Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 2015 Dec 22;82(1):95–105. doi: 10.1128/AEM.02723-15

Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei

Diego J Mercanti a,✉, Geneviève M Rousseau b, María L Capra a, Andrea Quiberoni a, Denise M Tremblay b, Simon J Labrie b, Sylvain Moineau b,✉
Editor: E G Dudley
PMCID: PMC4702644  PMID: 26475105

Abstract

Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: CL1, CL2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (CL1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages CL1, CL2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages CL1, CL2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts.

INTRODUCTION

The Lactobacillus casei group includes the species L. casei, L. paracasei, and L. rhamnosus, which are very closely related. They are grouped within the larger casei group because their species boundaries have not always been clear and, historically, their nomenclature has been sometimes controversial (1–3). Nonetheless, progress has been made in their classification (2, 4, 5).

Strains within this group are used in many fermented dairy products, where they contribute to flavor development (6). They can also be introduced into foods at a specific final concentration to provide a functional characteristic (probiotic). They are also used as starter cultures and propagate during the fermentation (7). In all of these cases, lactobacilli cells are targets for phage attacks, since virulent bacterial viruses are natural contaminants of food processing factories (8). In addition, it is well known that lysogeny is widespread in the genus Lactobacillus (9, 10). It was even suggested that these Lactobacillus prophages may evolve to become virulent phages (11). Within the L. casei group, high frequencies of prophage induction by mitomycin C treatment were observed in 10 out of 11 commercial strains, supporting the potential risks of introducing new phages within manufacturing facilities (10).

Genome sequencing has become necessary for the classification of prokaryote viruses and for understanding their evolution (12). Complete genome sequences of several phages infecting lactic acid bacteria (LAB) are now available in public databases. Most of these infect Lactococcus lactis (13) and Streptococcus thermophilus (14), as well as Lactobacillus spp. More than 40 Lactobacillus prophage and phage genome sequences are available, including phages infecting diverse Lactobacillus species, such as L. gasseri (15–18), L. johnsonii (19–21), L. plantarum (22–27), L. casei, L. paracasei, L. rhamnosus, and L. zeae (16, 28–35), L. helveticus (36), L. delbrueckii (37–42), L. salivarius (16), L. sanfranciscensis (43), L. fermentum (44, 45), L. brevis (46), and L. jensenii (47).

Morphology is the historical and still useful parameter for classifying phages. More than 40 phages of the L. casei group have been reported to date, mostly belonging to the Siphoviridae family (isometric capsid, long noncontractile tail) (48). The first casei phages were isolated from abnormal production of the fermented milk (Yakult): phages J-1 in 1965 (49) and PL-1 two years later (50). Their genome sequences were released only recently (32). A few other genome sequences of L. casei group phages are available, namely, A2 (28, 33, 34), phiAT3 (29), Lc-Nu (31), and Lb338-1 (35), as well as prophages Lca1 (16) and Lrm1 (30). Among these fully sequenced phage genomes, Lb338-1 is the only myophage (contractile tail) and possesses a much larger genome with a particularly low-GC content (35).

We analyzed five L. paracasei phages: CL1, CL2, iA2, iLp84, and iLp1308. Phages CL1 and CL2 were previously isolated from a spontaneously lysed culture of the probiotic strain L. paracasei A used in commercial milk beverages (51). Phage iA2 was previously induced by mitomycin C from the same probiotic strain, L. paracasei A (51). Phage iLp84 was isolated from a mitomycin C-induced strain, L. paracasei 84, and propagated on the indicator strain, L. paracasei INL3 (10). Similarly, phage iLp1308 was isolated from mitomycin C treatment of L. paracasei CNRZ 1308 and replicated in L. paracasei A14 (10). Phage iLp1308 has the same host range as phages CL1 and CL2, while the host range of phage iLp84 is restricted to fewer strains. We sequenced the genomes of these five phages to shed light on their genetic relationships, as well as to increase our understanding of phages infecting probiotic L. paracasei strains.

MATERIALS AND METHODS

Bacterial strains, phages, and culture conditions.

L. paracasei strains A, A14, and INL3 were grown at 37°C in MRS broth (Difco). Identification of the strains used in the present study was previously confirmed (10) by species-specific PCR (5). Prophage iA2 was induced from L. paracasei strain A using mitomycin C (Sigma-Aldrich) at a final concentration of 0.6 μg/ml, as described previously (51). Phage iA2 could not be propagated on any indicator strain. Phages CL1 and CL2, originally isolated from L. paracasei A, were propagated on that strain. Phages iLp84 and iLp1308 were propagated on the indicator strains L. paracasei INL3 and A14, respectively (10). Phages were then purified by three rounds of plaque purification using their respective indicator strain. All five phages were confirmed to belong to the Siphoviridae family by electron microscopy, as shown elsewhere (51, 52). To amplify the phages, host bacteria were grown to an optical density (600 nm) of 0.2 in MRS broth supplemented with 10 mM CaCl2 (MRS-Ca) and infected with phages at ∼104 PFU/ml, followed by incubation at 37°C until lysis. Phage lysates were filtered through a 0.45-μm-pore-size filter (Sarstedt) and kept at 4°C until use. Phage enumeration was assessed through the double-layer method (53), using MRS-Ca supplemented with 100 mM glycine (MRS-Ca-Gly) to increase the plaque size (54). Plates were incubated at 37°C for 18 h. The phages and bacterial strains used in the present study were stored at −80°C in MRS broth supplemented with 15% (vol/vol) glycerol at both the INLAIN Collection (Santa Fe, Argentina) and the Félix d'Hérelle Reference Center for Bacterial Viruses of the Université Laval (Québec, Canada). The identification numbers at the Félix d'Hérelle Reference Center are HER510, HER511, HER512, and HER513 (phages CL1, CL2, iLp84, and iLp1308) and HER1510, HER1512, and HER1513 (L. paracasei strains A, INL3, and A14).

DNA isolation and sequencing.

Phage genomic DNA was isolated using a Qiagen lambda maxi kit (Qiagen). To determine the genome extremities, phage DNA was cleaved with BglII (Roche Diagnostics) according to the manufacturer recommendations. DNA fragments were resolved in a 0.8% agarose gel in 40 mM Tris-acetate–1 mM EDTA buffer, stained with EZ-Vision Three (Amresco), and visualized under UV light. An aliquot of each digested phage DNA was heated at 75°C for 10 min prior to gel electrophoresis. The presence of an extra phage genomic fragment in the heated samples indicated the presence of cos-type genome extremities.

Sequencing libraries were prepared with the Nextera XT DNA library preparation kit (Illumina) according to the manufacturer's instructions. The libraries were sequenced using a MiSeq reagent kit v2 (Illumina; 500 cycles) on a MiSeq system. De novo assembly was performed with Ray assembler versions 2.1.1-devel and 2.2.0-devel using a k-mer size of 31 (55). A single contig was obtained for all phages with mean coverage depths of 2,536, 2,396, 2,828, 938, and 2,923 for phages CL1, CL2, iA2, iLp84, and iLp1308, respectively. Genome extremities were amplified using converging primers, and the PCR products were sequenced by the Sanger method using an ABI 3730xl apparatus at the sequencing and genotyping platform of the Centre Hospitalier de l'Université Laval. These latter phage sequences were assembled with Staden software (v2.0.0b9) (56).

Bioinformatics analysis.

Complete phage genomes were analyzed with BioEdit (57). Open reading frames (ORFs) were predicted with the command line version of GeneMarkS (version 4.29) using the setting for phage genomes (58). The identified ORFs were confirmed with ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). ORFs were considered candidates for evaluation when they encoded 25 or more amino acids (aa), and possessed both a conserved Shine-Dalgarno sequence (5′-AAGGAGGT-3′) and a start codon (AUG, UUG, or GUG). BLASTp was used to compare translated ORF products with known proteins. Hits were considered valid when the E value was lower than 10−3. Conserved domains in protein sequences were identified with the NCBI CD-search interface to search the Conserved Domain Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Physicochemical parameters of predicted proteins were calculated with the ProtParam Tool from the ExPASy proteomics server (http://web.expasy.org/protparam/). The bioinformatic tools tRNAscan-SE 1.21 (http://lowelab.ucsc.edu/tRNAscan-SE/) and ARAGORN (59) were used for tRNA gene detection. Phage codon usage was calculated using the Countcodon program (http://www.kazusa.or.jp/codon/) and compared to the codon usage of L. paracasei, obtained from the same database. For the alignment figures, phage and prophage protein sequences were compared using BLASTp 2.2.28+ (60). The percent identity between proteins was calculated by dividing the number of identical residues by the size of the largest protein. The genome maps were generated using the GenoPlotR package (61).

