In present study, we aimed to express T4 Lysozyme gene (gene e) in Streptococcus salivarus subsp. thermophilus to create better probiotics for poultry. The Esherichia coli plasmid, Bluescript II SK +/-harboring gene e named pL1, was converted to a new E. coli-Streptococcus sp. shuttle vector (pL2) by cloning and inserting Streptococcal replication origin of pTRW10 vector into pL1. pL2 plasmid isolated from E. coli was introduced into S. salivarius subsp. thermophilus and Lactococcus lactis cells by electro-transformation. The lysozyme enzymes expressing by these bacteria were found to be active on Micrococcus luteus cells and thereby preventing their growth on assay plates. Thermostability of these enzymes from the recombinant bacteria was also found different from each other. The lysozyme expressed by S. salivarius subsp. thermophilus cells seemed to increase its capacity for thermoresistance and was not denaturated at 70°C for 15 min. In contrast, the enzyme expressed by L.lactis and E. coli cells were easily denaturated when exposed to the same temperature treatment

Akinalp, AS

Asan, M

Ozcan, N

English

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African Journal of Biotechnology Vol. 6 (8), pp. 963-966, 16 April 2007 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2007 Academic Journals    Full Length Research Paper  Expression of T4 Lysozyme Gene (gene e) in Streptococcus salivarius subsp. Thermophilus  Ayca S. Akinalp1, Meltem Asan2 and Numan Ozcan3*  1Department of Animal Science, Faculty of Agriculture, Kahramanmaras Sutcuimam University, Kahramanmaras-Turkey. 2Department of Animal Science, Faculty of Agriculture, Çukurova University, 01330, Adana-Turkey. 3Department of Animal Science, Faculty of Agriculture, Çukurova University, 01330, Adana-Turkey.  Accepted 5 March, 2007  In present study, we aimed to express T4 Lysozyme gene (gene e) in Streptococcus salivarus subsp. thermophilus to create better probiotics for poultry. The Esherichia coli plasmid, Bluescript II SK +/- harboring gene e named pL1, was converted to a new E. coli-Streptococcus sp. shuttle vector (pL2) by cloning and inserting Streptococcal replication origin of pTRW10 vector into pL1. pL2 plasmid isolated from E. coli was introduced into S. salivarius subsp. thermophilus and Lactococcus lactis cells by electro-transformation. The lysozyme enzymes expressing by these bacteria were found to be active on Micrococcus luteus cells and thereby preventing their growth on assay plates. Thermostability of these enzymes from the recombinant bacteria was also found different from each other. The lysozyme expressed by S. salivarius subsp. thermophilus cells seemed to increase its capacity for thermoresistance and was not denaturated at 70°C for 15 min. In contrast, the enzyme expressed by L. lactis and E. coli cells were easily denaturated when exposed to the same temperature treatment.   Key words: Expression, lysozyme, Streptococcus salivarius subsp. thermophilus, thermostability.   INTRODUCTION  Lysozyme (EC 3.2.1.17) is an enzyme that catalyzes the hydrolysis of glycosidic bonds between N-acetyl glucose-mine (NAG) and N-acetyl muramic acid (NAM), which are present in peptidoglycan heteropolymers of prokaryotic cell wall (Nicklin et al., 1999). T4 lysozyme is a murami-dase which facilitates lysis of T4 phage-infected cells, thereby releasing replicated phage particles (Tsugita et al., 1968; Tsugita, 1971). Although it is similar to hen egg white lysozyme in the structure of its active site and its site of attack on the peptidoglycan cell wall (Mathews et al., 1981), T4 lysozyme presumably lyses T4-infected cells by attack on the cell wall from the cytoplasmic side (Perry et al., 1985). The phage lysozyme exhibits a much greater specific activity when assayed with Escherichia coli as a substrate than egg white lysozyme (Tsugita et al., 1968). In addition to its anti-bacterial action, lysozyme may have analgesic, anti-tumor, anti-metastatic and  anti-inflammatory  activities (Sava et al., 1989).  Lysozyme  is     *Corresponding author. E-mail: nozcan@cu.edu.tr. Tel: +90 3223386813, Fax: +90322 3386576. of widespread distribution in prokaryotes and eukaryotes such as bacteriophages, bacteria, plant and animals (Schindler et al., 1977). The gene encoding lysozyme is cloned from mammalian tissues and secretions, insects, plant, protozoa, bacteria and viruses (Schindler et al., 1977; Eschenlauer et al., 2000) and then expressed in some bacteria, fungi, yeast and plants (Eschenlauer et al., 2000; Oberto and Davison, 1985; Castanon et al., 1988; Cui et al., 1990; Tsuchia et al., 1992; Kim et al., 2004; Re-Vega et al., 2004). Gene e encoding lysozyme E cloned from bacteriophage T4 has been expressed in E. coli (Owen et al., 1983; Perry et al., 1985) and then its nucleotide sequence has been determined (Owen et al., 1983). Recombinant E. coli cells harboring pUC18L and pRS16L, which both expressing gene e, are grown in L and LB medium without lysis but burst out easily in hypotonic environment such as distilled water or 10 mM Tris-HCl, pH 8 buffer (Guzel et al., 2002). Probiotics are defined as viable microorganism which, upon ingestion in sufficient amounts, exert health benefits include alleviation of symptoms of lactose intolerance, enhancement of the immune system, reduction of the duration of rotavirus diarrhea, a decrease of faecal bacte- 964           Afr. J. Biotechnol.    