Screening for the presence of biosynthetic genes for antimicrobial lipopeptides in natural isolates of Bacillus sp.

Arch. Biol. Sci., Belgrade, 64 (4), 1425-1432, 2012 DOI:10.2298/ABS1204425S SCREENING FOR THE PRESENCE OF BIOSYNTHETIC GENES FOR ANTIMICROBIAL LIPOPEPTIDES IN NATURAL ISOLATES OF BACILLUS SP. S. STANKOVIĆ1, SANJA MIHAJLOVIĆ2, V. DRAGANIĆ1, I. DIMKIĆ1, G. VUKOTIĆ1, TANJA BERIĆ1 and Đ. FIRA1* University of Belgrade, Faculty of Biology, 11000 Belgrade, Serbia Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, 11000 Belgrade, Serbia 1 2 Abstract - A collection of 205 natural isolates of Bacillus was tested for the presence of genes for biosynthesis of antimicrobial lipopeptides, iturin, surfactin, fengycin and bacillomycin D. For the detection of iturin producers by PCR screening, we used forward ITUP1-F and reverse ITUP2-R primers which are capable of detecting a 2-kb region that includes the intergenic sequence between the ituA and ituB genes. A 675-bp fragment from the gene sfp from B. subtilis encoding 4’-phosphopantetheinyl transferase involved in the biosynthesis of surfactin was targeted for amplification by using primers P17 and P18. Other two pairs of primers were BACC1F and BACC1R for bacillomycin D and FEND1F and FEND1R for potential fengycin producers, respectively. The results of the screening showed that the majority of tested strains had more than one biosynthetic operon, since 81% possessed the genes for bacillomycin D production, 54% for surfactin, 38% for iturin and 25% for fengycin production. Key words: Bacillus, iturin, surfactin, fengycin, bacillomycin INTRODUCTION lished their place in the biological control of plant pathogenic bacteria and fungi (Pengnoo et al., 2000; Abanda-Nkpwatt et al., 2006). Many species of the genus Bacillus produce lipopeptides with antagonistic activity against bacteria, fungi or animal cells. Bacillus lipopeptides, which can be cyclic or linear, mainly consist of 7 to 11 amino-acid residues linked to β-amino or β-hydroxy fatty acids. Because of their amphiphilic nature, most act as biosurfactants. The length of the hydrocarbon chain of fatty acids can be different, and the cyclic structure of lipopeptides prevents the cleavage of their peptide bonds by proteolytic enzymes. The mechanism of the antagonistic action of lipopeptides is based on their interaction with the cell membrane and formation of pores or, at higher concentrations, the solubilization of the membrane, (Deleu et al., 2005). Antimicrobial lipopeptides are synthesized in a nonribosomal manner. The strains that produce them have estab- Lipopeptides of the iturin family, such as iturin, mycosubtilin and bacillomycin, consist of seven amino-acid residues circularized with β-amino fatty acid (Peypoux et al., 1978). Iturins are characterized by the chiral sequence LDDLLDL within the aminoacid ring and D-Tyr2 (Magnet-Dana and Peypoux, 1994). Beside its antimicrobial effect, Iturin A shows a high degree of thermostability, retaining 100% of biological activity after heating at 100°C for 30 min (Yu et al., 2002). The antagonistic effects of the iturin cyclic peptides are the result of their interaction with the cell membrane and formation of pores (Magnet-Dana and Peypoux, 1994). A similar effect was observed in the action of mycosubtilin which in1425 1426 S. STANKOVIĆ ET AL. fluences the permeability of the membranes of yeast cells (Besson and Michel, 1989). The Tyr residue at position 2 in the peptide ring of peptides from the iturin family has a significant role in the mechanism of pore formation in target cells (Harnois et al., 1989, Volpon et al., 1999). The Bacillus strains that produce iturins showed antifungal activity against some important plant pathogens, proving their potential in the application of biological control (Han et al., 2005; Arrebola et al., 2010). Strains of other Bacillus species also produce lipopeptides that belong to the surfactin family. This group consists of surfactin and its analogs lichenysins and pumilacidins. Similar to iturins, they have a cyclic structure of seven amino acids, however, the ring is closed with β-hydroxy fatty acid with a different length of hydrocarbon chain, and they act as very powerful biosurfactants (Arima et al., 1968; Nagai et al., 1996). Lichenysin A is produced by Bacillus licheniformis, and beside the determination of its structure, the biosynthetic operon of this lipopeptide has also been identified (Yakimov et al., 1995; 1998). The fengycin family of lipopeptides also includes plipastatins. Their structure is somewhat different to that of other lipopeptides. They consist of 10 aminoacid residues, and their structure contains a ring of eight amino-acid residues linked to a dipeptide associated with the β-hydroxy fatty acid. Within this family of lipopeptides, the composition of the stereoisomers of amino-acid residues can be different (Volpon et al., 2000). Some of the bacterial strains producing these lipopeptides display strong antagonistic effects against different organisms. Bacillus amyloliquefaciens strain GA1, which exhibits strong antifungal activity, contains the biosynthetic genes for eight antimicrobial agents, including lipopeptides from the surfactin, fengycin and iturin families, as well as macrolactin, difficidin, bacillaene, bacilysin and bacillibactin (Arguelles-Arias et al., 2009). The strain Bacillus thuringiensis CMB26 produces the analog of fengycin with bactericidal, fungicidal and insecticidal effect (Kim et al., 2004). These and many other strains have a significant potential in the biological control of plant diseases. In this study, we performed a screening for the presence of biosynthetic genes for the antimicrobial lipopeptides iturin, surfactin, fengycin and bacillomycin D in natural isolates of Bacillus from soil samples that were taken from different locations in Serbia, in order to identify the strains with the capacity for application in biological control. MATERIALS AND METHODS Media for bacterial growth and culture conditions Natural isolates of Bacillus sp. were grown aerobically in Luria Bertani (LB) broth, containing 1.0% Bacto-tryptone, 0.5% yeast extract, 1.0% NaCl, and maintained on LB plates. Agar plates were made by adding 1.5 % (wt/vol) agar (Torlak, Belgrade, Serbia) to the liquid medium. Bacteria were incubated at 30°C for 24 h. All strains used in this study are given in Table 2. Isolation of DNA Genomic DNA from the Bacillus strains was isolated as described earlier (Le Marrec et al., 2000). After centrifugation of the culture and two washes in TE buffer (10 mmol Tris-HCl (pH 8·0), 1 mmol EDTA), cells were resuspended in 1 ml lysis buffer (50 mmol Tris (pH 8·0), 1 mmol EDTA; 25% sucrose) containing 20 μg ml of lysozyme (Serva) and incubated for 45 min at 37°C. The reaction was stopped with 1ml EDTA (250 mmol pH 8·0) for 5 min. The samples were then treated with 400 μl of 20% (w/vol) SDS and 20 μl of a 20 mg/ml of Proteinase K (Sigma) solution. The mixture was incubated at 65°C until it became clear and less viscou (...truncated)