Escherichia coli variants for gas and indole production at elevated incubation temperatures.

Two strains of Escherichia coli were subjected to heat and cold-storage treatments to determine the stability of the fecal E. coli characteristics of gas production from lactose and indole production at elevated incubation temperatures. No variants were detected with repeated sublethal heat treatment. A high incidence of variants was observed with extended cold storage of the organisms in liquid and semisolid media, especially with poor nutrient composition, and in the absence of cryoprotective agents. The indole characteristic at elevated temperature was more stable than the production of gas from lactose. The critical temperature at which both gas production from lactose and the indole characteristic were lost was 44.5 degrees C. It appeared that the variants resulted from increased temperature sensitivity of the formic hydrogen lyase and tryptophanase enzymes, respectively.     

Confirmation of Escherichia coli and its distinction from Klebsiella species by gas and indole formation at 44 and 44.5 degrees C

AIMS: In the enumeration of coliform bacteria, confirmation of Escherichia coli has been based upon gas and indole production at the elevated incubation temperature. The test for gas production has recently been questioned. The aim of this study was to investigate the impact of gas production test on the reliability of confirmation of E. coli. METHODS AND RESULTS: The impact of several media on growth, gas and/or indole formation was tested at 44 and 44.5 degrees C using 547 environmental isolates. These were mainly E. coli, Klebsiella pneumoniae, K. oxytoca and Enterobacter cloacae strains. Another set of 250 faecal and environmental klebsiellae were tested for their maximum temperature for growth (Tmax) and for gas formation. Escherichia coli and even K. pneumoniae grew well in all the media, but gas production was more dependent on the medium used. Growth of the mainly gas negative Ent. cloacae and K. oxytoca strains was still more sensitive to the medium and incubation conditions. Tryptophan salt broth was the most productive medium for the indole test, followed by lauryl tryptose mannitole and tryptone mannitol ricinoleate broth (TRM). Tmax of K. oxytoca was clearly lower than Tmax of K. pneumoniae but a rather high fraction of its isolates produced indole at 44.5 degrees C. CONCLUSIONS: False-positive E. coli confirmation is possible if gas production is not tested for and the confirmation is based on indole test only. SIGNIFICANCE AND IMPACT OF THE STUDY: Erroneous positive results on routine analysis for E. coli can occur.     

Influence of ambient air temperature on the cooling/heating load of a single cell protein jacketed fermenter operating on cheese whey under continuous conditions

The heat generated by mixing and lactose metabolism, during the continuous production of single cell protein from cheese whey lactose using a jacketed fermenter with running cooling water, was calculated using a heat balance equation. The technique quantified the heat produced in and lost from the fermentation unit. Most of the heat generated by mixing in the cell-free system (97.47%) was lost with exhaust gas, while a very small amount (2.53%) was lost through the fermenter lid, wall, and bottom. The heat generated by mixing was significant (26.31% of the total heat generated in the fermentation system with an active yeast population present) and, therefore, cannot be ignored in heat balance calculations. About 19.71% of the total heat generated in the reactor was lost through the coolant at an ambient temperature of 22 +/- 0.5 degrees C, showing the need for a cooling system. A yeast population size of 986 million cells/mL and a lactose removal efficiency of 95.6% were observed. About 72.5% and 27.5% of the lactose consumed were used for growth and respiration, respectively. A yield of 0.66 g of cells/g of lactose was achieved. The heat released by unit biomass was 7.05 kJ/g of cells. The results showed the significant impact of ambient air temperature on the cooling load. The heat to be removed from the medium by the cooling system varied from 3.46 to 281.56 kJ/h when the temperature increased from 16 to 30 degrees C. A heating system is needed to maintain the medium temperature at 34 degrees C when the ambient air temperature is below 16 degrees C.     

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 degrees 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.     

A Membrane Filtration Technique for the Enumeration of Escherichia coli in Seawater

S ummary . Membrane filtration has become an accepted method for enumerating Escherichia coli in water, but little published evidence could be found to judge the specificity of the method to assess faecal contamination in either fresh or saline waters. The method is used in our laboratory to monitor the extent and degree of sewage pollution in coastal areas, but there is need for information on what proportion of lactose-fermenting colonies from seawater, developing at 44° on a 4% enriched Teepol medium, are E. coli type I. A total of 1352 colonies from seawater was tested for production of indole and for gas from lactose at 44°. In addition, 46% of the colonies were screened by the IMVEC series of tests. The proportion of colonies tested ranged from 10–100%, depending on the number of colonies on the membrane. Many of the colonies (81.9%) to which IMVEC tests were applied were E. coli type I; a further 10.9% were Irregular type I. The practical implications of these findings are discussed.     

