Abstract

The complete genome sequences of three lactococcal 936-type bacteriophages, 712, jj50 and P008, were determined. Comparative genomic analysis of these phages with the previously sequenced 936-type phages, sk1 and bIL170, reveals a strict conservation of the overall genetic organization of this geographically diverse phage group. Genetic divergence was mainly observed in the early expressed region of the phage genomes, where a number of deletions, exchanges and insertions appear to have occurred. These genetic differences may be responsible for the observed differential sensitivity to the lactococcal DNA injection blocking protein, Sie2009, and the abortive infection system, AbiA.

Introduction

Bacteriophages infecting Lactococcus lactis disrupt dairy fermentation processes, which may result in considerable economic losses. These bacteriophages are classified into several genetically distinct groups, the three most prevalent of which are the c2, 936 and P335 species. This practical relevance has resulted in these three species receiving more focused research attention, particularly at the level of sequence and functional analyses. Members of the c2 and 936 phage groups are virulent phages, while members of the P335 group may be virulent or temperate. More recent phage population studies have indicated that 936-type phages account for most of the isolates obtained from industrial settings (Josephsenet al,1994; Moineauet al,1996; Bissonnetteet al,2000). Yet, only two complete phage genomes from the 936 group are currently available in GenBank, namely sk1 (Chandryet al,1997) and bIL170 (Crutz-Le Coqet al,2002).

Some members of the 936 phage type, such as sk1, jj50 and P008, appear to infect a wider range of lactococcal strains unlike many others, including φjw31, jw32 and the prototype phage, φ936 (Dupontet al,2005). Host range studies in conjunction with phylogenetic analysis of the receptor-binding proteins (Dupontet al,2004b) of many phages of this group have permitted a tripartite subgrouping of the 936 phages, namely the sk1-type, the bIL170-type and the 712-type (Dupontet al,2005), although a recent study showed additional RBP subgroupings within this lactococcal phage group (Tremblayet al,2006).

Nonetheless, this general divide is also observed when testing the sensitivity of lactococcal phages against antiviral defence mechanisms naturally present in some L. lactis strains. For example, Sie2009 is a membrane-associated DNA injection blocking protein that provides resistance to the expressing lactococcal host against some 936 phages capable of infecting Lactococcus lactis ssp. cremoris strains but not those infecting Lactococcus lactis ssp. lactis strains (McGrathet al,2002). The ability of the sk1-like and bIL170-like phages to infect L. lactis ssp. cremoris and lactis strains, respectively, is due, at least in part, to the variable C-terminal domain of the receptor-binding proteins as deduced by Dupont (2004b).

Another type of phage defence mechanism found in L. lactis is called abortive infection or Abi. These systems, also called phage exclusion, block phage multiplication and cause cell death upon phage infection. This outcome decreases the number of progeny virions and limits their spread, allowing the bacterial population to survive (Chopinet al,2005). AbiA is an abortive infection system that is known to affect the early stages of the phage lytic cycle (Dinsmore & Klaenhammer, 1994). The protein exhibits increased hydrophobicity at its amino terminus, in a manner comparable to Sie2009. Its early-acting nature, coupled with the similarity in predicted membrane topology, encouraged a study of the effect of the lactococcal abortive infection system, AbiA (Dinsmore & Klaenhammer, 1994). The sensitivity pattern of the assayed phages to these resistance mechanisms appeared to coincide with the host specificity of the phages identified by Dupont (2004b).

This observation and the limited number of complete genomic sequences for 936-like phages prompted the sequencing of the lactococcal phages 712, jj50 and P008. These three virulent lactococcal phages of the 936 group have been isolated from geographically distinct regions throughout Europe, some of which have evolved the ability to overcome phage-defence mechanisms to a varying extent. This article increases our knowledge on this significant phage group and provides insights into their adaptive genome evolution.

Materials and methods

Phages and bacterial strains

The lactococcal strains, plasmids and 936-type phages used in this study are listed in Table 1. Lactococcal host strains were grown at 30°C in M17 broth (Oxoid Ltdet al, Hampshire, England) supplemented with 0.5% glucose. Chloramphenicol and erythromycin were added at a final concentration of 5μgmL−1 where appropriate.

