Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Novel D-glutamate catabolic pathway in marine Proteobacteria and halophilic archaea

The ISME Journal volume 17, pages 537–548 (2023)Cite this article

Subjects

Abstract

D-glutamate (D-Glu) is an essential component of bacterial peptidoglycans, representing an important, yet overlooked, pool of organic matter in global oceans. However, little is known on D-Glu catabolism by marine microorganisms. Here, a novel catabolic pathway for D-Glu was identified using the marine bacterium Pseudoalteromonas sp. CF6-2 as the model. Two novel enzymes (DgcN, DgcA), together with a transcriptional regulator DgcR, are crucial for D-Glu catabolism in strain CF6-2. Genetic and biochemical data confirm that DgcN is a N-acetyltransferase which catalyzes the formation of N-acetyl-D-Glu from D-Glu. DgcA is a racemase that converts N-acetyl-D-Glu to N-acetyl-L-Glu, which is further hydrolyzed to L-Glu. DgcR positively regulates the transcription of dgcN and dgcA. Structural and biochemical analyses suggested that DgcN and its homologs, which use D-Glu as the acyl receptor, represent a new group of the general control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) superfamily. DgcA and DgcN occur widely in marine bacteria (particularly Rhodobacterales) and halophilic archaea (Halobacteria) and are abundant in marine and hypersaline metagenome datasets. Thus, this study reveals a novel D-Glu catabolic pathway in ecologically important marine bacteria and halophilic archaea and helps better understand the catabolism and recycling of D-Glu in these ecosystems.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Metabolic pathways for D-Glu in bacteria.
Fig. 2: Identification of the gene cluster responsible for D-Glu catabolism in strain CF6-2.
Fig. 3: Identification of the key genes in the DGC.
Fig. 4: Analysis of the regulatory mechanism of DgcR on the transcriptions of the DGC genes.
Fig. 5: Functional analysis of the key enzymes DgcA and DgcN in the DGC from strain CF6-2.
Fig. 6: Structural and phylogenetic analyses of DgcN-25328.
Fig. 7: Analyses of the diversity and distribution of DgcAN sequences and validation of the functions of the DgcA and DgcN homologs in marine bacteria.
Fig. 8: Analyses of the diversity of archaea containing DgcAN homologs and validation of DgcA and DgcN enzyme activities from halophilic archaeal strains.

Data availability

All the RNA-seq read data have been deposited in NCBI’s sequence read archive (SRA) under project accession number PRJNA829977. The structure of DgcN-25328 has been deposited in the PDB under the accession code 7XRJ.

References

  1. Park JT, Strominger JL. Mode of action of penicillin-biochemical basis for the mechanism of action of penicillin and for its selective toxicity. Science. 1957;125:99–101.

    Article  CAS  PubMed  Google Scholar 

  2. Yang CC, Leong J. Structure of pseudobactin 7sr1, a siderophore from a plant-deleterious Pseudomonas. Biochemistry. 1984;23:3534–40.

    Article  CAS  PubMed  Google Scholar 

  3. Ghuysen JM. Data on the structure of disaccharide-peptide complexes liberated from the wall of Micrococcus lysodeikticus by the action of beta(1-4)N-acetyl-hexosaminidases. Biochim Biophys Acta. 1961;47:561–8.

    Article  CAS  PubMed  Google Scholar 

  4. Ashiuchi M, Misono H. Biochemistry and molecular genetics of poly-gamma-glutamate synthesis. Appl Microbiol Biotechnol. 2002;59:9–14.

    Article  CAS  PubMed  Google Scholar 

  5. Jorgensen NO, Stepanaukas R, Pedersen AG, Hansen M, Nybroe O. Occurrence and degradation of peptidoglycan in aquatic environments. FEMS Microbiol Ecol. 2003;46:269–80.

    Article  CAS  PubMed  Google Scholar 

  6. Kandler O, Konig H. Cell wall polymers in Archaea (Archaebacteria). Cell Mol Life Sci. 1998;54:305–8.

    Article  CAS  PubMed  Google Scholar 

  7. Nagata Y, Tanaka K, Iida T, Kera Y, Yamada R, Nakajima Y, et al. Occurrence of D-amino acids in a few archaea and dehydrogenase activities in hyperthermophile Pyrobaculum islandicum. Biochim Biophys Acta 1999;1435:160–6.

    Article  CAS  PubMed  Google Scholar 

  8. Jorgensen NOG, Middelboe M. Occurrence and bacterial cycling of D amino acid isomers in an estuarine environment. Biogeochemistry. 2006;81:77–94.

