|Albrecht, Jeffrey - UCLA|
|Gunsalus, Robert - UCLA|
Submitted to: Biomed Central (BMC) Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 12, 2002
Publication Date: June 12, 2002
Interpretive Summary: Escherichia coli like many enteric and soil bacteria can respire anaerobically (without oxygen) by using a variety of compounds as electron acceptors. The respiratory genes that are expressed during anaerobiosis are regulated based upon which compounds are present. Although nitrate is the preferred electron acceptor during anaerobic growth, in its absence, other enzymes such as the DMSO (dimethylsulfoxide) reductase enzyme can utilize DMSO and/or TMAO trimethylamine N-oxide as electron acceptors. The DMSO reductase enzyme is encoded by the dmsABC genes. This study investigated the regulation of the dmsABC genes by the anaerobic regulator, Fnr and the nitrate regulatory protein, NarL. The Fnr and NarL regulatory DNA binding sites for the dmsABC genes involved in anaerobic respiration were identified. DNA sequence changes in the Fnr-recognition site confirmed the contribution of Fnr in the 100-fold anaerobic activation of dmsABC expression. The NarL binding sites located within the dmsABC sequence suggest that multiple NarL molecules bind the DNA and may compete with Fnr and/or RNA polymerase for occupancy on the DNA, indicative of a negative regulator. Furthermore, only the activated form of NarL (phosphorylated) was capable of binding the dmsABC regulatory sequence. Since NarL is phosphorylated in the presence of nitrate, this suggests that only in the presence of the preferred electron acceptor, nitrate, does NarL bind to and repress the expression of dmsABC. This study investigated the genetic regulation of the dmsABC genes involved in anaerobic respiration by defining the Fnr and NarL DNA binding interactions within the dmsABC regulatory region. Together, the two regulators provide for the oxygen and nitrate regulated expression of these respiratory pathway genes in Escherichia coli. Knowledge of these genetic regulatory mechanisms will provide an understanding of how bacteria grow in anaerobic conditions, an environment that enteric bacteria such as E. coli often encounter especially in the animal host.
Technical Abstract: Escherichia coli can respire anaerobically using dimethyl sulfoxide (DMSO) or trimethylamine-N-oxide (TMAO) as the terminal electron acceptor for anaerobic energy generation. Expression of the dmsABC genes that encode the membrane-associated DMSO/TMAO reductase is positively regulated during anaerobic conditions by the Fnr protein and negatively regulated by the NarL protein when nitrate is present. The regions of dmsA regulatory DNA required for Fnr and NarL interactions in response to anaerobiosis and nitrate, respectively, were examined. Mutations within the Fnr site that deviated from the wild-type sequence, TTGATnnnAACAA, or that removed an entire half-site, abolished the anaerobic activation of dmsA-lacZ expression. The region for phosphorylated NarL binding at the dmsA promoter was identified by DNase I and hydroxyl radical footprinting methods: a large 97 bp region that overlaps the Fnr and RNA polymerase recognition sites was protected by NarL-phosphate but not by the nonphosphorylated form of NarL. Hydroxyl radical footprinting analysis revealed that NarL-phosphate protection of both dmsA strands occurred at ten bp intervals and was offset by 3 bp in the 3' direction. These findings suggest that multiple molecules of phosphorylated NarL bind along one face of the DNA with equivalent protein interactions across the minor groove and may interfere with Fnr and/or RNA polymerase interactions at the dmsA regulatory region. The interplay of these transcription factors ensures a hierarchical expression of the dmsABC genes when respiration of the preferred electron acceptors, oxygen and nitrate, is not possible.