A step into the rare biosphere: genomic features of the new genus Terrihalobacillus and the new species Aquibacillus salsiterrae from hypersaline soils

Abstract

Hypersaline soils are a source of prokaryotic diversity that has been overlooked until very recently. The phylum Bacillota, which includes the genus Aquibacillus, is one of the 26 phyla that inhabit the heavy metal contaminated soils of the Odiel Saltmarshers Natural Area (Southwest Spain), according to previous research. In this study, we isolated a total of 32 strains closely related to the genus Aquibacillus by the traditional dilution-plating technique. Phylogenetic studies clustered them into two groups, and comparative genomic analyses revealed that one of them represents a new species within the genus Aquibacillus, whereas the other cluster constitutes a novel genus of the family Bacillaceae. We propose the designations Aquibacillus salsiterrae sp. nov. and Terrihalobacillus insolitus gen. nov., sp. nov., respectively, for these two new taxa. Genome mining analysis revealed dissimilitude in the metabolic traits of the isolates and their closest related genera, remarkably the distinctive presence of the well-conserved pathway for the biosynthesis of molybdenum cofactor in the species of the genera Aquibacillus and Terrihalobacillus, along with genes that encode molybdoenzymes and molybdate transporters, scarcely found in metagenomic dataset from this area. In-silico studies of the osmoregulatory strategy revealed a salt-out mechanism in the new species, which harbor the genes for biosynthesis and transport of the compatible solutes ectoine and glycine betaine. Comparative genomics showed genes related to heavy metal resistance, which seem required due to the contamination in the sampling area. The low values in the genome recruitment analysis indicate that the new species of the two genera, Terrihalobacillus and Aquibacillus, belong to the rare biosphere of representative hypersaline environments.

Keywords: Aquibacillus; Bacillota; Terrihalobacillus; genome mining; hypersaline soils; osmoregulation mechanism; phylogenomics; rare biosphere.

Figure 1

Top hit identity values (%) of the 32 isolates against EzBioCloud database. Best hit was either the species Aquibacillus koreensis BH30097^T (strain names purple-colored) or Aquibacillus albus YIM 93624^T (strain names green-colored). Dot size is proportional to the length of the sequenced 16S rRNA gene. Dashed line indicates the 98.7% identity cutoff for species delineation.

Figure 2

BLASTn identity matrix among the partial or almost complete 16S rRNA gene sequences of the 32 strains isolated in this study.

Figure 3

Neighbor-joining phylogenetic tree based on the comparison of the 16S rRNA gene sequences showing the relationships among the new strains and species of the closely related genera Amphibacillus, Radiobacillus, Sediminibacillus, and Virgibacillus. Bootstrap values ≥70%, based on 1,000 pseudoreplicates, are indicated above branches. Nodes conserved across the three tree-constructing methods are marked with a filled circle. The species Thalassomonas viridans was used as an outgroup. Bar, 0.05 substitutions per nucleotide position.

Figure 4

Genome size (A) and G + C content (B) boxplots of the genome sequences belonging to the family Bacillaceae included into this study. The three new isolates exhibited a very similar value among them and with respect to the single species of the genus Radiobacillus.

Figure 5

Approximately maximum-likelihood phylogenomic tree based on 739 concatenated core protein sequences showing the relationships between the novel isolates and 79 closely related species of the family Bacillaceae. Bootstrap values ≥70% are indicated above the respective branch. Bar, 0.1 substitutions per nucleotide position.

Figure 6

OrthoANI (upper triangle)/GGDC (lower triangle) (A) and AAI (B) values (%) among strains 3ASR75-54^T, 3ASR75-11^T, 3ASR75-286, and the closest related members of the genera Aquibacillus, Radiobacillus, and Sediminibacillus. OrthoANI/GGDC results confirmed that strains 3ASR75-54^T, 3ASR75-11^T, and 3ASR75-286 cannot be affiliated to any of the currently described species of these genera and also proved that strains 3ASR75-11^T and 3ASR75-286 constituted a single species. AAI data unequivocally assigned strain 3ASR75-54^T to the genus Aquibacillus and suggested the placement of strains 3ASR75-11^T and 3ASR75-286 into a new genus within the family Bacillaceae.

