Complete genome sequence of Thermaerobacter composti strain Ins1, a spore-forming filamentous bacterium isolated from a deep geothermal reservoir

TL;DR

This paper presents the first complete genome sequence of a spore-forming bacterium from deep geothermal fluids.

Contribution

The study provides the first complete circular genome sequence for Thermaerobacter composti strain Ins1.

Findings

The complete genome of Thermaerobacter composti strain Ins1 was sequenced.

This is the first complete (circular) genome assigned to the species Thermaerobacter composti.

Abstract

We report the complete genome sequence of Thermaerobacter composti strain Ins1, a gram-positive filamentous spore-forming bacterium, isolated from deep geothermal fluids used for electricity production. This is the first complete (circular) genome assigned to the species Thermaerobacter composti.

Full text

ANNOUNCEMENT

Members of the Thermaerobacter genus are thermophilic bacteria isolated from high-temperature environments including compost (Thermaerobacter composti JCM 15650 strain Ni80^T^ (1), the Mariana Trench (Thermaerobacter marianensis 7p75a^T^) (2), shallow hydrothermal vents (Thermaerobacter nagasakiensis Ts1a^T^) (3), a bore run-off channel (Thermaerobacter subterraneus C21^T^) (4), and a hydrothermal beach (Thermaerobacter litoralis KW1^T^) (5). Strain Ins1 was isolated from fluids of the production well of the deep geothermal reservoir (163°C; 3,650 m depth) of an electricity production plant in the Upper Rhine Graben (49.1571411 N, 8.1480501 E). After enrichment in Marine Broth 2216 (BD Difco, USA) at 60°C without agitation, only one bacterium grew upon isolation on solid Marine Broth 2216 (1.5% agar).

Genomic DNA was extracted from a 10-day-old culture using the Wizard HMW DNA Extraction Kit (Promega, USA). Sequencing was performed on a Pacific Biosciences Sequel II instrument after DNA shearing, size selection, and HiFi SMRTbell Library construction (chemistry version: 11.1.0.154383; average insert size: 7,718 bp). A genome was assembled from 34,272 HiFi reads using flye (v. 2.9.2) (6) with 2,000 bp minimum overlap and three polishing rounds (average coverage 89×); the chromosome was circularized by flye based on the assembly graph. One consistently methylated motif (GC^m6^ANNNNNNGTT/A^m6^ACNNNNNNTGC, >99% detection rate) was identified by Smrtlink (v. 11). Assembly quality was assessed using CheckM (estimated completeness: 97.03%; contamination rate: 0.53%). The circular assembled genome corresponded to 3,001,239 bp with a 73% G + C content.

The genome was annotated using NCBI Prokaryotic Genome Annotation Pipeline (PGAP v.6.5). It included 2,363 protein-coding genes, 2 copies of each ribosomal RNA gene, 47 tRNAs, and 4 non-coding RNAs (signal recognition particle sRNA, 6S RNA, RNA component of RNase P, and the transfer-messenger RNA); 54 additional putative protein-coding genes were predicted to be non-functional (pseudogenes).

Strain Ins1 was assigned to the Thermaerobacter composti species. The 16S rRNA gene was 99.94% identical with the sequence of T. composti JCM 15650 based on pairwise alignment (NCBI blast). Digital DNA-DNA hybridization (https://ggdc.dsmz.de, dDDH, formula 2) was 86.2%, which falls within the limits of the same bacterial species (7). A phylogenetic tree based on 28 concatenated ribosomal/conserved proteins (elongation factor G/elongation factor Tu/‍L14/‍L15/‍L16/‍L17/‍L18/‍L2/‍L22/‍L23/‍L24/L29/L3‍/L4‍/L5‍/L6‍/S10‍/S11‍/S12‍/S13‍/S14‍/S17‍/S19‍/S3‍/S4‍/S5‍/S7‍/S8‍) aligned using muscle (v. 3.8.31) (8) was constructed by iqtree (v. 2.2.5) (9) with automated model selection, confirming that strain Ins1 clustered within the Thermaerobacter genus, closest to strain FW80 (10).

The amino acid composition of the full proteome showed a limited ERK bias around −15 (11), suggesting that the strain is moderately thermophilic [50°C–60°C predicted optimal growth temperature; 3.08 hours doubling time based on ribosomal protein genes, by gRodon v. 2.3.0 (12)]. Presence in geothermal water is likely due to the production of heat-resistant endospores. Accordingly, the genome encodes 58 out of 66 proteins considered essential for sporulation in Clostridiales (13), and endospores were observed in old cultures (1). Metabolic pathway analysis using gapseq v. 1.2 (14) suggested that the strain requires exogenous thiamine and may be auxotrophic for several amino acids (Try, His, Ser, Phe, Pro, Thr, Tyr, Val).