Coccidioides posadasii contains a single 1,3-beta-glucan synthase gene that appears to be essential for growth

Abstract

1,3-beta-Glucan synthase is responsible for the synthesis of beta-glucan, an essential cell wall structural component in most fungi. We sought to determine whether Coccidioides posadasii possesses genes homologous to known fungal FKS genes that encode the catalytic subunit of 1,3-beta-glucan synthase. A single gene, designated FKS1, was identified, and examination of its predicted protein product showed a high degree of conservation with Fks proteins from other filamentous fungi. FKS1 is expressed at similar levels in mycelia and early spherulating cultures, and expression decreases as the spherules mature. We used Agrobacterium-mediated transformation to create strains that harbor DeltaFKS1::hygB, a null allele of FKS1, and hypothesize that Fks1p function is essential, due to our inability to purify this allele away from a complementing wild-type FKS1 allele in a heterokaryotic strain. The heterokaryon appears normal with respect to growth rate and arthroconidium production; however, microscopic examination of strains with DeltaFKS1::hygB alleles revealed abnormal swelling of hyphal elements.

FIG. 1.

The C. posadasii Fks1 protein sequence is similar to Fks protein sequences from related pathogenic fungi. A graphic representation of C. posadasii Fks1p is shown. Features indicated are the glycine repeat region, the catalytic core domain, and the UDP-glucose consensus binding site (RITG). A multiple sequence alignment of selected fungal FKS catalytic domains is shown in the bottom half of the figure (26). The alignment was generated using ClustalW (version 1.4) and the BLOSUM (blocks substitution matrix) similarity matrix with an open gap penalty of 10, an extended gap penalty of 0.05, and a delay divergent of 40%. Shaded residues indicate amino acid identities. The predicted protein sequences of P. brasiliensis Fks1p (amino acids 754 to 1331) (accession number AF148715) and A. fumigatus Fks1p (amino acids 755 to 1332) (accession number U79728) are aligned with the C. posadasii Fks1p predicted protein sequence (amino acids 760 to 1337).

FIG. 2.

FKS1 RNA levels decrease during spherule maturation relative to the level in mycelia. FKS1 RNA levels were determined using real-time RT-PCR, normalized to actin as an internal control, and then expressed as severalfold changes during spherule development relative to the levels found in mycelia. The two differently shaded columns for each time point represent biological replicates.

FIG. 3.

FKS1 gene structure and deletion plasmid. (A) The genomic structure of the FKS1 locus is depicted, with a scale bar below in 1-kb increments. Primers used to generate PCR fragments containing the upstream and downstream flanking sequences with added restriction sites for cloning are indicated. (B) pAM1253 is the broad-host-range plasmid used to transform C. posadasii and create the FKS1 deletion.

FIG. 4.

Southern analysis of C. posadasii pAM1253 transformants. (A) Genomic DNA (10 μg) from transformant strains VFC10, VFC12, VFC13, VFC15, VFC16, and VFC43 and wild-type strain Silveira was digested with NdeI and separated on a 0.8% agarose gel. The gel was transferred to nitrocellulose, and the blot was probed with a radiolabeled 1.3-kb EcoRI-PstI fragment from the upstream region of FKS1. An autoradiograph of the resulting blot is shown on the left, and the predicted fragment sizes and genomic structures of possible recombinants are shown on the right. (B) The blot depicted in panel A was stripped of the 1.3-kb EcoRI-PstI FKS1 probe and reprobed with a radiolabeled 1.4-kb fragment containing the hphB gene, as shown on the diagram at the right side of the figure.

FIG. 5.

Transformant strains with ΔFKS1::hphB alleles show hyphal defects. (A to C) Transformant strains were grown for 72 h in 2× GYE agar medium with hygromycin at 50 μg/ml, stained with DAPI and calcofluor, and visualized using a Leica compound microscope with fluorescent optics. The strains in the micrographs are as follows: VFC12 (ΔFKS1::hphB; FKS1^WT) (A), VFC16 (ΔFKS1::hphB; FKS1^WT) (B), and VFC13 (ectopic hphB; FKS1^WT) (C). Scale bars are indicated in the upper left corners of the micrographs. Arrows indicate abnormally shaped hyphal compartments. (D to F) Transformant strains and the wild-type Silveira strain were grown as described above, with the inclusion of caspofungin (20 μg/ml) and the omission of hygromycin. The strains shown are as follows: Silveira (D), VFC13 (E), and VFC12 (F).