Fig. 9.
Influence of zrfC or aspf2 expression on the growth ability in acidic media of the AF801 (zaPR → zrfC) and AF891 (zaPR → aspf2) strains, respectively, and transcription of zrfC and aspf2 in both strains. (A) Growth of AF801 and AF891 on both acid (SDAE; pH 4.5) and alkaline (SDNE; pH 7.5) zinc-limiting agar media not supplemented with Zn2+ or supplemented with 1 or 10 μM Zn2+, as indicated at the top of each panel. (B) Analysis by Northern blotting of the transcription of zrfC and aspf2 under the control of the zaPR region in the AF801 and AF891 strains. These strains were constructed following the same strategy used previously to construct prototrophic pyrG+ strains at the pyrG1 locus of A. fumigatus but using a fragment contained in either the pZRF320 (for zrfC) or pASPF361 (for aspf2) plasmid as transforming DNA. The expression levels of zrfC in the AF891 strain and aspf2 in AF801 were analyzed as endogenous controls for the correct functioning of the wild-type zaP regions carried by these strains. Expression of zrfC in the AF731 strain and that of aspf2 in AF881 were also analyzed as additional controls, since these strains also express at the pyrG loci zrfC and aspf2, respectively, but under the control of a wild-type zaP region. The zaPR region that drives transcription of the tested gene is depicted on the right side of each blot. All fungal strains were grown in either the acid SDA or alkaline SDN medium for 20 h at 37°C with a supplement of 100 μM Zn2+ (+) or without a supplement of Zn2+ (−), as indicated at the top of each lane.