Genomic analysis revealed adaptive mechanism to plant-related fermentation of Lactobacillus plantarum NCU116 and Lactobacillus spp.

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Original Article

Genomic analysis revealed adaptive mechanism to plant-related fermentation of Lactobacillus plantarum NCU116 and Lactobacillus spp. Tao Huanga,b, Tao Xionga,b, Zhen Penga,b, Yang-sheng Xiaoa,b, Zhang-gen Liua,b, Min Hua,b, ⁎ Ming-yong Xiea,b, a b

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China College of Lifee Science & Food Engineering, Nanchang University, eNo. 235 Nanjing East Road, Nanchang, Jiangxi 330047, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Comparative genomics Lactobacillus plantarum NCU116 Adaptive mechanism Plantaricin

Lactobacillus plantarum NCU116 is the first sequenced strain derived from traditional Chinese sauerkraut (TCS). Since NCU116 manifested outstanding probiotic effects in vitro and in vivo, it is crucial to comprehend a clear genetic background for NCU116. Functional re-annotation and comparative analysis were performed to excavate the unique and representative genes in NCU116, in order to investigate its metabolic preference and adaptive mechanism. Horizontal gene transfer (HGT) seemed to occur frequently, which endows NCU116 with a strong ability to transport carbohydrates, as a strain-specific fructose/mannose-PTS was identified, and opu and osmC coding genes were retrieved as NCU116-specific. In addition, a strain-specific type I R/M system and several prophage loci were found in NCU116, which could play vital roles in self-defense mechanism. Pathways of bacterial metabolism on plant-related substrates fermentation were then generated by reconstruction of associated pathways. Moreover, a unique potential plantaricin-producing locus with high homology to that of JDM1 was defined in the genome of NCU116, which could be very important for the preservation of fermented-food. Our results would provide critical basis for the application of NCU116 in food and pharmaceuticals industries.

1. Introduction Lactic acid bacteria (LAB) are gram-positive bacteria that mainly produce lactic acid during fermentation [1], and are commonly found in naturally fermented foods including sauerkraut, beer, wine, juices, etc. Several species have been grouped into “Generally Regarded As Safe” (GRAS) by the Food and Drug Administration (FDA) [2,3] which are closely associated with humans and animals (oral cavity, intestine and vagina) [4–6]. Various specific LAB strains have been verified as “probiotics” which benefit human health. For instance, L. acidophilus NCFM was found to relieve intestinal inflammation [7]. L. rhamnosus GG was associated with decrease risk of necrotizing enterocolitis in infants [8]. Immunomodulatory potential of L. casei Zhang was studied through peanut allergy murine model [9]. As an important branch, L. plantarum is a widespread member of LAB, primarily associated with fermented-foods and acts as commensal bacterium in human intestine [4,6,10]. Generally, LAB play a crucial role in the fermentation process especially in traditional Chinese sauerkraut (TCS). A previous study reported that heterofermentative LAB (L. mesenteroides) initiated the fermentation process, yielding carbon dioxide to create an oxygen-depletion condition, and subsequently homo-fermenters (mainly L. ⁎

plantarum, L. lactis, L. casei and E. faecalis) intervened and dominated the maturation of TCS [11]. As an efficient carbohydrates converter, L. plantarum possesses both homo- and heterolactic fermentation pathways and contributes the most to the fermentation of TCS [12]. Various fermented food-derived LAB have been well characterized to better understand their roles in the corresponding fermentation process. Comparative analysis of genome sequence can be an effective way to elucidate the adaption to specific environments, which has been performed for L. kefiranofaciens ZW3 and L. mucosae LM1 [13,14]. As the first sequenced strain from TCS, L. plantarum NCU116 was isolated on account of in vitro studies including the potential of survival in simulated gastric fluid and tolerance to bile salt [15], as well as in vivo studies, including cholesterol-lowering effect [16], constipation alleviation effect [17] and modulation effect on immune system and intestinal microbiota [18,19]. NCU116 has been used as Directed Vat Set (DVS) starter for vegetables and fruits fermentation for nearly ten years because of its excellent fermentation performance, which makes NCU116 a unique and immensely valuable strain. Thus, in the study, reannotation was performed to obtain more detailed and accurate genetic information about NCU116, meanwhile, comparative analysis with those of seven Lactobacillus strains was performed to explore

Corresponding author at: State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China. E-mail address: [email protected] (M.-y. Xie).

https://doi.org/10.1016/j.ygeno.2019.05.004 Received 10 December 2018; Received in revised form 23 March 2019; Accepted 8 May 2019 0888-7543/ © 2019 Published by Elsevier Inc.

