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Genetics and Molecular Biology

Identification of a Replicon from pTXL1, a Small Cryptic Plasmid from Leuconostoc mesenteroides subsp. mesenteroides Y110, and Development of a Food-Grade Vector

Franck Biet, Yves Cenatiempo, Christophe Fremaux
Franck Biet
1Institut de Biologie Moléculaire et d'Ingénierie Génétique, Equipe d’Accueil 2224, Université de Poitiers, 86022 Poitiers Cedex
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  • For correspondence: franck.biet@pasteur-lille.fr
Yves Cenatiempo
1Institut de Biologie Moléculaire et d'Ingénierie Génétique, Equipe d’Accueil 2224, Université de Poitiers, 86022 Poitiers Cedex
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Christophe Fremaux
2Rhodia Food, Zone d’Activités de Buxières, 86220 Dangé Saint-Romain, France
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DOI: 10.1128/AEM.68.12.6451-6456.2002
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  • FIG. 1.
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    FIG. 1.

    Identification of the minimal replicon from pTXL1. The restriction map of pTXL1 is shown with the positions of the restriction sites. Partial enzymatic deletion by exonuclease III is denoted by ΔExoIII with the size of the deletion.

  • FIG. 2.
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    FIG. 2.

    Nucleotide sequences of the pTXL1 minimum replicon-linearized sequence and the deduced amino acid sequences for putative ORF1 and ORF2. The arrows with solid lines indicate inverted repeats (IRI, IRII, and IRIII), and the arrows with dashed lines indicate direct repeats (DRI, DRII, and DRIII).

  • FIG. 3.
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    FIG. 3.

    Detection of single-stranded intermediates of pFBYC18E (a), pGhost9:ISS1 (b), and the pTXL1 derivative (c) in L. mesenteroides subsp. dextranicum DSM20484. A nonalkaline Southern blot hybridization analysis was performed with radiolabeled pFBYC18E (a), pGhost9:ISS1 (b), and pTXL1 (c) probes. Whole-cell DNA was prepared from MRS cultures containing rifampin. DNA preparations treated with endonuclease S1 are indicated by a +, and untreated preparations are indicated by a −. OC, open circular DNA; CCC, covalently closed circular DNA; SS, ssDNA.

  • FIG. 4.
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    FIG. 4.

    Bacteriocin activity assays against Listeria ivanovii BUG 497 overlaying Leuconostoc strains. (A) L. mesenteroides subsp. mesenteroides Y105 (a); nonrecombinant L. mesenteroides subsp. dextranicum DSM20484 (b), and nonrecombinant L. cremoris LC (c); (B) L. mesenteroides subsp. mesenteroides Y105; (a) Leuconostoc mesenteroides subsp. dextranicum DSM20484 containing pFBYC070 (b), and Leuconostoc cremoris LC containing pFBYC070 (c).

Tables

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  • TABLE 1.

