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Applied and Environmental Microbiology, March 2005, p. 1283-1290, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1283-1290.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Novel Activator of Mannose-Specific Phosphotransferase System Permease Expression in Listeria innocua, Identified by Screening for Pediocin AcH Resistance

Junfeng Xue,¹ Ian Hunter,^1, Tori Steinmetz,^1, Adam Peters,^1, Bibek Ray,² and Kurt W. Miller^1*

Departments of Molecular Biology,¹ Animal Science, University of Wyoming, Laramie, Wyoming²

Received 30 July 2004/ Accepted 8 October 2004

To identify genes that are important for class IIa bacteriocin interaction and resistance in Listeria species, transposon Tn917 knockout libraries were constructed for Listeria innocua strain Lin11 and screened for mutants that are resistant to pediocin AcH. A highly resistant mutant (G7) (MIC > 20 µg/ml; 1,000-fold less susceptible than the wild type), in which the transposon integrated into the putative promoter of the lin0142 gene, was isolated. lin0142 is located immediately upstream of the mpt operon (mptA/mptC/mptD) that encodes the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system permease EII_t^Man, which serves as a docking protein for class IIa bacteriocins. The transcription of the mpt operon is known to be positively controlled by ⁵⁴ factor and ManR (a ⁵⁴-associated activator). Transcripts for lin0142 and mpt were undetectable in the G7 mutant, based on quantitative real-time reverse transcriptase PCR analysis. When the wild-type lin0142 gene was expressed at a 7.9-fold-elevated level in the mutant via a multicopy-number plasmid, the level of mpt mRNA became 70% higher than that in the wild-type strain. In addition, the complementation strain reverted back to the pediocin AcH-susceptible phenotype. The levels of manR and rpoN (⁵⁴) mRNAs were not directly influenced by the level of lin0142 transcription. lin0142 is the only one of the three mpt regulatory genes whose transcription is induced, albeit slightly (1.2-fold), by glucose. The combined results show that the lin0142 gene encodes a novel activator of the mpt operon. The Lin0142 protein contains a winged-helix DNA-binding motif and is distantly related to the Crp-Fnr family of transcription regulators.

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* Corresponding author. Mailing address: Department of Molecular Biology, University of Wyoming, P.O. Box 3944, Laramie, WY 82071-3944. Phone: (307) 766-2037. Fax: (307) 766-5098. E-mail: kwmiller{at}uwyo.edu.

Present address: Department of Medical Education, Scripts Mercy Hospital, San Diego, CA 92103.

Present address: School of Veterinary Medicine, University of Oregon, Eugene, OR 97403.

Present address: School of Medicine, University of Washington, Seattle, WA 98195.

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Applied and Environmental Microbiology, March 2005, p. 1283-1290, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1283-1290.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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