Fig 1.
Yeast phylogeny and centromeres.
The centromeres are signified by coloured ovals. Our cloned D. bruxellensis centromeres have features of both “regional” and “point” centromeres, and they appear in green—blue. The “conventional point” centromeres appear in blue; the “unconventional point” centromeres in red; the “regional” centromeres in green. A black oval displays the whole-genome duplication event (WGD). The phylogeny is adapted from Curtin and Pretorius, 2014, and the phylogeny and centromeres are adapted from Kobayashi et al., 2015.
Table 1.
Yeast strains used in this study.
Table 2.
Plasmids used in this study.
Fig 2.
Southern hybridization of the total DNA of the transformants carrying CEN1 or CEN2 plasmids.
(a) The gel and hybridization membrane for the CEN2 plasmid transformants P950 and P1210. (b) The gel and hybridization membrane obtained for the CEN2-5 (P1038) and CEN1 (P1172) transformants. The total DNA was digested either with EcoRI (present on the plasmids) or BglII (absent on the plasmids). Digested with EcoRI, the control plasmids were P950 (CEN2 of Y881), P1210 (CEN2 of Y879), P1038 (CEN2-5 deletion plasmid of CEN2) and P1172 (CEN1). Linearized by PstI (P950, P1210 and P1038) or HindIII (P1172) and labeled with [γ-32P] dCTP, the whole plasmid was used as the hybridization probe. In the figure, the EcoRI restriction-fragments of the corresponding plasmids are shown with arrows. As all the plasmids have a pUC57 segment, the probes also hybridized with 1 kb ladder (10787018, Invitrogen, Life Technologies).
Fig 3.
The analysis of CEN1 and CEN2 properties.
(a) The CEN1 locus has 65.6% AT content and carries the ser-tRNA gene; it carries part of a gene homologous to the RPT2 gene, the TVP15 gene, and the COPI-coated vesicle-protein encoding gene. The CEN2 locus has 62.4% AT content and it carries the gene coding for the MATE efflux family protein. (b) The AT-peaks occur at the intergenic region in CEN1 and within a 3’-region of CEN2. This subfigure was drawn using the DNA/RNA GC Content Calculator (http://www.endmemo.com/bio/gc.php). (c) The transformation efficiency of three plasmid types. (1) The plasmid with CEN1. (2) Two plasmids with CEN2, specifically the Y881 strain possessing the transposon and the Y879 strain lacking the transposon. (3) The deletion plasmids of CEN2, specifically CEN2-1, CEN2-2, CEN2-3, CEN2-4, CEN2-5; the plasmid CEN2-2 has a transposon. The P892/BI plasmid, which carried the URA3 gene and which had been linearized by BamHI (BI), was used as a control. (d) The schematic representation of the CEN1, CEN2 and CEN2 deletion-plasmids. The sequences that promote autonomous replication and stable maintenance are marked with a plus sign (+); the remaining sequences are marked with a minus sign (-). (e) The MITE-like transposon found within the CEN2 fragment (from 1925 to 2120 bp), named CEN2-2, was used as a query in a search through the D. bruxellensis genome (Y879 strain). 10 sequences (and their surroundings) were randomly sampled from the 51 present in the genome (S2 Table) and aligned with CEN2-2 (reference sequence) to present the “structural” parts of the transposon: inverted and direct repeats. The bases identical to the reference sequence are shown in grey. Inverted repeats are 7 bp long and they are at the beginning and end of the transposon; marked with red arrows only on the reference sequence, inverted repeats are present in all sequences in the genome. Direct repeats are marked with blue arrows; they are 5 bp long but have different sequences, and they immediately flank the transposon (in the genome of Y879 strain).
Table 3.
CEN1 and CEN2 plasmids stability in Y997 transformants.
Fig 4.
Hybridization of CEN1 and CEN2 to chromosomes.