THE RAPID IDENTIFICATION OF ESCHERICHIA COLI I BY THE PRODUCTION AT 44° OF BOTH INDOLE AND GAS FROM LACTOSE

SUMMARY: The modification of the 44° test proposed by Mackenzie, Taylor & Gilbert (1948) is useful for the rapid identification of Escherichia coli I in water and foods. False positive tests caused by other coli-aerogenes bacteria, or by their association with other organisms, can be considered rare. Only a few E. coli I fail to produce indole or ferment lactose at 44°, and further confirmation for routine purposes is necessary only when the results at 44° are discordant, e.g. indole positive-lactose negative or indole negative-lactose positive.     

Elevated serine catabolism is associated with the heat shock response in Escherichia coli.

The biochemical events associated with the heat shock response are not well understood in any organism, nor have the signals that initiate the induction of heat shock protein synthesis been identified. In this work, we demonstrate that the rate of serine catabolism of Escherichia coli cells grown in glucose minimal medium supplemented with serine is elevated three- to sevenfold when the growth temperature is shifted from 37 to 44 degrees C. Elevations in growth temperature and mutations or treatments that lead to elevated basal rates of serine catabolism at 37 degrees C result in the excretion into the culture medium of acetate derived from exogenous serine. Increases in the basal level of serine catabolism at 37 degrees C do not per se induce a heat shock response but are associated with abnormalities in the pattern of induction of heat shock polypeptides following a temperature shift. We postulate that the events responsible for or resulting from the elevation in serine catabolism associated with a shift-up in temperature modulate the induction of 3 of the 17 heat shock polypeptides identified in E. coli. These observations suggest that heat shock diverts serine away from the production of glycine and C1 units, which are required for initiation of protein synthesis and for nucleotide biosynthesis, and towards acetyl coenzyme A and acetate.     

A Rapid and Direct Plate Method for Enumerating Escherichia coli biotype I in Food

Direct enumeration of Escherichia coli biotype 1 in foods within 24 h has been achieved by a development of the method of Delaney, McCarthy & Grasso (1962) which is based on the production of indole at 44°. Indole positive E. coli growing at this temperature on a cellulose acetate membrane overlaying tryptone bile agar can be demonstrated. Characterization of 555 indole positive colonies from 843 samples of food showed that 95% were E. coli biotype 1 and a further 3·4% were 'faecal coliforms'. Anaerogenic and non-lactose fermenting E. coli are detected and the significance of these strains is discussed.     

Variability in gas production by Escherichia coli in enrichment media and its relationship to pH.

Variability in gas production in multiple subcultures of Escherichia coli was assessed in two selective enrichment media and in lactose peptone water. Considerable variability occurred with all media at 37 and 44 degrees C. Addition of buffer increased gas production and decreased variability. The relationships between pH, growth, and gas production were complex. In buffered media, viable counts increased by 269 x 10(6) to 382 x 10(6)/U of pH fall, whereas in unbuffered media, they increased by 9.45 x 10(6) to 30.37 x 10(6)/U of pH fall. In buffered and unbuffered media, pH fell as gas production rose. However, variability in gas production among individual subcultures was not associated with changes in pH.     

Requirement for phosphoglucomutase in exopolysaccharide biosynthesis in glucose- and lactose-utilizing Streptococcus thermophilus

To study the influence of phosphoglucomutase (PGM) activity on exopolysaccharide (EPS) synthesis in glucose- and lactose-growing Streptococcus thermophilus, a knockout PGM mutant and a strain with elevated PGM activity were constructed. The pgmA gene, encoding PGM in S. thermophilus LY03, was identified and cloned. The gene was functional in Escherichia coli and was shown to be expressed from its own promoter. The pgmA-deficient mutant was unable to grow on glucose, while the mutation did not affect growth on lactose. Overexpression of pgmA had no significant effect on EPS production in glucose-growing cells. Neither deletion nor overexpression of pgmA changed the growth or EPS production on lactose. Thus, the EPS precursors in lactose-utilizing S. thermophilus are most probably formed from the galactose moiety of lactose via the Leloir pathway, which circumvents the need for a functional PGM.     