1

Lactococcal stains, plasmids and bacteriophages used in this study

 Relevant features Reference/Source 
Lactococcus lactis strains 
ssp. cremoris NZ9000 Host to sk1, jj50, 712 & p2 Kuipers (1998) 
ssp. lactis IL1403 Host to P008, bIL170, P113G, P272 & bIL66 Chopin (1984) 
ssp. cremoris W34 Host to jw31 & jw32 Josephsen (1994) 
Plasmids 
pNZ44sie2009 pNZ44 containing sie2009, Cmr McGrath (2002) 
pCI816 pSA3 containing 6.2kb StuI fragment from pCI829. Emr AbiA+ Coffey (1991) 
Bacteriophages 
sk1 Propagated on NZ9000 Chandry (1997) 
jj50 Propagated on NZ9000 Josephsen & Vogensen (1989) 
712 Propagated on NZ9000 Rince (2000) 
p2 Propagated on NZ9000 Higgins (1988) 
P008 Propagated on IL1403 Loof (1983) 
bIL170 Propagated on IL1403 Crutz-Le Coq (2002) 
bIL66 Propagated on IL1403 Bidnenko (1995) 
P113G Propagated on IL1403 Loof & Teuber (1986) 
P272 Propagated on IL1403 Loof & Teuber (1986) 
jw31 Propagated on W34 Josephsen (1999) 
jw32 Propagated on W34 Josephsen (1999) 
 Relevant features Reference/Source 
Lactococcus lactis strains 
ssp. cremoris NZ9000 Host to sk1, jj50, 712 & p2 Kuipers (1998) 
ssp. lactis IL1403 Host to P008, bIL170, P113G, P272 & bIL66 Chopin (1984) 
ssp. cremoris W34 Host to jw31 & jw32 Josephsen (1994) 
Plasmids 
pNZ44sie2009 pNZ44 containing sie2009, Cmr McGrath (2002) 
pCI816 pSA3 containing 6.2kb StuI fragment from pCI829. Emr AbiA+ Coffey (1991) 
Bacteriophages 
sk1 Propagated on NZ9000 Chandry (1997) 
jj50 Propagated on NZ9000 Josephsen & Vogensen (1989) 
712 Propagated on NZ9000 Rince (2000) 
p2 Propagated on NZ9000 Higgins (1988) 
P008 Propagated on IL1403 Loof (1983) 
bIL170 Propagated on IL1403 Crutz-Le Coq (2002) 
bIL66 Propagated on IL1403 Bidnenko (1995) 
P113G Propagated on IL1403 Loof & Teuber (1986) 
P272 Propagated on IL1403 Loof & Teuber (1986) 
jw31 Propagated on W34 Josephsen (1999) 
jw32 Propagated on W34 Josephsen (1999) 

Sie2009 and AbiA activity spectra determination

Plaque assays were performed using the protocol described by Lillehaug (1997). The effectiveness of AbiA was examined as previously described by Coffey (1991), while Sie2009 was assayed against lactococcal phages as previously described by McGrath (2002).

DNA sequencing and sequence analysis

DNA of phages 712 and jj50 was isolated as described previously (Moineauet al,1994). The DNA was mechanically sheared and the resultant fragments were shotgun-cloned in pGem-Teasy (Promega, Madison, WI) to generate a plasmid library in Escherichia coli DH10b. The cloned fragments were subsequently sequenced, providing a fivefold sequence coverage (MWG Biotech AG, Ebersberg, Germany), representing c. 60% of the phage genomes. Sequence assembly was performed using the Seqman programe, version 5.05, of the DNAStar software package (DNAStar Incet al, Madison WI). Alignment to the publicly available sequences of bIL170 and sk1 permitted size estimation of the remaining gaps, which, in the case of small gaps, were closed by primer walking directly on phage DNA. Larger gaps were PCR-amplified and sequenced by primer walking. DNA of phage P008 was isolated with the Maxi Lambda DNA purification kit (Qiagen) according to the modifications described previously by Deveau (2002). This DNA was sequenced on both strands by primer walking using an ABI Prism 3700 apparatus from the genomic platform at the research center of the Centre Hospitalier de l'Université Laval. To determine the cos region, phage DNA was ligated and then, the region was amplified by PCR and the purified PCR products were sequenced. Phage terminal ends were also defined by sequence drop-off using unligated phage DNA as a template.

