As humans, it is customary to think of us interacting with our environment through the five senses sight, sound, smell, touch and taste. However, this information processing actually takes place through molecular signaling systems. Bacteria also process signals at the molecular level but they utilize a two-component system in which one protein a histidine kinase - senses an environmental signal, which it then transfers to a second protein a response regulator - that controls the reaction.
"These microbial systems are difficult to identify and study because they don't become active until they sense a specific environmental signal and we don't know what most of those signals are," Mukhopadhyay says. "We had to figure a way around this conundrum."
Mukhopadhyay and her co-authors were able to bypass the need to know the signal activation conditions and map virtually the entire D. vulgaris gene response network through genome-wide in vitro experimental determinations. They accomplished this using a "DNA-Affinity-Purified-chip (DAP-chip) strategy" they devised, in which purified response regulator proteins are incubated with genomic DNA and used to enrich DNA regions that bind to them. Both the enriched and the starting input DNA are amplified, pooled and hybridized in a customized D. vulgaris microarray to determine enriched gene targets.
"To our knowledge, this is the first extensive use of a genome-wide method to map all bacterial two component system response regulator binding sites in a single study," Mukhopadhyay says.
Mukhopadhyay and her colleagues have already used their new gene map to predict the functions of several response regula
|Contact: Lynn Yarris|
DOE/Lawrence Berkeley National Laboratory