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Journal of Bacteriology, April 2009, p. 2776-2782, Vol. 191, No. 8
0021-9193/09/$08.00+0     doi:10.1128/JB.01314-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Putrescine Importer PuuP of Escherichia coli K-12

Shin Kurihara,^1, Yuichi Tsuboi,¹ Shinpei Oda,¹ Hyeon Guk Kim,¹ Hidehiko Kumagai,² and Hideyuki Suzuki^3*

Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan,¹ Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi-cho, Ishikawa-gun, Ishikawa 921-8836, Japan,² Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan³

Received 17 September 2008/ Accepted 25 January 2009

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The Puu pathway is a putrescine utilization pathway involving gamma-glutamyl intermediates. The genes encoding the enzymes of the Puu pathway form a gene cluster, the puu gene cluster, and puuP is one of the genes in this cluster. In Escherichia coli, three putrescine importers, PotFGHI, PotABCD, and PotE, were discovered in the 1990s and have been studied; however, PuuP had not been discovered previously. This paper shows that PuuP is a novel putrescine importer whose kinetic parameters are equivalent to those of the polyamine importers discovered previously. A puuP⁺ strain absorbed up to 5 mM putrescine from the medium, but a puuP strain did not. E. coli strain MA261 has been used in previous studies of polyamine transporters, but PuuP had not been identified previously. It was revealed that the puuP gene of MA261 was inactivated by a point mutation. When E. coli was grown on minimal medium supplemented with putrescine as the sole carbon or nitrogen source, only PuuP among the polyamine importers was required. puuP was expressed strongly when putrescine was added to the medium or when the puuR gene, which encodes a putative repressor, was deleted. When E. coli was grown in M9-tryptone medium, PuuP was expressed mainly in the exponential growth phase, and PotFGHI was expressed independently of the growth phase.

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Polyamines (putrescine, spermidine, and spermine) are aliphatic amines that have two or more amino groups in their chemical structure. Polyamines are distributed widely, from prokaryotic (28) to eukaryotic (17) cells, and are found at especially high concentrations in proliferating cells such as cancer cells (15) and bacteria in the exponential growth phase (5). In vivo, polyamines play important roles as growth factors. For example, polyamines, as cations, bind to nucleic acid to stabilize its structure (30) and promote the synthesis of protein and nucleic acid (4). In addition, polyamines are involved in the modulation of cellular functions by interacting with glutamate receptors such as the N-methyl-D-aspartate receptor (29). Recently it was described that Proteus mirabilis, a gram-negative bacterium and a common urinary tract pathogen of humans, used putrescine as a cell-to-cell signaling molecule to induce differentiation into swarm cells (19, 23). If the synthesis of polyamines is blocked, then cell growth is stopped or profoundly slowed (27). The supplementation of exogenous polyamines restores the growth of these cells (4).

All polyamines have putrescine as a backbone structure, and putrescine is the precursor of all polyamines. The concentration of putrescine in Escherichia coli cells is very high (30 mM) (4) and is regulated by synthesis, transport, and degradation. We previously reported that the putrescine degradation pathway, the Puu pathway, involves -glutamylated intermediates in E. coli (12). All members of the Puu pathway are encoded by genes neighboring each other, which are named the puu gene cluster. The puu gene cluster includes an open reading frame encoding a putrescine transporter, PuuP, which was identified in the previous study (12).

The transport of putrescine in E. coli has been studied for many years. Four transporters that are able to take up putrescine into E. coli cell have been identified (Fig. 1). PotFGHI (18) was previously identified as an ATP-dependent putrescine transporter that is a member of the ATP-binding cassette (ABC) transporter family. PotABCD (3) is a spermidine transporter and is also a member of the ABC transporter family, and PotABCD can take up putrescine with lower affinity. PotE is active in both the excretion and uptake of putrescine (8, 9). Excretion is based on a putrescine-ornithine antiporter activity (8), and uptake is dependent on the membrane potential (9). PuuP is the most recently discovered putrescine transporter, which depends on proton motive force (12). It is thought that the presence of PuuP has a large influence on putrescine homeostasis in E. coli, because deletion of the puuP gene abolished the utilizability of putrescine as a sole nitrogen source and the complementation of puuP gene on the plasmid restored this utilizability (12). However, the properties and regulation of PuuP remain to be investigated. Furthermore, the significance of the multiplicity of genes encoding putrescine transporters should be studied to understand polyamine homeostasis in E. coli.