Assignment of ORF was firstly based on minimal size lengths (at least 30 amino acids) and secondly, the putative start codon (ATG, TTG or GTG) should be preceded by a potential ribosomal-binding site or translationally coupled to a preceding ORF. Automatic ORF prediction was carried out using Artemis software or OrfFinder available on the NCBI web site, followed by a manual check of identified ORFs. Computer-assisted DNA analyses were performed using a Genetics Computer Group sequence analysis software package, version 10.3, including GenBank release 146.0, GenPept release 146.0, UniProt release 4.3, Swiss-Prot release 46.3, TrEMBL release 29.3, NREF release 1.64, PROSITE release 18.45, Pfam release 14.0, REBASE release 503 and ClustalW web site (http://www.ebi.ac.uk/clustalw/). psi-blast and Advanced Blast Search 2.1 were also used for sequence comparisons with databases (Altschulet al,1997).

Nucleotide sequence accession numbers

The complete genomic sequences of lactococcal phages analysed in this study are available under the following GenBank accession numbers: phage bIL170 (NC001909), sk1 (NC001835), 712 (DQ227763), jj50 (DQ227764) and P008 (DQ054536).

Results and discussion

Activity Spectra of Sie2009 and AbiA

McGrath (2002) previously established that expression of Sie2009 confers a phage-resistant phenotype on the lactococcal host against 936-like phages sk1, jj50 and 712, while no such effect was observed against phage P008. In this study, Sie2009 was shown not to affect proliferation of phage bIL170, bIL66, P272 or P113G (Table 2). Interestingly, bacteriophages (P008, bIL170, bIL66, P272, P113G) infecting L. lactis ssp. lactis IL1403 were unaffected by Sie2009 while those infecting L. lactis ssp. cremoris (sk1, jj50, 712, p2, jw32) were more sensitive to the effects of this phage-encoded resistance system (Table 2). This suggests that Sie2009 is specific in its activity to a subgroup of the 936-type phages and perhaps exclusively active against a subgroup of phages infecting specific L. lactis ssp. cremoris strains. This may be further evidenced by the activity of Sie2009 against φjw32 and p2, which both infect L. lactis ssp. cremoris strains (Table 2). The apparent inactivity of this system against φjw31 and the previous observation of inactivity against another 936-type phage, 18-16, may point to the phage-specific activity of this system. It cannot be precluded that Sie2009 may interact with the host receptor for the sk1-like phages or another host-encoded factor in order to exert its effect. The host receptors for the sk1- and bIL170-like phages are different, which may explicate the selective action of Sie2009 (Dupontet al,2004a). However, it is interesting to note that in both cases, a putative glycosyltransferase and a membrane-associated protein of the operon encoding cell wall polysaccharide biosynthesis are apparently necessary for phage adsorption (Dupontet al,2004a).

2

Phage characteristics and responses to Sie2009 and AbiA

  Genome EOP 
Phage Host Length
(kb) % G+C
content # ORFs
predicted Sie2009 AbiA 
sk1 NZ9000 28451 34.51 54 <10−9 10−3 
jj50 NZ9000 27453 34.94 49 <10−9 10−2 
712 NZ9000 30510 33.89 55 <10−9 <10−9 
P008 IL1403 28538 34.68 58 
bIL170 IL1403 31754 34.35 64 
bIL66 IL1403 – – – 
P272 IL1403 – – – 
P113G IL1403 – – – 
p2 NZ9000 – – – 0.5 10−3 
jw31 W34 – – – 
jw32 W34 – – – 10−2 
  Genome EOP 
Phage Host Length
(kb) % G+C
content # ORFs
predicted Sie2009 AbiA 
sk1 NZ9000 28451 34.51 54 <10−9 10−3 
jj50 NZ9000 27453 34.94 49 <10−9 10−2 
712 NZ9000 30510 33.89 55 <10−9 <10−9 
P008 IL1403 28538 34.68 58 
bIL170 IL1403 31754 34.35 64 
bIL66 IL1403 – – – 
P272 IL1403 – – – 
P113G IL1403 – – – 
p2 NZ9000 – – – 0.5 10−3 
jw31 W34 – – – 
jw32 W34 – – – 10−2 

Efficiencies of plaquing (EOP) values are the mean values of five independent assays.

AbiA exhibited an activity pattern similar to that of Sie2009, albeit to various extents, against sk1, jj50 and 712 (Table 2). Interestingly, this abortive infection system was unable to prevent proliferation of bIL170 and P008 or bIL66, P272, P113G, jw31 and jw32. The variation in sensitivity observed between P008/bIL170 and sk1/jj50/712 to Sie2009 and AbiA provoked an interest in studying the genetic diversity within the 936 phage group.

