The Escherichia coli MG1655 genome has been completely sequenced. The annotated sequence, biochemical information, and other information were used to reconstruct the E. coli metabolic map. The stoichiometric coefficients for each metabolic enzyme in the E. coli metabolic map were assembled to construct a genome-specific stoichiometric matrix. The E. coli stoichiometric matrix was used to define the system's characteristics and the capabilities of E. coli metabolism. The effects of gene deletions in the central metabolic pathways on the ability of the in silico metabolic network to support growth were assessed, and the in silico predictions were compared with experimental observations. It was shown that based on stoichiometric and capacity constraints the in silico analysis was able to qualitatively predict the growth potential of mutant strains in 86% of the cases examined. Herein, it is demonstrated that the synthesis of in silico metabolic genotypes based on genomic, biochemical, and strain-specific information is possible, and that systems analysis methods are available to analyze and interpret the metabolic phenotype.

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The Escherichia coli MG1655 in silico metabolic genotype: Its definition, characteristics, and capabilities

Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology, the in vitro substrates recognized by most yeast protein kinases: we identified over 4,000 phosphorylation events involving 1,325 different proteins. These substrates represent a broad spectrum of different biochemical functions and cellular roles. Distinct sets of substrates were recognized by each protein kinase, including closely related kinases of the protein kinase A family and four cyclin-dependent kinases that vary only in their cyclin subunits. Although many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, indicating possible new roles for several kinases. Furthermore, integration of the phosphorylation results with protein-protein interaction and transcription factor binding data revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast. Because many yeast proteins and pathways are conserved, these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.

Global Analysis of Protein Phosphorylation in Yeast

Energetics of bacterial growth: balance of anabolic and catabolic reactions.

Lactic acid bacteria (LAB) constitute a heterogeneous group of bacteria that are traditionally used to produce fermented foods. The industrialization of food bio-transformations increased the economical importance of LAB, as they play a crucial role in the development of the organoleptique and hygienic quality of fermented products. Therefore, the reliability of starter strains in terms of quality and functional properties (important for the development of aroma and texture), but also in terms of growth performance and robustness has become essential. These strains should resist to adverse conditions encountered in industrial processes, for example during starter handling and storage (freeze-drying, freezing or spray-drying). The development of new applications such as life vaccines and probiotic foods reinforces the need for robust LAB since they may have to survive in the digestive tract, resist the intestinal flora, maybe colonize the digestive or uro-genital mucosa and express specific functions under conditions that are unfavorable to growth (for example, during stationary phase or storage). Also in nature, the ability to quickly respond to stress is essential for survival and it is now well established that LAB, like other bacteria, evolved defense mechanisms against stress that allow them to withstand harsh conditions and sudden environmental changes. While genes implicated in stress responses are numerous, in LAB the levels of characterization of their actual role and regulation differ widely between species. The functional conservation of several stress proteins (for example, HS proteins, Csp, etc) and of some of their regulators (for example, HrcA, CtsR) renders even more striking the differences that exist between LAB and the classical model micro-organisms. Among the differences observed between LAB species and B. subtilis, one of the most striking is the absence of a σB orthologue in L. lactis ssp. lactisas well as in at least two streptococci and probably E. faecalis. The overview of LAB stress responses also reveals common aspects of stress responses. As in other bacteria, adaptive responses appear to be a usual mode of stress protection in LAB. However, the cross-protection to other stress often induced by the expression of a given adaptive response, appears to vary between species. This observation suggests that the molecular bases of adaptive responses are, at least in part, species (or even subspecies) specific. A better understanding of the mechanisms of stress resistance should allow to understand the bases of the adaptive responses and cross protection, and to rationalize their exploitation to prepare LAB to industrial processes. Moreover, the identification of crucial stress related genes will reveal targets i) for specific manipulation (to promote or limit growth) , ii) to develop tools to screen for tolerant or sensitive strains and iii) to evaluate the fitness and level of adaptation of a culture. In this context, future genome and transcriptome analyses will undoubtedly complement the proteome and genetic information available today, and shed a new light on the perception of, and the response to, stress by lactic acid bacteria.

