Effectiveness of the lactococcal abortive infection systems AbiA, AbiE, AbiF and AbiG against P335 type phages

FEMS Microbiology Letters 210 (2002) 67^72 www.fems-microbiology.org E¡ectiveness of the lactococcal abortive infection systems AbiA, AbiE, AbiF and AbiG against P335 type phages Mark Tangney a a;b , Gerald F. Fitzgerald a;b;
Received 3 December 2001; received in revised form 20 February 2002; accepted 25 February 2002 First published online 22 March 2002 Abstract Four lactococcal abortive infection mechanisms were introduced into strains which were sensitive hosts for P335 type phages and plaque assay experiments performed to assess their effect on five lactococcal bacteriophages from this family. Results indicate that AbiA inhibits all five P335 phages tested, while AbiG affects PP335 itself and PQ30 but not the other P335 species phages. AbiA was shown to retard phage Q30 DNA replication as previously reported for other phages. It was also demonstrated that AbiG, previously shown to act at a point after DNA replication in the cases of c2 type and 936 type phages, acts at the level of, or prior to phage Q30 DNA replication. AbiE and AbiF had no effect on the P335 type phages examined. 1 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : P335; AbiA; AbiE; AbiF; AbiG ; Abortive infection mechanism 1. Introduction The susceptibility of lactococcal starter cultures to the action of a wide variety of bacteriophages is of considerable economic importance within the dairy fermentation industry. Bacteriophage infection is considered to be the most signi¢cant cause of decreased starter activity in commercial practice, with lytic lactococcal phages cited as the primary cause of fermentation failures [1,2]. The means by which the industry has addressed the phage problem has included the use of both physical barriers and biological precautions. The latter have largely focused on the selection and use of naturally resistant strains of lactococci and the nature of these resistance systems has been studied extensively [3^5]. Four principal naturally occurring phage resistance mechanisms have been described for Lactococcus species to date: adsorption inhibition, phage DNA penetration blocking, restriction^modi¢cation and abortive infection (for reviews see [1,5,6]). Of these mecha- * Corresponding author, at address b. Tel.: +353 (21) 490 2730; Fax : +353 (21) 427 6318. E-mail address : g.¢ (G.F. Fitzgerald). nisms, it has been suggested that abortive infection (Abi) is the most powerful [7]. Lactococcal phages have been classi¢ed into 12 groups on the basis of morphology and DNA homology [8]. Two of these species are most commonly found in dairy fermentations, i.e. the small isometric-headed 936 species (particularly in New Zealand, the USA and Ireland) [9^ 11] and the more virulent but less prevalent prolate-headed c2 species, while members of a third species (P335) have been isolated with increasing frequency [10,11]. This latter species includes both virulent and temperate types. Whereas all lactococcal Abi mechanisms published to date have been demonstrated to be e¡ective against 936 type phages, only AbiA, AbiC, AbiK and AbiU are known to have an e¡ect on members of the P335 family [12^15]. AbiA provides resistance to all three of the phage types mentioned and is described as an ‘early acting’ mechanism since it interferes with phage DNA replication within the host in the case of the P335 type P31 [12,16]. AbiK has been shown to be highly e¡ective against P335 species, preventing both phage ul36 and phage P335 DNA replication [14,17]. However, the DNA replication of phages p2 and p008 of the 936 group, which are also sensitive to AbiK, was shown to be una¡ected in its presence, providing the ¢rst report of di¡erent phage responses towards an Abi system [14]. 0378-1097 / 02 / $22.00 1 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 5 7 5 - X FEMSLE 10423 14-5-02 National Food Biotechnology Centre, University College, Cork, Ireland b Department of Microbiology, University College, Cork, Ireland 68 M. Tangney, G.F. Fitzgerald / FEMS Microbiology Letters 210 (2002) 67^72 A P335 phage/host interaction system was not available at the time of the initial discovery of the AbiE, AbiF and AbiG mechanisms in this laboratory; hence, these have previously been tested only against phages of the 936 and c2 species. Due to the increasing prevalence of P335 type phages in milk fermentations in Ireland (A. Forde, personal communication) and elsewhere [10,11] and because of the more recent availability of phage/host systems with which the interaction between these mechanisms and P335 type phages can be examined, a study was undertaken to assess their e¡ectiveness against several members of this phage family, and to partially characterise any observed e¡ects. 2.1. Bacterial strains, bacteriophages and plasmids The bacterial strains, bacteriophages and plasmids used in this study are listed in Table 1. Lactococcal strains were grown at 30‡C in M17 medium [23] containing 0.5% glucose. Stocks of all cultures were maintained at 320‡C in their growth medium containing 15% glycerol. Bacteriophages were propagated on their homologous hosts at 30‡C in GM17 broth. Plasmids were transformed into competent cells with the Bio-Rad Gene Pulser apparatus (Bio-Rad Corp., Richmond, CA, USA) using the conditions outlined in the Bio-Rad manual. Positive selection of recombinant plasmids was e¡ected using chloramphenicol (Cm, 10 Wg ml31 ). Restriction endonucleases were purchased from Roche Diagnostics Ltd. (East Sussex, UK) and utilised according to the manufacturer’s instructions. Lactococcal plasmid DNA for use as template in polymerase chain reactions was isolated as described by Anderson and McKay [24]. Oligonucleotide primers (Table 2) were synthesised using an Applied Biosystems PCR-MATE DNA synthesiser (Applied Biosystems Inc., Foster City, CA, USA). PCR reagents were purchased from Promega (Madison, WI, USA) and reactions were executed using an Omnigene thermal cycler (Hybaid Ltd., Middlesex, UK). The annealing temperatures for PCR programmes varied according to the melting temperatures of the speci¢c primers used. PCR products were separated on agarose gels in TAE bu¡er (40 mM Tris^acetate, 1 mM EDTA), stained with ethidium bromide, and visualised under UV light and photographed using a UVP Imagestore 5000 gel documentation system (UV Products Ltd., Cambridge, UK). DNA was transferred from agarose gels to nylon membranes (Hybond Nþ , Amersham International, Bucks., UK) by the method of Southern [25] as modi¢ed by Wahl et al. [26]. DNA was labelled using the enhanced chemiluminescence (ECL) gene detection system (Amersham International, Bucks., UK). Probe labelling, hybridisation conditions and washing steps were performed according to the instructions issued by the manufacturer (Amersham International, Bucks., UK). (...truncated)