A novel putrescine importer required for type 1 pili-driven surface motility induced by extracellular putrescine in Escherichia coli K-12

Abstract

Recently, many studies have reported that polyamines play a role in bacterial cell-to-cell signaling processes. The present study describes a novel putrescine importer required for induction of type 1 pili-driven surface motility. The surface motility of the Escherichia coli ΔspeAB ΔspeC ΔpotABCD strain, which cannot produce putrescine and cannot import spermidine from the medium, was induced by extracellular putrescine. Introduction of the gene deletions for known polyamine importers (ΔpotE, ΔpotFGHI, and ΔpuuP) or a putative polyamine importer (ΔydcSTUV) into the ΔspeAB ΔspeC ΔpotABCD strain did not affect putrescine-induced surface motility. The deletion of yeeF, an annotated putative putrescine importer, in the ΔspeAB ΔspeC ΔpotABCD ΔydcSTUV strain abolished surface motility in putrescine-supplemented medium. Complementation of yeeF by a plasmid vector restored surface motility. The surface motility observed in the present study was abolished by the deletion of fimA, suggesting that the surface motility is type 1 pili-driven. A transport assay using the yeeF(+) or ΔyeeF strains revealed that YeeF is a novel putrescine importer. The K(m) of YeeF (155 μM) is 40 to 300 times higher than that of other importers reported previously. On the other hand, the V(max) of YeeF (9.3 nmol/min/mg) is comparable to that of PotABCD, PotFGHI, and PuuP. The low affinity of YeeF for putrescine may allow E. coli to sense the cell density depending on the concentration of extracellular putrescine.

FIGURE 1.

Surface motility is induced by extracellular putrescine. Bacterial cultures were diluted in LB medium to an A₆₀₀ of 0.4, and 3 μl of the diluted cultures were dropped onto the center of LBGS plates, supplemented with or without 400 μm putrescine, and incubated at 37 °C. The plates were photographed 40 h after inoculation.

FIGURE 2.

The influence of gene disruptions of putrescine importers or putative putrescine importers on extracellular induced surface motility. Bacterial cultures were diluted in LB medium to an A₆₀₀ of 0.4, and 3 μl of the diluted cultures were spotted onto the center of LBGS plates, supplemented with or without 400 μm putrescine, and incubated at 37 °C. The plates were photographed 40 h after inoculation.

FIGURE 3.

The role of yeeF in surface motility induced by extracellular putrescine. Bacterial cultures were diluted in LB medium to an A₆₀₀ of 0.4, and 3 μl of the diluted cultures were spotted onto the center of LBGS plates, supplemented with or without 400 μm putrescine, and incubated at 37 °C. The plates were photographed 40 h after inoculation. The medium was supplemented with chloramphenicol (15 μg/ml) to maintain the plasmids.

FIGURE 4.

The uptake of extracellular putrescine depends on yeeF. Overnight precultures of SK591, SK595, and SK594 were inoculated in 100-ml LBG supplemented with (gray bars) or without (white bars) 400 μm putrescine in 500-ml Erlenmeyer flasks. The medium was supplemented with chloramphenicol (15 μg/ml) to maintain the plasmids. The initial optical density of the culture, measured at 600 nm, was adjusted to 0.001. The flasks were shaken at 150 rpm at 37 °C. Bacterial cells were harvested at A₆₀₀ = 1.1–1.3 and washed once with cold M9 glucose. The assays were performed three times with independent bacterial cultures. Values are expressed as the mean ± S.D. A, [¹⁴C]putrescine uptake by strains. The cells were incubated with 50 μm [¹⁴C]putrescine, and the reaction was terminated after 0, 1, 7, 13, or 20 min. The linearity between the uptake of [¹⁴C]putrescine and the incubation time was confirmed. B, concentration of intracellular putrescine in the different strains.

FIGURE 5.

YeeF is a major putrescine importer in E. coli grown in LBG medium. The ΔyeeF single knock-out mutant shows an impaired putrescine uptake. The strains MG1655 and SK496 were inoculated in 100-ml LBG medium in 500-ml Erlenmeyer flasks. The initial optical density of the culture, measured at 600 nm, was adjusted to 0.001. The flasks were shaken at 150 rpm at 37 °C. Bacterial cells were harvested at A₆₀₀ = 1.2–1.3 and washed once with cold M9-glucose. The cells were incubated with 500 μm [¹⁴C]putrescine, and the reaction was terminated after 2, 4, 6, 8, or 10 min. The linearity between the uptake of [¹⁴C]putrescine and the incubation time was confirmed. The assays were performed three times with independent bacterial cultures. Values are expressed as the mean ± S.D.

FIGURE 6.

fimA, not fliC, is indispensable in the surface motility observed in the present study. Bacterial cultures were diluted in LB medium to an A₆₀₀ of 0.4, and 3 μl of the diluted culture were spotted onto the center of LBGS plates, supplemented with or without 400 μm putrescine, and incubated at 37 °C. The plates were photographed 40 h after inoculation. In C, the medium was supplemented with chloramphenicol (15 μg/ml) to maintain the plasmids. A, the surface motility induced by putrescine and yeeF is fimA-dependent, not fliC-dependent. B, the surface motility depended on fimA, not fliC, of the strains in the wild-type background. C, complementation experiment with fimA.