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dc.contributor.authorSchu, Daniel J.
dc.contributor.authorScruggs, Jessica M.
dc.contributor.authorGeissenger, Jared S.
dc.contributor.authorMichel, Katherine G.
dc.contributor.authorStevens, Ann M.
dc.identifier.citationSchu DJ, Scruggs JM, Geissinger JS, Michel KG, Stevens AM (2014) Acyl-Homoserine Lactone Recognition and Response Hindering the Quorum-Sensing Regulator EsaR. PLoS ONE 9(9): e107687. doi:10.1371/journal.pone.0107687en_US
dc.description.abstractDuring quorum sensing in the plant pathogen Pantoea stewartii subsp. stewartii, EsaI, an acyl-homoserine lactone (AHL) synthase, and the transcription factor EsaR coordinately control capsular polysaccharide production. The capsule is expressed only at high cell density when AHL levels are high, leading to inactivation of EsaR. In lieu of detailed structural information, the precise mechanism whereby EsaR recognizes AHL and is hindered by it, in a response opposite to that of most other LuxR homologues, remains unresolved. Hence, a random mutagenesis genetic approach was designed to isolate EsaR* variants that are immune to the effects of AHL. Error-prone PCR was used to generate the desired mutants, which were subsequently screened for their ability to repress transcription in the presence of AHL. Following sequencing, site-directed mutagenesis was used to generate all possible mutations of interest as single, rather than multiple amino acid substitutions. Eight individual amino acids playing a critical role in the AHL-insensitive phenotype have been identified. The ability of EsaR* variants to bind AHL and the effect of individual substitutions on the overall conformation of the protein were examined through in vitro assays. Six EsaR* variants had a decreased ability to bind AHL. Fluorescence anisotropy was used to examine the relative DNA binding affinity of the final two EsaR* variants, which retained some AHL binding capability but remained unresponsive to it, perhaps due to an inability of the N-terminal domain to transduce information to the C-terminal domain.en_US
dc.description.sponsorshipNational Science Foundation grant MCB-0919984en_US
dc.description.sponsorshipThe publication was supported by Virginia Tech's Open Access Subvention Funden_US
dc.publisherPublic Library of Scienceen_US
dc.rightsCreative Commons Attribution 4.0 International (CC BY 4.0)*
dc.subjectamino acid substitutionen_US
dc.subjectbinding analysisen_US
dc.subjectchaperone proteinsen_US
dc.subjectDNA-binding proteinsen_US
dc.subjectpolymerase chain reactionen_US
dc.subjectsite-directed mutagenesisen_US
dc.subjectsubstitution mutationen_US
dc.titleAcyl-Homoserine Lactone Recognition and Response Hindering the Quorum-Sensing Regulator EsaRen_US
dc.typeArticle - Refereeden_US
dc.title.serialPLoS Oneen_US

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Creative Commons Attribution 4.0 International (CC BY 4.0)
License: Creative Commons Attribution 4.0 International (CC BY 4.0)