Terminase phylogeny.

The phylogenetic tree of the terminase was generated with a data set including sequences representing the main phage groups (62). The sequences were aligned using MAFFT with the automatic settings. The alignment was converted to the PHYLIP format with compatible name using in-house Python script. The most probable amino acid substitution model was determined using ProtTest 3.2 (63). The best model was then implemented in PhyML 3.0 to calculate the best tree (64). Branch support value was established using the Shimodaira-Hasegawa-like procedure (65). The leaves of the tree were renamed using the Newick utility package (66). Finally, the tree was rendered using the web interface ITOL (67).

Nucleotide sequence accession numbers.

The complete genome sequences of phages CL1, CL2, iA2, iLp84, and iLp1308 have been deposited in GenBank under accession numbers KR905066, KR905067, KR905068, KR905069, and KR905070, respectively.

RESULTS AND DISCUSSION

Phage genome overview.

The five phage genomes analyzed in the present study were double-stranded DNA molecules, and their general features are shown in Table 1. The genome sizes of phages CL1, CL2, and iLp84 ranged from 38,751 to 39,474 bp and were similar to most L. casei group phages already described (Table 1). The genomes of phages iA2 (34,155 bp) and iLp1308 (34,176 bp) were ∼2 kb shorter than the genome of phage Lc-Nu (36,466 bp), the smallest genome of L. casei phage reported before this study (Table 1). The GC content (44.8 to 45.6%) of the five phages was similar to the other L. casei group phages (Table 1). The previously characterized L. paracasei myophage ϕLb338-1 has a much lower GC content (37.4%), but it differs in structure from the L. casei phages and has a much larger genome (35). The genome of prophage iA2 has cohesive extremities with 3′ overhangs that are 10 nucleotides (nt) long (CGGCATGCAA). Of note, when this sequence is ligated, it generates an orf coding for a HNH endonuclease. Hence, we elected to end the map of prophage iA2 after this orf, and we located the cos site at positions 34135 to 34144.

TABLE 1.

General features of phage genomes of the L. casei group

Phage Genome size (kb) % GC Total no. of ORFs (plus strand/minus strand) Source or reference
CL1 39,474 44.8 62 (48/14) This study
CL2 38,751 44.9 60 (59/1) This study
iA2 34,155 45.6 50 (43/7) This study
iLp84 39,399 45.1 60 (49/11) This study
iLp1308 34,176 45.1 50 (42/8) This study
A2 43,411 44.9 61 (55/6) 34
J-1 40,931 44.8 63 (57/6) 32
Lca1 46,986 44.8 71 (56/15) 16
Lc-Nu 36,466 44.3 51 (50/1) 31
Lrm1 39,989 45.5 54 (51/3) 30
phiAT3 39,166 44.6 53 (50/3) 29
PL-1 38,880 44.9 59 (57/2) 32
Lb338 1 142,111 37.4 199 (128/71) 35
Open in a new tab

Genome organization.

Table 2 provides the analysis of the predicted ORFs for phage CL1. Homologous ORFs predicted for phages CL2, iLp84, and iLp1308 and their respective amino acid identities with the deduced proteins of phage CL1 are indicated as well. Phage CL1 was chosen as the reference because it has the highest identity with the other three phages (CL2, iLp84, and iLp1308; see Fig. 1, discussed below). Because prophage iA2 does not have significant similarity to these four phages, Table 3 presents the analysis of its 50 predicted ORFs, including 24 with a probable function. The organization of these genomes is modular and resembles that reported for other L. casei phages. Their genomes have clusters of genes involved in DNA packaging, morphogenesis, lysis, lysogeny, and replication (Fig. 1 and Tables 2 and 3). Still, as illustrated in Fig. 1, the five phages described here are clearly distinct from the other phages of the L. casei group characterized to date.

TABLE 2.

Features of ORFs of phage CL1 and homology with ORFs of phages CL2, iLp84, and iLp1308

