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  ![](media/image1.jpeg){width="1.4791666666666667in"   ***Advanced Scientific\                 ![](media/image2.png){width="0.6666666666666666in"
  height="0.4791666666666667in"}                        Computing Research***                   height="0.4930555555555556in"}

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**Biochemical Pathways Identified in Microbial Community (2008)**

## Marina G. Kalyuzhnaya^1^, Alla Lapidus^3^, Natalia Ivanova^3^, Alex C. Copeland^3^, Alice C. McHardy^4^, Ernest Szeto^5^, Asaf Salamov^3^, Igor V. Grigoriev^3^, Dominic Suciu^6^, Samuel R. Levine^2^, Victor M. Markowitz^5^, Isidore Rigoutsos^4^, Susannah G. Tringe^3^, David C. Bruce^7^, Paul M. Richardson^3^, Mary E. Lidstrom^1,2^, and Ludmila Chistoserdova^2^ ^1^Department of Microbiology, University of Washington; ^2^Department of Chemical Engineering, University of Washington; ^3^Production Genomics Facility, DOE Joint Genome Institute; ^4^IBM Thomas J. Watson Research Center; ^5^Lawrence Berkeley National Laboratory; ^6^Combimatrix Corporation; ^7^DOE Joint Genome Institute, Los Alamos National Laboratory

***Today's powerful sequencing machines can rapidly read the genomes of
entire communities of microbes, but the challenge is to extract
meaningful information from the jumbled reams of data. In a paper posted
online in Nature Biotechnology, researchers describe a novel approach
for extracting single genomes and discerning specific microbial
capabilities from mixed community ("metagenomic") sequence data. This
analysis of an entire community of Lake Washington microbes shows the
power of metagenomic approaches and IMG/M software***

For the first time, using an enrichment technique applied to microbial
community samples, the research team explored the sediments in Lake
Washington, bordering Seattle, WA, and characterized biochemical
pathways associated with nitrogen cycling and methane utilization,
important for understanding methane generation and consumption by
microbes. Methane is both a greenhouse gas and a potential energy
source.

"Even if you have lots of sequence, for complex communities it still
doesn't tell you which organism is responsible for which function," said
the paper's senior author Ludmila Chistoserdova, a microbiologist at the
University of Washington. "This publication presents an approach, via
simplification and targeted metagenomic sequencing, of how you can go
after the function in the environment."

Chistoserdova and colleagues study microbes that oxidize single-carbon
compounds such as methane, methanol and methylated amines, which are
compounds contributing to the greenhouse effect and are part of the
global carbon cycle.

"To utilize these single-carbon compounds, organisms employ very
specialized metabolism," said Chistoserdova. "We suspect that in the
environment, there are novel versions of this metabolism, and possibly
completely novel pathways."

Most of the microbes that oxidize single-carbon compounds are
unculturable and therefore unknown, as are the vast majority of microbes
on Earth. To find species of interest, the researchers sequenced
microbial communities from Lake Washington sediment samples,
Chistoserdova said, because lake sediment is known to be a site of high
methane consumption. However, these sediment samples contained over
5,000 species of microbes performing a complex, interconnected array of
biochemical tasks.

To enrich the samples for the microbes of interest, the researchers
adapted a technique called stable isotope probing. This is the first
time the technique has been used on a microbial community, Chistoserdova
said. The researchers used five different single-carbon compounds
labeled with a heavy isotope of carbon, and fed each compound to a
separate sediment sample. The microbes that could consume the compound
incorporated the labeled carbon into their DNA, Chistoserdova said,
while organisms that couldn't use the compound did not incorporate the
label. The labeled DNA was then separated out and sequenced. In this
way, microbial "subsamples" were produced that were highly enriched for
organisms that could metabolize methane, methanol, methylated amines,
formaldehyde and formate.

The functionally enriched samples contained far fewer microbes than the
total sample, Chistoserdova said. The sample that was fed methylated
amines (Fig. 1) was simple enough that the group was able to extract the
entire genome of a novel microbe, Methylotenera mobilis, that normally
comprises less than half a percent of the community, but appears to be a
first responder to methylated amines in the environment. The researchers
were able to construct much of M. mobilis' biochemistry, and predict
that it is also involved in nitrogen cycling, demonstrating the utility
of metagenomic analysis.

The DOE JGI performed the sequencing and assembly of these complex
metagenomic data sets. The complexity of the community's sequence
samples created new challenges for genome assembly. "It is very
important for metagenomic assemblies to rely on high-quality reads,"
said Alla Lapidus, microbial geneticist at the DOE JGI and co-author on
the paper. If some of the sequence is of low quality, she said, it can
lead to errors in assembly and gene annotation.

Because of the need for higher quality control, Lapidus said, the DOE
JGI developed a new quality control approach that involves a computer
tool called LUCY to trim out low-quality sequence in combination with
the Paracel Genome Assembler, which appeared to be more appropriate for
metagenomic assemblies. This approach was pioneered on the Lake
Washington project, Lapidus said, and due to its superior results it is
now the standard metagenomic assembly method at the DOE JGI.

"The DOE JGI's unique Integrated Microbial Genomics with Microbiome
Samples (IMG/M) data management system was used for detailed annotation,
and was instrumental for efficient comparative analysis and metabolic
reconstruction of the samples," Lapidus said.

IMG/M was jointly developed by DOE JGI and Berkeley Lab's Biological
Data Management and Technology Center (BDMTC). BDMTC Department Head
Victor Markowitz and staff scientist Ernest Szeto performed the data
processing for this study and were co-authors. Other DOE JGI authors
include Natalia Ivanova, Alex Copeland, Asaf Salamov, Igor Grigoriev,
Susannah Tringe, David Bruce (Los Alamos National Laboratory), and Paul
Richardson.

Michael Galperin, a microbial geneticist at the National Center for
Biotechnology Information at the National Institutes of Health, who was
not involved in the study, said in an email that the paper describes

![](media/image3.png){width="3.0in" height="3.529861111111111in"}

*Fig. 1. Methylamine-enriched community of Lake Washington sediment
featuring Methylotenera cells. (Photo: D. Kunkel; color, E. Latypova.)*

"an interesting novel approach" and the results "constitute a
significant advance in the emerging discipline of metagenomics."

"I think other people can use the same approach in different
environments, as long as they have an enrichment technique,"
Chistoserdova said. "For us this work is just the beginning, because now
we will be using this metagenomic sequence as a scaffold for downstream
experiments in our lake."

IMG/M is available at http://img.jgi.doe.gov/m.

**Publications:**

Marina G. Kalyuzhnaya et al., "High-resolution metagenomics targets
specific functional types in complex microbial communities," Nature
Biotechnology, published online 17 August 2008, doi:10.1038/nbt.1488.

**Funding:**

DOE Office of Biological and Environmental Research, Office of Advanced
Scientific Computing Research, and Community Sequencing Program;
National Science Foundation.