Shell Disease Syndrome Gene Experiment

Introduction

 Shell Disease Syndrome (SDS) is a disease that affects crustaceans and results in unappealing dark spots on the shells of the infected creatures. The diseased crustaceans are unappealing and hard to sell; therefore, the aquafarming business is greatly affected. SDS can progress and eventually lead to the death of the infected by degradation of chitin through the procuticle layer of the shell. During these SDS outbreaks, the bacteria Vibrio harveyi has been found in high concentrations and was analyzed for further analysis. It has been shown that the V. harveyi specimen contained mutations in the chb which lead to an increase to the severity of the SDS disease by catalyzing the degradation of the shell chitin with the chitin-degrading enzyme, chitinase. The wildtype genetic sequence was taken from the NCBI GenBank database which contained the accession number of J05004. This accession number was used on multiple occasions in order to have a constant wildtype genetic sequence. The gene has been successfully cloned and the gene product, chitobiase, has been transformed into Escherichia coli cells. According to a study, the protein was manly localized on the outer membrane band of E. coli and while being accompanied by the cleavage of a single peptide. [1] This also showed light on the structure, regulation of expression, and promoter regions. The result shows similarity to the outer membrane lipoprotein of the E. coli and chitobiase undergoes a similar maturation process. In another study, the promoter regions were also analyzed and the results indicated that the E. coli and V. harveyi shared two transcription initiation sites of the chb gene. [2] The chitobiase enzyme is classified as a glycosyl hydrolase which catalyze the hydrolysis of a glycosidic bonds in sugar complexes. The classification of glycosyl hydrolases has been broken down into 45 different families based on similarity between amino acid sequences. [3] The 20-member glycosyl hydrolases family contain an alpha/beta TIM-barrel as a catalytic domain and contain the conserved amino acid pairing of Aspartate539-Glutamate540 in Serratia marcescens. Mutations found in the conserved amino acid pair has been shown to cause changes in the catalytic domain function. [2]

 A previous experiment was conducted to create a rapid screening kit to identify the SDS outbreaks containing the mutated chb gene, called “Category 1” SDS Outbreaks. The unpublished experiment resulted in the use of Restriction Fragment Length Polymorphisms (RFLP) by Southern Blot to rapidly screen the Category 1 mutation and any unknown mutations. Category 1 contained a specific mutation in the chb gene where its product contained chitinase and chitobiase activity. However, another significant mutation was identified that did not align with the genetic composition of the wildtype or Category 1 mutation. This mutation was classified as “Category 2” SDS Outbreaks and was subjected to further analysis. The following experiment resulted in the Category 2 isolation and amplification by PCR and the purified PCR product was cloned into the plasmid pUC19 by the use of restriction enzymes AccI65 and SalI-HF to create a recombinant plasmid called pBMB2. Blue/White Screening and Colony PCR verification methods were conducted to obtain the pBMB2 plasmid containing the mutant chb vector which was used for further analysis that was completed during this experiment. The goal of the experiment was to use the pBMB2 recombinant plasmid to sequence the Category 2 chb gene in order to make predictions on the effect of the identified mutations and to express the mutant chb protein in order to determine the specific activity of chitobiase activity.

The sequencing of the chb gene allowed for accurate determination of the mutations in the nucleotide and amino acid sequences by the use of bioinformatic tools. This analysis allowed for predictions on how mutations affect the chitobiase activity by comparing the results to previous publications. The sequenced mutant was compared to the V. harveyi wildtype by the alignment program BLAST in order to find the mutations in the nucleotide sequence. The programs CLUSTAL and BLASTp were used to compare the identified mutations to conserved regions found in other members of the protein family. This not only allowed to identify if the mutations occurred in the conserved domains but also if the mutations would affect the catalytic function of the chitobiase enzyme.

The cell extraction and enzyme activity assay methods were derived from the original methods published in the Soto – Gil paper (1988). [4] However, this experiment was time sensitive, therefore, High Performance Liquid Chromatography (HPLC) was replaced with a faster method, crude Sarkosyl cell extract (SCE), for isolation and determination of chitobiase activity. Since the chitobiase enzymes were shown to translocate to the outer membrane of the DH5α E. coli specimen, a specific concentration of the Sarkosyl detergent (1%) was used to disrupt and isolate the crude periplasm and outer membrane which contained the chitobiase enzyme as well as other proteins. Before the chitobiase enzyme activity assay could be conducted, a Bicinchoninic Acid (BCA) assay was completed to determine the total protein content of each SCE sample and to ensure each sample had the same protein concentration as the others. The BCA assay was chosen over others because of unique characteristic of being compatible with detergents and detergent agents like Sarkosyl. The BCA assay determines protein concentration by forming a BCA-CuI product after the reduction of CuII when it comes in contact with the protein. The absorbance of the purple product can be determined at 562nm.