ORF Strand Position
 Size (aa) Size (kDa) pI SD sequence (5′–3′)a Putative function Representative ORF (% aa identity) in the indicated phage
Start Stop CL2 iLp1308 iLp84
1 + 31 522 163 18.2 5.7 CGGGAGGTgtggtgatatGTG Small subunit terminase ORF1 (100) ORF1 (25.6) ORF1 (25.8)
2 + 506 1759 417 48.3 7.7 AAGGATGGtgatgtcaATG Terminase large subunit ORF2 (80.7) ORF2 (99.8) ORF2 (98.3)
3 + 1719 3191 490 54.3 4.5 ACGGAGAAATG Portal protein ORF4 (99.8) ORF3 (100) ORF3 (97.6)
4 + 3157 4155 332 37.6 6.9 ACGGTGGTggcaatgATG Mu protein F-like protein ORF5 (100) ORF4 (96.4) ORF4 (92.5)
5 + 4165 4491 108 12.5 4.8 TAGGAGGAaacgATG ORF6 (100)
6 + 4662 5300 212 23.8 5.0 AAGGAGTTcttagcATG ORF7 (100) ORF5 (99.5) ORF5 (97.6)
7 + 5313 5627 104 10.8 5.2 AAGGAGGTacttttATG ORF8 (99.0) ORF6 (99.0) ORF6 (95.2)
8 + 5641 6660 339 37.6 4.9 AAGGAGGAttaacttATG ORF9 (100) ORF7 (100) ORF7 (95.7)
9 + 6747 6926 59 5.4 3.7 TATCCGCTgaccctGTG ORF10 (100) ORF8 (100)
10 + 6996 7370 124 14.2 4.8 AAGGAGGCatgaaATG ORF11 (100) ORF9 (100) ORF10 (87.9)
11 + 7375 7677 100 11.4 4.8 AAAGAGGTgatcatATG ORF12 (100) ORF10 (100) ORF11 (90.0)
12 + 7674 8039 121 13.5 10.5 TTGTGGGTgagacgaaATG ORF13 (100) ORF11 (100) ORF12 (86.0)
13 + 8040 8444 134 15.4 4.7 AAGCAGCAaagtagGTG Major structural protein ORF14 (99.3) ORF12 (99.3) ORF13 (97.0)
14 + 8456 9061 201 21.7 4.4 TAGGAGGCcataacATG Major tail protein ORF15 (100) ORF13 (100) ORF14 (84.6)
15 + 9147 9479 110 11.9 5.7 AAGGATTTtaaatcATG ORF16 (100) ORF14 (100) ORF15 (75.7)
16 + 9584 9937 117 13.7 9.3 CGAGACATtgaacgtATG ORF17 (100) ORF15 (100) ORF16 (88.9)
17 + 9930 13019 1029 108.5 9.0 AAGGAGGGagcatATG Tape measure ORF18 (99.8) ORF16 (99.9) ORF17 (15.4)
18 + 13022 15133 703 77.9 4.5 AAGGGGCTattttagttTTG Tail tip protein ORF19 (99.1) ORF17 (99.7) ORF18 (73.1)
19 + 15130 17595 821 89.6 4.9 CAGGAGGCatggctATG Host interaction protein ORF20 (99.4) ORF18 (99.3) ORF19 (47.0)
20 + 17605 17928 107 11.7 4.2 TAGGAGGTtgttATG ORF21 (99.1) ORF19 (100) ORF20 (95.3)
21 + 17921 18052 43 4.8 4.1 AAGGCGGCgaaatcaaGTG ORF22 (100) ORF20 (100)
22 + 18083 18469 128 14.4 6.0 AAGGAAGTgatgacaATG Holin ORF23 (87.7) ORF21 (78.9)
23 + 18450 18659 69 8.0 9.8 AAGGAGTGacagccGTG ORF24 (92.8) ORF22 (81.2)
24 + 18656 19651 331 34.5 4.5 AAGGAGACaagcaagcaATG ORF25 (99.4) ORF23 (98.5)
25 + 19656 20162 168 18.3 6.0 ATTGAGGGgtgattgaATG ORF26 (94.0) ORF24 (100)
26 + 20155 20616 153 15.9 5.0 GAGGAGGTgaaatagtATG Holin ORF27 (99.3) ORF25 (100)
27 + 20618 21670 350 37.5 5.3 GAGGAGGTgaaatagtATG Lysin ORF28 (98.6) ORF26 (98.6)
28 − 22319 21834 161 17.5 6.1 AAGGAGACtaaaaATG ssDNA-binding protein ORF27 (99.4)
29 − 23008 22334 224 25.9 8.6 CAGGTGGCgaacgtgaATG ORF28 (54.5)
30 − 23760 23005 251 27.3 8.5 TTGGAGGGtaaatgATG Single-strand annealing protein ORF31 (100)
31 − 24668 23760 302 33.7 5.6 AAGGAAGCaaacaATG ORF32 (99.7)
32 − 24886 24665 73 8.1 5.8 AAGGGGGTaacgtgATG ORF38 (80.8)
33 − 25043 24891 50 5.7 10.3 TTGGAGGCagtggaTTG ORF37 (92.0)
34 − 25128 25036 30 3.3 6.7 AAGAGGGCggagaaATG ORF36 (100)
35 − 26436 25294 380 44.3 9.9 AAAGAGGTgaaataagGTG Transposase
36 + 26530 26952 140 16.3 10.1 ATTGAGGTaatcatATG Transposase
37 − 27227 26997 76 8.6 7.9 GAGGCGGTatagatATG Antirepressor ORF34 (56.6) ORF35 (12.0)
38 − 27484 27230 84 9.6 9.5 AAGGAGAAtcatatATG ORF33 (100)
39 − 28128 27766 120 12.9 9.8 TTGGAGGGattcatcATG ORF32 (100)
40 − 29212 28535 225 24.4 4.2 TCGGAGGGaaataATG tcdA-tcdE operon negative regulator
41 − 29505 29296 69 7.9 9.3 AATGGGATgacggtATG Antirepressor
42 − 29755 29528 75 8.5 9.8 AACGAGGTgattacATG ORF34 (100)
43 + 29910 30020 36 4.3 9.3 CCGTAGAActgcgATG
44 + 30218 30445 75 8.6 9.0 AAAGAGGTaaacaaCATG ORF36 (97.4)
45 + 30438 30761 107 12.4 5.7 AAGGAGGTaacgatcATG Replication protein ORF37 (100)
46 + 30761 31705 314 36.4 8.6 AAGAAGGGtgATG Replication protein ORF38 (99.4)
47 + 31702 32964 420 47.4 5.6 AGGGAGGCaacgagCTTG DNA helicase ORF39 (100)
48 + 32966 33310 114 13.6 4.9 AAGGTGGCgtattgaaATG ORF40 (99.1) ORF47 (13.7)
49 + 33323 33610 95 10.9 5.4 GAGGAGGCaaaaacATG ORF48 (40.0) ORF41 (98.9)
50 + 33597 33851 84 9.4 9.6 CAGGAGGAatcttcaaATG ORF49 (98.8) ORF42 (98.8) ORF51 (88.1)
51 + 33848 34213 121 14.4 6.7 AAGGACGTgaagccagcATG Endodeoxyribonuclease RusA ORF50 (80.2) ORF43 (99.2) ORF52 (98.3)
52 + 34225 34428 67 7.8 6.8 AAGGAGAAaaatcATG ORF51 (98.5) ORF44 (97.0) ORF46 (86.6)
53 + 34547 34930 127 15.0 10.1 CGAGTCGTtagccATG ORF52 (100)
54 + 35239 35772 177 20.0 9.6 AAAGAAAAaataacaATG Putative endodeoxyribonuclease ORF53 (99.4)
55 + 35769 35951 60 7.2 8.9 TTGGAGGGaaaacaATG ORF54 (96.7)
56 + 35948 36025 25 2.9 10.3 AGGGAGGAgcggcaATG ORF55 (100) ORF45 (96.0) ORF35 (17.5)
57 + 36038 36256 72 8.1 5.7 TAGGAGGTgaataatTTG Transcriptional regulator ORF56 (95.8) ORF46 (93.1)
58 + 36330 36758 142 16,5 8.4 AAGGAGTGgacgcaATG Transcriptional regulator ORF57 (99.3) ORF47 (71.4) ORF56 (72.1)
59 + 37121 37375 84 9.4 4.2 CAGGAGGTaaagaacATG
60 + 37399 38616 405 46.5 6.2 TTGGAGGCgagtagATG ORF59 (97.5) ORF56 (37.3)
61 + 38603 39157 184 20.9 10.1 AAGGCTACggtgcacttATG HNH homing endonuclease
62 + 39150 39470 106 12.6 7.7 ACGGGCGAgtgtccccATG Ribonucleoside-diphosphate reductase ORF60 (80.7) ORF50 (66.4) ORF60 (65.4)
Open in a new tab
a

Start codons are indicated in boldface. Uppercase letters without boldface are SD positions, and matches to the SD sequence (AAGGAGGT) are underlined.

FIG 1.

FIG 1

Open in a new tab

Schematic representation of the genome organization of different Lactobacillus phages. Each line corresponds to a different genome and starts from its physical end. Predicted ORFs are represented by arrows. ORFs with the same color in different phages possess amino acid identity higher than 80%. The white ORFs have <80% identity with any another ORF. Asterisks indicate transposases in the genomes of phages CL1 and CL2.

TABLE 3.

Features of the ORFs of prophage iA2, predicted functions of proteins, and best matches with databases

