Abstract
During 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.
Highlights
Pantoea stewartii subsp. stewartii is a phytopathogen that causes Stewart’s vascular wilt and leaf blight of maize by producing an abundance of stewartan exo/capsular polysaccharides (EPS) in the xylem of an infected plant [1,2]
To determine which specific amino acids are important for EsaR-acyl-homoserine lactone (AHL) interactions that lead to a conformation change in EsaR, esaR was randomly mutagenized through error-prone PCR
15 AHL-independent variants (EsaR*) were initially identified that constitutively repressed a lacZ fusion, controlled by the esa box located at 210 within the esaR promoter upstream of lacZ, in either the presence or absence of AHL. This repression assay indicates that EsaR* is capable of binding to the esa box causing repression of lacZ and since this occurs despite the presence of AHL it serves as a functional screen for EsaR* variants as opposed to other possible types of variants
Summary
Pantoea stewartii subsp. stewartii is a phytopathogen that causes Stewart’s vascular wilt and leaf blight of maize by producing an abundance of stewartan exo/capsular polysaccharides (EPS) in the xylem of an infected plant [1,2]. The production of EPS is regulated by the EsaR/EsaI quorum-sensing system. EsaR is the best-studied example of a subset of AHL-hindered LuxR homologues, including but not limited to ExpR [6,7], YenR [8] and EanR [9], which bind to DNA in the absence of AHL and are deactivated when bound to AHL. This mechanism of AHL control is opposite to that of the majority of the LuxR protein family quorum-sensing regulators. Given the lack of structural information available as well as the relatively low sequence similarity of LuxR proteins [16], it is difficult to accurately predict which EsaR residues are responsible for AHL recognition and response
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