A Single-cell genome for Thiovulum sp

Abstract

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 μm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.

Fig 1

Phase-contrast micrographs of Thiovulum sp. cells from an enrichment from Elkhorn Slough, California. (A) Typical Thiovulum cell from this environment; (B) a cell undergoing division. Note the highly refractile polar sulfur granules in both cells. Micrographs are courtesy of Marie B. Lund.

Fig 2

16S rRNA phylogenetic tree of Thiovulum sp. ES, other colorless sulfur bacteria, and other Epsilonproteobacteria. Shown is a maximum likelihood tree bootstrapped 100 times; branch labels show numbers of trees with displayed branching pattern.

Fig 3

Phylogenetic tree of Thiovulum sp. ES and related Epsilonproteobacteria based on concatenated alignment of 35 single-copy genes (see Table ST1 in the supplemental material). Shown is a maximum likelihood tree bootstrapped 1,000 times; branch labels show numbers of trees with displayed branching pattern.

Fig 4

Genomically predicted catabolic electron flow network in Thiovulum sp. ES. Locus tags are sulfide:quinone oxidoreductase (ThvES_00003240), formate dehydrogenase (ThvES_00014980), ubiquinol cytochrome bc₁ complex (ThvES_00002990 to ThvES_00003010), cytochrome c₅₅₃ (ThvES_00003480, ThvES_00009980, and ThvES_00017420), nitrate reductase (ThvES_00000060 to ThvES_00000070), cytochrome c peroxidase (ThvES_00008770 and ThvES_00012320), cbb₃-type cytochrome c oxidase (ThvES_00014560 to ThvES_00014590), and polysulfide reductase (ThvES_00006260).

Fig 5

Genomically identified enzymes for the operation of a complete reverse TCA cycle and pyruvate synthase for autotrophic CO₂ fixation in Thiovulum sp. ES. The cofactors are indicated as predicted based on genome annotation and are not experimentally verified. Locus tags are isocitrate dehydrogenase (ThvES_00010990), aconitate hydratase (ThvES_00009840), ATP-citrate lyase (ThvES_00016940 and ThvES_00016970), malate dehydrogenase (locus tag no. ThvES_00001160), fumarate hydrolyase (ThvES_00001170 and ThvES_00007390), fumarate reductase (ThvES_00007350, ThvES_00007360, ThvES_00012790, ThvES_00017900, ThvES_00017910, and ThvES_00017920), succinyl coenzyme A synthase (ThvES_00013960 and ThvES_00014000), 2-oxoglutarate synthase (ThvES_00012430, ThvES_00012420, and ThvES_00012410), and pyruvate:ferredoxin oxidoreductase (ThvES_00006810, ThvES_00006820, ThvES_00006790, and ThvES_00006800).