Factors affecting the biotransformation of trimethylammonium compounds into l-carnitine by Escherichia coli

The biotransformation of d-carnitine and crotonobetaine into l-carnitine with wild and transformed E. coli strains under batch and continuous operation was optimised. In batch, the best conditions for the transformed strain were 30% oxygen saturation, a temperature of 41 °C and a minimal medium, whereas anaerobic cultures in either complex or minimal media at 37 °C and pH 7.5 were optimal for the wild strain. Studies on the expression of the enzymes involved in trimethylammonium metabolism showed that l-carnitine dehydratase activity was always higher than that of d-carnitine racemase. Experiments with the transformed strain in continuous cell-recycle reactors showed that, despite the higher productivity that could be achieved (0.65–1.2 g/L h), plasmid-bearing cells were segregated even when a selective medium was used. This fact was also confirmed by studying the evolution of the d-carnitine racemase level. Immobilization of the transformed strain in κ-carrageenan gels allowed continuous operation for l-carnitine production with no plasmid loss. In continuous processes with cell-retention systems, the wild strain showed higher productivity and stability than the transformed strain. Moreover, crotonobetaine was a better substrate for both strains used. Recycling with hollow-fiber cartridges provided the highest biomass level even though the l-carnitine dehydratase/biomass ratio was lower. However, membrane composition and cut-off had less influence on reactor performance as similar levels of productivity were attained. In spite of this, continuous processes attained a l-carnitine production as high as 11.5 g/L h as a result of the high enzyme induction and biomass levels.     

Factors affecting the Recovery of Gamma Irradiated Escherichia coli

S ummary . Two methods of viable counting have been compared for unirradiated and γ irradiated Escherichia coli NCTC 5933. The spread plate method has been shown to be more reproducible than a serial dilution method in a liquid medium, but the survivor-dose curves of γ irradiated bacteria obtained by the two methods were identical. The spread plate method was used to compare the survival of γ irradiated E. coli on peptone agar and on a synthetic glucose-salts agar over a range of survivor levels of c. 9 log cycles. The log % survivor-dose curve was linear for the synthetic medium but had a double exponential shape for the peptone agar. Recovery was much higher on peptone than on a synthetic medium. Using a tube dilution method in a liquid glucose-salts medium, 19 l -amino acids were tested for their effect on recovery. The slight increases in number of survivors attributable to some amino acids were associated with a change in the first part (100–1% survivors) of the curve rather than an alteration in final slope. The phosphate concentration of the solid synthetic medium markedly affected its ability to recover γ irradiated cells.     

Factors affecting the level of alanine racemase in Escherichia coli

Alanine racemase occupies a key position in the alanine branch of peptidoglycan biosynthesis. The level of this enzyme in Escherichia coli W is a function of the carbon source. For example, growth on l-alanine causes a 25-fold higher level of alanine racemase when compared with growth on glucose. When potential inducers of this enzyme are added to either a glucose or succinate medium, a low specificity is observed with those compounds that cause higher levels of enzyme. Growth of E. coli W on either pyruvate, d-alanine, or l-alanine resulted in lower levels of l- and d-alanine in the internal pool. With each of these carbon sources, the level of alanine racemase was markedly elevated when compared to glucose-grown cells; thus, with single carbon sources, the concentration of alanine in the pool is inversely related to the specific activity of alanine racemase. These observations support derepression as a possible mechanism that gives rise to higher levels of alanine racemase. Since multiple forms of the alanine racemase were not detected in extracts from E. coli W grown on various carbon sources, it would appear that this type of heterogeneity is not a consideration in interpreting the above results.     

Factors affecting the biosynthesis of L-tryptophan by genetically modified strains of Escherichia coli

Derivatives of Escherichia coli strain W3110 with increased tryptophan synthase (TS) activity were constructed. The biosynthesis of serine was shown to limit tryptophan production in minimal medium with indole as precursor. In the presence of serine and indole we obtained a correlation between the specific activity of TS and the specific productivity (qp) of tryptophan. Supplementation of the growth medium with glycine enhanced qp two-fold. In a strain with high serine hydroxymethyltransferase (SHMT) activity no such increase of tryptophan productivity was observed, although crude extracts from these cells were shown to produce tryptophan with indole, one-carbon units and glycine as precursors. Growth of the strain with high SHMT activity was inhibited in a medium with high glycine concentration. This inhibition could not be released by addition of isoleucine and valine. In a buffer system with permeabilized cells high in specific TS and SHMT activities we did not obtain any tryptophan production in presence of indole, glycine, one-carbon units and cofactors. On the other hand, in a buffer system with indole and serine as precursors we obtained high qp of tryptophan [13.3 g tryptophan (g dry wt cells)-1 h-1], which was correlated to the TS specific activity.     