Please cite this article as: Tao Huang, et al., Genomics, https://doi.org/10.1016/j.ygeno.2019.05.004

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core genome by using EDGAR software. According to the NeighborJoining phylogenetic tree, NCU116 was grouped into the species of L. plantarum, and the closetst strain was L. pentosus KCA1 as they share a branch node in the tree. The species of L. plantarum almost formed a single, monophyletic branch excepting L. pentosus KCA1. Thus, four strains from the species of L. plantarum (WCFS1, ST-III, JDM1 and 5-2) and three strains from the close species (L. brevis ATCC367, L. buchneri CD034 and L. reuteri DSM20016) were selected for the comparative analysis (Table 1). WCFS1 was isolated from human saliva, which is the first sequenced L. plantarum spp. strain and is most frequently used as a reference genome. ST-III was isolated from kimchi and has been used commercially. JDM1 was a Chinese commercial LAB, while 5-2 was isolated from fermented soybean and used as a probiotic LAB. The other three LABs used in the present study were all isolated from fermented foods. The genome of NCU116 is the largest, thus more coding sequences were predicted, inferring richer functionality. The G + C contents of these eight genomes were moderated, thus all selected strains were compatible for comparative analysis.

evolutional diversity at the genome level. This study will benefit the fundamental research and industrial application of NCU116. 2. Results and discussion 2.1. General genome features of L. plantarum NCU116 In the present study, the complete genome of NCU116 comprising one circular chromosome and three plasmids were re-annotated using SEED-based RAST server [20], which are composed of 3,245,809 bp, 11,618 bp, 48,567 bp and 48,695 bp, respectively (Fig. S1A). By manual curation in the re-annotation project, a total of 3396 genes were predicted in contrast to 3252 genes in a previous study (unpublished data). Despite 810 genes remain to be defined, 2586 genes were annotated with biological functions, which were classified into 30 functional categories according to RAST-subclass. (Fig. S1B). 2.2. Overview of functional genes in L. plantarum NCU116

3.2. Collinearity analysis

Genes involved in carbohydrates metabolism and membrane transport were the most abundant (408 and 403, respectively), indicating the great ability of NCU116 in sugar transport and metabolism, which is consistent with its physiological property. In addition, 11 genes involved in glycoside hydrolase family 13 (GH13) [21,22] were more explicitly annotated, including one α-amylase (L.p_NCU116_956), one trehalose-6-phosphate hydrolase (L.p_NCU116_901), one glucan 1,6-αglucosidase (L.p_NCU116_1174), two neopullulanase (L.p_NCU116_1110 and L.p_NCU116_1928), two α-glucosidases (L.p_NCU116_956 and L.p_NCU116_1273), and four oligo-1,6-glucosidases (L.p_NCU116_959, L.p_NCU116_974, L.p_NCU116_1187 and L.p_NCU116_2263), which were also known as the α-amylase family. These genes enable NCU116 to decompose macromolecules, such as starch in carrot or other vegetables. Such genes were presumably obtained from Lactococcus lactis through horizontal gene transfer (HGT), as they were commensal bacteria in TCS and shared high homology. A high number of genes (228 genes) were grouped into cell wall and capsule, of which 48 genes were found to encode cell capsular and extracellular polysaccharides, and 21 encode necessary nan cluster in sialic acid metabolism [23]. Genes involved in nan cluster, such as one nanA (N-acetylneuraminate lyase, L.p_NCU116_1241), one nanE (N-acetylmannosamine-6-phosphate 2epimerase, L.p_NCU116_1240) and several nanK (N-acetylmannosamine kinase, L.p_NCU116_745, L.p_NCU116_1184, L.p_NCU116_1242 and L.p_NCU116_1752) are deemed to be helpful in adaption to surroundings in the hosts, which was firstly reported in L. sakei 23 K [24]. Sialic acid is a kind of natural nine-carbon amino sugars, commonly found in vertebrates but erratically distributed in bacteria, and its related genes were repoeted to be obtained during interaction with animal cells, especially for bacteria colonized in the intestines of vertebrates [25]. Thus, it's speculated that NCU116 may live in the gut of human or animal before it was isolated from TCS. Besides, numerous protein and amino acid metabolism encoding genes (220 and 158, respectively) were retrieved in the genome of NCU116. Twenty different kinds of aminoacyl-tRNA synthetase were found, enabling NCU116 to take in organic nitrogen source when necessary. Moreover, large numbers of genes (115) associated with stress-responses, including 8 osmotic-regulators and 10 choline/betaine transporters (Table S1). In summary, more abundant genetic information was obtained by the re-annotation of NCU116, which may be very helpful for targeted study.

