The Aerogenic Response of Escherichia coli and Strains of Aerobacter in EC Broth and Selected Sugar Broths at Elevated Temperatures

Gas formation by 116 strains of Escherichia coli and 104 strains of Aerobacter was determined in a specially constructed and accurately controlled water bath employing EC, lactose, maltose, sucrose, glucose, levulose, and galactose broths at temperatures ranging from 44.5 to 46.5 C.

Greatest gas activity occurred in EC broth. In the range 44.9 to 45.5 C over 92% of the E. coli cultures formed gas, but the Aerobacter strains dropped from 68 to 2%. A natural point of separation of the two groups occurred at 45.5 C.

Inhibition of the gas-forming mechanism rather than death is the universal response of the Escherichia organisms to these temperatures. The inhibition increases with rising temperatures and is readily reversible. At 46.5 C, 64.5% of all the Escherichia cultures were inhibited and 69.1% of all the cultures were actually viable.

In EC broth it was found that as a group atypical E. coli (-+--) were the most resistant gas-positive types. Least resistant in EC broth was a group of known typical fecal isolates of E. coli (++--). Of intermediate resistance between the two groups was the large body of typical E. coli (++--) organisms.

Certain individual strains of E. coli excelled in the production of gas in the variety of sugar broths tested at elevated temperatures. The Aerobacter strains did not exhibit this property.

Finally it is suggested that elevated temperature incubation studies of this type be conducted in critically controlled water baths with an ascertained accuracy in the vicinity of 45.5 ± 0.1 C under full load.

     

Inhibition of growth of Escherichia coli by lactose and other galactosides

A study has been made of the inhibition of growth caused by the addition of lactose or other galactosides to lac constitutive Escherichia coli growing in glycerol minimal medium. The effect was greater at pH 5.9 and pH 7.9 than at pH 7.0. Inhibition of growth by lactose was observed also in the case of a β-galactosidase negative mutant. However, a lacY mutant, which has a defect in the entry of protons normally coupled with galactoside transport, showed only slight inhibition of growth on the addition of galactosides. In the case of the parental strain the addition of lactose resulted in a sharp fall in ΔpH across the cell membrane and a reduction in intracellular ATP, and the recovery was slow. Under the same conditions the lacY mutant showed a smaller and only transient effect. It is postulated that the sudden entry of protons associated with lactose uptake lowers the protonmotive force, reducing the ATP levels and inhibiting growth of the cells. This hypothesis would account also for the selection of lacY mutants found when E. coli is grown in the presence of isopropyl-β-d-thiogalactoside.     

Production and characterization of Escherichia coli glycerol dehydrogenase as a tool for glycerol recycling

NAD⁺-dependent glycerol (Gro) dehydrogenase (GroDHase) catalyzes the conversion of Gro into dihydroxyacetone (DHA), the first step for fermentative Gro metabolism in Escherichia coli. In this work, we cloned the gldA gene that codes for the E. coli GroDHase and homologously expressed, purified, and kinetically characterized the recombinant protein. To achieve this, the enzyme was over-produced using Gro supplemented growth medium and lactose as the inducer. The enzyme was highly purified using either pseudo-affinity chromatography or a simple heat-shock treatment, which is potentially valuable for industrial production of GroDHase. We detected efficient oxidation of Gro derived from biodiesel production to DHA by gas chromatography. The results presented in this work support recombinant GroDHase production in a biorefinery setting as a relevant tool for converting Gro into DHA for future biotechnological applications.     

