Abstract
Glycosyltransferases are key enzymes involved in the assembly of repeating units of exopolysaccharides (EPS). A glycosyltransferase generally consists of the N-terminal and the C-terminal domain, however, the functional role of these domains in EPS biosynthesis remains largely unknown. In this study, homologous overexpression was employed to investigate the effects of EpsFN, a truncated form of rhamnosyltransferase EpsF with only the N-terminal domain, on EPS biosynthesis in Streptococcus thermophilus 05-34. Reverse transcription qPCR and Western blotting analysis confirmed the successful expression of epsFN in 05-34 at the transcription and translation level, respectively. Further analysis showed that the monosaccharide composition and yield of EPS were not affected by the overexpression of epsFN, whereas the molecular mass decreased by 5-fold. Accordingly, the transcription levels of genes involved in EPS biosynthesis, including chain-length determination gene epsC, were down-regulated by 5- to 6-fold. These results indicated that the N-terminal domain of EpsF alone could influence the molecular mass of EPS, probably via lowering the concentration of sugar precursors, which may lead to decreased expression of genes responsible for chain-length determination.
Highlights
Microbial exopolysaccharides (EPS) are a wide group of secreted polymers that can be assembled as capsular polysaccharides (CPS) tightly associated with cell surface, or released as extracellular slime in the surrounding of the cell (Kleerebezem et al, 1999; Nwodo, Green & Okoh, 2012)
A 16.3 kb region encoding enzymes required for EPS biosynthesis was identified on the chromosome (Fig. 1)
Four genes (DIS31_02520~DIS31_02535) at the 5′-end are highly conserved in S. thermophilus species, and were assigned for regulation and chain-length control functions in EPS biosynthesis
Summary
Microbial exopolysaccharides (EPS) are a wide group of secreted polymers that can be assembled as capsular polysaccharides (CPS) tightly associated with cell surface, or released as extracellular slime in the surrounding of the cell (Kleerebezem et al, 1999; Nwodo, Green & Okoh, 2012). Heteropolysaccharides are commonly synthesized by the Wzx/Wzy-dependent pathway reported for EPS and O-antigen biosynthesis in Gram-negative bacteria (Whitfield, 1995). In this pathway, first, a priming glycosyltransferase catalyzes the transfer of a sugar-1-phosphate from a nucleotide diphospho-sugar to the undecaprenyl (C55) phosphate lipid moiety. In Gram-positive bacteria, a membrane protein complex Wzd/Wze (designated EpsC/EpsD in LAB) is postulated to be responsible for chain length determination and secretion of the mature EPS (Bentley et al, 2006; Lebeer et al, 2009)
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