    Article  CAS  Google Scholar 

  9. Kubota T, Kobayashi T, Nunoura T, Maruyama F, Deguchi S. Enantioselective utilization of D-amino acids by deep-sea microorganisms. Front Microbiol. 2016;7:511.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Yu Y, Yang J, Zheng LY, Sheng Q, Li CY, Wang M, et al. Diversity of D-amino acid utilizing bacteria from Kongsfjorden, Arctic and the metabolic pathways for seven D-amino acids. Front Microbiol. 2020;10:2983.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Sakai A, Xiang DF, Xu C, Song L, Yew WS, Raushel FM, et al. Evolution of enzymatic activities in the enolase superfamily: N-succinylamino acid racemase and a new pathway for the irreversible conversion of D- to L-amino acids. Biochemistry. 2006;45:4455–62.

    Article  CAS  PubMed  Google Scholar 

  12. Uo T, Yoshimura T, Tanaka N, Takegawa K, Esaki N. Functional characterization of alanine racemase from Schizosaccharomyces pombe: a eucaryotic counterpart to bacterial alanine racemase. J Bacteriol. 2001;183:2226–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Moore BC, Leigh JA. Markerless mutagenesis in Methanococcus maripaludis demonstrates roles for alanine dehydrogenase, alanine racemase, and alanine permease. J Bacteriol. 2005;187:972–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Troy FA. Chemistry and biosynthesis of the poly(-D-glutamyl) capsule in Bacillus licheniformis. II. Characterization and structural properties of the enzymatically synthesized polymer. J Biol Chem. 1973;248:316–24.

    Article  CAS  PubMed  Google Scholar 

  15. Thorne CB, Gómez CG, Noyes HE, Housewright RD. Production of glutamyl polypeptide by Bacillus subtilis. J Bacteriol. 1954;68:307.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Pedersen AGU, Thomsen TR, Lomstein BA, Jorgensen NOG. Bacterial influence on amino acid enantiomerization in a coastal marine sediment. Limnol Oceanogr. 2001;46:1358–69.

    Article  CAS  Google Scholar 

  17. Van Heijenoort J. Recent advances in the formation of the bacterial peptidoglycan monomer unit. Nat Prod Rep. 2001;18:503–19.

    Article  PubMed  Google Scholar 

  18. He WQ, Li GQ, Yang CK, Lu CD. Functional characterization of the dguRABC locus for D-Glu and D-Gln utilization in Pseudomonas aeruginosa PAO1. Microbiology. 2014;160:2331–40.

    Article  CAS  PubMed  Google Scholar 

  19. Tang BL, Yang J, Chen XL, Wang P, Zhao HL, Su HN, et al. A predator-prey interaction between a marine Pseudoalteromonas sp. and Gram-positive bacteria. Nat Commun. 2020;11:285.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Yu ZC, Tang BL, Zhao DL, Pang X, Qin QL, Zhou BC, et al. Development of a cold-adapted Pseudoalteromonas expression system for the Pseudoalteromonas proteins intractable for the Escherichia coli system. PLoS ONE. 2015;10:e0137384.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Yu ZC, Zhao DL, Ran LY, Mi ZH, Wu ZY, Pang X, et al. Development of a genetic system for the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913. Micro Cell Fact. 2014;13:13.

    Article  Google Scholar 

  22. Zhu Y, Zhang P, Lu T, Wang X, Li A, Lu Y, et al. Impact of MtrA on phosphate metabolism genes and the response to altered phosphate conditions in Streptomyces. Environ Microbiol. 2021;23:6907–23.

    Article  CAS  PubMed  Google Scholar 

  23. Williams JW, Northrop DB. Kinetic mechanisms of gentamicin acetyltransferase I. Antibiotic-dependent shift from rapid to nonrapid equilibrium random mechanisms. J Biol Chem. 1978;253:5902–7.

    Article  CAS  PubMed  Google Scholar 

  24. Noda M, Matoba Y, Kumagai T, Sugiyama M. A novel assay method for an amino acid racemase reaction based on circular dichroism. Biochem J. 2005;389:491–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Doi E, Shibata D, Matoba T. Modified colorimetric ninhydrin methods for peptidase assay. Anal Biochem. 1981;118:173–84.