Production of β-carotene and acetate in recombinant Escherichia coli with or without mevalonate pathway at different culture temperature or pH

Natural β-carotene has received much attention as consumers have become more health conscious. Its production by various microorganisms including metabolically engineered Escherichia coli or Saccharomyces cerevisiae has been attempted. We successfully created a recombinant E. coli with an engineered whole mevalonate pathway in addition to β-carotene biosynthetic genes and evaluated the engineered cells from the aspects of metabolic balance between central metabolism and β-carotene production by comparison with conventional β-carotene producing recombinant E. coli (control) utilizing a native methylerythritol phosphate (MEP) pathway using bioreactor cultures generated at different temperatures or pHs. Better production of β-carotene was obtained in E. coli cultured at 37°C than at 25°C. A two-fold higher titer and 2.9-fold higher volumetric productivity were obtained in engineered cells compared with control cells. Notably, a marginal amount of acetate was produced in actively growing engineered cells, whereas more than 8 g/L of acetate was produced in control cells with reduced cell growth at 37°C. The data indicated that the artificial operon of the whole mevalonate pathway operated efficiently in redirecting acetyl-CoA into isopentenyl pyrophosphate (IPP), thereby improving production of β-carotene, whereas the native MEP pathway did not convert a sufficient amount of pyruvate into IPP due to endogenous feedback regulation. Engineered cells also produced lycopene with a reduced amount of β-carotene in weak alkaline cultures, consistent with the inhibition of lycopene cyclase.     

Improving the Expression of Recombinant Proteins in E. coli BL21 (DE3) under Acetate Stress: An Alkaline pH Shift Approach

Excess acetate has long been an issue for the production of recombinant proteins in E. coli cells. Recently, improvements in acetate tolerance have been achieved through the use of genetic strategies and medium supplementation with certain amino acids and pyrimidines. The aim of our study was to evaluate an alternative to improve the acetate tolerance of E. coli BL21 (DE3), a popular strain used to express recombinant proteins. In this work we reported the cultivation of BL21 (DE3) in complex media containing acetate at high concentrations. In the presence of 300 mM acetate, compared with pH 6.5, pH 7.5 improved cell growth by approximately 71%, reduced intracellular acetate by approximately 50%, and restored the expression of glutathione S-transferase (GST), green fluorescent protein (GFP) and cytochrome P450 monooxygenase (CYP). Further experiments showed that alkaline pHs up to 8.5 had little inhibition in the expression of GST, GFP and CYP. In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5–8.0. Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0±0.5 to minimize the effects caused by acetate stress. The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.     

Enhanced Conversion of Lactose to Glycerol by Kluyveromyces fragilis Utilizing Whey Permeate as a Substrate

Kluyveromyces fragilis (CBS 397) is a nonhalophilic yeast which is capable of lactose utilization from whey permeate and high glycerol production under anaerobic growth conditions. However, the optimum yields of glycerol (11.6 mg/ml of whey permeate medium) obtained in this study occurred only in the presence of 1% Na₂SO₃ as a steering agent. The use of other concentrations of Na₂SO₃, as well as 5% NaCl and 1% ascorbic acid, had no or detrimental effects on cell growth, lactose utilization, and glycerol production. Glycerol yields were greater in cultures grown from a light inoculum of K. fragilis than in cultures in which a resuspended mass of cells was introduced into the medium. The results of this study suggest that this strain of K. fragilis may be useful commercially in the utilization of cheese whey lactose and the concomitant production of glycerol.     

Cellular Growth Arrest and Persistence from Enzyme Saturation

Metabolic efficiency depends on the balance between supply and demand of metabolites, which is sensitive to environmental and physiological fluctuations, or noise, causing shortages or surpluses in the metabolic pipeline. How cells can reliably optimize biomass production in the presence of metabolic fluctuations is a fundamental question that has not been fully answered. Here we use mathematical models to predict that enzyme saturation creates distinct regimes of cellular growth, including a phase of growth arrest resulting from toxicity of the metabolic process. Noise can drive entry of single cells into growth arrest while a fast-growing majority sustains the population. We confirmed these predictions by measuring the growth dynamics of Escherichia coli utilizing lactose as a sole carbon source. The predicted heterogeneous growth emerged at high lactose concentrations, and was associated with cell death and production of antibiotic-tolerant persister cells. These results suggest how metabolic networks may balance costs and benefits, with important implications for drug tolerance.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. I) Isolation and characterization of lactate dehydrogenases from thermophilic and mesophilic bacilli.

