©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Decrease in Cell Viability Due to the Accumulation of Spermidine in Spermidine Acetyltransferase-deficient Mutant of Escherichia coli(*)

(Received for publication, April 24, 1995; and in revised form, June 2, 1995)

Jun-ichi Fukuchi (1) Keiko Kashiwagi (1) Masahiro Yamagishi (2) Akira Ishihama (2) Kazuei Igarashi (1)(§)

From the (1)Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263, Japan and the (2)Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Physiological functions of spermidine acetyltransferase in Escherichia coli have been studied using the spermidine acetyltransferase (speG) gene-deficient mutant CAG2242 and the cloned speG gene. The growth of E. coli CAG2242 in the defined M9 medium was normal in the presence and absence of 0.5 mM spermidine. However, cell viability of E. coli CAG2242 at 48 h after the onset of growth decreased greatly by the addition of 0.5 mM spermidine. The amount of spermidine accumulated in the cells was approximately 3-fold that in the cells grown in the absence of spermidine. Transformation of the cloned speG gene to E. coli CAG2242 recovered the cell viability. Decrease in cell viability of E. coli CAG2242 was observed even when 0.5 mM spermidine was added at 24 h after the onset of growth. The results indicate that accumulated spermidine functions at the late stationary phase of growth. The accumulation of spermidine caused a decrease in protein synthesis but not in DNA and RNA synthesis at 28 h after the onset of growth. The synthesis of several kinds of proteins was particularly inhibited. They included ribosome modulation factor and OmpC protein. Since the ribosome modulation factor is essential for cell viability at the stationary phase of growth (Yamagishi, M., Matsushima, H., Wada, A., Sakagami, M., Fujita, N., and Ishihama, A.(1993) EMBO J. 12, 625-630), the decrease in the protein was thought to be one of the reasons for the decrease in cell viability. The decrease in the ribosome modulation factor mainly occurred at the translational level.


INTRODUCTION

Polyamines, aliphatic cations present in almost all living organisms, are known to be necessary for normal cell growth(1, 2, 3) . Thus, it is important to understand the mechanism by which the cellular polyamine concentration is regulated. Polyamine content is regulated by biosynthesis, degradation, uptake, and excretion. Although a rate-limiting enzyme of polyamine degradation in eukaryotes, spermidine/spermine N^1-acetyltransferase, has been recently studied in detail(4) , that in prokaryotes, spermidine acetyltransferase (SAT), (^1)has not. In eukaryotes, spermidine/spermine N^1-acetyltransferase is induced strongly in the presence of polyamines (5) and bis(ethyl)polyamine derivatives(6) , and it probably plays an important role in the decrease in polyamine toxicity by its accumulation(7, 8) . In Escherichia coli, spermidine acetylation increased in response to high pH (9) and cold shock(10) , but SAT activity did not change significantly under these conditions(11) . To help understand the characteristics of polyamine degradation in E. coli, we purified SAT and isolated the gene for SAT (speG)(12) . We found that SAT is present in very small amounts in cells and increases only by 2.5-3.5-fold under poor nutrient conditions. In this study, the physiological functions of spermidine acetyltransferase in E. coli were studied with E. coli CAG2242 (speG) and the cloned speG gene. We found that the accumulated spermidine due to the lack of SAT caused a decrease in cell viability at the stationary phase of growth through its inhibition of protein synthesis. The synthesis of several kinds of proteins was particularly inhibited. The decrease in cell viability paralleled the decrease in ribosome modulation factor (RMF), which is essential for cell viability(13) . RMF is a protein associated with 100 S ribosome dimers (14) and is synthesized during transition from exponential growth to the stationary phase(13) . We also recently found that RMF is a stationary phase-specific inhibitor of protein synthesis and that 100 S ribosome dimers are dormant forms of ribosomes. (^2)


EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Culture Conditions

E. coli C600 (supE44, hsdR, thi, thr, leu, lacY1, tonA21) and CAG2242 (speG, supE44, hsdR, thi, thr, leu, lacY1, tonA21), provided by Dr. E. W. Gerner (University of Arizona Health Sciences Center), were grown in M9 medium (15) containing 1% potassium lactate, 2 µg/ml thiamine, and 100 µg/ml each of leucine and threonine. To obtain pMWSAT, the 1.3-kilobase BamHI-EcoRI fragments were amplified by polymerase chain reaction using pSAT2 (12) as template. The primers used for polymerase chain reaction were 5`-d(AGCGGATAACAATTTCACACAGGA)-3` (24-mer) and 5`-d(CCGAATTCCGCAGTGGATGGTGT)-3` (23-mer). The 1.3-kilobase BamHI-EcoRI fragments treated with BamHI and EcoRI were then inserted into the BamHI-EcoRI sites of pMW119, a low copy number of plasmid(16) . E. coli CAG2242 transformed with pMWSAT or pMW119 was grown in the presence of ampicillin (100 µg/ml). Cell growth was followed by measuring absorbance at 540 nm. Cell viability was determined by counting colony numbers in aliquots of the culture grown on LB-containing 1.5% agar plates (15) at 37 °C.

Measurement of Polyamine and Protein Contents

Polyamine levels in E. coli were determined by HPLC as previously described (17) after homogenization and extraction of the polyamines with 5% trichloroacetic acid and centrifugation at 27,000 g for 15 min at 4 °C. Protein was determined by the method of Lowry et al.(18) .

Measurement of Macromolecular Synthesis in E. coli

E. coli CAG2242 cells (1 ml) were cultured in the presence and absence of 0.5 mM spermidine. At 12 or 28 h after the onset of cell growth, cells were labeled with 222 kBq of [^3H]thymidine (0.9 µM), 74 kBq of [^3H]uridine (1 µM), or 92.5 kBq of [S]methionine (30 µM). For measurement of [^3H]thymidine and [^3H]uridine incorporated into DNA and RNA, respectively, cold trichloroacetic acid insoluble radioactivity in 0.1-ml aliquots was counted in a liquid scintillation counter. For measurement of [S]methionine incorporated into protein, hot trichloroacetic acid insoluble radioactivity was counted using 0.1-ml aliquots.

Western Blot Analysis of Initiation Factors, Ribosomal Proteins, and RMF

Initiation factors (IF2 and 3) and ribosomal proteins (S1, L20, and L35) were prepared according to the previous publications(19, 20, 21) . RMF was synthesized chemically.^2 Ribosomes and antibody for each protein were prepared as previously described(22) . Western blotting was performed with 10 µg of ribosome-associated proteins (the 100,000 g precipitate fraction) by the method of Neilsen et al.(23) .

Sucrose Density Gradient Centrifugation of Ribosomes

The 30,000 g supernatant of E. coli cells (5 A units) was loaded on a 5-ml linear sucrose gradient (5-20%) containing 20 mM Tris-HCl, pH 7.5, 15 mM magnesium acetate, and 100 mM ammonium acetate. The tube was centrifuged for 80 min at 40,000 rpm in a Hitachi P55ST2. The sucrose gradients were fractionated into 31-33 tubes. Absorbance at 260 nm was measured after 4-fold dilution of each fraction.

Two-dimensional Electrophoresis of Proteins Synthesized

E. coli CAG2242 cells (0.5 ml) were cultured in the presence and absence of 0.5 mM spermidine. At 28 h after the onset of cell growth, cells were labeled with 7 MBq of [S]methionine (30 µM) at 37 °C for 30 min. Proteins synthesized (500,000 cpm) were analyzed by two-dimensional electrophoresis (24) and fluorography(25) .

Measurement of RMF mRNA by Primer Extension

Primer extension was performed according to the method of McKnight and Kingsbury(26) . Synthetic oligonucleotides, which hybridize 10-28 nucleotides upstream from the initiation codon AUG (primer 3 in (13) ) and 75-95 nucleotides downstream from the initiation codon AUG of RMF mRNA (primer 4 in (13) ), were end-labeled with [-P]ATP using T4 polynucleotide kinase. The P-labeled oligonucleotides were then hybridized with total RNA (50 µg) isolated from E. coli cells (27) to prime the synthesis of cDNA with Moloney murine leukemia virus reverse transcriptase. The product of this reaction was analyzed by electrophoresis on 5% acrylamide sequencing gel.

