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Sequencing-Based Approach Helps Map Genetic Interactions in Bacteria

NEW YORK (GenomeWeb News) – Researchers from the Howard Hughes Medical Research Institute and the Tufts University School of Medicine reported in the advance, online edition of Nature Methods this week that they have come up with a method for identifying genes needed for bacterial survival — and mapping genetic interactions in bacteria — using a combination of jumping genes and high-throughput sequencing.

The team's method, dubbed Tn-seq, uses a transposon library to disrupt sequences in the bacterial genome followed by high-throughput sequencing to track which gene disruptions are deleterious, neutral, or even advantageous. When they applied Tn-seq to Streptococcus pneumoniae, the bug behind conditions such as pneumonia and meningitis, the researchers found that disruptions to around 16 percent of the organism's genes prevented or severely compromised growth.

The researchers also demonstrated that combining Tn-seq with targeted gene deletions in S. pneumoniae offers a window into genetic interactions in the cell.

"[W]e present Tn-seq, a robust and sensitive method for the discovery of quantitative genetic interactions in microorganisms through massively parallel sequencing," senior author Andrew Camilli, a molecular biology and microbiology researcher affiliated with HHMI and Tufts University, and his colleagues wrote. "The approach does not depend on preexisting genomic microarray or an array of gene knockout strains but is instead based on the assembly of a saturated transposon insertion library."

To develop Tn-seq, the team exploited the properties of transposons, bits of DNA that jump around the genome, creating a genome-wide transposon library using magellan6, a derivative of the Mariner family transposon Himar1.

By sequencing the regions on either side of transposon insertion sites using an Illumina Genome Analyzer II, the team determined how disrupting each of the genes affected S. pneumoniae growth.

"We determined the change in frequency over time for each mutant by en masse sequencing of the regions flanking the transposon insertions, such that a change in fitness translated in a change in the number of reads," Camilli and his co-authors wrote.

The team's findings suggest that only about 16 percent of S. pneumoniae's genes are essential. The bacteria had less severe growth problems when another two percent of genes were affected. In contrast, disrupting six percent of genes actually improved the bug's growth. Fiddling with the remaining 76 percent or so of the S. pneumoniae genes had no obvious growth effects.

The team also demonstrated that they could use Tn-seq to find genetic interactions in S. pneumoniae. By individually knocking out five genes — three transcriptional regulators and two sugar transporters — and then doing Tn-seq, the researchers were able to find networks of genes that interacted with their genes of interest.

That search turned up 97 genes that apparently interact with the transcriptional regulator or sugar transport genes tested, with one gene in particular — ccpA — falling in the center this web of interacting genes.

The researchers noted that the Tn-seq approach could have applications not only for identifying essential genes and networks in other S. pneumoniae strains, but also for gleaning such genetic information from other bacterial species as well.

Camilli and his team hope that such studies will speed up the discovery of bacterial vulnerabilities that could be targeted for vaccine and drug development.

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