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FIG. 1. Transporters of putrescine of E. coli that are discussed in this paper. PuuP and PotFGHI are putrescine importers described previously. PotABCD comprises an uptake system that is preferential for spermidine but also takes up putrescine, albeit with lower affinity. YdcSTUV is annotated as a putative putrescine transporter in several databases, but this activity has never been confirmed experimentally. PotE is putrescine-ornithine antiporter that was reported to take up putrescine depending on the culture conditions.

This report examines the properties of PuuP, regulation of puuP expression, and significance of PuuP among the putrescine transporters in putrescine homeostasis.

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Strain and plasmid construction. The ggt gene of SH639 (Table 1) was disrupted as described previously (25). P1 transduction, DNA manipulation, and transformation were performed by standard methods (16, 20) In SK212, disruption of the puuP gene was performed as follows. pSK207, including the DNA sequence of puuR::kan⁺, was linearized using SalI. The resulting 6.4-kb fragment was used to transform strain JC7673 by electroporation, and kanamycin-resistant SK210 was obtained. puuR::kan⁺ was transducted into SH639 to obtain SK212. In SO22, disruption of the puuP gene was carried out as follows. pSO13, including the DNA sequence of puuP::tet⁺, was linearized using XbaI and SacI. The resulting 4.9-kb fragment was used to transform strain JC7673 by electroporation, and tetracycline-resistant SO22 was obtained. To construct YT20, the potABCD genes of KJ101 were disrupted by a method described previously by Datsenko and Wanner (2) using the PCR product amplified from pKD3 with the primers potABCD-1 and potABCD-2. As a result of homologous recombination, the region from the start codon of potA to the termination codon of potD was replaced by Flp recombination target (FRT)-cat⁺-FRT in YT20. In the construction of SK337, the potFGHI genes of KJ101 were disrupted as described previously (2) using the PCR product amplified from pKD13 with the primers potFGHI-1 and potFGHI-2. As a result of homologous recombination, in SK337 the region from the start codon of potF to the termination codon of potI was replaced by FRT-kan⁺-FRT. In the construction of SK359, the ydcSTUV genes of KJ101 were disrupted by the same method (2) using the PCR product amplified from pKD13 with the primers ydcSTUV-1 and ydcSTUV-2. Homologous recombination replaced the region from the start codon of ydcS to the termination codon of ydcV with FRT-kan⁺-FRT. To construct YT1, the potE gene of KJ101 was disrupted by the same method (2) using the PCR product amplified from pKD13 with the primers potE-1 and potE-2. After homologous recombination, the FRT-kan⁺-FRT sequence replaced the region from 45 bp downstream of the start codon of potE to 47 bp upstream of the termination codon of potE (whole length, 1.3 kb). To construct the multiple mutants, P1 transductions using P1(SO22, puuP::tet⁺), P1(SK337, potFGHI::FRT-kan⁺-FRT), P1(SK359, ydcSTUV::FRT-kan⁺-FRT), P1(YT1, potE::FRT-kan⁺-FRT), or P1(YT20, potABCD::FRT-cat⁺-FRT) were performed. In the elimination of the kan⁺ and cat⁺ genes from FRT-kan⁺-FRT and FRT-cat⁺-FRT, respectively, the recombination reaction by Flp flippase carried by pCP20 was used. Colony PCRs were performed with primers which annealed upstream and downstream of the genomic regions containing mutations, and the sizes of DNA amplified were measured to confirm that the intended insertion or deletion mutations were obtained.

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TABLE 1. Strains, phage, plasmids, and oligonucleotides used in this study

Media and reagents. In all experiments, unless otherwise noted, strains were grown at 37°C with shaking at 140 rpm in 60 ml of medium in a 300-ml Erlenmeyer flask. M9-tryptone (14) was used in the analysis of extracellular polyamine concentration and in bacterial cultures for real-time reverse transcription-PCR (RT-PCR) analysis. The compositions of W-Glc-Put (including 0.2% putrescine as the sole nitrogen source) and W-Put-AS (including 0.4% putrescine as the sole carbon source) were described previously (14). The composition of medium B was described previously (6). In the growth experiments on M9-Glc-Put and M9-Put-AS, the strains were precultured on an LB plate at 37°C and then streaked on a nutrient-limited plate and incubated at 20°C.

