Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. II. Dynamic response to famine and feast, activation of the methylglyoxal pathway and oscillatory behaviour
The metabolic dynamics of the Escherichia coli K-12 strain TG1 to feast and famine were studied in glucose-limited steady-state cultures by up- and downshifts of the dilution rate, respectively. An uncoupling of anabolic and catabolic rates was observed upon dilution rate upshifts, apparent through immediately increased glucose uptake rates which were not accompanied by an immediate increase of the growth rate but instead resulted in the temporary excretion of methylglyoxal, d- and l-lactate, pyruvate and, after a delay, acetate. The energetic state of the cell during the transient was followed by measuring the adenylate energy charge, which increased within 2 min after the upshift and declined thereafter until a new steady-state level was reached. In the downshift experiment, the adenylate energy charge behaved inversely; no by-products were formed, indicating a tight coupling of anabolism and catabolism. Both dilution rate shifts were accompanied by an instantaneous increase of cAMP, presaging the subsequent changes in metabolic pathway utilization. Intracellular key metabolites of the Embden–Meyerhof–Parnas (EMP) pathway were measured to evaluate the metabolic perturbation during the upshift. Fructose 1,6-diphosphate (FDP) and dihydroxyacetone phosphate (DHAP) increased rapidly after the upshift, while glyceraldehyde 3-phosphate decreased. It is concluded that this imbalance at the branch-point of FDP induces the methylglyoxal (MG) pathway, a low-energy-yielding bypass of the lower EMP pathway, through the increasing level of DHAP. MG pathway activation after the upshift was simulated by restricting anabolic rates using a stoichiometry-based metabolic model. The metabolic model predicted that low-energy-yielding catabolic pathways are utilized preferentially in the transient after the upshift. Upon severe dilution rate upshifts, an oscillatory behaviour occurred, apparent through long-term oscillations of respiratory activity, which started when the cytotoxic compound MG reached a threshold concentration of 1·5 mg l−1 in the medium.
AckermanR. S.,
CozzarelliN. R.,
EpsteinW.
1974; Accumulation of toxic concentrations of methylglyoxal by wild-type Escherichia coli K-12. J Bacteriol 119:357–362
AtkinsonD. E.
1968; The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7:4030–4034[CrossRef]
ChaplenF. W. R.,
FahlW. E.,
CameronD. C.
1996a; Detection of methylglyoxal as a degradation product of DNA and nucleic acid components treated with strong acid. Anal Biochem 236:262–269[CrossRef]
ChaplenF. W. R.,
FahlW. E.,
CameronD. C.
1996b; Method for determination of free intracellular and extracellular methylglyoxal in animal cells grown in culture. Anal Biochem 238:171–178[CrossRef]
ChassagnoleC.,
Noisommit-RizziN.,
SchmidJ. W.,
MauchK.,
ReussM.
2002; Dynamic modeling of the central carbon metabolism of Escherichia coli
. Biotechnol Bioeng 79:53–73[CrossRef]
FergusonG. P.,
ChackoA. D.,
LeeC.,
BoothI. R.
1996; The activity of the high-affinity K+ uptake system Kdp sensitizes cells ofEscherichia coli to methylglyoxal. J Bacteriol 178:3957–3961
FergusonG. P., TötemeyerS., MacLeanM. J.,
BoothI. R.
1998; Methylglyoxal production in bacteria: suicide or survival?. Arch Microbiol 170:209–219[CrossRef]
FravalH. N. A.,
McBrienD. C. H.
1980; The effect of methyl glyoxal on cell division and the synthesis of protein and DNA in synchronous and asynchronous cultures of Escherichia coli B/r. J Gen Microbiol 117:127–134
HopperD. J.,
CooperR. A.
1971; The regulation of Escherichia coli methylglyoxal synthase: a new control site in glycolysis?. FEBS Lett 13:213–216[CrossRef]
KadnerR. J.,
MurphyG. P.,
StephensC. M.
1992; Two mechanisms for growth inhibition by elevated transport of sugar phosphates in Escherichia coli
. J Gen Microbiol 138:2007–2014[CrossRef]
KalaposM. P.
1999; Methylglyoxal in living organisms: chemistry, biochemistry, toxicology and biological implications. Toxicol Lett 110:145–175[CrossRef]
LoT. W. C.,
WestwoodM. E.,
McLellanA. C.,
SelwoodT.,
ThornallyP. J.
1994; Binding and modification of proteins by methylglyoxal under physiological conditions. A kinetic and mechanistic study with Nα-acetylarginine,Nα-acetylcysteine, and Nα-acetyllysine, and bovine serum albumin. J Biol Chem 269:32299–32305
LowryO. H.,
CarterJ.,
WardJ. B.,
GlaserL.
1971; The effect of carbon and nitrogen sources on the level of metabolic intermediates in Escherichia coli
. J Biol Chem 246:6511–6521
NeijsselO. M.,
HuetingS.,
TempestD. W.
1977; Glucose transport capacity is not the rate-limiting step in the growth of some wild-type strains of Escherichia coli and Klebsiella aerogenes in chemostat culture. FEMS Microbiol Lett 2:1–3[CrossRef]
PapoulisA.,
Al-AbedY.,
BucalaR.
1995; Identification of N2-(1-carboxyethyl)guanine (CEG) as a guanine advanced glycosylation end product. Biochemistry 34:648–655[CrossRef]
PramanikJ.,
KeaslingJ. D.
1997; Stoichiometric model of Escherichia coli metabolism: incorporation of growth-rate dependent biomass composition and mechanistic energy requirements. Biotechnol Bioeng 56:398–421[CrossRef]
RussellJ. B.
1993; Glucose toxicity in Prevotella ruminicola: methylglyoxal accumulation and its effect on membrane physiology. Appl Environ Microbiol 59:2844–2850
SaierM. H.Jr, RamseierT. M.,
ReizerJ.
1996; Regulation of carbon utilization. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology vol 1 pp 1325–1343 Edited by
NeidhardtF. C.,
CurtisR.III,
IngrahamJ. L.,
LinE. C. C.,
LowK. B.,
MagasanikB.,
ReznikoffW. S.,
RileyM.,
SchaechterM.,
UmbargerH. E.
Washington, DC: American Society for Microbiology;
SauerU.,
LaskoD. R.,
FiauxJ.,
HochuliM.,
GlaserR.,
SzyperskiT.,
BaileyJ. E, WüthrichK.1999; Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism. J Bacteriol 181:6679–6688
Teixeira de MattosM. J.,
NeijsselO. M.
1997; Bioenergetic consequences of microbial adaptation to low-nutrient environments. J Biotechnol 59:117–126[CrossRef]
TempestD. W.,
NeijsselO. M.
1992; Physiological and energetic aspects of bacterial metabolite overproduction. FEMS Microbiol Lett 100:169–176[CrossRef]
TötemeyerS.,
BoothN. A.,
NicholsW. W.,
DunbarB.,
BoothI. R.
1998; From famine to feast: the role of methylglyoxal production in Escherichia coli. Mol Microbiol 27:553–562[CrossRef]
Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. II. Dynamic response to famine and feast, activation of the methylglyoxal pathway and oscillatory behaviour