Indian Journal of Biotechnology Vol 7, July 2008, pp 307-312

Amplification, cloning and sequencing of Enterococcus feacalis enolase gene A Shivaraj1,3, S Madhusudan1,2, C Ashok Kumar1, S Isloor3, Veere Gowda3, H J Dechamma1,

G R Reddy1 and V V S Suryanarayana1* 1Molecular Virology Laboratory, Indian Veterinary Research Institute, Hebbal, Bangalore 560 024, India

2Department of Biotechnology, Indian Academy Centre for Research & Post Graduate Studies, Hennur Cross Kalyananagar, Bangalore 560 043, India

3Department of Microbiology, Veterinary College, Hebbal, Bangalore 560 024, India

Received 26 April 2007; revised 12 November 2007; accepted 25 January 2008

α-Enolase, a key glycolytic enzyme, belongs to a novel class of surface proteins, which do not possess classical machinery for surface transport and transported on the cell surface through an unknown mechanism. It is a multifunctional protein and its ability to serve as a plasminogen receptor on the surface of a variety of hematopoetic, epithelial and endothelial cells suggest that it may play an important role in the intravascular and pericellular fibrinolytic system. Authors have amplified and cloned α-enolase gene of Enterococcus feacalis in a prokaryotic cloning vector, and then transferred it into Escherichia coli. The recombinant enolase vector (r-pBEnol) was isolated and sequenced. The sequence of the cloned enolase from E. feacalis was found identical to that of the E. feacalis V583. The sequence was submitted to NCBI nucleotide data bank and accession number (AM279410) was obtained. Keywords: Enolase, Enterococcus feacalis, cloning vector, sequence, nucleotide

Introduction The enzyme α-enolase (2-phospho-D-glycerate

hydrolyase) is a metallo enzyme that catalyzes the dehydration of 2-phospho-D-glycerate (PGA) to phosphoenol pyruvate (PEP) in the forward or catabolic direction in the second half of the Emden-Mayerhoff-Parmas glycolytic pathway1. In the reverse reaction (anabolic pathway) which occurs during gluconeogenesis, the same enolase catalyses hydration of PEP to PGA (phospho pyruvate hydratase). Enolase occupies a key position in the metabolic pathway of fermentation in general and glycolytic pathway in particular1. Enolase is one of the most abundantly expressed cytosolic protein in many organisms; more recently, it was found on the cell surfaces2-4. In vertebrates, this enzyme occurs as three isoenzymes—α-enolase is found in variety of tissues including liver, whereas β-enolase is almost exclusively found in muscle tissue, and γ-enolase is found in neurons and neuroendocrine tissues5. Besides its importance in glycolytic pathway, enolase has been usually identified to have role in systemic and autoimmune disorders. In addition to enzyme activity, enolase acts as a heat shock protein and, hence, may play a crucial role in transcription and other

pathophysiological functions6. Due to its abundance in most of the cells, it has become useful for studying basic mechanism of enzyme action and structural analysis. The accumulating evidence makes clear that enolase is a multifunctional protein. Streptococcus feacalis, now grouped as Enterococcus feacalis, is a common bacteria found in the oral cavity and is mostly involved in the dental caries7. Since dental caries is a major public health concern, inhibiting the growth of Streptococcus may be an ideal method to control the disease and the possible target may be enolase enzyme. Hence, the present research work aims at amplifying and sequencing of enolase gene from E. feacalis. Materials and Methods Isolation and Characterization of E. feacalis

Thirty five oral swabs were collected from healthy animals that had come to Veterinary College Hospital Bangalore, India. Swabs were directly streaked onto nutrient agar and brain heart infusion (BHI) agar plates. After an overnight incubation at 37oC, the colonies grown on the plates were further streaked onto a new agar plates until individual colonies were distinctly seen. The predominant bacterial colonies were individually streaked on slants and culture smears were Gram stained and observed under microscope. The bacterial isolates were characterized

—————— *Author for correspondence: Tel: 91-80-23418078; Fax: 91-80-23412509 E-mail: veluvarthy@yahoo.com

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by biochemical tests, growth of the colonies on 6.5% NaCl, growth at 10ºC and 40ºC and growth on Maconkey medium using laboratory manual of IMTECH8 and Bergey’s9. The tests confirmed that the isolates belonged to Streptococcus sp.

Isolation of Genomic DNA from E. feacalis E. feacalis genomic DNA was isolated as per the

standard protocol10. Single colony of the bacteria was inoculated in BHI broth and grown overnight. Cells were harvested from 5 mL of culture. 200 µL of genomic DNA extraction buffer [2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA and 100 µg/mL of Proteinase K], 200 µL of phenol:chloroform:isoamylalcohol (25:24:1) mixture, 0.3 g of acid washed beads were added and vortexed at the maximum setting and then 200 µL of TE [10 mM Tris-HCl (pH 8.0), 1 mM EDTA] and the DNA extracted from the aqueous layer was ethanol precipitated. The DNA pellet was dried and dissolved in 200 µL of TE and 3 µL RNase A to a final concentration of 10 mg/mL and once extracted with phenol:chloroform (25:24) and once by chloroform:isoamylalcohol (24:1). The DNA in the aqueous layer was ethanol precipitated with 1/10th volume of 3 M sodium acetate (pH 5.2). The DNA was washed with 70% alcohol, dried and dissolved in 100 µL of filtered quartz water (FQW). The quality of the DNA was checked by running on 0.8% agarose gel stained with ethidium bromide 0.5 µg/mL. A single intense band without smearing was noted. The extracted genomic DNA of the bacteria was used as template DNA for amplification of enolase gene.

