From famine to feast: the role of methylglyoxal production in Escherichia coli

The enzyme methylglyoxal synthase (MGS) was partially purified from Escherichia coli extracts, and the amino-terminal sequence of candidate proteins was determined, based on the native protein being a tetramer of about 69 kDa. Database analysis identified an open reading frame in the E . coli genome, YccG, corresponding to a protein of 16.9 kDa. When amplified and expressed from a controlled promoter, it yielded extracts that contained high levels of MGS activity. MGS expressed from the trc promoter accumulated to approximately 20% of total cell protein, representing approximately 900-fold enhanced expression. This caused no detriment during growth on glucose, and the level of methylglyoxal (MG) in the medium rose to only 0.08 mM. High-level expression of MGS severely compromised growth on xylose, arabinose and glycerol. A mutant lacking MGS was constructed, and it grew normally on a range of carbon sources and on low-phosphate medium. However, the mutant failed to produce MG during growth on xylose in the presence of cAMP, and growth was inhibited.     

Differential expression of mal genes under cAMP and endogenous inducer control in nutrient-stressed Escherichia coli

LamB glycoporin has an important general role in carbohydrate uptake during growth at low extracellular sugar concentrations. lamB and mal regulon induction during glucose starvation and glucose-limited continuous culture was investigated using lacZ fusions. A low-level burst of lamB induction occurred upon entry into glucose starvation-induced stationary phase but returned to basal levels during continued nutrient deprivation. Glucose-limited continuous culture elicited much higher expression of transporter genes in the mal regulon, as well as [¹⁴C]-maltose-transport activity; malEFG and malKlamB operons in glucose-limited chemostats were expressed to close to half of the level of maltose-induced batch cultures. Limitation-induced expression was dependent on both Crp-cAMP and MalT activation but was independent of RpoS function. As expected for a gene with a Crp-controlled promoter, malT expression was maximal under conditions which elicited the highest cAMP levels, but lamB induction did not behave in a corresponding fashion. Rather, maximal lamB induction occurred at rapid but suboptimal growth rates with micromolar or submicromolar medium glucose. Maximal transport and lamB induction coincided with increased endogenous maltotriose (inducer) concentrations during growth on glucose. Hence regulation of glycoporin and the maltose-transport system is not a starvation- or stationary-phase response but facilitates the adaptation of Escherichia coli to low-nutrient environments through endoinduction.     

Accumulation of pyrophosphate in Escherichia coli. Relationship to growth and nucleotide synthesis

Pyrophosphate (PPi) content of Escherichia coli is increased manyfold when the growth of the cells is limited by inhibition of the synthesis of nucleotides. The accumulated PPi is immediately degraded when inhibition is released and growth allowed to resume. Other tested nutritional deficiencies (starvation of carbon source, sulfate or an amino acid) fail to induce PPi accumulation.     

Bacterial adaptation: Macromolecular biosynthesis during diauxic growth of Escherichia coli

Abstract Synthesis of DNA, RNA and protein was studied during a diauxic adaptation (glucose- to acetate-utilization) of Escherichia coli B. The multiphasic transition described illuminates certain features of the mechanisms regulating metabolism of the 3 macromolecules involved in the genetic flow of information.     

Screening and characterization of proteorhodopsin color-tuning mutations in Escherichia coli with endogenous retinal synthesis

Proteorhodopsin is photoactive 7-transmembrane protein, which uses all-trans retinal as a chromophore. Proteorhodopsin subfamilies are spectrally tuned in accordance with the depth of habitat of the host organisms, numerous species of marine picoplankton. We try to find residues critical for the spectral tuning through the use of random PCR mutagenesis and endogenous retinal biosynthesis. We obtained 16 isolates with changed color by screening in Escherichia coli with internal retinal biosynthesis system containing genes for beta-carotene biosynthesis and retinal synthase. Some isolates contained multiple substitutions, which could be separated to give 20 single mutations influencing the spectral properties. The color-changing residues are distributed through the protein except for the helix A, and about a half of the mutations is localized on the helices C and D, implying their importance for color tuning. In the pumping form of the pigment, absorption maxima in 8 mutants are red-shifted and in 12 mutants are blue-shifted compared to the wild-type. The results of flash-photolysis showed that most of the low pumping activity mutants possess slower rates of M decay and O decay. These results suggest that the color-tuning residues are not restricted to the retinal binding pocket, in accord with a recent evolutionary analysis.     

