ASSESSMENT OF AN ESCHERICHIA COLI METHIONINE AUXOTROPH GROWTH ASSAY FOR QUANTIFYING CRYSTALLINE METHIONINE SUPPLEMENTED IN POULTRY FEEDS

Estimating availability of methionine is relevant to feed formulation since diets can be supplemented with crystalline methionine to meet the minimum requirements of rapidly growing birds. Bacterial assays have been developed to measure the bioavailable levels of several essential amino acids in feeds, including methionine. The E. coli methionine auxotroph strain used in this study exhibited a linear extent of growth response to increasing concentrations of methionine added to the minimal test media, in the range of 0 to 4 μg/mL. In addition the growth rates of the E. coli auxotroph were significantly (P < 0.01) different when the methionine concentrations were varied (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 μg/mL) in minimal media. To assay feeds, feed grade methionine was added to poultry feed mixtures and samples were diluted with M9 media. Using this assay for estimating crystalline methionine added to feed, the extent of growth of the methionine auxotroph was correlated with the levels of crystalline methionine supplemented in the feed (R²= 0.9873). For all supplementation levels methionine recovery percentages ranged from 71 to 80% indicating that the bacterial assay response to crystalline methionine was relatively constant in the presence of the feed matrix. The overall results indicate that the rapid detection of crystalline methionine added to feeds is possible using this E. coli methionine auxotroph growth-based assay.     

CONSTRUCTION AND GROWTH KINETICS OF A BIOLUMINESCENT METHIONINE AUXOTROPH ESCHERICHIA COLI STRAIN FOR POTENTIAL USE IN A METHIONINE BIOASSAY

Methionine is one of the essential and first limiting amino acids in animal nutrition. In this study, an Escherichia coli methionine auxotroph bacterial strain that exhibits a linear growth response to methionine concentrations was transformed with a plasmid containing genes encoding ampicillin resistance and bioluminescence in order to develop a microbiological technique for methionine quantitation. Transformants were selected based on antibiotic resistance and plasmid containing candidates were confirmed by restriction enzyme digestion and gel electrophoresis. To confirm the bioluminescent phenotype, video imaging of the strain using long exposure photography yielded colonies exhibiting bioluminescence. The strain was also tested in the presence of ampicillin supplemented media with increasing methionine concentrations and growth response (measured as optical density, OD), growth rates and methionine affinities were compared before and after transformation. Although the transformed E. coli methionine auxotroph exhibited somewhat different growth kinetic responses than the nontransformed strain, the standard curves used for estimating methionine concentrations were not different. Based on the results in this study the transformed bioluminescent strain could be used as an OD-based assay if bioluminescence equipment and materials are not available.     

POTENTIAL RAPID BIOASSAY FOR ALIMET® USING A METHIONINE ESCHERICHIA COLI AUXOTROPH

A potential rapid bioassay for methionine hydroxy analog (MHA) feed additive (ALIMET®) was examined using a methionine auxotroph E. coli strain. Bacterial cells were grown in minimal media containing a concentration range of 0 to 26.8 μM of either L-methionine or MHA as ALIMET®. Increasing either methionine or MHA concentration increased the growth rate of the methionine auxotroph. The estimated substrate affinities for methionine compared to MHA were not significantly different (P > 0.13) and the maximum growth rate estimates were also similar (P > 0.34). Methionine and MHA standard curves yielded linear responses (R²= 0.96) to increasing concentrations of the respective substrate. Based on these results it appears that the E. coli methionine auxotroph would have potential utility for further development of a rapid bioassay of ALIMET®.     

EVOLUTIONARY ADAPTATION TO TEMPERATURE. III. ADAPTATION OF ESCHERICHIA COLI TO A TEMPORALLY VARYING ENVIRONMENT

Six lines of the bacterium Escherichia coli were propagated for 2,000 generations in a temporally varying environment. The imposed environmental regime consisted of alternating days at 32°C and 42°C, with rapid transitions between them. These derived lines are competitively superior to their ancestor in this variable temperature regime. We also measured changes in the fitness of these lines, relative to their common ancestor, in both the constant (32°C and 42°C) and transition (from 32°C to 42°C and from 42°C to 32°C) components of this temporally varying environment, to determine whether the bacteria had adapted to the particular constant temperatures or the transitions between them, or both. The experimentally evolved lines had significantly improved fitness in each of the constant environmental components (32°C and 42°C). However, the experimental lines had not improved in making the sudden temperature transitions that were a potentially important aspect of the temporally variable environment. In fact, fitness in making at least one of the transitions (between 32°C and 42°C) unexpectedly decreased. This reduced adaptation to the abrupt transitions between these temperatures is probably a pleiotropic effect of mutations that were responsible for the increased fitness at the component temperatures. Among the six experimental lines, significant heterogeneity occurred in their adaptation to the constant and transition components of the variable environment.     

