Current Genetics最新文献

Insect pathogenic fungi, also known as entomopathogenic fungi, are one of the largest insect pathogenic microorganism communities, represented by Beauveria spp. and Metarhizium spp. Entomopathogenic fungi have been proved to be a great substitute for chemical pesticide in agriculture. In fact, a lot of functional genes were also already characterized in entomopathogenic fungi, but more depth of exploration is still needed to reveal their complicated pathogenic mechanism to insects. Metarhizium rileyi (Nomuraea rileyi) is a great potential biocontrol fungus that can parasitize more than 40 distinct species (mainly Lepidoptera: Noctuidae) to cause large-scale infectious diseases within insect population. In this study, a comparative analysis of transcriptome profile was performed with topical inoculation and hemolymph injection to character the infectious pattern of M. rileyi. Appressorium and multiple hydrolases are indispensable constituents to break the insect host primary cuticle defense in entomopathogenic fungi. Within our transcriptome data, numerous transcripts related to destruction of insect cuticle rather growth regulations were obtained. Most importantly, some unreported ribosomal protein genes and novel unannotated protein (hypothetical protein) genes were proved to participate in the course of pathogenic regulation. Our current data provide a higher efficiency gene library for virulence factors screen in M. rileyi, and this library may be also useful for furnishing valuable information on entomopathogenic fungal pathogenic mechanisms to host.

pET expression plasmids are widely used for producing recombinant proteins in Escherichia coli. Selection and maintenance of cells harboring a pET plasmid are possible using either a Tn3.1-type genetic fragment (which encodes a ß-lactamase and confers resistance to ß-lactam antibiotics) or a Tn903.1-type genetic fragment (which encodes an aminoglycoside-3'-phosphotransferase and confers resistance aminoglycoside antibiotics). Herein we have investigated how efficiently pET plasmids are maintained using these two fragments. The study reveals that pET plasmids are efficiently maintained with both Tn3.1 and Tn903.1 genetic fragments prior to the induction of recombinant protein production, and over short induction times (i.e., 2 h). However, over longer induction times (i.e., 20 h), the efficiency of plasmid maintenance depends on the host strain used, and the type of antibiotic selection cassette used. Based on our collective observations, we have 2 general tips for efficiently maintaining pET plasmids during recombinant production experiments. Tip #1: Use a strain with lowered levels of the T7 RNA polymerase, such as C41(DE3). pET plasmids will be efficiently maintained over long induction times with both the Tn3.1 and Tn903.1 genetic fragments, regardless of whether antibiotics are present during cultivation. Tip #2: If a strain with higher levels of T7 RNA polymerase strain is necessary, such as BL21(DE3)), keep induction times short or use a plasmid containing a Tn903.1-type fragment and select with kanamycin.

The demand for and acceptance of probiotics is determined by their quality and safety. Illumina NGS sequencing and analytics were used to examine eight marketed probiotics. Up to the species level, sequenced DNA was taxonomically identified, and relative abundances were determined using Kaiju. The genomes were constructed using GTDB and validated through PATRICK and TYGS. A FastTree 2 phylogenetic tree was constructed using several type strain sequences from relevant species. Bacteriocin and ribosomally synthesized polypeptide (RiPP) genes were discovered, and a safety check was performed to test for toxins, antibiotic resistance, and genetic drift genes. Except for two products with unclaimed species, the labeling was taxonomically correct. In three product formulations, Lactobacillus acidophilus, Limosilactobacillus reuteri, Lacticaseibacillus paracasei, and Bifidobacterium animalis exhibited two to three genomic alterations, while Streptococcus equinus was found in one. TYGS and GDTB discovered E. faecium and L. paracasei in distinctly different ways. All the bacteria tested had the genetic repertoire to tolerate GIT transit, although some exhibited antibiotic resistance, and one strain had two virulence genes. Except for Bifidobacterium strains, the others revealed a variety of bacteriocins and ribosomally synthesized polypeptides (RiPP), 92% of which were unique and non-homologous to known ones. Plasmids and mobile genetic elements are present in strains of L. reuteri (NPLps01.et_L.r and NPLps02.uf_L.r), Lactobacillus delbrueckii (NPLps01.et_L.d), Streptococcus thermophilus (NPLps06.ab_S.t), and E. faecium (NPLps07.nf_E.f). Our findings support the use of metagenomics to build better and efficient production and post-production practices for probiotic quality and safety assessment.

