Isolation of Candida glabrata homologs of the Saccharomyces cerevisiae KRE9 and KNH1 genes and their involvement in cell wall beta-1,6-glucan synthesis

Abstract

The Candida glabrata KRE9 (CgKRE9) and KNH1 (CgKNH1) genes have been isolated as multicopy suppressors of the tetracycline-sensitive growth of a Saccharomyces cerevisiae mutant with the disrupted KNH1 locus and the KRE9 gene placed under the control of a tetracycline-responsive promoter. Although a cgknh1Delta mutant showed no phenotype beyond slightly increased sensitivity to the K1 killer toxin, disruption of CgKRE9 resulted in several phenotypes similar to those of the S. cerevisiae kre9Delta null mutant: a severe growth defect on glucose medium, resistance to the K1 killer toxin, a 50% reduction of beta-1,6-glucan, and the presence of aggregates of cells with abnormal morphology on glucose medium. Replacement in C. glabrata of the cognate CgKRE9 promoter with the tetracycline-responsive promoter in a cgknh1Delta background rendered cell growth tetracycline sensitive on media containing glucose or galactose. cgkre9Delta cells were shown to be sensitive to calcofluor white specifically on glucose medium. In cgkre9 mutants grown on glucose medium, cellular chitin levels were massively increased.

FIG. 1

Disruption of CgKRE9 and CgKNH1 and morphological effects of the deletions. (A) Disruption of CgKRE9. A PCR-amplified fragment (double-headed arrow) from pCGK9ΔT (Materials and Methods) was used for the one-step gene replacement. (B) Disruption of CgKNH1. A KpnI-SacI fragment of pCGK1ΔH (Materials and Methods) was used for the one-step gene replacement. Homologous recombination between the two regions (hatched boxes) resulted in disruption of the chromosomal copy. The wild-type strain, 2001HTU (C), cgkre9Δ deletion strain SNBG1-7-7 (D), and cgknh1Δ deletion strain SNBG2-26 (E) as viewed by Normarski optics are shown. Cells precultured on galactose medium were cultured on glucose medium.

FIG. 2

Construction of a tetracycline-sensitive mutant of CgKRE9 (Tet^s CgKRE9). (A) Scheme for replacement of the cognate CgKRE9 promoter region with the tetracycline-responsive promoter. A PCR-amplified fragment (double-headed arrow) from pCGK9tetAB (Materials and Methods) was used for the one-step gene replacement. The solid arrow indicates the ORF of CgKRE9. Open and shaded boxes indicate the S. cerevisiae URA3 gene and the tetracycline-responsive promoter, 97t, respectively. Homologous recombination between the two regions (hatched boxes) resulted in generation of the Tet^s CgKRE9 mutant. (B) Growth inhibition by tetracycline on the Tet^s CgKRE9 mutants. A total of 10⁴ cells were inoculated and were cultured on YPD (solid bars) or on YPGal (open bars) for 20 h at 30°C. Growth of cells with tetracycline (50 μg/ml) is expressed as percent of optical density at 600 nm of cells without tetracycline. As the wild type (WT), strain ACG22 (Table 1) was used. Error bars, standard deviations.

FIG. 3

Restriction maps and deletional analysis of inserts of C. glabrata genomic DNA on pSB2-1 and pSBG9-1. Open bars indicate the inserts on pSB2-1 (A) and pSBG9-1 (B). Fragments used for deletional analysis are represented by solid bars. The presence and absence of complementation activity in Tet^s KRE9 knh1Δ cells are indicated as + and −, respectively. Arrows indicate ORFs of CgKRE9 (A) and CgKNH1 (B). Hatched bars indicate regions with homology to the syntenic S. cerevisiae genes.

FIG. 4

Growth of S. cerevisiae Tet^s KRE9 knh1Δ cells harboring either pSB2-1 or pSBG9-1. About 10⁴ cells were inoculated and cultured on YNB-glucose for 20 h (solid bars) or on YNB-galactose for 40 h (open bars) at 30°C. Cells were grown with or without tetracycline (50 μg/ml), and growth on tetracycline is expressed as the percentage of optical density at 600 nm of cells grown without tetracycline. The strain FAHAP4 (Table 1) was used as the wild type (WT). Error bars, standard deviations.

FIG. 5

Sequence comparisons of CgKre9p and CgKnh1p with their S. cerevisiae counterparts. (A) Alignment of the putative Kre9p and Knh1p amino acid sequences deduced from the C. glabrata (CgKRE9 and CgKNH1) and S. cerevisiae (KRE9 and KNH1) nucleotide sequences. The residues with conserved identity in all proteins are underlined in the consensus sequence. The putative N-terminal signals for secretion are underlined in each protein. Gaps (shown as dashes) were introduced to improve alignment. (B) Sequence identities between Kre9p and Knh1p proteins.

FIG. 6

Growth of S. cerevisiae Tet^s KRE9 knh1Δ cells harboring a single copy of either CgKRE9 or CgKNH1. About 10⁴ cells were inoculated and cultured on YNB-glucose for 20 h (solid bars) or on YNB-galactose for 40 h (open bars) at 30°C. Growth of cells with tetracycline (50 μg/ml) is expressed as percent of optical density at 600 nm of cells without tetracycline. FAHAP4 (Table 1) was used as the wild-type. Error bars, standard deviations.

FIG. 7

Cellular chitin levels in cgkre9 mutants of C. glabrata. (A) Effects of addition of tetracycline on cellular chitin levels in Tet^s CgKRE9 mutants. About 10⁶ cells were cultured on YPD with (solid bars) or without (open bars) tetracycline (50 μg/ml) at 30°C for 20 h, and the cellular chitin levels were measured. As the wild type (WT), strain ACG22 (Table 1) was used. (B) Effect of switching the carbon source on cellular chitin levels in cgkre9Δ mutant. Cells precultured on YPGal were inoculated onto either YPD (solid bars) or YPGal (hatched bars) and cultured at 30°C for 20 h, and the cellular chitin levels were measured. As the wild type (WT), strain 2001HTU (Table 1) was used. Error bars, standard deviations.