Method:

The following is a step-by-step protocol for measuring the chitobiase activity as described above in general. The protocol contains the reagents list and the corresponding amount used, step-by-step directions, and any calculations used:

Reagents List

0.39 ml of PNAG, 12mM in Tris HCl (10mM, pH 7.3)

 6.7 ml of Tris-HCl buffer

0.24 ml of SCE (30 µl for each reaction)

26 ml of Tris base

Six 1.6 ml Eppendorf tubes

One 12 ml test tube

Five 1.5 ml cuvettes

Protocol

1. Make 3 SCEs (pUC19, pBMB, pBMB2) as described in Part Z of lab manual.

2. Determine protein concentrations for the 3 SCEs using the BCA assay as described in Part AB of lab manual.

3. Dilute the SCEs so that they are all at the same concentration. 

4. Label two sets of three 1.6 ml Eppendorf tubes:

 1. pBMB (positive control)

2. pUC19 (negative control)

3. pBMB2 (experimental)

4. pBMB (positive control)

5. pUC19 (negative control)

6. pBMB2 (experimental)

5. Create enough master mix for 26 samples (includes blank) by adding 389.61 µl of PNAG, 12mM in Tris HCl (10mM, pH 7.3) and 6.6304 ml of Tris- HCl buffer (10mM, pH 7.3) to a 12 ml test tube to dilute the PNAG in Tris-HCl to a concentration of 666 µM

6. Add 1.08 ml of master mix to each Eppendorf tube

7. Add 1 ml of Tris Base into 5 labeled (blank, t0, t5, t10, and t15) 1.5 ml cuvettes

8. Add 30 ul of SCE to one Eppendorf tube and mix well by pipetting. (It’s recommended to complete one reaction at a time to minimize error. However, multiple reactions can be completed at once if desired.)

9. At time 0, 5, 10, and 15 mins transfer a 0.3 ml aliquot from the appropriate Eppendorf tube to the corresponding labeled cuvette. Mix by inverting the cuvette with parafilm covering the top.

10. Measure product absorbance at 400nm in a Genesys Spec with a blank containing 0.27 ml of master mix and 1 ml of Tris base.

11. After recording absorbance, discard solution in waste and thoroughly wash cuvettes so they can be used for the next reaction.

12. Complete steps 7 through 11 until all reactions have been completed and absorbance values have been recorded.

*Doubled the master mix and had two sets of tubes in order to repeat the experiment under the same conditions to demonstrate reproducibility

Results:

 After amplifying and cloning the Category 2 chb gene, the pBMB2 plasmid was sent to DNA sequencing to determine the mutant’s sequence of genes. Four primers, M13 forward, M13 reverse, and two (5 forward and 6 reverse) primers in the middle of the gene, were used to attempt to cover the entirety of the 3660bp mutant gene. After receiving the sequenced mutant, it was aligned with the wildtype gene by the use of the program BLAST and analyzed by the program CLUSTAL. This allowed for not only for the nucleotide sequences to be compared but also the amino acid sequences. In the following table, Table 1, contains the data collected from the comparisons previously mentioned. The data entails information such as primer used in sequencing, nucleotide mutation, region of mutation, amino acids changed, category of coding mutation, amino acid that aligns in the S. marcescens chitobiase, and amino acid that aligns in Human Hex A. It is important to note that only high-quality data was used and therefore, the regions containing “N” were ignored.

Table1. Analyzed Mutations in Category 2 chb Gene with BLAST and CLUSTAL

The program BLASTp was also used to identify changes in the amino acid sequence when the mutant was compared to other proteins in its family. The purpose of identifying these changes was to see if the conserved sequence was mutated which could change catalytic activity.

