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The response of Escherichia coli challenged by hyperosmotic stress can be separated into three phases (2, 10). (i) Immediately after hyperosmotic shock the cells shrink due to massive water efflux. (ii) Subsequently, this process is counteracted by an increase in internal solute concentration due to uptake of potassium ions and synthesis of glutamate. Additionally, compatible solutes like glycine betaine, proline, and trehalose are accumulated. (iii) After establishing stable osmotic conditions, DNA replication, protein synthesis, and growth are resumed. The fast influx of potassium ions increases not only osmolarity, but also the number of positive charges within the cell. Although this is partially balanced by synthesis of glutamate, polyamine efflux may occur to compensate for the charges transported into the cell due to potassium uptake (7). Since we are interested in osmoregulation and related transport processes, we reexamined polyamine transport in E. coli. To circumvent possible complications of previous studies, which required preloading of cells with labeled polyamines, preparation of inside-out vesicles, or overproduction of transport proteins (5), here we analyzed polyamines directly via reversed-phase high-pressure liquid chromatography (HPLC). Thus, we were able to examine polyamine fluxes under physiological conditions.

Growth-dependent polyamine excretion. While polyamine excretion by proliferating eukaryotic cells is widely accepted, it is still under dispute in prokaryotes. In contrast to Tabor and Tabor (8), who deny polyamine excretion by bacteria growing in minimal medium, Kashiwagi et al. (5) exactly propose this flux. We investigated polyamine transport in E. coli wild-type AN387 (9) during growth in M9 minimal medium. Putrescine was excreted in amounts strictly correlated to the cell mass (Fig. 1A), while release of other polyamines like spermidine and cadaverine was not detected. The reason for putrescine efflux during growth is unclear.

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Excretion of putrescine by strain AN387 grown in M9 medium. (A) Growth-dependent increase of external putrescine (solid circles, OD600; open squares, external putrescine). (B) Putrescine concentrations depending on hyperosmotic shock (solid squares, external putrescine after osmotic upshift; open squares, untreated control). NaCl (0.5 M) was added at time zero (indicated by an arrow).

Excretion of polyamines after hyperosmotic shock. To examine polyamine transport upon hyperosmotic stress, cells grown in M9 minimal medium were washed with warmed medium to remove putrescine excreted during growth, resuspended in fresh medium, and subsequently subjected to a hyperosmotic shock by addition of 0.5 M NaCl. As a consequence, strain AN387 started immediately to excrete putrescine, elevating its concentration in the supernatant from 0 to 25 µM within 30 min, while in an untreated control culture, an increase of only 8 µM due to growth-dependent excretion was observed (Fig. 1B).

Polyamine transport and accumulation of glutamate and potassium. To study if the hyperosmotic shift-dependent polyamine excretion is necessary to maintain the charge balance of the cell, internal polyamine, glutamate, and potassium concentrations were determined. For this purpose, cells were grown in K5 medium (3), washed and resuspended in warmed K5 medium, and subjected to an osmotic upshift. Following hyperosmotic shock, strain AN387 started immediately to excrete putrescine, and in the course of the experiment, internal putrescine decreased with a rate of approximately 1 nmol per mg (dry weight) per min from 45 to 5 nmol per mg (dry weight), while external putrescine concentrations rose from 0 to 53 µM (Fig. 2A). It was calculated that the increase in external putrescine exactly corresponded to its decrease in the cell. Changes in spermidine concentrations were not detected.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Solute fluxes in strain AN387 grown in K5 medium. The time of addition of 0.5 M NaCl (upshift) and an equal volume of distilled water (downshift) is indicated by arrows. (A) Polyamine concentrations depending on osmotic stress (solid squares, external putrescine; solid triangles, internal putrescine; open diamonds, internal spermidine). dw, dry weight. (B) Internal glutamate and potassium concentrations depending on osmotic stress (open circles, glutamate; open triangles, potassium).

Role of potE. Polyamines are highly hydrophilic, and passive diffusion through the cytoplasmic membrane is negligible. Two ABC transporters were described for the uptake of putrescine and spermidine besides an ornithine/putrescine antiporter, encoded by potE, which was considered for putrescine uptake and excretion (4). Since we were interested to identify the putrescine efflux system, we investigated the role of the potE gene. Transcription of potE was described to be acid inducible (4). Therefore, we first tested the expression of potE in different media by reverse transcription (RT)-PCR. A potE transcript was detected when RNA was used as template prepared from AN387 cells grown in M9, K5, or K5 medium adjusted to pH 5.5 and supplemented with 0.5 mM ornithine (Fig. 3). The absence of contaminating chromosomal DNA was verified in a control PCR without RT (data not shown). The increased amount of RT-PCR product found when RNA prepared from cells grown at pH 5.5 in the presence of ornithine was used as a template indicated that potE expression is enhanced under these growth conditions.

View larger version (61K): [in this window] [in a new window]   FIG. 3.   Transcription of potE. Products of RT-PCR using total RNA as the template prepared from cells grown in M9, K5, and K5 medium adjusted to pH 5.5 (lanes 1 to 3, respectively), and BstEII-digested  DNA (lane 4). The 0.6-kb potE gene product is indicated by an arrow.

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Solute fluxes in strain potE grown in K5 medium. For explanations, see the legend to Fig. 2.