Cloning and Expression of Glutamate Dehydrogenase A (gdhA) from the Endosymbiont Spp.

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Link to Item http://hdl.handle.net/10150/146916 CLONING AND EXPRESSION OF GLUTAMATE DEHYDROGENASE A (gdhA)

FROM THE COCKROCH ENDOSYMBIONT BLATTABACTERIUM SPP.

by

RYAN CHRISTOPHER WONG

A Thesis Submitted to The Honors College

In Partial Fulfillment of the Bachelors of Science degree with Honors in

Biology – Biomedical Sciences

THE UNIVERSITY OF ARIZONA May 2010

Approved by:

Dr. Nancy A. Moran Department of Ecology and Evolutionary Biology Date: May 5, 2010 STATEMENT BY AUTHOR

I hereby grant to the University of Arizona Library the nonexclusive worldwide right to reproduce and distribute my dissertation or thesis and abstract (herein, the "licensed materials"), in whole or in part, in any and all media of distribution and in any format in existence now or developed in the future. I represent and warrant to the University of Arizona that the licensed materials are my original work that I am the sole owner of all rights in and to the licensed materials, and that none of the licensed materials infringe or violate the rights of others. I further represent that I have obtained all necessary rights to permit the University of Arizona Library to reproduce and distribute any nonpublic third party software necessary to access, display, run or print my dissertation or thesis. I acknowledge that University of Arizona Library may elect not to distribute my dissertation or theses in digital format if, in its reasonable judgment, it believes all such rights have not been secured.

Signed: Date: May 5, 2010 ABSTRACT

Cockroaches (Dictyoptera:Blattaria) have a unique ability to store excess nitrogen as uric acid in their fat bodies. It has been suggested that cockroach bacterial endosymbionts may be responsible for using stored nitrogen in fat bodies and turning it into usable nitrogen (e.g. amino acids). Recent sequence data has suggested that the cockroach endosymbiont Blattabacterium spp. could have nitrogen recycling ability through urease (ureA) and glutamate dehydrogenase

(gdhA). To determine whether Blattabacterium gdhA is able to use ammonia, an assay was developed to first express the protein in E. coli and then observe ammonia activity through spectroscopy as well as ammonia activity of the purified protein. Blattabacterium gdhA was cloned into E. coli expression vectors, and the protein expression levels were assessed using SDS protein gels and Bradford’s Reagent assays. Detectable expression levels were not found in the

SDS gel or Bradford Reagent, suggesting that the gdhA protein may be expressed at levels difficult to detect with the current assays. This work invites further exploration of this exceptional system through improved techniques and revised experimental design. INTRODUCTION

The study of bacterial mutualism has become an increasingly popular and fruitful area of research. Insect-bacterial symbiosis are quite ubiquitous (Buchner 1965), with many insects – including, aphids, , tsetse flies, and psyllids – having ancient associations with their mutualistic . Bacterial symbionts have been associated with a wide range of functions: nutritional complementation (Shigenobu et al. 2000), tissue development (Koropatnick et al.

2004), immune system regulation (MacDonald and Monteleone 2005), reproduction and sex determination (Stouthamer et al. 1999; Hurst and Jiggins 2000; Haselkorn 2010), and protection against harmful infection (Oliver et al. 2003; Scarborough et al. 2005). With the introduction of molecular techniques, information regarding the evolutionary history and the functionality of bacterial symbionts has become increasingly more accessible (Moran et al. 2005).

In obligate endosymbiotic systems, the endosymbiont often produces nutrients and molecules that the host cannot obtain from its diet. In most cases, the endosymbiont genome loses non-essential genes that are not required for bacterial maintenance or required for the synthesis of nutrients required by the host (Moran et al. 2005). In the pea aphid endosymbiont

Buchnera, the amino acids that are not obtained through the aphid diet or synthesized by the aphid are complemented by the biosynthetic pathways encoded by a significantly reduced

Buchnera genome (Shigenobu et al. 2000). The Buchnera endosymbiont assisted aphids in circumventing nutritional barriers allowing for increased fitness and survival only feeding on plant phloem sap. Studying the fascinating roles of endosymbiosis in various species helps unveil some of the mechanisms that allow species to thrive in nutrient-limited environments.

