The analysis of <i>CEN1</i> and <i>CEN2</i> properties.

<p>(a) The <i>CEN1</i> locus has 65.6% AT content and carries the ser-tRNA gene; it carries part of a gene homologous to the <i>RPT2</i> gene, the <i>TVP15</i> gene, and the <i>COPI</i>-coated vesicle-protein encoding gene. The <i>CEN2</i> 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 <i>CEN1</i> and within a 3’-region of <i>CEN2</i>. This subfigure was drawn using the DNA/RNA GC Content Calculator (<a href="http://www.endmemo.com/bio/gc.php" target="_blank">http://www.endmemo.com/bio/gc.php</a>). (c) The transformation efficiency of three plasmid types. (1) The plasmid with <i>CEN1</i>. (2) Two plasmids with <i>CEN2</i>, specifically the Y881 strain possessing the transposon and the Y879 strain lacking the transposon. (3) The deletion plasmids of <i>CEN2</i>, specifically <i>CEN2-1</i>, <i>CEN2-2</i>, <i>CEN2-3</i>, <i>CEN2-4</i>, <i>CEN2-5</i>; the plasmid <i>CEN2-2</i> has a transposon. The P892/BI plasmid, which carried the <i>URA3</i> gene and which had been linearized by <i>Bam</i>HI (BI), was used as a control. (d) The schematic representation of the <i>CEN1</i>, <i>CEN2</i> and <i>CEN2</i> 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 <i>CEN2</i> fragment (from 1925 to 2120 bp), named <i>CEN2-2</i>, was used as a query in a search through the <i>D</i>. <i>bruxellensis</i> genome (Y879 strain). 10 sequences (and their surroundings) were randomly sampled from the 51 present in the genome (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161741#pone.0161741.s012" target="_blank">S2 Table</a>) and aligned with <i>CEN2-2</i> (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).</p

Chromosomal-break assay and association with the centromere histone H3.

<p>(a) Karyotype analysis of <i>D</i>. <i>bruxellensis</i> transformants carrying <i>CEN1</i> and <i>CEN2</i> 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 <i>CEN1</i> (OP98 and OP99) and the sub-cloned fragment of <i>CEN2</i> (<i>CEN2-5</i>, 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.</p

Yeast phylogeny and centromeres.

<p>The centromeres are signified by coloured ovals. Our cloned <i>D</i>. <i>bruxellensis</i> centromeres have features of both “regional” and “point” centromeres, and they appear in <i>green—blue</i>. The “conventional point” centromeres appear in <i>blue</i>; the “unconventional point” centromeres in <i>red</i>; the “regional” centromeres in <i>green</i>. 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.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Hybridization of <i>CEN1</i> and <i>CEN2</i> to chromosomes.

<p>The chromosome-sized DNA of the strains (Y879, Y881, Y891, Y880, Y865, Y883, Y900, and Y901) was separated on PFGE gel and hybridized with <i>CEN</i> probes. For the <i>CEN1</i> locus, the full fragment was used as a probe (amplified with primers OP98 and OP99). For the <i>CEN2</i> locus, the <i>CEN2-1</i> 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 <i>CEN1</i> and <i>CEN2</i> were marked with yellow and purple stars on the PFGE gel.</p

<i>CEN1</i> and <i>CEN2</i> plasmids stability in Y997 transformants.

<p><i>CEN1</i> and <i>CEN2</i> plasmids stability in Y997 transformants.</p

Biofilm, mat formation, and colony morphology of Y997 transformants carrying <i>CEN1</i> and <i>CEN2</i> plasmids.

<p>(a) Biofilm and total biomass of transformants grown in YNB with 0.2% glucose for 48 h. Transformants analysed: (1) integrative (P892/<i>Pst</i>I (control strain carrying empty vector); P950/<i>Pst</i>I (<i>CEN2</i> with transposon); P1210/<i>Pst</i>I (<i>CEN2</i> without transposon, from Y879 strain); P1038/<i>Pst</i>I (<i>CEN2-5</i> fragment with transposon); P1172/<i>Hind</i>III (plasmid carrying <i>CEN1</i>)); (2) replicative (P950 (<i>CEN2</i> with transposon); P1210 (<i>CEN2</i> without transposon, from Y879 strain); P1038 (<i>CEN2-5</i> fragment with transposon); P1172 (<i>CEN1</i>)). (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/<i>Pst</i>I (P892 integrated into the genome). P950; P1210; P1038 and P1172.</p