Measurement of RMF Synthesized

Cells were labeled with [S] methionine for 30 min as described above at the specified phases of growth. The amount of RMF synthesized was determined as previously described(22) . The 500,000 cpm of [S]methionine-labeled protein was treated with anti-RMF antiserum and then with 10% formalin-fixed Staphylococcus aureus Cowan I cells (Calbiochem). Immunoprecipitated RMF was then subjected to SDS-polyacrylamide gel electrophoresis on 13% acrylamide gel, and the protein was detected by fluorography.


RESULTS

Decrease in Cell Viability Due to the Accumulation of Spermidine

To clarify the physiological functions of SAT, a SAT-deficient mutant, E. coli CAG2242 cells, were grown in the presence and absence of 0.5 mM spermidine. As shown in Fig.1A, cell growth of E. coli CAG2242 with or without spermidine was equal to that of E. coli C600 (parent strain). The effect of spermidine on cell viability was then examined (Fig.1B). Cell viability of E. coli CAG2242 at 48 h after the onset of cell growth was decreased greatly by spermidine, but that of E. coli C600 was not. The transformant of E. coli CAG2242 with pMWSAT recovered cell viability.


Figure 1: Effects of spermidine and SAT on cell growth (A) and viability (B) of E. coli C600 and CAG2242. Cell growth (A) and viability (B) were measured as described under ``Experimental Procedures.'' , C600; bullet, C600 grown in the presence of 0.5 mM spermidine; , CAG2242; up triangle, filled, CAG2242 grown in the presence of 0.5 mM spermidine; , CAG2242/pMWSAT; black square, CAG2242/pMWSAT grown in the presence of 0.5 mM spermidine. Each value is the average of duplicate determinations.



Next, polyamine contents in E. coli cells were measured (Fig.2). When E. coli C600 cells were cultured in the presence of spermidine, spermidine accumulation was not observed. However, when E. coli CAG2242 cells were cultured in the presence of spermidine, spermidine accumulation was observed. The amount of spermidine accumulated in the cells was approximately 3-fold that in the cells grown in the absence of spermidine. The transformant of E. coli CAG2242 with pMWSAT did not cause the spermidine accumulation. The results strongly suggest that the accumulation of spermidine decreased the cell viability of E. coli cells.


Figure 2: Effects of spermidine and SAT on polyamine contents. E. coli cells grown in the presence and absence of 0.5 mM spermidine were harvested at the times shown in the figure, and polyamines were analyzed by HPLC. &cjs2090;, putrescine; shaded square, spermidine. Each value is the average of duplicate determinations.



Correlation between the Decrease in Cell Viability and the Decrease in Protein Synthesis

When macromolecular synthesis of the mutant CAG2242 cells was measured at 12 h after the onset of cell growth, DNA, RNA, and protein syntheses did not change significantly in the presence and absence of spermidine (Fig.3). Macromolecular synthesis was then measured at 28 h after the onset of cell growth. DNA and RNA syntheses decreased greatly, but the accumulated spermidine had no effect on these two syntheses. Although protein synthesis did not decrease significantly in the absence of spermidine, it was greatly decreased by its presence (Fig.3C). The results suggest that the syntheses of proteins necessary for cell viability are inhibited by spermidine.


Figure 3: Effect of spermidine on macromolecular synthesis at the stationary phase of growth of E. coli CAG2242. DNA (A), RNA (B), and protein (C) syntheses of E. coli CAG2242 grown in the presence (bullet) and absence () of 0.5 mM spermidine were measured by labeling of cells with [^3H]thymidine, [^3H]uridine, and [S]methionine, respectively, and by counting cold (A, B) and hot (C) trichloroacetic acid-insoluble radioactivity as described under ``Experimental Procedures.'' Each value is the average of duplicate determinations.



