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. 2018 Aug 20:9:1919.
doi: 10.3389/fmicb.2018.01919. eCollection 2018.

Transcriptomic Insights Into the Growth Phase- and Sugar-Associated Changes in the Exopolysaccharide Production of a High EPS-Producing Streptococcus thermophilus ASCC 1275

Aparna Padmanabhan  1 , Ying Tong  2 , Qinglong Wu  1 , Jiangwen Zhang  2 , Nagendra P Shah  1
Affiliations

Transcriptomic Insights Into the Growth Phase- and Sugar-Associated Changes in the Exopolysaccharide Production of a High EPS-Producing Streptococcus thermophilus ASCC 1275

Aparna Padmanabhan et al. Front Microbiol. .

Abstract

In a previous study, incorporation of high exopolysaccharide (EPS) producing dairy starter bacterium Streptococcus thermophilus ASCC 1275 was found to improve functionality of low fat mozzarella cheese and yogurt. This bacterium in its eps gene cluster has a unique pair of chain length determining genes, epsC- epsD, when compared to other sequenced S. thermophilus strains. Hence, the aim of this study was to understand the regulatory mechanism of EPS production in this bacterium using transcriptomic analysis to provide opportunities to improve the yield of EPS. As sugars are considered as one of the major determinants of EPS production, after preliminary screening, we selected three sugars, glucose, sucrose and lactose to identify the EPS producing mechanism of this bacterium in M17 medium. Complete RNA-seq analysis was performed using Illumina HiSeq 2000 sequencing system on S. thermophilus 1275 grown in three different sugars at two-time points, 5 h (log phase) and 10 h (stationary phase) to recognize the genes involved in sugar uptake, UDP-sugar formation, EPS assembly and export of EPS outside the bacterial cell. S. thermophilus 1275 was found to produce high amount of EPS (∼430 mg/L) in sucrose (1%) supplemented M17 medium when compared to other two sugars. Differential gene expression analysis revealed the involvement of phosphoenolpyruvate phosphotransferase system (PEP-PTS) for glucose and sucrose uptake, and lacS gene for lactose uptake. The pathways for the formation of UDP-glucose and UDP-galactose were highly upregulated in all the three sugars. In the presence of sucrose, eps1C1D2C2D were found to be highly expressed which refers to high EPS production. Protein homology study suggested the presence of Wzx/Wzy-dependent EPS synthesis and transport pathway in this bacterium. KEGG pathway and COG functional enrichment analysis were also performed to support the result. This is the first report providing the transcriptomic insights into the EPS production mechanism of a common dairy bacterium, S. thermophilus.

Keywords: RNA-seq; S. thermophilus ASCC 1275; exopolysaccharide; sugars; transcriptomics.

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Figures

FIGURE 1
FIGURE 1
(A) EPS production of S. thermophilus 1275 in glucose (G-1%), sucrose (S-1%), lactose (L-1%) supplemented Ml7 media at different time points. Sugar utilization and lactic acid production in glucose (B), sucrose (C) and lactose (D) media.
FIGURE 2
FIGURE 2
Venn diagram showing the number of differently expressed genes (>1.5 fold change, P < 0.01) during growth in M17 media supplemented with different sugars (glucose, sucrose, lactose) at logarithmic growth phase (5 h) and stationary growth phase (10 h).
FIGURE 3
FIGURE 3
Volcano plots showing DEGs of S. thermophilus 1275 at G10h vs. G5h (a), S10h vs. S5h (b), L10h vs. L5h (c), G5h vs. S5h (d), G5h vs. L5h (e), S5h vs. L5h (f), G10h vs. S10h (g), G10h vs. L10h (h), S10h vs. L10h (i).
FIGURE 4
FIGURE 4
The DEGs in PTS transport system under die influence of different sugars at the two-time points (A) Heat map of DEGs involved in sugar transport at 5 h. (B) Heat map of DEGs involved in sugar transport at 10 h.
FIGURE 5
FIGURE 5
EPS biosynthesis pathway of S. thermophilus 12 75 in the presence of three selected sugars, glucose, sucrose and lactose. Gene annotations:T303_09510 p-fructofuranosidase, T303_07865 p-galactosidase, T303_04850 glucokinase. T303_05140 phosphoglucomutase, T303_00105 UDP-glucose pyrophosphorylase, T303_07880 UDP-glucose 4-epinierase, T303_06690 UDP-galactose-4-epimerase, T303_07885 Galactose-1-phosphate uridylyltransferase, T303_07875 Galactose mutarotase, T303_07890 Galactokinase, T303_09500 Fructokinase, T303_06845 6-phosphofructokinase, T303_02195 Phosphoglucoseisomerase, T303_05515 glucosamine-fructose-6-phosphate aminotransferase, T303_07195 Phosphoglucosamine mutase, T303_03955 N-acetylglucosamine-1-phosphate uridyltransferase (bifunctional), T303_06336 UDP-galactopyranose mutase.
FIGURE 6
FIGURE 6
Gene expression pattern of EPS assembly genes, (A) EPS gene cluster of S. thermophilus 1275 (Wu et al., 2014). (B) Heat map of DEGs in EPS gene cluster at 5 h in the presence of glucose, sucrose and lactose, (C) Heat map of DEGs in EPS gene cluster at 10 h in the presence of glucose, sucrose, and lactose.
FIGURE 7
FIGURE 7
Changes in expression of genes associated with nucleotide sugar synthesis and carbohydrate metabolism. (A) Heat map of DEGs involved in nucleotide sugar synthesis and carbohydrate metabolism at 5 h in the presence of glucose, sucrose, and lactose. (B) Heat map of DEGs involved in nucleotide sugar synthesis and carbohydrate metabolism at 10 h in the presence of glucose, sucrose, and lactose.
FIGURE 8
FIGURE 8
Changes in expression of genes associated with ammo acid metabolism. (A) Heat map of DEGs involved in amino acid metabolism at 5 h in the presence of glucose, sucrose and lactose. (B) Heat map of DEGs involved in amino acid metabolism at 10 h in the presence of glucose, sucrose, and lactose.
FIGURE 9
FIGURE 9
KEGG pathway enrichment analysis of DEGs 5h vs. G10h (A), L5h vs. L10h (B), S5h vs. S10h (C), G5h vs. S5h (D), G5h vs. L5h (E), S5h vs. L5h (F), G10h vs. S10h (G), G10h vs. L10h (H), S10h vs. L10h (I).
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