Robustness analysis of the Escherichia coli metabolic network

Genomic, biochemical, and strain-specific data can be assembled to define an in silico representation of the metabolic network for a select group of single cellular organisms. Flux-balance analysis and phenotypic phase planes derived therefrom have been developed and applied to analyze the metabolic capabilities and characteristics of Escherichia coli K-12. These analyses have shown the existence of seven essential reactions in the central metabolic pathways (glycolysis, pentose phosphate pathway, tricarboxylic acid cycle) for the growth in glucose minimal media. The corresponding seven gene products can be grouped into three categories: (1) pentose phosphate pathway genes, (2) three-carbon glycolytic genes, and (3) tricarboxylic acid cycle genes. Here we develop a procedure that calculates the sensitivity of optimal cellular growth to altered flux levels of these essential gene products. The results indicate that the E. coli metabolic network is robust with respect to the flux levels of these enzymes. The metabolic flux in the transketolase and the tricarboxylic acid cycle reactions can be reduced to 15% and 19%, respectively, of the optimal value without significantly influencing the optimal growth flux. The metabolic network also exhibited robustness with respect to the ribose-5-phosphate isomerase, and the ribose-5-phosephate isomerase flux was reduced to 28% of the optimal value without significantly effecting the optimal growth flux. The metabolic network exhibited limited robustness to the three-carbon glycolytic fluxes both increased and decreased. The development presented another dimension to the use of FBA to study the capabilities of metabolic networks.     

Determination of antibiotics using luminescent Escherichia coli and serum

The methodical bases for detecting antibiotics using a bioluminescent assay and blood serum are briefed. Antibiotics inhibit the luminescence of a genetically engineered Escherichia coli strain. The degree of inhibition depended on the type of antibiotic, its concentration, and the time of cell incubation with antibiotic. The highest cell sensitivity was recorded towards the aminoglycoside antibiotics, which amounted to 85 +/- 10 ng/ml for gentamicin and streptomycin. The sensitivity of this system to a number of antibiotics essentially increased when the cells were previously activated with blood serum. The sensitivity of this method for gentamicin and streptomycin in the presence of blood serum amounted to 2.5 +/- 0.5 ng/ml; for tetracycline, 45 +/- 8 ng/ml. Use of the sera containing specific antibodies to the antibiotic detected provided a high sensitivity of the biosensor tested. Comparison of the luminescences of E. coli cells activated with normal and specific antisera upon incubation with an antibiotic allows the type of antibiotic and its quantitative content in the sample to be determined. Characteristic of the analysis of antibiotics with the help of recombinant E. coli are a high accuracy, sensitivity, specificity, simplicity, and a short time needed for measurement.     

Genetic and biochemical analysis of the biotin loci of Escherichia coli K-12

Some 60 biotin auxotrophs of Escherichia coli K-12 were isolated and classified into four groups according to their cross-feeding patterns, excertion products, and their ability to show a growth response to various biotin vitamers. Since all the mutants could be transduced with lambdadbio phages known to carry the entire bioA locus, it was concluded that all of the mutation sites were located in this locus. It was also possible to derive a gene order for the different mutant groups on the basis of transduction studies with various lambdadbio phages that carry portions of the bioA locus. A possible biochemical pathway for the biosynthesis of biotin in E. coli K-12 is discussed.     

Deletion analysis of the Escherichia coli taurine and alkanesulfonate transport systems

The Escherichia coli tauABCD and ssuEADCB gene clusters are required for the utilization of taurine and alkanesulfonates as sulfur sources and are expressed only under conditions of sulfate or cysteine starvation. tauD and ssuD encode an alpha-ketoglutarate-dependent taurine dioxygenase and a reduced flavin mononucleotide-dependent alkanesulfonate monooxygenase, respectively. These enzymes are responsible for the desulfonation of taurine and alkanesulfonates. The amino acid sequences of SsuABC and TauABC exhibit similarity to those of components of the ATP-binding cassette transporter superfamily, suggesting that two uptake systems for alkanesulfonates are present in E. coli. Chromosomally located in-frame deletions of the tauABC and ssuABC genes were constructed in E. coli strain EC1250, and the growth properties of the mutants were studied to investigate the requirement for the TauABC and SsuABC proteins for growth on alkanesulfonates as sulfur sources. Complementation analysis of in-frame deletion mutants confirmed that the growth phenotypes obtained were the result of the in-frame deletions constructed. The range of substrates transported by these two uptake systems was largely reflected in the substrate specificities of the TauD and SsuD desulfonation systems. However, certain known substrates of TauD were transported exclusively by the SsuABC system. Mutants in which only formation of hybrid transporters was possible were unable to grow with sulfonates, indicating that the individual components of the two transport systems were not functionally exchangeable. The TauABCD and SsuEADCB systems involved in alkanesulfonate uptake and desulfonation thus are complementary to each other at the levels of both transport and desulfonation.     