Stress responses in lactic acid bacteria

Proton channels exist in a wide variety of membrane proteins where they transport protons rapidly and efficiently. Usually the proton pathway is formed mainly by water molecules present in the protein, but its function is regulated by titratable groups on critical amino acid residues in the pathway. All proton channels conduct protons by a hydrogen-bonded chain mechanism in which the proton hops from one water or titratable group to the next. Voltage-gated proton channels represent a specific subset of proton channels that have voltage- and time-dependent gating like other ion channels. However, they differ from most ion channels in their extraordinarily high selectivity, tiny conductance, strong temperature and deuterium isotope effects on conductance and gating kinetics, and insensitivity to block by steric occlusion. Gating of H+ channels is regulated tightly by pH and voltage, ensuring that they open only when the electrochemical gradient is outward. Thus they function to extrude acid from cells. H+ch...

https://www.physiology.org/doi/pdf/10.1152/physrev.00028.2002

Voltage-Gated Proton Channels and Other Proton Transfer Pathways

The membrane-bound H(+)-ATPase plays a key role in free-energy transduction of biological systems. We report how the carbon and energy metabolism of Escherichia coli changes in response to deletion of the atp operon that encodes this enzyme. Compared with the isogenic wild-type strain, the growth rate and growth yield were decreased less than expected for a shift from oxidative phosphorylation to glycolysis alone as a source of ATP. Moreover, the respiration rate of a atp deletion strain was increased by 40% compared with the wild-type strain. This result is surprising, since the atp deletion strain is not able to utilize the resulting proton motive force for ATP synthesis. Indeed, the ratio of ATP concentration to ADP concentration was decreased from 19 in the wild type to 7 in the atp mutant, and the membrane potential of the atp deletion strain was increased by 20%, confirming that the respiration rate was not controlled by the magnitude of the opposing membrane potential. The level of type b cytochromes in the mutant cells was 80% higher than the level in the wild-type cells, suggesting that the increased respiration was caused by an increase in the expression of the respiratory genes. The atp deletion strain produced twice as much by-product (acetate) and exhibited increased flow through the tricarboxylic acid cycle and the glycolytic pathway. These three changes all lead to an increase in substrate level phosphorylation; the first two changes also lead to increased production of reducing equivalents. We interpret these data as indicating that E. coli makes use of its ability to respire even if it cannot directly couple this ability to ATP synthesis; by respiring away excess reducing equivalents E. coli enhances substrate level ATP synthesis.

Carbon and energy metabolism of atp mutants of Escherichia coli.

The C and D cell cycle periods of seven Escherichia coli K-12 strains and three E. coli B/r strains were determined by computer simulation of DNA histograms obtained by flow cytometry of batch cultures grown at several different generation times. To obtain longer generation times two of the K-12 strains were cultivated at several different dilution rates in glucose-limited chemostats. The replication period (C period) was found to be similar in K-12 and B/r strains grown at similar generation times. At generation times below 60 min the C period was constant; above 60 min it increased linearly with increasing generation time. The period from termination of replication to cell division (D period) was more variable. It was much shorter in B/r than in K-12 strains. Like the C period it was relatively constant at generation times below 60 min and it increased with increasing generation times at longer generation times. In glucose-limited chemostats good correlation was found between D periods and generation times, whereas batch cultures exhibited carbon-source-dependent variations. Chemostat cultures showed cell cycle variations very similar to those obtained in batch cultures. These flow cytometric determinations of cell cycle periods confirm earlier determinations of the C period and establish that the D period also varies with generation time in slowly growing cultures. In addition they extend the range of growth rates at which cell cycle periods have been determined in E. coli K-12.

Precise determinations of C and D periods by flow cytometry in Escherichia coli K-12 and B/r.