Cycles of famine and feast: the starvation and outgrowth strategies of a marineVibrio

Studies of starvation survival in non-differentiating bacteria have largely focused on physiological changes and regulatory aspects of a few master regulators such as the signal molecule ppGpp and the stationary phase alternative sigma factor, sigma S. Recent findings have implicated a series of novel key events for the entry as well as exit from starvation. The importance of alternative sigma factors other than sigma S is emerging. In addition, low molecular weight extracellular signals have been demonstrated to be essential for the induction and mediation of several adaptive responses. The importance of mRNA modification and stability for starvation survival as well as outgrowth is receiving renewedinterest. In this paper, we present the results obtained from studies of starvation survival and recovery ofVibrio sp. strain S14.     

Isolation and characterization of an endogenous inhibitor of protein synthesis in Escherichia coli K-12.

A low-molecular-weight factor was isolated from cell extracts of Escherichia coli K-12. The concentration of the factor in cells was dependent upon nutritional conditions, the concentration being higher in faster growing cells. Treatment of cells with colicin K caused an increase in concentration of the factor. The factor inhibited protein synthesis in E. coli. This inhibition was reversible, apparently because of metabolism of the factor. The inhibition of synthesis of beta-galactosidase lasted longer than the inhibition of protein synthesis; cyclic AMP eliminated this difference. The factor inhibited the synthesis of beta-galactosidase from preformed lac mRNA, indicating an inhibition of translation. Kinetic studies of the onset of inhibition of beta-galactosidase synthesis by the factor suggested that the factor may inhibit protein synthesis at the initiation of translation.     

Macromolecule synthesis in Escherichia coli BB under various growth conditions.

The kinetic behavior of the macromolecule synthesis of Escherichia coli during balanced growth in various media at different temperatures as investigated. The results indicate that macromolecule contents per cell can be expressed as exponential functions of the specific growth rate at a given temperature. It was shown that the content per cell at the zero growth rate was constant in each macromolecule component, irrespective of the growth temperature. The rate of ribonucleic acid (RNA) synthesis per unit weight of deoxyribonucleic acid and that of protein synthesis per unit weight of RNA were taken as efficiencies of RNA and protein synthesis, respectively; both of them were found to be dependent on the growth rate and temperature. The efficiency of RNA synthesis was found to be very high at a high growth rate, whereas that of protein synthesis was found to decrease above certain growth rate. At the same growth rate, an increase in the growth temperature resulted in a decrease in the efficiency of RNA synthesis but an increase in that of protein synthesis.     

Changes of Escherichia coli cell cycle parameters during fast growth and throughout growth with limiting amounts of thymine

It is generally accepted that during fast growth of Escherichia coli, the time (D) between the end of a round of DNA replication and cell division is constant. This concept is not consistent with the fact that average cell mass of a culture is an exponential function of the growth rate, if it is also accepted that average cell mass per origin of DNA replication (M_i) changes with growth rate and negative exponential cell age distribution is taken into account. Data obtained from cell composition analysis of E. coli OV-2 have shown that not only (M_i) but also D varied with growth rate at generation times () between 54 and 30 min. E. coli OV-2 is a thymine auxotroph in which the replication time (C) can be lengthened, without inducing changes in , by growth with limiting amounts of thymine. This property has been used to study the relationship between cell size and division from cell composition measurements during growth with different amounts of thymine. When C increased, average cell mass at the end of a round of DNA replication also increased while D decreased, but only the time lapse (d) between the end of a replication round and cell constriction initiation appeared to be affected because the constriction period remained fairly constant. We propose that the rate at which cells proceed to constriction initiation from the end of replication is regulated by cell mass at this event, big cells having shorter d times than small cells.Abbreviations OD₄₅₀ and OD₆₃₀ Optical density at a given wavelength in nm Dedicated to Dr. John Ingraham to honor him for his many contributions to Science     

Effects of varying the carbon source limiting growth on yield and maintenance characteristics of Escherichia coli in continuous culture.

