Sporulation Defect Research Articles

Suppression of Vps13 adaptor protein mutants reveals a central role for PI4P in regulating prospore membrane extension.

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71-Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation.

Genetic Dissection of Vps13 Regulation in Yeast Using Disease Mutations from Human Orthologs.

The VPS13 family of proteins have emerged as key players in intracellular lipid transport and human health. Humans have four different VPS13 orthologs, the dysfunction of which leads to different diseases. Yeast has a single VPS13 gene, which encodes a protein that localizes to multiple different membrane contact sites. The yeast vps13Δ mutant is pleiotropic, exhibiting defects in sporulation, protein trafficking, endoplasmic reticulum (ER)-phagy and mitochondrial function. Non-null alleles resulting from missense mutations can be useful reagents for understanding the multiple functions of a gene. The exceptionally large size of Vps13 makes the identification of key residues challenging. As a means to identify critical residues in yeast Vps13, amino acid substitution mutations from VPS13A, B, C and D, associated with human disease, were introduced at the cognate positions of yeast VPS13, some of which created separation-of-function alleles. Phenotypic analyses of these mutants have revealed that the promotion of ER-phagy is a fourth, genetically separable role of VPS13 and provide evidence that co-adaptors at the endosome mediate the activity of VPS13 in vacuolar sorting.

Characterization of the mbsA Gene Encoding a Putative APSES Transcription Factor in Aspergillus fumigatus.

The APSES family proteins are transcription factors (TFs) with a basic helix-loop-helix domain, known to regulate growth, development, secondary metabolism, and other biological processes in Aspergillus species. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus, five genes predicted to encode APSES TFs are present. Here, we report the characterization of one of these genes, called mbsA (Afu7g05620). The deletion (Δ) of mbsA resulted in significantly decreased hyphal growth and asexual sporulation (conidiation), and lowered mRNA levels of the key conidiation genes abaA, brlA, and wetA. Moreover, ΔmbsA resulted in reduced spore germination rates, elevated sensitivity toward Nikkomycin Z, and significantly lowered transcripts levels of genes associated with chitin synthesis. The mbsA deletion also resulted in significantly reduced levels of proteins and transcripts of genes associated with the SakA MAP kinase pathway. Importantly, the cell wall hydrophobicity and architecture of the ΔmbsA asexual spores (conidia) were altered, notably lacking the rodlet layer on the surface of the ΔmbsA conidium. Comparative transcriptomic analyses revealed that the ΔmbsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes, which was corroborated by elevated levels of GT production in the mutant. While the ΔmbsA mutant produced higher amount of GT, ΔmbsA strains showed reduced virulence in the murine model, likely due to the defective spore integrity. In summary, the putative APSES TF MbsA plays a multiple role in governing growth, development, spore wall architecture, GT production, and virulence, which may be associated with the attenuated SakA signaling pathway.

CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research.

The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious agents in wound botulism, infant botulism, and intestinal toxemia in adults. The identification of factors that contribute to spore formation is, therefore, essential to the development of strategies to control related health risks. Accordingly, development of a straightforward and versatile gene manipulation tool and an efficient sporulation-promoting medium is pivotal. Our strategy was to employ CRISPR-Cas9 and homology-directed repair (HDR) to replace targeted genes with mutant alleles incorporating a unique 24-nt “bookmark” sequence that could act as a single guide RNA (sgRNA) target for Cas9. Following the generation of the sporulation mutant, the presence of the bookmark allowed rapid generation of a complemented strain, in which the mutant allele was replaced with a functional copy of the deleted gene using CRISPR-Cas9 and the requisite sgRNA. Then, we selected the most appropriate medium for sporulation studies in C. botulinum Group II strains by measuring the efficiency of spore formation in seven different media. The most effective medium was exploited to confirm the involvement of a candidate gene in the sporulation process. Using the devised sporulation medium, subsequent comparisons of the sporulation efficiency of the wild type (WT), mutant and “bookmark”-complemented strain allowed the assignment of any defective sporulation phenotype to the mutation made. As a strain generated by complementation with the WT gene in the original locus would be indistinguishable from the parental strain, the gene utilized in complementation studies was altered to contain a unique “watermark” through the introduction of silent nucleotide changes. The mutagenesis system and the devised sporulation medium provide a solid basis for gaining a deeper understanding of spore formation in C. botulinum, a prerequisite for the development of novel strategies for spore control and related food safety and public health risk management.