    Article  CAS  PubMed  Google Scholar 

  26. Mccoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr. 2007;40:658–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Cao SN, Zhang WP, Ding W, Wang M, Fan S, Yang B, et al. Structure and function of the Arctic and Antarctic marine microbiota as revealed by metagenomics. Microbiome. 2020;8:47.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Bansal M, Alm E, Kellis M. Efficient algorithms for the reconciliation problem with gene duplication, horizontal transfer and loss. Bioinformatics 2012;28:i283–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Henikoff S, Haughn GW, Calvo JM, Wallace JC. A large family of bacterial activator proteins. Proc Natl Acad Sci USA. 1988;85:6602–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Tyrrell R, Verschueren KH, Dodson EJ, Murshudov GN, Addy C, Wilkinson AJ. The structure of the cofactor-binding fragment of the LysR family member, CysB: a familiar fold with a surprising subunit arrangement. Structure. 1997;5:1017–32.

    Article  CAS  PubMed  Google Scholar 

  31. Liu XX, Liu L, Song X, Wang GQ, Xiong ZQ, Xia YJ, et al. Determination of the regulatory network and function of the LysR-type transcriptional regulator of Lactiplantibacillus plantarum, LpLttR. Micro Cell Fact. 2022;21:65.

    Article  CAS  Google Scholar 

  32. Eisfeld J, Kraus A, Ronge C, Jagst M, Brandenburg VB, Narberhaus F. A LysR-type transcriptional regulator controls the expression of numerous small RNAs in Agrobacterium tumefaciens. Mol Microbiol. 2021;116:126–39.

    Article  CAS  PubMed  Google Scholar 

  33. Liu XX, Xiong ZQ, Wang GQ, Wang LF, Xia YJ, Song X, et al. LysR family regulator LttR controls production of conjugated linoleic acid in Lactobacillus plantarum by directly activating the cla operon. Appl Environ Microbiol. 2021;87:e02798–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Schwartz CJ, Giel JL, Patschkowski T, Luther C, Ruzicka FJ, Beinert H, et al. IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins. Proc Natl Acad Sci USA. 2001;98:14895–900.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ke Z, Zhou Y, Jiang W, Zhang M, Wang H, Ren Y, et al. McbG, a LysR family transcriptional regulator activates the mcbBCDEF gene cluster involved in the upstream pathway of carbaryl degradation in Pseudomonas sp. XWY-1. Appl Environ Microbiol. 2021;87:e02970–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Schmidt DMZ, Hubbard BK, Gerlt JA. Evolution of enzymatic activities in the enolase superfamily: functional assignment of unknown proteins in Bacillus subtilis and Escherichia coli as L-Ala-D/L-Glu epimerases. Biochemistry. 2001;40:15707–15.

    Article  CAS  PubMed  Google Scholar 

  37. Vandecandelaere I, Nercessian O, Segaert E, Achouak W, Faimali M, Vandamme P. Ruegeria scottomollicae sp. nov., isolated from a marine electroactive biofilm. Int J Syst Evol Microbiol. 2008;58:2726–33.

    Article  CAS  PubMed  Google Scholar 

  38. Vetting MW, Hegde SS, Javid-Majd F, Blanchard JS, Roderick SL. Aminoglycoside 2’-N-acetyltransferase from Mycobacterium tuberculosis in complex with coenzyme A and aminoglycoside substrates. Nat Struct Biol. 2002;9:653–8.

    Article  CAS  PubMed  Google Scholar 

  39. Klein DC. Arylalkylamine N-acetyltransferase: “the timezyme”. J Biol Chem. 2007;282:4233–7.

    Article  CAS  PubMed  Google Scholar 

  40. Mio T, Yamada-Okabe T, Arisawa M, Yamada-Okabe H. Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis. J Biol Chem. 1999;274:424–9.

    Article  CAS  PubMed  Google Scholar 

  41. Lee KK, Workman JL. Histone acetyltransferase complexes: one size doesn’t fit all. Nat Rev Mol Cell Biol. 2007;8:284–95.

    Article  CAS  PubMed  Google Scholar 

  42. Forouhar F, Lee IN, Vujcic J, Vujcic S, Shen JW, Vorobiev SM, et al. Structural and functional evidence for Bacillus subtilis PaiA as a novel N-1-spermidine/spermine acetyltransferase. J Biol Chem. 2005;280:40328–36.

    Article  CAS  PubMed  Google Scholar 

  43. Vetting MW, Park CH, Hegde SS, Jacoby GA, Hooper DC, Blanchard JS. Mechanistic and structural analysis of aminoglycoside N-acetyltransferase AAC(6’)-Ib and its bifunctional, fluoroquinolone-active AAC(6’)-Ib-cr variant. Biochemistry. 2008;47:9825–35.

