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Journal of Bacteriology, April 2009, p. 2218-2227, Vol. 191, No. 7
0021-9193/09/$08.00+0     doi:10.1128/JB.01636-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Both Thiamine Uptake and Biosynthesis of Thiamine Precursors Are Required for Intracellular Replication of Listeria monocytogenes{triangledown}

Kristina Schauer,1,3 Jürgen Stolz,2 Siegfried Scherer,1,3 and Thilo M. Fuchs1,3*

Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL), Abteilung Mikrobiologie,1 Abteilung Biochemie,2 Lehrstuhl für Mikrobielle Ökologie, Technische Universität München, Weihenstephaner Berg 3, and Am Forum 5, 85354 Freising, Germany3

Received 18 November 2008/ Accepted 20 January 2009

Thiamine pyrophosphate is an essential cofactor involved in central metabolism and amino acid biosynthesis and is derived from thiamine (vitamin B1). The extent to which this metabolite is available to bacterial pathogens replicating within host cells is still little understood. Growth studies using modified minimal Welshimer's broth (mMWB) supplemented with thiamine or the thiamine precursor hydroxymethylpyrimidine (HMP) showed that Listeria monocytogenes, in agreement with bioinformatic prediction, is able to synthesize thiamine only in the presence of HMP. This appears to be due to a lack of ThiC, which is involved in HMP synthesis. The knockout of thiD (lmo0317), which probably catalyzes the phosphorylation of HMP, inhibited growth in mMWB supplemented with HMP and reduced the replication rate of L. monocytogenes in epithelial cells. Mutation of a predicted thiamine transporter gene, lmo1429, led to reduced proliferation of L. monocytogenes in mMWB containing thiamine or thiamine phosphates and also within epithelial cells but had no influence on the expression of the virulence factors Hly and ActA. The toxic thiamine analogue pyrithiamine inhibited growth of wild-type strain EGD but not of the transporter mutant EGD{Delta}thiT. We also demonstrated that ThiT binds thiamine, a finding compatible with ThiT acting as the substrate-binding component of a multimeric thiamine transporter complex. These data provide experimental evidence that Lmo1429 homologs including Bacillus YuaJ are necessary for thiamine transport in gram-positive bacteria and are therefore proposed to be annotated "ThiT." Taken together, these data indicate that concurrent thiamine uptake and biosynthesis of thiamine precursors is a strategy of L. monocytogenes and possibly other facultative intracellular pathogens to enable proliferation within the cytoplasm.

* Corresponding author. Mailing address: Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL), Abteilung Mikrobiologie, Technische Universität München, Weihenstephaner Berg 3, D-85354 Freising, Germany. Phone: 49-8161-713859. Fax: 49-8161-714492. E-mail: thilo.fuchs{at}wzw.tum.de

{triangledown} Published ahead of print on 30 January 2009.

Journal of Bacteriology, April 2009, p. 2218-2227, Vol. 191, No. 7
0021-9193/09/$08.00+0     doi:10.1128/JB.01636-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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