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Characterization of a Leuconostoc bacteriophage infecting flavor 1
producers of cheese starter cultures 2
3
4
Hans Petter Kleppen*, Inglof F. Nes and Helge Holo. 5
6
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life 7
Sciences, P.O. Box 5003, N-1432 Aas, Norway. 8
9
*Corresponding author. Mailing address: Department of Chemistry, Biotechnology and Food 10
Science, Norwegian University of Life Sciences, P.O. Box 5003, NO-1432 Aas, Norway. Phone: 11
(+47) 6496 5885. E-mail: [email protected]. 12
13
14
Key words: Dairy, fermentation, cheese, lactic acid bacteria, Leuconostoc, bacteriophage, 15
complete genome sequence. 16
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00562-12 AEM Accepts, published online ahead of print on 13 July 2012
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Abstract 17
Dairy siphovirus φLmd1 which infects starter culture isolate Leuconostoc mesenteroides subsp. 18
dextranicum A1, showed resistance to pasteurization, and was able to grow on 3 of the 4 19
commercial starter cultures tested. Its 26,201 bp genome was similar to a Leuconostoc phage of 20
vegetable origin but not to dairy phages infecting Lactococcus. 21
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Introduction 34
Bacteria of the genus Leuconostoc (L.) are incorporated into dairy starter cultures due to their 35
ability to produce important metabolites such as diacetyl and CO2 from citric acid (6, 9). Diacetyl 36
is the primary source of aroma and flavor compounds in a variety of fermented milk products 37
including buttermilk, butter, quarg and various cheese types (6). Leuconostocs are important 38
flavor producers in L-type and DL-type mesophilic starter cultures, in the latter together with 39
Lactococcus lactis subsp. lactis biovar. diacetylactis. The different leuconostocs associated with 40
dairy starters include L. mesenteroides subsp. cremoris, L. mesenteroides subsp. dextranicum, L. 41
lactis and L. pseudomesenteroides (5, 10). 42
Bacteriophages negatively affect dairy fermentations by inhibiting the growth of key 43
lactic acid bacteria (LAB). Bacteriophages infecting Lactococcus have been extensively studied 44
for decades due to their dramatic effect on milk acidification rates (24). Lactococcal phages are 45
ubiquitous in dairy environments (26, 28, 32) and it has been shown that phages resident in the 46
dairy plant are responsible for killing lactococcal starter bacteria early in the fermentation (18). 47
Before phages become dairy residents they are likely to enter dairies through contaminated 48
milk (18, 20) and since natural habitats of Leuconostoc include green vegetation and silage (30) 49
a similar route of entry is likely for Leuconostoc phages. Atamer and co-workers studied the 50
thermal resistance of 77 Leuconostoc phages and found that commonly applied pasteurization 51
conditions were insufficient to ensure complete inactivation of dairy Leuconostoc phages (5). 52
Accordingly, Leuconostoc phages have been shown to be widely distributed in dairy products (5, 53
29). Phages infecting dairy Leuconostoc have previously been characterized (5, 11) and the 54
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genome sequence of a virulent L. mesenteroides phage (φ1-A4) and a temperate L. 55
pseudomesenteroides phage (φMH1), both isolated from vegetable fermentation have been 56
characterized (17, 19). 57
Knowledge on bacteriophages infecting dairy starter cultures is important for the 58
continued improvement of phage counter measures. In this study we analyzed the complete 59
genomic sequence of a Leuconostoc phage isolated from a Norwegian dairy producing Dutch-60
type cheese, and characterized the phage with respect to its ability to affect dairy fermentation. 61
Genomes of Leuconostoc phages from vegetable fermentations have been previously described, 62
but none from dairy fermentations. 63
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Host strain L. mesenteroides subsp. dextranicum A1. 73
The host bacterium, L. mesenteroides subsp. dextranicum A1, was isolated from a commercial 74
mixed mesophilic DL starter culture commonly employed in the industrial production of 75
cultured butter and various cheese types. The bacterium was grown at 30 °C in MRS (Oxoid, 76
Baskingstoke, UK). The partial 16s rRNA gene sequence of isolate A1 (corresponding to position 77
55 to 1387 in the Escherichia coli 16s rRNA gene) was 100.0 % identical to that of leuconostocs 78
belonging to ribospecies CHCC 2114 (27). Strains of this ribospecies have repeatedly been 79
isolated from fermented dairy products and have been assigned to both L. mesenteroides and L. 80
pseudomesenteroides species (27). The API50 CHL (BioMérieux, Lyon, France) sugar 81
fermentation pattern of the host bacterium (acid production from D-ribose, D-galactose, D-82
glucose, D-fructose, D-mannose, methyl-αD-glucopyranoside, N-acetylglucosamine, salicin, D-83
cellobiose, D-maltose, D-lactose, D-melibiose, sucrose, D-trehalose, D-raffinose, starch, 84
gentibiose and D-turanose) as well as its ability to grow in 6.5 % NaCl were in best accordance 85
with L. mesenteroides subspecies dextranicum (6, 13, 14). In the following the host strain name 86
will be shortened to L. mesenteroides A1. 87
Bacteriophage isolation and characterization. 88
Bacteriophage Lmd1 was isolated from brine used in the production of Dutch-type cheese in a 89
Norwegian dairy. Phage isolation and quantification was done by standard plaque assays 90
performed in MRS soft-agar supplemented with 5 mM CaCl2 (MRS-C). Before phage assays, the 91
brine sample was dialyzed against phage storage buffer TM (10mM Tris-HCl pH 7.4, 100mM 92
NaCl, 10mM MgCl2, 10mM CaCl2). 93
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Bacteriophage Lmd1 belongs to the Siphoviridae family of tailed phages and is of the B1 94
morphotype (1, 2) (Fig. 1). It has a capsid diameter of 41 nm and a tail measuring 115 by 10 nm. 95
The tail consists of 30 or 31 tail segments and a distinct baseplate could be observed at the tail 96
tip. The B1 morphotype is the most frequently encountered morphotype among the described 97
Leuconostoc phages, and also among dairy phages infecting Lactococcus lactis (2). φLmd1 98
produced large clear plaques on L. mesenteroides A1 lawns and had an average burst size (16) of 99
about 50. Lysis was completed 30 minutes after adsorption. Thermal inactivation studies on 100
φLmd1 revealed that the phage is unaffected by pasteurization but its titer was reduced by 101
more than 7-log when exposed to a thermal inactivation scheme resembling commonly 102
employed bulk starter vat sterilization schemes (96°C, 30 minutes). This was in accordance with 103
thermal resistance of other Leuconostoc phages (5). Since pasteurization does not affect φLmd1, 104
there is no barrier for the bacteriophage to enter cheese fermentation vats through 105
contaminated milk. Entry into bulk starter vats would, however, require contamination during 106
or after bulk starter milk cooling. 107
Many dairies practice rotation of different phage-unrelated starter cultures in order to 108
reduce impact of bacteriophages (26). We tested the ability of φLmd1 to multiply on 4 109
commercial starter cultures commonly used in the production of Dutch-type cheese, and found 110
that 3 of the four starters contained hosts for φLmd1 proliferation (supplementary figure S1). 111
This finding emphasizes the importance of assaying for Leuconostoc phages during selection of 112
starter cultures for rotation schemes. 113
The genome of φLmd1 114
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The sequence of the 26,201 base pair linear genome of Leuconostoc phage Lmd1 was found by a 115
combination of shotgun sequencing and primer walking. Briefly, genomic DNA was isolated from 116
purified phage particles (7) by standard phenol/chloroform extraction, and a shotgun library 117
prepared after partial digestion with AluI. Sequencing was performed using BigDye 3.1 118
chemistry (Applied Biosystems, Foster City, CA), and sequence assembly and analysis were done 119
using CLC Main Workbench version 6.5 (CLC bio, Aarhus, Denmark). Homology searches were 120
done using BLASTP and PSI-BLAST build 2.2.26+ (3, 4), and conserved domains were found by 121
searching the Conserved Domains Database (21-23) (June 2012). 122
Cohesive genome ends (23 bp: 5’-TCGTGCAATAGTAGGCGTTTTAA-3’) were identified by 123
restriction analysis and sequencing as described by others (8, 19). The G+C content of the 124
φLmd1 genome is 36.4%. A putative origin of replication (ori) was found between positions 125
1639 and 1873. This region comprises an A-T rich region and multiple repeats and hairpin 126
structures typical of phage replication origins (33). Forty open reading frames (ORFs) were 127
predicted using Prodigal (15). These constitute 91.7 % of the genomic sequence. Starting with 128
the ORF immediately downstream of ori, ORFs were given numbers consecutively (Fig. 2). By 129
homology searches, putative functions were assigned to 24 ORFs. Eight proteins, ORF9 and 130
ORF14 through 20, were identified as structural proteins by SDS-PAGE and mass spectrometry 131
performed essentially as described elsewhere (25) (Fig. 2 and Fig. S2). Similar to Leuconostoc 132
phage 1-A4 (19), none of the major protein bands seen by SDS-PAGE were identified as the in 133
silico predicted major capsid protein. The function of this protein remains to be elucidated. 134
Predicted ribosomal binding sites, start codons and putative gene functions are shown in 135
supplementary table 1. Similar to most characterized bacteriophage genomes, the genome of 136
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φLmd1 is organized in functional modules. Four modules are clearly identifiable: the DNA 137
replication module, DNA packaging module and the head and tail morphogenesis modules (Fig. 138
2). 139
The genome of φLmd1 closely resembles that of L. mesenteroides phage 1-A4 (19), both 140
with respect to sequence similarity and genome organization (Fig. 3). Through a functional 141
distribution analysis, Lu and co-workers showed that Leuconostoc phage 1-A4 cluster most 142
closely with several lactococcal phages including Q54-like, c2-like and 936-like phages (12), but 143
they suggested that φ1-A4 should form a separate functional cluster based on the relatively 144
large distance between it and its closest relatives (19). This is in agreement with the low number 145
of significant BLAST hits we found to phage sequences other than φ1-A4. 146
Almost half of the predicted proteins in φLmd1 did not show any similarity to φ1-A4 147
ORFs (Fig. 3). The dissimilar ORFs were mostly found on the negative strand in both phages, in 148
modules possibly involved in transcription regulation or host interaction. This putative 149
functional assignment is supported by the presence homologs of conserved Leuconostoc and 150
Weissella prophage genes in this region. 151
There was generally low sequence similarity at the DNA level between φLmd1 and φ1-152
A4, even within orfs encoding homologous proteins (not shown). The genome sequence of 153
φLmd1 might thus be useful in the development of DNA-based detection methods for dairy 154
Leuconostoc phages. 155
156
Database accession number 157
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The complete genome sequence of Leuconostoc phage Lmd1 has been deposited in GenBank 158
under accession number JQ659259. 159
ACKNOWLEDGEMENTS 160
This work was supported by TINE SA and The Research Council of Norway. We are grateful to 161
Morten Skaugen for excellent technical assistance with the mass spectrometry analysis. 162
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257
FIGURE LEGENDS 258
Figure 1 259
Transmission electron micrograph of φLmd1. Phage particles were purified on CsCl gradients 260
according to Boulanger (7), negatively stained with 2% (w/v) uranyl acetate on a carbon-formvar 261
membrane grid and examined on a FEI Morgagni 268 (FEI Company B.V., Eindhoven, The 262
Netherlands) microscope at an accelerating voltage of 100 kV. Scale bar indicates 100 nm. 263
Figure 2 264
Genome map of φLmd1. Positions of the predicted open reading frames are indicated by 265
arrows. Putative functions and functional modules are indicated above. Structural proteins 266
identified by mass spectrometry in this study are indicated by grey arrows. The putative origin 267
of replication is indicated by a black square, the three EcoRI recognition sites. The scale bar 268
marks genome positions at 2000 bp intervals. 269
Figure 3 270
Genome comparison between Leuconostoc mesenteroides phages Lmd1 and 1-A4 (Genbank 271
accession GQ451696). ORFs are indicated by numbered arrows. Grey connecting lines between 272
ORFs indicate identities. Light grey indicate 20% identities and black lines 100%, according to 273
the greyscale bar on the right. For detailed BLASTP scores between ORFs see supplementary 274
table 1. The locations of putative origins of replication (ori) and cos-sites (cos) are indicated. The 275
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scale bar below indicates 5000 bp. Genome comparison was carried out using Easyfig software 276
version 1.2.1 (31) with the following cutoff settings: minimum alignment length = 20, maximum 277
tblastx e-value = 0.001. This corresponded to a minimum sequence identity of 19.23 %. 278
279
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