Decrease in the Amount of RMF Due to the Accumulation of Spermidine

Since protein synthesis was inhibited by 80% by spermidine, the proteins synthesized were analyzed by two-dimensional electrophoresis to examine whether the synthesis of all proteins was equally inhibited by spermidine. Proteins were labeled with [S]methionine at 28 h after the onset of cell growth, and the same amount of radioactivity (500,000 cpm) was used for electrophoresis. As shown in Fig.4, the syntheses of some specific proteins (No. 1-3) were particularly repressed by spermidine. Since the protein termed No. 1 was identified as OmpC protein (outer membrane protein) by comparison with the protein analysis of the OmpC protein-deficient mutant KY2201(28) , we examined whether cell viability of the mutant at 24 and 48 h after the onset of cell growth decreased or not. However, there was no significant difference in cell viability between E. coli C600 and the OmpC protein-deficient mutant KY2201 cells (data not shown). The other two proteins (No. 2 and 3) have not been identified yet.


Figure 4: Two-dimensional analysis of proteins synthesized in E. coli CAG2242 grown in the presence and absence of 0.5 mM spermidine. A, without spermidine; B, with 0.5 mM spermidine. , proteins of which syntheses were particularly inhibited by 0.5 mM spermidine.



Next, the ribosomal proteins and their associated proteins were analyzed by Western blotting because of the strong inhibition of protein synthesis by spermidine (Fig.5). The amounts of ribosomal proteins S1, L20, and L35 (21, 29) in E. coli CAG2242 grown in the presence of 0.5 mM spermidine were nearly equal to those in E. coli CAG2242 grown in its absence. There were also no significant differences in the amounts of initiation factors 2 and 3. However, the amount of RMF, which is essential for cell viability(13) , decreased greatly in the cells grown in the presence of spermidine. When Western blot analysis was performed using the 30,000 g supernatant instead of ribosome-associated proteins, essentially the same results were obtained (data not shown). The results indicate that the amounts of ribosomal proteins and initiation factors in E. coli CAG2242 grown in the presence and absence of spermidine are nearly equal and the amount of RMF decreased greatly in the cells cultured with spermidine.


Figure 5: Western blot analysis of ribosomal proteins and their associated proteins. E. coli CAG2242 grown in the presence and absence of 0.5 mM spermidine was harvested at the times shown in the figure, and Western blot analysis was performed as described under ``Experimental Procedures.''



Since RMF is a stationary phase-specific inhibitor of protein synthesis and forms temporarily inactive 100 S ribosome dimers (14) ,^2 ribosome patterns were analyzed by sucrose gradient centrifugation. As shown in Fig.6, the relative amount of 100 S ribosome dimers decreased greatly in E. coli CAG2242 cells grown in the presence of spermidine. At 44 h after the onset of cell growth, there were no 100 S dimers in the spermidine- accumulated cells, and the amount of ribosomes (30, 50, and 70 S) decreased greatly. These results suggest that RMF is not only a stationary phase-specific inhibitor of protein synthesis but also an anti-degradation factor of ribosomes.


Figure 6: Sucrose density gradient centrifugation of ribosomes. E. coli CAG2242 was harvested at the times shown in the figure, and sucrose density gradient centrifugation was performed using 5 A units of the 30,000 g supernatant.



Accumulated Spermidine Functions at the Late Stationary Phase

RMF is known to be synthesized at the stationary phase (13) . Thus, we tested whether spermidine functions at the late stationary phase. Even when spermidine was added at 24 h after the onset of cell growth, cell viability decreased greatly at 48 h (Fig.7). However, the decrease in cell viability was delayed compared with that of cells cultured with spermidine at the onset of cell growth (Fig.1B). Ribosome patterns and the amount of RMF were analyzed at 32 and 44 h after the onset of cell growth. As shown in Fig.8, only small amounts of 100 S ribosome dimers and RMF existed in the 30,000 g supernatant at 44 h, indicating that the accumulated spermidine was operational at the late stationary phase.


