Article Figures & Data
Figures
- FIG 1
Copy numbers of five plasmids of L. lactis FM03-V1 in retentostat (left) and chemostat (right) cultures. Red, black, blue, and green represent independent retentostat cultures 1 and 2 and chemostat cultures 3 and 4, respectively. Every point represents the average plasmid copy number per DNA extraction calculated from triplicate qPCR measurements. These plasmid copy numbers were corrected for slight changes in the amount of DNA extracted, which significantly affected the copy number measurement of most plasmids (Table 2). The dashed lines represent predictions calculated from a linear mixed-effects model as explained in Materials and Methods.
- FIG 2
Correlation between the copy number and the size of the plasmids. Filled circles, theta-type replicating plasmids; open circle, rolling-circle replicating plasmid. Plasmid copy numbers were calculated at a growth rate of 0.2 h−1 on the basis of the obtained model for chemostat cultivations.
- FIG 3
Copy numbers of five plasmids of L. lactis FM03-V1 during chemostat cultivation at a constant growth rate (0.135 h−1) under different conditions: pH 5.5 or 7, under lactose or amino acid limitation, and with (+) or without (−) supplementation of citrate. Boxplots were made with the average plasmid copy numbers per DNA extraction, which were calculated from triplicate qPCR measurements. These plasmid copy numbers were corrected for slight changes in the amount of DNA extracted, which significantly affected the copy number measurements of most plasmids (Table 3). Triangles represent the predictions of linear mixed-effects models as explained in Materials and Methods. P values of significant effects are indicated in the plots.
Tables
- TABLE 1
Characteristics of the plasmids in L. lactis FM03-V1 used in this study
Plasmid Accession no. Size (kb) Replication type Putative function(s) pLd1 CP020605.1 8.3 Theta Citrate uptake pLd3 CP020607.1 4.0 RCRa Unknown pLd9 MF150537.1 15.3 Theta Phage defense/d-lactate dehydrogenase pLd10 MG813924.1 39.6 Theta Exopolysaccharide production pLd12 MG813926.1 3.3 Theta Restriction-modification system ↵a RCR, rolling-circle replication.
- TABLE 2
Significance of effects of DNA extraction and the growth rate on the copy numbers of five plasmids of L. lactis FM03-V1 during retentostat and chemostat cultivations
Cultivation Plasmid Fixed factorsa PCN as predicted by modelb Extraction Growth rate df t value P value df t value P value Minimum Maximum Fold change Retentostat (0.0003–0.025 h−1) pLd1 77 5.5 4.4 × 10−7 77 −3.3 0.0016 24.5 40.5 1.7 pLd3 77 2.6 0.011 77 −4.5 2.0 × 10−5 2.6 9.1 3.5 pLd9 77 6.0 7.1 × 10−8 77 −2.9 0.0056 14.3 22.7 1.6 pLd10 40 0.52 0.61 1.5 1.8c 1.2 pLd12 77 4.3 5.2 × 10−5 77 −1.6 0.12 28.2 39.6 1.4 Chemostat (0.05–0.6 h−1) pLd1 42 4.1 1.9 × 10−4 42 −0.44 0.66 17.2 18.0 1.0 pLd3 42 4.6 3.8 ×10−5 42 −2.0 0.057 2.9 4.2 1.5 pLd9 42 3.2 0.0029 80 −0.54 0.59 12.6 13.8 1.1 pLd10 42 3.8 4.2 × 10−4 42 −0.095 0.92 7.3 7.5 1.0 pLd12 42 −0.05 0.96 21.8 22.0 1.0 ↵a Statistical analyses were performed with R by regression with a linear mixed-effects model as explained in Materials and Methods.
↵b PCN, plasmid copy number. The model was used to predict the minimum and maximum number of plasmid copies in the tested range and the maximum fold change was calculated.
↵c Higher predicted plasmid copy number at higher growth rates.
- TABLE 3
Significance of effects of DNA extraction, pH, and nutrient limitation on the copy numbers of five plasmids of L. lactis FM03-V1 during chemostat cultivation at a constant growth rate (0.135 h−1) with and without supplementation of citratea
Plasmid Extraction pHb Nutrient limitationc df t value P value df t value P value Fold change df t value P value Fold change pLd1 50 8.7 1.4 × 10−11 50 −3.4 0.0013 2.0 pLd3 54 7.0 3.4 × 10−9 54 −6.8 8.6 × 10−9 2.2 pLd9 56 9.8 1.0 × 10−13 pLd10 55 8.5 1.4 × 10−11 55 −4.8 1.4 × 10−5 1.9 pLd12 56 6.5 2.2 × 10−8 - TABLE 4
Primers used for plasmid copy number determination by qPCR
Target Target gene Primer sequence (5′→3′) Amplicon size (bp) Forward Reverse Chromosome hisB, BSR25_0747a TCAAGCGGACATTAGTACAGGTATTGGTT CGAGCTTATTACCTAATGCTTCGCTGATAC 194 pLd1 citP, BSR25_2464 CATTGCTATGCCAATCATGG TTCCCGAGGATAACTGTTGG 132 pLd3 BSR25_2483 ATGGTCTAACTCACGGCTTCGCAATA GTTAGAACGTCGTGAGAGCGTCTTAGAT 136 pLd9 pLd9_6 CAACGTGACCGGACGATCCAGATA GGTTGTGCGTGACGTTGAATCTGATAA 92 pLd10 mobB, pLd10_21 AACTGCCATAAATTCCAGCACCGATTAAA ATTGTCTGCCTGTTCTGTTTCTTAGTTGTC 193 pLd12 repB, pLd12_1 GCGTTTAGAATAGGCTTCTTGTTTGCTAGA ATGCAAAGCCCTTATACTAAGCTACTGTCT 198 ↵a The gene hisB, located with one copy on the chromosome, was targeted to quantify the amount of chromosomal DNA.
