Temperature dependence of action of polymyxin B on Escherichia coli

The temperature dependence of the action of polymyxin B on Escherichia coli was studied by using K+, Ca2+, and tetraphenylphosphonium (TPP+) ion-selective electrodes. At room temperature (27 degrees C), Ca2+ was released immediately after addition of polymyxin, while the efflux of K+ occurred after 30 s. The rapid release of Ca2+ was not affected by incubation temperature, while the efflux of K+ was significantly lowered at temperatures below about 25-30 degrees C. The uptake of TPP+ also increased after polymyxin addition. The release of Ca2+ and the uptake of TPP+ supported the disruption of the outer membrane structure reported previously. In experiments with isolated membrane vesicles (the cytoplasmic membrane being exposed), the efflux of K+ was not delayed, but was lowered at temperatures below about 15-20 degrees C. This temperature range differed significantly from that of whole cells, and was interpreted as representing a difference in membrane fluidity between the outer and cytoplasmic membranes. The phase transition temperature of the outer membrane is known to be higher than that of the cytoplasmic membrane; and the temperature dependence of efflux of K+ from membrane vesicles was compatible with the phase transition temperature of liposomes prepared with phospholipids (not containing lipopolysaccharides) extracted from E. coli. Thus, it was speculated that, with whole cells, polymyxin molecules passed through the outer membrane at temperatures above the phase transition and reached the cytoplasmic membrane, increasing its K+ permeability. The mechanism of the permeability change is discussed in terms of deformation of the cytoplasmic membrane structure induced by polymyxin molecules.     

Cell-wall abnormalities of a Chlamydomonas reinhardtii mutant strain under suboptimal growth conditions

Sporangia were accumulated in autotrophically and mixotrophically growing cultures of the Chlamydomonas reinhardtii mutant strain ls entering the stationary phase. Such an accumulation of sporangia was never observed in stationary-phase cultures of wildtype strains. Sporangia harvested from stationary-phase cultures of the mutant strain ls released their zoospores after being resuspended in fresh culture medium. Liberation of zoospores was also observed during fixation of these sporangia with glutaraldehyde and OsO₄. Release of zoospores during fixation was prevented by pretreatment with 3 mol·l^–1 LiCl. Ultrastructural analyses of these LiCl-pretreated sporangia revealed that they contained abnormal sporangial walls: sporangia containing sporangia and sporangia surrounded by additional multilayered cell walls have been observed. Similar abnormal cell-wall structures were found in sporangia accumulated at the end of the dark period, when the mutant strain ls was grown photoautotrophically under a 12 h light-12 h dark regime with suboptimal aeration. When grown under optimal conditions, this particular mutant did not show any abnormal wall structures.This work has been supported by a grant from the Deutsche Forschungsgemeinschaft. The authors thank Mrs. C. Adami for the photographic work.     

Transductional construction of aspartase-hyperproducing strain of Escherichia coli B

Two aspartase-overproducing mutants of Escherichia coli B were characterized. Strain EAPc7 had a mutation enhancing aspartase formation in the region of aspartase gene. This mutation did not affect catabolite repression by aspartase. Strain EAPc244 showed a high cAMP content and an elevated adenylate cyclase activity. This mutation was closely linked to the ilv locus and caused the release of catabolite repression for various catabolite repression-sensitive enzymes, resulting in overproduction of adenylate cyclase. This mutation was transduced to an Ile⁻ strain derived from strain EAPc7 using the Ile⁺ selective marker. The constructed strain AT202, having the above 2 mutations, produced about 3-fold and 18-fold more aspartase than did the 2 parent strains and the wild-type strain, respectively, when cultured in the medium used for industrial production of aspartase. Strain AT202 maintained stably high aspartase activity after 30 cell generations. On the other hand, in E. coli K-12 harboring the aspA⁺ recombinant plasmid pYT471 (pBR322-aspA⁺), the activity decreased to the E. coli K-12 level. Hence, strain AT202 is more advantageous for industrial production of l-aspartic acid than cells harboring the aspA⁺-recombinant plasmid pYT471.     

Transport of vitamin B12 in Escherichia coli: energy dependence.

This paper presents some evidence that the osmotic shock-sensitive, energy-dependent transfer of vitamin B12 from outer membrane receptor sites into the interior of cells of Escherichia coli requires an energized inner membrane, without obligatory intermediation of adenosine 5'-triphosphate (ATP). The experiments measured the effects of glucose, D-lactate, anaerobiosis, arsenate, cyanide, and 2,4-dinitrophenol upon the rates of B12 transport by starved cells of E. coli KBT001, which possesses a functional Ca2+, Mg2+-stimulated adenosine triphosphatase (Ca,MgATPase), and of E. coli AN120, which lacks this enzyme. Both strains were able to utilize glucose and D-lactate aerobically to potentiate B12 transport, indicating that the Ca,MgATPase was not essential for this process. When respiratory electron transport was blocked, either by cyanide or by anaerobic conditions, and the primary source of energy for the cells was presumably ATP from glucose fermentation, the rate of B12 transport was much reduced in E. coli AN120 but not in E.coli KBT001. These results support the view that the CaMgATPase can play a role in B12 transport but only when the energy for this process must be derived from ATP. The results of experiments with arsenate also supported the conclusion that the generation of phosphate bond energy was not absolutely required for B12 transport.     

