Temperature-Sensitive Osmotic Remedial Mutants of Escherichia coli

A collection of temperature-sensitive mutants of Escherichia coli K-12 was examined for ability to grow at the restrictive temperature when the osmotic pressure of the medium was increased. Five of the fourteen mutants were found to be osmotic remedial. Four strains containing temperature-sensitive, osmotic-remedial mutations affecting aminoacyl-transfer ribonucleic acid synthetases were found to have altered permeability characteristics which may be attributable to changes in the lipopolysaccharide layer of the cell envelope at restrictive temperatures.     

Construction of Pta-Ack Pathway Deletion Mutants of Escherichia coli and Characteristic Growth Profiles of the Mutants in a Rich Medium

Escherichia coli grown in a rich medium excreted acetate and reused the acetate. Using cloned genes and a plasmid with a temperature-sensitive replication origin, three kinds of Pta-Ack pathway deletion mutants were constructed. Acetate production and reuse by wild-type cells grown in the rich medium was confirmed to largely occur through the Pta-Ack pathway. The deletion mutants of the gene encoding phosphotransacetylase secreted pyruvate before the secretion of acetate into the medium. A deletion mutant of the gene endocing acetate kinase grew at a slow rate, but its secretion and use of acetate were rapid. These results indicated that a pathway(s), other than the Pta-Ack pathway, functions in the control of excess carbon flow in the mutants.     

Calorimetrie Study of Escherichia coli Growth on Bouillon Medium

Heat evolution during the growth of Escherichia coli in a stationary culture on bouillon medium was continuously monitored with a conduction-type batch calorimeter at different pHs and temperatures. The growth thermograms were found to be reproducible within percent errors of ±2.1%, ±0.8% and ±1.5% for the peak time of a thermogram, the peak height and the total heat evolution, respectively. The mean heat evolution for the formation of a unit E. coli cell was co = (1.69 ±0.08) X 10^—8 J cell^{— 1} at pH 6.2 and 37°C. The mean heat evolution rate per unit cell during growth was </ = 3.6ρwcell^_1, which was consistent with values calculated for other microbial cells. The growth rate constant calculated on kinetic analysis of the growth thermograms was μ = 0.532 ± 0.068 hr ^-1 at pH 6.2 and 37°C. The pH dependence of the growth rate constant showed that the growth activity of E. coli cells on bouillon medium is maximum in the pH range of 5.5 to 6.5. From the temperature-dependent variations in the growth rate constant, the apparent activation energy of E. coli growth was found to be E_a =65.3 ±7.1 kJ mol^-1.     

Growth of Escherichia coli on Short-Chain Fatty Acids: Growth Characteristics of Mutants

The parent Escherichia coli K-12 is constitutive for the enzymes of the glyoxylate bypass and adapts to growth on long-chain fatty acids (C(12) to C(18)). It does not utilize medium-chain (C(6) to C(11)) or short-chain (C(4), C(5)) n-monocarboxylic acids. Several mutants of this strain which grow using short- or medium-chain acids, or both, as the sole carbon source were selected and characterized. One mutant (D(1)) synthesizes the beta-oxidation enzymes constitutively and grows on medium-chain but not on short-chain acids. A second (N(3)) is partially derepressed for synthesis of these enzymes and grows both on medium-chain and on short-chain acids. Secondary mutants (N(3)V(-), N(3)B(-), N(3)OL(-)) were derived from N(3). N(3)V(-) grows on even-chain but not on odd-chain acids and exhibits a lesion in propionate oxidation. N(3)B(-) grows on odd-chain but not on even-chain acids and exhibits no crotonase activity as assayed by hydration of crotonyl-CoA. N(3)OL(-) grows on acetate and propionate but does not utilize fatty acids C(4) to C(18); it exhibits multiple deficiencies in the beta-oxidation pathway. Growth on acetate of N(3), but not of the parent strain, is inhibited by 4-pentenoate. Revertants of N(3) which are resistant to growth inhibition by 4-pentenoate (N(3)PR) exhibit loss of ability to grow on short-chain acids but retain the ability to grow on medium-chain and long-chain acids. The growth characteristics of these mutants suggest that in order to grow at the expense of butyrate and valerate, E. coli must be (i) derepressed for synthesis of the beta-oxidation enzymes and (ii) derepressed for synthesis of a short-chain fatty acid uptake system.     

