{"paper_id":"201a2828-66a7-4f75-8fbd-54f4d6d85d7c","body_text":"Identification and Characterization of Putative Functional Cryptic Prophages in the Starter Strain Lactiplantibacillus plantarum WCFS1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification and Characterization of Putative Functional Cryptic Prophages in the Starter Strain Lactiplantibacillus plantarum WCFS1 Kwangjun Lee, Sooyeon Song This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8937826/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Prophages are widespread in lactic acid bacteria (LAB) and drive genome evolution and stress adaptation, making their analysis crucial for developing robust starter cultures in fermented food production. Here, we conducted comprehensive prophage analysis of the fully sequenced Lactiplantibacillus plantarum WCFS1 genome using three complementary prediction tools: PHASTEST, Phigaro, and PhageBoost. Cross-tool integration identified nine non-redundant prophage-associated regions, classified as two intact prophages (IP1, IP2), two cryptic prophages (CP1, CP2), and five genomic islands (GI1–GI5). Intact prophages contained complete structural and regulatory modules, while cryptic prophages lacked structural genes but retained recombinases, CI-like repressors, toxin-antitoxin systems, and exopolysaccharide (EPS) biosynthetic genes. Genomic islands were enriched in carbohydrate metabolism, bacteriocin, bile acid transformation, and DNA repair genes. This functional gene composition, consistent with cryptic prophage studies in other species, reveals that cryptic prophages contribute to L. plantarum WCFS1's stress tolerance and genome plasticity, offering targets for strain engineering to enhance fermentation stability and shelf-life in livestock-derived fermented foods. Lactiplantibacillus plantarum WCFS1 prophage cryptic prophage starter culture stress tolerance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Lactic acid bacteria (LAB) are essential microorganisms in the fermented food and probiotics industries. Representative starter strains such as Lactiplantibacillus plantarum , Levilactobacillus brevis , Streptococcus thermophilus , and Leuconostoc lactis are widely utilized (Wang et al., 2021). Among these, L. plantarum has attracted particular attention because of its antioxidant, anti-allergic, and anti-inflammatory properties, making it a promising candidate for the production of functional fermented foods (Aljohani et al., 2025; Ayivi & Ibrahim, 2022). Moreover, L. plantarum serves as a representative model probiotic owing to its high genomic diversity and remarkable environmental resilience (Zhang et al., 2025). The genomic diversity of L. plantarum not only reflects variations in gene content and structure but is also shaped by mobile genetic elements such as prophages. Whole-genome sequencing (WGS) of the reference strain WCFS1 revealed the presence of multiple bacteriophage-derived genes (Ventura et al., 2003), and similar findings have been reported in other Lactobacillus species (Brüssow et al., 2004; Kim & Park, 2022). Prophages typically exist in a dormant state within the host genome but can be activated under stress conditions—such as exposure to antibiotics, UV light, or oxidative stress—to enter the lytic cycle and produce infectious phage particles (Beamud et al., 2024; Howard-Varona et al., 2017; Little & Mount, 1982; Kaiser & Jacob, 1957). In contrast, cryptic prophages are inactive prophages that cannot generate viable phage particles due to the loss or inactivation of structural genes essential for the lytic cycle (Casjens, 2003; Wang et al., 2010). Once regarded as “genomic junk”, cryptic prophages are now recognized as functional genetic elements contributing to host physiology and stress adaptation (Hu et al., 2021). Even without inducibility, cryptic prophages can enhance host defense by harboring genes encoding Sie proteins or orphan methyltransferases, thereby conferring phage resistance (da Silva Duarte et al., 2018). For instance, studies on S. thermophilus M17PTZA496 demonstrated that a non-inducible cryptic prophage can provide superinfection immunity through immunity-related gene expression (da Silva Duarte et al., 2018). Bobay et al. (2013) categorized cryptic prophages as a subset of defective prophages and highlighted their role in shaping bacterial evolution by encoding various molecular systems such as gene transfer agents (GTAs), bacteriocins, and type VI secretion systems (T6SSs), all of which contribute to microbial competition (Bobay et al., 2013; Touchon et al., 2016; Wang et al., 2010). Thus, even though cryptic prophages have lost the ability to enter the lytic cycle, they are increasingly being viewed as functional reservoirs that enhance host survival and adaptability. The most comprehensive understanding of cryptic prophages to date comes from studies of Escherichia coli K-12, which contains nine cryptic prophages (DLP12, rac, Qin, e14, CP4-6, CP4-57, CPS-53, CP4-44, and CP4-1). Deletion of all nine elements (Δ9 strain) resulted in reduced growth rate, stress tolerance, and biofilm formation (Wang et al., 2010). Each cryptic prophage of E. coli has been associated with beneficial host traits such as environmental adaptability (Wang et al., 2010), stress resilience (Kim & Park, 2022), persistence regulation (Song et al., 2021), and cell–cell recognition (Song et al., 2019). In contrast to the extensive studies in E. coli , research on prophages and cryptic prophages in LAB remains limited. Recent genome surveys have revealed that more than 60% of Lactobacillus strains contain at least one intact prophage, suggesting that prophage regions are widespread in this genus (Pei et al., 2021). Furthermore, incomplete prophage regions, which may correspond to cryptic prophages, have been identified in several Lactobacillus genomes (Ventura et al., 2003). In GRAS (Generally Recognized As Safe) microorganisms such as Bifidobacterium, most prophage-related studies have focused on inducibility and phage contamination control rather than exploring prophage functions (Brüssow et al., 2004; Ventura et al., 2005). However, recent studies have reported that prophages with deletions in structural genes, as observed in S. thermophilus and L. lactis , may contribute to stress resistance and protection against superinfection in food-related environments (Kim & Park, 2022; Lavelle et al., 2023). Notably, Lavelle et al. (2023) identified incomplete prophage regions in all 83 S. thermophilus genomes analyzed using PHAST (Zhou et al., 2011), suggesting that cryptic prophages, together with orphan phage-derived genes, CRISPR-Cas, and restriction–modification systems, likely play cooperative roles in phage defense and host fitness. Although the biological functions of cryptic prophages in LAB remain poorly characterized, they may provide similar adaptive benefits to those observed in E. coli , enhancing starter culture survival and fermentation stability. In this study, we performed prophage prediction analyses on the fully sequenced genome of L. plantarum WCFS1 to identify cryptic prophage regions and explore the functional roles of their genes. The findings are expected to offer new insights into how cryptic prophages contribute to the adaptability and stability of LAB under viral stress conditions. MATERIALS AND METHODS Bacterial Strain and Whole Genome Data The complete genome sequence of L. plantarum WCFS1 (GenBank accession no. AL935263.2) was obtained from the National Center for Biotechnology Information (NCBI) database. The FASTA (.fna) and GenBank (.gbk) file formats were subsequently used for prophage prediction and functional analysis. Prophage Prediction Tools (PHASTEST, PHIGARO, PHAGEBOOST) To predict phage-related genomic regions, the following three prophage prediction tools were used. PHASTEST was used to evaluate the structural completeness of prophages and to classify regions as “Intact (> 90),” “Questionable (70 ≤ score ≤ 90),” or “Incomplete (< 70)” It predicts prophage regions by calculating similarity scores based on the presence of core structural genes such as capsid, tail, and integrase (Wishart et al., 2023). Phigaro was used to identify prophage regions within bacterial genomes by analyzing gene annotations and GC-content variation. It predicts prophage region by detecting clusters of phage hallmark genes through a hidden Markov model (HMM) and calculating the density of phage-like domains (Starikova et al., 2020). PhageBoost was used to detect short, degenerated prophages or cryptic fragments, employing a gene probability score based prediction approach across the genome without a fixed threshold (Sirén et al., 2021). Because of the limited interpretability of small prophage regions predicted by PhageBoost, open reading frames (ORFs) from each predicted region were extracted, and their functional similarity and conserved phage protein domains were validated using NCBI BLAST analysis. Classification of Genomic Regions into IP, CP, and GI To establish a unified classification framework, all regions predicted by PHASTEST, Phigaro, and PhageBoost were comparatively evaluated based on gene content, structural integrity, and cross-tool concordance. Each region was assigned to one of three categories: intact prophages (IP), cryptic prophages (CP), or genomic islands (GI). Intact prophages (IP) were defined as regions detected by two or more prediction tools that preserved a complete phage structural module, including portal proteins, terminase subunits, capsid and tail components, and tape-measure proteins, together with phage regulatory elements. Cryptic prophages (CP) were defined as phage-derived regions retaining integrases, CI-like repressors, Cro, or LexA/Xre regulators, as well as partially conserved or fragmented hallmark domains, but lacking the structural genes required for virion assembly. Genomic islands (GI) were defined as regions predicted exclusively by PhageBoost, lacking phage hallmark components or integrases and instead enriched in genes associated with host adaptation, such as carbohydrate metabolism, exopolysaccharide (EPS) biosynthesis, bacteriocin production, bile resistance, and DNA repair. Functional Annotation of Predicted Regions Functional annotation of each predicted region was performed using Prokka and Bakta, followed by manual curation of mobile genetic element (MGE)–related genes. Phage hallmark genes (integrase, terminase large/small subunits, portal, capsid, tail, holin), regulatory genes (CI, Cro, LexA/Xre, AlpA), and lysis-associated genes were assessed to determine phage origin and structural completeness. RESULTS Prediction and Classification of Prophage Regions in L. plantarum WCFS1 Three prophage prediction tools (PHASTEST, Phigaro, and PhageBoost) were applied to the complete genome of L. plantarum WCFS1 to obtain a comprehensive set of phage-associated regions ( Figure 1 ). PhageBoost predicted nine regions, whereas PHASTEST and Phigaro detected three and two regions, respectively ( Table 1 ). Because several predictions overlapped in genomic position, all outputs were consolidated into nine non-redundant regions distributed across the genome. Based on cross-tool comparison and structural integrity, these nine regions were classified into two intact prophages (IP1 and IP2), two cryptic prophages (CP1 and CP2), and five genomic islands (GI1 to GI5) ( Table 2 ). IP1 was located at 589–632 kb, supported by overlapping predictions from PHASTEST Region 1, Phigaro Region 1, and PhageBoost Region 3. IP2 was located at 2.16–2.22 Mb, corresponding to PHASTEST Region 3, Phigaro Region 2, and PhageBoost Region 7. In addition, CP1 (1.08–1.10 Mb) was supported by PHASTEST Region 2 and PhageBoost Region 5, whereas CP2 (approximately 2.99–3.01 Mb) was detected solely by PhageBoost Region 9. The five PhageBoost-only predictions, located at 0.34–0.35 Mb, 0.36–0.37 Mb, 1.08–1.09 Mb, 1.80–1.82 Mb, and 2.82–2.83 Mb, lacked hallmark phage structural genes and were therefore classified as genomic islands (GI1 to GI5). Characterization of Intact Prophages in L. plantarum WCFS1 Two intact prophages were identified based on the strong agreement among the three prediction tools and the complete preservation of hallmark structural and regulatory modules ( Table 2, Table 3 ). These regions also contained accessory genes with potential roles in host interaction, genome maintenance, and phage–host communication, which further supports their classification as structurally intact prophages. The IP1, located at 589 to 632 kb, harbored a full array of phage particle assembly genes, including the terminase large and small subunits, portal protein, major capsid and tail proteins, a tape measure protein, and holin. In addition, this region encoded transcriptional regulators such as CI, Cro, and LexA or Xre. The coexistence of these repressors indicates that IP1 may retain the capacity to modulate the lysogenic to lytic transition and to regulate prophage gene expression through SOS-associated transcriptional control (Erill et al., 2007; Quinones et al., 2005). This region was positioned adjacent to tRNA-Gly, tRNA-Leu, and tRNA-Asn, which is consistent with classical tRNA-mediated integration hotspots in temperate phages. Accessory genes were also