Fission Yeast Methylenetetrahydrofolate Reductase Ensures Mitotic and Meiotic Chromosome Segregation Fidelity.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the folate metabolic pathway, and its loss of function through polymorphisms is often associated with human conditions, including cancer, congenital heart disease, and Down syndrome. MTHFR is also required in the maintenance of heterochromatin, a crucial determinant of genomic stability and precise chromosomal segregation. Here, we characterize the function of a fission yeast gene met11+, which encodes a protein that is highly homologous to the mammalian MTHFR. We show that, although met11+ is not essential for viability, its disruption increases chromosome missegregation and destabilizes constitutive heterochromatic regions at pericentromeric, sub-telomeric and ribosomal DNA (rDNA) loci. Transcriptional silencing at these sites were disrupted, which is accompanied by the reduction in enrichment of histone H3 lysine 9 dimethylation (H3K9me2) and binding of the heterochromatin protein 1 (HP1)-like Swi6. The met11 null mutant also dominantly disrupts meiotic fidelity, as displayed by reduced sporulation efficiency and defects in proper partitioning of the genetic material during meiosis. Interestingly, the faithful execution of these meiotic processes is synergistically ensured by cooperation among Met11, Rec8, a meiosis-specific cohesin protein, and the shugoshin protein Sgo1, which protects Rec8 from untimely cleavage. Overall, our results suggest a key role for Met11 in maintaining pericentromeric heterochromatin for precise genetic inheritance during mitosis and meiosis.

The Fission Yeast RNA-Binding Protein Meu5 Is Involved in Outer Forespore Membrane Breakdown during Spore Formation.

In Schizosaccharomyces pombe, the spore wall confers strong resistance against external stress. During meiosis II, the double-layered intracellular forespore membrane (FSM) forms de novo and encapsulates the nucleus. Eventually, the inner FSM layer becomes the plasma membrane of the spore, while the outer layer breaks down. However, the molecular mechanism and biological significance of this membrane breakdown remain unknown. Here, by genetic investigation of an S. pombe mutant (E22) with normal prespore formation but abnormal spores, we showed that Meu5, an RNA-binding protein known to bind to and stabilize more than 80 transcripts, is involved in this process. We confirmed that the E22 mutant does not produce Meu5 protein, while overexpression of meu5+ in E22 restores the sporulation defect. Furthermore, electron microscopy revealed that the outer membrane of the FSM persisted in meu5∆ spores. Investigation of the target genes of meu5+ showed that a mutant of cyc1+ encoding cytochrome c also showed a severe defect in outer FSM breakdown. Lastly, we determined that outer FSM breakdown occurs coincident with or after formation of the outermost Isp3 layer of the spore wall. Collectively, our data provide novel insights into the molecular mechanism of spore formation.

The membrane mucin Msb2 regulates aflatoxin biosynthesis and pathogenicity in fungus Aspergillus flavus.

As a pathogenic fungus, Aspergillus flavus can produce carcinogenic aflatoxins (AFs), which poses a great threat to crops and animals. Msb2, the signalling mucin protein, is a part of mitogen-activated protein kinase (MAPK) pathway which contributes to a range of physiological processes. In this study, the roles of membrane mucin Msb2 were explored in A. flavus by the application of gene disruption. The deletion of msb2 gene (Δmsb2) caused defects in vegetative growth, sporulation and sclerotia formation when compared to WT and complement strain (Δmsb2C ) in A. flavus. Using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis, it was found that deletion of msb2 down-regulated aflatoxin B1 (AFB1 ) synthesis and decreased the infection capacity of A. flavus. Consistently, Msb2 responds to cell wall stress and osmotic stress by positively regulating the phosphorylation of MAP kinase. Notably, Δmsb2 mutant exhibited cell wall defect, and it was more sensitive to inhibitor caspofungin when compared to WT and Δmsb2C . Taking together, these results revealed that Msb2 plays key roles in morphological development process, stresses adaptation, secondary metabolism and pathogenicity in fungus A. flavus.

A Novel Mycovirus Evokes Transcriptional Rewiring in the Fungus Malassezia and Stimulates Beta Interferon Production in Macrophages.