Cockroaches (Dictyoptera:Blattaria) provide another example of a symbiotic relationship that overcomes nutrient limitations on the insect host. Cockroach diet usually consists of plant matter, but may also include decaying animal matter when available (Bell et al. 2007). Although plant material is readily accessible, it is relatively low in nitrogen. Cockroaches must therefore employ mechanisms to efficiently use, retain and acquire dietary nitrogen, which is essential for synthesizing amino acids, the building blocks of proteins. Generally, animals excrete excess nitrogen as waste products, but cockroaches store excess nitrogen as uric acid in fat bodies. It has been suggested that this uric acid can be recovered for future use when nitrogen sources are lacking (Cochran 1985); however, the pathway allowing cockroaches to utilize the stored uric acid has not been defined. There is evidence that this ability may be linked to the cockroach obligate endosymbiont, Blattabacterium spp.

Blattabacterium is a member of the phylum (Bandi et al. 1994) and has been associated with cockroaches for at least 140 million years. Only found elsewhere in a basal extant termite species (Lo et al. 2003), Blattabacterium spp. exists in all species of cockroaches examined to date (Dasch et al. 1984). Blattabacterium exist at high concentrations within bacteriocytes, which are located in the fat bodies containing the primary uric acid stores (Gier 1936). This physical location and close association does not seem coincidental. It has been suggested that Blattabacterium assists in recycling uric acid into usable nitrogen (Lo et al. 2003). When antibiotics or lysozyme were injected into the fat bodies to reduce the amount of Blattabacterium, the amount of uric acid increased dramatically and the cockroaches, especially in the nymph stage, showed inhibited growth or even death (Brooks and

Richards 1955; Cochran 1985). This suggests that Blattabacterium exists as a potential link in the use of stored uric acid and is necessary for insect growth and development. Unfortunately,

Blattabacterium spp. cannot be cultured in the laboratory (Brooks 1970) and as a result, measuring its potential uricolytic activity directly remains a challenge. The recent sequencing of the full Blattabacterium genome from two cockroach species has revealed genes coding for urease (ureA) and glutamate dehydrogenase (gdhA) (Sabree et al.

2009). Both genes suggest nitrogen recycling functions of the bacterial endosymbiont (Lopez-

Sanchez et al. 2009; Sabree et al. 2009). While the ability to degrade uric acid in the fat bodies remains unclear, both urease and glutamate dehydrogenase requires urea, a by-product of uric acid degradation. Urease can degrade urea and release ammonia that can be used by glutamate dehydrogenase to synthesize glutamate. In this process (Figure 1), glutamate dehydrogenase takes 2-oxoglutarate and ammonia and forms glutamate which serves at the precursor for glutamine and other amino acids. Glutamate dehydrogenase is especially important in cases where sources of energy are restricted because it is ATP-independent and requires ammonia

(Reitzer 2003).

The Blattabacterium-containing bacteriocytes exist among the fat bodies, an area potentially high in ammonia. A study showing that gdhA in E. coli is more active when ammonia concentrations are high (Helling 1994) makes gdhA a candidate for exploring cockroach nitrogen utilization. To demonstrate its function experimentally, the ability of

Blattabacterium gdhA to utilize ammonia will be tested using heterologous gene expression in E. coli and observing the activity of the construct to use ammonia. This study will attempt to uncover the ability of Blattabacterium gdhA to use ammonia to form useable glutamate. This

would further support the hypothesis that Blattabacterium spp. is involved in removing toxic

ammonia and recycling uric acid-stored nitrogen into useful glutamate.

MATERIALS AND METHODS

Bacterial Strains and Growth Conditions

Cultures of E. coli BL21 (DE3), E. coli ∆gdhA:Kan, and E. coli DP340 ∆gdhA were

grown at 37°C in Luria Broth media (1% bacto-tryptone, 0.5% yeast extract, 1%NaCl). Minimal

media conditions contained minimal salts plus 0.4% glucose as described by J. Brenchley

(Brenchley 1973). E. coli cultures were supplemented with their respective antibiotics in the

following concentrations: ampicillin (100μg/ml) and kanamycin (50 mg/ml). Single gene

knockout E. coli strains were obtained from Coli Genetic Stock Center (Baba et al. 2006).