Oligonucleotide Primers Primers encompassing full length enolase gene was

designed from the published sequence. The oligonucleoties were reconstituted to 1 nmol/µL stocks in sterile TE buffer. The primers were used at working dilution of 20 pmol/µL of sterile, filtered distilled water (FQW). The sequences of the primers were as follows;

Forward primer – 5�GCGGGATCCATGTCAAT-TATTACTGATATTTAC3� (34)

Reverse primer - 5�GCGGGATCCTTATAAGT-TGTAGAAAGATTTTAAGCCTTTGTA 3� (42)

Amplification of Enolase Gene by PCR Optimum annealing temperature was determined

by employing gradient PCR. Amplification reaction was performed in 0.5 mL tubes. Individual reactions (50 µL) contained 100 ng of the extracted DNA, 1× PCR assay buffer (250 mM Tris-HCl, 10 mM KCl,

1.5 mM MgCl2), 0.1 mM dNTP’s, 20 pmol each of forward and reverse primers, 1 unit of Taq DNA polymerase (Sigma, USA). Positive and negative samples were also run along with the DNA sample of interest to avoid the detection of false positive or false negative.

Reactions were thermally cycled in a Biometra-T Gradient (USA) thermal cycler system. PCR performed with forward and reverse primers involved an initial denaturation for 3 min at 94oC, followed by 30 cycles of 94oC for 1 min, 46-56oC gradient for annealing for 1 min, and extension at 72oC for 1 min 30 sec. Finally, the reactions were held at 72oC for 10 min. Specific and optimum amplification of the gene was seen at 52oC of annealing temperature. Subsequently, the gene was amplified at 52oC and the amplified PCR product (1.3 kb) was purified from low melting agarose gel, stained with ethidium bromide as per the standard protocol11 for further cloning. Cloning of Enolase Gene in Prokaryotic Cloning Vector

The purified PCR fragment was digested with BamHI (MBI Fermentas, USA) as per standard protocol. The purified DNA in a total reaction of 50 µL reaction mix contained 5 µL 10× buffer (supplied by the manufacturer) and one unit of enzyme. The digestion was carried out at 37oC for 3 h. Plasmid vector pBKS+ was also digested with BamHI enzyme in the similar way. The digested fragments were purified by phenol:chloroform extraction and used for ligation. Ligation reaction was carried out as per standard protocol using T4 DNA Ligase (MBI Fermentas, USA). Briefly 40 ng of insert DNA was mixed with 20 ng of linearized vector in presence of 1× ligase buffer (supplied by manufacturer). The ligated mixture (1 µL) was transferred into competent E. coli DH5α cells and plated on LB agar containing 50 µg/mL ampicillin, 1 mM IPTG and 0.02% X-gal. The white colonies observed after 16 h incubation were screened for the presence of the vector with insert by colony lysis and release of the insert by restriction enzyme digestion. Sequencing and Sequence Analysis

Sequencing of enolase gene from the recombinant plasmid (r-pBEnol) was done at sequencing facility of Bioserve Biotech India (P) Ltd, Hyderabad, India from both the directions. The sequence obtained was subjected to BLAST search. The sequence matched 98% with enolase gene of E. feacalis V583 (Fig. 1).

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Fig. 1—Sequence alignment showing homology between the cloned enolase gene and corresponding gene of E. feacalis V583

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Results and Discussion

Bacterial Isolation and Identification Of the 35 oral swabs streaked on BHI agar plates,

most of the plates showed the presence of mixed bacterial colonies. Five out of three showed the presence of the morphologically single type of colonies with the characteristic features of Streptococcus sp. One of the 5 plates was considered for the further isolation and purification of the bacteria. Single colony was re-streaked on BHI agar plate for characterization. On staining and microscopic observation it was found that Gram-positive cocci were predominant in single, pairs and chains. Further biochemical characterization confirmed the isolates as Enterococcus. Presence of the growth on crystal violet blood agar indicated that the bacteria belonged to Enterococcus, as crystal violet inhibits growth of Staphylococci and other Gram-positive bacteria. Growth was also seen in 6.5% NaCl but not in 12.5% NaCl as other Streptococci do not grow in the presence of NaCl and Staphylococci grows even in higher salt concentration upto 12.5%. Hence, the isolates were considered to belong to Enterococci. Further confirmation has come by the ability of the isolates to grow both at 10°C and 45°C. It has been reported that characteristic features like hydrolysis of esculin and deamination of arginine belong to group-D Streptococci12. The isolates could grow on Maconkey agar suggesting its ability to utilize bile salts.

