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The Cloning and Analysis of the Vibrio harveyi chb Gene from Shell Disease Syndrome Category 2 Isolates

Paul Hook BMB 445W Section 002 TA: Han Zheng

Introduction Shell Disease Syndrome (SDS) is an affliction that affects crustaceans around the world. The most widely reported form of SDS is characterized by exoskeleton degradation caused by bacteria and fungi occurring on living crustaceans that can lead to lesions and death [1]. The main reason SDS is of concern is because it can affect aquaculture farms growing crustaceans, ruining the product and costing those companies not only in lost profits, but also in time spent decontaminating. It has been found that chitin degradation in the exoskeletons by extracellular chitin degrading enzymes released by the microbial populations found in the lesions are the main cause of the syndrome [1]. Some of the most commonly isolated bacteria from the black SDS lesions are those of the genus Vibrio, including marine bacterium Vibrio harveyi [1, 2]. Bacteria, like Vibrio harveyi must break down chitin in two steps with two different enzymes, chitinase and N,N-diacetylchitobiase (chitobiase) [2]. Chitinase first breaks down chitin into dimers of chitobiose and then chitobiose is broken down into N-acetylglucosamine monomers by chitobiase [2]. The chitobiase gene of Vibrio harveyi has been identified and named chb [3]. Through study of outbreaks of SDS, it has been observed that there are three categories of SDS that require different treatments, which may be linked to mutations in the Vibrio harveyi chb gene. Our goal is to eventually develop an assay that would allow aquaculture farmers to identify the category of SDS outbreaks and the correct treatment for that outbreak. This experiment represents the first step in developing an assay for Category 2 SDS by cloning the Vibrio harveyi chb gene from a Category 2 SDS lesion and sequencing the gene. This was done by first amplifying the chb gene engineered with

compatible restriction enzyme sites and ligating it into a pUC19 vector, then selecting for the correct product in bacteria and elucidating the sequence. Methods PCR Reaction A PCR reaction was set up in order to amplify the chb gene from the genomic DNA from an SDS lesion. The PCR reaction included 30 L sterile water, 50 L PCR Master Mix (2x), 5 L forward primer (5-CGTGGTACCGAATTCAATAAGCAC-3) with a Kpn1-HF restriction site that ends up on the 3 end of the product (10 M), 5 L reverse primer (5-GCATCTAGAGAATTCGACCGTCATC-3) with a Xba1 restriction site that ends up on the 5 end of the product, 10 L genomic DNA from a SDS isolate to serve as the template DNA, and 2 units of Pfu DNA polymerase. The thermocycler settings for the initial cycle consisted of 4 minutes at 94oC, 1 minute at 55oC, and lastly 4 minutes at 72oC. The initial cycle was then followed by 21 cycles consisting of 1 minute at 94oC, 1 minute at 55oC, and then 7 minutes at 72oC. All of this was then followed by 24 hours at 4oC in the thermocycler and then storage at -20oC for approximately a week. Restriction Digests Digests with both restriction enzymes were set up for the pUC19 vector and the purified PCR chb gene product. The vector digest was set up with 500 ng pUC19, 2.5 L 10x NEBuffer 4, 2.5 L 10x BSA, and 15 units of both Kpn1-HF and Xba1. The PCR product digest was set up identically to the pUC19 digest except that 1,850 ng of purified PCR product replaced the pUC19 vector. The vector digest was incubated at 37oC for approximately 99 minutes. The PCR product digest was incubated at 37oC for approximately 76 minutes.

DNA Purifications After the PCR reaction and restriction digests, DNA was purified using solution purification according to the manufacturers (GelElute, 5 Prime) protocol (both PCR and digests) and gel purification with 1% agarose gel (only restriction digests). The PCR purification was done in order to purify the amplified chb gene with the two added restriction enzyme sites from the template DNA, buffer, and Pfu enzyme, that were present in the PCR reaction solution so that the product could be used in restriction digests. The restriction digest purifications were done in order to separate the added 12.5 L of digested pUC19 and PCR product from the salts and enzymes used in the restriction digests so that they would not interfere with the ligation reactions. Ligation Reactions Two separate ligation reactions were performed: a ligation reaction including the insert (ligation reaction 1) and a control ligation reaction. The ligation reaction 1 (20 L total) included purified, digested insert DNA (2.2 L), gel-purified, digested pUC19 vector (8 L), T4 DNA ligase (1 L), T4 DNA ligase buffer (2 L), and sterile water (6.8 L). The control ligation reaction (20 L total) included purified, digested pUC19 (4 L), T4 DNA ligase (1 L), T4 DNA ligase buffer (2 L), and sterile water (13 L). The purpose of running the control reaction was to provide a negative control in which we knew the ligation reaction could not produce the desired plasmid because of the lack of insert DNA. Transformation and Plating The cells that were used in the transformation reactions were NEB 5-alpha Competent E. coli cells (120 L total). Four different transformation reactions were set