Factors affecting the fermentative production of a lysozyme-binding antibody fragment in Escherichia coli

Fed-batch fermentation for production of a single-chain Fv antibody fragment (scFv) expressed as a recombinant periplastic protein from Escherichia coli was investigated. A high cell density of 50 g dry cell weight per liter was routinely achieved in a 14-L vessel by controlled exponential feeding of glucose to impose a constant specific growth rate. Following biomass accumulation, induction of the tac promoter by addition of IPTG was accompaied by a linear feed of yeast extract. The concentration of yeast extract feed was found to be highly influential upon both concentration and location of active product. Although scFv fragments were specifically targeted to the periplasmic space, at yeast extract feed rates of 0.72 g/h the final location was largely extracellular (68% to 79%). Total concentrations (extracellular + periplasmic) were of the order of 5 to 8 mg/L. A ten-fold increase in yeast extract supply increased total scFv concentration to almost 200 mg/L and 78% of this yield was retained in the periplasm. Control of such leakage of the recombinant product is fundamental to process design of downstream operations for product recovery. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 611-622, 1997.     

Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli

Based on the results of a systematic study of factors affecting plasmid yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular DNA from Streptomyces lividans and Escherichia coli was developed. The method consists of lysis of lysozyme-treated bacteria combined with alkaline denaturation of DNA at high temperature. Renaturation of CCC DNA and precipitation of single-stranded DNA together with protein is achieved by the addition of a minimal amount of phenol/chloroform. The screening procedure uses only a single tube and the samples can be analyzed by agarose gel electrophoresis about 30 min after lysis. Removal of phenol and further purification of the plasmid preparation is achieved by consecutive precipitations with isopropanol and spermine, followed by extraction with ethanol, producing samples suitable for restriction endonuclease digestion, ligation, and transformation of S. lividans protoplasts or competent E. coli cells in about 2 h. All steps of the procedure are explained in detail with information about the effects of changing parameters. This should help the experimenter to obtain reproducible results and may be useful if the method has to be adapted to new strains or plasmids.     

Factors affecting the heat resistance of Escherichia coli O157 : H7

Escherichia coli O157 : H7 has been reported as being not particularly heat resistant. However, several factors which might increase its heat resistance have been investigated in this study using five strains. Increase in growth temperature to 40 °C, as found in the cow gut, heat-shock at sub-lethal temperatures of 42, 45, 48 and 50 °C, and variable heating rate (1 °C min⁻¹ to 23 °C min⁻¹) had no dramatic effect on heat resistance. Growth phase had a marked impact on heat resistance ; late stationary phase cells were more heat-resistant than were log phase cells. The difference in heat resistance between the two phases of growth became more pronounced when cells were resuspended in fresh nutrient broth ; heat resistance of late stationary phase cells increased dramatically whereas no such effect was observed with log phase cells. The addition of polyphosphates to the heating medium did not increase heat resistance. A reduction in water activity of the heating medium from 0·995 to levels between 0·980 and 0·960 also resulted in a marked increase in heat resistance. This effect was more pronounced under conditions of extremely low water activity created by resuspending late stationary phase cells in sunflower oil. Survivors were detected even after a heat treatment at 60 °C for 1 h or 70 °C for 5 min. It can be confirmed that this serotype has no unusual heat resistance and that the heating environment markedly affects resistance.     

Factors affecting the intracellular parasitic growth of Bdellovibrio bacteriovorus developing within Escherichia coli

A procedure for one-step growth experiments on Bdellovibrio bacteriovorus growing parasitically in Escherichia coli B was developed. The resulting one-step growth curves showed that, under defined conditions at 30 C, each singly infected E. coli host cell, on the average, gave rise to 5.7 Bdellovibrio cells. This value was confirmed by single-burst experiments and by microscopic observations. In the temperature range of 25 to 38 C, the average burst size and the duration of the latent period were inversely proportional to the temperature. The effect of hydrogen ion concentration on the one-step growth kinetics in this system indicated a broad pH optimum, ranging from neutrality to slightly alkaline pH values. After Bdellovibrio cells and host cells were mixed, there was always a delay (the so-called "lag phase") before the parasite titer increased in terms of plaque-forming units. Phase-contrast microscopic observations indicated that this delay stems in part from the polyphasic nature of the Bdellovibrio life cycle. We propose the following five terms to make explicit the sequence of events in this life cycle: "attachment," "penetration," "elongation," "fragmentation," and "burst." Nutritional experiments revealed that Bdellovibrio obtains a major fraction of its cellular components from host-cell material. Infection of E. coli by Bdellovibrio without added Mg(++) or Ca(++) (0.003 m Mg(++), 0.002 m Ca(++)) resulted in partial or total lysis of the host cell soon after infection. Protoplast integrity was necessary for the normal completion of the intracellular growth phase of Bdellovibrio in E. coli; normal development of the parasite took place only in the presence of Mg(++) or Ca(++).     