    Bacterial strains and plasmids

    Strain or plasmidRelevant characteristic(s)Source or referencea
    Strains
        Escherichia coli DH5α recA endA1 gyrA96 thi-1 hsdR17 supE44 Δlac U169(φ80 ΔlacZΔM15) deoR F− λ−Gibco-BRL
        Leuconostoc mesenteroides subsp. mesenteroides
            Y105Wild-type strain 18
            Y110Wild-type strainCNCM I-1936a
            FR52Wild-type strain 24
        Leuconostoc mesenteroides subsp. dextranicum DSM20484Wild-type strainDSMZb
        Leuconostoc cremoris LCWild-type industrial strainRhodia Food
        Lactococcus lactis
            IL1403Lab strain 8
            MG1363Lab strain 13
            RD230Wild-type industrial strainRhodia Food
        Lactobacillus sake 23KPlasmid cured 3
        Listeria ivanovii BUG 497Lab strainPasteur Institute
        Pediococcus acidilactici P120Wild-type industrial strainRhodia Food
    Plasmids
        pBSSKII+pBluescript SK II+ cloning vector, 2.96 kb, AprlacZStratagene
        pGhost9:ISS1pWV01 derivative replicon 23
        pFR181.8-kb cryptic plasmid from L. mesenteroides FR52 5
        pFBYC018pBSSKII+::1.8-kb HindIII pFR18, 4.8 kb, Apr Ems 5
        pFBYC018EpFBYC018::1.1-kb BamHI pGhost9:ISS1 (Emr), 5.9 kb, Apr Emr 5
        pFBYC18EpFBYC018E with 2.5-kb PvuII pBSSKII+ deleted, 3.5 kb, Emr 5
        pTXL12.66-kb cryptic plasmid from L. mesenteroides Y110 5
        pFBYC050pBSSKII+::2.66-kb SalI pTXL1, 5.62 kb, Apr EmsThis study
        pFBYC051pBSSKII+::2.66-kb SpeI pTXL1, 5.62 kb, Apr EmsThis study
        pFBYC052pBSSKII+::1.8-kb SspI pFR18, 4.8 kb, Apr EmsThis study
        pFBYC050EpFBYC050::1.1-kb BamHI pGhost9:ISS1, 7.72 kb, Apr EmrThis study
        pFBYC50EpFBYC050E with 2.5-kb PvuII fragment deleted, EmrThis study
        pFBYC065pFBYC050E with 312-kb ExoIIIc fragment deleted, Apr EmrThis study
        pFBYC060KpFBYC050E with 482-kb KpnI-KpnI fragment deleted, Apr EmrThis study
        pFBYC062SpFBYC050E with 1.4-kb SalI-SspI fragment deleted, Apr EmrThis study
        pFBYC068SpFBYC050E with 1.9-kb SalI-AflIII fragment deleted, Apr EmrThis study
        pFBYC051EpFBYC051::1.1-kb BamHI pGhost9:ISS1, 7.72 kb, Apr EmrThis study
        pFBYC06pRW5.6 with HindIII deleted 4
        pFBYC069pFBYC051E::669-bp P59, dvnA fragment from pFBYC06This study
        pFBYC070pFBYC069::485-bp dvnA::mesY, mesI PCR productThis study
    • ↵ a CNCM, Collection Nationale de Cultures de Microorganisms, France.

    • ↵ b DSMZ, Deutsche Sammlung von Mikroorganismen and Zellkulturen, Germany.

    • ↵ c ExoIII, exonuclease III.

  • TABLE 2.

    Stability of pTXL1 derivatives during extended growth under nonselective conditions

    PlasmidReplicon% of DSM20484 clones containing the plasmid after 100 generationsaReference
    pGhost9:ISS1pWV0176 23
    pFBYC050EpTXL1100This study
    pFBYC50EpTXL1100This study
    pFBYC018EpFR183 5
    pFBYC18EpFR18100 5
    • ↵ a Percentages are averages of results from duplicate experiments and were determined as percentages of erythromycin-resistant colonies.

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Identification of a Replicon from pTXL1, a Small Cryptic Plasmid from Leuconostoc mesenteroides subsp. mesenteroides Y110, and Development of a Food-Grade Vector
Franck Biet, Yves Cenatiempo, Christophe Fremaux
Applied and Environmental Microbiology Dec 2002, 68 (12) 6451-6456; DOI: 10.1128/AEM.68.12.6451-6456.2002

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Identification of a Replicon from pTXL1, a Small Cryptic Plasmid from Leuconostoc mesenteroides subsp. mesenteroides Y110, and Development of a Food-Grade Vector
Franck Biet, Yves Cenatiempo, Christophe Fremaux
Applied and Environmental Microbiology Dec 2002, 68 (12) 6451-6456; DOI: 10.1128/AEM.68.12.6451-6456.2002
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KEYWORDS

Genetic Vectors
Leuconostoc
plasmids
Replicon

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