[1,4-¹⁴C]putrescine dihydrochloride (107 mCi/mmol) was purchased from GE Healthcare, Buckinghamshire, England.

Transport assay. The transport assay was performed as described previously (11) using [¹⁴C]putrescine dihydrochloride, except that M9-tryptone medium was used instead of M63 medium. In the kinetic analysis of PuuP, YT75 was grown in M9-tryptone and harvested in the exponential phase (A₆₀₀ = 0.5). The cells were incubated with 0.1, 0.5, 2, 5, or 10 µM [¹⁴C]putrescine dihydrochloride.

Analysis of polyamine concentration. The polyamines in the samples were measured by high-pressure liquid chromatography as described previously (14).

Real-time RT-PCR analysis. Real-time RT-PCR analysis was performed as described previously (14). puuP-specific primers puuP-RT1 and puuP-RT2 were designed using PRIMER 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) to amplify a 95-nucleotide fragments. The number of transcripts in a sample was determined by comparing the number of cycles required for the reaction to reach a common threshold, with a plot of threshold cycle values against standards (pSO19). The relative expression levels of puuP compared to the controls were calculated using OPTICON (Bio-Rad). The relative amounts of transcripts in the samples were further standardized by amplification of the gapA gene as an internal control using primers gapA-RT1 and gapA-RT2.

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Uptake of putrescine from the medium by PuuP. To analyze the uptake of extracellular putrescine by PuuP during growth in the M9-tryptone, the putrescine concentration in the culture supernatant was measured at different growth phases (Fig. 2). In the puuP⁺ strain, the putrescine concentration in the culture supernatant increased to 30 µM at 3.75 h, dropped to 0 µM at 4.5 h, and did not increase again before the end of the incubation at 28 h after inoculation (Fig. 2A). On the other hand, in the puuP strain, the putrescine concentration increased to around 60 µM at 4 h, was maintained at between 50 and 60 µM until 6.75 h after inoculation, and then gradually decreased to 2 µM at 28 h (Fig. 2A). It is thought that the increase of putrescine in the early growth phase was caused by the export of putrescine to the medium by E. coli and that the rapid drop in the concentration of extracellular putrescine in the puuP⁺ strain from 3.75 to 4.5 h (the exponential phase) after inoculation was caused by the vigorous putrescine uptake from the medium by PuuP expressed at this stage. Furthermore, it is thought that a transporter(s) other than PuuP functioned after 7 h after inoculation (the stationary phase) to gradually decrease the putrescine concentration in the medium.

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FIG. 2. Putrescine concentrations in culture supernatants with and without PuuP. Open circles, putrescine concentration in the culture supernatant of SH639 (puuP+); closed circles, putrescine concentration in the culture supernatant of SO23 (SH639 but puuP); open squares, growth of SH639; closed squares, growth of SO23. (A) Change in putrescine concentration in the culture supernatants of puuP+ and puuP strains. (B) Change in putrescine concentration in the culture supernatant of puuP+ and puuP strains grown in M9-tryptone medium supplemented with 1 mM putrescine.

To assess what quantity of putrescine is taken up from the medium by PuuP, putrescine was added to the medium at a concentration of 1 mM (Fig. 2B). A rapid fall in the concentration of putrescine from 0.82 to 0 mM was observed between 4 and 7 h after inoculation only in the case of puuP⁺ cells. The puuP strain imported less than 0.1 mM until 24 h after inoculation (stationary phase), although putrescine was added to the medium at a concentration of 1 mM (Fig. 2B). The PuuP-dependent decrease in the putrescine concentration in the culture supernatant was up to 5 mM putrescine in the medium (data not shown).

