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Method reveals parts of bacterium genome essential to life

By R&D Editors | August 30, 2011

A team at the Stanford University School of Medicine has
cataloged, down to the letter, exactly what parts of the genetic code are
essential for survival in one bacterial species, Caulobacter crescentus.

They found that 12% of the bacteria’s
genetic material is essential for survival under laboratory conditions. The
essential elements included not only protein-coding genes, but also regulatory
DNA and, intriguingly, other small DNA segments of unknown function. The other
88% of the genome could be disrupted without harming the bacteria’s ability to
grow and reproduce.

The study, which was enabled by the team’s
development of an extremely efficient new method of genetic analysis, paves the
way for better understanding of how bacterial life evolved and for improving
identification of DNA elements that are essential for many bacterial processes,
including the survival of pathogenic bacteria in an infected person. It was
published online in Molecular Systems
Biology
.

“This work addresses a fundamental question in biology: What
is essential for life?” says Beat Christen, PhD, one of the co-first authors of
the new paper and a postdoctoral scholar in developmental biology. “We came up
with a method to identify all the parts of the genome required for life.”

The bacteria studied is a non-pathogenic
freshwater species that has long been used in molecular biology research. Its
complete genome was sequenced in 2001, but knowing the letters in its genetic
code did not tell the researchers which bits of DNA were important to the
bacteria.

“There were many surprises in the analysis of the essential
regions of Caulobacter’s genome,”
says Lucy Shapiro, PhD, the paper’s senior author. “For instance, we found 91
essential DNA segments where we have no idea what they do. These may provide
clues to lead us to new and completely unknown bacterial functions.” Shapiro is
a professor of developmental biology and the director of the Beckman Center
for Molecular and Genetic Medicine at Stanford.

Caulobacter’s
DNA, like that of most bacteria, is a single, ring-shaped chromosome. To
perform their experiment, the researchers mutated many Caulobacter cells so that each cell incorporated
one piece of artificial DNA at a random location in its chromosome. The
artificial DNA, which was labeled so the scientists could find it later,
disrupted the function of the region of bacterial DNA where it landed. Over two
days, the researchers grew these mutants until they had about 1 million
bacterial cells, and then sequenced their DNA. After intensive computer
analysis, they created a detailed map of the entire bacterial genome to show
exactly where the artificial DNA segments had been inserted in the chromosome
of the surviving cells.

This mutation map contained many gaps—the
regions of the DNA where no living bacteria had survived with an artificial DNA
insertion. These regions, the researchers reasoned, must be essential for
bacterial life since disrupting them prevented bacterial survival.

“We were looking for the dog that didn’t
bark,” Shapiro says.

Scientists have used a similar mapping
strategy to find essential genetic elements before, but the Stanford team added
several innovations that greatly improved the speed and resolution of the
method.

“Our method is very streamlined,” Christen
says. “We can do an analysis that would have taken years in a few weeks. We can
immediately go to the answer.”

The new method collapses into a single
experiment work that used to take dozens of experimental steps, and shifts the
majority of the time needed for the research from laboratory work to data
analysis.

In total, the essential Caulobacter genome was 492,941 base pairs
long and included 480 protein-coding genes that were clustered in two regions
of the chromosome. The researchers also identified 402 essential promoter
regions that increase or decrease the activity of those genes, and 130 segments
of DNA that do not code for proteins but have other roles in modifying
bacterial metabolism or reproduction. Of the individual DNA regions identified
as essential, 91 were non-coding regions of unknown function and 49 were genes
coding proteins whose function is unknown. Learning the functions of these mysterious
regions will expand our knowledge of bacterial metabolism, the team says.

The research team anticipates that the new
technique will have several interesting uses in both basic and applied
research. For instance, the technique provides a rapid and economical method to
learn which genetic elements are essential in any microbial species.

“This would give fundamental information so we could
determine which essential genetic elements are conserved through evolution,”
says coauthor Harley McAdams, PhD, professor of developmental biology.

The scientists also pointed out that the
method could be used to examine which DNA segments are essential for bacterial
survival in specific circumstances, such as when pathogenic bacteria invade a
host animal or plant. Developing a comprehensive list of genetic elements that
make a bacterial species infectious could lead to the identification of new
anti-infective agents including new antibiotics.

SOURCE

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