up containing 30 L of bacteria each. They were transformed with undigested pUC19 vector (5 ng) with no insert (positive control), no DNA (negative control), 10 L of the ligation reaction 1, and 10 L of the control ligation reaction, respectively. The transformation reactions were first incubated on ice for 20 minutes, then they were heat shocked at 42oC for 30 seconds and chilled immediately after the heat shock for 5 minutes. The cells were then allowed to recover for 60 minutes at 37oC with 0.96 mL SOC. The transformation reactions were plated on 6 LB-AMP plates, 5 of which were coated with 100 L of a X-gal/IPTG mixture (Chromomax). The lone LB-AMP nonChromomax coated plate was used for the negative control reaction. Of the 5 coated plates, 3 were inoculated with 300 L of the ligation reaction 1 transformation, 1 was inoculated with the positive control, and 1 was inoculated with the control ligation reaction. Plasmid Isolation Two positive bacteria colonies were isolated from the ligation reaction 1 transformation plates and were grown in two separate liquid cultures containing TB and AMP. The two cultures were incubated overnight in a 37oC shaker. The plasmid DNA was then isolated from 3 mL of each culture (isolated plasmid 1 and isolated plasmid 2) according the manufacturers protocol (Stratagene). Analysis of Recombinant DNA In order to successfully analyze the isolated plasmids, a separate restriction digest was set-up for isolated plasmid 1, isolated plasmid 2, and pRSG129. The pRSG129 digest contained 80 ng of pRSG129 DNA, while the two isolated plasmid digests contained 4

L of their respective plasmid. All three were digested with 20 units of EcoR1. After digestion, the digested plasmids were run along with their undigested versions on a 0.8% gel at 175 V. Results PCR Reaction The PCR reaction was run in order to amplify the Vibrio harveyi chb gene and to engineer restriction enzyme sites on each end of the gene. The PCR products were then purified and analyzed on an agarose gel, the results of which can be seen in Figure 1. Both bands were located approximately halfway between the 3 kb and 4 kb ladder markers, giving the PCR product an approximate size of 3.5 kb. Both PCR bands were also extremely pure as shown by the fact that lanes 2 and 3 contained only a single band each. The intensity of the PCR-1 (lane 2) and purified PCR-1 (lane 3) were compared to the intensity of the 3.0 kb band in the ladder (lane 1) in order to determine the concentration of the samples, the approximate yield of the reaction, and the % yield of purification. It was estimated that the PCR-1 band was 2x brighter than the 3.0 kb band that contained 125 ng of DNA. The concentration was determined by multiplying 125 ng by 2, dividing by the amount of sample loaded (10 L), and then multiplying by the dilution factor (2). The concentration for PCR-1 was found to be 50 ng/L and the approximate yield of PCR-1 was found to be 5,000 ng by multiplying the concentration by the original sample volume (100 L). The purified PCR-1 sample band was determined to be 4x as bright as the 3.0 kb band and the concentration was calculated just as it was above. The concentration of the purified PCR-1 was found to be 100 ng/L and

since the total purified sample was approximately 30 L, the total yield of purified PCR1 was 3,000 ng. Since it was known that 3750 ng of DNA was purified, % yield of purification was calculated by the following equation: Equation: [Recovered DNA (ng)/DNA Added for Purification (ng)] x 100 = % yield Calculation: [3000 ng/3750 ng] x 100 = 80% yield

Figure 1. Gel Analysis of Vibrio harveyi chb gene PCR Products

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Figure 1. This 0.8% agarose gel includes a linear DNA ladder in lane 1, the PCR product in lane 2, and the solution purified PCR product in lane 3. The PCR product in both lanes 2 and 3 was found to have an approximate size of 3.5 kb. The PCR product in lane 2 was found to have a concentration of 50 ng/L and the pure product in lane 3 was found to have a concentration of 100 ng/L. Both product lanes were considered to be extremely pure because of the absence of any other bands.

