Sialic acid (N-acetyl neuraminic acid) utilization by Bacteroides fragilis requires a novel N-acetyl mannosamine epimerase - PubMed (original) (raw)

Sialic acid (N-acetyl neuraminic acid) utilization by Bacteroides fragilis requires a novel N-acetyl mannosamine epimerase

Christopher Brigham et al. J Bacteriol. 2009 Jun.

Abstract

We characterized the nanLET operon in Bacteroides fragilis, whose products are required for the utilization of the sialic acid N-acetyl neuraminic acid (NANA) as a carbon and energy source. The first gene of the operon is nanL, which codes for an aldolase that cleaves NANA into N-acetyl mannosamine (manNAc) and pyruvate. The next gene, nanE, codes for a manNAc/N-acetylglucosamine (NAG) epimerase, which, intriguingly, possesses more similarity to eukaryotic renin binding proteins than to other bacterial NanE epimerase proteins. Unphosphorylated manNAc is the substrate of NanE, while ATP is a cofactor in the epimerase reaction. The third gene of the operon is nanT, which shows similarity to the major transporter facilitator superfamily and is most likely to be a NANA transporter. Deletion of any of these genes eliminates the ability of B. fragilis to grow on NANA. Although B. fragilis does not normally grow with manNAc as the sole carbon source, we isolated a B. fragilis mutant strain that can grow on this substrate, likely due to a mutation in a NAG transporter; both manNAc transport and NAG transport are affected in this strain. Deletion of the nanE epimerase gene or the rokA hexokinase gene, whose product phosphorylates NAG, in the manNAc-enabled strain abolishes growth on manNAc. Thus, B. fragilis possesses a new pathway of NANA utilization, which we show is also found in other Bacteroides species.

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Figures

FIG. 1.

FIG. 1.

Schematic of the nanLET/nanR operon. The nanR gene product is a ROK family repressor protein. The nanL gene product is a NANA lyase (aldolase). The nanE gene product possesses similarity to the mammalian RnBP, also known to be an _N_-acetylglucosamine 2-epimerase. The nanT gene product is a transport protein of the major facilitator superfamily. The hyp gene encodes a hypothetical protein. Relevant deletion constructs and the schematics of the deletions are listed.

FIG. 2.

FIG. 2.

Growth phenotypes of nanR and nanLET gene knockout strains, and manNAc-utilizing strains. (A) Strains were grown in SAMM broth containing thymine, 0.5% NANA, and 0.02% glucose as described in Materials and Methods. Strains: ADB77 (wild type, ▪) and RC122 (Δ_nanR_, ▴), ΔL1 (Δ_nanL_, ▾), RC201 (Δ_nanE_, ⧫), RC140 (Δ_nanT_, •). (B) Growth on manNAc. Strains were inoculated in SAMM with thymine and 0.5% manNAc as described in Materials and Methods. Strains: ADB77 (▪), ADB77M (▴), ADB77MΔL1 (▾), ADB77MΔT (•), ADB77MΔE (⧫). (C) Growth of manNAc-utilizing strain and derivatives on NANA. Symbols represent the same strains as in panel B. Strains were inoculated into SAMM with thymine, 0.5% NANA, and 0.02% glucose as described in Materials and Methods. All curves shown here are representative of three separate growth experiments.

FIG. 3.

FIG. 3.

NANA lyase (aldolase) activities of B. fragilis strains. All strains were grown in SAMM broth with thymine and 0.5% of the indicated sugar. Extracts were prepared from cells and NANA lyase (aldolase) activity was measured as indicated in the supplemental material. ADB77 (wild type) cells were grown in xylose (X), glucose (G), and NANA (N). ADB77M cells were grown in glucose, NANA, and manNAc (M). RC122 (Δ_nanR_) cells were grown in xylose, glucose, and NANA.

FIG. 4.

FIG. 4.

NanE enzymatic activity. (A) manNAc is converted to NAG in an ATP-dependent manner. Extracts of B. fragilis cells with (ADB77, lanes 2 and 4) or without (RC201, lanes 1 and 3) an intact nanE gene were incubated with manNAc with or without ATP as described in Materials and Methods. Lane 5 is manNAc incubated in buffer without added extract. Arrows indicate the positions of NAG, manNAc, and NAG6-P standards, as determined in separate experiments. (B) manNAc→NAG steady-state ratio is 1:3. Purified NanEHis6 was incubated with ATP and [14C]manNAc (specific activity, 20 μCi/mmol) for the times indicated. (C) ATP, but not ATP, hydrolysis is required for manNAc epimerase activity. Purified NanEHis6 was incubated with or without ATP or ATP-γ-P and [14C]manNAc (specific activity, 20 μCi/mmol) as indicated. Each reaction in panel C was incubated for 18 h at 37°C. In panels B and C, the positions of manNac and NAG were determined in separate experiments.

FIG. 5.

FIG. 5.

NANA accumulation in RC122, RC122T, and the nanL disruption strain ADB77::pRAG210. Cells were incubated with labeled sugar (specific activity, 10 μCi/mmol) for a time course of 30 min as described in Materials and Methods. Aliquots were harvested at the times indicated on the x axis. The [14C]NANA specific activity was 0.1 μCi/mmol. The experiment with ADB77::pRAG210 cells was incubated for 15 min as described in Materials and Methods.

FIG. 6.

FIG. 6.

manNAc and NAG uptake in ADB77 (wild type) and ADB77M. Cells were incubated with labeled sugar (specific activity, 2.5 μCi/mmol) as described in Materials and Methods. Accumulation experiments were performed in triplicate.

FIG. 7.

FIG. 7.

Comparison of the NANA and amino sugar utilization pathways of E. coli (A) and B. fragilis (B). (A) Pathway adapted from Vimr et al. (36). GlcN,

d

-glucosamine. (B) Enzymes in boldface have been assayed by our laboratory. The NagA reaction has not been experimentally verified by us; however, there is an annotated sequence of such a gene in the B. fragilis 638R partial genome sequence. X designates the transporter, probably a NAG transporter, that contains the enabling mutation present in the strain ADB77M.

FIG. 8.

FIG. 8.

Phylogenetic tree of NanE proteins and RnBPs, including B. fragilis NanE (boxed). The tree was constructed by using the neighbor-joining method and was visualized with MEGA 3.1. hyp prot, hypothetical protein.

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References

    1. Allen, S., A. Zaleski, J. W. Johnston, B. W. Gibson, and M. A. Apicella. 2005. Novel sialic acid transporter of Haemophilus influenzae. Infect. Immun. 735291-5300. - PMC - PubMed
    1. Angata, T., and A. Varki. 2002. Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. Chem. Rev. 102439-469. - PubMed
    1. Baughn, A. D., and M. H. Malamy. 2002. A mitochondrial-like aconitase in the bacterium Bacteroides fragilis: implications for the evolution of the mitochondrial Krebs cycle. Proc. Natl. Acad. Sci. USA 994662-4667. - PMC - PubMed
    1. Berg, J. O., L. Lindqvist, and C. E. Nord. 1980. Purification of glycoside hydrolases from Bacteroides fragilis. Appl. Environ. Microbiol. 4040-47. - PMC - PubMed
    1. Bravo, I. G., S. Garcia-Vallve, A. Romeu, and A. Reglero. 2004. Prokaryotic origin of cytidylyltransferases and alpha-ketoacid synthases. Trends Microbiol. 12120-128. - PubMed

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