identified, including members of the SGNH/GDSL hydrolase family. These enzymes are part of a broad superfamily of carbohydrate-modifying hydrolases known to remodel extracellular polysaccharides and cell-surface glycans (Akoh et al., 2004; Anderson et al., 2022). Their presence within IP1 suggests that this prophage region carries additional host-associated enzymes, although the specific functional relevance of these hydrolases in L. plantarum WCFS1 remains to be experimentally verified. Additional recombination-related elements, such as integrase and recT, further imply that IP1 may facilitate localized genome plasticity or recombination-based prophage maintenance (Lopes et al., 2010; Xue et al., 2023). Taken together, the structural, regulatory, and accessory gene composition of this locus indicates that IP1 retains the capacity for induction and may influence host physiology through both phage-related and phage-derived auxiliary functions. The IP2 was identified between 2.16 and 2.22 Mb. This region represented the largest prophage-derived sequence in the genome and contained the complete structural module required for virion assembly. Key genes included the portal protein, terminase subunits, major and minor capsid proteins, head to tail connector proteins, tail fibers, and a tape measure protein. A holin gene was also present, together with multiple regulators such as CI, Cro, and LexA or Xre, which may coordinate prophage maintenance and control the transition into the lytic state. IP2 contained an extensive tRNA cluster, including tRNA-Trp, tRNA-Asn, tRNA-Ile, tRNA-Leu, tRNA-Met, and tRNA-Gly. The presence of these tRNA genes supports a tRNA-mediated integration mechanism (Tan et al., 2007) and may also contribute to translation optimization during phage gene expression (Kang et al., 2017). Accessory elements, such as DNA methyltransferase, type I site-specific DNase, 3-methyladenine DNA glycosylase, and SNF2- or DnaB-family replication proteins, were also present. These enzymes suggest that IP2 may carry the machinery required for replication initiation, recombination, epigenetic modification, and prophage genome protection (Murphy et al., 2013; Takahashi et al., 2002). Both IP1 and IP2 display complete structural integrity and contain a combination of hallmark phage genes, transcriptional regulators, and accessory functional elements. These characteristics support their classification as intact prophages within the L. plantarum WCFS1 genome. Although previous studies reported that prophages in this strain were not induced by mitomycin C (Ventura et al., 2003), more recent findings indicate that even genetically complete prophages are not necessarily induced under this chemical stress (Ambros & Ehrmann, 2022; Goerke et al., 2006). Considering evidence from L. reuteri , where RecA and LexA dependent SOS responses were shown to regulate prophage gene expression, the presence of LexA or Xre regulators in IP1 and IP2 suggests that these prophages may remain in a transcriptionally silent state under standard laboratory conditions but may be activated under specific environmental or physiological stress (Caggianiello et al., 2016). Therefore, both regions can be considered intact prophages at the structural and regulatory levels, with the potential for induction in response to appropriate triggers. Features of Cryptic Prophage Elements in L. plantarum WCFS1 Two genomic regions were identified as cryptic prophages based on their preservation of phage-derived regulatory and recombination elements despite the complete loss of structural modules required for virion assembly. Both CP1 and CP2 lacked hallmark structural genes, including capsid, tail, terminase, portal, tape measure protein, holin, and endolysin, confirming that neither region can undergo a productive lytic cycle. However, each cryptic prophage retained distinct functional remnants that may contribute to host adaptation, transcriptional regulation, and genomic plasticity. The first cryptic prophage, CP1, corresponds to PHASTEST Region 2 and PhageBoost Region 5 and is located at 1.08 to 1.10 Mb. CP1 retained several recombination-related enzymes, including insK, a catalytic integrase-domain protein, a XerC-family recombinase, and multiple transposase genes ( Table 4 ). These elements suggest that CP1 may retain partial site-specific recombination activity and contribute to localized genome rearrangements, consistent with the roles of integrases, Xer recombinases, and insertion sequences in prophage-associated genome plasticity (Ramisetty & Sudhakari, 2019; Touchon et al., 2016; Vandecraen et al., 2017). CP1 also carried a comprehensive set of exopolysaccharide biosynthesis genes, including mshA , cps2G , cpsC , and cpsD . These genes govern cell surface polysaccharide production and may influence adhesion, colonization, and environmental stress tolerance in the host bacterium (Caggianiello et al., 2016; Lebeer et al., 2012). In addition, CP1 included a RelB–DinJ toxin–antitoxin system, which is associated with growth regulation, stress adaptation, and persistence phenotypes (Christensen et al., 2004). A tyrosine phosphatase gene ( ywqE_1 ) was also identified. This enzyme is known to regulate carbohydrate metabolism and cell wall biosynthesis through protein dephosphorylation (Mijakovic et al., 2005). Together, these features support the interpretation that CP1, although structurally degraded, may contribute to cell envelope remodeling, metabolic regulation, and genomic flexibility as a domesticated cryptic prophage. The second cryptic prophage, CP2, corresponds to PhageBoost Region 9 and is located at 2.99 to 3.01 Mb. CP2 preserved a xerC -type integrase and a CI-like transcriptional repressor, suggesting residual regulatory capacity or responsiveness to stress-associated signals (Bobay et al., 2013; Castillo et al., 2017). Fragmented phage replication components, including an SF3 helicase domain protein and DNA replication-associated proteins, were also identified. CP2 additionally encoded regulatory and membrane-associated factors, including arpU , which is linked to cell envelope regulation, and pucI , a cytosine permease that may participate in membrane transport or nucleotide salvage pathways. Several domesticated phage-derived remnants were present, such as a bacteriocin immunity protein, a DUF1398 domain protein, and an integron-associated gene cassette protein. The coexistence of integrase, regulatory proteins, and replication-associated fragments suggests that CP2 may retain partial transcriptional or stress-responsive functions despite the loss of its structural module. Taken together, CP1 and CP2 represent structurally degraded but functionally enriched cryptic prophages within the L. plantarum WCFS1 genome. Both regions have lost the ability to form virions, yet each preserves distinct combinations of recombination-related enzymes, regulatory factors, EPS biosynthesis pathways, toxin–antitoxin modules, membrane transporters, and replication remnants. These domesticated prophage elements may contribute to host stress adaptation, cell envelope modification, interbacterial interactions, and genomic plasticity, consistent with the roles of cryptic prophages described in other bacterial species. Overview of Non-phage Genomic Island Regions in L. plantarum WCFS1 Several PhageBoost-specific regions lacked phage structural modules and were therefore interpreted as genomic islands rather than prophage remnants ( Figure 1, Table 4 ). These regions formed five functionally distinct islands, designated GI1 through GI5, and their compositions revealed coordinated contributions to carbohydrate metabolism, ecological competition, genome maintenance, and chemical stress adaptation in L. plantarum WCFS1. A group of islands enriched in carbohydrate- and energy-associated genes consisted of GI1 and GI3. GI1 was primarily characterized by a complete glycerol-utilization system, including glpK, glpO, and glpF (Lin, 1976), indicating a dedicated metabolic module for glycerol uptake and catabolism. In addition to these core metabolic genes, GI1 encoded MucBP-domain–containing proteins and multiple membrane-associated proteins, suggesting a potential role in host interaction or cell-surface adaptation. The remaining genes in this island were dominated by DUF-containing, orphan, and hypothetical proteins, consistent with an accessory or niche-adaptive genomic island rather than a prophage-derived region. GI2 displayed a more complex functional architecture, combining metabolic, regulatory, and bacteriocin-associated modules within the same genomic island. This region encoded yidA, brnQ, and napA, which are associated with HAD-family phosphatase activity (Burroughs et al., 2006), branched-chain amino acid transport (Dutta et al., 2022), and ion homeostasis, respectively. Notably, GI2 also contained multiple transcriptional regulators, including the rhaS/rhaR pair, a T-box leader, and two agrA homologs, indicating tight regulatory control. In addition, an expanded plantaricin bacteriocin operon (plnA, plnB, plnJ, plnK, plnL, plnQ, sunS, and a bacteriocin immunity protein) was co-localized within this region, suggesting coordinated regulation of metabolic adaptation and ecological competition. GI3 harbored genes involved in rhamnose biosynthesis, including rfbA, rmlB, and rfbC, along with a cellulase-domain–containing protein. These genes are associated with surface polysaccharide synthesis and carbohydrate-active pathways commonly implicated in cell-envelope modification and environmental adaptation (Giraud & Naismith, 2000; Raetz & Whitfield, 2002). Genomic islands associated with DNA repair, regulatory turnover, and genome stability were found in GI4. This region encoded enzymes such as prmA and a putative 3-methyladenine DNA glycosylase, as well as multiple regulatory proteins including HTH Cro/C1-type proteins and DUF-containing elements. Also, GI4 region contained IS1182- and ISL3-family transposases. The presence of these mobile genetic elements is consistent with genomic regions that undergo insertion or rearrangement events, indicating that GI4 includes components typically associated with local genome mobility (Siguier et al., 2014). GI5 contained genes associated with bile acid metabolism, including baiE, along with glutamyl-tRNA reductase and several regulatory and DUF-containing proteins. The presence of baiE indicates that this region includes components linked to bile acid transformation. However, the functional relevance of the additional genes to bile tolerance or digestive-environment adaptation remains to be determined. Based on their gene content, GI1 through GI5 contain clusters associated with glycerol utilization, amino-acid transport, transcriptional regulation, bacteriocin operons, rhamnose biosynthesis, DNA-repair enzymes, mobile genetic elements, and bile acid–related functions. These genomic islands differ in composition from the cryptic prophages CP1 and CP2, which lack these metabolic and regulatory gene sets and instead retain recombination-related or phage-derived regulatory remnants. DISCUSSION Prophage research is crucial for understanding bacterial genome evolution, stress adaptation, and the genetic basis of industrial strain robustness, particularly for starter cultures used in food fermentation. In this study, we identified two cryptic prophage regions (CP1 and CP2) and two intact prophages in the genome of L. plantarum WCFS1, and we showed that CP1 and CP2 retain diverse recombination-, EPS-, and stress-related genes that are likely to contribute to host fitness rather than phage production. Although CP1 and CP2 lack the structural components required for phage particle formation, the presence of conserved functional remnants suggests that cryptic prophages in L. plantarum WCFS1 may still influence host physiology and genome evolution. Both regions encode integrase-family recombinases, with CP1 carrying insK and CP2 encoding a xerC -type integrase (Tables 2 and 4 ). The retention of these recombination enzymes implies a potential for site-specific recombination and localized genome plasticity, which is consistent with reports on cryptic prophages in other bacteria (Canchaya et al., 2003). In addition, CP2 encodes a CI-like transcriptional repressor, indicating that regulatory functions can be maintained even after structural genes required for virion assembly have been lost (Bobay et al., 2013; Touchon et al., 2016). Insertion sequence elements and transposase-related genes are enriched within CP1 and its flanking regions, which is characteristic of mosaic genetic islands shaped through repeated insertion, deletion, and recombination (Siguier et al., 2014). Such genomic architectures are typical of prophage remnants that have undergone long-term domestication within bacterial chromosomes and are often associated with traits that enhance robustness in complex environments (Bobay et al., 2013; Touchon et al., 2016). This feature is particularly relevant for L. plantarum WCFS1, which is used as a starter culture in fermented foods and is required to withstand diverse stresses during fermentation and storage. Beyond recombination-associated components, CP1 carries an extended set of EPS biosynthesis genes, including mshA and cps2G , which are