The role of regulatory gene products in alkali sensitization by extracellular medium components in Escherichia coli

Organisms of Escherichia coli 1829 become alkali sensitized on transfer from pH 7·0 to pH 5·5 but they also secrete extracellular agents which induce alkali sensitivity when added (in neutralized filtrates) to organisms growing at pH 7·0. In contrast, filtrates from cultures grown at pH 7·0 have no effect. Filtrates were inactivated by protease but not by heat treatment in a boiling water-bath, suggesting that a very heat-stable protein is involved in alkali sensitivity induction. A heatstable low molecular weight component (or components) may also be needed for induction, or the induction protein itself may be of low molecular weight. Strains with lesions in hns, fur or himA produced almost inactive filtrates and it therefore appears that H-NS, Fur and IHF are involved in synthesis of the induction components. As the presence of protease during incubation at pH 5·5 totally abolished alkali sensitization of strain 1829 while inhibition of sensitization induction occurred if the induction components were removed by filtration or dialysis during pH 5·5 incubation, it is proposed that the extracellular induction components (EICs) are essential for the original sensitization response. These results suggest that sensitization induction occurs by a different mechanism to that which is believed to occur for most bacterial inducible response systems; these are claimed to involve exclusively intracellular reactions and components whereas the present response involves functioning of extracellular components.     

Glucose-induced acid tolerance appearing at neutral pH in log-phase Escherichia coli and its reversal by cyclic AMP

Escherichia coli shifted from broth at external pH (pH₀) 7·0 to pH₀ 7·0 broth plus glucose rapidly induced marked acid tolerance which also appeared, albeit to a lesser extent, plus maltose, sucrose or lactose. Tolerance appeared without the medium pH becoming acidic. Tolerance was most substantial when glucose was added at pH₀ 7·0 but was also appreciable at pH₀ 7·5, 8·0 and 8·5. Induction of tolerance by glucose was markedly reduced by cyclic AMP and essentially abolished plus NaCl or sucrose ; the induction process was also reduced but not fully inhibited by chloramphenicol, tetracycline and nalidixic acid. Glucose-induced organisms showed less acid damage to DNA and β-galactosidase and it is likely that this is because glucose induces a new pH homeostatic mechanism which keeps internal pH close to neutrality at acidic pH₀. In conclusion, it is clear that glucose induces a novel acid tolerance response in log-phase E. coli at pH₀ 7·0 ; it is now known that induction of this response involves the functioning of extracellular induction components including an extracellular induction protein.     

Novel acid sensitivity induced in Escherichia coli at alkaline pH

Transfer of pH 7.0-grown Escherichia coli to pH 9.0 led to rapid acid sensitivity induction (ASI), the response being fully accomplished within 15 min at 37°C in broth. Only a slight increase in acid sensitivity occurred at pH 8.2 but the response was substantial at pH 8.4 and complete at pH 9.0 with no further sensitization at pH 9.5–10.5. ASI was not prevented by lesions in rpoH, katF, ompR, relA, spoT, fur, phoU, phoM (CreC), phoB/R, unc(atp), phoP or cadA and was unaffected by nalidixic acid, L-leucine or iron starvation or excess. Full acid sensitivity was maintained for at least 2 h after a shift from pH 9.0 back to pH 7.0. ASI did not depend to a major extent on PhoE derepression and increased acid sensitivity of alkali-induced strain C75a ( phoE⁺ ) probably did not involve use of a new outer membrane proton pore.     

Induction of the PhoE porin by NaCI as the basis for salt-induced acid sensitivity in Escherichia coli

Z. LAZIM, T.J. HUMPHREY AND R.J. ROWBURY. 1996. Organisms grown in low salt broth (LSB) are acid resistant but become sensitive on growth for 30-60 min with 300 mmol 1⁻¹ added NaCl. Salt-induced acid sensitivity only occurs in relA⁺ strains and sensitization is abolished by glucose, this catabolite repression effect being reversed by cAMP. The finding that sensitization did not occur in a phoE strain but did occur in a phoE⁺ derivative of it suggested that the response might result from PhoE induction, since PhoE acts as the major outer membrane (OM) proton pore under most conditions. In agreement with this, low-salt broth (LSB)-grown cells of a chromosomally lac⁻ strain carrying pJP102 ( phoE-lacZ ) produced low levels of β-galactosidase but growth with added NaCl led to rapid and appreciable induction. Also, a phoA mutant carrying a phoE-phoA fusion produced little alkaline phosphatase after growth in LSB but much more in LSB with added NaCl. Increased β-galactosidase synthesis (in phoE-lacZ strains) in the presence of NaCl was abolished by glucose, this effect being reversible by cAMP, and there was more NaCl-induced synthesis of this enzyme in relA⁺ strains.
Accordingly, it appears that addition of NaCl to LSB leads to acid sensitivity because it induces synthesis of the OM proton pore PhoE.     

