Genomic analysis of Lactobacillus reuteri WHH1689 reveals its probiotic properties and stress resistance

Abstract Lactobacillus reuteri (L. reuteri) WHH1689, which was isolated from Chinese traditional highland barley wine, exhibited high survival period at room temperature in drinkable probiotic yogurt. This article aimed to indicate the genes involved in probiotic function of WHH1689 and reveal potential stress resistance based on genomic analysis. Analysis of comparative genome with closely related L. reuteri strains identified special stress adaptation. MUMmer and ACT softwares were applied for collinear analysis, and OrthoMCL program was used for sequence alignment involved in distribution of protein cluster. We identified genes coding for carbohydrate transport and enzymes, carbon metabolism pathway, gastrointestinal tract resistance, adhesive ability, and folic acid biosynthesis, etc. Genome sequence and comparative genome analysis of L. reuteri WHH1689 demonstrated specific genes for genetic adaptation and stress resistance. Tolerance, adhesion, and folate test indicated the strain had multiple probiotics. L. reuteri WHH1689 has the potential to be a probiotic candidate in dairy foods.


| Bacterial strain and cell line
The whole genome sequence of L. reuteri WHH1689 have been previously reported and deposited at Gen Bank with accession number CP027805 (Chen, Chen, Chen, Ren, Ge, Kang, et al., 2018).
The genomic sequences of other four L. reuteri strains are available from NCBI data base (http://www.ncbi.nlm.nih.gov/): L. reuteri
Based on the results of homologous gene analysis, single copy was selected for multi-sequence comparison and quality control using MAFFT software (Katoh & Toh, 2008). Then the phylogenetic tree was constructed with the RAxML software (Stamatakis, 2006).

| Bioinformatic analyses
OrthoMCL v2.0.3 software was used to compare the amino acid or nucleotide sequences of all the species involved in the analysis (Li, Stoeckert, & Roos, 2003). The threshold value was selected for similarity clustering to obtain gene homology (Fischer et al., 2011). The distribution of species in each proteome cluster can be counted to conduct genomic analysis within the core genomes of genus or species. Co-linear analysis of more genome sequences was performed using MUMmer 3.0 or ACT software (Hu et al., 2006;Toropov, Vakhitov, Shalaeva, Roshchina, & Sitkin, 2018).

| Tolerance test
Tolerance test was examined by pH, bile salt, and osmotic pressure.
Sodium chloride has strong water-reducing activity could effect on osmotic pressure. For resistance to osmotic stress, bacterial culture was collected as above and resuspended in 6.0%, 7.0%, 8.0% sodium chloride solution. Bacterial cells were cultured at 37°C for 24 hr.
Stress resistance was assessed by bacterial survival.

| Effect of high temperature on Lactobacillus strains
Lactobacillus reuteri WHH1689 and L. rhamnosus LGG were independently propagated and inoculated 2 ml into 200 ml of MRS.
Bacteria suspension were, respectively, cultured at 45 and 50°C for 0-30 days. Effect of high temperature on Lactobacillus strains were determined by bacterial counts.

| Scavenging of hydroxyl radical
The ability of the hydroxyl radical scavenging assay was determined using a Fenton reaction method (Rao, Giri, Goud, & Golder, 2016). L. reuteri WHH1689 and L. rhamnosus LGG were prepared at concentration ranging from 10 7 to 10 9 CFU/ml. Intracellular extracts were obtained by ultrasonic broken in ice bath. The reaction mixture containing 1.5 ml bright green reagent (0.5 mM), 1.0 ml FeSO4 (1.0 mM), 1.0 ml H 2 O 2 (2.5%, w/v) and 0.5 ml of bacteria extract in different concentration was immediately mixed and cultured at room temperature for 30 min. The scavenging ability for hydroxyl radical of stains was measured by the absorbance at 625 nm.
A i shown the absorbance of the sample, A 0 illustrated absorbance of the control without sample, and A represented the absorbance without Fenton reaction system and e sample.

