45NISR Research GRANT
2009 Research Grant

Studies on Novel Regulatory Mechanism For Nucleotide
Biosynthesis Through the Amino Acid Signal in Bacteria
Kan TANAKA
Graduate School of Horticulture, Chiba University

Research aims
Amino acids and nucleotides are important constituentsof every organism, and intracellular levels of these compounds are strictly regulated in cells at various levels. Thus,
to optimally produce these molecules by fermentation, it isvery important to understand the mechanisms of homeostasis of each biomolecule. In general, when concentration ofa specific amino acid dropped below the usual level, relevant uncharged tRNA increases and binds to ribosome,which results in the activation of stringent response provoked by alarmone nucleotide, (p)ppGpp. (p)ppGpp inhibits de novo ribosome biosynthesis and peptide elongation to suppress the amino acid consumption, and on theother hand, activate transcription of amino acid biosynthetic
genes to increase the amino acid biosynthesis. These effects
indicate that (p)ppGpp plays a central role for the aminoacid homeostasis. Moreover, (p)ppGpp inhibits initiationand elongation of DNA replication, which suppress theconsumption of nucleotides, while the effects on the nucleotide biosynthesis have not been well evaluated yet.
Recently, using luciferase genes of Vibrio fischeri as thereporter, we constructed a real-time monitoring system of
intracellular redox level in Escherichia coli (1). In a seriesof studies, we found the induction of stringent response,
which was mediated by addition of a certain amino acidsuch as serine or isoleucine, results in significant decreaseof the intracellular redox pool. Since this decrease was notobserved in ppGpp0 strain lacking the (p)ppGpp synthetic
activity, it was likely mediated by (p)ppGpp (data notshown). Another finding was that the decrease of redox
pool was disappeared or alleviated by addition of one-carbon-metabolites (OCM; glycine, methionine, thymidine,adenosine and panthotenate (2)) into the medium. OCM aremetabolites that are synthesized by the help of folate, andthus, (p)ppGpp was suggested to inhibit folate metabolism
which results in the inhibition of nucleotide biosynthesis
and the decrease of redox pool. This could be a critical regulatory point to couple amino acid metabolism and nucleotide metabolisms, and this study was performed tomake clear the underlying molecular mechanism.

pLXUV5, was previously constructed by inserting the constitutive lacUV5 promoter to drive the luxCDABE operonof pUCD615 (1, 3). Bacterial strains harboring pLXUV5
were cultivated in MOPS medium, and the luciferase activity (RLU) was monitored by luminometer (Berthold Lumat
LB9507) in 100 ml for 10 seconds. 0.4% glucose or galactose was added to the medium as the carbon source. Aminoacids were added at 50 mg/ml. A crr::kan allele was obtained from KO collection at National Institute of Genetics,
Japan (4), and strains were constructed by P1 transduction.
Measurements of intracellular metabolites were performedin cooperation with Keio University.
Results
1. CK8 strain harboring pLXUV5 was cultivated in
MOPS medium containing glucose as the carbon source,
and isoleucine was added to the medium during the logarithmic growth phase. Since excess isoleucine represses theacetohydroxy acid synthase (AHAS) that catalyzes the firstcommon step of isoleucine/valine biosynthesis, thisisoleucine addition was expected to induce starvation of valine and thus weak stringent response. Examining the intracellular metabolite concentrations revealed significant decreases of glycolytic intermediates, such as glucose-6-phosphate and glyceraldehyde-3-phosphate. Consistently, similar decreases of glycolytic intermediates were observed incase of stringent response caused by sudden removal ofamino acids from the rich cultivating medium (Dr. N. Saito,
personal communication). Thus, (p)ppGpp was suggested

Methods
In this study, a relA derivative of E. coli MC4100 strain,
CK8, was routinely used. Luciferase reporter plasmid,Fig. 1.

Structure of pLXUV5 (PlacUV5::luxCDABE)

46

Fig. 3.

Model for Redox Pool ControlConclusion

Fig. 2. Effect of carbon source on the valine-induced redox decline.
Valine (50 mg/ml was added to the medium at time 0, and luciferase activity was monitored.

to inhibit glycolytic flux and generation of reducing equivalents, and induce decrease of the intracellular redox pools.
2. CK8 (pLXUV5) was cultivated in MOPS medium inthe presence of either glucose or galactose as the sole carbon source, and the luciferase activity was monitored afteraddition of valine into the medium. While the valine addition greatly decreased the luciferase activity in case of glucose culture, the same valine addition made almost noeffect on the luciferase activity in case of galactose cultivation (Fig. 2). Other carbon sources such as maltose andglycerol resulted in similar results with the galactose case.
Thus, it was suggested that the glucose culture was the special case.
3. Given that the effect of stringent response was specifically observed in case of glucose culture, involvement ofPTS (PhosphoTransferase System) for glucose transport
was considered. Thus, we examined the effect of a null mutation of the crr gene that encodes IIAGlc for the phosphotransfer reaction to glucose. Other than the PTS function, it
is known that IIAGlc activates adenylate cyclase in its phosphorylated form and repress non-PTS lactose transporter in
its non-phosphorylated form. We constructed the crr mutant of CK8, and examined for the valine effect. As theresults, we found that weak decline of luciferase activity
was observed similarly in both carbon sources. This resultwas explained if IIAGlc regulates PTS activity cooperatingwith (p)ppGpp either directly or indirectly.

To explain the obtained results, we constructed a working
model as in Fig. 3. Amino acid starvation response affects
sugar catabolic flux through (p)ppGpp, and thus results inthe decline of cell redox pools. In this pathway, IIAGlc ofglucose PTS system has an important role. With respect tothe relationship with one carbon metabolism, (p)ppGpp islikely has an inhibitory effect on the folate metabolism andsubsequent PTS inhibition, while the direct target has notbeen identified. In case of folate starvation induced bytrimethoprim, starvation of glycine and methionine isinduced and thus stringent response and decline of redox
pools are also induced (5). In conclusion, this study has
revealed close relationship among amino acid metabolism,
one carbon metabolism and sugar metabolism.
Acknowledgment
I acknowledge Dr. Natsumi Saito (Keio University) for
collaboration on the metabolomic analysis and for sharing
unpublished results. I also thank National BioResource Project (E. coli) of National Institute of Genetics.
References
1.

Koga, K., Harada, T., Shimizu, H. and Tanaka, K. (2005) Bacterial
luciferase activity and the intracellular redox pool in Escherichia
coli. Mol. Genet. Genomics, 274, 180–188.
2. Harvery, R. J. (1973) Growth and initiation of protein synthesis inEscherichia coli in the presence of trimethoprim. J. Bacteriol., 114,309–322.
3. Rogowsky, P. M., Close, T. J., Chimera, J. A., Shaw, J. J. and Kado,
C. I. (1987) Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58. J. Bacteriol., 169, 5101–5112.
4. Yamamoto, N., et al. (2009) Update on the Keio collection of Escherichia coli single-gene deletion mutants. Mol. Sys. Biol., 5, 335.
5. Kwon, Y. K., Higgins, M. B. and Rabinowitz, J. D. (2010) Antifolateinduced depletion of intracellular glycine and purines inhibits
thymineless death in E. coli. ACS Chem. Biol., 5, 787–795.