Analysis of theEscherichia coliresponse to glycerol pulse in continuous, high-cell density culture using a multivariate approach
- 11 September 2008
- journal article
- research article
- Published by Wiley in Biotechnology & Bioengineering
- Vol. 102 (3), 910-922
- https://doi.org/10.1002/bit.22120
Abstract
Pulse experiments in continuous-culture are a valuable tool in microbial physiology research. However, inferences become difficult when the cell response is followed by monitoring many biochemical variables or when several types of perturbations are compared. Moreover, there is no objective criterion to delimit the time-window, so that the recorded responses will render valid inferences. Hence, we have investigated the capability of a multivariate approach to deal with complex data from a previously described series of pulse experiments. Data are concerned with 12 biochemical variables that were monitored when an anaerobic, steady-continuous culture of E. coli O74K74 was disturbed by six types of pulses (glycerol, fumarate, acetate, crotonobetaine, hypersaline plus high-glycerol basal medium and crotonobetaine plus hypersaline basal medium). Our analysis determined the instantaneous uptake rate for the pulsed metabolite (Dynamical Chemical-Balances), reduced the multivariate observations to one response curve (Principal Component Analysis) and determined the optimal time-window (Cluster Analysis). Finally, input-output data were filtered (Orthogonal Signal Correction) while both blocks were mathematically connected (Partial Least-squares Regression). This systematic approach allowed us to detect several relevant patterns not previously revealed: (i) Glycerol uptake rate did not follow a Michaelian kinetics but showed a biphasic dependence on glycerol concentration; noticeably, net uptake decreased 136-fold despite the high availability of glycerol in the milieu. (ii) The structure of the bacterial response changed during time the glycerol-disturbance lasted (2 h), hence analyses had to be limited to the early response (time from 0 to 5 min). (iii) By mathematically relating the input (glycerol uptake rate) with the output (12 biochemical responses) it was possible to identify which of the monitored variables were primary targets of the glycerol disturbance (namely: ATP, formate, acetyl-CoA synthase, isocitrate dehydrogenase, and isocitrate lyase), which were secondarily responsive (ethanol) and those that were independent (acetate, carnitine, lactate, and NADH/NAD ratio). Identification was achieved even though all the analyzed variables were affected by the pulse. (iv) Some variables exhibited uncorrelated dynamics despite their close functional relationship (ATP and NADH/NAD ratio, ethanol and lactate; carnitine and the crotonobetaine hydratase complex; acetate and the enzymes phosphotransacetylase, acetyl-CoA synthase and isocitrate lyase). The results are discussed in terms of E. coli transcriptional control, and it is concluded that glycerol pulse produces a stressing effect. The consequent activation of the polyamine-dependent mechanisms involved in such stressing effect provides a unified explanation for how glycerol uptake is down-regulated in the presence of high glycerol availability and how acetate can be produced without de novo biosynthesis. Biotechnol. Bioeng. 2009; 102: 910–922.Keywords
This publication has 42 references indexed in Scilit:
- Role of energetic coenzyme pools in the production of l-carnitine by Escherichia coliMetabolic Engineering, 2006
- Salt stress effects on the central and carnitine metabolisms ofEscherichia coliBiotechnology & Bioengineering, 2006
- How does gene expression clustering work?Nature Biotechnology, 2005
- Hierarchical Control of Anaerobic Gene Expression in Escherichia coli K-12: the Nitrate-Responsive NarX-NarL Regulatory System Represses Synthesis of the Fumarate-Responsive DcuS-DcuR Regulatory SystemJournal of Bacteriology, 2005
- Using chemometrics for navigating in the large data sets of genomics, proteomics, and metabonomics (gpm)Analytical and Bioanalytical Chemistry, 2004
- Application of Multivariate Analysis to Optimize Function of Cultured HepatocytesBiotechnology Progress, 2003
- Modeling of the biotransformation of crotonobetaine intoL-(−)-carnitine byEscherichia colistrainsBiotechnology & Bioengineering, 2002
- Polyamines decrease Escherichia coli outer membrane permeabilityJournal of Bacteriology, 1996
- The domains of slow bacterial growthCanadian Journal of Microbiology, 1988
- Partial least-squares regression: a tutorialAnalytica Chimica Acta, 1986