| Scavenging of DPPH radical
The DPPH scavenging assay was investigated according to the method (Lin & Chang, 2000). Intracellular extracts of Lactobacillus strains were collected as above. The reaction mixture was added 2 ml DPPH free radical ethanol solution (0.1 mM) and intracellular extracts of different concentration. Then the solution were quickly mixed and reacted at room temperature in the dark for 60 min. The supernatant was collected by centrifugation (10,000 × g for 10 min) and measured for absorbance at 517 nm. Phosphate buffer (PBS) was used as a blank control.
A i demonstrated the absorbance of the sample, A 0 shown the absorbance of the control.

| Adherence assay
Adhesion of the strains was assayed according to the reported method (Kim, Oh, Park, & Kim, 2009). HT-29 cell was prepared in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum in 24-well tissue culture plates at 1.0 × 10 6 cells/well concentration. L. reuteri WHH1689 and L. rhamnosus LGG at concentration ranging from 10 6 to 10 9 CFU/ml were added into cell solution. The plates were incubated at 37°C for 2 hr under aerobic condition (5% CO 2 /95% air atmosphere). The monolayer was washed three times with sterile PBS. For detaching, 0.05% Triton X-100 diluted in sterile solution which was used for pipetting adherent bacteria. L. rhamnosus LGG was control stain and every assay was performed in three times.
Hydroxyl radical scavenging rate (%) = Phylogenetic tree of L. reuteri WHH1689 constructed from Lactobacillus strains based on MAFFT

| Folic acid assay
The ability of folate produced by strains was determined by folic acid assay medium. L. plantarum LZ227 and L. reuteri WHH1689 were propagated in MRS broth at 37°C for 18 hr. The bacterial cells were collected by centrifugation (10,000 × g for 10 min) and washed twice with 0.01 M PBS (pH 7.2). A 0.5% inoculum of culture was, respectively, distributed into folic acid assay medium at 37°C in dark for 24 hr. The folate content of cell supernatant and intracellular extracts was assayed by Vita Fast ® Folic acid (IFP, R-Biopharm, Germany).

| Genome features of L. reuteri WHH1689
The circular genome of WHH1689 is comprised of 2,196 genes which were predicted with the average length of 814 bp. GC content accounts for 34.5% in inter genetic region. A total of 24 genomic islands involved in hundreds of functional genes including transporter, membrane protein, some enzyme, heavy metal resistance, and putative protein gene (Liu et al., 2015;Yoo et al., 2017). A phylogenetic F I G U R E 2 Circular genome map of L. reuteri WHH1689. From the inner circle: the first circle presents the GC skew (G + C/G−C), values >0 in green and values <0 in purple. The second circle depicts the GC content. The third circle depicts CRISPR repeats in black. The fourth circle highlights rRNA and tRNA, the color related to COG functional classification. The fifth to seventh circles denotes the sites of CDS tree of WHH1689 revealed the genetic evolution between different L. reuteri strains ( Figure 1). It showed that different branch with L. reuteri strains. L. reuteri ZLR003 and I5007 formed the closest genetic relation with WHH1689. The circular genome map of L. reuteri WHH1689 was showed the genome distribution ( Figure 2). The protein coding genes of WHH1689 were predicted by KEGG annotation and functionally categorized ( Figure 3). In the live organism, gene products do not exist in isolation. Different gene products perform specific biological functions together through orderly coordination.

| Carbon metabolism and carbohydrate transporter
Lactobacillus reuteri belongs to functional LAB and is able to metabolize carbohydrate to produce lactic acid. The carbon metabolism of Lactobacillus include pathway and complex enzyme was important to the utilization of carbohydrates. WHH1689 genome annotation were adopted by GO function categories, and 118 genes involved in carbohydrate transport and metabolism.
The 54 genes encoding energy production and conversion were F I G U R E 3 Histogram of KEGG. Histogram presents the number of genes involved in KEGG pathway. The colors indicates different systems, mazarine refers to cellular processes, bottle green depicted metabolism, red illustrates genetic information processing, purple denotes human diseases, blue highlights organismal system, and reseda shows environmental information processing indentified. These transporters consists variety pathway, PEP-PTS (phosphoenolpyruvate-phosphotransferase systems), ABC transporter, and Permease. PEP-PTS transporter was related to the majority of sugars including sucrose, fructose, glucose (Liu et al., 2015;Oberholzer et al., 2005). ABC transporter mainly could make ATP hydrolysis energy with substrate transfer into or out of cells (Roos et al., 2010;Santos et al., 2018). Carbohydrate transporter WHH1689 encodes 15 genes involved in phosphoenolpyruvate synthase and 13 genes for protein phosphotransferase. The 73 genes of ABC transporter in WHH1689 was involved in transporting, including amino acid, ATP-binding protein, and ABC transporter permease.
It is notable that the 29 genes of encoding major facilitator superfamily (MFS) transporter in WHH1689 were indentified. MFS belongs to the secondary transporter family which has effect on physiological processes (Yan, 2013). These results indicate WHH1689 has extensive carbohydrate transporter which could adapt to metabolism pathways.