Eukaryotic DNA replication is accompanied by the disassembly and reassembly of nucleosomes and the transmission of epigenetic marks to the newly assembled chromatids. Several histone chaperones, including CAF-1 and Asf1p, are central to these processes. On the other hand, replication forks pause at numerous positions throughout the genome, but it is not known if and how this pausing affects the reassembly and maintenance of chromatin structures. Here, we applied drug-free gene silencing assays to analyze the genetic interactions between CAC1, ASF1, and two genes that regulate the stability of the paused replisome (TOF1) and the resumption of elongation (RRM3). Our results show that TOF1 and RRM3 differentially interact with CAF-1 and ASF1 and that the deletions of TOF1 and RRM3 lead to reduced silencing and increased frequency of epigenetic conversions at three loci in the genome of S. cerevisiae. Our study adds details to the known activities of CAF-1 and Asf1p and suggests that the pausing of the replication fork can lead to epigenetic instability.

Binding of general transcription factors TFIID and TFIIA to basal promoters is rate-limiting for transcriptional initiation of eukaryotic protein-coding genes. Consequently, activator proteins interacting with subunits of TFIID and/or TFIIA can drastically increase the rate of initiation events. Yeast transcriptional activator Ino2 interacts with several Taf subunits of TFIID, among them the multifunctional Taf1 protein. In contrast to mammalian Taf1, yeast Taf1 lacks bromodomains which are instead encoded by separate proteins Bdf1 and Bdf2. In this work, we show that Bdf1 not only binds to acetylated histone H4 but can also be recruited by Ino2 and unrelated activators such as Gal4, Rap1, Leu3 and Flo8. An activator-binding domain was mapped in the N-terminus of Bdf1. Subunits Toa1 and Toa2 of yeast TFIIA directly contact sequences of basal promoters and TFIID subunit TBP but may also mediate the influence of activators. Indeed, Ino2 efficiently binds to two separate structural domains of Toa1, specifically with its N-terminal four-helix bundle structure required for dimerization with Toa2 and its C-terminal β-barrel domain contacting TBP and sequences of the TATA element. These findings complete the functional analysis of yeast general transcription factors Bdf1 and Toa1 and identify them as targets of activator proteins.

BRCA2 is a tumor-suppressor gene that is normally expressed in the breast and ovarian tissue of mammals. The BRCA2 protein mediates the repair of double-strand breaks (DSBs) using homologous recombination, which is a conserved pathway in eukaryotes. Women who express missense mutations in the BRCA2 gene are predisposed to an elevated lifetime risk for both breast cancer and ovarian cancer. In the present study, the efficiency of human BRCA2 (hBRCA2) in DSB repair was investigated in the budding yeast Saccharomyces cerevisiae. While budding yeast does not possess a true BRCA2 homolog, they have a potential functional homolog known as Rad52, which is an essential repair protein involved in mediating homologous recombination using the same mechanism as BRCA2 in humans. Therefore, to examine the functional overlap between Rad52 in yeast and hBRCA2, we expressed the wild-type hBRCA2 gene in budding yeast with or without Rad52 and monitored ionizing radiation resistance and DSB repair efficiency. We found that the expression of hBRCA2 in rad52 mutants increases both radiation resistance and DSB repair frequency compared to cells not expressing BRCA2. Specifically, BRCA2 improved the protection against ionizing radiation by at least 1.93-fold and the repair frequency by 6.1-fold. In addition, our results show that homology length influences repair efficiency in rad52 mutant cells, which impacts BRCA2 mediated repair of DSBs. This study provides evidence that S. cerevisiae could be used to monitor BRCA2 function, which can help in understanding the genetic consequences of BRCA2 variants and how they may contribute to cancer progression.