Mycoviruses infect fungi, and while most persist asymptomatically, there are examples of mycoviruses having both beneficial and detrimental effects on their host. Virus-infected Saccharomyces and Ustilago strains exhibit a killer phenotype conferring a growth advantage over uninfected strains and other competing yeast species, whereas hypovirus-infected Cryphonectria parasitica displays defects in growth, sporulation, and virulence. In this study, we identify a double-stranded RNA (dsRNA) mycovirus in five Malassezia species. Sequence analysis reveals it to be a totivirus with two dsRNA segments: a larger 4.5-kb segment with genes encoding components for viral replication and maintenance, and a smaller 1.4-kb segment encoding a novel protein. Furthermore, transcriptome sequencing (RNA-seq) of virus-infected versus virus-cured Malassezia sympodialis revealed an upregulation of dozens of ribosomal components in the cell, suggesting the virus modifies the transcriptional and translational landscapes of the cell. Given that Malassezia is the most abundant fungus on human skin, we assessed the impact of the mycovirus in a murine epicutaneous infection model. Although infection with virus-infected strains was not associated with an increased inflammatory response, we did observe enhanced skin colonization in one of two virus-infected M. sympodialis strains. Noteworthy, beta interferon expression was significantly upregulated in bone marrow-derived macrophages when challenged with virus-infected, compared to virus-cured, M. sympodialis, suggesting that the presence of the virus can induce an immunological response. Although many recent studies have illuminated how widespread mycoviruses are, there are relatively few in-depth studies about their impact on disease caused by the host fungus. We describe here a novel mycovirus in Malassezia and its possible implications in pathogenicity.IMPORTANCEMalassezia species represent the most common fungal inhabitant of the mammalian skin microbiome and are natural skin commensal flora. However, these fungi are also associated with a variety of clinical skin disorders. Recent studies have reported associations of Malassezia with Crohn's disease and pancreatic cancer, further implicating this fungal genus in inflammatory and neoplastic disease states. Because M. sympodialis has lost genes involved in RNA interference (RNAi), we hypothesized Malassezia could harbor dsRNA mycoviruses. Indeed, we identified a novel mycovirus of the totivirus family in several Malassezia species and characterized the MsMV1 mycovirus of M. sympodialis We found conditions that lead to curing of the virus and analyzed isogenic virus-infected/virus-cured strains to determine MsMV1 genetic and pathogenic impacts. MsMV1 induces a strong overexpression of transcription factors and ribosomal genes, while downregulating cellular metabolism. Moreover, MsMV1 induced a significantly higher level of beta interferon expression in cultured macrophages. This study sheds light on the mechanisms of pathogenicity of Malassezia, focusing on a previously unidentified novel mycovirus.

Rho factor mediates flagellum and toxin phase variation and impacts virulence in Clostridioides difficile.

The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the “flagellar switch” upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained single-nucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5’ leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile.

Sexual reproduction potential implied by functional analysis of SPO11 in Phaeodactylum tricornutum.

Phaeodactylum tricornutum is a model microalgae that is widely used to study diatom physiology and ecology. Since the meiotic process and sexual cycle have never been observed directly, P. tricornutum has been considered to be an asexual species. However, phylogenetic analysis of the P. tricornutum genome has revealed a series of meiosis-specific gene homologues in this species. We identified two copies of differently transcribed SPO11 homologs that contain the conserved motifs of Winged-helix and Toprim domains. The homolog PtSPO11-3 interacts with TopoVIB in yeast two-hybrid analysis, whereas the homolog PtSPO11-2 could rescue the sporulation defect of a Spo11 yeast mutant strain. PtSPO11-2 was also found to be significantly up-regulated at low temperatures in P. tricornutum and its key catalytic residue was important to the homolog's function in sporulation. The results herein provide positive clue that meiosis and sexual reproduction could exist in this diatom.

The intersection between stress responses and inositol pyrophosphates in Saccharomyces cerevisiae.

Saccharomyces cerevisiae adapts to oxidative, osmotic stress and nutrient deprivation through transcriptional changes, decreased proliferation, and entry into other developmental pathways such as pseudohyphal formation and sporulation. Inositol pyrophosphates are necessary for these cellular responses. Inositol pyrophosphates are molecules composed of the phosphorylated myo-inositol ring that carries one or more diphosphates. Mutations in the enzymes that metabolize these molecules lead to altered patterns of stress resistance, altered morphology, and defective sporulation. Mechanisms to alter the synthesis of inositol pyrophosphates have been recently described, including inhibition of enzyme activity by oxidation and by phosphorylation. Cells with increased levels of 5-diphosphoinositol pentakisphosphate have increased nuclear localization of Msn2 and Gln3. The altered localization of these factors is consistent with the partially induced environmental stress response and increased expression of genes under the control of Msn2/4 and Gln3. Other transcription factors may also exhibit increased nuclear localization based on increased expression of their target genes. These transcription factors are each regulated by TORC1, suggesting that TORC1 may be inhibited by inositol pyrophosphates. Inositol pyrophosphates affect stress responses in other fungi (Aspergillus nidulans, Ustilago maydis, Schizosaccharomyces pombe, and Cryptococcus neoformans), in human and mouse, and in plants, suggesting common mechanisms and possible novel drug development targets.