Figure 7: Effect of spermidine added at the late stationary phase on cell viability of E. coli CAG2242. Spermidine was added at 24 h after the onset of cell growth, and cell viability was measured. , without spermidine; bullet, with 0.5 mM spermidine. Each value is the average of duplicate determinations.




Figure 8: Effects of spermidine added at the late stationary phase on ribosomal patterns (A) and the amount of RMF (B). Sucrose density gradient centrifugation and Western blot analysis of RMF were performed as described in the legends of Fig.5and Fig. 6.



Mechanism of Decrease in the Amount of RMF

The molecular mechanism involved in the decrease in the amount of RMF was studied. The amount of RMF mRNA was first measured by primer extension (Fig.9A). It was found that the amount of mRNA in the spermidine-accumulated cells was nearly equal to that in normal cells at 20 h, and it decreased slightly at 28 h after the onset of cell growth. Essentially the same results were obtained with Northern blot analysis (data not shown). Then, the synthesis of RMF was analyzed by immunoprecipitation of [S]methionine-labeled protein (500,000 cpm) with its antibody and S. aureus Cowan I cells. As shown in Fig.9B, the amount of RMF synthesized in E. coli CAG2242 grown in the absence of spermidine was maximal at 20 h after the onset of cell growth, and this was much larger than the amounts synthesized in the cells grown in the presence of spermidine at 12, 20, and 28 h after the onset of cell growth. These results indicate that the amount of RMF accumulated in a 30-min labeling period diminished more rapidly than its mRNA level in the presence of spermidine.


Figure 9: Effect of spermidine on the amount of RMF mRNA (A) and RMF synthesis (B). A, the amount of mRNA was measured by primer extension. Primers 3 and 4 in (13) were used in the experiments of a and b, respectively. 1 and 3, E. coli CAG2242 was grown in the absence of spermidine, and RNA was prepared from the cells harvested at 20 and 28 h, respectively; 2 and 4, E. coli CAG2242 was grown in the presence of 0.5 mM spermidine, and RNA was prepared from the cells harvested at 20 and 28 h, respectively. Arrows indicate the initiation sites of transcription. B, RMF synthesis was measured as described under ``Experimental Procedures'' using E. coli CAG2242 labeled with [S]methionine. Numbers on the right represent molecular mass in kDa.




DISCUSSION

The physiological functions of SAT were examined in this study. We found that SAT is important for the maintenance of cell viability at the stationary phase of growth through the inhibition of spermidine accumulation. When SAT was deficient in E. coli cells, spermidine accumulating in the cells grown in the presence of 0.5 mM spermidine was approximately three times more than that in the cells grown in the absence of spermidine. In eukaryotic cells, accumulated polyamines and bis(ethyl)polyamines inhibited cell growth(7, 30) . The inhibition of cell growth paralleled the inhibition of protein synthesis, especially of mitochondrial protein synthesis due to the existence of unstable tRNAs in mitochondria. In E. coli, however, the accumulated spermidine neither decreased the cell growth nor inhibited protein synthesis at the logarithmic phase of cell growth. At the stationary phase, the accumulated spermidine due to the lack of SAT started to inhibit protein synthesis and caused a decrease in cell viability. Since almost all kinds of protein synthesis were inhibited (Fig.4), the amount of spermidine bound to ribosomes may increase at the stationary phase of growth and cause the inhibition of protein synthesis, which was observed in eukaryotic cells. Actually, the amount of ribosomes decreased at the stationary phase of growth. Accordingly, the relative amount of spermidine to ribosomes increased at the stationary phase of growth. These phenomena were recovered by the transformation of speG gene to SAT-deficient cells. The results clearly show that deficiency of SAT caused the accumulation of spermidine followed by the inhibition of protein synthesis and the decrease in cell viability.