PuuP-dependent uptake of putrescine in E. coli cells. In previous work (12), putrescine uptake was measured by counting ¹⁴C label in cells incubated with ¹⁴C-labeled putrescine. To confirm that putrescine was absorbed without degradation outside the cell, SH639 (puuP⁺), SO23 (puuP), and SO24 (pBR322-puuP⁺/puuP) were grown for 5 h in M9-tryptone and in M9-tryptone supplemented with 5 mM putrescine, and the putrescine concentration in the cells was measured by high-pressure liquid chromatography. The intracellular putrescine concentrations in SH639, SO23, and SO24 grown in M9-tryptone supplemented with 5 mM putrescine were 33, 8, and 89% higher than those in strains grown in M9-tryptone, respectively. This result suggests that PuuP takes up intact putrescine without external degradation.

Comparison of the kinetic parameters of PuuP with those of PotFGHI and PotABCD. In previous work (7), the kinetic values for putrescine uptake by PotFGHI and PotABCD were determined using bacteria harboring pACYC184 with potABCD or potFGHI grown in medium B. To compare the kinetic parameters of PuuP with those values reported in the previous work (7), the K_m and V_max values for putrescine uptake by PuuP were measured using YT75 (pACYC184-puuP/SK425) grown in M9-tryptone. When the kinetic parameters were determined, bacteria were grown in the media in which each transporter was induced well, because PotFGHI activity of the bacteria grown in M9-tryptone was very weak and PuuP activity of SK421 grown in medium B was weaker than that in M9-tryptone (data not shown). The K_m and the V_max of PuuP were 3.7 µM and 19.9 nmol/min/mg of protein, respectively. This result showed that the putrescine uptake capability of PuuP was equivalent to that of PotFGHI and PotABCD (Table 2), although the substrate affinity of PuuP was lower than that of PotFGHI.

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TABLE 2. Kinetic parameters of polyamine importersa

The function of the puuP gene of MA261 is impaired. Although PuuP had an extremely large influence on the uptake of extracellular putrescine (Fig. 2B), PuuP was not found during studies of other putrescine importers. In the study of other putrescine importers, potFGHI, potABCD, and potE were expressed from plasmid vectors introduced into derivatives of a polyamine-requiring strain, MA261 (speB speC) (7). SpeB and SpeC comprise pathways of putrescine synthesis, and MA261 is defective in putrescine synthesis because of speB and speC mutations. The growth of MA261 is stimulated by the addition of putrescine to medium B because extracellular putrescine is imported into the cells by a putrescine importer(s). In the present findings, a large amount of putrescine taken up from the medium by E. coli was dependent on PuuP. Therefore, it was expected that the PuuP-dependent import of putrescine would stimulate the growth of MA261, and YT4 (MA261 but puuP) was constructed. However, growth stimulation depending on PuuP in MA261 was not observed in M9-tryptone medium supplemented with 1 mM putrescine (Fig. 3). Surprisingly, the decrease of putrescine in the culture supernatant that was observed in the genetic background of SH639 (Fig. 2A) was not observed in MA261 (Fig. 3). To elucidate the reason for this discrepancy, the nucleic acid sequence of puuP of MA261 was determined. The open reading flame and the 384-bp upstream region of puuP including the puuP promoter were sequenced, and a mutation of adenine-329 to guanine was found. This mutation resulted in a replacement of Tyr-110 by Cys in PuuP. A transport assay was performed to confirm that the Y110C mutation resulted in a deficiency of putrescine uptake activity by PuuP. The [¹⁴C]putrescine uptake activity of the PuuP Y110C mutant was reduced to one-eighth of that of wild-type PuuP (data not shown). These results suggested that puuP of MA261 was mutated at some step during strain construction and that this mutation impaired the putrescine uptake activity of PuuP. It is suggested that one of the reasons that PuuP had not been discovered during the study of other polyamine importers was because of the Y110C mutation in PuuP of MA261.

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FIG. 3. Impairment of the puuP gene of MA261. Putrescine concentrations in the culture supernatants of MA261 (speB speC) and YT4 (MA261 but puuP) are shown. M9-tryptone medium was supplemented with 1 mM putrescine. Open circles, putrescine concentration in the culture supernatant of MA261 (speB speC); closed circles, putrescine concentration in the culture supernatant of YT4 (MA261 but puuP); open squares, growth of MA261; closed squares, growth of YT4.