Restriction Digests The vector, pUC19, and the purified PCR product were both digested under the same conditions with Kpn1-HF and Xba1 in order to allow for eventual ligatation of the 7

two together. Figure 2 shows the gel analysis of only the pUC19 vector. The double digest pUC19 band (lane 4) was compared to a DNA ladder (lane 1), and undigested pUC19 band (lane 2). A PCR product from another experiment was also analyzed on the same gel (lane 3) and can be ignored at this time. By comparing the undigested and digested pUC19 bands, it was determined that the digest did in fact work. This conclusion was reached by observing the differences in approximate size of the two bands for the same vector. The apparent increase in size when moving from lane 2 to lane 4 (from closer to the 2.0 kb band to closer to the 3.0 kb band) indicated that the plasmid had been transformed from supercoiled to linear, since linear DNA fragments run higher than supercoiled on gel. This transformation was caused by digestion. Even though it appeared that the digest was success, it could not be fully determined that is was complete because the difference between incomplete and complete digestion would only be less than 10 bases. The band in lane 4 was cut out and purified.

Figure 2. Gel Analysis of double digest of pUC19

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Figure 2. This 1% agarose gel contained a linear DNA ladder (lane 1), undigested pUC19 vector (lane 2), PCR product from another experiment (lane 3), and double digested pUC19 vector with Kpn1-HF and Xba1 (lane 4). It was determined that the digest 8

performed worked because linear DNA runs higher than supercoiled DNA and that expected difference was seen by the different apparent sizes of pUC19 in lane 2 (supercoiled) and 4 (cut). However the completeness of the digestion could not be confirmed.

Ligation Reactions In order to determine the amounts of pUC19 vector and PCR-1 chb gene insert needed for effective ligation reactions, both were run on an agarose gel (Figure 3) and analyzed. The PCR-1 chb insert band (Lane 2) was determined to be 15 times brighter than the pUC19 vector band (Lane 3). After analysis of the gel, the ratio of insert to vector was determined by the following equation, in which Si/Sv represent the sizes of the insert and vector, Iv/Ii represents the intensities of the vector and insert on the gel, and Vi/Vv represents the volume of the insert and vector loaded on the gel: Volume of insert in ligation= (3)(Volume of vector in ligation)(Si/Sv)(Iv/Ii)(Vi/Vv) Since the insert was estimated as 15 times brighter and the volume of vector that was desired to be used was 8 L, the ratio used was calculated as follows: Volume of insert in ligation = (3)(8 L)(3.66 KB/2.68 KB)(1/15)(12.5 L/12.5 L) = 2.2 L When analyzing the gel, it was observed that the lane containing the insert (Lane 2) could have been overloaded because of the smudges observed around the band in that lane. This could have lead to an over-estimation of the brightness of the insert band, which would have led to an under-estimation of the amount of insert needed in the ligation reaction. This possible under-estimation of insert needed could have led to less efficient ligation in ligation reaction 1.

Figure 3. Gel Used to Determine Vector:Insert Ratio for Ligation Reaction

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Figure 3. This 0.8% agarose gel contains DNA ladder (Lane 1), double digested, pure PCR-1 product (Lane 2), gel purified, double digested pUC19 (Lane 3), and PCR product from another experiment that can be ignored (Lane 4). It was determined that the PCR-1 product band (insert) was approximately 15x brighter than the pUC19 band (vector), however the brightness may have been over-estimated because of possible overloading in lane 2.

Transformation and Plating The transformed and plated E. coli cells were counted and the results were tabulated below (Table 1). Table 1. Blue and White Screening Plate Counts Plate Number of Blue Colonies Number of While Colonies Positive Control >300 0 Negative Control 0 0 Ligation Reaction 1 0 4 (3 plates) Control Ligation Reaction 0 0

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Table 1. This table includes the plate counts for the 6 plates inoculated with E.coli. All the plates except for the Negative Control were coated with a X-gal/IPTG mixture. White colonies were expected to contain the desired chb inserted plasmid, while the blue colonies should indicate bacteria not containing the desired plasmid. Two of the white colonies from the Ligation Reaction 1 plates were picked and the plasmid DNA was extracted for analysis. Based on the theory of blue/white screening, the white and the blue colonies should represent two specific transformation outcomes. The white colonies found should represent those bacteria that contain pUC19 plasmids including the desired insert (chb gene). The blue colonies found should represent those bacteria that contain pUC19 plasmids without the desired insert. Analysis of Recombinant DNA The gel with the isolated plasmids run on it was analyzed in order to determine if any of the isolated plasmids from the picked white colonies contained the desired insert (Figure 4).