Preparation of BL21 + pGEM-T : gdhA

The gdhA gene was amplified from previously isolated Blattabacterium spp. DNA with

Taq polymerase (New England Biolabs, Massachusetts, USA) using sets of gene-specific

primers (Table 1). The Table 1: Primers gdhA_ZS_F4 GAA TTC ATG TCA AAA AAA AAC PCR product was purified gdhA_ZS_R4 AAG CTT TGG AAA AAT ACC TA M13F TGT AAA ACG ACG GCC AGT using Qiagen PCR M13R AGG AAA CAG CTA TGA CCA TG T7 Promoter TAA TAC GAC TCA CTA TAG GG purification kit and then T7 Reverse GCT AGT TAT TGC TCA GCG G gdhA+ GGA ATT CAT GTC AAA AAA AAA CCA AAG cloned into the pGEM-T gdhA_R1 GCG AAG CTT TGG AAA AAT ACC TAA TTT TTC vector containing gdhA_F3 GAA TCC AAA CTA ACA ATT GAG G gdhA_R3 GAA TTC GTT ATT TCC CAT AAG AG ampicillin resistance using gdhA F2 CGA ATT CGA TTT GTA TCT GGT CAT TCC G gdhA F1 CGA ATT CCG ATG TTT GAT GAA AAT TTT ATT GTG Bolded type indicates HinD III or EcoRI restriction site the pGEM-T cloning kit (Promega, Wisconsin, USA). The plasmid construct was transformed into freshly prepared chemically competent BL21(DE3) (Chung et al. 1989). Positive transformants were selected on LB:Amp + IPTG+Xgal via blue/white screening. PCR, using vector specific primers, M13F and M13R, and gene-specific primers confirmed presence of the insert. The plasmid was extracted using the Qiagen Plasmid Extraction kit (Qiagen, CA, USA), sequenced through UAGC Gene Core. The sequence was used as a query in BLASTX searches against the NR protein database (www.ncbi.nlm.nih.gov) to further confirm the identity of the insert.

Preparation of BL21 + pET21b(+):gdhA

Plasmid from BL21 + pGEM-T:gdhA and pET21b(+) were each double digested using

EcoRI and HindIII (New England Biolabs) at 37°C overnight. The restriction enzymes were deactivated for 20 minutes at 65°C. Digested pGEM-T:gdhA plasmid was run through 1% agarose electrophoresis gel and the released gdhA gene was gel purified by Qiagen Gel

Extraction Kit (Qiagen). The pET21B vector was purified with the Qiagen PCR purification kit. gdhA and pET21B were ligated with T4 DNA ligase (Fermentas, MD, USA) and transformed as above. Positive transformants were selected from LB:Amp plates (100μg/ml). The insert was confirmed as above with T7 promoter and T7 reverse primers as well as gene specific primers.

Preparation of E. coli ∆gdhA:Kan + pGEM-T:gdhA

Purified pET21b(+):gdhA plasmid and pGEM-T were double digested with NdeI and StyI overnight as above (New England Biolabs) to ensure directional cloning. Purification, ligation, and transformation into E. coli ∆gdhA:Kan were performed as above. The insert was verified by PCR using vector-specific (M13F/M13R), gene-specific (gdhA_F3/R3), and combinations of vector- and gene-specific primers.

Protein Overexpression

A starter culture of BL21 + pET21B+:gdhA and BL21+pET21B was grown overnight in ampicillin. The overnight culture was diluted 1/100 in 5ml of fresh LB/ampicillin and allowed to grow to log-phase (O.D. 0.3-0.6). The culture was induced with 0mM, 0.1mM, 0.5mM, or 1mM

IPTG. After 2 hours, 4 hours and overnight post-induction, a 250μL sample was removed and centrifuged at 4,000rpm for 4 minutes from each sample. The supernatant was poured off and the sample was frozen at -20°C. The samples were tested for over-expression of gdhA protein.

