Functional diversity of nanohaloarchaea within xylan-degrading consortia

Abstract

Extremely halophilic representatives of the phylum Candidatus Nanohaloarchaeota (members of the DPANN superphyla) are obligately associated with extremely halophilic archaea of the phylum Halobacteriota (according to the GTDB taxonomy). Using culture-independent molecular techniques, their presence in various hypersaline ecosystems around the world has been confirmed over the past decade. However, the vast majority of nanohaloarchaea remain uncultivated, and thus their metabolic capabilities and ecophysiology are currently poorly understood. Using the (meta)genomic, transcriptomic, and DNA methylome platforms, the metabolism and functional prediction of the ecophysiology of two novel extremely halophilic symbiotic nanohaloarchaea (Ca. Nanohalococcus occultus and Ca. Nanohalovita haloferacivicina) stably cultivated in the laboratory as members of a xylose-degrading binary culture with a haloarchaeal host, Haloferax lucentense, was determined. Like all known DPANN superphylum nanoorganisms, these new sugar-fermenting nanohaloarchaea lack many fundamental biosynthetic repertoires, making them exclusively dependent on their respective host for survival. In addition, given the cultivability of the new nanohaloarchaea, we managed to discover many unique features in these new organisms that have never been observed in nano-sized archaea both within the phylum Ca. Nanohaloarchaeota and the entire superphylum DPANN. This includes the analysis of the expression of organism-specific non-coding regulatory (nc)RNAs (with an elucidation of their 2D-secondary structures) as well as profiling of DNA methylation. While some ncRNA molecules have been predicted with high confidence as RNAs of an archaeal signal recognition particle involved in delaying protein translation, others resemble the structure of ribosome-associated ncRNAs, although none belong to any known family. Moreover, the new nanohaloarchaea have very complex cellular defense mechanisms. In addition to the defense mechanism provided by the type II restriction-modification system, consisting of Dcm-like DNA methyltransferase and Mrr restriction endonuclease, Ca. Nanohalococcus encodes an active type I-D CRISPR/Cas system, containing 77 spacers divided into two loci. Despite their diminutive genomes and as part of their host interaction mechanism, the genomes of new nanohaloarchaea do encode giant surface proteins, and one of them (9,409 amino acids long) is the largest protein of any sequenced nanohaloarchaea and the largest protein ever discovered in cultivated archaea.

Keywords: CRISPR; ecology of nanohaloarchaea; methylomics; nanohaloarchaeal-haloarchaeal symbioses; ncRNA.

Figure 1

Pairwise genome comparison between Ca. Nanohalococcus occultus SVXNc, Ca. Nanohalovita haloferacivicina BNXNv, and Ca. Nanohalobium constant LC1Nh. Circos-based genome alignments and Dot-plot alignments are present in the left and right columns, respectively. Links indicate pairs of orthologous genes between the genomes, and the color is scaled to the percentage of amino acid identity levels (shown as the bottom-left inserts). The genomic islands predicted with IslandViewer4 are shown as black septa, while the positions of giant SPEARE proteins found in the BNXNv and SVXNc genomes are shown as red septa. For the SVXNc and LC1Nh couple, the similarity was too low for the two-way ANI index calculation, and only one-way ANI was shown.

Figure 2

Structure of the CRISPR-Cas systems found in the Ca. Nanohalococcus occultus SVXNc genome and MAGs belonging to other two nanohaloarchaea (A) and phylogenetic analysis of Cas1 proteins (B). The tree was constructed using the PhyML 3.0 plugin inside Geneious 7.1 with BLOSUM62 substitution model and 1,000 bootstrap replicates. Bootstrap values are shown as filled circles at the nodes.

Figure 3

Atlas view of the circular chromosome of the strain SVXNc. Histogram graphs show GC-skew and GC-composition fluctuations in a 5,000 bp sliding window. Locations of motifs GDGCHC with bipartite methylated cytosine residues on the direct and reverse complement DNA strands are depicted by blue triangles outside and inside of the outermost ring representing the chromosome. In the motif sequence GDGCHC, the methylated cytosine (C) is shown red, and the guanine residues (G) opposing the methylated cytosine on the complementary strand are italicized. Locations of the two identified genomic islands are depicted by numbered pink boxes.

Figure 4

Field emission scanning electron micrographs of Ca. Nanohalovita haloferacivicina BNXNv (A, B) and Ca. Nanohalococcus occultus SVXNc (C, D) attached to their respective hosts, Haloferax lucertense BNX82 and SVX82. The images show a strong difference in the median multiplicity of host-attached nanosymbionts: Ca. Nanohalovita BNXNv−1–2 cells/host cell; Ca. Nanohalococcus SVXNc−4–7 cells/host cell. Dividing BNXNv cells are indicated by white arrows (A). Scale bars represent 1,000 nm.

Figure 5

Domain structure of giant surface proteins BNXNv_0298 and SVXNc_0300. The giant protein of Ca. Nanohalovita BNXNv is 8,880 aa-long (gene is 26,643 bp-long) with a calculated molecular weight of 974,524 Da and an estimated pI of 4.11. The giant protein of Ca. Nanohalococcus SVXNc is 9,409 aa-long (gene is 28,230 bp-long) with a calculated molecular weight of 1,030,728 Da and an estimated pI of 4.03.

Figure 6

Schematic diagram of the BNXNv phosphotransferase (PTS) system (A) and the proposed pathway for sialic acid synthesis by SVXNc inferred from its genome analysis and transcriptome data (B). The BNXNv PTS system seems to be responsible for the simultaneous transport and phosphorylation of sugar substrates. A series of enzyme intermediates, including EI (BNXNv_0770), HPr (BNXNv_0769), EIIA (BNXNv_0768), EIIB (BNXNv_0765), and EIIC (BNXNv_0767), were predicted to be phosphorylated.