Effect of growth conditions on glutamate transport in the wild-type strain and glutamate-utilizing mutants of Escherichia coli.

The effects of growth conditions on the glutamate transport activity of intact cells and membrane vesicles and on the levels of glutamate-binding protein in wild-type Escherichia coli K-12 CS101 and in two glutamate-utilizing mutants, CS7 and CS2TC, were studied. Growth of CS101 on aspartate as the sole source of carbon or nitrogen resulted in a severalfold increase in glutamate transport activity of intact cells and membrane preparations to levels characteristic of the operator-constitutive mutant CS7. The high glutamate transport activity of mutant CS7 was not depressed further by growth on aspartate. Synthesis of glutamate-binding protein was not enhanced by aspartate in either strain. Mutant CS2TC produces a heat-labile repressor of glutamate permease synthesis and is therefore able to grow on glutamate at 42 C but not at 30 C. CS2TC cells grown in a glycerol-minimal medium at the restrictive temperature (30 C) exhibit low glutamate transport activity. Growth on aspartate at 30 C results in derepressed synthesis of glutamate permease. Cells grown on glycerol at 42 C have high glutamate transport activity. No further derepression is obtained upon growth on aspartate. Growth of CS101 and CS7 in "rich broth" greatly reduces the levels of glutamate-binding protein but does not appreciably affect glutamate transport by whole cells or membrane preparations. The identity of the carrier and the role of the binding protein in glutamate transport are discussed in the light of these findings.     

Radiation sensitization of Escherichia coli B/r by 2'-chloro-2'-deoxythymidine under various irradiation conditions

In order to elucidate the mechanism of sensitization of E. coli B/r cells to X-irradiation by 2'-chloro-2'-deoxythymidine (2'Cl-TdR), the survival curves of the cells in which 2'Cl-TdR was incorporated into DNA were obtained following X-irradiation under various conditions. The marked sensitization of E. coli cells by 2'Cl-TdR to the killing action of X-rays was observed, when E. coli cells labelled with 2'Cl-TdR were exposed to X-rays in the absence of oxygen as well as in the presence of oxygen. The sensitization factor calculated from inactivation constants from survival curves irradiated in the absence of O2 was about a half of that obtained in the presence of O2. Under the conditions where 2'Cl-TdR was not incorporated into the DNA of E. coli cells, the presence of 2'Cl-TdR in the cell suspension fluid at the time of irradiation caused no sensitization of the cells to X-irradiation. The sensitization factor for 2'Cl-TdR obtained under N2O was almost same as that obtained under N2. It was also observed that the sensitization factor obtained in the presence of glycerol at a concentration of 1 mol dm-3 under N2 was similar to that obtained in the absence of glycerol. These results indicated that the direct effect of ionizing radiation on DNA was closely associated with the sensitization of E. coli B/r cells by 2'Cl-TdR and that the radical at the C-2' position of the deoxyribose moiety in DNA produced by X-irradiation was transformed into lethal damage for E. coli cells even in the absence of O2. However, this transformation occurred more efficiently in the presence of O2 than in the absence of O2.     

Beta-lactamase production by Escherichia coli under different growth conditions

Facultative anaerobic bacteria, such as Escherichia coli, are more resistant to cephalosporin antibiotics during anaerobic growth. Strict anaerobic ambience reduces beta-lactamase production or the enzyme affinities for their substrates. A different balance between DNA gyrase and topoisomerase I activity, during aerobic and anaerobic growth condition, could be related to the bacteria behavior.     

Effect of growth conditions on peptidoglycan content and cytoplasmic steps of its biosynthesis in Escherichia coli.

In an attempt to bring some insight into how peptidoglycan synthesis is controlled in Escherichia coli, simple parameters, such as cell peptidoglycan content, the pool levels of its seven uridine nucleotide precursors, and the specific activities of five enzymes involved in their formation, were investigated under different growth conditions. When exponential-phase cells with generation times ranging from 25 to 190 min were examined, the peptidoglycan content apparently varied as the cell surface area changed, and no important variations in the pool levels of the nucleotide precursors or in the specific activities of the five enzymes considered were observed. The peptidoglycan of exponential-phase cells accounted for 0.7 to 0.8% of the dry cell weight, whereas that of stationary-phase cells accounted for 1.4 to 1.9%. Depending on the growth conditions, the number of peptidoglycan disaccharide peptide units per cell varied from 2.4 X 10(6) to 5.6 X 10(6). The levels of the nucleotide precursor pools as well as the specific activities of the D-glutamic acid- and D-alanyl-D-alanine-adding enzymes varied little with the growth phase. The specific activities of UDP-N-acetylglucosamine transferase, UDP-N-acetylglucosamine-enolpyruvate reductase, and the diaminopimelic acid-adding enzymes decreased by 20 to 50% at most in the late stationary phase. The results are discussed in terms of the possible importance for cell survival of the maintenance of a high capacity for peptidoglycan synthesis, whatever its rate under various growth conditions, and of a balance between the synthesis and breakdown of peptidoglycan during active growth.