 The noncoding region mutations at g.214>G and g.3277G>C do not affect the level of expression of the chitobiase because these noncoding regions are not in terminator sites or dyad symmetry sites. The coding region mutation of g.493C>G can be categorized as a nonconservative missense mutation that results in the substitution of Alanine to Glycine. However, by using CLUSTAL and BLASTp, the mutation does not appear to alter the chitobiase activity due to the position of the amino acid, 16, which is not considered to be a conserved domain. The g.2721C>G mutation in the sequencing reaction can be categorized as a nonconservative missense mutation that results in the substitution of Glycine to Glutamic Acid. The chemical properties of the two amino acids are significant, however, there was no evidence of conservation in this region when the CLUSTAL program was used. The BLASTp data resulted in identification of this mutation in the TIM barrel folding region. The substitution could result in improper TIM barrel folding which would alter the chitobiase activity. The g.2053_2058delACGAAG mutation resulted in the deletion of Aspartic Acid 536 and Glutamic Acid 537. According to the CLUSTAL data, both of these amino acids are highly conserved in the glycosal hydrolases family and loss of these amino acids would result in the loss of the catalytic function in the chitobiase enzyme. [2]

 In order to accurately determine the activity of the Category 2 chitobiase mutant, in vitro assays were used to calculate the specific activity. The protein concentration in each SCE sample were determined by using a BCA assay and comparing the absorbance of the non-diluted SCE and 1:10 diluted SCE samples to the BSA standard curve. Dilutions of the sample were made to obtain absorbance values in the standard curve’s range in case the non-diluted SCE samples had concentrations that were too high. The following table, Table 2, gives the absorbance values and calculated concentration of their respectable samples. Also shown below, Figure 1, is the standard curve created to calculate the SCE concentrations. The 1:10 dilutions contained concentrations that were slightly lower compared to the non-diluted SCE samples. This could error could be due to human error such as pipetting.

Table 2. Absorbance Values and Concentrations of BSA Standards, Non-Diluted and Diluted SCE Samples from BCA Assay

Figure 1. BSA Standard Curve from BCA Assay. The best fit trendline was used to create an equation that could then be used to calculate the approximate protein concentration in each SCE sample.

 After determining the protein concentration of the SCE samples, the samples were diluted to the same concentration as mentioned in the Method section and were subjected to the enzyme activity assay. The following table, Table 2, gives the absorbance values of each SCE and the normalizes absorbance of pBMB SCE and pBMB2 SCE. The normalized absorbance values were given by using the pUC19 as a background control. This allowed an accurate level of enzyme activity to be determined. Since both assays were conducted in an identical matter, the absorbance values at each time point were averaged together and were graphed, as shown below in Figure 2, with error bars based on standard deviation.

Table 3. Chitobiase Assay With PNAG

*SCE Concentration of 0.534 mg/ml and 30µl of each sample was used*

Figure 2. Averaged Absorbance of pBMB SCE and pBMB2 SCE. Absorbance values at 15 minutes were excluded in making the graph because of the extremely low which resulted in an illegible graph. The extremely low absorbance values are due to pipetting not enough SCE sample as indicated.

 The normalized absorbance values of the pBMB and pBMB2 SCE samples were used to calculate the units and the specific activity of the wildtype and category 2 mutant chitobiase. The change in absorbance at the time point 10 minutes was selected for the calculations. The molar absorptivity coefficient of 18,100 liter/mol-cm was used due to the coefficient being pH dependent and the protocol contains the use of Tris Base, as done in the Soto – Gil paper (1988) publication. The following table, Table 4, shows the calculated units of enzyme activity and specific activity of the pBMB and pBMB2 SCE samples. The enzyme and specific activity of the pBMB2 SCE sample was determined to be zero because of the lack of significant product formed by the enzyme.

Table 4. Calculated Enzyme and Specific Activities of pBMB and pBMB2 SCE Samples at 10 minutes

 A mock Western Blot analysis was also used to differentiate the chitobiase from other proteins in the sample. This was needed because the BCA Assay only determines the total protein concentration in the sample. The Western Blot contained GAPDH protein served as the internal loading control and is shown below, Figure 3. The intensity measurements indicate that the chitobiase enzyme was present in both samples, thus verifying the specific activity of each sample to be accurate.

Figure 3. Mock Western Blot with GAPDH used as the internal loading control

Example Calculations:

Below are the example calculations of the Units of enzyme activity and specific activity for the pBMB SCE sample at time point 10 mins.