Overexpression and functional analysis of cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2

Cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2 was overexpressed in Escherichia coli using the Cold expression system and the recombinant enzyme, rBglAp, was characterized. The purified rBglAp exhibited similar enzymatic properties to the native enzyme, e.g., (i) it had high activity at 0 degrees C, (ii) its optimum temperature and pH were 10 degrees C and 8.0, respectively, and (iii) it was possible to rapidly inactivate the rBglAp at 50 degrees C in 5 min. Moreover, rBglAp was able to hydrolyze both ONPG and lactose with K(m) values of 2.7 and 42.1mM, respectively, at 10 degrees C. One U of rBglAp could hydrolyze about 70% of the lactose in 1 ml of milk in 24h, and the enzyme produced trisaccharide from lactose. We conclude that rBglAp is a cold-active enzyme that is extremely heat labile and has significant potential application to the food industry.     

Supplemental ascorbic acid does not affect inferred heat loss in broiler chickens exposed to elevated temperature

The provision of supplemental ascorbic acid has been reported to lower the body temperature of chickens maintained at elevated environmental temperatures. Since body temperature is the net effect of heat production and heat loss, it is not known if the reductions in body temperature were due to a lower heat production or an increase in heat loss. The purpose of this work was to determine if supplemental ascorbic acid facilitates heat loss in chickens exposed to an elevated temperature. On day 12 post-hatch broiler chickens were implanted intra-abdominally with a thermo-sensitive radio transmitter. The following day, birds were placed inside an indirect calorimeter maintained at 34 C for 24 h and provided water containing 0 or 400 ppm ascorbic acid. Oxygen consumption, carbon dioxide production, heat production, respiratory exchange ratio, and body core temperature were measured for 3 h; beginning 21 h after the birds were placed inside the calorimeter. No differences were observed in heat production or body core temperature between birds provided or not and 400 ppm ascorbic acid. This suggests that ascorbic acid has no effect on heat loss. Birds provided ascorbic acid did exhibit a significantly lower respiratory exchange ratio suggesting a greater utilization of lipid for energy production. Although lipid has a lower heat increment compared with protein and carbohydrate, the significance of this finding to birds exposed to elevated temperature is not known. In conclusion, under the conditions of this study the provision of supplemental ascorbic acid to broiler chickens maintained at an elevated temperature did not affect heat loss as inferred from measured heat production and body core temperature.     

Construction of Lactose-Utilizing Xanthomonas campestris with a Mini-Mu Derivative

Xanthomonas campestris is not able to grow in lactose media. The lactose operon from Escherichia coli as part of a mini-Mu phage was integrated at random sites in the chromosome of this bacterium. Clones expressing (beta)-galactosidase were selected. The resulting strain X. campestris 204, is suitable for production of xanthan gum directly from lactose.     

High-efficiency synthesis of oligosaccharides with a truncated β-galactosidase from Bifidobacterium bifidum

An exceptionally large beta-galactosidase, BIF3, with a subunit molecular mass of 188 kDa (1,752 amino acid residues) was recently isolated from Bifidobacterium bifidum DSM20215 [M?ller et al. (2001) Appl Environ Microbiol 67:2276-2283]. The BIF3 polypeptide comprises a signal peptide followed by an N-terminal beta-galactosidase region and a C-terminal galactose-binding motif. We have investigated the functional importance of the C-terminal part of the BIF3 sequence by deletion mutagenesis and expression of truncated enzyme variants in Escherichia coli. Deletion of approximately 580 amino acid residues from the C-terminal end converted the enzyme from a normal, hydrolytic beta-galactosidase into a highly efficient, transgalactosylating enzyme. Quantitative analysis showed that the truncated beta-galactosidase utilised approximately 90% of the reacted lactose for the production of galacto-oligosaccharides, while hydrolysis constituted a 10% side reaction. This 9:1 ratio of transgalactosylation to hydrolysis was maintained at lactose concentrations ranging from 10% to 40%, implying that the truncated beta-galactosidase behaved as a "true" transgalactosylase even at low lactose concentrations.     