Osmotic Pressure in Escherichia coli as Rendered Detectable by Lysozyme Attack

The enhanced susceptibility of plasmolyzed Escherichia coli to lysozyme attack was used to estimate the internal osmotic pressure of these cells under various conditions. Differences were detected between strains, culture media, stages in the growth cycle, and the osmotically active material used to produce plasmolysis. Lysozyme also was found to attack unplasmolyzed cells at 0 C and between 50 and 70 C.     

Enhanced Trehalose Production Improves Growth of Escherichia coli under Osmotic Stress

The biosynthesis of trehalose has been previously shown to serve as an important osmoprotectant and stress protectant in Escherichia coli. Our results indicate that overproduction of trehalose (integrated lacI-P_tac-otsBA) above the level produced by the native regulatory system can be used to increase the growth of E. coli in M9-2% glucose medium at 37°C to 41°C and to increase growth at 37°C in the presence of a variety of osmotic-stress agents (hexose sugars, inorganic salts, and pyruvate). Smaller improvements were noted with xylose and some fermentation products (ethanol and pyruvate). Based on these results, overproduction of trehalose may be a useful trait to include in biocatalysts engineered for commodity chemicals.     

Transketolase Mutants of Escherichia coli

Transketolase mutants have been selected after ethyl methane sulfonate mutagenesis of Escherichia coli. These strains are unable to grow on any pentose and, in addition, require a supplement of aromatic amino acids or shikimic acid for normal growth on any other carbon source. Revertants are normal in both respects and also contain transketolase. Transketolase mutants do not require exogenous pentose for growth. Preliminary genetic mapping of the locus is presented.     

Nonchemotactic Mutants of Escherichia coli

We have isolated 40 mutants of Escherichia coli which are nonchemotactic as judged by their failure to swarm on semisolid tryptone plates or to make bands in capillary tubes containing tryptone broth. All the mutants have normal flagella, a fact shown by their shape and reaction with antiflagella serum. All are fully motile under the microscope and all are sensitive to the phage chi. Unlike its parent, one of the mutants, studied in greater detail, failed to show chemotaxis toward oxygen, glucose, serine, threonine, or aspartic acid. The failure to exhibit chemotaxis does not result from a failure to use the chemicals. The swimming of this mutant was shown to be random. The growth rate was normal under several conditions, and the growth requirements were unchanged.     

Mutants of Escherichia coli with High Minimal Temperatures of Growth.

O'Donovan, Gerard A. (University of California, Davis), Catherine L. Kearney, and John L. Ingraham. Mutants of Escherichia coli with high minimal temperatures of growth. J. Bacteriol. 90:611-616. 1965.-Three general classes of mutants showing increased minimal temperatures of growth have been isolated from Escherichia coli. These mutants do not grow at temperatures below 20 C, although their parents can grow at temperatures as low as 8 C. The first class of mutants (K-I) cannot grow below 20 C in either complex or minimal medium, but grows at nearly normal rates at 37 C on both types of media. Normal growth rate at 20 C can be conferred on these mutants by infection at a low multiplicity with a transducing phage grown on the parent. The second class of mutants (K-II) fails to grow only in minimal medium at 20 C. These mutants are characterized by their singular response to specific nutrients in minimal medium at 20 C. The third class of mutants (K-III) grows normally in minimal medium at all temperatures with either glucose or glycerol as the carbon source, but does not grow at 20 C with lactose as the carbon source.     