一种快速、精确构建大肠杆菌组氨酸营养缺陷型的方法

将表达Red体内重组蛋白的质粒pKD46转化大肠杆菌：DH5α,用5′端与组氨酸基因同源,3′端与卡那霉素抗性基因同源的引物获得具有卡那霉素抗性基因的PCR产物,然后电击转化DH5α,在λRed重组系统的帮助下,通过卡那霉素抗性基因两侧的组氨酸基因序列在体内与大肠杆菌染色体上的组氨酸基因发生同源重组,置换了DH5α组氨酸操纵元中的hisDCB基因,最后利用卡那霉素抗性基因两端的FRT位点,通过FTP位点专一性重组将卡那霉素抗性基因去除,最终获得了不具抗性的大肠杆菌组氨酸营养缺陷型菌株。为在大肠杆菌及其他菌株中快速、精确的构建营养缺陷型菌株提供了有益的参考。     

EXPERIMENTS ON THE ADAPTATION OF ESCHERICHIA COLI TO SODIUM CHLORIDE

1. It has been shown that a fairly constant fraction of the total number of bacteria in a fresh-water culture of E. coli can reproduce on direct transfer to a saline medium with a definite NaCl concentration, as judged from the viable count determinations in such a medium. 2. The absolute value of this fraction depends on a number of factors other than the salt content of the test medium, such as the hydrogen ion and yeast autolysate concentrations, aeration, and the physiological condition of the bacteria. 3. A method for testing the degree and rate of adaptation of the bacteria to saline environment, depending on the analysis of changes in the value of the salt-viable fraction, was developed. 4. Maximum adaptability to saline environments was found during the early stationary phase of NaCl-free cultures. Low adaptability accompanied the logarithmic phase and the senescence of the cultures. 5. The limits of variation could be extended by treatment of non-dividing cells with gradually increasing concentrations of salt or by subjecting them to a single intermediate NaCl concentration. This acclimatization was independent of reproduction. The number of bacteria becoming capable of reproducing in a hitherto unfavorable environment increased with the period of exposure to intermediate salt concentrations until a maximum value was reached. 6. This maximum value was shown to depend on the salinity of the test medium, the age of the bacterial culture, and the method of preliminary treatment. "Optimal acclimatization" could be effected by subjecting the organisms to a single fairly low intermediate NaCl concentration. 7. The rate of the individual acclimatization process was shown to be greater at higher than at lower temperatures. 8. Acclimatized bacteria rapidly lost their increased ability to reproduce in saline media upon return to a salt-free environment, although no reproduction of the cells could be detected. This was interpreted as an indication that the processes involved are readily reversible. 9. Studies on the reproduction of E. coli in strongly saline broth indicated that only those cells originally acclimatized to the salt concentration of the medium could divide. All cells produced in such a medium could continue to reproduce. The propagation in the altered medium was not accompanied by any further acclimatization throughout five subcultures. 10. Both the division rate and the maximum crop of cultures in saline broth were considerably lower than of those in a fresh-water medium. No change in either occurred throughout five successive subcultures. The morphology of the organisms was also altered by the presence of salt. 11. The division rate, maximum crop, morphology, and adaptive power returned immediately to normal on re-transfer of bacteria grown in an NaCl-containing medium to "salt-free" broth. 12. The entire adaptive response of the bacteria to a considerable increase in the salinity of the environment could thus be separated into two components: an acclimatization, independent of reproduction, and a selection of those cells with the widest range of potentialities.     