Growth phase-dependent regulation of two putrescine importers, PuuP and PotFGHI, in M9-tryptone medium. There are four putrescine importers (PotFGHI, PotE, PotABCD, and PuuP) that were described previously and one putative putrescine importer (YdcSTUV) in E. coli. To clarify which importer functions in specific growth phases in E. coli, five strains that have only one putrescine importer gene and SK425, in which all genes encoding known or annotated putrescine importers were deleted, were constructed. Transport assays with strains that were harvested in the exponential phase (A₆₀₀ = 0.5) and in the stationary phase (A₆₀₀ = 3 to 3.5) were performed (Fig. 4A). In the exponential phase (Fig. 4A), SK421, which had only puuP⁺ as a putrescine importer, exhibited the highest [¹⁴C]putrescine transport activity. However, in the stationary phase (Fig. 4A), the transport activity of SK421 was decreased significantly. In the stationary phase, the [¹⁴C]putrescine transport activity of SK422 (only potF⁺G⁺H⁺I⁺) was the highest among these strains, although the transport activity was low (Fig. 4A). To evaluate the influence of the activity of PuuP and PotFGHI on the environment, the concentrations of putrescine in the culture supernatant of SK421 (only puuP⁺), SK422 (only potF⁺G⁺H⁺I⁺), and SK425 (all genes encoding known or annotated as putrescine importers were deleted) were measured (Fig. 4B). The concentration of putrescine in the culture supernatant of SK421 (only puuP⁺) decreased from 25 µM to almost 0 µM in the exponential phase but increased gradually to 15 µM in the stationary phase because SK421 lacks potFGHI, which are expressed in the stationary phase. On the other hand, the putrescine concentration in the culture supernatant of SK422 (only potF⁺G⁺H⁺I⁺) increased to 60 µM because SK422 lacks puuP, which is expressed in the exponential phase, and then decreased to 50 µM. This result was consistent with the [¹⁴C]putrescine uptake activity of SK421 (only puuP⁺) being high in the exponential phase and the [¹⁴C]putrescine uptake activity of SK422 (only potF⁺G⁺H⁺I⁺) being relatively high in the stationary phase (Fig. 4B). The accumulation of putrescine in the culture supernatant of SK425 (all genes encoding known or annotated as putrescine importers were deleted) was highest in these three strains (Fig. 4B). The concentration of putrescine in the culture supernatant of SK425 was 90 µM at 30 h after inoculation (Fig. 4B).

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FIG. 4. Growth phase-dependent regulation of two putrescine importers, PuuP and PotFGHI, in M9-tryptone medium. (A) Putrescine import activities of various putrescine importers (PuuP, PotFGHI, PotE, and PotABCD) and of a putative putrescine importer (YdcSTUV) at the exponential and stationary growth phases. SK421 (only puuP+, shown as PuuP), SK422 (only potF+G+H+I+, shown as PotFGHI), SK365 (only potE+, shown as PotE), SK423 (only potA+B+C+D+, shown as PotABCD), SK424 (only ydcS+T+U+V+, shown as YdcSTUV), and SK425 (all genes encoding known or annotated putrescine importers were deleted, shown as all) were grown in M9-tryptone medium and harvested in the exponential growth phase (A600 = 0.5, black bars) or in the stationary phase (A600 = 3 to 3.5, white bars). Cells were incubated with 10 µM [14C]putrescine. The linearity between the uptake of [14C]putrescine and the incubation time was confirmed. (B) Putrescine concentrations in the culture supernatants of strains SK421 (only puuP+, closed circles), SK422 (only potF+G+H+I+, open squares), and SK425 (all genes encoding known or annotated putrescine importers were deleted, closed triangles).

Relationship between putrescine transporters and the ability to utilize putrescine. To compare the importance of the transporters in the uptake of putrescine, the strains that had only one remaining putrescine importer were grown on plates (Fig. 5A) with putrescine as a sole carbon or nitrogen source (Fig. 5B). Only SK421, which has only PuuP as a putrescine transporter, could grow on putrescine as a sole source of nitrogen or carbon. This result clearly indicates that only PuuP out of five putrescine importers which are known or annotated is essential for utilization of putrescine as a sole nitrogen or carbon source and can support the growth of E. coli.