Synteny of the chromosomes in LABs used in this study were determined, with NCU116 as a reference (Fig. S3A). Dots in the synteny plot denoted physical co-localization against the reference sequence. Species from L. plantarum showed overall conservation because few rearrangements were found, while ATCC367, CD034 and DSM20016 showed less collinearity against NCU116 according to the scattered dots. These results are consistent with the results of phylogenetic tree. Although the evolution distance between NCU116 and the other three strains (not belonging to L. plantarum spp.) were relatively large, considerable amounts of collinear orthologous genes were identified, suggesting the conservation between different species. Furthermore, rearrangements were observed through the intrinsically diagonals when compared within L. plantarum spp. (in the lower left and upper right of the plots). Results from analysis of full chromosome alignments by progressiveMAUVE (Fig. 1B) supported the rearrangements. The embedded local collinear blocks (LCBs) with different colors showed an overall conservation among different L. plantarum strains as they were mostly linked up with each other. Intersections of connection lines indicated the more intuitive rearrangements in different strains, which agreed with the findings from synteny plots mentioned above. More remarkably, regions without LCB outlines were considered to be strainspecific, because no collinearity was found between them. Thereby we notice a large blanked region (from 3,245,807 bp to the end) located at the end of the NCU116 genome (Fig. 1A), and this strain-specific region (see detailed in Core-genes) happened to cover a fraction of chromosome and the whole region of three plasmids. Such strain-specific genes were proposed to be attributed to HGT evocable gene acquisition events. Besides, when the NCU116 genome was removed in the MAUVE-comparison, LCBs between different strains increased with the decrease of connecting lines. Restructuration occurred during evolution, indicating the plasticity of genome of NCU116. In addition, average nucleoside identity (ANI) and average amino acid identity (AAI) were determined. From phylogenetic perspective, ANI and AAI are indispensable approaches to taxonomy for microbial organisms [26]. In view of the need for biological classification in this study, AAI could be a more appropriate method in microbial taxonomy as amino acid sequences are more conserved when compare to nucleoside sequences among close strains. Therefore, AAI was calculated for every genome pair of the eight strains, and results were shown by a heat map presented as a matrix (Fig. S4A). The values of AAI in five L. plantarum were around 99.5%, suggesting the highly close evolutionary relationship. And yet, as expected, AAI values of three strains (not belonging to L. plantarum spp.) were approximately 61–67%, indicating large difference of amino acid and big genetic distance. Results showed that WCFS1 was the closest strain to NCU116, proved by both AAI and

3. Phylogenetic analysis of L. plantarum NCU116 and Lactobacillus strains 3.1. Whole-genome phylogenetic analysis Forty different species of LAB which have been completely sequenced were used to construct a phylogenetic tree (Fig. S2), based on 2

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was constructed for a better understanding of the five L. plantarum strains, pan-genome (3,690) and core-genes (2,409) were generated, and the unique genes were identified to shed light of the similarities and differences between NCU116 and other strains. Core-genes refers to the essential genes which are highly conserved within L. plantarum spp., and are involved in cell cycle, carbohydrate and protein metabolism, etc. WCFS1 and NCU116 seemed to harbour more luxuriant genome because the numbers of unique genes were high (216 and 171 genes, respectively). Most of the unique genes from NCU116 were hypothetical proteins, but still, quite a few genes (97) were annotated. Thirtynine of the annotated genes were authenticated in the three plasmids of

ANI (Fig. S4B). In brief, findings in phylogenetic analysis of NCU116 were reciprocally authenticated for its evolutionary status. 3.3. Core-genes and pan-genome Next, core- and pan-genome were performed for five (only L. plantrum strains) and eight (all strains used in this study) LAB strains. A set of orthologous genes (2,409) represent the highly conserved gene family among L. plantarum spp., and the number decreased to 769 while the comparison extended to eight genomes (Fig. S3B), which described the diversity among them at the genome level. A venn diagram (Fig. 2)

Fig. 1. MAUVE alignment of genomes from different L. plantarum strains. Prophage elements were identified as P1-P5, the black boxes represent intact prophage, while the red and blue boxes represent incomplete and questionable prophage. NCU116 was set as reference. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 3

