Conditional lethal disruption of TATA-binding protein gene in Penicillium marneffei
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.
References (39)
- et al.
Optimized vectors and selection for transformation of Neurospora crassa and Aspergillus nidulans to bleomycin and phleomycin resistance
Gene
(1990) - et al.
Identification and distinct regulation of yeast TATA box-containing genes
Cell
(2004) Multiple TATA-binding factors come back into style
Cell
(1997)- et al.
Dimerization of the TATA binding protein
J. Biol. Chem.
(1995) The induction and repression of nitrate reductase in the fungus Aspergillus nidulans
Biochem. Biophys. Acta
(1966)- et al.
An amdS–lacZ fusion for studying gene regulation in Aspergillus
Gene
(1988) - et al.
SPT15, the gene encoding the yeast TATA binding factor TFIID, is required for normal transcription initiation in vivo
Cell
(1989) - et al.
Isolation of the gene encoding the yeast TATA binding protein TFIID: a gene identical to the SPT15 suppressor of Ty element insertions
Cell
(1989) - et al.
Transcription properties of a cell type-specific TATA-binding protein, TRF
Cell
(1997) - et al.
Cloning and structural organization of a xylanase-encoding gene from Penicillium chrysogenum
Gene
(1993)
A role for TBP dimerization in preventing unregulated gene expression
Mol. Cell
Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes
Gene
Control of morphogenesis in the human fungal pathogen Penicillium marneffei
Int. J. Med .Microbiol.
Current Protocols In Molecular Biology
The abaA homologue of Penicillium marneffei participates in two developmental programmes: conidiation and dimorphic growth
Mol. Microbiol.
An STE12 homolog from the asexual, dimorphic fungus Penicillium marneffei complements the defect in asexual development of an Aspergillus nidulans steA mutant
Genetics
The basic helix–loop–helix protein with similarity to the fungal morphological regulators, Phd1p, Efg1p and StuA, controls conidiation but not dimorphic growth in Penicillium marneffei
Mol. Microbiol.
The CDC42 homolog of the dimorphic fungus Penicillium marneffei is required for correct cell polarization during growth but not development
J. Bacteriol.
Biochemistry and structural biology of transcription factor IID (TFIID)
Annu. Rev. Biochem.
Cited by (12)
Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species
2022, Metabolic Engineering CommunicationsCitation Excerpt :The xylP promoter (pxylP) controlling expression of a xylanase from Penicillium chrysogenum allows high induction by xylan or its degradation product xylose with low basal activity in the absence of an inducer (Haas et al., 1993; Zadra et al., 2000). pxylP was demonstrated to permit conditional gene expression of diverse genes in various mold species including P. chrysogenum (Bugeja et al., 2010, 2013; Huber et al., 2019; Janus et al., 2009; Kopke et al., 2013; Pongsunk et al., 2005; Sigl et al. 2010, 2011), Penicillium marneffei (Bugeja et al., 2010, 2013; Pongsunk et al., 2005), Aspergillus nidulans (Monahan et al., 2006; Wong et al., 2007; Wong et al. 2008; Wong et al. 2009; Tribus et al., 2010; Ma et al., 2018; Pidroni et al., 2018; Wang et al., 2021; Li et al., 2021; Unkles et al., 2014), Aspergillus fumigatus (Yasmin et al., 2012; Gsaller et al., 2012; Fazius et al., 2012; Altwasser et al., 2015; Baldin et al. 2015, 2021; Vaknin et al., 2016; Misslinger et al., 2019; Bauer et al., 2019; López-Berges et al., 2021; Handelman et al., 2021; Fabri et al., 2021) and Sordaria macrospora (Kopke et al., 2010). A. fumigatus is a ubiquitous saprobic fungus but at the same time the most common mold pathogen of humans.
A new variant of self-excising β-recombinase/six cassette for repetitive gene deletion and homokaryon purification in Neurospora crassa
2014, Journal of Microbiological MethodsCitation Excerpt :To date, it is possibly the best regulatable promoter in Aspergilli. It has been also successfully used in Penicillium marneffei (Monahan et al., 2006; Pongsunk et al., 2005). Hartmann et al. (2010) used this promoter in A. fumigatus to regulate expression of β-recombinase in a self-excising marker cassette, which was tightly shut off during the selection of transformants and highly expressed during eviction of the cassette.
The interplay between iron and zinc metabolism in Aspergillus fumigatus
2009, Fungal Genetics and BiologyAn Overlooked and Underrated Endemic Mycosis—Talaromycosis and the Pathogenic Fungus Talaromyces marneffei
2023, Clinical Microbiology ReviewsThe Lysine Deacetylase RpdA Is Essential for Virulence in Aspergillus fumigatus
2019, Frontiers in Microbiology