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FIG. 5. Utilizability of putrescine as a sole source of nitrogen or carbon depending on PuuP. (A) Left panel, arrangement of strains on the plates. SK421 (only puuP+), SK422 (only potF+G+H+I+), SK365 (only potE+), SK423 (only potA+B+C+D+), SK424 (only ydcS+T+U+V+), and SK425 (all genes encoding known or annotated putrescine importers were deleted) were streaked (see also Fig. 4A) on W-Glc-Put (including putrescine as a sole nitrogen source, center panel) or M9-Put-AS (including putrescine as a sole carbon source, right panel). (B) Compositions of W-Glc-Put and M9-Put-AS.

Expression of puuP is repressed by puuR at the transcription level. It was reported previously that the expression of puuA and puuD was strongly enhanced by the deletion of puuR (13, 14). Therefore, it was expected that the expression of puuP would also be enhanced by the deletion of puuR. Both the [¹⁴C]putrescine uptake activity and the transcription of puuP were strongly enhanced by deletion of the puuR gene (Fig. 6).

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FIG. 6. Regulation of puuP by puuR. White bars, regulation of [14C]putrescine uptake activity of PuuP by deletion of the puuR gene. puuR+ (SH639) and puuR (SK212) strains were grown in M9-tryptone medium and harvested at an A600 of 0.5 (exponential phase). The cells were incubated with 10 µM [14C]putrescine. The linearity between the uptake of [14C]putrescine and the incubation time was confirmed. Gray bars, regulation of the transcription of the puuP gene by deletion of the puuR gene. puuR+ (SH639) and puuR (SK212) strains were grown in M9-Tryptone medium and harvested at an A600 of 0.5, and extracted RNA was subjected to real-time RT-PCR analysis. The transcription level of puuP was quantitated and compared with the puuP expression in the puuR+ strain, which was taken as 1. Error bars indicate standard deviations.

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This study revealed that PuuP is an efficient putrescine importer whose kinetic parameters are not dramatically different from those of PotFGHI (18) and PotABCD (3) (Table 2). This is clear because among all of the genes for putrescine transporters, puuP is indispensable for E. coli to grow on medium containing putrescine as a sole source of nitrogen or carbon (Fig. 5). In fact, E. coli took up a large amount of putrescine from the medium using PuuP (Fig. 2B). MA261, which has been used in studies of polyamine importers, has a mutation in puuP, which decreased the putrescine uptake activity of PuuP severely.

This study revealed that PuuP functions in the exponential growth phase and PotFGHI in the stationary growth phase in the uptake of putrescine from M9-tryptone medium (Fig. 4). Because the activity of PotFGHI was four times higher than that of PuuP when the strains were grown in medium B, which has been used in the study of PotFGHI (3, 7), with shaking at 100 rpm in the stationary phase (data not shown), PotFGHI appears to work more actively than PuuP under some growth conditions.

When a large amount of putrescine was added to the medium, the amount of the putrescine uptake dependent on other transporters in the stationary phase was very small (only 100 µM), compared with up to 5 mM uptake that was dependent on PuuP (Fig. 2B). Furthermore, puuP was highly induced by the addition of putrescine to the medium (data not shown). It seems that only puuP was induced, while other putrescine importers were not induced. These results were consistent with the result that E. coli could grow on putrescine as a sole nitrogen or carbon source in a PuuP-dependent manner (Fig. 5). This indicates that since PuuP is induced by putrescine and takes up a large amount of putrescine in the early stage growth, it is essential for E. coli to form colonies by utilizing putrescine as a nutrient source.

The results described above indicate that PuuP takes up a large amount of putrescine from the medium, followed by the degradation of putrescine by the Puu pathway, and PuuP allows E. coli to grow on putrescine as a sole nitrogen or carbon source. Two putrescine degradation systems are known: the Puu pathway via -glutamylated metabolites (12) and the YdcW-YgjG pathway without -glutamylation (14, 21, 22). Multiple putrescine uptake and degradation systems exist in E. coli. No influence on the growth on putrescine as a sole carbon or nitrogen source was observed when ydcW and ygjG were deleted, while the deletion of genes encoding the Puu pathway abolished the ability to utilize putrescine as a sole carbon or nitrogen source (14), which was consistent with the result that only PuuP among the putrescine importers is effective in the utilization of putrescine as a sole nitrogen or carbon source (Fig. 5). The results indicate that when E. coli grows on putrescine, the Puu pathway is the mainstream putrescine degradation pathway in the exponential growth phase, and PuuP is engaged as a major importer of putrescine.