Among the proteins synthesized at the stationary phase, synthesis of OmpC protein and RMF was strongly inhibited by the accumulated spermidine. As expected, the inhibition of RMF synthesis mainly occurred at the translational level. We recently reported that most polyamines exist as a polyamine-RNA complex in cells and contribute to the stimulation of some kinds of protein synthesis under normal conditions(31, 32) . In other words, the degree of stimulation of protein synthesis by polyamines differs in each protein. In a eukaryotic cell-free system, we also reported that low concentrations of spermidine strongly stimulate protein synthesis directed by the mRNA having a GC-rich 5`-untranslated region and that high concentrations of spermidine inhibit the protein synthesis greatly (33) . Thus, it is presumed that the degree of inhibition of protein synthesis by the accumulated spermidine also differs in each protein. Our present results may be in accordance with the concept that the polyamine regulation of protein synthesis depends on the base composition of mRNA, that is, the tertiary structure of mRNA. Further studies will be necessary to provide a clear explanation for the late disappearance of RMF in cells accumulating spermidine.

RMF is known to be essential for cell viability (13) and is synthesized only at the stationary phase of growth. Accumulated spermidine also functions at the stationary phase (Fig.7). A decrease in OmpC protein did not influence cell viability. It has been reported that the stationary phase sigma factor ^S is also involved in cell viability(34) , and its synthesis is positively regulated by guanosine tetrasphosphate (ppGpp)(35) . The amounts of ^S measured by Western blotting and of ppGpp measured by thin layer chromatography did not change significantly in the cells grown in the presence and absence of spermidine (data not shown). Thus, the decrease in RMF by the accumulated spermidine is probably the major reason for the decrease in cell viability.

Although the rates of RMF synthesis were different in cells grown in the presence and absence of spermidine (Fig.9), the amounts of RMF existing in the cells were nearly equal until 28 h after the onset of cell growth (Fig.5). The reason for this remains to be clarified. It may be that excess amounts of RMF that cannot be bound to 100 S ribosome dimers are degraded rapidly.

RMF is essential for cell viability at the stationary phase of growth (13) , but we do not know yet how RMF is involved in cell viability. We suggest that RMF may function as an anti-degradation factor of ribosomes since the amount of ribosomes decreased greatly in the spermidine-accumulated cells harvested at 44 h after the onset of cell growth. However, the possibility that degradation of ribosomes may be the result of cell death can also not be ruled out at present.


FOOTNOTES

*
This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan and by Research Aid from the Uehara Memorial Foundation (Japan). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

^1
The abbreviations used are: SAT, spermidine acetyltransferase; RMF, ribosome modulation factor; HPLC, high performance liquid chromatography; SPD, spermidine.

^2
A. Wada, K. Igarashi, S. Yoshimura, S. Aimoto, and A. Ishihama, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. E. W. Gerner for the kind gifts of E. coli C600 and CAG2242.


