Closing the genome of unculturable cable bacteria using a combined metagenomic assembly of long and short sequencing reads

Abstract

Many environmentally relevant micro-organisms cannot be cultured, and even with the latest metagenomic approaches, achieving complete genomes for specific target organisms of interest remains a challenge. Cable bacteria provide a prominent example of a microbial ecosystem engineer that is currently unculturable. They occur in low abundance in natural sediments, but due to their capability for long-distance electron transport, they exert a disproportionately large impact on the biogeochemistry of their environment. Current available genomes of marine cable bacteria are highly fragmented and incomplete, hampering the elucidation of their unique electrogenic physiology. Here, we present a metagenomic pipeline that combines Nanopore long-read and Illumina short-read shotgun sequencing. Starting from a clonal enrichment of a cable bacterium, we recovered a circular metagenome-assembled genome (5.09 Mbp in size), which represents a novel cable bacterium species with the proposed name Candidatus Electrothrix scaldis. The closed genome contains 1109 novel identified genes, including key metabolic enzymes not previously described in incomplete genomes of cable bacteria. We examined in detail the factors leading to genome closure. Foremost, native, non-amplified long reads are crucial to resolve the many repetitive regions within the genome of cable bacteria, and by analysing the whole metagenomic assembly, we found that low strain diversity is key for achieving genome closure. The insights and approaches presented here could help achieve genome closure for other keystone micro-organisms present in complex environmental samples at low abundance.

Comparative genomic analysis of nickel homeostasis in cable bacteria

“…To create clonal enrichment cultures of cable bacteria, we followed the same steps as described previously [ 31 ]. In brief, natural sediment from the site was autoclaved under N 2 atmosphere, filled into cores and exposed to air.…”

“…Two clonal enrichment cultures (HY10‑6 and GW3‑4) resulted from this inoculation procedure. To evaluate if a single cable bacterium strain was present in the enrichment cultures, we used V3‑V4 16S rRNA amplicon sequencing as described before [ 31 ].The two clonal cultures were subsequently maintained in the lab by regularly transferring a small amount of the clonal enrichment culture to freshly autoclaved sediment .…”

“…Filaments were washed in MilliQ (mQ) water droplets (~ 20 µL) to remove sediment particles and salts. Fluorescence in situ hybridization (FISH) and Raman microscopy were performed following the same procedure as described in detail previously [ 31 ]. Raman spectra were collected using a Renishaw inVia™ Qontor® Confocal Raman microscope with a 532 nm excitation laser.…”

“…Polishing as described previously [ 31 ] did not result in properly polished genomes for GW3‑4 and HY10‑6 (the 16S rRNA sequences changed in composition and length with every polishing step). Therefore, to polish the circular cable bacteria contigs, two rounds of polishing with the Illumina reads, acquired by single‑filament amplification, were performed using Pilon v1.24 [ 41 ].…”

“…Including the two closed genomes generated in this study, the initial dataset included a total of 51 publicly available genomes of cable bacteria, of which 45 were incomplete genomes [ 18 , 19 , 42 , 45 ] and 6 closed genomes [ 19 , 31 , 46 ]. Genomes were downloaded from the NCBI genome database (accessed 21 august 2023) and evaluated for genome completeness using CheckM2 v1.02 [ 47 ].…”