Isolation of Fucosyltransferase-Producing Bacteria from Marine Environments

Fucose-containing oligosaccharides on the cell surface of some pathogenic bacteria are thought to be important for host-microbe interactions and to play a major role in the pathogenicity of bacterial pathogens. Here, we screened marine bacteria for glycosyltransferases using two methods: a one-pot glycosyltransferase assay method and a lectin-staining method. Using this approach, we isolated marine bacteria with fucosyltransferase activity. There have been no previous reports of marine bacteria producing fucosyltransferase. This paper thus represents the first report of fucosyltransferase-producing marine bacteria.

Because the fucosylated oligosaccharides of glycoconjugates play important roles in many biological processes, an abundant supply of fucosides is essential for a detailed investigation of their biological functions. To produce these fucosides enzymatically, many kinds of fucosyltransferases in large quantities are essential. Generally, bacterial enzymes are stable and show broad acceptor substrate specificity compared with their mammalian counterparts. For these reasons, we have been screening bacteria for glycosyltransferase activities, including fucosyltransferase activity and galactosyltransferase activity.
The bacterial isolates used in the screening were prepared from samples of seawater, sand, mud, small animals, and seaweed collected from the shore of various locations in Japan. Bacteria that grew on marine agar 2216 at 15,25,28 or 30°C were isolated from the samples. Then, these bacteria were inoculated in a 15-mL test tube containing 6 mL of marine broth 2216 (Becton-Dickinson, Franklin Lakes, NJ, USA) and cultivated at 15, 25, 28, or 30°C for 18 h on a rotary shaker (180 rpm). Bacteria were harvested from 2 to 4 mL of the culture broth by centrifugation, suspended in 200 μL of 20 mM bis-Tris buffer (pH 6.0) that contained 0.2% Triton X-100, lysed by sonication on ice, and used immediately for the one-pot glycosyltransferase assay. To assess the enzymatic activities of various glycosyltransferases simultaneously, a one-pot glycosyltransferase assay was performed by mixing various donor substrates of glycosyltransferases with a mixture of acceptor substrates.
, and 4-nitrophenyl β-Dmannopyranoside (Man-β-pNp). All of the acceptor substrates were purchased from Sigma. The reaction mixture for the one-pot glycosyltransferase assay (50 μL) consisted of the following: an enzyme sample, a mixture of 0.5 mM acceptor substrates consisting of 4-nitrophenyl compounds, a mixture of 0.5 mM donor substrates consisting of sugar nucleotides that included 4,620 Bq UDP-[U-14 C]-Gal, 4,620 Bq UDP-[U-14 C]-GlcNAc, and 4,620 Bq GDP-[U-14 C]-Fuc, 100 mM bis-Tris buffer (pH 6.0), 10 mM MnCl2, and 3 mM ATP. The reaction was carried out at 25°C for 16 to 18 h. After the reaction, 100 μL water was added to the reaction mixture, and the mixture was applied to a Sep-Pak Vac C18 cartridge 1 cc/50 mg 55-105 μm (Waters, Milford, MA, USA) that was conditioned with ethanol and equilibrated with water. The column was washed twice with 1 mL water, and the reaction product was eluted with 1 mL of 70% ethanol. One milliliter of scintillation cocktail was added to the eluate, and the radioactivity of the mixture was measured using a liquid scintillation counter.
More than 1,000 bacterial isolates were examined for glycosyltransferase activity, some of which tested positive in the first screening using the one-pot glycosyltransferase assay (Table 1). Table 1 showed the results of the assay with a mixture of acceptor substrates and donor substrates in the reaction mixture. With regard to DOT-118-2 and OKI-895, radioactivities of the eluates containing the mixture of acceptor substrates in the reaction mixture were over 2,000 cpm and approximately 1,100 cpm, respectively. On the other hand, radioactivities of the eluates without the mixture of acceptor substrates in the reaction mixture were about 100 cpm and about 300 cpm, respectively; however, with or without the mixture of acceptor substrates in the reaction mixture, almost the same radioactivities of the eluates were observed in other bacterial cases. Thus, we assumed DOT-118-2 and OKI-895 to be candidate glycosyltransferase producers among the tested bacteria.
Then, DOT-118-2 and OKI-895 were tested for their ability to transfer fucose, galactose, and Nacetylglucosamine to acceptor substrates, independently. DOT-118-2 and OKI-895 were isolated from samples obtained from the shore of the Okhotsk Sea, Hokkaido, Japan and the Eastern China Sea, Okinawa, Japan, respectively. As a result, we confirmed that DOT-118-2 exhibited fucosyltransferase activity ( Table 2). No galactosyltransferase activity or N-acetylglucosaminyltransferase activity was observed in the lysate prepared from DOT-118-2. With regard to OKI-895, very weak fucosyltransferase activity was observed under the test conditions used in this study. No galactosyltransferase activity or N-acetylglucosaminyltransferase activity was observed in the lysate prepared from OKI-895.
A partial DNA sequence (accession number: AB71119) of the 16S rRNA gene of DOT-118-2 showed the highest similarity, over 98%, to the DNA sequence of the 16S rRNA gene from Polaribacter sp. S-6 (accession number DQ978987). The result of phylogenetic tree analysis of the 16S rRNA gene is shown in Supplementary Figure 1. Further biochemical characterization of this bacterium is underway. Recently, it has been reported that marine bacteria including Polaribacter produce exopolysaccharides, EPS, as a strategy for growth, adhering to solid surfaces, and to survive adverse conditions (21). It has been also reported that EPS are composed of monosaccharide such as D-arabinose, D-xylose, D-glucose, D-galactose, D-mannose, L-fucose, Dglucosamine, D-galactosamine, and so on; however, biosynthesis of EPS has not been understood comprehensively. The structure and composition of EPS produced by DOT-118-2 and OKI-895 are still unknown, and there is a possibility that the fucosyltransferase activities reported in this paper might be involved in the biosynthesis of EPS.
We screened marine bacteria for glycosyltransferase activities using two methods, the one-pot glycosyltransferase assay and lectin staining. As a result, we obtained two fucosyltransferase-producing bacteria from marine environments. Although enzymatic characterization of the fucosyltransferases produced by DOT-118-2 and OKI-895 has not been performed yet, there is a possibility that these enzymes would be good tools for producing fucosides enzymatically in the near future.