Editor: Robert Burne
Dependence of swarming in Escherichia coli K-12 on spermidine and the spermidine importer
Version of Record online: 10 MAR 2009
© 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiology Letters
Volume 294, Issue 1, pages 97–101, May 2009
How to Cite
Kurihara, S., Suzuki, H., Tsuboi, Y. and Benno, Y. (2009), Dependence of swarming in Escherichia coli K-12 on spermidine and the spermidine importer. FEMS Microbiology Letters, 294: 97–101. doi: 10.1111/j.1574-6968.2009.01552.x
- Issue online: 2 APR 2009
- Version of Record online: 10 MAR 2009
- Received 10 December 2008; accepted 16 February 2009.First published online 10 March 2009.
In a previous work, it was observed that the swarming of polyamine-deficient Proteus mirabilis (speB::sm) was severely inhibited on Luria–Bertani (LB) swarming plates (LBSw) (LB, 0.5% glucose, 0.5% agar), and it was clarified that extracellular putrescine was important as a signaling molecule for the induction of swarming in P. mirabilis. However, a polyamine-deficient strain (delta-speAB delta-speC) of Escherichia coli swarmed as well as the parental strain on LBSw plates. We report that the swarming phenotype of a polyamine-deficient E. coli strain is dependent on spermidine and PotABCD, a spermidine importer.
Polyamines (putrescine, spermidine, and spermine) are aliphatic amines that are widely distributed in bacteria, plants, and animals (Tabor & Tabor, 1984). They play important roles in cell proliferation, tissue growth, and differentiation (Tabor & Tabor, 1984). In bacteria, polyamines bind to DNA to stabilize its structure (Tabor & Tabor, 1985), and they modulate the translation of several genes by binding to their mRNA (Igarashi & Kashiwagi, 2006). Polyamines are synthesized and exported by bacteria, and they are present in the environment where bacteria live. For example, in the intestinal tract of animals, the concentration of polyamines originating from intestinal bacteria can reach micromolar levels (Noack et al., 1998). During the growth of Escherichia coli in broth, putrescine is exported by E. coli in a manner proportional to cell density (Schiller et al., 2000).
Escherichia coli synthesizes two polyamines, putrescine and spermidine. There are two pathways by which putrescine is synthesized. One is the ornithine decarboxylation pathway catalyzed by SpeC, and the other is the pathway in which putrescine is synthesized from arginine by a two-step reaction: decarboxylation of arginine to yield agmatine, which is catalyzed by SpeA, and hydrolysis of agmatine to produce putrescine and urea, which is catalyzed by SpeB. Spermidine is synthesized from putrescine by SpeE by the addition of the aminopropyl moiety derived from decarboxylated S-adenosylmethionine. The decarboxylation of S-adenosylmethionine is catalyzed by SpeD.
The transport systems for polyamines have been well studied in E. coli. PotABCD (Pistocchi et al., 1993) is a spermidine importer that imports putrescine too, and it was previously reported (Higashi et al., 2008) that MdtJI exports spermidine. As described above, E. coli has both import and export systems for polyamines; therefore, it is thought that E. coli exports or imports polyamines according to need by altering the expression of the genes encoding the polyamine transporters. However, the function of extracellular polyamines is not well known.
Swarming is a bacterial process that allows cells to move in a coordinated manner and expand the population to new locations. The process of swarming is distinct from swimming in that swarming is a multicellular process that occurs on solid surfaces and requires the differentiation of vegetative cells into a specialized cell type called a swarmer cell (Rather, 2005). In a previous work, it was demonstrated that in Proteus mirabilis, a gram-negative bacterium and a common urinary tract pathogen in humans (Mobley et al., 1996; Rozalski et al., 1997), putrescine was used as an inducer of differentiation into swarm cells (Sturgill & Rather, 2004; Rather, 2005). In the study (Sturgill & Rather, 2004), it was observed that putrescine rescued the induction of swarming in a putrescine-deficient mutant of P. mirabilis (speB::sm), but spermidine did not. Because polyamines are highly hydrophilic, the import of polyamines through the hydrophobic membrane into the cell requires transporters; however, studies focusing on polyamine transporters as importers of the signaling molecule still need to be conducted. In this study, we report that in E. coli, spermidine as well as putrescine is important for swarming of the polyamine-deficient mutant (ΔspeAB::FRT ΔspeC::FRT), and that the induction of swarming is dependent on the spermidine importer PotABCD.
