No abstract

Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This ‘suicide’ CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans.

microRNAs (miRNAs), endogenous posttranscriptional repressors by base-pairing of their cognate mRNAs in plants and animals, have mostly been thought lost in the kingdom of fungi. Here, we report the identification of miRNAs from the fungus Cryptococcus neoformans. With bioinformatics and Northern blotting approaches, we found that these miRNAs and their hairpin precursors were present in this fungus. The size of miR1 and miR2 is 22 nt and 18 nt, respectively. The precursors are about ∼70 nt in length that is close to mammalian pre-miRNAs. Characteristic features of miRNAs are also found in miR1/2. We demonstrated that the identified miRNAs, miR1 and miR2, caused transgene silencing via the canonical RNAi pathway. Bioinformantics analysis helps to reveal a number of identical sequences of the miR1/2 in transposable elements (TEs) and pseudogenes, prompting us to think that fungal miRNAs might be involved in the regulation of the activity of transposons and the expression of pseudogenes. This study identified functional miRNAs in C. neoformans, and sheds light on the diversity and evolutionary origin of eukaryotic miRNAs.

The multicopper laccase is a major virulence factor in Cryptococcus neoformans. Its expression regulation is complex. We presented molecular evidence to show that laccase expression was induced by high concentrations of exogenous copper. Melanin production and laccase enzymatic activity increased dramatically in response to the addition of copper to the media. Reverse transcription-PCR amplification of the laccase gene LAC1 mRNA revealed that the induction occurred at the transcriptional level, which required the copper-responsive factor-encoding gene CUF1. Disruption of CUF1 demolished the activation of LAC1 transcription by copper, whereas the reconstituted strain restored the phenotypic defects. Furthermore, copper induction was shown to be independent of derepression by glucose starvation, a well-established activation factor for laccase expression. These results demonstrate a role of the copper-responsive factor gene CUF1 in the expression of laccase in C. neoformans.

Chaetomium globosum is one of the most common fungi in nature. It is best known for producing chaetoglobosins; however, the molecular basis of chaetoglobosin biosynthesis is poorly understood in this fungus. In this study, we utilized RNA interference (RNAi) to characterize a polyketide synthase gene, pks-1, in C. globosum that is involved in the production of chaetoglobosin A. When pks-1 was knocked down by RNAi, the production of chaetoglobosin A dramatically decreased. Knock-down mutants also displayed a pigment-deficient phenotype. These results suggest that the two polyketides, melanin and chaetoglobosin, are likely to share common biosynthetic steps. Most importantly, we found that pks-1 also plays a critical role in sporulation. The silenced mutants of pks-1 lost the ability to produce spores. We propose that polyketides may modulate cellular development via an unidentified action. We also suggest that C. globosum pks-1 is unique because of its triple role in melanin formation, chaetoglobosin biosynthesis and sporulation. This work may shed light on chaetoglobosin biosynthesis and indicates a relationship between secondary metabolism and fungal morphogenesis.

Lipids, commonly including phospholipids, sphingolipids, fatty acids, sterols, and triacylglycerols (TAGs), are important biomolecules for the viability of all cells. Phospholipids, sphingolipids, and sterols are important constituents of biological membranes. Many lipids play important roles in the regulation of cell metabolism by acting as signaling molecules. Neutral lipids, including TAGs and sterol esters (STEs), are important storage lipids in cells. In view of the importance of lipid molecules, this review briefly summarizes the metabolic pathways for sterols, phospholipids, sphingolipids, fatty acids, and neutral lipids in fungi and illustrates the differences between fungal and human (or other mammalian) cells, especially in relation to lipid biosynthetic pathways. These differences might provide valuable clues for us to find target proteins for novel antifungal drugs. In addition, the development of lipidomics technology in recent years has supplied us with a shortcut for finding new antifungal drug targets; this ability is important for guiding our research on pathogenic fungi.

Wooden shipwrecks are a significant part of the underwater cultural heritage. In 2007, the Nanhai No. 1 shipwreck was salvaged from the seabed and moved into the Marine Silk Road Museum, where it is still stored in a water tank. We analysed the microbial communities colonizing the hull surface of the Nanhai No. 1 shipwreck during storage. Six samples exposed to air were collected from different spots of the ship that exhibited obvious microbial plaques. High-throughput sequencing revealed the bacterial community includes both aquatic and terrestrial species, while in the fungal community, Fusarium was the most abundant genus across all samples and accounted for 84.91% to 98.40% of the total community composition. Two Fusarium species were isolated from the samples and were identified as F. solani and F. oxysporum. Both of the isolates were able to degrade cellulose, but only F. solani had the ability to degrade lignin. Antimicrobial efficacy in inhibiting the growth of Fusarium was assessed with five kinds of biocides, and isothiazolinones exhibited specific inhibition of Fusarium growth. These results provide critical background information to protect and reduce the biodegradation and destruction of this important historical shipwreck, and inform efforts to protect other similar artifacts.

A new gene (named AP gene) encoding an alkaline serine protease with dehairing function was cloned from Bacillus pumilus UN-31-C-42 and its nucleotide sequence was determined. The expression of AP gene was induced with IPTG in Escherichia coli after the mature protease region was cloned into pET15b and SDS-PAGE showed expressed product clearly, but no alkaline protease activity was detected. In order to express the AP gene in B. subtilis, a recombinant expression plasmid was constructed which contained a promoter Bp53 (also from B. pumilus), the AP gene and an E. coli-B. subtilis shuttle vector pSUGV4. This plasmid was introduced into B. subtilis WB600 and the transformant displayed the hydrolyzed zone on a milk plate. The expressed product can be easily detected with SDS-PAGE and the fermentation fluid of the transformant showed low alkaline protease activity and dehairing activity. This is the first report of a gene cloned from B. pumilus, encoding an alkaline serine protease, which can alone accomplish the whole dehairing process.