known to improve environmental resilience and surface-associated fitness in Lactobacillus species (Douillard et al., 2013). Prophage-derived EPS genes can be transcriptionally activated under nutrient limitation or stress, thereby enhancing survival and persistence under harsh conditions that resemble those encountered in animal-based fermented products, such as low pH, high salt, or oxidative stress (Douillard et al., 2013; Touchon et al., 2016). CP1 also encodes a RelB–DinJ toxin–antitoxin module and a tyrosine phosphatase homolog, both of which have been implicated in stress protection, growth regulation, and metabolic signaling in bacteria (Wang et al., 2010; Touchon et al., 2016). These features suggest that cryptic prophage remnants may contribute to the long-term viability and functional stability of L. plantarum WCFS1 during extended fermentation and chilled storage, not only through recombination, but also via metabolic and regulatory integration. Comparative genomics has shown that prophage regions in Lactobacillus can harbor antibiotic resistance genes, virulence-associated factors, and mobile elements, indicating a potential contribution to horizontal gene transfer and adaptive genome evolution (Douillard et al., 2013; Touchon et al., 2016). The genetic composition of CP1 and CP2, which retain domesticated phage-derived components capable of modulating gene expression or facilitating genomic rearrangements, is consistent with this view (Bobay et al., 2013; Touchon et al., 2016). In the context of starter culture application, such adaptive potential may support the long-term stability and performance of L. plantarum WCFS1 in dynamic meat or dairy matrices and under variable processing conditions, including temperature shifts, osmotic stress, and competition with other microorganisms. Taken together, these functional signatures support the view that CP1 and CP2, although structurally incapable of producing phage particles, remain integrated genetic elements that may influence stress responses, persistence-like states, metabolic regulation, and genomic adaptability in L. plantarum WCFS1. From an applied perspective, cryptic prophage-derived traits in this strain may enhance robustness, survival, and technological reliability as a starter culture, contributing to consistent fermentation and extended shelf-life of livestock-derived fermented foods. Future studies should validate CP1 and CP2 functions through stress induction (mitomycin C, UV), excision PCR or qPCR, stress transcriptomics/proteomics, and CRISPR deletion during model fermentation (Otsuji et al., 1959; Lunde et al., 2003; Touchon et al., 2016; Wang et al., 2010). These approaches will clarify cryptic prophages' contributions to L. plantarum WCFS1's fermentation performance and storage stability. Declarations Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (NRF-2020R1F1A1072397; RS-2023-00210305) for SYS. The authors declare no conflicts of interest. Ethics statement This study used publicly available data and did not involve human participants or live vertebrate animals. Therefore, ethical approval and informed consent were not required. References Akoh, C. C., Lee, G. C., Liaw, Y. C., Huang, T. H., & Shaw, J. F. (2004). GDSL family of serine esterases/lipases. Prog Lipid Res , 43 (6), 534-552. https://doi.org/10.1016/j.plipres.2004.09.002 Aljohani, A., Rashwan, N., Vasani, S., Alkhawashki, A., Wu, T. T., Lu, X., Castillo, D. A., & Xiao, J. (2025). The Health Benefits of Probiotic Lactiplantibacillus plantarum: A Systematic Review and Meta-Analysis. Probiotics Antimicrob Proteins , 17 (5), 3358-3377. https://doi.org/10.1007/s12602-024-10287-3 Ambros, C. L., & Ehrmann, M. A. (2022). Distribution, inducibility, and characterisation of prophages in Latilactobacillus sakei. BMC Microbiol , 22 (1), 267. https://doi.org/10.1186/s12866-022-02675-y Anderson, A. C., Stangherlin, S., Pimentel, K. N., Weadge, J. T., & Clarke, A. J. (2022). The SGNH hydrolase family: a template for carbohydrate diversity. Glycobiology , 32 (10), 826-848. https://doi.org/10.1093/glycob/cwac045 Ayivi, R. D., & Ibrahim, S. A. (2022). Lactic acid bacteria: an essential probiotic and starter culture for the production of yoghurt. International Journal of Food Science and Technology , 57 (11), 7008-7025. https://doi.org/10.1111/ijfs.16076 Beamud, B., Benz, F., & Bikard, D. (2024). Going viral: The role of mobile genetic elements in bacterial immunity. Cell Host Microbe , 32 (6), 804-819. https://doi.org/10.1016/j.chom.2024.05.017 Bobay, L. M., Rocha, E. P., & Touchon, M. (2013). The adaptation of temperate bacteriophages to their host genomes. Mol Biol Evol , 30 (4), 737-751. https://doi.org/10.1093/molbev/mss279 Brüssow, H., Canchaya, C., & Hardt, W. D. (2004). Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiol Mol Biol Rev , 68 (3), 560-602, table of contents. https://doi.org/10.1128/mmbr.68.3.560-602.2004 Burroughs, A. M., Allen, K. N., Dunaway-Mariano, D., & Aravind, L. (2006). Evolutionary genomics of the HAD superfamily: understanding the structural adaptations and catalytic diversity in a superfamily of phosphoesterases and allied enzymes. J Mol Biol , 361 (5), 1003-1034. https://doi.org/10.1016/j.jmb.2006.06.049 Caggianiello, G., Kleerebezem, M., & Spano, G. (2016). Exopolysaccharides produced by lactic acid bacteria: from health-promoting benefits to stress tolerance mechanisms. Appl Microbiol Biotechnol , 100 (9), 3877-3886. https://doi.org/10.1007/s00253-016-7471-2 Canchaya, C., Fournous, G., Chibani-Chennoufi, S., Dillmann, M. L., & Brüssow, H. (2003). Phage as agents of lateral gene transfer. Curr Opin Microbiol , 6 (4), 417-424. https://doi.org/10.1016/s1369-5274(03)00086-9 Casjens, S. (2003). Prophages and bacterial genomics: what have we learned so far? Mol Microbiol , 49 (2), 277-300. https://doi.org/10.1046/j.1365-2958.2003.03580.x Castillo, F., Benmohamed, A., & Szatmari, G. (2017). Xer Site Specific Recombination: Double and Single Recombinase Systems. Front Microbiol , 8 , 453. https://doi.org/10.3389/fmicb.2017.00453 Christensen, S. K., Maenhaut‐Michel, G., Mine, N., Gottesman, S., Gerdes, K., & Van Melderen, L. (2004). Overproduction of the Lon protease triggers inhibition of translation in Escherichia coli: involvement of the yefM‐yoeB toxin‐antitoxin system. Molecular microbiology , 51 (6), 1705-1717. da Silva Duarte, V., Giaretta, S., Campanaro, S., Treu, L., Armani, A., Tarrah, A., Oliveira de Paula, S., Giacomini, A., & Corich, V. (2018). A Cryptic Non-Inducible Prophage Confers Phage-Immunity on the Streptococcus thermophilus M17PTZA496. Viruses , 11 (1). https://doi.org/10.3390/v11010007 Douillard, F. P., Ribbera, A., Järvinen, H. M., Kant, R., Pietilä, T. E., Randazzo, C., Paulin, L., Laine, P. K., Caggia, C., von Ossowski, I., Reunanen, J., Satokari, R., Salminen, S., Palva, A., & de Vos, W. M. (2013). Comparative genomic and functional analysis of Lactobacillus casei and Lactobacillus rhamnosus strains marketed as probiotics. Appl Environ Microbiol , 79 (6), 1923-1933. https://doi.org/10.1128/aem.03467-12 Dutta, S., Corsi, I. D., Bier, N., & Koehler, T. M. (2022). BrnQ-Type Branched-Chain Amino Acid Transporters Influence Bacillus anthracis Growth and Virulence. mBio , 13 (1), e0364021. https://doi.org/10.1128/mbio.03640-21 Erill, I., Campoy, S., & Barbé, J. (2007). Aeons of distress: an evolutionary perspective on the bacterial SOS response. FEMS Microbiol Rev , 31 (6), 637-656. https://doi.org/10.1111/j.1574-6976.2007.00082.x Giraud, M. F., & Naismith, J. H. (2000). The rhamnose pathway. Curr Opin Struct Biol , 10 (6), 687-696. https://doi.org/10.1016/s0959-440x(00)00145-7 Goerke, C., Köller, J., & Wolz, C. (2006). Ciprofloxacin and trimethoprim cause phage induction and virulence modulation in Staphylococcus aureus. Antimicrob Agents Chemother , 50 (1), 171-177. https://doi.org/10.1128/aac.50.1.171-177.2006 Howard-Varona, C., Hargreaves, K. R., Abedon, S. T., & Sullivan, M. B. (2017). Lysogeny in nature: mechanisms, impact and ecology of temperate phages. The ISME Journal , 11 (7), 1511-1520. https://doi.org/10.1038/ismej.2017.16 Hu, J., Ye, H., Wang, S., Wang, J., & Han, D. (2021). Prophage Activation in the Intestine: Insights Into Functions and Possible Applications. Front Microbiol , 12 , 785634. https://doi.org/10.3389/fmicb.2021.785634 Kaiser, A. D., & Jacob, F. (1957). Recombination between related temperate bacteriophages and the genetic control of immunity and prophage localization. Virology , 4 (3), 509-521. https://doi.org/https://doi.org/10.1016/0042-6822(57)90083-1 Kang, H. S., McNair, K., Cuevas, D. A., Bailey, B. A., Segall, A. M., & Edwards, R. A. (2017). Prophage genomics reveals patterns in phage genome organization and replication. bioRxiv , 114819. https://doi.org/10.1101/114819 Kim, S. H., & Park, J. H. (2022). Characterization of Prophages in Leuconostoc Derived from Kimchi and Genomic Analysis of the Induced Prophage in Leuconostoc lactis. J Microbiol Biotechnol , 32 (3), 333-340. https://doi.org/10.4014/jmb.2110.10046 Lavelle, K., McDonnell, B., Fitzgerald, G., van Sinderen, D., & Mahony, J. (2023). Bacteriophage-host interactions in Streptococcus thermophilus and their impact on co-evolutionary processes. FEMS Microbiol Rev , 47 (4). https://doi.org/10.1093/femsre/fuad032 Lebeer, S., Claes, I., Tytgat, H. L., Verhoeven, T. L., Marien, E., von Ossowski, I., Reunanen, J., Palva, A., Vos, W. M., Keersmaecker, S. C., & Vanderleyden, J. (2012). Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells. Appl Environ Microbiol , 78 (1), 185-193. https://doi.org/10.1128/aem.06192-11 Lin, E. C. (1976). Glycerol dissimilation and its regulation in bacteria. Annu Rev Microbiol , 30 , 535-578. https://doi.org/10.1146/annurev.mi.30.100176.002535 Little, J. W., & Mount, D. W. (1982). The SOS regulatory system of Escherichia coli. Cell , 29 (1), 11-22. https://doi.org/10.1016/0092-8674(82)90085-x Lopes, A., Amarir-Bouhram, J., Faure, G., Petit, M.-A., & Guerois, R. (2010). Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs. Nucleic Acids Research , 38 (12), 3952-3962. https://doi.org/10.1093/nar/gkq096 Lunde, M., Blatny, J. M., Lillehaug, D., Aastveit, A. H., & Nes, I. F. (2003). Use of real-time quantitative PCR for the analysis of phiLC3 prophage stability in lactococci. Appl Environ Microbiol , 69 (1), 41-48. https://doi.org/10.1128/aem.69.1.41-48.2003 Mijakovic, I., Musumeci, L., Tautz, L., Petranovic, D., Edwards, R. A., Jensen, P. R., Mustelin, T., Deutscher, J., & Bottini, N. (2005). In vitro characterization of the Bacillus subtilis protein tyrosine phosphatase YwqE. J Bacteriol , 187 (10), 3384-3390. https://doi.org/10.1128/jb.187.10.3384-3390.2005 Murphy, J., Mahony, J., Ainsworth, S., Nauta, A., & van Sinderen, D. (2013). Bacteriophage orphan DNA methyltransferases: insights from their bacterial origin, function, and occurrence. Appl Environ Microbiol , 79 (24), 7547-7555. https://doi.org/10.1128/aem.02229-13 Otsuji, N., Sekiguchi, M., Iijima, T., & Takagi, Y. (1959). Induction of Phage Formation in the Lysogenic Escherichia coli K-12 by Mitomycin C. Nature , 184 (4692), 1079-1080. https://doi.org/10.1038/1841079b0 Pei, Z., Sadiq, F. A., Han, X., Zhao, J., Zhang, H., Ross, R. P., Lu, W., & Chen, W. (2021). Comprehensive Scanning of Prophages in Lactobacillus: Distribution, Diversity, Antibiotic Resistance Genes, and Linkages with CRISPR-Cas Systems. mSystems , 6 (3), e0121120. https://doi.org/10.1128/mSystems.01211-20 Quinones, M., Kimsey, H. H., & Waldor, M. K. (2005). LexA Cleavage Is Required for CTX Prophage Induction. Molecular Cell , 17 (2), 291-300. https://doi.org/10.1016/j.molcel.2004.11.046 Raetz, C. R., & Whitfield, C. (2002). Lipopolysaccharide endotoxins. Annu Rev Biochem , 71 , 635-700. https://doi.org/10.1146/annurev.biochem.71.110601.135414 Ramisetty, B. C. M., & Sudhakari, P. A. (2019). Bacterial 'Grounded' Prophages: Hotspots for Genetic Renovation and Innovation. Front Genet , 10 , 65. https://doi.org/10.3389/fgene.2019.00065 Siguier, P., Gourbeyre, E., & Chandler, M. (2014). Bacterial insertion sequences: their genomic impact and diversity. FEMS Microbiol Rev , 38 (5), 865-891. https://doi.org/10.1111/1574-6976.12067 Sirén, K., Millard, A., Petersen, B., Gilbert, M Thomas P., Clokie, M. R. J., & Sicheritz-Pontén, T. (2021). Rapid discovery of novel prophages using biological feature engineering and machine learning. NAR Genomics and Bioinformatics , 3 (1). https://doi.org/10.1093/nargab/lqaa109 Song, S., Guo, Y., Kim, J.