BACTERIOCIN ASSOCIATED GENES IN FRENCH
AND UKRAINIAN FERMENTED VEGETABLE
ISOLATES OF LACTIPLANTIBACILLUS
PLANTARUM

Nataliia Limanska, Andrii Merlich, Maryna Zlatohurska, Mykola Galkin,
Nadiia Korotaieva, Tetiana Ivanytsia, Yvan Choiset, Volodymyr Ivanytsia,
Thomas Haertlé, Thomas Haertlé

► To cite this version:

Nataliia Limanska, Andrii Merlich, Maryna Zlatohurska, Mykola Galkin, Nadiia Korotaieva, et al. BACTERIOCIN ASSOCIATED GENES IN FRENCH AND UKRAINIAN FERMENTED VEGETABLE ISOLATES OF LACTIPLANTIBACILLUS PLANTARUM. Journal of Microbiology, Biotechnology and Food Sciences, 2021, pp.e5871. 10.55251/jmbfs.5871. hal-03721586

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# BACTERIOCIN ASSOCIATED GENES IN FRENCH AND UKRAINIAN FERMENTED VEGETABLE ISOLATES OF LACTIPLANTIBACILLUS PLANTARUM

Nataliia Limanska<fnref n="1" />, Andrii Merlich<fnref n="1" />,<fnref n="2" />, Maryna Zlatohurska<fnref n="1" />, Mykola Galkin<fnref n="1" />, Nadiia Korotaieva<fnref n="1" />, Tetiana Ivanytsia<fnref n="1" />, Yvan Choiset<fnref n="2" />, Volodymyr Ivanytsia<fnref n="1" />, Thomas Haertlé<fnref n="2" />

Address(es): PhD Nataliia Limanska,

## ARTICLE INFO

Received 22. 2. 2022

Revised 20. 4. 2022

Accepted 4. 5. 2022

Published xx.xx.201x

Regular article

OPEN ACCESS

## ABSTRACT

Lactiplantibacillus plantarum species is known for the high diversity of the loci encoding bacteriocin (plantaricin) production. The aim of this work was to study the distribution of pln genes in French and Ukrainian isolates of L. plantarum originated from fermented plant material. PCR with the primers to plnA, plnB, plnC, plnD, plnEF, plnI, plnK, plnJ, plnG, plnN, plnW genes was performed to find out the peculiarities of L. plantarum strains. French and Ukrainian isolates shared none of the identical combinations of genes from plantaricin regulon indicating the diversity of pln loci in L. plantarum of fermented plant origin from the distant geographical regions. Certain combinations of genes from plantaricin regulon could be proposed as indicators marking the geographical origin of L. plantarum strains. Among the studied isolates, some resembled the described in the literature L. plantarum strains, and others contained unique combinations of pln genes.

Keywords: Lactiplantibacillus plantarum, plantaricin, pln loci, diversity, geographical areas

## INTRODUCTION

Lactiplantibacillus plantarum species is characterized by the high diversity of the strains (Martino et al., 2016; Choi et al., 2018; Araujo et al., 2020) including genetic organization of pln loci encoding bacteriocin production (Basa et al., 2020; Choi et al., 2021; Wu et al., 2021). As the result, bacteriocins belong to the most variable sub-categories of secretome (Martino et al., 2016). All pln loci are similar in the presence of a two-component regulatory system consisting of a histidine protein kinase and response regulators, in encoding inducible peptides, proteins of an ABC transport systems and some other products (Saenz et al., 2009; Diep et al., 2009). Synthesis of plantaricins is mediated by quorum-sensing (Kwak et al., 2016).

Phylogenetic analysis of 54 L. plantarum strains from different geographical zones including 22 strains of plant origin, showed the presence of three groups encompassing strains with various combinations of pln genes (Choi et al., 2021). Martino et al. (2016) analyzed draft and complete genomes of 54 L. plantarum strains and found that the strains with the same geographical origin could share similar orthologous groups in their genomes as it was described for L. plantarum isolated from Vietnamese fermented fruits, vegetables and pickled meat products. At the same time, no correlation between the habitat origin of L. plantarum strains (animal or plant organisms, products) and genetic specificity was found (Martino et al., 2016; Choi et al., 2018).

We have previously described the absence of association between geographical origin of the L. plantarum strains isolated from grape must and pickles in France and Ukraine by means of their RAPD-profiles (Limanska et al., 2019). The aim of this work was to study the distribution of pln genes in French and Ukrainian isolates of L. plantarum.

## MATERIAL AND METHODS

### Bacterial strains

L. plantarum originated from grape collected in France (13 isolates) and from grape and pickles collected in Ukraine (17 isolates, Tab 1) were studied for the presence of pln genes.

