Supplementary document 1:
acdA was originally annotated as AN2264.3 at the Broad Institute (http://www.broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html) or as AN2264 at the Aspergillus Genome Database (http://www.aspergillusgenome.org/). The sequences were fusions of three genes on both databases and the Broad Institute has re-annotated the sequence (acdA is now ANID_12335.1). However, the new annotation failed to annotate the correct stop codon. Therefore, a re-annotation was performed with the new coordinates shown below.
acdA
Chromosome VII
3,371, 3,371,029 – 3,369,484 (Crick strand)
1546 nucleotides
Relative Coordinates / Chromosomal CoordinatesCDS / 1 – 75 / 3,371,029 – 3,370,955
Intron / 76 – 148 / 3,370,954 – 3,370882
CDS / 149 – 394 / 3,370,881 – 3,370,636
Intron / 395 – 467 / 3,370,635 – 3,370,563
CDS / 468 – 680 / 3,370,562 – 3,370,350
Intron / 681 – 736 / 3,370,349 – 3,370,294
CDS / 737 - 1546 / 3,370,293 – 3,369,484
acdB was originally annotated as AN10834.3 at the Broad Institute or as AN10834 at the Aspergillus Genome Database. Re-sequencing of the gene detected an error in the original genome sequence. The sequence obtained from both databases contain an additional C at position 393, which leads to a frameshift and an inability to establish the correct start codon at position 364. Re-annotation was performed with the new coordinates shown below.
acdB
Chromosome I
2,574,866 – 2,573,332 (Crick strand)
1528 nucleotides
Relative Coordinates / Chromosomal CoordinatesCDS / 1 – 951 / 2,574,866 – 2,573,916
Intron / 952 – 1006 / 2,573,917 – 2,573,854
CDS / 1007 – 1528 / 2,573,853 – 2,573,332
aoxB was originally annotated as AN6765.3 at the Broad Institute or as AN6765 at the Aspergillus Genome Database. Alignments with S. cerevisiae FOX1 and A. nidulans aoxA suggested the occurrence of an additional intron. Re-annotation was performed with the new coordinates shown below.
aoxB
Chromosome I, 3,097,788 – 3,100,194 (Crick strand)
2407 nucleotides
Relative Coordinates / Chromosomal CoordinatesCDS / 1 – 776 / 3,100,194 – 3,099,419
Intron / 777 – 875 / 3,099,418 – 3,099,320
CDS / 876 – 1273 / 3,099,319 – 3,098,922
Intron / 1274 – 1315 / 3,098,921 – 3,098,880
CDS / 1316 – 1659 / 3,098,879 – 3,098,536
Intron / 1660 – 1718 / 3,098,535 – 3,098,477
CDS / 1719 – 1800 / 3,098,476 – 3,098,395
Intron / 1801 – 1842 / 3,098,394 – 3,098,353
CDS / 1843 – 1983 / 3,098,352 – 3,098,212
Intron / 1984 – 2042 / 3,098,211 – 3,098,153
CDS / 2043 – 2407 / 3,098,152 – 3,098,017
Intron / 2408 – 2238 / 3,098,016 – 3,097,957
CDS / 2239 – 2407 / 3,097,956 – 3,097,788
Figure S1: Phylogenetic tree of Fox1 and homologues including the putative FA-CoA oxidases AoxA and AoxB
The phylogenetic tree was produced using ClustalW (accurate) (Thompson et al. 1994), Seqboot and Protpars (Felsenstein 1989) in Biomanager by ANGIS (http://www.angis.org.au). The tree was edited using TreeView (Page 1998) and is shown as a radial tree. Bootstrap values and scale bar (amino acid substitutions/site) are displayed. No out-group has been selected.
The tree of AoxA and AoxB was produced using alignments of following homologous proteins: A. clavatus 1: ACLA_007790, A. clavatus 2: ACLA_094690 re-annotated; A. flavus 1: AFL2G_00480.2, A. flavus 2: AFL2G_06552.2 re-annotated; A. flavus 3: AFL2G_11284.2; A. fumigatus: Afu7g06090 and Afu7g06100 re-annotated; A. nidulans AoxA: AN6752.3, A. nidulans AoxB: AN6765.3 re-annotated; A. niger: e_gw1_5.1444.1; A. oryzae 1: AO090005000479, A. oryzae 2: AO090010000014, A. terreus 1: ATEG_04376.1, A. terreus 2: ATEG_06288.1, A. terreus 3: ATEG_06308.1, A. terreus 4: ATEG_09798.1; B. cinerea: BC1G_08482.1; C. albicans 1: orf19.1652, C. albicans 2: orf19.1655, C. albicans 3: orf19.5723; C. immitis 1: CIMG_01877.3; C. immitis 2: CIMG_10832.3, C. immitis 3: CIMG_11894.3 re-annotated; F. graminearum: FGSG_02287.3; N. fischeri: NFIA_027340; S. cerevisiae Fox1: YGL205W; Y. lipolytica 1: YALI0C23859, Y. lipolytica 2: YALI0D24750, Y. lipolytica 3: YALI0E06567, Y. lipolytica 4: YALI0E27654, Y. lipolytica 5: YALI0E32835, Y. lipolytica 6: YALI0E10857.
Figure S2: Alignment of N. crassa NCU01181.3 and the A. nidulans putative FA-CoA dehydrogenases AcdA and AcdB
The amino acid sequence of N. crassa NCU01181.3 and the re-annotated protein sequences of A. nidulans AcdA (AN2264.3) and AcdB (AN10834.3) have been aligned using the programs ClustalW (accurate) (Thompson et al. 1994) and Boxshade in Biomanager (http://www.angis.org.au). Identical amino acids are shaded in black, similar amino acids in grey. The acyl-CoA dehydrogenase middle domain (first box) and C-terminal domain (second box) are marked.
Figure S3: Localisation of the GFP-AcdA and GFP-AcdB
Spore suspensions of strains expressing the GFP fusion proteins in a wild-type (WT) background, MH11914 (GFP-AcdA) and MH11915 (GFP-AcdB), or in pexE background, MH11920 (GFP-AcdA) and MH11921 (GFP-AcdB) were inoculated in supplemented minimal media and incubated overnight at 24ºC. Microscope slides were prepared, viewed and photographed. Hyphae were visualised by DIC and GFP expression was detected as described in Material and Methods. The magnification is 125x; the scale bar represents 20µm. Each construct is listed above to the appropriate panel of photos.
Figure S4: Occurrence of the CCGAGG sequence in the promoters of acdA and acdB and induction of acdA(p)lacZ and acdB(p)lacZ by different carbon sources
1kb of the promoter (p) of acdA (a) and acdB (b) (-1000 to –1) is shown as a schematic overview in intervals of 100bp. CCGAGG sequences are represented by triangles. The numbers above the triangles indicate location of the CCGAGG sequence.
ß-galactosidase assays of acdA(p)lacZ (a) and acdB(p)lacZ (b) in different wild-type, farA∆, farB∆, scfA∆ and farA∆farB∆ background were performed. All strains were grown in supplemented glucose-minimal media for 16h at 37ºC. Mycelia were washed with and transferred to supplemented media containing either 1% glucose, 50mM proline, 10mM butyrate or 2.5mM oleate as sole carbon source for an additional 4h. Standard error was calculated from three independent experiments. All assays are recorded in units per minute per mg protein, but the scales differ between the two figures. The strains used for acdA(p)lacZ were MH11566, MH11648, MH11649, MH1150 and MH12008. For acdB(p)lacZ, the strains used were MH12055, MH12129, MH12130, MH12131.
1