Figure 4. Recombinant DNA analysis Gel

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Figure 4. This 0.8% agarose gel contains DNA ladder (Lane 1), undigested recombinant plasmid 1 (Lane 2), digested recombinant plasmid 1 (Lane 3), undigested recombinant plasmid 2 (Lane 4), digested recombinant plasmid 2 (Lane 5), undigested pRSG192 plasmid (lane 6), and digested pRSG192. All digestions were done with EcoR1. Through

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analysis, it was determined that neither recombinant plasmid was the desired recombinant plasmid. After analysis of the gel, it was determined that neither of the isolated plasmids were the desired recombinant plasmid and both were false positives. This was concluded because it was expected that if either of the isolated plasmids were the desired plasmid, the undigested versions would run near a size of 6 KB. That result was not observed and both of the undigested isolated plasmids (Lane 2 and 4) ran near 3 KB. It was also expected that the digested desired plasmids would show two fragments that ran at about 3.6 and 2.6 KB, respectively. That result was not observed in either of the digested lanes (Lane 3 and 5). Our conclusion was further confirmed by the observation that neither of the isolated plasmids showed similar patterns to the pRSG192, positive control lanes (Lanes 6 and 7). Even though the digested lanes (3 and 5) did not seem to contain the desired plasmid, they did seem to contain two distinct lanes that migrated close to each other with one fragment appearing below its undigested counterpart and one appearing close to the undigested counterpart. It was concluded that these lanes contained a mixture of cut, linear DNA, and uncut, supercoiled DNA. DNA Sequencing When comparing the sequence of the Category 2 SDS chb gene to the wild-type (WT), eight total mutations were found, all of which occurred in the coding region and caused amino acid changes or deletions (Table 2).

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Table 2. Summary of Mutations Found in Category 2 SDS chb gene Nucleotide Nucleotide Regulatory or Amino Acid Amino Acid Number Change Coding Number Change Changed from WT 493 CG Coding 16 Alanine Glycine 2721 CG Coding 759 Glutamine Glutamic Acid 2053 Deletion of A Coding 536 The deletion of Asparagine-539 2059 Deletion of C Coding 538 and Glutamic 2060 Deletion of G Coding 538 Acid-540 2063 Deletion of G Coding 539 2064 Deletion of A Coding 540 2065 Deletion of A Coding 540 Table 2. Mutations found in the category 2 SDS chb gene when compared to the WT chb gene. Eight mutations were found, all within the coding region and all causing amino acid changes or deletions. Discussion Recombinant Plasmid Analysis It was found that both isolated, positive recombinant plasmids were false positives and did not contain the desired plasmid as the blue/white screening indicated. There are two main possibilities as to why the false positives were observed. The first possibility is that the plasmid that was isolated was simply religated pUC19 containing no insert, which had a mutation in the Lac-z alpha gene. This would have disrupted the gene in the same way an insert would have and could have been introduced during gel purification of the vector, in which the vector was exposed to two mutagens in UV light and ethidium bromide. The second possibility is that exonucleases were introduced through the use of multiple pipette tips, different buffers, and water. If exonucleases were introduced, they would have chewed away at the sticky ends of both the vector and insert,

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leaving the blunt ends to be ligated together causing a frame shift mutation in the Lac-z alpha gene. Effect of Mutations Found in Category 2 SDS Isolates on Chitobiase Activity Eight total mutations were found in the Category 2 SDS chb gene, when compared to the WT gene (Table 2). While all of the mutations were in coding regions, the set of six deletion mutations that led to the deletion of Asp-539 and Glu-540 were determined to have to biggest possible effect on chitobiase activity. As described in Tews, et al., both Asp-539 and Glu-540 are located in the active site of chitobiase and are extremely important residues for its activity [4]. Asp-539 is vital to stabilizing the chitobiose molecule through a polar interaction in a bent conformation essential for cleavage [4]. Glu-540 forms a hydrogen bond with the glycosidic oxygen of chitobiose and is the key catalytic residue for the acid-base reaction used for carrying out cleavage [4]. Tews, et. al., mention that when the structure of chitobiases were compared to chitinases, one main difference was that chitinases have a grove that binds polysaccharides randomly, while chitobiases have a specific docking site [4]. Since both of the deleted residues interact directly with chitobiose, their deletion may have increased the activity of chitobiase by eliminating the specificity of the enzyme for only chitobiose. This may have allowed the mutated chitobiose to be able to interact and cleave the larger polysaccharide chitin in the same way that a chitinase enzyme does. The second mutation (Nucleotide 2721) caused an amino acid change from a hydrophilic residue (glutamine) to a negatively charged residue (glutamic acid). The affected residue (amino acid 759) is found next to a positively charged amino acid (Arginine 760) and both residues are found in between 5 hydrophobic residues (754-758)