 
We are grateful to Kazuei Igarashi, Graduate School of Pharmaceutical Sciences, Chiba University, for providing strain MA261. We thank Fumihiko Sato and Nobuhiro Ikezawa, Graduate School of Biostudies, Kyoto University, for the real-time PCR equipment and advice on the real-time RT-PCR analysis of puuP.

S.K., Y.T., and S.O. were supported by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

 
* Corresponding author. Mailing address: Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan. Phone: 81-75-724-7763. Fax: 81-75-724-7766. E-mail: hideyuki{at}kit.ac.jp

Published ahead of print on 30 January 2009.

Present address: Japan Collection of Microorganisms, Microbe Division, RIKEN, BioResource Center, Wako, Saitama, 351-0198, Japan.

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1
1. Cunningham-Rundles, S., and W. K. Maas. 1975. Isolation, characterization, and mapping of Escherichia coli mutants blocked in the synthesis of ornithine decarboxylase. J. Bacteriol. 124:721-729.
2
2. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645.[Abstract/Free Full Text]
3
3. Furuchi, T., K. Kashiwagi, H. Kobayashi, and K. Igarashi. 1991. Characteristics of the gene for a spermidine and putrescine transport system that maps at 15 min on the Escherichia coli chromosome. J. Biol. Chem. 266:20928-20933.[Abstract/Free Full Text]
4
4. Igarashi, K., and K. Kashiwagi. 2006. Polyamine modulon in Escherichia coli: genes involved in the stimulation of cell growth by polyamines. J. Biochem. (Tokyo) 139:11-16.[Abstract/Free Full Text]
5
5. Igarashi, K., and K. Kashiwagi. 2000. Polyamines: mysterious modulators of cellular functions. Biochem. Biophys. Res. Commun. 271:559-564.[CrossRef][Medline]
6
6. Igarashi, K., K. Kashiwagi, K. Kishida, Y. Watanabe, A. Kogo, and S. Hirose. 1979. Defect in the split proteins of 30-S ribosomal subunits and under-methylation of 16-S ribosomal RNA in a polyamine-requiring mutant of Escherichia coli grown in the absence of polyamines. Eur. J. Biochem. 93:345-353.[Medline]
7
7. Kashiwagi, K., N. Hosokawa, T. Furuchi, H. Kobayashi, C. Sasakawa, M. Yoshikawa, and K. Igarashi. 1990. Isolation of polyamine transport-deficient mutants of Escherichia coli and cloning of the genes for polyamine transport proteins. J. Biol. Chem. 265:20893-20897.[Abstract/Free Full Text]
8
8. Kashiwagi, K., S. Miyamoto, F. Suzuki, H. Kobayashi, and K. Igarashi. 1992. Excretion of putrescine by the putrescine-ornithine antiporter encoded by the potE gene of Escherichia coli. Proc. Natl. Acad. Sci. USA 89:4529-4533.[Abstract/Free Full Text]
9
9. Kashiwagi, K., S. Shibuya, H. Tomitori, A. Kuraishi, and K. Igarashi. 1997. Excretion and uptake of putrescine by the PotE protein in Escherichia coli. J. Biol. Chem. 272:6318-6323.[Abstract/Free Full Text]
10
10. Kohara, Y., K. Akiyama, and K. Isono. 1987. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell 50:495-508.[CrossRef][Medline]
11
11. Koyanagi, T., T. Katayama, H. Suzuki, and H. Kumagai. 2004. Identification of the LIV-I/LS system as the third phenylalanine transporter in Escherichia coli K-12. J. Bacteriol. 186:343-350.[Abstract/Free Full Text]
12
12. Kurihara, S., S. Oda, K. Kato, H. G. Kim, T. Koyanagi, H. Kumagai, and H. Suzuki. 2005. A novel putrescine utilization pathway involves -glutamylated intermediates of Escherichia coli K-12. J. Biol. Chem. 280:4602-4608.[Abstract/Free Full Text]
13
13. Kurihara, S., S. Oda, H. Kumagai, and H. Suzuki. 2006. -Glutamyl--aminobutyrate hydrolase in the putrescine utilization pathway of Escherichia coli K-12. FEMS Microbiol. Lett. 256:318-323.[CrossRef][Medline]