-S., Wang, X., & Wood, T. K. (2019). Phages Mediate Bacterial Self-Recognition. Cell Reports , 27 (3), 737-749.e734. https://doi.org/10.1016/j.celrep.2019.03.070 Song, S., Kim, J. S., Yamasaki, R., Oh, S., Benedik, M. J., & Wood, T. K. (2021). Escherichia coli cryptic prophages sense nutrients to influence persister cell resuscitation. Environ Microbiol , 23 (11), 7245-7254. https://doi.org/10.1111/1462-2920.15816 Starikova, E. V., Tikhonova, P. O., Prianichnikov, N. A., Rands, C. M., Zdobnov, E. M., Ilina, E. N., & Govorun, V. M. (2020). Phigaro: high-throughput prophage sequence annotation. Bioinformatics , 36 (12), 3882-3884. https://doi.org/10.1093/bioinformatics/btaa250 Takahashi, N., Naito, Y., Handa, N., & Kobayashi, I. (2002). A DNA methyltransferase can protect the genome from postdisturbance attack by a restriction-modification gene complex. J Bacteriol , 184 (22), 6100-6108. https://doi.org/10.1128/jb.184.22.6100-6108.2002 Tan, Y., Zhang, K., Rao, X., Jin, X., Huang, J., Zhu, J., Chen, Z., Hu, X., Shen, X., Wang, L., & Hu, F. (2007). Whole genome sequencing of a novel temperate bacteriophage of P. aeruginosa: evidence of tRNA gene mediating integration of the phage genome into the host bacterial chromosome. Cell Microbiol , 9 (2), 479-491. https://doi.org/10.1111/j.1462-5822.2006.00804.x Touchon, M., Bernheim, A., & Rocha, E. P. (2016). Genetic and life-history traits associated with the distribution of prophages in bacteria. Isme j , 10 (11), 2744-2754. https://doi.org/10.1038/ismej.2016.47 Vandecraen, J., Chandler, M., Aertsen, A., & Van Houdt, R. (2017). The impact of insertion sequences on bacterial genome plasticity and adaptability. Crit Rev Microbiol , 43 (6), 709-730. https://doi.org/10.1080/1040841x.2017.1303661 Ventura, M., Canchaya, C., Kleerebezem, M., de Vos, W. M., Siezen, R. J., & Brüssow, H. (2003). The prophage sequences of Lactobacillus plantarum strain WCFS1. Virology , 316 (2), 245-255. https://doi.org/10.1016/j.virol.2003.08.019 Ventura, M., Lee, J. H., Canchaya, C., Zink, R., Leahy, S., Moreno-Munoz, J. A., O'Connell-Motherway, M., Higgins, D., Fitzgerald, G. F., O'Sullivan, D. J., & van Sinderen, D. (2005). Prophage-like elements in bifidobacteria: insights from genomics, transcription, integration, distribution, and phylogenetic analysis. Appl Environ Microbiol , 71 (12), 8692-8705. https://doi.org/10.1128/aem.71.12.8692-8705.2005 Wang, X., Kim, Y., Ma, Q., Hong, S. H., Pokusaeva, K., Sturino, J. M., & Wood, T. K. (2010). Cryptic prophages help bacteria cope with adverse environments. Nat Commun , 1 , 147. https://doi.org/10.1038/ncomms1146 Wishart, D. S., Han, S., Saha, S., Oler, E., Peters, H., Grant, J. R., Stothard, P., & Gautam, V. (2023). PHASTEST: faster than PHASTER, better than PHAST. Nucleic Acids Res , 51 (W1), W443-w450. https://doi.org/10.1093/nar/gkad382 Wiull, K., Haugen Lisa, K., Eijsink Vincent, G. H., & Mathiesen, G. (2025). CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1. Microbiology Spectrum , 13 (2), e02025-02024. https://doi.org/10.1128/spectrum.02025-24 Xue, F., Ma, X., Luo, C., Li, D., Shi, G., & Li, Y. (2023). Construction of a bacteriophage-derived recombinase system in Bacillus licheniformis for gene deletion. AMB Express , 13 (1), 89. https://doi.org/10.1186/s13568-023-01589-w Zhang, Z., Niu, H., Qu, Q., Guo, D., Wan, X., Yang, Q., Mo, Z., Tan, S., Xiang, Q., Tian, X., Yang, H., & Liu, Z. (2025). Advancements in Lactiplantibacillus plantarum: probiotic characteristics, gene editing technologies and applications. Crit Rev Food Sci Nutr , 65 (29), 6623-6644. https://doi.org/10.1080/10408398.2024.2448562 Zhou, Y., Liang, Y., Lynch, K. H., Dennis, J. J., & Wishart, D. S. (2011). PHAST: a fast phage search tool. Nucleic Acids Res , 39 (Web Server issue), W347-352. https://doi.org/10.1093/nar/gkr485 Tables Tables 1 to 5 are available in the supplementary files section Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.docx Tables.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 10 May, 2026 Reviewers agreed at journal 30 Apr, 2026 Reviewers agreed at journal 29 Apr, 2026 Reviewers invited by journal 29 Apr, 2026 Editor assigned by journal 25 Feb, 2026 Submission checks completed at journal 24 Feb, 2026 First submitted to journal 22 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-8937826\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":632149275,\"identity\":\"f87f39ae-e376-4623-bdee-d90bc590f647\",\"order_by\":0,\"name\":\"Kwangjun Lee\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Jeonbuk National University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Kwangjun\",\"middleName\":\"\",\"lastName\":\"Lee\",\"suffix\":\"\"},{\"id\":632149277,\"identity\":\"0071d8e5-dae6-42b5-bb12-a24924187150\",\"order_by\":1,\"name\":\"Sooyeon Song\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYPACG2YDKMsArzokkEa6lsMMxGuRb+89/PJn23l2c+keA4YfNQzG5g0EtBicOZdmzdt2m9lyzhkDxp5jDGYyBwhpkcgxM2YEajG4kWPAwNvAYCNB0GEzcswMf7adA2th/EuMFoYbOcYPeNsOgLUwA20xI6jF4MwZM2aec8nMljPSCg7LHJMwJuyw9h7jjz/K7JLNJZI3PnxTY2M4g6DDGBjYJBjZGJJBrAMMDIR9AgLMHxj+MNgRpXQUjIJRMApGJgAA/Z45V0lVekIAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Jeonbuk National University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Sooyeon\",\"middleName\":\"\",\"lastName\":\"Song\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-02-22 08:23:16\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-8937826/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-8937826/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":108798460,\"identity\":\"a115e057-749a-486a-aac0-97eb5761a5b4\",\"added_by\":\"auto\",\"created_at\":\"2026-05-08 13:49:58\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":123196,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003ePrediction of prophage regions in \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eLactiplantibacillus plantarum\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e WCFS1. \\u003c/strong\\u003eCircular genome map illustrates prophage regions predicted by three independent tools: PHASTEST (blue), PhageBoost (purple), and Phigaro (red). Each colored block represents a predicted prophage region, and overlapping regions indicate consensus predictions across tools. The accompanying table summarizes the genomic coordinates and sizes of all predicted regions. (PHASTEST-3, PhageBoost-7, and Phigaro-2).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8937826/v1/9a4a9159add6ff881b130d5a.png\"},{\"id\":108807672,\"identity\":\"1fa8bd63-0fbb-4856-87cb-af510d55dbfc\",\"added_by\":\"auto\",\"created_at\":\"2026-05-08 15:31:07\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":72520,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eIntegration of PhageBoost, PHASTEST, and Phigaro predictions to define prophage regions. \\u003c/strong\\u003eFigure showing the integration of prophage prediction results obtained using three independent tools and the consolidation of overlapping regions into unified prophage or genomic island categories. Each column represents a prediction tool, and colored blocks indicate regions predicted by the respective method.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8937826/v1/a8020117356c0f22fc13757d.png\"},{\"id\":108798462,\"identity\":\"0d1f21ce-41bf-4c63-bbde-d48e54c3835d\",\"added_by\":\"auto\",\"created_at\":\"2026-05-08 13:49:58\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":328365,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eLinear genome map of intact prophage (IP) regions predicted by PhageBoost\\u003c/strong\\u003e. Linear genome maps of intact prophage (IP) regions predicted using PhageBoost. Genes are displayed as directional arrows scaled to their genomic length. The mapped regions contain complete prophage architectures, including modules associated with integration, packaging, region assembly, and lysis, supporting their classification as intact prophages.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8937826/v1/c69180c2e262718f6361f82c.png\"},{\"id\":108807554,\"identity\":\"89e0f45a-5b76-439a-8308-8c6841bffc52\",\"added_by\":\"auto\",\"created_at\":\"2026-05-08 15:30:37\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":118910,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eLinear genome map of cryptic prophage (CP) regions predicted by PhageBoost. \\u003c/strong\\u003eLinear genome maps of cryptic prophage (CP) regions predicted using PhageBoost. Genes are shown as directional arrows proportional to their genomic length. These regions retain partial prophage-associated genes but lack complete sets of structural and lysis modules required for functional phage particle formation, consistent with their designation as cryptic prophages.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8937826/v1/91f6dd320a4731dcdb340f75.png\"},{\"id\":108798465,\"identity\":\"7442b55e-ed00-4582-ab86-cc2189dc73af\",\"added_by\":\"auto\",\"created_at\":\"2026-05-08 13:49:58\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":290138,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eLinear genome map of genomic island (GI) regions predicted by PhageBoost. \\u003c/strong\\u003eLinear genome maps of genomic island (GI) regions predicted using PhageBoost. Genes are represented as directional arrows scaled to genomic length. The depicted regions lack conserved prophage organization and do not encode complete phage-related modules, indicating that they correspond to genomic islands rather than intact or cryptic prophages.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8937826/v1/d7de1a53e3b4499753236611.png\"},{\"id\":108810031,\"identity\":\"77536c19-1d07-4f3b-b9f3-bca81a4a520e\",\"added_by\":\"auto\",\"created_at\":\"2026-05-08 15:57:00\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1062923,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8937826/v1/1b522894-e509-4ee8-93e3-2cbc9b94318f.pdf\"},{\"id\":108807756,\"identity\":\"e5e774ca-89cb-42f3-8461-a4a5038bce2e\",\"added_by\":\"auto\",\"created_at\":\"2026-05-08 15:31:27\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":136893,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementaryinformation.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8937826/v1/4824f7346d04c8c3e8238631.docx\"},{\"id\":108807749,\"identity\":\"c9b35b2c-a037-4513-aaff-0dd18e524a1f\",\"added_by\":\"auto\",\"created_at\":\"2026-05-08 15:31:27\",\"extension\":\"docx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":73627,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Tables.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8937826/v1/c8a802ff1d62b7259af81b58.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Identification and Characterization of Putative Functional Cryptic Prophages in the Starter Strain Lactiplantibacillus plantarum WCFS1\",\"fulltext\":[{\"header\":\"INTRODUCTION\",\"content\":\"\\u003cp\\u003eLactic acid bacteria (LAB) are essential microorganisms in the fermented food and probiotics industries. Representative starter strains such as \\u003cem\\u003eLactiplantibacillus plantarum\\u003c/em\\u003e, \\u003cem\\u003eLevilactobacillus brevis\\u003c/em\\u003e, \\u003cem\\u003eStreptococcus thermophilus\\u003c/em\\u003e, and \\u003cem\\u003eLeuconostoc lactis\\u003c/em\\u003e are widely utilized (Wang et al., 2021). Among these, \\u003cem\\u003eL. plantarum\\u003c/em\\u003e has attracted particular attention because of its antioxidant, anti-allergic, and anti-inflammatory properties, making it a promising candidate for the production of functional fermented foods (Aljohani et al., 2025; Ayivi \\u0026amp; Ibrahim, 2022). Moreover, \\u003cem\\u003eL. plantarum\\u003c/em\\u003e serves as a representative model probiotic owing to its high genomic diversity and remarkable environmental resilience (Zhang et al., 2025).\\u003c/p\\u003e \\u003cp\\u003eThe genomic diversity of \\u003cem\\u003eL. plantarum\\u003c/em\\u003e not only reflects variations in gene content and structure but is also shaped by mobile genetic elements such as prophages. Whole-genome sequencing (WGS) of the reference strain WCFS1 revealed the presence of multiple bacteriophage-derived genes (Ventura et al., 2003), and similar findings have been reported in other Lactobacillus species (Br\\u0026uuml;ssow et al., 2004; Kim \\u0026amp; Park, 2022).\\u003c/p\\u003e \\u003cp\\u003eProphages typically exist in a dormant state within the host genome but can be activated under stress conditions\\u0026mdash;such as exposure to antibiotics, UV light, or oxidative stress\\u0026mdash;to enter the lytic cycle and produce infectious phage particles (Beamud et al., 2024; Howard-Varona et al., 2017; Little \\u0026amp; Mount, 1982; Kaiser \\u0026amp; Jacob, 1957). In contrast, cryptic prophages are inactive prophages that cannot generate viable phage particles due to the loss or inactivation of structural genes essential for the lytic cycle (Casjens, 2003; Wang et al., 2010). Once regarded as \\u0026ldquo;genomic junk\\u0026rdquo;, cryptic prophages are now recognized as functional genetic elements contributing to host physiology and stress adaptation (Hu et al., 2021). Even without inducibility, cryptic prophages can enhance host defense by harboring genes encoding Sie proteins or orphan methyltransferases, thereby conferring phage resistance (da Silva Duarte et al., 2018). For instance, studies on \\u003cem\\u003eS. thermophilus\\u003c/em\\u003e M17PTZA496 demonstrated that a non-inducible cryptic prophage can provide superinfection immunity through immunity-related gene expression (da Silva Duarte et al., 2018).\\u003c/p\\u003e \\u003cp\\u003eBobay et al. (2013) categorized cryptic prophages as a subset of defective prophages and highlighted their role in shaping bacterial evolution by encoding various molecular systems such as gene transfer agents (GTAs), bacteriocins, and type VI secretion systems (T6SSs), all of which contribute to microbial competition (Bobay et al., 2013; Touchon et al., 2016; Wang et al., 2010). Thus, even though cryptic prophages have lost the ability to enter the lytic cycle, they are increasingly being viewed as functional reservoirs that enhance host survival and adaptability.