<table><caption>Table 1 Origin of L. plantarum strains</caption><thead><tr><td>Strain</td><td>Source of isolation</td><td>Country of origin</td></tr></thead><tbody><tr><td>L. plantarum ONU 351, ONU 352, ONU 353, ONU 354, ONU 355, ONU 356, ONU 357, ONU 359, ONU 362, ONU 363, ONU 364, ONU 365, ONU 471</td><td>Grape must</td><td>France</td></tr><tr><td>L. plantarum ONU 12, ONU 311, ONU 312, ONU 313, ONU 333, ONU 335, ONU 337, ONU 339, ONU 340, ONU 342, ONU 345, ONU 348, ONU 349, ONU 350</td><td>Grape must</td><td>Ukraine</td></tr><tr><td>L. plantarum ONU 472, ONU 475</td><td>Sauerkraut</td><td>Ukraine</td></tr><tr><td>L. plantarum ONU 476</td><td>Fermented pickled tomatoes</td><td>Ukraine</td></tr></tbody></table>

### PCR amplification of the genes from pln loci

DNA from overnight cultures of L. plantarum was isolated using Qiagen DNA purification kit (Germany). PCR was carried out with the primers to plnA, plnB, plnC, plnD, plnEF, plnI, plnK, plnJ, plnG, plnN, plnW genes as described and reviewed in Ben Omar et al. (2008) according to the wide distribution of these genes in fermented vegetable material (Ben Omar et al., 2008; Rojo-Bezares et al., 2008; Sáenz et al., 2009; Diep et al., 2009; Choi et al., 2021).

## RESULTS AND DISCUSSION

Our results are in agreement with the data indicating that pln loci are widespread in oenological L. plantarum (Sáenz et al., 2009; Knoll et al., 2008). However, French and Ukrainian isolates shared none of the identical combinations of genes from plantaricin regulon indicating the diversity of pln loci in L. plantarum strains of fermented plant origin from distant geographical regions (Tab 2).

<footnote n="1">¹Odessa National I.I. Mechnikov University (ONU), Biological Faculty, Microbiology, Virology and Biotechnology Department, Dvorianska str., 2, 65082, Odessa, Ukraine, phone number:+38 048 731 71 51.</footnote>

<footnote n="2">²French National Institute for Agriculture Research (INRA), UR1268, Biopolymères, Interactions, Assemblages, BP 71627, rue de la Géraudière, 44316 Nantes Cedex 3, France.</footnote>

<footnote>*Corresponding author: limanska@onu.edu.ua</footnote>

<footnote>https://doi.org/10.55251/jmbfs.5871</footnote>
<table><caption>**Table 2** Presence of pln genes in genomes of the French and Ukrainian isolates of *L. plantarum*</caption><thead><tr><th>Strains and their origin</th><th>plnA</th><th>plnB</th><th>plnC</th><th>plnD</th><th>plnEF</th><th>plnG</th><th>plnI</th><th>plnJ</th><th>plnK</th><th>plnN</th><th>plnW</th></tr></thead><tbody><tr><td><strong>France</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>351, 352, 357, 471</td><td>-</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>353, 354</td><td>-</td><td>+</td><td>-</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>355</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>356, 365</td><td>-</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>359</td><td>-</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>362</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>363</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>364</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td><strong>Ukraine</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>12, 311, 313</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>312</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>333</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>335</td><td>-</td><td>+</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>337</td><td>-</td><td>+</td><td>+</td><td>-</td><td>+</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td>-</td></tr><tr><td>339</td><td>-</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>340, 472, 475, 476</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td></tr><tr><td>342, 345, 348, 349, 350</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td></tr></tbody></table>

Thus, 16 unique combinations of pln genes were found among French and Ukrainian isolates – 8 in each geographical group. Within each geographical group, 2-5 strains could share identical set of pln genes; however, no combinations were identical in *L. plantarum* from distant geographical areas.

The complete plnABCD locus was detected in 15.4% of the French isolates, and in 36.3% of Ukrainian *L. plantarum* (Fig 1).

Our strain *L. plantarum* ONU 362 containing plnA, plnB, plnD genes but missing plnC by the combination of the studied genes was identical to *L. plantarum* EC80 strain from fermented maize described by Ben Omar et al. (2008).

Gene plnN indicating the presence of plnMNOP operon was found in 100% of French, and in 94.1% of Ukrainian strains (Fig 1).

Strains *L. plantarum* ONU 355 and ONU 364 from France and ONU 333, 340, 345, 348, 349 and ONU 350 from Ukraine contained the complete plnABCD operon and the gene plnN which allowed to characterize them as C11- and WCFS1-like strains. Strains WCFS1 and C11 with an exception of two genes are similar in pln loci (Diep et al., 2009; Kleerebezem et al., 2003). Sáenz et al. (2009) investigated strains isolated in Spain from must and wines for the presence of 27 pln genes and clustered them in groups with well described strains C11, WCFS1, J51, J23 and NC8. The group of the strains resembling C11 and WCFS1 in composition of pln regulon was the largest – 60,6%.