| Carbohydrate-active enzymes (CAZymes)
Carbohydrate-Active Enzymes (CAZymes) database was used to analyze genome getting biological information on carbohydrate enzymes. It plays a crucial role in host carbohydrates and intestinal microbe with encoding genes. CAZymes can be found in organisms and nature product, particularly abundant in microorganism (Ardèvol & Rovira, 2015). The CAZymes of WHH1689 contains 21 genes encode glycoside hydrolases (GHs) and 18 genes encode glycosyl transferases (GTs

| Stress resistance analysis
Lactic acid bacteria are beneficial for human because they have strong tolerance when strains entering gastrointestinal could survive and colonize. L. reuteri WHH1689 exhibited higher resistance in simulating gastrointestinal tract and viable counts could be tested after 4 weeks Chen, Chen, Chen, Ren, Ge, Kang, et al., 2018). Stress regulation mechanism was involved in different aspects, including pH, temperature, osmotic pressure, bile salt, oxidation. The stress-related proteins not only can reveal genetic adaptation but also regulate evolution resistance (Boden & Merali, 2001). Analysis of stress-related proteins of L. reuteri WHH1689 was shown in Table 2. WHH1689 contains two genes related to alkaline phosphatase and two genes encode alkaline shock protein Asp23 which is linked to cell membrane improving Gram-positive bacteria tolerance. Moreover, five genes encode sodium-proton antiporter which evaluated the energy of the Na + and H + movement for converting the function of transporters, five genes encoded F0F1 ATP synthase which was related with ATP synthesis utilizing ion translocation . The potential functional information was obtained from genome of WHH1689.
Tolerance test was necessary to verify the functionality of the strain. We studied the stress resistance of L. reuteri WHH1689 which showed stable vitality to different treatment (Table 3)
Two genes encoded that choloylglycine hydrolase and inorganic pyrophosphatase which were involved in CBAs. In this paper, effect of bile LGG was used as control strain. Figure 4   consistent with the results, which showed that WHH1689 may be a potential strain that can survive in high temperature.
Oxidative stress refers to the essential balance in aerobic metabolism. Seven genes encoded oxidase, reductase, and dehydrogenase were identified which may have potential oxidation resistance (Sachan, Johnsen, & Hongu, 2012). We studied the scavenging for hydroxyl radical and DPPH radical of strains. Figure 5a showed scavenging for hydroxyl radical of LGG and WHH1689. L. rhamnosus GG (LGG) proved to have significant effect on antioxidants and superoxide dismutase which was used as for contrast strain (Goyal, Rishi, & Shukla, 2013). Both the strains demonstrated scavenging activity of hydroxyl radical in the concentration range of 10 7 -10 9 CFU/ml. With the counts of bacteria increased, the scavenging rate showed an upward trend. The maximum rate was occurred at the concentration range of 10 9 CFU/ml. The strains WHH1689 and LGG, respectively, had scavenging of 48.68% ± 0.16 and 49.06% ± 0.09. The results were not significant between two strains indicated WHH1689 was capable for scavenging hydroxyl radical.
For DPPH radical in Figure 5b,

| Adhesion ability
The adhesion of LAB indicated that cells can adhere to small intestinal epithelial cell surface, and the mechanism was related to hydrophobicity and potentially surface exposed (PSE) proteins.
PSE protein play crucial role in adhesion or binding to cell surface (Barinov et al., 2009). The genome analysis showed that WHH1689 contained gene encode fibronectin-binding protein (orf00991), lipoprotein signal peptidase (orf00987, orf01257), maltose phosphorylase (orf00060), triosephosphate isomerase (TPI, orf00265, orf00451, orf00189). TPI was associated with glycolysis which can be released to organism for acclimatization and improve adhesion ability (Helfert, Estévez, Bakker, Michels, & Clayton, 2001)  We examined the adhesion ability of WHH1689 and LGG at different concentrations to HT-29 epithelial cells in Table 4. From 10 6 to 10 9 CFU/ml, both of the strains showed high adhesion rate. The