14
14. Kurihara, S., S. Oda, Y. Tsuboi, H. G. Kim, M. Oshida, H. Kumagai, and H. Suzuki. 2008. -Glutamylputrescine synthetase in the putrescine utilization pathway of Escherichia coli K-12. J. Biol. Chem. 283:19981-19990.[Abstract/Free Full Text]
15
15. Linsalata, M., M. G. Caruso, S. Leo, V. Guerra, B. D'Attoma, and A. Di Leo. 2002. Prognostic value of tissue polyamine levels in human colorectal carcinoma. Anticancer Res. 22:2465-2469.[Medline]
16
16. Miller, J. H. 1992. A short course in bacterial genetics. A laboratory mannual and handbook for Escherichia coli and bacteria, p.263-274, 437. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
17
17. Pegg, A. E. 1986. Recent advances in the biochemistry of polyamines in eukaryotes. Biochem. J. 234:249-262.[Medline]
18
18. Pistocchi, R., K. Kashiwagi, S. Miyamoto, E. Nukui, Y. Sadakata, H. Kobayashi, and K. Igarashi. 1993. Characteristics of the operon for a putrescine transport system that maps at 19 minutes on the Escherichia coli chromosome. J. Biol. Chem. 268:146-152.[Abstract/Free Full Text]
19
19. Rather, P. N. 2005. Swarmer cell differentiation in Proteus mirabilis. Environ. Microbiol. 7:1065-1073.[CrossRef][Medline]
20
20. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed., p.1.1-2.110. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
21
21. Samsonova, N. N., S. V. Smirnov, I. B. Altman, and L. R. Ptitsyn. 2003. Molecular cloning and characterization of Escherichia coli K12 ygjG gene. BMC Microbiol. 3:2-11.[CrossRef][Medline]
22
22. Samsonova, N. N., S. V. Smirnov, A. E. Novikova, and L. R. Ptitsyn. 2005. Identification of Escherichia coli K12 YdcW protein as a -aminobutyraldehyde dehydrogenase. FEBS Lett. 579:4107-4112.[CrossRef][Medline]
23
23. Sturgill, G., and P. N. Rather. 2004. Evidence that putrescine acts as an extracellular signal required for swarming in Proteus mirabilis. Mol. Microbiol. 51:437-446.[CrossRef][Medline]
24
24. Suzuki, H., T. Koyanagi, S. Izuka, A. Onishi, and H. Kumagai. 2005. The yliA, -B, -C, and -D genes of Escherichia coli K-12 encode a novel glutathione importer with an ATP-binding cassette. J. Bacteriol. 187:5861-5867.[Abstract/Free Full Text]
25
25. Suzuki, H., H. Kumagai, T. Echigo, and T. Tochikura. 1989. DNA sequence of the Escherichia coli K-12 -glutamyltranspeptidase gene, ggt. J. Bacteriol. 171:5169-5172.[Abstract/Free Full Text]
26
26. Suzuki, H., H. Kumagai, and T. Tochikura. 1987. Isolation, genetic mapping, and characterization of Escherichia coli K-12 mutants lacking -glutamyltranspeptidase. J. Bacteriol. 169:3926-3931.[Abstract/Free Full Text]
27
27. Tabor, C. W., and H. Tabor. 1984 Polyamines. Annu. Rev. Biochem. 53:749-790.[CrossRef][Medline]
28
28. Tabor, C. W., and H. Tabor. 1985. Polyamines in microorganisms. Microbiol. Rev. 49:81-99.[Free Full Text]
29
29. Traynelis, S. F., M. Hartley, and S. F. Heinemann. 1995. Control of proton sensitivity of the NMDA receptor by RNA splicing and polyamines. Science 268:873-876.[Abstract/Free Full Text]
30
30. van Dam, L., N. Korolev, and L. Nordenskiold. 2002. Polyamine-nucleic acid interactions and the effects on structure in oriented DNA fibers. Nucleic Acids Res. 30:419-428.[Abstract/Free Full Text]

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Journal of Bacteriology, April 2009, p. 2776-2782, Vol. 191, No. 8
0021-9193/09/$08.00+0     doi:10.1128/JB.01314-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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