The magnitudes of Yo (grams [dry weight] formed per gram of atom O) and mo, the maintenance respiration (milligram-atoms of O per gram [dry weight] per hour), of Escherichia coli B have been determined by growing the organism in aerobic continuous culture limited by a number of different substrates. The value found were as follows: glucose--tyo = 12.5, mo = 0.9; glucose plus 2.7 mM cyclic adenosine 3',5'-monophosphate (cAMP)--Yo = 31.2, mo = 9.3; galactose--Yo = 13.2, mo = 1.8; mannitol--Yo = 20.1, mo = 6.1; L-glutamate--Yo = 25.5, mo = 17.7; glycerol--Yo = 14.9, mo = 10.0; succinate--Yo = 11.2, mo = 12.1; and acetate--Yo = 14.7, mo = 25.4. During growth in anaerobic continuous culture with limiting glucose YATP was found to be 10.3 g (dry weight)/mol of adenosine 5'-triphosphate (ATP) and m ATP was 18.9 mmol of ATP/g (dry weight) per h. The aerobic growth yields of cells growing on glucose, glucose plus cAMP, mannitol, and glutamate were consistent with the hypothesis that carbohydrates partially repress oxidative phosphorylation, but the yields of cells growing on glycerol, succinate, acetate, and galactose were all lower than expected. We conclude that, like the efficiency of oxidative phosphorylation, both the maintenance respiration and the amount of ATP necessary to serve maintenance processes are determined by the identity of the growth substrates. Yields smaller than expected may be explained by the absence of respiratory control exerted by phosphate acceptors.     

Control of membrane lipid synthesis in Escherichia coli during growth and during the stringent response.

The regulation of phospholipid synthesis in cells of Escherichia coli was studied in vivo during growth and during the stringent response to amino acid starvation. Strains harboring the hybrid plasmid pLC44-14 (Clark, L., and Carbon, J. (1976) Cell 9, 91-99), which had increased levels of glycerophosphate acyltransferase, were used to study the involvement of this enzyme in the control of phospholipid synthesis. In addition, regulation was studied by measuring the levels of three early intermediates of phospholipid synthesis:phosphatidic acid, CDP-diglyceride, and dCDP-diglyceride. The liponucleotides were measured by a new enzymatic method which allows determinations to be made on crude lipid extracts. Results from experiments on growing cells are consistent with regulation of membrane lipid synthesis occurring in fatty acid synthesis or at the level of glycerophosphate acylation, but not at any later step. Experiments on the inhibition of lipid synthesis during the stringent response make it possible to rule out explanations which involve the inhibition of a single enzyme; enzymes both before and after the liponucleotides in phospholipid synthesis must be affected.     

Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 1. Readjustment of metabolic enzyme synthesis

The metabolic burden and the stress load resulting from temperature-induced production of human basic fibroblast growth factor is connected to an increase in the respiratory activity of recombinant Escherichia coli, thereby reducing the biomass yield. To study the underlying changes in metabolic enzyme synthesis rates, the radiolabeled proteom was subjected to two-dimen- sional gel electrophoresis. After temperature-induction, the cAMP-CRP controlled dehydrogenases of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle (LpdA and SdhA) were induced four times, reaching a maximum 1 h after the temperature upshift. The more abundant tricarboxylic acid cycle dehydrogenases (Icd and Mdh) were initially produced at reduced rates but regained preshift rates within 30 min. The adenylate energy charge dropped immediately after the temperature upshift but recovered within 1 h. Similar profiles in dehydrogenase synthesis rates and adenylate energy charge were found in a control cultivation of a strain carrying the "empty" parental expression vector. Although both strains exhibited significant differences in growth pattern and respiration rates after the temperature upshift, the adaptation of the energetic state of the cells and the synthesis of enzymes from the energy-generating catabolic pathway did not seem to be affected by the strong overproduction of the recombinant growth factor. In contrast, the synthesis rates of enzymes belonging to the biosynthetic machinery, e.g., translational elongation factors, decreased more strongly in the culture synthesizing the recombinant protein. In control and producing culture, synthesis rates of elongation factors paralleled the respective growth rate profiles. Thus, cells seem to readjust their metabolic activities according to their energetic requirements and, if necessary, at the cost of their biosynthetic capabilities.