Protein Overexpression Assays

Bradford Reagent

To qualitatively determine if protein was over-expressed, a Bradford reagent assay was conducted. Frozen samples from above were thawed in ice and lysed using 50μL B-PER

Bacterial Protein Extraction Reagent (Pierce Biotechnology, Illinois, USA). The mixture was vortexed for at least 1 minute on high. A protein standard was created using various concentrations of BSA from 10mg/ml to 5μg/ml. In a 48 or 64 well plate, 5μL of sample or standard was added to 250μL of Bradford reagent. The plate was gently mixed for 30 seconds by light shaking and allowed to equilibrate for at least 5 minutes at room temperature prior to visual analysis. 6x His Tag protein samples collected by Qiagen Ni-NTA Spin Kit protocol were also tested with Bradford Reagent (Sigma Co., Missouri, USA). SDS Protein Gel

An SDS protein gel was conducted using protein expression assay frozen samples to identify the correct protein was being expressed. Bacterial cells were lysed with 50μL B-PER

Bacterial Protein Extraction Reagent as above. 20 μL of cell lysate was added to 20 μL Laemmli

Buffer + 20% DTT (Bio-Rad, California, USA) and incubated for 5 minutes at 95°C. The SDS gel was run for 1.5 hours at 100mV. The gel was placed in destain solution (30% methanol, 10% glacial acetic acid) and allowed to equilibrate for 30 minutes and then in Coomassie brilliant blue

R250 solution (0.25% Coomassie Brilliant Blue, 50% methanol, 10% glacial acetic acid) for 1 hour. The gel was then placed in destain solution and alternated between 30 seconds in the microwave on high and shaking. Destain solution was exchanged every 3rd or 4th time until a clear background was revealed (Sambrook et al. 1989).

Results and Discussion

Amplification of Blattabacterium gdhA gene

Amplification of Blattabacterium gdhA from previously isolated DNA was challenging requiring 4 sets of primers and multiple optimizations including PCR additives. The primer combination gdhA_F1 and gdhA_R1 bound to template DNA inconsistently resulting in little to no amplication. To account for variation in primer binding properties and template DNA secondary structure, optimization was attempted through an annealing temperature gradient and the addition of the PCR additive dimethyl sulfoxide (DMSO) were implemented. To check the effect of annealing temperature on the reaction, a gradient from 42°C to 55°C was conducted, but amplicfication was not achieved. The addition of 5% DMSO to the PCR reaction mixture was intended to break up disulfide bonds and make regions of the DNA more accessible to primer binding (de Miranda et al. 2010). Again, amplification could not be with the addition of DMSO, suggesting that the annealing temperature and template DNA secondary structure were not the cause of inconsistent amplification.

The inability to achieve PCR amplification with the primers gdhA_F1, +, F2, R1, and R2 prompted a reanalysis of the gdhA sequence and the corresponding primers. The beginning of the gdhA sequence contains includes a rather lengthy string of adenine and cytosine bases, and an overall %GC of 29.6, which is generally considered low compared to the reported optimal %GC between 39%-58% for primer design (Buck et al. 1999). Suboptimal %GC reduces the binding strength of the primer to the template DNA, and without effective and specific binding, the primer is inefficient and may not amplify the gene fragment. In addition, the primers used had

rather low melting temperatures (Tm) between 42°C and 50°C, which are below the ideal Tm for primers range from 53°C to 65°C (Buck et al. 1999). Lower melting temperatures can lead to less specific primer-template binding and variation in amplicon length. To resolve these complications, primers (gdhA_F3, gdhA_R3) were designed outside of the gdhA coding region

(~100bp from each end) in an area more suitable for designing primers. With this fragment cloned into a vector, the original primers were used to amplify the gdhA region inside the larger fragment. Use of the resulting plasmid permitted amplification with the lower annealing temperatures of the original primers without compromising primer specificity and without the added interference from competing genomic DNA. The original primers, designed to amplify from the start codon, ensure in-frame translation and reduce the likelihood of introducing nonsense mutations that would terminate protein expression.

The primers gdhA_F3/R3 yielded amplicons, but amplified non-specifically from

Blattabacterium DNA resulting in two main bands. The band with the expected size was gel purified and cloned into pGEM-T cloning vector and transformed into E. coli BL21(DE3). The insert was sequenced and then compared to GenBank protein sequence database, using

BLASTX, to confirm that the amplified product encodes Blattabacterium GdhA.