Fig. 2—Agarose gel electrophoresis of PCR product of E. feacalis enolase: Lane M, 1 kb DNA ladder; lane 2, PCR amplified enolase gene (1.3 kb); and lane 3, Negative control.

Amplification of 1.3 kb Enolase Gene by PCR

Genomic DNA of the isolated Enterococcus was extracted as described above. The primers selected for amplification corresponded to α-enolase gene of Enterococcus. Initial standardization by many gradient-PCR has facilitated the specific amplification as observed by high intense band. The optimum annealing temperature was found to be 52oC. An intense single band of size approximately 1.3 kb was visible on 1% agarose gel stained with ethidium bromide (Fig. 2). No bands were visible in negative control, indicating that the amplified DNA was a copy of the specific gene of the template and the primers were highly specific for α-enolase. The use of specific primers coupled with size of amplified product of 1.3 kb indicated that the amplification might be α-enolase gene. Earlier amplified α-enolase gene reported from other sources, such as Onchocerca volvulus13 (1615 bp), latex of the rubber tree Hevea brasiliensis14 (1651 bp), Streptococcus sorbinus7 (1303 bp) and

Trypanosoma brucei (2500 bp), 15 were bigger than the present Enterococcus α-enolase gene. Cloning of Enolase in Prokaryotic Cloning Vector

The purified PCR product was ligated into BamHI site of pBKS+ cloning vector. The ligated mixture was transferred into competent E. coli DH5α cells and plated on X-gal-IPTG plate. Of 15 transforments appeared on X-gal-IPTG plate, 10 blue and 5 white colonies were observed. White colonies were streaked on fresh LB-ampicillin plate for further screening. Colonies were subjected to lysis and analyzed by 1% agarose gel stained with ethidium bromide to check their mobility (Fig. not shown). Plasmids from all the five were found to be migrating slower than the control vector, indicating that these are positive for the presence of inserted DNA. Further plasmid DNA from all the 5 isolates were prepared and subjected to BamHI enzyme digestion. The products were analyzed by 1% agarose gel stained with ethidium bromide using molecular size marker (Fig. 3). One of the recombinant plasmid DNA was further studied for the presence of enolase gene by sequence analysis. Sequence Analysis

The purified plasmid from one of the positive clones was sequenced using T7 and T3 primers from both the directions at sequencing facility of Bioserve Biotech India (P) Ltd., Hyderabad, India. A sequence

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Fig. 3—Agarose gel electrophoresis of BamHI digested pBKS+vector with and without enolase: Lane M, 1 kb DNA ladder; lanes 1-5, BamHI digested plasmid with enolase; and lane 6, BamHI digested control plasmid vector without insert.

Fig. 4—Nucleotide sequence of the cloned enolase gene [initiation (ATG) and termination sites (TAA) underlined]

of 1299 bp could be read from the ladder sequence (Fig. 4). As shown, the sequence has an ATG codon at nucleotide position 1-3 and a termination codon at nucleotide position 1297-1299. The sequence was subjected to restriction enzyme site computer programme www.neb.com (Fig. not shown). As observed in reported sequence of the enolase gene from data base, the sequence lacks BamHI site. The sequence was compared with the published sequence (Fig. 1), which showed homology of 98%. The sequence was submitted to NCBI nucleotide data bank and acc.no. (AM279410) was obtained. The GC content of the amplified gene was 40% and AT content was 60%. The derived amino acids from the sequenced nucleotides were found to be of 433 amino acids (Fig. 5) with the stop codon TAA at 1296 bp from ATG (start codon). The derived amino acid sequence had total of 70 acidic amino acids including 39 glutamic acid residues and 31 aspartic acid residues, and 35 basic amino acids including 12

Fig. 5—Deduced amino acid sequence of E. feacalis α-enolase gene (plasminogen binding motifs underlined)

Arginine residues and 23 lysine, yielding a net negative charge on protein rendering the protein acidic. In Pneumococcal α-enolase, an internal binding motifs 248-FYDKERKVY-256 is a major site for binding to plasminogen as reported earlier16. The derived amino acid sequence reveals that the above motif is present at the position 248-FYDKEKGGYV-257. C-terminal lysines in the epitope 428-KSFYNLKK-435 are important for binding to plasminogen in case of Streptococcal surface α-enolase protein as reported earlier17. However, in derived amino acid sequence, this epitope 426-KSFYNLKK-433 comes two amino acids earlier in the C- terminal end; hence, the binding epitopes are intact with mainly lysine residues, which are important for plasminogen binding. Further studies are being carried out to express the enolase gene in expression vector for the study of its enzyme

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activity. Acknowledgement

The authors wish to thank the Director, Indian Veterinary Research Institute, Hebbal, Bangalore for providing necessary facilities for carrying out the work. References

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