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L.p_NCU116_plas3_10) were found only in the GIs of NCU116, which could be activated in osmotic environment. These genes were presumed to increase resistance to osmotic pressure in the hypertonic TCS brine which was usually salted. What's more, a fructose-/mannose-inducible PTS complex (L.p_NCU116_plas3_52 - L.p_NCU116_plas3_53) was defined to be strain-specific, and could pump more carbohydrates into cytoplasm of NCU116. Prophage-like elements are prevalent for bacteria, and were postulated to carry genes that increase adaptiveness in specific ecological niches or hosts [28]. Prophages integrated in bacterial genome are important vectors for lateral transfer among strains whether or not organized in the same species, which increased genetic diversity and resulted in strain specificities [29,30]. Prophage remnants were defined by PHASTER webserver for the eight strains (Table S3, Fig. S5B). Interestingly, the genome of DSM 20016 is the smallest, but the largest number of prophage elements (7) were found, with a total length of 215.3 Kbp, representing the maximum ratio (10.77%) of its genome. There were 3 intact and 2 residual prophages found in the NCU116 genome, and the total length was 146.6 Kbp (4.37% of its genome), which represented the maximum among L. plantarum spp. Remarkably, a propahge (approximately 40 Kbp) was found to be intraspecific conserved within L. plantarum spp. The conserved prophages (NCU116P3, ST-III-P1, JDM1-P3 and 5-2-P1) shared high homology to Lactobacillus phage Sha1 (temperate phage of L. plantarum, derived from kimchi), while it was absent in the genome of WCFS1 (Fig. 1B). And the conserved prophages were proposed to be beneficial for plant-related substrates fermentation, because all of them expect WCFS1 were derived from fermented foods. Subsequently BLAST was used to validate the conservation of these prophages (Fig. 3A). Rearrangements seemed to occur motr frequently when compared to Sha1, and inversion was found in ST-III (thus the fragment was inverted to compare with others). All of the compared prophages harboured two short-repeat regions, which were defined as attL and attR, representing the start and the end of an intact prophage. The two core components of prophage were found, including lysogeny module and replication module. Interestingly, the replication module was formed by discrete element in Sha1, while the continuous elements were found in the other compared strains. Hence, it's suggested that Sha1 other than the rest four strains may experience rearrangement. Although Sha1 was reported to be mitomycin-C-inducible, to our knowledge, it should be clearly noted that such temperate prophage cannot spill out from the host bacteria naturally [31], which has been demonstrated by the experience of using NCU116 as DVS starter in fruit and vegetable fermentation for nearly ten years. In addition, a transport operon comprising two ABC transporters were defined as conserved genes among three of analyzed strains (NCU116, WCFS1 and 5–2), which shared homology with Bacillus virus SPbeta (belonging to Siphoviridae). It seemed to be common for prophage to carry immunity proteins because such proteins were identified within five of the analyzed strains, including superinfection immunity and cognate repressor response for phage invasions. However, though prophages distributed remarkably within bacteria and some of them were well characterized before, it is still not clear

Fig. 2. Venn diagram of core-gene and pan-genome for L. plantarum strains.

NCU116, which was consistent with Mauve alignment. Besides, 51 of the annotated genes were identified as phage-associated genes (Table S2), suggesting that the unique genes were overall related to mobile elements. The unique genes may endow NCU116 with greater capacity of carbohydrates metabolism, because 12 genes including an integrated fructose/mannose transporters were found in the unique genes. NCU116 was found in the complicated living system (TCS), where a diverse range of microorganisms co-existed, suggesting that NCU116 may gain genes and features via HGT, and infections from temperate phage during evolution.

4. Genomic islands (GIs) and prophage sequences Gene gain and loss occurred frequently in bacteria, some of the obtained genes cluster together to form a short region which do not belong to themselves originally, and adaptive functions are commonly found in these regions, called GIs [27]. GIs were generated by IslandViewer and illustrated in circular plots for strains used in this study. NCU116 harboured the highest number of GIs (16) among the eight strains, with length from 4014 bp to 55,437 bp, total length of GIs reached 259,374 bp, representing 7.73% of the genome (Table S3). Meanwhile, phage-like regions were defined by PHASTER, almost all of the phage-like sequence were located in GIs (Fig. S5A). Despite the phage-like proteins which constituted the majority of GIs, it is interesting to note that several opp (oligopeptide ABC transport) clusters were species-specific for L. plantarum spp. as none were found in ATCC 367, CD 034 and DSM 20016. Besides, one opu complex (glycine betaine transport system, L.p_NCU116_plas2_3 and L.p_NCU116_plas2_5 - 7) and two osmC family peroxiredoxin (L.p_NCU116_plas3_9 and

Table 1 Genomic information used in the comparative genome analysis collected in public databases. Isolation

Genome Size

GC%

CDSs

Source

Accession Numb.

L. L. L. L. L. L. L. L.

3,354,689 bp 3,308,273 bp 3,254,376 bp 3,197,759 bp 3,237,652 bp 2,291,220 bp 2,500,564 bp 1,999,618 bp

44.40 44.44 44.50 44.70 44.70 46.04 44.24 38.90

3396 3181 3194 3084 3148 2180 2352 2049

Chinese sauerkraut Human saliva [40] Kimchi [6] Chinese commercial LAB [60] Fermented soybean [47] Wine [61] grass silage [62] Sourdough [63]

NZ_CP016071.1 NC_004567.2 NC_014554.1 NC_012984.1 NZ_CP009236.1 NC_008497.1 NC_018610.1 NC_009513.1

plantarum NCU116 plantarum WCFS1 plantarum ST-III plantarum JDM1 plantarum 5–2 brevis ATCC 367 buchneri CD034 reuteri DSM 20016

4

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Fig. 3. Comparative analysis of prophage and plantaricin-producing loci for L. plantarum strains. (A) The integration of Lactobacillus phage Sha1 and its collinear loci from L. plantarum strains. Corresponding locus of ST-III (ST-III P1) was inverted to a better matching, and no similar locus was defined in WCFS1. (B) Comprehensive analysis of plantaricin-producing loci from L. plantarum strains. Corresponding locus of NCU116 was reversed to a better matching.