\\u003c/p\\u003e \\u003cp\\u003eThe most comprehensive understanding of cryptic prophages to date comes from studies of \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e K-12, which contains nine cryptic prophages (DLP12, rac, Qin, e14, CP4-6, CP4-57, CPS-53, CP4-44, and CP4-1). Deletion of all nine elements (Δ9 strain) resulted in reduced growth rate, stress tolerance, and biofilm formation (Wang et al., 2010). Each cryptic prophage of \\u003cem\\u003eE. coli\\u003c/em\\u003e has been associated with beneficial host traits such as environmental adaptability (Wang et al., 2010), stress resilience (Kim \\u0026amp; Park, 2022), persistence regulation (Song et al., 2021), and cell\\u0026ndash;cell recognition (Song et al., 2019).\\u003c/p\\u003e \\u003cp\\u003eIn contrast to the extensive studies in \\u003cem\\u003eE. coli\\u003c/em\\u003e, research on prophages and cryptic prophages in LAB remains limited. Recent genome surveys have revealed that more than 60% of \\u003cem\\u003eLactobacillus\\u003c/em\\u003e strains contain at least one intact prophage, suggesting that prophage regions are widespread in this genus (Pei et al., 2021). Furthermore, incomplete prophage regions, which may correspond to cryptic prophages, have been identified in several \\u003cem\\u003eLactobacillus\\u003c/em\\u003e genomes (Ventura et al., 2003).\\u003c/p\\u003e \\u003cp\\u003eIn GRAS (Generally Recognized As Safe) microorganisms such as Bifidobacterium, most prophage-related studies have focused on inducibility and phage contamination control rather than exploring prophage functions (Br\\u0026uuml;ssow et al., 2004; Ventura et al., 2005). However, recent studies have reported that prophages with deletions in structural genes, as observed in \\u003cem\\u003eS. thermophilus\\u003c/em\\u003e and \\u003cem\\u003eL. lactis\\u003c/em\\u003e, may contribute to stress resistance and protection against superinfection in food-related environments (Kim \\u0026amp; Park, 2022; Lavelle et al., 2023).\\u003c/p\\u003e \\u003cp\\u003eNotably, Lavelle et al. (2023) identified incomplete prophage regions in all 83 \\u003cem\\u003eS. thermophilus\\u003c/em\\u003e genomes analyzed using PHAST (Zhou et al., 2011), suggesting that cryptic prophages, together with orphan phage-derived genes, CRISPR-Cas, and restriction\\u0026ndash;modification systems, likely play cooperative roles in phage defense and host fitness.\\u003c/p\\u003e \\u003cp\\u003eAlthough the biological functions of cryptic prophages in LAB remain poorly characterized, they may provide similar adaptive benefits to those observed in \\u003cem\\u003eE. coli\\u003c/em\\u003e, enhancing starter culture survival and fermentation stability.\\u003c/p\\u003e \\u003cp\\u003eIn this study, we performed prophage prediction analyses on the fully sequenced genome of \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1 to identify cryptic prophage regions and explore the functional roles of their genes. The findings are expected to offer new insights into how cryptic prophages contribute to the adaptability and stability of LAB under viral stress conditions.\\u003c/p\\u003e\"},{\"header\":\"MATERIALS AND METHODS\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBacterial Strain and Whole Genome Data\\u003c/h2\\u003e \\u003cp\\u003eThe complete genome sequence of \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1 (GenBank accession no. AL935263.2) was obtained from the National Center for Biotechnology Information (NCBI) database. The FASTA (.fna) and GenBank (.gbk) file formats were subsequently used for prophage prediction and functional analysis.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eProphage Prediction Tools (PHASTEST, PHIGARO, PHAGEBOOST)\\u003c/h3\\u003e\\n\\u003cp\\u003eTo predict phage-related genomic regions, the following three prophage prediction tools were used. PHASTEST was used to evaluate the structural completeness of prophages and to classify regions as \\u0026ldquo;Intact (\\u0026gt;\\u0026thinsp;90),\\u0026rdquo; \\u0026ldquo;Questionable (70\\u0026thinsp;\\u0026le;\\u0026thinsp;score\\u0026thinsp;\\u0026le;\\u0026thinsp;90),\\u0026rdquo; or \\u0026ldquo;Incomplete (\\u0026lt;\\u0026thinsp;70)\\u0026rdquo; It predicts prophage regions by calculating similarity scores based on the presence of core structural genes such as capsid, tail, and integrase (Wishart et al., 2023). Phigaro was used to identify prophage regions within bacterial genomes by analyzing gene annotations and GC-content variation. It predicts prophage region by detecting clusters of phage hallmark genes through a hidden Markov model (HMM) and calculating the density of phage-like domains (Starikova et al., 2020). PhageBoost was used to detect short, degenerated prophages or cryptic fragments, employing a gene probability score based prediction approach across the genome without a fixed threshold (Sir\\u0026eacute;n et al., 2021). Because of the limited interpretability of small prophage regions predicted by PhageBoost, open reading frames (ORFs) from each predicted region were extracted, and their functional similarity and conserved phage protein domains were validated using NCBI BLAST analysis.\\u003c/p\\u003e\\n\\u003ch3\\u003eClassification of Genomic Regions into IP, CP, and GI\\u003c/h3\\u003e\\n\\u003cp\\u003eTo establish a unified classification framework, all regions predicted by PHASTEST, Phigaro, and PhageBoost were comparatively evaluated based on gene content, structural integrity, and cross-tool concordance. Each region was assigned to one of three categories: intact prophages (IP), cryptic prophages (CP), or genomic islands (GI).\\u003c/p\\u003e \\u003cp\\u003eIntact prophages (IP) were defined as regions detected by two or more prediction tools that preserved a complete phage structural module, including portal proteins, terminase subunits, capsid and tail components, and tape-measure proteins, together with phage regulatory elements.\\u003c/p\\u003e \\u003cp\\u003eCryptic prophages (CP) were defined as phage-derived regions retaining integrases, CI-like repressors, Cro, or LexA/Xre regulators, as well as partially conserved or fragmented hallmark domains, but lacking the structural genes required for virion assembly.\\u003c/p\\u003e \\u003cp\\u003eGenomic islands (GI) were defined as regions predicted exclusively by PhageBoost, lacking phage hallmark components or integrases and instead enriched in genes associated with host adaptation, such as carbohydrate metabolism, exopolysaccharide (EPS) biosynthesis, bacteriocin production, bile resistance, and DNA repair.\\u003c/p\\u003e\\n\\u003ch3\\u003eFunctional Annotation of Predicted Regions\\u003c/h3\\u003e\\n\\u003cp\\u003eFunctional annotation of each predicted region was performed using Prokka and Bakta, followed by manual curation of mobile genetic element (MGE)\\u0026ndash;related genes. Phage hallmark genes (integrase, terminase large/small subunits, portal, capsid, tail, holin), regulatory genes (CI, Cro, LexA/Xre, AlpA), and lysis-associated genes were assessed to determine phage origin and structural completeness.\\u003c/p\\u003e\"},{\"header\":\"RESULTS\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003ePrediction and Classification of Prophage Regions in \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThree prophage prediction tools (PHASTEST, Phigaro, and PhageBoost) were applied to the complete genome of \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1 to obtain a comprehensive set of phage-associated regions (\\u003cstrong\\u003eFigure 1\\u003c/strong\\u003e). PhageBoost predicted nine regions, whereas PHASTEST and Phigaro detected three and two regions, respectively (\\u003cstrong\\u003eTable 1\\u003c/strong\\u003e). Because several predictions overlapped in genomic position, all outputs were consolidated into nine non-redundant regions distributed across the genome. Based on cross-tool comparison and structural integrity, these nine regions were classified into two intact prophages (IP1 and IP2), two cryptic prophages (CP1 and CP2), and five genomic islands (GI1 to GI5) (\\u003cstrong\\u003eTable 2\\u003c/strong\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003eIP1 was located at 589\\u0026ndash;632 kb, supported by overlapping predictions from PHASTEST Region 1, Phigaro Region 1, and PhageBoost Region 3. IP2 was located at 2.16\\u0026ndash;2.22 Mb, corresponding to PHASTEST Region 3, Phigaro Region 2, and PhageBoost Region 7.\\u003c/p\\u003e\\n\\u003cp\\u003eIn addition, CP1 (1.08\\u0026ndash;1.10 Mb) was supported by PHASTEST Region 2 and PhageBoost Region 5, whereas CP2 (approximately 2.99\\u0026ndash;3.01 Mb) was detected solely by PhageBoost Region 9.\\u003c/p\\u003e\\n\\u003cp\\u003eThe five PhageBoost-only predictions, located at 0.34\\u0026ndash;0.35 Mb, 0.36\\u0026ndash;0.37 Mb, 1.08\\u0026ndash;1.09 Mb, 1.80\\u0026ndash;1.82 Mb, and 2.82\\u0026ndash;2.83 Mb, lacked hallmark phage structural genes and were therefore classified as genomic islands (GI1 to GI5).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCharacterization of Intact Prophages in \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTwo intact prophages were identified based on the strong agreement among the three prediction tools and the complete preservation of hallmark structural and regulatory modules (\\u003cstrong\\u003eTable 2, Table 3\\u003c/strong\\u003e). These regions also contained accessory genes with potential roles in host interaction, genome maintenance, and phage\\u0026ndash;host communication, which further supports their classification as structurally intact prophages.\\u003c/p\\u003e\\n\\u003cp\\u003eThe IP1, located at 589 to 632 kb, harbored a full array of phage particle assembly genes, including the terminase large and small subunits, portal protein, major capsid and tail proteins, a tape measure protein, and holin. In addition, this region encoded transcriptional regulators such as CI, Cro, and LexA or Xre. The coexistence of these repressors indicates that IP1 may retain the capacity to modulate the lysogenic to lytic transition and to regulate prophage gene expression through SOS-associated transcriptional control (Erill et al., 2007; Quinones et al., 2005). This region was positioned adjacent to tRNA-Gly, tRNA-Leu, and tRNA-Asn, which is consistent with classical tRNA-mediated integration hotspots in temperate phages. Accessory genes were also identified, including members of the SGNH/GDSL hydrolase family. These enzymes are part of a broad superfamily of carbohydrate-modifying hydrolases known to remodel extracellular polysaccharides and cell-surface glycans (Akoh et al., 2004; Anderson et al., 2022). Their presence within IP1 suggests that this prophage region carries additional host-associated enzymes, although the specific functional relevance of these hydrolases in \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1 remains to be experimentally verified. Additional recombination-related elements, such as integrase and recT, further imply that IP1 may facilitate localized genome plasticity or recombination-based prophage maintenance (Lopes et al., 2010; Xue et al., 2023). Taken together, the structural, regulatory, and accessory gene composition of this locus indicates that IP1 retains the capacity for induction and may influence host physiology through both phage-related and phage-derived auxiliary functions.\\u003c/p\\u003e\\n\\u003cp\\u003eThe IP2 was identified between 2.16 and 2.22 Mb. This region represented the largest prophage-derived sequence in the genome and contained the complete structural module required for virion assembly. Key genes included the portal protein, terminase subunits, major and minor capsid proteins, head to tail connector proteins, tail fibers, and a tape measure protein. A holin gene was also present, together with multiple regulators such as CI, Cro, and LexA or Xre, which may coordinate prophage maintenance and control the transition into the lytic state. IP2 contained an extensive tRNA cluster, including tRNA-Trp, tRNA-Asn, tRNA-Ile, tRNA-Leu, tRNA-Met, and tRNA-Gly. The presence of these tRNA genes supports a tRNA-mediated integration mechanism (Tan et al., 2007) and may also contribute to translation optimization during phage gene expression (Kang et al., 2017). Accessory elements, such as DNA methyltransferase, type I site-specific DNase, 3-methyladenine DNA glycosylase, and SNF2- or DnaB-family replication proteins, were also present. These enzymes suggest that IP2 may carry the machinery required for replication initiation, recombination, epigenetic modification, and prophage genome protection (Murphy et al., 2013; Takahashi et al., 2002).