AUTHOR=Naganuma Takeshi , Iinuma Yoshiakira , Nishiwaki Hitomi , Murase Ryota , Masaki Kazuo , Nakai Ryosuke 

TITLE=Enhanced Bacterial Growth and Gene Expression of D-Amino Acid Dehydrogenase With D-Glutamate as the Sole Carbon Source

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.02097

DOI=10.3389/fmicb.2018.02097

ISSN=1664-302X

ABSTRACT=<p>In a search for life-supporting, not life-assisting, <sc>D</sc>-amino acid metabolism, an environmental strain that grows better with <sc>D</sc>-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 <sc>D</sc>-glutamate than with <sc>L</sc>-glutamate. Conversely, the D/L ratio of total cellular glutamate was as low as 4/96, which suggests that <sc>D</sc>-glutamate is more likely catabolized than anabolized. Strain A25 was phylogenetically most closely related to the gamma-proteobacterial species <italic>Raoultella ornithinolytica</italic>, with a 16S rRNA gene sequence similarity of 100%. A standard strain, <italic>R. ornithinolytica</italic> JCM 6096<sup>T</sup>, also showed similarly enhanced growth with <sc>D</sc>-glutamate, which was proven for the first time. Gene expression of the enzymes involved in <sc>D</sc>-amino acid metabolism was assayed by reverse-transcription quantitative PCR (RT-qPCR) using specifically designed primers. The targets were the genes encoding <sc>D</sc>-amino acid dehydrogenase (DAD; EC 1.4.99.1), glutamate racemase (EC 5.1.1.3), <sc>D</sc>-glutamate oxidase (EC 1.4.3.7 or EC 1.4.3.15), and UDP-N-acetyl-α-<sc>D</sc>-muramoyl-<sc>L</sc>-alanyl-<sc>D</sc>-glutamate ligase (EC 6.3.2.9). As a result, the growth of strains A25 and <italic>R. ornithinolytica</italic> JCM 6096<sup>T</sup> on <sc>D</sc>-glutamate was conspicuously associated with the enhanced expression of the DAD gene (<italic>dadA</italic>) in the exponential phase compared with the other enzyme genes. <italic>Pseudomonas aeruginosa</italic> is also known to grow on <sc>D</sc>-glutamate as the sole carbon source but to a lesser degree than with <sc>L</sc>-glutamate. A standard strain of <italic>P. aeruginosa</italic>, JCM 5962<sup>T</sup>, was tested for gene expression of the relevant enzymes by RT-qPCR and also showed enhanced <italic>dadA</italic> expression, but in the stationary phase. Reduction of ferricyanide with <sc>D</sc>-glutamate was detected in cell extracts of the tested strains, implying probable involvement of DAD in the <sc>D</sc>-glutamate catabolizing activity. DAD-mediated catalysis may have advantages in the one-step production of α-keto acids and non-production of H<sub>2</sub>O<sub>2</sub> over other enzymes such as racemase and <sc>D</sc>-amino acid oxidase. The physiological and biochemical importance of DAD in <sc>D</sc>-amino acid metabolism is discussed.</p>