Department of Biochemistry and Microbiology, Faculté des Sciences et de Génie, Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine Dentaire, Université Laval, Québec, Canada G1K 7P41
Agriculture and Agri-Food Canada, Food Research and Development Centre (FRDC), 3600 Casavant Blvd, St-Hyacinthe, Québec, Canada J2S 8E32
Author for correspondence: Sylvain Moineau. Tel: +1 418 656 3712. Fax: +1 418 656 2861. e-mail: Sylvain.Moineau{at}bcm.ulaval.ca
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ABSTRACT |
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Keywords: bacteriophages, abortive infection mechanism, Lactococcus
Abbreviations: c.o.i., centres of infection; e.c.o.i., efficiency at which centres of infection form; e.o.p., efficiency of plaquing
a Present address: Department of Food Science and Nutrition and Centre de Recherche en Science et Technologie du Lait (STELA), Faculté des Sciences de lAgriculture et de lAlimentation, Université Laval, Québec, Canada G1K 7P4.
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INTRODUCTION |
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Abortive infection mechanisms (Abi) form a particular class of natural anti-phage systems. These phage resistance mechanisms are generally plasmid-encoded and act after phage adsorption, DNA penetration and early gene expression. Their intracellular activities result in reduced burst size, efficiency of plaquing (e.o.p.) and efficiency at which centres of infection form (e.c.o.i.). One of the distinctive aspects of Abi systems is the massive cellular death observed in infected cells (Durmaz et al., 1992 ; Geis et al., 1992
; Sing & Klaenhammer, 1990
). To date, 17 Abi systems have been cloned and sequenced in L. lactis.
The first lactococcal Abi system, AbiA, was isolated (Klaenhammer & Sanozky, 1985 ) from the highly resistant industrial strain L. lactis ME2 (Klaenhammer, 1989
; Hill et al., 1990
). In P335-infected AbiA+ cells, phage DNA replication and major capsid protein production are absent (Hill et al., 1991
; Moineau et al., 1993
). Site-directed mutagenesis of a leucine repeat motif in AbiA showed that this structure is essential to the phage resistance phenotype (Dinsmore et al., 1998
). A specific DNA fragment isolated from a mutant phage, insensitive to AbiA, reduced the resistance phenotype towards the wild-type phage when provided in trans in the Abi+ cells (Dinsmore & Klaenhammer, 1997
). A similar phenomenon was observed for AbiD1 (Bidnenko et al., 1995
), which contains two helixturnhelix motifs in its sequence (Anba et al., 1995
). A helixturnhelix motif was also identified at the N-terminal end of AbiJ (Deng et al., 1997
). Amino acid motifs characteristic of DNA-binding functions were not detected in AbiD and AbiF. However, homologies were found between AbiD1 and AbiF (47% identity), between AbiD and AbiD1 (28% identity), and between AbiD and AbiF (26% identity) (Garvey et al., 1995
; McLandsborough et al., 1995
).
The genetic determinants of AbiB (Cluzel et al., 1991 ), AbiH (Prévots et al., 1996
) and AbiN (Prévots et al., 1998
) are encoded on the chromosome. The action of AbiB is possibly induced by an early phage transcript and leads to the degradation of phage mRNAs 1015 min after infection (Parreira et al., 1996
). For AbiE (Garvey et al., 1995
), AbiG (OConnor et al., 1996
) and AbiL (Deng et al., 1999
), a DNA region containing two ORFs is associated with the phage resistance phenotype. The requirement of the two peptides to generate full resistance has yet to be established in the case of AbiE. Two putative integral membrane-spanning helices were identified near the N-terminal end of AbiC (Durmaz et al., 1992
). This system caused a reduction in the synthesis of the major capsid protein of a P335 phage (Moineau et al., 1993
). The AbiQ mechanism appears to result in defective assembly of 936 and c2 phages (Émond et al., 1998
). Three other Abi genes were also sequenced, namely, AbiI (Su et al., 1997
), AbiO (Prévots & Ritzenthaler, 1998
) and AbiP (GenGank accession no. U90222).
In a previous study, we showed that plasmid pSRQ800 coded for the phage abortive infection system AbiK and conferred strong resistance against small isometric phages of the 936 and P335 species (Émond et al., 1997 ). A database query, based on amino acid composition, suggested that AbiK and AbiA might be in the same protein family. Furthermore, as with AbiA, replication of phage ul36 (P335 species) DNA and production of its major capsid protein were not observed in the AbiK+-infected cells (Émond et al., 1997
). The characterization of Abi systems is generally perfomed using only one phage species and it is generally assumed that the observations will be similar towards the other phage species. Here, we report distinct observations on the lytic cycle of 936 and P335 phages resulting from the presence of AbiK.
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METHODS |
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DNA isolation and manipulation.
Routine DNA manipulations were carried out as described by Sambrook et al. (1989) . Phage DNA was isolated according to the method of Chibani Azaïez et al. (1998)
with the following modifications: concentrated phages were treated with proteinase K (1 mg ml-1) for 1 h at 37 °C. Phenol/chloroform (24:1, v/v) extractions were repeated until a white interface was absent. DNA was precipitated using 2 vols ethanol and dissolved in 50 µl 2H2O. Purified DNA was digested with restriction enzymes as recommended by the manufacturer (Boehringer Mannheim).
Phage p2 restriction map.
Phage p2 DNA was digested with the restriction enzymes EcoRI, EcoRV, HindIII, NcoI, SalI, ScaI and SphI in single and double digestions. Restriction fragments were separated by electrophoresis on agarose gel, stained with ethidium bromide and photographed under UV illumination. The profiles were used to establish the EcoRV restriction map.