5

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Fig. 4. Overview of proposed pathways present for Lactobacillus strains. Blue represents the homo- and heterolactic fermentation pathways. Green represents the truncated citrate metabolism pathways. Orange represents the proteolysis and amino acid metabolism pathways. And green squares embedded in the membrane (gray) represent PTS and ABC transporters. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

associated niches (e.g., pear and kimchi). Presumably, phage attacks are common in the fermentation of TCS, due to the use of unsterilized raw vegetables with multiple bacteria. As LABs have been used for vegetable fermentation for a long time, there has been a strong selective pressure towards phage-resistant strains. The strain-specific R/M system proteins may endow TCS-derived NCU116 with increased phage resistance especially for plant-related habitat, which could be implied by the homology comparison. CRISPR-Cas systems can provide protective immunity cooperatively with R/M systems against invasive mobile genetic elements (MGEs) [35,36]. But unfortunately, only 5 CRISPRs in L. buchneri CD034 were found, nothing but few CRISPR-residues were detectable in the other strains. It was also reported that CRISPR were rarely found in L. plantarum and L. reuteri species [35]. Thus, it is proposed that R/M systems may be more important than CRISPR in NCU116 when resisting the invasion of MGEs.

how prophages affect the regulation and metabolism of its host. Clearly more information is needed to lift the veil on the symbiosis and antagonism relationship between such adaptive organism and prophages. 5. Restriction/modification (R/M) systems and CRISPR-Cas systems Multiple R/M systems were frequently found in bacteria, and found to play an essential biological role for innate immunity [32,33]. Foreign invasive DNA may be cleaved at well-defined restriction sites by restriction endonucleases, while the autologous methyltransferase forestalls to cleave endogenous restriction sites [34]. R/M systems were determined for all of the eight strains by referring to REBASE database. Interestingly, a type I R/M complex (L.p_NCU116_plas3_31–33) was identified in the last GI of NCU116 (in the third plasmid), comprising a restriction (R) subunits, a modification (M) subunits and a specificity (S) subunit, which have been designated as host-specificity determinants. And the complex was identified as intraspecific genes among L. plantarum spp. A broader BLAST search found that these genes overall occurred in organisms derived from brewery-associated environments, sharing homology with Pediococcus claussenii ATCC BAA-344, L. brevis strain TMW 1.2113 and L. plantarum strain ZFM55, etc. The results suggested that these three genes might be specific for plant-fermentation. Two more type II R/M system methylases (including L.p_NCU116_12, L.p_NCU116_1084 and L.p_NCU116_1085) were found in NCU116 genome, which encodes the second higher number of R/M systems, followed DSM 20016 encoding 4 R/M systems, while few were found in the rest strains. All of the methylases detected in NCU116 were also strain-specific, sharing homology with L. plantarum strains SRCM102022, SN35N and KC3, which were all isolated from plant-

6. Proposed pathways in Lactobacillus spp 6.1. Carbohydrate metabolism Genes involved in carbohydrates transportation and decomposion were used to reconstruct carbohydrate metabolism for the strains used in this study (Fig. 4; Table S4). Extracellular carbohydrates could be pumped into bacterial cytoplasm through PTS systems, and various transporters were found in Lactobacilli. As expected, L. plantarum strains obviously had stronger ability to transport, and degrade macromolecules such as starch and amylose. Genes encoding 1,4-alpha-glucan branching enzyme (glgB) and glycogen phosphorylase (glgP) were found in L. plantarum strains, which degrade big sugars into small sugars such 6

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plantarum spp., however, these proteinases play important roles in protein metabolism [5,40,41]. Various genes associate to oligopeptide ABC transport (opp and dtpT) system were found in all Lactobacilli strains, as well as various peptidases including dipeptidase (pepDEV), oligoendopeptidase (pepF), aminopeptidase (pepNC) and endopeptidase (pepO). Besides, genes encoding livFGHKM were present, enabling NCU116 to capture amino acids from TCS for nitrogen metabolism (Fig. 4; Table S4). Genes encoding serine dehydratases, such as dsdA and sdh (catalyzing the interconversion of pyruvate and serine), and glyA gene encoding hydroxymethyltransferase (converting serine into glycine) were present. In addition, a gene encoding succinate-semialdehyde dehydrogenase (gabD) was present, enabling NCU116 to convert succinate into GABA (γ-aminobutyric acid). Meanwhile, a glutamate decarboxylase encoded by gadB is able to remove carboxygroup from glutamate, leading to carbon dioxide production, which is then turned into proline by glutamate 5-kinase (proABC). Glycine, glutamate and proline described above endow fermented carrot with umami and sweet taste, concentration of such amino acid almost doubled after fermentation by NCU116, and the content of amino acid also increased significantly after fermentation (data not published). Another pathway depending on aspartate aminotransferase (asp) was found in NCU116, which could transfer aspartate and 2-oxoglutarate into glutamate and oxaloacetate, suggesting that NCU116 may accommodate asparate-oxaloacetate transformation, as more copies were found. An argininosuccinate metabolism operon comprising one lyase (argH) and one synthase (argG) clustered together was present, and could subsequently be used for energy production and NADH regeneration. Asp combing with argHG could constitute a synergistic effect on coordination of global metabolism for Lactobacilli strains, while such genes were absent in ATCC367. Bitter amino acids, such as valine (Val), leucine (Leu) and isoleucine (Ile) were all degraded, because the level of these amino acids decreased significantly after fermentation by NCU116 in mango [42], carrot and pear (data not published). However, related pathways for degradation of Val, Leu and Ile remains unclear.