Materials and methods
Media and culture conditions
The Luria–Bertani (LB) (BD, Franklin Lakes, NJ) swarming plates (LBSw) used in this study comprise LB supplemented with 0.5% glucose and 0.5% agar (Eiken, Tokyo, Japan). The M9 (Miller, 1992) swarming plates (M9Sw) contained M9 supplemented with 0.5% glucose, instead of the 0.2% glucose described in Miller (1992), and 0.5% agar (Eiken). The water content of the plates was equalized in each experiment by incubation of the autoclaved medium in a waterbath of 50 °C for 30 min; the medium was then poured into a Petri dish (∅=10 cm) and dried with the lid open on a clean bench for 30 min. The strains were precultured in 5 mL LB in a tube (∅=18 mm) at 37 °C overnight. In the experiments using LBSw, the overnight culture was diluted to an A600 nm of 0.4 in LB, and in the experiments using M9Sw, the overnight culture was washed once with M9 buffer (M9 medium without glucose) and diluted to an A600 nm of 0.4 in M9 medium. Then, 3 μL of the diluted culture was dropped onto the center of the plate. The plate was sealed with a paraffin film and incubated at 37 °C. The compositions of the LBSw liquid medium (LBSwL) and M9Sw liquid medium (M9SwL) are LB+0.5% glucose and M9+0.5% glucose, respectively.
Strain and plasmid construction
The 4130-bp fragments, including the entire region of potABCD, the 58-bp region downstream of potABCD, and the 232-bp region upstream of potABCD was amplified by PCR. KOD-plus-DNA polymerase (Toyobo, Osaka, Japan), the genome of SH639 (Table 1) that was used as a template, and the primers ‘potABCD up SacI’ and ‘potABCD down SmaI’ were used for the PCR, following the manufacturer's instructions. The primers were designed so as to add SacI and SmaI restriction sites to the 5′-end of the amplified region. The amplified fragment was ligated to pMW119 digested with SmaI and SacI, and this plasmid was designated pMW119-potA+B+C+D+. The cloned region including potABCD was sequenced to confirm that no sequence changes had occurred. pMW119-potA+B+C+D+ was digested with SmaI and SacI and blunt ended with a DNA blunting kit (Takara, Otsu, Japan). The obtained fragment including the potABCD gene was ligated to pBelobac11 cleaved by HpaI, and this plasmid was designated pBelobac11-potA+B+C+D+. In the construction of SK479 and YT16, speAB, speC, and potABCD were disrupted by the method described previously (Datsenko & Wanner, 2000). P1 transduction (Miller, 1992) was used for constructing the multiple mutants.
|Characteristics or sequences||Sources or references|
|SH639||F−Δggt-2||Suzuki et al. (1987, 2005)|
|SK479||SH639 but ΔspeAB::FRT ΔspeC::FRT ΔpotABCD::FRT||This study|
|YT16||SH639 but ΔspeAB::FRT ΔspeC::FRT||This study|
|pBelobac11||Mini-F replicon cat+||New England Biolabs|
|pBelobac11-potA+B+C+D+||Mini-F replicon cat+potA+B+C+D+||This study|
|pMW119||pSC101 replicon bla+rep+lacZ+||New England Biolabs|
|pMW119-potA+B+C+D+||pSC101 replicon bla+rep+potA+B+C+D+||This study|
|potABCD up SacI||5′-TTTGAGCTCGTAGTCACCCTCACTTTTTG-3′||This study|
|potABCD down SmaI||5′-TTTCCCGGGTAGCCACATCCTTGCTAACT-3′||This study|
The swarming motility of the putrescine-deficient strain of E. coli is similar to that of the parental strain
As described previously, on the LBSw, the swarming ability of the speB::sm mutant of P. mirabilis was inhibited compared with that of the wild-type strain (Sturgill & Rather, 2004). However, the putrescine-deficient E. coli mutant YT16 (ΔspeAB::FRT ΔspeC::FRT) containing pBelobac11 swarmed as well as strain SH639 (speA+B+speC+) containing pBelobac11 did (Fig. 1).