Porphyrin-Accumulating Mutants of Escherichia coli

Four mutants (pop-1, pop-6, pop-10, and pop-14) which accumulate a red water-insoluble pigment were obtained in Escherichia coli K-12 AB1621. For each mutant, the red pigment was shown to be protoporphyrin IX, a late precursor of heme. Mutagenic treatment of mutant pop-1 yielded a secondary mutant, pop-1 sec-20, which accumulated a brown water-soluble pigment. The brown pigment was shown to be coproporphyrin III. Mutant pop-1 resembled the parental strain in its cytochrome absorption spectrum, catalase activity, and ability to grow on nonfermentable carbon and energy sources; therefore, its ability to produce and utilize heme was unimpaired. Judged on the same criteria, the secondary mutant, pop-1 sec-20, was partially heme and respiratory deficient. Growth in anaerobic conditions decreased by 25% the accumulation of protoporphyrin by pop-1; under the same conditions, pop-1 sec-20 did not accumulate coproporphyrin or coproporphyrinogen. The mutations causing protoporphyrin accumulation in all four pop mutants were found to map in the lac to purE (10-13 min) region of the E. coli chromosome. In the case of mutant pop-1, the mutation was shown to be strongly linked to the tsx locus (12 min). In mutant pop-1 sec-20, the second mutation causing coproporphyrin accumulation was co-transducible with the gal locus at a frequency of 88 to 96%. The mechanism of porphyrin accumulation by the mutants is discussed.     

Effect of Helium Gas at Elevated Pressure on Iron Transport and Growth of Escherichia coli

Helium at an ambient pressure of 68 at m with 0.2 atm of O(2) shortened by 1 to 1.5 h the lag phase for growth of Escherichia coli in minimal medium supplemented with 2 muliters of cell-free culture filtrate (CFF) per ml or with 1 muM 2,3-dihydroxybenzoylserine (DHBS), an iron chelator. The lag phase of cultures not exposed to helium could be shortened by use of supplements, but higher concentrations were required-10 to 30 muliters of CFF per ml or 10 to 50 muM DHBS. Strain AN 193 of E. coli, which requires the DHBS precursor 2,3-dihydroxybenzoic acid (DHBA), grew well in media with 10 muM DHBA when exposed to helium at 68 atm, whereas 100 muM DHBA was required for growth in unexposed cultures. In the presence of 100 muM DHBA plus 1.0 muM ethylenediaminetetraactic acid, growth was inhibited at 1 and 68 atm. Growth was restored, however, by the addition of 0.1 muM FeSO(4) at 68 atm and 1.0 muM FeSO(4) at 1 atm, but lag times were invariably shorter in the pressurized cultures. Hydrostatic pressures of 68 atm did not reduce the lag phase in the presence of CFF, DHBS, or DHBA. Our results suggest that 68 atm of helium pressure, but not hydrostatic pressure, elicited a more rapid transport of iron into the cells.     

Effect of Nutrient Concentration on the Growth of Escherichia coli

The relationship between specific growth rate of Escherichia coli and the concentration of limiting nutrient (glucose or phosphate or tryptophan) has been determined for populations in a steady state. At high concentrations the specific growth rate is independent of the concentration of nutrient, but at low concentrations the specific growth rate is a strong function of the nutrient concentration. Such a relationship was predicted by Monod; however, Monod's equation does not predict the relationship over the entire range of nutrient concentration. If parameters of the equation are estimated from the results obtained at low concentrations, then at high concentrations of nutrient, the specific growth rate is significantly higher than that predicted by Monod's equation. These results were interpreted on the basis that the rate of growth is controlled by at least two parallel reactions and that the affinities of the enzymes catalyzing these reactions are different. The relationship between specific growth rate and mean cell volume was also measured, and the results indicate that mean cell volume depends not only on the specific growth rate but also on the nature of the limiting nutrient. There are different mean cell volumes at the same specific growth rate established by different limiting nutrients. Therefore, the mean cell volume is not uniquely determined by the specific growth rate.     