ORF Strand Position
 Size (aa) Size (kDa) pI SD sequence (5′–3′)a Putative function Best BLAST hit
Start Stop Protein (organism) (extentb; % aa identity) E value Size (aa) GenBank accession no.
1 + 58 444 128 14.5 5.2 AAGGGGATgacgaaaTTG Lpp229_06031 (L. paracasei) (128/128; 100%) 1E–88 131 EPC46831.1
2 + 447 2177 576 66.2 5.7 AGCGAGGCgaatagcaATG Terminase Terminase (L casei BL23) (576/576; 100%) 0 576 CAQ66068.1
3 + 2196 3431 411 45.8 5.0 AAGGAGGTgattatTTG Portal protein Phage portal protein (L casei BL23) (410/411; 99%) 0 411 CAQ66069.1
4 + 3403 4116 237 25.5 4.6 ACGGAGGGaggtgATG ATP-dep. Clp protease Clp protease (L. casei BL23) (235/235; 100%) 3E–168 235 CAQ66070.1
5 + 4121 5350 409 43.7 5.7 AAAGGGGAataacctcATG Major capsid protein Phage capsid protein (L. casei BL23) (409/409; 100%) 0 409 CAQ66071.1
6 + 5424 5672 82 8.3 4.3 AAGGAGCTagaacATG LCABL_09860 (L. casei BL23) (82/82; 100%) 2E–48 82 CAQ66072.1
7 + 5686 6012 108 12.2 4.3 TAGGCGGTgatcaaagATG LCABL_09870 (L. casei BL23) (108/108; 100%) 1E–71 108 CAQ66073.1
8 + 5951 6289 112 13.2 8.0 TTGGAGGTgagaacagATG Head-tail adaptor LCABL_09880 (L. casei BL23) (112/112; 100%) 3E–74 112 CAQ66074.1
9 + 6273 6602 109 12.6 4.6 ATGGAGTTgagtttggactATG LCABL_09890 (L. casei BL23) (109/109; 100%) 3E–72 109 CAQ66075.1
10 + 6592 6975 127 14.1 9.2 ATCGAGGTgattgcggGTG Tail component LCABL_09900 (L. casei BL23) (127/127; 100%) 1E–88 127 CAQ66076.1
11 + 6987 7634 215 23.6 5.4 TAGGAGGAatcgctATG Major tail protein Tail protein (L. casei BL23) (215/215; 100%) 4E–157 215 CAQ66077.1
12 + 7711 8076 121 13.7 6.7 TAGGAGGAcaaaggaatATG LCABL_09920 (L. casei BL23) (121/121; 100%) 4E–83 121 CAQ66078.1
13 + 8136 8318 60 7.2 9.0 CTGGGCCTtttatTTG AF91_09510 (L. paracasei N1115) (58/60; 98%) 1E–33 60 AHJ33419.1
14 + 8338 11310 990 105.5 9.4 AAGGAGGAaaataATG Tape measure protein Tape measure (L. casei BL23) (990/990; 100%) 0 990 CAQ66080.1
15 + 11317 12012 231 25.3 5.4 AAGGAGGCtttgatgacggATG Tail protein Gp13 protein (L. casei BL23) (231/231; 100%) 3E–166 231 CAQ66081.1
16 + 12009 16415 1,468 158.8 4.9 TAGGTGGTgttatgtATG Tail protein, endopeptidase PblB (L. casei LC2W) (1468/1468; 100%) 0 1,468 AEA53317.1
17 + 16444 16869 141 15.8 4.7 TGGGAGGAaaacATG LCABL_09970 (L. casei BL23) (141/141; 100%) 4E–95 141 CAQ66083.1
18 + 16872 17141 89 9.7 4.5 AAGGAGAGaagtaatcATG Orf59 (L. casei BL23) (89/89; 100%) 8E–55 89 CAQ66084.1
19 + 17187 17480 97 10.8 5.6 AAGAAGTGagaaaGTG LCABL_09990 (L. casei BL23) (96/97; 99%) 4E–63 97 CAQ66085.1
20 + 17470 17904 144 14.8 4.8 AAGGAGAAaataaccATG Holin Holin (L. casei BL23) (144/144; 100%) 1E–92 144 CAQ66086.1
21 + 17904 18092 62 6.9 5.0 CAGGAGGCcaagtaATG LCABL_10010 (L. casei BL23) (62/62; 100%) 1E–34 62 CAQ66087.1
22 + 18079 19053 324 34.8 4.8 GAGGAGGCgtacacgaATG Lysin Amidase (L. casei BL23) (324/324; 100%) 0 324 CAQ66088.1
23 – 19435 20574 379 43.8 9.7 ATGGGGGCaagtgacATG Integrase Integrase (L. casei BL23) (379/379; 100%) 0 379 CAQ66041.1
24 – 20692 21759 355 40.6 6.3 AGTGGGGTaagataaTTG Abi family protein CAAX protease (L. casei BL23) (355/355; 100%) 0 355 CAQ66042.1
25 – 21904 22335 143 15.8 5.1 AAGGAGGTaatagcATG LCABL_09570 (L. casei BL23) (143/143; 100%) 7E–98 143 CAQ66043.1
26 – 22405 22887 160 19.0 4.9 AAGGAGGAttagctgATG LCABL_09580 (L. casei BL23) (160/160; 100%) 7E–110 160 CAQ66044.1
27 – 22891 23184 97 11.6 8.6 AAGGACCCtcgactATG LCABL_09590 (L. casei BL23) (97/97; 100%) 5E–63 97 CAQ66045.1
28 – 23368 24228 286 33.0 5.2 AGGGAAAAgcaaaATG LCABL_09600 (L. casei BL23) (286/286; 100%) 0 286 CAQ66046.1
29 – 24215 24583 122 13.5 6.6 TTGGAGGGatttttATG Cro/CI regulator Cro/CI regulator (L. casei BL23) (122/122; 100%) 1E–81 122 CAQ66047.1
30 + 24839 25036 65 7.3 10.0 TAGGAGGTgaccactggtATG LC2W_0948 (L. casei LC2W) (65/65; 100%) 2E–38 65 AEA53282.1
31 + 25033 25755 240 26.7 9.8 AAGGAGGAatccaaATG Putative antirepressor LCABL_09620 (L. casei BL23) (240/240; 100%) 8E–175 240 CAQ66048.1
32 + 25763 25975 70 7.7 8.2 AAGGAGTGaagtacgtTTG BN194_09360 (L. casei W56) (69/70; 99%) 4E–40 70 CCK21883.1
33 + 26084 26308 74 9.1 8.9 AACAGCGTgccgacATG LCABL_09630 (L. casei BL23) (74/74; 100%) 3E–46 74 CAQ66049.1
34 + 26308 26400 30 3.3 8.0 AAGGAATTggtgaataATG LCABL_09640 (L. casei BL23) (30/30; 100%) 1E–11 30 CAQ66050.1
35 + 26397 26609 70 8.1 4.2 GAGGCAGTggatcgaATG LCABL_09650 (L. casei BL23) (70/70; 100%) 2E–42 70 CAQ66051.1
36 + 26619 27494 291 33.7 5.9 TAGGGGGTtgttATG Recombinase Protein YqaJ (L. casei BL23) (291/291; 100%) 0 291 CAQ66052.1
37 + 27497 27691 64 7.1 4.3 AAGAAGATtgaatATG LCABL_09670 (L. casei BL23) (64/64; 100%) 1E–37 64 CAQ66053.1
38 + 27691 28578 295 32.8 5.4 AAGGAGGAtgaagactaATG RecT DNA repair protein RecT protein (L. casei BL23) (295/295; 100%) 0 295 CAQ66054.1
39 + 28587 28832 81 9.2 8.9 AAGTAGGTgagctgATG HTH transcription regulator Orf8 (L. casei BL23) (81/81; 100%) 3E–51 81 CAQ66055.1
40 + 28837 29682 281 32.7 9.1 GCAGAGGTgatctgATG DNA damage inducible DnaD (L. casei BL23) (281/281; 100%) 0 281 CAQ66056.1
41 + 29720 30541 273 30.6 9.6 AACGAGGTgaaacATG DNA replication protein Prophage pi3 protein46 (L. casei BL23)(273/273; 100%) 0 273 CAQ66057.1
42 + 30538 30837 99 11.6 4.9 AAGGAAGGcatgtcATG LCABL_09720 (L. casei BL23) (99/99; 100%) 6E–65 99 CAQ66058.1
43 + 30800 31084 94 10.6 4.8 AAGCGGGAaagttcgATG LC2W_0960 (L. casei LC2W) (94/94; 100%) 2E–61 94 AEA53294.1
44 + 31053 31400 115 14.0 10.0 AATGAGGTgactgaaaATG LCBD_0957 (L. casei BD-II) (115/115; 100%) 8E–79 115 AEA56455.1
45 + 31393 31971 192 22.1 10.4 GAGGAGCTggcgctATG LCABL_09750 (L. casei BL23) (192/192; 100%) 2E–138 192 CAQ66061.1
46 + 31988 32407 139 15.6 9.0 AGGGAGGAttgatgATG Holliday junction resolvase Resolvase (L. casei BL23) (139/139; 100%) 2E–95 140 CAQ66062.1
47 + 32412 32675 87 9.6 5.1 AAGGAGGTctaacgccATG LCABL_09770 (L. casei BL23) (87/87; 100%) 2E–53 87 CAQ66063.1
48 + 32699 33022 107 13.1 9.8 GCGGAGGTtaagaaATG LCABL_09780 (L. casei BL23) (107/107; 100%) 3E–71 107 CAQ66064.1
49 + 33101 33550 149 17.9 5.1 AAGGAGTGgggccgTTG Transcriptional regulator LCABL_09790 (L. casei BL23) (148/149; 99%) 6E–101 149 CAQ66065.1
50 + 33775 34155 126 14.7 8.7 TTGGAGGTggatatgATG HNH endonuclease LCBD_0963 (L. casei BD-II) (126/126; 100%) 2E–89 127 AEA56461.1
Open in a new tab
a

Start codons are indicated in boldface. Uppercase letters without boldface are SD positions, and matches to the SD sequence (AAGGAGGT) are underlined.

b

That is, the number of identical amino acids/the total number of amino acids.

Phylogeny and comparative analysis of the phages.

We looked at the relationships of these five phages to other phages using one of the most conserved genes: the large terminase subunit. This gene was used previously in attempts to classify phages (68). Casjens et al. (62) also proposed to use the terminase phylogeny for classification of phages into different groups according to their encapsidation mechanism. Figure 2 presents the phylogenic analysis of a data set of terminase sequences, including the five phages of this study and others present in public databases. Based on terminase phylogeny, the phages CL1, CL2, iLp84, and iLp1308 are highly related and possess a P22-like headful mechanism. Among Lactobacillus phages, only phages Lj965 (infecting L. johnsonii) and LL-H (L. delbrueckii) have this feature, and only Lj965 is related to the five L. paracasei phages. The other L. casei group phages sequenced thus far have a 3′-extended cos packaging system similar to phage iA2. These findings highlight the diversity in this group of phages.