The chromosome-sized DNA of the strains (Y879, Y881, Y891, Y880, Y865, Y883, Y900, and Y901) was separated on PFGE gel and hybridized with CEN probes. For the CEN1 locus, the full fragment was used as a probe (amplified with primers OP98 and OP99). For the CEN2 locus, the CEN2-1 part (amplified with primers OP91 and OP127) was used as a probe; this was performed to avoid the transposon part, which is present in the genome in several copies. The hybridization signals for both CEN1 and CEN2 were marked with yellow and purple stars on the PFGE gel.
Fig 5.
Direct and inverted repeats and motif sequences in CEN1 and CEN2.
(a) CDE-, ARS-, CB-like elements, D. bruxellensis specific motifs, ser-tRNA gene, and transposon-like sequences are shown. Sc–S. cerevisiae; Db–D. bruxellensis; Nc–N. castellii; Yl–Y. lipolytica (b) Direct and inverted repeats are shown on the CEN1 and CEN2 sequence. Repeats of the same type are shown with the same colour pattern.
Fig 6.
Chromosomal-break assay and association with the centromere histone H3.
(a) Karyotype analysis of D. bruxellensis transformants carrying CEN1 and CEN2 plasmids integrated into the genome by PFGE. Y879 and Y997 strains were used as controls. Transformants with changed karyotypes are arrowed. (b) Co-immunoprecipitation with Cse4 antibodies. ChIP samples were used as a template for PCR performed at two annealing temperatures (64 and 65°C) using specific primers for CEN1 (OP98 and OP99) and the sub-cloned fragment of CEN2 (CEN2-5, primers SW9 and SW10). Samples: 1) 1 kb ladder; 2) ChIP Ca antibodies with Y879 chromatin (primers OP98 and OP99); 3) ChIP Ca antibodies with Y879 chromatin (primers OP98 and OP99); 4) ChIP Mouse Ig, negative control antibody with Y879 chromatin (primers OP98 and OP99); 5) no antibody, negative control with Y879 chromatin (primers OP98 and OP99); 6) 1 kb ladder; 7) input of ChIP assay, Y879 chromatin (primers OP98 and OP99); 8) 1 kb ladder; 9) ChIP Ca antibodies with Y879 chromatin (primers SW9 and SW10); 10) ChIP Ca antibodies with Y879 chromatin (primers OP98 and OP99); 11) 1 kb ladder; 12) input of ChIP assay, Y879 chromatin (primers SW9 and SW9); 13) input of ChIP assay, Y879 chromatin (primers OP98 and OP99); 14) ChIP Mouse Ig, negative control antibody with Y879 chromatin (primers SW9 and SW10); 15) no antibody, negative control with Y879 chromatin (primers SW9 and SW10); 16) ChIP Mouse Ig, negative control antibody with Y879 chromatin (primers OP98 and OP99); 17) no antibody, negative control with Y879 chromatin (primers OP98 and OP99); 18) 1kb ladder.
Fig 7.
Biofilm, mat formation, and colony morphology of Y997 transformants carrying CEN1 and CEN2 plasmids.
(a) Biofilm and total biomass of transformants grown in YNB with 0.2% glucose for 48 h. Transformants analysed: (1) integrative (P892/PstI (control strain carrying empty vector); P950/PstI (CEN2 with transposon); P1210/PstI (CEN2 without transposon, from Y879 strain); P1038/PstI (CEN2-5 fragment with transposon); P1172/HindIII (plasmid carrying CEN1)); (2) replicative (P950 (CEN2 with transposon); P1210 (CEN2 without transposon, from Y879 strain); P1038 (CEN2-5 fragment with transposon); P1172 (CEN1)). (b) The biofilm normalized by biomass. (c) The colony morphology of corresponding transformants on YNB medium with 2% glucose and 2% agar. (d) Mat formation by Y997 transformants on YPD with 0.3% agar grown 30 days at room temperature. (e) The DNA content of each transformant carrying circular plasmid estimated by flow cytometry and compared to that of the control strain P892/PstI (P892 integrated into the genome). P950; P1210; P1038 and P1172.