Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

Abstract

cDehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with 13 C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the ␣-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia. Dehalococcoides species are strictly anaerobic bacteria known for the ability to use a variety of halogenated aliphatic and aromatic compounds as respiratory electron acceptors. Many of these organohalides are persistent and toxic groundwater pollutants. Dehalococcoides isolates use hydrogen as the sole electron donor and acetate plus bicarbonate as carbon sources. While some biochemical details of the respiratory electron chain have been studied, knowledge of the carbon metabolism of Dehalococcoides spp. is scarce. Sequenced and annotated genomes of several Dehalococcoides strains provide a basis for the generation of hypotheses for carbon metabolism but also highlight gaps in our understanding (12,15,21). For instance, the genome annotations lack key genes for the biosynthesis of methionine, alanine, serine, glycine, and threonine. In addition, genes may be annotated incorrectly as evidenced by the recent identification of a gene encoding a Recitrate synthase previously annotated as homocitrate synthase (14). A modeling approach has used a pan-genome of all available Dehalococcoides sequences to develop a model for the central metabolism and growth of Dehalococcoides species (2). This study emphasi...

Identification and Characterization ofRe-Citrate Synthase in Syntrophus aciditrophicus

“…1). Glutamate derived from [1-13 C]acetate was mainly labeled on C-1 (␣-carboxyl group) in Dehalococcoides strains (19,51,52), and this labeling pattern was consistent with that of glutamate in Thermoanaerobacter sp. (16) and Desulfovibrio vulgaris (18).…”

The restricted metabolism of the obligate organohalide respiring bacterium Dehalobacter restrictus: lessons from tiered functional genomics

“…Several homologues of the latter enzyme were also detected in the proteome (see the electronic supplementary material, table S5). The role of the WL pathway and more generally the assimilation of CO 2 have been questioned for other OHRB such as various isolates of Desulfitobacterium [18,19,21,64] or of Dehalococcoides [65][66][67]. In Desulfitobacterium hafniense strains, components of the WL pathway have been shown to participate in the use of phenyl methyl ethers as electron donor [64].…”

Incomplete Wood–Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring Dehalococcoides mccartyi

“…One of the missing genes, metF, corresponds to methylene-tetrahydrofolate reductase (MTHFR) in the C1 metabolism pathway, which reduces 5-, 10-methylene-tetrahydrofolate (CH 2 -THF) to 5-methyl-tetrahydrofolate (CH 3 -THF), a required methyl donor for methionine biosynthesis (14). Although deletion of metF in bacteria often leads to methionine auxotrophy, D. mccartyi exhibits the ability of de novo methionine biosynthesis without this gene, suggesting the existence of an alternate mechanism for generation of the methyl donor for methionine biosynthesis (15)(16)(17)(18).…”

“…1). Indeed, D. mccartyi strains 195 and CBDB1 have been found to be incapable of reducing CO 2 to either the methyl or carbonyl group of acetylCoA in 13 C-labeled tracer experiments (16,18). However, as the reactions catalyzed by MeTr, FDH, and CODH are often…”