as glucose and fructose. Similar genes were absent in the other strains (ATCC367, CD034 and DSM20016). In addition, genes involved in CAZymes categories were defined for a better understanding of carbohydrate-active enzymes in Lactobacilli strains (Fig. S4C). A carbohydrate transport and degradation operon (from gene locus L.p_NCU116_959 to L.p_NCU116_1119) comprising one sucrose-specific PTS system, four maltose and maltodextrin ABC transporters, five glycoside hydrolase (GH)-13 family genes, as well as genes related to sugar-phosphorylase, was identified. Macromolecular carbohydrates, such as starch and glucan, could be transported across membrane via PTS and ABC transport systems by consuming ATP, then degraded into oligosaccharides (e.g., glucose and fructose) by α-amylase (L.p_NCU116_969), neopullulanase (L.p_NCU116_1110) and α-glucan branching enzyme (L.p_NCU116_1115). As NCU116 was obtained from TCS, starch could be the most abundant soluble sugar of vegetables, which may be a reason that NCU116 was more suitable for plant-associated fermentation. As the richest mono- and oligosaccharides in fermentation substrates (such as radish and cabbage), fructose, glucose and sucrose could be used as direct carbon source, as ptsG, ptsS and fruA responsible for transporting these small molecular carbohydrates were all identified in NCU116, and the other four L. plantarum strains. Numbers of genes involved in PTS systems within L. plantarum strains were roughly similar, with NCU116 having a bit more (82 PTS genes). All enzymes involved in homo- and heterolactic fermentation pathways were found in all L. plantarum strains, which enabled this species to switch between both metabolic pathways. Results suggested that L. plantarum spp. may have a stronger ability to convert pyruvate into lactate under anaerobic conditions, because more lactate dehydrogenase (ldh) copies and genes involved in glycolysis were identified, and that's exactly why L. plantarum matured sauerkraut in the post fermentation period [11,37]. NCU116 may be more suitable for vegetable-fermentation because more gene copies involved in this pathway were identified. Furthermore, pyruvate can be converted into formate by formate C-acetyltransferase (pflD) and pyruvate formate lyase (pfl), and acetyl phosphate catalyzed by pyruvate oxidase (poxL) under oxic condition and then yielding carbon dioxide, accelerating the maturation of sauerkraut by reducing oxygen in the jar. Pyruvate dehydrogenase (aceEF) and dihydrolipoamide dehydrogenase (pdhD) could catalyze pyruvate to form acetyl-CoA, and then convert into aldehyde by acetaldehyde dehydrogenase (adhA). Pyruvate could also be converted into α-acetolactate by acetolactate synthase (als), then decarboxylate to acetoin through enzymolysis of acetolactate decarboxylase (aldB). However, acetoin may be the end-product for most Lactobacillus strains except for CD034, which exclusively possessed 2,3butanediol dehydrogenase, proving L. plantarum spp. could not produce 2,3-butanediol, which was the major volatile flavor compound of fermented yogurt. Aldehyde and acetoin were the main volatile flavor substances of fermented foods, and further, aldehyde could be used for biosynthesis of other flavor compounds [38]. Oxaloacetate could be formed as an important intermediate, promoted by pyruvate carboxylase (pyc). Another pathway suggested that oxaloacetate could be obtained via citrate lyase-catalyzed (citDEF) citrate assimilation, yielding acetate. Malate dehydrogenase (mdh), fumarate hydratase (fum) and fumarate reductase (frd) were found in NCU116, indicating that this strain could use citrate as an external electron acceptor, which has been proved experimentally in Acetobacter spp., with succinate as the end product of the reductive TCA pathway [39]. Interestingly, an oxaloacetate decarboxylase (oad) coding gene was found in NCU116, and was absent in the other strains. This elaborated carbohydrates uptake and degradation machinery suggested that NCU116 may thrive and dominate the plant-related fermentation especially for TCS.

