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PMID:12533468

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Citation

Nothaft, H, Parche, S, Kamionka, A and Titgemeyer, F (2003) In vivo analysis of HPr reveals a fructose-specific phosphotransferase system that confers high-affinity uptake in Streptomyces coelicolor. J. Bacteriol. 185:929-37

Abstract

HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS), serves multiple functions in carbohydrate uptake and carbon source regulation in low-G+C-content gram-positive bacteria and in gram-negative bacteria. To assess the role of HPr in the high-G+C-content gram-positive organism Streptomyces coelicolor, the encoding gene, ptsH, was deleted. The ptsH mutant BAP1 was impaired in fructose utilization, while growth on other carbon sources was not affected. Uptake assays revealed that BAP1 could not transport appreciable amounts of fructose, while the wild type showed inducible high-affinity fructose transport with an apparent K(m) of 2 microM. Complementation and reconstitution experiments demonstrated that HPr is indispensable for a fructose-specific PTS activity. Investigation of the putative fruKA gene locus led to identification of the fructose-specific enzyme II permease encoded by the fruA gene. Synthesis of HPr was not specifically enhanced in fructose-grown cells and occurred also in the presence of non-PTS carbon sources. Transcriptional analysis of ptsH revealed two promoters that are carbon source regulated. In contrast to what happens in other bacteria, glucose repression of glycerol kinase was still operative in a ptsH background, which suggests that HPr is not involved in general carbon regulation. However, fructose repression of glycerol kinase was lost in BAP1, indicating that the fructose-PTS is required for transduction of the signal. This study provides the first molecular genetic evidence of a physiological role of the PTS in S. coelicolor.

Links

PubMed PMC142823

Keywords

Amino Acid Sequence; Bacterial Proteins; Base Sequence; Carbon/metabolism; Codon; Fructose/metabolism; Molecular Sequence Data; Phosphoenolpyruvate Sugar Phosphotransferase System/genetics; Phosphoenolpyruvate Sugar Phosphotransferase System/physiology; Streptomyces/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

STRCO:PTHP

involved_in

GO:0009401: phosphoenolpyruvate-dependent sugar phosphotransferase system

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

STRCO:PTHP

GO:0009401: phosphoenolpyruvate-dependent sugar phosphotransferase system

ECO:0000315:

P

Fig 1B and Table 1:

To investigate the role of HPr in vivo, the ptsH gene was inactivated by gene replacement with the apramycin resistance gene aacC4. The derived mutant was verified by Southern blotting and was designated BAP1 (ΔptsH::aacC4) (see Materials and Methods). Western blotting was performed to determine whether the ptsH gene product was absent.

BAP1 did not grow on fructose, while growth on glucose, mannitol, glycerol, glutamate, and other carbon sources tested was unchanged.

Phosphorylation of fructose was not detectable in extracts of BAP1 grown in the presence or absence of fructose.

complete
CACAO 2597


See also

References

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