rial enzyme activity and mutagenicity, prevention of recurrence of superficial bladder cancer, and prevention of atrophic diseases (Fuller, 1992). Probiotic Lactic Acid Bacteria (LAB) are commercialized mainly in dairy food and feed supplements (Salminen and Wright, 1993; Wallace and Chesson, 1995).  The present study is aimed to express the gene e in Streptococcus salivarius subsp. thermophilus to create recombinant probiotic for poultry, which combines bene-ficial effects of both probiotic and lysozyme enzyme.   MATERIALS AND METHODS  Bacterial strains, plasmids and growth media  S. salivarius subsp. thermophilus FI8976, L. lactis IL1403 strains and pTRW10 plasmid DNA (E. coli-Streptococcus sp. shuttle vector) were obtained from M. Sait EKNC (University of Kahramanmaras Sütcü Imam, Kahramanmaras, Turkey). The host bacterium E. coli DH5α and the indicator bacteria, Micrococcus luteus, were purchased from Stratagene and Sigma-Aldrich, respectively. Recombinant plasmid DNA pL2 was created from pL1 (pBluescript II SK +/- plus gene e, not published) in this study. S. salivarius subsp. thermophilus and Lactococcus lactis were cultured in M17 at 42oC and 37oC, respectively. E. coli and M. luteus cells were cultured in LB (Luria Bertani) at 37oC. Growth media were supplemented with 10 µg/mL ampicillin to select recombinant S. salivarius subsp. thermophilus and L. lactis and 50 µg/ml ampicillin for recombinant E. coli cells. All recombinant techniques were performed according to Sambrook et al. (1989) if otherwise is stated.    Construction, transformation and isolation of plasmid DNA  To construct pL2 plasmid, the coding sequences of the Streptococcal origin of replication from pTRW10 were inserted into HindIII restriction site of the pL1 plasmid. The plasmid pL2 constructed was transformed into S. salivarius subsp. thermophilus FI8976 (Wei et al., 1995), L. lactis IL1403 (McIntyre and Harlender, 1989; Holo and Nes, 1989) and E. coli DH5α (Dower et al., 1988) by electroporation. Plasmid DNA was isolated from E. coli, S. salivarius subsp thermophilus and L. lactis according to Birnboim and Doly (1979).   Plate growth inhibition assay  The lysozyme activity of recombinant bacteria was screened by growth inhibition of lysozyme sensitive M. luteus on the plates. All the recombinant bacteria were grown until 1.2 (OD600nm). The cultures were harvested by centrifugation. The bacterial supernatant containing lysozyme was exposed to 37, 50 and 70°C for 15 min. Indicator strain M. luteus (500 µl) was grown until 0.6 (OD450) (Anonymous, 2007) and then added into the supernatant (1000 µl). The mixture was incubated at 37°C for 1 h. After incubation, the mixture was spread on the surface of LB plates and incubates overnight at 37°C. The growing M. luteus colonies on the plates were then accounted.   Thermostability of lysozyme enzyme on SDS-PAGE  To determine the thermostability of lysozyme enzymes, the supernatant of recombinant bacteria is exposed to various tempera-     tures (37, 40, 50, 60, 70, 80, 90 and 100°C) for 15 min before centrifugation at 15.000 rpm for 15 min to remove denaturated proteins. The supernatant was then mixed with equal volume of tricholoroacetic acid. Protein was then collected by centrifugation. Protein analysis was performed by using a denaturating polyacryla-mide gel (SDS-PAGE, 12% (w/v)). After electrophoresis, protein bands were visualized by comassie blue staining procedure (Laemli, 1970).   RESULTS   To construct pL2 plasmid vector, 2300 bp HindIII cut DNA fragment of E. coli-Streptococus shuttle vector (pTRW10), carrying Streptococcus origin of replication, was trans-fered into HindIII site of pL1 vector harboring of pBluescript II SK +/- and lysozyme gene (gene e) of Bacteriophage T4 (Figure 1).  Ligation mixture was used to introduce into E. coli cells and resulting E. coli/pL1 and E. coli/pL2 transformants were grown as mixed population in LB-Agar medium including 50 µg/mL of ampicillin. Plasmid DNA was isolated from mixed population of E. coli harboring either pL1 or pL2 was directly used to transform S. salivarius subsp. thermophilus and L. lactis cells by electro-transformation. S. salivarius subsp. thermophilus/pL2 and L. lactis/pL2 colonies could grow on M17 agar medium supplemented with 10 µg/mL ampicillin because of its Streptococcal origin of replication carried on the plasmid. The pL2 plasmid was isolated from S. salivarius subsp. thermophilus and L. lactis cells. It was then re-electro-transformed into E. coli to construct E. coli/pL2 cells. Lysozyme E expressed by three recombinant bacteria (S. salivarius subsp. thermophilus/pL2, L. lactis/pL2 and E. coli/pL2) were tested for lysis on assay plates. The recombinant bacteria yielded clear zones around colonies by lysing M. luteus cells grown on all the surface of the test plates.  