The proppin Bcas3 and its interactor KinkyA localize to the early phagophore and regulate autophagy

ABSTRACT To resolve the signaling mechanisms that mediate the starvation-induced processes of Dictyostelium sporulation and encystation, we performed insertional mutagenesis on cells harboring an mRFP-tagged spore gene. We isolated a mutant in kinkyA (knkA), a gene without known function, which formed fruiting bodies with a kinked stalk and lacking viable spores. Immunoprecipitation of lysates of KnkA-YFP-transformed knkA− cells yielded a mammalian BCAS3 homolog as a KnkA interactor. bcas3− phenocopied knkA− and Bcas3 colocalized with KnkA to puncta. Bcas3 shares sequence similarity with proppins (beta-propellors that bind phosphoinositides). Mutation of 2 Bcas3 residues that are essential for PtdIns3P binding in proppins prevented Bcas3 binding to PtdIns3P as well as punctate Bcas3 and KnkA localization. KnkA puncta also colocalized with small but not large vesicles that contain the autophagy protein Atg8 and were contiguous with the endoplasmic reticulum. knkA− and bcas3− cells showed a pronounced decrease of RFP-GFP-Atg8 in neutral early autophagosomes, indicating that KnkA and Bcas3 are required for macroautophagy/autophagy. Knockouts in atg7, atg5 or atg9 substantiated this finding by showing similar sporulation defects as knkA− and bcas3− . Defective Dictyostelium sporulation is evidently a useful diagnostic tool for the discovery of novel autophagy genes. Abbreviations: Atg: Autophagy-related; BCAS3: BCAS3 microtubule associated cell migration factor; cAMP: 3ʹ,5ʹ-cyclic adenosine monophosphate; ER: endoplasmic reticulum; GFP: green fluorescent protein; PAS: phagophore assembly site; PRKA/PKA: protein kinase cAMP-dependent; Proppin: beta‐propellers that bind phosphoinositides; PtdIns3P: phosphatidylinositol 3-phosphate; REMI: restriction enzyme-mediated insertional mutagenesis; RFP: red fluorescent protein; RT-qPCR: reverse transcriptase - quantitative polymerase chain reaction; WIPI: WD repeat domain, phosphoinositide interacting; YFP: yellow fluorescent protein

Genetic Analysis of Four Sexual Differentiation Process Proteins (isp4/SDPs) in Chaetomium thermophilum and Thermomyces lanuginosus Reveals Their Distinct Roles in Development

Fungal sexual development requires the involvement of a large number of functional genes. Fungal genes encoding sexual differentiation process proteins (SDPs), isps, have been known for decades. isp4/SDP and its homologs function as oligopeptide transporters (OPTs), yet their roles in reproduction are unknown. Here, we genetically analyzed all four isp4/SDP homologs in the sexual species Chaetomium thermophilum and asexual species Thermomyces lanuginosus. Using single gene deletion mutants, we found that T. lanuginosus SDP (TlSDP) participated in asexual sporulation, whereas the other homologs participated in sexual morphogenesis. In complementary tests, C. thermophilum SDPs (CtSDP1-3) restored sporulation defects in TlSDP deletion strains (ΔTlSDP), and their translated proteins, which were localized onto the cytomembrane, possessed OPT activity. Interestingly, CtSDP2 accumulated at the top of the hyphae played a distinct role in determining the sexual cycle, glutathione transport, and lifespan shortening. A unique 72nt-insertion fragment (72INS) was discovered in CtSDP2. Biological analysis of the 72INS deletion and DsRED-tagged fusion strains implied the involvement of 72INS in fungal growth and development. In contrast to TlSDP, which only contributes to conidial production, the three CtSDPs play important roles in sexual and asexual reproduction, and CtSDP2 harbors a unique functional 72INS that initiates sexual morphogenesis.

Slx5p-Slx8p Promotes Accurate Chromosome Segregation by Mediating the Degradation of Synaptonemal Complex Components during Meiosis.