Phage genome sequence analysis

The complete nucleotide sequences of the bacteriophages jj50, 712 and P008 were determined by a dual strategy of shotgun cloning and primer walking. The genomes of jj50, P008 and 712 were found to be 27453, 28538 and 30510bp in length, respectively. The genomes of phages jj50, P008 and 712 contain 49, 58 and 55 ORFs, respectively, with the same overall genetic organization as previously determined for both sk1 and bIL170 (Chandryet al,1997; Crutz-Le Coqet al,2002). Table 3 outlines the identified ORFs of P008 and its homologues in the other completely sequenced 936-type phages. Each of the functional modules appears to be maintained in these newly described phages (Fig. 1).

3

Coordinates of phage P008 ORFs and representative ORFs in other 936-type phages

    Homologous ORF (% aa identity)  
ORF Start Stop Predicted
protein Size
(kDa) sk1 bIL170 712 jj50 Putative function 
Late expressed region 
254 778 19.90 ORF 1 (96) L1 (98) ORF 1 (98) ORF 1 (97) Terminase small subunit 
779 2401 63.01 ORF 2 (98) L2 (96) ORF 2 (98) ORF 2 (99) Terminase large subunit 
2391 2675 11.18 ORF 3 (97) L3 (97) ORF 3 (98) ORF 3 (98) HNH endonuclease 
2688 3824 43.32 ORF 4 (95) L4 (97) ORF 4 (96) ORF 4 (96) Portal protein 
3805 4341 19.90 ORF 5 (98) L5 (96) ORF 5 (99) ORF 5 (98) Protease 
4334 5515 43.73 ORF 6 (99) L6 (97) ORF 6 (99) ORF 6 (99) Structural protein 
4559 5515 35.16 Non-annotated (99) L7 (98) − − Structural protein 
5536 5799 10.01 ORF 7 (100) L8 (100) ORF7 (99) ORF 7 (98) Structural protein 
5799 6113 11.87 ORF 8 (94) L9 (94) ORF8 (96) ORF 8 (97) Structural protein 
10 6110 6451 12.88 ORF 9 (96) L10 (98) ORF 9 (94) ORF 9 (99) Structural protein 
11 6442 6807 13.65 ORF 10 (97) L11 (96) ORF 10 (97) ORF 10 (97) Major tail structural protein 
12 6836 7273 15.82 − L12 (80) ORF 12 (40) − Putative collar structure 
13 7297 8202 32.35 ORF 11 (97) L13 (97) ORF 11 (87) ORF 11 (94)  
14 8257 8532 10.61 ORF 12 (98) L14 (100) ORF 13(100) ORF 12 (100)  
15 8552 9064 19.86 ORF 13 (97) L15 (98) ORF 14 (97) ORF 13 (99)  
16 9064 11814 96.21 ORF 14 (71) L16 (94) ORF 15 (78) ORF 14 (80) Tail tape measure 
17 11814 12710 34.53 ORF 15 (91) L17 (97) ORF 16 (88) ORF 15 (95)  
18 12710 13837 42.82 ORF 16 (94) L18 (98) ORF 17 (95) ORF 16 (95)  
19 13827 14120 11.31 ORF 17 (97) L19 (100) ORF 18 (98) ORF 17 (94)  
20 14110 14913 28.65 ORF 18 (72) L20 (95) ORF 19 (60) ORF 18 (56) Receptor-binding protein 
21 14935 15288 13.34 ORF 19 (96) L21 (94) ORF 20 (96) ORF 19 (95) Holin 
22 15285 15986 26.26 ORF 20 (77) L22 (97) ORF 21 (70) ORF 20 (70) Lysin 
Early expressed region 
23 16630 16094 19.93 Non-annotated (93) E36 (91) ORF 23 (74) −  
24 16977 16720 10.12 − E35 (99) − −  
25 17267 16998 10.32 ORF 21 (93) E33 (96) ORF 24 (90) ORF 21 (94)  
26 17787 17440 13.59 ORF 23 (67) E31 (98) ORF 26 (82) ORF 22 (67)  
