Subcellular distribution of enzyme I of the Escherichia coli phosphoenolpyruvate:glycose phosphotransferase system depends on growth conditions

Abstract

The phosphoenolpyruvate:glycose phosphotransferase system (PTS) participates in important functions in the bacterial cell, including the phosphorylation/uptake of PTS sugars. Enzyme I (EI), the first protein of the PTS complex, accepts the phosphoryl group from phosphoenolpyruvate, which is then transferred through a chain of proteins to the sugar. In these studies, a mutant GFP, enhanced yellow fluorescent protein (YFP), was linked to the N terminus of EI, giving Y-EI. Y-EI was active both in vitro (>/=90% compared with EI) and in vivo. Unexpectedly, the subcellular distribution of Y-EI varied significantly. Three types of fluorescence were observed: (i) diffuse (dispersed throughout the cell), (ii) punctate (concentrated in numerous discrete spots throughout the cell), and (iii) polar (at one or both ends of the cell). Cells from dense colonies grown on agar plates with LB broth or synthetic (Neidhardt) medium showed primarily bipolar or punctate fluorescence. In liquid culture, under carefully defined carbon-limiting growth conditions [ribose (non-PTS), mannitol (PTS sugar), or dl-lactate], cellular levels of enzymatically active Y-EI remain essentially constant for each carbon source, but fluorescence distribution depends on C source, cell density, growth phase, and apparently on "conditioned medium." Fluorescence was diffuse during exponential growth on LB or ribose/Neidhardt medium. On ribose they became punctate in the stationary phase, reverting to diffuse when more ribose was added. In LB, both Y-EI and a nonphosphorylatable mutant, H189Q-Y-EI, showed a diffuse fluorescence during growth, but, shortly after the addition of isopropyl beta-d-thiogalactopyranoside, Y-EI became bipolar; H189Q-Y-EI did not. The functions of EI sequestration remain to be determined.

Fig. 1.

Subcellular distribution of FP under different growth conditions. Fluorescence micrographs of BL21-DE3-ΔEI cells harboring pRSETB-Y-EI plasmid grown on agar plates containing LB medium (A) or Neidhardt medium supplemented with 20 mM d-mannitol (B), 20 mM d-ribose (C), or 40 mM dl-lactate (D). (E and F) Cells in liquid culture. Shown are 20 mM d-ribose in Neidhardt medium (E) and control cells harboring pRSETB-YFP (no enzyme I) in LB broth in the presence of 0.5 mM IPTG (F). See Results.

Fig. 2.

Growth of BL21-DE3-ΔE1 containing pRSETB-Y-EI in Neidhardt medium/ribose (1.89 mM). When cells reached stationary phase (at an OD₄₂₀ of 1), more ribose (1.89 mM; indicated by left-most arrow) was added. This cycle was repeated twice. After the third addition, cells showed a punctate distribution (80% of the population) in stationary phase. When more ribose (10 mM) was added (right-most arrow), the population of punctate cells decreased progressively for 60–90 min, when ≈10% of cells were punctate and the remainder were diffuse.

Fig. 3.

Growth curves of BL21-DE3-ΔE1 harboring pRSETB-Y-EI cells in Neidhardt medium at the indicated ribose concentrations. In stationary phases, the percentages of punctate cells were as follows: in 1.89 mM ribose, 5–10%; 3.78 mM, 15%; 5.67 mM, 30%; 7.56 mM, ≈35%; 30 mM, >80%. During the growth phases, no punctate cells were observed; only a diffuse distribution of fluorescence was seen.

Fig. 4.

Fluorescence micrographs of BL21-DE3-ΔE1 cells containing either plasmid pRSETB-Y-EI (A) or pRSETB-Y-EI H189Q (B) in LB broth. The samples were taken 2 h after the addition of 0.5 mM IPTG. Virtually all cells expressing Y-EI (A) exhibited a polar distribution of the fluorescence, whereas all cells that expressed the H189Q mutant (B) displayed a diffuse distribution.

Fig. 5.

Growth curves of BL21-DE3-ΔE1 cells harboring either pRSETB-Y-EI (Upper) or pRSETB-Y-EI H189Q (Lower). The cells were grown in LB medium, and when the OD₄₂₀ was 1.0 for each culture, IPTG (0.5 mM) was added as denoted by the arrows. Samples were removed at the indicated times to determine the OD₄₂₀ (left ordinate, □, ▵) and the percentage of cells that exhibited a bipolar distribution of fluorescence (right ordinate, ▪, ▴). Although ≈99% of Y-EI-containing cells showed a polar distribution at 2 h after addition of IPTG, essentially all H189Q-containing cells showed a diffuse distribution.