EVOLUTIONARY ADAPTATION TO TEMPERATURE. IV. ADAPTATION OF ESCHERICHIA COLI AT A NICHE BOUNDARY

Following an environmental change, the course of a population's adaptive evolution may be influenced by environmental factors, such as the degree of marginality of the new environment relative to the organism's potential range, and by genetic factors, including constraints that may have arisen during its past history. Experimental populations of bacteria were used to address these issues in the context of evolutionary adaptation to the thermal environment. Six replicate lines of Escherichia coli (20°C group), founded from a common ancestor, were propagated for 2000 generations at 20°C, a novel temperature that is very near the lower thermal limit at which it can maintain a stable population size in a daily serial transfer (100-fold dilution) regime. Four additional groups (32/20, 37/20, 42/20, and 32–42/20°C groups) of six lines, each with 2000 generation selection histories at different temperatures (32, 37, 42, and daily alternation of 32 and 42°C), were moved to the same 20°C environment and propagated in parallel to ascertain whether selection histories influence the adaptive response in this novel environment. Adaptation was measured by improvement in fitness relative to the common ancestor in direct competition experiments conducted at 20°C. All five groups showed improvement in relative fitness in this environment; the mean fitness of the 20°C group after 2000 generations increased by about 8%. Selection history had no discernible effect on the rate or final magnitude of the fitness responses of the four groups with different histories after 2000 generations. The correlated fitness responses of the 20°C group were measured across the entire thermal niche. There were significant tradeoffs in fitness at higher temperatures; for example, at 40°C the average fitness of the 20°C group was reduced by almost 20% relative to the common ancestor. We also observed a downward shift of 1–2°C in both the upper and lower thermal niche limits for the 20°C selected group. These observations are contrasted with previous observations of a markedly greater rate of adaptation to growth near the upper thermal limit (42°C) and a lack of trade-off in fitness at lower temperatures for lines adapted to that high temperature. The evolutionary implications of this asymmetry are discussed.     

FLUORESCENT DNA BINDING DYE-BASED APPROACH FOR MEASURING THE GROWTH OF AN ESCHERICHIA COLI LYSINE AUXOTROPH AND QUANTIFYING LYSINE

Microbiological assays for determination of bioavailable lysine appear to have many advantages. However, since the developed assay is based on bacterial growth and considerable optical density (OD) is required to detect distinguishable differences in extent of growth, it can be time consuming. The purpose of this study was to explore the fluorescence as an alternative method to measure bacterial growth instead of OD and examine the possibility to shorten the time required for the lysine assay. An assay based on SYTO 9 green fluorescent DNA binding dye (Live/Dead BacLight Protocol, Molecular Probes) was used to stain all bacteria in a population. Additional experiments were carried out to determine the ability of fluorescence based on SYTO 9 to overcome problems associated with high nonbacterial background that contributes to OD. From this study it appears that using fluorescence based on SYTO 9 green fluorescent staining, the E. coli lysine auxotroph growth assay can be completed in 9 h instead of 11 h and has the advantage of improved detection sensitivity. Problems associated with interference by high background nonbacterial OD can be partially resolved by fluorescence.     

EVOLUTIONARY ADAPTATION TO TEMPERATURE II. THERMAL NICHES OF EXPERIMENTAL LINES OF ESCHERICHIA COLI

Groups of replicated lines of the bacterium Escherichia coli were propagated for 2,000 generations at constant 32, 37, or 42°C, or in an environment that alternated between 32 and 42°C. Here, we examine the performance of each group across a temperature range of 12-44°C measuring the temperatures over which each line can maintain itself in serial dilution culture (the thermal niche). Thermal niche was not affected by selection history: average lower and upper limits remained about 19 and 42°C for all groups. In addition, no significant differences among groups were observed in rate of extinction at more extreme temperatures. Within the thermal niche, we measured the mean fitness of the evolved groups relative to their common ancestor. Increases in mean fitness were temperature specific, with the largest increase for each group occurring near its selected temperature. Thus, the temperature at which mean fitness relative to the ancestor was greatest (the thermal optimum) diverged by about 10°C for the groups selected at constant 32°C versus constant 42°C. Tradeoffs in relative fitness (decrements relative to the ancestor elsewhere within the thermal niche) did not necessarily accompany fitness improvements, although tradeoffs were observed for a few of the lines. We conclude that adaptation in this system was quite temperature specific, but substantial divergence among groups in thermal optima had little effect on the limits of their thermal niches and did not necessarily involve tradeoffs in fitness at other temperatures.     

