1.0 Introduction
The genetic material of many organisms comprises of DNA that encompasses functional units referred to as genes. Agene is either a DNA or RNA segment that codes the information needed to produce a functional biological product (Nelson, 2008). The dissimilar ways genes transmit anatomical, behavioral, and biochemical characteristics from parents to offspring are regarded to as hereditary (Hartwell et al., 2008). The traits of a species are encoded by genes and hereditary variations can be as a result of different alleles as well as environmental disparities which influences the phenotypic expressions. Phenotypic expressions are the observable characteristics that correspond to the organisms gene expression under particular environmental conditions (Gilbride and Victorio, 2009)
Gene mutation is as a result of changes or errors in DNA sequence in an organism that are heritable. Gene mutation can either be spontaneous or induced. Spontaneous mutations are very rare and they are the result of errors in DNA replication or the direct action of transposons (Hartwell et al., 2008). Because of the infrequency of spontaneous mutations, mutations are frequently induced to escalate the possibility of attaining definite changes. Mutations can as well be induced by changes in DNA chemistry or direct interference with repair mechanisms. Chemical mutagens, such as deaminating agents are most frequently used to promote mutations in DNA of an organism (Prescott et al., 2005).
Bacterial exposure to environmental or other chemical agents can result into phenotypic alterations similar to mutations in alleles of a gene. Phenotypic change caused by environmental agents that mimics a mutation is regarded to as a phenocopy. Since phenocopies do not induce changes in genes they are therefore not heritable (Hartwell et al., 2008). Introducing variations in bacteria via environmental agents is easy and permits for rapid assessment of the change a specific environmental factor has educed in the organisms phenotype. Phenotypic changes due to environmental factors tend to affect all cells in a specified culture. Genotypic changes, unlike phenotypic changes that are reversible in the absence of environmental factors. These are permanent changes and only affect a small portion of the cells in a given culture. Genotypic changes that lead to phenotype amongst specified cell culture are the result of mutations taking place in the DNA (Gilbride and Victorio, 2009).
Mutations are readily observed if they result in a noticeable phenotype and a mutation from the common gene form considered as the wild type to a mutant form is regarded to as a forward mutation. Some mutants are unstable and undergo a second mutation to produce an organism that seems to be wild type and this is again called a reversion mutation (Prescott et al., 2005). Thus, an organism that has reverted back to original phenotype is known as a revertant. Tracing genotypic alterations that have taken place in a bacterium can be done by using differential media. This way revertant and mutant could be identified based on phenotypes on the culture media. Besides, nutritional mutants as well exist in bacteria (Prescott et al., 2005). These deficiencies are the result of mutations in genes which create a metabolic pathway that are under the operons control. An operon is a DNA sequence of bases which encompasses a promoter, operator, and one or several structural genes. The expression of genes is usually controlled by an operon. Mutations in a metabolic pathway of a bacterium never permit the microorganism to be grown on a culture medium lacking enough supply of the end product (Prescott et al., 2005). A mutation in a gene encoding a result of the pathway like an enzyme that synthesizes the next intermediate to the pathway will lead to a nonfunctional pathway and growth either the next intermediate within the pathway or end product is needed. Mutants of this kind are known as auxotrophs and need the end product of the pathway growth. Auxotrophs selectivity is just by culturing them on minimal media. Contrarily, microorganisms that grow on minimal medium devoid of supplementation of other growth enhancing factors are called prototrophs. When the metabolic pathway that the microorganism takes for final product synthesis is known, it could be determined at which point of the pathway the mutation has taken place (Prescott et al., 2005).
During the growth of bacterial culture, spontaneous mutations do rarely occur and usually result in a genotypic single nucleotide alteration. There is no definite source of mutation away from spontaneity and the mutant organisms will have diverse traits than the parent strain. A considerable number of spontaneous mutants lead to bacterial antibiotic resistance that enable bacteria to even grow in presence of antibiotics. This is a selective advantage that allows isolation and selection of resistant mutants (Gilbride and Victorio, 2009). The aim of the experiment conducted was to gain an understanding of differences between genotypic and phenotypic changes, and as to whether certain phenotypic changes were as a result of environmental or genotype changes. Nutritional mutants were cultured and the point of mutations in the metabolic pathway was determined. Finally, bacterial cultures were grown on streptomycin plates to isolate resistant mutants.
