig. 4). The highest expression level of cps4E was 1.8-fold upon exposure to 8-hour CO 2 treatment, while its expression returned to 1.5-fold upon exposure to 24-hour CO 2 treatment (Fig. 4d).

Discussion
Changes in environmental conditions may alter extracellular polysaccharide production (Lloret et al. 1998) and induce the synthesis of new type of EPSs in bacteria (Nandal et al. 2005). In addition, the overexpression of a certain gene involved in EPS synthesis can increase or decrease the level of a speci c sugar component in EPSs (Nguyen et al. 2021). Hence, based on gene expression pro les, it is possible to predict the alteration of monosaccharide composition in EPSs.
In this study, glmU was only upregulated under CO 2  The pgmB1 encodes for β-phosphoglucomutase which catalyzes the interconversion of D-glucose 6phosphate and D-glucose 1-phosphate to form beta-D-glucose 1,6-(bis)phosphate. pgmB1 plays an important role in the formation of sugar nucleotides as UDP-glucose (Li et al. 2019). Thus, the overexpression of pgmB1 may result in increased glucose content in EPS composition. In our study, the expression of pgmB1 increased when L. plantarum VAL6 was exposed to CO 2 intensi cation culture but decreased in exposure to stresses at pH 3 and at 7-10% NaCl. Summary, our results revealed that different environmental conditions can alter the expression level of genes involved in EPS synthesis. Based on achieved results, we propose a pro le for the changes in the expression of EPS-related genes in L. plantarum VAL6 by applying environmental challenges ( Table 2). The expression of these genes may lead to changes in the monosaccharide composition of EPSs. However, it is necessary to further study the expression levels of the respective proteins in order to better understand the potential relationships between these regulatory systems and environmental stresses.