乳糖诱导木聚糖酶基因在大肠杆菌中的可溶性表达

以构建好的大肠杆菌工程菌BL21(DE3)/xylanase为研究对象,研究了以IPTG和乳糖作为诱导剂时重组蛋白的表达规律。在摇瓶发酵条件下研究了诱导剂浓度、诱导时机、诱导培养时间和诱导培养温度对目标蛋白表达的影响。实验结果表明,乳糖作为诱导剂时,重组菌产酶活力33.9 U/mg略高于IPTG作为诱导剂时重组菌产酶活力28.10 U/mg,这为乳糖作为诱导剂应用于重组大肠杆菌生产木聚糖酶提供了参考依据。     

Production L-tryptophan by Escherichia coli cells

Whole cells of Escherichia coli B 10 having high tryptophan synthetase activity were used directly as an enzyme source to produce L-tryptophan from indole and L- or D,L-serine. This strain is tryptophan auxotrophic, which is tryptophanase negative and, in addition, L- and D-serine deaminase negative under production conditions. To avoid inhibition of tryptophan synthetase by a high concentration of indole, nonaqueous organic solvents, Amberlite XAD-2 adsorbent, and nonionic detergents were used as reservoirs of indole in the reaction mixture for the production of L-tryptophan. As a result, different effects were observed on the production of L-tryptophan. Particularly, among the nonionic detergents, Triton X-100 was very efficient. Using Triton X-100 for production of L-tryptophan from indole and L- or D,L-serine by whole cells of Escherichia coli B 10, 14.14 g/100 mL and 14.2 g/100 mL of L-tryptophan were produced at 37 degrees C for 60 h.     

Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima

AIMS: Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima for the hydrolysis of lactose and the production of galacto-oligosaccharides. METHODS AND RESULTS: A putative beta-galactosidase gene of Thermotoga maritima was expressed in Escherichia coli as a carboxyl terminal His-tagged recombinant enzyme. The gene encoded a 1100-amino acid protein with a calculated molecular weight of 129,501. The expressed enzyme was purified by heat treatment, His-tag affinity chromatography, and gel filtration. The optimum temperatures for beta-galactosidase activity were 85 and 80 degrees C with oNPG and lactose, respectively. The optimum pH value was 6.5 for both oNPG and lactose. In thermostability experiments, the enzyme followed first-order kinetics of thermal inactivation and its half-life times at 80 and 90 degrees C were 16 h and 16 min, respectively. Mn2+ was the most effective divalent cation for beta-galactosidase activity on both oNPG and lactose. The Km and Vmax values of the thermostable enzyme for oNPG at 80 degrees C were 0.33 mm and 79.6 micromol oNP min(-1) mg(-1). For lactose, the Km and Vmax values were dependent on substrate concentrations; 1.6 and 63.3 at lower concentrations up to 10 mm of lactose and 27.8 mm and 139 micromol glucose min(-1) mg(-1) at higher concentrations, respectively. The enzyme displayed non-Michaelis-Menten reaction kinetics with substrate activation, which was explained by simultaneous reactions of hydrolysis and transgalactosylation. CONCLUSIONS: The results suggest that the thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galacto-oligosaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of hydrolysis and transgalactosylation performed by beta-galactosidase of hyperthermophilic bacteria.     

Prolonged production of tryptophan using immobilized bacteria

Tryptophan production of a genetically modified E. coli strain possessing elevated tryptophan synthetase activity but sensitive to indole could be prolonged by the immobilization of cells in calcium alginate gel beads with a calculated half-life time of 12.2 days.     

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase was studied. The tryptophan synthase (EC 4.2.1.20) of Escherichia coli catalyzed beta-substitution reaction of L-serine into L-tryptophan and the amino acid racemase (EC 5.1.1.10) of Pseudomonas putida catalyzed the racemization of D-serine simultaneously in one reactor. Under optimal conditions established for L-tryptophan production, a large-scale production of L-tryptophan was carried out in a 200-liter reactor using intact cells of E. coli and P. putida. After 24 h of incubation with intermittent indole feeding, 110 g liter-1 of L-tryptophan was formed in molar yields of 91 and 100% for added DL-serine and indole, respectively. Continuous production of L-tryptophan was also carried out using immobilized cells of E. coli and P. putida. The maximum concentration of L-tryptophan formed was 5.2 g liter-1 (99% molar yield for indole), and the concentration decreased to 4.2 g liter-1 after continuous operation for 20 days.