Abstract
In a search for life-supporting, not life-assisting, D-amino acid metabolism, an environmental strain that grows better with D-glutamate as the sole carbon source was isolated from an ordinary river. The strain, designated as A25, exhibited a faster growth rate and greater cell yield with D-glutamate than with L-glutamate. Conversely, the D/L ratio of total cellular glutamate was as low as 4/96, which suggests that D-glutamate is more likely catabolized than anabolized. Strain A25 was phylogenetically most closely related to the gamma-proteobacterial species Raoultella ornithinolytica, with a 16S rRNA gene sequence similarity of 100%. A standard strain, R. ornithinolytica JCM 6096T, also showed similarly enhanced growth with D-glutamate, which was proven for the first time. Gene expression of the enzymes involved in D-amino acid metabolism was assayed by reverse-transcription quantitative PCR (RT-qPCR) using specifically designed primers. The targets were the genes encoding D-amino acid dehydrogenase (DAD; EC 1.4.99.1), glutamate racemase (EC 5.1.1.3), D-glutamate oxidase (EC 1.4.3.7 or EC 1.4.3.15), and UDP-N-acetyl-α-D-muramoyl-L-alanyl-D-glutamate ligase (EC 6.3.2.9). As a result, the growth of strains A25 and R. ornithinolytica JCM 6096T on D-glutamate was conspicuously associated with the enhanced expression of the DAD gene (dadA) in the exponential phase compared with the other enzyme genes. Pseudomonas aeruginosa is also known to grow on D-glutamate as the sole carbon source but to a lesser degree than with L-glutamate. A standard strain of P. aeruginosa, JCM 5962T, was tested for gene expression of the relevant enzymes by RT-qPCR and also showed enhanced dadA expression, but in the stationary phase. Reduction of ferricyanide with D-glutamate was detected in cell extracts of the tested strains, implying probable involvement of DAD in the D-glutamate catabolizing activity. DAD-mediated catalysis may have advantages in the one-step production of α-keto acids and non-production of H2O2 over other enzymes such as racemase and D-amino acid oxidase. The physiological and biochemical importance of DAD in D-amino acid metabolism is discussed.
Highlights
MATERIALS AND METHODSD-amino acids are minor components of living organisms, but they occur in a wide range of natural environments such as soils, riverine, lacustrine, and marine systems (e.g., Wu et al, 2007; Tremblay and Benner, 2009; Barbaro et al, 2014), snow and ice (Barbaro et al, 2017), aerosols (Wedyan and Preston, 2008), and precipitation (Yan et al, 2015)
Our results showed the enhanced expression of the gene encoding D-amino acid dehydrogenase in all three strains, but at different growth phases between the species
∗1D-amino acid dehydrogenase gene; ∗2glutamate racemase gene; ∗3D-glutamate oxidase or D-glutamate; ∗4gene of UDP-N-acetyl-α-D-muramoyl -L-alanyl-D-glutamate ligase involved in the synthesis of a cell-wall peptide in bacteria; and, ∗516S rRNA gene (16S)
Summary
D-amino acids are minor components of living organisms, but they occur in a wide range of natural environments such as soils (a recent review by Vranova et al, 2012), riverine, lacustrine, and marine systems (e.g., Wu et al, 2007; Tremblay and Benner, 2009; Barbaro et al, 2014), snow and ice (Barbaro et al, 2017), aerosols (Wedyan and Preston, 2008), and precipitation (Yan et al, 2015). Another reason was that the 16S rRNA sequences of 6 environmental strains out of the 20 sequenced isolates (excluding A25) were most closely related to that of P. aeruginosa at 97–100% similarities, as described later The enzymes and their coding genes involved in D-glutamate metabolism were surveyed by PATHWAY map00471 “DGlutamine and D-glutamate metabolism” of KEGG1 (Kanehisa and Goto, 2000) and the complete genomes of R. ornithinolytica (Shin et al, 2013; Thijs et al, 2014; Bao et al, 2015; Al-Bayssari et al, 2016) and P. aeruginosa (Stover et al, 2000). As the method is applicable when the amplification efficiencies are close to 100%, we checked that the efficiencies were 90–100% with artificially synthesized target genes (synthetic DNA) and our designed primers described above
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