Phage DNA replication.
Intracellular phage DNA replication was monitored by the method of Hill et al. (1991) . In short, total DNA was isolated from cultures infected at a m.o.i. of 1, digested with EcoRV and heated at 65 °C for 10 min. The heat treatment prior to migration on gel allowed the identification of the fragments carrying the cos sites. The fragments were then electrophoretically separated on a 0·8% agarose gel. After ethidium bromide staining, DNA was visualized under UV and photographed. For phage p2, DNA was transferred to Hybond-N nylon membranes (Amersham) by capillary blotting (Southern, 1975
). A probe was prepared by labelling the EcoRV fragments from the phage p2 genome with the DIG-High Prime Kit (Boehringer Mannheim). Prehybridization, hybridization and posthybridization washes as well as detection were performed as directed by the manufacturer. The standard hybridization buffer (50% formamide) and CSPD were used for the hybridization steps and chemiluminescent detection, respectively.
Electron microscopy.
Phage morphology was observed as described previously (Moineau et al., 1992 ). The lytic cycle was monitored by electron microscopy according to the following protocol. The sensitive and resistant hosts were grown in GM17 to an optical density of 0·5. Calcium chloride was then added at a final concentration of 10 mM, followed by phage p2 at a m.o.i. of 1. The infected cells were incubated at 30 °C. At various time points, 1·7 ml culture was transferred to an Eppendorf tube and cells were fixed using a modification of the method of Strausbauch et al. (1985)
. The cell pellets were resuspended in a glutaraldehyde solution (2% in 0·1 M sodium cacodylate, pH 7·3) and incubated at room temperature for 1·5 h. The cells were centrifuged and the pellets were embedded in 3% agar dissolved in 0·1 M sodium cacodylate. After gelification on ice, samples were cut into 0·51·0 mm3 pieces with a razor blade, rinsed five times in cacodylate buffer and post-fixed in 1% OsO4 in water for 1·5 h at room temperature. The samples were rinsed in water, gradually dehydrated in increasing concentrations of ethanol solutions (10100%) and embedded in Spurr resin. After sectioning, the specimens were observed with a transmission electron microscope Phillips 420 at 80 kV.
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RESULTS |
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DISCUSSION |
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The absence of accumulation of P335 phage DNA in infected cells suggests an interference of AbiK with an early stage of the lytic cycle. The fact that the concatenated DNA of 936 phages was not efficiently processed into mature genome suggests that AbiK halted the packaging process, possibly as a consequence of the disrupted early stages. These molecular consequences are likely to result from an interaction of AbiK with analogous phage components of both species, or with a host product necessary for a common early step of phage development. A better understanding of the molecular interactions between AbiK and phages of the 936 and P335 species will provide valuable information on the similarities and differences in the lytic cycle of these phages. The absence of the mature processed form of the 936 phage genome in the AbiK+ cells could also be the result of defective concatemeric DNA intermediates that cannot serve as a substrate for the synthesis of mature phage DNA, as observed in the case of choleraphage 149 infecting Vibrio cholerae biotype El Tor (Chowdhury et al., 1989
). However, in that case, the concatemeric DNA formed during the infection was unstable and rapidly degraded (Majumdar et al., 1988
).
A direct interference of AbiK with the head morphogenetic pathway of 936 phages cannot be excluded. In phage lambda, mutations affecting late genes involved in head morphogenesis can provoke similar accumulation of concatenated DNA (Catalano et al., 1995 ; Murialdo, 1991
; Murialdo & Becker, 1978
; Murialdo & Tzamtzis, 1997
). Recently, a similar organization of the late genes was observed between lambda and the lactococcal phage sk1, a close relative of P008 and p2 (Chandry et al., 1997
). This finding suggests that the mechanism of action of AbiK on 936 and P335 phages is likely to be different. The large size of the abiK gene (1789 bp) supports the possibility of a multifunctional protein with more than one active site. In Escherichia coli, a high proportion of proteins of more than 300 amino acids are estimated to be multifunctional (Gething, 1997
).
We previously demonstrated that the impact of AbiK on phage resistance was substantially improved when cloned in a high-copy vector (Émond et al., 1997 ). Furthermore, the infection of an AbiK+ cell with multiple phages reduced the efficacy of this anti-phage system (data not shown). Therefore, it appears that the balance between the number of invading phages and the intracellular concentration of AbiK molecules is an important variable in experimental procedures on phagehost interactions. Our results showed that AbiK is potent enough to resist multiple phage infections, up to a threshold value, above which the resistance factor is most likely outnumbered and the proportion of viruses escaping AbiK is enhanced. Similarly, the codominance of phages p2 and p2K in the coinfection experiment supports the idea that competition occurs at the molecular level for factors needed for the AbiK phenotype.
In conclusion, AbiK has diverse consequences on the lytic cycle of 936 and P335 phages. This is the first report of such a dual effect of an Abi on two phage species. The molecular basis for the different kinds of interactions is not yet understood but further characterization of variant phages resistant to AbiK, such as p2K, could be a useful tool in investigating the AbiK mode of action. However, our results emphasize the importance of conducting mechanistic studies on both phage species. Finally, the determination of e.c.o.i. with extended m.o.i. values could represent an additional tool to evaluate the robustness of any given Abi system.
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ACKNOWLEDGEMENTS |
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Received 18 May 1999;
revised 14 September 1999;
accepted 22 October 1999.