Enumeration of stressed cells of Escherichia coli.

Cells of Escherichia coli K-12 were stressed by heating at 48 degrees C or by acid treatment at pH 4.2 for periods up to 1h. The addition of catalase to the selective medium increased the count of heat-stressed cells by 2.3-fold and acid-stressed cells by 4.8-fold. However, these values represented only a small percentage (3% for heat-stressed and 6% for acid-stressed cells respectively) of the population of injured but still viable cells. The addition of mannitol to the selective medium used to count acid-stressed cells did not increase the count. Whilst the presence of H2O2 in media may cause significant errors in the estimation of E. coli in certain situations these errors are unlikely to be significant in physiological studies of populations of cells injured by stress.     

Cytoplasmic protein mobility in osmotically stressed Escherichia coli

Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably, Escherichia coli grows in minimal medium over a wide range of external osmolalities (0.03 to 1.8 osmol). The mean cytoplasmic biopolymer volume fraction ((phi)) for such adapted cells ranges from 0.16 at 0.10 osmol to 0.36 at 1.45 osmol. For cells grown at 0.28 osmol, a similar phi range is obtained by plasmolysis (sudden osmotic upshift) using NaCl or sucrose as the external osmolyte, after which the only available cellular response is passive loss of cytoplasmic water. Here we measure the effective axial diffusion coefficient of green fluorescent protein (D(GFP)) in the cytoplasm of E. coli cells as a function of (phi) for both plasmolyzed and adapted cells. For plasmolyzed cells, the median D(GFP) (D(GFP)(m)) decreases by a factor of 70 as (phi) increases from 0.16 to 0.33. In sharp contrast, for adapted cells, D(GFP)(m) decreases only by a factor of 2.1 as (phi) increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by (phi) alone. By comparison with quantitative models, we show that the data cannot be explained by crowding theory. We suggest possible underlying causes of this surprising effect and further experiments that will help choose among competing hypotheses. Recovery of the ability of proteins to diffuse in the cytoplasm after plasmolysis may well be a key determinant of the time scale of the recovery of growth.     

Effects of Dimethylsulfoxide on the Lactose Operon in Escherichia coli

Fowler, Audree V. (University of California, Los Angeles), and Irving Zabin. Effects of dimethylsulfoxide on the lactose operon in Escherichia coli. J. Bacteriol. 92:353-357. 1966.-Dimethylsulfoxide (DMSO) at a concentration of 5% (v/v) in the culture medium inhibits the growth of Escherichia coli to only a slight extent, and does not affect the differential rate of synthesis of beta-galactosidase. Resting cells remain viable after shaking in the presence of 20% DMSO for 3 hr at 37 C. Both beta-galactosidase and thiogalactoside transacetylase retain almost all activity after incubation in even higher concentrations of the solvent for many hours. DMSO decreases the permeability barrier. The rate of hydrolysis of o-nitrophenyl-beta-d-galactoside (ONPG) in whole cells containing beta-galactosidase but lacking permease is increased in cells treated with 5% DMSO. Several permeaseless strains preinduced for beta-galactosidase will grow on lactose in the presence, but not in the absence, of 5% DMSO. When permeaseless strains are grown on tetrazolium-lactose-agar, the presence of 5% DMSO causes a definite but not marked shift toward the lactose-positive character.     

乳糖诱导甜蛋白Monellin在大肠杆菌中的表达

根据已报道的单链Monellin甜蛋白的氨基酸序列,按大肠杆菌基因偏爱密码子,设计和人工合成了单链monellin基因。将单链monellin基因克隆到大肠杆菌表达载体pET-28a中,构建了重组表达载体pET28a-mon,转化大肠杆菌BL21(DE3),得到表达Monellin的大肠杆菌工程菌株。借助SDS-PAGE分析方法,研究了乳糖代替IPTG诱导大肠杆菌表达甜蛋白Monellin。通过对乳糖作为诱导剂表达条件进行优化,Monellin的表达量可占细胞总蛋白的33.09%,与IPTG诱导表达量接近。本研究结果为乳糖作为诱导剂应用于重组大肠杆菌生产甜蛋白Monellin提供了参考依据。     

Restoration of colony-forming activity in osmotically stressed Escherichia coli by betaine.

Exposure of Escherichia coli to 0.8 M NaCl caused a rapid and large decrease in colony-forming activity. When such osmotically upshocked cells were exposed to betaine, colony-forming activity was restored. Betaine was able to restore colony-forming activity even when chloramphenicol inhibited protein synthesis. Thus, restoration was not the result of cell turnover. The cells were not killed by exposure to 0.8 M NaCl, because during exposure they accumulated ATP intracellularly. Betaine treatment caused this cellular ATP to decrease to a lower level. This work may provide the foundation for a simple plating procedure to quantitatively detect nonculturable E. coli in ocean beach recreational waters.     

Lactose permease of Escherichia coli catalyzes active beta-galactoside transport in a gram-positive bacterium.