FIG 2.

FIG 2

Open in a new tab

Maximum-likelihood tree for the taxonomy of several phages and prophages according to amino acid sequences of the large terminase subunit. Sequences were obtained in the present study and from multiple phage genomes available in databases. Phage names are indicated at the end of each terminal branch. Colors categorize phages with different encapsidation systems: blue, 3′-extended cos ends; orange, Mu-like headful; violet, lambda-like 5′-extended cos; light blue, T7-like direct terminal repeats; light green, T4-like headful; dark green, P2-like 5′-extended cos ends; pink, GTA headful; and red, P22-like headful.

Among the other phages of the L. casei group presenting 3′-extended cos ends, the large terminase subunits of phages A2, J-1, PL-1, and prophage Lrm1 are closely related. As indicated previously, phages J-1 and PL-1 were isolated during the manufacture of Yakult in the mid-1960s, and they are almost identical (49, 50). However, phage PL-1 infects a strain insensitive to J-1 (49, 50). Prophage iA2 shares a low level of similarity to these phages, but it falls within the same cluster based on terminase phylogeny (Fig. 2).

Genomic comparison of phages CL1 and CL2.

As shown in Fig. 1, phages CL1 and CL2 are highly related (>80% identity), and they were both amplified on the same strain, L. paracasei A. The differences were mainly found in genes coding for nonstructural proteins, probably involved in replication, regulation, and lysogeny. Both phages have homing endonucleases of similar size (ORF61/CL1 and ORF3/CL2), but with low genetic identity. The presence of at least three genes encoding putative transposases in CL1 (ORF33, ORF35, and ORF36) and one in CL2 (ORF37) (Table 2) was somewhat surprising and may be responsible for phage diversity since transposases can promote genome rearrangements and genetic exchange. In CL1, however, only ORF35 and ORF36 seem to be functional, based on their protein size. The former (ORF35) has 381 amino acid residues, and transposases of similar length (and with >71% identity) were reported for diverse species of Lactobacillus, including L. paracasei phage Lb338-1, but also strains of L. brevis, L. kisonensis, L. pentosus, L. malefermentans, and Sporolactobacillus laevolacticus. The ORF36 transposase has 141 amino acid residues and also seems to be complete, although the identity to enzymes of similar size was lower and corresponded mainly to transposases of L. versmoldensis, L. salivarius, and several species of Staphylococcus. Phage CL2's ORF37, which has a very high amino acid identity to ORF33 of CL1, has only 50 amino acid residues, and this gene product is likely not functional. Based on BLAST analysis, similarities were found only to partial sequences of reported transposases of Staphylococcus aureus and S. epidermidis.

Genomic comparison of the phages CL1 and iLp1308.

The genome of phage iLp1308 is ∼5 kb smaller than CL1 and CL2 but retains a high level of identity with them, especially in genes involved in DNA packaging, morphogenesis, and cell lysis. Inverted repetitions (285 bases) flank orf29 and orf34 (4.3 kb apart) in the genome of iLp1308 (Fig. 3), but they are absent in the other four phages. The region between these repetitions in the genome of phage iLp1308 contains six genes encoded on the opposite strand, probably as a result of a recombination event. Similarly, phage CL1 has a 4.9-kb region (from orf32 to orf40) that is missing between orf32 and orf33 in phage iLp1308 (Fig. 3). This 4.9-kb region contains genes encoding the antirepressor protein, the tcdA-tcdE operon negative regulator, and transposases, as discussed previously. These DNA rearrangements may have occurred during past integration/excision events from bacterial genomes.

FIG 3.

FIG 3

Open in a new tab

Comparison of the DNA and amino acid identity (blue-filled arrows) between the phages CL1 and iLp1308. The level of amino acid identity is shown with a white-blue gradient where ORFs represented by white arrows share <90% identity. The ORFs represented by blue arrows share 90% identity or more, with the shade of blue increasing with the level of identity as indicated by the legend. DNA identity between both genomes is also indicated for sequences running in the same (red shadows) or in opposite directions (blue shadows). Asterisks indicate transposases in the genome of phage CL1. Black triangles indicate 285-nt inverted repeats in the genome of phage iLp1308.

Analysis of phage iLp84 genome.

Phage iLp84 has a genome size similar to those of CL1 and CL2, but the identity is limited to genes and proteins involved in DNA packaging and morphogenesis. The tail tape measure, tail fiber, and tail-host interaction proteins are the largest structural proteins among these phages. Some of them share high amino acid identities (>80%). The tail fiber and tail-host interaction protein of phage iLp84 possesses 72 to 78% amino acid identity with the corresponding proteins in phages CL1, CL2, and iLp1308. In support of the similarities between the tail-host interaction proteins, these four phages have the same host range (10). Conversely, the tail tape measure protein of phage iLp84 does not have significant identity with the other phages.

Analysis of phage iA2 genome.

The genome of phage iA2 is completely different from the other phages infecting the L. casei group, suggesting a different origin. The endolysin (ORF22) is the only protein that has similarity with proteins of phages CL1, CL2, and iLp1308, whereas ORF38 (putative DNA repair protein) has similarity with a protein of phage iLp84. These differences are in accordance with other distinct phenotypes. For example, phage iA2 is readily induced at high level after treatment with mitomycin C but we could not find an indicator strain for this phage. As discussed below, phage iA2 is highly similar to putative prophages extracted from the genomic sequences of strains of the L. casei group available in GenBank (Fig. 4).

FIG 4.

FIG 4

Open in a new tab

Genome organization of prophage iA2 and prophages extracted from the available genomic sequences of L. casei group strains. Genomes start from the attachment sites. ORFs are indicated by arrows. ORFs with the same color in different prophages possess an amino acid identity higher than 99%. White ORFs are unique. Groups indicate prophages that are highly conserved in different strains.

Comparison with prophages.

The occurrence of prophage DNA within bacterial genomes is common and the presence of such sequences can be identified using bioinformatic tools such as PHAST. Bacterial genomes can harbor inducible prophages but also altered/remnants of prophages displaying insertions, deletions, or rearrangements (16). The BLAST analyses of the five phage genomes characterized in the present study revealed that they are more closely related to prophage sequences found in bacterial chromosomes (data not shown). These prophage sequences were found in the genome of L. casei strains but also in strains of the other two species of the casei group, L. paracasei and L. rhamnosus. This is likely due to the high degree of relatedness among species included in the casei group (5). Some Lactobacillus phages were previously assumed to be able to cross the species barrier. For example, L. rhamnosus phage Lc-Nu is homologous to the L. casei temperate phages phiAT3 and A2 (31).

Phages CL1 and CL2 showed high identity with sequences found in L. rhamnosus strains ATCC 53103, GG, and LOCK900, and L. casei strains 12A and ATCC 334 (data not shown). However, alignments were always fragmented and covered, in the best cases, just 48 to 50% of phage CL1 and CL2 genomes. Homology between the genomes of CL1/CL2 and the genome of L. casei ATCC 334 was detected in two different regions, one being the prophage Lca1 (16). The genome of phage iLp1308 aligned relatively well with sequences in the genomes of three L. casei strains: W56, BD-II, and BL-23 (data not shown). Sequences identical to the phage iLp84 genome were found in the genomes of the same L. casei strains (data not shown).

Interestingly, the genome of the prophage iA2 was also observed in the L. casei strains discussed above (W56, BD-II, and BL-23) and also in L. casei LC2W (Fig. 4). Unlike the other four phages studied, the identity to prophage iA2 sequences found in these four bacterial genomes was very high (from 99.90 to 99.98%) and included virtually the entire prophage genome, although with some rearrangements. Three other fully sequenced Lactobacillus strains contained very large fragments of iA2 prophage (L. paracasei N1115 and 8700:2 and L. casei LOCK919) (Fig. 4).

We extracted these prophage sequences from the genomes of strains of the L. casei group for a comparative analysis with prophage iA2. The comparison is presented in Fig. 4. As expected from the nucleotide identity levels, most of the deduced ORFs of prophages from L. casei strains W56, BD II, and BL-23 (group 1) share high amino acid identity (>99%) with the predicted proteins of prophage iA2. A second prophage, found in each of these four bacterial strains, was found to be very well conserved (prophage group 2) but with noticeably lower identity to group 1. About half of the deduced ORFs of L. casei LOCK919 and L. paracasei N1115 prophages, and only 4 ORFs of L. paracasei 8700:2 prophage possess amino acid identity over 99% (Fig. 4). Nonetheless, for most ORFs of these three phages, identity with prophages of group 1 is still high, indicating that they could belong to the same group (data not shown). All prophages of group 1 and prophages of L. casei LOCK919 and L. paracasei N1115 are integrated at the same site, in a region coding for a tRNA-Leu. Prophage of group 2 are integrated in a region coding for a tRNA-Arg.