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and 2 hydrophobic residues (761-762). The affected amino acid is also found near an important tryptophan residue (Tryptophan 737) that is included in a group of tryptophan residues in the active site that pack around chitobiose and stabilize it for cleavage [4]. The mutation found would allow the negatively charged glutamic acid to pair with the positively charged arginine next to it and the charges would be neutralized. This would allow the hydrophobic residues around those two amino acids to pack more tightly together. This could lead to an upstream effect of allowing a key, stabilizing residue, tryptophan 737, to pack more tightly around the substrate. The tighter packing potentially caused by this mutation could have offset the loss of the docking interactions that helped stabilize the substrate in the WT enzyme. This would have allowed for no major loss of activity due to potentially weaker binding of the new substrate, chitin. The last mutation (nucleotide 493) caused a change in amino acid from alanine to glycine and was not thought to have any effect on chitobiase activity because it was a change from one simple, hydrophobic residue to another simple, hydrophobic residue. The amino acid that was mutated is also found outside of the active site domain (Domain III), further confirming our conclusion [4]. Mutated Chitobiase, the Promotion of SDS, and Vibrio harveyi in SDS lesions The deletion mutations that potentially cause chitobiase to be more active and degrade chitin could promote SDS as well as promote Vibrio harveyis survival in the SDS microniches. It is proposed that SDS arises through destruction of the outer epicuticle followed by infection by microbes [1]. The role of the increased activity of chitobiase would come into play after the epicuticle is damaged and the procuticle made of chitin is exposed. The increased activity caused by the mutation would eliminate the

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need for a chitinase enzyme, which would lead to quicker and more efficient degradation of chitin and quicker progression of the disease. The quicker progression of the disease would lead to the epidermis being reached sooner, leading to increased severity of the disease and even death. The ability for more chitin to be broken down could also promote the disease by attracting and allowing for the survival of more scavenger microbes in the lesions [1]. The more viable scavenger microbes in the legions could lead to more predator microbes moving into the region as well. Therefore, the more efficient breakdown of chitin would not only lead to a quicker progression of the disease, but also to a larger amount of potentially infectious microbes in the lesion. Besides disease cause and progression, Vibrio harveyi would also gain an evolutionary advantage over the other microbes in the microniches through the mutation. The mutated enzyme would allow Vibrio harveyi to use the chitin available instead of just the chitobiose, leading to increased propagation and survival in the microniche when compared to scavenger microbes, predator microbes, or microbes that need for chitin to be broken down first by other organisms before they can utilize it. Possible Experimental Improvements Even though there were no major problems associated with this experiment, there were still possible improvements that could be implemented. One major improvement could be made by unifying all the purification protocols so that the purifications were either all solution or all gel. Gel solution proved more difficult than solution purification, so solution purification would be recommended, not only for ease, but also for a better purification yield in future experiments. Even though it was found that solution purification was easier and more efficient, the use of gel purification in this case was

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necessary because of the need to separate DNA fragments with sizes above 40 bp, which is only possible through gel purification. Gel purification allowed for identification and separation of digested pUC19 from the incompletely digested pUC19, allowing for the use of only digested pUC19 in the ligation reaction, which made the ligation reaction more efficient. This could not have been accomplished with solution purification methods. Conclusion The successful cloning and sequencing of the Vibrio harveyi chb gene from Category 2 SDS will allow for identification of differences between the three SDS categories and the wild type. It could also possibly lead to a quick identification assay that could be used by aquaculture farms. The sequencing of the category 2 SDS chb gene led to the identification of specific mutations that may lead to chitobiase being able to break down chitin and promote SDS. The next step would be to use the found data to analyze enzyme activity by site directed mutagenesis, which could lead to an even greater understanding of the role of chitobiase in SDS. References [1] Vogan C.L., Powell A., Rowley, AF. (1998) Shell disease in crustaceans just chitin recycling gone wrong? Environ. Microbial. 10: 826-35. [2] Jannatipour, M., Soto-Gil, R.W., Childers, L.C., Zyskind, J.W. (1987) Translocation of Vibrio harveyi N,N'-Diacetylchitobiase to the Outer Membrane of Escherichia coli. J. Bacteriol. 169: 3785-91. [3] Soto-Gil, R.W., Zyskind, J.W. (1989) N,N-Diacetylchitobiase of Vibrio harveyi: Primary Structure, Processing, and Evolutionary Relationships. J. Biol. Chem. 264: 14778-83. [4] Tews, I., Perrakis, A., Oppenheim, A., Dauter, Z., Wilson, K.S., and Vorigas, C.E. (1996) Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease. Nature Structural Biology 3: 638 648.

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