Article Figures & Data
Figures
- 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.
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.
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.
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
- TABLE 1.
Bacterial strains and plasmids
Strain or plasmid Relevant 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 Y105 Wild-type strain 18 Y110 Wild-type strain CNCM I-1936a FR52 Wild-type strain 24 Leuconostoc mesenteroides subsp. dextranicum DSM20484 Wild-type strain DSMZb Leuconostoc cremoris LC Wild-type industrial strain Rhodia Food Lactococcus lactis IL1403 Lab strain 8 MG1363 Lab strain 13 RD230 Wild-type industrial strain Rhodia Food Lactobacillus sake 23K Plasmid cured 3 Listeria ivanovii BUG 497 Lab strain Pasteur Institute Pediococcus acidilactici P120 Wild-type industrial strain Rhodia Food Plasmids pBSSKII+ pBluescript SK II+ cloning vector, 2.96 kb, AprlacZ Stratagene pGhost9:ISS1 pWV01 derivative replicon 23 pFR18 1.8-kb cryptic plasmid from L. mesenteroides FR52 5 pFBYC018 pBSSKII+::1.8-kb HindIII pFR18, 4.8 kb, Apr Ems 5 pFBYC018E pFBYC018::1.1-kb BamHI pGhost9:ISS1 (Emr), 5.9 kb, Apr Emr 5 pFBYC18E pFBYC018E with 2.5-kb PvuII pBSSKII+ deleted, 3.5 kb, Emr 5 pTXL1 2.66-kb cryptic plasmid from L. mesenteroides Y110 5 pFBYC050 pBSSKII+::2.66-kb SalI pTXL1, 5.62 kb, Apr Ems This study pFBYC051 pBSSKII+::2.66-kb SpeI pTXL1, 5.62 kb, Apr Ems This study pFBYC052 pBSSKII+::1.8-kb SspI pFR18, 4.8 kb, Apr Ems This study pFBYC050E pFBYC050::1.1-kb BamHI pGhost9:ISS1, 7.72 kb, Apr Emr This study pFBYC50E pFBYC050E with 2.5-kb PvuII fragment deleted, Emr This study pFBYC065 pFBYC050E with 312-kb ExoIIIc fragment deleted, Apr Emr This study pFBYC060K pFBYC050E with 482-kb KpnI-KpnI fragment deleted, Apr Emr This study pFBYC062S pFBYC050E with 1.4-kb SalI-SspI fragment deleted, Apr Emr This study pFBYC068S pFBYC050E with 1.9-kb SalI-AflIII fragment deleted, Apr Emr This study pFBYC051E pFBYC051::1.1-kb BamHI pGhost9:ISS1, 7.72 kb, Apr Emr This study pFBYC06 pRW5.6 with HindIII deleted 4 pFBYC069 pFBYC051E::669-bp P59, dvnA fragment from pFBYC06 This study pFBYC070 pFBYC069::485-bp dvnA::mesY, mesI PCR product This study - TABLE 2.
Stability of pTXL1 derivatives during extended growth under nonselective conditions
Plasmid Replicon % of DSM20484 clones containing the plasmid after 100 generationsa Reference pGhost9:ISS1 pWV01 76 23 pFBYC050E pTXL1 100 This study pFBYC50E pTXL1 100 This study pFBYC018E pFR18 3 5 pFBYC18E pFR18 100 5 ↵ a Percentages are averages of results from duplicate experiments and were determined as percentages of erythromycin-resistant colonies.