The following several lines of evidence demonstrate that lactose permease (LacY) of Escherichia coli is assembled into the cytoplasmic membrane of gram-positive Corynebacterium glutamicum, expressing the lacY gene, as a functional carrier protein. (i) LacY was detected immunologically in the cytoplasmic membrane fraction of the heterologous host. (ii) Recombinant C. glutamicum cells bearing the lacY gene displayed an increased influx of o-nitrophenyl-beta-D-galactopyranoside, which was inhibited by N-ethylmaleimide. (iii) Washed cells were capable of accumulating methyl-beta-D-thiogalactoside about 60-fold. (iv) The uptake of methyl-beta-D-thiogalactoside was energy dependent and could be inhibited by the addition of 10 microM carbonyl cyanide-m-chlorophenylhydrazone. LacY of E. coli was active in the recombinant C. glutamicum cells despite the different membrane lipid compositions of these organisms.     

Lactose Variability of Escherichia coli in Thermally Stressed Reactor Effluent Waters

Lactose-utilizing and nalidixic acid-resistant populations of Escherichia coli, having an optimum growth temperature of 37°C, were placed in modified diffusion chambers. The chambers were submerged in the epilimnion and hypolimnion of a 1,100-hectare lake (Par Pond) which receives cooling water from a nuclear production reactor. Control chambers were placed in a deep-water reservoir and a Flowing-Streams Laboratory, both of which had comparable temperatures to Par Pond. The populations of E. coli were sampled regularly for up to 3 weeks. Viability of the bacteria was determined by dilution plating to nutrient agar followed by replicate plating onto selective medium to determine lactose utilization and nalidixic acid sensitivity. Initial populations of E. coli were lactose positive but changed to lactose negative in Par Pond when the reactor was operating (i.e., cooling water from the heat exchangers was being discharged to the lake). This alteration occurred most rapidly in the chambers closest to the cooling-water discharge point. Such changes did not occur in a deep-water reservoir, in Par Pond when the reactor was not operating, or in the Flowing-Streams Laboratory. The nalidixic acid-resistant characteristic remained stable regardless of the chambers' placement or reactor operations. Although the reasons for such alterations are unclear, it appears that lactose-negative populations of E. coli are selected for in these reactor effluent waters. The loss of the lactose characteristic prevents the recognition and identification of E. coli in this cooling lake (when the reactor is operating) and may prevent the assessment of water quality based on coliform recognition.     

Superoxide dismutase activity in thermally stressed Staphylococcus aureus.

The effects of heat and NaCl on the activity of superoxide dismutase from Staphylococcus aureus were examined. A linear decrease in superoxide dismutase activity occurred when S. aureus MF-31 cells were thermally stressed for 90 min at 52% C in 100 mM potassium phosphate buffer (pH 7.2). After 20 min of heating, only 5% of the superoxide dismutase activity was lost. Heating for 60, 90 and 120 min resulted in decreases of approximately 10, 22, and 68%, respectively. The rates of thermal inactivation of superoxide dismutase from S. aureus strains 196E and 210 were similar and slightly greater than those of strains MF-31, S-6, and 181. The addition of NaCl before or after heating resulted in increased losses of superoxide dismutase activity.     

Diluent sensitivity in thermally stressed cells of pseudomonas fluorescens.

Thermally injured cells of Pseudomonas fluorescens were unable to produce colonies on Trypticase soy agar (TSA) after dilution with 0.1% peptone. Nutritional exigency could not be used as the criterion for this injury, since varying the composition of the plating medium had little effect on the number of colonies that developed. The injured cells had no requirement for compounds known to leak out during the heat treatment in order to recover. The cells did not exhibit injury if dilution preceded heat treatment on the plating medium, demonstrating that the heat treatment sensitized the cells to the trauma of dilution. Substitution of 0.1% peptone with growth medium as the diluent largely offset the previously observed drop in TSA count. Little difference in survival was observed when monosodium glutamate or the balance of the defined medium was used as the diluent. The diluent effect was ionic rather than osmotic. The presence of cations was important in maintaining the integrity of the injured cell, and divalent cations enhanced this protective effect. The role of these cations at the level of the cell envelope is discussed.     

Diluent sensitivity in thermally stressed cells of pseudomonas fluorescens.

Thermally injured cells of Pseudomonas fluorescens were unable to produce colonies on Trypticase soy agar (TSA) after dilution with 0.1% peptone. Nutritional exigency could not be used as the criterion for this injury, since varying the composition of the plating medium had little effect on the number of colonies that developed. The injured cells had no requirement for compounds known to leak out during the heat treatment in order to recover. The cells did not exhibit injury if dilution preceded heat treatment on the plating medium, demonstrating that the heat treatment sensitized the cells to the trauma of dilution. Substitution of 0.1% peptone with growth medium as the diluent largely offset the previously observed drop in TSA count. Little difference in survival was observed when monosodium glutamate or the balance of the defined medium was used as the diluent. The diluent effect was ionic rather than osmotic. The presence of cations was important in maintaining the integrity of the injured cell, and divalent cations enhanced this protective effect. The role of these cations at the level of the cell envelope is discussed.     

