Isolation, Detection and Inactivation of a Myoviridae Bacteriophage Infecting Bacillus Amyloliquefaciens FB11
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Ann Microbiol (2015) 65:1841–1846 DOI 10.1007/s13213-014-1022-9 ORIGINAL ARTICLE Isolation, detection and inactivation of a Myoviridae bacteriophage infecting Bacillus amyloliquefaciens FB11 Chetsadaporn Pitaksutheepong & Mattika Abhisingha & Jureeporn Dumnin & Wonnop Visessanguan Received: 19 August 2014 /Accepted: 9 December 2014 /Published online: 4 January 2015 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015 Abstract Bacillus amyloliquefaciens is commonly used as a fermentation. These fermentation industries consider phage starter culture for fermentation of soybeans and soybean attacks a major threat. For instance, production of meals. Like other starter cultures, B. amyloliquefaciens fermented soybean products, like natto (Japan) and FB11 faces threats from phage infection. Frequent phage chungkookjang (Korea), has been disrupted by phage attacks during large-scale cell culture cause serious yield attacks (Nagai and Yamasaki 2009; Umene et al. 2009; losses. To alleviate this problem, phage detection, identifica- Kim et al. 2011), with a number of phages belonging to tion and inactivation are required. The isolation and prelim- the Caudovirales order (Myoviridae and Siphoviridae fam- inary characterization of a phage designated BA01 revealed ilies) being isolated from contaminated nattos and that it is a Myoviridae phage, with some regions of its ge- chungkookjang. Subsequently, extensive studies on the nome sharing sequence similarity with the genome of Bacil- characterization and inactivation of these phages [particu- lus subtilis phage SPO1. BA01 showed strong lytic activity larly those attacking B. subtilis (natto)] have been essential against B. amyloliquefaciens, B. circulans and B. pumilus.A in managing phage decontamination in natto production PCR-based method was developed to detect BA01 in large- factories (Nagai 2012). scale cell culture of B. amyloliquefaciens FB11,whereapair Unfortunately, although B. amyloliquefaciens is used of degenerate primers could detect BA01 with a limit of widely as a starter culture for fermentation of soybeans or detection of 104 PFU/mL. Inactivation of BA01 was soybean meals (Meerak et al. 2007; Seo et al. 2013), there is achieved by either thermal treatment at 70 °C for 5 min or little information on phages of B. amyloliquefaciens. De- treatment with peracetic acid-based disinfectant (0.3 % v/v) cades ago, there was a report on induction of a temperate, for 5 min. or lysogenic, phage named H2 from B. amyloliquefaciens strain H (Zahler et al. 1987). The morphology suggested that it belongs to the Caudovirales order, Siphoviridae family Keywords Bacteriophage . Bacillus amyloliquefaciens . while its genome shares sequence similarity with Group III Caudovirales . Myoviridae . Isolation . Inactivation B. subtilis temperate phages. The most recent report on a B. amyloliquefaciens phage was by Muyombwe et al. Introduction (1999), where a phage was isolated from sewage during an attempt to isolate an endolysin gene. There were no details Fermented soybean meal is commonly used as a protein on phage morphology, classification or host range. source in swine feed, with Bacillus subtilis and Bacillus Despite the relative lack of information, phage infec- tion remains a problem, particularly for a strain used lo- amyloliquefaciens generally being used as starter cultures for cally in Thailand, B. amyloliquefaciens FB11. To control C. Pitaksutheepong (*) : M. Abhisingha : J. Dumnin : phage infections, the phage was isolated and classified W. Visessanguan morphologically into the Caudovirales order, Myoviridae Food Biotechnology Research Unit, National Center for Genetic family. A PCR-based method was then developed for the Engineering and Biotechnology, 113 Thailand Science Park, early detection of phage attack. Finally, chemical and Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand thermal treatments were examined for fast and efficient e-mail: [email protected] ways to inactivate this phage. 