AGITATION DURING INCUBATION REDUCES THE TIME REQUIRED FOR A LYSINE MICROBIOLOGICAL GROWTH ASSAY USING AN ESCHERICHIA COLI AUXOTROPHIC MUTANT

Microbiological assays involving Escherichia coli lysine auxotrophs must be optimized to facilitate routine use. Our objectives in this study were to characterize growth of an auxotrophic E. coli lysine mutant (American Type Culture Collection strain #23812) and examine the effect of agitation on E. coli mutant growth. A defined minimal salts basal medium was used and supplemented with various lysine concentrations. The E. coli lysine auxotroph responded to increasing lysine concentration with increasing optical density. When maximum optical density (MOD) was determined for the auxotroph, a linear increase was obtained as lysine concentrations were increased (R²± 0.96) for both agitation and static cultures. Growth rates were not significantly (p > 0.05) affected by lysine concentrations, cultural conditions or their combined effect. However, growth with agitation significantly (p < 0.05) reduced the assay time by shortening the lag phase and causing stationary phase to occur earlier. The values of R² (± 0.96) relatively remained constant over the range while the bacterial population were in the stationary phase. In conclusion, the lysine growth assay using the E. coli lysine auxotroph can be made more rapid by agitating the culture during incubation.     

EVOLUTIONARY ADAPTATION TO TEMPERATURE. VII. EXTENSION OF THE UPPER THERMAL LIMIT OF ESCHERICHIA COLI

What factors influence the ability of populations to adapt to extreme environments that lie outside their current tolerance limits? We investigated this question by exposing experimental populations of the bacterium Escherichia coli to lethally high temperatures. We asked: (1) whether we could obtain thermotolerant mutants with an extended upper thermal limit by this selective screen; (2) whether the propensity to obtain thermotolerant mutants depended on the prior selective history of the progenitor genotypes; and (3) how the fitness properties of these mutants compared to those of their progenitors within the ancestral thermal niche. Specifically, we subjected 15 independent populations founded from each of six progenitors to 44°C; all of the progenitors had upper thermal limits between about 40°C and 42°C. Two of the progenitors were from populations that had previously adapted to 32°C, two were from populations adapted to 37°C, and two were from populations adapted to 41–42°C. All 90 populations were screened for mutants that could survive and grow at 44°C. We obtained three thermotolerant mutants, all derived from progenitors previously adapted to 41–42°C. In an earlier study, we serendipitously found one other thermotolerant mutant derived from a population that had previously adapted to 32°C. Thus, prior selection at an elevated but nonlethal temperature may predispose organisms to evolve more extreme thermotolerance, but this is not an absolute requirement. It is evidently possible to obtain mutants that tolerate more extreme temperatures, so why did they not become prevalent during prior selection at 41–42°C, near the upper limit of the thermal niche? To address this question, we measured the fitness of the thermotolerant mutants at high temperatures just within the ancestral niche. None of the four thermotolerant mutants had an advantage relative to their progenitor even very near the upper limit of the thermal niche; in fact, all of the mutants showed a noticeable loss of fitness around 41°C. Thus, the genetic adaptations that improve competitive fitness at high but nonlethal temperatures are distinct from those that permit tolerance of otherwise lethal temperatures.     

EVOLUTIONARY ADAPTATION TO TEMPERATURE. IX. PREADAPTATION TO NOVEL STRESSFUL ENVIRONMENTS OF ESCHERICHIA COLI ADAPTED TO HIGH TEMPERATURE

Abstract Stressful environments may be considered as those that reduce fitness, sometimes due in part to the increased metabolic expenditure required to sustain life. Direct adaptation to a stressor is expected to increase fitness and reduce maintenance metabolism, with the latter leading to increased biomass production. In this study, we test the general hypothesis that such adaptation to one stressor can preadapt organisms to novel stressful environments. Six lines of Escherichia coli propagated for 2000 generations at 41–42°C (42 group), a stressful temperature, were compared to six control lines propagated for 2000 generations at 37°C (37 group) and to the common ancestor of both groups. We assayed biovolume yield (a measure of growth efficiency) and competitive fitness in the 42 group's selective high temperature environment as well as five novel stressful environments–acid, alkali, ethanol, high osmolarity and peroxide. As previously reported, at high temperature the 42 group had both higher yield and fitness than the 37 group and ancestor. In the novel environments, the 42 group generally produced yields higher than the 37 group (and marginally higher than the ancestor), but we found no differences in competitive fitness among the 37 and 42 groups and the ancestor. We also found that the performance of lines within groups was not correlated across stressful environments for either yield or relative fitness. Because previous adaptation to one stressor did not improve our measure of Darwinian fitness in novel stressful environments, we conclude that the 42 group shows no useful preadaptation, or cross‐tolerance, to these types of environments.     

