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

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.

The Capability of Utilizing Abiotic Enantiomers of Amino Acids by Halomonas sp. LMO_D1 Derived From the Mariana Trench

“…This is likely correlated with the transcription level of genes or the tolerance upper limit of the substrates. A similar phenomenon in which D-AAs supported higher biomass yield than L-enantiomers has also been reported previously in experiments examining Raoultella ornithinolytica A25; however, the mechanism remains unclear (Naganuma et al, 2018). Further investigation may be required to fill the knowledge gap in regard to the metabolism of amino acid enantiomers.…”

Imaging of Actively Proliferating Bacterial Infections by Targeting the Bacterial Metabolic Footprint with d-[5-11C]-Glutamine

“…26−30 This scavenging and modification ability enables bacteria to use DAAs as carbon and/or nitrogen sources for metabolism, antimicrobial peptides production, and essential molecules for diverse functions such as cell growth, biofilm dispersion, and spore germination. 31,32 Moreover, unlike mammals, bacteria utilize DAAs extensively for the synthesis of the peptidoglycan (PG), an indispensable component for bacterial cell growth and multiplication process. 33,34 Indeed, this mechanism has been exploited to develop PET imaging methods using D-[ 11 C]alanine and D-[ 11 C]methionine for bacterial infection imaging.…”

“…Although DAAs may be acquired through dietary intake and through in vivo conversion by host amino acid racemases, , the utilization of DAAs in mammals (e.g., d -serine, d -aspartate, d -alanine, and d -cysteine) is limited to the central nervous and endocrine systems. In contrast, bacteria can utilize both enantiomeric types of amino acids ( d and l forms) via specialized amino acid transport proteins and broad-spectrum bacterial racemase enzymes. − This scavenging and modification ability enables bacteria to use DAAs as carbon and/or nitrogen sources for metabolism, antimicrobial peptides production, and essential molecules for diverse functions such as cell growth, biofilm dispersion, and spore germination. , Moreover, unlike mammals, bacteria utilize DAAs extensively for the synthesis of the peptidoglycan (PG), an indispensable component for bacterial cell growth and multiplication process. , Indeed, this mechanism has been exploited to develop PET imaging methods using d -[ 11 C]­alanine and d -[ 11 C]­methionine for bacterial infection imaging. Convincing results have been reported in dual-infection myositis mouse models. − …”

Advanced “Green” Prebiotic Composite of Bacterial Cellulose/Pullulan Based on Synthetic Biology-Powered Microbial Coculture Strategy

“…The growth without any supplement of carbon sources observed in this experiment could be attributed to other ingredients in MRS medium, such as beef and yeast extracts, which contain some carbohydrates and can be used for bacterial growth. Naganuma et al [ 35 ] reported that microorganisms were able to utilize amino acids for growth. This could explain why probiotic could grow in the negative control sample.…”