The lysozyme of S. salivarius subsp. thermophilus was found to be active between pH 6.0 and 9.0 on assay plates (data is not shown). Lysozyme E expressed by S. salivarius subsp. thermophilus/pL2 cells maintained its activity on test plates (data is not shown) and SDS-PAGE when subject to the temperature at 70oC for 15 min. In contrast, the enzyme expressed by both E. coli/pL2 and L. lactis/pL2 cells could not withstand temperatures above 50oC (Figures 2, 3 and 4). Live M. luteus cells counts on assay plates with heat-treated lysozyme was also consistent with the results of the SDS-PAGE (Figure 5).   DISCUSSION  The expression in S. salivarius subsp. thermophilus of bacteriophage T4 lysozyme gene was studied. To express lysozyme gene in the bacterium, a shuttle vector called pL2 was constructed using Streptococcal replica-tion origin. Bacteriophage T4 lysozyme gene was actively expressed in the three  bacteria.  It  was  not  known  that        Figure 1. Construction of pL2 plasmid (pBluscript II SK +/- plus lysozyme plus replication origin of pTRW10).      Figure 2. Thermostability of lysozyme E from S. salivarius subsp. thermophilus/pL2 in supernatant at different temperatures on SDS-PAGE.       Figure 3. Thermostability of lysozyme E from E. coli/pL2 in supernatant at different temperatures on SDS-PAGE. Akinalp et al.        965      Figure 4. Thermostability of lysozyme E from L. lactis/pL2 in supernatant at different temperatures on SDS-PAGE.     gene e or lacZ promoter is recognized by the cells.  Guchte et al. (1989) constructed two lactococcal expres-sion vectors and expressed a fusion gene containing hen egg white lysozyme gene in L. lactis subsp. lactis. On the other hand, three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in L. lactis subsp. lactis. Hen egg white lysozyme was not active but bacteriophage T4 and λ lysozyme were produced actively in L. lactis (Guchte et al., 1992). Non-toxic expression of gene e was observed previously in E. coli (Perry et al., 1985; Guzel et al., 2002). Although both S. salivarius subsp. thermophilus and L. lactis colonies harboring pL2 were grown in liquid and on solidified growth media and seemed to be unaffected by the action of lysozyme, the weakened cell wall of the bacteria actually rendered the cell to burst out easily under osmotic pressure upon treated with distilled water or 10 mM Tris. HCl, pH 8.0. T4 lysozyme presumably lyses T4-infected E. coli cells by attack on the cell wall from the cytoplasmic side (Perry et al., 1985). However, the attack of lysozyme on LAB might be expected from both side of the cell wall. The lysozyme E purified from Bacteriophage T4 has been reported to have a single polypeptide chain of molecular weight 19 kDa and the pH optimum between 7.0 and 8.0. The lysozyme of S. salivarius subsp. thermo-philus was also found active between pH 6.0 and 9.0 on assay plates. When incubating the purified enzyme various temperature for 5 min in 0.1 M phosphate buffer, pH 6.5, it retained 20% of its original activity at 65oC (Tsugita et al., 1968). However, lysozyme E expressed by S. salivarius subsp. thermophilus/pL2 cells seemed to increase its capacity for thermoresistance and was not denatured when subjected to temperature of 70oC for 15 min. The introduction, by directed mutagenesis, of a 3 - 97 disulfide bond into the normally disulfide-free  enzyme T4  966           Afr. J. Biotechnol.      Figure 5. Bacteriphage T4 lysozyme enzyme activity on Micrococcus luteus cells after heat-treated from various bacterial sources.    lysozyme stabilized the protein against irreversible thermal inactivation in vitro (Perry and Wetzel, 1984; 1986). The crosslinked variant T4 lysozyme (13C-97C/C54V) is more stable to inactivation at 70oC and pH 6.5 than non-crosslinked T4 lysozyme (C54V/C97S) (Perry and Wetzel, 1986). However, thermostability of T4 lysozyme expressed by S. salivarius subsp. thermophi-lus/pL2 cells may be caused by different mechanism of protein folding in thermophilic hosts. Lysozyme was not protected and thereby degraded after incubation of the supernatant both from L. lactis/pL2 and E.coli/pL2 sourc-es above 50oC (Figure 5). Although it is common practice to express the genes coding for thermostable proteins in mesophilic hosts such as E. coli and Bacillus subtilis for their easy purification by heat treatment, this is the first report to the our knowledge that a gene from mesophilic source was expressed in moderately thermophilic host to produce moderately thermostable protein. 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Expression of T4 Lysozyme Gene (gene e) in Streptococcus salivarius subsp. Thermophilus

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Expression of T4 Lysozyme Gene (gene e) in Streptococcus salivarius subsp. Thermophilus

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