2.0 Methods and Materials
In the first experiment, two nutrient agar plates, one containing phenol and the other with no phenol were streaked with Proteus vulgaris and incubated at 37oC for 24 hours. Extra two nutrient agar plates were streaked with Serratia marcescens. One plate was incubated at room temperature and the other at 37oC for a period of 24 hours. After 24 hours, the plates incubated were examined for growth, swarming, and pigmentation. Additionally, in the experiment two, one white and one red isolated colonies of Escherichia coli (ATCC 15939) were streak on two different MacConkey agar plates. Both plates were incubated for 24 hours at 37oC. After incubation the plates were examined for revertants and mutants. The third experiment, employed 5 dissimilar strains of E. coli (EMG23, KS463, EMG4, EMG5, EMG6) each were cultured on different minimal media having different trytophan biosynthetic pathway intermediates(Anthrantic acid, Tryptophan, and Indole) as well as one control group. 4 drops of 1 casein hydrolysate was added to all plates and spread plated (Gilbride and Victorio, 2009). The plates were later incubated for 24 hour at 37oC and then examined for point of mutation in pathway and wild type strains. The final experiment involved preparation of 3 streptomycin gradient agar plates, which were incubated with 0.1mL of E. coli strain (EMG23) through spread plating method. Plates were labeled in accordance to the concentration of streptomycin (low and high). They were then incubated for 2-4 days at 37oC and were colonies were observed on both high and low concentration streptomycin sides.
3.0 Results
3.1 Experiment 1 Bacterial Variation Because of Environmental Change
When P. vulgaris was streaked on nutrient agar, swarming colonies were observed which was characteristic of this bacterium. However, when it was streaked on nutrient agar with phenol no growth was observed. Besides, when S. marcescens was streaked on the nutrient agar and incubated for 24hour at room temperature, the colonies had a distinct red color. Though, when S. marcescens was incubated for 24 hours at 37oC the colonies observed lacked the red pigment rather they appeared pink pale in color.
3.2 Experiment 2 Bacterial Variation Because of Genotypic Change
When a red colony of E. coli (ATCC 15939) mutant was streak on MacConkey agar plate the colonies were observed to be red in color and fermented lactose (Lac). However, when a white colony of the E. coli mutant was streak on MacConkey agar plate, a mixture of white and red colonies were observed showing that some of the colonies were incapable of fermenting lactose (white colonies), while others were able to ferment lactose (red colonies)
3.3 Experiment 3 Nutritional Mutants
The table below shows the growth or no growth of five strains of E. coli provided with 3 different supplements of Indole, Trytophan, and Anthranilic acid as well as one control group devoid of supplements. Growth implies that the strain is capable of growing on medium comprising of supplements, whereas no growth shows that the strain is unable to grow is the presence of supplement. It was thus noted that all strains of E. coli were able to grow on the medium with trytophan, EMG6 was able grow with all supplements except the control. EMG5 was able to grow with Tryptophan and Indole and both EMG4 and KS463 were unable to grow with no tryptophan. EMG23, a strain of E. coli was able to grow on both non supplemented and supplemented medium as shown in Table 1
3.4 Experiment 4 Isolation of Streptomycin-Resistant Mutant of E. coli
When EMG23 strain was plated on a gradient agar plate encompassing streptomycin and tryptic soy agar no colony growth was observed on all 3 plates. This therefore indicated that no streptomycin resistant mutations occurred in the strain of E. coli.
4.0 Discussion
P. vulgaris is a bacterium which under Enterobacteriaceae class of bacteria. It is as well flagellated bacterium and thus, sometimes is known as peritrichous bacteria (Prescott, 2005). From the results obtained, when P. vulgaris was streak on nutrient agar with no phenol it indicated the swarming characteristic of this bacterium. High motility of P. vulgaris bacteria bring about the swarming characteristic that was observed on the media plate. The phenotype of swarming is easy to identify on a nutrient agar plate, since swarming bacteria cause colonies to come close to each other thus making it hard to isolate a single colony. Though, there was no observable growth of P. vulgaris due to the presence of phenol on the nutrient agar plate that is not the occurrence theoretically. In low concentration of phenol P. vulgaris is not able to swarm and thus, it would encompass usual streaking visible. The presence of phenol in the media makes the Proteus spp flagella not to function properly (Costerton et al., 1974). This is because phenol binds to the proteins and phospholipids that are present on the exterior membrane of the cell wall, inhibiting P. vulgaris to exhibit the swarming characteristics. This experiment once again, it was carried out to signify the difference between phenotypic and genotypic changes. Swarming inhibition in P. vulgaris was due to environmental agent (phenol). Although, the flagella were not functional in phenol presence, the alteration was just a direct result of the environment and the phenotypic alteration was short lived.
The disparities in the color of colonies produced by the same strain of S. marcescens can be ascribed to the environmental conditions. As clearly indicated, the two nutrient agar plates were incubated at different temperatures one at 37oC and the other at room temperature for 24 hour period. S. marcescens colonies coloration was as a result of prodigiosin a secondary metabolite that results to production of a red pigment (Williams, 1973). Prodigiosin biosynthesis by S. marcescens takes place on a narrow temperature range, though the bacteria itself can grow at a broader temperature range (Francia et al., 1997). The utmost prodigiosin production occurs at between 28 oC and 30 oC and thus its synthesis in S. marcescens is normally inhibited at 37 oC (Khanafari et al., 2006). This is in agreement with the obtained results in this experiment because when S. marcescens was plated at room temperature prodigiosin biosynthesis was not inhibited. The inhibition was noted at higher temperature of 37 oC. The significance of this experiment was to prove that alterations in phenotype were not as result of gene mutations, because if alterations were due to genotype then as both plates were streaked with similar strain and similar stock of S. marcescens, then at least colonies with the similar phenotypic changes were expected on both plates. However, this was not evident and the differences can only be ascribed to change in temperatures (an environmental condition). It imperative to note that this is not a genotype permanent change of species, because if the pale pink colonies are restreaked on a nutrient agar plate and incubated at room temperature they will produce prodigiosin and attaining a distinct red color. From the obtained results it can therefore be concluded that higher temperatures of 37 oC and above inhibit the expression of prodigiosin, while lower temperature near room temperatures encourages the prodigiosin expression (Anandkumar et al., 2004).
Examining the results of isolated colonies of E. coli mutant incubated on MacConkey agar both red and white colonies were observed on the plate which was originally streak with white colonies. The logic behind color differences of these colonies lies in the idea that lactose fermenting bacteria (Lac) are able to ferment lactose. Acid is usually produced during lactose fermentation, when acid is produced by bacteria on MacConkey agar that contains pH indicator (neutral red) come in contact with each other they lead to red colonies (Prescott et al, 2005). On contrary, when non lactose fermenting bacteria (Lac-) grow on MacConkey agar they produce no acid and thus, no contact with pH indicator and the colonies grow white in color. It was observed that when red colonies of E. coli are streak the resulting colonies are all red on the plate. This shows that all these colonies are lactose fermenting ((Lac). This as well implies that red colonies undergo forward mutation where the genotype of the colonies does not change from its initial state (Lac) to a mutant (Lac-). However, as observed when white colonies were streak on MacConkey agar the resulting colonies were a mixture of both Lac-Lac, this is because the mutation in the mutant white colony of E. coli is unstable and it often reverts back to initial state (Gilbride and Victorio, 2009). Reversions in the bacterium are permanent alterations in the genotype and in this case depict visible phenotype red colonies.
Table 1 presents the list of E. coli mutant strains grown in minimal media with dissimilar tryptophan intermediates supplementation. This table assists in identifying the strains which grow in different supplementations, thereby allowing detection of a gene in which mutations have taken place in the metabolic pathway for trytophan synthesis. All E. coli strains grow in the presence of tryptophan. This is expected since tryptophan is the end product in the pathway bypassing all the conservations in the intermediates needed to produce tryptophan. As results indicated EMG23 is the wild type of all E. coli strains examined this experiment. This implies that EMG23 is the sole strain which can grow the control plate that had no supplementation. This further implies that the strain is a prototroph and thus does not need supplements to grow on the minimal medium. It can synthesize the end product needed.
E. coli (EMG6) strain has a mutation occurring at point A (trpD, trpE). The logic for this is that if EMG6 is supplied with any other intermediate its capable of growing on the minimal media except in the absence of an intermediate. This implies EMG6 is unable to convert the first Chroismate to Anthranilic acid because of a mutation at point A. However, when the strain is offered with consecutive intermediates it can synthesize tryptophan. From the results its evident that E. coli strain EMG5 encompasses a mutation occurring at point B (tryC), because when its offered with Anthranilic acid it never grows on the media thus this indicates that point B is mutated and the conversion of Anthranilic acid to Indole is not occurring. Consequently, when an E. coli strain is provided with the next intermediate Indole it grows and becomes evident that the mutation had taken place in point B. KS463 and EMG4 strains of E. coli are unable to grow with no tryptophan presence and can as well not grow in the presence of any other supplementation. However, with the results obtained it still can not be determined at which point in the pathway the mutations have occurred. Mutations identification in anabolic pathway contains a major application and being that a range of these mutants can be employed to establish the identity of pathway intermediates (Prescott, 2005).
Evaluating and examining the final experimental results that cultured E. coli in a gradient agar plate with streptomycin it was evident that there was no observable growth realized by plating. This shows that E. coli colonies underwent spontaneous mutations occurring to become streptomycin resistant hence they were all inhibited in their growth in streptomycin presence. This is a rational outcome because the possibility of achieving spontaneous mutation is very low, approximately one per 107 to 1911 cells. If there was growth on the gradient agar, to be sure that the colonies are real mutants, growth would have to take place on the side with the higher streptomycin concentration. The explanation for this is that, if there was growth on the side with lower streptomycin concentration it could be because of the fact that not adequate streptomycin has reached that part of the plate. However, the presence of bacteria on the side of higher streptomycin concentration then concludes that these bacteria are streptomycin resistant, since the area is hostile for their growth.