渗透压对硝化作用的影响

在气升式内循环硝化反应器中研究了渗透压对硝化作用的影响。保持进水氨氮浓度420mg·L-1,将进水渗透压逐渐从4.3×105Pa提高到18.8×105Pa,硝化反应器的氨氮转化率稳定在93%～100%。将进水渗透压进一步提高到19.2×105Pa,氨氮转化率降至69.2%。渗透压对硝化作用的影响具有突发性,临界值在18.8×105～19.2×105Pa之间。受高渗透压胁迫时,活性污泥中硝化细菌的形态趋向单一,个体变小,内膜数量减少,并产生许多不明成分的颗粒状内含物。解除渗透压胁迫后,细胞结构恢复。添加钾离子能够缓解高渗压对硝化作用的影响。高渗透压胁迫以及解除渗透压胁迫可增强污泥硝化活性,比污泥氨氮转化率(污泥以SS计)分别从0.083kg·kg-1·d-1升至0.509kg·kg-1·d-1和2.569kg·kg-1·d-1,同比提高5.1倍和30.0倍。     

Rapid Growth Mutants of Escherichia Coli

If rapid growth (rap) mutants of Escherichia coli could be obtained, these might prove a valuable contribution to fields as diverse as growth rate control, biotechnology and the regulation of the bacterial cell cycle. To obtain rap mutants, a dnaQ mutator strain was grown for four and a half days continuously in batch culture. At the end of the selection period, there was no significant change in growth rate. This result means that selecting rap mutants may require an alternative strategy and a number of such alternatives are discussed.     

Response of Cell Walls of Escherichia coli to a Sudden Reduction of the Environmental Osmotic Pressure

The rate of survival after osmotic shocks was found to be dependent on the state of growth. When growing logarithmically, Escherichia coli was about 20 to 100 times more sensitive to an abrupt decrease of the environmental osmotic pressure than when it was in the stationary phase. Subjecting rapidly growing cells to such a treatment caused fingerlike extrusions to emerge from the bacterial wall. Our results suggest that underneath these extrusions the rigid layer of the wall contains weak areas which appear as discontinuities or gaps when viewed in an electron microscope. After exposure to osmotic shock, the gaps became wider. We concluded that the gaps represent sites of mucopolymer synthesis where the rigid structure has temporarily been opened by hydrolytic enzymes to allow for the insertion of new wall material into the older portions of the wall.     

Phosphoglucomutase Mutants of Escherichia coli K-12

Bacteria with strongly depressed phosphoglucomutase (EC 2.7.5.1) activity are found among the mutants of Escherichia coli which, when grown on maltose, accumulate sufficient amylose to be detectable by iodine staining. These pgm mutants grow poorly on galactose but also accumulate amylose on this carbon source. Growth on lactose does not produce high amylose but, instead, results in the induction of the enzymes of maltose metabolism, presumably by accumulation of maltose. These facts suggest that the catabolism of glucose-1-phosphate is strongly depressed in pgm mutants, although not completely abolished. Anabolism of glucose-1-phosphate is also strongly depressed, since amino acid- or glucose-grown pgm mutants are sensitive to phage C21, indicating a deficiency in the biosynthesis of uridine diphosphoglucose or uridine diphosphogalactose, or both. All pgm mutations isolated map at about 16 min on the genetic map, between purE and the gal operon.     

Mutants of Escherichia coli Sensitive to Antibiotics

Mutants of Escherichia coli sensitive to the antibiotic synergistin A, an inhibitor of protein synthesis, were isolated. These mutants were pleiotropic, being also sensitive to a large number of unrelated antibiotics and to lysis by detergents. These pleiotropic responses indicated that the mutations affected cell wall or membrane synthesis. Consequently, selection for antibiotic-sensitive mutants constitutes a useful means for isolating cell wall or membrane mutants.     

Biochemical Characterization of the ctr Mutants of Escherichia coli

A test procedure based on complementation in mixed extracts is described for the assay of heat-stable protein and enzyme I of the phosphoenolpyruvate-dependent phosphotransferase system. The test was used to assay a collection of pleiotropic carbohydrate mutants of Escherichia coli (ctr mutants) and revertants of these mutants. All mutants were found to lack enzyme I of the phosphoenolpyruvate-dependent transferase system. Revertants of these mutants to complete wild phenotype regained enzyme I-forming ability. Reversion to partial wild type was not accompanied by restoration of enzyme I-forming ability.