Restoration of colony-forming activity in osmotically stressed Escherichia coli by betaine

Exposure of Escherichia coli to 0.8 M NaCl caused a rapid and large decrease in colony-forming activity. When such osmotically upshocked cells were exposed to betaine, colony-forming activity was restored. Betaine was able to restore colony-forming activity even when chloramphenicol inhibited protein synthesis. Thus, restoration was not the result of cell turnover. The cells were not killed by exposure to 0.8 M NaCl, because during exposure they accumulated ATP intracellularly. Betaine treatment caused this cellular ATP to decrease to a lower level. This work may provide the foundation for a simple plating procedure to quantitatively detect nonculturable E. coli in ocean beach recreational waters.     

Accumulation of glutamate by osmotically stressed Escherichia coli is dependent on pH.

In the present study, we measured the accumulation of glutamate after hyperosmotic shock in Escherichia coli growing in synthetic medium. The accumulation was high in the medium containing sucrose at a pH above 8 and decreased with decreases in the medium pH. The same results were obtained when the hyperosmotic shock was carried out with sodium chloride. The internal level of potassium ions in cells growing at a high pH was higher than that in cells growing in a neutral medium. A mutant deficient in transport systems for potassium ions accumulated glutamate upon hyperosmotic stress at a high pH without a significant increase in the internal level of potassium ions. When the medium osmolarity was moderate at a pH below 8, E. coli accumulated gamma-aminobutyrate and the accumulation of glutamate was low. These data suggest that E. coli uses different osmolytes for hyperosmotic adaptation at different environmental pHs.     

Lactose Permeation Via the Arabinose Transport System in Escherichia coli K-12

This paper describes the isolation and characterization of a mutant of Escherichia coli that transports lactose and its analog thiomethylgalactoside via the arabinose permeation system. Unlike transport via the lactose permease, this transport is not inhibited by thiodigalactoside, but was inhibited by arabinose, xylose, and fucose. The site of the mutation was in the arabinose C gene and confers constitutivity on the entire arabinose operon. Furthermore, this transport was found in 24 independently isolated arabinose-constitutive strains, and in strains which had been induced with arabinose and then starved to remove all traces of it. It was therefore concluded that lactose and thiomethylgalactoside are low-affinity substrates of at least one component of the normal arabinose permeation system.     

Effect of sediments on the survival of Escherichia coli in marine waters.

Escherichia coli, a fecal coliform, was found to survive for longer periods of time in unsterile natural seawater when sediment material was present than in seawater alone, and at least on one occasion growth was observed to occur. This enteric bacterium was found to increase rapidly in number in autoclaved natural seawater and autoclaved sediment taken from areas receiving domestic wastes, even when the seawater had salinities as high as 34 g/kg. However, in autoclaved seawater, growth was always more gradual and never reached numbers as high as those observed when sediment was present. It was found that nutrients were easily eluted from the sediment after autoclaving or upon addition to artificial seawater, but little elution occured during mixing of the sediments with unsterile natural seawater. The longer survival of E. coli in the sediment is attributed to the greater content of organic matter present in the sediment than the sweater. These laboratory results, in part, could explain why on a volume basis larger numbers of coliforms and fecal coliforms and fecal coliforms were found in estuarine sediments than the overlaying water at field sites.     

Survival of Vibrio cholerae and Escherichia coli in estuarine waters and sediments.

In in vitro estuarine water and sediment chambers, the survival of Vibrio cholerae and Escherichia coli was determined by plate counting and direct counting techniques. V. cholerae strains included environmental, clinical, and serotype O1 and non-O1 isolates, whereas E. coli strains included ATCC 25922 and a freshly cultured human isolate. Recovery of V. cholerae varied significantly with incubation temperature. Growth and extended periods of survival occurred in sterile sediments, sterile waters, and nonsterile waters, but not in nonsterile sediments. In contrast to V. cholerae, viable cells of E. coli decreased rapidly in both sterile and nonsterile estuarine waters. Direct counts revealed that E. coli cells were intact in the estuarine water, but attempts to resuscitate them were unsuccessful. The data suggest that V. cholerae survives better in estuarine waters than E. coli. The results may explain the recent observations that V. cholerae levels do not correlate well with fecal coliform concentrations in estuarine waters. Furthermore, the results add increasing evidence to support the theory that V. cholerae is an autochthonous bacterium in estuaries.     

Phenotypic and genotypic comparison of Escherichia coli from pristine tropical waters.

Nine fecal-coliform-positive strains were isolated from pristine sites in a tropical rain forest. These sites included nonpolluted rivers and water from bromeliads (epiphytes) which were 30 ft (ca. 910 cm) above the ground. Phenotypically, all of these isolates were identified as Escherichia coli. Their DNA was isolated and purified, and the base composition (G + C content) was determined and compared with that of E. coli B (ATCC 11303). The DNA from the environmental isolates was also hybridized to radiolabeled DNA from E. coli B. Eight strains had a DNA base composition similar to that of E. coli B and gave more than 75% homology with E. coli B. One strain had a different DNA base composition and a relatively low percentage of homology with the reference strain. The finding of E. coli in pristine tropical waters suggests that this bacterium could be a natural inhabitant in these environments and is not a reliable indicator of recent human fecal contamination in tropical waters. The indicators that are currently used in the tropics to test the biological quality of water should be reevaluated.