Taken altogether, these comparisons highlighted the widespread occurrence of phage-related sequences in strains of the casei group. These prophage sequences significantly contribute to their diversity. It is tempting to speculate that perhaps prophage sequences could be used as markers for either strain identification or tracking.

Presence of tRNA in phage genomes.

Although the importance of tRNAs in phage genomes is not fully understood, their presence is relatively common, particularly in large genomes (69). One tRNA was detected in the genomes of phages CL2 and iLp84. No tRNA was detected in the smaller genomes of phages iLp1308 and iA2 (Table 4). Table 4 lists the tRNA present in the available phage genomes of the L. casei group. Phage Lb338-1 is the only one with more than one tRNA in its genome. Phage iLp84 tRNA provides the amino acid isoleucine (Ile, ATA). This tRNA is similar to the one found in phage A2, but the A2 tRNA provides the amino acid leucine (TTA). The isoleucine codon ATA is used by L. paracasei at frequencies of 3.7%, while for phage iLp84 it is 13.2%. This observation supports the hypothesis that phages encode tRNAs corresponding to codons less frequently used by the host bacteria to favor the expression of phage proteins (61).

TABLE 4.

tRNA found in L. casei group phages using the bioinformatic tool ARAGORN

Phage Accession no. Genome size (bp) No. of tRNAs tRNA position
 %GC tRNA type Anticodon
Start Stop
Lb338-1 FJ822135.1 142,111 5 21370 21442 47.9 Undetermined (Asn|Stop)
23807 23880 41.9 Arg cct
24706 24779 48.6 Arg tct
25603 25691 51.7 Ile tat
27758 27832 44.0 Thr tgt
CL2 KR905067 38,751 1 36764 36836 53.4 Undetermined (Asp|seC)
iLp84 KR905069 39,399 1 36908 36981 52.7 Ile tat
A2 AJ251789.2 43,411 1 38396 38466 62.0 Leu taa
Lc-Nu AY131267.2 36,466 1 32542 32614 53.4 Undetermined (Asp|seC)
Lrm1 EU246945.1 39,989 1 36665 36737 53.4 Met cat
Open in a new tab

Conclusion.

Lactobacillus phages have been studied less than other phages of lactic acid bacteria (48). The interest in this genus has been eclipsed by substantial studies on Lactococcus lactis and Streptococcus thermophilus, the main species of LAB used as starters in the dairy industry (70). However, strains of the L. casei group (L. casei, L. paracasei, and L. rhamnosus) are now widely used as probiotics in foods. It is recognized that increased used of LAB will eventually lead to infection by virulent phages (70). Therefore, Lactobacillus phages deserve attention, considering the potential risk of losses associated with phage infections of probiotic bacteria, whose replacement with suitable strains is difficult to achieve.

In previous studies we demonstrated the high occurrence of prophages in probiotic strains added to dairy foods (10, 51). Here, we characterize five new phages of the increasingly important L. casei group, including four that can replicate in indicator strains. These phages are distinct from the previously characterized Lactobacillus phages and can be divided into two groups (CL1/CL2/iLp1308/iLp84 and iA2). Our results also point out what appears to be a high frequency of recombination events between phages and their host prophages, leading to phage and host diversity. These findings also suggest that new Lactobacillus phages likely remain to be discovered.

Our comparative genomic analyses also suggest ample distribution of prophages in the genomes of strains across the L. casei group. This offers a reservoir of genes that could be used by virulent phages to rapidly evolve (71, 72). The impact of these phage-host interactions on the properties (including probiotics), phenotypes, and stability of Lactobacillus strains is currently unknown. Future research should look to discover the frequency of these interactions and their impact on strain activities. It would also be of interest to study the different molecular mechanisms used by Lactobacillus to defend against these phages (73).

ACKNOWLEDGMENTS

We thank Barbara-Ann Conway for editorial assistance. We are grateful to Pier-Luc Plante and Jacques Corbeil for preliminary genome assembly and access to the Illumina platform.

This study was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET; project PIP 112-200801-01206 [Argentina]). D.J.M. acknowledges funding from the CONICET and by the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) (project PICT 2010-0138 [Argentina]). S.M. holds a Tier 1 Canada Research Chair in Bacteriophages.