| Folic acid biosynthesis genes
Folic acid is commonly found in all kinds of food such as plant, vegetable, fruit, and meat food which is an important substance involved in nucleic acid synthesis and cell differentiation. The most important physiological function of folic acid is the influence of deficiency on the development of fetal nervous system. The majority of LAB was folic acid deficient strains, however, some have the ability to synthesize it. A series of enzymes catalyzed guanine nucleoside triphosphate (GTP) production by purine metabolism could form folic acid (Bolin & Cardozo-Pelaez, 2007). In WHH1689 genome, we found that enzymes related to GTP pathway, folA (orf00877), folB (orf01353), folC (orf01350, orf00595), folD (orf01235), folE (orf01351), folk (orf01352), folP (orf01348), suggesting potential folic acid biosynthesis of the strain (Licciardi, Tang, Billingham, Armes, & Lewis, 2005). We tested the ability of folic acid production by L. reuteri WHH1689. L. plantarum LZ227 has been reported as probiotic strain producing B-group vitamins used as the comparative strain in Figure 6 (Li, Zhou, & Gu, 2016). Both of the two strains grew well on folic acid assay medium indicated WHH1689 could produce folate. The folic acid content of cell supernatants produced by WHH1689 was 476.0 μg/L, and intracellular extracts had a folate content of 27.5 μg/L.

| Comparative genomic analysis of special genes
We selected L. reuteri DSM 20016, L. reuteri TD1, L. reuteri SD2112, L. reuteri I5007 for comparative genomic, which have been whole genome-sequenced and deposited in GenBank. The amount and annotation of special genes have been showed base on comparative genome (  (Szabó, Kiss, & Olasz, 2010). The IS30 family present in most bacteria including Gram-positive and Gram-negative bacteria and they could be distributed in various Lactobacillus spp (Kumar, Grover, Kaushik, & Batish, 2014). Furthermore, IS30 elements have been proved to be associated with environmental adaption and stress resistance, due to hypothetical genes involved in carbohydrate metabolism which was benefit for gastrointestinal microflora colonization (El et al., 2012). Special genes of WHH1689  (Lysnyansky et al., 2009). Transposition of ISL3 could potentially influence on the expression of adjacent genes (Morel et al., 2017).
Therefore, specific genes mostly related to the ISs family speculated WHH1689 could be of benefit for genetic adaptation and stress resistance.

| Diversity of adaptation
Comparative genome of L. reuteri display diversity of lifestyle in cellular component, molecular function, and biological progress. The 285 kb region from 105,600 to 125,400 exclusively encodes special genes of WHH1689. The region is much longer than other L. reuteri strains (Figure 7). Moreover, this whole region has the higher GC and metQ could provide various transport pathways and improve adaptation (Gunnewijk & Poolman, 2000;Hollenstein, Frei, & Locher, 2007). This special region reveals WHH1689 could be capable for stress resistance and adapt effectively to different environmental conditions.

| CON CLUS IONS
Lactobacillus reuteri WHH1689 isolated from Chinese traditional which has high viability and low postacidification in a roomtemperature-storage drinkable. In this paper, we revealed genes related to carbon metabolism pathway, folic acid biosynthesis, stress resistance and adaptation diversity based on comparative genomic analysis. Functional assay confirmed gene prediction identified probiotic properties of WHH1689. These results could provide genetic basis for long-term survival and probiotic function, especially in environment adaptation. Further work we will be needed to research on transcriptome and metabonomics. Transcriptome profiling analysis may reveal metabolic changes across various growth conditions in Lactobacillus strains.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interests to declare.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.
F I G U R E 7 Diversity of adaptation region genes in L. reuteri strains. Functional gene of adaptation diversity based on comparative genome. The red and blue region illustrated high sequence identity and reverse direction. The genes including fructosyltransferase (sacB), hydroxyethylthiazole kinase (thiM), glycosyl transferase family (glt), PTS sugar transporter (pts), and methionine ABC transporter (ABC)