Sodium chloride induces an NhaA/NhaR-independent acid sensitivity at neutral external pH in Escherichia coli.

Escherichia coli previously grown in low-salt broth, pH 7.0, produced organisms which were markedly more acid sensitive when subsequently cultured in the same broth with 200 mM or more salt (NaCl) added. Induction of acid sensitivity occurred rapidly at both 37 and 30 degrees C, with a substantial effect within 15 min. Sensitization was partially inhibited by chloramphenicol and tetracycline and may depend on both protein synthesis-dependent and -independent physiological changes in the NaCl-induced organisms; sensitization did not result from osmotic shocking on transfer to challenge medium. Induction of acid sensitivity was affected by neither the sodium ion pore inhibitor amiloride nor the DNA synthesis inhibitor nalidixic acid; rifampin had a small effect, similar to that of chloramphenicol. Chlorides of other monovalent cations, especially Li+ and NH4+, also produced sensitization to acid, although CsCl was ineffective but did not interfere with sensitization by NaCl. Other sodium salts were also active as sensitizers, as were chlorides of divalent cations, but although sucrose (but not glycerol) was a good inducer, the results were not fully in accord with triggering of induction solely by the NaCl-associated increase in osmotic pressure. Sensitization was not prevented by deletion of the nhaA, nhaR, or nhaB gene. Acid sensitivity of NaCl-induced cells was slightly reduced after 90 min of growth at 37 degrees C in low-salt broth but was completely lost after 240 min. For NaCl-induced cells, acid killing in challenge media was not inhibited by amiloride. The NaCl-induced sensitization is distinct from the phenomenon of acid sensitivity induction in E. coli at alkaline external pH.     

Effects of pH on expression and stabilization of β-galactosidase by recombinantE. coli with a thermally-inducible expression system

Summary Effects of pH on -galactosidase expression and stabilization were investigated using recombinantE. coli harboring an expression vector with a thermally-inducible P_L promoter. Expression of -galactosidase was strongly promoted by lowering culture pH when culture temperature was raised to the induction temperature. Optimal pH for induction ranged from 5.4–5.8. The degradation of expressed -galactosidase could be reduced by lowering the culture temperature while at the same time slightly increasing the culture pH in the -gal degradation stage.     

Characterization of Thermoanaerobacter Glucose Isomerase in Relation to Saccharidase Synthesis and Development of Single-Step Processes for Sweetener Production

Regulation of glucose isomerase synthesis was studied in Thermoanaerobacter strain B6A, which fermented a wide variety of carbohydrates including glucose, xylose, lactose, starch, and xylan. Glucogenic amylase activities and β-galactosidase were produced constitutively, whereas the synthesis of glucose isomerase was induced by either xylose or xylan. Production of these saccharidase activities was not significantly repressed by the presence of glucose or 2-deoxyglucose in the growth media. Glucose isomerase production was optimized by controlling the culture pH at 5.5 during xylose fermentation. The apparent temperature and pH optima for these cell-bound saccharidase activities were as follows: glucose isomerase, 80°C, pH 7.0 to 7.5; glucogenic amylase, 70°C, pH 5.0 to 5.5; and β-galactosidase, 60°C, pH 6.0 to 6.5 Glucose isomerase, glucogenic amylase, and β-galactosidase were produced in xylose-grown cells that were active and stable at 60 to 70°C and pH 6.0 to 6.5. Under single-step process conditions, these saccharidase activities in whole cells or cell extracts converted starch or lactose directly into fructose mixtures. A total of 96% of initial liquefied starch was converted into a 49:51 mixture of glucose and fructose, whereas 85% of initial lactose was converted into a 40:31:29 mixture of galactose, glucose, and fructose.     

Purification and characterization of a sodium-stimulated β-galactosidase from Bifidobacterium longum CCRC 15708

Summary β-galactosidase from Bifidobacterium longum CCRC 15708 was first extracted by ultrasonication then purified by Q Fast-Flow chromatography and gel chromatography on a Superose 6 HR column. These steps resulted in a purification of 15.7-fold, a yield of 29.3%, and a specific activity of 168.6 U mg⁻¹ protein. The molecular weight was 357 kDa as determined from Native-PAGE. Using o-nitrophenyl-β-d-galactopyranoside (ONPG) as a substrate, the pH and temperature optima of the purified β-galactosidase were 7.0 and 50 °C, respectively. The enzyme was stable at a temperature up to 40 °C and at pH values of 6.5–7.0. K _m and V _max for this purified enzyme were noted to be 0.85 mM and 70.67 U/mg, respectively. Na⁺ and K⁺ stimulated the enzyme up to 10-fold, while Fe³⁺, Fe²⁺, Co²⁺, Cu²⁺, Ca²⁺, Zn²⁺, Mn²⁺ and Mg²⁺ inhibited the activity of β-galactosidase. Furthermore, although glucose, galactose, maltose, or raffinose exerted little or no effect on the β-galactosidase activity, lactose and fructose inhibited the enzyme activity. The effect of lactose on the enzyme activity for ONPG is probably a case of competitive inhibition. A relatively high specific activity of β-galactosidase from B. longum CCRC 15708 could be obtained by Q Fast-Flow chromatography and gel chromatography on a Superose 6 HR column. In some aspects, particularly the activation by monovalent cations, the properties of β-galactosidase of B. longum CCRC 15708 are different from those obtained from other sources. Data collected in the present study are of value and indispensable when β-galactosidase from B. longum CCRC 15708 is employed in practical application.     

Heterologous expression of a gene encoding a thermostable β-galactosidase from <Emphasis Type="Italic">Alicyclobacillus acidocaldarius</Emphasis>

A genomic DNA library screen yielded the nucleotide sequence of a 12 kb fragment containing a gene (2067 bp) coding a thermostable β-galactosidase from Alicyclobacillus acidocaldarius ATCC 27009. The β-galactosidase gene was expressed in Pichia pastoris, and up to 90 mg recombinant β-galactosidase/l accumulated in shake flask cultures. Using o-nitrophenyl-β-d-galactopyranoside as a substrate, the optimum pH and temperature of the purified recombinant β-galactosidase were 5.8–6.0 and 70°C, respectively. The enzyme retained 90% of its activity when heated at 70°C for 30 min. Approximately 48% of lactose in milk was hydrolyzed following treatment with the recombinant enzyme over 60 min at 65°C.     

Effect of Radiation Environment on the Thermal Resistance of Irradiated Spores of Bacillus subtilis

The degree of heat sensitization induced in spores of Bacillus subtilis by irradiation with gamma rays was not changed when irradiation was carried out at low oxygen tension (1 mm of Hg) as compared with irradiation in an air atmosphere. The degree of heat sensitization appeared to be slightly greater at pH 7.0 than at pH 4.5. The substrate was found to influence the degree of induced heat sensitivity with less of an effect produced in complex organic substrates.     

Acid sensitivity induction (ASI) at alkaline pH in Escherichia coli involves two major sensitization components, induction of both being switched on by increased internal pH

Acid sensitivity induction (ASI) at alkaline pH in Escherichia coli 1157 ( phoE ) is only fully mounted if protein synthesis occurs for the first 5 min of the 12–15 min induction period, but appreciable sensitization occurs in the presence of chloramphenicol indicating that there are protein synthesis-independent and -dependent components (components 1 and 2). Component 1 sensitization is in place after 10 min at pH_o 9.0 but the dependent process is induced slightly more slowly. Collapsing ΔpH at pH_o 9.0 did not prevent full ASI, indicating that component 1 and 2 induction does not depend on increased ΔpH. The two induction (or activation) processes responded differently to intermediate levels of external alkalinization; component 1 was induced at pH_o 8.3 but component 2 needed pH_o 8.4 or greater. Collapsing ΔpH at pH_o 8.0 led to full induction of component 1 and appreciable induction of component 2, indicating that both processes are triggered if pH_i rises to 8.0 or greater (presumably at pH_o 8.3–8.4). Component 1 appears less important for ASI in 1157-4 ( phoE ⁺) than in 1157 ( phoE ).     