REFERENCES

  1. Tabor, C. W., and Tabor, H. (1984) Annu. Rev. Biochem. 53,749-790 [CrossRef][Medline] [Order article via Infotrieve]
  2. Tabor, C. W., and Tabor, H. (1985) Microbiol. Rev. 49,81-99
  3. Pegg, A. E. (1988) Cancer Res. 48,759-774 [Abstract]
  4. Casero, R. A., and Pegg, A. E. (1993) FASEB J. 656,653-661
  5. Persson, L., and Pegg, A. E. (1984) J. Biol. Chem. 259,12364-12367 [Abstract/Free Full Text]
  6. Libby, P. R., Bergeron, R. J., and Porter, C. W. (1989) Biochem. Pharmacol. 38,1435-1442 [CrossRef][Medline] [Order article via Infotrieve]
  7. He, Y., Kashiwagi, K., Fukuchi, J., Terao, K., Shirahata, A., and Igarashi, K. (1993) Eur. J. Biochem. 217,89-96 [Abstract]
  8. Poulin, R., Coward, J. K., Lakanen, J. R., and Pegg, A. E. (1993) J. Biol. Chem. 268,4690-4698 [Abstract/Free Full Text]
  9. Dubin, D. T., and Rosenthal, S. M. (1960) J. Biol. Chem. 235,776-782 [Medline] [Order article via Infotrieve]
  10. Tabor, C. W., and Dobbs, L. G. (1970) J. Biol. Chem. 245,2086-2091 [Abstract/Free Full Text]
  11. Carper, S. W., Willis, D. G., Manning, K. A., and Gerner, E. W. (1991) J. Biol. Chem. 266,12439-12441 [Abstract/Free Full Text]
  12. Fukuchi, J., Kashiwagi, K., Takio, K., and Igarashi, K. (1994) J. Biol. Chem. 269,22581-22585 [Abstract/Free Full Text]
  13. Yamagishi, M., Matsushima, H., Wada, A., Sakagami, M., Fujita, N., and Ishihama, A. (1993) EMBO J. 12,625-630 [Abstract]
  14. Wada, A., Yamazaki, Y., Fujita, N., and Ishihama, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,2657-2661 [Abstract]
  15. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual , pp. 440-442, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  16. Yamaguchi, K., and Masamune, Y. (1985) Mol. & Gen. Genet. 200,362-367
  17. Igarashi, K., Kashiwagi, K., Hamasaki, H., Miura, A., Kakegawa, T., Hirose, S., and Matsuzaki, S. (1986) J. Bacteriol. 166,128-134 [Medline] [Order article via Infotrieve]
  18. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275 [Free Full Text]
  19. Hershey, J. W. B., Yanov, J., Johnston, K., and Fakunding, J. L. (1977) Arch. Biochem. Biophys. 182,626-638 [Medline] [Order article via Infotrieve]
  20. Igarashi, K., Kashiwagi, K., Kishida, K., Kakegawa, T., and Hirose, S. (1981) Eur. J. Biochem. 114,127-131 [Abstract]
  21. Kashiwagi, K., and Igarashi, K. (1987) Biochim. Biophys. Acta 911,180-190 [Medline] [Order article via Infotrieve]
  22. Kashiwagi, K., Sakai, Y., and Igarashi, K. (1989) Arch. Biochem. Biophys. 268,379-387 [Medline] [Order article via Infotrieve]
  23. Neilsen, P. J., Manchester, K. L., Towbin, H., Gordon, J., and Thomas, G. (1982) J. Biol. Chem. 257,12316-12321 [Abstract/Free Full Text]
  24. O'Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021 [Abstract]
  25. Laskey, R. A., and Mills, A. D. (1975) Eur. J. Biochem. 56,335-341 [Abstract]
  26. McKnight, S. L., and Kingsbury, R. (1982) Science 217,316-324 [Medline] [Order article via Infotrieve]
  27. Emory, S. A., and Belasco, J. (1990) J. Bacteriol. 172,4472-4481 [Medline] [Order article via Infotrieve]
  28. Sato, T., and Yura, T. (1981) J. Bacteriol. 145,88-96 [Medline] [Order article via Infotrieve]
  29. Wada, A., and Sako, T. (1987) J. Biochem. (Tokyo) 101,817-820 [Abstract]
  30. He, Y., Suzuki, T., Kashiwagi, K., Kusama-Eguchi, K., Shirahata, A., and Igarashi, K. (1994) Eur. J. Biochem. 221,391-398 [Abstract]
  31. Watanabe, S., Kusama-Eguchi, K., Kobayashi, H., and Igarashi, K. (1991) J. Biol. Chem. 266,20803-20809 [Abstract/Free Full Text]
  32. Miyamoto, S., Kashiwagi, K., Ito, K., Watanabe, S., and Igarashi, K. (1993) Arch. Biochem. Biophys. 300,63-68 [CrossRef][Medline] [Order article via Infotrieve]
  33. Ito, K., Kashiwagi, K., Watanabe, S., Kameji, T., Hayashi, S., and Igarashi, K. (1990) J. Biol. Chem. 265,13036-13041 [Abstract/Free Full Text]
  34. McCann, M. P., Kidwell, J. P., and Matin, A. (1991) J. Bacteriol. 173,4188-4194 [Medline] [Order article via Infotrieve]
  35. Gentry, D. R., Hernandez, V. J., Nguyen, L. H., Jensen, D. B., and Cashel, M. (1993) J. Bacteriol. 175,7982-7989 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.