27 17954 17787 6.86 ORF 24 (40) E30 (96) ORF 27 (41) ORF 23 (41)  
28 18142 17954 7.23 − E29 (95) − −  
29 18389 18135 9.98 ORF 25 (75) E28 (98) ORF 28 (73) ORF 24 (74)  
30 18656 18453 8.22 ORF 26 (55) E27 (96) − ORF 25 (56)  
31 18860 18717 5.59 − E25 (95) − −  
32 19297 18857 17.57 − − − −  
33 19604 19299 12.10 ORF 26 (85) E24 (83) − ORF 25 (84)  
34 19732 19604 4.41 ORF 27 (56) E23 (59) − ORF 26 (60)  
35 20106 19792 11.86 ORF 31 (80) E17 (86) − ORF 30 (78)  
36 20597 20172 16.56 − E20 (73) ORF 31 (50) − HNH endonuclease 
37 21185 20676 20.06 ORF 32 (91) E15 (92) ORF 32 (94) ORF 31 (92)  
38 21397 21182 8.30 ORF 33 (97) E14 (97) ORF 33 (93) ORF 32 (97)  
39 21769 21410 12.99 ORF 34 (94) E13 (92) ORF 34 (94) ORF 33 (94)  
40 22348 21773 22.13 ORF 35 (54) E12 (50) ORF 35 (88) ORF 34 (59) sak 
41 22650 22324 12.71 ORF 36 (63) − − ORF 35 (55)  
42 23044 22637 15.71 ORF 38 (80) E10 (88) ORF 38 (81) ORF 37 (82) DNA polymerase subunit 
43 23147 22857 11.87 ORF 39 (97) E9 (94) − ORF 38 (94)  
44 23524 23144 14.67 ORF 40 (52) E8 (83) ORF 39 (72) ORF 39 (59)  
45 23840 23577 10.36 ORF 41 (89) E7 (94) ORF 40 (87) ORF 40 (87)  
46 24021 23812 8.03 − E6 (100) ORF 41 (44) −  
47 24910 24014 34.24 ORF 43 (41) E5 (40) ORF 42 (96) ORF 42 (44)  
48 25115 24954 6.16 E4 bIL66M1 (95) − ORF 45 (77) −  
49 25386 25162 8.62 − − − −  
50 26031 25912 4.52 − E2 (100) ORF 46 (98) −  
51 26169 26315 5.53 − ORF 0 (89) − −  
52 26610 26443 6.83 ORF 50 (87) E1 (83) ORF 49 (88) ORF 45 (84)  
Middle expressed region 
53 26761 26889 4.81 ORF 51 (97) M1 (97) ORF 51 (93) ORF 46 (98)  
54 26894 27025 5.12 ORF 52 (95) M2 (97) ORF 52(100) ORF 47 (95)  
55 27022 27504 18.04 ORF 53 (95) M3 (96) ORF 53 (97) ORF 48 (94) Holliday junction endonuclease 
56 27501 27668 6.14 ORF 54 (98) M4 (96) ORF 54 (91) ORF 49 (98)  
57 27882 28004 4.26 − − − −  
58 28298 28426 4.82 − − − −  
    Homologous ORF (% aa identity)  
ORF Start Stop Predicted
protein Size
(kDa) sk1 bIL170 712 jj50 Putative function 
Late expressed region 
254 778 19.90 ORF 1 (96) L1 (98) ORF 1 (98) ORF 1 (97) Terminase small subunit 
779 2401 63.01 ORF 2 (98) L2 (96) ORF 2 (98) ORF 2 (99) Terminase large subunit 
2391 2675 11.18 ORF 3 (97) L3 (97) ORF 3 (98) ORF 3 (98) HNH endonuclease 
2688 3824 43.32 ORF 4 (95) L4 (97) ORF 4 (96) ORF 4 (96) Portal protein 
3805 4341 19.90 ORF 5 (98) L5 (96) ORF 5 (99) ORF 5 (98) Protease 
4334 5515 43.73 ORF 6 (99) L6 (97) ORF 6 (99) ORF 6 (99) Structural protein 
4559 5515 35.16 Non-annotated (99) L7 (98) − − Structural protein 
5536 5799 10.01 ORF 7 (100) L8 (100) ORF7 (99) ORF 7 (98) Structural protein 
5799 6113 11.87 ORF 8 (94) L9 (94) ORF8 (96) ORF 8 (97) Structural protein 
10 6110 6451 12.88 ORF 9 (96) L10 (98) ORF 9 (94) ORF 9 (99) Structural protein 
11 6442 6807 13.65 ORF 10 (97) L11 (96) ORF 10 (97) ORF 10 (97) Major tail structural protein 
12 6836 7273 15.82 − L12 (80) ORF 12 (40) − Putative collar structure 