EVOLUTIONARY ADAPTATION TO TEMPERATURE. I. FITNESS RESPONSES OF ESCHERICHIA COLI TO CHANGES IN ITS THERMAL ENVIRONMENT

We used bacteria to study experimentally the process of genetic adaptation to environmental temperature. Replicate lines of Escherichia coli, founded from a common ancestor, were propagated for 2,000 generations in 4 different thermal regimes as 4 experimental groups: constant 32, 37, or 42°C (thermal specialists), or a daily alternation between 32 and 42°C (32/42°C: thermal generalists). The ancestor had previously been propagated at 37°C for 2,000 generations. Adaptation of the groups to temperature was measured by improvement in fitness relative to the ancestor, as estimated by competition experiments. All four experimental groups showed improved relative fitness in their own thermal environment (direct response of fitness). However, rates of fitness improvement varied greatly among temperature groups. The 42°C group responded most rapidly and extensively, followed by the 32 and 32/42°C groups, whose fitness improvements were indistinguishable. The 37°C group, which experienced the ancestral temperature, had the slowest and least extensive fitness improvement. The correlated fitness responses of each group, again relative to the common ancestor, were measured over the entire experimental range of temperatures. No necessary tradeoff between direct and correlated responses of fitness was apparent: for example, the improved fitness of the 42°C group at 42°C was not accompanied by a loss of fitness at 37°C or 32°C. However, the direct fitness responses were usually greater than the correlated responses, judged both by comparing direct and correlated responses of a single group at different temperatures and by comparing direct and correlated responses of different groups at a single temperature. These comparisons indicate that the observed adaptation was, in fact, largely temperature specific. Also, the fitness responses of the generalist group across a range of temperatures were less variable than those of the thermal specialist groups considered as whole.     

THE ASSOCIATED GROWTH OF STREPTOCOCCUS LACTIS AND ESCHERICHIA COLI

SUMMARY: The possibility that contamination of farmhouse starters by coli-aerogenes bacteria may be a factor in producing the subtle flavour of farmhouse Cheddar cheese has been discussed. The associated growth of Strep. lactis and E. coli I at 30° and 37° resulted in the rapid disappearance of E. coli from the mixtures, even though it had been the dominant organism in some of them originally. Mixtures containing Strep. lactis and an anaerogenic strain of E. coli still contained this variant at the end of a month, although in no definite ratio and in a very much reduced proportion. It is concluded that the components of coli-lactic starters to be used in the manufacture of cheese should be combined together in the vats.     

EVOLUTIONARY ADAPTATION TO TEMPERATURE. VI. PHENOTYPIC ACCLIMATION AND ITS EVOLUTION IN ESCHERICHIA COLI

Acclimation refers to reversible, nongenetic changes in phenotype that are induced by specific environmental conditions. Acclimation is generally assumed to improve function in the environment that induces it (the beneficial acclimation hypothesis). In this study, we experimentally tested this assumption by measuring relative fitness of the bacterium Escherichia coli acclimated to different thermal environments. The beneficial acclimation hypothesis predicts that bacteria acclimated to the temperature of competition should have greater fitness than do bacteria acclimated to any other temperature. The benefit predicted by the hypothesis was found in only seven of 12 comparisons; in the other comparisons, either no statistically demonstrable benefit was observed or a detrimental effect of acclimation was demonstrated. For example, in a lineage evolutionarily adapted to 37°C, bacteria acclimated to 37°C have a higher fitness at 32°C than do bacteria acclimated to 32°C, a result exactly contrary to prediction; acclimation to 27°C or 40°C prior to competition at those temperatures confers no benefit over 37°C acclimated forms. Consequently, the beneficial acclimation hypothesis must be rejected as a general prediction of the inevitable result of phenotypic adjustments associated with new environments. However, the hypothesis is supported in many instances when the acclimation and competition temperatures coincide with the historical temperature at which the bacterial populations have evolved. For example, when the evolutionary temperature of the population was 37°C, bacteria acclimated to 37°C had superior fitness at 37°C to those acclimated to 32°C; similarly, bacteria evolutionarily adapted to 32°C had a higher fitness during competition at 32°C than they did when acclimated to 37°C. The more surprising results are that when the bacteria are acclimated to their historical evolutionary temperature, they are frequently competitively superior even at other temperatures. For example, bacteria that have evolved at either 20°C or 32°C and are acclimated to their respective evolutionary temperatures have a greater fitness at 37°C than when they are acclimated to 37°C. Thus, acclimation to evolutionary temperature may, as a correlated consequence, enhance performance not only in the evolutionary environment, but also in a variety of other thermal environments.