Meiosis increases genetic diversity, yet the genome complement needs to be stable to ensure offspring viability. Both small ubiquitin‐like modifier (SUMO) and ubiquitin have been reported to participate in meiotic regulation, yet functions of the SUMO‐ubiquitination crosstalk in meiosis remain unclear. Here, it is reported that a SUMO‐targeted ubiquitin ligase, Slx8p, promotes accurate chromosome segregation during meiosis, since the deletion of SLX8 leads to increased aneuploidy due to a defect in synaptonemal complex (SC) component degradation. Both the RING domain and SUMO interacting motifs of Slx8p are essential for meiotic progression and maintaining spore viability, and the expression of tetraubiquitin fused with SUMO partially rescues meiotic defects in the SLX8‐deletion strain. Furthermore, Slx5p‐Slx8p can directly add ubiquitin to SUMOylated Zip1p and Ecm11p, and forced degradation of Ecm11p partially rescues the sporulation defects of the SLX8 deletion strain. These findings provide a mechanism for SC disassembly and reveal that the crosstalk between SUMOylation and ubiquitination facilitates accurate chromosome segregation by promoting SC component degradation during meiosis.

Construction of a Codon-Adapted Nourseotricin-Resistance Marker Gene for Efficient Targeted Gene Deletion in the Mycophenolic Acid Producer Penicillium brevicompactum.

Penicillium brevicompactum is a filamentous ascomycete used in the pharmaceutical industry to produce mycophenolic acid, an immunosuppressant agent. To extend options for genetic engineering of this fungus, we have tested two resistance markers that have not previously been applied to P. brevicompactum. Although a generally available phleomycin resistance marker (ble) was successfully used in DNA-mediated transformation experiments, we were not able to use a commonly applicable nourseothricin resistance cassette (nat1). To circumvent this failure, we constructed a new nat gene, considering the codon bias for P. brevicompactum. We then used this modified nat gene in subsequent transformation experiments for the targeted disruption of two nuclear genes, MAT1-2-1 and flbA. For MAT1-2-1, we obtained deletion strains with a frequency of about 10%. In the case of flbA, the frequency was about 4%, and this disruption strain also showed reduced conidiospore formation. To confirm the deletion, we used ble to reintroduce the wild-type genes. This step restored the wild-type phenotype in the flbA deletion strain, which had a sporulation defect. The successful transformation system described here substantially extends options for genetically manipulating the biotechnologically relevant fungus P. brevicompactum.

Alternative Oxidase Is Involved in the Pathogenicity, Development, and Oxygen Stress Response of Botrytis cinerea.

Alternative oxidase (AOX) is a ubiquinol terminal oxidase that is involved in fungal mitochondrial oxidative phosphorylation. In this study, we analyzed the roles of AOX in Botrytis cinerea by generating BcAOX deletion mutants. The mutants exhibited defects in mycelial growth, sporulation, spore germination, and virulence. Furthermore, the sensitivity of the mutants to quinone outside inhibitor fungicides and oxidative stress were increased. All phenotypic variations could be restored in the complemented strain. In summary, these results showed that BcAOX is involved in the regulation for vegetative development, adaptation to environmental stress, and virulence of B. cinerea.

The Velvet Proteins VosA and VelB Play Different Roles in Conidiation, Trap Formation, and Pathogenicity in the Nematode-Trapping Fungus Arthrobotrys oligospora

The velvet family proteins VosA and VelB are involved in growth regulation and differentiation in the model fungus Aspergillus nidulans and other filamentous fungi. In this study, the orthologs of VosA and VelB, AoVosA, and AoVelB, respectively, were characterized in the nematode-trapping fungus Arthrobotrys oligospora, which captures nematodes by producing trapping devices (traps). Deletion of the AovelB gene resulted in growth defects in different media, and the aerial hyphae from the ΔAovelB mutant lines were fewer in number and their colonies were less dense than those from the wild-type (WT) strain. The ΔAovelB mutants each displayed serious sporulation defects, and the transcripts of several sporulation-related genes (e.g., abaA, flbC, rodA, and vosA) were significantly down-regulated compared to those from the WT strain. Furthermore, the ΔAovelB mutant strains became more sensitive to chemical reagents, including sodium dodecyl sulfate and H2O2. Importantly, the ΔAovelB mutants were unable to produce nematode-capturing traps. Similarly, extracellular proteolytic activity was also lower in the ΔAovelB mutants than in the WT strain. In contrast, the ΔAovosA mutants displayed no obvious differences from the WT strain in these phenotypic traits, whereas conidial germination was lower in the ΔAovosA mutants, which became more sensitive to heat shock stress. Our results demonstrate that the velvet protein AoVelB is essential for conidiation, trap formation, and pathogenicity in A. oligospora, while AoVosA plays a role in the regulation of conidial germination and heat shock stress.