13 7297 8202 32.35 ORF 11 (97) L13 (97) ORF 11 (87) ORF 11 (94)  
14 8257 8532 10.61 ORF 12 (98) L14 (100) ORF 13(100) ORF 12 (100)  
15 8552 9064 19.86 ORF 13 (97) L15 (98) ORF 14 (97) ORF 13 (99)  
16 9064 11814 96.21 ORF 14 (71) L16 (94) ORF 15 (78) ORF 14 (80) Tail tape measure 
17 11814 12710 34.53 ORF 15 (91) L17 (97) ORF 16 (88) ORF 15 (95)  
18 12710 13837 42.82 ORF 16 (94) L18 (98) ORF 17 (95) ORF 16 (95)  
19 13827 14120 11.31 ORF 17 (97) L19 (100) ORF 18 (98) ORF 17 (94)  
20 14110 14913 28.65 ORF 18 (72) L20 (95) ORF 19 (60) ORF 18 (56) Receptor-binding protein 
21 14935 15288 13.34 ORF 19 (96) L21 (94) ORF 20 (96) ORF 19 (95) Holin 
22 15285 15986 26.26 ORF 20 (77) L22 (97) ORF 21 (70) ORF 20 (70) Lysin 
Early expressed region 
23 16630 16094 19.93 Non-annotated (93) E36 (91) ORF 23 (74) −  
24 16977 16720 10.12 − E35 (99) − −  
25 17267 16998 10.32 ORF 21 (93) E33 (96) ORF 24 (90) ORF 21 (94)  
26 17787 17440 13.59 ORF 23 (67) E31 (98) ORF 26 (82) ORF 22 (67)  
27 17954 17787 6.86 ORF 24 (40) E30 (96) ORF 27 (41) ORF 23 (41)  
28 18142 17954 7.23 − E29 (95) − −  
29 18389 18135 9.98 ORF 25 (75) E28 (98) ORF 28 (73) ORF 24 (74)  
30 18656 18453 8.22 ORF 26 (55) E27 (96) − ORF 25 (56)  
31 18860 18717 5.59 − E25 (95) − −  
32 19297 18857 17.57 − − − −  
33 19604 19299 12.10 ORF 26 (85) E24 (83) − ORF 25 (84)  
34 19732 19604 4.41 ORF 27 (56) E23 (59) − ORF 26 (60)  
35 20106 19792 11.86 ORF 31 (80) E17 (86) − ORF 30 (78)  
36 20597 20172 16.56 − E20 (73) ORF 31 (50) − HNH endonuclease 
37 21185 20676 20.06 ORF 32 (91) E15 (92) ORF 32 (94) ORF 31 (92)  
38 21397 21182 8.30 ORF 33 (97) E14 (97) ORF 33 (93) ORF 32 (97)  
39 21769 21410 12.99 ORF 34 (94) E13 (92) ORF 34 (94) ORF 33 (94)  
40 22348 21773 22.13 ORF 35 (54) E12 (50) ORF 35 (88) ORF 34 (59) sak 
41 22650 22324 12.71 ORF 36 (63) − − ORF 35 (55)  
42 23044 22637 15.71 ORF 38 (80) E10 (88) ORF 38 (81) ORF 37 (82) DNA polymerase subunit 
43 23147 22857 11.87 ORF 39 (97) E9 (94) − ORF 38 (94)  
44 23524 23144 14.67 ORF 40 (52) E8 (83) ORF 39 (72) ORF 39 (59)  
45 23840 23577 10.36 ORF 41 (89) E7 (94) ORF 40 (87) ORF 40 (87)  
46 24021 23812 8.03 − E6 (100) ORF 41 (44) −  
47 24910 24014 34.24 ORF 43 (41) E5 (40) ORF 42 (96) ORF 42 (44)  
48 25115 24954 6.16 E4 bIL66M1 (95) − ORF 45 (77) −  
49 25386 25162 8.62 − − − −  
50 26031 25912 4.52 − E2 (100) ORF 46 (98) −  
51 26169 26315 5.53 − ORF 0 (89) − −  
52 26610 26443 6.83 ORF 50 (87) E1 (83) ORF 49 (88) ORF 45 (84)  
Middle expressed region 
53 26761 26889 4.81 ORF 51 (97) M1 (97) ORF 51 (93) ORF 46 (98)  
54 26894 27025 5.12 ORF 52 (95) M2 (97) ORF 52(100) ORF 47 (95)  
55 27022 27504 18.04 ORF 53 (95) M3 (96) ORF 53 (97) ORF 48 (94) Holliday junction endonuclease 
56 27501 27668 6.14 ORF 54 (98) M4 (96) ORF 54 (91) ORF 49 (98)  
57 27882 28004 4.26 − − − −  
58 28298 28426 4.82 − − − −  

