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Researchers Sequence Genome of Single Marine Bacterial Cell

By Andrea Anderson

NEW YORK (GenomeWeb News) – Researchers from the US and UK have used single-cell genome amplification, sequencing, and assembly to begin characterizing an uncultured bacterial species from an ocean sample.

The team, which included investigators from the J. Craig Venter Institute, the University of California at San Diego, and Illumina Cambridge, sequenced the genome of an individual bacterial cell from an uncharacterized species found in a California ocean sample. The findings, which the team presented online in Nature Biotechnology yesterday, hint that the Deltaproteobacterium has the genetic wherewithal to help break down chlorophyll components and may contribute to biomass degradation processes in its ocean environment.

"Our approach enables acquisition of genome assemblies for uncultivated bacteria using only short reads," the study authors wrote, "providing cell-specific genetic information absent from metagenomic studies."

Though there is a great deal of interest in characterizing the microbes that live in and on the human body and in the environment, most bacterial species can't be grown in the lab, hindering researchers' ability to sequence and study pure populations of these bacteria.

"There are huge numbers of bacteria in the environment and in the human body that we knew nothing about, simply because we don't know how to culture them," JCVI researcher Roger Lasken, corresponding author on the study, told GenomeWeb Daily News.

Efforts have been made to metagenomically sequence bacterial communities — an approach that provides a peek at the genetic capabilities of the community as a whole, but which does not offer details about the genetic characteristics of members of that community.

"Metagenomics provides information about a few genes across many bacteria," co-author Pavel Pevzner, a computer science researcher at UCSD, told GWDN. "Metagenomics cannot reach many genes in a single bacteria."

In an effort to look at more of these genes, he explained, the team used single-cell sequencing to access individual bacterial cell genomes — an approach that they hope will help in unraveling the genetics behind bacterial compounds that microbes use to kill one another, to exploit materials in their environments, and more.

For instance, Pevzner said, to understand how bacteria kill each other, it's important to sequence the genes coding for so-called non-ribosomal peptide synthetases, enzymes that can produce compounds with antibiotic properties. With traditional metagenomics, it's difficult to sequence these and other genes from specific bacteria or do proteomic studies on the resulting proteins.

As such, Pevzner said the single-cell methods described in the new study complements metagenomics by bridging the gap between those community-based studies and more detailed genetic and proteomic studies.

He and his colleagues used a single-cell genome sequencing strategy to tackle the genome of an uncultured marine bacterium dubbed SAR324 that had been collected at a La Jolla ocean site and classified as a Deltaproteobacteria based on 16S ribosomal RNA sequencing.

Their single-cell sequencing method relied on a combination of multiple displacement amplification, Illumina GAIIx paired-end sequencing, and a new single cell assembly algorithm called EULER+Velvet-SC, designed to address amplification bias associated with the MDA method.

After demonstrating the feasibility of their sequencing and assembly strategy on cells from two genetically characterized bacterial species, Escherichia coli and Staphylococcus aureus, the researchers turned their attention to SAR324, generating single-cell genome sequence that was assembled into 4.3 million bases of contig sequence housing 3,811 open reading frames.

From their findings so far, the team estimates that the overall SAR324 genome is between 4.95 million and 6.42 million base pairs.

While more research is needed to tease apart the details of SAR324's role in its environment, patterns in its genome suggest the bug is aerobic and mobile. Along with transfer RNA, amino acid, and vitamin-related genes, for instance, the researchers identified genes involved in aerobic metabolism, flagella formation, and chemotaxis.

Based on the presence of genes that appear to code for enzymes involved in breaking down a component of chlorophyll, the team speculated that SAR324, which is found in both shallow and deep ocean samples, might help to degrade photosynthetic organisms as they sink below the parts of the ocean receiving sunlight.

In the future, researchers plan to use a similar strategy to sequence individual bacterial cells found in a range of environments — from the far reaches of the ocean to hospital wards and human body sites.

"The cost-effective approach demonstrated here should contribute to exploration of microbial taxonomy and evolution and facilitate the mining of environmental organisms for genes and pathway of interest to biotechnology and biomedicine," the study authors wrote.

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