Functional amyloids have been identified in a wide variety of organisms including bacteria, fungi, plants, and vertebrates. Intracellular and extracellular amyloid fibrils of different proteins perform storage, protective, structural, and regulatory functions. The structural organization of amyloid fibrils determines their unique physical and biochemical properties. The formation of these fibrillar structures can provide adaptive advantages that are picked up by natural selection. Despite the great interest in functional and pathological amyloids, questions about the conservatism of the amyloid properties of proteins and the regularities in the appearance of these fibrillar structures in evolution remain almost unexplored. Using bioinformatics approaches and summarizing the data published previously, we have shown that amyloid fibrils performing similar functions in different organisms have been arising repeatedly and independently in the course of evolution. On the other hand, we show that the amyloid properties of a number of bacterial and eukaryotic proteins are evolutionarily conserved. We also discuss the role of protein-based inheritance in the evolution of microorganisms. Considering that missense mutations and the emergence of prions cause the same consequences, we propose the concept that the formation of prions, similarly to mutations, generally causes a negative effect, although it can also lead to adaptations in rare cases. In general, our analysis revealed certain patterns in the emergence and spread of amyloid fibrillar structures in the course of evolution.

In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen species formation by flow cytometry confirmed that Itraconazole clearly induced an internal oxidative stress, which can be significantly abolished in rotenone-exposed cells. Analysis of the antioxidant enzymatic activities of catalase and superoxide dismutase pointed to a moderate decrease of catalase in trehalase-deficient mutant cells compared to the wild type, with an additional increase upon addition of rotenone. These enzymatic changes were imperceptible in the case of superoxide dismutase. Alternative assays with Voriconazole led to a similar profile in the results regarding cell growth and antioxidant activities. Collectively, our data suggest that the antifungal action of Itraconazole on C. parapsilosis is dependent on a functional mitochondrial activity. They also suggest that the central metabolic pathways in pathogenic fungi should be considered as preferential antifungal targets in new research.

In fungi, the cell wall plays a crucial role in morphogenesis and response to stress from the external environment. Chitin is one of the main cell wall components in many filamentous fungi. In Aspergillus nidulans, a class III chitin synthase ChsB plays a pivotal role in hyphal extension and morphogenesis. However, little is known about post-translational modifications of ChsB and their functional impacts. In this study, we showed that ChsB is phosphorylated in vivo. We characterized strains that produce ChsB using stepwise truncations of its N-terminal disordered region or deletions of some residues in that region and demonstrated its involvement in ChsB abundance on the hyphal apical surface and in hyphal tip localization. Furthermore, we showed that some deletions in this region affected the phosphorylation states of ChsB, raising the possibility that these states are important for the localization of ChsB to the hyphal surface and the growth of A. nidulans. Our findings indicate that ChsB transport is regulated by its N-terminal disordered region.

The cAMP-dependent protein kinase (PKA) pathway in Schizosaccharomyces pombe plays an important role in microtubule organization and chromosome segregation. Typically, loss of functional Pka1 induces sensitivity to the microtubule-destabilizing drug thiabendazole (TBZ) and chromosome mis-segregation. To determine the mechanism via which Pka1 is involved in these events, we explored the relevance of transcription factors by creating a double-deletion strain of pka1 and 102 individual genes encoding transcription factors. We found that rst2∆, tfs1∆, mca1∆, and moc3∆ suppressed the TBZ-sensitive phenotype of the pka1∆ strain, among which tfs1∆ was the strongest suppressor. All single mutants (rst2∆, tfs1∆, mca1∆, and moc3∆) showed a TBZ-tolerant phenotype. Tfs1 has two transcriptional domains (TFIIS and Zn finger domains), both of which contributed to the suppression of the pka1∆-induced TBZ-sensitive phenotype. pka1∆-induced chromosome mis-segregation was rescued by tfs1∆ in the presence of TBZ. tfs1 overexpression induced the TBZ-sensitive phenotype and a high frequency of chromosome mis-segregation, suggesting that the amount of Tfs1 must be strictly controlled. However, Tfs1-expression levels did not differ between the wild-type and pka1∆ strains, and the Tfs1-GFP protein was localized to the nucleus and cytoplasm in both strains, which excludes the direct regulation of expression and localization of Tfs1 by Pka1. Growth inhibition by TBZ in pka1∆ strains was notably rescued by double deletion of rst2 and tfs1 rather than single deletion of rst2 or tfs1, indicating that Rst2 and Tfs1 contribute independently to counteract TBZ toxicity. Our findings highlight Tfs1 as a key transcription factor for proper chromosome segregation.