Mutants defective in the production of encapsulin show a tan-phase-locked phenotype in Myxococcus xanthus.

Myxococcus xanthus, a myxobacterium, displays phase variation between yellow phase and tan phase. We found that deletion of the encA gene encoding encapsulin and the encF gene encoding a metalloprotease causes formation of tan colonies that never transform into yellow colonies. The encA and encF mutants were defective in the production of DK-xanthene and myxovirescin. They did not produce extracellular polysaccharides; hence, the cells did not aggregate in liquid and showed reduced swarming on agar plates. The mutants had defective sporulation, but were rescued extracellularly by wild type cells. All these traits indicate that the encA and encF mutants are likely to be tan-phase-locked, and encapsulin has a close relationship with phase variation in M. xanthus. The encA and encF genes are localized in the same gene cluster, encBAEFG (MXAN_3557~MXAN_3553). Unlike the encA and encF genes, deletion of other genes in the cluster did not show tan-phase-locked phenotype.

Deletion and Characterization of Two Signaling Genes in Streptomyces

Streptomyces coelicolor is a filamentous, high G‐C (guanine‐cytosine), gram‐positive bacterium that produces an aerial mycelium. The genome consists of a 8,667,507 base pair linear chromosome for this organism and contains the largest number of genes so far discovered in a bacterium. It belongs to the phylum Actinobacteria. In addition, streptomycetes are used to produce the majority of antibiotics in the medical field. This study focuses on the effect of the deletion of Sco5218, which encodes a putative integral membrane protein and Sco5219, which encodes a putative lipoprotein in Streptomyces coelicolor. Both of these genes are found in a potential operon. The goal of this research is to construct the double mutant and discern differences in phenotype compared to the single deletion mutants of each gene. We hypothesize that the double mutant will have an enhanced effect on the sporulation defect of Streptomyces. Sco5218 is believed to contain an HD‐GYP domain through bioinformatics analysis and is highly conserved throughout the Streptomyces genus. The HD‐GYP domain is typically associated with cyclic di‐GMP specific phosphodiesterase activity. Following REDIRECT protocol, the Streptomyces coelicolor cosmid clone was introduced into E. coli via electroporation. PCR was then utilized for the deletion of Sco5218 and Sco5219. This PCR product was run onto a gel and purified. This pure PCR product was then transformed into the plasmid vector which will then be mated into Streptomyces coelicolor. The relevance of this research allows us to see the importance of the HD‐GYP domain in bacteria. We witnessed a slight delay in sporulation with the Sco5218 mutant deletion. With the added deletion of Sco5219 we hypothesize that there will be an enhanced delay in sporulation. If we witness a delay in the sporulation with the double mutant in Streptomyces we can deduce that both genes play a role in bacterial development. These findings will allow us to further delve into the study of other bacteria and have a basis for other orthologs containing the HD‐GYP domain.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Involvement of the two L-lactate dehydrogenase in development and pathogenicity in Fusarium graminearum.

Lactate dehydrogenase (LDH) widely exists in organisms, which catalyzes the interconversion of pyruvate into lactate with concomitant interconversion of NADH and NAD+. In this study, two L-type lactate dehydrogenase genes FgLDHL1 and FgLDHL2 were characterized in an ascomycete fungus Fusarium graminearum, a causal agent of wheat head blight. Both the single-gene deletion mutants of FgLDHL1 or FgLDHL2 exhibited phenotypic defects in vegetative growth, sporulation, spore germination, L-lactate biosynthesis and activity. Additionally, the two L-lactate dehydrogenases were involved in the utilization of carbon sources and maintenance of redox homeostasis during spore germination. Pathogenicity assays showed that ΔFgLDHL1 exhibits reduced virulence on wheat spikelets and on corn stigmas, suggesting that it was indirectly correlated with a reduced level of deoxynivalenol accumulation. These results indicate that FgLDHL1 and FgLDHL2 play multiple roles in the developmental processes and pathogenesis in F. graminearum, and help understand the functional diversity of D-/L-lactate dehydrogenase in phytopathogenic fungi.