Factors affecting the synthesis of penicillins by the immobilized penicillin acylase from Escherichia coli

The effect of pH, temperature, reactant concentration, and reaction time has been investigated for the synthesis of N-benzhydryl-N′-acetamidopiperazyl-6-penicillanic acid and N-benzyl-N′-acetamidopiperazyl-6-penicillanic acid from 6-aminopenicillanic acid by the immobilized penicillin acylase from Escherichia coli. The synthesis of penicillins from carboxylic acids proceeds most rapidly at pH 5; with ethyl ester derivatives of carboxylic acids the pH optimum is higher (6–7). The most rapid synthesis of penicillins was obtained with ethyl ester derivatives of carboxylic acids. The optimum temperatures were 25–35°C.     

Incomplete entry of bacteriophage T7 DNA into F plasmid-containing Escherichia coli.

The penetration of bacteriophage T7 DNA into F plasmid-containing Escherichia coli cells was determined by measuring Dam methylation of the entering genome. T7 strains that cannot productively infect F-containing cells fail to completely translocate their DNA into the cell before the infection aborts. The entry of the first 44% of the genome occurs normally in an F-containing cell, but the entry of the remainder is aberrant. Bypassing the normal mode of entry of the T7 genome by transfecting naked DNA into competent cells fails to suppress F exclusion of phage development. However, overexpression of various nontoxic T7 1.2 alleles from a high-copy-number plasmid or expression of T3 1.2 from a T7 genome allows phage growth in the presence of F.     

Factors affecting the formation of alkaline phosphatase isozymes in Escherichia coli K-12.

Physiological and genetic factors affecting the formation of isozymes of alkaline phosphatase in Escherichia coli K-12 were studied. Our results are compatible with the hypothesis proposed by Schlesinger and his co-workers (Schlesinger et al., 1975) that the multiple forms of the enzyme are produced by converting a newly synthesized one (the least negatively charged one) into less negatively charged forms. Neither energy source nor de novo synthesis of the enzyme was necessary for the conversion. It is also confirmed that the conversion is effectively inhibited by externally added arginine (Piggott et al., 1972) but only partially by canavanine (arginine analog) or casamino acids. We isolated a mutant strain which did not form isozyme(s), if any, under the condition in which the wild type strain formed isozymes. The mutation(s) was proved to be mapped in the locus (or loci) other than alkaline phosphatase structure gene in the E. coli genetic map. We tentatively proposed to designate this the iap gene(s), an abbreviation for isozyme of alkaline phosphatase, which plays a role in isozyme formation.     

Factors affecting the survival of Salmonella and Escherichia coli in anaerobically fermented pig waste

The survival of Salmonella typhimurium was investigated in acidogenic, anaerobically fermented pig wastes and in synthetic media, each containing volatile fatty acids (VFA). Salm. typhimurium survived at pH 6·8, but not at pH 4·0, when incubated at 37°C for 24 h in either fermented or synthetic medium containing VFA. The minimum inhibiting concentration of VFA for Salm. typhimurium after 48 h incubation at 30°C at pH 4·0 was 0·03 mol/l and for Escherichia coli it was 0·09 mol/l. Fermented pig wastes in a digester, maintained at pH 5·9, were inoculated with Salm. typhimurium and then incubated at 37°C for 24 h. The pH was adjusted to either 4·0 or 5·0 and after a further 48 h at 30°C, Salm. typhimurium survived at pH 5·0 but not at pH 4·0. It was concluded that pH is critical in determining the survival of this organism in acidogenic anaerobically fermented pig waste.     

Factors affecting the survival of Salmonella and Escherichia coli in anaerobically fermented pig waste

The survival of Salmonella typhimurium was investigated in acidogenic, anaerobically fermented pig wastes and in synthetic media, each containing volatile fatty acids (VFA). Salm. typhimurium survived at pH 6.8, but not at pH 4.0, when incubated at 37 degrees C for 24 h in either fermented or synthetic medium containing VFA. The minimum inhibiting concentration of VFA for Salm. typhimurium after 48 h incubation at 30 degrees C at pH 4.0 was 0.03 mol/l and for Escherichia coli it was 0.09 mol/l. Fermented pig wastes in a digester, maintained at pH 5.9, were inoculated with Salm. typhimurium and then incubated at 37 degrees C for 24 h. The pH was adjusted to either 4.0 or 5.0 and after a further 48 h at 30 degrees C, Salm. typhimurium survived at pH 5.0 but not at pH 4.0. It was concluded that pH is critical in determining the survival of this organism in acidogenic anaerobically fermented pig waste.     

Adhesion and entry of uropathogenic Escherichia coli

To effectively colonize a host animal and cause disease, many bacterial pathogens have evolved the mechanisms needed to invade and persist within host cells and tissues. Recently it was discovered that uropathogenic Escherichia coli, the primary causative agent of urinary tract infections, can invade and replicate within uroepithelial cells. This can provide E. coli with a survival advantage, allowing the microbes to better resist detection and clearance by both innate and adaptive immune defence mechanisms. Adhesive organelles, including type 1, P, and S pili along with Dr adhesins, promote both bacterial attachment to and invasion of host tissues within the urinary tract. Interactions mediated by these adhesins can also stimulate a number of host responses that can directly influence the outcome of a urinary tract infection.