Transcriptome analysis of phosphate starvation response in Escherichia coli

Escherichia coli has a PhoR-PhoB two-component regulatory system to detect and respond to the changes of environmental phosphate concentration. For the E. coli W3110 strain growing under phosphate-limiting condition, the changes of global gene expression levels were investigated by using DNA microarray analysis. The expression levels of some genes that are involved in phosphate metabolism were increased as phosphate became limited, whereas those of the genes involved in ribosomal protein or amino acid metabolism were decreased, owing to the stationary phase response. The upregulated genes could be divided into temporarily and permanently inducible genes by phosphate starvation. At the peak point showing the highest expression levels of the phoB and phoR genes under phosphate-limiting condition, the phoB- and/or phoR-dependent regulatory mechanisms were investigated in detail by comparing the gene expression levels among the wild-type and phoB and/or phoR mutant strains. Overall, the phoB mutation was epistatic over the phoR mutation. It was found that PhoBR and PhoB were responsible for the upregulation of the phosphonate or glycerol phosphate metabolism and high-affinity phosphate transport system, respectively. These results show the complex regulation by the PhoR-PhoB two-component regulatory system in E. coli.     

Structure and biochemical activities of Escherichia coli MgsA

Bacterial "maintenance of genome stability protein A" (MgsA) and related eukaryotic enzymes play important roles in cellular responses to stalled DNA replication processes. Sequence information identifies MgsA enzymes as members of the clamp loader clade of AAA+ proteins, but structural information defining the family has been limited. Here, the x-ray crystal structure of Escherichia coli MgsA is described, revealing a homotetrameric arrangement for the protein that distinguishes it from other clamp loader clade AAA+ proteins. Each MgsA protomer is composed of three elements as follows: ATP-binding and helical lid domains (conserved among AAA+ proteins) and a tetramerization domain. Although the tetramerization domains bury the greatest amount of surface area in the MgsA oligomer, each of the domains participates in oligomerization to form a highly intertwined quaternary structure. Phosphate is bound at each AAA+ ATP-binding site, but the active sites do not appear to be in a catalytically competent conformation due to displacement of Arg finger residues. E. coli MgsA is also shown to form a complex with the single-stranded DNA-binding protein through co-purification and biochemical studies. MgsA DNA-dependent ATPase activity is inhibited by single-stranded DNA-binding protein. Together, these structural and biochemical observations provide insights into the mechanisms of MgsA family AAA+ proteins.     

Gene expression analysis of the response by Escherichia coli to seawater

Gene expression of Escherichia coli cells exposed to seawater for 20 h was compared to that of exponentially growing cells (mops-glucose 0.2%) using DNA microarray technology. The expression of most (ca. 3000) of the 4228 open reading frames on the microarray remained unchanged; the relative expression of about 320 genes decreased in seawater, whereas that of ca. one fourth (937) increased. Clearly coherent expression patterns were observed for several functional gene groups. Induced genes were numerous in groups specifying the degradation of small molecules (carbon compounds, amino acids and fatty acids), energy metabolism (aerobic and anaerobic respiration, pyruvate dehydrogenase and TCA cycle), chemotaxis and mobility, flagella biosynthesis, surface structures and phage related functions. Repressed genes were clustered in two groups, cell division and nucleotides biosynthesis, indicating a cessation of growth. This revised version was published online in August 2006 with corrections to the Cover Date.     

Physiological and biochemical analysis of the effects of alkaline phosphatase overproduction in Escherichia coli.