Accounting for errors in the original primer design suggested by absent gene fragments, the primers were reviewed using Gene Runner 3.05 software (Hastings Software); a program designed to predict the type and strength of any primer-primer interactions. It was discovered that the primer combinations, except for gdhA_F3 and R3, preferentially formed dimers with a

∆G between -5 to -11 which would be difficult, if not impossible to break, under typical PCR conditions or with optimization additives.

A new set of primers were designed (gdhA_F4/R4), analyzed, and used for PCR. Instead of amplifying off of the pGEM-T construct, the gene fragment was amplified from

Blattabacterium DNA to limit the accumulation of PCR errors. The fragment was cloned into

pGEM-T, and then into pET-

21b(+). The resulting plasmid pET-

21b(+):gdhA (Figure 2) was

created. Following inconclusive

SDS-PAGE gels, the pET-

21b(+):gdhA insert was sequenced

to ensure that gdhA was cloned in

the correct reading frame. It was

discovered that a PCR-based error

resulted in a nonsense mutation in

the 17th codon. A nonsense

mutation this early in the sequence can only result in small, presumably nonfunctional protein that is undetectable by the Bradford’s reagent or SDS protein gel. A second strain from the original transformation was also sequenced contained only other synonymous substitutions. This construct was used for protein assays.

Evaluating Protein Expression

To determine optimal protein expression conditions, incubation temperature, IPTG induction concentrations, and induction length were varied. GdhA protein was expected to weigh approximately 53 kilodaltons, determined by EditSeq (Lasergene, DNAstar, Wisconsin, USA).

Varying the growth conditions, the gdhA construct at 4 hours post induction with 1mM IPTG revealed a promising band at the expected size (Figure 3). Measuring total protein concentration in 6xHis-tag/ Ni-NTA purification fractions using the Bradford protein assay lead to inconclusive results. There were no observable differences in color among samples (Figure 4), even among protein extracts from different induction temperatures (25°C and 37°C). At 25°C,

Figure 3: SDS Protein Gel (200mV for , stained with Coomassie Brilliant Blue – R250, Destain with 30% Methanol/10% glacial acetic acid solution. Arrow indicates the expected gdhA size on the protein ladder. The double sided arrow indicates possible gdhA protein. the growth rate of E. coli was inhibited requiring more than 5 hours to reach an OD600 of 0.3 which is optimal for induction. An O/N induction was tested at room temperature growth, which showed a reduced amount of protein when separated on an SDS gel (not shown).

Altering growth conditions was necessary to find optimal conditions that produced the most protein. Regulating the amount of protein expression through IPTG concentrations, growth temperature and length of induction can impact protein expression significantly. Temperature variation, in a Streptococcus pneumoniae study (Pandya et al. 2005), induced protein expression variation in 29% of the 1717 genes when grown at different temperatures. Blattabacterium gdhA is expressed in the cockroach host approximately at room temperature. Expressing these proteins at 37°C may lead to misfolding, sequestering into inclusion bodies or reducing protein activity

(Ideno et al. 2004; Martinez-Alonso et al. 2009). E. coli BL21 shows increased solubility of protein expressed from a plasmid when grown at 25°C (de Groot and Ventura 2006). In the current study, the SDS gels only indicated a reduction of total protein content when bacteria was grown at 25°C, as opposed to a reduction in the expression levels of individual proteins. Limitations of Bradford’s Reagent

It is important to mention the limitations of the Bradford’s assay. Bradford’s reagent has

a limited range of sensitivity and is variable depending on total protein concentration. Protein

concentration was only controlled through OD600 normalization when inoculating the new culture

from the overnight culture. At the initial time point before induction, equal concentrations of

bacteria were present in the cultures. However, the introduction of IPTG has proven to be

relatively harmful to bacteria (Hansen et al. 1998). This could cause differences in the

concentration of bacteria during induction causing inaccurate protein expression results. In

addition, slight differences in

bacterial concentration and variation

in total protein concentration may

overshadow any colorimetric

differences that may be seen with

Bradford’s reagent. Looking at 4

hours post induction and overnight

post induction (Figure 5), there

seem to be differences in the

intensity of blue compared to the Figure 5: Bradford Reagent Assay with gdhA:pET21b(+) in BL21. Grown at 37°C. Negative Control: pET21B control. However, a Qiagen 6xHis-

tag/ Ni-NTA protein purification did not reveal any recoverable gdhA protein containing the

6xHis tail. Since the protein content of the eluent was tested through Bradford’s, protein may

have been expressed but only at low levels. An SDS gel may be necessary to further examine protein expression due to its increased sensitivity. It is suggested that gdhA protein may only be required in small quantities and cannot manifest its presence in a qualitative test.

