Conditional lethal disruption of TATA-binding protein gene in Penicillium marneffei

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Abstract

Problems can arise in studying the regulation of transcription in fungi if gene disruption is employed to evaluate the role of essential transcription factors. Herein, we have developed a method to characterize the essential genes of Penicillium marneffei. This has been used to examine the significance of P. marneffei TATA-binding protein (TBP) in growth and development. Strains in which the expression of TbpA could be regulated were constructed by placing tbpA under the control of the xylP promoter. The construct was introduced into P. marneffei and the resulting strains were used to produce P. marneffei tbpA deletion strains. Phenotypic examination of growth of the tbpA overexpressing strains revealed that high levels of TbpA expression inhibit fungal growth at conidial germination in both filamentous and yeast forms. Under repressing conditions, the tbpA deletion strains failed to grow at 25 °C whilst showing reduced growth at 37 °C. The results suggested that TbpA is essential for P. marneffei filamentous growth, but plays a less significant role in growth and development during the yeast phase.

Introduction

Penicillium marneffei is an opportunistic human pathogen endemic to southeast Asia and southern China (Sirisanthana and Sirisanthana, 1995). P. marneffei differs from all other members of its genus in that it displays a temperature dependent dimorphism (Andrianopoulos, 2002). When cultured at 25 °C, P. marneffei grows in a hyphal form and produces a red pigment that diffuses into agar. At 37 °C, P. marneffei hyphae undergo a process known as arthroconidiation to produce uninucleate arthroconidia which divide by fission to form the uninucleate yeast cells (Chan and Chow, 1990, Garrison and Boyd, 1973). The yeast form of P. marneffei is of pathological relevance and is predominant intracellularly during host infection. The regulatory mechanisms which control dimorphic switching and pathogenicity in dimorphic pathogenic fungi, such as P. marneffei, are of much interest.

TATA-binding protein (TBP) is a general transcription factor required for initiation of transcription in eukaryotes. It is a small protein of approximately 30 kDa. During transcription initiation, TBP acts as a common subunit that interacts with other specific transcription factors resulting in the formation of transcription pre-initiation complex by all three RNA polymerases (Hernandez, 1993). The TBP core domain folds into a symmetrical structure that resembles a saddle. The symmetry extends beyond the direct sequence repeats, such that the two halves are similar in structure, even though they differ in sequence (Hernandez, 1993). This core domain also binds activators, TBP-associated factors (TAFs), repressors, and general transcription factors (Hernandez, 1993).

TBP encoding genes have been cloned and characterized in a number of fungi. Mutations in TBP-encoding SPT15 gene from Saccharomyces cerevisiae produce pleiotropic effects, causing defects in mating, sporulation, and growth. Loss-of-function mutations of SPT15 result in lethality, showing that SPT15 is essential for growth (Eisenmann et al., 1989, Hahn et al., 1989). Although Spt15 can function interchangeably with mammalian TBP in RNA polymerase II in vitro transcription assays, human TBP cannot suppress the cell viability defect of Δspt15 strains. This is due to an inability of human TBP to fully substitute for Spt15 in RNA polymerase III-dependent transcription. The findings demonstrate that the activities of this general transcription factor are partially conserved among eukaryotic organisms (Cormack et al., 1994). Other fungal TBP-encoding genes were cloned from Candida albicans (TBP1) (Leng et al., 1998) and Aspergillus nidulans (tbpA) (Kucharski and Bartnik, 1997). Both could suppress the lethality associated with Δspt15 mutations in S. cerevisiae. Nevertheless, the effect of deleting the C. albicans or A. nidulans gene was not examined.

Understanding the transcriptional mechanisms which control the dimorphic program is central to understanding this developmental phenomenon, as well as the capacity of dimorphic fungi to infect the host and cause disease. Several studies have identified transcription factors which control various aspects of development in P. marneffei, including conidiation and dimorphic switching, and most of these factors are believed to be RNA polymerase II-dependent (Borneman et al., 2000, Borneman et al., 2001, Borneman et al., 2002). In this study, the TBP-encoding gene, tbpA, of P. marneffei was identified, cloned, and characterized. The data suggest that the TbpA is required for filamentous growth at 25 °C, but is less relevant to the growth of the pathogenic yeast form at 37 °C. Given that TBP loss-of-function mutations are lethal in S. cerevisiae, a combined conditional allele, and overexpression strategy was developed to examine the function of TbpA in P. marneffei using tbpA null mutants. This approach will permit the study of other essential genes in P. marneffei and can be applied to a range of fungal systems.

Section snippets

Fungal strains, plasmids, and libraries

Penicillium marneffei strains used in this study are listed in Table 1. FRR2161 was kindly provided by Dr. J. Pitt (CSIRO Food Industries, Sydney, Australia). A cDNA library was constructed in pSPORT1 by using mRNA from P. marneffei strain RT-72 grown at 37 °C for 14 days (Pongsunk and Chaiyaroj, unpublished). The flanking regions of the tbpA gene for homologous recombination was selected from a λ-BlueSTAR genomic library of P. marneffei strain FRR2161 (Andrianopoulos, unpublished).

To generate a

Cloning of the P. marneffei tbp A homologue

A P. marneffei cDNA library was screened for genes differentially expressed in the hyphal form as compared to the yeast form. A clone, pSPORT-733, containing a 1.7 kb DNA insert was identified. DNA sequence analysis of the insert revealed an open reading frame (ORF) of 768 bp with high homology to genes encoding TATA-binding proteins (TBP), including SPT15 from S. cerevisiae and tbpA from A. nidulans. The deduced amino acid sequence of the tbpA ORF is predicted to encode a 255-amino acid

Discussion

TBP is an important constituent of the transcriptional machinery for all eukaryotic RNA polymerases examined to date. Comparison of the predicted amino acid sequence of the P. marneffei tbpA gene among different eukaryote species revealed strong conservation of the C-terminal domain with more than 75% identity between human TBP and other TBP (Hernandez, 1993). In contrast to the C-terminus, the N-terminus of TBP is more divergent and has unclear function. The deduced amino acid sequence of P.

Acknowledgments

We thank Drs. M.J. Hynes, M.A. Davis, and R.B. Todd for their valuable scientific suggestions, and Dr. J. Svasti for critical reading of the manuscript. We acknowledge A. Blanchfield and Q. Lang for their technical assistance. This work was supported by BIOTEC, National Science and Technology Development Agency (NSTDA) and the Chulabhorn Research Institute. S.P. was supported by The Royal Golden Jubilee Ph.D. Program of the Thailand Research Fund.

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