YjhS (NanS) is required for Escherichia coli to grow on 9-O-acetylated N-acetylneuraminic acid

Abstract

The nanATEK-yhcH, yjhATS, and yjhBC operons in Escherichia coli are coregulated by environmental N-acetylneuraminic acid, the most prevalent sialic acid in nature. Here we show that YjhS (NanS) is a probable 9-O-acetyl N-acetylneuraminic acid esterase required for E. coli to grow on this alternative sialic acid, which is commonly found in mammalian host mucosal sites.

FIG. 1.

nanS is required for E. coli to grow on Neu5,9Ac₂ as the sole carbon source. Overnight cultures of BW30270 and its isogenic nanS mutant derivative grown on glycerol as the carbon source were diluted 1:50 into fresh medium containing either glycerol or Neu5,9Ac₂ as follows: BW30270 with glycerol (closed squares); nanS mutant with glycerol (closed triangles); BW30270 with Neu5,9Ac₂ (open squares); nanS mutant with Neu5,9Ac₂ (open triangles).

FIG. 2.

Chemical analysis of Neu4,5Ac₂ in spent culture medium. (A) Neu4,5Ac₂ at a 0.1% final concentration was incubated in minimal glycerol medium without cells for 7 h and then subjected to DMB analysis as described in the text. (B) Neu4,5Ac₂ in the spent culture supernatant of wild type was grown for 7 h in the presence of glycerol. (C) Neu4,5Ac₂ in the spent culture supernatant of the nanS mutant was grown in glycerol medium for 7 h. Note that the same profiles were obtained after comparable chemical analyses of 24-h cultures, indicating the stability of Neu4,5Ac₂ and its resistance to metabolism by E. coli K-12. RF, relative fluorescence.

FIG. 3.

Growth of the wild type and the yjhS and nanA mutants on different sialic acids. In panels A to D, the indicated wild-type strains (open boxes) and their isogenic yjhS deletion derivatives (shaded boxes) were diluted 50-fold from stationary-phase cells grown in M63 medium plus glycerol (0.4%) into 0.1 ml of M63 medium containing 0.1% of the indicated sialic acids or glycerol in 96-well plates. (A) Growth on Neu5Ac. (B) Growth on Neu5Gc. (C) Growth on Neu5,9Ac₂. (D) Growth on glycerol. The plate was incubated without shaking at 37°C for 16 h, and A₆₀₀ values were recorded with a microplate reader. Data represent the means of three independent experiments ± standard deviations. (E) S. enterica serovar Typhimurium wild-type strain 14028, nanA strain LT2 derivative M2, and E. coli nanA mutant EV78 were diluted 60-fold into 0.5 ml of M63 medium containing 0.4% glycerol, 0.1% Neu5Ac, or 0.1% Neu5,9Ac₂. Cultures were grown at 37°C with vigorous aeration; the A₆₀₀ values were recorded after 7 h.

FIG. 4.

Utilization of Neu5Ac or Neu5,9Ac₂ by BW30270 and its yjhS deletion derivative growing simultaneously in the presence of glycerol. BW30270 or its yjhS derivative was diluted 60-fold into 0.5 ml of M63 medium plus glycerol containing either 0.1% Neu5Ac or Neu5,9Ac₂ and incubated at 37°C with vigorous aeration for 7 h unless indicated otherwise. (A) Neu5,9Ac₂ incubated without cells. (B) Neu5Ac incubated without cells. (C) Wild type grown with Neu5Ac. (D) Mutant grown with Neu5Ac. Note that the minor peak eluting at about 10 min as shown in these panels and in the succeeding panel is likely to represent a trace amount of 2-deoxy-d-manno-octulosonic acid derived from sloughed lipopolysaccharide during the incubation that was not removed by the centrifugation step used to produce the spent culture media for the analyses (22). (E) Wild type grown with Neu5,9Ac₂. (F) Mutant grown with Neu5,9Ac₂. (G) Neu5,9Ac₂ incubated without cells for 17 h. (H) Mutant grown with Neu5,9Ac₂ for 17 h.