\\u003c/p\\u003e\\n\\u003cp\\u003eBoth IP1 and IP2 display complete structural integrity and contain a combination of hallmark phage genes, transcriptional regulators, and accessory functional elements. These characteristics support their classification as intact prophages within the \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1 genome. Although previous studies reported that prophages in this strain were not induced by mitomycin C (Ventura et al., 2003), more recent findings indicate that even genetically complete prophages are not necessarily induced under this chemical stress (Ambros \\u0026amp; Ehrmann, 2022; Goerke et al., 2006). Considering evidence from \\u003cem\\u003eL. reuteri\\u003c/em\\u003e, where RecA and LexA dependent SOS responses were shown to regulate prophage gene expression, the presence of LexA or Xre regulators in IP1 and IP2 suggests that these prophages may remain in a transcriptionally silent state under standard laboratory conditions but may be activated under specific environmental or physiological stress (Caggianiello et al., 2016). Therefore, both regions can be considered intact prophages at the structural and regulatory levels, with the potential for induction in response to appropriate triggers.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFeatures of Cryptic Prophage Elements in \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTwo genomic regions were identified as cryptic prophages based on their preservation of phage-derived regulatory and recombination elements despite the complete loss of structural modules required for virion assembly. Both CP1 and CP2 lacked hallmark structural genes, including capsid, tail, terminase, portal, tape measure protein, holin, and endolysin, confirming that neither region can undergo a productive lytic cycle. However, each cryptic prophage retained distinct functional remnants that may contribute to host adaptation, transcriptional regulation, and genomic plasticity.\\u003c/p\\u003e\\n\\u003cp\\u003eThe first cryptic prophage, CP1, corresponds to PHASTEST Region 2 and PhageBoost Region 5 and is located at 1.08 to 1.10 Mb. CP1 retained several recombination-related enzymes, including insK, a catalytic integrase-domain protein, a XerC-family recombinase, and multiple transposase genes (\\u003cstrong\\u003eTable 4\\u003c/strong\\u003e). These elements suggest that CP1 may retain partial site-specific recombination activity and contribute to localized genome rearrangements, consistent with the roles of integrases, Xer recombinases, and insertion sequences in prophage-associated genome plasticity (Ramisetty \\u0026amp; Sudhakari, 2019; Touchon et al., 2016; Vandecraen et al., 2017). CP1 also carried a comprehensive set of exopolysaccharide biosynthesis genes, including \\u003cem\\u003emshA\\u003c/em\\u003e, \\u003cem\\u003ecps2G\\u003c/em\\u003e, \\u003cem\\u003ecpsC\\u003c/em\\u003e, and \\u003cem\\u003ecpsD\\u003c/em\\u003e. These genes govern cell surface polysaccharide production and may influence adhesion, colonization, and environmental stress tolerance in the host bacterium (Caggianiello et al., 2016; Lebeer et al., 2012). In addition, CP1 included a RelB\\u0026ndash;DinJ toxin\\u0026ndash;antitoxin system, which is associated with growth regulation, stress adaptation, and persistence phenotypes (Christensen et al., 2004). A tyrosine phosphatase gene (\\u003cem\\u003eywqE_1\\u003c/em\\u003e) was also identified. This enzyme is known to regulate carbohydrate metabolism and cell wall biosynthesis through protein dephosphorylation (Mijakovic et al., 2005). Together, these features support the interpretation that CP1, although structurally degraded, may contribute to cell envelope remodeling, metabolic regulation, and genomic flexibility as a domesticated cryptic prophage.\\u003c/p\\u003e\\n\\u003cp\\u003eThe second cryptic prophage, CP2, corresponds to PhageBoost Region 9 and is located at 2.99 to 3.01 Mb. CP2 preserved a \\u003cem\\u003exerC\\u003c/em\\u003e-type integrase and a CI-like transcriptional repressor, suggesting residual regulatory capacity or responsiveness to stress-associated signals (Bobay et al., 2013; Castillo et al., 2017). Fragmented phage replication components, including an SF3 helicase domain protein and DNA replication-associated proteins, were also identified. CP2 additionally encoded regulatory and membrane-associated factors, including \\u003cem\\u003earpU\\u003c/em\\u003e, which is linked to cell envelope regulation, and \\u003cem\\u003epucI\\u003c/em\\u003e, a cytosine permease that may participate in membrane transport or nucleotide salvage pathways. Several domesticated phage-derived remnants were present, such as a bacteriocin immunity protein, a DUF1398 domain protein, and an integron-associated gene cassette protein. The coexistence of integrase, regulatory proteins, and replication-associated fragments suggests that CP2 may retain \\u0026nbsp;partial transcriptional or stress-responsive functions despite the loss of its structural module.\\u003c/p\\u003e\\n\\u003cp\\u003eTaken together, CP1 and CP2 represent structurally degraded but functionally enriched cryptic prophages within the \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1 genome. Both regions have lost the ability to form virions, yet each preserves distinct combinations of recombination-related enzymes, regulatory factors, EPS biosynthesis pathways, toxin\\u0026ndash;antitoxin modules, membrane transporters, and replication remnants. These domesticated prophage elements may contribute to host stress adaptation, cell envelope modification, interbacterial interactions, and genomic plasticity, consistent with the roles of cryptic prophages described in other bacterial species.\\u003cstrong\\u003e\\u003cbr clear=\\\"all\\\"\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eOverview of Non-phage Genomic Island Regions in \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSeveral PhageBoost-specific regions lacked phage structural modules and were therefore interpreted as genomic islands rather than prophage remnants (\\u003cstrong\\u003eFigure 1, Table 4\\u003c/strong\\u003e). These regions formed five functionally distinct islands, designated GI1 through GI5, and their compositions revealed coordinated contributions to carbohydrate metabolism, ecological competition, genome maintenance, and chemical stress adaptation in \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1.\\u003c/p\\u003e\\n\\u003cp\\u003eA group of islands enriched in carbohydrate- and energy-associated genes consisted of GI1 and GI3.\\u003cbr\\u003eGI1 was primarily characterized by a complete glycerol-utilization system, including glpK, glpO, and glpF (Lin, 1976), indicating a dedicated metabolic module for glycerol uptake and catabolism. In addition to these core metabolic genes, GI1 encoded MucBP-domain\\u0026ndash;containing proteins and multiple membrane-associated proteins, suggesting a potential role in host interaction or cell-surface adaptation. The remaining genes in this island were dominated by DUF-containing, orphan, and hypothetical proteins, consistent with an accessory or niche-adaptive genomic island rather than a prophage-derived region.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eGI2 displayed a more complex functional architecture, combining metabolic, regulatory, and bacteriocin-associated modules within the same genomic island. This region encoded yidA, brnQ, and napA, which are associated with HAD-family phosphatase activity (Burroughs et al., 2006), branched-chain amino acid transport (Dutta et al., 2022), and ion homeostasis, respectively. Notably, GI2 also contained multiple transcriptional regulators, including the rhaS/rhaR pair, a T-box leader, and two agrA homologs, indicating tight regulatory control. In addition, an expanded plantaricin bacteriocin operon (plnA, plnB, plnJ, plnK, plnL, plnQ, sunS, and a bacteriocin immunity protein) was co-localized within this region, suggesting coordinated regulation of metabolic adaptation and ecological competition.\\u003c/p\\u003e\\n\\u003cp\\u003eGI3 harbored genes involved in rhamnose biosynthesis, including rfbA, rmlB, and rfbC, along with a cellulase-domain\\u0026ndash;containing protein. These genes are associated with surface polysaccharide synthesis and carbohydrate-active pathways commonly implicated in cell-envelope modification and environmental adaptation (Giraud \\u0026amp; Naismith, 2000; Raetz \\u0026amp; Whitfield, 2002).\\u003c/p\\u003e\\n\\u003cp\\u003eGenomic islands associated with DNA repair, regulatory turnover, and genome stability were found in GI4. This region encoded enzymes such as prmA and a putative 3-methyladenine DNA glycosylase, as well as multiple regulatory proteins including HTH Cro/C1-type proteins and DUF-containing elements. Also, GI4 region contained IS1182- and ISL3-family transposases. The presence of these mobile genetic elements is consistent with genomic regions that undergo insertion or rearrangement events, indicating that GI4 includes components typically associated with local genome mobility (Siguier et al., 2014).\\u003c/p\\u003e\\n\\u003cp\\u003eGI5 contained genes associated with bile acid metabolism, including baiE, along with glutamyl-tRNA reductase and several regulatory and DUF-containing proteins. The presence of \\u003cem\\u003ebaiE\\u003c/em\\u003e indicates that this region includes components linked to bile acid transformation. However, the functional relevance of the additional genes to bile tolerance or digestive-environment adaptation remains to be determined.\\u003c/p\\u003e\\n\\u003cp\\u003eBased on their gene content, GI1 through GI5 contain clusters associated with glycerol utilization, amino-acid transport, transcriptional regulation, bacteriocin operons, rhamnose biosynthesis, DNA-repair enzymes, mobile genetic elements, and bile acid\\u0026ndash;related functions. These genomic islands differ in composition from the cryptic prophages CP1 and CP2, which lack these metabolic and regulatory gene sets and instead retain recombination-related or phage-derived regulatory remnants.\\u003c/p\\u003e\"},{\"header\":\"DISCUSSION\",\"content\":\"\\u003cp\\u003eProphage research is crucial for understanding bacterial genome evolution, stress adaptation, and the genetic basis of industrial strain robustness, particularly for starter cultures used in food fermentation. In this study, we identified two cryptic prophage regions (CP1 and CP2) and two intact prophages in the genome of \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1, and we showed that CP1 and CP2 retain diverse recombination-, EPS-, and stress-related genes that are likely to contribute to host fitness rather than phage production. Although CP1 and CP2 lack the structural components required for phage particle formation, the presence of conserved functional remnants suggests that cryptic prophages in \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1 may still influence host physiology and genome evolution. Both regions encode integrase-family recombinases, with CP1 carrying \\u003cem\\u003einsK\\u003c/em\\u003e and CP2 encoding a \\u003cem\\u003exerC\\u003c/em\\u003e-type integrase (Tables\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e and \\u003cspan refid=\\\"Tab4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). The retention of these recombination enzymes implies a potential for site-specific recombination and localized genome plasticity, which is consistent with reports on cryptic prophages in other bacteria (Canchaya et al., 2003). In addition, CP2 encodes a CI-like transcriptional repressor, indicating that regulatory functions can be maintained even after structural genes required for virion assembly have been lost (Bobay et al., 2013; Touchon et al., 2016).\\u003c/p\\u003e \\u003cp\\u003eInsertion sequence elements and transposase-related genes are enriched within CP1 and its flanking regions, which is characteristic of mosaic genetic islands shaped through repeated insertion, deletion, and recombination (Siguier et al., 2014). Such genomic architectures are typical of prophage remnants that have undergone long-term domestication within bacterial chromosomes and are often associated with traits that enhance robustness in complex environments (Bobay et al., 2013; Touchon et al., 2016). This feature is particularly relevant for \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1, which is used as a starter culture in fermented foods and is required to withstand diverse stresses during fermentation and storage.\\u003c/p\\u003e \\u003cp\\u003eBeyond recombination-associated components, CP1 carries an extended set of EPS biosynthesis genes, including \\u003cem\\u003emshA\\u003c/em\\u003e and \\u003cem\\u003ecps2G\\u003c/em\\u003e, which are known to improve environmental resilience and surface-associated fitness in \\u003cem\\u003eLactobacillus\\u003c/em\\u003e species (Douillard et al., 2013). Prophage-derived EPS genes can be transcriptionally activated under nutrient limitation or stress, thereby enhancing survival and persistence under harsh conditions that resemble those encountered in animal-based fermented products, such as low pH, high salt, or oxidative stress (Douillard et al., 2013; Touchon et al., 2016). CP1 also encodes a RelB\\u0026ndash;DinJ toxin\\u0026ndash;antitoxin module and a tyrosine phosphatase homolog, both of which have been implicated in stress protection, growth regulation, and metabolic signaling in bacteria (Wang et al., 2010; Touchon et al., 2016). These features suggest that cryptic prophage remnants may contribute to the long-term viability and functional stability of \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1 during extended fermentation and chilled storage, not only through recombination, but also via metabolic and regulatory integration.