For control analysis, it is necessary to modulate the activity of an enzyme around its normal level and measure the changes in steady-state fluxes or concentrations. We describe an improved method for effecting the modulation, as elaborated for Escherichia coli. The chromosomal gene, encoding the enzyme of interest, is put under the control of a lacUV5 or a tacI promoter. The alternative use of the two promoters leads to an expression range which should make it suitable for the use in control analysis of many enzymes. The lacUV5 promoter should be used when the wild-type expression level is low, the tacI promoter when the latter is high. The endogenous lac operon is placed under the control of a second copy of the lacUV5 promoter and a lac Yam mutation (eliminating lactose permease, the transport system for the inducer isopropyl-thio-β-D-galactoside) is introduced.



The method was demonstrated experimentally by constructing E. coli strains, in which the chromosomal atp operon is transcribed from the lacUV5 and the tacI promoter. We measured the concentration of the c subunit of H+-ATPase, and found that the expression of this enzyme could be modulated between non-detectable levels and up to five times the wild-type level. Thus, in the absence of inducer, no expression of atp genes could be detected when the atp operon was controlled by the lacUV5 promoter, and we estimate that the expression was less than 0.0025 times the wild-type level. We show that the introduction of a lac Y mutation facilitated the attainment of steady induction levels of partially induced cells. The mutation also reduced positive cooperativity in the dependence of expression on the concentration of isopropyl-thio-β-D-galactoside (the inducer) and shifted the concentration of inducer needed for half maximum induction to higher values. These properties should facilitate the experimental modulation of the enzyme activity by varying the concentration of the inducer.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1993.tb19885.x

The use of lac‐type promoters in control analysis

Integration into the cytoplasmic membrane and function of the three F0 subunits, a, b and c, of the membrane-bound ATP synthase of Escherichia coli K12 were analysed in situations where synthesis of only one or two types of subunits was possible This was achieved by combined use of atp mutations and plasmids carrying and expressing one or two of the atp genes coding for ATP synthase subunits AU three F0 subunits were found to be required for the establishment of efficient H+ conduction Subunits a and b individually as well as together were found to bind F1 ATPase to the membrane while subunit c did not The ATPase activity bound to either of these single subunits, or in pairwise combinations, was not inhibited by N,N'-dicyclohexylcarbodiimide Also ATP-dependent H+ translocation was not catalysed unless all three F0 subunits were present in the membrane The integration into the membrane of the subunits a and b was independent of the presence of other ATP synthase subunits

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1983.tb01388.x

Membrane integration and function of the three F0 subunits of the ATP synthase of Escherichia coli K12.

Transcriptional fusions between the phage lambda promotor pR and ATP synthase genes, atp, on plasmid pBR322 were constructed in order to study the effects upon growth and physiology of Escherichia coli of induced overproduction of H+-ATPase subunits. Constitutive overproduction of the complete enzyme had earlier been found to result in decreased growth rate and cytological defects. When a 15-fold overproduction of subunit a alone, or together with subunit c, or with all other ATP synthase subunits was suddenly induced, the following effects were observed. Inhibition of growth and protein synthesis within 10 min of induction, which effect was suppressed by N,N'-dicyclohexylcarbodiimide, also when the chromosomal atp genes coding for the Fo subunits a, b and c were deleted. Partial collapse of the membrane potential delta psi at 4-6 min after induction paralleled by inhibition of thiomethylgalactoside and guanosine transport. Respiration and alpha-methylglucoside transport was not affected. The partial collapse of delta psi, and the specific inhibition of proton-driven transport systems is taken to show that the subunit a has--when suddenly overproduced and inserted into the membrane--a protonophoric activity. It is suggested that this protonophoric activity of subunit a is related to the function of this subunit in the Fo sector in H+-ATPases.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1985.tb03939.x

Ole Michelsen

Papers

Carbon and energy metabolism of atp mutants of Escherichia coli.

Precise determinations of C and D periods by flow cytometry in Escherichia coli K-12 and B/r.

The use of lac‐type promoters in control analysis

Membrane integration and function of the three F0 subunits of the ATP synthase of Escherichia coli K12.

Proton conduction by subunit a of the membrane-bound ATP synthase of Escherichia coli revealed after induced overproduction.