Symbiosis between nanohaloarchaeon and haloarchaeon is based on utilization of different polysaccharides

Abstract

Nano-sized archaeota, with their small genomes and limited metabolic capabilities, are known to associate with other microbes, thereby compensating for their own auxotrophies. These diminutive and yet ubiquitous organisms thrive in hypersaline habitats that they share with haloarchaea. Here, we reveal the genetic and physiological nature of a nanohaloarchaeon-haloarchaeon association, with both microbes obtained from a solar saltern and reproducibly cultivated together in vitro. The nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh is an aerotolerant, sugar-fermenting anaerobe, lacking key anabolic machinery and respiratory complexes. The nanohaloarchaeon cells are found physically connected to the chitinolytic haloarchaeon Halomicrobium sp. LC1Hm. Our experiments revealed that this haloarchaeon can hydrolyze chitin outside the cell (to produce the monosaccharide N-acetylglucosamine), using this beta-glucan to obtain carbon and energy for growth. However, LC1Hm could not metabolize either glycogen or starch (both alpha-glucans) or other polysaccharides tested. Remarkably, the nanohaloarchaeon's ability to hydrolyze glycogen and starch to glucose enabled growth of Halomicrobium sp. LC1Hm in the absence of a chitin. These findings indicated that the nanohaloarchaeon-haloarchaeon association is both mutualistic and symbiotic; in this case, each microbe relies on its partner's ability to degrade different polysaccharides. This suggests, in turn, that other nano-sized archaeota may also be beneficial for their hosts. Given that availability of carbon substrates can vary both spatially and temporarily, the susceptibility of Halomicrobium to colonization by Ca Nanohalobium can be interpreted as a strategy to maximize the long-term fitness of the host.

Keywords: haloarchaea; nanohaloarchaea; polysaccharide utilization; solar salterns; symbiosis.

Fig. 1.

Phylogenetic placement of Ca. Nanohalobium based on concatenated partial amino acid sequences of the 122 proteins conserved in Archaea (archaeal marker genes). Bootstrap values are shown at the nodes; the bar indicates 0.20 changes per position. Cultivated and uncultured members of the candidate phylum Nanohaloarchaeota are highlighted in red and blue, respectively. A detailed list of DPANN genomes and the methods used for the tree construction are given Materials and Methods.

Fig. 2.

Scanning and transmission electron micrographs of Halomicrobium sp. LC1Hm and Ca. Nanohalobium constans LC1Nh coculture growing in the LC medium supplemented with chitin. Images depict tiny coccoidal nanohaloarchaeal cells (285 ± 50 nm in diameter) either detached or adhered to the host haloarchaeal cells. Up to 17 nanohaloarchaeota cells can closely interact with the single host cell (A–D); nanohaloarchaeal cells express the pilus-like structures (thick and long protein stalks of the archaella) (A, D, and E), which can unwind to thin filaments at the points indicated by red arrows.

Fig. 3.

Transmission electron micrographs of chitin-growing coculture of Ca. Nanohalobium constans LC1Nh and Halomicrobium sp. LC1Hm. (A) symbiont-free Halomicrobium sp. LC1Hm cell. (B–F) Images of pronounced interaction (fusion) between Halomicrobium sp. LC1Hm and Ca. Nanohalobium constans LC1Nh cells. The ectosymbiont causes evident membrane stretching (red arrow) and is often engulfed by extracellular matrix (blue arrows). PHA: granules of polyhydroxyalkanoate. (Scale bars: 200 nm.)

Fig. 4.

Growth of Halomicrobium sp. LC1Hm in pure (axenic) culture and in coculture with Ca. Nanohalobium constans LC1Nh: (A) Growth on chitin. (B) Growth on starch. (C) The oxygen consumption of axenic LC1Hm and LC1Hm + LC1Nh cultures during growth on chitin. (D and E) The concentration of acetate and reduced sugars, N-acetylglucosamine, and glucose in the supernatants during the midexponential and stationary phases of growth on chitin and starch, respectively. The overall significance level P < 0.01 is shown by single asterisks. Plotted values are means, and error bars (SDs) are based on three culture replicates.