REFERENCES

  • 1.Tindall BJ. 2008. The type strain of Lactobacillus casei is ATCC 393, ATCC 334 cannot serve as the type because it represents a different taxon, the name Lactobacillus paracasei and its subspecies names are not rejected and the revival of the name “Lactobacillus zeae” contravenes rules 51b (1) and (2) of the International Code of Nomenclature of Bacteria. Opinion 82. Int J Syst Evol Microbiol 58:1764–1765. doi: 10.1099/ijs.0.2008/005330-0. [DOI] [PubMed] [Google Scholar]
  • 2.Dicks LM, Du Plessis EM, Dellaglio F, Lauer E. 1996. Reclassification of Lactobacillus casei subsp. casei ATCC 393 and Lactobacillus rhamnosus ATCC 15820 as Lactobacillus zeae nom. rev., designation of ATCC 334 as the neotype of L. casei subsp. casei, and rejection of the name Lactobacillus paracasei.. Int J Syst Bacteriol 46:337–340. doi: 10.1099/00207713-46-1-337. [DOI] [PubMed] [Google Scholar]
  • 3.Dellaglio F, Felis GE, Torriani S. 2002. The status of the species Lactobacillus casei (Orla-Jensen 1916) Hansen and Lessel 1971 and Lactobacillus paracasei Collins et al. 1989. Request for an opinion. Int J Syst Evol Microbiol 52:285–287. doi: 10.1099/00207713-52-1-285. [DOI] [PubMed] [Google Scholar]
  • 4.Collins MD, Phillips BA, Zanoni P. 1989. Deoxyribonucleic acid homology studies of Lactobacillus casei, Lactobacillus paracasei sp. nov., subsp. paracasei and subsp. tolerans, and Lactobacillus rhamnosus sp. nov., comb nov. Int J Syst Evol Microbiol 39:105–108. [Google Scholar]
  • 5.Desai AR, Shah NP, Powell IB. 2006. Discrimination of dairy industry isolates of the Lactobacillus casei group. J Dairy Sci 89:3345–3351. doi: 10.3168/jds.S0022-0302(06)72371-2. [DOI] [PubMed] [Google Scholar]
  • 6.Liu M, Nauta A, Francke C, Siezen RJ. 2008. Comparative genomics of enzymes in flavor-forming pathways from amino acids in lactic acid bacteria. Appl Environ Microbiol 74:4590–4600. doi: 10.1128/AEM.00150-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Saarela M, Mogensen G, Fondén R, Mättö J, Mattila-Sandholm T. 2000. Probiotic bacteria: safety, functional and technological properties. J Biotechnol 84:197–215. doi: 10.1016/S0168-1656(00)00375-8. [DOI] [PubMed] [Google Scholar]
  • 8.Capra ML, Binetti AG, Mercanti DJ, Quiberoni A, Reinheimer JA. 2009. Diversity among Lactobacillus paracasei phages isolated from a probiotic dairy product plant. J Appl Microbiol 107:1350–1357. doi: 10.1111/j.1365-2672.2009.04313.x. [DOI] [PubMed] [Google Scholar]
  • 9.Suarez V, Zago M, Quiberoni A, Carminati D, Giraffa G, Reinheimer J. 2008. Lysogeny in Lactobacillus delbrueckii strains and characterization of two new temperate prolate-headed bacteriophages. J Appl Microbiol 105:1402–1411. doi: 10.1111/j.1365-2672.2008.03876.x. [DOI] [PubMed] [Google Scholar]
  • 10.Mercanti DJ, Carminati D, Reinheimer JA, Quiberoni A. 2011. Widely distributed lysogeny in probiotic lactobacilli represents a potentially high risk for the fermentative dairy industry. Int J Food Microbiol 144:503–510. doi: 10.1016/j.ijfoodmicro.2010.11.009. [DOI] [PubMed] [Google Scholar]
  • 11.Brussow H. 2001. Phages of dairy bacteria. Annu Rev Microbiol 55:283–303. doi: 10.1146/annurev.micro.55.1.283. [DOI] [PubMed] [Google Scholar]
  • 12.Ackermann HW, Kropinski AM. 2007. Curated list of prokaryote viruses with fully sequenced genomes. Res Microbiol 158:555–566. doi: 10.1016/j.resmic.2007.07.006. [DOI] [PubMed] [Google Scholar]
  • 13.Mahony J, van Sinderen D. 2014. Current taxonomy of phages infecting lactic acid bacteria. Front Microbiol 5:7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Quiberoni A, Moineau S, Rousseau G, Reinheimer J, Ackermann H-W. 2010. Streptococcus thermophilus bacteriophages. Int Dairy J 20:657–664. doi: 10.1016/j.idairyj.2010.03.012. [DOI] [Google Scholar]
  • 15.Altermann E, Klaenhammer TR. 2011. Group-specific comparison of four lactobacilli isolated from human sources using differential blast analysis. Genes Nutr 6:319–340. doi: 10.1007/s12263-010-0191-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Ventura M, Canchaya C, Bernini V, Altermann E, Barrangou R, McGrath S, Claesson MJ, Li Y, Leahy S, Walker CD, Zink R, Neviani E, Steele J, Broadbent J, Klaenhammer TR, Fitzgerald GF, O'Toole PW, van Sinderen D. 2006. Comparative genomics and transcriptional analysis of prophages identified in the genomes of Lactobacillus gasseri, Lactobacillus salivarius, and Lactobacillus casei.. Appl Environ Microbiol 72:3130–3146. doi: 10.1128/AEM.72.5.3130-3146.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Altermann E, Klein JR, Henrich B. 1999. Primary structure and features of the genome of the Lactobacillus gasseri temperate bacteriophage (phi)adh. Gene 236:333–346. doi: 10.1016/S0378-1119(99)00236-X. [DOI] [PubMed] [Google Scholar]
  • 18.Baugher JL, Durmaz E, Klaenhammer TR. 2014. Spontaneously induced prophages in Lactobacillus gasseri contribute to horizontal gene transfer. Appl Environ Microbiol 80:3508–3517. doi: 10.1128/AEM.04092-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Ventura M, Canchaya C, Pridmore D, Berger B, Brussow H. 2003. Integration and distribution of Lactobacillus johnsonii prophages. J Bacteriol 185:4603–4608. doi: 10.1128/JB.185.15.4603-4608.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Ventura M, Canchaya C, Pridmore RD, Brussow H. 2004. The prophages of Lactobacillus johnsonii NCC 533: comparative genomics and transcription analysis. Virology 320:229–242. doi: 10.1016/j.virol.2003.11.034. [DOI] [PubMed] [Google Scholar]
  • 21.Desiere F, Pridmore RD, Brussow H. 2000. Comparative genomics of the late gene cluster from Lactobacillus phages.. Virology 275:294–305. doi: 10.1006/viro.2000.0498. [DOI] [PubMed] [Google Scholar]
  • 22.Kodaira KI, Oki M, Kakikawa M, Watanabe N, Hirakawa M, Yamada K, Taketo A. 1997. Genome structure of the Lactobacillus temperate phage phi g1e: the whole genome sequence and the putative promoter/repressor system. Gene 187:45–53. doi: 10.1016/S0378-1119(96)00687-7. [DOI] [PubMed] [Google Scholar]
  • 23.Ventura M, Canchaya C, Kleerebezem M, de Vos WM, Siezen RJ, Brussow H. 2003. The prophage sequences of Lactobacillus plantarum strain WCFS1. Virology 316:245–255. doi: 10.1016/j.virol.2003.08.019. [DOI] [PubMed] [Google Scholar]
  • 24.Briggiler Marco M, Garneau JE, Tremblay D, Quiberoni A, Moineau S. 2012. Characterization of two virulent phages of Lactobacillus plantarum.. Appl Environ Microbiol 78:8719–8734. doi: 10.1128/AEM.02565-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Lu Z, Altermann E, Breidt F, Predki P, Fleming HP, Klaenhammer TR. 2005. Sequence analysis of the Lactobacillus plantarum bacteriophage ϕJL-1. Gene 348:45–54. doi: 10.1016/j.gene.2004.12.052. [DOI] [PubMed] [Google Scholar]
  • 26.Chibani-Chennoufi S, Dillmann ML, Marvin-Guy L, Rami-Shojaei S, Brussow H. 2004. Lactobacillus plantarum bacteriophage LP65: a new member of the SPO1-like genus of the family Myoviridae.. J Bacteriol 186:7069–7083. doi: 10.1128/JB.186.21.7069-7083.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Yoon BH, Jang SH, Chang HI. 2011. Sequence analysis of the Lactobacillus temperate phage Sha1. Arch Virol 156:1681–1684. doi: 10.1007/s00705-011-1048-2. [DOI] [PubMed] [Google Scholar]
  • 28.Garcia P, Ladero V, Suarez JE. 2003. Analysis of the morphogenetic cluster and genome of the temperate Lactobacillus casei bacteriophage A2. Arch Virol 148:1051–1070. doi: 10.1007/s00705-003-0008-x. [DOI] [PubMed] [Google Scholar]
  • 29.Lo TC, Shih TC, Lin CF, Chen HW, Lin TH. 2005. Complete genomic sequence of the temperate bacteriophage PhiAT3 isolated from Lactobacillus casei ATCC 393. Virology 339:42–55. doi: 10.1016/j.virol.2005.05.022. [DOI] [PubMed] [Google Scholar]
  • 30.Durmaz E, Miller M, Azcarate-Peril M, Toon S, Klaenhammer T. 2008. Genome sequence and characteristics of Lrm1, a prophage from industrial Lactobacillus rhamnosus strain M1. Appl Environ Microbiol 74:4601–4609. doi: 10.1128/AEM.00010-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Tuohimaa A, Riipinen KA, Brandt K, Alatossava T. 2006. The genome of the virulent phage Lc-Nu of probiotic Lactobacillus rhamnosus, and comparative genomics with Lactobacillus casei phages. Arch Virol 151:947–965. doi: 10.1007/s00705-005-0672-0. [DOI] [PubMed] [Google Scholar]
  • 32.Dieterle ME, Jacobs-Sera D, Russell D, Hatfull G, Piuri M. 2014. Complete genome sequences of Lactobacillus phages J-1 and PL-1. Genome Announc 2:e00998-13. doi: 10.1128/genomeA.00998-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Herrero M, de los Reyes-Gavilán CG, Caso JL, Suárez JE. 1994. Characterization of ϕ393-A2, a bacteriophage that infects Lactobacillus casei.. Microbiology 140:2585–2590. doi: 10.1099/00221287-140-10-2585. [DOI] [Google Scholar]
