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Identification of Neurosporra Crassa Mutants In The Arginine Biosynthetic Pathway
Abstract

This project identified five unknown mutant extracts and a wildtype sample through measuring intermediate levels, high pressure liquid chromatography (HPLC), enzyme assays, and nuclear magnetic resonance (NMR). The N. Crassa strains consisted of six different enzymes: arg 1, arg 6, arg 10, arg 12, and arg 14. Steady state levels were determined using HPLC and enzyme assays. Also, NMR was performed as another technique to analyze the profiles of the samples. Sample E was the known wildtype. Measuring steady state levels proved that sample A was arg 12, B was arg 14, C was arg 1, D was arg 6, and F was arg 10.

Introduction
Neurospora crassa has been instrumental for the understanding of several basic and fundamental aspects of biology. N. crassa is a red bread mold that belongs to the phylum Ascomycota and are species with largely spreading colonies that are easy to culture and can survive in minimal media. Studies with these fungi pioneered the use of microorganisms and has become the most important model organisms in modern biology (2).
The first studies of sexual reproduction of the common bread mold, Neurospora, was done by Bernard O. Dodge, an American botanist and researcher on heredity of fungi. He first began his
research by investigating the taxonomy and reproduction of the fungi Ascobolaceae. This early research proved to be important for his future study of Neurospora. Through the work of Bernard O. Dodge, N. crassa was found to
reproduce sexually and asexually. Dodge s findings led to more research by Edward Tatum and George Wells Beadle. Their research exposed N. crassa to x-rays, causing mutations. They also observed the metabolic pathways that caused errors in specific enzymes. This finding led Tatum and Beadle to propose the one gene, one enzyme hypothesis, which suggests that specific genes code specific proteins (3).
Figure 1: Arginine Biosynthetic Pathway
The arginine biosynthetic pathway of N. crassa has interest for several reasons. Some enzymes in the pathway are seen in the urea cycle, which is used in terrestrial animals. In animals, arginine is considered an essential amino acid. There is a pathway with enzymes that synthesize arginine, but it does not produce enough to provide the needs of cells, so it is consumed through diet. The available intermediates in the pathway are glutamate, citrulline, arginine, and ornithine.
In figure 1, the pathway represents the intermediates and the enzymes that catalyze the biochemical reaction (arg #). These enzymes are proteins that are encoded by genes that can be mutated. For example if there was a mutation in the gene arg 12, the mutant will not be able to convert Ornithine to Citrulline, there will be decreased levels of arginosuccinate and arginine compared to the wild-type. Also, there would be increased levels of ornithine and the reaction will run in the reverse direction causing increased levels of glutamate. The enzymes only influence the rate of the reaction, allowing the product to substrate ratio to be at an equilibrium. When mutants are blocked, they are unable to grow in minimal medium and require supplementation. However, all mutants can grow if the proper intermediate is provided in the media.
Six samples of unknowns were provided that had a complete set of functional biosynthetic enzymes (wild-type) or have defective enzymes in their sets (mutants). The N. crassa strain provided consisted of a wild-type and five mutants: arg 1, arg 6, arg10, arg 12, and arg 14. Two potential methods to determine unknown identities of the samples are growth test and measuring intermediate levels. In the growth test, the unknowns are inoculated onto minimal medium which contained either none or one of the arginine biosynthetic pathway intermediates (1). It is expected that the mutants will grow in the intermediates that come after the blocked enzyme reaction, but not those that come before the block. For the measurement of level of intermediates, the unknowns will be grown, the amino acids will be extracted, and separated by reverse phase high pressure liquid chromatography (HPLC). It is expected that the mutants will have a decreased level of those intermediates that come after the blocked step and an increased level for those that come before the blocked step compared to wild type(1).
HPLC is an ideally suited technique for separation and identification of amino acids, carbohydrates, lipids, proteins and other biologically active molecules (2). HPLC has many advantages such as the speed and resolution of analysis is far greater than the classical methods like thin layer chromatography (TLC). This machinery is very reproducible and the instrument operation and data analysis are easily automated (2). The two major advances of the HPLC are the development of stationary supports with small particle sizes and large surface, and has improved elution rates by applying high pressure to the solvents. HPLC depends on the hydrophobic binding interactions between the solute molecule in the mobile phase and the immobilized hydrophobic ligand, the stationary phase (2). It is used to identify and quantify each component in a mixture. The challenges in HPLC are highly efficient and fast separation with high resolution and ideally low back pressure for various types of samples (9). The distribution of the solute between the two phases depends on the binding properties of the medium, the hydrophobicity of the solute and the composition of the mobile phase (2).
Nuclear magnetic resonance (NMR) is a phenomenon which occurs when the nuclei of certain atoms are immersed in a static magnetic field and exposed to a second oscillating magnetic field. It determines the physical and chemical properties of atoms or the molecules in which they are contained. NMR can provide detailed information about the structure, dynamics, reaction state, and chemical environment of molecules (7). The vast majority of NMR-based metabolomics studies have used biological fluids as the sample of choice. This is because these are relatively easy to collect; sample preparation for NMR is generally straightforward, and they provide a wealth of biochemical information that is sensitive to the dynamic metabolic status of the organism they have been collected from (8).
This experiment identified all extracts as being either wild-type or one of the arg mutants: arg 1, arg 6, arg10, arg 12, arg 14. This was accomplished through growth tests, measuring intermediate levels, HPLC, and nuclear magnetic resonance (NMR).
This experiment identified all extracts as being either wild-type or one of the arg mutants: arg 1, arg 6, arg10, arg 12, arg 14. This was accomplished through growth tests, measuring intermediate levels, HPLC, and nuclear magnetic resonance (NMR).