sak, Gene identified to be involved in AbiK-sensitivity (Bouchard & Moineau, 2004); E4bIL66M1, Early expressed ORF E4 of the mutant derivative of the 936-type phage, bIL66 (Bidnenkoet al,1995).

Note: ORFs 1A, 25, 44, 45 and 50 of φ712 possess no homologous counterparts in the other sequenced phages. ORF 22 of φ712 possesses 39% aa identity to E37 of bIL170, which is an HNH endonuclease not identified on the table above.

1

Schematic representation of the genomic organization of the 936-type phages, jj50, sk1 (Chandryet al,1997), 712, P008 and bIL170 (Crutz-Le Coqet al,2002), highlighting functional modules within the temporally transcribed regions. Genomic regions connected by a grey block are indicative of those possessing at least 60% identity at the amino acid level. The putative origins of replication (ori) of the phages are indicated below the relevant region of the individual phages. ORFs of the same colour represent those with at least 80% identity at the amino acid level.

1

Schematic representation of the genomic organization of the 936-type phages, jj50, sk1 (Chandryet al,1997), 712, P008 and bIL170 (Crutz-Le Coqet al,2002), highlighting functional modules within the temporally transcribed regions. Genomic regions connected by a grey block are indicative of those possessing at least 60% identity at the amino acid level. The putative origins of replication (ori) of the phages are indicated below the relevant region of the individual phages. ORFs of the same colour represent those with at least 80% identity at the amino acid level.

Owing to the genetic similarities, it seems obvious that phages bIL170, 712 and P008 have evolved from the same ancestral core as sk1 and jj50 but with greater divergence occurring throughout their evolution. It is hypothesized that the ancestors of the phages used in this study and other 936-like phages were, at some point, capable of infecting the same host strain. This would have provided the opportunity for genetic recombination to occur, resulting in diversification of this phage species. The frequency of homologous recombination of 936-like phages was observed to be relatively high in vitro and is proposed to mimic the natural situation (Crutz-Le Coqet al,2006).

Notwithstanding the high degree of similarity of these phages, it should be highlighted that some features of these phages vary and fall into subgroups within this closely related phage group. The most noteworthy of these are the proposed origin of replication, the cos terminal regions and the presence/absence of a homologue of the putative collar protein of bIL170 (L12).

Replication origin

Previously, the origin of replication of phage sk1 was experimentally determined to be within orf47 (Chandryet al,1997). Phage bIL170 does not possess a homologue of this gene. However, an additional orf (orf0) was found in this phage at a corresponding position, with a high A+T content (71mol%) and a short conserved region between the two phages (Crutz-Le Coqet al,2002). Consequently, orf0bIL170 was assigned as the putative replication origin of bIL170 (Crutz-Le Coqet al,2002). Phage P008 possesses a homologue (orf51P008) to orf 0bIL170 and consequently, both phages possibly have a similar origin of replication. Sequence analysis revealed that jj50 carries an ORF with 93% homology to ORF47sk1 and it is, therefore, assumed that this phage has the same replication origin (Fig. 1). The genome of 712 does not possess a homologue of either orf47sk1 or orf0bIL170. However, phage 712 possesses a homologue of e2bIL170, which is absent in sk1 and jj50. This gene is located just downstream of orf0 on the genome of bIL170 and its homologous equivalent in 712 possesses an A+T composition similar to that of orf0bIL170 (71.4%). This orf of phage 712, and possibly the A+T-rich flanking regions, could be implied as being necessary as the replication origin. In summary, three types of putative origin of replication are found within these five 936-like phages, namely sk1/jj50, bIL170/P008 and 712.

Genomic termini

With respect to the terminal genomic regions, Parreira (1996) observed a maintenance of the cos site, two of the four putative terminase binding sites (R1 and R3) and five AATCT sequences postulated to be involved in DNA bending and the 10bp inverted repeats positioned on either side of the cos site between four 936-like phages, namely bIL66, P008, sk1 and bIL41. A similar observation was made for phages jj50, 712 and bIL170. Additionally, the deletion of R2 observed in P008 and bIL66 is also found in bIL170, thus compounding the relatedness of these phages (Fig. 2).

2

Comparison of the cos terminal regions of the 936-type phages. Shaded are the cos sites, inverted repeats (IR), direct repeats (D1–5) and the putative terminase binding sites (R1–4).

2

Comparison of the cos terminal regions of the 936-type phages. Shaded are the cos sites, inverted repeats (IR), direct repeats (D1–5) and the putative terminase binding sites (R1–4).

