Effect of Spermidine Analogues on Cell Growth of Escherichia coli Polyamine Requiring Mutant MA261

Abstract

The effects of spermidine analogues [norspermidine (NSPD, 33), spermidine (SPD, 34), homospermidine (HSPD, 44) and aminopropylcadaverine (APCAD, 35)] on cell growth were studied using Escherichia coli polyamine-requiring mutant MA261. Cell growth was compared at 32°C, 37°C, and 42°C. All four analogues were taken up mainly by the PotABCD spermidine-preferential uptake system. The degree of stimulation of cell growth at 32°C and 37°C was NSPD ≥ SPD ≥ HSPD > APCAD, and SPD ≥ HSPD ≥ NSPD > APCAD, respectively. However, at 42°C, it was HSPD » SPD > NSPD > APCAD. One reason for this is HSPD was taken up effectively compared with other triamines. In addition, since natural polyamines (triamines and teteraamines) interact mainly with RNA, and the structure of RNA is more flexible at higher temperatures, HSPD probably stabilized RNA more tightly at 42°C. We have thus far found that 20 kinds of protein syntheses are stimulated by polyamines at the translational level. Among them, synthesis of OppA, RpoE and StpA was more strongly stimulated by HSPD at 42°C than at 37°C. Stabilization of the initiation region of oppA and rpoE mRNA was tighter by HSPD at 42°C than 37°C determined by circular dichroism (CD). The degree of polyamine stimulation of OppA, RpoE and StpA synthesis by NSPD, SPD and APCAD was smaller than that by HSPD at 42°C. Thus, the degree of stimulation of cell growth by spermidine analogues at the different temperatures is dependent on the stimulation of protein synthesis by some components of the polyamine modulon.

Fig 1. Structure of triamines.

Fig 2. Effect of triamines on growth of E. coli MA261.

A. Growth of E. coli MA261. Cell growth at 32°C, 37°C and 42°C in the presence and absence of 0.1 mM triamines was followed by measuring A₅₄₀. B. Polyamine content in cells at A₅₄₀ = 0.3 was measured as described in Materials and Methods. Values are mean ± S. E. of triplicate determinations. Student’s t test was performed for the value obtained in the presence and absence of triamine. * p < 0.05; ** p < 0.01; *** p < 0.001. The growth of E. coli MA261 in the presence of triamine was significantly faster than that in the absence of triamine.

Fig 3. Effect of triamines on cell growth of E. coli MA261 potD::Km.

A and B. Cell growth of E. coli MA261 potD::Km and polyamine content were measured as described in the legend of Fig 2. ns, p ≥ 0.05; * p < 0.05; ** p < 0.01.

Fig 4. Levels of potABCD mRNA and PotD protein in E. coli MA261 cultured in the presence and absence of triamines.

Cells were harvested at A₅₄₀ = 0.3. A. The level of potABCD mRNA was measured by dot blotting [12]. The probe for dot blotting was prepared by PCR using primers, 5’-CGACTTACGAGTCGAACGAAACCATGTACG-3’ and 5’-AGAACCGTTCCAGATCATGCCGAGGTTAAC-3’. B. The level of PotD protein was measured by Western blotting using 10 μg protein. Values are mean ± S. E. of triplicate determinations. ns, p ≥ 0.05; *, p < 0.05; **, p < 0.01.

Fig 5. Levels of various proteins encoded by the polyamine modulon in E. coli MA261 cultured in the presence and absence of triamines.

The levels of OppA, RpoE (σ²⁴), StpA and EmrR were measured together with a control protein, RpoD (σ⁷⁰) by Western blotting using 10 μg protein. Values are mean ± S. E. of triplicate determinations. The degree of stimulation of protein synthesis by triamines was measured using a Las-3000 luminescent image analyzer. ns, p ≥ 0.05; * p < 0.05; ** p < 0.01.

Fig 6. Stabilization of OppA and RpoE WT RNAs, and more stable modified RNAs measured by CD.

A, D, G and J. Structures of OppA WT RNA, OppA + U RNA, RpoE WT RNA, and RpoE -20(ΔU) RNA are shown. B and C (OppA WT RNA), E and F (OppA + U RNA), H and I (RpoE WT RNA), and K and L (RpoE -20(ΔU) RNA). Concentration-dependent shifts induced by Mg²⁺ or a triamine with 1 mM Mg²⁺ at 37°C and 42°C in elliplicity at 208 nm are shown. Values are mean ± S. E. of triplicate determinations. The K_d values of Mg²⁺ and a triamine with 1 mM Mg²⁺ were determined by the double reciprocal equation plot.