Materials & Methods
Measuring levels of Intermediates:
A 500 mL solution of Vogel s minimal with 1.5% sucrose and 0.2 mM arginine was prepared. The solution was then divided into five 100 mL portions and transferred into 250 mL Erlenmeyer flasks. The medium was then inoculated by adding 1-2 mL ultrapure water to the VM+ Arg slants. They were then shaken to suspend the conidia in the water and settled for a few minutes. The sterile technique was used again and 1-2 mL of the sterile water was added to the Erlenmeyer flask that contained the liquid medium. The slants were then disposed of by autoclaving. The mutants/KO were then grown for 24-48 hours on the shaker at room temperature. After the shaking was completed, a large amount of mycelia was grown and collected by filtration on a Buchner funnel under gentle vacuum. The mycelia pad was washed with ultrapure water. The mycelia was then transferred to a 15 mL conical glass centrifuge tube with 3 mL of ultrapure water. The conical tubes were then racked and placed in boiling water for 15-20 minutes then cooled to room temperature. The tubes were then placed in a table top centrifuge at 2,000 rpm for 5 minutes. The supernatant was then transferred to microfuge tubes and if they were not used, they were stored to freeze at -20 C. The remaining liquid was removed from the pellet, 3.0 mL of 0.5 M NaOH was added and the tubes were parafilmed and stored at room temperature.
Nuclear Magnetic Resonance:
After the mycelia was grown the supernatant was removed which contained the amino acids. About 600 uL was saved in separate microfuge tubes for each sample. Each sample was freeze dried and then mixed with a solvent, deuterated chlorophorm. All samples were tested using the Bruker 400 MHz, for H-NMR. There were 36 samples tested and the data was displayed using principal component analysis (PCA).
Growth in Intermediate
Mutant A B C D E F
1 + + + + + +
2 + + + + + +
3 + + + + + +
4 + + + + + +
5 + + + + + +
Wildtype + + + + + +
Results
Table 1: Growth Tests of Unknown Mutants and Wildtype
Samples Glutamate Citrulline Arginine Ornithine
A 0.850 0.565 0.636 N/D
B 1.865 N/D 0.662 N/D
C 1.328 0.826 0.707 N/D
D 0.811 0.160 0.372 N/D
E 0.258 N/D 0.248 N/D
F 1.225 N/D 0.997 N/D
In table 1 the growth tests for the mutant extracts and wildtype are displayed. Growth occurred on all slants which resulted in a failure of determining the wildtype extract. Following the growth tests, amino acids were separated by reverse phase HPLC which provided the retention times, glutamate being the first one eluted at 2.9 minutes. Citrulline, arginine, and ornithine eluted next with retention times of 7.3, 7.9, and 13.9. Using the retention times can assist in identifying the amino acids in the samples. Also, all the samples showed traces of arginine which could be the reason as to why growth occurred on all slants.

Table 2: Steady State Levels of Amino Acids in the samples. (N/D=Not detectable).
(Units: LU*s amino acid/mg protein).
Table 2 shows steady state levels which indicates the concentration of amino acids in each of the N. Crassa metabolite fractions. Each sample was derived from a N. crassa strain, being a mutation in the arginine biosynthetic pathway. All samples had similar values for the area of amino acids with the exception of sample A. Citrulline was present in one chromatogram of data for sample A but failed to appear in the second set of data. The steady state level of citrulline in sample A is based on the area of only one peak. Sample E was the N. crassa wild-type and having this knowledge allowed a comparison with the other five samples. Sample B had resulted in a higher glutamate steady state level (1.865 LU*s /mg protein) which is about 16-folds above the wild-type. The wildtype did not show any peaks for the amino acid citrulline but extract C had the highest steady state level for citrulline (0.826 LS*s/mg protein) which is about 7-folds above extract D and 2-folds above extract A. Sample F had a buildup of glutamate with a steady state level of 1.225 LM*s/mg protein and did not show any traces of citrulline.