6.3. Potential to produce plantaricin As a model strain of L. plantarum spp., the entire plantaricin (pln) producing locus of WCFS1 has been studied previously. The pln-cluster in L. plantarum C11 was well authenticated which was almost identical to that of WCFS1 [40,43]. Thus comparative analysis was focused on pln loci, and results showed that there were more diversified plantaricin producing mechanisms, especially for NCU116 and JDM1 (Fig. 3B). The pln loci from NCU116 (from genome sequence of 794,688 bp to 807,471 bp) and JDM1 were almost identical to each other. Four operons were found in both chromosomes, including a two-peptide bacteriocin operon plnEFI, enabling NCU116 to produce a plantaricin with double-glycine leader and a cognate immunity protein containing CAAX-like protease. A truncated bacteriocin operon containing only plnLR complex was identified in NCU116. A similar reductive operon was found in L. plantarun J51 [44]. A highly conserved transport operon plnGHSTUVW comprising an ABC transporter (plnG) and an accessory protein (plnH) committing to secretion of active EF-type plantaricin was identified in NCU116, while the rest four genes encoding type II CAAXlike proteases were less-characterized [43–46]. Furthermore, a regulatory operon plnD as well as a plnB-like histidine kinase, in strain NCU116 and JDM1 were different from the other strains (5-2, ST-III and WCFS1). Further analyses of the upstream plnB and plnB-like (L.p_NCU116_766) genes were performed by BLAST. Results showed that WCFS1 and ST-III harboured the same kind of plnB as percentage of identity reached almost 100% even when extended to all pln loci, while genes encoding plnNC8IF and plnNC8HK were found in 5-2 which shared high homology with that of L. plantarum strain NC8 and J51 [44,46]. Interestingly, nucleic acid sequence analysis of plnB-like (from NCU116 and JDM1) showed only 88% identity of 55% coverage when compared to its counterpart from 5 to 2. However, protein sequence

6.2. Proteolysis and amino acid metabolism Information on extracellular proteinases was not clear enough for L. 7

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9. Additional analysis

analysis revealed that L.p_NCU116_766 belongs to GHKL domain-containing family sensor histidine kinase, which dedicates to the threecomponent quorum-sensing regulatory system along with plnD. Besides, a unique region encoding another bacteriocin operon plnNC8αβc flanking upstream plnR was found via BLAST search in the genome of 52, suggesting its potential to produce three different kinds of bacterioncin (plnEF, plnJK and plnNC8αβ), which has not been well-characterized for 5-2 [47]. In brief, core components for plantaricin-producing genes were present in NCU116, indicating its potential to produce a type II plantaricin plnEF. These properties may enable NCU116 to thrive and dominant the fermentation process till the final stage, and as a culture starter it may comply with the no-additive approach for preservation.

Genomic islands was investigated by using IslandViewer webserver, which integrates three prediction tools (IslandPick, SIG-HMM and IslandPath-DIMOB) [52]. Multiple R/M systems were determined by refering to the REBASE database [53]. Prophage elements and potential CRISPRs in the genomes were identified by PHASTER [54] and CRISPRFinder pipeline [55]. Sequence and amino acid similarity searches were conducted by BLAST+ suit or webserver [56], and visualized by Easyfig software [57] if necessary. The glycobiome of the investigated strains was defined based on the CAZy database [58]. The central carbohydrate, proteolysis and amino acid metabolism were investigated through the KEGG database [59]. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ygeno.2019.05.004.

7. Conclusion Competing interests In this study, we performed a re-annotation for the first sequenced genome from TCS-derived L. plantarum NCU116. NCU116 has been proven to be a probiotic strain and used as industrial DVS starter for fruit/vegetable fermentation. The accuracy and abundance of the genetic information have been improved significantly. Comparative genomics of eight Lactobacillus spp. genome from various species and niches provided a better opportunity for understanding the niche adaption and metabolic preference of the L. plantarum spp., especially for NCU116. Phylogenetic analysis including synteny, AAI and ANI analysis all showed that the closest strain to NCU116 was the reference strain WCFS1. Then core-genes and pan-genome were generated, and whole sequence alignment and GIs analysis found the gene gain and loss against selective pressure. Furthermore, multiple R/M systems and prophage elements were defined and investigated, which would be relevant to a strong self-defense mechanism associated with thriving growth in plant-fermentation. What's more, proposed pathways were reconstructed for the investigated strains, aiming at finding out the differences in carbohydrate and amino acid metabolism between intraand interspecies. Genotype exploration elaborated a proposed mechanism by which NCU116 thrives and influences the fermentation process, as well as the potential of producing a novel plantaricin different from other analyzed strains. In addition, more detailed and accurate genetic information for the probiotic NCU116 were obtained, which will be indispensable for the direction of application in industrial foods or pharmaceutics.

