Flanking direct repeats of hisG alter URA3 marker expression at the HWP1 locus of Candida albicans

HWP1 encodes an adhesin of Candida albicans and has been implicated in filamentation and virulence. URA3, an often-used transformation selection marker, is apparently incorrectly expressed when integrated at the HWP1 locus, which results in an attenuated virulence phenotype. Expression of URA3 is compromised by ectopic integration at other loci as well. In contrast, prior studies from the authors' laboratory had demonstrated that the filamentation deficiency and attenuated virulence of hwp1 Delta mutants were fully restored in rescued strains in which URA3 was integrated at the HWP1 locus. This discrepancy prompted a reinvestigation of these mutants. A series of congenic strains were constructed which demonstrated that the filamentation and virulence defects of a homozygous hwp1 Delta mutant could be rescued without introduction of a functional HWP1 allele. Despite the absence of detectable differences in URA3 expression, analysis of suppressor mutations suggested that reduced URA3 expression gave rise to the mutant phenotypes. Several independent spontaneous suppressor mutations that restored filamentation to strains of genotype hwp1 Delta:: hisG-URA3-hisG/hwp1 Delta:: hisG had acquired a tandem duplication of the hisG-URA3-hisG marker cassette. The hwp1 null mutant and rescued strains differed by the presence or absence of flanking hisG sequence. Substitution of the hisG-URA3-hisG insert of the hwp1 null mutant with URA3 alone largely rescued the filamentation and virulence phenotypes. The presence of a single copy of hisG adjacent to URA3 had no effect. It is concluded that flanking direct repeats of hisG, present as part of a recyclable disruption cassette, negatively influenced URA3 expression and are responsible for the previously reported phenotypes of the hwp1 mutants.