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Published on: November 29, 2014
The Phn system of Mycobacterium smegmatis: a second high-affinity ABC-transporter for phosphate
Susanne Gebhard1, Sieu L Tran1, Gregory M Cook1
1Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, PO Box 56, Dunedin, New Zealand.
Abstract:
Uptake of inorganic phosphate, an essential but often limiting nutrient, in bacteria is usually accomplished by the high-affinity ABC-transport system Pst. Pathogenic species of mycobacteria contain several copies of the genes encoding the Pst system (pstSCAB), and two of the encoded proteins, PstS1 and PstS2, have been shown to be virulence factors in Mycobacterium tuberculosis. The fast-growing Mycobacterium smegmatis contains only a single copy of the pst operon. This study reports the biochemical and molecular characterization of a second high-affinity phosphate transport system, designated Phn. The Phn system is encoded by a three-gene operon that constitutes the components of a putative ABC-type phosphonate/phosphate transport system. Expression studies using phnD- and pstS-lacZ transcriptional fusions showed that both operons were induced when the culture entered phosphate limitation, indicating a role for both systems in phosphate uptake at low extracellular concentrations. Deletion mutants in either phnD or pstS failed to grow in minimal medium with a 10 mM phosphate concentration, while the isogenic wild-type strain mc(2)155 grew at micromolar phosphate concentrations. Analysis of the kinetics of phosphate transport in the wild-type and mutant strains led to the proposal that the Phn and Pst systems are both high-affinity phosphate transporters with similar affinities for phosphate (i.e. apparent K(m) values between 40 and 90 muM P(i)). The Phn system of M. smegmatis appears to be unique in that, unlike previously identified Phn systems, it does not recognize phosphonates or phosphite as substrates.
Insights
Mycobacterium smegmatis possesses two high-affinity phosphate transport systems, Pst and Phn. The Phn system, previously uncharacterized, functions alongside Pst to ensure bacterial growth under phosphate-limiting conditions.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Physiology
Background:
- Inorganic phosphate is an essential nutrient for bacterial growth, often limiting in various environments.
- The high-affinity ABC-transport system Pst is the primary mechanism for phosphate uptake in most bacteria.
- Mycobacterium tuberculosis pathogenic strains utilize multiple Pst system copies, with PstS1 and PstS2 acting as virulence factors.
Purpose of the Study:
- To biochemically and molecularly characterize a second high-affinity phosphate transport system in Mycobacterium smegmatis, designated Phn.
- To investigate the role of the Phn system in phosphate uptake and bacterial growth under nutrient limitation.
Main Methods:
- Construction and analysis of deletion mutants in the phnD and pstS genes.
- Expression studies using phnD- and pstS-lacZ transcriptional fusions.
- Kinetic analysis of phosphate transport in wild-type and mutant strains.
Main Results:
- A novel three-gene operon, Phn, encoding a putative ABC-type phosphonate/phosphate transport system was identified in M. smegmatis.
- Both Pst and Phn operons are induced under phosphate-limiting conditions, suggesting a cooperative role in phosphate acquisition.
- Mutants lacking either phnD or pstS exhibited impaired growth at low phosphate concentrations, while the wild-type grew efficiently.
- Kinetic studies revealed that both Phn and Pst systems function as high-affinity phosphate transporters with similar affinities (apparent K(m) 40-90 μM P(i)).
- The M. smegmatis Phn system uniquely does not transport phosphonates or phosphite, distinguishing it from other known Phn systems.
Conclusions:
- Mycobacterium smegmatis utilizes two distinct high-affinity phosphate transport systems, Pst and Phn, to ensure nutrient acquisition.
- The Phn system in M. smegmatis is specialized for phosphate transport and does not recognize phosphonates or phosphite.
- Both systems are crucial for bacterial survival and growth when extracellular phosphate concentrations are low.
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