Guanine crystal formation by bacteria

Abstract

Background: Guanine crystals are organic biogenic crystals found in many organisms. Due to their exceptionally high refractive index, they contribute to structural color and are responsible for the reflective effect in the skin and visual organs in animals such as fish, reptiles, and spiders. Occurrence of these crystals in animals has been known for many years, and they have also been observed in eukaryotic microorganisms, but not in prokaryotes.

Results: In this work, we report the discovery of extracellular crystals formed by bacteria and reveal that they are composed of guanine monohydrate. This composition differs from that of biogenic guanine crystals found in other organisms, mostly composed of β anhydrous guanine. We demonstrate the formation of these crystals by Aeromonas and other bacteria and investigate the metabolic traits related to their synthesis. In all cases studied, the presence of the bacterial guanine crystals correlates with the absence of guanine deaminase, which could lead to guanine accumulation providing the substrate for crystal formation.

Conclusions: Our finding of the hitherto unknown guanine crystal occurrence in prokaryotes extends the range of organisms that produce these crystals to a new domain of life. Bacteria constitute a novel and more accessible model to study the process of guanine crystal formation and assembly. This discovery opens countless chemical and biological questions, including those about the functional and adaptive significance of their production in these microorganisms. It also paves the road for the development of simple and convenient processes to obtain biogenic guanine crystals for diverse applications.

Keywords: Aeromonas; Bacterial guanine crystals; Biogenic guanine crystals; Biomaterial; Guanine monohydrate; Melanin.

Fig. 1

SEM micrographs of the crystalline material produced by 34mel. Crystalline aggregates and individual nanocrystals found in bacterial cultures grown in solid (A–C) and liquid (D–F) LB medium (increasing levels of magnification from left to right)

Fig. 2

Characterization of guanine monohydrate crystals produced by 34mel. A ESI–MS spectrum of biogenic guanine compared to the simulated data for guanine. B ESI MS/MS spectrum of the [M + H]⁺ ion m/z 152.0574. Solvent: methanol: H₂O. In color, proposed structures for MS/MS obtained ions are shown. C Powder X-ray diffraction pattern of the biogenic guanine crystals and the simulated powder X-ray diffraction pattern obtained from the guanine monohydrate single-crystal X-ray data [12]. D FT-IR spectra of biogenic crystals and commercial guanine, signals associated with water molecules are highlighted in green and those of carbonyl and amine groups in orange

Fig. 3

Purine metabolism in Aeromonas. A Purine metabolic pathway in 34mel, showing de novo formation of purines (blue arrows) and salvage reactions (green arrows). Enzymes absent in 34mel with the corresponding E.C. numbers are shown with crossed out arrows. B Comparison of the genomic region containing guaD and xdhABC in A. hydrophila showing their absence in representative Aeromonas. Homologous flanking genes are shown: auxin efflux carrier family transporter (green) and ExeM/NucH family extracellular endonuclease (blue) along with the locus tags in each genome. C Phylogenomic tree of Aeromonas species showing the presence (black) or the absence (blue) of the gene encoding the guanine deaminase. The numbers below branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 94.4%. The tree was rooted at the midpoint [29]. The strains used and the accession numbers of their genomes are indicated in Additional file 4: Table S3

Fig. 4

Experimental analysis of guanine crystal formation and melanin production in selected bacterial species. Comparative information related to the presence of genes encoding guanine deaminase (guaD) and xanthine dehydrogenase (xdh) is included. The symbol * indicates that the genome for the corresponding strain is not available, but the gene(s) is (are) present or absent in the type strain of the species (genome information in Additional file 4: Table S3). Presence of melanin and/or guanine crystals is indicated by colored circles or diamonds

Fig. 5

Characterization of guanine monohydrate crystals produced by several bacteria. A–C SEM micrographs of the crystals produced by the bacteria A. salmonicida subsp. masoucida (A), S. oneidensis (B), and A. media (C). D Powder X-ray diffraction patterns of the crystals produced by the different bacteria and the simulated powder X-ray diffraction pattern obtained from the guanine monohydrate single-crystal X-ray data [12]