Figure 2: Principal Component Analysis (PCA) Scores Plot
Figure 2 displays the scores plot which was used for metabolic profiling in N.Crassa. There were 36 samples loaded into the nuclear magnetic resonance (NMR) based on six different extracts. The multivariable data was reduced to a smaller number of important dimension by PCA. Rotation of the data into this new coordinate system means that the first axis (principle component 1), is the most important direction of variability and likely reflects important underlying processes. Amino acids highly expressed in some samples and not expressed in others will have a lot of variation and influence on the principal components. Each datapoint in the scores plot represents a spectrum. The data presented in figure 2 does not show strong support for similar samples clustering together since the wildtype samples are scattered throughout the graph.
Discussion
Growth Tests
As shown in table 1, the growth tests for this experiment did not produce accurate results because growth occurred on all slants tested. Five mutants were provided with the addition of a wildtype extract. The mutants are blocked at different steps meaning that each sample would require supplementation if grown in an unsupplemented medium. Each mutant can grow in a particular medium that provides the proper amino acid that occurs after the blocked step for a particular mutant. Therefore, once all extracts were inoculated into the unsupplemented medium, the wildtype should result in growth solely, therefore distinguishing this sample from the mutants. In addition, these tests can determine which step is blocked based on which mutant grows in the presence of a particular intermediate. In figure 1 (arginine biosynthetic pathway), if a mutant grows while citrulline is supplemented but not when ornithine is provided, that would prove the mutant to be identified as arg 12. This mutant is blocked in the formation of citrulline meaning that the enzyme, ornithine carbamoyltransferase is defective. This mutant will grow in the presence of citrulline or arginine, but will not grow in the presence of any intermediate before that blocked step.
Identification of Mutant Enzymes:
Determining mutants was accomplished by comparing the wild-type (sample E), which was the control, to the other five samples. The amino acid steady state levels helped to identify N. Crassa mutant strains. The results proved that the acetylglutamate synthase mutant (Arg 14) was sample B. This was determined based on the glutamate levels in the sample having a steady state level of 1.865 LU*s /mg protein. This was the largest value due to excess glutamate produced when arg 14 is blocked.
Determining the mutants for samples F was completed by comparing the amounts of citrulline and ornithine because they are the next set of locations for the mutant gene. Due to the enzymes being blocked, citrulline levels would be very low. Sample F did not show any detection for the amino acid citrulline and displayed a high steady state level for glutamate (1.225 LU*s/mg protein). Sample F having no detection for citrulline and showing excess levels of glutamate prove this extract to be the acetylglutamate kinase mutant (arg 6).
Sample C was evaluated by comparing the sample to the wild-type. The steady state level for citrulline was 0.826 LU*s/mg protein, being the highest value among all mutants. The wildtype did not have any traces of citrulline. A buildup of citrulline indicates sample C as being the best candidate for the arginosuccinate synthase mutant (arg 1).
Steady state levels for samples A and D raised the most uncertainty. The results for sample A did not point to a definitive conclusion linked to any mutants. Thus, there were not any characteristics in A that stood out or raised awareness towards this extract being linked to one of the mutants. Sample D could possibly be linked to the ornithine trancarbamylase mutant (arg 12) based on low steady state levels of citrulline (0.160 LU*s/mg protein). Ultimately, the results are not strong enough to point to any definitive conclusions.
Nuclear Magnetic Resonance:
Nuclear magnetic resonance was performed to examine the profiles of the six extracts. Thirty-six samples were tested which generated data using principal component analysis (PCA), which is a method for compressing a lot of data into something that captures the essence of the original data (5). The PCA took the data of 36 dimensions and flattened it to 2 dimensions to make the interpretation easier. Figure 2 shows that principle component one captures the most variation in the data while principle component two captured the second most variation. The scores plot demonstrates data points clustering together which indicates samples with similar profiles. Unfortunately, these data points did not cluster together to successfully support the sample profiles for the wildtypes. For example, all of the wildtype samples should be clustered together to represent similar profiles. The scores plot shows data points clustering together based on group similarity. The next factor observed was analyzing whether the mutants formed clusters between one another to show similarities. Whether the individual mutants clustered properly or not is difficult to contradict. For example, the difference between arg 6 and arg 12 can be so small because it is only one mutation out of hundreds. This small difference may not show any variation in two particular mutants. Therefore, the results in figure 2 clearly indicate that the expression in the wildtype extracts are not correlated and may or may not be correlated for the mutant extracts.
In conclusion, five N. crassa mutants were identified along with a wildtype extract. This was completed using HPLC, enzyme assays, measuring intermediate levels, and metabolic profiling. Sample E was the known wildtype and was used as a control for the five mutants. Extract B was the acetylglutamate synthase mutant because it contained the highest steady state level for glutamate (1.865 LU*s/mg protein). Extract F was the acetylglutamate kinase mutant (arg 6) because of having excess glutamate and showing no traces of citrulline. Extract C could have possibly been the ornithine carbamoyltransferase mutant (arg 12) based on the highest steady state level for citrulline (0.826 LU*s/mg protein). Extracts A and D did not result in any key factors linking these samples to any mutants.

References
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