Arab Republic of Egypt

1. Heterologous Expression of pctA Gene Expressing Propionicin T1 by Some Lactic Acid Bacterial Strains using pLEB590 Arab Republic of Egypt Alexandria University Sameh E. Mohamed and Mahmoud K. Tahoun Department of Dairy Science and Technology, Faculty of Agriculture, Alexandria University. Aflatoon Street, El-Shatby21545, Alexandria, Egypt. ABSTRACT Cloning and expression of pctA gene of P. thoenii 419 into LAB pctA gene of P. thoenii 419 was earlier transferred to P. freudenreichii [9] using pAMT1 to study the heterologous expression of the PT1-encoding gene. In the present study, pctA gene was cloned into a food-grade cloning vector [pLEB590, 10] and transferred to different LAB strains. Propionicin T1 [PT1] is a bacteriocin secreted by Propionibacterium thoenii 419 that inhibits the closely – related propionibacteria except P. freudenreichii. The antimicrobial activity of PT1 extends to inhibit the growth of Bacillus subtilis DB100 host as well as Clostridium sporogenes DSM1446. In the present work, pctA gene expressing PT1 was isolated and transferred to different lactic acid bacterial [LAB] strains using pLEB590 as an expression vector, where pctA gene worked under the effect of P45 promoter in the modified pLEB590 [named: pLEBA]. LAB transformants showed strong antimicrobial activity against B. subtilis DB100 host and C. sporogenes DSM1446. The new LAB strains can be used as starters in dairy industry to control spore formers and improve the shelf-life of dairy products. Fig. 1: DNA manipulations and the construction of pLEBA Keywords: Propionicin T1 – Propionibacteria – pLEB590 – LAB– Spore formers. Aim of The Present Work The present work was carried out to transform lactic starter cultures with pctA gene that expresses PT1 capable of controlling the growth of spore formers that can survive milk heat treatments. Electroporation and stability of pLEBA into LAB strains The electroporation efficiency was 1.4 X 106 cfu/µg DNA for L.lactis LL108, while that for S. thermophilus was found to be 2.0 x 104 cfu/µg DNA. On the other hand, L. bulgaricus DSM20080 and L. plantarum TF103 recorded 8.1 x 108 and 5.2 x 106 cfu/µg DNA, respectively.It was also observed that 96% of cells retained pLEBA and showed nisin – resistant phenotype on nisin – containing media. According to Takala and Saris [10], after 18 successive replications, 97.8% of cells retained pLEB590 and showed nisin – resistant phenotype. MATERIALS AND METHODS Screening of P. thoenii 419 for its antimicrobial activity, PT1 isolation, partial purification and extraction from PAGE gel Spot agar assay was performed [1]. PT1 was isolated and partially purified [2] with some modifications [1]. PAGE in the presence of 0.1% SDS was performed, PT1 was extracted through the reverse staining of the PAGE gel [3]. PT1 assays and direct detection of PT1 on SDS – PAGE Antimicrobial activity of the producer strains was measured either by well diffusion or by critical dilution followed by measuring the bactericidal action of PT1. PT1 activity was detected directly on SDS – PAGE [4]. Total crude protein isolated from transformants media, partial purification, SDS - PAGE, and direct detection on gel LAB transformants antimicrobial activity was determined using direct detection method. The inhibition zones of PT1 secreted by L.bulgaricus DSM20080 [pLEBA], L. plantarum TF103 [pLEBA], and L. lactis LL108 [pLEBA] were wider than that of P. thoenii 419. PT1 over expression was expected to the effect of P45 promoter available in pLEB590. Genomic DNA isolation, extraction from gel, primers designing, and specific PCR Genomic DNA was isolated using BioBasic EZ-10 g DNA spin column kit and DNA bands were extracted using AxyPrepTM DNA Gel Extraction Kit. Suitable primers [BGPT1F (A T A G G A T C C C A C T C A A A C C C A) and XGPT1R2 (A T A C T C G A G G T G A A T T C C A A G)] were selected from several primers designed using Geneious software 4.0.2 to isolate the pctA gene [290 bp] of P. thoenii419. PCR program were performed [1] using a thermal cycler [Techne, UK]; the Master Mix® and Pfu DNA polymerase were used as recommended by the manufacturer [Bioron, Germany]. Fig. 2.(a) PAGE of LAB transformants and their parental strains, (b) Direct detection against B. subtilis DB100 host, (c) Direct detection against C. sporogenes DSM1446 DNA manipulations Isolation of plasmids from LAB was performed [5].Purification of DNA samples was performed using BioBasic EZ-10 DNA purification spin column kit. DNA digestions, blunting reactions, DNA ligations, and electroporation of Escherichia coli strains were carried out [6], while electroporation of lactobacilli strains was carried out [7] and electroporation of lactococci strains was performed [8]. RESULTS AND DISCUSSION ACKNOWLEDGEMENT PT1, screening and partial characterization P. thoenii 419 total crude proteins were partially purified using ammonium sulphate [75 %]. After dialysis, the protein sample was concentrated 100 times using PEG [Mw 6000]. Such concentrated protein was examined against different microorganisms. B. subtilis DB100 host and C. sporogenes DSM1446 were highly inhibited by the antimicrobial peptide. Faye et al. [1] stated that P. thoenii 419 secretes two antimicrobial peptides namely protease – activated antimicrobial peptide [PAMP] and PT1. Since the former is secreted in an inactive form, thus PT1 is responsible for the antimicrobial activity of the crude protein extract. The present work was supported using the fund of the International Center for Genetic Engineering and Biotechnology [ICGEB, Italy]. REFERENCES Faye, T; Langsrud, T; Nes, I F; and Holo, H[2000] Appl Environ Microbiol 66: 4230 – 4236. Paik, H D; and Glatz, B A [1995] Lait75: 367 – 377. Fernandez – Patron, C; Castellanos – Serra, L; and Rodriguez, P [1992] Biotechniques 12: 564 – 573. Ben – Shushan, G; Zakin, V; and Gollop, N [2003] Peptides 24: 1733 – 1740. O’Sullivan, D J; and Klaenhammer, T R [1993] Appl Environ Microbiol 59: 2730 – 2733. Sambrook, J; and Russell, D W [2001] Molecular cloning: a laboratory manual, [Third Ed], Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA [ISBN: 0879695765]. Serror, P; Sasaki, T; Ehrlich, S D; and Maguin, E [2002] Appl Environ Microbiol 68: 46 – 52. Holo, H; and Nes, I F [1989] Appl Environ Microbiol 55: 3119 – 3123. Brede, D A; Faye, T; Stierli, M P; Dasen, G; Theiler, A; Nes, I F; Meile, L; and Holo, H [2005] Appl Environ Microbiol 71: 8077 – 8084. Takala, T M; and Saris, P E J [2002] ApplMicrobiolBiotechnol 59: 467 – 471. Isolation of pctA gene of P. thoenii 419 using specific PCR and sequencing of the PCR product After PCR, gel electrophoresis was performed on agarose gel [1%]. Furthermore, a sample of the purified PCR product was sequenced using ABI Prism 377 DNA sequencer [Perkin – Elmer, Applied Biosystems, USA]. Our sequencing data was similar to the published sequence of pctAgene [1].