Putative collar structure

The middle and late genes of all five phages are well conserved and overall, there is little divergence in these regions (Fig. 1), with the exception of the putative collar protein (located between the capsid and the tail) of bIL170 (L12), which has truncated homologues in 712 and P008 but no homologous counterpart in either sk1 or jj50. A collar structure with protruding whiskers has been observed for φP008 in previous reports, although with limited ability to demonstrate the whisker structure due to the staining technique (Loofet al,1983; Jarviset al,1991). This collar structure was also evidenced in φbIL170 while electron micrographs of φsk1 demonstrate no such structure (Dupontet al,2004b). Under the conditions used in this study, electron micrographs of φjj50, 712 and P008 are comparable to those of φsk1, revealing no discernible collar structure. Previous studies also demonstrate that φP113G and P272 do not possess a collar/whisker structure, while both structures are clearly visible in another 936-type phage, φ853 (Jarvis & Meyer, 1986; Loof & Teuber, 1986). Heteroduplex analysis of the genomes of φP008 and P272/P113G revealed that P113G differs from P008 only in the region encoding the collar/whisker structure (Loof & Teuber, 1986). However, a deletion within the gene coding for L12 of phage bIL41 has shown that it is a structural component that does not influence phage propagation or tail assembly but is hypothesized to play a nonessential role in host-range determination/extension (Crutz-Le Coqet al,2006). The presence of a host-recognition domain (found in other phages eg. the P335 phages bIL309 and BK5-T) provides a persuasive argument to support the theory of its involvement in additional roles apart from its structural function.

Early expressed region

The greatest level of divergence between the five phages compared in this study was apparent in the early expressed region. Crutz-Le Coq (2002) observed a similar finding when genomic comparisons of sk1 and bIL170 were made. Phages sk1 and jj50 share the greatest homology, with the majority of the proteins possessing over 90% aa identity. The numerous insertions/deletions, termed indels, observed in the early expressed region of the phages may have arisen through the acquisition of mobile DNA as suggested by the presence of the homing endonucleases observed in the early expressed regions of bIL170, P008 and 712. It is believed that these ORFs have arisen independently in bIL170, although the conservation of the relative positioning of these ORFs may indicate a similar evolutionary pathway of bIL170, P008 and 712.

Relationship between genome analysis and sensitivity to AbiA or Sie2009

Sie2009 is highly effective against phages sk1, jj50, 712 (EOP<10−9) while AbiA also confers resistance but to a lesser extent against the same three phages. Of note, despite numerous trials, we failed to obtain insensitive phage mutants derived from these three phages (data not shown), possibly indicating that ORFs involved in sensitivity (or required for activation) to this defence system may be absent on the genomes of the phages (P008 and bIL170) insensitive to these systems. This ORF, if present in phages sensitive to the phage defence systems, would be proposed to reside in the early expressed region as the middle and late expressed regions are well conserved. An early expressed gene of the P335 phage, φ31(ORF245), was identified as the ORF involved in sensitivity to AbiA (Dinsmore & Klaenhammer, 1997). No such gene was identified in φsk1 although 12% of the genome, covering part of the early expressed region, could not be cloned in this study and it was postulated that if φsk1 possesses such a gene, it would be in this region (Dinsmore & Klaenhammer, 1997).

While this is currently conjecture, the information provided by the genome sequences represents an excellent basis and comprehensive approach for studies relating to the sensitivity of this industrially problematic group of phages to a spectrum of phage defence mechanisms. This work also represents the first broad-ranging study of the genetic diversity within this group of phages of distinct geographical backgrounds. Given the recent resurgence of interest in phage biology, most notably the recent studies of the receptor binding and structural proteins of the 936-type phages (Crutz-Le Coqet al,2006; Spinelliet al,2006), the sequences embody a valuable resource to evolutionary and biological studies of dairy phages.

Acknowledgements

The authors wish to thank J. Josephsen (The Royal Veterinary and Agricultural University, Denmark) for kindly providing lactococcal phages and strains used as part of this study. We also thank Dr Ackermann for his excellent assistance with the electron micrographic work. J. Mahony is in receipt of research funding by the Irish Research Council for Science Engineering and Technology under the Embark Initiative. D. van Sinderen is a recipient of a Science Foundation of Ireland Investigatorship award (01/IN1/B198). H. Deveau is a recipient of a graduate student scholarship from the Fonds Québécois de Recherche sur la Nature et les Technologies (FQRNT). This study was funded, in part, by a strategic grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to S. Moineau.

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