The low-affinity phosphate transporter PitA is dispensable for in vitro growth of Mycobacterium smegmatis

Abstract

Background: Mycobacteria have been shown to contain an apparent redundancy of high-affinity phosphate uptake systems, with two to four copies of such systems encoded in all mycobacterial genomes sequenced to date. In addition, all mycobacteria also contain at least one gene encoding the low-affinity phosphate transporter, Pit. No information is available on a Pit system from a Gram-positive microorganism, and the importance of this system in a background of multiple other phosphate transporters is unclear.

Results: The aim of this study was to determine the physiological role of the PitA phosphate transporter in Mycobacterium smegmatis. Expression of pitA was found to be constitutive under a variety of growth conditions. An unmarked deletion mutant in pitA of M. smegmatis was created. The deletion did not affect in vitro growth or phosphate uptake of M. smegmatis. Expression of the high-affinity transporters, PstSCAB and PhnDCE, was increased in the pitA deletion strain.

Conclusion: PitA is the only low-affinity phosphate transport system annotated in the genome of M. smegmatis. The lack of phenotype of the pitA deletion strain shows that this system is dispensable for in vitro growth of this organism. However, increased expression of the remaining phosphate transporters in the mutant indicates a compensatory mechanism and implies that PitA is indeed used for the uptake of phosphate in M. smegmatis.

Figure 1

Expression of a transcriptional pitA-lacZ fusion construct in M. smegmatis. Wild-type M. smegmatis harbouring the pitA-lacZ construct pAH1 was grown in ST medium containing 100 mM phosphate (Control), followed by 2 h starvation in phosphate-free (-P_i) or Mg²⁺-free (-Mg²⁺) ST medium, or 2 h exposure to 5 mM EDTA (+ EDTA), pH 4 or pH 9. β-Galactosidase (β-Gal) activities were assayed and are expressed in Miller Units (MU). Results are the mean ± standard deviation of three independent experiments.

Figure 2

Construction of an unmarked pitA deletion mutant of M. smegmatis mc²155. A: Schematic diagram of the two-step approach for deletion of pitA. The knock-out construct consisted of two fragments flanking pitA on the left (LF) and right (RF) in pX33. Integration of the vector (thick grey line) into the chromosome (thin black line) via the left flank (Int LF) or right flank (Int RF) and subsequent deletion of pitA (KO) are shown. Restriction sites of BamHI (B) and fragment sizes as detected in Southern hybridization are indicated. Drawing not to scale. WT, wild-type. B: Southern hybridization analysis of the integration event. BamHI-digests of genomic DNA of wild-type mc²155 (lane 1) and a candidate colony (lane 2) were probed with radiolabeled right flank PCR product of the deletion construct. C: Southern hybridization analysis of pitA deletion. Analysis of wild-type mc²155 (lane 1) and the pitA deletion strain (lane 2) was performed as in panel B. Molecular masses are indicated in kb.

Figure 3

Kinetics of phosphate uptake. Initial uptake rates of ortho-phosphate (³³P, > 92.5 TBq mmol^-1) into LBT-grown whole cells of M. smegmatis mc²155 (solid squares) and the pitA deletion strain (open squares) were measured over 60 s at phosphate concentrations between 25 μM and 500 μM. Rates are expressed as nmol phosphate min^-1 mg mycobacterial protein extract^-1, and data are shown as the mean ± standard error of the mean from two to five independent measurements per point.

Figure 4

Expression from the pst and phn promoters in the pitA deletion background. Transcriptional phnD-lacZ and pstS-lacZ fusion constructs were introduced into wild-type M. smegmatis (open bars), the pitA deletion strain (black bars) and the pitA complemented strain (hatched bars). β-Galactosidase (β-Gal) activities, expressed as Miller Units (MU), were determined from cultures grown in ST medium with 100 mM phosphate and are shown as the mean ± standard deviation from three independent experiments. Significant differences between samples in one-way ANOVA followed by Bonferroni post-test analyses are indicated by two (p < 0.01) or three (p < 0.001) asterisks.