\\u003c/p\\u003e \\u003cp\\u003eComparative genomics has shown that prophage regions in \\u003cem\\u003eLactobacillus\\u003c/em\\u003e can harbor antibiotic resistance genes, virulence-associated factors, and mobile elements, indicating a potential contribution to horizontal gene transfer and adaptive genome evolution (Douillard et al., 2013; Touchon et al., 2016). The genetic composition of CP1 and CP2, which retain domesticated phage-derived components capable of modulating gene expression or facilitating genomic rearrangements, is consistent with this view (Bobay et al., 2013; Touchon et al., 2016). In the context of starter culture application, such adaptive potential may support the long-term stability and performance of \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1 in dynamic meat or dairy matrices and under variable processing conditions, including temperature shifts, osmotic stress, and competition with other microorganisms.\\u003c/p\\u003e \\u003cp\\u003eTaken together, these functional signatures support the view that CP1 and CP2, although structurally incapable of producing phage particles, remain integrated genetic elements that may influence stress responses, persistence-like states, metabolic regulation, and genomic adaptability in \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1. From an applied perspective, cryptic prophage-derived traits in this strain may enhance robustness, survival, and technological reliability as a starter culture, contributing to consistent fermentation and extended shelf-life of livestock-derived fermented foods.\\u003c/p\\u003e \\u003cp\\u003eFuture studies should validate CP1 and CP2 functions through stress induction (mitomycin C, UV), excision PCR or qPCR, stress transcriptomics/proteomics, and CRISPR deletion during model fermentation (Otsuji et al., 1959; Lunde et al., 2003; Touchon et al., 2016; Wang et al., 2010). These approaches will clarify cryptic prophages' contributions to \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1's fermentation performance and storage stability.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (NRF-2020R1F1A1072397; RS-2023-00210305) for SYS. The authors declare no conflicts of interest.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study used publicly available data and did not involve human participants or live vertebrate animals. Therefore, ethical approval and informed consent were not required.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eAkoh, C. C., Lee, G. C., Liaw, Y. C., Huang, T. H., \\u0026amp; Shaw, J. F. (2004). GDSL family of serine esterases/lipases. \\u003cem\\u003eProg Lipid Res\\u003c/em\\u003e,\\u003cem\\u003e 43\\u003c/em\\u003e(6), 534-552. https://doi.org/10.1016/j.plipres.2004.09.002 \\u003c/li\\u003e\\n\\u003cli\\u003eAljohani, A., Rashwan, N., Vasani, S., Alkhawashki, A., Wu, T. T., Lu, X., Castillo, D. A., \\u0026amp; Xiao, J. (2025). The Health Benefits of Probiotic Lactiplantibacillus plantarum: A Systematic Review and Meta-Analysis. \\u003cem\\u003eProbiotics Antimicrob Proteins\\u003c/em\\u003e,\\u003cem\\u003e 17\\u003c/em\\u003e(5), 3358-3377. https://doi.org/10.1007/s12602-024-10287-3 \\u003c/li\\u003e\\n\\u003cli\\u003eAmbros, C. L., \\u0026amp; Ehrmann, M. A. (2022). Distribution, inducibility, and characterisation of prophages in Latilactobacillus sakei. \\u003cem\\u003eBMC Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 22\\u003c/em\\u003e(1), 267. https://doi.org/10.1186/s12866-022-02675-y \\u003c/li\\u003e\\n\\u003cli\\u003eAnderson, A. C., Stangherlin, S., Pimentel, K. N., Weadge, J. T., \\u0026amp; Clarke, A. J. (2022). The SGNH hydrolase family: a template for carbohydrate diversity. \\u003cem\\u003eGlycobiology\\u003c/em\\u003e,\\u003cem\\u003e 32\\u003c/em\\u003e(10), 826-848. https://doi.org/10.1093/glycob/cwac045 \\u003c/li\\u003e\\n\\u003cli\\u003eAyivi, R. D., \\u0026amp; Ibrahim, S. A. (2022). Lactic acid bacteria: an essential probiotic and starter culture for the production of yoghurt. \\u003cem\\u003eInternational Journal of Food Science and Technology\\u003c/em\\u003e,\\u003cem\\u003e 57\\u003c/em\\u003e(11), 7008-7025. https://doi.org/10.1111/ijfs.16076 \\u003c/li\\u003e\\n\\u003cli\\u003eBeamud, B., Benz, F., \\u0026amp; Bikard, D. (2024). Going viral: The role of mobile genetic elements in bacterial immunity. \\u003cem\\u003eCell Host Microbe\\u003c/em\\u003e,\\u003cem\\u003e 32\\u003c/em\\u003e(6), 804-819. https://doi.org/10.1016/j.chom.2024.05.017 \\u003c/li\\u003e\\n\\u003cli\\u003eBobay, L. M., Rocha, E. P., \\u0026amp; Touchon, M. (2013). The adaptation of temperate bacteriophages to their host genomes. \\u003cem\\u003eMol Biol Evol\\u003c/em\\u003e,\\u003cem\\u003e 30\\u003c/em\\u003e(4), 737-751. https://doi.org/10.1093/molbev/mss279 \\u003c/li\\u003e\\n\\u003cli\\u003eBr\\u0026uuml;ssow, H., Canchaya, C., \\u0026amp; Hardt, W. D. (2004). Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. \\u003cem\\u003eMicrobiol Mol Biol Rev\\u003c/em\\u003e,\\u003cem\\u003e 68\\u003c/em\\u003e(3), 560-602, table of contents. https://doi.org/10.1128/mmbr.68.3.560-602.2004 \\u003c/li\\u003e\\n\\u003cli\\u003eBurroughs, A. M., Allen, K. N., Dunaway-Mariano, D., \\u0026amp; Aravind, L. (2006). Evolutionary genomics of the HAD superfamily: understanding the structural adaptations and catalytic diversity in a superfamily of phosphoesterases and allied enzymes. \\u003cem\\u003eJ Mol Biol\\u003c/em\\u003e,\\u003cem\\u003e 361\\u003c/em\\u003e(5), 1003-1034. https://doi.org/10.1016/j.jmb.2006.06.049 \\u003c/li\\u003e\\n\\u003cli\\u003eCaggianiello, G., Kleerebezem, M., \\u0026amp; Spano, G. (2016). Exopolysaccharides produced by lactic acid bacteria: from health-promoting benefits to stress tolerance mechanisms. \\u003cem\\u003eAppl Microbiol Biotechnol\\u003c/em\\u003e,\\u003cem\\u003e 100\\u003c/em\\u003e(9), 3877-3886. https://doi.org/10.1007/s00253-016-7471-2 \\u003c/li\\u003e\\n\\u003cli\\u003eCanchaya, C., Fournous, G., Chibani-Chennoufi, S., Dillmann, M. L., \\u0026amp; Br\\u0026uuml;ssow, H. (2003). Phage as agents of lateral gene transfer. \\u003cem\\u003eCurr Opin Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 6\\u003c/em\\u003e(4), 417-424. https://doi.org/10.1016/s1369-5274(03)00086-9 \\u003c/li\\u003e\\n\\u003cli\\u003eCasjens, S. (2003). Prophages and bacterial genomics: what have we learned so far? \\u003cem\\u003eMol Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 49\\u003c/em\\u003e(2), 277-300. https://doi.org/10.1046/j.1365-2958.2003.03580.x \\u003c/li\\u003e\\n\\u003cli\\u003eCastillo, F., Benmohamed, A., \\u0026amp; Szatmari, G. (2017). Xer Site Specific Recombination: Double and Single Recombinase Systems. \\u003cem\\u003eFront Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 8\\u003c/em\\u003e, 453. https://doi.org/10.3389/fmicb.2017.00453 \\u003c/li\\u003e\\n\\u003cli\\u003eChristensen, S. K., Maenhaut‐Michel, G., Mine, N., Gottesman, S., Gerdes, K., \\u0026amp; Van Melderen, L. (2004). Overproduction of the Lon protease triggers inhibition of translation in Escherichia coli: involvement of the yefM‐yoeB toxin‐antitoxin system. \\u003cem\\u003eMolecular microbiology\\u003c/em\\u003e,\\u003cem\\u003e 51\\u003c/em\\u003e(6), 1705-1717. \\u003c/li\\u003e\\n\\u003cli\\u003eda Silva Duarte, V., Giaretta, S., Campanaro, S., Treu, L., Armani, A., Tarrah, A., Oliveira de Paula, S., Giacomini, A., \\u0026amp; Corich, V. (2018). A Cryptic Non-Inducible Prophage Confers Phage-Immunity on the Streptococcus thermophilus M17PTZA496. \\u003cem\\u003eViruses\\u003c/em\\u003e,\\u003cem\\u003e 11\\u003c/em\\u003e(1). https://doi.org/10.3390/v11010007 \\u003c/li\\u003e\\n\\u003cli\\u003eDouillard, F. P., Ribbera, A., J\\u0026auml;rvinen, H. M., Kant, R., Pietil\\u0026auml;, T. E., Randazzo, C., Paulin, L., Laine, P. K., Caggia, C., von Ossowski, I., Reunanen, J., Satokari, R., Salminen, S., Palva, A., \\u0026amp; de Vos, W. M. (2013). Comparative genomic and functional analysis of Lactobacillus casei and Lactobacillus rhamnosus strains marketed as probiotics. \\u003cem\\u003eAppl Environ Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 79\\u003c/em\\u003e(6), 1923-1933. https://doi.org/10.1128/aem.03467-12 \\u003c/li\\u003e\\n\\u003cli\\u003eDutta, S., Corsi, I. D., Bier, N., \\u0026amp; Koehler, T. M. (2022). BrnQ-Type Branched-Chain Amino Acid Transporters Influence Bacillus anthracis Growth and Virulence. \\u003cem\\u003emBio\\u003c/em\\u003e,\\u003cem\\u003e 13\\u003c/em\\u003e(1), e0364021. https://doi.org/10.1128/mbio.03640-21 \\u003c/li\\u003e\\n\\u003cli\\u003eErill, I., Campoy, S., \\u0026amp; Barb\\u0026eacute;, J. (2007). Aeons of distress: an evolutionary perspective on the bacterial SOS response. \\u003cem\\u003eFEMS Microbiol Rev\\u003c/em\\u003e,\\u003cem\\u003e 31\\u003c/em\\u003e(6), 637-656. https://doi.org/10.1111/j.1574-6976.2007.00082.x \\u003c/li\\u003e\\n\\u003cli\\u003eGiraud, M. F., \\u0026amp; Naismith, J. H. (2000). The rhamnose pathway. \\u003cem\\u003eCurr Opin Struct Biol\\u003c/em\\u003e,\\u003cem\\u003e 10\\u003c/em\\u003e(6), 687-696. https://doi.org/10.1016/s0959-440x(00)00145-7 \\u003c/li\\u003e\\n\\u003cli\\u003eGoerke, C., K\\u0026ouml;ller, J., \\u0026amp; Wolz, C. (2006). Ciprofloxacin and trimethoprim cause phage induction and virulence modulation in Staphylococcus aureus. \\u003cem\\u003eAntimicrob Agents Chemother\\u003c/em\\u003e,\\u003cem\\u003e 50\\u003c/em\\u003e(1), 171-177. https://doi.org/10.1128/aac.50.1.171-177.2006 \\u003c/li\\u003e\\n\\u003cli\\u003eHoward-Varona, C., Hargreaves, K. R., Abedon, S. T., \\u0026amp; Sullivan, M. B. (2017). Lysogeny in nature: mechanisms, impact and ecology of temperate phages. \\u003cem\\u003eThe ISME Journal\\u003c/em\\u003e,\\u003cem\\u003e 11\\u003c/em\\u003e(7), 1511-1520. https://doi.org/10.1038/ismej.2017.16 \\u003c/li\\u003e\\n\\u003cli\\u003eHu, J., Ye, H., Wang, S., Wang, J., \\u0026amp; Han, D. (2021). Prophage Activation in the Intestine: Insights Into Functions and Possible Applications. \\u003cem\\u003eFront Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 12\\u003c/em\\u003e, 785634. https://doi.org/10.3389/fmicb.2021.785634 \\u003c/li\\u003e\\n\\u003cli\\u003eKaiser, A. D., \\u0026amp; Jacob, F. (1957). Recombination between related temperate bacteriophages and the genetic control of immunity and prophage localization. \\u003cem\\u003eVirology\\u003c/em\\u003e,\\u003cem\\u003e 4\\u003c/em\\u003e(3), 509-521. https://doi.org/https://doi.org/10.1016/0042-6822(57)90083-1 \\u003c/li\\u003e\\n\\u003cli\\u003eKang, H. S., McNair, K., Cuevas, D. A., Bailey, B. A., Segall, A. M., \\u0026amp; Edwards, R. A. (2017). Prophage genomics reveals patterns in phage genome organization and replication. \\u003cem\\u003ebioRxiv\\u003c/em\\u003e, 114819. https://doi.org/10.1101/114819 \\u003c/li\\u003e\\n\\u003cli\\u003eKim, S. H., \\u0026amp; Park, J. H. (2022). Characterization of Prophages in Leuconostoc Derived from Kimchi and Genomic Analysis of the Induced Prophage in Leuconostoc lactis. \\u003cem\\u003eJ Microbiol Biotechnol\\u003c/em\\u003e,\\u003cem\\u003e 32\\u003c/em\\u003e(3), 333-340. https://doi.org/10.4014/jmb.2110.10046 \\u003c/li\\u003e\\n\\u003cli\\u003eLavelle, K., McDonnell, B., Fitzgerald, G., van Sinderen, D., \\u0026amp; Mahony, J. (2023). Bacteriophage-host interactions in Streptococcus thermophilus and their impact on co-evolutionary processes. \\u003cem\\u003eFEMS Microbiol Rev\\u003c/em\\u003e,\\u003cem\\u003e 47\\u003c/em\\u003e(4). https://doi.org/10.1093/femsre/fuad032 \\u003c/li\\u003e\\n\\u003cli\\u003eLebeer, S., Claes, I., Tytgat, H. L., Verhoeven, T. L., Marien, E., von Ossowski, I., Reunanen, J., Palva, A., Vos, W. M., Keersmaecker, S. C., \\u0026amp; Vanderleyden, J. (2012). Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells. \\u003cem\\u003eAppl Environ Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 78\\u003c/em\\u003e(1), 185-193. https://doi.org/10.1128/aem.06192-11 \\u003c/li\\u003e\\n\\u003cli\\u003eLin, E. C. (1976). Glycerol dissimilation and its regulation in bacteria. \\u003cem\\u003eAnnu Rev Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 30\\u003c/em\\u003e, 535-578. https://doi.org/10.1146/annurev.mi.30.100176.002535 \\u003c/li\\u003e\\n\\u003cli\\u003eLittle, J. W., \\u0026amp; Mount, D. W. (1982). The SOS regulatory system of Escherichia coli. \\u003cem\\u003eCell\\u003c/em\\u003e,\\u003cem\\u003e 29\\u003c/em\\u003e(1), 11-22. https://doi.org/10.1016/0092-8674(82)90085-x \\u003c/li\\u003e\\n\\u003cli\\u003eLopes, A., Amarir-Bouhram, J., Faure, G., Petit, M.-A., \\u0026amp; Guerois, R. (2010). Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs. \\u003cem\\u003eNucleic Acids Research\\u003c/em\\u003e,\\u003cem\\u003e 38\\u003c/em\\u003e(12), 3952-3962. https://doi.org/10.1093/nar/gkq096 \\u003c/li\\u003e\\n\\u003cli\\u003eLunde, M., Blatny, J. M., Lillehaug, D., Aastveit, A. H., \\u0026amp; Nes, I. F. (2003). Use of real-time quantitative PCR for the analysis of phiLC3 prophage stability in lactococci. \\u003cem\\u003eAppl Environ Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 69\\u003c/em\\u003e(1), 41-48. https://doi.org/10.1128/aem.69.1.41-48.2003 \\u003c/li\\u003e\\n\\u003cli\\u003eMijakovic, I., Musumeci, L., Tautz, L., Petranovic, D., Edwards, R. A., Jensen, P. R., Mustelin, T., Deutscher, J., \\u0026amp; Bottini, N. (2005). In vitro characterization of the Bacillus subtilis protein tyrosine phosphatase YwqE. \\u003cem\\u003eJ Bacteriol\\u003c/em\\u003e,\\u003cem\\u003e 187\\u003c/em\\u003e(10), 3384-3390. https://doi.org/10.1128/jb.187.10.3384-3390.2005 \\u003c/li\\u003e\\n\\u003cli\\u003eMurphy, J., Mahony, J., Ainsworth, S., Nauta, A., \\u0026amp; van Sinderen, D. (2013). Bacteriophage orphan DNA methyltransferases: insights from their bacterial origin, function, and occurrence. \\u003cem\\u003eAppl Environ Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 79\\u003c/em\\u003e(24), 7547-7555. https://doi.org/10.1128/aem.02229-13 \\u003c/li\\u003e\\n\\u003cli\\u003eOtsuji, N., Sekiguchi, M., Iijima, T., \\u0026amp; Takagi, Y. (1959). Induction of Phage Formation in the Lysogenic Escherichia coli K-12 by Mitomycin C. \\u003cem\\u003eNature\\u003c/em\\u003e,\\u003cem\\u003e 184\\u003c/em\\u003e(4692), 1079-1080. https://doi.org/10.1038/1841079b0 \\u003c/li\\u003e\\n\\u003cli\\u003ePei, Z., Sadiq, F. A., Han, X., Zhao, J., Zhang, H., Ross, R. P., Lu, W., \\u0026amp; Chen, W. (2021). Comprehensive Scanning of Prophages in Lactobacillus: Distribution, Diversity, Antibiotic Resistance Genes, and Linkages with CRISPR-Cas Systems. \\u003cem\\u003emSystems\\u003c/em\\u003e,\\u003cem\\u003e 6\\u003c/em\\u003e(3), e0121120. https://doi.org/10.1128/mSystems.01211-20 \\u003c/li\\u003e\\n\\u003cli\\u003eQuinones, M., Kimsey, H. H., \\u0026amp; Waldor, M. K. (2005). LexA Cleavage Is Required for CTX Prophage Induction. \\u003cem\\u003eMolecular Cell\\u003c/em\\u003e,\\u003cem\\u003e 17\\u003c/em\\u003e(2), 291-300. https://doi.org/10.1016/j.molcel.2004.11.046 \\u003c/li\\u003e\\n\\u003cli\\u003eRaetz, C. R., \\u0026amp; Whitfield, C. (2002). Lipopolysaccharide endotoxins. \\u003cem\\u003eAnnu Rev Biochem\\u003c/em\\u003e,\\u003cem\\u003e 71\\u003c/em\\u003e, 635-700. https://doi.org/10.1146/annurev.biochem.71.110601.135414 \\u003c/li\\u003e\\n\\u003cli\\u003eRamisetty, B. C. M., \\u0026amp; Sudhakari, P. A. (2019). Bacterial \\u0026apos;Grounded\\u0026apos; Prophages: Hotspots for Genetic Renovation and Innovation. \\u003cem\\u003eFront Genet\\u003c/em\\u003e,\\u003cem\\u003e 10\\u003c/em\\u003e, 65. https://doi.org/10.3389/fgene.2019.00065 \\u003c/li\\u003e\\n\\u003cli\\u003eSiguier, P., Gourbeyre, E., \\u0026amp; Chandler, M. (2014). Bacterial insertion sequences: their genomic impact and diversity. \\u003cem\\u003eFEMS Microbiol Rev\\u003c/em\\u003e,\\u003cem\\u003e 38\\u003c/em\\u003e(5), 865-891. https://doi.org/10.1111/1574-6976.12067 \\u003c/li\\u003e\\n\\u003cli\\u003eSir\\u0026eacute;n, K., Millard, A., Petersen, B., Gilbert, M Thomas P., Clokie, M. R. J., \\u0026amp; Sicheritz-Pont\\u0026eacute;n, T. (2021). Rapid discovery of novel prophages using biological feature engineering and machine learning. \\u003cem\\u003eNAR Genomics and Bioinformatics\\u003c/em\\u003e,\\u003cem\\u003e 3\\u003c/em\\u003e(1). https://doi.org/10.1093/nargab/lqaa109 \\u003c/li\\u003e\\n\\u003cli\\u003eSong, S., Guo, Y., Kim, J.-S., Wang, X., \\u0026amp; Wood, T. K. (2019). Phages Mediate Bacterial Self-Recognition. \\u003cem\\u003eCell Reports\\u003c/em\\u003e,\\u003cem\\u003e 27\\u003c/em\\u003e(3), 737-749.e734. https://doi.org/10.1016/j.celrep.2019.03.070 \\u003c/li\\u003e\\n\\u003cli\\u003eSong, S., Kim, J. S., Yamasaki, R., Oh, S., Benedik, M. J., \\u0026amp; Wood, T. K. (2021). Escherichia coli cryptic prophages sense nutrients to influence persister cell resuscitation. \\u003cem\\u003eEnviron Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 23\\u003c/em\\u003e(11), 7245-7254. https://doi.org/10.1111/1462-2920.15816 \\u003c/li\\u003e\\n\\u003cli\\u003eStarikova, E. V., Tikhonova, P. O., Prianichnikov, N. A., Rands, C. M., Zdobnov, E. M., Ilina, E. N., \\u0026amp; Govorun, V. M. (2020). Phigaro: high-throughput prophage sequence annotation. \\u003cem\\u003eBioinformatics\\u003c/em\\u003e,\\u003cem\\u003e 36\\u003c/em\\u003e(12), 3882-3884. https://doi.org/10.1093/bioinformatics/btaa250 \\u003c/li\\u003e\\n\\u003cli\\u003eTakahashi, N., Naito, Y., Handa, N., \\u0026amp; Kobayashi, I. (2002). A DNA methyltransferase can protect the genome from postdisturbance attack by a restriction-modification gene complex. \\u003cem\\u003eJ Bacteriol\\u003c/em\\u003e,\\u003cem\\u003e 184\\u003c/em\\u003e(22), 6100-6108. https://doi.org/10.1128/jb.184.22.6100-6108.2002 \\u003c/li\\u003e\\n\\u003cli\\u003eTan, Y., Zhang, K., Rao, X., Jin, X., Huang, J., Zhu, J., Chen, Z., Hu, X., Shen, X., Wang, L., \\u0026amp; Hu, F. (2007). Whole genome sequencing of a novel temperate bacteriophage of P. aeruginosa: evidence of tRNA gene mediating integration of the phage genome into the host bacterial chromosome. \\u003cem\\u003eCell Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 9\\u003c/em\\u003e(2), 479-491. https://doi.org/10.1111/j.1462-5822.2006.00804.x \\u003c/li\\u003e\\n\\u003cli\\u003eTouchon, M., Bernheim, A., \\u0026amp; Rocha, E. P. (2016). Genetic and life-history traits associated with the distribution of prophages in bacteria. \\u003cem\\u003eIsme j\\u003c/em\\u003e,\\u003cem\\u003e 10\\u003c/em\\u003e(11), 2744-2754. https://doi.org/10.1038/ismej.2016.47 \\u003c/li\\u003e\\n\\u003cli\\u003eVandecraen, J., Chandler, M., Aertsen, A., \\u0026amp; Van Houdt, R. (2017). The impact of insertion sequences on bacterial genome plasticity and adaptability. \\u003cem\\u003eCrit Rev Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 43\\u003c/em\\u003e(6), 709-730. https://doi.org/10.1080/1040841x.2017.1303661 \\u003c/li\\u003e\\n\\u003cli\\u003eVentura, M., Canchaya, C., Kleerebezem, M., de Vos, W. M., Siezen, R. J., \\u0026amp; Br\\u0026uuml;ssow, H. (2003). The prophage sequences of Lactobacillus plantarum strain WCFS1. \\u003cem\\u003eVirology\\u003c/em\\u003e,\\u003cem\\u003e 316\\u003c/em\\u003e(2), 245-255. https://doi.org/10.1016/j.virol.2003.08.019 \\u003c/li\\u003e\\n\\u003cli\\u003eVentura, M., Lee, J. H., Canchaya, C., Zink, R., Leahy, S., Moreno-Munoz, J. A., O\\u0026apos;Connell-Motherway, M., Higgins, D., Fitzgerald, G. F., O\\u0026apos;Sullivan, D. J., \\u0026amp; van Sinderen, D. (2005). Prophage-like elements in bifidobacteria: insights from genomics, transcription, integration, distribution, and phylogenetic analysis. \\u003cem\\u003eAppl Environ Microbiol\\u003c/em\\u003e,\\u003cem\\u003e 71\\u003c/em\\u003e(12), 8692-8705. https://doi.org/10.1128/aem.71.12.8692-8705.2005 \\u003c/li\\u003e\\n\\u003cli\\u003eWang, X., Kim, Y., Ma, Q., Hong, S. H., Pokusaeva, K., Sturino, J. M., \\u0026amp; Wood, T. K. (2010). Cryptic prophages help bacteria cope with adverse environments. \\u003cem\\u003eNat Commun\\u003c/em\\u003e,\\u003cem\\u003e 1\\u003c/em\\u003e, 147. https://doi.org/10.1038/ncomms1146 \\u003c/li\\u003e\\n\\u003cli\\u003eWishart, D. S., Han, S., Saha, S., Oler, E., Peters, H., Grant, J. R., Stothard, P., \\u0026amp; Gautam, V. (2023). PHASTEST: faster than PHASTER, better than PHAST. \\u003cem\\u003eNucleic Acids Res\\u003c/em\\u003e,\\u003cem\\u003e 51\\u003c/em\\u003e(W1), W443-w450. https://doi.org/10.1093/nar/gkad382 \\u003c/li\\u003e\\n\\u003cli\\u003eWiull, K., Haugen Lisa, K., Eijsink Vincent, G. H., \\u0026amp; Mathiesen, G. (2025). CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1. \\u003cem\\u003eMicrobiology Spectrum\\u003c/em\\u003e,\\u003cem\\u003e 13\\u003c/em\\u003e(2), e02025-02024. https://doi.org/10.1128/spectrum.02025-24 \\u003c/li\\u003e\\n\\u003cli\\u003eXue, F., Ma, X., Luo, C., Li, D., Shi, G., \\u0026amp; Li, Y. (2023). Construction of a bacteriophage-derived recombinase system in Bacillus licheniformis for gene deletion. \\u003cem\\u003eAMB Express\\u003c/em\\u003e,\\u003cem\\u003e 13\\u003c/em\\u003e(1), 89. https://doi.org/10.1186/s13568-023-01589-w \\u003c/li\\u003e\\n\\u003cli\\u003eZhang, Z., Niu, H., Qu, Q., Guo, D., Wan, X., Yang, Q., Mo, Z., Tan, S., Xiang, Q., Tian, X., Yang, H., \\u0026amp; Liu, Z. (2025). Advancements in Lactiplantibacillus plantarum: probiotic characteristics, gene editing technologies and applications. \\u003cem\\u003eCrit Rev Food Sci Nutr\\u003c/em\\u003e,\\u003cem\\u003e 65\\u003c/em\\u003e(29), 6623-6644. https://doi.org/10.1080/10408398.2024.2448562 \\u003c/li\\u003e\\n\\u003cli\\u003eZhou, Y., Liang, Y., Lynch, K. H., Dennis, J. J., \\u0026amp; Wishart, D. S. (2011). PHAST: a fast phage search tool. \\u003cem\\u003eNucleic Acids Res\\u003c/em\\u003e,\\u003cem\\u003e 39\\u003c/em\\u003e(Web Server issue), W347-352. https://doi.org/10.1093/nar/gkr485 \\u003c/li\\u003e\\n\\u003c/ol\\u003e\"},{\"header\":\"Tables\",\"content\":\"\\u003cp\\u003eTables 1 to 5 are available in the supplementary files section\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"food-science-of-animal-resources\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"Learn more about [Food Science of Animal Resources](https://link.springer.com/journal/44463)\",\"snPcode\":\"44463\",\"submissionUrl\":\"https://submission.springernature.com/new-submission/44463/3?\",\"title\":\"Food Science of Animal Resources\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Open\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Lactiplantibacillus plantarum WCFS1, prophage, cryptic prophage, starter culture, stress tolerance\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8937826/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8937826/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eProphages are widespread in lactic acid bacteria (LAB) and drive genome evolution and stress adaptation, making their analysis crucial for developing robust starter cultures in fermented food production. Here, we conducted comprehensive prophage analysis of the fully sequenced \\u003cem\\u003eLactiplantibacillus plantarum\\u003c/em\\u003e WCFS1 genome using three complementary prediction tools: PHASTEST, Phigaro, and PhageBoost. Cross-tool integration identified nine non-redundant prophage-associated regions, classified as two intact prophages (IP1, IP2), two cryptic prophages (CP1, CP2), and five genomic islands (GI1–GI5).\\u003cstrong\\u003e \\u003c/strong\\u003eIntact prophages contained complete structural and regulatory modules, while cryptic prophages lacked structural genes but retained recombinases, CI-like repressors, toxin-antitoxin systems, and exopolysaccharide (EPS) biosynthetic genes. Genomic islands were enriched in carbohydrate metabolism, bacteriocin, bile acid transformation, and DNA repair genes. This functional gene composition, consistent with cryptic prophage studies in other species, reveals that cryptic prophages contribute to \\u003cem\\u003eL. plantarum\\u003c/em\\u003e WCFS1's stress tolerance and genome plasticity, offering targets for strain engineering to enhance fermentation stability and shelf-life in livestock-derived fermented foods.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Identification and Characterization of Putative Functional Cryptic Prophages in the Starter Strain Lactiplantibacillus plantarum WCFS1\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-05-08 13:49:49\",\"doi\":\"10.21203/rs.3.rs-8937826/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-11T01:07:06+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"110770702998803309346245233810199206157\",\"date\":\"2026-04-30T04:39:41+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"83423304460224079974878823477697075226\",\"date\":\"2026-04-30T02:12:24+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-04-30T02:11:43+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-02-25T07:32:57+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-02-25T01:43:56+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Food Science of Animal Resources\",\"date\":\"2026-02-22T08:11:47+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"food-science-of-animal-resources\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"Learn more about [Food Science of Animal Resources](https://link.springer.com/journal/44463)\",\"snPcode\":\"44463\",\"submissionUrl\":\"https://submission.springernature.com/new-submission/44463/3?\",\"title\":\"Food Science of Animal Resources\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Open\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"3f9bf8fa-36ec-45a2-9855-40651136af3c\",\"owner\":[],\"postedDate\":\"May 8th, 2026\",\"published\":true,\"recentEditorialEvents\":[{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-11T01:07:06+00:00\",\"index\":12,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"110770702998803309346245233810199206157\",\"date\":\"2026-04-30T04:39:41+00:00\",\"index\":11,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"83423304460224079974878823477697075226\",\"date\":\"2026-04-30T02:12:24+00:00\",\"index\":10,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"2\",\"date\":\"2026-04-30T02:11:43+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-05-08T13:49:49+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-05-08 13:49:49\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8937826\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8937826\",\"identity\":\"rs-8937826\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}