Abstract
Bacterial genome sequencing has provided a wealth of genetic data. However, the definitive functional characterization of hypothetical open reading frames and novel biosynthetic genes remains challenging. This is particularly true for genes involved in protein glycosylation because the isolation of their glycan moieties is often problematic. We have developed a focused metabolomics approach to define the function of flagellin glycosylation genes in Campylobacter jejuni 81-176. A capillary electrophoresis-electrospray mass spectrometry and precursor ion scanning method was used to examine cell lysates of C. jejuni 81-176 for sugar nucleotides. Novel nucleotide-activated intermediates of the pseudaminic acid (Pse5NAc7NAc) pathway and its acetamidino derivative (PseAm) were found to accumulate within select isogenic mutants, and use of a hydrophilic interaction liquid chromatography-mass spectrometry method permitted large scale purifications of the intermediates. NMR with cryo probe (cold probe) technology was utilized to complete the structural characterization of microgram quantities of CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-L-glycero-alpha-L-manno-nonulosonic acid (CMP-Pse5NAc7Am), which is the first report of Pse modified at C7 with an acetamidino group in Campylobacter, and UDP-2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose, which is a bacillosamine derivative found in the N-linked proteinglycan. Using this focused metabolomics approach, pseB, pseC, pseF, pseI, and for the first time pseA, pseG, and pseH were found to be directly involved in either the biosynthesis of CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am. In contrast, it was shown that pseD, pseE, Cj1314c, Cj1315c, Cjb1301, Cj1334, Cj1341c, and Cj1342c have no role in the CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am pathways. These results demonstrate the usefulness of this approach for targeting compounds within the bacterial metabolome to assign function to genes, identify metabolic intermediates, and elucidate novel biosynthetic pathways.
Highlights
The availability of bacterial genomic sequences has provided unprecedented opportunity for comparative studies
Using CE-ESMS and precursor ion scanning for fragment ions characteristic of CMP, we were able to identify CMP-Pse5NAc7NAc and CMP-pathway and its acetamidino derivative (PseAm) as nucleotide-activated precursors related to the biosynthesis of Pse5NAc7NAc and PseAm in C. jejuni 81–176 (21)
Lysates from 15 mutants in the flagellin glycosylation locus of strain 81–176 were compared with the wild-type by CE-ESMS for sugar nucleotide intermediates of the Pse5NAc7NAc/PseAm biosynthetic pathway
Summary
Bacterial Strains and Growth Conditions—Parent strain C. jejuni 81–176 and isogenic mutants described by Guerry et al (10) (see Table 1) were grown in Mueller-Hinton broth (500 ml) under microaerophilic conditions for 24 h at 37 °C. Intracellular sugar-nucleotides were extracted from the cell lysates using ENVI-Carb (12 ml, 1 g) solid phase extraction cartridges from Supelco (Bellefonte, PA). Using commercially available ENVI-Carb SPE cartridges, intracellular sugar-nucleotides were extracted from the cell lysates of C. jejuni 81–176 and the isogenic mutant pseC using an optimized SPE method based on that of Rabinaet al. Cell lysates (2 ml) were loaded on the ENVI-Carb cartridges and washed with 4 ml of water, 4 ml of 5% acetonitrile, and with 4 ml of triethylammonium acetate (10 mM; pH 7.0). The extracts were evaporated to dryness on a SpeedVac concentrator to remove the TEAA and reconstituted in either de-ionized water for CE-ESMS analysis or acetonitrile-ammonium acetate (6.5 mM; pH 5.5) (70 –30, v/v) for HILIC-MS. For proton and carbon experiments, the methyl resonance of acetone was used as an internal reference (␦H 2.225 ppm and ␦C 31.07 ppm)
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