Lactate dehydrogenases from thermophilic bacilli (Bacillus stearothermophilus, Bacillus caldotenax) and from mesophilic bacilli (Bacillus X1, Bacillus subtilis) have been isolated by a two-step purification procedure. Only one type (LDH-P4) composed of four identical subunits (Mr 34 000 or 36 000) was found in each bacillus. The tetrameric enzymes were characterized with respect to thermostability, pH and temperature dependence of the pyruvate reduction and the L-lactate oxidation, substrate specificity, saturation kinetics (Km values of pyruvate, lactate, NAD, NADH), pyruvate and oxamate inhibition, and activation by fructose bisphosphate. The thermophilic and mesophilic enzymes differ characteristically in these parameters. Preliminary structural data (amino acid composition, comparative N-terminal sequence analysis) show the expected close phylogenetic relationship (high degree of sequence homology), but also typical differences between thermophilic and mesophilic dehydrogenases, a suitable basis for further comparative studies.     

Substitution of an Alanine Residue for Glycine 146 in TMP Kinase from Escherichia coli Is Responsible for Bacterial Hypersensitivity to Bromodeoxyuridine

The wild-type TMP kinases from Escherichia coli and from a strain hypersensitive to 5-bromo-2′-deoxyuridine were characterized comparatively. The mutation at codon 146 causes the substitution of an alanine residue for glycine in the enzyme, which is accompanied by changes in the relative affinities for 5-Br-UMP and TMP compared to those of the wild-type TMP kinase. Plasmids carrying the wild-type tmk gene from Escherichia coli or Bacillus subtilis, but not the defective tmk gene, restored the resistance to bromodeoxyuridine of an E. coli mutant strain.     

Selection of lactose-adapted cells in vinca minor and Dantura innoxia cultures; location and characterization of β-galactosidase and lactase activities

Lactose-adapted cells were obtained from Datura innoxia sucrose growing calli cultures and from Vinca minor glucose growing calli cultures. Lactose adaptation process points out the homogeneity of the cell population towards lactose uptake in V. minor cultures while it reveals the presence of heterogeneous population in D. innoxia cultures.In both species, lactose hydrolysis was only occurring in the cells; no lactase activity was detected in the culture medium. An intermittent lactase activity was determined in a cell-free extract during the culture period. Lactase activity was detected in Vinca glucose grown cells as well in Datura lactose-adapted cells cultured in absence of lactose; so lactase is a constitutive enzyme. Galactose liberated during lactose hydrolysis was not toxic for thecells; it was released into the culture medium and not metabolized in Vinca cultures while it was metabolized in Datura cultures at the end of the culture period.     

Enhanced 2′-Fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate

2′-Fucosyllactose (2′-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Bacillus subtilis have been engineered to produce 2′-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2′-FL, but accumulation of by-products due to overflow metabolism of glucose hampered efficient production of 2′-FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2′-FL by engineered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to produce 2′-FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2′-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from Escherichia coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2′-FL production. The resulting engineered yeast produced 25.5 g/L of 2′-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2′-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2′-FL was secreted when a complex medium was used, 82% of the produced 2′-FL remained inside the cells when a minimal medium was used. As yeast extract is already used as food and animal feed ingredients, 2′-FL enriched yeast extract can be produced cost-effectively using the 2′-FL-accumulating yeast cells.     

Lactose utilization in a cryptic strainEscherichia coli ML 35

The permease-negative strainE. coli ML 35 utilized lactose at 30°C 5–6 times more slowly than glucose or permease-positive strains. The rate of utilization could be raised by cultivating the cells in the presence of streptomycin, acriflavine or actinomycin C. Phenethyl alcohol and the basic proteins ribonuclease, protamine or histone stimulated lactose utilization by washed cells of the same strain not precultivated in the presence of these substances. Ribonuclease did not influence lactose utilization by the permease-positive strain ML 308. Under the given experimental conditions, the presence of basic proteins did not cause β-galactosidase to be released into the medium as a result of lysis of the cells. Streptomycin and ribonuclease did not influence TMG transport into the cells. Basic proteins increased the rate of ONPG hydrolysis by intact cells almost to the level observed in the permease-positive strain. Guanidine, spermine, spermidine, putrescine and cadaverine did not influence lactose utilization by strain ML 35. Spermine antagonized the stimulant effect of ribonuclease or histone on lactose utilization. The experiments were supplemented by comparing the effect of temperature on lactose and glucose utilization by a permease-positive and-negative strain ofE. coli.     