  • 34.Proux C, van Sinderen D, Suarez J, Garcia P, Ladero V, Fitzgerald GF, Desiere F, Brussow H. 2002. The dilemma of phage taxonomy illustrated by comparative genomics of Sfi21-like Siphoviridae in lactic acid bacteria. J Bacteriol 184:6026–6036. doi: 10.1128/JB.184.21.6026-6036.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Alemayehu D, Ross RP, O'Sullivan O, Coffey A, Stanton C, Fitzgerald GF, McAuliffe O. 2009. Genome of a virulent bacteriophage Lb338-1 that lyses the probiotic Lactobacillus paracasei cheese strain. Gene 448:29–39. doi: 10.1016/j.gene.2009.08.008. [DOI] [PubMed] [Google Scholar]
  • 36.Zago M, Scaltriti E, Rossetti L, Guffanti A, Armiento A, Fornasari ME, Grolli S, Carminati D, Brini E, Pavan P, Felsani A, D'Urzo A, Moles A, Claude JB, Grandori R, Ramoni R, Giraffa G. 2013. Characterization of the genome of the dairy Lactobacillus helveticus bacteriophage ϕAQ113. Appl Environ Microbiol 79:4712–4718. doi: 10.1128/AEM.00620-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Vasala A, Dupont L, Baumann M, Ritzenthaler P, Alatossava T. 1993. Molecular comparison of the structural proteins encoding gene clusters of two related Lactobacillus delbrueckii bacteriophages. J Virol 67:3061–3068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Mikkonen M, Alatossava T. 1994. Characterization of the genome region encoding structural proteins of Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H. Gene 151:53–59. doi: 10.1016/0378-1119(94)90632-7. [DOI] [PubMed] [Google Scholar]
  • 39.Mikkonen M, Raisanen L, Alatossava T. 1996. The early gene region completes the nucleotide sequence of Lactobacillus delbrueckii subsp. lactis phage LL-H. Gene 175:49–57. doi: 10.1016/0378-1119(96)00119-9. [DOI] [PubMed] [Google Scholar]
  • 40.Riipinen KA, Forsman P, Alatossava T. 2011. The genomes and comparative genomics of Lactobacillus delbrueckii phages. Arch Virol 156:1217–1233. doi: 10.1007/s00705-011-0980-5. [DOI] [PubMed] [Google Scholar]
  • 41.Casey E, Mahony J, O'Connell-Motherway M, Bottacini F, Cornelissen A, Neve H, Heller KJ, Noben JP, Dal Bello F, van Sinderen D. 2014. Molecular characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages Appl Environ Microbiol 80:5623–5635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Casey E, Mahony J, Neve H, Noben JP, Bello FD, van Sinderen D. 2014. Genome and proteome analysis of bacteriophage Ldl1 reveals the existence of a novel phage group infecting Lactobacillus delbrueckii subsp. lactis.. Appl Environ Microbiol 81:1319–1326. doi: 10.1128/AEM.01268-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Ehrmann MA, Angelov A, Picozzi C, Foschino R, Vogel RF. 2013. The genome of the Lactobacillus sanfranciscensis temperate phage EV3. BMC Res Notes 6:514. doi: 10.1186/1756-0500-6-514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Yoon BH, Chang HI. 2011. Complete genomic sequence of the Lactobacillus temperate phage LF1. Arch Virol 156:1909–1912. doi: 10.1007/s00705-011-1082-0. [DOI] [PubMed] [Google Scholar]
  • 45.Zhang X, Wang S, Guo T, Kong J. 2011. Genome analysis of Lactobacillus fermentum temperate bacteriophage ϕPYB5. Int J Food Microbiol 144:400–405. doi: 10.1016/j.ijfoodmicro.2010.10.026. [DOI] [PubMed] [Google Scholar]
  • 46.Jang SH, Yoon BH, Chang HI. 2011. Complete nucleotide sequence of the temperate bacteriophage LBR48, a new member of the family Myoviridae.. Arch Virol 156:319–322. doi: 10.1007/s00705-010-0841-7. [DOI] [PubMed] [Google Scholar]
  • 47.Martin R, Escobedo S, Suarez JE. 2010. Induction, structural characterization, and genome sequence of Lv1, a prophage from a human vaginal Lactobacillus jensenii strain. Int Microbiol 13:113–121. [DOI] [PubMed] [Google Scholar]
  • 48.Villion M, Moineau S. 2009. Bacteriophages of Lactobacillus.. Front Biosci 14:1661–1683. [DOI] [PubMed] [Google Scholar]
  • 49.Hino M, Ikebe N. 1965. Studies on the lactic acid bacteria employed for beverage production II. Isolation and some properties of a bacteriophage infected during the fermentation of lactic acid beverage. J Agric Chem Soc Jpn 39:472–476. [Google Scholar]
  • 50.Watanabe K, Takesue S, Jin-Nai K, Yoshikawa T. 1970. Bacteriophage active against the lactic acid beverage-producing bacterium Lactobacillus casei.. Appl Microbiol 20:409–415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Capra ML, Mercanti DJ, Reinheimer JA, Quiberoni AL. 2010. Characterisation of three temperate phages released from the same Lactobacillus paracasei commercial strain. Int J Dairy Technol 63:396–405. doi: 10.1111/j.1471-0307.2010.00600.x. [DOI] [Google Scholar]
  • 52.Mercanti DJ, Ackermann HW, Quiberoni A. 2015. Characterization of two temperate Lactobacillus paracasei bacteriophages: morphology, kinetics, and adsorption. Intervirology 58:49–56. doi: 10.1159/000369207. [DOI] [PubMed] [Google Scholar]
  • 53.Svensson U, Christiansson A. 1991. Methods for phage monitoring. Bull FIL-IDF 263:29–39. [Google Scholar]
  • 54.Lillehaug D. 1997. An improved plaque assay for poor plaque-producing temperate lactococcal bacteriophages. J Appl Microbiol 83:85–90. doi: 10.1046/j.1365-2672.1997.00193.x. [DOI] [PubMed] [Google Scholar]
  • 55.Boisvert S, Laviolette F, Corbeil J. 2010. Ray: simultaneous assembly of reads from a mix of high-throughput sequencing technologies. J Comput Biol 17:1519–1533. doi: 10.1089/cmb.2009.0238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Staden R. 1996. The Staden sequence analysis package. Mol Biotechnol 5:233–241. doi: 10.1007/BF02900361. [DOI] [PubMed] [Google Scholar]
  • 57.Hall TA. 1999. BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98. [Google Scholar]
  • 58.Lukashin AV, Borodovsky M. 1998. GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res 26:1107–1115. doi: 10.1093/nar/26.4.1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Laslett D, Canback B. 2004. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res 32:11–16. doi: 10.1093/nar/gkh152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009. BLAST+: architecture and applications. BMC Bioinformatics 10:1471–2105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Guy L, Roat Kultima J, Andersson SGE. 2010. genoPlotR: comparative gene and genome visualization in R. Bioinformatics 26:2334–2335. doi: 10.1093/bioinformatics/btq413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Casjens SR, Gilcrease EB, Winn-Stapley DA, Schicklmaier P, Schmieger H, Pedulla ML, Ford ME, Houtz JM, Hatfull GF, Hendrix RW. 2005. The generalized transducing Salmonella bacteriophage ES18: complete genome sequence and DNA packaging strategy. J Bacteriol 187:1091–1104. doi: 10.1128/JB.187.3.1091-1104.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Darriba D, Taboada GL, Doallo R, Posada D. 2011. ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 27:1164–1165. doi: 10.1093/bioinformatics/btr088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321. doi: 10.1093/sysbio/syq010. [DOI] [PubMed] [Google Scholar]
  • 65.Shimodaira H. 2002. An approximately unbiased test of phylogenetic tree selection. Syst Biol 51:492–508. doi: 10.1080/10635150290069913. [DOI] [PubMed] [Google Scholar]
  • 66.Junier T, Zdobnov EM. 2010. The Newick utilities: high-throughput phylogenetic tree processing in the UNIX shell. Bioinformatics 26:1669–1670. doi: 10.1093/bioinformatics/btq243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Letunic I, Bork P. 2011. Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucleic Acids Res 39:W475–W478. doi: 10.1093/nar/gkr201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Lopes A, Tavares P, Petit MA, Guerois R, Zinn-Justin S. 2014. Automated classification of tailed bacteriophages according to their neck organization. BMC Genomics 15:1027. doi: 10.1186/1471-2164-15-1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Samson JE, Moineau S. 2010. Characterization of Lactococcus lactis phage 949 and comparison with other lactococcal phages. Appl Environ Microbiol 76:6843–6852. doi: 10.1128/AEM.00796-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Garneau J, Moineau S. 2011. Bacteriophages of lactic acid bacteria and their impact on milk fermentations. Microb Cell Fact 10:S20. doi: 10.1186/1475-2859-10-S1-S20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Labrie S, Moineau S. 2007. Abortive infection mechanisms and prophage sequences significantly influence the genetic makeup of emerging lytic lactococcal phages. J Bacteriol 189:1482–1487. doi: 10.1128/JB.01111-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72.Capra ML, Quiberoni A, Ackermann HW, Moineau S, Reinheimer JA. 2006. Characterization of a new virulent phage (MLC-A) of Lactobacillus paracasei.. J Dairy Sci 89:2414–2423. doi: 10.3168/jds.S0022-0302(06)72314-1. [DOI] [PubMed] [Google Scholar]
  • 73.Labrie S, Samson J, Moineau S. 2010. Bacteriophage resistance mechanisms. Nat Rev Microbiol 8:317–327. doi: 10.1038/nrmicro2315. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES