J Ind Microbiol Biotechnol DOI 10.1007/s10295-016-1739-5

GENETICS AND MOLECULAR BIOLOGY OF INDUSTRIAL ORGANISMS

A novel chimeric prophage vB_LdeS‑phiJB from commercial Lactobacillus delbrueckii subsp. bulgaricus Tingting Guo1 · Chenchen Zhang1 · Yongping Xin1 · Min Xin1 · Jian Kong1 

Received: 21 October 2015 / Accepted: 12 January 2016 © Society for Industrial Microbiology and Biotechnology 2016

Abstract  Prophage vB_LdeS-phiJB (phiJB) was induced by mitomycin C and UV radiation from the Lactobacillus delbrueckii subsp. bulgaricus SDMCC050201 isolated from a Chinese yoghurt sample. It has an isometric head and a non-contractile tail with 36,969 bp linear doublestranded DNA genome, which is classified into the group a of Lb. delbrueckii phages. The genome of phiJB is highly modular with functionally related genes clustered together. Unexpectedly, there is no similarity of its DNA replication module to any phages that have been reported, while it consists of open-reading frames homologous to the proteins of Lactobacillus strains. Comparative genomic analysis indicated that its late gene clusters, integration/lysogeny modules and DNA replication module derived from different evolutionary ancestors and integrated into a chimera. Our results revealed a novel chimeric phage of commercial Lb. delbrueckii and will broaden the knowledge of phage diversity in the dairy industry. Keywords  Lactobacillus delbrueckii subsp. bulgaricus · Comparative genomic · Chimeric phage

Electronic supplementary material  The online version of this article (doi:10.1007/s10295-016-1739-5) contains supplementary material, which is available to authorized users. * Jian Kong [email protected] 1



State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nanlu, Jinan 250100, People’s Republic of China

Introduction Lactic acid bacteria (LAB) are important industrial organisms used for the manufacture of fermented dairy products. During fermentation processes, LAB initiate the rapid acidification of raw milk, contribute to flavor and texture, and may confer health benefits on the end products [22, 35]. However, when specific LAB cells come into contact with a virulent phage, the productivity of starter cultures can significantly decrease, and the fermentation process is delayed or even halted. In consequence, low quality products are obtained with the following economic losses for the dairy industry [16]. Although several precautionary strategies have been adopted, including improved sanitation, process changes, strain rotation and isolation of phage-resistant strains, phage contamination is still a persistent and costly problem in modern mega-scale processes [16, 17, 29]. Phages are ubiquitous in dairy plants because of the non-sterile environment of raw or pasteurized milk and whey emission [16]. Moreover, starter strains are a hidden source of temperate phages [28]. Comparative genomic analysis revealed that many LAB are equipped with one or more prophages in their genome [8, 14, 24, 47]. Different bacterial stresses can induce the prophage and trigger the lytic cycle, and therefore the use of lysogenic strains in a starter culture may lead to cell lysis during fermentation [16, 24]. Moreover, prophages could reconstruct the viral genes and produce novel virulent phages with broadened host ranges and undesirable properties [14, 18, 21]. Therefore, lysogenic starters represent a potential high risk for the dairy industry. On the other hand, as mobile genetic elements, prophage integration can regulate the bacterial populations, inactivate or alter the expression of some bacterial genes [1]. Additionally, the presence or absence of

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prophages can generate variation within a bacterial species, and phages are also important vehicles for horizontal transfer of genetic information between bacterial strains [6, 11]. Lactobacillus delbrueckii is one of the most widely used LAB in the dairy industry for the manufacture of yogurt and hard cheeses [40]. Considering the economic relevance, Lb. delbrueckii phage research has progressed significantly. Over 20 Lb. delbrueckii phages have been characterized [2, 3, 9, 10, 19, 26, 30, 34, 36, 42]. And the absorption receptor and resistant mutants have been identified and constructed for Lb. delbureckii phages [31–33, 37, 38, 41]. According to virion morphology and DNA homology, the Lb. delbrueckii phages are classified into five groups. Phages in group a have isometric heads and pac-sites, while group b phages possess isometric heads and cos-sites. JCL1032 belongs to group c with a prolate head. Group d consists of isometric phages that do not share DNA homology with groups a or b [34]. The newly created group e consists of phage Ldl1 which bears little resemblance to other characterized phages infecting Lb. delbrueckii [10]. To date, eight genome sequences of Lb. delbrueckii virulent phages are available in the GenBank database (LL-H, LL-Ku, c5, phiLdb, Ld3, Ld17, Ld25A and Ldl1) [9, 10, 26, 34, 42], while only one Lb. delbrueckii temperate phage JCL1032 has been completely sequenced [34]. Our genetic knowledge about Lb. delbrueckii temperate phages remains limited. Lactobacillus delbrueckii subsp. bulgaricus SDMCC050201 is a commercial starter strain for yoghurt fermentation in China. The present study reported the isolation and characterization of an inducible prophage vB_LdeS-phiJB (referred to as phiJB) from Lb. delbrueckii SDMCC050201, the phiJB morphology and DNA restriction profile, its genome sequence and comparative genomic analysis with related Lb. delbrueckii phages.

Materials and methods Induction of Lb. delbrueckii phage The commercial starter Lactobacillus delbrueckii subsp. bulgaricus SDMCC050201 was isolated from a Chinese yoghurt sample and submitted to Shandong university organism culture collection. To induce the prophages, Lb. delbrueckii SDMCC050201 was incubated in de Man, Rogosa and Sharpe (MRS) broth statically overnight at 37 °C. Subsequently, 2 % overnight cultures were transferred into MRS-Ca (MRS supplemented with 10 mM CaCl2) broth for cultivation until the optical density at 600 nm (OD600) reached 0.5. Mitomycin C (MMC, SigmaAldrich, St. Louis, MO, USA) was then added to a final concentration of 0.25, 0.5, 0.75, 1.0 or 1.25 μg/mL. For

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UV radiation, the exponential growth culture (OD600 of 0.4) of strain SDMCC050201 was collected by centrifugation at 6000×g for 5 min, washed with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4). A total of 2.0 × 108 cells were resuspended in 2 mL phosphate-buffered saline, poured into the glass petri dishes (75 × 15 mm) and illuminated by 19 W UV light (intensity 60 μW/cm2) at 56 cm for 30, 60, 90 and 120 s. After recovering the cells from the plate, an equivalent volume of twofold MRS-Ca broth was added. Decreases of the OD600 were monitored at regular intervals over 8 h after the addition of MMC or treatment by UV radiation. To confirm the release of prophage, DNA extraction was carried out from the supernatant of strain SDMCC050201 treated with MMC and UV radiation based on the method described previously [47], and digested with restriction enzyme BamHI (Takara, Tokyo, Japan). Seven Lb. delbrueckii strains collected in our lab, including SDMCC050201, SDMCC050203, SDMCC050210, SDMCC050211, S, SDW1, SDW2 and ATCC11842 kind gift of Prof. van de Guchte [40] were screened for sensitivity to phiJB by the double-layer plaque assay method [42]. Electron microscopy The lysates produced by MMC induction were centrifuged at 8000×g for 10 min to remove the cell debris, and then filtered through a 0.22 μm membrane. The filtrate was treated with DNase I and RNase A both at a final concentration of 1 μg/mL at 37 °C for 1 h. Phage particles were concentrated with 0.5 M NaCl and 10 % polyethylene glycol 6000 overnight on ice, centrifuged at 12,000×g for 20 min and resuspended in SM buffer (0.58 % NaCl, 0.2 % MgSO4·7H2O, 1 M Tris–HCl, pH 7.5). The concentrated phage particles were sedimented at 25,000×g for 60 min, washed twice in 0.1 M neutral ammonium acetate, and then resuspended in 50 μL SM buffer. Phages were placed on 300 mesh carbon-coated copper grids and then stained with phosphotungstic acid (2 % w/v, pH 7.2) for 30 s. The grid was dried for 10 min and examined under a FEI TF20 transmission electron microscope at 200 kV. Restriction enzyme analysis To determine the phage packaging type, phage DNA aliquots ligated with T4 DNA ligase (Takara, Tokyo, Japan) or not, were digested with the restriction enzymes PstI and BamHI (Takara, Tokyo, Japan) according to the manufacturer’s instructions. The digested DNA samples were treated for 10 min at 70 °C, and then separated on a 1 % (w/v) agarose gel.

J Ind Microbiol Biotechnol Table 1  Primers used in this study

Analysis and primer Tail-PCR  AD  dSP1-R  dSP2-R  dSP3-R  dSP1-L  dSP2-L  dSP3-L Confirmation  aRF4  aRR4  aLF4  aLR4  aBF  aBR

Sequence (5′–3′)a

Description

(G/C/A)N(G/C/A)NNNGGAA GCACCTTTTTCACACGCTTGATG ACTTTTGCGTCTTTGCTGATTATGG AAGAGGGCCAACAAAACGTAAAACG TGGAGATACTTCCCACAGTTTCGTC ATTGCGTCATCAGCTAATTTGGC TGGTGTAGTGGTCTATCCGTCAAAC

Arbitrary degenerate primer Specific primer, internal ORF12 Specific primer, internal ORF12 Specific primer, internal ORF12 Specific primer, internal ORF11 Specific primer, internal ORF11 Specific primer, internal ORF11

TGCGTCTTTGCTGATTATGG TATCACCAAGGAAGATTGCA TTGTCATTGACTTTTTGGCA TGGTGTAGTGGTCTATCCGT GAAGATAAAAATTCCACAAA

Amplifies attR site Amplifies attR site Amplifies attL site Amplifies attL site Amplifies attB site

GTTAGCCGTGGCTGTTTG

Amplifies attB site

a

 N = A, T, G or C

DNA sequencing and bioinformatics analysis DNA sequencing of the phiJB genome was performed at the Chinese National Human Genome Center (Shanghai) using a 454 genome sequencer (454 GS FLX) and the sequence was assembled using the Newbler Assembler. The assembled sequence was scanned for open-reading frames (ORFs) using AUG, UUG, or GUG as start codons. An ORF was accepted if it contained at least 29 amino acids with a potential ribosomal binding site (RBS, with the core consensus sequence AGGAGG) preceding the start codon. Putative ORFs were identified using ORFinder (http://www. ncbi.nlm.nih.gov/gorf/gorf.html). Functional annotation to an ORF was performed using Blast2go (http://blast2go. bioinfo.cipf.es/start_blast2go) and BLASTp (http://blast. ncbi.nlm.nih.gov/Blast.cgi). The conserved domain search at the NCBI Conserved Domain Database (http://www. ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and EMBL InterProScan (http://www.ebi.ac.uk/Tools/InterProScan/) was used to support the annotations. Molecular weights and theoretical isoelectric points were calculated using the Expasy Computer pI/Mw Tool (http://www.expasy. ch/tools/pi_tool.html). Signal peptide was predicted using the SignalP 4.1 Server (http://www.cbs.dtu.dk/services/ SignalP/). Transmembrane domains were predicted using TMHMM (http://www.cbs.dtu.dk/services/TMHMM). The tRNAs were searched using the tRNAscan-SE server (http://lowelab.ucsc.edu/tRNAscan-SE). Collinear analysis of the genome of phiJB with three Lb. delbrueckii phages was performed using the Mauve genome alignment software [12]. Phylogenetic tree was constructed with MEGA 6.06.

To obtain the attL and attR sequences of strain SDMCC050201, thermal asymmetric interlaced PCR (TAIL-PCR) was adopted [20]. PCR products were obtained from strain SDMCC050201 genomic DNA using each of three primers that specifically bound to the predicted ORF 11 or ORF 12 combined with random primers (see Table 1). To confirm the attL and attR sequences, primers aLF4/aLR4 and aRF4/aRR4 were used for PCR amplification and the products were sequenced. To detect the attB site, primers aBF/aBR were used for PCR amplification using genomic DNA of strain SDMCC050201 as the template. Nucleotide sequence accession number The complete nucleotide sequence of the prophage phiJB genome was deposited in the GenBank database under accession number KF188409.

Results Prophage induction When various concentrations of MMC were added into the cultures of Lb. delbrueckii SDMCC050201, the culture turbidity decreased with increasing MMC concentration (Fig. 1a). To confirm the release of prophage from Lb. delbrueckii SDMCC050201, phage DNA was extracted from the supernatant of MMC treated culture, and digested by restriction enzyme BamHI (Fig. 1b). A prophage was present and designated as vB_LdeS-phiJB (phiJB). For UV radiation treatment for 30 or 60 s, a

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Fig. 1  Prophage induction by MMC and UV radiation from Lb. delbrueckii SDMCC050201. a Growth curves of Lb. delbrueckii SDMCC050201 under different concentrations of MMC. b Restriction enzyme analysis of DNA extracted from the supernatant of MMC treated culture of strain SDMCC050201. The DNA was digested with BamHI. Lanes 1, 2, 3, 4 and 5 represent samples treated with MMC at a final concentration of 0.25, 0.5, 0.75, 1.0 and 1.25 μg/mL, respectively. M and m, λ-HindIII DNA ladder

typical induction curve showing initial growth followed by cell lysis was observed for strain SDMCC050201 (Fig.  1c). The restriction map of genomic DNA extracted from the supernatant of UV radiation treated SDMCC050201 culture was same as that extracted from the MMC treated culture, which confirmed the release of phiJB by UV radiation (Fig. 1d). Lactobacillus delbrueckii SDMCC050201 and other seven Lb. Lb. delbrueckii strains were screened for sensitivity to phiJB, but no plaques were detected by the doublelayer plaque assay method. Thus, a propagation host for phiJB was not identified in this work. Electron microscopy PhiJB has an icosahedral capsid with an estimated diameter of 53 ± 2 nm (n  = 12), a flexible non-contractile tail of 183 ± 4 nm in length (n = 10), and several fibers (Fig. 2). Based on its morphology, phiJB belongs to the Siphoviridae family of the International Committee on Taxonomy of Viruses [23]. Genome characterization and functional assignment PhiJB has a linear double-stranded DNA genome comprising 36,969 bp with an overall GC content of 47.7 %. The GC content is 48.3 % in coding sequences (CDS) and 54.6 % at codon position 3 (GC3). No evidence could be found for the presence of cohesive, protruding ends (cos) in

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(Takara, Tokyo, Japan) and 1 kb DNA ladder (Thermo Fisher Scientific, Waltham, MA, USA). c Growth curves of Lb. delbrueckii SDMCC050201 after treated with UV radiation. d Restriction enzyme analysis of DNA extracted from the supernatant of UV radiation treated SDMCC050201 culture. The DNA was digested with BamHI. Lanes 1 and 2 represent samples treated by UV radiation for 30 and 60 s. M and m, λ-HindIII DNA ladder and 1 kb DNA ladder

Fig. 2  Electron micrograph of phage phiJB

the genome of phiJB, and ligation of its DNA did not alter the restriction patterns (Fig. 3). Therefore, phiJB is packaged in a headful mechanism (pac-type). Bioinformatic analysis revealed that the phiJB genome contained 46 ORFs covering 92.3 % of the whole genome length (Table S1). 43 ORFs are oriented in the same direction, while ORFs 12, 13 and 14 are located on the opposite strand. Most of the RBS sequences matched the core consensus sequence (AGGAG) of LL-H [25], and 89 % of the starting codons were AUG, while UUG and GUG were also observed. No tRNA was found in the phiJB genome. A putative function was attributed to 25 ORFs that showed homology to annotated phage proteins or bacterial proteins in public databases. Like many LAB phages, the phiJB genome was organized into the following functional modules: DNA packaging, head and tail morphogenesis, cell

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lysis, integration, lysogeny and DNA replication modules (Fig. 4a). The late gene clusters of phiJB. Orf30JB to orf40JB, comprising the predicted DNA packaging and head modules, are almost identical to the corresponding modules of Lb. delbrueckii phage LL-H. Apart from several point mutations, the most remarkable difference in these modules between the two phages is the product of orf36JB, showing

homology to a hypothetical protein of Lb. delbrueckii (3.0E−23), and therefore, it is proposed that this gene may be acquired from the bacterial genome. The tail module of phiJB bears resemblance in its gene organization and the deduced amino acid sequences of its products to phage LL-H. A notable difference in this region between phages phiJB and LL-H is located in orf43JB and orf44JB. Orf43JB represents a fusion of two successive orfs encoding ORF125LL-H and ORF75LL-H. Sequence alignment showed that the intensive gene mutations occurred in the residues from 84 to 124 of ORF43JB, corresponding to the C-terminal of ORF125LL-H. Similarly, orf44JB is a fusion of the next six genes of LL-H. The C-terminus of ORF44JB contains an N-acetyl-d-glucosamine binding site and murein transglycosylase domain, which might be involved in the process of adsorption and penetration of the cell wall [48]. The residues 529–1138 of ORF44JB show sequence similarity to the putative tape measure protein of Lactobacillus phage Lc-Nu. In many double-stranded DNA phages, a conserved frameshift occurs in the tail assembly chaperones between the major tail protein and the tape measure protein [45]. However, a specific slippery sequence was not identified in the N-terminus of orf44JB, and phage phiJB does not contain frameshift in orf44JB. The lysis module comprises the putative holin and endolysin encoded by orf 6JB and orf 7JB. ORF6JB shows 88 % identity with the holin protein of LL-H and contains one transmembrane domain between residues 6 and 28, with an N-out, C-in topology. ORF7JB exhibits sequence similarity to the muramidase of LL-H and the lytic enzyme encoded by phage mv4. The N-terminus of ORF7JB contains a Glyco_25 domain, which is classified as an endoN-acetylmuramidase based on a search of the NCBI Conserved Domain Database. Multiple sequence alignment and evolution analyses of 12 putative endolysins of Lb. delbrueckii phages and 4 muramidases of Lb. delbrueckii indicated that these peptidoglycan hydrolyses could be divided

Fig. 4  Map of phiJB genome. a Genomic organization of phiJB. Each arrow represents an open reading frame (ORF) and numbering refers to Table S1. Arrows are orientated according to the direc-

tion of transcription. b Prophage integration in Lb. delbrueckii SDMCC050201. The aligned nucleotide sequences of regions contain the attB, attP, attL and attR, and the 15-bp core is highlighted

Fig. 3  Agarose gel electrophoresis of PstI- (lane 1 and 2) and BamHI- (lane 3 and 4) generated DNA fragments of phiJB. Lanes 1 and 3 ligated DNAs. Lanes 2 and 4 non-ligated DNAs. M and m, λ-HindIII DNA ladder and 1 kb DNA ladder

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Fig. 5  Phylogenetic map of 12 putative endolysins of Lb. delbrueckii phages and four muramidases of Lb. delbrueckii strains. The tree was constructed using the Neighbor-Joining method with MEGA6.06

into two major groups, and the evolution of Lb. delbrueckii phage PGHs may have a close relationship with that in the same group of Lb. delbrueckii phages (Fig. 5). Genes involved in integration and lysogeny modules. ORF12JB shares 85 % identity with the integrase from phage mv4 and is predicted to be involved in DNA integration. Commonly, the integrase gene is located downstream of the endolysin gene in LAB phage genomes [46]. In phiJB, the putative integrase (ORF12JB) is separated from the putative endolysin (ORF7JB) by four putative ORFs. ORF8JB, ORF9JB and ORF10JB absent in other phages are homologous with unknown proteins of Lactobacillus. The G + C content of this region (44.6 %) is slightly lower than that of the phiJB genome (47.7 %). The attP site is often adjacent to the gene encoding integrase, and the attB site is commonly found near tRNA or tmRNA genes [44]. In the present study, a physical map of the attB region of the host strain Lb. delbrueckii SDMCC050201 was determined by Tail-PCR (Fig. 4b). The attP site of phiJB is close to the 3′ end of the putative integrase gene, and the attB sequence appears at the 3′ end of the tmRNA, suggesting a different attB location from the related phage mv4, of which the core region overlaps the 3′ end of a tRNASer [13]. In Lactobacillus phages, the lysogenic/lytic switch module frequently consists of a cI-like repressor, a Cro-like protein and an antirepressor [15, 46]. Orf14JB and orf15JB encoded the putative transcriptional regulators and orf16JB encoded the antirepressor, which are almost identical to the corresponding proteins of phage mv4. Genes involved in DNA replication and regulation. The DNA replication and regulation module does not exhibit similarity to the corresponding modules of known phages, while most of the genes have sequence homology

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with those from Lactobacillus. ORF18JB and ORF19JB have several characteristics in common with the type III restriction enzyme Res subunit and the NTP-binding protein. ORF20JB exhibits homology to single-stranded DNAbinding protein, ORF21JB to DNA primase, ORF22JB to ATPase, ORF25JB to endonuclease, and ORF29JB to transcriptional regulator. The functions of the predicted proteins of ORFs 23, 24, 26, 27 and 28 are unknown, and they lack any identifiable conserved protein domains. To date, two types of DNA replication modules in Lb. delbrueckii phages have been reported [34]. The arrangement of the phiJB replication module closely resembles to that of phage JCL1032, consisting of NTP-binding, helicase, single strand binding protein and primase. Therefore, the DNA replication of phiJB belongs to the ϕP4α-type helicase-primase type. Comparative genome analysis of phiJB with the related Lb. delbrueckii phages We investigated the evolutionary relatedness and genetic diversity by comparative genome analysis of phiJB with LL-H, mv4 and c5. As shown in Fig. 6, the gene order of the head and tail morphogenesis, cell lysis and packaging modules of phiJB was largely correlated with LL-H, and these two phages shared a high level of sequence similarity in the above modules. However, the integration, lysogeny and DNA replication modules showed high diversity between these two phages. Compared with mv4, phiJB exhibited significant similarity in the integration and lysogeny modules. In the case of the sequence alignment between phiJB and c5, nucleotide sequence similarity was quite limited (data not shown), confirming that these two phages represented different taxonomic groups of Lb.

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Fig. 6  Schematic representation of comparison of the gene order and orientation in the genomes of phiJB with LL-H and the early gene cluster of mv4. Genes are shown as rectangles; gray rectangles indi-

cate genes that are transcribed rightward and white rectangles indicate genes that are transcribed leftward

delbrueckii phages. The DNA replication module of phiJB was composed of ORFs homologous with proteins from Lactobacillus, rather than being homologous with proteins of phage origin. It is clear that the late and early gene clusters of phiJB originated from different evolutionary lines. In addition, large non-coding region also exists among the late gene cluster, lysogeny module and DNA replication module. Therefore, it is suggested that phiJB is a novel chimeric Lb. delbrueckii phage with genetic diversity.

higher than that of the overall GC content (47.7 %), which is consistent with the host strain Lb. delbrueckii subsp. bulgaricus [40]. Second, horizontal gene transfer frequently occurred in the phiJB genome, and these insertion fragments all appeared to be of the Lactobacillus sp. origin. It indicated that the precursor of phiJB might propagate in a relatively wide host ranges. In addition, gene fusion was observed, which may achieved by point mutations at the start (or stop) codons and a cascade of related genes. The generated fusion proteins contained multiple domains, which were previously suggested for the predicted TMP protein of Lb. delbrueckii phage Ldl [10]. These phenomena confirm that prophages are not silent in the host genome; instead they are undergoing frequent insertion, mutation and rearrangement events [24]. Another interesting feature of this genome sequence is the DNA replication module, which was reassembled from ten bacterial genes and two viral genes. This confirmed that phiJB had taken habitat in various Lactobacillus strains, from which it plucked certain sequences. Interestingly, although phiJB belongs to the group a of Lactobacillus phages, there are distinct differences between phiJB and phage LL-H deriving from the same group, particularly in the type of DNA replication module [34]. The phages LL-H, mv4, LL-Ku and c5 have the “initiator-helicase loader” type, while phiJB adopts the “ϕP4α-type helicase-primase” type, similar to the group c phage JCL1032 [34, 43]. The composition and type of DNA replication module greatly reflects the genetic diversity of phiJB. Comparative genomic analysis indicated that the genome of phiJB is chimeric, with the integration/ lysogeny module, the DNA replication module and the late gene cluster having distinct evolutionary origins.

Discussion In comparison with the virulent phages infecting Lb. delbrueckii during dairy fermentation processes, our knowledge of Lb. delbrueckii temperate phages is limited and only a few of them have been studied in detail [3, 13, 19, 34, 36]. The possible reason is that most starter culture suppliers will test their strains for the presence of prophages. Usually, lysogenic strains carrying easily inducible prophage will not find their way into commercial products [16]. Here, a novel prophage phiJB was obtained from the commercial starter Lb. delbrueckii SDMCC050201 isolated from a Chinese yoghurt sample, which represented a high potential risk for dairy fermentation processes. PhiJB has an icosahedral capsid and a non-contractible tail. Its genome DNA shares homology with Lb. delbrueckii phage LL-H. According to its morphological and genetic characteristics, phiJB belongs to the group a of Lb. delbrueckii phages. The genome sequence of phiJB exhibits several interesting features. First, phiJB may be undergoing rapid evolution along with its host. The GC3 (54.6 %) is significantly

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The phenomenon of chimeric phage formation has also been found in Streptococcus phage [7]. In S. pyogenes phage, genetic chimerism has been suggested to increase phage fitness and the dissemination of the genes located within the shuffled modules [4]. Recently, it has been pointed that the recombination-based formation of a chimeric genome served as an alternative mechanism to escape CRISPR immunity for S. thermophilus phage [5, 27]. Considering that the CRISPR-Cas system also exists in the genome of Lb. delbrueckii [39, 40], the chimeric phage phiJB might have been assembled under the pressure of CRISPR-Cas adaptive immunity, to escape specific sequences targeting and keep survival in natural system. Therefore, the formation of a chimeric genome might represent a novel strategy to keep up with host evolution and increase fitness for Lb. delbrueckii phages. In conclusion, this study provides knowledge on a novel chimeric prophage of Lb. delbrueckii. Its induction from commercial starter represents a potential risk for the usage of lysogenic starter strain during dairy fermentation processes. Its genome information will facilitate better understanding of phage diversity in the dairy industry. Acknowledgments  We would like to thank M. van de Guchte for the kindness of providing Lb. delbrueckii subsp. bulgaricus ATCC11842. This work was supported by a grant of National Natural Science Foundation of China (no. 31271905, 31471715) and China Postdoctoral Science Foundation Funded Project (no. 2014M550364). Compliance with ethical standards  Conflict of interest  The authors declare that they have no conflict of interest. Human and animal rights and informed consent  This article does not contain any studies with human participants or animals performed by any of the authors.

References 1. Akhter S, Aziz RK, Edwards RA (2012) PhiSpy: a novel algorithm for finding prophages in bacterial genomes that combines similarity- and composition-based strategies. Nucleic Acids Res 40:e126. doi:10.1093/nar/gks406 2. Aleksandrova V, Ishlimova D, Urshev Z (2013) Classification of Lactobacillus delbrueckii ssp. bulgaricus phage Gb1 into group “b” Lactobacillus delbrueckii bacteriophages based on its partial genome sequencing. Bulg J Agric Sci 19(Supplement 2):90–93 3. Auad L, Räisänen L, Raya RR, Alatossava T (1999) Physical mapping and partial genetic characterization of the Lactobacillus delbrueckii subsp. bulgaricus bacteriophage lb539. Arch Virol 144:1503–1512 4. Aziz RK, Edwards RA, Taylor WW, Low DE, McGeer A, Kotb M (2005) Mosaic prophages with horizontally acquired genes account for the emergence and diversification of the globally disseminated M1T1 clone of Streptococcus pyogenes. J Bacteriol 187:3311–3318. doi:10.1128/JB.187.10.3311-3318.2005

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J Ind Microbiol Biotechnol 5. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero D, Horvath P (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315:1709–1712. doi:10.1126/science.1138140 6. Baugher JL, Durmaz E, Klaenhammer TR (2014) Spontaneously induced prophage in Lactobacillus gasseri contribute to horizontal gene transfer. Appl Environ Microbiol 80:3508–3517. doi:10.1128/AEM.04092-13 7. Brüssow H, Desiere F (2001) Comparative phage genomics and the evolution of Siphoviridae: insights from dairy phages. Mol Microbiol 39(2):213–222. doi:10.1046/j.1365-2958.2001.02228.x 8. Canchaya C, Proux C, Fournous G, Bruttin A, Brüssow H (2003) Prophage genomics. Microbiol Mol Biol Rev 67:238–276. doi:10.1128/MMBR.67.2.238-276.2003 9. Casey E, Mahony J, O’Connell-Motherway M, Bottacini F, Cornelissen A, Neve H, Heller KJ, Noben J-P, Bello FD, van Sinderen D (2014) Molecular characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages. Appl Environ Microbiol 80:5623–5635. doi:10.1128/AEM.01268-14 10. Casey E, Mahony J, Neve H, Noben JP, Bello FD, van Sinderen D (2015) Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1. Appl Environ Microbiol 81:1319–1326. doi:10.1128/AEM.03413-14 11. Casjens S (2003) Prophages and bacterial genomics: what have we learned so far? Mol Microbiol 49:277–300. doi:10.1046/j.1365-2958.2003.03580.x 12. Darling ACE, Mau B, Blattner FR, Perna NT (2004) Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res 14:1394–1403. doi:10.1101/ gr.2289704 13. Dupont L, Boizet-Bonhoure B, Coddeville M, Auvray F, Ritzenthaler P (1995) Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum. J Bacteriol 177:586–595 14. Durmaz E, Miller MJ, Azcarate-Peril MA, Toon SP, Klaenhammer TR (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 15. García P, Ladero V, Alonso JC, Suárez JE (1999) Cooperative interaction of CI protein regulated lysogeny of Lactobacillus casei by bacteriophage A2. J Virol 73:3920–3929 16. Garneau JE, Moineau S (2011) Bacteriophages of lactic acid bacteria and their impact on milk fermentations. Microb Cell Fact 10(Suppl 1):S20. doi:10.1186/1475-2859-10-S1-S20 17. Guglielmotti D, Marcó MB, Vinderola C, de Los Reyes Gavilán C, Reinheimer J, Quiberoni A (2007) Spontaneous Lactobacillus delbrueckii phage-resistant mutants with acquired bile tolerance. Int J Food Microbiol 119:236–242. doi:10.1016/j. ijfoodmicro.2007.08.010 18. Labrie SJ, 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 19. Lahbib-Mansais Y, Boizet B, Dupont L, Mata M, Ritzenthaler P (1992) Characterization of a temperate bacteriophage of Lactobacillus delbrueckii subsp. bulgaricus and its interactions with the host cell chromosome. Microbiology 138:1139–1146 20. Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674–681

J Ind Microbiol Biotechnol 21. Lunde M, Blatny JM, Lillehaug D, Aastveit AH, Nes IF (2003) Use of real-time quantitative PCR for the analysis of ϕLC3 prophage stability in lactococci. Appl Environ Microbiol 69:41– 48. doi:10.1128/AEM.69.1.41-48.2003 22. Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B, Koonin E, Pavlov A et al (2006) Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci USA 103:15611–15616. doi:10.1073/pnas.0607117103 23. Matthews REF (1982) Classification and nomenclature of viruses. Fourth report of the international committee on taxonomy of viruses. Intervirology 17:1–200 24. 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 25. Mikkonen M, Alatossava T (1994) Characterization of the genome region encoding structural proteins of Lactobacillus delbrueckii subsp. lactis lacteriophage LL-H. Gene 151:53–59 26. Mikkonen M, Räisänen L, Alatossava T (1996) The early gene region completes the nucleotide sequence of Lactobacillus delbrueckii subsp. lactis phage LL-H. Gene 175:49–57 27. Paez-Espino D, Sharon I, Morovic W, Stahl B, Thomas B, Barrangou R, Banfield J (2015) CRISPR immunity drives rapid phage genome evolution in Streptococcus thermophilus. mBio 6(2):e00262-15. doi:10.1128/mBio.00262-15 28. Piotr J, Podles´ny M, Pawelec J, Malinowska A, Kowalczyk S, Targn´ski Z (2013) Spontaneous release of bacteriophage particles by Lactobacillus rhamnosus Pen. J Microbiol Biotechnol 23:357–363. doi:10.4014/jmb.1207.07037 29. Quiberoni A, Guglielmotti D, Reinheimer JA (2003) Inacti vation of Lactobacillus delbrueckii bacteriophages by heat and biocides. Int J Food Microbiol 84:51–62. doi:10.1016/ S0168-1605(02)00394-X 30. Quiberoni A, Guglielmotti D, Binetti A, Reinheimer J (2004) Characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages and the physicochemical analysis of phage adsorption. J Appl Microbiol 96:340–351. doi:10.1046/j.1365-2672.2003.02147.x 31. Räisänen L, Schubert K, Jaakonsaari T, Alatossava T (2004) Characterization of lipoteichoic acids as Lactobacillus delbrueckii phage receptor components. J Bacteriol 186:5529–5532 32. Räisänen L, Draing C, Pfitzenmaier M, Schubert K, Jaakonsaari T, von Aulock S, Hartung T, Alatossava T (2007) Molecular interaction between lipoteichoic acids and Lactobacillus delbrueckii phages depends on d-Alanyl and alpha-glucose substitution of poly(glycerophosphate) backbones. J Bacteriol 189:4135–4140 33. Ravin V, Räisänen L, Alatossava T (2002) A conserved C-terminal region in Gp71 of the small isometric-head phage LL-H and ORF474 of the prolate-head phage JCL1032 is implicated in specificity of adsorption to its host, Lactobacillus delbrueckii. J Bacteriol 184:2455–2459 34. Riipinen K-A, Forsma 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 35. Saxelin M, Tynkkynen S, Matilla-Sandholm T, de Vos WM (2005) Probiotic and other functional microbes: from markets to mechanisms. Curr Opin Biotechnol 16:204–211. doi:10.1016/j. copbio.2005.02.003

36. Suárez V, Zago M, Quiberoni A, Carminati D, Giraffa G, Reinheimer J (2008) Lysogeny in Lactobacillus delbrueckii strains and characterization of two new temperate prolateheaded bacteriophages. J Appl Microbiol 105:1402–1411. doi:10.1111/j.1365-2672.2008.03876.x 37. Suárez VB, Maciel N, Guqiielmotti D, Zago M, Giraffa G, Reinheimer J (2008) Phage-resistance linked to cell heterogeneity in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1. Int J Food Microbiol 128:401–405 38. Trucco V, Reinheimer J, Quiberoni A, Suárez VB (2011) Adsorption of temperate phages of Lactobacillus delbrueckii strains and phage resistance linked to their cell diversity. J Appl Microbiol 110:935–942 39. Urshev Z, Ishlimova D (2015) Distribution of clustered regularly interspaced palindrome repeats CRISPR2 and CRISPR3 in Lactobacillus delbrueckii ssp. bulgaricus strains. Biotechnol Biotec Eq 29:541–546 40. van de Guchte M, Penaud S, Grimaldi C, Barbe V, Bryson K, Nicolas P et al (2006) The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution. Proc Natl Acad Sci USA 103:9274–9278. doi:10.1073/ pnas.0603024103 41. Vinderola G, Marcó MB, Guglielmotti DM, Perdigón G, Giraffa G, Reinheimer J, Quiberoni A (2007) Phage-resistant mutants of Lactobacillus delbrueckii may have functional properties that differ from those of parent strains. Int J Food Microbiol 116:96–102 42. Wang S, Kong J, Gao C, Guo T, Liu X (2010) Isolation and characterization of a novel virulent phage (phiLdb) of Lactobacillus delbrueckii. Int J Food Microbiol 137:22–27. doi:10.1016/j. ijfoodmicro.2009.10.024 43. Weigel C, Seitz H (2006) Bacteriophage replication modules. FEMS Microbiol Rev 30:321–381. doi:10.1111/j.1574-6976.2006.00015.x 44. Williams KP (2002) Integration sites for genetic elements in prokaryotic tRNA and tmRNA genes: sublocation preference of integrase subfamilies. Nucleic Acids Res 30:866–875. doi:10.1093/nar/30.4.866 45. Xu J, Hendrix RW, Duda RL (2004) Conserved translational frameshift in dsDNA bacteriophage tail assembly genes. Mol Cell 16:11–21. doi:10.1016/j.molcel.2004.09.006 46. 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 J-B, 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 47. Zhang X, Kong J, Qu Y (2006) Isolation and characteri zation of a Lactobacillus fermentum temperate bacteriophage from Chinese yogurt. J Appl Microbiol 101:857–863. doi:10.1111/j.1365-2672.2006.03007.x 48. Zhang X, Wang S, Guo T, Kong J (2011) Genome analy sis of Lactobacillus fermentum temperate bacteriophage ϕPYB5. Int J Food Microbiol 144:400–405. doi:10.1016/j. ijfoodmicro.2010.10.026

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A novel chimeric prophage vB_LdeS-phiJB from commercial Lactobacillus delbrueckii subsp. bulgaricus.

Prophage vB_LdeS-phiJB (phiJB) was induced by mitomycin C and UV radiation from the Lactobacillus delbrueckii subsp. bulgaricus SDMCC050201 isolated f...
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