Unformylated initiator tRNA is not a signal for the stringent control of RNA synthesis.

When Escherichia coli MRE 600 or Bacillus subtilis W 23 are grown in glucose-salt medium supplemented with purines, thymidine and glycine, trimethoprim stops the synthesis of protein by causing a specific lack of methionyl-tRNA. The synthesis of RNA is simultaneously restricted by the stringent control mechanism. Guanosine tetraphosphate (ppGpp) largely accumulates. The addition of methionine abolishes the level of ppGpp and relieves the inhibition of RNA synthesis. The aminoacylation of methionine-specific tRNAs was found to be completely restored. The methionyl-tRNA_f^Met however does not become formylated. These results indicate that unformylated initiator tRNA is not a sufficient condition for the accumulation of ppGpp and the onset of stringent control.     

Author index

The effect of the accumulation of β-galactosides on the uptake of P_i into cells and cell nucleotides was examined in ML strains of Escherichia coli. Nonmetabolizable sulfur analogs of lactose, which are accumulated only in the presence of the product of y gene of the Lac operon, inhibited the uptake of P₁ into whole cells and into cell nucleotides. This inhibition was most pronounced in starved cells, those with a low rate of ATP production. When the cell membrane was disrupted by sonication or detergents, the inhibition was lost. No significant inhibition was seen in y^? strains or in inducible y⁺ strains which were not induced. Hence. inhibition of the uptake of phosphate into nucleotides is dependent on the presence of the product of the y gene and a β-galactoside.A technique using ³²P_i and ³³P₁ was developed for simultaneously measuring the turnover and level of nucleotides. β-Galactosides inhibited ATP synthesis in aerobic cells, but stimulated ATP synthesis in anaerobic cells, indicating that an intermediate of oxidative phosphorylation was the source of energy for β-galactoside accumulation.     

Lactose biosynthesis: A sensitive radiochemical assay

A method is presented for the determination of lactose biosynthesis from labeled glucose, galactose, or other precursors based upon the addition of samples of the reaction mixture (after removal of the tissue or biosynthetic enzymes) to each of two strains of Escherichia coli. While both strains can metabolize glucose and galactose, only one is able to hydrolyze lactose. The sugars are converted by the bacteria largely to cell material and carbon dioxide. The difference between the residual, nonvolatile, soluble radioactivity in the medium from the two bacterial cultures represents the lactose unused by the strain unable to hydrolyze it.     

Role of Glucose in Enhancing the Temperature-Dependent Growth Inhibition of Escherichia coli O157:H7 ATCC 43895 by a Pseudomonas sp.

Growth of Escherichia coli O157:H7 strain ATCC 43895 was monitored at 5, 10, 15, and 25°C in both pure and mixed (1:1) cultures with a gluconate-producing Pseudomonas sp. found in meat to evaluate the effect of the absence and presence of 1% glucose in broth on temperature-dependent competition. The number of colonies of the Pseudomonas strain exceeded 9 log CFU/ml under all conditions tested. The pathogen grew better as the temperature increased from 10 to 15 and 25°C and grew better in pure culture than in mixed cultures. Pseudomonas sp. inhibited E. coli O157:H7 in cocultures with glucose at 10°C, while at 15°C the pathogen exhibited a biphasic pattern of growth with an intermediate inactivation period. Pathogen inhibition was much weaker in cocultures grown without glucose at 10 to 15°C and, irrespective of glucose, at 25°C. These results indicate that glucose enhances the growth inhibition of E. coli O157:H7 by some Pseudomonas spp., potentially due to its rapid uptake and conversion to gluconate, at low (≤15°C) temperatures.     