The spermidine importer PotABCD is important for the swarming of the putrescine-deficient strain on the LBSw
Unlike in the case of P. mirabilis, in E. coli, the ability to synthesize putrescine was not involved in the swarming ability on LBSw. This result suggested that spermidine present in the medium induced the swarming of the putrescine-deficient strain YT16 containing pBelobac11 because the LB medium contains c. 100 μM of spermidine. Therefore, to examine the role of the spermidine importer encoded by potABCD, we constructed a potABCD deletion mutant in the speABC mutant background (Fig. 2). As shown in Fig. 2a, both the strain YT16 (ΔspeABΔspeC potA+B+C+D+) containing pBelobac11 and the strain SK479 (ΔspeABΔspeCΔpotABCD) complemented with pBelobac11-potA+B+C+D+ swarmed well on LBSw plate. In contrast, the swarming of SK479 (ΔspeABΔspeCΔpotABCD) containing pBelobac11 was severely inhibited (Fig. 2a). We determined the influence of cell growth on the extent of swarming by growing three strains in LBSwL (Fig. 2b) and measuring the A600 nm values. Growth of SK479 containing pBelobac11 was partially inhibited (Fig. 2b) and its swarming activity was severely inhibited, compared with YT16 containing pBelobac11 and SK479 containing pBelobac11-potA+B+C+D+, which showed good swarming. This result suggested that although the growth inhibition partially inhibited the swarming activity, other factors also existed.
PotABCD and spermidine are essential for swarming on M9Sw minimal medium
To clearly demonstrate that spermidine and PotABCD are important for the swarming of E. coli, we used M9Sw minimal medium. YT16 containing pBelobac11, SK479 containing pBelobac11, and SK479 containing pBelobac11-potA+B+C+D+ did not show swarming activity on M9Sw minimal medium. However, YT16 containing pBelobac11 (carrying potA+B+C+D+ in the genome) and SK479 containing pBelobac11-potA+B+C+D+ showed good swarming on M9Sw supplemented with 1 μM spermidine. In contrast, SK479 (ΔpotABCD) containing pBelobac11 did not show swarming activity on the same plates (Fig. 3a). In order to determine the influence of growth on the extent of swarming, three strains were grown in M9SwL supplemented with or without 1 μM spermidine, and the A600 nm values were measured (Fig. 3b). In M9SwL supplemented with 1 μM spermidine, the growth of YT16 containing pBelobac11 and SK479 containing pBelobac11-potA+B+C+D+, which swarmed well on the semisolid medium, was better than the growth of SK479 containing pBelobac11. However, the inhibition of swarming was not mainly due to the growth inhibition, because in M9SwL supplemented with 1 μM spermidine, SK479 containing pBelobac11, which did not swarm, showed 80% of the growth observed in YT16 containing pBelobac11 and SK479 containing pBelobac11-potA+B+C+D+, which swarmed well. In addition, SK479 containing pBelobac11 showed the best growth among the three strains in M9SwL and the growth of SK479 containing pBelobac11 in M9SwL (closed triangles) was comparable to that of YT16 containing pBelobac11 (open squares) and SK479 containing pBelobac11-potA+B+C+D+ (open diamonds) in M9SwL supplemented with 1 μM spermidine at 45 h after inoculation. Therefore, it is thought that growth is not related directly to the swarming activity, although the growth speed in the initial growth phase may partially affect the activity of swarming.
In the previous study on the swarming of P. mirabilis (Sturgill & Rather, 2004), the swarming of the putrescine-deficient mutant (speB::sm) was significantly inhibited on the LBSw. In this study, however, the ΔspeABΔspeC mutant of E coli swarmed as well as the parental strain on the LBSw (Fig. 1). Furthermore, it was clarified that the swarming of the ΔspeABΔspeC E. coli mutant was induced by spermidine imported by the spermidine importer PotABCD (Figs 2 and 3). In the previous study on P. mirabilis (Sturgill & Rather, 2004), putrescine was identified as an extracellular signaling molecule that induced swarming, but the importer responsible for the uptake of the signaling molecule was not identified. In this study, spermidine was identified as a molecule that induces swarming; moreover, PotABCD was identified as a spermidine importer that is indispensable for the induction of swarming. In E. coli, polyamine oxidase activity, by which putrescine can be formed from N1-acetylspermidine yielded by the acetylation reaction of catalyzed spermidine by SpeG, has not been detected (Ignatenko et al., 1996). Therefore, it is thought that the induction of swarming on media containing spermidine is not the consequence of the conversion of spermidine to putrescine but the direct effect of spermidine itself. In the previous study on P. mirabilis (Sturgill & Rather, 2004), the polyamine-deficient mutant (speB::Sm) absorbed the extracellular putrescine exported by the speB+ strain adjacently cultured on the LBSw, and it showed swarming activity. This experiment clearly showed that putrescine can be physiologically characterized as an extracellular signaling molecule that is transferred from cell to cell during the induction of swarming. However, it was impossible to show that spermidine exported by E. coli induced swarming of the polyamine-deficient mutant, because the effect of spermidine is indistinguishable from the effect of putrescine on swarming; their effects are indistinguishable because spermidine is synthesized from putrescine in E. coli cells and E. coli cells have both spermidine and putrescine. However, spermidine exported from bacteria is present in the environment; for example, in the intestinal tract, several tens of micromolar spermidine (Matsumoto & Benno, 2007) is synthesized and exported by intestinal bacteria (Noack et al., 1998). Furthermore, the spermidine exporter of E. coli has been identified previously (Higashi et al., 2008). All the results obtained in this study indicate that spermidine as well as putrescine plays a role in the induction of swarming as an extracellular signaling molecule, which is transferred from cell to cell in E. coli.
We are grateful to Dr Philip N. Rather, Department of Microbiology and Immunology, Emory University School of Medicine, for the helpful discussions.
- 2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. P Natl Acad Sci USA 97: 6640–6645. & (
- 2008) Identification of a spermidine excretion protein complex (MdtJI) in Escherichia coli. J Bacteriol 190: 872–878. , , , , , , & (
- 2006) Polyamine modulon in Escherichia coli: genes involved in the stimulation of cell growth by polyamines. J Biochem 139: 11–16. & (
- 1996) Expression of the human spermidine/spermine N1-acetyltransferase in spermidine acetylation-deficient Escherichia coli. Biochem J 319: 435–440. , , , & (
- 2007) The relationship between microbiota and polyamine concentration in the human intestine: a pilot study. Microbiol Immunol 51: 25–35. & (
- 1992) A short course in bacterial genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, pp. 263–274, 437. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (
- 1996) Construction of a flagellum-negative mutant of Proteus mirabilis: effect on internalization by human renal epithelial cells and virulence in a mouse model of ascending urinary tract infection. Infect Immun 64: 5332–5340. , , , , , & (
- 1998) Dietary guar gum and pectin stimulate intestinal microbial polyamine synthesis in rats. J Nutr 128: 1385–1391. , , , & (
- 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. , , , , , & (
- 2005) Swarmer cell differentiation in Proteus mirabilis. Environ Microbiol 7: 1065–1073. (
- 1997) Potential virulence factors of Proteus bacilli. Microbiol Mol Biol R 61: 65–89. , & (
- 2000) Polyamine transport and role of potE in response to osmotic stress in Escherichia coli. J Bacteriol 182: 6247–6249. , , , & (
- 2004) Evidence that putrescine acts as an extracellular signal required for swarming in Proteus mirabilis. Mol Microbiol 51: 437–446. & (
- 1987) Isolation, genetic mapping, and characterization of Escherichia coli K-12 mutants lacking gamma-glutamyltranspeptidase. J Bacteriol 169: 3926–3931. , & (
- 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. , , , & (
- 1984) Polyamines. Annu Rev Biochem 53: 749–790. & (
- 1985) Polyamines in microorganisms. Microbiol Rev 49: 81–99. & (