1842 Ann Microbiol (2015) 65:1841–1846 Materials and methods Table 1 Host range of phage BA01 Bacterial strain Inhibition zone Bacterial strains (mm)a/degree of lysisb Bacillus amyloliquefaciens FB11 was used as the host for Bacillus amyloliquefaciens BCC 18049 14.0/++ phage isolation. Indicator strains used for determination of phage host range were obtained from the BIOTEC Culture B. amyloliquefaciens FB11 9.3/++ − Collection (BCC), National Center for Genetic Engineering Bacillus cereus BCC 27609 and Biotechnology, Thailand and from the Japan Collection Bacillus circulans JCM 2504 9.2/++ − of Microorganisms (JCM). Bacteria were grown in Luria Bacillus licheniformis ATCC 53757 − Bertani (LB) medium at 37 °C for 16–18 h. Bacillus megaterium BCC 18046 Bacillus pumilus BCC 6493 9.2/+ Bacillus subtilis subsp. subtilis JCM 1465 − Isolation of bacteriophage B. subtilis BCC 6327 − Bacillus thuringiensis subsp. aizawai − A culture of B. amyloliquefaciens FB11 naturally infected by BCC 14313 bacteriophages was collected from a local factory and centri- a Diameter of inhibition zone is the mean of two determinations fuged. The supernatant was filtered through a 0.2-μmmem- b − No inhibition zone, + turbid inhibition zone, ++ clear inhibition zone brane. A 200 μl aliquot of the filtrate was used to perform double-layer-agar plating (Adams 1959). Single plaques were Partial characterization of the phage genome picked and placed in 10 mL LB medium containing 100 μL B. amyloliquefaciens FB11. The culture was incubated for Three pairs of degenerate primers named BA44F/BA44R (5′- complete lysis at 37 °C and centrifuged at 10,000 g for CTTSAAGMRTG GKKTGARYTRGAAGG-3′/5′-CCYG μ 10 min. The supernatant was filtered through a 0.2- mmem- CYTCMYKWARYGCSATKATRGT-3′), BA31F/BA31R brane and the filtrate was used for double-layer-agar plating as (5′- AAGGACATTAAAGGRTATGAG-3′/5′- CTTT above. To obtain the pure phage strain, single-plaque isolation YKRGWACCCC ATCTYAAATT-3′), and BA19F/BA19R was repeated twice. (5′- GGHTAYTCHKBWTTYTAYRTNGG-3′/5′-TCNS CRTANCC YTCWAWRTCWATWCC-3′)wereusedfor PCR amplification of phage BA01genomic DNA. The Large scale preparation of bacteriophage and DNA extraction primers were designed based on conserved regions of coding sequences of Bacillus phage SPO1 and other Bacillus phages. Phage multiplication and DNA extraction were performed ac- Primers BA44F and BA44R were designed based on three cording to Binetti et al. (2005). transcriptional regulation proteins (gp44, gp51 and gp25.1) of Bacillus phage SPO1 (accession number YP_002300288, Host range determination YP_002300295 and YP_002300396, respectively). Primers BA19F and BA19R were based on the conserved regions of The indicator strains used in this study are listed in Table 1. gp19.1 (Bacillus phage SPO1, YP_002300377) and hypothet- The lytic activity of BA01 was tested as follows: 100 μL ical proteins of Bacillus phages SPP1 and SPBc2 (NP_690719 overnight host culture (1×108 CFU/mL) was mixed with and NP_046674). Primer set BA31F/BA31R was designed 0.7 % melted LB agar. The melted agar was poured on top based on gp31.2 of Bacillus phage SPO1 (YP_002300418) of 2 % solidified LB agar to make double-layer agar. After and endoribonuclease of Bacillus phage SP82 (AAA56884). solidification, 10 μL phage suspension (1×109 PFU/mL) The PCR mixture contained 50 ng phage genomic DNA, 1× μ were dropped onto a lawn of each indicator strain. Plates were PCR buffer, 1.25 mM MgCl2,100 M dNTPs (Fermentas, incubated at 37 °C for 16–18 h and inhibition zone diameters Vilnius, Lithuania), 450 nM of each primer (Biodesign, Klong recorded. Luang Pathumthani, Thailand) and 1 U Taq polymerase (Invitrogen, São Paulo, Brazil). PCR amplifications were per- formed for 30 cycles of 94 °C for 1 min, 45 °C for 30 sec and Electron microscopy 72 °C for 30 sec. PCR products were analyzed on 1.2 % aga- rose gels by ethidium bromide staining before cloning into Phage suspensions were dropped onto carbon-coated copper pDrive (Qiagen, Hilden, Germany). The recombinant plas- grids and stained with 2 % uranyl acetate. Phage morphology mids were individually transformed into E. coli JM109. Plas- was viewed under a JOEL transmission electron microscope mid DNA was extracted using QIAprep® (Qiagen) and sub- (JEM-1220, Japan) at an accelerating voltage of 80 kV. jected to nucleotide sequencing. BLAST analysis was Ann Microbiol (2015) 65:1841–1846 1843 performed using the BLASTN algorithm available at http:// single-plaque isolation. Electron micrographs revealed a www.ncbi.nlm.nih.gov. phage with a head and a contractile tail with a base plate (Fig. 1).Theheadhadadiameterofapproximately118nm Detection of phage BA01 by PCR analysis and the tail was about 72 nm long and 30 nm wide. The baseplate was 54 nm wide. The inner tube was extended AcultureofB. amyloliquefaciens FB11 that was suspected of 70 nm from the tail shaft and base plate. Based on its mor- phage attack was collected and centrifuged. The supernatant phology, BA01 was classified into the Caudovirales order, was filtered through a 0.2-μm membrane. The filtrate was Myoviridae family. Determination of the host range revealed then boiled for 5 min and 1 μL was used for PCR analysis that BA01 showed strong lytic activity against with primer set BA44F/BA44R under the conditions detailed B. amyloliquefaciens, B. circulans and B. pumilus, but was above. unable to infect B. subtilis, B. megaterium, B. cereus, B. licheniformis and B. thuringiensis (Table 1). Determination of the