    Article  CAS  PubMed  Google Scholar 

  44. Krtenic B, Drazic A, Arnesen T, Reuter N. Classification and phylogeny for the annotation of novel eukaryotic GNAT acetyltransferases. PLoS Comput Biol. 2020;16:e1007988.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Arima J, Isoda Y, Hatanaka T, Mori N. Recombinant production and characterization of an N-acyl-D-amino acid amidohydrolase from Streptomyces sp. 64E6. World J Microbiol Biotechnol. 2013;29:899–906.

    Article  CAS  PubMed  Google Scholar 

  46. Tsai YC, Tseng CP, Hsiao KM, Chen LY. Production and purification of D-Aminoacylase from Alcaligenes denitrificans and taxonomic study of the strain. Appl Environ Microbiol. 1988;54:984–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Mendes MI, Smith DE, Pop A, Lennertz P, Fernandez Ojeda MR, Kanhai WA, et al. Clinically distinct phenotypes of Canavan disease correlate with residual aspartoacylase enzyme activity. Hum Mutat. 2017;38:524–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kawakami R, Ohmori T, Sakuraba H, Ohshima T. Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by D-amino acids. Amino Acids. 2015;47:1579–87.

    Article  CAS  PubMed  Google Scholar 

  49. Matsumoto M, Homma H, Long ZQ, Imai K, Iida T, Maruyama T, et al. Occurrence of free D-amino acids and aspartate racemases in hyperthermophilic archaea. J Bacteriol. 1999;181:6560–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Kawakami R, Ohshida T, Sakuraba H, Ohshima T. A novel PLP-dependent alanine/serine racemase from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Front Microbiol. 2018;9:1481.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Satomura T, Sakuraba H, Suye S, Ohshima T. Dye-linked D-amino acid dehydrogenases: biochemical characteristics and applications in biotechnology. Appl Microbiol Biotechnol. 2015;99:9337–47.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank Xiaoju Li and Haiyan Sui from Life Science General Research Technology Platform of SKLMT (State Key Laboratory of Microbial Technology, Shandong University) for the assistance in X-ray diffraction experiment. This work was supported by the National Science Foundation of China (U2006205, 91851205, 31961133016, U1706207), Major Scientific and Technological Innovation Project (MSTIP) of Shandong Province (2019JZZY010817), and the Program of Shandong for Taishan Scholars (tspd20181203).

Author information

Authors and Affiliations

  1. State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China

    Yang Yu, Hai-Yan Cao, Zhao-Jie Teng, Yanping Zhu, Xiu-Lan Chen & Yu-Qiang Zhang

  2. College of Marine Life Sciences & Frontiers Science Center for Deep Ocean Multispheres and Earth System, Ocean University of China, Qingdao, China

    Peng Wang, Min Wang, Andrew McMinn, Yin Chen & Yu-Zhong Zhang

  3. Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, China

    Peng Wang, Yu-Zhong Zhang & Xiu-Lan Chen

  4. Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, TAS, Australia

    Andrew McMinn

  5. School of Life Sciences, University of Warwick, Coventry, UK

    Yin Chen

  6. University of Chinese Academy of Sciences, Beijing, China

    Hua Xiang

  7. State Key Laboratory of Microbial Resources, the Institute of Microbiology CAS, Beijing, China

    Hua Xiang

  8. Marine Biotechnology Research Center, State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China

    Yu-Zhong Zhang

Authors
  1. Yang Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hai-Yan Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhao-Jie Teng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yanping Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Min Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Andrew McMinn
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yin Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hua Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yu-Zhong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Xiu-Lan Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Yu-Qiang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

YY performed all experiments. PW and H-YC helped in protein structure determination and molecular docking simulations. Z-JT helped in bioinformatic analysis. YZ helped in EMSAs experiments. YY and X-LC wrote the manuscript. MW, AM and YC revised the manuscript. HX helped in experiments. Y-ZZ, X-LC and Y-QZ designed the experiments and directed the study.

Corresponding authors

Correspondence to Yu-Zhong Zhang, Xiu-Lan Chen or Yu-Qiang Zhang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yu, Y., Wang, P., Cao, HY. et al. Novel D-glutamate catabolic pathway in marine Proteobacteria and halophilic archaea. ISME J 17, 537–548 (2023). https://doi.org/10.1038/s41396-023-01364-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41396-023-01364-6

Search

Advanced search

Quick links