Activation by gene amplification of pitB, encoding a third phosphate transporter of Escherichia coli K-12

Abstract

Two systems for the uptake of inorganic phosphate (P(i)) in Escherichia coli, PitA and Pst, have been described. A revertant of a pitA pstS double mutant that could grow on P(i) was isolated. We demonstrate that the expression of a new P(i) transporter, PitB, is activated in this strain by a gene amplification event.

FIG. 1

Characterization of the pstS mutation in C86. (A) Amplification of the pstS gene and its promoter region by PCR using chromosomal DNA of strains K10, C86, C78, and MC4100 (5) and the primers pst4 (5′-GAGTAATAAATGGATGCCC-3′) and pst5 (5′-CGGTGGGTTAAAAGCAGGC-3′). Strains K10 (pitA10), C86 (pstS21 derivative of K10), and C78 (pstS28 derivative of K10) were kindly provided by the E. coli Genetic Stock Center (Department of Biology, Yale University, New Haven, Conn.). The positions of the molecular size markers are indicated at the left. (B) Position of the IS2 element in the promoter region of the pstS gene in strain C86. The two pho boxes, the −10 region and the Shine-Dalgarno sequence (SD), are indicated. The start of the coding region of pstS is indicated by an arrow.

FIG. 2

Expression of the PstS and the PhoU proteins in pitA mutant strain K10 (lane 1), pitA pstS mutant strain CE1485 (lane 2), and the pseudorevertant strain CE1487 (lane 3). Cells were grown in a peptone-based, phosphate-poor medium (11) supplemented with 0.5% glucose, 660 μM K₂HPO₄, and 1 mM G3P (HP_i medium) (A) or with no K₂HPO₄ or G3P added (LP_i medium) (B). The alternative phosphate source G3P was omitted from the LP_i medium, since it may be degraded by alkaline phosphatase, thereby generating P_i. Although growth of CE1485 in the LP_i medium was very poor, enough cells could be collected for this analysis. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12) and, for Western blotting (1), transferred to a nitrocellulose membrane (Schleicher & Schuell). Immunodetection was performed with polyclonal antisera directed against the P_i-binding protein (PstS) and PhoU. The positions of molecular size standard proteins are indicated on the left in kilodaltons.

FIG. 3

Growth and ³³P_i uptake. (A) Growth curve of pitA mutant strain K10 (○), pitA pstS double-mutant CE1485 (□), and the pseudorevertant CE1487 (▵). Cells were grown overnight in Luria broth supplemented with G3P, pelleted, and resuspended in HEPES-buffered synthetic medium (25) supplemented with 0.5% glucose and 660 μM K₂HPO₄. Growth was monitored for 7 h. (B) Uptake of ³³P_i by cells of strains K10 (○), CE1485 (□), and CE1487 (▵). Cells were grown in Luria broth supplemented with 20 mM glucose and 1 mM G3P to an optical density at 660 nm (OD₆₆₀) of approximately 0.9, washed, and resuspended in a solution of 20 mM potassium piperazine-N,N′-bis(2-ethanesulfonate) (PIPES) (pH 7.0)–10 mM MgSO₄. These cells were stored on ice, and, within 2 h, transport assays were performed at 30°C with 50 μM ³³P-labeled potassium phosphate as described previously (27). The experiments were repeated three times with essentially the same results, and data from a representative experiment are shown.

FIG. 4

(A and B) Maps of the pitB chromosomal region of strains CE1485 (A) and CE1487 (B). Only the relevant BamHI, ClaI, and EcoRI sites are depicted. At the top of panel A, the probes used for Southern hybridization are indicated. At the top of panel B, the arrowheads indicate PCR primers with the following sequences: pr1, 5′-GGAAGATCGATGCGCTGG-3′; pr2, 5′-CCATTACCAGCCTTGGGG-3′; pr3, 5′-GGGGAAATTCTTCTCGGC-3′; pr4, 5′-GGATATCGTCAGCGGCGC-3′; pr5, 5′-CCTGTGTATATATCAAGGCC-3′; pr6, 5′-CAGGTAACGATGGTGCGG-3′; and pr7, 5′-CCTGCTCGGCACTCTCGG-3′. The numbers between the restriction sites indicate the lengths of the fragments in kilobases. (C) Nucleotide sequence of the pitB-gsp intercistronic region. Coding sequences for the glutathionylspermidine synthetase (Gsp), including the stop codon, and PitB proteins are indicated in bold italics and boxed. Dashed arrows indicate inverted repeats, which may function as the transcriptional terminator of the gsp gene. Putative −35 and −10 sequences of the pitB promoter are indicated. The insertion in strain CE1487 of the amplified DNA fragment containing IS5-pitB is indicated.

FIG. 5

Southern blot analysis of chromosomal DNA of CE1485 and CE1487. Blots were hybridized with the pitB probe (A) and probe 3 (B) (see Fig. 4). DNA was digested with BamHI or ClaI, and fragments were separated by electrophoresis on a 0.8% agarose gel. The DNA was transferred from the gel to Hybond-N⁺ membranes (Amersham) with a vacuum blotter (Bio-Rad model 785). After transfer, the filter was washed in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate [pH 7.0]) for 5 min, and DNA was cross-linked by UV irradiation for 2 min. Labeling of the probes, hybridization, and detection were done with digoxigenin labeling and detection kits (Boehringer Mannheim). Hybridization and stringency washes were carried out at 68°C. The positions of molecular size standard DNA fragments are indicated in kilobases.

FIG. 6

Uptake of ³³P_i in right-side-out membrane vesicles of strain CE1491 expressing pitB from plasmid pSL41. Membrane vesicles were diluted to a final protein concentration of 0.1 to 0.5 mg of protein/ml in air-saturated 50 mM potassium PIPES (pH 7.0)–10 mM MgSO₄. The membrane vesicles were preincubated for 3 min at 30°C with 2 μM pyrroloquinoline quinone in the absence (▵) or presence (■) of 20 mM glucose to generate the PMF and in the presence of glucose in combination with 2.5 μM valinomycin (□) or 2.5 μM nigericin (▴). Transport was initiated on addition of 50 μM ³³P-labeled potassium phosphate and analyzed by rapid filtration (27). The experiments were repeated twice with essentially the same results, and data from a representative experiment are shown.