The authors declare no competing interests. Acknowledgments This study was funded by the National Natural Science Foundation of China (No. 31560449) and the Target-Directing Foundation of SKLF (No. SKLF-ZZA-201610). References [1] M.I. Masood, M.I. Qadir, J.H. Shirazi, I.U. Khan, Beneficial effects of lactic acid bacteria on human beings, Critical Reviews in Microbiology 37 (2011) 91. [2] D.C. Donohue, S. Salminen, A. Von Wright, A. Ouwehand, Safety of novel probiotic bacteria, (2004). [3] M. Bernardeau, J.P. Vernoux, S. Henri-Dubernet, M. Guéguen, Safety assessment of dairy microorganisms: The Lactobacillus genus, International Journal of Food Microbiology 126 (2008) 278–285. [4] M. Kleerebezem, J. Boekhorst, R. van Kranenburg, D. Molenaar, O.P. Kuipers, R. Leer, R. Tarchini, S.A. Peters, H.M. Sandbrink, M.W.E.J. Fiers, W. Stiekema, R.M.K. Lankhorst, P.A. Bron, S.M. Hoffer, M.N.N. Groot, R. Kerkhoven, M. de Vries, B. Ursing, W.M. de Vos, R.J. Siezen, Complete genome sequence of < em > Lactobacillus plantarum WCFS1, Proc. Natl. Acad. Sci. 100 (2003) 1990–1995. [5] C.-J. Liu, R. Wang, F.-M. Gong, X.-F. Liu, H.-J. Zheng, Y.-Y. Luo, X.-R. Li, Complete genome sequences and comparative genome analysis of Lactobacillus plantarum strain 5-2 isolated from fermented soybean, Genomics 106 (2015) 404–411. [6] Y. Wang, C. Chen, L. Ai, F. Zhou, Z. Zhou, L. Wang, H. Zhang, W. Chen, B. Guo, Complete genome sequence of the probiotic Lactobacillus plantarum ST-III, J. Bacteriol. 193 (2011) 313–314. [7] A.S. Andreasen, N. Larsen, T. Pedersenskovsgaard, R.M.G. Berg, K. Møller, K.D. Svendsen, M. Jakobsen, B.K. Pedersen, Effects of Lactobacillus acidophilus NCFM on insulin sensitivity and the systemic inflammatory response in human subjects, Br. J. Nutr. 104 (2010) 1831–1838. [8] A.F. Kane, A.D. Bhatia, P.W. Denning, A.L. Shane, R.M. Patel, Routine supplementation of Lactobacillus rhamnosus GG and risk of necrotizing enterocolitis in very low birth weight infants, J. Pediatr. 195 (2018) 73–79. [9] Q. Zhang, M. Hu, C. Ren, W. Chen, Immunomodulatory effects of Lactobacillus casei on a murine model of peanut allergy, Acta Microbiol Sin. 58 (2018) 73–82. [10] Z.Y. Zhang, C. Liu, Y.Z. Zhu, Y. Zhong, Y.Q. Zhu, H.J. Zheng, G.P. Zhao, S.Y. Wang, X.K. Guo, Complete genome sequence of Lactobacillus plantarum JDM1, J. Bacteriol. 191 (2009) 5020–5021. [11] T. Xiong, Q. Guan, S. Song, M. Hao, M. Xie, Dynamic changes of lactic acid bacteria flora during Chinese sauerkraut fermentation, Food Control 26 (2012) 178–181. [12] K. Illeghems, V.L. De, S. Weckx, Comparative genome analysis of the candidate functional starter culture strains Lactobacillus fermentum 222 and Lactobacillus plantarum 80 for controlled cocoa bean fermentation processes, BMC Genomics 16 (2015) 766. [13] Z. Xing, W. Geng, C. Li, Y. Sun, Y. Wang, Comparative genomics of Lactobacillus kefiranofaciens ZW3 and related members of Lactobacillus. Spp reveal adaptations to dairy and gut environments, Sci. Rep. 7 (2017) 12827. [14] V. Valeriano, J.K. Oh, B.B. Bagon, H. Kim, D.K. Kang, Comparative genomic analysis of Lactobacillus mucosae LM1 identifies potential niche-specific genes and pathways for gastrointestinal adaptation, Genomics 111 (2019) 24–33. [15] T. Xiong, S. Song, X. Huang, C. Feng, G. Liu, J. Huang, M. Xie, Screening and identification of functional Lactobacillus specific for vegetable fermentation, J. Food Sci. 78 (2013) M84–M89. [16] C. Li, S.P. Nie, Q. Ding, K.X. Zhu, Z.J. Wang, T. Xiong, J. Gong, M.Y. Xie, Cholesterol-lowering effect of Lactobacillus plantarum NCU116 in a hyperlipidaemic rat model, J. Funct. Foods 8 (2014) 340–347.

8. Method 8.1. Re-annotation and comparative genomics of different Lactobacillus spp. strains The coding sequences of L. plantarum NCU116 were predicted by Genemarks, and then used to re-annotate through RASTtk pipeline [20]. Predictions from RAST and information from NCBI were merged into a single annotation file after manual curation, as well as the classification of functional genes. And circular maps of chromosome and plasmids were performed by Artemis and DNAPlotter software [48,49], G + C content and GC skew were adjusted appropriately for different regions of the genome. Comparison between L. plantarum NCU116 and those of various available strains, namely, L. plantarum strain WCFS1, ST-III, JDM1 and 5–2, L. brevis ATCC 367, L. buchneri CD034 and L. reuteri DSM 20016 were performed by different means. Whole genome alignment was used to visualize the conserved genomic regions and large-scale rearrangements through progressiveMAUVE software [50]. And the EDGAR software framework [51] was used to perform the differential gene content across the genomes, as well as the phylogenetic analysis including venn diagrams, syntney plot, AAI, ANI and phylogenetic tree. 8

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