Role of cyclic AMP on differentiation of T- and B-lymphocytes during the immune induction.

Spleen cell cultures from genetically thymus-deficient nude mice were restored with a T-cell replacing factor obtained from normal spleen cells of Balb/c-Ig^b mice stimulated with concanavalin A. Treatment of these cultures with an inhibitory dose of cyclic AMP did not result in reduction of the number of specific antibody-forming cells after stimulation by antigen, whereas the same treatment led to inhibition in cultures restored with normal hydrocortisone-resistant thymus lymphocytes. Further experiments lead to the conclusion that the early effect of cAMP on the immune induction seen in vitro reflects inhibition of the production or secretion of a T-cell factor which is a prerequisite for triggering B-cells with a thymus-dependent antigen.     

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.     

Evaluation of Antimicrobial Activity of Bauhinia purpurea Leaves Under In Vitro Conditions

This study was undertaken with an objective of testing the antibacterial and antifungal activities of Bauhinia purpurea leaves and identifying the bioactive compounds. The antimicrobial activity of leaf extract was determined in aqueous and organic extracts and the minimum inhibitory concentration (MIC) against six species of pathogenic and non-pathogenic microorganisms: Bacillus subtilis, Staphylococcus aureus, Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa and Candida albicans using the disk diffusion method. The chemical constituents of organic plant extract were separated by thin layer chromatography and purified by column chromatography and further identified by gas chromatography–mass spectrometry (GC–MS) analysis. Significant inhibitory activity was observed with methanol extracts of plant against the test microorganisms while less antibacterial activity was observed in hexane, acetone and aqueous extracts. MIC of B. purpurea extract was ≤1,500 μg/ml against S. aureus and B. subtilis while this extract showed no inhibition against Gram-negative S. typhi, E. coli and P. aeruginosa or against fungus C. albicans. Eleven compounds were identified in B. purpurea leaf extract by GC–MS analysis. The composition of B. purpurea revealed the presence of lupeol, stigmasterol, lanosterol, ergosterol, beta-tocopherol, phytol, hexadeconic acids, hexadeconic acids methyl esters, octadecadienoic acids and octadecatrienoic acid. Stigmasterol and lupeol were the most abundant (34.48 and 15.63 %). Other phytosterols like lanosterol (4.15 %) and ergosterol (2.82 %) were also found to be present in this extract.     

Comparison of Enterococci and Coliform Microorganisms in Commercially Produced Pecan Nut Meats

Pecan nut meats in the unbroken shell are sterile for enteric microorganisms. Recovery of coliform microorganisms or enterococci from finished pecan nut meats indicated contact contamination, assuming the tempering procedures to be satisfactory. Results of specific studies, designed toward developing background data on the sanitary significance of enterococci and coliform microorganisms in the production of pecan meats are reported. Unbroken pecan nuts or nut meats from various stages of shelling operations were diluted with a phosphate-buffered diluent. Serial dilutions were inoculated into Lactose Broth and Azide Dextrose Broth. The lactose fermentors were carried through indole, methyl red, Voges-Proskauer, and citrate reactions; the positive Azide Dextrose cultures were confirmed in Ethyl Violet Azide Broth and microscopically. Viable plate counts were obtained. Enterococci were found resistant to many deterrent factors affecting coliforms. Recoveries of enterococci were detected long after pollution had occurred. Little correlation was found between enterococcal recovery and observed insanitary practices in commercial shelling operations. Using the coliaerogenes group and, specifically, Escherichia coli as a sanitation index, microorganisms allowed accurate appraisal of tempering, personnel practices, and contact surface contaminating factors. It is felt this was due, in part, to the more delicate growth characteristics of E. coli. The fact that other pathogenic microorganisms, capable of causing gastrointestinal upsets, are associated with the presence of E. coli introduces a health factor which is important to regulatory agencies concerned with consumer protection.