Effect of seawater on Escherichia coli β-galactosidase activity

An investigation of β-galactosidase activity of Escherichia coli strain H10407, under different physiological and environmental conditions, e.g. induced and uninduced osmotic stress, light, etc., was undertaken. In this study E. coli was employed as a model for faecal coliforms in waste water. β-Galactosidase activity was induced by isopropyl-β-D-thiogalactoside (IPTG). Enzyme activity (U cell^-1)/cell for sewage bacteria and for induced E. coli was similar, i.e. log U cell^-1= -8.5 whereas uninduced E. coli yielded log U cell^-1= -12.1. Initial enzyme activity was not dependent on phase of growth of the cell (exponential vs stationary phase) or whether marine or fresh water at the time of initial dilution. However, osmotic change resulted in a decrease in culturable cells, even though enzyme activity remained constant. A significant decrease in the number of culturable bacteria, followed by a decrease in β-galactosidase activity, was observed after exposure of cells to visible light radiation. It is concluded that β-galactosidase enzyme is retained in viable but non-culturable E. coli. Furthermore, β-galactosidase appears to offer a useful and rapid (25 min) measure of the viability of faecal coliforms, and therefore, of the water quality of bathing and shellfishing areas.     

Utilization of protein-hydrolyzed cheese whey for production of β-galactosidase by Kluyveromyces marxianus

We studied the utilization of protein-hydrolyzed sweet cheese whey as a medium for the production of β-galactosidase by the yeasts Kluyveromyces marxianus CBS 712 and CBS 6556. The conditions for growth were determined in shake cultures. The best growth occurred at pH 5.5 and 37°C. Strain CBS 6556 grew in cheese whey in natura, while strain CBS 712 needed cheese whey supplemented with yeast extract. Each yeast was grown in a bioreactor under these conditions. The strains produced equivalent amounts of β-galactosidase. To optimize the process, strain CBS 6556 was grown in concentrated cheese whey, resulting in a higher β-galactosidase production. The β-galactosidase produced by strain CBS 6556 produced maximum activity at 37°C, and had low stability at room temperature (30°C) as well as at a storage temperature of 4°C. At −4°C and −18°C, the enzyme maintained its activity for over 9 weeks. Received 20 January 1999/ Accepted in revised form 30 April 1999     

A pH-regulated promoter for the expression of recombinant proteins inEscherichia coli

Summary A series of plasmid vectors have been developed which allows control of recombinant gene expression by altering the pH of the growth medium. Expression is controlled by the regulatory region of thecadA gene ofE. coli. Experiments using -galactosidase as the expressed gene have resulted in an induced expression of up to 60-fold when the pH of the growth medium is lowered from pH 7 to 5.5. Expression can also be induced by switching from aerobic to anaerobic growth environment. The pH and anaerobic effects are additive boosting the expression level of -galactosidase to a dramatic value of 200 fold. Finally, the pH-induction effect is fully reversible, a unique property which allows continuous control of gene expression using exiting pH monitoring and control equipment.     

Effects of Carbon Source and Vitreoscilla Hemoglobin (VHb) on the Production of β-Galactosidase in Enterobacter aerogenes

At fixed concentration (0.5%), lactose and galactose acted as inducers while glucose and other tested carbon sugars showed repression effects on β-galactosidase production in Enterobacter aerogenes strain. The expression of Vitreoscilla hemoglobin gene (vgb) in this bacterial strain managed to overcome the repression effects as well as improving the induction of β-galactosidase formation by carbon sources. In parallel, the bacterial O₂ consumption was increased correspondingly to the vgb induction of β-galactosidase synthesis. When Enterobacter aerogenes strains were grown at the incubation temperature 42°C, about 5-fold higher enzyme productivity was obtained than with a similar incubation at 37°C. The bacterial growth expressed as biomass yield had a different optimum temperature and was not influenced to the same extent by variations in the carbon sources. These data are discussed in terms of proposed enhancement in β-galactosidase productivity by vgb expression as well as its significance to improve the technology of whey processing.