Overexpression of the Escherichia coli phoA gene, coding for alkaline phosphatase (PhoA), on multicopy plasmids caused a severe defect in the precursor processing (secretion) of PhoA, beta-lactamase, and the outer membrane protein OmpA. This secretion defect continued even after the repression of phoA expression, indicating that protein secretion was irreversibly impaired in cells. Among the secretory proteins, only OmpA gradually secreted posttranslationally. The inverted inner membrane vesicles prepared from cells with the secretion defect showed appreciably reduced translocation activity in vitro. But the membrane vesicles retained the ability to generate a proton motive force which, together with ATP, is essential as an energy source for the efficient secretion of proteins in E. coli. An appreciable amount of incompletely translocated PhoA molecules was detected in the inner membranes of cells with the secretion defect.     

Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli.

Mutants of Escherichia coli K-12 requiring high concentrations of branched-chain amino acids for growth were isolated. One of the mutants was shown to be defective in transport activity for branched-chain amino acids. The locus of the mutation (hrbA) was mapped at 8.9 min on the E. coli genetic map by conjugational and transductional crosses. The gene order of this region is proC-hrbA-tsx. The hrbA system was responsible for the uptake activity of cytoplasmic membrane vesicles. It was not repressed by leucine. The substrate specificities and kinetics of the uptake activities were studied using cytoplasmic membrane vesicles and intact cells of the mutants grown in the presence or absence of leucine. Results showed that there are three transport systems for branched-chain amino acids, LIV-1, -2, and -3. The LIV-2 and -3 transport systems are low-affinity systems, the activities of which are detectable in cytoplasmic membrane vesicles. The systems are inhibited by norleucine but not by threonine. The LIV-2 system is also repressed by leucine. The LIV-1 transport system is a high-affinity system that is sensitive to osmotic shock. When the leucine-isoleucine-valine-threonine-binding protein is derepressed, the high-affinity system can be inhibited by threonine.     

Accuracy of alternative representations for integrated biochemical systems

The Michaelis-Menten formalism often provides appropriate representations of individual enzyme-catalyzed reactions in vitro but is not well suited for the mathematical analysis of complex biochemical networks. Mathematically tractable alternatives are the linear formalism and the power-law formalism. Within the power-law formalism there are alternative ways to represent biochemical processes, depending upon the degree to which fluxes and concentrations are aggregated. Two of the most relevant variants for dealing with biochemical pathways are treated in this paper. In one variant, aggregation leads to a rate law for each enzyme-catalyzed reaction, which is then represented by a power-law function. In the other, aggregation produces a composite rate law for either net rate of increase or net rate of decrease of each system constituent; the composite rate laws are then represented by a power-law function. The first variant is the mathematical basis for a method of biochemical analysis called metabolic control, the latter for biochemical systems theory. We compare the accuracy of the linear and of the two power-law representations for networks of biochemical reactions governed by Michaelis-Menten and Hill kinetics. Michaelis-Menten kinetics are always represented more accurately by power-law than by linear functions. Hill kinetics are in most cases best modeled by power-law functions, but in some cases linear functions are best. Aggregation into composite rate laws for net increase or net decrease of each system constituent almost always improves the accuracy of the power-law representation. The improvement in accuracy is one of several factors that contribute to the wide range of validity of this power-law representation. Other contributing factors that are discussed include the nonlinear character of the power-law formalism, homeostatic regulatory mechanisms in living systems, and simplification of rate laws by regulatory mechanisms in vivo.     

Identification of an ITPase/XTPase in Escherichia coli by structural and biochemical analysis

Inosine triphosphate (ITP) and xanthosine triphosphate (XTP) are formed upon deamination of ATP and GTP as a result of exposure to chemical mutagens and oxidative damage. Nucleic acid synthesis requires safeguard mechanisms to minimize undesired lethal incorporation of ITP and XTP. Here, we present the crystal structure of YjjX, a protein of hitherto unknown function. The three-dimensional fold of YjjX is similar to those of Mj0226 from Methanococcus janschii, which possesses nucleotidase activity, and of Maf from Bacillus subtilis, which can bind nucleotides. Biochemical analyses of YjjX revealed it to exhibit specific phosphatase activity for inosine and xanthosine triphosphates and have a possible interaction with elongation factor Tu. The enzymatic activity of YjjX as an inosine/xanthosine triphosphatase provides evidence for a plausible protection mechanism by clearing the noncanonical nucleotides from the cell during oxidative stress in E. coli.     

Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems

Bacillus spp. are Gram-positive bacteria that secrete a large number of extracellular proteins of industrial relevance. In this report, three Bacillus extracellular hydrolytic enzymes, i.e., alpha-amylase, mannanase and chitinase, were cloned and over-expressed in Gram-negative Escherichia coli. We found that both the native signal peptides and that of E. coli outer membrane protein, OmpA, could be used to direct the secretion of the recombinant enzymes. The expressed enzymes were observed as clearing zones on agar plates or in zymograms. Determination of enzyme activities in different cell compartments suggested that the ability of the enzymes to be secreted out into the culture medium depends on the time of induction, the type of the signal peptides and the molecular mass of the enzymes. After overnight induction, most of the enzyme activities (85-96%) could be harvested from the culture supernatant. Our results suggest that various signal peptides of Bacillus spp. can be recognized by the E. coli secretion machinery. It seems possible that other enzymes with similar signal peptide could be secreted equally well in E. coli expression systems. Thus, our finding should be able to apply for cloning and extracellular production of other Bacillus hydrolytic enzymes as well as other proteins.     

The role of thiol redox systems in the peroxide stress response of Escherichia coli

The effect of mutations in the genes encoding glutathione, glutaredoxin, thioredoxin, and thioredoxin reductase on the response of growing Escherichia coli to oxidative stress was studied. The gshA mutants defective in glutathione synthesis had the lowest resistance to high doses of H2O2, whereas the trxB mutants defective in thioredoxin reductase synthesis had the highest resistance to this oxidant, exceeding that of the parent strain. Among the studied mutants, the trxB cells demonstrated the highest basic levels of catalase activity and intracellular glutathione; they were able to rapidly reach the normal GSH level after oxidative stress. At the same time, these bacteria showed high frequency of induced mutations. The expression of the katG and sulA genes suggests that, having different sensitivity to high oxidant concentrations, the studied mutants differ primarily in their ability to induce the antioxidant genes of the OxyR and SOS regulons.     

Escherichia coli one- and two-hybrid systems for the analysis and identification of protein-protein interactions

Genetic methods based on fusion proteins allow the power of a genetic approach to be applied to the self-assembly of proteins or protein fragments, regardless of whether or not the normal function of the fused assembly domains is either known or amenable to selection or screening. The widespread adoption of variations of the yeast two-hybrid system originally described by S. Fields and O. Song (1989, Nature 340, 245-246) demonstrates the usefulness of these kinds of assays. This review describes some of the many systems used to select or screen for protein-protein interactions based on the regulation of reporter constructs by hybrid proteins expressed in bacteria, including recent implementations of generalizable two-hybrid systems for Escherichia coli.     

Genome-wide analysis of the general stress response network in Escherichia coli: sigmaS-dependent genes, promoters, and sigma factor selectivity

The sigmaS (or RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli. While nearly absent in rapidly growing cells, sigmaS is strongly induced during entry into stationary phase and/or many other stress conditions and is essential for the expression of multiple stress resistances. Genome-wide expression profiling data presented here indicate that up to 10% of the E. coli genes are under direct or indirect control of sigmaS and that sigmaS should be considered a second vegetative sigma factor with a major impact not only on stress tolerance but on the entire cell physiology under nonoptimal growth conditions. This large data set allowed us to unequivocally identify a sigmaS consensus promoter in silico. Moreover, our results suggest that sigmaS-dependent genes represent a regulatory network with complex internal control (as exemplified by the acid resistance genes). This network also exhibits extensive regulatory overlaps with other global regulons (e.g., the cyclic AMP receptor protein regulon). In addition, the global regulatory protein Lrp was found to affect sigmaS and/or sigma70 selectivity of many promoters. These observations indicate that certain modules of the sigmaS-dependent general stress response can be temporarily recruited by stress-specific regulons, which are controlled by other stress-responsive regulators that act together with sigma70 RNA polymerase. Thus, not only the expression of genes within a regulatory network but also the architecture of the network itself can be subject to regulation.