Change in absorbance in 10 minutes:

$∆\mathit{Abs}=\frac{0.962–0}{10\mathit{mins}}=0.0962/\mathit{min}$

Concentration per minute was determined by the use of Beer’s Law:

$\mathrm{C}=\frac{\mathrm{A}}{\mathrm{\epsilon l}}= \frac{0.0962/\mathit{min}}{(18,100/\mathrm{ }\mathrm{M\; cm})\left(1\mathrm{ cm}\right)}=0.0000053149\mathit{ M}/\mathit{min}$

Units of Enzyme Activity:

$\mathit{Units}= \frac{C}{\mathit{min}}\times \mathit{Total\; Volume\; of\; Assay\; Reaction}\times \frac{{10 }^6\mathit{\mu moles}}{\mathit{moles}}= 0.0000053149\frac{\mathit{moles}}{\mathit{L\; min}}\times 0.0013L\times \frac{{10 }^6\mathit{\mu moles}}{\mathit{mole}}$

$\mathit{Units}=0.0691 \frac{\mathit{\mu moles}}{\mathit{min}}$

Specific Activity:

$\mathit{SA}=\frac{\mathit{Units}}{\mathit{mg\; Total\; Protein}}=\frac{\mathit{Units}}{\mathit{Diluted\; Concentration}\times \mathit{Volume\; of\; Sample\; Added\; to\; Assay}}=\frac{0.0691 \frac{\mathit{\mu moles}}{\mathit{min}}}{0.536\frac{\mathit{mg}}{\mathit{ml}}\times 0.03\mathit{ml}}$

$\mathit{SA}=4.30\frac{\mathit{Units}}{\mathit{mg}}$

Discussion:

 As mentioned above, the category 2 mutant contained deletions of Aspartic Acid and Glutamic Acid in positions 536 and 537, respectively. When compared to other family protein members, these amino acids were highly conserved and were found to be part of the catalytic domain in the chitobiase found in S. marcescens. Therefore, the little to no activity of the category 2 mutant was expected due to the loss of the catalytic function of the chitobiase.

 Even though the category 2 mutation was determined to have lost its catalytic function, the same mutation was found to cause a more severe SDS outbreak. This change in chitobiase activity could lead to a more severe SDS outbreak by the mutated enzyme having a possibly increased expression of extracellular proteases and lipases that assist in destroying the epicuticle layer of the shell. [5] After the destruction of the epicuticle, the newly exposed chitin could cause a chemosensory response in nearby chitinolytic microorganisms that are in the open water or from sedimentation. Also, chitin and especially chitobiose are strong inducers of proteins in the chitin catabolism found in Vibrio furnissii. [6] The induction of the cascade would result in a more severe outbreak because of the chemo-attractants. However, the use of probiotic bacteria could be used as a controlling agent to possibly treat the severe SDS outbreak. [7] The probiotics have been used in aquatic environments and would compete for adhesion sites and metal ions, as well as produce compounds that inhibit a selected protein.

 The experiment could have been improved by excluding the crude protein extraction method used and replace it with HPLC like was done in the Soto – Gil paper (1988). HPLC would take a few more weeks to complete however, the method is by far more accurate with less sample needed.

References:

1. M Jannatipour, R W Soto-Gil, L C Childers, J W Zyskind Journal of Bacteriology Aug 1987, 169 (8) 3785-3791; DOI: 10.1128/jb.169.8.3785-3791.1987

2. Soto-Gil, R W, and J W Zyskind. “N,N’-Diacetylchitobiase of Vibrio Harveyi. Primary Structure, Processing, and Evolutionary Relationships.” Journal of Biological Chemistry, 5 Sept. 1989, www.jbc.org/content/264/25/14778.short.

3. Henrissat, B and A Bairoch. “New families in the classification of glycosyl hydrolases based on amino acid sequence similarities” Biochemical journal vol. 293 ( Pt 3),Pt 3 (1993): 781-8.
4. N, N-diacetylchitobiase of Vibrio Harveyi. Soto-Gil R, Childers L, Huisman W, Dahms A, Jammatipour M, Hedjran F, Zyskind J. Methods in Enzymology (1988) 161:534-529.
5. Shell disease in crustaceans – just chitin recycling gone wrong? Claire L. Vogan, Adam Powell, Andrew F. Rowley

6. Lee and Saul Roseman Nemat O. Keyhani, Lai-Xi Wang, Yuan C. doi: 10.1074/jbc.271.52.33409 J. Biol. Chem. 1996, 271:33409-33413

7. Laurent Verschuere, Geert Rombaut, Patrick Sorgeloos, Willy Verstraete Microbiol Mol Biol Rev. 2000 Dec; 64(4): 655–671.