New Directions

Many factors can account for reduced expression of protein in a foreign host. A codon bias may exist where specific organism may respond better to a different set of synonymous sequence. Foreign protein may be under expressed especially in highly divergent sequences. For example, E. coli shows a relatively high degree of codon bias that relates to level of expression

(McPherson and Wootton 1983). In addition, codon bias seems to reduce expression especially in systems with high %AT or low %GC (Teller et al. 1992). The Blattabacterium gdhA gene lies at the lower range of GC content at 32%GC (GeneRunner). Endosymbiont genomes typically have an increased %AT (Moran et al. 2009) which may help explain the low amounts of

Blattabacterium gdhA expressed in E. coli.

Since a low protein concentration will not manifest itself in an SDS gel or in a Bradford

Assay clearly, a complementation assay with Blattabacterium gdhA in an E. coli strain missing gdhA may provide a better method for revealing functionality of gdhA. If gdhA is expressed at low concentrations suggested by the Bradford results, a change in growth rate of the complemented E. coli strain compared to the non-complemented strain could be observed. This would in turn test the functionality of gdhA independent of protein concentration. Two E. coli gdhA mutant strains have been obtained from the Coli Genetic Stock Center: JW1750-2, which has had its endogenous gdhA inactivated by the insertion of a kanamycin resistance gene cassette

(Baba et al.), and PA340, which has a single, enzyme-inactivating Lys-92 to Gln-92 substitution in gdhA (Helling 1994). The introduction of pGEM-T:gdhA plasmid into either E. coli gdhA knockout is still in progress.

Before initiating complementation, it is important to recognize that E. coli has access to the glutamine oxoglutarate aminotransferase (GOGAT) pathway as well as glutamate dehydrogenase. GOGAT (Figure 6) uses ammonia, glutamate and ATP to synthesize glutamine and eventually more glutamate. This pathway exists as an alternate mechanism to synthesize glutamate. It complements glutamate dehydrogenase as GOGAT is most active in an energy rich environments due to ATP requirements (Helling 1994). This presents a problem because conducting this assay in normal growth media would allow the E. coli strains the liberty of using either glutamate synthesizing pathway. Glutamate could be produced through either pathway, skewing complementation results; however, GOGAT can only work if glutamate is already present. Growth in minimal media containing only ammonia as the nitrogen source and glucose as a carbon source would force the bacteria to take advantage of gdhA. Any growth that exceeds growth of the negative control can therefore be attributed to complementation by the introduced

Blattabacterium gdhA and will suggest ammonia recycling ability.

As a final way to improve the experimental design, it would be suggested to re-amplify the gdhA insert using a polymerase with a higher fidelity. The Taq polymerase (New England

Biolabs) has an error rate 6 times higher than the Pfu Turbo polymerase (New England Biolabs) which has proofreading capabilities (Cline et al. 1996). The reasoning behind this would be to construct a vector with an insert that is as similar to the original coding sequence as possible. In order conduct this efficiently, gdhA_F4 and gdhAA_R4 would need to be revised to include necessary base pairs tails at the ends of the restriction sites. This enables the cleaned PCR product to be double-digested and then ligated directly into the vector. Once cloned into both pET-21b and pGEM-T, protein expression and complementation assays should be performed again.

The implications of discovering the ability of Blattabacterium to use ammonia for synthesis of usable amino acids would provide the insight into the cockroach’s unique system of combating a limited nitrogen environment. From an evolutionary and ecological perspective, understanding this pathway would further support the importance of bacterial symbionts in the general biology of different insects and their contribution to the processes of adaption, co- evolution and ecological persistence.

Acknowledgements

I would like to thank Zakee Sabree for being the main source of advice throughout this project and John Stavrinides for technical support, consultations, and assistance in the preparation of the manuscript. References

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