Fig. 5.

Reconstruction of central metabolic and homeostatic functions of Ca. Nanohalobium constans LC1Nh based on genomic, proteomic, targeted metabolomic, and physiological analyses. Enzymes involved in energy production and in reactive oxygen species (ROS) homeostasis/redox regulation are highlighted in yellow. Chitin degradation enabled by seven extracellularly expressed GH18 endochitinases and one GH20 chitodextrinase of the host, is indicated by the gray arrow in the lower-right part of the figure. Depolymerization of (1 → 4)- and (1 → 6)-alpha-d-glucans by two experimentally confirmed extracellularly expressed glucoamylases of Ca. Nanohalobium constans LC1Nh is shown with the green arrow in the upper-left part of the figure. The mutualistic uptake of the formed sugars is indicated by the red arrow. Compounds abbreviations: Asp, aspartate; a-KG, alpha ketoglutarate; 1,3BPG, 1,3-biphosphoglycerate; CoA, coenzyme A; DHAP, dihydroxyacetone phosphate; F-6P, fructose-6-phosphate; F1,6BP, fructose-1,6-biphosphate; Glc-6P, glucose-6-phosphate; Glc-1P, glucose-1-phosphate; GlcNAc, N-acetyl-glucosamine; GlcNAc-6P, N-acetyl-glucosamine-6-phosphate; GlcN-6P, N-glucosamine-6-phosphate; Glu, glutamate; Gln, glutamine; GAP, glyceraldehyde-3-phosphate; OA, oxaloacetate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenol pyruvate; Prx, peroxiredoxin; Trx, thioredoxin. Genes and systems abbreviations: ACL, acetate-CoA ligase; ADH, alcohol dehydrogenase; AK, adenylate kinase; AST, aspartate aminotransferase; AVD, diversity-generating retroelement protein; CLec, C-type lectin fold; DGR, diversity-generating retroelements system; Dsb, disulfide bond formation; GNFAT, glucosamine–fructose-6-phosphate aminotransferase; ENO, enolase; FBPA, fructose-1,6-bisphosphatase; G1P, glucose-1-phosphatase; GAPD, glyceraldehyde-3-phosphate dehydrogenase; GDE, glycogen debranching enzyme; GH, glycogen hydrolase; GNPAT, bifunctional UDP-N-acetylglucosamine pyrophosphatase; GPUT, UTP-glucose-1-phosphateuridylyltransferase; GS, glycogen synthase; HK, gluco(hexo)kinase; LamG, laminin G domain; LDH, lactate dehydrogenase; MaE, malic enzyme; MFS, major facilitator superfamily; MSRA, peptide-methionine (S)-S-oxide reductase; MSRB, peptide-methionine (R)-S-oxide reductase; NOX, pyridine nucleotide-disulphide oxidoreductase; PDH, pyruvate dehydrogenase; PEPS, phosphoenolpyruvate synthase; PGI, glucose-6-phosphate isomerase; PGM, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase; PGK, phosphoglycerate kinase; PF/GK, phosphofructokinase/glucokinase; PKD, polycystic kidney disease; ROS, reactive oxygen species; RT, reverse transcriptase; SOD, superoxide dismutase; TIM, triosephosphate isomerase; TrxR, thioredoxin reductase. Details on genes and systems abbreviations are provided in SI Appendix, Table S7.

Fig. 6.

Comparative metabolic analysis of the 19 nanohaloarchaeal genomes thus far sequenced. Proteins of Ca. Nanohalobium constans LC1Nh proteins are shown in magenta. The A-type H⁺ translocating ATPase complex (nine subunits) is intact in 13 nanohaloarchaeal genomes, whereas incomplete complexes are shown in gray with the numbers of subunits found. The list is not mutually exclusive, as a given protein can have more than one function or domain and was counted in each appropriate category.