Selective Utilization of Exogenous Amino Acids by Dehalococcoides ethenogenes Strain 195 and Its Effects on Growth and Dechlorination Activity

Abstract

Bacteria of the genus Dehalococcoides are important members of bioremediation communities because of their ability to detoxify chloroethenes to the benign end product ethene. Genome-enabled studies conducted with Dehalococcoides ethenogenes 195 have revealed that two ATP-binding cassette (ABC)-type amino acid transporters are expressed during its exponential growth stages. In light of previous findings that Casamino Acids enhanced its dechlorination activity, we hypothesized that strain 195 is capable of importing amino acids from its environment to facilitate dechlorination and growth. To test this hypothesis, we applied isotopomerbased dilution analysis with 13 C-labeled acetate to differentiate the amino acids that were taken up by strain 195 from those synthesized de novo and to determine the physiological changes caused by the significantly incorporated amino acids. Our results showed that glutamate/glutamine and aspartate/asparagine were almost exclusively synthesized by strain 195, even when provided in excess in the medium. In contrast, phenylalanine, isoleucine, leucine, and methionine were identified as the four most highly incorporated amino acids, at levels >30% of respective proteinogenic amino acids. When either phenylalanine or all four highly incorporated amino acids were added to the defined mineral medium, the growth rates, dechlorination activities, and yields of strain 195 were enhanced to levels similar to those observed with supplementation with 20 amino acids. However, genes for the putative ABC-type amino acids transporters and phenylalanine biosynthesis exhibited insignificant regulation in response to the imported amino acids. This study also demonstrates that using isotopomer-based metabolite analysis can be an efficient strategy for optimizing nutritional conditions for slow-growing microorganisms.

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

“…In the light of this discrepancy, we reassessed our data; however, the incorporation of a single unlabeled amino acid into an accurately labeled peptide would change the mass of the precursor ion and also the masses of y-type ions, and therefore, we do not see an alternative to our conclusions. On the other hand, we recognized a correlation of the nonlabeled mass fraction in protein hydrolysates from strain 195 with the hydrophobicity of the respective amino acids (29). In fact, the four amino acids phenylalanine, isoleucine, leucine, and methionine reported to be taken up most by strain 195 are among the most hydrophobic amino acids.…”

“…The protein amount roughly correlates with that from 10 7 cells of Escherichia coli (2). In contrast, standard numbers of Dehalococcoides cells used for GC-MS analysis after protein hydrolysis were 1.5 ϫ 10 11 to 2.3 ϫ 10 11 cells (24,29). The high sensitivity of our approach is of utmost importance when analyzing very slowly growing organisms or organisms in mixed cultures.…”

“…It would therefore be important to confirm that such unlabeled hydrophobic amino acids do indeed stem from proteinintegrated amino acids and not from unlabeled free amino acids that were dissolved in or attached to the lipid membrane. A contamination of protein hydrolysates with free unlabeled amino acids would explain (i) the finding that phenylalanine but not tyrosine was highly diluted, although both share a largely common biosynthesis pathway, and (ii) the reported insignificant transcriptional feedback from phenylalanine onto pheA (29). Our approach avoids such effects as all amino acids are measured as a part of intact peptides.…”

“…A recent isotopomer-based dilution study, in which Dehalococcoides strain 195 was incubated with 13 C-labeled acetate and unlabeled amino acids and harvested proteins were completely hydrolyzed and analyzed by GC-MS, reported incorporation of many amino acids from the medium, including phenylalanine, isoleucine, leucine, and methionine at percentages of more than 30% (29). In the light of this discrepancy, we reassessed our data; however, the incorporation of a single unlabeled amino acid into an accurately labeled peptide would change the mass of the precursor ion and also the masses of y-type ions, and therefore, we do not see an alternative to our conclusions.…”

Versatility in Corrinoid Salvaging and Remodeling Pathways Supports Corrinoid-Dependent Metabolism in Dehalococcoides mccartyi

“…Cl Ϫ released was calculated from metabolic dechlorination from TCE to vinyl chloride (VC) as described previously (11). Cell number in the cultures was determined by quantitative real-time PCR with primers specific to the D. mccartyi strain 195 tceA gene, using a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA) as described previously (56). Dechlorination and cell yield are reported as the percentage of the average level observed in cultures containing cobalamin.…”