Strain *L. plantarum* ONU 337 with the absent plnN gene could resemble the strain *L. plantarum* J51 described in the literature (Diep et al., 2009; Navarro et al., 2008).

Pln E/F was found in 30.8% of the French strains and in 94.1% of Ukrainian isolates. Complete operon pln EFI was detected in 30.8% of the French isolates and 53% of the Ukrainian strains.

plnK was detected in 100% of French strains, and in 65% of Ukrainian isolates. Strains ONU 12, ONU 311, ONU 312 and ONU 313 could resemble the described *L. plantarum* J23 strain (Rojo-Bezares et al., 2008) by absence of plnABCD and plnK.

Gene plnJ was present in 53% of Ukrainian isolates, among them both genes of plnJKLR operon – plnJ and plnK – were found in 35.3%. In French isolates plnJ genes were absent (Fig 1). Loss of the sequences of some genes resulting in negative PCR result could probably happen due to integration of mobile genetic elements found in the pln clusters (Evanovich et al., 2019; Choi et al., 2021).

The absence of plnJ in ONU 333, 355 and 364 was unusual for the WCFS1 and C11 types of plantaricin regulons. The similar situation was described in Sáenz et al. (2009) for an oenological *L. plantarum* strain missing plnJ and moreover, missing plnD but with the similarity > 89% ranged in one group with C11 and WCFS1. Two strains from fermented vegetables isolated in Korea were also characterized by the presence of plnK and absence of plnJ (Choi et al., 2021).

Operons plnEFI and plnJKLR encode two-peptide bacteriocins, PlnEF and PlnJK and their immunity proteins (Anderssen et al., 1998; Diep et al., 2009; Havarstein et al., 1995; Kleerebezem et al., 2003). Among Ukrainian strains, 76.4% carried genes encoding two bacteriocins – plnEF and plnJK. Strains with the potential synthesis of two types of bacteriocins are more adaptable to the environment and indeed, they are more abundant among the strains of fermented food origin (Choi et al., 2021). Use of such strains is the most perspective for probiotic and food production. plnG from plnGHSTUVW operon was found in 69.2% of the French strains, and in 53% of Ukrainian isolates (Fig 1).

Five from all the tested *L. plantarum* strains – ONU 342, 345, 348, 349 and 350 (of Ukrainian origin) carried all the tested genes except the plantaricin W (Tab 2).

None of the studied French and Ukrainian strains encoded the plnW. The plantaricin W structural gene encoding two-peptide bacteriocin PLW was initially described for the strain *L. plantarum* LMG 2379 isolated from fermenting wine in the USA (Holo et al., 2001). Our results coincide with the previously reported rare occurrence of this gene in *L. plantarum* (Diep et al., 2009; Ben Omar et al., 2008), although strains encoding plnW were also described (Sogandi et al., 2019).

<figure><img src="image_2.png" /><figcaption>**Figure 1** Percentage of *L. plantarum* strains originated from France (a) and Ukraine (b)</figcaption></figure>

The regulatory operon plnABCD encodes an inducing pheromone peptide PlnA, a histidine protein kinase PlnB and the response regulators PlnC and PlnD (Anderssen et al., 1998; Diep et al., 2009; Havarstein et al., 1995; Kleerebezem et al., 2003).
The results of our study describing various combinations of pln genes confirm the data of the previous investigators about the high genetic diversity of *L. plantarum* species (Saenz et al., 2009; Diep et al., 2009; Ben Omar et al., 2008; Sogandi et al., 2019).

Although we could not find differences between *L. plantarum* originated from distant geographical areas in our previous work based on RAPD-analysis (Limanska et al., 2019), study of distribution of pln genes allowed us to distinguish the unique genetic combinations in French and Ukrainian isolates.

CONCLUSION

French and Ukrainian strains of *L. plantarum* of plant origin shared no identical combinations of pln genes showing the high diversity of pln loci in isolates from distant geographical regions.

Certain combinations of genes from plantaricin regulon could be proposed as indicators marking the geographical origin of *L. plantarum* strains.

Among the studied isolates, some resembled those described in the literature for *L. plantarum* strains, while others encoded the unique combinations of pln genes.

**Acknowledgments:** This work was supported by a bilateral French-Ukrainian project "Dnipro" granted by the Ministry of Foreign and European Affairs of France and the Ministry of Education and Science of Ukraine, and by the individual grant for Andrii Merlich (Campus-France) supported by the French Embassy in Ukraine.

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