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Thiamin transport in Helicobacter pylori lacking the de novo synthesis of thiamin.
- Source :
-
Microbiology (Reading, England) [Microbiology (Reading)] 2019 Feb; Vol. 165 (2), pp. 224-232. Date of Electronic Publication: 2019 Jan 08. - Publication Year :
- 2019
-
Abstract
- Helicobacter pylori lacks the genes involved in the de novo synthesis of thiamin, and is therefore a thiamin auxotroph. The PnuT transporter, a member of the Pnu transporter family, mediates the uptake of thiamin across the membrane. In the genome of H. pylori, the pnuT gene is clustered with the thiamin pyrophosphokinase gene thi80. In this study, we found that [ <superscript>3</superscript> H]thiamin is incorporated into the H. pylori SS1 strain via facilitated diffusion with a Km value of 28 µM. The incorporation of radioactive thiamin was inhibited to some extent by 2-methyl-4-amino-5-hydroxymethylpyrimidine or pyrithiamine, but was largely unaffected by thiamin phosphate or thiamin pyrophosphate. RT-PCR analysis demonstrated that the pnuT and thi80 genes are cotranscribed as a single transcript. The estimated Km value for thiamin in the thiamin pyrophosphokinase activity exerted by the recombinant Thi80 protein was 0.40 µM, which is much lower than the Km value of thiamin transport in H. pylori cells. These findings suggested that the incorporated thiamin from the environment is efficiently trapped by pyrophosphorylation to make the transport directional. In addition, the thiamin transport activity in the pnuT-deficient H. pylori strain was less than 20 % of that in the wild-type strain at extracellular thiamin concentration of 1 µM, but the incorporated scintillation signals of the pnuT-deficient strain with 100 nM [ <superscript>3</superscript> H]thiamin were nearly at the background level. We also found that the pnuT-deficient strain required 100-times more thiamin to achieve growth equal to that of the wild-type. These findings reflect the presence of multiple routes for entry of thiamin into H. pylori, and PnuT is likely responsible for the high-affinity thiamin transport and serves as a target for antimicrobial agents against H. pylori.
- Subjects :
- Bacterial Proteins genetics
Bacterial Proteins metabolism
Biological Transport drug effects
Biological Transport genetics
Escherichia coli genetics
Escherichia coli metabolism
Helicobacter pylori drug effects
Helicobacter pylori genetics
Membrane Transport Proteins deficiency
Membrane Transport Proteins genetics
Mutation
Operon
Pyrimidines pharmacology
Pyrithiamine pharmacology
Recombinant Proteins genetics
Recombinant Proteins metabolism
Thiamin Pyrophosphokinase genetics
Helicobacter pylori metabolism
Membrane Transport Proteins metabolism
Thiamin Pyrophosphokinase metabolism
Thiamine metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1465-2080
- Volume :
- 165
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Microbiology (Reading, England)
- Publication Type :
- Academic Journal
- Accession number :
- 30620266
- Full Text :
- https://doi.org/10.1099/mic.0.000765