Figure 7

Scatter plot displaying AAI-orthoANI pairs of values for the studied genomes within and between genera assuming the placement of strains 3ASR75-11^T and 3ASR75-286 into the new genus Terrihalobacillus (A) and into the genus Radiobacillus (B). Dotted lines denote the 65–72% AAI cutoff for genus delineation. Strain 3ASR75-54^T has been included among the species of the genus Aquibacillus. Circles and crosses indicate intra-and inter-genus values, respectively.

Figure 8

Upset plot showing the intersecting orthologous genes of the isolated strains 3ASR75-54^T, 3ASR75-11^T, and 3ASR75-286, and the species of the closely related genera Aquibacillus, Radiobacillus, and Sediminibacillus. The core genome encoded a total of 1,451 genes out of a pangenome of 18,524 genes. Isolated strains 3ASR75-54^T, 3ASR75-11^T, and 3ASR75-286 harbored an accessory genome with 197, 112, and 153 exclusive genes, respectively. A total of 458 genes were solely present in strains 3ASR75-11^T and 3ASR75-286, while 82 genes where exclusively shared between the three isolates.

Figure 9

Conserved biosynthesis pathway for molybdenum cofactor in prokaryotes. Enzymes catalyzing each reaction are indicated next to the arrow. 5′-GTP, 5′-guanosine triphosphate; cPMP, cyclic pyranopterin monophosphate; MPT, molybdopterin; Mo-MTP, molybdenum inserted in MTP; MCD, MPT cytosine dinucleotide; bis-MGD, MPT guanosine dinucleotide.

Figure 10

(A) Distribution of the isoelectric point of the proteomes under study. Haloarcula vallismortis DSM 3756^T (GCF_900106715.1) and Salinibacter ruber DSM 13855^T (GCF_000013045.1) were also included as representatives of extreme halophiles with salt-in osmoregulation strategy. The proteomes of members of the family Bacillaceae were less acidic than those of the extreme halophiles, suggesting a salt-out mechanism. (B) Reconstruction of the different strategies employed for the studied strains to cope with osmotic stress. Purple filled arrows indicate uptake, green filled arrows indicate extrusion, purple empty arrows indicate biosynthesis, and green empty arrows indicate degradation. Enzymes and transporters are colored key as follows: K+ uptake (dark blue), Na+ extrusion (red), unspecific osmoprotectant uptake (green), unspecific extrusion (light purple), glycine betaine biosynthesis and uptake (light blue), ectoine metabolism and uptake (orange). (C) Heatmap of presence (light blue)/absence (light yellow) of osmoregulation-related genes in the new strains 3ASR75-54^T, 3ASR75-11^T, and 3ASR75-286, and the closely related species of the genera Aquibacillus, Radiobacillus, and Sediminibacillus.

Figure 11

Circular genomic DNA map of strains 3ASR75-11^T, 3ASR75-286, and 3ASR75-54^T. Circles indicate, from the inside outward: G + C content; location of the ectABC operon (orange) and lysC and asd genes (blue); coding sequences in the lagging strand (green); coding sequence in the leading strand (red). A closer insight of the ectABC operon context is displayed below each genome map.

Figure 12

Fragment recruitment of the three new isolated strains from relevant hypersaline metagenomic datasets. Further information about these metagenomes is detailed in Supplementary Table S1. (A) Relative abundance represented as RPKG of strains 3ASR75-11^T, 3ASR75-286, and 3ASR75-54^T together to three reference halophilic species. Squared root transformation was performed for Y-axis in order to better visualize low values. (B) Plots show read recruitment across genome length of the new isolates from Gujarat desert soil (left) and Odiel Saltmarshes hypersaline soil (right) metagenomes.