Identifikacija i genotipizacija kolekcije Bacillus spp. izolata: karakterizacija bakteriocina liheniocina 50.2

Abstract

Filogenetska raznovrsnost kolekcije 166 izolata roda Bacillus je analizirana upotrebom PFGE metode profilisanja kombinovane sa sekvenciranjem tuf gena odabranih izolata. Analiza PFGE profila je pokazala veliku genetičku raznovrsnost izolata koja je grupisana u 21 grupu. Po jedan izolat, predstavnik svake grupe, identifikovan je na osnovu sekvence tuf gena. Pokazano je veoma jasno i nedvosmisleno grupisanje izolata. Ukupno je identifikovano osam vrsta: Bacillus megaterium, Bacillus pumilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus safensis, Bacillus thuringiensis, Bacillus cereus i Bacillus anthracis. Posebno zadovoljavajući rezultati su postignuti u razdvajanju B. subtilis i B. amyloliquefaciens izolata kao i B. thuringiensis i B. cereus u posebne klade na filogenetskom stablu. Ispitivanje antibakterijske aktivnosti čitave kolekcije Bacillus spp. je pokazalo da samo izolat VPS50.2, tada identifikovan kao B. licheniformis, proizvodi antimikrobno jedinjenje tipa bakteriocina aktivno protiv indikatorskog soja B. subtilis 168 (Berić, 2010). U ovom radu je bakteriocin liheniocin 50.2 okarakterisan kao termostabilni protein, koji zadržava svoju aktivnost i na visokim temperaturama (30 min na 121°C); tolerantan je na vrlo širok opseg pH vrednosti (najveću aktivnost pokazao je na pH 9); delimično je otporan na dejstvo proteolitičkih enzima i i zadržava aktivnosti i u prisustvu različitih katjona. Na osnovu određenih osobina, liheniocin 50.2 predstavlja novi bakteriocin koji pripada klasi II.3. Sinteza liheniocina 50.2 zavisi od faze rasta soja producenta B. licheniformis VPS50.2 i koincidira sa početkom sporulacije. Testiranjem je utvrđeno da su optimalni uslovi za proizvodnju bakteriocina: gajenje 42h od početka inkubacije, u LB hranljivom medijumu na 37°C, uz aeraciju. Antibakterijski spektar liheniocina 50.2 pokriva samo Gram-pozitivne bakterije, ali pokazuje aktivnost protiv velikog broja različitih vrsta, uključujući i Listeria monocytogenes, meticilin-rezistentne Staphylococcus aureus, β- hemolitičke streptokoke i koagulaza negativne stafilokoke (KNS)...

The phylogenetic diversity of the Bacillus collection, containing 166 isolates, was analyzed by using the PFGE fingerprinting method combined with the sequencing of the tuf gene of the selected isolates. According to the PFGE analysis which showed a high genetic diversity, the isolates were grouped into 21 clusters. A representative of each group was identified based on the sequence of the tuf gene. Very clear and unambiguous grouping of the isolates was demonstrated. In total, eight species were identified: Bacillus megaterium, Bacillus pumilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus safensis, Bacillus thuringiensis, Bacillus cereus and Bacillus anthracis. Particularly satisfactory results have been achieved in the separation of B. subtilis and B. amyloliquefaciens isolates, as well as B. thuringiensis and B. cereus, into the individual clades on the phylogenetic tree. Examination of the antibacterial activity of whole collection of Bacillus spp. showed that only isolate VPS50.2, identified as B. licheniformis produces a bacteriocin which has antimicrobial activity against indicator strain B. subtilis 168 (Berić, 2010). In this research, the bacteriocin licheniocin 50.2 was characterized as the thermostable protein, which retains its activity even at the higher temperatures (30 min at 121 ° C); tolerates a very wide range of pH values (the highest activity was shown at pH 9); it is partially resistant to the action of proteolytic enzymes and it also maintains activity in the presence of various cations. Based on certain properties, licheniocine 50.2 represents a new bacteriocin belongs to the II.3 bacteriocin class. Production of the licheniocine 50.2 depends on the stage of the growth of the B. licheniformis VPS50.2 strain and it coincides with the onset of sporulation. The optimal conditions for the production of this bacteriocin were as followed: 42h cultivation in the LB medium at 37° C, with aeration. The antibiotic spectrum of licheniocine 50.2 covers only Gram-positive bacteria. However, it shows activity against the large number of different species, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, β-hemolytic streptococci and coagulase-negative staphylococci (CNS)...