Enhancement of the synthesis of RpoN, Cra, and H-NS by polyamines at the level of translation in Escherichia coli cultured with glucose and glutamate

Abstract

Proteins whose synthesis is enhanced by polyamines at the level of translation were identified in a polyamine-requiring mutant cultured in the presence of 0.1% glucose and 0.02% glutamate instead of 0.4% glucose as an energy source. Under these conditions, enhancement of cell growth by polyamines was almost the same as that in the presence of 0.4% glucose. It was found that synthesis of RpoN, Cra, and H-NS was enhanced by polyamines at the level of translation at the early logarithmic phase of growth (A(540) of 0.15). The effects of polyamines on synthesis of RpoN, H-NS, and Cra were due to the existence of unusual Shine-Dalgarno sequences (RpoN and H-NS) and an inefficient GUG initiation codon (Cra) in their mRNAs. Thus, rpoN, cra, and hns genes were identified as new members of the polyamine modulon. Because most of the polyamine modulon genes thus far identified encode transcription factors (RpoS [sigma(38)], Cya, FecI [sigma(18)], Fis, RpoN [sigma(54)], Cra, and H-NS), DNA microarray analysis of mRNA expressed in cells was performed. At the early logarithmic phase of growth, a total of 97 species of mRNAs that were up-regulated by polyamines more than twofold were under the control of seven polyamine modulon genes mentioned above.

FIG. 1.

Growth and polyamine content of E. coli MA261. (A) Culture of E. coli MA261 cells. •, 0.1% glucose and 0.02% glutamate with 100 μg/ml putrescine; ▴, 0.1% glucose and 0.02% glutamate without putrescine; ○, 0.4% glucose with 100 μg/ml putrescine; ▵, 0.4% glucose without putrescine. (B) Polyamine content was measured as described in Materials and Methods. ▪, polyamine content of cells cultured in the presence of 0.1% glucose and 0.02% glutamate; □, polyamine content of cells cultured in the presence of 0.4% glucose. Data are shown as means ± SEs of triplicate determinations. PUT, putrescine; SPD, spermidine.

FIG. 2.

Effect of polyamines on synthesis of RpoN in E. coli MA261. (A) Cells were cultured in the presence and absence of 100 μg/ml putrescine and harvested at an A₅₄₀ of 0.15. Western blotting of RpoN was performed using 10 μg of protein of cell lysate. (B) Measurement of [³⁵S]methionine-labeled RpoN was performed using 1,000,000 cpm of [³⁵S]methionine-labeled protein and antibody to RpoN. (C) Dot blot analysis of rpoN mRNA was performed as described in Materials and Methods. (D) Schematic of the rpoN-lacZ fusion genes. The rpoN gene containing a 259-nucleotide 5′ upstream region with an unmodified or modified SD sequence and a 135-nucleotide open reading frame was fused to the lacZ gene. (E) Western blotting of RpoN-β-Gal fusion protein was performed using 20 μg of protein of cell lysate. The levels of rpoN-lacZ mRNA in cells cultured with and in those cultured without 100 μg/ml putrescine were nearly equal judging from the dot blot analysis. Values are means ± SEs of triplicate determinations. PUT, putrescine.

FIG. 3.

Effect of polyamines on synthesis of Cra in E. coli MA261. (A) Western blotting of Cra was performed using 10 μg of protein of cell lysate. (B) Dot blot analysis of cra mRNA was performed as described in Materials and Methods. (C) Schematic of cra-lacZ fusion genes. The cra gene containing a 346-nucleotide 5′ upstream region and a 114-nucleotide open reading frame with a GUG or AUG initiation codon was fused to the lacZ gene. (D) Western blotting of Cra-β-Gal fusion protein was performed using 20 μg of protein of cell lysate. The levels of cra-lacZ mRNA in cells cultured with and in those cultured without 100 μg/ml putrescine were nearly equal judging from the dot blot analysis. Values are means ± SEs of triplicate determinations. PUT, putrescine.

FIG. 4.

Levels of OppA, Cya, RpoS, FecI, Fis, and RF2 in E. coli MA261 cultured with and without putrescine. Western blotting of OppA, Cya, RpoS, FecI, Fis, and RF2 was performed using 1, 10, 10, 50, 10, and 10 μg of protein of cell lysate, respectively. Values are means ± SEs of triplicate determinations. PUT, putrescine.

FIG. 5.

Measurement of cya mRNA (A), cAMP (B), and CRP and RpoF (C) levels in E. coli MA261 cultured with or without putrescine. These were measured as described in Materials and Methods. Western blotting of CRP and RpoF (σ²⁸) was performed using 10 and 20 μg of protein of cell lysate, respectively. The arrow indicates the position of intact RpoF, and the second band is a degradation product of RpoF. Values are means ± SEs of triplicate determinations. PUT, putrescine.

FIG. 6.

Effect of polyamines on synthesis of H-NS in E. coli MA261. (A) Genes that regulate σ²⁸ synthesis. (B) Western blotting of H-NS was performed using 10 μg of protein of cell lysate. (C) Dot blot analysis of hns mRNA was performed as described in Materials and Methods. (D) Schematic of hns-lacZ fusion genes. The hns gene containing a 287-nucleotide 5′ upstream region with an unmodified or a modified SD sequence and a 123-nucleotide open reading frame was fused to the lacZ gene. (E) Western blotting of H-NS-β-Gal fusion protein was performed using 20 μg of protein of cell lysate. The levels of hns-lacZ mRNA in cells cultured with and in those cultured without 100 μg/ml putrescine were nearly equal judging from the dot blot analysis. Values are means ± SEs of triplicate determinations. PUT, putrescine.